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THE PRINCIPLES

OF

BACTERIOLOGY:

A PRACTICAL MANUAL FOR STUDENTS AND
PHYSICIANS.

BY
A. C. ABBOTT, M.D.,

FIRST ASSISTANT, LABORATORY OF HYGIENR, UNIVERSITY OF PENNSYLVANIA,
PHILADELPHIA.

SECOND EDITION, ENLARGED AND THOROUGHLY
REVISED.

With 94 Illustrations, of which |7 are Colored.

PHILADELPHIA:
LEA BROTHERS & CO.

1894.
&

A.7596(
1241 C24

Entered according to Act of Congress, in the year 1894, by
LEA BROTHERS & CO.,
In the Office of the Librarian of Congress at Washington, D.C.

DORNAN, PRINTER.

PREFACE TO THE SECOND EDITION.

THE cordial reception with which this book has met, -
and the demand for a second edition, afford the author
no small degree of gratification. In revising The Prin-
ciples of Bacteriology advantage has been taken of the
valuable suggestions kindly offered by the reviewers of
the first edition, for which the writer here acknowledges
his indebtedness.

The section of the work devoted to descriptive bac-
teriology has been somewhat extended, but no effort has
been made to cover the entire field, only those species
being introduced that are comparatively common, or of
importance in enabling the student to acquire a funda-
mental working knowledge capable of wider application.
Wherever practicable, these descriptions have been sup-
plemented by illustrations, for the majority of which
the author is responsible. The introduction of colored
figures in the text is a new feature in this edition, and
one which should increase its usefulness. A sketch of
the evolution of our knowledge upon immunity and
infection has been introduced, and an outline of appa-
ratus necessary for a beginner’s laboratory has been

appended.

iv PREFACE TO THE SECOND EDITION,

The original purpose of the book has been main-
tained, and it is hoped that the second edition, contain-
ing double the letterpress and treble the number of
illustrations found in its predecessor, will in some cor-
responding measure improve upon the service which
the work has apparently rendered to students and

physicians.
A.C, A,

PHILADELPHIA, July, 1894.

PREFACE TO THE FIRST EDITION.

Iv preparing this book the author has kept in mind
the needs of the student and practitioner of medicine,
for whom the importance ,of an acquaintance with
practical bacteriology cannot be overestimated.

It is to advances made through bacteriological re-
search that we are indebted for much of our knowledge
of the conditions underlying infection, and for the
elucidation of many hitherto obscure problems con-
cerning the etiology, the modes of transmission, and
the means of prevention of infectious maladies.

Only within a comparatively short time have students
and physicians been enabled to obtain the systematic
instruction in this science that is of value in aiding
them in their efforts to check disease. The rapid in-
crease in the number who are availing themselves of
these opportunities speaks directly for the practical
value of the science.

As the majority of those undertaking the study of
bacteriology do so with the view of utilizing it in
medical practice, and as many of these can devote to it
but a portion of their time, it is desirable that the
subject-matter be presented in as direct a manner as

possible.

Ed

V1 PREFACE TO THE FIRST EDITION,

Presuming the reader to be unfamiliar with the sub-
ject, the author has restricted himself to those funda-
mental features that are essential to its understanding.
The object has been to present the important ideas and
methods as concisely as is compatible with clearness,
and at the same time to accentuate throughout the
underlying principles which govern the work.

With the view of inducing independent thought on
the part of the student, and of diminishing the fre-
quency of that oft-heard query, ‘“ What shall I do
next?” experiments have been suggested wherever it
is possible. These have been arranged to illustrate the
salient points of the work and to attract attention to
the minute details, upon the observation of which: so
much in bacteriology depends.

A.C, A.

PHILADELPHIA, December, 1891.

CONTENTS.

INTRODUCTION.

The overthrow of the doctrine qf spontaneous generation
and the further application of the law: “Omne vivum ex
vivo ”’—The birth of modern bacteriology

CHAPTER I.

Definition of bacteria—Their place in nature—Differ-
ence between parasites and saprophytes—Nutrition of
bacteria—Products of bacteria—Their relation to oxygen
—Influence of temperature upon their growth

CHAPTER II.
Morphology of bacteria—Grouping—Mode of multi-
plication—Spore-formation—Motility .
CHAPTER III.

Principles of sterilization by heat—Methods employed
—Discontinued sterilization—Sterilization under pressure
—Apparatus employed—Chemical disinfection and steril-
ization

CHAPTER IV.

The principles involved in the methods of isolating bac-
teria in pure cultures by the plate method of Koch—
Materials employed .

CHAPTER V.

Preparation of nutrient media—Bouillon, gelatin, agar-
agar, potato, blood-serum, etc. .

PAGES

13-26

27-35

36-46

47-67

68-72

73-96

Vili . CONTENTS.

CHAPTER VI.

Preparation of tubes, flasks, etc., in which media are to
be preserved

CHAPTER VII.
Technique of sia plates—Esmarch tubes, Petri
plates, etc. :
CHAPTER VIII.
The incubating oven—Gas pressure regulator—Thermo-
regulator—Safety burner for heating the incubator
CHAPTERINX.

The study of colonies—Their naked-eye appearances
and their variations under different conditions—Difler-
ences in the structure of colonies of different species of
bacteria—Stab cultures—Slant cultures

CHAPTER X.

Methods of staining—Solutions employed—Preparation
and staining of cover-slips—Preservation and preparation
of tissues for section cutting—Staining of tissues, general
and special methods .

CHAPTER XI.

Systematic study of an organism—Points to be con-
sidered in identifying an organism as a definite species

CHAPTER XII.

Inoculation of animals—The various methods: subcu-
taneous ; intra-venous ; into the great serous cavities; into
the anterior chamber of the eye—Observation of animals
after inoculation

CHAPTER XIII

Post-mortem examination of animals—Bacteriological
examination of tissues at autopsy — Disposal of tissues and
disinfection of instruments after the examination

PAGES

97-100

101-110

111-118

119-123

124-158

159-184

185-203

204-208

CONTENTS. a

APPLICATION OF THE METHODS OF
BACTERIOLOGY. DESCRIPTIONS
OF SOME OF THE MORE IM-
PORTANT SPECIES.

CHAPTER XIV.

PAGES

To obtain material with which to begin work. . 209-212

CHAPTER XV.

Various experiments in sterilization, by steam and by
hotair ‘ : ‘ ; 5 : : . 213-217

CHAPTER XVI.

Suppuration — The staphylococcus pyogenes aureus—
Staphylococcus pyogenes albus and citreus—Streptococcus
pyogenes—Bacillus pyocyanus—General remarks . . 218-236

CHAPTER XVII.

Sputum septicemia—Septicemia caused by the micro-
coccus tetragenus—Tuberculosis 3 : ; ‘ . 237-248

CHAPTER XVIII.

Tuberculosis—Microscopic appearance of miliary tuber-
cles—Encapsulation of tuberculous foci—Diffuse caseation
—Cavity formation—Primary infection—Modes of infec-
tion—Location of the bacilli in the tissues—Staining pecu-
liarities of the bacillus tuberculosis—Organisms with which
the bacillus tuberculosis may be confounded—Points of
differentiation . : : ‘ : : f F . 249-268

CHAPTER XIX.

Glanders—Characteristics of the disease—Histological
structure of the glanders nodule—Susceptibility of differ-
ent animals to glanders—The bacillus of glanders, its
morphological and cultural peculiarities—Diagnosis of
glanders . : : 4 2 j : ‘ . 269-278

x CONTENTS.

CHAPTER XX.

The bacillus diphtherie—Its isolation and cultivation
—Morphological and cultural peculiarities—Pathogenic
properties—Variations in virulence .

CHAPTER XXII.

Typhoid fever—The bacillus typhi abdominalis, its mor-
phological, biological and pathogenic peculiarities—The
bacterium coli commune—Its resemblance to the bacillus of
typhoid fever—Its morphological, cultural, and pathogenic
properties—Differentiation between the two organisms

CHAPTER XXII.

The spirillum (comma bacillus) of Asiatic cholera—Its
morphological, biological, and pathogenic peculiarities—
The bacteriological diagnosis of Asiatic cholera

CHAPTER XXIII.

Organisms of interest, historic and otherwise, that have
been confounded with the spirillum of Asiatic cholera—
Their peculiarities and points of differentiation— Vibrio
proteus or bacillus of Finkler and Prior—=Spirillum tyroge-
num or cheese spirillum of Deneke—Spirillum of Miller—
Vibrio Metchnikovi . , i : i ‘ ‘

CHAPTER XXIV.

The bacillus anthracis and its effects upon animals—Its
peculiarities under varying conditions of surroundings

CHAPTER XXV.

The more important of the organisms found in the soil
—The nitrifying organisms—The bacillus of tetanus—
Bacillus of malignant cdema—Bacillus of symptomatic
anthrax

CHAPTER XXVI.

Infection and immunity—The types of infection, inti-
mate nature of the same—Septicemia —Toxemia—Vari-
ations in infectious processes—Immunity, natural and
aequired—The hypotheses that have been advanced in
explanation of immunity—Conclusions

PAGES

279-294

295-312

313-339

340-356

357-369

370-394

395-417

CONTENTS. si

CHAPTER XXVIII.

PAGES
Bacteriological study of water, air, soil—Methods em-
ployed—Precautions to be observed—Apparatus used and
methods of using them. : : : ; ; . 418-441
CHAPTER XXVIII.
Methods of testing disinfectants and antiseptics—Ex-
periments illustrative of the precautions to be taken—
Experiments in skin-disinfection . . : ; . 442-454

APPENDIX.

Apparatus necessary in a beginner’s bacteriological
laboratory . ‘ : ‘ : : ; ‘ : . 455-460

BACTERIOLOGY.

INTRODUCTION.

“Omne vivum ex vivo”—The overthrow of the doctrine of spontaneous
io bacteriological studies—The birth of modern bacteri-

THE study of Bacteriology may be said to have had
its beginning with the observations of Antony van
Leeuwenhoek in the year 1675. Though it is during
the past decade and a half that this line of research has
received its greatest impulse, yet by a review of the
developmental stages through which it has passed in its
life of more than two centuries we see that it has a
most interesting and instructive history. From the
very outset its history is inseparably connected with
that of medicine, and as it now stands its relations to
hygiene and preventive medicine are of the utmost im-
portance. It is, indeed, toa more intimate acquaintance
with the biological activities of the unicellular, vege-
table micro-organisms that modern hygiene owes much
of its value and our knowledge of infectious diseases
has reached the position it now occupies. Though the
contributions which have done most to place bacteriology
on the footing of a science are those of recent years, still,
during the earlier stages of its development, many obser-
vations were made which formed the foundation work

for much that was to follow. Before regularly begin-
2

14 BACTERIOLOGY.

ning our studies, therefore, it may be of advantage to
acquaint ourselves with the more prominent of these
investigations.

Antony van Leeuwenhoek, the first to describe the
bodies now recognized as bacteria, was born at Delft,
in Holland, in 1632. He was not considered a man of
liberal education, having been during his early years an
apprentice to a linendraper. During his apprenticeship
he learned the art of lens-grinding, in which he became
so proficient that he eventually perfected a simple lens
by means of which he was enabled to see objects of much
smaller dimensions than any hitherto seen with the best
compound microscopes in existence at that date. At
the time of his discoveries he was following the trade
of linendraper in Amsterdam.

In 1675 he published the fact that he had succeeded
in perfecting a lens by means of which he could detect
in a drop of rain-water living, motile “animalcules” of
the most minute dimensions—smaller than anything that
had hitherto been seen. Encouraged by this discovery,
he continued to examine various substances for the
presence of what he considered animal life in its most
minute form. He found in sea-water, in well-water, in
the intestinal canal of frogs and birds, and in his own
diarrhoeal evacuations, objects that differentiated them-
selves the one from the other, not only by their shape
and size, but also by the peculiarity of movement which
some of them were seen to possess. In the year 1683
he discovered in the tartar scraped from between the
teeth a form of micro-organism upon which he laid
special stress. This observation he embodied in the
form of a contribution which was presented to the
Royal Society of London on September 14, 1683. This

INTRODUCTION. 15

paper is of particular importance, not only because of
the careful, objective nature of the description given
of the bodies seen by him, but also for the illustrations
which accompany it. From a perusal of the text and
an inspection of the plates there remains little room
for doubt that Leeuwenhoek with his primitive lens
had seen the bodies now recognized as bacteria.

Upon seeing these bodies he was apparently very
much impressed, for he writes: “With the greatest
astonishment I saw that everywhere through the ma-
terial which I was examining were distributed animal-
cules of the most microscopic dimensions, which moved
themselves about in a remarkably energetic way.”

This observation was shortly followed by others of
an equally important nature. His field of observation
appears to have increased rapidly, for after a time he
speaks of bodies of much smaller dimensions than those
at first described by him.

Throughout all of Leeuwenhoek’s work there is a
egnspicuous absence of the speculative. His contribu-
tions are remarkable for their purely objective nature.

After the presence of these organisms in water, in the
mouth, and in the intestinal evacuations was made
known to the world, it is hardly surprising that they
were immediately seized upon as the explanation of the
origin of many obscure diseases. So ‘universal became the
belief in a causal relation between these “ animalcules”
and disease, that it amounted almost to a germ mania.
It became the fashion to suspect the presence of these
organisms in all forms and kinds of disease, simply
because they had been demonstrated in water.

Though nothing of value at the time had been done
in the way of classification, and still less in separating

16 BACTERIOLOGY.

and identifying the members of this large group, still,
the foremost men of the day did not hesitate to ascribe
to them not only the property of producing disease
conditions, but some even went so far as to hold that
variations in the appearance of symptoms of disease
were the result of differences in the behavior of the
organisms in the tissues.

Marcus Antonius Plenciz, a physician of Vienna in
1762, declared himself a firm believer in the work of
Leeuwenhoek, and based the doctrine which he taught
upon the discoveries of the Dutch observer and upon
observations of a confirmatory nature which he himself
had made. The doctrine of Plenciz assumed a causal re-
lation between the micro-organisms discovered and de-
scribed by Leeuwenhoek and all infectious diseases. He
claimed that the material of infection could be nothing
else than a living substance, and endeavored on these
grounds to explain the variations in the period of incuba-
tion of the different infectious diseases. He likewise
believed the living contagium to be capable of multipli-
cation within the body, and spoke of the possibility of
its transmission through the air. He claimed a special
germ for each disease, holding that just as from a given
cereal only one kind of grain can grow, so by the special
germ for each disease only that disease can be pro-
duced.

He found in all decomposing matters innumerable
minute “animalcule,” and was so firmly convinced of
their etiological relation to the process that he formu-
lated the law: that decomposition can only take place
when the decomposable material becomes coated with a
layer of the organisms, and can proceed only when they
increase and multiply.

INTRODUCTION. 17

However convincing the arguments of Plenciz appear,
they seem to have been lost sight of in the course of
subsequent. events, and by a few were even regarded
as the productions of an unbalanced mind. For ex-
ample, as late as 1820 we find Ozanam expressing him-
self on the subject as follows: “ Many authors have
written concerning the animal nature of the contagion
of infectious diseases ; many have indeed assumed it to
be developed from animal substances and that it is itself
animal, and possesses the property of life. I shall not
waste time in efforts to refute these absurd hypotheses.”

Similar expressions of opinion were heard from many
other medical men of the time, all tending in the same
direction, all doubting the possibility of these micro-
scopic creatures belonging to the world of living
things.

It was not until between the fourth and fifth decade
of the present century that by the fortunate coincidence
of a number of important discoveries the true relation
of the lower organisms to infectious diseases was scien-
tifically pointed out. With the investigations of Pasteur
upon the cause of putrefaction in beer and the souring
of wine; with the discovery by Pollender and Davaine
of the presence of rod-shaped organisms in the blood of
all animals dead of splenic fever, and with the progress of
knowledge upon the parasitic nature of certain diseases
of plants, the old question of “contagium animatum ”
again began to receive attention. It was taken up by
Henle, and it was he who first logically taught this
doctrine of infection.

The main point, however, which had occupied the
attention of scientific men from time to time for a
period of about two hundred years subsequent to

18 BACTERIOLOGY.

Leeuwenhoek’s discoveries, was the origin of these
bodies. Do they generate spontaneously, or are they
the descendants of pre-existing creatures of the same
kind? was the all-important question. Among the
participants in this discussion were many of the most
distinguished men of the day.

In 1749 Needham, who held firmly to the opinion
that the bodies which were creating such a general
interest developed spontaneously, as the result of vege-
tative changes in the substances in which they were
found, attempted to demonstrate by experiment the
grounds upon which he held this view. He maintained
that the bacteria which were seen to appear around a
grain of barley which was allowed to germinate in a
watch-crystal of water, which had been carefully cov-
ered, were the result of changes in the barley-grain
itself, incidental to its germination.

Spallanzani, in 1769, drew attention to the laxity of
the methods employed by Needham, and demonstrated
that if infusions of decomposable vegetable matter were
placed in flasks, which were then hermetically sealed,
and the flasks and their contents allowed to remain for
some time in a vessel of boiling water, neither living
organisms could be detected nor would decomposition
appear in the infusions so treated. The objection raised
by Treviranus, viz., that the high temperature to which
the infusions had been subjected had so altered them
and the air about them, that the conditions favorable to
spontaneous generation no longer existed, was met by
Spallanzani by gently tapping one of the flasks that
had been boiled, against some hard object until a
minute crack was produced; invariably organisms and
decomposition appeared in the flask thus treated.

INTRODUCTION. 19

From the time of the experiments of Spallanzani
until as late as 1836 but little advance was made in the
elucidation of this obscure problem.

In 1836 Schulze attracted attention to the subject by
the convincing nature of his investigations. He showed
that if the air which gained access to boiled infusions
was robbed of its living organisms by being caused to
pass through strong acid or alkaline solutions no de-
composition appeared and living organisms could not
be detected in the infusions. Following quickly upon
this contribution came Schwann, in 1837, and somewhat
later (1854) Schroder and Dusch, with similar resnlts
obtained by somewhat different means. Schwann de-
prived the air which passed to his infusions of its living
particles by passing it through highly-heated tubes ;
whereas Schréder and Dusch, by means of cotton-wool
interposed between the boiled infusion and the outside
air, robbed the air passing to the infusions of its-organ-
isms by the simple process of filtration. In 1860
Hoffmann and in 1861 Chevreul and Pasteur demon-
strated that the precautions taken by the preceding in-
vestigators for rendering the air which entered these
flasks free from bacteria were not necessary; that all
that was necessary to prevent the access of bacteria to
the infusions in the flasks was to draw out the neck of
the flask into a fine tube, bend it down along the side
of the flask and then bend it up again a few inches
from its extremity, and leave the mouth open. The
infusion was then to be boiled in the flask thus prepared
and the mouth of the tube left open. The organisms
which now fall into the tube will be arrested by the
drop of water of condensation which collects at its
lowest angle, and none can enter the flask.

20 BACTERIOLOGY.

Conclusive as this work may appear, there still ex-
isted a number of doubters who required further proof
that “spontaneous generation ” was not the explanation
for the mysterious appearance of these minute living
objects, and it was not until some time later that Tyn-
dall, in his well-known investigations upon the floating
matters in the air, demonstrated again that the presence
of living organisms in decomposing fluids was always
to be explained either by the pre-existence of similar
living forms in the infusion or upon the walls of the
vessel containing it, or by the infusion having been ex-
posed to air which had not been deprived of its organisms.

Throughout all the work bearing upon this subject,
from the time of Spallanzani to that of Tyndall, certain
irregularities were constantly appearing. It was found
that particular substances required to be heated for a
much longer time than was necessary to render other
substances free from living organisms, and even under
the most careful precautions decomposition would occa-
sionally appear.

In 1762 Bonnet, who was deeply interested in this
subject, suggested, in reference to the results obtained
by Needham, the possibility of the existence of “ germs,
or their eggs,” which have the power to resist the tem-
perature to which some of the infusions employed in
Needham’s experiments had been subjected.

More than a hundred years after Bonnet had made
this purely speculative suggestion it became the task of
Ferdinand Cohn, of Breslau, to demonstrate its accuracy.

Cohn repeated the foregoing experiments with like
results. He concluded that the irregularities could only
be due to either the existence of more resistant species
of bacteria or to more resistant stages into which certain

INTRODUCTION. 21

bacteria have the property of passing. After much
work he demonstrated that certain of the rod-shaped
organisms possess the power of passing into a resting
or spore stage in the course of their life history, and
when in this stage they are much less susceptible to the
deleterious action of high temperatures than when they
are growing as normal vegetative forms. With the
discovery of these more resistant spores the doctrine of
spontaneous generation received its final blow. It was
no longer difficult to explain the irregularities in the
foregoing experiments, nor was it any longer to be
doubted that putrefaction and fermentation were the
result of bacterial life and not the cause of it, and that
these bacteria were the offspring from pre-existing
similar forms. In other words, the law of Harvey,
Omne vivum ex ovo, or its modification, Omne vivum ex
vivo, was shown to apply not only to the more highly -
organized members of the animal and vegetable king-
doms, but to the most microscopic, unicellular creatures
as well.

The establishment of this point served as an impetus
to further investigations, and as the all-important ques-
tion was that concerning the relation of these micro-
scopic organisms to disease, attention naturally turned
to this channel of study. Even before the hypothesis of
spontaneous generation had received its final refutation
a number of observations of a most important nature
had been made by investigators who had long since
ceased to consider spontaneous generation as a tenable
explanation of the origin of the microscopic living
particles.

In the main, these studies had been conducted upon

wounds and the infections to which they are liable ; in
oi

22 BACTERIOLOGY.

fact, the evolution of our knowledge of: bacteriology to
the point it now occupies is so intimately associated
with this particular line of investigation that a few
historical facts in connection with it may not be without
interest.

The observations of Rindfleisch, in 1866, in which he
describes the presence of small, pin-head points in the
myocardium and general musculature of individuals
that have died as a result of infected wounds offer,
probably, the first reliable contribution to this subject.
He studied the tissue changes round about these points
to the stage of miliary abscess formation. He refers
to the organisms as “ vibrios.” Almost simultaneously
Von Recklinghausen and Waldeyer described similar
changes that they had observed in pyemia and occa-
sionally secondary to typhoid fever. Von Reckling-
hausen believed the granules seen in the abscess-points
to be micrococci and not tissue detritus, and gave as
the reason that they were regular in size and shape,
and gave specific reactions with particular staining
fluids. Birch-Hirschfeld was able to trace bacteria
found in the blood and organs to the wound as the
point of entrance, and believed both the local and con-
stitutional condition to stand in direct ratio to the
number of spherical bacteria present in the wound.
He observed also that as the organisms increased in
number they could often be found within the bodies ot
pus corpuscles. His studies of pyzmia led him to the
important conclusion that in this condition micro-
organisms were always present in the blood.

Of immense importance to the subject were the in-
vestigations of Klebs, made at the Military Hospital
at Carlsruhe in 1870-71. He not only saw, as others

INTRODUCTION. 23

before him had done, that bacteria were present in dis-
eases following upon the infection of wounds, but
described the manner in which the organisms had
gained entrance from the point of injury to the internal
organs and blood. His opinion was that the spherical
and rod-shaped bodies that he saw in the secretions of
wounds were closely allied, and gave to them the designa-
tion “ microsporon septicum.” His opinion was that the
organisms gained access to the tissues round about the
point of injury both by the aid of the wandering leuco-
cytes and by being forced through the connective-tissue
lymph spaces by the mechanical pressure of muscular
contraction.

On erysipelatous inflammations secondary to injury
important investigations were also being made. Wilde,
Orth, Von Recklinghausen, Lukomsky, Billroth, Ehr-
lich, Fehleisen, and others agreeing that in these condi-
tions micro-organisms could always be detected in the
lymph channels of the subcutaneous tissues; and
through the work of Oertel, Nassiloff, Classen, Letze-
rich, Klebs,, and Eberth the constant presence of
bacteria in the diphtheritic deposits at times seen
on open wounds was established. Simple and natural
as all this may seem to us now, the stage to which the
subject had developed when these observations were
recorded did not admit of their meeting with uncondi-
tional acceptance. The only strong argument in favor
of the etiological relation of the organisms that had been
seen, in production of the diseases with which they were
associated, was the constancy of this association. No
efforts had been made to isolate them, and few or none
to reproduce the pathological conditions by inoculation.
Moreover, not a small number of investigators were

24 BACTERIOLOGY.

skeptical as to the importance of these observations ;
many claimed that micro-organisms were normally
present in the blood and tissues of the body, and some
even believed that the organisms seen in the diseased
conditions were the result rather than the cause of the
maladies. Itis hardly necessary to do more than say
that both of these views were purely speculative, and
have never had a single reliable experimental argu-
ment in their favor. Billroth and Tiegel, who held to
the former opinion, did endeavor to prove their posi-
tion through experimental means, but the methods
employed by them were of such an untrustworthy
nature that the fallacy of deductions drawn from them
was very quickly demonstrated by subsequent investi-
gators. Their method for demonstrating the presence
of micro-organisms in normal tissue was to remove bits
of tissue from the healthy animal body with heated in-
struments and drop them into hot melted paraffin, hold-
ing that all living organisms on the surface of the tissues
would be destroyed by the high temperature, and that
if decomposition should subsequently occur, it would
prove that it was the result of the growth of bacteria in
the depths of the tissue to which the heat had not pene-
trated. Decomposition did usually set in, and they
accepted this as proof of the accuracy of their view.
Attention was, however, shortly called to the fact that
in cooling the contraction of the paraffin caused small
rents and cracks into which dust, and bacteria lodged
upon it, could accumulate and finally gain access to
the tissues with the occurrence of decomposition as a
consequence. Their results were thus explained after a
manner analogous to that employed by Spallanzani, in
1769, in demonstrating to Treviranus the fallacy of the

INTRODUCTION. 25

opinion held by him and the accuracy of his own
views, viz., that it was always through the access of
organisms from without that decomposition primarily
originates. (See page 18.)

Under the most careful precautions, against which
no objection could be raised, these experiments of Bill-
roth and Tiegel were repeated by Pasteur, Burdon-
Sanderson, and Klebs, but with failure in each and
every instance to demonstrate the presence of bacteria
in the healthy living tissues.

The fundamental researches of Koch (1881) upon
pathogenic bacteria and their relation to the infectious
diseases of animals differed from those of preceding
investigators in many important respects. The scien-
tific methods of analysis with which each and every
obscure problem was met as it arose served at once to
distinguish the worker as a pioneer in this hitherto
but partly cultivated domain. The outcome of these
experiments was the establishment of a foundation upon
which bacteriology of the future was to rest. He, for
the first time, demonstrated that distinct varieties of
infection, as evidenced by anatomical changes, are due
in many cases to the activities of particular specific
organisms, and that by proper methods it is possible to
isolate these organisms in pure culture, to cultivate them
indefinitely, to reproduce the conditions by inoculation
of these pure cultures into susceptible animals, and, by
continuous inoculation from an infected to a healthy ani-
mal, to continue the disease at will. By the methods
that he employed he demonstrated a series of separate
and distinct diseases that can be produced in mice and
rabbits by the injection into their tissues of putrid
substances. The diseases known as septicemia of mice,

26 BACTERIOLOGY.

a disease characterized by progressive abscess forma-
tion, and pyzemia and septicemia of rabbits, are among
the affections produced by him in this way. It was in
the course of this work that the Abbe system of sub-
stage condensing apparatus was first used in bacteriology ;
that the aniline dyes suggested by Weigert were brought
into general use; that the isolation and cultivation of
bacteria in pure culture on solid media was shown to
be possible; and that animals were employed as a means
of obtaining pure cultures of pathogenic bacteria.

With the bounteous harvest of original and impor-
tant suggestions that was reaped from Koch’s classical
series of investigations bacteriology reached an epoch
in its development, and at this period modern bacteri-
ology may justly be said to have had its birth.

Nore.—I have presented only the most prominent
investigations that will serve to indicate the lines along
which the subject has developed. For a more detailed
account of the historical development of the work the
reader is referred to Loefiler’s Vorlesungen iiber die
geschichtliche Entwickelung der Lehre von den’ Bacterien,
upon which I have drawn largely in preparing the
foregoing sketch.

CHAPTER I.

Definition of bacteria—Their place in nature—Difference between parasites
and saprophytes—Nutrition of bacteria—Products of bacteria—Their relation
to oxygen—Influence of temperature upon their growth.

By the term bacteria is understood that large group
of minute vegetable organisms which multiply by a
process of transverse division. They are spherical,
oval, rod-like, and spiral in shape, and are commonly
devoid of chlorophyll. Owing to the absence of
chlorophyll from their composition the bacteria are
forced to obtain their nutritive materials from organic
matters as such, and lead, therefore, either a sapro-
phytic? or parasitic® form of existence.

Their life processes are so rapid and energetic that
they result in the most profound alterations in the
structure and composition of the materials in and upon
which they are developing.

Decomposition, putrefaction, and fermentation result
from the activities of the saprophytic bacteria, while
the changes brought about in the tissues of their host

1 Chlorophyll is the green coloring matter possessed by the higher plants
by means of which they are enabled in the presence of sunlight to decom-
pose carbonic acid (CO,) and ammonia (NHs;) into their elementary constitu-
ents.

2 A saprophyte is an organism that obtains its nutrition from dead organic
matter.

3 A parasite lives always at the expense of some other living, organic crea-,
ture, known as its host, and in the strictest sense of the word cannot develop
upon dead matter. There is, however, a group of so-called “facultative” —
saprophytes and parasites which possess the power of accommodating them-
selves to existing surroundings—at one time leading a parasitic, at another
time a saprophytic form of existence.

28 BACTERIOLOGY.

by the pure parasitic forms, find expression in disease
processes and not infrequently complete death.

The rdle played in nature by the saprophytic bacteria
is a very important one. Through their presence the
highly complicated tissues of dead animals and vegeta-
bles are resolved into the simpler compounds, carbonic
acid, water, and ammonia, in which form they may be
taken up and appropriated as nutrition by the more
highly organized members of the vegetable kingdom.
It is through this ultimate production of carbonic acid,
ammonia, and water by the bacteria, as end-products in
the processes of decomposition and fermentation of the
dead animal and vegetable tissues, that the demands
of growing vegetation for these compounds are sup-
plied.

The chlorophyll plants do not possess the power of
obtaining their carbon and nitrogen from such highly
organized and complicated substances as serve for the
nutrition of bacteria, and as the production of these
simpler compounds (CO,, NH;, H,O) by the animal
world is not sufficient to meet the demands of the chlo-
rophyll plants, the importance of the part played by
bacteria in making up this deficit cannot be overesti-
mated. Were it not for the activity,of these micro-
scopic living particles, all life upon the surface of the
earth would undoubtedly cease. Deprive higher vegeta-
tion of the carbon and nitrogen supplied to it as a result
of bacterial activity, and its development comes rapidly
to an end; rob the animal kingdom of the food-stuffs
supplied to it by the vegetable world, and life is no
longer possible.

It is plain, therefore, that the saprophytes, which
represent the large majority of all bacteria, must be

PARASITES AND SAPROPHYTES. 29

looked upon by us in the light of benefactors, without
which existence would be impossible.

With the parasites, on the other hand, the conditions
are far from analogous. Through their activities there
is constantly a loss, rather than a gain, to both the
animal and vegetable kingdoms. Their host must
always be a living body in which exist conditions favor-
able to their development and from which they appro-
priate substances necessary to the health and life of the
organism to which they may have found access ; at the
same time they eliminate substances as products of their
nutrition that are directly poisonous to the tissues in
which they are growing.

In their relations to humanity the positions occupied
by the two biologically different groups, the saprophytes
on the one hand and the parasites on the other, are
diametrically opposite ; the saprophytic forms standing
in the relation of benefactors, in resolving dead animal
and vegetable bodies into their component parts, which
serve for food for living vegetation, and, at the same
time, removing from the surface of the earth the re-
mains of all dead organic substances ; while the parasitic
group exists only at the expense of the more highly
organized members of both kingdoms. It is to the
parasitic group that the pathogenic’ organisms belong.

In addition to the saprophytes that are concerned
in the changes to which allusion has just been made,
there exist other saprophytic forms whose life processes
result in specific changes of most interesting and im-
portant natures. Some of these are characterized by
their property of producing pigments of different color ;

1 Pathogenic organisms are those which posséss the property of producing
disease.

30 BACTERIOLOGY.

these are known as the chromogenic! forms. Just what
their exact réle in Nature is, it is difficult to say ; but it
is probable that in addition to their most conspicuous
function of color production, they are also in some
way concerned in the great process of disintegration
which is constantly going on in all dead organic sub-
stances.

Others, the so-called photogenic, or phosphorescent
bacteria, possess the property of producing light or of
illuminating the medium on which they grow by a
peculiar phosphorescence. These are found in sea-water
and in decomposing phosphorescent fish and meat.

Still others, the so-called zymogenic bacteria, are con-
cerned in the various fermentations, while the putrefac-
tive or saprogenic bacteria are those that produce the
particular fermentation that we know as putrefaction.
Another very important saprophytic group comprises
the so-called nitrifying and denitrifying bacteria, whose
activities result in specific forms of fermentation—the
former oxidizing ammonia to nitrous and nitric acid,
the latter reducing nitric acid to nitrous acid and am-
monia. The so-called thiogenic bacteria convert sul-
phuretted hydrogen into higher sulphur compounds.

We have said that through the agency of chlorophyll,
in the presence of sunlight, the green plants are enabled to,
obtain the amount of nitrogen and carbon which is neces-
sary to their growth from such simple bodies as carbon
dioxide and ammonia, which they decompose into their
elementary constituents. The bacteria, on the other
hand, owing to the absence of chlorophyll from their
tissues, do not possess this power. They must, there-

+ Chromogenic, possessing the property of generating color.

NUTRITION OF BACTERIA. 81

fore, have their carbon and nitrogen presented as such,
in the form of decomposable organic substances.

In general, the bacteria obtain their nitrogen most
readily from soluble albumins, and, to a certain degree,
but by no means so easily, from salts of ammonia. In
some of Nigeli’s experiments it appeared probable that
they could obtain the necessary amount of nitrogen
from salts of nitric acid. At all events, he was able
in certain cases to demonstrate a reduction of nitric to
nitrous acid, and ultimately toammonia. Nevertheless,
in all of these experiments circumstances point to the
probability that the nitrogen obtained by the bacteria
for building up their tissues in the course of their
development, was derived from some source other than
that of the nitric acid or the nitrates, and fhat the
reduction of this acid was most probably a secondary
phenomenon.

For the supply of carbon, many of the carbon com-
pounds serve as sources upon which the bacteria can
draw. The carbon deficit, for example, can be obtained
from sugar and bodies of like composition; from glyce-
rine and many of the fatty acids; and from the alkaline
salts of tartaric, citric, malic, lactic, and acetic acids.
In some instances carbon compounds, which when pres-
ent in concentrated form inhibit the growth of bac-
teria, may, when highly diluted, serve as nutrition for
them. Salicylic acid and ethyl alcohol come under this
head.

In addition to carbon and nitrogen, water is essential
to the life and development of bacteria. Without it
no development occurs, and in many cases drying the
organisms results in their death. Certain forms, on the
contrary, though incapable of multiplying when in the

32 BACTERIOLOGY.

dry stage, may be completely deprived of their water
without causing them to lose the power of reproduction
when favorable conditions reappear.

The closer study of the bacteria, and a more intimate
acquaintance with their nutritive changes, demonstrate
an appreciable variability in the character of the sub-
stances best suited for the nutrition of different species,
one requiring a tolerably concentrated form of nutri-
tion, while another needs but a very limited amount of
proteid substance for its-development. Certain mem-
bers bring about most profound alterations in the media
in which they exist, while others produce but little
apparent change. In one case alterations in the reac-
tion of the media will be conspicuous, while in an-
other no such variation can be detected. With the
growth of some forms products resulting from pro-
cesses of fermentation appear. Other varieties produce
poisons of remarkable degrees of toxicity, while the
growth of others may be accompanied by the bodies
characteristic of putrefaction.

For the normal development of bacteria it is not only
essential that the sources from which they can obtain the
necessary nutritive elements should exist, but account
must also be taken of the products of growth of the
organism in these substances. Nitrogen and carbon
compounds in the proper form to be taken up and
appropriated by the organism may exist in sufficient
quantities, and still the growth of the organism after a
very short time be entirely checked, owing to the pro-
duction during their growth of substances inhibitory to
their further development. Most conspicuous are the
changes produced by the growing bacteria in the chemical
reaction of the media. Since the majority of them grow

PRODUCTS OF BACTERIA, 83

best in media of a neutral or very slightly alkaline
reaction, any excessive production of alkalinity or
acidity, as a product of growth, arrests development,
and no evidence of life or further multiplication can be
detected until this deviation from the neutral reaction
has been corrected.

Most favorable for the development of bacteria are
neutral or very slightly alkaline solutions of albumin in
one form or another.

Of considerable importance and interest in the study
of the nutritive changes of’ bacteria is the difference in
their relation to oxygen. With certain forms oxygen
is essential for the proper performance of their func-
tions, while with another group no evidence of life can
be detected under the access of oxygen, and in a third
group oxygen appears to play but an unimportant part,
for development occurs as well with as without it. It
was Pasteur who first demonstrated the existence of
species in the bacteria family which not only grow and
multiply and perform definite physiological functions
without the aid of oxygen, but to the existence of
which oxygen is positively harmful. To these he gave
the name anaérobic bacteria, in contradistinction to
another group for the proper performance of whose
functions oxygen is essential; these he called aérobic
bacteria. In addition to these, there is a third group
for the maintenance of whose existence the absence or
presence of oxygen is apparently of no moment—their
development progresses as well with as without it ; these
represent the class known as facultative in their re-
lation to this gas. It is in this third group, the faculta-
tive, that the majority of bacteria belong. Though the
multiplication of the facultative varieties is not inter-

34 BACTERIOLOGY.

fered with by either the presence or absence of oxygen,
yet experiments demonstrate that the products of their
growth are different under the varying conditions of
absence or presence.of this gas.

For example: in the case of certain of the chromo-
genic forms the presence or absence of oxygen has a
very decided effect upon the production of the pigments
by which they are characterized.

Notr.—Observe the difference between the intensity
of color produced upon the surface of the medium and
that along the track of the needle in stab-cultures of
the bacillus prodigiosus and of the spirillum rubrum.
With the former the red color is apparently a product
dependent upon the presence of oxygen, while in the
latter the greatest intensity of color occurs at the point
farthest removed from the action of oxygen.

Another element which plays a most important part
in the biological functions of these organisms is the
temperature under which they exist. The extremes of
temperature under which most bacteria are known to
grow range from 5.5° C. to 48° C. At the former
temperature development is hardly appreciable; it be-
comes more and more active until 38° C. is reached,
when it is at its optimum, and, as a rule, ceases with
43° C.; though it is said that species exist that will
multiply at as high a temperature as 60° C. and others
as low as 0° C. The most favorable temperature for
the development of pathogenic bacteria is that of the
human body, viz., 37.5° C. There are a number of
bacteria commonly present in water, the so-called
normal water bacteria, that grow best at about 20° C.

INFLUENCE OF TEMPERATURE ON BACTERIA. 85

In general then, from what has been learned, it may
be said that for the growth and development of bacteria
organic matter of a neutral or slightly alkaline reac-
tion, in the presence of moisture and at a suitable tem-
perature, is necessary. From this can be formed some
idea of the omnipresence in Nature of these minute
vegetable forms. Everywhere that these conditions
obtain bacteria can be found.

a iad a

CHAPTER II+

Morphology! of bacteria—Grouping—Mode of multiplication—Spore-forma-
tion—Motility.
ey,
In structure the bacteria are unicellular, and are seen

to exist as spherical, rod- or spiral-shaped bodies.
They always develop from pre-existing cells of the same
character and never appear spontaneously.

The classifications of the older authors and of the
botanists are usually upon purely morphological pecu-
liarities, and in consequence are more or less compli-
cated. The present tendency is to simplify this mor-
phological classification, and to bring the bacteria into
three great groups, with their subdivisions ; each group
comprising those members whose individual outline is
that either of a sphere, a rod, or a spiral.

To these three grand divisions are given the names
cocci or micrococci, bacilli, and spirilla.

In the group micrococct belong all spherical forms, '
i. e., all those forms the isolated individual members of
which are of equal diameter in all directions. (See Fig.
l,abed.)

The bacilli comprise all oval or rod-formed bacteria.
(See Fig. 2.)

To the spirilla belong all organisms that are curved
when seen in short segments, or when in longer threads
are twisted in the form of a corkscrew. (See Fig. 3.)

The micrococci are subdivided according to their
grouping, as seen in growing cultures, into staphylococci

1 Morphology, pertaining to shape ; outline.

MORPHOLOGY. 387

Fie. 1,

re}
pod ; og
0
8 Bede BE %o,
g a af
lo 0000
fe)
68 os} SaQ00
a b
aD a
eo
Pa age % wG of e Q
o%
© ye g % OG Eo So
ge, Yo oe g & 4)
Oe oo
ye e W o% of
c ad

i. 6. Streptococci. e¢. Diplococei. d. Tetrads.

. ¢, Sarcine.
Fig. 2.
A ‘ oy ~\ \ | XN
Sas ~ NM Ss
Sanna \s NN) aoe Fh
ee v4 ~
precy 4 “oS.
(77 b ¢
= ue
IN Ny
2 - .
--\ | . LY (
d e tf :
a. Bacilli in pairs. b. Single bacilli. ¢ and d. Bacilli in threads. eand/.
Bacilli of variable morphology.
Fic. 8
a, “ ,

‘Ps eere, ) San (
Fy » / A 1
Py, x
ad Pa \ ‘\ MA <
a
é
a ne d. Spirilla in short eee and longer threads—the so-called

comma forms and spirals. 6. The forms known as spirochete. c. The thick
spirals sometimes known as vibrios.

—those growing in masses like clusters of grapes (see
Fig. 1, a); streptococci—those growing in chains con-
3

38 BACTERIOLOGY.

sisting of a number of individual cells strung together
like beads or- pearls upon a string (see Fig. 1, 0);
diplococci-—those growing in pairs (Fig. 1, ¢); tetrads
—those developing as fours (Fig. 1, d); and sarcine—
those dividing into fours, eights, etc., as cubes—that is,
in contra-distinction to all other forms, the segmenta-
tion, which is rarely complete, takes place in three
directions of space, so that when growing the bundle of
segmenting cells presents somewhat the appearance of
a bale of cotton (Fig. 1, e).

To the bacilli belong all rod-shaped organisms, 1. ¢.,
those in which one diameter is always greater than the
other.

o9 ie y- \~
<P rX one
, aN a ae XZ Are
. a Pan eS J

a b c d

a. Bacillus subtilis with spores. b. Bacillus anthracis with spores. c. Clos-
tridium form with spores. d. Bacillus of tetanus with end spores,

In this group are found those organisms the life cycle
of many of which present deviations from the simple
rod shape. Many of them in the course of development
increase in length into long threads, along the course of
which traces of segmentation may usually be found—
the anthrax bacillus and bacillus subtilis are conspicuous
examples of this. Again, under certain conditions,
many of them possess the property of forming within
the body of the rods oval, glistening spores (see Fig. 4),
and if the conditions are not altered the rods may
entirely disappear, so that nothing may be left in the
culture but these oval forms. In some of them this

MORPHOLOGY. 39

phenomenon of spore-formation is accompanied by an
enlargement or swelling of the bacillus at the point at
which the spore is located (see Fig. 4, c and d). Again,
many of them, from unfavorable conditions of nutrition,
aération, or temperature undergo pathological changes
—that is, the individuals themselves experience altera-
tions in their protoplasm which result in distortion of
their outline, and the appearance of the so-called “ in-
volution forms.” (See Fig. 5, a and 6.) In all of

Fie. 5.
@
ibe 4 ae OD 5 ¢
ee ? (Ce 2
a, | 4 As 6
ch ¢ @ 4
oF
a t)

a, Spirillum of Asiatic cholera (comma bacillus). b. Involution forms of
this organism as seen in old cultures.

these conditions, however, so long as death has not
actually occurred, it is possible under favorable condi-
tions to cause these forms to revert to the rod-shaped
ones from which they originated.

It must be borne in mind, though, that it is never
possible by any means to bring about changes in these
organisms that will result in the permanent conversion
of the morphology of the members of one group into
that of another—that is, one can never produce bacilli
from micrococci or vice versa, and any evidence which
may be presented to the contrary is based upon untrust-
worthy methods of observation.

Not infrequently bacteria may be observed irregu-
larly massed together as a pellicle. When in this
condition they are held together by a gelatinous

AO BACTERIOLOGY.

material, and are known as zooglea of bacteria. (See
Fig. 6.)

Very short oval bacilli may sometimes be mistaken
for micrococci, and at times micrococci in the stage of
segmentation into diplococci may be mistaken for short
bacilli; but by careful inspection it will always be
possible to detect a continuous outline along the sides of
the former and a slight transverse indentation or par-
tition-formation between the segments of the latter.
The high index of refraction of spores, the property

Zoogloea of bacilli.

which gives to them their glistening appearance, will
always serve to distinguish them from micrococci. This
difference in refraction will be especially noticed if the
illumination from the reflector of the microscope with
which they are to be examined is reduced to the smallest
possible bundle of light-rays. The spores, moreover,
take up the staining reagents much less readily than
do the micrococci. The most reliable differential point,
however, is the property, possessed by the spores, of
developing into bacilli; and of the spherical organism
with which it has been confounded, of producing other
micrococci of the same round form.

For convenience, a common classification of the
bacilli is that based upon constant characteristics which
are seen to appear in the course of their development

MORPHOLOGY. 41

under special conditions—certain of them possessing the
power of forming spores, while from others this pecu-
liarity is absent.
_ As yet but little is known of the life history of the
spiral forms. Efforts toward their cultivation under
artificial conditions have thus far been successful in
only a few cases. Morphologically, they are thread-
or rod-like bodies which are twisted into the form of
spirals, In some of them the turns of the spiral -are
long, in others quite short. They are motile, and
multiply apparently by the simple process of fission.’
The micrococci develop by simple fission. When
development is in progress a single cell will be seen to
elongate slightly in one of its diameters. Over the
centre of the long axis thus formed will appear a slight
indentation in the outer envelope of the cell; this
indentation will increase in extent until there exist
eventually two individuals which are distinctly spheri-
cal, as was the parent from which they sprang, or they
will remain together for a time as diplococci. The sur-
faces now in juxtaposition are flattened against one
another, and not infrequently a fine, pale dividing line
may be seen between the two cells. (See Fig. 1, c and
d.) A similar division in the other direction will
now result in the formation of a group of forms as
tetrads.
\ In the formation of staphylococci such division occurs
irregularly in all directions, resulting in the produc-
tion of the clusters in which these organs are com-
monly seen. (See Fig. 1, a). With the streptococci,
however, the tendency is for the segmentation to con-
tinue in one direction only, resulting in the production

1 Dividing into two transversely.

‘49 BACTERIOLOGY.

of long chains of 4, 8, and 12 individuals. (See Fig.
1, 6).

The sarcine divide more or. less regularly in three
directions of space, but instead of becoming separated
the one from the other as single cells, the tendency is
for the segmentation to be incomplete ; the cells remain-
ing together in masses. 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 cotton
or a packet of rags. (See Fig. 1, e.)

The multiplication of bacilli is in the main similar
to that given for the micrococei. A dividing cell will
elongate slightly in the direction of its long axis ; an
indentation will appear about midway between its poles,
and will become deeper and deeper until eventually two
daughter cells will be formed. This process may occur
in such a way that the two young bacilli will adhere
together by their adjacent ends in much the same way
that sausages are seen to be held together in strings
(Fig. 2, f), or the segmentation may take place more at
right angles to the long axis, so that the proximal ends
of the young cells are flattened while the distal extremi-
ties may be rounded or slightly pointed (Fig. 2, e).
The segmentation of the anthrax bacillus, with which
we are subsequently to become acquainted, results, when
completed, in an indentation of the adjacent extrem-
ities of the young segments, so that by the aid of
high magnifying powers these surfaces are seen to be
actually concave. (See Fig. 76.) Bacilli never divide
longitudinally.

With the spore-forming bacilli, under favorable con-
ditions of nutrition and temperature, the same is seen

SPORE-FORMATION. 43

to occur during vegetation, but as soon as these condi-
tions become altered, either by the exhaustion of nutri-
tion, the presence of detrimental substances, unfavorable
temperatures, etc., there appears the stage in their life
cycle to which we have referred as “spore-formation.”
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 than is possible for them when
in the growing or vegetative condition.

In the spore, resting, or permanent stage, as it is
called, no evidence of life whatever is given by the
spores, though as soon as the conditions which favor
their germination have been renewed, these spores de-
velop again into the same kind of cells as those from
which they originated, and the appearances observed in
the vegetative or growing stage of their history are
repeated.

Multiplication of spores, as such, does not occur ;
they possess the power of developing into individual
rods of the same nature as those from which they were
formed, but not of giving vise to a direct reproduction of
spores.

When the conditions which favor spore-formation
present, the protoplasm of the vegetative cells is seen
to undergo a change. It loses its normal homogeneous
appearance and becomes marked by granular, refrac-
tive points of irregular shape and size. These eventu-
ally coalesce, leaving the remainder of the cell clear
and transparent. When this coalescence of highly
refractive particles is complete the spore is perfected.
In appearance, the spore is oval or round, very highly
refractive, and of a glistening appearance. It is easily
differentiated from the remainder of the cell, which now

44 BACTERIOLOGY.

consists only of a cell-membrane and a transparent,
clear fluid which surrounds the spore. Eventually
both the cell-membrane and its fluid contents disappear,
leaving the oval spore free.

The spore, when perfectly developed, is highly glis-
tening, oval in contour, and has the appearance of being
surrounded by a dark, sharply defined border. It pos-
sesses no motion other than the mechanical tremor com-
mon to all insoluble microscopic particles suspended in
fluids, and it remains quiescent until there appear con-
ditions favorable to its subsequent development into the
vegetative form from which it originated. Occasionally
the membrane of the vegetative cell in which the spore
is formed does not disappear from around it, and the
spore may then be seen lying in a very delicate tubular
envelope. Now and then remnants of the envelope may
be noticed adhering to the spore which has not yet be-
come completely free.

When stained, the spore-containing cells do not take
up the dyes in a homogeneous way. By the ordinary
methods the spores do not stain, so that they appear in
the stained cells as pale, transparent, oval bodies, sur-
rounded by the remainder of the cell, which has taken
up the staining.

A single cell produces but one spore. This may be
located either at an extremity or in the centre of the
cell. (Fig. 4.)

Occasionally spore-formation is accompained by an -
enlargement of the cell at the point at which the process
is in progress. As a result, the outline of the cell loses
its regular rod shape and becomes that of a club, a
drum-stick, or a lozenge, depending upon whether the

MOTILITY. 45

location of the spore is to be at the pole or in the centre
of the cell. (See Fig. 4, e and d.)

In addition to the property of spore-formation there is
another striking difference between the members of the
rod-shaped organisms, namely, the property of motility
which many of them are seen to possess. This power
of motion is due to the possession by the motile bacilli
of very delicate, hair-like appendages or flagella, by the
lashing motions of which the rods possessing them are
propelled through the fluid. In some cases the flagella

ey Fig. 7.

a, spiral forms with a flagellum at only one end; 8, bacillus of typhoid
fever with flagella given off from all sides; c, large spirals from stagnant
water with wisps of flagella at their ends (spirillum undula).

are located at but one end of a bacillus, either singly or
in a bunch ; again, they may be seen at both poles, and
in some cases, especially with the bacillus of typhoid
fever, they are given off from the whole surface of the
rod. (See Fig. 7.)

For a long time the motility of bacteria was only
supposed to be due to the possession of some such form
of locomotive apparatus because similar appendages
had been seen in certain of the large, motile spirilla
found in stagnant water, and it was not until very

recently that the accuracy of this suspicion was actually
3*

46 BACTERIOLOGY.

demonstrated. By a special method of staining, Léffler
has been able, in a number of cases, to render visible
these hair-like appendages. His method consists in the
employment of a mordant, by the aid of which the
flagella are caused to retain the staining, and thus be-.
come visible. Léffler’s method of staining will be found
in the chapter devoted to this part of the technique.

CHAPTER III.

Principles of sterilization by heat—Methods employed—Discontinued
sterilization—Sterilization under pressure—Apparatus employed—Chemical
disinfection and sterilization.

In the laboratory it is often common to employ the
term sterilization for the destruction of bacteria by
heat and the term disinfection for the accomplishment
of the same end through the use of chemical agents.
This distinction in the use of the terms is not strictly
correct, as we shall endeavor to explain.

The laboratory application of the word sterilization for
the destruction of bacteria by high temperatures proba-
bly arose from the circumstance that culture media and
certain other articles that it is desirable to render abso-
lutely free from bacterial life are not treated by chemical
agents for this purpose, but are exposed to the influ-
ence of heat in various forms of apparatus known as
sterilizers; and the process is, therefore, known as
sterilization. On the other hand, cultures no longer
useful, bits of infected tissue, and apparatus generally
that it is desirable to render free from danger are
commonly subjected for a time to the action of com-
pounds possessing germicidal properties, 7. ¢., to the
action of disinfectants ; and the process is, therefore,
known as disinfection, though the same end can be
reached by the application of heat to these articles also.
Strictly speaking, sterilization implies the complete
destruction of the vitality of all micro-organisms
that may be present in or upon the substance to be

48 BACTERIOLOGY.

sterilized, and can be accomplished by the proper appli-
cation of both thermal and chemical agents; while
disinfection, though it may, need not, of necessity,
insure the destruction of all living forms that are pres-
ent, but only those possessing the power of infecting ;
it may or may not, therefore, be incomplete in the sense
of sterilization. From this we see it is possible to
accomplish both sterilization and disinfection as well
by chemical as by thermal means.

In practice the employment of these means is gov-
erned by circumstances. In the laboratory it is essen-
tial that all culture media with which the work is
to be conducted are free from all living bacteria or
their spores—they must be sterile—and it is equally
important that their original chemical composition
should remain unchanged. It is evident, therefore,
that sterilization of these substances by means of
chemicals is out of the question, for, while the media
could be thus sterilized, it would be necessary, in order
to accomplish this, to add to them substances capable
not only of destroying all micro-organisms present,
but the presence of such substances would prevent the
growth of bacteria that are to be subsequently culti-
vated in these media—that is to say, after performing
their sterilizing or germicidal function, they would, if
present, exhibit their antiseptic properties. The cir-
cumstances under which chemical sterilization or disin-
fection is practised in the laboratory are ordinarily either
those in which it is desirable to render materials free
from danger that are not affected by the chemical action
of the agents used, such as glass apparatus, etc., or where
destructive changes in the composition of the substances

.to be treated, as in the cases of old cultures, infected

STERILIZATION BY HEAT. 49

tissues, etc., are a matter of no consequence. On the
other hand, for the sterilization of all materials to be
used as culture media heat only is employed.

The two processes will be explained in this chapter,
beginning with

STERILIZATION BY HEAT.

Sterilization by means of high temperature is accom-
plished in several ways, viz., by subjecting the sub-
stances to be treated to a high temperature in a properly
constructed oven, this is known as dry sterilization ; by
subjecting them to the action of streaming or live steam
at the temperature of 100° C.; and by subjecting them
to the action of steam under pressure, under which
circumstances the temperature to which they are ex-
posed becomes more and more elevated as the pressure
increases.

Experiments have taught us that the process of steril-
ization by dry heat has a relatively limited application
because of its many disadvantages. For successful
sterilization by the method of dry heat, not only is a
relatively high temperature essential, but the substances
under treatment must be exposed to this temperature
for a comparatively long time. Its penetration into
the substances which are to be sterilized is, more-
over, much less energetic than that of steam. Many
substances of vegetable and animal origin are rendered
useless by subjection to the dry method of sterilization.
For these reasons there are comparatively few materials
that can be sterilized in this way without seriously
impairing their further usefulness.

Successful sterilization by dry heat cannot usually be
accomplished at a temperature lower than 150° C., and

50 BACTERIOLOGY.

to this degree of heat the objects should be subjected for
not less than one hour. For the sterilization, therefore,
of the organic materials of which the media employed
in bacteriological work are composed, and of domestic
articles, such as cotton, woollen, wooden, and leather
articles, this method is entirely unsuitable. In bac-
teriological work its application is limited to the ster-
ilization of glassware principally—such, for example,
as flasks, plates, small dishes, test-tubes, pipettes—and
such metal instruments as are not seriously injured by
the high temperature.

Sterilization by moist heat—steam—offers conditions
much more favorable. The penetrating action of the
steam is not only more energetic, but the temperature
at which sterilization is ordinarily accomplished is, as a
rule, not destructive to the objects under treatment. This
is conspicuously seen in the work of the laboratory ; the
culture media, composed in the main of decomposable or-
ganic materials that would be rendered entirely worthless
if exposed to the dry method of sterilization, sustain
no injury whatever when intelligently subjected to an
equally effective sterilization with steam. The same
may be said of cotton and woollen fabrics, bedding,
clothing, ete.

Aside from the relations of the two methods to the
materials to be sterilized, their action toward the organ-
isms to be destroyed is quite different. The penetrating
action of the steam renders it by far the more efficient
agent of the two. The spores of several organisms
which are killed by an exposure of but a few moments
to the action of steam resist the destructive action of
dry heat at a higher temperature for a much greater
length of time.

STERILIZATION BY HEAT. 51

These differences will be strikingly brought out in
the experimental work on this subject. For our pur-
poses it is necessary to remember that the two methods
have the following applications :

The dry method, at a temperature of 150°-180° C.,
for one hour, is employed for the sterilization of glass-
ware: flasks, test-tubes, culture dishes, pipettes, plates,
ete.

‘The sterilization by steam is practised with all cul-
ture media, whether fluid or solid. Bouillon, milk,
gelatin, agar-agar, potato, etc., are under no circum-
stances to be subjected to dry heat.

The way in which heat is employed in processes
of sterilization varies with circumstances. In its em-
ployment as dry heat its application is always contin-
uous—i. e., the objects to be sterilized are simply exposed
to the proper temperature for the length of time neces-
sary to destroy all living organisms which may be upon
them. With the use of steam, on the other hand, the
objects to be sterilized are frequently of such a nature that
a prolonged application of the heat would materially
injure them. For this and other reasons steam is usually
applied intermittently and for short periods of time.
The principles involved in this method of sterilization
depend upon differences of resistance toward heat which
the organisms to be destroyed are seen to possess at
different stages of their development. During the life
histo#y of many of the bacilli there is a time in which
the resistance of the organism toward the action of both
chemical and thermal agents is much higher than at
other stages of its development. This increased power
of resistance is seen to exist when these organisms are
in the spore or resting stage, to which reference has

52 BACTERIOLOGY.

already been made. When in the vegetative or grow-
ing stage, most bacteria are killed in a short time by a
relatively low temperature, whereas, when conditions
have arisen which favor the production of spores the
spores are seen to be capable of resisting very much
higher temperatures for an appreciably longer time.
These differences in resistance toward heat which the
spore-forming organisms are seen to possess at their
different stages of development are taken advantage of
in that process of sterilization by steam known as the
fractional or intermittent method, and form the principle
on which the method is based.

As the culture media to be sterilized are of a more
or less unstable nature, being dependent for their value
upon the presence of more or less unstable organic com-
pounds, the object aimed at in this method is to destroy
the organisms in the shortest time and with the least
amount of heat. It is accomplished by subjecting them
to the elevated temperature at a time when they are
in the vegetating or growing stage—. e., the stage at
which they are least resistant. In order to accomplish
this it is necessary that there should exist conditions of
temperature, nutrition, and moisture which favor the
vegetation of the bacilli, and the germination of any
spores that may be present. When, as in freshly pre-
pared nutrient media, these surroundings are found, the
spore-forming organisms are not only less likely to
enter the spore stage than when their environments are
less favorable to their vegetation, but spores which may
already exist develop very quickly into mature cells.

It is plain, then, that with the first application of the
steam to the substance to be sterilized the mature vege-
tative forms of these organisms are destroyed, while cer-

“ FRACTIONAL” STERILIZATION. 53

tain spores that might have been present resist this
treatment, providing the sterilization has not been con-
tinued for too long a time. If now the sterilization is
discontinued, and the material which presents conditions
favorable to the germination of the spores is allowed to
stand for a time, usually for about twenty-four hours,
at a temperature of from 30°-35° C., those spores which
resisted the action of the steam will in the course of this
interval germinate into the less resistant vegetative cells.
A second short exposure to the steam kills these forms
in turn, and bya repetition of this process all organisms
which were present may be destroyed without the appli-
cation of the steam having been of long duration at
any time. In this process the usual plan is to subject
the materials to be sterilized to the action of steam,
under the normal conditions of temperature and pres-
sure, for fifteen minutes on each of three successive
days, and during the intervals to retain them at a
temperature of about 25°-30°C. At the end of this
time all living organisms which were present will
have been destroyed, and unless opportunity is given
for the access of new organisms from without, the
substances thus treated remain sterile.

It must be borne in mind that this method of sterili-
zation is only applicable in those cases which present
conditions favorable to the germination of the spores
into mature vegetative cells. Dry substances or organic
materials in which decomposition is far advanced, where
the conditions of nutrition favorable to the germination
of spores are not present, cannot be successfully sterilized
by the intermittent method.

The process of fractional sterilization at low tempera-
tures is based upon exactly the same principle, but dif-

54 BACTERIOLOGY.

fers, in two respects, viz., it requires a greater number
of exposures for its accomplishment, and the tempera-
ture at which it is conducted is not raised above 68°—
70° C. It is employed for the sterilization of easily
decomposable materials, which would be rendered use-
less. by the temperature of steam, but which remain
intact at the temperature employed. This process
requires that the material to be sterilized should be sub-
jected to a temperature of 68°-70° C. for one hour on
each of six successive days, the interval of twenty-four
hours between the exposures admitting of the germina-
tion of spores into mature cells. During this interval
the substances under treatment are kept at about 25°-
30° C. The temperature employed in this process
suffices to destroy the vitality of almost all organisms in
the vegetative stage in about one hour. Until recently
blood-serum was always sterilized by the intermittent
method at low temperature.

Sterilization by steam is also practised by what may
be called the direct method. That is to say, both the
mature organisms and the spores which may be present
in the material to be sterilized are destroyed by a single
exposure to the steam. In this method steam at its
ordinary temperature and pressure—live steam or stream-
ing steam as it is called—is employed just as in the first
method described, but it is allowed to act for a much
longer time, usually not less than one hour; or, steam
under pressure, and consequently of a higher tempera-
ture, is now frequently employed. In this method a
single exposure of fifteen minutes is sufficient for the
destruction of all bacilli and their spores, providing the
pressure of the steam is not less than one atmosphere
over and above that of normal—this is approximately

*
STERILIZATION APPARATUS, 55

equivalent to a temperature of 122° C. to which the
organisms are exposed.

The objection to both of these methods of direct
sterilization by steam is that many substances that it
is desirable to retain in as near their normal condition
as possible are materially altered by this energetic form
of treatment. Gelatin is not only rendered cloudy, but
often loses the power of gelatinizing. Many of the other
media contain always a fine precipitate after this method ;
in fact, for most of the media which are employed the
discontinued method at the temperature of streaming
steam gives the most satisfactory results.

For sterilization by steam the apparatus commonly
employed has, until recently, been the cylindrical boiler
recommended by Koch. (See Fig. 8.)

Its construction is very simple. It consists of a
copper cylinder, the lower fourth of which is somewhat
larger in diameter than the remaining three-fourths, and
acts as a reservoir for the water from which the steam
is to be generated. Covering this section of the cylinder
is a wire rack or grating through which the steam
passes, and which serves as a bottom upon which the
objects to be sterilized rest. Above this, comprising
the remaining three-fourths of the cylinder, is the cham-
ber for the reception of the materials over and through
which the steam is to pass. The cylinder is closed by
a snugly-fitting cover through which are usually two
perforations into which a thermometer and a manometer
may be inserted. The whole of the outer surface of
the apparatus is encased in a non-conducting mantle of
asbestos or felt.

The water is heated by a gas-flame placed in an en-
closed chamber, upon which the apparatus rests, which

56 BACTERIOLOGY.

serves to diminish the loss of heat and deflection of the
flame through the action of draughts. The apparatus
is simple in construction, and the only point which is to
be observed while using it is the level of the water in

Fig. 8.

Steam sterilizer, pattern of Koch.

the reservoir. On the reservoir is a water-gauge which
indicates at all times the amount of water in the appa-
ratus. The amount of water should never be too small
to be indicated by the gauge, otherwise there is danger
of the reservoir becoming dry and the bottom of the
apparatus being destroyed by the direct action of the
flame.

A sterilizer that has come into very general use in
bacteriological laboratories is one originally intended
for use in the kitchen. It is the so-called “ Arnold

STERILIZATION UNDER PRESSURE. 57

Steam Sterilizer.” It is very ingenious in its construc-
tion as well as economical in its employment.

The difference between this apparatus and that just
described is that it provides for the condensation of the
steam after its escape from the sterilizing chamber, and
returns the water of condensation automatically to the
reservoir, so that in practice the apparatus requires but
little attention, as with moderate care there is no fear of
the water in the reservoir becoming exhausted and the
consequent destruction of the sterilizer.

Fig. 9 shows a section through this apparatus.

Arnold steam sterilizer.

STERILIZATION UNDER PRESSURE.

For sterilization by steam under pressure several spe-
cial forms of apparatus exist. The principles involved in
them all are, however, the same. They provide for the

58 BACTERIOLOGY.

generation of steam in a chamber from which it cannot
escape when the apparatus is closed. Upon the cover
of this chamber is a safety-valve, which can be regulated
so that any degree of pressure (and coincidently of tem-
perature) that is desirable can be maintained within the

Autoclave or digester for sterilizing by steam under pressure.

sterilizing chamber. These sterilizers are known as
“digesters” and as “autoclaves.” Their construction
can best be understood by reference to Fig. 10.

ORDINARY DRY STERILIZER. 59

STERILIZATION BY HOT AIR.

The hot-air sterilizers used in laboratories are simply
double-walled boxes of Russian or Swedish iron (Fig.
11), having a double-walled door, which closes tightly,
and a heavy copper bottom. They are arranged with
ventilating openings for the escape of the contained air
and the entrance of the heated air. The flame, usually
from a rose burner (Fig. 12), is applied directly to the
bottom. The heat circulates from the lower surface
around about the apparatus through the space between
its walls.

Fig. 12,

The construction of the copper bottom of the appa-
ratus upon which the flame impinges is designed to
prevent the direct action of the flame upon the sheet-
iron bottom of the chamber. It consists of several

60 BACTERIOLOGY.

copper plates placed one above the other, but with a
space of about 4 to 5 mm. between the plates. These
copper bottoms after a time become burned out, and
unless they are replaced the apparatus is useless. The
older form of hot-air sterilizers are so constructed that
their repair is a matter involving some time and expense.
To meet this objection I have had constructed a steril-
izer in all respects similar to the old form except in the
arrangement of this copper bottom. This is so made
that it can be easily slipped in and out, so that by
keeping several sets of copper plates on hand a new one
can readily be slipped into the apparatus when the old
one is burned out.

In the employment of the hot-air sterilizer care should
always be given to the condition of the copper bottom ;
for the direct application of the heat to the sheet-iron plate
upon which the substances to be sterilized stand results
not only in destruction of the apparatus, but frequently
in destruction of the substances undergoing sterilization.

Since the temperature at which this form of steriliza-
tion is usually accomplished is high—150° to 180° C.
—it is well to have the apparatus encased in asbestos
boards, to diminish the radiation of heat from its sur-
faces. This not only confines the heat to the apparatus,
but guards against the destructive action of the radiated
heat on woodwork, furniture, etc., that may be in the
neighborhood.

CHEMICAL STERILIZATION AND DISINFECTION.

As has already been stated, it is possible by means
of certain chemical substances to destroy all bacteria
and their spores that may be within or upon various

STERILIZATION AND DISINFECTION. 61

materials and objects, i. e., to sterilize them; and it is
also possible by the same means to rob infected objects
of their dangerous infective properties without at the
same time sterilizing them—i. e., to disinfect them.
This latter process depends upon the fact that the
vitality of many of the less resistant pathogenic organ-
isms is easily destroyed by an exposure to particular
chemical substances, while a similar exposure may be
without effect upon the more resistant saprophytes and
their spores that are present.

The use of chemicals for sterilization is not to be
considered in connection with substances that are to be
employed as culture media, and their employment is
restricted in the laboratory to materials that are of no
further value and to infected articles that are not in-
jured by the action of the agents used. In short, they
are mainly of value in rendering infected waste materials
free from danger. For the successful performance of
this form of disinfection there is one fundamental rule
always to be borne in mind, viz., it is absolutely essen-
tial to success that the disinfectant used should come in
direct contact with the bacteria to be destroyed, other-
wise there is no disinfection.

For this reason, one should always remember, in
selecting the disinfecting agent, the nature of the
materials containing the bacteria upon which it is to
act, for the majority of disinfectants, and particularly
those of an inorganic nature, vary in the degree of
their potency with the chemical nature of the mass to
which they areapplied. Often the materials containing
the bacteria to be destroyed are of such a character that
they combine with the disinfecting agent to form insol-

uble precipitates ; these so interfere with the penetration
4

62 BACTERIOLOGY.

of the disinfectant that many bacteria may escape its
destructive action entirely and no disinfection be accom-
plished, though an agent might have been employed
that would, under other circumstances, have given
entirely satisfactory results.

In the destruction of bacteria by means of chemical sub-
stances, there occurs, most probably, a definite chemical
reaction ; that is to say, the characteristics of both the
bacteria and the agent employed in their destruction are
lost in the production of a third body, the result of their
combination. It is impossible to say with absolute cer-
tainty, as yet, that this is the case, but the evidence that
is rapidly accruing from the more recent studies upon
disinfectants and their mode of action points strongly to
the accuracy of this belief. This reaction, in which the
typical structures of both bodies concerned is lost, takes
place between the agent employed for disinfection and
the protoplasm of the bacteria. For example, in the
reaction that is seen to take place between the salts
of mercury and albuminous bodies there results a third
compound, which has neither the characteristics of mer-
cury nor of albumin, but partakes of the peculiarities
of both; it is a combination of albumin and mercury
known by the indefinite term “albuminate of mercury.”
Some such reaction as this occurs when the soluble
salts of mercury are brought in contact with bacteria.
This view has recently been strengthened by the experi-
ments of Geppert, in which the reaction was caused to
take place between the spores of the anthrax bacillus
and a solution of mercuric chloride, the result being
the apparent destruction of the living properties of the
spores by the formation of this third compound. In
these experiments it was shown that though this com-

DISINFECTION AND ANTISEPTICS. 68

bination had taken place, still it did not of necessity
imply the complete death of the protoplasm of the
spores, for if by proper means the combination of mer-
cury with their protoplasm was broken up, many of
the spores returned from their condition of apparent
death to that of life, with all their previous disease-
producing and cultural peculiarities. Geppert employed
a solution of ammonium sulphide for the purpose of
destroying the combination of spore-protoplasm and
mercury ; the mercury was -precipitated from the pro-
toplasm as an insoluble sulphide, and the protoplasm
of the spores returned to its original condition. These
and other somewhat similar experiments have given an
entirely new impulse to the study of disinfectants,
and in the light shed by them many of our previ-
ously formed ideas concerning the action of disinfecting
agents must be modified. The process is not a cata-
lytic one—i. e., occurring simply as a result of the
presence of the disinfecting body which is not itself
destroyed in its process of destruction—but is, as said, a
definite chemical reaction which takes place within cer-
tain more or less fixed limits; that is to say, with a given
amount of the disinfectant employed, just so much work,
expressed in terms of disinfection—destruction of bac-
teria—can be accomplished.

Another point in favor of this view is the increased
energy of the reaction with elevation of temperature.
Just as in many other chemical phenomena, the in-
tensity of the reaction becomes greater under the
influence of heat, so in the process of disinfection the
combination between the disinfectant and the organisms
to be destroyed is much more energetic at a temperature
of 87° to 39° C. than it is at 12° to 15° C.

64 BACTERIOLOGY.

What has been said refers more particularly to the
inorganic salts which are employed for this purpose.
It is probable that the organic bodies which possess dis-
infectant properties owe this power to some such similar
reaction, though, as yet, these substances have not been
so thoroughly studied in this relation.

The reaction between these inorganic salts and albu-
minous bodies is not a selective action ; they combine
in most instances with any or all protoplasmic bodies
present. For this reason the results of the practical
application of many of the commonly employed dis-
infectants is a matter of grave doubt. For example,
the disinfection of excreta, sputum, or blood containing
-pathogenic organisms, by means of corrosive sublimate,
is a procedure of very questionable success. The amount
of sublimate employed may be entirely used up and
rendered inactive as a disinfectant by the ordinary
protoplasmic substances present without having any
appreciable effect upon the bacteria which may be in the
mass.

These remarks are introduced in order to guard
against the implicit confidence so often placed in the
disinfecting value of corrosive sublimate. In bacterio-
logical laboratories, where there is constantly more or
less of infectious material, it is the custom, with few
exceptions, to have vessels containing solutions of corro-
sive sublimate at hand, by which infectious materials
may be rendered harmless. The value of this pro-
cedure, as we have just learned, is always more or less
questionable, especially in those cases in which the
substance to be disinfected is of an albuminous nature.
With the introduction of such substances into the sub-
limate solution the mercury is quickly precipitated by

HEAT. 65

the albumin and its disinfecting properties may be
entirely destroyed ; we may in a very short time have
little else than water containing a precipitate of albumin
and mercury, in so far as its value as a disinfectant is
concerned,

Though the other inorganic salts have not been so
thoroughly studied in this connection, it is nevertheless
probable that the same precautions should be taken in
their employment as we now know to be necessary in
the use of the salts of mercury.

Where it is desirable to use chemical disinfectants in
the laboratory, much more satisfactory results can
usually be obtained through the employment of carbolic
acid in solution. A 3 or 4 per cent. solution of com-
mercial carbolic acid in water requires a somewhat longer
time for disinfection of such resistant objects as the spores
of the bacillus anthracis, but it is at the same time open
to fewer objections than are solutions of the inorganic
salts. For less resistant organisms its action is usually
complete in from twenty minutes to one-half hour.

In the laboratory heat is the surest agent to employ.
All tissues containing infectious organisms should be
burned, and all cloths, test-tubes, flasks, and dishes
should be boiled in 2 per cent. soda solution for fifteen
to twenty minutes, or placed in the steam sterilizer for
half an hour.

Intestinal evacuations may best be disinfected with
milk of lime, a mixture composed of lime in solution and
in suspension, ordinary fluid “ white-wash.” This
should be thoroughly mixed with the evacuations until
the mass reacts distinctly alkaline, and should remain
in contact with the infective substance for one or two
hours.

66 BACTERIOLOGY.

Sputum in which tubercle bacilli are present, as well
as the vessel containing it, must be boiled in soda solu-
tion for fifteen minutes or steamed in the sterilizer for
at least half an hour.

On the whole, in the laboratory we should as yet
rely more upon the destructive properties of heat than
upon that of chemical agents.

From what has been said, the absurdity of sprinkling
about, here and there, a little carbolic acid or in placing
about apartments in which infectious diseases are in
progress little vessels of carbolic acid, must be plain.
The disinfection of water-closets and cesspools by allow-
ing now and then a few cubic centimetres of some so-
called disinfectant to trickle through the pipes is absurd.
A disinfectant must be applied to the bacteria, and must
be in contact with them for a long enough time to insure
the destruction of their life.

In the light of the latest experiments upon disin-
fectants, the place formerly occupied by many agents in
the list of substances employed for the purpose will most
likely be changed as they are studied more closely.

