ft.

THE PRINCIPLES

OF

BACTERIOLOGY:

A PRACTICAL MANUAL FOR STUDENTS
AND PHYSICIANS.

BY

A. C. ABBOTT, M. D.,

PROFESSOR OF HYGIENE AND BACTERIOLOGY, AND DIRECTOR OF THE LABO-
RATORY OF HYGIENK, UNIVERSITY OF PENNSYLVANIA.

SEVENTH EDITION,

ENLARGED AND THOROUGHLY REVISED.
With 100 Illustrations, of which 24 are colored.

K. LEWIS,

.\(;xi:«; cow K I! ST I! K v.'\
V. W. ( '.

ALL RIGHTS RESERVED, 1905

PRINTED IN AMERICA.

PREFACE TO SEVENTH EDITION.

DURING the interval that has elapsed since the appear-
ance of the Sixth Edition of this book the problem that
has chiefly engaged the attention of the foremost workers
in the field of Bacteriology is the old one of Infection
and Immunity.

In its modern development this has become a question
of the broadest biological bearing, and, as knowledge
accumulates, we find it to be no longer of exclusive
bacteriological interest.

Ramifying, as it does, in many diverse fields it grows
correspondingly complex, and a complete presentation
of the conceptions and methods would be inappropriate
to a work of this character. However, an effort has
been made to present in this edition sufficient to acquaint
the reader with the manifold directions taken by these
studies.

Furthermore, the practical results, often of the utmost
importance to Preventive Medicine, have been included.
This, we believe, adds materially to the value of the book.

The section on Methods has been brought fully up to
date, and, while not covering all the new technique,

iv PREFACE TO SEVENTH EDITION.

contains those procedures which have been tried and found
trust worthy.

In the descriptive section certain less important species
and varieties have been eliminated, and those of more
frequent occurrence substituted.

Nomenclature has been altered to conform to the
suggestions of the advanced systematists.

In presenting this edition it gives me great pleasure
to acknowledge the valuable assistance rendered by
Prof. D. H. Bergey, of this Laboratory.

A. C. A.

LABORATORY OF HYGIENE,
UNIVERSITY OF PENNSYLVANIA.

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.

vi 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.

PHILADELPHIA, December, 1891.

CONTENTS.

INTRODUCTION.

PAGES

"Orane vivum ex vivo" — The overthrow of the doctrine of
spontaneous generation — Earlier bacteriological studies —
The birth of modern bacteriology 17-30

CHAPTER I.

Definition of bacteria— Difference between parasites and
saprophytes — Their place in nature — Bacterial enzymes —
Products of bacteria — Nutrition of bacteria — Their relation
to oxygen — Influence of temperature upon their growth —
Cheinotaxis 31-48

CHAPTER II.

Morphology of bacteria — Chemical composition of bacteria —
Classification of bacteria into families and genera— Group-
ing— Mode of multiplication — Spore-formation — Motility —
The thermal death-point of bacteria 49-68

CHAPTER III.

Principles of sterilization by heat — Methods employed — Dis-
continued sterilization — Fractional sterilization — Appa-
ratus employed— Sterilization by hot air — Sterilization
under pressure— Chemical disinfection and sterilization—
Mode of action of disinfectants— Practical disinfection . . GO-!Mi

CHAPTER IV.

Principles involved in the methods of isolation of bacteria
in pure culture by tin- plate method of Koch— Materials
employed 97-103

CHAPTER V.

Preparation of nutrient media— Bouillon, gelatin, agar-atrar.

potato, blood-serum, etc 104-137

vii

viii CONTEXTS.

CHAPTER VI.

PAGES

Preparation of the tubes, flasks, etc., iu which the media are
to be preserved 138-141

CHAPTER VII.
Technique of making plates — Petri plates, Esmarch tubes, etc. 142-151

CHAPTER VIII.

The incubating-ovtjn — Gas-pressure regulator— Thermo-regu-
lator— Safety burner employed in heating the incubator . 152-159

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 bac-
teria— Stab-cultures — Slant-cultures 1GO-1G6

CHAPTER X.

Methods of staining — Impression cover-slip preparations —
Solutions employed — Preparation and staining of cover-
slips — Staining solutions — Staining in general — Special
staining-methods 167-192

CHAPTER XI.

Systematic study of an organism — Points to be considered in
determining the morphologic and biologic characters of a
culture — Methods by which the various biologic and chem-
ical characters of a culture may lie ascertained — Facts
necessary to permit the identification of an organism as a
definite species 193-234

CHAPTER XII.

Inoculation of animals — Subcutaneous inoculation — Intra-
venous injection — Inoculation into the great serous cavities
and into the anterior chamber of the eye — Observation of
animals after inoculation 235-257

CHAPTER XIII.

Post-mortem examination of animals — Bacteriological exami-
nation of the tissues— Disposal of tissues and disinfection
of instruments after the examination — Study of tissues and
exudates during life ... 258-266

CONTENTS. ix

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

CHAPTER XIV.

PAGES

To obtaiu material with which to begin work 267-270

CHAPTER XV.

The pyogenic organisms — Suppuration — Mirococcus nureus —
Micrococcua pyogenes and citreus — Staphylococcus epidermi-
dis albuit — Streptococcus pyogenes — Micrococcus gonorrhcese —
Mii-rococcus intracellularis — Pseudomonas aeruginosa — Bacil-
lus of bubonic plague — Bacterium pseudodiphtheriticum . . 271-326

CHAPTER XVI.

Sputum septicaemia — Septicaemia resulting from the presence
of sarcina tetragena, or bacterium pneurnonite in the spu-
tum of apparently healthy persons — The occurrence of
bacterium influenza} in sputum 327-345

CHAPTER XVII.

Tuberculosis — Microscopic appearance of miliary tubercles —
Diffuse caseation — Cavity formation — Encapsulation of
tuberculous foci — Primary infection — Modes of infection —
Location of the bacilli in the tissues— Stain ing-peculiarities
— Organisms with which bacterium tuberculosis may be con-
founded : Itiiflrr'nim lt'i>r;v : bacterium, smegmatis — Points of
differentiation— Acid-proof bacteria — Actinomycetes — Acti-
nonii/rt's bnrix. A<-titi<>myces Israeli, Actinomyces madurx,
Ai-thunni/ccs farcinicus, Actinomyces Eppingeri — Actinomyces
pseudotuberculosis 346-388

CHAPTER XVIII.

Glanders — Characteristics of the disease — Histological struc-
ture of the glanders nodule — Susceptibility of different
aniniiils to glanders — The bacterium of glanders; its mor-
phological and cultural peculiarities — Diagnosis of glanders . 389-398

X CONTENTS.

CHAPTER XIX.

PAGES

Bacterium diphtherias — Its isolation and cultivation — Mor-
phological and cultural peculiarities— Pathogenic properties
— Variations in virulence — Bacterium pseudodiphtheriti-
cum — Bacterium xerosis — Diphtheria antitoxin 399-425

CHAPTER XX.

Typhoid fever— Study of the organism concerned in its pro-
duction— Bacillus coli— Its resemblance to the bacillus of
typhoid fever— Its morphological, cultural, and pathogenic
properties — Itsdiffentiation from bacillus (yphosiis — Methods
of isolating the typhoid bacillus— Bacilli^ paratyphoids — Its
resemblance to bacillus typhosns — Its morphological, cul-
tural, and pathogenic properties 426-463

CHAPTER XXI.

Bacillus dysenterise — The group of bacilli found in cases of
epidemic, endemic, and sporadic dysentery — The morpho-
logical, biological, and pathogenic characters of the several
members of the group — The differentiation of the different
types of bacilli 464-472

CHAPTER XXII.

The Cholera Group of Organisms — The spirillum (comma
bacillus) of Asiatic cholera — Its morphological and cultural
pecularities — Pathogenic properties — The bacteriological
diagnosis of Asiatic cholera — Microspira Metchnikovi —
Microspira (" vibrio") Schuylkilliensis — Its morphological,
cultural, and pathogenic characters 473-509

CHAPTER XXIII.

Study of bacterium anthracis, and the effects produced by its
inoculation into animals — Peculiarities of the organism
under varying conditions of surroundings — Anthrax vac-
cines—Anthrax immune serum 510-527

CHAPTER XXIV.

The most important of the organisms found in the soil— The
nitrifying bacteria— The bacillus of tetanus— The bacillus
of malignant oedema — The bacillus of symptomatic anthrax
— Bacterium Welchii— Bacillus sporogenes 528-558

CONTENTS. xi

CHAPTER XXV.

PAGES

Infection and immunity — The types of infection : intimate
nature of infection — Septicaemia, toxfemia, variations in in-
fectious processes— Immunity, natural and acquired, active
and passive — The hypotheses that have been advanced in
explanation of immunity — Conclusions 559-615

CHAPTER XXVI.

Bacteriological study of water — Methods employed — Pre-
cautions to be observed — Apparatus used, and methods
of using it — Methods of investigating air and soil — liac
teriological study of milk — Methods employed 616-648

CHAPTER XXVII.
Various experiments in sterilization by steam and by hot air . 649-653

CHAPTER XXVIII.

Methods of testing disinfectants and antiseptics — Experiments
illustrating the precautions to be taken — Experiments in
skin-disinfection 654-667

APPENDIX.
Apparatus necessary in a beginner's bacteriological laboratory . 669-674

BACTERIOLOG-Y.

INTRODUCTION.

" Omne vivum ex vivo " — The overthrow of the doctrine of spontaneous
generation — Earlier bacteriological studies — The birth of modern
bacteriology.

BACTERIOLOGY may be said to have had its begin-
ning with the observations of Leeuwenhoek in the
latter part of the seventeenth century. Though its
most rapid and important development has taken place
since about 1880, still, a review of the various evo-
lutionary phases through which it has passed in the
course of more than two hundred years reveals an
entertaining and instructive history. From the very
outset its history is inseparably connected with that
of medicine, and from the outcome of bacteriological
research preventive medicine, in its modern concep-
tion, received its primary impulse. Through a more
intimate acquaintance with the biological activities
of the unicellular vegetable micro-organisms modern
hygiene has attained almost the dignity of an exact
cc, and properly merits the importance and promi-
now generally accorded to it. From studies in
the domain of bacteriology our knowledge of the causa-
tion, course, and prevention of infectious diseases is
daily becoming more accurate, and it is needless to em-
phasize the relation of such knowledge to the manifold
problems that present themselves to the student of
2 17

18 BACTERIOLOGY.

modern medicine. Though the contributions which
have done most to place bacteriology on the footing of
a science are those of recent years, still, during the
earlier stages of its development, many 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 our-
selves with the more prominent of those investigations.

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

In 1675 he published the fact that he had succeeded
in perfecting a lens by means of which he could detect
in a drop of rain-water living, motile "animalcules"
of the most minute dimensions — smaller than anything
that had hitherto been seen. Encouraged by this 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 diarrhoBal evacuations, objects that differenti-
ated themselves the one from the other, not only by
their shape and size, but also by the peculiarity of
motility which some of them were seen to possess.

INTR OD UCTION. 1 9

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 to the Royal Society
of London on September 14, 1683. This paper is of
peculiar 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 accom-
pany 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.1

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
animalcules 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 contri-
butions 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 not surprising that they
were immediately seized upon as the explanation of the

1 See Arcana Naturse detecta ab ANTONIO VAN LEEUWENHOEK ;
Delphis Batavorum, 1695.

20 BACTERIOLOGY.

origin of many obscure diseases. So universal became
the belief in a causal relation between the " 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 even 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 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 maintained that the material of infection could be
nothing else than a living substance, and endeavored on
these grounds to explain the variations in the period of
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 believed
in the existence of a special germ for each disease, hold-
ing that just as from a given cereal only one kind of

INTRODUCTION. 21

grain can grow, so by the special germ for each disease
only that disease can be produced.

He found in all decomposing matters innumerable
minute "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 ah 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 con-
tagion of disease ; 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 effort to refute these absurd
hypotheses."

Similar expressions of opinion were heard from many
other investigators 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 nineteenth century that by the fortunate coinci-
dence of a number of important discoveries the true rela-
tion of the lower organisms to infectious diseases was
scientifically pointed out. With the fundamental inves-
tigations of Pasteur upon the souring and putrefaction of
beer and wine; with the discovery by Pol lender and
Davaine of the pre-enre of rod-shaped organisms in the

22 B A CTERIOL OG Y.

blood of animals dead of splenic fever, and with the
progress of knowledge upon the parasitic nature of cer-
tain diseases of plants, the old question of "contagium
aniraatum" 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 the "animalcules." Do
they generate spontaneously, or are they the descendants
of pre-existing creatures of the same kind ? was the all-
important question. Among the earlier 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 his reasons for
holding this view. He maintained that the bacteria
which appeared alxjut a grain of barley germinating in
a carefully covered watch-crystal of water were the
result of changes going on in the barley-grain itself,
incidental to its germination.

Spallanzani, in 1769, drew attention to the laxity of
Needham's experimental methods, and demonstrated that
if infusions of decomposable vegetable matter be placed
in flasks, which, after being hermetically sealed, were
heated for a time in boiling water, no living organisms
would be detected in them, nor would decomposition
appear in the infusions so treated. The objection
raised by Treviranus, viz., that the high temperature

INTRODUCTION. 23

to which the infusions had been subjected had so al-
tered them and the air about them that the conditions
favorable to spontaneous generation no longer existed,
was promptly met by Spallanzani when he gently tapped
one of the flasks that had been boiled against a hard
object until a minute crack was produced ; invariably
organisms and decomposition appeared in the flask thus
treated.

From the time of the experiments of Spallanzani
until as late as 1836 but little advance was made in the
elucidation of this, at that time, 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
be robbed of its living organisms by first passing it
through strong acid or alkaline solutions no decom-
position occurred, 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 infusions 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 preceding investigators
for rendering the air which entered these flasks free
from bacteria were not necessary; that all that was
required to prevent the access of bacteria to the infu-
sions in the flasks was to draw out the neck of the flask

24 BACTERIOLOGY.

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 by
the drop of water of condensation which collected at its
lowest angle, and none could enter the flask.

While, from our modern standpoint, the results of
these investigations seem to be of a most convincing
nature, yet there were many at the time who required
additional proof that "spontaneous generation" was
not the explanation for the mysterious appearance
of these minute living creatures. The majority, if
not all, of such doubts were subsequently dissipated
through the well-known investigations of Tyndall upon
the floating matters of the air. In these studies he
demonstrated by numerous ingenious and instructive
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 viable organisms.

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

In 1762 Bonnet, who was deeply interested in this

INTRODUCTION. 25

subject, suggested, in reference to the results obtained
by Needham, the possibility of the existence of " germs
or their eggs," which had 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 in-
dulged in this pure speculation it became the happy
privilege of Ferdinand Cohn, of Breslau, to demonstrate
its accuracy and importance.

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.
He demonstrated that some of the rod-shaped organ-
isms possess the power of passing into a resting- or
spore-stage in the course of their life-cycle, analogous
to the seeding stage of higher plants, 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-blow. It was no
longer difficult to explain the inconsistencies in the re-
sults of former investigations, nor was it any longer to be
doubted that putrefaction and fermentation were the re-
sult of bacterial life and not the cause of it, and that these
bacteria were the offspring of 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.

26 BACTERIOLOGY.

The establishment of this point gave an impetus to
further investigations, and as the all-important ques-
tion was that concerning the relation of the 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
its present development 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 Ilindfleisch, 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, represent,
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 pyaemia and, occa-
sionally, secondary to typhoid fever. Von Reckling-
hausen believed the granules seen in the abscess-points
to be micrococci and not tissue-detritus, and gave as
the reason that they were regular in size and shape, and
gave specific reactions with particular stain ing-flu ids.
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 the constitutional

INTRODUCTION. 27

conditions 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 pyaemia 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 seen, that bacteria were present in dis-
eases following infection of wounds, but described the
manner in which the organisms had gained entrance
from the point of injury to the internal organs and
blood. He expressed the opinion that the spherical
and rod-shaped bodies which he saw in the secretions of
wounds were closely allied, and he gave to them the
designation " microsporon septicum." He believed that
the organisms gained access to the tissues round about
the point of injury both by the aid of the wandering
leucocytes and by being forced through the connec-
tive-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.

28 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 urged that the organisms seen in dis-
eased 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 experimental 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 made manifest 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, they holding that
all living organisms on the surface of the tissues would be
destroyed by the high temperature, and that if decom-
position should subsequently occur it would prove that
it was the result of the growth of bacteria in the depths
of the tissues to which the heat had not penetrated.

INTRODUCTION 2&

Decomposition did usually set in, and they accepted
this as proof of the accuracy of their view. Attention
was, however, shortly called to the fact that in cooling
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 originated. (See page 22.)

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

The fundamental researches of Koch (1881) upon
pathogenic bacteria and their relation to the infectious
diseases of animals differed from those of preceding
investigators in many important respects. The scien-
tific methods of analysis with which each and every
obscure problem was met as it arose served at once to
distinguish him as a pioneer in this hitherto but imper-
fectly cultivated domain. The outcome of these inves-
tigations was the establishment of a foundation upon
which bacteriology of the future was to rest. He, for the
first time, demonstrated that distinct varieties of infection,
as evidenced by anatomical changes, are due in many
cases to the activities of specific micro-organisms, and
that by proper methods it is possible to isolate these

30 BACTERIOLOGY.

organisms in pure culture, to cultivate them indefinitely
under artificial conditions, to reproduce the lesions by
inoculation of these pure cultures into susceptible ani-
mals, and to continue the disease at will by continuous
inoculation from an infected to a healthy animal. By
the methods that he employed he demonstrated a series
of separate and distinct diseases that can be produced in
mice and rabbits by the injection of putrid substances
into their tissues. The disease known as septicaemia of
mice ; likewise a disease characterized by progressive
abscess-formation, and pyaemia and septicaemia of rab-
bits, were among the affections first produced by him in
this way. It was in the course of this work that the
Abbe system of substage condensing apparatus was first
used in bacteriology ; that the aniline dyes suggested by
Weigert were brought into general use ; that the isola-
tion and cultivation of bacteria in pure culture on solid
media were shown to be possible ; and that animals
were employed as a means of obtaining 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.

NOTE. — 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 Loffler's instructive and enter-
taining Vorlesungen fiber die gcschichtliche Entmckelung
der Lehre von den Bacterien, upon which I have drawn
freely in preparing the foregoing sketch.

CHAPTER I.

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

BACTERIA (more properly bacteriacese or schizomy-
cetes) were regarded by the older writers as infusoria.
This was because of their capacity for developing in
infusions, their property of spore-formation, their resist-
ance to drying, their power of independent motion, and
the absence of chlorophyll from their tissues. In the
modern conception, however, this classification is unten-
able, and bacteria, by virtue of their distinguishing
peculiarities, are now treated as a group by themselves
that may briefly be defined as comprising microscopic,
unicellular, vegetable organisms that multiply by the
process of transverse division.

Inasmuch as bacteria are not possessed of chloro-
phyll,1 their metabolic processes are fundamentally dif-
ferent from those of the higher plants in which it is
present. They cannot, as in the case of the green
plants, obtain carbon and nitrogen from such simple
bodies as carbon dioxide and ammonia, but are forced
to secure these essential elements from organic matter
as such. This power to decompose and assimilate

1 Chlorophyll is the green coloring-matter possessed by the higher
plants by means of w'.iich they are enabled in the presence of sunlight
to decompose carbonic acid (COs) and ammonia (NHs) into their ele-
mentary constituents.

31

32 BACTERIOLOGY.

organic matters is signally different in different species
of bacteria, and, singular to say, there is a small group
(to be described later) from which this function is appar-
ently absent, in spite of the fact that no compensatory
chlorophyll is discernible in their tissues.

SAPROPHYTES AND PARASITES. — In the case of
certain bacteria, in fact, the majority, the source of food-
supply must of necessity be dead organic matters of either
animal or vegetable origin. They cannot exist in the
presence of living tissues. To the members of this
group the designation saprophytic or mdatropJtic (A.
Fischer) is given. To that group that can exist only
upon living organic matters, and herein belong many
(not all) of the disease-producing bacteria, the appella-
tion parasitic or paratrophic (A. Fischer) is applied ;
while for the few species that either do not require
organic matters, or do not, so far as is known, have the
faculty of decomposing and assimilating proteid stuffs
at all, the name prototrophic is suggested by Fischer.
In the strict sense of the word, a parasite or paratroph
can exist only in the body of a living host, and a sapro-
phyte or metatroph only upon lifeless organic matters,
and such obligate parasites and saphmphytes are known,
but in the majority of cases such nutritive conditions are
not obligatory, many of both metatrophs and paratrophs
having the power to adapt themselves to conditions other
than those for which they are by nature best fitted. For
instance, certain species that exhibit their most impor-
tant properties under conditions of parasitism may,
nevertheless, lead a metatrophic existence when circum-
stances demand it, and, on the other hand, particular
species usually metatrophic by nature may find condi-
tions favorable to their development in a living host.

THEIR PL A CE IN NA TURE. 33

To such adaptable species the designation " facultative "
is given, and, when employed, signifies that the species
in question has the faculty of adapting itself to en-
vironments other than those in which it is usually en-
countered. In this sense all of the disease-producing
bacteria that can be cultivated artificially are manifestly
facultative metatrophs or saprophytes.

The life-processes of bacteria are so rapid, complex,
and energetic that they result in the most profound
alterations in the structure and composition of the
materials in and upon which they are developing.

Decomposition, putrefaction, and fermentation result
from the activities of the metatrophic bacteria ; while
the changes brought about in the tissues of their living
host by the purely parasitic forms find expression in
disease-processes, and not infrequently in complete death.

THEIR PLACE IN NATURE. — The role played in
nature by the metatrophs is a very important one.
Through their functional activities the highly compli-
cated tissues of dead animals and vegetables are resolved
into the simpler compounds, carbonic acid, water, and
ammonia, in which form they may be taken up and ap-
propriated as nourishment by the more highly organized
members of the vegetable kingdom. It is through this
ultimate production of carbonic acid, ammonia, and
water by bacteria, as end-products in the processes of
decomposition and fermentation of dead animal and
vegetable tissues, that the demands of growing vegeta-
tion 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 the simpler
compounds, carbon dioxide and ammonia, by the animal

34 BACTERIOLOGY.

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 creatures 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 pos-
sible. It is plain, therefore, that the saprophytes, which
represent the large majority of all bacteria, must be
looked upon in the light of benefactors, without which
existence would be impossible.

With the parasites, on the other hand, the conditions
are far from analogous. Through their metabolic activ-
ities 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 terrestrial life, the positions oc-
cupied by the two functionally different groups, the sap-
rophytes on the one hand, and the parasites on the other,
are diametrically opposed : the saprophytic forms stand-
ing as benefactors, in resolving dead animal and vege-
table bodies into their component parts, which serve as
food for living vegetation, and at the same time remov-
ing from the surface of the earth the remains of all dead

THEIR PLACE IN NATURE. 35

organic substances ; while the parasitic group exists
only at the expense of the more highly organized mem-
bers of both the animal and vegetable kingdoms. It is
to the parasitic group that the pathogenic1 organisms
belong.

In addition to the metatrophs that are concerned in
the changes to which allusion has just been made, there
exist other species whose life-processes result in specific
changes of great interest and importance. Some of
these are characterized by their property of producing
pigments of different color; these are known as the
chromogenic2 species. Just what the biological signifi-
cance of the pigment-producing function is cannot
be said, but the fact that many of the chromogens
are richly endowed with proteolytic3 activity makes it
probable that they are, in common with other meta-
trophs, concerned in the omnipresent process of disinte-
gration in progress in all dead organic matters, and
that, after all, their power to produce colors, though
conspicuous, is of but subordinate importance. From
the investigations of Beyerinck it would seem that in
some of the chromogenic forms the pigment is an
integral part of the bacteria themselves ; in others that
it is an excretory product of species that are them-
selves colorless; while in still others, that it is an
excretory product which remains intimately associated
with the bacterial cells and in part or wholly stains
them.

Others, the so-called photogenic or phosphorescent

1 Pathogenic organisms are those which possess the property of pro-
ducing disease.

* Chromogenic: possessing the property of generating color.
! Proteolytic : the power of dissolving or digesting proteids.

36 BACTERIOLOGY.

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

Still others, the so-called zymogenic bacteria, are con-
cerned in the various fermentations, such, for instance,
as acetic-, lactic-, and butyric-acid fermentations ; and
many of the industries, such, for example, as those
concerned in the making of \vine, beer, cheese, butter,
and indigo, are more or less directly dependent upon
the fermentation that accompanies the growth of pecu-
liar species of bacteria in those materials.

The saprogenic bacteria are those that produce the
particular fermentation that we know as putrefac-
tion.

Another very important saprophytic group comprises
the so-called nitrifying and denitrifying bacteria, whose
activities 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 am-
monia. It is through their association (symbiosis) with
the nitrifying bacteria that certain plants, the legumi-
nous, 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 nitro-
gen a biological significance that had hitherto been
denied it.

The so-called thiogcnic bacteria convert sulphuretted
hydrogen into higher sulphur compounds.

The bacteria concerned in the foregoing changes per-
form the functions, as said, by virtue of special fer-
ments or enzymes that are elaborated by them.

BACTERIAL ENZYMES. 37

BACTERIAL ENZYMES. — The enzymes arc, in gen-
eral, amorphous products of living protoplasm, that
are able to split up large quantities of complex or-
ganic and inorganic substances into smaller, simpler,
more soluble, and diffusible combinations. Bacteria
produce a variety of enzymes, by means of which
they are able to derive their nutrition from complex
molecular substances. The enzymes are to some extent
dialysable. The principal enzymes produced by bacteria
are proteolytic, diastatic, in verting, coagulating, and sugar
splitting.

The proteolytic or albumin-dissolving enzymes are
formed by a great many bacteria. The most familiar
indications of the formation of a proteolytic enzyme are
seen in the liquefaction of gelatin, of coagulated blood-
serum, and of casein. Most frequently the proteolytic
enzyme is allied to trypsin, as shown by Abbott and
Gildersleeve,1 since the liquefaction or digestion pro-
ceeds only under an alkaline reaction. Some bacteria,
however, produce a proteolytic enzyme analogous to
pepsin, and this enzyme is active under an acid reac-
tion. The proteolytic enzymes of different bacteria
vary considerably with regard to their resistance to
heat, some being destroyed in a few minutes when
heated to 60° or 70° C., while others may be boiled for
a short time without suffering marked deterioration (see
Abbott and Gildersleeve, loc. cit.). The proteolytic
enzymes also differ in respect to their susceptibility to
the action of acids and other chemicals.

The formation of proteolytic enzymes is one of the
functions of bacteria that is easily disturbed by external
conditions. Long-continued cultivation on media in

1 Abbott and (Hldersleevc : Jonrn. of Med. Research, vol. v., 1903.

38 BACTERIO L OG Y.

which this function is not required may lead to marked
deterioration, while continued cultivation under condi-
tions calling forth this function may result in the pro-
duction of a race of organisms in which the function is
unusually prominent.

The addition of carbohydrates and of glycerine to
culture-media interferes with production of the proteo-
lytic enzyme by many species of bacteria, as shown by
Auerbach.1

Diastatic enzymes convert starch into sugar. This
function is best studied on media containing starch, as
potato infusion or solutions of starch. By appropriate
tests the intermediate steps in the conversion of the
starch into sugar may be traced by testing a portion of
the culture-medium from time to time. Fermi 2 found
this function in a large number of bacteria studied,
especially in organisms of the subtilis group and in the
microspira of the cholera group.

Inverting enzymes convert saccharose into dextrose.
These enzymes are produced by comparatively few
bacteria. Fermi found this function manifested by
bacillus megatherium, pseudomonas fluorescens, bacillus
vulgaris, microspira comma, microspira Metchnikovi,
and others.

Coagulating enzymes are those which coagulate milk.
Rennet may be taken as the typical form. This altera-
tion is quite common in association with an acid reac-
tion, but in such instances it is not always certain that
the coagulation has not been induced by the acid formed.
Gorini3 found that cultures of bacillus prodigiosus, ster-

1 Auerbach: Arcbiv fur Hygiene, Bd. xxxi. p. 311.
1 Fermi : Archiv fiir Hygiene, Bd. xi., and Centralblatt fur Bacte-
rinlngie, Bd. xii.
• Gorini : Centralblatt fur Bacteriologie, Bd. xii. p. 666.

BACTERIAL ENZYMES. 39

ilizecl by heating to 60° C., caused a solid coagulation of
sterile milk in a few days.

A small number of bacteria have also been encoun-
tered that bring about coagulation of milk with a dis-
tinctly alkaline reaction. This function has been
noticed in bacteria isolated from milk, and especially
in bacterium pseudodiphtheriticum isolated from cows'
milk (Bergey).

Sugar-splitting enzymes are very common in bacteria.
This function varies in different species as seen in the
different end-products that are formed. Buchner suc-
ceeded in isolating the sugar-splitting enzyme (zymase)
of yeast-cells, and when thus isolated it still possesses
the power of inducing active fermentation of sugar. It
is believed that the sugar-splitting enzymes of bacteria
are similar in character to the zymase of yeast-cells.
The splitting up of carbohydrates appears to be brought
about by the bacteria for the purpose of obtaining oxygen,
a- indicated by the nature of the end-products formed,
and also by the conditions under which it may be car-
ried out — i. c., the absence of atmospheric oxygen.

The splitting of the carbohydrate molecule may be
illustrated as follows :

C,HW06 = 2C2H60 + 2C02
Grape sugar = 2 alcohol -f 2 carbon dioxid.

or C6H,A = 2CSH603

Grape sugar = 2 lactic acid.

or C6HI2O6 = 3C,H4O2

Grape sugar = 3 acetic acid.

According to Theobald Smith J all facultative anaero-
bic bacteria form acids from carbohydrates, while the
strict aerobic bacteria do not have this function, or

'Theobald Smith: Centralblatt fur Bacteriologie, Bd. xviii.

40 BACTERIOLOGY.

bring about the alteration so slowly that it is concealed
by the simultaneous production of alkali. Among the
acids formed by bacteria, besides carbon dioxid, we have
lactic, acetic, butyric, proprionic, and formic ; and fre-
quently there is also produced ethyl alcohol, aldehyd,
and acetone.

The lactic acid formed by the action of different bac-
teria on carbohydrates may be either dextrorotatory or
laevorotatory, or almost equal quantities of both forms
may be present and the mixture be optically inactive.

PRODUCTS OF BACTERIA. — As stated, bacteria that
produce disease are known as pathogenic. They induce
disease by their poisonous action upon the tissues in
which they are located. The materials of which cer-
tain species of bacteria are constructed, and the products
of growth of certain others, are of the greatest import-
ance in their relation to animal and human pathology.
Particular species, while not eliminating soluble poisons
as a product of metabolism, are nevertheless themselves
built up of poisonous proteids, or of proteids with
which toxic materials are so intimately associated that
they can only be isolated by the most refined and elabo-
rate chemical manipulations. Others produce in the
course of their growth soluble poisons that can readily
be separated, by very simple methods, from the bacteria
that produced them. The proteid matters making up
the bodies of many species of bacteria, even those not
conspicuously pathogenic, have been shown by Buchner
to induce disease when isolated and injected into the
tissues of animals ; in some cases causing only rise of
body-temperature, in others acute inflammatory proc-
esses with pus-formation. To such proteids Buchner
has given the name bacterial proteins.

NUTRITION OF BACTERIA. 41

The poisonous soluble products of bacterial growth
are known as toxins and ptoma'ins: toxins being, in gen-
eral, uncrystallizable poisons, secreted or excreted by

the bacteria, of whose intimate chemical nature little or

§

nothing is known ; while ptoma'ins are crystallizable prod-
ucts of their metabolic activity which, physically speaking,
are analogous to the ordinary vegetable alkaloids.

NUTRITION OF BACTERIA. — 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 ele-
mentary constituents. The bacteria, on 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 presented as such, in the form
of decomposable organic substances.

In general, bacteria obtain their nitrogen most
readily from soluble albumins, and to a certain extent,
but by no means so easily, from salts of ammonium. In
some of Nageli'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 by the bac-
teria for building up their tissues in the course of
their development was derived from some source
other than the nitric acid or the nitrates, and that the
reduction of this acid was most probably a secondary
phenomenon. It must be borne in mind, however, that

42 BACTERIOLOGY.

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 glyc-
erin and many of the fatty acids ; and from the alka-
line salts of tartaric, citric, malic, lactic, and acetic
acids. In some instances carbon compounds, which
when present in concentrated form inhibit the growtli
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 species and
developmental forms, on the contrary, though incapable
of multiplying when in the dry state, may be com-
pletely deprived of their water without causing them
to lose the power of reproduction under favorable con-
ditions.

Closer study of bacteria, and a more intimate ac-
quaintance 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

NUTRITION OF BACTERIA. 43

of proteid substance for its development. Certain
of them 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 evolution
of compounds characteristic of putrefaction.

In considering the normal development of bacteria
we must not lose sight of the fact that this is influenced
both by the quality and the quantity of the nutritive mate-
rials to which they have access, and by the character of
the metabolic products that accumulate in these materials
as a result of their vital processes. Nitrogen and carbon
compounds may be present in amount and kind entirely
suitable to normal bacterial growth, and yet this may
be checked, after a comparatively short time, by the
accumulated products of bacterial metabolism, some of
which possess the property of inhibiting growth and
ultimately of even destroying the bacteria that produced
them. The most common and conspicuous examples
of the inhibiting conditions that are coincident with
bacterial growth are alterations in the chemical reaction
of the matters in which the bacteria are developing. Since
the majority of them grow best in media of a neutral or
very slightly alkaline reaction, any excessive development
of alkalinity or acidity, as a result of growth, arrests
development, and no evidence of life or further multi-
plication can be detected until this deviation from the
neutral (or the suitable) reaction has been corrected.

44 BACTERIOLOGY.

THEIR RELATION TO OXYGEN. — Of considerable
importance and interest in the study of the nutritive
changes of bacteria is the difference in their relation to
oxygen. For certain species free oxygen is essential to
the proper performance of their functions ; in another
group no evidence of life can be detected under its
access ; while in a third group free 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 bac-
teria which not only grow and multiply and perform
definite physiological functions without the aid of free
oxygen, but to the existence of which it is positively
harmful. To these he gave the name anaerobic bac-
teria, in contradistinction to the aerobic group, for the
proper performance of whose functions free oxygen is
essential. The anaerobic bacteria derive their oxygen
entirely from oxygen compounds in the materials in
which they are growing. In addition to these there is a
third group, for the maintenance of whose existence the
absence or presence of uncombined oxygen is apparently
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. Since all growing bacteria, anaerobic
as well as aerobic, generate carbonic acid in the course
of their development, it is evident that oxygen must
in reality be obtained by them from some source, and
must be regarded as essential to their life-processes;
but the manner in which it is appropriated by them
varies, the aerobic species taking it from the air as free
oxygen, while the anaerobic species, not possessed of

INFLUENCE OF TEMPERATURE UPON GROWTH. 45

this power, obtain it through the decomposition of more
or less stable oxygen-containing compounds.

Though the multiplication of the facultative varieties
is not interfered with by either the presence or absence
of free oxygen, yet experiments demonstrate that the
products of their growth are different under the varying
conditions of absence or presence of this gas. For ex-
ample : in the case of certain of the chromogenic forms
the presence or absence of oxygen has a very decided
effect upon the production of the pigments by which
they are characterized.

NOTE. — 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
bacillus prodigiosus and of spirillum rubrum. In the
former the red color is apparently a product dependent
upon the presence of oxygen, while in the latter the
greatest intensity of color occurs at the point furthest
removed from the action of oxygen.

INFLUENCE OF TEMPERATURE UPON THE GROWTH.
—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,
when it is at its 'optimum, and, as a rule, ceases at
43° C. ; though species exist that multiply at as high
a temperature as 70° C. and others at as low as 0° C.

46 BACTERIOLOGY.

The investigations of Globig,1 Miquel,2 and Macfadyen
and Bloxall3 have revealed the existence in the soil,
in water, in faeces, in sewage, in dust, and, in fact, prac-
tically everywhere, of bacteria that under artificial culti-
vation show no evidence of life at a temperature lower
than 60° to 65° C., and will even grow at such high
temperatures as 70° and 75° C., a state of affairs almost
paradoxical, inasmuch as these are temperatures that suf-
fice for the coagulation of albumin, and, in consequence,
are generally incompatible with life. 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 pre-
ceding investigators.

The most favorable temperature for the development
of pathogenic bacteria is that of the human body, viz.,
37.5° C. There are a number of bacteria commonly
present in water, the so-called normal water bacteria.,
that grow best at about 20° C.

Under natural conditions it frequently occurs that
the development of one species or group of species of
bacteria is directly dependent upon the functional ac-
tivities of another totally distinct species, the growth of
one group resulting in conditions that are of vital im-
portance to the existence of the other. This interde-
pendence of species is known as symbiosis. It is observed,
for instance, in the course of putrefaction, where,

1 Globig : Zeitschrift fur Hygiene, Bd. iii. S. 294.

2 Miquel : Annales de Micrographie, 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.

CHEMOTAX1S. 47

through exhaustion of free oxygen by the actively
germinating aerobic varieties, the conditions are sup-
plied that enable the anaerobic species to develop and
exercise their biological activities. Again, through the
proteolytic activity of enzymes produced by certain
species of bacteria, other species are supplied with nu-
trition that would otherwise be unassimilable or only
imperfectly so. Similar symbiotic relations between
bacteria and higher plants are also noticed, notably that
between certain bacteria of the soil and the group of
leguminous plants, whereby the latter are enabled,
through the assistance of the former, to make tip their
nitrogen deficit in large part from the free nitrogen of
the atmosphere. (See page 36.)

CHEMOTAXIS. — Another interesting biological pecu-
liarity of bacteria is that discovered by Engelmann and
by Pfeffer, known as chemotaxis. This term applies to
the peculiar phenomena of attraction and of repulsion
that are exhibited by motile bacteria when in the pres-
ence of solutions of bodies of various chemical compo-
sition. Engelmann demonstrated that the bacteria in
decomposing infusions accumulate in great numbers in
the neighborhood of the sources of oxygen. In a hang-
ing-drop of such an infusion the bacteria will be seen to
accumulate in a dense mass along the edge or around the
edge of small bubbles of air in the fluid. Even plant
cells in the infusion, whose chlorophyll sets free oxygen
in the light, are surrounded by large numbers of bacteria.
The positive chemotactic affinity between oxygen and bac-
teria was employed by Engelmann as a basis for the dem-
onstration of small quantities of oxygen in studying the
assimilative action of various kinds of light upon the
plant-cell. Pfeffer showed that when a neu-

48 BACTERIOLOGY.

tral fluid (a drop of water) containing motile bacteria
is brought in contact with a weak solution of either
peptone, sodium chloride, or dextrin, the bacteria are at.
once attracted toward the solution ; this reaction is
designated " positive chemotaxis." On the other hand,
if brought in contact with an acid, an alkaline, or an
alcoholic solution, the bacteria are repelled or driven
from the point at which the two fluids are diffusing;
that is, they exhibit " negative chemotactic " affinities.
The significance of these . reactions is not understood,
but it has been aptly suggested that they may be funda-
mentally analogous to the specific positive and negative
affinities exhibited by the ions (see page 91) resulting
from the dissociation of electrolytes, and that they may
" have their explanation in the forces of ionic attraction
and repulsion."1 In this connection it is important to
note that the wandering cells of the animal body, the
leucocytes, exhibit also these chemotactic phenomena;
and it is especially necessary to a complete comprehen-
sion of the process of suppuration to bear in mind that
among the substances which have the greatest attraction
for these wandering cells, are the products of growth of
certain bacteria in some cases, and the protoplasmic con-
stituents of the bacteria themselves in others.

From what has been learned, it may be said, in
general, that for the growth and development of bacteria
organic matter of a neutral or slightly alkaline reaction,
in the presence of moisture and at a suitable temperature,
is all that is necessary. From this can be formed some idea
of the omnipresence in nature of these minute vegetables.
Bacteria may be found wherever these conditions obtain.

'Read Sewall on "Some Relations of Osmosis and Ionic Action in
Clinical Medicine," International Clinics, vol. xi., Eleventh Series.

CHAPTER II.

Morphology l of bacteria — Chemical composition of bacteria — Classi-
fication of bacteria into families and genera — Grouping — Mode of
multiplication — Spore-formation — Motility— The thermal death-
point of bacteria.

IN structure the bacteria are unicellular, always de-
veloping from pre-existing cells of the same character
and never appearing spontaneously. They are seen to
occur as spherical, rod- and spiral-shaped bodies that
multiply by the simple process of transverse division,
belonging, therefore, to the schizomycetes or fission
fungi.

Under what we are accustomed to regard as normal
conditions of development, and by the ordinary methods
of examination, bacteria appear very simple in form and
structure. They are cells consisting of a protoplasmic
mass within a membranous hull that is discernible with
more or less difficulty. The protoplasmic body is of
material closely allied, chemically speaking, to ordinary
vegetable proteid. It is often homogeneous, but in par-
ticular species and under various conditions of growth
the central mass in stained specimens is commonly
marked by the presence of very dark granules, the
so-called metachromatic granulations. Again, in other
>pccies paraplastic granules giving the microchemical
reactions of fat, starch, sulphur, etc., are to be seen.
Under certain physical conditions the protoplasmic body
presents irregular rents or retractions, the result of pro-
teolytic or of osmotic disturbances dependent upon the

1 Morphology : pertaining to shape, outline, structure.
4 49

50 BACTERIOLOGY.

character of the fluid in which the bacteria are located ; in
fact, the deeply staining granules, other than those of fat,
starch, and sulphur, that are often observed, are regarded
by some writers (especially A. Fischer) as but altered or
condensed protoplasm due to the same influences.

In certain species the protoplasmic body is always
more dense at the poles of the cells than at the middle,
so that when stained the ends are much darker than the
intervening portion. Sometimes in other species the
reverse is the case.

By some investigators the protoplasmic central mass
is regarded as a nucleus, and, functionally speaking,
possibly it is to all intents and purposes, but this cannot
be certainly decided. In the great majority of cases,
however, with the ordinary methods of examination, it
is not seen to possess any of the structural peculiarities
that we are accustomed to regard as the distinguishing
attributes of cell-nuclei.

