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http://www.archive.org/details/cu381924000245310

THE PRINCIPLES

OF

BACTERIOLOGY:

A PRACTICAL MANUAL FOR STUDENTS
AND PHYSICIANS.

BY

A. OC. ABBOTT, M.D.,

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

WITH ILLUSTRATIONS.

PHILADELPHIA:

LEA BROTHERS & CoO.
1892.

i)

eR ae

To] 4 2
Entered according to Act of Congress, in the year 1891, by
LEA BROTHERS & CO.,
In the Office of the Librarian of Congress at Washington, D. C.

GK
AG

PORNAN, PRINTER,

PREFACE.

In 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 prac-
tical 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 clu-
cidation of many hitherto obscure problems concerning
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

iv PREFACE.

subject-matter be presented in as direct a manner as
possible.

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.
ALC. A.

PHILADELPHIA, December, 1891.

CONTENTS.

INTRODUCTION.

“Omne vivum ex vivo””—The overthrow of the doctrine
of spontaneous generation

CHAPTER IT.

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 multipli-
cation—Spore-formation—Motility

CHAPTER III.

Principles of sterilization by heat—Different methods
employed—Principles of discontinued sterilization—Ster-
ilization under pressure—Apparatus employed

CHAPTER IV.

Disinfection—Antiseptics—Inorganic salts as disinfec-
tants—The value of corrosive sublimate—Heat

CHAPTER V.

The principles involved in the methods of isolation of
bacteria in pure culture by the plate method of Koch—
Materialsemployed . . . ,

PAGE

13-21

22-28

29-36

37-48

49-53

54-58

vi CONTENTS.

CHAPTER VI.

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

CHAPTER VII.

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

CHAPTER VIII.

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

CHAPTER IX.

The incubator used in bacteriological work—Gas-press-
ure regulator—Thermo-regulator—The form of burner
employed in heating the incubator

CHAPTER X.

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

CHAPTER XI.
Systematic study of an organism—Steps necessary in
identifying an organism as a definite species
CHAPTER XII.

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

CHAPTER XIII.

Inoculation of animals—Subcutaneous inoculation—
Intra-venous injection

PAGE

59-76

81-90

91-98

99-103

104-121

122-150

151-158

CONTENTS. vu

CHAPTER XIV.
PAGE
Post-mortem examination of animals—Bacteriological

examination of the tissues—Disposal of tissues and dis-
infection of instruments after the examination : . 159-163

CHAPTER XV.
Scheme for the complete study of an organism 164, 165

PRACTICAL APPLICATION OF THE
METHODS OF BACTERIOLOGY.

CHAPTER XVI.

To obtain material upon which to begin work . 167-170

CHAPTER XVII.

Various experiments in sterilization—Steam and hot-air
methods of sterilizing . F 171-175

CHAPTER XVIII.

Bacteriological study of water, air, and soil—Methods
of counting the colonies on the plates—Wolffhiigel’s
counting apparatus—Sedgwick’s method . 176-191

CHAPTER XIX.

Inceulation experiments with sputum—Sputum sep-
ticeemia—Septicemia resulting from the presence of the
micrococcus tetragenus in the tissues—Tuberculosis 192-200

CHAPTER XX.

Tuberculosis—Microscopic appearance of miliary tuber-
cles—Diffuse caseation—Cavity-formation—Encapsulation
of tuberculous foci—Primary infection—Modes of infec-
tion—Location of the bacilli in the tissues—Staining

peculiarities 201-216

vill CONTENTS.

CHAPTER XXI.

Suppuration—The staphylococcus pyogenes aureus

CHAPTER XXII.

Typhoid fever—Study of the organism concerned in its
production

CHAPTER XXIII.

Study of the bacillus of anthrax, and the effects pro-
duced by its inoculation into animals—Peculiarities of the
organism under varying conditions of surroundings

CHAPTER XXIV.

Bacteriology of diphtheria—Behavior of the bacillus

diphtheriz in the tissues of susceptible animals

CHAPTER XXYV.

Experiments illustrating precautions to be taken in the
study of disinfectants and antiseptics—Skin-disinfection .

PAGE

217-224

225-231

243-252

253-257

BACTERIOLOGY.

INTRODUCTION.

“Omne vivum ex vivo”’—The overthrow of the doctrine of
spontaneous generation.

THE study of Bacteriology may be said to have had
its birth with the observations made by. 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
beginning its history is inseparably connected with the
history of medicine, and as it now stands its relations to
hygiene and preventive medicine are of the most im-
portant nature. It is, indeed, to a more intimate ac-
quaintance with the biological activities of the micro-
organisms that modern hygiene owes much of its value,
and our knowledge upon infectious diseases has been
developed to 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 f?

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 lens by
means of which he was enabled to see objects of much
smaller dimensions than any hitherto seen with the
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 diar-
rheeal evacuations, objects that differentiated themselves
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 dis-
covered in the tartar scraped from between the teeth)
a form of microdrganism upon which he laid special
stress. This observation he embodied in the form of’
a contribution which was presented te the Royal So-
ciety of London on September 14, 1683. This paper is

INTRODUCTION. 15

of particular importance, not only because of the careful,
objective nature of the description given for the bodies
seen by him, but also for the illustrations which accom-
pany 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 astonished, 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
conspicuous absence of the speculative. His contribu-
tions are marked by 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 for the
origin of many obscure diseases. So universal was 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 con-
ditions, but some even went so far as to hold that varia-
tions in the appearance of the 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, expressed 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 him-
self had made. The doctrine of Plenciz assumed a
causal relation between the microdrganisms discovered
and deseribed by Leeuwenhoek and all infectious dis-
eases. He claimed that infection could be nothing else
than a living substance, and endeavored on these grounds
to explain the variations in the period of incubation for
the different infectious diseases. He likewise believed
the living contagium to be capable of multiplication
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 produced.

He found in all decomposing matters innumerable
minute “animalculee,” 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.

However convincing the arguments of Plenciz appear,

INTRODUCTION. 17

they seem to have been lost sight of in the course of
subsequent events, and by a few were even held in the
light of productions from an unbalanced mind. For
example, as late as 1820 we find Ozanam expressing
himself 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 absurb 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 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 doc-
trine 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
Leeuwenhock’s discoveries, was the origin of these
bodies. Do they generate spontancously, or are they

18 BACTERIOLOGY.

the descendants of preéxisting creatures of the same
kind? was the all-important question. Among the
participants in this discussion were many of the most
prominent 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 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 spon-
taneous generation no longer existed, was met by Spal-
lanzani 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.

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.

INTRODUCTION. 19

In 1836 Schulz 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) Schréder and Dusch, with similar results ob-
tained by somewhat different means. Schwann deprived
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 organisms by the
simple process of filtration. In 1860 Hoffmann and in
1861 Chevreul and Pasteur demonstrated that the pre-
cautions taken by the preceding investigators for ren-
dering 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 condensa-
tion which collects at its lowest angle, and none can
enter the flask.

Convincing though this work may seem, there still
existed a number of doubters who required further proof
that “spontaneous generation” was not the explanation

20 BACTERIOLOGY.

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 preéxistence of similar liv-
ing forms in the infusion or upon the walls of the vessel
containing it, or by the infusion having been exposed
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 certain substances required to be heated for a much
longer time than was necessary to render other sub-
stances free from living organisms, and even under the
most careful precautions decomposition would occasion-
ally appear.

In 1762 Bonnet, who was deeply interested in this
subject, had suggested, in reference to the results ob-
tained by Needham, the possibility of the existence of
“germs, or their eggs,” which have the power to resist
the temperature 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
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

INTRODUCTION. 21

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 fore-
going experiments, or 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 preéxisting 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 kingdoms, but to the most micro-
scopic, unicellular creatures as well.

Nore.—I have presented only the most prominent
investigations which aided in overthrowing the doctrine
of spontaneous generation. For a more detailed account
of this work the reader is referred to Léffler’s Vorle-
sungen iiber die geschichtliche Entwickelung der Lehre
von den Bacterien, upon which I have drawn largely in
preparing the foregoing sketch.

Q*

CHAPTER I.

Definition of bacteria—Their place in nature—Difference between
parasites and saprophytes—Nutrition of bacteria—-Products of bac-
teria—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 pro-
cess 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 either a
saprophytic” 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 upon which
they are developing.

Decomposition and fermentation result from the pres-
ence of the saprophytic bacteria, while the changes

_' Chlorophyll is the green coloring matter possessed by the higher
plants by means of which they are enabled in the presence of sun-
light to decompose carbonic acid (CO,) and ammonia (NH,) into
their elementary constituents.

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

* A parasite lives always at the expense of some other living,
organic creature, and in the strictest sense of the word cannot exist
upon dead matter. There exist, however, a group of so-called
“facultative” saprophytes and parasites which possess the power of
accommodating themselves to existing surroundings—at one time
leading a parasitic, at another time a saprophytic form of existence,

PARASITES AND SAPROPHYTES. 23

brought about in the tissues of their host by the pure
parasitic forms, find expression in disease processes and
not unfrequently complete death.

The réle 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 and ammonia, in which form they may be taken up
and appropriated as nutrition by the more highly organ-
ized members of the vegetable kingdom. It is to this
ultimate production of carbonic acid, ammonia, and water
by the bacteria, as end-products in the processes of de-
composition and fermentation of the dead animal and
vegetable tissues, that the demands of growing vegeta-
tion for these compounds can be supplied.

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 the 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 chloro-
phyll plants, the importance of the part played by the
bacteria in making up this deficit cannot be overestimated.
Were it not for the activity of these microscopic living
particles, all life upon the surface of the earth would
certainly cease. Deprive higher vegetation of’ the car-
bon 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 re-
present by far the large majority of all bacteria, must be

24 BACTERIOLOGY.

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 existence 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 which are necessary to the health and
life of the tissues of the organism to which they may
have found access. At the same time the substances
which they form as products of their nutrition are direct
poisons for the surrounding tissues.

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 exist 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 which are concerned
in the changes to which allusion has just been made,
there exist other saprophytic forms which are recognized
by their property of producing pigments of different
color. These are known as the chromogenic? forms.

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

» Chromogenic, possessing the property of producing color.

NUTRITION OF BACTERIA. 25

Just what their exact rdle 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 disintegra-
tion which is constantly going on in all dead organic
substances.

We know 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 have
their carbon and nitrogen presented as such, in the
form of decomposable organic compounds.

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
the salts of nitric acid. At all events, he was able in
certain cases to demonstrate a reduction of nitric
to nitrous acid, and ultimately to ammonia. Neverthe-
less, 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 devel-
opment, was derived from some source other than that of
the nitric acid or the nitrites, and that 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

a

26 BACTERIOLOGY.

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 present
in concentrated form inhibit the growth of the lower
organisms, may, when highly diluted, serve as nutrition
for these bodies. Salicylic acid and ethyl alcohol come
under this head.

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

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 more concentrated form of nutrition,
while another needs but a very limited amount of pro-
teid substance for its development. Certain members
bring about most profound alterations in the media in
which they exist, while others produce but little appa-
rent change, In one case alterations in the reaction of
the media will be most conspicuous, while in another no
such variation can be detected. With certain forms
oxygen is essential for the proper performance of their
functions, 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

°

BACTERIAL RELATION TO OXYGEN. 27

it. In the case of certain of the chromogenic forms
the presence or absence of oxygen has a very decided
effect upon the production of the pigments by which
they are characterized.

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 substauces. Nitrogen and carbon
compounds in the proper form to be taken up and appro-
priated by the organism may exist in sufficient quanti-
ties, and still the growth of the organism after a very
short time be entirely checked, owing to the production
during their growth of substances inhibitory to their fur-
ther development. Most conspicuous are the changes
produced by the growing bacteria in the reaction of the
media, Since the majority of these bodies grow best in
media of a neutral or very slightly alkaline reaction, any
excessive production of alkalinity or acidity, as a pro-
duct 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. It was Pasteur who first
demonstrated the existence of species in the bacteria
family which not only grow and multiply and_per-
form definite physiological functions without the aid
of oxygen, but to the existence of which oxygen is
positively harmful. To these he gave the name of

28 BACTERIOLOGY.

“anaérobic” bacteria in contradistinction to another
group for the proper performance of whose functions
oxygen is essential, which he called “aérobic” bacteria.
In addition to these, there is a third group for the main-
tenance 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 relation to
this gas. Itis in this third group, the facultative, that
the majority of bacteria belong. Though the multipli-
cation of the facultative varieties is not interfered with
by either the presence or absence of oxygen, yet experi-
ments show that the products of their growth are differ-
ent under the varying conditions of absence or presence
of this gas.

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 bacteria are known to grow
range from 5.5° C. to 48° C. At the former tempera-
ture development is hardly appreciable, it becomes more
and more active until 38° C. is reached, when it is at its
optimum, and, as a rule, ceases with 43° C. Neither of
the extremes can be considered normal temperatures for
the growth of these organisms. The most favorable
temperature for the development of the majority of
bacteria is that of the human body, viz., 37.5° C.

In general it may be said that for the growth and
development of bacteria, organic matter of a neutral or
slightly alkaline reaction, in the presence of moisture
and at a suitable temperature, is necessary. From this
can be formed some idea of the omnipresence in nature
of these minnte vegetable forms. Everywhere that
these conditions exist, bacteria can be found,

CHAPTER II.

Morphology! of the bacteria—Grouping—Mode of multiplication
—Spore-formation—Motility.

In form the bacteria are unicellular, and are seen to
exist as spherical, rod- or spindle-shaped bodies. They
always develop from preéxisting cells of the same char-
acter and never appear spontaneously.

The classifications of the older authors were upon
purely morphological peculiarities, and in consequence,
were more or less complicated. The present tendency -
is to simplify this morphological 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 spirilli,

In the group microcoect belong all spherical forms,
i.e., all those forms the individual members of which
are of equal diameter in all directions.

The bacilli comprise all oval or rod-formed bacteria.

To the spirilli belong all organisms which are twisted
in the form of a corkscrew.

The micrococci are subdivided according to their
grouping, as seen in growing cultures: into staphylo-
cocci—those growing in masses like fish-roe or clusters
of grapes; streptococci—those growing in chains con-

1 Morphology, pertaining to shape; outline.

30 BACTERIOLOGY.

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

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

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 others, under certain condi-
tions, possess the property of forming within the body
of the rods oval, glistening spores, 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.
Again, many of them, from unfavorable conditions of
nutrition, aération, or temperature undergo pathological
changes—that is, the individuals themselves experience
alterations in their protoplasm which result in distor-
tion of their outline. This is the production of the
so-called “involution forms.” But in all of these con-
ditions, so long as death has not actually occurred, it is
possible under favorable conditions to cause these furms
to revert to the original rod-shaped bacilli from which
they originated,

MORPHOLOGY. 81

It must be borne in mind, however, 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 inaccu-
rate methods of work.

Not infrequently bacilli may be observed irregularly
massed together as a pellicle. When in this condition
they are held together by a gelatinous material, and are
known as zodgloea of bacilli.

Very short oval bacilli may sometimes be mistaken for
micrococci, and at times micrococci in the stage of segmen-
tation into diplococci may be mistaken for short bacilli ;
but by careful inspection it will always be possible to de-
tect a continuous outline along the sides of the former
and a slight transverse indentation or partition-forma-
tion between the segments of the latter. The high index
of refraction of spores, the property 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
crucial test, however, is the property, in the case of the
spores, of growing out intu bacilli; and of the spherical
organism with which it has been confounded, of develop-
ing only into another micrococcus of the same round
form.

For convenience, a common classification of the bacilli

82 BACTERIOLOGY.

is that based upon constant characteristics which are
seen to appear in the course of their development under
special conditions—certain of them possessing the power
of forming spores, while from others this peculiarity 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 unsuccessful.
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 micrococei 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
center 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. A similar division
in the other direction will now result in the formation
of a group of forms as tetrads. This, in short, is the
method of multiplication of the micrococci.

In the formation of the staphylococci such division
occurs irregularly in all directions, resulting in the pro-
duction of the clusters in which these organs are com-

1 Dividing into two transversely,

MODE OF MULTIPLICATION. 33

monly seen. With the streptococci, however, the tendency
is for the segmentation to continue in one dircction only,
resulting in the production of the long chains of 4, 8,
and 12 individuals.

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
together in masses and 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 packet of
rags.