The agents, then, which will prove of most value in
the laboratory for the purpose of rendering infectious
materials harmless are: Heat, either by burning, by
steaming for from half an hour to an hour, or by boil-
ing in a 2 per cent. soda solution for fifteen minutes; a
solution of chlorinated lime (“chloride of lime”), in
which the percentage of chlorine is high; 3 to 4 per
cent. solution of commercial carbolic acid ; and milk of
lime. The materials to be disinfected in either of the
lime solutions should remain in them for about two
hours. The solutions should be freshly prepared when
needed, as they rapidly decompose upon standing.

DISINFECTANT AGENTS, 67

Antiseptic. An antiseptic is a body which, by its
presence, prevents the growth of bacteria without of
necessity killing them. A body may be an antiseptic
without possessing disinfecting properties to any very
high degree, but a disinfectant is always an antiseptic
as well.

CHAPTER IV.

Principles involved in the methods of isolation of bacteria in pure culture
by the plate method of Koch—Materials employed.

As was stated in the introductory chapter, the isola-
tion in pure cultures of the different species of bacteria
from mixtures of these organisms was rendered possible
only through the methods suggested by Koch. Since
the adoption of these methods they have undergone
many modifications, but the principle involved has
remained unaltered. The observation which led to
their development was a very simple one, and one that
is commonly before us. Koch noticed that on solid sub-
stances, such, for example, as a slice of potato or of bread,
whieh had been exposed for a time to the air and which
afforded proper nourishment for the lower organisms,
there developed after a short time small patches of
material which proved to be colonies of bacteria. Each
of these colonies on closer examination showed itself
to be, as a rule, composed of but a single species.
There was no tendency on the part of these colonies to
become confluent, and from the differences in their
naked-eye appearances it was easy to see that they were
mostly the outgrowth of different species of bacteria.

The question that then presented itself was: If from
a mixture of organisms floating in the air it is possible
in this way to obtain in pure cultures the individual
organisms composing the mixture, what means can be
employed for obtaining the same results at will from
mixtures of different organisms when found under other
conditions ?

PLATE METHOD OF KOCH. 69

It was plain that the organisms were to be distin-
guished, the one from the other, only by the structure
and general appearance of the colonies growing from
them, for by their morphology alone this is impossible.

What means could be devised, then, for separating the
individual members of a mixture in such a way that
they would remain in a fixed position, and be so widely
separated, the one from the other, as not to inter-
fere with the production of colonies of characteristic
appearance, which would, under the proper conditions,
develop from each individual cell?

If one takes in the hand a mixture of barley, rye,
corn, oats, etc., and attempts to separate the mass into
its constituents by picking out the different grains, much
difficulty is experienced ; but if the handful of grain be
thrown upon a large flat surface, as upon a table, the
grains become more widely separated and the task is
considerably simplified; or, if sown upon proper soil
the various grains will develop into growths of entirely
different external appearance by which they can readily
be recognized as unlike in nature. Similarly, if a test-
tube of decomposed bouillon be poured out upon a large
flat surface, the individual bacteria in the mass are very
much more widely separated the one from the other
than they were when the bouillon was in the tube ; but
they are in a fluid medium, and there is no possibility
of their either remaining separated or of their forming
colonies under these conditions, so that it is impossible by
this means to pick out the individuals from the mixture.

If, however, it is possible to find some substatice
which possesses the property of being at one time fluid
and at another time solid, which can be added to this

bouillon without in any way interfering with the life-
4*

70 BACTERIOLOGY.

functions of the bacteria, then, as solidification sets in,
the organisms will be fixed in their positions and the
conditions will be analogous to that seen on the bit of
pptato.

Gelatin possesses this property. At a temperature
which does not interfere with the life of the organisms
it is quite fluid, whereas when subjected to a lower
temperature it solidifies. When once solid it may be
kept at a temperature favorable to the growth of the
bacteria and retain its solid condition.

Gelatin was added to the fluids containing mixtures
of bacteria, and the whole was then poured upon a large
flat surface, allowed to solidify, and the results noted.
It was found that the conditions seen on the slice of
potato could be reproduced, that the individuals in the
mixture of bacteria grew well in the gelatin, and, as on the
potato, grew in colonies of typical macroscopic structure,
so that they could easily be distinguished the one from the
other by their naked-eye appearances. It was necessary,
however, to use a more dilute mixture of bacteria than
that seen in the original decomposed bouillon. The
number of individuals in the tube was so enormous
that on the gelatin plate they were so closely packed
together that it was not only impossible to pick them
out because of their proximity the one to the other,
bul also because this packing together materially inter-
fered with the production of those characters by means
of which differences can be. seen with the naked eye.
The numbers of organisms were then diminished by a
process of dilution, consisting of transferring a small
portion of the original mixture into a second tube of
sterilized bouillon to which gelatin had been added and
liquefied; from this a similar portion: was added to a

CULTURE MATERIALS EMPLOYED, 71

third gelatin-bouillon tube, and so on. These were then
poured upon large surfaces and allowed to solidify.
The results were entirely satisfactory. On the gelatin
plates from the original tube, as was expected, the
colonies were too numerous to be of any use; on the
plates made from the first dilution they were much
fewer in number, but still they were usually too numer-
ous and too closely packed to permit of characteristic
growth ; but on the second dilution they were, as a rule.
fewer in number and widely separated, so that the in-
dividuals of each species were in no way prevented by
the proximity of its neighbors from growing in its own
typical way. There was then no difficulty in picking
out the colonies resulting from the growth of the differ-
ent individual bacteria.

Such, then, is the principle underlying Koch’s method
for the isolation of bacteria by means of solid media.

The fundamental part of the media employed is the
bouillon, which contains all the elements necessary for
the nutrition of most bacteria, the gelatin being em-
ployed simply for the purpose of rendering the bouillon
solid. The medium on which the organisms are growing
is, therefore, simply solidified bouillon, or beef tea.

In practice, two forms of gelatin are employed—the
. one an animal or bone gelatin, the ordinary table gelatin
of good quality; and the other a vegetable gelatin,
known as agar-agar, or Japanese gelatin, which is
obtained from a group of alge growing in the sea along
the coast of Japan, where it is employed as an article of
diet by the natives.

Aside from these differences in origin of the two forms
of gelatin employed, their behavior toward heat and
toward bacteria renders them of different application

72 BACTERIOLOGY.

in bacteriological work. The animal gelatin liquefies
at a much lower temperature, and likewise solidifies at
a very much lower temperature, than does the agar-
agar. Ordinary gelatin, in the proportion commonly
used in this work, liquefies at about 24° C., and
becomes solid at from 8°-10° C. It may be employed
for those organisms which do not require a higher tem-
perature for their development than 22°C. Agar-agar,
on the other hand, does not liquefy until the tempera-
ture has reached about 98°-99° C. It remains fluid
ordinarily until the temperature has fallen to 38°-39°
C., when it rapidly solidifies. For our purposes, only
that form of agar-agar can be used which remains fluid
at from 38°-40° C. Agar-agar which remains fluid
only at a temperature above this point would be too
hot, when in a fluid state, for use; many of the organ-
isms which would be introduced into it would either be
destroyed or checked in their development by so high
a temperature. Agar-agar, therefore, is for use in those
cases in which the cultivation must be conducted at a
temperature above that at which gelatin remains solid.

In addition to the differences toward temperature,
the relations of these two gelatins to bacteria are differ-
ent. Many bacteria bring about alterations in gelatin
which cause it to become liquid (a process of peptoniza-
tion), in which state it remains. There are no known
organisms that bring about such a change in agar-agar.

As a rule, the colony-formations seen upon gelatin
are much more characteristic than those which develop
on agar-agar, and for this reason gelatin is to be pre-
ferred when circumstances will permit. Both gelatin
and agar-agar may be used in the preparation of plates
and Esmarch tubes, subsequently to be described.

CHAPTER V.

Preparation of media—Bouillon, gelatin, agar-agar, potato, blood-serum, etc.

As has been stated, the fundamental part of our cul-
ture media is beef tea, or bouillon.

Boviiton.—The formula of Koch for the prepara-
tion of this medium has undergone many modifications
to meet special cases, but for general use his original
formula is still retained. It is as follows: Five hundred
grammes of finely-chopped lean beef, free from fat and
tendons, is to be soaked in one litre of water for
twenty-four hours. During this time it is to remain in
the ice-chest or to be otherwise kept at a low tempera-
ture. It is then to be strained through a coarse towel and
pressed until a litre of fluid is obtained. To this is to be
added ten grammes (1.0 per cent.) of dried peptone and
five grammes (0.5 per cent.) of common salt (NaCl). It
is then to be rendered exactly neutral or very slightly
alkaline, with a few drops of saturated soda solution.
The flask containing the mixture is then to be placed
either in the steam sterilizer or in a water-bath, or over
a free flame, and kept at the boiling-point until all the
albumin is coagulated, and the fluid portion is of a
clear, pale, straw-color. It is then filtered through a
folded paper filter, and sterilized in the steam sterilizer
by the fractional method. Certain of the modifications
of this method are of sufficient value to justify mention.
Most important is the neutralization. Ordinarily, this
is accomplished with the saturated soda solution, and

74 BACTERIOLOGY.

the reaction is determined with the ordinary red and
blue litmus paper.

Soda solution is not so good as a strong solution of
caustic soda or potash, because the carbonic acid libe-
rated from the sodium carbonate is frequently seen to
give rise to a confusing temporary acid reaction which
disappears on heating. To obviate this, Schultz (Cen-
tralbl. f. Bakt. u. Parasitenkunde, Bd. x., Nos. 2 and 3,
1891) recommends exact titration with a solution of
caustic soda. For this purpose a 4 per cent. solution
of caustic soda is prepared. From this a 0.4 per cent.
solution is made, and with it the titration is practised.
After the bouillon has been deprived of all coagulable
albumin and blood-coloring matter by boiling and filtra-
tion, and has cooled down to the temperature of the air,
its whole volume is exactly measured.

' From it a sample of exactly 5 or 10 ec. is then
taken, and to it a few drops of one of the indicators com-
monly employed in analytical work is added. Schultz
recommends 1 drop of phenolphthalein solution (1
gramme phenolphthalein in 300 c.c. of alcohol) to 1 ec.
of bouillon. The beaker containing the sample is
placed upon white paper, and the dilute caustic soda
solution is then allowed to drop into it, very slowly,
from a burette, until there appears a very delicate rose
color, which indicates the beginning of alkaline reaction.
A second sample of the bouillon is treated in the same
way. If the amounts of soda solution required for
each sample deviate but very slightly or not at all
the one from the other, the mean of these amounts is
taken as the amount of the soda solution necessary to
neutralize the quantity of bouillon employed. If 10c.c.
of bouillon were employed, then, for the whole amount

BOUILLON. 95

of 1 litre, just 100 times as much, minus that for the
two samples used in titration, will be needed. For
example: To neutralize 10 c.c. of bouillon, 2 c.c. of the
diluted (0.4 per cent.) caustic soda solution were em-
ployed. For the remaining 980 c.c. of the litre of
bouillon, then, 196 c.c. (200 c.c.—4 e.c., the amount
employed for the two samples of 10 c.c. each of bouil-
lon) is needed of the 0.4 per cent. solution, or one-
tenth of this amount of the 4 per cent. solution.

For the neutralization of the whole bulk of the
bouillon it is better to employ the strong alkaline
solution, as by its use the volume is not increased
to so great an extent as when the dilute solution is
used.

It is evident that this method is much more exact
than that ordinarily employed, but at the same time it
must be remembered that for its success it requires
exactness in the measurement of the volumes and the
preparation of the dilutions. To obviate error, it is
better to employ this method when the solutions are all
cool and of nearly the same temperature, so that rapid
fluctuations in temperature, and consequent alterations
in volume, will not materially interfere with the accu-
racy of the results.

This method of neutralization, which is employed by
Schultz, is to be recommended for those experiments in
which slight inaccuracies in the reaction of the media
play an important part.

For the ordinary purposes of the beginner, however,
results quite satisfactory in their nature may be obtained
by the employment of the saturated soda solution for
neutralization and the litmus paper as the indicator.
For some time, however, it has been our practice to

76 BACTERIOLOGY.

employ the yellow curcuma paper for the detection of
alkalinity rather than the red litmus paper.

Not infrequently the filtered bouillon, neutralized and
sterilized, will be seen to contain a fine, flocculent pre-
cipitate. This may be due either to excess of alkalinity
or to incomplete precipitation of the albumin. The
former may be corrected with dilute acetic or hydro-
chloric acid, and the bouillon again boiled, filtered, and
sterilized ; or, if due to the latter cause, subsequent
boiling and filtration usually results in ridding the
bouillon of the precipitate.

Another modification now generally employed is the
use of meat extracts instead of the infusion of meat.
Almost any of the meat extracts of commerce answer
the purpose, though we usually prefer Liebig’s. It is
employed in the strength of from two to four grammes
to the litre of water. Peptone and sodium chloride are
added as in the bouillon made from the meat infusion.
The advantage of this method is that it takes less time
than when the infusion of meat is used, affords a solu-
tion of more uniform composition if used in fixed pro-
portions, and for general use it answers just as well.

Nutrient GELATIN.—For the preparation of gela-
tin the bouillon is first prepared in exactly the same
way as has just been described, except that the neutral-
ization takes place after the gelatin has been completely
dissolved, which occurs very rapidly in hot bouillon.
The reaction of the gelatin as it comes from the manu-
factories is usually quite acid, so that a much larger
amount of alkali is needed for its neutralization than
for other media. The gelatin is added in the propor-
tion of 10 to 12 per cent. The complete solution of
the gelatin may be accomplished either over the water-

NUTRIENT GELATIN. 77

bath, in the steam sterilizer, or over a free flame. If
the latter method is practised, care must be taken that
the mixture is constantly stirred to prevent burning at
the bottom and consequent breaking of the flask, if a
flask is employed.

For some time it has been our practice to use, for the
purpose of making both gelatin and agar-agar, enamelled
iron saucepans instead of glass flasks; by this means
the free flame may be employed without danger of
breaking the vessel, and, with a little care, without fear
of burning the media. Under any conditions it is
better to protect the bottom of the vessel from the
direct action of the flame by the interposition of several
layers of wire gauze, a thin sheet of asbestos-board, or
an ordinary cast-iron stove-plate.

When the gelatin is completely melted, it may be
filtered through a folded paper filter on an ordinary
funnel ; if the solution is perfect, this should be very
quickly accomplished.

Fig. 18.

For the filtration of such substances as gelatin and
agar-agar it is of much importance to have a properly
folded filter. To fold a filter correctly, proceed as fol-
lows: A circular piece of filter paper is folded exactly
through its centre, forming the fold 1, 1 (Fig. 13); the

78 BACTERIOLOGY.

end 1 is then folded over to 1’, forming the fold 5;
1 and 1’ are each then brought to 5, thus forming the
folds 3 and 7; 1 is then carried to the point 7, and the
fold 4 is formed, and by carrying 1’ to 3 the fold 6 is
produced ; and by bringing 1 to 3 and 1’ to 7 the folds
2 and 8 result.

Thus far the ridges of all folds are on the side of the
paper next to the table on which we are folding. The
paper is now taken up, and each space between the
seams just produced is to be subdivided by a seam or
fold through its centre, as indicated by the dotted lines
in Fig. 13, but with the creases on the side opposite to

Fic. 14.

that occupied by the creases 1, 2, 3, 4, ete., first made.
As each of these folds is made the paper is gradually
folded into a wedge-shaped bundle (Fig. 14, a), which,
when opened, assumes the form of a properly folded
filter seen in b, Fig. 14. Before placing it upon the
funnel it is well to go over each crease again and see
that it is as tightly folded as possible, without tearing it.
The advantage of the folded filter is that by its use a
much greater filtering surface is obtained, as it is in con-
tact with the funnel only at the points formed by the
ridges, leaving the majority of the flat surface free for
filtration.

FILTRATION. 79

The employment of the hot-water funnel, so often
recommended, has been dispensed with in this work to
a very large extent, as we know that, if the solution
of the gelatin is complete, filtration is so rapid as not
to necessitate the use of an apparatus for maintaining
the high temperature. The temperature at which the
hot-water funnel retains the gelatin is so high that evap-
oration and concentration rapidly occur, and in conse-
quence the filtration is, as a rule, retarded. The filtra-
tion is frequently done in the steam sterilizer, but this
too is unnecessary if the gelatin is quite dissolved. At
the ordinary temperature of the room and by the means
commonly employed for the filtration of other sub-
stances, both gelatin and agar-agar may be rapidly fil-
tered if they are completely dissolved.

It not infrequently occurs that, even under the most
careful treatment, the filtered gelatin is not perfectly
transparent (the condition in which it must exist, other-
wise it is useless), and clarification becomes necessary.
For this purpose the mass must be redissolved, and
when at a temperature between 60° C. and 70° C., the
whites of two eggs, which have been beaten up with
about 50 ec.c. of water, are added. The whole is then
thoroughly mixed together and again brought to the
boiling-point, and kept at this point until coagulation of
the albumin occurs. It is better not to break up the
large masses of coagulated albumin if it can be avoided,
as when broken up into fine flakes they clog the filter
and materially retard filtration.

The practice sometimes recommended of removing
these albuminous masses by first filtering the gelatin
through a cloth, and then finally through paper, is not
only superfluous, but in most instances renders the pro-

80 BACTERIOLOGY.

cess of filtration much more difficult, because of the
disintegration of these masses into the finer particles,
which have the effect just mentioned.

Under no circumstances is a filter to be used
without first having been moistened with water. If
this is not done, the pores of the paper, which are
relatively large when in a dry state, when moistened by
the gelatin not only diminish in size, but in contract-
ing are often entirely occluded by the finer albuminous
flakes which become fixed within them, and filtration
practically ceases. The preliminary moistening with
water causes the diminution of the size of the pores
to such an extent that the finer particles of the precipi-
tate rest on the surface of the paper, instead of becoming
fixed in its meshes.

During boiling it is well to filter, from time to time,
a few cubic centimetres of the gelatin into a test-tube
and boil it over a free flame for a minute or so; in this
way one can detect if all the albumin has been coagu-
lated and when the solution is ready for filtration.

Gelatin should not, as a rule, be boiled over ten or
fifteen minutes at one time, or left in the steam sterilizer
for more than thirty to forty-five minutes, otherwise its
property of solidifying is materially diminished.

As soon as the gelatin is complete, whether it is
retained in the flask into which it has been filtered or
decanted off into sterilized test-tubes, it should be steril-
ized in the steam sterilizer on three successive days, for
fifteen minutes each day—the mouth of the flask or the
test-tubes containing it having been previously closed
with cotton plugs.

Nutrient AGAR-AGAR.—The preparation of nu-
trient agar-agar by the beginner is, as a rule, a some-

NUTRIENT AGAR-AGAR. 81

7

what tedious and time-taking experience. This is owing
mainly to lack of patience and failure to adhere strictly
to the rules laid down for the preparation of this medium.
Many methods are recommended for its preparation ;
almost every worker has some slight modification of his
own.

The methods that have given us the best results,
and from which there are no grounds for deviating, are
as follows:

Prepare the bouillon in the usual way. Agar-agar
reacts neutral, so that the bouillon may be neutralized
before the agar is added. Then add finely-chopped agar
in the proportion of 1 to 1.5 per cent. Place the mix-
ture in a porcelain-lined iron vessel and make a mark
on the sides of the vessel at which the level of the fluid
stands, add about 250 c.c. to 300 c.c. of water and allow
the mass to boil slowly, occasionally stirring, over a free
flame, for from one and one-half to two hours. Care
must be taken that it does not boil over the sides of the
vessel. From time to time observe if the fluid has
fallen below the mark of its original level; if it has,
add water until its original volume is restored. At the
end of the time given remove the flame and place the
vessel containing the mixture in a large dish of cold
water ; stir the agar continuously until it has cooled down
to about 68°—70° C., and then add the whites of two
eggs which have been beaten up in about 50 cc. of
water; or the ordinary dried albumin of commerce
may be dissolved in cold water in the proportion of
about 10 per cent., and used. The results are equally
as good as where eggs are used. Mix this carefully
throughout the agar, and allow the mass to boil slowly
for about another half-hour, observing all the while the

82 BACTERIOLOGY.

level of the fluid. It is necessary to reduce the tempera-
ture of the mass to the point given, 68°-70° C., other-
wise the coagulation of the albumin will occur in lumps
and masses as soon as it is added, and its clearing action
will not be homogeneous. The process is a purely
mechanical one—the finer particles, which would other-
wise pass through the pores of the filter, being taken
up by the albumin as it coagulates and retained in the
coagula.

At the end of one-half hour the boiling mass may be
easily and quickly filtered through a heavy, folded
paper filter at the room temperature, and, as a rule, the
filtrate is as clear and as transparent as agar-agar
usually appears.

Another plan that insures complete solution of the
agar without causing the precipitates that are com-
monly seen when all the ingredients are added at
first and boiled for a long time, is to weigh oui the
necessary amount of agar-agar, 10 or 15 grains, and
place this in 1300 or 1400 c.c. of water and boil down
over a free flame to 1000 c.c. The peptone, salt, and
beef-extract are then added and the boiling again con-
tinued until they are dissolved. The clarification with
egg albumen may then be done, and usually the mass
filters quite clear and does not show the presence of
precipitates upon cooling. If the mixture is positively
alkaline, it is not only cloudy but it filters with diffi-
culty ; if it is acid, it is usually quite clear; but, as
Schultz has pointed out, it loses at the same time some
of its gelatinizing properties. The bouillon should
always be neutralized before the agar-agar is added to
it, for if the bouillon be acid, from the acid of the meat,
it robs the agar, under the influence of heat, of some of

PREPARATION OF POTATOES. 83

its gelatinizing powers, which cannot be regained by
subsequent neutralization.

Another method by which the agar-agar can easily
and quickly be melted, is by steam under pressure. If
the flask containing the mixture of bouillon and agar
be kept in the digester or autoclav, with the steam
under a pressure of one-half to one atmosphere, as
shown by the gauge, for ten minutes, the agar-agar
will be found at the end of this time completely melted,
and filtration may then be accomplished with but little
difficulty.

If glycerin is to be added to the agar-agar, it is done
after filtration and before sterilization. The nutritive
properties of the media for certain organisms, particu-
larly the tubercle bacillus, is improved by the addition
of glycerin in the proportion of 5 to 7 per cent.

If after filtration a fine flocculent precipitate is seen,
look to the reaction of the medium. If it is quite alka-
line, neutralize, boil, and filter again. If the reaction
is neutral or only very slightly acid, dissolve and clarify
again with egg albumin by the method given.

The most important point in all the media, aside
from the correct proportion of the ingredients, is their
reaction. They must be neutral or very slightly alka-
line. But few organisms develop well on media of an
acid reaction. In all of the above media the meat ex-
tracts now on the market may usually be substituted for
the meat itself in preparing the bouillon. In this case
the preparation known as Liebig’s Meat Extract may
be employed in the proportion of from two to four
grammes to the litre of water.

PREPARATION OF PoTaToEs.—Potatoes are pre-
pared for use in two ways:

84 BACTERIOLOGY.

1. They are taken as they come to the market—old
potatoes being usually recommended, and carefully
scrubbed under the water-tap with a stiff brush until
all adherent dirt has been removed ; “the eyes” and all
discolored or decayed parts are carefully removed with
a pointed knife. They are then to be placed in a solu-
tion of corrosive sublimate of the strength of 1 : 1000
and allowed to remain there for twenty minutes ; at the
end of this time, without rinsing off the sublimate, they
are placed in a covered tin bucket with a perforated
bottom and sterilized in the steam sterilizer for forty-
five minutes. On the second and third days the sterili-
zation is repeated for fifteen to twenty minutes each
day. They must not be removed from the sterilizing
bucket until sterilization is complete. At the end of
this time they are ready for use. When prepared in
this way, they are usually intended to be cut into two
halves, and the cultivation of the organisms is to be
conducted upon the flat surfaces of the sections.

This method requires some care to prevent con-
tamination during manipulation. The hand which is
to take up the potato from the bucket, which until
now has remained covered, is first disinfected in the
sublimate solution for ten minutes, the potato is then
taken up between the thumb and index finger, and sev-
ered into two by a knife which has just been sterilized
in the free flame until it is quite hot. The blade of the
knife is passed not quite through the potato, but nearly
so. A large glass culture-dish for the reception of the
two halves of the potato having been disinfected for
twenty minutes with 1: 1000 sublimate solution and
then drained of all the adherent solution, is at hand
ready for the bits of potato; the cover is removed, and

PREPARATION OF POTATOES. 85

by twisting the knife gently the two halves of the
potato may be caused to fall apart in the dish and
usually to fall upon their convex surfaces, leaving the
flat sections uppermost. The cover is placed upon the
dish and the potatoes are ready for inoculation.

2. Preparation of potatoes for test-tube cultures. Method
of Bolton.’ If the potatoes are to be employed for test-
tube cultures, one simply scrubs off the coarser particles
of dirt with water and a brush, and with a cork-borer
punches out cylindrical bits of potato which will fit
loosely into the test-tubes to be used.
On each bit of potato is then to be cut a
slanting surface running diagonally from
about the junction of the first and second
third of the cylinder to the diagonally
opposite end. These cylinders of potato
are now to be left in running water over
night, otherwise they are very much dis-
colored by the sterilization to which they
are to be subjected. At the end of this
time they are placed in previously pre-
pared test-tubes, one piece in each tube,
with the slanting surface up, the cotton
plugs of the tubes replaced, and they
are then to be sterilized in the steam
for forty-five minutes. On the second
and third days they are to be sterilized
for fifteen to twenty minutes each day.

The entire sterilization may be accom-
plished in the autoclave with the steam under a pres-
sure of one atmosphere, by a single exposure of twenty

Fig. 15.

Potato in test-tube.

1 Medical News, 1887, vol. i. p. 188.
5

86 BACTERIOLOGY.

to twenty-five minutes. When finished they have the
appearance seen in Fig. 15, except that there is no
growth upon the surface as is shown in the cut.

Care must be given to the sterilization of potatoes,
because they always have adhering to them the or-
ganisms commonly found in the ground, the spores of
which are among the most resistant of all known
organisms. The so-called “ potato bacillus” is one of
this group; it is an organism which not infrequently
is more or less of an obstacle to the work of the
beginner.

Bioop-Serum.—Blood-serum requires special care
in its preparation ; it is desirable under all conditions
to reduce the unavoidable contamination, which to a
certain extent occurs during manipulation, to its min-
imum degree.

It is possible to collect serum from small animals and
in small quantities under such precautions that it is
perhaps not contaminated, but ordinarily for laboratory
purposes a larger quantity is needed, so that the
slaughter-houses form the sources from which it is
usually obtained, and here a certain amount of contami-
nation is unavoidable, though its degree may be limited
by proper precaution. The animal from which the
blood is to be collected should be drawn up to the ceil-
ing by the hind legs, the head should be held well back,
and with one pass of a very sharp knife the throat
should be completely cut through. The blood which
will be spurting from the severed vessels should be col-
lected in large glass jars which have been previously
cleaned, disinfected, and all traces of the disinfectant
removed with alcohol and finally ether. The latter
evaporates very quickly and leaves the jar quite dry.

BLOOD-SERUM. 87

The jars should be provided with covers which close
hermetically—these too should be carefully disinfected.
The best form of glass vessels for the purpose are the
large glass museum jars of about one gallon capacity,
which close by a cover that can be tightly screwed
down upon a rubber joint. From two such jarfuls of
blood one can recover quite a large quantity of clear
serum, ordinarily from 500-700 c.c. The jars having
been filled with blood, their covers are placed loosely
upon them and they are allowed to stand for about
fifteen minutes until clotting has begun. At the end
of this time a clean glass rod is passed around the edges
of the surface of the clot to break up any adhesions to
the wall of the jar that might have formed, and which
would prevent the sinking of the clot to the bottom.
The covers are then tightly replaced, and with as little
agitation as possible the jars are placed in an ice-chest,
where they remain for twenty-four to forty-eight hours.
The temperature should, however, not be low enough
to prevent coagulation, but should be sufficiently low to
interfere with the development of any living organisms
that may be present. The temperature of the ordinary
domestic refrigerator is sufficient for the purpose. After
twenty-four to forty-eight hours the clot will have
become firm, and will be seen at the bottom of the jar.
Above it is a quantity of dark straw-colored serum.
The serum may then be drawn off with a sterilized
pipette and placed in tall cylinders that have previ-
ously been plugged with cotton wadding and sterilized.
After treating all the serum in this way, care having
been taken to get as little of the coloring matter of the
blood as possible, it may be placed again in the
ice-chest for twenty-four hours during which time the

88 BACTERIOLOGY.

corpuscular elements will sink to the bottom, leaving
the supernatant fluid quite clear. This may then be
pipetted off, either into sterilized test-tubes, about 8 ¢.c.
to each tube, or into small sterilized flasks of about 100
c.c. capacity. It is then to be sterilized by the inter-
mittent method at low temperatures, viz., for one hour on
each of five consecutive days at a temperature of
68°-70° C. During the intervening days it is to be
kept at the room temperature to permit of the de-
velopment of any spores that may be present in their
vegetative forms, in which condition they are killed by
an hour’s exposure to the temperature of 70° C.

Fia. 16.

At the end of this time the serum in the tubes may
either be retained as fluid serum or solidified at between
76°-80° C. In solidifying the serum the tubes should
be placed in an inclined position so that as great
a surface as possible may be given to the serum. The

BLOOD-SERUM. 89

process of solidification requires constant attention if
good results are to be obtained, i. e., if a translucent,
solid medium is to result. If the old, small form of
apparatus is employed (Fig. 16), then the solidification
can be accomplished in a shorter time than if the larger
forms, which are now frequently employed, are used.
No definite rule for the time that will be required can
be laid down, for this is not constant. If the small
solidifying apparatus is used, very good results may be
obtained in about two hours at 78° C. It frequently
requires a longer time at a higher temperature than
has been mentioned. This is especially the case with
Léffler’s serum mixture.

The best results are obtained when a low temperature
is employed for a long time. Under any circumstances
the tubes must be observed from time to time through
the glass door or cover with which the solidifying oven
is provided, and each time the oven should be slightly
jarred with the hand to see if solidification, as indi-
cated by the disappearance of tremors from the serum,
is beginning. If the temperature gets too high, or the
exposure is too long, an opaque medium results. The
temperature to be observed is that of the air inside the
chamber, and also that of the water surrounding it.
The latter is usually a degree or two higher than the
former. The tubes should not rest directly upon the
heated bottom or against the heated sides of the chamber,
but should lie upon racks of wood or wire, and be pro-
tected from the sides by a wire screen of gauze; in this
way the tubes are all exposed to about the same tem-
perature. The thermometer which indicates the tem-
perature inside the chamber should not touch the
surfaces, but should either be suspended free from above

90 BACTERIOLOGY.

through a cork in the top of the apparatus, if the large
form of apparatus is used; or should lie upon a rack of
cork or wood, its bulb being free and a little lower than
the other extremity, if the small, old-fashioned appa-
ratus of Koch is employed. The latter form is prefer-
able, as it is more easily managed.

When solidification is complete, the tubes are to be
retained in the erect position and, unless they are
intended for immediate use, must be prevented from
drying. The superfluous ends of the cotton plugs should
be burned off, and the mouths of the tubes may then be
covered by sterilized rubber caps, or, as Ghriskey sug-
gests, they may be closed with sterilized corks pushed
in on top of the cotton plugs. Even with the greatest
care it not uncommonly happens that one or two of the
lot of tubes thus prepared and protected will become
contaminated. This is usually due to spores of moulds
that have fallen into the rubber caps or on the cotton
plugs during manipulation, and, finding no means of
outward growth, have thrown their hyphe downward
through the cotton into the tube, and their spores have
fallen on the surface of the serum and developed there.

It is often desirable to obtain blood-serum in small
quantities, either for culture purposes or for the study
of the serum of different animals in its relation to bac-
teria, and for this purpose Nuttall (Centralbl. Bakt. u.
Parasitenkunde, 1892, Bd. xi. p. 539) suggests a very
convenient method. By the use of a sterilized vessel,
of the shape given in Fig. 17, from ten to one hundred
cubic centimetres of blood can be collected, and if proper
precautions are observed no contamination by bacteria
need occur. The collecting bulb is used in the fol-
lowing way: An artery, either femoral or carotid, is

BLOOD-SERUM. 91

exposed, and around it two ligatures are placed ; that
distant from the heart is tightened, while the one nearest
the heart is left loose; between the latter and. the -
heart the artery is clamped. A small slit is then made
in its wall, into which the point a of the bulb is intro-
duced and the artery bound tightly around it with the
hitherto loose ligature ; the clamp is removed and the

Fic. 17.

a
Nuttall’s bulb for collecting blood-serum under antiseptic precautions.

bulb quickly fills with blood. The clamp is now again
put in position, the point of the bulb removed and
sealed in the gas-flame, the loose ligature tightened, the
wound closed, and the vessel containing the blood is set
aside in a cool place until coagulation has occurred.
The serum is most easily withdrawn from the bulb by
means of a pipette, closed above with a cotton plug and
supplied with a bit of rubber tubing about one-half
metre long with glass mouth-piece. By holding the
pipette in the hand and sucking upon the rubber tube
one can more easily direct the point of the pipette than
if it is used in the ordinary way.

92 BACTERIOLOGY.

The bulbs are easily blown, and after having been

sealed at the point and plugged with cotton can be kept
- on hand just as are sterilized test-tubes.

Spectra, Mepra.—The media just described—bou-
illon, nutrient gelatin, nutrient agar-agar, potato, and
blood-serum—are those in general use in the laboratory
for purposes of isolation and study of the ordinary forms
of bacteria. For the finer points of differentiation
special media have been suggested ; a few of them will
be mentioned.

Milk. Fresh milk should be allowed to stand over
night in the ice-chest, the cream then removed, and the
remainder of the milk pipetted into test-tubes, about
8 c.c. to each tube, and sterilized by the intermittent
process, at the temperature of steam, for three successive
days.

The cream is best separated from the milk by the use
of a cylindrical vessel with stopcock at the bottom,
by means of which the milk, devoid of cream, may be
drawn off. A Chevalier creamometer with stopcock at
the bottom serves the purpose very well. It should be
covered while standing.

Milk may be used as a culture medium without any
addition to it, or, before sterilizing, a few drops of
litmus tincture may be added, just enough to give it a
pale blue color. By this means it may be seen that dif-
ferent organisms bring about different reactions in the
medium ; some producing alkalies which cause the blue
color to be intensified, others producing acids which
change it to red, while others bring about neither of
these changes. Similarly litmus solution is often added
to gelatin and agar-agar for the same purpose.

Milk may also be employed as a solid culture medium

SPECIAL MEDIA. 93

by the addition to it of gelatin or agar-agar in the pro-
portions given for the preparation of the ordinary nutri-
ent gelatin or agar-agar. It has, however, in this form
the disadvantage of not being transparent, and can there-
fore best be used for the study of those organisms which
grow upon the surface of the medium without causing
liquefaction.

Nutrient gelatin and agar-agar can also be prepared
from neutral milk whey, obtained from milk by pre-
cipitation of the casein.

Dunham’s peptone solution, The medium usually
known as Dunham’s solution is prepared according to
the following formula :

Dried peptone . f ; $ e i ‘ § a 1 part.
Sodium chloride . b 2 ‘ < ‘i a 0.5.48
Distilled water . : : ‘ ° 7 - 100 parts.

It is usually of a neutral or slightly alkaline reaction,
and neutralization is not, therefore, necessary. It is
filtered, decanted into tubes or flasks, and sterilized in
the steam sterilizer in the ordinary way. The most
common use to which this solution is put is in deter-
mining if the organism under consideration possesses
the property of producing indol as one of its products
of nutrition. It is essential for accuracy that the prep-
aration of dried peptone employed should be of as
nearly chemical purity as is possible, and indeed the
other ingredients should be correspondingly free from
impurities. Gorini (Centralblatt fiir Bakteriologie und
Parasitenkunde, 1893, Bd. xiii. p. 790) calls attention
to the fact that impurities in the peptone, particularly
the presence of carbohydrates, so interfere with the
production of indol by certain bacteria that otherwise

produce it that it is ofttimes impossible, when such prep-
5*

94 BACTERIOLOGY,

arations have been employed, to obtain the characteristic
color reaction of this body, and where it is obtained it
is always after a much longer time than is the case
where peptone free from these substances has been used.
He suggests the advisability of testing the purity of all
peptone preparations before using them, by means of
the reaction that they exhibit when acted upon by
Fehling’s alkaline copper solution. Under the influence
of this agent pure peptone in solution gives a violet
color (the biuret reaction), which remains permanent
even after boiling for five minutes. If instead of a.
violet color there appears a red or reddish-yellow pre-
cipitate the peptone should be discarded, as in his
experience no indol is produced from peptone giving
this reaction. Both the peptone solution and that of
the copper (particularly the latter) should be relatively
dilute in order for the reaction to be successful.

Peptone rosalic acid solution. Peptone solution to
which rosalic acid is added serves well for the detection
of alterations in reaction. It consists of the peptone
solution of Dunham, to each 100 c.c. of which 2 c.c. of
the following solution is added :

Rosalic acid (coralline) . 5 : ‘ f 0.5 gramme.
Alcohol (80 per cent.) : < a ‘ - 100 ae

This is to be boiled, filtered, and decanted into clean,
sterilized test-tubes, about 8 to 10 c.c. to each tube. The
tubes are then to be sterilized in the usual way by steam.
When sterilization is completed and the tubes cooled, the
solution will be of a very pale rose color, which disap-
pears entirely under the action of acids, and becomes
much more intense when alkalies are produced. We
have used this solution for some time for the study of
the reactions produced by different organisms, and find

SPECIAL MEDIA, 95

it a valuable addition to our means of differentiation of
bacteria.

Rosalie acid cannot be used with safety in solutions
containing glucose, as the reducing action of the latter
deprives it of its color.

Lactose-litmus-agar, or gelatin of Wurtz. A medium
of much use in the differentiation of bacteria is that
recommended by Wurtz, consisting of ordinary nutrient,
slightly alkaline agar-agar to which from 2 to 3 per
cent. of lactose and sufficient litmus tincture to give it
a pale blue color have been added. Bacteria capable of
causing fermentation of lactose when grown on this
medium develop into colonies of a pale pink color and
cause, likewise, a reddening of the surrounding medium,
owing to the production of acid as a result of their
action upon the lactose; while other bacteria, incapable
of such fermentative activities, grow as pale blue colonies
and cause no reddening of the surrounding medium.
It is especially useful for the differentiation of the
bacillus of typhoid fever, which does not possess the
property of bringing about fermentation of lactose, from
other organisms that simulate it in many other respects
but which possess this property.

Its preparation is as follows: To nutrient agar-agar
or gelatin, the alkalinity of which is such that one cubic
centimetre will require 0.1 c.c. of a 1: 20 normal sul-
phuric acid solution to neutralize it, lactose is added in.
the proportion of 2 or 3 per cent. ; it is then decanted
into test-tubes and sterilized in the usual way. When
sterilization is complete there is to be added to each
tube enough sterilized litmus tincture to give a decided
though not very intense blue color. This must be done
carefully, to avoid contamination of the tubes during

96 BACTERIOLOGY.

manipulation. It is better not to add the litmus tinc-
ture before sterilizing the tubes, as its color character-
istics are in some way altered by its contact with
organic matters under the influence of heat.

When ready it may be used as ordinary agar-agar or
gelatin, either for plates or slant cultures.

Léffler’s blood-serum mixture. Léffler’s blood-serum
mixture consists of one part of neutral meat-infusion
bouillon containing 1 per cent. of grape-sugar, and three
parts of blood-serum. This mixture is placed in test-
tubes, sterilized, and solidified in exactly the way given
for blood-serum.

Guarniari’s agar-gelatin :

Meat-infusion . . . a as 4 3 . 950 ¢.¢.
Sodium chloride . Z : ‘ . . & grammes.
Peptone . : 5 c ‘ . " - 25-30
Gelatin . 3 ‘ a i i : . 40-60 *f
Agar-agar . c ws a = 6 : 3 34
Water ‘ m zi . eS i . 50c¢

The point in the preparation of this medium is its
reaction, which should be exactly neutral.

The list of special media is too great to be given in a
work of -this size. Their description must be seen in
the original. Those that have been given above will
suffice for obtaining a clear understanding of the prin-
ciples of the work.

Nore.—The term “ meat-infusion ” always implies a
watery extract of meat made by mixing 500 grammes of
finely-chopped lean meat and 1 litre of water together,
and allowing them to stand in a cool place for twenty-
four hours. At the end of this time the fluid portion
is strained off through a coarse towel. This represents
the infusion.

CHAPTER VI.

Preparation of the tubes, flasks, etc., in which the media are to be pre-
served.

WHILE the media are in course of preparation it is
well to get the test-tubes and flasks ready for their
reception, and it is essential that they should be as clean
as is possible to make them. For this purpose it is
advisable that both new tubes and those which have
previously been used should be boiled for some time,
about thirty to forty-five minutes, in a strong solution
of common soda, about a 4 or 6 per cent. solution ; it
is not necessary to be exact as to the strength, but it
should not be weaker than this. At the end of this
time they are to be carefully swabbed out with a cylin-
drical bristle brush, preferably one having a reed handle
Fig. 18), as those with wire handles are apt to break

Brush for cleaning test-tubes.

through the bottoms of the tubes. All trace of adherent
material should be carefully removed. When the tubes
are quite clean they may be rinsed in a warm solution
of commercial hydrochloric acid of the strength of about
1 per cent. This is to remove the alkali. They are
then to be thoroughly rinsed in clear, running water,
and stood top down until the water has drained from

98 BACTERIOLOGY.

them. When dry they are to be plugged with raw
cotton. The plugging with the cotton requires a little
practice before it can be properly done. The cotton
should be introduced into the mouths of the tubes in
such a way that no cracks or creases exist, but should
fill them quite regularly all around. The plugs should
fit neither too tightly nor too loosely, but should be
just firmly enough in position to sustain the weight of
the tube into which it is placed when held up by the
portion which projects from and overhangs the mouth
of the tube. The tubes thus plugged with cotton are
now to be placed upright in a wire basket and hedted
for one hour in thé hot-air sterilizer at a temperature
of about 150° C. A very-good rule for this process of
sterilizing is to observe the tubes from time to time, and
as soon as the cotton has become a very light brown
color, not deeper than a dark-cream tint, to consider
sterilization complete. The tubes are then removed
and allowed to cool down.

The cotton used for this purpose should be the
ordinary cotton batting of the shops, and not absorbent
cotton ; the latter becomes too tightly packed, and is,
moreover, much too expensive for this purpose.

Care should be taken not to burn the cotton, other-
wise the tubes will become coated with a dark-colored
oily deposit which renders them unfit for use, and they
will have to be cleaned again.

FILLinc THE TuBES.— When the tubes are cold they
may be filled. This is best accomplished by the use of
a spherical form of funnel, such as is shown in Fig. 19.
The liquefied medium is poured into this funnel, which
has been carefully washed, and by pressing the pinch-
cock with which the funnel is provided, the desired

. FILLING THE TUBES. 99

amount of material (5-10 c.c.) may be allowed to flow
into the tubes held under its opening.

Fig. 19.

Funnel for filling tubes with culture media.

Jt is not necessary to sterilize the funnel, for the
medium is to be subjected to this process as soon as it
is in the test-tubes.

Care should be taken that none of the medium is
dropped upon the mouth of the test-tube, otherwise the
cotton plugs become adherent to it and are not only
difficult to remove, but present a very untidy appear-
ance, and interfere, indeed, with the proper manipula-
tions.

As soon as the tubes have been filled they are to be
sterilized in the steam sterilizer for fifteen minutes on

100 BACTERIOLOGY. Y

each of three successive days. During the intervening
days they may be kept at the ordinary room tempera-
ture.

When sterilization is complete, and the medium in
the tubes is still liquid, some of them may be placed in
a slanting position, at an angle of about ten degrees
with the surface on which they rest, and the medium
allowed to solidify in this position. These are for the
so-called slant cultures. The balance may solidify in
the erect position ; these serve for the plate cultures.

For Esmarch tubes not more than 5 c.c. of material
should be placed in each tube, as more than this renders
the rolling difficult and irregular.

CHAPTER VII.

Technique of making plates—Esmarch tubes, Petri plates, etc.

Puates.—The plate method can be practised with
both agar-agar and gelatin. It cannot be practised
with blood-serum, because the serum, when once solidi-
fied, cannot be again liquefied.

Plates are usually referred to as “a set.” This term
implies three individual plates, each representing the
mixture of organisms in a higher stage of dilution.
The first plate is known usually as “the original,” or
“plate 1,” the first dilution from this as “ plate 2,” and
the second as “ plate 3.”

In the preparation of a set of plates the following
are the steps to be observed :

Three tubes, each containing from 7 to 9 c.c. of gela-
tin or agar-agar, are placed in the warm water bath
until the medium has become liquid. If agar-agar is
employed, this is accomplished at the boiling-point of
water ; if gelatin is used, a much lower temperature
suffices (35°-40° C.). When liquefaction is complete
the temperature of the water, in the case of agar-agar,
must be reduced to 41°-42° C., at which temperature
the agar-agar remains liquid, and the organisms may
be introduced into it without fear of destroying their
vitality. The medium being now liquid and of the
proper temperature, a very small portion of the mixture
of organisms to be studied is taken up with a steril-

102 BACTERIOLOGY.

ized, looped platinum wire, Fig. 20. This is nothing
more than a piece of platinum wire of about 5 cm.
long, twisted into a small loop at one end and fused
into a bit of glass rod, which acts as a handle, at the
other extremity. This loop is one of the most useful
of bacteriological instruments, as there is hardly a
manipulation in the work into which it does not enter.
Under no conditions is it to be employed without having

Looped and straight platinum wires in glass handles.

been passed through the gas-flame until quite hot; this
is for the purpose of sterilization. One should form a
habit of never taking up one of these platinum-wire
needles, as they are called, for they are both looped and
curved or straight (Fig. 20, 6), without passing it
through the flame, and the sooner the beginner learns
to do this as a matter of reflex, the sooner does he rid
himself of one of the possible sources of error in his
work. It must be remembered, though, that it should
not be used when hot, otherwise the organisms taken
upon it are killed by the high temperature ; after steril-
ization in the flame one waits for a few seconds until it
is cool before using.

The bit of material under consideration is transferred
with the sterilized loop into tube No. 1, “ the original,”
where it is carefully disintegrated by gently rubbing it

TECHNIQUE OF MAKING PLATES. 103

against the sides of the tube. The more carefully this
is done the more homogeneous will be the distribution
of the organisms and the better the results. The loop
is then again sterilized, and three of its loopfuls are
passed, without touching the sides of the tube, from “the
original” into tube No. 2, where they are carefully
mixed. Again the loop is sterilized and again three
dips are made from tube 2 into tube 3. This completes
the dilution. The loop is now sterilized before laying
it aside.

Levelling tripod with glass chamber for plates.

During this manipulation, which must be done
quickly if agar-agar is employed, the temperature of
the water in the bath in which the tubes stand should
never get lower than 39° C., and never higher than
43° C. If it falls too low, below 38° C., the agar-agar
gelatinizes, and can only be redissolved by a tempera-
ture that would be destructive to the organisms which
may have been introduced into the tubes. This is not

104 BACTERIOLOGY.

of so much moment with gelatin, as it may readily be
redissolved at a temperature not detrimental to the
organisms with which the tubes may have been inocu-
lated.

THE CooLING-sTAGE AND LEVELLING TRIPOD.—
While the medium of which the plates are to be made
is melting, it is well to arrange the cooling-stage (Fig.
21) upon which it is to be subsequently solidified.

This stage consists of a glass dish filled with ice-
water and covered with a ground-glass plate, which in
turn has a dome-shaped cover. The dish rests upon a
tripod which can be brought to an exact level, as indi-
cated by the spirit-level, by raising or lowering its legs
by means of screws, with which they are provided.
Three stages are usually employed. When ready for
use they should be exactly level.

Russia iron box for holding plates, etc., during sterilization in dry heat.

THE Gass PLATEsS.—On each of the stages is to be
placed a glass plate upon which the liquefied gelatin or
agar-agar is to be poured and allowed to solidify. It
is, therefore, necessary that the plates should not only
be sterile when placed upon the stages, but should be
carefully protected by a cover against dust and bacteria
from outside sources during manipulation.

TECHNIQUE OF MAKING PLATES, 105

A number of plates at a time are usually sterilized in
the dry sterilizer at a temperature of 150° to 180° C.
for one hour. During sterilization and until used they
are retained in an iron box (Fig. 22), which is especially
designed for the purpose.

They should never be placed upon the stage until
cold ; otherwise they crack.

When the plates which have been placed upon the
stages are quite cold, the melted gelatin or agar-agar in
the tubes which represent the three dilutions should be
poured upon them, each tube being emptied upon a
separate plate. If the medium is quite fluid it spreads
over the surface of the plates in a thin, even layer.
Sometimes it may be more evenly spread as it flows
from the tube by the aid of a sterilized glass rod.

Fia. 23.

Glass benches for supporting plates.

As the contents of each tube are emptied upon a plate
the cover of the cooling-stage is quickly replaced and
the plate allowed to stand until the gelatin or agar-agar
is quite solid. This takes longer with gelatin than with
agar. When quite solid they are placed upon little
glass benches (Fig. 23), and each bench is labelled with
the number of the plate in the series of dilutions. The
benches, with the plates upon them, are then piled one
above the other in a glass dish, the so-called “ culture-
dish,” in which the plates are to be kept during the

106 BACTERIOLOGY.

growth of the bacteria. The benches are sterilized
before using, in the way given for the plates.

CULTURE-DISH.—This dish, which is about 22 cm.
in diameter and has vertical sides of about 6 cm. in
height, is provided with a cover of exactly the same
design, but of a little larger diameter. This cover,
when placed upon the dish containing the plates, fits
over it and prevents the access of dust. Prior to using,
the dish and cover should have been disinfected for
one-half hour with 1: 1000 sublimate, and then all the
sublimate solution allowed to drain from it.

Into the bottom of this dish is sometimes placed a
disk of sterilized filter-paper moistened with sterilized
water, which serves to prevent the drying of the plates.
This, however, is not necessary.

If agar-agar is employed, the dish and its contents
may be placed at a temperature of 37°-38° C.; if
gelatin, the temperature at which the plates are now to
be kept should not be over 22° C., otherwise the gela-
tin becomes liquefied and the plates are rendered useless.

When development has occurred, the object of the
dilution will easily be seen, and the different species of
bacteria in the mixture will be recognized by differences
in the character of the colonies growing from them.

This, in short, is the plate method of Koch for the
separation of the individual species contained in a mix-
ture of bacteria. Many modifications of this method
exist ; all, however, are based upon the same principles.
The modifications have for their object the accomplish-
ment of the same end, but with a smaller armamenta-
rium of apparatus.

Perri’s MopIFIcATION OF THE PLATE METHOD.—
The modification which approaches nearest to the original

ESMARCH’S TUBES. 107

method, and at the same time lessens very materially the
number of steps in the process, is that suggested by Petri.
It consists in substituting for the plates small, round,
double glass dishes, which have about the same surface-
area as the plates. The liquid medium may be poured
directly into these little dishes without their being exactly
level. Each dish acts as a plate. Their covers are then
to be replaced, and they are set aside for observation.
In all other respects the steps are the same as those given
for Koch’s original method. Petri’s dishesare flat, double
dishes of glass (Fig. 24). They are of about 8 cm. in

Fia. 24.

Weil

TT i, ce

Petri double dish, now generally used instead of plates.

diameter and about 1.5 to 2 cm. in height, the walls
being vertical. They may readily be sterilized either
by the hot-air or steam methods of sterilization. They
are very useful for this work, as they do away with the
necessity for the cooling-stage and levelling tripods,
though in warm weather the cooling-stage may be used
to hasten the solidification of gelatin.

Esmarcu’s Tuses.—The modification of Koch’s
method which insures the greatest security from con-
tamination by outside organisms and requires the small-
est supply of apparatus, is that suggested by v. Esmarch.
It differs from the other methods thus: The dilutions
having been prepared in tubes containing a smaller
amount of medium than usual—as a rule not more than

108 BACTERIOLOGY.

5 to 6 c.c.—are, instead of being poured out upon plates
or into dishes, spread over the inner surface of the tube
containing them, and without removing the cotton plugs
are caused to solidify in this position. The tubes then
present a thin cylindrical lining of gelatin or agar-agar,
upon which the colonies develop. In all other respects
the conditions for the growth of the organisms are the
same as in flat plates.

Esmarch directs that after completion of the dilutions
the tops of the cotton plugs in the tubes should be cut
off flush with the mouth of the test-tube and a steril-
ized rubber cap be placed over this. They are then to
be held in the horizontal position and twisted between
the fingers upon their long axes under ice-water. The
- gelatin becomes solidified thereby and adheres to the
sides of the tube. When the gelatin is quite hard the
tubes are removed from the water, wiped dry, the.rubber
caps removed, and they are set aside for observation.

For some time past we have deviated from the direc-
tion given by v. Esmarch for this part of his method,
and instead of rolling the tubes under ice-water, we roll
them upon a block of ice (Fig. 25), after the method
devised by Booker in the Pathological Laboratory of
the Johns Hopkins University in 1887. In this method
a small block of ice only is needed. It is arranged
nearly level, and is held in position by being placed in
a dish upon a cloth. A horizontal groove is melted in
the surface of the ice with a test-tube of hot water. The
tubes to be rolled are then held in an almost, not quite,
horizontal position and twisted between the fingers
until the sides are moistened by the contents to within
about 1 em. of the cotton plug, care being taken that
the gelatin does not touch the cotton; otherwise the latter

ESMARCH’S TUBES, 109

becomes adherent to the sides of the tube and is difficult
to remove. The tube is then placed in the groove in
the ice and rolled, neither rubber cap nor cutting off of
the cotton plug being necessary.

Fie. 25,

Demonstrating Booker’s method of rolling Esmarch tubes on a block of ice.

The advantages of this process over that followed by
v. Esmarch are that it requires less time, is cleaner, no
rubber caps are needed, the rolled tubes are more regu-
lar, and the gelatin does not touch the cotton plug, as
is always the case in the tubes rolled under water,
because of the impossibility of holding them steady at
one level.

There is an impression that Esmarch tubes are not
a success when made from ordinary nutrient agar-agar
because of the tendency of this medium to collapse and
fall into the bottom of the tube. This slipping down
of the agar-agar is due to the water that is squeezed
from it during solidification getting between the medium
and the walls of the tube. ‘This can easily be overcome

by allowing the rolled tubes to remain in nearly a hori-
6

110. BACTERIOLOGY.

zontal position, the cotton end of the tube about 1.5 to
2 cm. higher than the bottom of the tube, for twenty-
four hours after rolling them. During this time the
edge of the agar-agar nearest the cotton plug becomes
dried and adherent to the walls of the tube, while the
water collects at the most dependent point, «. ¢., the
bottom of the tube. After this they may be retained
in the upright position without fear of the agar-agar
slipping down. We have followed this process for
several years with entire satisfaction.)

In all these processes, if the dilutions of the number
of organisms have been properly conducted, the results
will be the same. The original plate or tube, as a rule,
will be of no use because of the great number of colonies
in it. Plate or tube No. 2 may be of service, but plate
or tube 3 will usually contain the organisms in such
small numbers that the colonies originating from them
will have nothing to prevent their characteristic de-
velopment.

For reasons of economy, the “original,” tube 1,
is sometimes substituted by a tube containing normal
salt solution (0.6 to 0.7 per cent. of sodium chloride
in water), which is thrown aside as soon as the dilu-
tions are completed and only plates or tubes 2 and 3
are made.

1 The impression that agar-agar is not suitable for roll tubes was shown to
be erroneous, and the above method was developed in the Pathological
Laboratory of the Johns Hopkins University.

CHAPTER VIII.

The incubating oven—Gas-pressure regulator —Thermo-regulator — The
safety burner employed in heating the incubator.

Tue IncuBaTor.— When the plates have been made,
it must be borne in mind that for the development of
certain forms of bacteria a higher temperature is neces-
sary than for the growth of others. The pathogenic
or disease-producing organisms all grow more luxuri-
antly at the temperature of the human body (37.5° C.)
than at lower temperatures; whereas, with the ordi-
nary saprophytic forms almost any temperature between
18° C. and that of the body is favorable. It therefore
becomes necessary to provide some place in which a
constant temperature suitable to the growth of the
pathogenic organisms can be maintained. For this
purpose there have been devised a number of different
forms of apparatus. Fundamentally they are all based
upon the same principles, however, and a general de-
scription of the essential points involved in their con-
struction will be all that is needed here.

This apparatus has the names thermostat, incubator,
and brooding oven. It is a copper chamber (Fig. 26)
with double walls, the space between which is filled
with water. The incubating chamber may be opened
or closed by a closely fitting double door, inside of
which is usually a false door of glass through which the
contents of the chamber may be inspected without
actually opening it. The whole apparatus is encased

112 BACTERIOLOGY.

in either asbestos boards or thick felt to prevent radia-
tion of heat and consequent fluctuations in temperature.
In the top of the chamber is a small opening through

Fig. 26.

ere

el

S

Incubator used in bacteriological work.

which a thermometer projects into its interior. At
either corner, leading into the space containing the
water, are other openings for the reception of another
thermometer and a thermo-regulator, and for refilling the
apparatus as the water evaporates. On the side is a

THE INCUBATOR,. 118

water-gauge for showing the level of the water between
the walls. The object of the water chamber, which is
formed by the double wall arrangement, is to insure, by
means of the warmed water, an equable temperature at
all parts of the apparatus—at the top as well as at the
sides, back, and bottom, and the apparatus should be
kept filled with water, otherwise the purpose for which
it is constructed will not be accomplished. When the
chamber between the walls is filled with water heat is
supplied from a gas-flame placed beneath it.

Fig. 27.

iy D

Vf)

.
Koch’s safety burner.

The burner employed in heating the incubator was
originally devised by Koch and is known as “ Koch’s
safety burner” (Fig. 27). It is a Bunsen burner pro-
vided with an arrangement for automatically turning
off the gas supply and thus preventing accidents should

114 BACTERIOLOGY.

the flame become extinguished at a time when no one
was near. The gas-cock by which the gas is turned on
and off is provided with a long arm which is weighted
and which, when the gas is turned on and burning, rests
upon the side of a revolving, horizontal plate that is
connected with the free ends of two metal spirals which -
are fixed in opposite directions on either side of the
flame and heated by it. If by draughts or any other
accident the flame becomes extinguished the metal
spirals cool, and in cooling contract, twist the horizontal
plate in the opposite direction and allow the weighted
arm of the gas-cock to fall. By its falling the gas
supply is turned off.

THERMO-REGULATORS.—The regulation and mainte-
nance of the proper temperature within the incubator
is accomplished by the employment of an automatic
thermo-regulator.

The form of thermo-regulator used for this purpose
is constructed upon principles involving the expansion
and contraction of fluid substances under the influence
of heat and cold. By means of this expansion and
contraction, the amount of gas passing from the source
of supply to the burner may be either diminished or
increased as the temperature of the substance in which
the regulator is placed either rises or falls.

The simplest form of thermo-regulator which serves
to illustrate the principles involved is seen in Fig. 28.

It consists of a glass cylinder e, having a communi-
cating branch tube 6, and rubber stopper f, through
which projects the bent tube a. The tube a is ground
to a slanting point at the extremity which projects into
the tube e, and is provided a short distance above this
point with a capillary opening g, in one of its sides.

THERMO-REGULATORS. 115

When ready for use the cylinder ¢ is filled with fluid,
either mercury, calcium chloride solution, alcohol and
ether, or a number of other substances depending upon
the temperature to which it is to be subjected, up to

Fic. 28.

Mercurial thermo-regulator.

about the level shown in the figure. It is then allowed
to stand or is suspended in the bath the temperature of
which it is to regulate. The rubber tubing coming
from the gas supply is attached to the outer end of the
glass tube uv, and the tube going to the burner is slipped
over the branch tube 6. The gas is turned on and the

116 BACTERIOLOGY.

burner lighted and placed under the bath. The gas
now streams through the tube a into the cylinder e and
out at 6 to the burner, but as the temperature of the
bath rises, the fluid contained in the cylinder e, under
the influence of the elevation of temperature begins to
expand, and as a continuous rise in temperature pro-
ceeds, the expansion of the fluid accompanies it and
gradually closes the slanting opening A of tube a. In
this way the supply of gas becomes diminished and
the rise in temperature of the bath will be less rapid,
until finally the opening at A will be closed entirely,
when the supply of gas to the burner will now be
limited to that passing through the capillary opening
g. This is not sufficient to maintain the highest tem-
perature reached, and a gradual contraction of the fluid
now occurs until there is again an outflow of gas from
the opening h, when again the temperature rises. This
contraction and expansion of the fluid in the regulator
continues until eventually a point is reached at which
the position of the fluid in the cylinder e allows of the
passage of just enough gas from the opening h to
maintain a constant temperature. This, in short, is the
principle on which thermo-regulators are constructed,
but it must be borne in mind that a great deal of detail
exists in the construction of an accurate instrument.
The number of different forms of this apparatus is
comparatively large, and each form has its special
merits.

The value, that is the delicacy, of the thermo-
regulator depends upon a number of factors, all of
which it would be useless to introduce into a book of
this kind, but in general it may be said that the essen-
tial points to be observed in selecting a thermo-regulator

GAS-PRESSURE REGULATORS. 117

depend in the main upon the temperatures to which it
is to be applied. For low temperatures such fluids as
ether, alcohol, and calcium chloride solution, which ex-
pand and contract rapidly and regularly under slight
variations in temperature, are commonly employed ;
whereas for temperatures approaching the boiling-point
of water mercury is most frequently used.

Fig, 29.

Moitessier’s gas-pressure regulator.

The temperature of the incubator is to be regulated,
then, by the use of some such form of apparatus as that
just described. It should be of sufficient delicacy to
prevent a fluctuation of more than 0.2° C. in the tem-
perature of the air within the chamber of the apparatus.

GAS-PRESSURE REGULATORS.—A gas-pressure regu-
lator is not rarely intervened between the gas supply

and the thermo-regulator. This apparatus has for its
6%

118 BACTERIOLOGY.

object the maintenance of a constant pressure of the gas
going to the thermo-regulator. There are several in-
struments of this form in use, but they do not accom-
plish the object for which they are designed.

The instrument most commonly employed, the appa-
ratus of Moitessier (Fig. 29), is based on somewhat the
same principles as the large regulators seen at the
manufactories of illuminating gas, which act very well
when employed on the large scale, as one sees them
there; but which, when applied to the limited and
sudden fluctuations seen in the gas coming from an
ordinary gas-cock, are practically useless. They are
too gross in their construction, and are only seen to act
under comparatively great and gradual fluctuations in
pressure. If a good form of thermo-regulator is em-
ployed there is no necessity for the use of any of the
forms of pressure-regulators thus far introduced.

CHAPTER IX.

The study of colonies—Their naked-eye peculiarities and their appearance
under different conditions—Differences in the structure of colonies from
different species of bacteria—Stab cultures—Slant cultures.

THE plates upon agar-agar which have been prepared
from a mixture of organisms and have been placed in
the incubator, and those of gelatin which have been
maintained at the ordinary temperature of the room, are
usually ready for examination after twenty-four to
forty-eight hours. They will be found to be marked
here and there by small points or little islands of more
or less opaque appearance. In some instances these
will be so transparent that it is with difficulty one can
see them with the naked eye. Again, they may be of
a dense opaque appearance, at one time sharply circum-
scribed and round, again irregular in their outline ;
here a point will present one color, there perhaps
another. On gelatin some of the points will be seen to
be lying on the surface of the medium, others will have
sunk into little depressions, while at still other points
the clear gelatin will be marked by more or less saucer-
shape pits containing opaque fluid.

Place the plate containing these points upon the stage
of the microscope and examine them with the lowest
power objective, and again differences will be observed.
Some of these minute points will be finely granular,
others coarsely so ; some will present a radiated appear-
ance, while a neighbor may be concentrically arranged.

120 BACTERIOLOGY.

Here nothing particularly characteristic will present,
there the point may resolve itself into a little mass hav-
ing somewhat the appearance of a very small pellicle of
raw cotton. All these differences, and many more, aid
us in saying that these little points must be different
in their nature. With a pointed platinum needle take
up a bit of one of these little islands, prepare it for
microscopic examination (see chapter on stained cover-
slip preparations), and examine it under the high power
oil-immersion objective, under access of the greatest
amount of light afforded by the illuminator of the
microscope. The preparation will be seen to be made
up entirely of bodies of the same shape ; they will all
be spheres, or ovals, or rods, but not a mixture of these
forms, if proper care in the manipulation has been
taken. Examine in the same way a neighboring spot
which possesses different naked-eye appearances, and it
will often be found to consist of bodies of an entirely
different appearance from those in the first preparation.

These spots or islands on the surface of the plates are
colonies of bacteria, differing severally, not only in out-
ward appearances, the one from the other, but, as our
cover-slip preparations show, in the morphological char-
acteristics of the individual organisms composing them.
If from one of these colonies a second set of plates be
prepared, the peculiarities which were at first observed
in this colony will be reproduced in all of the new set
of colonies which develop ; each will be found to con-
sist of the same organisms as the colony from which the
plates were made. In other words, these peculiarities
are constant under constant conditions.

With all organisms differences in the appearance of
the colonies depending upon their location in the medium

STAB AND SMEAR CULTURES. 121

can usually be detected. When deep down in the
medium, owing to surrounding pressure, they are quite
round, oval, or lozenge-shape ; whereas, when they are
on the surface of the gelatin or agar, they may take
quite a different form. This is purely a mechanical
effect, and is always to be borne in mind, otherwise
errors are apt to arise.

Pure Cuttures.—If from one of these small colonies
a bit be taken upon the point of a sterilized platinum
needle and introduced into a tube of sterilized gelatin
or agar-agar, the growth that results will be what is
known as a “ pure culture,” the condition in which all
organisms must be before a systematic study of their
many peculiarities is begun. Sometimes several series
of plates are necessary before the organism can be ob-
tained pure, but by patiently following this plan the
results will ultimately be satisfactory.

TrEsT-TUBE CULTURES; SraB CULTURES; SMEAR
CuLtures.—After separating the organisms, the one
from the other by the plate method just described, they
must be isolated from the plates as pure stab or smear
cultures.

This is done in the following way: Decide upon the
colony from which the pure culture is to be made.
Select preferably a small colony and one as widely sep-
arated from other colonies as possible. Sterilize in the
gas-flame a straight platinum-wire needle. The glass
handle of the needle should be drawn through the flame
as well as the needle itself, otherwise contamination
from this source may occur. When it is cool, which is
in five or ten seconds, take up carefully a portion of the
colony. Guard against touching anything but the colony.
If, during manipulation, the needle touches anything

122 BACTERIOLOGY.

else whatever than the colony from which the culture is
to be made, it must be sterilized again. This holds not
only for the time before touching the colony, but also
during its passage into the test-tube from the colony,
otherwise there is no guarantee that the growth resulting
from the inoculation of this bit of colony into a fresh
sterile medium will be pure.

In the meantime have in the other hand a test-tube
of sterile medium : gelatin, agar-agar, or potato. This
tube is held across the palm of the hand in an almost
horizontal position with its mouth pointing out between
the thumb and index finger and its contents toward the
body of the worker. With the disengaged fingers of
the hand holding the needle, the cotton plug is removed
from the tube by a twisting motion and placed between
the index and second fingers of the hand holding the
tube in such a way that the portion of the plug which
fits into the mouth of the test-tube looks toward the
dorsal surface of the hand and does not touch any portion
of the hand—this is accomplished by placing on/y the
overhanging portion of the plug between the fingers.
The needle containing the bit of colony is now to be
thrust into the medium in the tube if a stab culture is
desired, or rubbed gently over its surface if a smear
culture is to be made. The needle is then withdrawn,
the cotton plug replaced, and the needle sterilized before
it is laid down. Neither the needle nor its handle
should touch the inner sides of the test-tube if it can be
avoided.

The tube is then labelled and set aside for observa-
tion. The growth which appears in the tube after
twenty-four to thirty-six hours will be a pure culture
of the organisms of which the colony was composed.

STAB AND SMEAR CULTURES. 123

Cultures of this form are not only useful as a means
of preserving pure cultures of the different organisms
with which we may be working, but serve also to bring
out certain characteristics of different organisms when
grown in this way.

If gelatin is employed and the organism which has
been introduced into it possesses the power of bringing
about liquefaction, this result is by no means of the same
appearance for all organisms. Some organisms cause a
liquefaction which spreads across the whole upper sur-
face of the gelatin and continues gradually downward ;
again it occurs in a funnel shape, the broad end of the
funnel being uppermost and the point downward, corre-
sponding to the track of the needle. At times a stock-
ing- or sac-formed liquefaction may be noticed.

Norte.— Obtain a number of organisms from different
sources in pure cultures by the method given. Plant
them as pure cultures, all at the same time, in gelatin
—preferably gelatin of the same making—retain them
under the same conditions of temperature, and sketch
the finer differences in the way in which liquefaction
occurs.

CHAPTER X.

Methods of staining—Solutions employed—Preparation and staining of
cover-slips—Preparation of tissues for section-cutting—Staining of tissues—
Special staining methods.

THE entire list of solutions and methods that are
recommended for the staining of bacteria are not essen-
tial to the work of the beginner, so that only those
which are of most common application will'be given in
this book. In general, it suffices to say, bacteria stain
best with watery solutions of the basic aniline dyes,
and of these, fuchsin, gentian-violet, and methylene-
blue are those most frequently employed.

In practical work bacteria require to be stained in
two conditions: either dried upon cover-slips and then
stained, or stained in sections of tissues in which they
have been deposited during the course of disease. In
both processes the essential point to be borne in mind is
that the bacteria, because of their microscopic dimen-
sions, require to be more conspicuously stained than
the surrounding materials upon the cover-slips or in
the sections, otherwise their differentiation is a matter
of the greatest difficulty, if not of impossibility. For
this reason, especially in the case of section staining, it
frequently becomes necessary to decolorize the tissues
after removing them from the staining solutions in
order to render the bacteria more prominent; and for
this purpose special methods, which provide for decolor-
ization of the tissues without robbing the bacteria of

COVER-SLIP PREPARATIONS. 125

their color, are employed. The ordinary method of
cover-slip examination of bacteria, constantly in use in
these studies, is performed in the following way:

CovER-SLIP PREPARATIONS.—In order that the dis-
tribution of the organisms upon the cover slips may be
uniform and in as thin a layer as possible, it is essential
that the slip should be clean and free from grease.
For cleansing the slips several methods may be em-
ployed.

The simplest plan with new cover-slips is to im-
merse them for a few hours in strong nitric acid, after
which they are rinsed in water, then in alcohol, ether,
and, finally, they may be kept in alcohol to which a
little ammonia has been added. When they are to be
used they should be wiped dry with a clean cotton or
silk handkerchief.

If the slips have been previously used, boiling in
strong soap solution, followed by rinsing in clean warm
water, then treated as above, renders them clean enough
for ordinary purposes.

A method commonly employed is to remove all
coarse adherent matter from slips and slides by allowing
them to remain for a time in strong nitric or sulphuric
acid. They are removed from the acid after several
days, rinsed off in water, and treated as above. Knauer
has recently suggested the boiling of soiled cover-slips
and slides for from twenty to thirty minutes in a 10 per
cent. watery solution of lysol, after which they are to be
carefully rinsed in water until all trace of the lysol has
disappeared. They are then to be wiped dry with a
clean handkerchief.

Liffler’s method, which provides for the complete
removal of all grease, is to warm the cover-slips in con-

126 BACTERIOLOGY.

centrated sulphuric acid for a time, then rinse them in
water, after which they are kept in a mixture of equal
parts of alevhol and ammonia. They are to be dried
on a cloth from which the fat has been extracted.

Sleps in making the preparations. Place upon the
centre of one of the clean, dry cover-slips a very small
drop of distilled water or physiological salt solution.
With a platinum needle, which has been sterilized in
the gas-flame just before using and allowed to cool, take
up a very small portion of the colony to be examined
and mix it carefully with the drop on the slip until
there exists a very thin homogeneous film over the
larger part of the surface. This is to be dried upon
the slip by either allowing it to remain upon the table
in the horizontal position under a cover, to protect it
from dust, or by holding it between the fingers (not with
the forceps), at some distance above the gas-flame, until
it is quitedry. If held with the forceps over the flame
at this stage, too much heat may be unconsciously applied,
and the morphology of the organisms in the preparation
distorted. When held between the fingers with the
layer of bacteria away from the flame no such accident
is likely to occur. When the whole pellicle is com-
pletely dried the slip is to be taken up with the forceps,
and, holding the side upon which the bacteria are de-
posited away from the direct action of the flame, is to
be passed through the flame three times, a little more
than one second being allowed for each transit. Unless
the preliminary drying at the low temperature has been
complete, the preparation will be rendered worthless by
the subsequent “fixing” at the higher temperature, for
the reason that the protoplasm of bacteria when moist
coagulates at these temperatures, and in doing so the

COVER-SLIP PREPARATIONS. 127

normal outline of the cells is altered. If carefully
dried before fixing, this does not occur and the mor-
phology of the organism remains unchanged. A better
plan for the process of fixing is to employ a copper
plate of about 35 em. long by 10 cm. wide by 0.3 cm.
thick. This plate is laid upon an iron tripod and a
small gas-flame is placed beneath one of its extremities.
By this arrangement one can get a graduated tempera-
ture, beginning at the point of the plate above the gas-
flame where it is hottest, and becoming gradually cooler
toward the other end of the plate, which may be of a
very low temperature. By dropping water upon the
plate, beginning at the hottest point and proceeding to-
ward the cooler end, it is easy to determine the point
at which the water just boils; it is at a little below this
point that the cover-slips are to be placed, bacteria side
up, and allowed to remain about ten minutes, when the
fixing will be complete. The same may be accomplished
in a small copper drying oven, which is regulated to
remain at the temperature of 95° to 98° C. In very
particular work this plan is to be preferred to the pro-
cess of passing the cover-slips through the flame, as the
organisms are always subjected to the same degree of
heat, and the distortions which sometimes occur from
the too great and irregular application of high tem-
peratures may in part be eliminated, or if not, will be
more nearly constant. The fixing consists in drying or
coagulating “the gelatinous envelope surrounding the
organisms, by which means they are caused to adhere
to the surface of the cover-slip. When fixed, the stain-
ing is usually a simple matter. The majority of bac-
teria with which the beginner will have to deal stain
readily with solutions of any of the basic aniline dyes.

128 BACTERIOLOGY.

To stain the fixed cover-slip preparation it is taken
by one of its edges between the forceps, and a few
drops of a watery solution of fuchsin, gentian-violet,
or methylene-blue are placed upon the film and are
allowed to remain there twenty to thirty seconds. The
slip is then carefully rinsed in water, and without dry-
ing is placed bacteria down upon a slide ; the excess of
water is taken up by covering it with blotting-paper
and gently pressing upon it, and the preparation is
ready for examination.