The enveloping hull or membrane is in some cases
apparently only a modification of the protoplasmic cen-
tral mass, at times being only a condensation of that
protoplasm ; again, it seems to be chemically different
from it. In a few instances it appears to be allied to
cellulose in its chemical composition. Sometimes it is
so thick as to be readily seen, while again it is discerni-
ble only by special methods of examination. In partic-
ular species it may, by appropriate methods, be seen as
a sharply defined capsule inclosing a clear zone in which
the deeply stained central mass lies. Occasionally the
central protoplasmic mass is surrounded by an ill-
defined slimy material that causes the individual cells
to adhere to one another in more or less compact masses
or pellicles (zooglcea, Fig. 1).

CHEMICAL COMPOSITION OF BACTERIA. 51

CHEMICAL COMPOSITION OF BACTERIA. — The bodies
of bacteria consist of water, salts, and albuminous sub-
stances, with smaller proportions of various extractive
substances soluble in alcohol or ether, such as triolein,
tripalmitin, tristearin, lecithin, and cholesterin. In
many varieties substances giving the reaction of starch
have been found, while others give the true reactions
of cellulose (jB. subtilis). Nuclein has not been found
in any of the bacteria, though the nuclein bases, xan-
thin, guanin, adenin, have been found.

The relative amounts of water in bacteria are influ-
enced to a large extent by the nature of the medium on

FIG. 1.

Zoogloea of bacilli.

which they have been grown. In like manner the con-
tent in albumin, extractive substances, and salts varies
with the conditions under which the bacteria have been
cultivated. E. Cramer1 has studied the chemical com-
position of bacteria in great detail. As the result of
his studies of microspira comma, he found its composi-
tion to be as follows : water 88.3 per cent., albumin 7.6
per cent., ash 3.6 per cent. The dry substance of the
bacteria contains the following : albumin 65 per cent,,
ash 31 per cent. From 76 to 80 per cent, of the ash
consists of sodium chloride and phosphate.

In size the bacteria are certainly the smallest living

1 E. Cramer: Avchiv fur Hygiene, Bd. xiii., xvi., xxii., and xxviii.

52 BACTERIOLOGY.

creatures with which we have acquaintance, being visible
only when very highly magnified. In order that some
conception of their microscopic dimensions might be
formed, it has been computed that of the average size
bacteria about thirty billion would be required to weigh
a gramme, and that about one billion seven hundred
million of the small spherical forms might readily be
suspended in a drop of water.

THE CLASSIFICATION OF BACTERIA IN FAMILIES.

The classification of bacteria into families, genera,
and species has been a subject of much labor and dis-
cussion. It is impossible, on account of limited knowl-
edge of certain species, to make the classification of the
bacteria accurate.

The basis for modern systems of classification has
been the most pronounced morphologic characters. The
system of classification which has proven of greatest
value is that projx>sed by Migula, in Engler and Prantl's
Die Nat'iirliehen Pflanzenfamilien, 1896, and elaborated
in his System der Eakterien, 1900. The nomenclature
employed in the description of the bacteria is that of
Migula.

SCHIZOMYCETES. — Bacteria. — Unicellular, chloro-
phyll-free organisms which reproduce by division into
one, two, or three directions of space. Sexual repro-
duction is absent. Many species develop endogenous
spores. Motility, occurring in some genera, is due to the
presence of flagella ; in Beggiatoa and Spirochata the
motile organs are unknown.
I. ORDER : EUBACTERIA.

Cells without central nuclei, sulphur, and bacterial
purpurin ; colorless or only feebly colored.

CLASSIFICATION OF BACTERIA IN FAMILIES. 53

1. Family: Coccacece (Zopf, Migula).

Cells in a free state, spherical, becoming slightly
elliptical before division. Division occurs in
one, two, or three directions of space. Motile
organs are present only in a few species.
Endospore formation not known to occur.

1 . Genus : Streptococcus (Billroth). — Cells spher-

ical and without motile organs. Division
only in one direction of space. After divi-
sion the cells separate or they remain for
a shorter or longer time in apposition and
frequently form long chains. Usually two
cells are seen lying close together with a
slightly greater interval between the next
two members in the chain.

2. Genus: Micrococcus (Hallier, Colin). — Cells

which in their free state are spherical.
Division in two directions of space. If the
cells after division remain for a shorter or
longer time in apposition they form simple
or flat aggregations of the cells in which the
opposing sides of the organisms are flat-
tened. Motile organs are absent. Endo-
spore formation has not been demonstrated.

3. Genus: Sarcina (Goodsir). — Cells which in

their free state are spherical. Division in
three directions of space, forming the well-
known packet-form aggregations. Besides
this, cells frequently occur singly and as
diplococci or tetracocci or in irregular
aggregations. Motile organs are absent.
Endospore formation has not been definitely
demonstrated.

BACTERIOLOGY.

4. Genus: Planocoeeus (Migula). — Single cells,

spherical, hot usually showing aggregations
of two of four cells. Division in two direc-
tions of space. Motile organs are present
in the form of one or two long wavy fla-
gella. Endospore formation does not occur.
Only a few species are known of this genus.

5. Genus : Plan&sarcina (Migula ». — Single cells,

spherical, division in three directions of
space. Usually the cells remain in appo-
sition as diplococci or tetracooci, less fre-
quently as distinct packet forms. Motile
organs are present in the form of shorter or
longer flagella. Endospore formation does
not occur. Only three species have been
described.
2. family: BaeteriaeeoR.

Cells which in their free state occur as cylin-
drical rods which divide only in one direction
of space, tliat is, at right angles to the long
axis of the cylinder. Cells may be very short,
so that it is difficult to differentiate them from
the coccaceae. Division occurs in some species,
as far as observation has demonstrated, not in
the division of a cell into two daughter eelK
but in each large rod a number of cell divi-
sions in various stages are usually seen.
1. Genus: Bacterium (Ehrenberg, Migula). —
shorter or longer cylindrical cells, some-
times threads of considerable length with-
out flagella-. Endospore formation has been
demonstrated in a number of species, in
others it is absent.

CLASSIFICATION OF li.irTKRIA IN FAMILIES. 55

2. Genus: I>ucif/nx (( \>lm, Migula). — Shorter or

longer rod forms, sometimes short ovoid
cells, and in other species long thread forms.
Motile, with flagella distributed over the
entire body. Endospore formation occurs
in many species.

3. Genus: l^finlomonas. — Shorter or longer

cylindrical cells, sometimes thread forms,
motile, with polar flagella. The number of
flagella varies in different species from one
to ten. Endospore formation is present in
a few of the species.
3. Family : Spirallacece.

Cells more or less curved, at times forming dis-
tinct spirals when a number are joined end to
end. Division of the cells in one direction of
space, that is, at right angles to the long axis
of the rod. Endospore formation is absent
except in a few species. Motility is usually
present ; where the motile organs are known
they are polar.

1. Genus: ${>iro#oinH (Migula). — Cells usually

twisted in rather large spirals, non-motile,
without flagella, stiff without flexibility.
The number of species of this genus is very
small.

2. Genus: Mi<'ru*i>ira (Schroter). — Cells mostly

t'oiiiniM-l'.inii or sausage shaped, cur vex! or
joined in S-shaped figures, or even in long
spiral chains. Motile, with one to three
polar flagella. Endospore formation has
not been demonstrated.

3. (Ifn a* : Spirillum (Khrcnherg). — Twisted rods

56 BA CTERIOLOG Y.

of variable thickness and length, frequently
forming long spirals. Endospore formation
has been observed in a few species. The
cells are motile and possess flagella at one
or both poles.

4. Genus: Spirochceta. — Cells of spiral form,
thin but usually quite long, motile and flexi-
ble, snake-like but also screw-like in motion.
Motile organs are unknown. Endospo re-
formation has not been observed.
4. Family: Chlamydobacteriacexje.

Cells cylindrical and thread forms, surrounded
with a sheath. Multiplication results through
motile and non-motile gonidia which arise
directly from vegetative cells and develop into
threads.

1. Genus: Chlamydothrlx. — Cells cylindrical,

non-motile, enclosed in a sheath. Fre-
quently the sheath is only apparent upon
applying reagents. Multiplication through
non-motile round or ovoid gonidia that are
derived directly from the vegetative cells.
Synonyms : Streptothrix (Cohn, Migula) ;
Leptothrix (Kiitzing).

2. Genus: Grenothrix (Cohn). — Thread-form

bacteria without branching, attached at one
end, showing a differentiation of base and
apex, and increasing in thickness toward
the free end. Sheath rather thick. The
sheaths of old threads in waters containing
iron are saturated with oxide of iron. The
cells are cylindrical, sometimes flattened.
Multiplication through non-motile gonidia,

CLASSIFICATION OF BACTERIA IN FAMILIES. 57

usually round in form, that are derived
from vegetative cells through fission. The
cells of the thicker rods divide in three
directions of space, those of the thinner
threads only at right angles to the long axis
of the threads.

3. Genus: Phmymidiotkrix (Engler). — Thread

bacteria with very delicate barely visible
sheath, sometimes 100 microns long and 3
to 12 microns wide. Cells cylindrical, later
flattened. Multiplication through non-
motile gonidia, which are derived from
vegetative cells through division in three
directions of space. Probably similar to
crenothrix.

4. Gmm: Sphcerotilus (Kiitzing). — Cells cylin-

drical, enclosed in the sheaths, dichoto-
mously branched threads, without differen-
tiation of base and apex. Multiplication
through gonidia, which swarm from the
sheaths and attach themselves to objects and
develop into new threads. The gonidia have
a bunch of flagella attached to one pole.
The present tendency is to simplify this morpholog-
ical classification, and to distribute the bacteria into
three great groups, with their subdivisions, the mem-
bers of each group being identified 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.

MODE OF MULTIPLICATION. — In the group micro-
coed belong all spherical forms — •/. ?., all those forms
the isolated individual members of which are practically

58

BACTERIOLOGY.

of the same diameter in all directions. (See Fig. 2, a,
b, <•, (1, e.)

FIG. 2.

00
00 ,o% <*> oO

n. Staphylococci. 6. Streptococci, c. Diplococci. d. Tetrads, e. Sarcinse.

FIG. 3.

\

rf e /

a. Bacilli in pairs. 6. Single bacilli, cand d. Bacilli in threads.
e and /. Bacilli of variable morphology. •

FIG. 4.

P,

s

a b c fl

annd rf. Spirilla in short segments and longer threads —the so-called comma
forms and spirals, b. The forms known as spirochseta. c. The thick spirals
sometimes known as vibrios.

CLASSIFICATION OF BACTERIA IN FAMILIES. 59

The hncilli comprise all oval or rod-formed bacteria.
(See Fig. 3.)

To the xpiriUa belong all organisms that are curved
when seen in short segments, or when in longer threads /
are twisted in the form of a corkscrew. (See Fig. 4.) /

The micrococci are subdivided according to their pre-/
vailing mode of grouping, as seen in growing cultures/
into staphylococci — those growing in masses like cluster^
of grapes (see Fig. 2, a) ; streptococci — those growing ii
chains consisting of a number of individuals strum
together like beads upon a string (see Fig. 2, 6) ; diplc
cocci — those growing in pairs (Fig. 2, c) ; tetrads — thode
developing as fours (Fig. 2, d) ; and sarcince — those
dividing into fours, eights, etc., as cubes — that is,
contradistinction to all other forms, the segmentatioJ
which is rarely complete, takes place regularly in thi
directions of space, so that when growing the bundle
segmenting cells presents somewhat the appearance ofia
bale of cotton (Fig. 2, e).

To the bacilli belong all straight, rod-shaped bacteria
— i. 6., those in which one diameter is always greater
than the other.

In this group are found those organisms the life-
cycle of many of which presents deviations from the
simple rod-shape. Many of them in the course of
development increase in length into long threads;
along which traces of segmentation may usually be
found — the anthrax bacillus and bacillus cereus are
conspicuous examples of this. Again, under certain
conditions, many of them possess the property of form-
ing within the body of the rods oval, glistening spores
(see Fig. 6), and, if the conditions are not altered, the
rods may entirely disappear and nothing be left in

60 BACTERIOLOGY.

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. 6, c and d).
Again, many of them, from unfavorable conditions of
nutrition, aeration, 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

FIG. 5.

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

all of these conditions, however, so long as death has
not occurred, it is possible to cause these forms to revert
to the typical rods from which they originated, by the
renewal of conditions favorable to their normal vege-
tation.

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, nor vice versa; and any evidence which
may be presented to the contrary is based upon untrust-
worthy methods of experimentation.

Very short oval bacilli may sometimes be mistaken

GROUPING. 61

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 illu-
mination of the microscope be reduced to the smallest
possible bundle of light-rays. The spores, moreover,
take up staining-reagents much less readily than do
the micrococci. The most reliable differential points,
however, are the infallible properties possessed by the
spores of developing into bacilli, and by the spherical
organism with which they may have been confounded
of always producing other micrococci of the same
spherical form.

A convenient classification of bacilli is that based
upon constant characteristics which are seen to ap-
pear 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
arc twisted into the form of spirals. In some of them
the turns of the spiral are long, in others quite short.

62 BA CTERIOL OGY.

In some the threads appear rigid, in others flexible.
They are motile and multiply apparently by the simple
process of fission.1 In most respects, save form and the
property of producing spores, they are analogous in
their mode of growth to the bacilli.

The micrococci multiply 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. 2, c and d.) A similar
division in the other direction will now result in the
formation of groups of fours 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. 2, 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. 2, 6.)

The sarcinae 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
1 Dividing into two transversely.

SPORE-FORMATION. 63

fission, give to the bundles of cells the appearance com-
monly ascribed to them, viz., that of a bale of cotton or
a packet of rags. (See Fig. 2, e.)

The mode of multiplication of bacilli is similar to
that of the micrococci — i. e., a dividing cell elongates
slightly in the direction of its long axis ; an indenta-
tion appears about midway between its poles, and
this becomes deeper and deeper, until eventually two
daughter-cells have formed. This process may occur
in such a way that the two young bacilli adhere
together by their adjacent ends in much the same way
that sausages are seen to be held together in strings
(Fig. 3,/), 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.
3, e). The segmentation of the anthrax bacillus, with
which we are to become acquainted later, results, when
completed, in an indentation of the adjacent extrem-
ities of the young segments, so that by the aid of
high magnifying powers these surfaces are seen to be
actually concave. Bacilli never divide longitudinally.

SPORE-FORMATION. — With the spore-forming bacilli,
under favorable conditions of nutrition and temperature,
the same mode of segmentation is seen to occur during
vegetation ; but as soon as these conditions become
altered by ilie exhaustion of nourishment, the presence
of detrimental substances, unfavorable temperatures, etc.,
they enter, in their life-cycle, the stage to which we
have referred as spore-formation. This is the process
by which the organisms are enabled to enter a state in
which they resist deleterious influences to a much higher
degree than is possible for them when in the growing or
vegetative condition.

64 BACTERIOLOGY.

In the spore, resting, or permanent state, as it is vari- .
ously 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

FIG. 6.

bed

a. Bacillus subtilis with spores. 6. Bacillus anthracis with spores, c. Clos-
tridium form with spores, d. Bacillus of tetanus with end spores.

possess only 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
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

SPORE-FORM A TION. 65

of a cell-membrane and a transparent, clear space
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. When
thus perfectly developed, the spore may be regarded as
analogous to the seeds of higher plants. Like the seed,
it evinces no evidence of life until placed under condi-
tions favorable to germination, when there develops
from it a cell identical in all respects with that from
which it originated. Its tenacity of life, as in the case
of seeds, is almost unlimited. It may be kept in a dry
state, and this has actually been done, for years without
losing the power of germination. The glistening, en-
veloping spore-membrane is not of uniform thickness
throughout, and in consequence when germination oc-
curs the growing bacillus, the so-called vegetative form
of the organism, protrudes through the thinnest part
of the spore-membrane — that is, through the point of
least resistance. This may be either the end or the side
of the spore, according to the species under observation.
In certain cases such a protrusion is not observed, but
in its place the spore in toto appears to be gradually
absorbed or in some way converted directly into a
vegetating cell. 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 conditions favorable to its subsequent
development. Occasionally the membrane of the vege-
tative cell in which the spore is formed does not disap-
pear from around it, and the spore may then be seen
lying in a very delicate tubular envelope. Now and
then, remnants of the envelope may be noticed ad-

5

66 BACTERIOLOGY.

hering to a spore which has not yet become com-
pletely free.

By the ordinary methods of staining, spores do not
become colored, so that they appear in the stained
cells as pale, transparent, oval bodies, surrounded by
the remainder of the cell, which has taken up the dye.

A single cell produces but one spore. This may be
located either at an extremity or in the centre of the
cell. (Fig. 6.)

Occasionally spore-formation is accompanied by an
enlargement of the cell at the point at which the proc-
ess is in progress. As a result, the outline of the cell
loses its regular rod shape and becomes that of a club,
a drum-stick, or a lozenge, depending upon whether
the location of the spore is to be at the pole or in the
centre of the cell. (See Fig. 6, e and d.)

MOTILITY. — In addition to the property of spore-for-
mation there is another striking difference between vari-
ous species of the rod-shaped organisms, namely, the prop-
erty of motility, by which some of them are distinguished.
This power of motion is due to very delicate, hair-
like appendages or flagella, by the lashing motions of
which the rods possessing them are propelled through
the fluid. In some cases the flagella are located at
but one end of a bacillus, either singly (monotrichic) or
in a tuft (lophotrichic) ; and in some cases, especially
with the bacillus of typhoid fever, they are given off
from the whole surface of the rod (peritrichic). (See Fig.
7.) In a few instances similar locomotive organs have
been detected on spherical bacteria — i. e., motile micro-
cocci have been observed.

For a long time this property of independent motion
could only be assumed to be due to the possession of

THERMAL DEATH-POINT OF BACTERIA.

67

some such form of locomotive apparatus, because similar
appendages had been seen upon some of the large motile
spirilla found in stagnant water, but it was not until a
few years ago that the accuracy of this assumption was
actually demonstrated. By a special method of staining
Loffler1 was the first to render visible these hair-like
appendages. His method, as well as the several modi-
fications that have been made of it, depends for success
upon the use of mordants, through the agency of which
the stains employed are caused to adhere with increased
tenacity to the objects under treatment.

FIG. 7.

a b c

a. Spiral forms with a flagellum at only one end. 6. Bacillus of typhoid
fever with flagella given off from all sides, c. Large spirals from stagnant
water with wisps of flagella at their ends (spirillum undula).

THERMAL DEATH-POINT OF BACTERIA. — By " ther-
mal death-point of bacteria " is meant the temperature
necessary to kill them in a given time. As this varies
with different species, it is an aid to identification. For
the practical purposes of the sanitarian the knowledge is
of fundamental importance. The thermal death-point of
an organism is ascertained by subjecting it to varying de-
grees of temperature for five or ten minutes until the point

1 Loffler's method of staining will be found iu the chapter devoted
to this part of the technique.

68 BACTERIOLOGY.

is reached where it is killed. The test is best carried
out by means of small glass bulbs, the so-called Stern-
berg bulbs, or through the use of capillary tubes con-
taining a small amount of fluid inoculated with the
organism to be studied. The bulb, or tube, is sealed in
the gas flame and placed in a water-bath kept at 50° C.
for five minutes. Sub-cultures are now made to learn
whether the bacteria have been killed or not. If the organ-
ism survives the test is repeated at 55°, 60°, 65°, and
70° C. Finally, the test is repeated for each degree of
temperature between the points where growth is still
apparent and where the organisms have been killed.
If the bacteria were killed when heated to 60° C. for
five minutes, but survived when heated to 55° C., then
similar tests are made for the same length of time for
each degree of temperature between 55° and 60° C. It
will usually be found that heating for ten minutes suf-
fices to kill the bacteria at a temperature one or two de-
grees lower than that required when heated for only five
minutes. All such tests should be made at least in
duplicate, and the mean of the results taken.

CHAPTER III.

Principles of sterilization by heat — Methods employed — Discontinued
sterilization — Fractional sterilization — Apparatus employed — Ster-
ilization under pressure — Sterilization by hotair — Chemical disin-
fection and sterilization — Mode of action of disinfectants — Practical
disinfection.

OF fundamental importance to successful bacterio-
logical manipulations are acquaintance with the prin-
ciples underlying the methods of sterilization and dis-
infection, 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
probably arose from the circumstance that culture-
media, and certain other articles that it is desirable
to render 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,
arc commonly subjected for a time to the action of chem-
ical compounds possessing germicidal properties — i. e.,

69

70 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 insure the destruction of all
living forms that are present, need not of necessity do
so, but may be limited in its action to those only that
possess the power of infecting ; it may or may not, there-
fore, 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 gov-
erned by circumstances. In the laboratory it is essen-
tial that all culture-media with which 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 would 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. 71

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 exhibiting 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, pathological exudates,
faeces, 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.1

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 An occasional exception to this is the use of chloroform, mentioned
above.

72 BA CTERIOLOG Y.

circumstance the temperature to which they are ex-
posed becomes more and more elevated as the pressure
increases.

Experience has taught us that the process of ster-
ilization by dry heat is of limited application because
of its many disadvantages. For successful sterilization
by the method of dry heat, not only is a relatively
high temperature needed, but the substances under
treatment must be exposed to this temperature for a
comparatively long time. The penetration of dry heat
into materials which are to be sterilized is, moreover,
much less thorough than that of steam. Many sub-
stances of vegetable and animal origin are rendered
valueless by subjection to the dry method of sterilization.
For these reasons comparatively few materials 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 pi e?
as flasks, plates, small dishes, test-tubes, pipettes — and
such metal instruments as are not seriously injured by
the high temperature.

METHODS EMPLOYED. — Sterilization by moist heat
— steam — offers conditions much more favorable. The

STERILIZATION BY HEAT. 73

penetrating power of the steam is not only more ener-
getic, but the temperature at which sterilization is ordi-
narily accomplished is, as a rule, not destructive to the
objects under treatment. This is conspicuously seen in
the work of the laboratory; the culture-media, com-
posed in the main of decomposable organic materials
that would be rendered entirely worthless if exposed to
the dry method of sterilization, sustain no injury what-
ever when intelligently subjected to an equally effective
sterilization with steam. The same may be said of cot-
ton and woollen fabrics, bedding, clothing, etc.

Aside from the relations of the two methods to the
materials to be sterilized, their action toward the or-
ganisms to be destroyed is quite different. The pene-
trating power of steam renders it by far the more effi-
cient 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, such as flasks, test-tubes, culture-dishes, pipettes,
plates, etc.

Sterilization by steam is practised with all culture-
media, whether fluid or solid. Bouillon, milk, gelatin,
a^ar-a<rar, potato, etc., are under no circumstances to
be subjected to dry heat.

DISCONTINUED STERILIZATION. — The manner in

74 BACTERIOLOGY.

which heat is employed in processes of sterilization
varies with circumstances. When used in the dry
form its application is always continuous — /. 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 in-
jure them. For this and other reasons steam is usually
applied intermittently and for short periods of time. The
principles involved in the intermittent method of sterili-
zation 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

STERILIZATION BY HEAT. 75

taken advantage of in the process of sterilization by
steam known as the discontinuous, fractional, or inter-
mittent method, and are the essential feature of the
principles on which the method is based.

As culture-media 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 sub-
jecting 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, this combination is found, the spore-forming or-
ganisms 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 be discontinued, and the material
which presents conditions favorable to the germination
of the spores be allowed to stand for a time, usually
for about twenty-four hours, at a temperature of from
20° to 22° C., those spores which resisted the action of
the steam will, in the course of this interval, germinate

76 BA GTERIOLOG Y.

into the less resistant vegetative cells. A second short
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 such an
exposure to heat as 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 vegetative 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, as a general rule, have
been destroyed, and, unless opportunity is given for
the access of new organisms from without, the sub-
stances thus treated remain sterile. As an exception
to this, certain species of spore-forming bacteria are
occasionally encountered that are not readily destroyed
by this mode of treatment. These species are found so

STERILIZATION BY HEAT. 77

uniformly in the soil that the customary designation for
them is that of " the soil bacteria." This group includes
a number of species that are endowed with remarkable
resistance to heat. Some of them are probably ther-
mophilic by nature, which would account not only for
the failure to destroy their spores by the ordinary ex-
posures to steam, but also for their slow and incomplete
development from the spore to the less resistant vege-
tative stage during the intervals between the heatings,
for, as a rule, the materials containing them are kept at
a temperature during these intervals that is too low to
favor the rapid germination of the species having ther-
mophilic tendencies.

As a result of the presence of these species, media
that have been subjected to the customary discontin-
uous method of sterilization may, after having been
kept for a time, reveal the presence of isolated col-
onies of bacteria distributed through them in such a
way as to preclude all likelihood of their having fallen
upon it from the air after sterilization was supposedly
complete.

Theobald Smith l has called attention to an instruc-
tive personal experience. He finds that when media
are present in vessels in only thin layers the spores of
anaerobic species do not develop into the vegetative
forms during the interval between the heatings, for the
reason that the shallow layer of medium does not suf-
ficiently exclude free oxygen to permit it ; and by sub-
jecting such materials, apparently sterilized by the inter-
mittent method, to strictly anaerobic conditions a devel-
opment of anaerobic species will often occur. On the

1 Theobald Smith: Journal of Experimental Medicine, vol. iii. No.
6, p. 647.

78 BACTERIOLOGY.

other hand, if the vessels be nearly filled with media,
and especially if the area of the surface be small, the
conditions are much more favorable to the germination
of anaerobic spores, and sterilization by this process after
such precautions is usually perfect.

Fortunately, these undesirable experiences are rare,
but that they do occur, and result in no small degree
of annoyance, will be admitted 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.

FRACTIONAL, STERILIZATION. — The process of frac-
tional sterilization at low temperatures is based upon ex-
actly the same principle, but differs in two respects from
the foregoing in the manner by which it is practised, viz.,
it requires a greater number of exposures for its accom-
plishment, and the temperature at which it is conducted is
not above (58°-70° C. It is employed for the sterilization
of easily decomposable materials, which would be rendered
useless by steam, but which are unaltered by the tem-
perature 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 higher temperatures. This process requires
that the material to be sterilized should be subjected to

STERILIZATION BY HP: AT. 79

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, in about one hour, the vitality of
almost all organisms in the vegetative stage. Formerly
blood-serum was always sterilized by the intermittent
method at a low temperature.

STERILIZATION UNDER PRESSURE. — 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 for 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
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 press-
ure 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

80 BACTERIOLOGY.

fine precipitates are said to result, and some believe
the reaction undergoes a change. In the experience
of those who have used steam under pressure not ex-
ceeding one atmosphere 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 violently, and, as a rule,
will boil quite out of the tubes, flasks, etc., containing
them. For this reason the apparatus must be kept
closed until cool, or until the gauge indicates that press-
ure no longer exists within the chamber, and even then
it should be opened very cautiously. It is patent from
this that the temperature and time of exposure of arti-
cles sterilized by this process cannot usually be con-
trolled with accuracy. It requires some time to reach a
given pressure after the apparatus is closed, and it also
requires time for cooling after the desired exposure to
such pressure before the apparatus can be opened.

It is manifest that during these three periods, viz.,
((t) reaching the pressure desired, (6) time during which
the pressure is maintained, and (e) time for fall of press-
ure 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.
Clearly, if the desired pressure and temperature have
been maintained for ten minutes, one cannot say that

STERILIZATION BY HEAT.

81

that is all the heat to which the articles have been sub-
jected during their stay in the chamber. In this light,
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.
Nevertheless, for general laboratory purposes, sterili-
zation by steam under pressure has so much to recom-
mend it in the way of economy of time and certainty
of accomplishment that it has practically superseded
the older methods of sterilization by streaming or live

FIG. 8.

Steam sterilizer, pattern of Koch.

steam ; and in most laboratories the original styles of
steam sterilizers are rapidly giving way to some one or
another of the modern forms of autoclave.

82 BACTERIOLOGY.

For sterilization by live steam the apparatus in com-
mon use was for a long time the cylindrical boiler rec-
ommended by Koch. (See Fig. 8.) Its construction
is very simple, essentially that of the ordinary domestic
potato-steamer. It consists of a copper cylinder, the
lower fifth, approximately, of which is somewhat larger
in circumference than the remaining four-fifths and
serves as a reservoir for the water from which the
steam is to be generated. Covering this section of the
cylinder is a wire rack or grating, through which the
steam passes, and which supports the articles to be
sterilized. Above this, comprising the remaining four-
fifths of the cylinder, is the chamber for the reception
of the materials over and through which the steam is to
pass. The cylinder is closed by a snugly fitting cover,
through which are usually two perforations, into which
a thermometer and a manometer may be inserted. The
whole of the outer surface of the apparatus is encased in
a non-conducting mantle of asbestos or felt.

The water is heated by a gas-flame placed in an en-
closed chamber, upon which the apparatus rests, which
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 small
to be indicated by the gauge ; otherwise there is danger
of the reservoir becoming dry and the bottom of the ap-
paratus 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

STERILIZATION UNDER PRESSURE. 83

for use in the kitchen. It is called the " Arnold steam
sterilizer." It is very ingenious in its construction as
well as economical in its employment.

The difference between this apparatus and that just
described is that it provides for the condensation of the
steam after its escape from the sterilizing chamber, and
returns the water of condensation automatically to the

FIG. 9.

Arnold steam sterilizer.

reservoir, so that in practice the apparatus requires but
little attention, as with ordinary care there is no likeli-
hood of the water in the reservoir becoming exhausted,
with the consequent destruction of the sterilizer.
Fig. 9 shows a section through this apparatus.

STERILIZATION UNDER PRESSURE.

The advantages of the use of steam under pressure
for the purposes of sterilization have received such

84

BACTERIOLOGY.

general recognition that almost everywhere this method
is supplanting the older one of intermittent sterilization
with streaming or live steam. By this plan one is able
to accomplish, by a single exposure of fifteen minutes to
steam under a pressure of one atmosphere, the same end
that would, with streaming steam, require three expos-
ures of fifteen minutes on each of three successive days.

FIG. 10.

JL

Autoclave, pattern of Wiesnegg. A. External appearance. B. Section.

For sterilization by steam under pressure several spe-
cial forms of apparatus exist. The principles involved
in them all are, however, the same. They provide for
the generation of steam in a chamber from which it
cannot escape when the apparatus is closed. Upon the
cover of this chamber is a safety-valve, which can be
regulated so that any degree of pressure (and coinci-

STERILIZATION BY HOT AIR. 85

dently of temperature) that is desirable may be main-
tained within the sterilizing chamber. These sterilizers
are known as " digesters " and as " autoclaves." Their

FIG. 11.

Autoclave or digester for sterilizing by steam under pressure.

construction can best be understood by reference to
Figs. 10 and 11.

STERILIZATION BY HOT AIR.

The hot-air stcrili/i-rs used in laboratories are sim-
ply doublr-xvalh'd boxes of Russian or Swedish iron

86

BACTERIOLOGY.

(Fig. 12), having a double- walled door, which closes
tightly, and a heavy copper bottom. They are pro-
vided with 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.

FIG. 12.

FIG. 13.

Laboratory hot-air sterilizer.

Rose-burner.

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
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

CHEMICAL STERILIZATION AND DISINFECTION. 87

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 had constructed some years ago
a sterilizer in all respects similar to the old form except
in the arrangement of the copper bottom. This latter
is made in such a way that it can easily be removed, so
that by keeping several sets of copper plates on hand a
new plate 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 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 been stated, it is possible by means of cer-
tain chemical substances to destroy all bacteria and
their spores that may be within or upon various mate-
rials and objects — i. e., to sterilize them ; and it is also
possible by the same means to rob objects of their

88 BACTERIOLOGY.

dangerous infective properties without at the same
time sterilizing them — i*. e., to disinfect them. This
latter process depends upon the fact that the vital-
ity 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 ex-
ceptionally such volatile germicides as chloroform and
ether are employed for the sterilization of special culture-
media. (See Preservation of Blood-serum with Chloro-
form.) In short, they are mainly of value in rendering
infected waste-materials innocuous. For the successful
performance of this form of disinfection there is one
fundamental rule always to be borne in mind, viz., it
is 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 to
l>e destroyed are of such a character that they combine
with the disinfecting agent to form insoluble, more or

CHEMICAL STERILIZATION AND DISINFECTION. 89

less inert precipitates ; these so interfere with the pene-
tration of the disinfectant that many bacteria may escape
its destructive action entirely and no disinfection be ac-
complished, although an agent may have been employed
that would, under other circumstances, have given en-
tirely satisfactory results.

An antiseptic is a body which, by its presence, pre-
vents 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.

MODE OF ACTION OF DISINFECTANTS. — In the de-
struction of bacteria by means of chemical substances
there occurs, most probably, a definite chemical reac-
tion— that is to say, the characteristics both of 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 state
with certainty, as yet, that this is in general the case ; but
the evidence that is rapidly accruing from 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 albuminous bodies there results a
third compound, which has neither all the character-
istics of mercury nor of albumin, but partakes of
some of the peculiarities of lx>th ; it is a combina-
tion of albumin and mercury, commonly known by the

90 BACTERIOLOGY.

indefinite term " albnrainate of mercury." Some such
reaction as this apparently occurs when the soluble silts
of mercury are brought in contact with bacteria. This
view has 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-
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 death of
the spores, for if by proper means the combination of
mercury with their protoplasm was broken up, many
of the spores resumed their vitality, with all their pre-
vious 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 experi-
ments have given a new impulse to the study of disin-
fectants, and in the light shed by them many of our
previously formed ideas concerning the action of disin-
fecting agents have been modified.

The process of disinfection is not a catalytic one —
i. 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 chem-
ical reaction occurring within more or less fixed limits ;
that is to say, with a given amount of the disinfect-
ant employed so much work, expressed in terms of disin-
fection— destruction of bacteria — can be accomplished.

CHEMICAL STERILIZATION AND DISINFECTION. 91

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 and rapidity of the reaction become greater under
the influence of heat, so in the process of disinfection
the combination between the disinfectant and the organ-
isms to be destroyed is much more energetic at a tem-
perature of 37° to 39° C. than it is at 12° to 15° C.

A number of important and novel suggestions with
regard to the modus operandi of disinfection were
brought out through the work of Kronig and Paul,1
who took up the subject from its physico-chemical
standpoint. The comprehensive nature of this elab-
orate investigation precludes more than a brief men-
tion of some of the conclusions reached, and in order
that these may be intelligible, certain beliefs (working
hypotheses) of the physical chemists should be borne in
mind. In 1887 Arrhenius proposed the theory that
when an electrolyte (a compound decomposable by an
electric current) is dissolved in water its molecules break
down, not simply into their component atoms, but into
ions, which are atoms or groups of atoms having electro-
positive and electro-negative characteristics. According
to this theory, salts, when dissolved in water, undergo
electrolytic dissociation into metallic and acidic ions,
the former being the electro-positive cation, the latter
the electro-negative anion ; sodium chloride, for exam-
ple, resolving itself, under these conditions, into its
sodium, or metal-ion, and its chlorine, or acidic ion.
The electro-positive cations, according to Ostwald, com-
prise the metals and metal-like radicals, such as am-

1 Kronig and Paul: Zeitschrift fur Hygiene und Infectionskrank-
heiten, 1897, vol. xxv. pp. 1-112.

92 BA CTERIOLOO Y.

monium (NH4) and hydrogen (H) ; while the electro-
negative anions include the halogens, the acidic radicals
(such as NO3 and SO4), and hydroxyl.1 Using this theory
as the basis of their investigations, Kronig and Paul
reached the following conclusions with regard to the
action of chemical disinfectants :

The germicidal value of a metallic salt depends not
only upon its specific character, but also upon that of its
anion.

Solutions of metallic salts in which the metallic part
is represented by a complex ion and in which the con-
centration of the metal ion is very slight, have but
feeble disinfecting activity.

The halogen compounds of mercury act according to
the degree of their dissociation.

The disinfecting power of the halogens — chlorine,
bromine, iodine — (as well as their compounds) is in in-
verse ratio to their atomic weights.

The disinfecting activity of watery solutions of mer-
curic chloride is diminished by the addition to them
of other halogen compounds of metals and of hydro-
chloric acid. It appears probable that this is due to
obstruction offered to electrolytic dissociation.

The disinfecting activities of watery solutions of mer-
curic nitrate, mercuric sulphate, and mercuric acetate are
increased by the moderate addition of sodium chloride.

In general, acids disinfect according to the degree
of their dissociation — i. e., according to the concentra-
tion of their hydrogen ions in the solution.

1 Consult Ostwald's Lehrbuch der Allg. Chemic ; or Muir's transla-
tion of Ostwald's Solutions, p. 189, published by Longmans, Green &
Co., London and New York, 1891. Also " The Rise of the Theory of
Electrolytic Dissociation," etc., by H. C. Jones, Ph. D., Johns Hopkins
Hospital Bulletin, No. 87, June, 1898, p. 136.

CHEMICAL STERILIZATION AND DISINFECTION. 93

The bases, potassium, sodium, lithium, and ammo-
nium hydroxide, disinfect according to the degree of
their dissociation — i. e., corresponding to the- concen-
tration of their hydroxyl ions in the solution.

The disinfecting activity of metallic salts is, in gen-
eral, less in albuminous fluids than in water. It is
probable that this is due to a diminution in the concen-
tration of metallic ions in the solution.

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 it is the custom to keep at
hand vessels containing solutions of corrosive sublimate,
into which infectious materials may be placed. The

94 BACTERIOLOGY.

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 where the solution used is not freshly pre-
pared and frequently replenished. On the introduction
of such substances into the sublimate solution the mer-
cury is quickly precipitated by the albumin, and its dis-
infecting properties may be in large part or entirely
destroyed ; we may in a very short time have little else
than water containing an inactive precipitate of albumin
and mercury, in so far as its value as a disinfectant is
concerned.

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

PRACTICAL DISINFECTION. — Where it is desirable
to use chemical disinfectants in the laboratory, much
more satisfactory results can usually be obtained from
the employment of carbolic acid in solution. A 3 or 4
per cent, solution of commercial carbolic acid in water
requires longer for disinfection ; but it is, at the same
time, open to fewrer objections than are solutions of the
inorganic salts ; though here, too, we find a somewhat
analogous 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 antitrade.

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-

CHEMICAL STERILIZATION AND DISINFECTION. 95

soda) solution for fifteen to twenty minutes, or placed
in the steam sterilizer for half an hour.

Intestinal evacuations may best be disinfected with
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 contains a considerable
excess of the lime, and should remain in contact with
them for one or two hours. Excreta may also be easily
disinfected by thoroughly mixing them with two or
three times their volume of boiling water, after which
they are kept covered until cool.

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 rely more
upon the destructive properties of heat than upon those
of chemical agents.

From what has been said, the absurdity of sprink-
ling here and there a little carbolic acid, or of placing
vessels of carbolic acid about apartments in which in-
fectious diseases are in progress, must be plain. Treat-
ment 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 bacteria, and must
be in contact with them for a long enough time to insure
the destruction of their life.

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

96 BACTERIOLOGY.

The agents, then, which will prove of greatest value in
the laboratory for the purpose of rendering infectious
materials harmless are : heat, either by burning, by
steaming for from half an hou^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.

CHAPTER IV.

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

As was stated in the introductory chapter, the isola-
tion in pure cultures of the different species that may
be present in mixtures of bacteria was rendered possible
only through the methods suggested by Koch. Since
the adoption of these methods they have undergone
many modifications, but the fundamental principle re-
mains the same. The observation which led to their
development was a very simple one, and one that is
frequently before us. Koch noticed that on solid sub-
stances, such, for example, as a slice of potato or of moist
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
which proved to be colonies of bacteria. Each of
these colonies on closer examination proved to be, as
a rule, composed of distinct species of micro-organisms.
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 rep-
resented, in the main, the development 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 in-
dividuals, what means can be employed for obtaining the
7 97

98 BACTERIOLOGY.

same results at will from mixture of different species of
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 might 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 favorable 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, the
task is tedious, to say the least of it ; but if the handful
of grain be thrown upon a large flat surface, as upon a
table, the grains become widely separated and the matter
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 upon a large,
flat surface, the individual bacteria in the mass are
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, some substance could be found which

METHODS OF ISOLATION.

99

possesses the property of being at one time fluid and
at another time solid, and which could be added to
this bouillon without in any way interfering with the
life-functions of the bacteria, then, as solidification set
in, the organisms would be fixed in their positions, and
the conditions would be analogous to those seen on the
bit of potato.

FIG. 14.

Showing certain macroscopic characteristics of colonies. Natural size.

Gelatin possesses this property, and it was, therefore,
used. At a temperature which does not interfere with
the life of the organisms it is quite fluid, whereas when
subjected to a lower temperature it solidifies. When

1 00 BA CTERIOLOG Y.

once solid it may be kept at a temperature favorable to
the growth of the bacteria and will remain in its solid
state.

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 pecu-
liarities, 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 the original decom-
posed 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 impossible
to pick them out, not only because of their proximity
the one to the other, but also because this packing
together materially interfered with the production of
those characteristic differences visible to the naked
eye. The numbers of the organisms were then dimin-
ished 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 portion was
added to a third gelatin-bouillon tube, and so on.
These were then poured upon large surfaces and allowed
to solidify. The result was entirely satisfactory. On
the gelatin plates from the original tube, as was ex-
pected, the colonies were too numerous to be of use ;
on the plates made from the first dilution they were
much fewer in number, but usually they were still too

METHODS OF ISOLATION. 101

numerous and too closely packed to permit of charac-
teristic growth ; 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 typical way. (See Fig. 15.) There
was then no difficulty in picking out the colonies result-
ing from the growth of the different individual bacteria.

FIG. 15.

C

Series of plates showing the results of dilution upon the number of colonies :
A. Plate No. 1, or " original." B. First dilution, or Plate No. 2. C. Second
dilution, or Plate No. 3. 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 constituent 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, gelatinous substances are employed — the
one an animal or bone gelatin, the ordinary table gelatin
of good quality; the other a vegetable gum, known
as agar-agar, the native name for CVylon moss or I Jen-

1 02 BACTERIOLOG Y.

gal isinglass, which is obtained from a group of algae
growing in the sea along the coast of Japan, China, and
many parts of the East, where it is employed as an
article of diet by the natives.

The behavior of the two gelatinous substances 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° to 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. It remains fluid ordinarily until the temperature has
fallen to 38°-39° C., when it rapidly solidifies. For
our purposes, only that form of agar-agar can be used
which remains fluid at from 38° to 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 employed in those cases in
which the cultivation must be conducted at a tempera-
ture above the melting-point of gelatin.

