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Cornell University Library
Sthaca, New York
THE
CHARLES EDWARD VAN CLEEF
MEMORIAL LIBRARY
FROM
The Department of Anatomy,
Cornell U..Medical. College,
Stimson Hall...
versity Library
The principles of bacteriology:a practic
Cornell University
Library
The original of this book is in
the Cornell University Library.
There are no known copyright restrictions in
the United States on the use of the text.
http :/Awww.archive.org/details/cu31924003692096
THE PRINCIPLES
Or
hACTE flO LOG Xs
A PRACTICAL MANUAL FOR STUDENTS
AND PHYSICIANS.
BY
A. C. ABBOTT, M.D.,
PROFESSOR OF HYGIENE, AND DIRECTOR OF THE LABORATORY OF HYGIENE,
UNIVERSITY OF PENNSYLVANIA.
FOURTH EDITION, ENLARGED AND THOROUGHLY
REVISED.
With 106 Illustrations, of which 19 are colored.
LEA BROTHERS & CO.,
PHILADELPHIA AND NEW YORK.
1897.
va e a AD Qt.
VRAREE! Ax,
A a
A S@ FBS
Entered according to the Act of Congress in the year 1897, by
LEA BROTHERS & CO.,
In the Office of the Librarian of Congress. All rights reserved.
PHILADELPHIA : ,
DORNAN, PRINTER.
£
of
é
*
MED 5
t
4
‘a
ANATOMY.
24, 6F oS
Sti we
pes
“2
)
PREFACE TO THE FOURTH EDITION.
Ir becomes again the pleasant duty of the author
to express his gratification at the favorable recognition
that this book continues to receive, and to acknowledge
his indebtedness to those of his readers who have kindly
criticized its shortcomings and offered suggestions for
its betterment.
In most cases such suggestions have been acted upon;
in certain others they have been of such a nature that
their adoption, while perhaps desirable, would have
increased the size of the book too greatly for its pur-
pose.
In this edition an effort has been made to include the
more important of the newer ideas bearing directly
upon the subjects under treatment, and, when deemed
necessary, opinions expressed in former editions have
been made to conform to later views. In addition to
the topics treated in the last edition there have been
introduced illustrated descriptions of the bacillus of
bubonic plague, of the bacillus of influenza, and of
the micrococcus of gonorrhcea, as well as a number of
new illustrations relating to descriptive passages in the
text.
A. GC. A.
PHILADELPHIA, May, 1897.
PREFACE TO THE SECOND EDITION.
THE cordial reception with which this book has met,
and the demand for a second edition, afford the author
no small degree of gratification. In revising The Prin-
ciples of Bacteriology advantage has been taken of the
valuable suggestions kindly offered by the reviewers of
the first edition, for which the writer here acknowledges
his indebtedness.
The section of the work devoted to descriptive bac-
teriology has been somewhat extended, but uo effort
has been made to cover the entire field, only those spe-
cies being introduced that are comparatively common
or of importance in enabling the student (o acquire a
fundamental working knowledge capable of wider ap-
plication. Wherever practicable, these descriptions have
been supplemented by illustrations, for the majority of
which the author is responsible. The introduction of
colored figures in the text is a new feature in this edi-
tion, and one which should increase its usefulness. A
sketch of the evolution of our knowledge upon immu-
nity and infection has been introduced, and an outline
of apparatus necessary for a beginner’s laboratory has
been appended.
vi PREFACE TO THE SECOND EDITION.
The original purpose of this book has been main-
tained, and it is hoped that the second edition, contain-
ing double the letterpress and treble the number of
illustrations found in its predecessor, will in some cor-
responding measure improve upon the service which
the work has apparently rendered to students and
physicians.
Ay Co As
PHILADELPHIA, July, 1894.
PREFACE TO THE FIRST EDITION.
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 elu-
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 med-
ical practice, and as many of these can devote to it but
a portion of their time, it is desirable that the subject-
matter be presented in as direct a manner as possible.
viii PREFACE TO THE FIRST EDITION.
Presuming the reader to be unfamiliar with the sub-
ject, the author has restricted himself to those funda-
mental features that are essential to its understanding.
The object has been to present the important ideas and
methods as concisely as is compatible with clearness,
and at the same time to accentuate throughout the
underlying principles which govern the work.
With the view of inducing independent thought on
the part of the student, and of diminishing the fre-
quency of that oft-heard query, ‘‘ What shall I do
next?’’ experiments have been suggested wherever it
is possible. These have been arranged to illustrate the
salient points of the work and to attract attention to
the minute details, upon the observation of which so
much in bacteriology depends.
A.C. A.
PHILADELPHIA, December, 1891.
CONTENTS.
INTRODUCTION.
The overthrow of the doctrine of spontaneous generation—‘‘ Omne
vivum ex vivo’’—Earlier bacteriological studies—The birth of mod-
ern bacteriology
CHAPTER TI.
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 .
CHAPTER II.
Morphology of bacteria—Grouping—Mode of multiplication—
Spore-formation—Motility . F
CHAPTER III.
Principles of sterilization by heat—Methods employed—Discon-
tinued sterilization—Sterilization under pressure—Apparatus em-
ployed—Chemical disinfection and sterilization ‘
CHAPTER IV..
Principles involved in the methods of isolation of bacteria in pure
culture by the plate method of Koch—Materials employed
CHAPTER V.
Preparation of nutrient media—Bouillon, gelatin, agar-agar, potato,
blood-serum, ete.
PAGE
13-26
27-35
36-46
47-71
79-108
x CONTENTS.
CHAPTER VI.
Preparation ot the tubes, flasks, ete., in which the media are to
be preserved .
CHAPTER VII.
Technique of making plates—Esmarch tubes, Petri plates, etc.
CHAPTER VIII.
The incubating-oven--Gas-pressure regulator—Thermo-regulator—
Safety burner employed in heating the incubator.
CHAPTER IX.
The study of colonies—Their naked-eye peculiarities and their ap-
pearance under different conditions—Differences in the structure
of colonies of different species of bacteria—Stab-cultures—Slant-
cultures .
CHAPTER X.
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
CHAPTER NI.
Systematic study of an organism—Points to be considered in iden-
tifying an organism as a definite species .
CHAPTER XII.
Inoculation of animals—Subcutaneous inoculation ; intravenous
injection—Inoculation into the great serous cavities, and into the an-
terior chamber of the eye—Observation of animals after inoculation
CHAPTER XIII.
Post-mortem examination of animals—Bacteriological examina-
tion of the tissues—Disposal of tissues and disinfection of instru-
ments after the examination
PAGE
109-112
113-124
125-132
133-138
139-176
177-205
206-227
228-233
CONTENTS.
APPLICATION OF THE METHODS
BACTERIOLOGY. DESCRIPTIONS
OF SOME OF THE MORE IM-
PORTANT SPECIES.
CHAPTER XIV.
To obtain material with which to begin work
CHAPTER XV
Various experiments in sterilization by steam and by hot air
CHAPTER XVI.
Suppuration—Staphyli Us pyog aureus—Staphyl CUS pyo-
genes albus and citreus—Streptococcus pyogenes - Gonococcus—Bacillus
pyocyaneus—Bacillus of Bubonic Plague.
CHAPTER XVII.
Sputum septicemia—Septicemia resulting from the presence of
micrococcus tetragenus in the tissues .
CHAPTER XVIII.
Tuberculosis—Microscopic appearance of miliary tubercles—En-
eapsulation of tuberculous foci—Diffuse caseation—Cavity-forma-
tion—Primary infection—Modes of infection—Location of the bacilli
in the tissues—Staining-peculiarities—Organisms with which bacillus
tuberculosis may be confounded—Points of differentiation—Bacillus
of influenza . ‘ .
CHAPTER XIX.
Glanders—Characteristies of the disease—Histological structure of
the glanders nodule—Susceptibility of different animals to glanders
—tThe bacillus of glanders ; its morphological and cultural peculiari-
ties—Diagnosis of glanders : z
CHAPTER XxX.
Bacillus diphtherix—Its isolation and cultivation—Morphological
and cultural peculiarities—Pathogenic properties—Variations in
virulence .
CHAPTER XXII.
Typhoid fever— Study of the organism concerned in its produc-
tion—Bacterium coli commune—Its resemblance to the bacillus of
typhoid fever—Its morphological, cultural, and pathogenic prop-
erties—Its differentiation from bacillus typhi abdominalis
x1
OF
PAGE
235-238
239-243
244-276
277-288
289-314
315-324
325-341
342-364
xii CONTENTS.
CHAPTER XXII.
The spirillum (comma bacillus) of Asiatic cholera—Its morphologi-
cal and cultural peculiarities—Pathogenic properties—The bacterio-
logical diagnosis of Asiatic cholera 3 é ‘
CHAPTER XXIII.
Organisms of interest, historically and otherwise, that have been
confounded with the spirillum of Asiatic cholera—Their peculiari-
ties and differential features— Vibrio proteus, or bacillus of Finkler
and Prior—Spirilium tyrogenum, or cheese spirillum of Deneke—The
spirillum of Miller— Vibrio Metchnikovi
CHAPTER XXIV.
Study of bacillus anthracis, and the effects produced by its in-
oculation into animals—Peculiarities of the organism under varying
conditions of surroundings .
CHAPTER XXV.
The most important of the organisms found in the soil—The nitri-
fying bacteria—The bacillus of tetanus—The bacillus of malignant
cedema—The bacillus of symptomatic anthrax
CHAPTER XXVI.
Infection and immunity—The types of infection ; intimate nature
of infection—Septicemia, toxemia, variations in infectious pro-
cesses—Immunity, natural and acquired—The hypotheses that have
been advanced in explanation of immunity—Conclusions .
CHAPTER XXVII.
Bacteriological study of water—Methods employed—Precautions
to be observed—Apparatus used, and methods of using them—
Methods of investigating air and soil
CHAPTER XXVIII.
Methods of testing disinfectants and antiseptics—Experiments
illustrating the precautions to be taken—Experiments in skin-dis-
infection
APPENDIX.
Apparatus necessary in a beginner’s bacteriological laboratory .
PAGE
365-393
394-411
412-427
428-452
453-483
484-511
512-525
527-532
BACTERIOLOGY.
INTRODUCTION.
“Omne vivum ex vivo’’—The overthrow ot the doctrine of spontaneous
generation—Earlier bacteriological studies—The birth of modern bacteri-
ology.
THE study of Bacteriology may be said to have had
its beginning with the observations of Antony van
Leeuwenhoek in the year 1675. Though it is during
the past decade and a half that this line of research
has received its greatest impulse, yet, by a review of
the developmental stages through which it has passed
in its life of more than two centuries, we see that it has
a most interesting and instructive history. From the
very outset its history is inseparably connected with
that of medicine, and as it now stands its relations to
hygiene and preventive medicine are of fundamental
importance. It is, indeed, through a more intimate
acquaintance with the biological activities of the uni-
cellular vegetable micro-organisms that modern hygiene
has attained the prominence and importance now justly
accorded to it. Through studies in the domain of bac-
teriology our knowledge of the causation, course, and
prevention of infectious diseases is daily becoming more
accurate, and it is needless to emphasize the relation of
such knowledge to the manifold problems that present
themselves to the student of preventive medicine.
2
14 BACTERIOLOGY.
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 de-
velopment, many observations were made which formed
the foundation-work for much that was to follow.
Before regularly beginning our studies, therefore, it
may be of advantage to acquaint ourselves with the
more prominent of these investigations.
Antony van Leeuwenhoek, the first to describe the
bodies now recognized as bacteria, was born at Delft, in
Holland, in 1632. He was not considered a man of
liberal education, having been during his early years an
apprentice to a linendraper. During his apprenticeship
he learned the art of lens-grinding, in which he became
so proficient that he eventually perfected a simple Jens
by means of which he was enabled to sce objects of
much smaller dimensions than any hitherto scen with
the best compound microscopes in existence at that date.
At the time of his discoveries he was following the
trade of linendraper in Amsterdam.
In 1675 he published the fact that he had succeeded
in perfecting a lens by means of which he could detect
in a drop of rain-water living, motile ‘ animaleules’’
of the most minute dimensions—smaller than anything
that had hitherto been seen. Encouraged by this dis-
covery, 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 diarrheal evacuations, objects that differenti-
ated 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.
INTRODUCTION. 15
In the year 1683 he discovered in the tartar scraped
from between the teeth a form of micro-organism upon
which he laid special stress. This observation he em-
bodied in the form of a contribution which was presented
to the Royal Society of London on September 14, 1683.
This paper is of particular importance, not only because
of the careful, objective nature of the description given
of the bodies seen by him, but also for the illustrations
which accompany it. From a perusal of the text and
an inspection of the plates there remains little room for
doubt that Leeuwenhoek saw with his primitive lens
the bodies now recognized as bacteria.'
Upon seeing these bodies he was apparently very
much impressed, for he writes: ‘‘ With the greatest
astonishment I observed that everywhere throughout
the material which I was examining were distributed
animaleules of the most microscopic dimensions, which
moved themselves about in a remarkably energetic
way.’’
This discovery 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 remarkable for their purely objective nature.
After the presence of these organisms in water, in
the mouth, and in the intestinal evacuations was made
known to the world, it is hardly surprising that they
were immediately seized upon as the explanation of the
1 See Arcana Nature detecta ab ANTONIO VAN LEEUWENHOEK ; Delphis
Batavorum, 1695.
16 BACTERIOLOGY.
origin of many obscure diseases. So universal became
the belief in a causal relation between these ‘‘ animal-
cules’? 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 the
mouth, intestinal evacuations, and water.
Though nothing of value at the time had been done
in the way of classification, and still less in separating
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 pathological
conditions, but some even went so far as to hold that
variations in the appearance of symptoms of disease
were the result of differences in the behavior of the
organisms in the tissues.
Marcus Antonius Plenciz, a physician of Vienna in
1762, declared himself a firm believer in the work of
Leeuwenhoek, and based the doctrine which he taught
upon the discoveries of the Dutch observer and upon
observations of a confirmatory nature which he himself
had made. The doctrine of Plenciz assumed a causal
relation between the micro-organisms discovered and
described by Leeuwenhoek and all infectious diseases.
He claimed that the material of infection could be noth-
ing else than a living substance, and cndeayored on
these grounds to explain the variations in the period of
incubation of the different infectious diseases. He like-
wise believed the living contagium to be capable of
multiplication within the body, and spoke of the possi-
bility of its transmission through the air. He claimed
a special germ for cach disease, holding that just as from
a given cereal only one kind of grain can grow, so by
INTRODUCTION. 17
the special germ for each disease only that disease can
be produced.
He found in all decomposing matters innumerable
minute ‘‘ animalcule,’’ and was so firmly convinced of
their etiological relation to the process that he formu-
lated the law: that decomposition can only take place
when the decomposable material becomes coated with a
layer of the organisms, and can proceed only when they
increase and multiply.
However convincing the arguments of Plenciz may
appear, they seem to have been lost sight of in the
course of subsequent events, and by a few were even
regarded as the productions of an unbalanced mind.
For example, as late as 1820 we find Ozanam express-
ing himself on the subject as follows: ‘‘ Many authors
have written concerning the animal nature of the conta-
gion of infectious diseases; many have indeed assumed
it to be developed from animal substances and that it
is itself animal and possesses the property of life; I
shall not waste time in efforts to refute these absurd
hypotheses.”’
Similar expressions of opinion were heard from many
other medical men of the time, all tending in the same
direction, all doubting the possibility of these micro-
scopic creatures belonging to the world of living things.
It was not until between the fourth and fifth decades
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 scienti-
fically 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
18 BACTERIOLOGY.
all animals dead of splenic fever, and with the progress
of knowledge upon the parasitic nature of certain dis-
eases of plants, the old question of ‘‘ contagium ani-
matum’’ again began to receive attention. It was taken
up by Henle, and it was he who first logically taught
this doctrine of infection.
The main point, however, that had occupied the atten-
tion of scientific men from time to time for a period of
about two hundred years subsequent to Leeuwenhoek’s
discoveries was the origin of these bodies. Do they
generate spontaneously, or are they the descendants of
pre-existing creatures of the same kind? was the all-
important question. Among the participants in this
discussion were many of the most distinguished men of
the day.
In 1749 Needham, who held firmly to the opinion
that the bodies which were attracting such general atten-
tion developed spontaneously, as the result of vegetative
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 covered, were
the result of changes in the barley-grain itself inci-
dental 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 a time in a vessel of boiling water, neither living
organisms could be detected nor would decomposition
INTRODUCTION. 19
appear in the infusions so treated. The objection raised
by Treviranus, viz., that the high temperature to which
the infusions had been subjected had so altered them
and the air about them that the conditions favorable to
spontaneous generation no longer existed, was met by
Spallanzani by gently tapping one of the flasks, that
had been boiled, against some hard object until a minute
crack was produced ; invariably organisms and decom-
position 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.
In 1836 Schulze attracted attention to the subject by
the convincing nature of his investigations. He showed
that if the air which gained access to boiled infusions
was robbed of its living organisms by being caused to
pass through strong acid or alkaline solutions no decom-
position appeared, and living organisms could not be
detected in the infusions. Following quickly upon
this contribution came Schwann, in 1837, and somewhat
later (1854) Schroder and Dusch, with similar results
obtained by somewhat different means. Schwann de-
prived the air which passed to his infusions of its living
particles by conducting it through highly heated tubes;
whereas Schroder and Dusch, by means of cotton-wool
interposed between the boiled infusion and the outside
air, robbed the air passing to the infusions of its organ-
isms by the simple process of filtration. In 1860 Hoff-
mann and in 1861 Chevreul and Pasteur demonstrated
that the precautions taken by the preceding investiga-
tors for rendering the air which entered these flasks free
from bacteria were not necessary; that all that was
necessary to prevent the access of bacteria to the infu-
20 BACTERIOLOGY.
sions 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 centimetres 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 fell into the open end of the tube were arrested hy
the drop of water of condensation which collected at its
lowest angle, and none could enter the flask.
Though from our present-day standpoint the results
of these investigations seem to be of a most convincing
nature, yet there existed at the time many who required
additional proof that ‘‘spontaneous generation ’’ was not
the explanation for the mysterious appearance of these
minute living objects. The majority, if not all, of such
doubts were subsequently dissipated through the well-
known investigations of Tyndall upon the floating mat-
ters of the air. In these studics he demonstrated by
experiments that the presence of living organisms in
decomposing fluids was always to. be explained either
by the pre-existence of similar living forms in the infu-
sion 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
irregularitics were constantly appearing. It was found
that particular substances required to be heated for a
much longer time than was necessary to render other
substances free from living organisms, and even under
the most careful precautions decomposition would occa-
sionally appear.
In 1762 Bonnet, who was deeply interested in this
INTRODUCTION. 21
subject, suggested, in reference to the results obtained
by Needham, the possibility of the existence of ‘‘ germs,
or their eggs,’’? which have the power to resist the tem-
perature to which some of the infusions employed in
Needham’s experiments had been subjected.
More than a hundred years after Bonnet had made
this purely speculative suggestion it became the happy
privilege of Ferdinand Cohn, of Breslau, to demon-
strate 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
or spore stage in the course of their life-cycle, and when
in this stage they are much less susceptible to the dele-
terious action of high temperatures than when they are
growing as normal vegetative forms. With the discov-
ery of these more resistant spores the doctrine of spon-
taneous generation received its death-hblow. It was no
longer difficult to explain the irregularities in the fore-
going experiments, nor was it any longer to be doubted
that putrefaction and fermentation were the result of
bacterial life and not the cause of it, and that these bac-
teria were the offspring from pre-existing similar forms.
In other words, the law of Harvey, Omne vivum ex ovo,
or its modification, Omne vivum ex vivo, was shown to
apply not only to the more highly organized members
of the animal and vegetable kingdoms, but to the most
microscopic, unicellular creatures as well.
The establishment of this point served as an impetus
Ox
22 BACTERIOLOGY.
to further investigations, and as the all-important ques-
tion was that concerning the relation of these micro-
scopic organisms to disease, attention naturally turned
into this channel of study. Even before the hypothesis
of spontaneous generation had received its final refutation
a number of observations of a most important nature had
been made by investigators who had long since ceased to
consider spontaneous generation as a tenable explanation
of the origin of the microscopic living particles.
In the main, these studies had been conducted upon
wounds and the infections to which they are liable; in
fact, the evolution of our knowledge of bacteriology to
the point it now occupies is so intimately associated with
this particular line of investigation that a few historical
facts in connection with it may not be without interest.
The observations of Rindfleisch, in 1866, in which
he describes the presence of small, pin-head points in
the myocardium and general musculature of individuals
that had died as a result of infected wounds, offer,
probably, the first reliable contribution to this subject.
He studied the tissue-changes round about these points
up to the stage of miliary abscess formation. He refers
to the organisms as ‘ vibrios.’? Almost simultaneously
Von Recklinghausen and Waldeyer described similar
changes that they had observed in pyzemia and occa-
sionally secondary to typhoid fever. Von Reckling-
hausen believed the granules seen in the abscess-points
to be micrococci and not tissuc-detritus, and gave as
the reason that they were regular in size and shape, and
gave specific reactions with particular staining-fluids.
Birch-Hirschfeld was able to trace bacteria found in
the blood and organs to the wound as the point of en-
trance, and believed both the local and constitutional
INTRODUCTION. 293,
condition to stand in direct ratio to the number of spher-
ical bacteria present in the wound. He observed also
that as the organisms increased in number they could
often be found within the bodies of pus corpuscles.
His studies of pyzemia led him to the important con-
clusion that in this condition micro-organisms were
always present in the blood.
Of immense importance to the subject were the in-
vestigations of Klebs, made at the Military Hospital
at Carlsruhe in 1870-71. He not only saw, as others
before him had done, that lhacteria were present in dis-
eases following upon the infection of wounds, but de-
scribed the manner in which the organisms had gained
entrance from the point of injury to the internal organs
and blood. His opinion was that the spherical and rod-
shaped bodies that he saw in the secretions of wounds
were closely allied, and gave to them the designation
‘“microsporon septicum.’’? His opinion was that the
organisms gained access to the tissues round about the
point of injury both by the aid of the wandering leuco-
cytes and by being forced through the connective-tissue
lymph-spaces by the mechanical pressure of muscular
contraction.
On erysipelatous inflammations secondary to injury
important investigations were also being made, Wilde,
Orth, Von Recklinghausen, Lukomsky, Billroth, Ehr-
lich, Fehleisen, and others agreeing that in these con-
ditions micro-organisms could always be detected in the
lymph-channels of the subcutaneous tissues; and through
the work of Oertel, Nassiloff, Classen, Letzerich, Klebs,
and Eberth the constant presence of bacteria in the
diphtheritic deposits at times seen on open wounds was
established.
24 BACTERIOLOGY.
Simple and natural as all this may seem to us now,
the stage to which the subject had developed when these
observations were recorded did not admit of their meet-
ing with unconditional acceptance. The only strong
argument in favor of the etiological relation of the
organisms that had been seen, to the diseases with which
they were associated, was the constancy of this associa-
tion. No efforts had been made to isolate them, and
few or none to reproduce the pathological conditions by
inoculation. Moreover, not a small number of inves-
tigators were skeptical as to the importance of these
observations; many claimed that micro-organisms were
normally present in the blood and tissues of the body,
and some even believed that the organisms seen in the
diseased conditions were the result rather than the cause
of the maladies. It is hardly necessary to do more
than say that both of these views were purely specula-
tive, and have never had a single reliable experimental
argument in their favor. Billroth and Tiegel, who held
to the former opinion, did endeavor to prove their posi-
tion through cxperimental means; but the methods em-
ployed by them were of such an untrustworthy nature
that the fallacy of deductions drawn from them was
very quickly demonstrated by subsequent investigators.
Their method for demonstrating the presence of micro-
organisms in normal tissues was to remove bits of organs
from the healthy animal body with heated instruments
and drop them into hot melted paraffin, holding that all
living organisms on the surface of the tissues would be
destroyed by the high temperature, and that if deeom-
position should subsequently occur it would prove that
it was the result of the growth of bacteria in the depths
of the tissue to which the heat had not penetrated.
INTRODUCTION. 25
Decomposition did usually set in, and they accepted
this as proof of the accuracy of their view. Atten-
tion was, however, shortly called to the fact that in
cooling there was contraction of the paraffin, resulting
usually in the production of small rents and cracks in
which dust, and bacteria lodged upon it, could accumulate
and finally gain access to the tissues, with the occurrence
of decomposition as a consequence. Their results were
thus explained after a manner analogous to that em-
ployed by Spallanzani, in 1769, in demonstrating to
Treviranus the fallacy of the opinion held by him and
the accuracy of his own views, viz., that it was always
through the access of organisms from without that de-
composition primarily originates. (See page 19.)
Under the most careful precautions, against which
no objection could be raised, the experiments of Billroth
and Tiegel were repeated by Pasteur, Burdon-Sander-
son, and Klebs, but with failure in each and every
instance to demonstrate the presence of bacteria in the
healthy living tissues.
The fundamental researches of Koch (1881) upon
pathogenic bacteria and their relation to the infectious
diseases of animals differed from those of preceding
investigators in many important respects. The scien-
tific methods of analysis with which each and every
obseure problem was met as it arose served at once to
distinguish the worker as a pioneer in this hitherto but
partly cultivated domain. The outcome of these ex-~
periments was the establishment of a foundation upon
which the bacteriology of the future was to rest. He, for
the first time, demonstrated that distinct varieties of infec-
tion, as evidenced by anatomical changes, are due in many
cases to the activities of specific micro-organisms, and
26 BACTERIOLOGY.
that by proper methods it is possible to isolate these
organisms in pure culture, to cultivate them indefinitely,
to reproduce the conditions by inoculation of these pure
cultures into susceptible animals, and, by continuous
inoculation from an infected to a healthy animal, to
continue the disease at will. By the methods that he
employed he demonstrated a series of separate and dis-
tinct diseases that can be produced in mice and rabbits
by the injection into their tissues of putrid substances.
The disease known as septicemia of mice; also a disease
characterized by progressive abscess-formation ; and
pyemia and septicemia of rabbits, are among the affec-
tions produced by him in this way. It was in the course
of this work that the Abbe system of substage condens-
ing apparatus was first used in bacteriology; that the
aniline dyes suggested by Weigert were brought into
general use; that the isolation and cultivation of bacteria
in pure culture on solid media were shown to be possible;
and that animals were employed as a means of obtain-
ing from mixtures pure cultures of pathogenic bacteria.
With the bounteous harvest of original and important
suggestions that was reaped from Koch’s classical series
of investigations bacteriology reached an epoch in its
development, and at this period modern bacteriology
may justly be said to have had its birth.
Nore.—I have presented only the most prominent
investigations that will serve to indicate the lines along
which the subject has developed. For a more detailed
account of the historical development of the work the
reader is referred to Leeffler’s Vorleswngen tiber die
geschichtliche Entwickelung der Lehre von den Bacterien,
upon which I have drawn largely in preparing the fore-
going sketch.
CHAPTER TI.
Definition of bacteria—Their place in nature—Difference between parasites
and saprophytes—Nutrition of bacteria—Products of bacteria—Their relation
to oxygen—Influence of temperature upon their growth.
By the term bacteria is understood that large group
of minute vegetable organisms the individual members
of which multiply by a process of transverse division.
They are spherical, oval, rod-like, and spiral in shape,
and are commonly devoid of chlorophyll. Owing to
the absence of chlorophyll from their composition, the
bacteria are forced to obtain their nutritive materials
from organic matters as such, and lead, therefore, either
a saprophytic’ or parasitic’ form of existence.
Their life-processes are so rapid, complex, and ener-
getic that they result in the most profound alterations
in the structure and composition of the materials in and
upon which they are developing.
Decomposition, putrefaction, and fermentation result
from the activities of the saprophytic bacteria, while the
changes brought about in the tissues of their host by the
1 Chlorophyll is the green coloring-matter possessed by the higher plants
by means of which they are enabled in the presence of sunlight to decom-
pose carbonic acid (CQ,) and ammonia (NH;) into their elementary constit-
uents.
2 A saprophyte is an organism that obtains its nutrition from dead organic
matter.
3 A parasite lives always at the expense of some other living, organic crea-
ture, known as its host, and in the strictest sense of the word cannot develop
upon dead matter. There is, however, a group of so-called ‘‘ facultative”
saprophytes and parasites which possess the power of accommodating them-
selves to existing surroundings—at one time leading a parasitic, at another
time a saprophytic form of existence.
28 BACTERIOLOGY.
pure parasitic forms find expression in disease-processes
and not infrequently in complete death.
The role played in nature by the saprophytic bacteria is
avery importantone. Through their functional activities
the highly complicated tissues of dead animals and vege-
tables are resolved into the simpler compounds, carbonic
acid, water, and ammonia, in which form they may be
taken up and appropriated as nutrition by the more
highly organized members of the vegetable kingdom.
It is through this ultimate production of carbonic acid,
ammonia, and water by the bacteria, as end-products in
the processes of decomposition and fermentation of the
dead animal and vegetable tissues, that the demands of
vrowing vegetation for these compounds are 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 bacteria, and as the production of these
simpler compounds (CO,, NH;, H,O) by the animal
world is not sufficient to meet the demands of the chlo-
rophyll plants, the importance of the part played by
bacteria in making up this deficit cannot be overesti-
mated. Were it not for the activity of these microscopic
living particles, all life upon the surface of the earth
would cease. Deprive higher vegetation of the carbon
and nitrogen supplied to it as a result of bacterial ac-
tivity, and its development comes rapidly to an end; rob
the animal kingdom of the food-stuffs supplied to it by
the vegetable world, and life is no longer possible.
It is plain, therefore, that the saprophytes, which
represent the large majority of all bacteria, must be
looked upon by us in the light of benefactors, without
which existence would be impossible.
THEIR PLACE IN NATURE. 29
With the parasites, on the other hand, the conditions
are far from analogous. Through thcir activities there
is constantly a loss, rather than a gain, to both the
animal and vegetable kingdoms. Their host must
always be a living body in which exist conditions
favorable to their development, and from which they
appropriate substances that are necessary to the health
and life of the organism to which they have found
access; at the same time they eliminate substances as
products of their nutrition that are directly poisonous
to the tissues in which they are growing.
In their relations to humanity, the positions occupied
by the two biologically different groups, the saprophytes
on the one hand and the parasites on the other, are dia-
metrically opposite:—the saprophytic forms stand in the
relation of benefactors, in resolving dead animal and
vegetable bodies into their component parts, which serve
as food for living vegetation, and, at the same time, they
remove from the surface of the earth the remains of all
dead organic substances; while the parasitic group exists
only at the expense of the more highly organized mem-
bers of both kingdoms. It is to the parasitic group that
the pathogenic’ organisms belong.
In addition to the saprophytes that are concerned in
the changes to which allusion has just been made, there
exist other saprophytic forms whose life-processes result
in specific changes of most interesting and important
natures. Some of these are characterized by their prop-
erty of producing pigments of different color; these are
known as the chromogenic? forms. Just what their
1 Pathogenic organisms are those which possess the property of producing
disease.
2 Chromogenic :—possessing the property of generating color.
30 BACTERIOLOGY.
exact role in nature is it is difficult to say;\but it is prob-
able that, in addition to their most conspicuous function
of color-production, they are also in some way concerned
in the omnipresent process of disintegration which is
constantly going on in all dead organic substances.
Others, the so-called photogenic or phosphorescent
bacteria, possess the property of producing light or of
illuminating the medium on which they grow by a pecu-
liar phosphorescence. These are found in sea-water and
in decomposing phosphorescent fish and meat.
Still others, the so-called zymogenic bacteria, are con-
cerned in the various fermentations; while the putrefac-
tive or saprogenic bacteria are those that produce the
particular fermentation that we know as putrefaction.
Another very important saprophytic group comprises the
so-called nitrifying and denitrifying bacteria, whose activi-
ties are concerned in specific forms of fermentation—the
former oxidizing ammonia to nitrous and nitric acids, the
latter reducing nitric acid to nitrous acid and ammonia.
It is through their association (symbiosis) with the nitri-
fying bacteria that certain plants, the leguminous, are
enabled to make up their nitrogen deficit in part from
the free nitrogen of the air. The discovery of this
phenomenon gave to free atmospheric nitrogen a biolog-
ical significance that had hitherto been denied it. The
so-called thiogenic bacteria convert sulphuretted hydro-
gen into higher sulphur compounds.
We have said that through the agency of chlorophyll,
in the presence of sunlight, the green plants are enabled
to obtain the amount of nitrogen and carbon which is
necessary to their growth from such simple bodies as
carbon dioxide and ammonia, which they decompose
into their elementary constituents. The bacteria, on
NUTRITION OF BACTERIA. 31
the other hand, owing to the absence of chlorophyll
from their tissues, do not possess this power. They
must, therefore, have their carbon and nitrogen pre-
sented as such, in the form of decomposable organic
substances.
In general, the bacteria obtain their nitrogen most
readily from soluble albumins, and, to a certain extent,
but hy no means so easily, from salts of ammonium. In
some of Nigeli’s experiments it appeared probable that
they could obtain the necessary amount of nitrogen
from inorganic nitrates, 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 hy the bac-
teria for building up their tissues in the course of their
development was derived from some source other than
that of the nitric acid or the nitrates, and that the
reduction of this acid was most probably a secondary
phenomenon. It must he borne in mind, however, that
there exists a specific group of bacteria, the nitrifying
bacteria, that apparently increase and multiply without
appropriating proteid nutrition. They are concerned
in the particular form of fermentation that results in
the oxidation of ammonia to nitrous and nitric acids, a
process everywhere in progress in the superficial layers
of the soil.
For the supply of carbon many of the carbon com-
pounds serve as sources upon which the bacteria can
draw. The carbon deficit, for example, can be obtained
from sugar and bodies of like composition; from gly-
cerine and many of the fatty acids; and from the alka-
line salts of tartaric, citric, malic, lactic, and acetic
32 BACTERIOLOGY.
acids. In some instances carbon compounds which,
when present in concentrated form, inhibit the growth
of bacteria, may, when highly diluted, serve as nutri-
tion for them. Salicylic acid and ethyl alcohol are of
this class.
In addition to carbon and nitrogen, water is essential
to the life and development of bacteria. Without it
no development occurs, and in many cases drying the
organisms results in their death. Certain forms, on
the contrary, though incapable of multiplying when in
the dry stage, may be completely deprived of their
water without causing them to lose the power of repro-
duction when favorable conditions reappear.
The closer study of the bacteria, and a more intimate
acquaintance with their nutritive changes, demonstrate
an appreciable variability in the character of the sub-
stances best suited for the nutrition of different species,
one requiring a tolerably concentrated form of nutri-
tion, while another needs but a very limited amount
of proteid substance for its development. Certain
members bring about most profound alterations in the
media in which they exist, while others produce but
little apparent change. In one case alterations in the
reaction of the media will be conspicuous, while in
another no such variation can be detected. With the
growth of some forms products resulting from specific
processes of fermentation appear. Other varieties pro-
duce poisons of remarkable degrees of toxicity, while
the growth of others may be accompanied by the bodies
characteristic of putrefaction.
For the normal development of bacteria it is not only
essential that the sources from which they can obtain
the necessary nutritive elements should exist, but ac-
NUTRITION OF BACTERIA. 33
count must also be taken of the products of growth of
the organisms in these substances. Nitrogen and carbon
compounds in the proper form to be appropriated by
bacteria may exist in sufficient quantities, and still their
growth may be checked after a very short time by the
accumulation of products of nutrition that are inhibitory
to their further development. Most conspicuous are the
changes that growing bacteria produce in the chemical
reaction of the media. Since the majority of them grow
best in media of a neutral or very slightly alkaline reac-
tion, any excessive production of alkalinity or acidity,
as a product of growth, arrests development, and no
evidence of life or further multiplication can be detected
until this deviation from the neutral reaction has been
corrected.
Most favorable for the development of bacteria are
neutral or very slightly alkaline solutions of proteid
materials in one form or another.
Of considerable importance and interest in the study
of the nutritive changes of bacteria is the difference in
their relation to oxygen. With certain forms oxygen
is essential to the proper performance of their func-
tions, while with another group no evidence of life can
be detected under the access of oxygen, and in a third
group oxygen appears to play but an unimportant role,
for development occurs as well with as without it.
It was Pasteur who first demonstrated the existence of
particular species of bacteria which not only grow and
multiply and perform definite physiological functions
without the aid of oxygen, but to the existence of which
oxygen is positively harmful. To these he gave the
name anaérobic bacteria, in contradistinction to the
aérobie group, for the proper performance of whose
34 BACTERIOLOGY.
functions oxygen is essential. In addition to these
there is a third group, for the maintenance of whose
existence the absence or presence of oxygen is appar-
ently of no moment—development progresses as well
with as without it; the members of this group comprise
the class known as facultative in their relation to this
gas. It is to this third group, the facultative, that the
majority of bacteria belong. Though the multiplication
of the facultative varieties is not interfered with by
either the presence or absence of oxygen, yet experi-
ments demonstrate that the products of their growth
are different under the varying conditions of absence or
presence of this gas.
For example: in the case of certain of the chromo-
genic forms the presence or absence of oxygen has a
very decided effect upon the production of the pigments
by which they are characterized.
Norr.—Observe the difference between the intensity
of color produced upon the surface of the medium and
that along the track of the needle in stab-cultures of
the bacillus prodigiosus and of the spirillum rubrum.
With the former the red color is apparently a product
dependent upon the presence of oxygen, while in the
latter the greatest intensity of color uccurs at the point
farthest removed from the action of oxygen.
Another factor which plays a highly important part
in the biological functions of these organisms is the
temperature under which they exist. The extremes of
temperature between which the majority of bacteria are
known to grow range from 5.5° to 43° C. At the
former temperature development is hardly appreciable;
it becomes more and more active until 38° C. is reached,
GROWTH AND DEVELOPMENT OF BACTERIA. 35
when it is at its optimum, and, as a rule, ceases with
43° C.; though species exist that will multiply at as high
a temperature as 70° C. and others at as low as 0° C.
The studies of Globig,' Miquel,’ and Macfadyen and
Bloxall? have demonstrated that there exist in the soil,
in water, in feces, in sewage, in dust, and, in fact, prac-
tically everywhere, bacteria that under artificial culti-
vation show no evidence of life at a temperature lower
than 60° to 65° C., and would even grow at as high
a temperature as 70° to 75° C., degrees of heat suffi-
cient for the coagulation of albumin. Rabinowitsch*
has likewise described a number of species of these
‘¢ thermophilic ’’ bacteria, as they are called, but states
that it was possible in her experiments to obtain evidence
of their growth at a lower temperature (34° to 44° C.),
as well as at the higher temperature mentioned by
preceding investigators. The most favorable tempera-
ture for the development of pathogenic bacteria is that
of the human body, viz., 37.5° C. There are a num-
ber of bacteria commonly present in water, the so-called
normal water bacteria, that grow best at about 20° C.
In general then, from what has been learned, it may
be said that for the growth and development of bacteria
organic matter of a neutral or slightly alkaline reaction,
in the presence of moisture and at a suitable tempera-
ture, is necessary. From this can be formed some idea
of the omnipresence in nature of these minute vegetable
forms. Everywhere that these conditions obtain bac-
teria can be found.
1 Globig: Zeitschrift fiir Hygiene, Bd. iii. S. 294.
2 Miquel: Annales de Micrographié, 1888, pp. 4 to 10.
3 Macfadyen and Bloxall: Journal of Path. and Bact., vol. iii. Part I.
4 Rabinowitsch : Zeitschrift fiir Hygiene u. Infectionskrankheiten, Bd. xx.
Heft 1, S. 154 to 164.
CHAPTER IL,
Morphology? of bacteria—Grouping—Mode of multiplication—Spore-forma-
tion—Motility.
In structure the bacteria are unicellular; they are seen
to occur as spherical, rod- or spiral-shaped bodies. They
always develop from pre-existing cells of the same char-
acter and never appear spontancously.
The classifications of the older authors and of the
botanists are usually upon purely morphological pecu-
liarities, and, because of slight variations that are seen
to occur in the size and shape of one and the same
species, are 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, the members of each group being
determined by their individual outline, viz., that of a
sphere, a rod, or a spiral.
To these three grand divisions are given the names
cocci or micrococci, bacilli, and spirilla.
In the group micrococct belong all spherical forms,
i. e., all those forms the isolated individual members of
which are practically of the same diameter in all direc-
tions. (See Fig. 1, a, 6, ¢, d.)
The bacilli comprise all oval or rod-formed bacteria.
(See Fig. 2.)
To the spirilla belong all organisms that are curved
1 Morphology :—pertaining to shape, outline, structure,
GROUPING
37
when seen in short segments, or when in longer threads
are twisted in the form of a corkscrew.
(See Fig. 3.)
Fie. 1. °
a
a
OO wm wu
oO.
%o @ °
© © o @ 7
op gy, 2 02.
m5 9 go o°
BoP ao e
a, Staphylococci
b. Streptococci.
ec. Diplococci
d, Tetrads. e. Sarcins
Fie, 2,
vas \
ah sa ag oer
Sha ey aS »; pee
B pa Pt \ \ = 5
-- \ SA
uw b
a4 g I
ai a
d é f
a. Bacilliin pairs. b. Single bacilli. e¢ andd. Bacilliin threads
eand/f. Bacilli of variable morphology.
Fic. 3.
4 (
eke fia re We Wy,
aN Fa “ee wn
a b
\(
¢
aandd. Spirilla in short segments and longer threads—the so-called comma
forms and spirals. 0b.
sometimes known as vibrios.
d
b. The forms Known as spirocheta.
e. The thick spirals
3
38 BACTERIOLOGY.
The micrococci are subdivided according to their
grouping, as seen in growing cultures, into staphylococet
—those growing in masses like clusters of grapes (see
Fig. 1, a); streptococcithose growing in chains con-
sisting of a number of individual cells strung together
like beads upon a string (see Fig. 1, 6); dtplococes
—those growing in pairs (Fig. 1, ¢); tetrads—those
developing as fours (Fig. 1, d); and sarcine—those
dividing into fours, eights, etc., as cubes—that is, in
contradistinction to all other forms, the segmentation,
which is rarely complete, takes place regularly in three
directions of space, so that when growing the bundle of
segmenting cells presents somewhat the appearance of a
bale of cotton (Fig. 1, e).
To the bacilli belong all straight, rod-shaped bacteria
—i.e., those in which one diameter is always greater
than the other.
Fie, 4.
0 oF 3 —_ £ \ oo
ie YAFe Po
a No a ae
a b e a
a. Baclllus subtilis with spores. 6. Bacillus anthracis with spores. c. Clos-
tridium form with spores. d. Bacillus of tetanus with end spores.
In this group are found those organisms the life-cycle
of many of which presents deviations from the simple
rod shape. Many of them in the course of develop-
ment increase in length into long threads, along the
course of which traces of segmentation may usually be
found—the anthrax bacillus and bacillus subtilis are
conspicuous examples of this. Again, under certain
conditions, many of them possess the property of form-
GROUPING. 39
ing within the body of the rods oval, glistening spores
(see Fig. 4), and, if the conditions are not altered, the
rods may entirely disappear, and nothing be left in
the culture but these oval spores. In some of them
this phenomenon of spore-formation is accompanied by
an enlargement or swelling of the bacillus at the point
at which the spore is located (see Fig. 4, ¢ and 4d).
Again, many of them, from unfavorable conditions of
nutrition, aération, or temperature, undergo pathological
changes—that is, the individuals themselves experience
degeneration of their protoplasm with coincident dis-
tortion of their outline; they are then usually referred
to as ‘‘ involution forms’’ (see Fig. 5, a and 6). In
Fie. 5.
ere ae
yy) cy —w ¢
ae 1 (e ‘
ta a
ef
a b
uw. Spirillum of Asiatic cholera (comma bacillus). 6. Involution forms of
this organism as seen in old cultures.
all of these conditions, however, so long as death has
not actually occurred, it is possible to cause these forms
to revert to the rod-shaped ones from which they orig-
inated, by the renewal of the conditions favorable to
their normal vegetation.
It must be borne in mind, though, that it is never
possible by any means to bring about changes in these
organisms that will result in the permanent conversion
of the morphology of the members of one group into
that of another—that is, one can never produce bacilli
from micrococci, or vice versa, and any evidence which
40 BACTERIOLOGY.
may be presented to the contrary is based upon untrust-
worthy methods of observation.
Not infrequently bacteria 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 zoogleea of bacteria. (See Fig. 6.)
bacilli.
f
Zooglcea 0:
Very short oval bacilli may sometimes be mistaken
for micrococci, and at times micrococci in the stage of
segmentation into diplococci may be mistaken for short
bacilli; but by careful inspection it will always be
possible to detect a continuous outline along the sides
of the former, and a slight transverse indentation or
partition-formation 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 is especially noticeable if the
illumination from the reflector of the microscope with
which they are examined be reduced to the smallest
possible bundle of light-rays. The spores, moreover,
take up the staining reagents much less readily than do
the micrococci. The most reliable differential points,
however, are: the infallible property possessed by the
spores of developing into bacilli, and that of the spher-
ical organism with which they may have been con-
GERMINATION. 41
founded of always producing other micrococci of the
same round form.
For convenience, a common classification of the bacilli
is that based upon constant characteristics which are seen
to appear in the course of their development under spe-
cial conditions—certain of them possessing the power
of forming spores, while from others this peculiarity
is absent.
We have less knowledge of the life-history of the
spiral forms. Efforts toward their cultivation under
artificial conditions have thus far been successful in
only a comparatively limited number of cases. Mor-
phologically, 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.' In most respects, save form and
the power of producing spores, they are analogous in
their mode of growth to the bacilli.
The micrococci develop by simple fission. When
development is in progress a single cell will be seen to
elongate slightly in one of its diameters. Over the
centre of the long axis thus formed will appear a slight
indentation in the outer envelope of the cell; this inden-
tation will increase in extent until there exist eventually
two individuals which are distinctly spherical, as was
the parent from which they sprang, or they will remain
together for a time as diplococci; the surfaces now in
juxtaposition are flattened against one another, and not
infrequently a fine, pale dividing-line may be seen
between the two cells. (See Fig. 1,¢ and d.) A similar
1 Dividing into two transversely.
42 BACTERIOLOGY.
division in the other direction will now result in the
formation of a group of forms as tetrads.
In the formation of staphylococci such division occurs
irregularly in all directions, resulting in the production
of the clusters in which these organisms are commonly
seen, (See Fig. 1, a.) With the streptococci, however,
the tendency is for the segmentation to continue in one
direction only, resulting in the production of long chains
of 4, 8, and 12 individuals. (See Fig. 1, 6.)
The sarcine divide more or less regularly in three
directions of space; but instead of becoming separated
the one from the other as single cells, the tendency is
for the segmentation to be incomplete, the cells remain-
ing together in masses. The indentations upon these
masses or cubes, which indicate the point of incomplete
fission, give to these bundles of cells the appearance
commonly ascribed to them—that of a bale of cotton
or a packet of rags. (See Fig. 1, e.)
The multiplication of bacilli is in the main similar to
that given for the micrococci. A dividing cell will elon-
gate slightly in the direction of its long axis; an inden-
tation will appear about midway between its poles, and
will become deeper and deeper until eventually two
daughter cells will be formed. This process may occur
in such a way that the two young bacilli will adhere
together by their adjacent ends in much the same way
that sausages are seen to be held together in strings
(Fig. 2, f), or the segmentation may take place more
at right angles to the long axis, so that the proximal
ends of the young cells are flattened, while the distal
extremities may be rounded or slightly pointed (Fig.
2,e). The segmentation of the anthrax bacillus, with
which we are to become acquainted later, results, when
SPORE-FORMATION. 43
completed, in an indentation of the adjacent extrem-
ities of the young segments, so that by the aid of
high magnifying powers these surfaces are seen to be
actually concave. Bacilli never divide longitudinally.
With the spore-forming bacilli, under favorable con-
ditions of nutrition and temperature, the same is seen
to occur during vegetation; but as soon as these condi-
tions become altered by the exhaustion of nutrition,
the presence of detrimental substances, unfavorable
temperatures, etc., there appears the stage in their life-
eycle to which we have referred as ‘‘ spore-formation.’’
This is the process by which the organisms are enalled
to enter a stage in which they resist deleterious influ-
ences to a much higher degree than is possible for them
when in the growing or vegetative condition.
In the spore, resting, or permanent stage, as it is
called, no evidence of life whatever is given by the
spores, though as soon as the conditions which favor
their germination have been renewed, these spores de-
velop again into the same kind of cells as those from
which they originated, and the appearances observed in
the vegetative or growing stage of their history are
repeated.
Multiplication of spores, as such, does not occur; they
possess the power of developing into individual rods of
the same nature as those from which they were formed,
but not of giving 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 by granular, refractive
points of irregular shape and size. These eventually
coalesce, leaving the remainder of the cell clear and
44 BACTERIOLOGY.
transparent. When this coalescence of highly refrac-
tive particles is complete the spore is perfected. In
appearance the spore is oval or round, and very highly
refractive—glistening. It is easily differentiated from
the remainder of the cell, which now consists only
of a cell-membrane and a transparent, clear fluid
which surrounds the spore. Eventually both the cell-
membrane and its fluid contents disappear, leaving the
oval spore free; it then gives the impression of being
surrounded by a dark, sharply defined border. It
evinces no motion other than the mechanical tremor
common to all insoluble microscopic particles suspended
in fluids, and it remains quiescent until there appear con-
ditions favorable to its subsequent development into a
vegetative form similar to that from which it originated.
Occasionally the membrane of the vegetative cell in
which the spore is formed does not disappear from
around it, and the spore may then be seen lying in a
very delicate tubular envelope. Now and then, rem-
nants of the envelope may be noticed adhering to a
spore which has not yet become completely free.
In staining, the spore-containing cells do not take
up the dyes in a homogeneous way. By the ordinary
methods the spores do not stain, so that they appear in
the stained cells as pale, transparent, oval bodies, sur-
rounded by the remainder of the cell, which has taken
up the staining.
A single cell produces but one spore. This may be
located either at an extremity or in the centre of the
cell. (Fig. 4.)
Occasionally spore-formation is accompanied by an
enlargement of the cell at the point at which the pro-
cess is in progress. As a result, the outline of the cell
MOTILITY. 45
loses its regular rod shape and becomes that of a club,
a 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. (See Fig. 4, e and d.)
In addition to the property of spore-formation there
is another striking difference between the rod-shaped
organisms, namely, the property of motility which
many of them are seen to possess. This power of mo-
tion is due to the possession by the motile bacilli of very
Fic. 7.
a. Spiral forms with a flagellum at only one end. b. Bacillus of typhoid
fever with flagella given off from ail sides. c. Large spirals from stagnant
water with wisps of flagella at their ends (spirillum undula).
delicate, hair-like appendages or flagella, by the lashing
motions of which the rods possessing them are propelled
through the fluid. In some cases the flagella are located
at but one end of a bacillus, either singly or in a bunch;
again, they may be seen at both poles, and in some
cases, especially with the bacillus of typhoid fever, they
are given off from the whole surface of the rod. (See
Fig. 7.) Ina few instances similar locomotive organs
have been detected on spherical bacteria—i. e., motile
micrococci have been observed.
For a long time this property of independent motion
that is peculiar to certain species of bacteria was sup-
8%
46 BACTERIOLOGY.
posed to be due to the possession of some such form
of locomotive apparatus, because similar appendages had
been seen in some of the large, motile spirilla found
in stagnant water, and it was not until recently that the
accuracy of this supposition was actually demonstrated.
By a special method of staining Loeffler has been able,
in a number of cases, to render visible these hair-like
appendages. His method consists in the employment
of a mordant, by the aid of which the flagella are caused
to retain the staining, and thus become visible. Leef-
fler’s method of staining will be found in the chapter
devoted to this part of the technique.
CHAPTER II.
Principles of sterilization by heat— Methods employed — Discontinued
sterilization—Sterilization under pressure—Apparatus employed—Chemical
disinfection and sterilization.
Most important for the proper performance of bac-
teriological manipulations are acquaintance with the
principles underlying the methods of sterilization and
disinfection, and familiarity with the approved methods
of applying these principles in practice.
In many laboratories it is customary to employ the
term sterilization for the destruction of bacteria by heat,
and the term disinfection for the accomplishment of the
same end through the use of chemical agents. This
distinction in the use of the terms is not strictly correct,
as we shall endeavor to explain.
The laboratory application of the word sterilization for
the destruction of bacteria by high temperatures prob-
ably arose from the circumstance that culture media,
and certain other articles that it is desirable to ren-
der absolutely free from bacterial life, are not treated
by chemical agents for this purpose, but are exposed to
the influence of heat in various forms of apparatus
known as sterilizers; and the process is, therefore,
known as sterilization. On the other hand, cultures
no longer useful, bits of infected tissue, and apparatus
generally, that it is desirable to render free from danger,
are commonly subjected for a time to the action of chem-
ical compounds possessing germicidal properties—i. e.,
48 BACTERIOLOGY.
to the action of disinfectants; and the process is, there-
fore, known as disinfection, though the same end can
also be reached by the application of heat to these arti-
cles. Strictly speaking, sterilization implies the com-
plete destruction of the vitality of all micro-organisms
that may be present in or upon the substance to be
sterilized, and can be accomplished by the proper appli-
cation of both thermal and chemical agents; while
disinfection, though it may, need not of necessity,
insure the destruction of all living forms that are pres-
ent, but only of those possessing the power of infecting ;
it may or may not, therefore, be complete in the sense
of sterilization. From this we see it is possible to
accomplish both sterilization and disinfection as well
by chemical as by thermal means.
In practice the employment of these means is goy-
erned by circumstances. In the laboratory it is essen-
tial that all culture media with which the work is to be
conducted should be free from living bacteria or their
spores—they must be sterile; and it is equally impor-
tant that their original chemical composition should
remain unchanged. It is evident, therefore, that ster-
ilization of these substances by means of chemicals is
out of the question, for, while the media could be thus
sterilized, it would be necessary, in order to accomplish
this, to add to them substances capable not only of de-
stroying all micro-organisms present, but whose pres-
ence wonld at the same time prevent the growth of
bacteria that are to be subsequently cultivated in these
media—that is to say, after performing their sterilizing
or germicidal function the chemical disinfectants would,
by their further presence, exhibit their antiseptic prop-
erties and thus render the material useless as a culture
STERILIZATION BY HEAT. 49
medium. Exceptions to this are seen, however, in the
case of certain volatile substances possessing disinfect-
ant powers—chloroform and ether, for instance; these
bodies, after performing their germicidal activities,
may be driven off by gentle heat, leaving the media
quite suitable for purposes of cultivation. They are
not, however, in general use in this capacity.
The circumstances under which chemical sterilization
or disinfection is practised in the laboratory are, ordi-
narily, either those in which it is desirable to render
materials free from danger that are not affected by the
chemical action of the agents used, such as glass appa-
ratus, etc., or where destructive changes in the compo-
sition of the substances to be treated, as in the case of
old cultures, infected tissues, etc., are a matter of no
consequence. On the other hand, for the sterilization
of all materials to be used as culture media heat only
is employed.'
The two processes will be explained in this chapter,
beginning with
STERILIZATION BY HEAT.
Sterilization by means of high temperature is accom-
plished in several ways, viz., by subjecting the articles
to be treated to a high temperature in a properly con-
structed oven—this is known as dry sterilization; by
subjecting them to the action of streaming or live steam
at the temperature of 100° C.; and by subjecting them
to the action of steam under pressure, under which
1 Anexception to this is the use of chloroform, a volatile disinfectant, that
may easily be eliminated after having exercised its germicidal properties.
This is, however, not a commonly employed method.
50 BACTERIOLOGY.
circumstance the temperature to which they are ex-
posed becomes more and more elevated as the pressure
increases.
Experiments have taught us that the process of ster-
ilization by dry heat is of limited application because
of its many disadvantages. For successful steriliz-
ation 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 penetration into
materials which are to be sterilized is, moreover, much
less thorough than that of steam. Many substances of
vegetable and animal origin are rendered useless by
subjection to the dry method of sterilization. For these
reasons there are comparatively few materials that can
be sterilized in this way without seriously impairing
their further usefulness.
Successful sterilization by dry heat cannot usually
be accomplished at a temperature lower than 150° C.,
and to this degree of heat the objects should be sub-
jected for not less than one hour. For the sterilization,
therefore, of the organic materials of which the media
employed in bacteriological work are composed, and of
domestic articles, such as cotton, woollen, wooden, and
leather articles, this method is wholly unsuitable. In
bacteriological work its application is limited to the
sterilization of glassware 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.
Sterilization by moist heat—steam—offers conditions
much more favorable. The penetrating power of the
steam is not only more complete, but the tempera-
STERILIZATION BY HEAT. 51
ture at which sterilization is ordinarily accomplished is,
as a rule, not destructive to the objects under treat-
ment, This is conspicuously seen in the work of the
laboratory; the culture media, composed in the main
of decomposable organic materials that would be ren-
dered entirely worthless if exposed to the dry method
of sterilization, sustain no injury whatever when intel-
ligently subjected to an equally effective sterilization
with steam. The same may be said of cotton and
woollen fabrics, bedding, clothing, etc.
Aside from the relations of the two methods to the
materials to be sterilized, their action toward the organ-
isms to be destroyed is quite different. The penetrating
power 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 pur-
poses it is necessary to remember that the two methods
have the following applications :
The dry method, at a temperature of 150°-180° C.
for one hour, is employed for the sterilization of glass-
ware: flasks, test-tubes, culture-dishes, pipettes, plates,
etc.
The sterilization by steam is practised with all culture
media, whether fluid or solid. Bouillon, milk, gelatin,
agar-agar, potato, etc., are under no circumstances to
be subjected to dry heat.
The manner in which heat is employed in processes
of sterilization varies with circumstances. When used
59 BACTERIOLOGY.
in the dry form 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 the use of steam, on the other hand, the
articles to be sterilized are frequently of such a nature
that a prolonged application of heat might materially
injure them. For this and other reasons steam is usually
applied intermittently and for short periods of time.
The principles involved in this method of sterilization
depend upon differences of resistance to heat which
the organisms to be destroyed are known to possess at
different stages of their development. During the life-
cycle of many of the bacilli there is a stage in which
the resistance of the organism to the action of both
chemical and thermal agents is much greater than at
other stages of their development. This increased
power of resistance appears when these organisms are
in the spore or resting stage, to which reference has
already been made. When in the vegetative or grow-
ing stage most bacteria are killed in a short time by a
relatively low temperature, whereas, under conditions
which favor the production of spores, the spores are
seen to be capable of resisting very much higher tem-
peratures for an appreciably longer time; indeed, spores
of certain bacilli have been encountered that retain the
power of germinating after an exposure of from five to
six hours to the temperature of boiling water. Such
powers of resistance have never been observed in the
vegetative stage of development. These differences in
resistance to heat which the spore-forming organisms
possess at their different stages of development are
taken advantage of in the process of sterilization by
STERILIZATION BY HEAT. 53
steam known as the fractional or intermittent method,
and are the essential feature of the principles on which
the method is based.
As the culture media to be sterilized are dependent
for their usefulness upon the presence of more or less
unstable organic compounds, the object aimed at in this
method is to destroy the organisms in the shortest time
and with the least amount of heat. It is accomplished
by subjecting them to the elevated temperature at a time
when the bacteria are in the vegetating or growing stage
—i.e., the stage at which they are most susceptible to
detrimental influences. In order to accomplish this it is
necessary that there should exist conditions of tempera-
ture, nutrition, and moisture which favor the vegetation
of the bacilli and the germination of any spores that
may be present. When, as in freshly prepared nutrient
media, these surroundings are found, the spore-forming
organisms are not only less likely to enter the spore-
stage than when their environments are less favorable
to their vegetation, but spores which may already exist
develop very quickly into mature cells.
It is plain, then, that with the first application of
steam to the substance to be sterilized the mature vege-
tative forms are destroyed, while certain spores that
may be present resist this treatment, providing the
sterilization is not continued 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
20°-30° C., those spores which resisted the action of
the steam will, in the course of this interval, germinate
into the less resistant vegetative cells. A second short
54 BACTERIOLOGY.
exposure to the steam kills these forms in turn, and
by a repetition of this process all bacteria that were
present may be destroyed without the application of
the steam having been of long duration at any time.
It should be remembered that while spores which
may be present are not directly killed by the ex-
posure to heat that they experience in the intermit-
tent method of sterilization, still their power of ger-
mination is somewhat inhibited by this treatment. In
this method, therefore, if the temperature of 100° C.
be employed for too long a time, it is possible so to
retard the germination of the spores as to render it
impossible for them to develop into the vegetating stage
during the interval between the heatings. By exces-
sively long exposures to high temperature, but not long
enough to destroy the spores directly, the object aimed
at in the method may be defeated, and in the end the
substance undergoing sterilization be found still to con-
tain living bacteria. In this process the plan that has
given most satisfactory results is to subject the materials
to be sterilized to the action of steam, under the ordi-
nary conditions of atmospheric pressure, for fifteen min-
utes on each of three successive days, and during the
intervals to maintain them at a temperature of about
25°-30° C. At the end of this time all living organ-
isms which were present will have been destroyed, and,
unless opportunity is given for the access of new organ-
isms from without, the substances thus treated remain
sterile.
As an exception to this, one occasionally encounters
certain species of spore-forming bacteria that are not
readily destroyed by this mode of treatment. They
are, presumably, of the group of so-called “ soil organ-
STERILIZATION BY HEAT. 55
isms,’’ and represent the forms most resistant to the
influence of heat. We are not as yet sufficiently familiar
with all their peculiarities to warrant our speaking with
certainty as to a means of sterilizing media in which
they are present. It does not seem unlikely that they are
of the thermophilic (possibly facultative thermophilic)
variety, and they show little tendency to develop into
the vegetative stage between the heatings, germinating
perhaps so slowly at the temperature under which they
find themselves as not to leave completely the spore
stage before another exposure to the steam, but mani-
festing after a time properties of life in the media that
are thought to be sterile and which have been placed
aside for subsequent use. This is a mere hypothesis,
however, and is as yet entirely wanting in experi-
mental proof.
Fortunately, these undesirable experiences are rare;
but that they do occur, and result in no small degree
of annoyance, is an experience that has probably been
had by most bacteriologists.
It must be borne in mind that this method of steril-
ization is only applicable in those cases which present
conditions favorable to the germination of the spores
into mature vegetative cells. Dry substances, such as
instruments, bandages, apparatus, etc., or organic ma-
terials in which decomposition is far advanced, where
conditions of nutrition favorable to the germination of
spores are not present, do not offer the conditions requi-
site for the successful operation of the principles under-
lying the intermittent method of sterilization.
The process of fractional sterilization at low temper-
atures is based upon exactly the same principle, but
differs from the foregoing in the method by which it is
56 BACTERIOLOGY.
practised in two respects, viz., it requires a greater
number of exposures for its accomplishment, and the
temperature at which it is conducted is not raised above
68°-70° C. It is employed for the sterilization of easily
decomposable materials, which would be rendered use-
less by steam, but which remain intact at the tempera-
ture employed, and for certain albuminous culture
media that it is desirable to retain in a fluid condition
during sterilization, but which would be coagulated if
exposed to high temperatures. 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, the interval of twenty-four hours
between the exposures admitting of the germination
of spores into mature cells. During this interval the
substances under treatment are kept at about 25°-30°
C. The temperature employed in this process suffices
to destroy the vitality of almost all organisms in the
vegetative stage in about one hour. Until recently
blood-serum was always sterilized by the intermittent
method at low temperature.
Sterilization by steam is also practised by what may
be called the direct method. That is to say, both the
mature organisms and the spores which may be present
in the material to be sterilized are destroyed by a single
exposure to the steam. In this method steam at its ordi-
nary temperature and pressure—live steam or streaming
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 an hour; or steam
under pressure, and consequently of a higher tempera-
ture, is now frequently employed. By the latter pro-
cedure a single exposure of fifteen minutes is sufficient
STERILIZATION BY HEAT. 57
for the destruction of practically all bacilli and their
spores, providing the pressure of the steam is not less
than one atmosphere over and above that of normal;
this is approximately equivalent to a temperature of
122° C. to which the organisms are exposed,
The objection that has been urged to both of these
methods, particularly that in which steam under pres-
sure is employed, is that the properties of the media
are altered. Gelatin is said to become cloudy and lose
the property of solidifying ; in bouillon and agar-agar
fine precipitates are thought to occur, and some think
the reaction undergoes a change. In the experience
of those who have used steam under pressure, not ex-
ceeding one or one and one-half atmospheres for ten
to fifteen minutes, these obstacles have rarely been
encountered. There is one point to be borne in mind,
however, in using steam under pressure, viz., it is not
possible to regulate the time of exposure to the same
degree of nicety as where ordinary live steam is used.
The reason for this is that if the apparatus be opened
to remove the objects being sterilized while the steam
within it is under pressure, the escape of steam will
be so rapid that all fluids within the chamber, thus
suddenly relieved of pressure, will begin to boil vio-
lently, and, as a rule, will boil quite out of the tubes,
flasks, etc., containing them. or this reason the
apparatus must be kept closed until cool, or until the
gauge indicates that pressure no longer exists within
the chamber, and even then it should be opened very
cautiously. It is patent from this that the tempera-
ture and time of exposure of articles sterilized by this
process cannot usually be controlled with accuracy.
It requires some time to reach a given pressure after
58 BACTERIOLOGY.
the apparatus is closed, and it also requires time for
cooling after the desired exposure to such pressure be-
fore the apparatus can be opened.
It is manifest that during these three periods, viz.,
(a) reaching the pressure desired, (4) time during which
the pressure is maintained, and (c) time for fall of pres-
sure before the chamber can be opened, it is difficult to
say certainly to what temperature and pressure the arti-
cles in the apparatus have, on the whole, been subjected.
Fia. 8.
Steam sterilizer, pattern of Koch.
Clearly, if the desired pressure and temperature have
been maintained for ten minutes, one cannot say that
this is all the heat to which the articles have been sub-
jected during their stay in the chamber. In this light,
STERILIZATION BY HEAT. 59
while steam under pressure may answer very well for
routine sterilization, still it presents insurmountable
obstacles to its use in more delicate experiments where
time-exposure to definite temperature is of importance.
For sterilization by live steam the apparatus com-
monly employed has, until recently, been the cylin-
drical boiler recommended by Koch. (See Fig. 8.)
Its construction is very simple, essentially that of the
ordinary potato-steamer used in the kitchen. It con-
sists of a copper cylinder, the lower fifth of which is
somewhat larger in circumference than the remaining
four-fifths, and acts as a reservoir for the water from
which the steam is to be generated. Covering this sec-
tion of the cylinder is a wire rack or grating through
which the steam passes, and which serves to support
the articles to be sterilized. Above this, comprising
the remaining four-fifths of the cylinder, is the cham-
ber for the reception of the materials over and through
which the steam is to pass. The cylinder is closed
by a snugly fitting cover through which are usually
two perforations into which a thermometer and a ma-
nometer may be inserted. The whole of the outer
surface of the apparatus is encased in a non-conducting
mantle of asbestos or felt.
The water is heated by a gas-flame placed in an en-
closed chamber, upon which the apparatus rests, which
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
apparatus. The amount of water should never be too
60 BACTERIOLOGY.
small to be indicated by the gauge; otherwise there is
danger of the reservoir becoming dry and the bottom
of the apparatus being destroyed by the direct action of
the flame.
A sterilizer that has come into very general use in
bacteriological laboratories is one originally intended for
use in the kitchen. It is the so-called ‘“‘Arnold Steam
Sterilizer.’ It is very ingenious in its construction as
well as economical in its employment.
Arnold steam sterilizer.
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 ordinary care there is no fear of
STERILIZATION UNDER PRESSURE. 61
the water in the reservoir becoming exhausted and the
consequent destruction of the sterilizer.
Fig. 9 shows a section through this apparatus.
STERILIZATION UNDER PRESSURE.
For sterilization by steam under pressure several spe-
cial forms of apparatus exist. The principles involved
in them all are, however, the same. They provide for
Fic. 10.
Autoclave, pattern of Wiesnegg. A. External appearance. B. Section.
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
4
62 BACTERIOLOGY.
regulated so that any degree of pressure (and coinci-
dently of temperature) that is desirable can be main-
tained within the sterilizing chamber. These sterilizers
Autoclave or digester for sterilizing by steam under pressure.
are known as “‘ digesters’? and as “autoclaves.’’
Their construction can best be understood by reference
to Figs. 10 and 11.
STERILIZATION BY HOT AIR. 63
STERILIZATION BY HOT AIR.
The hot-air sterilizers used in laboratories are simply
double-walled boxes of Russian or Swedish iron (Fig.
12), having a double-walled door, which closes tightly,
and a heavy copper bottom. They are arranged with
ventilating openings for the escape of the contained air
and the entrance of the heated air. The flame, usually
from a rose burner (Fig. 13), is applied directly to the
bottom. The heat circulates from the lower surface
around about the apparatus through the space between
its walls.
Fie. 12.
]
|
fe
The construction of the copper bottom of the appa-
ratus upon which the flame impinges is designed to pre-
vent the direct action of the flame upon the sheet-iron
bottom of the chamber. It consists of several copper
64 BACTERIOLOGY.
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
forms of hot-air sterilizers are so constructed that their
repair is a matter involving some time and expense.
To meet this objection I have had constructed a steril-
izer in all respects similar to the old form except in the
arrangement of this copper bottom. This is made in
such a way that it can be easily removed, so that by
keeping several sets of copper plates on hand a new one
can readily be inserted when the old one is burned out.
In the employment of the hot-air sterilizer care
should always be given to the condition of the copper
bottom; for the direct application of the heat to the
sheet-iron plate upon which the substances to be steril-
ized stand results not only in destruction of the appa-
ratus, but frequently in destruction of the substances
undergoing sterilization.
Since the temperature at which this form of steril-
ization is usually accomplished is high, from 150° to
180° C., it is well to have the apparatus encased in
asbestos boards, to diminish the radiation of heat from
its surfaces. This not only confines the heat to the
apparatus, but guards against the destructive action of
the radiated heat on woodwork, furniture, etc., that
may be in the neighborhood.
CHEMICAL STERILIZATION AND DISINFECTION.
As has already been stated, it is possible by means
of certain chemical substances to destroy all bacteria
and their spores that may be within or upon various
CHEMICAL STERILIZATION, ETC. 65
materials and objects—i. ¢., to sterilize them; and it is
also possible by the same means to rob infected objects
of their dangerous infective properties without at the
same time sterilizing them—i.e., to disinfect them.
This latter process depends upon the fact that the
vitality of many of the less resistant pathogenic organ-
isms is easily destroyed by an exposure to particular
chemical substances that may be without effect upon
the more resistant saprophytes and their spores that are
present.
In general, the use of chemicals for sterilization is
not to be considered in connection with substances that
are to be employed as culture media, and their employ-
ment is restricted in the laboratory to materials that
are of no further value, and to infected articles that are
not injured by the action of the agents used, though for
particular purposes such volatile germicides as chloro-
form and ether may serve as exceptions to this. (See
Preservation of Blood-serum with Chloroform.) In
short, they are mainly of value in rendering infected
waste materials free from danger. Tor the successful
performance of this form of disinfection there is one
fundamental rule always to be borne in mind, viz., it
is absolutely essential to success that the disinfectant
used should come in direct contact with the bacteria to
be destroyed, otherwise there is no disinfection.
For this reason, one should always remember, in
selecting the disinfecting agent, the nature of the mate-
rials containing the bacteria upon which it is to act, for
the majority of disinfectants, and particularly those of
an inorganic nature, vary in the degree of their potency
with the chemical nature of the mass to which they are
applied. Often the materials containing the bacteria
66 BACTERIOLOGY.
to be destroyed are of such a character that they com-
bine with the disinfecting agent to form insoluble pre-
cipitates; these so interfere with the penetration of the
disinfectant that many bacteria may escape its destruc-
tive action entirely and no disinfection be accomplished,
though an agent might have been employed that would,
under other circumstances, have given entirely satisfac-
tory results.
In the destruction of bacteria by means of chemical
substances there occurs, most probably, a definite chem-
ical reaction—that is to say, the characteristics of both
the bacteria and the agent employed in their destruction
are lost in the production of an inert third body, the
result of their combination. It is impossible to say
with absolute certainty, as yet, that this is the case; but
the evidence that is rapidly accruing from the more
recent studies upon disinfectants and their mode of
action points strongly to the accuracy of this belief.
This reaction, in which the typical structures of both
bodies concerned are 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 albumi-
nous bodies there results a third compound, which has
the characteristics neither of mercury nor of albumin,
but partakes of the peculiarities of both; it is a com-
bination of albumin and mercury known by the indefi-
nite term ‘‘albuminate of mercury.’? Some such
reaction as this occurs when the soluble salts of mer-
cury are brought in contact with bacteria. This view
has recently been strengthened by the experiments of
Geppert, in which the reaction was caused to take place
between the spores of the anthrax bacillus and a solu-
CHEMICAL STERILIZATION, ETC. 67
tion of mercuric chloride, the result being the apparent
destruction of the vitality of the spores by the forma-
tion of this third compound. In these experiments it
was shown that though this combination had taken
place, still it did not of necessity imply the complete
death of the spores, for if by proper means the com-
bination of mercury with their protoplasm was broken
up, many of the spores returned from their condition
of apparent death to that of life, with all their previ-
ous disease-producing and cultural peculiarities. Gep-
pert employed a solution of ammonium sulphide for
the purpose of destroying the combination of spore-
protoplasm and mercury; the mercury was precipi-
tated from the protoplasm as an insoluble sulphide,
and the protoplasm of the spores returned to its original
condition. These and other somewhat similar ex-
periments have given an entirely new impulse to the
study of disinfectants, and in the light shed by them
many of our previously formed ideas concerning the
action of disinfecting agents must be modified. The
process is not a catalytic one—7. e., occurring simply as
a result of the presence of the disinfecting body, which
is not itself decomposed during its process of destruction
—but is, as said, a definite chemical reaction occurring
within 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—des-
truction of bacteria—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 inten-
sity of the reaction becomes greater under the influence
of heat, so in the process of disinfection the combination
68 BACTERIOLOGY.
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 possessing dis-
infectant properties owe this power to some such similar
reaction, though, as yet, these substances have not been
so thoroughly studied in this relation.
The reaction between the inorganic salts and albu-
minous bodies is not selective; they combine in most
instances with any or all protoplasmic bodies present.
For this reason the employment of many of the com-
moner disinfectants in general practice is a matter of
doubtful advantage. For example, the disinfection of
excreta, sputum, or blood, containing pathogenic organ-
isms, by means of corrosive sublimate, is a procedure
of questionable success. The amount of sublimate em-
ployed may be entirely used up and rendered inactive
as a disinfectant by the ordinary protoplasmic sub-
stances 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 many
bacteriological laboratories, where there is constantly
more or less of infectious material, it is the custom,
with few exceptions, to keep vessels containing solu-
tions of corrosive sublimate at hand, into which in-
fectious materials may be placed. The value of this
procedure, as we have just learned, may be more or
less questionable, especially in those cases in which the
substance to be disinfected is of a proteid nature and
CHEMICAL STERILIZATION, ETC. 69
where the solution used is not freshly prepared. With
the introduction 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 con-
cerned,
Though the other inorganic salts have not been so
thoroughly studied in this connection, it is nevertheless
probable that the same precautions should be taken in
their employment as we now know to be necessary in
the use of the salts of mercury.
Where it is desirable to use chemical disinfectants
in the laboratory much more satisfactory results can
usually be obtained through the employment of carbolic
acid in solution, A three or four per cent. solution of
commercial carbolic acid in water requires a somewhat
longer time for disinfection; but it is, at the same time,
open to fewer objections than are solutions of the inor-
ganic salts, though here, too, we find a somewhat anal-
ogous reaction between the carbolic acid and proteid
matters. Under ordinary circumstances its action is
complete in from twenty minutes to one-half hour. It
is not reliable for the disinfection of resistant spores,
such, for instance, as those of bacillus anthracis.
In the laboratory heat is the surest agent to employ.
All tissues containing infectious organisms should be
burned, and all cloths, test-tubes, flasks, and dishes
should be boiled in 2 per cent. soda (ordinary washing-
soda) solution for fifteen to twenty minutes, or placed
in the steam sterilizer for half an hour.
Intestinal evacuations may best be disinfected with
4*
70 BACTERIOLOGY.
boiling water or with milk of lime, a mixture composed
of lime in solution and in suspension—ordinary fluid
‘‘ white-wash.’’? This should be thoroughly mixed
with the evacuations until the mass reacts distinctly
alkaline, and should remain in contact with the infective
substance for one or two hours. If boiling water be
used, the amount should be about double the volume of
the mass to be disinfected. They should be thoroughly
mixed and allowed to stand, covered, until cold.
Sputum in which tubercle bacilli are present, as well
as the vessel containing it, must be boiled in 2 per cent.
soda solution for fifteen minutes, or steamed in the ster-
ilizer for at least half an hour.
On the whole, in the laboratory we should as yet
rely more upon the destructive properties of heat than
upon those of chemical agents.
From what has been said, the absurdity of sprink-
ling 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 allowing now and then a few cubic centimetres of
some so-called disinfectant to trickle through the pipes
is ridiculous. A disinfectant must be applied to the bac-
teria, and must be in contact with them for a long enough
lime to insure the destruction of their life.
In the light of the latest experiments upon disin-
fectants, the place formerly occupied by many agents
in the list of substances employed for the purpose will
most likely be changed as they are studied more closely.
The agents, then, which will prove of most value in
the laboratory for the purpose of rendering infectious
materials harmless are: heat, either by burning, by
CHEMICAL STERILIZATION, ETC. 71
steaming for from half an hour to an hour, or by boil-
ing in a 2 per cent. sodium carbonate solution for fifteen
minutes; 3 to 4 per cent. solution of commercial car-
bolic acid; milk of lime, and a solution of chlorinated lime
(‘‘ chloride of lime’’) containing not less than 0.25 per
cent. of free chlorine. The chloride of lime from
which such a solution is to be made should be fresh
and of good quality. Good chlorinated lime, as pur-
chased in the shops, should contain not less than 25 to
30 per cent. of available chlorine. The materials to be
disinfected in either of the lime solutions should remain
in them for about two hours. The solutions should be
freshly prepared when needed, as they rapidly decom-
pose 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. A germicide is a body possessing the property
of killing bacteria.
CHAPTER IV.
Principles involved in the methods of isolation of bacteria in pure culture
py the plate method of Koch—Materials employed.