Another plan that is sometimes used is to bring the
slip upon the slide, bacteria down, without rinsing off
the staining fluid; the excess of fluid is removed with
blotting-paper and the preparation is ready for examina-
tion with the microscope. This method is satisfactory
and time-saving, but must always be practised with
care. The staining fluid should always be carefully
filtered before using, to rid it of insoluble particles
which might mislead the examiner into mistaking them
for bacteria. If upon examination the preparation
proves to be of particular interest, so that it is desirable
to preserve it, then it is to be mounted permanently.
The drop of immersion oil is to be removed from the
surface of the slip with blotting-paper, and the slip
loosened, or rather floated, from the slide by allowing
water to flow around its edges. It is then taken up
with the forceps, carefully deprived of the water adher-
ing to it by means of blotting-paper, and then allowed
to dry. When dry it is mounted in xylol-Canada-
balsam by placing a small drop of the balsam upon
the surface of the film, and then inverting the slip upon
a clean glass slide. It is sometimes desirable to have
the balsam harden quickly, and a method that is com-

THE ORDINARY STAINING SOLUTIONS. 129

monly employed to induce this is as follows: The slide,
held by one of its ends between the fingers, is warmed
over the gas-flame until quite hot; a drop of balsam is
then placed on the centre of it, and it is again warmed ;
the cover-slip is then placed in position, and when the
balsam is evenly distributed the temperature is rapidly
reduced by rubbing the bottom of the slide with a
towel soaked in cold water. Usually the preparation
is firmly fixed after this treatment ; but a little practice
is necessary in order not to overheat and not to crack
the slide. The method is applicable only to cover-slip
preparations and cannot be used with tissues.

IMPRESSION COVER-SLIP PREPARATIONS.—The im-
pression preparations differ in value from the ordinary
cover-slip preparations only in one respect: they pre-
sent an impression of the organisms as they were ar-
ranged in the colony from which the preparation is
made. They are made by gently covering the colony
with a thin, clean cover-slip, lightly pressing upon it,
and, without moving the slip laterally, lifting it up by
one of its edges. The organisms adhere to the slip in
the same relation to one another that they had in the
colony. The subsequent steps of drying, fixing, stain-
ing, and mounting are the same as those just given for
the ordinary cover-slip preparations.

By this method, constancies in the arrangement and
grouping of the individuals in a colony can often be
made out. Some will always appear irregularly massed
together, others will grow in parallel bundles, while
others, again, will be seen as long twisted threads.

Tue Orpinary Srarnine Soiutions.—The solu-
tions commonly employed in staining cover-slip prepa-
rations are, as has been stated, watery solutions of the

130 BACTERIOLOGY.

basic aniline dyes—fuchsin, gentian-violet, and methy-
lene-blue. These solutions may be prepared either by
directly dissolving the dyes in substance in water until
the proper degree of concentration has been reached, or
by preparing them from concentrated watery or alco-
holiec solutions of the dyes which may be kept on hand
as stock. The latter method is that commonly prac-
tised.

The solutions of the colors which are in constant use
in staining are prepared as followed :

Prepare as stock, saturated alcoholic or watery solu-
tions of fuchsin, gentian-violet, and methylene-blue.
These solutions are best prepared by pouring into clean
bottles enough of the dyes in substance to fill them to
about one-fourth of their capacity. The bottle should
then be filled with alcohol or with water, tightly corked,
well shaken, and allowed to stand for 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 will then be labelled
saturated alcoholic or watery solution of fuchsin, gen-
tian-violet, or methylene-blue, as the case may be.
These alcoholic solutions are not employed for staining
purposes.

The solutions with which the staining is accomplished
are made from these alcoholic solutions in the follow-
ing way :

An ordinary test-tube of about 13 mm. diameter is
three-fourths filled with distilled water and the concen-
trated alcoholic or watery solution of the dye is then

SPECIAL STAINING SOLUTIONS. 181

added, little by little, until one can just see through the
solution. It is then ready for use. Care must be
taken that the color does not become too dense. The
best results are obtained when it is just transparent as
viewed through a layer of about 12 to 14 mm. thick.

These represent the staining solutions in everyday
use. They are kept in bottles supplied with stoppers
and pipettes (Fig. 30), and when used are dropped upon
the preparation to be stained. After remaining upon
the preparation for from twenty to thirty seconds, they
are washed off in water and the preparation can then
be examined.

Rack of bottles for staining solutions.

For certain bacteria which stain only imperfectly
with these simple solutions it is necessary to employ
some agent that will increase the penetrating action of
the dyes. Experience has taught us that this can be
accomplished by the addition to the solutions of small
quantities of alkaline substances or by dissolving the
staining materials in strong watery solutions of either
aniline oil or carbolic acid, instead of simple water—in
other words, by employing mordants with the stains.

Of the solutions thus prepared which may always be
employed upon bacteria that show a tendency to stain

132 BACTERIOLOGY.

imperfectly, there are three in common use—Léoffler’s
alkaline methylene-blue solution, the Koch-Ehrlich ani-
line-water solution of either fuchsin, gentian-violet, or
methylene-blue, and Ziehl’s solution of fuchsin in car-
bolic acid. These solutions are as follows :

Liffler’s alkaline methylene-blue solution :

Concentrated alcoholic solution of methylene-blue . 30 ¢.c.
Caustic potash in 1: 10,000 solution 3 F a . 100 ce.

Koch-Ehrlich aniline-water solutions. To about 100
c.c. of distilled water aniline oil is added, drop by drop,
and the solution thoroughly shaken after each addition,
until it is of an opaque appearance. It is then filtered
through moistened filter-paper until the filtrate is per-
fectly clear. To 100 c.c. of the clear filtrate add 10 cc.
of absolute alcohol and 11 c.c. of the concentrated al-
coholic solution of either fuchsin, methylene-blue, or
gentian-violet, preferably fuchsin or gentian-violet.

Ziehl’s carbolic-fuchsin solution :

Distilled water f a - . 100 ec.
Carbolic acid (erystaine) < es F ‘ 5 grammes.
Alcohol . ‘i ? ‘ F 2 - ec.
Fuchsin in substance . * ‘ = 1 gramme.

Or it may be prepared "= ddliting to a 5 per cent.
watery solution of carbolic acid the saturated alcoholic
solution of fuchsin until a metallic lustre appears on the
surface of the fluid.

Both the Koch-Ehrlich and the Ziehl solutions
decompose after having been made for a time; par-
ticularly is this the case with the former, so that it is
better to prepare them when needed in small quantities
than to employ old solutions. Solutions older than
fourteen days should not be used.

The three solutions just given may be used for cover-
glass preparations in the ordinary way.

STAINING THE TUBERCLE BACILLUS. 183

In some manipulations it becomes necessary to stain
the bacteria very intensely, so that they may retain their
color when exposed to the action of decolorizing agents.
These methods are usually employed for the purpose of
depriving surrounding objects or tissues of their color in
order that the stained bacteria may stand out in greater
contrast. It is in these cases that the staining solu-
tion with which the bacteria are being treated is to be
warmed, and in some cases boiled, so as to further
increase its penetrating action. When so treated, cer-
tain of the bacteria will retain their color, even when
exposed to very strong decolorizers. The tubercle
bacillus is characterized from all other bacteria, except
the bacillus of leprosy, by the tenacity with which it
retains its color when treated in this way. It is an
organism that is difficult to stain, but when once stained
is equally difficult to rob of its color.

METHOD OF STAINING THE TUBERCLE BACILLUS.—
Select from the sputum of a tuberculous subject one of
the small, white, cheesy masses which it is seen to con-
tain. Spread this upon a cover-slip and dry and fix
it in the usual way. The slip is now to be taken by
its edge with the forceps and the film covered with a
few drops of either the solution of Koch-Ehrlich or of
Zieh]. It is then held over the gas-flame, at first some
distance away, gradually being brought nearer, until the
fluid begins to boil. After it has bubbled up once or
twice it is removed from the flame, the excess of stain-
ing washed away in a stream of water, and it is then
immersed in a 30 per cent. solution of nitric acid in
water and allowed to remain there until all the color
has disappeared. In some cases this takes longer than

in others. One can always determine if decolorization
7

134 BACTERIOLOGY.

is complete by washing off the acid in a stream of water.
If the preparation is still quite colored it should be again
immersed in the acid; if of only a very faint color it
may be dipped in alcohol, again washed off in water, and
may now be stained with some contrast color. If, for
example, the tubercle bacilli have been stained with
fuchsin, methylene-blue forms a good contrast stain. In
making the contrast stain the steps in the process are
exactly those followed in the ordinary staining of cover-
slip preparations in general: the slip containing the
stained tubercle bacilli is rinsed off carefully in water
and a few drops of the methylene-blue svlution are
placed upon it and allowed to remain for thirty to forty
seconds, when it is again rinsed in water and examined
microscopically. For the purpose of observing the
difference between the behavior of the tubercle bacilli
and the other organisms present in the preparation
toward this method of staining, it is well to examine
the preparation microscopically before the contrast stain
is made, then remove it, give it the contrast color, and
examine it again. It will be seen that before the con-
trast color has been given to the preparation the
tubercle bacilli will be the only stained objects to be
made out, and the preparation will appear devoid ot
other organisms, but upon examining it after it has
received the contrast color, a great many other or-
ganisms will now appear; these will take on the second
color employed, while the tubercle bacilli will retain
their original color. Before decolorization all organisms
in the preparation were of the same color, but during the
application of the decolorizing solution all except the
tubercle bacilli gave up their color. This characteristic,
as said, serves to differentiate the tubercle bacillus from

GABBETT’S METHOD. 135

other organisms with which it might be confounded.
A number of different methods have been suggested
for the staining of tubercle bacilli, but the original
method as employed by Koch is so satisfactory in
its results that it is not advisable to substitute others
for it. The above differs from the original Koch-
Ehrlich method for the staining of tubercle bacilli in
sputum only in the occasional employment of Ziehl’s
carbolic-fuchsin solution and in the method of heating
the preparation with the staining fluid upon it.

As Nuttall has pointed out, however, the strong acid
decolorizer used in this method can be with advantage
replaced by much more dilute solutions, as a certain
number of the bacilli are entirely decolorized by the
too energetic action of the strong acids. He recom-
mends the following method of decolorization: After
staining the slip or section in the usual way, pass it
through three alcohols; it is then to be washed out in a
solution composed of

Water. 2. wwe eee 150 AC,
ALCOR. we lm lw Oe
Concen. sulphuricacid . . . . . 20 to 30 drops.

From this they are removed to water and carefully
rinsed. The remaining steps in the process are the
same as those given in the other methods.

Gapperr’s Metnop for the staining of tubercle
bacilli recommends itself because of its simplicity arid
the rapidity with which it can be performed. By
many it is considered the best method for routine
employment. It consists in staining the cover-slips,
prepared in the manner given, for from two to five
minutes in a cold carbolic-fuchsin solution, after which
they are subjected to the action of Gabbett’s methy-

136 BACTERIOLOGY.

lene-blue sulphuric acid solution. This latter con-
sists of

Sulphuric acid, strength 25 percent... . . 100¢.c.
Methylene-blue, in substance . . : : 1to 2 grammes.

They are then rinsed off in water and are ready for
examination. The tubercle bacilli will be stained red
by the fuchsin, while all other bacteria, cell nuclei, etc.,
will be tinted blue.

Gramw’s Meruop.—Another differential method of
staining. which is very commonly employed is that
known as Gram’s method. In this method the objects
to be stained are treated with an aniline-water solution
of gentian-violet made after the formula of Koch-
Ehrlich. After remaining in this for twenty to thirty
minutes they are immersed in an iodine solution com-
posed of

Iodine : 7 . ; . é . 5 e 1 gramme.
Potassium iodide . ‘ i 3 ar é 2 grammes,
Distilled water : 3 7 7 6 is - 800c.¢.

In this they remain for about five minutes; they are
then transferred to alcohol and thoroughly rinsed. It
they are still of a violet color they are again treated
with the iodine solution followed by alcohol, and this is
continued until no trace of violet color is visible to the
naked eye. They may then be examined, or a contrast
color of carmine or Bismarck-bréwn may be given
them.

This method is particularly useful in demonstrating
the capsule which is seen to surround some bacteria,
particularly the micrococcus lanceolatus of pneumonia.

GuLAcIAL Acetic Actd MEeruop.—Another method
which may be employed for demonstrating the presence
of the capsule surrounding certain organisms, is to

STAINING OF SPORES. 137

prepare the cover-slips in the ordinary way, then cover
the layer of bacteria upon them with glacial acetic acid,
which is instantly poured off (not washed off in water),
and the aniline-water gentian-violet solution dropped
upon them; this is allowed to remain three or four
minutes, is poured off, and a few drops more are added,
and lastly the slip is washed off in water. A very clear,
sharply-cut picture usually follows this method of pro-
cedure.

STAINING OF SpoRES.—We have learned that one of
the points by which spores may be recognized is their
refusal to take up staining substances when applied in
the ordinary way. They may, however, be stained by
special methods ; of these one that has given very satis-
factory results in our hands is as follows: The cover-
slip is to be prepared from the material containing the
spores in the ordinary way, dried, and fixed. It is then
to be held by its edge with the forceps, and its surface
covered with Léffler’s alkaline methylene-blue solu-
tion. Itis then held over the Bunsen flame until the
fluid boils ; it is then removed, and after a few seconds
is heated again. This is continued for about one
minute, after which it is washed off in water and
dipped five or six times in alcohol containing about 0.2
to 0.8 per cent. of hydrochloric acid. This is rinsed off
in water and the preparation is now stained for from
eight to ten seconds in aniline-water fuchsin solution
(Koch-Ehrlich solution), and finally again washed in
water. By this method the spores are of a blue color
and the body of the cell red.

By another process the cover-slip is floated, bacteria
down, upon the surface of a watch-crystalful of freshly
prepared Koch-Ehrlich solution of fuchsin. This is

1388 BACTERIOLOGY.

then held by its edge with the forceps about 2 em. above
a very small flame of a Bunsen burner, care being taken
that the flame touches only the centre of the bottom of
the crystal. After a few seconds the crystal is elevated
gradually until it is about 6 to 8 cm. above the flame,
then it is slowly moved down to the flame again, and
this up-and-down movement is continued until the
staining fluid begins to boil. As soon as a few bubbles
have been given off it is held aside for a minute or two
and the process of heating is repeated. When the
boiling begins, the crystal is held aside again for a
minute or two. The crystal is heated in this way for
about five or six consecutive times. When the fluid
has stood for about five minutes after the last boiling,
the preparation is transferred, without washing in
water, into a second watch-crystal containing the fol-
lowing decolorizing solution :

Absolute alcohol. z soe 6 100 Ge.

Hydrochloric acid . é 5 i Z é . 3 ¢.c.

In this solution it is placed, bacteria up, and the
vessel is tilted from side to side for about one minute.
It is then removed, washed in water, and stained with
the methylene-blue solution. The spores will be stained
red and the body of the cells will be blue.

Moe.tter’s MetTHop ror Sraininc Spores.—A
method that has recently been published by Moeller is
designed to favor the penetration of the coloring mate-
rial through the spore membrane by macerating the
spores in a solution of chromic acid before staining
them. It is as follows: ;

The cover-slips are prepared in the usual way, or the
fixing may be accomplished with absolute alcohol in-
stead of high temperatures. The preparation is then

METHOD FOR STAINING FLAGELLA. — 139

held for two minutes in chloroform, then washed off in
water, then placed for from one-half to two minutes in
a 5 per cent. solution of chromic acid; again washed
off in water, and now stained in carbolic fuchsin. In
the process of staining, the slip is taken by the corner
with the forceps, and carbolic fuchsin is dropped upon
the side containing the spores. It is then held over the
flame until it boils, and then held some distance above
the flame for one minute. The staining fluid is then
poured off and the preparation is completely decolorized
in 5 per cent. sulphuric acid, again washed off in water,
and finally stained for thirty seconds in the watery
methylene-blue solution. The spores will be red, the
body of the cells blue.

In this method the object of the preliminary ex-
posure to chloroform is to dissolve away any crystals of
lecithin, cholesterin, or fat that may be in the prepara-
tion, and which when stained might give rise to con-
fusion.

It must be remembered: that there are conspicuous
differences in the behavior of spores of different bac-
teria to staining methods. Some stain readily by
either of the methods especially devised for this pur-
pose, while others can hardly be stained at all, or only
with the greatest difficulty, by any of the known pro-
cesses.

Lorrier’s METHOD FoR STAINING FLAGELLA.—
For the demonstration of the locomotive apparatus
possessed by motile bacteria we are indebted to Léffler.
By a special method of staining in which the use of
mordants played the essential part, he has shown that
these organisms possess very delicate, hair-like appen-
dages, by the lashing movements of which they propel

140 BACTERIOLOGY.

themselves through the fluid in which they are located.
The method as given by Léffler is as follows :

(1) It is essential that the bacteria be evenly and
not too numerously distributed upon the cover-slip. The
slips must therefore be carefully cleansed. (See Léffler’s
method of cleaning cover-slips.) Five or six of the
carefully cleansed cover-slips are to be placed in a line
on the table, and on the centre of each slip a very small
drop of tap-water is placed. From the culture to be
examined a minute portion is transferred to the first
slip and carefully mixed with the drop of water ; from
this mixture a small portion is transferred to the
second, and from the second to the third slip, and so
on—in this way insuring a dilution of the number of
organisms present in the preparation.

These slips are then dried and fixed in the ordinary
way. They are next to be warmed in the following
solution :

Tannic acid solution in water (20 acid, 80 water) . 10c¢
Cold saturated solution of ferro-sulphate ‘ . 5e¢
Saturated watery or alcoholic solution of fuchsin . lee.

This solution represents the mordant. A few drops
of it are to be placed upon the film of bacteria on the
cover-slip, which is then to be held over the flame until
the solution begins to steam. It should not be boiled.
After steaming, the mordant is washed off in water and
finally in alcohol. The bacteria are then to be stained
in a saturated aniline-water fuchsin solution.

When treated in this way different bacteria behave
differently : the flagella of some stain readily in the
above solutions; others require the addition of an alkali
in varying quantities; while others stain best after the
addition of acids. To meet these conditions an exact

METHOD FOR STAINING FLAGELLA. 141

1 per cent. solution of caustic soda in water must be
prepared, and also a solution of sulphuric acid in water
of such strength that one cubic centimetre will be
exactly neutralized by one cubic centimetre of the
alkaline solution.

For different bacteria which have been studied by
this method, the one or the other of these solutions is
to be added to the mordant in the following propor-
tions.

Of the acid solution :

For the bacillus of Asiatic cholera. F . %toldrop.
For the spirillum rubrum ss. - ri ‘ . 9 drops.

Of the alkaline solution :

For the bacillus of typhoid fever “ : » lac
For the bacillus subtilis $ - ‘i - . 28 to 30 drops.
For the bacillus of malignant oedema . 386t037 “

For other organisms one must determine whether the
results are better after the addition of acid or alkali,
and how much of either is required. In general it may
be said that bacteria which produce acids in the media
in which they are growing require the addition of alka-
lies to the mordant, while those that produce alkalies
require acids to be added. By following Léffler’s direc-
tions the delicate, hair-like flagellee on motile organisms
may be rendered plainly visible.

There are several points and slight modifications in
connection with this method that require to be empha-
sized in order to insure success: The solutions should
be perfectly fresh; after mixing the tannic acid, iron,
and fuchsin solutions together they should be filtered
before using ; when placed on the cover-slip and held
over the flame never heat to the boiling-point ; indeed, the

best results are obtained when the preparation is held high
7*

142 BACTERIOLOGY.

above the flame and removed from it at the first evidence
of vaporization, or, better still, a little before this point is
reached. We have derived no advantage from the
addition of acids or alkalies to the mordant as recom-
mended by Léffler, but obtain with a fair degree of
regularity satisfactory results through the use of the
neutral mordant alone.’

STAINING IN GENERAL.

The physics of staining and decolorization is hardly
a subject to be discussed in a book of this character,
but, as Kiihne has pointed out, solutions which favor
the production of diffusion currents facilitate intensity
of staining, and by a similar process increase the energy
of decolorizing agents. For example, tissues which are
transferred from water into watery solutions of the
coloring matters are less intensely stained and more
easily decolorized than when transferred from alcohol
into watery staining fluids; for the same reason tissues
stained in watery solutions of the dyes do not become
decolorized so readily when placed in water as when
placed in alcohol.

The diffusion of staining solutions into the proto-
plasm of dried bacteria, as found upon cover-slip prep-
arations, is much greater and more rapid than when the
same bacteria are located in the interstices of tissues.
These differences are not in the bacteria themselves, but
in the obstruction to diffusion offered by the tissues in
which they are located.

The result of absence of diffusion may easily be illus-

1I am indebted to Dr. James Homer Wright, Thomas Scott Fellow in

Hygiene, 1892-93, University of Pennsylvania, for the suggestions in connec-
tion with the modification of this method.

DECOLORIZING . SOLUTIONS. 148

trated. Prepare a cover-slip preparation, dry it care-
fully, fix it, and without allowing water to get on it
from any source, attempt to stain it with a solution
of the dyes in absolute alcohol, washing it out subse-
quently with absolute alcohol; the result is negative.
The absolute alcohol does not possess the property of
diffusing into the dried tissues, and hence, as has been
stated before, alcoholic solutions of the staining dyes
should not be employed. ‘The staining dyes should
always be watery.!

Deco.orizine SoLutTions.—As regards the employ-
ment of decolorizing agents, it must always be borne in
mind that objects which are easily stained are also easily
decolorized, and those that can be caused to take up the
staining material only with difficulty are also very diffi-
cult to rob of their color. The most common decolor-
izer in use is probably alcohol—not absolute alcohol,
but alcohol containing more or less of water. Water
alone has this property, but in a much lower degree
than dilute alcohol. On the other hand, a much more
energetic decolorization than that possessed by either
alone can be obtained by alternate exposures to alcohol
and water. More energetic in their decolorizing action
than either water or alcohol, are solutions of the acids.
They appear, particularly when they are alcoholic solu-
tions, to diffuse rapidly into tissues and bacteria and
very quickly extract the staining materials which have
been deposited there. For this reason these solutions
should be employed with much care.

1 In the beginning of this chapter it was stated that the saturated alcoholic
solutions of the dyes do not serve as stains for bacteria, It must be remem-
bered that this holds only when absolute alcohol and perfectly dry coloring
matters have been used. If but a small proportion of water is present, the
bacteria may be stained with these solutions.

144 BACTERIOLOGY.

Very dilute acetic acid robs tissues and bacteria of
their staining with remarkable activity ; still more ener-
getic are solutions of the mineral acids, and particularly,
as has been said, when this action is accompanied by
the decolorizing properties of alcohol.

The acid solutions that are commonly employed are:

Acetic acid in from 0.1 per cent. to 5 per cent. watery
solution.

Nitric acid in from 20 per cent. to 30 per cent. watery
solution.

Hydrochloric acid in 3 per cent. solution in alcohol.

STAINING OF BACTERIA IN TISSUES.

In staining tissues for the purpose of demonstrating
the bacteria which they may contain, a number of points
must be borne in mind: the conditions which favor the
diffusion of the staining fluids into the bacteria are now
not so favorable to rapid staining as they were when
the bacteria alone were present upon cover-slips ; the
staining of tissues, therefore, requires a longer exposure
to the dyes than does that of cover-slips. In tissues, too,
there are other substances beside the bacteria which
become stained, and these, unless robbed in whole or in
part of their color, may so mask the stained bacteria as
to render them difficult, if not impossible of detection.
Tissues must, therefore, always be subjected to some
degree of decolorization, and this must be practised
without depriving the bacteria of their color.

The details of the methods of decolorization will be
described in the section on the technique of staining.

Another point to be remembered in staining tissues
is that they can never be heated and retain their struc-

STAINING OF BACTERIA IN TISSUES. 145

ture, in the same way that one heats cover-slips. The
best results are not obtained in efforts to hasten the
staining by subjection to high temperatures, but rather
by longer exposures at lower temperatures.

HARDENING THE TissuEs.—The bits of tissue—not
greater than 1 cm. cube—are to be placed, as fresh as
possible, in absolute alcohol. The bit of tissue should
rest upon a pad of cotton or filter-paper in the bottle
containing the alcohol, in order that it may be elevated
and surrounded by the part of the alcohol which is
specifically the lightest, and consequently contains least
water. Thealcohol abstracts water from the tissue, and,
as the dehydration proceeds, the tissue becomes accord-
ingly more and more dense. When of about the con-
sistency of fresh solid rubber, or preferably not quite
so dense, it is ready to cut. A small portion of about
0.5 em. cube should be cemented to a bit of cork with
ordinary mucilage, and allowed to remain in the open
air for a minute or two for the mucilage to harden.
Alcohol should be dropped upon it occasionally, to pre-
vent drying of the tissue. When the mucilage is hard,
the cork with the piece of tissue upon it may be left in
alcohol over night, and on the following day the sections
may be cut.

SEcTION-cUTTING.—This is accomplished by the use
of an instrument known as a microtome. In Fig. 31
is seen the form now commonly employed. It is
known by the name of the maker, as Schanze’s micro-
tome. It is an apparatus provided with a clamp for
holding the cork upon which the tissue is cemented and
also a sliding clamp which carries a knife. The tissue
is clamped horizontally, and the knife is caused to slide
across its upper surface, also in the horizontal direction.

146 BACTERIOLOGY.

Beneath the clamp for holding the tissue is a milled
disk, by means of which a screw is caused to revolve,
and in revolving raises or lowers the clamp holding the
tissue, so that the tissue may be brought closer to or
farther from the plane in which the knife slides. By
this arrangement sections of any desired thickness can.
be cut by turning the milled disk with the one hand and
causing the knife to traverse the tissue with the other.

Fie. 31.

call

=
:

Schanze’s microtome.

The tissue and the knife-blade should be kept wet
with alcohol, so that the sections may float upon the
blade of the knife, from which they can be easily
removed without tearing, with a curved needle or a
camel-hair pencil. As the sections are cut they are
placed in a dish containing alcohol.

There are some tissues which, by reason of their his-
tological structure, do not become sufficiently dense

STAINING OF BACTERIA IN TISSUES. 147

when exposed to alcohol to permit of their being cut in
the above way. It becomes necessary to render them
, more solid by filling their interstices with some sub-
stance that neither interferes with their structure nor
prevents their being cut into sections. They must be
“imbedded,” as this process is called.

Imbedding in celloidin. Most convenient for this
purpose is celloidin, a body somewhat similar to col-
lodion, soluble in a mixture of equal parts of alcohol
and ether, as well as in absolute alcohol.

After hardening in alcohol the tissue to be imbedded
is placed into a-mixture of equal parts of absolute
alcohol and ether and left there for twenty-four hours.
It is then transferred to celloidin. Two solutions of
celloidin are to be employed, the one a thin solution in
a mixture of equal parts of absolute alcohol and ether,
the other a thick solution in the same solvent. Into
the thin solution, which should be of about the consist-
ence of very thin syrup, the tissue is placed from the
absvlute alcohol and ether, and allowed to remain there
for twenty-four hours. It is then placed in the thick
solution for about a day. From this it may be removed
and placed immediately upon a bit of cork or a block
of wood. The adherent celloidin will act as a cement,
and as it hardens rapidly, the tissue is soon fast to the
cork. It is then left in 60 per cent. alcohol for twenty-
four hours to complete the solidification of the celloidin
after which sections may be cut in the way just described
for tissues not so treated.

Imbedding in paraffin. After bits of the tissue not
larger than a cubic centimetre have been hardened in
the usual way, they are placed in fresh absolute alcohol
for twenty-four hours to complete the process. From

148 BACTERIOLOGY.

this they are transferred to pure turpentine, and kept
in a warm oven at a temperature not exceeding 35° to
38° C. Here they remain for a time sufficient for them
to become thoroughly saturated with the turpentine, as
is recognized by the transparent appearance that they
assume. From this they are placed into paraffin that
is melted at 53° C. and allowed to remain in this for
three or four hours. They are then transferred to a
small paper or metal mould, or a pill-box, and melted
paraffin is poured over them. When the paraffin has
become solid the mould or pill-box is removed from
around it, the excess of paraffin removed from about
the imbedded tissue, and the latter is ready for cutting.

When the sections are cut they are freed from par-
affin by exposing them to turpentine ; the latter is re-
moved by washing in alcohol and the sections can now
be stained in the ordinary way. In cutting sections
from tissues that have been imbedded in paraffin the
long axis of the knife should be at nearly right angles
to the direction in which the knife travels. For bac-
teriological purposes the method of imbedding in par-
affin does not as a rule give such good results as when
the celloidin method is employed. In this work,
therefore, the latter is usually preferred.

STAINING OF THE SEcTIONS.—The sections when cut
may be stained in a variety of ways. The ordinary
watery solutions of the three common basic aniline dyes
—fuchsin, gentian-violet, or methylene-blue—or, what
is better, the alkaline methylene-blue solution of Léffler,
may be employed for general use.

The acid aniline dyes, as well as some of the vege-
table coloring matters, are essentially nuclear stains, and
are not applicable to the staining of bacteria.

STAINING OF BACTERIA IN TISSUES. 149

Into a watch-glass containing either of the staining
solutions mentioned, the sections are to be placed after
having been in water for about one minute. They
remain in the staining solutions for from five to eight
minutes. They are then removed, rinsed in water, and
partly decolorized in 0.1 per cent. acetic acid for only a
few seconds; again washed out in water, then in abso-
lute alcohol for a few seconds, and from this again into
absolute alcohol for the same time, and finally into cedar
oil or xylol. Here they remain for from one-half to
three-fourths of a minute. They are now to be carefully
spread ‘out upon a spatula, which is held in the fluid
under them, and without draining off the fluid are trans-
ferred to a clean glass slide. This must be done care-
fully to avoid tearing. The easiest way to do this is to
hold the spatula on which the section floats in one hand,
with its point just touching the surface of the glass slide,
and then with a needle pull the section gently off upon
the slide. The fluid comes with it, and the floating sec-
tion may be easily spread out into a flat surface. The
excess of fluid is taken up with blotting-paper, after
which a drop of xylol-balsam is placed upon the centre
of the section, and is then covered with a thin, clean
cover-slip. It is now ready for examination.

Each step in the above process has its definite object.
The sections are placed in water before staining in order
that the diffusion of the staining solution into the tissues
may be diminished ; otherwise our efforts at rendering
the bacteria more conspicuous by decolorizing the tissues
in which they are located would rob the bacteria of
their color as well.

The acetic acid and also the alcohol are decolorizers,
and are directed toward the excess of staining in the

150 BACTERIOLOGY.

tissues. The cedar oil or xylol are bodies which mix
on the one hand with alcohol, on the other with balsam.
They are known as “clearing fluids,” and not only serve
to differentiate the component parts of the tissue but fill
up the gap that would otherwise be left in the process,
for a section cannot be mounted in balsam directly from
alcohol ; the two bodies do not mix perfectly.

A number of clearing agents are in general use; in
fact, almost all the essential oils come under this head.
There is one—oil of cloves—which is very commonly
used in histological work, but it must not be employed
in tissues containing bacteria. It not only extracts too
much color from the bacteria, but causes them to fade
after the sections have been mounted for a time.

When the section thus stained and mounted is ex-
amined microscopically, it may be found that the tissues
still possess so much color that the bacteria are not
visible, in which case they have not been decolorized
sufficiently; or, on the other hand, both bacteria and
tissues may have parted with their stains—then decol-
orization has been carried too far. In either case the
fault must be remedied in the manipulation of the next
section to be mounted.

In short, the steps in the process of staining sections
in general are these: .

a. From alcohol into distilled water for one minute.

b. Into the staining fluid for from five to eight
minutes.

¢c. Into water for from three to five minutes.

d. Into 0.1 per cent. acetic acid for about one-half
minute.

e. Absolute alcohol for a few seconds.

f. Absolute alcohol for a few seconds.

STAINING OF BACTERIA IN TISSUES. 151

g. Xylol for about one-half minute.

h. Removal with spatula or section-lifter to slide.

t. Removal of excess of xylol.

)» Mounting in xylol-balsam.

The section must be lifted from one vessel to the other
by means of either a curved needle or a glass rod drawn
out to a fine end and bent in the form of a curved needle.

By the above process of staining, which can be prac-
tised as a general method for most bacteria in tissues,
the nuclei of the tissue cells, as well as the bacteria, will
be more or less deeply stained.

SpecIAL Mersops oF Strarinrna Bacrerra IN
TissuEs.—For purposes of contrast stains it some-
times becomes necessary to completely, or nearly com-
pletely, decolorize the tissues and leave the bacteria
unaltered in color. For this purpose special methods
depending on the staining peculiarities of the bacteria
under consideration have been devised.

Gram’s method with tissues. One of the most com-
monly employed differential stains is that of Gram. In
general, it is practised in the way given for its employ-
ment on cover-slip preparations with some slight
modifications.

In this method the sections are to be placed from
water into a solution of aniline-water gentian-violet,
as prepared by the Koch-Ehrlich formula, but which has
been diluted with about one-third its volume of water.
In this the sections remain for about ten minutes, prefer-
ably in a warm place, at a temperature of about 40° C.
They should never, under any conditions, be boiled.

From .this they are washed alternately in the iodine
solution and alcohol, occasionally renewing the stained
with clean alcohol, until all color has been extracted

152 BACTERIOLOGY.

from them. They are then brought for one minute into
a dilute watery solution of eosin or safranin, or Bismarck-
brown; again washed out for a few seconds in alcohol,
and finally for one-fourth minute in absolute alcohol.
From this they are transferred to xylol for a half-
minute. The remaining steps in the process are the
same as those given in the general method. In some
cases better results are obtained by reversing the steps
in the process and staining the bacteria last, for then
the frequent decolorizing action of the alcohol on the
bacteria is diminished; thus, place the sections from
alcohol into eosin, safranin or Bismarck-brown for a few
minutes, then wash out in 50 per cent. alcohol, then for
from three to five minutes in the dilute aniline-water
gentian-violet solution, then into the iodine bath, after
three minutes wash out in alcohol, and, finally, for one-
fourth minute in absolute alcohol, and then into the
xylol, from which they may be mounted, The organ-
isms which may be stained by this method are mic.
tetragenus, b. diphtherie, b. anthracis, staph. pyogenes
aureus, and a few others. It cannot be successfully
employed with the bacillus of typhoid fever.
Staining with dahlia and decolorizing with soda solu-
-tion. Another method that is not very commonly em-
ployed, though the results obtained by its use are in
many cases very satisfactory, is to stain the tissues in a
strong watery solution of dahlia (about one-fourth satu-
rated) for from ten to fifteen minutes ; from this they
are brought into a 2 per cent. solution of sodium or
potassium carbonate, and from this into alcohol, alter-
nating from the one to the other, until the section is
almost colorless. From the alcohol they are rinsed out
in water and then brought into a dilute watery solution

STAINING OF BACTERIA IN TISSUES. 153

of either eosin, Bismarck-brown, or safranin for one
minute, then washed out in alcohol, finally in absolute
alcohol, and then into xylol, from which they may be
mounted in the manner given.

Especially brilliant results are obtained when tissues
containing anthrax bacilli are stained by this process ;
the bacilli will be of a deep blue color, while the sur-
rounding tissues will be of the color used as contrast.

Kiihne’s carbolic methylene-blue method. Stain the
sections in the following solution for from one-half to
one hour:

Methylene-blue, in substance i ‘ : . 1.5 grammes.
Absolute alcohol. f fi ‘ - 10¢.c.

Rub up thoroughly in a mortar, and when the blue
is completely dissolved, add gradually 100 c.c. of a 5
per cent. solution of carbolic acid. (The solution de-
composes after a short time ; it should be made fresh
when needed.) From this the sections are washed out
in water, then in 1.5 to 2 per cent. hydrochloric acid in
water, from this into a solution of lithium carbonate of
the strength of six to eight drops of a concentrated
watery solution of the salt to ten drops of water, and
from this again thoroughly washed in water ; then into
absolute alcohol containing enough methylene-blue in
substance to give it a tolerably dense color, then for a
few minutes into aniline oil to which a little methylene-
blue in substance has been added; then completely
rinse out in pure aniline oil, from this into thymol or
oil of turpentine for two minutes, and then into xylol,
from which they are mounted in xylol-balsam. The
advantages of this method are that it is generally ap-
plicable, and by its use the bacteria are not robbed of
their color, whereas the tissues are sufficiently decolor-

154 BACTERIOLOGY.

ized to render the bacteria, visible and admit of the use
of contrast stains.

Weigert’s modification of Gram’s method for sections.
Stain the sections in the Koch-Ehrlich aniline-water
gentian-violet solution for five or six minutes ; wash out
in water or physiological salt solution (0.6 to 0.7 per cent.
solution of sodium chloride in distilled water) ; transfer
them with the section-lifter to the slide; take up the
excess of fluid by gently pressing upon the flat section
with blotting-paper ; treat the section with the iodine
solution used by Gram ; take up the excess of the solu-
tion with blotting-paper ; cover the section with aniline
oil—this not only differentiates the component parts of
the section, but dehydrates as well; wash out the
aniline oil with xylol, and mount in the usual way in
xylol-balsam. Or, decolorization with iodine may be
omitted, and the sections, after staining in the aniline-
water gentian-violet for five or six minutes or longer, if
necessary, are transferred to the slide without being
washed in water or salt solution, or if so only very
slightly and rapidly, dried as completely as possible
with filter-paper, then are decolorized with a mixture
of aniline oil (one part) and xylol (two parts). This is
the delicate part of the process and can be watched
under the low power of the microscope ; when decolor-
ization is sufficient (repeated applications of the aniline
oil and xylol mixture are generally necessary), pure
xylol replaces the mixture, and the specimen is finally
mounted in xylol balsam. Unless all the aniline oil is
replaced by the xylol the specimen will not keep well.
In this process the aniline oil is really the decolorizer
and has the valuable property of absorbing a certain
amount of water, so that dehydration with alcohol is

STAINING OF BACTERIA IN TISSUES. 155

avoided. This method, while it stains certain bac-
teria in tissues very satisfactorily, is nevertheless
designed especially for the staining of fibrin. Fibrin
and hyaline material will be stained deep blue, bacteria
a dark violet.

STAINING OF TUBERCLE BACILLI IN TissuEs.—As
for the staining of cover-slips, only those methods most
commonly employed will be given.

The method of Ehrlich. Stain the sections in aniline-
water fuchsin or gentian-violet for twenty-four hours ;
decolorize in 20 per cent. nitric acid for a few seconds
only, the color need not be entirely extracted; then into
70 per cent. alcohol until no more color can be ex-
tracted by the alcohol; stain as contrast color in dilute
watery methylene-blue, malachite-green, or Bismarck-
brown solution ; wash out in 90 per cent. alcohol, then
in absolute alcohol for a few seconds ; clear up in xylol
and mount in xylol-balsam.

Method of Zieht-Neelsen. Stain the sections in
warmed carbol-fuchsin solution for one hour ; tempera-
ture to be about 45° to 50°C. Decolorize for a few
seconds in 5 per cent. sulphuric acid, then in 70 per
cent. alcohol, and from this on as by the Ehrlich
method.

Dry method. For the tubercle bacilli, as for many
other organisms in tissues, the following method may
be employed if only the presence of organisms is to be
detected and the histological condition of the tissues is
a matter of no consequence: Bring the sections from
water upon a slide or cover-slip, dry, fix, and stain by
. the methods for cover-slip preparations.

Gray's method. The method employed by Gray at
the Army Medical Museum, Washington, D. C., a

156 BACTERIOLOGY.

description of which is given by Borden, is as follows :
The tissue to be stained should be hardened, preferably
in alcohol, in pieces not exceeding 1.5 by 1.5 by 1 cm.
in size, though tissues hardened by any other of the
regular methods can be stained. Alcohol is to be pre-
. ferred, however, and after its use the bacilli stain more
quickly and brilliantly than when one of the other
hardening fluids, Miller’s, for instance, is employed.
After the tissue has been hardened it is imbedded in
paragin, and cut in the usual manner. The sections
are then cemented to the slides with a filtered 4 per
cent. solution of gold label gelatin, to which is added
chloral hydrate, in the proportion of 1 per cent., as
preservative. Several drops of this are placed on a
slide, a section laid on top, and the slide placed in a
warming oven, kept at a temperature slightly below the
melting-point of the paraffin. In about five minutes
all wrinkles will have been taken out of the section,
which will lie perfectly flat and smooth on the surface
of the gelatin solution. The slide is then removed from
the oven and the surplus fluid poured from it, thus
bringing the section down into contact with its surface,
after which it is set aside in a place protected from
dust, to remain until the section is firmly cemented to
it by the drying of the gelatin solution. The drying
may be hastened by keeping the slides in an oven
“below the melting-point of the paraffin, but it is best
to set the slides aside until the next day, when the sec-
tions will be found to be perfectly cemented to them.
The paraffin is then removed from the section by tur-
pentine, the turpentine by absolute alcohol, the absolute
alcohol by 50 per cent. alcohol, and this by water, after
which the slides are placed in a 5 per cent. aqueous

STAINING OF BACTERIA IN TISSUES. 157

solution of potassium bichromate for five minutes.
This renders the gelatin insoluble, and prevents the
sections from leaving the slides during their necessarily
more or less prolonged immersion in the fuchsin stain.
The potassium bichromate is washed out with water,
and the slides are then placed in a fuchsin stain, which
is prepared as follows:

Fucebsin i " a ‘ A . F ‘ 1.5 grammes,
Absolute alcohol F , ie of » » 14 Ge,
Carbolic acid crystals, pure . ; z 3 6 grammes.
Water . : reer - 100 ce.

Dissolve the fuchsin in the alcohol and the carbolic
acid in the water. Mix the two solutions and let stand
for twelve hours, with occasional shaking or stirring,
then filter.

The slides are left in this solution a sufficient length
of time. In tissues properly hardened in alcohol the
tubercle bacilli stain very quickly, generally five min-
utes being sufficient to stain them deeply.

Prolonged immersion in the fuchsin does no harm
and insures certainty of results. After a section has
been in the stain a sufficient length of time it, with the
slide to which it is cemented, is washed in water until
the surplus stain is removed ; it is then subjected to
the action of a combined decolorizer and contrast stain
made as follows :

Methyl-blue : ‘ 7 . i r . 2.25 grammes.
Absolute aleohol . 5 30. .e.
Sulphuricacid . A 7 7 ‘ 12 a

Water (distilled) . ‘ ‘ x 4 » 100 *

Dissolve the methyl-blue in the alcohol, add the acid
to the water, mix the two solutions, and let stand, with
occasional shaking, for twelve hours, then filter.

This solution is allowed to act upon the tissue for a
8

158 BACTERIOLOGY.

few seconds, and as soon as the blue color predominates
over the red, as seen by transmitted light, the section is
immediately washed in water. Generally the red color
reappears and the section must be again subjected to the
action of the blue solution and again washed in water.
This must be repeated until the blue almost, if not quite
completely and permanently, replaces the red stain.
This is the most difficult part of the process and entirely
satisfactory results are only obtained after some practice.
The tendency is usually to not sufficiently replace the
fuchsin with the methyl-blue, and in consequence the
red color of the bacilli is masked by the red of the
surrounding tissues. Unless all acid is thoroughly
removed by the final washing in water the stain is not
permanent. The section is then completely dehydrated
- with absolute alcohol, after taking up the excess of
water on the slide with blotting-paper. The alcohol is
followed by turpentine, and the process is completed by
mounting in xylol-balsam.

In case it is desired to stain sections cut by the
freezing method, they are placed upon a slide on which
a few drops of the gelatin fixative have been placed, and
after about five minutes, during which the fixative will
have penetrated the section, the surplus is poured from
beneath the section. The slides are then set aside for
the gelatin to harden by drying, and after drying they
are placed in bichromate fluid to render the gelatin
insoluble. They are then manipulated in exactly the
same manner as the sections cut by the paraffin method.

This method gives equally good results with tissues
containing the lepra bacillus as with those containing
tubercle bacilli.

CHAPTER XI.

Systematic study of an organism—Points to be considered in identifying an
organism as a definite species.

AFTER isolating an organism by the plate method,
considerable work is necessary in order to establish its
identity as a definite species.

It must possess certain morphological and cultural
peculiarities, which must be constant under constant
conditions.

Its form at different stages must always be the same.
Its ability or inability to produce spores must not vary
under proper conditions. Its growth upon the different
media under constant conditions of temperature and
reaction must always present the same outward appear-
ances. The reactions given by it to the media in which
it is growing must follow a fixed rule. Its power to
produce liquefaction of the gelatin, or to grow upon it
without bringing about this change, must always be the
same. Its motility or non-motility, and, if motile, the
number and position of its organs of locomotion, must
be determined. Its production of certain chemical pro-
ducts must be detected by chemical analysis. Its be-
havior toward oxygen—i. e., does it require this gas for
its growth? is this gas an indifferent factor? or by its
presence are the life processes of the organism checked ?
—must be decided. Its behavior under varying condi-
tions of temperature and under the influence of different
chemical bodies, as well as its growth in media of

160 BACTERIOLOGY.

different reactions, are to be studied. The property of
producing fermentation with the liberation of gases
must be ascertained ; if it produces pigment, what are
the conditions favorable and unfavorable to this func-
tion; and lastly, we must consider its behavior when
introduced into the bodies of animals used for experi-
mental work—i. ¢., is it a disease-producing organism,
or does it belong to the group of innocent saprophytes ?
We have learned the methods of obtaining colonies,
and have acquainted ourselves with some of the pecu-
liarities by which they are distinguished from one
another. The next important step is to determine the
morphology of the individuals composing these colo-
nies as well as their relation to each other in the colony.
These points are decided by microscopic examination
of bits of the colony which are transferred to thin
glass cover-slips, upon which they are dried, stained,
and mounted. Cover-slips for this purpose are pre-
pared in two ways: either by taking up a bit of the
colony on a platinum needle, smearing it upon a cover-
slip, staining it, and examining it—by which only the
morphology of the individuals can be made out—or by
the method of “impression cover-slip preparations,”
by which not only the morphology, but also the relation
of the organisms to one another in the colony can be
determined. The details of these methods will be
found in the chapter on the methods of staining.

MICROSCOPIC EXAMINATION OF PREPARATIONS.

4
THe DirreRENT Parts OF THE MicroscoPE.—
Before describing the process of examining prepara-
tions microscopically, a few definitions of the terms

MICROSCOPIC EXAMINATIONS. 161

used in referring to the microscope may not be out of
place.

The ocular or eye-piece is the lens at which the eye is
placed in looking through the instrument.

The objective is the lens which is at the distal end of
the barrel of the instrument, and which serves to mag-
nify the object to be examined.

The stage is the shelf or platform of the microscope
on which the object rests.

The reflector is the mirror placed beneath the stage,
which serves to direct the light to the object examined.

The coarse adjustment is the rack-and-pinion arrange-
ment by which the barrel of the microscope can be
quickly raised or lowered.

The fine adjustment serves to raise and lower the
barrel of the instrument very slowly and gradually.

For the microscopic study of bacteria it is essential
that the microscope be provided with an oil-immersion
system and a sub-stage condensing apparatus.

The Oil-immersion or Homogeneous System consists in
an objective so constructed that it can only be used
when the media through which the light passes in
entering it are all of the same index of refraction—. e.,
are homogeneous. This is accomplished by interposing
between the face of the lens and the cover slip covering
the object to be examined a body which refracts the
light in the same way as do the glass slide, the cover-
slip, and the glass of which the objective is made. For
this purpose a drop of oil of the same index of refrac-
tion as the glass is placed upon the face of the lens, and
the examinations are made through this oil. There is
thus no loss of light from deflection, as is the case in
the dry systems.

162 BACTERIOLOGY.

The sub-stage condensing apparatus is a system of
lenses situated beneath the central opening of the stage.
They serve to condense the light passing from the

‘reflector to the object in such a way that it is focussed
upon the object, thus furnishing the greatest amount of
illumination. Between the condenser and reflector is
placed an adjustable diaphragm, the aperture of which
can be regulated, as circumstances require, to permit of
either a very small or very large amount of light pass-
ing to the object.

Microscopic EXAMINATION OF COVER-SLIPS.—
The stained cover-slip is to be examined with the oil-
immersion objective, and with the diaphragm of the
sub-stage condensing apparatus open to its full extent.
The object gained by allowing the light to enter in such
a large volume is that the contrast produced by the
colored bacteria in the brightly illuminated field is
much more conspicuous than when a smaller amount of
light is thrown upon them. This is true not only for
stained bacteria on cover-slips, but likewise for their
differentiation from surrounding objects when they are
located in tissues. With unstained bacteria and tissues,
on the contrary,.the structure can best be made out by
reducing the bundle of light-rays to its smallest
amount, and in this way favoring, not color contrast,
but contrasts which appear as lights and shadows due to
the differences in permeability to light of the various
parts of the material under examination.

Steps In EXAMINING STAINED PREPARATIONS
WITH THE OIL-IMMERSION SysTEM.—Place upon the
centre of the cover-slip which covers the preparation a
small drop of immersion oil. Place the slide upon the
centre of the stage of the microscope. With the coarse

MICROSCOPIC EXAMINATIONS, 163

adjustment lower the oil-immersion objective until it
just touches the drop of oil. Open the illuminating
apparatus to its full extent. Then, with the eye to the
microscope and the hand on the fine adjustment, turn
the adjusting-screw toward the right until the field be-
comes somewhat colored in appearance. When this is
seen, proceed more slowly in the same direction, and,
after one or two turns, the object will be in focus. Do
not remove the eye from the instrument until this has been
accomplished.

Then, with one hand upon the fine adjustment and
the thumb and index finger of the other hand holding
lightly the slide by its end, the slide may be moved
about under the objective. At the same time the screw
of the fine adjustment must be turned back and forth so
that the different levels of the preparation may one after
the other be brought into focus. In this way the whole
section or preparation may be inspected. When the
examination is finished, raise the objective from the
preparation by turning the screw of the coarse adjust-
ment toward you. Remove the preparation from the
stage, and, with a fine silk cloth or handkerchief, wipe
very gently and carefully the oil from the face of the lens.
The lens is then unscrewed from the microscope and
placed in the case intended for its reception.

During work, of course, the lens need not be cleaned
and put away after each examination; but when the
work for the day is over, an immersion lens must always
be protected in this way. Under no circumstances should
it be allowed to remain in the immersion oil or exposed
to dust for any length of time.

EXAMINATION OF UNSTAINED PREPARATIONS.—
“ Hanging drops.” It frequently becomes necessary to

164 BACTERIOLOGY.

examine bacteria in the unstained condition. The cir-
cumstances calling for this arise while studying the
multiplication of cells, the germination of spores, the
formation of spores, and the absence or presence of
motility.

In this method the organisms to be studied are sus-
pended in a drop of salt solution or bouillon in the
centre of acover-slip. This is then placed, drop down,
upon an object-glass in the centre of which a hollow or
depression is ground (Fig. 32). The slip is held in posi-
tion by a thin layer of vaselin, which may be painted

Fie. 32.

= ——— 7

Hollow ground-glass slide for observing bacteria in hanging drops.

around the margins of the depression. This not only
prevents the slip from moving from its position during
examination, but also prevents drying by evaporation if
the preparation is to be observed for any length of
time. This is known as the “hanging-drop” method of
examination or cultivation. It is indispensable for the
purposes mentioned, and at the same time requires con-
siderable care in its manipulation. The fluid is so trans-
parent that the cover-slip is often broken and the face
of the objective injured by its being brought down upon
the preparation before one is aware that the focal dis-
tance has been reached. This may be avoided by grasp-
ing the slide with the left hand and moving it back
and forth under the objective as it is brought down
toward the object. As soon as the least pressure is felt
upon the slide the objective must be raised, otherwise

MICROSCOPIC EXAMINATIONS. 165

the cover-slip will be broken and the lens may be ren-
dered worthless.

A safer plan is to bring the edge of the drop into the
centre of the field with one of the higher power dry
lenses. When this is accomplished, substitute the im-
mersion for the dry system, and the edge of the drop
can now easily be found.

In examining bacteria by this method there is a pos-
sibility of error that must be guarded against. All
microscopic insoluble particles in suspension in fluids
possess a peculiar tremor or vibratory motion, the so-
called “Brownian motion.” This is very apt to give
the impression that the organisms under examination
are motile, when in truth they are not so, their move-
ment in the fluid being due only to this molecular
tremor.

The difference between the motion of bodies under-
going this molecular tremor and that possessed by cer-
tain living bacteria is that the former particles never
move from their place in the field, while the living
bacteria alter their position in relation to the surround-
ing organisms, and may dart from one position in the
field to another. With some cases the true movement
of bacteria is very slow and undulating, while in others
it is rapid and darting. The molecular tremor may be
seen with non-motile and with dead organisms.

Note.—Prepare three hanging-drop preparations—
one from a drop of dilute India-ink, a second from a
culture of micrococci, and a third from a culture of
the bacillus of typhoid fever. In what way do they
differ ?

8*

166 BACTERIOLOGY.

Srupy oF SporE-roRMATION.—The hanging-drop
method just mentioned is not only employed for the
detection of the motility of an organism, but also for
the study of its spore-forming properties.

Since with aérobic organisms spore-formation occurs,
as a rule, only in the presence of oxygen, and is induced
more by limitation of the nutrition of the organisms
than by any other factor, it is essential that these two
points should be borne in mind in preparing the drop
cultures in which the process is to be studied. For
this reason the drop of bouillon should be small and
the air-chamber relatively large.

The cover-slip and hollow-ground slide should be
carefully sterilized, and with a sterilized platinum loop
a very small drop of bouillon is placed in the centre of
the cover-slip. The slip is then inverted over the hollow
depression in the sterilized object-glass and sealed with
vaselin, The most convenient method of performing
this last step in the process is to paint a ring of vaselin
around the edges of the hollow in the slide, and then,
without taking the cover-slip up from the table upon
which it rests, invert the hollow over the drop and
press it gently down upon the cover-slip. The vaselin
causes the slip to adhere to the slide, so that it can be
easily taken up. The drop now hangs in the centre of
the small air-tight chamber which exists between the
depression in the slide and the cover-slip. (See Fig.
32, page 164.)

A very thin drop of sterilized agar-agar may be sub-
stituted for the bouillon. It serves to retain the organ-
isms in a fixed position, and the process may be more
easily followed.

As soon as finished, the preparation is to be exam-

\

STUDY OF SPORE-FORMATION. 167

ined microscopically, and the condition of the organ-
isms noted. It is then to be retained in a warm
chamber especially devised for the purpose and kept
under continuous observation. The form of chamber
best adapted for the purpose is one which envelops the
whole microscope. It is provided with a window
through which the light enters, and an arrangement for
moving the slide about from the outside. The forma-
tion of spores requires a much longer time than the
germination of spores into bacilli, but with patience
‘both processes may be satisfactorily observed.

It will be noticed that the description of this process
is very much like that which has just been given, but
differs from it in one respect, viz., that in this manip-
ulation we are not making a preparation which is simply
to be examined and then thrown aside, but it is an actual
pure culture, and must be kept as such, otherwise the
observation will be worthless. For this reason the
greatest care must be observed in the sterilization of all
objects employed. Studies upon spore-formation by this
method frequently continue over hours, and sometimes
days, and contamination must, therefore, be carefully
guarded against. The study should be begun with the
vegetative form of the organisms; the hanging-drop
preparation should, for this reason, always be made
from a perfectly fresh culture of the organism under
consideration, before time has elapsed for spores to
form.

The simple detection of the presence or,absence of
spore-formation can in many cases be made by other
methods. For example, many species of bacteria which
possess this property form spores most readily upon
media from which it is somewhat difficult for them to

168 BACTERIOLOGY,

obtain the necessary nutrition; potatoes and agar-agar
that have become a little dry offer very favorable
conditions, because of the limited area from which the
growing bacteria can draw their nutritive supplies and
because of the free access which they have to oxygen;
for, their growth being on the surface, they are sur-
rounded by this gas unless means are taken to prevent
it. By the hanging-drop method, however, more than
this simple property may be determined. It is possible
not only to detect the stages and steps in the formation
of endogenous spores, but when the spores are completely
formed by transferring them to a fresh bouillon-drop or
drop of agar-agar, preserved in the same way, their ger-
mination into mature rods may be seen. The word
rods is used because as yet we have no evidence that
endogenous spore-formation occurs in any of the other
morphological groups of bacteria.

Srupy or GELatin CuLrurEs.—As has been pre-
viously stated, the behavior of bacteria toward gelatin
differs—some of them producing apparently no altera-
tion in the medium, while others bring about a form of
peptonization which results in liquefaction of the gela-
tin at and around the place at which the colonies are
growing. In some instances this liquefaction spreads
laterally and downward, causing a saucer-shaped exca-
vation, while in others the colony sinks directly down
into the gelatin and may be seen lying at the bottom of
a funnel-shaped depression. These differences are con-
stantly employed as one of the means of differentiating
otherwise closely allied members of the same family of
bacteria. Studies upon the spirillum of Asiatic cholera
and a number of closely allied species, for example,
reveal a decided difference in the form of liquefac-

STUDY OF CULTURES ON POTATO. 169

tion produced by these different organisms. The
slightest detail in this respect must be noted, and its
frequency or constancy under different conditions
determined.

CuntuRES ON Potato.—A very important feature
in the study of an organism is its growth on sterilized
potato. Many organisms present appearances under
this method of cultivation which alone can almost be
considered characteristic. In some cases coarsely lobu-
lated, elevated, dry or moist patches of development
oceur after a few hours; again, the growth may be finely
granular and but slightly elevated above the surface of
the potato; at one time it will be dry and dull in
appearance, again it may be moist and glistening.
Sometimes there is a production of bubbles, owing to
fermentation brought about by the growth of the organs.

A most striking form of development on potato is
that possessed by the bacillus of typhoid fever and the
bacillus of diphtheria. After the inoculation of a potato
with either of these organisms there is no naked-eye
evidence of a growth in either instance, though micro-
scopic examination of scrapings from the surface of the
potato reveals an active multiplication of the organisms
which had been planted there. The potato is one of
the most important differential media which we possess
for this work.

REACTION PRODUCED BY ORGANISMS IN THEIR
GrowTH.—The reactions produced in the media by
different organisms in the course of their growth are
very valuable as means of differentiation.

In some cases these changes are so marked that they
are readily detected by the coarser reagents ; again they
are so slight as to require the employment of the most

170 BACTERIOLOGY.

delicate indicators. They are sometimes seen to produce
at one period of their growth an alkaline, at another
period an acid reaction. This is seen in the cultures of
the bacillus diphtheria of Loffler.

These differences are best seen after the addition to the
media in which the organisms are to grow of some of
the chemical substances which do not interfere with the
development of the organisms, but which under one
reaction are of one color, and with an alteration of’ the
reaction become a different color, the change being in-
dicated by the play of colors. Such substances as litmus,
in the form of the so-called “litmus tincture,’ and
coralline (rosolic acid) in alcoholic solution have been
employed for this purpose. They may be added to the
media in the proportions given in the chapter on media,
and the alterations in their colors studied with different
bacteria. Milk and litmus tincture or peptone solution
to which rosolic acid has been added are very favorable
media for this experiment.

In milk, coagula will now and then appear as a result
of acids produced during the bacterial life, while again
acids may be produced and yet no coagulation be noticed.

ANILINE Dyes FoR DIFFERENTIAL DIAGNOSIS.—
The addition to solid media of some of the aniline
dyes, fuchsin, methylene-blue, methylene-green, and
several others, as well as combinations of these dyes,
have been recommended as a means of differentiation of
organisms, the differences claimed to be produced con-
sisting of alterations in the color of the media due to
oxidizing or reducing properties of the growing bacteria.
As yet but little has come from this method of work.
It cannot at present be recommended as a reliable means
of diagnosis.

ANILINE DYES. 171

Benavion TowarDd STAIninc ReEAGEN'Ts.—The
behavior of certain organisms toward the different dyes
and their reactions under special methods of after-treat-
ment serve as aids to their diagnosis. With very few
exceptions all bacteria stain readily with the common
aniline dyes, but they differ materially in the tenacity
with which they retain these colors under the subse-
quent treatment with decolorizing agents.

The tubercle bacillus and the bacillus of leprosy, for
example, are difficult to stain, but when once stained
retain their color under the action of such energetic de-
colorizing agents as alcohol, nitric acid, oxalic acid, ete.

Certain other organisms when stained with a solution
of gentian violet in aniline-water, retain their color when
treated with such decolorizing bodies as iodine solution
and alcohol (Gram’s method), while again others are
completely decolorized by this method

Many of them can only be treated with water, or but
for a few seconds with alcohol, without losing their
color.

It is essential that these peculiarities should be care-
fully noted in studying an organism.

FERMENTATION.—The production of gas as an indica-
tion of fermentation is an accompaniment of the growth
of some organisms. This is best studied in media to
which 1 to 2 per cent. of grape sugar has been added.

In this experiment the test-tube should be filled to
about one-half its volume with agar-agar. The me-:
dium is then liquefied, and when at the proper tem-
perature, a small quantity of a pure culture of the
organism under consideration should be carefully dis-
tributed through it. The tube is then placed into ice-
water and rapidly solidified in the vertical position.

172 BACTERIOLOGY.

When solid it is placed in the incubator. After twenty-
four to thirty-six hours, if the organism possesses the
property of causing fermentation of sugar, the medium
will be dotted everywhere with very small cavities con-
taining the gas that has resulted.

This property of fermentation with production of gas
is of such importance as a differential means that latterly
considerable attention has been given to it, and those
who have been most intimately concerned in the-devel-
opment of our knowledge on the subject do not consider
it enough to say that the growth ofan organism “is
accompanied by the production of gas-bubbles,” but
that under given conditions we should determine not
only the amount of gas produced by the organism under
consideration but also its quality. For this purpose
Smith! recommends the employment of the fermenta-
tion-tube used by Einhorn in the quantitative fermenta-
tion test for sugar in the urine. It is a tube bent at an
acute angle, closed at one end and enlarged with a bulb
at the other. At the bend the tube is constricted. To
ita glass foot is attached so that the tube may stand
upright. (See Fig. 33.) To fill the tube the fluid
(they are only used with fluid media) is poured into the
bulb until this is about half full. The tube is then
tilted until the closed arm is nearly horizontal, so that
the air may flow out into the bulb and the fluid flow
into the closed arm to take its place. When this
has been completely filled enough fluid should be
added to cover the lowest expanding portion of the
bulb and the opening of the bulb plugged with cotton.

1 An excellent and exhaustive contribution to this subject has been made
by Theobald Smith in “The Wilder Quarter-Century Book,’’ Ithaca, N. Y.,
1893.

FERMENTATION. 173

They are then to be sterilized. During sterilization
they are to be maintained in the upright position.
Under the influence of heat the
tension of the aqueous vapors in the
closed arm forces most of the fluid
into the bulb. As the tubes cool
the fluid returns to its place in the
closed arm and fills it again with the
exception of a small space at the top
which is occupied by the air origi-
nally dissolved in the liquid and
which has been driven out by the
heat. The air-bubble should be
tilted out after each sterilization, and
finally, after the third exposure to
steam this arm of the tube will be
free from air.

The medium employed is bouillon
containing some fermentable carbo-
hydrate, as glucose, lactose, or sac-
charose. After inoculation the flasks are placed in the
incubator and the amount of gas that collects in the
closed arm is, from day to day, noted.

From studies that have been made this gas consists
usually of about one part by volume of carbonic acid and
two parts by volume of an explosive gas consisting
largely of hydrogen. For determining the nature and
quantitative relations of these gases Smith! recommends
the following procedure: “The bulb is completely
filled with a 2 per cent. solution of sodium hydroxide
(NaOH) and closed tightly with the thumb. The fluid

Fig. 33.

Einhorn’s fermentation-
tube.

1 Loe. cit., p. 196.

174 BACTERIOLOGY.

is shaken thoroughly with the gas and allowed to flow
back and forth from bulb to closed branch and the re-
verse several times to insure intimate contact of the
CO, with the alkali. Lastly, before removing the thumb
all the gas is allowed to collect in the closed branch, so
that none may escape when the thumb is removed. If
CO, be present a partial vacuum in the closed branch
causes the fluid to rise suddenly when the thumb is
removed. After allowing the layer of foam to subside
somewhat the space occupied by gas is again measured,
and the difference between this amount and that meas-
ured before shaking with the sodium hydroxide solution
gives the proportion of CO, absorbed. The explosive
“character of the residue is determined as follows: The
cotton plug is replaced and the gas from the closed
branch is allowed to flow into the bulb and mix with
the air there present. The plug is then removed and a
lighted match inserted into the mouth of the bulb.
The intensity of the explosion varies with the amount
of air present in the bulb.”

CuLtivaTion Wirnour OxyGEn.—As we have
already learned, there is a group of organisms to which
the name “anaérobic organisms” has been given, which
are characterized by their inability to grow in the pres-
ence of oxygen. For the cultivation of the members
of this group a number of devices are employed for the
exclusion of oxygen from the cultures.

' Koch’s method. Koch covered the surface of a gelatin
plate, which had been previously inoculated, with a thin
sheet of sterilized isinglass. The organisms which
grew beneath it were supposed to grow without oxygen.

Hesse’s method. Hesse poured sterilized oil upon the
surface of a culture made by stabbing into a tube of

CULTIVATION WITHOUT OXYGEN 175

gelatin. The growth that occurred along the track of
the needle was supposed to be anaérobic in nature.
Methods of Liborius. Liborius has suggested two
useftil methods for this purpose. The one is to fill a
test-tube about three-quarters
full of gelatin or agar-agar,
which, after having been ster-
ilized, is to be kept in a vessel
of boiling water for ten min-
utes to expel all air from it.
It is then rapidly cooled in
ice-water, and when between
30° and 40° C., still fluid, is
to be inoculated and very rap-
idly solidified. It is then
sealed up in the flame. An-
aérobice bacteria develop only
in the lower layers of the
medium. Another method is
that in which he employs a
special tube, known as “the Pear earn ae
Liborius tube.” Its construc- cultures,
tion is shown in Fig. 34.
Through the side tube hydrogen is passed until all air
is expelled ; the contracted parts, both of the neck of
the tube and the side arm, are then sealed in the flame.!
This tube can be used for either solid or liquid media,
but, owing to its usual small capacity, gives better re-
sults with fluid media. (For precautions in using the hy-
drogen method sec note to Frinkel’s method, page 178.)

1 As the tubes come from the maker the contracted parts marked x in the
cut are usually so thick as to render the sealing in the flame during the pas-
sage of hydrogen somewhat troublesome ; it is better to draw them out in the
flame quite thin before passing the hydrogen into the tube. This makes the
final sealing a matter of no difficulty.

176 BACTERIOLOGY.

Method of Buchner. The plan suggested by Buchner
of allowing the cultures to develop in an atmosphere
robbed of its oxygen by pyrogallic acid gives very good
results. In this method the culture, which is either a
slant or stab culture in a test-tube, is placed—tube,
cotton plug, and all—into a larger tube in the bottom
of which has been deposited 1 gramme of pyrogallic
acid and 10 ce. of {5 normal’ caustic potash solution.
The larger tube is then tightly plugged with a rubber
stopper. The oxygen is quickly absorbed by the pyro-
gallic acid, and the organisms develop in the remaining
constituents of the atmosphere — nitrogen, a small
amount of CO,, and a trace of ammonia.

Method of C. Frankel. Carl Frinkel suggests the
following as a modification of or substitute for the
tubes of Liborius: The tube is first prepared as if for
an ordinary Esmarch tube. The cotton plug is then
replaced by a rubber stopper, through which pass two
glass tubes. These must all have been sterilized in the
steam sterilizer before using. On the outer side of the
stopper these two tubes are bent at right angles to the
long axis of the test-tube into which they are to be
placed, and both are slightly drawn out in the gas
flame. At the outer extremity of both of these tubes

1 A normal solution is one that contains in a litre as many grammes of the
dissolved substance as are indicated by its molecular equivalent. The equiva-
lent is that amount of a chemical compound which possesses the same chem-
ical value as does one atom of hydrogen. For example: One molecule of
hydrochlorie acid (HCl) has a molecular weight and also an equivalent
weight of 36.5; a molecule of this acid has the same chemical value as one
atom of hydrogen, Its normal solution is therefore 36.5 grammes to the litre.
On the other hand, sulphuric acid (H,SO,) contains in each molecule two
replaceable hydrogen atoms; its normal solution is not, therefore, 80 grammes
(its molecular weight) to the litre, but that amount which would be equiva-
lent chemically to one hydrogen atom, viz., 40 grammes (one-half its molecu-
lar weight) to the litre. A normal solution of caustic potash contains as
many grammes to the litre as the number of its molecular weight—56.1
grammes to the litre of water.

CULTIVATION WITHOUT OXYGEN. 177

a plug of cotton is placed ; this is to prevent access of
foreign organisms during manipulation. At the inner
side of the rubber stopper—that is, the end which is to
be inserted into the test-tube—the glass tubes are of
different lengths: one reaches to within 0.5 cm. of the
bottom of the test-tube, the other is cut off flush with

Friinkel’s method for the cultivation of anaérobic bacteria.

the under surface of the stopper. This rubber stopper,
with its glass tubes, is to replace the cotton plug of the
test-tube ; the outer end of the longer glass tube is then
connected with a hydrogen generator and hydrogen is
allowed to bubble through the gelatin (Fig. 35, a) in
the tube until all contained air has been expelled and

178 BACTERIOLOGY.

its place taken by the hydrogen.’ When the hydrogen
has been bubbling through the gelatin for about five
minutes (at least) one can be reasonably sure that all
oxygen has been expelled. The drawn-out portions of
the tubes can then be sealed in the gas-flame without
fear of anexplosion. The protruding end of the rubber
stopper is then painted around with melted paraffin and
the tube rolled in the way given for ordinary Esmarch
tubes. A tube thus prepared and containing growing
colonies is shown in Fig. 35, B.

The development that now occurs is in an atmosphere
of hydrogen, all oxygen having beenexpelled. During
the operation the tube containing the liquefied gelatin
should be kept in a water-bath at a temperature suffi-
ciently high to prevent its solidifying and at the same
time not high enough to kill the organisms with which
it has been inoculated.

One of the obstacles to the successful performance of
this method is the bubbling of the gelatin, the foam
from which will often fill the exit tube and sometimes be

1 Before beginning the experiment it is always wise to test the hydrogen,
i. ¢., tosee that itis free from oxygen and thereis no danger of an explosion, for
unless this is done the entire apparatus may be blown to pieces and a serious
accident occur. The agents used should be pure zinc, and pure sulphuric acid
of about 25 to 30 per cent. strength. When the gas is beginning to be given
off, the outlet of the generator should be closed and kept closed until the gas
reservoir is quite filled with hydrogen. The outlet should then be opened
and the entire volume of gas allowed to escape, care being taken that no
flame is in the neighborhood. This should be repeated again, after which a
sample of the hydrogen generated should be collected in an inverted test-tube
in the ordinary way for collecting gases over water, viz., by filling a test-tube
with water, closing its mouth with the thumb, inverting it, and placing its
mouth under water, when, after removing the thumb, the water will be kept
in it by atmospheric pressure. The hydrogen which is flowing from the open
generator may be conveyed to the test-tube by a bit of rubber tubing. When
the water has been replaced try the gas, by holding a flame near the open mouth
of the test-tube. If no explosion occurs, the hydrogen is safe to use. Should

there be an explosion the process must be repeated until the hydrogen simply
burns with a colorless flame.

ESMARCH’S METHOD. 179

forced from it. This may be obviated by reversing the
order of proceeding, viz.: Prepare the Esmarch roll
tube in the ordinary way with the organism to be
studied, using a relatively small amount of gelatin, so
as to have as thin a layer as possible when it is rolled.
Then replace the cotton plug with the sterilized rubber
stopper carrying the glass tubes through which the
hydrogen is to be passed, and allow the hydrogen to
flow through just as in the method first given. The
gas now passes over the solid gelatin instead of through
it, and consequently no bubbling results. In all other
respects the procedure is the same as that given by
Frankel.

Method of Kitasato and Weil. For favoring the
anaérobic conditions, Kitasato and Weil have suggested
the addition to the culture media of some strong re-
ducing agent. They recommend formic acid in 0.3 to
0.5 per cent.; glucose in 1.5 to 2 per cent., or blue
litmus tincture in 5 per cent. by volume. This is, of
course, in addition to an atmosphere from which all
oxygen has been expelled.

Ksmarch’s method. Esmarch’s plan is to prepare in
the usual way a roll tube of the organisms; subject it
to a low temperature, and while quite cold fill it with
liquefied gelatin, which is caused to solidify rapidly.
In this method the colonies develop along the sides of
the tubes, and can more easily be studied than where
they are mixed through the gelatin, as in the method
of Liborius.

By some workers the oxygen is removed by actual
pumping with the air-pump.

Many other methods exist for this special purpose,
but for the beginner those given will suffice.

180 BACTERIOLOGY.

From what has been said it may be inferred that the
cultivation of anaérobic bacteria is a simple matter and
attended with but little difficulty. Such an inference
will, however, be quickly dispelled when the beginner
attempts this part of his work for the first time, and
particularly when his efforts are directed toward the
isolation of these forms from other organisms with
which they are associated. The presence of spore-
forming, facultative anaérobes in mixed cultures is
always to be suspected, and it is this group that renders
the task so difficult. At best the work requires undi-
vided attention and no small degree of skill in bacterio-
logical technique.

Inpot Propuction.—The production of products
other than those which give rise to alterations in the
reaction of the media, and whose presence may be de-
tected by chemical reactions, is now a recognized step
in the identification of different species of bacteria.
Among these chemical products there is one which is
produced by a number of organisms, and whose presence
may easily be detected by its characteristic behavior
when treated with certain substances. I refer to the
body nitroso-indol, the reactions of which were described
by Beyer in 1869, and the presence of which as a pro-
duct of growth of certain bacteria has since furnished a
topic for considerable discussion.

Indol, the name by which this body is now generally
known, when acted upon by reducing agents, is seen to
become of a more or less conspicuous rose color. This
body was recognized as one of the products of growth
of the spirillum of Asiatic cholera first by Poel, and
a short time subsequently by Bujwid and by Dunham,
and for a time was thought to be peculiarly character-

INDOL PRODUCTION. 181

istic of the growth of this organism. It has since been
found that there exist other bacteria which also possess
the property of producing indol in the course of their
development.

The method employed for its detection is as follows:
Cultivate the organism for twenty-four to forty-eight
hours at a temperature of 37° C., in the simple peptone
solution known as “ Dunham’s solution” (see formula
for this medium). This solution is preferred because
its pale color does not mask the rose color of the reac-
tion when the amount of indol present is very small.

Four tubes should always be inoculated and kept
under exactly the same conditions for the same length
of time.

At the end of twenty-four or forty-eight hours the
test may be made. Proceed as follows: To a tube con-
taining 7 c.c. of the peptone solution, but which has not
been inoculated, add 10 drops of concentrated sulphuric
acid. To another similar tube add 1 ¢.c. of a 0.01 per
cent. solution of sodium nitrite, and afterward 10 drops
of concentrated sulphuric acid. Observe the tubes for
five to ten minutes. No alteration in their color appears,
or at least there will be no production of a rose color.
They contain no indol.

Treat in the same way, with the acid alone, two of
the tubes which have been inoculated. If no rose color
appears after five or ten minutes, add 1 c.c. of the sodium
nitrite solution. If now no rose color is produced, the
indol reaction may be considered as negative. No indol
is present. :

If indol is present, and the rose color appears after
the addition of the acid alone, it is plain that not only

indol has been formed, but likewise a reducing body.
9

182 BACTERIOLOGY.

This is found, by proper means, to be salts of nitrous
acid. The sulphuric acid liberates this acid from its
salts and permits of its reducing action being brought
into play.

If the rose color appears only after the addition of
both the acid and the nitrite solution, then indol has
been formed during the growth of the organisms, but no
nitrites.

Control the results obtained by treating the two re-
maining cultures in the same way.