In addition to their thermal reactions, these two
gelatinous substances are affected very differently by
different species of bacteria. As we shall learn later,
certain bacteria elaborate in the course of their growth
digestive (proteolytic) enzymes or ferments that, in their
action upon proteid matters, are strikingly like pepsin

METHODS IN ISOLATION. 103

in some and trypsin in other instances. When bacteria
endowed with this physiological property are cultivated
upon bone gelatin their growth is accompanied by the
progressive digestion (liquefaction) of the gelatin, which
liquefied gelatin cannot again be brought to a solid con-
dition. We know of no bacteria capable of producing
a similar liquefaction of agar-agar or vegetable gum.
This striking difference between the two gelatinous sub-
stances under the influence of bacterial activity is one
of the most important and commonly employed differ-
ential reactions in the identification of species.

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
culture-media is beef-tea, or bouillon.

BOUILLON. — 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 : 500 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 which time the mixture is to remain in
an ice-chest or to be otherwise kept at a low tempera-
ture. At the end of twenty-four hours it is to be strained
through a coarse towel and pressed until a litre of fluid is
obtained. To this are to be added 10 grammes (1.0 per
cent.) of dried peptone and 5 grammes (0.5 per cent.) of
common salt (Nad). It is then to be rendered exactly
neutral or very slightly alkaline to litmus with a few
drops of saturated sodium carbonate solution. The flask
containing the mixture is then to be placed either in a
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 modifications of this method
are of sufficient value to justify mention. Most im-

104

BOUILLON. 105

portant 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 ; 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 frequently gives rise to a confusing, tem-
porary acid reaction which disappears on heating;
nor is litmus the most reliable indicator to employ.
To obviate this, Schultz1 recommends exact titration
with a solution of caustic soda. For this purpose a 4
per cent, solution of caustic soda is prepared. From this
a 0.4 per cent, solution is made, and with it the titration
is practised. After the bouillon has been deprived of all
coagulable albumin and blood-coloring-matter by boiling
and filtration, and has cooled down to the temperature
of the air, 'its volume is exactly measured.

From this a sample of exactly 5 or 10 c.c. is then taken,
and to it 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 of phenolphtalein in 300 c.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 very slowly into it
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

1 Schultz: Ccntralbl. f. Bakt. u. Parasitenkmide, 1891, vol. x. Nos.

106 BACTERIOLOGY.

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.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. less 4 c.c., the amount employed for the
two samples of 10 c.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, should be adopted for experiments in which it
is desirable to have the reaction of the medium accurate
and constantly of the same degree.

BOUILLON. 107

For the purposes of the beginner, however, results
quite satisfactory in their nature may be obtained by the
employment of the saturated sodium carbonate solution
for neutralization, with litmus paper as the indicator.

In the exhaustive paper of Fuller l on the question of
reaction it was shown that the results obtained by titrating
the same culture-medium with the same alkaline solu-
tion diifered 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 c.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 bac-
teriologists 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 c.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 c.c. of phenolphtalein
solution 2 is added and titration with the one-twentieth
normal caustic alkali solution is quickly made. The

1 Fuller : " On the Proper Reaction of Nutrient Media for Bacterial
Cultivation," Public Health (Journal of the American Public Health
Association), Quarterly Series, 1895, vol. i. p. 381.

2 A 0.5 per cent, solution of the powder in 50 per cent, alcohol.

1 08 EA CTERIOLOG Y.

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
c.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 ex-
perience gained from 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 c.c. — i. e., from 15 to 20 c.c. less than the
amount needed to bring the reaction up to the neutral
point.

Not infrequently the filtered bouillon, neutralized

NUTRIENT GELATIN. 109

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, fil-
tered, and sterilized ; or, if due to the latter cause, sub-
sequent 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 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 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.

NUTRIENT GELATIN. — For the preparation of gelatin
the bouillon is first made in the way given, except
that its reaction is corrected after the gelatin has
been completely dissolved, which occurs very rapidly
in hot bouillon. The reaction of the gelatin of com-
merce is frequently quite acid, so that a much larger
amount of alkali is needed for its neutralization than
for other media. It is possible, however, to obtain
from the makers an excellent grade of gelatin from
which all acid has been carefully washed.1 The gelatin
is added in the proportion of 10 to 12 per cent. Its

1 Hesteberg's acid-free, gold-label gelatin Las given us entire satis-
faction in this respect. It is an imported article.

110 BACTERIOLOGY.

complete solution may be accomplished either over a
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.

It is now almost the universal practice to use enam-
elleled iron saucepans, instead of glass flasks, for the
purpose of making both gelatin and agar-agar ; by this
means the free flame may be employed without danger
of breaking the vessel, and, with a little care, without
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 ordi-
nary 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 solution is complete, 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. Inability to fold a filter properly is so
common with beginners that a detailed description of
the steps may not be out of place. To fold a filter cor-
rectly, proceed as follows : 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
V to 3 the fold 6 is produced ; and by bringing 1 to 3
and 1' to 7 the folds 2 and 8 result.

NUTRIENT GELATIN.

Ill

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

FIG. 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), wrhich when
opened assumes the form of a properly folded filter
(seen in 6, Fig. 17). Before placing it upon the fun-
nel it is well to go over each crease and see that it is
as closely folded as possible, care being taken not to
tear it. The advantage of the folded filter is that by

112 BACTERIOLOGY.

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 greater part of the flat surface
free for filtration.

The employment of the hot-water funnel, so often
recommended, has been dispensed with in this work to
a very large extent, for the reason that if solution of
the gelatin is complete, filtration is so rapid as not to
necessitate the use of an apparatus for maintaining a
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
filtration is, as a rule, retarded. The filtration is fre-
quently 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 to which it must be brought,
otherwise it is useless), and clarification becomes neces-
sary. For this purpose the mass must be redissolved,
and when at a temperature between 60° and 70° C. an
egg, 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. The
albumin coagulates as large flocculent masses, and it is
better not to break them up if it can be avoided, as
when broken up into fine flakes they clog the filter and
materially retard filtration.

NUTRIENT GELATIN. 313

The practice sometimes recommended of removing
these albuminous coagula by first filtering the gelatin
through a cloth, and then through paper, is not only
superfluous, but in most instances renders the process
of filtration much more difficult, because of the disin-
tegration of the masses into finer particles, which have
the effect just mentioned, viz., of clogging the filter.

Under no circumstances should a filter be used with-
out 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
surface of the paper, instead of becoming fixed in its
meshes.

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

Gelatin should not, as a rule, be boiled more than
ten or fifteen minutes at one time, or be left in the
steam sterilizer for more than thirty minutes ; other-
wise its property of solidifying may be impaired.

As soon as the preparation of the gelatin is complete,
whether it is retained in the flask into which it has
been filtered or decanted into sterilized test-tubes, it
should be sterilized, the mouth of the flask or the test-
tubes containing it having previously tx«n closed with

8

114 BACTERIOLOGY.

cotton plugs. It may be sterilized by either the inter-
mittent method with streaming steam or by a single
application of steam under pressure in the autoclave.
If the latter method be selected, the pressure should
not exceed one atmosphere and the time of exposure be
not over fifteen minutes.

NUTRIENT AGAR-AGAE. — The preparation of nutrient
agar-agar by the beginner is far too frequently a tedious
and time-consuming operation. 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 having
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 on the side
of the vessel make a mark at the height at w^hich the level
of the fluid stands ; if a litre of medium is being made,
add about 250 to 300 c.c. more of water and allow the
mass to boil slowly, occasionally stirring, over a free
flame, from one and a half to two hours ; or until
the excess of water — i. e., the 250 or 300 c.c. that
were added — has evaporated. Care must be taken
that the mixture does not boil over the sides of the

NUTRIENT AGAR-AGAR. 115

vessel. From time to time observe if the fluid has
fallen below its original level ; if it has, add water
until its volume of 1 litre is restored. At the end of
the time given remove the flame and place the vessel
containing the mixture in a large dish of cold water ;
stir the agar-agar continuously until it has cooled 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 commerce may be dis-
solved 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 carefully 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 temperature of the mass
to the point given, 68°-70° C., otherwise the coagula-
tion of the albumin will occur suddenly in lumps and
masses as soon as it is added, and its clarifying action
will not be uniform. The process of clarification
with the egg is purely mechanical ; the finer particles,
which would otherwise pass through the pores of the
filter, being taken up by the albumin as it coagulates
and being 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; as a rule, the
filtrate is as clear and transparent as agar-agar usually
appears.

It may 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 entirely unnecessary. Agar-agar prepared after

116 BACTERIOLOG Y.

the methods just given should pass 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
agar-agar without causing the precipitates often seen
when all the ingredients are added at once 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 c.c. The peptone, salt, and beef-extract
are then added and the boiling 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, and filters more quickly,
but, as Schultz has pointed out, it loses at the same time
some of its gelatinizing properties. The bouillon should
always be neutralized before the agar-agar is added to
it ; for if the bouillon be acid from the juices of the
meat, it robs the agar-agar, under the influence of
heat, of part of its gelatinizing power, which will not
be regained by subsequent neutralization.

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

If glycerin is to be added to the agar-agar, it is done

PREPARATION OF POTATOES. 117

after filtration and before sterilization. The nutritive
properties of the media for certain organisms, particu-
larly the tubercle bacillus, are increased by the addition
of glycerin in the proportion of 5 to 7 per cent.

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

The most important feature of all the media, aside
from the correct proportion of the ingredients, is their
reaction. 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 media mentioned above 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 POTATOES. — Potatoes are prepared
for use in two \vays :

1. They are taken as they come to market — old
potatoes being usually recommended — and carefully
scrubbed under a 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 placed in a solu-
tion of corrosive sublimate of the strength of 1 : 1000,
where they are allowed to remain 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
Iwttom and sterilized in the steam sterilizer for forty-

118 BA CTERIOLOG Y.

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, when they are ready for
use. When prepared in this way they are usually in-
tended 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 in
two by a knife which has just been sterilized in a free
flame until it is quite hot. The knife is passed not
quite through the potato, but nearly so. A large glass
culture-dish for the reception of the halves of the
potato, having been disinfected for twenty minutes with
1 : 1000 sublimate solution and then drained of all ad-
herent solution, should be at hand ready for the potato ;
the cover is removed, and by twisting the knife gently
the halves of the potato may be caused to fall apart in
the dish and usually to fall upon their convex surfaces,
leaving the flat sections uppermost. The cover of
the dish is replaced and the potatoes are ready for
inoculation.

2. Preparation of potatoes far test-tube cultures. Method
of Bolton.1 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

1 Medical News, vol. i. of 1887, No. 12, p. 318.

PREPARATION OF POTATOES.

119

FIG. 18.

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-
ders of potato are 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. After
being thus washed they are placed in pre-
viously prepared test-tubes, one piece in each
tube, with the slanting surface up, the cot-
ton plugs of the tubes replaced, and they
are then to be sterilized in steam for fifteen
to twenty minutes on each of three successive
days. Or the entire sterilization may be ac-
complished in the autoclave, with the steam
under a pressure of one atmosphere, by a
single exposure of twenty to twenty-five
minutes. Potatoes thus prepared 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 discs 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

Potato in test-
tube.

1 20 BA CTERIOLOG Y.

the mass a mean of the composition of the several pota-
toes, or bits of potatoes, used in making it, an advan-
tage where uniformity is desired.

Care must be given to the sterilization of potatoes,
because they always have adhering to them the organ-
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 organism
which is not infrequently more or less of an obstacle
to the work of the beginner.

BLOOD-SERUM. — By the original method of preparing
blood-serum a great many precautions were taken that
have been found unnecessary to the success of the more
modern plans.

It is possible to collect serum 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 slaughter-
houses are the source from which it is usually obtained,
and here a certain amount of contamination is unavoid-
able, though its extent may be limited by proper pre-
cautions.

The precautions to be taken at the slaughter-house in
the collection of blood and the preparation of serum for
culture purposes are 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 cut. The blood
which spurts from the severed vessels should be col-
lected in large glass jars which have been previously
cleaned and disinfected, and all traces of the disin-
fectant removed with alcohol and, finally, with ether.

BLOOD-SERUM. 121

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 care-
fully disinfected. The best form of vessels for the
purpose is the large 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 to 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 may 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 Avith cotton Avadding
and sterilized. After treating all the serum in this way,
care having been taken to exclude as much as possible of

122

BACTERIOLOGY.

the coloring-matter of the blood, it may be placed again
in the ice-chest for twenty-four hours, during which
time the corpuscular elements will sink to the bottom,
leaving the supernatant fluid quite clear. This may
then be pipetted off, either into sterilized test-tubes,
about 8 c.c. to each tube, or into small sterilized flasks
of about 100 c.c. capacity. It is then to be sterilized
by the 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.

FIG. 19.

Chamber for sterilizing and solidifying blood-serum. (Kocn.)

At the end of this time the serum in the tubes may
either be retained as fluid serum or solidified at between

BLOOD-SERUM. 123

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 serum. The proc-
ess of solidification requires constant attention if good
results are to be obtained — i. e., if a translucent, solid
medium is the result. If the old, small form of appa-
ratus be employed (Fig. 19), 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
Loffler's serum-mixture.

The best results are obtained when a low temperature
is employed for a long time. Under any circumstances
the tubes should 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 or gauze : in
this way all the tubes are exposed to about the same

124 BACTERIOLOGY.

temperature. The thermometer which indicates the
temperature inside the chamber should not touch the
surfaces, bat should either be suspended free from
above through a cork in the top of the apparatus, if
a 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 a 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 hyphae
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 diagnosis of diphtheria by the method of Loftier has
become so general, and large quantities of serum-tubes
have been found necessary, modifications have been sug-
gested that have, in this country at least, almost entirely

BLOOD-SERUM. 125

supplanted the method of Koch. These modifications
comprehend both the source and the manner of obtain-
ing the serum, and the method of subsequently steriliz-
ing it. In the first place, it is becoming more and more
the custom to obtain serum from horses, not because it
possesses any nutritive advantages over that from
bovines, but because it can, in some large cities at
least, be easily obtained from the laboratories in which
horses are used in the production of antitoxins. In
these places the blood is drawn direct from the jugular or
some other large vein by means of a trocar thrust through
the skin into the vessel. The result is that the animal
is not injured, the blood is obtained in a cleanly manner,
and when due precautions are taken it is almost free
from bacterial contamination, so that the sterilization
of the serum obtained from it offers little or no diffi-
culty. For particular purposes blood and serum are
often obtained in a somewhat similar manner from other
animals.

For the sterilization of serum the method now in
vogue is that of Councilman and Mallory. Its popu-
larity is due to the following facts : by it the serum is
more quickly and easily prepared ; rigid precautions
against contamination 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

126 BACTERIOLOGY.

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.

METHODS OF OBTAINING BLOOD-SERUM. — It is often
desirable to obtain small quantities of blood-serum
under strictly aseptic precautions, and for this purpose
Nuttall1 suggests a very convenient method. By the
use of a sterilized vessel, of the shape shown in Fig.
20, from 10 to 100 c.c. of blood can be collected, and if
proper precautions are observed no contamination by
bacteria need occur. The collecting bulb is used in the
following way : an artery, either femoral or carotid, is
exposed, 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
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

1 Nuttall : Centralbl. f. Bakt. u. Parasitenkunde, 1892, vol. xi. p. 539.

BLOOD-SERUM. 127

sealed in a 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 piece of rubber-tubing about one-half
metre long, with glass mouth-piece. By holding the
pipette in the hand and sucking upon the rubber tube
one can more easily direct the point of the pipette than

FIG. 20.

a

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

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. An ordinary test-tube drawn
out at the bottom to a fine point may be substituted
for the pear-shaped bulb with equally satisfactory
results.

Latapie ' describes an apparatus for the collection of

1 Latapie : Aunales de 1'Institut Pasteur, 1900, vol. xiv. p. 106.

1 28 BA CTER IOL <)<;} '.

small amounts of blood-serum from experimental ani-
mals which has been found very useful. The amount
of serum that can be obtained in this manner from a
small quantity of blood is much greater than in the
Ntittall bulb. The Latapie apparatus has been very
much improved and simplified by Rivas.1 The advan-
tao-es of the Rivas modification are that it is much more

O

easily constructed and is far more durable — that is, it is
less liable to break during sterilization and in subsequent
manipulations.

The Rivas apparatus is constructed from two test-
tubes about 15 x 180 mm. in size. The mouth of
one test-tube is drawn out into a long narrow neck
1 cm. in diameter and about 5 cm. in length. Three
or four points on the side of the tube are softened in
the flame of a blowpipe, and the softened glass driven
inward by means of a piece of pointed wood. This
gives supports on the interior of the tube to hold the
coagulated blood in place. Between the long narrow
neck and the body of the tube a constriction is formed
by drawing out the tube while heated. The second tube
also has a similar constriction about 20 cm. from its
mouth.

The two tubes are now fitted together by inserting the
one with the long narrow neck into the second tube ; a
small amount of cotton being first carefully folded
around the neck of the first tube, so as to prevent the
entrance of dust. The two tubes are then fastened to-
gether by means of a wire twisted around the constric-
tion at the neck of each tube, and the apparatus is then
wrapped in cotton and sterilized in a hot-air sterilizer.

1 Rivas: University of Pennsylvania Medical Bulletin, vol. xvii.
1904, p. 295.

BLOOD-SERUM.

129

Before using the apparatus the extremity of the first
tube is heated in the gas-flame, and by touching this
point with a piece of pointed glass rod it is gently drawn

FIG. 21.

Rivas apparatus for collecting blood-serum : A, long narrow neck on first
tube; B, constriction on tubes near mouth ; C, invaginations on first tube;
D, small cannula drawn out on extremity of first tube ; E, blood-clot, and F,
blood-serum collected in bottom of second tube.

out into a fine cannula. When the animal has been pre-
pared for the operation and a vessel exposed, the point

130 BACTERIOLOGY.

of the cannula is snipped off with a sterile scissors, when
the point of the cannula is inserted into the vessel. The
pressure of blood is sufficient to fill the first tube. The
point of the cannula is now removed from the vessel and
sealed in a gas-flame. The apparatus is laid aside in an
almost horizontal position until the blood has become
completely coagulated. It is then inverted and set
aside for the serum to separate and trickle down through
the narrow neck of the first tube and collect in the
second tube. When this has occurred, the wire holding
the two tubes together is unwound, and the first tube is
removed and the second plugged with a well-fitting
sterile cotton plug, when the serum may be preserved
in the tube for several days without danger of con-
tamination.

PRESERVATION OF BLOOD-SERUM. — It is some-
times 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 very satisfactory. In
the course of Kirschner's investigations chloroform was
shown to possess decided disinfectant properties ; as it is
quite volatile, it is easily got rid of when its disinfectant
or antiseptic properties 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. This
latter provision is one on which success depends. If
the vessel containing the mixture of chloroform and
serum be not tiyhtly corked, the chloroform vapor

SPECIAL MEDIA. 131

escapes pretty rapidly and exerts no preservative action.
In fact, bacteria will grow uninterruptedly in a cotton-
stoppered test-tube containing bouillon to which chloro-
form has been added. When required for use, the
serum is decanted into test-tubes, which are then placed
in a water-bath at about 50° C. until all the chloro-
form has been driven off; this can be determined
by the absence of its characteristic odor. The serum
may then be solidified, sterilized by heat, and em-
ployed for culture purposes. We have found serum
so preserved to answer all requirements as a culture-
medium.

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

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

The separation of the cream may be accelerated and
rendered more complete if the cylinder containing the
milk be placed in the steam sterilizer for fifteen minutes
before it is placed in the ice-chest.

The cream is best separated from the milk by the use
of a cylindrical vessel with a stopcock at the bottom, by
means of which the milk, devoid of cream, may be
drawn off. A Chevalier creamometer with a stopcock

132 BACTERIOLOGY.

at the bottom serves the purpose very well. It should
be covered while standing.1

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 ordinary nutri-
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.

Litmus-milk-whey. An important differential medium
is milk-whey to which litmus tincture has been added.
A number of methods for its preparation are in use, but
the one employed by Durham seems to be the most sat-
isfactory. Briefly it is as follows : fresh milk, free
from antiseptic adulterations, is gently warmed and
clotted with essence of rennet. The whey is strained

1 For some time past we have been using what is technically known
as " separator milk " — i. e., the fluid left after milk has been deprived
of its fat (cream) by centrifugal force.

SPECIAL MEDIA. 133

off and the clot hung up to drain in a piece of muslin.
The whey, which is somewhat turbid and yellow, is
then cautiously neutralized with a 4 per cent, citric acid
solution, neutral litmus solution being used as the indi-
cator. It is then heated upon a water-bath to 100° C.
for about half an hour; thereby nearly the whole of the
proteid is coagulated. It is then filtered clear and neu-
tral litmus solution is added until it is of a distinct pur-
ple color. If the filtered whey is cloudy, let it stand in
a cold place for a day or two and decant off the clear
supernatant portion or pass it through a Berkfeld filter.
The whey should never be heated above 100° C. or
neutralized with mineral acids, otherwise there is a
danger of so modifying the milk-sugar present as seri-
ously to impair the usefulness of the medium. When
properly prepared, the medium is free from proteid, and
contains only water, lactose, the salts of the milk, and
a small quantity of a body suggestive of dextrose or
galactose. The medium is of great utility in detecting
the power of bacteria to cause acid fermentation in a
non-proteid medium containing a fermentable sugar;
and for observing the variations of this power in closely
allied though not identical species.

DunJuim's peptone solution. The medium usually
known as Dunham's solution is prepared according to
the following formula :

Dried peptone 1 part.

Sodium chloride 0.5 "

Distilled water 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-

134 BACTERIOLOGY.

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 metabolic products. It is essential for accu-
racy that the preparation of dried peptone employed
should be as nearly chemically pure as is possible,
and indeed the other ingredients should be corre-
spondingly free from impurities. Gorini l calls attention
to the fact that impurities in the peptone, particularly
the presence of carbohydrates, so interfere with the
production of indol by certain bacteria that otherwise
produce it, that it is ofttimes impossible, under such cir-
cumstances, to obtain the characteristic color-reaction of
this body, and where it is obtained it is always after a
much longer time than is the case where peptone free
from these substances has been used.

Peckham has also demonstrated that where bacteria
have the property of forming indol and also of fer-
menting carbohydrates, their proteolytic function, as
evidenced by the appearance of indol as a product
of metabolism, may be completely suppressed by the
addition of such fermentable carbohydrates as glucose,
saccharose, and lactose to the proteid solution in which
they are developing.*

Gorini suggests the advisability of testing the purity
of all peptone preparations before using them, by means
of the reaction that they exhibit with Fehling's alka-
line copper solution. Under the influence of this re-
agent pure peptone in solution gives a violet color (the

1 Gorini : Centralblatt fur Bakteriologie und Parasitenkunde, 1893,
vol. xiii. p. 790.
* See Journal of Experimental Medicine, 1897, vol. ii. p. 549.

SPECIAL MEDIA. 135

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 (par-
ticularly the latter) should be relatively dilute in order
for the reaction to be successful.

Lactose litmus-agar, or litmus-gelatin of Wurtz. A
medium of much use in the differentiation of bacteria
is that recommended by Wurtz, consisting of slightly
alkaline nutrient agar-agar, to which from 2 to 3 per
cent, of lactose and sufficient litmus tincture to give
it a pale-blue color have been added. Bacteria capable
of causing fermentation of lactose when grown on this
medium develop into colonies of a pale-pink color and
cause, likewise, a reddening of the surrounding medium,
owing to the production of acid as a result of their
action upon the lactose ; while other bacteria, incapable
of such fermentative activities, grow as pale-blue colonies
and cause no reddening of the surrounding medium.
It is especially useful 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 1 c.c.
will require 0.1 c.c. of a 1 : 20 normal sulphuric-acid
solution to neutralize it, lactose is added in the propor-
tion of 2 or 3 per cent. ; it is then decanted into test-
tubes and sterilized in the usual way. When ster-
ilization is complete enough sterilized litmus tincture

136 BACTERIOLOGY.

should be added to each tube to give a decided, though
not very intense, blue color. This must be done care-
fully, to avoid contamination of the tubes during ma-
nipulation. It is better not to add the litmus tincture
before sterilizing the tubes, as its color-characteristics
are altered by contact with organic matters under the
influence of heat. This medium is used for both test-
tube and plate cultivation, just as is ordinary agar-agar
and gelatin.

Loffler's blood-serum mixture. Loffler'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.)

The Serum-water Media of Hiss. — A medium which
has been found very serviceable in the differentiation
between closely related bacteria is prepared by mixing
one part of blood-serum (either horse or bovine) and
three parts of distilled water. This is neutralized, and
heated in a water-bath or an Arnold steam sterilizer
until it becomes opalescent. A 5 per cent, aqueous
solution of litmus is then added in the proportion of 1
per cent. Any one of the carbohyd rates, as dextrose,
•lactose, saccharose, levulose, mannite, etc., is then added
in the proportion of 1 per cent. The finished medium
is then placed in test-tubes. The medium must be ster-
ilized in an Arnold steam sterilizer, and it is advisable
to allow the sterilizer to remain uncovered during the

SPECIAL MEDIA. 137

process of sterilization to avoid excessive heating of the
medium.

The relative degree of acidity produced, with or with-
out coagulation, with or without gas-production, and
with or without reduction of the litmus, in a series of
tubes of this medium containing the different carbohy-
drates serves to differentiate between related species of
bacteria. For instance, the colon bacillus produces an
acid reaction with coagulation and gas-formation with
some of the carbohydrates, while the typhoid bacillus
produces a lower degree of acidity with coagulation, but
without gas-production. Similarly, the different types
of the dysentery bacillus may be differentiated by means
of their effects on the different carbohydrates in this
medium.

A complete list of the special media would be too
voluminous for a book of this size. For their descrip-
tion the reader is referred to the current literature.
Those that have been given above will suffice for ob-
taining a clear understanding of the principles of the
subject. In the chapters upon the Pathogenic Bacteria
such special media as have proved of use for purposes
of identification and differentiation are described in
detail.

CHAPTER VI.

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

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 about thirty
to forty-five minutes in a 2 to 3 per cent, solution of
common soda ; it is not necessary 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 with
a reed handle (Fig. 22, a), as those with wire handles

FIG. 22.
a

Brushes for cleaning test-tubes.

ure apt to break through the bottoms of the tubes,
though Messrs. Lentz & Sons, of this city, have in
large part eliminated this objection from the wire-handle
brush depicted in Fig. 22, 6. All traces of adherent

138

FILLING THE TUBES. 139

material should be carefully removed. When the tubes
are quite clean they may be rinsed in a warm solution
of commercial hydrochloric acid of the strength of
about 1 per cent. This is to remove the alkali. They
are then to be thoroughly rinsed in clear, running water,
and stood top down until the water has drained from
them. When dry they are to be plugged with raw
cotton ; this 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. 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 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 guide
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 necessitates recleans-

in£- .

FILLING THE TUBES. — When the tubes are cold

they may be filled. This is best accomplished by the

140

BACTERIOLOGY.

use of a separating funnel, such as is shown in Fig.
23. The liquefied medium is poured into this funnel,
which has been carefully washed, and by pressing the
pinchcock with which the funnel is provided the desired
amount of material (5-10 c.c.) may be allowed to flow
into the tubes held under its opening. It is not neces-
sary to sterilize the funnel, for the medium is to be sub-
jected to this process as soon as it is in the test-tubes.

FIG. 28.

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 materially with the manipulations.

FILLING THE TUBES. 141

As soon as the tubes have been filled they are to be
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 -tempera-
ture.

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

PLATES. — The plate method can be employed 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
a mixture of organisms in a higher state of dilution.
The first plate is known usually as " the original," or
" plate No. 1," the first dilution from this as " plate
No. 2," and the second as " plate No. 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 a 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
142

TECHNIQUE OF MAKING PLATES. 143

platinum wire (Fig. 24, a) about 5 cm. long, twisted
into a small loop at one end and fused into a bit
of glass rod, which serves 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 into which it does not enter. Under no circum-
stances is it to be employed without having been
passed through a gas-flame until quite hot, for the
purpose of sterilization. One should form a habit

FIG. 24.
a

b
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 (Fig.
24, 6) as well as looped, without passing it through
a flame ; and the sooner the beginner learns to do this
as a reflex action, the sooner does he eliminate 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 will be killed
by the high temperature ; after sterilization in the flame
one waits for a few seconds until it is cool before using.
A minute portion of the material under consideration
is transferred with the sterilized loop into tube No. 1,
" the original," where it is thoroughly disintegrated by
gently rubbing it against the sides of the tube. The
more carefully this is done the more uniform will be

144 SA CTERIOLOG Y.

the distribution of the organisms and the better the
results. The loop is then again sterilized and three
of its loopfuls are passed, without touching the sides of
the tube, from " the1 original " into tube No. 2, where
they are carefully mixed. Again the loop is sterilized,
and again three dips are made from tube No. 2 into
tube No. 3. This completes the dilution. The loop is
now sterilized before laying it aside.

FIG. 25.

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 be lower than 39° C., and never higher than
43° C. If it falls below 38° C., the agar-agar solid-
ifies, and can only be redissolved at a temperature
that would be destructive to the organisms which may
have been introduced into the tubes. This is not of
so much moment with gelatin, since it may readily be

TECHNIQUE OF MAKING PLATES. 145

redissolved at a temperature not detrimental to the
organisms with which the tubes may have been inocu-
lated.

THE COOLING-STAGE AND LEVELLING-TRIPOD. —
While the medium of which the plates are to be made
is melting it is well to arrange the cooling-stage (Fig.
25) 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 PLATES. — In the original plate method
devised by Koch the contents of each of the tubes of
agar-agar or gelatin are poured out in a thin layer upon
the surface of a sterile glass plate. As soon as pre-
pared, the glass plates containing the culture are placed
in a large glass culture dish.

PETRI'S MODIFICATION OF THE PLATE METHOD. —
The modification, now in general use, that approaches
nearest to the original method, and at the same time
lessens very materially the number of steps in the proc-
ess, is that suggested by Petri. It consists in substitut-
ing for the plates small, round, double glass dishes,
having about the same surface-area as the plates (Fig.
26). The liquid medium is poured directly into these
little dishes and their covers replaced ; they are then
set aside for observation. In all other respects the
process is the same as Koch's original method. Petri's
10

146 BACTERIOLOGY.

dishes are about 8 cm. in diameter and about 1.5 to 2
cm. in height, the sides being vertical. They may
readily be sterilized either by hot air or steam. They
are very useful for this work, as they do away with the
necessity for the cooling-stage and levelling-tripod,
though in warm weather the cooling-stage may be used
to hasten the solidification 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 round or square in shape, so arranged
that it can be filled with cold water, or that cold water
can constantly be passed through it by means of a
rubber tube connected with a spigot. The inlet for the

FIG. 26.

Petri double dish, now generally used instead of plates.

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 filling the
space with water. The box should be sufficiently strong
to resist the pressure of the water. A convenient size
is from 20-25 cm. in diameter and about 1.5 to 2 cm.
high. It is simple in construction, and can be made by
any copper-spinner. An idea of its construction is
given in Fig. 27.

When gelatin or agar-agar is to be cooled, it
is only necessary to place the dishes containing it

ESMARCH TUBES. M7

on top of this box and keep cold water circulating
through it.

ESMARCH TUBES. — The modification of Koch's
method which insures the greatest security from con-
tamination by extraneous organisms and requires the
least amount of apparatus is that suggested by v.
Esmarch. It differs from the other methods thus : the
dilutions haying been prepared in tubes containing a
smaller amount of medium than usual — as a rule, not
more than 5 to 6 c.c. — arc, instead of being poured upon
plates or into dishes, spread over the inner surface of

FIG. 27.

Metal cooling-stage.

the tubes containing them, and, without removing the
cotton plugs, solidified in this position. The tubes then
present a thin cylindrical lining of gelatin or agar-agar,
upon which the colonies develop. In all other respects
the conditions for the growth of the organisms are the
same as in flat plates.

Esmarch directs that after completion of the 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

148 E A CTERIOLOG Y.

then to be held in a horizontal position and twisted
between the fingers upon their long axis under ice-
water. The gelatin becomes solidified thereby and
adheres to the sides of the tube. When the gelatin is
quite hard the tubes are removed from the water, wiped
dry, the rubber caps removed, and 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. 28), after the method

FIG. 2&

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

devised by Booker in 1887 in the Pathological Labora-
tory of the Johns Hopkins University. In this method
a small block of ice only is needed. It is levelled
and held in position by being placed upon a towel in
a dish. A horizontal groove is melted in the upper
surface of the ice with a test-tube of hot water. The
tubes to be rolled are then held in an almost — not quite

ESMARCH TUBES. 149

— 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
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.

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
uniform, and the gelatin does not touch the cotton plug,
as is always the case in tubes rolled under water, be-
cause of the impossibility of keeping them at one level.

There is an erroneous impression that Esmarch tubes
are not a success when made from ordinary nutrient
agar-agar because of the tendency of this medium to
shrink and slip to the bottom of the tube. This slip-
ping down of the agar-agar is due to the water, which is
squeezed from it during solidification, getting between
the medium and the walls of the tube. This can easily
be overcome by allowing the rolled tubes to remain
in a nearly horizontal position for twenty-four hours
after rolling them, the mouth of the tube being about
1 cm. higher than the bottom. During this time the
margin of the agar-agar nearest the cotton plug dries
and becomes adherent to the walls of the tube, while
the water collects at the most dependent point — i. e., the
bottom of the tubes. After this they may be retained
in the upright position without danger of the agar-
agar slipping down. In all these manipulations,
if the dilutions of the number of organisms have been
properly conducted, the results will be the same. The

150 BACTERIOLOGY.

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 No. 3
will usually contain the organisms in such small num-
bers that there will be nothing to prevent the charac-
teristic development of the colonies originating from
them.

For reasons of economy the " original," tube No. 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 Nos. 2 and 3
are made.

THE SERIAL TUBE METHOD OF SEPARATION. —
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.
In pursuance of this method one selects a number of
tubes containing the medium set in 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 in succession over the surface
of the medium in tubes Nos. 2, 3, 4, etc. When de-
velopment has occurred essentially the same conditions
as regards separation of the colonies will be found as
when plates are poured. If a slanted medium be em-
ployed, 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

ESMARCH TUBES. 151

should be kept in an upright position to prevent the
fluid flowing over the surface. When sufficiently de-
veloped, single colonies may be isolated with compara-
tive ease from tubes prepared in this manner. (See also
method for the isolation of bacillus diphtherice on blood-
serum.)

CHAPTER VIII.

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

THE INCUBATOE. — 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 grow more luxuriantly
at the temperature of the human body (37.5° C.) than
at lower temperatures ; whereas for the ordinary sap-
rophytic forms almost any temperature between 18°
and 37° C. is suitable. It therefore becomes neces-
sary to provide a place in which a constant tem-
perature favorable to the growth of the pathogenic
organisms can be maintained. For this purpose a num-
ber of different forms of apparatus have been devised.
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.

The apparatus known as thermostat, incubator, or
brooding-oven, is a copper chamber (Fig. 29) with
double walls, the space between which is filled with
water. The incubating-chamber has a closely fitting
double door, inside of which is usually a door of glass
through which the contents of the chamber may be in-
spected without actually opening it. The whole appa-
ratus is encased in either asbestos-boards or thick felt,

152

THE INCUBATOR.

153

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 corner, leading into the space con-
taining the water, are other openings for the reception

FIG. 29.

Incubator used in bacteriological work.

of another thermometer and a thermo-regulator, and for
refilling the apparatus as the water evaporates. On the
side is a water-gauge for showing the level of the water

154

BACTERIOLOGY.

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 tem-
perature in all parts of the apparatus — at the top as well
as at the sides, back, and bottom ; the apparatus should
be kept filled with water, otherwise the purpose for
which it is constructed will not be accomplished. When
the chamber between the walls is filled with water heat
is supplied by a gas-flame placed beneath it.

FIG. 30.

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. 30). It is a Bunsen burner pro-
vided with an arrangement for automatically turning
off the gas-supply and thus preventing accidents should

THERMO-REGULATORS. 155

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 automatic
thermo-regulator.

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

The simplest form of thermo-regulator which serves
to illustrate the principles involved is seen in Fig. 31.
It consists of a glass cylinder, e, having a communi-
cating branch tube 6, and rubber stopper /, through
which projects the bent tube a. The tube a is ground
to a slanting point at the extremity which projects into
the tube e, and is provided ;i short distance above this
point with a capillary opening, </, in one of its sides.

156

BACTERIOLOGY.

When ready for use the cylinder e is filled with mer-
cury up to about the level shown in the figure. It is
then allowed to stand, or is suspended, in the bath the
temperature of which it is to regulate. The rubber
tubing coming from the gas-supply is attached to the

FIG. 31.

Mercurial thermo-regulator.

outer end of the glass tube a, and the tube going to the
burner is slipped over the branch tube 6. The gas is
turned on and the burner lighted and placed under the
bath. The gas now streams through the tube a into the
cylinder e and out at b to the burner ; but as the tern-

THERMO-REGULATORS. 157

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 mercury
accompanies it and gradually closes the slanting opening
h of tube a. In this way the supply of gas becomes
diminished and the rise in temperature of the bath will
be less rapid, until finally the opening at h will be closed
entirely, when the supply of gas to the burner will now
be limited to that passing through the capillary 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 the tem-
perature again 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 h 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 diifer-
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 uport a number of factors, all of which it
would be useless to describe in a book of this kind ;
but in general it may be said that the essential points to
be observed in selecting a thcrmo-regulator depend in the
main upon the temperatures at which it is to be used.
For low temperatures, regulators containing such fluids

158

BACTERIOLOGY.

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.

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

FIG. 32.

Moitessier's gas-pressure regulator.

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

GAS-PRESSURE REGULATORS. 159

forms of regulator in use, but they do not accomplish
the object for which they are designed.

The instrument most commonly employed, the appa-
ratus of Moitessier (Fig. 32), 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 colouies — Their naked-eye peculiarities and their appear-
ance 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 from 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 circum-
scribed and round, again irregular in their outline ; here
a point will present one color, there perhaps another.
On gelatin some of the points will be seen to be lying
on the surface of the medium, others will have sunk
into little depressions, while at still other points the
clear gelatin will be marked by more or less saucer-
shaped pits containing opaque fluid.

Place the plate containing these points upon the
stage of a 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 ;

160

THE STUDY OF COLONIES. 161

here nothing particularly characteristic will present,
there the point may resolve itself into a mass having
somewhat the appearance of a little pellicle of raw cot-
ton. All these differences, and many more, aid us in
saying that these objects must be different in their
nature. With a pointed platinum needle take up a
bit of one of these small islands, prepare it for micro-
scopic examination (see chapter on Stained CWer-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 their
gross 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
it will be reproduced in all of the new 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 uniform conditions.

The appearance of the colonies developing from
all organisms is regulated by their location in the
11

1 62 BACTERIOLOG Y.

medium in which they are growing. When deep
down in the medium they are usually round, oval, or
lozenge-shaped ; whereas when 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 CULTURES. — If from one of these small col-
onies a bit be taken upon the point of a sterilized plati-
num needle and introduced into a tube of sterilized
gelatin or agar-agar, the growth that results will be
what is known as a " pure culture," the condition 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
organisms can be obtained pure, but by patiently fol-
lowing this plan the results will ultimately be satis-
factory.

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 a
gas-flame a straight platinum-wire- needle. The glass
handle of the needle should be drawn through the flame
as well as the needle itself, otherwise contamination from
this source may occur. When it is cool, which is in five
or ten seconds, take up carefully a portion of the colony.

TEST-TUBE, STAB- AND SMEAR-CULTURES. 163

Guard against touching anything but the colony. If
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- or
stroke-culture is to be made. The needle is then with-
drawn, 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 observation. The growth which appears in the tube
after twenty-four to thirty-six hours should be a pare

164 BACTERIOLOGY.

culture of the organisms of which the colony was
composed.

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. 33.

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

TEST-TUBE, STAB- AND SMEAR-CULTURES. 165

been introduced into it possesses the power of bringing
about liquefaction — i. e., of digesting it — it will soon be
discovered that the mode of liquefaction differs with
different organisms and is practically constant for the
same organism. Some bacteria cause a liquefaction
which spreads across the whole upper surface of the
gelatin and continues gradually downward ; with others
it occurs in a funnel-shape, the broad end of the funnel
being uppermost and the point downward, correspond-
ing to the track of the needle ; at times a stocking- or
sac-like liquefaction may be noticed. (See Fig. 33.)

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

Select from your collection a non-spore-bearing, ac-
tively liquefying species. Cultivate it as a pure culture
in nutrient bouillon for three days. Then heat this
bouillon culture to 68° C. on a water-bath for ten
minutes. In the meantime prepare several tubes con-
taining each about 10 c.c. of:

Gelatin 7 grammes.

Phenol 0.25 gramme.

Water 100 c.c.

Let the carbolized gelatin in one tube remain solid, and
bring that in another to a liquid state by gentle heat. On
the surface of the gelatin in the first tube place 0.5 c.c. of
the heated (and cooled) culture, and mark on the side of
the tube the point of contact between the fluid culture and

166 BACTERIOLOGY.

the solid gelatin. To the tube of liquefied gelatin add
likewise 0.5 c.c. of the heated culture, mix it thoroughly
with the gelatin, and place the tube containing the
mixture in cold water until the mass becomes solid.
Set both tubes aside at a temperature not above 20° C.
Note what occurs at the end of an hour, by next day,
and after three days. Alter the experiment by filtering
the three-day-old bouillon culture through a porcelain
or a Berkefeld filter, instead of heating it as directed
above. Are the results modified ? How do you inter-
pret these results?

CHAPTER X.

Methods of staining — Impression cover-slip preparations — Solutions
employed — Preparation and staining of cover-slips — Staining
solutions — Staining in general — Special staining methods.

A COMPLETE 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 the 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 are 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
dimensions, require to be more conspicuously stained
than the surrounding materials upon the cover-slips or
in the sections, otherwise their recognition is a matter of
the greatest difficulty, if not of impossibility. For this
reason, especially in section-staining, it frequently be-
comes 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 employed.
The ordinary method of cover-slip examination of b#c-

187

1 68 BACTERIOLOG Y.

teria, constantly in use in these studies, is performed in
the following way :

COVER-SLIP PREPARATIONS. — In order that the dis-
tribution of the organisms upon the cover-slips may
be uniform and in as thin a layer as possible it is es-
sential 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, and then treating as above, renders them clean
enough for ordinary purposes.