The multiplication of the bacilli is in the main similar
to that given for the micrococci. 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 aud 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, 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 extremities may be
rounded or slightly pointed. In the anthrax bacillus,
with which we are subsequently to become more inti-
mately acquainted, the segmentation, when completed,
results in an indentation of the adjacent extremities of
the young segments, so that by.the aid of high magni-
fying powers these surfaces are seen to be actually con-
cave in their outline. Bacilli never divide longitudinally.

With the spore-forming bacilli, under favorable con-

34 BACTERIOLOGY.

ditions of nutrition and temperature, the same process is
seen to occur, but so soon as these conditions become
altered, either by the exhaustion of the nutrition, the
presence of detrimental substances, unfavorable tempera-
tures, 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 again
to be seen.

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 rise 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 here and there by
granular, refractive points of irregular shape and size.
These eventually coalesce and leave the remainder of
the cell clear and transparent. When this coalescence
of highly refractive particles is complete the spore is per-
fected. In appearance, the spore is oval or round, very
highly refractive, and of a glistening appearance. It is

SPORE—-FORMATION. 35

easily differentiated from the remainder of the cell,
which now consists only of a cell-membrane and a per-
fectly transparent, clear fluid which surrounds the spore.
Eventually both the cell-membrane and its fluid con-
tents disappear, leaving the oval spore free in the medium
in which it has been formed.

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 conditions favor-
able to its subsequent development into the vegetative
form from which it originated, appear. 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.

Occasionally spore-formation is accompanied 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

36 BACTERIOLOGY.

drum-stick, or a lozenge, depending upon whether the
location of the spore is to be at the pole or in the centre
of the cell.

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 flagellee
come off from 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.

For a long time the motility of certain of the bacteria
was 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 spirillee found
in stagnant water, but it was not until very recently
that the accuracy of this suspicion was actually demon-
strated. 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 em-
ployment of a mordant, by the aid of which the flagelle
are caused to retain the staining, and thus become
visible. Loffler’s method of staining will be found in
the chapter devoted to this part of the technique.

CHAPTER IIT.

Principles of sterilization by heat—Different methods employed-—
Principles of discontinued sterilization—Sterilization under pressure
—Apparatus employed.

By the term sterilization, as employed here, is under-
stood the destruction of bacteria by heat. It is accom-
plished in two ways: either by dry heat, or by moist
heat in the form of steam.

Experiments have taught us that the process of steril-
ization by dry heat has a relatively limited application
because of its many disadvantages. or successful ster-
ilization by the method of dry heat, not only is a rela-
tively high temperature essential, but the substances
under treatment must be exposed to this temperature for
a comparatively long time. Its penetrating action into
the substances which are to be sterilized is, moreover,
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 substances which
may be sterilized in this way without serious damage to
their further usefulness.

Successful sterilization by dry heat cannot usually be
accomplished at a temperature lower than 150° C.,
and to this degree of heat must the objects 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,
3

38 BACTERIOLOGY.

and leather articles, this method is entirely impracticable.
In bacteriological work its application is limited to the
sterilization of glass-ware principally—such, for exam-
ple, as flasks, plates, small dishes, test-tubes, pipettes—
and such metal instruments as are not seriously injured
by the high temperature.

With sterilization by moist heat—stearn—the condi-
tions are much more favorable. The penetrating action
of the steam is not only much more energetic, but the
temperature at which this is ordinarily accomplished is,
as a rule, not destructive in its action. This is conspic-
uously seen in the work of the laboratory. The culture
media, composed in the main of decomposable organic
materials, which 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 he 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.

These differences will be strikingly brought out in
the experimental work on this subject. For our purposes
it is necessary to say that the two methods have the fol-
lowing applications :

The dry method, at a temperature of 150°-180° ©.,
for one hour, is employed for the sterilization of since

PRINCIPLES OF STERILIZATION BY HEAT. 39

ware: flasks, test-tubes, culture dishes, pipettes, plates,
ete.

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

The methods by which heat is employed in processes
of sterilization vary with circumstances. In its em-
ployment as dry heat its application is always continuous
—i. e., the objects to be sterilized are simply exposed to
the proper temperature for the length of time necessary
to destroy all living organisms which may be upon them.
With steam, on the other hand, the objects to be steril-
ized are frequently of such a nature that a prolonged
application of the heat would materially injure them.
For this reason 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 history 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 devel-
opment. This increased power of resistance is seen to
exist when these organisms are in the spore or resting
stage, to which reference has already been made.
When in the vegetative or growing stage, most of
these organism are killed in a short time by a relatively
low temperature, whereas, when conditions have arisen
which favor the production of spores, these spores are
seen to be capable of resisting very much higher tem-
peratures for an appreciably longer time. These differ-

40 BACTERIOLOGY.

ences in resistance toward heat which the spore-forming
organisms are seen to possess at their different stages of
development, are taken advantage of in the process of
sterilization by steam which is known as the fractional
or intermittent method, and form the principle on which
the method is based.

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 the time when they are in the
vegetating or growing stage—t.e., the stage at which
they are least resistant. In order to accomplish this it
is necessary that there should exist conditions of tem-
perature, nutrition, and moisture which favor the vege-
tation of the bacilli, and the germination of any spores
that may be present. When these surroundings are
fuund, the spore-forming organisms are not only less
likely to go into the spore stage than when their en-
vironments 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-
tain spores which 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 steain will in the course of this
time germinate into the less resistant vegetative cells

“PRACTIONAL’’ STERILIZATION. 41

A second short exposure to the steam kills these forms
in turn, and by a repetition of this process all organisms
which were present may be destroyed without the appli-
cation of the steam having been at any time of long
duration. 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 pressure,
for fifteen minutes on each of three successive days, and
during the intervening days to retain them at a temper-
ature of about 25°-30° C. At the end of this time all
living organisms which were present will have been de-
stroyed, 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 steriliza-
tion 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 proper conditions for 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-
fers in two respects, viz., it requires a longer time for
its accomplishment, and the temperature at which it is
conducted is not raised above 68°-70° C. It is em-
ployed for the sterilization of easily decomposable mate-
rials, which would be rendered useless by the tempera-
ture of steam, but which remain intact at the temperature
employed. This process requires that the material to be
sterilized should be subjected to a temperature of 68°—
70° C. for one hour on each of six successive days, an

42, BACTERIOLOGY.

interval of twenty-four hours being allowed between the
exposures to this temperature for the germination of
spores into mature cells. During this interval the sub-
stances 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 vege-
tative stage in about one hour. Blood-serum is 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 equivalent to
an approximate 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 which 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
loses the power of gelatinzing. Many of the other
media contain always a fine precipitate after this method ;
in fact, for most of the media which we employ, the

STERILIZATION APPARATUS. 43

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, a cut of which may be seen in
Fig. 1.

Fic. 1.

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

Af BACTERIOLOGY.

materials 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. he cylinder is closed by a
snugly-fitting cover through which are usually two per-
forations into which a thermometer and a manometer may
be inserted. The whole of the outer surface of the appa-
ratus 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
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
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 appa-
ratus being destroyed by the direct action of the flame.

A sterilizer now gaining favor for use in laboratories
is an apparatus originally intended for use in the kitchen.
It is the so-called “Arnold Steam Sterilizer.” It is very
ingenious in its construction 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

STERILIZATION UNDER PRESSURE. 45

the water in the reservoir becoming exhausted and the
consequent destruction of the sterilizer.
Fig. 2 gives an illustration of this apparatus.

For sterilization by steam under pressure several
special forms of apparatus exist. The principles of
them all are, however, the same. They provide for the
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 desirable can be maintained
within the sterilizing chamber. These sterilizers are
known as “digestors” and also by the French name
“autoclav.” Their construction can best be understood
by reference to Fig. 3.
The dry sterilizers used in laboratorics are simply

double-walled boxes of Russian or Swedish iron (ig. 4),
3¢

46 BACTERIOLOGY.

i

| = e ‘

having a double-walled door, which closes tightly, and
a heavy copper bottom. They are arranged with venti-
lating openings for the escape of the contained air and
the entrance of the heated air. The flame, usually from
a rose burner (Fig. 5), is applied directly to the bottom.
The heat circulates from the lower surface around about
the apparatus through the space between its walls.

The construction of the copper bottom of the appa-
ratus upon which the flame impinges, is designed to
prevent the direct action of the Hame upon the sheet-

ORDINARY DRY STERILIZER. 47

iron bottom of the chamber. It consists of several
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

‘i LA

io

of sterilizers are so constructed that their repair is a.
matter involving some time and expense. To meet this
objection I have had constructed a sterilizer in all re-
spects 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 dry sterilizer care should

48 BACTERIOLOGY.

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, ete., that may be in the
neighborhood.

CHAPTER IV.

Disinfection—Antiseptics—Inorganic salts as disinfectants—The
value of corrosive sublimate—Heat.

In contradistinction to sterilization, disinfection im-
plies the destruction of bacteria by chemical processes.
In the destruction of bacteria by means of chemical
substances, 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 point 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

50 BACTERIOLOGY

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-
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—t.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 intensity

DISINFECTION AND ANTISEPTICS. 51

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 de-
stroyed is much more energetic at a temperature of 37°
to 39° C. than it is at 12° to 15° C.

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 disin-
fectant 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 efficacy 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 procedure,

52 HEAT. «

as we have just learned, is always more or less question-
able, especially in those cases in which the substance to
be disinfected is of an albuminous nature. With the in-
troduction of such substances into the sublimate solution
the mercury is quickly precipitated by 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.

In the laboratory, then, 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 at least half an hour.

Intestinal evacuations may best be disinfected with
milk of lime, a mixture composed of lime in solution and
in suspension. This should be thoroughly mixed with
the evacuations until the mass reacts distinctly alkaline,
and should remain in contact with the infective sub-
stance for several hours.

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, for 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-

DISINFECTANT AGENTS. 53

ing now and then a few cubic centimetres of some so-
called disinfectant to trickle through the pipes is a
failure. <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 disinfectants, the place formerly
occupied by many agents in the list of substances em-
ployed 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 boiling
in a 2 per cent. soda solution for fifteen minutes; a solu-
tion of chlorinated lime (“chloride of lime”), in which
the percentage of chlorine is high; and mi/k of lime.
The materials to be disinfected in either of the lime
solutions should remain in them for several hours. The
solutions should be freshly prepared when needed, as
they rapidly decompose upon standing.

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 V.

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

Since the introduction of the plate method for iso-
lating in pure culture the individual species from mix-
tures of bacteria, a number of modifications have been
adopted, but the principle of them all is the same.
The observation which led to their development was
a very simple one, and one that is commonly before
us. Koch noticed that on solid substances, such, for
example, as a slice of potato, which had been exposed
for a time to the air and which afforded proper nourish-
ment for the lower organisms, there developed after a
short time small patches of material which proved to be
colonics of bacteria. Each of these colonies on closer ex-
amination showed itself to be, as a rule, composed of but
a single species. There was no tendency toward a con-
fluence of these colonics, 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. 55

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 upon 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 ina fixed position, and be sufficiently
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 a test-tube of decomposed bouillon were poured out
upon a large flat surface, the individual bacteria in the
mass would be 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 in-
dividuals in the mixture.

If, however, it is possible to find some substance
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
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 secn on the bit of
potato.

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 he

56 BACTERIOLOGY.

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 separated 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, but
also because this close 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 pro-
cess 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 third galatin-
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 numerous and too closely
packed to permit of characteristic growth; but on the

CULTURE MATERIALS EMPLOYED. 57

second dilution they were, as a rule, fewer in number
and widely separated, so that the individuals 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 result-
ing from the growth of the different individual bacteria.

Such, then, are the principles upon which Koch’s
method for the isolation of bacteria by means of solid
media is based.

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 in
the 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. Ordi-
nary gelatin liquefies at about 24° C., and becomes solid
at from 8°-10° C. It may be employed for those organ-
isms which do not require a higher temperature for their
development than 22° C. Agar-agar, on the other hand,
does not liquefy until the temperature has reached about

58 BACTERIOLOGY

98°-99° C. It remains fluid ordinarily until the tem-
perature 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° ©. Agar-agar which remains fluid only at a tem-
perature above this point would be too hot, when in a
fluid state, for use; many of the organisms 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 (probably a process of
peptonization), in which state it remains. There are no
known organisms which bring about such a change in
the agar-agar.

Asarule, the colony-formations seen upon gelatine
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 VI.

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

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

Bovititon.—The formula of Koch for the preparation
of this medium has undergone many modifications to
meet special cases, but for general use his original for-
mula 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 kept at a low temperature. 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 on 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

60 BACTERIOLOGY.

is accomplished with the saturated soda solution, and
the reaction is detected with the ordinary red and blue
litmus paper.

Soda solution is not so good asa strong solution of
caustic soda or potash, because the carbonic acid liberated
from the sodium carbonate is frequently seen to give rise
to a confusing temporary acid reaction which disappears
on heating. To obviate this, Schultz (Centralbl. f. Bact.
u. Parasitenkunde, Bd. x., Nos. 2 and 3, 1891) recom-
mends 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 filtration, 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 cc. 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 phenolphtalein solution (1
gramme phenolphtalein in 800 c.c. of alcohol) to 1 cc.
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 10 c.c.

BOUILLON. 61

of bouillon were employed, then, for the whole amount
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 cc. of bouillon, 2 c.c. of the diluted
(0.4 per cent.) caustic soda solution were employed. For
the remaining 980 c.c. of the litre of bouillon, then,
200 ec. (—4 ec, the amount employed for the two
samples of 10 c.c. each of bouillon) 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 tem-
perature, and consequent alterations in volume, will not
materially interfere with the accuracy 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
4

62 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.

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 proportion of
from 10 to 12 per cent. The complete solution of the
gelatin may be accomplished either over the water-bath,
in the steam sterilizer, or over a free flame. If the latter
method is practised, care must be given that the mixture
is constantly stirred to prevent burning at the bottom
and consequent breaking of the flask, if a flask is em-
ployed.

For some time it has been our practice to use, for the
purpose of making both gelatin and 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

NUTRIENT GELATIN. 63

the bottom of the vessel from the direct action of the
flame by the interposition of several layers of wire gauze
or a thin sheet of asbestos-board.

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.

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 sv high that evap-
oration and condensation 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 is
unnecessary if the gelatin is quite dissolved. At the
ordinary temperature of the room and by the means com-
monly employed for the filtration of other substances,
both gelatin and agar-agar may be rapidly filtered 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 «.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

64 BACTERIOLOGY.

large masses of coagulated albumin if it can be avoided,
as when broken up into fine flakes they clog the filter
and render filtration very difficult.

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-
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 for these
purposes 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 albumin-
ous flakes which become fixed within them. In this
way the filter may become almost entirely occluded.
The preliminary moistening with water causes the
diminution of the size of the pores to such an extent
that the finer particles of the precipitate now 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
coagulated 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

NUTRIENT AGAR—AGAR. ; 65

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 the
nutrient agar-agar by the beginner is, as a rule, a some-
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 method which has given the best results in our
hands, and from which there are no grounds for devi-
ating, is 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 mixture 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 e.c. of water and allow the
mass to boil slowly, occasionally stirring, over a free
flame for three or four hours. Care must be given 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 re-
move the flame and place the vessel containing the mix-
ture in a large dish of cold water; stir the agar con-
tinously until it has cooled down to about 68°-70° C.,

66 BACTERIOLOGY.

and then add the whites of two eggs which have been
beaten up in about 50 cc. of water. Mix this carefully
throughout the agar, and allow the mass to boil slowly
for about one-half hour, observing all the while the level
of the fluid. It is necessary to reduce the temperature
of the mass to the extent given, 68°-70° C., otherwise
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 mechani-
cal one—the finer particles, which would otherwise 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.
If the mixture is positively alkaline, it is not only cloudy,
but it filters with difficulty ; 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 the bouillon, which is normally
acid, from the acid of the meat, robs the agar, under
* the influence of heat, of some of its gelatinizing powers ;
this 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 digestor or autoclav, with the steam under
a pressure of one atmosphere, as shown by the gauge, for
from twenty to thirty minutes, the agar-agar will be
found at the end of this time completely melted, and fil-
tration may then be accomplished with but little difficulty.

PREPARATION OF POTATOES, 67

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 extracts
now on the market may occasionally 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 three to five grammes
to the litre of water.

PREPARATION OF PoTaToOES.—Potatoes are prepared
for use in two ways:

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 into a covered tin bucket with a perforated
bottom and sterilized in the steam sterilizer for forty-

68 BACTERIOLOGY.

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
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 up-
permost. The cover is placed upon the dish and the
potatoes are ready for inoculation.