The test is sometimes made by allowing cencentrated
acid to flow down the sides and collect at the bottom of
the tube ; the reaction is then seen as a rose-colored
zone overlying the line of contact of the acid and cul-
ture medium. This method is open to the objection
that if indol is present in only a very limited amount,
the rose color produced by it is apt to be masked by a
brown color that results from the charring action of the
concentrated acid on the other organic matters in the
culture medium, so that its presence may in this way
escape detection. In view of this, Petri recommends
the use of dilute sulphuric acid. He states that when
indol is present the characteristic rose color appears a
little more slowly with the dilute acid, but is more per-
manent, and there is never any danger of its presence
being masked by the occurrence of other color reactions.

POINTS TO BE OBSERVED IN DESCRIBING AN ORGANISM.

The following is an outline of points to be considered
in describing a new organism or in identifying an or-
ganism with one already described :

1. Its source—as air, water, or soil. If found in the

DESCRIBING NEW ORGANISMS. 188

animal body, is it normally present or only in patholog-
ical conditions?

2. Its form, size, mode of development, occurrence of
involution forms or other variations in morphology.
Grouping, asin pairs, chains, clumps, zodgloea ; presence
of capsule; development and germination of spores ;
arrangement of flagella.

3. Staining peculiarities—especially its reactions with
Gram’s (or Weigert’s fibrin) stain, and peculiar or
irregular modes of staining.

4. Motility—to be determined on very fresh cultures
and on cultures in different media.

5. Its relation to oxygen—is it aérobic, anaérobic, or
facultative? Does it develop in other gases, as car-
bonic acid, hydrogen, etc.?

6. Both the macroscopic and microscopic appearance
of its colonies on nutrient gelatin and on nutrient agar-
agar.

7. The appearance of its growth in stab and slant
cultures on gelatin, agar-agar, blood-serum, and on
potato.

8. The character of its growth in fluid media, as in
bouillon, milk, litmus milk, rosolic-acid-peptone solu-
tion, and in bouillon containing glucose.

9. Does it grow best in acid, alkaline, or neutral
media?

10. Is the normal reaction of the medium altered by
its growth? Is its growth accompanied by the produc-
tion of indol ; is the indol associated with the coincident
production of nitrites?

11. Is its growth accompanied by the production of
gas, as evidenced by the appearance of gas-bubbles in
the media—both in media containing fermentable sugars

184 BACTERIOLOGY.

and those from which these bodies are absent? When
cultivated in sugar-bouillon in the fermentation tube,
what proportion of gas is evolved under known con-
ditions? How much of this gas is carbonic acid and
how much is explosive?

12. At what temperature does it thrive best, and the
lowest and highest temperatures at which it will de-
velop? What is its thermal death-point, both by
steam and dry-air methods of determining this point?

13. What is its behavior when exposed to chemical
disinfectants and antiseptics? does it withstand drying
and other injurious influences, both in the vegetative
and spore stages? The germicidal value of the blood-
serum of different animals may also be tried upon it.

14. Its pathogenic powers—modes of inoculation by
which these are demonstrated; quantity of material
used in inoculation; duration of the disease and its
symptoms ; lesions produced, and distribution of the
bacteria in the inoculated animal; which animals are
susceptible and which immune, and the character of
its pathogenic activities? Variations in virulence, and
the probable cause to which they are due. Can they
be produced artificially and at will?

15. The detection of specific, toxic, and immunizing
products of growth.

CHAPTER XII.

Inoculation of animals—Subcutaneous inoculation ; intra-venous injection
—Inoculation into the great serous cavities; and into the anterior chamber of
the eye—Observation of animals after inoculation.

AFTER subjecting an organism to the methods of
study that we have thus far reviewed, there remains to
be tested its action upon animals—i. e., to determine if
it possesses the property of producing disease or not,
and, if so, what are the pathological results of its
growth in the tissues of these animals, and in what way
must it gain entrance to the tissues in order to produce
these results.

The means of deciding these points is by inoculation,
which is practised in different ways according to cir-
cumstances. Most commonly a bit of the culture to be
tested is simply introduced beneath the skin of the
animal, but in other cases it may be necessary to intro-
duce it directly into the vascular circulation or into one
or the other of the great serous cavities; or, for still
other purposes of observation, into the anterior chamber
of the eye, upon the iris.

SupcuTaNEous INocULATION oF ANIMALS.—The
animals usually employed in the laboratory for purposes
of inoculation are white mice, gray house-mice, guinea-
pigs, rabbits, and pigeons.

For simple subcutaneous inoculation the steps in the
process are practically the same in all cases. The hair
or feathers are to be carefully removed. If the skin

186 BACTERIOLOGY.

is very dirty it may be scrubbed with soap and water.
Disinfection of the skin is impossible, so that it need
not be attempted. IPf thé inoculation is to be by means.
of a hypodermatic syringe, then a fold of the skin may
be lifted up and the needle inserted in the way common
to this procedure. If asolid culture is to be inoculated,
a fold of the skin may be taken up with the forceps
and a pocket cut into it’'with scissors which have previ-
ously been sterilized. This pocket must be cut large
enough to admit the end of the needle without its
touching the sides of the opening as it is inserted.
Beneath the skin will be found the superficial and deep
connective-tissue fascie. These must be taken up with
sterilized forceps, and with sterilized scissors incised in
a way corresponding to the opening in the skin. The
pocket is then to be held open with the forceps and the
substance to be inserted is introduced as far back under
the skin and fasciz as possible, care being taken not to
touch the edges of the wound if it can be avoided. The
wound may be then simply pulled together and allowed
to remain. No stitching or efforts at closing it are
necessary.

During manipulation the animal must be held still.
For this purpose special forms of holders have been
devised, but if an assistant is to be obtained for the
operation, the simple subcutaneous inoculation may be
made without the aid of a mechanical holder.

For mice, however, a holder is of much convenience.
This piece of apparatus consists of a bit of board of about
7x10 cm. and 2 em. thick, upon which is tacked a hol-
low, tapering roll of wire gauze, a truncated cone of
about 6 cm. long and of about 1.5 cm. in diameter at
one end and 2 cm. at its other end.

SUBCUTANEOUS INOCULATION. 187

This is tacked upon the board in such a position that
its long axis runs in the long axis of the board,
being equidistant from its two sides. Its small end is
placed at the edge of the board. The mouse is taken up
by the tail by means of a pair of tongs and allowed to
crawl into the smaller end of this wire cone. When so
far in that only the root of the tail projects, the animal
is then fixed in this position by a clamp and thumb-
screw, with which the apparatus (Fig. 36) is provided.
The animal usually remains perfectly quiet and may be
handled without difficulty.

Fic. 36.

Mouse-holder, with mouse in proper position.

The hair from over the root of the tail is to be care-
fully cut away with the scissors, and a pocket cut
through the skin at this point. The inoculation is
then made into the loose tissues under the skin over
this part of the back in the way that has just been
described. It is best always to insert the needle some
distance along the spinal column, and thus deposit the
material as far from the surface-wound as possible.

As the subcutaneous operation is very simple and
takes only a few moments, guinea-pigs, rabbits, and
pigeons are best held by an assistant. The front legs
in the one hand and the hind legs in the other, with

188 BACTERIOLOGY.

the animal stretched upon its back on a table, is the
usual position for the operation when practised upon
guinea-pigs and rabbits. The point at which the inoc-
ulations are commonly made is in the abdominal walls
either to the right or left of the median line and about
3 cm. distant. When pigeons are used they are held
with the legs, tail, and ends of the wings in the one
hand, and the head and anterior portion of the body
in the other, leaving the area occupied by the pectoral
muscles, over which the inoculation is to be made, free
for manipulation. The hair should be closely cut with
the scissors in the case of the guinea-pigs and rabbits,
and the feathers pulled out in the case of the pigeon.

INJECTION INTO THE CIRCULATION.—It is not infre-
quently desirable to inject the material under considera-
tion directly into the circulation of an animal. If the
rabbit is to be employed for the purpose, the operation
is usually done upon one of the veins in the ear.

To those who have had no practice in this procedure
it offers a great many difficulties; but if the directions
which will be given are strictly observed, the greatest
of these obstacles to the successful performance of the
operation may be overcome.

When viewing the circulation in the ear of the rabbit
by transmitted light, three conspicuous branches of the
main vessel (vena auricularis posterior) will be seen.
One runs about centrally in the long axis of the ear,
one runs along its anterior margin, and one along its
posterior margin. The central branch (the ramus ante-
rior of the vena auricularis posterior) is the largest and
most conspicuous vessel of the ear, and is, therefore,
selected by the inexperienced as the branch into which
it would appear easiest to insert a hypodermatic needle.

INJECTION INTO THE CIRCULATION. 189

This, however, is fallacious. This vessel lies very loosely
imbedded in connective tissue, and in efforts to intro-
duce a needle into it, rolls about to such an extent that
only after a great deal of difficulty does the experiment
succeed. On the other hand the posterior branch
(ramus lateralis posterior of the vena auricularis poste-
rior) is a very fine, delicate vessel which runs along the
posterior margin of the ear, and which is so firmly fixed
in the dense tissues which surround it that it is pre-
vented from rolling about under the point of the needle.
The further away from the mouth of the vessel—that
is, the nearer we approach its capillary extremity—
the more favorable become the conditions for the success
of the operation.

Select, then, the very delicate vessel lying quite close
to the posterior margin of the ear, and make the injec-
tion as near to the apex of the ear as possible. The
injection is always to be made from the dorsal surface
of the ear.

Of no less importance than the selection of the proper
vessel, is the shape of the point of the needle employed.

The hypodermatic needles as they come from the
makers are not suited at all for this operation because
of the way in which their points are ground. If one
examines carefully the point of a new hypodermatic
needle it will be seen that the long point, instead of
presenting a flat, slanting surface, when viewed from
the side, is more or less of a curved surface. Now, in
efforts to introduce such a needle into a vessel of very
small calibre, it is commonly seen that the extreme
point of the needle, instead of remaining in the vessel,
as it would do were it straight, very commonly projects

into the opposite wall, and as the needle is inserted
9*

190 BACTERIOLOGY.

further and further into the tissues, it is usually pushed
through the vessels into the loose tissues beyond, and
the material to be injected is deposited into these tissues
instead of into the circulation. If, on the contrary, the
slanting point of the needle is ground down until its
surface is perfectly flat, and when viewed from the side
and no more curvature exists, then when once inserted
into a vessel it usually remains there, and there is no ten-
dency to penetrate through the opposite wall. We never
use a new hypodermatic needle until its point is carefully
ground down to a perfectly flat, slanting surface and no
more curvature exists.

These differences may perhaps come out clearer if
represented diagrammatically.

Fic. 37.

Hypodermatic needles. a, Improper point; b, proper shape of point,

‘In Fig. 37, a, the needle has the point usually seen
when new.

In Fig. 37, 6, the point has been ground down to the
shape best suited for this operation.

The needles need not be returned to the maker. One
can grind them to the shape desired in a few minutes
upon an oilstone.

The size of the needle is that commonly employed for
subcutaneous injections.

When the operation is to be performed, an assistant

INJECTION INTO THE CIRCULATION. 191

holds the animal gently but firmly in the crouching
position upon a table. If the animal does not remain
quiet it is best to wrap it in a towel so that nothing but
its head protrudes, though in the most cases we have
not found this necessary, and particularly if the animal
has not been excited prior to the beginning of the
operation.

The animal should be placed so that the ear upon
which the operation is to be performed comes between
the operator and the source of light. This renders
visible by transmitted light not only the coarser vessels
of the ear, but also their finer branches. The point at
which the injection is to be made is to be shaved clean
of hair, by means of a razor and soap.

The filled hypodermatic syringe is taken in one hand
and with the other hand the ear is held firmly. The
point of the needle is then inserted through the skin
and into the finest part of the ramus posterior, the part
nearest the apex of the ear, where the course of the
vessel is nearly straight. When the point of the
needle is in this vessel it gives to the hand a sensation
quite different from that felt when it is in the midst of
connective tissue. As soon as one thinks the point of
the needle is in the vessel, a drop or two of the fluid
may be injected from the syringe, and if his suspicions
are correct the circulation in the small ramifications and
their anastomoses will quickly alter in appearance.
Instead of their containing blood, the colorless fluid
which is being injected will now be seen to circulate.
This must be carefully observed, for sometimes when the
needle-point is not actually in the vessel, but is in the
lymph-spaces surrounding it, an appearance somewhat
similar is to be seen. It may always be differentiated,

1 92 BACTERIOLOGY.

however, by continuing the injection, when the circu-
lation of clear fluid through the vessels will not only
fail to take the place of the circulating blood but
there will at the same time appear a localized swelling
under the skin about the point of the needle. The
needle must then be withdrawn and inserted into the
vessel at a point a little nearer to its proximal end.

Care must be taken that no air is injected.

The hypodermatic syringe and needle must, previous
to operation, have been carefully sterilized in the steam
sterilizer. The animal must be kept under close observa-
tion for about an hour after injection.

Fie. 38.

Forms of hypodermatic syringe.

A, Koch’s syringe; B, syringe of Strohschein ; C, Overlack’s form.
AJ

The form of syringe best suited for this operation is
of the ordinary design, but one that permits of thorough
sterilization by steam. It should be made of glass and
metal, with asbestos packings. The syringes commonly
employed are those shown in Fig. 38—A, Koch’s; B,
Strohschein’s ; C, Overlack’s.

INOCULATION INTO THE SEROUS CAVITIES. 193

The operation is one that cannot be learned from
verbal description. It can only be successfully per-
formed after actual practice.

If the precautions which have been mentioned are
observed, but little difficulty in performing the opera-
tion will be experienced.

Its convenience and simplicity over other methods for
the introduction of substances into the circulation com-
mend it as an operation with which to make oneself
familiar. The animals sustain practically no wound,
they experience no pain—at least they give no evidence
of pain—and no anesthesia is required.

INOCULATION INTO THE GREAT SEROUS CAVITIES.

Inoculation into the peritoneum presents no difficulties
if fluids are to be introduced. In this case one makes,
with a pair of hot scissors, a small nick through the
skin down to the underlying fascia, and, taking up a
fold of the abdominal wall between the fingers, plunges
the hypodermatic needle through the opening just made
directly into the peritoneal cavity. There is no fear
of penetrating the intestines or other internal viscera if
the puncture is made along the median line at about
midway between the end of the sternum and the sym-
physis pubis. Though this may seem a rude method, it
is, nevertheless, the rarest of accidents to find that the
intestines have been penetrated. The object of the
primary incision is to lessen the chances of contaminat-
ing the inoculation by bacteria located in the skin,
some of which would adhere to the needle if it were
plunged directly through the skin, and might complicate
the results.

194 BACTERIOLOGY,

If solid substances, bits of tissue, etc., are to be intro-
duced into the peritoneum it becomes necessary to con-
duct the operation upon the lines of a laparotomy. The
hair should be shaved from a small area over the median
line, after which the skin is to be thoroughly washed.
A short longitudinal incision (about 2 em. long) is then,
to be made in the median line through the skin, and
down to the fascie. Two subcutaneous sutures, as.
employed by Halsted, are then to be introduced trans-
verse to the line of incision at about 1 cm. apart, and

their ends left loose. This particular sort of suture
does not pass through the skin, but the needle is intro-
duced into the subcutaneous tissues along the edge of
the incision. In this case they are to pass into the
abdominal cavity and out again, entering at one side of
the line of incision and leaving at the other as indicated
by the solid and dotted lines in Fig. 39. (This figure
indicates the primary opening through the skin. By the
longitudinal dotted line is seen the opening to be made into

INOCULATION INTO THE SEROUS CAVITIES. 195

the abdomen ; by the transverse dotted lines, with their
loose ends, the sutures as placed in position before the
abdomen is opened ; it will be seen that these sutures in
all cases pass through the subcutaneous tissues only and
do not penetrate the skin proper.) The opening through
the remaining layers may now be completed ; the bit of
tissue deposited in the peritoneal cavity, under precau-
tions that will exclude all else ; the edges of the wound
drawn evenly and gently together by tying the sutures,
and the lines of incision dressed with collodion. It
should be needless to say that this operation must be
conducted under the strictest precautions to avoid com-
plications. All instruments, sutures, ligatures, etc.,
roust be carefully sterilized either in the steam sterilizer
for twenty minutes, or by boiling in 2 per cent. soda
solution for ten minutes; the hands of the operator,
though they should not touch the wound, should be
carefully cleansed and the material to be introduced
into the abdomen should be handled with only sterilized
instruments.

Inoculation into the pleural cavity is much less fre-
quently called for—in fact, it is not a routine method
employed in this work. It is not easy to enter the
pleural cavity with a hypodermatic needle without injur-
ing the lung, and it is rare that conditions call for the
introduction of solid particles in this locality.

Inoculation into the anterior chamber of the eye is per-
formed by making a puncture through the cornea just
in front of its junction with the sclerotic, the knife being
passed into the anterior chamber in a plane parallel
to the plane of the iris. By the aid of a fine pair of
forceps the bit of tissue is passed through the opening
thus made and is deposited upon the iris, where it is

196 BACTERIOLOGY.

allowed to remain, and where its pathogenic properties
upon the iris can be conveniently studied. It is a mode
of inoculation of very limited application, and is there-
fore but rarely practised. It was employed by Cohnheim
in demonstrating the infectious nature of tuberculous
tissues, tuberculosis of the iris being the constant result
of the introduction of tuberculous tissue into the
anterior chamber of the eye of rabbits.

OBSERVATION OF ANIMALS AFTER INOCULATION.

After either of these methods of inoculation, particu-
larly when unknown species of bacteria are being
tested, the animal is to be kept under constant observa-
tion and all that is unusual in its conduct noted—as, for
instance, elevation of temperature ; loss of weight ; pecu-
liar position in its cage; loss of appetite ; roughening
of the hair; excessive secretions, either from the air-
passages, conjunctiva, or kidneys ; looseness of or hemor-
rhage from the bowels; tumefaction or reaction at site of
inoculation, etc. If death ensues in from two to four
days it may reasonably be expected that at autopsy
evidence of either acute septic or toxic processes will
be found. It sometimes occurs, however, that inocula-
tion results in the production of chronic conditions, and
the animal must be kept under observation, often for
weeks. In these cases it is important to note the pro-
gressive changes that are in progress by their effect
upon the physical conditions of the animal, viz., upon the
nutritive processes as evidenced by fluctuation in weight
and upon the body temperature. For this purpose the
animal is to be weighed daily, always at about the same
hour and always about midway between the hours of

OBSERVATION OF INOCULATED ANIMALS, 19%

feeding ;+at the same time its temperature as indi-
cated by a thermometer placed in the rectum is to be
recorded,’ By the comparison of these daily observa-
tions with one another, one is aided in observing the
course the infection is taking.

Too much stress must not, however, be laid upon
moderate and sudden daily fluctuations in either tem-
perature or weight, as it is a common observation that
presumably normal animals when confined in cages and
fed regularly often present very striking temporary gains
and losses in weight, often amounting to 50 or 100
grammes in twenty-four hours, even in animals whose
total weight may not exceed 500 or 600 grammes;
similarly they are seen to experience unexplainable rises
and falls of temperature, often as much as a degree
from one day to another. Such fluctuations have appa-
rently no bearing upon the general condition of the
animal, but are probably due to transient causes, such
as overfeeding or scarcity of food, improper feeding,
lack of exercise, excitement, fright, etc.

The accompanying charts (Figs. 40, 41, 42, 43) will
serve to illustrate some of these points. The animals,
two rabbits and two guinea-pigs, were taken at random
from among the stock animals and placed each in a clean
cage, the kind used for animals under experiment, and
kept under as good general conditions as possible. For
the first week the rabbits received each 100 grammes of
green food (cabbage and turnips) daily, and the guinea-
pigs 30 grammes each of the same food. During the
second week ‘this daily amount of food was doubled ;

1 The thermometer must be inserted into the rectum beyond the grasp of
the sphincter, otherwise pressure upon its bulb by contraction of this muscle
may give results higher than the actual body temperature.

198 BACTERIOLOGY.

Fic. 40. bg
o st NI
mol
a o | |q Ns
a
o ay H
.
uae b
Tan N
or | fe Dy
6 ||: y :
2
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3 ‘ 5
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a] 4 ry 4
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a
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6e q
sz | |¢ By] 3
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ra = ; 3
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aE 3
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wa Le 3
g| er 8 YT g
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Alu is s &
VBA 3 3 8 g
Yeas 5

Showing diurnal fluctuations of temperature and weight of a rabbit receiv-
ing varying amounts of food. Animal otherwise under normal conditions.

during the third week it was quadrupled, and for the
fourth and fifth weeks they each received an excess of

OBSERVATION. OF INOCULATED ANIMALS. 199

Fic. 41.
cL ~|
a
EF ot | fs
:
6 et | {i ,
at | yi fl
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6 >
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9% 6 qT &,
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a : LTS ba
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6L 4 rm bs
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ry g gy
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+ = es
zB eb << q
ay zw BT Ts fe
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wey, 3 3 3 g z
panqwisduas| o 225 8

Showing diurnal fluctuations of temperature and weight of a rabbit receiv-
ing varying amounts of food. Animal otherwise under normal conditions.

food daily, consisting of green vegetables and grain
(oats and corn). By reference to jthe charts sudden

200 BACTERIOLOGY.

Fig. 42.

Chart.3
=
J

2S Ee ol oe

Se oo
Excess of food daily.

April 1894,

SS Ee ee ee

120 grams food daily,

GO grams food daily.

ea as os Sa
a4 == 7Jo 5 3=b3K

ee

80 grams fobd daily.

March,1994,
et
x

qT MN,

510
i)

Jounqeatinag| 2 225

Showing diurnal fluctuations of temperature and weight of a guinea-pig
receiving varying amounts of food. Animal otherwise under normal condi-
tions.

diurnal fluctuations in weight will be observed that do
not correspond in all instances with scarcity or sufficiency

OBSERVATION OF INOCULATED ANIMALS. 9201

Fie, 43.
” st
a
E +L 2 }
eh ' KJ
N
a H ne
uli 7
ot
oth
8
L
9 ° A
: ‘
q L q a
+ ‘ h 5
# ‘ 2
Z] os é €
3 i 3
al z te 2
a é 3
a] o4 f a
te :
TE }

3
6z a FE
= LJ
8B } 3
lz b 2
TF N H
az | |t 7 a

Y i
+4
4
a yi
we q
Wz Bi 4 g
0% [ie 3
et | {d s 6
aL fi 8
aL
ot $
i
4 :
gL e pu Fy
D
att b LA i
g| et i | 3
a Lf
iB tee i Lt B
afu os 8
aoe B g é x.
e385

Showing diurnal fluctuations of temperature and weight of a guinea-pig
receiving varying amounts of food. Animal otherwise under normal condi-
tions.

of food. With the rabbits there is a gradual loss of
weight with the smaller amounts of food, which losses

202 BACTERIOLOGY.

are not totally recovered as the food is increased. With
the guinea-pigs there is likewise at first a loss, but after
a short time the weight remains tolerably constant, and
is not as conspicuously affected by the increase in’ food
as one might expect. From the recorded temperatures
one sees the peculiar fluctuations mentioned. To just
what they are due it is impossible to say. It is mani-
fest that the normal temperature of these animals, if we
can speak of a normal temperature for animals present-
ing such fluctuations, is about a degree or more, Centi-
grade, higher than that of human beings. The animals
from which these charts were made were not inoculated,
nor were they subjected to any operative procedures
whatever, the only deviations from normal conditions
being the variations in the daily amount of food given.

In certain instances, however, there will be noticed a
constant tendency to diminution in weight, notwith-
standing the daily fluctuations, and after a time a con-
dition of extreme emaciation may be reached, the animal
often being reduced to from 50 to 60 per cent. of its
original weight. In other cases, after inoculations to
which the animal is not susceptible, rabbits in particular,
if properly fed, will frequently gain steadily in weight.
The condition of progressive emaciation just mentioned
is conspicuously seen after intra-venous inoculation of
rabbits with cultures of the bacillus typhi abdominalis and
of the bacterium coli commune referred to in the chapter
on the latter organism, and if looked for will doubtless
be seen to follow inoculation with other organisms
capable of producing chronic forms of infection, but
which are frequently considered non-pathogenic because
of their inability to induce acute conditions. Not infre-
quently in chronic infections there may be hardly any

OBSERVATION OF INOCULATED ANIMALS. 203

marked and constant temperature variations until just
before death, when there will sometimes be a rise and
at other times a fall of temperature.

In the majority of cases, however, one must be very
cautious as to the amount of stress laid upon changes
in weight and temperature, for unless they are progres-
sive or continuous in one or another direction they
may have little or no significance in indicating the
existence or absence of disease.

CHAPTER XIII.

Post-mortem examination of animals—Bacteriological examination of the
tissues—Disposal of tissues and disinfection of instruments after the exam-
ination.

Durine the bacteriological examination of the tis-
sues of dead animals, certain rigid precautions must be
observed in order to avoid error.

The autopsy should be made as soon as possible after
death. If delay cannot be avoided, the animal should
be kept on ice until the examination can be made, other-
wise decomposition sets in, and the saprophytic bacteria
now present may interfere with the accuracy of results.

When the autopsy is to be made, the animal is first
inspected externally, and all visible lesions noted. It
is then to be fixed upon its back upon a board with
nails or tacks. The four legs and the end of the nose,
through which the tacks are driven, are to be moder-
ately extended. Plates are now to be made from
the site of inoculation, if this is subcutaneous. The
surfaces of the thorax and abdomen are then to be
moistened to prevent the fine hairs, dust, etc., from
floating about in the air and interfering with the work.
An incision is then made through the skin from.
the chin to the symphysis pubis. This is only a skin
incision, and does not reach deeper than the muscles.
It is best done by first making a small incision with a
scalpel, just large enough to permit of the introduction
of one blade of a blunt-pointed scissors. It is then
completed with the scissors. The whole of the skin is

POST-MORTEM EXAMINATION OF ANIMALS. 905

now to be carefully dissected away, not only from the
abdomen and thorax, but from the axillary, inguinal,
and cervical regions, and the fore and hind legs as well.
The skin is then pinned back to the board so as to keep
it as far from the abdomen and thorax as possible, for
it is from the skin that the chances of contamination
are greatest.

It now becomes necessary to proceed very carefully.
All incisions from this time on are to be made only
through surfaces that have been sterilized. The sterili-
zation is best accomplished by the use of a broad-bladed
common knife that has been heated in the gas-flame.
The blade, made quite hot, is to be held upon the
region of the linea alba until the skin at that region
begins to burn ; it is then held transverse to this line
over about the centre of the abdomen, thus making two
sterilized tracks through which the abdomen may be
opened by a crucial incision. The sterilization thus ac-
complished is, of course, directed only against organisms
that may have fallen upon the surface from without, and it
therefore need not extend deep down through the tissues.

In the same way two burned lines may be made from
either extremity of the transverse line up to the top of
the thorax.

With a hot scissors the central longitudinal incision,
extending from the point of the sternum to the genitalia,
is to be made without touching the internal viscera. The
abdominal wall must therefore be held up during the
operation with sterilized forceps or hook.

The cross incision is made in the same way. When
this is completed, an incision through the ribs with a pair
of heavy, sterilized scissors, is made along the scorched

tracks on either side of the thorax.
10

206 BACTERIOLOGY.

After this the whole anterior wall of the thorax may
easily be lifted up, and by severing the connections with
the diaphragm it may be completely removed.

When this is done and the abdominal flaps laid back,
the contents of both cavities are to be inspected and their
condition noted without disturbing them.

After this, the first steps to be taken are to prepare
plates or Esmarch tubes from the blood, liver, spleen,
kidneys, and any exudates that may exist.

This is best done as follows:

Heat a scalpel quite hot and apply it to a small sur-
face of the organ from which the cultures are to be
made. Hold it upon the organ until the surface directly
beneath it is visibly scorched. Then remove it, heat it
again, and while quite hot insert its point through the
capsule of the organ. Into the opening thus made insert
a sterilized platinum-wire loop, made of wire a little
heavier than that commonly employed. Project: this
deeply into the tissues of the organ; by twisting it
about enough material from the centre of the organ can
be obtained for making the cultures.

As the resistance offered by the tissues is sometimes
too great to permit of a puncture with the ordinary
wire loop, Nuttall (Centralblatt fiir Bakteriologie und
Parasitenkunde, 1892, Bd. xi. p. 538) has devised for
the purpose a platinum-wire spear which possesses con-
siderable advantage over the loop. It is of the form
seen in Fig. 44. It is easily made by beating a piece
of heavy platinum wire into a spear-head at one end,
and perforating this with a small drill, as seen in the
cut. It is attached by the other end to either a metal
or glass handle, preferably the former. It can be
readily thrust into the densest of the soft tissues, and

EXAMINATION OF TISSUES, 9207

by twisting it about after its introduction particles of
the tissue sufficient for examination are withdrawn in
the eye of the spear-head.

Fig. 44.

Nuttall’s platinum spear for use at autopsies.

The cultures from the blood are usually made from
one of the cavities of the heart, which is always entered
through a surface which has been burned in the way
given.

Tn addition to cultures, cover-slips from the site of in-
oculation, from each organ and from any exudates that
may exist, must be made. These, however, are preparcd
after the materials for the cultures have been obtained.

They need not be examined immediately, but may be
placed aside, under cover, on bits of paper upon which
the name of the organ from which they were prepared
is written.

When the autopsy is complete and the gross appear-
ances have been carefully noted, small portions of each
organ are to be preserved in 95 per cent. aleohol for
subsequent examination. Throughout the entire autopsy
it must be borne in mind that all cultures, cover-slips,
and tissues must be carefully labelled, not only with the
name of the organ from which they originate, but with
the date, designation of the animal, etc., so that an
account of their condition after closer study may be
subsequently inserted in the protocol.

The cover-slips are now to be stained, mounted, and
examined microscopically, and the results carefully noted.

The same may be said for the subsequent study of the

208 BACTERIOLOGY.

cultures and the hardened tissues which are to be stained
and subjected to microscopic examination. The results
of microscopic study of the cover-slip preparations
and those obtained by cultures should in most cases cor-
respond, though it not rarely occurs that bacteria are
present in such small numbers in the tissues that their
presence may be overlooked microscopically, and still
they may appear in the cultures.

If the autopsy has been performed in the proper way,
under the precautions given, and sufficiently soon after
death, the results of the bacteriological examination
should be either negative or the organisms which
appear should be in pure cultures.

This is particularly the case with the cultures made
from the internal viscera.

Both the cover-slips and cultures made from the point
of inoculation are apt to contain a variety of organisms.

If the organism obtained in pure culture from the
internal viscera, or those predominating at the point of
inoculation of the animal, have caused its death, then
subsequent inoculation of pure cultures of this organ-
ism into the tissues of a second animal should produce
sinailar results.

When the autopsy is quite finished, the remainder of
the animal should be burned ; all instruments subjected
to either sterilization by steam or boiling for fifteen
minutes in a 1 to 2 per cent. soda solution, and the board
upon which the animal was tacked, as well as the tacks,
towels, dishes, and all other implements used at the
autopsy, are to be sterilized by steam. All cultures,
cover-slips, and, indeed, all articles likely to have
infectious material upon them, must be thoroughly
sterilized as soon as they are of no further service.

APPLICATION OF THE METHODS OF
BACTERIOLOGY.

CHAPTER XIV.

To obtain material with which to begin work.

Expose to the air of an inhabited room a slice of
freshly steamed potato or a bit of slightly moistened
bread upon a plate for about one hour. Then cover it
with an ordinary water-glass and place it in a warm spot
(temperature not to exceed that of the human body—
37.5° C.), and allow it to remain unmolested. At the
end of twenty-four to thirty-six hours there will be
seen upon the cut surface of the bread or potato small,
round, oval, or irregularly round patches which present
various appearances.

These differences in macroscopic appearance are due,
in some cases, to the presence or absence of color; in
others to a higher or lower degree of moisture ; in some
instances a patch will be glistening and smooth while its
neighbor may be dull and rough or wrinkled; here
will appear an island regularly round in outline and
there an area covered by an irregular ragged deposit.
All of these gross appearances are of value in aiding us
to distinguish between these colonies—for colonies they
are—and under the same conditions the organisms com-

210 BACTERIOLOGY.

posing each of them will always produce growths of ex-
actly the same appearance. It was just such an experi-
ment as this, accidentally performed, that suggested to
Koch a means of separating and isolating from mix-
tures of bacteria the component individuals in pure
cultures, and it was from this observation that the
methods of cultivation on solid media were evolved.

If, without molesting our experiment, we continue the
observation from day to day, we shall notice changes in
the colonies due to the growth and multiplication of the
individuals composing them. In some cases the colo-
nies will always retain their sharply cut, round, or oval
outline, and will increase but little in size beyond that
reached after forty-eight to seventy-two hours, whereas
others will spread rapidly, and will very quickly over-
run the surface upon which they are growing, and in-
deed, grow over the smaller, less rapidly developing
colonies. In a number of instances, if the observation
be continued long enough, many of these rapidly grow-
ing colonies will, after a time, lose their lustrous and
smooth or regular surface and will show, at first here and
there, elevations which will continue to appear until the
whole surface takes on a wrinkled appearance. Again
bubbles may be seen scattered through the colonies.
These are due to the escape of gas resulting from fer-
mentation which the organisms bring about in the
medium upon which they are growing. Sometimes
peculiar odors resulting from the same cause will be
noticed.

Note carefully all these changes and appearances, as
they must be employed subsequently in identifying the
individual organisms from which each colony on the
medium has developed.

EXPOSURE AND CONTACT. 211

If now we examine these points upon our bread or
potato with a hand-lens of low magnifying power we
will be enabled to detect differences not noticeable to the
naked eye. In some cases we shall still see nothing more
than a smooth non-characteristic surface ; while in others,
minute, sometimes regularly arranged, corrugations may
be observed. In one colony they may appear as toler-
ably regular radii, radiating from a central spot; and
again they may appear as concentric rings; and if by
the methods which have been described we obtain from
these colonies their individual components in pure cul-
ture, we shall see that this characteristic arrangement
in folds, radii, or concentric rings, or the production of
color, is under normal conditions constant.

So much for the simplest naked-eye experiment that
can be made in bacteriology, and which serves to furnish
the beginner with material upon which to begin his
studies. It is not necessary at this time for him to
burden his mind with names for these organisms ; it is
sufficient for him to recognize that they are mostly of
different species and that they possess characteristics
which will enable him to differentiate the one from the
other.

In order now for him to proceed it is necessary that
he should have familiarized himself with the methods
by which his media are prepared and the means em-
ployed in sterilizing them and retaining them sterile—
i.e., of preventing the access of foreign germs from
without—otherwise his efforts to obtain and retain his
organisms as pure cultures will be in vain.

EXPposuRE AND Contact.—Make a number of plates
from bits of silk used for sutures, after treating them
as follows :

212 BACTERIOLOGY.

Place some of these pieces (about 5 centimetres long)
into a sterilized test-tube, and sterilize them by steam
for one hour. At the end of the sterilization remove
one piece with sterilized forceps and allow it to brush
against your clothing, then make a plate from it; draw
another piece across the table and then plate it. Sus-
pend three or four pieces upon a sterilized wire hook
and let them hang for thirty minutes free in the air,
being sure that they touch nothing but the hook ; then
plate them separately.

Note the results.

In what way do these experiments differ and how can
the differences be explained ?

Expose to the air six Petri dishes into which either
sterilized gelatin or agar-agar has been poured and allowed
to solidify ; allow them to remain exposed for five, ten,
fifteen, twenty, twenty-five, and thirty minutes, in a
room where no one is at work. ‘Treat a second set in
the same way in a room where several persons are
moving about. Be careful that nothing touches them,
and that they are exposed only to the air. Each dish
must be carefully labelled with the time of its exposure.

Do they present different results? What is the rea-
son for this difference ?

Which predominate, colonies resulting from the
growth of bacteria, or those from common moulds?

How do you account for this condition ?

CHAPTER XV.

Various experiments in sterilization by steam and by hot air.

PLACE in one of the openings in the cover of the steam
sterilizer an accurate thermometer ; when the steam has
been streaming for a minute or two the thermometer will
register 100° C.; wrap in a bundle of towels or rags or
pack tightly in cotton a maximum thermometer ; let this
thermometer be in the centre of a bundle large enough
to quite fill the chamber of the sterilizer. At the end
of a few minutes’ exposure to the streaming steam
remove it; it will be found to indicate a temperature
of 100° C, i

Closer study of the penetration of steam has taught
us, however, that the temperature which is found at the
centre of such a mass may sometimes be that of the
air in the meshes of the material, and not that of
steam, and for this reason the sterilization at that
point may not be complete, because hot air at 100° C.
has not the sterilizing properties that steam at the
same temperature possesses. It it necessary, there-
fore, that this air should be expelled from the meshes
of the material and its place taken by the steam be-
fore sterilization is complete. This is insured by allow-
ing the steam to stream through the substances a few
minutes before beginning to calculate the time of ex-
posure. There is as yet no absolutely sure means of
saying that the temperature at the centre of the mass is

that of hot air or of steam, so that the exact length of
10*

214 BACTERIOLOGY.

time that is required for the expulsion of the air from
the meshes of the material cannot be given.

Determine if the maximum thermometer indicates a
temperature of 100° C. at the centre of a moist bundle
in the same way as when a dry bundle was employed.

To about 50 cc. of bouillon add about one gramme of
chopped hay, and allow it to stand in a warm place for
twenty-four hours. At the end of this time it will be
found to contain a great variety of organisms. Continue
the observation, and a pellicle will be seen to form on
the surface of the fluid. This pellicle will be made up
of rods which grow as long threads in parallel strands.
In many of these rods glistening spores will be seen.
After thoroughly shaking, filter the mass through a fine
cloth to remove coarser particles.

Pour into each of several test-tubes about 10 ce. of
the filtrate. Allow one tube to remain unmolested in
a warm place. Place another in the steam sterilizer for
five minutes; a third for ten minutes; a fourth for
one-half hour; a fifth for one hour.

At the end of each of these exposures inoculate a tube
of sterilized bouillon from each tube. Likewise make
a set of plates or Esmarch tubes upon both gelatin and
agar-agar from each tube, and note the results. At the
same time prepare a set of plates or Esmarch tubes on
agar-agar and on gelatin from the tube which has not
been exposed to the action of the steam.

The plates or tubes from the unmolested tube will
present colonies of a variety of organisms ; separate and
study these.

Those from the tube which has been sterilized for five
minutes will present colonies in moderate numbers, but,

STEAM AND HOT-AIR STERILIZING. -915

as a rule, they will represent but a single organism.
Study this organism in pure cultures.

The same may be predicted for the tube which has
been heated for ten minutes, though the colonies will be
fewer in number.

The thirty-minute tube may or may not give one or
two colonies of the same organism.

The tube which has been heated for one hour is
usually sterile.

The bouillon tubes from the first and second tubes
which were heated will usually show the presence of
only one organism—the bacillus which gave rise to the
pellicle-formation in our original mixture. This organ-
ism is the bacillus subtilis, and will serve as an object
upon which to study the difference in resistance toward
steam between the vegetative and spore stages of the
same organism.

Inoculate about 100 c.c. of sterilized bouillon with a
very small quantity of a pure culture of this organism,
and allow it to stand in a warm place for about six
hours. Now subject this culture to the action of steam
for five minutes; it will be seen that sterilization, as a
rule, is complete.

Treat in the same way a second flask of bouillon, in-
oculated in the same way with the same organism, but
after having stood in a warm place for from forty-eight
to seventy-two hours, that is, until the spores have
formed, and it will be found that sterilization is not
complete—the spores of this organism have resisted
the action of steam for five minutes.

To determine if sterilization is complete always resort
to the culture methods, as the macroscopic and micro-
scopic methods are deceptive; cloudiness of the media

216- BACTERIOLOGY.

or the presence of organisms microscopically does not
always signify that the organisms possess the property
of life.

Inoculate in the same way a third flask of bouillon
with a very small drop from one of the old cultures
upon which the pellicle has formed ; mix it well and
subject it to the action of steam for two minutes; then
place it to one side for from twenty to twenty-four
hours, and again heat for two minutes ; allow it to stand
for another twenty-four hours, and repeat the process on
the third day. No pellicle will be formed, and yet spores
were present in the original mixture, and, as we have
seen, the spores of this organism are not killed by an
exposure of five minutes to the steam. How can this
result be accounted for?

Saturate several pieces of cotton thread, each about
2 cm. long, in the original decomposed bouillon, and dry
them carefully at the ordinary temperature of the room,
then at a little higher temperature—about 40° C.—to
complete the process. Regulate the temperature of the
hot-air sterilizer for about 100° C., and subject several
pieces of this infected and dried thread to this tempera-
ture for the same lengths of time that we exposed the
same organisms in bouillon to the steam, viz. : five, ten,
thirty, and sixty minutes. At the end of each of these
periods remove a bit of thread, and prepare a set of
plates or Esmarch tubes from it. Are the results anal-
ogous to those obtained when steam was employed ?

Increase the temperature of the dry sterilizer and
repeat the process. Determine the temperature and
time necessary for the destruction of these organisms
by the dry heat. These threads should not be simply

STEAM AND HOT-AIR STERILIZING. - 217

laid upon the bottom of the sterilizer, but should be sus-
pended from a glass rod, which may be placed inside
the oven, extending across its top from one side to the
other.

Place several of the infected threads in the centre of
a bundle of rags. Subject this to a temperature neces-
sary to sterilize the threads by the dry method. Treat
another similar bundle to sterilization by steam. In
what way do the two processes differ ?

CHAPTER XVI.

Suppuration—The staphylococcus pyogenes aureus—Staphylococcus pyo-
genes albus and citreus—Streptococcus pyogenes—Bacillus pyocyanus—Gen-
eral remarks.

PREPARE from the pus of an acute abscess or boil,
that has been opened under antiseptic precautions, a
set of plates of agar-agar. Care must be taken that
none of the antiseptic fluid gains access to the culture
tubes, otherwise its antiseptic effect may be seen and
the development of the organisms interfered with. It
is best, therefore, to take up a drop of the pus upon the
platinum-wire loop after it has been flowing for a few
seconds ; even then it must be taken from the mouth
of the wound and before it has run over the surface of
the skin. At the same time prepare two or three cover-
slips from the pus.

Microscopic examination of these slips will reveal
the presence of a large number of pus-cells, both multi-
nucleated and with horseshoe-shaped nuclei, some
threads of disintegrated and necrotic connective tissue,
and, lying here and there throughout the preparation,
small round bodies which will sometimes appear-_singly,
sometimes in pairs, and frequently will be seen grouped
together somewhat like clusters of grapes. (See Fig.
45.) They stain readily and are commonly located in
the material between the pus-cells; very rarely they
may be seen in the protoplasmic body of the cell.
(Compare the preparation with a similar one made
from the pus of gonorrhea—see Fig. 46. In what

SUPPURATION. 219

Fie. 45,

Preparation from pus, showing pus-cells, A, and staphylococci, C

way do the two preparations differ the one from the

other ?)
After twenty-four hours in the incubator the plates

will be seen to be studded here and there with yellow or

Fic. 46.
: cae
ge? fe
: ? gr
ay 7
Cisse 2
to BT Mame
A bb pe, Ge :

Pus of gonorrhcea, showing diplococci in the bodies of the pus-cells.

orange-colored colonies, which are usually round, moist,
and glistening in their naked-eye appearances. When
located in the depths of the medium they are commonly

220 BACTERIOLOGY.

seen to be lozenge or whetstone in shape, while often
they appear as irregular stars with blunt points, and
again as irregularly lobulated dense masses. In struc-
ture they aré conspicuous for their density. Under the
low objective they appear, when on the surface, as
coarsely granular, irregularly round patches, with more
or less ragged borders and a dark irregular central
mass, which has somewhat the appearance of masses of
coarser clumps of the same material as that composing
the rest of the colony. Microscopically, these colonies
are composed of small round cells, irregularly grouped
together. They are in every way of the same appear-
ance as those seen upon the original cover-slip prepa-
rations.

Prepare from one of these colonies a pure stab culture
in gelatin. After thirty-six to forty-eight hours lique-
faction of the gelatin along the track of the needle, and
most conspicuous at its upper end, will be observed.
As growth continues the liquefaction becomes more or
less of a stocking-shape, and gradually widens out at its
upper end into an irregular funnel. This will continue
until the whole of the gelatin in the tube eventually
becomes fluid. There can always be noticed at the
bottom of the liquefying portion an orange-colored or
yellow mass composed of a number of the organisms
which have sunk to the bottom of the fluid.

On potato the growth is quite luxuriant, appearing
as a brilliant orange-colored layer, somewhat lobulated
and a little less moist than when growing upon agar.
It does not produce fermentation with gas-production.
It belongs to the group of facultative aérobes.

In milk it rapidly brings about coagulation with
acid reaction.

SUPPURATION, 221

a

It is not motile, and being of the family of micro-
cocci, does not form endogenous spores. It possesses,
however, a marked resistance toward detrimental
agencies.

In bouillon it causes a diffuse clouding, and after a
time presents a yellow sedimentation.

This organism is the commonest of the pathogenic
bacteria with which we shall meet. It is the staphylo-
coccus pyogenes aureus, and is the organism most
frequently concerned in the production of acute, cir-
cumscribed, suppurative inflammations. It is almost
everywhere present, and is the organism most dreaded
by the surgeon.

In studying its effects upon lower animals a number
of points are to be remembered. While it is the etio-
logical factor in the production of most of the suppura-
tive processes in man, still it is with no little difficulty
that these conditions can be reproduced in the lower
animals. Its subcutaneous introduction into their tis-
sues does not always result in abscess-formation, and
when it does, there seems to have been some coincident
interference with the circulation in these tissues which
renders them less able to resist its inroads. When
introduced‘ into the great serous cavities of the lower
animals it is not always followed by the production of
inflammation. If the abdominal cavity of a dog, for
example, be carefully opened so as to make as slight a
wound as possible, and no injury be done to the intes-
tines, large quantities of bouillon cultures or watery
suspensions of this organism may, and repeatedly have
been introduced into the peritoneum without the slight-
est injury to the animal. On the contrary, if some
substance which acts as a direct irritant to the intes-

222 BACTERIOLOGY.

tines—such, for example, as a small bit of potato upon
which the organisms are growing—is at the same time
introduced, or the intestines be mechanically injured,
so that there is a disturbance in their circulation, then
the introduction of these organisms is promptly fol-
lowed by acute and fatal peritonitis. (Halsted.)

On the other hand, the results which follow their
introduction into the circulation are practically constant.
If one injects into the circulation of the rabbit through
one of the veins of the ear, or in any other way, from
0.1 to 0.3¢.¢ of a bouillon culture or watery suspension
of a virulent variety of this organism, a fatal pyeemia
always follows in from two and one-half to three days.
A few hours before death the animal is frequently seen
to have severe convulsions. Now and then excessive
secretion of urine is noticed. The animal may appear
in moderately good condition until from eight to ten
hours before death. At the autopsy a typical picture
presents ; the voluntary muscles are seen to be marked
here and there by yellow. spots, which average the size
of a flaxseed, and are of about the same shape. They
lie usually with their long axis running longitudinally
between the muscle fibres. As the abdominal and
thoracic cavities are opened the diaphragm is often seen
to be studded by them. Frequently the pericardial sac
is distended with a clear gelatinous fluid, and almost
constantly the yellow points are to be seen in the
myocardium. The kidneys are rarely without them ;
here they appear on the surface scattered about as
single yellow points, or again, are seen as conglomerate
masses of small yellow points which occupy, as a rule,
the area fed by a single vessel. If one makes a section
into one of these yellow points it will be seen to extend

STUDY OF COVER-SLIPS AND SECTIONS. 993

deep down through the substance of the kidney as a
yellow, wedge-shaped mass, the base of the wedge being
at the surface of the organ.

Itis very rare that these abscesses—for abscesses these
yellow points are, as we shall see when we come to
study them more closely—are found either in the liver,
spleen, or brain ; their usual location being, as said, in
the kidney, myocardium, and voluntary muscles.

These minute abscesses contain a dry, cheesy, necrotic
centre, in which the staphylococci are present in large
numbers, as may be seen upon cover-slips prepared
from them. They may also be obtained in pure culture
from these suppurating foci.

Preserve in Miiller’s fluid and in alcohol duplicate
bits of all the tissue in which the abscesses are located.

When these tissues are hard enough to cut, sections
should be made through the abscess-points, and the
histological changes carefully studied.

Microscopic StuDY OF COVER-SLIPS AND SECTIONS.
—TIn cover-slip preparations this organism stains readily
with the ordinary dyes.

In tissues, however, it is best toemploy some method
by means of which contrast stains may be employed
and the location and grouping of the organisms in the
tissues rendered more conspicuous.

When stained, sections of tissues containing these
small abscesses present the following appearances :

To the naked eye will be seen here and there in the
section, if the abscesses are very numerous, small, darkly
stained areas which range in size from that of a pin-
point up to those having a diameter of from 1 to 2 mm.
These points, when in the kidney, may be round or oval
in outline, or may appear wedge-shaped, with the base

224 BACTERIOLOGY.

of the wedge toward the surface of the organ. The
differences in shape depend frequently upon the direc-
tion in which the section has been made through the
kidney. In the muscles they are irregularly round or
oval.

When quite small they appear to the naked eye as
simple, round, or oval, darkly stained points, but when
they are more advanced a pale centre can usually be
made out.

When magnified, they appear in the earliest stages
as minute aggregations of small cells, the nuclei of
which stain intensely. Almost always there can be
seen about the centre of these cell-accumulations evi-
dences of progressing necrosis. The normal structure
of the cells of the tissue will be more or less destroyed ;
there will be seen a granular condition due to cell-frag-
mentation ; at different points about the centre of this
area the tissue will appear cloudy and the tissue-cells
will not stain readily. All about and through this spot
will be seen the nuclei of pus-cells, many of which are
undergoing disintegration. In the smallest of these
beginning abscesses the staphylococci are to be seen
scattered about the centre of the necrotic tissue, but in
a more advanced stage they are commonly seen massed
together in very large numbers in the form commonly
referred to as emboli of micrococci.

The localized necrosis of the tissues which is seen at
the centre of the abscess is the direct result of the
action of a poison produced by the bacteria, and is the
starting-point for all abscess-formations.

When the process is somewhat advanced the different
parts of the abscess are more easily detected. They then
present in sections somewhat the following conditions :

STUDY OF COVER-SLIPS AND SECTIONS. 995

At the centre can be seen a dense, granular mass, which
stains readily with the basic aniline dyes and, when
highly magnified, is found to be made up of staphy-
lococci. Sometimes the shape of this mass of staphylo-
cocci corresponds to that of the capillary in which the
organisms became lodged and developed. Immediately
about the embolus of cocci the tissues are seen to be in
an advanced stage of necrosis. Their structure is
almost completely destroyed, though it is seen to be
more advanced in some of the elements of the tissues
than in others. As we approach the periphery of this
faintly stained necrotic area, it becomes marked here
and there with granular bodies, irregular in size and
shape, which stain in the same way as do the nuclei of
the pus-cells and represent the result of disintegration
going on in these cells.

Beyond this we come upon a dense, deeply stained
zone, consisting of closely packed pus-cells; of granular
detritus resulting from destructive processes acting upon
these cells ; and of the normal cellular and connective-
tissue elements of the part. Here and there through
this zone will be seen localized areas of beginning death
of the tissues. This zone gradually fades away into the
healthy surrounding tissues. It constitutes the so-called
“abscess-wall,”’

Such is the picture presented by the miliary abscess
when produced experimentally in the rabbit, and it
corresponds throughout with the pathological changes
which accompany the formation of larger abscesses in
the tissues of human beings.

From these small abscesses in the tissues of the rabbit
the staphylococcus pyogenes aureus may again be ob-
tained in pure culture, and will present identically the

226 BACTERIOLOGY.

same characteristics that were possessed by the culture
with which the animal was inoculated.

Tue Less Common PyocEenic ORGANISMS.—
The pus of an acute abscess in the human being may
sometimes contain other organisms beside the staphylo-
coccus pyogenes aureus. The staphylococcus pyogenes
albus and citreus may be found. The colonies of the
former are white, those of the latter are lemon-color.
With these exceptions they are in all essential cultural
peculiarities similar to the staphylococcus aureus. As
a rule they are not virulent for animals, and when they
do possess pathogenic properties, it is in a much lower
degree than is commonly the case with the golden
staphylococcus. The streptococcus pyogenes is also
sometimes present. The commonest of the pyogenic
organisms however is that just described—the staphylo-
coccus pyogenes aureus.

Tue Srrerrococcus PyoGENEs.—From a spread-
ing phlegmonous inflammation prepare cover-slips and
cultures. What is the predominating organism? Does
it appear as irregular clusters of grapes, or has its in-
dividuals a definite regular arrangement? Are its
colonies like those of the staphylococcus pyogenes
aureus ?

Isolate this organism in pure cultures. | In these cul-
tures it will be found on microscopic examination to
present an arrangement somewhat like a chain of beads.
(Fig. 47.)

Determine its peculiarities and describe them accu-
rately. They should be as follows:

Upon microscopic examination a micrococcus should
be found, but differing in its arrangement from the
staphylococci just described. The single cells are not

THE STREPTOCOCCUS PYOGENES. 997

scattered irregularly or arranged in clumps similar to
bunches of grapes, but are joined together in chains like
strands of beads. These strands are sometimes regular
in the arrangement and size of the individual cells com-
posing them, but more commonly certain irregular parts
may be seen in them. Here they appear as if two or

Streptococcus pyogenes.

three cells had fused together to form a link, so to
speak, in the chain that is somewhat longer than the
remaining links; again, portions of the chain may be
thinner than the rest, or may appear broken or ragged.
Commonly the individuals comprising this chain of cocci
are not round, but appear flattened on the sides adjacent
to one another, The chains are sometimes short, con-
sisting of four to six cells, or again they may be much
longer, and extend from a half to two-thirds across the
field of the microscope.

Under artificial conditions it sometimes grows well,
and can be cultivated through many generations, while
again it rapidly loses its vitality. When isolated from
the diseased area upon artificial media it seems to retain
its vitality for a longer period if replanted upon fresh
media every day or two for a time; but if the first
generation is not treated in this way, but allowed to re-

228 BACTERIOLOGY.

main upon the original medium, it is not uncommon to
find the organism incapable of further cultivation after
a week or ten days. :

Under no conditions is the growth of this organism
very luxuriant.

On gelatin plates its colonies appear after forty-eight
to seventy-two hours as very small, flat, round points, of
a bluish-white or opalescent appearance. They do not
cause liquefaction of the gelatin, and in size they rarely
exceed 0.6—-0.8 mm. in diameter. Under low magnify-
ing power they have a brownish or yellowish tinge by
transmitted light, and are finely granular. As the
colonies become older their regular border may become
slightly irregular or notched.

In stab cultures in gelatin they grow along the entire
needle-track as a finely granular line, the granules re-
presenting minute colonies of the organism. On the
surface the growth does not usually extend beyond the
point of puncture.

On agar-agar plates the colonies appear as minute
pearly points, which when slightly magnified are seen
to be finely granular, of a light-brownish color, and
regular at their margins.

When smeared upon the surface of agar-agar or gela-
tin slants the growth that results is a-thin, pearly,
finely granular layer, consisting of minute colonies
growing closely side by side. Its growth is most
luxuriant on glycerin agar-agar at the temperature of
the incubator (37.5° C.), and least on gelatin.

On blood-serum its colonies present little that is
characteristic ; they appear as small, moist, whitish
points, from 0.6 to 0.8 mm. in diameter, that are
slightly elevated above the surface of the serum. They

THE STREPTOCOCCUS PYOGENES. 9229

do not coalesce to form a layer over the surface, but
remain as isolated colonies.

On potato no visible development appears, but after
a short time (thirty-six to seventy-two hours) there is a
slight increase of moisture about the point inoculated,
and microscopic examination shows that a multiplication
of the organisms placed at this point has occurred.

In milk its conduct is not always the same, some
cultures causing a separation of the milk into a firm
clot and colorless whey, while others do not produce this
coagulation. The latter, when cultivated in milk of a
neutral or slightly alkaline reaction, to which a few
drops of litmus tincture has been added, produce a very
faint pink color after twenty-four hours at 37.5° C.;
this change in color is not apparently due to the pro-
duction of acidity, as there is no coagulation, and the
reaction of the milk when tested by delicate litmus or
by curcuma paper is sometimes seen to be still slightly
alkaline, and to remain so for several weeks.

In bouillon it grows as tangled masses or clumps,
which upon microscopic examination are seen to consist
of long chains of cocci twisted or matted together.

It grows best at the temperature of the body (37.5°
C.), and develops, but less rapidly, at the ordinary room
temperature.

It is a facultative anaérobe.

It stains with the. ordinary aniline dyes, and is not
decolorized when subjected to Gram’s method.

It is not motile, and, being a micrococcus, does not
form spores. Under artificial conditions we have no
reason to believe that it enters a stage where its
resistance to detrimental agencies is increased. In

the tissues of the body, however, it appears to possess a
11

230 BACTERIOLOGY.:

marked tenacity to vitality, for it is not rare to ob-
serve recurrences of inflammatory conditions due to this
organism, often at a relatively long time after the
primary site of infection is healed :

When introduced into the tissues of lower animals its
effects are uncertain. Rosenbach and Passet claimed
that protracted, progressive, erysipelatoid inflammations
were produced, and Fehleisen, who described a strepto-
coccus in erysipelas that is in all probability identical
with the streptococcus pyogenes now under considera-
tion, stated that it produced in the tissues of rabbits
(the base of the ear) a sharply defined migratory red-
dening without pus-formation. Asa rule, it is difficult
to obtain any definite pathological alterations in the tis-
sues of animals through the introduction into them of
cultures of this organism by any of the methods of in-
oculation ordinarily practised.

This is the streptococcus pyogenes, and is the organ-
ism most commonly found in rapidly spreading suppu-
ration in contradistinction to the staphylococcus pyogenes
aureus, which is most frequently found in circumscribed
abscess-formations ; they may be found together.

If the opportunity presents, obtain cultures from a
case of erysipelas. Compare the organism thus obtained
with the streptococcus just mentioned. Inoculatea rabbit
both subcutaneously and into the circulation with about
0.2 cc. of a pure culture of these organisms in bouillon.
Do the results correspond, and do they in any way
suggest the results obtained with the staphylococcus
pyogenes aureus when introduced into animals in the
same way? Do these streptococci flourish readily on
ordinary media?

The organisms that have just been described are com-

THE STREPTOCOCCUS PYOGENES. 231

monly known as the “pyogenic cocci” of Ogston,
Rosenbach, and Passet, and up to as late as 1885 were
believed to be the specific factors concerned in the pro-
duction of suppurative inflammations. Since that time,
however, considerable modification of this view has
taken place, and while they are still known to be the
most common causes of suppuration, they are also known
not to be the only causes of this process.

With the more general application of bacteriological
methods to the study of the manifold conditions coming
under the eye of the physician, the surgeon, and the
pathologist, results are constantly being obtained that
do not accord with the view formerly held in regard to
the specific relation of the pyogenic cocci to all forms
of suppuration. There is an abundance of evidence
now at command to justify the opinion that there are
a number of organisms not commonly classed as pyo-
genic which may, under peculiar circumstances, assume
this property. For example:

The bacillus of typhoid fever has been found in pure
culture in osteomyelitis of the ribs; in acute purulent
otitis media ; in abscess of the soft parts; in the pus of
empyema, and in localized fibro-peritonitis, either dur-
ing its course or as a sequela of typhoid fever.

The bacterium coli commune has been found to be pres-
ent in pure culture in acute peritonitis ; in liver abscess ;
in purulent inflammation of the gall-bladder and ducts;
in appendicitis; and Welch’ has found it in pure cul-
ture in fifteen different inflammatory conditions.

The micrococcus lanceolatus (pneumococcus) has been
found to be the only organism present in abscess of the

1 Welch: ‘Conditions Underlying the Infection of Wounds,’’ American
Journal of the Medical Sciences, November, 1891.

232 BACTERIOLOGY.

soft parts; in purulent infiltration of the tissues about
a fracture; in purulent cerebro-spinal meningitis ; in
suppurative synovitis ; in acute pericarditis, and in acute
inflammation of the middle ear.

Moreover, many of the less common organisms have
been detected in pure cultures in inflammatory con-
ditions with which they were not previously thought to
be concerned, and to which they are not usually related
etiologically.

In consideration of such evidence as this, it is plain
that we can no longer adhere rigidly to the opinions
formerly held upon the etiology of suppuration, but
must subject them to modifications in conformity with
this newer evidence. We now know that there exist
bacteria other than the “ pyogenic cocci,” which, though
not normally pyogenic, may give rise to tissue changes
indistinguishable from those produced by the ordinary
pus organisms.’

THE BACILLUS PYOCYANUS (BACILLUS OF GREEN PUS).

Another common organism that may properly be
mentioned at this place, though perhaps not strictly
pyogenic, is a bacillus frequently found in discharges
from wounds, viz., the bacillus pyocyanus, or bacillus of
green pus, or of blue pus, or of blue-green pus, as it
is commonly called. The bacillus pyocyanus is a
delicate rod with rounded or pointed ends. It is
actively motile; does not form spores. As seen in
preparations made from cultures it is commonly clus-

1 Fora more detailed discussion of the subject, see ‘‘The Factors Concerned
in the Productlon of Suppuration,” International Med. Mag., Phila., May,
1892,

THE BACILLUS PYOCYANUS. 933

tered together in irregular masses. It does not form
long filaments, there being rarely more than four joined
together end to end, and most frequently not even two.

It grows readily on all artificial media, and gives to
some of them a bright-green color, that is most con-
spicuous where it is in contact with the air. This green
color is not seen in the growth itself to any extent, but
is diffused through the medium on which the organism
“is developing. With time this color becomes much
darker, and in very old agar cultures may become
almost black (sometimes very dark blue-green, at others
brownish-black).

Its growth on gelatin in stab cultures is accompanied
by liquefaction, and the diffusion of a bright-green color
throughout the unliquefied medium. As liquefaction
continues and the entire gelatin ultimately becomes
fluid, the green color is confined to the superficial
layers that are in contact with the air. The form
taken by the liquefying portion of the gelatin in the
earliest stages of development is somewhat that of an
irregular, slender funnel. (See Fig. 48.)

On gelatin plates the colonies develop rapidly ; they
are not sharply circumscribed, but usually present at
first a fringe of delicate filaments about their periphery
(see Fig. 49); as growth progresses and liquefaction be-
comes more advanced the central mass of the colony
sinks into the liquefied depression, while at the same
time there is an extension of the colony laterally. At
this stage the colony, when slightly magnified, may pre-
sent various appearances, the most common being that
shown in Fig. 50.

The gelatin between the growing colonies takes on a
bright yellowish-green color, but as growth is compar-

234. BACTERIOLOGY.

atively rapid, it is quickly entirely liquefied, and one
often sees the colonies floating about in the pale-green
fluid.

Fie. 49.

Colony of b. pyocyanus after twenty-four
hours on gelatin at 20°-22° C,

Fie. 50.

Stab culture of b.
pyocyanus in gel-
atin after twenty- Colony of b. pyocyanus after forty-two hours
eight hours at 22°C. on gelatin at 20°-22° C,

On agar-agar the growth is dry, sometimes with a
slight metallic lustre, and is of a whitish or greenish-
white color, while the surrounding agar is bright green.
With time this bright green becomes darker, passing to
blue-green, and finally turns almost black.

On potato the growth is brownish, dry, and slightly
elevated above the surface. With some cultures the
potato about the growth becomes green; with others
this change is not so noticeable. With many cul-

THE BACILLUS PYOCYANUS. 935

tures a peculiar phenomenon may be produced by
lightly touching the growth with a sterilized platinum
needle. This phenomenon consists in a change of color
from brown to green at the point touched. It is best
seen in cultures that have been kept in the incubator
for from seventy-two to ninety-six hours. It occurs
in from one to three minutes after touching with the
needle, and may last from ten minutes to half an hour.
This is the “chameleon phenomenon” of Paul Ernst.

In bouillon the green color appears, and the growth
is seen in the form of delicate flocculi. A very delicate
mycoderma is also produced.

In milk it causes an acid reaction, with coincident
coagulation of the casein. :

On blood serum and egg albumin its growth is accom-
panied by liquefaction. The growth on coagulated egg
albumin is seen as a dirty-gray deposit surrounded by a
narrow brownish zone; the remaining portion of the
medium is bright green in color. As the culture becomes
older the green may give way to a brown discoloration.

In peptone solution (double strength) it causes a
bluish-green color. In one of four cultures from dif-
ferent sources there was only a blue color produced.

It produces indol.

It stains with the ordinary dyes, and its flagella may
be readily demonstrated by Loffler’s method of staining.

Inoculation into animals. As a rule, cultures of this
organism obtained directly from the discharges of a
wound are capable, when introduced into animals, of
lighting up diseased conditions; but cultures that are
kept on artificial media for a long time may in part,
or completely, lose this power.

When guinea-pigs or rabbits are inoculated subcuta-

236 BACTERIOLOGY.

neously with 1 cc. of virulent fluid cultures of this
organism death usually results in from eighteen to
thirty-six hours. At the seat of inoculation there is
found an extensive purulent infiltration of the tissues
and a marked zone of inflammatory cedema.

When introduced directly into the peritoneal cavity
the results are also fatal, and at autopsy a genuine
fibrinous peritonitis is found. There is usually an
accumulation of serum in both the peritoneal and pleural
cavities. At autopsies after both methods of inoculation
the organisms will be found in the blood and internal
viscera in pure cultures.

When animals are inoculated with small doses (less
than 1 ce. of a bouillon culture) of this organism, death
may not ensue, and only a local inflammatory reaction
(abscess formation) may be set up. In these cases the
animals are usually protected against subsequent inocu--
lation with doses that would otherwise prove fatal.

Most interesting in connection with the bacillus pyo-
cyanus is the fact, as brought out in the experiments of
Bouchard, and of Charrin and others, that its products
possess the power of counteracting the pathogenic activ-
ities of the bacillus anthracis. That is to say, if an
animal is inoculated with a virulent anthrax culture,
and soon after is inoculated with a culture of the bacillus
pyocyanus, the fatal effects of the former inoculation
may be prevented.

In the literature upon the green-producing organisms
that have been found in inflammatory conditions, sev-
eral varieties—believed to be distinct species—have been
described, but when cultivated side by side their bio-
logical differences are seen to be so slight as to render it
probable that they are but modifications of one and the
same species.

CHAPTER XVII.

Sputum septicemia—Septiceemia resulting from the presence of the micro-
coccus tetragenus in the tissues—Tuberculosis.

OsraIn from a tuberculous patient a sample of fresh
sputum—that of the morning is preferable. Spread it
out in a thin layer upon a black glass plate and select
one of the small, white, cheesy masses or dense mucous
clumps that will be seen scattered through the sputum.
With a pointed forceps smear it carefully upon two
or three thin cover-slips, dry and fix them in the
way given for ordinary cover-slip preparations. Stain
one in the ordinary way with Loffler’s alkaline methyl-
lene blue solution, the other by the Gram method, the
third after the method given for tubercle bacilli in fluids
or sputum.

In that stained by Loffler’s method—slip No. 1—
will be seen a great variety of organisms—round cells,
ovals, short and long rods, perhaps spiral forms. But
not infrequently will be seen diplococci, having more
or less of a lancet shape; they will be joined together
by their broad ends, the points of the lancet being away
from the point of juncture of the two cells. There may
also be seen masses of cocci which are conspicuous for
their arrangement into groups of fours, the adjacent
surfaces being somewhat flattened. They are not sar-
cina, as one can see by the absence of the division in
the third direction of space—they divide only in two

directions.
11*

238 BACTERIOLOGY.

In the slip stained by the Gram method the same
groups of the cocci which grow as threes and fours will
be seen, but our lancet-shaped diplococci will now pre-
sent an altered appearance—there can now be detected
a capsule surrounding them. This capsule is very
delicate in structure, and though a frequent accompani-
mentis notconstant. Itcan sometimes be demonstrated
by the ordinary methods of staining, though the method
of Gram is most satisfactory. (Fig. 52.)

In the third slip, which has been stained by the
method given for tubercle bacilli in sputum, if de-
colorization has been properly conducted and no con-
trast stain has been employed, the field will be color-
less or of only a very pale rose color. None of the
numerous organisms seen in the first slip can now be
detected, but instead there will be seen scattered through
the field very delicate stained rods, which present, in
most instances, a conspicuous beaded arrangement of
their protoplasm—that is, the staining is not homoge-
neous, but at tolerably regular intervals along each rod
there is seen alternating intervals of light and color.
These rods may be found singly, in groups of twos or
three, or sometimes in clumps consisting of large num-
bers. When in twos or threes it is not uncommon to
find them describing an X or a V in their mode of ar-
rangement, or again they will be seen lying parallel the
one to the other.

If contrast stains are used, these rods will be detected
and recognized by their retaining the original color with
which they have been stained, whereas all other bac-
teria in the preparation, as well as the tissue-cells which
are in the sputum, will take up the contrast color.
(Fig. 51.)

INOCULATION OF SPUTUM. 939

These delicate beaded rods are the bacillus tubercu-
losis. The lancet-shaped diplococci with the capsule
are the micrococcus lanceolatus.

Fie. 51,

Tuberculous sputum stained by Gabbet’s method. Tubercle bacilli seen as
red rods; all else is stained blue.

The cocci grouped in fours are the micrococcus tetra-
genus.

InocuLaTion EXPERIMENT. — Inoculate into the
subcutaneous tissues of a guinea-pig one of the small
white caseous masses similar to that which has been
examined microscopically. If death ensues it will be
the result of one of the three following forms of
infection :

a. Septiceemia’ resulting from the introduction into
the tissues of an organism frequently present in the
sputum. It exists under the various names: micro-
coccus of sputum septicemia; diplococcus pneumonie ;
pneumococeus of Frankel; meningococcus ; strepto-

1 Septiceemia is that form of infection in which the blood is the chief field
of activity of the organisms.

240 BACTERIOLOGY.

coccus lanceolatus Pasteuri; micrococcus lanceolatus ;
micrococcus Pasteuri; coccus lanceolatus ; bacillus sali-
varius septicus; bacillus septicus sputigenous; diplo-
coccus lanceolatus capsulatus ; micrococcus pneumoniz
croupose.

b. A form of septicemia resulting from the invasion
of the tissues by an organism frequently seen in the
sputum of tuberculous subjects. It is characterized by
its tendency to divide into fours. It is the micrococcus
tetragenus.

c. General or local tuberculosis.

a. SPUTUM SEPTICAMIA.

Tf at the end of twenty-four to thirty-six hours the
animal is found dead, we may safely suspect that the
result was produced by the introduction into the tissues
of the organism of sputum septicemia above mentioned,
viz., the micrococcus lanceolatus, which is not uncom-
monly found in the mouth of healthy individuals as
well as in other conditions.

Inspection of the seat of inoculation usually reveals
a local reaction. “This may be of a serous, fibrinous,
hemorrhagic, necrotic, or purulent character. Fre-
quently we may find combinations of these conditions,
such as fibro-purulent, fibrino-serous, or sero-hemor-
rhagic.”* The most conspicuous naked-eye change
undergone by the internal organs will be enlargement
of the spleen. It is usually swollen, but may at times
be normal in appearance. It is sometimes hard, dark
red, and dry, or it may be soft and rich in blood. Fre-
quently there is a limited fibrinous exudation over por-
tions of the peritoneum.

’ Welch : Johns Hopkins Hospital Bulletin, December, 1892, vol, iii, No. 27.

SPUTUM SEPTICEMIA, 241

Except in the exudations, the organisms are found
only in the lumen of the bloodvessels, where they are
usually present in enormous numbers..

In the blood they are practically always free and are
but rarely found within the bodies of leucocytes.

In stained preparations from the blood and exudates
a capsule is not infrequently seen surrounding the
organisms. (Fig. 52.) This, however, is not constant.

Fie. 52,

nH

Micrococcus lanceolatus in blood of rabbit. Stained by method of Gram.
Decolorization not complete.

If a drop of blood from this animal be introduced
into the tissues of a second animal (mouse or rabbit)
identically the same conditions will be reproduced.

If the organism be isolated from the blood of the
animal in pure culture, and a portion of this culture be
introduced into the tissues of a susceptible animal, again
we shall see the same pathological picture.

It must be remembered, however, that this organism
when cultivated for a time on artificial media rapidly
loses its virulent properties. If, therefore, failure to re-
produce the disease after inoculation from old cultures

242 BACTERIOLOGY.

should occur, it is in all probability due to a disappear-
ance of virulence from the organism.

This organism was discovered by Sternberg in 1880.
It was subsequently described by A. Frankel as the
etiological factor in the production of acute fibrinous
pneumonia.

It is not uncommonly present in the saliva of healthy
individuals, having been found by Sternberg in the oral
cavity of about 20 per cent. of healthy persons examined
by him. It is constantly to be detected in the rusty
sputum of patients suffering from acute fibrinous pneu-
monia. Its presence has been detected in the middle
ear, in the pericardial sac, in the pleura, in the serous
cavities of the brain, and indeed it may penetrate from
‘its primary seat in the mouth to almost any of the
more distant organs.

The organism is commonly found as a diplococcus,
though here and there short chains of four to six indi-
viduals joined together may be detected. (Fig. 51,
page 239.) The morphology of the individual célls is
more or less oval, or, more strictly speaking, lancet-
shaped, for at one end it is commonly pointed. When
joined in pairs the junction is always between the
broad ends of the ovals, never between the pointed
extremities.

As already stated, in preparations directly from the
sputum or from the blood of animals, a delicate cap-
sule may frequently be seen surrounding them. Though
fairly constant in preparations directly from the blood
of animals and from the sputum or lungs of pneumonic
patients, the capsule is but rarely observed in artificial
cultures. Occasionally in cultures on blood-serum, in
milk, and on agar-agar they can, according to some

SPUTUM SEPTICZMIA, 943

authors, be detected ; but this is by no means constant
or frequent.

This organism grows under artificial conditions very
slowly, and frequently not at all.

When successfully grown upon the different media it
presents somewhat the following appearance :

On gelatin it grows very slowly, if at all, probably
owing in part to the low temperature at which gelatin
cultures must be kept. If development occurs it ap-
pears as minute whitish or blue-white points on the
plates. These very small colonies are round, finely
granular, sharply circumscribed, and slightly elevated
above the surface of the gelatin. The growth is very
slow and no liquefaction of the gelatin accompanies it.

If grown in slant or stab cultures the surface devel-
opment is very limited; along the needle-track tiny
whitish or bluish-white granules appear.

On nutrient agar-agar the colonies are almost trans-
parent; they are more or less glistening and very deli-
cate in structure. On blood-serum development is
more marked, though still extremely feeble. Here it
also appears as a cluster of isolated fine points growing
closely side by side.

A growth on potato is not usually observed. When
grown in milk it commonly causes an acid reaction
with coincident coagulation of the casein. Some
varieties, especially non-virulent ones, do not coagulate
milk.’ It is not motile.

It grows best at a temperature of from 35° to 38° C.
Under 24° C. there is usually no development, but in
a few cases it has been seen to grow at as low a tem-

12 Welch, loc. cit.

244 BACTERIOLOGY.

perature as 18° C. From 42° C. on the development is
checked.

Under most favorable conditions the growth is very
slow. It grows as well without as with oxygen. It
is, therefore, one of the facultative anaérobic forms.

The most successful efforts at the cultivation of this
organism are those seen when the agar-gelatin mixture
of Guarniari is employed. (See this medium.)

It may be stained with the ordinary aniline staining
reagents. For demonstration of the capsule the method
of Gram gives the best results. (See Stainings.)

This organism is conspicuous for the irregularity of
its behavior when grown under artificial conditions ;
usually it loses its pathogenic properties after a few
generations ; but again this peculiarity may be retained
for a much longer time. Not rarely it fails to grow
after three or four transplantations on artificial media,
though at times it may be carried through many gen-
erations.