As was stated in the introductory chapter, the isola-
tion in pure cultures of the different species that may
be present in mixtures of bacteria was rendered possi-
ble only through the methods suggested by Koch. Since
the adoption of these methods they have undergone
many modifications, but the principle originally involved
has remained unaltered. The observation which led to
their development was a very simple one, and one that
is commonly before us. Koch noticed that on solid
substances, such, for example, as a slice of potato or of
bread, which had been exposed for a time to the air and
which afforded proper nourishment for the lower organ-
isms, there developed after a short time small patches
of material which proved to be colonies of bacteria.
Each of these colonies on closer examination showed
itself to be, as a rule, composed of but a single species.
There was little tendency on the part of these colonies
to become confluent, and from the differences in their
naked-eye appearances it was easy to see that they were
mostly the outgrowth of different species of bacteria.
The question that then presented itself was: If from
a mixture of organisms floating in the air it is possible
in this way to obtain in pure cultures the component
individuals, what means can be employed to obtain the
same results at will from mixtures of different species of
METHODS OF ISOLATION. 73
bacteria when found together under other conditions?
It was plain that the organisms were to be distinguished
primarily, the one from the other, only by the structure
and general appearance of the colonies growing from
them, for by their morphology alone this is impossible.
What means could be devised, then, for separating the
individual members of a mixture in such a way that
they would remain in a fixed position, and be so widely
separated, the one from the other, as not to interfere
with the production of colonies of characteristic appear-
ance, which would, under the proper conditions, develop
from each individual cell ?
If one take in the hand a mixture of barley, rye,
corn, oats, etc., and attempt to separate the mass into
its constituents by picking out the different grains,much
difficulty is experienced ; but if the handful of grain be
thrown upon a large flat surface, as upon a table, the
grains become more widely separated and the task is
considerably simplified; or, if sown upon proper soil,
the various grains will develop into growths of entirely
different external appearance by which they can readily
be recognized as unlike in nature. Similarly, if a test-
tube of decomposed bouillon be poured out upon a large
flat surface, the individual bacteria in the mass are very
much more widely separated the one from the other
than they were when the bouillon was in the tube; but
they are in a fluid medium, and there is no possibility
of their either remaining separated or of their forming
colonies under these conditions, so that it is impossible
by this means to pick out the individuals from the
mixture.
If, however, it is possible to discover some substance
which possesses the property of being at one time fluid
74 BACTERIOLOGY.
and at another time solid, and 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 those seen on the bit
of potato.
Fig, 14,
Plate showing certain macroscopic characteristics of colonies. Natural size.
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 tem-
perature it solidifies. When once solid it may be kept
METHODS OF ISOLATION. 15
at a temperature favorable to the growth of the bacteria
and will remain in 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 struc-
ture, so that they could easily be distinguished the one
from the other by their naked-eye appearances. (See
Fig. 14.) 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 im-
possible to pick them out because of their proximity the
one to the other, but also because this packing together
materially interfered with the production of those char-
acters by means of which differences can be seen with
the naked eye. The numbers of organisms were then
diminished by a process of dilution, consisting of trans-
ferring 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 gelatin-bouillon tube, and so on.
These were then poured upon large surfaces and allowed
to solidify. The results were entirely satisfactory. On
the gelatin plates from the original tube, as was ex-
pected, 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 charac-
76 BACTERIOLOGY.
teristic growth; but on the second dilution they were,
as a rule, fewer in number and widely separated, so that
the individuals of each species were in no way pre-
vented by the proximity of their neighbors from grow-
ing each in its own typical way. (Fig. 15.) There
was then no difficulty in picking out the colonies result-
ing from the growth of the different individual bacteria.
Fie. 15.
A B Oo
Series of plates showing the results of dilution upon the number of colonies:
A, Plate No. 1, or “original.” 2B. First dilution, or Plate No.2. C. Second
dilution, or Plate No. 8. About one-fourth natural size.
This, then, is the principle underlying Koch’s method
for the isolation of bacteria by means of solid media.
The fundamental part of the media employed is the
bouillon, which contains all the elements necessary for
the nutrition of most bacteria, the gelatin being em-
ployed simply for the purpose of rendering the bouillon
solid. The medium on which the organisms are grow-
ing 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
METHODS OF ISOLATION. 77
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 under the
influence of heat and of bacterial growth renders them
of different application in bacteriological work. The
animal gelatin liquefies at a much lower temperature,
and also requires a lower temperature for its solidifica-
tion, than does the agar-agar. Ordinary gelatin, in the
proportion commonly used in this work, liquefies at
about 24°-26° C., and becomes solid at from 8°-10° C,
It may be employed for those organisms which do not
require a higher temperature for their development than
22°- 24° C. Agar-agar, on the other hand, does not
liquefy until the temperature has reached about 98°-99°
C. Itremains fluid ordinarily until the temperature has
fallen to 88°-39° C., when it rapidly solidifies. For
our purposes, only that form of agar-agar can be used
which remains fluid at from 38°-40° C. Agar-agar
which remains fluid only at a temperature above this
point would be too hot, when in a fluid state, for use;
many of the organisms introduced into it would either
be destroyed or checked in their development by so high
a temperature. Agar-agar is for use in those cases in
which the cultivation must be conducted at a temperature
above the melting-point of gelatin.
In addition to their differences when under the in-
fluence of various temperatures, the relations of these
two gelatins to bacteria are quite distinct. Many bac-
teria bring about alterations in gelatin which cause it to
become liquid (a process analogous to peptonization), in
which state it remains. There are no known organisms
that bring about such a change in agar-agar.
78 BACTERIOLOGY.
As a rule, the colony-formations seen upon gelatin
are much more characteristic than those which develop
on agar-agar, and for this reason gelatin is to be pre-
ferred when circumstances will permit. Both gelatin
and agar-agar may be used in the preparation of plates
and Esmarch tubes, subsequently to be described.
CHAPTER V.
Preparation of media—Bouillon, gelatin, agar-agar, potato, blood-serum, etc.
As has been stated, the fundamental constituent of
our culture media is beef tea, or bouillon.
BourLton.—The directions of Koch for the prepara-
tion of this medium have undergone many modifications
to meet special cases, but for general use his original
formula is still retained. It is as follows: five hun-
dred grammes of finely chopped lean beef, free from
fat and tendons, are to be soaked in one litre of water
for twenty-four hours. During this time the mixture
is to remain in the ice-chest or to be otherwise 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 are to be added ten grammes (1.0 per cent.) of
dried peptone and five grammes (0.5 per cent.) of com-
mon salt (NaCl). It is then to be rendered exactly
neutral or very slightly alkaline with a few drops of
saturated sodium carbonate solution. The flask con-
taining the mixture is then to be placed either in the
steam sterilizer or in a water-bath, or over a free flame,
and kept at the boiling-point until all the albumin is
coagulated and the fluid portion is of a clear, pale
straw color. It is then filtered through a folded paper
filter, and sterilized in the steam sterilizer by the frac-
tional method. Certain of the modifications of this
method are of sufficient value to justify mention. Most
80 BACTERIOLOGY.
important is the neutralization. Ordinarily, this is ac-
complished with the saturated sodium carbonate solu-
tion, and the reaction is determined with red and blue
litmus papers, and for the beginner this method serves
most purposes.
The sodium carbonate solution is not so good, how-
ever, as a strong solution of caustic soda or potash,
because the carbonic acid liberated from the sodium
carbonate is frequently seen to give rise to confusing,
temporary acid reaction which disappears on heating,
nor is litmus the most reliable indicator to employ.
To obviate this, Schultz (Centralb. f. Bakt. u. Parasi-
enkunde, 1891, Bd. x., Nos. 2 and 3) recommends exact
titration with a solution of caustic soda. For this pur-
pose 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 ex-
actly measured.
From it a sample of exactly 5 or 10 ¢.c.is then taken,
and to this a few drops of one of the indicators com-
monly employed in analytical work are added. Schultz
recommends 1 drop of phenolphtalein solution (1
gramme phenolphtalein in 300 ¢.c. of alcohol) to 1
c.c. 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 caustic soda solution required
BOUILLON. 81
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 alkali necessary to neutralize
the quantity of bouillon employed. If 10 c.c. 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 ¢.c. 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,
196 c.c. (200 c.c.—4 c.c., the amount employed for the
two samples of 10 ¢.c. each of bouillon) are needed of
the 0.4 per cent. solution, or one-tenth of this amount
of the 4 per cent. caustic soda solution.
For the neutralization of the whole bulk of the
bouillon it is better to employ the stronger 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 exactness in
the measurement of the volumes and in the preparation
of the dilutions is required. To obviate error, it is
better to employ this method when the solutions are all
cool and of nearly the same temperature, so that rapid
fluctuations in temperature, and consequent alterations
in volume, will not materially interfere with the accu-
racy of the results.
This method of neutralization, as suggested by
Schultz, is to be adopted for those experiments in which
it is desirable to have the reaction of the medium accur-
ate and constantly of the same degree.
For the ordinary purposes of the beginner, however,
82 BACTERIOLOGY.
results quite satisfactory in their nature may be obtained
by the employment of the saturated sodium carbonate
solution for neutralization and litmus paper as the indi-
cator. For some time, however, it has been our practice
to employ the yellow curcuma paper for the detection of
alkalinity, rather than the red litmus paper.
In the exhaustive paper of Fuller' on this point it
was shown that the results obtained by titrating the
same culture medium with the same alkali solution
differed very markedly with the indicator employed.
For instance, a litre of ordinary meat-infusion nutrient
agar-agar required 47 c.c. of a normal caustic alkali
solution to neutralize it when phenolphtalein was the
indicator used, 28 c.c. when blue litmus was employed,
and 5 e.c. when rosolic acid was substituted. It is
manifest from this that the actual reactions of media,
in the neutralization of which different indicators have
been used, may differ very widely from one another,
and that the results of cultivation on a medium neu-
tralized by one method are not fairly comparable with
those obtained when another indicator has been used.
For the sake of uniformity Fuller suggests that bacte-
riologists should agree upon some one trustworthy
method of neutralization and employ it to the exclusion
of other methods. He recommends, as the procedure
that has given the most satisfactory results in his hands,
a modification of Schultz’s method, viz., 5 e.c. of the
culture medium are to be mixed with 45 c.c. of distilled
water in a porcelain evaporating dish and boiled for
three minutes, after which 1 e.c. of phenolphtalein
1 Fuller: On the Proper Reaction of Nutrient Media for Bacterial Cultiva-
tion. “Public Health” (Journal of the American Public Health Association),
Quarterly Series, 1895, vol. i. p. 881.
BOUILLON. 83
solution’ is added and the titration with one-twentieth
normal caustic alkali solution is quickly made. The
neutral point (slightly on the side of alkalinity) is indi-
cated by the appearance of a pink color, the effect of
the alkali on the phenolphtalein. From the amount
of one-twentieth normal alkali solution needed for 5 e.c.
of the medium it is easy to calculate the number of cubic
centimetres of the normal solution that will be required
to neutralize the entire mass.
The phenolphtalein neutral point lies so high, aver-
aging 47 c.c. of normal caustic alkali solution per litre
for nutrient meat-infusion agar-agar, and 56 c¢.c. per
litre for nutrient gelatin, that it is improbable from
experience gained by the older methods that the condi-
tions offered by media neutral to this indicator are suit-
able for the growth of all bacteria, so that with particular
species it may be necessary to determine by experiment
the degree of deviation from the neutral point that is
best suited for development. In Fuller’s experience the
degree of deviation from the phenolphtalein neutral
point that gives in general the best results is represented
by from 15 to 20 of his scale—i. e., there should remain
enough uncombined acid in a litre of the finished me-
dium to require the further addition of caustic alkali to
the extent of from 15 to 20 c.c. of a normal solution to
bring the reaction of the mass up to the phenolphtalein
neutral point. Thus, for example, if upon titration it
should be found that to neutralize a litre of nutrient
meat-infusion gelatin by the phenolphtalein process 55
c.c, of normal caustic alkali solution would be needed,
the amount actually added would be from 35 to 40 e.¢.—
1 A 0.5 per cent. solution of the powder in 50 per cent. alcohol.
84 BACTERIOLOGY.
i.e, from 15 to 20 cc. less than the amount needed
to bring the reaction up to the neutral point.
Not infrequently the filtered bouillon, neutralized
and sterilized, will be seen to contain a fine, flocculent
precipitate. This may be due either to excess of alka-
linity or to incomplete precipitation of the albumin.
The former may be corrected with dilute acetic or
hydrochloric acid, and the bouillon again boiled, filtered,
and sterilized; or, if due to the latter cause, subsequent
boiling and filtration usually result in ridding the
bouillon of the precipitate.
Another modification now generally employed is the
use of meat-extracts instead of the infusion of meat.
Almost any of the meat-extracts of commerce answer
the purpose, though we usually employ Liebig’s. It
is used in the strength of from two to four grammes to
the litre of water. Peptone and sodium chloride are
added as in the bouillon made from the meat-infusion.
The advantages of meat-extract are: it takes less time;
affords a solution of more uniform composition if used
in fixed proportions, and in general use gives results
that are equally as satisfactory as those obtained from
the employment of infusion of meat.
Nurrient GELATIN.—For the preparation of gelatin
the bouillon is first prepared in exactly the same way
as has just been described, except that the neutralization
takes place after the gelatin has been completely dis-
solved, which occurs very rapidly in hot bouillon. The
reaction of the gelatin as it comes from the manufac-
tories is frequently quite acid, so that a much larger
amount of alkali is need for its neutralization than
for other media. It is possible, however, to obtain
from the makers an excellent grade of gelatin from
NUTRIENT GELATIN. 85
which all acid has been carefully washed.’ The gelatin
is added in the proportion of 10 to 12 percent. Its
complete solution may be accomplished either over the
water-bath, in the steam sterilizer, or over a free flame.
If the latter method be practised, care must be taken
that the mixture is constantly stirred to prevent burn-
ing at the bottom and consequent breaking of the flask,
if a flask is employed.
For some time it has been our practice to use, for the
purpose of making both gelatin and agar-agar, enam-
elled iron saucepans instead of glass flasks; by this
means the free flame may be employed without danger
of breaking the vessel, and, with a little care, without
fear of burning the media. Under any conditions it is
better to protect the bottom of the vessel from the
direct action of the flame by the interposition of several
layers of wire gauze, a thin sheet of asbestos-board, or
an ordinary cast-iron stove-plate.
When the gelatin is completely melted it may be
filtered through a folded paper filter supported on an
ordinary funnel; if the solution is perfect, this should
be very quickly accomplished.
For the filtration of such substances as gelatin and
agar-agar it is of much importance to have a properly
folded filter. To fold a filter correctly, proceed as fol-
lows: a circular piece of filter paper is folded exactly
through its centre, forming the fold 1, 1’ (Fig. 16); the
end 1 is then folded over to 1’, forming the fold 5; 1
and 1’ are each then brought to 5, thus forming the
folds 3 and 7; 1 is then carried to the point 7, and the
fold 4 is formed, and by carrying 1’ to 3 the fold 6 is
1 Hesteberg’s acid-free, gold label gelatin has given us entire satisfaction
in this respect. 5
86 BACTERIOLOGY.
produced; and by bringing 1 to 3 and 1’ to 7 the folds
2 and 8 result.
Fig. 16.
Thus far the ridges of all folds are on the side of the
paper next to the table on which we are folding. The
paper is now taken up, and each space between the seams
just produced is to be subdivided by a seam or fold
through its centre, as indicated by the dotted lines in
Fig. 16, but with the creases on the side opposite to that
Fic. 17.
occupied by the creases 1, 2, 3, 4, etc., first made. As
each of these folds is made the paper is gradually folded
into a wedge-shaped bundle (Fig 17, a), which when
opened assumes the form of a properly folded filter
(seen in 6, Fig. 17). Before placing it upon the funnel
it is well to go over each crease again and see that it is
NUTRIENT GELATIN, 87
as tightly folded as possible, without tearing it. The
advantage of the folded filter is that by its use a much
greater filtering surface is obtained, as it is in contact
with the funnel only at the points formed by the ridges,
leaving the majority of the flat surface free for filtra-
tion.
The employment of the hot-water funnel, so often
recommended, has been dispensed with in this work to
a very large extent, as we know that, if the solution of
the gelatin is complete, filtration is so rapid as not to
necessitate the use of an apparatus for maintaining the
high temperature. The temperature at which the hot-
water funnel retains the gelatin is so high that evapora-
tion and concentration rapidly occur, and in consequence
the filtration is, as a rule, retarded. The filtration is
frequently done in the steam sterilizer, but this too is
unnecessary if the gelatin is quite dissolved. At the
ordinary temperature of the room, and by the means
commonly employed for the filtration of other sub-
stances, both gelatin and agar-agar may be rapidly
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° and 70° C. an
ego, which has been beaten up with about 50 c.c. of
water, is added. The whole is then thoroughly mixed
together and again brought to the boiling-point, and
kept there until coagulation of the albumin occurs. It
is better not to break up the large masses of coagulated
albumin if it can be avoided, as when broken up into
88 BACTERIOLOGY.
fine flakes they clog the filter and materially retard
filtration.
The practice sometimes recommended of removing
these albuminous masses by first filtering the gelatin
through a cloth, and then finally through paper, is not
only superfluous, but in most instances renders the pro-
cess of filtration much more difficult, because of the dis-
integration of these masses into finer particles, which
have the effect just mentioned, viz., of clogging the filter.
Under no circumstances is a filter to be used without
first having been moistened with water. If this is not
done, the pores of the paper,which are relatively large
when in a dry state, when moistened by the gelatin not
only diminish in size, but in contracting are often en-
tirely occluded by the finer albuminous flakes which
become fixed within them, and filtration practically
ceases. The preliminary moistening with water causes
diminution of the size of the pores to such an extent
that the finer particles of the precipitate rest on the sur-
face of the paper, instead of becoming fixed in its meshes.
During boiling it is well to filter, from time to time,
a few cubic centimetres of the gelatin into a test-tube
and boil it over a free flame for a minute or so; in this
way one can detect if all the albumin has been coagu-
lated—i. ¢., if 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 may be impaired.
As soon as the gelatin is complete, whether it is re-
tained in the flask into which it has been filtered or
decanted off into sterilized test-tubes, it should be ster-
ilized in the steam sterilizer on three successive days,
NUTRIENT AGAR-AGAR. 89
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 nutrient
agar-agar by the beginner is far too frequently a tedious
and time-taking experience. This is due mainly to
lack of patience and to deviation from the rules laid
down for the preparation of this medium. If the
directions given below for the preparation of nutrient
agar-agar be strictly observed, no difficulty whatever
should be encountered. Many methods are recom-
mended for its preparation; almost every worker has
some slight modification of his own.
The methods that have given us the best results, and
from which we have no good grounds for departing,
are as follows:
Prepare the bouillon in the usual way. Agar-agar
reacts neutral or very slightly alkaline, so that the
bouillon may be neutralized before the agar-agar is
added. Then add finely chopped or powdered agar-
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 side of the vessel at which the level of
the fluid stands; if a litre of medium is being made,
add about 250 c.c. to 300 cc. more of water and
allow the mass to boil slowly, occasionally stirring,
over a free flame, for from one and one-half to two
hours; or, in other words, until the excess of water
—it.e, the 250 or 300 c.c. that were added — has
evaporated. Care must be taken that it does not boil
over the sides of the vessel. From time to time observe
if the fluid has fallen below the mark of its original
level; if it has, add water until its volume of 1 litre is
90 BACTERIOLOGY.
restored. At the end of the time given remove the flame
and place the vessel containing the mixture in a large
dish of cold water; stir the agar-agar continuously until
it has cooled down to about 68°-70° C., and then add
the white of one egg which has been beaten up in about
50 c.c. of water; or the ordinary dried albumin of com-
merce may be dissolved in cold water in the proportion
of about 10 per cent., and used; the results are equally
as good as when eggs are employed. Mix this care-
fully throughout the agar-agar, and allow the mass to
boil slowly for about another half-hour, observing all
the while the level of the fluid, which should not fall
below the litre mark. It is necessary to reduce the tem-
perature of the mass to the point given, 68°-70° C.;
otherwise the coagulation of the albumin will occur
suddenly in lumps and masses as soon as it is added,
and its clearing action will not be homogeneous. The
process of clarification with the egg is purely mechani-
cal—the finer particles, which would otherwise pass
though 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 transparent as agar-agar usually appears.
It might be well to emphasize the fact that for the
filtration of agar-agar a hot-water funnel, or any other
special device for maintaining the temperature of the
mass, is totally unnecessary. Agar-agar prepared after
the methods just given should filter through a properly
folded paper filter at the rate of a litre in from twelve
to fifteen minutes.
Another plan that insures complete solution of the
NUTRIENT AGAR-AGAR. 91
agar-agar without causing the precipitates that are com-
monly seen when all the ingredients are added at first
and boiled for a long time, is to weigh out the necessary
amount of agar-agar, 10 or 15 grammes, and place this
in 1300 or 1400 c.c. of water and boil down over a free
flame to 1000 e.c. The peptone, salt, and beef-extract
are then added and the boiling again continued until
they are dissolved. The clarification with egg-albumin
may then be done, and usually the mass filters quite
clear and does not show the presence of precipitates
upon cooling. If the mixture is positively alkaline,
it is not only cloudy, but it filters with difficulty; if it
is acid, it is usually quite clear, filters more quickly, but,
as Schulze has pointed out, loses at the same time some
of its gelatinizing properties. The bouillon should al-
ways be neutralized before the agar-agar is added to
it, for if the bouillon be acid, from the acid of the
meat, it robs the agar-agar, under the influence of heat,
of some of its gelatinizing powers, which cannot be re-
gained 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-
agar be kept in the digester or autoclave, with the steam
under a pressure of about one atmosphere, as shown
by the gauge, for ten minutes, the agar-agar will be found
at the end of this time completely melted, and filtration
may then be accomplished with but little difficulty.
If glycerin is to be added to the agar-agar, it is done
after filtration and before sterilization. The nutritive
properties of the media for certain organisms, particu-
larly the tubercle bacillus, are improved by the addition
of glycerin in the proportion of 5 to 7 per cent:
92 BACTERIOLOGY.
If after filtration a fine flocculent precipitate is
seen, look to the reaction of the medium. If it is quite
alkaline, 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 reac-
tion. They must be neutral or very slightly alkaline
‘to litmus. (See remarks on Neutralization of Media.)
Only a few organisms develop well on media of an acid
reaction. In all of the above media the meat-extracts
now on the market may usually be substituted for the
meat itself in preparing the bouillon. They may be
employed in the proportion of from two to four grammes
to the litre of water.
PREPARATION OF PoTatToEs.—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 in a covered tin bucket with a perforated
bottom and sterilized in the steam sterilizer for forty-
five minutes. On the second and third days the steril-
ization 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
PREPARATION OF POTATOES. 93
they are usually intended to be cut in half, and the
cultivation of the organisms is to be conducted upon the
flat surfaces of the sections. (Koch’s original method.)
This method requires some care to prevent contam-
ination 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 severed 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 sec-
tions uppermost. The cover is placed upon the dish
and the potatoes are ready for inoculation.
2. Preparation of potatoes for test-tube cultures. Method
of Bolton.’ If the potatoes are to be employed for test-
tube cultures, one simply scrubs off the coarser particles
of dirt with water and a brush, and with a cork-borer
punches out cylindrical bits of potato which will fit
loosely into the test-tubes to be used. On each bit of
potato is then to be cut a slanting surface running from
about the junction of the first and second thirds of the
cylinder to the diagonally opposite end. These cylin-
1 Medical News, 1887, vol. i. p. 138.
5*
94
BACTERIOLOGY.
ders of potato are now to be left in running water over
night, otherwise they will be very much discolored by the
sterilization to which they are to be subjected. At the
end of this time they are placed in previously prepared
test-tubes, one piece in each tube, with the slanting sur-
face up, the cotton plugs of the tubes replaced, and they
are then to be sterilized in the steam for fifteen to
twenty minutes on each of three successive days.
Or the entire sterilization may be accomplished in
the autoclave, with the steam under a pressure of one
ha yy
Potato in test-
tube,
atmosphere, by a single exposure of twenty
to twenty-five minutes. When finished
they have the appearance seen in Fig. 18,
except that there is no growth upon the
surface as is shown in the cut.
For some purposes potatoes may be ad-
vantageously peeled, sliced into disks of
about 1 cm. in thickness, and placed in
small glass dishes provided with covers,
similar to the ordinary Petri dishes. The
dish and its contents are then sterilized by
steam in the usual way (method suggested
by von Esmarch). By this plan a relatively
large area for cultivation is obtained.
Potatoes may also be boiled, or steamed,
and mashed, and the mass placed in covered
dishes, test-tubes, or flasks, and sterilized.
By this method one obtains in the mass a
mean of the composition of the several pota-
toes, or bits of potatoes, used in making it,
an advantage where uniformity is desired.
Care must be given to the sterilization of potatoes,
because they always have adhering to them the organ-
BLOOD-SERUM., 95
isms commonly found in the ground, the spores of which
are among the most resistant known. The so-called
‘* potato bacillus’’ is one of this group; it is an organ-
ism which is not infrequently more or less of an obstacle
to the work of the beginner.
BLoop-seRuM. — Originally blood-serum required
special care in its preparation; it was always necessary
to reduce the unavoidable contamination, which to a
certain extent occurs when the blood is obtained, to the
minimum degree.
It is possible to collect seram from small animals and
in small quantities under such precautions that it is per-
haps not contaminated; but, ordinarily, for laboratory
purposes a larger quantity is needed, so that the
slaughter-houses form the source from which it is usu-
ally obtained, and here a certain amount of contamina-
tion is unavoidable, though its degree may be limited
by proper precaution.
The steps that were formerly thought to be essential
to the successful collection of blood and the preparation
of serum for culture purposes were about as follows :
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 completely cut
through. The blood which spurts 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 provided with
covers which close hermetically—these, too, should be
carefully disinfected. The best form of glass vessels
96 BACTERIOLOGY.
for the purpose is the large glass museum jar of about
one gallon capacity, which closes by a cover that can be
tightly screwed down upon a rubber joint. From two
such jarfuls of blood one can recover quite a large quan-
tity of clear serum, ordinarily from 500-700 c.c. The
jars having been filled with blood, their covers are placed
loosely upon them and they are allowed to stand for
about fifteen minutes until clotting has begun. At the
end of this time a clean glass rod is passed around the
edges of the surface of the clot to break up any adhe-
sions to the side of the jar that might have formed, and
which would prevent the sinking of the clot to the
bottom. The covers are then replaced and tightly
clamped in position, and with as little agitation as pos-
sible the jars are placed in an ice-chest, where they
remain for twenty-four to forty-eight hours. The
temperature should, however, not be low enough to
prevent coagulation, but should be sufficiently low to
interfere with the development of any living organ-
isms that may be present. The temperature of the
ordinary domestic refrigerator is sufficient for the
purpose. After twenty-four to forty-eight hours the
clot will have become firm, and will be seen at the
bottom of the jar. Above it is a quantity of dark
straw-colored serum. The serum may then be drawn
off with a sterilized pipette and placed in tall cylinders
that have previously 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
BLOOD-SERUM. 97
then be pipetted off, either into sterilized test-tubes,
about 8 ¢.c. to each tube, or into small sterilized flasks
of about 100 c.c. capacity. It is then to be sterilized
by the intermittent method at low temperatures, viz., for
one hour on each of five consecutive days at a tempera-
ture 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,
Chamber for sterilizing and solidifying blood-serum. (Kocu.)
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 sur-
face as possible may be given to the seram. The pro-
cess of solidification requires constant attention if good
98 BACTERIOLOGY.
results are to be obtained—i. ¢., if a translucent, solid
medium is to result. If the old, small form of appa-
ratus be employed (Fig. 19), then the solidification can
be accomplished in a shorter time than if the larger
forms commonly 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 solidify-
ing apparatus be used, very good results may be ob-
tained 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
Leeffler’s serum mixture.
The best results are obtained when a low temperature
is employed for a long time. Under any circumstances
the tubes must be observed from time to time through
the glass door or cover with which the solidifying oven
is provided, and each time the oven should be slightly
jarred with the hand to see if solidification, as indi-
cated by the disappearance of tremors from the serum,
is beginning. If the temperature gets too high, or the
exposure is too long, an opaque medium results. The
temperature to be observed is that of the air inside
the chamber, and also that of the water surrounding it.
The latter is usually a degree or two higher than the
former. The tubes should not rest directly upon the
heated bottom or against the heated sides of the cham-
ber, 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
BLOOD-SERUM. 99
the large form of apparatus be 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-fash-
ioned apparatus of Koch be employed. The latter form
is preferable, as it is more easily managed.
When solidification is complete the tubes are to be
retained in the erect position, and, unless they are
intended for immediate use, must be prevented from
drying. The superfluous ends of the cotton plugs
should be burned off, and the mouths of the tubes may
then be covered by sterilized rubber caps, or, as Ghris-
key suggests, they may be closed with sterilized corks
pushed in on top of the cotton plugs. Even with the
greatest care it not uncommonly happens that one or
two of the lot of tubes thus prepared and protected will
become contaminated. This is usually due to spores of
moulds that have fallen into the rubber caps or on the
cotton plugs during manipulation, and, finding no
means of outward growth, have thrown their hyphe
downward through the cotton into the tube, and their
spores have fallen on the surface of the serum and
developed there.
The foregoing is, in the main, the plan originally
recommended by Koch for the preparation of this
medium. In recent times, however, particularly since
the study of diphtheria by the method of Leeffler has
become so general, and large quantities of serum tubes
were found to be necessary, a modification has been
suggested that has, in this country at least, almost en-
tirely supplanted the method by Koch. The popularity
of the Councilman-Mallory method is due to the fol-
lowing facts: by it the serum is more quickly and
easily prepared; rigid precautions against contamination
100 BACTERIOLOGY.
during collection of the serum are not so necessary, and
the resulting medium, while not transparent or even
translucent (points aimed at in the original method),
fully meets all the requirements.
The special points in the method are: the serum is
decanted into test-tubes as soon as obtained; it is then
firmly coagulated in a slanting position in the dry-air
sterilizer at from 80° to 90° C.; it is then sterilized in
the steam sterilizer at 100° C. on three successive days,
as in the case of other culture media. It may then be
protected against evaporation by sterilized rubber caps
or sterilized corks in the way already described, and set
aside until needed.
Unless the coagulation in the dry sterilizer be com-
plete, the surface of the serum will be found to be blis-
tered and pitted by bubbles and cavities after it has
been subjected to the steam sterilization. A similar
formation of cavities over the surface of the serum will
occur if the temperature of the hot-air sterilizer, in
which it is solidified, is allowed to get above 90° C.,
or if it be elevated to this point too quickly.
It is of no special advantage to have the serum clear,
as the admixture of blood-coloring-matter does not
affect its nutritive properties.
It is often desirable to obtain blood-serum in small
quantities, either for culture purposes or for the study
of the serum of different animals in its relation to bac-
teria, and for this purpose Nuttall (Centralb. fiir Bakt.
u. Parasitenkunde, 1892, Bd. xi. p. 539) suggests a very
convenient method. By the use of a sterilized vessel, of
the shape given in Fig. 20, from ten to one hundred
cubic centimetres of blood can be collected, and if proper
precautions are observed no contamination by bacteria
BLOOD-SERUM. 101
need occur. The collecting bulb is used in the follow-
ing way: an artery, either femoral or carotid, is ex-
posed, and around it two ligatures are placed; that
distant from the heart is tightened, while the one near-
est the heart is left loose; between the latter and the
heart the artery is clamped. A small slit is then made
in its wall, into which the point a of the bulb is intro-
duced and the artery bound tightly around it with the
Ww
Nuttall’s bulb for collecting blood-serum under antiseptic precautions.
hitherto loose ligature; the clamp is removed and the
bulb quickly fills with blood. The clamp is now again
put in position, the point of the bulb removed and
sealed in the gas-flame, the loose ligature tightened,
the wound closed, and the bulb containing the blood is
set aside in a cool place until coagulation has occurred.
The serum is most easily withdrawn from the bulb by
means of a pipette, closed above with a cotton-plug, and
supplied with a bit of rubber tubing, about one-half
metre long, with glass mouth-piece. By holding the
102 BACTERIOLOGY.
pipette in the hand and sucking upon the rubber tube
one can more easily direct the point of the pipette than
if it is used in the ordinary way.
The bulbs are easily blown, and after having been
sealed at the point and plugged with cotton can be kept
on hand just as are sterilized test-tubes.
It is sometimes desirable to preserve blood-serum in
a fluid state. This can be done by the fractional method
of sterilization at low temperatures, already described,
or with much less effort, and without the use of heat,
by a method that we have found to be very satisfactory.
In the studies of Kirchner chloroform was shown to
possess decided disinfectant properties; as it is quite
volatile, it is easily removed when its disinfectant or
antiseptic functions are no longer required. If, there-
fore, the serum to be preserved be placed in a closely
stoppered flask and enough chloroform added to form a
thin layer, about 2 mm., on the bottom, the serum may
be kept indefinitely without contamination, so long as
the chloroform is not permitted to evaporate. When
required for use the seram is decanted into test-tubes,
which are then placed in a water-bath at about 50° C.
until all the chloroform has been driven off; this can
be determined by the disappearance of its characteristic
odor. The serum may then be solidified, sterilized by
heat, and employed for culture purposes. We have
found serum so preserved to answer all requirements as
a culture medium.
SpeciAL MeEp1a.—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. or the finer points of differentia-
SPECIAL MEDIA. 103
tion 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 e.c. to each tube, and sterilized by the intermittent
process, at the temperature of steam, for three succes-
sive days.
The separation of the cream may be accelerated and
rendered more complete by one sterilization of the milk
in the cylinder before it is placed in the ice-chest.
The cream is best separated from the milk by the use
of a cylindrical vessel with stopcock at the bottom, by
means of which the milk, devoid of cream, may be
drawn off. A Chevalier creamometer with stopcock
at the bottom serves the purpose very well. It should
be covered while standing.’
Milk may be used as a culture medium without any
addition to it, or, before sterilizing, a few drops of
litmus tincture may be added, just enough to give it a
pale blue color. By this means it will be seen that
different organisms bring about different reactions in
the medium; some producing alkalies which cause the
blue color to be intensified, others producing acids which
change it to red, while others bring about neither of
these changes. Similarly litmus solution is often added
to gelatin and agar-agar for the same purpose.
Milk may also be employed as a solid culture medium
by the addition to it of gelatin or agar-agar in the pro-
portions given for the preparation of the ordinary nutri-
1 For some time past we have been using what is technically known as
“separator milk’’—i. ¢., the fluid left after milk has been deprived of its fat
(cream) by centrifugal force.
104 BACTERIOLOGY.
ent gelatin or agar-agar. It has, however, in this form
the disadvantage of not being transparent, and can
therefore 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 after pre-
cipitation of the casein.
Dunham’s peptone solution. The medium usually
known as Dunham’s solution is prepared according to
. the following formula:
Dried peptone . , ‘ i . ‘ fs 1 part.
Sodium chloride ‘ . ~ 05 ©
Distilled water . x ~ o 2 A 100 parts.
It is usually of a neutral or slightly alkaline reac-
tion, and neutralization is not, therefore, necessary.
It is filtered, decanted into tubes or flasks, and ster-
ilized in the steam sterilizer in the ordinary way.
The most common use to which this solution is put
is in determining if the organism under considera-
tion possesses the property of producing indol as one
of its products of nutrition. It is essential for accu-
racy that the preparation of dried peptone employed
should be of as nearly chemical purity as is possi-
ble, and indeed the other ingredients should be
correspondingly free from impurities. Gorini (Central-
blatt fiir Bakteriologie und Parasitenkunde, 1893, Bd.
xiii. p. 790) calls attention to the fact that impurities
in the peptone, particularly the presence of carbohy-
drates, so interfere with the production of indol by
certain bacteria that otherwise produce it, that it is
ofttimes impossible, when such preparations have been
employed, to obtain the characteristic color-reaction of
SPECIAL MEDIA. 105
this body, and where it is obtained it is always after a
much longer time than is the case where peptone free
from these substances has been used. He suggests the
advisability of testing the purity of all peptone prep-
arations before using them, by means of the reaction
that they exhibit when acted upon by Fehling’s alka-
line copper solution. Under the influence of this
agent pure peptone in solution gives a violet color (the
biuret reaction), which remains permanent even after
boiling for five minutes. If, instead of a violet color,
there appears a red or reddish-yellow precipitate, the
peptone should be discarded, as in his experience no
indol is produced from peptone giving this reaction.
Both the peptone solution and that of the copper (partic-
ularly the latter) should be relatively dilute in order
for the reaction to be successful.
Peptone rosolic acid solution. Peptone solution con-
taining rosolic acid serves well for the detection of alter-
ations in reaction. It consists of the peptone solution
of Dunham, to each 100 ¢.c. of which 2 c.c. of the
following solution are added:
Rosolic acid (coralline) . " . 0.5 gramme.
Alcohol (80 per cent.) A s - 100 ce.
This is to be boiled, filtered, and decanted into clean,
sterilized test-tubes, about 8 to 10 ¢.c. to each tube.
The tubes are then to be sterilized in the usual way by
steam. When sterilization is completed and the tubes
cooled the solution will be of a very pale rose color,
which disappears 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
106 BACTERIOLOGY.
find it a valuable addition to our means of differentiat-
ing bacteria.
Rosolic acid cannot be used with safety in solutions
containing glucose, as the reducing action of the latter
deprives it of its color.
Lactose-litmus-agar, or gelatin of Wurtz. A medium
of much use in the differentiation of bacteria is that
recommended by Wurtz, consisting of ordinary nutri-
ent, slightly alkaline agar-agar, to which from 2 to 3
per cent. of lactose and sufficient litmus tincture to give
it a pale blue color have been added. Bacteria capable
of causing fermentation of lactose when grown on this
medium develop into colonies of a pale pink color and
cause, likewise, a reddening of the surrounding medium,
owing to the production of acid as a result of their
action upon the lactose; while other bacteria, incapable of
such fermentative activities, grow as pale blue colonies
and cause no reddening of the surrounding medium.
It is an especially useful aid in the differentiation of
the bacillus of typhoid fever, which does not possess
the property of bringing about fermentation of lactose,
from other organisms that simulate it in many other
respects, but which do possess this property.
Its preparation is as follows: to nutrient agar-agar
or gelatin, the alkalinity of which is such that one cubic
centimetre will require 0.1 ¢.c. of a 1:20 normal sul-
phuric acid solution to neutralize it, lactose is added in
the proportion of 2 or 3 per cent.; it is then decanted into
test-tubes and sterilized in the usual way. When ster-
ilization is complete there is to be added to each tube
enough sterilized litmus tincture to give a decided though
not very intense blue color. This must be done care-
fully, to avoid contamination of the tubes during ma-
SPECIAL MEDLA, 107
nipulation. It is better not to add the litmus tincture
before sterilizing the tubes, as its color-characteristics
are in some way altered by its contact with organic
matters under the influence of heat.
When ready it may be used as ordinary agar-agar or
gelatin, either for plates or slant-cultures.
Leeffler’s blood-serum mixture. Lieeffler’s blood-serum
mixture consists of one part of neutral meat-infusion
bouillon, containing 1 per cent. of grape-sugar, and
three parts of blood-serum. This mixture is placed in
test-tubes, sterilized, and solidified in exactly the way
given for blood-serum. It requires for its solidification
a somewhat higher temperature and a longer exposure
to this temperature than does blood-serum to which no
bouillon has been added. (See also the Councilman-
Mallory method. )
Guarniari’s agar-gelatin :
Meat-infusion 950 ¢.c.
Sodium chloride 5 grammes.
Peptone . é c c é . 25-30
Gelatin 40-60 “
Agar-agar ‘ : 3-4 “f
Water . ; 50 ¢.¢.
The point in the preparation of this medium is its
reaction, which should be exactly neutral.
The full list of special media is too great to be given
in a work of this size. For their description the reader
is referred to the current literature on the subject.
Those that have’been given above will suffice for ob-
taining a clear understanding of the principles of the
work.
Note.—The term ‘‘meat-infusion ’’ always implies a
watery extract of meat made by mixing 500 grammes
108 BACTERIOLOGY.
of finely chopped lean meat and 1 litre of water to-
gether, and allowing them to stand in a cool place for
twenty-four hours. At the end of this time the fluid
portion is strained off through a coarse towel. This
represents the infusion.
CHAPTER VI.
Preparation of the tubes, flasks, etc., in which the media are to be pre-
served.
WHILE the media are in course of preparation it is
well to get the test-tubes and flasks ready for their
reception, and it is essential that they should be as clean
as 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 2 to 3 per cent.
solution of common soda; it is not necesgary to be exact
as to strength, but it should not be weaker than this.
At the end of this time they are to be carefully
swabbed out with a cylindrical bristle brush, preferably
one having a reed handle (Fig. 21), as those with wire
Fig. 21.
Brusb for cleaning test-tubes.
handles are apt to break through the bottoms of the
tubes. All traces of adherent material should be care-
fully removed. When the tubes are quite clean they
may be rinsed in a warm solution of commercial hydro-
chloric acid of the strength of about 1 per cent. This
is to remove the alkali. They are then to be thor-
oughly rinsed in clear, running water, and stood top
down until the water has drained from them. When
6
110 BACTERIOLOGY.
dry they are to be plugged with raw cotton. The plug-
ging 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 regu-
larly all around. The plug should fit neither too
tightly nor too loosely, but should be just firmly enough
in position to sustain the weight of the tube into which
it is placed when held up by the portion which projects
from and overhangs the mouth of the tube. The tubes
thus plugged with cotton are now to be placed upright
in a wire basket and heated for one hour in the hot-air
sterilizer at a temperature of about 150° C. A very
good rule for this process of sterilization 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.
The cotton used for this purpose should be the ordi-
nary 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,
empyreumatic, oily deposit, which renders them unfit
for use until they have been cleaned again.
Fitting THE Tuses.—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. 22. The liquefied medium is poured into this
funnel, which has been carefully washed, and by
pressing the pinchcock with which the funnel is pro-
vided the desired amount of material (5-10 c.c.)
FILLING THE TUBES. 111
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.
Funnel for filling tubes with culture media.
Care should be taken that none of the medium is
dropped upon the mouth of the test-tube, otherwise the
cotton plug becomes adherent to it, and is not only
difficult to remove, but presents a very untidy appear-
ance, and interferes, indeed, with the proper manipula-
tions.
As soon as the tubes have been filled they are to be
112 BACTERIOLOGY.
sterilized in the steam sterilizer for fifteen minutes on
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 remainder may solidify in
the erect position; these serve for making plates.
For Esmarch tubes not more than 5 c.c. of material
should be placed in each tube, as more than this renders
it difficult to distribute the gelatin evenly over the inner
surface of the tubes when they are rolled.
CHAPTER VII.
Technique of making plates—Esmarch tubes, Petri plates, etc.
PLatTEs.—The plate method can be practised with
both agar-agar and gelatin. It cannot be practised with
blood-serum, because the serum, when once solidified,
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 state of dilution.
The first plate is known usually as ‘“‘the original,’’ or
“plate 1,” the first dilution from this as ‘‘ plate 2,”’
and the second as ‘‘plate 3.”’
In the preparation of a set of plates the following
are the steps to be observed:
Three tubes, each containing from 7 to 9 c.c. of gela-
tin or agar-agar, are placed in the warm water-bath
until the medium has become liquid. If agar-agar is
employed, this is accomplished at the boiling-point of
water; if gelatin is used, a much lower temperature
suffices (35°-40° C.). When liquefaction is complete
the temperature of the water, in the case of agar-agar,
must be reduced to 41°-42° C., at which temperature
the agar-agar remains liquid, and the organisms may
be introduced into it without fear of destroying their
vitality. The medium being now liquid and of the
proper temperature, a very small portion of the mixture
of organisms to be studied is taken up with a sterilized,
114 BACTERIOLOGY.
looped platinum wire (Fig. 23, a). This is nothing
more than a piece of platinum wire about 5 cm. long,
twisted into a small loop at one end and fused into a
bit of glass rod, which acts as a handle, at the other
extremity. This loop is one of the most useful of bac-
teriological instruments, as there is hardly a manipula-
tion in the work into which it does not enter. Under
no conditions is it to be employed without having been
passed through the gas-flame until quite hot; this is for
the purpose of sterilization. One should form a habit
Fig. 23.
Looped and straight platinum wires in glass handles.
of never taking up one of these platinum-wire needles,
as they are called, for they are curved and straight as
well as looped (Fig. 23, 6), without passing it through
the flame, and the sooner the beginner learns to do this as
a matter of reflex, the sooner does he rid himself of one
of the possible sources of error in his work. It must
be remembered, though, that it should not be used when
hot, otherwise the organisms taken upon it are killed
by the high temperature; after sterilization in the flame
one waits for a few seconds until it is cool before using.
The bit of material under consideration is transferred
with the sterilized loop into tube No. 1, ‘‘the original,”’
where it is carefully disintegrated by gently rubbing it
against the sides of the tube. The more carefully this
is done the more homogeneous will be the distribution
TECHNIQUE OF MAKING PLATES. 115
of the organisms and the better the results. The loop
is then again sterilized, and three of its loopfuls are
passed, without touching the sides of the tube, from ‘‘the
original’? into tube No. 2, where they are carefully
mixed. Again the loop is sterilized, and again three
dips are made from tube 2 into tube 3. This completes
the dilution. The loop is now sterilized before laying
it aside.
Fig, 24.
Levelling-tripod with glass chamber for plates.
During this manipulation, which must be done
quickly if agar-agar be employed, the temperature of
the water in the bath in which the tubes stand should
never get lower than 39° C., and never higher than
43°C. If it falls too low, below 38° C., the agar-agar
gelatinizes, and can only be redissolved by a tempera-
ture that would be destructive to the organisms which
may have been introduced into the tubes. This is not
of so much moment with gelatin, as it may readily be
redissolved at a temperature not detrimental to the
116 BACTERIOLOGY.
organisms with which the tubes may have been inocu-
lated.
THE CooLInG-sfaGE 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.
24) upon which the gelatin or agar-agar is to be subse-
quently solidified.
This stage consists of a glass dish filled with ice-
water and covered with a ground-glass plate, which in
turn has a dome-shaped cover. The dish rests upon a
tripod which can be brought to an exact level, as indi-
cated by the spirit-level, by raising or lowering its legs
by means of thumb-screws, with which they are pro-
vided. Three stages are usually employed. When
ready for use they should be exactly level.
THE GLAss PLAtTEs.—On each of the stages is to be
placed a glass plate upon which the liquefied gelatin or
Russia iron box for holding plates, etc., during sterilization in dry beat.
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 they should
be carefully protected by a cover against dust and bac-
teria from outside sources during manipulation.
A number of plates ata time are usually sterilized in
GLASS BENCHES. 117
the dry sterilizer at a temperature of 150° to 180° C.
for one hour. During sterilization and until used
they are retained in an iron box (Fig. 25), 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.
Fic. 26.
Glass benches for supporting plates.
As the contents of each tube are emptied upon a plate
the cover of the cooling-stage is quickly replaced and the
plate allowed to stand until the gelatin or agar-agar is
quite solid. This takes longer with gelatin than with
agar. When quite solid they are placed upon little
glass benches (Fig. 26), and each bench is labelled with
the number of the plate in the series of dilutions. The
benches, with the plates upon them, are then piled one
above the other in a glass dish, the so-called ‘‘culture-
dish,’’ in which the plates are to be kept during the
growth of the bacteria. The benches are sterilized
before using, in the way given for the plates.
6*
118 BACTERIOLOGY.
CULTURE-DISH.—This dish, which is about 22 cm.
in diameter and has vertical sides of about 6 cm. in
height, is provided with a cover of exactly the same
design, but of a little larger diameter. ‘This cover,
when placed upon the dish containing the plates, fits
over it and prevents the access of dust. Prior to using,
the dish and cover should have been disinfected for one-
half an hour with 1 : 1000 sublimate, and then all the
sublimate solution allowed to drain from it.
In the bottom of this dish is sometimes placed a disk
of sterilized filter-paper moistened with sterilized water,
which serves to prevent the drying of the medium. This,
however, is not necessary.
If agar-agar be employed, the dish and its contents
may be kept at a temperature of 37°-388° C.; if gel-
atin, the temperature at which the plates are to be
maintained 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 be easily 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
mixture of bacteria. Many modifications of this method
exist; all, however, are based upon the same _ prin-
ciples. The modifications have for their object the
accomplishment of the same end, but with a smaller
armamentarium of apparatus, and in general the one
or the other of these modifications has entirely sup-
planted the original plate method as practised and
recommended by Koch.
PETEIS MODIFIED PLATE METHOD. 119
Perri’s MopIricaTIoNn OF THE PLATE MerHop.—
The modification which approaches nearest to the orig-
inal method, and at the same time lessens very mate-
rially the number of steps in the process, is that sug-
gested by Petri. It consists in substituting for the
plates small, round, double glass dishes, which have
about the same surface-area as the plates. The liquid
medium may be poured directly into these little dishes
without their being exactly level. Each dish acts as a
plate. Their covers are then to be replaced, and they
are set aside for observation. In all other respects the
steps are the same as those given for Koch’s original
method. Petri’s dishes are flat, double dishes of glass
Te
ve
Lu
Petri double dish, now generally used instead of plates.
eT
IMT
(Fig. 27). They are about 8 cm. in diameter and about
1.5 to 2 cm. in height, the walls being vertical. They
may readily be sterilized either by the hot-air or steam
methods of sterilization. They are very useful for this
work, as they do away with the necessity for the cool-
ing-stage and levelling-tripod, though in warm weather
the cooling-stage may be used to hasten the solidifica-
tion of gelatin. A cooling-stage of very convenient
design for use with these dishes consists of a closed, flat
metal box, either of copper or block tin, and either
round or square in shape, so arranged that it can be
_
120 BACTERIOLOGY.
filled with cold water, or that cold water can constantly
be passed through it by means of a rubber connection
with a spigot. The inlet for the water should be just
above the bottom of the box, and the outlet just beneath
the top and slightly turned upward and then downward,
so as to insure the complete filling of the space with water.
The box should be sufficiently strong to resist the pres-
sure of the water. A convenient size is from 20 to 25
em. in diameter, and of about 1.5 to 2cm.high. It
is simple in construction, and can be made by any cop-
per spinner. An idea of its construction is given in
Fig. 28,
Fie. 28,
Metal cooling-stage.
When gelatin or agar-agar is to be cooled it is only
necessary to place the dishes containing it on top of this
box and start cold water circulating through it.
Esmarcu 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
ESMARCH TUBES. 121
plates or into dishes, spread over the inner surface of
the tube containing them, and without removing the
cotton plugs are caused to solidify in this position. The
tubes then present a thin cylindrical lining of gelatin
or agar-agar, upon which the colonies develop. In all
other respects the conditions for the growth of the organ-
isms are the same as in flat plates.
Esmarch directs that after completion of the dilu-
tions the tops of the cotton plugs in the test-tubes
should be cut off flush with the mouths of the tubes and
sterilized rubber caps be placed over them. They are
then to be held in the horizontal position and twisted
between the fingers upon their long axis under ice-
water. The gelatin becomes solidified thereby and
adheres to the sidés of the tube. When the gelatin is
quite hard the tubes are removed from the water, wiped
dry, the rubber caps removed, and the tubes set aside
for observation.
For some time past we have deviated from the direc-
tion given by v. Esmarch for this part of his method,
and instead of rolling the tubes under ice-water, we roll
them upon a block of ice (Fig. 29), after the method
devised by Booker in the Pathological Laboratory of
the Johns Hopkins University in 1887. In this method
a small block of ice only is needed. It is arranged
nearly level, and is held in position by being placed in
a dish upon a towel. A horizontal groove is melted in
the surface of the ice with a test-tube full of hot water.
The tubes to be rolled are then held in an almost, not
quite, horizontal position and twisted between the fingers
until the sides are moistened by the contents to within
about 1 cm. of the cotton plug, care being taken that
the gelatin does not touch the cotton; otherwise the latter
122 BACTERIOLOGY.
becomes adherent to the sides of the tube and is difficult
to remove. ‘The tube is then placed in the groove in
the ice and rolled, neither rubber cap nor cutting off of
the cotton plug being necessary.
Fig. 29.
Se
Demonstrating Booker’s method of rolling Esmarch tubes on a block of ice.
The advantages of this process over that followed by
v. Esmarch are that it requires less time, is cleaner,
no rubber caps are needed, the rolled tubes are more
regular, 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 to 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 over-
come by allowing the rolled tubes to remain in nearly
ESMARCH TUBES. 123
a horizontal position, the cotton end of the tube about
1 em. 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 tube. After this they may be retained in
the upright position without fear of the agar-agar slip-
ping down. We have followed this process for several
years with entire satisfaction.’ In all these processes,
if the dilutions of the number of organisms have been
properly conducted, the results will be the same. The
original plate or tube, as a rule, will be of no use be-
cause of the great number of colonies in it. Plate or
tube No. 2 may be of service, but plate or tube 3 will
usually contain the organisms in such small numbers
that the colonies originating from them will have noth-
ing 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 dilutions
are completed, and only plates or tubes 2 and 3 are
made.
Another method for the separation of bacteria and
their isolation as single colonies consists in the making
of dilutions upon the surface of solid media, such as
potato, coagulated blood-serum, agar-agar, and gelatin.
For the performance of this method one selects a num-
ber of tubes containing the medium to be employed in
1 The impression that agar-agar is not suitable for rolled tubes was shown
to be erroneous, and the above method was developed in the Pathological
Laboratory of the Johns Hopkins University.
124 BACTERIOLOGY.
a slanting position. With a platinum needle a bit of
the substance to be studied is smeared upon tube No. 1;
without sterilizing the needle it is passed thoroughly
over the surface of the medium in tubes 2, 3, 4, ete.,
etc., in succession. When development has occurred
essentially the same conditions as regards separation of
the colonies will be found as is the case when plates are
poured. If a slanted medium be employed, about the
most dependent angle of which water of condensation has
accumulated, as blood-serum, agar-agar, and potato, the
dilutions may be made in this fluid, and this is then to be
carefully smeared over the solid surface of the medium.
The tubes thus treated should be kept in an upright
position to prevent the fluid from flowing over the sur-
face. When sufficiently developed, single colonies may
be isolated from tubes prepared in this manner with
comparative ease. (See also method for the isolation
of bacillus diphtherie on blood-serum. )
CHAPTER VIII.
The incubating oven —Gas-pressure regulator — Thermo-regulator —The
safety burner employed in heating the incubator.
Tue IycuBpator.—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 luxuriantly
at the temperature of the human body (37.5° C.) than at
lower temperatures; whereas,with the ordinary sapro-
phytic forms almost any temperature between 18° C.
and that of the body is suitable. It therefore becomes
necessary to provide some place in which a constant
temperature favorable to the growth of the pathogenic
organisms can be maintained. For this purpose there
have been devised a number of different forms of appa-
ratus. Fundamentally they are all based upon the same
principles, however, and a general description of the
essential points involved in their construction will be all
that is needed here.
This apparatus has the names thermostat, incubator,
and brooding-oven. It is a copper chamber (Fig. 30)
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 con-
tents of the chamber may be inspected without actually
opening it. The whole apparatus is encased in either
126 BACTERIOLOGY.
asbestos boards or thick felt, to prevent radiation of
heat and consequent fluctuations in temperature. In
the top of the chamber is a small opening through which
a thermometer projects into its interior. At either cor-
ner, leading into the space containing the water, are
Fig. 30.
|
2),
te
= as uly
[ i Ee Dae
\ f
ri |
| i
“ie (isl
a fil uit
oe
Incubator used in bacteriological work.
other openings for the reception of anothcr thermometer
and a thermo-regulator, and for refilling the apparatus
as the water evaporates. On the side is a water-gauge
THE INCUBATOR. 127
for showing the level of the water between the walls.
The object of the water chamber, which is formed by
the double wall arrangement, is to insure, by means of
the warmed water, an equable temperature at all parts
of the apparatus—at the top as well as at the sides,
back, and bottom—and the apparatus should be kept
filled with water, otherwise the purpose for which it is
constructed will not be accomplished. When the cham-
ber between the walls is filled with water heat is sup-
plied from a gas-flame placed beneath it.
Fic. 31.
Koch’s safety burner.
The burner employed in heating the incubator was
originally devised by Koch, and is known as “‘ Koch’s
safety burner’? (Fig. 31). It is a Bunsen burner pro-
vided with an arrangement for automatically turning
128 BACTERIOLOGY.
off the gas-supply and thus preventing accidents should
the flame become extinguished at a time when no one
is near. The gas-cock by which the gas is turned on
and off is provided with a long arm which is weighted,
and which, when the gas is turned on and burning, rests
upon an arm attached to the side of a revolving, hori-
zontal disk that is connected with the free ends of two
metal spirals which are fixed by their other ends in oppo-
site directions on either side of the flame and heated by
it. If by draughts or any other accident the flame be-
comes extinguished, the metal spirals cool, and in cool-
ing contract, twist the horizontal disk in the opposite
direction, and allow the weighted arm of the gas-cock
to fall. By its falling the gas-supply is turned off.
THERMO-REGULATORS.—The regulation and main-
tenance of the proper temperature within the incubator
are accomplished by the employment of an antomatic
thermo-regulator.
The common 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 ex-
pansion and contraction the amount of gas passing
from the source of supply to the burner may be either
diminished or increased as the temperature of the
substance in which the regulator is placed either rises
or falls.
The simplest form of thermo-regulator which serves
to illustrate the principles involved is seen in Fig. 32.
It consists of a glass cylinder e, having a communi-
eating branch tube }, and rubber stopper jf, through
which projects the bent tube a. The tube a is ground
to a slanting point at the extremity which projects into
THERMO-REGULATORS. 129
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 mer-
cury up to about the level shown in the figure. It is
Fie, 32,
Mercurial thermo-regulator.
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 the tube going to the
burner is slipped over the branch tube b. The gas is
130 BACTERIOLOGY.
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 tem-
perature of the bath rises the mercury contained in
the cylinder e, under the influence of the elevation of
temperature, begins to expand, and, as a continuous rise
in temperature proceeds, the expansion of the fluid ac-
companies 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 / will be closed
entirely, when the supply of gas to the burner will now
be limited to that passing through the capillary open-
ing g. This is not sufficient to maintain the highest
temperature reached, and as cooling begins a gradual
contraction of the mercury occurs until there is again
an outflow of gas from the opening A, when again the
temperature rises. This contraction and expansion of
the mercury in the regulator continues until eventually
a point is reached at which its position in the cylinder
e allows of the passage of just enough gas from the
opening A to maintain a constant temperature; and,
therefore, a constant degree of expansion of the mercury
in the tube e. 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 construc-
tion of an accurate instrument. The number of differ-
ent forms of this apparatus is comparatively large, and
each form has its special merits.
The value—that, is, the delicacy—of the thermo-reg-
ulator 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
GAS-PRESSURE REGULATORS. 181
be observed in selecting a thermo-regulator depend in the
main upon the temperatures to which it is to be applied.
For low temperatures, regulators containing such fluids
as ether, alcohol, and calcium chloride solution, which
expand and contract rapidly and regularly under slight
variations in temperature, are commonly employed;
whereas for temperatures approaching the boiling-point
of water mercury is most frequently used.
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.
Fie. 33.
Moitessier’s gas-pressure regulator.
GAS-PRESSURE REGULATORS.—A gas-pressure reg-
ulator is not rarely intervened between the gas-supply
132 BACTERIOLOGY.
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
instruments of this form in use, but they do not ac-
complish the object for which they are designed.
The instrument most commonly employed, the appa-
ratus of Moitessier (Fig. 33), is based on somewhat the
same principles as the large regulators seen at the manu-
factories of illuminating gas. Such apparatus act very
well when employed on the large scale, as one sees them
at the gas-works; but when applied to the limited and
sudden fluctuations seen in the gas coming from an
ordinary gas-cock are practically useless. They are too
gross in their construction, and act only under compar-
atively great and gradual fluctuations in pressure. If
a good form of thermo-regulator be employed, there is
no necessity for the use of any of the pressure-regulators
thus far introduced.
CHAPTER IX.
The study of colonies—Their naked-eye peculiarities and their appearance
under different conditions—Differences in the structure of colonies from
different species of bacteria—Stab-cultures—Slant-cultures.
THE plates of agar-agar which have been prepared
from a mixture of organisms and have been placed in
the incubator, and those of gelatin which have been
maintained at the ordinary temperature of the room,
are usually ready for examination after twenty-four to
forty-eight hours. They will be found 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 cireum-
scribed and round, again irregular in their outline; here
a point will present one color, there perhaps another.
On gelatin some of tbe points will be seen to be lying
on the surface of the medium, others will have sunk
into little depressions, while at still other points the
clear gelatin will be marked by more or less saucer-
shaped pits containing opaque fluid.
Place the plate containing these points upon the
stage of the microscope and examine them with a low-
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;
i
134 BACTERIOLOGY.
here nothing particularly characteristic will present,
there the point may resolve itself into a little mass
having somewhat the appearance of a very small pellicle
of raw cotton. All these differences, and many more,
aid us in saying that these little points must be different
in their nature. With a pointed platinum needle take up
a bit of one of these little islands, prepare it for micro-
scopic examination (see chapter on stained cover-slip
preparations), and examine it under the high-power oil-
immersion objective, with 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 dif-
ferent naked-eye appearances, and it will often be found
to consist of bodies of an entirely different appearance
from those seen 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 first observed
in this colony will be reproduced in all of the new set
of colonies which develop; each will be found to consist
of the same organisms as the colony from which the
plates were made. In other words, these peculiarities
are constant under like conditions.
With all organisms differences in the appearance of
the colonies dependent upon their location in the me-
TEST-TUBE, STAB- AND SMEAR-CULTURES. 135
dium can usually be detected. When deep down in the
medium, owing to surrounding pressure, they are quite
round, oval, or lozenge-shaped; 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 due to the pressure of, or resistance offered by,
the medium surrounding them, and is always to be
borne in mind, otherwise errors are apt to arise.
Pure Cuutures.—If from one of these small col-
onies a bit be taken upon the point of a sterilized plati-
num needle and introduced into the tube of sterilized
gelatin or agar-agar, the growth that results will be
what is known as a ‘‘ pure culture,’”’ the condition to
which all organisms must be brought before a system-
atic study of their many peculiarities is begun. Some-
times several series of plates are necessary before the
organism can be obtained pure, but by patiently follow-
ing 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 sep-
arated from other colonies as possible. Sterilize in the
gas-flame a straight platinum-wire needle. The glass
handie of the needle should be drawn through the flame
as well as the needle itself, otherwise contamination from
this source may occur. When it is cool, which is in five
or ten seconds, take up carefully a portion of the colony.
Guard against touching anything but the colony. If
136 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 result-
ing 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
other 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 por-
tion of the hand; this is accomplished by placing only
the overhanging portion of the plug between the fingers.
The needle containing the bit of colony is now to be
thrust into the medium in the tube if a stab-culture is
desired, or rubbed gently over its surface if a smear-
culture is to be made. The needle is then withdrawn,
the cotton plug replaced, and the needle sterilized before it
is laid down. Neither the needle nor its handle should
touch the inner sides of the test-tube if it can be avoided.
The tube is then labelled and set aside for observa-
tion. The growth which appears in the tube after
twenty-four to thirty-six hours will be a pure culture
of the organisms of which the colony was composed.
TEST-TUBE, STAB- AND SMEAR-CULTURES. 137
Cultures of this form are not only useful as a means
of preserving the different organisms with which we
may be working, but serve also to bring out certain
Fig, 34,
H
i
|
y 4
! i
ie
Series of stab-cultures in gelatin, showing modes of growth of different
species of bacteria.
characteristics of different organisms when grown in
this way.
If gelatin be employed and the organism which has
been introduced into it possesses the power of bringing
138 BACTERIOLOGY.
about liquefaction, it will soon be discovered that this
result is by no means of the same appearance for all
organisms. Some organisms cause a liquefaction which
spreads across the whole upper surface of the gelatin
and continues gradually downward; again, it occurs in
a funnel-shape, the broad end of the funnel being upper-
most and the point downward, corresponding to the
track of the needle. At times a stocking- or sac-like
liquefaction may be noticed. (See Fig. 34.)
Nore.—Obtain a number of organisms from differ-
ent sources in pure cultures by the method given. Plant
them as pure cultures, all at the same time, in gelatin—
preferably gelatin of the same making—retain them
under the same conditions of temperature, and sketch
the finer differences in the way in which liquefaction
occurs.
CHAPTER XX.
Methods of staining — Solutions employed— Preparation and staining of
cover-slips—Preparation of tissues for section-cutting—Staining of tissues—
Special staining-methods.
THE entire list of solutions and methods that are
recommended for the staining of bacteria is not essen-
tial to the work of the beginner, so that only those
which are of most common application will be given
in this book. In general, it suffices to say that bac-
teria stain best with watery solutions of the basic ani-
line 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 fre-
quently 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 decolorization of the
tissues without robbing the bacteria of their color, are
140 BACTERIOLOGY.
employed. The ordinary method of cover-slip exam-
ination of bacteria, constantly in use in these studies, is
performed in the following way:
Cover-sLip Preparations.—In order that the dis-
tribution of the organisms upon the coyer-slips may be
uniform and in as thin a layer as possible it is essential
that the slips 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 immerse
them for a few hours in strong nitric acid, after which
they are rinsed in water, then in alcohol, then ether,
and, finally, they may be kept in alcohol to which a
little ammonia has been added. When about 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 acid 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.
Leeffler’s method, which provides for the complete
removal of all grease, is to warm the cover-slips in con-
centrated sulphuric acid for a time, then rinse them in
COVER-SLIP PREPARATIONS. 141
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 all 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 un-
consciously applied, and the morphology of the organ-
isms in the preparation distorted. When held between
the fingers with the thin 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 protoplasm
of bacteria when moist coagulates at these tempera-
tures, and in doing so the normal outline of the cells is
7*
142 BACTERIOLOGY.
altered. If carefully dried before fixing, this does not
occur and the morphology of the organism remains un-
changed. A better plan for the process of fixing is to
employ a copper plate about 35 cm. long by 10 em.
wide by 0.3 em. 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 hottest, and becom-
ing gradually cooler toward the other end of the plate,
which may be of a very low temperature. . By dropping
water upon the plate, beginning at the hottest point and
proceeding: 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 min-
utes, when the fixing will be complete. The same may
be accomplished in a small copper drying-oven, which
is regulated to remain at the temperature of 95° to
98°C. In very particular work this plan is to be pre-
ferred 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 some-
times occur from the too great and irregular application
of high temperatures may in part be elimiuated, or, if
not, will be more nearly constant. The fixing consists
in drying or coagulating the gelatinous envelope sur-
rounding 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.
COVER-SLIP PREPARATIONS. 143
To stain the fixed cover-slip preparation it is taken
by one of its edges between the forceps, and a few
drops of a watery solution of fuchsin, gentian-violet, or
methylene-blue are placed upon the film and 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 by covering it with blotting-paper and gently press-
ing upon it, and the preparation is ready for examina-
tion.
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 exam-
ination with the microscope. This method is satisfac-
tory 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 be taken for bacteria. If upon examina-
tion 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 blot-
ting-paper, and the slip loosened, or rather floated, from
the slide by allowing water to flow around its edges.
It is then taken up with the forceps, carefully deprived
of the water adhering to it by means of blotting-paper
and then allowed to dry. When dry it is mounted in
xylol-Canada-balsam by placing a small drop of the
balsam upon the surface of the film, and then inverting
the slip upon a clean glass slide. It is sometimes de-
sirable to have the balsam harden quickly, and a method
that is commonly employed to induce this is as follows:
144 BACTERIOLOGY.
the slide, held by one of its ends between the fingers, is
warmed over the gas-flame until quite hot; a drop of
balsam is then placed on the centre of it, and it is again
warmed; the cover-slip is then placed in position, and
when the balsam is evenly distributed the temperature
is rapidly reduced by rubbing the bottom of the slide
with a towel soaked in cold water. Usually the prepara-
tion is firmly fixed after this treatment; a little practice
is necessary, however, in order not to overheat and not
to crack the slide. The method is applicable only to
cover-slip preparations, and cannot be safely used with
tissues.
IMPRESSION COVER-SLIP PREPARATIONS.—The im-
pression preparations differ in value from the ordinary
cover-slip preparations only in one respect: they pre-
sent an impression of the organisms as they were
arranged in the colony from which the preparation is
made. They are made by gently covering the colony
with a thin, clean cover-slip, lightly pressing upon it,
and, without moving the slip laterally, lifting it up by
one of its edges. The organisms adhere to the slip in
the same relation to one another that they had in the
colony. The subsequent steps of drying, fixing, stain-
ing, and mounting are the same as those just given for
the ordinary cover-slip preparations.
By this method constancies in the arrangement and
grouping of the individuals in a colony can often be
made out. Some will always appear irregularly massed
together, others will grow in parallel bundles, while
others, again, will be seen as long, twisted threads.
Nore.—From a colony of bacillus subtilis make a
cover-slip preparation in the ordinary way; now make
ORDINARY STAINING-SOLUTIONS. 145
an impression cover-slip preparation of another colony
of the same organism. Compare the results.
THE ORDINARY STAINING-SOLUTIONS.—The solu-
tions commonly employed in staining cover-slip prepa-
rations are, as has been stated, watery solutions of the
basic aniline dyes—fuchsin, gentian-violet, and meth-
ylene-blue. These solutions may be prepared either by
directly dissolving the dyes in substance in water until
the proper degree of concentration has been reached,
or by preparing them from concentrated watery or alco-
holic solutions of the dyes which may be kept on hand
as stock. The latter method is that commonly prac-
tised.
The solutions of the colors which are in constant use
in staining are prepared as follows:
Prepare as stock, saturated alcoholic or watery solu-
tions of fuchsin, gentian-violet, and methylene-blue.
These solutions are best prepared by pouring into clean
bottles enough of the dyes in substance to fill them to
about one-fourth of their capacity. Each bottle should
then be filled with alcohol or with water, tightly corked,
well shaken, and allowed to stand for twenty-four hours.
If at the end of this time all the staining-material has
been dissolved, more should be added, the bottle being
again shaken and allowed to stand for another twenty-
four hours; this must be repeated until a permanent
sediment of undissolved coloring-matter is seen upon
the bottom of the bottle. The bottles are then to be
labelled saturated alcoholic or watery solution of fuch-
sin, gentian-violet, or methylene-blue, as the case may
be. he alcoholic solutions are not directly employed for
staining-purposes.
146 BACTERIOLOGY.
The solutions with which the staining is accom-
plished are made from these stock solutions in the fol-
lowing 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
taken that the color does not become too dense. The
best results are obtained when it is just transparent as
viewed through a layer of about 12 to 14 mm. thick.
These represent the staining-solutions in everyday
use. They are kept in bottles supplied with stoppers
and pipettes (Fig. 35), and when used are dropped upon
Fic. 35.
Rack of bottles for staining-solutions.
the preparation to be stained. After remaining upon
the preparation for from twenty to thirty seconds they
are washed off in water, and the preparation can then
be examined.
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
ORDINARY STAINING-SOLUTIONS. 147
accomplished by the addition to the solutions of small
quantities of alkaline substances, or by dissolving the
staining-materials in strong watery solutions of either
aniline oil or carbolic acid, instead of simple water—in
other words, by employing special solvents and mor-
dants with the stains.
Of the solutions thus prepared which may always be
employed upon bacteria that show a tendency to stain
imperfectly, there are three in common use—Leeffler’s
alkaline methylene-blue solution; the Koch-Ehrlich
aniline-water solution of either fuchsin, gentian-violet,
or methylene-blue; and Ziehl’s solution of fuchsin in
carbolic acid. These solutions are as follows:
Leeffler’s alkaline methylene-blue solution :
Concentrated alcoholic solution of methylene-blue 30 ¢.c.
Caustic potash in 1:10,000 solution . é fi - 100 ce.
Koch-Ehrlich aniline-water solution. 'To about 100
c.c. of distilled water aniline oil is added, drop by drop,
and the solution thoroughly shaken after each addition,
until it is of an opaque appearance. It is then filtered
through moistened filter-paper until the filtrate is per-
fectly clear. To 100 c.c. of the clear filtrate add 10 c.c.
of absolute alcohol and 11 c¢.c. of the concentrated alco-
holic solution of either fuchsin, methylene-blue, or gen-
tian-violet, preferably fuchsin or gentian-violet.
Ziehl’s carbol-fuchsin solution :
Distilled water . a 100 ¢.c.
Carbolic acid (crystalline) . 5 grammes.
Alcobol . c ri 5 - 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.
148 BACTERIOLOGY.
The Koch-Ehrlich solution decomposes after having
been made for a time, so that it is better to prepare it
fresh in small quantities when needed than to employ
old solutions. Solutions older than fourteen days should
not be used.
The three solutions just given may be used for cover-
glass preparations in the ordinary way.
In some manipulations it becomes necessary to stain
the bacteria very intensely, so that they may retain
their color when exposed to the action of decolorizing
agents. These methods are usually employed when it
is desirable to deprive 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-solution 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, certain of the bacteria will retain their color,
even when exposed to very strong decolorizers. The
tubercle bacillus is distinguished from the great ma-
jority of other bacteria 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.
MerHop 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
contain. 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
that of Ziehl. Itis then held over the gas-flame, at first
some distance away, gradually being brought nearer,
STAINING THE TUBERCLE BACILLUS. 149
until the fluid begins to boil. After it has bubbled
up once or twice it is removed from the flame, the
excess of stain washed away in a stream of water, then
immersed in a 30 per cent. solution of nitric acid in
water, and allowed to remain there until all the color
has disappeared. In some cases this takes longer than
in others. One can always determine if decolorization
is complete by washing off the acid in a stream of water.
If the preparation is still quite colored, it should be
again immersed in the acid; if of only a very faint color,
it may be dipped in alcohol, again washed off in water,
and may now be stained with some contrast-color. If,
for example, the tubercle bacilli have been stained with
fuchsin, methylene-blue forms a good contrast-stain.
In making the contrast-stain the steps in the process
are exactly those followed in the ordinary staining of
cover-slip preparations in general: the slip containing
the stained tubercle bacilli is rinsed off carefully in
water, and a few drops of the methylene-blue solution
are placed upon it and allowed to remain for thirty or
forty seconds, when it is again rinsed in water and ex-
amined microscopically. For the purpose of observing
the difference between the behavior of the tubercle
bacilli and the other organisms present in the prepara-
tion toward this method of staining, it is well to exam-
ine the preparation microscopically before the contrast-
stain is made, then remove it, give it the contrast-color,
and examine it again. It will be seen that before the
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 examining it after it has re-
ceived the contrast-color a great many other organisms
150 BACTERIOLOGY.
will now appear; these will take on the second color
employed, while the tubercle bacilli will retain their
original color. Before decolorization all organisms in
the preparation were of the same color, but during the
application of the decolorizing solution all except the
tubercle bacilli gave up their color. This characteristic,
together with reactions to be described, as said, serves
to differentiate the tubercle bacillus from other organ-
isms with which it might be confounded. A number
of different methods have been suggested for the stain-
ing of tubercle bacilli, but the original method as em-
ployed 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 occa-
sional employment of Ziehl’s carbol-fuchsin solution
and in the method of heating the preparation with the
staining-fluid upon it.
As Nuttall has pointed out, however, the strong acid
decolorizer used in this method can, with advantage, be
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 recommends
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 Fi . ; . 150 ¢.¢.
Alcohol ; a F ‘4 . d0ac
Concentrated sulphuric acid ‘ 20 to 30 drops.
From this it is removed to water and carefully rinsed.
The remaining steps in the process are the same as those
given in the other methods.
GRAM’S METHOD. 151
Gassetr’s Meruop for the staining of tubercle
bacilli recommends itself because of its simplicity and
the rapidity with which it can be performed. By many
it is considered the best method for routine employ-
ment. It consists in staining the cover-slips, prepared
in the manner given, for from two to five minutes in
acold carbol-fuchsin solution, after which they are sub-
jected to the action of Gabbett’s methylene-blue sul-
phuric acid solution. This latter consists of
Sulphuric acid, strength 25 per cent. ‘i - 100 ce.
Methylene-blue, in substance . é < a 1 to 2 grammes.
They are then rinsed off in water and are ready for
examination. The tubercle bacilli will be stained red
by the fuchsin, while all other bacteria, cell nuclei,
etc., will be tinted blue.
Gram’s Mernuop.—Another differential method of
staining which is very commonly employed is that
known as Gram’s method. In this method the objects
to be stained are treated with an aniline-water solution
of gentian-violet made after the formula of Koch-
Ehrlich. After remaining in this for twenty to thirty
minutes they are immersed in an iodine solution com-
posed. of
Iodine . . z 1 gramme.
Potassium iodide E ‘i 2 grammes.
Distilled water. 7 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.
152 BACTERIOLOGY.
This method is particularly useful in demonstrating
the capsule which is seen to surround some bacteria,
particularly the micrococcus lanceolatus of pneumonia.
GuaciaL Acetic Actip MetHop.—Another method
which may be employed for demonstrating the presence
of the capsule surrounding certain organisms is to pre-
pare the cover-slips in the ordinary way, then cover the
layer of bacteria upon them with glacial acetic acid,
which is instantly poured off (not washed off in water),
and the aniline-water gentian-violet solution dropped
upon them; this is allowed to remain three or four
minutes, is poured off, and a few drops more are added,
and lastly the slip is washed off in a solution of sodium
chloride. Usually this is of the strength of the ordinary
physiological salt-solution, viz., 0.6 to 0.7 per cent., but
at times the strength must be greater, sometimes in-
creased to from 1.5 to 2 per cent. of salt. The reason
for this is that if the slips be washed in water, or in
salt-solution that is too weak, the mucin capsule that
has been coagulated by the acetic acid is redissolved
and rendered invisible. This does not occur when the
salt-solution is of the proper strength—a point that can
be determined only after a few trials with solutions of
different strengths. (Welch.) A very clear, sharply cut
picture usually follows this method of procedure.
STAINING oF SporEs.—We have learned that one of
the points by which spores may be recognized is their
refusal to take up staining-substances when applied in
the ordinary way. They may, however, be stained by
special methods; of these, one that has given very satis-
factory results in our hands is as follows: the cover-
slip is to be prepared from the material containing the
spores in the ordinary way, dried, and fixed. It is then
STAINING OF SPORES. 153
to be held by its edge with the forceps, and its surface
covered with Leeffler’s alkaline methylene-blue solu-
tion. It is then held over the Bunsen flame until the
fluid boils; it is then removed, and after a few seconds
is heated again. This is continued for about one min-
ute, after which it is washed off in water and dipped
five or six times in alcohol containing about 0.2 to 0.3
per cent. of hydrochloric acid. This is rinsed off in
water and the preparation is now stained for from eight
to ten seconds in aniline-water fuchsin solution (Koch-
Ehrlich solution), and finally again washed in water.