2. Preparation of potatoes for test-tube cultures. 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

BLOOD—SERUM. 69

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 discolored by the sterilization to
which they are to be subjected. At the end of this time
' they are placed into previously prepared test-tubes, one
piece in each tube, with the slanting surface up, the cot-
ton plugs of the tubes replaced and they are then to be
sterilized in the steam for forty-five minutes. On the
second or third day they are to be sterilized
for fifteen to twenty minutes each day.

The entire sterilization may be accom-
plished in the autoclav with the steam under
a pressure of one atmosphere, by a single
exposure of twenty to twenty-five minutes.
When finished they have the appearance seen
in Fig. 6.

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

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

It is possible to collect serum from small animals and
4*

70 BACTERIOLOGY.

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 ob-
tained, and here’a certain amount of contamination 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 ceiling by the hind
legs, the head should be held well back, and with one
pass of a very sharp knife the throat should be com-
pletely cut through. The blood which will be spurting
from the severed vessels should be collected 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. The jars should be pro-
vided 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
which can be tightly screwed down upon a rubber joint.
From two such jarfuls of blood one can recover quite
a large quantity of 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

BLOOD—SERUM. 71

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 or-
ganisms 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 which 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
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.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 development of any
spores that may be present into their vegetative forms,
in which condition they are killed by an hour’s exposure
to the temperature of 70° C.

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
process of solidification requires constant attention if

72 BACTERIOLOGY.

good results are to be obtained, 7. ¢., if a translucent,
solid medium is to result. If the old, small form of
apparatus is employed (Fig. 7), 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

BLOOD—-SERUM. 73

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 ex-
posure is too long, an opaque medium results. The tem-
perature to be observed is that of the air inside the cham-
ber, 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 protected from the
sides by a wire screen of gauze; in this way the tubes
are all exposed to about the same temperature. The
thermometer which indicates the temperature inside the
chamber should not touch the surfaces but should either
be suspended free from above 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 apparatus of Koch is employed.
The latter form is preferable, as it is more easily man-
aged.

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 dry-
ing. The superfluous ends of the cotton plugs should be
burned off, and the mouths of the tubes should then be
covered by sterilized rubber caps. Even with the greatest,
care, it not uncommonly happens that one or two of the
lot of tubes thus prepared and protected will become con-
taminated. 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 hyphen downward through

74 BACTERIOLOGY.

the cotton into the tube, and their spores have fallen upon
the surface of the serum and gone on to develop.

SpecraL Mxrpra.—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 ce. 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 stop-cock at the bottom, by
means of which the milk, devoid of cream, may be drawn
off. A Chevallier creamometer with stop-cock at the
bottom serves the purpose very well. It should he
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.

Milk may also be employed as a solid culture medium
by the addition to it of gelatin or agar-agar in the pro-
portions given for the preparation ot the ordinary nutri-

SPECIAL MEDIA. 75

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 solution and peptone-rosalic-acid solution.
Peptone solution, to which rosalic acid has been added,
also serves very well for the detection of alterations in
reaction. The peptone solution of Dunham is the form
that we have usually employed. It consists of

Distilled water : . 100 parts.
Dried peptone . 1 part.
Sodium chloride : : 0.5 “

and 4 cc. of the following solution :

Rosalic acid (coralline) 0.5 gramme.
Alcohol (80 per cent.) 100 c.c.

This is to be boiled, filtered, and decanted into clean,
sterilized test-tubes, about 8 to 10 cc. 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 it
a valuable addition to our means of differentiation of
bacteria.

Léffler’s blood-serum mixture. Loffler’s blood-serum
mixture consists of one part of neutral meat-infusion

76 BACTERIOLOGY.

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 ; 950 e.c.
Sodium chloride ; : 5 grammes.
Peptone . : . 25-80 “
Gelatin . 40-60 “
Agar-agar ‘ ; 3-4“
Water . 50 cc.

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 which have been given above will
suffice for obtaining a clear understanding of the prin-
ciples of the work.

Norr.—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 VII.

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

WSILE the media are in course of preparation it is
-well to get the test-tubes and flasks ready for their recep-
tion. It is essential that the tubes for this purpose should
be as clean as it 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. solu-
tion; 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. 8), as those with wire handles are apt to break

Fig. 8.

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 them.
When dry they are to be plugged with raw cotton. The

78 BACTERIOLOGY.

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 be neither too tight nor too
loose, the regular rule being that when in position the
plug should fit tight enough to just 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 heated
for one hour in the 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.

FILLING THE Tupes.—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. 9, The liquefied medium is poured into this funnel,
which has been carefully washed, and by pressing the
pinch-cock with which the fetvucl is provided, the

FILLING THE TUBES. 79

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

It 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 given that none of the medium is
dropped upon the mouth of the test-tube, otherwise the
cotton plugs become adherent to the tube and are not
only difficult to remove, but present a very untidy ap-
pearance, and interfere, indeed, with the proper manipu-
lations.

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

80 BACTERIOLOGY.

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

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. of material
should be placed in each tube, as more than this renders
the rolling difficult and irregular.

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

PiatEs.—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-
tied, 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 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 re-
mains 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 sterilized, looped platinum wire, an

82 BACTERIOLOGY.

“ese” as it is called, Fig. 10. This is nothing more than
a piece of platinum wire of about 5 ¢.m. 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
“oese” is one of the most useful of bacteriological instru-
ments, as there is hardly a manipulation in the work into
which it does not enter. Under no conditions is it to be

Fie. 10.

employed without having been passed through the gas-
flame until quite hot; this is for the purpose of sterili-
zation. One should form a habit of never taking up
one of these “ oeses,” or platinum-wire needles, as they
are also called, for they are both looped and curved or,
straight, 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 pos-
sible sources of error in his work. It must be remem-
bered, though, that the “ oese” should not be used when
hot, otherwise the organisms taken up with it are killed
by the high temperature; after sterilizing it in the flame
one waits for a few seconds before using it.

The bit of material under consideration is transferred
with the sterilized “oese” into tube No. 1, “ the
original,” where it is carefully disintegrated by gently
rubbing it against the sides of the tube. The more
carefully this is done the more homogeneous will be the

TECENIQUE OF MAKING PLATES, 83

distribution of the organisms and the better the results.
The “oese” 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 “oese” is sterilized
and again three dips are made from tube 2 into tube 3.
This completes the dilution. The “oese” is now steril-
ized before laying it aside.

Fig. 11.

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 gelatinizes, and can
only be redissolved by a temperature which would be
destructive to the organisms which may have been in-
troduced into the tubes. This is not of so much
moment with gelatin, as it may readily be redissolved

84 BACTERIOLOGY.

at a temperature not detrimental to the organisms with
which the tubes may have been inoculated

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. 11)
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 indicated 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.

THE Gass PLatEs.—On the stages are to be placed
the glass plates 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.

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

TECHNIQUE OF MAKING PLATES. 85

are retained in an iron box (Fig. 12), 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.

As the contents of each tube is 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. 13), and each bench is

Fie. 13.

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 growth of the bacteria. The benches are
sterilized before using, in the way given for the plates,
CuuruReE-DIsH.—This dish, which is about 22 cm. in

diameter and has vertical sides of about 6 cm. in height,
5

86 ' BACTERIOLOGY.

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 disc
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 gelatin 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 Metuop.—
The modification which approaches nearest to the original
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-

ESMARCH’S TUBES. 87

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 dishes are flat, double
dishes of glass (Fig. 14). They are of about 8 cm. in

Fig. 14.

cme a |
ST

diameter and about 1.5 to 2 em. 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
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

88 BACTERIOLOGY.

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 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 be-
comes 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.

Fria. 15.

ron
|
aN q

|

il i

" 7

For some time past we have deviated from the direc-
tions given by v. Esmarch for this part of his method.
Instead of rolling the tubes under ice-water, we roll
them upon a block of ice (Fig. 15). In this method a
small block of ice only is needed. It is arranged nearly

ESMARCH’S TUBES. 89

level, and is held in position by being placed in a dish
upon cloths. A horizontal groove is melted in the sur-
face of the ice with a test-tube of hot water. The tubes
to be rolled are then held in an almost, not quite, hori-
zontal position and twisted between the fingers until the
sides are moistened by the contents to within about
1 cm. of the cotton plug, care being taken that the
gelatin does not touch the cotton; otherwise the latter
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, no rubber cap or cutting off of the
cotton plug being necessary.

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 at nearly a hori-
zontal position, the cotton end of the tube about 1.5 to
2m. 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, i.e., the bottom of the

90 BACTERIOLOGY.

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 num-
ber 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 contained 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 development.

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.

CHAPTER IX.

The incubator used in bacteriological work—Gas-pressure regu-
lator—Thermo-regulator—The form of burner employed in heating
the incubator.

Tue Incuzator.—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 (87.5° C.)
than at lower temperatures ; whereas, with the ordinary
saprophytic forms almost any temperature between 18°
to 20° C. and that of the body is favorable. It there-
fore 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 isa copper chamber (Fig. 16)
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
chambers may be inspected without actually opening it.

92 BACTERIOLOGY.

The whole apparatus is encased in either abestos boards
or thick felt to prevent radiation of heat and consequent
fluctuations in temperature. In the top of the chamber

Fig. 16.

3 id a

Tin
tubal

is a small opening through which a thermometer pro-
jects 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.

Ward ates a

THE INCUBATOR. 93

On the side is a 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 arrange-
ment, 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. The
apparatus should be kept filled with water, otherwise
the object for which it is constructed will not be accom-
plished. When the chamber between the walls is filled
with water the apparatus is heated from a gas-flame
which is placed beneath it.”

Fic. 17.

ci ‘

ia

The burner employed in heating the incubator is
known as “Koch’s safety burner” (Fig. 17). It is a
Bunsen burner provided with an arrangement for auto-

matically turning off the gas supply and thus preventing
5*

94 BACTERIOLOGY.

accidents should the flame become extinguished at atime
when no one was near. The gas-cock is provided with
a long arm which is weighted and which, when the gas
is turned on and burning, rests upon the end of a metal
spiral which is heated by the flame. If by draughts or
any other accident the flame becomes extinguished the
metal spiral cools, and in cooling contracts and allows
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 con-
traction, the amount of gas passing from the source of
supply to the burner may be either diminished or in-
creased 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, 18.

It consists of a glass cylinder e, having a communicat-
ing branch tube 6, and rubber stoopper 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.

“When ready for use the cylinder e 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 about

THERMO—REGULATORS. 95

the level shown in the cut. 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 a, and

Fig. 18.

the tube going to the burner is slipped over the branch
tube b. The gas is turned on and the 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

96 BACTERIOLOGY.

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 h 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 h 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 A, when again the temperature rises.
This contraction and expansion of the fluid in the regu-
lator 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 com-
paratively large, and form has each its special merits.
The value, that is, the délicacy 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 essential points to be
observed in selecting a thermo-regulator 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 expand and contract
rapidly and regularly under slight variations in temper-
ature, are commonly employed ; whereas for temperatures

GAS—PRESSURE REGULATORS. 97

approaching the boiling-point of water mercury is most
frequently used.

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.

Fro. 19.

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
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 none of them accom-
plishes the object for which they are designed.

98 BACTERIOLOGY.

The instrument most commonly employed, the appar-
atus of Moitessier (Fig. 19), is based on somewhat the
same principles as the large regulators seen at the manu-
factories 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 fluc-
tuations 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 compara-
tively great and gradual fluctuations in pressure. If a
good form of thermo-regulator is employed, there is no
necessity for the use of any of the forms of pressure-
regulators thus far introduced.

CHAPTER X.

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—Stah 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 main-
tained 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, while others will
be in the centre of a slight depression, the result of
liquefaction of the gelatin about this point.

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.

100 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 a stained cover-
slip preparation (see chapter on cover-slip preparations)
from it, and examine it under the high power oil-immer-
sion 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 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 the new set of colonies
which develop. In other words, these peculiarities are
constant under constant conditions. The colonies will
be found to consist of the same organisms as the colony
from which the plates were made, and colonies of no
other organisms will be present.

With all organisms differences in the appearance of

STAB AND SMEAR CULTURES. 101

the colonies depending upon their location in the medium
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 pecularities is begun. Sometimes several series of
plates are necessary before the organism can be obtained
pure, but by patiently following this plan the results
will ultimately be satisfactory.

TEsT-TUBE CuLTURES; StaB 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 separated
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 three
to five seconds, take up carefully a portion of the colony.
Guard against touching anything but the colony. If,

102 BACTERIOLOGY.

during manipulation, the needle touches anything 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 only the over-
hanging 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. 103

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.

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 note the finer differences
in the way in which liquefaction occurs.

CHAPTER XI.

Systematic study of an organism—Steps necessary 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 con-
ditions.

Its form at certain 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 must
always present the same outward appearances. The
reactions given by it to the media in which it is growing
must follow a fixed rule. Its power to produce liquefac-
tion of the gelatin, or to grow upon it without bringing
about this change, must always be thesame. Its motility
or non-motility must be determined. Its production of
certain chemical products must be detected by chemical
analysis. Its behavior toward oxygen—i. e., does it re-
quire 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 determined. Its behavior
under varying conditions of temperature and under the
influence of different chemical bodies as well as its growth
in media of different reactions are to be studied. The
property of producing fermentation with the libera-

MICROSCOPIC EXAMINATIONS. 105

tion of gases must be ascertained ; and lastly, we must
consider its behavior when introduced into the bodies of
animals used for experimental work—i. ¢., is it a disease-
producing organism, or does it belong to the group of
innocent saprophytes ?

We have learned the methods for obtaining colonies,
and have studied some of the peculiarities which are to
distinguish them from one another. The next important
step is to determine the morphology of the individuals
composing these colonies as well as their relation to each
other in the colony. These points are decided by micro-
scopic examination of bits of the colony which have been
transferred to thin glass cover-slips, upon which they are
dried, stained, and mounted. Cover-slips for this pur-
pose are prepared in two ways: either by taking up a
bit of the colony on a 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 deter-
mined. The details of these methods will be found in
the chapter on the methods of staining.

MICROSCOPIC EXAMINATION OF PREPARATIONS.

Ture DirrerENT Parts oF THE MIcroscopE.—
Before describing the process of examining preparations
microscopically, a few definitions of the terms 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

106 BACTERIOLOGY.

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 to be
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 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—i. e., are homogeneous.
This is accomplished by interposing between the face of
the lens and the cover-slip covering the object to be ex-
amined 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 refraction as the glass
is placed upon the face of the lens, and the examina-
tions are made through this oil. There is thus no loss
of light from deflection, as is the case in the dry systems.

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. Between the condenser and reflector

MICROSCOPIC EXAMINATIONS. 107

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 passing
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 fullextent. 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 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 Examinina Srarvep 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
adjustment lower the oil-immersion objective until it
just touches the drop of oil. Open the illuminating appa-
ratus to its full extent. Then, with the eye to the micro-
scope and the hand on the fine adjustment, turn. the
adjusting-screw toward the right until the field becomes
somewhat colored in appearance. When this is seen,

108 BACTERIOLOGY.

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 accom-
plished.

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
examine bacteria in the unstained condition. The circum-
stances calling for this arise while studying the multipli-
cation 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

MICROSCOPIC EXAMINATIONS. 109

centre of a cover-slip. This is then placed, drop down,
upon an object-glass in the centre of which a hollow or
depression is ground (Fig. 20). The slip is held in posi-
tion by a thin layer of vaselin, which may be painted

Fig. 20.

| es ———=E ]

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
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.

110 BACTERIOLOGY.

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 which are
undergoing this molecular tremor and that possessed by
certain 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.

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 ?

Stupy or SPorE-FORMATION.—The hanging-drop
method just mentioned is not only employed for the de-
tection of the motility of an organism, but 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

STUDY OF SPORE-—FORMATION. 111

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 de-
pression in the slide and the cover-slip. (See Fig. 20.)

A drop of sterilized agar-agar may be substituted for
the bouillon. It serves to retain the organisms in a fixed
position, and the process may be more easily followed.

As soon as finished, the preparation is to be examined
microscopically, and the condition of the organisms
noted. ,It is then to be retained in a warm chamber
especially devised for the purpose and kept under con-
tinuous observation. The form of chamber best adapted
for the purpose is one which envelops the whole micro-
scope. It is provided with a window through which the
light enters, and an arrangement for moving the slide
about from the outside. Theformation of spores requires
a much longer time than the germination of spores into
bacilli, but with patience both processes may be satis-
factorily observed.