Inoculation into animals. The results of inocula-
tions with pure cultures of this organism are also
conspicuous for their irregularity. Most commonly
when the organism is of full virulence the form of
septicemia just described is produced, but at times
it is found to be totally devoid of pathogenic powers;
between these extremes cultures may be obtained pos-
sessing all variations in the intensity of their disease-
producing properties. The principal pathological con-
ditions that may be produced by this organism by
inoculations into animals, according to the degree of
its virulence, are acute septicemia, spreading inflam-
matory exudations, and circumscribed abscesses. All
three of these conditions may sometimes be produced

MICROCOCCUS TETRAGENUS, 945

by inoculating the same culture into rabbits in varying
amounts.

Rabbits, mice, guinea-pigs, dogs, rats, cats, and sheep
are susceptible to infection by this organism. Chickens
and pigeons are insusceptible. Young animals, as a
rule, are more easily infected than old ones. Rabbits
and mice are the most susceptible of the animals used
for experimental purposes, and in testing the virulence
of a culture it is well to inoculate one of each, for with
the same cultures it sometimes occurs that it may be
virulent for mice and not for rabbits, and vice versa.

If the culture is virulent, intra-vascular or intra-
peritoneal injections into rabbits may produce rapid
and fatal septicemia, while subcutaneous inoculation of
the same material may result in only a localized in-
flammatory process. On the other hand, subcutaneous
inoculation of less virulent cultures may produce a local
process, while intra-venous inoculation may be without
result. This organism is the cause of a number of
pathological conditions in human beings that have not
hitherto been considered as related to one another etio-
logically. It is always present in the inflamed area of
the lung in acute fibrinous or lobar pneumonia; it is
known to cause acute cerebro-spinal meningitis, endo-
and pericarditis, certain forms of pleuritis, arthritis
and peri-arthritis, and otitis media.

b. SEPTICEMIA CAUSED BY THE MICROCOCCUS
TETRAGENUS.

Should the death of the animal not occur within the
first twenty-eight to thirty hours after inoculation, but
be postponed until between the fourth and the eighth

246 BACTERIOLOGY,

day, it may occur as a result of invasion of the tissues
by the organism now to be described, viz., the micro-
coccus tetragenus.

This organism was discovered by Gaffky and was
subsequently described by Koch in the account of his
experiments upon tuberculosis. It is often present in
the saliva of healthy individuals and is commonly
present in the sputum of tuberculous patients. Koch
found it very frequently in the lung cavities of
phthisical patients. It, however, plays no part in the
etiology of tuberculosis.

It is a small round coccus of about 1 , transverse
diameter. It is seen as single cells, joined in pairs and
in threes, but its most conspicuous grouping is in fours,
from which arrangement it takes its name. In prepa-
rations made from cultures of this organism it is not
rare to find, here and there, single bodies which are
much larger than the other individuals in the field.
Close inspection reveals them to be cells in the ini-
tial stage of division into twos and fours. A pecu-
liarity of this organism is that the cells are seen to be
bound together by a transparent gelatinous substance.

When cultivated artificially it grows very slowly.:

Upon gelatin plates the colonies appear as round,
sharply circumscribed, punctiform masses which are
slightly elevated above the surface of the surrounding
medium. Under a low magnifying power they are
seen to be slightly granular and present a more or less
glassy lustre.

The colonies increase but little in size after the third
or fourth day. If cultivated as stab cultures in gelatin
there appears upon the surface at the point of inocula-
tion a circumscribed white point, slightly elevated above

MICROCOCCUS TETRAGENUS. DAT

the surface and limited to the immediate neighborhood
of the point of inoculation. Down the needle-track
the growth is not continuous, but appears in isolated,
round, dense white clumps or beads, which do not
develop beyond the size of very small points.

Tt does not liquefy gelatin.

Upon plates of nutrient agar-agar the colonies appear
as small, almost transparent, round points, which have
about the same color and appearance as a drop of egg
albumin; they are very slightly opaque. They are
moist and glistening. They rarely develop to an extent
exceeding 1 to 2 mm. in diameter.

Upon agar-agar as stab or slant cultures, the surface
growth has more or less of a mucoid appearance. It is
moist, glistening, and irregularly outlined. The outline
of the growth depends upon the moisture of the agar.
It is slightly elevated above the surface of the medium.

In contradistinction to the gelatin stab-cultures, the
growth in agar-agar is continuous along the track of the
needle.

The growth on potato is a thick, irregular, slimy-
looking patch.

The presence of the transparent gelatinous substance
which is seen to surround these organisms renders them
coherent, so that efforts to take up a portion of a colony
from the agar-agar or potato cultures result usually in
drawing out fine silky threads consisting of organisms
imbedded in this gelatinous material.

The organism grows best at from 35° C. to 38° C.,
but can be cultivated at the ordinary room temperature
—about 20° C.

The growth under all conditions is slow.

It grows both in the presence of and without oxygen.

248 BACTERIOLOGY.

It is not motile.

It stains readily with all the ordinary aniline dyes.
In tissues its presence is readily demonstrated by the
staining method of Gram.

The grouping into fours is particularly well seen in
sections from the organs of animals dead of this form
of septicemia.

In such sections the organisms will always be found
within the capillaries.

Inoculation into animals, To the naked eye no alter-
ation can be seen in the organs of animals that have
died as a result of inoculation with the micrococcus
tetragenus ; but microscopic examination of cover-slip
preparations from the blood and viscera reveals the
presence of the organisms throughout the body—espe-
cially is this true of preparations from the spleen.
White mice and guinea-pigs are susceptible to the
disease. Gray mice, dogs, and rabbits are not suscep-
tible to this form of septicemia. Subsequent inocu-
lation of healthy animals with a drop of blood, a bit
of tissue, or a portion of a pure culture of this organ-
ism from the body of an animal dead of the disease,
results in a reproduction of the conditions found in the
dead animal from which the tissues or cultures were
obtained. —

It sometimes occurs that in guinea-pigs which have
been inoculated with this organism there results local
pus-formations instead of a general septicemia. The
organisms will then be found in the pus-cavity.

CHAPTER XVIII.

Tuberculosis—Microscopic appearance of miliary tubercles—Encapsulation
of tuberculous foci—Diffuse caseation—Cavity-formation—Primary infection
—Modes of infection—Location of the bacilli in the tissues—Staining pecu-
liarities—Organisms with which the bacillus tuberculosis may be confounded
—Points of differentiation.

SHOULD the animal succumb to neither of the septic
processes just described, then its death from tuberculosis
may be reasonably expected.

When this disease is in progress, alterations in the
lymphatic glands nearest the seat of inoculation may be
detected by the touch in from two to four weeks. They
will then be found to be enlarged. Though not constant,
tumefaction and subsequent ulceration at the point of
inoculation may sometimes be observed. Progressive
emaciation, loss of appetite, and difficulty in respiration
point to the existence of the tubercular process. Death
ensues in from four to eight weeks after inoculation.
At autopsy either general or local tuberculosis may be
found. The expressions of the tubercular process are
so manifold and in different animals differ so widely the
one from the other, that no rigid law as to what will
appear at autopsy can @ priori be laid down.

The guinea-pig, which is best suited for this experi-
ment, because of the greater regularity of its suscepti-
bility to the disease over that of other animals usually
found in the laboratory, presents, in the main, changes
that are characterized by a condition of coagulation-
necrosis and caseation. This is particularly the case

250 BACTERIOLOG F.

when the infection is general, i.e, when the process is
of the acute miliary type. This pathological-anatomical
alteration is best seen in the tissues of the liver and
spleen of these animals, where the condition is most
pronounced,

In general, the tubercular lesions can be divided into
those of strictly focal character—the miliary and the
conglomerate tubercles, and those which are more diffuse
in theirnature. The latter lesions, although of the same
fundamental nature as the miliary tubercles, are much
greater in extent and not so sharply circumscribed.

These latter lesions play a greater rdle in the path-
ology of the disease than do the miliary nodules, although
it is to the presence of the latter that the disease owes
its name.

At autopsy the pathological manifestations of the
disease are not infrequently seen to be confined to the
seatof inoculation and the neighboring lymphatic glands.
These tissues will then present all the characteristics
of the tuberculous process in the stage of cheesy de-
generation. When the disease is general the degree
of its extension varies. Sometimes the small gray
nodules—the miliary tubercles—are only to be seen with
the naked eye in the tissues of the liver and spleen.
Again, they may invade the lungs, and commonly
they are distributed over the serous membranes of
the intestines, the lungs, the heart, and the brain.
These simple gray nodules, as seen by the naked eye,
vary in size from that of a pin-point to that of a hemp-
seed, and as arule are, in this stage, the result of the
fusion of two or more smaller miliary foci. Though
the two terms, “miliary” and “ conglomerate,” exist
for the description of the macroscopic appearance of

MILIARY TUBERCULOSIS. 251

these nodules, yet it is very rare that any condition
other than that due to the fusion together of several of
these minute foci can be detected by the naked eye.
The miliary tubercles are of a pale gray color, with a
white centre, are slightly elevated above the surface of
the tissue in which they exist, and, as stated, vary con-
siderably in dimensions, usually appearing as points
which range in size from that of a pin-point to that of a
pin-head. They are not only located upon the surface of
the organs, but are distributed through the depths of
the tissues. To the touch they sometimes present nothing
characteristic, but may frequently, when closely packed
together in large numbers, give a mealy or sandy sensa-
tion to the fingers. Stained sections of these miliary
tubercles present an entirely characteristic appearance,
and the disease may bediagnosticated by these histologi-
cal changes alone, though the crucial test in the diagnosis
is the finding of tubercle bacilli in these nodules.
Microscopic APPEARANCE OF MILIARY TUBER-
cLES.—The simple miliary tubercles under the low mag-
nifying power of the microscope presentsomewhat the fol-
lowing appearance: Thereis a central pale area, evidently
composed of necrotic tissue because of its incapacity for
taking up the nuclear stains commonly employed.
Scattered here and there through this necrotic area may
be seen granular masses irregular in size and shape;
they take up the stains employed and are evidently the
fragments of cell-nuclei in the course of destruction.
Through the necrotic area may here and there be seen
irregular lines, bands, or ridges, the remains of tissues
not yet completely destroyed by the necrotic process.
Around the periphery of this area may sometimes be
noticed large multi-nucleated cells, the nuclei of which

252 BACTERIOLOGY.

are arranged about the periphery of the cell or grouped
irregularly at its poles. The arrangement of these
nuclei appears in the sections sometimes as ovals, again
they are somewhat crescentic in their grouping. In the
tubercles from the human subject these large “giant-
cells,” as they are called, are quitecommon. They are
much less frequent in the tubercular tissues from the
lower animals.

Round about this central focus of necrosis is seen a
more or less broad zone of closely packed small round
and oval bodies which stain readily but not homogene-
ously. They vary in size and shape, and are seen to
be imbedded in a delicate network of fibrinous-looking
tissue.

This fibrin-like network in which these bodies lie,
and which is a common accompaniment of giant-cell
formation, is in part composed of fibrin, but is in the
main, most probably, the remains of the interstitial
fibrous tissue of the part. This zone of which we are
speaking is the zone of so-called “ granulation tissue,”
and consists of leucocytes, granulation cells, fibrin and
the fibrous remains of the organ ; the irregularly oval,
granular bodies which take up the staining are the
nuclei of these cells. The zone of granulation tissue
surrounds the whole of the tubercular process, and at
its periphery fades gradually into the healthy surround-
ing tissue or fuses with a similar zone surrounding
another tubercular focus. This may be taken as a
description of the typical miliary tubercle.

DirrvusE Caseation.—The diffuse caseation, as said,
plays a more important rdle in the tuberculous lesion,
both in the human and experimental forms, than does
the formation of miliary tubercles. In this a large

CAVITY-FORMATION. 253

area of tissue undergoes the same process of necrosis
and caseation as the centre of the miliary tubercle. In
some tissues it is more marked than in others. These
tissues are the lungs and the lymph-glands. In rab-
bits, particularly, all the changes in the lung frequently
come under this head. When this is the case solid
masses are found, sometimes as large as a pea, or in-
volving even an entire lobe or the whole lung in some
cases. They are of a whitish-yellow, opaque color, and
on section are peculiarly dry and hard. Entire lym-
phatic glands may be changed in this way. The con-
ditions for this caseation of the tissues are probably
given when a large number of tubercle bacillus enter the
tissue simultaneously and a wide area is involved, in-
stead of the small centre of the miliary tubercle. Ne-
crosis is so rapid that time is not given for those reactive
changes to take place in the tissues which result in the
formation of the outer zone of the miliary tubercle.
In other instances the entire caseous area is surrounded
by a granulation zone similar to that around the caseous
centre of the miliary tubercles. It is of special impor-
tance to recognize the connection between this diffuse
caseation of the tissue and the tubercle bacillus because
until its nature was accurately determined the caseous
pneumonia of the lungs formed the chief obstacle which
many had in recognizing the infectiousness of tubercu-
losis.
CAVITY-FORMATION.—The production of cavities
which forms such a prominent feature in human tuber-
culosis, particularly in the lungs, is due to the soften-
ing of the necrotic caseous masses or of aggregations of
miliary tubercles. The material softens and is expelled,
and a cavity remains. In the wall of this cavity
12

254 BACTERIOLOGY.

the tuberculous changes still proceed, both as diffuse
caseation and formation of miliary tubercles. The
whole cavity, with the reactive changes in the tissues
of its walls, may be considered as representing a single
tubercle, its wall forming a tissue very analogous to
the outer zone of the single tubercle, the cavity itself
corresponding to the caseous centre. In animals used
for experiment cavity-formation of this sort is very
rare, owing to the greater resistance of the caseous
tissue.

In the contents and in the walls of tubercular cavi-
ties bacteria other than the tubercle bacilli are found.
It is to the influence of some of these, as we have just
seen, that diseases other than tuberculosis may some-
times be produced by the inoculation of animals with
the sputum from such cases.

ENCAPSULATION OF TUBERCULAR Foct.—It not
uncommonly occurs that round about a necrotic tuber-
cular focus there is formed a fibrous capsule which may
completely cut off the diseased from the healthy tissue
surrounding it. Or a tubercular focus may, through
the resistance of the tissue in which it is located, be
more or less completely isolated. In this condition
the diseased foci may lie dormant for a long time and
give no evidence of their existence, until by some in-
tercurrent interference they are caused to break through
their envelopes. With the passage of the bacilli or their
spores from the central foci into the vascular or lym-
phatic circulation the disease may then become general.

It is to some such accident as this that the sudden
appearance of general tubercular infection in subjects
supposed to have recovered from the primary local
manifestations may often be attributed. The breaking-

TUBERCULAR INFECTION. 255

down of old caseous lymphatic glands is a common
example of this condition.

Primary Inrection.—The primary infection occurs
through either the vascular or lymphatic circulation.
Through these channels the bacilli gain access to the
tissues and become lodged in the finer capillary ramifi-
cations or in the more minute lymph-spaces. Here
they find conditions favorable to their development, and
in the course of this process produce substances of a
chemical nature which act directly in bringing about the
death of the tissues in their immediate neighborhood.
This tissue-death is probably the very first effect of the
bacilli in the body, and represents the necrotic centre,
which can always be seen in even the most minute
tubercles. With the production of this progressive
necrosis—for progressive it is, as it continues as long
as the bacilli live and continue to produce their poison-
ous products—there is in addition a reactive change in
the surrounding tissues, which consists in the formation
of the granulation zone at the outer margins of the
dying and dead tissue. This zone consists of small,
round granulation-cells and of leucocytes, all of which
are seen in the meshes of the finer fibrous tissues of the
part. At the same time alterations are produced in the
walls of the vessels of the locality ; this tends to oc-
clude them, and thus the process of tissue-death is
favored by a diminution of the amount of nutrition
brought to them. These changes continue until eventu-
ally the life processes of the bacilli are checked, or
conglomerate tubercles, widespread caseation, or cavity-
formation results.

Mopes or Inrection.—Experimentally, tuberculosis
may be produced in susceptible animals by subcutaneous

256 BACTERIOLOGY,

inoculation ; by direct injection into the circulation ;
by injection into the peritoneal cavity ; by feeding of
tuberculous material ; by the introduction of the bacilli
into the air-passages, and by inoculation into the an-
terior chamber of the eye.

In the human subject the most common portals of
infection are, doubtless, the air-passages, the alimentary
tract, and cutaneous wounds. When introduced sub-
cutaneously the resulting process finds its most pro-
nounced expression in the lymphatic system. The
growing bacilli make their way into the lymphatic
spaces of the loose cellular tissue, are taken up in the
lymph stream and deposited in the neighboring lymph-
atic glands. Here they may remain and give rise to
no alteration further than that seen in the glands them-
selves, or they may pass on to neighboring glands, and
eventually be disseminated throughout the whole lymph-
atic system, ultimately reaching the vascular system.

When having gained access to the bloodvessels, the
results are the same as those following upon intra-
vascular injection of the bacilli, namely : general tuber-
culosis quickly follows, with the most conspicuous pro-
duction of miliary tubercles in the lungs and kidneys,
less numerous in the spleen, liver, and bone marrow.

When inhaled into the lungs, if conditions are favor-
able, multiplication of the bacilli quickly follows. With
their growth they are mechanically pressed into the tis-
sues of the lungs. As multiplication continues some are
transported from the primary seat of infection to healthy
portions of the lung tissue, there to give rise to a further
production of the tubercular process.

In the same way infection through the alimentary
tract is in the main due to mechanical pressure of

TUBERCLE BACILLI IN TISSUES. 957

the bacilli upon the walls of the intestines. Investiga-~
tion has shown that lesions of the intestinal coats are
not necessary for the entrance of tubercle bacilli from
the intestines into the body. They may be trans-
ported from the intestinal tract into the lymphatics in
the same way that the fat droplets of the chyle find
entrance into the lymphatic circulation.

The evidence produced by Cornet’ points to the lungs
as the most common portals of natural infection for the
human being. Unlike most pathogenic organisms, the
tubercle bacillus has the property of forming spores
within the tissues. These spores, which are highly re-
sistant and are not destroyed by drying, are thrown off
from the lungs in the sputum of tuberculous patients in
large number, and unless special precautions are taken
to prevent it the sputum becomes dried, is ground
into dust, and sets free in the atmosphere the spores
of tubercle bacilli which came with it from the lungs.
The frequency of pulmonary tuberculosis points to this
as one of the commonest sources and modes of infection.

LocaTION OF THE BACILLI IN THE TissuEs.—The
bacilli will be found to be most numerous in those
tissues which are in the active stage of the process.

In the very initial stage of the disease the bacilli will
be fewer in number than later. At this time only here
and there single rods may be found; later they will be
more numerous, and, finally, when the process has ad-
vanced to a stage easily recognizable by the naked eye,
they will be found in the granulation zones in clumps
and scattered about in large numbers.

In the central necrotic masses, which consist of cell

1 Cornet: Zeit. fur Hygiene, 1889, Bd. v., 8. 191.

258 BACTERIOLOGY.

detritus, it is rare that the organisms can be demon-
strated microscopically. It is at the periphery of these
areas and in the progressing granular zone that they are
most frequently to be seen.

This apparent absence of the bacilli from the central
necrotic area must not be taken, however, as evidence
that this tissue does not contain them. As bacilli, they
are difficult to demonstrate here because the probabili-
ties are that in this locality, owing to conditions unfa-
vorable to their further growth, they are in the spore
stage, a stage in which it is as yet impossible, with our
present methods of staining, to render them visible.
The fact that this tissue is infective, and with it the
disease can be reproduced in susceptible animals, speaks
for the accuracy of this assumption. A conspicuous
example of this condition is seen in old scrofulous
glands. These glands usually present a slow process,
are commonly caseous, and always possess the property
of producing the disease when introduced into the tis-
sues of susceptible animals, and yet they are the most
difficult of all tissues in which to demonstrate micro-
scopically the presence of tubercle bacilli.

In tubercles containing giant-cells the bacilli can
usually be demonstrated in the granular contents of
these cells. Frequently they will be found accumu-
lated at the pole of the cell opposite to that occupied
by the nuclei, as if there existed an antagonism between
the nuclei and the bacilli. In some of these cells,
however, the distribution of the bacilli is seen to be
irregular, and they will be found scattered among the
nuclei as well as in the necrotic centre of the cell. As
the number of bacilli in the giant-cell increases the
cell itself is ultimately destroyed.

CULTIVATION OF TUBERCLE BACILLUS. 59

Tubercular tissues always contain the bacilli or their
spores, and are always capable of reproducing the dis-
ease when introduced into the body of a susceptible
animal. From the tissues of this animal the bacilli
may again be obtained and cultivated artificially, and
these cultures are capable of again producing the
disease when further inoculated. Thus the postulates
which are necessary to prove the etiological role of the
organism in the production of this malady are all ful-
filled.

THE TUBERCLE Baciiius.—Of the three pathogenic
organisms liable to occur in the sputum of a tuberculous
subject, the tubercle bacillus will give us most difficulty
in our efforts at cultivation.

It is, in the strict sense of the word, a parasite and
finds conditions entirely favorable to its development
only in the animal body. On ordinary artificial media
the bacilli taken directly from the animal body grow
only very imperfectly or in many cases not at all.
From this it seems probable that there is a difference
in the nature of individual tubercle bacilli—some ap-
pearing to be capable only of growth in the animal
tissues, while others are apparently possessed of the
power to lead a limited saprophytic existence. It may be,
therefore, that those bacilli which we obtain as artificial
cultures from the animal body are offsprings from the
more saprophytic varieties. At best, one never sees
with the tubercle bacillus a saprophytic condition in
any way comparable to that possessed by many of the
other organisms with which we have to deal.

In efforts to cultivate this organism directly from the
tissues of the animal, the method by which one obtains
the best results is that recommended by Koch—cultiva-

260 BACTERIOLOGY.

tion upon blood-serum. So strictly is this organism a
parasite that very limited alterations in the conditions
under which it is growing may result in failure to suc-
cessfully study it. It is, therefore, necessary that the
injunctions for obtaining it in pure culture should be
carefully observed.

The blood-serum upon which the organism is to be
cultivated should be comparatively freshly prepared—
that is, it should not be dry.

PREPARATION OF CULTURES FROM TIssUES.—
Under strictest antiseptic precautions, remove from the
animal the tubercular tissue—the liver, spleen, or a
lymphatic gland being preferable. Place the tissue in
a sterilized Petri dish and dissect out with sterilized
scissors and forceps the small tubercular nodules. Place
each nodule upon the surface of the blood-serum, one
nodule in each tube, and with a heavy, sterilized, looped
platinum needle or spatula, rub it carefully over the
surface. It is best to dissect away twenty to thirty such
tubercles and treat each in the same way. Some of the
tubes will remain sterile, others may be contaminated by
outside organisms during the manipulation, while a few
may give the result desired—a growth of the bacilli
themselves.

After inoculating the tubes they should be carefully
sealed up to prevent evaporation and consequent dry-
ing. This is done by burning off the superfluous
overhanging cotton plug in the gas-flame, and then im-
pregnating the upper layers of the cotton with either
sealing-wax or paraffin of a high melting-point; or by
inserting over the burned end of the cotton plug a soft,
closely fitting cork that has been sterilized in the steam
sterilizer just before using (Ghriskey). This precaution

CULTURES OF TUBERCLE BACILLUS. 961

is necessary because of the slow growth of the organism.
Under the most favorable conditions tubercle bacilli
directly from the animal body show no- evidence of
growth for about twelve days after inoculation upon
blood-serum, and, as they must be retained during this
time at the body temperature—37.5 C.—evaporation
would take place very rapidly and the medium would
become too dry for their development.

If these primary efforts result in the appearance of a
culture of the bacilli, further cultivations may be made
by taking up a bit of the colony, preferably a moderately
large quantity, and transferring it to fresh serum, and
this in turn is sealed up and retained at the same tem-
perature. Once having obtained the organism in pure
culture its subsequent cultivation may be conducted
upon the glycerin-agar-agar mixture—ordinary neutral
nutrient agar-agar to which 6 or 7 per cent. of glycerin
has been added. This is a very favorable medium for
the growth of this organism after once having estab-
lished its saprophytic form of existence, though blood-
serum is perhaps the best medium to be employed in
obtaining the first generation of the organism from the
tubercular tissues.

The organism may be cultivated also on neutral
milk to which 1 per cent. of agar-agar has been added,
also upon the surface of potato, and likewise in meat
infusion bouillon containing 6 or 7 per cent of
glycerin.

Cultures of the tubercle bacillus are characteristic
in appearance—after once having seen them there is
but little probability of subsequent mistake.

They appear as dry masses, which may develop upon

the surface of the medium either as flat scales or as
12*

262 BACTERIOLOGY.

lumps of mealy-looking granules. They are never
moist, and frequently have the appearance of coarse
meal which has been spread upon the surface of the
medium. In the lower part of the tube in which they
are growing, i.e. that part occupied by a few drops of
fluid which has in part been squeezed from the medium
during the process of solidification, and is in part water
of condensation, the colonies may be seen to float as a
thin pellicle upon the surface of the fluid.

The individuals making up the growth adhere so
tenaciously together that it is with the greatest difficulty
that they can be completely separated. In even the
oldest and dryest cultures pulverization is impossible.
The masses can only be separated and broken up by
grinding in a mortar with the addition of some foreign
substance, such.as very fine, sterilized sand, dust, etc.

The cultures are of a dirty-drab or brownish-gray
color when seen on serum or on glycerin-agar-agar.

On potato they grow in practically the same way,
though the development is much more limited. They
are here of nearly the same color as the potato on which
they are growing.

On milk-agar-agar they are of so nearly the same
color as the medium that, unless they are growing as the
mealy-looking masses considerably elevated above the
surface, their presence is less conspicuous than when on
the other media.

In bouillon they grow as a thin pellicle on the sur-
face. This may fall to the bottom of the fluid and con-
tinue to develop, its place on the surface being taken
by a second pellicle.

Under all conditions of artificial development the
cultures of this organism are always very dry and

STAINING PECULIARITIES OF B. TUBERCULOSIS. 263

brittle in appearance, though in truth the individuals
adhere tenaciously together by a very glutinous sub-
stance.

The tubercle bacillus does not develop on gelatin,
because of the low temperature at which this medium
must be used.

Microscopic APPEARANCE OF THE TUBERCLE
BaciiLus.—Microscopically the organism itself is a
delicate rod, usually somewhat beaded in its structure,
though rarely it is seen to be homogeneous. It is either
quite straight or somewhat curved or bent on its long
axis. In some preparations involution-forms, consist-
ing of rods a little clubbed at one extremity or slightly
bulging at different points, may be detected. It varies
in length—sometimes being seen in very short segments,
again much longer, though never as long threads. On
an average its length is seen to vary from 2 to5v. It
is commonly described as being in length about one-
fourth to one-half the diameter of a red blood-corpuscle.
It is very slender. (Fig. 51, page 239.)

These rods usually present, as has been said, an
appearance of alternate stained and colorless portions.
Tt is the latter portions which are believed to be the
spores of the organism, though as yet no absolute proof
of this opinion has been established.

At times these colorless portions are seen to bulge
slightly beyond the contour of the rod, and in this way
give to the rods the beaded appearance so commonly
ascribed to them. ;

Srarninc PEcuLIARITIES.—A peculiarity of this
organism is its behavior toward staining reagents, and
by this means alone it may be easily recognized. The
tubercle bacillus does not stain by the ordinary methods.

264 BACTERIOLOGY.

It possesses some peculiarity in its composition that
renders it more or less proof against the simpler
dyes. It is therefore necessary that more energetic
and penetrating reagents than the ordinary watery
solutions should be employed. Experience has taught
us that certain substances not only increase the solu-
bility of the aniline coloring substances, but by their
presence the penetration of the coloring agents is very
much increased. .Two of these substances are aniline
oil and carbolic acid. They are employed in the
solutions to about the point of saturation. (For the
exact proportions see chapter on Staining Reagents.)

Under the influence of heat these solutions are seen
to stain all bacteria very intensely—the tubercle bacilli
as well as the ordinary forms. If we subject our prep-
aration, which may contain a mixture of tubercle
bacilli and other forms, to the action of decolorizing
agents, another peculiarity of the tubercle bacillus will
be observed. While all other organisms in the prep-
aration will give up their color and: become invisible,
the tubercle bacillus retains it with marked tenacity.
It stains with great difficulty, but once stained it
retains the color even under the influence of strong
decolorizing agents.

ORGANISMS WITH WHICH THE BACILLUS TUBERCU-
LOSIS MAY BE CONFUSED.

DIFFERENTIAL Draenosis.—While this peculiar
micro-chemical reaction is usually considered to be
diagnostic of the bacillus tuberculosis, it is well to
remember that there are at least three other species of
bacilli which, when similarly treated, react in the same
way. It is of importance to bear this point in mind,

DIFFERENTIAL DIAGNOSIS OF BACILLI. 9265

particularly in the microscopic examination of urine
and pathological secretions from the genito-urinary
tract and from the rectum, for of the three species two
are frequently found in these localities, viz., the so-called
smegma bacillus, located in the smegma and often seen
beneath the prepuce and upon the vulva, both normally
and in disease, and the bacillus of syphilis, described by
Lustgarten as contained in syphilitic manifestations,
particularly in primary sores. The third organism of
this group —the bacillus of leprosy—because of its rarity,
is not so likely to cause error in diagnosis of troubles
occurring in these localities.

According to Hueppe, the differential diagnosis
between the four organisms depends upon the following
reactions: When stained by the carbol-fuchsin method
commonly employed in staining the tubercle bacillus the
syphilis bacillus becomes almost instantly 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
bacillus 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
conspicuously retentive of their color even after treat-
ment with both acids and alcohol.

To differentiate, then, between the four organisms he
recommends the following order of procedure, based on
the above reactions :

1. Treat the stained preparation with sulphuric
acid; the syphilis bacillus becomes decolorized, the
reaction being almost instantaneous.

2. If it is not at once decolorized, treat with alcohol ;

266 BACTERIOLOGY.

if it is the smegma bacillus this will rob it of its
color.

3. If it is still not decolorized it is either the lepra
or tubercle bacillus.

The differential diagnosis between the last two
organisms is less satisfactory ; they both take on the
same stains and both retain them or give them up under
treatment with the same decolorizers. The results of
investigations, however, indicate differences in the rate
of staining and decolorization, and it is accepted by
many of those who have compared the two organisms
that the lepra bacillus takes up staining very much
more readily than does the tubercle bacillus, often
staining perfectly by an exposure of only a few minutes
to cold watery solutions of the dyes, but when once
stained it retains its color much more tenaciously when
acted upon by decolorizing agents than does the latter
organism. 5

According to Baumgarten, the lepra bacillus is stained
by an exposure of six to seven minutes to a cold, satu-
rated watery solution of fuchsin, and retains the stain
when subsequently treated with acid alcohol (nitric acid,
1 part; alcohol, 10 parts). By similar treatment for
the same length of time the bacillus tuberculosis does
not become’stained.

These points, particularly what has been said with
reference to the smegma bacillus and the bacillus of
syphilis, are of much practical importance and should
always be borne in mind in connection with microscopic
examination of materials to which these organisms are
liable to gain access. It is hardly necessary to say that
in the examination of sputa and pathological fluids from
other parts of the body the tubercle bacillus is, of the

TUBERCULOSIS IN ANIMALS. 967

four organisms, always the one most commonly encoun-
tered.

TUBERCULIN.—The filtered products of growth from
old fluid cultures of the tubercle bacillus represent what
is known as tuberculin—a group of proteid substances
possessing most interesting properties. When injected
subcutaneously into healthy subjects tuberculin has no
effect, but when introduced into the body of the tuber-
culous person or animal a pronounced systemic reaction
results, consisting of sudden but temporary elevation of
temperature with, at the same time, the occurrence of
marked hyperemia round about the tuberculous focus, a
change histologically analogous to that seen in the
primary stages of acute inflammation. This zone of
hyperemia, with the coincident exudation and infiltra-
tion of cellular elements, probably aids in the isolation
or casting off of the tuberculous nodule, the inflamma-
tory zone forming, so to speak, a line of demarcation
between the diseased and healthy tissue.

As a curative agent for the treatment of tuberculosis,
tuberculin has not merited the confidence that was at
first accorded to it. Its greatest field of usefulness is
now admitted to be as an aid to the diagnosis of obscure
cases, and more particularly those occurring in cattle,
where it has proven itself to be of inestimable value in
this particular application.

SUSCEPTIBILITY OF ANIMALS TO TUBERCULOSIS.—
The animals which are known to be susceptible to the
tubercular processes are man, apes, cattle, horses, sheep,
guinea-pigs, pigeons, rabbits, cats, and field mice.

White mice, dogs, and rats possess immunity against
the disease.

We have reviewed the three common pathogenic

268 BACTERIOLOGY.

organisms with which we may come in contact in the
sputum of tuberculous individuals. Occasionally other
forms may be present. The pyogenic forms are not
rarely found, and for some time after diphtheria the
bacillus of Léffler is demonstrable in the pharynx, so
that it too may be present under exceptional circum~-
stances. These latter organisms will be described under
their proper heads.

CHAPTER XIX.

Glanders—Characteristics of the disease—Histological structure of the
glanders nodule—Susceptibility of different animals to glanders—The bacillus
of glanders; its morphological and cultural peculiarities—Diagnosis of
glanders.

Synonyms: Rotz (Ger.), Morve (Fr.).

Though most commonly seen in the horse and ass,
glanders is not rarely met with in other animals, and is
occasionally encountered in man. When occurring
spontaneously in the horse its primary seat is usually
upon the mucous membrane of the nostrils. It appears
in the form of small gray nodules, about which the
membrane is congested and swollen. These nodules
ultimately coalesce to form ulcers. There is a profuse
slimy discharge from the nostrils during the course of
the disease. It may extend from its primary seat in
the nose to the mouth, larynx, trachea, and ultimately
to the lungs. Its secondary manifestations are observed
along the lymphatics that communicate with the primary
focus, in the lymphatic glands, and as metastatic foci in
the internal organs. Less frequently the disease is seen
to begin in the skin, particularly in the region of the
neck and breast. When in this locality the subcuta-
neous lymphatics become involved, and are converted
into indurated, knotty cords, easily discernible from
without.

When occurring in man it is usually in individuals
who have been in attendance upon animals affected with
the disease. It may occur upon the mucous membrane

270 BACTERIOLOGY.

of the nares, but its most conspicuous expressions are in
the skin and muscles, where appear abscesses, phleg-
mons, erysipelas-like inflammations, and local necrosis
closely resembling carbuncles. Metastases to the lungs,
kidneys, and testicles, as in the horse, may also be
seen.

When occurring upon the mucous membrane glanders
is characterized by the presence of small gray nodules
about as large as a pin-head, that closely resemble
miliary tubercles in their naked-eye appearance. These
consist histologically of granulation tissue, 7. e., of small
round cells, very similar to proliferating leucocytes, of
some lymph-cells, and, in the earliest stages, of a small
proportion of necrotic tissue. As they grow older, and
the process advances, there is a tendency toward central
necrosis, with the ultimate formation of a soft, yellow,
creamy, pus-like material. Though strikingly like
miliary tubercles in certain respects in the early stages,
there are, nevertheless, decided points of difference be-
tween them.

The round-cell infiltration of the glanders nodules
consists essentially of polynuclear leucocytes, while
that of the miliary tubercle partakes more of the nature
of a lymphocytic infiltration; in the later stages of
the process the glanders nodule breaks down into a soft
creamy matter, very analogous to ordinary pus, while
in the later stages of the miliary tubercle the tendency
is toward an amalgamation of its histological constitu-
ents, and ultimately to necrosis with caseation. The
giant-cell formation common to tuberculosis is never
seen in the glanders nodule. As Baumgarten aptly
puts it: “The pathological manifestations of glanders,
from the histological aspect, stand midway between the

GLANDERS. 271

acute purulent and the chronic inflammatory processes.” !
Evidently these differences are only to be explained by
differences in the nature of the causes that underlie the
several affections. We have studied the characteristics
of the bacillus tuberculosis; we shall now take up the
bacillus of glanders and note the striking differences
between them.

THE Baciiius oF GLANDERS (bacillus mallei).—In
1882 Loffler and Schiitz discovered in the diseased
tissues of animals suffering from glanders a bacillus
that, when isolated in pure culture and inoculated into
susceptible animals, possessed the property of reproduc-
ing the disease with all its clinical and pathological
manifestations. It is therefore the cause of the disease.

Bacillus of glanders (bacillus mallet).

It isa short rod, with rounded or slightly pointed
ends, that usually takes up the staining somewhat
irregularly. (See Fig. 53.) When examined in stained
preparations its continuity is marked by alternating
darkly and lightly stained areas. It is. usually seen as
a single rod, but may occur in pairs, and less frequently
in longer filaments.

1 For a further discussion of the pathology and pathogenesis of this disease,
see Lehrbuch der pathologischen Mykologie, by Baumgarten, 1890.

272 BACTERIOLOGY.

The question as to its spore-forming property is still
an open one, though the weight of evidence is in oppo-
sition to the opinion that it possesses this peculiarity.
Certain observers claim to have demonstrated spores
in the bacilli by particular methods of staining, but this
statement can have but little weight when compared
with the behavior of the organism when subjected to
more conclusive tests. For example, it does not resist
exposure to 3 per cent. carbolic acid solution for longer
than five minutes, nor to 1 : 5000-sublimate solution for
more than two minues. It is destroyed in ten minutes
in some experiments, and in five in others, by a tem-
perature of 55° C., and when dried it loses-its vitality,
according to different observers, in from thirty to forty
days ; all of which speak directly against this being a
spore-bearing bacillus.

It is not motile, and does not, therefore, possess
flagella.

Tt grows readily on the ordinary nutrient media at
from 25° C. to 38° C.

Upon nutrient agar-agar, both with and without
glycerin, it appears as a moist, opaque, glazed layer,
with nothing characteristic about it. This is true both
for smear cultures and for single colonies.

Its growth on gelatin is much less voluminous than
on media that can be kept at higher temperature, though
it does grow on this media at room temperature without
causing liquefaction.

Its growth on blood-serum is seen in the form of a
moist, opaque, slimy layer, inclining to a yellowish or
dirty brownish-yellow tinge. It does not liquefy the
serum,

On potato its growth is moderately rapid, appearing

THE BACILLUS OF GLANDERS, 273

at the end of from twenty-four to thirty-six hours at
37° C.as a moist, amber-yellow, transparent deposit
which becomes deeper in color and denser in consistence
as growth progresses. It finally takes on a reddish-
brown color, and the potato about it becomes dark-
ened.

In bouillon it causes diffuse clouding, with ultimately
the formation of a more or less tenacious or ropy sedi-
ment.

In milk to which a little litmus has been added, it
causes the blue color to become red or reddish in from
four to five days, and quite red after two weeks at 37°
C. At the same time the milk is separated into a firm
clot of casein and clear whey.

Its reactions to heat are very interesting—at 42° C.
it will often grow for twenty days or more. It will not
grow at 43° C., and is killed, by exposure to this tem-
perature for forty-eight hours. Itis killed in five hours
when exposed to 50° C., and in five minutes by 55° C.

It grows both with and without oxygen ; it is there-
fore facultative as regards its relation to this gas.

On cover-slips it stains readily with all the basic
aniline dyes, and, as a rule, as stated, presents conspicu-
ous irregularities in the way that it takes up the dyes,
being usually marked by deeply stained areas that
alternate with points at which it either does not stain
at all or only slightly.

The animals that are susceptible to infection by this
organism are horses, asses, field mice, guinea-pigs, and
cats. Baumgarten records cases of infection in lions
and tigers that have been fed, in menageries, with flesh
from horses affected with the disease. Rabbits are but
slightly susceptible; dogs and sheep still less so. Man

274 BACTERIOLOG Y.

is susceptible, and infection not rarely terminates fatally.
White mice, common gray house mice, rats, cattle, and
hogs are insusceptible.

InocuLATION EXPERIMENTS.-—The most favorable
animal upon which to study the pathogenic properties
of this organism in the laboratory is the common field
mouse. When inoculated subcutaneously with a small
portion of a pure culture of the glanders bacillus death
ensues in about seventy-two hours. The most con-
spicuous tissue changes will be enlargement of the spleen,
which is at the same time almost constantly stndded
with minute gray nodules, the typical glanders nodule.
They are rarely present in the lungs, but may frequently
be seen in the liver. From these nodules the glanders
bacillus may be obtained in pure culture. With the
exception of the characteristic nodule the disease, as
seen in this animal, presents none of the characteristics
that it displays in the horse and ass. The clinical and
pathological manifestations resulting from inoculation of
guinea-pigs are much more characteristic. The animal
lives usually from six to eight weeks after inoculation,
and in this time becomes affected with a group of most
interesting and peculiar pathological processes. The
specific inflammatory condition of the mucous mem-
brane of the nostrils is almost always present. The
joints become swollen and infiltrated to such an extent
as often to interfere with the use of the legs. In male
animals the testicles become enormously distended with
pus, and on closer examination a true orchitis and
epididymitis is seen to be present. The internal organs,
particularly the lungs, kidneys, spleen, and liver, are
usually the seat of the nodular formations character-
istic of the disease. From all of these disease foci

B, MALLEI: STAINING IN TISSUES. 9275

the bacillus causing them can be isolated in pure
culture.

STAINING IN TissuEs.—Though always present in
the diseased tissues, considerable trouble is usually ex-
perienced in demonstrating the bacteria by staining
methods. The difficulty lies in the fact that the bacilli
are very easily decolorized, and in tissues stained by
the ordinary processes are robbed of their color even by
the alcohol with which the tissue is rinsed out and de-
hydrated. If we will remember not to employ concen-
trated stainings and not to expose the reactions to them
for too long a time, but little treatment with decoloriz-
ing agents is necessary, and very satisfactory prepara-
tions will be obtained. A number of good methods
have been suggested for staining the glanders bacilli in
tissues, and if what has been said will be borne in mind
no difficulty should be experienced.

Two satisfactory methods that we have used for this
purpose, though perhaps no better than some of the
others, are as follows :

(a) Transfer the sections from alcohol to distilled
water. This lessens the violence with which the stain-
ing subsequently takes hold of the tissues by diminish-
ing the activity of the diffusion that would occur if
they were placed from alcohol into watery solutions of
the dyes. Transfer from distilled water to the slide,
absorb all water with blotting-paper, and stain with
two or three drops of

Carbolic fuchsin . ; ‘ ‘i . Wee,
Distilled water ‘ ne ot t 4 - 100 cc.

for thirty minutes ; absorb all superfluous staining with
blotting-paper, and wash the section three times with
0.3 per cent. acetic acid, not allowing the acid to act for

276 BACTERIOLOGY.

more than ten seconds each time. Remove all acid
from the section by carefully washing in distilled water ;
absorb all water by gentle pressure with blotting-paper,
and finally, at very moderate heat, or with a small bel-
lows (Kithne), dry the section completely on the slide.

When dried clear up in xylol, and mount in xylol
balsam.

(6) The other method is as follows:

Transfer sections from alcohol to distilled water ;
from water to the dilute fuchsin solution, and gently
warm (about 50° C.) for fifteen to twenty minutes.

- Transfer section from the staining solution to the slide,
absorb. all superfluous staining with blotting-paper, and
then treat them with 1 per cent. acetic acid from one-
half to three-quarters of a minute. Remove all trace
of acid with distilled water, absorb all water by gentle
pressure with blotting-paper, and then treat the sec-
tions with absolute alcohol by allowing it to flow over
them drop by drop. For small sections three or four
drops are sufficient. Under no circumstances should
the alcohol be allowed to act for more than one-
quarter of a minute. Clear up in xylol and mount in
xylol balsam.

In method 6 the tissues are better preserved than in
a, where they were dried.

Very good preparations are also obtained by the use
of Léffler’s alkaline methylene-blue, if care be taken
not to stain for too long a time or to decolorize with
alcohol too energetically.

No method of contrast-stain for this organism in tis-
sues has been devised.

In properly stained tissues the bacilli will be found
most numerous in the centre of the nodules, becoming

MALLEIN. 217

fewer as we approach the periphery. They usually lie
between the cells, but at times may be seen almost fill-
ing some of the epithelial cells, of which the nodule
contains more or less. They are always present in
these nodules in the tissues; they are rarely present in
the blood, and, if so, in only small numbers.

Diagnosis OF THE DISEASE BY THE METHOD OF
Srravuss.—From what has been said the diagnosis of
glanders by routine bacteriological methods is certain and
relatively easy, but requires time. In clinical work it is
of great importance for the diagnosis to be established
as quickly as possible. With this in view Strauss has
devised a method that has given entirely satisfactory
results. It consists in introducing into the peritoneal
cavity of a male guinea-pig a bit of the suspected tissue
or culture. If it is from a genuine case of glanders
the testicles begin to swell in about thirty hours, and as
this proceeds the skin over them becomes red and shin-
ing, desquamation occurs, evidences of pus-formation
are seen, and, indeed, the abscess (purulent . orchitis)
often breaks through the skin. The diagnostic sign is
the tumefaction of the testicles.

Ma.ein.—The filtered products of growth of the
glanders bacillus in fluid media represent what is known
as mallein—a group of compounds that bear to glanders
pretty much the same relation that tuberculin bears to
tuberculosis. It is used with considerable success as
a diagnostic aid in detecting the existence or absence of
deep-seated manifestations of the disease, the glanderous
animal reacting in from four to ten hours to subcuta-
neous injections of mallein, while the animal not so

affected gives no such reactions.
13

278 BACTERIOLOGY.

It is prepared from old glycerin-bouillon cultures of
the glanders bacillus by steaming them for several hours
in the sterilizer, after which they are filtered through
unglazed porcelain.

CHAPTER XX.

Bacillus diphtherix—tts isolation and cultivation—Morphological and cul-
tural peculiarities—Pathogenic properties—Variations in virulence.

From the gray-white deposit on the fauces of a diph-
theritic patient prepare a series of cultures in the fol-
lowing way :

Have at hand five or six tubes of Léffler’s blood-
serum mixture. (See article on Media.)

Pass a stout platinum needle, which has been steril-
ized, into the membrane and twist it around once or
twice or brush it gently over the surface of the mem-
brane. Without touching it against anything else
rub it carefully over the surface of one of the serum
tubes ; without sterilizing it pass it over the surface of
the second, then the third, fourth, and fifth tube. Place
these tubes in the incubator. Then prepare cover-slips
from scrapings from the membrane on the fauces. If
the case is true diphtheria the tubes will be ready for
examination on the following day.

The reason that plates are not made in the regular
way in this examination is that the bacillus of diphtheria
develops much more luxuriantly on the serum mixture,
from which plates cannot be made, than it does on the
media from which they can be made. The method em-
ployed, however, insures a dilution in the number of
organisms present, and this, in addition to the fact that
the bacillus diphtheriz grows much more quickly on
the serum mixture than do other organisms, makes

280 BACTERIOLOGY.

its isolation by this method a matter of but little diffi-
culty.

After twenty-four hours in the incubator the tubes
will present a characteristic appearance. Their surfaces
will be marked at different points by more or less
irregular patches of a white or cream-colored growth
which is usually more dense at the centre than at its
irregular periphery.

Except now and then, when a few orange-colored
colonies may be seen, these large irregular patches are
the most conspicuous objects on the surface of the
serum. Occasionally, almost nothing else appears.

The cover-slips made from the membrane at the
time the cultures were prepared will be found on micro-
scopic examination to present, in many cases, a great
variety of organisms, but conspicuous among them
will be noticed slightly curved bacilli of irregular size
and outline. In some cases they will be more or less
clubbed at one or both ends; sometimes they appear
spindle in shape, again as curved wedges ; now and then
they will be seen irregularly segmented. They arerarely
or never regular in outline. If the preparation has
been stained with Léffler’s alkaline methylene-blue
solution many of these irregular rods are seen to be
marked by circumscribed points in their protoplasm
which stain very intensely ; they appear almost black.
This irregularity in outline is the morphological char-
acteristic of the bacillus diphtheriew of Léffler. It must
be remembered, however, that the diagnosis of diphtheria
cannot be made from the examination of cover-slip
preparations alone, for there are other organisms present
in the mouth cavity, particularly in the mouth of persons
having decayed teeth, the morphology of which is so

MORPHOLOGY OF B. DIPHTHERLZ. 981

like that of the bacillus of diphtheria that they might
easily be mistaken for that organism if subjected to
microscopic examination only.

The bacillus diphtheric of Léffler (its discoverer) can
readily be identified by its cultural peculiarities in con-
nection with its pathogenic activity when introduced
into tissues of susceptible animals. In guinea-pigs and
kittens the results of its growth are identical with those
found in the bodies of human beings who have died of
diphtheria.

When studied in pure culture, its morphological and
cultural peculiarities are as follows:

MorpHoLtogy.—As obtained directly from the
diphtheritic deposit in the throat of an individual sick
of the disease, it is sometimes comparatively regular
in shape, appearing as straight or slightly curved rods
with more or less pointed ends. More frequently, how-
ever, spindle and club shapes occur and not rarely many
of these rods take up the staining irregularly ; in some
of them very deeply staining round or oval points can
be detected.

When cultures are examined microscopically it is
especially characteristic to find irregular, bizarre forms,
such as rods with one or both ends swollen, and very
frequently rods broken at irregular intervals into short,
sharply marked segments, either round, oval, or with
straight sides. Some forms stain uniformly, others in
various irregular ways, the most common being the
appearance of deeply stained granules in a lightly stained
bacillus.

By a series of studies upon this organism when cul-
tivated under artificial conditions, we have found that
its form depends very largely upon the nature of its

282 BACTERIOLOGY.

environment. That is to say, its morphology is always
more regular, and it is smaller on glycerin-agar-agar
than on other media used for its cultivation ; while
upon Loffler’s blood-serum the other extremes of devel-
opment appear; here one sees, instead of the very short,
spindle, lancet, club-shaped, always segmented and
regularly staining forms as seen upon glycerin-agar,
long, irregularly staining threads, that are sometimes
clubbed and sometimes pointed at their extremities.
They are usually marked by areas that stain more
intensely than do the rest of the rod, and at times they
may bea little swollen at the centre. These differences
are so conspicuous that microscopic preparations from
cultures from the same source upon glycerin-agar-agar
and upon blood-serum, when placed side by side, would
hardly be recognized as of the same organism, unless
its peculiar behavior under these circumstances was
already known.

Fig. 54.
we
be ¢ —s eae
9 jo ee "1 7 o
woe, “me AX
a6: ov”,
og ae few fed me.
6 oe 4Ge
a GA

Bacillus diphtheriz. u. Its morphology when cultivated on glycerin-agar.
b. Its morphology as seen in cultures on Liffler’s blood-serum.

On plain nutrient agar-agar (that is, nutrient agar-
agar without glycerin) ; on solidified egg-albumin ; on
a medium consisting of dried albumin, as found in
commerce, dissolved in bouillon (about 10 grammes
albumin to 100 cc. of bouillon containing 1 per cent.
of grape-sugar) ; in bouillon without glycerin, and in

B. DIPHTHERIA ON SERUM MIXTURE. 283.

bouillon to which a bit of hard-boiled egg has been
added, the morphology of the organism is about inter-
mediate in both size and outline between the forms seen
upon glycerin-agar-agar and upon Loffler’s blood-serum.
There will appear about an equal number of short
segmented and longer irregularly staining forms, but in
general the longest are rarely as long as the long forms
seen on blood-serum, and throughout they are not
so conspicuous for the irregularity of their staining.

In cultures made upon two sets of nutrient agar-agar
tubes, differing only in the fact that one set contains
glycerin to the extent of 6 per cent., while the others
contain none, a noticeable difference in morphology
can usually be made out; while the forms on the gly-
cerin-agar cultures are throughout small, pretty regular
in size, shape, and staining, those on the plain agar are
larger, stain more irregularly, vary more in shape, and
when stained by Léffler’s blue are not so uniformly
marked by the pale transverse lines that give to them
the appearance of being made up of numerous short
segments.

Though the outline of this organism is more regular
under some circumstances than others, it is nevertheless
always conspicuous for its manifold variations in
shape.

GrowTH on SeruM Mixture.—The medium upon
which it grows most rapidly and luxuriantly, and which
is best adapted for determining its presence in diphthe-

_Yitic exudations is, as has been stated, the blood-serum
mixture of Léffler. (See chapter on Media.) On the
blood-serum mixture the colonies of the bacillus diph-
therie grow so much more rapidly than the other
organisms usually present in secretions and exudations

284 BACTERIOLOGY.

in the throat that at the end of twenty-four hours they
are often the only colonies that attract attention, and if
others of similiar size are present, they are generally of
quite a different aspect. Its colonies are large, round,
elevated, grayish-white, or yellowish with a centre more
opaque than the slightly irregular periphery. The sur-
face of the colony is at first moist, but after a day or
two becomes rather dry in appearance.

A. blood-serum tube studded over with coalescent or
scattered colonies of this organism is so characteristic
that one familiar with the appearance can anticipate
with tolerable certainty the results of microscopic ex-
amination.

GLYCERIN-AGAR-AGAR.—Upon nutrient glycerin-
agar-agar the colonies likewise present an appearance
that may readily be recognized. They are in every way
more delicate in their structure than when on the serum
mixture. They appear at first, when on the surface, as
very flat, almost transparent, dry, non-glistening, round
points which are not elevated above the surface upon
which they are growing. When slightly magnified
they are seen to be granular, and present an irregular
central marking which is more dense and darker by
transmitted light than the thin, delicate zone which
surrounds it. As the colony increases in size the thin
granular peripheral zone becomes broader, is usually
marked by ridges or cracks, and its periphery is notched
or scalloped. (Fig 55, ¢.) These colonies are always
quite dry in appearance. When deep down in the agar-
agar they are coarsely granular. (Fig. 55, a.) They
rarely exceed 3 mm. in diameter.

GELATIN.—On gelatin the colonies develop much
more slowly than on the other media which can be

B. DIPHTHERIA ON GELATIN. 285

retained at a higher temperature. They rarely present
their characteristic appearances on gelatin in less than
seventy-two hours.

Fic. 55,

&” 9 KOS es
»°s a, &
a &

Colonies of bacillus diphtheriz on glycerin-agar. a. Colonies located in the
depths of the medium. 06. Colonies just breaking out upon the surface of the
medium. c. Fully developed surface colony.

They then appear as flat, dry, translucent points,
usually round in outline.

When magnified slightly, the centre is seen to be
more dense than the surrounding zone or zones, for they
are sometimes marked by a concentric arrangement of
zones. The periphery is irregularly notched. Like the
colonies seen on agar-agar, they are granular, but are
much more granular when seen in the depths of the
gelatin than when on its surface. On gelatin the colonies
rarely become very large; usually they do not reach a
diameter of over 1.5 mm.

Boviitton.—In bouillon it usually grows in fine
clumps, which fall to the bottom of the tube, or become
deposited on its sides without causing a diffuse clouding
of the bouillon. There are sometimes exceptions to this
naked-eye appearance: the bouillon may appear dif-
fusely clouded, but if one inspects it very closely,
particularly if one examines it microscopically in the

form of a hanging drop, the arrangement in clumps will
13*

286 BACTERIOLOGY.

still be seen, but they are so small as not to be detect-
able by the unaided eye.

In bouillon which is kept at a temperature of
35°-37° C. for a long time, a soft, whitish pellicle
often forms over a part of the surface,

Changes in reactions of the bouillon. The reaction
of the bouillon becomes at first acid, and, subsequently,
again alkaline, changes which can be observed in
cultivations in bouillon to which a little rosolic acid
has been added. .

Porato.—On potato at a temperature of 35°-37° C,
its growth after several days in entirely invisible ; only
a thin, dry glaze appearing at the point at which the
potato was inoculated. Microscopic examination of
the potato after twenty-four hours at 35°-37° C. shows
a decided increasein the number of individual organisms
planted.

Stas AND SiaAnt CuLrures.—In stab and slant
cultures on both gelatin and glycerin-agar-agar, the
surface growth is seen to predominate over that along
the track of the needle in the depths of the media.

Isolated colonies on the surface of either of the media
in this method of cultivation present the same charac-
teristics that have been given for the colonies on
plates.

The growth in simple stab cultures does not extend
laterally very far beyond the point at which the needle
entered the medium.

It is a non-motile organism.

It does not form spores.

It is killed in ten minutes by a temperature of
58° C.

It grows at temperatures ranging from 22° C. to

INOCULATIONS WITH B. DIPHTHERIA. 287

37° C., but most luxuriantly at the latter tempera-
ture.

Its growth in the presence of oxygen is more active
than when this gas is excluded.

Srarninc.—In cover-slip preparations made either
from the fauces of a diptheritic patient or from a pure
culture of the organism, it is seen to stain readily with
the ordinary aniline dyes. It stains also by the method
of Gram, but the best results are those obtained by the
use of Loffler’s alkaline methylene-blue solution ; this
brings out the dark points in the protoplasmic body of
the bacilli and thus aids in their identification.

For the purpose of demonstrating the Léffler bacillus
in sections of diphtheritic membrane, both the Gram
method and the fibrin method of Weigert give excellent
results.

PATHOGENIC PROPERTIES.— When inoculated sub-
cutaneously into the bodies of susceptible animals the
result is not the production of a septicemia, as is seen
to follow the introduction into animals of certain other
organisms with which we shall have to deal, but the
bacillus of diphtheria remains localized at the point
of inoculation, rarely disseminating further than the
nearest lymphatic glands. It develops at the point in
the tissues at which it is deposited, and during its
development gives rise to changes in the tissues which
result entirely from the absorption of poisonous albu-
mins produced by the bacilli in the course of their
development.

In a certain number of cases’ diphtheria bacilli have
been found in the blood and internal organs of individ-

1 Frosch : Die Verbreitung des Diphtherie-bacillus im Kérper des Menschen.
Zeit. fir Hygiene und Infectionskrankheiten, 1893, Bd. xiii. p. 49-52

288 BACTERIOLOGY.

uals dead of the disease, but all that has been learned
from careful study of the secondary manifestations of
diphtheria tends to the opinion that they are in no way
dependent upon the immediate presence of bacteria, and
that the occasional appearance of diphtheria bacilli in
the internal organs is in all probability accidental, and
usually unimportant.

By special methods of inoculation’ (the injection of
fluid cultures into the testicles of guinea-pigs) diphtheria
bacilli can be caused to appear in the omentum, but this
is purely an artificial manifestation of the disease and
one that is probably never encountered in the natural
course of events. Very rarely similar results follow
upon subcutaneous inoculation.

If a very minute portion of either a solid or fluid
pure culture of this organism be introduced into the
subcutaneous tissues of a guinea-pig or kitten, death
of the animal ensues in from twenty-four hours to
five days. The usual changes are an extensive local
cedema with more or less hyperemia and ecchymosis
at the site of inoculation; swollen and reddened
lymphatic glands ; increased serous fluid in the perito-
neum, pleura, and pericardium ; enlarged and hemor-
rhagic supra-renal capsules ; occasionally slightly swollen
spleen ; and sometimes fatty degeneration in the liver,
kidney, and myocardium. In guinea-pigs, especially, the
liver often shows numerous macroscopic dots and lines on
the surface and penetrating the substance of the organ.
They vary in size from a pin-point to a pin-head and
may be even larger. They are white and do not
project above the surface of the capsule.

1 Abbott and Ghriskey: A Contribution to the Pathology of Experimental
Diphtheria. Johns Hopkins Hospital Bulletin, No. 30, April, 1893.

INOCULATIONS WITH B. DIPHTHERIA. 989

The bacilli are always to be found at the seat of in-
oculation, most abundant in the grayish-white fibrino-
purulent exudate. They become fewer at a distance
from this, so that the more remote parts of the cede-
matous tissues do not contain them. They are found
not only free, but contained in large number in leuco-
cytes, some of which have fragmented nuclei or have
lost their puclei. The bacilli within leucocytes, as well
as some outside, frequently stain very faintly and irreg-
ularly, and may appear disintegrated and dead.

Culture-tubes inoculated from the blood, spleen,
liver, kidneys, supra-renal capsules, distant lymphatic
glands, and serous transudates generally yield negative
results; and negative results are also obtained when
these organs are examined microscopically for the
bacilli.

Microscopic examination of the tissues at the seat of
inoculation, as well as of the liver, spleen, kidneys,
lymphatic glands and elsewhere, reveals the presence of
localized foci of cell death, characterized by a peculiar
fragmentation of the nuclei of the cells of these
parts.

This destruction of nuclei results in the occurrence
of groups of irregularly shaped, deeply staining bodies,
having at some times the appearance of particles of
dust, while again they may be much larger. Some of
them are tolerably regular in outline, while others are
irregularly crescentic, dumb-bell, flask-shape, whet-
stone-shape, or bladder-like in form. Occasionally
nuclei having the appearance of being pinched or drawn
out can be seen. Atsome points the fragments are
grouped into isolated masses indicating the location
of the nucleus from the destruction of which they

290 BACTERIOLOGY.

originated. These particles always stain much more
intensely than do the normal nuclei of the part. *

These peculiar alterations, as Oertel has shown, in
their distribution, are characteristic of human diphtheria,
and the demonstration of similar changes in animals
inoculated with this organism is important additional
proof that diphtheria is caused by it.

An affection may be produced by the inoculation of
certain animals that is in all respects identical with the
disease diphtheria as it exists in man. If one opens the
trachea of a kitten and rubs upon the mucous membrane
asmall portion of a pure culture of this organism, the
death of the animal usually ensues in from two to four
days. At autopsy the wound will be found covered with
a grayish, adherent, necrotic, distinctly diphtheritic layer.
Around the wound the subcutaneous tissues will be
cedematous. The lymphatic glands at the angle of the
jaws will be swollen and reddened. The mucous mem-
brane of the trachea at the point upon which the bacilli
were deposited will be covered with a tolerably firm,
grayish-white, loosely attached psendo-membrane in all
respects identical with the croupous membrane observed
in the same situation in cases of human diphtheria.
In the pseudo-membrane and in the cdematous fluid
about the skin-wound, bacilli diphtherie may be found
both in cover-slips and in cultures.

From what we have seen—the localization of the bacilli
at the point of inoculation, their absence from the internal
organs, and the changes brought about in the cellular
elements of the internal organs—there is but one inter-

1 See “ The Histological Changes in Experimental Diphtheria,’’ also “ The
Histological Lesions produced by the Toxalbumin of Diphtheria,” by Welch

and Flexner. Johns Hopkins Hospital Bulletin, August, 1891, and March,
1892,

°

INOCULATIONS WITH B. DIPHTHERLZ. 991

pretation for this process, viz.: that it is due to the pro-
duction of a soluble poison by the bacteria growing at
the seat of inoculation, which, gaining access to the cir-
culation, produces the changes that we observe in the
tissues of the internal viscera.

_ This poison has been isolated from cultures of the
bacillus diphtheriw, and is found to belong, not to the
crystallizable ptomaines, but to the toxic albumins—
bodies which, in their chemical composition, are anal-
ogous to the poison of certain venomous serpents.
By the introduction of this toralbwmin, as it is called,
into the tissues of guinea-pigs and rabbits, the same
pathological alterations may be produced that we have
seen to follow the result of inoculation with the bacilli
themselves, except, perhaps, the production of false
membrane.

Under certain circumstances with which we are not
acquainted the bacillus diphtherie becomes diminished
in virulence or may lose it entirely, so that it is no
longer capable of producing death of susceptible ani-
mals, but may cause only a transient local reaction
from which the animal entirely recovers. Sometimes
this reaction is so slight as to be overlooked, and again
careful search may fail to reveal evidence of any reac-
tion at all. The production of the extremes of its
pathogenic properties, viz., death of the animal, on the
one hand, and only very slight local effects on the other,
were at one time thought to indicate the existence of two
separate and distinct organisms that were alike in cultural
and morphological peculiarities, but which differed in
their disease-producing power. Further studies on this
point have, however, shown that the genuine bacillus
diphtherie may possess almost all grades in the degree

292 BACTERIOLOGY.

of its virulence, and that absence of or diminution in
virulence can hardly serve to distinguish as separate
species these varieties that are otherwise alike; more-
over, the histological conditions found at the site of
inoculation in animals that have not succumbed, but in
which only the local reaction has appeared, are in most
cases characterized by the same changes that are seen
at autopsy in animals in which the inoculation has
proven fatal.

In the course of their observations upon a large
number of cases, Roux and Yersin found that it was
not difficult to detect in the diphtheritic deposits of one
and the same individual bacilli of identical cultural
and morphological peculiarities, but of very different
degrees of virulence, and that with the progress of the
disease toward recovery the less virulent varieties often
became quite frequent.’

There is, moreover, a mild form of diphtheria affecting
only the mucous membrane of the nares, known as
membranous rhinitis, from which it is very common to
obtain cultures in all respects identical with those from
typical diphtheria, save for their power to kill suscepti-
ble animals. On inoculation these cultures produce
only local reactions, but these are characterized histo-
logically by the same tissue changes that follow inocu-
lation with the fully virulent organism.

Clinically, membranous rhinitis is never such an
alarming disease as is laryngeal or pharyngeal diph-
theria, and, as stated, the organisms causing it are often
of a low degree of virulence, though they are, never-
theless, genuine diphtheria bacilli.

1 It must not be assumed from this that the bacilli lose their virulence

entirely, or that they all become attenuated with the establishment of con-
valescence. :

INOCULATIONS WITH B. DIPHTHERIA. 993

For those organisms that are in all respects identical
with the virulent bacillus diphtheria, save for their in-
ability to kill guinea-pigs, the designation “ pseudo-
diphtheritic bacillus” is usually employed, but from
such observations as those just cited we are inclined to
the opinion that pseuco-diphtheritic, as applied to an
organism in all respects identical with the genuine
bacillus, except that it is not fatal to susceptible ani-
mals, is a misnomer, and that it would be more nearly
correct to designate this organism as the attenuated or
non-virulent diphtheritic bacillus, reserving the term
“pseudo-diphtheritic” for that organism or group of
organisms (for there are probably several) that is enough
like the diphtheria bacillus to attract attention, but is
distinguishable from it by certain morphological and
cultural peculiarities aside from the question of viru-
lence.

It is a well-known fact that many pathogenic organ-
isms—conspicuous among: these being the micrococcus
lanceolatus, the staphylococcus pyogenes aureus, and
the group of so-called hemorrhagic septicemia organ-
isms—undergo marked variations in the degree of their
pathogenic properties; and yet these organisms, when
found either devoid of this peculiarity, or possessing it
to a diminished degree, are not designated as “pseudo”
forms of these organisms, but simply as the organisms
themselves, the virulence of which, from various causes,
has been modified.

@

Nore.—Prepare cover-slip preparations from the
mouth-cavity of healthy individuals and from those
having decayed teeth. Do they correspond in any way
with those made from diphtheria? Do the same with

294 BACTERIOLOGY.

different forms of sore-throat. Do the peculiarities of
any of the organisms suggest those of the bacillus of
diphtheria? Wherein is the difference ?

In cultures and cover-slips made from both diphtheria
and from innocent sore-thrvats are there any organisms
which are almost constantly present? Which are they,
and what are their characteristics ?

Which are the predominating organisms in the an-
ginas of scarlet fever?

Do these organisms simulate, in their cultural and
morphological peculiarities, any of the different species
with which you have been working?

CHAPTER XXI.

Typhoid fever—Study of the organism concerned in its production—The
bacterium coli commune—Its resemblance to the bacillus of typhoid fever—
Its morphological, cultural, and pathogenic properties—Its differentiation
from the bacillus typhi abdominalis.

THE organism, discovered by Eberth and by Gaffky,
generally recognized as the etiological factor in the
production of typhoid fever, may be described as
follows :

It is a bacillus about three times as long as it is broad,
with rounded ends. It may appear at one time as very
short ovals, at another time as long threads, and both
forms may occur together. Its breadth remains toler-
ably constant. Its morphology presents little that will

Fia. 56. ‘ Fria. 57,

4

we oS
gn ihean y
i") NW

7
: HOY <S

Bacillus typhi abdominalis from Bacillus typhi abdominalis show-
culture twenty-four hours old, on ing flagella stained by Lioffler’s
agar-agar, method.

aid in its identification (see Fig. 56). It stains a trifle
less readily with the aniline dyes than do most of the
other organisms. It is very actively motile, and when

296 BACTERIOLOGY.

stained by the special method of Loffler (see this
method in chapter on Stainings) is seen to possess very
delicate locomotive organs in the form of fine, hair-like
flagella, which are given off in large numbers from all
parts of its surface (see Fig. 57). These flagella are
not seen in unstained preparations, nor are they ren-
dered visible by the ordinary methods of staining.

In patients suffering from this disease it has been
found during life in the blood, urine, and feces, and at
autopsies in the tissues of the spleen, liver, kidneys,
intestinal lymphatic glands, and intestines.

GELATIN PLATESs.—Its growth, when seen in the
depths of the medium, presents nothing characteristic, ap-
pearing simply as round or oval, finally granular points.
On the surface it develops as very superficial, blue-white
colonies, with irregular borders. They are a little denser
at the centre than at the periphery. When magnified,

Fia. 58.

oe)

Colony of bacillus typhi

inalis on gelatin.

the colonies present wrinkles or folds, which give to
them, in miniature, the appearance seen in the relief
maps made to represent mountainous districts. These
colonies have sometimes the appearance of flattened
pellicles of glass-wool, and usually present more or less
of a pearl-like lustre.

On AGAR-AGAR the colonies present nothing typical.

TYPHOID BACILLI IN MILK. 297

Stas CuLrures.—In stab cultures the growth is
mostly on the surface, there being only a very limited
development down the track made by the needle. The
surface growth has the same appearance in general as
that given for the colonies.

Porato.—The growth on potato is usually described
as luxuriant but invisible, making its presence evident
only by the production of a slight increase of moisture
at the inoculated point, and by a limited resistance
offered to the needle when scraped across the track
of Srowth. While this is true in most cases, yet it
cannot be considered as constant, for at times this
organism is seen to develop more or less visibly on
potato.

Porato GELATIN.—The growth is similar to that
upon ordinary nutrient gelatin.

Miix.—It does not cause coagulation when grown in
sterilized milk.

It does not liquefy gelatin.

It grows both with and without oxygen.

It does not produce indol. ,

In bouillon it causes a uniform clouding of the
medium and brings about a slightly acid reaction.

It does not grow rapidly.

It does not produce fermentation, and on lactose-
litmus-agar grows as pale-blue colonies, causing no
reddening of the surrounding medium. In the fermen-
tation tube, in glucose or lactose bouillon, no evolution
of gas as a result of fermentation occurs.

It does not form spores. The irregularities of stain-
ing so commonly seen in this organism have in some
instances led to the belief that the pale, unstained por-
tions of the bacilli indicate the presence of spores.

298 BACTERIOLOGY.

More reliable tests, however, have demonstrated the
error of this opinion.

It grows at any temperature between 20° C. and 38°
C., though more favorably at the latter point.

It is very sensitive to high temperatures, being killed
by an exposure of ten minutes to 60° C., and in a much
shorter time to slightly higher temperatures.

Owing to a tendency toward retraction of its proto-
plasm from the cell envelope and the consequent pro-
duction of vacuoles in the bacilli, the staining of this
organism is usually more or less irregular. At &me
points in a single cell marked differences in the intensity
of the staining will be seen, and here and there areas
quite free from color can commonly be detected. These
colorless portions are often so cleanly cut in outline as
to look as if they had been punched out with a sharp
instrument. (Diagrammatically represented in Fig. 59.)

Fic. 59,

Diagrammatic representation of retraction of protoplasm, with production
of pale points, in the bacillus typhi abdominalis.

PRESENCE IN TissuES.—It is not easy to demonstrate
this organism in tissues unless it is present in large
numbers. The manipulations to which the sections are
subjected in being mounted often rob the bacilli of their
staining, and render them invisible, or nearly so. If,
however, sections be stained in the carbolic-fuchsin solu-
tion, either at the ordinary temperature of the room
or at a higher temperature (40° to 45° C.), then washed

TYPHOID INOCULATION IN ANIMALS. 999

out in absolute alcohol, and cleared up in xylol and
mounted in balsam, the bacilli (particularly if the tissue
is the liver or spleen) can readily be detected massed
together in their characteristic clumps. If used in the
same way, the alkaline methylene-blue solution gives
also very satisfactory results.

In searching for the typhoid bacilli in tissues, their
mode of growth under these circumstances must always
be borne in mind, otherwise much labor will be ex-
pended in vain. In tissues the typhoid bacilli do not
lie scattered about in the same way as do the organisms
in tissues from cases of septicemia; they are not
regularly distributed along the course of the capillaries,
but are localized in small clumps through the tissues,
and it is for these clumps, which are easily detected
under the low-power objective, that one should search.
When the section is prepared for examination, if it be
gone over with the low-power objective, one will notice
at irregular intervals little masses that look in every
respect like particles of staining-matter which have been
precipitated upon the section at that point. When these
little masses are examined with a higher power objective
they will be found to consist of small ovals or short
rods so closely packed together that the individuals
composing the clump can often be seen only at the very
periphery of the mass. This is the characteristic ap-
pearance of the typhoid organism in tissues. The little
masses are usually in the neighborhood of a capillary.

Resuut oF INocuLation into LOWER ANIMALS.—
A great many experiments have been made with the
view of reproducing the pathological conditions of this
disease, as seen in man, in the tissues of lower animals,
but with limited success. Fatal results without the

300 BACTERIOLOGY.

appearance of the typical pathological changes have fre-
-quently followed these attempts, but in most cases they
could easily be traced to the toxic,’ rather than to the
truly infective? action of the materials introduced into
the animals.

The most successful efforts for the production of the
typical typhoid lesions in lower animals are those
reported by Cygneus. By the introduction of the
typhoid bacilli into the tissues of dogs, rabbits, and mice
he was able to produce in the small intestines conditions
that were histologically and to the naked eye analogous
to those found in the human subject.

Of a number of experiments made by the writer
with the same object in view, only one positive result
followed the introduction of typhoid bacilli into the
circulation of rabbits. In this case the ulcer in the
ileum was macroscopically and microscopically identical
with those found at autopsy in the small intestine of
the human subject dead of this disease. The typhoid
bacilli were not only obtained from the spleen of the
animal by culture methods, but were also demonstrated
microscopically in their characteristic clumps in sections
of the organ.

In connection with the inoculation of animals with
the bacillus typhi abdominalis, observations of a most
important nature have recently been made upon the
artificial induction of susceptibility to its pathogenic
action by Sanarelli.? He found that rabbits, guinea-
pigs, and mice could be rendered susceptible to in-

4
1 Toxic—Poisonous results not necessarily accompanied by the growth of
organisms throughout the tissues.
2 Infective or septic—Poisoning of the tissues as a result of the growth of
bacteria in them.
8 Sanarelli: Annales de l'Institut Pasteur, 1892, tome vi.

TYPHOID INOCULATION IN ANIMALS. 801

fection by this organism by injecting into them the
products of growth of certain saprophytes—proteus
vulgaris, bacillus prodigiosus, and bacterium coli com-
mune—and that by whatever means the animal was sub-
sequently inoculated with fresh cultures of the typhoid
bacillus, either into the circulation or into the perito-
neal cavity, death resulted in from twelve to forty-
eight hours, with the most conspicuous pathological
alterations in the digestive tract, and particularly in the
small intestines. In these cases the infection is general
and the organisms may be recovered from the blood
and internal organs. It is the opinion of Sanarelli
that the toxic conditions produced by the preliminary
injections of the products of growth of the saprophytic
organisms may be considered as analogous to a sim-
ilar condition that may occur in man from the ab-
sorption of abnormal products of fermentation from
the intestinal canal—an auto-intoxication that so re-
duces the resistance of the individual as to render him
susceptible to infection by the bacillus of typhoid fever,
should it gain access to his alimentary tract.