By this method the spores are of a blue color and the
body of the cell red.
By another process the cover-slip is floated, bacteria
down, upon the surface of freshly prepared Koch-
Ehrlich solution of fuchsin contained in a watch-crys-
tal. This is then held by its edge with the forceps about
2 cm. above a very small flame of a Bunsen burner,
care being taken that the flame touches only the centre
of the bottom of the crystal. After a few seconds the
crystal is elevated gradually until it is about 6 to 8 cm.
above the flame, then it is slowly moved down to the
flame again, and this up-and-down movement is con-
tinued 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, when the process of heating is repeated.
When the boiling begins the crystal is held aside again
for a minute ortwo. ‘The crystal is heated in this way
for about five or six consecutive times. When the fluid
has stood for about five minutes after the last boiling
the preparation is transferred, without washing in water,
into a second watch-crystal containing the following
decolorizing solution:
154 BACTERIOLOGY.
Absolute alcohol . é . e F 100 c.c.
Hydrochloric acid c : . ¥ a 7 P 3 ¢.c.
In this solution it is placed, bacteria up, and the
vessel is tilted from side to side for about one minute.
Tt 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.
MoeLuer’s Metsop ror STArninc 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 in-
stead 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 carbol-fuchsin. In the
process of staining, the slip is taken by the corner with
the forceps, and carbol-fuchsin is dropped upon the
side containing the spores. It is then held over the
flame until it boils, and then held some distance above
the flame for one minute. The staining-fluid is then
-poured off and the preparation is completely decolorized
in 5 per cent. sulphuric acid, again washed off in water,
and finally stained for thirty seconds in the watery
methylene-blue solution. The spores will be red, the
body of the cells blue.
In this method the object of the preliminary ex-
posure to chloroform is to dissolve away any crystals
of lecithin, cholesterin, or fat that may be in the pre-
METHODS FOR STAINING FLAGELLA. 155
paration, and which when stained might give rise to
confusion.
It must be remembered that there are conspicuous
differences in the behavior of spores of different bacteria
to staining-methods. Some stain readily by either of
the methods especially devised for this purpose, while
others can hardly be stained at all, or only with the
greatest difficulty, by any of the known processes.
Loarr.er’s Mernop ror Sraining FLAGELLA.—
For the demonstration of the locomotive apparatus pos-
sessed by motile bacteria we are indebted to Leefiler.
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 as given by Loeffler is as follows:
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 Leeffler’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:
156 BACTERIOLOGY.
Tannic acid solution in water (20 acid, 80 water) ‘ . 10 ce.
Cold saturated solution of ferro-sulphate . e . » 54¢
Saturated watery or alcoholic solution of fuchsin , » lee
This solution represents the mordant. A few drops
of it are to be placed upon the film of bacteria on the
cover-slip, which is then to be held over the flame until
the solution begins to steam. It should not be boiled.
After steaming, the mordant is washed off in water and
finally in alcohol. The bacteria are then to be stained
in a saturated aniline-water-fuchsin solution.
When treated in this way different bacteria behave
differently: the flagella of some stain readily in the
above solutions; others require the addition of an alkali
in varying quantities; while others stain best after the
addition of acids. To meet these conditions an exact
1 per cent. solution of caustic soda in water must be
prepared, and also a solution of sulphuric acid in water
of such strength that one cubic centimetre will be ex-
actly neutralized by one cubic centimetre of the alkaline
solution.
For different bacteria which have been studied by
this method the one or the other of these solutions is
to be added to the mordant in the following propor-
tions.
Of the acid solution:
For the bacillus of Asiatic cholera ‘ ¥ to 1 drop.
For the spirillum rubrum . 9 drops.
Of the alkaline solution:
For the bacillus of typhoid fever . : lee.
For the bacillus subtilis 5 3 28 to 30 drops.
For the bacillus of malignant cedema 361037
For other organisms one must determine whether the
results are better after the addition of acid or alkali,
and how much of either is required. In general, it may
BUNGE’S METHOD. 157
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 Leeffler’s direc-
tions the delicate, hair-like flagella on motile organisms
may be rendered plainly visible.
There are several points and slight modifications in
connection with this method that require to be empha-
sized in order to insure success: the culture to be em-
ployed should be young, not over 18-20 hours old. It
should have developed for this time on fresh agar-agar
at 37° to 38° C.; the mordant should not be perfectly
fresh, as the best results are obtained from the use of
old solutions that have stood exposed to the air, and
that have been filtered just before using; when placed
on the cover-slip and held over the flame never heat the
mordant to the boiling-point ; indeed, the best results are
obtained when the preparation is held high above the flame
and removed from it at the first evidence of vaporization,
or, better still, a little before this point is reached. We
have derived no advantage from the addition of acids
or alkalies to the mordant, as recommended by Jeeffler;
but obtain, with a fair degree of regularity, satisfactory
results through the use of the neutral mordant alone.'
Bunce’s MEetuop.—A useful modification of Leef-
fler’s method is that recommended by Bunge: prepare
a saturated solution of tannin, and a solution of liquor
ferri sesquichlor. of the strength of 1:20 of distilled
water. To 3 parts of the tannin solution add 1 part of
the dilute iron solution. To 10 c.c. of such a mixture
1 Jam indebted to Dr. James Homer Wright, Thomas Scott Fellow in Hy-
giene, 1892-'93, University of Pennsylvania, for some of the suggestions in
connection with the modification of this method.
8
158 BACTERIOLOGY.
add 1 e.c. of concentrated watery solution of fuchsin.
This mordant is not to be used fresh, but only after
standing exposed to the air for several days (better for
several weeks). After preparing the cover-slip with all
precautions necessary to cleanliness the filtered mordant
is allowed to act cold for about five minutes, after which
it is slightly warmed; the slip is then washed off in
water, dried, and faintly stained with carbol-fuchsin.
No addition of acid or alkali to the mordant is neces-
sary.
Toe Mersop or Van ERMENGEM. — Another
method of demonstrating the presence of flagella is that
suggested by Van Ermengem. It is somewhat more
complicated than either of the preceding methods. The
steps in the process are as follows:
In the centre of a perfectly cleaned cover-slip place
a drop of a very dilute suspension, in physiological salt-
solution, of a 10- to 18-hour old agar-agar culture of
the organism to be studied. The suspension of the
organisms in the salt-solution should be very dilute in
order to favor the isolation of single cells on the slip
and also to obviate the occurrence of excessive precip-
itation. The slips are then to be dried in the air and
in the gas-flame in the usual manner.
The mordant used consists of:
Osmic acid (2 per cent. solution) 1 part.
Tannin (10-25 per cent. solution) z 2 parts.
To this 4 or 5 drops of glacial acetic acid may be
added, but experience has shown this to be hardly
necessary.
Place a drop or two of this mordant on the cover-slip
to be stained, and allow it to act for one-half hour at
room temperature, or for five minutes at 50° to 60° C.
STAINING IN GENERAL. 159
Wash carefully in water and alcohol, and then im-
merse for a few seconds in the ‘ sensitizing bath,’ viz.,
a 0.25-0.5 per cent. solution of silver nitrate. With-
out washing, bring the slip into a watch-crystalful of
the ‘‘ reducing and reinforcing bath,’’ viz.:
Gallic acid 5 grains,
Tannin é . . ‘ 3
Fused pot. acetate . 10 46
Dist. water 4 H 350“
After a few seconds pass the slip back into a watch-
erystal containing the dilute silver bath (0.25-0.5 per
cent. solution of silver nitrate in water) and keep it
in constant motion until the solution begins to take on
a brown or blackish:color. Wash in water thoroughly y
dry with blotting-paper, and mount in balsam.
STAINING IN GENERAL.
The physics of staining and decolorization is hard]
a subject to be discussed at length in a book of this
character; but, as Kithne has pointed out, it may be
said that solutions which favor the production of diffu-
sion 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 mat-
ters are less intensely stained and more easily decolor-
ized 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 protoplasm
of dried bacteria, as found upon cover-slip preparations,
160 - BACTERIOLOGY.
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-
trated. Prepare a cover-slip preparation, dry it care-
fully, fix it, and, without allowing water to get on it
from any source, attempt to stain it with a solution of
the dyes in absolute alcohol, washing it out subsequently
with absolute alcohol; the result is negative. The abso-
lute 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.’
DECOLORIZING-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 decolor-
izer in use is probably aleohol—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 ener-
getic decolorization than that possessed by either alone
can be obtained by alternate exposures to aleohol and
1 In the beginning of this chapter it was stated that the saturated alcoholic
solutions of the dyes do not serve as stains for bacteria. It must be remem-
bered that this holds only when absolute alcohol and perfectly dry coloring
matters have been used. If but asmall proportion of water is present, the
bacteria may be stained with these solutions, though the results are, as a
rule, unsatisfactory.
STAINING OF BACTERIA IN TISSUES. 161
water. More energetic in their decolorizing action than
either water or alcohol are solutions of the acids. They
appear, 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 employed with much care.
Very dilute acetic acid robs tissues and bacteria of
their stain with remarkable activity; still more ener-
getic are solutions of the mineral acids, and particularly,
as has been said, when this action is accompanied by
the decolorizing-properties of alcohol.
The acid solutions that are commonly employed are:
Acetic acid in from 0.1 per cent. to 5 per cent. watery
solution.
Nitric acid in from 20 per cent. to 30 per cent. watery
solution.
Hydrochloric acid in 3 per cent. solution in alcohol.
STAINING OF BACTERIA IN TISSUES.
In staining tissues for the purpose of demonstrating
the bacteria which they may contain a number of points
must be borne in mind: the conditions which favor the
diffusion of the staining-fluids into the bacteria are now
not so favorable to rapid staining as they were when
the bacteria alone were present upon cover-slips; the
staining of tissues, therefore, requires a longer exposure
to the dyes than does that of cover-slips. In tissues,
too, there are other substances beside the bacteria which
become stained, and these, unless robbed in whole or in
part of their color, may so mask the stained bacteria as
to render them difficult, if not impossible, of detection.
162 BACTERIOLOGY.
Tissues must, therefore, always be subjected to some
degree of decolorization, and this must be accomplished
without depriving the bacteria of their color.
The details of the method of decolorization will be
described in the section on the technique of staining.
Another point to be remembered in staining tissues
is that they cannot be heated and retain their structure
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 to lower temperatures.
HARDENING THE TissuEs.—The bits of tissue—not
greater than one cubic centimetre—are to be placed, as
fresh as possible, in absolute alcohol. The bit of tissue
should rest upon a pad of cotton or filter-paper in the
bottle containing the alcohol, in order that it may be ele-
vated 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, about half
a cubic centimetre, should be cemented to a bit of cork
with ordinary mucilage, and allowed to remain in the
open air for a minute or two for the mucilage to harden,
Alcohol should be dropped upon it occasionally to pre-
vent drying of the tissue. When the mucilage is hard
the cork with the piece of tissue upon it may be left in
alcohol over night, and on the following day the sec-
tions may be cut.
SECTION-CUTTING.—This is accomplished by the use
of an instrument known asa microtome. In Fig. 36
SECTION-CUTTING. 163
is seen the form now commonly employed. It is known
by the name of the maker, as Schanze’s microtome.
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 a horizontal plane. Be-
neath the clamp for holding the tissue is a milled disk,
Fia, 36.
han i
ie a ut
=>)
Schanze’s microtome.
by means of which a screw is caused to revolve, and in
revolving raises or lowers the clamp holding the tissue,
so that the tissue may be brought closer to or farther
from the plane in which the knife slides. By this
arrangement sections of any desired thickness can be
cut by turning the milled disk with the one hand and
causing the knife to traverse the tissue with the other.
The tissue and the knife-blade should be kept wet
164 BACTERIOLOGY.
with alcohol, so that the sections may float upon the
blade of the knife, from which they can be easily re-
moved, without tearing, with a curved needle or a
camel-hair pencil. As the sections are cut they are
placed in a dish containing alcohol.
There are some tissues which, by reason of their
histological 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 sub-
stance that neither interferes with their structure, nor
prevents their being cut into sections. They must be
““imbedded,’’ as this process is called.
Imbedding in celloidin. Most convenient for this
purpose is celloidin, a body somewhat similar to collo-
dion, soluble in a mixture of equal parts of alcohol
aud ether, as well as in absolute alcohol.
After hardening in alcohol the tissue to be imbedded
is placed in a mixture of equal parts of absolute alcohol
and ether and left there for twenty-four hours. It is
then transferred to celloidin. Two solutions of celloidin
are to be employed, the one a thin solution in a mixture
of equal parts of absolute alcohol and ether, the other a
thick solution in the same solvent. Into the thin solu-
tion, which should be of about the consistence of very
thin syrup, the tissue is placed from the absolute alcohol
and ether, and allowed to remain there for twenty-four
hours. It is then placed in a thick solution for about
aday. From this it may be removed and placed imme-
diately upon a bit of cork or a block of wood. The
adherent celloidin will act as a cement, and as it hardens
rapidly the tissue is soon fast to the cork. It is then
left in 60 per cent. alcohol for twenty-four hours to
STAINING OF THE SECTIONS. 165
complete the solidification of the celloidin, after which
sections may be cut in the way just described for tissues
not so treated.
Imbedding in paraffin. After bits of the tissue not
larger than a cubic centimetre have been hardened in
the usual way they are placed in fresh absolute alcohol
for twenty-four hours to complete the process. From
this they are transferred to pure turpentine, and kept in
a@ warm oven at a temperature not exceeding 35° to
38° C. Here they remain for a time sufficient for them
to become thoroughly saturated with the turpentine, as
is recognized by the transparent appearance that they
assume. From this they are placed in paraffin that is
melted at 53° C., and allowed to remain in this for
three or four hours. They are then transferred to a
small paper or metal mould, or a pill-box, and melted
paraffin is poured over them. When the paraffin has
become solid the mould or pill-box is removed from
around it, the excess of paraffin removed from about
the imbedded tissue, and the latter is ready for cutting.
When the sections are cut they are freed from par-
affin by exposing them to turpentine; the latter is re-
moved by washing in alcohol and the sections can now
be stained in the ordinary way. In cutting sections
from tissues that have been imbedded in paraffin the
long axis of the knife should be at nearly right angles
to the direction in which the knife travels. For bacte-
riological purposes the method of imbedding in paraffin
does not, as a rule, give such good results as when the
celloidin method is employed. In this work, therefore,
the latter is usually preferred.
STAINING OF THE Sections.—The sections when cut
may be stained in a variety of ways. The ordinary
8X
166 BACTERIOLOGY.
watery solutions of the three common basic aniline dyes
—tfuchsin, gentian-violet, and methylene-blue—or, what
is better, the alkaline methylene-blue solution of Leef-
fler may be employed for general use.
Some of the acid aniline dyes, as well as some of the
vegetable coloring matters, are essentially nuclear stains,
and are not applicable to the staining of bacteria.
Into a watch-glass containing either of the staining-
solutions mentioned the sections are to be placed after
having been in water for about one minute. They re-
main 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. solution of acetic acid
for only a few seconds; again washed out in water, then
in absolute 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 care-
fully 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 floats in one
hand, with its point just touching the surface of the
glass slide, and then with a needle pull the section
gently off upon the slide. The fluid comes with it, and
the floating section may be easily spread out into a flat
surface. The excess of fluid is taken up with blot-
ting-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 examina-
tion.
Each step in the above process has its definite object.
STAINING OF THE SECTIONS. 167
The sections are placed in water before staining in order
that the diffusion of the staining-solution into the tis-
sues may be diminished; otherwise our efforts at render-
ing 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 stain in the
tissues, though they have also some decolorizing action
upon the bacteria. The cedar oil and xylol are bodies
which mix on the one hand with alcohol, and on the
other with balsam. They are known as ‘clearing
fluids,’’ and not only serve to differentiate the compo-
nent parts of the tissue, but fill up the gap that would
otherwise be left in the process, for a section cannot
be mounted in balsam directly from alcohol; the two
bodies do not mix perfectly.
A number of clearing agents are in general use; in
fact, almost all the essential oils come under this head.
There is one—oil of cloves—which is very commonly
used in histological work; but it must not be employed
in tissues containing bacteria. It not only extracts too
much color from the bacteria, but causes them to fade
after the sections have been mounted for a time.
When the section thus stained and mounted is exam-
ined microscopically it may be found that the tissues
still possess so much color that the bacteria are not vis-
ible, in which case they have not been decolorized suffi-
ciently; or, on the other hand, both bacteria and tissues
may have parted with their stains—then decolorization
has been carried too far. In either case the fault must
be remedied in the manipulation of the next section to
be mounted.
168 BACTERIOLOGY.
In short, the steps in the process of staining sections
in general are these:
a. From aleohol into distilled water for one minute.
b. Into the staining-fluid for from five to eight min-
utes.
c. Into water for from three to five minutes.
d. Into 0.1 per cent. acetic acid for about one-half
minute.
Into absolute alcohol for a few seconds.
Into absolute alcohol again for a few seconds.
. Xylol for about one-half minute.
. Removal with spatula or section-lifter to slide.
i. Removal of excess of xylol.
j- Mounting in xylol-balsam.
The section must be lifted from one vessel to the other
by means of either a curved needle or a glass rod drawn
out to a fine end and bent in the form of a curved needle.
By the above process of staining, which can be prac-
tised as a routine method for most bacteria in tissues,
the nuclei of the tissue cells, as well as the bacteria, will
be more or less deeply stained.
SeecraL Metuops or Sraininc BacrTEeria IN
Tissues. —For purposes of contrast-stains it sometimes
becomes necessary to decolorize completely, or nearly
completely, the tissues and leave the bacteria unaltered
in color. For this purpose special methods depending
on the staining-peculiarities of the bacteria under con-
sideration have been devised.
Gram’s method with tissues. One of the most com-
monly employed differential stains is that of Gram.
In general, it is practised in the way given for its em-
ployment on cover-slip preparations, with some slight
modifications.
~Q Ko
STAINING OF THE SECTIONS. 169
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, pref-
erably 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 Bis-
marck-brown, again washed out for a few seconds in
alcohol, and finally for one-fourth minute in absolute
alcohol. From this they are transferred to xylol for a
half-minute. The remaining steps in the process are
the same as those given in the general method. In
some cases better results are oktained by reversing the
steps in the process and staining the bacteria last, for
then the frequent decolorizing action of the alcohol on
the bacteria is diminished; thus, place the sections from
alcohol into eosin, safranin, or Bismarck-brown for a few
minutes, then wash out in 50 per cent. alcohol, then for
from three to five minutes in the dilute aniline-water
gentian-violet solution, then into the iodine bath, after
three minutes wash out in alcohol, and, finally, for one-
fourth minute in absolute alcohol, and then into the
xylol, from which they may be mounted. Some of the
organisms which may be stained by this method are
micrococcus tetragenus, b. diphtherie, b. anthracis, and
staph. pyogenes aureus. It cannot be successfully em-
ployed with the bacillus of typhoid fever.
170 BACTERIOLOGY.
Staining with dahlia and decolorizing with sodium car-
bonate solution. Another method that is not very com-
monly employed, though the results obtained by its use
are in many cases very satisfactory, is to stain the tis-
sues in a strong watery solution of dahlia (about one-
fourth saturated) for from ten to fifteen minutes; from
this they are transferred into a 2 per cent. solution of
sodium or potassium carbonate, and from this into alco-
hol, alternating from the one to the other until the sec-
tion is almost colorless. From the aleohol they are
rinsed out in water and then put into a dilute watery
solution of either eosin, Bismarck-brown, or safranin for
one minute, then washed out in alcohol, finally in abso-
lute alcohol, and then in xylol, from which they may be
mounted in the manner given.
Especially brilliant results are obtained when tissues
containing anthrax bacilli are stained by this process;
the bacilli will be of a deep blue color, while the sur-
rounding tissues will be,of the color used as contrast.
Kiihne’s carbolic methylene-blue method. Stain the
sections in the following solution for from one-half to
one hour:
Methylene-blue, in substance 1.5 grammes.
Absolute alcohol 10 «Ge.
Rub up thoroughly in a mortar, and when the blue
is completely dissolved add gradually 100 ¢.c. of a 5
per cent. solution of carbolic acid. (The solution de-
composes after a short time; it should be made fresh
when needed.) From this the sections are washed out
in water, then in 1.5 to 2 per cent. hydrochloric acid
in water, from this they are transferred to a solution of
lithium carbonate of the strength of six to eight drops
of a concentrated watery solution of the salt to ten drops
STAINING OF THE SECTIONS. 171
of water, and from this they are again thoroughly
washed in water, then in absolute alcohol containing
enough methylene-blue in substance to give it a toler-
ably dense color, then for a few minutes in aniline oil
to which a little methylene-blue in substance has been
added, then completely rinse out in pure aniline oil;
from this they are passed into thymol or oil of turpen-
tine 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 decolorized to render
the bacteria visible and admit of the use of contrast-
stains.
Weigert’s modification of Gram’s method for sections.
Stain the sections in the Koch-Ehrlich aniline-water
gentian-violet solution for five or six minutes; wash
out in water or physiological salt-solution (0.6 to 0.7
per cent. solution of sodium chloride in distilled water) ;
transfer them with the section-lifter to the slide; take
up the excess of fluid by gently pressing upon the flat
section with blotting-paper; treat the section with the
iodine solution used by Gram; take up the excess of
the 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 ani-
line-water gentian-violet for five or six minutes—or
longer, if necessary—are transferred to the slide without
being washed in water or salt-solution (or, if so, only
very slightly and rapidly), dried as completely as possi-
172 BACTERIOLOGY.
ble with filter-paper, and then decolorized with a mix-
ture 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 (repeated applications of the
aniline oil and xylol mixture are generally necessary)
pure xylol replaces the mixture, and the specimen is
finally mounted in xylol-balsam. Unless all the ani-
line 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 satisfactorily, is nevertheless de-
signed especially for the staining of fibrin. Fibrin and
hyaline material will be stained deep blue, bacteria a
dark violet.
SraIninG OF TUBERCLE BAcILLI IN TissuEs.—As
for the staining of cover-slips, only those methods most
commonly employed will be given.
The method of Ehrlich. Stain the sections in aniline-
water fuchsin or gentian-violet for twenty-four hours;
decolorize in 20 per cent. nitric acid for a few seconds
only—the color need not be entirely extracted; then into
70 per cent. alcohol until no more color can be extracted
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 abso-
lute alcohol for a few seconds; clear up in xylol and
mount in xylol-balsam.
Method of Zehl-Neelsen. Stain the sections in warmed
carbol-fuchsin solution for one hour; temperature to be
about 45° to 50° C. Decolorize for a few seconds in 5
STAINING OF TUBERCLE BACILLIIN TISSUES. 173
per cent. sulphuric acid, then in 70 per cent. alcohol,
and from this on as by the Ehrlich method.
Dry method. For tubercle bacilli, as for many other
organisms in tissues, the following method may be
employed if only the presence of organisms is to be
detected and the histological condition of the tissues is
a matter of no consequence: bring the sections from
water upon a slide or cover-slip, dry, fix, and stain by
the methods for cover-slip preparations.
Gray’s method. The method employed by Gray at
the Army Medical Museum, Washington, D. C., a de-
scription of which is given by Borden, is as follows:
the tissue to be stained should be hardened, preferably
in alcohol, in pieces not exceeding 1.5 by 1.5 by 1 em.
in size, though tissues hardened by any other of the
regular methods can be stained. Alcohol is to be pre-
ferred, however, as after its use the bacilli stain more
quickly and brilliantly than when one of the other
hardening fluids—Miiller’s, for instance—is employed.
After the tissue has been hardened it is imbedded in
paraffin, and cut in the usual manner. The sections
are then cemented to the slides with a filtered $ per cent.
solution of gold-label gelatin, to which is added chloral
hydrate in the proportion of 1 percent., as a preservative.
Several drops of this are placed on each slide, a section
laid on top, and the slides placed in a warming-oven
kept at a temperature slightly below the melting-point
of the paraffin. In about five minutes all wrinkles will
have been taken out of the sections, which will lie per-
fectly flat and smooth on the surface of the gelatin solu-
tion. The slides are then removed from the oven and
the surplus fluid poured from them, thus bringing the
sections in contact with their surface, after which they
174 BACTERIOLOGY.
are set aside in a place protected from dust, to remain
until the sections are firmly cemented to them by the
drying of the gelatin solution. The drying may be
hastened by keeping the slides in an oven below the
melting-point of the paraffin, but it is best to set the
slides aside until the next day, when the sections will
be found to be perfectly cemented to them. The par-
affin is then removed from the sections by turpentine,
the turpentine by absolute alcohol, the absolute alcohol
by 50 per cent. alcohol, and this by water, after which
the slides are placed in a 5 per cent. aqueous solution of
potassium bichromate for five minutes. This renders
the gelatin insoluble, and prevents the sections from
leaving the slides during their necessarily more or less
prolonged immersion in the fuchsin stain. The potas-
sium bichromate is washed out with water, and the slides
are then placed in a fuchsin stain, which is prepared as
follows:
Fuchsin ‘ ‘ 1.5 grammes.
Absolute alcohol 14 ee.
Carbolic acid crystals (pure) 6 grammes.
Water : - 100 Ge.
Dissolve the fuchsin in the alcohol and the carbolic
acid in the water. Mix the two solutions and let stand
for twelve hours, with occasional shaking or stirring;
then filter.
The length of time that the slide remains in this solu-
tion varies with circumstances. The tubercle bacilli
stain very quickly; in tissues properly hardened in
alcohol five minutes are generally sufficient to stain
them deeply.
Prolonged immersion in the fuchsin does no harm
and insures certainty of results. After a section has
STAINING OF TUBERCLE BACILLI IN TISSUES. 175
been in the stain a sufficient length of time it, with the
slide to which it is cemented, is washed in water until
the surplus stain is removed; it is then subjected to the
action of a combined decolorizer and contrast-stain made
as follows:
Methyl-blue 2.25 grammes.
Absolute alcohol 30 c.e,
Sulphuric acid . : me ee
Water (distilled) ‘ loo
Dissolve the methyl-blue in the alcohol, add the acid
to the water, mix the two solutions, and Jet stand, with
occasional shaking, for twelve hours; then filter.
This solution is allowed to act upon the tissue for a
few seconds, and as soon as the blue color predominates
over the red, as seen by transmitted light, the section is
immediately washed in water. Generally the red color
reappears, and the section must be again subjected to
the action of the blue solution and again washed in
water. This must be repeated until the blue almost,
if not quite completely and permanently, replaces the
red stain. This is the most important part of the pro-
cess, and entirely satisfactory results are only obtained
after some practice. The tendency is usually not to
replace sufficiently the fuchsin with the methyl-blue,
and in consequence the red color of the bacilli is masked
by the red of the surrounding tissues. Unless all acid
is thoroughly removed by the final washing in water
the stain is not permanent. ‘The section is then com-
pletely dehydrated with absolute alcohol, after taking
up the excess of water on the slide with blotting-paper.
The alcohol is followed by turpentine, and the process
is completed by mounting in xylol-balsam.
In case it is desired to stain sections cut by the freez-
176 BACTERIOLOGY.
ing method, they are placed upon a slide on which a
few drops of the gelatin fixative have been placed, and
after about five minutes, during which the fixative will
have penetrated the section, the surplus is poured from
beneath the section. The slides are then set aside for
the gelatin to harden by drying, and after drying they
are placed in bichromate fluid to render the gelatin
insoluble. They are then manipulated in exactly the
same manner as the sections cut by the paraffin method.
This method gives equally as good results with tissues
containing the lepra bacillus as with those containing
tubercle bacilli.
CHAPTER XI.
Systematic study of an organism—Points to be considered in identifying
an organism as a definite species.
AFTER isolating an organism by the plate method
considerable work is necessary in order to establish its
identity as a definite species.
It must possess certain morphological and cultural
peculiarities, which must be constant under constant
conditions.
Its form at different stages must always be the same.
Its ability or inability to produce spores must not vary
under proper conditions. Its growth upon the different
media under constant conditions of temperature and
reaction must always present the same outward appear-
ances. The changes brouglit about bv it in the reaction
of the media in which it is growing must follow a fixed
rule. Its power to produce liquefaction of the gelatin,
or to grow upon it without bringing about this change,
must always be the same. Its motility or non-motility,
and, if motile, the approximate number and position of
its organs of locomotion, must be determined. Its pro-
duction of certain chemical products must be detected
by chemical analysis. Its behavior toward oxygen—
i.e., Does it require this gas for its growth? Is this gas
an indifferent factor? or, By its presence are the life-
processes of the organism checked ?—must be decided.
Its behavior under varying conditions of temperature
and under the influence of different chemical bodies, as
178 BACTERIOLOGY.
well as its growth in media of different reactions, is
to be studied. The property of producing fermentation
with the liberation of gases, and the character and quan-
titative relations of these gases, must be ascertained; if
it produces pigment, what are the conditions favorable
and unfavorable to this function; and, lastly, we must
consider its behavior when introduced into the bodies
of animals used for experimental work—4.e., Is it a dis-
ease-producing organism, or does it belong to the group
of innocent saprophytes ?
We have learned the methods of obtaining colonies,
and have acquainted ourselves with some of the pecu-
liarities by which they are distinguished from one
another. The next important steps are 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 microscopic examination of
bits of the colony which are transferred to thin glass
cover-slips, upon which they are dried, stained, and
mounted, Cover-slips for this purpose are prepared in
two ways: either by taking up a bit of the colony on a
platinum needle, smearing it upon a cover-slip, staining
it, and examining it—by which only the morphology of
the individual bacteria 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 method of staining.
MICROSCOPIC EXAMINATION OF PREPARATIONS.
Tuer Dirrerent Parts or THE MIcROscOoPE.—
Before describing the process of examining prepara-
DIFFERENT PARTS OF THE MICROSCOPE. 179
tions microscopically, a few definitions of the terms
used in referring to the microscope may not be out of
Fic, 37.
place. (The different parts of the microscope referred
to below are indicated by letters in Fig. 37.)
180 BACTERIOLOGY.
The ocular or eye-piece (A) is the lens at which the eye
is placed in looking through the instrument. It serves
to magnify the image projected through the objective.
The objective (B) is the lens which is at the distal end
of the barrel of the instrument, and which serves to
magnify the object to be examined.
The stage (c) is the shelf or platform of the micro-
scope on which the object to be examined rests.
The diaphragms are the perforated stops that fit in
the centre of the stage. They vary in size, so that dif-
ferent amounts of light may be admitted to the object
by using diaphragms with larger or smaller openings.
The “Iris” diaphragm (pD) opens and closes like the
iris of the eye. It is so arranged that its opening for
admission of light can be increased or diminished by
moving a small lever in one or another direction.
The reflector (E) is the mirror placed beneath the stage,
which serves to direct the light to the object to be ex-
amined.
The coarse adjustment (F) is the rack-and-pinion ar-
rangement by which the barrel of the microscope can
be quickly raised or lowered.
The fine adjustment (G) serves to raise and lower the
barrel of the instrument very slowly and gradually.
For the microscopic study of bacteria it is essential
that the microscope be provided with an oil-immersion
system and a sub-stage condensing apparatus.
The oil-immersion or homogeneous system consists of
an objective so constructed that it can only be used when
the transparent 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
EXAMINATION OF COVER-SLIPS. 181
the object to be examined a body which refracts the
light in the same way as do the glass slide, the cover-
slip, and the glass of which the objective is made. For
this purpose a drop of oil of the same index of refrac-
tion as the glass is placed upon the face of the lens,
and the examinations are made through this oil. There
is thus no loss of light from deflection, as is the case in
the dry system.
The sub-stage condensing apparatus (H) is a system
of lenses situated beneath the central opening of the
stage. They serve to condense the light passing from
the reflector to the object in such a way that it is
focussed upon the object, thus furnishing the greatest
amount of illumination. Between the condenser and
reflector is placed the ‘‘Iris’’ 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.
The nose-piece (I) consists of a collar, or group of
collars joined together (two or more), that is attached to
the distal end of the tube of the microscope. It enables
oné to attach several objectives to the instrument in
such a way that by simply rotating the nose-piece the
various lenses of different power may be conveniently
used in succession.
Microscopic EXAMINATION OF CovER-SLIPS.—The
stained cover-slip is to be examined with the oil-immer-
sion objective, and with the diaphragm of the sub-stage
condensing apparatus open to itsfull extent. The object
gained by allowing the light to enter in such a large vol-
ume is that the contrast produced by the colored bacteria
in the brightly illuminated field is much more conspic-
uous than when a smaller amount of light is thrown upon
9
182 BACTERIOLOGY.
them. This is true not only for stained bacteria on
cover-slips, but likewise for their differentiation from
surrounding objects when they are located in tissues.
With unstained bacteria and tissues, on the contrary, the
structure can best be made out by reducing the bundle
of light-rays to the smallest amount compatible with
distinct vision, and in this way favoring, not color-con-
trast, 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.
Sreps in Examiyinc STAINED PREPARATIONS
WITH THE OIL-IMMERSION SysTEM.—Place upon the
centre of the cover-slip which covers the preparation a
small drop of immersion oil. Place the slide upon the
centre of the stage of the microscope. With the coarse
adjustment lower the oil-immersion objective until it
just touches the drop of oil. Open the illuminating
apparatus to its full extent. Then, with the eye to the
ocular 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
proceed more slowly in the same direction, and, after
ove or two turns, the object will be in focus. Do not
remove the eye from the instrument until this has been
accomplished.
Then, with one hand upon the fine adjustment and
the thumb and index finger of the other hand holding
the slide lightly 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 planes of the preparation may be
brought into focus one after the other. In this way the
whole section or preparation may be inspected. When
UNSTAINED PREPARATIONS. 183
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 circum-
stances 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 cir-
cumstances calling for this arise while studying the
multiplication of cells, the germination of spores, the
formation of spores, and the absence or presence of
motility.
In this method the organisms to be studied are sus-
pended in a drop of physiological salt-solution or bou-
illon in the centre of a cover-slip. This is then placed,
drop down, upon a slide in the centre of which a hollow
or depression is ground (Fig. 38). The slip is held in
Fig. 38.
(none SS 4
Longitudinal section of hollow-ground glass slide for observing bacteria in
hanging drops.
position by a thin layer of vaselin, which may be
painted around the margins of the depression. This
184 BACTERIOLOGY.
not only prevents the slip from moving from its posi-
tion 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 indispen-
sable for the purposes mentioned, and at the same time
requires considerable care in its manipulation. The
fluid is so transparent that the cover-slip is often broken
by the objective being brought down upon the prepara-
tion before one is aware that the focal distance has been
reached. This may be avoided by grasping 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 rendered 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
should now be somewhere near the centre of the field.
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 only this molecular tremor.
The difference between the motion of bodies under-
going this molecular tremor and that possessed by cer-
tain living bacteria is that the former particles never
move from their place in the field, while the living
STUDY OF SPORE-FORMATION. 185
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.
Nore.—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 oF SpoRgE-FORMATION.—The hanging-drop
method just mentioned is not only employed for detect-
ing the motility of an organism, but also for the study
of its spore-forming properties.
Since with aérobic organisms spore-formation occurs,
as a rule, only in the presence of oxygen, and is induced
more by limitation of the nutrition of the organisms
than by any other factor, it is essential that these two
points should be borne in mind in preparing the drop
cultures in which the process is to be studied. Jor 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
186 BACTERIOLOGY.
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 be-
tween the depression in the slide and the cover-slip.
(See Fig. 38.)
A very thin drop of sterilized agar-agar may be sub-
stituted for the bonillon. It serves to retain the organ-
isms in a fixed position, and the process may be more
easily followed.
As soon as finished the preparation is to be 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. The formation of spores
requires a much longer time than the germination of
spores into bacilli, but with patience both processes may
be satisfactorily observed.
Tt will be noticed that the description of this process
is very much like that which immediately precedes, but
differs from it in one respect, viz., that in this manipu-
lation we are not making a preparation which is simply
to be examined and then thrown aside, but it is an
actual pure culture, and must be kept as such, otherwise
the observation will be worthless. For this reason the
greatest care must be observed in the sterilization of
all objects employed. Studies upon spore-formation by
this method frequently continue over hours, and some-
STUDY OF GELATIN CULTURES. 187
times days, and contamination must, therefore, be care-
fully guarded against. The study should be begun with
the vegetative form of the organisms ; the hanging-drop
preparation should, for this reason, always be made
from a perfectly fresh culture of the organism under
consideration before time has elapsed for spores to form.
The simple detection of the presence or absence of
spore-formation can in many cases be made by other
methods. For example, many species of bacteria which
possess this property form spores most readily upon
media from which it is somewhat difficult for them to
obtain the necessary nutrition; potatoes and agar-agar
that have become a little dry offer very favorable con-
ditions, because of the limited area from which the
growing bacteria can draw their nutritive supplies and
because of the free access which they have to oxygen;
for, their growth being on the surface, they are sur-
rounded by this gas unless means are taken to prevent
it. By the hanging-drop method, however, more than
this simple property may be determined. It is possible
not only to detect the stages and steps in the formation
of endogenous spores, but when the spores are com-
pletely formed by transferring them to a fresh bouillon-
drop or drop of agar-agar, preserved in the same way,
their germination 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 oF GELATIN CuLTureEs.—.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 the growth of others is
accompanied by an enzymotic action that results in
188 BACTERIOLOGY.
liquefaction of the gelatin at and around the place at
which the colonies are growing. In some instances
this liquefaction spreads laterally and downward, caus-
ing a saucer-shaped excavation, while in others the
colony sinks directly down into the gelatin and may be
seen lying at the bottom of a funnel-shaped depression.
These differences are constantly employed as one of
the means of differentiating otherwise closely allied
members of the same family of bacteria. (See Fig.
34.) Studies upon the spirillum of Asiatic cholera
and a number of closely allied species, for example,
reveal a decided difference in the form 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.
CuLtTuRES oN Porato.—A very important feature
in the study of an organism is its growth on sterilized
potato. Many organisms present appearances under
this method of cultivation which alone can almost be
considered characteristic. In some cases coarsely lob-
ulated, elevated, dry or moist patches of development
occur 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 ap-
pearance, again it may be moist and glistening. Some-
times there is a production of bubbles, owing to fermen-
tation brought about by the growth of the organisms.
A most striking form of development on potato is
that possessed by the bacillus of typhoid fever and the
bacillus of diphtheria. After the inoculation of a potato
with either of these organisms there is usually no naked-
eye evidence of a growth in either instance, though
microscopic examination of scrapings from the surface
REACTIONS PRODUCED BY BACTERIA. 189
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.
REACTIONS PRODUCED BY BACTERIA DURING THEIR
GrowrH.—The reactions produced in the media by
different species of bacteria 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 pro-
duce at one period of their growth an alkaline, at
another period an acid reaction. This is seen in the
cultures of the bacillus diphtherie of Loeffler.
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 indi-
cated by the play of colors. Such substances as litmus
in the form of the so-called “‘ litmus tincture,’’ and co-
ralline (rosolic acid) in alcoholic solution, are commonly
employed for this purpose. They may be added to the
media in the proportions given in the chapter on media,
and the alterations in their colors studied with different
bacteria. Milk and litmus tincture or peptone solution
to which rosolic acid has been added are very favorable
media for this experiment.
In milk coagula will now and then appear as a result
of the influence of acids, produced from milk sugar by
bacterial action, upon the casein of the milk, while
g*
190 BACTERIOLOGY.
again acids may be produced and yet no coagulation be
noticed.
ANILINE Dyes ror DirFERENTIAL DIAGNOsIS.—
The addition to solid media of some of the aniline
dyes, fuchsin, methylene-blue, methylene-green, and
several others, as well as combinations of these dyes,
has been recommended as a means of differentiation
of bacteria. The differences that are said to be pro-
duced consist of alterations in the color of the media due
to oxidizing or reducing properties of the growing bac-
teria. As yet but little has come from this method of
work. It cannot at present be recommended as a reli-
able means of diagnosis.
BEHAVIOR TOWARD STAINING-REAGENTS. — The
behavior of certain bacteria toward the different dyes
and their reactions under special methods of after-
treatment serve as aids to their diagnosis. With very
few exceptions 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
decolorizing-agents as alcohol, nitric acid, oxalic acid, ete.
Certain other organisms when stained with a solu-
tion of gentian-violet in aniline-water retain their color
when treated with such decolorizing-bodies as iodine
solution and alcohol (Gram’s method), while again
others are completely decolorized by this method.
Many of them can only be treated with water, or but
for a few seconds with alcohol, without losing their
color.
FERMENTATION. 191
It is essential that these peculiarities should be care-
fully noted in studying an organism.
FERMENTATION. —The production of gas as an in-
dication of fermentation is an accompaniment of the
growth of some bacteria. This is best studied in
media to which 1 to 2 per cent. of grape sugar (glucose)
has been added.
In this experiment the test-tube should be filled to
about one-half its volume with agar-agar. The medium
is then liquefied, and when reduced to the proper tem-
perature a small quantity of a pure culture of the organ-
ism under consideration should be carefully distributed
through it. The tube is then placed in 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 glucose, the medium will
be dotted everywhere with very small cavities contain-
ing the gas that has resulted.
This property of fermentation with production of
gas is of such importance as a differential means that
latterly considerable attention has been given to it, and
those who have been most intimately concerned in the
development of our knowledge on the subject do not
consider it enough to say that the growth of an organ-
ism ‘‘is accompanied by the production of gas-bub-
bles,’’ but that under given conditions we should deter-
mine not only the amount of gas or gases produced by
the organism under consideration, but also their nature
and quality. For this purpose Smith’ recommends the
1 An excellent and exhaustive contribution to this subject has been made
by Theobald Smith in ‘‘ The Wilder Quarter-Century Book,” Ithaca, N. Y.,
1893.
192 BACTERIOLOGY.
employment of the ffermentation-tube. It is a tube
bent at an acute angle, closed at one end and enlarged
with a bulb at the other. At the bend the tube is
constricted. To it a glass foot is attached so that
the tube may stand upright. (See Fig. 39.) To fill
the tube the fluid (it is only used with fluid media)
Fie. 39.
Fermentation-tube.
is poured into the bulb until this is about half full.
The tube is then tilted until the closed arm is nearly
horizontal, so that the air may flow out into the bulb
and the fluid flow into the closed arm to take its
place. When this has been completely filled enough
fluid should be added to cover the lowest expanding
portion of the bulb, and the opening of the bulb plugged
with cotton. The.tubes thus filled are then to be ster-
ilized. During sterilization they are to be maintained
FERMENTATION. 193
in the upright position. Under the influence of heat the
tension of water-vapor in the closed arm forces most
of the fluid into‘the bulb. As the tube cools the fluid
returns to its place in the closed arm and fills it again,
with the exception of a small space at the top, which is
occupied by the air originally dissolved in the liquid
and which has been driven out by the heat. The air-
bubble should be tilted out after each sterilization, and
finally, after the third exposure to steam, this arm of
the tube will be free from air.
The medium employed is bouillon containing some
fermentable carbohydrate, as glucose, lactose, or saccha-
rose. After inoculation the flasks are placed in the
incubator and the amount of gas that collects in the
closed arm is, from day to day, noted.
From studies that have been made this gas is found
to consist usually of about one part by volume of car-
bonic acid and two parts by volume of an explosive
gas consisting largely of hydrogen. For determining the
nature and quantitative relations of these gases Smith’
recommends the following procedure: ‘‘ The bulb is
completely filled with a 2 per cent. solution of sodium
hydroxide (NaOH) and closed tightly with the thumb.
The fluid is shaken thoroughly with the gas and allowed
to flow back and forth from bulb to closed branch and
the reverse several times to insure intimate contact of
the CO, with the alkali. Lastly, before removing the
thumb all the gas is allowed to collect in the closed branch,
so that none may escape when the thumb is removed.
If CO, be present, a partial vacuum in the closed branch
causes the fluid to rise suddenly when the thumb is re-
1 Loe. cit., p. 196.
194 BACTERIOLOGY.
moved, After allowing the layer of foam to subside
somewhat the space occupied by gas is again measured,
and the difference between this amount and that meas-
ured before shaking with the sodium hydroxide solution
gives the proportion of CO, absorbed. The explosive
character of the residue is determined as follows: the
cotton plug is replaced and the gas from the closed
branch is allowed to flow into the bulb and mix with
the air there present. The plug is then removed and
a lighted match inserted into the mouth of the bulb.
The intensity of the explosion varies with the amount
of air present in the bulb.’’
CULTIVATION WITHOUT OxyGEN.—As we have
already learned, there is a group of organisms to which
the name ‘‘anaérobic organisms’’ has been given, which
are characterized by their inability to grow in the pres-
ence of oxygen. For the cultivation of the members
of this group a number of devices are employed for the
exclusion of oxygen from the cultures.
Koch’s method. Koch covered the surface of a gel-
atin 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.
Methods of Liborius. Liborius has suggested two
useful methods for this purpose. The one is to fill a
test-tube about three-quarters full of gelatin or agar-
agar, which, after having been sterilized, is to be kept
in a vessel of boiling water for ten minutes to expel all
CULTIVATION WITHOUT OXYGEN. 195
air from it. It is then rapidly cooled in ice-water, and
when between 30° and 40° C., still fluid, is to be inoc-
ulated and very rapidly solidified. It is then sealed up
in the flame. Anaérobic bacteria develop only in the
lower layers of the medium. His other method is that
in which he employs a special tube, known as ‘‘ the
Liborius tube.’’ Its construction is shown in Fig. 40.
Liborius tube for anaerobic cultures.
Through the side tube hydrogen is passed until all
air is expelled; the contracted parts, both of the neck
of the tube and the side arm, are then sealed in the
flame.’ This tube can be used for either solid or liquid
1 As the tubes come from the maker the contracted parts marked x in the
cut are usually so thick as to render the sealing in the flame during the pas-
sage of hydrogen somewhat troublesome ; it is better to draw them out in the
flame quite thin before passing the hydrogen intothe tube This makes the
final sealing a matter of no difficulty.
196 BACTERIOLOGY.
media, but, owing to its usual small capacity, gives
better results with fluid media. (For precautions in
using hydrogen see note to Frinkel’s method, page
198.)
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 have been deposited 1 gramme of pyrogallic
acid and 10 c.c. of 34; normal’ caustic potash solution.
The larger tube is then tightly plugged with a rubber
stopper. The oxygen is quickly absorbed by the pyro-
gallic acid, and the organisms develop in the remaining
constituents of the atmosphere, viz., nitrogen, a small
amount of CO,, and a trace of ammonia.
Method of C. Frénkel. Carl Frinkel suggests the
following as a modification of or substitute for the tubes
of Liborius: the tube is first inoculated as if it were
to be poured as a plate or rolled as an ordinary Esmarch
tube. The cotton plug is then replaced by a rubber
stopper, through which pass two glass tubes. These
1 A normal solution is one that contains in a litre as many grammes of the
dissolved substance as are indicated by its molecular equivalent, The equiv-
alent is that amount of a chemical compound which possesses the same
chemical value as does one atom of hydrogen. For example: 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 (H,SO,) contains in each molecule two re-
placeable hydrogen atoms; its norma] solution is not, therefore, 80 grammes
(its molecular weight) to the litre, but that amount which would be equiva-
lent chemically to one hydrogen atom, viz., 40 grammes (one-half its molecu-
lar weight) to the litre. A normal solution of caustic potash contains as
many grammes to the litre as the number of its molecular weight—56.1
grammes to the litre of water.
CULTIVATION WITHOUT OXYGEN. 197
must all have been sterilized in the steam sterilizer
before using. On the outer side of the stopper these
two tubes are bent at right angles to the long axis of
the test-tube into which they are to be placed, and both
are slightly drawn out in the gas-flame. Both of these
Frinkel’s method for the cultivation of anaérobic bacteria.
tubes must be provided, before sterilization, with a
plug of cotton; this is to prevent the access of foreign
organisms to the medium during manipulations. At
the inner side of the rubber stopper—that is, the end
which is to be inserted into the test-tube—the glass
tubes are of different lengths: one reaches to within
0.5 em. of the bottom of the test-tube, the other is cut
off flush with the under surface of the stopper. The
198 BACTERIOLOGY.
outer end of the longer glass tube is then connected
with a hydrogen generator and hydrogen is allowed
to bubble through the gelatin (Fig. 41, 4) in the tube
until all contained air has been expelled and its place
taken by the hydrogen.'| When the hydrogen has been
bubbling through the gelatin for about five minutes
(at least) one can be reasonably sure that all oxygen
has been expelled. The drawn-out portions of the
tubes can then be sealed in the gas-flame without fear
of an explosion. The protruding end of the rabber
stopper is then painted around with melted paraffin
and the tube rolled in the way given for ordinary
Esmarch tubes. A tube thus prepared and containing
growing colonies is shown in Fig. 41, B.
The development that now occurs is in an atmos-
phere of hydrogen, all oxygen having been expelled.
During the operation the tube containing the liquefied
gelatin should be kept in a water-bath at a temperature
sufficiently high to prevent its solidifying, and at the
1 Before beginning the experiment it is always wise to test the hydrogen—
i. e., to see that it is free from oxygen and there is no danger of an explosion,
for unless this be done the entire apparatus may be blown to pieces and a
serious accident occur. The agents used should be pure zinc, and pure sul-
phuric acid of about 25 to 30 per cent. strength. With the primary evolu-
tion of the gas the outlet of the generator should be closed and kept closed
until the gas reservoir is quite filled with hydrogen. The outlet should then
be opened and the entire volume of gas allowed to escape, care being taken
that no flameisin the neighborhood, This shou!d be repeated again, after
which a sample of the hydrogen generated should be collected in an inverted
test-tube in the ordinary way for collecting gases over water, viz., by filling a
test-tube with water, closing its mouth with the thumb, inverting it, and
placing its mouth under water, when, after removing the thumb, the water
will be kept in it by atmospheric pressure. The hydrogen which is flowing
from the open generator may be conducted to the test-tube by a bit of rubber
tubing. When the water has been replaced try the gas by holding a flame
near the open mouth of the test-tube. If no explosion occurs, the hydrogen
is safe to use. Should there be an explosion the generation of hydrogen must
be continued in the apparatus until it simply burns with a colorless flame
when tested in a test-tube.
CULTIVATION WITHOUT OXYGEN. 199
same time not high enough to kill the organisms with
which it has been inoculated.
One of the obstacles to the successful performance of
this method is the bubbling of the gelatin, the foam
from which will often fill the exit tube and sometimes
be forced from it. This may be obviated by reversing
the order of proceeding, viz.: roll the Esmarch tube
in the ordinary way with the organisms to be studied,
using a relatively small amount of gelatin, so as to
have as thin a layer as possible when it is rolled.
Then replace the cotton plug with the sterilized rubber
stopper carrying the glass tubes through which the
hydrogen is to be passed, and allow the hydrogen to
flow through just as in the method first given. The
gas now passes over the gelatin instead of through it, and
consequently no bubbling results. In all other respects
the procedure is the same as that given by Frankel.
Method of Kitasato and Weil. For favoring the an-
aérobic conditions Kitasato and Weil have suggested
the addition to the culture media of some strong re-
ducing agent. They recommend formic acid in 0.3 to
0.5 per cent.; glucose in 1.5 to 2 per cent.; or blue
litmus tincture in 5 per cent. by volume. This is, of
course, in addition to an atmosphere from which all
oxygen has been expelled.
Esmarch’s method. Esmarch’s plan is to prepare in
the usual way a roll tube of the organisms; subject it
to a low temperature, and while quite cold fill it with
liquefied gelatin, which is caused to solidify rapidly.
In this method the colonies develop along the sides of
the tubes, and can more easily be studied than where
they are mixed through the gelatin, as in the method of
Liborius.
200 BACTERIOLOGY.
By some workers the oxygen is removed from the
culture medium by the use of the air-pump.
Many other methods exist for this special purpose,
but for the beginner those given will suffice.
From what has been said it may be inferred that the
cultivation of anaérobic bacteria is a simple matter and
attended with but little difficulty. Such an inference
will, however, be quickly dispelled when the beginner
attempts this part of his work for the first time, and
particularly when his efforts are directed toward the
separation of these forms from other organisms with
which they are associated. The presence of spore-
forming, facultative anaérobes in mixed cultures is
always to be suspected, and it is this group that renders
the task so difficult. At best the work requires undi-
vided attention and no small degree of skill in bacteri-
ological technique.
Inpot Propuction.—The production of products
other than those that give rise to alterations in the reac-
tion of the media, and whose presence may be detected
by chemical reactions, is now a recognized step in the
identification of different species of bacteria. Among
these chemical products there is one that is produced by
a number of organisms, and whose presence may easily
be detected by its characteristic behavior when treated
with certain substances. I refer to the body nitroso-indol,
the reactions of which were described by Beyer in 1869,
and the presence of which ax a product of the growth of
certain bacteria has since furnished a topic for consid-
erable discussion.
Indol, the name by which this body is now generally
known, when acted upon by reducing agents, is seen to
become of « more or less conspicuous rose color. This
INDOL PRODUCTION. 201
body was recognized as one of the products of growth
of the spirilltm of Asiatic cholera first by Poel, and a
short time subsequently by Bujwid and by Dunham,
and for a time was thought to be peculiarly charac-
teristic of the growth of this organism. It has since
been found that there are many other bacteria which
also possess the property of producing indol in the
course of their development.
The method employed for its detection is as follows:
cultivate the organism for twenty-four to forty-eight
hours at a temperature of 37° C., in the simple pep-
tone 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 reaction when the amount of indol present is very
small.
Four tubes should always be inoculated and kept
under exactly the same conditions for the same length
of time.
At the end of twenty-four or forty-eight hours the
test may be made. Proceed as follows: to a tube con-
taining 7 c.c. of the peptone solution, but which has not
been inoculated, add 10 drops of concentrated sulphuric
acid. To another similar tube add 1 c.c. of a 0.01 per
cent. solution of sodium nitrite, and afterward 10 drops
of concentrated sulphuric acid. Observe the tubes for
five to ten minutes. No alteration in their color ap-
pears, or at least there will be no production of a rose
color. They contain no indol.
Treat in the same way, with the acid alone, two of
the tubes which have been inoculated. If no rose color
appears after five or ten minutes, add 1 c.c. of the
sodium nitrite solution. If now no rose color is pro-
202 BACTERIOLOGY.
duced, 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 salts of nitrous
acid. The sulphuric acid liberates this acid from its
salts and permits of its reducing action being brought
into play.
If the rose color appears only after the addition of
both the acid and the nitrite solution, then indol has
been formed during the growth of the organisms, but
no nitrites.
Control the results obtained by treating the two re-
maining cultures in the same way.
The test is sometimes made by allowing concentrated
acid to flow down the sides and collect at the bottom of
the tube; the reaction is then seen as a rose-colored
zone overlying the line of contact of the acid and cul-
ture medium. This method is open to the objection
that, if indol is present in only a very limited amount,
the rose color produced by it is apt to be masked by a
brown color that results from the charring action of the
concentrated acid on the other organic matters in the
culture medium, so that its presence may in this way
escape detection. In view of this, Petri recommends
the use of dilute sulphuric acid. He states that when
indol is present the characteristic rose color appears a
little more slowly with the dilute acid, but is more per-
manent, and there is never any danger of its presence
being masked by the occurrence of other color reactions.
Test for Nitrites. For this purpose Lunkewicz has
recently recommended the employment of Tlosvay’s
DESCRIBING AN ORGANISM. 203
modification of the method of Griess. As reagents
the following solutions are employed:
u. Naphthylamine Q 0.1 gramme.
Dist. water. - 20.0 ¢.c.
Acetic acid (25 per cent. sol.) 150.0 ¢.c.
b. Sulfanilic acid Z Fi 7 0.5 gramme,
Acetic acid (25 per cent. sol.) 150.0 ¢ ¢.
In preparing solution a the naphthylamine is dis-
solved in 20 c¢.c. of boiling water, filtered, allowed to
cool, and mixed with the dilute acetic acid.
Solutions a and 6 are then mixed. The resulting
mixture should be colorless. It is best to prepare it
fresh as it is needed, though if kept in a closely stop-
pered flask it retains it virtues for some time.
When added to cultures containing nitrites, in the
proportion of one volume to five volumes of the cul-
ture, a deep red color appears in a few seconds. If the
nitrites are not present, no color reaction occurs. In
making the test on cultures always control the results
by tests on the same medium no inoculated, as some of
the ingredients of which the medium is composed may
contain nitrites. Lunkewicz recommends the use of
Merck’s peptone for this test, claiming that nitrites are
always to be found in Witte’s peptones.
POINTS TO BE OBSERVED IN DESCRIBING AN ORGANISM.
The following is an outline of points to be considered
in describing a new organism or in identifying an
organism with one already described:
1. Its source—as air, water, or soil. If found in the
animal body, is it normally present or only in patholog-
ical conditions ?
2. Its form, size, mode of development, occurrence of
204 BACTERIOLOGY.
involution-forms or other variations in morphology.
Grouping, as in pairs, chains, clumps, zoogloea; pres-
ence of capsule; development and germination of spores;
arrangement of flagella.
3. Staining-peculiarities—especially its reactions with
Gram’s (or Weigert’s fibrin) stain, and peculiar or irreg-
ular modes of staining.
4. Motility—to be determined on very fresh cultures
and on cultures in different media.
5. Its relation to oxygen—is it aérobic, anaérobic,
or facultative? Does it develop in other gases, as car-
bonic acid, hydrogen, ete. ?
6. Both the macroscopic and microscopic appearance
of its colonies on nutrient gelatin and on nutrient agar-
agar.
7. The appearance of its growth in stab- and slant-
cultures on gelatin, agar-agar, blood-serum, and on
potato.
8. The character of its growth in fluid media, as in
bouillon, milk, litmus milk, rosolic-acid-peptone solu-
tion, and in bouillon containing glucose.
9. Does it grow best in acid, alkaline, or neutral
media ?
10. Is the normal reaction of the medium altered by
its growth? Is its growth accompanied by the produc-
tion of indol; is the indol associated with the coincident
production of nitrites?
11. Is its growth accompanied by the production of
gas,as evidenced by the appearance of gas-bubbles in
the media—both in media containing fermentable sugars
and those from which these bodies are absent? When
cultivated in sugar-bouillon in the fermentation-tube,
what production of gas is evolved under known condi-
DESCRIBING AN ORGANISM. 205
tions? How much of this gas is carbonic acid and how
much is explosive ?
12. At what temperature does it thrive best, and the
lowest and highest temperatures at which it will de-
velop? What is its thermal death-point, both by steam
and dry-air methods of determining this point?
13. What is its behavior when exposed to chemical
disinfectants and antiseptics? Does it withstand dry-
ing and other injurious influences, both in the vegeta-
tive and spore stages? The germicidal value of the
blood-serum of different animals may also be tried
upon it.
14. Its pathogenic powers—modes of inoculation by
which these are demonstrated; quantity of material used
in inoculation; duration of the disease and its symp-
toms; lesions produced, and distribution of the bacteria
in the inoculated animal; which animals are susceptible
and which immune, and the character of its pathogenic
activities? Variations in virulence, and the probable
cause to which they are due. Can they be produced
artificially and at will?
15. The detection of specific, toxic, and immunizing
products of growth.
16. Its behavior when exposed to the influence of
blood-serum of animals immunized from it; also its
behavior when mixed with serum from an animal in
the height of infection by it. Are the relations be-
tween the organism and the serum constant and spe-
cific?
10
CHAPTER XII.
Inoculation of animals—Subcutaneous inoculation ; intravenous injection
—Inoculation into the great serous cavities, and into the anterior chamber of
the eye—Observation of animals after inoculation,
AFTER subjecting an organism to the methods of
study that we have thus far reviewed there remains to
be tested its action upon animals—i. e., to determine if
it possesses the property of producing disease or not,
and, if so, what are the pathological results of its
growth in the tissues of these animals, and in what way
must it gain entrance to the tissues in order to produce
these results? The mode of deciding these points is by
inoculation, which is practised in different ways accord-
ing to circumstances. Most commonly a bit of the
culture to be tested is simply introduced beneath the
skin of the animal, but in other cases it may be neces-
sary to introduce it directly into the vascular or lym-
phatic circulation or into one or the other of the great
serous cavities; or, for still other purposes of observa-
tion, into the anterior chamber of the eye, upon the iris.
SuscuranEous InocuLation or 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.
SUBCUTANEOUS INOCULATION OF ANIMALS. 207
Sterilization of the skin is impossible, so that it need
not be attempted. If the inoculation is to be by means
of a hypodermic 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 for-
ceps and a pocket cut into it with scissors which have
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 incised in
a way corresponding to the opening in the skin. The
pocket is then to be held open with the forceps and the
substance to be inserted is introduced as far back under
the skin and fascize as possible, care being taken not to
touch the edges of the wound if it can be avoided.
The wound may then be simply pulled together and
allowed to remain. No stitching or efforts at closing it
are necessary, though a drop of collodion over the point
of operation may serve to lessen contamination.
During manipulation the animal must be held still.
For this purpose special forms of holders have been
devised, but, if an assistant is to be obtained for the
operation, the simple subcutaneous inoculation may be
made without the aid of a mechanical holder.
Tt is at times, however, more convenient to dispense
with the presence of an assistant, and several forms of
apparatus have been devised for holding guinea-pigs,
rats, rabbits, etc. For small animals, such as mice and
rats, the holder suggested by Kitasato is very useful.
It is simply a metal plate attached to a stand by a
208 BACTERIOLOGY.
clamped ball-and-socket joint, so that it can be fixed in
any position. It is provided with a spring-clip at one
Fig. 42.
Kitasato’s mouse holder.
Fig. 43.
Holder for larger animals.
end that holds the animal by the skin of the neck, and
at the other end with another clamp that holds the tail
of the animal. This holder is shown in Fig. 42.
SUBCUTANEOUS INOCULATION OF ANIMALS. 209
For larger animals the form of holder shown in Fig.
43 is commonly used.
A very simple and useful holder for guinea-pigs
consists of a metal cylinder of about 5 em. in diameter
Fie. 44.
The Voges-Rabinowitsch holder for guinea-pigs.
and about 13 cm. long; closed at one end by a perfor-
ated cap of either tin or wire netting. Along the side
of this box is a longitudinal slit of 12 mm. wide that
runs for 9.5 cm. from within 0.5 mm. of the open ex-
tremity of the cylinder.
210 BACTERIOLOGY.
The animal is placed in such a cylinder with its head
toward the perforated bottom. It is then easily pos-
sible to make subcutaneous inoculation by taking up a
bit of skin through the slit in the side of the box, or
to make intraperitoneal injection by drawing the pos-
terior extremities slightly from the box and holding
them steady between the index and second finger, as
seen in Fig. 44. It is also very convenient for use
when the rectal temperature of these small animals is to
be taken. The manipulations can easily be made with-
out the aid of an assistant. Its construction is best
seen in Fig. 44,7
For ordinary subcutaneous inoculations at the root
of the tail in mice a simple piece of apparatus consists
of a bit of board about 7 x 10 cm. and 2 em. thick,
upon which is tacked a hollow, tapering roll of wire
gauze, a truncated cone, about 6 em. long and about
1.5 cm. in diameter at one end and 2 cm. at its other
Fie. 45.
Mouse-holder, with mouse in proper position.
end. This is tacked upon the board in such a position
that its long axis runs in the long axis of the board, being
equidistant from its two sides. Its small end is placed
1 Ceutralblatt fiir Bakteriologie und Parasitenkunde, Bd. 18, 1895, p. 530.
SUBCUTANEOUS INOCULATION OF ANIMALS. 211
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-serew, with which the apparatus (Fig. 45) is
provided. The animal usually remains perfectly quiet
and may be handled without difficulty.
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 tissue under the skin over this part
of the back in the way that has just been described.
It is always best 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 may be 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 commonly made is in the abdominal wall either to
the right or left of the median line and about 3 cm.
distant. When pigeons are used they are held with the
legs, tail, and ends of the wings in the one hand, and
the head and anterior portion of the body in the other,
leaving the area occupied by the pectoral muscles, over
which the inoculation is to be made, free for manipu-
lation. The hair over the point selected for the in-
oculation should be closely cut with the scissors in
the case of guinea-pigs and rabbits, and from a small
212 BACTERIOLOGY.
area the feathers should be plucked in the case of the
pigeon.
INJECTION INTO THE CIRCULATION.—It is not in-
frequently desirable to inject the material under consid-
eration directly into the circulation of an animal. If
a rabbit is to be employed for the purpose, the opera-
tion is usually done upon one of the veins in the ear. .
To those who have had no practice in this procedure
it offers a great many difficulties; but if the directions
which will be given are strictly observed, the greatest
of these obstacles to the successful performance of the
operation may be overcome.
When viewing the circulation in the ear of the rabbit
by transmitted light three conspicuous branches of the
main vessel (vena auricularis posterior) will be seen.
One runs about centrally in the long axis of the ear,
one runs along its anterior margin, and one along its
posterior margin. The central branch (ramus anterior
of the vena auricularis posterior) is the largest and most
conspicuous vessel of the ear, and is, therefore, selected
by the inexperienced as the branch into which it would
appear easiest to insert a hypodermic needle. This,
however, is fallacious. This vessel lies very loosely
imbedded in connective tissue, and, in efforts to intro-
duce a needle into it, rolls about to such an extent that
only after a great deal of difficulty does the experiment
succeed, On the other hand, the posterior branch (ramus
lateralis posterior of the vena auricularis posterior) is a
very fine, delicate vessel which runs along the posterior
margin of the ear, and which is so firmly fixed in the
dense tissues which surround it that it is prevented from
rolling about under the point of the needle. The fur-
ther away from the mouth of the vessel—that is, the
INJECTION INTO THE CIRCULATION. 213
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. From
time to time the vessels of the ear will be found to con-
tain so little blood that they are hardly distinguishable,
making it very difficult to introduce the needle. This
is sometimes overcome by pressure at the root of the
ear, causing, thereby, stasis of the blood and distention
of the vessels. A very satisfactory method of causing
the veins to become more prominent is to lightly press
or gently prick with the point of a needle the skin over
the vessel to be used. In a few seconds, as a result of
this irritation, the vessel will have become dilated, dis-
tended with blood, readily distinguished from the sur-
rounding tissues, and may then be easily punctured by
the needle of the syringe. 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 hypodermic needles as they come from the
makers are not suited at all for this operation, because
of the way in which their points are ground. If one
examine carefully the point of a new hypodermic
needle, it will be seen that the long point, instead of pre-
senting a flat, slanting surface when viewed from the
side, has a more or less curved surface. Now, in efforts
to introduce such a needle into a vessel of very small
calibre, it is commonly seen that the extreme point of
the needle, instead of remaining in the vessel, as it
would do were it straight, very commonly projects into
10*
214 BACTERIOLOGY.
the opposite wall, and as the needle is inserted further
and further into the tissues it is usually pushed through
the vessels into the loose tissues beyond, and the mate-
rial to be injected is deposited into these tissues, instead
of into the circulation. If, on the contrary, the slanting
point of the needle be ground down until its surface is
perfectly flat when viewed from the side, and no more
curvature exists, then when once inserted into a vessel it
usually remains there, and there is no tendency to pene-
trate through the opposite wall. We never use a new
hypodermic 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.
Hypodermic needles magnified. u. Improper point. b. Proper shape of point.
In Fig. 46, a, the needle has the point usually seen
when new.
In Fig. 46, 6, the point has been ground down to the
shape best suited for this operation.
The needles need not be returned to the maker. One
can grind them to the shape desired in a few minutes
upon an oilstone.
The size of the needle is that commonly employed
for subcutaneous injections.
INJECTION INTO THE CIRCULATION. 215
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 most cases we have not
found this necessary, and particularly if the animal has
not been excited prior to the beginning of the operation.
The animal should be placed so that the ear upon
which the operation is to be performed comes between
the operator and the source of light. This renders vis-
ible 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 hypodermic syringe is taken in one hand
and with the other hand the ear is held firmly. The
point of the needle is then inserted through the skin
and into the finest part of the ramus posterior, the part
nearest the apex of the ear, where the course of the
vessel is nearly straight. When the point of the needle
is in this vessel it gives to the hand a sensation quite
different from that felt when it is in the midst of con-
nective 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 bis 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 some-
what similar is to be seen. It may always be differen-
216 BACTERIOLOGY.
tiated, however, by continuing the injection, when the
circulation of clear fluid through the vessels will not
only fail to take the place of the circulating blood, but
there will at the same time appear a localized swelling
under the skin about the point of the needle. The
needle must then be withdrawn and inserted into the
vessel at a point a little nearer to its proximal end.
Care must be taken that no air is injected.
The hypodermic syringe and needle must, previous
to operation, have been carefully sterilized in the steam
sterilizer or in boiling water. The animal must be
kept under close observation for about an hour after
injection.
The operation is one that cannot be learned from
verbal description. It can only be successfully per-
formed after actual practice.
If the precautions which have been mentioned are
observed, but little difficulty in performing the opera-
tion will be experienced.
Its greater convenience and simplicity as compared
with other methods for the introduction of substances
into the circulation commend it as an operation with
which to make one’s self familiar. The animals sustain
practically no wound, they experience no pain—at least
they give no evidence of pain—and no anesthetic is
required.
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 packings that may be sterilized by steam
without injury. The syringes commonly employed are
those shown in Fig. 47—A, Kxoch’s; B, Strohschein’s;
C, Overlack’s.
INJECTION INTO THE CIRCULATION. 9217
For operations requiring exact dosage experience
has led me to prefer a syringe after the pattern of C,
in Fig. 47—4. ¢., of the form commonly used by physi-
cians. The reason for this is as follows: in making
hypodermic injections or injections into the circulation
there is a certain amount of resistance to the passage of
fluid from the needle. If one overcomes this resistance
Fic. 47.
Forms of hypodermic syringe.
A, Koch’s syringe. B. Syringe of Strohschein. C. Overlack’s form.
by means of a cushion of compressed air, as is the case
in syringes A and B of Fig. 47, the sudden expansion
of the air in the body of the syringe when resistance is
overcome frequently causes a larger amount of fluid
to be ejected from the needle than is desired. No
such accident is likely to occur when the fluid is forced
from the barrel of the syringe by the head of a close-
fitting piston, with no air intervening between the fluid
and the head of the piston. With such an instrument,
properly manipulated, the dose can always be controlled
with accuracy.
218 BACTERIOLOGY.
INocULATION INTO THE LYMPHATIC CIRCULATION.
—Fluid cultures or suspensions of bacteria may be in-
jected into the lymphatics by way of the testicles. The
operation is a simple one. One simply plunges the
point of the hypodermic needle directly into the sub-
stance of the testicle and then injects the amount desired.
Injections made in this manner are sometimes fol-
lowed by interesting pathological lesions of the lym-
phatic apparatus of the abdomen.
INOCULATION INTO THE GREAT SEROUS CAVITIES.
Inoculation into the peritoneum presents no difficulties
if fluids are to be introduced. In this case one makes,
with a pair of sterilized scissors, a small nick through
the skin down to the underlying fascie, and, taking up a
fold of the abdominal wall between the fingers, plunges
the hypodermic needle through the opening just made
directly into the peritoneal cavity. There is no fear of
penetrating the intestines or other internal viscera if
the puncture be made along the median line at about
midway between the end of the sternum and the sym-
physis pubis. Though this may seem a rude method,
it is, nevertheless, the rarest of accidents to find that
the intestines have been penetrated. The object of the
primary incision is to lessen the chances of contaminat-
ing the inoculation by bacteria located in the skin, some
of which would adhere to the needle if it were plunged
directly through the skin, and might complicate the re-
sults.
If solid substances, bits of tissue, etc., are to be intro-
duced into the peritoneum, it becomes necessary to con-
duct the operation upon the lines of a laparotomy.
INOCULATION BY GREAT SEROUS CAVITIES. 219
The hair should be shaved from a small area over the
median line, after which the skin is to be thoroughly
washed. A short longitudinal incision (about 2 em.
long) is then to be made in the median line through the
skin, and down to the fascia. Two subcutaneous
sutures, as employed by Halsted, are then to be intro-
duced transversely to the line of incision at about 1 em.
apart, and their ends left loose. This particular sort of
suture does not pass through the skin, but, instead, the
needle is introduced into the subcutaneous tissues along
the edge of the incision. In this case they are to pass
into the abdominal cavity and out again, entering at one
side of the line of incision and leaving at the other, as
indicated by the solid and dotted lines in Fig. 48.
(This figure indicates the primary opening through the
skin. By the longitudinal dotted line is seen the open-
ing to be made into the abdomen; by the transverse
dotted lines, with their loose ends, the sutures as placed
220 BACTERIOLOGY.
in position before the abdomen is opened ; it will be seen
that these sutures in all cases pass through the subcuta-
neous tissues only and do not penetrate the skin proper.)
The opening through the remaining layers may now
be completed; the bit of tissue deposited in the perito-
neal cavity, under precautions that will exclude all else;
the edges of the wound drawn evenly and gently to-
gether by tying the sutures, and the lines of incision
dressed with collodion. It should be needless to say
that this operation must be conducted under the strictest
precautions, to avoid complications. All instruments,
sutures, ligatures, etc., must be carefully sterilized either
in the steam sterilizer for twenty minutes, or by boiling
in 2 per cent. sodium carbonate solution for ten min-
utes; the hands of the operator, though they should not
touch the wound, should be carefully cleansed, and the
material to be introduced into the abdomen should be
handled with only sterilized instruments.
Inoculation into the plewral cavity is much less fre-
quently called for—in fact, it is not a routine method
employed in this work. It is not easy to enter the
pleural cavity with a hypodermic needle without injur-
ing the lung, and it is rare that conditions call for the
introduction of solid particles in this locality.
Inoculation into the anterior chamber of the eye is per-
formed by making a puncture through the cornea just
in front of its junction with the sclerotic, the knife being
passed into the anterior chamber in a plane parallel to
the plane of the iris. By the aid of a fine pair of for-
ceps the bit of tissue is passed through the opening thus
made and is deposited upon the iris, where it is allowed
to remain, and where its pathogenic properties upon the
iris can be conveniently studicd. It is a mode of inoc-
ANIMALS AFTER INOCULATION. 221
ulation of very limited application, and is therefore but
rarely practised. It was employed in the classical
experiments of Cohnheim in demonstrating the infec-
tious nature of tuberculous tissues, tuberculosis of the
iris being the constant result of the introduction of
tuberculous tissue into the anterior chamber of the eye
of rabbits.
OBSERVATION OF ANIMALS AFTER INOCULATION.
After either of these methods of inoculation, particu-
larly when unknown species of bacteria are being tested,
the animal is to be kept under constant observation and
all that is unusual in its conduct noted—as, for instance,
elevation of temperature; loss of weight ; peculiar posi-
tion in its cage; loss of appetite; roughening of the
hair ; excessive secretions, either from the air-passages,
conjunctiva, or kidneys; looseness of or hemorrhage
from the bowels; tumefaction or reaction at site of inoc-
ulation, etc. If death ensue in from two to four days,
it may reasonably be expected that at autopsy evidence
of either acute septic or toxic processes will be found.
It sometimes occurs, however, that inoculation results
in the production of chronic conditions, and the animal
must be kept under observation often for weeks. In
these cases it is important to note the progress of the
changes by their effect upon the physical conditions of
the animal, viz., upon the nutritive processes as evi-
denced by fluctuation in weight, and upon the body
temperature. For this purpose the animal is to be
weighed daily, always at about the same hour and
always about midway between the hours of feeding;
at the same time its temperature as indicated by a ther-
222 BACTERIOLOGY.
mometer placed in the rectum is to be recorded.’ By
the comparison of these daily observations with one
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223
ANIMALS AFTER INOCULATION.
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BACTERIOLOGY.
224
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ANIMALS AFTER INOCULATION.
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and falls of temperature, often as much as a degree from
one day to another. Such fluctuations have apparently
226 BACTERIOLOGY.
no bearing upon the general condition of the animal,
but are probably due to transient causes, such as over-
feeding or scarcity of food, improper feeding, lack of
exercise, excitement, fright, ete.
The accompanying charts (Figs. 49, 50, 51, 52) will
serve to illustrate some of these points. The animals,
two rabbits and two guinea-pigs, were taken at random
from among the stock animals and placed each in a
clean cage, the kind used for animals under experiment,
and kept under as good general conditions as possible.
For the first week the rabbits received each 100 grammes
of green food (cabbage and turnips) daily, and the
guinea-pigs 30 grammes each of the same food. During
the second week this daily amount of food was doubled;
during the third week it was quadrupled ; and for the
fourth and fifth weeks they each received an excess of
food daily, consistiug of green vegetables and grain (oats
and corn). By reference to the charts sudden diurnal
fluctuations in weight will be observed that do not
correspond in all instances with scarcity or sufliciency
of food. With the rabbits there is a gradual loss of
weight with the smaller amounts of food, which losses
are not totally recovered as the food is increased. With
the guinea-pigs there is likewise at first a loss, but after
a short time the weight remains tolerably constant, and
is not so conspicuously affected by the increase in food
as one might expect. From the recorded temperatures
one sees the peculiar fluctuations mentioned. To just
what they are due it is impossible to say. It is mani-
fest that the normal temperature of these animals, if we
can speak of a normal temperature for animals present-
ing such fluctuations, is about a degree or more, Centi-
grade, higher than that of human beings. The animals
ANIMALS AFTER INOCULATION. 927
from which these charts were made were not inoculated,
nor were they subjected to any operative procedures
whatever, the only deviations from normal conditions
being the variations in the daily amount of food given.
In certain instances, however, there will be noticed
a constant tendency to diminution in weight, notwith-
standing the daily fluctuations, and after a time a con-
dition of extreme emaciation may be reached, the
animal often being reduced to from 50 to 60 per cent.
of its original weight. In other cases, after inoculations
to which the animal is not susceptible, rabbits in par-
ticular, if properly fed, will frequently gain steadily in
weight. The condition of progressive emaciation just
mentioned is conspicuously seen after intravenous inoc-
ulation of rabbits with cultures of the bacillus typhi
abdominalis and of the bacterium coli commune referred
to in the chapter on the latter organism, and if looked
for will doubtless be seen to follow inoculation with
other organisms capable of producing chronic forms of
infection, but which are frequently considered non-
pathogenic because of their inability to induce acute
conditions. Not infrequently in chronic infections there
may be hardly any marked and constant temperature
variations until just before death, when there will some-
times be a rise and at other times a fall of temperature.
In the majority of cases, however, one must be very
cautious as to the amount of stress laid upon changes
in weight and temperature, for unless they are progres-
sive or continuous in one or another direction they may
have little or no significance in indicating the existence
or absence of disease.
CHAPTER XIII.
Post-mortem examination of animals—Bacteriological examination of the
tissues—Disposal of tissues and disinfection of instruments after the exarn-
ination.