It will be noticed that the description of this process

112 BACTERIOLOGY,

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, other-
wise 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 some-
times days, and contamination must, therefore, be care-
fully guarded against, The study should be begun with
the vegetative form of the organism; the hanging-drop
preparation should, for this reason, always be made from
a perfectly fresh culture of the organism under consider-
ation, 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. Jor example, many species of bacteria which
possess this property form spores most readily upon
media from which it is somewhat difficult for them to
obtain the necessary nutrition ; potatoes and agar.agar
which 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 surrounded
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 en-
dogenous 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-

STUDY OF CULTURES ON POTATO. 113

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.

Stupy or GELATIN CuLturEs.—<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
gelatin at and around the place at which the colonies
are growing. In some instances this liquefaction spreads
laterally, in others it sinks directly down into the gelatin.
These differences have been conspicuously shown and em-
ployed as one of the means of differentiation of otherwise
closely allied members of the same family of bacteria.
Studies upon the organism of Asiatic cholera and a
number of closely allied forms reveal a decided differ-
ence in the manner of liquefaction produced by these
different organisms. The slightest detail in this respect
must be noted, and its frequency or constancy under
different conditions determined.

CULTURES ON Potato.—A very important feature
in the study of an organism is its growth on sterilized
potato. Many organisms present appcaiances 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 he moist and glistening.
Sometimes there is a production of bubbles, owing to
fermentation brought about by the growth of the organs.

114 BACTERIOLOGY.

A most striking form of development on potato is
that possessed by the organism 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.

Reacrion PRopUCED 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
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 diphtherize of Lofier.

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 tincture, and coralline (rosolic
acid) in alcoholic solution have been employed for this
purpose. They may be added to the media in the pro-
portions given in the chapter on media, and the altera-
tions in their colors studied with different bacteria.

ANILINE DYES. 115

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.

AwiuinE Dyes FoR DIFFERENTIAL D1agnosis.—
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 consisting in alterations in the
color of the media due to oxidizing or reducing proper-
ties 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.

Bewavior Towarp Srarninc Reacents.—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.

116 BACTERIOLOGY.

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.
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.

Where it is important that the nature of the gas thus
produced should be studied, it must be collected, and a
special form of apparatus is employed. The cultivation
is now to be conducted ina fluid medium. The fermen-
tation flasks of somewhat the pattern of that used by
Einhorn in the fermentation test for sugar in the urine
serve very well for this purpose.

CuLtivation Wirnour OxycEen.—<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 ot

CULTIVATION WITHOUT UXYGEN. 117

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.

Flesse’s method. Hesse poured sterilized oil upon the
surface of a culture made by stabbing into a tube of
gelatin. The growth that occurred along the track of
the needle was supposed to be anaérobic in nature.

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 e.c. of 2, normal! caustic potash solution.
The larger tube is then tightly plugged with a rubber

1 A normal solution is one that contains in a litre as many
grammmes of the dissolved substance as are indicated by its molec-
ular equivalent. The equivalent is that amount of a chemical com-
pound which possesses the same chemical value as does one atom of
hydrogen. Forexample: One molecule of hydrochloric 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 (II,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 equivalent chemically to one hydrogen
atom, viz., 40 grammes (one-half its molecular 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.

6%

118 BACTERIOLOGY.

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 Frankel suggests the
following. method: 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 have all 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
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 dif-
ferent lengths: one reaches to within 0.5 cm. of the
bottom of the test-tube, the other is cut off flush with 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 in the tube
until all contained air has been expelled and its place
taken by the hydrogen. The organisms are to grow,
then, in an atmosphere of hydrogen. When all air
has been expelled, the two external ends of the glass
tubes are to be sealed in the gas-flame at the portions
where they have been drawn out. In sealing the tubes
in the gas-flame one must be sure that all air has been
expelled, otherwise an explosion is inevitable. This

ESMARCH’S METHOD. 119

may be accomplished by allowing the hydrogen to
bubble through the gelatin for about ten minutes. The
rubber stopper is then painted around with melted
paraffin and the tube rolled in the way given for ordi-
nary Esmarch tubes.

During the operation the tube containing the liquefied
gelatin should be kept in a water-bath of a temperature
which will prevent its solidifying and at the same time

‘not kill the organisms with which it has been inoculated.

Method of Laborius. Another method, that of Libo-
rius, is to fill a test-tube about three-quarters full of
gelatin, This is kept at the temperature of boiling water
for ten minutes to expel all air from it. It is then to be
rapidly cooled in ice-water, and, when between 30° C.
and 40° C., is to be inoculated, and the gelatin rapidly
solidified. It is then to be sealed up in the flame.

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 0.8 to
0.5 per cent.

Esmarch’s method. Esmarch’s plan is to prepare in
the usual way an Esmarch tube of the organisms, and
this is to be subjected to a low temperature, and while
quite cold is filled with liquefied gelatin, and the whole
allowed 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 gela-
tin, 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.

120 BACTERIOLOGY.

Inpot Propucrion.—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 simple 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.

Indol, when acted upon by reducing agents, is seen
to become of a more or less conspicuous rose color. This
body was recognized some time ago as one of the pro-
ducts of growth of the comnia bacillus of Asiatic cholera,
and for a time was thought to characterize the changes
produced in the media through the growth of this organ-
ism. It has since been found that there exist other
bacteria which also possess the property of producing
this body 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 cc. of a 0.01 per

INDOL PRODUCTION. 121

cent. solution of sodium nitrite, aud 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. Ifnow 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.
This is found, by proper means, to be nitrous acid.
The sulphuric acid liberates it from its salts and permits
of its reducing action being seen.

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.

CHAPTER XII.

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

THE entire list of solutions and methods which have
been recommended for the staining of bacteria are not
essential to the work of the beginner, so that only those
methods 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. 123

their color, are employed. The ordinary method of
cover-slip examination of bacteria, which is 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 employed.

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.

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

124 BACTERIOLOGY.

centrated sulphuric acid for a time, then rinse them
in water, after which they are kept in a mixture of
equal parts of alcohol and ammonia. They are to be
dried on a cloth from which the fat has been extracted.
Steps 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 quite dry. If held with the forceps over
the flame at this stage, too much heat may be uncon-
sciously applied, and the morphology of the organisms in
the preparation distorted. When held between the fin-
gers with the layer of bacteria away from the flame no
such accident is likely to occur, When the whole
pellicle is completely dried the slip is to be taken
up with the forceps, and, holding the side upon which
the bacteria are deposited 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 proto-
plasm of bacteria when moist coagulates at these tem-

COVER-SLIP PREPARATIONS, 125

peratures, and in doing so the normal outline of the
cells is altered. If ‘carefully dried before fixing, this
does not occur and the morphology of the organism
remains unchanged. A better plan for the process of
fixing is to employ a copper plate of about 35 cm. 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 temperature, beginning at the
point of the plate above the gas-flame where it is hot-
test, and becoming gradually cooler toward the other
end of the plate, which may be of a very low tempera-
ture. By dropping water upon the plate, beginning at
the hottest point and proceeding toward 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. This plan is to
be preferred to the process 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 appli-
cation of high temperatures may in part be eliminated.
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 staining is usually a simple matter.
The majority of bacteria with which the beginner will
have to deal stain readily with solutions of any of the
basic aniline dyes.

126 BACTERIOLOGY.

To stain the fixed preparation it is taken 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 drying is placed bacteria down upon a slide, the
excess of water is taken up with blotting-paper, 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
from the slide by allowing water to flow around its
edges. It is then taken up with the forceps, carefully
deprived of the water adhering to it by means of blot-
ting-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.

Impression CovER-SLIP PREPARATIONS.—The im-
pression preparations differ in value from the ordinary
cover-slip preparations only in one respect : they present
an impression of the organisms as they were arranged in

THE ORDINARY STAINING SOLUTIONS. 127

the colony from which the preparation was 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, staining, 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.

THe Orpinary Srarsine SoLurions.—The solu-
tions commonly employed in staining cover-slip prepara-
tions are, as has been stated, watery solutions of the basic
aniline dyes— fuchsin, gentian-violet, and methylene-
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 prepar-
ing them from concentrated watery or alcoholic solutions
of the dyes which may be kept on hand as stock. The
latter method is that commonly practised.

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

Prepare as stock, saturated alcoholic or watery solutions
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 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

128 BACTERIOLOGY..

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 satu-
rated alcoholic or watery solution of fuchsin, gentian-
violet, or methylene-blue, as the case may be. The
alcoholic solutions will not answer for staining purposes.

The solutions with which the staining is accomplished
are made from these alcoholic solutions in the following
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

added, little by little, until one can just see through
the solution. It is then ready for use. Care must be
given 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

SPECIAL STAINING SOLUTIONS. 129

and pipettes (Fig. 21),and when used are dropped upon
the preparation to be stained. After remaining upon
the preparation for about thirty seconds, they are
washed off in water and the preparation can then be
examined.

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 accom-
plished by the addition to the solutions of small quan-
tities of alkaline substances or by dissolving the staining
materials in strong watery solutions of either aniline oil
or carbolic acid, instead of simple water.

Of the solutions thus prepared which may always be
employed upon bacteria which show a tendency to stain
imperfectly, there are three in common use—Léfiler’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.c.
Caustic potash in 1: 10,000 solution . : 100 ce.

Koch-Ehvlich aniline-water solutions. To about 100
ec. 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 c.c.
of absolute alcohol and 11 cc. of the concentrated al-
coholic solution of either fuchsin, methylene-blue, or
gentian-violet, preferably fuchsin or gentian-violet.

130 BACTERIOLOGY.

Ziehl’s carbolic-fuchsin solution :

Distilled water. : i F . 100 ce.
Carbolic acid (crystalline) . ‘ ‘ 5 grammes.
Alcohol - . ‘ » 10 ce.
Fuchsin in substance . 1 gramme.

Or it may be prepared by adding 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 very quickly after having been made, so
that it is better to prepare them when needed in small
quantities than to employ old solutions. Solutions older
than seven to nine days should not be used.

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

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 are usually employed for the purpose of depriv-
ing 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 stroug 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.

STAINING THE TUBERCLE BACILLUS. 181

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
Ziehl. 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 ina 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 decoloriza-
tion 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 con-
trast. 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 solution 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

132 BACTERIOLOGY.

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 con-
trast color, and examine it again. It will-be seen
that before the contrast 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 of other organisms, but upon ex-
amining it after it has received the contrast color, a
great many other organisms 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 decolor-
izing solution all except the tubercle bacilli gave up their
color. This characteristic, as said, serves to differentiate
the tubercle bacillus from all other organisms, except
the bacillus of leprosy, which stains in the same way as
does the bacillus of tuberculosis. A number of different
methods have been suggested for the staining of tuber-
cle 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 em-
ployment of Ziehl’s carbolic-fuchsin solution and 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-

GLACIAL ACETIC ACID METHOD. 133

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 4 a s - 150 cc.
Alcohol. ‘ : - 50 c.c.
Concen. sulphuric acid. ‘ 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.

Gram’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 composed of

Iodine . ; : ‘ : 1 gramme.
Potassium iodide Add ‘ 2 grammes.
Distilled water. é , 300 c.c.

In this they remain for about five minutes ; they are
then transferred to alcohol and thoroughly rinsed. If
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-brown may be given them.

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

GuacraL Acetic Acip Metaop.—Another method

which may be employed for demonstrating the presence
7

134 BACTERIOLOGY.

of the capsule which surrounds certain organisms, is to
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 again a few drops 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 the following will prove use-
ful: The cover-slip is to be prepared from the material
containing the spores in the ordinary way, dried, and
fixed. It is then floated, bacteria down, upon the sur-
face of a watch-crystalful of freshly prepared Koch-
Ehrlich solution of fuchsin. This is then held by its
edge with the forceps about 2 cm. above a very small
flame of a Bunsen burner, care being given that the
flame touches only the centre of the bottom of the erys-
tal. After a few seconds the crystal is elevated gradu-
ally 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, again the crystal is held aside 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

STAINING OF SPORES. 135

about five minutes after the last boiling, the preparation
is transferred, without washing in water, into a second
watch-crystal containing the following decolorizing solu-
tion:

Absolute alcohol . 3 ‘ , - 100 ec.
Hydrochloric acid . ‘ : F ‘ ; 3 ce.

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.

MoeELLEr’s MeErnop For Srarnrnc Spores.—A
method that has recently been published by Moeller is
designed to favor the penetration of the coloring material
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 instead
of high temperatures. The preparation is then 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 methy-

136 BACTERIOLOGY.

lene-blue solution. The spores will be red, the body of
the cells blue.

In this method the object of the preliminary exposure
to chloroform is to dissolve away any crystals of lecithin,
cholesterin, or fat that may be in the preparation, and
which when stained might give rise to confusion.

L6rrier’s METHOD FoR STAINING FLAGELLA.—
For the demonstration of the locomotive apparatus pos-
sessed 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
themselves through the fluid in which they are located.
The method of 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 :

METHOD FOR STAINING FLAGELLA. 137

Tannic acid solution in water (20 acid, 80

water). . ‘ . : 10 ce.
Cold saturated solution of ferro-sulphate . 5 cc.
Saturated watery or alcoholic solution of

fuchsin. ‘ : ; . ‘ e “aseie:

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 to be stained in a
saturated aniline-water fuchsin solution.

‘When treated in this way different bacteria behave
differently: the flagelle 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
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 centimentre 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 proportions.

Of the acid solution :

For the bacillus of Asiatic cholera . . 4 tol drop.
For the spirillum rubrum. : . 9 drops.

Of the alkaline solution :

For the bacillus of typhoid fever . . lee.
For the bacillus subtilis ‘ ‘ . 28 to 30 drops.
For the bacillus of malignant edema . 36 to 37 “

1388 BACTERIOLOGY.

For other organisms one must determine whether the
Tesults 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 Loffler’s direc-
tions the delicate, hair-like flagellee on motile organisms
may be rendered plainly visible.

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-

DECOLORIZING SOLUTIONS. 139

trated. Prepare a cover-slip preparation, dry it care-
fully, fix it, and without allowing water to get upon it
from any source, attempt to stain it with a solution of
the dyes in 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.’

DrcoLorizine SoLuTions.—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 decolorizer
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 de-
colorization 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 ap-
pear, particularly when they are alcoholic solutions, 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 em-
ployed with much care,

" 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 remembered 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.

140 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 0.1 per cent. to 5 per cent. watery
solution.

Nitric acid in 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 a 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 with the 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 structure,

STAINING OF BACTERIA IN TISSUES. 14]

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.

Harpenine 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 con-
taining 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. The
alcohol abstracts water from the tissue, and, as the
dehydration proceeds, the tissue becomes accordingly
more and more dense. When of about the consistency
of fresh solid rubber, or preferably not quite so dense, it
is ready to cut. A small portion of about 0.5 cm. 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 prevent 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 it may be cut.

SEcTIon-curTTinc.—This is accomplished by the use
of an instrument known as a microtome (Fig. 22). 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. Beneath
the clamp for holding the tissue is a milled disc, by
means of which a screw is caused to revolve, and in

revolving raises or lowers the clamp holding the tissue,
7%

142 BACTERIOLOGY.

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 disc with the one hand and causing
the knife to traverse the tissue with the other.

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.

Fig. 22.

> lh

ie
fives

iad ie:
——" _
Hite Al

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

STAINING OF BACTERIA IN TISSUES, 143

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.

Two solutions of celloidin are to be employed, the one
a thin solution in a mixture of absolute alcohol and
ether, equal parts, the other a thick solution in absolute
alcohol. Into the thin solution the tissue is placed from
absolute alcohol, and allowed to remain for twenty-four
or forty-eight hours. It is then placed in the thick
solution for one or two days. From this it may be
removed and placed immediately upon a bit of cork,
The adherent celloidin will act as a cement, and as it
hardens rapidly, the tissue is soon fast to the cork ; after
remaining in 60 per cent. alcohol for twenty-four hours
to complete the solidification of the celloidin, sections
may be cut as in the way just described for tissues not
so treated.

The paraffin method of imbedding is not to be recom-
mended for bacteriological purposes.

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 Loffler,
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.

Into a watch-glass containing either of the staining

144 BACTERIOLOGY.

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 transferred
to a clean glass slide. This must be done carefully to
avoid tearing ‘The easiest way to do this is to hold the
spatula on which the section fluats 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 section
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
tissues. The cedar oil or xylol are bodies which mix
on the one hand with alcohol, on the other with balsam.