More recently it was found by Alessi! that rats,
guinea-pigs, and rabbits, when permitted to breathe the
gaseous products of decomposition from the contents of
a cesspool, or from other decomposing matters, grad-
ually became susceptible to infection by the typhoid
bacillus. After an exposure of from five to seventy-
two days in the case of rats, seven to fifty-eight days in
the case of guinea-pigs, and three to eighteen days in
the case of rabbits, the resistance of the animals was so
diminished that inoculation with relatively small

1 Alessi: Centralblatt fir Bakteriologie u. Parasitenkunde, 1894, Bd. xv.,
No. 7, p. 228.
14

302 BACTERIOLOGY.

amounts of cultures of the typhoid bacillus proved fatal
in from twelve to thirty-six hours. Autopsies upon
these animals revealed the presence of hemorrhagic
enteritis, hypertrophy of Peyer’s patches, and enlarge-
ment of the spleen. The bacilli were found in the
blood, liver, and spleen.

The importance of these observations in their bearing:
upon the etiology of typhoid fever, if they are demon-
strated by subsequent experiment to be trustworthy, is
too obvious to necessitate emphasis, and it is greatly to
be desired that they may not be permitted to pass
unnoticed, but that others interested may find occasion
to institute experiments in the same direction with the
hope that some light may be shed upon the mooted
question concerning the influence of gaseous products
of decomposition upon the health of individuals, and
particularly upon the part played by them in dimin-
ishing natural resistance to infection. —

Because of the variations in the morphology and cul-
tural peculiarities of this organism, and because of the
difficulty experienced in efforts to reproduce in lower
animals the conditions found in the human subject,
typhoid fever is bacteriologically one of the most un-
satisfactory of the infectious diseases.

There are a number of other organisms which botani-
cally appear to be so closely related to the typhoid bacil-
lus, and which, with our present methods for studying
them, so closely simulate it that the difficulty of identify-
ing this organism sometimes is very great. In addition
to this, the variability constantly seen in pure cultures
of the typhoid bacillus itself in no way renders the -
task more simple.

For example, the morphology of the typhoid bacillus

TYPHOID INOCULATION IN ANIMALS. 803

is conspicuously inconstant; its growth on potato,
which is usually given as characteristic, may, with the
same culture, at one time appear as the typical invisible
development, at another time it may grow in a way
easily to be seen with the naked eye; and the change of
reaction which it is said to produce in bouillon is some-
times much more intense than at others.

The only properties possessed by it that may be said
to be constant are its motility, its inability to cause
fermentation of glucose, lactose, and saccharose, its
incapacity for coagulating milk, the absence of indol-
production, and its growth on gelatin plates ; but there
are other organisms which approach these same charac-
teristics to a degree that renders their differentiation
from the typhoid organism often a matter that requires
the careful application of all these differential tests.

These points should be borne in mind in the exami-
nation of drinking-water supposed to be contaminated
by typhoid dejections, for the organisms which most
nearly approach the typhoid bacillus in growth and
morphology are just those organisms which would
appear in water contaminated from cesspools, i. e., the
organisms constantly found in the normal intestinal
tract. Even in the stools of typhoid-fever patients
the presence of these normal inhabitants of the intes-
tinal tract renders the isolation of the typhoid organisms
somewhat troublesome.

The spleen of a patient dead of typhoid fever is the
safest place from which to obtain cultures of this organ-
ism for study. But it must always be remembered that
the same channels through which the typhoid bacillus
gains access to this viscus are likewise open to other
organisms present in the intestines, and for this reason

304 BACTERIOLOGY.

the bacterium coli commune, a normal inhabitant of
the colon, may also be found in this locality.

Note.—Obtain a pure culture of typhoid bacilli, and
from this make inoculations upon a series of potatoes of
different age and from different sources. Do they all
grow alike?

Before sterilizing, render another lot of potatoes
slightly acid with a few drops of very dilute acetic
acid; render others very slightly alkaline with dilute
caustic soda. Do any differences in the growth result ?

Make a series of twelve tubes of peptone solution to
which rosolic acid has been added. Inoculate them all
with as near the same amount of material as possible
(one loopful from a bouillon culture into each tube) ;
place them all in the incubator. Is the color-change,
as compared with the control tube, the same in all cases?

Compare the morphology of cultures of the same age
on gelatin, agar-agar, and potato.

Select a culture in which the vacuolations are quite
marked. Examine this culture unstained. Do the or-
ganisms look as if they contained spores? How would
you demonstrate that the vacuolations are not spores ?

Obtain from the normal feces a pure culture of the
commonest organism present. Write a full description
of it. Now make parallel cultures of this organism and
of the typhoid organism on all the different media.
How do they differ? In what respects are they
similar ?

Bacrertum Cont Commune (colon bacillus ; bacillus
Neapolitanus of Emmerich).—This organism was found
by Escherich, in 1885, in the intestinal discharges of
milk-fed infants. It has since been demonstrated to

BACTERIUM COLI COMMUNE. 305

be a normal inhabitant of the intestines of man and of
certain domestic animals (cattle, hogs, dogs).

For a time after its discovery it was considered of
but little importance and attracted attention only
because of its resemblance, in certain respects, to the
bacillus of typhoid fever, with which it was occasionally
confounded. In this particular it is still a subject for
study, and some have even gone so far as to regard
them as modifications of the same species, though in
the present state of our knowledge this is certainly
overstepping the mark. That they possess in common
certain general points of resemblance and often approach
one another in some of their biological peculiarities is
true; but,as we shall learn, they each possess peculiarities
which, when taken together, render their differentiation
from one another a matter of but little difficulty.

With the wider application of bacteriological methods
to the study of pathological processes it was occasion-
ally observed that, under favorable circumstances, this
organism was disseminated from its normal habitat and
appeared in remote organs, often associated with diseased
conditions. This was also, at first, considered of but
trifling moment, and its presence in these localities was
usually explained as accidental. Its repeated appearance,
however, in different parts of the body outside of the
intestines and the frequency of its association with
pathological conditions ultimately attracted attention to
it, and in consequence during the past two or three
years a great deal has been written concerning the
possible pathogenic nature of this organism.

The fact that it is always with us in most intimate
association with certain of our life processes, together
with the fact that it is known to appear in organs other

306 BACTERIOLOGY.

than that in which it is normally located, and that its
occurrence in diseased conditions is not rare, justifies
the opinion that it is one of the most important of the
micro-organisms with which we have to deal.

While not generally considered to be a pathogenic
organism, there is, nevertheless, sufficient evidence to
warrant the statement that, under favorable conditions,
with which we are not entirely familiar, this organism
may assume pathogenic properties and that its presence
in diseased conditions is not always to be considered as
accidental, though this is frequently the case.

The morphological and cultural peculiarities of the
bacterium coli commune are as follows :

Morphology. In shape it is a rod with rounded
ends, sometimes so short as to appear almost spherical,
while again it is seen as very much longer threads.
Often both forms will be associated in the same culture.
It may occur as single cells or as pairs, joined end-to-
end. There is nothing to be said of its morphology
that can aid in its identification, for in this respect it
simulates many other organisms. It is usually said to
be motile, and undoubtedly is motile in the majority of
cases, but its movements are so sluggish that a positive
opinion is often difficult.

By Loffler’s method of staining, flagella can be
demonstrated, though not in such numbers as are seen
to occur on the typhoid fever bacillus.

It does not form spores.

Tt grows both with and without oxygen.

On gelatin. When on the surface its colonies appear
as small, dry, irregular, flat, blue-white points that are
commonly somewhat dentated at the margin. They
are a trifle denser at the centre than at the periphery,

BACTERIUM COLI COMMUNE. 807

and are often marked at or near the middle by an oval
or round nucleus-like mass—the original colony from
which the layer on the surface developed. When located
in the depths of the gelatin, and examined with a low-
power lens, they are at first seen to be finely granular
and of a very pale greenish-yellow color; later they
become denser, darker, and much more markedly
granular. In shape they are round, oval, and lozenge-
like. When the surface colonies are viewed under a
low power of the microscope they present essentially
the same appearance as that given for the bacillus of
typhoid fever, viz.: they resemble flattened pellicles of
glass-wall, or patches of finely ground colorless glass.
Colonies of this organism on gelatin are frequently
encountered that cannot be distinguished from those
resulting from the growth of the bacillus of typhoid
fever.

In stab and smear cultures on gelatin the surface
growth is flat, dry, and blue-white or pearl color.
Limited growth occurs along the track of the needle in
the depths of the gelatin. ‘As the culture becomes
older the gelatin round about the surface growth may
gradually lose its transparency and become cloudy,
often quite opaque. In still older cultures small roots,
or branch-like projections from the surface growth into
the gelatin are sometimes seen to occur.

Tt does not cause liquefaction of gelatin.

Its growth on nutrient agar-agar and on _blood-
serum is luxuriant but not characteristic.

In bouillon it causes diffuse clouding with sedi-
mentation. In some bouillon cultures an attempt at
pellicle formation on the surface may be seen, but this
is not always the case. In old bouillon cultures the

308 BACTERIOLOGY.

reaction is seen to have become alkaline, and a decided
ecalf odor may be detected.

It produces indol in bouillon and in peptone solution.

Its growth on potato is rapid and voluminous, appear-
ing after twenty-four to thirty-six hours in the incubator
as a more or less lobulated layer of a drab, dark-cream,
or brownish-yellow color.

In neutral milk containing a little litmus tincture
the blue color is changed to red after from eighteen to
twenty-four hours in the incubator, and in addition the
majority of cultures cause a firm coagulation of the
casein in about thirty-six hours, though frequently
this takes longer. Very rarely, the litmus may indicate
the production of acid and no coagulation occur.

In media containing glucose it grows rapidly and
causes active fermentation with liberation of carbonic
acid and hydrogen. If cultivated in solid media to
which glucose (2 per cent.) has been added, the gas-
formation is recognized by the appearance of numerous
bubbles along and about the track of the growth. If
cultivated in fluid media, also containing glucose, in the
fermentation tube, evidence of fermentation is given
by the collection of gas in the closed arm of the tube.

On lactose-litmus-agar its colonies are pink and the
color of: the surrounding medium is changed from blue
to red.

In Dunham’s peptone solution it produces indol in
from forty-eight to seventy-two hours.

It stains with the ordinary aniline dyes. It is de-
colorized when treated with iodine after having been
stained by the method of Gram.

By comparing what has been said of the bacillus typhi
abdominalis and of the bacterium coli commune it will

ANIMAL INOCULATIONS. 309

be seen that while they simulate each other in certain
respects they still possess individual characteristics by
which they may readily be differentiated. The most
important of the differential points are :

1. Motility of the bacillus typhi abdominalis is much
more conspicuous, as a rule, than is that of the bacterium
coli commune.

2. On gelatin the colonies of the typhoid bacillus
develop more slowly than do those of the colon
bacillus.

3. On potato the growth of the typhoid bacillus is
usually invisible (though not always), while that of the
colon bacillus is rapid, luxuriant and always visible.

4, The typhoid bacillus does not cause coagulation of
milk with acid reaction. The colon bacillus does this
in from thirty-six to forty-eight hours in the incubator.

5. The typhoid bacillus never causes fermentation,
with liberation of gas, in media containing glucose,
lactose, or saccharose. The colon bacillus is conspicuous
for its power of causing fermentation in such solu-
tions.

6. In nutrient agar-agar or gelatin containing lactose
and litmus tincture, and of a slightly alkaline reaction,
the color of the colonies of typhoid bacillus is blue,
and there is no reddening of the surrounding medium,
while the colonies of the colon bacillus are pink and the
medium round about them becomes red.

7. The typhoid bacillus does not possess the property
of producing indol in solutions of peptone; the
growth of the colon bacillus in these solutions is
accompanied by the production of indol in from forty-
eight to seventy-two hours at 37° to 38° C.

Animal inoculations. As with the bacillus of typhoid

14*

310 BACTERIOLOGY.

fever, the results of inoculation of animals with cultures
of this organism cannot be safely predicted. According
to the observations of Escherich, Emmerich, Weisser, and
others, the results that do appear are in most instances
to be attributed to the toxic rather than to the infective
properties of the culture used.

When introduced into the subcutaneous tissues of
mice it has no effect, while similar inoculations of
guinea-pigs are sometimes (not always) followed by
abscess formation at the point of injury, or by altera-
tions very similar to those produced by intra-vascular
inoculation, viz.: death in less than twenty-four hours,
accompanied by redness of the peritoneum and marked
hyperemia and ecchymoses of the small intestine ;
together with swelling of Peyer’s patches. The cecum
and colon may remain unchanged or present enlarged
follicles. There may or may not be an accumulation
of fluid in the abdominal cavity, but peritonitis is
rarely present. The small intestines may contain
bloody mucus.

Intra-venous inoculation of rabbits may be followed
by similar changes with often the occurrence of diar-
rhcea before death, which may, in the acute cases, result
in from three to forty hours. In another group of
cases acute fatal intoxication does not result, and the
animal lives for weeks or months, dying ultimately of
what appears to be the effects of a slow or chronic form
of infection. For a few hours after inoculation these
animals present no marked symptoms; exceptionally
somnolence and diarrhoea have been observed at this
period, indicating acute intoxication from which the
animal has recovered. The affection is unattended by
fever. The most marked symptom is loss of weight.

’

ANIMAL INOCULATIONS. 311

This is usually progressive from the first or second day
after inoculation, with slight fluctuations until death.

At autopsy the animal is found to be emaciated.
The subcutaneous tissues and the muscles appear pale
and dry. The serous cavities, particularly the peri-
cardial, may contain some excess of serum. The viscera
are anemic. The spleen is small, thin, and pale.
Exceptionally ulcers and ecchymoses are observed in
the cecum, but generally there are no lesions of the
intestinal tract.

The most striking and constant lesions, those most
characteristic of the affection, are in the bile and in the
liver ; the quantity of bile may not exceed the normal,
but in other cases the gall-bladder may be abnormally
distended with bile. The bile is nearly colorless or has
a pale yellowish or brownish tint, with little or none of
a greenish color. Its consistence is much less viscid
than normal, being often thin and watery. It usually
contains small, opaque, yellowish particles or clumps
which can be seen floating in it, even through the walls
of the gall-bladder. These clumps consist microscopi-
cally of bile-stained, apparently necrotic, epithelial
cells ; leucocytes in small numbers; amorphous masses
of bile pigment, and bacteria often in zodgloea-like
clumps. Similar material is found in the larger bile-
ducts.

The liver frequently contains opaque, whitish or
yellowish-white spots and streaks of irregular size and
shape, which give a peculiar mottling to the organ when
present in large numbers. These areas may be numer-.
ous, or only one or two may be found. In size they
range from minute points to areas of from 2 to 3 cm.
in extent.

312 BACTERIOLOGY.

By microscopic examination they are found to repre-
sent places where the liver cells have undergone necrosis
accompanied with emigration of leucocytes, and the
cells about them are ina condition of fatty degeneration.

In sections of the liver, masses of the bacilli may be
discovered in and about the necrotic foci just described.

At these autopsies the colon bacillus is not found
generally distributed through the body, but is only to
be detected in the bile, liver, and occasionally in the
spleen.’

1 Consult paper by Blachstein on this subject. Johns Hopkins Hospital
Bulletin, July, 1891.

CHAPTER XXII.

The spirillum (comma bacillus) of Asiatic cholera—Its morphological and
cultural peculiarities—Pathogenic properties—The bacteriological diagnosis of
Asiatic cholera.

Av the conference held in Berlin in 1884 for the
purpose of discussing the cholera question, it was
announced by Koch’ that he had discovered in the
intestinal evacuations of individuals suffering from
Asiatic cholera a micro-organism that he believed to
be the cause of the malady. The importance of this
statement necessarily attracted widespread attention to
the subject, and as one of the results there existed,
for a short time following, some skepticism as to the
accuracy of Koch’s claim. These doubts arose as a
result of a series of contributions from other observers
who endeavored to prove that the organism found by
Koch in cholera evacuations was one that is common
to other localities, and not a specific accompaniment
of this disease. It was not very long, however,
before it was evident that the objectiors raised by
the opponents of Koch were based upon untrustworthy
observations, and that by reliable methods of investi-
gation the organism to which he had called attention
could be easily differentiated from either and all of those
with which it was claimed to be identical.

This organism, known as the spirillum of Asiatic
cholera and as Koch’s “comma bacillus,” because of its
morphology, is identified by the following peculiarities :

1 Verhandlungen der Conferenz zur Eriérterung der Cholerafrage, 1884,
Berlin.

314 BACTERIOLOGY.

THE MORPHOLOGICAL AND BIOLOGICAL PECULIARITIES
OF THE SPIRILLUM OF ASIATIC CHOLERA.

_ Morphology. It is a slightly curved rod of about
from 0.8 to 2.0 » in length and from one-sixth to one-
third in thickness ; that is to say, it is from about one-
half to two-thirds the length of the tubercle bacillus,
but is thicker and plumper. Its curve is usually not
more marked than that of a comma, and, indeed, it isoften
almost straight; at times, though, the curve is much
more pronounced, and may even describe a semicircle.
Occasionally the curve may be double, one comma
joining another, with their convexities pointing in
opposite directions, so that a figure similar to the letter
S is produced. In cultures, long spiral or undulating
threads may often be seen. From these appearances
this organism cannot be considered a bacillus, but rather
an intermediate type between the bacilli and the spirilla.
Koch thinks it not improbable that the short comma
forms represent segments of a true spirillum, the normal
form of the organism. (Fig. 60.)

Fig. 60,
: cans 4 .
Ne A WW <y
Ka W () as N
er A gis KS
oth wg es
BING oe
Spirillum of Asiatic cholera, Impression cover-slip from a
colony thirty-four hours old.
It does not form spores, and we have no reliable
evidence that it possesses the property of entering at

‘CHOLERA SPIRILLUM: MORPHOLOGY. 815

any time a stage when its powers of resistance to detri-
mental agencies are increased.

It is a flagellated organism, but has only a single
flagellum attached to one of its ends.

It is actively motile, especially in the comma stage,
though the long spiral forms also possess this property.

Grouping. As found in the slimy flakes in the intes-
tial discharges from cholera patients, Koch likens its
mode of grouping to that seen in a school of small fish
when swimming up stream, #. ¢., they all point in nearly
the same direction and lie in irregularly parallel, linear
groups that are formed by one comma being located
behind the other without being attached to it.

Fre, 61.
B.
8 fo
a A e
Gir. ne
’ awe
An (> We ~ §
=> a ca
4% Marty
| ae ae 2
Yeh Sg ¢! ee
wae fg a:
€ = wal Te

Involution forms of the spirillum of Asiatic cholera, as seen in old cultures.

On cover-slip preparations made from cultures in the
ordinary way there is nothing characteristic about the
grouping, but in impression cover-slips made from
young cultures the short commas will nearly always be
seen in small groups of three or four, lying together in
such a way as to have their long axes nearly parallel
to one another. (See Fig. 60.)

In old cultures in which development has ceased, it
undergoes degenerative changes, and the characteristic

.

316 BACTERIOLOGY.

comma and spiral shapes may entirely disappear, their
place being taken by irregular involution forms that
present every variety of outline. (See Fig. 61.) In
this stage they take on the staining very feebly, and
often not at all.

Cultural peculiarities. On plates of nutrient gelatin
that have been prepared from a pure culture of this
organism and kept at a temperature of from 20° to 22°
C., development can often be observed after as short a
period as twelve hours, but frequently not before sixteen
to eighteen hours. This is especially true of the first
or “original” plate, containing the largest number of
colonies. At this time the plate will present_to the
naked eye an appearance that has been likened to a
ground-glass surface, or to a surface that has been
stippled with a very finely pointed needle, or one
upon which very fine dust has been sprinkled. This
appearance is due to the presence of minute colonies
closely packed together upon the surface of the gelatin.
In the depth of the gelatin can also be seen, closely
packed, small points, likewise representing growing
colonies. As growth progresses liquefaction occurs
around the superficial colonies, and in consequence this
plate is usually entirely liquid after from twenty-four
to thirty hours; the developmental phases through
which the colonies pass cannot, therefore, be studied
upon it.

On plates 2 and 3, where the colonies are more widely
separated, they can be seen after twenty-four to thirty
hours as small, round, or oval, white or cream-white
points, and whey located superficially there can be
detected around them a narrow transparent zone of
liquefaction. As growth continues, this liquefaction

CULTURE OF THE CHOLERA SPIRILLUM. 317

extends downward rather than laterally, and the colony
ultimately assumes the appearance of a dense, white
mass lying at the bottom of a sharply-cut pit or funnel
containing transparent fluid. This liquefaction is never
very widespread nor rapid, and rarely extends for more
than one millimetre beyond the colony proper. On
plates containing few colonies there is but little or no
tendency for them to become confluent, and, as a rule,
they do not exceed 2 to 3 mm. as an average diameter.

Fic. 62.

Developmental stages of colonies of the spirillum of Asiatic cholera at
20° to 22°C. on gelatin. X about 75 diameters.

a. After sixteen to eighteen hours. b. After twenty-four to twenty-six
hours. c. After thirty-eight to forty hours. d. After forty-eight to fifty
hours. e. After sixty-four to seventy hours.

When examined under a low magnifying lens the
very young colonies (sixteen to eighteen hours) appear
as pale, translucent, granular globules of a very delicate
greenish or yellowish-green color, sharply outlined and
not perfectly round. (See a, Fig. 62.) As growth
progresses, this homogeneous granular appearance is
replaced by an irregular lobulation, and ultimately the
sharply-cut margin of the colony becomes dentated or

818 BACTERIOLOGY.

scalloped. (See b and ¢, Fig. 62.) After forty-eight
hours (and frequently sooner), liquefaction of the gelatin
has taken place to such an extent that the appearance.
of the colony is entirely altered. Under the magnify-
ing glass the colony proper is now seen to be torn and
ragged about its edges, while here and there shreds of
the colony can be detected scattered through the liquid
into which it is sinking. These shreds evidently repre-
sent portions of the colony that have become detached
from its margin as it gradually sank into the liquefied
area.

At d, in Fig. 62, will be seen a representation of the
several appearances afforded by the colonies at this
stage. At the end of the second, or during the early
part of the third day the sinking of the colonies into
the liquefied pits, resulting from their growth, is about
complete, and under a low lens they now appear as
dense, granular masses, surrounded by an area of lique-
faction through which can be seen granular prolonga-
tions of the colony, usually extending irregularly between
the periphery and the central mass. (See e, Fig. 62.) If
the periphery be examined, it will be seen to be fringed
with delicate, cilia-like lines that radiate from it in
much the same way that cilia radiate from the ends of ..
certain columnar epithelial cells.

These are the more marked phases through which
he colonies of this organism pass in their development
on gelatin plates. With some cultures the various ap-
pearances here given appear more quickly, while in
cultures from other sources they may be somewhat
retarded.

On plates of nutrient agar-agar the appearance of the
colonies is not characteristic. They appear as round or

CULTURE OF THE CHOLERA SPIRILLUM. 319

oval patches of growth that are moist and tolerably
transparent. The colonies on this medium at 37° C,
naturally grow to a larger size than do those upon
gelatin at 22° C.

In stab cultures in gelatin there appears at the top
of the needle track after thirty-six to forty-eight hours

Stab cultures of the spirillum of Asiatic cholera in gelatin,
at 18° to 20° C.

a. After twenty-four hours. 6. After forty-eight hours. ¢. After seventy-
two hours. d, After ninety-six hours.

at 22° C. a small, funnel-shaped depression. As the
growth progresses, liquefaction will be seen to occur
about this point. In the centre of the depression can
be distinguished a small, dense, whitish clump, the

320 BACTERIOLOGY.

colony itself. As growth continues the depression
increases in extent and ultimately assumes an appear-
ance that consists in the apparent sinking of the liquefied
portion in such a way as to leave a perceptible air-space
between the top of the liquid and the surface of the solid
gelatin. The growth now appears to be capped by a
small air-bubble. The impression given by it at this
stage is not only that there has been a liquefaction, but
also a coincident evaporation of the fluid from the
liquefied area and a constriction of the superficial open-
ing of the funnel. (See a, b,c, and d, Fig. 63.) Lique-
faction is not especially active along the deeper portions
of the track made by the needle, though in stab cultures
in gelatin the liquefaction is much more extensive than
that usually seen around colonies on plates. It spreads
laterally at the upper portion, and after about a week
a large part of the gelatin in the tube may have become
fluid, and the growth loses its characteristic appearance.

Stab- and smear-cultures on agar-agar present noth-
ing characteristic. They are usually only an exaggera-
tion of the appearance afforded by the single colonies
on this medium.

Its growth in bouillon is luxuriant, causing a diffuse
clouding and the ultimate production of a delicate film
upon the surface.

In sterilized milk of a neutral or amphoteric reac-
tion at a temperature of 36°-38°C. it develops actively,
and gradually produces an acid reaction with coagula-
tion of the casein. It retains its vitality under these
conditions for about three weeks or more. The blue
color of milk to which neutral litmus tincture has been
added is changed to pink after thirty-six or forty-eight
hours at body temperature.

CULTURE OF THE CHOLERA SPIRILLUM. 321

Its growth in peptone solution, either that of Dun-
ham (see Special Media) or the one preferred by Koch,
viz., 2 parts Witte’s peptone, 1 part sodium chloride,
and 100 parts distilled water, is accompanied by the
production of both indol and nitrites, so that after
eight to twelve hours in the incubator at 37° C. the
rose color characteristic of indol appears upon the
addition of sulphuric acid alone. (See Indol Reac-
tion.)

In peptone solution to which rosolic acid has been
added the red color is very much intensified after four
or five days at 37° C.

Its growth on potato of a slightly acid reaction is
seen after three or four days at 37° C. as a dull, whit-
ish, non-glistening patch at and about the site of inoc-
ulation. It is not elevated above the surface of the
potato, and can only be distinctly seen when held to
the light in a particular position. Growth on acid
potato occurs, however, only at or near the body tem-
perature, owing probably to the acid reaction, which is
sufficient to prevent development at a lower tempera-
ture, but does not have this effect when the temperature
is more favorable.

On solidified blood-serum the growth is usually said
to be accompanied by slow liquefaction. I have not
succeeded in obtaining this result on Léffler’s serum,
nor have I detected anything characteristic about its
growth on this medium.

The temperature most favorable for its growth is
between 35° and 38°C. It grows, but more slowly,
at 17° C. Under 16° C. no growth is visible.

It is not destroyed by freezing. When exposed to
65° C. its vitality is destroyed in five minutes.

322 BACTERIOLOGY.

It is strictly aérobic, its development ceasing if the
supply of oxygen is cut off.

Tt does not grow in an atmosphere of carbonic acid,
but is not killed by a temporary exposure to this gas.
It does not grow in acid media, but flourishes best in
media of neutral or slightly alkaline reaction. It is so
sensitive to the action of acids that, at 22° C., its devel-
opment is arrested when an acid reaction equivalent to
0.066 to 0.08 per cent. hydrochloric or nitric acid is
present (Kitasato).

In cultures, the development of this organism
reaches its maximum relatively quickly, then remains
stationary for a short period, after which degeneration
begins. The dying comma bacilli become altered in
appearance and assume the condition known as “involu-
tion forms.” (See Fig. 61.) When in this state they
take up coloring reagents very faintly or not at all, and
may lose entirely their characteristic shape.

When present with other bacteria, under conditions
favorable to growth, the comma bacillus at first grows
much more rapidly than do the others; in twenty-four
hours it will often so outnumber the other organisms
present that microscopic examination would lead one
to think that the material under consideration was a
pure culture of this organism. This, however, does not
last longer than two or three days; they then begin to
die, and the other organisms gain the ascendency. This
fact has been taken advantage of by Schottelius' in the
following method devised by him for the bacteriological
examination of dejections from cholera patients :

In dejections that are not examined immediately

1 Deutsche med. Wochenschrift, 1885, No. 14.

CULTURE OF THE CHOLERA SPIRILLUM. 393

after being passed it is often difficult, because of the
large number of other bacteria that may be present, to
detect with certainty the cholera organism by micro-
scopical examjnation. It is advantageous in these cases
to mix the dejections with about double their volume
of slightly alkaline beef tea, and allow them to stand
for about twelve hours at a temperature of between 30°
and 40° C. There appears at the end of this time,
especially upon the surface of the fluid, a conspicuous
increase in the number of comma bacilli, and cover-slip
preparations made from the upper layers of the fluid
will reveal an almost pure culture of this organism.

It is not improbable that a similar process occurs in the
intestines of those suffering from Asiatic cholera, viz.:
a rapid multiplication of the comma bacilli that haye
gained access to the intestines takes place, but lasts for
only a short time, when the comma bacilli begin to dis-
appear, and after a few days their place is taken by
other organisms.

In connection with his experiments upon the poison
produced by the cholera organism, Pfeiffer’ states that
in very young cultures, grown under the access of
oxygen, there is present a poisonous body that possesses
intense toxic properties. This primary cholera-poison
stands in very close relation to the material com-
posing the bodies of the bacteria themselves, and is
probably an integral constituent of them, for the
vitality of the cholera spirilla can be destroyed by
means of chloroform and thymol, and by drying, with-
out, apparently, any alteration of this poisonous body.
Absolute alcohol, concentrated solutions of neutral

1 Zeitschrift f. Hygiene u. Infectionskrankheiten, Bd. xi, p. 398.

324 BACTERIOLOGY.

salts, and a temperature of 100° C., decompose this
substance, leaving behind secondary poisons which
possess a similar physiological activity, but only when
given in from ten to twenty times the dose necessary
to produce the same effects with the primary poison.

Other members of the vibrio family also, namely,
the vibrio Metchnikovi and that of Finkler and
Prior (see description of these species), contain, accord-
ing to Pfeiffer, closely related poisons.

Experiments upon animals. As a result of ex-
periments for the purpose of determining if the disease
can be produced in any of the lower animals, it is
found that white mice, monkeys, cats, dogs, poultry,
and many other animals, are not susceptible to in-
fection by the methods usually employed in inoculation
experiments. When animals are fed on pure cultures
of the comma bacillus no effect is produced, and the
organisms cannot be obtained from the stomach or
intestines; they are destroyed in the stomach and do
not reach the intestines; they are not demonstrable in
the feces of these animals. Intra-vascular injections
of pure culture into rabbits are followed by a temporary
illness, from which the animals usually recover in from
two to three days; intra-peritoneal injections into
white mice are, as a rule, followed by death in from
twenty-four to forty-eight hours; the conditions in
both instances most probably resulting from the toxic
activities of the poisonous products of growth of the
organism that are present in the culture employed.
None of the lower animals have ever been known to
suffer from cholera spontaneously.

The experiments of Nicati and Rietsch, in which the
common bile-duct was ligated, and fluid cultures of

EXPERIMENTS WITH CHOLERA SPIRILLUM. 395

the organism were injected directly through the walls
of the duodenum, demonstrated the fact that the acid
reaction of the gastric juice destroyed the cholera
organisms and prevented their access to the small in-
testine when they had been administered by the mouth ;
at the same time it was seen that the interference with
the flow of bile diminished intestinal peristalsis, and
permitted the organisms to remain for a longer time
where they had been deposited. By. this method
Nicati and Rietsch,’ Van Ermengen,’ Koch,3 and others
were enabled to produce in the animals upon which they
operated a condition that was, if not identical, at all
events very similar pathologically to that seen in the
intestine of subjects dead of the disease.

At a subsequent conference held in Berlin in 1885,
Koch‘ described the following method by means of which
he had been able to obtain a relatively high degree of
constancy in all his efforts to produce cholera in lower
animals : Bearing in mind the point made by Nicati and
Rietsch as to the effect produced by the acid reaction of
the gastric juice, this reaction was first to be neutralized
by injecting, through a soft catheter passed down the
cesophagus into the stomach, 5 c.c. of a 5 per cent.
solution of sodium carbonate. Ten or fifteen minutes
later this was to be followed by the injection into the
stomach (also through a soft cathether) of 10 cc. of a
bouillon culture of the cholera spirillum. For the
purpose of arresting peristalsis and permitting the
organisms to remain in the stomach and upper.part of the

1 Archiv de Phys. norm. et path., 1885, xvii., 3e sér., t. vi. Compt.-rend.,
xcix. p. 928. Rev. de Hygiéne, 1885. Rev. de Méd., 1885. v.

2 « Recherches sur le Microbe du Choléra Asiatique,” Paris-Bruxelles, 1885.
Bull. de l’Acad roy. de Méd. de Belgique, 3e sér., xviii.

3 Loe. cit. is + Loc. cit., 1885,

326 BACTERIOLOGY.

duodenum for as long a time as possible, the animal was
to receive, immediately following the injection of the
culture, an intra-peritoneal injection, by means of a
hypodermatic syringe, of 1 c.c. of tincture of opium
for each 200 grammes of its body weight. Shortly
after this last injection a deep narcosis sets in and lasts
from a half to one hour, after which the animal is
again as lively as ever. Of 35 guinea-pigs inoculated
in this way by Koch, 30 died of a condition that was,
in general, very similar to that seen in Asiatic cholera.

The condition of these animals before death is de-
scribed as follows: Twenty-four hours after the opera-
tion the animal appears sick ; there is a loss of appetite,
and the animal remains quiet in its cage. On the
following day a paralytic condition of the hind extre-
mities appears, which, as the day goes on, becomes more
pronounced ; the animal lies quite flat upon its ab-
domen or on its side, with its legs extended ; respiration
is weak and prolonged, and the pulsations of the heart
are hardly perceptible; the head and extremities are
cold, and the body temperature is frequently subnormal.

The animal usually dies after remaining in this
condition for a few hours.

At autopsy the small intestine is found to be deeply
injected and filled with a flocculent, colorless fluid.
The stomach and intestines do not contain solid masses,
but fluid; when diarrhcea does not occur, firm scybala
may be expected in the rectum. Both by microscopic
examination and by culture methods, comma bacilli are
found to be present in the small intestine in practically
pure culture.

More recently Pfeiffer’ has determined that essentially

1 Zeitschrift fiir Hygiene, Bd., xi. and xiv.

CHOLERA: GENERAL CONSIDERATIONS. 397

similar constitutional effects may be produced in guinea-
pigs by the intra-peritoneal injection of relatively large
numbers of this organism. His plan is to scrape
from the surface of a fresh culture on agar-agar as
much of the growth as can be held upon a moderate
sized wire loop. This is then finely divided in 1 e.c.
of bouillon and, by means of a hypodermatic syringe,
is injected directly into the peritoneal cavity. When
virulent cultures have been used this is quickly followed
by a fall in the temperature of the animal; this is
gradual and continuous until death ensues, which is
usually in from eighteen to twenty-four hours after the
operation, though exceptionally cases do occur in which
the animal recovers, even after having exhibited marked
symptoms of most profound toxemia.

General considerations. In all cases of Asiatic cholera,
and only in this disease, the organism just described can
be detected in the intestinal evacuations. The more
acute the case.and the more promptly the examination
is made after the evacuations have been passed from the
patient, the less will be the difficulty experienced in
detecting the organism.

In some cases it can be detected in the vomited
matters, though by no means so constantly as in the
intestinal contents.

As a rule, bacteriological examination fails to reveal
the presence of the organisms in the blood and internal
organs in this disease, though Nicati and Rietsch claim
to have obtained them from the common bile-duct in
rapidly fatal cases, and in two out of five cases they
were present in the gall-bladder. Doyen and Rasst-
schewsky' found them in the liver in pure cultures,

1 Reference to Vratch, 1885, in Allg. Med. Central. Zeitung, Berlin.

328 BACTERIOLOGY.

and Tizzoni and Cattani! in both the blood and the
gall-bladder.

The cholera spirillum is a facultative parasite; that
is to say, it apparently finds in certain portions of the
world, particularly in those countries in which Asiatic
cholera is endemic, conditions that are not entirely un-
favorable to its development outside of the body. This
has been found to be the fact not only by Koch, who
detected the presence of the organism in the water-tanks
in India, but by many other observers who have suc-
ceeded in demonstrating its growth under conditions not
embraced in the ordinary methods that are employed
for the cultivation of bacteria.

The results of experiments having for their object
the determination of the length of time during which
this organism may retain its vitality in water are con-
spicuous for their irregularity. In the transactions ot
the congress in Berlin, for the discussion of the cholera
question, it is stated, in connection with this point,
that the experiments made with tank-water in India
sometimes resulted in demonstrating the multiplication
of the organisms introduced into it, while in other cases
they died very quickly.

On February 8, 1884, comma bacilli were found in
the tank at Saheb-Began, in Calcutta, and it was pos-
sible to demonstrate them in a living condition up to
February 23d.

Koch states that in ordinary spring-water or well-
water the organisms retained their vitality for thirty
days, whereas in the canal-water (sewage) of Berlin they
died after six or seven days; but if this latter were mixed
with fecal matters, the organisms retained their vitality

1 Centralblatt f. die med. Wissenschaften, 1886, No. 43.

CHOLERA: GENERAL CONSIDERATIONS. 3929

for but twenty-seven hours; and in the undiluted con-
tents of cesspools it is impossible to demonstrate them
after twelve hours. In the experiments of Nicati and
Rietsch they retained their vitality in sterilized distilled
water for twenty days; in Marseilles canal-water (sew-
age), for thirty-eight days; in sea-water, sixty-four
days; in harbor-water, eighty-one days, and in bilge-
water, thirty-two days.

In the experiments of Hochstetter, on the other
hand, they died in distilled water in less than twenty-
four hours in five of seven experiments; in one of the
two remaining experiments they were alive after a day,
and in the other after seven days.

In one experiment with the domestic water supply ot
Berlin the organism retained its vitality for 267 days ;
in another for 382 days, notwithstanding the fact that
many other organisms were present at the same time.
There is no single ground upon which these variations
can be explained, for they depend apparently upon a
number of factors which may act singly or together.
For example, in general it may be said that the higher
the temperature of the water in which these organisms
are present, up to 20° C., the longer do they retain their
vitality ; the purer the water, that is, the poorer in
organic matters, the more quickly do the organisms die,
whereas the richer it is in organic matter the longer do
they retain their vitality.

Still another point that must be considered ‘in this
connection is the antagonistic influences under which
they find themselves when placed in water con-
taining large numbers of organisms that are, so to
speak, at home in water—the so-called normal water
bacteria.

330 BACTERIOLOGY.

The effect of light upon growing bacteria must not
be lost sight of, for it has been shown that a surpris-
ingly large number of these organisms are robbed of
their vitality by a relatively short exposure to the rays
of the sun, and it is, therefore, not unlikely that the
non-observance of this fact may be, in part at least,
accountable for some of the discrepancies that appear in
the results of these experiments.

In his studies upon the behavior of pathogenic and
other micro-organisms in the soil, Carl Fraenkel’ found
that the cholera spirillum was not markedly susceptible
to those deleterious influences that cause the death of
a number of other pathogenic organisms. During the
months of August, September, and October, cultures
of the comma bacillus that had been buried in the
ground at a depth of three metres retained their
vitality ; on the other hand, in other months, particu-
larly from April to July, they lost their vitality when
buried to the depth of only two metres. Ata depth
of one and a half metres vitality was not destroyed,
and there was a regular development in cultures so
placed.

Asa result of experiments performed in the Imperial
Health Bureau, at Berlin, it was found that the bodies
of guinea-pigs that had died of cholera induced by
Koch’s method of inoculation contained no living
cholera spirilla when exhumed after having been buried
for nineteen days in wooden boxes, or for twelve days
in zinc boxes. In a few that had been buried in moist
earth, without having been encased in boxes, when ex-
humed after two or three months, the results of exami-
nations for cholera spirilla were likewise negative.

1 Zeitschrift f. Hygiene, Bd. ii. p. 521.

CHOLERA: GENERAL CONSIDERATIONS. 33]

Kitasato,’ in his experiments with the cholera organ-
ism, found that when mixed with the intestinal evacua-
tions of human beings under ordinary conditions they
lost their vitality in from a day and a half to three
days. If the evacuations were sterilized before the
cultures were mixed with them, the organisms retained
their vitality up to from twenty to twenty-five days.
He was unable to come to any definite conclusion as
to the cause of these phenomena.

It was demonstrated by Hesse* and by Celli® that
many substances commonly employed as food-stufts
offer a favorable nidus upon which the cholera organism
may develop. In his experiments upon its behavior in
milk, Kitasato* found that at a temperature of 36° C.
the cholera spirillum developed very rapidly during the
first three or four hours, and outnumbered the other
organisms commonly found in milk. They then dimin-
ished in number from hour to hour as the acidity of
the milk increased, until finally their vitality was lost ;
at the same time the common saprophytic bacteria
increased in number. Relatively the same process
occurs at a lower temperature, from 22° to 25° C., but
the process is slower, the maximum development of the
cholera organism being reached at about the fifteenth
hour, after which time they were overgrown by the
ordinary saprophytes present. .

From this it would seem that the vitality of the
cholera spirillum in milk depends largely upon the
reaction: the more quickly the milk becomes sour,
the more qtickly does the organism become inert,

1 Zeitschrift fir Hygiene, Bd. v. p. 487. 2 Thid., Bd. v. p. 527.
% Bolletino della R. Accad. Med. di Roma, 1888,
4 Zeitschrift f. Hygiene, Bd. y. p. 491.

332 BACTERIOLOGY.

while the longer the milk retains its neutral or only
very slightly acid reaction, the longer do the cholera
organisms that may be present in it retain their power
of multiplication.

According to Laser,' the cholera organism retains its
vitality in butter for about seven days; it is therefore
possible for the disease to be contracted by the use of
butter that has in any way been in contact with cholera
material.

In regard to the antagonism between the cholera
spirillum and other organisms with which it may come
in contact, the experiments of Kitasato? led him to
conclude that no organism has been found which,
when growing in the same culture medium with it,
possessed the power of depriving it of its vitality
within a short time. On the other hand, the experi-
ments showed that there were quite a number of other
organisms the development of which was checked, and
in some cases their vitality was completely destroyed,
when growing in the same medium with the cholera
spirillum.

From this it would appear that the disappearance
of the cholera spirillum from mixed cultures and from
the evacuations in so short a time as has been men-
tioned, is due more to unfavorable nutritive condi-
tions than to the direct action of the other organisms
present.

When completely dried, according to Koch’s experi-
ments, the cholera spirillum does not retain its vitality
for longer than twenty-four hours, but’ by others its
vitality is said to be destroyed by an absolute drying of

1 Zeitschrift fir Hygiene, Bd. x. p, 513. 2 Ibid., Bd. vi. p. 1.

CHOLERA: GENERAL CONSIDERATIONS. 3383

three hours. In the moist condition, as in artificial
cultures, vitality may be retained for many months,
though repeated observations lead us to believe that,
under these circumstances, the virulence is dimin-
ished. According to Kitasato,' they retain their vitality
when smeared upon thin glass cover-slips and kept in
the moist chamber for from 85 to 100 days, and for as
long as 200 days when deposited upon bits of silk
thread.

In the course of his studies upon the destiny of
pathogenic micro-organisms in the dead body, Von
Esmarch* found that, when the cadaver of a guinea-
pig dead from the introduction of cholera organisms
into the stomach, was immersed in water and decom-
position allowed to set in, after eleven days, when de-
composition was far advanced, it was impossible to find
any living cholera spirilla by the ordinary plate
methods.

A similar experiment resulted in their disappearance
after five days. In another experiment, in which de-
composition was allowed to go on without the animal
being immersed in water, none could be detected after
the fifth day.

Carl Fraenkel*® has shown that an atmosphere of
carbonic acid is directly inhibitory to the development
of the cholera spirillum, and Percy Frankland‘ states
that in an atmosphere of this gas it dies in about
eight days. In an atmosphere of carbon monoxide its
vitality is lost in nine days, and in general the same
may be said for it when under the influence of an
atmosphere of nitrous oxide gas.

1 Zeitschrift fiir Hygiene, Bd. v. p. 134. 2 Ibid., Bd. vii. p.1.
8 Ibid., Bd. v. p. 332. 15% 4 Tbid., Bd. vi. p. 18.

334 BACTERIOLOGY.

From what has been said we see that the spirillum
of Asiatic cholera, while possessing the power of pro-
ducing in human beings one of the most rapidly fatal
forms of disease with which we are acquainted, is still
one of the least resistant of the pathogenic organisms
known to us. Under conditions most favorable to its
growth its development is self-limited ; it is conspicu-
ously susceptible to acids, alkalies, other chemical disin-
fectants, and heat ; but when partly dried upon clothing,
food, or other objects, it may retain its vitality for a rela-
tively long period of time, and it is more than probable
that it is in this way that the disease is often carried
from points in which it is epidemic or endemic into
localities that are free from the disease.

THE DIAGNOSIS OF ASIATIC CHOLERA BY BACTERIO-
LOGICAL METHODS.

Because of the manifold channels that are open for
the dissemination of this disease it is of the utmost
importance that its true nature should be recognized as
quickly as possible, for with every moment of delay in
its recognition opportunities for its spread are multi-
plying. It is essential, therefore, when employing bac-
teriological means in making the diagnosis, to bear in
mind those biological and morphological features of the
organism that appear most quickly under artificial
methods of cultivation, and which, at the same, time
may be considered as characteristic of it, viz., its peculiar
morphology and grouping; the much greater rapidity
of its growth over that of other bacteria with which it
may be associated; the characteristic appearance of its
colonies on gelatin-plates and of its growth in stab

DIAGNOSIS OF ASIATIC CHOLERA. 335

cultures in gelatin ; its property of producing indol and
coincidently nitrites in from six to eight hours in pep-
tone solution at 37° to 38° C.; and its power of
causing the death of guinea-pigs in from sixteen to
twenty-four hours when introduced into the peritoneal
cavity, death being preceded by symptoms of extreme
toxemia, characterized by prostration and gradual and
continuous fall in temperature of the animal’s body.

In a publication recently made by Koch! he called
attention to a plan of procedure that is employed in
this work in the Institute for Infectious Diseases at
Berlin. In this scheme the points that have been
enumerated are taken into account, and by its employ:
ment the diagnosis can be established in the majority
of cases of Asiatic cholera in from eighteen to twenty-
two hours. In general the steps to be taken and points
to be borne in mind are as follows. The material
should be examined as early as possible after it has
been passed.

I. Microscopic examination. Fyrom one of the small
slimy particles that will be seen in the semi-fluid evac-
uations prepare a cover-slip preparation in the ordinary
way and stain it. If, upon microscopic examination,
only curved rods, or curved rods greatly in excess of
all other forms, are present, the diagnosis of Asiatic
cholera is more than likely correct; and particularly is
this true if these organisms are arranged in irregular
linear groups with the long axes of all the rods point-
ing in nearly the same direction, that is to say, some-
what as minnows arrange themselves when swimming
in schools up stream. (Koch.)

In 1886 Weisser and Frank? expressed their opinion

1 Zeitschrift fir Hygiene, 1893, Bd. xiv. 2 Tbid., Bd. i., p. 397.

336 BACTERIOLOGY.

upon the value of microscopic examination in these
cases in the following terms :

(a) In the majority of cases microscopic examination
is sufficient for the detection of the presence of the
comma bacillus in the intestinal evacuations of cholera
patients.

(6) Even in the most acute cases, running a very
rapid course, the comma bacillus can always be found
in the evacuations.

(c) In general, the number of cholera spirilla present
is greater the earlier death occurs; when death is
postponed, and the disease continues for a longer
period, their number is diminished.

(d) Should the patient not die of cholera, but from
some other disease, such as typhoid fever, that may be
engrafted upon it, the comma bacilli may disappear
entirely from the intestines.

IJ. With another slimy flake prepare a set of gelatin
plates. Place them at a temperature of from 20° to
22° C., and at sixteen, twenty-two, and thirty-six hours
observe the appearance of the colonies. Usually at
about twenty-two hours the colonies of this organism
_ can easily be identified by one familiar with them.

III. With another slimy flake start a culture in a tube
of peptone solution—either the solution of Dunham
or, as Koch proposes, a solution of double the strength
of that of Dunham (Witte’s peptone is to be used,
as it gives the best and most constant results). Place
this at 37° to 38° C., and at the end of from six to
eight hours prepare cover-slips from the upper layers
(without shaking) and examine them microscopically.
If comma bacilli are present and capable of multipli-
cation they will be found in this locality in almost

DIAGNOSIS OF ASIATIC CHOLERA. 337

pure culture. After doing this prepare a second
peptone culture from the upper layers, also a set of
gelatin plates, and with what remains make the test
for indol by the addition of ten drops of concentrated
sulphuric acid for each ten cubic centimetres of fluid
contained in the tube. If comma bacilli are growing
in the tube the rose color characteristic of the presence
of indol should appear.

By following this plan “a bacteriologist who is
familiar with the morphological and biological pecu-
liarities of this organism should make a more than
probable diagnosis at once by microscopic examination
alone, and a positive diagnosis in from twenty to, at the
most, twenty-four hours after beginning the examina-
tion.” (Koch.)

There are certain doubtful cases in which the organ-
isms are present in the intestinal canal in very small
numbers, and microscopic examination is not, therefore,
of so much assistance. In these cases plates of agar-
agar, of gelatin, and cultures in the peptone solution
should be made. ;

The plates of agar-agar should not be prepared in
the usual way, but the agar-agar should be poured
into Petri dishes and allowed to solidify, after
which one of the slimy particles may be smeared
over its surface. The comma bacillus, being mark-
edly aérobic, develops very much more readily when
its colonies are located upon the surface than when
they are in the depths of the medium. A point
to which Koch calls attention, in connection with this
step in the manipulation, is the necessity for having the
surface of the agar-agar free from the water that is
squeezed from it when it solidifies, as the presence of

338 BACTERIOLOGY.

the water interferes with the development of the colonies
as isolated points and causes them to become confluent.
To obviate this he recommends that the agar-agar be
poured into the plates and the water allowed to separate
from the surface at the temperature of the incubator
before they are used. It is wise, therefore, when one
is liable to be called on for such work as this to keep a
number of sterilized plates of agar-agar in the incubator
ready for use, just as sterilized tubes of media are
always ready and at hand. The advantage of using
the agar plates is the higher temperature at which they
can be kept, and consequently a more favorable condi-
tion for the development of the colonies. As soon as
isolated colonies appear they should be examined mi-
croscopically for the presence of organisms having the
morphology of the one for which we are seeking, and
as soon as such is detected gelatin plates and cultures
in peptone solution (for the indol reaction) should be
made. The peptone cultures started from the original
material should be examined microscopically from hour
to hour after the sixth hour that they have been in the
incubator. The material taken for examination should
always come from near the surface of the fluid, and
care should be taken not to shake the tube. As soon
as comma bacilli are detected in anything like consid-
erable numbers in the upper layers of the fluid agar-
agar plates and fresh peptone cultures should be made
from them. The colonies will develop on the agar-agar
plates at 37° C. in from ten to twelve hours to a size
sufficient for recognition by microscopic examination,
and from this examination an opinion can usually be
given. This opinion should always be controlled by
cultures in the peptone solution made from each of

DIAGNOSIS OF ASIATIC CHOLERA, 339

several single colonies, and finally the test for the pres-
ence or absence of indol in these cultures.

In all doubtful cases in which only a few curved
bacilli are present, or in which irregularities in either
the rate or mode of their development occurs, pure cul-
tures should be obtained by the agar-plate method and
the method of cultivation in peptane solution, as soon
as possible, and their virulence tested upon animals.
For this purpose cultures upon agar-agar from single
colonies must be made. From the surface of one of such
cultures a good sized wire-loopful should be scraped
and this broken up in about one cubic centimetre of
bouillon, and the suspension thus made injected by
means of a hypodermatic syringe directly into the peri-
toneal cavity of a guinea-pig of about 350 to 400
grammes weight. For larger animals more material
should be used. If the material injected is from a
fresh culture of the cholera organism toxic symptoms
at once begin to appear; these have their most pro-
nounced expression in the lowering of temperature,
and if one follows this decline in temperature from time
to time with the thermometer it will be seen to be gradual
and continuous from the time of injection to the death
of the animal (Pfeiffer'), which occurs in from eighteen
to twenty-four hours after the operation.

In general, this is the procedure employed in the
Institute for Infectious Diseases, at Berlin, under

Koch’s direction.
1 Loe. cit.

CHAPTER XXIII.

Organisms of interest historically and otherwise, that have been confounded.
with the spirillum of Asiatic cholera—Their peculiarities and differential
features—The vibrio proteus. or bacillus of Finkler and Prior—The spirillum
tyrogenum, or cheese spirillum of Deneke—The spirillum of Miller—The vibrio
Metschnikovi.

\ VIBRIO PROTEUS (FINKLER-PRIOR BACILLUS).

Finkler and Prior were the first to contest experi-
mentally the significance of the presence of Koch’s
comma bacillus in Asiatic cholera, claiming to have
found it in the dejections of individuals suffering from
other maladies, particularly cholera nostras. The
morphological and biological differences between the
organism that Finkler and Prior had discovered and
those of the comma bacillus described by Koch are,
however, so pronounced as to warrant the opinion
that the confusion had arisen through imperfect and
untrustworthy methods of experimentation. At a some-
what later period Finkler and Prior retracted their
claims of identity for the two organisms, and held that
the bacterium with which they were dealing was pecu-
liar to cholera nostras—an opinion which, in the light
of subsequent work was also proved to be without
foundation in fact.

The characteristics of the spirillum of Finkler and
Prior are as follows :

MorpHoLocy.—It is thicker and longer than the
spirillum of Asiatic cholera; it is often thicker at the

VIBRIO PROTEUS: CULTURAL PECULIARITIES. 341

middle than at the poles; it forms, like the “comma
bacillus,” screw-like, twisted threads (Fig. 64).

It is supplied with a single flagellum at one of its ends,
and is, therefore, motile.

It, like the comma bacillus, readily undergoes degener-
ative changes under conditions unfavorable to growth
and presents the variety of shapes grouped under the
head “involution forms.” According to Buchner this
is especially the case when the medium in which they
are growing contains glucose (6 per cent.) or glycerin
(2 per cent.).

Fig. 64,
oe
SY 5 : i
go 6 LK
= G Mg ee &
(4 ( i
wn ef #

-
¢

Vibro proteus, Finkler-Prior bacillus, from culture on agar-agar twenty-
four hours old.

CULTURAL PECULIARITIES.—On gelatin plates the
development of its colonies is far more rapid, and
liquefaction much more extensive, than in the case of
the cholera spirillum. After twenty-two to twenty-
four hours in this medium at 20° to 22° C. the average
size of the colonies is about double that of the comma
bacillus. The colonies are darker and denser and do
not present under the low lens the same degree of
granulation and subsequent lobulation, and they do not
become serrated or scalloped around the margin as is
the case with Koch’s organism. After twenty-two to
twenty-four hours they are usually nearly round, regu-
larly granular, and more or less sharply defined. (See

3842 BACTERIOLOGY.

Fig. 65, a.) At times they may show indefinite mark-
ings or creases, somewhat suggestive of lobulations.
After forty-eight hours on gelatin they usually range
from one to three millimetres (some even larger) in
diameter, and will appear as sharply cut saucer-shaped
pits of liquefaction, in the most dependent portion of
which lies a dense, irregular mass, the colony proper.
Under low magnifying power they present at this stage
an appearance similar to that shown in Fig. 65, 6, the

Fie. 65.

Colonies of the Finkler-Prior bacillus on gelatin. > about 75 diameters.

w. After twenty-two hours on gelatin at 20° to 22°C. 0b. After forty-eight
hours on gelatin at 20° to 22° C.

central dense mass representing the éolony and the irreg-
ular ragged lines surrounding it being shreds that have
become torn away as it sank into the liquid caused
by its growth. The zone surrounding it, extending to
the periphery, is somewhat cloudy, and is simply lique-
fied gelatin. There is a marked tendency for the lique-
faction to spread laterally and for the colonies to run
together, so that, even on plates containing few colo-
nies, in sixty to seventy-two hours at from 20° to
22° C., the entire gelatin is usually converted into a

VIBRIO PROTEUS: CULTURAL PECULIARITIES. 343

yellowish—white fluid. Under these conditions its
growth is accompanied by a marked aromatic odor im-
possible to describe; this is especially the case when
the liquefaction is far advanced.

Fie. 66,

Stab culture of the Finkler-Prior bacillus in gelatin, at 18° to 20° C.

a. After twenty-four hours. b. After forty-eight hours. c. After seventy-two
hours. d. After ninety-six hours.

In stab cultures in gelatin at the room temperature
liquefaction is noticed about the upper part of the
needle-track in twenty-four hours. This condition
gradually increases, and at the end of two or three days
the entire upper portion of the gelatin has become con-
verted into a cloudy fluid, whereas at the lower part

344 BACTERIOLOGY.

of the canal the liquefaction progresses less rapidly but
is still much more marked than that seen as a result of
the growth of Koch’s spirillum. Indeed, under these
circumstances there is no similarity whatever between
the growth of the two organisms (see a, , c, d, Fig. 66,
and compare these with corresponding cuts in Fig. 63).

It is customary to see, scattered through the cloudy
liquefied gelatin, ragged, more or less dense masses, frag-
ments of the colony proper.

On nutrient agar-agar there is nothing particularly
characteristic about its growth, appearing only as a
moist, grayish or yellowish-gray deposit.

On potato after forty-eight to seventy-two hours there
appears a pale yellowish-gray deposit; this is moist,
glazed, and marked by lobulations, and is surrounded
by an irregular colorless zone of growth that is much
less moist than that forming the central area. It grows
well on potato at the ordinary temperature of the room.

It causes liquefaction of solidified blood-serum and
of coagulated egg albumin.

In milk to which neutral litmus tincture has been
added the blue color takes on a pink tinge in from two
to three days at 37° to 38° C.

It does not form indol nor does it cause fermentation
of glucose.

In peptone solution containing rosolic acid the color
is somewhat deepened after four or five days at 37° C.

EXPERIMENTS UPON ANIMALS.—By ordinary methods
of inoculation this organism is without pathogenic
properties. Injections, subcutaneous and intra-vascular
and directly into the stomach, give negative results.
When introduced into the stomach of guinea-pigs by
the method employed by Koch in his cholera experi-

SPIRILLUM TYROGENUM. 845

ments, Finkler and Prior had 3 out of 10 animals, and
Koch 5 out of 15 animals so treated to die.

The claim of Finkler and Prior that this organism
was related etiologically to cholera nostras has been
shown by subsequent work to have been unjustifiable.

In 1885, 1886, and 1887 Franck! examined seven
cases that clinically presented the condition of cholera
nostras ; in none of these seven cases was the organism
of Finkler and Prior, which they claimed to be the
cause of the disease, found. In all cases the results
of bacteriological examination, in so far as the constant
presence of an organism that might stand in causal
relation to the disease was concerned, were negative.
Only the ordinary intestinal bacteria were found.

SPIRILLUM TYROGENUM (CHEESE SPIRILLUM OF
DENEKE).

Deneke'’s cheese spirillum, spirillum tyrogenum. From agar culture twenty-
four hours old.

Another spiral form, likewise forming short, comma-
shaped segments in the course of its growth (Fig. 67),
is that found by Deneke in old cheese. In morphology
this organism is a little smaller than Koch’s spirillum,

1 Zeitschrift f. Hygiene, Bd. iv, p. 207.

346 BACTERIOLOGY.

It is motile and has but a single flagellum attached to
one of itsends. It liquefies gelatin more rapidly than
does Koch’s organism. It possesses no characteristic
grouping, as can be seen in impression cover-slips of its
colonies. It does not form spores. On gelatin plates
its colonies develop very rapidly as saucer-shaped
depressions ; after twenty-four hours they vary from
1 to 4 mm. in transverse diameter. To the naked
eye they are almost transparent and are usually marked
by a denser centre and peripheral zone, the space
between being quite clear. They are not regularly
round in all cases. A peculiar aromatic odor accom-
panies their growth on gelatin. Under a low magnify-
ing power the smallest colonies are irregularly round
in outline, their borders being often rough and broken,
and the body of the colony is frequently marked by
creases or ridges that give to it a lobulated appearance
The larger colonies under the same lens appear as

Colony of spirillum tyrogenum on gelatin, twenty-four hours old.

granular patches, a little denser at the periphery and
centre than at the intermediate portions. The periphery
gradually fades away and no distinct circumference
can be made out. (See Fig. 68.) The colonies of an
intermediate size in which liquefaction is just beginning

SPIRILLUM TYROGENUM. 8347

to be apparent, show a dense granular centre, the col-
ony itself, and about it a delicate, granular develop-
mental zone. ;

In stab cultures in gelatin liquefaction is rapid,
causing at the end of twenty-four hours a cup-shaped

Fia,. 69.

Stab culture of Deneke’s cheese spirillum in gelatin, at 18° to 20° C.

a. After twenty-four hours. 6b. After forty-eight hours. c. After seventy-two
hours. d. After ninety-six hours.

depression at the top of the needle-track, the superficial
area of which is about half that of the gelatin in the
tube. (Fig. 69, a.) The liquefying process spreads
laterally and at the end of forty-eight hours the whole
upper portion of the gelatin may have become liquid.
(Fig. 69, 6.) This process continues along the

348 BACTERIOLOGY.

track of the needle and after seventy-two and ninety-
six hours the appearances shown in Fig. 69, ¢ and d,
will be produced.

There is nothing particularly characteristic about its
growth upon agar-agar.

On potato there appears a moist, glazed, yellowish,
and, at points, brownish-yellow growth that is sur-
rounded by a drier, colorless zone. It is not lobulated.

In milk containing neutral litmus tincture a pink
color appears after two to three days at 37° C.; after
four days the milk is almost decolorized and there is
beginning to appear coagulation of the casein with a
layer of clear whey above it. During the subsequent
twenty-four hours there is complete separation of the
contents of the tube into clot and whey.

In Dunham’s peptone solution it does not form indol
and the reaction for this body does not appear with
either sulphuric acid alone or plus sodium nitrite.

It causes liquefaction of both coagulated blood-serum
and egg albumin.

There is no pellicle formed as a result of its growth
in bouillon.

It does not produce fermentation of glucose.

In rosolic-acid-peptone solution its growth causes
the red color to become deepened after four or five days
at 87° C.

By Koch’s method of introducing cultures into the
stomach of guinea-pigs this organism produced the
death of three out of fifteen animals experimented
upon—the deaths resulting, most probably, more from
the toxic action of the products of growth that were
introduced with the organisms than to any pathogenic
powers possessed by the organism itself.

MILLER'S SPIRILLUM. 349

MILLER’S SPIRILLUM.

Another spirillum that has been likened to that of
Koch is the one obtained by Miller from a carious
tooth. It has so many characteristics in common with
the organism of Finkler and Prior that Miller was
inclined to consider them identical. In morphology
they are indistinguishable. (See Fig. 70.) It grows

Spirillum of Miller. From agar culture twenty-four hours old.

rapidly, and, like the spirillum of Finkler and Prior,
causes rapid liquefaction of gelatin with the coincident
production of a peculiar aromatic odor.

The colonies on gelatin plates appear after twenty-four
hours as small, transparent pits of liquefaction in the
centre of which can be seen a minute white point, the
colony itself. Under a low lens the largest of these
points are uniformly granular and regularly round,
and as a rule are surrounded by a peripheral zone that
is a little darker than the central portion of the colony.
The circumference is delicately fringed by short, cilia-
like prolongations of growth whieh are not as a rule
straight, but are twisted in all directions and can only
be detected upon very careful examination. (See a, Fig.
71.) When located deep in the gelatin the colonies

are round, sharply circumscribed, of a pale-yellowish or
16

350 BACTERIOLOGY.

greenish-yellow color, and marked by very delicate
irregular lines or ridges. After forty-eight hours the
plate containing many colonies is entirely liquefied,
while that containing only a few shows the presence of
round, sharply cut, shallow pits of liquefaction that
measure from two to ten mm. in diameter. They are
a little denser at the centre than at the periphery, and
the dense centre is not sharply circumscribed but fades
off into what has the appearance of a delicate film.
(See 6, Fig. 71.) As the colonies become older they

Fic. 71.

Colonies of Miller's spirillum on gelatin, at 20° to 22°C. x about fifty-seven
diameters,
a. Colony just beneath the surface of the gelatin. b. Colony on the surface
of the gelatin.

are sometimes marked by irregular radii extending
from periphery to centre like the spokes of a wheel.

In stab cultures in gelatin it rapidly produces lique-
faction, both at the surface and along the needle-track,
and in most respects gives rise to a condition very like
that resulting from the growth of Finkler and Prior’s
spirillum, though differing from it in certain details.
(See a, 6, ¢, d, Fig. 72.)

On agar-agar nothing of special interest appears as a
result of its development.

On potato its growth is very like that of the cholera
spirillum, viz., it appears at 37° C. as a dry, white

MILLER’S SPIRILLUM. 351

patch that lies quite flat upon the surface and can
often only be seen when the tube is held to the light
in a special way.

Stab culture of Miller’s spirillum in gelatin, at 18° to 20° C,

a. After twenty-four hours. b. After forty-eight hours. c. After seventy-two
hours. d. After ninety-six hours.

Its growth in bouillon is not characteristic. It does
not form a pellicle.

It causes liquefaction of both coagulated blood-serum
and egg albumin.

It does not produce indol.

It does not cause fermentation of glucose.

It is non-motile.

In milk containing blue litmus tincture it causes

352 BACTERIOLOGY.

almost complete decolorization in from three to four
days at 37° C., with coincident coagulation of the
casein and the formation of a layer of clear whey
above it.

It causes the red color of rosolic-acid-peptone solu-
tion to become somewhat intensified after four or five
days at 37° C.

Of twenty-one animals treated with this organism
by Koch’s method of inoculation only four died.

VIBRIO METCHNIKOVI.

The spirillum that simulates very closely the comma
bacillus of cholera in its morphological and cultural
peculiarities, but which is still easily distinguished from
it, is that described by Gamaleia under the name of
vibrio Metchnikovi. It was found post-mortem in a
number of fowls that had died in the poultry market
of Odessa, and the experiments of the discoverer de-
monstrate that it is related etiologically to the gastro-
enteritis with which the chickens had been suffering.

Vibrio Metchnikovi from agar culture, twenty-four hours old.

In morphology it is seen as short, curved rods and
as longer, spiral-like filaments. It is usually thicker
than Koch’s spirillum and is at times much longer,

VIBRIO METCHNIKOVI. 353

while again it is seen to be shorter. It is usually
more distinctly curved than the “comma bacillus.”
(Fig. 73.)

It is supplied with a single flagellum at one of its
extremities and is, therefore, motile.

It does not form spores.

It is aérobie.

Its growth upon gelatin plates is usually character-
ized, according to Pfeiffer, by the appearance of two
kinds of liquefying colonies, one strikingly like those
of the Finkler-Prior organism, the other very similar
to those produced by Koch’s comma bacillus, though in
both cases the liquefaction resulting from the growth
of this organism is more energetic than that common to
the spirillum of Asiatic cholera. After from twenty-
four to thirty hours the medium-sized colonies, when
examined under a low power of the microscope, show a
yellowish-brown, ragged central mass surrounded by a
zone of liquefaction that is marked by a border of
delicate radii. (Fig. 74.)

Colony of vibrio Metohnikovi in gelatin after thirty hours at 20° to 22° C.
X about 75 diameters.

In gelatin stab cultures the growth has much the same
general appearance as that of the cholera spirillum, but
is very much exaggerated in degree. The liquefaction is
far more rapid and the characteristic appearance of the
growth is lost in from three to four days. (See a, b,
ce, d, Fig. 75.) Development and liquefaction along

354 BACTERIOLOGY.

the deeper parts of the needle-track is much more pro-
nounced than is the case with the “ comma bacillus.”

Fie. 75.

Stab culture of vibrio Metchnikovi in gelatin, at 18° to 20° C.

a. After twenty-four hours. b, After forty-eight hours. c. After seventy-
two hours. d. After ninety-six hours.

Its growth on agar-agar is rapid, and after twenty-
four to forty-eight hours there appears a grayish deposit
having a tendency to take on a yellowish tone.

On potato at 37° C. its growth is seen as a moist,
coffee-colored patch surrounded by a much paler zone.
The whole growth is so smooth and glistening that it
has almost the appearance of being varnished.

In bouillon it quickly causes opacity, with the ulti-

VIBRIO METCHNIKOVI. 355

mate production of a delicate pellicle upon the
surface. a :

It causes liquefaction of blood-serum, the liquefied
area being covered by a dense, wrinkled pellicle.

When grown in peptone solution it produces indol
and coincidently nitrites, so that the rose-colored reac-
tion characteristic of indol is obtained by the addition
of sulphuric acid alone. The production of indol by
this organism is usually greater than that common to
the comma bacillus under the same circumstances.

In milk it causes an acid reaction with coagulation
of the casein. The coagulated casein collects at the
bottom of the tube in irregular masses, above which is
a layer of clear whey. If blue litmus has been added
to the milk the color is changed to pink by the end of
twenty-four to thirty hours, and after forty-eight hours
decolorization and coagulation occurs. The clots of
casein are not re-dissolved. After about a week the
acidity of the milk is at its maximum, and the organ-
isms quickly die.

It causes the red color of rosolic-acid-peptone solu-
tion to become very much deeper after four or five days
at 87° C.

It does not cause fermentation of glucose with pro-
duction of gas.

It is killed in five minutes by a temperature of 50°
C. (Sternberg.)

It is pathogenic for chickens, pigeons, and guinea-
pigs. Rabbits and mice are affected only by very large
doses.

Chickens affected with the choleraic gastro-enteritis,
of which this organism is the cause, are ususlly seen
sitting quietly about with ruffled feathers. They are

356 BACTERIOLOGY.

afflicted with diarrhoea, but do not have any elevation
of temperature. A hyperemia of the entire gastro-
intestinal tract is seen at autopsy. The internal
organs do not, as a rule, present anything abnormal to
the naked eye. The intestinal canal contains yellowish
fluid with which blood may be mixed. In adult
chickens the spirilla are not found in the blood, but in
young ones they are usually present in small numbers.
By subcutaneous inoculation pigeons succumb to the
pathogenic activities of this organism in from eight to
twelve hours. At autopsy pretty much the same con-
dition is seen as was described for chickens, except that
large numbers of the spirilla are usually present in
the blood. Guinea-pigs usually die in from twenty
to twenty-four hours after subcutaneous inoculation.
At autopsy an extensive oedema of the subcutaneous
tissues about the seat of the inoculation is seen, and
there is usually a necrotic condition of the tissues
in the vicinity of the point of puncture. As the
blood and internal organs contain the vibrios in large
numbers, the infection in these animals takes, therefore,
the form of acute general septicemia.

Gastro-enteritis may be produced in both chickens
and guinea-pigs by feeding them with food in which
cultures of this organism have been mixed.

Nore.—More recently, particularly since the late
epidemic in Hamburg, quite a number of curved or
spiral organisms, somewhat like the cholera spirillum,
have been discovered. For the descriptions of these
the reader is referred to the current bacteriological
literature.

CHAPTER XXIV.

Study of the bacillus anthracis, and the effects produced by its inoculation
into animals—Peculiarities of the organism under varying conditions of sur-
roundings.

THE discovery that the blood of animals suffering
from splenic fever, or anthrax, always contained minute
rod-shaped bodies (Pollender, 1855; Davaine, 1863),
led to a closer study of this disease, and has resulted
probably, in contributing more to our knowledge of
bacteriology in general than work upon any of the
other infectious maladies.

The outcome of these investigations is that a rod-
shaped micro-organism, now known as the bacillus
anthracis, is always present in the blood of animals
suffering from this disease; that this organism can be
obtained from the tissues of these animals in pure
cultures, and that these artificial cultures of the bacillus
anthracis when introduced into the body of susceptible
animals can again produce a condition identical to that
found in the animal from which they were obtained.

The disease is a true septicemia, and the capillaries
throughout the body after death will always be found
to contain the typical rod-shaped organism in larger or
smaller numbers.

This organism, when isolated in pure cultures, is
seen to be a bacillus which varies considerably in its
length, ranging from short rods of 2 to 3, in length to

longer threads of 20 to 25, in length. In breadth it is
16*

308 BACTERIOLOGY.

from 1 to 1.25». Frequently very long threads made
up of several rods, joined end to end, are seen.

When obtained directly from the body of an animal, it
is usually in the form of short rods square at the ends.
If highly magnified the ends are seen to be a trifle
thicker than the body of the cell and somewhat in-
dented or concave, peculiarities that help to distinguish
it from certain other organisms that are somewhat like
it morphologically. (See Fig. 76.)

Fic. 76.

“Ay
Fae

Bacillus anthracis highly magnified to show swellings and concavities
at extremities of the single cells.

When cultivated artificially at the temperature of the
body, the bacillus of anthrax presents a series of very
interesting stages.

The short rods develop into long threads, which may
be seen twisted or plaited together after the manner of
ropes, each thread being marked by the points of junc-
ture of the short rods composing it. (Fig. 77, a and b.)

In this condition it remains until alterations in its
surroundings, the most conspicuous being diminution in
its nutritive supply, favor the production of spores.
When this stage begins, changes in the protoplasm
of the bacilli may be noticed ; they become marked by
irregular, granular bodies, which eventually coalesce
into glistening, oval spores, one of which lies in nearly

BACILLUS ANTHRACIS. 359

every segment of the long thread, and gives to the
thread the appearance of a string of glistening beads.
(Fig. 78.) In this stage they remain but a short time.
The chains of spores, which are held together by the

Fic. 77,

Bacillus anthracis. Plaited and twisted threads seen in fresh growing
cultures. about 400 diameters.

remains of the cells in which they formed, become
broken up, and eventually nothing but free oval spores,
and here and there the remains of mature bacilli
which have undergone degenerative changes can be

Fic. 78.

Threads of bacillus anthracis containing spores. about 1200 diameters.

found. In this condition the spores, capable of resist-
ing deleterious influences, remain and, unless their sur-
roundings are altered, have been seen to continue in
this living, though inactive, condition for a very long

360 BACTERIOLOGY.

time. If again placed under favorable conditions
each spore will germinate into a mature cell, and the
same series of changes will be repeated until the favor-
able surroundings become again gradually unfavorable
to development, when spore-formation is again seen.
Spore-formation takes place only at temperatures
ranging from 18° to 43° C., 37.5° C. being the most
favorable temperature. Under 12° C. they are not
formed. With this organism spore-formation does not
occur in the tissues of the living animal, its usual con-
dition at this time being that of short rods. Occa-
sionally, however, somewhat longer forms may be seen.

The bacillus of anthrax is not motile.

GrowTH ON AGAR-AGAR.—The colonies of this or-
ganism, as seen upon agar-agar, present a very typical
appearance, from which they have been likened unto
the head of Medusa. From a central point which is
more or less dense, consisting of a felt-like mass of long
threads matted irregularly together, the growth con-
tinues outward upon the surface of the agar. (Fig. 79.)

Colony of bacillus anthracis on agar-agar.

It is made up of wavy bundles in which the threads are
seen to lie parallel side by side or are twisted in strands
like those of a rope—sometimes they have a plaited

B. ANTHRACIS: CULTURES. 361

arrangement. (See Fig. 76.) These bundles twist about
and cross in all directions, and eventually disappear at
the periphery of the colony. At the extreme periphery
of the colonies it is sometimes possible to trace single
bundles of these threads for long distances across the
surface of the agar-agar. The colony itself is not cir-
cumscribed in its appearance, but is more or less irregu-
larly fringed or ragged, or scalloped. To the naked
eye they look very much like minute pellicles of raw
cotton that have been pressed into the surface of the
agar-agar.

As the colonies continue to grow, they become more
and more dense, opaque, and granular and rough on
the surface. When touched with a sterilized needle,
one experiences a sensation that suggests, somewhat,
the matted structure of these colonies. The bit that
may thus be taken from a colony is always more or less
ragged.