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

Loffler'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 and then rinse them

COVER-SLIP PREPARATIONS. 169

in water, after which they are kept in a mixture of
equal parts of alcohol and ammonia. They are to be
dried on a cloth from which 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 water or physiological salt-solution.
With a platinum needle, which has been sterilized in
a 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
forceps), at some distance above a gas-flame, until it
is quite dry. If held with the forceps over the flame
at this stage, too much heat may be unconsciously ap-
plied, and the morphology of the organisms in the prep-
aration 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 forceps, and, holding the side upon which the bac-
teria are deposited away from the direct action of the
flame, it 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 tem-
perature 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

1 70 BA CTERIOLOG Y.

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 cm. wide by
0.3 cm. thick. This plate is laid upon an iron tripod
and a small gas-flame is placed beneath one of its
extremities. By this arrangement one can get a gradu-
ated temperature, beginning at the part 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 a temperature of from 95° to
98° 0. In very particular comparative studies this plan
is to be preferred to the process of passing the cover-
slips through a flame, as the organisms are always sub-
jected to the same degree of heat, and the distortions
which sometimes occur from too great and irregular ap-
plication of high temperatures may be eliminated. The
fixing consists in drying or coagulating the gelatinous en-
velope surrounding the organisms, by which means they
are caused to adhere to the surface of the cover-slip. It
is sometimes desirable to fix the preparations without the
use of heat, as in the case of pus or other exudates. In
this event, after drying the thinly spread material care-
fully in the air, the cover-slip on which it is placed is

COVER-SLIP PREPARATIONS. 171

immersed in a mixture of equal parts of absolute alco-
hol and ether for about 15 minutes. At the end of
this time it may be removed and stained. The advan-
tage of this method is that there is less distortion and,
as a rule, less precipitation (or, perhaps better, no char-
ring) of extraneous matter. When fixed, staining is
usually a simple matter. The majority of bacteria with
which the beginner will have to deal stain readily with
watery solutions of any of the basic aniline dyes, such,
for instance, as fuchsin, methylene-blue, or gentian-
violet.

To stain the fixed cover-slip preparation, it is taken
by one of its edges between forceps, and a few drops
of a watery solution of either of the dyes named are
placed upon the film and allowed to remain 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 pressing
upon it, after which the preparation is ready, for ex-
amination.

Another plan 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 satis-
factory and time-saving, but must always be prac-
tised with care. The staining-fluid should always be
filtered before using, to rid it of insoluble particles
which might be taken for bacteria. If upon exami-
nation the preparation proves of particular interest,
so that it is desirable to preserve it, then it may be
mounted permanently. The drop of immersion oil

172 BACTERIOLOGY.

is to be removed from the surface of the slip with
blotting-paper, and the slip loosened, or rather floated,
from the slide by allowing water to flow around its
edges. It is then taken up with forceps, carefully de-
prived of the water adhering to it by means of blotting-
paper, and 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 :
the slide, held by one of its ends between the fingers, is
warmed over a 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 wet with cold water. Usually the prepara-
tion is firmly fixed after this treatment ; a little practice
is necessary, however, in order not to overheat and
crack the slide. The method is applicable only to
cover-slip preparations, and cannot be safely used with
tissues.

IMPRESSION COVER-SLIP PREPARATIONS. — Impres-
sion preparations differ from ordinary cover-slip prep-
arations in only one respect : they present 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 by one of its edges. The organ-
isms adhere to the slip in the same relation to one
another that they had in the colony. The subsequent

ORDINARY STAINING-SOLUTION& 173

steps of drying, fixing, staining, and mounting are the
same as those just given for ordinary cover-slip prep-
arations.

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,
others show growth in parallel bundles, while others,
again, will be seen as long, twisted threads.

NOTE. — From a colony of bacillus subtilis make a
cover-slip preparation in the ordinary way ; now make
an impression cover-slip preparation of another colony
of the same organism. Compare the results.

ORDINARY STAINING-SOLUTIONS. — The solutions
commonly employed in staining cover-slip preparations
are, as has been stated, watery solutions of the basic
aniline dyes — fuchsin, gentian-violet, and methylene-
blue. These solutions may be made either by directly
dissolving the dyes in substance in water until the
proper degree of concentration has been reached, or by
using concentrated watery or alcoholic solutions of the
dyes which may be kept on hand as stock. The latter
method is the one commonly practised.

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

Prepare as stock, saturated alcoholic or watery solu-
tions of fuchsin, gentian-violet, and methylene-blue.
These solutions are best made 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,
\vell shaken, and allowed to stand for twenty-four hours.

174

BACTERIOLOGY.

If by then all the staining-matcrial 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. The alcoholic solutions are not directly employed for
staining-purposes.

The solutions with which staining is accomplished
are made from the stock solutions in the following
way :

An ordinary test-tube of about 13 mm. diameter is
three-fourths filled with distilled water and the con-
centrated alcoholic or watery solution of the dye
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 this dilution is just

FIG. 34.

Rack of bottles for staining-solutions.

transparent as viewed through a layer about 12 to
14 min. thick. A solution consisting of 5 parts of the

ORDINARY STAIN ING-SOLUTIONS. 175

saturated alcoholic solution and 95 parts of distilled
water proves, as a general thing, to he satisfactory.

The above represent the staining-solutions in every-
day use. They may be kept in bottles supplied with
stoppers and pipettes (Fig. 34), and when used are
dropped upon the preparation to be stained.

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

Loffler's alkaline methylene-blue solution :

Concentrated alcoholic solution of methylene-blue . . 30 c.c.
Caustic potash in 1 : 10,000 solution 100 c.c.

Koch-Ehrlich aniline-water solution. To about 100
c.c. of distilled water aniline oil is added drop by drop
until the solution has an opaque appearance, the vessel
containing the solution being thoroughly shaken after
the addition of each drop. It is then filtered through
moistened filter-paper until the filtrate is perfectly

176 BACTERIOLOGY.

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.
ZiehVs carbol-fuchsin solution :

Distilled water 100 c.c.

Carbolic acid (crystallized) 5 grammes.

Alcohol 10 c.c.

Fucbsin 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.

The Koch-Ehrlich solution decomposes after 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
further to 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-

STAINING IN GENERAL. 177

jority of other bacteria by the tenacity with which it
retains the color when treated in this way ; it is an
organism difficult to stain, but when once stained is
equally difficult to rob of its color.

STAINING IN GENEKAL.

The physics of staining and decolorization is hardly
a subject to be discussed at length in a book of this
character; but, as Kuhne has pointed out, it may be
briefly said that solutions which favor the production of
diffusion-currents facilitate intensity of staining, and by
a similar process increase the energy of decolorizing-
agents. For example, tissues which are transferred
from water into watery solutions of the coloring-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 in the protoplasm
of dried bacteria, as found upon cover-slip preparations,
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 illustrated :

Prepare a cover-slip preparation, dry it carefully, 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 subsequently with

12

178 BA CTERIOLOG Y,

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,
alcoholic solutions of the staining-dyes cannot be satis-
factorily employed. The staining-dyes should always
be watery.1

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 made to take up the
staining-material only with difficulty are also very diffi-
cult to rob of their color. The most common decolor-
izer in use is probably alcohol — not absolute alcohol,
but alcohol containing more or less of water. Water
alone has this property, but in a much less 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 alcohol and
water. More energetic in their decolorizing action than
either water or alcohol are solutions of the acids. They
appear, particularly when they are alcoholic 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,

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 remembered that this holds only when absolute alcohol and
perfectly dry coloring-matters have been used. If but a small propor-
tion of water is present, the bacteria may be stained with these solu-
tions, though the results are, as a rule, unsatisfactory.

STAINING THE TUBERCLE BACILLUS. 179

as has been said, when this action is accompanied by
the decolorizing-properties of alcohol.

The acid solutions commonly employed are :

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

Nitric acid in from 20 to 30 per cent, watery solu-
tion.

Sulphuric acid in from 5 to 10 per cent, solution in
water.

Hydrochloric acid in from 1 to 3 per cent, sqlution in
alcohol.

NOTE. — For details as to the technique of hardening
and cutting .sections and staining bacteria in tissues, the
student is referred to Mallory and Wright's Pathological
Technique.

METHOD or 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, dry and
fix it in the usual way. The slip is now to be taken
by its edge with forceps and the film covered with a
few drops of either the solution of Koch-Ehrlich or
that of Ziehl. It is then held over a gas-flame, at first
some distance away, gradually being brought nearer
until the fluid begins to boil. After it has bubbled
once or twice it is removed from the flame, the excess
of stain washed away in a stream of water, then im-
mersed in a 30 per cent, solution of nitric acid in
water, and allowed to remain until all color has dis-
ap}>eared. This takes longer in some cases 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 distinctly colored, it should
be again immersed in the acid ; if of only a very faint

180 B A CTERIOL OG Y.

color, it may be dipped in alcohol, again washed in
water, and 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 contain-
ing the stained tubercle bacilli is carefully rinsed in
water, and a few drops of the methylene-blue solution
placed upon it and allowed to remain for thirty or
forty seconds, when it is again rinsed in water and
examined microscopically. For this purpose of observ-
ing the difference in behavior of the tubercle bacilli
and the other organisms present in the preparation
toward this method of staining, it is well to exam-
ine the preparation microscopically before the con-
trast-stain is made; then give it the contrast-color,
and again examine. It will be seen that before the
contrast-color has been given to the preparation the
tubercle bacilli are the only stained objects to be
made out, and the preparation appears devoid of
other organisms ; but upon examining it after it has
received the contrast-color a great many other or-
ganisms will appear; these take on the second color
employed, while the tubercle bacilli retain their orig-
inal color. Before decolorization all organisms in the
preparation were of the same color, but during the appli-
cation of the decolorizing solution all except the tubercle
bacilli gave up their color. This micro-chemical charac-
teristic, together with reactions to be described, 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-

STAINING THE TUBERCLE BACILLUS. 181

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 hi the occa-
sional employment of Ziehl's earbol-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 number
of the bacilli are entirely decolorized by the too energetic
action of the strong acids. He recommends the follow-
ing method of decolorization : after staining the slip or
section in the usual way, pass it through three alcohols ;
it is then to be washed in a solution composed of

Water 150 c.c.

Alcohol 50 c.c.

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.

GABBETT'S METHOD 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
a cold earbol-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.) .... 100 c.c.
Methylene-hlue, in substance 1 to 2 grammes.

182 BACTERIOLOGY.

The cover-slips are then rinsed 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 METHOD. — Another important differential
method of staining which is very commonly employed
is that recommended by Gram. In this method the
objects 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 a solution composed of

Iodine 1 gramme.

Potassium iodide 2 grammes.

Distilled water 300 c.c.

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

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

GLACIAL ACETIC ACID METHOD. — Another method
that 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

STAINING OF SPORES. 183

minutes, is poured off, and a few drops more are added,
and lastly the slip is washed in a solution of sodium
chloride. Usually this is of the strength of physio-
logical salt-solution, viz., 0.6 to 0.7 per cent. ; but at
times it must be stronger, occasionally as concen-
trated as 1.5 to 2 per cent. 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.

Ribbert also recommends for the staining of capsu-
latcd bacteria the momentary immersion of the cover-
slips in a saturated solution of dahlia in a mixture of
100 parts of water, 50 parts of alcohol, and 12^ parts
of glacial acetic acid ; after which the excess of color
is removed by washing in water.

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 fairly satis-
factory results in our hands is as follows : the cover-
slip is to be prepared from the material containing the
spores in the ordinary way, dried, and fixed. It is then
to be held by its edge with forceps, and its surface cov-
ered with Loffler's alkaline methylene-blue solution. 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 minute, after
whioh it is washed in water and then dccolori/ed in

184 BA CTERIOLOQ Y.

Alcohol (80 per cent.) • • 98 c.c.

Nitric acid 2 c.c.

until all visible blue color has disappeared. It is then
rinsed in water and dipped for from 3 to 5 seconds in

Saturated alcoholic solution of eosin 10 c.c.

Water 90 c.c.

after which it is again rinsed in water and finally
mounted for examination. If the decolorization in
the acid alcohol be not carried too far, the preparation
will show the spores stained blue and the bodies of the
cells to have taken on the rose color characteristic of eosin.
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 forceps about
2 cm. above a very small flame of a Bnnsen burner,
care being taken that the flame touches only the centre
of the bottom of the crystal. After a few seconds the
crystal is gradually elevated 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 stain ing-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 heating is repeated.
When the boiling begins the crystal is again held aside
for a minute or two. The crystal is heated in this way
five or six times. When the fluid has stood for about five
minutes after the last boiling the preparation is trans-
ferred, without washing in water, to a second watch-
crystal containing the following decolorizing solution :

Absolute alcohol 100 c.c.

Hydrochloric acid 3 c.c.

MOELLER'S METHOD FOR STAINING SPORES. 185

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

MOELLER'S METHOD FOR STAINING 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 held
for two minutes in chloroform, washed in water, placed
for from one-half to two minutes in a 5 per cent, solu-
tion of chromic acid, again washed in water, and stained
with carbol-fuchsin. In the process of staining, the slip
is taken by the corner with forceps, and carbol-fuchsin
is dropped upon the side containing the spores. It is
then held over a 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 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 any crystals of leci-
thin, cholestcrin, or fat that may be in the preparation,
and which when stained might cause confusion.

It must be remembered that there are conspicuous
differences in the behavior of spores of different bacteria
to staining-methods and of the spores of a single species

186 BACTERIOLOGY.

in different stages of development. 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 ; some stain readily when fully developed, but
with difficulty when only partly developed ; others have
this peculiarity reversed.

LOFFLER'S METHOD FOR STAINING FLAGELLA.—
For the demonstration of the locomotive apparatus pos-
sessed by motile bacteria we are indebted to Loffler.
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 growing.
The method as given by Loffler 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 perfectly clean. (See Loffte^s
method of cleaning cover-slips.) Five or six of the
carefully cleansed cover-slips are to be placed in a line
on a 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 preparations. These slips are then dried
and fixed in the ordinary way. They are next to be
warmed in the following solution :

Tannicacid solution in water (20 acid, 80 water) .... 10 c.c.

Cold saturated solution of ferrous sulphate 5 c.c.

Saturated watery or alcoholic solution of fuchsin . 1 c.c.

METHOD FOR STAINING FLAG ELL A. 187

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 a 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 Loffler recommends the one or the other of
these solutions to be added to the mordant in the follow-
ing proportions.

Of the acid solution :

For spirillum choleras Asiaticse, i to 1 drop of acid to 16 c.c. of mordant.

For spirillum rnbrum, 9

For spirillum Metschnikoffi, 4 " "

For bacillus pyocyaneus, 5

For spirillum concentricum, no addition of either acid or alkali.

Of the alkaline solution :

For bacillus mesentericiis mdgatus, 4 drops of alkali to 16 c.c. of mordant.

For micrococcus agilis, 20

For bacillus typhomts, 22

For bacillus subtilis. 28-30 " " " "

For bacillus nedematis maligni, 36-37 "
For bacillus anthracis symp- )
tomatici,

188 BACTERIOLOGY.

The drops used run 22 to the cubic centimetre.

For other organisms one must determine whether the
results are better after the addition of acid or alkali,
and how much of either is required. In general, it may
be said that bacteria which produce acids in the media
in which they are growing require the addition of alka-
lies to the mordant, while those that produce alkalies
require acids to be added. By following Loffler'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 Loffler ;
but obtain, with a fair degree of regularity, satisfactory
results through the use of the neutral mordant alone.1

BUNGE'S METHOD. — A useful modification of Lof-
fler's method is that recommended by Bunge : prepare

1 I am indebted to Dr. James Homer Wright, Thomas Scott Fellow
in Hygiene, 1892-'93, University of Pennsylvania, for some of the
suggestions in connection with the modification of this method.

DUCKWALL'S METHOD. 189

a saturated solution of tannin, and a solution of liquor
fcrri 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
add 1 c.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 in
water, dried, and faintly stained with carbol-fuchsin.
No addition of acid or alkali to the mordant is neces-
sary.

DUCKWALL'S METHOD l is a modification of the Lof-
fler method, and the results obtained thereby are very
satisfactory.

Preparation of the Staining Agents. — The fixing agent
is mordant, and the stain is carbol-gentian-violet or,
preferably, carbol-fuchsin.

The Mordant.

Desiccated tannic acid 2 grammes.

Cold saturated solution ferrous sulphate (aqueous) 5 "

Distilled water 15 c.c.

Saturated alcoholic solution of fuchsin 1 "

The tannic acid is dissolved in the water first by the
application of gentle heat, then the ferrous sulphate,
and then the alcoholic solution of fuchsin are added.
To these ingredients it is advisable to add a certain
amount of sodium hydroxide, a 1 per cent, solution,
varying from 0.5 to 1 c.c. The best grade of filter-

1 The Cauncr, vol. xx. p. C3.

1 90 BA CTERIOLOG Y.

paper is used for filtering the mordant, and there should
be left a heavy precipitate. After filtering, the color
of this mordant should be of a reddish-brown hue, not
clear, but somewhat cloudy, and this mordant must be
used within five hours after it is made. After that time
it loses its fixing power. This is indicated by its
gradual clarification and darkened color. It gives the
best results when strictly fresh, and accomplishes its
work in a much shorter time, so that very little if any
heating is required when it is placed on the cover-glass
preparation.

Carbol-fuchsin Stain. — Take about 1 gramme of
granulated fuchsin (not the acid fuchsin), put it in a
bottle, and pour over it about 25 c.c. of warm absolute
alcohol. Shake vigorously and let it stand for several
hours before using. The carbol-fuchsin is made by
diluting the saturated alcoholic solution four or five
times with a 5 per cent, solution of carbolic acid. Car-
bol-fuchsin should be freshly made, heated, and filtered
before using.

The application of this method of demonstrating the
flagella varies with different organisms with regard to
the length of time the mordant and stain are allowed to
act, and the amount of sodium hydroxide solution used.
Usually, it is well to heat the mordant on the cover-slip
to steaming, and allow it to act from one-half to one
minute. It is then washed off with water and a small
quantity of alcohol poured over the surface and washed
off instantly. The water on the cover-slip is now
absorbed from the edge of the cover-slip with clean
filter-paper. The carbol-fuchsin stain is now applied
and heated just enough to generate a thin vapor. The
stain should not act for more than from one-half to one

METHOD OF VAN ERMENGEM. 191

minute. The cover-slip is now dried, then xylol is
poured over the surface, the excess being removed with
filter-paper. The cover-slip is now mounted in xylol
balsam.

THE METHOD OF 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
fixed over a gas-flame in the usual manner.

The mordant used consists of:

Osmic acid (2 per cent, solution) 1 part.

Tanniii (10-25 per cent, solution) 2 parts.

Place a drop or two of the 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.
Wash carefully in water and in 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. Without
washing, bring the slip into a watch-crystalful of the
" reducing and reinforcing bath," viz. :

Gallic acid 5 grammes.

Tannin 3 "

Fused potassium acetate ,. 10 "

Distilled water . . 350 "

192 BACTERIOLOGY.

After a few seconds pass the slip back into a watch-
crystal 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 ;
dry with blotting-paper, and mount in balsam.

CHAPTER XI.

Systematic study of an organism — Points to be considered in determin-
ing the morphologic and biologic characters of a culture — Methods
by which the various biologic and chemical characters of a cul-
ture may be ascertained — Facts necessary to permit the identifi-
cation of an organism as a definite species.

AFTER isolating an organism in pure culture by the
plate method, considerable work is necessary in order to
establish its identity. Small portions of the pure cult-
ure are taken upon the point of a sterile platinum wire
and transplanted into the various culture-media. These
sub-cultures of the organism are then placed under suit-
able conditions of temperature and environment, and
examined from day to day to note the alterations that
occur in the different media. In the systematic study
of an organism no one character can be relied upon to
the exclusion of others. It is necessary to note the
microscopic appearance of the individual organism and
its behavior toward different staining solutions and
other reagents ; in addition it is necessary to note the
gross appearance of the culture on the different media
as shown by naked-eye (macroscopic) examination as
well as under a lens of low magnifying power (micro-
scopic) ; while equal importance must be given to the
chemical alterations produced by the bacteria in the dif-
ferent media, and the influence of different reagents,
when added to the media, to show the presence of cer-
tain metabolic products. In this manner the entire life
history of an organism, outside the animal body, may
be ascertained.

13 193

194 BACTERIOLOGY.

The different characters of an organism may be
grouped as : (a) morphologic, those ascertained by exam-
ination of the individual organism under a lens of high
magnifying power; (6) biologic, those ascertained by
macroscopic and microscopic study of the gross appear-
ance of the culture in the different media; (c) biochemic,
the alterations produced in the different media as shown
by direct examination or by the use of different reagents ;
and (d) pathogenic, the effects of the inoculation of the
culture into susceptible animals.

SCHEME OF STUDY. — Record the source from whence
the organism was derived. Was this the normal habitat
of the organism, or was it present accidentally ?

MORPHOLOGIC CHARACTERS. — Note the shape, size,
and grouping of the organism as it occurs in the differ-
ent media. Observe the nature of the ends of the indi-
vidual organism. Determine the presence or absence
of motility in very young cultures. If motility is
observed, apply one of the special methods for demon-
strating flagella to note their relative number and loca-
tion. Stain young cultures by means of the different
staining solutions, and note the effect of each. Do the

O 7

organisms stain deeply and uniformly, or are they
stained in a peculiar manner? Apply the Gram method
of staining, and note whether or not the organisms
are decolorized by the alcohol. Stain the organisms
deeply with carbol-fuchsin staining solution, and note
the effect of different decolorizing agents ; and ascertain
whether the organisms are capable of resisting the
decolorizing effects of dilute acids. Do the organisms
show the presence of a capsule when taken from the
blood or tissues of an animal, or when taken from cult-
ures in milk or blood-serum ? Examine cultures that

BIOLOGIC CHARACTERS. 195

are several days old, and note whether spores are being
formed. Note particularly the position of the spore
within the cell. Is the spore of smaller or greater
diameter than the cell in which it is forming ? Exam-
ine cultures that are a week or more old, and note
whether the organisms have undergone any definite
alterations in form (involution forms), or whether they
present evidences of fragmentation or granulation of
their protoplasm (degeneration forms).

BIOLOGIC CHARACTERS. — Colony-formation. — Ob-
serve the character of the colonies formed in gelatin
and agar-agar plates. Describe a typical surface colony
and a typical deep colony, both as to their macroscopic
and microscopic appearance. What is the relative size
of the colonies formed on each of these media when
they are sufficiently separated from one another to permit
unhindered development ? Note the color and internal
structure of the colonies as well as their relative density.
What is the nature of the surface contour and arrange-
ment of the colonies ? Note their general character, as
to whether they are moist or dry, compact or loosely
constructed, sharply circumscribed or spreading over the
surface of the medium. Do the gelatin colonies show
evidences of liquefaction ?

Agar-slant Inoculations. — Observe the nature of the
growth on the surface of an agar-agar slant inoculation.
Describe the color, texture, and optical characters of the
growth. Is the growth confined to the line of inocula-
tion, or has it a tendency to spread over the surface of
the medium ? Is it smooth or rough, moist or dry,
glistening or dull in character ? If the organism forms
pigment, note whether the pigment is confined to the
area of growth or whether it extends into the medium

196 BACTERIOLOGY.

itself. Record the manner in which the culture changes
in its appearance on successive days.

Agar-stab Inoculations. — Observe the nature of the
growth in an agar-agar-stab inoculation. Note whether
the growth is most voluminous at or near the surface
or in the depth of the stab. If the organism produces
pigment, note whether the pigment-formation is most
marked at or near the surface or at the bottom of the
stab. Record the alterations that are observed on sev-
eral successive days.

Gelatin-stab Inoculations. — Observe the nature of the
growth in a gelatin-stab inoculation. Is the growth
most voluminous at or near the surface or at the bottom
of the stab? Note the general character of the growth
on the surface, especially as to its contour, extent, and
color. Note the character of the growth in the stab.
Is it continuous along the whole line of inoculation, or
is it confined to isolated areas? If the organism has
the property of liquefying gelatin, note carefully the
manner in which the liquefaction proceeds. How soon
does liquefaction begin, and in what length of time is a
tube of gelatin completely liquefied?

Potato Culture. — Observe the nature of the growth on
potato. This is an important differentiating medium,
since some organisms grow very sparingly or without
producing a visible growth. Other organisms grow
very characteristically. Some organisms have the prop-
erty of breaking up the starch of the potato into sim-
pler compounds. This is sometimes manifested by the
formation of gas. Many of the chromogenic bacteria
find the potato a most suitable pabulum on Avhich to
form their pigment, the pigment formed on this medium
having at times an especial brilliancy. Note in detail

BIOLOGIC CHARACTERS. 197

all the changes that occur in the growth on successive
days.

Growth in Bouillon. — Observe the nature of the
growth in bouillon. Note whether the fluid shows tur-
bidity or not, as well as the extent and distribution of
this alteration. Note whether any sediment is being
formed, as well as the nature and amount of such sedi-
ment. Does the organism form a definite growth (pel-
licle or scum) on the surface of the bouillon ? What is
the character of the pellicle ? Is is readily dislodged,
and, when dislodged, is it replaced by a new pellicle ?
Note whether the color of the medium has become
altered. Note the manner in which the appearance of
the culture changes on several successive days.

Growth in Litmus-milk. — Observe the nature of the
growth in litmus-milk. Has the reaction of the medium
become altered? To what is such alteration attribu-
table? Note whether there is precipitation of casein.
Record the extent and rapidity with which this altera-
tion takes place, as well as the reaction of the fluid
while the change is being produced. Is there any evi-
dence of the subsequent liquefaction of the precipitated
casein ? Has the litmus been altered in any manner
except as shown by altered reaction of the medium?
In what part of the tube has such alteration of the
litmus commenced? If the litmus has been decolorized,
is it possible to restore its color by the admixture of air
with the fluid ? Note the manner in which the appear-
ance of the medium changes on successive days.

Growth in Special Media. — The special culture-media
may be employed to ascertain additional biologic char-
acters of an organism, such as the production of indol,
reduction of nitrates to nitrites, the formation of ammo-

198 BACTERIOLOGY.

nia, production of gas in media containing different car-
bohydrates, or the reducing power of the organism on
aniline dyes, etc.

Influence of External Agencies. — Note the vitality of
the organism under the influence of various physical and
chemical agents. Determine the temperature at which
it thrives best, as well as the lowest and highest tem-
peratures at which growth is possible. Determine the
thermal death-point of the organism by subjecting it to
various degrees of temperature from 55° to 75° C. for
ten minutes. Determine its resistance to drying; to
the influence of light; to the influence of germicidal
substances. Determine the influence of different gases
upon the growth of the organism, such as hydrogen,
nitrogen, or carbon dioxide. Determine the chemical
reaction of the culture-media best adapted for its
growth.1

BIOCHEMIC CHARACTERS. — If the organism exhibits
chromogenic properties, ascertain whether the pigment
is intra- or extracellular. Ascertain under what condi-
tions of temperature, reaction, and constitution of media,
or under what atmospheric conditions this function is
best exhibited. Note the influence of different reagents
upon the pigment, such as chloroform, ether, alcohol,
water, acids, or alkalies. Note whether the organism
exhibits photogenic properties, and if so, ascertain what
conditions are most suitable for the manifestation of this
phenomenon.

Ascertain whether or not the organism produces en-
zymes. Does it manifest a proteolytic function, as shown

1 For more detailed description of the variations in the character
of the macroscopic and microscopic appearance of the cultures in the
different media, the student is referred to Chester's Determinative
Bacteriology and Eyre's Bacleriologic Technique.

BIOCHEMIC CHARACTERS. 199

by the liquefaction of gelatin, casein, or blood-serum ?
Note whether this function is manifested in alkaline or
in acid condition of the medium. Does it manifest a
precipitating effect (rennet ferment ?) upon casein ?
Note whether this is manifested in alkaline or in acid
condition of the medium. Does the organism have the
property of breaking up any of the carbohydrates into
simpler compounds ? Is this alteration accompanied or
not by the liberation of gas ? If so, ascertain the rela-
tive amount of gas formed from a given quantity of
carbohydrate. Analyze the gas formed, and state the
relative proportion of carbon dioxide and residual (ex-
plosive) gas formed. If the carbohydrates are broken
up without the evolution of gas, then ascertain what
intermediary and end-products are formed. Are acids,
aldehyde, or alcohol formed? Ascertain the nature
of the acids produced. If lactic acid is formed
from lactose, ascertain its character by means of the
polariscope.

Ascertain whether the organism produces indol,
phenol, or skatol. Are these substances formed with
the simultaneous reduction of nitrates to nitrites ? Are
the nitrites reduced further into ammonia?

Pathogenic Properties. — Ascertain whether any of the
animals used for experimental purposes are susceptible
when inoculated with the organism. Are all species of
laboratory animals equally susceptible, or are some
immune? Note the size of the dose and the manner
of inoculation that gives the most constant and charac-
teristic results. What are the symptoms and post-
mortem appearances produced? What is the location
of the organisms in the body of the dead animal? Are
they confined to the seat of inoculation (toxaemia), or are

200 BACTERIOLOGY.

they distributed more or less generally throughout the
body (bacterisemia) ?

Note whether the virulence of the organism is main-
tained when grown for several generations on artificial
media, or whether it soon becomes attenuated. Which
culture-medium is best suited to conserve the virulence
of the organism ? In what manner does its environ-
ment influence the virulence ? If the virulence is readily
lost, may it be regained by any of the known methods ?

Ascertain whether the organism forms a soluble toxin
when grown in fluid media, as sugar-free bouillon. If
toxin is formed, ascertain whether the antitoxic state is
readily induced in susceptible animals.

If no soluble toxin is formed, ascertain whether ani-
mals may be immunized by the injection of sub-lethal
doses of dead or living cultures. Is* a bactericidal immu-
nity induced by this means? Does the serum of immune
animals possess protective and curative properties when
administered to susceptible animals before or after inoc-
ulation with the living organism ? Does the serum of
immune animals possess the property of agglutinating
the organisms in relatively higher dilutions than the
serum of normal animals of the same species ?

The majority of the bacteria may be identified with-
out resorting to such a detailed study of the biochemic
and pathogenic properties as given in the foregoing out-
line, but for some of the pathogenic bacteria it has been
necessary to apply all the known tests in order to defi-
nitely establish their identity. By means of such
detailed studies on related organisms, it has been possi-
ble to differentiate varieties whose characters are con-
stant, yet in genera] they are so closely related that it is
impossible from the clinical manifestations produced to

DIFFERENT PARTS OF THE MICROSCOPE. 201

state definitely which particular variety of organism is
responsible for the conditions. This is especially true
of the different varieties of bacillus dysenterise, and of
the group of typhoid and paratyphoid organisms. Fur-
ther study will, no doubt, reveal variations in other
pathogenic bacteria, which varieties are to-day regarded
as a distinct species.

MICROSCOPIC EXAMINATION OF PREPARATIONS.

THE DIFFERENT PARTS OF THE MICROSCOPE. —
Before describing the method of examining prepara-
tions microscopically, a few definitions of the terms
used in connection with the microscope may not be out
of place. (The different parts of the microscope referred
to below are indicated by letters in Fig. 35.)

The ocular or eye-piece (A) is the lens at which the
eye is placed when looking through the instrument. It
serves to magnify the image projected through the ob-
jective.

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 (D) 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

202

BACTERIOLOGY.

stage, which serves to illuminate the object to be exam-
ined.

The coarse adjustment (F) is the rack-and-pinion ar-

FlG. 35.

!— G

rangement by which the barrel of the microscope can
be quickly raised or lowered.

DIFFERENT PARTS OF THE MICROSCOPE. 203

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
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 little or 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 a 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
one to attach several objectives to the instrument in

204 BA CTERIOLOG Y.

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 its full 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 conspicu-
ous than when a smaller amount of light is thrown upon
them. This is true not only for stained bacteria on
cover-slips, but likewise for their differentiation from
surrounding objects when they are located in tissues.
With unstained bacteria and tissues, on the contrary, the
structure can best be made out by reducing the bundle
of light-rays to the smallest amount compatible with
distinct vision, and in this way favoring, not color-con-
trast, but contrasts which appear as lights and shado/r*,
due to the differences in permeability to light of the
various parts of the material under examination.

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

UNSTAINED PREPARATIONS. 205

one or two turns, the object will be in focus. Do not
remove the eye from the instrument until this has been
accomplished.

Then, with one hand upon the fine adjustment and
the thumb and index finger of the other hand hold-
in"; the slide lightly by its end, it 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
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
rrri/ gently and carefully the oil from the face of the lens.
The lens is then unscrewed from the microscope and
placed iu 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-
<-u in stances 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-

206 £- 1 CTERIOLOG Y.

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. 36). The slip is held in

FIG. 36.

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
not only prevents the slip changing its position during
examination, but also prevents drying by evaporation
if the preparation is to be observed for any length
of time. This is known as the " hanging-drop "
method of examination or cultivation. It is 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 moved 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, when the edge of the drop
is readily detected with the higher power lens some-
where near the centre of the field.

In examining bacteria by this method there is a pos-

STUDY OF SPORE-FORMATION. 207

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 living bac-
teria alter their position in relation to the surround-
ing organisms, and may dart from one position in the
field to another. In some cases the true movement of
bacteria is very slow and undulating, while in others it
is rapid and darting. The molecular tremor may be
seen with non-motile and with dead organisms.

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

STUDY OF SPORE-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 mode of spore-formation.

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

208 BACTERIOLOGY.

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 from the
table upon which it rests, invert the hollow over the
drop and press it gently down upon the cover-slip. The
vaselin causes the slip to adhere to the slide, so that it
can be easily taken up. The drop now hangs in the
centre of the small air-tight chamber which exists be-
tween the depression in the slide and the cover-slip.
(See Fig. 36.)

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

As soon as finished the preparation is to be 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
to the purpose is one which envelops the whole micro-
scope. It is provided with a window through which
the light enters, and an arrangement by which the slide
may be moved from the outside. The formation of
spores requires a much longer time than the germina-

STUDY OF SPORE-FORMATION. 209

tion of spores into bacilli, but with patience both proc-
esses may be satisfactorily observed.

It 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-
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 nourishment ; potatoes and agar-agar
that have become a little dry oifer 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 specific property may be determined. It is possible
not only to detect the stages and steps in the formation
14

210 BACTERIOLOGY.

of endogenous spores, but when the spores are com-
pletely formed their germination into mature rods may
be seen by transferring them to a fresh bouillon-drop
or drop of agar-agar preserved in the same way. The
word rods is used because we have as yet no evidence
that endogenous spore-formation occurs in any of the
other morphological groups of bacteria.

HANGING-BLOCK CULTURES. — Hill1 has devised a
method for observing the development of individual
bacteria, which consists in the substitution for the ordi-
nary " hanging drop " of liquid or jelly a cube of solid-
ified agar-agar, on the surface of which the bacteria are
distributed.

The " hanging block " is prepared as follows : " Pour
melted nutrient agar into a Petri dish to the depth of
one-eighth to one-quarter inch. Cool this agar and cut
from it a block about one-quarter to one-third inch
square and of the thickness of the layer of agar in the
dish. This block has a smooth upper and under sur-
face. Place it, under surface down, on a slide and
protect it froni dust. Prepare an emulsion in sterile
water of the organism to be examined if it has been
grown on a solid medium, or use a broth culture ; spread
the emulsion or broth upon the upper surface of the
block, as if making an ordinary cover-slip preparation.
Keep the slide and block in an incubator at 37° C. for five
to ten minutes to dry slightly. Then lay a clean sterile
cover-slip on the inoculated surface of the block in
close contact with it, avoiding, if possible, the forma-
tion of air-bubbles. Remove the slide from the lower
surface of the block, and invert the cover-slip so that the
agar block is uppermost. With a platinum loop run a
1 Hill : Journal of Medical Research, vol. vii., 1902, p. 202.

CULTIVATION IN COLLODION CAPSULES. 211

drop or two of melted agar along each side of the agar
block where it is in contact with the cover-slip. This
seal hardens at once, preventing slipping of the block.
Place the preparation in the incubator again for
five or ten minutes to dry the agar seal. Invert this
preparation over a moist chamber and seal the cover-
slip in place with white wax or paraffin. Vaselin
softens too readily at 37° C., allowing shifting of the
cover-slip. The preparation may then be examined at
leisure."

Aerobic bacteria receive sufficient oxygen by dif-
fusion, and for anaerobic bacteria it will suffice to
expose the block to the action of alkaline pyrogallic
solution.

CULTIVATION OF BACTERIA IN COLLODION CAP-
SULES.— The use of small capsules of collodion for the
cultivation of bacteria was first introduced by Metschni-
koff, Roux, and Salembini. The bacteria under study are
placed in a small collodion capsule containing a nutritive
fluid, as bouillon, and, after sealing the capsule, it is
placed in the peritoneal cavity of an animal. Under
these conditions the bacteria are protected against the
phagocytic action of the body-cells, though the metabolic
products of the bacteria diffuse out and act upon the
animal.

Collodion capsules are easily constructed by the method
of McCrae, as follows : Fuse a short piece of narrow,
thin-walled glass tubing into the top of a No. 12 gelatin
capsule. This is done by simply heating one end of the
glass tubing in the flame of a Bunsen burner and push-
ing it through the top of the capsule. Both halves of the
gelatin capsule are now fitted together, and the closed
capsule is dipped repeatedly into liquid collodion. When

212 BACTERIOLOGY.

the covering of collodion on the exterior of the capsule
is of sufficient thickness — i. e., about that of writing
paper — it is allowed to dry for a short time, and is then
tilled with water and placed in a pan of hot water. This
will dissolve out the gelatin frame-work and leave the
collodion envelope. The water is now removed from
the capsule by means of a capillary pipette. The nutri-
tive fluid is introduced by the same means, and the filled
capsule is dropped into a tube of bouillon and sterilized,
after which the fluid is inoculated and the glass tube is
sealed off in the gas-flame. Before using the inoculated
capsule its tightness must be tested. This is done by
dropping it into a tube of sterile bouillon and placing in
the incubator for twenty-four hours. If growth appears
in the bouillon of the tube, then either the wall of the cap-
sule is permeable to bacteria, or the bouillon has been con-
taminated during the manipulation. As the wall of the
capsule is to possess the physical qualities of an osmotic
membrane, through which only dialyzable substances can
pass, it must, naturally, be free from cracks or air-holes,
and in making the capsules the greatest care must be
taken to eliminate these defects.

STUDY OF GELATIN CULTURES. — As has been pre-
viously stated, the behavior of bacteria toward gelatin
differs — some of them producing apparently no altera-
tion in the medium, while the growth of others is
accompanied by an enzymotic action that results in
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 almost vertically into the gelatin and may
be seen lying at the bottom of a funnel-shaped depres-

CULTURES ON POTATO. 213

sion. These differences are constantly employed as
one of the means of differentiating otherwise closely
allied members of the same family of bacteria. (See
Fig. 33.) Studies upon the spirillum of Asiatic
cholera and a number of closely allied species, for
example, reveal decided differences in the form of
liquefaction produced by these various organisms.
The minutest detail in this respect must be noted, and
its frequency or constancy under varying conditions
determined.

CULTURES ON POTATO. — A very important feature
in the study of an organism is its growth on sterilized
potato. Many organisms present appearances under
this method of cultivation which alone can almost be
considered characteristic. In some cases coarsely 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 bubbles, due to the fermentative action of the
growing bacteria on the carbohydrates of the potato,
are produced.

A most striking form of development on potato is that
often exhibited by the bacillus of typhoid fever and the
bacillus of diphtheria. After inoculation of a potato
%vith either of these organisms there is usually no naked-
eye evidence of growth, though microscopic examina-
tion of scrapings from the surface of the potato reveals
an active multiplication of the organisms which had
been planted there. The potato is one of the most im-
portant differential media that we possess for this work.

214 BACTERIOLOGY.

CHANGES IN THE REACTION OF MEDIA AS A RESULT
OF BACTERIAL ACTIVITY.

For purposes of differentiation, much stress is laid
upon the reaction assumed by media as a result of bac-
terial growth. Under the influence of certain species
the medium will become acid, under that of others it is
alkaline, while some cause little or no change. In
media of particular composition — i. e., those containing
traces of fermentable carbohydrates, notably muscle-
sugar, as seen in infusions of fresh meat — the reaction
may become acid with the beginning of growth and
subsequently change to alkaline after the supply of
fermentable sugar is exhausted. These changes of reac-
tion are most conveniently observed through the use of
indicators — bodies that either lose or change their usual
color as the reaction of the medium to which they are
added changes.

Such substances as litmus, in the form of the so-called
"litmus tincture," and coralline (rosolic acid) in alco-
holic 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 changes in their
colors studied with different bacteria. Milk and litmus
tincture or peptone solution to which rosolic acid has
been added are excellent media for this experiment.

ANILINE DYES FOR DIFFERENTIAL DIAGNOSIS. —
The addition to solid media of the aniline dyes, fuchsin,
methylene-blue, methylene-green, saffranin, neutral red,
and several others, as well as combinations of these
dyes, has been recommended as a means of differentia-
tion of bacteria. The changes that occur as a result of
bacterial growth in media so treated consist of alterations

FERMENTA TION. 21 5

in the color of the media, due to the oxidizing or reduc-
ing properties of the growing bacteria. It is doubtful
if this is, in general, an important differential method ;
at all events, it has been pretty well abandoned, after
having enjoyed at one time some degree of popularity,
though a number of investigators still regard saffranin
and neutral red as useful agents for the differentiation
of allied species, and as handy aids in the identification
of those species capable of reducing them.

BEHAVIOR TOWARD STAINING-REAGENTS. — The be-
havior of certain bacteria toward the different dyes
and their reactions under special methods of after-
treatment aid materially in their identification. With
very few exceptions bacteria stain readily with the com-
mon aniline dyes ; but they differ markedly in the te-
nacity with which they retain these colors under the
subsequent treatment with decolorizing-agents.

The tubercle bacillus and the bacillus of leprosy, for
example, are difficult to stain ; but when once stained
they retain their color under the action of such energetic
decolorizing-agents as alcohol, nitric acid, oxalic acid,
etc. 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 washed in water, or but
for a few seconds in alcohol, without losing their
color. It is essential that all these peculiarities should
be carefully noted in studying an organism.