STAINING OF BACTERIA IN TISSUES. 145

They are known as “clearing fluids,” and fill up the
gap that would otherwise be left in the process, for a
section cannot be mounted in balsam directly from alco-
hol; 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.

Ff. Absolute alcohol for a few seconds.

g. Xylol for about one-half minute.

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

zt. Removal of excess of xylol.

j. Mounting in xylol-balsam.

146 BACTERIOLOGY.

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.

SpectAL Meruops oF STAINING BACTERIA IN
TissuESs.—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 commonly
employed differential stains is that of Gram. In gene-
ral, it is practised in the way given for its employment
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
from them. They are then brought for one minute into
a dilute watery solution of eosin or safranin or into picro-
carmine ; again washed out for a few seconds in alcohol,
and finally for one-fourth minute in absolute alcohol.

STAINING OF BACTERIA IN TISSUES. 147

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
picro-carmine for one-half hour, 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 abso-
lute alcohol, and then into the xylol, from which they
may be mounted. The organisms 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 two 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
of either eosin, Bismarck-brown, or safranin for one
minute, then washed out in alcohol, finally into absolute
alcohol, and then into xylol, from which they may be
mounted in the manner given.

148 BACTERIOLOGY.

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.

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

Methylene-blue, in substance a . 1.5 grammes.
Absolute alcohol . : ‘ A . ce.

Rub up thoroughly in a mortar, and when the blue
is completely dissolved, add gradually 100 cc. 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 methy-
lene-blue in substance has been added; then com-
pletely rinse out in pure auiline 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
applicable, and by its use the bacteria are not robbed of
their color, whereas the tissues are sufficiently decolor-
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 Ehrlich’s aniline-water gentian-violet

STAINING OF BACTERIA IN TISSUES. 149

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 solu-
tion used by Gram; take up the excess of the solution
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 gen-
tian-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 decolorization is sufficient (re-
peated 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 avoided. This method,
while it stains certain bacteria in tissues very satisfac-
torily, is nevertheless designed especially for the stain-
ing of fibrin. Fibrin and hyaline materal will be
stained deep blue, bacteria a dark violet.

150 BACTERIOLOGY.

STAINING FoR TUBERCLE BactLui 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 Ziehl-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
isa 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.

CHAPTER XIII.

Inoculation of animals—Subcutaneous inoculation, intra-venous
injection.

AFTER subjecting an organism to the methods of study
that we have just reviewed, it remains that its action
upon the lower animals should be tested—i. ¢., to deter-
mine 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.

This is commonly determined by both subcutaneous
and intra-venous inoculation.

SuscuTanEous 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
is very dirty it may be scrubbed with soap and water.
Disinfection of the skin is impossible, so that it need not
be attempted. If the 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 a solid culture is to be inocu-
lated, a fold of the skin may be taken up with the
forceps and a pocket cut into it with scissors which have

152 BACTERIOLOGY.

previously 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, and in-
cised in a way corresponding to the skin. The pocket
is then to be held open with the forceps and the sub-
stance to be inserted is introduced as far back under
the skin and fascie 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 quiet.
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 cm. 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 em. in diameter at
one end and 2 cm. at its other end.

This is tacked upon the board in such a position that
its long axis runs in the long diameter 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-

SUBCUTANEOUS INOCULATION. 153

screw, with which the apparatus (Fig. 23) is provided.
The animal usually remains perfectly quiet and may be
handled without difficulty.

Fig. 23.

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 same 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 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 inoculations are com-
monly made is in the abdominal walls either to the right
or left of the median line and about 8 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

154 BACTERIOLOGY.

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 be strictly observed, the greatest of
these obstacles to the successful performance of the oper-
ation 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 iuto which
it would appear easiest to insert a hypodermatic needle.
This, however, is fallacious. This vessel lies very loosely
imbedded in connective tissue, and in efforts to introduce
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

INJECTIUN INTO THE CIRCULATION. 155

in the dense tissues which surround it that it is prevented
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 be-
cause of the way in which their points are ground.
If one examines carefully the point of a new hypo-
dermatic needle it will be seen that the long point, in-
stead 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 vessel a
of very small calibre, it is commonly seen that the ex-
treme 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 in-
serted 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 con-
‘trary, the slanting point of the needle is ground down
until its surface is perfectly flat, and when viewed from
the side no more curvature exists, then when once in-
serted into a vessel it usually remains there, and there

156 “BACTERIOLOGY.

is no tendency 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.

Fie. 24.

In Fig. 24, a, the needle has the point usually seen
when new.

In Fig. 24, B, 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
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

INJECTION INTO THE CIRUULATION. 157

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
aud into the finest part of the ramus posterior, the part
nearest the apex of the ear. 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,
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 given that no air is injected.

The hypodermatic syringe and needle must, previous
8

158 BACTERIOLOGY.

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.

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
auaplayed are those shown in Fig. 25—A, Koch’s ; B,
Strohschein’s ; C, Overlack’s.

Fia. 25.

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 pupae 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.

CHAPTER XIV.

Post-mortem examination of animals—Bacteriological examina-
tion of the tissues—Disposal of tissues and disinfection of instru-
ments after the examination.

For the purpose of examining bacteriologically the
tissues 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
which will now be present may interfere with the accu-
racy of the 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 moderately
extended. 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 genitalia, 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 then carefully dissected

160 BACTERIOLOGY.

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. This is best
accomplished by the use of a broad-bladed common knife
which can be heated in the gas-flame. The blade is to be
heated quite hot, and 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 accomplished 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 scissors, which have been sterilized, is made
along the scorched tracks on either side of the thorax.

After this the whole anterior wall of the thorax may

EXAMINATION OF ANIMALS. 161

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 point of inoculation,
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 plates or Esmarch tubes.

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.

In addition to cultures, cover-slips from each organ
and from any exudates that may exist, must be made.
These, however, are prepared 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,

162 BACTERIOLOGY.

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. alcohol for sub-
sequent 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, name 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
in the protocol.

The same may be said for the subsequent study of the
cultures and the hardened tissues which are to be stained
and subjected to microscopic examination. The results
of the microscopic study of the cover-slip preparations
and those obtained by cultures should in most cases corre-
spond, 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 in-
ternal viscera, or those predominating at the point of

EXAMINATION OF ANIMALS. 163

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
similar 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 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 in-
fectious material upon them, must be thoroughly steril-
ized as soon as they are of no further service.

CHAPTER XV.

Scheme for the complete study of an organism.

THE following scheme will serve as a guide for the
systematic study of an organism :

1. Its form and grouping as seen when discovered.

2. Where discovered.

3. The appearance of its colonies on gelatin plates or
Esmarch tubes.

4, The appearance of its colonies on agar-agar plates
or Esmarch tubes.

5. The appearance of its growth in stab and slant
cultures on gelatin.

6. The appearance of its growth in stab and slant
cultures on agar-agar.

7. Its growth on potato.

8. Its growth on blood-serum.

9. Its behavior in bouillon.

10. Its behavior in milk, plain.

11. Its behavior in peptone-rosolic-acid solution.

12. Its behavior in milk containing litmus solution.

13. What is its normal morphology? What mor-
phological changes does it pass through under varying
conditions of life?

14. Does it form spores?

15. Is it motile? Are the flagellaee demonstrable by
Léffler’s method of staining? Is an acid or an alkali
to be added to the mordant; if so, the quantity? In

at

SCHEME FOR STUDY OF AN ORGANISM. 165

what way are the flagelle given off from the body of the
organism ?

16. Does it produce gas-bubbles in ordinary agar-
agar or gelatin ?

17. Does it produce gas-bubbles in agar-agar or gel-
atin to which 1 to 2 per cent. grape sugar has been
added ?

18. Does it produce indol? Is the indol accom-
panied by a coincident production of nitrites ?

19. At what temperature does it grow most luxu-
riantly ?

20. What is the lowest temperature at which it
grows?

21. Is it aérobic, anaérobic, or facultative in its rela-
tions to oxygen?

22. What are its staining peculiarities ?

23. Will it withstand drying?

24, At what temperature is it killed by heat—both
by steam and the hot-air method ?

25. Is it pathogenic when introduced either subcu-
taneously or directly into the circulation of animals?
If so, for which animals? Do any of the animals used
for this work possess natural immunity against infection
by this organism ?

26. What are the histological appearances seen in the
tissues of animals for which this organism is pathogenic ?

g*

PRACTICAL APPLICATION OF THE METHODS
OF BACTERIOLOGY.

CHAPTER XVI.

To obtain material upon 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 can 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 con-
sist in some cases in the presence or absence of color ;
again in 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-

168 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 is upon this observation that the
methods of cultivation on solid media are based.

Tf, without molesting our experiment, we continue the
observation from day to day, we may notice changes in
the colonies due to the growth and multiplication of the
individuals composing them. In some cases the colo-
pies 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. Ina 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 here and there through the colo-
nies. These are due to the escape of gas resulting from
fermentation 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 is growing.

EXPOSURE AND CONTACT, 169

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.

ExposurE AND Contact.—Make a number of plates
from bits of silk used for sutures, after treating them
as follows :

170 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; an-
other piece draw across the table and then plate it. Sus-
pend upon a sterilized wire hook three or four pieces
and let them hang for thirty mintues 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 labled 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 XVII.

Various experiments in sterilization—Steam and hot-air methods
of sterilizing.

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 tempera-
ture of 100° C.

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 destructive properties that steam at the
same temperature possesses. It is 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

172 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 c.c. 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 c.c. of
the filtrate. Allow one tube to remain unmolested in
a warm place. Place another in the steam sterilizer for
five minutes. A third should remain 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. 173

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 to illustrate the
difference in resistance toward steam between the vege-
tative and spore stages of the same organism.

Inoculate about 100 ¢.c. of sterilized bouillon with a
very smal] 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
or the presence of organisms microscopically does not

174 BACTERIOLOGY.

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
upou 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 analogous 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
laid upon the bottom of the sterilizer, but should be sus-

STEAM AND HOT—AIR STERILIZING. 175

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 necessary
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 XVIII.

Bacteriological study of water, air, and soil—Methods of counting
the colonies on the plates—Wolff hiigel’s counting apparatus—Sedg-
wick’s method.

THE possible spread of infectious diseases by means
of water-supplies has formed a topic of discussion by
sanitarians for a long time.

The school of Von Pettenkofer has always taken the
ground that the appearance and spread of epidemic
diseases, of which typhoid fever and Asiatic cholera are
types, is due more to alterations in the soil, resulting
from fluctuations in the level of the soil-water, than
to any part that the drinking-water may play ; in op-
position to this Koch and his pupils hold that these
epidemics can, in most instances, be traced directly to the
influence of the water-supply.

The weight of evidence as it now stands favors the
opinion that these diseases are frequently the result of
imperfections in the supply of water intended for do-
mestic purposes, and there exists sufficient proof of this
to necessitate our controlling all such supplies by careful
quantitative and qualitative bacteriological analyses.

THE QUALITATIVE BACTERIOLOGICAL ANALYSIS OF
Water,—The qualitative bacteriological analysis of
water entails much labor, as it requires that not only
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,

ANALYSIS OF WATER. 177

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 15 to 20 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
should be made as quickly after collecting the sample as
is possible.

Where circymstances 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.

It is therefore advisable that where this work is to be
done, the Esmarch tubes or Petri plates should be pre-
pared on the spot.

For the purpose of qualitative analysis it is necessary
that a small portion of the water—one, 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

178 BACTERIOLOGY.

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 at the body-temperature,
and one set upon gelatin, which are to be kept at a tem-
perature of 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 and number of
organisms on corresponding plates exist, and if so on
which plates the larger number of colonies have de-
veloped. In this way the temperature most favorable
for the growth of most of these organisms may be
determined. The opinion has been advanced that many
of the organisms constantly present in water, which
make up its normal flora, develop better at a lower than
at a higher temperature. This will not be the case,
however, if pathogenic forms are present, because they,
as a rule, require the body-temperature for their most
favorable development, though some of them do grow
very well at a lower temperature.

The isolation of the different species and their sys-
tematic study is to be conducted in the way given for all
bacteria.

THE QUANTITATIVE EsTIMATION OF BACTERIA IN
Water.—The quantitative analysis requires more care in
the measurement of the exact volume of water employed,
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

ANALYSIS OF WATER. 179

made. after the water has been in a vessel for a day or
two are often very different from those which would
have been obtained on the spot.

Where it is not possible, however, to make the analysis
on the spot, the sample of water should be collected and
packed in ice and kept on ice until ready for use, which
should in all cases be as soon after its collection as
possible.

For the collection of water for this purpose, the best
vessel to be employed is a glass bulb (Fig. 26) or
balloon, which one soon learns to make for himself
from glass tubing.

It consists simply of a round glass sphere blown on
the end of a glass tube, which latter is subsequently

Fic. 26.

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
bulb is quickly filled because of the existence of the
negative pressure within it.

‘A number of them may be blown, 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 taken.
It rapidly fills with water. This may serve as a sample.

180 BACTERIOLOGY.

from which to prepare plates or Esmarch tubes on the
spot, or the tip of the stem may be re-sealed in the
flame of an alcohol Jamp, the bulb packed in ice, and
transported in this condition to the laboratory.

In beginning the quantitative analysis of water with
which one is not acquainted, there are certain preliminary
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 done usually by making pre-
liminary plates from one drop, two drops, 0.25 e.c.,
0.5 cc, and 1 ¢.c. 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
oceurred, one selects the plate upon which the colonies
are only moderate in number—about 200 to 300 colonies
presenting—and employs in the subsequent 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, two, or three volumes, as the case
may be, of sterilized distilled water. This dilution must
be accurate, and its exact extent noted, so that subse-
quently 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.

COUNTING COLONIES ON PLATES, 181

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 ce. is
added to a tube of liquefied gelatin, carefully mixed and
poured out asa 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 prevent con-
tamination from without, 99 e.c. of sterilized distilled
water—that is, we have now a dilution of 1: 100.
Again, 0.25 ec. 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 per-
haps not strictly true, we had 180 organisms in 0 25 ¢.¢.
of our 1: 100 dilution, therefore in 0.25 c.c. of the
original water we had 180 x 100 = 18,000 bacteria,
which-will be 72,000 bacteria per cubic centimetre (0.25
= 18,000, 1 cc. = 18,000 x 4 = 72,000). The re-
sults are always to be expressed in terms of the number
of bacteria per cubic centimetre of the original water.

Throughout this part of the work it is to be borne in
mind that when one refers to plates it is not to a set, as
in the isolation experiments, but to a single plate.

METHOD OF CoUNTING THE COLONIES ON THE
Piates.— For convenience in counting colonies on
plates or in tubes, it is of advantage to divide the whole
area of the gelatin occupied by colonies into smaller areas
of exact size For this purpose several very convenient
devices exist.

182 BACTERIOLOGY.

WoLFFHUGEL’s CounTING APPARATUS.—This ap-
paratus (Fig. 27) consists of a flat wooden stand, the

Fic. 27.

RSX
WSS
WN

SEK
SS SS

Se
Se

centre of which is cut out in such a way that either a
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-
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 facilitated by the use of a
small hand-lens.

ESMARCH’S COUNTER. 183

In Fig. 28 is seen the form of hand-lens commonly
employed.

A plan that is frequently given for the counting of
colonies by the use of these devices is to count the num-
ber of colonies in each of a number (eight or ten) of the
squares, and take the average of these counts as the

= =

ll lit"

fe —— M
—— =

average for each square on the whole surface of the
gelatin. The result is then obtained by multiplying
this average by the number of squares taken up by the
whole surface of the gelatin.

The results vary so much in different counts of the
same plate, when made in this way, that they can hardly
be considered approximate.

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.

Esmarcu’s CounTer.—Esmarch has devised a coun-
ter (Fig. 29) for estimating the number of colonies pres-
ent 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 Wolff hiigel’s
apparatus. If the number of colonies in an Esmarch
tube is to be determined, a simpler method to the use of

184 BACTERIOLOGY.

his apparatus may be employed. It consists in dividing
the tube by lines into four or six longitudinal areas which
are subdivided by transverse 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,

or exactly straight. Beginning at one of these squares
at one end of the tube, which 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 rotation is counted, care being taken not
to count a square more than once; they 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-

ESMARCH’S COUNTER. 185

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
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 shaking
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.