During the bacteriological examination of the tissues
of dead animals certain rigid precautions must be ob-
served 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, otherwise decomposition sets in, and the sapro-
phytic bacteria now present may interfere with the
accuracy of results. When the autopsy is to be made
the animal is first inspected externally, and all visible
lesions noted. It is then to be fixed upon its back
upon a board with nails or tacks. The four legs and
the end of the nose, through which the tacks are driven,
are to be moderately extended. Plates are now to be
made from the site of inoculation, if this is subcuta-
neous. The surfaces of the thorax and abdomen are
then to be moistened to prevent the fine hairs, dust,
etc., from floating about in the air and interfering with
the work. An incision is then made through the skin
from the chin to the symphysis pubis. This is only a
skin incision, and does not reach deeper than the mus-
cles. It is best done by first making a small incision
with a scalpel, just large enough to permit of the intro-
duction of one blade of a blunt-pointed scissors. It is
POST-MORTEM EXAMINATION OF ANIMALS. 299
then completed with the scissors. The whole of the
skin is now to be carefully dissected away, not only
from the abdomen and thorax, but from the axillary,
inguinal, and cervical regions, and the fore and hind
legs as well. The skin is then pinned back to the board
so as to keep it as far from the abdomen and thorax as
possible, for it is from the skin that the chances of
contamination are greatest.
It now becomes necessary to proceed very carefully.
All incisions from this time on are to be made only
through surfaces that have been sterilized. The sterili-
zation is best accomplished by the use of a broad-bladed
common table knife that has been heated in the gas-
flame. The blade, made quite hot, is to be held upon
the region of the linea alba until the skin at that region
begins to burn; it is then held transverse to this line
over about the centre of the abdomen, thus making two
sterilized tracks through which the abdomen may be
opened by a crucial incision. The sterilization thus
accomplished is, of course, directed only against organ-
isms that may have fallen upon the surface from with-
out, 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 geni-
talia, is to be made without touching the internal vis-
cera. 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
ll
230 BACTERIOLOGY.
pair of heavy, sterilized scissors is made along the
scorched tracks on either side of the thorax.
After this the whole anterior wall of the thorax may
easily be lifted up, and by severing the connections with
the diaphragm it may be completely removed.
When this is done and the abdominal flaps laid back,
the contents of both cavities are to be inspected and
their condition noted without disturbing them.
After this the first steps to be taken are to prepare
plates or Esmarch tubes from the blood, liver, spleen,
kidneys, and any exudates that may exist.
This is best done as follows:
Heat a scalpel quite hot and apply it to a small sur-
face of the organ from which the cultures are to be
made. Hold it upon the organ until the surface directly
beneath it is visibly scorched. Then remove it, heat it
again, and while quite hot insert its point through the
capsule of the organ. Into the opening thus made
insert a sterilized platinum-wire loop, made of wire a
little heavier than that commonly employed. Project
this deeply into the tissues of the organ; by twisting
it about enough material from the centre of the organ
can be obtained for making the cultures.
As the resistance offered by the tissues is sometimes
too great to permit of a puncture with the ordinary
wire loop, Nuttall (Centralblatt fiir Bakteriologie und
Parasitenkunde, 1892, Bd. xi. p. 538) has devised for
the purpose a platinum-wire spear which possesses con-
siderable advantage over the loop. It is of the form
seen in Fig. 53. It is easily made by beating a piece
of heavy platinum wire into a spear-head at one end,
and perforating this with a small drill, as seen in the
cut. It is attached by the other end to either a metal
POST-MORTEM EXAMINATION OF ANIMALS. 9231
or glass handle, preferably the former. It can readily
be thrust into the densest of the soft tissues, and by
twisting it about after its introduction particles of the
tissue sufficient for examination are withdrawn in the
eye of the spear-head.
Fig. 53,
ORES
Nuttall’s platinum spear for use at autopsies.
The cultures from the blood are usually made from
one of the cavities of the heart, which is always entered
through a surface which has been burned in the way
given.
In addition to cultures, cover-slips from the site of
inoculation, 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.
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
subsequent examination. Throughout the entire au-
topsy it must be borne in mind that all cultures,
cover-slips, and tissues must be carefully labelled,
not only with the name of the organ from which
they originate, but with the date, designation of the
animal, etc., so that an account of their condition
232 BACTERIOLOGY.
after closer study may be subsequently inserted in the
protocol.
The cover-slips are now to be stained, mounted, and
examined microscopically, and the results carefully
noted.
The same may be said for the subsequent study of
the cultures and the hardened tissues which are to be
stained and subjected to microscopic examination. The
results of microscopic study of the cover-slip prepara-
tions and of those obtained by cultures should in most
cases correspond, though it not rarely occurs that bac-
teria 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 ap-
pear should be in pure cultures.
This is particularly the case with cultures made from
the internal viscera.
Both the cover-slips and cultures made from the point
of inoculation are apt to contain a variety of organisms.
If the organism obtained in pure culture from the
internal viscera, or those predominating at the point of
inoculation of the animal, have caused its death, then
subsequent inoculation of pure cultures of this organism
into the tissues of a second animal should produce sim-
ilar 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 min-
utes in a 1 to 2 per cent. soda solution, and the board
POST-MORTEM EXAMINATION OF ANIMALS. 233
upon which the animal was tacked, as well as the tacks,
towels, dishes, and all other implements used at the au-
topsy, are to be sterilized by steam. All cultures, cover-
slips, and, indeed, all articles likely to have infectious
material upon them, must be thoroughly sterilized as
soon as they are of no further service.
APPLICATION OF THE METHODS OF
BACTERIOLOGY.
CHAPTER NIV.
To obtain material with which to begin work.
Expose to the air of an inhabited room a slice of
freshly steamed potato or a bit of slightly moistened
bread upon a plate for about one hour. Then cover it
with an ordinary water-glass and place it in a warm
spot (temperature not to exceed that of the human body
—37.5° C.), and allow it to remain unmolested. At
the end of twenty-four to thirty-six hours there will be
seen upon the cut surface of the bread or potato small,
round, oval, or irregularly round patches which present
various appearances.
These differences in macroscopic appearance are due,
in some cases, to the presence or absence of color; in
others to a higher or lower degree of moisture; in
some instances a patch will be glistening and smooth,
while its neighbor may be dull and rough or wrinkled;
here will appear an island regularly round in outline,
and there an area covered by an irregular ragged de-
posit. 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 composing each of them will always produce
236 BACTERIOLOGY.
growth of exactly the same appearance. It was just
such an experiment as this, accidentally performed, that
suggested to Koch a means of separating and isolating
from mixtures of bacteria the component individuals in
pure cultures, and it was from this observation that the
methods of cultivation on solid media were evolved.
If, without molesting our experiment, we continue
the observation from day to day, we shall notice changes
in the colonies due to the growth and multiplication of
the individuals composing them. In some cases the
colonies 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 overrun the surface upon which they are grow-
ing, and, indeed, grow over the smaller, less rapidly
developing colonies. In a number of instances, if the
observation be continued long enough, many of these
rapidly growing colonies will, after a time, lose their
lustrous and smooth or regular surface and will show,
at first here and there, elevations which will continue
to appear until the whole surface takes on a wrinkled
appearance. Again, bubbles may be seen scattered.
through the colonies. These are due to the escape of
gas resulting from fermentation which the organisms
bring about in the medium upon which they are grow-
ing. Sometimes peculiar odors resulting from the same
cause will be noticed.
Note carefully all these changes and appearances, as
they must be employed subsequently in identifying the
individual organisms from which each colony on the
medium has developed.
If now we examine these points upon our bread or
MATERIAL WITH WHICH TO BEGIN WORK. 9237
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 noth-
ing 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 tolerably regular radii, radiating from a cen-
tral spot; and again they may appear as concentric
rings; and if by the methods which have been de-
scribed we obtain from these colonies their individual
components in pure culture, we shall see that this char-
acteristic 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:
11*
238 BACTERIOLOGY.
Place some of these pieces (about 5 centimetres long)
into a sterilized test-tube, and sterilize them by steam
for one hour. At the end of the sterilization remove
one piece with sterilized forceps and allow it to brush
against your clothing, then make a plate from it; draw
another piece across the table and then plate it. Sus-
pend three or four pieces upon a sterilized wire hook
and let them hang for thirty minutes free in the air,
being sure that they touch nothing but the hook; then
plate them separately.
Note the results.
In what way do these experiments differ and how
can the differences be explained ?
Expose to the air six Petri dishes into which either
sterilized gelatin or agar-agar has been poured and
allowed to solidify; allow them to remain exposed for
five, ten, fifteen, twenty, twenty-five, and thirty min-
utes in a room where no one is at work. Treat a sec-
ond set in the same way in a room where several persons
are moving about. Be careful that nothing touches
them, and that they are exposed only to the air. Each
dish must be carefully labelled with the time of its
exposure.
Do they present different results? What is the rea-
son for this difference ?
Which predominate, colonies resulting from the
growth of bacteria, or those from common moulds?
How do you account for this condition ?
CHAPTER XV.
Various experiments in sterilization by steam and by hot air.
PLACE in one of the openings in the cover of the
steam sterilizer an accurate thermometer; when the
steam has been streaming for a minute or two the ther-
mometer 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 indi-
cate a temperature 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
sterilizing properties that steam at the same temperature
possesses. It is necessary, therefore, that this air should
be expelled from the meshes of the material and its place
taken by the steam before sterilization is complete. This
is insured by allowing the steam to stream through the
substances a few minutes before beginning to calculate
the time of exposure. 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 time that is required for the expulsion
240 BACTERIOLOGY.
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. Con-
tinue 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 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 gel-
atin 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,
STERILIZATION BY STEAM AND HOT AIR. 241
but, as a rule, they will represent but a single organism.
Study this organism in pure cultures.
The same may be predicted for the tube which has
been heated for ten minutes, though the colonies will be
fewer in number.
The thirty-minute tube may or may not give one or
two colonies of the same organism.
The tube which has been heated for one hour is
usually sterile.
The bouillon tubes from the first and second tubes
which were heated will usually show the presence of
only one organism—the bacillus which gave rise to the
pellicle-formation in our original mixture. This organ-
ism is the bacillus subtilis, and will serve as an object
upon which to study the difference in resistance toward
steam between the vegetative and spore stages of the
same organism.
Inoculate about 100 c.c. of sterilized bouillon with
a very small quantity of a pure culture of this organism,
and allow it to stand in a warm place for about six
hours. Now subject this culture to the action of steam
for five minutes; it will be seen that sterilization, as a
rule, is complete.
Treat in the same way a second flask of bouillon,
inoculated 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
242 BACTERIOLOGY.
or the presence of bacteria microscopically does not
always signify that the organisms possess the property
of life,
Inoculate in the same way a third flask of bouillon
with a very small drop from one of the old cultures upon
which the pellicle has formed; mix it well and subject
it to the action of steam for two minutes; then place it
to one side for from twenty to twenty-four hours, and
again heat for two minutes; allow it to stand for another
twenty-four hours, and repeat the process on the third
day. No pellicle will be formed, and yet spores were
present in the original mixture, and, as we have seen,
the spores of this organism are not killed by an exposure
of five minutes to the steam. How can this result be
accounted for ?
Saturate several pieces of cotton thread, each about 2
em. 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 temper-
ature for the same lengths of time that we exposed the
same organisms in bouillon to the steam, viz., five, ten,
thirty, and sixty minutes. At the end of each of these
periods remove a bit of thread, and prepare a set of
plates or Esmarch tubes from it. Are the results anal-
ogous to those obtained when steam was employed ?
Increase the temperature of the dry sterilizer and
repeat the process. Determine the temperature and
time necessary for the destruction of these organisms
by the dry heat. These threads should not be simply
STERILIZATION BY STEAM AND HOT AIR. 243
laid upon the bottom of the sterilizer, but should be
suspended from a glass rod, which may be placed inside
the oven, extending across its top from one side to the
other.
Place several of the infected threads in the centre of
a bundle of rags. Subject this to a temperature neces-
sary to sterilize the threads by the dry method. Treat
another similar bundle to sterilization by steam. In
what way do the results of the two processes differ ?
CHAPTER XVI.
Suppuration—The staphylococcus pyogenes aureus—Staphylococcus pyo-
genes albus and citreus—Streptococcus pyogenes—Bacillus pyocyaneus—Gen-
eral remarks.
Prepare from the pus of an acute abscess or boil
that has been opened under antiseptic precautions a set
of plates of agar-agar. Care must be taken that none
of the antiseptic fluid gains access to the culture tubes,
otherwise its antiseptic effect may be seen and the devel-
opment of the organisms interfered with. It is best,
therefore, to take up a drop of the pus upon the plati-
num-wire loop after it has been flowing for a few sec-
onds; even then it must be taken from the mouth of
the wound and before it has run over the surface of the
skin. At the same time prepare two or three cover-
slips from the pus.
Microscopic examination of these slips will reveal the
presence of a large number of pus-cells, both multi-
nucleated and with horseshoe-shaped nuclei, some
threads of disintegrated and necrotic connective tissue,
and, lying here and there throughout the preparation,
small round bodies which will sometimes appear singly,
sometimes in pairs, and frequently will be seen grouped
together somewhat like clusters of grapes. (See Fig.
54.) They stain readily and are commonly located in
the material between the pus-cells; very rarely they
may be seen in the protoplasmic body of the cell.
(Compare the preparation with a similar one made from
SUPPURATION. 245
the pus of gonorrhcea (see Fig. 56, page 259). In what
way do the two preparations differ, the one from the
other ?)
Fie. 54,
Preparation from pus, showing pus-cells, A, and staphylococci, C.
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 the naked-eye appearances.
When located in the depths of the medium they are
commonly seen to be lozenge or whetstone in shape,
while often they appear as irregular stars with blunt
points, and again as irregularly lobulated dense masses.
In structure they are conspicuous for their density.
Under the low objective they appear, when on the sur-
face, as coarsely granular, irregularly round patches,
with more or less ragged borders and a dark irregular
central mass, which has somewhat the appearance of
masses of coarser clumps of the same material as that
composing the rest of the colony. Microscopically,
these colonies are composed of small round cells, irreg-
246 BACTERIOLOGY.
ularly grouped together. They are in every way of
the same appearance as those seen upon the original
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,
most conspicuous at its upper end, will be observed.
As growth continues the liquefaction becomes more or
less of a stocking-shape, and gradually widens out at its
upper end into an irregular funnel. This will continue
until the whole of the gelatin in the tube eventually
becomes fluid. There can always be noticed at the
bottom of the liquefying portion an orange-colored or
yellow mass composed of a number of the organisms
which have sunk to the bottom of the fluid.
On potato the growth is quite luxuriant, appearing as
a brilliant, orange-colored layer, somewhat lobulated
and a little less moist than when growing upon agar-
agar. It does not produce fermentation with gas-pro-
duction. 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 micro-
cocci does not form endogenous spores. It possesses,
however, a marked resistance toward detrimental agen-
cies.
In bouillon it causes a diffuse clouding, and after a
time presents a yellow sedimentation.
This organism is the commonest of the pathogenic
bacteria with which we shall meet. It is the staphylo-
coccus pyogenes aureus, and is the organism most fre-
quently concerned in the production of acute, cireum-
scribed, suppurative inflammations. It is almost every-
STAPHYLOCOCCUS PYOGENES AUREUS. 9247
where present, and is the organism that causes the
surgeon so much annoyance.
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 suppu-
rative processes in man, still it is with no little difficulty
that these conditions can be reproduced in lower ani-
mals. Its subcutaneous introduction into their tissues
does not always result in abscess-formation, and when it
does there seems to have been some coincident interfer-
ence with the circulation and nutrition of these tissues
which renders them less able to resist its inroads. When
introduced into the great serous cavities of the lower
animals its presence here, too, 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 intestines, large quantities of bouillon cul-
tures or watery suspensions of this organism may be,
and repeatedly have been introduced into the peritoneum
without the slightest injury to the animal. On the con-
trary, 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—be at
the same time introduced, or the intestines be mechani-
cally injured, so that there is a disturbance in their cir-
culation, then the introduction of these organisms is
promptly followed by acute and fatal peritonitis. (Hal-
sted.*)
On the other hand, the results which follow their in-
troduction into the circulation are practically constant.
1 Halsted: The Johns Hopkins Hospital Reports. Report in Surgery No. 1,
1891, Vol. II., No. 5, pp. 301-308.
248 BACTERIOLOGY.
If one inject 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 ¢.c. of a bouillon culture or watery suspension
of a virulent variety of this organism, a fatal pyemia
always follows in from two and one-half to three days.
A few hours before death the animal is frequently seen
to have severe convulsions. Now and then excessive
secretion of urine is noticed. The animal may appear
in moderately good condition until from eight to ten
hours before death. At the autopsy a typical picture
presents; the voluntary muscles are seen to be marked
here and there by yellow spots, which average the size
of a flaxseed, and are of about the same shape. They
lie usually with their long axis running longitudinally
between the muscle fibres. As the abdominal and tho-
racic cavities are opened the diaphragm is often seen to
be studded by them. Frequently the pericardial sac is
distended with a clear gelatinous fluid, and almost con-
stantly the yellow points are to be seen in the myocar-
dium. The kidneys are rarely without them; here they
appear on the surface, scattered about as single yellow
points, or, again, are seen as conglomerate masses of
small yellow points which occupy, as a rule, the area
fed by a single vessel. If one make 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 the
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 kid-
ney, myocardium, and voluntary muscles.
COVER-SLIPS AND SECTIONS. QA9
These minute abscesses contain a dry, cheesy, necrotic
centre, in which the staphylococci are present in large
numbers, as may be seen upon cover-slips prepared from
them. They may also be obtained in pure culture from
these suppurating foci.
Preserve in Miiller’s fluid and in alcohol duplicate
bits of all the tissue in which the abscesses are located.
When these tissues are hard enough to cut sections
should be made through the abscess-points, and the his-
tological 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 utilized, and
the location and grouping of the organisms in the
tissues rendered more conspicuous.
When stained, sections of tissues containing these
small abscesses present the following appearances:
To the naked eye will be seen here and there in the
section, if the abscesses are very numerous, small, darkly
stained areas which range in size from that of a pin-
point up to those having a diameter of from 1 to 2 mm.
These points, when in the kidney, may be round or oval
in outline, or may appear wedge-shaped, with the base
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 in stained sections, 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.
250 BACTERIOLOGY.
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 evidences of pro-
gressing necrosis. The normal structure of the cells of
the tissues will be more or less destroyed; there will be
seen a granular condition due to cell-fragmentation; 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 ab-
scesses the staphylococci are to be seen scattered about
the centre of the necrotic tissue, but in a more advanced
stage they are commonly seen massed together in very
large numbers in the form commonly referred to as
emboli of micrococet.
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 repre-
sents the starting-point for all abscess-formations.
When the process is farther developed the different
parts of the abscess are more easily detected. They
then present in sections somewhat the following condi-
tions: at the centre can be seen a dense, granular mass
which stains readily with the basic aniline dyes and,
when highly magnified, is found to be made up of
staphylococci. Sometimes 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 destroyed, though it is
COVER-SLIPS AND SECTIONS. 251
seen to be more advanced in some of the elements of
the tissues than in others. .As we approach the periph-
ery 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 dis-
integration 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 cor-
responds 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 rab-
bit the staphylococcus pyogenes aureus may again be
obtained in pure culture, and will present identically
the same characteristics that were possessed by the cul-
ture with which the animal was inoculated.
THe Less Common Pyocenic OrGAnNisms.—The
pus of an acute abscess in the human being may some-
times contain other organisms beside the staphylococcus
pyogenes aureus. The staphylococcus pyogenes albus
and citreus may be found. The colonies of the former
are white, those of the latter are lemon-color. With
these exceptions they are in all essential cultural peculi-
252 BACTERIOLOGY.
arities similar to the staphylococcus aureus. As a rule,
they are not virulent for animals, and when they do pos-
sess pathogenic properties it is in a much lower degree
than is commonly the case with the golden staphy-
lococeus. The streptococcus pyogenes is also sometimes
present. The commonest of the pyogenic organisms,
however, is that just described, viz.: the staphylococcus
pyogenes aureus, An organism that is almost univer-
sally present in the skin, and is often concerned in pro-
ducing mild forms of inflammation, is the staphylococcus
epidermidis albus (Welch), an organism that may readily
be confused with the staphylococcus albus. It is distin-
guished from the latter by the slowness with which it
liquefies gelatin and by the comparative absence of
pathogenic properties when injected into the circulation
of rabbits. Welch regards this organism as a variety
of the staphylococcus pyogenes albus. He suggests
the above designation for it because of its very limited
pyogenic properties.
Tue Srreprococcus PyoGENnes.—From a spread-
ing phlegmonous inflammation prepare cover-slips and
cultures. What is the predominating organism? Does
it appear in the form of regular clusters like those of
grapes, or have its individuals a definite regular ar-
rangement? Are its colonies like those of the staphy-
lococcus pyogenes aureus ?
Tsolate this organism in pure cultures. In these cul-
tures it will be found on microscopic examination to
present an arrangement somewhat like a chain of beads.
(Fig. 55.)
Determine its peculiarities and describe them accu-
rately. They should be as follows:
Upon microscopic examination a micrococcus should
THE STREPTOCOCCUS PYOGENES. 253
be found, but differing in its arrangement from the
staphylococci just described. The single cells are not
scattered irregularly or arranged in clumps similar to
bunches of grapes, but are joined together in chains like
strands of beads. These strands are sometimes regular
in the arrangement and size of the individual cells com-
posing them, but more commonly certain irregular parts
may be seen in them. Here they appear as if two or
Fig. 55,
Streptococcus pyogenes.
three cells had fused together to form a link, so to speak,
in the chain, that is somewhat longer than the remain-
ing links; again, portions of the chain may be thinner
than the rest, or may appear broken or ragged. Com-
monly the individuals comprising this chain of cocci
are not round, but appear flattened on the sides adjacent
to one another. The chains are sometimes short, con-
sisting of four to six cells, or again they may be much
longer, and extend from a half to two-thirds of the way
across the field of the microscope.
Under artificial conditions it sometimes grows well,
and can be cultivated through many generations, while
again it rapidly loses its vitality. When isolated from
the diseased area upon artificial media it seems to retain
its vitality for a longer period if replanted upon fresh
media every day or two for a time; but if the first gen-
12
254 BACTERIOLOGY.
eration is transplanted and is allowed to remain upon
the original medium, it is not uncommon to find the
organism incapable of further cultivation after a week
or ten days.
Under no conditions is the growth of this organism
very luxuriant.
On gelatin plates its colonies appear after forty-eight
to seventy-two hours as very small, flat, round points, of
a bluish-white or opalescent appearance. They do not
cause liquefaction of the gelatin, and in size they rarely
exceed 0.6-0.8 mm. in diameter. Under low magnify-
ing power they have a brownish or yellowish tinge by
transmitted light, and are finely granular. As the col-
onies become older their regular border may become
slightly irregular or notched.
In stab-cultures in gelatin they grow along the entire
needle-track as a finely granular line, the granules rep-
resenting minute colonies of the organism. On the
surface the growth does not usually extend beyond the
point of puncture.
On agar-agar plates the colonies appear as minute
pearly points, which when slightly magnified are seen
to be finely granular, of a light-brownish color, and
regular at their margins.
When smeared upon the surface of agar-agar or gel-
atin slants the growth that results is a thin, pearly,
finely granular layer, consisting of minute colonies
growing closely side by side. Its growth is most lux-
uriant on glycerin agar-agar at the temperature of the
incubator (87.5° C.), and least on gelatin.
On blood-serum its colonies present little that is char-
acteristic; they appear as small, moist, whitish points,
from 0.6 to 0.8 mm. in diameter, that are slightly ele-
THE STREPTOCOCCUS PYOGENES. 955
vated above the surface of the serum. They do not
coalesce to form a layer over the surface, but remain as
isolated colonies.
On potato no visible development appears, but after
a short time (thirty-six to seventy-two hours) there is
a slight increase of moisture about the point inoculated,
and microscopic examination shows that a multiplication
of the organisms placed at this point has occurred.
In milk its conduct is not always the same, some cul-
tures causing a separation of the milk into a firm clot
and colorless whey, while others do not produce this
coagulation. The latter, when cultivated in milk of a
neutral or slightly alkaline reaction, to which a few
drops of litmus tincture have been added, produce a
very faint pink color after twenty-four hours at 37.5°
C.; there is no coagulation.
In bouillon it grows as tangled masses or clumps,
which upon microscopic examination are seen to consist
of long chains of cocci twisted or matted together.
It grows best at the temperature of the body (37.5°
C.), and develops, but less rapidly, at the ordinary room
temperature. When virulent, its virulence is said by
Petruschky to be preserved by retaining cultures in
the ice-chest after they have been growing on gelatin
for two days at 22° C.
It is a facultative anaérobe.
It stains with the ordinary aniline dyes, and is not
decolorized when subjected to Gram’s method.
Jt is not motile, and, being a micrococcus, does not
form endogenous spores. Under artificial conditions
we have no reason to believe that it enters a stage
where its resistance to detrimental agencies is increased.
In the tissues of the body, however, it appears to pos-
256 BACTERIOLOGY.
sess a marked tenacity to vitality, for it is not rare to
observe recurrences of inflammatory conditions due to
this organism, often at a relatively long time after the
primary site of infection is healed.
When introduced into the tissues of lower animals its
effects are uncertain. Rosenbach and Passet claimed
that protracted, progressive, erysipelatoid inflammations
were produced, and Fehleisen, who described a strep-
tococcus in erysipelas that is in all probability identical
with the streptococcus pyogenes now under considera-
tion, stated that it produced in the tissues of rabbits
(the base of the ear) a sharply defined, migratory red-
dening without pus-formation. ‘The writer bas encoun-
tered a culture of this organism that possessed the prop-
erty of inducing erysipelas when introduced into the
skin of the ear, and disseminated abscess-formation
when injected into the circulation of rabbits. This
observation has an important bearing upon the ques-
tion concerning the identity of streptococci found in
inflammatory conditions. As a rule, it is difficult to
obtain any definite pathological alterations in the tis-
sues of animals through the introduction into them of
cultures of this organism by any of the methods of
inoculation ordinarily practised. Occasionally, how-
ever, cultures are encountered that are conspicuous for
their pathogenic powers.
This is the streptococcus pyogenes, and is the organism
most commonly found in rapidly spreading suppura-
tion 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
TAE STREPTOCOCCUS PYOGENES. 257
with the streptococcus just mentioned. Inoculate rab-
bits both subcutaneously and into the circulation with
about 0.2 ¢.c. of pure cultures of these organisms in
bouillon. Do the results correspond, and do they in
any way suggest the results obtained with the staphylo-
coccus pyogenes aureus when introduced into animals in
the same way? Do these streptococci flourish readily
on ordinary media?
The organisms that have just been described are
commonly known as the ‘‘ pyogenic cocci’’ of Ogston,
Rosenbach, and Passet, and up to as late as 1885 were
believed to be the specific factors concerned in the pro-
duction of suppurative inflammations. Since that time,
however, considerable modification of this view has
taken place, and while they are still known to be the
most common causes of suppuration, they are also
known not to be the only causes of this process.
With the more general application of bacteriological
methods to the study of the manifold conditions coming
under the eye of the physician, the surgeon, and the
pathologist, observations are constantly being made
that do not accord with the view formerly held with
regard to the specific relation of the pyogenic cocci to
all forms of suppuration. There is an abundance of
evidence now at command to justify the opinion that
there are a number of organisms not commonly classed
as pyogenic which may, under peculiar circumstances,
assume this property. For example:
The bacillus of typhoid fever has been found in pure
culture in osteomyelitis of the ribs; in acute purulent
otitis media; in abscess of the soft parts; in the pus
of empyema, and in localized fibro-peritonitis, either
during its course or as a sequela of typhoid fever.
258 BACTERIOLOGY.
The bacterium coli commune has been found to be
present in pure culture in acute peritonitis; in liver
abscess; in purulent inflammation of the gall-bladder
and ducts; in appendicitis; and Welch’ has found it in
pure culture in fifteen different inflammatory conditions.
The micrococcus lanceolatus (pneumococcus) has been
found to be the only organism present in abscess of the
soft parts; in purulent infiltration of the tissues about
a fracture; in purulent cerebro-spinal meningitis; in
suppurative synovitis; in acute pericarditis, and in acute
inflammation of the middle ear.
Moreover, many of the less common organisms have
been detected in pure cultures in inflammatory condi-
tions with which they were not previously thought to
be concerned, and to which they are not usually related
etiologically.
In consideration of such evidence as this it is plain
that we can no longer adhere rigidly to the opinions
formerly held upon the etiology of suppuration, but
must subject them to modifications in conformity with
this newer evidence. We now know that there exist
bacteria other than the ‘‘ pyogenic cocci,’’ which, though
not normally pyogenic, may give rise to tissue-changes
indistinguishable from those produced by the ordinary
pus organisms.’
GONOCOCCUS. MICROCOCCUS GONORRHG.
One observes upon microscopic examination of cover-
slips prepared from the pus of acute gonorrhcea that
1 Welch: ‘Conditions underlying the Infection of Wounds,’ American
Journal of the Medical Sciences, November, 1891.
2 For a more detailed discussion of the subject see ‘‘ The Factors Concerned
in the Production of Suppuration,” International Medical Magazine, Phila-
delphia, May, 1892.
GONOCOCCUS. 259
many of the pus-cells contain within their protoplasm
numerous small, stained bodies that are usually arranged
in pairs. Occasionally a cell is seen that contains only
one or two pairs of such bodies; again, a cell will be
encountered that is packed with them. Occasionally
masses of these small bodies will be seen lying free in
the pus. (See Fig. 56.) The majority of the pus-cells
do not contain them.
Fig. 56,
Pus of gonorrhcea, showing diplococci in the bodies of the pus-cells.
These small, round, or oval bodies are the so-called
“* gonococci’’ discovered by Neisser, and more fully
studied subsequently by Bumm, to whom we are in-
debted for much of our knowledge concerning them.
As the name implies, this organism is a micrococcus,
and as it is commonly arranged in pairs (flattened at
the surface in juxtaposition) it is often designated as
diplococcus of gonorrhea. It is always to be found in
gonorrhceal pus, and often persists in the urethral dis-
charges and secretions far into the stage of conva-
lescence. It is not present in inflammatory conditions
other than those of gonorrheal origin.
260 BACTERIOLOGY.
It is easily detected microscopically in the secretions
of acute gonorrhea. In secondary lesions and in very
old, chronic cases it is difficult of detection and fre-
quently eludes all efforts to find it. It is stained by the
ordinary methods, but perhaps most satisfactorily with
the alkaline solution of methylene-blue. Most impor-
tant as a differential test is its failure to stain by the
method of Gram. (How does this compare with the
behavior of the other pyogenic cocci when treated in
the same way ?)
It does not grow upon the ordinary nutrient media,
and has only been isolated in culture through the em-
ployment of special methods. Its growth under arti-
ficial circumstances seems to depend upon some par-
ticular nutrient substance that is supplied by blood or
blood-serum, and in all the media that have been suc-
cessfully used for its cultivation this substance is
apparently an essential constituent. By the majority
of investigators it is thought that only human blood
possesses this important ingredient.
It was first isolated in culture by Bumm, who used
for this purpose coagulated human blood-serum ob-
tained from the placenta.
Wertheim improved the method of Bumm by using
a mixture of equal parts of sterile human blood-serum
and ordinary sterilized nutrient agar-agar, the latter
having been liquefied and kept at 50° C. until after
the mixture was made, when it was allowed to cool and
solidify.
Other investigators have substituted for human blood-
serum certain pathological fluids from the human body,
such as ascites fluid, fluid from ovarian cysts, and serous
effusions from the pleura and from the joint-cavities.
GONOCOCOUS. 261
The method used by Pfeiffer for the cultivation of
the bacillus of influenza is also said to have been suc-
cessfully employed. Abel recommends a needle-prick
in the finger as a most convenient source from which to
obtain the necessary amount of human blood that is to
be smeared over the surface of the slanting agar-agar
when Pfeiffer’s method is employed.
Wright’s modification of Steinschneider’s method has
given such satisfactory results in his hands that it will
be given here with more or less detail. The medium
consists of a mixture of urine, blood-serum (human or
bovine, either serving the purpose), and nutrient agar-
agar. The urine and blood-serum are collected with-
out special aseptic precautions, and subsequently freed
from bacteria by filtration through unglazed porcelain.
Frequently this is the tedious part of the process, as
the serum and urine pass very slowly through the por-
celain filters that are generally employed in laborato-
ries. Wright recommends a filtering cylinder manu-
factured by the Boston Filter Company as an apparatus
that not only strains out all bacteria, but also permits of
a very rapid passage of the fluid.
The details of the method as given by Wright are as
follows: ‘‘A litre of nutrient agar is prepared in the
usual manner, and after filtration it is evaporated to
about 600 c.c. This concentration is desirable, so that,
after the dilution with the urine and serum, the medium
may be sufficiently firm. This concentrated agar is then
run into test-tubes and the whole sterilized by steam on
three successive days. The quantity of agar placed in
each tube is smaller than is usual; this is in order to
allow for the subsequent addition of the urine and
serum.
12*
262 BACTERIOLOGY.
‘¢The blood-serum, which need not be free from cor-
puscles, is first passed through white sand, which is
supported in a funnel by filter-paper, in order to re-
move as much as possible any particles in suspension,
and is then mixed with half its volume of fresh urine.
The mixture of urine and blood-serum is next filtered
by suction through an unglazed porcelain cylinder into
a receiving flask, such as chemists use for similar pur-
poses by means of a water vacuum pump. This frees
the mixture from bacteria.
“The usual precautions are, of course, taken to pre-
vent the contamination of the filtrate, such as the previous
sterilization by steam of the cylinder and receiving flask,
besides others which will occur to any bacteriologist.
‘To the agar in each test-tube, which is fluid and of
a temperature of about 40° C., there is added about
one-third to one-half its volume of the filtered mixture
of urine and blood-serum. This is conveniently accom-
plished by pouring the mixture from the receiving flask
through the lateral tube, inserted near its neck directly
into the tubes. The preliminary melting of the agar
is best effected in the steam sterilizer in order that any
organisms which have found lodgement in the cotton
plugs of the tubes may be destroyed. When the agar
is melted it is cooled and kept fluid by placing the
tubes in a water-bath at 40° C. Each tube, after the
addition of the urine and serum to the fluid agar, is
quickly shaken to insure a good mixture, and is then
placed in an inclined position to allow the agar to
solidify with a slanting surface. When the medium
in the tubes has solidified the tubes are placed in the
incubator for about twenty-four hours to test for con-
taminations, after which they are ready for use.”’
GONOcOocCUSs. 263
The successive dilutions are now to be made upon
the slanting surface of this mixture, as the mass in the
tubes cannot be redissolved without exposure to a de-
gree of heat that apparently interferes with the nutri-
tive value of the serum contained in the medium.
When inoculated with gonorrheal pus, by smearing
a loopful over the surface, the tubes are to be kept at
from 37° to 38° C. The organism does not develop
properly at a temperature below this point.
After twenty-four hours the colonies of the gono-
coceus appear on the surface of the medium, accord-
ing to Wright, as very tiny, grayish, semi-translucent
points. After forty-eight hours they may be about
1 millimetre or so in diameter, slightly elevated, with
a rounded outline, grayish in color, and by transmitted
light semi-translucent. By reflected light their sur-
face has the appearance of frosted glass. Later, if few
in number, so that their growth is unimpeded, the colo-
nies may attain a diameter of 2 millimetres or more,
become thicker and denser, with a faintly brownish
tinge about their centres, and a slightly irregular out-
line.
Under a low power of the microscope a fully de-
veloped colony is seen to consist of a general circular
expansion, with thin, translucent, smooth, sharply de-
fined margin, but becoming brownish, granular, and
thicker toward the central portion, which is made up
of coarse, granular, brown-colored clumps closely
packed together.
The appearances coincide with the figure of such a
colony given by Wertheim.
If transplanted from the original culture to either
glycerin agar-agar or to Leeffler’s serum mixture, a
264 BACTERIOLOGY.
growth is sometimes observed, more often in the latter
than in the former, but of so feeble a nature that these
substances cannot be regarded as suitable for its culti-
vation. As a rule, development does not occur on
glycerin agar.
Microscopic examination of colonies of this organism
reveals the presence of a diplococcus somewhat larger
than the ordinary pyogenic cocci. The opposed sur-
faces of the individual cells that comprise the couplets
are flattened and separated by a narrow slit. At times
the cocci are arranged as tetrads.
This organism cannot be grown at a temperature
lower than that of the human body, and cultures that
have been obtained by either of the favorable methods
are said to lose their vitality when kept at ordinary
room temperature for about two days.
It is killed in a few hours by drying.
Cultures retain their vitality under favorable condi-
tions of nutrition, temperature, and moisture for from
three to four weeks.
It is without pathogenic properties for monkeys,
dogs, and horses, as well as for the ordinary smaller
animals used for this purpose in the laboratory.
In man typical gonorrhea has been produced on
several occasions by the introduction into the urethra
of pure cultures of this organism.
In addition to its causal relation to specific ure-
thritis, it is the cause of gonorrhceal prostatitis in
man, of gonorrheeal proctitis in both sexes, and of gon-
orrheeal inflammation of the urethra, of Bartholin’s
glands, of the cervix uteri, and of the vagina in
women and young girls. It is etiologically related to
the specific conjunctivitis (ophthalmia neonatorum) of
GONOCOCOUS. 265
young infants, and also occasionally of ophthalmia in
adults.
Secondarily, it is concerned in specific inflammations
of the tubes and ovaries, of the lymphatics communi-
cating with the genitalia, of the serous surfaces of joints,
and of those of the heart, lungs, and abdominal cavity.
Positive AND NeGATIVE DisTINGUISHING PECULI-
ARITIES OF THE Gonococcus.—Since gonorrheeal dis-
charges may be contaminated with pyogenic cocci other
than those causing the specific inflammation, it is im-
portant in efforts to isolate this organism that the dif-
ferential tests be borne in mind and put into practice.
The gonococcus is differentiated from the commoner
pyogenic organisms by the following peculiarities:
First, it is practically always seen in the form of dip-
lococci, the pair of individual cells having the appear-
ance of two hemispheres, with the diameters opposed
and separated from one another by a narrow, colorless
slit. (Is this the case with the staphylococcus or strep-
tococcus pyogenes ?)
Second, in the pus it is practically always within the
protoplasmic bodies of pus cells. (How does this com-
pare with the conditions found in ordinary pus?)
Third, it stains readily with the ordinary staining-
reagents, but loses its color when treated by the method of
Gram. (Treat a cover-slip from ordinary pus by this
method and note the result.)
Fourth, it does not develop upon any of the ordinary
media used in the laboratory; while the common pus-
organisms, with perhaps the exception of the strepto-
cocci, are vigorous growers and are not markedly fas-
tidious as to their nutritive medium,
Fifth, when obtained in pure culture by either of the
266 BACTERIOLOGY.
special procedures noted above, its cultivation may be
continued upon the same medium; but growth will
usually not be observed if it is transplanted to ordi-
nary nutrient gelatin, agar-agar, bouillon, or potato;
should it grow under these circumstances its develop-
ment will be very feeble. (Is this the case with com-
mon pus-producers ?)
Sixth, it has no pathogenic properties for animals,
while several of the pyogenic cocci, notably staphylo-
coccus aureus and streptococcus pyogenes, are usually
capable of exciting pathological conditions. (This is
less commonly true of streptococcus pyogenes than of
staphylococcus aureus.)
BACILLUS PYOCYANEUS (BACILLUS OF GREEN PUS).
Another common organism that may properly be
mentioned at this place, though perhaps not strictly
pyogenic, is a bacillus frequently found in discharges
from wounds, viz., the bacillus pyocyaneus, or bacillus
of green pus, or of blue pus, or of blue-green pus, as it
is commonly called. The bacillus pyocyaneus is a deli-
cate rod with rounded or pointed ends. It is actively
motile; does not form spores. As seen in preparations
made from cultures it is commonly clustered together
in irregular masses. It does not form long filaments,
there being rarely more than four joined together end
to end, and most frequently not even two.
Jt grows readily on all artificial media, and gives to
some of them a bright-green color that is most conspic-
uous where it is in contact with the air. This green
color is not seen in the growth itself to any extent, but
is diffused through the medium on which the organism
BACILLUS PYOCYANEUS. 267
is developing. With time this color becomes much
darker, and in very old agar-agar cultures may become
almost black (sometimes very dark-blue green, at others
brownish-black).
Fia. 57. Fic. 58.
Colony of b. pyocyaneus after twenty-four
hours on gelatin at 20°-22° C.
Stab-culture of b.
pyocyaneus in gel-
atin after twenty- Colony of b. pyocyaneus after forty-two hours
eight hours at 22°C. on gelatin at 209-22° C,
Its growth on gelatin in stab-cultures is accompanied
by liquefaction and the diffusion of a bright-green color
throughout the unliquefied medium. As liquefaction
continues, and the entire gelatin ultimately becomes
fluid, the green color is confined to the superficial layers
that are in contact with the air. The form taken by the
liquefying portion of the gelatin in the earliest stages of
268 BACTERIOLOGY.
development is somewhat that of an irregular, slender
funnel. (See Fig. 57.)
On gelatin plates the colonies develop rapidly; they
are not sharply circumscribed, but usually present at
first a fringe of delicate filaments about their periphery
(see Fig. 58). As growth progresses and liquefaction
becomes more advanced, the central mass of the colony
sinks into the liquefied depression, while at the same
time there is an extension of the colony laterally. At
this stage the colony, when slightly magnified, may
present various appearances, the most common being
that shown in Fig. 59.
The gelatin between the growing colonies takes on a
bright yellowish-green color; but as growth is compar-
atively rapid, it is quickly entirely liquefied, and one
often sees the colonies floating about in the pale-green
fluid.
On agar-agar the growth is dry, sometimes with a
slight metallic lustre, and is of a whitish or greenish-
white color, while the surrounding agar-agar is bright
green. With time this bright green becomes darker,
passing to blue-green, and finally turns almost black.
On potato the growth is brownish, dry, and slightly
elevated above the surface. With some cultures the
potato about the growth becomes green; with others this
change is not so noticeable. With many cultures a pecu-
liar phenomenon may be produced by lightly touching
the growth with a sterilized platinum needle. This
phenomenon consists in a change of color from brown
to green at the point touched. It is best seen in cul-
tures that have been kept in the incubator for from
seventy-two to ninety-six hours. It occurs in from one
to three minutes after touching with the needle, and
BACILLUS PYOCYANEUS. 269
may last from ten minutes to half an hour. This is
the ‘‘ chameleon phenomenon’’ of Paul Ernst.
In bouillon the green color appears, and the growth
is seen in the form of delicate flocculi. A very deli-
cate mycoderma is also produced.
In milk it causes an acid reaction, with coincident
coagulation of the casein.
On blood-serum and egg-albumin its growth is ac-
companied by liquefaction. The growth on coagulated
egg-albumin is seen as a dirty-gray deposit surrounded
by a narrow brownish zone; the remaining portion of
the medium is bright green in color. As the culture
becomes older the green may give way to a brown dis-
coloration.
In peptone solution (double strength) it causes a
bluish-green color. In one of four cultures from differ-
ent sources there was a distinct blue color produced.
It produces indol.
Tt stains with the ordinary dyes, and its flagella may
be readily demonstrated by Leeffler’s method of staining.
Inoculation into animals. As a rule, cultures of this
organism obtained directly from the discharges of a
wound are capable, when introduced into animals, of
lighting up diseased conditions; but cultures that are
kept on artificial media for a long time may in part, or
completely, lose this power.
When guinea-pigs or rabbits are inoculated subcuta-
neously with 1 c.c. of virulent fluid cultures of this
organism, death usually results in from eighteen to
thirty-six hours. At the seat of inoculation there is
found an extensive purulent infiltration of the tissues
and a marked zone of inflammatory cedema.
When introduced directly into the peritoneal cavity
270 BACTERIOLOGY.
the results are also fatal, and at autopsy a genuine
fibrinous peritonitis is found. There is usually an ac-
cumulation of serum in both the peritoneal and pleural
cavities. At autopsies after both methods of inocula-
tion the organisms will be found in the blood and inter-
nal viscera in pure cultures.
When animals are inoculated with small doses (less
than 1 ¢.c. of a bouillon culture) of this organism
death may not ensue, and only a local inflammatory
reaction (abscess-formation) may be set up. In these
cases the animals are usually protected against subse-
quent inoculation with doses that would otherwise
prove fatal.
Most interesting in connection with bacillus pyocy-
aneus is the fact, as brought out in the experiments of
Bouchard, and of Charrin and others, that its products
possess the power of counteracting the pathogenic ac-
tivities of bacillus anthracis. That is to say, if an
animal be inoculated with a virulent anthrax culture,
and soon after be inoculated with a culture of bacillus
pyocyaneus, the fatal effects of the former inoculation
may be prevented.
In the literature upon the green-producing organisms
that have been found in inflammatory conditions sev-
eral varieties—believed to be distinct species—have
been described, but when cultivated side by side their
biological differences are seen to be so slight as to ren-
der it probable that they are but modifications of one
and the same species.
THE BACILLUS OF BUBONIC PLAGUE.
Before passing from the subject of suppuration it
may not be inappropriate to call attention to the light
THE BACILLUS OF BUBONIC PLAGUE. 9271
that modern methods of investigation have shed upon
the etiology of bubonic plague, an epidemic disease
characterized by suppuration of the lymphatic glands,
and accompanied by a very high rate of mortality.
This pestilence, probably endemic in certain sections
of the Orient, is one of the most conspicuous epidemic
diseases of history. Since early in the Christian era
epidemics and pandemics of plague have made their
appearance in Europe at different times. During and
after the Middle Ages it was more or less frequent in
India, China, Arabia, Northern Africa, Italy, France,
Germany, and Great Britain. In history it is vari-
ously known as the ‘‘ Justinian Plague’’ of the sixth
century, the ‘‘ Black Death’? of the fourteenth cen-
tury, and the ‘‘ Great Plague of London” of the sev-
enteenth century, though it is difficult to say to what
extent these pestilences were uncomplicated manifesta-
tions of genuine bubonic plague. During the existence
of the Justinian Plague 10,000 people are said to have
died in Constantinople in a single day, and Hecker esti-
mates that during the pandemic of the Black Death
25,000,000 people (a quarter of the entire population
of Europe) succumbed to the disease. During the Great
Plague of London (1664-65) the total mortality for one
year was 68,596, out of an estimated population of
460,000 souls.
It is not surprising to learn that it was to guard
against the plague that quarantine regulations were
first established.
For the most recent, and probably the most exact
information concerning the cause and pathology of the
plague we are indebted to the investigations of Yersin,
272 BACTERIOLOGY.
of Kitasato, and of Aoyama, conducted during the epi-
demic of 1894 in Hong-Kong, China. The results of
these studies indicate that bubonic plague is an infec-
tious, not markedly contagious, disease that depends for
its existence upon the presence in the tissues of a spe-
cifie micro-organism —the so-called plague or pest
bacillus.
Bacillus of bubonic plague: A, in pus from suppurating bubo ; B, the
bacilli very much enlarged to show peculiar polar staining.
This organism is described as a short, oval bacillus,
usually seen single, sometimes joined end to end in pairs
or threes, less commonly as longer threads. It stains
more readily at its ends than at its centre. It is some-
times capsulated; is non-spore-forming; is aérobic, and
THE BACILLUS OF BUBONIC PLAGUE. 273
is non-motile. It is found in large numbers in the
suppurating glands, and in much smaller numbers in
the circulating blood. (Fig. 60.)
It is demonstrable in cover-slip preparations made
from the pus and in sections of the glands by the ordi-
nary staining-methods. Yersin states that it retains
its color when treated by the method of Gram, while
Kitasato says that it at one time stains by this method
and at another it becomes decolorized. Aoyama observed
that those bacilli within the suppurating glands were
decolorized, while those in the blood retained the stain
when treated by Gram’s method.
Since there is often a mixed infection in these cases
it appears likely that the above discrepancy may be
attributed to individual peculiarities of different species
of bacteria that were under examination.
It may be cultivated upon ordinary nutrient media,
The most favorable temperature for its growth is
between 36° and 39° C. Its colonies on glycerin agar-
agar and on coagulated blood-serum are described as
iridescent, transparent, and whitish. On gelatin at
18°-20° C. it develops as small, sharply defined, white
colonies. In stab-cultures it develops both on the sur-
face and along the track of the needle. Its growth is
slow. It does not cause a diffuse clouding of bouillon,
but grows rather as irregular, flocculent clumps that
adhere to the sides or sink to the bottom of the vessel,
leaving the fluid clear.
It is pathogenic for mice, rats, guinea-pigs, rabbits,
and sheep. Pigeons are immune. The animals suc-
cumb to subcutaneous inoculation in from two to three
days. According to Yersin, the site of subcutaneous
inoculation becomes edematous and the neighboring
274 BACTERIOLOGY.
lymphatics enlarged in a few hours. After twenty-four
hours the animal is quiet, the hair is rumpled, tears
stream from the eyes, and later convulsions set in which
last till death. The results found at autopsy are: blood-
stained oedema at the site of inoculation, reddening and
swelling of the lymphatic glands, bloody extravasation
into the abdominal walls, serous effusion into the pleu-
ral and peritoneal cavities; the intestine is occasionally
hypereemic, the adrenal bodies congested, and the spleen
is enlarged, often showing the presence of grayish points
suggestive of miliary tubercles. The plague, or pest,
bacillus is to be detected in large numbers in the local
cedema, the lymph glands, the blood, and the internal
organs.
As is the case with the group of hemorrhagic septi-
cemia bacteria, when death does not result promptly
after infection there is usually only local evidence of
the inoculation, the distribution of the micro-organisms
throughout the body being considerably diminished.
It is said that when virulent cultures are employed
animals may sometimes be infected by way of the ali-
mentary tract.
This organism is killed by drying at ordinary room
temperature in four days. It is killed in three to four
hours by direct sunlight. It is destroyed in a half hour
by 80° C., and in a few minutes by 100° C. (steam).
It is killed in one hour by 1 per cent. carbolic acid
and in two hours by 1 per cent. milk of lime.
The bacilli apparently lose their virulence after long-
continued cultivation under artificial circumstances,
and it is said that from slowly developing, chronic
buboes non-virulent or feebly virulent cultures are
often obtained. Variations in the degree of virulence
THE BACILLUS OF BUBONIC PLAGUE. 275
have been observed in different colonies from the same
source,
In man the bacilli are most numerous in the en-
larged, suppurating lymphatics. They are present, but
in smaller numbers, in the blood and the internal organs.
It has been observed that in the suppurating lym-
phatic glands of man a variety of organisms may be
present, conspicuous among them being the so-called
plague bacillus. Occasionally, micrococci predominate.
In these cases of mixed infection the pest bacilli are
said to stain less intensely with alkaline methylene-blue
than do the streptococci, and more intensely than do the
staphylococci that are present. Also, in this event, the
streptococci retain the Gram stain, while the pest bacilli
and the staphylococci do not. It has been suggested
that possibly the organisms found by Kitasato in the
blood, and which he describes as pest bacilli, that re-
tained the color when treated by the method of Gram,
were pairs of micrococci and not bacilli at all.
It is the opinion of Aoyama that the suppuration of
the glands is not caused by the plague bacillus, but is
rather the result of the action of the pyogenic cocci
with which it is so often associated.
Again, according to Aoyama, the most important and
frequent mode of infection in man is through wounds
of the skin. He does not regard either the air-pas-
sages or the alimentary tract as frequent portals of
infection.
The order in which the lymphatics manifest disease
appears to depend upon the location of the primary
infection. That is to say, if it is upon the feet, as of
persons who go barefooted, the superficial and deep
inguinal glands are the first to show signs of the dis-
276 BACTERIOLOGY.
ease; while if infection occurs through wounds of the
hand, the buboes appear first in the axillary region.
As a rule,the wound through which infection is re-
ceived shows little or no inflammatory reaction.’
1 The works of Yersin, of Kitasato, and of Aoyama have been exhaustively
reviewed by Flexner in the Bulletin of the Johns Hopkins Hospital, vol. v.,
1894, p. 96, and vol. vii., 1896, p. 180. I am indebted to these reviews for much
that is here presented on this subject.
CHAPTER XVII.
Sputum septicemia—Septicemia resulting from the presence of micro-
coccus tetragenus in the tissues—Tuberculosis.
Oxsrtarin 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 it. 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 Leeffler’s alkaline methylene-blue
solution, the other by the Gram method, the third after
the method given for tubercle bacilli in fluids or spu-
tum.
In that stained by Leeffler’s method—slip No. 1—
will be seen a great variety of organisms—round cells,
ovals, short and long rods, perhaps spiral forms. But
not infrequently will be seen diplococci, having more or
less of a lancet shape; they will be joined together by
their broad ends, the points of the lancet being away
from the point of juncture of the two cells. There may
also be seen masses of cocci which are conspicuous for
their arrangement into groups of fours, the adjacent
surfaces being somewhat flattened. They are not sar-
cina, as one can see by the absence of the division in
the third direction of space—they divide in only two
directions.
13
278 BACTERIOLOGY.
In the slip stained by the Gram method the same
groups of the cocci which grow as threes and fours will
be seen, but our lancet-shaped diplococci will now pre-
sent an altered appearance—there can now be detected
a capsule surrounding them. This capsule is very deli-
cate in structure, and, though a frequent accompani-
ment, is not constant. It can sometimes be demon-
strated by the ordinary methods of staining, though
the method of Gram is most satisfactory. (ig. 62.)
In the third slip which has been stained by the
method given for tubercle bacilli in sputum, if decol-
orization has been properly conducted and no contrast-
stain has been employed, the field will be colorless 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 in-
stances, a conspicuous beaded arrangement of their pro-
toplasm—that is, the staining is not homogeneous, but
at tolerably regular intervals along each rod there are
seen alternating intervals of light and color. These rods
may be found singly, in groups of twos and threes, or
sometimes in clumps consisting of large numbers.
When in twos or threes it is not uncommon to find
them describing an X ora V in their mode of arrange-
ment, 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 bacteria
in the preparation, as well as the tissue-cells which are
in the sputum, will take up the contrast-color. (Fig. 61.)
These delicate beaded rods are the bacillus tubercu-
SPUTUM SEPTICEMIA. 279
losis. The lancet-shaped diplococci with the capsule
are the micrococcus lanceolatus.
Fic. 61.
Tuberculous sputum stained by Gabbett’s method. Tubercle bacilli seen as
red rods; all else is stained blue.
The cocci grouped in fours are the micrococcus tetra-
genus.
InocuLaTIoN Experiment. — Inoculate into the
subcutaneous tissues of a guinea-pig one of the small
white caseous masses similar to that which has been
examined microscopically. If death ensues, it will be
the result of one of the three following forms of infec-
tion:
a. Septicemia' 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 Frinkel; meningococcus; strepto-
coccus lanceolatus Pasteuri; micrococcus lanceolatus;
micrococcus Pasteuri; coccus lanceolatus; bacillus sali-
1 Septiceemia is that form of infection in which the blood is the chief field
of activity of the organisms.
280 BACTERIOLOGY.
varius septicus; bacillus septicus sputigenus; diplo-
coccus lanceolatus capsulatus; micrococcus pneumonize
croupose.
b. A form of septicemia resulting from the invasion
of the tissues by an organism frequently seen in the
sputum of tuberculous subjects. It is characterized
by its tendency to divide into fours. It is the miero-
coccus tetragenus.
e. Local or general tuberculosis.
a. SPUTUM SEPTICEMIA.
If at the end of twenty-four to thirty-six hours the
animal be found dead, we may safely suspect that the
result was produced by the introduction into the tissues
of the organism of sputum septicemia above mentioned,
viz., the micrococeus lanceolatus, which is not uncom-
monly found in the mouths of healthy individuals as
well as in other conditions.
Inspection of the seat of inoculation usually reveals
a local reaction. ‘‘ This may be of a serous, fibrinous,
hemorrhagic, necrotic, or purulent character. Fre-
quently we may find combinations of these conditions,
such as fibro-purulent, fibrino-serous, or sero-hemor-
rhagic.”’? The most conspicuous naked-eye change
undergone by the internal organs will be enlargement
of the spleen. It is usually swollen, but may at times
be normal in appearance. It is sometimes hard, dark
red, and dry, or it may be soft and rich in blood. Fre-
quently there is a limited fibrinous exudation over por-
tions of the peritoneum.
1 Welch: Johns Hopkins Hospital Bulletin, December, 1892, vol. iii, No. 27.
SPUTUM SEPTICEMIA. 28]
Except in the exudations, the organisms are found
only in the lumen of the bloodvessels, where they are
usually present in enormous numbers.
In the blood they are practically always free and are
but rarely found within the bodies of leucocytes.
In stained preparations from the blood and exudates
a capsule is not infrequently seen surrounding the organ-
isms. (Fig. 62.) This, however, is not constant.
Fic. 62.
Micrococcus lanceolatus in blood of rabbit. Stained by method of Gram.
Decolorizatiou not complete.
If a drop of blood from this animal be introduced
into the tissues of a second animal (mouse or rabbit),
identically the same conditions will be reproduced.
If the organism be isolated from the blood of the
animal in pure culture, and a portion of this culture be
introduced into the tissues of a susceptible animal,
again we shall see the same pathological picture.
It must be remembered, however, that this or-
ganism when cultivated for a time on artificial media
rapidly loses its pathogenic properties. If, therefore,
failure to .reproduce the disease after inoculation
282 BACTERIOLOGY.
from old cultures should occur, it is in all probability
due to a disappearance of virulence from the or-
ganism.
This organism was discovered by Sternberg in 1880.
Tt 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
individuals, having been found by Sternberg in the oral
cavities of about 20 per cent. of healthy persons examined
by him. It is constantly to be detected in the rusty
sputum of patients suffering from acute fibrinous pnen-
monia. Its presence has been detected in the middle
ear, in the pericardial sac, in the pleura, in the serous
cavities of the brain, and indeed it may penetrate from
its primary seat in the mouth to almost any of the more
distant organs.
The organism is commonly found as a diplococcus,
though here and there short chains of four to six indi-
viduals joined together may be detected. (Fig. 62, page
281.) The individual cells are more or less oval, or,
more strictly speaking, lancet-shaped, for at one end
they are commonly pointed. When joined in pairs the
junction is always between the broad ends of the ovals,
never between the pointed extremities.
As already stated, in preparations directly from the
sputum or from the blood of animals, a delicate capsule
may frequently be seen surrounding them. Though
fairly constant in preparations directly from the blood
of animals and from the sputum or lungs of pneumonic
patients, the capsule is but rarely observed in artificial
cultures. Occasionally in cultures on blood-serum, in
milk, and on agar-agar they can, according to some
SPUTUM SEPTICEMIA. 283
authors, be detected; but this is by no means constant,
or even frequent.
This organism grows under artificial conditions very
slowly, and frequently not at all.
When successfully grown upon the different media it
presents somewhat the following appearance ;
On gelatin it grows very slowly, if at all, probably
owing in part to the low temperature at which gelatin
cultures must be kept. If development occurs, it ap-
pears as minute whitish or blue-white points on the
plates. These very small colonies are round, finely
granular, sharply circumscribed, and slightly elevated
above the surface of the gelatin. The growth is very
slow, and no liquefaction of the gelatin accompanies it.
If grown in slant- or stab-cultures, the surface-devel-
opment is very limited; along the necdle-track tiny
whitish or bluish-white granules appear.
On nutrient agar-agar the colonies are almost trans-
parent; they are more or less glistening and very deli-
cate in structure. On blood-serum development is more
marked, though still extremely feeble. Here it also ap-
pears as a cluster of isolated fine points growing closely
side by side.
A growth on potato is not usually observed. When
grown in milk it commonly causes an acid reaction with
coincident coagulation of the casein. Some varieties,
especially non-virulent ones, do not coagulate milk.*
It is not motile.
It grows best at a temperature of from 35° to 38° C.
Under 24° C. there is usually no development, but in a
few cases it has been seen to grow at as low a tempera-
1 Welch, loc. cit.
984 BACTERIOLOGY.
ture as 18° C. From 42°C. on the development is
checked.
Under most favorable conditions the growth is very
slow. It grows as well without as with oxygen. It is,
therefore, one of the facultative anaérobic forms.
The most successful efforts at the cultivation of this
organism are those seen when the agar-agar-gelatin
mixture of Guarniari is employed. (See this medium. )
It may be stained with the ordinary aniline staining-
reagents. For demonstrating the capsule the method
of Gram and the acetic acid method give the best
results. (See Stainings.)
This organism is conspicuous for the irregularity
of its behavior when grown under artificial conditions;
usually it loses its pathogenic properties after a few
generations; but again this peculiarity may be re-
tained fora much longer time. Not rarely it fails to
grow after three or four transplantations on artificial
media, though at times it may be carried through many
generations.
Inoculation into animals. The results of inoculations
with pure cultures of this organism are also conspicuous
for their irregularity. When the organism is of full
virulence the form of septiceemia just described is
usually produced, but at times it is found to be totally
devoid of pathogenic powers; between these extremes
cultures may be obtained possessing all variations in
the intensity of: their disease-producing properties.
The principal pathological conditions that may be pro-
duced by this organism by inoculations into animals,
according to the degree of its virulence, are acute septi-
cemia, spreading inflammatory exudations, and cir-
cumscribed abscesses. All three of these conditions
MICROCOCCUS TETRAGENUS. 285
may sometimes be produced by inoculating the same
cultures into rabbits in varying amounts.
Rabbits, mice, guinea-pigs, dogs, rats, cats, and sheep
are susceptible to infection by this organism. Chickens
and pigeons are insusceptible. Young animals, as a
rule, are more easily infected than old ones, Rabbits
and mice are the most susceptible of the animals used
for experimental purposes, and in testing the virulence
of a culture it is well to inoculate one of each, for with
the same cultures it sometimes occurs that it may be
virulent for mice and not for rabbits, and vice versa.
If the culture is virulent, intravascular or intra-
peritoneal injections into rabbits may produce rapid and
fatal septicemia, while subcutaneous inoculation of the
same material may result in only a localized infamma-
tory process. On the other hand, subcutaneous inocula-
tion of less virulent cultures may produce a local process,
while intravenous inoculation may be without result.
This organism is the cause of a number of pathological
conditions in human beings that have not hitherto been
considered as related to one another etiologically. It
is always present in the inflamed area of the lung in
acute fibrinous or lobar pneumonia; it is known to cause
acute cerebro-spinal meningitis, endo- and peri-carditis,
certain forms of pleuritis, arthritis and peri-arthritis,
and otitis media.
b. SEPTICAMIA CAUSED BY THE MICROCOCCUS
TETRAGENUS.
Should the death of the animal not occur within the
first twenty-eight to thirty hours after inoculation, but
be postponed until between the fourth and eighth day,
13*
286 BACTERIOLOGY.
it may occur as a result of invasion of the tissues by
the organism now to be described, viz., the mierococcus
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 pulmonary cavities of
phthisical patients.
It, however, plays no part in the etiology of tuber-
culosis.
It isa small round coccus of about 1 y 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
preparations made from cultures of this organism it
is not rare to find, here and there, single bodies which
are much larger than the other individuals in the field.
Close inspection reveals them to be cells in the 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 to 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-
MICROCOCCUS TETRAGENUS. 287
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 be-
yond the size of very small points.
Tt does not liquefy gelatin.
Upon plates of nutrient agar-agar the colonies appear
as small, almost transparent, round points, which have
about the same color and appearance as a drop of egg-
albumin; they are very slightly opaque. They are
moist and glistening. They rarely develop to an
extent exceeding 1 to 2 mm. in diameter.
Upon agar-agar as stab- or slant-cultures the surface-
growth has more or less of a mucoid appearance. It
is moist, glistening, and irregularly outlined. The out-
line of the growth depends upon the moisture of the
agar-agar. It is slightly elevated above the surface of
the medium.
In contradistinction to the gelatin stab-cultures, the
growth in agar-agar is continuous along the track of
the needle.
The growth on potato is a thick, irregular, slimy-
looking patch.
The presence of the transparent gelatinous substance
which is seen to surround these organisms renders them
coherent, so that efforts to take up a portion of a colony
from the agar-agar or potato cultures result usually in
drawing out fine, silky threads consisting of organisms
imbedded in this gelatinous material.
The organism grows best at from 35° C. to 38° C.,
but can be cultivated at the ordinary room temperature
—about 20° C.
288 BACTERIOLOGY.
The growth under all conditions is slow.
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.
Inoculation into animals. To the naked eye no alter-
ation can be seen in the organs of animals that have
died as a result of inoculation with the micrococcus tet-
ragenus; but microscopic examination of cover-slip
preparations from the blood and viscera reveals the
presence of the organisms throughout the body—espe-
cially is this true of preparations from the spleen.
White mice and guinea-pigs are susceptible to the dis-
ease. Gray mice, dogs, and rabbits are not susceptible
to this form of septicemia. Subsequent 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 re-
production 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 result local
pus-formations, instead of a general septicemia. The
organisms will then be found in the pus-cavity.
CHAPTER XVIII.
Tuberculosis—Microscopic appearance of miliary tubercles—Encapsulation
of tuberculous foci—Diffuse caseation—Cavity-formation—Primary infection
—Modes of infection—Location of the bacilli in the tissues—Staining-pecu-
liarities—Organisms with which bacillus tuberculosis may be confounded—
Points of differentiation.
SHOULD the animal succumb to neither of the septic
processes just described, then its death from tuberculosis
may be reasonably expected.
When this disease is in progress alterations in the
lymphatic glands nearest the seat of inoculation may
be detected by the touch in from two to four weeks.
They will then be found to be enlarged. Though not
constant, tumefaction and subsequent ulceration at the
point of inoculation may sometimes be observed. Pro-
gressive emaciation, loss of appetite, and difficulty in
respiration point to the existence of the general tuber-
cular 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 ani-
mals vary so widely the one from the other, that no
rigid law as to what will appear at autopsy can @ priori
be laid down.
The guinea-pig, which is best suited for this experi-
ment because of the greater regularity of its suscepti-
bility to the disease over that of other animals usually
found in the laboratory, presents, in the main, changes
that are characterized by a condition of coagulation-
290 BACTERIOLOGY.
necrosis and caseation. This is particularly the case
when the infection is general—i.e., when the process is
of the acute miliary type. This pathological-anatom-
ical alteration is best seen in the tissues of the liver
and spleen of these animals, where the condition is most
pronounced.
In general, the tubercular lesions can be divided into
those of strictly focal character —i.e., 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 cireum-
scribed.
These latter lesions play a greater réle in the pathol-
ogy of the disease than do the miliary nodules, although
it is to the presence of the miliary nodules that the
disease owes its name.
At autopsy the pathological manifestations of the dis-
ease are not infrequently seen to be confined to the seat
of inoculation and to the neighboring lymphatic glands.
These tissues will then present all the characteristics of the
tuberculous process in the stage of cheesy degeneration.
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 hempseed, and, as a rule, are, in this stage, the
result of the fusion of two or more smaller miliary foci.
Though the two terms ‘‘ miliary’? and ‘‘ conglomerate’?
MICROSCOPIC APPEARANCE OF TUBERCLES. 291
exist for the description of the macroscopic appearance
of these nodules, yet it is very rarely that any condition
other than that due to the fusion together of several of
these minute foci can be detected by the naked eye.
The miliary tubercles are of a pale gray color, with a
white centre, are slightly elevated above the surface of
the tissue in which they exist, and, as stated, vary con-
siderably in dimensions, usually appearing as points
which range in size from that of a pin-point to that of
a pin-head. ‘hey 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 noth-
ing characteristic, but may frequently, when closely
packed together in large numbers, give a mealy or
sandy sensation to the fingers. Stained sections of these
miliary tubercles present a distinctly characteristic
appearance, and the disease may be diagnosticated by
these histological changes alone, though the crucial test
in the diagnosis is the finding of tubercle bacilli in these
nodules.
Microscopic APPEARANCE OF MILIARY TUBER-
cLes.— The simple miliary tubercles under a low
magnifying power of the microscope present somewhat
the following appearance: there is a central pale area,
evidently composed of necrotic tissue because of its in-
capacity for taking up the nuclear stains commonly
employed. Scattered here and there through this ne-
crotic area may be seen granular masses irregular in
size and shape; they take up the stains employed, and
are evidently the fragments of cell-nuclei in the course
of destruction. Through the necrotic area may here
and there be seen irregular lines, bands, or ridges, the
remains of tissues not yet completely destroyed by the
292 BACTERIOLOGY.
necrotic process. Around the periphery of this area
may sometimes be noticed large multinucleated cells,
the nuclei of which are arranged about the periphery
of the cell or grouped irregularly at its poles. The
arrangement of these nuclei as observed in sections
is usually oval, or somewhat ecrescentic. In the tuber-
cles from the human subject these large ‘‘ giant-cells,”
as they are called, are quite common. They are much
less frequent in tubercular tissues from lower ani-
mals.
Round about the 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 homoge-
neously. They vary in size and shape, and are seen to
be imbedded in a delicate network of fibrinous-looking
tissue.
This fibrin-like network in which these bodies lie, and
which is a common accompaniment of giant-cell forma-
tion, 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 speak-
ing is the zone of so-called ‘‘ granulation tissue,’ and
consists of leucocytes, granulation cells, fibrin, and the
fibrous remains of the organ; the irregularly oval, gran-
ular bodies which take up the stain 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 tissues or
fuses with a similar zone surrounding another tubercu-
lar focus. This may be taken as a description of the
typical miliary tubercle.
Dirruse CasEATION.—The diffuse caseation, as said,
plays a more important ré/e in the tuberculous lesion,
CAVITY-FORMATION. 293
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 tissues it is more marked than in others. These
tissues are the lungs and lymph-glands. In rabbits,
particularly, all the changes in the lung frequently come
under this head. When this is the case solid masses
are found, sometimes as large as a pea, or involving
even an entire lobe or the whole lung in some cases.
They are of a whitish-yellow, opaque color, and on sec-
tion 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 simul-
taneously 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 forma-
tion of the outer zone of the miliary tubercle. In
other instances the entire caseous area is surrounded by
a granulation zone similar to that around the caseous
centre of the miliary tubercles. It is of special im-
portance to recognize the connection between this dif-
fuse caseation and the tubercle bacillus, because until
its nature was accurately determined the caseous pneu-
monia of the lungs formed the chief obstacle which
many encountered in recognizing the specific infectious-
ness of tuberculosis.
CAVITY-FORMATION.—The production of cavities,
which form such a prominent feature in human tuber-
culosis, particularly in the lungs, is due to softening
of the necrotic, caseous masses or of aggregations of
294 BACTERIOLOGY.
miliary tubercles. The material softens and is ex-
pelled, and a cavity remains. In the wall of this
cavity the tuberculous changes still proceed, both as
diffuse caseation and formation of miliary tuber-
cles. The whole cavity with the reactive changes
in the tissues of its walls may be considered as rep-
resenting a single tubercle, its wall forming a tissue
very analogous to the outer zone of the single tuber-
cle, the cavity itself corresponding to the caseous .
centre.
In animals used for experiment cavity-formation of
this sort is very rare, owing to the greater resistance of
the caseous tissue. That it is, however, possible to pro-
duce in rabbits pulmonary cavities in all physical re-
spects similar to those seen in the human being has
been most beautifully demonstrated by Prudden. He
showed that when he had injected into the trachea of
rabbits, already affected with tubercular consolidation
of the lungs, fluid cultures of streptococcus pyogenes,
the result of the mixed infection thus brought about was
cavity-formation in eight out of nine lungs subjected
to the conditions of the experiment; while in only one
out of eleven did cavities form under the influence of
the tubercle bacillus alone.’
In the contents and in the walls of tubercular cavi-
ties in man bacteria other than the tubercle bacillus are
found. It is to the influence of some of these, as we
have seen, that diseases other than tuberculosis may
sometimes be produced by the inoculation of animals
with the sputum from such cases.
1 Prudden: Experimental Phthisis in Rabbits, with the Formation of Cavi-
ties, ete. Transactions of the Association of American Physicians, 1894, vol.
ix, p. 166.
PRIMARY INFECTION. 295
ENCAPSULATION OF TuBERCULAR Foct.—It not
uncommonly occurs that round about a necrotic tuber-
cular focus there is formed a fibrous capsule which may
completely cut off the diseased from the healthy tissue
surrounding it. Or a tubercular focus may, through
the resistance of the tissue in which it is located, be
more or less completely isolated. In this condition the
diseased foci may lie dormant for a long time and give
no evidence of their existence, until by some intercur-
rent interference they are caused to break through their
envelopes. With the passage of the bacilli or their
spores from the central foci into the vascular or lym-
phatic circulation the disease may then become general.
It is to some such accident as this that the sudden
appearance of general tubercular infection in subjects
supposed to have recovered from the primary local
manifestations may often be attributed. The breaking-
down of old caseous lymphatic glands is a common
example of this condition.
Primary [nrecrion.—The primary infection occurs
through either the vascular or lymphatic circulation.
Through these channels the bacilli gain access to the
tissues and become lodged in the finer capillary ramifi-
cations or in the more minute lymph-spaces. Here
they find conditions favorable to their development,
and in the course of tbeir life-processes produce sub-
stances of a chemical nature which act directly in
bringing about the death of the tissues in their imme-
diate neighborhood. This tissue-death is probably the
very first effect of the bacilli in the body, and repre-
sents 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
296 BACTERIOLOGY.
proceeds as long as the bacilli live and continue to
produce their poisonous 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 leuco-
eytes, all of which are seen in the meshes of the finer
fibrous tissues of the part. At the same time altera-
tions are produced in the walls of the vessels of the
locality; these tend to occlude them, and thus the pro-
cess of tissue-death is favored by a diminution of the
amount of nutrition brought to them. These changes
may continue until eventually conglomerate tubercles,
widespread caseation, or cavity-formation results; or
from one cause or another the life-processes of the
bacilli may be checked and recovery occur.
Mopes 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
tuberculous material; by the introduction of the bacilli
into the air-passages, and by inoculation into the ante-
rior chamber of the eye.
In the human subject the most common portals of
infection are, doubtless, the air-passages, the alimentary
tract, and cutaneous wounds. When introduced subcu-
taneously the resulting process finds its most pronounced
expression in the lymphatic system. The growing
bacilli make their way into the lymphatic spaces of
the loose cellular tissue, are taken up in the lymph
stream and deposited in the neighboring lymphatic
glands. Here they may remain and give rise to no
alteration further than that seen in the glands them-
MODES OF INFECTION. 297
selves, or they may pass on to neighboring glands, and
eventually be disseminated throughout the whole lymph-
atic system, ultimately reaching the vascular system.
After having gained access to the bloodvessels, the
results are the same as those following upon intravas-
cular injection of the bacilli, namely, general tubercu-
losis quickly follows, with the most conspicuous pro-
duction of miliary tubercles in the lungs and kidneys,
less numerous in the spleen, liver, and bone marrow.
When inhaled into the lungs, if conditions are favor-
able, multiplication of the bacilli quickly follows. With
their growth they are mechanically pressed into the
tissues of the lungs. As multiplication continues some
are transported from the primary seat of infection to
healthy portions of the lung tissue, there to give rise
to a further production of the tubercular process.
In the same way infection through the alimentary
tract is in the main due to mechanical pressure of the
bacilli upon the walls of the intestines. Investigation
has shown that lesions of the intestinal coats are not
necessary for the entrance of tubercle bacilli from the
intestines into the body. They may be transported
from the intestinal tract into the lymphatics in the
same way that the fat-droplets of the chyle find
entrance into the lymphatic circulation.
The evidence produced by Cornet,' together with
general statistical evidence, points to the lungs as the
most common portal of natural infection for the human
being. Unlike most pathogenic organisms, the tubercle
bacillus is believed to have the property of forming
spores within the tissues. These spores, which are pre-
1 Cornet: Zeit. fiir Hygiene, 1889, Bd. vy. 8. 191.
298 BACTERIOLOGY.
sumably highly resistant and not destroyed by drying,
are thrown off from the lungs in the sputum of tuber-
culous patients in large numbers, and unless special
precautions be taken to prevent it the sputum becomes
dried, is ground into dust, and sets free in the atmos-
phere the spores of tubercle bacilli which came with it
from the lungs. The frequency of pulmonary tuber-
culosis points to this as one of the commonest sources
and modes of infection.
Location OF THE BACILLI IN THE TissuEs.—The
bacilli will be found to be most numerous in those
tissues which are in the active stage of the process.
In the very initial stage of the disease the bacilli will
be fewer in number than later. At this time only here
and there single rods may be found; later they will be
more numerous, and, finally, when the process has ad-
vanced to a stage easily recognizable by the naked eye,
they will be found in the granulation zones in clumps
and scattered about in large numbers.
In the central necrotic masses, which consist of cell
detritus, it is rare that the organisms can be demon-
strated microscopically. It is at the periphery of these
areas and in the progressing granular zone that they
are most frequently to be seen.
This apparent absence of the bacilli from the central
necrotic area must not be taken, however, as evidence
that this tissue does not contain them. As bacilli,
they are difficult to demonstrate here because the
probabilities are that in this locality, owing to con-
ditions unfavorable to their farther growth, they are
in the spore-stage, a stage in which it is as yet impos-
sible, with our present methods of staining, to render
them visible. The fact that this tissue is infective,
THE TUBERCLE BACILLUS. 299
and with it the disease can be reproduced in suscepti-
ble animals, speaks for the accuracy of this assump-
tion. .\ conspicuous example of this condition is seen
in old scrofulous glands. These glands usually pre-
sent a slow process, are commonly caseous, and always
possess the property of producing the disease when
introduced into the tissues of susceptible 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 accumu-
lated at the pole of the cell opposite to that occupied
by the nuclei, as if there existed an antagonism between
the nuclei and the bacilli. In some of these cells,
however, the distribution of the bacilli is seen to be
irregular, and they will be found scattered among the
nuclei as well as in the necrotic centre of the cell. As
the number of bacilli in the giant-cell increases the cell
itself is ultimately destroyed.
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
formulated by Koch, which are necessary to prove the
etiological ré/e of an organism in the production of a
malady, are all fulfilled.
Tur TuBERCLE Bacriuus.—Of the three patho-
genic organisms liable to occur in the sputum of a tu-
300 BACTERIOLOGY.
berculous subject, the tubercle bacillus will give us
most difficulty in our efforts at cultivation.
It is, in the strict sense of the word, a parasite, and
finds conditions entirely favorable to its development
only in the animal body. On ordinary artificial media
the bacilli taken directly from the animal body grow
only very imperfectly, or, in many cases, not at all.