FERMENTATION. — The production of gas as an indi-
cation of fermentation is an accompaniment of the
growth of certain bacteria. This is best studied in

216 BACTERIOLOGY.

media to which 1 to 2 per cent, of grape-sugar (glucose)
has been added. A convenient method of demonstrat-
ing this property is to employ a tube about half full of
agar-agar containing the necessary amount of grape-
sugar. The medium is to be liquefied on a water-bath,
and then cooled to about 42° C., when a small quantity
of a pure culture of the organism under consideration
should carefully be 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 incu-
bator. 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 containing the gas that has resulted.
This property of fermentation with evolution of gas
is of such importance as a differential characteristic
that 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 sufficient 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. For this purpose, Smith1 recommends the
employment of the fermentation-tube. This 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. 37.) To fill

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

FERMENTA TION. 217

the tube, the fluid (it is used only with fluid media)
is poured into the bulb until this is about half full.
The tube is then tilted until the closed arm is nearly
horizontal, so that the air may flow out into the bulb
and the fluid flow into the closed arm to take its place.
When this has been completely filled sufficient fluid
should be added to bring its level within the bulb just
beyond the bend, and the opening of the bulb plugged

Fermentation-tube.

with cotton. The tubes thus filled are then to be ster-
ilized. During sterilization they are to be maintained
in the upright position. Under the influence of heat the
tension of the 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

218 BACTERIOLOGY.

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 saccharose. After inoculation the
flasks are placed in the incubator, and the amount of
gas that collects in the closed arm is noted from day to
day. From studies that have been made this gas is
found to consist usually of about one part by volume of
carbonic acid and two parts by volume of an explosive
gas consisting largely of hydrogen. For determining the
nature and quantitative relations of these gases Smith l
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 CO2 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 CO2 be present, a partial vacuum in the closed branch
causes the fluid to rise suddenly when the thumb is re-
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 CO2 absorbed. The explosive
character of the residue is determined as follows : the
thumb is placed again over the mouth of the bulb and
the gas from the closed branch is allowed to flow into

1 Loc. cit., p. 196.

FERMENTATION.

219

FIG. 38.

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."

Durham's Fermentation-tube. — Dur-
ham employs a convenient modification
of the ordinary fermentation-tube, which
is constructed in the following man-
ner: test-tubes of about 10 or 12 c.c.
capacity are placed in an inverted posi-
tion within a larger test-tube, and the
latter plugged with cotton in the usual
way and sterilized. (See Fig. 38.) The
small tube should fit loosely within the
larger one. The medium to be used is
run into the larger tube until there is
present about 50 per cent, more than the
volume of the smaller tube. The whole
is then sterilized in streaming steam by
the fractional method. After the first
sterilization the small tube will be found
almost filled with fluid, over which a
small air-bubble lies. After the second
or third sterilization this air-bubble is
completely expelled, and the small tube
contains nothing but the liquid.

The medium that Durham employs
for the fermentation-test is a 1 per cent,
solution of Witte's peptone in distilled
water, to which have been added known amounts of
some such fermentable sugar as glucose, saccharose,
lactose, mannite, etc., as the case may demand. He

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'• -

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tat

m's f
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ermen-
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220 BACTERIOLOC Y.

prefers peptone to meat-infusion bouillon for the reason
that the latter often contains traces of muscle-sugar,
and thereby is likely to complicate the results. He
prefers neutralization with organic acids rather than
mineral acids, and uses citric acid by preference, the
reason for this being that where sugars such as those
mentioned are acted upon by mineral acids under the
influence of heat their composition is apt to be altered.

NOTE. — Prepare two fermentation-tubes as follows :
Fill one with a 1 per cent, watery solution of peptone
to which 2 per cent, of glucose has been added ; fill
the other with a similar peptone solution, but to which
only 0.3 per cent, of glucose has been added. Sterilize
and inoculate with bacillus coli communis. How do
the two tubes differ from one another after eighteen to
twenty-four hours in the incubator? First, as regards
the reaction of the fluid in the open arms of the tubes.
Second, as to accumulation of gas in closed arms of the
tubes. Third, as to the capacity of each solution for
reducing copper in Fehling's solution. What differ-
ences are observed, and how may they be explained?

CULTIVATION WITHOUT OXYGEN. — As we have
already learned, there is a group of bacteria to which
the designation "anaerobic" has been given, which
are characterized by inability to grow in the presence
of free oxygen. For the cultivation of the members of
this group, a number of devices are employed for the
exclusion of free oxygen from the cultures.

Koch's method. Koch covered the surface of a gela-
tin plate, which had been previously inoculated, with
a thin sheet of sterilized isinglass. The organisms

CULTIVATION WITHOUT OXYGEN.

221

which grew beneath it were supposed to develop with-
out oxygen.

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

FIG. 39.

Liborius tube for anaerobic cultures.

Methods of Liborius. Liborius has suggested two
useful methods for this purpose. One is to nearly (about
three-quarters) fill a test-tube with 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 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 in
the flame. Anaerobic bacteria develop only in the lower

222 BACTERIOLOGY.

layers of the medium. In his other method he employs
a special tube, known as " the Liborius tube." Its con-
struction is shown in Fig. 39.

Through the side tube hydrogen is passed until it re-
places all the air ; the contracted parts, both of the neck
of the tube and the side arm, are then sealed in the
flame.1 This tube can be used for either solid or liquid
media, but, owing to its usual small capacity, gives
better results with fluid media. (For precautions in
using hydrogen, see note to Frankel's method, page
223.)

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 -fa normal 2 caustic-potash solution.

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 passage of hydrogen somewhat troublesome ; it is better
to draw them out in the flame quite thin before passing the hydrogen
into the tube. This makes the final sealing a matter of no difficulty.

2 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 equivalent 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 (HC1) 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
(H2SCU) contains in each molecule two replaceable hydrogen atoms ;
its normal solution is not, therefore, 80 grammes (its molecular weight)
to the litre, but that amount which would be equivalent chemically to
one hydrogen atom, viz., 40 grammes (one-half its molecular weight)
to the litre. A normal solution of caustic potash contains as many

CULTIVATION WITHOUT OXYGEN.

223

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 CO2, and a trace of ammonia.

FIG. 40.

Frankel's method for the cultivation of anaerobic bacteria.

Method of C. Frdnkel. Carl Frankel suggests the
following as a modification of or substitute for the tube
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
must all have been sterilized in the steam sterilizer

grammes to the litre as the number of its molecular weight — 56.1
grammes to the litre of water.

224 HACTERIOLOGY.

before using. On the outer side of the stopper these
two tubes are bent at right angles to the long axis of
tbe test-tube into which they are to be placed, and both
are slightly drawn out in a gas-flame. Both of these
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 cm. of the bottom of the test-tube, the other is cut
off flush with the under surface of the stopper. The
outer end of the longer glass tube is then connected
with a hydrogen generator and hydrogen is allowed
to bubble through the gelatin (Fig. 40, A) in the tube
until all contained air has been expelled and its place
taken by the hydrogen.1 When the hydrogen has been

1 Before beginning the experiment it is always wise to test the hydro-
gen— i. e., to see that it is free from oxygen and that 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 sulphuric acid of about 25 to 30 per cent,
strength. With the primary evolution 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 flame
is in the neighborhood. This should be repeated, 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 rubber tubing. When the water has been replaced test the gas
by holding a flame near the open mouth of the test-tube. If no explo-
sion occurs, the hydrogen is safe to use. Should there be an explosion,
the generation of hydrogen must be continued ia the apparatus until
it burns with a colorless flame when tested in a test-tube.

CULTIVATION WITHOUT OXYGEN. 225

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 rubber
stopper is then painted around with melted paraffin
and the tube rolled in the way given for ordinary
Esmarch tubes. A tube thus prepared and containing
growing colonies is shown in Fig. 40, 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
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 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 an-

15

226 BACTERIOLOGY.

aerobic conditions Kitasato and Weil have suggested
the addition to the culture-media of some strong re-
ducing-agent. They recommend formic acid or sodium
formate in 0.3 to 0.5 per cent. ; glucose in 1.5 to 2 per
ce'nt. ; 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. As a reducing-
agent for this purpose, Theobald Smith regards a weaker
solution of glucose, 0.3 to 0.5 per cent., as more ad-
vantageous ; and Wright obtains better results when
glucose is added if the primary reaction of the media
is about neutral to phenolphtalein.

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 when they are
scattered through the gelatin, as in the method of Liborius.

Method of Park. A very simple, convenient, and
efficient method is employed by Park. It consists in
covering the medium in which the anaerobic species are
to be cultivated with liquid paraffin (albolene). The
best results are obtained when the amount of paraffin
added is about half that of the liquid in the tube or
flask. The liquid paraffin has the advantage over the
solid paraffin in not retracting from the walls of the
vessel on cooling. All air is expelled from flasks or
tubes prepared in this way, by heating them in the auto-
clave. The layer of paraffin prevents the reabsorption
of oxygen driven off by the heat. After cooling, the
inoculation is made by passing the needle through the
paraffin well down into the media.

INDOL PRODUCTION. 227

By some workers the oxygen is removed from the
culture-medium by the use of the air-pump.

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

From what has been said, it may be inferred that tlie
cultivation of anaerobic bacteria is a simple matter
attended with but little difficulty. Such an opinion
will, however, be quickly abandoned 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 anaerobes in mixed cultures is
always to be suspected, and it is this group that renders
the task so difficult. At best the work requires undi-
vided attention and no small, degree of skill in bacterio-
logical technique.

INDOL PRODUCTION. — The generation 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 products is one that is produced by a number
of organisms, and whose presence may easily be de-
tected by its characteristic behavior when treated with
certain substances. I refer to nitroso-indol, the reac-
tions of which were described by Beyer in 1869, and
the presence of which as 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 becomes
of a more or less decided rose color. This body was

228 B A CTERIOLOG Y.

recognized as one of the products of growth of the
spirillum of Asiatic cholera first by Poel, and a short
time subsequently by Bujvvid and by Dunham, and for
a time was believed to be peculiarly characteristic 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. It is constantly present in putrefying
matters, and is one of the aromatic bodies that give to
faeces their characteristic odor.

The methods employed for its detection are 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 is 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

INDOL PRODUCTION. 229

appears after five or ten minutes, add 1 c.c. of the
sodium nitrite solution. If now no rose color is pro-
duced, the indol reaction may be considered as negative
— i. e., no indol has been formed as a product of the
growth of the bacteria.

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 coincidently a reducing-
body. This is found, by proper means, to be 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
remaining cultures in the same way.

The test is sometimes made by allowing concentrated
sulphuric 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
culture-medium. This method is open to the objection
that, if indol is present in only a very small amount,
the faint rose tint 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 recom-
mends the use of dilute sulphuric acid. He states that
when indol is present the characteristic rose color ap-
pears a little more slowly wHh the dilute acid, but
it is more permanent, and there is never any like-

230 BACTERIOLOGY.

lihood of its presence being masked by other color-
reactions.

Muir and Ritchie recommend the use of ordinary
fuming or yellow nitric acid for this test. In this
method two or three drops of the acid are added to
the culture under consideration. If indol be present,
the red color appears as a result of the reducing action
of the nitrous acid upon it. The defect in this method
is that it reveals only the presence of indol, and fails to
indicate whether or not reducing-bodies were coinci-
dently formed with the indol. As a test for indol alone
it is convenient and entirely trustworthy.

REDUCING POWER OF BACTERIA. — The power to
reduce chemical compounds from a higher to a lower
state may be said to be common to all bacteria. In
some bacteria, perhaps the majority, it is most conspicu-
ously manifested in connection with substances contain-
ing sulphur, hydrogen sulphide being formed. In other
bacteria it is best seen in connection with the alterations
produced in certain pigments, as litmus, methylene-
blue, indigo, etc., the normal color disappearing in part
or entirely according to the nature and activity of the
process. Other bacteria have the property of reducing
certain salts, as in the reduction of nitrates to nitrites,
or even to ammonia by the denitrifying bacteria. In
some instances these reductions result from the fact that
the bacteria liberate hydrogen from the compounds, in
others it results from the fact that the bacteria abstract
oxygen from such compounds, while in still other instances
the reduction is of a more complex nature. Each of these
changes, therefore, indicates the nature of some of the me-
tabolic activities manifested by the bacteria in question.

Some of these reductions may be detected by the

REDUCING POWER OF BACTERIA. 231

application of comparatively simple tests for the pres-
ence of the end-products.

Test with Pigments. — The reduction of various pig-
ments and aniline dyes is usually manifested by altera-
tions in the depth of color or in the complete decoloriza-
tion of the pigment. In some instances the reduction
is first manifested in the depth of the medium, and in
such instances the natural color of the pigment may
frequently be restored on shaking the medium. This
is manifestly a deoxidation of the pigment arising from
the avidity of the bacteria for oxygen when growing in
the depth of the medium. In other instances the reduc-
tion is more complete, and simple agitation of the
medium fails to restore the original color.

Test for Hydrogen Sulphide. — The reduction of sul-
phur compounds may be determined by growing the
bacteria in peptone solution containing ferric tartrate,
when the presence of hydrogen sulphide will be indi-
cated by the brownish-black or jet-black color of the
precipitated iron-sulphide.

The complete reduction of nitrates is brought about
by many bacteria. Other bacteria are capable of carry-
ing the reducing action as far as the formation of ammo-
nia, while still others merely reduce the nitrates to
nitrites. These reducing functions are encouraged and
may be demonstrated by cultivating the bacteria in pep-
tone solution containing potassium nitrate.

Test for Nitrites. — The method of Griess,as modified by
Ilosvay, is quite satisfactory. These reagents are required :

(a) Naphthylamine, 0.1 gramme.

Distilled water, 20.0 c.c.

Acetic acid (25 per cent, solution), 1 50.0 "

(6) Sulfanilic acid, 0.5 gramme.

Accticacid(25percent. solution), 150.0 c.c.

232 BACTERIOLOGY.

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 b are then mixed. It is best prepared as needed,
though it may be preserved for some time in a glass-
stoppered bottle.

In testing for nitrites the reagent is added in the
proportion of one volume of reagent to five volumes
of culture. When nitrites have been formed a deep-
red color appears in a few seconds. If no nitrites have
been formed the culture remains colorless. In testing
cultures it is always necessary to control the results by
blank tests on a portion of the same medium that had
not been inoculated, as some of the ingredients of the
medium may have contained nitrites.

Another test for the formation of nitrites is a mixt-
ure of starch and potassium iodide, as follows :

Starch, 2.0 grammes.

Potassium iodide, 0.5

Water, 100.0 c.c.

Warm the mixture until the starch is completely dis-
solved.

In testing for nitrites add 0.5 c.c. of the reagent to a
tube of culture, and follow this by the addition of 2 or
3 drops of pure sulphuric acid. If nitrites have been
formed, a dark-blue or purple color will appear. Con-
trol-tubes of the medium show no color reaction, or
merely a trace of blue coloration.

Test for Ammonia. — The formation of ammonia may
be detected by testing with Nessler's reagent. The
most satisfactory results are obtained by cultivating

REDUCING POWER OF BACTERIA. 233

the organisms in a litre of culture fluid and then
distilling off portions of the culture, collecting in
Nessler tubes, and applying 1 c.c. of the reagent to
each 50 c.c. of the distillate. The presence of ammo-
nia in the distillate is shown by the yellow coloration
resulting from the addition of the reagent.

The direct application of the reagent to the culture
will give satisfactory results if a great deal of ammonia
has been formed. In this instance the mercury in
the reagent will be precipitated as mercurous oxide.
Another rough test for the formation of ammonia is to
place a strip of filter-paper — moistened with the Ness-
ler reagent — over the mouth of a test-tube containing
the culture, and then gently heating the culture. As
the ammonia is driven off by the heat, it will react on
the reagent on the strip of paper.

EXAMINATION OF CULTURES FOR BACTERIAL TOX-
INS.— In the systematic study of a pathogenic organism
it is necessary to know whether it is capable of pro-
ducing a soluble toxin when growing in culture-media.
This is done by filtering cultures of various ages and
testing the effect of the filtrate upon susceptible
animals.

FILTRATION OF CULTURES. — A variety of filters
have been devised for the purpose of filtering liquid
cultures and other fluids to obtain sterile filtrates.
These filters are usually constructed of unglazed porce-
lain or of infusorial earth, and are made in the form of
hollow cylinders or bulbs. The best-known forms of
bacterial filters are those of Chamberland and of Berke-
feld. All the filters used for this purpose require some
motive power to force the fluid through the filter. Com-
pressed air may be employed to force the fluid through

234 BACTERIOLOGY.

the filter, or atmospheric pressure may be utilized by
creating a negative pressure on the distal side of the
filter by the use of an air-pump.

It is always necessary to test the sterility of the fil-
trate by making cultures from it into nutritive media
and noting whether growth takes place or not.

CHAPTER XII.

Inoculation of animals — Subcutaneous inoculation — Intravenous in-
jection— Inoculation into the great serous cavities, and into the
anterior chamber of the eye — Observation of animals after inocu-
lation.

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 cult-
ure to be tested is simply introduced beneath the skin
of the animal ; but in other cases it may be necessary
to introduce it directly into the vascular or lymphatic
circulation, or into one or the other of the great serous
cavities ; or, for still other purposes of observation, into
the anterior chamber of the eye, upon the iris or within
the skull cavity, upon the dura or brain substance.

SUBCUTANEOUS INOCULATION OP 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

235

236 BA CTERIOLOG Y.

dirty, it may be scrubbed with soap and water. Steriliza-
tion of the skin is practically impossible, so it need not
be attempted. If the inoculation is to be made by means
of a hypodermic syringe, then a fold of the skin may be
lifted up and the needle inserted in the usual way. If a
solid culture is to be inoculated, a fold of skin may be
taken up with forceps 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 super-
ficial and deep connective-tissue fasciae. These must be
taken up with sterilized forceps, and with sterilized scis-
sors incised in a way corresponding to the opening in the
skin. The pocket is then to be held open with the for-
ceps and the substance to be inserted is introduced as
far under the skin and fasciae as possible, care being
taken not to touch the edges of the wound if it can
be avoided. The edges of 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 at hand, the simple sub-
cutaneous inoculation may be made without the aid of
a mechanical holder.

It is at times, however, more convenient to dispense
with an assistant ; one of several forms of apparatus that
have been devised for holding mice, guinea-pigs, rats,
rabbits, etc., may then be used. For small animals, such
as mice and rats, the holder suggested by Kitasato is very

SUBCUTANEOUS INOCULATION OF ANIMALS. 237

useful. It is simply a metal plate attached to a stand by a
clamped ball-and-socket joint, so that it can be fixed in

FIG. 41.

Kitasato's mouse-holder.

any position. It is provided with a spring-clip at one
end that holds the animal by the skin of the neck, and

FIG. 42.

Holder for larger animals.

at the other end with another clamp that holds the tail
of the animal. This holder is shown in Fig. 41. For

238

BACTERIOLOGY.

larger animals the form of holder shown in Fig. 42 is
commonly used.

The holder devised by Sweet,1 which can be made of
any size, from that suitable to a guinea-pig up to that
large enough to secure a dog, is in every way the most
convenient that we have encountered and, from the
standpoint of the animal, is the most humane. It con-

FiG. 43.

sists of four pieces of heavy round wire so bent that two
engage the animal immediately behind the lower jaw
while the remaining two close over the muzzle. All
are held in position by a single clamp controlled by a
single thumb-screw. When the screw is reversed and

1 Sweet: " A simple, humane holder for small animals under exper-
iment," University of Penna. Med. Bull., 1903, No. 2, p. 78.

SUBCUTANEOUS INOCULATION OF ANIMALS. 239

the clump opened the anterior and posterior wire of each
puir falls away from the median line, thereby liberating
the animal. To secure the animal it is placed upon its
back, the head laid in the cradle formed by the bent
wires, the latter are adjusted to the proper position, and
all secured by the turn of the single set-screw. Of
course, the extremities of the animal are to be secured.
This is done by means of cords securely held by a
patent fastener made by the Tie Co., of Unadilla, N. Y.
These fasteners are in every way more convenient than
the elects in common use. An idea of the apparatus is
given in Fig. 43.

A very simple and useful holder for guinea-pigs con-
sists of a metal cylinder of about 5 cm. diameter and
about 13 cm. long, closed at one end by a perforated
cap of either tin or wire netting. Along the side of
this box is a longitudinal slit 12 mm. wide that runs for
9.5 cm. from within 0.5 cm. of the open extremity of
the cylinder. The animal is placed in such a cylinder
with its head toward the perforated bottom. It is then
easily possible 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 posterior 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 ani-
mals is to be taken. The manipulations can easily be
made without the aid of an assistant. Its construction
is seen in Fig. 44.1

For ordinary subcutaneous inoculations at the root

1 Centralblatt fur Bacteriologie und Parasiteukuude, 1895, vol. xviii.
p. 530.

240

BACTERIOLOGY.

of the tail in mice a simple apparatus consists of a
piece of board about 7 x 10 cm. and 2 cm. thick,
upon which is tacked a hollow truncated cone of wire
gauze, about 6 cm. long and about 1.5 cm. in diameter
at one end and 2 cm. at its other end. This is tacked

FIG. 44.

The Voges holder for guinea-pigs.

upon the board in such a position that its long axis is
in the long axis of the board, being equidistant from its
sides. Its small end is placed at the edge of the board.
The mouse is taken up by the tail by means of a pair

INJECTION INTO THE CIRCULATION. 241

of tongs and allowed to crawl into the smaller end of
the wire cone. When so far in that only the root of
the tail projects the animal is fixed in this position by
a clamp and thumb-screw, with which the apparatus
(Fig. 45) is provided. The animal usually remains per-
fectly quiet and may be handled without difficulty.

The hair over the root of the tail is to be care-
fully cut away with 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

FIG. 45.

Mouse-holder, with mouse in proper position.

spinal column, and thus deposit the material as far from
the surface- wound as possible.

As the subcutaneous inoculation 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.

18

242 BACTERIOLOGY.

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. In the case of fur-bearing animals the hair over
the point selected for the inoculation should be closely
cut with scissors, and from a small area the feathers
should be plucked in the case of birds.

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 employed for the purpose, the operation is
usually done upon one of the veins in the ear. To those
who have had no practice with 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 (rawws anterior
of the vena auricularis posterior) is the largest and most
conspicuous vessel of the ear, and is, therefore, believed
by the inexperienced to be 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

INJECTION INTO THE CIRCULATION. 243

lateralis posterior of the vena auricularis posterior} is a
very fine, delicate vessel which runs along the posterior
margin of the ear, and 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
further away from the mouth of the vessel — that is, the
nearer we approach its capillary extremity — the more
favorable become the conditions for the success of the
operation.

After shaving the ear and carefully washing it with
clean water select the very delicate vessel lying quite
close to the posterior margin of the ear, and make the
injection as near to the apex of the ear as possible. At
times the vessels of the ear will be found to contain
so little blood that they are hardly distinguishable,
making it very difficult to introduce the needle into
them. This is sometimes overcome by pressure at the
root of the ear, causing stasis of the blood and distention
of the vessels. A very satisfactory method of causing
the veins to become prominent is to press lightly or
prick gently 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 distended
with blood, and readily distinguishable from the sur-
rounding tissue ; it may then be easily punctured by
the needle of the syringe. A sharp flick with the
finger will often produce the same result. 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 manner in which their points are ground. If
one examine carefully the point of a new hypodermic

244 BA CTER10LOG Y.

needle, it will be seen that the long point, instead of
presenting 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 usually seen that the point of the needle,
instead of remaining in the vessel, as it would do were
it straight (or "chisel pointed"), very commonly pro-
jects into the opposite wall ; and as the needle is inserted
further and further it is usually pushed through the
vessel-walls into the loose tissues beyond, and the
material to be injected is deposited in these tissues,
instead of into the circulation. If, on the contrary,
the slanting point of the needle be ground until its sur-
face is perfectly flat when viewed from the side, and no
curvature exists, then when once inserted it usually
remains within the vessel, and there is no tendency to
penetrate the opposite wall. We never use a new hypo-
dermic needle until its point is carefully ground to a per-
fectly flat, slanting surface with no curvature whatever.
These differences may perhaps be more easily under-
stood if represented diagrammatically. In Fig. 46, a,

FIG. 46.

Hypodermic needles, magnified, a. Improper point, b. Proper shape of point.

the needle has the point usually seen when new. In Fig.
47, 6, the point has been ground 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

INJECTION INTO THE CIRCULATION. 245

needle is that commonly employed by physicians for
subcutaneous injections in human beings.

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 only its
head protrudes; though in most cases we have not
found this necessary, particularly if the animal has not
been excited prior to beginning the operation.

The ear in which the injection is to be made should be
shaved clean of hair by means of a razor and soap and
then washed with water. It is unnecessary to attempt
disinfection of the skin.

The animal should be placed so that the prepared
ear comes between the operator and the source of light.
This renders visible by transmitted light not only the
coarser vessels of the ear, but also their finer branches.

The 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 supposes the point of
the needle is in the vessel a drop or two of the fluid may
be injected from the syringe, and, if his suspicions are
correct, the circulation in the small ramifications and
their anastomoses will rapidly alter in appearance —
i. e., the circulating blood will be displaced very quickly
by the clear, transparent fluid that is being injected. At
this stage one must proceed very carefully, for some-
times when the needle-point is not actually in the ves-

246 BA CTERIOL OGY.

sel, but is in the lymph-spaces surrounding it, an ap-
pearance somewhat similar is seen. This may always
be differentiated, however, by continuing the injection,
when the flow of clear fluid through the vessels will not
only fail to take the place of the circulating blood, but
at the same time a localized swelling, due to an accu-
mulation of the fluid injected, will appear 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 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 operation will be experienced.

Its greater convenience and simplicity, as compared
with other methods for the introduction of substances
into the circulation, commend it as a technical procedure
with which to make one's self familiar. The animals
sustain practically no wound, they experience no suffer-
ing— at least they give no evidence of pain — and no
anaesthetic 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.

For operations requiring exact dosage experience

INOCULATION INTO LYMPHATIC CIRCULATION. 247

lias led me to prefer a syringe after the pattern of C,
in Fig. 47 — i. e., the form commonly used by physi-
cians. The reason for this is as follows : in making
injections, either into the circulation or under the skin,
there is a certain amount of resistance to the passage of
fluid from the needle. If one overcomes this resistance
by means of a cushion of compressed air, as is the case
in syringes A and £, 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 injected than is desired. No such accident is likely

FIG. 47.

Forms of hypodermic syringe.
A. Koch's syringe. B. Syringe of Strohschein. C. Overlack's form.

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.

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 in no wise complicated. One simply plunges
the point of the hypodermic needle directly into the sub-

248 BACTERIOLOGY.

stance of the testicle and then injects the amount desired.
Injections made in this manner are sometimes followed
by interesting pathological lesions of the lymphatic
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 fascia?, and, taking 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 little or no
danger 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 symphysis pubis. Though this may seem a rude
method, it is rare that the intestines are penetrated or
otherwise injured. The object of the primary incision
is to lessen the chances of contamination by bacteria
located in the skin, some of wrhich might adhere to the
needle if it were plunged directly through the skin, and
thus complicate the results.

If solid substances, bits of tissue, etc., are to be intro-
duced into the peritoneum, it becomes necessary to con-
duct the operation upon the lines of a laparotomy.
The hair should be shaved from a small area over the
median line, after which the skin is to be thoroughly
washed. A short longitudinal incision (about 2 cm.
long) is then to be made in the median line through the
skin and down to the fasciae. Two subcutaneous
sutures, as employed by Halsted, are then to be intro-
duced transversely to the line of incision about 1 cm.
apart, and their ends left loose. This particular sort of
suture does not pass through the skin, but, instead, the

INOCULATION INTO GREAT SEROUS CAVITIES. 249

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.
(The figure indicates the primary opening through the
skin. The longitudinal dotted line shows the opening to

FIG. 48.

Diagram of skin incision and sutures in laparotomy on animals.

be made into the abdomen ; the transverse dotted lines,
with their loose ends, represent the sutures as placed
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 is deposited in the peri-
toneal cavity, under precautions that will exclude all
else, the edges of the wound drawn evenly and gently
together by tying the sutures, and the lines of incision
dressed with collodion. It should be needless to say
that this operation must be conducted under the strictest

250 BACTERIOLOGY.

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, must be carefully cleansed, and the
material to be introduced into the abdomen should be
handled with only sterilized instruments.

Inoculation into the pleural cavity is much less fre-
quently required — in fact, it is not a routine method.
It is not easy to enter the pleural cavity with a hypo-
dermic needle without injuring the lung, and it is rare
that conditions call for the introduction of solid parti-
cles into 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 activities upon the
iris can be conveniently studied. It is a mode of inoc-
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 tuber-
culous tissue into the anterior chamber of the eyeof 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 deviations from the normal are to be carefully noted

ANIMALS AFTER INOCULATION.

251

— as, for instance, elevation of temperature ; loss of
weight ; peculiar position in the cage ; loss of appetite ;

roughening of the hair; excessive secretions, from either
the air-passages, conjunctiva, or kidneys ; looseness of or
hemorrhage from the bowels ; tumefaction or reaction at

252

BACTERIOLOGY.

site of inoculation, etc. If death ensue in from two to
four days, it may reasonably be expected that at autopsy

;J

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

AXIMALS AFTKIl INOCULATION.

253

animal must be kept under observation often for weeks.
In these cases it is important to note the progress of

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254

BACTERIOLOGY.

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weighed daily, always at about the same hour and
always about midway between the hours of feeding;

ANIMALS AFTER INOCULATION. 255

at the same time its temperature, as indicated by a
thermometer placed in the rectum, is to be recorded.1
By comparison of these daily observations the ob-
server is aided in determining the course the infection
is taking.

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

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 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

1 The thermometer must be inserted into the rectum beyond the
grasp of the sphineter, otherwise pressure upon its bulb by contraction
of this muscle may force up the mercurial column to a point higher
than that resulting from the actual body-temperature.

2 56 BA GTE 'RIO L 0 G Y.

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, consisting of green vegetables and grains
(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 suf-
ficiency of food. With the rabbits there is a gradual
loss of weight with the smaller amounts of food, which

O '

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 con-
stant, and is not so conspicuously affected by the increase
in food as one might expect. From the recorded tem-
peratures one sees the peculiar fluctuations mentioned.
To just what they are due it is impossible to say. It
is manifest that the normal temperature of these animals,
if we can speak of a normal temperature for animals
presenting such fluctuations, is about a degree or more,
Centigrade, higher than that of human beings. The
animals from which these charts were made were not
inoculated, nor were they subjected to any operative
procedures whatever, the only deviations from normal
conditions being the variations in the daily amount of
food given.

In certain instances, however, there will be noticed
a constant tendency to diminution in weight, notwith-
standing the daily fluctuations, and after a time a con-
dition of extreme emaciation may be reached, the
animal often being reduced to from 50 to 60 per cent,
of its original weight. In other cases, after inoculations
to which the animal is not susceptible, rabbits in par-
ticular, if properly fed, will frequently gain steadily in

ANIMALS AFTER INOCULATION. 257

weight. The condition of progressive emaciation just
mentioned is conspicuously seen after intravenous inoc-
ulation of rabbits with cultures of bacillus typhosus and
of bacillus coli, referred to in the chapter on the latter
organism, and if looked for will doubtless be seen to fol-
low inoculation with other organisms capable of producing
chronic forms of infection, but which are frequently con-
sidered 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 sometimes there
will 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 as indicating the existence
or absence of disease.

17

CHAPTER XIII.

Post-mortem examination of animals — Bacteriological examination of
the tissues — Disposal of tissues and disinfection of instruments
after the examination— Study of tissues and exudates during life.

DURING bacteriological examination of the tissues
of dead animals certain precautions must be rigidly
observed in order to arrive at correct conclusions.

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
muscles. It is best done by first making with a scalpel
an incision just large enough to permit of the intro-
duction of one blade of a blunt-pointed scissors. It is

258

POST-MORTEM EXAMINATION OF ANIMALS. 259

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. It is then pinned flat upon 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
table-knife that has been heated in a gas-flame. The
blade, made quite hot, is to be held upon the region of
the linea alba until the tissues of that region begin to
burn ; it is then held transversely to this line over about
the centre of the abdomen, thus making two sterilized
tracks, through which the abdomen may be opened by
a crucial incision. The sterilization thus accomplished
is, of course, directed only against organisms that may
have fallen upon the surface from without, and there-
fore it 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 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 hooks.
The cross-incision is made in the same way. When
this is completed an incision through the ribs with a
pair of heavy, sterilized scissors is made along the

260 BACTERIOLOGY.

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 surface of the organ from which cultures are
to be made. Hold it upon the organ until the surface
directly beneath 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 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 tissue is sometimes
too great to permit of puncture with the ordinary wire
loop, Nuttall l has devised for the purpose a platinum-
wire spear which possesses great advantage over the
loop. It has 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 or glass handle, preferably the former.
It can readily be thrust into the densest of the soft tissues,

1 Centralbatt f-iir Bakteriologie und Parasitenkunde, 1892, Bd. xi.
p. 538.

POST-MORTEM EXAMINATION OF ANIMALS. 261

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.

Nuttall's platinum spear for use at autopsies.

Cultures from the blood are usually made from
one of the cavities of the heart, which is always punct-
ured at a point 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 be present 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
after closer study may be subsequently inserted in the
protocol.

The cover-slips are now to be stained, mounted, and

26 2 BA CTERIOLOG Y.

examined microscopically, and the results carefully
noted.

The same care with regard to noting, labelling,
etc., should be exercised in the subsequent study of
the cultures and the hardened tissues, which arc 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, with the precautions given, and sufficiently soon
after death, the results of the bacteriological exami-
nation should be either negative or the organisms
which are isolated should be in pure cultures. This is
particularly the case with cultures made from the inter-
nal 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 remains of
the animal should be burned ; all instruments subjected
to either sterilization by steam or boiling for fifteen
minutes in a 1 to 2 per cent, soda solution ; and the
board upon which the animal was tacked, as well as the

STUDIES OF TISSUES DURING LIFE. 263

ticks, towels, dishes, and all other implements used at
the autopsy, be sterilized by steam. All cultures, cover-
slips, and, indeed, all articles likely to have infectious
material upon them, must be sterilized as soon as they
are of no further service.

What has been said with regard to the study of dead
tissues obtained at autopsy applies equally well to the
bacteriological study of tissues and exudates obtained
during life. In the latter case, however, certain pre-
cautions are always to be observed. In the first place,
it is desirable to obtain the materials under aseptic pre-
cautions, care being taken that no disinfectant fluids
are mixed with them. They should be subjected to
study as soon as possible after removal from the body.
In the case of tissues that cannot be examined on the
spot, they should be placed in a sterile Petri dish or
in a stoppered, sterile, wide-mouthed bottle and taken
at once to the laboratory. The surface should then be
seared with a hot knife and an incision through the
seared area into the centre made with a knife that has
been sterilized and allowed to cool. From the depths
of this incision enough material may be obtained for
microscopic examination and for the preparation of
cultures. Fluid exudates that must be taken to the
laboratory should be collected in either a sterile test-
tube, or, better, in a sterile capillary tube that is
subsequently sealed at both ends in a gas-flame.
When bacteriological examination of the blood dur-
ing life is required, it is customary to obtain the neces-
sary sample of blood by pricking the skin. It must
be remembered, in this connection, that the skin usu-
ally contains a number of species of bacteria that

264 BA CTERIOLOG Y.

are of no pathological significance and have nothing
to do with the disease from which the individual
may be suffering. It is manifestly essential to ex-
clude these. It is not possible to exclude them cer-
tainly and completely under all circumstances, without
a more or less elaborate procedure ; but an effort to do
so should always be made. As a rule, the greater num-
ber of them may be removed from the skin by careful
washing with warm water and soap and a sterile brush,
after which the skin should be rinsed with alcohol and
allowed to dry spontaneously. The drop of blood may
then be obtained from the skin thus cleaned by a prick
with a sharp, sterilized lancet. The presence in the
cultures of a staphylococcus, growing slowly, with
white colonies, is a frequent experience, and does not
necessarily imply that this organism bears an etiological
relation to the disease from which the individual may
be suffering (see staphylococcus epidermidis albus, page
281).

In the study of many of the common diseases,
notably the exanthemata, both at autopsy and during
life, by the methods above outlined, the investigation
often yields negative results, and yet there is every
reason for believing these diseases to be dependent for
their existence upon invasion of the body by some form
or another of living micro-organisms, capable of growth
in the tissues and susceptible of being transmitted from
individual to individual, either directly or indirectly.
In this connection it is appropriate to call attention
to the novel and important technical procedures that
have been employed by Nocard and Roux * in their
investigations conducted in the Pasteur Institute at

1 Nocard and Roux : Annales de 1'Iustitut Pasteur, April 25, 1898.

STUDIES OF TISSUES DURING LIFE. 265

Paris. In the course of their studies upon the pleuro-
pneumonia of cattle, a disease in which all investigators
had hitherto failed to detect by either microscopic or
culture-methods any species of bacteria that might rea-
sonably be regarded as the causative agent, they detected
a group of bodies, apparently bacteria, of such infinitesi-
mally small dimensions as entirely to escape detection
by the usual methods of examination.

The results of Nocard and Roux were obtained both
through the adoption of special methods of cultivation
and the use of very high amplifying powers for micro-
scopic examination. The method of cultivation was that
suggested in 1896 by Metehnikoff, Roux, and Salimbeni,1
and is essentially as follows : very thin-walled, small
sacs of collodion are sterilized by steam, filled with
bouillon, inoculated with the exudate or tissue to be
tested, sealed with sterile collodion, and placed in the
abdominal cavity of an animal — rabbit, guinea-pig,
chicken, dog, sheep, or calf, as the case may require.
The wall of the collodion sac is impermeable to bac-
teria or to leucocytes, but is an osmotic membrane
through which fluids and some of their dissolved
contents readily diffuse. This diffusion supplies the
bacteria within the sacs with such matters from the
living fluids of the animal as are apparently essential to
their development, while at the same time the bacteria
develop uninterruptedly, being protected by the collodion
membrane from the antagonistic action of the fixed and
wandering cells of the tissues. After a period of from a
few days to several months the animal is sacrificed, and
the sac removed from the peritoneal cavity and its con-

1 Metehnikoff, Roux, and Salimbeni: Annales de 1'Institnt Pasteur,
1896, p. 257.

266 BA CTERIOLOG Y.

tents subjected to microscopic examination. This latter
part of the work was unsatisfactory when conducted with
the usual combinations of lenses employed in bacterio-
logical work. Satisfactory examinations could only be
made by the use of very high magnifying powers, about
2000 diameters, and unusually brilliant illumination.
When conducted under these conditions the sacs inoc-
ulated with matters from the pulmonary exudates of
pleuro-pneumonia were found to contain numerous mo-
tile points or dots of such extremely small size that it
was often impossible to decide as to their exact form.
Control-^acs not inoculated, but kept in the peritoneal
cavity of the same animal, manifestly under similar con-
ditions, did not reveal the presence of the minute bodies.
In referring at length to this investigation it is not
my purpose to discuss the object of it, but only to
direct attention to this novel technique, which seems
capable of much wider application. There is a group
of common maladies, such as measles, scarlet fever,
smallpox, etc., of the etiology of which we know noth-
ing, and on which it has hitherto been impossible
to shed important light by the usual bacteriological
procedures. Nocard and Roux have apparently re-
vealed to us a world of micro-organisms whose existence
has hitherto been unsuspected, and it is not unreason-
able to suppose that it is among this group that we are
to seek for the causative agents of many specific dis-
eases whose etiology is as yet obscure.1

1 An excellent review of the paper of Nocard and Eonx is to be
found in the Philadelphia Medical Journal of June 11, 1898.

APPLICATION OF THE METHODS OF
BACTERIOLOGY.

CHAPTER XIV.

To obtain material with which to begin work.

EXPOSE to the air of an inhabited room a slice of
freshly steamed potato or a bit of slightly moistened
bread upon a plate for about one hour. Then cover it
with an ordinary water-glass, place it in a warm spot
(temperature not to exceed that of the human body
— 37.5° C.), and allow it to remain undisturbed. In
from 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 of
irregular, ragged deposit. All these gross appear-
ances 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 growth of exactly the same ap-
pearance. It was just such an experiment as this,

267

268 BACTERIOLOGY.

accidentally performed, that suggested to Koch a means
of separating and isolating in pure cultures the com-
ponent individuals from mixtures of bacteria, and it
was from this observation that the methods of cultiva-
tion on solid media were evolved.

If, without molesting these objects, we continue the
observations 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 quickly overrun
the surface upon which they are growing, and, indeed,
grow over the smaller, less rapidly developing colonies.
In a number of instances, if the observation be con-
tinued long enough, many of these rapidly growing
colonies will, after a time, lose their lustrous and smooth
or regular surface and will show here and there eleva-
tions, which will continue to appear until the whole
surface becomes conspicuously wrinkled. Again, bub-
bles 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 growing. Sometimes peculiar odors due
to 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 colonies upon the bread or
potato with a hand-lens of low magnifying power, we
will be enabled to detect differences not noticeable to
the naked eye. In a few cases we may still see nothing

MATERIAL WITH WHICH TO BEGIN WORK. 269

more than a smooth, non-characteristic surface ; while
in others minute, sometimes regularly arranged tiny
corrugations may be observed. In one colony they may
appear as tolerably regular lines, 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
characteristic arrangement in folds, radii, or concentric
rings, or the production of color, is characteristic of the
growth of the organism under the conditions first
observed, and by a repetition of those conditions may
be reproduced at will.

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 commence his
studies. It is not necessary at this time for him to bur-
den his mind with names for these organisms ; it is suffi-
cient for him to recognize that they are mostly of differ-
ent 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 :

Place some of the pieces (about 5 centimetres long)
in a sterilized test-tube, and sterilize them by streaming
steam for one hour or in the autoclave for fifteen min-

270 BACTERIOLOGY.

utes at one atmosphere pressure. 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 a dusty table
and then plate it. Suspend three or four pieces upon a
sterilized wire hook and let them hang for twenty min-
utes 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
should 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 ?

Sprinkle a little fine dust over the surface of a plate
of sterile gelatin or agar-agar ; examine the dust-par-
ticles with the microscope immediately after depositing
them on the medium, and again after eighteen to twenty-
four hours. What differences do you detect? What
information of sanitary importance does this give?

Under the description of each of the pathogenic bacte-
ria more or less detailed directions will be found for the
discovery and isolation of each of the pathogenic bacteria.

CHAPTER XV.