In considering water from a bacteriological standpoint,
it must always be borne in mind that comparisons of the
water with any general fixed standard are not of much
value, for just as normal waters from different sources are
seen to present differences in their chemical composition
without being unfit for use, so may the number of
bacteria per volume in water from one source always be
greater or smaller than that from another locality, and yet
no differences can be seen to result from their employ-
ment. For this reason the proper study of any water,
from this point of view, means the establishment of what
may be termed its normal proportion of bacteria, as well
as a study of the organisms most commonly present. For
this purpose experiments covering a long period of time,
made at short intervals, must be conducted, and from
these observations the means for that water at the dif-
ferent seasons of the year calculated. Marked devia-
tions from these means, either in quantity or quality of
the bacteria present, are the only comparisons that are of
any value.

A sudden variation from the normal mean in the
number of bacteria in any water calls at once for a

186 BACTERIOLOGY.

quantitative chemical analysis as well as a thorough
inspection of the supply; at the same time the character
of the organisms should be subjected to most careful
study.

BacTEeRIOLoGicaL 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, Wtirz, 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 pro-
cedure. An objection raised against it is that organisms
may be lost, and not come into the calculation, by pass-
ing 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
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. 30.) When the aspiration is finished the sand
is mixed with liquefied gelatin, plates are made, and

SEDGWICK’S METHOD. 137

the number of developing colonies counted, the results
giving the number of organisms contained in the volume
of air aspirated through the sand.

Fig. 30.

t
NI

NWI
WON

D
y

The tube packed with sand is seen at the point a.

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
granuated 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.

Sepewick’s MretHop.—On the whole, the method
proposed by Sedgwick gives such uniform results that
it is to be recommended above the others. It is as fol-
lows :

The apparatus employed consists essentially of three
parts :

(1) A glass tube of a special form to which the name

aérobioscope has been given.

188 BACTERIOLOGY.

(2) A stout copper cylinder of about sixteen litres
capacity, provided with a vacuum-gauge.

(3) An air-pump.

The aérobioscope (Fig. 31) is about 35 cm. in its en-
tire length ; itis 15 cm. long and 4.5 em. in diameter at
its expanded part; one end of the expanded part is nar-
rowed down to a neck 2.5 cm. in diameter and 2.5 cm.
long. To the other end is fused a glass tube 15 cm.
long and 0.5 cm. inside diameter, in which is to be
placed the filtering material.

Fic. 31.

Upon this narrow tube, 5 cm. 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 (b), 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 cm. (d) of the narrow tube above
the wire gauze. This column of sugar is the filtering
material employed to engage and retain the microérgan-
isms. 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. 189

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 corresponding to the amount of air
required.

A sterilized aérobioscope is now to be fixed in the up-
right position and its small end connected by a rubber
tube with a stop-cock on the cylinder. The cotton plug
is then removed from the upper end of the aérobioscope,
and the desired amount of air is aspirated through the
sugar. The organisms will be held back by the sugar.
During manipulation the cotton plug is to be protected
from contamination with germs from without.

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 in an almost
horizontal position, the sugar, and with it the bacteria,
are brought into the large part (e) of the apparatus.
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
c, the sugar dissolves, and the whole is then rolled on
ice, just as is done in the preparation of an ordinary
Esmarch tube.

The gelatin is most easily poured into the aérobioscope

by the use of a small, sterilized cylindrical funnel (Fig.
g*

190 BACTERIOLOGY.

32), 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 con-
tamination, carry the tubes to the laboratory, where the
cultivation part of the work may be done.

Aside from this advantage, the use of a vacuum-
cylinder permits a known volume of air to be aspirated
with great ease, and its rate of flow through the filter
regulated to a nicety.

The filter being soluble, only the insoluble bacteria
are left imbedded in the gelatin.

For general use this method is to be preferred to the
others that have been mentioned.

BACTERIOLOGICAL STUDY OF SOIL. 191

BacrerioLoeicaL Stupy or THE Sori.—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
for investigation in sterilized water and then making
plates 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 these organisms practised in con-
nection with the ordinary methods.

CHAPTER XIX.

Inoculation experiments with sputum—Sputum septicemia—
Septicemia resulting from the presence of the micrococcus tetra
genus in the tissues—Tuberculosis.

Opsratin 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 Léffler’s alkaline methy-
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 in-
frequently 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 ar-
rangement into groups of fours, the adjacent surfaces
being somewhat flattened. They are not sarcing, as one
can see by the absence of the division in the third direc-
tion—they divide only in two directions.

INOCULATION WITH SPUTUM. 193

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 deli-
cate in structure, and though a frequent accompaniment
is not constant. It can sometimes be demonstrated by
the ordinary methods of staining, though the method of
Gram is most satisfactory.

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
threes, 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.

These delicate beaded rods are the tubercle bacilli.

194 BACTERIOLOGY.

The lancet-shaped diplococci with the capsule are the
diplococei of sputum septicaemia.

The cocci grouped in fours are the micrococcus tetra-
genus.

InocuLtaTion ExprerimMEent.—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 ;
pneumococcus of Frankel; diplococcus lanceolatus—
lancet-formed diplococcus ; meningococcus ; streptococcus
lanceolatus Pasteuri.

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.

ce. General or local tuberculosis.

a. SPUTUM SEPTICEMIA.

If 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,
which is not uncommonly found in the mouth of healthy
individuals as well as in other conditions.

1 Septicemia is that form of infection in which the blood is the
chief field of activity of the organisms.

SPUTUM SEPTICAMIA. 195

Inspection reveals nothing abnormal at the seat of
inoculation, except when death is postponed for a longer
time, when some cedema may be present. At autopsy
the most conspicuous naked-eye change will be en-
largement of the spleen. Frequently there is a limited
fibrinous exudation over portions of the peritoneum.

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 always free and are not found
in the body of leucocytes.

In stained preparations from the blood and exudates
a capsule is not unfrequently seen surrounding the organ-
isms. This, however, is not constant.

If a drop of blood from this animal be introduced
into the tissues of a second animal (mouse, rabbit, or
guinea-pig), identically the same conditions will be re-
produced.

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
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. Frinkel as the
etiological factor in the production of acute fibrinous

pneumonia.
It is not uncommonly present in the saliva of healthy

196 BACTERIOLOGY.

individuals, having been found by Sternberg in the oral
cavity of about 20 per cent. of healthy persons exam-
ined by him. It is constantly to be detected in the
rusty sputum of patients suffering from acute fibrinous
pneumonia. 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 probably
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. The mor-
phology of the individual cells is more or less oval, or
more strictly speaking, lancet-shaped, for at one end
there is commonly a pointed appearance. When joined
in pairs the junction is always between the broad ends
of the ovals, never between the pointed extremities.

In preparations directly from the sputum or from the
blood of animals, a delicate capsule may frequently be
seen surrounding them. This occurs only in prepara-
tions directly from the body and is not seen in pre-
parations from cultures.

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, probably owing in
part to the low temperature at which gelatin cultures
must be kept. If development occurs it appears as
very small whitish or blue-white points on the plates.
These very small colonies are round, finely granular,
sharply circumscribed, and slightly elevated above the

SPUTUM SEPTICAMIA. 197

surface of the gelatin. The growth is very slow and no
liquefaction.of the gelatin occurs.

If grown in slant or stab cultures, the surface devel-
opment is very limited; along the needle track very
small, whitish granules appear. This organism is con-
spicuous for the rapidity with which it loses its patho-
genic properties and the fact that after a very few gen-
erations it can no longer be caused to grow.

On agar-agar the colonies are almost transparent; they
are more or less glistening and very delicate in their
structure.

On blood-serum the growth is more marked, though
still extremely feeble. Here it appears as a very deli-
cate film, consisting of fine points growing closely side
by side.

A growth on potato has not been observed.

The organism is not motile.

It grows best at a temperature between 35° C.—38° C.

Under 24° C. no growth has been observed, and 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.)

b. SEPTICEMIA OF THE MICROCOCCUS TETRAGENUS.

Should the death of the animal not occur within
the first twenty-eight to thirty hours after inoculation,

198 BACTERIOLOGY.

but be postponed until between the fourth and the eighth
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 in-
spection reveals these bodies to be cells in the initial
stage of division into twos and fours. A peculiarity 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-

SEPTICEMIA OF M. TETRAGENUS. 199

tion a circumscribed white point, slightly elevated above
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,

It 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 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.
Tt is slightly elevated above the surface of the medium.

In contradistinction to the gelatin stab-cultures, the
growth is continuous along the track of the needle in
the stab cultures upon agar-agar.

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 results usually in
drawing out fine silky threads consisting of organisms
imbedded in this gelatinous material.

The organism grows best at from 35° C. to 88° C.,
but can be cultivated at the ordinary room temperature
—about 20° C.

The growth under all conditions is not rapid.

200 BACTERIOLOGY.

It grows both in the presence of and without oxygen.

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.

To the naked eye no alteration can be seen in the
organs of animals which have died as a result of inocu-
lation with the micrococcus tetragenus; but micro-
scopic examination of cover-slip preparations from the
blood and viscera reveals the presence of the organisms
throughout the body—especially is this true of prepara-
tions from the spleen." White mice and guinea-pigs are
susceptible to the disease. Gray mice, dogs, and rabbits
are not susceptible to this form of septicemia. Subse-
quent inoculation of healthy animals with a drop of
blood, a bit of tissue, or a portion of a pure culture of
this organism 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 XX.

Tuberculosis—Microscopic appearance of miliary tubercles—En-
capsulation of tuberculous foci—Diffuse caseation—Cavity-formation
—Primary infection—Modes of infection—Location of the bacilli
in the tissues—Staining peculiarities.

SHOULD the animal succumb to neither of the septic
processes just described, then its death from tuberculosis
may be reasonably expected.

When this process 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 be
found at autopsy can a 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
which are characterized by a condition of coagulation,
necrosis, and caseation. This is particularly the case
when the infection is general, i. ¢., when the process is

202 BACTERIOLOGY.

of the acute miliary type. This pathological-anatomical
alteration is best seen in the tissue 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 their nature. 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 role 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 confined to the seat of
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 asa rule are, in this stage, the result of the
fusion between two or more smaller miliary foci. Though
the two terms, “miliary” and “conglomerate,” exist
for the description of the macroscopic appearance of
these nodules, yet it is very rare that any condition

MILIARY TUBERCLES. 203

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 be diagnosticated 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 Mitiary TUBER-
cLES.—The simple miliary tubercles under the low mag-
nifying power of the microscope preseitt somewhat the fol-
lowing appearance: There isa central pale area, evidently
composed of necrotic tissue because of its incapacity for
taking up the staining 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 com-
pletely destroyed by the necrotic process. Around
the periphery of this area may sometimes be noticed
large multi-nucleated cells, the nuclei of which are
arranged about the periphery of the cell or grouped

204 BACTERIOLOGY.

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 quite common. 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 fibrous-looking tissue.

This fibrous-looking 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, and the
fibrous remains of the part ; the irregularly oval, granu-
lar 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 surrounding tissue or
fuses with a similar zone surrounding another tuber-
cular focus. This may be taken as the description of a
typical miliary tubercle.

DirrvusE CasEeation.—The diffuse caseation, as said,
plays a more important role in the tuberculous lesion,
both in the human and. experimental forms, than does
the formation of miliary tubercles. In this a large area
of tissue undergoes the same process of necrosis and
caseation as the centre of the miliary tubercle. In some

DIFFUSE CASEATION,. 205

tissues it is more marked than in others. These tissues
are the lungs and the lymph-glands. In rabbits, par-
ticularly, 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 involving 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 lymphatic glands
may be changed in this way. The conditions for this
caseation of the tissues are probably given when a large
number of tubercle bacilli enter the tissue simultaneously
and a wide area is involved, instead of the small centre
of the miliary tubercle. Necrosis 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 en-
tire caseous area is surrounded by a zone similar to that
around the caseous centre of the miliary tubercles. It
is of special importance to recognize the connection be-
tween this diffuse caseation of the tissue and the tubercle
bacilli, because until its nature was accurately deter-
mined the caseous pneumonia of the lungs formed the
chief obstacle which many had in recognizing the infec-
tiousness of tuberculosis.

Cavity-FoRMATION.—The production of cavities
which forms such a prominent feature in human tuber-
culosis, particularly in the lungs, is due to the softening 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 the tuber-
culous changes still proceed, both as a diffuse caseation
and formation of miliary tubercles. The whole cavity

with the reactive changes in the tissues of its walls may
10

206 BACTERIOLOGY.

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 the lower animals 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 cavities
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 sometimes be
produced by the inoculation of lower animals with the
sputum from such cases.

ENCAPSULATION OF TUBERCULAR Focr.—It not un-
commonly occurs that round about a necrotic tubercular
focus there is formed a fibrous capsule which may com-
pletely cut off the diseased from the healthy tissue sur-
rounding 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 intercurrent interfer-
ence 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 lymphatic circula-
tion 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-
down of old caseous lymphatic glands is a common
example of this condition.

Primary Inrecrion.——The primary infection occurs
through either the vascular or lymphatic circulation:

MODES OF TUBERCULAR INFECTION. 207

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 going to the part; this tends to
occlude 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.

Mopses or Inrecrion.—Experimentally, tuberculosis
may be produced in susceptible animals by subcutaneous
inoculation, by direct injection into the circulation, by
injection into the peritoneal cavity, by feeding of tuber-_
culous material, and by the introduction of the bacilli
into the air-passages.

908 BACTERIOLOGY.

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 fine 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 themselves,
or they may pass on to neighboring glands and eventually
be disseminated throughout the whole lymphatic 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: general tuberculosis
quickly follows, with the most conspicuous production
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 the mechanical pressure of
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 the tubercle bacilli
_ from the intestines into the body. They may be trans-
ported from the intestinal tract into the lymphatics in

LOCATION OF BACILLI IN THE TISSUES. 209

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 of infection.

Location OF THE BAcILLI IN THE TissuEs.—The
bacilli will be found to be most numerous in those tis-
sues 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 stage 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
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

210 BACTERIOLOGY.

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 present usually a slow process, are commonly
caseous, and always possess the property of producing
the disease when introduced into the tissues of suscepti-
ble animals, and yet they are the most difficult of all
tissues in which to demonstrate microscopically 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 accumulated at the pole of the cell oppo-
site 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.

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 dis-
ease when further inoculated. Thus the postulates

CULTIVATION OF TUBERCLE BACILLUS. 211

which are necessary to prove the etiological rdle of the
organism in the production of this malady are all ful-
filled.

Tue TuBercLe BacttLus.—Of the three pathogenic
organisms of which we are speaking, 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 the individual bacilli of this group—some
appearing 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 members of the group. At best, one
never sees with the tubercle bacillus a saprophytic con-
dition 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—cultivation
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 successfully 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.

212 BACTERIOLOGY.

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 sur-
face of the blood-serum. 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 manipu-
lation, 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 best 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. This
precaution 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 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

CULTURES FROM TISSUES. 213

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 haying 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 infu-
sion bouillon to which 6 or 7 per cent. of glycerin has
been added.

In appearance the cultures of the tubercle bacilli are
characteristic—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
clumps of mealy-looking granules. They are never
moist, and frequently have the appearance of coarse
meal which had been spread upon the surface of the
medium. In the lower part of the tube in which they
are growing (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.

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
10*

214 BACTERIOLOGY.

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, ete.

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 developnient 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 appear 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
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
Bacriius.—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-

STAINING PECULIARITIES. 215

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 seg-
ments, again much longer. On an average its length is
seen to vary from 2 to 5 y». 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.

These rods usually present, as has been said, an
appearance of alternate stained and colorless portions.
It 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.

Stainine Prcuiariries.——A peculiarity of this
organism is its behavior toward staining reagents, and
by this means alone it may be easily recognized. The
tubercle bacilli do not stain by the ordinary methods.
They possess some peculiarity in their composition
which renders them more or less proof against the
simpler dyes. It is therefore necessary that more ener-
getic 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. These substances are aniline oil and
carbolic acid. They are both present in the point of
the solutions to about saturation. (For the exact
proportions see chapter on Staining Reagents.)

Under the influence of heat, these solutions are seen

216 BACTERIOLOGY.

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 bacilli will be ob-
served. While all other organisms in the preparation
will give up their color and become invisible, the tubercle
bacilli retain it with marked tenacity. They stain with
great difficulty, but once stained they retain the color
even under the influence of strong decolorizing agents.

The only other organism possessing a similar pecu-
liarity is the bacillus of leprosy, with which, under
ordinary conditions, we are not likely to come in con-
tact. This micro-chemical reaction therefore serves as
a means of differentiating this organism in sputum and
other fluids from the body of suspected subjects from
all other bacteria that are likely to be present.