From this it seems probable that there is a difference
in the nature of individual tubercle bacilli—some
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 arti-
ficial cultures from the animal body are offsprings from
the more saprophytic varieties. At best, one never sees
with the tubercle bacillus a saprophytic condition in
any way comparable to that possessed by many of the
other organisms with which we have to deal.
In efforts to cultivate this organism directly from the
tissues of the animal, the method by which one obtains
the best results is that recommended by Koch, viz., cul-
tivation 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 study
it successfully. It is, therefore, necessary that the
injunctions for obtaining it in pure culture should be
carefully observed.
PREPARATION OF CULTURES FROM TissuESs.— Under
strictest antiseptic precautions remove from the animal
the tubercular organ—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
PREPARATION OF CULTURES FROM TISSUES. 301
upon the surface of the blood-serum, one nodule in each
tube, and with a heavy, sterilized, looped platinum needle
or spatula, rub it carefully over the surface. It is best
to dissect away twenty to thirty such tubercles and
treat each in the same way. Some of the tubes will
remain sterile, others may be contaminated by outside
organisms during the manipulation, while a few may
give the result desired, viz., a growth of the tubercle
bacilli themselves.
The blood-serum upon which the organism is to be
cultivated should be comparatively freshly prepared—
that is, should not be dry.
After inoculating the tubes they should be carefully
sealed up to prevent evaporation and consequent dry-
ing. This is done by burning off the superfluous over-
hanging cotton plug in the gas-flame, and then impreg-
nating the upper layers of the cotton with either
sealing-wax or paraffin of a high melting-point; or
by inserting over the burned end of the cotton plug a
soft, closely fitting cork that has been sterilized in the
steam sterilizer just before using (Ghriskey). This
precaution 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 inocu-
lation upon blood-serum, and, as they must be retained
during this time at the body temperature—37.5° C.—
evaporation would take place very rapidly and the
medium would become too dry for their development.
If these primary efforts result in the appearance of a
culture of the bacilli, further cultivations may be made
by taking up a bit of the colony, preferably a moder-
ately large quantity, and transferring it to fresh serum,
14
302 BACTERIOLOGY.
and this in turn is sealed up and retained at the same
temperature. Once having obtained the organism in
pure culture, its subsequent cultivation may be con-
ducted 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 it has
accommodated itself to its saprophytic mode of exist-
ence, 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 containing 6 or 7 per cent. of glycerin.
Cultures of the tubercle bacillus are characteristic in
appearance—after once having seen them there is but
little probability of subsequent mistake.
They appear as dry masses, which may develop upon
the surface of the medium either as flat scales or as.
lumps of mealy looking granules. They are never
moist, and frequently have the appearance of coarse
meal which has been spread upon the surface of the
medium. In the lower part of the tube in which they
are growing—i.e., that part occupied by a few drops of
fluid which has in part been squeezed from the medium
during the process of solidification, and is in part water
of condensation—the colonies may be seen to float as a
thin pellicle upon the surface of the fluid.
The individuals making up the growth adhere so
tenaciously together that it is with the greatest diffi-
culty that they can be completely separated. In even
the oldest and dryest cultures pulverization is impos-
APPEARANCE OF TUBERCLE BACILLUS. 303
sible. 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 development is much more limited. They
are here of nearly the same color as the potato on which
they are growing. When cultivated for a time on
potato they are said to lose their pathogenic properties.
On milk-agar-agar they are of so nearly the same
color as the medium that, unless they are growing as
the mealy looking masses, considerably elevated above
the surface, their presence is less conspicuous than
when on the other media.
In bouillon they grow as a thin pellicle on the sur-
face. This may fall to the bottom of the fluid and
continue 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. :
Microscopi: 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
304 BACTERIOLOGY.
axis. In some preparations involution-forms, consisting
of rods a little clubbed at one extremity or slightly
bulging at different points, may be detected. Branch-
ing forms of this organism have been described. It
varies in Jength—sometimes being seen in very short
segments, again much longer, though never as long as
threads. On an average, its length is seen to vary
from 2 to 5 yw. It ix commonly described as being in
length about one-fourth to one-half the diameter of a
red blood-corpuscle. It is very slender. (Fig. 61,
page 279.)
These rods usually present, as has been said, an ap-
pearance 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.
STAINING-PECULIARITIES.—A_ peculiarity of this
organism is its behavior toward staining-reagents, and
by this means alone it may be easily recognized. The
tubercle bacillus does not stain by the ordinary
methods. It possesses some peculiarity in its com-
position that renders it more or less proof against the
simpler dyes. It is therefore necessary that more
energetic and penetrating reagents than the ordinary
watery solutions should be employed. Experience has
taught us that certain substances not only increase the
solubility of the aniline coloring substances, but by
their presence the penetration of the coloring agents is
very much increased. Two of these substances are
DIFFERENTIAL DIAGNOSIS. 305
aniline oil and carbolie acid. They are employed in
the solutions to about the point of saturation. (For
the exact proportions see chapter on Staining-reagents. )
Under the influence of heat these solutions are seen
to stain all bacteria very intensely—the tubercle bacilli
as well as the ordinary forms. If we subject our prep-
aration, which may contain a mixture of tubercle bacilli
and other forms, to the action of decolorizing-agents,
another peculiarity of the tubercle bacillus will be ob-
served. While all other organisms in the preparation
will give up their color and become invisible, the
tubercle bacillus retains it with marked tenacity. It
stains with great difficulty, but once stained it retains
the color even under the influence of strong decolor-
izing-agents.
ORGANISMS WITH WHICH THE BACILLUS TUBERCU-
LOSIS MAY BE CONFUSED.
DirFerentTiAL Diacenosis.—While its peculiar
micro-chemical reaction is usually considered to be
diagnostic of the bacillus tuberculosis, it is well to
remember that there are at least three other species
of bacilli which, when similarly treated, react in the
same way. It is of importance to bear this point in
mind, particularly in the microscopic examination of
urine and pathological secretions from the genito-
urinary tract and from the rectum, for of the three
species two are frequently found in these localities,
viz., the so-called smegma bacillus, located in the
smegma and often seen beneath the prepuce and upon
the vulva, both normally and in disease, and the so-
called bacillus of syphilis, described by Lustgarten as
306 BACTERIOLOGY.
contained in syphilitic manifestations, particularly in
primary sores. The third organism of this group—the
bacillus of leprosy—because of its rarity is not so likely
to cause error in the diagnosis of pathological conditions
occurring in these localities.
According to Hueppe, the differential diagnosis be-
tween the four organisms depends upon the following
reactions: when stained by the carbol-fuchsin method
commonly employed in staining the tubercle bacillus
the syphilis bacillus becomes almost instantly decolor-
ized by treatment with mineral acids, particularly sul-
phuric acid, whereas the smegma bacillus resists such
treatment fora much longer time, and the lepra and
tubercle bacillus for a still longer time. On the other
hand, if decolorization is practised with alcohol, instead
of acids, the smegma bacillus is the first to lose its color.
The bacillus tuberculosis and the bacillus of leprosy are
conspicuously retentive of their color even after treat-
ment with both acids and alcohol.
To differentiate, then, between the four organisms he
recommends the following order of procedure, based on
the above reactions :
1. Treat the preparation, stained with carbol-fuchsin,
with sulphuric acid ; the syphilis bacillus becomes de-
colorized, the reaction being almost instantaneous.
2. If it is not at once decolorized, treat with alcohol;
if it is the smegma bacillus, this will rob it of its color.
3. If it is still not decolorized, it is either the lepra
or tubercle bacillus,
Grethe (Fortschritte der Med., 1896, No. 9) recom-
mends the following as a trustworthy means of distin-
guishing between the tubercle bacillus and the smegma
bacillus: stain in hot carbol-fuchsin solution, wash off
ANIMALS SUSCEPTIBLE TO TUBERCULOSIS. 307
in water, and treat the preparation with a saturated
solution of methylene-blue in alcohol. If the ques-
tionable organism is the tubercle bacillus, it retains its
red color; if the smegma bacillus, the red color is dis-
solved out by the alcohol and the organism becomes
stained blue.
The differential diagnosis between the tubercle bacil-
lus and the lepra bacillus is less satisfactory; they both
take on the same stains and both retain them or give
them up under treatment with the same decolorizers.
The results of investigations, however, indicate differ-
ences in the rate of staining and decolorization, and it is
accepted by many of those who have compared the two
organisms that the lepra bacillus takes up stain very
much more readily than does the tubercle bacillus,
often staining perfectly by an exposure of only a few
minutes to cold watery solutions of the dyes; but when
once stained it retains its color much more tenaciously
when acted upon by decolorizing-agents than does the
latter organism.
According to Baumgarten, the lepra bacillus is stained
by an exposure of six to seven minutes to a cold, satu-
rated watery solution of fuchsin, and retains the stain
when subsequently treated with acid alcohol (nitric
acid, 1 part; alcohol, 10 parts). By similar treatment
for the same length of time the bacillus tuberculosis
does not ordinarily become stained.
These points, particularly what has been said with
reference to the smegma bacillus and the bacillus of
syphilis, are of much practical importance, and should
always be borne in mind in connection with microscopic
examination of materials to which these organisms are
liable to gain access. It is hardly necessary to say that
3808 BACTERIOLOGY.
in the examination of sputum and pathological fluids
from other parts of the body the tubercle bacillus is,
of the four organisms, always the one most commonly
encountered, while the organism described by Lust-
garten as the bacillus of syphilis is seen so rarely that
many trustworthy investigators question its existence
as a species distinct from the ordinary smegma bacillus.
TupercuLin.—The filtered products of growth from
old fluid cultures of the tubercle bacillus represent what
is known as tuberculin—a group of proteid substances
possessing most interesting properties. When injected
subcutaneously into healthy subjects tuberculin has no
effect; but when introduced into the body of the tuber-
culous person or animal a pronounced systemic reaction
results, consisting of sudden but temporary elevation of
temperature, with, at the same time, the occurrence of
marked hyperemia round about the tuberculous focus,
a change histologically analogous to that seen in the
primary stages of acute inflammation. This zone of
hyperemia, with the coincident exudation and infiltra-
tion of cellular elements, probably aids in the isolation
or casting off of the tuberculous nodule, the inflamma-
tory zone forming, so to speak, a line of demarcation
between the diseased and healthy tissue.
Asa curative agent for the treatment of tuberculosis,
tuberculin has not merited the confidence that was at
first accorded to it. Its greatest field of usefulness is
now admitted to be as an aid to the diagnosis of obscure
cases, and more particularly those occurring in cattle,
where it has proved itself to be of inestimable value in
this particular application.
SUSCEPTIBILITY OF ANIMALS TO TUBERCULOSIS.—
The animals which are known to be susceptible to the
ANIMALS SUSCEPTIBLE TO TUBERCULOSIS. 309
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 some time after diphtheria the
bacillus of Leffler is demonstrable in the pharynx, so
that it, too, may be present under exceptional circum-
stances. These latter organisms will be described under
their proper heads.
From time to time fowls are known to suffer from
a form of tuberculosis that is in many respects similar
to human tuberculosis both as regards pathological
lesions and etiology. The bacillus causing the dis-
ease, while very much like the genuine bacillus tuber-
culosis morphologically, differs from it in cultural pecu-
liarities, notably in its inability to produce general
tuberculosis in rabbits and guinea-pigs; in its growth
into long branched forms at 45° to 50° C.; and in its
never having been detected in human or mammalian
tuberculosis.
Anatomical lesions very suggestive of those produced
by bacillus tuberculosis have also from time to time been
observed in certain rodents. They do not appear to be
of specific nature as regards etiology, for the reason that
different authors have described different species of
bacilli as the causative agents. The disease suggests
tuberculosis only by the more superficial character of
its lesions, for in no instance have the organisms de-
tected been in any way similar to the genuine bacillus
14*
310 BACTERIOLOGY.
tuberculosis. These affections usually pass under the
name pseudo-tuberculosis.
THE BACILLUS OF INFLUENZA.
An important historic epidemic disease, on the nature
of which much light has been shed through modern
methods of investigation, is influenza. Quoting Hirsch:
the first trustworthy literary records that we have of
this disease date from the early part of the twelfth
century.
Between 1173 and 1874 it made its epidemic or pan-
demic appearance on eighty-six different occasions. Its
first appearance in this country was in Massachusetts in
1627; since that time there have been twenty-two vis-
itations of influenza to the United States. The recent
epidemic, namely, that of 1889~90, appears to have
originated in Central Asia and to have spread pretty
much over the entire civilized world. The occurrence
of influenza is always remarkable for the rapidity with
which it spreads.
During the recent pandemic investigations, having
for their object the discovery of its cause, were insti-
tuted, with the result of demonstrating in the catarrhal
secretions from the air-passages a micro-organism that
is claimed to stand in causal relation to influenza.
This organism, a bacillus, bacillus influenze, as it is
called, was discovered, isolated, cultivated, and deseribed
by R. Pfeiffer.
It is a very small, slender, non-spore-forming, non-
motile, aérobic bacillus, occurring singly and in pairs,
joined end to end. It stains with watery solutions of
the ordinary basic aniline dyes; somewhat better with
THE BACILLUS OF INFLUENZA. 311
alkaline-methylene-blue, but best when treated for five
minutes with a dilution of Ziehl’s carbol-fuchsin in
water (the color of the solution should be pale red).
(Fig. 63.) It is decolorized by the method of Gram.
Bacillus of influenza in sputum.
It develops only at temperatures ranging from 26°
to 48° C. Its optimum temperature for growth is
37°C. It possesses the peculiarity of developing upon
only those artificial culture-media to which blood or
blood-coloring-matter has been added. Its cultivation
is best conducted and its development most satisfac-
torily observed by the following procedure: over the
surface of a slanted agar tube or over agar-agar solid-
ified in a Petri dish smear a small quantity of sterile
blood (not blood-serum). A. bit of the mucus from the
sputum of the influenza patient is then taken up with
sterilized forceps or on a sterilized wire loop, rinsed
off in sterile bouillon or water and rubbed over the
312 BACTERIOLOGY.
surface of the prepared agar-agar. The plate or tube
is then placed in the incubator at 37° to 38° C. If in-
fluenza bacilli be present, they will develop as minute,
transparent, watery colonies that are without structure
and which resemble somewhat minute drops of dew.
They are discrete and show little or no tendency to
coalesce.
If a small bit of mucus be rubbed over the surface
of ordinary nutrient agar-agar, no such colonies de-
velop. In making the diagnosis by this method cul-
tures on both agar-agar containing blood (not blood-
serum) and agar-agar containing no blood should
always be made, for the reason that growth of these
peculiar colonies in the former and no such growth in
the latter are evidence that one is dealing with mate-
rials from a case of influenza.
It may also be cultivated in bouillon to which blood
has been added, if kept at body temperature. The
growth appears as whitish flakes. Since this organism
is a strict aérobe, its cultivation can only be conducted
on the surface of the medium used—i. ¢., where it has
freest access to oxygen. It is therefore inadvisable to
prepare plates in the usual way. When its cultivation
ig attempted in bouillon it is recommended, in order to
favor the free diffusion of oxygen, that the depth of
fluid be very shallow.
Contrary to what might be supposed, the bacillus of
influenza has very little tenacity to life outside of the
diseased body. It is destroyed by rapid drying in
from two to three hours, and when dried more slowly
in from eight to twenty-four hours. Cultures retain
their vitality for from two to three weeks. The
organism dies in water in a little over a day. As a
THE BACILLUS OF INFLUENZA. 313
result of these observations, Pfeiffer does not believe
the disease to be disseminated by either the air or the
water, but rather by direct infection from the catarrhal
secretions of the patients.
This organism has not been found outside of the
human body. In the influenza patient it is present in
the catarrhal secretions, bronchial mucous membrane,
and the diseased lung tissues. It may be demonstrated
microscopically in the mucus by cover-slip prepara-
tions made in the usual way and stained with diluted
carbol-fuchsin, referred to above. In the tissues it
may be demonstrated in sections stained in the same
solution. In the sputum the bacilli are found as masses
and as scattered cells. (See Fig. 63.) They are also
found within the bodies of leucocytes, especially in the
later stages of the disease when convalescence has set
in; at this time they appear as very small, irregular,
evidently degenerated bacilli within white blood-
corpuscles. They are also present in the nasal secre-
tions.
At autopsies it is advisable to cut out small pieces of
the diseased tissue of about the size of a pea or a bean,
rub them well in a small quantity of sterile water or
bouillon, and make the cultures from this infusion.
By this procedure two advantages are gained: first,
a dilution of the number of bacteria present; and,
secondly, the tissue furnishes the amount of hemoglo-
bin that is necessary for the growth of the organism.
Under these circumstances it is, of course, not neces-
sary to make a further addition of blood to the culture
medium.
The only animal that has been found to be suscept-
ible to inoculation with this organism is the monkey.
314 BACTERIOLOGY.
By intratracheal injection Pfeiffer succeeded in causing
a toxic condition that proved fatal. He does not re-
gard the death of the animals as due to infection, but
rather to intoxication. The disease, as seen in man,
has not been reproduced in animals.
CHAPTER XIX.
Glanders—Characteristics of the disease—Histological structure of the
glanders nodule—Susceptibility of different animals to glanders—The ba-
cillus of glanders; its morphological and cultural peculiarities—Diagnosis of
glanders.
Synonyms: Rotz (Ger.), Morve (Fr.).
The disease is generally known as glanders when
the mucous membrane of the nostrils is affected, and
as farcy when the skin is the principal site of involve-
ment.
Though most commonly seen in the horse and ass,
glanders is not rarely met with in other animals, and is
occasionally encountered in man. When occurring
spontaneously in the horse its primary seat is usually
upon the mucous membrane of the nostrils. It appears
in the form of small gray nodules, about which the
membrane is congested and swollen. These nodules
ultimately coalesce to form ulcers. There is a profuse
slimy discharge from the nostrils during the course of
the disease. Jt may extend from its primary seat in
the nose to the mouth, larynx, trachea, and ultimately
to the lungs. Its secondary manifestations are observed
along the lymphatics that communicate with the primary
focus; in the lymphatic glands, and as metastatic foci
in the internal organs. Less frequently the disease is
seen to begin in the skin, particularly in the region of
the neck and breast. When in this locality the sub-
cutaneous lymphatics become involved, and are con-
316 BACTERIOLOGY.
verted into indurated, knotty cords—‘“‘ farey buds’’—
easily discernible from without.
When occurring in man it is usually in individuals
who have been in attendance upon animals affected
with the disease. It may occur upon the mucous
membrane of the nares, but its most conspicuous ex-
pressions are in the skin and muscles, where appear
abscesses, phlegmons, erysipelas-like inflammations, and
local necrosis closely resembling carbuncles. Metas-
tases to the lungs, kidneys, and testicles, as in the horse,
may also be seen.
When occurring upon the mucous membrane glan-
ders is characterized by the presence of small gray
nodules, about as large as a pin-head, that closely
resemble miliary tubercles in their naked-eye appear-
ance. These consist histologically of granulation tissue—
i.e, of small round cells, very similar to proliferating
leucocytes, of some lymph-cells, and, in the earliest
stages, of a small portion of necrotic tissue. As they
grow older, and the process advances, there is a ten-
dency toward central necrosis, with the ultimate for-
mation of a soft, yellow, creamy, pus-like material.
Though strikingly like miliary tubercles in certain
respects in the early stages, they present, nevertheless,
decided points of difference when examined more
minutely.
The round-cell infiltration of the glanders nodules
consists essentially of polynuclear leucocytes, while
that of the miliary tubercle partakes more of the
nature of a lymphocytic infiltration; in the later
stages of the process the glanders nodule breaks down
into a soft creamy matter, very analogous to ordinary
pus, while in the later stages of the miliary tubercle
THE BACILLUS OF GLANDERS. 317
the tendency is toward an amalgamation of its histo-
logical constituents, and ultimately to necrosis with
caseation. The giant-cell formation common to tuber-
culosis is never seen in the glanders nodule. As
Baumgarten aptly puts it: ‘‘The pathological mani-
festations of glanders, from the histological aspect, stand
midway between the acute purulent and the chronic in-
flammatory processes.’’' Evidently these differences are
only to be explained by differences in the nature of the
causes that underlie the several affections. We have
studied the characteristics of bacillus tuberculosis ; we
shall now take up the bacillus of glanders and note the
striking differences between them.
THE BaciLius OF GLANDERS (bacillus mallei).—In
1882 Loeffler and Schiitz discovered in the diseased tis-
sues of animals suffering from glanders a bacillus that,
Fic. 64.
Bacillus of glanders (bacillus mallei).
when isolated in pure culture and inoculated into sus-
ceptible animals, possesses the property of reproducing
the disease with all its clinical and pathological mani-
festations. It is therefore the cause of the disease.
1 For a further discussion of the pathology and pathogenesis of this disease
see Lehrbuch der pathologischen Mykologie, by Baumgarten, 1890. See, also,
Wright: The Histological Lesions of Acute Glanders in Man. Journal of Ex-
perimental Medicine, vol. i. p. 577.
318 BACTERIOLOGY.
It is a short rod, with rounded or slightly pointed
ends, that usually takes up the stain somewhat irreg-
ularly. (See Fig. 64.) When examined in stained
preparations its continuity is marked by alternating
darkly and lightly stained areas. It is usually seen as
a single rod, but may occur in pairs, and less frequently
in longer filaments.
The question as to its spore-forming property is still
an open one, though the weight of evidence is in oppo-
sition to the opinion that it possesses this peculiarity.
Certain observers claim to have demonstrated spores in
the bacilli by particular methods of staining, but this
statement can have but little weight when compared
with the behavior of the organism when subjected to
more conclusive tests. For example, it does not, at
any stage of development, resist exposure to 3 per cent.
cearbolic acid solution for longer than five minutes, nor
to 1: 5000 sublimate solution for more than two min-
utes. It is destroyed in ten minutes in some experi-
ments, and in five in others, by a temperature of 55° C.,
and when dried it loses its vitality, according to dif-
ferent observers, in from thirty to forty days; all of
which speak directly against this being a spore-bearing
bacillus.
It is not motile, and does not, therefore, possess
flagella.
It grows readily on the ordinary nutrient media at
from 25° to 38° C.
Upon nutrient agar-agar, both with and without gly-
cerin, it appears as a moist, opaque, glazed layer, with
nothing characteristic about it. This is true both for
smear-cultures and for single colonies.
Its growth on gelatin is much less voluminous than
THE BACILLUS OF GLANDERS. 319
on media that can be kept at bigher temperature,
though it does grow on this media at room temperature
without causing liquefaction.
Its growth on blood-serum is seen in the form of a
moist, opaque, slimy layer, inclining to a yellowish or
dirty, brownish-yellow tinge. It does not liquefy the
serum.
On potato its growth is moderately rapid, appearing
at the end of from twenty-four to thirty-six hours at
37° C. as a moist, amber-yellow, transparent deposit
which becomes deeper in color and denser in consistence
as growth progresses. It finally takes on a reddish-
brown color, and the potato about it becomes darkened.
In bouillon it causes diffuse clouding, with ultimately
the formation of a more or less tenacious or ropy sedi-
ment.
In milk to which a little litmus has been added it
causes the blue color to become red or reddish in from
four to five days, and quite red after two weeks at 37° C.
At the same time the milk is separated into a firm clot
of casein and clear whey.
Its reactions to heat are very interesting—at 42° C.
it will often grow for twenty days or more. It will
not grow at 43° C., and is killed by exposure to this
temperature for forty-eight hours. It is killed in five
hours when exposed to 50° C., and in five minutes by
55° C,
It grows both with and without oxygen; it is there-
fore facultative as regards its relation to this gas.
On cover-slips it stains readily with all the basic
aniline dyes, and, as a rule, as stated, presents conspic-
uous irregularities in the way that it takes up the dyes,
being usually marked by deeply stained areas that alter-
320 BACTERIOLOGY.
nate with points at which it either does not stain at all
or only slightly.
The animals that are susceptible to infection by this
organism are horses, asses, field mice, guinea-pigs, and
cats. Baumgarten records cases of infection in lions
and tigers that have been fed, in menageries, with flesh
from horses affected with the disease. Rabbits are but
slightly susceptible; dogs and sheep still less so. Man
is susceptible, and infection not rarely terminates fatally.
White mice, common gray house-mice, rats, cattle, and
hogs are insusceptible.
InocuLaTion EXPERIMENTS.—The most favorable
animal upon which to study the pathogenic properties
of this organism in the laboratory is the common field
mouse. When inoculated subcutaneously with a small
portion of a pure culture of the glanders bacillus death
ensues in about seventy-two hours. The most conspicu-
ous tissue-changes will be enlargement of the spleen,
which is at the same time almost constantly studded
with minute gray nodules, the typical glanders nodule.
They are rarely present in the lungs, but may frequently
be seen in the liver. From these nodules the glanders
bacillus may be obtained in pure culture. With the
exception of the characteristic nodule, the disease as
seen in this animal presents none of the characteristics
that it displays in the horse and ass. The clinical and
pathological manifestations resulting from inoculation
of guinea-pigs are much more characteristic. The ani-
mal lives usually from six to eight weeks after inocu-
lation, and in this time becomes affected with a group
of most interesting and peculiar pathological processes.
The specific inflammatory condition of the mucous
membrane of the nostrils is almost always present. The
°
STAINING IN TISSUES. 321
joints become swollen and infiltrated to such an extent
as often to interfere with the use of the legs. In male
animals the testicles become enormously distended with
pus, and on closer examination a true orchitis and epi-
didymitis are seen to be present. The internal organs,
particularly the lungs, kidneys, spleen, and liver, are
usually the seat of the nodular formations characteristic
of the disease. From all of these disease-foci the
bacillus causing them can be isolated in pure culture.
Srarnixe in TissvEs.—Though always present in
the diseased tissues, considerable trouble is usually ex-
perienced in demonstrating the bacteria by staining-
methods. The difficulty lies in the fact that the bacilli
are very easily decolorized, and in tissues stained by the
ordinary processes are robbed of their color even by
the alcohol with which the tissue is rinsed out and de-
hydrated. If we will remember not to employ con-
centrated stains, and not to expose the sections to the
stains for too long a time, but little treatment with
decolorizing-agents is necessary, and very satisfactory
preparations will be obtained. A number of good
methods have been suggested for staining the glanders
bacilli in tissues, and if what has been said will be
borne in mind, no difficulty should be experienced.
Two satisfactory methods that we have used for this
purpose, though perhaps no better than some of the
others, are as follows:
(a) Transfer the sections from alcohol to distilled
water. This lessens the violence with which the stain
subsequently takes hold of the tissues, by diminish-
ing the activity of the diffusion that would occur if
they were placed from alcohol into watery solutions of
the dyes. Transfer from distilled water to the slide,
322 BACTERIOLOGY.
absorb all water with blotting-paper, and stain with two
or three drops of
Carbol-fuchsin . é . ce.
Distilled water 100 ¢.c.
for thirty minutes; absorb all superfluous stain with
blotting-paper, and wash the section three times with
0.3 per cent. acetic acid, not allowing the acid to act
for more than ten seconds each time. Remove all acid
from the section by carefully washing in distilled water;
absorb all water by gentle pressure with blotting-paper,
and finally, at very moderate heat, or with a small bel-
lows (Kiihne), dry the section completely on the slide.
When dried clear up in xylol, and mount in xylol
balsam.
(6) Transfer sections from alcohol to distilled water;
from water to the dilute fuchsin solution, and gently
warm (about 50° C.) for fifteen to twenty minutes.
Transfer sections from the staining-solution to the slide,
absorb all superfluous stain with blotting-paper, and
then treat them with 1 per cent. acetic acid from one-
half to three-quarters of a minute. Remove all trace
of acid with distilled water, absorb all water by gentle
pressure with blotting-paper, and then treat the sections
with absolute alcohol by allowing it to flow over them
drop by drop. For small sections three or four drops
are sufficient. Under no circumstances should the
alcohol be allowed to act for more than one-quarter of
a minute. Clear up in xylol and mount in xylol-balsam.
In method 6 the tissues are better preserved than in
a, where they are dried.
Very good preparations are also obtained hy the use
of Leeffler’s alkaline methylene-blue, if care be taken
MALLEIN. 393
not to stain for too long a time or to decolorize with
alcohol too energetically.
No method of contrast-stain for this organism in
tissue has been devised.
In properly stained tissues the bacilli will be found
most numerous in the centre of the nodules, becoming
fewer as we approach the periphery. They usually lie
between the cells, but at times may be seen almost fill-
ing some of the epithelial cells, of which the nodule
contains more or less. They are always present in these
nodules in the tissues; they are rarely present in the
blood, and, if so, in only small numbers.
Diaenosis oF THE Disease BY THE METHOD OF
Srrauss.—From what has been said the diagnosis of
glanders by routine bacteriological methods is certain
and relatively easy, but requires time. In clinical work
it is of great importance for the diagnosis to be estab-
lished as quickly as possible. With this in view Strauss
devised a method that has given entirely satisfactory
results. It consists in introducing into the peritoneal
cavity of a male guinea-pig a bit of the suspected tissue
or culture. If it be from a genuine case of glanders,
the testicles begin to swell in about thirty hours, and
as this proceeds the skin over them becomes red and
shining, desquamation occurs, evidences of pus-forma-
tion are seen, and, indeed, the abscess (purulent orchitis)
often breaks through the skin. The diagnostic sign is
the tumefaction of the testicles.
Ma.uein.—The filtered products of growth of the
glanders bacillus in fluid media represent what is known
as mallein—a group of compounds that bear to glanders
pretty much the same relation that tuberculin bears to
tuberculosis. It is used with considerable success as a
324 BACTERIOLOGY.
diagnostic aid in detecting the existence or absence of
deep-seated manifestations of the disease, the glanderous
“animal reacting in from four to ten hours to subcuta-
neous injections of mallein, while an animal not so
affected gives no such reactions.
It is prepared from old glycerin-bouillon cultures of
the glanders bacillus by steaming them for several hours
in the sterilizer, after which they are filtered through
unglazed porcelain.
CHAPTER XX.
Bacillus diphtherix—Its isolation and cultivation—Morphological and cul-
tural peculiarities—Pathogenic properties—Variations in virulence.
From the gray-white deposit on the fauces of a diph-
theritic patient prepare a series of cultures in the fol-
lowing way:
Have at hand five or six tubes of Leeffler’s blood-
serum mixture. (See chapter 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 membrane.
Without touching it against anything else rub it care-
fully over the surface of one of the serum tubes; with-
out 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 scrap-
ings from the membrane on the fauces. If the case is
true diphtheria, the tubes will be ready for examination
on the following day.
The reason that plates are not made in the regular
way in this examination is that the bacillus of diph-
theria 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
employed, however, insures a dilution in the number of
organisms present, and this, in addition to the fact that
bacillus diphtherie grows much more quickly on the
15
326 BACTERIOLOGY.
serum mixture than do other organisms, makes its iso-
lation 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 at different points by more or less irreg-
ular patches of a white or cream-colored growth which
is usually more dense at the centre than at its irregular
periphery.
Except now and then, when a few orange-colored col-
onies may be seen, these large irregular patches are the
most conspicuous objects on the surface of the serum.
Occasionally, almost nothing else appears.
The cover-slips made from the membrane at the time
the cultures were prepared will be found on microscopic
examination to present, in many cases, a great variety of
organisms, but conspicuous among them will be noticed
slightly curved bacilli of irregular size and outline.
In some cases they will be more or less clubbed at one
or both ends; sometimes they appear spindle in shape,
again as curved wedges; now and then they will be seen
irregularly segmented. They are rarely or never reg-
ular in outline. If the preparation has been stained
with Leeffler’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 irregularity
in outline is the morphological characteristic of the
bacillus diphtherice of Luceffler.
It must be remembered, however, that the diagnosis
of diphtheria should not under all circumstances be
made from the examination of cover-slip preparations
alone, for there are other organisms present in the mouth
cavity, particularly in the mouths of persons having
MORPHOLOGY. 327
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 only; and again, the genuine diphtheria
bacillus is sometimes found in the mouth cavities of
healthy persons in attendance upon diphtheria cases,
who were at the time insusceptible to the pathogenic
activities of the organism. In the vast majority of
instances, however, where the clinical condition of the
patient justifies a suspicion of diphtheria, a microscopic
examination alone of the deposit in the throat will serve
to confirm or contradict this opinion.
Bacillus diphtherie, discovered microscopically by
Klebs, and isolated in pure culture and proved to
stand in causal relation to diphtheria by Loeffler, can
readily be identified by its cultural peculiarities and
by its pathogenic activity when introduced into tissues
of susceptible animals. In guinea-pigs and kittens the
results of its growth are histologically 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 :
MorpHotocy.—As obtained directly from the diph-
theritic deposit in the throat of an individual sick of
the disease, it is sometimes comparatively regular in
shape, appearing as straight or slightly curved rods with
more or less pointed ends. More frequently, however,
spindle and club shapes occur, and not rarely many of
these rods take up the stain irregularly; in some of
them very deeply stained, round or oval points can be
detected.
When cultures are examined microscopically it is
328 BACTERIOLOGY.
especially characteristic to find irregular, bizarre forms,
such as rods with one or both ends swollen, and very
frequently rods broken at irregular intervals into short,
sharply marked segments, either round, oval, or with
straight sides. Some forms stain uniformly, others in
various irregular ways, the most common being the
appearance of deeply stained granules in a lightly
stained bacillus.
By a series of studies upon this organism when cul-
tivated under artificial conditions we have found that
its form depends very largely upon the nature of its
environment. That is to say, its morphology is always
more regular, and it is smaller on glycerin-agar-agar
than on other media used for its cultivation; while
upon Leefiler’s blood-serum the other extremes of de-
velopment appear: here one sees, instead of the very
short, spindle, lancet, club-shaped, always segmented
and regularly staining forms as seen upon glycerin-agar-
agar, long, irregularly staining threads that are some-
times clubbed and sometimes pointed at their extremi-
ties. They are usually marked by areas that stain more
intensely than does the rest of the rod, and at times they
may be a little swollen at the centre. These differences
are so conspicuous that microscopic preparations from
cultures from the same source, but cultivated in the one
case on glycerin-agar-agar and in the other upon blood-
serum, when placed side by side would hardly be recog-
nized as of the same organism, unless its peculiar be-
havior under these circumstances was already known.
During the past year or so various authors have called
attention to branching forms of this organism that are
occasionally encountered, especially when cultivated
upon albumin. We have never seen the branching
MORPHOLOGY. 329
diphtheria bacilli; and in approximately 6000 blood-
serum cultures from cases of diphtheria that have been
examined during the past two years by three competent
bacteriologists at the laboratory of the Board of Health
of Philadelphia, the branching forms of this organism
were not observed in a single instance. It is fair to
assume, therefore, that this peculiar morphological
variation of bacillus diphtherie is comparatively rare.
Fie. 65.
Fg
sf 4, 2a f - ae
a Lae, va
eae woVR
46, ed o e “ ae :
ae: G%&@ “oe ae
6 oe ’ = ss
a ; b Ac
Bacillus diphtheriz. a. Its morphology when cultivated on glycerin-agar-
agar. .b. Its morphology as seen in cultures on Leeffler’s blood-serum.
On plain nutrient agar-agar (that is, nutrient agar-
agar without glycerin); on solidified egg-albumin; on
a medium consisting of dried albumin, as found in com-
merce, dissolved in bouillon (about 10 grammes albumin
to 100 e.c. of bouillon containing 1 per cent. of grape-
sugar); in bouillon without glycerin, and in bouillon to
which a bit of hard-boiled egg has been added, the mor-
phology of the organism is about intermediate, in both
size and outline, between the forms seen upon glycerin-
agar-agar and upon Leeffler’s blood-serum. There
will appear about an equal number of short segmented
and longer irregularly staining forms, but in general
the longest are rarely as long as the long forms seen on
blood-serum, and throughout they are not so conspicu-
ous for the irregularity of their staining.
330 BACTERIOLOGY.
In cultures made upon two sets of nutrient agar-agar
tubes, differing only in the fact that one set contains
glycerin to the extent of 6 per cent., while the others
contain none, a noticeable difference in morphology can
usually be made out; while the forms on the glycerin-
agar-agar cultures are throughout small, and pretty
regular in size, shape, and staining, those on the plain
agar-agar are larger, stain more irregularly, vary more in
shape, and when stained by Leeffler’s blue are not so
uniformly marked by pale transverse lines that give to
them the appearance of being made up of numerous
short segments.
Though the outline of this organism is more regular
under some circumstances than others, it is nevertheless
always conspicuous for its manifold variations in shape.
Growta on Serum Mrixturs.—The medium upon
which it grows most rapidly and luxuriantly, and which
is best adapted for determining its presence in diphther-
itic exudation, is, as has been stated, the blood-serum
mixture of Loeffler. (See chapter on Media.) On the
blood-serum mixture the colonies of the bacillus diph-
therize grow so much more rapidly than the other or-
ganisms usually present in 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 or yellowish, with a centre more
opaque than the slightly irregular periphery. The sur-
face of the colony is at first moist, but after a day or
two becomes rather dry in appearance.
A blood-serum tube studded over with coalescent or
seattered colonies of this organism is so characteristic
GLYCERIN AGAR-AGAR. 331
that one familiar with the appearance can anticipate
with tolerable certainty the results of microscopic exam-
ination.
GLYCERIN AGAR-AGAR.—Upon nutrient glycerin
agar-agar the colonies likewise present an appearance
that may readily be recognized. They are in every
way more delicate in their structure than when on the
serum mixture. They appear at first, when on the sur-
face, as very flat, almost transparent, dry, non-glisten-
ing, round points which are not elevated above the
surface upon which they are growing. When slightly
magnified they are seen to be granular, and to present
an irregular central marking which is denser and darker
Fic, 66.
r
a
os & . «
a é
Colonies ot bacillus diphtheriz on glycerin-agar-agar. a. Colonies located
in the depths of the medium. b. Colonies just breaking out upon the surface
of the medium. c. Fully developed surface-colony.
by transmitted light than the thin, delicate zone which
surrounds it. As the colony increases in size the thin
granular peripheral zone becomes broader, is usually
marked by ridges or cracks, and its periphery is notched
or scalloped. (Fig. 66, ¢.) These colonies are always
quite dry in appearance. When deep down in the agar-
agar they are coarsely granular. (Fig. 66,a.) They
rarely exceed 3 mm. in diameter.
332 BACTERIOLOGY.
GELATIN.—On gelatin the colonies develop much
more slowly than on the other media that can be re-
tained at a higher temperature. They rarely present
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 col-
onies rarely become very large; usually they do not
reach a diameter of over 1.5 mm.
Bovuiitton.—In bouillon it usually grows in fine
clumps, which fall to the bottom of the tube, or become
deposited on its sides without causing a diffuse clouding
of the bouillon. There are sometimes exceptions to
this naked-eye appearance: the bouillon may be dif-
fusely clouded; but if one inspect it very closely, par-
ticularly if one examine it microscopically as a hanging
drop, the arrangement in clumps will always be de-
tected, but they are so small as not to be discernible
by the unaided eye.
In bouillon which is kept at a temperature of 385°—
37° C. for a long time a soft, whitish pellicle often
forms over a part of the surface.
Changes in reactions of the bouillon. The reaction of
the bouillon frequently becomes at first acid, and, sub-
sequently, again alkaline, changes which can be observed
in cultivations in bouillon to which a little rosolic acid
STAINING. 333
has been added. This play of reactions has been attrib-
uted to the primary fermentation of muscle sugar that
is often present in the bouillon.
Porato.—On potato at a temperature of 35°-37° C.
its growth after several days is entirely invisible, there
being only a thin, dry glaze appearing at the point at
which the potato was inoculated. Microscopic examin-
ation of scrapings from the potato, after twenty-four
hours at 35°-37° C., reveals a decided increase in the
number of individual organisms planted.
STaB- AND SLANT-CULTURES.—In stab- and slant-
cultures on both gelatin and glycerin-agar-agar the
surface-growth is seen to predominate over that along
the track of the needle in the depths of the media.
Isolated colonies on the surface of either of the media
in this method of cultivation present the same charac-
teristics that have been given for the colonies on plates.
The growth in simple stab-cultures does not extend
laterally very far beyond the point at which the needle
entered the medium.
It is a non-motile organism.
It does not form spores.
It is killed in ten minutes by a temperature of 58° C.
It grows at temperatures ranging from 22° C. to 37°
C., but most luxuriantly at the latter temperature.
Its growth in the presence of oxygen is more active
than when this gas is excluded.
Srarnine.—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 Leeffler’s alkaline methylene-blue solution; this
15*
BRE BACTERIOLOGY.
brings out the dark points in the protoplasmic body of
the bacilli and thus aids in their identification.
For the purpose of demonstrating the Loeffler bacil-
lus in sections of diphtheritie membrane, both the Gram
method and the fibrin method of Weigert give excellent
results.
ParHocenic Properties.—When inoculated sub-
cutaneously into the bodies of susceptible animals the
result is not the production of septiceemia, as is seen to
follow the introduction into animals of certain other
organisms with which we shall have to deal, but the
bacillus of diphtheria remains localized at the point
of inoculation, rarely disseminating further than the
nearest lymphatic glands. It develops at the point in the
tissues at which it is deposited, and during its develop-
ment gives rise to changes in the tissues which result
entirely from the absorption of poisonous albumins pro-
duced by the bacilli in the course of their development.
In a certain number of cases! diphtheria bacilli have
been found in the blood and internal organs of individ-
uals dead of the disease; but all that has been learned
from careful study of the secondary manifestations of
diphtheria tends to the opinion that they are in no way
dependent upon the immediate presence of bacteria, and
that the occasional appearance of diphtheria bacilli in
the internal organs is in all probability accidental, and
usually unimportant.
By special methods of inoculation’ (the injection of
1 Frosch: Die Verbreitung des Diphtherie-bacillus im Kirper des Menschen
Zeit. fiir Hygiene und Infectionskrankheiten, 1893, Bd. xiii. pp. 49-52. Booker:
Archives of Pediatrics, Aug. 18938. Wright and Stokes : Boston Med, and Surg.
Journ., March and April, 1895.
2 Abbott and Ghriskey: A Contribution to the Pathology of Experimental
Diphthetria. The Johns Hopkins Hospital Bulletin, No. 30, April, 1893.
PATHOGENIC PROPERTIES. 335
fluid cultures into the testicles of guinea-pigs) diph-
theria bacilli can be caused to appear in the omentum,
but this is purely an artificial manifestation of the dis-
ease and one that is probably never encountered in the
natural course of events. More rarely similar results
follow upon subcutaneous inoculation.
If a very minute portion of either a solid or fluid
pure culture of this organism be introduced into the
subcutaneous tissues of a guinea-pig or kitten, death of
the animal ensues in from twenty-four hours to five
days. The usual changes are an extensive local edema,
with more or less hyperemia and ecchymosis at the
site of inoculation; swollen and reddened lymphatic
glands; increased serous fluid in the peritoneum, pleura,
and pericardium; enlarged and hemorrhagic adrenal
bodies; occasionally slightly swollen spleen; and some-
times fatty degeneration in the liver, kidney, and myo-
cardium. In guinea-pigs, especially, the liver often
shows numerous macroscopic dots and lines on the sur-
face and penetrating the substance of the organ. They
vary in size from « pin-point to a pin-head, and may be
even larger. They are white and do not project above
the surface of the capsule.
The bacilli are always to be found at the seat of inoc-
ulation, most abundant in the grayish-white, fibrino-
purulent exudate. They become fewer at a distance
from this, so that the more remote parts of the edema-
tous tissues do not contain them. They are found not
only free, but contained in large number in leucocytes,
some of which have fragmented nuclei, or have lost
their nuclei. The bacilli within leucocytes, as well as
some outside, frequently stain very faintly aud irregu-
larly, and may appear disintegrated and dead.
336 BACTERIOLOGY.
Culture-tubes inoculated from the blood, spleen, liver,
kidneys, adrenal bodies, distant lymphatic glands, and
serous transudates, generally yield negative results; and
negative results are also obtained when these organs are
examined microscopically for the bacilli.
Microscopic examination of the tissues at the seat of
inoculation, as well as of the liver, spleen, kidneys,
lymphatic glands and elsewhere, reveals the presence
of localized foci of cell-death, characterized by a pecu-
liar fragmentation of the nuclei of the cells of these
parts.
This destruction of nuclei results in the occurrence
of groups of irregularly shaped, deeply staining bodies,
having at times the appearance of particles of dust,
while again they may be much larger. Some of
them are tolerably regular in outline, while others are
irregularly crescentic, dumb-bell, flask-shape, whet-
stone shape, or bladder-like in form. Occasionally
nuclei having the appearance of being pinched or drawn
out can be seen. At some points the fragments are
grouped into isolated masses, indicating the location of
the nucleus from the destruction of which they orig-
inated. These particles always stain much more in-
tensely than do the normal nuclei of the part.?
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 important
additional proof that diphtheria is caused by it.
1 See ‘The Histological Changes in Experimental Diphtheria,” also ‘‘ The
Histological Lesions produced by the Toxalbumin of Diphtheria,” by Welch
and Flexner. The Johns Hopkins Hospital Bulletin, August, 1891, and March,
1892.
PATHOGENIC PROPERTIES. 337
An affection may be produced by the inoculation of
certain animals that is in all respects identical with the
disease diphtheria as it exists in man. If one open the
trachea of a kitten and rub upon the mucous mem-
brane a small portion of a pure culture of this organ-
ism, the death of the animal usually ensues in from two
to four days. At autopsy the wound will be found
covered with a grayish, adherent, necrotic, distinctly
diphtheritic layer. Around the wound the subcuta-
neous tissues will be edematous. The lymphatic glands
at the angle of the jaws will be swollen and reddened.
The mucous membrane of the trachea at the point upon
which the bacilli were deposited will be covered with
a tolerably firm, 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 bacillus
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 interpretation for this process, viz., that it is due to
the production of a soluble poison by the bacteria grow-
ing at the seat of inoculation, which, gaining access to
the circulation, produces the changes that we observe in
the tissues of the internal viscera.
This poison has been isolated from cultures of the
bacillus diphtheria, and is found to belong, not to the
crystallizable ptomaines, but to the toxic albumins—
bodies which, in their chemical composition, are analo-
338 BACTERIOLOGY.
gous to the poison of certain venomous serpents. By
the introduction of this ovalbumin, as it is called, into
the tissues of guinea-pigs and rabbits the same patho-
logical alterations may be produced that we have seen
to follow the result of inoculation with the bacilli them-
selves, except, perhaps, the production of false mem-
branes.
Under the influence of certain circumstances with
which we are not acquainted the bacillus diphtherie be-
comes diminished in virulence or may lose it entirely,
so that it is no longer capable of producing death of
susceptible animals, but may cause only a transient local
reaction from which the animal entirely recovers. Some-
times this reaction is so slight as to be overlooked, and
again careful search may fail to reveal evidence of any
reaction at all. This exhibition of the extremes of its
pathogenic properties, viz., death of the animal, on the
one hand, and only very slight local effects on the other,
was at one time thought to indicate the existence of two
separate and distinct organisms that were alike in cul-
tural and morphological peculiarities, but which differed
in their disease-producing power. Further studies on
this point have, however, shown that the genuine bacil-
lus diphtherice may possess almost all grades in the
degree of its virulence, and that absence of or diminu-
tion in virulence can hardly serve tu distinguish as sep-
arate species these varieties that are otherwise alike;
moreover, the histological conditions found at the site
of inoculation in animals that have not succumbed, but
in which only the local reaction has appeared, are in
most cases characterized by the same changes that are
seen at autopsy in animals in which the inoculation has
proved fatal.
PATHOGENIC PROPERTIES. 339
In the course of their observations upon a large num-
ber of cases Roux and Yersin found that it was not
difficult to detect, in the diphtheritic deposits of one and
the same individual, bacilli of identical cultural and
morphological peculiarities, but of very different degrees
of virulence, and that with the progress of the disease
toward recovery the less virulent varieties often became
quite frequent. '
There is, moreover, a mild form of diphtheria affect-
ing only the mucous membrane of the nares, known as
membranous rhinitis, from which it is very common to
obtain cultures in all respects identical with those from
typical diphtheria, save for their inability to kill suscep-
tible animals. On inoculation these cultures produce
only local reactions, but they are characterized histolog-
ically by the same tissue-changes that follow inoculation
with the fully virulent organism.
Clinically, membranous rhinitis is never such an
alarming disease as is laryngeal or pharyngeal diph-
theria, and, as stated, the organisms causing it are often
of a low degree of virulence, though they are, never-
theless, genuine diphtheria bacilli.
For those organisms that are in all respects identical
with the virulent bacillus diphtherie, save for their ina-
bility to kill guinea-pigs, the designation ‘‘pseudo-diph-
theritic bacillus’’ is usually employed; but from such
observations as those just cited we are inclined to the
opinion that pseudo-diphtheritic, as applied to an organ-
ism in all respects identical with the genuine bacillus,
except that it is not fatal to susceptible animals, is a
1 It must not be assumed from this that the bacilli lose their virulence
entirely, or that they ali become attenuated with the establishment of con-
valescence.
340 BACTERIOLOGY.
misnomer, and that it would be more nearly correct to
designate this organism as the attenuated or non-viru-
lent diphtheritic bacillus, reserving the term ‘‘pseudo-
diphtheritic’’ for that organism or group of organisms
(for there are probably several) that is enough like the
diphtheria bacillus to attract attention, but is distin-
guishable from it by certain morphological and cultural
peculiarities aside from the question of virulence.
It is a well-known fact that many pathogenic organ-
isms—conspicuous among these being the micrococcus
lanceolatus, the staphylococcus pyogenes aureus, and the
group of so-called ‘‘ hemorrhagic septicemia’’ organ-
isms—undergo marked variations in the degree of their
pathogenic properties, and yet these organisms, when
found either devoid of this peculiarity, or possessing it
to a diminished degree, are not designated as ‘‘pseudo”’
forms, but simply as the organisms themselves, the viru-
lence of which, from various causes, has been modified.
Nore.—Prepare cover-slip preparations from the
mouth-cavities 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 organisms suggest those of the bacillus of
diphtheria? Wherein is the difference ?
In cultures and cover-slips made from both diph-
theria and from innocent sore-throats are there any
organisms which are almost constantly present? Which
are they, and what are their characteristics ?
Which are the predominating organisms in the an-
ginas of scarlet fever?
Do these organisms simulate, in their cultural and
PATHOGENIC PROPERTIES. 341
morphological peculiarities, any of the different species
with which you have been working?
Do the diphtheria bacilli disappear from the throat
with the disappearance of the membrane? How long
do they persist? When obtained from the throat of
convalescents are they still pathogenic for guinea-pigs ?
CHAPTER XXI.
Typhoid fever—Study of the organism concerned in its production.
Bacterium coli commune—Its resemblance to the bacillus of typhoid fever—
Its morphological, cultural, and pathogenic properties—Its differentiation
from bacillus typhi abd
lic
THE organism, discovered by Eberth and by Gaffky,
generally recognized as the etiological factor in the pro-
duction of typhoid fever, may be described as follows:
It is a bacillus about three times as long as it is
broad, with rounded ends. It may appear at one time
as very short ovals, at another time as long threads,
and both forms may occur together. Its breadth remains
Fie, 67.
f
Bacillus typhi abdominalis from Bacillus typhi abdominalis show-
culture twenty-four hours old, on -ing flagella stained by Leeffler’s
agar-agar. method.
tolerably constant. Its morphology presents little that
will aid in its identification (see Fig. 67). It stains a
trifle 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 Leeffler (see
STAB-CULTURES. 343
this method in chapter on Staining) is seen to possess
very delicate locomotive organs in the form of fine,
hair-like flagella, which are given off in large numbers
from all parts of its surface (see Fig. 68). These
flagella are not seen in unstained preparations, nor are
they rendered visible by the ordinary methods of
staining.
In patients suffering from this disease it has been
found during life in the blood, urine, and feces, and at
autopsies in the tissues of the spleen, liver, kidneys,
intestinal lymphatic glands, and intestines.
GELATIN PLATES.—Its growth, when seen in the
depths of the medium, presents nothing characteristic,
appearing simply as round or oval, finely granular
points. On the surface it develops as very superficial,
blue-white colonies, with irregular borders. They are
a little denser at the centre than at the periphery.
Fie. 69,
Colony of bacillus typhi abdominalis on gelatin.
When magnified, the colonies present wrinkles or folds,
which give to them, in miniature, the appearance seen
in the relief maps made to represent mountainous dis-
tricts (Fig. 69). These colonies have sometimes the
appearance of flattened pellicles of glass-wool, and
usually present more or less of a pearl-like lustre.
On AGAR-AGAR the colonies present nothing typical.
Srap-cuLTURES.—In stab-cultures the growth is
344 BACTERIOLOGY.
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 a needle when it is scraped across the track
of growth. While this is true in most cases, yet it
cannot be considered as constant, for at times this
organism is seen to develop more or less visibly on
potato.
Porato GELATIN.—The growth is similar to that
upon ordinary nutrient gelatin.
Mixx.—It does not cause coagulation when grown
in sterilized milk.
Tt does not liquefy gelatin.
It grows both with and without oxygen.
In bouillon it causes a uniform clouding of the me-
dium and brings about a slightly acid reaction.
It does not grow rapidly.
Inpot Formation.—It is customary to regard this
organism as devoid of the power of forming indol; in
fact, this has hitherto been considered as one of its im-
portant differential peculiarities. By the usual methods
of cultivation and testing the indol reaction is not ob-
served in cultures of the typhoid bacillus. It has
recently been shown, however, by Dr. Peckham, that
by repeated transplantation, at short intervals, into
either Dunham’s peptone solution, or, preferably, a
freshly prepared alkali-tryptone solution, made from
tryptonized beef muscle, that the indol-producing func-
INDOL FORMATION. 345
tion may be induced in the genuine typhoid bacillus
obtained directly from the spleens of typhoid cadavers.
It does not produce gaseous fermentation. On lactose-
litmus-agar-agar it grows as pale-blue colonies, causing
no reddening of the surrounding medium; though if
glucose be substituted for lactose, both the colonies and
the surrounding medium become red. In the fermen-
tation-tube, in glucose or lactose bouillon, no evolution
of gas as a result of fermentation occurs.
It does not form spores. The irregularities of stain-
ing so commonly seen in this organism have in some
instances led to the belief that the pale, unstained por-
tions of the bacilli indicate the presence of spores.
More reliable tests, however, have demonstrated the
error of this opinion. (What is the most trustworthy
test of spore-formation ?)
It grows at any temperature between 20° 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.
-
iy:
Diagrammatic representation of retraction of protoplasm, with production of
pale points, in bacillus typhi abdominalis
Owing to a tendency to retraction of its protoplasm
from the cell envelope and the consequent produc-
1 See A. W. Peckham: The Influence of Environment upon the Biological
Functions of the Colon Group of Bacilli. Journal of Experimental Medicine,
vol, ii. 1897.
346 BACTERIOLOGY.
tion of vacuoles in the bacilli, the staining of this
organism is usually more or less irregular, At some
points in a single cell marked differences in the inten-
sity of the staining will be seen, and here and there
areas quite free from color can commonly be detected.
These colorless portions are often so cleanly cut that
they look as if they had been punched out with a sharp
instrument. (Diagrammatically represented in Fig. 70.)
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 often rob the bacilli of their
staining, and render them invisible, or nearly so. If,
however, sections be stained in the carbol-fuchsin solu-
tion, either at the ordinary temperature of the room or
at a higher temperature (40° to 45° C.), then washed
out in absolute alcohol, and cleared up in xylol and
mounted in balsam, the bacilli (particularly if the tissue
be the liver or spleen) can readily be detected, massed
together in their characteristic clumps. If used in the
same way, the alkaline methylene-blue solution gives
also very satisfactory results.
In searching for the typhoid bacilli in tissues their
mode of growth under these circumstances must always
be borne in mind, otherwise much labor will be ex-
pended in vain. In tissues the typhoid bacilli do not
lie scattered about in the same way as do the organisms
in tissues from cases of septicemia; they are not regu-
larly distributed along the course of the capillaries, but
are localized in small clumps through the tissues, and
it is for these clumps, which are easily detected under
a low-power objective, that one should search. When
the section is prepared for examination, if it be gone
INOCULATION INTO LOWER ANIMALS. 347
over with a low-power objective, one will notice at
irregular intervals little masses that look in every re-
spect like particles of staining-matter which have been
precipitated upon the section at that point. When
these little masses are examined with a higher power
objective they will be found to consist of small ovals or
short rods so closely packed together that the individuals
composing the clump can often be seen only at the very
periphery of the mass. This is the characteristic ap-
pearance of the typhoid organism in tissues. The little
masses are usually in the neighborhood of a capillary.
Resut or InocuLatTion 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
frequently followed these attempts, but in most cases
they could easily be traced to the toxic,' rather than to
the truly infective’ action of the materials introduced
into the animals.
The most successful efforts for the production of the
typical typhoid lesions in lower animals are those re-
ported 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 intestine con-
ditions that were histologically and to the naked eye
analogous to those found in the human subject.
Of a large number of experiments made by the writer
with the same object in view, only one positive result
1 Toxic—Poisonous results not necessarily accompanied by the growth of
organisms throughout the tissues.
2 Infective or septic—Pvisoning of the tissues as a result of the growth of
bacteria within them,
348 BACTERIOLOGY.
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 method, but were also demonstrated microscopi-
cally in their characteristic clumps in section of the organ.
In connection with the inoculation of animals with
bacillus typhi abdominalis observations of a most im-
portant nature have been made by Sanarelli' upon the
artificial induction of susceptibility to its pathogenic
action. He found that rabbits, guinea-pigs, and mice
could be rendered susceptible to infection by this organ-
ism by preliminary injections into them of the products
of growth of certain saprophytes—proteus vulgaris,
bacillus prodigiosus, and bacterium coli commune—and
that by whatever means the animal was subsequently
inoculated with fresh cultures of the typhoid bacillus,
either into the circulation or into the peritoneal cavity,
death resulted in from twelve to forty-eight hours, with
the most conspicuous pathological alterations in the
digestive tract, and particularly in the small intestine.
In these cases the infection is general, and the organisms
may be recovered from the blood and internal organs.
It is the opinion of Sanarelli that the toxic conditions
produced by the preliminary injections of the products
of growth of the saprophytic organisms may be consid-
ered as analogous to a similar condition that may occur
in man from the absorption of abnormal products of
fermentation from the intestinal canal—an auto-intoxi-
1 Sanarelli: Annales de l'Institut Pasteur, 1892, tome vi.
INOCULATION INTO LOWER ANIMALS. 349
cation that so reduces the resistance of the individual as
to render him susceptible to infection by the bacillus of
typhoid fever, should it gain access to his alimentary
tract.
More recently it was reported by Alessi’ that rats,
guinea-pigs, and rabbits, when compelled to breathe the
gaseous products of decomposition from the contents of
a cesspool, or from other decomposing matters, gradu-
ally became susceptible to infection by the typhoid
bacillus; but unfortunately for the integrity of this
observation the description given by Alessi of the two
cultures of so-called typhoid bacilli used by him’ for
inoculation, was in one case certainly not that of the
typhoid organism, and in the other the culture used had
been kept under artificial conditions so long as hardly
to be reliable for tests of this character.
The importance of these observations in their bearing
upon the etiology of typhoid fever, if they are demon-
strated by subsequent experiment to be trustworthy, is
too obvious to necessitate emphasis, and it is greatly to
be desired that they may not be permitted to pass un-
noticed, but that others interested may find occasion to
institute experiments in the same direction with the
hope that some light may be shed upon the mooted
question concerning the influence of gaseous products
of decomposition upon the health of individuals, and
particularly upon the part played by them in diminish-
ing natural resistance to infection.”
1 Alessi: Centralblatt fiir Bakteriologie u. Parasitenkunde, 1894, Bd. xv.,
No. 7, p. 228.
2 See paper by the author: ‘‘ The Effects of the Gaseous Products of Decom -
position upon the Health, and Resistance to Infection, of Certain Animals
that are forced to Respire Them.” Transactions of the Association of Amer-
ican Physicians, 1895, vol. x. pp. 16-44.
16
350 BACTERIOLOGY.
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 unsat-
isfactory of the infectious diseases.
There are a number of other organisms which botan-
ically appear to be nearly related to the typhoid bacillus,
and which, with our present methods for studying them,
so closely simulate it, that the difficulty of identifying
this organism is sometimes 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
was formerly described as characteristic, may,with the
same organism, at one time appear as the typical invis-
ible development, at another time it may grow in a way
easily to be seen with the naked eye; and the change of
reaction which it is said to produce in bouillon is some-
times much more intense than at others. The indol-
producing function, hitherto regarded as absent from
this organism, is now known to be occasionally demon-
strable by ordinary methods, and frequently demonstra-
ble by special methods of cultivation. (Peckham, J. ¢.)
The only properties possessed by it that may be said
to be constant are its motility, its inability to cause gas-
eous fermentation of glucose, lactose, or saccharose, its
incapacity for coagulating milk, and its growth on
gelatin plates; but there are other organisms which
approach these same characteristics to a degree that
renders their differentiation from the typhoid organisms
INOCULATION INTO LOWER ANIMALS. 351
often a matter that requires the careful application of
all the different tests.
Probably the most trustworthy, certainly the most
recently described, reaction of the typhoid bacillus is
that seen when it is brought in contact with the blood-
serum from human beings sick of typhoid fever, or from
animals that have survived inoculation with cultures of
this organism. This reaction consists of a peculiar
alteration in the relation of the organisms to one
another in the fluid. As ordinarily seen in a hanging
drop of bouillon the typhoid bacillus appears as single,
actively motile cells; when to such a drop a drop of
dilute serum from a case of typhoid fever is added the
motility of the organism gradually becomes lessened,
and finally ceases, and the bacteria congregate together
in larger and smaller clumps. The reaction may also
be made in another way, viz., by adding to about 4 or
5 c.c. of a twenty-four-hour-old bouillon culture of
typhoid bacilli in a narrow test-tube about eight drops
of serum from a case of typhoid fever, after which the
tube is placed in the incubator. After a few hours the
normally clouded culture is seen to have undergone a
change; instead of the diffuse cloud caused by the
growth, the fluid is found to be clear and to contain
within it flocculent masses of the bacteria that have
agglutinated together asa result of the specific action of
the serum used. When employed conversely—i.e., for
deciding if the serum used is from a case of typhoid
fever or not—the reaction constitutes what is known as
“¢ Widal’s serum diagnosis of typhoid fever.” For this
latter purpose it is often necessary to test several cul-
tures of genuine typhoid bacilli, from different sources
and of varying degrees of vitality, before a culture is
352 BACTERIOLOGY.
finally encountered that gives the reaction most conspic-
uously and quickly with genuine typhoid serum. This
culture is then to be set aside to be used for this test
with serums from doubtful cases of the disease. In the
hands of all those who have employed this method care-
fully for the diagnosis of typhoid fever the results are
reported to have been almost uniformly satisfactory.
The reaction is, so far as experience indicates, specific—
i.¢., a typical reaction does not occur between typhoid
serum and organisms other than the typhoid bacillus,
nor between the typhoid bacillus and serums other than
those of typhoid fever. There are, however, confusing
reactions—so-called pseudo-reactions—in which more or
less clumping of the bacilli anda diminution of motion,
without complete cessation, are observed. These have
been seen to occur with normal blood and with blood
from other febrile conditions. It is said by Johnston
and McTaggart' that they can be prevented if cultures
of just the proper degree of vitality areemployed. The
method is yet in the experimental stage, and there are
still numerous features that are not entirely clear. It
is, however, in the light of present experience, fair
presumptive evidence that the serum is from a case of
typhoid fever when unmistakable agglutination and
cessation of motion are seen in typhoid bacilli that are
mixed with the serum of a suspicious febrile condition.
For the hanging-drop test sufficient serum may be ob-
tained from a needle-prick in the finger, while for the
test-tube reaction a larger amount is needed; this may
be obtained from blood drawn from a superficial vein
by means of a hypodermic syringe, or from the
cleansed skin by a wet-cup, or, better still, from a
small cantharides blister.
1 Johnston and McTaggart: Montreal Medical Journal, March, 1897,
ISOLATING THE TYPHOID BACILLUS. 353
All the preceding points should be borne in mind in
the examination of drinking-water supposed to be con-
taminated by typhoid dejections, for the organisms
which most nearly approach the typhoid bacillus in
growth and morphology are just those organisms which
would appear in water contaminated from cesspools—
i.e, the organisms constantly found in the normal intes-
tinal tract. Even in the stools of typhoid-fever patients
the presence of these normal inhabitants of the intes-
tinal tract renders the isolation of the typhoid organisms
somewhat troublesome.
BACILLUS.
A number of special methods for the isolation of the
typhoid bacillus from mixtures, such as water, feces,
etc., that contain it have been recommended, but none
of them has given general satisfaction, and many have
proved to be entirely untrustworthy. That which has
perhaps attracted the most attention is the recently de-
vised medium of Elsner. It is an acid mixture of
gelatin, potato juice, and iodide of potash. It contains
no peptone, and no sodium chloride is added. On this
medium it is claimed that the ordinary, rapidly grow-
ing, liquefying saprophytes do not develop, and that the
colon bacillus and typhoid bacillus find it favorable for
growth. These are differentiated from one another by
the macroscopic and microscopic character of their
colonies—i. e., the growth of the colon colonies differs
little or not at all from that seen on ordinary nutrient
gelatin, while that of the typhoid colonies is so slow
that they are hardly visible at the end of twenty-four
hours. After forty-eight hours they appear under the
354 BACTERIOLOGY.
low power of the microscope as small, pale, finely gran-
ular, almost transparent bodies that are easily distin-
guished from the coarser, brownish colonies of the colon
bacillus.
While the method is useful, it has its limitations, and
is not always reliable. At times colon colonies will
develop in a way that would readily cause one to mis-
take them for typhoid colonies, while again typhoid col-
onies will take on the characteristics of those due to the
growth of the colon bacillus. This is especially the
case in plates over forty-eight hours old that have been
kept at ordinary room temperature.
In our experience the most serviceable feature of this
method is the elimination of many of the common sap-
rophytes usually present in mixtures containing the
typhoid and colon bacilli. The majority of them do
not grow upon gelatin made by this method, which will
now be described.
The description given by Elsner’ for the mode of
preparation of the medium is so incomplete and unsat-
isfactory in most of the important details that practi-
cally all those who have used the method have been
obliged to develop their own technique from the general
suggestions made in his original communication. The
‘Elsner medium” that has given satisfaction in our
hands is prepared as follows: grate 1 kilogram of
peeled potatoes and allow to stand in the refrige-
rator over night. Then press out all the juice, using
an ordinary meat-press for the purpose. Filter this
fresh juice cold, to remove as much of the starch gran-
ules as possible; if this is not done the starch when
1 Elsner: Zeit. fur Hygiene und Infectionskrankheiten, 1896, Bd. 21, p. 25.
ISOLATING THE TYPHOID BACILLUS. 355
heated swells to such an extent as to render filtration
almost impracticable. Boil the filtrate and filter again.
Test the filtrate for acidity by titrating 10 c.c. with a
decinormal solution of sodium hydroxide, the indicator
used being 6 drops of the ordinary 4 per cent. solution
of phenolphtalein in 50 per cent. alcohol. The acidity
of the juice should be such as to require 3 ¢.c. of a deci-
normal sodium hydroxide solution to neutralize 10 c.c. of
the juice (Potter). If the acidity is found to be greater
than this, which is usually the case, dilute with water
until the proper degree is reached. If less than this,
the juice may be concentrated by evaporation. It is
desirable that this acidity should arise from the acids
normally present in the potato, and should not be arti-
ficially obtained by the addition of other acids. Now
add 10 per cent. of gelatin (no peptone and no sodium
chloride), dissolve by boiling and again test the acidity,
using 10 c.c. of the mixture and phenolphtalein as
before. Deduct 3 c.c. (the acidity of the potato juice,
that is to be maintained) from the number of c.c. of the
decinormal sodium hydroxide solution required to neu-
tralize the 10 c. c. of the gelatin mixture, and from the
resulting figure calculate the amount of normal solution
of sodium hydroxide needed for the entire volume, and
add it. Boil, clarify with an egg, and filter through
paper in the usual manner. To the filtrate add potas-
sium iodide in the proportion of 1 per cent. Decant
into tubes and sterilize.
The spleen of a patient dead of typhoid fever is the
safest source from which to obtain cultures of the
typhoid bacillus for study. But it must always be re-
membered that the same channels through which the
356 BACTERIOLOGY.
typhoid bacillus gains access to this viscus are likewise
open to other organisms present in the intestines, and
for this reason the bacterium coli commune, a normal
inhabitant of the colon, may also be found in this
locality.
Nots.—Obtain a pure culture of typhoid bacilli, and
from this make inoculations upon a series of potatoes
of different ages and from different sources. Do they
all grow alike?
Before sterilizing render another lot of potatoes
slightly acid with a few drops of very dilute acetic
acid; render others very slightly alkaline with dilute
caustic soda. Do any differences in the growth result ?
Make a series of twelve tubes of peptone solution to
which rosolic acid has been added. Inoculate them all
with as near the same amount of material as possible
(one loopful from a bouillon culture into each tube);
place them all in the incubator. Is the color-change,
as compared with that of 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
organisms 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 ?
BACTERIUM COLI COMMUNE. 357
Bacterium Coit Communk (colon bacillus; bacillus
Neapolitanus of Emmerich).—This organism was dis-
covered by Escherich, in 1885, in the intestinal dis-
charges of milk-fed infants. It has since been demon-
strated to be a normal inhabitant of the intestines of
man and of certain domestic animals (cattle, hogs, dogs).
For a time after its discovery it was considered of
but little importance and attracted attention only be-
cause of its resemblance, in certain respects, to the bacil-
lus of typhoid fever, with which it was occasionally
confounded. In this particular it still serves as a sub-
ject for study. Some have even gone so far as to regard
them but as varieties of one and the same species,
though in the present state of our knowledge this is
certainly an assumption for which, as yet, there are not
sufficient grounds. That they possess in common cer-
tain general points of resemblance and often approach
one another in some of their biological peculiarities is
true; but, as we shall learn, they each possess peculiari-
ties which, when taken together, render their differenti-
ation from one another a matter of but little difficulty.
With the wider application of bacteriological methods
to the study of pathological processes it was occasionally
observed that, under favorable circumstances, this or-
ganism was disseminated from its normal habitat and
appeared in remote organs, often associated with dis-
eased conditions. This was also, at first, considered as
of but trifling moment, and its presence in these locali-
ties was usually explained as accidental. Its repeated
appearance, however, in different parts of the body out-
side of the intestines, and the frequency of its association
with pathological conditions, ultimately attracted atten-
tion to it, and in consequence during the past few
16*
358 BACTERIOLOGY.
years a great deal has been written concerning the
possible pathogenic nature of this organism.
The fact that it is always with us in most intimate
association with certain of our life-processes, together
with the fact that it is known to appear in organs other
than that in which it is normally located, and that its
occurrence in diseased conditions is not rare, justifies the
opinion that it is one of the most important of the
micro-organisms with which we have to deal.
While not generally considered to be a pathogenic
organism, there is, nevertheless, sufficient evidence to
warrant the statement that, under favorable conditions,
with which we are not entirely familiar, this organism
may assume pathogenic properties and that its presence
in diseased conditions is not always to be considered as
accidental, though this is frequently the case.
The morphological and cultural peculiarities of the
bacterium coli commune are as follows :
Morphology. In shape it is a rod with rounded ends,
sometimes so short as to appear almost spherical, while
again it is seen as very much longer threads. Often
both forms will be associated in the same culture. It
may occur as single cells or as pairs, joined end-to-end.
There is nothing to be said of its morphology that
can aid in its identification, for in this respect it simu-
lates many other organisms. It is usually said to be
motile, and undoubtedly is motile in the majority of
cases, but its movements are so sluggish that a positive
opinion is often difficult.
By Leeffler’s method of staining, flagella can be
demonstrated, though not in such numbers as are seen
to occur on the typhoid fever bacillus.
It does not form spores.
BACTERIUM COLI COMMUNE. 359
It grows both with and without oxygen.
On gelatin. When on the surface its colonies appear
small, dry, irregular, flat, blue-white points that are
commonly somewhat denuded at the margin. They are
a trifle denser at the centre than at the periphery, and
are often marked at or near the middle by an oval or
round nucleus-like mass— the original colony from
which the layer on the surface developed. When
located in the depths of the gelatin, and examined with
a low-power lens, they are at first seen to be finely gran-
ular and of a very pale greenish-yellow color; later
they become denser, darker, and much more markedly
granular. In shape they are round, oval, and lozenge-
like. When the surface colonies are viewed under a
low power of the microscope they present essentially
the same appearance as that given for the bacillus of
typhoid fever, viz., they resemble flattened pellicles of
glass-wall, or patches of finely ground colorless glass.
Colonies of this organism on gelatin are frequently en-
countered that cannot be distinguished from those result-
ing from the growth of the bacillus of typhoid fever,
though, as a rule, their growth isa little more luxu-
riant.
In stab- and smear-cultures on gelatin the surface-
growth is flat, dry, and blue-white or pearl color. Lim-
ited growth occurs along the track of the needle in the
depths of the gelatin. As the culture becomes older,
the gelatin round about the surface-growth may grad-
ually lose its transparency and become cloudy, often
quite opaque. In still older cultures small root- or
branch-like projections from the surface-growth into the
gelatin are sometimes seen to occur.
Tt does not cause liquefaction of gelatin.
360 BACTERIOLOGY.
Its growth on nutrient agar-agar and on blood-serum
is luxuriant but not characteristic.
In bouillon it causes diffuse clouding with sedimen-
tation. In some bouillon cultures an attempt at pellicle
formation on the surface may be seen, but this is not
always the case. In old bouillon cultures the reaction
‘is seen to have become alkaline, and a decided fecal
odor may be detected.
It produces indol in bouillon and in peptone solution.
Its growth on potato is rapid and voluminous, ap-
pearing after twenty-four to thirty-six hours in the
incubator as a more or less lobulated layer of a drab,
dark-cream, or brownish-yellow color.
In neutral milk containing a little litmus tincture
the blue color is changed to red after from eighteen to
twenty-four hours in the incubator, and, in addition, the
majority of cultures cause a firm coagulation of the
casein in about thirty-six hours, though frequently this
takes longer. Very rarely the litmus may indicate the
production of acid and no coagulation occur.
In media containing glucose it grows rapidly and
causes active fermentation with liberation of carbonic
acid and hydrogen. If cultivated in solid media to
which glucose (2 per cent.) has been added, the gas-
formation is recognized by the appearance of numerous
bubbles along and about the points of growth. If cul-
tivated in fluid media, also containing glucose, in the
fermentation-tube, evidence of fermentation is given by
the collection of gas in the closed arm of the tube.
On lactose-litmus-agar-agar its colonies are pink and
the color of the surrounding medium is changed from
blue to red.
In Dunham’s peptone solution it produces indol in
BACTERIUM COLI COMMUNE. 361
from forty-eight to seventy-two hours. It stains with
the ordinary aniline dyes. It is decolorized when
treated by the method of Gram.
By comparing what has been said of the bacillus typhi
abdominalis and of the bacterium coli commune it will
be seen that while they simulate each other in certain
respects they still possess individual characteristics by
which they may readily be differentiated. The most
important of the differential points are :
1. Motility of the bacillus typhi abdominalis is much
more conspicuous, as a rule, than is that of the bacterium
coli commune.
2. On gelatin the colonies of the typhoid bacillus
develop more slowly than do those of the colon bacillus.
3. On potato the growth of the typhoid bacillus is
usually invisible (though not always), while that of the
colon bacillus is rapid, luxuriant, and always visible.
4, The typhoid bacillus does not cause coagulation of
milk with acid reaction. The colon bacillus does this
in from thirty-six to forty-eight hours in the incubator.
5. The typhoid bacillus never causes fermentation,
with liberation of gas, in media containing glucose, lac-
tose, or saccharose. The colon bacillus is conspicuous
for its power of causing fermentation in such solutions.
6. In nutrient agar-agar or gelatin containing lactose
and litmus tincture, and of a slightly alkaline reaction,
the color of the colonies of typhoid bacillus is pale blue,
and there is no reddening of the surrounding medium,
while the colonies of the colon bacillus are pink and
the medium round about them becomes red.
7. The typhoid bacillus does not, as a rule, possess the
property of producing indol in solutions of peptone; the
growth of the colon bacillus in these solutions is accom-
362 BACTERIOLOGY.
panied by the production of indol in from forty-eight
to seventy-two hours at 37° to 38° C.
8. When twenty-four hours old bouillon cultures of
both organisms are brought in contact with the blood-
serum from a case of genuine typhoid fever (after the
fifth day of the disease), the characteristic agglutination
(clumping) of the bacilli occurs in the typhoid culture
and not in that of the colon bacillus (Widal’s reaction).
Animal inoculations. As with the bacillus of typhoid
fever, the results of inoculation of animals with cultures
of this organism cannot be safely predicted. According
to the observations of Escherich, Emmerich, Weisser,
and others, the results that do appear are in most in-
stances to be attributed to the toxic rather than to the
infective properties of the culture used.
When introduced into the subcutaneous tissues of
mice it has no effect, while similar inoculations of guinea-
pigs are sometimes (not always) followed by abscess-
formation at the point of injury, or by alterations very
similar to those produced by intravascular inoculation,
viz., death in less than twenty-four hours, accompanied
by redness of the peritoneum and marked hyperemia
and ecchymoses of the small intestine; together with
swelling of Peyer’s patches. The cecum and colon
may remain unchanged or present enlarged follicles.
There may or may not be an accumulation of fluid in
the abdominal cavity, but peritonitis is rarely present.
The small intestine may contain bloody mucus.
Intravenous inoculation of rabbits may be followed
by similar changes, with often the occurrence of diar-
rheea before death, which may, in the acute cases, result
in from three to forty hours. In another group of
cases acute fatal intoxication does not result, and the
BACTERIUM COLI COMMUNE. 363
animal lives for weeks or months, dying ultimately of
what appears to be the effects of a slow or chronic form
of infection For a few hours after inoculation these
animals present no marked symptoms; exceptionally
somnolence and diarrhea have been observed at this
period, indicating acute intoxication from which the
animal has recovered. The affection is unattended by
fever. The most marked symptom is loss of weight.
This is usually progressive from the first or second day
after inoculation, with slight fluctuations until death.
At autopsy the animal is found to be emaciated.
The subcutaneous tissues and the muscles appear pale
and dry. The serous cavities, particularly the pericar-
dial, may contain some excess of serum. ‘The viscera
are anemic. The spleen is small, thin, and pale. Ex-
ceptionally ulcers and ecchymoses are observed in the
cecum, but generally there are no lesions of the intes-
tinal tract.