Suppuration — Micrococcus aureus — Micrococcus pyogenes and citreus
— Staphylococcus epidermidis albus — Streptococcus pyogenes —
Micrococcus gonorrhoeas — Micrococcus intracellularis — Pseudomo-
nas asruginosa — Bacillus of bubonic plague — Bacterium pseudo-
diphtheriticum.

MICROCOCCUS AUREUS (ROSENBACH), MIGULA, 1900.

Synonyms: Staphylococcus pyogenes aureus, Eosenbach, 1884; Micro-
coccus pyogenes aureus, Migula, 1895 ; Micrococcus pyogenes, Leh-
inanii and Neumann, 1896.

PREPARE a set of plates of agar-agar from the pus
of an acute abscess or boil that has been opened under
antiseptic precautions. Care must be taken that none
of the antiseptic used gains access to the culture-tubes,
otherwise its antiseptic effect may be operative and the
development of the organisms interfered with. It is
best, therefore, to take a drop of the pus upon a 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 incision 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

271

272 BACTERIOLOGY.

from the pus of gonorrhoea. (See Fig. 57.) In what
way do the two preparations differ, the one from the
other?)

FIG. 54.

, '5V A

t * ' t

Preparation from pus, showing pus-cells, A, and micrococci, 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 their 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-
ularly grouped together. They are in every way of

MICROCOCCUS AUREUS. 273

the same appearance as those seen upon the original
cover-slip preparation.

Prepare from one of these colonies a pure stab-culture
in gelatin. After thirtj-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 liquefied portion becomes more
or less of a stocking-shape, and gradually widens 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-production.

It belongs to the group of facultative anaerobes.

In milk it rapidly brings about coagulation with acid
reaction.

It is not motile, and being of the family of micrococci
does not form endogenous spores. It possesses, how-
ever, a degree of resistance to detrimental agencies that
is somewhat greater than that common to non-spore-
bearing bacteria.

In bouillon it causes a diffuse clouding, and after a
time a yellow or orange-colored sedimentation.

This organism is the commonest of the pathogenic
bacteria with which we shall meet. It is micrococcus
aureus, and is the organism most frequently concerned
in the production of acute, circumscribed, suppurative
inflammations. It is almost everywhere present, and

18

274 BACTERIOLOGY.

is the organism causing continuous annoyance to the
surgeon.

In studying its effects upon lower animals a number
of points are to be remembered. While it is the etio-
logical factor in the production of most of the 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 is likewise not always accompanied
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 cult-
ures 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.1)

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 Sur-
gery, No. 1, 1891, vol. ii. No. 5, pp. 301-303.

MICROCOCCUS AUREUS. 275

If one inject into the circulation of the rabbit through
a vein of the ear, or in any other way, from 0.1 to
0.3 c.c. of a bouillon culture or watery suspension of
a virulent variety of this organism, a fatal pyaemia
always follows in from two and one-half to three days.
A few hours before death the animal suifers frequently
from 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 with them. Frequently the pericardial sac
is distended with a clear gelatinous fluid, and almost
constantly the yellow points are seen in the myocar-
dium. The kidneys are rarely without them ; here
they appear on the surface as isolated 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.

276 BACTERIOLOGY.

These minute abscesses contain a dry, cheesy, necrotic
centre, in which the ruicrococci are present in large
numbers, as may be seen upon cover-slips prepared
from them. This organism may also be obtained in
pure culture from the suppurating foci.

Preserve in Muller's fluid and in alcohol duplicate
bits of all the tissues 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 STUDY 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 the 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 in a more advanced stage a
pale centre can usually be made out.

When magnified they appear in the earliest stages as

COVER-SLIPS AND SECTIONS. 277

minute aggregations of small cells, the nuclei of which
stain intensely. Almost always evidences of progress-
ing necrosis can be seen about the centre of these cell-
accumulations. The normal structure of the cells of
the tissues is more or less destroyed ; there is seen
a granular condition due to cell-fragmentation ; at dif-
ferent points about the centre of this area the tissue
appears cloudy and the tissue-cells do not stain read-
ily. Round about and through this spot are seen
the nuclei of pus-cells, many of which are undergoing
disintegration. In the smallest of these beginning ab-
scesses the micrococci are to be seen scattered about
the centre of the necrotic tissue ; but in a more advanced
stage they are commonly seen massed together in very
large numbers in the form commonly referred to as
emboli of micrococci.

When the process is well advanced, the different parts
of the abscess are more easily detected. They then pre-
sent in sections somewhat the following conditions : 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 micro-
cocci. Sometimes the shape of this mass of micro-
cocci corresponds to that of the capillary in which the
organisms became lodged and developed. Immediately
about the embolus of cocci the tissues are in an advanced
stage of necrosis. Their structure is almost completely
destroyed, although the destruction is seen to be more
advanced in some of the elements of the tissues than in
others. As we approach the periphery of this faintly
stained necrotic area it becomes marked here and there
with granular bodies, irregular in size and shape, which
stain in the same way as do the nuclei of the pus-cells

278 BACTERIOLOGY.

and represent the result of disintegration going on in
these cells.

Beyond this area 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 from beginning to end with the pathological
changes which accompany the formation of larger ab-
scesses in the tissues of human beings.

From these small abscesses in the tissues of the
rabbit micrococcus aureus may again be obtained in pure
culture, and will present identically the same character-
istics that were possessed by the culture with which the
animal was inoculated.

A characteristic of all staphylococcus abscesses, small
as well as large, is centralized death of tissue ; even in
those of microscopic dimensions this area of necrosis
is always discernible by appropriate methods of exami-
nation. It represents the very starting-point of the
destructive process, and is referable to the action upon
the tissues of poisons elaborated by living bacteria that
have gained access to them.

As a result of the studies of van de Velde, Krauss,
von Lingelsheim, Neisser and \Vecnsberg, and others,
our knowledge of the poison that causes the destruction
— staphylotoxin, as it is called — has been greatly ex-

COVER-SLIPS AND SECTIONS. 279

tended. Through the work of these investigators we now
know that the pathogenic properties of staphylococcus
pyogenes aureus are due to a definite soluble toxin elab-
orated by it : that this poison is produced under arti-
ficial Conditions of cultivation, and may be separated
from the living organisms by filtration ; that when
injected into the living animal body its effects upon the
tissues are essentially reproductions of those accompany-
ing the growth of the organism itself; that when this
action is tested upon particular cells, such as erythro-
cytes and leucocytes, two distinct properties are exhib-
ited, one a hamolytic, through which the red corpuscles
of the blood are dissolved, the other a leucocidic, through
which the white blood-corpuscles are destroyed ; that
the hsemolytic and leucocidic properties are distinct
from one another, and are due to the activities of two
lysins, of which the staphylotoxin is (in part?) com-
posed, and which may be separated from one another by
appropriate methods of analysis ; that the result of the
treatment of animals with gradually increasing non-fatal
doses of staphylotoxin is the appearance in the blood of
the animals of antitoxic bodies (antilysins) that inhibit
the action of the toxins (lysins) ; and, finally, that in
the serum of certain animals (man and horse) similar
antilysins in varying amounts are normally present.1

Petersen, Paltchikowsky, Proscher, and others have
recently attempted to prepare an antistaphylococcus
serum. The serum of patients recovering from severe
staphylococcus infections contains protective substances

1 See van de Veld e: Annales de 1'Institut Pasteur, tome x. p. 580.
Krauss : Wiener klin. Wochenschrift, 1900, No. 3. Von Lingelsheim :
Etiologie uud Therapie der Staphylokoken Infektion (monograph),
Berlin- Wien, 1900. Neisser and Wechsberg: Zeitschrift fur Hygiene
und Infektionskraukheiten, 1901, Bd. xxxvi. 8. 299.

280 BA CTERIOL 0 G Y.

which serve to protect rabbits from twice the fatal close
of a staphylococcus culture. Similarly the serum of
immunized rabbits and goats, as shown by the exper-
iments of Petersen, possesses about the same degree of
protective powers. No antitoxic power could be dem-
onstrated in the serum of the treated animals. The
extremely limited degree of the protective power of the
antistaphylococcus serums prepared thus far makes it
impossible to employ them for curative purposes in
human beings, as Petersen calculated that an adult
would require from 350 to 700 c.c. of the serum at a sin-
gle dose, as judged by its effects on the lower animals.

OTHER COMMOX PYOGENIC ORGANISMS.

MICROCOCCUS PYOGENES (Rosenbacli), Migula, 1900. Synonyms:
Staphylococcus pyogenes albus, Eosenbach, 1884 ; Micrococcus pyogenes
albus, Lehmann and Neumann, 1896.

MICROCOCCUS CITREUS (Passet), Migula, 1900. Synonym: Staphylo-
coccus pyogenes citreus, Passet, 1885.

The pus of an acute abscess in the human being
may sometimes contain organisms other than micro-
coccus aureus. Micrococcus pyogenes and citreus may be
found. The colonies of the former are white, those of
the latter are lemon color. With these exceptions they
are in all essential cultural peculiarities similar to micro-
coccus aureus. As a rule, they are not virulent for
animals, and when they do possess pathogenic proper-
ties, it is in a much lower degree than is commonly the
case with the golden staphylococcus. Streptococcus
pyogenes is also sometimes present. The commonest
of the pyogenic organisms, however, is that just de-
scribed, viz., micrococcus aureus. An organism that is
almost universally present in the skin, and is often con-
cerned in producing mild forms of inflammation, is

OTHER COMMON PYOGENIC ORGANISMS. 281

staphylococcus epidermidis albus (Welch), an organism
that may readily be confused with micrococcus pyogenes.
It is distinguished from the latter by the slowness with
which it liquefies gelatin and by the comparative absence
of pathogenic properties when injected into the circula-
tion of rabbits. Welch regards this organism as a
variety of micrococcus pyogenes.

FIG. 55.

Streptococcus pyogenes in pus.

STREPTOCOCCUS PYOGENES (ROSENBACH), MIGTJLA,
1900.

Synonyms: Streptococcus, Billroth, 1874; Streptococcus pyogenes,
Rosenbach, 1884.

From a spreading phlegmonous inflammation prepare
cover-slips and cultures. What is the predominating
organism ? Does it appear in the form of irregular clus-
ters like those of grapes, or have its individuals a definite,
regular arrangement ? (See Fig. 55.) Are its colonies
like those of micrococcus aureus?

Isolate this organism in pure cultures. In these cul-

282 BACTERIOLOGY.

tures it will be found on microscopic examination to
present an arrangement somewhat like a chain of beads.
(Fig. 560

Determine its peculiarities and describe them accu-
rately. They should be as follows :

Upon microscopic examination a micrococcus should
be found, but differing in its arrangement from the
staphylococci just described. The single cells are not
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-

FIG. 56.

Streptococcus pyogenes.

posing them, but more commonly certain irregular groups
may be seen in them. Here they appear as if two or
three cells had fused together to form a link, so to speak,
in the chain, that is somewhat longer than the remaining
links ; again, portions of the chain may be thinner than the
rest, or it may appear broken or ragged. Commonly the
individuals comprising this chain of cocci are not round,
but appear flattened on the sides adjacent to one another.
The chains are sometimes short, consisting of four to six
cells ; or, again, they may be much longer, and extend from
a half to two-thirds across the field of the microscope.

Under artificial conditions it sometimes grows well,
and can be cultivated through many generations, while

STREPTOCOCCUS PYOGENES. 283

ut other times it rapidly loses its vitality. When isolated
from the diseased area upon artificial media it seems to
retain its vitality for a longer period if replanted upon
fresh media every day or two for a time ; but if the
first generation is transplanted and is allowed to re-
main upon the original medium, it is not uncommon
to find the organism incapable of further cultivation
after a week or ten days.

Under no conditions is the growth of this organism
very luxuriant.

On gelatin plates its colonies appear after forty-eight
to seventy-two hours as very small, flat, round points of
a bluish-white or opalescent appearance. They do not
cause liquefaction of the gelatin, and in size they rarely
exceed 0.6-0.8 mm. in diameter. Under low magnify-
ing power they have a brownish or yellowish tinge by
transmitted light, and are finely granular. As the 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 most luxuriant
growth is seen on glycerin-agar-agar at the tempera-

284 li.irrERIOLOGY.

ture of the body (37.5° C.), and its least on gelatin at
from 18° to 20° C.

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-
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 of inocula-
tion, and microscopic examination shows that multiplica-
tion of the organisms placed at this point has occurred.

In milk its conduct is not always the same, some cult-
ures 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, as a
rule, only a very faint pink color after twenty-four hours
at 37.5° C.

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 this property is said by
Petruschky to be preserved by retaining the cultures in
the ice-chest after they have been growing on gelatin
for two days at 22° C.

Its virulence may often be increased by passing it
through a series of susceptible animals.

It is a facultative anaerobe.

STREPTOCOCCUS PYOGENES. 285

It stains with the ordinary aniline (lyes, and is not
decolorized when subjected to Gram's method.

It 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 in
which its resistance to detrimental agencies is increased.
In the tissues of the body, however, it appears to pos-
sess marked 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 has healed.

Streptococcus pyogenes is the organism most commonly
found in rapidly spreading suppurations, while micrococ-
cus aureus is most frequently found in circumscribed
abscess formations ; they may also be found together.

The results of its inoculation into the tissues of
lower animals are described by Rosenbach and Passet
as protracted, progressive, erysipelatoid inflammations ;
and Fehleisen, who described a streptococcus in erysip-
elas that is in all probability identical with the strepto-
coccus pyogenes under consideration, stated that it pro-
duced in the tissues of rabbits (the base of the ear)
a sharply defined, migratory reddening without pus-
lornuition. The writer has encountered a culture of
this organism that possessed the property 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 im-
portant bearing upon the question concerning the iden-
tity of streptococci found in various inflammatory con-
ditions, such, for instance, as the spreading erysipelatoid
manifestations on the one hand, and the circumscribed
abscess-formations on the other.

286 BACTERIOLOGY.

The results that follow upon the inoculation of ani-
mals with cultures of streptococci obtained from various
inflammatory lesions are, as a rule, inconstant. At
times cultures will be encountered that are apparently
without virulence, no matter how tested ; while again
cultures from other sources exhibit the most marked
pathogenic properties, even when employed in almost
infinitesimal quantities. Between these extremes every
gradation may be expected. The virulence of a culture
is not necessarily proportional to the intensity of the
pathological process from which it is derived.

There is never any certainty of faithfully repro-
ducing, by inoculation into susceptible animals, the
pathological lesion from which a culture of the organ-
ism may have been obtained. The introduction into a
susceptible animal of a culture derived from either a
spreading phlegmon or an erysipelatous inflammation
may result in erysipelas, general septicaBmia, local ab-
scess-formation, or, as said, may have no effect at all.
Cultures may be encountered that are pathogenic for
one susceptible species of animals and not for another.

Under the ordinary conditions of artificial cultiva-
tion fully virulent varieties of streptococcus pyogenes
usually lose their virulence after a short time.
Petruschky1 preserves this property by cultivation
upon nutrient gelatin for two days at 22° C., keeping
the cultures after this time in the refrigerator, and
transplanting upon fresh gelatin every five or six days.
Marmorek2 finds the virulence preserved by growing
the organism in a mixture of 2 parts of horse or

1 Petruschky : Centralblatt fur Bakteriologie und Parasitenkunde,
1895, Abth. i. Bd. xvii.

2 Marmorek : Auuales de 1'Iustitut Pasteur, 1895.

ANTISTREPTOCOCCUS SERUM. 287

human blood-serum and 1 part of nutrient bouillon, or
of 1 part of ascites-fluid and 2 parts of bouillon.

Certain authors are of the opinion that a relation
exists between virulence and the length of the chains
formed by streptococci when growing in fluid media.
It is held that those forming the long chains, strepto-
coccus longus, are the only ones concerned in animal
pathology, and hence the only ones by which patho-
genic powers may be exhibited ; while those form-
ing the short chains, streptococcus brevis, are not, as a
rule, pathogenic, and may often be readily differentiated
from the other variety by more or less gross cultural
characteristics, such as slow liquefaction of gelatin,
visible growth on potato, etc.1

ANTISTREPTOCOCCUS SERUM. — For a time a great
deal of interest was created by the announcement of
Marmorek that he had succeeded in inducing in ani-
mals a state of immunity from streptococcus infection,
and that the blood-serum of such animals when injected
into other susceptible animals and human beings pos-
sessed not only the property of rendering them insus-
ceptible to this particular form of infection, but even
exhibited curative powers in cases already infected.
This serum was obtained from horses or asses that had
been rendered immune by the gradual introduction into
their tissues of increasing amounts of virulent strepto-
cocci.

A great deal of experimental work has been done
during the past decade on the perfection of an antistrep-
tococcus serum. The views of the different expcri-

l V. Lingelsheim: Zeitschrift fur Hygiene, 1891, Band x., and 1892,
Band xii. Behring: Oentralblatt fiir Bakteriologie und Parasiten-
kunde, 1892, Band xii.

288 BACTERIOLOGY.

menters differ materially on certain fundamental points.
Some regard the streptococci encountered in different
diseases as possessing specific relations to such diseases ;
as, for instance, the streptococcus found in cases of
scarlet fever is believed by Moser and others to be specific
for that disease, and consequently the antistreptococcus
serum obtained by immunization with such an organism
is believed to possess far less curative properties against
other streptococcus infections. If this idea should
prove correct then it will be necessary to obtain serum
from animals that have been simultaneously immun-
ized with a number of different streptococci derived
from various disease conditions — a so-called polyvalent
serum.

Other experimenters believe that the frequent passage
of a culture of streptococcus through the lower animals
renders it less virulent, or at least alters its virulence
for human beings, and that the serum obtained through
the immunization of animals with such cultures is less
efficacious than when the original virulence of the
organisms is maintained by cultivation on suitable
media.

Though all experimental evidence contraindicates the
production of soluble toxins in large amounts by the
streptococcus when grown in artificial media, Marmorek
still believes that by special methods of cultivation the
toxin-forming powers can be augmented, and that the
immunization of animals with such cultures serves a use-
ful purpose in giving the serum of the treated animal a
more definite antitoxic power.

Aronson prepares his antistreptococcus serum by
immunizing horses with streptococcus cultures that have
been rendered highly virulent by repeated passage

ANTISTREPTOCOCCUS SERUM, 289

through animals. By this means he secures a 20-fold
normal scrum, a " normal " serum being one of which
0.01 c.'c. protects a mouse from 100 times the lethal dose
of highly virulent streptococci. Besides this the horses
are subsequently immunized with streptococcus cultures
derived from severe cases of infection in human beings
without passage through animals, and in this way he
believes it possible to overcome the objections of those
who regard the passage through animals as useless.

Baginsky, Louis Fischer, Charlton, and others report
having obtained favorable results in the treatment of
cases of scarlet fever complicated with severe streptococ-
cus infection. After several doses of 20 c.c. of the serum
the fever declined steadily and continuously, with the
rapid disappearance of necrotic membranes in the throat,
and subsidence of the swelling of the glands of the
neck.

Foulerton l employed the antistreptococcus serum in
the treatment of cases of puerperal fever. The serum
employed was a polyvalent one derived from a horse
immunized with five strains of streptococcus. He
states that apart from the failure of the serum treat-
ment in puerperal fever arising from the uncertainty as
to the particular strain of streptococcus which is pres-
ent, the question of the dose of the serum to be employed
is of considerable importance. He advises to com-
mence treatment with an injection of at least 20 c.c.,
and if necessary this is repeated every twenty-four
hours. If no improvement results from two doses of
20 c.c. each, administered within twelve hours, it is
ii-ck'ss to persist in administering it. Large doses are
necessary for success.

1 Foulertou : The Lancet, Dec. 31, 1904.
19

290 BACTERIOLOGY.

Walker1 finds that an injection of antistreptococcus
serum in cases of pure streptococcus infection has been
followed by strikingly beneficial results. He believes
the variability in the results of the serum treatment
to be due to a specific affinity of a serum for the par-
ticular strain of streptococci used in producing it. He
states that more uniform results are likely to be obtained
from a polyvalent serum ; from the prompt injection of
serum at the commencement instead of near the close of a
severe infection ; and from the use of recently prepared
serum. He also advises the administration of the serum
for some days after the general symptoms have disap-
peared, in order to avoid a recrudescence. The question
of dose must be judged by the nature of each case and
the effect obtained by the injection, but it is important
to know that large doses spread over several days have
been used without ill effect. The most rational method
would seem to be that of a large injection (from 20 to
25 c.c.) on the first occasion, followed by smaller doses
as the case may require.

NOTE. — If the opportunity presents, obtain cultures
from a case of erysipelas. Compare the organism
thus obtained with streptococcus pyogenes. Inocu-
late rabbits both subcutaneously and into the circula-
tion with about 0.2 c.c. of pure cultures of these organ-
isms in bouillon. Do the results correspond, and do they
in any way suggest the results obtained with staphylo-
coccus pyogenes aureus when introduced into animals in
the same way? Do these streptococci flourish readily
on ordinary media?

1 Walker : The Lancet, Dec. 31, 1904.

LESS COMMON PYOGENIC ORGANISMS. 291

THE LESS COMMON PYOGENIC ORGANISMS.

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, there has been considerable modification of
this view, 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
tinder the eye of the physician, the surgeon, and the
pathologist, observations are constantly being made
that do not accord with the earlier ideas upon the
specific relation of the pyogenic cocci to all forms of
suppuration. There is an abundance of evidence to
justify the opinion that a number of organisms not
commonly classed as pyogenic may, under certain cir-
cumstances, 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 sequel of typhoid fever.

Bacillus coli has been found in pure culture in acute
peritonitis, in liver-abscess, in purulent inflammation
of the gall-bladder and ducts, and in appendicitis ; and
Welch l has found it in pure culture in fifteen different
inflammatory conditions.

1 Welch : " Conditions Underlying the Infection of Wounds," Ameri-
can Journal of the Medical Sciences, Novemher, 1891.

292 BACTERIOLOGY.

MicroeocGus lanceolaius (pneumococcus) has been found
alone in abscess of the soft parts, in purulent infiltra-
tion 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.

Organisms of the bacterium pseudodiphtheriticum
group are frequently encountered in large numbers in
the pus of superficial wounds, and especially in ulcera-
tions of the skin and mucous membranes.

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.1

MICROCOCCUS GONORRHCEJE (NEISSER), 1879.
Synonym : Gonococcus Neisser, Bumm, 1887.

One observes upon microscopic examination of cover-
slips prepared from the pus of acute gonorrhoea that
many of the pus-cells contain within their protoplasm

1 Fora more detailed discussion of the subject, see " The Factors Con-
cerned in the Production of Suppuration," International Medical
Magazine, Philadelphia, May, 1892.

MICROCOCCUS GONORRHCE^E. 293

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. 57.) The majority of the pus-cells
do not contain them.

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,

.;•<* T- fiw,

tt*i

Put of gonorrhoea, showing diplococci in the bodies of the pus-cells.

and as it is commonly arranged in pairs (flattened at
the surfaces in juxtaposition) it is often designated as
diplococcus of gonorrhoea. It is always to be found in
gonorrhosal 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 gonorrhceal origin.

It is easily detected microscopically in the secretions

294 BA CTERIOLOG Y.

of acute gonorrhoea. 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 ordinary nutrient media,
and has only been isolated in culture through the em-
ployment of special methods. Its growth under arti-
ficial conditions seems to depend upon some partic-
ular 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 many investi-
gators it is thought that only human blood possesses
this important ingredient, though this opinion is not
universal.1

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-

1 An instructive article on this subject is that by Foulerton : " On
Micrococcus Gonorrhcese and Gonorrhceal Infection," Transactions of
the British Institute of Preventive Medicine, 1897, series L

MICROCOCCUS GONORRHCEjE. 295

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.

The method used by Pfeiffer for the cultivation of
bacterium influenzse is also said to have been success-
fully 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 somewhat in detail. The medium con-
sists 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
porcelain filters generally employed in laboratories.
Wright recommends a filtering cylinder manufactured
by the Boston Filter Company as an apparatus that not
only gives a sterile filtrate, but also permits of 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 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

296 BACTERIOLOGY.

each tube is smaller than is usual ; this is in order to
allow for the subsequent addition of the urine and
serum.

" 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 far as is 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 pre-
vious sterilization by steam of the cylinder and receiv-
ing-flask, besides others which will occur to any bacteri-
ologist.

" 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 uniform mixture, and is
then placed in an inclined position to allow the agar to

MICROCOCCUS GONORRH(E;E. 297

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-
tain i nations, after which they are ready for use."

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 gonorrho3al 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-
coccus 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 semi-translu-
cent by transmitted light. 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, be-
come thicker and denser, with a faintly brownish tinge
about their centres, and a slightly irregular outline.

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.

298 BACTERIOLOGY.

The appearances coincide with the figure of such a
colony given by Wertheim.1

Wassermann 2 calls attention to the success he has
had in cultivating this organism upon a mixture of
swine-serum and nitrose, the latter being a com-
mercial product chemically known as casein-sodium
phosphate.

The preparation of the medium and its composition
are as follows :

In an Erlenmeyer flask mix 15 c.c. of swine-serum, as
free as possible from hemoglobin ; 30 to 35 c.c. of water ;
2 to 3 c.c. of glycerin ; and finally 0.8 to 0.9 gramme
(i. e., about 2 per cent.) of nitrose. This is boiled, with
gentle agitation, over a free flame, until all ingredients
are dissolved and the cloudy fluid has become quite
clear. After such boiling the mixture can be sterilized
by steam without precipitating the albumin, and may
then be kept indefinitely ready for use.

When needed, the flask and its contents are heated to
50° C. ; from six to eight tubes of 2 per cent, peptone-
agar-agar are dissolved by boiling, brought to 50° C.,
and then mixed with the solution in the flask and the
mass poured into Petri dishes. Upon the surface of this
serum-nitrose-agar the cultivation is to be conducted.
Wassermann lays particular stress upon two points that
are essential to success, viz., the preliminary boiling of
the serum-nitrose mixture before steam sterilization, as
this prevents precipitation of the albumin ; and the
necessity of having both the serum-nitrose mixture and
the agar-agar, to be mixed with it, at not over 50° C.,

1 Deutsche med. Wochenschrift, 1891, No. 50 ; Centralblatt fur Gyna-
kologie, 1891, No. 24.

2 Zeitschrift fur Hygiene und Jnfektionskrankheiten, Bd. xvii. j>. 298,

MICROCOCCUS GONORRHCEJE. 299

for if they are at boiling temperature when mixed, or if
they are brought to the boiling temperature after mixing,
the albumin will be precipitated notwithstanding the
presence of the nitrose, which otherwise prevents this.

Wassermann further observes that some samples of
serum require to be more highly diluted with water than
in the proportions given above ; that the agar-agar
should be feebly, but distinctly, alkaline to litmus,
causing no reddening whatever of blue litmus paper ;
and, finally, that the Petri dishes containing the solidi-
fied medium on which the cultures are growing are best
kept bottom upward, so as to prevent water of con-
densation collecting on the surface. By the use of the
above medium he has cultivated the gonococcus from
about one hundred different cases.

LIPSCHUTZ'S MEDIUM. — Lipschiitz J publishes a new
medium for the cultivation of micrococcus gonorrhoeas.
He sought to find a medium that could be prepared
easily from substances occurring in commerce. After
testing a number of albuminous preparations of vege-
table and animal origin, he selected the pulverized egg-
albumen of Merck for this purpose. The culture-
medium is prepared as follows : A 2 per cent, solution
of the egg-albumin is made in water, to which is added
20 c.c. of a tenth-normal caustic soda solution per 100
c.c. of fluid, and this is allowed to stand for one-half hour,
being agitated from time to time. It is then filtered
and placed in Erlenmeyer flasks in amounts of 30 to 50
c.c., and sterilized by the intermittent method. The
medium, when thus prepared, is colorless, transparent,
of a light-yellow color, and reacts distinctly alkaline to
iitmus-|>aper. To this medium nutrient agar-agar or

1 Lipschiitz: Ccntralblatt fiir Bacteriologie, Originale, Bd. 36, 1904.

300 BA CTERIOL OG Y.

the ordinary bouillon may be added in the proportion
of one part of the egg-albumin medium to two or three
parts of the agar medium or the bouillon, and this he
calls the " egg-albumin-agar " or the " egg-albumin-
bouillon" media, on which micrococeus gonorrhoea? grows
very satisfactorily. The special advantages claimed for
this medium are that it can be prepared at any time and
without difficulty, is quite clear and transparent, and
permits, where agar-agar is used, the employment of
the medium for the study of colony formations.

If transplanted from the original culture to either
glycerin-agar-agar or to Loffler's serum-mixture, a
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.

MICROCOCCUS GONORRH(E&. 301

This organism 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 gonorrhcea 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 gonorrhoeal prostatitis in
man, of gonorrhoeal proctitis in both sexes, and of gon-
orrhoeal 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 young
infants, and also occasionally to 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.

Other species of micrococci have from time to time
been described as occurring in the pus of acute urethritis
and of other purulent inflammations. Many of these
are of no significance. Some of them possess peculiarities
that might lead to confusion. The diplococcus described
by Heiman1 has certain points of resemblance to the
gonococcus, such as its location in the bodies of pus-
cells, its grouping as diplococci, its size and general ap-
pearance ; but it is still readily distinguished from the
gonococcus by its retention of color when stained by
Gram's method. The diplococcus detected by Bumm
in puerperal cystitis is likewise often found within pus-
cells, but it is readily differentiated from the gono-
coccus by its growth upon ordinary nutrient media.

1 New York Modical Record, June 22, 1895.

302 BA CTERIOL OGY.

Jficrococcus intmcettularis of Weichselbaum, isolated
from the pus of cerebro-spinal meningitis, is micro-
scopically also strikingly like the gonococcus as it is
seen in pus ; but, unlike the latter organism, may be
cultivated by the ordinary methods.

POSITIVE AND NEGATIVE DISTINGUISHING PECU-
LIARITIES OF MICROCOCCUS GONORRHCE;E. — Since gon-
orrhoeal discharges may be contaminated with pyogenic
cocci other than those causing the specific inflammation,
it is important in efforts to isolate this organism that
the differential tests be borne in mind and put into
practice. The gonococcus is differentiated from the
commoner pyogenic organisms by the following pecu-
liarities :

First, it is practically always seen in the form of
diplococci, the pair of individual cells having the
appearance of two hemispheres, with the diameters
opposed, and separated from one another by a narrow,
colorless slit. (Is this the case with micrococcus aureus
or streptococcus pyogenes ?)

Second, in gonorrhoeal pus it is practically always to
be found icithin the protoplasmic bodies of pus-cells.
(How does this compare 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.

MICROCOCCUS IXTRACELLULARIS. 303

Fifth, when obtained in pure culture by either of the
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 micrococcus
aureus and streptococcus pyogenes, are usually capable
of exciting pathological conditions. (This is less com-
monly true of streptococcus pyogenes than of micrococcus
aureus.)

MICROCOCCUS INTRACELLULARIS (WEICHSELBUAlf),
MIGULA, 1900.

Synonyms : Diplococcus intracellularis meningitidis, Weichselbaum,
1887; Streptococcus iiitracellularis (Weichselbaum), Lehmauu and
Neumann, 1896.

Of the several organisms mentioned that might be
mistaken for the gonococcus, no one of them is as sug-
gestive and none, per se, so important as that concerned
in the causation of epidemic cerebrospinal meningitis.

This organism, described by Weichselbaum in 1887
under the name "diplococcus intracellularis meningi-
tidis," was found by him in the exudations of the brain and
spinal cord in six cases of acute cerebrospinal meningitis.

As its name implies, it is a diplococcus, practically
always seen within the bodies of pus-cells (polymorpho-
nuclear leucocytes) in the exudations characteristic of
this disease. It is not seen within the other cells of the
morbid process. It slains readily with any of the ordi-

304 £ A CTERIOL OO Y.

nary aniline dyes, but is decolorized by the method of
Gram. It is conspicuous for the irregular way in which
it takes up the dye, some cells in a preparation (either
from the exudate or from cultures) being brightly and
intensely colored, others being much less so, or, indeed,
often nearly colorless. There is also a marked variation
in the size of individual cocci, some being normal, others
being apparently swollen. These latter are often pale,
with a deeply staining centre, giving the appearance of
a coccus surrounded by a capsule. It is not improbable
that these are degenerated or involuted cells. The
irregularities here noted are more common in cultures
than in fresh exudates from acute cases, and more
common in old than in young cultures. As seen in
cultures, it is commonly arranged in pairs with the
individuals flattened at the surfaces of juxtaposition.
Sometimes it is seen grouped as four and occasionally as
short chains of three or four cells, but never as long
chains. Its size is that of the common pyogenic micro-
cocci, and its outline and arrangement in the pus-cells
are so like those of the gonococcus that the figure depict-
ing gonorrhceal pus answers equally well to illustrate the
appearance of the exudate from acute meningitis.

While a facultative saprophyte, still its parasitic
nature is so marked that it can only be cultivated with
some trouble and uncertainty. The most satisfactory
medium for its isolation in pure culture from the dis-
eased meninges is coagulated blood-serum (Loffler's
mixture), and even here one is not successful with each
attempt. So uncertain is its growth under artificial
conditions that it is always advisable to inoculate a
number of tubes with relatively large quantities of the
exudate, and even then growth often occurs in only a

MICROCOCCUS INTRACELLULARIS. 305

part of them, notwithstanding the fact that on micro-
scopic examination the organism may have been readily
detected in large numbers in the exudate. Illustrative
of this difficulty, the following experience of Council-
man, Mallory, and Wright may properly be quoted:1

"As showing the difficulty in growing the organisms
in cultures made from the meninges at the post-mortem
examination, ten cultures were made in one case from
the exudation on the brain and six from the cord, cover-
slip examinations showing abundant organisms in the
cells. Only two of the cultures from the brain and one
from the cord showed a growth. As a rule, the organ-
isms were more easily obtained in cultures made from
the acute cases than from the chronic."

When successfully isolated in pure culture its growth
is never profuse on any medium. On the serum mixt-
ure of Loffler the isolated colonies appear as round,
viscid, smooth, sharply defined points that may attain
a diameter of,,! to 1.5mm. There is no liquefaction
of the medium. Cultures from very acute cases occa-
sionally present an abundant growth of fine, transparent
colonies strongly suggestive of those of micrococcus
lanceolatus.

On glycerin-agar the colonies are round, pearly, trans-
lucent, flat, and viscid in appearance. They tend to
become confluent. Under low magnifying power they
are homogeneous, sem {transparent, faintly brownish,
with well-defined smooth margins. On plain agar the
growth is feeble and uncertain.

Its growth in bouillon is slow and uncertain. It does
not cause clouding of the fluid, but collects at the bottom

1 See " Epidemic Cerebrospinal Meningitis," etc., Report of the State
Board of Health, Mass., 1898, by Councilman, Mallory, and W'ight.
20

306 BA CTERIOLOG Y.

of the tube as a scanty grayish sediment, that when dis-
turbed gives the impression of having a mucoid con-
sistency.

It does not grow on potato and causes no change in
litmus-milk.

It grows only at the temperature of the body, and
can be kept growing only by being transplanted to fresh
media about every two days, and even then growth
often ceases after a comparatively small number of trans-
plantations. If from a fresh growing culture a number
of tubes be inoculated and kept under favorable condi-
tions, it is a common experience to have growth on only
a part of them. It is sometimes impossible to obtain
a second growth on agar-agar.

In addition to its presence in the meningeal exuda-
tion of epidemic cerebrospinal meningitis, this organism
may appear as a secondary invader of the lung, causing
more or less extensive pneumonic exudation ; of the
joints; the ear; the eye; and the nose and throat.
Though rarely, its presence in the circulating blood may
sometimes be demonstrated (Gwynn).

By none of the ordinary methods of inoculation can
the disease be reproduced in animals. Subcutaneous
inoculation with pure cultures has no effect. Injections
into the great serous cavities may or may not result in
serofibrinous or fibrinopurulent inflammation. Intra-
venous inoculations are equally unsatisfactory.

The only successful attempts to reproduce the morbid
conditions from which the organism is obtained are
those in which the living cultures have been injected
directly into the meninges. Weichselbaum produced
congestion with pus formation in the meninges of dogs
and rabbits by direct injection through openings made

MICROCOCCUS INTRACELLULARIS. 307

in the skulls; and Councilman, Mallory, and Wright
caused the death of a goat by the injection into the
spinal canal of 1 c.c. of a bouillon suspension of a pure
culture of the organism. The autopsy revealed intense
congestion of the meninges of both brain and cord, with
slight clouding of the meninges and slight increase of
meningeal fluid. Microscopically there was deep injec-
tion of the vessels of the cerebral meninges, accompanied
by an exudation composed principally of pus-cells.
There was very little fibrin and only small numbers of
diplococci in the pus-cells. The purulent infiltration
extended along the vessels into the cerebral cortex.
The morbid condition was less marked in the cord than
in the brain. The micrococci were recovered in pure
culture both from cord and brain.

While the portal of entry for this organism to the
system is not known, it is still of importance to note
that it often makes its exit from the body by way of the
organs that are secondarily involved, and that open to
without, as the eai'j nose, eye, and lungs.

It is of equal importance to note that the organism is
of very low power of resistance, being destroyed in
twenty-four hours by direct sunlight and by drying at
body-temperature, and in seventy-two hours by drying
in the dark at ordinary room-temperature.

For the diagnosis during life of epidemic cerebrospinal
meningitis by bacteriological methods it is desirable that
the meningeal fluid be obtained during the most acute
stage of the disease. This is best done by the operation
of lumbar puncture, a de.scription of which, as given by
Mallory and Wright, is as follows :

" The operation and the subsequent examination of
the fluid should be as carefully performed as any other

308 BA CTERIOLOG Y.

bacteriological investigation, in order to obtain accurate
results. The back of the patient and the operator's
hands should be made sterile. The needle should be
boiled for ten minutes. The patient should lie on the
right side, with the knees drawn up, and with the
uppermost shoulder so depressed as to present the
spinal column to the operator. This position permits
the operator to thrust the needle directly forward rather
than from side to side. An antitoxin needle, 4 cm. in
length, with a diameter of 1 mm., is well adapted for
infants and young children. A longer needle is neces-
sary for adults and children over ten years of age.

"Aspiration of the fluid is not necessary, but some
operators prefer to attach a hypodermic syringe to the
needle, to aiford a better grasp for the hand. In this
case the syringe would have to be detached to allow
the fluid to flow. The additional manipulation, and
possibly the defective sterilization of the syringe,
might impair the subsequent bacteriological examina-
tion.

" The puncture is generally made between the third
and the fourth lumbar vertebrae, sometimes between the
second and third. The thumb of the left hand is
pressed between the spinous processes, and the point of
the needle is entered about 1 cm. to the right of the
median line. Care must be exercised to prevent the
point of the needle from passing to the left of the
median line and striking the bone. At a depth of 3 or
4 cm. in children and 7 or 8 cm. in adults the needle
enters the subarachnoid space, and the fluid flows usually
by drops. If the point of the needle meets with a bony
obstruction, it is advisable to withdraw the needle some-
what, and to thrust again, directing the point of the

PSEUDOMONAS ^RUGINOSA. 309

needle toward the median line, rather than to make
lateral movements, with the danger of breaking the
needle or causing a hemorrhage. The smallest quantity
of blood obscures the macroscopic appearance of the
fluid by rendering it cloudy. The fluid is allowed to
drop into an absolutely clean test-tube, which previously
has been sterilized by dry heat to 150° C. and stop-
pered with cotton. The fluid should be allowed to
drop into the tube without running down the sides.
From 5 to 15 c.c. of fluid is a sufficient quantity for
examination." l

PSEUDOMONAS JERUGINOSA (SCHROTER, 1872),
MIGULA, 1900.

Synonyms : Bacterium seruginosum, Schroter, 1872 ; Bacillus aeru-
ginosus, Schroter, 1872; Bacillus pyocyaneus, Gessard, 1882; Pseudo-
iiionas pyocyanea, Migula, 1896.

Another common organism that may properly be
mentioned at this place, though perhaps not strictly
pyogenic, is a pseudomouas frequently found in dis-
charges from wounds, viz., pseudomonas ceruffinosa, or
" bacillus of green pus," or of blue pus, or of blue-green
pus, as it is variously designated. Pseudomonas ceru-
ginosa is a delicate rod with rounded or pointed ends.
It is actively motile ; does not form spores. As seen
in preparations made from cultures, it is commonly clus-
tered in irregular masses. It does not form long fila-
ments, there being rarely more than four joined end to
end, and most frequently occurs as single cells.

It grows readily on all artificial media, and gives to

1 For a comprehensive treatment of this subject from its etiological
and pathological standpoints, see the monograph of Councilman, Mai-
lory, and Wright, to which reference was made ahove, and to which
we are indebted for much contained in the foregoing sketch.

310

BACTERIOLOGY.

some of them a bright-green color that is most conspic-
uous where it is in contact with the air. This green
color, which becomes more and more marked as growth
advances, is not seen in the growth itself to any extent,
but is diffused through the medium on which the organ-
ism is developing. Ultimately 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).

FIG. 58.

Colony of ps. aeruginosa after twenty-four hours
on gelatin at 20°-22° C.

FIG. 60.

Colony of ps. smiginosa after forty-two hours on
gelatin at 20°-22° C.

. — To a fresh agar culture of this organism, in
which the green coloration of the medium is especially
marked, add about 2 c.c. of chloroform. Shake gently, and

Stab-cnlture of
ps. aeruginosa in gel-
atin after twenty-
eight hours at 22° C.

PSEUDOMONAS sERUQINOSA. 311

note that the chloroform extracts a blue coloring-matter
from the culture, leaving the latter more or less yellow.

Its growth on gelatin in stab-cultures is accompanied
by liquefaction and the diffusion of a bright-green color
throughout the surrounding unliquefied medium. As
liquefaction continues, and the whole of the gelatin
ultimately becomes fluid, the green color is confined
to the superficial layers in contact with the air. The
form taken by the liquefying portion of the gelatin in
the earliest stages of development is somewhat that of
an irregular slender funnel. (See Fig. 58.)

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. 59.) As growth progresses and liquefaction
becomes more advanced the central mass of the colony
sinks into the liquid, 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. 60.

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 into blue-green, and finally turns almost black.

On potato the growth is brownish, dry, and slightly

312 B A GTERIOLOG Y.

elevated above the surface. In some cultures the
potato about the line of growth becomes green ; in
others this change is not so noticeable. With many
cultures a peculiar phenomenon, consisting of a change
of color from brown to green, may be produced by
lightly touching the growth with a sterile platinum
needle. The change occurs only at the point touched.
It is best seen in cultures that have been kept in the
incubator for from seventy-two to ninety-six hours. It
occurs in from one to three minutes after touching with
the needle, and may last from ten minutes to half an
hour. This is the " chameleon phenomenon " of Paul
Ernst.