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
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 a long time after diphtheria the
bacillus of Loffler is known to be demonstrable in the
pharynx. These latter organisms will be described
under their proper heads.

CHAPTER XXI.

Suppuration—The staphylococcus pyogenes aureus.

PREPARE from the pus of an acute abscess or boil,
which has been opened under antiseptic precautions, a
set of plates of agar-agar. Care must be given 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 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
after the manner of clusters of grapes. They stain
readily and are commonly located in the material be-
tween 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 gonorrhoea. In
what way do the two preparations differ the one from
the other ?)

218 - BACTERIOLOGY.

After twenty-four hours in the incubator the plates
-will be seen to be studded here and there with yellow or
orange-colored colonies, which are usually round, moist,
and glistening in their naked-eye appearances. Under
the low-power hand-lens they are frequently irregularly
star-shaped or lobulated in outline and appear very dense
in structure. 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 appear-
ance of masses of coarser clumps of the same material
as that composing the rest of the colony. When deep
down in the culture medium they present ‘but little that
is typical. Microscopically, these colonies are composed
of small round cells, irregularly grouped together. They
are in every way of the same appearance as those seen
upon the cover-slip preparations.

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 then when growing upon agar. It

SUPPURATION. 219

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.

It is not motile, and being of the family of micrococci,
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. g

This organism is the commonest of the pathogenic
bacteria with which we shall meet. It is the staphy-
lococcus 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
render 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

220 BACTERIOLOGY.

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 intestines
—such, for example, as a small bit of potato upon which
the organisms are growing—is at the same time intro-
duced, or the intestines be mechanically injured, so that
there is a disturbance in their circulation, then the in-
troduction of these organisms is promptly followed by
acute and fatal peritonitis.

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 cc. of a bouillon culture or watery suspension
of this organism, a fatal pysemia always follows in from
two and one-half to three days. A few hours before
death the animal is frequently seen to have severe con-
vulsions. 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 vol-
untary muscles are seen to be marked here and there
by yellow spots, which average the size of a flax-seed,
and are of about the same shape. They lie usually with
their long axis running longitudinally between the mus-
cle fibres. As the abdominal and thoracic cavities are
opened the diaphragm is not rarely seen to be studded by
them. Frequently the pericardial sac is distended with
a clear gelatinous fluid, and almost constantly the yel-
low points are to be seen in the myocardium. The kid-
neys 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

STUDY OF COVER-SLIPS AND SECTIONS, 221

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 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.

It is 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 points.

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 Stupy oF CovER-SLIPS AND SECTIONS.
—In cover slip preparations this organism stains readily
with the ordinary dyes.

In tissues, however, it is best to employ 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.

222 BACTERIOLOGY.

These points, when in the kidney, may be round or oval
in outline, or may appear wedge-shaped, with the base
of the wedge toward the surface of the organ. The
differences in shape depend frequently upon the direction
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 here and there 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. 223

At the centre can be seen a dense, granular mass, which
stains readily with the aniline dyes and, when highly
magnified, is found to be made up of staphylococci. Some-
times the shape of this mass of staphylococci 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 de-
stroyed, 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 disintregation 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
same characteristics that were possessed by the culture
with which the animal was inoculated.

224 BACTERIOLOGY.

Tue Less Common PyoGEnic 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.
The streptococcus pyogenes is also sometimes present.
The commonest of the pyogenic organisms however is
that just described—the staphylococcus pyogenes aureus.

Tue Srreprococcus PyogEnes.—F rom a spreading
phlegmonous inflammation prepare cultures. What is
the predominating organism? Does it appear as irregu-
lar clusters of grapes, or has its individuals 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 to present an arrangement some-
what like a chain of beads. Determine its cultural
peculiarities and describe them accurately.

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 cirewmscribed
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. Inoculate a rabbit
both subcutaneously and into the circulation with about
0.2 c.c. of a pure bouillon culture of these organisms.
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?

CHAPTER XXII.
Typhoid fever—Study of the organism concerned in its production.

THE organism, discovered by Eberth and by Gaffky,
which is recognized as the etiological factor in the
production of typhoid fever, may be described as
follows :

In patients suffering from this disease it has been
found during life in the blood, urine, and feces, and at
autopies in the tissues of the spleen, liver, kidneys,
intestinal lymphatic glands, and intestines.

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. Its breadth
remains tolerably constant. Its morphology presents
nothing that will aid in its identification. It stains a
little less readily with the aniline dyes than do most of
the other organisms. It is very actively motile, and
when stained by the special method of Léoffler (see this
method in chapter on Stainings) is seen to possess very
delicate locomotive organs in the form of fine, hair-like
flagellee, which are given off in large numbers from all
parts of its surface. These flagellee are not seen in
unstained preparations, or are they rendered visible by
the ordinary methods of staining.

GeLatin Pxates.—Its growth, when seen in the
depths of the medium, has nothing characteristic, ap-
pearing simply as round or oval, finely granular points.
On the surface it develops as very superficial, blue-white

226 BACTERIOLOGY.

colonies, with irregular borders. They are a little denser
at the centre than at the periphery. When magnified,
the colonies present wrinkles or folds, which give to
them, in miniature, the appearance seen in the relief
maps which represent mountainous districts. These
colonies have sometimes the appearance of flattened
pellicles of glass-wool, and glisten with more or less
of a bronze color.

On AGAR-AGAR the colonies present nothing typical.

Stag Curtures.—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
growth.

Poraro GELATIN.—The growth is similar to that
upon ordinary nutrient gelatin.

MiLx.—It does not cause coagulation when grown in
sterilized milk.

Tt 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 with liberation of gas.

It does not form spores. The irregularities of stain-
ing so commonly seen in this organism have in some

TYPHOID BACILLI IN TISSUES. 227

instances led to the belief that the pale, unstained portions
of the bacilli indicate the presence of spores. More
exact 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 some
po‘nts 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.

PRESENCE IN TissuEs.—It is not easy to demonstrate
this organism in tissues unless it is present in large num-
bers. The manipulations to which the sections are sub-
jected in being mounted rob the bacilli of their staining,
and render them invisible, or nearly so. If, however,
sections be stained in the carbolic-fuchsin solution, either
at the ordinary temperature of the room for from eigh-
teen to twenty hours, or at a higher temperature
(40° to 45° C.) for a shorter time, then washed out in
absolute alcohol, and cleared up in xylol and mounted
in balsam, as a rule, 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 methylene-blue solution gives also very
satisfactory results.

In searching for the typhoid bacilli in tissues, their

228 BACTERIOLOGY.

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 ;
they are localized in small clumps through the tissues,
and it is for thes: clumps, which are easily detected
under the low-power objective, that one should search.
When the section is prepared for examination, if it is
gone over with the low-power objective, one will notice
here and there little masses that look in every respect
like particles of staining-matter which have been pre-
cipitated upon the section at that point. If 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 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 or [NocuLATION 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
appearance of the typical pathological changes have fre-
quently followed these attempts, but in most cases they
could be easily traced to the toxic,’ rather than to the

1 Toxic—poisonous results not necessarily accompanied by the
growth of organisms in the tissues.

TYPHOID INOCULATION IN ANIMALS. 229

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
recently reported by Cygnus. 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 which 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.

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
these organisms, so closely simulate it, that the difficulty

1 Infective or septic—poisoning of the tissues as a result of the

growth of bacteria in them.
11

230 BACTERIOLOGY.

of identifying this organism 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
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 de-
velopment, at another time it may grow in a way easily
to be seen with the naked eye; the change of reaction
which it is said to produce in bouillon is sometimes
much more intense than at others.

The only properties possessed by it that may be said
to be constant are its motility, the absence of indol-
production, and its growth on gelatin plates; but there
are other organisms which possess these same character-
istics in a degree that renders their differentiation from
the typhoid organism a matter of.extreme difficulty, if
not of impossibility.

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, ¢.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
no small task.

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

TYPHOID INOCULATION IN ANIMALS. 231

even here the bacterium coli communis, the normal
organism of the colon, is not unlikely to be found. This
organism, however, always grows visibly on potato, co-
agulates milk, produces a more pronounced pink color in
litmus-milk than does the typhoid bacillus, and is much
less actively motile.

Obtain a pure culture of typhoid bacilli and from
this make inoculations upon a series of potatoes of dift
ferent age and from different sources. Do they all grow
alike ?

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?

CHAPTER XXIII.

Study of the bacillus of anthrax, and the effects produced by its
inoculation into animals — Peculiarities of the organism under
varying conditions of surroundings.

THE discovery that the blood of animals suttering
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 microédrganism, now known as the bacillus of
anthrax, is always present in the blood of animals suf-
fering from this disease; that this organism can be ob-
tained from the tissues of these animals in pure cultures,
and that these artificial cultures of the bacillus of anthrax
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 septicaemia, 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
rods of 20 to 25 in length. In breadth it is from 1

STUDY OF THE BACILLUS OF ANTHRAX. 283

to 1.25 4. Frequently very long threads made up of
several rods, joined end to end, are seen.

As it is obtained from the body of the animal, it is
usually in the form of short rods square at the ends.

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-
tion of the short rods composing it.

In this condition it remains until alterations in its
surroundings, most conspicuously diminution in its nutri-
tive supply, favor the production of spores. When this
stage begins, alterations in the protoplasm of the bacilli
may be noticed; they become marked by irregular, gran-
ular bodies, which eventually coalesce into glistening,
oval spores, one of which lies in nearly every seement
of the long thread, and gives to the thread the appear-
ance of a string of glistening beads. In this stage they
remain but a short time. The chains of spores, which
are held together by the 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 found. In this condition the spores,
capable of resisting deleterious influences, remain and,
unless their surroundings are altered, have been seen to
continue in this living, though inactive, condition for a
very long time. When placed under favorable condi-
tions again, each spore will germinate into a mature cell,
and the same series of changes will be repeated until
the favorable surroundings become again gradually

234 BACTERIOLOGY.

unfavorable to development, when the spore-formation
is again seen. The spore-formation takes place only at
temperatures ranging from 18° to 48° C., 37.50° C,
being the most favorable temperature. Under 12° C.
they are not formed. With this organism, spore-forma-
tion does not occur in the tissues of the living animal,
its usual condition at this time being that of short rods.
Occasionally, 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 hair of a 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 continues
outward upon the surface of the 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 arrangement. These
bundles twist about and cross in all directions, and
eventually disappear at the periphery of the colony.
The colony itself is not circumscribed in its appearance,
but is more or less irregularly fringed and ragged, or
scalloped. To the naked eye they look very much like
minute pellicles of raw cotton which had been pressed
into the surface of the agar-agar. At the extreme peri-
phery of the colonies it is sometimes possible to trace
single bundles of these threads for long distances across
the surface of the agar-agar.

As the colonies continue to grow, they become more
and more dense; they become opaque in color, and gran-
ular and rough on the surface. When touched with a

ANTHRAX STAB AND SLANT CULTURES. 235

sterilized needle, one experiences a sensation that sug-
gests, 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.

Bourtiton.—In bouillon the growth is characterized
by the formation of flaky masses, which also have very
much the appearance of bits of cotton. Microscopic
examination of one of these flakes shows the twisted
and plaited arrangement of the long threads.

Potato.—It develops rapidly as a dry, granular,
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 observed.

Stas AND SLANT CuLTURES.—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 pronounced
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.

236 BACTERIOLOGY.

Under favorable conditions of oxygen, 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 one source would read-
ily be killed by an exposure of one minute to the tem-
perature of steam, whereas those from other sources
resisted this temperature for longer times, reaching in
some cases as long as twelve minutes.

Srarnine.—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,
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.

InNocuLATION 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

ANTHRAX INOCULATION IN ANIMALS. 237

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 oedematous 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, soft and
brittle. 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 the short rods in
large numbers. Nowhere can spore-formation be de-
tected. Upon microscopic examination of sections of the
organs which have been hardened in alcohol, the capilla-
ries 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 distributed through
the spleen. The glomeruli of the kidneys and the capilla-
ries of the lungs are frequently quite packed with them.
The capillaries of the liver contain them in large numbers,
Hemorrhages, probably due to rupture of capillaries by
the mechanical pressure of the bacilli which are develop-
ing in 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.
11*

238 BACTERIOLOGY. =

Cultures from the different organs or from the cedema-
tous fluid about the point of inoculation, result in growth
of the bacillus anthracis.

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 the air-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 a strong gentian-violet solu-
tion, and another cover-slip preparation which will be
drawn through the flame twelve to fifteen times, stained
with aniline gentian-violet, washed off in iodine solu-

EXPERIMENTS. 239

tion, and then in water. Examine these microscopically.
Do they all present the same appearance? To what are
the differences due ?

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 steps.

Prepare a 1: 1000 solution of carbolic acid in bouillon.
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

240 BACTERIOLOGY.

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
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 ; the other allow to grow at the same temperature
for three or four days. Remove the first after the time
mentioned and subject it to a 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 ce. 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 oese of the contents of this
tube. Are the results on the plates alike?

EXPERIMENTS. 241

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.

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 cc. 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.

Are 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

242 BACTERIOLOGY.

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
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 XXIV.

Bacteriology of diphtheria—Behavior of the bacillus diphtheriz
in the tissues of susceptible animals.

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 smear
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 experiment 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 diphtherie grows much more quickly on

244 BACTERIOLOGY.

the serum mixture than do other organisms, makes its
isolation by this method a matter of but little difficulty.

After twenty-four hours in the incubator the tubes
will present a characteristic appearance. Their surfaces
will be marked here and there by more or less irregular
patches of a dense 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 col-
onies may be seen, these large irregular patches are the
most conspicuous objects on the surface of the serum.

-Now and then almost nothing else can be made out on
the tubes.

The cover-slips made from the membrane at the
time the cultures were prepared will be found in
many cases to present a great variety of organisms,
but conspicuous among them will be noticed slightly
curved bacilli of very 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 are rarely or never reg-
ular in outline. If the preparation has been stained
with Loffler’s alkaline methylene-blue solution many
of these irregular rods are seen to be marked by cir-
cumscribed points in their protoplasm which stain
very intensely; they appear almost black. This ir-
regularity in outline is the morphological character-
istic of the bacillus diphtheriz of Loffler. 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 those

B. DIPHTHERIA ON SERUM—MIXTURE. 245

having decayed teeth, the morphology of which is so
like that of the bacillus of diphtheria that they might
easily be mistaken for that organism if subjected to
microscopic examination alone.

The bacillus diphtherize of Loffler (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:

In morphology it varies greatly in size and shape,
averaging 2.5 to 3 # in length and 0.5 to 0.8 v in thick-
ness. Its morphological characters are so peculiar as te
render its detection on cover-slip preparations, and in
sections from diphtheritic membranes, in most cases an
easy matter. Sometimes appearing as a regular straight
or slightly bent rod, with rounded ends, 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.

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-
ritic 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-

246 BACTERIOLOGY.

therize grow so much more rapidly than other organisms
usually present in the secretions and exudations in the
throat that at the end of twenty-four hours they are
often the only colonies that attract attention, and if
others of similar size are present, they are generally of
quite a different aspect Its colonies are large, round,
elevated, grayish-white, with a centre more opaque than
the slightly irregular periphery. The surface of the
colony is at first moist, but after a day or two rather
dry in appearance.

A blood-serum tube studded over with coalescent or
scattered colonies of this organism is so characteristic
in appearance that one can anticipate with tolerable cer-
tainty the results of microscopic examination.

GLYCERIN AGAR-AGAR—Upon nutrient glycerin-
agar-agar the colonies likewise present an appearance
which 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 sur-
rounds it. The periphery of the colony is marked by an
irregularly notched appearance. These colonies are al-
ways quite dry in appearance. When deep down in the
agar-agar they are coarsely granular. They rarely exceed
3 mm. in diameter.

GELATIN.—On gelatin the colonies develop much
more slowly than on the other media which can be
retained at a higher temperature. They rarely present

B. DIPHTHERI® ON POTATO. 247

their characteristic appearances on gelatin in less than
seventy-two hours.

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.

Bourtion.—In bouillon it usually grows in fine
clumps, which fal] 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 diffusely
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 still be
seen, but they are so small as not to have been detected
by the unaided eye.

In bouillon which is kept at a temperature of
35°-37° C. for a long time, a soft, whitish membrane
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 well observed in
cultivations in bouillon to which a little rosolic acid
has been added.