The most striking and constant lesions, those most
characteristic of the affection, are in the bile and in the
liver; the quantity of bile may not exceed the normal,
but in other cases the gall-bladder may be abnormally
distended with bile. The bile is nearly colorless or has
a pale yellowish or brownish tint, with little or none of
a greenish color. Its consistence is much less viscid
than normal, being often thin and watery. It usually
contains small, opaque, yellowish particles or clumps
which can be seen floating in it, even through the walls
of the gall-bladder. These clumps consist microscop-
ically of bile-stained, apparently necrotic, epithelial
cells; leucocytes in small numbers; amorphous masses of
bile pigment, and bacteria often in zodglea-like clumps.
Similar material is found in the larger bile-ducts.
364 BACTERIOLOGY.
The liver frequently contains opaque, whitish or yel-
lowish-white spots and streaks of irregular size and
shape, which give a peculiar mottling to the organ when
present in large number. These areas may be numer-
ous, or only one or two may be found. In size they
range from minute points to areas of from 2 to 3 em. in
extent.
By microscopic examination they are found to repre-
sent places where the liver cells have undergone necrosis
accompanied by emigration of leucocytes, and the cells
about them are in a condition of fatty degeneration.
In sections of the liver masses of the bacilli may be
discovered in and about the necrotic foci just described.
At these autopsies the colon bacillus is not found
generally distributed through the body, but is only to
be detected in the bile, liver, and occasionally in the
spleen.'
1 Consult paper by Blachstein on this subject. Johns Hopkins Hospital”
Bulletin, July, 1891.
CHAPTER XXIT.
The spirillum (comma bacillus) of Asiatic cholera—Its morphological and
cultural peculiarities—Pathogenie properties—The bacteriological diagnosis
of Asiatic cholera.
At the conference held in Berlin in 1884 for the
purpose of discussing the cholera question, it was an-
nounced by Koch’ that he had discovered in the intes-
tinal evacuations of individuals suffering from Asiatic
cholera a micro-organism that he believed to be the
cause of the malady. The importance of this state-
ment necessarily attracted widespread attention to the
subject, and as one of the results there existed, for a
short time following, some skepticism as to the accuracy
of Koch’s claim. These doubts arose as a result of a
series of contributions from other observers who en-
deayored to prove that the organism found by Koch in
cholera evacuations was one that is common to other
localities, and not a specific accompaniment of this dis-
ease. It was not very long, however, before it was
evident that the objections raised by the opponents of
Koch were based upon untrustworthy observations, and
that by reliable methods of investigation the organism
to which he had called attention could be easily differ-
entiated from either and all of those with which it was
claimed to be identical.
This organism, known as the spirillum of Asiatic
1 Verhandlungen der Conferenz zur Erérterung der Cholerafrage, 1884,
Berlin.
366 BACTERIOLOGY.
cholera, and as Koch’s ‘‘ comma bacillus,’’ because of
its morphology, is identified by the following peculi-
arities :
THE MORPHOLOGICAL AND BIOLOGICAL PECULIARITIES
OF THE SPIRILLUM OF ASIATIC CHOLERA.
Morphology. Tt isa slightly curved rod varying from
about 0.8 to 2.0 in length and from 0.3 to 0.4 » in
thickness—that is to say, it is usually from about one-
half to two-thirds the length of the tubercle bacillus,
but is thicker and plumper. Its curve is frequently
not more marked than that of a comma, and, indeed, it
is often almost straight; at times, though, the curve is
much more pronounced, and may even describe a semi-
circle. Occasionally the curve may be double, one
comma joining another, with their convexities pointing
in opposite directions, so that a figure similar to the
letter S is produced. In cultures long spiral or undu-
lating threads may often be seen. rom these appear-
ances this organism cannot be considered as a bacillus,
but rather as an intermediate type between the bacilli
and the spirilla. Koch thinks it not improbable that
the short comma forms represent segments of a true
spirillum, the normal form of the organism. (Fig. 71.)
It does not form spores, and we have no reliable evi-
dence that it possesses the property of entering, at any
time, a stage when its powers of resistance to detrimental
agencies are increased.
It is a flagellated organism, but has only a single
flagellum attached to one of its ends.
It is actively motile, especially in the comma stage,
though the long spiral forms also possess this property.
SPIRILLUM OF ASIATIC CHOLERA. 367
Grouping. As found in the slimy flakes in the intes-
tinal discharges from cholera patients, Koch likens its
mode of grouping to that seen in a school of small fish
when swimming up stream—i.e., they all point in nearly
Fie. 71.
fiv'\ , oe
y ‘ws mi (
i
Oa ‘i We \\ Sy
( g VY VA
y iV SiN
Magen
Tae Ne ;
Spirillum of Asiatic cholera. Impression cover-slip from a
colony thirty-four hours old.
the same direction and lie in irregularly parallel, linear
groups that are formed by one comma being located
behind the other without being attached to it,
Fig. 72.
8. :
aerlu™N
Involution-forms of the spirillum of Asiatic cholera, as seen in old cultures.
On cover-slip preparations made from cultures in the
ordinary way there is nothing characteristic about the
grouping, but in impression cover-slips made from
young cultures the short commas will nearly always be
368 BACTERIOLOGY.
seen in small groups of three or four, lying together in
such a way as to have their long axes nearly parallel to
one another. (See Fig. 71.)
Tn old cultures in which development has ceased it
undergoes degenerative changes, and the characteristic
comma and spiral shapes may entirely disappear, their
place being taken by irregular involution-forms that
present every variety of outline. (See Fig. 72.) In
this stage they take on the stain very feebly, and often
not at all.
Cultural peculiarities. On pe of nutrient gelatin
that have been prepared from a pure culture of this
organism and kept at a temperature of from 20° to 22°
C., development can often be observed after as short a
period as twelve hours, but frequently not before sixteen
to eighteen hours. This is especially true of the first
or ‘‘ original’’ plate, containing the largest number of
colonies. At this time the plate will present to the
naked eye an appearance that has been likened to a
ground-glass surface, or to a surface that has been stip-
pled with a very finely pointed needle, or one upon
which very fine dust has been sprinkled. This appear-
ance is due to the presence of minute colonies closely
packed together upon the surface of the gelatin. In
the depth of the gelatin can also be seen, closely packed,
small points, likewise representing growing colonies.
As growth progresses liquefaction occurs around the
superficial colonies, and in consequence this plate is
usually entirely liquid after from twenty-four to thirty
hours; the developmental phases through which the
colonies pass cannot, therefore, be studied upon it.
On plates 2 and 3,where the colonies are more widely
separated, they can be seen after twenty-four to thirty
SPIRILLUM OF ASIATIC CHOLERA. 369
hours as small, round, or oval, white or cream-white
points, and when located superficially there can be de-
tected around them a narrow transparent zone of lique-
faction. As growth continues this liquefaction extends
downward rather than laterally, and the colony ulti-
mately assumes the appearance of a dense, white mass
lying at the bottom of a sharply cut pit or funnel con-
taining transparent fluid. This liquefaction is never
very widespread nor rapid, and rarely extends for more
than one millimetre beyond the colony proper. On
plates containing few colonies there is but little or no
tendency for them to become confluent, and, as a rule,
they do not exceed 2 to 3 mm. as an average diameter.
Fie. 73.
Me
Developmental! stages of colonies of the spirillum otf Asiatic cholera at
20° to 22° C. on gelatin. X about 75 diameters.
a. After sixteen to eighteen hours. 6. After twenty-four to'twenty-six hours,
ec. After thirty-eight to forty hours. d, After forty-eight to fifty hours. c.
After sixty-four to seventy hours.
When examined under a low magnifying lens the
very young colonies (sixteen to eighteen hours) appear
as pale, translucent, granular globules of a very delicate
greenish or yellowish-green color, sharply outlined and
not perfectly round. (See a, Fig. 73.) As growth pro-
370 BACTERIOLOGY.
gresses this homogeneous granular appearance is re-
placed by an irregular lobulation, and ultimately the
sharply cut margin of the colony becomes dentated or
scalloped. (See 6 and ¢, Fig. 73.) After forty-eight
hours (and frequently sooner) liquefaction of the gelatin
has taken place to such an extent that the appearance
of the colony is entirely altered. Under the magnify-
ing glass the colony proper is now seen to be torn and
ragged about its edges, while here and there shreds of
the colony can be detected scattered through the liquid
into which it is sinking. These shreds evidently
represent portions of the colony that became detached
from its margin as it gradually sank into the liquefied
area.
At d, in Fig. 73, will be seen a representation of the
several appearances afforded by the colonies at this stage.
At the end of the second, or during the early part of the
third day, the sinking of the colonies into the liquefied
pits resulting from their growth is about complete, and
under a low lens they now appear as dense, granular
masses, surrounded by an area of liquefaction through
which can be seen granular prolongations of the colony,
usually extending irregularly between the periphery and
the central mass. (See e, Fig. 73.) If the periphery be
examined, it will be seen to be fringed with delicate,
cilia-like lines that radiate from it in much the same
way that cilia radiate from the ends of certain columnar
epithelial cells.
These are the more marked phases through which the
colonies of this organism pass in their development on
gelatin plates. With some cultures the various appear-
ances here given appear more quickly, while in cultures
from other sources they may be somewhat retarded.
SPIRILLUM OF ASIATIC CHOLERA. 371
On plates of nutrient agar-agar the appearance of
the colonies is not characteristic. They appear as round
or oval patches of growth that are moist and tolerably
transparent. The colonies on this medium at 37° C.
naturally grow to a larger size than do those upon gel-
atin at 22° C,
Stab-cultures of the spirillum of Asiatic cholera in gelatin,
at 18° to 20° C.
a, After twenty-four hours. b. After forty-eight hours. c. After seventy-
two hours. d. After ninety-six hours,
In stab-cultures in gelatin there appears at the top
of the needle track after thirty-six to forty-eight hours
at 22°C. a small, funnel-shaped depression. As the
growth progresses liquefaction will be seen to occur
372 BACTERIOLOGY.
about this point. In the centre of the depression can
be distinguished a small, dense, whitish clump, the col-
ony itself. As growth continues the depression increases
in extent and ultimately assumes an appearance that
consists in the apparent sinking of the liquefied portion
in such a way as to leave a perceptible air-space between
the top of the liquid and the surface of the solid gelatin.
The growth now appears to be capped by a small air-
bubble. The impression given by it at this stage is not
only that there has been a liquefaction, but also a coin-
cident evaporation of the fluid from the liquefied area
and a constriction of the superficial opening of the
funnel. (See a, 6, ¢, and d, Fig. 74.) Liquefaction is
not especially active along the deeper portions of the
track made by the needle, though in stab-cultures in
gelatin the liquefaction is much more extensive than
that usually seen around colonies on plates. It spreads
laterally at the upper portion, and after about a week a
large part of the gelatin in the tube may have become
fluid, and the growth will have lost its characteristic
appearance.
Stab- and smear-cultures on agar-agar present noth-
ing characteristic. They are usually only an exagger-
ation of the appearance afforded by the single colonies
on this medium.
Its growth in bouillon is luxuriant, causing a diffuse
clouding and the ultimate production of a delicate film
upon the surface.
In sterilized milk of a neutral or amphoteric reaction
at a temperature of 36°-38° C. it develops actively,
and gradually produces an acid reaction with coagula-
tion of the casein. It retains its vitality under these
conditions for about three weeks or more. The blue
SPIRILLUM OF ASIATIC CHOLERA. 373
color of milk to which neutral litmus tincture has been
added is changed to pink after thirty-six or forty-eight
hours at body temperature.
Its growth in peptone solution, either that of Dun-
ham (see Special Media) or the one preferred by Koch,
viz., 2 parts Witte’s peptone, 1 part sodium chloride,
and 100 parts distilled water, is accompanied by the
production of both indol and nitrites, so that after
eight to twelve hours in the incubator at 37° C. the
rose color characteristic of indol appears upon the
addition of sulphuric acid alone. (See Indol Reac-
tion. )
(What does the presence of nitrites in these cultures
signify ?)
In peptone solution to which rosolic acid has been
added the red color is very much intensified after four
or five days at 37° C.
Its growth on potato of a slightly acid reaction is
seen after three or four days at 37° C. as a dull,whitish,
non-glistening patch at and about the site of inocula-
tion. It is not elevated above the surface of the potato,
and can only be distinctly seen when held to the light
in a particular position. Growth on acid potato occurs,
however, only at or near the body temperature, owing
probably to the acid reaction, which is sufficient to pre-
vent development at a lower temperature, but does not
have this effect when the temperature is more favorable.
On solidified blood-serum the growth is usually said to
be accompanied by slow liquefaction. I have not suc-
ceeded in obtaining this result on Leeffler’s serum, nor
have I detected anything characteristic about its growth
on this medium.
The temperature most favorable for its growth is
17
374 BACTERIOLOGY.
between 35° and 38° C. It grows, but more slowly,
at 17°C. Under 16° C. no growth is visible.
It is not destroyed by freezing. When exposed to
65° C, its vitality is destroyed in five minutes.
It is strictly aérobic, its development ceasing if the
supply of oxygen be cut off.
It does not grow in an atmosphere of carbonic acid,
but is not killed by a temporary exposure to this gas.
It does not grow in acid media, but flourishes best in
media of neutral or slightly alkaline reaction. It is so
sensitive to the action of acids that at 22° C. its devel-
opment is arrested when an acid reaction equivalent to
0.066 to 0.08 per cent. hydrochloric or nitric acid is
present (Kitasato).
In cultures the development of this organism reaches
its maximum relatively quickly, then remains stationary
for a short period, after which degeneration begins.
The dying comma bacilli become altered in appearance
and assume the condition known as ‘‘involution-forms.’’
(See Fig. 72.) When in this state they take up color-
ing-reagents very faintly or not at all, and may lose
entirely their characteristic shape.
When present with other bacteria, under conditions
favorable to growth, the comma bacillus at first grows
much more rapidly than do the others; in twenty-four
hours it will often so outnumber the other organisms
present that microscopic examination would lead one
to take the material under consideration to be a pure
culture of this organism. This, however, does not last
longer than two or three days; they then begin to die,
and the other organisms gain the ascendency. This
fact has been taken advantage of by Schottelius' in the
1 Deutsche med, Wochenschrift, 1885, No. 14.
SPIRILLUM OF ASIATIC CHOLERA. 375
following method devised by him for the bacteriological
examination of dejections from cholera patients:
In dejections that are not examined immediately after
being passed it is often difficult, because of the large
number of other bacteria that may be present, to detect
with certainty the cholera organism by microscopic ex-
amination. It is advantageous in these cases to mix
the dejections with about double their volume of slightly
alkaline beef-tea, and allow them to stand for about
twelve hours at a temperature of between 30° and 40°
C. There appears at the end of this time, especially
upon the surface of the fluid, a conspicuous increase in the
number of comma bacilli, and cover-slip preparations
made from the upper layers of the fluid will reveal an
almost pure culture of this organism.
It is not improbable that a similar process occurs in
the intestines of those suffering from Asiatic cholera,
viz., a rapid multiplication of the comma bacilli that
have gained access to the intestines takes place, but lasts
for only a short time, when the comma bacilli begin to
disappear, and after a few days their place is taken by
other organisms.
In connection with his experiments upon the poison
produced by the cholera organism Pfeiffer’ states that
in very young cultures, grown under the access of oxy-
gen, there is present a poisonous body that possesses
intense toxic properties. This primary cholera-poison
stands in very close relation to the material composing
the bodies of the bacteria themselves, and is probably
an integral constituent of them, for the vitality of the
cholera spirilla can be destroyed by means of chloro-
1 Zeitschrift f. Hygiene u. Infectionskrankheiten, Bd. xi. p. 393.
376 BACTERIOLOGY.
form and thymol, and by drying, without apparently
any alteration of this poisonous body. Absolute alco-
hol, concentrated solutions of neutral salts, and a tem-
perature of 100° C., decompose this substance, leaving
behind secondary poisons which possess a similar physi-
ological activity, but only when given in from ten to
twenty times the dose necessary to produce the same
effects with the primary poison.
Other members of the vibrio family also, namely,
vibrio Metchnikovt and that of Finkler and Prior (see
description of these species), contain, according to Pfeif-
fer, closely related poisons.
Experiments upon animals. As a result of experi-
ments for the purpose of determining if the disease can
be produced in any of the lower animals it is found
that white mice, monkeys, cats, dogs, poultry, and many
other animals are not susceptible to infection by the
methods usually employed in inoculation experiments.
When animals are fed on pure cultures of the comma
bacillus no effect is produced, and the organisms cannot
be obtained from the stomach or intestines; they are
- destroyed in the stomach and do not reach the intes-
tines; they are not demonstrable in the feces of these
animals. Intravascular injections of pure culture into
rabbits are followed by a temporary illness, from which
the animals usually recover in from two to three days;
intraperitoneal injections into white mice are, as a rule,
followed by death in from twenty-four to forty-eight
hours; the conditions in both instances most probably
resulting from the toxic activities of the poisonous pro-
ducts of growth of the organisms that are present in the
culture employed. None of the lower animals have ever
been known to suffer from Asiatic cholera spontaneously.
SPIRILLUM OF ASIATIC CHOLERA. 377
The failure to induce cholera in animals by feeding,
or by injection of cultures into the stomach,was shown
by Nicati and Rietsch' to be due to the destructive
action of the acid gastric juice on the bacilli. They
showed that if cultures of this organism were intro-
duced into the alimentary tract of certain animals in
such a manner that they would not be subjected to the
influence of the gastric juice, a condition pathologically
closely simulating cholera as it occurs in man could be
produced. For this purpose the common bile duct was
ligated, after which the cultures were injected directly
into the duodenum. Such interference with the flow of
bile lessens intestinal peristalsis, and thus permits the
development of the bacilli at the point at which they
are deposited—that is, the portion of the intestine hav-
ing an alkaline reaction and beyond the influence of the
acid stomach-juice.
By this method Nicati and Rietsch, Van Ermengem,’
Koch,’ and others were enabled to produce in the ani-
mals upon which they operated a condition that was, if
not identical, at all events very similar pathologically to
that seen in the intestines of subjects dead of the disease.
Ata subsequent conference held in Berlin in 1885
Koch* described the following method by means of
which he had been able to obtain a relatively high de-
gree of constancy in all his efforts to produce cholera in
lower animals: bearing in mind the point made by
Nicati and Rietsch as to the effect produced by the acid
reaction of the gastric juice, this reaction was first to be
1 Archiy. de Phys. norm. et path., 1885, xvii., 3e sér., t. vi. Compt.-rend.,
xcix. p. 928, Rev. de Hygiéne, 1885, Rev. de Méd , 1885, v.
2 « Recherches sur le Microbe du Choléra Asiatique,” Paris-Bruxelles, 1885,
Bull. de l’Acad. roy. de Méd. de Belgique, 3esér., xviii.
3 Loe. cit. 4 Loe. cit., 1885.
378 BACTERIOLOGY.
neutralized by injecting through a soft catheter passed
down the cesophagus into the stomach 5 ¢.c. of a 5 per
cent. solution of sodium carbonate. Ten or fifteen min-
utes later this was to be followed by the injection into
the stomach (also through a soft catheter) of 10 c.c. of a
bouillon culture of the cholera spirillum. For the pur-
pose of arresting peristalsis and permitting the bac-
teria to remain in the stomach and upper part of the
duodenum for as long a time as possible, the animal was
to receive, immediately following the injection of the
culture, an intraperitoneal injection, by means of a
hypodermic syringe, of 1 c.c. of tincture of opium for
each 200 grammes of its body weight. Shortly after
this last injection a deep narcosis sets in and lasts from
a half to one hour, after which the animal is again as
lively as ever. Of 35 guinea-pigs inoculated in this
way by Koch, 30 died of a condition that was, in gen-
eral, very similar to that seen in Asiatic cholera.
The condition of these animals before death is de-
scribed as follows: twenty-four hours after the opera-
tion the animal appears sick; there is a loss of appetite,
and the animal remains quiet in its cage. On the fol-
lowing day a paralytic condition of the hind extremities
appears, which, as the day goes on, becomes more pro-
nounced; the animal lies quite flat upon its abdomen or
on its side, with its legs extended; respiration is weak
and prolonged, and the pulsations of the heart are hardly
perceptible; the head and extremities are cold, and the
body temperature is frequently subuormal.
The animal! usually dies after remaining in this con-
dition for a few hours.
At autopsy the small intestine is found to be deeply
injected and filled with a flocculent, colorless fluid. The
SPIRILLUM OF ASIATIC CHOLERA. 379
stomach and intestines do not contain solid masses, but
fluid; when diarrhcea does not occur, firm scybala may
be expected in the rectum. Both by microscopic exam-
ination and by culture methods comma bacilli are found
to be present in the small intestine in practically pure
culture.
More recently Pfeiffer' has determined that essen-
tially similar constitutional effects may be produced in
guinea-pigs by the intraperitoneal injection of rela-
tively large numbers of this organism. His plan is to
scrape from the surface of a fresh culture on agar-agar
as much of the growth as can be held upon a moderate-
sized wire loop. ‘This is then finely divided in 1 cc.
of bouillon and, by means of a hypodermic syringe, is
injected directly into the peritoneal cavity. When vir-
ulent cultures have been used this is quickly followed
by a fall in the temperature of the animal; this is grad-
ual and continuous until death ensues, which is usually
in from eighteen to twenty-four hours after the opera-
tion, though exceptionally cases do occur in which the
animal recovers, even after having exhibited marked
symptoms of most profound toxemia.
In pursuance of his studies upon this disease Pfeiffer’
has demonstrated that it is possible to render an animal
tolerant or immune to the poisonous properties of this
organism by repeated injections of non-fatal doses of
dead cultures (cultures that have been killed by the
vapor of chloroform or by heat). He also demon-
strated that animals so immuned possess a specific
germicidal action toward the cholera spirillum—i. e., if
into the peritoneal cavity of an animal immunized from
1 Zeitschrift fiir Hygiene, Bd. xi. and xiv.
2 Zeit. fiir Hyg. u. Infectionskrankheiten, Bd. ix. Heft. i.
380 BACTERIOLOGY.
Asiatic cholera living cholera spirilla be introduced,
they will all be destroyed (disintegrated) within a rela-
tively short time. Furthermore, if the serum of an
animal immunized from cholera be injected into the
peritoneal cavity of a similar animal not so protected,
and immediately afterward living cholera spirilla be
introduced, a similar disintegration and destruction of
the bacteria will also result. He shows that a more or
less definite relation exists between the amount of serum
and the number of organisms introduced. Such a de-
struction of the comma bacillus by the serum of an
immunized animal does not occur outside the animal
body—that is, cannot be demonstrated in a test-tube.
The specificity of this reaction is suggested by Pfeiffer
as a means of differentiating the cholera spirillum from
other suspicious species, for no such disintegration of
bacterial cells is observed if species other than the
cholera spirillum be introduced into the peritoneal cavity
of animals immunized from Asiatic cholera.
Pfeiffer has demonstrated that the serum of animals
artificially immunized from Asiatic cholera has an agglu-
tinating effect upon fluid cultures of the cholera spi-
rillum similar to that seen when typhoid bacilli are
mixed with the serum from typhoid cases, or from
animals artificially immunized from typhoid infection
or intoxication. (See Agelutinin.)
General considerations. In all cases of Asiatic chol-
era, and only in this disease, the organism just described
can be detected in the intestinal evacuations. The more
acute the case and the more promptly the examination
is made after the evacuations have been passed from
the patient, the less will be the difficulty experienced in
detecting the organism.
SPIRILLUM OF ASIATIC CHOLERA. 381
In some cases it can be detected in the vomited
matters, though by no means so constantly as in the
intestinal contents.
As a rule, bacteriological examination fails to reveal
the presence of the organisms in the blood and internal
organs in this disease, though Nicati and Rietsch claim
to have obtained them from the common bile-duct in
rapidly fatal cases, and in two out of five cases they
were present in the gall-bladder. Doyen and_Rasst-
schewsky' found them in the liver in pure culture, and
Tizzoni and Cattani’ in both the blood and the gall-
bladder.
The cholera spirillum is a facultative parasite; that
is to say, it apparently finds in certain portions of the
world, particularly in those countries in which Asiatic
cholera is endemic, conditions that are not entirely un-
favorable to its development outside of the body. This
has been found to be the fact not only by Koch, who
detected the presence of the organism in water-tanks
in India, but by many other observers who have suc-
ceeded in demonstrating its growth under conditions
not embraced in the ordinary methods that are em-
ployed for the cultivation of bacteria.
The results of experiments having for their object
the determination of the length of time during which
this organism may retain its vitality in water are con-
spicuous for their irregularity. In the transactions of
the congress in Berlin, for the discussion of the cholera
question, it is stated, in connection with this point, that
the experiments made with tank-water in India some-
times resulted in demonstrating the multiplication of
1 Reference to Vratch, 1885, in Allg. Med. Central Zeitung, Berlin.
2 Centralblatt f. die med. Wissenschaften, 1886, No. 43.
17*
382 BACTERIOLOGY.
the organisms introduced into it, while in other cases
they died very quickly.
On February 8, 1884, comma bacilli were found in
the tank at Saheb-Began, in Calcutta, and it was possi-
ble to demonstrate them in a living condition up to
February 23d.
Koch states that in ordinary spring-water or well-
water the organisms retained their vitality for thirty
days, whereas in the canal-water (sewage) of Berlin they
died after six or seven days; but if this latter were
mixed with fecal matters, the organisms retained their
vitality for but twenty-seven hours; and in the undi-
luted contents of cesspools it is impossible to demon-
strate them after twelve hours. In the experiments of
Nicati and Rietsch they retained their vitality in steril-
ized distilled water for twenty days; in Marseilles canal-
water (sewage), for thirty-eight days; in sea-water,
sixty-four days; in harbor-water, eighty-one days; and
in bilge-water, thirty-two days.
In the experiments of Hochstetter, on the other hand,
they died in distilled water in less than twenty-four
hours in five of seven experiments; in one of the two
remaining experiments they were alive after a day, and
in the other after seven days.
In one experiment with the domestic water-supply of
Berlin the organism retained its vitality for 267 days;
in another for 382 days, notwithstanding the fact that
many other organisms were present at the same time.
There is no single ground upon which these variations
can be explained, for they depend apparently upon a
number of factors which may act singly or together.
For example, in general it may be said that the higher
the temperature of the water in which these organisms
SPIRILLUM OF ASIATIC CHOLERA. 383
are present, up to 20° C., the longer do they retain their
vitality; the purer the water—that is, the poorer in
organic matters—the more quickly do the organisms
die, whereas the richer it is in organic matter the longer
do they retain their vitality.
Still another point that must be considered in this
connection is the antagonistic influences under which
they find themselves when placed in water containing
large numbers of organisms that are, so to speak, at
home in water—the so-called normal water-bacteria.
The effect of light upon growing bacteria must not
be lost sight of, for it has been shown that a surprisingly
large number of these organisms are robbed of their
vitality by a relatively short exposure to the rays of
the sun, and it is therefore not unlikely that the non-
observance of this fact may be, in part at least, account-
able for some of the discrepancies that appear in the
results of these experiments.
In his studies upon the behavior of pathogenic and
other micro-organisms in the soil Carl Frankel! found
that the cholera spirillum was not markedly susceptible
to those deleterious influences that cause the death of a
number of other pathogenic organisms. During the
months of August, September, and October cultures of
the comma bacillus that had been buried in the ground
at a depth of three metres retained their vitality; on
the other hand, in other months, particularly from April
to July, they lost their vitality when buried to the depth
of only two metres. At a depth of one and a half
metres vitality was not destroyed, and there was a_reg-
ular development in cultures so placed.
1 Zeitschrift fiir Hygieve, Bd. ii. p. 521.
384 BACTERIOLOGY.
Asa result of experiments performed in the Imperial
Health Bureau, at Berlin, it was found that the bodies
of guinea-pigs that had died of cholera induced by
Koch’s method of inoculation contained no living chol-
era spirilla when exhumed after having been buried for
nineteen days in wooden boxes, or for twelve days in
zine boxes. Ina few that had been buried in moist
earth, without having been encased in boxes, when ex-
humed after two or three months, the results of exam-
inations for cholera spirilla were likewise negative.
Kitasato,' in his experiments with the cholera organ-
ism, found that when mixed with the intestinal evacu-
ations of human beings under ordinary conditions they
lost their vitality in from a day and a half to three days.
If the evacuations were sterilized before the cultures
were mixed with them, the organisms retained their
vitality up to from twenty to twenty-five days. He
was unable to_come to any definite conclusion as to the
cause of these phenomena.
It was demonstrated by Hesse’ and by Celli* that
many substances commonly employed as food-stuffs
serve as favorable materials for the development of the
cholera organism. In his experiments upon its behavior
in milk Kitasato’ found that at a temperature of 36° C.
the cholera spirillum developed very rapidly during the
first three or four hours, and outnumbered the other
organisms commonly found in milk. They then dimin-
ished in number from hour to hour as the acidity of the
milk increased, until finally their vitality was lost; at
the same time the common saprophytic bacteria in-
1 Zeitschrift fur Hygiene, Bd. v. p. 487.
2 Thid , Ba. v. p. 527.
8 Bolletino della R. Acad. Med. di. Roma, 1888.
4 Zeitschrift fir Hygiene, Bd. v. p. 491.
SPIRILLUM OF ASIATIC CHOLERA. 385
creased in number. Relatively the same process occurs
at a lower temperature, from 22° to 25° C., but the
process is slower, the maximum development of the
cholera organisms being reached at about the fifteenth
hour, after which time they were overgrown by the
ordinary saprophytes present.
From this it would seem that the vitality of the
cholera spirillum in milk depends largely upon the re-
action: the more quickly the milk becomes sour the
more quickly does the organism become inert, while the
longer the milk retains its neutral, or only very slightly
acid reaction, the longer do the cholera organisms that
may be present in it retain their power of multiplica-
tion.
According to Laser,’ the cholera organism retains its
vitality in butter for about seven days; it is therefore
possible for the disease to be contracted by the use of
butter that has in any way been in contact with cholera
material.
In regard to the antagonism between the cholera
spirillum and other organisms with which it may come
in contact, the experiments of Kitasato’ led him to
conclude that no organism has been found which,
when growing in the same culture medium with it, pos-
sessed the power of depriving it of its vitality within
a short time. On the other hand, the experiments showed
that there were quite a number of other organisms the
development of which was checked, and in some cases
their vitality was completely destroyed, when growing
in the same medium with the cholera spirillum.
From this it would appear that the disappearance of
1 Zeitschrift fiir Hygiene, Bd. x. p. 513. 2 Tbid., Bd. vi. p. 1.
886 BACTERIOLOGY.
the cholera spirillum from mixed cultures and from the
evacuations in so short a time as has been mentioned,
is due more to unfavorable nutritive conditions than to
the direct action of the other organisms present.
When completely dried, according to Koch’s experi-
ments, the cholera spirillum does not retain its vitality
for longer than twenty-four hours, but by others its
vitality is said to be destroyed by an absolute drying of
three hours. In the moist conditions, as in artificial
cultures, vitality may be retained for many months,
though repeated observations lead us to believe that,
* under these circumstances, the virulence is diminished.
According to Kitasato,' they retain their vitality when
smeared upon thin glass cover-slips and kept in the
moist chamber for from 85 to 100 days, and for as long
as 200 days when deposited upon bits of silk thread.
In the course of his studies upon the destiny of path-
ogenic micro-organisms in the dead body yon Esmarch?
found that, when the cadaver of a guinea-pig dead from
the introduction of cholera organisms into the stomach
was immersed in water and decomposition allowed to
set in, after eleven days, when decomposition was far
advanced, it was impossible to find any living cholera
spirilla by the ordinary plate methods.
A similar experiment resulted in their disappearance
after five days. In another experiment, in which de-
composition was allowed to go on without the animal
being immersed in water, none could be detected after
the fifth day.
Carl Frankel* has shown that an atmosphere of car-
bonie acid is directly inhibitory to the development of
1 Zeitschrift fiir Hygiene, Bd. v. p. 184. 2 Thid., Bd. vii. p, 1.
8 Tbid., Bd. v. p. 382.
THE DIAGNOSIS OF ASIATIO CHOLERA. 387
the cholera spirillum, and Perey Frankland! states that
in an atmosphere of this gas it dies in about eight days.
In an atmosphere of carbon monoxide its vitality is
lost in nine days, and in general the same may be said
for it when under the influence of an atmosphere of
nitrous oxide gas.
From what has been said we see that the spirillum of
Asiatic cholera, while possessing the power of producing
in human beings one of the most rapidly fatal forms of
the disease with which we are acquainted, is still one of
the least resistant of the pathogenic organisms known
tous. Under conditions most favorable to its growth
its development is self-limited; it is conspicuously sus-
ceptible to acids, alkalies, other chemical disinfectants,
and heat; but when partly dried upon clothing, food,
or other objects, it may retain its vitality for a relatively
long period of time, and it is more than probable that
it is in this way that the disease is often carried from
points in which it is epidemic or endemic into localities
that are free from the disease.
THE DIAGNOSIS OF ASIATIC CHOLERA BY BACTERIO-
LOGICAL METHODS.
Because of the manifold channels that are open for
the dissemination of this disease it is of the utmost im-
portance that its true nature should be recognized as
quickly as possible, for with every moment of delay in
its recognition opportunities for its spread are multiply-
ing. It is essential, therefore, when employing bacteri-
ological means in making the diagnosis, to bear in mind
1 Tbid., Bd. vi. p. 13.
388 BACTERIOLOGY.
those biological and morphological features of the organ-
ism that appear most quickly under artificial methods
of cultivation, and which, at the same time, may be
considered as characteristic of it, viz., its peculiar mor-
phology and grouping; the much greater rapidity of its
growth over that of other bacteria with which it may
be associated; the characteristic appearance of its col-
onies on gelatin plates and of its growth in stab-cultures
in gelatin; its property of producing indol and coinci-
dently nitrites in from six to eight hours in peptone
solution at 37° to 38° C.; and its power of causing the
death of guinea-pigs in from sixteen to twenty-four
hours when introduced into the peritoneal cavity, death
being preceded by symptoms of extreme toxemia, char-
acterized by prostration and gradual and continuous
fall in the temperature of the animal’s body.
In a publication made by Koch! he called atten-
tion to a plan of procedure that is employed in this
work in the Institute for Infectious Diseases at Ber-
lin. In this scheme the points that have been enume-
rated are taken into account, and by its employment
the diagnosis can be established in the majority of
cases of Asiatic cholera in from eighteen to twenty-two
hours. In general, the steps to be taken and points to
be borne in mind are as follows: the material should
be examined as early as possible after it has been passed.
I. Microscopic examination, From one of the small
slimy particles that will be seen in the semi-fluid evac-
uations prepare a cover-slip preparation in the ordinary
way and stain it. If, upon microscopic examination,
only curved rods, or curved rods greatly in excess of all
1 Zeitschrift fur Hygiene, 1893, Bd. xiv.
THE DIAGNOSIS OF ASIATIC CHOLERA. 389
other forms, are present, the diagnosis of Asiatic cholera
is more than likely correct; and particularly is this true
if these organisms are arranged in irregular linear
groups with the long axes of all the rods pointing in
nearly the same direction—that is to say, somewhat as
minnows arrange themselves when swimming in schools
up stream. (Koch.)
In 1886 Weisser and Frank’ expressed their opinion
upon the value of microscopic examination in these cases
in the following terms:
(a) In the majority of cases microscopic examination
is sufficient for the detection of the presence of the
comma, bacillus in the intestinal evacuations of cholera
patients.
(6) Even in the most acute cases, running a very rapid
course, the comma bacillus can always be found in the
evacuations.
(c) In general, the number of cholera spirilla present
is greater the earlier death occurs; when death is post-
poned, and the disease continues for a longer period,
their number is diminished.
(d) Should the patient not die of cholera, but from
some other disease, such as typhoid fever, that may be
engrafted upon it, the comma bacilli may disappear en-
tirely from the intestines.
II. With another slimy flake prepare a set of gelatin
plates. Place them at a temperature of from 20° to
22° C., and at sixteen, twenty-two, and thirty-six hours
observe the appearance of the colonies. Usually at
about twenty-two hours the colonies of this organism
can easily be identified by one familiar with them.
1 Zeitschrift fiir Hygiene, Bd. i. p. 397.
390 BACTERIOLOGY.
III. With another slimy flake start a culture in a
tube of peptone solution—either the solution of Dun-
ham or, as Koch proposes, a solution of double the
strength of that of Dunham (Witte’s peptone is to be
used, as it gives the best and most constant results).
Place this at 37° to 38° C., and at the end of from six
to eight hours prepare cover-slips from the upper layers
(without shaking) and examine them microscopically ;
if comma bacilli were present in the original material,
and are capable of multiplication, they will be found in
this locality in almost pure culture. After doing this
prepare a second peptone culture from the upper layers
of the one just examined, also a set of gelatin plates,
and with what remains make the test for indol by the
addition of ten drops of concentrated sulphuric acid
for each ten cubic centimetres of fluid contained in the
tube. If comma bacilli are growing in the tube, the
rose color characteristic of the presence of indol should
appear.
By following this plan ‘a bacteriologist who is
familiar with the morphological and biological peculi-
arities of this organism should make a more than prob-
able diagnosis at once by microscopic examination alone,
and a positive diagnosis in from twenty to, and at the
most, twenty-four hours after beginning the examina-
tion.”’ (Koch.)
There are certain doubtful cases in which the organ-
isms are present in the intestinal canal in very small
numbers, and microscopic examination is not, therefore,
of so much assistance. In these cases plates of agar-
agar, of gelatin, and cultures in the peptone solution
should be made.
The plates of agar-agar should not be prepared in
THE DIAGNOSIS OF ASIATIC CHOLERA. 391
the usual way, but the agar-agar should be poured into
Petri dishes and allowed to solidify, after which one of
the slimy particles may be smeared over its surface.
The comma bacillus, being markedly aérobic, develops
very much more readily when its colonies are located
upon the surface than when they are in the depths of
the medium. A point to which Koch calls attention,
in connection with this step in the manipulation, is the
necessity for having the surface of the agar-agar free
from the water that is squeezed from it when it solid-
ifies, as the presence of the water interferes with the
development of the colonies at isolated points and causes
them to become confluent. To obviate this he recom-
mends that the agar-agar be poured into the plates and
the water allowed to separate from the surface at the
temperature of the incubator before they are used. It
is wise, therefore, when one is liable to be called on for
such work as this to keep a number of sterilized plates
of agar-agar in the incubator ready for use, just as ster-
ilized tubes of media are always ready and at hand.
The advantage of using the agar plates is the higher
temperature at which they can be kept, and consequently
a more favorable condition for the development of the
* colonies. As soon as isolated colonies appear they
should be examined microscopically for the presence of
bacteria having the morphology of the one for which
we are seeking, and as soon as such is detected gelatin
plates and cultures in peptone solution (for the indol
reaction) should be made. The peptone cultures started
from the original material should be examined micro-
scopically from hour to hour after the sixth hour that
they have been in the incubator. The material taken
for examination should always come from near the sur-
392 BACTERIOLOGY.
face of the fluid, and care should be taken not to shake
the tube. As soon as comma bacilli are detected in
anything like considerable numbers in the upper layers
of the fluid, agar-agar plates and fresh peptone cultures
should be made from them. The colonies will develop
on the agar-agar plates at 37° C. in from ten to twelve
hours to a size sufficient for recognition by microscopic
examination, and from this examination an opinion can
usually be given. This opinion should always be con-
trolled by cultures in the peptone solution made from
each of several single colonies, and finally the test for
the presence or absence of indol in these cultures.
In all doubtful cases in which only a few curved
bacilli are present, or in which irregularities in either
the rate or mode of their development occur, pure cul-
tures should be obtained by the agar-agar plate method
and by the method of cultivation in peptone solution,
as soon as possible, and their virulence tested upon ani-
mals. For this purpose cultures upon agar-agar from
single colonies must be made. From the surface of one
of such cultures a good sized wire-loopful should be
seraped and this broken up in about one cubic centi-
metre of bouillon, and the suspension thus made injected
by means of a hypodermic syringe directly into the peri-
toneal cavity of a guinea-pig of about 350 to 400
grammes weight. For larger animals more material
should be used. If the material injected is from a
fresh culture of the cholera organism, toxic symptoms
at once begin to appear; these have their most pro-
nounced expression in the lowering of temperature, and
if one follows this decline in temperature from time to
time with the thermometer it will be seen to be gradual
and continuous from the time of injection to the death
THE DIAGNOSIS OF ASIATIC CHOLERA 393
of the animal (Pfeiffer), which occurs in from eighteen
to twenty-four hours after the operation.
In general, this is the procedure employed in the
Institute for Infectious Diseases, at Berlin, under
Koch’s direction.
+ Loe. cit.
CHAPTER XXIII.
Spirilla of interest, historically and otherwise, that have been confounded
with the spirillum of Asiatic cholera—Their peculiarities and differential
features— Vibrio proteus, or bacillus of Finkler and Prior—Spirillum tyrogenum,
or cheese spirilim of Deneke—The spirillum of Miller— Vibrio Metchnikovi.
VIBRIO PROTEUS (FINKLER-PRIOR BACILLUS).
Finkler and Prior were the first to contest experi-
mentally the significance of the presence of Koch’s
comma bacillus in Asiatic cholera, claiming to have
found it in the dejections of individuals suffering from
other maladies, particularly cholera nostras. The mor-
phological and biological differences between the organ-
ism that Finkler and Prior discovered and those of
the comma bacillus described by Koch are, however,
so pronounced as to warrant the opinion that the
confusion arose through imperfect and untrustworthy
methods of experimentation. At a somewhat later
period Finkler and Prior retracted their claims of iden-
tity for the two organisms, and held that the bacterium
with which they were dealing was peculiar to cholera
nostras—an opinion which, in the light of subsequent
work, was also proved to be without foundation in fact.
The characteristics of the spirillum of Finkler and
Prior are as follows:
MorprHo.toey.—lIt is thicker and longer than the
spirillum of Asiatic cholera; it is often thicker at the
middle than at the poles; it forms, like the ‘‘ comma
bacillus,” screw-like, twisted threads (Fig. 75).
VIBRIO PROTEUS. 395
It is supplied with a single flagellum at one of its
ends, and is, therefore, motile.
Fic. 75.
Vibrio proteus, Finkler-Prior bacillus, from culture on agar-agar twenty-
four hours old.
It, like the comma bacillus, readily undergoes degen-
erative changes under conditions unfavorable to growth,
and presents the variety of shapes grouped under the
head ‘‘ involution-forms.’’ According to Buchner, this
is especially the case when the medium in which they
are growing contains glucose (5 per cent.) or glycerin
(2 per cent. ).
CULTURAL PECULIARITIES.—On gelatin plates the
development of its colonies is far more rapid, and lique-
faction far more extensive, than in the case of the
cholera spirillum. After twenty-two to twenty-four
hours in this medium at 20° to 22° C. the average size
of the colonies is about double that of the comma bacil-
lus. The colonies are darker and denser and do not
present under the low lens the same degree of granula-
tion and subsequent lobulation, and they do not become
serrated or scalloped around the margin as is the case
with Koch’s organism. After twenty-two to twenty-
four hours they are usually nearly round, regularly
granular, and more or less sharply defined. (See Fig.
76,a.) At times they may show indefinite markings
396 BACTERIOLOGY.
or creases, somewhat suggestive of lobulations. After
forty-eight hours on gelatin they usually range from
one to three millimetres (some even larger) in diameter,
and will appear as sharply cut, saucer-shaped pits of
liquefaction, in the most dependent portion of which
lies a dense, irregular mass, the colony proper. Under
low magnifying power they present at this stage an ap-
pearance similar to that shown in Fig. 76, 6, the central
dense mass representing the colony and the irregular
Fig. 76.
Colonies of the Finkler-Prior bacillus on gelatin. > about 75 diameters.
a. After twenty-two hours at 20° to 22°C. b. After forty-eight hours at 20°
to 22°C.
ragged lines surrounding it being shreds that have be-
come torn away as it sank into the liquid caused by its
growth. The zone surrounding it, extending to the
periphery, is somewhat cloudy, and is simply liquefied
gelatin. There is a marked tendency for the liquefac-
tion to spread laterally and for the colonies to run
together, so that, even on plates containing few colonies,
in sixty to seventy-two hours at from 20° to 22° C., the
entire gelatin is usually converted into a yellowish-
VIBRIO PROTEUS. 397
white fluid. Under these conditions its growth is ac-
companied by a marked aromatic odor, impossible to
describe; this is especially the case when the liquefac-
tion is far advanced.
Stab-culture of the Finkler-Prior bacillus in gelatin at 18° to 20° C,
a. After twenty-four hours. 6. After forty-eight hours. ¢. After seventy-two
hours. d. After ninety-six hours.
In stab-cultures in gelatin at the room temperature,
liquefaction is noticed about the upper part of the
needle-track in twenty-four hours. This condition
gradually increases, and at the end of two or three days
the entire upper portion of the gelatin has become con-
verted into a cloudy fluid, whereas at the lower part of
the canal the liquefaction progresses less rapidly, but is
18
398 BACTERIOLOGY.
still much more marked than that seen as a result of
the growth of Koch’s spirillum. Indeed, under these
circumstances there is no similarity whatever between
the growth of the two organisms (see a, }, ¢, d, Fig. 77,
and compare these with corresponding cuts in Fig. 74).
It is customary to see scattered through the cloudy
liquefied gelatin, ragged, more or less dense masses,
fragments of the colony proper.
On nutrient agar-agar there is nothing particularly
characteristic about its growth, appearing only as a
moist, grayish or yellowish-gray deposit.
On potato, after forty-eight to seventy-two hours,
there appears a pale, yellowish-gray deposit; this is
moist, glazed, and marked by lobulations, and is sur-
rounded by an irregular, colorless zone of growth that
is much less moist than that forming the central area.
It grows well on potato at the ordinary temperature of
the room.
Tt causes liquefaction of solidified blood-serum and
of coagulated egg-albumin.
In milk to which neutral litmus tincture has been
added the blue color takes on a pink tinge in from two
to three days at 37° to 38°C.
It does not form indol nor does it cause fermentation
of glucose.
In peptone solution containing rosolic acid the color
is somewhat deepened after four or five days at 37° C.
EXPERIMENTS UPON ANIMALS.—By ordinary meth-
ods of inoculation this organism is without pathogenic
properties. Injections, subcutaneous and intravascular
and directly into the stomach, give negative results.
When introduced into the stomach of guinea-pigs by
the method employed by Koch in his cholera experi-
SPIRILLUM TYROGENUM. 399
ments, Finkler and Prior had 3 out of 10 animals, and
Koch 5 out of 15 animals so treated to die.
The claim of Finkler and Prior that this organism
was related etiologically to cholera nostras has been
shown by subsequent work to be unjustifiable.
In 1885, 1886, and 1887 Franck’ examined seven
cases that clinically presented the condition of cholera
nostras ; in none of these seven cases was the organism
of Finkler and Prior, which they claimed to be the
cause of the disease, found. In all cases the results of
bacteriological examination, in so far as the constant
presence of an organism that might stand in causal
relation to the disease was concerned, were negative.
Only the ordinary intestinal bacteria were found.
SPIRILLUM TYROGENUM (CHEESE SPIRILLUM OF
DENEKE).
Another spiral form, likewise forming short, comma-
shaped segments in the course of its growth (Fig. 78),
is that found by Deneke in old cheese. It is a little
smaller than Koch’s spirillum. It is motile and has
but a single flagellum, attached to one of its ends. It
liquefies gelatin more rapidly than does Koch’s organ-
ism. It possesses no characteristic grouping, as can be
seen in impression cover-slips of its colonies. It does
not form spores. On gelatin plates its colonies develop
very rapidly as saucer-shaped depressions; after twenty-
four hours they vary from 1 to 4 mm. in transverse
diameter. To the naked eye they are almost trans-
parent, and are usually marked by a denser centre and
1 Zeitschrift f, Hygiene, Bd. iv. p. 207.
400 BACTERIOLOGY.
peripheral zone, the space between being quite clear.
They are not regularly round in all cases. A peculiar
aromatic odor accompanies their growth on gelatin.
Deneke’s cheese spirillum, spirillum tyrogenum. From agar agar culture
twenty-four hours old.
Under low magnifying power the smallest colonies are
irregularly round in outline, their borders being often
rough and broken, and the body of the colony is fre-
quently marked by creases or ridges that give to it a
lobulated appearance. The larger colonies under the
Fie. 79.
Colony of spirillum tyrogenum on gelatin, twenty-four hours old.
same lens appear as granular patches, a little denser at
the periphery and centre than at the intermediate por-
tions. The periphery gradually fades away and no dis-
tinct circumference can be made out. (See Fig. 79.)
The colonies of an intermediate size, about which lique-
SPIRILLUM TYROGENUM. 401
faction is just beginning to be apparent, show a dense
granular centre, the colony itself, and round about it a
delicate, granular developmental zone.
In stab-cultures in gelatin liquefaction is rapid, caus-
ing at the end of twenty-four hours a cup-shaped depres-
sion at the top of the needle-track, the superficial area
of which is about half that of the gelatin in the tube.
Fie. 80.
Stab-culture of Deneke's cheese spirillum in gelatin, at 18° to 20° C.
a. After twenty-four hours. b. After forty-eight hours. c¢. After seventy-two
hours. d. After ninety-six bours.
(Fig. 80, a.) The liquefying process spreads laterally,
and at the end of forty-eight hours the whole upper
portion of the gelatin may have become liquid. (Fig.
80, 6.) This process continues along the track of the
402 BACTERIOLOGY.
needle, and after seventy-two and ninety-six hours the
appearances shown in Fig. 80, ¢ and d, will be produced.
There is nothing particularly characteristic about its
growth upon agar-agar.
On potato there appears a moist, glazed, yellowish,
and, at points, brownish-yellow growth that is sur-
rounded by a drier, colorless zone. It is not lobu-
lated.
In milk containing neutral litmus tincture a pink color
appears after two to three days at 37° C.; after four
days the milk is almost decolorized and there is begin-
ning to appear coagulation of the casein with a layer of
clear whey above it. During the subsequent twenty-
four hours there is complete separation of the contents
of the tube into clot and whey.
In Dunham’s peptone solution it does not form indol,
and the reaction for this body does not appear with
either sulphuric acid alone or plus sodium nitrite.
It causes liquefaction of both coagulated blood-serum
and egg-albumin.
There is no pellicle formed as a result of its growth
in bouillon.
It does not produce fermentation of glucose.
In rosolic-acid-peptone solution its growth causes the
red color to become deepened after four or five days at
37°C,
By Koch’s method of introducing cultures into the
stomachs of guinea-pigs this organism produced the
death of three out of fifteen animals experimented
upon—the deaths resulting, most probably, more from
the toxic action of the products of growth that were
introduced with the organisms than to any pathogenic
powers possessed by the organism itself.
MILLER’S SPIRILLUM. 403
MILLER’S SPIRILLUM.
Another spirillum that has been likened to that of
Koch is the one obtained by Miller from a carious tooth.
It has so many characteristics in common with the or-
ganism of Finkler and Prior that Miller was inclined
to consider them identical. In morphology they are
indistinguishable. (See Fig. 81.) It grows rapidly,
and, like the spirillum of Finkler and Prior, causes
rapid liquefaction of gelatin with the coincident pro-
duction of a peculiar aromatic odor.
Spirillum of Miller, From agar-agar culture twenty-four hours old.
The colonies on gelatin plates appear after twenty-
four hours as small, transparent pits of liquefaction, in
the centre of which can be seen a minute white point,
the colony itself. Under a low lens the largest of these
points are uniformly granular and regularly round, and,
as a rule, are surrounded by a peripheral zone that is a
little darker than the central portion of the colony.
The circumference is delicately fringed by short, cilia-
like prolongations of growth which are not, as a rule,
straight, but are twisted in all directions and can only
be detected upon very careful examination. (See a,
Fig. 82.) When located deep in the gelatin the col-
onies are round, sharply circumscribed, of a pale yel-
404 BACTERIOLOGY.
lowish or greenish-yellow color, and marked by very del-
icate irregular lines or ridges. After forty-eight hours
the plate containing many colonies is entirely liquefied,
while that containing only a few shows the presence of
round, sharply cut, shallow pits of liquefaction that
measure from 2 to 10 mm. in diameter. They are a
little denser at the centre than at the periphery, and
the dense centre is not sharply circumscribed, but fades
off into what has the appearance of a delicate film.
(See 5, Fig. 82.) As the colonies become older they
are sometimes marked by irregular radii extending from
periphery to centre like the spokes of a wheel.
Fie. 82.
Colonies of Miller’s spirillum on gelatin, at 20° to 22°C. about 57
diameters.
a, Colony just beneath the surface of the gelatin. b. Colony on the surface
of the gelatin.
In stab-cultures in gelatin it rapidly produces lique-
faction, both at the surface and along the needle-track,
and in most respects gives rise to a condition very like
that resulting from the growth of Finkler and Prior’s
spirillum, though differing from it in certain details.
(See a, b, ¢, d, Fig. 83.)
On agar-agar nothing of special interest appears as a
result of its development.
On potato its growth is very like that of the cholera
spirillum,viz., it appears at 37° C. as a dry, white patch
MILLERS SPIRILLUM. 405
that lies quite flat upon the surface and can often only
be seen when the tube is held to the light in a special
way.
Stab-culture of Miller’s spirillum in gelatin, at 18° to 20° C.
a. After twenty-four hours. b. After forty-eight hours. c. After seventy-two
hours. d. After ninety-six hours.
Its growth in bouillon is not characteristic. It does
not form a pellicle.
Tt causes liquefaction of both coagulated blood-seruam
and egg-albumin.
Tt does not produce indol.
It does not cause fermentation of glucose.
Tt is non-motile.
In milk containing blue litmus tincture it causes
almost complete decolorization in from three to four
18*
406 BACTERIOLOGY.
days at 37° C.,with coincident coagulation of the casein
and the formation of a layer of clear whey about it.
It causes the red color of rosolic-acid-peptone solution
to become somewhat intensified after four or five hours
at 37° C.
Of twenty-one animals treated with this organism by
Koch’s method of inoculation only four died.
VIBRIO METCHNIKOVI.
The spirillum that simulates very closely the comma
bacillus of cholera in its morphological and cultural
peculiarities, but which is still easily distinguished from
it, is that described by Gamaleia under the name of
Fig. 84.
Vibrio Metchnikovi from agar-agar culture, twenty-four hours old.
vibrio Metchnikovi. It was found post mortem in a num-
ber of fowls that had died in the poultry market of
Odessa, and the experiments of the discoverer led him
to believe that it was related etiologically to the gastro-
enteritis with which the chickens had been suffering.
In morphology it is seen as short, curved rods and as
longer, spiral-like filaments. Jt is usually thicker than
Koch’s spirillum and is at times much longer, while
VIBRIO METCHNIKOVI. 407
again it is seen to be shorter. It is usually more dis-
tinctly curved than the ‘“‘ comma bacillus.”’ (Fig. 84.)
It is supplied with a single flagellum at one of its
extremities, and is, therefore, motile.
It does not form spores.
It is aérobic.
Its growth upon gelatin plates is usually character-
ized, according to Pfeiffer, by the appearance of two
kinds of liquefying colonies, one strikingly like those
of the Finkler-Prior organism, the other very similar
to those produced by Koch’s comma bacillus, though in
both cases the liquefaction resulting from the growth of
this organism is more energetic than that common to
the spirillum of Asiatic cholera. After from twenty-
four to thirty hours the medium-sized colonies, when
examined under a low power of the microscope, show a
yellowish-brown, ragged central mass surrounded by a
zone of liquefaction that is marked by a border of deli-
cate radii. (Tig. 85.)
Colony of vibrio Metchnikovi in gelatin, after thirty hours at 20° to 22°C.
X about 75 diameters.
In gelatin stab-cultures the growth has much the
same general appearance as that of the cholera spiril-
lum, but is very much exaggerated in degree. The lique-
faction is far more rapid, and the characteristic appear-
ance of the growth is lost in from three to four days.
(See a, 6, ¢, d, Fig. 86.) Development and liquefaction
408 BACTERIOLOGY.
along the deeper parts of the needle-track are much
more pronounced than is the case with the ‘‘ comma
bacillus.’’
Stab-culture of vibrio Metchnikovi in gelatin, at 18° to 20° C.
a. After twenty-four hours. b. After forty-eight hours. ec. After seventy-two
hours. d, After ninety-six hours,
Its growth on agar-agar is rapid, and after twenty-
four to forty-eight hours there appears a grayish de-
posit having a tendency to take on a yellowish tone.
On potato at 37°C. its growth is seen as a moist,
coffee-colored patch surrounded by a much paler zone.
The whole growth is so smooth and glistening that it
has almost the appearance of being varnished.
In bouillon it quickly causes opacity, with the ulti-
VIBRIO METCHNIKOYVI, 409
mate production of a delicate pellicle upon the surface.
It causes liquefaction of blood-serum, the liquefied area
being covered by a dense, wrinkled pellicle.
When grown in peptone solution it produces indol
and coincidently nitrites, so that the rose-colored reac-
tion characteristic of indol is obtained by the addition
of sulphuric acid alone. The production of indol by
this organism is usually greater than that common to
the comma bacillus under the same circumstances.
In milk it causes an acid reaction with coagulation of
the casein. The coagulated casein collects at the bot-
tom of the tube in irregular masses, above which is a
layer of clear whey. If blue litmus has been added
to the milk, the color is changed to pink by the end of
twenty-four to thirty hours, and after forty-eight hours
decolorization and coagulation occur. The clots of
casein are not re-dissolved. After about a week the
acidity of the milk is at its maximum, and the organ-
isms quickly die.
It causes the red color of the rosolic-acid-peptone
solution to become very much deeper after four or five
days at 37° C.
It does not cause fermentation of glucose with pro-
duction of gas.
It is killed in five minutes by a temperature of 50° C.
(Sternberg. )
It is pathogenic for chickens, pigeons, and guinea-
pigs. Rabbits and mice are affected only by very large
doses.
Chickens affected with the choleraic gastro-enteritis,
of which this organism is the cause, are usually seen
sitting quietly about with ruffled feathers. They are
afflicted with diarrhoea, but do not have any elevation
410 BACTERIOLOGY.
of temperature. A hyperemia of the entire gastro-
intestinal tract is seen at autopsy. The other internal
organs do not, as a rule, present anything abnormal to
the naked eye. The intestinal canal contains yellowish
fluid with which blood may be mixed. In adult chickens
the spirilla are not found in the blood, but in young
ones they are usually present in small numbers. By
subcutaneous inoculation pigeons succumb to the path-
ogenic activities of this organism in from eight to
twelve hours. At autopsy pretty much the same con-
dition is seen as was described for chickens, except that
large numbers of the spirilla are usually present in the
blood. Guinea-pigs usually die in from twenty to
twenty-four hours after subcutaneous inoculation. At
autopsy an extensive cedema of the subcutaneous tissues
about the seat of inoculation is seen, and there is usually
a necrotic condition of the tissues in the vicinity of the
point of puncture. As the blood and internal organs
contain the vibrios in large numbers, the infection in
these animals takes, therefore, the form of acute, gen-
eral septicemia.
Gastro-enteritis may be produced in both chickens
and guinea-pigs by feeding them with food in which
cultures of this organism have been mixed.
In the autumn of 1896 the author isolated from the
Schuylkill River at Philadelphia a spirillum that is
pathogenic for pigeons and for guinea-pigs, and that
possesses so many of the other characteristics com-
mon to the group of spirilla of which the cholera
spirillum forms the most important member, as to
justify the opinion that they are members of the same
family. That it is not identical with the cholera
VIBRIO SCHUYLKILIENSIS. 411
spirillum is evident, for the reason that the latter pro-
duces cholera, while the vibrio Schuylkiliensis manifestly
does not.}
Nore.—Since the late epidemic of cholera in Ham-
burg quite a number of curved or spiral organisms,
somewhat like the cholera spirillum, have been discov-
ered. or the descriptions of these the reader is re-
ferred to the current bacteriological literature.
1 For the detailed description of this organism see Journal of Experimen-
tal Medicine, vol. i. p. 419; also Transactions of the Association of American
Physicians, 1896, vol. xi. p. 394.
CHAPTER XXTY.
Study of bacillus anthracis, and the effects produced by its inoculation
into animals—Peculiarities of the organism under varying conditions of sur-
roundings.
THE discovery that the blood of animals suffering
from splenic fever, or anthrax, always contained minute
rod-shaped bodies (Pollender, 1855; Davaine, 1863),
led to a closer study of this disease, and has resulted
probably in contributing more to our knowledge of
bacteriology in general than work upon any of the
other infectious maladies.
The outcome of these investigations is that a rod-
shaped micro-organism, now known as bacillus an-
thracis, is always present in the blood of animals suffer-
ing from this disease; that this organism can be obtained
from the tissues of these animals in pure cultures, and
that these artificial cultures of bacillus anthracis when
introduced into the body of susceptible animals can
again produce a condition identical with that found in
the animal from which they were obtained.
The disease is a true septicemia, and after death the
capillaries throughout the body will always be found to
contain the typical rod-shaped organism in larger or
smaller numbers.
This organism, when isolated in pure culture, is seen
to be a bacillus which varies considerably in its length,
ranging from short rods of 2 to 5 # in length to longer
threads of 20 to 25 # in length. In breadth it is from
BACILLUS ANTHRACIS. 413
1 to 1.25 4. Frequently very long threads made up of
several rods, joined end to end, are seen.
When obtained directly from the body of an animal
it is usually in the form of short rods square at the ends.
If highly magnified, the ends are seen to be a trifle
thicker than the body of the cell and somewhat indented
or concave, peculiarities that help to distinguish it from
certain other organisms that are somewhat like it mor-
phologically. (See Fig. 87.)
Bacillus anthracis highly magnified to show swellings and concavities
at extremities of the single cells.
When cultivated artificially at the temperature of the
body the bacillus of anthrax presents a series of very
interesting stages.
The short rods develop into long threads, which may
be seen twisted or plaited together after the manner of
ropes, each thread being marked by the points of junc-
ture of the short rods composing it. (Fig. 88, a and 6.)
In this condition it remains until alterations in its
surroundings, the most conspicuous being diminution in
its nutritive supply, favor the production of spores.
When this stage begins, changes in the protoplasm of
the bacilli may be noticed; they become marked by
irregular, granular bodies, which eventually coalesce into
glistening, oval spores, one of which lies in nearly every
414 BACTERIOLOGY.
segment of the long thread, and gives to the thread the
appearance of a string of glistening beads. (Fig. 89.)
Fia. 88.
4
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Bacillus anthracis. Plaited and twisted threadg seen in fresh growing
cultures. >< about 400 diameters,
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 under-
gone degenerative changes, can be found. In this con-
dition the spores capable of resisting deleterious influ-
Fie. 89.
Threads of bacillus anthracis containing spores. X< about 1200 diameters.
ences remain, and, unless their surroundings are altered,
have been seen to continue in this living, though inac-
BACILLUS ANTHRACIS. 415
tive, condition for a very long time. If again placed
under favorable conditions, each spore will germinate
into a mature cell, and the same series of changes will
be repeated until the favorable surroundings become
again gradually unfavorable to development, when
spore-formation is again seen. Spore-formation takes
place only at temperatures ranging from 18° to 43° C.,
37.5° C. being the most favorable temperature. Under
12° C. they are not formed. With this organism spore-
formation does not occur in the tissues of the living
animal, its usual 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 head of Medusa. From a central point which is
Colony of bacillus anthracis on agar-agar.
more or less dense, consisting of a felt-like mass of long
threads matted irregularly together, the growth con-
tinues outward upon the surface of the agar-agar. (Fig.
90.) It is made up of wavy bundles in which the
threads are seen to lie parallel side by side or are twisted
416 BACTERIOLOGY.
in strands like those of a rope—sometimes they have a
plaited arrangement. (See Fig. 88.) These bundles
twist about and cross in all directions, and eventually
disappear at the periphery of the colony. At the ex-
treme periphery of the colonies it is sometimes possible
to trace single bundles of these threads for long dis-
tances across the surface of the agar-agar. The colony
itself is not circumscribed in its appearance, but is more
or less irregularly fringed or ragged, or scalloped. To
the naked eye they look very much like minute pellicles
of raw cotton that have been pressed into the surface
of the agar-agar.
As the colonies continue to grow they become more
and more dense, opaque, and granular and rough on the
surface. When touched with a sterilized needle one
experiences a sensation that suggests, somewhat, the
matted structure of these colonies. The bit that may
thus be taken from a colony is always more or less
ragged.
GELATIN.—The colonies on gelatin at the earliest
stages also present the same wavy appearance; but this
characteristic soon becomes in part destroyed by the
liquefaction of the gelatin which is produced by the
growing organisms. This allows them to sink to the
bottom of the fluid, where they lie as an irregular mass.
Through the fluid portion of the gelatin may be seen
small clumps of growing bacilli, which look very much
like bits of cotton-wool.
Bovurtion.—In bouillon the growth is characterized
by the formation of flaky masses, which also have very
much the appearance of bits of raw cotton. Micro-
scopic examination of one of these flakes reveals the
twisted and plaited arrangement of the long threads.
BACILLUS ANTHRACIS. 417
Porato.—It develops rapidly as a dull, dry, gran-
ular, whitish mass, which is more or less limited to the
point of inoculation. On potato, at the temperature of
the incubator, its spore-formation may easily be ob-
served.
STab- 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 pro-
nounced on the surface than down the track of the
needle.
On gelatin it causes liquefaction, which begins on the
surface at the point inoculated, and spreads outward and
downward.
It grows best with aceess to oxygen, and very poorly
when the supply of oxygen is interfered with.
Under favorable conditions of aération, nutrition, and
temperature its growth is rapid.
Under 12° C. and above 45° C. no growth occurs.
The temperature of the body is most favorable to its
development.
The spores of the anthrax bacillus are very resistant
to heat, though the degree of resistance is seen to vary
with spores. of different origin. von Esmarch found
that anthrax spores from some sources would readily be
killed by an exposure of one minute to the temperature
of steam, whereas those from other sources resisted this
temperature for longer times, reaching in some cases as
long as twelve minutes.
Srarnrne.—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
418 BACTERIOLOGY.
stained in tissues with a strong watery solution of
dahlia, after which the tissue is decolorized in 2 per
cent. sodium carbonate solution, washed in water, dehy-
drated 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—with eosin, for example—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 organisms will be of a purple
color, and will be seen in the capillaries of the tissues,
while the tissues themselves will be of a pale rose color.
InocuLaTIon Into ANIMALS.—Introduce into the
subcutaneous tissues of the abdominal wall of a guinea-
pig or rabbit a portion of a pure culture of bacillus
anthracis. In about forty-eight hours the animal
will be found dead. Immediately at the point of in-
oculation 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
edematous. Here and there, scattered through this
cedematous tissue, small ecchymoses will be seen. The
underlying muscles are pale in color. Inspection of
the internal viscera reveals no very marked macro-
scopic changes except in the spleen. This is enlarged,
dark in color, and soft. The liver may present the
appearance of cloudy swelling; the lungs may be red
or pale red in color; the heart is usually filled with
blood. There are no changes to be seen by the naked
eye.
Prepare cover-slip preparations from the blood and
other viscera. They will all be found to contain short
BACILLUS ANTHRACIS. 419
rods in large numbers. Nowhere can spore-formation
be detected. Upon microscopic examination of sec-
tions of the organs which have been hardened in alco-
hol the capillaries are seen to be filled with the bacilli;
in some places closely packed together in large num-
bers, 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 moder-
ately evenly distributed through the spleen. The
glomeruli of the kidneys and the capillaries of the
Fie. 91.
% \ \
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4 \ \ \ Ny ;
; \ 5
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1 Wo N f
nN ~~ S o AN
\ \ ANY i Ny]
VW \
\ \
\ ’ \y My a
M4 ye \ CAN
. Na \ ve 3
\ a poh!
Anthrax bacilli in liver of mouse. XX about 450 diameters, Bacilli stained
by Gram’s method ; tissue stained with Bismarck-brown.
lungs are frequently quite packed with them. The
capillaries of the liver contain them in large numbers.
(Fig. 91.) Hemorrhages, probably due to rupture of
capillaries by the mechanical pressure of the bacilli
which are developing within them, not uncommonly
occur. When this occurs in the mucous membranes
of the alimentary tract the blood may escape through
the mouth or anus; when in the kidneys, through the
uriniferous tubules.
€
ATOMY. :
ra
420 BACTERIOLOGY.
Cultures from the different organs or from the cedema-
tous fluid about the point of inoculation result in growth
of 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.
PROTECTIVE INOCULATION.
The most noteworthy application of artificially pre-
pared living vaccines to the protection of animals
against infection is seen in connection with anthrax
in sheep and in bovines.
By a variety of procedures the virulent anthrax
bacillus may be in part or totally robbed of its patho-
genic properties. It is through the very mild consti-
tutional disturbance, caused in animals vaccinated with
such weakened cultures, that protection is often afforded
against the severer, frequently fatal, form of the infec-
tion.
Without reviewing the various methods that have
been employed for attenuating the virulence of this
organism to a degree suitable for protective vaccina-
tion, it will suffice to say that the most satisfactory
results have been obtained by long-continued cultiva-
tion (ten to thirty days) at a temperature of from 42°
to 43° C. In this procedure the spore-free, virulent
bacillus anthracis, obtained directly from the blood of
a recently dead animal, is brought at once into sterile
BACILLUS ANTHRACIS. 421
nutrient bouillon in about twenty test-tubes, which
are immediately placed in an incubator that is care-
fully regulated to maintain a temperature of 42.5° C.
There should not be a fluctuation of over 0.1° C.
After about a week a tube is removed from the incu-
bator on each successive day and its virulence tested at
once on animals. The degree of attenuation experienced
by the cultures grown under these circumstances is deter-
mined by tests upon rabbits, guinea-pigs, and mice. The
first culture removed may or may not kill rabbits, the
most resistant of the three animals used for the test,
while it will certainly kill the guinea-pigs and mice; after
another two or three days rabbits will no longer succumb
to inoculation with the culture last removed from the
incubator, while no diminution will as yet be noticed
in its pathogenesis for the other two species. After
four to seven days more a culture may be encountered
that kills only mice, the guinea-pigs escaping; while
ultimately, if the experiment be continued, a degree of
attenuation may be reached in which the organism has
not even the power of killing a mouse, though it still
retains its vitality. Investigation of these attenuations
shows them to possess all the characteristics of enfeebled
anthrax bacillus; they grow slowly and less vigorously
when transplanted; they do not form spores while
under a high temperature; and microscopically they
present evidences of degeneration. When introduced
beneath the skin of animals they disseminate but
slightly beyond the site of inoculation, and do not, as
a rule, cause the general septicemia that occurs in sus-
ceptible animals after inoculation with normal cultures
of this organism. In the practical employment of these
attenuated cultures for protective purposes two vaccines
19
422 BACTERIOLOGY.
are employed. These were designated by Pasteur as
‘first’? and ‘second’? vaccines. The “ first’’ is the
one that killed only the mice in the preliminary tests,
while the ‘‘ second”’ is that which killed both mice and
guinea-pigs, but failed to kill the rabbit. When larger
animals, such as sheep or cattle, are to be protected by
vaccination with these vaccines, a subcutaneous inocu-
lation of about 0.3 ¢.c. of the first vaccine is usually
given. This should be practically without noticeable
effect, causing neither rise of body-temperature nor
other constitutional or local symptoms. After a period
of about two weeks the second vaccine is injected in
the same way; this may or may not cause disturbance.
In the event of its doing so the symptoms are rarely
alarming, and, if the vaccines have been properly pre-
pared and tested before use, they disappear within a
short time after the injection.
In the large majority of cases sheep, bovines, horses,
and mules may be safely protected against anthrax by
the careful practice of this method.
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 tempera-
ture of the room, place the agar-agar culture in the in-
cubator, 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 maintain the other at a
BACILLUS ANTHRACIS. 423
temperature 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 (which is the surest
source from which to obtain non-spore-bearing anthrax
bacilli), prepare a hanging-drop preparation; also a
cover-slip preparation in the usual way and stain it
with a strong gentian-violet solution, and another
cover-slip preparation which is to be drawn through
the flame twelve to fifteen times, stained with aniline
gentian-violet, washed off in iodine solution 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 48° 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 a spore into a
mature cell. Describe carefully the developmental
stages.
424 BACTERIOLOGY.
Prepare a 1: 1000 solution of carbolic acid in bouil-
lon. Inoculate this with virulent anthrax spores. If
no development occurs after two or three days at the
temperature 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 permits
of the development 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 development 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, etc. After five or six generations 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 to 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. (Why from the
blood of an animal and not from a culture?) Allow one
of them to grow for from fourteen to eighteen hours in
the incubator; allow the other to grow at the same tem-
perature 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
develop from the two different cultures compare? Was
BACILLUS ANTHRACIS. 425
there any difference in the time required for their de-
velopment 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 c.c.
of sterilized physiological 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 min-
utes make a plate upon agar-agar with one loopful of
the contents of this tube. Are the results on the plates
alike ?
Determine the exact time necessary to sterilize ob-
jects, such as silk or cotton threads, on which anthrax
spores have been dried, by the steam method and by
the hot-air method.
Prepare from the blood of an animal just dead of
anthrax 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 charac-
teristic ?
Inoculate six Erlenmeyer flasks of sterile bouillon,
each containing about 35 ¢.c. of the medium, from
either the blood of an animal just dead of anthrax or
from a fresh virulent culture in which no spores are
formed.
426 BACTERIOLOGY.
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 tempera-
ture of 42.5° C. Study each flask carefully, both in
its culture-peculiarities and in its pathogenic properties
when employed 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 ex-
periment, note carefully which one it is, and after ten
to twelve days repeat the inoculation, using the same
culture; if it again survives, inoculate it with the cul-
ture preceding the one just used in the order of removal
from the incubator; if it still survives, inoculate it with
virulent 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 ani-
mals 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
BACILLUS ANTHRACIS. 427
rabbit and a guinea-pig from the culture made on the
ninth day. Should the animals survive, inoculate them
again after three or four days with a culture made on
the sixth day. Do the results appear in any way
peculiar ?
CHAPTER XXV.
The most important of the organisms found in the soil—The bitrifying
bacteria—The bacillus of tetanus—The bacillus of malignant cedema--The
bacillus of symptomatic anthrax.
By the employment of bacteriological methods in the
study of the soil much light has been shed upon the
cause and nature of the interesting and momentous
biological phenomena that are there constantly in
progress. Of these, the one that is of the greatest
importance comprises those changes that accompany
the widespread process of disintegration and decompo-
sition, to which reference has already been made (see
Chap. I.). This resolution of dead, complex, organic
compounds into simpler structures that are assimilable
as food for growing vegetation is dependent upon the
activities of bacteria located in the superficial layers of
the ground, It is not throughout a simple process,
brought about by a single, specific species of bacteria;
but represents a series of metabolic alterations, each
definite step of which is most probably the result of
the activities of different species or groups of species,
acting singly or together (symbiotically). Our knowl-
edge upon the subject is not sufficient to permit of our
following in detail the manifold alterations undergone
by dead organic material in the process of decomposi-
tion that results in its conversion into inorganic com-
pounds, with the formation of carbonic acid, ammonia,
and water as conspicuous end-products. It suffices to
NITRIFYING BACTERIA. 429
say that, wherever dead organic matters are exposed to
the action of the great group of saprophytic bacteria,
in which are found many different species, the altera-
tions through which they pass are ultimately character-
ized by the appearance of these three bodies. When the
process of decomposition occurs in the soil, however, it
does not cease at this point, but we find still further
alterations—alterations concerning more particularly
the ammonia. This change in ammonia is character-
ized by the products of its oxidation, viz., by the for-
mation of nitrous and nitric acids and their salts; it is
not a result of the direct action of atmospheric oxygen
upon the ammonia, but occurs through the instrumen-
tality of a special group of saprophytes known as the
nitrifying organisms. They are found in the most super-
ficial layers of the ground, and though more common
in some places than in others, they are, nevertheless,
present over the entire earth’s surface. The most con-
spicuous example of the functional activity of this spe-
cific form of soil organism is that seen in the immense
saltpetre beds of Chili and Peru, where, through the
activities of these microscopic plants, nitrates are pro-
duced from the ammonia of the fecal evacuations of
sea-fowls in such enormous quantities as to form the
source of supply of this article for the commercial
world. A more familiar example, though hardly upon
such a great scale, is that seen in the decomposition
and subsequent nitrification of the organic matters of
sewage and other impure waters, in the process of puri-
fication by filtration through the soil, a process in which
it is possible to follow, by chemical means, the organic
matters from their condition as such through their con-
spicuous modifications to their ultimate conversion into
19*
430 BACTERIOLOGY.
ammonia, nitrous and nitric acids. In fact, the same
breaking down and building up, resulting ultimately
in nitrification, occurs in all nitrogenous matters that
are thrown upon the soil and allowed to decay. It is
largely through this means that growing vegetation
obtains the nitrogen necessary for the nutrition of its
tissues, and when viewed from this standpoint we ap-
preciate the importance of this process to all life, ani-
mal as well as vegetable, upon the earth.
These very important and interesting nitrifying
organisms, of which there appear to be several, have
been subjected to considerable study, and are found to
possess peculiarities of sufficient interest to justify a
more or less detailed description. For a long time all
efforts to isolate them from the soils in which they were
believed to be present, and to cultivate them by the
processes commonly employed in bacteriological work,
resulted in failure, and it was not until it was found
that the ordinary methods of bacteriological research
were in no way applicable to the study of these bacteria
that other, and ultimately successful, methods were de-
vised. By these special devices nitrifying bacteria,
capable of oxidizing ammonia to nitric acid, have been
isolated and cultivated, and the more important of their
biological peculiarities recorded by Winogradsky in
Switzerland, by G. C. and P. F. Frankland in Eng-
land, and by Jordan and Richards in this country.
From the similarity of the properties, given by these
several observers, of the nitrifying organisms isolated
by them, it seems likely that they have all been work-
ing with either the same organism or very closely allied
species.
The organism generally known as the nitro-monas of
NITRIFYING BACTERIA. 431
Winogradsky is a short, oval, and frequently almost
spherical cell. It divides as usual for bacteria, but
there is little tendency for the daughter-cells to adhere
together or to form chains. In cultures they are com-
monly massed together, by a gelatinous material, in the
form of zooglea. They do not form spores, and are
probably not motile, though Winogradsky believes he
has occasionally detected them in active motion. As
has been stated, they do not grow upon the ordinary
nutrient media, and cannot, therefore, be isolated by the
means commonly employed-in separating different spe-
cies of bacteria. The most astonishing property of this
organism is its ability to grow and perform its specific
fermentative function in solutions absolutely devoid of
organic matter. It is believed to be able to obtain its
necessary carbon from carbonic acid. Yor its isolation
and cultivation Winogradsky recommends the following
solution:
Ammonium sulphate ‘ i P é é 1 gramme
Potassium eaceasid . : : 1 se
Pure water . a ‘ . 5 + 1000 ¢.c.