In bouillon the green color appears, and the growth
is seen in the form of delicate flocculi. A very delicate
mycoderrna is also produced. As growth progresses,
the bouillon becomes darker and darker in color, until
it finally is about comparable in this respect with crude
petroleum ; at the same time it assumes a peculiar ropi-
ness, and very old cultures (four to six weeks in the
incubator) may attain about the consistency of egg-
albumin. This is due to the production of a substance
closely allied, chemically speaking, to mucin. Whether
it is a metabolic product or one resulting from the
degeneration or the auto-digestion, so to speak, of the
bacteria, cannot now be said ; at all events, in cultures
presenting this peculiarity very few bacteria of normal
appearance — indeed, very few bacteria at all — are to be
seen on microscopic examination.

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

PSEUDOMONAS jERUGINOSA. 313

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 we observed the production of a distinct
blue color.

It produces indol.

It stains with the ordinary dyes, and its flagella may
readily be demonstrated by Loffler's method of staining.

It is an active producer of a proteolytic enzyme that,
may readily be separated and its digestive properties
observed by the following simple method : Prepare a
bouillon culture of about 70 to 80 c.c. volume, and
allow it to grow at 37° to 38° C. for four or five days.
Filter through a Berkfeld filter into a sterile receiver.
Under aseptic precautions decant the filtrate into sterile
test-tubes, about 7 c.c. to each tube. Then under aseptic
precautions make the following tests : To one tube add a
small bit of hard-boiled egg (about one-half the size of
a pea) and place in an incubator. Render another tube
slightly acid with dilute hydrochloric acid, and add a
bit of the white of egg to it also. Do the results differ?

Heat another tube to 80° C. for fifteen minutes, and
repeat the experiment. Has the heating had any effect?

To another tube add carbolic acid to the extent of 2
or 3 per cent. Is the digestive activity of the solution
modified ?

To two ordinary tubes of gelatin add carbolic acid
until it is present to the extent of 0.25 per cent, in each
tube. Solidify the gelatin in one tube in the upright

314 BACTERIOLOGY.

position ; lot that in the other remain fluid. On the
surface of the former pour 0.5 c.c. of the pyocyaneus
filtrate, and mark the point of contact between the
gelatin and filtrate. To the other tube add a similar
amount of the filtrate, mix thoroughly, and solidify in
a glass of cold water.

At the end of eighteen to twenty hours note result.
Is it possible to solidify again the gelatin through which
the filtrate was mixed, by placing the tube in cold water?

Do the activities of this enzyme suggest those of any
of the enzymes encountered in the animal body ?
Which? and Why?

INOCULATION INTO ANIMALS. — As a rule, cultures
of this organism obtained directly from the discharges
of the wound are capable, when introduced into ani-
mals, of producing diseased conditions ; but cultures
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 are
found an extensive purulent infiltration of the tissues
and a marked zone of inflammatory oedema.

When introduced directly into the peritoneal cavity
the results are also fatal, and at autopsy a genuine
fibrinous peritonitis is found. There is usually an 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 pure cultures in
the blood and internal viscera.

When animals are inoculated with small doses (less
than 1 c.c. of a bouillon culture) of this organism death

PSEUDOMONAS jERUGINOSA. 315

0

may not ensue, and only a local inflammatory reaction
(abscess-formation) may be set up. In these cases the
animals are usually protected against subsequent inocu-
lation with doses that would otherwise prove fatal.

Most interesting in connection with pseudomonas
ceruginosa is the fact, as brought out in the experiments
of Bouchard, and of Charrin and others, that its prod-
ucts possess the power of counteracting the pathogenic
activities of bacterium anthrads. That is to say, if an
animal be inoculated with a virulent anthrax culture,
and soon after be inoculated with a culture of pseudo-
monas wruginosa, the fatal effects of the former inocu-
lation may be prevented. Emmerich and Low ' are
inclined to attribute this to the direct bacteriolytic action
of the enzymes upon the anthrax bacteria introduced
into the tissues.

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.

BACILLUS PESTIS, YERSIN, 1894.
THE BACILLUS OF BUBONIC PLAGUE.

Before passing from the subject of suppuration it
may not be inappropriate to call attention to the light
that modern methods of investigation have shed upon
the etiology of bubonic plague, an epidemic disease

1 Munchener mcd. Wochenschrift, 1898, No. 40 ; Ontralblatt fur
Bakteriologie und Parasitenkundc, 1899, Abt. i. No. 1, p. 33.

316 BACTERIOLOGY.

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 epi-
demics and pandemics of plague have made their appear-
ance in Europe at different times. During and for a time
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 variously
known as the " Justinian Plague " of the sixth century,
the " Black Death " of the fourteenth century, and the
" Great Plague of London " of the seventeenth century,
though it is difficult to say to what extent these pesti-
lences were uncomplicated manifestations of genuine
bubonic plague. During the existence of the Justinian
Plague 10,000 people are said to have died in Con-
stantinople in a single day, and Hecker estimates 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.

The first and certainly the most exact information
up to date concerning the cause and pathology of the
plague resulted from the investigations of Yersin, of
Kitasato, and of Aoyama, conducted during the epi-
demic of 1894 in Hong-Kong, China; although since
then numerous other investigators have made addi-

THE BACILLUS OF BUBONIC PLAGUE. 317

tional important contributions to our knowledge of
the subject. The results of these studies demonstrate
that bubonic plague is an infectious, not markedly con-
tagious, disease that depends for its existence upon the
presence in the tissues of a specific micro-organism —
the so-called plague or pest bacillus.

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 aerobic, and
is non-motile. It is found in large numbers in suppu-
rating glands, and in much smaller numbers in the cir-
culating blood. (Fig. 61.)

It is demonstrable in cover-slip preparations made
from the pus and in sections of the glands by the ordi-
nary staining-methods.

Wilm l has found it by culture methods in the spleen,
lungs, liver, kidneys, stomach, walls of the intestine,
urine, and intestinal contents of fresh cadavers; and
during life in the blood, expectoration, faces, and urine
of patients sick of plague. He failed to find it in the
perspiration.

Yersin states that it does not retain the 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

1 Wilm: Hygicnische Rundschau, 1897, p. 217.

318 i:.\< Ti:ilIOLOGY.

attributed to individual peculiarities of different species
of bacteria that were under examination, an opinion
that is borne out by more recent studies, from which it
has been decided that the genuine plague or pest bacillus
does not stain by Gram's method.

It may be cultivated upon ordinary nutrient media,

Bacillus of bubonic plague: A, in pus from suppurating bubo; B, the
bacillus very much enlarged to show peculiar polar staining.

although preference is given by some to a neutral or
slightly alkaline 2 per cent, peptone solution containing
from 1 to 2 per cent, of gelatin.

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

'////•: ' BA CILLUS OF BUBONIC PLAGUE. 319

iridescent, Iran spa rent, and whitish. On gelatin at
18°-20° C. it develops as small, sharply defined, white
colonies without liquefaction of the medium. In stab-
cultures it develops both on the surface 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 shows but limited growth on potato. It does
not ferment glucose with production of gas, nor does it
form indol. It coagulates milk.

This organism is killed by drying at ordinary room-
temperature in four days. It is killed in three or 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.1

It is pathogenic for rats, mice, guinea-pigs, rabbits,
hogs, horses, monkeys, cats, chickens, and sparrows.
Pigeons, hedgehogs, and frogs are immune, and dogs
and bovines are apparently so.2 Animals succumb to
subcutaneous inoculation in from two to three days.
According to Yersin, the site of subcutaneous inocu-
lation becomes osdematous and the neighboring lym-
phatics are 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

1 Sou " Viability of the Bacillus Pestis," by M. J. Rosenau, U. S.
Marine-Hospital Service, Bulletin No. 4, of the Hygienic Laboratory,
U. S. M.-H., Washington, I). C., 1901.

2 Nuttall : Centralblat fiir Bakteriologie uud Parasiteiikunde, 1897,
Abt. i. Bd. xxii. S. 97.

320 BACTERIOLOGY.

swelling of the lymphatic glands, bloody extravasation
into the abdominal walls, serous effusion into the pleu-
ra 1 and peritoneal cavities ; the intestine is occasionally
hypersemic, the adrenal bodies congested, and the spleen
enlarged, often being studded with grayish points, sug-
gestive of miliary tubercles. The plague, or pest, ba-
cillus is detected in large numbers in the local oedema,
the lymph-glands, the blood, and the internal organs.

As is the case in general with the group of hemor-
rhagic septicaemia bacteria, the members of which it
resembles in certain other respects, 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 considera-
bly diminished.

Animals that survive inoculation with this organism
usually exhibit a certain degree of immunity from sub-
sequent infection.

Nuttall l notes that feeding-experiments have resulted
in fatal infection in gray and white rats, house- and
field-mice, guinea-pigs, rabbits, hogs, apes, cats, chick-
ens, sparrows, and flies. He also calls attention to the
fact that flies may live for several days after being in-
fected with this organism, and if at liberty to fly about
may manifestly infect persons or food-stuffs on which
they alight or fall.

The bacilli apparently lose their virulence after
long-continued cultivation under artificial conditions,
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
have been observed in different colonies from the same
1 Nuttall : loc. cit.

TIIK BACILLUS OF BUBONIC PLAGUE. 321

source. Virulence is said by Yersin, Calmctte, and
Borrel l to be accentuated by passing the organism
through a series of susceptible animals.

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, but among them are always the plague bacilli.
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 staphylo-
cocci that are present. Also, in this event, the strepto-
cocci retain the Gram stain, while the pest bacilli do not
and the staphylococci may or may not. It has been
suggested that possibly the organisms found by Kitasato
in the blood, and which he describes as pest bacilli,
that retained 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. It is also his
belief that the most important and frequent mode of
infection in man is through wounds of the skin. He
does not regard either the air-passages or the alimentary
tract as frequent portals of infection. "VVilm, on the
contrary, is inclined to regard the alimentary tract as a
frequent portal of infection ;2 and subsequent investiga-

1 Annales do I'lnstitut Pasteur, 1895, p. 589.

2 \Vilm : loc. cit.
21

322 BACTERIOLOGY.

tions leave little doubt that infection occasionally occurs
through the respiratory tract.

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-
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

\Vyssokowitz and Zabolotny2 call attention to the
fact that the blood of patients convalescing from plague
has an agglutinating action upon fluid cultures of the
plague bacillus analogous to that observed when the
blood-serum of typhoid or of cholera patients is mixed
with similar cultures of the typhoid or the cholera
bacillus.

Yersin, Calmette, and Borrel 3 have demonstrated that
the general principles underlying the establishment of
artificial immunity apply as well to this disease as to
a number of others. They have shown that by the use
of dead cultures (destroyed by heat) of the plague
bacillus animals may be rendered immune from infec-
tion by the virulent living organism. They have also
shown that the serum of the blood of these animals is
not only capable of conferring immunity upon other ani-
mals into which it is injected, but it has curative proper-

1 The works of Yersin, of Kitasato, aud of Aoyama have been ex-
haustively reviewed by Flexner in the Bulletin of the Johns Hopkins
Hospital, 1894, vol. v. p. 96, and 1896, vol. vii. p. 180. I am indebted
to these reviews for much that is here presented on this subject.

1 Annales de 1'Institut Pasteur, 1897, p. 663.

5 Ibid., 1895, p. 589.

ANTI-PLAGUE SERUM. 323

ties as well, providing it be employed at an early stage
of the disease. In 1896 Yersin1 used the serum of arti-
ficially immunized horses in the treatment of plague
in human beings. Of 26 persons (3 in Canton and 23
in Amoy, China) who received injections of the serum
during the early stages of the disease, in no case later
than the fifth day, only 2 died. Comparing this mor-
tality of 7.6 per cent, with the mortality of 80 per cent,
among persons in this epidemic who were treated in
other ways, he feels justified in regarding the method as
worthy of consideration.

ANTI-PLAGUE SERUM. — During recent years a great
deal of further investigation has been made in order to
ascertain the pathogenic properties of bacillus pestis,
and to obtain a more detailed knowledge of immunity
against infection by this organism. The studies of the
latter class include the estimation of the quantitative
value of the pest-serum when tested on different species
of animals, its protective and curative properties when
employed on such animals, especially its protective
properties against experimental inhalation pneumonia
and infection by feeding plague-infected materials.
These observations have been carried out by several
|x-t commissions — namely, those of Germany, Austria,
and Egypt — as well as the detailed investigations of the
Institutes for Infectious Diseases at Berlin, at Berne,
and the Pasteur Institute at Paris.2 The preparation
of the pest-serum is conducted as follows : Horses are
injected at first with dead cultures of bacillus pestis,
then with increasing doses of living agar cultures of the

1 Annales de 1'Institut Pasteur, 1897, p. 81.

* The important literature bearing on this subject is appended to
tin- report of Kolle, Hetsch, and Otto (Zeitechr. f. Hygiene, Bd. 38,
p. 368).

324 BA CTERIOLOG Y.

highly virulent bacillus. These organisms are injected at
intervals of from eight to twelve clays. By means of this
treatment a serum is obtained which is of limited cura-
tive value, but of very marked protective value. Poly-
valent serums do not seem to give any better results.
Kolle questions whether it will ever be possible to pre-
pare antitoxic pest-serum, because so far we are unable
to demonstrate a true pest-toxin.

The unsatisfactory results with serum therapy in
plague in a number of epidemics cannot be attributed,
according to the investigations of Hetsch and Rimpau,
to the absence of specific amboceptors in the pest-serum
for bacillus pestis. Even in the earlier investigations
on the estimation of the value of the pest-serum, Kolle
was lead to doubt whether the serum conformed exactly
with the laws of bactericidal serums ; in other words,
whether the pest-serum acted as a purely bactericidal
serum or not. It is true that some bacteriolytic action
can be demonstrated in the pest-serum ; but Markl was
able to show that with virulent cultures the leucocytes
play a most important part in the destruction of the
bacteria when injected into the peritoneal cavity of an
animal. Kolle concludes, therefore, that the pest-serum
is neither a purely antitoxic serum, like that of diph-
theria and tetanus, nor a purely bactericidal serum, like
that of cholera and typhoid, but that its action rests
probably much more upon substances the biological
character of which is as yet undetermined, in addition
to some bactericidal action. In this respect it corre-
sponds somewhat with the anthrax serum, as shown by
the experiments of Sobernheim. For this reason Kolle
calls the pest-serum an " anti-infectious " serum, as he
believes that by this designation its biological action is

THE HAFFKINE VACCINE AGAINST PLAGUE. 325

expressed more definitely than by the term bactericidal
serum.

THE HAFFKINE VACCINE AGAINST PLAGUE. — A
great deal of work has also been done in recent years
in conferring active immunity against bacillus pestis by
the Haffkine method — that is, by the injection of dead
cultures of the organism, and also by the injection of
organisms of low degree of virulence, whereby an active
immunity is conferred. Especially satisfactory immuniz-
ing results have been obtained by combining the vacci-
nation with dead cultures or cultures of low virulence
with the administration of the immune serum. While
the vaccination is entirely harmless for animals suscep-
tible to bacillus pestis, even in large doses, the experi-
ments on human beings could only be carried out in
combination with the administration of the immune
serum or after previous injection of dead cultures.

In 1897 Haffkine reported to the Indian government
that he had prepared an inoculation fluid for the protec-
tion of human beings against plague infection. This
fluid is now prepared by the Plague Research Labora-
tory in Bombay, and up to December, 1900, they had
distributed 1,628,696 doses. It has been found that
this fluid does not give the same degree of protection
against plague as does vaccinia against smallpox, but
the general results obtained in India indicate that the
individuals vaccinated with this material are not only
far less liable to infection by the plague organism, but
if they become infected they are more likely to recover.

Bannerman, of the Plague Research Laboratory in
Bombay, gives several instances of the value of the
inoculation as a protection against plague infection, and
cites the personnel of the Southern Mahratta Railway,
which were as follows :

326 BACTERIOLOG Y.

Of 990 persons inoculated twice, 6 contracted plague,
of which 1, or 0.1+ per cent, of those inoculated, died.

Of 270 persons inoculated once, 5 contracted plague,
and 1, or 0.3+ per cent, of those inoculated, died.

Of 760 persons not inoculated, 35 contracted plague,
and 21, or 2.7+ per cent, of those not inoculated, died.

These results indicate a reduction in the mortality of
94.1 per cent, in those who had been inoculated.

In Hubli there were, in the summer of 1898, 24,631
inoculated, compared with 17,786 uninoculated persons.
In that city the mortality from plague for the inoculated
was 89.6 per cent, lower than for the uninoculated, and
Bannerman states that in practically all instances a
reduction in mortality of about 90 per cent, is brought
about by the anti-plague vaccination.

Forsyth l reports on 30,609 cases in India that had
been vaccinated by the Haffkine method, of which
number 329 were subsequently attacked by plague, 50
of whom died, a case mortality of 15.1 per cent. He
gives a table showing the relative behavior of uninocu-
lated and inoculated persons toward plague.

No in OasMof Attack- Death- Case mor-

Classes. ^J" iSe rate P6* ***«». rate per tality per

(a) Uninoculated, 31,874 1457 4.5 659 2.06 4-V2
(6) Inoculated, 12,886 171 1.3 29 0.22 16.9

The anti-plague vaccine is administered in doses of
5 c.c. The reaction following the inoculation differs in
different individuals, especially with regard to the tem-
]K-rature. Haffkine recommends for immunizing pur-
poses the employment of smaller doses for the first
injection, and after the subsidence of the reaction the
use of a larger second injection. Even under these
conditions the reactions are sometimes quite marked.

1 Forsyth : The Lancet, vol. ii., 1903, p. 1646.

CHAPTER XVI.

Sputum septicaemia— Septicaemia resulting from the presence of sarcina
tetragena, or from bacterium pneumonia in the sputum of appar-
ently healthy persons — The occurrence of bacterium influenza
in the sputum.

IT is not infrequent that apparently healthy persons
harbor in the mouth cavity, nose, or throat a variety of
pathogenic organisms without manifesting any symptoms
of their presence. Some of these pathogenic organisms
may be readily detected by appropriate methods of cul-
tivation. This is especially true of the bacterium diph-
theriae, as will be shown in a subsequent chapter. Some
of the other pathogenic bacteria are not so readily isolated
by the cultural method, but may be demonstrated by ap-
propriate methods of staining. On staining cover-glass
preparations of sputum by the Gram method and counter-
staining with eosin it is often possible to detect bacterium
pneumonias and bacterium influenza? by reason of their
peculiar morphology and staining reactions.

The most satisfactory results, however, are obtained
by the subcutaneous or intravenous injection of the
sputum into guinea-pigs or rabbits. By this means the
non-pathogenic organisms are quickly eliminated and
the pathogenic organisms, if present, produce their
characteristic lesions. Probably the most frequent re-
sult of such inoculation of sputum is the production of
a general septicaemia.1

Obtain from a tuberculous patient a sample of fresh

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

327

328 BACTERIOLOGY.

sputum — that of the morning is preferable. Spread
it in a thin layer upon a black glass plate and select
one of the small, white, cheesy masses or dense mu-
cous clumps scattered through it. With a pointed
forceps smear this carefully upon two or three thin
cover-slips, dry and fix them in the way given for
ordinary cover-slip preparations. Stain one in the
ordinary way with Loffler's alkaline methylene-blue
solution, one other by the Gram method, and a third
after the method given for bacterium tuberculosis in
fluids or sputum.

In that stained by Loffler's method — slip No. 1 —
will be seen a great variety of organisms — round cells,
ovals, short and long rods, perhaps spiral forms. But
not infrequently will be seen diplococci having more
or less of a lancet shape, 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 by
their arrangement into groups of fours, the adjacent
surfaces being somewhat flattened. They are not sar-
cinse, as one can see by the absence of the division in
the third direction of space — they divide in only two
directions.

In the slip stained by the Gram method the same
groups of cocci which grow as threes and fours
will be seen; but the lancet-shaped diplococci now
present an altered appearance — they are usually sur-
rounded by a capsule. 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. (Fig. 63.)

SPUTUM SEPTIC JEMI A. 329

In the third slip, which has been stained by the
method given for tubercle bacteria in sputum, if decolor-
ization 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 instances, a conspicuous beading of their pro-
toplasm— that is, the ' staining is not homogeneous,
but at tolerably regular intervals along each rod are
seen alternating stained and unstained points. These
rods may be found singly, in groups of twos and threes,
and sometimes in clumps consisting of large numbers.
When in twos or threes it is not uncommon to find
them describing an X or a V in their mode of arrange-
ment, or again they may 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. 62.)

This delicate, beaded rod is bacterium tuberculosis.
The lancet-shaped diplococcus with the capsule is bac-
terium pneumonice. The cocci grouped in fours are
sarcina tdragena.

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 ensue, it will, in

330

BACTERIOLOGY.

all probability, be the result of one of the three follow-
ing forms of infection :

a. Septicaemia resulting from the introduction into
the tissues of bacterium pneumonice.

b. A form of septicaemia resulting from the introduc-
tion of sarcina tetragena, an organism frequently seen in
the sputum of tuberculous subjects.

c. Local or general tuberculosis.

FIG. 62.

Tuberculous sputum stained by Ciabbett's method. Tubercle bacteria seen
as red rods ; all else is stained blue.

SPUTUM SEPTICAEMIA.

BACTERIUM PNEUMONIA (WEICHSELBAUM), MIGULA,
1900.

Synonyms: Diplococcus pueumonise, Weichselbaum, 1886; Pneumo-
coccus, Friinkel, 1886 ; Micrococcus of sputum septicsemia ; Diplococcus
lanceolatus ; Streptococcus lauccolatus ; Streptococcus pasteuri ; Micro-
coccus lauceolatus.

If at the end of twenty-four to thirty-six hours the
animal be found dead, we may reasonably predict that
the result was produced by the introduction into the tis-
sues of the organism of sputum septicaemia above men-
tioned, viz., bacterium pneumonice, which is not uncom-

BACTERIUM PNEUMONIA. 331

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.

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. 63.) This, however, is not constant.

1 1' a drop of blood from the dead animal be intro-
duced into the tissues of a second animal (mouse or
rabbit), identically the same conditions will be repro-
duced.

If the organism be isolated in pure culture from the
blood of the animal, and a portion of this culture be
introduced into the tissues of a susceptible animal,
we shall see again the same pathological picture.

It must be remembered, however, that this or-

1 Welch : Johns Hopkins Hospital Bulletin, December, 1892, vol. iii.
No. 27.

332 BA CTERIOL OGY.

ganism when cultivated for a time on artificial media
rapidly loses its pathogenic properties. If, therefore,
failure to reproduce the disease after inoculation
with old cultures should occur, it is in all probability
due to a loss of virulence of the organism.

FIG. 63.

Bacterium pneumonia; in blood of rabbit. Stained by method of Gram.
Decolorization not complete.

This organism was discovered by Sternberg in 1880.
It was subsequently described by A. Frankel as the
etiplogical 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, and certain authors are of the opinion that it
occurs in the oral or nasal cavities of all individuals
at various times during life. It is constantly to be
detected in the rusty sputum of patients suffering from
acute fibrinous pneumonia. Its presence has been de-
tected in the middle ear, in the pericardial sac, in the
pleura, and in the serous cavities of the brain ; and

BACTERIUM PNEUMONIA. 333

indeed it may penetrate from its usual site of develop-
ment in the mouth to 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 may be detected. (Fig. 63.) 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 at the
broad ends of the ovals, never at the pointed extremities.
When in chains only the terminal cells are pointed, and
then at their distal 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 it can, according to some
authors, be detected ; but this is by no means constant,
or even frequent.

Even under the most favorable artificial conditions
this organism grows but slowly and frequently not at
all.

When successfully grown upon the different media it
presents somewhat the following appearances :

On gelatin its development is very limited and often
no growth at all occurs. This is probably due in part
to the low temperature at which gelatin cultures must
be kept. If development occurs, the growth appears
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

334 BA CTERIOLOGY.

surface of the gelatin. They do not cause liquefaction
of the gelatin.

If grown in slant- or stab-cultures, the surface-devel-
opment is very limited ; along the needle-track tiny
whitish or bluish-white granules appear.

On nutrient agar-agar the colonies are almost trans-
parent, more or less glistening, and very delicate in
structure. On blood-serum development is more
marked, though still extremely feeble, appearing as
a cluster of isolated fine points growing closely side by
side.

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.1

It is not motile.

It grows best at a temperature of from 35° to 38° C.
Below 24° C. there is usually no development, but in a
few cases it has been seen to grow at as low a tempera-
ture as 18° C. Above 42° C. development is checked.

It grows as well without as with oxygen. It is
therefore one of the facultative anaerobic forms.

Cultivation of this organism is most successful 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 re-
sults. (See Stainings.)

This organism is conspicuous for the irregularity of
its behavior when grown under artificial conditions :

1 Welch : loc. cit.

BACTERIUM PNEUMONIA. 335

usually it loses its pathogenic properties after a few
generations ; but again this peculiarity may be retained
for a much longer time. Not rarely it fails to grow
after three or four transplantations on artificial media,
though at times it may be carried through many gen-
erations.

INOCULATION INTO ANIMALS. — The results of inocu-
lations with pure cultures of this organism are also con-
spicuous for their irregularity. When the organism is
of full virulence the form of septicaemia 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 every variation in
the intensity of their disease-producing properties.
The principal pathological conditions that may be pro-
duced by the inoculation of susceptible animals with
this organism are, according to the degree of its viru-
lence, acute septicaemia, spreading inflammatory exuda-
tions, and circumscribed abscesses. All three of these
conditions may sometimes be produced by inoculating
rabbits with the same cultures 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 the
same culture may sometimes 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 septicaemia ; while subcutaneous inoculation of the

336 BACTERIOLOGY.

same material may result in only a localized inflamma-
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.

ANTIPNEUMONIC SERUM. — The recent experiments
of Panichi1 on the serum therapy of pneumonia are of
great interest. In order to obtain a high grade of anti-
pneumonic serum, he first perfected a special culture
medium (a special bouillon) in which the organisms
produced their specific toxin outside the body. Cultures
grown in this special medium killed rabbits acutely with
moderate toxic action, while ordinary blood-cultures
killed by inducing septicaemia.

In testing the curative properties of his antipneumonic
serum, Panichi injected rabbits subcutaneously with 0.2
c.c. of his virulent culture, and subsequently a dose of
serum was injected into the ear veins, in proportion to
the body-weight of the animal. A dose of 0.25 per cent,
of the body-weight sufficed to save the animal if adminis-
tered not later than the elapse of five-sixths of the dura-
tion of the disease, while the control-animals died in
from twenty to fifty-six hours. If the curative dose of
serum is delayed longer, 2 per cent, of the body-weight
is required instead of only 0.25 per cent. Panichi con-
1 Panichi : Cent. f. Bact., Bd. xxxv., Referat.

SARCINA TETRAGENA. 337

eludes that the serum does not possess any bactericidal
power, but that it acts through its antitoxic properties.

Panichi employed his serum in the treatment of seven
cases of pneumonia. The serum was injected intrave-
nously, and was followed by lysis — that is, there was a
reduction of the temperature, of the pulse, and of the
respiration, as well as improvement in the general ap-
pearance of the patient. The bright red exudate be-
came rusty and, later, catarrhal in character. Diminished
crepitation was noticed when a sufficient dose had been
reached (15 to 30 c.c.). Of the seven cases, six expe-
rienced a helpful action. In one the result was negative.
In the latter the treatment was undertaken at five-
sevenths of the total duration of the disease. The treat-
ment of human beings corresponded entirely with the
results of the experimental study of the serum in ani-
mals, in the quantity of serum required for treatment, as
well as in the time period within which the injection was
of value.

INFECTION WITH SARCINA TETRAGENA (GAFFKY),
MIGULA, 1900.

Synonym : Micrococcus tetragenus, Gaffky, 1883.

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, it
may result from the invasion of the tissues by the organ-
ism now to be described, viz., sarcina tetragena.

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

22

338 BACTERIOLOGY.

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 tuberculosis.

It is a small round coccus of about 1 ft 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 bound together
by a transparent gelatinous mass.

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-
tion a circumscribed white point, slightly elevated above
the surface and limited to the immediate neighborhood
.of the point of inoculation. Down the needle-track the
growth is not continuous, but appears in isolated, round,
dense white clumps or beads, which do not develop
beyond very small points.

It does not liquefy gelatin.

Upon plates of nutrient agar-agarthe colonies appear

SARCINA TETRAGENA. 339

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 transparent mucilaginous 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 the mucoid material.

The organism grows best at from 35° to 38° C., but
can be cultivated at the ordinary room-temperature —
about 20° C.

The growth under all conditions is slow.

It grows both in the presence of and without oxygen.

It is not motile.

It stains readily with all the ordinary aniline dyes.

In tissues its presence is readily demonstrated by the
stabling-method of Gram.

The grouping into fours is particularly well seen in
sections from the organs <>i' animals dead of this form

340 BACTERIOLOGY.

of septicaemia. In such sections the organisms will
always be found within the capillaries.

INOCULATION INTO ANIMALS. — To the naked eye
no alteration can be seen in the organs of animals that
have died as a result of inoculation with sarcina
tetragena ; 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 septicaemia. 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 this disease, results in a re-
production of the conditions found in the dead animal
from which the tissues or cultures were obtained.

It sometimes happens that in guinea-pigs which have
been inoculated with this organism local pus-formations
result, instead of a general septicaemia. The organisms
will then be found in the pus-cavity.

BACTERIUM INFLUENZA (R. PFEIFFER) LEHMANN
AND NEUMANN, 1896.

Synonym : Influenza bacillus, R. Pfeiffer, 1892.

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-

BACTERIUM INFLUENZA. 341

domic 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 pan-
demic of 1889-'90, the most severe for a long time,
appears to have originated in Central Asia and to have
spread pretty much over the entire civilized world. The
advent of influenza in a community is always remark-
able for its astonishing rate of transmission from per-
son to person and its dissemination over wide areas.

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.

Auerbach l examined over 700 cultures prepared from
cases of diphtheria and scarlet fever for the presence
of the influenza bacillus, and had 38 positive results, of
which 12 were from cases of diphtheria, 3 from scarlet
fever, 6 from diphtheria and scarlet fever, 7 from diph-
theria and measles, and 10 from suspicious diphtheria
angina. He employed the culture, and also the staining,
methods on pigeon-blood agar, the latter consisting in
the staining of the preparations with the Gram-Weigert
stain, followed with a diluted carbol-glycerine-fuchsin
stain. By this staining method it was not at all difficult
to detect the presence of bacterium influenzas. It ap-
peared as thin reddish-violet rods in contrast to the dark
blue diphtheria organisms and streptococci and pneu-
mococci.

By appropriate methods of staining it is also fre-
quently possible to demonstrate the presence of this
1 Auerbach : Zeitschr. f. Hygiene, Bd. 47, 1904, p. 259.

342

BACTERIOLOGY.

organism in the secretions of the nose, mouth, and
throat of apparently healthy persons.

This organism, bacillus influenzce, as it is called, was
discovered, isolated, cultivated, and described by R.
Pfeiffer.

It is a very small, slender, non-spore-forming, non-

FIG. 64.

Bacterium influenza in sputum.

motile, aerobic bacillus, occurring singly and in pairs,
joined end to end. It stains with watery solutions of
the ordinary basic aniline dyes ; somewhat better with
alkaline methylene-blue, but best when treated for five
minutes with a dilution of Ziehl's carbol-fuchsiu in
water (the color of the solution should be pale red).
(Fig. 64.) It is decolorized by the method of Gram.

It develops only at temperatures ranging from 26°
to 43° 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

BACTERIUM INFLUENZA. 343

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
in sterile bouillon or water and rubbed over the sur-
face 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 cult-
ures 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 materials from a
case of influenza.

The organism may also be cultivated in bouillon to
which blood has been added, if kept at body-tempera-
ture. The growth appears as whitish flakes. Since this
organism is a strict ae'robe, its cultivation can only be
conducted on the surface of the medium used — i. e.y
where it has freest access to oxygen. It is therefore
inadvisable to prepare plates in the usual way. When
its cultivation is attempted in bouillon it is recom-

344 BA CTERIOLOG Y.

mended, in order to favor the free diffusion of oxygen,
that the depth of fluid be very shallow.

Contrary to what might be supposed, bacterium
influenzse has very little tenacity of life outside of the
diseased body. It is destroyed in from two to three
hours by rapid drying, and in from eight to twenty-
four hours when dried more slowly. Cultures retain
their vitality for from two to three weeks. The organ-
ism dies in water in a little over a day. As a 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 bacteria are found as
masses and as scattered cells. (See Fig. 64.) 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 bacteria within white blood-
corpuscles. They are also present in the nasal secre-
tions.

At autopsies it is advisable to cut out pieces of
the diseased tissue about the size of a pea or a bean,
break them up in a small quantity of sterile water or
bouillon, and make the cultures from this infusion.

BACTERIUM INFLUENZA. 345

By this procedure two advantages are gained : first,
a dilution of the number of bacteria present; and,
secondly, the tissue furnishes the amount of haemo-
globin 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 susceptible
to inoculation with this organism is the monkey. By
intratracheal injection Pfeiffer succeeded in causing a
toxic condition that proved fatal. He does not regard
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 XVII.

Tuberculosis — Microscopic appearance of miliary tubercles — Diffuse
caseation — Cavity-formation — Encapsulation of tuberculous foci —
Primary infection — Modes of infection — Location of the bacilli in
the tissues — Staining-peculiarities — Organisms with which bac-
terium tuberculosis may be confounded: bacterium lepne ; bacterium
smegmatis — Points of differentiation — Acid-proof bacteria — Actino-
myces — Actinomyces bovis, Actinomyces Israeli, Actinomyces madnne,
Actinomyces farcinicus, Actinomyces Eppingeri, Actinomyces pseudo-
tuberculosis.

BACTERIUM TUBERCULOSIS (KOCH), MIGULA, 1900.
Synonym : Bacillus tuberculosis, Koch, 1882.

LOCAL OR GENERAL TUBERCULOSIS. — Should the
animal succumb to neither of the septic processes just
described, then its death from tuberculosis may reason-
ably be expected.

When this disease is in progress alterations in the
lymphatic glands nearest the site of inoculation may
be detected by the touch in from two to four weeks.
They will then be found enlarged. Though not con-
stant, tumefaction and subsequent ulceration at the
point of inoculation may be observed. Progressive
emaciation, loss of appetite, and difficulty in respiration
point to the existence of the general tuberculous process.
Death ensues in from four to eight weeks after inocula-
tion. At autopsy either general or local tuberculosis
may be found. The expressions of tuberculosis are so
manifold and in different animals vary so widely the
one from the other, that no fixed law as to what will
appear at autopsy can d. priori be laid down.

The guinea-pig, which is best suited for this experi-
ment because its susceptibility to tuberculosis is greater
and more constant than that of other animals usually

346

LOCAL OR GENERAL TUBERCULOSIS. 347

found in the laboratory, presents, in the main, changes
that are characterized by coagulation-necrosis and case-
ation. This is particularly the case when the infec-
tion is general — i. e.} when the process is of the acute
miliary type ; then the tissues of the liver and spleen
present the most favorable field for the study of this
pathological-anatomical alteration.

In general, the tubercular lesions can be divided into
those of strictly focal character — L e., the miliary and
the conglomerate tubercles — and those which are more
diffuse. The latter lesions, although fundamentally
of the same nature as the miliary tubercles, are much
greater in extent and not so sharply circumscribed.
These latter lesions play a more conspicuous rdle in the
pathology 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 then present all the characteristics of the
tuberculous process in the stage of cheesy degeneration.
When the disease is more general the degree of its exten-
sion varies. Sometimes the small gray nodules — mili-
ary tubercles — are only to be seen with the naked eye in
the tissues of the liver and spleen. Again, they may in-
vade the lung, and frequently they are distributed over
the serous membranes of the intestines, the lungs, the
heart, and the brain. These 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"

348 BA CTERIOLOG Y.

are employed for the description of the microscopic
appearance of these nodules, yet it is very rarely that
any condition other than that due to the fusion 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 are located, and, as stated, vary
considerably in dimensions, usually appearing as points
which range in size from that of a pin-point to that of
a pin-head. They are not only located upon the surface
of the organs, but are distributed through the depths of
the tissues. To the touch they sometimes present noth-
ing characteristic, but when closely packed together in
large numbers they usually give a mealy or sandy sen-
sation to the hand passed over them. Stained sections
of 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 demonstration of tubercle bacilli
within the nodules.

MICROSCOPIC APPEARANCE OF MILIARY TUBER-
CLES.— A miliary tubercle under a low magnifying
power of the microscope presents somewhat the follow-
ing appearance : there is a central pale area, evidently
composed of necrotic tissue because of its incapacity
for taking up the nuclear stains commonly employed.
Scattered through this necrotic area may be seen granular
masses irregular in size and shape ; they take up the stains
employed and are evidently fragments of cell-nuclei in
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

LOCAL OR GENERAL TUBERCULOSIS. 349

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 crescentic. 'In 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 for-
mation, 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 tuberculous 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.

DIFFUSE CASEATION. — The diffuse caseation, as said,
plays a more important role in the tuberculous lesion,
both in the human and experimental forms, than does

350 S A CTERIOLOG Y.

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 certain tissues, notably the lungs and lymphatics,
it is more marked than in others. In rabbits, par-
ticularly, all the changes in the lung frequently come
under this head. When this is the case solid masses
are found, sometimes as large as a pea, or involving
even an entire lobe or the whole lung in some cases.
They are of a whitish-yellow, opaque color, and on sec-
tion are peculiarly dry and hard. Entire lymphatic
glands may be changed in this way. The conditions
which appear to be most favorable to the occurrence
of this widespread caseation of the tissues are the
simultaneous deposition of a large number of tubercle
bacilli in them, and the involvement of a wide area in-
stead of a single isolated point, as in the miliary tubercle.
Necrosis is so rapid that time does not suffice for those
reactive changes to take place in the tissues which result
in the formation of the outer zone of the miliary tubercle.
In other instances the entire caseous area is surrounded
by a granulation-zone similar to that around the caseous
centre of the miliary tubercles. It is of special im-
portance to recognize the etiological connection between
this diffuse caseation and the tubercle bacillus, because
until its nature was accurately determined 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, a
prominent feature in human tuberculosis, particularly
of the lungs, is due to softening of the necrotic,
caseous masses or of aggregations of miliary tuber-

LOCAL OR GENERAL TUBERCULOSIS. 351

cles. The material softens and is expelled, and a
cavity remains. In the wall of this cavity the tuber-
culous changes still proceed, both as diffuse caseation
and formation of miliary tubercles. The whole cavity
with the reactive changes in the tissues .of its walls
may be properly conceived as a single gigantic tuber-
cle, its wall forming a tissue very analogous to the outer
/one of the single tubercle, the cavity itself correspond-
ing 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
produce in rabbits pulmonary cavities in all physical
respects similar to those seen in the human being
has been beautifully demonstrated by Prudden. He
showed that when he had injected fluid cultures of strep-
tococcus pyogenes into the trachea of rabbits already af-
fected with tubercular consolidation of the lungs, 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.1

In the contents and in the walls of tubercular cavi-
ties in man bacteria other than bacillus tuberculosis 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; and it is also to the
absorption of their toxic products that some of the con-

1 Prudden : " Experimental Phthisis in Rabbits, with the Formation
of Cavities," etc., Transactions of the Association of American Physi-
cians, 1894, vol. ix. p. 166.

352 BA CTERIOLOG Y.

stitutional manifestations commonly seen in cases of
advanced pulmonary tuberculosis are attributed.

ENCAPSULATION OF TUBERCULAR Foci. — It not
uncommonly occurs that round about a necrotic tuber-
culous focus there is formed a fibrous capsule which may
completely shut off the diseased from the healthy tissue
surrounding it ; or a tuberculous focus may, through the
resistance of the tissue in which it is located, be more
or less completely isolated. In this condition the dis-
eased foci may lie dormant for a long time and give no
evidence of their existence, until they are caused to
break through their envelopes by some disturbing
cause. With the passage of the bacilli or their spores
from such a focus into the vascular or lymphatic cir-
culation the disease may 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 recurrence of tuberculosis.

PRIMARY INFECTION. — 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 their life-processes produce sub-
stances of a chemical nature which serve to bring
about characteristic changes in the immediate neighbor-
hood. In the beginning the fixed cells, particularly the
endothelial cells of the capillaries and lymph-spaces,
are stimulated to proliferation. With the onset of this

LOCAL OR GENERAL TUBERCULOSIS 353

phenomenon karyokinesis, evidence of cell multiplica-
tion may readily be detected in and about the affected
focus. As proliferation continues and as the local cir-
culation becomes more and more impaired, the centre of
the diseased area gradually assumes a condition of in-
activity, and ultimately presents all the characteristics
of dead and dying tissue. This death of tissue is one
of the earliest, the most easily recognized, and the most
characteristic results of tubercular infection, and may
usually be detected, in greater or less degree, even in
the youngest and most minute tubercles. With the pro-
duction of this progressive necrosis — for progressive it is,
as it 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 a granulation-zone at the
outer margins of the dying and dead tissue. This zone
consists of small, round granulation-cells and of leuco-
cytes, 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 proc-
ess 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.

MODES OF INFECTION. — 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
83

354 BACTERIOLOGY.

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 other than that seen in the glands them-
selves; or they may pass on to neighboring glands,
and eventually be disseminated throughout the lym-
phatic system, ultimately reaching the vascular system.

Having gained access to the bloodvessels the results
are the same as those following intravascular injection
of the bacilli, namely : general tuberculosis quickly
follows, with the production of miliary tubercles most
conspicuous 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. Co-
incident with their growth they are mechanically pressed
into the tissues of the lungs. As multiplication con-
tinues some are transported from the primary site of
infection to healthy portions of the lung-tissue, where,
through their development, the process is repeated.

In the same way infection by way of the alimentary
tract is in the main due to the bacilli being forced by
mechanical pressure into the walls of the intestines. In-
vestigation has shown that lesions of the intestinal coats
are not necessary for the entrance of tubercle bacilli

LOCAL OR GENERAL TUBERCULOSIS. 355

from the lumen of the intestines into the internal
organs and tissues. 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.