GeLATIN.—The colonies on gelatin at the earliest
stages also present the same wavy appearance ; but this
characteristic soon becomes in part destroyed by the
liquefaction of the gelatin which is produced by the
growing organisms. This allows them to sink to the
bottom of the fluid, where they lie as an irregular mass.
Through the fluid portion of the gelatin may be seen
small clumps of growing bacilli, which look very much
like bits of cotton-wool.

BovrLion.—In bouillon the growth is characterized
by the formation of flaky masses, which also have very
much the appearance of bits of raw cotton. Microscopic
examination of one of these flakes reveals the twisted
and plaited arrangement of the long threads.

Porato.—It develops rapidly as a dull, dry, gran-

362 BACTERIOLOGY.

war, whitish mass, which is more or less limited to the
point of inoculation. On potato, at the temperature of
the incubator, its spore-formation may easily be ob-
served.

Stas AND SLtanr CuitureEs.—Stab and slant cul-
tures on agar-agar present in general the appearances
given for the colonies, except that the growth is much
more extensive. The growth is always more pro-
nounced on the surface than down the track of the
needle.

On gelatin it causes liquefaction, which begins on the
surface at the point inoculated, and spreads outward
and downward.

It grows best with access to oxygen, and very poorly
when the supply of oxygen is interfered with.

Under favorable conditions of aération, nutrition,
and temperature its growth is rapid.

Under 12° C. and above 45° C. no growth occurs.
The temperature of the body is most favorable to its
development.

The spores of the anthrax bacillus are very resistant
to heat, though the degree of resistance is seen to vary
with spores of different origin. Esmarch found that
anthrax spores from some sources would readily be killed
by an exposure of one minute to the temperature of
steam, whereas those from other sources resisted this
temperature for longer times, reaching in some cases
as long as twelve minutes.

Srarninc.—The anthrax bacilli stain readily with
the ordinary aniline dyes. In tissues their presence
may also be demonstrated by the ordinary aniline stain-
ing fluids, or by Gram’s method. They may also be
stained in tissues with a strong watery solution of dahlia,

B. ANTHRACIS: INOCULATION INTO ANIMALS. 368

after which the tissue is decolorized in 2 per cent. soda
solution, washed in water, dehydrated in alcohol, cleared
up in xylol, and mounted in balsam. This leaves the
bacilli stained, while the tissues are decolorized ; or the
tissues may be stained a contrast color—eosin, for ex-
ample—after the dehydration in alcohol, and before the
clearing up in xylol. In this case they must be washed
out again in alcohol before using the xylol. In the
preparation treated in this way, the rod-shaped organ-
isms will be of a purple color, and will be seen in the
capillaries of the tissues, while the tissues themselves
will be of a pale rose color.

InocuLATion INTO ANIMALS.—Introduce into the
subcutaneous tissues of the abdominal wall of a guinea-
pig or rabbit, a portion of a pure culture of the bacillus
anthracis. In about forty-eight hours the animal will
be found dead. Immediately at the point of inoculation
but little or no reaction will be noticed, but beyond
this, extending for a long distance over the abdomen
and thorax, the tissues will be markedly cedematous.
Here and there, scattered through this cedematous
tissue, small ecchymoses will be seen. The underlying
muscles are pale in color. Inspection of the internal
viscera reveals no very marked macroscopic changes
except in the spleen. This is enlarged, dark in color,
and soft. The liver may present the appearance
of cloudy swelling; the lungs may be pale or pale-red
in color; the heart is usually filled with blood. There
are no other changes to be seen by the naked eye.

Prepare cover-slip preparations from the blood and
other viscera. They will all be found to contain short
rods in large numbers. Nowhere can spore-formation
be detected. Upon microscopic examination of sections

364 BACTERIOLOGY.

of the organs which have been hardened in alcohol, the
capillaries are seen to be filled with the bacilli; in some
places closely packed together in large numbers, at
other points fewer in number. Usually they are present
in largest numbers in those tissues having the greatest
capillary distribution and at those points at which the
circulation is slowest. They are moderately evenly dis-
tributed through the spleen. The glomeruli of the
kidneys and the capillaries of the lungs are frequently
quite packed with them. The capillaries of the liver
contain them in large numbers. (Fig. 80.) Hemor-

Fig. 80.

Anthrax bacilli in liver of mouse; < about 450 diameters. Bacilli stained
by Gram’s method; tissue stained with Bismarck-brown.

rhages, probably due to rupture of capillaries by the
mechanical pressure of the bacilli which are developing
within them, not uncommonly occur. When this occurs
in the mucous membranes of the alimentary tract, the
blood may escape through the mouth or anus ; when in
the kidneys, through the uriniferous tubules.

Cultures from the different organs or from: the cedema-
tous fluid about the point of inoculation, result in growth
of the bacillus anthracis.

B. ANTHRACIS: EXPERIMENTS. 865

The amphibia, dogs, and the majority of birds are
not susceptible to this disease. Rats are difficult to infect.
Rabbits, guinea-pigs, white mice, gray house-mice, sheep,
and cattle are susceptible. Infection may occur either
through the circulation, through theair-passages, through
the alimentary tract, or, as we have just seen, through
the subcutaneous tissues.

EXPERIMENTS.

Prepare three cultures of anthrax bacilli—one upon
gelatin, one upon agar-agar, and one upon potato. Allow
the gelatin culture to remain at the ordinary temperature
of the room, place the agar culture in the incubator, and
the potato culture at a temperature not above 18° to
20° C. Prepare cover-slips from each from day to day.
What differences are observed ?

Prepare two potato cultures of the anthrax bacillus.
Place one in the incubator and retain the other at a tem-
perature of from 18° to 20° C. Examine them each
day. Do they develop in the same way?

From a fresh culture of anthrax bacilli, in which
spore-formation is not yet begun, prepare a hanging-
drop preparation ; also a cover-slip preparation in the
usual way and stain it with astrong gentian-violet solu-
tion, and another cover-slip preparation which is to be
drawn through the flame twelve to fifteeen times, stained
with aniline gentian-violet, washed off in iodine solu-
tion and then in water. Examine these microscopically.
Do they all present the same appearance? To whatare
the differences due?

366 BACTERIOLOGY.

Do the anthrax threads, as seen in a fresh, growing,
hanging drop, present the same morphological appear-
ance as when dried and stained upon a cover-slip?
How do they differ ?

Liquefy a tube of agar-agar, and when it is at the
temperature of 40° to 43° C., add a very minute quan-
tity of an anthrax culture which is far advanced in the
spore stage. Mix it thoroughly with the liquid agar-
agar and from this prepare several hanging drops under
strict antiseptic precautions, using the fluid agar-agar
for the drops instead of bouillon or salt solution. Select
from among these preparations that one in which the
smallest number of spores are present. Under the
microscope observe the development of this spore into a
mature cell. Describe carefully the developmental
stages.

Prepare a 1: 1000 solution of carbolic acid in houillon.
Inoculate this with virulent anthrax spores. If no de-
velopment occurs after two or three days at the tempera-
ture of the thermostat, prepare a solution of 1: 1200,
and continue until the point is reached at which the
amount of carbolic acid present just admits of the de-
velopment of the spores. When the proper dilution is
reached prepare a dozen of such tubes and inoculate one
of them with virulent anthrax spores. As soon as de-
velopment is well advanced, transfer a loopful from this
tube into a second of the carbolic acid tubes ; when this
has developed, then from this into a third, ete. After
five or six generations which have been treated in this
way, study the spore-production of the organisms in
that tube. If it is normal, continue to inoculate from

B. ANTHRACIS: EXPERIMENTS. 367

one carbolic acid tube into another, and see if it is
possible by this means to influence in any way the
production of spores by the organism with which you
are working. What is the effect, if any ?

Prepare two bouillon cultures, each from one drop of
blood of an animal dead of anthrax. Allow one of them
to grow for from fourteen to eighteen hours in the incu-
bator ; allow the other to grow at the same temperature
for three or four days. Remove the first after the time
mentioned and subject it toa temperature of 80° C. for
thirty minutes. At the end of this time prepare four
plates from it. Make each plate with one drop from
the heated bouillon culture. At the end of three or four
days treat the second tube in identically the same way.
How do the number of colonies which developed from
the two different cultures compare? Was there any
difference in the time required for their development on
the plates ?

From a potato culture of anthrax bacilli which has’
been in the incubator for three or four days, scrape
away the growth and carefully break it up in 10 e.c. of
sterilized normal salt solution. The more carefully it
is broken up the more accurate will be the experiment.
Place this in a bath of boiling water and at the end of
one, three, five, seven, and ten minutes make a plate
upon agar-agar with one loopful of the contents of this
tube. Are the results on the plates alike ?

Determine the exact time necessary. to sterilize ob-
jects, such as silk or cotton threads, on which anthrax
spores have been dried, by the steam method and by the
hot-air method.

368 BACTERIOLOGY.

Prepare from the blood of an animal just dead of an-
thrax a bouillon culture. After this has been in the
incubator for from three to four hours, subject it to a
temperature of 55° C. for ten minutes. At the end of
this time make plates from it, and also inoculate a rabbit
subcutaneously with it. What are the results? Are
the colonies on the plates in every way characteristic?

Inoculate six Erlenmeyer flasks of sterile bouillon,
each containing about 35 c.c. of the medium, from either
the blood of an animal just dead of anthrax or from a
fresh virulent culture in which no spores are formed.

Place these flasks in the incubator at a temperature
of 42.5° C. At the end of five, ten, fifteen, twenty,
twenty-five, etc., days remove a flask. Label each flask
as it is taken from the incubator with the exact number
of days for which it had been at the temperature of
42.5° C. Study each flask carefully, both in its cultural
peculiarities and its pathogenic properties when em-
ployed on animals.

Aye these cultures identical in all respects with those
that have been kept at 37° C.?

If they differ, in what respect is the difference most
conspicuous ?

Should any of the animals survive the inoculations
made from the different cultures in the foregoing exper-
iment, note carefully which one it is, and after ten to
twelve days repeat the inoculation, using the same cul-
ture ; if it again survives, inoculate it with the culture
preceding the one just used in the order of removal from
the incubator ; if it still survives, inoculate it with vir-
ulent anthrax. What is the result? How is the result

B. ANTHRACIS: EXPERIMENTS. 869

to be explained? Do the cultures which were made
from these flasks at the time of their removal from the
incubators act in the same way toward animals as the
organisms growing in the flasks? Is the action of each
of these cultures the same for mice, guinea-pigs, and
rabbits ?

Prepare a 2 per cent. solution of sulphuric acid in
distilled water ; suspend in this a number of anthrax
spores ; at the end of three, six, and nine days at 35° C.
inoculate both a guinea-pig and a rabbit. Prepare cul-
tures from this suspension on the third, sixth, and ninth
days ; when the cultures have developed inoculate a rab-
bit and a guinea-pig from the culture made on the ninth
day. Should the animal survive, inoculate it again
after three or four days with a culture made on the
sixth day. Do the results appear in any way peculiar ?

CHAPTER XXYV.

The most important of the organisms found in the soil—The nitrifying
bacteria—The bacillus of tetanus—The bacillus of malignant edema—The
bacillus of symptomatic anthrax.

By the employment of bacteriological methods in
the study of the soil much light’ has been shed upon
the cause and nature of the interesting and momentous
biological phenomena that are there constantly in pro-
gress. Of these, the one that is of greatest importance
comprises those changes that accompany the widespread
process of disintegration and decomposition, to which
reference has already been made. This resolution of
dead, complex, organic compounds into simpler struc-
tures that are assimilable as food for growing vege-
tation is dependent upon the activities of bacteria
located in the superficial layers of the ground. It is
not throughout a simple process, brought about by
a single, specific, species of bacteria, but represents a
series of metabolic alterations, each definite step of which
is most probably the result of the activities of different
species or group of species, acting singly or together
(symbiotically), Our knowledge upon the subject
is not sufficient to permit of our following in detail
the manifold alterations undergone by dead organic
material in the process of decomposition that results in
its conversion into inorganic compounds, with the forma-
tion of carbonic acid, ammonia, and water as conspicuous
end-products; it suffices to say that, wherever dead
organic matters are exposed to the action of the great

THE NITRIFYING BACTERIA. 8371

group of saprophytic bacteria, in which are found many
different species, the alterations through which they
pass are ultimately characterized by the appearance of
these three bodies. When the process of decomposition
occurs in the soil, however, it does not cease at this
point, but we find still further alterations—alterations
concerning more particularly the ammonia This
change in ammonia is characterized by the products of
its oxidation, viz., by the formation of nitrous and
nitric acids and their salts; it is not a result of the
diréct action of atmospheric oxygen upon the ammonia,
but occurs through the instrumentality of a special
group of saprophytes known as the nitrifying organ-
isms. They are found in the most superficial layers
of the ground, and though more common in some
places than in others, they are, nevertheless, present
over the entire earth’s surface. The most conspicuous
example of the functional activity of this specific form
of soil organism is that seen in the immense saltpetre
beds of Chili and Peru, where, through the activities of
these microscopic plants, nitrates are produced from the
ammonia of the fecal evacuations of sea-fowls in such
enormous quantities as to form the source of supply of
this article for the commercial world. A more familiar
example, though hardly upon such a great scale, is that
seen in the decomposition and subsequent nitrification of
the organic matters of sewage and other impure waters
in the process of purification by filtration through the
soil; a process in which it is possible to follow, by
chemical means, the organic matters from their condition
as such through their conspicuous modifications to their
ultimate conversion into ammonia, nitrous and nitric
acids. In fact the same breaking down and building up,

372 BACTERIOLOGY.

resulting ultimately in nitrification, occurs in all nitro-
genous matters that are thrown upon the soil and
allowed to decay. It is largely through this means that
growing vegetation obtains the nitrogen necessary for
the nutrition of its tissues, and when viewed from this
standpoint we appreciate the importance of this process
to all life, animal as well as vegetable, upon the earth.

These very important and interesting nitrifying
organisms, of which there appear to be several, have
been subjected to considerable study and are found to
possess peculiarities of sufficient interest to justify a
more or less detailed description. For a long time all
efforts to isolate them from the soils in which they were
believed to be present, and to cultivate them by the
processes commonly employed in bacteriological work,
resulted in failure, and it was not until it was found
that the ordinary methods of bacteriological research
were in no way applicable to the study of these bacteria
that other, and ultimately successful, methods were
devised. By these special devices nitrifying bacteria,
capable of oxidizing ammonia to nitric acid, have been
isolated and cultivated, and the more important of their
biological peculiarities recorded by Winogradsky in
Switzerland, by G. and P. F. Frankland in England,
and by Jordan and Richards in this country. From
the similarity of the properties, given by these several
observers, of the nitrifying organisms isolated by
them, it seems likely that they have all been working
with either the same organism or very closely allied
species.

The organism generally known as the nitro-monas
of Winogradsky is a short, oval, and frequently almost
spherical cell. It divides as usual for bacteria, but

THK NITRIFYING BACTERIA. 373

there is little tendency for the daughter cells to adhere
together or to form chains. In cultures they are com-
monly massed together, by a gelatinous material, in the
form of zodglea. They do not form spores, and are
probably not motile, though Winogradsky believes he
_has occasionally detected them in active motion. As
has been stated, they do not grow upon the ordinary
nutrient media, and cannot, therefore, be isolated by
the means commonly employed in separating different
species of bacteria. The most astonishing property of
this organism is its ability to grow and perform its
specific fermentive function in solutions absolutely
devoid of organic matter.. It is believed to be able to
obtain its necessary carbon from carbonic acid. For
its isolation and cultivation Winogradsky recommends
the following solution :

Ammonium sulphate . ‘ é . 1 gramme.
Potassium phosphate . ‘ 1 sf
Pure water . 5 7 - 1000 ¢.c.

To each flask containing 100 cc. of this fluid is
added from 0.5 to 1.0 gramme of basic magnesium car-
bonate suspended in a little distilled water and steril-
ized by boiling. One of the flasks is then to be inocu-
lated with a minute portion of the soil under investiga-
tion, and after four to five days a small portion is to be
withdrawn by means of a capillary pipette from over
the surface of the layer of magnesium carbonate and
transferred to a second flask, and similarly after four or
five days from this to a third flask,and soon. As this
medium does not offer conditions favorable to the growth
of bacteria requiring organic matter for their develop-
ment, those that were originally introduced with the
soil quickly disappear, and ultimately only the nitrify-

17

B74 BACTERIOLOGY.

ing organisms remain. These are to be seen as an
almost transparent film attached to the clumps and
granules of magnesium carbonate on the bottom of the
flask.

For their cultivation upon a solid medium he em-
ploys a mineral gelatin, the gelatinizing principle of
which is silicic acid. A solution of from 3 to 4 per
cent. of silicic acid in distilled water, and having a
specific gravity of 1.02, remains fluid and can be pre-
served in flasks in this condition (Kiihne). By the
addition of certain salts to such a solution gelatiniza-
tion occurs and will be more or less complete, according
to the proportion of salts added. The salts that have
given the best results, and the method of mixing them
are as follows:

Ammonium sulphate . 0.4 gramme.
a { Magnesivin sulphate . 005
Calcium chloride . trace.
Potassium phosphate . 0.1 gramme.
b~ Sodium carbonate . - 0.6 to 0.9 au
(pistittea water . Fi : é 100 ¢.c.

The sulphates and chloride (a) are mixed in 50 cc.
of the distilled water, and the phosphate and carbonate
(6) in the remaining 50 ¢.c. in separate flasks.

Each flask with its contents is then sterilized and
after cooling are mixed together. This represents the
solution of mineral salts that is to be added to the
silicic acid, little by little, until the proper degree of
consistence is obtained (that of ordinary nutrient gela-
tin). This part of the process is best conducted in the
culture dish. If it is desired to separate the colonies,
as in an ordinary plate, the inoculation and mixing of
the material introduced must be done before gelatiniza-
tion is complete ; if the material is to be distributed

THE NITRIFYING BACTERIA, 8375

only over the surface of the medium, then the mixture
must first be allowed to solidify.

By the use of this silicate-gelatin Winogradsky has
isolated from the gelatinous film in the bottom of fluids
undergoing nitrification a bacillus which he believes to
be associated with the nitro-monas in the nitrifying
process.

Our knowledge of these organisms is as yet too in-
complete to permit of a satisfactory description of all
their morphological and biological peculiarities. What
has been said will serve to indicate the direction in
which further studies of the subject should be prose-
cuted.

For further details the reader is referred to the
original contributions.'

In addition to the bacteria concerned in putrefac-
tion and nitrification there are occasionally present in
the soil micro-organisms possessing disease-producing
properties. Conspicuous among these may be men-
tioned the bacillus of malignant cedema (vibrion sep-
tique of the French), the bacillus of tetanus, and the
bacillus of symptomatic anthrax (Rauschbrand, Ger-
man ; charbon symptomatique, French). It is some-
times due to the presence of one or the other of
these organisms that wounds to which soil has had
access (crushed wounds from the wheels of cars or
wagons, wounds received in agricultural work, etc.)
are followed by such grave disturbances of the con-
stitution.

1 Winogradsky: Annales de l'Institut Pasteur, tomesiv., 1890, and v., 1891.
Jordan and Richards: Rep. State Board of Health, Mass. “ Purification
of Sewage and Water.” 1890, vol. ii. p. 864,
Frankland, G. C. and P, F.: Proc, Royal Soc. London, 1890, xlvii.

376 BACTERIOLOGY.

THE BACILLUS OF TETANUS.

In 1884 Nicolaier produced tetanus in mice and rab-
bits by the subcutaneous inoculation of particles of
garden earth, and demonstrated that the pus produced
at the point of inoculation was capable of reproducing
the disease in other mice and rabbits. He did not
succeed in isolating the organism in pure culture. In
1884 Carle and Rattone, and in 1886 Rosenbach de-
monstrated the infectious nature of tetanus as it occurs
in man by producing the disease in animals through
the inoculation of them with the secretions from the
wounds of individuals affected with the disease. In
1889 Kitasato obtained the bacillus of tetanus in pure
culture, and described his method of obtaining it and
its biological peculiarities as follows :

Method of obtaining it. Inoculate several mice sub-
cutaneously with the secretions from the wound of a
case of typical tetanus. This material usually contains
not only tetanus bacilli, but other organisms as well,
so that at autopsy, if tetanus results, there may be
more or less of suppuration at the seat of inoculation
in the mice. In order to separate the tetanus bacillus
from the others that are present, the pus is smeared
upon the surface of several slanted blood-serum or
agar-agar tubes and placed at 37° to 38° C. After
twenty-four hours all the organisms will have devel-
oped and microscopic examination will usually reveal
the presence of a few tetanus bacilli, recognizable by their
shape, viz., that of a small pin, with a spore serving
as the head. After. forty-eight hours at 38° C. the
culture is subjected to a temperature of 80° C. in a water-
bath for from three-quarters to one hour. Atthe end

THE BACILLUS OF TETANUS. 377

of this time, series of plates or Esmarch tubes in slightly
alkaline gelatin are made with very small amounts of
the culture and kept in an atmosphere of hydrogen (see
pages 175-178). They are then kept at from 18° to 20°
C., and at the end of about one week the tetanus bacillus
begins to appear in the form of colonies. After about
ten days the colonies should not only be examined
microscopically, but each colony that had developed in
the hydrogen atmosphere should be obtained in pure
culture and again grown under the same conditions.
The colonies that grow only without oxygen, and
which are composed of the pin-shaped organisms,
must be tested upon mice. If they represent growths
of the tetanus bacillus, the typical clinical manifesta-
tions of the disease will be produced in these animals.

In obtaining the organism from the soil much diffi-
culty is experienced. There are a number of spore-
bearing organisms here that are facultative in their
relation to oxygen, and are, therefore, very difficult to
eliminate ; and there is, moreover, one in particular,
that, like the tetanus bacillus, forms a polar spore. This
spore is, however, less round and much more oval than
that of the tetanus bacillus, and gives to the organism
containing it more the shape of a javelin (or clos-
tridium, properly speaking) than that of a pin, the
characteristic shape of the tetanus organism. It is non-
pathogenic, and grows both with and without oxygen,
and should, consequently, not be mistaken for the latter
bacillus. It must also be borne in mind that there are
occasionally present in the soil still other bacilli which
form polar spores, and which, when in this stage, are
almost identical in appearance with the tetanus bacillus,
but they will usually be found to differ from it in their

378 BACTERIOLOGY.

relation to oxygen, and they are also without disease-
producing properties.

Morphology. It isa slender rod with rounded ends.
It may appear as single rods, or, in cultures, as long
threads. It is motile, though not actively so. The

\ 3
BR Vor
7, ~/ ‘ 7a
/ ot ap Vp
; 604A

Tetanus bacillus. a. Vegetative stage, from gelatin culture, us. Spore
stage, showing pin shapes.

motility is somewhat increased by observing the organ-
ism upon a warm stage.

At the temperature of the body it rapidly forms
spores. These are round, thicker than the cell, and
usually occupy one of its poles, giving to the rod
the appearance of a small pin. (Fig. 81.) When in
the spore stage it is not motile.

It is stained by the ordinary aniline staining re-
agents. It remains colored under the employment of
Gram’s method.

Cultural peculiarities. It is an exquisite anaérobe
and cannot be brought to development under the access
of oxygen. It grows well in an atmosphere of pure
hydrogen, but does not grow under the influence of
carbonic acid.

Tt grows in ordinary nutrient gelatin and agar-agar
of a slightly alkaline reaction. Gelatin is slowly lique-
fied, with the coincident production of a small amount

THE BACILLUS OF TETANUS. 379

of gas. Neither agar-agar nor blood-serum are lique-

fied by its growth.

The addition to the media of
from 1.5 to 2 per cent. of glucose,
0.1 per cent. of indigo-sodium-
sulphate, or 5 per cent. by volume
of blue litmus tincture favors its
growth.

It grows well in alkaline bouil-
lon under an atmosphere of hy-
drogen.

It may be cultivated through
numerous generations under arti-
ficial conditions without loss of
virulence.

Appearance of the colonics. The
colonies on gelatin under an at-
mosphere of hydrogen have, in
their early stages, somewhat the
appearance of the common bacillus
subtilis, viz., they have a dense,
felt-like centre surrounded by a
fringe of delicate radii. The
liquefaction is so slow that the
appearance is retained for a rela-
tively long time, but eventually
becomes altered. In very old
colonies the entire mass is made
up of a number of distinct threads
that give to it the appearance of a
common mould. (See Fig. 82.)

In stab cultures. In stab eul-

Fre, 82.

Colonies of the tetanus
bacillus four days old, made
by distributing the organ-
isms through a tube nearly
filled with glucose-gelatin.
Cultivation under an at-
mosphere of hydrogen.
(From FRANKEL and
PFEIFFER )

tures made in tubes about three-quarters filled with
gelatin, growth begins at about 1.5 to 3 em. below the

380 BACTERIOLOGY.

surface and gradually assumes the appearance of a
cloudy linear mass with prolongations radiating into
the gelatin from all sides. Liquefaction with coinci-
dent gas-production results, and may reach almost to
the surface of the gelatin.

Relations to temperature. It grows best under a
temperature of from 36° to 38° C.; gelatin cultures
kept at from 20° to 25° C. begin to grow after three or
four days. In an atmosphere of hydrogen at from 18°
to 20° C., growth does not usually occur before one
week. No growth occurs under 14° C. At the tem-
perature of the body, spores are formed in cultures in
about thirty hours, whereas in gelatin cultures at from
20° to 25° C. they do not usually appear before a week,
when the lower part of the gelatin is quite fluid. —

Spores of the tetanus bacillus when dried upon bits
of thread over sulphuric acid in the desiccator and
subsequently kept exposed to the air, retain their vitality
and virulence for a number of months. Their vitality
is not destroyed by an exposure of one hour to 80° C.;
on the other hand, an exposure of five minutes to
100° C. in the steam sterilizer kills them. They resist
the action of 5 per cent. carbolic acid for ten hours, but
succumb when exposed to it for fifteen hours. In the same
solution, plus 0.5 per cent. hydrochloric acid, they are
no longer active after two hours. They are killed
when acted upon for three hours by corrosive subli-

“mate, 1: 1000, and in thirty minutes by the same solu-
tion plus 0.5 per cent. hydrochloric acid.

Action upon animals. After subcutaneous inoculation
of mice with minute portions of a pure culture of this
organism tetanus develops in twenty-four hours and
ends fatally in from two to three days. Rats, guinea-

THE BACILLUS OF TETANUS. 381

pigs, and rabbits are similarly affected, but only by
larger doses than are required for mice ; the fatal dose
for a rabbit being from 0.3 to 0.5 c.c. of a well-developed
bouillon culture. The period of incubation for rats
and guinea-pigs is twenty-four to thirty hours, and for
rabbits from two to three days. Pigeons are but
slightly, if at all, susceptible.

The tetanic convulsions always appear first in the
parts nearest the seat of inoculation and subsequently
become general.

At autopsies upon animals that have succumbed to
inoculations with pure cultures of the tetanus bacillus
there is little to be seen by either macroscopic, micro-
scopic, or culture examination. At the seat of inocu-
lation there is usually only a hyperemic condition.
There is no suppuration. The internal organs do not
present any change, and culture methods of examina-
tion show them to be free from bacteria. The death of
the animal results from the absorption of a soluble
poison, either produced by the bacteria at the seat of
inoculation or, which seems more probable, produced
by the bacteria in the culture from which. they are
obtained and introduced with them into the tissues of
the animal at the time of the inoculation. In support
of the latter hypothesis: Mice have been inoculated
with pure cultures of this organism; after one hour
the point at which the inoculation was made was excised
and the tissues cauterized with the hot iron; notwith-
standing the short time during which the organisms

1 Animals and human beings that have become infected with this organism
in the natural way commonly present a condition of suppuration at the site
of infection; this is probably not due, however, to the tetanus bacillus, but
to other bacteria that have also gained access to the wound at the time of
infection.

17

382 BACTERIOLOGY.

were in contact with the tissues and the subsequent
radical treatment, the animals died after the usual
interval and with the regular symptoms of tetanus.

The poison produced by the tetanus bacillus, and to
which the symptoms of the disease are due, has been
isolated and subjected to detailed study ; some of its
peculiarities, as given by Kitasato, are as follows :’

“When cultures of this organism are robbed of their
bacteria by filtration through porcelain, the filtrate
contains the soluble poison and is capable, when injected
into animals, of causing tetanus.

“Tnoculations of other animals with bits of the
organs of the animal dead from the action of the
tetanus poison produce no result ; but similar inocula-
tions with the blood or with the serous exudate from
the pleural cavity always result in the appearance of
tetanus. The poison is, therefore, largely present in
the circulating fluids.

“The greatest amount of poison is produced by
cultivation in fresh neutral bouillon of a very slightly
alkaline reaction.

“The activity of the poison is destroyed by an
exposure of one and one-half hours to 55° C.; of twenty
minutes to 60° C.; and of five minutes to 65° C.

“By drying at the temperature of the body under
access of air the poison is destroyed, but by drying at
the ordinary temperature of the room, or at this tem-
perature in the desiccator over sulphuric acid, it is not
destroyed.

“Diffuse daylight diminishes the intensity of the
poison. Its intensity is preserved for a much longer
time when kept in the dark.

1 Zeitschr. fiir Hygiene, 1891, Bd. x. p. 267.

THE BACILLUS OF MALIGNANT G@DEMA, 383

“Direct sunlight robs it of its poisonous properties
in from fifteen to eighteen hours.

“Tts activity is not diminished by diluting a fixed
amount with water or nutrient bouillon.

“Mineral acids and strong alkalies lessen its inten-
sity.”

The chemical nature of this poison is not positively
known, but from the recent observations of Brieger
and Cohn it is not to be classed with the albumins in
the sense in which the word is commonly uscd. When
obtained in a pure, concentrated form its toxic proper-
ties are seen to be altered by acids, by alkalies, by sul-
phuretted hydrogen, and by temperatures above 70° C.
Even when carefully protected from light, moisture,
and air it gradually becomes diminished in strength.
When freshly prepared by the methods of the authors
just cited its potency is almost incredible, 0.000,05
milligramme being sufficient to cause fatal tetanus in
a mouse weighing fifteen grammes.

THE BACILLUS OF MALIGNANT GDEMA.

The bacillus of malignant cedema, also known as the
vibrion septique is another pathogenic form almost
everywhere present in the soil. In certain respects it
is a little like the bacillus of anthrax, and was at one
time confounded with it, but it differs in the marked
peculiarity of being a strict anaérobe. It was first
observed by Pasteur, but it was not until later that
Koch, Liborius, Kitt, and others, described its pecu-
liarities in detail. It can usually be obtained by
inserting under the skin of rabbits or guinea-pigs small
portions of garden earth, street dust, or decomposing

384 BACTERIOLOGY.

organic substances. There results a widespread oedema,
with more or less of gas production in the tissues. In
the cedematous fluid about the seat of inoculation the
organism under consideration may be detected. (Fig.
83, A.)

Fic. 83,

Bacillus of malignant cedema.

A. Bacilli in short and long threads in cedematous fluid from site of inocu-
lation of guinea-pig. (After Kocn.) .
B. Spore stage of the organism ; from culture.

It is a rod of about 3 to 3.5 long and from 1 to
1.1 » thick, i. @, it is about as long as the bacillus
anthracis but is a trifle more slender. It is usually
found in pairs, joined end to end, but may occur as
longer threads ; particularly is this the case in cultures.
When in pairs, the ends that approximate are squarely
cut, while the distal extremities are rounded. When
occurring singly, both ends are rounded. (How does it
differ in this respect from the bacillus anthracis?) It
is slowly motile and its flagella are located both at the
ends and along the sides of the rod. It forms spores
that are usually located in or near the middle of the
body of the cell. These may cause a swelling of the

THE BACILLUS OF MALIGNANT GiDEMA. 385

cell at the point at which they are located and give to
it a more or less oval, spindle, or lozenge shape. (Fig.

83, B.)

It is a strict anaérobe, growing on
all the ordinary media, but not under
the access of oxygen. It grows well
in a hydrogen atmosphere. It causes
liquefaction of gelatin.

In tubes containing about 20 to 30
c.c. of gelatin that has been liquefied,
inoculated with a small amount of the
culture, and then rapidly solidified in
ice-water, growth appears in the form
of isolated colonies at or near the bot-
tom of the tube in from two to three
days at 20° C. These colonies, when
of from 0.5 to 1 mm. in diameter, ap-
pear as little spheres filled with clear
liquid, and are difficult, for this reason,
to detect. (Fig. 84.)

As they gradually increase in size
the contents of the spheres become
cloudy and are marked by fine radi-
ating stripes, easily to be detected
with the aid of a small hand-lens. In
deep-stab cultures in agar-agar and
gelatin, development only occurs along
the track of puncturé at a distance
below the surface. Growth is fre-
quently accompanied by the produc-
tion of gas-bubbles.

Fic. 84.

Colonies of the
bacillus of wmalig-
nant cedema in deep
gelatin culture. (Af-
ter FRANKEL and
PFEIFFER.)

It causes rapid liquefaction of blood-serum with pro-
duction of gas-bubbles, and in two or three days the

386 BACTERIOLOGY.

entire medium may have become converted into a yel-
lowish, semi-fluid mass.

The most satisfactory results in the study of the
colonies are obtained by the use of plates of nutrient
agar-agar kept in a chamber in which all oxygen has
been replaced by hydrogen. The colonies appear as
dull-whitish points, irregular in outline, and when
viewed with a low-power lens are seen to be marked
by a network of branching and interlacing lines that
radiate in an irregular way from the centre toward the
periphery.

It grows well at the ordinary temperature of the
room, but reaches its highest development at the tem-
perature of the body.

It stains readily with the ordinary aniline dyes. It
is decolorized when treated by Gram’s method.

Pathogenesis. ‘The animals that are known to be
susceptible to inoculation with this organism are
man, horses, calves, dogs, goats, sheep, pigs, chickens,
pigeons, rabbits, guinea-pigs, and mice. Cases are
recorded in which men and horses have developed the
disease after injuries, doubtless due to the introduction
into the wound, at the time, of soil or dust containing
the organism.

If one introduces into a pocket beneath the skin of a
susceptible animal about as much garden earth as can
be held upon the point of a penknife, the animal fre-
quently dies in from twenty-four to forty-eight hours.
The most conspicuous result found at autopsy isa wide-
spread cedema at and about the seat of inoculation. The
cedematous fluid is at places clear, while again it may be
marked with blood; it is usually rich in bacilli (Fig.
83, A) and contains gas-bubbles. Of the internal

THE BACILLUS OF MALIGNANT EDEMA. 387

organs, only the spleen shows much change. It is
large, dark in color, and contains numerous bacilli. If
the autopsy is made immediately after death, bacilli are
not commonly found in the blood of the heart, but if
deferred for several hours the organisms will be found
in this locality also, a fact that speaks for their multi-
plication in the body after death. At the moment of
death they are present in all the internal viscera and on
the serous surfaces of the organs.

Mice are probably most susceptible to the action of
this organism, and it is not rare to find the organisms
in the heart’s blood, even immediately after death.
They die, as a result of these inoculations, in from six-
teen to twenty hours.

Where pure cultures are used for inoculation a
relatively large amount must be employed, and it
should be introduced into a deep pocket in the subcu-
taneous tissues some distance from the surface, In
continuing the inoculations from animal to animal
small portions of organs or a few drops of the cedema-
fluid should be used. The inoculation may also be
successfully made by introducing into a pocket in the
skin bits of sterilized thread or paper upon which
cultures have been dried.

The methods for obtaining the organism in pure
culture, from the cadaver of an animal dead from in-
oculation, are in all essential respects the same as those
given for obtaining cultures from tissues in general,
but it must be remembered that the organism is a strict
anaérobe and will not grow under the influence of oxy-
gen (see methods of cultivating anaérobic species).

In some respects this bacillus suggests the bacillus
anthracis, but differs from it in so many important

388 BACTERIOLOGY.

details that there is no excuse for confounding the
two.

Nore.—From what has been said of this organism,
what are the most important differential points between
it and the bacillus anthracis? Inoculate several mice
with small portions of garden earth and street dust.
Isolate the organism that agrees most nearly with the
description here given for the bacillus of malignant
cedema. Compare its morphological, biological, and
pathogenic peculiarities with those of the bacillus
anthracis under similar circumstances.

Still another pathogenic organism that may be
present in the soil is

THE BACILLUS OF SYMPTOMATIC ANTHRAX ;

bactérie du charbon symptomatique (French); Bacillus
des Rauschbrand (German). It is the organism con-
cerned in the production of the disease of young cattle
and sheep commonly known as “ black leg,” “ quarter
evil,” and “ quarter ill,” a disease that prevails in cer-
tain localities during the warm months, and which is
characterized by a peculiar emphysematous swelling
of the muscular and subcutaneous cellular tissues over
the quarters. The muscles and cellular tissues at
the points affected are seen on section to be saturated
with bloody serum, and the muscles, particularly, are
of a dark, almost black color. In these areas, in the
bloody trausudates of the serous cavities, in the bile,
and, after death, in the internal organs, the organism
to be described can always be detected. It is manifest
from this that the soil of localities over which infected
herds are grazing may readily become contaminated

THE BACILLUS OF SYMPTOMATIC ANTHRAX. 389

through a variety of channels, and thus serve as a
source of further dissemination of the disease.

The organism was first observed by Feser, and sub-
sequently by Bollinger and others. The most complete
description of its morphological and biological pecu-
culiarities is that of Kitasato (Zeitschr. fiir Hygiene,
Bd. vi. p. 105; Bd. viii. p. 55). The following is from
Kitasato’s contributions: It is an actively motile rod
of about 3 to 5 long by 0.5 to 0.6% thick. It is
rounded at its ends, and, as a rule, is seen singly,
though now and then pairs joined end to end may :
occur. It has no tendency to form very long threads.
(Fig. 85, A.)

\ (~ ue

oN ye 28
N

haa \ Pp

[72

sal ia ~ 4

Bacillus of symptomatic anthrax. (After KITASATO.)

A. Vegetating forms from a gelatin culture. 8B. Spore forms from an agar
culture.

It forms spores, and when in this stage is seen to
be slightly swollen at or near one of its poles, the loca-
tion in which the spore usually appears. (Tig. 85, B)

It is conspicuously prone to undergo degenerative
changes, and involution forms are commonly seen, not
only in fresh cultures but in the tissues of affected
animals as well.

Though actively motile when in the vegetative stage,
it loses this property and becomes motionless when
spores are forming.

390

BACTERIOLOGY.

It is strictly anaérobic and cannot be cultivated in an
atmosphere in which oxygen is present. It grows best
under hydrogen, and does not grow under carbonic acid.

Fie. 86.

Colonies of the
bacillus of symp-
tomatic anthrax,
in deep gelatin
culture. (After
FRANKEL and
PFEIFFER.)

20° to 25° C.

The media most favorable to its
growth are those containing glucose (1.5
to 2 per cent.), glycerin (4 to 5 per cent.),
or some other reducing body, such as
indigo-sodium-sulphate, sodium formate,
ete,

When cultivated upon gelatin plates
in an atmosphere of hydrogen the col-
onies appear as irregular, slightly lobu-
lated masses. After a short time lique-
faction of the gelatin occurs and the
colony presents a dark, dense, lobu-
lated and broken centre, surrounded by
a much more delicate fringe-like zone.

When distributed through a deep layer
of liquefied gelatin that is subsequently
caused to solidify, colonies develop at
only the lower portions of the tube.
The single colonies appear as discrete
globules that cause rapid liquefaction
of the gelatin and ultimately coalesce —
into irregular, lobulated, liquid areas.
In some of the larger colonies an ill-
defined, concentric arrangement of alter-
nate clear and cloudy zones can be made
out. (Fig. 86.)

In deep-stab cultures in gelatin growth
begins after about two to three days at

It begins usually at about one or two

centimetres below the surface and causes slow liquefac-

THE BACILLUS OF SYMPTOMATIC ANTHRAX, 39}

tion at and around the track of its development.
During the course of its growth gas-bubbles are pro-
duced.

In deep-stab cultures in agar-agar at 37° to 38° C.
growth begins in from twenty-four to forty-eight hours,
also at about one or two centimetres below the surface,
and is accompanied by the production of gas-bubbles.
There is produced at the same time a peculiar, penetrat-
ing odor somewhat suggestive of that of rancid butter.
Under these conditions spores are formed after about
thirty hours.

It grows well in bouillon of very slightly acid re-
action under hydrogen, but does not retain its viru-
lence for so long a time as when cultivated upon solid
media. In this medium it develops in the form of white
flocculi that sink ultimately to the bottom of the glass
and leave the supernatant fluid quite clear. If the vessel
is now gently shaken these delicate flakes are distributed
homogeneously through it. In bouillon cultures there
is often seen a delicate ring of gas-bubbles around the
point of contact of the tube and the surface of the
bouillon. There is produced also a peculiar penetrating
sour or rancid odor.

It grows best at the body temperature, 7. e., from 37°
to 38° C., but can also be brought to development at
from 16° to 18° C. Under 14° C. no growth is seen.
Spore formation appears much sooner at the higher than
at the lower temperatures. When its spores are dried
upon bits of thread in the desiccator over sulphuric
acid and then kept under ordinary conditions they
retain their vitality and virulence for many months.
Similarly, bits of flesh from the affected areas of ani-
mals dead of this disease, when completely dried, are

392 BACTERIOLOGY.

seen to retain the power of reproducing the disease for
a long time. The spores are tolerably resistant to the
influence of heat: when subjected to a temperature of
80° C. for one hour their virulence is not affected, but
an exposure to 100° C. for five minutes completely
destroys them. They are also seen to be somewhat
resistant to the action of chemicals: when exposed to
5 per cent. carbolic acid they retain their disease-pro-
ducing properties for about ten hours, whereas the vege-
tative forms are destroyed in from three to five minutes ;
in corrosive sublimate solution of the strength of
1: 1000 the spores are killed in two hours.

When gelatin cultures are examined microscopically
the organisms are usually seen as single rods with
rounded ends. When cultivated in agar-agar at a
higher temperature spores are formed after a short
time; these spores are oval, slightly flattened on their
sides, thicker than the bacilli, and, as stated, frequently
occupy a position inclining to one of the poles of the
bacillus, though they are as often seen in the middle.
The bacillus containing a spore has usually a clubbed
or spindle shape.

It stains readily with the ordinary aniline dyes. It
is decolorized by Gram’s method. Its spores may be
stained by the methods usually employed in spore-
staining.

Pathogenesis.—When susceptible animals, especially
guinea-pigs, are inoculated in the deeper subcutaneous
cellular tissues with pure cultures of this organism, or
with bits of tissue from the affected area of another
animal dead of the disease, death ensues in from one to
two days. It is preceded by rise of temperature, loss of
appetite, and general indisposition. The seat of inocula-

THE BACILLUS OF SYMPTOMATIC ANTHRAX. 393

tion is swollen and painful, and drops of bloody serum
may sometimes be seen exuding from it. At autopsy the
subcutaneous cellular tissues and underlying muscles
present a condition of emphysema and extreme cedema.
The cedematous fluid is often blood-stained and the
muscles are of a blackish or blackish-brown color.
The lymphatic glands are markedly hyperemic. The
internal viscera present but little alteration visible to the
naked eye. In the blood stained serous fluid about the
point of inoculation short bacilli are present in large
numbers. These often present slight swellings at the
middle or near the end. They are not seen as threads
but lie singly in the tissues. Occasionally two will be seen
joined end to end. If the autopsy is made immediately
after death these organisms may not be detected in the
internal organs, but if not made until after a few hours
they will be found there also. In fresh autopsies
only vegetative forms of the organism may be found,
but later (in from twenty to twenty-four hours) spore-
bearing rods may be detected. (How does this compare
with the bacillus anthracis?) By successive inoculations
of susceptible animals with the serous fluid from the seat
of inoculation of the dead animal, the disease may be
reproduced.

Cattle, sheep, goats, guinea-pigs, and mice are sus-
ceptible to infection with this organism, and present the
conditions above described ; whereas horses, asses, and
white rats present only local swelling at the site of in-
oculation. Swine, dogs, cats, rabbits, ducks, chickens,
and pigeons are, as a rule, naturally immune to the
disease. ;

Though closely simulating the bacillus of malignant
oedema in many of its peculiarities, this organism can,

394 BACTERIOLOGY.

nevertheless, be readily distinguished from it. It is
smaller ; it does not develop into long threads in the tis-
sues ; it is more actively motile, and forms spores more
readily in the tissues of the animal than does the bacillus
of malignant oedema. In their relation to animals they
also differ, viz.: cattle, while conspicuously susceptible
to symptomatic anthrax, are practically immune toward
malignant cdema; and while swine, dogs, rabbits,
chickens, and pigeons are readily infected with malig-
nant cedema, they are not, as a rule, susceptible to
symptomatic anthrax. Horses are affected only locally,
and not seriously, by the bacillus of symptomatic an-
thrax, but they are conspicuously susceptible to both
artificial inoculation and natural infection by the
bacillus of malignant cedema.

The distribution of the two organisms over the earth’s
surface is also quite different. The cedema bacillus is
present in almost all soils, while the bacillus of symp-
tomatic anthrax appears to be confined to certain locali-
ties, especially places over which infected herds have
been pastured.

A single attack of symptomatic anthrax, if not fatal,
affords subsequent protection, while infection with the
malignant cedema bacillus appears to predispose to re-
currence of the disease. (Baumgarten.)

CHAPTER XXVI.

Infection and immunity—The types of infection; intimate nature of in-
fection—Septicemia, toxemia, variations in infectious processes—Immunity,
natural and acquired—The hypotheses that have been advanced in explaua-
tion of immunity—Conclusions.

AN organism capable of producing disease we call
pathogenic or infective, and the process by which it
produces disease we know as infection. Diseases, there-
fore, that depend for their existence upon the presence
of bacteria in the tissues are infectious diseases.

What is the intimate nature of this process we call
infection? Is it due to the mechanical presence of
living bacteria in the body, or does it result from the
deposition in the tissues of substances produced by these
bacteria, that are either locally or generally incompatible
with life? Or, is the group of pathological alterations
and constitutional symptoms seen in these diseases the
result of abstraction from the tissues, by the bacteria
growing in them, of substances essential to their vitality ?
These are some of the more important of the questions
that present themselves in the course of analysis of this
interesting phenomenon.

Let us look into several typical infectious diseases,
note what we find, and see how far the results will
aid us in formulating an opinion. We begin with a
study of those diseases in which there is a general infec-
tion, 7. e., in which there is a general distribution of the
infective agents throughout the body. This group com-
prises the “septicemias,” and of them the disease of

396 BACTERIOLOGY.

animals known as anthrax represents a type of the
condition. If the cadaver of an animal dead of anthrax
be examined by bacteriological methods it will be dis-
covered that there is present in all the organs and
tissues an organism, a bacillus, of definite form and
biological characteristics ; and if the organs and tissues
generally be subjected to microscopic examination this
same organism will be found always present and always
located within the capillaries. At many points it will
be seen crowded in the capillaries in such numbers as
to almost, if not quite, burst them, and very commonly
their lumen for a considerable extent is entirely occluded
by the growing bacilli. In such a case as this we might
be tempted to conclude that death had resulted from
mechanical interference with the capillary circulation.
Suppose, however, we subject the cultures obtained
from this animal to conditions, either chemical or
thermal, that are not particularly favorable to their
normal development, and from time to time inoculate
susceptible animals with the cultures so treated. The
result will be that as we continue to expose our cul-
tures to unfavorable surroundings, the period of time
that is required for them to cause the death of animals
will, in some cases, gradually become extended, until
finally death will not ensue at all after inoculation. If,
as these animals die, a careful record of the conditions
found at autopsy be kept and compared, it will ulti-
mately be noticed that the animals that die a longer
time after inoculation present conditions more or less
at variance with those seen in the original animal.
These differences usually consist in a diminution of
the number of bacilli that appear upon culture plates
from the blood and internal organs, and in a lessening

INFECTION AND IMMUNITY. 397

in the amount of mechanical obstruction offered to
the circulation through plugging of the capillaries by
masses of bacilli, as detected by microscopic exami-
nation of sections of the organs ; indeed, this latter con-
dition may often have almost, if not quite, disappeared.
We see here an animal dead from the invasion of the
same organism that produced death in the first animal,
but with little or none of the appearances to which we
were inclined to attribute the death of that animal. It
is apparent then, that this organism, with which we
have been working, can destroy the vitality of an
animal in a way other than by mechanically obstruct-
ing its bloodvessels; it possesses some other means of
destroying life. Possibly its growth in the tissues is
accompanied by the production of soluble poisons, which
when present in the blood are not compatible with life.

Let us see if the study of another group of infec-
tions can shed any light upon the subject. Introduce
into the subcutaneous tissues of a guinea-pig a small
amount of a pure culture of the bacillus of diphtheria.
In three or four days the animal dies. We proceed
with our autopsy in exactly the same way that we
did with the animals dead of anthrax, and will be
astonished to find that the organs, blood, and tissues
generally are sterile,| in so far as the presence of the
organism with which the animal was inoculated is con-
cerned, and by both culture and microscopic methods
it is only possible to detect them at the site of inocula-
tion, where they were deposited. It is very evident
that we have here a condition with which mechanical
plugging of the capillaries could have had nothing to

1 In by far the greater number of cases this is true, but under particular
circumstances there are exceptions.

18

398 BACTERIOLOGY.

do, for there are no organisms in the blood to interfere
with its circulation. Our hypothesis then in regard to
the condition found in our first case of anthrax is again
not tenable. Similarly, if an animal that has died of
tetanus be examined we do not find the bacilli in the
tissues and circulating fluids generally, and, indeed,
often fail to find them at the point of injury. Plainly,
these fatal results with their accompanying tissue
changes occur from the presence of a something that
cannot be detected by either cultural or microscopic
methods, and this something can be only a soluble sub-
stance that is produced at the site of inoculation, gains
access to the circulation and through this channel causes
death, for it is hardly to be imagined that the insignifi-
cant wound made in the course of inoculation could
per se have had this effect. In other words, these latter
animals have died from what is called toxemia (poison
in the blood), a condition conspicuously different from
septicemia, as seen in our first animal dead of an-
thrax.

There are, again, other infectious diseases, many of
which are known to present variations from what might
be considered a typical course, that may still further
serve to support the view that infection is a process in
which the mechanical effect of organisms in the cir-
culating fluids is of little consequence. _ Conspicuous
among these are the infections that follow upon the
introduction into the tissues of susceptible animals of
cultures of the micrococcus lanceolatus (pneumococcus),
of the bacillus of chicken cholera, and of the organ-
isms concerned in the production of the so-called
hemorrhagic septicemias. When running their normal
course these organisms cause typical septicemias wher

INFECTION AND IMMUNITY. 399

introduced into animals, but often, from causes not en-
tirely clear, the animals die with only local lesions, or
with but very few organisms in the internal viscera. We
see here conditions analogous to those observed in the two
experiments with anthrax, viz., we find a group of dis-
eases that are normally classed as septiceemias, because of
the general invasion of the body by the organisms con-
cerned in their production, but which frequently assume
a purely local character—in both instances proving
fatal to the animal infected. From what we have seen
it is manifestly probable, whether these diseases be
designated as septiceemias or septic troubles, or toxeemias
or toxic troubles, that death is produced in all instances
by the poisonous products resulting from the growth of
the infecting bacteria. In the case of typical anthrax,
and other varieties of septicemia, the production of this
poison is associated with the general dissemination of
the organisms throughout the body, while in those infec-
tions often referred to as toxemias, of which diph-
theria may be taken as a type, the poison is produced
by the organisms that remain localized at the site of in-
vasion, and is from thence disseminated throughout the
body by the circulating fluids.

Infection thus far, then, appears to be a chemical
process. Through special investigations that have been
made upon the products of growth of certain pathogenic
bacteria, this opinion has received further confirmation ;
it has been found possible by the use of appropriate
methods to isolate, from among the mass of material in
which certain of these organisms have been artificially
cultivated, substances which, when separated from the
bacteria by which they were produced, possess the
power of causing in animals all the constitutional symp-

‘

400 BACTERIOLOGY.

toms and pathological tissue changes that are seen to
occur in the course of infection by the organisms them-
selves.

In some instances the production of the poisonous
principles, even under artificial conditions of cultiva-
tion, is of a most astonishing nature, and poisons result
that, in the degree of their toxicity, exceed anything
hitherto known to us. For instance, the potencies
of the poisons that have been isolated from cultures of
the bacillus diphtherie and the bacillus of tetanus have
been carefully determined by experiments upon animals,
and it has been found that 0.4 milligramme of the
former is capable of killing eight guinea-pigs, each
weighing 400 grammes, or two rabbits, each weighing
three kilogrammes (Roux and Yersin') ; and that 0.0001
milligramme of the latter will produce tetanus in a
mouse, with all the characteristic manifestations of the
disease (Brieger and Cohn”).

In short, infection may be best conceived as a contest
between the invading organisms on the one side and the
resisting tissues of the animal body on the other, the
weapons of offense of the former being the poisonous
products of their growth, and the means of defense
possessed by the latter being substances which are, so
to speak, antidotal to these poisons. If the tissue ele-
ments are not of sufficient vigor to neutralize the bac-
terial poisons, the bacteria are victorious, and infection
results, while, if there is failure to establish a condi-
tion of disease, the tissues are victorious, and are said to
be resistant or to possess immunity to this particular
form of infection.

1 Annales de )’Institut Pasteur, tome iii., 1889, p. 287.
2 Zeitschr. fiir Hygiene u. Infektionskrankheiten, Bd. xv., Heft i., 1893.

INFECTION AND IMMUNITY. 401

It isa common observation that certain human beings
and animals are more susceptible to the different forms
of infection than are others, and that some are apparently
not at all liable to particular diseases ; in other words,
they are naturally immune to the maladies.

Again, it is often observed that an individual or
animal after having recovered from certain forms of
infection has thereby acquired protection against subse-
quent attacks of like character; in other words, they
are said to have acquired immunity to this trouble.

The problem involving the explanation of these in-
teresting observations has afforded material for reflection
and hypothesis for a long time, but it is only through
investigations that have been conducted during the past
few years that it has met with anything approaching
reasonable solution.

Conspicuous among the observers who have endeav-
ored to explain the modus operandi of immunity may
be mentioned Chauveau, Pasteur, Metchnikoff, Buch-
ner, Fliigge, and his pupils (Smirnow, Sirotinin,
Bitter, Nuttall) Fodor, and Hankin, and in the follow-
ing pages we will present briefly the results of investi-
gations by these various authors.

In 1880 Chauveau’ suggested an explanation for the
phenomenon of immunity that has since been known
as the retention hypothesis. It is, in short, as fol-
lows: That the immunity commonly seen to exist in
animals that have passed through an attack of infection,
against a subsequent outbreak of the same malady, and
likewise the immunity that has been produced artificially
by vaccination, exists by virture of some bacterial pro-
duct that has been retained or deposited in the tissues of

1 Comptes-rendus, etc., No. 91. July, 1880.

402 BACTERIOLOGY.

those animals, and that this product by its presence
prevents the development of the same organisms if they
should subsequently gain access to the body.

Bearing upon this view the experiments of Sirotinin,!
made with cultures of various pathogenic bacteria,
demonstrated that in so far as culture experiments were
concerned the only substances produced by growing
bacteria that could be in any way inimical to their
further development were substances that gave rise to
alterations in the reaction of the medium in which
they were developing, 7. ¢., acids or alkalies produced
by the bacteria themselves. So long as the organisms
were not actually dead from exposure to these substances,
correction of the abnormal reaction was followed by
further development of the organisms. Sirotinin, also
states that materials containing the products of growth
of bacteria, so long as they are maintained at a neutral
or only slightly alkaline reaction, serve very well as
media upon which to cultivate again the same organism
that produced them, providing the nutritive elements
have not been entirely exhausted. He remarks: that if
in such a concentrated form as we find the life-products
of bacteria in the medium in which they are growing,
no inhibitory compounds beyond acids and alkalies are
to be detected, it is hardly probable that they are pro-
duced in the tissues of the living animal, and retained
there, to a degree sufficient to prevent the growth of bac-
teria that may subsequently gain entrance to these
tissues, after the disappearance of the organisms con-
cerned in the primary invasion. On the other hand,
Salmon and Smith,’ Roux and Chamberland,’ and

1 Zeitschr. fiir Hygiene, Bd. iv., 1888.
2 Proc. of the Biol. Soc., Washington, D. C., 1886, vol. iii.
3 Annales de l'Institut Pasteur, tomes i , ii., 1888-89.

INFECTION AND IMMUNITY. 403

others had demonstrated that a sort of immunity
against certain forms of infection may be afforded to
susceptible animals by the injection into their tissues of
the products of growth of particular organisms which,
if themselves introduced into the animal body would
produce fatal results. In the light of subsequent experi-
ments, however, the interpretation of this phenomenon
is not that claimed by the supporters of this hypothesis.

As opposed to the view of Chauveau, Pasteur' and
certain of his pupils believed that the immunity fre-
quently afforded to the tissues by an attack of infection,
or following upon vaccination against infection, was
due rather to an abstraction from the tissues, by the
organisms that were concerned in the primary attack,
of a something that is necessary to the growth of the
infecting organism should it gain entrance to the body
at any subsequent time. This view is known as the
exhaustion hypothesis.

As to the exhaustion hypothesis of Pasteur, there is,
as yet, no evidence whatever for its support. The
work of Bitter,? which was undertaken with the view
of determining if, in the process of acquiring immunity,
there occurred this exhaustion from the tissues of
material necessary to the growth of bacteria that might
gain entrance to them at some later date, gave only
negative results. The flesh of animals in which im-
munity had been produced contained all the elements
necessary for the growth and nutrition of the bacter‘a
against which the animals had been protected, just as
did the flesh of non-vaccinated animals.

In 1884 Metchnikoff’® published the first of a serics

1 Bull. de l’Acad. de Med., 1880. 2 Zeitschr. fiir Hygiene, Bd.iv., 1888.
8 Arbeiten aus dem Zodlogischen Institut der Universitit Wien., 1884, Bd. v.
Fortschritte der Med., Bd. ii., 1884.

404 BACTERIOLOGY.

of observations upon the relation that is seen ‘to exist
between certain of the mesodermal cells of lower
animals and insoluble particles that may be present in
the tissues of these animals. The outcome of these
investigations was the establishment of his well-known
doctrine of phagocytosis, the principle of which is that
the wandering cells of the animal organism, the leuco-
cytes, possess the property of taking up, rendering
inert, and digesting micro-organisms with which they
may come in contact in the tissues. Metchnikoff be-
lieved that in this way immunity against infection may
in many, if not all, cases be explained. He believed
that susceptibility to or immunity against infection was
essentially a matter between the invading bacteria on
the one hand and the leucocytes of the tissues on the
other. The success or failure of the leucocytes in pro-
tecting the animal against infection depends, according
to this doctrine, entirely upon the efficiency of the
means possessed by them for destroying bacteria.
When these means are of sufficient vigor to bring
about the death of the bacteria, the tissues are victori-
ous, but when the poisons generated by the bacteria
are potent to arrest the phagocytic action of the leuco-
cytes, then the tissues succumb and infection results.

Has this doctrine of phagocytosis, as advanced by
Metchnikoff, stood the test of experimental criticism?
Evidence that has accrued since the time of its sugges-
tion has rendered questionable the advisability of its
general application.

The first severe blow that this theory received was
given by Nuttall,’ in his work upon the anti-bacterial
action of the animal economy. In these experiments

1 Zeitschrift fiir Hygiene, vol. iv., 1888,

INFECTION AND IMMUNITY. 405

Nuttall showed positively that the part played by the
leucocytes was not essential to the destruction of viru-
lent bacteria in the blood of animals, but that the serum
of the blood, when quite free from cellular elements,
possessed this power to a degree equal to that of the.
blood when all the constituent parts were present. In
the blood, as such, phagocytosis could be seen, but, as a
rule, the bacteria presented evidence of having under-
gone degenerative changes before they had been taken
up by the wandering cells.

Contrary to the notions in existence at the time,
Traube and Gscheidlen,’ as far back as 1874, demon-
strated that considerable quantities of septic material
could be injected into the circulating blood without
apparently any effect upon the animal. As a result
of these experiments, the question that naturally
presented itself was: Does the animal organism
possess the power of rendering septic organisms inert,
and if so, to what extent? Their further work showed
that appeciable numbers of living bacteria could be in-
jected into the circulation of warm-blooded animals
without producing any noticeable effect. Particularly
was this the case with dogs. If they injected into the
circulation of a dog as much as 1.5 c.cem. of decompos-
ing fluid, the blood drawn from the animal after from
twenty-four to forty-eight hours showed no especial
tendency to decompose, though it was kept under obser-
vation for a long time. They believed this power, of
rendering living organisms inert, to be possessed by the
circulating blood to only a limited degree, for, after the
injection of much larger amounts of the putrid fluid
into the blood of the animal, death usually ensued in

1 Jahresbericht der Schlesischen as sue Cultur, 1874; Jahr, lii. p. 179.

406 BACTERIOLOGY.

from twenty-four to forty-eight hours. The blood
drawn from the animal just before death contained the
living bacteria of putrefaction, and underwent decom-
position. They attributed the germicidal phenomenon
to the action of the “ozonized oxygen of the corpuscles
of the blood.”

In 1882 Rauschenbach! demonstrated that, in the
process of coagulation, fibrin was formed not as a
specific product of the action of the colorless elements
of the blood alone, but also as a result of the combined
action between all animal protoplasm and healthy
blood-plasma, and that in the process there was always
a disintegration of the leucocytes that were present.
In 1884 Groth? demonstrated further that such a dis-
integration of leucocytes occurred in normal circulating
blood, though here it was not accompanied by coagu-
lation. The results of these observations suggested
the question: Does such a disintegration occur when
vegetable protoplasm is introduced into the blood? For
the purpose of answering this question, Grohmann,’ a
pupil of Alexander Schmidt, undertook to study the
action of the circulating blood upon the vegetable pro-
toplasm of bacteria.

He noticed that clotting of the blood of the horse
was very much accelerated by the addition to it of cer-
tain bacteria, and that at the same time the develop-
ment of the bacteria was checked, and in the case of
the pathogenic varieties their virulence was diminished.

1 Ueber die Wechselwirkung zwischen Protoplasma und Blutplasma. Dis-
sertation, Dorpat, 1882,

2 Ueber die Schicksale der farblosen Elemente in kreisendem Blut. Disser-
tation, Dorpat, 1884.

3 Ueber die Einwirkung des zellenfreien Blutplasma auf einige pflanzliche
Mikro-organismen. Dissertation, Dorpat, 1884.

INFECTION AND IMMUNITY. 407

This was particularly the case when the anthrax bacillus
was employed.

Grohmann seems to have appreciated the significance
of this observation, though he took no steps to study it
more closely. He remarks that the system probably
possesses, in the plasma of the blood, a body having
disinfectant properties (Joc. cit., pp. 6 and 33). This
work, however, was not conducted according to the
more exact methods of modern bacteriological research,
so that the complete demonstration of this phenomenon
must be attributed to Nuttall.

Since the publication of Nuttall’s work his results
have received confirmation from all sides. Fodor,!
Buchner,’ Lubarsch,? Nissen,‘ Stern,’ Prudden,® Charrin
and Roger,’ and others have continued in the same
line, and have all made practically the same observa-
tion. ,

After the demonstration by Nuttall that the serum of
the blood was directly detrimental to the vitality of
certain pathogenic bacteria, it became the work of a
number of investigators to determine to which element
of the serum this property is due, or if it is a function
of the serum only as a whole.

In the course of Buchner’s experiments it was de-
monstrated that the serum was robbed of this power by
an exposure to a temperature of 55° C. for half an
hour ; that its efficacy as a germicide was not diminished
by alternate freezing and thawing; that by dialysis

1 Centr. f. Bakteriologie u. Parasitenkunde, 1890, vol. vii., No. 24.
2 Archiv fiir Hygiene, 1890, vol x. parts 1 and 2.

3 Centr. f. Bakt. u. Parasitenkunde, 1889, vol. vi., No. 18.

4 Zeitschr. fiir Hygiene, 1889, vol. vi. part 3.

5 Zeitschr. fiir klin. Med., 1890, vol. viii. parts 1 and 2.

6 N. Y. Med. Record, 1890, vol. xxxvii., pp. 85, 86.
7 Soc. de Biol. de Paris.

408 BACTERIOLOGY,

or extreme dilution with distilled water, its germicidal
activity was diminished, or completely checked ; but
that an equal dilution could be made, if sodium chloride
solution (0.6-0.7 per cent.) was substituted for the dis-
tilled water, without the bactericidal action of the serum
losing any of its power. From this he concluded that the
active element in this phenomenon is a living albumin,
an essential constituent of which is sodium chloride, and
which, when robbed of this salt, either by dialysis or
dilution, becomes inert in its behavior toward bac-
teria.

He found, moreover, that the activity of the serum
alone against bacteria was greater than when the cellular
elements of the blood were present. This he explains
-by the assumption that in the serum alone the germi-
cidal element predominates, whereas in the blood, as
such, outside of the body, it is still present, but is over-
balanced by the nutrition offered by the disintegrated
cellular elements; so that here the nutritive element is
most conspicuous, and the destructive activity toward
bacteria is less effectual.

A closer study of the nature of this germicidal ele-
ment in the body of animals was made by Hankin and
Martin." The former isolated from the spleen and
lymphatic glands a body—a globulin—which in solu-
tion possesses germicidal properties.

Similar germicidal, ferment-like globulins have been
isolated from the blood by Ogata,? and in their studies
upon tetanus Tizzoni and Cattani® found a body
that was antagonistic to the poison produced by the
organism of this disease.

1 British Medical Journal, May 31, 1890.

2 Centr. f. Bakt. u. Parasitenkunde, 1891, vol. ix., p. 599.
8 Tbid., p. 685.

INFECTION AND IMMUNITY. 409

Hankin believes the globulins or “ defensive pro-
teids ” that he has discovered and the albuminoid bodies
studied by Buchner to be identical. The most interest-
ing and, in the light of work that has appeared since,
the most important, of Hankin’s observations were not
those upon the power of these globulins to destroy the
vitality of living organisms, but rather those upon the
relation between them and the poisonous proteid pro-
ducts of the organisms. For example, if the poisonous
products of virulent anthrax bacilli be isolated and
mixed with the globulin extracted from normal tissues,
the experiments of Hankin showed a directly destruc-
tive action on the part of the bacterial products. He
found that the amount of poisonous albumose produced
by the attenuated anthrax bacilli, that are employed as
vaccines, was much less than that produced by the
organisms possessing full virulence, and he suggests
that perhaps the protective influence of vaccinations
that are practised by introducing into the animal the
organisms that have been attenuated in virulence is due
to a gradual tolerance acquired by the cells of the tis-
sues to the action of the poison when produced in these
small quantities ; in the same way that a tolerance was
acquired by the tissues for the venom of the rattlesnake
in the experiments of Sewall,' and similar to that fol-
lowing the injection into the tissues of small quantities
of hemialbumose, which in large amounts rapidly
proves fatal.

Of utmost importance to these studies of the blood
and fluids of the body are the experiments of Behring
and Kitasato’ upon the production of immunity to

1 Journal of Physiology, 1887, vol. viii. p. 208.
2 Behring and Kitasato: Deutsche med. Woch., 1890, Bd. xvi. p. 1113.

410 BACTERIOLOGY.

tetanus. In their studies upon the blood of animals
subjected to these experiments it was found that it was
not only possible to render animals immune from this
disease, but that the serum of the blood of these im-
munified animals affords immunity when injected into
the peritoneal cavity of other animals that had not been
so protected ; and moreover, that this serum possesses
curative powers over the disease after it has, in some
cases, been in progress fora time. They found, further,
that the serum of animals that had been rendered im-
mune to tetanus, when brought in contact with the
poison of tetanus, completely destroyed its poisonous
properties, and that the serum from animals or from
human beings that do not possess immunity to this
disease has no such power.

Another hypothesis in explanation of the immunity
acquired by the tissues of the animal organism is that
advanced by Buchner,' who suggests that in the primary
infection, from which the ‘animal has recovered, there
has been produced a reactive change in the integral
cells of the body that enables them to protect them-
selves against subsequent inroads of the same organism.
Though somewhat more vague at first glance than the
other theories in regard to this phenomenon, it is, never-
theless, in the light of subsequent research, most prob-
ably the correct explanation of the establishment of
immunity in many, if not all, cases. Experiments
that bear directly upon this idea have demonstrated
that, if animals are subjected to injections of the poison-
ous products of growth of certain virulent bacteria,
they respond to this treatment by more or less pro-

1 Buchner: Eine neue Theorie iiber Erzielung von Immunitit gegen In-
fektionskrankheiten. Munich, 1883,

INFECTION AND IMMUNITY. All

nounced constitutional reactions, and that during this
period, and for a short time following, they possess pro-
tection against the invasion of the virulent bacteria
themselves. This observation has, moreover, not been
confined to those cases in which injections of the pro-
ducts of growth have been followed by inoculations with
the bacteria by which they were produced, but what is
still more interesting, and confirmatory of Buchner’s
view, it is claimed that a sort of protection against
certain specific infections can also be afforded to animals
by the injection into them of cultures of entirely dif-
ferent species of bacteria, or their products, and that in
some cases these are not of necessity of the disease-pro-
ducing variety. For instance, Emmerich and Mattei!
claim to have rendered rabbits insusceptible to anthrax
through injections into them of cultures of the strepto-
coccus of erysipelas.

This, they claim, is not due to any antagonism
between the organisms themselves, for in culture experi-
ments the two organisms grew well together, without
any alteration in their pathogenic properties, but rather
to the production of a tissue-change by which resistance
to the inroads of the virulent bacilli was established.
Emmerich and Mattei interpret this “ reactive tissue-
change” as a power acquired by the integral cells of
the body, of eliminating a product that is detrimental
to the pathogenic activity of the anthrax bacilli.

Pawlowsky,’? who obtained similar results from the
introduction into the animal of cultures of the bacillus
prodigiosus, of staphylococcus pyogenes aureus, and of
the micrococcus lanceolatus, believes them to be due to

1 Emmerich und Mattei: Fortschritte der Medizin, 1887, p. 653,
2 Pawlowski: Virchow’s Arch., vol. cviii. p. 494.

412 BACTERIOLOGY,

the induction of increased energy on the part of the
wandering cells, preparing them thus for the more
difficult task of destroying the more virulent organisms
with which the animal is subsequently to be inoculated.

The experiments of G. and F. Klemperer’ upon acute
fibrinous pneumonia, though too limited in extent to
be accepted as conclusive, have, nevertheless, offered a
number of most significant suggestions, not only in
connection with several obscure features of this disease,
but also in relation to the establishment of tissue
resistance.

They found but little difficulty in affording immunity
to animals that are otherwise susceptible to the patho-
genic action. of the organisms concerned in the pro-
duction of this disease,’ by the introduction into their
tissues of the products of growth of the organisms from
which the latter had been separated. The immunity.
thus produced is seen in some cases to last as long as
six months; again it is seen to disappear suddenly in a
way not to be explained. It was seen in one case to be
hereditary.

The energy of the substance that has the power of
affording immunity was seen to be very much increased
by subjecting it to temperatures somewhat higher than
that at which it was produced by the bacteria. The
Klemperers found that if this substance was heated
to a temperature of from 41° to 42°C. for three or
four days, or to 60° C. for from one to two hours,
intravenous injection was followed by complete im-
munity in from three to four days; whereas, if the

1G, and F. Klemperer : Berliner klin. Wochenschr., 1891, Nos. 34 and 35.
2 Animals do not, as a rule, present the pneumonie changes seen in human

beings. The introduction of the micrococeus lanceolatus into their tissues
results, in the case of susceptible animals, in the production of septicemia,

INFECTION AND IMMUNITY. 418

unwarmed material was used, immunity did not appear
until fourteen days, and then only after the employ-
ment of relatively large amounts. Moreover, when the
previously heated products are introduced into the cir-
culation of the animal, the systemic reaction is of but
short duration, but if the unwarmed substance is em-
ployed, immunity is manifest only after the appearance
of considerable elevation of temperature, which lasts for
along time. In explanation of these differences, they
suggest that, in the latter case, the high fever that is
seen to occur in the animal may serve to replace the
warming to which the bacterial products had not pre-
viously been subjected, and which is necessary before
they are in a position to bring about the condition
of immunity. They claim that the bacterial pro-
ducts employed in producing immunity in this case
are not, in reality, the immunity-affording substance,
but that they are only the agents that bring about in
the tissues of the animals alterations that result in the
production of another body that protects the animal.
In support of this, their argument is that several days
are necessary for the production of immunity by the
introduction into the animal of the bacterial products ;
whereas, if the blood-serum of this animal, which is
now protected, be introduced into the circulation of
another animal, no such delay is seen, but instead, the
animal is forthwith protected. In the former case the
actual protecting body had first to be manufactured
by the tissues ; whereas, in the second it is already
prepared, and is introduced as such into the second
animal.

They found the serum of immunified animals to be not
only capable of rendering other animals immune, but that

414 BACTERIOLOGY.

it possessed curative powers when the disease is already
in progress. The serum of immunified animals, when
injected into the circulation of animals in which this
form of infection was in progress, and in which there
was a body-temperature of from 40.4° to 41° C., reduced
this temperature to normal (37.5° C.) in twelve con-
secutive experiments during the first twenty-four hours
following its employment.

In their opinion, the crisis, seen in pneumonia
in human beings, indicates the moment at which the
poisonous products, manufactured by the bacteria located
in the lungs, are present in the circulation in amounts
sufficient to call forth in the tissues the reactive change
that results in the production of the antidotal substance
that has the power of rendering the poisons inert.

At the time of the crisis in pneumonia the bacteria
themselves are in no way affected. They remain in
the lungs, and can be detected, in full vigor and viru-
lence, in the sputum of patients a long time after the
disease is cured. They have lost none of their power
of producing poisonous products, and still possess their
original pathogenic relations toward susceptible animals.
It is only after the crisis that their poisons are neutral-
ized by this antidotal proteid that has been eliminated
by the cells of the tissues, and as this occurs the sys-
temic manifestations gradually disappear. The Klem-
perers claim to have isolated from cultures of the micro-
coceus lanceolatus a proteid body that is the agent con-
cerned in producing the tissue reaction which results in
the formation of the protecting substance. They likewise
isolated from the serum of immunified animals a pro-
teid that possesses the same powers as the serum itself
—viz., of affording immunity and curing the disease.

INFECTION AND IMMUNITY. 415

Here, again, it appears that the processes of infection
and immunity are chemical in their nature, the active
poisons of the invading organisms—‘“the pneumo-
toxines ”—being instrumental in producing the dis-
eased condition, while the antidotal or resisting body of
the tissues—“ the anti-pneumotoxine ”—is the agent by
which the poison is neutralized.

Results in general analogous to those of G. and F.
Klemperer have also been obtained by Emmerich and
Fowitzky.’

In the light of these experiments, the hypothesis
advanced by Buchner, that the establishment of im-
munity is to be explained by reactive changes in the
integral cells of the body, receives additional support,
and when we consider the observations of Bitter,? who
found that in protective vaccinations against anthrax
the vaccines do not disseminate themselves through the
body, as is the case when the virulent organisms are
introduced, but remain at the point of inoculation, and
from this point produce, by the absorption of their
chemical products, the systemic changes through which
the animal is protected against subsequent infection by
the virulent organisms, we feel justified in concluding that
the weight of evidence is strongly in favor of this view.