To each flask containing 100 c.c. of this fluid is added
from 0.5 to 1.0 gramme of basic magnesium carbonate
suspended in a little distilled water and sterilized by
boiling. One of the flasks is then to be inoculated with
a minute portion of the soil under investigation, and
after four to five days a small portion is to be with-
drawn, by means of a capillary pipette, from over the
surface of the layer of magnesium carbonate and trans-
ferred to a second flask, and similarly after four or five
days from this to a third flask, and so on. As this
medium does not offer conditions favorable to the
growth of baeteria requiring organic matter for their
432 BACTERIOLOGY.
development, those that were originally introduced with
the soil quickly disappear, and ultimately only the nitri-
fying organisms remain. These are to be seen as an
almost transparent film attached to the clumps and gran-
ules of magnesium carbonate on the bottom of the flask.
For their cultivation upon a solid medium he employs
a mineral gelatin, the gelatinizing principle of which is
silicic acid. A solution of from 3 to 4 per cent. of silicic
acid in distilled water, and having a specific gravity of
1.02, remains fluid and can be preserved in flasks in
this condition (Kiihne). By the addition of certain
salts to such a solution gelatinization occurs, and will be
more or less complete according to the proportion of
salts added. The sults that have given the best results
and. the method of mixing them are as follows:
Ammonium sulphate é ¢ . 0.4 gramme.
ai Magnesium sulphate 2 % 0.05 = «
( calcium chloride . . ‘ . - js . trace.
Potassium phosphate a ‘ . 0.1 gramme.
b { Sodium carbonate . . .06to09 «
Distilled water 100 c.c.
The sulphates and chloride (a) are mixed in 50 c.c. of
the distilled water, and the phosphate and carbonate (6)
in the remaining 50 c.c., in separate flasks.
Each flask is then sterilized with its contents, which
after cooling are mixed together. This represents the
solution of mineral salts that is to be added to the silicic
acid, little by little, until the proper degree of consist-
ency is obtained (that of ordinary nutrient gelatin).
This part of the process is best conducted in the culture
dish. If it is desired to separate the colonies, as in an
ordinary plate, the inoculation and mixing of the mate-
rial introduced must be done before gelatinization is
NITRIFYING BACTERIA. 433
complete; if the material is to be distributed over only
the surface of the medium, then the mixture must first
be allowed to solidify.
By the use of this silicate-gelatin Winogradsky has
isolated from the gelatinous film in the bottom of fluids
undergoing nitrification a bacillus which he believes to
be associated with the nitro-monas in the nitrifying
process.
Our knowledge of these organisms is as yet too in-
complete to permit of a satisfactory description of all
their morphological and biological peculiarities. What
has been said will serve to indicate the direction in
which further studies of the subject should be prose-
cuted,
For further details the reader is referred to the orig-
inal contributions.*
In addition to the bacteria concerned in decomposition
and nitrification there are occasionally present in the
soil micro-organisms possessing disease-producing prop-
erties. Conspicuous among these may be mentioned
the bacillus of malignant cedema (vibrion septique of
the French), the bacillus of tetanus, and the bacillus of
symptomatic anthrax (Rauschbrand, German; charbon
symptomatique, French). It is sometimes due to the
presence of one or the other of these organisms that
wounds to which soil has had access (crushed wounds
from the wheels of cars or wagons, wounds received in-
agricultural work, etc.) are followed by such grave
disturbances of the constitution.
1 Winogradsky: Annales de l'Institut Pasteur, tomes iv., 1890, and v., 1891.
Jordan and Richards: Rep. State Board of Health, Mass., ‘‘ Purification
of Sewage and Water,” 1890, vol. ii. p. 864.
Frankland, G. C. and P. F.: Proc. Royal Soc. London, 1890, xvii.
434 BACTERIOLOGY.
THE BACILLUS OF TETANUS.
In 1884 Nicolaier produced tetanus in mice and rab-
bits by the subcutaneous inoculation of particles of
garden earth, and demonstrated that the pus produced
at the point of inoculation was capable of reproducing
the disease in other mice and rabbits. He did not suc-
ceed in isolating the organism in pure culture. In 1884
Carle and Rattone, and in 1886 Rosenbach, demon-
strated the infectious nature of tetanus as it occurs in
man by producing the disease in animals through the
inoculation of them with the secretions from the wounds
of individuals affected with the disease. In 1889 Kita-
sato obtained the bacillus of tetanus in pure culture,
and described his method of obtaining it and its bio-
logical peculiarities as follows :
Method of obtaining it. Inoculate several mice sub-
cutaneously with the secretions from the wound of a case
of typical tetanus. This material usually contains not
only tetanus bacilli, but other organisms as well, so that
at autopsy, if tetanus results, there may be more or less
of suppuration at the seat of inoculation in the mice.
In order to separate the tetanus bacillus from the others
that are present the pus is smeared upon the surface of
the several slanted blood-serum or agar-agar tubes and
placed at 37° to 38° C. After twenty-four hours all
the organisms will have developed, and microscopic
examination will usually reveal the presence of a few
tetanus bacilli, recognizable by their shape, viz., that of
a small pin, with a spore representing the head. After
forty-eight hours at 38° C. the culture is subjected to a
temperature of 80° C. in a water-bath for from three-
THE BACILLUS OF TETANUS. 435
quarters to one hour. At the end of this time series of
plates or Esmarch tubes in slightly alkaline gelatin are
made with very small amounts of the culture and kept
in an atmosphere of hydrogen (see pages 194-199).
They are then kept at from 18° to 20° C., and at the
end of about one week the tetanus bacillus begins to
appear in the form of colonies. After about ten days
the colonies should not only be examined microscopic-
ally, but each colony that has developed in the hydro-
gen atmosphere should be obtained in pure culture and
again grown under the same conditions. The colonies
that grow only without oxygen, and which are com-
posed of the pin-shaped organisms, must be tested upon
mice. If they represent growths of the tetanus bacillus,
the typical clinical manifestations of the disease will be
produced in these animals.
In obtaining the organism from the soil much diffi-
culty is experienced. There are a number of spore-
bearing organisms here that are facultative in their
relation to oxygen, and are, therefore, very difficult to
eliminate; and there is, moreover, one in particular
that, like the tetanus bacillus, forms a polar spore.
This spore is, however, less round and much more oval
than that of the tetanus bacillus, and gives to the
organism containing it more the shape of a javelin (or
clostridium, properly speaking) than that of a pin, the
characteristic shape of the spore-bearing tetanus organ-
ism. It is non-pathogenic, and grows both with and
without oxygen, and should, consequently, not be mis-
taken for the latter bacillus. It must also be borne in
mind that there are occasionally present in the soil still
other bacilli which form polar spores, and which, when
in this stage, are almost identical in appearance with
436 BACTERIOLOGY.
the tetanus bacillus; but they will usually be found to
differ from it in their relation to oxygen, and they are
also without disease-producing properties.
Morphology. It is a slender rod with rounded ends.
It may appear as single rods, or, in cultures, as long
threads. It is motile, though not actively so. The
motility is rendered somewhat more conspicuous by
examining the organism upon a warm stage.
Fic, 92.
Tetanus bacillus. a. Vegetative stage, from gelatin culture. B. Spore-
stage, showing pin-shapes.
At the temperature of the body it rapidly forms
spores. These are round, thicker than the cell, and
usually occupy one of its poles, giving to the rod the
appearance of a small pin. (Fig. 92.) When in the
spore-stage it is not motile.
It is stained by the‘ordinary aniline staining-reagents.
It remains colored under the employment of Gram’s
method.
Cultural peculiarities. It is an exquisite anaérobe,
and cannot be brought to development under the access
of oxygen. It grows well in an atmosphere of pure
hydrogen, but does not grow under the influence of
carbonic acid.
THE BACILLUS OF TETANUS. 437
It grows in ordinary nutrient gelatin and agar-agar
of a slightly alkaline reaction. Gelatin is slowly lique-
fied, with the coincident production of a small amount
of gas. Neither agar-agar nor
blood-serum is liquefied by its
growth.
The addition to the media of
from 1.5 to 2 per cent. of glucose,
0.1 per cent. of indigo-sodium
sulphate, or 5 per cent. by volume
of blue litmus tincture favors its
growth.
It grows well in alkaline bouil-
lon under an atmosphere of hy-
drogen.
It may be cultivated through
numerous generations under arti-
ficial conditions without loss of
virulence,
Appearance of the colonies. The
colonies on gelatin under an at-
mosphere of hydrogen have, in
their early stages, somewhat the
appearance of the colonies of the
common bacillus subtilis, viz., they
have a dense, felt-like centre sur-
rounded by a fringe of delicate
radii. The liquefaction is so slow
that the appearance is retained for
a relatively long time, but eventu-
ally becomes altered. In very old
colonies the entire mass is made
up of a number of distinct threads
Colonies of the tetanus
bacillus four days old,made
by distributing the organ-
isms through a tube nearly
filled with glucose-gelatin.
Cultivation under an at-
mosphere of hydrogen,
(From FRANKEL and
PFEIFFER.)
438 BACTERIOLOGY.
that give to it the appearance of a common mould. (See
Fig. 93.) '
In stab-cultures. In stab-cultures made in tubes
about three-quarters filled with gelatin growth begins
at about 1.5 to 3 em. below the surface, and gradually
assumes the appearance of a cloudy, linear mass,
with prolongations radiating into the gelatin from all
sides. Liquefaction with coincident gas production
results, and may reach almost to the surface of the
gelatin.
Relation to temperature and to chemical agents. It
grows best under a temperature of from 36° to 38° C.;
gelatin cultures kept at from 20° to 25° C. begin to
grow after three or four days. In an atmosphere of
hydrogen at from 18° to 20° C. growth does not usu-
ally occur before one week. No growth occurs under
14° C. At the temperature of the body spores are
formed in cultures in about thirty hours, whereas in
gelatin cultures at from 20° to 25° C. they do not usu-
ally appear before a week, when the lower part of the
gelatin is quite fluid.
Spores of the tetanus bacillus when dried upon bits
of thread over sulphuric acid in the desiccator and sub-
sequently kept exposed to the air, retain their vitality
and virulence for a number of months. Their vitality
is not destroyed by an exposure of one hour to 80° C.;
on the other hand, an exposure of five minutes to
100° C, in the steam sterilizer kills them. They resist
the action of 5 per cent. carbolic acid for ten hours, but
succumb when exposed to it for fifteen hours. In the
same solution, plus 0.5 per cent. hydrochloric acid,
they are no longer active after two hours. They are
killed when acted upon for three hours by corrosive
THE BACILLUS OF TETANUS. 439
sublimate, 1: 1000, and in thirty minutes by the same
‘solution plus 0.5 per cent. hydrochloric acid.
Action upon animals. After subcutaneous inocula-
tion of mice with minute portions of a pure culture of
this organism tetanus develops in twenty-four hours
and ends fatally in from two to three days. Rats,
guinea-pigs, and rabbits are similarly affected, but only
by larger doses than are required for mice: the fatal
dose for a rabbit being from 0.3 to 0.5 e.c. of a well-
developed bouillon culture. The period of inoculation
for rats and guinea-pigs is twenty-four to thirty hours,
and for rabbits from two to three days. Pigeons are
but slightly, if at all, susceptible.
The tetanic convulsions always appear first in the
parts nearest the seat of inoculation, and subsequently
become general.
At autopsies upon animals that have succumbed to
inoculations with pure cultures' of the tetanus bacillus
there is little to be seen by either macroscopic or micro-
scopic examination, and cultures from the seat of inocu-
lation are usually negative in so far as finding the teta-
ous bacillus is concerned. At the seat of inoculation
there is usually only a hyperemic condition. In un-
complicated cases there is no suppuration. The internal
organs do not present any change, and culture-methods
of examination show them to be free from bacteria.
The death of the animal results from the absorption of
a soluble poison, either produced by the bacteria at the
seat of inoculation or, which seems more probable, pro-
1 Animals and human beings that have become infected with this organism
in the natural way commonly present a condition of suppuration at the site
of infection ; this is probably not due, however, to the tetanus bacillus, but
to other bacteria that have also gained access to the wound at the time of
infection.
440 BACTERIOLOGY.
duced by the bacteria in the cultwre from which they are
obtained and introduced with them into the tissues of |
the animal at the time of the inoculation. In support
of the latter hypothesis: mice have been inoculated with
pure cultures of this organism; after one hour the point
at which the inoculation was made was excised and the
tissues cauterized with the hot iron; notwithstanding
the short time during which the organisms were in
contact with the tissues and the subsequent radical
treatment, the animals died after the usual interval
and with the regular symptoms of tetanus.
The poison produced by the tetanus bacillus, and
to which the symptoms of the disease are due, has
been isolated and subjected to detailed study; some
of its peculiarities, as given by Kitasato, are as
follows :'
‘© When cultures of this organism are robbed of their
bacteria by filtration through porcelain the filtrate con-
tains the soluble poison, and is capable, when injected
into animals, of causing tetanus.
‘‘Tnoculations of other animals with bits of the
organs of the animal dead from the action of the teta-
nus poison produce no result; but similar inoculations
with the blood or with the serous exudate from the
pleural cavity always result in the appearance of teta-
nus. The poison is, therefore, largely present in the
circulating fluids.
“« The greatest amount of poison is produced by culti-
vation in fresh neutral bouillon of a very slightly alka-
line reaction.
‘The activity of the poison is destroyed by an ex-
1 Zeitschr. fiir Hygiene, 1891, Bd. x. p. 267.
THE BACILLUS OF MALIGNANT @DEMA. 44]
posure of one and one-half hours to 55° C.; of twenty
minutes to 60° C.; and of five minutes to 65° C.
‘« By drying at the temperature of the body under
access of air the poison is destroyed ; but by drying at the
ordinary temperature of the room, or at this temperature
in the desiccator over sulphuric acid, it is not destroyed.
‘Diffuse daylight diminishes the intensity of the
poison. Its intensity is preserved for a much longer
time when kept in the dark.
‘Direct sunlight robs it of its poisonous properties
in from fifteen to eighteen hours.
‘* Its activity is not diminished by diluting a fixed
amount with water or nutrient bouillon.
‘“ Mineral acids and strong alkalies lessen its inten-
sity.’”
The chemical nature of this poison is not positively
known, but according to the recent observations of
Brieger and Cohn it is not to be classed with the albu-
mins in the sense in which the word is commonly used.
When obtained in a pure, concentrated form its toxic
properties are seen to be altered by acids, by alkalies,
by sulphuretted hydrogen, and by temperatures above
70° C. Even when carefully protected from light,
moisture, and air it gradually becomes diminished in
strength. When feshly prepared by the methods of
the authors just cited its potency is almost incredible,
0.000,05 milligramme being sufficient to cause fatal
tetanus in a mouse weighing fifteen grammes.
THE BACILLUS OF MALIGNANT GDEMA.
The bacillus of malignant cedema, also known as
vibrion septique, is another pathogenic form almost
449 BACTERIOLOGY.
everywhere present in the soil. In certain respects it
is a little like the bacillus of anthrax, and was at one
time confounded with it; but it differs in the marked
peculiarity of being a strict anaérobe. It was first
observed by Pasteur, but it was not until later that
Koch, Liborius, Kitt, and others described its peculi-
arities in detail. It can usually be observed by insert-
ing under the skin of rabbits or guinea-pigs small por-
tions of garden earth, street dust, or decomposing
organic substances. There results a widespread oedema,
with more or less of gas production in the tissues. In
the cedematous fluid about the seat of inoculation the
organism under consideration may be detected. (Fig.
94, A.).
Fig. 94.
Bacillus of malignant cedema.
A. Bacilli in short and long threads in cedematous fluid from site of inocu-
lation of guinea-pig. (After Kocu.)
B. Spore-stage of the organism; from culture.
It is a rod of about 3 to 3.5 yu long and from 1 to
1.1 y» thick—i.e., it is about as long as the bacillus
anthracis, but is a trifle more slender. It is usually
found in pairs, joined end to end, but may occur as
longer threads; particularly is this the case in cultures,
THE BACILLUS OF MALIGNANT G@DEMA. 443
When in pairs the ends that approximate are squarely
cut, while the distal extremities are rounded. When
occurring singly both ends are round-
ed. (How does it differ in this respect
from bacillus anthracis?) It is slowly
motile, and its flagella are located
both at the ends and along the sides
of the rod. It forms spores that are
usually located in or near the middle
of the body of the cell. These may
cause a swelling of the cell at the point
at which they are located and give to
it a more or less oval, spindle, or lozenge
shape. (Fig. 94, B.)
It is a strict anaérobe, growing on
all the ordinary media, but not under
the access of oxygen. It grows well
ina hydrogen atmosphere. It causes
liquefaction of gelatin.
In tubes containing about 20 to 30
c.c. of gelatin that has been liquefied,
inoculated with a small amount of the
culture, and then rapidly solidified in
ice-water, growth appears in the form
of isolated colonies at or near the bot-
tom of the tube in from two to three
days at 20° C. These colonies, when
of from 0.5 to 1 mm. in diameter, ap-
pear as little spheres filled with clear
liquid, and are difficult, for this reason,
to detect. (Fig. 95.)
Fie. 95.
Colonies of the ba-
cillus of malignant
cedema in deep gela-
tin culture. (After
FRANKEL and PFEIF-
FER.)
As they gradually increase in size the contents of the
spheres become cloudy and are marked by fine radiating
444 BACTERIOLOGY.
stripes, easily to be detected with the aid of a small
hand-lens. In deep stab-cultures in agar-agar and gel-
atin development occurs only along the track of punc-
ture at a distance below the surface. Growth is fre-
quently accompanied by the production of gas-bubbles.
It causes rapid liquefaction of blood-serum with
production of gas-bubbles, and in two or three days
the entire medium may have become converted into a
yellowish, semi-fluid mass.
The most satisfactory results in the study of the col-
onies are obtained by the use of plates of nutrient agar-
agar kept in a chamber in which all oxygen has been
replaced by hydrogen. The colonies appear as dull
whitish points, irregular in outline, and when viewed
with a low-power lens are seen to be marked by a net-
work of branching and interlacing lines that radiate in
an irregular way from the centre toward the periphery.
It grows well at the ordinary temperature of the
room, but reaches its highest development at the tem-
perature of the body.
It stains readily with the ordinary aniline dyes. It
is decolorized when treated by Gram’s method.
Pathogenesis. The animals that are known to be
susceptible to inoculation with this organism are man,
horses, calves, dogs, goats, sheep, pigs, chickens, pig-
eons, rabbits, guinea-pigs, and mice. Cases are recorded
in which men and horses have developed the disease
after injuries, doubtless due to the introduction into the
wound, at the time, of soil or dust containing the or-
ganism.
If one introduce into a pocket beneath the skin of a
susceptible animal about as much garden earth as can
be held upon the point of a penknife, the animal fre-
THE BACILLUS OF MALIGNANT GIDEMA. 445
quently dies in from twenty-four to forty-eight hours.
The most conspicuous result found at autopsy is a wide-
spread cedema at and about the seat of inoculation.
The cedematous fluid is at places clear, while again it
may be marked with blood; it is usually rich in bacilli
(Fig. 94, 4) and contains gas-bubbles. Of the internal
organs only the spleen shows much change. It is large,
dark in color, and contains numerous bacilli. If the
autopsy be made immediately after death, bacilli are not
commonly found in the blood of the heart; but if de-
ferred for several hours, the organisms will be found in
this locality also, a fact that speaks for their multiplica-
tion in the body after death. At the moment of death
they are present in all the internal viscera and on the
serous surfaces of the organs.
Of all animals mice are probably the most suscepti-
ble to the action of this organism, and it is not rare to
find the organisms in the heart’s blood, even immedi-
ately after death. They die, as a result of these inocu-
lations, in from sixteen to twenty hours.
Where pure cultures are used for inoculation a rela-
tively large amount must be employed, and it should be
introduced into a deep pocket in the subcutaneous tissues
some distance from the surface. In continuing the in-
oculations from animal to animal small portions of
organs or a few drops of the cedema-fluid should be
used. The inoculation may also be successfully made
by introducing into a pocket in the skin bits of steril-
ized thread or paper upon which cultures have been
dried.
The methods for obtaining the organism in pure cul-
ture, from the cadaver of an animal dead from inocula-
tion, are in all essential respects the same as those given
20
446 BACTERIOLOGY.
for obtaining cultures from tissues in general, but it
must be remembered that the organism is a strict anaé-
robe, and will not grow under the influence of oxygen
(see methods of cultivating anaérobic species).
In certain superficial respects this bacillus suggests
bacillus anthracis, but differs from it in so many impor-
tant details that there is no excuse for confounding the
two.
Norr.—From what has been said of this organism,
what are the most important differential points between
it and bacillus anthracis? Inoculate several mice with
small portions of garden earth and street dust. Isolate
the organism that agrees most nearly with the descrip-
tion here given for the bacillus of malignant cedema.
Compare its morphological, biological, and pathogenic
peculiarities with those of bacillus anthracis under sim-
ilar circumstances,
Still another pathogenic organism that may be present
in the soil is
THE BACILLUS OF SYMPTOMATIC ANTHRAN;
bactérie du charbon symptomatique (French); Bacillus
des Rauschbrand (German). It is the organism con-
cerned in the production of the disease of young cattle
and sheep commonly known as ‘‘ black leg,’’ ‘‘quarter
evil,”’ and ‘‘quarter ill,’’ a disease that prevails in cer-
tain localities during the warm months, and which is
characterized by a peculiar emphysematous swelling of
the muscular and subcutaneous cellular tissues over the
quarters, The muscles and cellular tissues at the points
THE BACILLUS OF SYMPTOMATIC ANTHRAX. 447
affected are seen on section to be saturated with bloody
serum, and the muscles, particularly, are of a dark,
almost black color. In these areas, in the bloody trans-
udates of the serous cavities, in the bile, and, after
death, in the internal organs, the organism to be de-
scribed can always be detected. It is manifest from
this that the soil of localities over which infected herds
are grazing may readily become contaminated through
a variety of channels, and thus serve as a source of
further dissemination of the disease.
The organism was first observed by Feser, and subse-
quently by Bollinger and others. The most complete
description of its morphological and biological peculi-
arities is that of Kitasato (Zeitschr. fiir Hygiene, Bd. vi.
p. 105; Bd. viii. p. 55). The following is from Kita-
sato’s contributions: it is an actively motile rod of
“ as wht Q
id ~S vo
Z,l b 4 (
LAl %~ i)
Bacillus of symptomatic anthrax. (After KirasaTo.)
a. Vegetating forms from a gelatin culture. 1. Spore-forms from an agar-
agar culture.
about 3 to 5 long by 0.5 to 0.6 y thick. It is
rounded at its ends, and, as a rule, is seen singly,
though now and then pairs joined end to end may occur.
It has no tendency to form very long threads. (Fig.
96, A.)
448
BACTERIOLOGY.
Tt forms spores, and when in this stage is seen to be
slightly swollen at or near one of its poles, the location in
Fic. 97.
Colonies of the
bacillus of symp-
tomatic anthrax,
in deep. gelatin
culture. (After
FRANKEL and
PFEIFFER.)
which the spore usually appears. (Tig.
96, B.) It is conspicuously prone to un-
dergo degenerative changes, and involu-
tion-forms are comnionly seen, not only
in fresh cultures, but in the tissues of
affected animals as well.
Though actively motile when in the
vegetative stage, it loses this property and
becomes motionless when spores are form-
ing.
It is strictly anaérobic and cannot be
cultivated in an atmosphere in which oxy-
gen is present. It grows best under hy-
drogen, and does not grow under carbonic
acid.
The media most favorable to its growth
are those containing glucose (1.5 to 2 per
cent.), glycerin (4 to 5 per cent.), or some
other reducing body, such as indigo-so-
dium sulphate, sodium formate, etc.
When cultivated upon gelatin plates
in an atmosphere of hydrogen the col-
onies appear as irregular, slightly lobu-
lated masses. After a short time lique-
faction of the gelatin occurs and the
colony presents a dark, dense, lobulated
and broken centre, surrounded by a
much more delicate, fringe-like zone.
When distributed through a deep layer of liquefied
gelatin that is subsequently caused to solidify colonies
develop at only the lower portions of the tube. The
THE BACILLUS OF SYMPTOMATIC ANTHRAX. 449
single colonies appear as discrete globules that cause
rapid liquefaction of the gelatin, and ultimately coalesce
into irregular, lobulated, liquid areas. In some of the
larger colonies an ill-defined, concentric arrangement of
alternate clear and cloudy zones can be made out.
(Fig. 97.)
In deep stab-cultures in gelatin growth begins after
about two or three days at 20° to 25°C. It begins
usually at about one or two centimetres below the sur-
face, and causes slow liquefaction at and around the
track of its development. During the course of its
growth gas-bubbles are produced.
In deep stab-cultures in agar-agar at 37° to 38° C.
growth begins in from twenty-four to forty-eight hours,
also at about one or two centimetres below the surface,
and is accompanied by the production of gas-bubbles.
There is produced at the same time a peculiar, penetrat-
ing odor somewhat suggestive of that of rancid butter.
Under these conditions spores are formed after about
thirty hours.
It grows well in bouillon of very slightly acid reac-
tion under hydrogen, but does not retain its virulence
for so long a time as when cultivated upon solid media.
In this medium it develops in the form of white flocculi
that sink ultimately to the bottom of the glass and leave
the supernatant fluid quite clear. If the vessel be now
gently shaken, these delicate flakes are distributed homo-
geneously through it. In bouillon cultures there is
often seen a delicate ring of gas-bubbles around the
point of contact of the tube and the surface of the
bouillon. There is produced also a peculiar, penetrat-
ing, sour or rancid odor.
It grows best at the body temperature—i.e., from 37°
450 BACTERIOLOGY.
to 38° C., but can also be brought to development at
from 16° to 18°C. Under 14° C. no growth is seen.
Spore-formation appears much sooner at the higher than
at the lower temperatures. When its spores are dried
upon bits of thread in the desiccator over sulphuric acid,
and then kept under ordinary conditions, they retain
their vitality and virulence for many months. Sim-
ilarly, bits of flesh from the affected areas of animals
dead of this disease, when completely dried, are seen to
retain the power of reproducing the disease for a long
time. The spores are tolerably resistant to the influence
of heat: when subjected to a temperature of 80° C. for
one hour their virulence is not affected, but an expo-
sure to 100° C. for five minutes completely destroys
them. They are also seen to be somewhat resistant to
the action of chemicals: when exposed to 5 per cent.
carbolic acid they retain their disease-producing prop-
erties for about ten hours, whereas the vegetative forms
are destroyed in from three to five minutes; in corro-
sive sublimate solution of the strength of 1: 1000 the
spores are killed in two hours.
When gelatin cultures are examined microscopically
the organisms are usually seen as single rods with
rounded ends. When cultivated in agar-agar at a
higher temperature spores are formed after a short
time; the spores are oval, slightly flattened on their
sides, thicker than the bacilli, and, as stated, frequently
occupy a position inclining to one of the poles of the
bacillus, though they are as often seen in the middle.
The bacillus containing a spore has usually a clubbed
or spindle shape.
It stains readily with the ordinary aniline dyes. It
is decolorized by Gram’s method. Its spores may be
THE BACILLUS OF SYMPTOMATIC ANTHRAX. 451
stained by the methods usually employed in spore-
staining.
Pathogenesis. When susceptible animals, especially
guinea-pigs, are inoculated in the deeper subcutaneous
cellular tissues with pure cultures of this organism, or
with bits of tissue from the affected area of another
animal dead of the disease, death ensues in from-one to
two days. It is preceded by rise of temperature, loss
of appetite, and general indisposition. The seat of
inoculation is swollen and painful, and drops of bloody
serum may sometimes be seen exuding from it. At
autopsy the subcutaneous cellular tissues and under-
lying muscles present a condition of emphysema and
extreme cedema. The cedematous fluid is often blood-
stained and the muscles are of a blackish or blackish-
brown color. The lymphatic glands are markedly
hyperemic. The internal viscera present but little
alteration visible to the naked eye. In the blood-
stained serous fluid about the point of inoculation short
bacilli are present in large numbers. These often pre-
sent slight swellings at the middle or near the end.
They are not seen as threads, but lie singly in the
tissues. Occasionally two will be seen joined end to
end. If the autopsy be made immediately after death,
these organisms may not be detected in the internal
organs; but if not made until after a few hours, they
will be found there also. In fresh autopsies only veg-
etative forms of the organism may be found, but later
(in from twenty to twenty-four hours) spore-bearing rods
may be detected. (How does this compare with bacillus
anthracis ?) By successive inoculations of susceptible
animals with the serous fluid from the seat of inoculation
of the dead animal the disease may be reproduced.
452 BACTERIOLOGY.
Cattle, sheep, goats, guinea-pigs, and mice are sus-
ceptible to infection with this organism, and present the
conditions above described; whereas horses, asses, and
white rats present only local swelling at the site of inoc-
ulation. Swine, dogs, cats, rabbits, ducks, chickens, and
pigeons are, as a rule, naturally immune from the disease.
Though closely simulating the bacillus of malignant
cedema in many of its peculiarities, this organism can,
nevertheless, be readily distinguished from it. It is
smaller; it does not develop into long threads in the
tissues; it is more actively motile, and forms spores
more readily in the tissues of the animal than does the
bacillus of malignant edema. In their relation to ani-
mals they also differ, viz., cattle, while conspicuously
susceptible to symptomatic anthrax, are practically im-
mune from malignant cedema; and while swine, dogs,
rabbits, chickens, and pigeons are readily infected with
malignant cedema, they are not, as a rule, susceptible to
symptomatic anthrax. Horses are affected only locally,
and not seriously, by the bacillus of symptomatic an-
thrax; but they are conspicuously susceptible to both
artificial inoculation and natural infection by the bacil-
lus of malignant cedema.
The distribution of the two organisms over the earth’s
surface is also quite different. The cedema bacillus is
present in almost all soils, while the bacillus of symp-
tomatic anthrax appears to be confined to certain local-
ities, especially places over which infected herds have
been pastured.
A single attack of symptomatic anthrax, if not fatal,
affords subsequent protection, while infection with the
malignant cedema bacillus appears to predispose to re-
currence of the disease. (Baumgarten. )
CHAPTER XXVI.
Infection and immunity—The types of infection; intimate nature of in-
fection—Septicemia, toxemia, variations in infectious processes—Immunity,
natural and acquired—The hypotheses that have been advanced in explana-
tion of immunity—Conclusions.
AN organism capable of producing disease we call
pathogenic or infective, and the process by which it pro-
duces disease we know as infection. Diseases, therefore,
that depend for their existence upon the presence of
bacteria in the tissues are infectious diseases.
What is the intimate nature of this process we call
infection? Is it due to the mechanical presence of
living bacteria in the body, or does it result from the
deposition in the tissues of substances produced by these
bacteria that are either locally or generally incompat-
ible with life? Or, is the group of pathological altera-
tions and constitutional symptoms seen in these diseases
the result of abstraction from the tissues, by the bacteria
growing in them, of substances essential to their vitality ?
These are some of the more important of the questions
that present themselves in the course of analysis of this
interesting phenomenon.
Let us look into several typical infectious diseases,
note what we find, and see how far the observations
thus made will aid us in formulating an opinion. We
begin with a study of those diseases in which there is
a general infection—i.e., in which there is a general dis-
tribution of the infective agents throughout the body.
This group comprises the “‘ septiceemias,’’ and of them
20*
454 BACTERIOLOGY.
the disease of animals known as anthrax represents a
type of the condition. If the cadaver of an animal
dead of anthrax be examined by bacteriological methods,
it will be discovered that there is present in all the
organs and tissues an organism, a bacillus, of definite
form and biological characteristics; and if the organs,
and tissues generally, be subjected to microscopic exam-
ination this same organism will be found always present
and always located within the capillaries. At many
points it will be seen crowded in the capillaries in such
numbers as almost, if not quite, to burst them, and very
commonly their lumen for a considerable extent is en-
tirely occluded by the growing bacilli. In such a case
as this we might be tempted to conclude that death had
resulted from mechanical interference with the capillary
circulation. Suppose, however, we subject the cultures
obtained from this animal to conditions, either chemical
or thermal, that are not particularly favorable to their
normal development, and from time to time inoculate
susceptible animals with the cultures so treated. The
result will be that, as we continue to expose our cultures
to unfavorable surroundings, the period of time that is
required for them to cause the death of animals will,
in some cases, gradually become extended, until finally
death will not ensue at all after inoculation. If, as
these animals die, a careful record of the conditions
found at autopsy be kept and compared, it will ulti-
mately be noticed that the animals that die a longer
time after inoculation present conditions more or less
at variance with those seen in the original animal that
died more quickly after having been inoculated. These
differences usually consist in a diminution of the num-
ber of bacilli that appear upon culture plates from the
INFECTION AND IMMUNITY. 455
blood and internal organs, and in a lessening in the
amount of mechanical obstruction offered to the circu-
lation through plugging of the capillaries by masses of
bacilli, as detected by microscopic examination of sec-
tions of the organs; indeed, this latter condition may
often have almost, if not quite, disappeared. We see
here an animal dead from the invasion of the same
organism that produced death in the first animal, but
with little or none of the appearances to which we were
inclined to attribute the death of that animal. It is
apparent, then, that this organism with which we have
been working can destroy the vitality of an animal in
a way other than by mechanically obstructing its blood-
vessels; it possesses some other means of destroying
life. Possibly its growth in the tissues is accompanied
by the production of soluble poisons, which when pres-
ent in the blood are not compatible with life.
Let us see if the study of another group of infections
will furnish any evidence in support of such an hypoth-
esis. Introduce into the subcutaneous tissues of a
guinea-pig a small amount of pure culture of the bacil-
lus of diphtheria. In three or four days the animal
dies. We proceed with our autopsy in exactly the same
way that we did with the animals dead of anthrax, and
will be astonished to find that the organs, blood, and
tissues generally are sterile,‘ in so far as the presence of
the organism with which the animal was inoculated is
concerned, and by both culture and microscopic methods
it is possible to detect them only at the site of inocula-
tion, where they were deposited. It is very evident
that we have here a condition with which mechanical
1 In by far the greater number of cases this is true, but under particular
circumstances there are exceptions.
456 BACTERIOLOGY.
plugging of the capillaries could have had nothing to
do, for there are no organisms in the blood to interfere
with its circulation. Our hypothesis then with regard
to the condition found in our first case of anthrax is
again not tenable. Similarly, if an animal that has
died of tetanus be examined, we do not find the bacilli
in the tissues and circulating fluids generally, and, in-
deed, often fail to find them at the point of injury.
Plainly, these fatal results following upon inoculations
with the diphtheria and the tetanus bacillus, with their
accompanying tissue-changes, occur from the presence
of a something that cannot be detected by either cul-
tural or microscopic methods, and this something can be
only a soluble substance that is produced by the growing
bacteria at the site of inoculation, gains access to the
circulation, and through this channel causes death, for
it is hardly to be imagined that the insignificant wound
made in the course of inoculation could per se have had
this effect. In other words, these latter animals have
died from what is called toxemia (poison in the blood),
a condition conspicuously different from septicemia, as
seen in our first animal dead of anthrax.
There are, again, other infectious diseases, many of
which are known to present variations from what might
be considered a typical course, that may still further
serve to support the view that infection is a process in
which the mechanical effect of organisms in the cireu-
lating fluids is of little conseyuence. Conspicuous
among these are the infections that follow upon the
introduction into the tissues of susceptible animals of
cultures of micrococcus lanceolatus (pneumococcus), of
the bacillus of chicken cholera, and of the organisms
concerned in the production of the so-called ‘‘ hemor-
INFECTION AND IMMUNITY. 457
rhagic septicemias.’’ When running their normal
course these organisms cause typical septiceemias in sus-
ceptible animals ; but often, from causes not entirely
clear, the animals die with only local lesions, or with
but very few organisms in the internal viscera. We
see here conditions analogous to those observed in
the two experiments with anthrax, viz., we find a
group of diseases that are properly classed as septi-
cemias, because of the usual general invasion of the
body by the organisms concerned in their production,
but which frequently assume a purely local character
—in both instances proving fatal to the animal in-
fected. From what we have seen it is manifestly
probable that, whether these diseases be designated as
septicemias or toxemias, death is produced in all in-
stances by the poisonous products resulting from the
growth of the infecting bacteria. In the case of typical
anthrax, and other varieties of septicemia, the produc-
tion of this poison is associated with the general dis-
semination of the organisms throughout the body, while
in those infections often referred to as toxeemias, of
which diphtheria may be taken as a type, the poison is
produced by the organisms that remain localized at the
site of invasion, and is thence disseminated throughout
the body by the circulating fluids.
Infection thus far, then, appears to be a chemical
process.
Through special investigations that have been made
upon the products of growth of certain pathogenic bac-
teria this opinion has received further confirmation;
it has been found possible by the use of appropriate
methods to isolate, from among the mass of material in
which certain of these organisms have been artificially
458 BACTERIOLOGY.
cultivated, substances which, when separated from the
bacteria by which they were produced, possess the power
of causing in animals all the constitutional symptoms
and pathological tissue-changes that are seen to occur
in the course of infection by the organisms themselves.
In some instances these poisons—toxins,'as they are
collectively called—appear to be the direct result of
metabolic changes brought about by bacteria in the
medium or tissues in which they may be developing —
i.e., they are products of nutrition that pass readily into
solution, as is conspicuously seen in the case of the
bacillus of diphtheria and of tetanus when under both
artificial cultivation and in the animal body. Many
bacteria which do not possess the power of generating
or secreting such poisons may, nevertheless, have inti-
mately associated with their protoplasmic bodies poison-
ous substances that can only be isolated by particular
methods; thus the toxins of bacillus tuberculosis and
of spirillum cholerce Asiatice are much more conspicu-
ously present in the protoplasm of these bacteria than
in the fluids in which they have grown, and Buchner
has isolated from several species of bacteria ‘‘ bacterio-
proteins’’ having the common properties of solubility in
alkalies, resistance to the boiling temperature, attraction
of leucocytes (positive chemotaxis), and pyogenic powers.
There is as yet little agreement of opinion as to the
chemical nature of toxins, but it is probable that the
group comprises different bodies of the nature of globu-
lins, nucleo-albumins, peptones, albumoses, and enzymes
or ferments.
1 “ Toxins’’ is the term commonly used to designate amorphous poisons of
a proteid nature ; while “ ptomaines”’ is the term used to signify nitrogenous
poisons that are erystallizable.
INFECTION AND IMMUNITY. 459
Toxic ptomaines are probably not conspicuously con-
cerned in producing the characteristic symptoms of
infection, as they are absent from cultures of certain
highly pathogenic bacteria.
In some instances the production of the poisonous
principles, even under artificial conditions of cultiva-
tion, is of a most astonishing nature, and poisons result
that, in the degree of their toxicity, exceed anything
hitherto known to us. For instance, the potencies of
the poisons that have been isolated from cultures of
bacillus diphtherie and of the bacillus of tetanus have
been carefully determined by experiments upon ani-
mals, and it has been found that 0.4 milligramme of
the former is capable of killing eight guinea-pigs, each
weighing 400 grammes, or two rabbits, each weighing 3
kilogrammes (Roux and Yersin'); and that 0.0001 mil-
ligramme of the latter will produce tetanus in a mouse,
with all the characteristic manifestations of the disease
(Brieger and Cohn’).*
In short, infection may be best conceived as a contest
between the invading organisms on the one side and the
resisting tissues of the animal body on the other, the
weapons of offence of the former being the poisonous
products of their growth, the toxins, and the means of
defence possessed by the latter being substances which
are,so to speak, antidotal to these poisons. To these
substances possessed by the animal body for resisting
infection the name ‘‘alexines’’ has been given by
Buchner, while the name “‘ defensive proteids’’ is sug-
1 Annales de l'Institut Pasteur, tome iii., 1889, p. 287.
2 Zeitschr. fiir Hygiene u. Infektionskrankheiten, 1893, Bd. xv. Heft i.
2 Through the use of more recently devised methods we are enabled to in-
crease still further the toxicity of these poisons; especially is this the case
with regard to the diphtheria toxin.
460 BACTERIOLOGY.
gested by Hankin. If the tisgue-elements are not of
sufficient vigor to neutralize the bacterial poisons, the
bacteria are victorious and infection results; while, if
there be failure to establish a condition of disease, the
tissues are victorious, and are said to be resistant or to
possess immunity from this particular form of infection.
It is a common observation that certain human beings
and animals are more susceptible to the different forms
of infection than are others, and that some species of
animals are apparently not at all liable to particular
diseases; in other words, they are naturally immune from
the maladies. The term ‘‘natural immunity,’’ as now
employed, implies a congenital condition of the individ-
ual or species, a condition peculiar to his idioplasm,
which has been transmitted to him as a tissue-char-
acteristic through generations of progenitors.
Again, it is often observed that an individual or ani-
mal after having recovered from certain forms of infec-
tion has thereby acquired protection from subsequent
attacks of like character; in other words, they are said to
have acquired immunity from this trouble. ‘Acquired
immunity ’’ implies, therefore, a condition of the tissues
of an individual, not of necessity peculiar to other mem-
bers of the race or species, that has resulted during his
life from the stimulation of his integral cells by one or
another of the specific, infective irritants that may have
been purposely introduced, or accidentally gained access
to his body.
The problem involving the explanation of these inter-
esting observations has afforded material for reflection
and hypothesis for a long time. It is only through
investigations that have been conducted during the past
few years that it has met with anything approaching
INFECTION AND IMMUNITY. 461
reasonable solution, and even now there remain a num-
ber of important points that are more or less veiled in
obscurity.
Conspicuous among the observers who have endeay-
ored to explain the modus operandi of immunity may
be mentioned Chauveau, Pasteur, Metclinikoff, Buch-
ner, Fligge and his pupils (Smirnow, Sirotinin, Bitter,
Nuttall), Fodor, Hankin, and Pfeiffer. In the follow-
ing pages we will present briefly the result of investi-
gations by these various authors.
In 1880 Chauveau' suggested an explanation for the
phenomenon of immunity that has since been known as
the “‘ retention hypothesis.’’? It is, in short, as follows:
that the immunity commonly seen to exist in animals
that have passed through an attack of infection from
a subsequent outbreak of the same malady, and likewise
the immunity that has been produced artificially by
vaccination, exist by virtue of some bacterial product
that has been retained or deposited in the tissues of those
animals, and that this product by its presence prevents
the development of the same organisms if they should
subsequently gain access to the body.
Bearing upon this view the experiments of Sirotinin,”
made with cultures of various pathogenic bacteria,
demonstrated that, in so far as culture-experiments were
concerned, the only substance produced by growing
bacteria that could be in any way inimical to their further
development were substances that gave rise to altera-
tions in the reaction of the medium in which they were
developing—i.e., acids or alkalies produced by the bac-
teria themselves. So long as the organisms were not
Comptes-rendus, etc., July, 1880, No. 91.
2 Zeitsch. fiir Hygiene, 1888, Bd. iv.
462 BACTERIOLOGY.
actually dead from exposure to these substances correc-
tion of the abnormal reaction was followed by further
development of the organisms. Sirotinin also states
that materials containing the products of growths of
bacteria, so long as they are maintained at a neutral or
only slightly alkaline reaction, serve very well as media
upon which to cultivate again the same organism that
produced them, providing the nutritive elements have
not been entirely exhausted. He remarks that, if in such
a concentrated form as we find the life-products of bac-
teria in the medium in which they are growing, no
inhibitory compounds beyond acids and alkalies are to
be detected, it is hardly probable that they are produced
in the tissues of the living animal, and retained there,
to a degree sufficient to prevent the growth of bacteria
that may subsequently gain entrance to these tissues,
after the disappearance of the organisms concerned in
the primary invasion. On the other hand, Salmon and
Smith,’ Roux and Chamberland,? and others had
demonstrated that a sort of immunity against certain
forms of infection may be afforded to susceptible ani-
mals by the injection into their tissues of the products
of growth of particular organisms which, if themselves
introduced into the animal body, would produce fatal
results. In the light of subsequent experiments, how-
ever, the interpretation of this phenomenon is probably
not that claimed by the supporters of this hypothesis.
As opposed to the view of Chauveau, Pasteur® and
certain of his pupils believed that the immunity fre-
quently afforded to the tissues by an attack of infection,
1 Proc, of the Biol. Soc., Washington, D. C., 1886, vol. iii.
2 Annales de l'Institut Pasteur, 1888-’89, tomes i., ii.
3 Bull. de l’Acad. de Méd., 1880.
INFECTION AND IMMUNITY. 463
or following upon vaccination against infection, was
due rather to an abstraction from the tissues, by the
organisms that were concerned in the primary attack,
of a something that is necessary to the growth of the
infecting organism should it gain entrance to the body
at any subsequent time. This view is known as the
“exhaustion hypothesis.’’
As to the exhaustion hypothesis of Pasteur, there is,
as yet, no evidence whatever for its support. The work
of Bitter,' which was undertaken with the view of de-
termining if, in the process of acquiring immunity, there
occurred this exhaustion from the tissues of material
necessary to the growth of bacteria that might gain
entrance to them at some later date, gave only negative
results. The flesh of animals in which immunity had
been produced contained all the elements necessary for
the growth and nutrition of the bacteria against which
the animals had been protected, just as did the flesh of
non-vaccinated animals.
In 1884 Metchnikoff? published the first of a series
of observations upon the relation that is seen to exist
between certain of the mesodermal cells of lower ani-
mals and insoluble particles that may be present in the
tissues of these animals. The outcome of these inves-
tigations was the establishment of his well-known doc-
trine of phagocytosis, the principle of which is that the
wandering cells of the animal organism, the leucocytes,
possess the property of taking up, rendering inert, and
digesting micro-organisms with which they may come
in contact in the tissues. Metchnikoff believed that
1 Zeitschr. fiir Hygiene, 1888, Bd. iv.
2 Arbeiten aus dem Zodlogischen Institut der Universitat Wien., 1884, Bd. v.
Fortschritte der Med., 1884, Bd. ii.
464 BACTERIOLOGY.
in this way immunity from infection may in many, if
not all, cases be explained. He believed that suscepti-
bility to or immunity from infection was essentially a
matter between the invading bacteria on the one hand,
and the leucocytes of the tissues on the other. The
success or failure of the leucocytes in protecting the
animal against infection depends, according to this doc-
trine, entirely upon the efficiency of the means possessed
by them for destroying bacteria. When these means
are of sufficient vigor to bring about the death of the
bacteria the tissues are victorious, but when the poisons
generated by the bacteria are potent to arrest the pha-
gocytic action of the leucocytes then the tissues succumb
and infection results.
Has this doctrine of phagocytosis, as advanced by
Metchnikoff, stood the test of experimental criticism?
Evidence that has accrued since the time of its sugges-
tion has rendered questionable the advisability of its
unconditional adoption in the strict sense that Metch-
nikoff propounded it. The later studies of a number
of investigators indicate that while the leucocytes play
a most important part in the phenomenon of immunity,
it is hardly likely that this always occurs through their
taking up within themselves and digesting invading
bacteria, as Metchnikoff believes, but rather that their
part in the process is to secrete protective chemical sub-
stances that are thrown into the circulating blood, and
which, in part at least, comprise the defensive bodies to
which Buchner has given the name “ alexines.’’!
The first severe blow that Metchnikoff’s theory of
phagocytosis received was given by Nuttall,’ in his
1 See Hahn. Arch. fiir Hygiene, 1895, Bd. xxv. p. 105.
2 Zeitschrift fiir Hygiene, 1888, vol. iv.
INFECTION AND IMMUNITY. AG5
work upon the bactericidal action of the animal econ-
omy. In these experiments Nuttall showed positively
that the destruction of virulent bacteria in the blood of
animals was not necessarily dependent upon the imme-
diate presence of living leucocytes, but that the serum
of the blood, when quite free from cellular elements,
possessed this power to a degree cqual to that of the
blood when all the constituent parts were present. In
the blood, as such, phagocytosis could be seen, but, as a
rule, the bacteria presented evidence of having under-
gone degenerative changes before they had been taken
up by the wandering cells.
Contrary to the notions in existence at the time,
Traube and Gscheidlen,' as far back as 1874, demon-
strated that considerable quantities of septic material
could be injected into the circulating blood without
apparently any effect upon the animal. As a result
of these experiments, the question that naturally pre-
sented itself was: Does the animal organism possess
the power of rendering septic organisms inert, and if
so, to what extent? Their further work showed that
appreciable numbers of living bacteria could be injected
into the circulation of warm-blooded animals without
producing any noticeable effect. Particularly was this
the case with dogs. If they injected into the circula-
tion of a dog as much as 1.5 cc. of decomposing
fluid, the blood drawn from the animal after from
twenty-four to forty-eight hours showed no especial
tendency to decompose, though it was kept under obser-
vation for along time. They believed this power, of
rendering living organisms inert, to be possessed by the
1 Jahresbericht der Schlesischen Ges. fiir Cultur., 1874; Jahr. iii. p. 179.
466 BACTERIOLOGY.
circulating blood to only a limited degree, for, after the
injection of much larger amounts of the putrid fluid
into the blood of the animal, death usually ensued in
from twenty-four to forty-eight hours. The blood
drawn from the animal just before death contained the
living bacteria of putrefaction, and underwent decom-
position. They attributed the germicidal phenomenon
to the action of the ‘‘ ozonized oxygen of the corpuscles
of the blood.’’
In 1882 Rauschenbach' demonstrated that, in the
process of coagulation, fibrin was formed not as a spe-
cific product of the action of the colorless elements of
the blood alone, but also as a result of the combined
action between all animal protoplasms and healthy blood
plasma, and that in the process there was always a dis-
integration of the leucocytes that were present. In
1884 Groth? demonstrated further that such a disinte-
gration of leucocytes occurred in normal circulating
blood, though here it was not accompanied by coagula-
tion. The results of these observations suggested the
question: Does such a disintegration occur when vege-
table protoplasm is introduced into the blood? For the
purpose of answering this question, Grohmann,’ a pupil
of Alexander Schmidt, undertook to study the action of
the circulating blood upon the vegetable protoplasm of
bacteria.
He noticed that clotting of the blood of the horse
was very much accelerated by the addition to it of cer-
1 Ueber die Wechselwirkung zwischen Protoplasma und Blutplasma. Dis-
sertation, Dorpat, 1882.
2 Ueber die Schicksale der farblosen Elemente in kreisendem Blut. Disser-
tation, Dorpat, 1884.
8 Ueber die Einwirkung des zellenfreien Blutplasma auf einige pflanzliche
Mikro-organismen. Dissertation, Dorpat, 1884.
INFECTION AND IMMUNITY. 467
tain bacteria; that at the same time the development
of the bacteria was checked, and in the case of the path-
ogenic varieties their virulence was diminished. This
was particularly the case when the anthrax bacillus was
employed.
Grohmann seems to have appreciated the significance
of this observation, though he took no steps to study the
subject more closely. He remarks that the system prob-
ably possesses, in the plasma of the blood, a body hav-
ing disinfectant properties (oc. cit., pp. 6 and 33). This
work, however, was not conducted according to the more
exact methods of modern bacteriological research, so
that the complete demonstration of this phenomenon
must be accredited to Nuttall.
Since the publication of Nuttall’s work his results
have received confirmation from all sides. Fodor,!
Buchner,? Lubarsch,*? Nissen,* Stern,> Prudden,® Char-
rin and Roger,’ and many others have continued in the
same line, and have all made practically the same obser-
vation.
After the demonstration by Nuttall that the serum
of the blood was directly detrimental to the vitality of
certain pathogenic bacteria, it became the work of a
number of investigators to determine to which element
of the serum this property is due, or if it is a function
of the serum only as a whole.
In the course of Buchner’s experiments it was demon-
strated that the serum was robbed of this property by
1 Centr. f. Bakteriologie u. Parasitenkunde, 1890, vol. vii. No. 24.
2 Archiv fiir Hygiene, 1890, vol. x. parts 1 and 2.
3 Centr f. Bakt. u. Parasitenkunde, 1889, vol, vi. No. 18.
+ Zeitschr. fiir Hygiene, 1889, vol. vi. part 3.
5 Zeitschr. fiir klin. Med., 1890, vol. viii. parts 1 and 2.
6 N. Y. Med. Record, 1890, vol, xxxvii. pp. 85, 86.
7 Soc. de Biol. de Paris,
468 BACTERIOLOGY.
an exposure to a temperature of 55° C. for half an
hour; that its efficacy as a germicide was not dimin-
ished by alternate freezing and thawing ; that by dialy-
sis or extreme dilution with distilled water its germicidal
activity was diminished, or completely checked; but
that an equal dilution could be made, if sodium chlo-
ride solution (0.6-0.7 per cent.) was substituted for the
distilled water, without the bactericidal action of the
serum losing any of its power. From this he con-
cluded that the active element in this phenomenon is a
living albumin, an essential constituent of which is
sodium chloride, and which, when robbed of this salt,
either by dialysis or dilution, becomes inert in its be-
havior toward bacteria. or this or these germicidal
constituents of the blood he suggested the name “ alex-
ines.’”’
He found, moreover, that the activity of the serum
alone against bacteria was greater than when the cellu-
lar elements of the blood were present. This he ex-
plains by the assumption that in the serum alone the
germicidal element predominates, whereas in the blood,
as such, outside of the body, it is still present, but its
influence is counteracted by the nutrition offered to the
bacteria by the disintegrated cellular elements; so that
here the nutritive feature is most conspicuous, and the
destructive activity toward bacteria is less effectual.
A closer study of the nature of this germicidal ele-
ment in the body of animals was made by Hankin and
Martin.' The former isolated from the spleen and
lymphatic glands a body—a globulin—which in solu-
tion possesses germicidal properties.
1 British Medical Journal, May 31, 1890,
INFECTION AND IMMUNITY. 469
Similar germicidal, ferment-like globulins have been
isolated from the blood by Ogata,’ and in their studies
upon tetanus Tizzoni and Cattani’ found a body that
was antagonistic to the poison produced by the organism
of this disease.
According to the observations of Vaughan,* the most
important germicidal or protective agents possessed by
the body are the nucleins; and Kossel has shown that
the cholera vibrio, streptococcus, staphylococcus, and
typhoid bacillus are destroyed by 0.5 per cent. solution
of nucleinic acid.
Hankin believes the globulins or ‘‘defensive pro-
teids’’ that he has discovered and the albuminoid
bodies studied by Buchner to be identical. The most
‘interesting and, in the light of work that has appeared
since, the most important, of Hankin’s observations
were not those upon the power of these globulins to
destroy the vitality of living organisms, but rather those
upon the relation between them and the poisonous me-
tabolic products of growth of the organisms. For ex-
ample, if the poisonous products of virulent anthrax
bacilli be isolated and mixed with the globulin extracted
from normal tissues, the experiments of Hankin showed
a directly destructive action on the part of the bacterial
products. He found that the amount of poisonous
albumose produced by the attenuated anthrax bacilli,
that are employed as vaccines, was much less than that
produced by the organisms possessing full virulence,
and he suggests that perhaps the protective influence of
vaccinations that are practised by introducing into the
animal the organisms that have been attenuated in vir-
1 Centr. f. Bakt. u. Parasitenkende, 1891, vol. ix. p. 599.
2 Tbid , p. 683. 3 Vaughan : Medical News, May 20, 1893. __
21
470 BACTERIOLOGY.
ulence is due to a gradual tolerance acquired by the
cells of the tissues to the action of the poison when
produced in these small quantities; in the same way
that a tolerance was acquired by the tissues for the
venom of the rattlesnake in the experiments of Sewall!
(and more recently in the work of Fraser, Calmette,
Weir Mitchell, and others), and similar to that follow-
ing the injection into the tissues of small quantities of
hemialbumose, which in large amounts rapidly proves
fatal.
Of utmost importance to these studies of the blood
and fluids of the body are the experiments of Behring
and Kitasato” upon the production of immunity to teta-
nus. In their investigations upon the blood of animals
subjected to these experiments it was found that it was
not only possible to render animals immune from this
disease, but that the serum of the blood of these immu-
nized animals afforded immunity when injected into the
peritoneal cavity of other animals that had not been so
protected; and moreover, that this serum possesses cura-
tive powers over the disease after it has, in some cases,
been in progress for a time. They found, further, that
the serum of animals that had been rendered immune
from tetanus, when brouyht in contact with the poison of
tetanus, completely destroyed its poisonous properties,
and that the serum from animals or from human beings
that do not possess immunity from this disease has no
such power.
The demonstration by Behring and Kitasato of the
fact that the serum of an immunized animal can not
only confer immunity to another susceptible animal,
1 Journal of Physiology, 1887, vol. vii. p. 203.
* Behring and Kitasato; Deutsche med. Woch., 1890, Bd. xvi. p. 1113,
INFECTION AND IMMUNITY. 471
but in the case of tetanus (and diphtheria, as subse-
quently demonstrated by Behring and his associates),
cure the disease after it is already in progress, is one of
the most important steps that has been made in this
entire field of study. The subsequent application of the
principle involved in that observation by Behring and
his colleagues, in their successful efforts to devise a cure
for diphtheria in man, has resulted in a triumph which
marks an epoch in modern scientific medicine. The
same principle has been employed for obtaining cura-
ative agents against other forms of infection and intoxi-
cation, notably, of Asiatic cholera, typhoid fever, lobar
pneumonia, streptococcus and staphylococcus infection,
rabies, tuberculosis, bubonic plague, syphilis, vaccinia,
and serpent venom; but unfortunately, as yet, with but
indifferent success ; certainly in no case to the same
favorable degree as has been seen in the treatment of
diphtheria with antitoxic serum.
Another hypothesis in explanation of the immunity
acquired by the tissues of the animal organism is that
advanced by Buchner," who suggests that in the primary
infection, from which the animal has recovered, there
has been produced a reactive change in the integral cells
of the body that enables them to protect themselves
against subsequent inroads of the same organism.
Though somewhat more vague at first glance than the
other theories in regard to this phenomenon, it is, never-
theless, in the light of subsequent research, most prob-
ably the correct explanation of the establishment of
immunity in many, if not all, cases. Experiments that
bear directly upon this idea have demonstrated that, if
1 Buchner : Eine neue Theorie iiber Erzielung von Immunitit gegen In-
fektionskrankheiten. Miinchen, 1883.
472, BACTERIOLOGY.
animals be subjected to injections of the poisonous pro-
ducts of growth of certain virulent bacteria, they re-
spond to this treatment by more or less pronounced
constitutional reactions, and that during this period,
and for a short time following, they are protected from
the invasion of the virulent bacteria themselves. This
observation has, moreover, not been confined to those
cases in which injections of the products of growth have
been followed by inoculations with the bacteria by
which they were produced, but, what is still more in-
teresting and confirmatory of Buchner’s view, it is
claimed that a sort of protection from certain specific
infections can also be afforded to animals by the injec-
tion into them of cultures of entirely different species of
bacteria, or their products, and that in some cases these
are not of necessity of the disease-producing variety.
For instance, Emmerich and Mattei’ claim to have
rendered rabbits insusceptible to anthrax through injec-
tions into them of cultures of the streptococcus of ery-
sipelas.
This, they claim, is not due to any antagonism be-
tween the organisms themselves, for in culture experi-
ments the two organisms grew well together, without
any alteration in their pathogenic properties, but rather
to the induction of a tissue-activity by which resistance
to the inroads of the virulent bacilli was established.
Emmerich and Mattei interpret this ‘‘ reactive tissue-
change’’ as a power acquired by the integral cells of the
body, through the influence of a stimulus, of generating
a product that is detrimental to the pathogenic activity
of the anthrax bacilli.
1 Emmerich und Matti: Fortschritte der Medizin, 1887, p. 653.
INFECTION AND IMMUNITY. 473
Pawlowsky,' who obtained similar results from the
introduction into the animal of cultures of bacillus
prodigiosus, of staphylococcus pyogenes aureus, and of
micrococcus lanceolatus, believes them to be due to the
induction of increased energy on the part of the wan-
dering cells, preparing them thus for the difficult task
of destroying the more virulent organisms with which
the animal is subsequently to be inoculated.
Protection that is afforded in this way apparently
contraindicates a specific relation between the morbific
elements of particular infections and the protective sub-
stances that are present in the body of the animal that
has been rendered insusceptible to them. It is proba-
ble, however, that this is only apparent, and that the
observations of Emmerich and Mattei and of Paw-
lowsky can be explained in another way: in the blood of
animals there is present what may be termed a normal
protective (Buchner’s alexines) having no specific rela-
tions to any particular variety of infection, but serving
to protect the animal more or less completely against all
bacterial invasion. By the methods employed in the
preceding experiments it seems likely, in the light of
more recent work, that this normal antidote was simply
temporarily accentuated through the tissue-stimulation
resultant upon the treatment that the animals had re-
ceived, for it has never been shown to be possible to
bring about as high or as permanent a degree of im-
munity in an animal from a particular disease as that
which can be obtained by the use of the specific micro-
organism causing the disease, or the products of its
growth, especially the latter.
1 Pawlowsky : Virchow’s Arch., vol. eviii. p. 494.
A474 BACTERIOLOGY.
A striking illustration of this protective reaction on
the part of the animal tissues is brought out in the
course of R. Pfeiffer’s' experiments on Asiatic cholera.
He found that it was possible to confer immunity to
animals from this disease; that the blood-serum of such
animals protected susceptible animals into which it was
injected against what would otherwise be a fatal dose of
the cholera spirillum; that the peritoneal fluids of the
artificially immunized animal had an almost instantane-
ous disintegrating, bactericidal action upon living cholera
spirilla that were injected into the peritoneal cavity; that
the serum from the immune animal had no such effect
upon cholera spirilla when tried in the test-tube; but if
virulent cholera spirilla be injected into the peritoneum
of an animal that was not immune, and this be at once
followed by an intraperitoneal injection of serum from
an immune animal, almost instantly the peculiar dis-
integration of the bacteria, as observed in the perito-
neum of the immune animal, could be detected. This
latter observation is of the utmost importance in its
bearing on Buchner’s hypothesis, for we see here a
serum from an immune animal that is capable of
conferring immunity; capable, on injection into a sus-
ceptible animal, of endowing its fluids with the peculiar
disintegrating, germicidal function noted in the perito-
neum of the immune animal from which the serum
may have originated ; incapable of bactericidal ac-
tivity outside the body, but the influence of which in
the peritoneum of a susceptible animal is to call forth
at once this interesting phenomenon. Manifestly the
germicidal substance in this case is neither contained
1 Pfeiffer: Zeit. f. Hyg. u. Infektionskrankheiten, Bd. xviii. p. 1; Ebenda,
Bd. xx. p. 198.
INFECTION AND IMMUNITY. A475
in the protective serum nor in the peritoneum of the
susceptible animal before receiving the serum, but is
generated by the tissues as a result of the specific irri-
tation of a something contained in this serum; in
other words, in consequence of a reaction on the part of
the peritoneal tissues, or possibly those of the entire
animal,
The experiments of G. and F. Klemperer! upon acute
fibrinous pneumonia, though too limited in extent to be
accepted as conclusive, have, nevertheless, offered a
number of most significant suggestions, not only in
connection with several obscure features of this disease,
but also in relation to the establishment of tissue-resist-
ance,
They found but little difficulty in affording immunity
to animals that are otherwise susceptible to the patho-
genic action of the organisms concerned in the produc-
tion of this disease,” by the introduction into their
tissues of the products of growth of the organisms from
which the latter had been separated. The immunity
thus produced is seen in some cases to last as long as
six months; again it is seen to disappear suddenly in a
way not to be explained. It was seen in one case to be
hereditary, probably having been conferred upon the
young, during the nursing-period, through the milk of
the mother, as Ehrlich? has shown to occur in animals
artificially immunized from abrin, ricin, and robin.
The energy of the substance that has the power of
affording immunity was seen to be very much increased
1 G. and F. Klemperer: Berliner klin. Wochenschr., 1891, Nos. 34 and 35,
2 Animals do not, as a rule, present the pneumonic changes seen in human
beings. The introduction of micrococcus lanceolatus into their tissues results,
in the case of susceptible animals, in the production of septicemia.
3 Ehrlich: Zeit. fiir Hygiene u. Infektionskrankheiten, 1892, Bd. xii. p. 183,
476 BACTERIOLOGY.
by subjecting it to temperatures somewhat higher than
that at which it was produced by the bacteria. The
Klemperers found that if this substance was heated to
a temperature of from 41° to 42° C. for three or four
days, or to 60° C. for from one to two hours, its intra-
venous injection was followed by complete immunity
in from three to four days; whereas, if the unwarmed
material was used, immunity did not appear before four-
teen days, and then only after the employment of rela-
tively large amounts. Moreover, when the previously
heated products are introduced into the circulation of
the animal the systemic reaction is of but short dura-
tion; but if the unwarmed substance is employed, immu-
nity is manifest only after the appearance of considerable
elevation of temperature, which lasts for a long time.
In explanation of these differences they suggest that,
in the latter case, the high fever that is seen to occur in
the animal may serve to replace the warming to which
the bacterial products had not previously been sub-
jected, and which is necessary before they are in a posi-
tion to bring about the condition of immunity. They
claim that the bacterial products employed to produce
immunity in this case are not, in reality, the immunity-
affording substance, but that they are only the agents
that bring about in the tissues of the animal alterations
that result in the production of another body that pro-
tects the animal. In support of this, their argument is
that several days are necessary for the production of
immunity by the introduction into the animal of the
bacterial products; whereas, if the blood-serum of this
animal, which is now protected, be introduced into the
circulation of another animal, no such delay is seen,
but instead, the animal is forthwith protected. In the
INFECTION AND IMMUNITY. 477
former case the actual protecting body had first to be
manufactured by the tissues; whereas, in the second it
is already prepared, and is introduced as such into the
second animal.
They found the serum of artificially immune animals
to be not only capable of rendering other animals im-
mune, but that it possessed curative powers when the
disease is already in progress. The serum of immu-
nized animals when injected into the circulation of ani-
mals in which there was a body-temperature of from
40.4° to 41° C. reduced this temperature to normal
(87.5° C.) in twelve consecutive experiments during
the first twenty-four hours following its employment.
In their opinion, the crisis seen in pneumonia in
human beings indicates the moment at which the pois-
onous products, manufactured by the bacteria located in
the lungs, are present in the circulation in amounts suffi-
cient to stimulate the tissues to the reaction that results
in the production of the antidotal substance that has
the power of rendering the poisons inert.
At the time of the crisis in pneumonia the bacteria
themselves are in no way affected. They remain in the
lungs, and can be detected, in full vigor and virulence,
in the sputum of patients a long time after the disease
is cured. They have lost none of their power of pro-
ducing poisonous products, and still possess their orig-
inal pathogenic relations toward susceptible animals.
It is only after the crisis that their poisons are neutral-
ized by this antidotal proteid that has been produced
by the cells of the tissues, and as this occurs the systemic
manifestations gradually disappear. The Klemperers
claim to have isolated from cultures of micrococcus
lanceolatus a proteid body that is the agent concerned in
21*
478 BACTERIOLOGY.
producing the tissue-reaction which results in the for-
mation of the protecting substance. They likewise
isolated from the serum of immunized animals a pro-
teid that possesses the same powers as the serum itself,
viz., of affording immunity and curing the disease.
Here, again, it appears that the processes of infection
and immunity are chemical in their nature, the active
poisons of the invading organisms—‘‘ the pneumo-
toxins’’—being instrumental in producing the diseased
condition, while the antidotal or resisting body of the
tissues—‘‘ the anti-pneumotoxin’’—is the agent by
which the poison is neutralized.
Results in general analogous to those of G. and F.
Klemperer have also been obtained by Emmerich and
Fowitzky."
In the light of these experiments the hypothesis ad-
vanced by Buchner, that the establishment of immunity
is to be explained by reactive changes in the integral
cells of the body, receives additional support, and when
we consider the observations of Bitter,? who found that
in protective vaccinations against anthrax the vaccines
do not disseminate themselves through the body, as is
the case when the virulent organisms are introduced,
but remain at the point of inoculation, and from this
point produce, by the absorption of their chemical pro-
ducts, the systemic changes through which the animal
is protected against subsequent infection by the virulent
organisms,we feel justified in concluding that the weight
of evidence is strongly in favor of this view.
The experiments of the past two or three years indi-
cate the probability of there being present in the blood
1 Emmerich and Fowitzky : Miinchener med. Wochenschr; 1891, No. 32.
2 Bitter: Zeitschrift fur Hygiene, 1888, Bd. iv.
INFECTION AND IMMUNITY. 479
of animals several different bodies having totally differ-
ent relations to bacteria and their products, according to
the conditions under which they exist. First, there is
present in the blood-serum of practically all animals
the normal defensive ‘‘alexines’’ already mentioned;
second, the antitoxins that are found in the blood of
animals artificially immunized from special sorts of
infection and intoxication, the functions of which are
susceptible of demonstration outside the body as well
as within the tissues of the living animal; third, a body
possessed of disintegrating bacteriolytic powers—1. e.,
having the property of actually breaking bacteria to
pieces, so that the phenomenon may be observed under
the microscope. This phenomenon is especially to be
seen within the peritoneum of guinea-pigs that have
been rendered immune from Asiatic cholera and from
the typhoid and colon infections or intoxications. It is
rarely or never seen outside the body, and is not to be
confounded with the ordinary bactericidal function of the
alexines that is demonstrable in most normal serums;
and fourth, a body, the so-called ‘‘agglutinin’’ (Gruber),
that is regarded by Widal to represent a ‘‘reaction of
infection,’? and not of immunity. The presence of
this body in a serum is announced by its peculiar influ-
ence on the activity and arrangement of bacteria with
which it is brought in contact. In the case of typhoid
fever in man, for instance, the serum obtained during
the early and middle stages of the disease, when mixed
with fluid cultures or suspensions of the typhoid bacil-
lus, causes the bacilli to lose their motility and to con-
gregate (agglutinate) together in masses and clumps, a
condition never seen in normal cultures of this organism,
and practically never observed when normal serum is
480 BACTERIOLOGY.
employed. There are evidences of the presence of
‘‘agelutinin’’ in certain of the antitoxic serums from
immune animals, viz., that of animals immune from
cholera, pyocyaneus, typhoid and colon infections. So
far as experience has gone, this agglutinating influence
is manifested in the great majority of cases only upon
the organisms from which the animal is protected. In
view of the fact that its presence is regarded as indica-
tive of a reaction of infection, its detection in the blood
of immune animals may at first sight appear paradoxi-
cal. We should not lose sight of the fact, however,
that it is assumed to be totally distinct from the other
substances that are concerned in immunity, and its pres-
ence in immune animals may, therefore, be reasonably
explained as a result of the ‘‘ reactions of infection’’
that were coincident with the primary injections into
the animal of infective or intoxicating matters neces-
sary to the establishment of the condition of immunity.
The experiments that have been cited afford but an
imperfect idea of the enormous amount of work that
has been done upon the manifold phases of these im-
portant subjects; they may, however, serve to indicate
the direction in which the lines of research have been
laid. As a result of such investigations, our knowledge
upon infection and immunity may at present be sum-
marized about as follows :
1. That infection may be considered as a contest be-
tween bacteria and living tissues, conducted on the part
of the former by means of the poisonous products of
their growth, and resisted by the latter through the
agency of proteid bodies normally present in and gen-
erated by their integral cells.
2. That when infection occurs it may be explained
INFECTION AND IMMUNITY. 481
either by the excess of vigor of the bacterial products
over the antidotal or protective proteids produced by
the tissues, or to some cause that has interfered with the
normal activity and production of these bodies.
3. That in the serum of the normal circulating blood
of many animals there exists a substance that is cap-
able, outside of the body, of rendering inert bacteria
that, if introduced into the body of the animal, would
prove infective.
4, That immunity is most frequently seen to follow
the introduction into the body of the products of growth
of bacteria that in some way or other have been mod-
ified. This modification may be artificially produced
in the products themselves of virulent organisms, and
then introduced into the tissues of the animal; or the
virulent bacteria may be so treated that they are no
longer of full virulence, and when introduced into
the body of the animal will produce poisons of a
much less vigorous nature than would otherwise be
the case.
5. That immunity following the introduction of bac-
terial products into the tissues is not in all cases the
result of the permanent presence of these substances,
per se, in the tissues, or of a tolerance acquired by the
tissues to them; but is probably, in certain instances,
due to the formation in the tissues of another body that
acts as a protecting antidote to the poisonous products
of invading organisms.
6. That this protecting proteid which is generated by
the cells of the tissues need not of necessity be antag-
onistic to the life of the invading organisms themselves,
but in some cases must be looked upon more as an anti-
dote to their poisonous products.
482 BACTERIOLOGY.
7. That immunity, as conceived by Ehrlich, may be
either “ active’’ or ‘‘passive.’? According to this in-
terpretation, it is ‘‘active’’? when resulting from an or-
dinary non-fatal attack of infectious disease; or from a
mild attack of infection purposely induced through the
use of living vaccines ; or from the gradual introduc-
tion of toxins into the tissues until a conspicuous stage
of tolerance is reached. It is ‘‘passive’’ when occur-
ring as a result of the direct transference of the per-
fected immunizing substance from an immune to a
susceptible animal, as by the injection of blood-serum
from the former into the latter. ‘‘ Passive immunity’’
is, in most cases, conferred at once, without the delay
incidental to the usual modes of establishing ‘active
immunity.’’ Asa rule, ‘active’ is more lasting than
“‘ passive’? immunity.
8. That phagocytosis, though frequently observed, is
not essential to the establishment of immunity, but is
more probably a secondary process, the bacteria being
taken up by the leucocytes only after having been mod-
ified in virulence through the normal germicidal activity
of the serum of the blood and of other fluids in the
body. It is, however, probable that important factors
in the establishment of immunity are substances secreted
and thrown into the circulating fluids by the living leu-
cocytes.
9. That, of the hypotheses advanced in explanation
of acquired immunity, the one worthy of greatest con-
fidence is that which assumes immunity to be due to
reactive changes on the part of the tissues that result
in the formation in these tissues of antitoxic substances
capable of neutralizing the poisons produced by the
bacteria against which the animal has been immunized;
INFECTION AND IMMUNITY. 483
though in certain cases the establishment of immunity
is accompanied by the assumption of germicidal as well
as antitoxic properties by the fluids and tissues, and in
a few instances the germicidal is more pronounced than
the antitoxic function.
CHAPTER XXVITI.
Bacteriological study of water— Methods employed — Precautions to be
observed—A pparatus used, and methods of using them—Methods of investi-
gating air and soil.
THE conditions that favor the epidemic outbreak of
typhoid fever, Asiatic cholera, and other maladies of
which these may be taken as types, have served as a
subject for discussion by sanitarians for a long time.
Of the hypotheses that have been advanced in ex-
planation of the existence and dissemination of these
diseases, two stand pre-eminent and are worthy of con-
sideration. They are the ‘‘ ground-water’ theory of
von Pettenkofer and his pupils, and the ‘ drinking-
water’’ theory of the school of bacteriologists of which
Koch stands at the head.
The adherents to the ‘‘ ground-water’’ view explain
the presence of these diseases in epidemic form through
alterations in the soil resulting from fluctuations in the
level of the soil-water, and assign to the drinking-water
either a very insignificant réle, or, as is most frequently
the case, ignore it entirely. On the other hand, those
who have been instrumental in developing the drinking-
water hypothesis claim that alterations in the soil play
little or no part in favoring the appearance of these dis-
eases in a neighborhood; but that, as a rule, they appear
as a result of direct infection, through the use of waters
that are contaminated with materials containing the
specific organisms that are known to cause such diseases.
BACTERIOLOGICAL STUDY OF WATER. 485
As a result of many observations on both sides of
the question, the evidence is greatly in favor of the
opinion that polluted water is primarily the underlying
cause of these epidemics, and this too, very often, when
the state of the soil-water, in the light of the ‘“‘ground-
water’’ hypothesis, is just the reverse of what it should
be in order to render it answerable for them. It is
manifest, therefore, that the careful bacteriological study
of water intended for domestic use is of the greatest
importance, and should be a routine procedure in all
communities receiving their water-supply from sources
that are liable to pollution.
The object aimed at in such investigations should be
to determine if the water approaches constancy in the
number and kind of bacteria contained in it—for all
waters, except deep ground-water, contain bacteria; if
sudden fluctuations in the number and kind of bacteria
occur in these waters, and if so, to what are they due;
and finally, and most important, does the water contain
constantly, or at irregular periods, bacteria that can be
traced to human excrement, not of necessity pathogenic
varieties, but bacteria that are known to be present
normally in the intestinal canal? For, if conditions
are favorable to the presence of these varieties, the same
conditions would favor the admission to the water of
other forms of bacteria that are concerned in the pro-
duction of diseases of the intestines.
In considering water from a bacteriological stand-
point it must always be borne in mind that compari-
sons with any general fixed standard are not of much
value, for just as normal waters from different sources
are seen to present variations in their chemical compo-
sition, without being unfit for use,so may the relative
486 BACTERIOLOGY.
number of bacteria in water from one source be always
greater or smaller than in that from another, and
yet no difference may be seen to result from their em-
ployment. For this reason the proper study of any
water, from this point of view, should begin with the
establishment of what may be called its normal mean
number of bacteria, as well as the character of the pre-
vailing species; and in order to do this the investiga-
tions must cover a long period of time through all the
seasonal variations of weather. From data obtained in
this way it may be possible to predict approximately
the normal bacteriological condition of water at any
season. Marked deviations from these ‘‘ means,”’’
either in the quantity or quality of the organisms pres-
ent, can then be considered as indicative of the existence
of some unusual, disturbing element, the nature of which
should be investigated. Similarly, it is impossible to
formulate an opinion of much value from a single chem-
ical analysis of a water, for the results thus obtained
indicate only the state of the water at the time the
sample was procured, and give no indication as to
whether it differed at that time from its usual condition,
or from the normal condition of the water throughout
the immediate neighborhood.
The interpretation of the results of both chemical
and bacteriological analysis of a sample of water ac-
quires its full value only through comparison, either
with ‘‘means’’ that have been determined for this
water, or with the results of simultaneous analyses of
a number of samples from the other sources of supply
of the locality.
The aid of the bacteriologist is frequently sought in
connection with investigations upon waters that are sup-
BACTERIOLOGICAL STUDY OF WATER. 487
posed to be concerned in the production of disease, partic-
ularly typhoid fever, either in isolated cases or in wide-
spread epidemic outbreaks, and almost as often do
reliable bacteriologists fail to detect the bacillus that is
the cause of typhoid fever in these waters.