Porato.—On potato at a temperature of 85°-37° C.
its growth after several days is entirely invisible; only

248 BACTERIOLOGY.

a thin, dry glaze can be noticed 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 increase in the number of individual organisms
planted.

Stas AnD Sianr CuLtures.—lIn 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.

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
87° C., but most luxuriantly at the latter temperature.

Its growth in the presence of oxygen is more active
than when the gas is excluded.

Srainrne.—In cover-slip preparations made either
from the fauces of a diphtheritic 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 Léffler’s alkaline methylene-blue solution ; this
brings out the dark points in the protoplasmic body of
the bacilli and aids thus in their identification.

For the purpose of demonstrating the Loffler bacilli
in sections of diphtheritic membrane, both the Gram

INOCULATIONS OF B. DIPHTHERIA. 249

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. The
bacillus of diphtheria remains localized at the point
of inoculation, never spreading 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 into the circulation of poisonous albumins
produced by the bacilli in the course of their develop-
ment.

If a very minute portion of either a solid or fluid
pure culture of this organism is introduced into the
subcutaneous tissues of a guinea-pig or kitten, death of
the animal is seen to ensue in from twenty-four hours
to five days. The usual changes are an extensive local
cedema with more or less hypereemia and ecchymosis at
the site of inoculation; frequently 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 ; sometimes fatty degeneration in the liver, kidney,
and myocardium. ‘The bacilli are always to be found
at the seat of inoculation, most abundant in the grayish-
white fibrino-purulent exudate present at the point of
inoculation, and becoming fewer at a distance from this,
so that the more remote parts of the edematous fluid do
not contain any bacilli. The bacilli are found not only
free, but contained in large number in leucocytes, some

250 BACTERIOLOGY.

of which have fragmented nuclei or have lost their nuclei.
The bacilli within the leucocytes as well as some outside
frequently stain very faintly and irregularly, and may
appear disintegrated and dead.

Tn all cases culture-tubes inoculated with the blood,
spleen, liver, kidneys, supra-renal capsules, distant
lymphatic glands, and serous transudates yield nega-
tive growths. Negative results are also always obtained
when these organs are examined microscopically for the
bacilli.

Microscopic examinations of the tissues about the seat
of inoculation, as well as of the liver, spleen, kidneys,
lymphatic glands, and elsewhere, show a condition of
extensive cell-death which is characterized by an extreme
degree of fragmentation of the nuclei of the cells of these
parts. These peculiar alterations, as Oertel has shown,
in their distribution are characteristic of human diph-
theria, and the demonstration of similar changes in
animals inoculated with this organism is no small
additional proof that diphtheria is caused by it.

An affection may be produced by the inoculation of
certain animals 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 a small
portion of a pure culture of this organism, the death of
the animal will ensue in from two to four days. At
autopsy the wound will be found covered with a gray-
ish, 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,

INOCULATIONS OF B. DIPHTHERIA. 251

grayish-white, loosely attached pseudo-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 cedematous fluid
about the skin-wound, bacilli diphtheria 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-
pretation for this process, and that is the production of
a soluble poison by the bacteria growing at the seat of
inoculation, which gains access to the circulation and
produces the changes that we observe in the tissues of
the internal viscera.

This poison has been isolated from cultures of the
bacillus of diphtheria by Brieger and Frankel. It is
found to belong, not to the crystallizable ptomaines, but
to the toxic albumins. By the introduction of this tox-
albumin, 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 inocu-
lation with the bacilli themselves, except, perhaps, the
production of false membrane.

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 different forms of
sore-throat. Do the peculiarities of any of the organ-
isms suggest those of the bacillus of diphtheria? Wherein
is the difference?

Tn cultures and cover-slips made from both diphtheria

252 BACTERIOLOGY.

and innocent sore-throats are there any organisms which
are almost constantly present? Which are they, and
what are their characteristics ?

In the anginas of scarlet fever which are the pre-
dominating organisms ?

In their cultural and morphological peculiarities, do
these organisms simulate any of the different species
with which you have been working?

CHAPTER XXY.

Experiments illustrating precautions to be taken in the study of
disinfectants and antiseptics—Skin disinfection.

Into each of three tubes containing 10 ¢.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 cc. 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 ?

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 ex-
actly 10 cc. of a 1:500 solution of corrosive subli-
mate. 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 incuba-
tor. Note the results at the end of twenty-four, forty-

eight, and seventy-two hours. How do you explain them ?
12

254. BACTERIOLOGY.

Make identically the same experiment with a spore-
containing culture of anthrax, except that the drop from
the mixture will be transferred to 10 cc. of a mixture
of equal parts of ammonium sulphide and sterilized dis-
tilled water. After remaining in this for about half a
“minute, a drop will be transferred to a tube of lique-
fied ‘agar-agar, poured into Petri dishes, labelled, and
placed in the incubator. Note the results. How are
they explained ?

Prepare a 1:1000 solution of corrosive sublimate.
To each of twelve bouillon tubes containing exactly 10
c.c. add: one drop to the first one, 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 staphylo-
coccus pyogenes aureus. Place them all in the incubator
after carefully labelling them. Note the order in which
growth appears.

Do the same with anthrax spores, with spores of bacillus
subtilis, with the typhoid bacillus, and see how the re-
sults compare. From these experiments what will be
the strength of corrosive sublimate necessary to act as
an antiseptic under these conditions for the organisms
employed?

Make a similar series of experiments, using a five per
cent. solution of carbolic acid.

Determine the antiseptic point in bouillon of the
common disinfectants 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-

EXPERIMENTS. 255

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 solution of am-
monium sulphide of the strength given.

To 10 ce. of a bouillon culture of staphylococcus
pyogenes aureus, or anthrax spores, add 10 c.c. of corro-
sive 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
which this has been determined under these conditions).
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 experiment in
which organisms which have been exposed to sublimate
for a much shorter time than is necessary to destroy

256 BACTERIOLOGY.

them, when transferred directly to a culture medium do
not grow, and yet, when the same organism which has
not been exposed to snblimate 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.

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
the one from the other ?

To what are these differences due ?

What have these experiments taught ?

SKIN-—DISINFECTION,. 257

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 which
has been sterilized in the flame and allowed to cool
down. 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.
Tt should be washed in hot water before using.

INDEX.

BSCESSES, miliary, 221
microscopic appearances of,
222, 223
Aérobic organisms, 28
aAgar-agar, 57, 65
clarification of, 65
filtration of, 66
peculiarities of, 57, 58
Air, bacteriological analy sisof, 186
Petri’s method for, 187
Sedgwick’s method for,
187-191
Anatrobic organisms, 27, 28
methods of cultivation, 116-
119
method of Buchner, 117
Esmarch, 119
Frankel, 118
Hesse, 117
Kitasato and Weil, 119
Koch, 117
Liborius, 119
Aniline dyes as aids in differenti-
ation, 115
Animals, inoculation of, 151-158
intra-venous, 154-158
instruments for, 156-158
subcutaneous, 151

post-mortem examination of, ,
1

159-163
cultures from tissues, 161
disinfection of imple-
ments, 163 |
disposal of remains, 163
external inspection, 159

incision through skin,

159
opening the cavities, 160
position of the animal,
159

|

Anthrax, 232
bacillus of, 232-238
cultivation of, 234-236
experiments with, 238—
242
inoculation with, 236-
238
staining of, 236
Antisepties, 53
experiments upon, 254
Apparatus for air analysis, Petri’s,
187

Sedgwick’s 188
for counting colonies,
march’s, 184
Wolffhigel’s, 182
Autoclay or digestor, 46

Es-

)ACILLI, 29-32
Bacillus subtilis, 178
of tuberculosis, 211
Bacteria, capsule surrounding,
13:

classification of, 29

constant characteristics of,
104

definition of, 22

flagelle upon, 136

sess of, on solid media,

Hoch’s observation, 64—
57
microscopic examination of,
105
morphology of, 29
constancy of, 31
motility of, 36
multiplication of, 32, 33
nutrition of, 25-27

260

Bacteria, reactions produced by,
114

relation to oxygen, 27, 28
to staining reagents, 115
to temperature, 28
role in nature, 23-25
systematic study of, 104-121
scheme for, 164, 165
Bacterium coli commune, 230, 231
Benches, glass, for holding plates,
85

Blood-serum, 69
Lofiler’s mixture, 75, 76
preparation of, 69
preservation of, 73, 74
solidification of, 71-73
sterilization of, 71

Bonnet, 20

Bottles for staining solutions, 128

Bouillon, 57-59
neutralization of, 60, 61

Box, iron, in which to sterilize

plates, 84

Brownian motion, 110

Bulb for water samples, 179

Burner, Koch’s safety, 93, 94
rose or crown, 47

HLOROPHYLL, definition
of, 22
Cohn, 20
Colonies, characteristic appear-
ance of, 168, 169
study of, 99-101
Cooling-stage, 84
Cover-slips, cleaning of, 123
impression, 126
microscopic examination of,
107
preparation of, 123-127
steps in making, 124
Culture-dish for plates, 85
Cultures, gelatin, 113
hanging-drop, 109
potato, 113
in tube, 69
pure, 101
stab and smear, 101-103
method of making, 101,
102

INDEX,

iW rs sees solutions, 139
Decomposition, 22, 23
Diphtheria, 243
bacillus of, 245-252
cultivation of, 245-248
pathogenic properties of,
249-252
preparation of cultures
from, 243-245
staining of, 248
Diplococci, 30
Diplococcus of pneumonia, 194
Disinfectants, experiments upon,
253-257
Disinfection, 49-53
agents employed for, 52, 53
in the laboratory, 52
inorganic salts for, 49-51
methods of, 52

{}RYSIPELAS, 224
Esmarch’s roll tubes, 87
modification of Esmarch’s
method, 88
advantages of this pro-
cess, 89
roll tubes of agar-agar, 89
tube being rolled on ice, 88
Experiments, 167
exposure and contact, 169,
170

pe aérobic and
anaérobic organisms, 28
Fermentation, 22, 28, 116
Filters, preparation of, 64
Flagella, Léffler’s method of
staining, 136
Flasks, etc., cleaning of, 77
Funnel for filling Sedgwick’s.
aérobioscope, 190
for filling test-tubes, etc., 79

(J ELATIN, 57-62

J clarification of, 63
filtration of, 63
peculiarities of, 57, 58

Guarniari’s agar-gelatin, 76

INDEX, 261

L ANGING-DROP _ prepara-
tions, 108
Henle, 17

Hoffmann, 19
Hypodermatic needles, 156
syringes, 158

NCUBATORS, 91-93
Indol, its production by bac-
teria, 120
method for detecting its pres-
ence, 120, 121
Introduction, 13

EEUWENHOER’S discover-
A ies, 13-15
Lens for counting colonies, 183
Levelling tripod, 84
Léffler’s blood-serum mixture,
75, 76

EDIA, 59-76
agar-agar, 65-67
clarification of, 65, 66
filtration of, 66
solution of, 65, 66
blood-serum, 69-74
precautions in obtain-
ing, 69, 70
preservation of, 73, 74
solidifying, 71-73
sterilization of, 71
bouillon, 59
neutralization of, 59-61
gelatin, 62-65
clarification of, 63, 64
filtration of, 63
neutralization of, 62
meat infusion, 76
potatoes, 67-69 ;
for test-tube cultures, 68
special, 74-76
agar-gelatin of Guarni-
ari, 76
blood-serum mixture of
Léfiler, 75
Dunham’s peptone solu-
tion, 75

Media, special: Dunham’s pep-
tone solution, with ro-
solic acid, 75
milk, 74
as a solid medium,

with litmus, 74
Micrococci, 29
Micrococcus of sputum septics-
mia, 194-197
tetragenus, 197
animals susceptible to,
200
description of, 198-200
results of inoculation
with, 197
Microscope, 105-107
adjustment, coarse, 106
fine, 106
condenser, sub-stage, 106
immersion system, oil, 106
steps in using, 107
objective, 105
ocular, 105
reflector, 106
stage, 106
Microtome, 142
Mouse-holder, 152, 153

EEDHAM, 18
Nitrites, 121

‘6 (\ESE,” platinum, 82
Oil-immersion system, steps
in using, 107
Ozanam, 17

ARASITES, 22
Pasteur, 17-19
Pasteur and Chevreul, 19
Peptone solution, Dunham’s, 75
-rosolic-acid solution, 75
Petri’s dish, 87
modification of Koch’s meth-
od, 86
Plates, technique of making, 81
Platinun: needles, loops (“oese”’),
81, 82
Plenciz, 16

262

Pneumococcus of Frankel, 194
Potatoes, preparation of, 67
for test-tube cultures, 68, 69
sterilization of, 69
Practical application of bacterio-
logical methods, 167
materials for starting,
167 :
Pus organisms, 219-224

2euEAtoS, gas-pressure, 97,
98
thermo-, 94-97

ae en definition of,
22

Sarcinize, 30
Schréder and Dusch, 19
Schulz, 19
Schwann, 19
Section-cutting, 141
Septicemia, definition of, 194
sputum, 194
organism concerned in,
1

its different names,
194
pathological alterations
in, 194, 195
Skin - disinfection, experiments
upon, 255-257
Soil, bacteriological study of, 191
Spallanzani, 18
Spirilli, 28-32
Spore-formation, 33-36
study of, 110
method for the, 111-113
Spores, staining of, 134
Moeller’s method, 134
Sputum, tubercular, 192
microscopic examination of,
1

results of inoculation with,
194-216
septicemia of micrococ-
cus tetragenus, 197
sputum septicemia, 194
tuberculosis, 201
Staining in general, 138
methods of, 122-150

INDEX.

Staining solutions employed, 127
bottles for, 128
special solutions, 129-133
acetic acid method, 133
Grams stain, 133
Koch-Ehrlich, 129
Loffler’s blue, 129
Ziehl’s carbol fuchsin,
130
Staphylococci, 29
i ene pyogenes albus,
22

aureus, 217
citreus, 224
Sterilization, 37
apparatus employed in, 43-47
dry heat, 37
its applications, 38
experiments in, 171-174
hot-air method, 174
precautions, 171
steam method, 171-174
temperature in steril-
izer, 171
methods of, 39
intermittent, at high tem-
perature, 40, 41
at low temperature,
41, 42
under pressure, 42
steam, 38
its application, 39
Sterilizer, blood-serum, 72
hot-air, 47
steam, Arnold’s, 45
Koch’s, 43
Streptococci, 29
Streptococcus pyogenes, 224
Suppuration, 217
organisms concerned in acute,

cultural peculiarities of,
217-219

results of inoculation
with, 219-223

EST-TUBE cleaner, 77
Test-tubes, cleaning of, 77
filling for Esmarch tubes, 80
with media, 78, 79

INDEX.

Test-tubes, plugging with cotton,
78

position after filling, 80
sterilization after filling, 79
sterilization of, 78

Tetrads, 30

Tissues, cultures from, 161
hardening of, 141
imbedding, 142
pbaeing, st bacteria in, 140-

steps in the process, 145
special methods of staining,
146
dahlia method, 147
dry method, 150
Erhlich’s method, 150
Gram’s method, 146
Kihne’s method, 148
Weigert’s method, 148
Zieh|-Neelsen, 150
Tripod for levelling plates, 83
Tuberculosis, 201
bacillus of, 211
cultivation from the tis-
sues, 212
cultural peculiarities of,
213
methods of staining, 131
Nuttall’s moditica-
tion, 132
dry method, 150
Erhlich, 150
Ziehl-Neelsen, 150
microscopic appearance
of, 214
staining peculiarities of,
215

- cavity-formation, 206
diffuse caseation, 205
encapsulation of tubercular

foci, 206

268

Tuberculosis, infection, modes of,
207

primary, 206
location of the bacilli in, 209
manifestations of, 201-203
miliary tubercles, 203
susceptibility of animals to,
216
Tyndall, 20
Typhoid fever, 225
bacillus of, 225-227
in tissues, 227, 228
results of inoculation
with, 228-230

ATER, 176
general observations upon
bacteriological analysis of,
185
qualitative bacteriological
analysis of, 176
precautions in obtaining
samples for, 177
preliminary steps in, 177
quantitative _ bacteriological
analysis of, 178
counting the colonies in,
181
apparatus for, 182,
183

dilution of water for,
180, 181
obtaining sample for, 179
preliminary steps in, 180
source of error in, 185
relation to epidemics, 176
typhoid organisms in, 230

[00GL@A, 31
A