The experiments tbat have been cited afford but an
imperfect idea of the enormous amount of work that has
been done upon these important subjects ; they may, how-
ever, serve to indicate the direction in which the lines of
research have been laid. As a result of such investiga-
tions, our knowledge upon infection and immunity may
at present be summarized about as follows:

1 Emmerich and Fowitzky : Miinchener med. Wochenschr., 1891, No. 32.
2 Bitter: Zeitschrift fir Hygiene, 1888, Bd. iv.

416 BACTERIOLOG F.

1. That infection may be considered as a contest
between bacteria and living tissues, conducted on the
part of the former by means of the poisonous products
of their growth, and resisted by the latter through the
agency of proteid bodies normally present in and
eliminated by their integral cells.

2. That when infection occurs it may be explained
either by the excess of vigor of the bacterial products
over the antidotal or protective proteids eliminated by
the tissues, or to some cause that has interfered with
the normal activity and production of these bodies.

3. That immunity is most frequently seen to follow
the introduction into the body of the products of growth
of bacteria that in some way or other have been modified.
This modification may be artificially produced in the
products themselves of virulent organisms, and then
introduced into the tissues of the animal; or the viru-
lent bacteria may be so treated that they are no longer
virulent, and when introduced into the body of the
animal will eliminate poisons of a much less vigorous
nature than would otherwise be the case.

4. That immunity following the introduction of
bacterial products into the tissues is not in all cases the
result of the permanent presence of these substances,
per se, in the tissues, or to a tolerance acquired by the
tissues to them, but is probably, in certain instances, due
to the formation in the tissues of another body that acts
as a protecting antidote to the poisonous products of
invading organisms.

5. That this protective proteid that is eliminated by
the cells of the tissues need not of necessity be antago-
nistic to the life of the invading organisms themselves,

INFECTION AND IMMUNITY. 417

but in some cases must be looked upon more as an anti-
dote to their poisonous products.

6. That in the serum of the normal circulating blood
of many animals there exists a substance that is capable,
outside of the body, of rendering inert bacteria that,
if introduced into the body of the animal, would prove
infective.

7. That phagocytosis, though frequently observed, is
not essential to the establishment of immunity, but is
more probably a secondary process, the bacteria being
taken up by the leucocytes only after having been
modified in virulence through the normal germicidal
activity of the serum of the blood and of other fluids in
the body.

8. That, of the hypotheses that exist for the explan-
ation of immunity, the one which assumes acquired
immunity to be due to reactive changes on the part of
the tissues has received the greatest support.

CHAPTER XXVII.

Bacteriological study of water—Methods employed—Precautions to be
observed—Apparatus used, and methods of using them—Methods of investi-
gating air and soil.

THE conditions that favor the epidemic outbreak of
typhoid fever, Asiatic cholera, and other maladies of
which these may be taken as types, have served as a
subject for discussion by sanitarians for a long time.

Of the hypotheses that have been advanced in explan-
ation of the existence and dissemination of these dis-
eases, two stand pre-eminent and are worthy of con-
sideration. They are the “ground-water” theory of
Von Pettenkofer and his pupils, and the “drinking-
water” theory of the school of bacteriologists of which
Koch stands at the head.

The adherents to the “ground-water” view explain
the presence of these diseases in epidemic form through
alterations in the soil resulting from fluctuations in the
level of the soil water, and assign to the drinking-water
either a very insignificant réle, or, as is most frequently
the case, ignore it entirely. On the other hand, those
who have been instrumental in developing the drinking-
water hypothesis, claim that alterations in the soil play
little or no’ part in favoring the appearance of these
diseases in a neighborhood, but that, as a rule, they
appear as a result of direct infection through the use of
waters that are contaminated with materials containing
the specific organisms known to be the cause of them.

As a result of many observations on both sides of the

STUDY OF WATER. 419

question, the evidence is greatly in favor of the opinion
that polluted drinking-water is primarily the under-
lying cause of these epidemics, and this too, very often,
when the state of the soil water, in the light of the
“ground-water” hypothesis, is just the reverse of what
it should be in order to render it answerable for them.
It is manifest, therefore, that the careful bacteriological
study of water intended for domestic use is of the
greatest importance, and should be a routine procedure
in all communities receiving their water supply from
sources that are liable to pollution.

The object aimed at in such investigations should be
to determine if the water approaches constancy in the
number and kind. of bacteria contained in it—for all
waters, except deep ground water, contain bacteria; if
sudden fluctuations in the number of bacteria occur in
these waters, and if so, to what they are due ; and finally,
and most important, Does the water contain constantly,
or at irregular periods, bacteria that can be traced to
human excrement, not of necessity pathogenic varieties,
but bacteria that are known to be present normally in
the intestinal canal? For, if conditions are favorable to
the presence of these varieties the same conditions would
favor the admission to the water of other forms of
bacteria that are concerned in the production of diseases
in the intestines.

In considering water from a bacteriological stand-
point, it must always be borne in mind that comparisons
with any general fixed standard are not of much value,
for just as normal waters from different sources are
seen to present variations in their chemical composition,
without being unfit for use, so may the number of
bacteria per volume in water from one source be always

420 BACTERIOLOGY.

greater or smaller than in that from another, and yet
no difference may be seen to result from their employ-
ment. For this reason the proper study of any water,
from this point of view, should begin with the establish-
ment of what may be called its normal mean number
of bacteria, as well as the character of the prevailing
species ; and in order to do this the investigations must
cover a long period of time through all the seasonal
variations of weather. From data obtained in this way
it may be possible to predict approximately the normal
bacteriological condition of water at any season. Marked
deviations from these “ means,” either in the quantity or
quality of the organisms present, can then be considered
as indicative of the existence of some unusual disturbing
element, the nature of which should be investigated.

Similarly, it is impossible to formulate an opinion of
much value from a single chemical analysis of a water,
for the results thus obtained indicate only the state of the
water at the time the sample was procured, and give no
indication as to whether it differed at that time from
its usual condition, or from the normal condition of the
water throughout the immediate neighborhood.

The interpretation of the results of both chemical
and bacteriological analysis of a sample of water ac-
quires its full nature only through comparison, either
with “means” that have been determined for this
water, or with the results of simultaneous analyses of a
number of samples from the other sources of supply of
the locality.

The aid of the bacteriologist is frequently sought in
connection with investigations upon waters that are
supposed to be concerned in the production of disease,
particularly typhoid fever, either in isolated cases or in

STUDY OF WATER. 421

widespread epidemic outbreaks, and almost as often do
reliable bacteriologists fail to detect the bacillus that is
the cause of typhoid fever in these waters.

The failure to find the organisms of typhoid fever in
water by the usual methods of analysis does not by
any means prove that they are not present or have not
been present. The means that are ordinarily employed in
the work admit of such a very small volume of water
being used in the test that we can readily understand how
these organisms might be present in moderate numbers
and yet none of them be included in the drop or two
of the water that are taken for study. The conditions
are not those of a solution, each drop of which contains
exactly as much of the dissolved material as do all
other drops of equal volume, but are rather those of a
suspension in each drop or volume of which the number
of suspended particles are liable to the greatest degree
of variation. Furthermore, there are other reasons
that would, @ priori, preclude our expecting to find
the typhoid bacilli in water in which we may have
reason to believe they had been deposited, viz., atten-
tion is not usually directed to the water until the
presence of the disease has become conspicuous, usually
in from three to four weeks after the time when the
pollution probably occurred. Three or four weeks is
ordinarily sufficient time for the delicate, non-resistant
bacillus of typhoid fever to succumb to the unfavorable
conditions under which it finds itself in water. By
unfavorable conditions is meant the absence of suitable
nutrition ; unfavorable temperature; probably the an-
tagonistic influence of more hardy saprophytic bacteria,
particularly the so-called “ water bacteria,” and of more

highly organized water plants ; the effect of mechanical
19

499 BACTERIOLOGY.

precipitation; and of great importance, the disinfecting
action of direct sunlight.

Though it is so rare as to be almost never, that
typhoid bacilli are found in drinking-water, it must,
nevertheless, not be supposed that bacteriological analy-
ses of suspicious waters shed no light upon the exist-
ence of pollution and the suitability or non-suitability
of the water for drinking purposes.

In the normal intestinal tract of all human beings,
and many other mammals, as well as associated with
the specific disease-producing bacterium in the intes-
tines of typhoid fever patients, is an organism that
is frequently found in polluted drinking-waters, and
whose presence is proof positive of pollution by
either normal or diseased intestinal contents; and
though efforts may result in failure to detect the specific
bacillus of typhoid fever, the finding of the other
organism, the bacterium coli commune, justifies one
in expressing the opinion that the water under con-
sideration has been polluted by intestinal evacuations
from either human beings or animals. Waters so
located as to be liable to such pollution can never be
considered as other than a continuous source of danger
to those using them.

Another point to be remembered is in connection with
the value of chlorine as indicative of contamination by
human excrement. It is commonly taught that an
excessive amount of chlorine in water points to con-
tamination by human excreta. This may or may not
be true according to circumstances. A high propor-
tion of this substance in a sample of water from a
locality the neighboring waters of which are poor in
chlorine, is unquestionably a suspicious indication, but

STUDY OF WATER. 423

in a district close to the sea or near salt deposits, for
instance, where the water generally is high in chlorine,
the value of the indications thus afforded is very much
diminished unless the amount found in the sample
under examination greatly exceeds the normal “‘ mean,”
previously determined, for the amount of chlorine in
the waters of the neighborhood.

A striking example of such a condition as the latter
recently occurred in the experience of the writer while
inspecting a group of water supplies on the east coast
of Florida. In each instance the water was obtained
from properly protected artesian wells, ranging from
200 to 400 feet deep, and located within a few hundred
yards of the sea. The first sample that was subjected
to chemical analysis revealed such an unusually high
proportion of chlorine that, had this sample alone been
considered, the opinion that it was polluted by human
excreta might have been advanced. To prevent such
an error samples of water from a number of wells in
the neighborhood were examined, and they were all
found to contain from ten to twelve times the amount
of chlorine that ordinarily appears in inland waters,
the excess being evidently due to leakage through the
soil into the wells of water from the sea. In short,
the presence of an excess of chlorine in water, while
often indicating pollution from human evacuations,
may, nevertheless, sometimes arise from other sources,
but the presence in water of bacteria normally found
in the intestinal canal can manifestly admit of but
one interpretation, viz., that fecal matters have at
some time and place been deposited in this water,
and that while no specific disease-producing organisms
may have been detected, still, waters in which such

424 ‘BACTERIOLOGY.

pollutions are possible are a constant menace to the
health of those who use them for domestic pur-
poses. :

A sudden variation from the normal, mean number
of bacteria, or from the normal chemical composition
of a water, calls at once for a thorough inspection of the
supply, while at the same time the characters of the
organisms present are to be subjected to the most careful
study.

THE QUALITATIVE BACTERIOLOGICAL ANALYSIS
oF WaTER.—The qualitative bacteriological analysis
of water entails much labor, as it requires not only that
all the different species of organism found in the water
should be isolated, but that each representative should
be subjected to systematic study, and its pathogenic or
non-pathogenic properties determined.

-For this purpose the methods for the isolation of
individual species, which have already been described,
and the means of studying these species when isolated,
are indispensable.

For this analysis certain precautions essential to
accuracy are always to be observed.

The sample is to be collected under the most rigid
precautions that will exclude organisms from sources
other than that under consideration. If drawn from a
spigot, it should never be collected until the water has
been flowing for fifteen to twenty minutes in a full
stream. If obtained from a stream or a spring, it
should be collected, not from the surface, but rather
from about one foot beneath the surface.

It should always be collected in vessels which have
previously been thoroughly freed from all dirt and
organic particles, and then sterilized. And the plates

STUDY OF WATER, 425

should be made as quickly as possible after collecting
the sample.

Where circumstances permit, all water analyses
should be made on the spot at which the sample is
taken, as it is known that during transportation, unless
the samples are kept packed in ice, a multiplication of
the organisms contained in it always occurs.

For the purpose of qualitative analysis it is necessary
that a small portion of the water—oné, two, three, five
drops—should first be employed as the amounts from
which plates are to be made. In this way one forms
some idea as to the approximate number of organisms
in the water, and can, in consequence, determine the
amount of water necessary to use for each set of plates.
Duplicate plates are always to be made—one set upon
agar-agar, which are to be kept in the incubator at
body temperature, and one set upon gelatin, to be kept
at from 18° to 20° C.

As soon as the colonies have developed the plates are
to be carefully compared and studied. It is to be
noted if any difference in the appearance of the organ-
isms on corresponding plates exists, and if so, to what
is it due? Itis to be particularly noted which plates
contain the greater number of colonies, those kept at
the higher or those at the lower temperature. In this
way the temperature best suited for the growth of the
majority of these organisms may be determined.

As a rule, the greater number of colonies appears
upon the gelatin plates that are kept at 18° to 20° C.,
and from this it would seem that many of the normal
water bacteria do not find the higher temperature so
favorable to their development as do the organisms
not naturally present in water, particularly the patho-
genic varieties,

426 BACTERIOLOGY.

Nore.—In determining if the organisms found are
possessed of pathogenic properties, in what way will
your tests be influenced by this observation ?

From recent investigations upon this subject it ap-
pears that the difference in behavior toward heat of
bacteria present in water may have a very important
application. Dr. Theobald Smith, of Washington, has
recently suggested a method by which it is easily pos-
sible to isolate, from waters in which they are present,
certain organisms that are of the utmost importance in
influencing our judgment upon the fitness of the water
for domestic use. By the addition of small quantities,
one, two, or three drops of the suspicious water to fer-
mentation tubes (see article on Fermentation Tube)
containing bouillon to which 2 per cent. of glucose has
been added, and keeping therfi at the temperature of
the body, 37° to 38° C., the growth of the intestinal
bacteria that may be present in the water is favored,
while that of the water organisms is not; in consequence,
after from thirty-six to forty-eight hours the fermenta-
tion, characteristic of most of these organisms, is evi-
denced by the accumulation of gas in the closed end of
the tube, From these tubes the growing bacteria can then
be easily isolated by the plate method, and it will not
be infrequent to find intestinal bacteria present in pure
culture.

Another method for the same object is to collect a
sample of about 100 c.c. of the water to be tested in a
sterilized flask, and add to this about 25 e.c. of steril-
ized bouillon of four times the usual strength. This
is then placed in the incubator at 37° to 38° C., for
thirty-six to forty-eight hours, after which plates are

STUDY OF WATER. 427

to be made from it in the usual way; the results will
often be a pure culture of some single organism, either
one of the intestinal variety or a closely allied species.
By a method analogous to the latter the spirillum of
Asiatic cholera has been isolated from water; and by
taking advantage of the effect of elevated temperature
upon the bacteria of water, Dr. Vaughan, of Michigan,
has succeeded in isolating from suspicious waters a
group of organisms very closely allied to the bacillus
of typhoid fever.

THE QUANTITATIVE EStIMATION OF BACTERIA IN
WaTER.—Quantitative analysis requires more care
in the measurement of the exact volume of water em-
ployed, for the results are to be expressed in terms of
the number of individual organisms to a definite volume.
The necessity for making the plates at the place at
which the sample is collected is to be particularly
accentuated in this analysis, for the multiplication of
the organisms during transit is so great that the results
of analyses made after the water has been in a vessel
for a day or two are often very different from those
that would have been obtained on the spot.

Nore.—Inoculate a tube containing about ten cubic
centimetres of sterilized distilled or tap water with a
very small quantity of a solid culture of some one of
the organisms with which you have been working,
taking care that none of the culture medium is intro-
duced into the water-tube and that the bacteria are
evenly distributed through it. Make plates at once,
and on each succeeding day, from this tube, and deter-
mine by counts whether there is an increase or diminu-
tion in the number of organisms-—i. ¢., if they are

428 BACTERIOLOGY.

growing or dying. Represent the results graphically,
and it will be noticed that in many cases there is at
first, during the first three or four days, a multiplica-
tion, after which there is a rapid diminution; and, if
the organism does not form spores, usually complete
death in from ten to twelve days. This is not true for
all organisms, but does hold for many.

Where it is not convenient, however, to make the
analysis on the spot, the sample of water should be col-
lected and packed in ice and kept on ice until ready for
use, which should in all cases be as soon after its collec-
tion as possible.

For the collection of water for this purpose, a con-
venient vessel to be employed is a glass bulb (Fig. 87)
or balloon, which one soon learns to make for oneself
from glass tubing.

Fig. 87.

Tt consists simply of a round glass sphere blown on
the end of a glass tube, which latter is subsequently
drawn out into a fine capillary stem and sealed while
hot. As it cools, the contraction of the air within the
bulb results in the production of a negative pressure.
If the point of the stem be broken off under water, the
water is pressed up into the bulb, because of the existence
of the negative pressure within. The negative pressure
obtained in this way is frequently not sufficient to permit
of the bulb being completely filled, and often only a few
drops of fluid can be obtained. To obviate this the

STUDY OF WATER. 429

bulbs may be blown and allowed to cool, but not sealed.
After a sufficient number of them are prepared they are
taken, one at a time, and gently warmed over the flame;
while still warm the extremity of the stem is dipped
into distilled water and held there until a few drops
have passed up into the bulb; this is then care-
fully boiled, or rather, completely vaporized, over the
flame, and while the steam is still escaping the point is
sealed in the gas flame. All air will have thus been
replaced by water vapor, and if the point of the stem
be now broken off under water the bulb will fill quickly
and completely. It is not desirable to fill them com-
pletely, but rather to only about three-fourths of their
capacity, as when full it is difficult to empty them with-
out contaminating the contents. They are emptied by
gently warming over a gas or alcohol flame.

A number of them may be made, sealed, and kept
on hand. They are sterile’so long as they are sealed,
because of the heat that is employed in their manu-
facture.

When a sample of water is to be taken, the point of
_ a bulb is simply broken off with sterilized forceps under
water at the place from which the sample is to be pro-
cured and held there until the necessary amount has been
obtained. This may serve as a sample from which to
prepare plates or Esmarch tubes on the spot, or the tip
of the stem may be resealed in the flame of an alcohol
lamp, the bulb packed in ice, and transported in this
condition to the laboratory.

Another very simple and useful device for collecting
water samples is that recommended by Kirschner. It
consists of a piece of glass tubing of about 5 or 6 mm.

inside diameter and 36 cm. long, bent in the form of a
19*

430 BACTERIOLOGY.

U, with either extremity of the arms bent again at
right angles in the same place and drawn out to a point
and sealed. ‘They are sterilized in the flame as they
are made. The sample is collected by breaking off
both points, immersing one of them into the water and
sucking on the other until the tube is filled. Then
both points are again sealed in the flame and the tube
packed in ice. The objection to this tube is the danger
of contaminating its contents with saliva during the
‘ act of filling by suction, though this danger is not so
great as might at first appear, as we shall learn in our
efforts to cultivate bacteria from the mouth cavity.

Nore.—Make cover-slips from your own mouth;
make plates on both gelatin and agar-agar, at the same
time. Compare the number of bacteria found by
microscopic examination of the cover-slips with the
number of colonies that develop on the plates.

In beginning the quantitative analysis of water with
which one is not acquainted, there are certain prelimi-
nary steps that are essential.

It is necessary to know approximately the number of
organisms contained in any fixed volume, so as to de-
termine the quantity of water to be employed for the
plates or tubes. This is usually done by making pre-
liminary plates from one drop, .two drops, 0.25 c.c.,
0.5 c.c., and 1 cc. of the water. After each plate has
been labelled with the amount of water used in making
it, it is placed aside for development. When this has
occurred, one selects the plate upon which the colo-
nies are only moderate in number—about 200 to 300
colonies presenting—and employs in the subsequent

STUDY OF WATER. 431

analysis the same amount of water that was used in
making this plate.

If the original water contained so many organisms
that there developed on a plate or tube made with one
drop too many colonies to be easily counted, then the
sample must be diluted with one, ten, twenty-five,
fifty, or one hundred volumes, as the case may require,
of sterilized distilled water. This dilution must be
accurate, and its exact extent noted, so that subsequently
the number of organisms per volume in the original
water may be calculated.

The use of a drop is not sufficiently accurate. The
dilution should therefore always be to a degree that
will admit of the employment of a volume of water
that may be exactly measured, 0.25, 0.5 c.c. being the
amounts most convenient for use.

Duplicate plates should always be made and the
mean of the number of colonies that develop upon
them taken as the basis from which to calculate the
number of organisms per volume in the original water.

For example: From a sample of water, 0.25 cc. is
added to a tube of liquefied gelatin, carefully mixed and
poured out as a plate. When development occurs, the
number of colonies are too numerous to be accurately
counted. One cubic centimetre of the original water is
then to have added to it, under precautions that pre-
vent contamination from without, 99 c.c. of sterilized
distilled water—that is, we have now a dilution of
1:100. Again, 0.25 c.c. of this dilution is plated and
we find 180 colonies on the plate. Assuming that each
colony develops from an individual bacterium, though
this is perhaps not strictly true, we had 180 organisms
in 0.25 «ec. of our 1:100 dilution, therefore in 0.25

432 BACTERIOLOGY.

c.c. of the original water we had 180 x 100 = 18,000
bacteria, which will be 72,000 bacteria per cubic centi-
metre (0.25 = 18,000, 1 ec. = 18,000 x 4 = 72,000).
The results are always to be expressed in terms of the
number of bacteria per cubic centimetre of the original
water.

Another point of very great importance (already
mentioned) is the effect of temperature upon the num-
ber of colonies of bacteria that will develop on plates
made from water. It must always be remembered
that a larger number of colonies appear on gelatin
plates made from water and kept at 18° to 20° C. than
on agar-agar plates kept in the incubator. The follow-
ing table, illustrative of this point, gives the results of
parallel analyses of the same waters, the one series of.
counts having been made upon gelatin plates at the
ordinary temperature of the room, the other upon
plates of agar-agar kept for the same length of time in
the incubator at from 37° to 38° C. It will be seen
from the table that much the larger number of colonies,
i. e., much higher results, are always obtained when
gelatin is employed. The importance of this point in
the quantitative bacteriological analysis of water is too
apparent to require further comment.

STUDY OF WATER. 433

TABLE ILLUSTRATING THE PROPORTION BETWEEN THE RE-
SULTS OBTAINED BY THE USE OF GELATIN AND AGAR-AGAR
IN QUANTITATIVE BACTERIOLOGICAL ANALYSIS OF WATER.
RESULTS RECORDED ARE THE NUMBER OF COLONIES THAT
DEVELOPED FROM THE SAME AMOUNT OF WATER IN EACH
SERIES.!

NUMBER OF COLONIES FROM WATER THAT DEVELOPED UPON—

Gelatin plates at 16° to 20° C, Agar-agar plates at 37° to 38° C.
310 . 170
280 140
310 180
340 160
650 210
630 320
380} 290
400 f 210

1000 { 100
890 130
340 280
370 210
490) 110
580 100

Throughout this part of the work it is to be borne
in mind that when one refers to plates it is not toa
set, as in the isolation experiments, but to a single
plate.

METHOD OF COUNTING THE COLONIES ON PLATES.
—For convenience in counting colonies on plates or in
tubes, it is customary to divide the whole area of the
gelatin occupied by colonies into smaller areas, and
either count all the colonies in each of these areas and
add the several sums together for the total ; or to count
the number of colonies in each of several areas, ten or
twelve, take the mean of the results and multiply this
by the number of areas containing colonies.

17 am indebted to Dr. James Homer Wright, Thomas Scott Fellow in
Hygiene (1892-93), University of Pennsylvania, for the results presented in
this table.

434 BACTERIOLOG F.

By this latter method, however, the results vary so
much in different counts of the same plate, that they
cannot be considered as more than rough approxima-
tions.

Norre.—Prepare a plate; calculate the number of
colonies upon it by this latter method. Now repeat
the calculation, making the average from another set of
squares. Now actually count the entire number of
colonies on the plate. Compare the results.

For facilitating the counting of colonies several very
convenient devices exist.

WOoLFFHUGEL’s CounTING APPARATUS.—This ap-
paratus (Fig. 88) consists of a flat wooden stand, the
centre of which is cut out in such a way that either a

Fic. 88.

Wolff hiigel’s apparatus for counting colonies.

black or white glass plate may be placed in it. These
form a background upon which the colonies may more
easily be seen when the plate to be counted is placed
upon it. When the gelatin plate containing the colo-

STUDY OF WATER. 435

nies has been placed upon this background of glass,
it is then covered by a transparent glass plate which
swings on a hinge. When this plate is in position, it
is just above the colonies without touching them. This
plate is ruled in square centimetres and subdivisions.

The gelatin plate is moved about until it rests under
the centre of the area occupied by the ruled lines.

The number of colonies in each square centimetre is
then counted, and the sum-total of the colonies in all
these areas gives the number of colonies on the plate.

Where the colonies are quite small, as is frequently
the case, the counting may be rendered easier by the
use of a small hand-lens.

Fic. 89.

Lens for counting colonies.

In Fig. 89 is seen the form of hand-lens commonly
employed.

Esmarcy’s Coun'rer.— Esmarch has devised a
counter (Fig. 90) for estimating the number of colonies
present when they are upon a cylindrical surface, as
when in rolled tubes. The principles and methods of
estimation are practically the same as those given for.
Wolffhiigel’s apparatus. If the number of colonies in
an Esmarch tube is to be determined, a simpler method
than the use of his apparatus may be employed. It
consists in dividing the tube by lines into four or six
longitudinal areas which are subdivided by transverse

436 BACTERIOLOGY.

lines drawn about 1 or 2 cm. apart. The lines may be
drawn with pen and ink. They need not be exactly
the same distance apart, nor exactly straight. Beginning
with one of these squares at one end of the tube, which

Fig. 90.

Esmarch’s apparatus for counting colonies in roll tubes.

may be marked with a cross, the tube is twisted with
the fingers, always in one direction, and the exact
number of colonies in each square as it appears in rota-
tion is counted, care being taken not to count a square
more than once ; the sums are then added together, and
the result gives the number of colonies in the tube. This
method may be facilitated by the use of a hand-lens.
In all these methods there is one error that is difficult
to eliminate: it is assumed that each colony represents
the outgrowth from a single organism. This is prob-
ably not always the case, as there may exist clumps of
bacteria which represent hundreds or even thousands of
individuals, but which still give rise to but a single

AIR ANALYSIS, 437

colony —this is usually estimated as a single organism
in the water under analysis.

Where grounds exist for suspecting the presence of
these clumps, they may in part be broken up by shak-
ing the original water with sterilized sand.

What has been said for the bacteriological examina-
tion of water holds good for all fluids which are to be
subjected to this form of analysis.

BacrTERIoLoGicaAL AIR ANALYSIS.—Quite a num-
ber of methods for the bacteriological study of the air
exist.

In the main they consist either of allowing air to pass
over solid nutrient media (Koch, Hesse) and observing
the colonies which develop upon the media, or of filtering
the bacteria from the air by means of porous and liquid
substances, and studying the organisms thus obtained.
(Miguel, Petri, Strauss, Wiirz, Sedgwick.)

The former methods have given place almost entirely
to the latter for reasons of greater exactness possessed
by the latter.

In some of the methods which provide for the filtra-
tion of bacteria from the air by means of liquid sub-
stances, a measured volume of air is aspirated through
liquefied gelatin ; this is then rolled into an Esmarch
tube, and the number of colonies counted, just as was
done in the water analysis. This is the simplest
procedure. An objection raised against it is that
organisms may be lost, and not come into the calcula-
tion, by passing through the medium in the centre of
an air-bubble without being arrested by the fluid—
an objection that appears more of speculative than of
real value.

The methods of filtration through porous substances

438 BACTERIOLOGY,

appear, on the whole, to give the best results. Petri
recommends the aspiration of a measured volume of air
through glass tubes into which sterilized sand is packed.
(Fig. 91.) When the aspiration is finished the sand
is mixed with liquefied gelatin, plates are made, and
the number of developing colonies counted, the resulis
giving the number of organisms contained in the volume
of air aspirated through the sand.

Fig. 91.

Petri’s apparatus for bacteriological analysis of air. The tube
packed with sand is seen at the point «.

The main objection to this method is the possibility
of mistaking a sand granule for a colony. This objec-
tion has been overcome by Sedgwick, who employs
granulated sugar instead of the sand ; this, when brought
into the liquefied gelatin, dissolves, and no such error
as that possible in the Petri method can be made.

SEDe@wick’s MerHop.—On the whole, the method
proposed by Sedgwick gives such uniform results that
it is to be recommended above the others. It is as
follows :

AIR ANALYSIS, 439

The apparatus employed by him consists essentially
of three parts:

(1) A glass tube of a special form to which the name
aérobioscope has been given.

(2) A stout copper cylinder of about sixteen litres
capacity, provided with a vacuum-gauge.

(3) An air-pump.

i

rt ee ee

ki

Sedgwick’s aérobioscope.

The aérobioscope (Fig. 92) is about 35 cm. in its
entire length ; it is 15 cm. long and 4.5 em. in diameter
at its expanded part; one end of the expanded part is
narrowed down to a neck 2.5 cm. in diameter and 2.5
em. long. To the other end is fused a glass tube 15
em. long and 0.5 em. inside diameter, in which is to be
placed the filtering material.

Upon this narrow tube, 5 em. from the lower end, a
mark is made with a file, and up to this mark a small
roll of brass-wire gauze (a) is inserted ; this serves as a
stop for the filtering material which is to be placed over
it. Beneath the gauze (at 5), and also at the large end
(c), the apparatus is plugged with cotton. When thor-
oughly cleaned, dried, and plugged, the apparatus is to
be sterilized in the hot-air sterilizer. When cool, the
cotton plug is removed from the large end (c), and
sterilized No. 50 granulated sugar is poured in until
it just fills the 10 em. (d) of the narrow tube above
the wire gauze. This column of sugar is the filtering
material employed to engage and retain the bacteria.

440 BACTERIOLOGY.

After pouring in the sugar, the cotton-wool plug is
replaced, and the tube is again sterilized at 120° C. for
several hours.

Taking the air sample. In order to measure the
amount of air used, the value of each degree on the
vacuum-gauge is determined in terms of air by means
of an air-meter, or by calculation from the known
capacity of the cylinder. This fact ascertained, the
negative pressure indicated by the needle on exhausting
the cylinder shows the volume of air which must pass
into it in order to fill the vacuum. By means of the
air-pump one exhausts the cylinder until the needle
reaches the mark ctorresponding to the amount of air
required.’

A sterilized aérobioscope is now to be fixed in the
upright position and its small end connected by a rubber
tube with a stopcock on the cylinder, or to a glass tube
tightly fixed in the neck of an aspirating bottle by
means of a perforated rubber stopper. The cotton plug
is then removed from the upper end of the aérobioscope,
and the desired amount of air is aspirated through the
sugar. Dust particles and bacteria will be held back
by the sugar. During manipulation the cotton plug is
to be protected from contamination.

When the required amount of air has been aspirated
through the sugar the cotton plug is replaced, and by
gently tapping the aérobioscope while held inan almost
horizontal position, the sugar, and with it the bacteria,
are brought into the large part (e) of the apparatus.

1 Such a cylinder and air-pump are not necessary. A pair of ordinary as-
pirating bottles of known capacity graduated into litres and fractions thereof
answer perfectly well. Or one can determine by the weight of water that has
flowed from the aspirator, the volume of air that has passed in to take its
place, 7. «., the volume of air that has passed through the aérobioscope.

AIR ANALYSIS, 441

When all the sugar is thus shaken down into this part
of the apparatus, about 20 c.c. of liquefied, sterilized
gelatin is poured in through the opening at the end ¢,
the sugar dissolves, and the whole is then rolled on ice,
just as is done in the preparation of an ordinary
Esmarch tube.

Bent funnel for use with aérobioscope.

The gelatin is most easily poured into the aérobio-
scope by the use of a small, sterilized, cylindrical funnel
(Fig. 93), the stem of which is bent to an angle of
about 110° with the long axis of the body.

The larger part of the aérobioscope is divided into
squares to facilitate the counting of the colonies.

By the employment of this apparatus one can make
these analyses at any place, and can, without fear of
contamination, carry the tubes to the laboratory, where
the cultivation part of the work may be done.

Aside from this advantage, the filter being soluble, only
the insoluble bacteria are left imbedded in the gelatin.

442 BACTERIOLOGY.

For general use this method is to be preferred to the
others that have been mentioned.

BacTERIOLOGICAL STUDY OF THE So1L.—Bacterio-
logical study of the soil may be made by either break-
ing up small particles of earth in liquefied media and
making plates directly from this, or by what is per-
haps a better method, as it gets rid of insoluble parti-
cles which may give rise to errors: breaking up the soil
in sterilized water and then making plates immediately
from the water.

It must be borne in mind that many of the ground
organisms belong to the anaérobic group, so that in
these studies this point should be remembered and the
methods for the cultivation of such organisms practised
in connection with the ordinary methods.

CHAPTER XXVIII.

Methods of testing disinfectants and antiseptics—Experiments illustrating
the precautions to be taken—Experiments in skin disinfection.

THERE are several ways of determining the germicidal
value of chemical substances, the most common being
to expose organisms dried upon bits of silk thread to
the disinfectant for different lengths of time, and then,
after removing, and carefully washing the threads in
water, to place them into nutrient media at a favorable
temperature, and notice if any growth appears. If no
growth results the disinfection is presumably success-
ful. Another method is to mix fluid cultures of bacteria
with the disinfectant in varying proportions, and, after
different intervals of time, to determine if disinfection
is in progress by transferring a portion of the mixture
to nutrient media, just as in the other method of work.

By the former process the bits of thread, usually
about 1 to 2cm. long, are placed in a dry test-tube pro-
vided with a cotton plug and carefully sterilized, either
by the dry method or in the steam sterilizer, before
using. They are then immersed in a pure bouillon
enlture or in a salt solution suspension of the organism
upon which the disinfectant is to be tested. I say pure
culture because it is always desirable in testing a new
germicide to determine its value as such on several
different resistant species of bacteria, both in the vege-
tating and in the spore stage. After the threads have
remained in the culture or suspension for, from five to

444 BACTERIOLOGY,

ten minutes they are removed under antiseptic precau-
tions and carefully separated and spread out upon the
bottom of a sterilized Petri dish. This is then placed
either in the incubator at a temperature not exceeding
38° C. until the excess of fluid has evaporated, or in a
desiccator over sulphuric acid, calcium chloride, or any
other drying agent, but they are not left there until
absolutely dry, only until the excess of moisture has
disappeared. When sufficiently dry they can then be
employed in the test. This is done by immersing them
in solutions of the disinfectant of different but known
strengths for a fixed interval of time, say one or two
hours, after which they are removed, rinsed off in
sterilized distilled water to remove the excess of disin-
fectant adhering to them, and placed into fresh, steril-
ized culture media, which is then placed in the incu-
bator at from 37° to 38°C. If after twenty-four,
forty-eight, or seventy-two hours a growth occurs at or
about the bit of thread, and this growth consists of the
organism upon which the test was made, manifestly
there has been no disinfection; if no growth occurs
after, at most, ninety-six hours, it is safe to presume
that the bacteria have been killed, unless our efforts at
rinsing off the excess of disinfectant from the thread
have not been successful, and a small amount of dis-
infectant is now active in preventing development, 7. e.,
is acting as an antiseptic.

By the latter process, in which cultures or suspen-
sions of the organisms are mixed with different but
known strengths of the disinfectant, a small portion of
the mixture, usually a loopful or a drop, is transferred
at the end of a definite time to the fresh medium which
is to determine whether the organisms have been killed

TESTING DISINFECTANTS AND ANTISEPTICS. 445

or not. This is commonly a tube of fluid agar-agar
which is poured out into a Petri dish, allowed to solidify,
and placed in the incubator, as in the other experiment.

After the minimum strength of disinfectant necessary
to destroy the vitality of the organisms with which we
are working has been determined, for any fixed time, it
then remains for us to decide what is the shortest time
in which this strength will have the same effect. We
then work with a constant dilution of the disinfectant,
but with different intervals of exposure—one, five, ten
minutes, etc.—until we have decided not only the
minimum amount of disinfectant required for the de-
struction of the bacteria, but the shortest time neces-
sary for this under known conditions.

A factor not to be lost sight of is the temperature
under which these experiments are conducted, for it
must always be borne in mind that the action of a dis-
infectant is usually more energetic at a higher than at
a lower temperature.

Now in both of these methods it is easy to see that
unless special precautions are taken a minute portion of
the disinfectant may be carried along with the thread,
or drop, into the medium which is to determine whether
the organisms do or do not possess the power of growth,
and here have a restraining or antiseptic action. For
organisms in their normal condition, that is, those which
have never been exposed to the action of a disinfectant,
the amount necessary to restrain growth, for certain dis-
infecting agents, is very small indeed, and for organisms
that have already been exposed for a time to such agents
this amount is even much less. It is plain, then, that
if the test is to be an accurate one, precautions must be

taken against admitting this minute trace of disin-
20

446 BACTERIOLOGY.

fectant to the medium with which we are to determine
if the bacteria that have been exposed to its action have
been killed or not.

The precautions that have hitherto been taken for
preventing this accident are, where the threads are
employed, washing in sterilized distilled water and then
in alcohol; or, where the fluid cultures were mixed
with the disinfectant in solution, an effort was usually
made to dilute the amount of disinfectant carried over,
to a point at which it loses its inhibiting power.

While these are sufficient in many cases, they do not
answer for all. Certain chemicals have the property
of combining so firmly with the threads upon which the
bacteria are located as to require other special means
of ridding the threads of them; and in solutions in
which proteid substances are present along with the
bacteria a similar union between them and the disin-
fectant may likewise take place. In both instances this
amount of disinfectant adhering to the silk threads or in
combination with the proteids must be gotten rid of,
otherwise the results of the test may be fallacious. A
partial solution of the problem comes from studies that
have been made upon corrosive sublimate in its various
applications for disinfecting purposes, and in this con-
nection it has been shown by Shaefer ' that it is impos-
sible to rid silk threads of the corrosive sublimate
adhering to them by simple washing, as the sublimate
acts as a mordant and forms a firm union with the
tissues of the threads. Braatz? found the same to hold
for catgut. For example, he found that catgut which
had been immersed in solutions of sublimate gave the

1 Shaefer: Berliner klin. Woch., 1890, No. 3, p. 50.
2 Braatz: Centr. f. Bakt. und Parasitenkunde, Bd. viii. No. 1, p. 8.

TESTING DISINFECTANTS AND ANTISEPTICS. 447

characteristic reactions of the salt after having been
immersed in distilled water, which had been repeatedly
renewed, for five weeks.

& He remarks that a similar firm combination between
sublimate and cotton will take place after a longer
time, but it occurs so slowly that it cannot interfere
with disinfection experiments in the same way as he
believes the employment of silk to act.

The most successful attempt at removing all traces
of sublimate from the threads or from the proteid sub-
stances in which are located the bacteria whose vitality
are to be tested, is that made by Geppert, who subjected
them to the action of ammonium sulphide in solution.
By this procedure the mercury is converted into in-
soluble sulphide and does not now have an inhibiting
effect upon the growth of those bacteria that may not
have succumbed to its action when in the form of
sublimate.

Tn the second method of testing disinfectants, men-
tioned above—that is, when cultures of bacteria and solu-
tions of the disinfectant are mixed, and after a time
a drop of the mixture is removed and added to sterile
nutrient media, the inhibiting amount of disinfectant
can readily be gotten rid of by dilution, that is to say,
instead of transporting the drop directly to the fresh
medium, add it to 10 or 12 cc. of sterilized salt solu-
tion (0.6-0.7 per cent. of NaCl in distilled water), or
distilled water, and after thoroughly shaking add a
drop of this to the medium in which the power of
development of the bacteria is to be determined

Another important point to be borne in mind in
testing disinfectants is the necessity of so arranging the
conditions that each individual organism will be ex-

448

BACTERIOLOGY.

posed to the action of the agent used. Where clumps
of bacteria exist we are not always assured of this, for

—

Cylindrical funnel
used for filtering cul-
tures on which dis-
infectants are to be
tested.

only those on the surface of the clump
may be affected, while those in the
centre of the mass may entirely escape,
being protected by those surrounding
them. Theseclumps and minute masses
are especially liable to be present in
fluid cultures and in suspensions of
the bacteria, and must be eliminated
before the test is begun, if it is to be
made by mixing them with solutions
of the agent to be tested. This is
best accomplished in the following
way: The organisms should be culti-
vated in bouillon containing sand
or finely divided particles of glass ;
after growing for a sufficient length of
time they are then to be shaken thor-
oughly, in order that all clumps may
be mechanically broken up by the sand.
The culture is then filtered through a
tube containing closely packed glass
wool.

The filtration may be accomplished
without fear of contamination of the
culture by the employment of an
Allihin tube, which is practically noth-
ing more than a thick-walled test-tube
drawn out to a finer tube at its blunt
end so as to convert it into a sort of

cylindrical funnel. The tube when finished and ready
for use has the appearance given in Fig. 94.

TESTING DISINFECTANTS. AND ANTISEPTICS. 449

The whole tube, after being plugged at the bottom
of its wide part with glass wool and at its wide open
extremity with cotton wool, is placed vertically, small
end down, into an Erlenmeyer flask of about 100 c.c.
capacity and sterilized in a steam sterilizer for the
proper time. It is kept in the covered sterilizer until
it is to be used, which should be as soon as possible
after sterilization.

The watery suspension or bouillon culture of the
organisms is now to be filtered repeatedly through the
glass wool into sterilized flasks until a degree of trans-
parency is reached which will permit the reading of
moderately fine print through a layer of the fluid of
about 2 em. thick, i.e. through an ordinary test-tube
full of it. It can then be subjected to the action of
the disinfectant, and, as a rule, the results are far more
uniform than when no attention is paid to the exist-
ence of clumps. It is hardly necessary to say that in
the practical employment of disinfectants outside the
laboratory no such precautions are taken, but in lab-
oratory work, where it is desirable to determine exactly
the value of different substances as germicides, all the
precautions that have been mentioned will be found
essential to success.

In determining the germicidal value of different
chemical agents upon certain pathogenic bacteria, sus-
ceptible animals are sometimes inoculated with the
organisms after they have been exposed to the disinfec-
tant. If no pathological condition results, disinfection is
presumed to have been successful, while if the condition
characteristic of the activities of the given organism in
the tissues of this animal appears, the reverse is the case.
The objections to this method that have been raised

450 BACTERIOLOGY.

are: “First. The test organisms may be modified as
regards reproductive activity without being killed ; and
in this case a modified form of the disease may result
from the inoculation, of so mild a character as to escape
observation. Second. An animal that has suffered
this modified form of the disease enjoys protection,
more or less perfect, from future attacks, and if used
for a subsequent experiment may, by its immunity from
the effects of the pathogenic test organism, give rise to
the mistaken assumption that this had been destroyed
by the action of the germicidal agent to which it had
been subjected.” (Sternberg.)

DETERMINATION OF ANTISEPTIC PROPERTIES.

In this test sterile media are employed and are usually
arranged in two groups: the one to remain normal in
composition and to serve as controls, while to the other
is to be added the substance to be tested in different but
known strengths. It is customary to employ test-tubes
each containing an exact amount of bouillon, gelatin,
or agar-agar, as the case may be. To each tube a
definite amount of the antiseptic is added, and if it is
not of a volatile nature or not injured by heat, they
may then be sterilized. After this they are to be in-
oculated with the organism upon which the test is to
be made, and at the same time one of the “control”
tubes (one of those to which no antiseptic has been
added) is inoculated. They are all then to be placed
in the incubator and kept under observation. If at the
end of twenty-four, forty-eight, or seventy-two hours
no growth appears in any but the “ control” tubes, it
is evident that the antiseptic must be added in smaller

EXPERIMENTS. 45]

amounts, for we are to determine the point at which it
as not as well as that at which it is capable of prevent-
ing development. The experiment is then repeated,
using smaller amounts of the antiseptic until we reach
a point at which growth just occurs notwithstanding
the presence of the antiseptic, and its antiseptic strength
falls a trifle above the amount present in this tube.
If, for example, there was development in the tubes in
which the antiseptic was present in the proportion of
1:1000 and no growth in the one in which it was
present in 1:1400, the experiment would be repeated
with strength of the antiseptic corresponding to 1:1000,
1:1100, 1:1200, 1:13800, 1:1400, and in this way one
gradually strikes the point at which growth is just pre-
vented. This point represents the antiseptic value of
the substance used for the organism upon which it has
been tested.

EXPERIMENTS.

Into each of three tubes containing 10 c.c.—one of
normal salt solution, another of bouillon, a third of
fluid blood-serum—add as much of a culture of the
staphylococcus pyogenes aureus as can be held upon
the looped platinum needle. Mix this thoroughly, so
that no clumps exist, and then add exactly 10 ce. of
1:500 solution of corrosive sublimate. Mix it thor-
oughly, and at the end of three minutes transfer a drop
from each tube into a tube of liquefied agar-agar, and
pour this into a Petri dish. Label each dish carefully
and place them in the incubator. Are the results the
same in all the plates? How are the differences to be
explained? To what strength of the disinfectant were
the organisms exposed in the experiment?

452 BACTERIOLOGY.

Into each of two tubes containing 10 c.c.—the one
of normal salt solution, the other of bouillon—add as
much of a spore-containing culture of anthrax bacilli
as can be held upon the loop of the platinum wire.
Mix this thoroughly so that no clumps exist, and then
add exactly 10 c.c. of a 1:500 solution of corrosive
sublimate. Mix thoroughly and at the end of five
minutes transfer a drop from each tube into a tube of
liquefied agar-agar. Pour this immediately into a
Petri dish. Label each dish carefully and place them
in the incubator. Note the results at the end of twen-
ty-four, forty-eight, and seventy-two hours. How do
you explain them ?

Make identically the same experiment with the same
spore-containing culture of anthrax bacilli, except that
the drop from the mixture is to be transferred to 10 c.c.
of a mixture of equal parts of ammonium sulphide and
sterilized distilled water. After remaining in this for
about half a minute, a drop is to be transferred to a
tube of liquefied agar-agar, poured into Petri dishes,
labelled, and place in the incubator. Note the results.
Do they correspond with those obtained in the preceding
experiment? How are the differences explained ?

Prepare a 1:1000 solution of corrosive sublimate.
To each of twelve tubes containing exactly 10 cc. of
bouillon add: one drop to the first, two drops to the
second, and so on until the last tube has had twelve
drops added to it. Mix thoroughly and then inoculate
each with one wire-loopful of a bouillon culture of
staphylococcus pyogenes aureus. Place them all in the
incubator after carefully labelling them. Note the
order in which growth appears.

EXPERIMENTS. 453

Do the same with anthrax spores, with spores of
bacillus subtilis, and with the typhoid bacillus, and see
how the results compare. From these experiments what
will be the strength of corrosive sublimate necessary to
act as an antiseptic under these conditions for the organ-
isms employed?

Make a similar series of experiments, using a five
per cent. solution of carbolic acid.

Determine the antiseptic point of the common dis-
infectants for the organisms with which you are working.

Determine the time necessary for the destruction of
the organisms with which you are working, by corro-
sive sublimate in 1:1000 solution, under different con-
ditions—with and without the presence of albuminous
bodies other than the bacteria, and under varying condi-
tions of temperature.

In making these experiments be careful to guard
against the introduction of enough sublimate into the
agar-agar from which the Petri plate is to be made to
inhibit the growth of the organisms which may not
have been destroyed by the sublimate. This may be
done by transferring two drops from the mixture of
sublimate and organisms into not less than 10 c.c. of
sterilized salt solution in which they may be thoroughly
shaken for from one to two minutes, or into the solu-
tion of ammonium sulphide of the strength given.

To 10 ec. of a bouillon culture of staphylococcus
pyogenes aureus, or anthrax spores, add 10 c.c. of cor-
rosive sublimate in 1:500 solution, and allow it to re-
main in contact with the organisms for only one-half the

time necessary to destroy them (use an organism for
20*

454 BACTERIOLOGY.

which this has been determined). Then transfer a drop
of the mixture to each of three liquefied agar-agar
tubes and pour them into Petri dishes. Place them
in the incubator and observe them for twenty-four,
forty-eight, and seventy-two hours. No growth occurs.
How is this to be accounted. for?

At the end of seventy-two hours inoculate all of
these plates with a culture of the same organism which
has not been exposed to sublimate, by taking up bits of
the culture on the needle and drawing it across the
plates. A growth now results. We have here an ex-
periment in which organisms which have been exposed
to sublimate for a much shorter time than necessary to
destroy them, when transferred directly to a favorable
culture medium do not grow, and yet, when the same
organism which has not been exposed to sublimate is
planted upon the same medium it does grow. How is
this to be accounted for ?

Skin-disinfection.— With a sterilized knife scrape
from the skin of the hands, at the root of the nails and
under the nails, small particles of epidermis. Prepare
plates from them. Note the results.

Wash the hands carefully for ten minutes in hot
water and scrub them during this time with soap and a
sterilized brush. Rinse them in hot water. Again
prepare plates from scrapings of the skin on the fingers,
at the root of the nails, and under the nails. Note the
results.

Again, wash as before in hot water with soap and
brush, rinse in, hot water, then soak the hands for five
minutes in 1:1000 corrosive sublimate solution, and, as
before, prepare plates from scrapings from the same
localities. Note the results.

SKIN-DISINFECTION EXPERIMENTS. 455

Repeat this latter procedure in exactly the same way,
but before taking the scrapings let someone pour am-
monium sulphide over the points from which the scrap-
ings are to be made. After it has been on the hands
about three minutes again scrape and note the results
upon plates made from the scrapings.

Wash as before in hot water and soap, rinse in clean
hot water, immerse for a minute or two in alcohol, after
this in 1:1000 sublimate solution, and finally in am-
monium sulphide, and then prepare plates from scrapings
from the points mentioned.

In what way do the results of these experiments
differ one from another?

To what are these differences due?

What have these experiments taught ?

In making the above experiments it must be remem-
bered that the strictest care is necessary in order to pre-
vent the access of germs from without into our media.
The hand upon which the experiment is being per-
formed must be held away from the body and must not
touch any object not concerned in the experiment. The
scraping should be done with the point of a knife that
has been sterilized in the flame and allowed to cool
The scrapings may be transferred directly from the
knife-point to the gelatin by means of a sterilized
platinum wire loop.

The brush used should be thoroughly cleansed and
always kept in 1:1000 solution of corrosive sublimate.
It should be washed in hot water before using.

APPENDIX.

List of apparatus and materials required in a be-
ginner’s bacteriological laboratory :

MICROSCOPE AND ACCESSORIES.

Microscope with coarse and fine adjustment and
heavy, firm base; Abbe sub-stage condensing system,
arranged either as the “simple” or as the regular Abbe
condenser, in either case to be provided with iris dia-
phragm ; objectives equivalent, in the English nomen-
clature, to about one-fourth inch, and one-sixth inch
dry, and one-twelfth inch oil-immersion system; a
triple, revolving nose-piece ; three oculars, varying in
magnifying power; and a bottle of immersion oil.

Glass slides, English shape and size and of colorless
glass.

Six slides with depressions in centre of about 6 to 8
mm. in diameter.

Cover-slips, 15 by 15 mm. square and from 0.15 to
0.18 mm. thick.

Forceps. One pair of fine-pointed forceps and one
pair of the Cornet pattern, for holding cover-slips.

Platinum needles in glass handles. One straight, of
about 4 cm. long; one looped at the end and of about
4 cm. long; and one straight of about 8 cm. long.

458 BACTERIOLOGY.

Glass handles to be of about 3 mm. thickness and from
15 to 17 em. long.

STAINING AND MOUNTING REAGENTS.

200 c.c. of saturated alcoholic solution of fuchsin.

200 cc. of saturated alcoholic solution of gentian
violet.

200 c.c. of saturated alcoholic solution of methylene-
blue.

200 grammes of pure aniline.

200 grammes of C. P. carbolic acid.

500 grammes of C. P. nitric acid.

500 grammes of C. P. sulphuric acid.

200 grammes of C. P. glacial acetic acid.

1 litre of ordinary 93-95 per cent. alcohol.

1 litre of absolute alcohol.

500 grammes of ether.

500 grammes of pure xylol.

50 grammes of Canada balsam dissolved in xylol.

100 grammes of Schering’s celloidin.

10 grammes of iodine and 30 grammes of iodide of
potassium in substance.

100 grammes of tannic acid.

100 grammes of ferrous sulphate.

Distilled water.

FOR NUTRIENT MEDIA.

4 pound Liebig’s or Armour’s beef’ extract.

250 grammes Witte’s peptone.

2 kilogrammes of gold label gelatin (Hesteberg’s).
100 grammes of agar-agar in substance.

APPENDIX. 459

200 grammes of sodium chloride (ordinary table
salt).

500 grammes of pure glycerin.

50 grammes of pure glucose.

50 grammes of pure lactose.

100 grammes of caustic potash.

200 c.c. of litmus tincture.

10 grammes of rosolic acid (corallin).

Blue and red litmus paper ; curcuma paper.

5 grammes of phenolphthalein in substance.

Filter paper, the quality ordinarily used by druggists.

100 grammes of pyrogallie acid.

1 kilogramme of C. P. granulated zine.

GLASSWARE.

200 best quality test-tubes, slightly -heavier than
those sold for chemical work, of about 12 to 13 cm.
long and 12 to 14 mm. inside diameter.

15 Petri double dishes of about 8 or 9 cm. in diam-
eter and from 1 to 1.5 cm. deep.

6 Florence flasks, Bohemiam glass, 1000 c.c. capacity.

6 Florence flasks, Bohemian glass, 500 c.c. capacity. -

12 Erlenmeyer flasks, Bohemian glass, 100 cc.
capacity.

1 graduated measuring cylinder, 1000 c.c. capacity.

1 graduated measuring cylinder, 100 c.c. capacity.

25 bottles, 125 cc. capacity, narrow necks, with
ground glass stoppers.

25 bottles, 125 c.c. capacity, wide mouths, with ground
glass stoppers.

1 anatomical or preserving jar, with tightly fitting
cover, of about 4 litres capacity, for collecting blood-
serum.

460 BACTERIOLOGY.

2 battery jars of about 2 litres capacity, provided
with loosely fitting, weighted, wire-net covers, for mice.

10 feet of soft glass tubing, 2 or 3 mm. inside diameter.

20 feet of soft glass tubing, 4 mm. inside diameter.

6 glass rods, 18 to 20 em. long and 3 or 4 mm. in
diameter.

6 pipettes of 1 ¢.c. each, divided into tenths.

2 pipettes of 10 cc. each, divided into cubic centi-
metres and fractions.

1 burette of 50 c.c. capacity, divided into cubic centi-
metres and fractions.

1 separating funnel of 750 c.c. capacity, for filling
tubes.

2 glass funnels, best quality, about 15 em. in diam-
eter.

2 glass funnels, best quality, about 8 cm. in diameter.

2 glass funnels, best quality, about 4 or 5 cm. in
diameter.

6 ordinary water tumblers for holding test-tubes.

1 ruled plate for counting colonies.

1 gas generator, 600 c.c. capacity, pattern of Kipp or
v. Wartha.

BURNERS, TUBING, ETC.

2 Bunsen burners, single flame.

1 Rose burner.

1. Koch safety burner, single flame.

6 feet of white rubber gas-tubing.

12 feet of pure, red rubber tubing of 5 to 6 mm. inside
diameter.

1 thermo regulator, pattern of L. Meyer or Reichert.

2 thermometers, graduated in degrees Centigrade
registering from 0° to 100°, graduated on the stem.

APPENDIX. 461

1 thermometer graduated in tenths and registering
from 0° to 50° C,
1 thermometer registering to 200° C.

INSTRUMENTS, ETC.

1 microtome, pattern of Schanze, with knife.

1 razor strop.

6 cheap quality scalpels, assorted sizes.

2 pair heavy dissecting forceps.

1 pair medium-size straight scissors.

1 pair small-size straight scissors.

1 hypodermatic syringe that will stand steam steril-
ization.

2 teasing needles.

1 pair long-handled crucible tongs for holding mice.

1 wire mouse-holder.

2 small pine boards on which to tack animals for
autopsy.

2 covered stone jars for disinfectants and for receiv-
ing infected materials.

INCUBATORS AND STERILIZERS.

1 incubator, simple square form, either entirely of
copper or of galvanized iron with copper bottom.

1 medium-size hot-air sterilizer with double walls,,
asbestos jacket, and movable false bottom of copper
plates.

1 medium size steam sterilizer; either the pattern
of Koch or that known as the Arnold steam sterilizer
preferably the latter.

462 BACTERIOLOGY.

MISCELLANEOUS.

1 pair of balances, capacity 1 kilogramme ; accurate
to 0.2 gramme.

1 set of cork borers.

1 hand-lens,

1 wooden filter-stand.

2 iron stands with rings and clamps.

3 round, galvanized iron wire baskets to fit loosely
into steam sterilizer.

3 square, galvanized iron wire baskets to fit loosely
into hot-air sterilizer.

1 sheet-iron box for sterilizing pipettes, etc.

1 covered agate-ware saucepan, 1200 c.c. capacity.

2 iron tripods.

1 yard of moderately heavy wire gauze.

2 test-tube racks, each holding 24 tubes, 12 in a row.

1 constant-level, cast-iron water-bath.

2 potato knives.

2 test-tube brushes with reed handles.

Cotton batting.

Copper wire, wire-nippers.

Round and triangular files.

Labels.

Towels and sponges.

INDEX.

Aa microscopic aay | Animals, post mortem examina-

of miliary, 223-225
Aérobic bacteria, 33
Aérobioscope, 438
Agar-agar, 80-83

clarification of, 81, 82

filtration of, 82

peculiarities of, 71, 72
Air, bacteriological analysis of,

436-441
methods used in, 436-441
method of Petri, 437
of Sedgwick, 438
Anaérobic bacteria, methods of
cultivating, 174-180
method of Buchner, 176
Esmarch, 179
Frankel, 176-179
Hesse, 174, 175
Kitasato and Weil, 179
Koch, 174
Liborius, 175
Aniline dyes as differential aids,
170
Animals, fluctuations in weight
and temperature, 197-203
inoculation of, 185-196
intra-ocular, 195-196
intra-peritoneal and
pleural, 193-195
intra-vascular, 188-193
subcutaneous, 185-188
observation of, after inocula-
tion, 196
post-mortem examination of,
204-208
cultures from tissues at,
206, 207
disinfection of imple-
ments after, 208
disposal of remains, 208

external inspection at,-

204

tion of, the incision
through the skin, 204
opening the cavities,
205, 206
position of animal, 204
precautions during, 204-
206
Anthrax, 357-369
bacillus of, 357-365
biology of, 358-365
experiments with, 365-
369
ee af in tissues,
363, 3:
mor falngy of, 357, 358
results of inoculations
with, 363-365
spore formation in, 358-

staining of, 362, 363
susceptibility of animals
to, 365
nature of, 357
Antiseptics, 67, 449, 450
experiments with, 451, 452,
453
method of testing, 449, 450
Apparatus i a analysis, Petri’s,
3

Sedgwick’s, 438
for counting colonies, 434, 435
Esmarch’s, 435
Wolffhiigel’s, 434
needed in beginner’s labora-
tory, 457-462

ACILLI, 38
differentiation from spores, 40
flagella upon, 45
involution forms of, 39
life cycle of, 38-39

464

Bacilli, motility of, 45
multiplication of, 42~43
spore formation in, 38, 39

Bacillus anthracis, 357-365
coli communis, 304-312
“comma,” 313-324
diphtheriz, 279-294
Finkler-Prior, 340-345
leprae, 265, 266
mallei, 271-274
Neapolitanus, 304-312
nitrifying, 371
cedematis maligni, 383
of smegma, 265, 266
of symptomatic anthrax, 388
of syphilis, 265, 266
pyocyanus, 232-236
subtilis, 215
tetani, 376
tuberculosis, 259-264
typhi abdominalis, 295-304

Bacteria, anaérobic species, 33,

174-180

capsule surrounding, 136

classification of, morphologi-
cal, 36

constant characteristics of, 39

definition of, 27

discovery of, 13-15

examination of, on cover-
slips, 162, 163

flagellated forms, 45, 46

isolation of, in pure culture,68

principles involved, 68—
7

microscopic examination of,
160-165

morphology of, 36-40

motility of, 45, 46

multiplication of, 36--45

nutrition of, 30-35

points to be observed in de-
scribing, 182-184

reactions produced by grow-
ing, 169

relation to temperature, 34,

results of their growth, 29, 30
role in Nature, 27-30

spore formation in, 42-44
staining reactions of, 171

INDEX.

Bacteria, systematic study of, 159
Bacteriology, application of the
methods of, 209
Bacterium coli commune, 304-
312
cultural peculiarities of, 306-

differentiation from b. typhi
abdominalis, 309
morphology of, 306
results of inoculation with,
309-312
where found, 304, 305
Behring and Kitasato, work on
immunity, 409
Billroth, 24
Birch-Hirschfeld, 22
Black leg (see Symptomatic an-
thrax).
Blood-serum, as culture medium,
86-92
collection of, by Nuttall’s
method, 90-92
germicidal properties of,
404

active principles to which
are due the, 408, 409
earlier observations on,
405-407
Léfiler’s mixture, 96
preparation of, 86-88
preservation of, 90
solidification of, 89, 90
sterilization of, 88
Bolton’s method for the prepara-
tion of potatoes, 85
Booker’s modification of Esmarch
method, 108, 109
Bonnet, 20
Bottles for staining solutions,
131
Bouillon, 73-76
neutralization of, 73-75
Bread and potatoes, exposed to
air, 209
Brooding oven, 111-113
Brownian motion, 165
Bulb for water samples, 428
Burdon-Sanderson, 25
Burner, Koch’s safety, 113
rose or crown, 59

INDEX.

HLOROPHYLL, 27
Cholera Asiatica, diagnosis
of, by bacteriological
means, 322, 334-339
method of Schottelius, 322
of Koch, 334-339
Cholere Asiatice, spirillum, 313
characteristics of, 314-324
biological, 315-324
morphological, 314
experiments upon animals
with, 324-327
general considerations upon,
327-334
behavior in butter, 332
in milk, 331
in soil, 330
destiny in the dead body,
330-333
effect of drying, 332, 333
existence outside the body,
328
life in water, 328, 329
location in the body, 327
relation to gases, 333
to other bacteria, 332
to putrefaction, 330, 331,
333
to sunlight, 330
poison produced by, 323
Cohn, 20, 21
Colon bacillus (see Bacterium
coli commune).
Colonies, counting of, 433
study of, 119-121
Comma bacillus (see Cholera
Asiatica).
Cooling stage and levelling tri-
pod, 104
Cover-slips, cleaning of, 125
impression preparations with,
129

preparation of, for micro-
scopic examination, 126-
129

Cultures, gelatin, 123, 168, 169

hanging drop, 164-168

potato, 169

pure, 121

reactions of, 169, 170

stab and smear, 121-123

465

ECOLORIZING
tions, 143
Decomposition, 27-29, 370, 371
Diphtheria, bacillus of, 279-294
cultural peculiarities of, 283-
287
location in the tissues, 289
method of obtaining, 279-281
modification in virulence,
291-293
morphology of, 281-283
pathogenesis of, 287-291
poison produced by, 291
Diplococci, 38
Disinfectants—
animals as test objects in ex-
periments with, 449, 450
experiments upon, 451-454
methods of testing, 443-450
precautions to be taken in
testing, 445-449
use of, in the laboratory, 65,
6

solu-

Disinfection, general remarks
upon, 47
influence of temperature
upon, 63

inorganic salts in, 63-65

in the laboratory, 65

modus operandi of, 62

reliable agents for purposes
of, 66

selection of agent to be used
in, 61

Dunhawm’s solution, 93

RYSIPELAS, 230
4 Esmarch’s tubes, 107
Booker’s method of roll-
ing, 108, 109
of agar-agar, 109
Exposure and contact experi-
ments, 211

ACULTATIVE, use of the
term, 27, 33
Fermentation, 171-174
gases resulting from, 174
particular forms of, 30

466

Fermentation-tube, 172

Filters, method of folding, 77, 78

Finkler-Prior bacillus, 340-345

Flagella, staining of, 189-142

Flagellated organisms, 45

Funnel for filling aérobioscope,
440

test-tubes, 99
for filtering cultures, 447

eee regulator,
11
Gelatin, 76-80
cultures in, 168
their characteristics, 168,
169
Glanders, pathological manifesta-
tions of, 269-271
bacillus of, 271-274
inoculation with, 274
staining of, in tissues,
275-277
diagnosis of, by use of mal-
lein, 277
by method of Strauss,
277 ;
susceptibility of animals to,
273, 274
Gonococcus, 219
Gonorrhea, pus from, 219
Green-pus bacillus, 232
Guarniari’s agar-gelatin, 96

ALSTED, 194 and 222
Hanging-drop preparations,
163-165
Henle, 17
Hoffmann, 19
Hydrogen, test for purity of, 178
Hypodermatic syringes and nee-
dles, 190-192

eeeeene of tissues, 147,
148
Immunity, 401-417
acquired, 401
conclusions concerning, 416,
417

INDEX.

Immunity, hypotheses in ex-
planation of, 401-415
exhaustion, 403
of Buchner, 410
value of, 415
of Metchnikoff, 403, 404
criticism of, 404, 405
retention, 401
natural, 401
studies upon the blood in
relation to, 404-410
work of Behring and Kita-
sato on, 409
of G. and F. Klemperer
on, 412
Incubators, 111-113
Indol, production of, by bacteria,
180
tests for, 181, 182
Infection, 395-400
chemical nature ot, 399
conclusions concerning, 400
modus operandi of, 398, 399
poisons present in certain
forms of, 400
Inoculation of animals, 185-196
intra-ocular, 195, 196
intra-peritoneal and pleural,
193-195
intra-vascular, 188-193
subcutaneous, 185-188

LEBS, 22, 23
Klemperer, F. and G., their
work on pneumonia, 412
Koch, 25, 26, 55, 68, 73, 106, 113,
135, 210, 246, 259, 313, 325, 332,
335, 837, 839, 345, 383, 384

[ ACTOSE LITMUS agar-agar
4 or gelatin, 95
Leeuwenhoek’s discoveries, 13-15
Lens for counting colonies, 434
Levelling tripod, 103
Lepra bacillus, staining peculiari-

ties of, 265, 266
Litmus-milk, 92
Loffler’s blood-serum, 96

stain for flagella, 139

INDEX,

1 pape aes edema, bacillus
of, 383-388
biolo; y of, 385, 386
morp ology of, 383-385
nallapenents of, 386-388
susceptibility of animals to,
386
Mallein, 277
Meat infusion, 96
Media, 73-96
agar-agar, 80-83
filtration of, 82
blood-serum, 86-92
Léffler’s mixture, 96
Nuttall’s method, 90
bouillon, 73-76
neutralization of, 73-75
Dunham's peptone solution,
93

gelatin, 76-80

Guarniari’s agar gelatin, 96

lactose-litmus agar or gela-
tin, 95

litmus-milk, 92

meat infusion, 96

milk, 92

milk-agar, 93

lial solution,

potatoes, 83-86
in test-tubes, 85
Micrococci, 36
mode of development, 41, 42
Micrococcus lanceolatus, 240-245
irregularities in development
of, 244
morphological peculiarities
of, 241, 242
results of inoculation with,
244, 245
staining of, 244
variations in virulence, 244
where found, 242
Micrococcus tetragenus, 240, 245—

susceptibilities of animals to,
248
where found, 246
Microscope, component parts of,
160-162
Microtome, 146

467

Milk, 92
-agar, 93
Morse “holder, 187

AEGELI, 31
Needham, 18
Nitrification, 371
Nitrifying bacteria, 371-375
methods of cultivating, 373-
375
peculiarities of, 372, 373
Nitrites, 182
Nitro-monas, 372

-| Normal solution, 176

Nuttall, 90, 135, 207, 404

IL-IMMERSION system, steps
in using, 162

Ozanam, 17
ARASITES, 27
Pasteur, 19, 25, 33, 388, 401,
403

Peptone solution, 93
with rosolic acid, 94
test for purity of, 93, 94
Phagocytosis, 404
Plates, 68-72, 101-106
apparatus used in making,
102-106
Koch’s fundamental observa-
tion, 68-71
principles involved, 68-
ap

materials used, 71, 72
Petri, 106
technique of making, 101-
106
Platinum needles and loops, 102
Plenciz, Marcus Antonius, teach-
ings of, 16
Post-mortem examination of ani-
mals, 204-208
cultures from the tissues at,
206, 207
Nuttall’s spear for mak-
ing, 207
disinfection after, 208

468

Post-mortem examination of ani-
mals, precautions necessary
at, 204-206
preparation of cover-slips at,
207

preservation of tissues, 207
Postulates to be fulfilled, 259
Potatoes, characteristics of cul-

tures on, 169
preparation of, 83-86
Bolton’s method in test-
tubes, 85
Pure cultures, 121
Pyocyanus, bacillus, 232-236
chameleon phenomenon of,
235
‘pathogenic properties of, 235,
236

protective inoculations with,
236

UARTER evil or quarter ill
(see Symptomatic anthrax).

ECKLINGHAUSEN, 22, 23
Regulator, gas-pressure, 117,
118

thermo-, 114-117
Rindfleisch, 22
Roux and Yersin, 292

APROPHYTES, definition, 27
Sarcina, 38
mode of development, 42
Schottelius, method of examin-
ing cholera evacuations, 322
Schréder and Dusch, 19
Schulze, 19
Schwann, 19
Section-cutting, 145
Septicemia, 239, 395, 398
micrococcus tetragenus, 245—
248
sputum, 239-245
Skin disinfection experiments,
454, 455
Smear cultures, 121, 123
Smegma bacillus, staining pecu-
liarities of, 265, 266

INDEX.

Soil, bacteriological analysis of,
442

organisms present in, 372—
375

phenomena in operation in,
370-372
Spallanzani, demonstrations of, 18
Spirilla, 41
Spirillum cholere Asiatice, 313
of Deneke, 345-349
biology, 346-348
morphology, 345, 346
pathogenesis, 348
of Finkler-Prior (see Vibrio
proteus).
Metchnikovi (see Vibrio
Metchnikovi).
of Miller, 349-352
biology, 349-352
morphology, 349
pathogenesis, 352
tyrogenum (see Spirillum of
Deneke).
undula, 45
Spores, formation of, 166
method of studying, 166-168
mode of development, 43, 44
staining of, 137, 138
Sputum, tubercular, 237
microscopic examination of,
237-239
pathogenic properties of, 239
septicemias, 239-248
tuberculosis 249-264
Stab cultures, 121-123
Staining, methods and solttions
employed, 124-158
acetic acid, 136
Gabbett’s, 135
general remarks upon, 142
Gram’s, 136
Gray’s, 155
Koch-Ehrlich’s, 132
Kihne’s, 153
Léffler’s alkaline methylene-
blue, 1382
staining of flagella, 139
Moeller’s, 138
ordinary solutions used in,
129-131
bottles for holding, 131

INDEX.

Staining, Weigert’s method, 154
Ziehl-Neelsen’s carbol-fuch-
sin, 132
Staphylococci, 36
ephy locog a: pyogenes albus,

aureus, 221
cultural _peculiari-
ties, 219-221

pathogenic proper-

ties, 221-223
where to be ex-
pected, 221
citreus, 226
Sterilization, 47-60
chemical, 60-67
fractional or
52, 58
by heat, 49-60
aa involved, 49-
5

by hot air, 59
apparatus used, 59
by steam, 50-55
apparatus used, 55-58
under pressure, 54
experiments, 213-217
with hot air, 216, 217
with steam, 213-216
Sternberg, 242 and 355
Streptococci, 37
Streptococcus pyogenes, 226
cultural peculiarities of, 227—
229
effects of inoculations with,
2380
microscopic appearance of,
226

where to be expected, 230
Suppuration, 218, 230-232 .
bacteria common to, 218, 219,
226
less common causes of, 231,
232
general remarks upon, 230-—
232
microscopic appearance of
pus, 218
Symptomatic anthrax, 388, 389
acillus of, 389-394
biology of, 390-392

intermittent, |

469

Symptomatic anthrax, bacillus of,
differentiation from
bacillus of malignant
oedema, 393, 394

morphology of, 389
pathogenesis of, 392, 393
susceptibility of animals
to, 393
Syphilis, bacillus of, staining pe-
culiarities of, 265, 266

| PEST TUBES, cleaner for, 97
| cleaning of, 98
filling with media, 98, 99
: apparatus used in, 99
plugging with cotton, 98
position after filling, 100
sterilization of, 98
after filling, $9, 100
Tetanus, bacillus of, 376-383
biology of, 378-380
method of obtaining, 376-
378
morphology of, 378
| pathogenesis, 380-382
poison produced by, 382, 383
Tetrads, 38
Thermo-regulator, 114-117
Thermostat, 111-113
Tissues—
cultures from, at autopsies,
206, 207
Nuttall’s spear for mak-
ing, 207
imbedding of, 147, 148
preservation of, for micro-
scopic examination, 145,
207
section-cutting, 145
staining of bacteria in, 148-

steps in the process of,
15

special methods of—
dahlia method of,
152
dry method of, 155
‘Ehrlich’s, 155
Gram’s, 151

Gray’s, 155

21

470

Tissues, staining bacteria in, spe-
cial methods —
Kiuhne’s, 153
; Weigert’s, 154
Zieh]-Neelsen’s, 155
Toxemia, 397, 398
Tripod for levelling plates, 103
Tuberculin, 267
Tuberculosis, 249-264
cavity formation in, 2538, 254
diffuse caseation of, 252, 253
encapsulation of tubercular
foci, 254
location of the bacilli in,
257-259
manifestations of, 249-255
miliary tubercles, histologi-
cal structure of, 251, 252
modes of infection, 255-257
primary infection, 255
sputum in 237
inoculation of animals
with, 289
microscopic appearance
-of, when stained, 237,
238
staining of, 133-136
susceptibility of animals to,
267, 268
Tuberculosis, bacillus, 259-264
cultivation of, from tissues,
259-261
appearance of cultures,
261-263
methods of staining, 133-136,
155-158
dry method, 155
Gabbett’s 135
Gray’s, 155
Koch-Ehrlich, 182, 155
Nuttall’s modification
of, 1385
Ziehl-Neelsen method,
132, 155
microscopic appearance of,
263, 264

organisms that simulate it, |.

differential diagnosis of,
265-267
staining in tissues, 155-158

INDEX.

Tuberculosis, bacillus, staining
peculiarities of, 263, 264
Tyndall, 20
Typhoid fever, bacillus of, 295-
4

biology of, 296-298

constant properties of, 303

differentiation from bacillus
coli commune, 309

difficulty in identifying, 302

experiments with, 304

inoculations with, 300

induced susceptibility to,
300, 301

location in the tissues, 299

morphology of, 295

water as a carrier of, 418,
421, 423

where to obtain the, 303

VIER Metchnikovi, 352-356
biology of, 353-355
morphology of, 352
pathogenesis of, 355, 356
proteus, 340 345
characteristics of, 340-

biological, 841-344
morphological, 340,
341
pathogenic properties of,
344, 345 :
relation to cholera nos-
tras, 340, 345

ATER, general observations
upon, 418-424
qualitative bacteriological
analysis of, 424
precautions in obtaining
sample for, 424, 425
preliminary steps in,
425
quantitative bacteriological
analysis of, 427
counting of colonies in,
3-436
apparatus for, 433-
435

INDEX.

Water, quantitative _ bacterial
analysis of, dilution of
sample in, 431

obtaining sample for,
428-430

preliminary tests, 430

selection of culture me-
dium, 431, 432

source of error, 436

relation to epidemics, 418,
419

typhoid bacilli in, 420-422

471

Water, value of bacteriological
examinations of, 421,
422 :

of chemical examina-

tions of, 422, 423

Welch, 231, 240, 243

Wound infection, studies upon,

21-26 ~

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MORRIS (HENRY). SURGICAL DISEASES OF THE KIDNEY.
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