The failure to find the organisms of typhoid fever in
water by the usual methods of analysis does not by
any means prove that they are not present or have not
been present. The means that are ordinarily employed
in the work admit of such very small volume of water
being used in the test that we can readily understand
how these organisms might be present in moderate num-
bers and yet none of them be included in the drop or
two of the water that are taken for study. The con-
ditions are not those of a solution, each drop of which
contains exactly as much of the dissolved material as
do all other drops of equal volume, but are rather those
of a suspension in each drop or volume of which the
number of suspended particles is liable to the greatest.
degree of variation. Furthermore, there are other rea-
sons that would, a priori, preclude our expecting to find
the typhoid bacilli in water in which we may have rea-
son to believe they had been deposited, viz., attention
is not usually directed to the water until the presence of
the disease has become conspicuous, usually in from
three to four weeks after the time when the pollution
probably occurred. Three or four weeks are ordinarily
sufficient time for the delicate, non-resistant bacillus of
typhoid fever to’ succumb to the unfavorable conditions
under which it finds itself in water. By unfavorable
conditions are meant the absence of suitable nutrition;
unfavorable temperature; probably the antagonistic in-
fluence of more hardy saprophytic bacteria, particu-
488 BACTERIOLOGY.
larly the so-called ‘‘ water-bacteria,’’? and of more
highly organized water-plants; the effect of mechanical
precipitation; and, of great importance, the disinfecting
action of direct sunlight.
Though it is so rare as to be almost never, that
typhoid bacilli are found in drinking-water, it must,
nevertheless, not be supposed that bacteriological analy-
ses of suspicious waters shed no light upon the exist-
ence of pollution and the suitability or non-suitability
of the water for drinking-purposes.
In the normal intestinal tract of all human beings,
and of many other mammals, as well as associated with
the specific disease-producing bacterium in the intes-
tines of typhoid-fever patients, is an organism that is
frequently found in polluted drinking-waters, and
whose presence is proof positive of pollution by either
normal or diseased intestinal contents; and though
efforts may result in failure to detect the specific bacil-
lus of typhoid fever, the finding of the other organism,
the bacterium coli commune, justifies one in expressing
the opinion that the water under consideration has been
polluted by intestinal evacuations from either human
beings or animals. Waters so located as to be liable to
such pollution can never be considered as other than a
continuous source of danger to those using them.
Another point to be remembered is in connection with
the value of chlorine as indicative of contamination by
human excrement. It is commonly taught that an ex-
cessive amount of chlorine in water points to contam-
ination by human excreta. This may or may not be
true according to circumstances. A high proportion of
this substance in a sample of water from a locality, the
neighboring waters of which are poor in chlorine, is
BACTERIOLOGICAL STUDY OF WATER. 489
unquestionably a suspicious indication, but in a district
close to the sea or near salt deposits, for-instance, where
the water generally is high in chlorine, the value of the
indications thus afforded is very much diminished
unless the amount found in the sample under examina-
tion greatly exceeds the normal ‘‘ mean,’’ previously
determined, for the amount of chlorine in the waters of
the neighborhood.
A striking example of such a condition as the latter
recently occurred in the experience of the writer while
inspecting a group of water-supplies on the east coast
of Florida. In each instance the water was obtained
from properly protected artesian wells, ranging from
200 to 400 feet deep, and located within a few hundred
yards of the sea. The first sample that was subjected
to chemical analysis revealed such an unusually high
proportion of chlorine that, had this sample alone been
considered, the opinion that it was polluted by human
excreta might have been advanced. To prevent such
an error samples of water from a number of wells in
the neighborhood were examined, and they were all
found to contain from ten to twelve times the amount
of chlorine that ordinarily appears in inland waters, the
excess being evidently due to leakage through the soil
into the wells of water from the sea. In short, the pres-
ence of an excess of chlorine in water, while often indi-
cating pollution from human evacuations, may, never-
theless, sometimes arise from other sources; but the
presence in water of bacteria normally found in the
intestinal canal can manifestly admit of but one inter-
pretation, viz., that fecal matters have at some time
and place been deposited in this water, and that while
no specific disease-producing organisms may have been
490 BACTERIOLOGY.
detected, still, waters in which such pollutions are pos-
sible are a constant source of menace to the health of
those who use them for domestic purposes.
A sudden variation from the normal, mean number
of bacteria, or from the normal chemical composition of
a water, calls at once for a thorough inspection of the
supply, while at the same time the characters of the
organisms present are to be subjected to the most care-
ful study.
THE QUALITATIVE BACTERIOLOGICAL ANALYSIS
or Water.—The qualitative bacteriological analysis
of water entails much labor, as it requires not only that
all the different species of organism found in the water
should be isolated, but that each representative should
be subjected to systematic study, and its pathogenic or
non-pathogenic properties determined.
For this purpose a knowledge of the methods for the
isolation of individual species, which have already been
described, and of the means of studying these species
when isolated, is indispensable.
For this analysis certain precautions essential to
accuracy are always to be observed.
The sample is to be collected under the most rigid
precautions that will exclude organisms from sources
other than that under consideration. If drawn from a
spigot, it should never be collected until the water has
been flowing for fifteen to twenty minutes in a full
stream. If obtained from a stream or a spring, it
should be collected, not from the surface, but rather
from about one foot beneath the surface.
It should always be collected in vessels which have
previously been thoroughly freed from all dirt and
organic particles, and then sterilized; and the plates
BACTERIOLOGICAL STUDY OF WATER. 491
should be made as quickly as possible after collecting
the sample.
When circumstances permit, all water analyses should
be made on the spot at which the sample is taken, as it
is known that during transportation, unless the samples
are kept packed in ice, a multiplication of the organ-
isms contained in it always occurs.
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
in the water, and can, in consequence, determine the
amount of water necessary to use for each set of plates.
Duplicate plates are always to be made—one set upon
agar-agar, which are to be kept in the incubator at
body temperature, and one set upon gelatin, to be kept
at from 18° to 20° C.
As soon as the colonies have developed the plates are
to be carefully compared and studied. It is to be noted
if any difference in the appearance of the organisms on
corresponding plates exists, and if so, to what is it due?
It is to be particularly noted which plates contain the
greater number of colonies, those kept at the higher or
those at the lower temperature. In this way the tem-
perature best suited for the growth of the majority of
these organisms may be determined.
Asa rule, the greater number of colonies appear upon
the gelatin plates that are kept at 18° to 20° C., and
from this it would seem that many of the normal water-
bacteria do not find the higher temperature so favorable
to their development as do the organisms not naturally
present in water, particularly the pathogenic varieties.
492 BACTERIOLOGY.
Nore.—In determining if the organisms found are
possessed of pathogenic properties, in what way will
your tests be influenced by this observation ?
From recent investigations upon this subject it ap-
pears that the difference in behavior toward heat of
bacteria present in water may have a very important
application. Dr. Theobald Smith has recently sug-
gested a method by which it is easily possible to isolate,
from waters in which they are present, certain organisms
that are of the utmost importance in influencing our
judgment upon the fitness of the water for domestic
use. By the addition of small quantities, one, two, or
three drops of the suspicious water to fermentation
tubes (see article on Fermentation Tube) containing
bouillon to which 2 per cent. of glucose has been added,
and keeping them at the temperature of the body, 37°
to 38° C., the growth of the intestinal bacteria that may
be present in the water is favored, while that of the
water-organisms is not; in consequence, after from
thirty-six to forty-eight hours the fermentation char-
acteristic of most of these organisms is evidenced by
the accumulation of gas in the closed end of the tube.
From these tubes the growing bacteria can then be
easily isolated by the plate method, and it will not be
infrequent to find intestinal bacteria present in pure
culture.
Another method for the same object is to collect a
sample of about 100 ¢.c. of the water to be tested in a
sterilized flask, and add to this about 25 c.c. of steril-
ized bouillon of four times the usual strength. This
is then placed in the incubator at 37° to 38° C., for
thirty-six to forty-eight hours, after which plates are to_
BACTERIOLOGICAL STUDY OF WATER. 493
be made from it in the usual way; the results will often
be a pure culture of some single organism, either one of
the intestinal variety or a closely allied species. By a
method analogous to the latter the spirillum of Asiatic
cholera has been isolated from water; and by taking
advantage of the effect of elevated temperature upon
the bacteria of water Dr. Vaughan, of Michigan, has
succeeded in isolating from suspicious waters a group of
organisms very closely allied to the bacillus of typhoid
fever.
THE QUANTITATIVE ESTIMATION OF BACTERIA IN
WaTerR.—Quantitative analysis requires more care in
the measurement of the exact volume of water em-
ployed, for the results are to be expressed in terms of the
number of individual organisms to a definite volume.
The necessity for making the plates at the place at
which the sample is collected is to be particularly
accentuated in this analysis, for the multiplication of
the organisms during transit is so great that the results
of analyses made after the water has been in a vessel
for a day or two are often very different from those that
would have been obtained on the spot.
Norte. —Inoculate a tube containing about ten cubic
centimetres of sterilized distilled or tap water with a
very small quantity of a solid culture of some one of
the organisms with which you have been working,
taking care that none of the culture medium is intro-
duced into the water-tube and that the bacteria are
evenly distributed through it. Make plates at once,
and on each succeeding day, from this tube, and deter-
mine by counts whether there is an increase or diminu-
tion in the number of organisms—i. e., if they are
22
494 BACTERIOLOGY.
growing or dying. Represent the results graphically,
and it will be noticed that in many cases there is at first,
during the first three or four days, a multiplication,
after which there is a rapid diminution; and, if the
organism does not form spores, usually complete death
in from ten to twelve days. This is not true for all
organisms, but does hold for many.
Where it is not convenient, however, to make the
analysis on the spot, the sample of water should be col-
lected and packed in ice and kept on ice until ready
for use, which should in all cases be as soon after its
collection as possible.
For the collection of water for this purpose, a con-
venient vessel to be employed is a glass bulb (Fig. 98)
or balloon, which one soon learns to make for one’s
self from glass tubing.
Fic, 98,
Glass bulb for collecting samples of water.
It consists simply of a round glass sphere blown on
the end of a glass tube, which latter is subsequently
drawn ont into a fine capillary stem and sealed while
hot. As it cools, the contraction of the air within the
bulb results in the production of a negative pressure.
If the point of the stem be broken off under water,
the water is pressed up into the bulb, because of the
existence of the negative pressure within. The nega-
tive pressure obtained in this way is frequently in-
sufficient to permit of the bulb being completely filled,
BACTERIOLOGICAL STUDY OF WATER. 495
and often only a few drops of fluid can be obtained.
To obviate this bulbs may be blown and allowed to
cool, but not sealed. After a sufficient number of
them are prepared they are taken, one at a time, and
gently warmed over the flame; while still warm the
extremity of the stem is dipped into distilled water
and held there until a few drops have passed up into
the bulb; this is then carefully boiled, or, rather, com-
pletely vaporized, over the flame, and while the steam is
still escaping the point is sealed in the gas-flame. All
air will thus have been replaced by water vapor, and if
the point of the stem be now broken off under the water
the bulb will fill quickly and completely. It is not
desirable to fill them completely, but rather to only
about three-fourths of their capacity, as when full it is
difficult to empty them without contaminating the con-
tents. They are emptied by gently warming over a gas
or alcohol flame.
A number of them may be made, sealed, and kept on
hand. ‘They are sterile so long as they are sealed, be-
cause of the heat that is employed in their manufacture,
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 procured and held there until the necessary amount
has been obtained. This may serve as a sample from
which to prepare plates or Esmarch tubes on the spot,
or the tip of the stem may be resealed in the flame of
an alcohol lamp, the bulb packed in ice, and transported
in this condition to the laboratory.
Another very simple and useful device for collecting
water samples is that recommended by Kirschner. It
consists of a piece of glass tubing of about 5 or 6 mm,
496 BACTERIOLOGY.
inside diameter, and 36 em. long, bent in the form of a
U, with either extremity of the arms bent again at right
angles in the same plane and drawn out to a point and
sealed. They are sterilized in the flame as they are
made. The sample is collected by breaking off both
points, immersing one of them in water and sucking on
the other until the tube is filled. Then both points are
again sealed in the flame and the tube packed in ice.
The objection to this tube is the danger of contaminat-
ing its contents with saliva during the act of filling by
suction, though this danger is not so great as might at
first appear, as we shall learn in our efforts to cultivate
bacteria from the mouth-cavity.
Nors.—Make cover-slips from your own mouth;
make plates on both gelatin and agar-agar, at the same
time. Compare the number of bacteria found by
microscopic examination of the cover-slips with the
number of colonies that develop on the plates.
In beginning the quantitative analysis of water with
which one is not acquainted there are certain prelim-
inary steps that are essential.
It is necessary to know approximately the number of
organisms contained in any fixed volume, so as to deter-
mine the quantity of water to be employed for the plates
or tubes. This is usually done by making preliminary
plates from one drop, two drops, 0.25 ¢.¢., 0.5 ¢.¢., and
1 c.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 occurred one
selects the plate upon which the colonies are only mod-
erate in number—about 200 to 300 colonies presenting
BACTERIOLOGICAL STUDY OF WATER. 497
—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, ten, twenty-five, fifty,
or one hundred volumes, as the case may require, of
sterilized distilled water. This dilution must be accu-
rate, and its exact extent noted, so that subsequently the
number of organisms per volume in the original water
may be calculated.
The use of a drop is not sufficiently accurate. The
dilution should therefore always be to a degree that will
admit of the employment of a volume of water that
may be exactly measured, 0.25 and 0.5 ¢.c. being the
amounts most convenient for use.
Duplicate plates should always be made and the
mean of the number of colonies that develop upon
them taken as the basis from which to calculate the
number of organisms per volume in the original water.
For example: from a sample of water 0.25 c.c. is
added to a tube of liquefied gelatin, carefuily mixed and
poured out as a plate. When development occurs the
number of colonies are too numerous to be accurately
counted. One cubic centimetre of the original water
is then to have added to it, under precautions that pre-
vent contamination from without, 99 c.c. of sterilized
distilled water—that is, we have now a dilution of
1:100. Again, 0.25 c.c. of this dilution is plated,
and we find 180 colonies on the plate. Assuming that
each colony develops from an individual bacterium,
though this is perhaps not strictly true, we had 180
organisms in 0.25 c.c. of our 1: 100 dilution, therefore
498 BACTERIOLOGY.
in 0.25 ¢.c. of the original water we had 180 x 100 =
18,000 bacteria, which will be 72,000 bacteria per eubic
centimetre (0.25 =18,000, 1 ec. =18,000 xX 4 =72,000).
The results are always to be expressed in terms of the
number of bacteria per cubic centimetre of the original
water.
Another point of very great importance (already men-
tioned) is the effect of temperature upon the number of
colonies of bacteria that will develop on plates made
from water. It must always be remembered that a
larger number of colonies appear on gelatin plates made
from water and kept at 18° to 20° C, than on agar-agar
plates kept in the incubator. The following table, illus-
trative of this point, gives the results of parallel anal-
yses of the same waters, the one series of counts having
been made upon gelatin plates at the ordinary tempera-
ture of the room, the other upon plates of agar-agar
kept for the same length of time in the incubator at
from 87° to 38° C. It will be seen from the table
that much the larger number of colonies—i.e., much
higher results, are always obtained when gelatin is
employed. The importance of this point in the quan-
titative bacteriological analysis of water is too apparent
to require further comment.
BACTERIOLOGICAL STUDY OF WATER. 499
TABLE ILLUSTRATING THE PROPORTION BETWEEN THE RE-
SULTS OBTAINED BY THE USE OF GELATIN AND AGAR-AGAR
IN QUANTITATIVE BACTERIOLOGICAL ANALYSIS OF WATER.
RESULTS RECORDED ARE THE NUMBER OF COLONIES THAT
DEVELOPED FROM THE SAME AMOUNT OF WATER IN EACH
SERIES.1
NUMBER OF COLONIES FROM WATER THAT DEVELOPED UroN—
Gelatin plates at 18° to 20° C. Agar-agar plates at 37° to 38° C.
BIO ey eo as. 7 70
230 — » «140
310 . ny os . $180
340 . 4160
650 . ~ £210
ot ee # ‘ { 320
380 ‘ es “es 1290
iol $ ee ee ee) 1)
1000 i oy 4c - « $100
8907 2. . ‘i . '130
340) . ‘ . & . £280
BIO) a ks i et { 210
4902. a) eh Saks ae pO)
580 | 100
Another point of equal importance in its influence
upon the number of colonies that develop is the reac-
tion of the gelatin. A marked excess of either alka-
linity or acidity always has a retarding effect upon
many species found in water. Experience at Law-
rence has shown that gelatin of such a degree of acidity
as to require the further addition of from 15 to 20 e.c.
per litre of a normal caustic alkali solution to bring it to
the phenolphtalein neutral point gives, on the whole,
the best results. Thus, by way of illustration, Fuller
found that a sample of Merrimac River water gave
5800 colonies per c.c. on phenolphtalein neutral gel-
atin, 15,000 colonies on gelatin that would need 20 c.c.
of normal alkali solution to bring it up to the phenol-
1I am indebted to Dr. James Homer Wright, Thomas Scott Fellow in
Hygiene (1892-’93), University of Pennsylvania, for the results presented in
this table.
500 BACTERIOLOGY.
phtalein neutral point—z.e., a feebly acid nutrient gel-
atin, and 500 colonies on a gelatin so alkaline as to
require 20 c.c. of a normal acid solution to bring it
back to the phenolphtalein neutral point.
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 PLATES.
—For convenience in counting colonies on plates or in
tubes it is customary to divide the whole area of the
gelatin occupied by colonies into smaller areas, and
either count all the colonies in each of these areas and
add the several sums together for the total, or to count
the number of colonies in each of several areas, ten
or twelve, take the mean of the results and multiply
this by the number of areas containing colonies. This
latter procedure obtains, of course, only when all the
areas are of the same size.
By this latter method, however, the results vary so
much in different counts of the same plate that they
cannot be considered as more than rough approxima-
tions.
Nore.—Prepare a plate; calculate the number of
colonies upon it by this latter method. Now repeat
the calculation, making the average from another set
of squares. Now actually count the entire number of
colonies on the plate. Compare the results.
For facilitating the counting of colonies several very
convenient devices exist.
BACTERIOLOGICAL STUDY OF WATER. 501
WoLFFHUGEL’s COUNTING-APPARATUS.—Thisappa-
ratus (Fig. 99) consists of a flat wooden stand, the 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.
Fig. 99.
La
eA
SERS
ST
Sok
Wolffhiigel’s apparatus for counting colonies.
When the gelatin plate containing the colonies. has been
placed upon this background of glass, it is then covered
by a transparent glass plate which swings on a hinge.
This plate, which is ruled in square centimetres and
subdivisions, when in position is just above the colo-
nies, without touching them.
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;
or, as has already been indicated, if the number of colo-
nies be very great a mean may be taken of the number
22%
502 BACTERIOLOGY.
in several (6 or 8) squares; this is to be multiplied by
the total number of squares occupied by the gelatin.
The result is an approximation of the total number of
colonies.
When the colonies are quite small, as is frequently
the case, the counting may be rendered easier by the
use of a small hand-lens.
Fic. 100.
ii;
oe
Gin
quid
SS ||
——— a
Lens for counting colonies.
In Fig. 100 is seen the form of hand-lens commonly
employed with this apparatus. Several useful modifi-
cations of this apparatus have been introduced. The
most important is that of Lafar (Cenlralblati fiir Bakte-
riologie und Parasitenkunde, 1891, Band xv. p. 331).
Lafar’s counter consists of a glass disk of the diameter
of ordinary size Petri dishes. It is supplied with a
collar or flange that fits around the bottom of the Petri
dish, and thus holds the counter in position. The disk
is ruled with concentric circles and its area divided into
sectors of such size that the spaces between the con-
centric circles and the radii forming the sectors are of
equal size. Three of the sectors are subdivided into
smaller areas of equal size for convenience in counting
when the colonies are very numerous. The principles
involved are similar to those of the preceding appara-
tus, but the circular form of the apparatus admits of
BACTERIOLOGICAL STUDY OF WATER. 503
more exactness when counting colonies on a circular
plate.’
Parks (Journ. Bact. and Path., 1896, vol. iv. No. 1)
has introduced a cheap and convenient modification of
Lafar’s apparatus.” It consists of a sheet of white
paper on which is printed a black disk that is ruled
Fra. 101.
Or
6 8
Park’s apparatus for counting colonies (reduced one-third).
with white lines, in somewhat the same fashion as is
Lafar’s counter, though the areas of the smallest sub-
divisions are not of one size and do not bear a constant
1 Lafar’s apparatus is to be obtained from F. Mollenkopf, 10 Thorstrasse,
Stuttgart, who holds the patent forit. Its price is about 8 or 9 marks.
2 Copies of this apparatus are to be had of Ash & Co., 42 Southwark Street,
London, or of Lentz & Sons, North Eleventh Street, Philadelphia, Pa. (The
cost is but a few cents per copy.)
504 BACTERIOLOGY.
relation to each other. To use this apparatus (Fig.
101) the Petri dish is placed centrally upon it, the
cover of the dish is removed, and the colonies are
counted as they lie over the spaces bounded by the
white lines on the black disk beneath. When the
plate is centred over the black disk the portion lying
over one sector is exactly one-sixteenth of the whole
plate.
Esmarcn’s Counter.—Esmarch has devised a
counter (Fig. 102) for estimating the number of colo-
nies present when they are upon a cylindrical surface,
as when in rolled tubes. The principles and methods
of estimation are practically the same as those given for
Wolff hiigel’s apparatus.
Fie. 102.
Esmarch’s apparatus for counting colonies in rolled tubes.
A simpler method than by the use of Esmarch’s appa-
ratus may be employed for counting the colonies in
BACTERIOLOGICAL AIR ANALYSIS. 505
rolled tubes. It consists in dividing the tube by lines
into four or six longitudinal areas, which are subdivided
by transverse lines drawn about 1 or 2cm. apart. The
lines may be drawn with pen and ink. They need not
be exactly the same distance apart nor exactly straight.
Beginning with one of these squares at one end of the
tube, which 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; the sums are then added
together, and the result gives the number of colonies in
the tube. This method may be facilitated by the use
of a hand-lens.
In all these methods there is one error that is diffi-
cult to eliminate: it is assumed that each colony repre-
sents the outgrowth from a single organism. This is
probably 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.
BACTERIOLOGICAL AIR ANALYSIS.—Quite a number
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,
506 BACTERIOLOGY.
or of filtering the bacteria from the air by means of
porous and liquid substances, and studying the organ-
isms thus obtained. (Miguel, Petri, Strauss, Wiirz,
Sedgwick-Tucker. )
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
Fig. 103.
we ———
Petri's apparatus for bacteriological analysis of air. The tube
packed with sand is seen at the point a.
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 to have more of speculative than of real value.
BACTERIOLOGICAL AIR ANALYSIS. 507
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. 103.) When the aspiration is finished the sand
is mixed with liquefied gelatin, plates are made, and
the number of developing colonies counted, the results
giving the number of organisms contained in the volume
of air aspirated through the sand.
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 and Tucker, who
employ granulated sugar instead of the sand; this, when
brought into the liquefied gelatin, dissolves, and no such
error as that possible in the Petri method can be made.
Sepewick-TuckerR Metitop.—On the whole, the
method proposed by Sedgwick and Tucker gives such
uniform results that it is to be recommended above the
others. It is as follows:
The apparatus employed by them consists essentially
of three parts:
1. A glass tube of a special form, to which the name
aérobioscope has been given.
2, A stout copper cylinder of about sixteen litres
capacity, provided with a vacuum-gauge.
3. An air-pump.
The aérobioscope (Fig. 104) is about 35 cm. in its
entire length; it is 15 em. long and 4.5 em. in diam-
eter at its expanded part; one end of the expanded
part is narrowed down to a neck 2.5 cm. in diameter
and 2.5 em. long. To the other end is fused a glass
tube 15 em. long and 0.5 em. inside diameter, in which
is to be placed the filtering material.
508 BACTERIOLOGY.
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 6), and also at the
large end (c), the apparatus is plugged with cotton.
The Sedgwick-Tucker aérobioscope.
When thoroughly 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 thoroughly dried and sterilized No. 50
granulated sugar is poured in until it just fills the 10
em. (d) of the narrow tube above the wire-gauze. This
column of sugar is the filtering material employed to
engage and retain the bacteria. After pouring in the
sugar the cotton-wool plug is replaced, and the tube is
again sterilized at 120° C. for several hours.
Taking the air sample. In order to measure the
amount of air used the value of each degree on the
vacuum-gauge is determined_in terms of air by means
of an air-meter, or by calculation from the known ca-
pacity of the cylinder. This fact ascertained, the nega-
tive 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
BACTERIOLOGICAL AIR ANALYSIS. 509
the mark corresponding to the amount of air re-
quired.?
A sterilized aérobioscope is now to be fixed in the
upright position and its small end connected by a rubber
tube with a stopcock on the cylinder, or to a glass tube
tightly fixed in the neck of an aspirating bottle by
means of a perforated rubber stopper. The cotton
plug is then moved from the upper end of the aérobio-
scope, and the desired amount of air is aspirated through
the sugar. Dust particles and bacteria will be held
back by the sugar. During manipulation the cotton
plug is to be protected from contamination.
When the required amount of air has been aspirated
through the sugar the cotton plug is replaced, and by
gently tapping the aérobioscope while held 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 ¢,
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 aérobio-
scope by the use of a small, sterilized, cylindrical funnel
(Fig. 105), 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.
1 Such a cylinder and air-pump are not necessary. A pair of ordinary as-
pirating bottles of known capacity graduated into litres and fractions thereof
answer perfectly well. Or one can determine by the weight of water that
has flowed from the aspirator the volume of air that has passed in to take
its place—i, e., the volume of air that has passed through the aérobioscope.
510 BACTERIOLOGY.
By the employment of this apparatus one can make
these analyses at any place, and can, without fear of
contamination, carry the tubes to the laboratory, where
the cultivation part of the work may be done.
Fic. 105.
Bent funnel for use with aérobioscope.
Aside from this advantage, 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.
BaAcTERIOLOGICAL Stupy oF THE Sort.—Bacterio-
logical study of the soil may be made by either breaking
up small particles of earth in liquefied media and mak-
ing plates directly from this, or by what is perhaps a
better method, as it gets rid of insoluble particles which
may give rise to errors: breaking up the soil in steril-
ized water and then making plates immediately from
the water.
BACTERIOLOGICAL STUDY OF THE SOIL. 511
It must be borne in mind that many of the ground
organisms belong to the anaérobic group, so that in
these studies this point should be remembered and the
methods for the cultivation of such organisms practised
in connection with the ordinary methods. It must also
be remembered that the nitrifying organisms, every-
where present in the ground, cannot be isolated by the
ordinary methods, and will not appear in plates made
after either of the above plans. The special devices
that have been arranged for their cultivation will be
found in the chapter on soil organisms.
CHAPTER XXVIII.
Methods of testing disinfectants and antiseptics—Experiments illustrating
the precautions to be taken—Experiments in skin disinfection.
THERE are several ways of determining the germicidal
value of chemical substances, the most common being
to expose organisms dried upon bits of silk thread to
the disinfectant for different lengths of time, and then,
after removing, and carefully washing the threads in
water, to place them in nutrient media at a favorable
temperature, and notice if any growth appears. If no
growth results, the disinfection is presumably successful.
Another method is to mix fluid cultures of bacteria with
the disinfectant in varying proportions, and, after dif-
ferent intervals of time, to determine if disinfection is
in progress by transferring a portion of the mixture to
nutrient media, just as in the other method of work.
By the former process the bits of thread, usually
about 1 to 2 em. long, are placed in a dry test-tube
provided with a cotton plug and carefully sterilized,
either by the dry method or in the steam sterilizer,
before using. They are then immersed in a pure
bouillon culture or in a salt solution suspension of the
organism upon which the disinfectant is to be tested.
I say “‘pure culture” because it is always desirable in
testing a new germicide to determine its value as such
on several different resistant species of bacteria, both
in the vegetating and in the spore stage. After the
threads have remained in the culture or suspension for
METHODS OF TESTING DISINFECTANTS. 513
from five to ten minutes they are removed under anti-
septic precautions and carefully separated and spread
out upon the bottom of a sterilized Petri dish. This is
then placed either in the incubator at a temperature not
exceeding 38° C. until the excess of fluid has evapor-
ated, or in a desiccator over sulphuric acid, calcium
chloride, or any other drying agent, but they are not
left there until absolutely dry, only until the ewcess of
moisture has disappeared. When sufficiently dry they
can then be employed in the test. This is done by
immersing them in solutions of the disinfectant of dif-
ferent but known strengths for a fixed interval of time,
say one or two hours, after which they are removed,
rinsed off in sterilized distilled water to remove the
excess of disinfectant adhering to them, and placed in
fresh sterilized culture media, which is then placed in
the incubator at from 37° to 38° C. If after twenty-
four, forty-eight, or seventy-two hours a growth occurs
at or about the bit of thread, and this growth consists
of the organism upon which the test was made, mani-
festly there has been no disinfection; if no growth
occurs after, at most, ninety-six hours, it is safe to pre-
sume that the bacteria have been killed, unless our
efforts at rinsing off the excess of disinfectant from
the thread have not been successful, and a small
amount of disinfectant is now active in preventing
development—i.¢., is acting as an antiseptic.
By the latter process, in which cultures or suspen-
sions of the organisms are mixed with different but
known strengths of the disinfectant, a small portion of
the mixture, usually a loopful or a drop, is transferred
at the end of a definite time to the fresh medium
which is to determine whether the organisms have
514 BACTERIOLOGY.
been killed or not. This is commonly a tube of fluid
agar-agar, which is poured out into a Petri dish,
allowed to solidify, and placed in the incubator, as in
the other experiment.
After the minimum strength of disinfectant necessary
to destroy the vitality of the organisms with which we
are working has been determined, for any fixed time, it
then remains for us to decide what is the shortest time
in which this strength will have the same effect. We
then work with a constant dilution of the disinfectant,
but with different intervals of exposure—one, five, ten
minutes, etc.—until we have decided not only the mini-
mum amount of disinfectant required for the destruction
of the bacteria, but the shortest time necessary for this
under known conditions.
A factor not to be lost sight of is the temperature
under which these experiments are conducted, for it
must always be borne in mind that the action of a dis-
infectant is usually more energetic at a higher than at a
lower temperature.
Now in both of these methods it is easy to seg that
unless special precautions are taken a minute portion of
the disinfectant may be carried along with the thread,
or drop, into the medium which is to determine whether
the organisms do or do not possess the power of growth,
and here have a restraining or antiseptic action. For
organisms jn their normal condition—that is, those
which have never been exposed to the action of a dis-
infectant, the amount of certain disinfectants that is
necessary to restrain growth is very small indeed, and
for organisms that have already been exposed for a
time to such agents this amount is even much less.
It is plain, then, that if the test is to be an accurate
METHODS OF TESTING DISINFECTANTS. 515
one, precautions must be taken against admitting this
minute trace of disinfectant to the medium with which
we are to determine if the bacteria that have been
exposed to the disinfectant have been killed or not.
The precautions that have hitherto been taken to
prevent this accident are, where the threads are em-
ployed, washing them in sterilized distilled water and
then in alcohol; or, where fluid cultures were mixed
with the disinfectant in solution, an effort was usually
made to dilute the amount of disinfectant carried over
to a point at which it loses its inhibiting power.
While such precautions are sufficient in many cases,
they do not answer for all. Certain chemicals have the
property of combining so firmly with the threads upon
which the bacteria are located as to require other special
means of ridding the threads of them; and in solutions
in which proteid substances are present along with the
bacteria a similar union between them and the disin-
fectant may likewise take place. In both instances this
amount of disinfectant adhering to the silk threads or
in combination with the proteids must be gotten rid of,
otherwise the results of the test may be fallacious. A
partial solution of the problem comes from studies that
have been made upon corrosive sublimate in its various
applications for disinfecting purposes, and in this con-
nection it has been shown by Shaefer' that it is impos-
sible to rid silk threads of the corrosive sublimate ad-
hering to them by simple washing, as the sublimate
acts as a mordant and forms a firm union with the tis-
sues of the threads. Braatz’ found the same to hold
good for catgut. For example, he found that catgut
1 Shaefer: Berliner klin. Woch., 1890, No. 3, p. 50.
2 Braatz: Ceutr. f. Bakt. und Parasitenkunde, Bd. viii. No. 1, p. 8.
516 BACTERIOLOGY.
which had been immersed in solutions of sublimate
gave the characteristic reactions of the salt after having
been immersed in distilled water, which had been re-
peatedly renewed, for five weeks.
He remarks that a similar firm combination between
sublimate and cotton will take place after a longer time,
but it occurs so slowly that it cannot interfere with dis-
infection experiments in the same way as he believes the
employment of silk to act.
The most successful attempt at removing all traces of
sublimate from the threads or from the proteid sub-
stances in which are located the bacteria whose vitality
is to be tested is that made by Geppert, who subjected
them to the action of ammonium sulphide in solution.
By this procedure the mercury is converted into insolu-
ble sulphide, and does not now have an inhibiting effect
upon the growth of those bacteria that may not have suc-
cumbed to its action when in the form of the bichloride.
In the second method of testing disinfectants, men-
tioned above—that is, when cultures of bacteria and
solutions of the disinfectant are mixed, and after a time
a drop of the mixture is removed and added to sterile
nutrient media, the inhibiting amount of disinfectant
can readily be gotten rid of by dilution—that is to say,
instead of transporting the drop directly to the fresh
medium, add it to 10 or 12 ¢.c. of sterilized salt-solu-
tion (0.6-0.7 per cent. of NaCl in distilled water), or
distilled water, and after thoroughly shaking add a drop
of this to the medium in which the power of develop-
ment of the bacteria is to be determined.
Another important point to be borne in mind in test-
ing disinfectants is the necessity of so arranging the
conditions that each individual organisms will be ex-
METHODS OF TESTING DISINFECTANTS. 517
posed to the action of the agent used.
When clumps
of bacteria exist we are not always assured of this, for
only those on the surface of the clump
may be affected, while those in the
centre of the mass may entirely escape.
being protected by those surrounding
them. These clumps and minute masses
are especially liable to be present in
fluid cultures and in suspensions of
the bacteria, and must be eliminated
before the test is begun, if this is to be
made by mixing them with solutions
of the agent to be tested. This is
best accomplished in the following
way: the organisms should be culti-
vated in bouillon containing sand
or finely divided particles of glass;
after growing for a sufficient length of
time they are then to be shaken thor-
oughly, in order that all clumps may
be mechanically broken up by the sand.
The culture is then filtered through a
tube containing closely packed glass
wool.
The filtration may be accomplished
without fear of contamination of the
culture by the employment of an
Allihin tube, which is practically noth-
ing more than a thick-walled test-tube
drawn out to a finer tube at its blunt
end so as to convert it into a sort of
Fie. 106.
Bs iN)
—
Cylindrical funnel
used for filtering cul-
tures on which dis-
infectants are to be
tested.
cylindrical funnel. The tube when finished and ready
for use has the appearance given in Fig. 106.
23
518 BACTERIOLOGY.
The whole tube, after being plugged at the bottom
with glass wool and at its wide open extremity with
cotton wool (a, Fig. 106), is placed vertically, small
end down, into an Erlenmeyer flask of about 100 ec.
capacity and sterilized in a steam sterilizer for the
proper time. It is kept in the covered sterilizer until
it is to be used, which should be as soon as possible
after sterilization.
The watery suspension or bouillon culture of the
organisms is now to be filtered repeatedly through the
glass wool into sterilized flasks until a degree of trans-
parency is reached which will permit the reading of
moderately fine print through a layer of the fluid
about 2 em, thick—z.e., through an ordinary test-tube
full of it. It can then be subjected to the action of the
disinfectant, and, as a rule, the results are far more
uniform than when no attention is paid to the exist-
ence of clumps. It is hardly necesgary to say that in
the practical employment of disinfectants outside the
laboratory no such precautions are taken, but in labor-
atory work, where it is desired to determine ewactly the
value of different substances as germicides, all the pre-
cautions that have been mentioned will be found essen-
tial to precision.
The disinfectant value of gases and vapors is deter-
mined by their influence upon test-objects in closed
chambers. The object is to determine the proportion
of the gas, when mixed with air, that is required to
destroy the bacteria exposed to its action in a given
time. For this purpose the test is commonly made as
follows: under a sterilized bell glass of known capacity
the test-objects are placed. Into the chamber is then
admitted sufficient of a known mixture of air and the
METHODS OF TESTING DISINFECTANTS. 519
gas under consideration to eliminate completely all the
air; or, the pure gas itself may be introduced in the
amount necessary to give the desired dilution when
mixed with the air in the chamber. After the time de-
cided upon for the test the infected articles are removed
and the vitality of the bacteria upon them is determined.
In the case of the vapors of volatile fluids, such, for
instance, as formaline, the fluid is placed under the bell
glass in an open dish; in another open dish the test-
objects are placed. The bell glass is then sealed to an
underlying ground-glass plate by vaseline or paraffin,
and the fluid is allowed to vaporize at ordinary room
temperature. The point here to be decided is the vol-
ume or weight of such a fluid that it is necessary to
expose in an air chamber of known cubic capacity in
order that bacteria may be destroyed by its vapor in a
given time.
In determining the germicidal value of different
chemical agents upon certain pathogenic bacteria, sus-
ceptible animals are sometimes inoculated with the
organisms after they have been exposed to the disin-
fectant. If no pathological condition results, disinfec-
tion is presumed to have been successful; while if the
condition characteristic of the activities of the given
organism in the tissues of this animal appears, the
reverse is the case. The objections to this method
that have been raised are: ‘‘ First. The test-organisms
may be modified as regards reproductive activity with-
out being killed; and in this case a modified form of
the disease may result from the inoculation, of so mild
a character as to escape observation. Second. An ani-
mal that has suffered this modified form of the disease
enjoys protection, more or less perfect, from future at-
520 BACTERIOLOGY.
tacks, and if used for a subsequent experiment may, by
its immunity from the effects of the pathogenic test-
organism, give rise to the mistaken assumption that
this had been destroyed by the action of the germicidal]
agent to which it had been subjected.” (Sternberg.)
DETERMINATION OF ANTISEPTIC PROPERTIES.
In this test sterile media are employed and are usu-
ally arranged in two groups: the one to remain normal
in composition and to serve as controls, while to the.
other is to be added the substance to be tested in dif-
ferent but known strengths. It is customary to employ
test-tubes each containing an exact amount of bouillon,
gelatin, or agar-agar, as the case may be. To each tube
a definite amount of the antiseptic is added, and if it is
not of a volatile nature or not injured by heat, they
may then be sterilized. After this they are to be in-
oculated with the organism upon which the test is to
be made, and at the same time one of the ‘‘ control’
tubes (one of those to which no antiseptic has been
added) is inoculated. They are all then to be placed
in the incubator and kept under observation. If at the
end of twenty-four, forty-eight, or seventy-two hours
no growth appears in any but the ‘‘ control’’ tubes, it
is evident that the antiseptic must be added in smaller
amounts, for we are to determine the point at which it
is not as well as that at which it is capable of prevent-
ing development. The experiment is then repeated,
using smaller amounts of the antiseptic until we reach
a point at which growth just occurs notwithstanding
the presence of the antiseptic ; its antiseptic strength
then lies a trifle above the amount present in this tube.
EXPERIMENTS. 521
If, for example, there was no development in the tubes
in which the antiseptic was present in the proportion
of 1: 1000 and growth in the one in which it was
present in 1 : 1400, the experiment would be repeated
with strength of the antiseptic corresponding to 1 : 1000,
1: 1100, 1 : 1200, 1: 1300, 1 : 1400, and in this way one
gradually strikes the point at which growth is just pre-
vented. This point represents the antiseptic value of
the substance used for the organism upon which it has
been tested.
EXPERIMENTS.
Into each of three tubes containing 10 c.c.—one of
normal salt-solution, another of bouillon, a third of fluid
blood-serum—add as much of a culture of the staphylo-
coccus pyogenes aureus as can be held upon the looped
platinum needle. Mix this thoroughly, so that no clumps
exist, and then add exactly 10 c.c. of 1 : 500 solution of
corrosive sublimate. Mix it thoroughly, and at the
end of three minutes transfer a drop from each tube
into a tube of liquefied agar-agar, and pour this into a
Petri dish. Label each dish carefully and place them
in the incubator. Are the results the same in all the
plates? How are the differences to be explained? To
what strength of the disinfectant were the organisms
exposed in the experiment ?
Into each of two tubes containing 10 ¢c.c.—the one
of normal salt-solution, the other of bouillon—add as
much of a spore-containing culture of anthrax bacilli
as can be held upon the loop of the platinum wire.
Mix this thoroughly so that no clumps exist, and then
add exactly 10 c.c. of a 1: 500 solution of corrosive
sublimate. Mix thoroughly, and at the end of five
522 BACTERIOLOGY.
minutes transfer a drop from each tube into a tube of
liquefied agar-agar. Pour this immediately into a
Petri dish. Label each dish carefully and place them
in the incubator. Note the results at the end of twenty-
four, forty-eight, and seventy-two hours. How do
you explain them ?
Make identically the same experiment with the same
spore-containing culture of anthrax bacilli, except that
the drop from the mixture is to be transferred to 10 c.c.
of a mixture of equal parts of ammonium sulphide and
sterilized distilled water. After remaining in this for
about half a minute, a drop is to be transferred to a
tube of liquefied agar-agar, poured into Petri dishes,
labelled, and placed in the incubator. Note the results.
Do they correspond with those obtained in the pre-
ceding experiment? How are the differences ex-
plained ?
Prepare a 1: 1000 solution of corrosive sublimate.
To each of twelve tubes containing exactly 10 c.c. of
bouillon add one drop to the first, two drops to the
second, and so on until the last tube has had twelve
drops added to it. Mix thoroughly and then inoculate
each with one wire-loopful of a bouillon culture of
staphylococcus pyogenes aureus. Place them all in the
incubator after carefully labelling them. Note the order
in which growth appears.
Do the same with anthrax spores, with spores of
bacillus subtilis and with the typhoid bacillus, and see
how the results compare. From these experiments
what will be the strength of corrosive sublimate neces-
EXPERIMENTS. 523
sary to act as an antiseptic under these conditions for
the organisms employed ?
Make a similar series of experiments, using a 5 per
cent. solution of carbolic acid.
Determine the antiseptic point of the common disin-
fectants for the organisms with which you are working.
Determine the time necessary for the destruction of
the organisms with which you are working, by corro-
sive sublimate in 1 : 1000 solution, under different con-
ditions—with and without the presence of albuminous
bodies other than the bacteria, and under varying con-
ditions 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 organism into not less than 10 c.c. of sterilized
physiological salt-solution, in which they may be thor-
oughly shaken for from one to two minutes, or into the
solution of ammonium sulphide of the strength given.
To 10 c.c. of a bouillon culture of staphylococcus
pyogenes aureus, or anthrax spores, add 10 ¢.c. of cor-
rosive sublimate in 1: 500 solution, and allow it to re-
main in contact with the organisms for only one-half
the time necessary to destroy them (use an organism
for which this has been determined). Then transfer a
drop of the mixture to each of three liquefied agar-agar
tubes and pour them into Petri dishes. Place them in
the incubator and observe them for twenty-four, forty-
524 BACTERIOLOGY.
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 cul-
ture 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 necessary to destroy them,
when transferred directly to a favorable culture medium
do not grow, and yet, when the same organism which has
not been exposed to sublimate at all is planted upon the
same medium it does grow. How is this to be ac-
counted 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 some one pour am-
monium sulphide over the points from which the scrap-
EXPERIMENTS. 525
ings are to be made. After it has been on the hands
about three minutes again scrape, and note the result
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
ammonium sulphide, and then prepare plates from
scrapings from the points mentioned.
In what way do the results of these experiments
differ one from another ?
To what are these differences due?
What have these experiments taught?
In making the above experiments it must be remem-
bered that the strictest care is necessary in order to
prevent the access of germs from without into our
media. ‘The hand upon which the experiment is being
performed must be held away from the body and must
not touch any object not concerned in the experiment.
The scraping should be done with the point of a knife
that has been sterilized in the flame and allowed to cool.
The scrapings may be transferred directly from the
knife-point to the gelatin by means of a sterilized plat-
inum wire loop.
The brush used should be thoroughly cleansed and
always kept in 1: 1000 solution of corrosive sublimate.
It should be washed in hot water before using.
23*
APPENDIX.
List of apparatus and materials required in a begin-
ner’s bacteriological laboratory:
MICROSCOPE AND ACCESSORIES.
Microscope with coarse and fine adjustment and
heavy, firm base; Abbe sub-stage condensing system,
arranged either as the ‘‘simple’’ or as the regular Abbe
condenser, in either case to be provided with iris dia-
phragm; objectives equivalent, in the English nomen-
clature, to about one-fourth inch and one-sixth inch
dry, and one-twelfth inch oil-immersion system; a
triple revolving nose-piece; three oculars, varying in
magnifying power; and a bottle of immersion oil.
Glass slides, English shape and size and of colorless
glass.
Six slides with depressions in centre of about 6 to 8
mm. in diameter.
Cover-slips, 15 by 15 mm. square and from 0.165 to
0.18 mm. thick.
Forceps. One pair of fine-pointed forceps and one
pair of the Cornet or Stewart pattern, for holding
cover-slips.
Platinum needles in glass handles. One straight,
of about 4 cm. long; one looped at the end of about 4
528 BACTERIOLOGY.
cm. long; and one straight of about 8 em. long. Glass
handles to be of about 3 mm. thickness and from 15 to
17 em. long.
STAINING- AND MOUNTING-REAGENTS.
200 e.c. of saturated alcoholic solution of fuchsin.
200 ¢.c. of saturated alcoholic solution of gentian
violet.
200 c.c. of saturated alcoholic solution of methylene-
blue.
200 grammes of pure aniline.
200 grammes of C. P. carbolic acid.
500 grammes of C. P. nitric acid.
500 grammes of C. P. sulphuric acid.
200 grammes of C. P. glacial acetic acid.
1 litre of ordinary 93-95 per cent. alcohol.
1 litre of absolute alcohol.
500 grammes of ether.
500 grammes of pure xylol.
50 grammes of Canada balsam dissolved in xylol.
100 grammes of Schering’s celloidin.
10 grammes of iodine andZ30 grammes of iodide of
potassium in substance.
100 grammes of tannic acid.
100 grammes of ferrous sulphate.
Distilled water.
FOR NUTRIENT MEDIA.
+ pound Liebig’s or Armour’s beef extract.
250 grammes Witte’s peptone.
2 kilogrammes of gold label gelatin (Hesteberg’s).
GLASSWARE. 529
100 grammes of agar-agar in substance.
200 grammes of sodium chloride (ordinary table salt).
500 grammes of pure glycerin.
50 grammes of pure glucose.
20 grammes of pure lactose.
100 grammes of caustic potash.
200 c.c. of litmus tincture.
10 grammes of rosolic acid (corallin).
Blue and red litmus paper; curcuma paper.
5 grammes of phenolphtalein in substance.
Filter paper, the quality ordinarily used by druggists.
100 grammes of pyrogallic acid.
1 kilogramme C. P. granulated zinc.
GLASSWARE.
200 best quality test-tubes, slightly heavier than those
sold for chemical work, about 12 to 13 em. long and
12 to 14 mm. inside diameter.
15 Petri double dishes about 8 or 9 em. in diameter
and from 1 to 1.5 em. deep.
6 Florence flasks, Bohemian glass, 1000 c.c. capacity.
6 Florence flasks, Bohemian glass, 500 c.c. capacity.
12 Erlenmeyer flasks, Bohemian glass, 100 c.c.
capacity.
1 graduated measuring cylinder, 1000 c.c. capacity.
1 graduated measuring cylinder, 100 c.c. capacity.
25 bottles, 125 c.c. capacity, narrow necks with
ground glass stoppers.
25 bottles, 125 c¢.c. capacity, wide mouths, with
ground glass stoppers.
1 anatomical or preserving jar, with tightly fitting
530 BACTERIOLOGY.
cover, of about 4 litres capacity, for collecting blood-
serum.
2 battery jars of about 2 litres capacity, provided
with loosely fitting, weighted, wire-net covers, for mice.
10 feet of soft glass tubing, 2 or 3 mm. inside diam-
eter.
20 feet of soft glass tubing, 4 mm. inside diameter.
6 glass rods, 18 to 20 cm. long and 3 or 4 mm. in
diameter.
6 pipettes of 1 c.c. each, divided into tenths.
2 pipettes of 10 c.c. each, divided into cubic centi-
metres and fractions.
1 burette of 50 c.c. capacity, divided into cubic cen-
timetres and fractions.
1 separating funnel of 750 c.c. capacity, for filling
tubes.
2 glass funnels, best quality, about 15 em. in diam-
eter. ;
2 glass funnels, best quality, about 8 cm. in diameter.
2 glass funnels, best quality, about 4 or 5 cm. in
diameter.
2 porcelain dishes, 200 ¢.c. capacity.
6 ordinary water tumblers for holding test-tubes.
1 ruled plate for counting colonies.
1 gas generator, 600 ¢.c. capacity, pattern of Kipp
or v. Wartha.
BURNERS, TUBING, ETC.
2 Bunsen burners, single flame.
1 Rose burner.
1 Koch safety burner, single flame.
6 feet of white rubber gas-tubing.
INCUBATORS AND STERILIZERS. 531
12 feet of pure red rubber tubing of 5 to 6 mm. inside
diameter.
1 thermo-regulator, pattern of L, Meyer or Reichert.
2 thermometers, graduated in degrees Centigrade,
registering from 0° to 100°, graduated on the stem.
1 thermometer graduated in tenths and registering
from 0° to 50° C,
1 thermometer registering to 200° C.
INSTRUMENTS, ETC.
1 microtome, pattern of Schanze, with knife.
1 razor strop.
6 cheap quality scalpels, assorted sizes. 2 pairs heavy
dissecting-forceps.
1 pair medium-size straight scissors.
1 pair small-size straight scissors.
1 hypodermic syringe that will stand steam steriliza-
tion.
2 teasing-needles.
1 pair long-handled crucible tongs for holding mice.
1 wire mouse-holder.
2 small pine boards on which to tack animals for
autopsy.
2 covered stone jars for disinfectants and for receiv-
ing infected materials.
INCUBATORS AND STERILIZERS.
1 incubator, simple square form, either entirely of
copper, or of galvanized iron with copper bottom.
1 medium-size hot-air sterilizer with double walls,
asbestos jacket, and movable false bottom of copper
plates.
532 BACTERIOLOGY.
1 medium-size steam sterilizer; either the pattern of
Koch, or that known as the Arnold steam sterilizer,
preferably the latter.
MISCELLANEOUS.
1 pair of balances, capacity 1 kilogramme; accurate
to 0.2 gramme.
1 set of cork borers.
1 hand-lens.
1 wooden filter-stand.
2 iron stands with rings and clamps.
3 round, galvanized iron wire baskets to fit loosely
into steam sterilizer.
3 square, galvanized iron wire baskets to fit loosely
into hot-air sterilizer.
1 sheet-iron box for sterilizing pipettes, etc.
1 covered, agate-ware saucepan, 1200 ¢.c. capacity.
2 iron tripods.
1 yard of moderately heavy wire gauze.
2 test-tube racks, each holding 24 tubes, 12 in a row.
1 constant-level, cast-iron water-bath.
2 potato-knives.
2 test-tube brushes with reed handles.
Cotton batting.
Copper wire, wire nippers.
Round and triangular files.
Labels.
Towels and sponges.
IND
Fee substage condensing sys-
tem of, 26
Abscess, histological study of, 249
production of, 247, 248, 249
Aérobic bacteria, 33
Aérobioscope, 508
Agar-agar, preparation of (see
edia).
properties of, 76, 77
Agglutinin, 497
Air, bacteriological analysis of,
505-510
Petri’s method for, 507
Sedgwick-Tucker meth-
od, 507-510
Alexines, 459, 464
Anaérobic bacteria, 33
methods of cultivating,
194-200
Buchner’s, 196
Esmarch’s 199
Frankel’s, 196
Hesse’s, 194
Kitasato and Weil’s,
199
Koch’s, 194
Liborius’s, 194
Aniline dyes for differentiating
bacteria, 190
Animals, fluctuations in weight
and temperature of, 221-
227
inoculation of, 206-221
apparatus used in, 208,
209, 210, 217
intralymphatic, 218
intraocular, 220 ;
intraperitoneal and pleu- |
ral, 218
intravascular, 212
EX.
Animals, inoculation of, subcuta-
neous, 206
observations of, after inocula-
tion, 221-227
post-mortem examination of,
228-233
cultures from tissues at,
230
disinfection of imple-
ments after, 232
disposal of remains from,
232
external inspection, 228
incision through skin,
228
Nuttall’s spear for use
at, 230
opening the body cavi-
ties, 229
position of animal, 228
precautions during, 228
preservation of tissues
from, 231
Anthrax, 412-427
animals that are susceptible
to, 420
bacillus of, 412-427
biology of, 415-418
discovery of, 17, 412
experiments with, 422-
427
morphology of, 412-
415
pathogenesis of, 418-
420
protective inoculation
against, 420-422
spore- formation, 413-
415
staining of, 417
534 INDEX.
Anthrax, symptomatic, bacillus ' Bacteria, constancy in morphol-
of, 446-452
Antiseptic, definition of, 71
Antiseptics, tests of, 520
)ACILLI, 36-38
Bacteria, aérobic, 33
Apparatus necessary to bacteri-
ological work, 526-532
preparation of, 109 /
Appendix, list of apparatus, 526 |
differentiation from spores, 40
flagella upon, 45
involution-forms of, 39
life-cycle of, 38 |
mode of multiplication, 41-:
spore-formation in, 38, 39
Bacillus anthracis, 412-427
i
motility of, 45
|
i
coli communis, 357-364
“comma,” 365-393
diphtheriz, 325-341
Finkler-Prior, 394-399
influenze, 310-314
lepree, 306, 307
mallei (of glanders),315-324 |
Neapolitanus, 357-364
nitrifying, 428-433
cedematis maligni, 441-446
of bubonic plague, 269-275 |
pyocyaneus, 265-269 {
pseudo-diphtheria, 339
smegma, 305-307
subtilis, 241
symptomatic anthrax, 446 |
syphilis, 305-307 ~
tetani, 434-441
tuberculosis, 299-308
typhi abdominalis, 342-356
anaérobic, 33
methods of cultivating,
194-200
behavior toward staining-re- |
agents, 190
capsule surrounding, 152
chromogenic, 29
classification of, 36
conditions necessary _ to!
growth of, 35
ogy of, 89
definition of, 27
denitrifying, 30
discovery of, 13-15
facultative, 34
fermentation by, 191
apparatus for testing,
192
gases resulting from,
flagellated forms of, 45
identification of, 177
involution-forms of, 39
isolation of, in pure culture,
principles of, 72-74
on slanted media, 123,
124
microscopic examination of,
178-185
modes of multiplication of,
41-43
morphology of, 36-46
motility of, 45-46
nitrifying, 30, 428-433
nutrition of, 31-33
photogenic, 30
points to be observed in de-
scribing, 203
reaction produced by, 189
relation to man, 28, 29
relation to temperature, 34,
results of growth, 29, 30
role in nature, 28
saprogenic, 30
spore -formation of, 38-41,
?
study of, 185
staining-reactions of, 190
systematic study of, 177
thermophilic, 34, 35
thiogenic, 30
zymogenic, 30
: Bacteriology, application of meth-
ods of, 235
Bacterium coli commune, 357-
characteristics of
cultural, 359-361
INDEX.
Bacterium coli commune, char-
acteristics of,
morphologi-
eal, 358
pathogenic, 362
differentiation of,
from bac. typh.
abdom., 361
where found, 357
Behring and Kitasato, 470
Billroth, 23
and Tiegel, 24
Bireh-Hirschfeld, 22
Black leg (see Symptomatic An-
thrax).
Blood, relations to bacteria and
to toxins, 467 ;
Blood-serum as culture medium |
(see Media).
germicidal element of, 467—
469
action of, 464
Bolton’s potato method, 93
Bonnet, 20
Booker’s modification of Es-
march’s method, 121
Bouillon (see Media).
Brieger and Cohn, 441-459
Brooding-oven, 125-127
Brownian motion, 184
Buchner, 467-471
Bulb for water samples, 494
Burdon-Sanderson, 25
Burner, Koch’s safety, for use
with incubator, 127
ARBOLIC acid as disinfect-
ant, 69
Chauveau, 461
Chevreul and Pasteur, 19
Chlorophyll, 27, 28
Cholera Asiatica, diagnosis of,
387-393
method of Schotte-
lius, 374, 375
spirillum of, 365
behavior of, in but-
ter, 385
in milk, 384
in soil, 383
535
Cholera Asiatica, behavior of
spirillum of, in
water, 381-383
characteristics of,
cultural, 368
-876
morphological,
366-368
destiny of, in dead
body, 384-386
effects of drying,
386
existence outside
the body, 381
experiments upon
oe with, 376
general considera-
tions upon, 380
location in the body,
380, 381
poisons produced
by, 375
relation to gases,
874, 386-387
to other bacte-
ria, 874, 385
putrefac-
tion, 384
to sunlight, 383
specific reaction of
immuned __ani-
mals to, 379
toxin of, 458
Chromogenic bacteria, 30
Classen, 23
Cohn, 21
Colon bacillus (see Bacterium
Colli Commune).
Colonies, counting of, 500
study of, 133-135
Comma bacillus (see Cholera
Asiatica).
Cornet, 297
Corrosive sublimate as disinfect-
ant, 66-68
Cooling-stage, 115, 120
Cover-slips, cleaning of, 140
impression, 144
microscopic examination of,
181
to
536
Cover-slips, preparation of, 141
steps in making, 141
Cultures, gelatin, 187
hanging-drop, 183
potato, 188
pure, 135
reactions of, 189
stab- and smear-, 135
Cygnzus, 347
D2 eee solutions,
160
Decomposition, 27
Defensive proteids, 469
Deneke’s cheese spirillum (see
Spirillum tyrogenum).
Denitrifying bacteria, 30
Diphtheria, bacillus of, 325-341
cultural peculiarities of,
330-334
experiments upon, 340
location in tissues, 3835-
337
method of obtaining,
825
modification in viru-
lence, 338
morphology of, 327
pathogenesis of, 3834-
340
poison produced by, 337
potency of, 459
principles of immuniz-
ing against, 430, 431
pseudo-diphtheria bac ,
839
histological changes accom-
panying, 336
Diplococci, 38
Disinfectants and antiseptics, ex-
periments with, 521
general considerations, 64-71
methods of testing, 512
precautions to be ob-
served, 514
use of animals as test-objects
for, 519
use in the laboratory, 70-71
Disinfection, general considera-
tions, 64-71
INDEX.
Disinfection, influence of temper-
ature on, 67
inorganic salts in, 65-67
in the laboratory, 70, 71
modus operandi, 66
reliable agents for purposes
of, 69, 70
selection of agents to be
used in, 65, 66
Dunham’s solution, 104
BERTH, 23
Ehrlich, 23
Emmerich and Fowitzky, 478
and Mattei, 472
Erysipelas, 256
Escherich, 357
Esmarch tubes, 120, 123
Booker’s method of roll-
ing, 121
made of agar-agar, 122
Exposure and contact-experi-
ments upon, 236
IACULTATIVE bacteria, 34
use of the term, 34
Fehleisen, 23
Fermentation, 27, 191
gases resulting from, 193
particular forms of, 30
-tube, 192
method of using, 192-
194
Filter, method of folding, 85-87
Finkler-Prior bacillus, 394-399
Flagella, 45, 46
methods of staining, 165--
159
Bunge’s, 157
Leefiler’s 155
; van Ermengem’s, 158
| Flagellated organisms, 45
Frankland, G. and P. F., 430
Funnel for filling aérobioscope,
510
for filling test tubes, 110,
| 111
for filtering cultures, 517
hot water, 87
INDEX.
eee eimai regulator,
1
Gelatin, cultures in, 187
their characteristics,
187, 188
preparation of (see Media).
properties, 76-78
Geppert, 66, 67, 516
Glanders, 315-324
bacillus of, 317
cultivation of, 318-320
inoculation with, 320
morphology, 317,318
staining of, in tissues,
321
diagnosis of, by use of mal-
lein, 323
by Strauss’s method, 323
manifestations of, 315-317
histology of, 316, 317
susceptibility of animals to,
0
synonyms, 315
Gonococcus, 258-266
appearance in pus, 258
cultivation of, 260
Bumm’s method for the,
260
Wertheim’s method for
the, 260
Wright’s method for
the, 261
distinguishing features of,
morphology of, 259
pathogenesis, 264-265
vitality of, 264
Gonorrheea. pus of, 259
Green pus bacillus (see Bacillus
pyocyaneus).
Guarniari’s agar-gelatin, 107
ALSTED, 219, 246
Hanging-drop, 183
Hankin, 468, 469
and Martin, 468
Henle, 18
Hoffmann, 19
Hot-water funnel, 87
Hydrogen, test for purity of, 198
5387
Hypodermic syringes and _nee-
dles, 213, 217
[BERING of tissues, 164,
165
Immunity, 460
acquired, 460
blood in, 467
conclusions concerning, 480,
483
earlier studies on blood rela-
tive to, 465
“exhaustion” hypothesis, 463
experiments of the Klem-
perers on, 475
humoral theory of, 464
hypothesis of Buchner, 471
evidence in favor of,
472
natural, 460
nature of protective bodies,
467, 469
observations of Behring and
Kitasato, 470
“retention ” hypothesis, 461
theory of Metchnikoff, 463
Incubator, 125, 127
burner for heating, 127
Indol, production of, by bacteria,
200
method of detecting, 201
Infection, 453, 460
chemical nature of, 457
conclusions concerning, 459
modus operandi, 457
poisons present in, 456, 459
study of types of, 453, 458
Influenza, bacillus of, 310, 314
cultivation of, 311, 312
dissemination of, 313
isolation of, from tissues,
313
morphology of, 310
occurrence in tissues, 313
staining of, 310, 311
susceptibility of animals
to, 313
vitality of, 312
Inoculation of animals, 206-221
intraocular, 220
538
Inoculation, intraperitoneal and |
pleural, 218
intravascular, 212
subcutaneous, 206
intralymphatic, 218
apparatus used in, 208, 209,
210, 217
Introduction, 138-26
Involution-forms of bacteria, 39
Isolation of colonies on slanted
media in tubes, 123, 124
t
H
jeepas and Richards, 430
| LEBS, 23-25, 327 |
Klemperer, F. and G., work '
on pneumonia, 475
Koch, fundamental researches of,
5, 26
postulates of, 298 |
safety burner of, 127 |
ACTOSE-LITMUS agar-agar
or gelatin (see Media).
Leeuwenhoek, 13-16
Lens for counting colonies, 502
Lepra bacillus, 306, 307
staining- -peculiarities of, |
306, 807
Letzerich, 23
Levelling-tripod, 115
Lime, chloride of, 71
milk of, 69, 71 |
Litmus milk, 103 |
Leeffler’s alkaline
blue, 147
blood-serum mixture, 107
isolation of the bacillus of
diphtheria, 327
stain for flagella, 46, 155
Leefller and Schtitz, discovery of
the bacillus of glanders, 317
Lukomsky, 23
methylene- :
ALIGNANT cedema, bacillus
of, 441, 446
cultural peculiari-
ties of, 443, 444
INDEX.
| Malignant cedema, bacillus of,
morphology of, 449,
pathogenesis of, 444
susceptibility of ani-
mals to, 444
i Mallein, 323
Meat-extracts in culture media,
84
-infusion, 107
Media, culture, 79
agar-agar, 89
clarification of, 90
filtration of, 90
glycerin, 91
neutralization
79-84
solution of, 89, 91
blood-serum, 95
Councilman - Mal-
lory method, 99
mixture of Leefiler,
107
Nuttall’s
100
original method of
Koch, 95-99
preservation of, 99,
2
of,
method,
by chloroform,
102
sterilization and so-
lidification of, 97-
99
bouillon, 79
neutralization
79-84
gelatin, 84
clarification of, 87
filtration of, 85-
87
solution of, 85
sterilization of, 84,
of,
Guarniari’s
gelatin, 107
lactose-litmus agar-agar
or gelatin, 106
litmus milk, 103
meat-infusion, 107
milk, 103
-agar-agar, 89
agar-agar
INDEX.
Media-, culture, peptone solution,
Dunham’s, 104
rosolic-acid- peptone so-
lution, 105
potatoes, 92
Bolton’s method, 98
Esmarch’s method,
94
mashed, 94
original method, 92
Metchnikoff, 463
Milk (sce Media).
Micrococci, 36, 37
mode of multiplication, 41, 42
Micrococcus lanceolatus, 280 -
285
irregularities in devel-
opment, 282, 284
morphological peculiar-
ities, 281
results of inoculation
with, 284, 285
staining of, 284
susceptibility of animals
to, 285
variations in virulence,
285 .
where found, 282
Micrococcus tetragenus, 279, 285-
cultural peculiarities of,
286-288
morphology of, 286
susceptibility of animals
to, 288
where found, 286
Microscope, parts of, 178-181
Microtome, 163
AGELT, 31
Nassiloff, 23
Needham, 18
Nitrification, 428
Nitrifying bacteria, 428-433
Nitrites, test for, 202
Nitro-monas of Winogradsky,
430-433
cultural peculiarities of,
431-433
morphology of, 431
539
Normal solution, 196
Nuttall, 230, 464-467
ae immersion system, use of,
Oertel, 38, 386
Ozanam, 17
ARASITE, 27
Pasteur, 17,19, 25, 442, 462
Peptone, test of purity of, 104,
105
with rosolic acid, 105
Peritonitis, production of, 247
Petri’s dishes, 119
Pfeiffer, 375, 379, 380
Phagocytosis, 463
Photogenic bacteria, 30
Plague, bubonic, bacillus of, 270-
276
cultivation of, 273
mode of infection with,
275
morphology of, 272
occurrence in tissues,
2738, 275
pathogenesis, 273, 274
vitality of, 274
Plates, apparatus employed in
making, 113-120
Esmarch’s modification, 120
Booker’s modification
of, 121
Koch’s fundamental obser-
vations, 72-73
materials used in making,
113
Petri’s modification, 119
principles involved, 72-76
technique of making, 113-
116
Platinum needles and loops, 114
Plenciz, 16
Post-mortem examination of ani-
mals, 228-233
cultures from tissues at,
230
disinfection of imple-
ments after, 232
540
Post-mortem examination, dis-
posal of remains from,
232
external inspection at,
228
incision through the
skin at, 228
Nuttall’s spear for use
at, 230
opening of the body cav-
ities, 229
position of animal dur-
ing, 228
precautions during, 228
preparation of cover-
slips at, 231
preservation of mate-
rials, 231
Postulates of Koch, 298
Potato, characteristics of cultures
on, 188
preparation for culture pur-
poses (see Media),
Prudden, 293, 467
Pseudo-diphtheria bacillus, 339
-tuberculosis, 309
Pure culture, 135
Pm i appearance of,
2
Putrefaction, 27
Pyemia, production of, 248
Pyocyaneus, bacillus, 266-270
chameleon phenomena
of, 269
pathogenic properties of,
269, 270
protective properties of,
270
Caeaces evil or quarter ill
(see Symptomatic Anthrax).
ECKLINGHAUSEN, 22, 23
Regulator, gas-pressure, 131
thermo-, 128-131
Rindfleisch, 22
Rosolic - acid - peptone solution
(see Media).
Roux and Yersin, 459
INDEX.
APROGENIC bacteria, 30
Saprophyte, 27
role in nature, 28
Sarcine, 38
mode of multiplication, 42
Schottelius’s method of examin-
ing cholera evacuations, 374,
375
Schréder and Dusch, 19}
Schulze, 19
Schwann, 19
Section-cutting, 162
Septiceemia, 279, 280, 285
from micrococcus tetragenus,
285
from sputum, 280
Skin-disinfection, experiments in,
524
Smear-cultures, 135
Smegma bacillus, staining-pecu-
liarities of, 306-308
Soil, bacteriological analysis of,
nitrifying bacteria in, 428
organisms present in, 433
phenomena in operation in,
428-430
Spallanzani, 18, 19
Spirilla, 36-41
Spirillum of Asiatic cholera see
Cholera .
of Deneke, 394-403
biology of, 399-402
morphology of, 399
pathogenesis of, 402
of Finkler-Prior (see Vibrio
Proteus).
of Metchnikoff (see Vibrio
Metchnikovi).
of Miller, 403-406
biology of, 403-406
morphology of, 403
pathogenesis of, 406
tyrogenum (see Spirillum of
Deneke).
undula, 45
Spores, formation of, 43-45
method of studying, 38-
41, 43, 44, 185
mode of development, 43, 44
recognition of, 40, 44
INDEX.
Spores, staining of, 152
Sputum, inoculations with, 279
microscopic examination of,
277, 278
pathogenic properties of, 279,
280
septicemias, 280, 285
tuberculosis, 289
tubercular, 277
Stab-cultures, 135
Staining, methods and solutions
used in, 139-162
acetic acid, 152
Bunge’s, 157
Gabbett’s, 151
general remarks on, 159
Gram’s, 151
Gray’s, 173
Koch - Ehrlich’s, 147, 148, :
172
Kuehne’s, 170
Leeffler’s blue, 147
Leeffler’s flagellar, 155
Meeller’s, 154
ordinary solutions used, 145
bottles for holding, 146
van Ermengem’s, 158
Weigeri’s, 171
Zieh|-Neelsen, 147, 172
Staphylococcus pyogenes albus,
251
aureus, 244-251
cultural peculiari-
ties of, 245-247
pathogenesis, 247
where to be ex-
pected, 246
citreus, 25]
Sterilization, chemical, 64-71
direct, 56-58
experiments upon, 239-243
by heat, 49-64
principles involved, 52
by hot air, 63, 64
apparatus used, 63
by steam, 51-61
apparatus used, 58-62
under pressure, 56, 61, 62
intermittent, 52-55
at low temperature, 55
principles involved, 47-70
541
| Sterilization, use of the term, 47-
|
Sternberg, 282
Strauss’s method for diagnosis of
glanders, 323
Streptococci, 38
mode of multiplication, 42
Streptococcus pyogenes, 252-257
biology of, 252-256
effects of inoculation
; with, 256
morphology of, 253
where to be expected,
25Y, 256
Subtilis bacillus, 241
Suppuration, 244
bacteria common to, 246
general remarks upon, 257
less common causes of, 251,
257, 258
microscopic appearance of
pus, 244
Symptomatic anthrax, bacillus of,
446-452
biology of, 448-451
differentiation from ba-
cillus of malignant
edema, 452
morphology of, 447
pathogenesis, 451
susceptibility of animals
to, 452
Syphilis bacillus, staining of,
304-307
EST-TUBES, cleaner for, 109
cleaning of, 109
filling with media, 110
apparatus for, 111
plugging with cotton, 110
position after filling, 112
sterilization of, 110
Tetanus, bacillus of, 434-441
biology of, 4836-439
effects on animals, 439
method of obtaining, 434
morphology of, 436
poison produced by, 440,
441
toxin, potency of, 441
24
642
Tetrads, 38
Thermophilic bacteria, 30, 54, 55
Thermo-regulator, 128
Thermostat (see Incubator).
Thiogenic bacteria, 30
Tissues, cultures from, at autop-
sies, 230
Nuittall’s spear for mak-
ing, 230
cutting sections of, 162
hardening of, 162
imbedding of, 164, 165
in celloidin, 164
in paraffin, 165
preservation of, 162
staining of bacteria in, 165-
176
special methods, 168-
176
dahlia, 170
dry, 173
Ehrlich’s, 172
Gram’s, 168
Gray’s, 178
Kuehne's, 170
Weigert’s, 171
Zieh|-Neelsen’s, 172
steps in the process, 168
Toxeemia, 457
Toxins, 456-459
Traube and Gscheidlen, 465
Treviranus, 19
Tripod for levelling plates, 115
Tube, Esmarch, 120, 123
Tuberculin, 308
Tuberculosis, 289-310
cavity-formation in, 293, 294
conditions simulating, 309
diffuse caseation of, 292
encapsulation of tubercular
foci, 295
giant cells in, 292
location of bacilli in, 298
manifestations in experimen-
tal, 290
miliary tubercles, structure
of, 291
modes of infection, 296
primary infection, 295
pseudo, 309, 310
sputum in, 277
INDEX.
Tuberculosis sputum, inoculation
of animals with, 279
microscopic appearance
of, 278, 279
staining of, 148
susceptibility of animals to,
308
Tuberculosis, bacillus of, 299-
308
appearance of cultures,
302
cultivation from tissues,
300
methods of staining, 148
dry method, 173
Gabbett’s, 151
Gray’s, 173
Koch-Ehrlich’s, 148,
172
Nuttall’s
tion, 150
Ziehl-Neelsen’s, 172
microscopic appearance
of, 303
organisms that simulate
it, 305
differential diagnosis of,
modifica-
staining of, in tissues,
172-176
staining-peculiarities of,
304s
toxin of, 458
Tyndall, 20
Typhoid fever, bacillus of, 342—
356
constant properties
of, 350
cultivation of, 343-
346
difficulty in identi-
fying, 350
differentiation from
bacillus coli com-
munis, 361
Elsner’s medium for
isolating, 353
experiments with,
356
inoculations
347
with,
INDEX.
Typhoid fever, bacillus of, loca-
tion of, in tissues,
346
morphology, 342
reaction of, with ty-
phoid serum,351
source from which
to obtain, 348, 355
water as a carrier of,
484, 488
Widal’s _ reaction
with, 351
AUGHAN, 469
Vibrio Metchnikovi, 406-
410
characteristics of, eit
ral, 407-409
morphological, 406 |
pathogenesis of, 409,
10
Vibrio proteus of Finkler and |
Prior, 394-399 |
cultivation of, 395 |
morphology of, 394
pathogenesis of, 398
relation to cholera nos- |
tras, 394, 399
Vibrion septique, 441-446
ALDEYER, 22
Water, general observa-
tions upon bacterio-
logical study of, 484
qualitative bacteriologi-
cal analysis of, 490
543
Water, qualitative bacteriologi-
cal analysis of,
precautions in ob-
taining sample,
490
prelimnary steps in,
491
quantitative bacteriological
analysis of, 493
counting of colo-
nies in, 500
apparatus for,
501-505
dilution of sample
in, 497
obtaining
for, 494
selection of proper
medium for, 498-
500
source of error, 505
relation to epidemics, 484,485
typhoid bacilli in, 486-488
value of bacteriological ex-
amination of, 487-490
value of chemical examina-
tion of, 486, 489
Weigert, 26
Welch, 258, 280, 283
Widal’s reaction, 351
Wilde, 23
Winogradsky,
430-433
sample
nitro-monas of,
’ Wound infection, 22-26
Wurtz’s agar-agar and gelatin,
106
LoS LEA of bacteria, 40
Zymogenic bacteria, 30
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INDEX,
ANATOMY. Gray, p. 11; Allen, 3; Treves, 30; Gerrish, 11; Ellis, 9.
DICTIONARIES. Dunglison, p. 8; Duane, 8; National, 4.
PHYSICS. Draper, p. 8; Robertson, 24. ({Schofield, 25.
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CHEMISTRY. Simon, p. 26; Attfield, 3; Fownes, 10; Chalres, 5;
PHARMACY. Caspari, p. 5. [Luff, 19; Remsen, 24.
MATERIA MEDICA. Culbreth, p.6; Maisch, 19; Farquharson, 9 ;
DISPENSATORY. National, p. 21. [Bruce, 4.
THERAPEUTICS. Hare, p. 13; Fothergill, 10; Whitla, 31; Year-
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DIAGNOSIS. Musser, p.21; Hare, 12; Simon, 25; Herrick, 15; Hutchi-
son & Rainey, 16.
CLIMATOLOGY. Solly, p. 26; Hayem & Hare, 14. [Hamilton, 12.
NERVOUS DISEASES. Dercum, p.7; Gray, 11; Mitchell, 20;
MENTAL DISEASES. Clouston, p. 6; Savage, 24; Folsom, 10.
BACTERIOLOGY. Abbott, p.2; Vaughan & Novy. 30; Senn’s
(Surgical), 25. [Dunhan, 8.
HISTOLOGY. Klein, p. 18; Schafer’s Essen., 25 ; Schafer’s Pract., 25;
PATHOLOGY. Green, p. 12; Gibbes, 10; Coats, 6; Pepper (Surgical), 23
SURGERY. Park, p. 22; Dennis, 7; Roberts, 24; Ashhurst, 3; Treves, 29;
Bryant, 5; Druitt, 8.
SURGERY—OPERATIVE. Stimson, p. 27; Smith, 26; Treves, 29.
SURGERY—ORTHOPEDIC. Young, p. 31; Gibney, 10.
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FRACTURES and DISLOCATIONS. Hamilton, p. 12; Stimson, 27.
OPHTHALMOLOGY. Norris & Oliver, p. 21; Nettleship, 21; Juler,17;
OTOLOGY. Politzer, p. 23; Burnett, 5; Field, 9; Bacon, 4. [Berry, 4.
LARYNGOLOGY and RHINOLOGY. Browne, p. 4, Coakley, 6.
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PEDIATRICS. J. Lewis Smith, p 26; Owen, 22; Thomson, 29.
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CULVER (E. M.) AND HAYDEN (J.R.). MANUAL OF VENE-
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Lea BrotHers & Co., PHILADELPHIA AND NEw York. 7
DALTON (JOHN C.). A TREATISE ON ITUMAN PHYSIOLOGY.
Seventh edition, thoroughly revised and greatly improved. In one
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DOCTRINES OF THE CIRCULATION OF THE BLOOD. In
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DAVENPORT (F.H.). DISEASES OF WOMEN. A Manual of
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ortly.
DAVIS (EDWARD P.). A TREATISE ON OBSTETRICS. FOR
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work unequalled in excellence. | work is all that could be desired. A
—The Chicago Clinical Review. thoroughly scientific and brilliant
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DAVIS (F. H.). LECTURES ON CLINICAL MEDICINE. Second
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DE LA BECHE’S GEOLOGICAL OBSERVER. In one large octavo
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DENNIS (FREDERIC S.) AND BILLINGS (JOHN S.). A SYS-
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Complete work in four very handsome octavo volumes, containin
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London Lancet. sidered as the rival of this.—The
It may be fairly said to represent | American Journal of the Medical
the most advanced condition of | Sciences.
DERCUM (FRANCIS X., EDITOR).