Unlike most pathogenic organisms, the tubercle ba-
cillus is believed to have the property of forming spores
within the tissues. These spores, which are presum-
ably 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, and which have the property of ex-
citing the disease in a certain number of persons who
inhale them. The frequency of pulmonary tuber-
culosis points to this as one of the commonest sources
and modes of infection in human beings. This opinion
is borne out by statistical studies upon the disease, as
well as by such evidence as Cornet * has produced upon
the infective nature of dust taken from apartments
occupied by persons suffering from pulmonary tuber-
culosis.

LOCATION OF THE BACILLI IN THE TISSUES. — The
bacilli will be found most numerous in those tissues
which are the seats of the active stage of the process.

In the initial stage of the disease the bacilli will
be fewer in number than later. At this time only
single rods may here and there be found ; later they
are more numerous ; and, finally, when the process
has advanced to a stage easily recognizable by the naked

1 Cornet: Zeitechrift fur Hygiene, 1889, Bd. v. S. 191.

356 BACTERIOLOGY.

eye, they are 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 and often from old caseous tissues must
not be taken, however, as evidence that these materials
are not infective. As bacilli, they are difficult to
demonstrate here because the probabilities are that at
this stage of the process, owing to conditions unfavor-
able to their further growth, they are in the spore-
stage, a stage in which it is as yet impossible, with
our present methods of staining, to render them visi-
ble. The facts that this tissue is infective, and that
with it the disease can be reproduced in susceptible
animals, speak for the accuracy of this assumption.
A conspicuous example of this condition is seen in
old scrofulous glands. These glands usually present
a slow process, are commonly caseous, and always
possess the property of producing the disease when
introduced into the 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,

BACTERIUM TUBERCULOSIS. 357

however, the distributiop 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,1 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 be obtained and cultivated artificially, and these
cultures are capable of again producing the disease
when further inoculated. Thus the postulates for-
mulated by Koch, which are necessary to prove the
etiological role of an organism in the production of a
malady, are fulfilled.

THE BACTERIUM TUBERCULOSIS.

Of the three pathogenic organisms liable to occur
in the sputum of a tuberculous subject, the tubercle
bacillus gives us the greatest 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 of growth in the animal tis-
sues only, 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-
1 The property of spore-formation is assumed, not proved.

358 BACTERIOL OG Y.

ficial cultures from the animal body are offsprings of
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.

For the cultivation of bacillus tuberculosis directly
from the tissues of the animal, the method by which one
obtains the best results is that recommended by Koch,
viz., cultivation upon blood-serum. Its parasitic ten-
dencies are so pronounced that even very slight variations
in the conditions under which one endeavors to isolate
bacillus tuberculosis from the tissues may cause total
failure. It is, therefore, necessary that the injunctions
for obtaining it in pure culture should be carefully
observed.

PREPARATION OF CULTURES FROM TISSUES. — Under
strictest antiseptic precautions remove from the animal
the diseased 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
upon the surface of the blood-serum, one nodule in
each tube, and without attempting to break it up or
smear it over the surface, leave it for four or five days
in the incubator. After this time it may be rubbed
over the surface of the serum. The object of this is to
give to the bacilli in the nodule an opportunity to
multiply, under the favorable conditions of temperature
and moisture, before an effort is made to distribute them
over the surface of the medium. 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 extraneous saprophytic

BACTERIUM TUBERCULOSIS. 359

organisms during the manipulation, while a few may
give the result desired, viz., a growth of the tubercle
bacillus itself.

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 to prevent evaporation and consequent dry-
ing. This is done by burning off the overhanging
cotton plug in a gas-flame, and then impregnating
the upper layers of the cotton with either sealing-
wax or paraffin of a high melting-point ; or by insert-
ing 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,
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 from 4 to 6 per cent,
of glycerin has been added. This is a very favorable

360 BACTERIOLOGY.

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 tuberculous 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 from 4 to 6 per cent, of
glycerin.

Cultures of the tubercle bacillus are characteristic
in appearance — once having seen them there is 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 coarse granular
masses. They are never moist, and frequently have the
appearance of dry meal 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 composing the growth adhere so
tenaciously together that it is with the greatest diffi-
culty they can be separated. In even the oldest and
dryest cultures pulverization is impossible. The masses
can only be separated and broken up by grinding in
a mortar with the addition of some foreign substance,
such as very fine, sterilized sand, dust, etc.

The cultures are of a dirty-drab or brownish-gray
color when seen on serum or glycerin-agar-agar.

On potato they grow in practically the same way,

BACTERIUM TUBERCULOSIS. 361

though the development is much more limited. On
this medium they are 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 patho-
genic properties.

On milk-agar-agar they are of so nearly the same
color as the medium that, unless they are growing as
characteristic mealy-looking masses, considerably ele-
vated above the surface, their presence is less conspicu-
ous than when on other media.

In bouillon they grow as a thin pellicle on the sur-
face. This may fall to the bottom of the fluid and con-
tinue to develop, its place on the surface being taken by
a second pellicle.

The tubercle bacillus does not develop on gelatin
because of the low temperature at which this medium
must be used.

MICROSCOPIC APPEARANCE OF BACTERIUM TUBER-
CULOSIS.— Microscopically the organism itself is a
delicate rod, usually somewhat beaded in its structure,
though rarely it is seen to be homogeneous. .It is either
quite straight, or somewhat curved or bent on its long
axis. In some preparations involution-forms, 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 length — sometimes being seen in very short
segments, again much longer, though never as long
threads. Usually its length varies from 2 to 5 p. It
is commonly described as being in length about one-
fourth to one-half the diameter of a red blood-corpuscle.
It is very slender. (See Fig. 63.)

These rods usually present, as has been said, an ap-

362 BACTERIOLOGY.

pearance of alternate stained and colorless portions.
The latter portions are believed to be the spores of
the organism, though as yet no incontestable proof of
this opinion has been presented. 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-PECULJARITTES. — 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 composition that
renders it proof against the simpler staining processes.
It is therefore necessary that more energetic and pene-
trating reagents than the ordinary watery solutions
should be employed. Experience has taught us that
certain substances not only increase the solubility of the
aniline dyes, but by their presence the penetration of
the coloring-agents is very much increased. Two of
these are aniline oil and carbolic acid. They are
employed in the solutions to about the point of satura-
tion. (For the exact proportions, see chapter on Stain-
ing-reagents.)

Under the influence of heat these solutions are seen
to stain all bacteria very intensely — the tubercle bacilli
as well as other forms. If we subject our prepara-
tion, which may contain a mixture of tubercle bacilli
and other species, to the action of decolorizing-agents,
another peculiarity of the tubercle bacillus will be
observed. AVhile all other organisms in the prepara-
tion 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

ORGANISMS RESEMBLING B. TUBERCULOSIS. 363

the color even under the action of strong decolorizing-
agents.

We have reviewed the three common pathogenic
organisms that may be encountered in the sputum of
tuberculous individuals. Occasionally other species may
be present. The pyogenic forms are not rarely found,
and for some time after an attack of diphtheria the
bacillus of Loffler is demonstrable in the pharynx, so
that it, too, may be present under exceptional circum-
stances.

ORGANISMS WITH WHICH BACTERIUM TUBERCULOSIS
MAY BE CONFUSED.

It is important to note that in the study of tubercu-
losis one may fall into error unless it be borne in mind
that there is a group of bacilli whose members are in
many respects so like the genuine bacillus tuberculosis
as easily to be mistaken for it. While its peculiar
micro-chemical reaction is usually sufficient for identifi-
cation, particularly in connection with human patho-
logical lesions, it is well to remember that the confusing
organisms are not only characterized by the same stain-
ing peculiarities as bacillus tuberculosis, but may readily
be mistaken for it on morphological grounds also.
Furthermore, while not all the members of this group
are capable of causing disease, some of them are patho-
genic for the same animals that are susceptible to true
tubercular infection ; and these may produce in those
animals lesions which are distinguishable from genuine
tubercles only by their finer histological structure. A
few words concerning some of these varieties, with a
brief summary of their more important peculiarities,
may not be out of place.

364 BACTERIOLOGY.

BACTERIUM LEPR.E. — Between 1879 and 1881 there
was described by Hansen and by Neisser an organism,
a bacillus, that was constantly to be found in the nodules,
characteristic of leprosy. For this organism the name ba-
cillus leprcB was suggested. Though very like bacterium
tuberculosis in both morphology and staining properties,
it is, however, a little shorter, thicker, and much less
homogeneously stained. Its presence in the tissues and
secretions is demonstrated by the same method as that
employed for detecting bacillus tuberculosis. In sec-
tions of leprous nodules, stained by the ordinary Koch-
Ehrlich process, the bacilli, crowded together in the
large so-called " lepra cells," are always to be seen in
great abundance. It is unlikely that bacillus leprse has
ever been cultivated artificially, and the disease has cer-
tainly never been reproduced in animals by inoculation
with bits of the diseased tissue, so that nothing can be
said of the life-history of this organism.

BACTERIUM SMEGMATIS. — In 1885 Alvarez and
Tavel discovered in the fatty secretions about the gen-
italia an organism that suggested the bacterium of
tuberculosis. It was found both in syphilitic and in
healthy persons. Their observation has been abundantly
confirmed by others, and the organism to which they
directed attention is now regarded as pretty commonly
present in the smegma. It is known, therefore, as the
smegma bacterium (bacterium smegmatis). In this
secretion it is found in clumps located upon or within
epithelial cells. It stains by the method used in staining
bacterium tuberculosis. It has no pathogenic po\uT.
It is said to have been artificially cultivated upon coag-
ulated hydrocele fluid and in milk.

In 1884 Lustgarten described an organism, the so-

THE ACID-PROOF BACTERIA. 365

/

called "bacillus of syphilis," that he had discovered in
primary syphilitic lesions and in the secretions from
syphilitic ulcers. In staining reactions, but more espe-
cially in morphology, this organism is said to be stri-
kingly like bacterium tuberculosis. He found it in the
tissues, usually within the bodies of large, apparently
swollen, cells. He found it not only in the primary sores
about the genitalia, but in the syphilitic lesions of the
remote organs as well. As this organism has never
been cultivated artificially, and as the majority of com-
petent observers, working upon the most promising
material, have failed to detect it, the prevailing opinion
is to the effect that the organism is not regularly asso-
ciated with syphilis and has nothing to do with its
causation.

It is not unlikely that bacterium smegmatis and bac-
terium syphilidis are identical.

THE ACID-PROOF BACTERIA. — In addition to the
species mentioned, quite a group of other "acid-proof"
bacteria, as they are called, have been described by dif-
ferent investigators. They are characterized by staining,
as does bacterium tuberculosis, by retaining the stain
to a greater or less extent when treated with acids and
alcohol, and by being in many instances strikingly like
bacterium tuberculosis in their morphology. The mem-
bers of this group seem to be distributed pretty widely
in nature. They have been detected in non-tuberculous
sputum, in gangrene of the lung, in the normal intestinal
contents of man and domestic animals, in the soil, in
fodder — i. e., grass, hay, and seed — in manure, and in
butter. They are not regularly found under any of these
conditions, and they are rarely present in very large

366 BA CTERIOLOG Y.

numbers. Inasmuch as they are occasionally encoun-
tered under circumstances that might lead one to look for
true tubercle bacilli, and since they possess certain pecu-
liarities through which it has been the custom to identify
bacillus tuberculosis — i. e., retention of the stain when
acted upon by acids or alcohol, and a more or less deli-
cate, beaded form — the possibility of their being con-
founded with that organism is obvious. In consequence
they have received a great deal of attention during the
past few years.

Space does not permit of a description of the twenty
odd species (?) that have been described by different in-
vestigators. It will suffice to say, from personal study
of the group, that in all probability not more than three }
perhaps only two, species are really represented, and
that the remainder may fairly be regarded as varieties.
As said, the characteristic common to all the mem-
bers of this group is that they are to greater or less
extent acid-proof — /. e., when once stained by the Koch-
Ehrlich or Ziehl process the color is not in all cases
removed by the ordinary acid decolorizers. In this par-
ticular, however, there is considerable variation. In
morphology some of them might readily be mistaken
for bacillus tuberculosis, though even these are usually
a trifle larger and less delicate than that organism ;
others are at once differentiated from normal tubercle
bacilli, but have somewhat the appearance of bacillus
tuberculosis when degenerated or involuted ; still others
have nothing in their general appearance to lead to con-
fusion.

When mixed with other bacteria, as is the case in the
soil, in manure, in intestinal contents, etc., their isola-
tion in pure culture is a matter of difficulty, and this is

THE ACID-PROOF BACTERIA. 367

by no means lessened by .the fact that under these cir-
cumstances they are always numerically in the minority.
When present in butter, their isolation offers fewer diffi-
culties, for by the injection of the butter containing
them into the peritoneal cavity of guinea-pigs conditions
are created that favor their development, and from ani-
mals so treated they may usually be recovered in pure
culture.

AVhen studied in pure culture, all of them are at once
distinguished from bacillus tuberculosis by the follow-
ing group characteristics : they are of relatively rapid
growth, there being usually an abundant development
on glycerin agar-agar after twenty-four to forty-eight
hours at body-temperature ; they grow well but less
rapidly at ordinary room-temperature — i. e., at 18° to
20° C. ; they grow well in litmus-milk, and, as a rule,
produce alkali that causes the color to become a deep
blue ; the growth on agar-agar is dry, shrivelled, and
wrinkled in appearance, and of a soft, mealy consistency
in some cases (Holler's grass bacillus II., Rabinowitsch
butter bacillus, for instance), while in others it is more
membranous, as in the case of Moller's timothy bacillus.
We have never seen in these cultures the hard, coarse
granules so common to cultures of bacillus tuberculosis ;
on glycerin agar-agar some of them, namely, the timothy
bacillus of MSller and its varieties, grow with a distinct
orange color, while others, the grass bacillus II. of
Moller, the butter bacillus of Rabinowitsch, and their
closely allied varieties, begin as a grayish-white deposit
which may ultimately become of a pale or even distinct
salmon color.

When pure cultures of them are injected into such
animals as rabbits or guinea-pigs, some of them have no

368 B A CTERIOL OG Y.

effect, and others cause lesions of more or less impor-
tance, the result being dependent upon the quantity em-
ployed and the mode of inoculation. By subcutaneous
or intraperitoneal injection of pure cultures the result is
usually negative. Occasionally the superficial lymphatic
glands in the neighborhood of the site of inoculation
may be inflamed and purulent. This we have seen only
with the subcutaneous inoculation. If pure cultures be
injected into the peritoneal cavity along with some
sterile, irritating substance, such as sterilized butter, a
widespread fibrinopurulent peritonitis is commonly the
result.

When injected directly into the circulation of rabbits,
the kidneys are almost uniformly affected, and in the
majority of instances they are, singularly enough, the
only organs in which lesions are to be detected. If, for
instance, a cubic centimetre of a carefully prepared
suspension in bouillon of, let us say, Moller's grass
bacillus II., be injected into the circulation of a rabbit,
and the animal be killed after twelve to fourteen days,
the kidneys will be found marked by gray or yellowish
points that range in size from that of a pin-point to
that of a pin-head. They are sometimes very few in
number, but in other cases both kidneys may be thickly
studded with them. Often they are not elevated above
the cortex of the organ, but in as many cases they are
sharply defined, yellow in color, and stand up promi-
nently from the cortical surface, being at the same time
so adherent to the capsule that the removal of the latter
tears them out bodily from the substance of the organ.
In the very early stages of development these nodules
are often difficult to distinguish from young tubercles, the
reaction of the tissues being, as in the case of tubercles,

THE ACID-PROOF BACTERIA. 369

characterized by proliferation of the fixed cells with
little evidence of leucocytic invasion ; later on, true
giant-cell formation is recorded by some observers. We
have not seen this. Clumps of endothelial nuclei or of
lymphoid cells that remotely suggest the arrangement
seen in giant cells are often encountered, but we have
not regarded them as true giant cells. When fully
developed, the nodule may present a mixed condition of
caseatioH and suppuration. The conditions, as a whole,
when advanced suggest a low grade of inflammatory
reaction. Occasionally nodules are encountered, espe-
cially in the kidney, that cannot be distinguished from
tubercles. The bacilli are always to be found within
the nodules ; most frequently as single rods or clumps
of rods, occasionally as rosette-like mycelia very sug-
gestive of the characteristic growth of the actinomyces
fungus in the tissues. This mode of development has
also been observed with bacillus tuberculosis.

It is important to note the difference between the re-
sults of intravenous inoculation of rabbits with bacillus
tuberculosis and with the organisms under consideration.
When bacillus tuberculosis is employed, the lungs, as
well as the kidneys, are always involved, while with the
grass bacillus II., the timothy bacillus, and the butter
bacillus, involvement of the lungs, in our experiments,
has been the exception rather than the rule.

Another point of interest is the lack of tendency on
the part of the non-tuberculous process to progress or
become disseminated.

That the members of this group are botanically
related to bacillus tuberculosis there seems little room
to doubt ; but from personal study and from available
evidence from other sources it appears unlikely that

24

370 BACTERIOLOGY.

they are, except experimentally, concerned in disease-
production or that they are of importance to either
human or animal pathology.1

In the microscopic examination, particularly of urine,
of secretions from about the anus, rectum, and genitalia,
and of butter, it is manifestly of importance to bear in
mind the existence of this confusing group, for it is in
such secretions and substances that they are most often
encountered. The smegma bacillus and the butter
bacillus are especially liable to lead one into error of
diagnosis. This is less apt to be the case with the com-
paratively rare lepra bacillus and the questionable
syphilis bacillus.

DIFFERENTIAL DIAGNOSIS. — According to Hueppe,
the differential diagnosis between bact. tuberculosis,
bact. smeginatis, and bact. leprse, depends upon the fol-
lowing reactions: when stained by the carbol-fuchsin
method commonly employed in staining the tubercle
bacillus the syphilis bacillus becomes almost instantly
decolorized by treatment with mineral acids, particularly
sulphuric acid, whereas the smegma bacillus resists such
treatment for a much longer time, and the lepra and
tubercle bacillus for a still longer time. On the other
hand, if decolonization 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 reac-
tions :

1For the literature on "acid-proof" bacilli, see Cowie, Journal of
Experimental Medicine, 1900, vol. v. p. 205.

DIFFERENTIAL DIAGNOSIS. 371

1. Treat the preparation, .stained with carbol-fuchsin,
with dilute sulphuric acid; the so-called syphilis bacillus be-
comes decolorized, 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 recommends 1 the following as a trustworthy
means of distinguishing between the tubercle bacillus and
the smegma bacillus : stain in hot carbol-fuchsin solution,
wash in water, and treat the preparation with a saturated
solution of methylene-blue in alcohol. If the question-
able organism is the tubercle bacillus, it retains its red
color ; if the smegma bacillus, the red color is dissolved
by the alcohol and the blue color is substituted for it.

The differential diagnosis between the tubercle bacil-
lus and the lepra bacillus is less satisfactory ; they both
take 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
stated 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 after 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
1 Fortschritte der Medicin, 1896, No. 9.

872 BACTERIOLOGY.

when subsequently treated with acid alcohol (nitric
acid, 1 part ; alcohol, 10 parts). By similar treatment
for the same length of time bacillus tuberculosis does
not ordinarily become stained.

These points, particularly what has been said with ref-
erence to the smegma bacillus, are of much practical
importance, and should always be borne in mind in con-
nection with the microscopic examination of materials
to which these organisms are likely to gain access. It
is hardly necessary to say that in the examination of
sputum and pathological fluids from other parts of the
body the tubercle bacillus is, of the organisms noted,
always the one most commonly encountered, while the
organism described by Lustgarten as the bacillus of
syphilis is seen so rarely that many trustworthy investi-
gators question its existence as a species distinct from
the ordinary smegma bacillus.

BACTERIUM TUBERCULOSIS AVIUM (MAFFUCCl),
MIGULA, 1900.

Synonyms: Bacillus tuberculosis avium, Maffucci, 1891 ; Mycobacte-
rium tuberculosis avium, Lehmanii and Neumann, 1896.

From time to time fowls are known to suffer from
a form of tuberculosis that in a number of ways sug-
gests human or mammalian tuberculosis. The bacillus
causing the disease, the so-called bacillus of fowl tuber-
culosis, bacillus tuberculosis avium, while simulating the
genuine bacillus tuberculosis morphologically, differs
from it both in cultural and pathogenic peculiarities.
Thus, for instance, it develops into much longer and
somewhat thinner threads; grows rapidly on media
without glycerin or glucose ; does not grow on potato ;
develops as well at from 42° to 43° C. as at 37° to 38°

BACTERIUM TUBERCULOSIS AVWM. 373

C. ;l its virulence is not diminished by cultivation at
43° C. ; development on artificial media begins in from
six to eight days after inoculation ; young cultures on.
solid media are whitish, soft, and moist, becoming yel-
lowish and slimy with age ; it is somewhat more resist-
ant to drying and high temperatures than the bacillus
of mammalian tuberculosis; the results of its patho-
genic activities are almost always chronic, are rarely
located in the lungs or intestines, but are especially fre-
quent in the liver and spleen ; the lesions are conspic-
uously rich in bacteria, do not show the central necrotic
area that characterizes the mammalian tubercle ; the
disease is transmissible from the hen to the embryo
chick ; the only susceptible mammal is the rabbit ; the
guinea-pig and dog are naturally immune ; it has the
same micro-chemical staining-reactions as mammalian
bacillus tuberculosis; it has never been certainly de-
tected in human tuberculosis.

Some are inclined to regard this organism as but a
variety of the genuine bacillus tuberculosis, and it is not
unreasonable to believe that the sojourn of that organ-
ism in the body of a refractory animal, whose normal
temperature is so high as that of the fowl, when not fatal
to the organism, might result in striking modifications
of certain of its biological functions. In fact, Nocard 2
li;i< shown that if the genuine bacillus tuberculosis from
man be left in the peritoneal cavity of chickens (by the
collodion-sac method of Mctschnikoflf, Roux, and Sal-
lembini, which see) for from five to eighth months, they
will, by the end of this time, have become so modified

1 The normal body-temperature of fowls ranges between 41.5° and
42.5° C.

2 Nocard: Aniiales de 1'Institut I'asteur, 1898, p. 561.

374 BACTERIOLOGY.

in their biological peculiarities as to simulate very
closely the bacillus of fowl tuberculosis.

Moore1 reports studies on bacterium tuberculosis
avium in an epidemic occurring in California. He ob-
tained pure cultures by inoculating glycerin-agar or blood-
serum tubes directly from tuberculous livers and spleens.
In the original cultures little difficulty was experienced
in cultivating the organism on glycerine-agar, fresh dog-
serum, Dorset's egg-medium, potato, and glycerine-
bouillon. The general cultural peculiarities observed
agreed with those described by Maffucci, Nocard, Straus
and Gamaleia, and others. He states that the tubercle
bacteria resemble quite closely those of the bovine and
human varieties in their size and general morphology
as they are found in the tissues of the fowl. The aver-
age length of a large number of measurements was 2.7
microns. Moore also tested the pathogenesis of the
freshly isolated avian tubercle bacteria on fowls, rabbits,
guinea-pigs, and pigeons. The results of these inocu-
lations, however, were unsatisfactory, as were also feed-
ing experiments of healthy fowls with human tubercu-
lous sputum rich in bacteria.

VARIETIES OF B. TUBERCULOSIS. — Theobald Smith2
has called attention to certain very conspicuous differences
that maybe observed between the bacilli obtained from hu-
man and those from bovine tuberculosis ; and in a series of
inoculation experiments Ravenel has shown that for a large
number of species tubercle bacilli of bovine origin were
constantly more virulent than those from human sources.

Anatomical lesions very suggestive of, though not

1 Moore: Journal of Medical Research, 1904, vol. vi.

2 Smith : Transactions of the Association of American Physicians,
1896, vol. xi. p. 75.

TUBERCULIN. 375

identical with, those produced by bacillus tuberculosis,
have also from time to time been observed in mice, rats,
guinea-pigs, rabbits, cats, goats, bovines, hogs, and man.
They do not appear to be of a specific nature as regards
etiology, for the reason that different authors have
described different organisms as the causative agents.
These affections are usually classed under the name
pseudotuberculosis.

SUSCEPTIBILITY OF ANIMALS TO TUBERCULOSIS. —
The animals that are known to be susceptible to tuber-
culosis are man, apes, cattle, horses, sheep, guinea-pigs,
pigeons, rabbits, cats, and field-mice. White mice,
dogs, and rats possess immunity from the disease.

TUBERCULIN. — The filtered products of growth from
old fluid cultures of the tubercle bacillus represent what
is known as tuberculin — a group of proteid substances
possessing most interesting properties. When injected
subcutaneously into healthy subjects tuberculin has no
effect ; but when introduced into the body of a 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 hyperscmia about the tuberculous focus,
a change histologically analogous to that seen in the
primary stages of acute inflammation. This zone of
hypersemia, with the coincident exudation and infiltra-
tion of cellular elements, probably aids in the isolation
or casting off of the tuberculous nodule, the inflamma-
tory zone forming, so to speak, a line of demarcation
between the diseased and healthy tissue.

As a curative agent for the treatment of tuberculosis,
tuberculin has not merited the confidence that was at

376 BACTERIOLOGY.

first accorded to it. Its field of usefulness is now
almost limited to the diagnosis of obscure cases, and
even for this purpose it is less frequently employed
than formerly.

In veterinary medicine it has proved much more
trustworthy as a diagnostic aid, and is practically
everywhere in use for the detection of incipient tuber-
culosis, especially in cattle.

VACCINATION AGAINST TUBERCULOSIS. — Recent
experiments by v. Behring, Pearson and Gilliland, and
others have shown that it is possible to immunize ani-
mals with lowly virulent tubercle bacteria of human
origin, and after one or two injections with such organ-
isms the animals show a marked degree of tolerance to
the more highly virulent bovine organisms. The
results of experiments in this direction have been so
encouraging that it is probable this method may be
utilized for the active immunization of cattle against
tuberculosis.

ACTINOMYCETES.

The term actinomycetes is restricted to a group of
organisms having morphological affinities with the bac-
teria on the one hand and the hyphomycetes on the
other. They resemble the bacteria in that they occur
as homogeneous threads which under artificial cultiva-
tion may become segmented into short bacillus- or
coccus-like fragments. Furthermore, they are unlike
the moulds in that they have not a double wall ; are not
filled with fluid containing granules, and the segments
are not separated from one another by a distinct parti-
tion. They simulate the moulds in that they develop
from spores into dichotomously branching threads,

ACTINOMYCETES. 377

which ultimately form colonies having more or less
resemblance to true mycelia. Certain of the threads
composing such a mycelium become fruit hyphae, break-
ing up into round, glistening, spore-like bodies. As a
rule, these spores are devoid of the high resistance to
heat exhibited by bacterial spores, and are stainable by
the ordinary methods.

The limits of this group are ill defined and its recog-
nized components are not as a whole well understood.

The longest known and most carefully studied acti-
nomycetes are act. bovis, act. madurse, act. farcinicus,
and act. Eppingeri, although many other varieties have
been encountered in association with important and
interesting pathological lesions.

The fact that certain bacteria, viz., b. tuberculosis, b.
mallei, b. diphtheria?, generally regarded as bacteria,
are, as a rule, segmented and occasionally show a ten-
dency to branch, has led to their being classified at
times with the actinomycetes ; on this point, however,
there is as yet no consensus of opinion.

It is interesting to note that the pathological lesions
in which actinomycetes have been detected show in many
cases certain similarities to true tubercular processes,
and in a few instances, save for the absence of tubercle
bacteria, as we usually see them, were indistinguishable
from tuberculosis.

More or less imperfectly studied varieties of actino-
mycetes have been encountered in abscess of the brain,
cerebrospinal meningitis, endocarditis, bronchopneumo-
nia, pleuropneumonia, pustular exanthemata, abscess of
the lung, bronchiectasis, pulmonary gangrene, necrosis
of the vertebra?, subphrenic abscess, noma, pseudotuber-
c,ulosis, etc.

378 BACTERIOLOGY.

In some cases the actinomycetes can be obtained in
culture from the diseased tissues ; almost as often they
cannot. Sometimes the inoculation of animals with
bits of the diseased tissue or with cultures results in the
production of pathological lesions referable to the organ-
ism ; again, no effect follows upon such inoculation. As
seen in the tissues by microscopic examination, actino-
mycetes may appear as long, convoluted, irregularly stain-
ing, beaded, branching threads, or as clumps of short,
markedly beaded, sometimes branched rods. At times
a clump of the short or longer threads is encountered
in the tissues that gives the distinct impression of
mycelial structure.

Some of the varieties that have been described are
best demonstrated in the tissues or exudates by the
Gram or Gram-Weigert method of staining ; others are
decolorized by this process, and are rendered visible
only by the simpler procedures. Some of them are to
a limited extent proof against the action of acid decolor-
izers. Though many accounts of the presence of these
morphological types in a variety of conditions have
been recorded, the descriptions in the main are meagre
and often insufficient for identification. A few, how-
ever, have been found so constantly in association with
more or less definite clinical and pathological conditions
that a brief description of them may be of service.

ACTINOMYCES Bovis (also commonly known as strep-
tothrix actinomyces, actinomyces fungus, ray fungus)
was first observed by von Langenbeck in a case of
vertebral caries in 1845. According to Bollinger, the
fungus had been seen by Hahn a number of years before
in museum specimens, but had been regarded by him
as a penicillium. The name actinomyces or ray fungus

ACTINC/MYCETES. 379

originated with Harz. It is constantly to be detected in
the tissues and exudates of the disease of cattle known
as actinomycosis, " lumpy jaw," " wooden tongue," etc.
The typical tumor of this disease is characterized by
inflammation, pus formation, excessive new formation
of connective tissue, abscesses, cavities, and sinuses.
Viewed as a whole, the tumor presents points of resem-
blance to the osteo-sarcomatous, the scrofulous or tuber-
culous, and the cancerous processes. The disease occa-
sionally occurs in man, and according to the point of
entrance of the parasite may arise in the mouth, the
pharynx, the lungs, the intestines, or the skin. In ani-
mals the disease is characterized by an excessive new
formation of connective tissue, so that tumefaction is
always a conspicuous peculiarity. In man, on the other
hand, suppuration is the most prominent feature.

If the purulent discharge from an actinomycotic
tumor be examined fresh, it will be found to contain
tiny yellow (sulphur color as a rule) clumps. If these
be examined, unstained, in a drop of physiological salt
solution or water under the microscope, they will be found
to be made up of a rosette-like mass of closely inter-
woven threads. (See Fig. 65.) At the centre the mass
may show the presence of spherical, coccus-like bodies
or granules, while at the periphery the free ends of the
threads are more or less distinctly bulbous or nodular,
or both, and they may show branching. Sometimes the
free ends of the threads are only slightly or not at all
swollen.

These mycelia — the actinomyces — may be stained by
the ordinary aniline dyes, or by the Weigert or the
Gram method, though by either of these procedures its
full structure is not, as a rule, brought out. The reason

380 BA CTERIOL OG Y.

for this is that the terminal bulbs are not due to enlarge-
ment of the thread itself, but rather to a colloid degen-
eration of its enveloping sheath. This colloid matter,
having different microchemical reactions from the
enclosed thread, requires different reagents to stain it.
The entire structure may be seen when the fungus is
stained first by the Gram method, and subsequently with
eosin or saifranin. For the demonstration of the fungus
in sections, the method of Mallory gives satisfaction.
It is as follows : Stain the section on the side with

FIG. 65.

Actinomycosis fungus in pus. Fresh, unstained preparation. Magnified
about 500 diameters.

gentian-violet ; clear and dehydrate with aniline oil in
which a little basic fuchsin has been dissolved ; remove
the aniline oil-fuchsin with xylol, and mount in xylol
balsam. In sections treated in this way the coccus-like
central masses and the filamentous threads making up
the mass of the mycelium are stained blue ; the club-
like extremities of the thread are red. Often the red-
stained hyaline material is seen to be penetrated to its
extremity by a sharply defined blue thread.

Cultivation of the fungus from the aetinomycotic pus

ACTINCfMYCETES. 381

presents difficulties for the following reasons: 'All the
mycelia seen by microscopic examination are not living ;
as a rule, they grow slowly even under the best of cir-
cumstances ; and generally there are many other, more
rapidly growing, living organisms in the pus. When
pure cultures are obtained, it grows (according to Bos-
trom) on all the ordinary artificial media. It develops
at room-temperature, but better at that of the body.

It grows both with and without oxygen.

The young colonies appear as grayish points com-
posed of a felt-work of fine threads. As the colonies
become older they become denser and more opaque.
Very old colonies are almost leathery in consistency.
On blood-serum the growth after a time assumes a
salmon, an orange, or a yellowish-red color. Growth
on gelatin is accompanied by slow liquefaction.

A yellowish-red growth, limited in extent, occurs on
potato. It causes no clouding of bouillon, but grows as
cottony clumps that sink to the bottom.

The bulbous extremities seen upon the mycelial
threads fresh from the pus are not usually seen under
conditions of artificial cultivation. They are sometimes
observed in colonies located in the depths of solid media.
The white, powdery coating seen on old colonies repre-
sents the so-called i( spores." They are not, however,
resistant to heat, being destroyed, according to Domec,
by 75° C. in five minutes.

Bovines are the animals most frequently affected.
The disease has been seen in swine, dogs, and horses.

The most common seat of the disease is the jaw, and
this, together with the fact that particles of fodder, such
as bits of grain, chaff, straw, and barley beard, have
been detected in the diseased tissues in association with

382 BACTERIOLOGY.

the causative fungus, has led to the belief that the par-
asite gains access to the tissues with such food-stuffs. It
has not, however, been recognized outside the animal body.
The disease is apparently not transmissible from animal
to animal or from animal to man. Inoculation of ani-
mals with pure cultures is usually negative, although
nodular formations have followed the injection of large
quantities into the peritoneal cavity of rabbits. In
Bostrom's cases the nodules presented only a few of the
club-shaped extremities of the threads, and there was
no evidence of multiplication of the fungus ; while in
the experiments of Israel and Wolf it is said there
developed, in from four to seven weeks after intraperi-
toneal inoculation, larger and smaller tumors in which
typical mycelia were present, and from which the fungus
was obtained in pure culture.

ACTINOMYCES MADURA. — This organism is sup-
posed to be concerned in the causation of mycetoma or
Madura foot. Two varieties of mycetoma are known,
viz., the pale or ochroid and the black or melanoid.
Save for its occurrence in the foot, mycetoma is, patho-
logically speaking, almost a counterpart of actinomy-
cosis; and the suspicion of their identity is by no
means lessened by the fact that the actinomyces con-
stantly associated with the ochroid variety is to all
intents and purposes identical with actinomyces bovis.
It differs from that organism only in such minor details
as to leave little doubt that they are very closely related,
if not identical, so that a description of the one serves
equally to aid in the identification of the other.

The investigations of Wright,1 conducted upon a case

1 Wright : " A Case of Mycetoma (Madura Foot)," Journal of Experi-
mental Medicine, 1898, vol. iii., p. 421.

ACTINOMYCETES. 383

encountered in Boston, point to another type of parasite as
the causative factor in the black mycetoma. Instead of an
actinomyces, Wright found a true mould. He expresses
the opinion that the pale mycetoma is, etiologically, actino-
mycosis, and that the black is a hyphomycetic infection.

The fungus encountered and isolated in pure culture
by Wright presented the following characteristics : As
obtained from the affected tissues, the mycelia under the
microscope appear as black or brown mulberry-like
masses less than one millimetre in diameter. They are
hard, rather brittle, and difficult to break up under the
cover-glass. On soaking them in a strong solution of
sodium hydroxide they become softened and the struct-
ure of the fungus-mass can be made out. Under high
magnifying power these masses are found to consist of
pigment-granules, ovoid translucent bodies, and dis-
tinctly branching separate hyphse. Sometimes these
latter exhibit dilatations or varicosities of their seg-
ments. The periphery of a granule shows the pres-
ence of club-shaped hyphse, closely set and radially
arranged. From such granules growth on artificial
culture-media may be obtained. When transferred
direct from the tissues to artificial media, growth in
every case starts from the granule about four or five
days after it is placed upon the culture-media.

On solid media it first appears as delicate tufts of
whitish filaments. These in the course of a few days
increase in number and length, and, in the case of the
potato, form a dense whitish or pale-brown felt-work
having a tendency to spread widely.

In pure cultivation it is seen as long, branching hyphse
with delicate transverse septa. In old forms the hyphse
may be swollen at the points marked by the septa, and

384 BA CTERIOL OGY.

may then appear as strings of plump oval segments.
The filaments have a definite wall, inclosing granules
and pale areas. No spore-bearing organs are seen.

On potato, it grows as a dense, widely spreading, vel-
vety membrane; pale brown at the centre and white at
the periphery. The potato takes on a dark-brown color
and becomes very moist and dark ; coffee-colored gran-
ules appear upon the surface of the growth.

In bouillon the growth assumes a puff-ball appear-
ance. The medium assumes a deep coffee-bro\vn color,
and ultimately a mycelium growth appears upon the
surface and throughout the fluid.

When grown in potato infusion (20 grammes of potato
boiled in water, filtered and made up to a litre), the
growth is characterized by the appearance of black
granules in the midst of the mycelium. The black
granules consist of closely packed spherical or polyhe-
dral cells, together with some short, thick segmented
hypha?. The walls of these cells have a black appear-
ance, and masses of them are black and opaque under
the microscope.

On agar-agar, growth appears as a grayish mesh-work
of widely spreading filaments. In old cultures black
granules (sclerotia) appear among the filaments. No
growth occurs in the depth of the medium.

No results were obtained by the inoculation of ani-
mals with either the material direct from the tissues or
with pure cultures.

The tissue from which this fungus was obtained con-
si-ted, briefly, of a more or less atypical connective-
tissue new-growth, with numerous areas of suppuration
marked by the black granules just described.

On histological study of the tumor the primary effect

ACTINOMYCETES. 385

produced by the parasite appears to be the development
of nodules of epithelial cells and of giant cells from the
tissues immediately about them. Later, suppuration of
the nodules and abscess formation occur.. This in time
gives rise to excessive development of granulation and
connective tissue.

ACTINOMYCES FARCiNicus (bacille du farcin des
boeuffs (Nocard) ; oospora farcinica; actinomyces .bovis
farcinicus). — This organism was discovered by Nocard
(1 888) in a disease of cattle that is suggestive of farcy
as seen in horses. The lesions consist of chains of en-
larged subcutaneous lymph-glands, which on examina-
tion are found to be in a condition somewhat simulating
tuberculosis. Similar nodules are sometimes encoun-
tered in the internal organs.

By microscopic examination the organism is seen as
long, branching threads consisting of short segments.

It is non-motile. Spore-formation is questionable,
Nocard having seen it, while Lehman and Neumann
have not. The organism may be stained by the ordi-
nary methods, and also by the Gram-Weigert process.
It grows on all the ordinary culture-media, and at both
room- and body-temperature, especially well at the
latter. It is aerobic.

Colonies in agar-agar reach a size of from 1 to 2 mm. ;
are yellowish white in color, irregular in outline, and
have the appearance of a glazed, membranous mass.

On gelatin, the growtli is much slower, so that after
ten days the colonies appear as tiny translucent round
glistening points. Under low power of the micro-
scope these colonies are sharply circumscribed, grayish
or greenish in color, and are without characteristic
structure.

25

386 BACTERIOLOGY.

Growth in bouillon is characterized by a tough, slimy
sediment, and at times by more or less of pellicle-forma-
tion. Pellicle-formation is encouraged by the addition
of glycerin. The bouillon is not uniformly clouded by
the growth.

In milk, it causes an alkaline reaction, solution of
casein, but no coagulation.

On potato, it grows slowly as a dull yellowish-white
dry membrane.

Bovines, sheep, and guinea-pigs are susceptible to in-
oculation ; rabbits, dogs, cats, horses, and asses are not.

When pure cultures are injected into either the circu-
lation or the peritoneal cavity of guinea-pigs, death en-
sues in from nine to twenty days. The autopsy reveals
diifuse pseudotuberculosis of the omentum. "Within
the pseudotubercles the organism is seen as long,
branching threads, often matted together as a true
mycelium.

By subcutaneous inoculation only the neighboring
lymph -glands are affected.

The disease farcin des boeufs is said to be more com-
mon in Guadeloupe than elsewhere.

ACTIXOMYCES EPPINGERI. — This organism was dis-
covered by Eppinger in an abscess of the brain. He
regarded it as a cladothrix, and gave to it the designa-
tion cladothrix asteroides. It grows well in pure cult-
ure under artificial conditions, and is pathogenic for
animals. In the case studied by Eppinger the organ-
ism was present not only in the abscess, but also in the
meninges of the brain and cord and in the bronchial
and supraclavicular lymph-glands. There is no doubt
of its causal relation to the conditions.

In pure culture it grows well on ordinary media. It

ACTINOMYCETES. 387

appears as long, branching threads, many of which are
composed of short quadratic segments. Spores are not
formed. Motility is doubtful ; it has been observed by
Eppinger, while Lehman and Neumann failed to detect
it. It stains both by the ordinary dyes and by the
method of Gram. It grows scarcely, if at all, under
anaerobic conditions. It grows at room-temperature,
but much better at the temperature of the body. The
best growth is observed on nutrient agar-agar containing
2 per cent, of glucose. The colonies on the surface of
glucose agar-agar appear as yellowish-white, round,
finely granular, dull patches that are surrounded by a
narrow paler zone. In the depths of the medium they
do not develop beyond very small points.

On gelatin the growth is very slow ; there is no
liquefaction, and after a time the colonies take on an
orange-red color.

Bouillon is not uniformly clouded. Growth takes
place on the surface in the form of a whitish pellicle,
in which dense white masses may be seen. These
latter increase in size, become detached, and fall to
the bottom of the vessel, to collect as mycelium-like
sediment.

On potato, growth begins as a coarsely granulated
white layer, which becomes gradually red in color. It
is ultimately covered by a fine, hair-like growth.

Both rabbits and guinea-pigs are susceptible to its
pathogenic action. When injected into either the circu-
lation, the peritoneal cavity, or beneath the skin, there
develop in from one to four weeks a condition closely
simulating tuberculosis (" pseudotuberculosis clado-
thrica"). The organism quickly loses its pathogenic
properties under artificial cultivation.

388 BA CTERIOLOG Y.

ACTINOMYCES PSEUDOTUBERCULOSIS. — In 1897

Flexner detected this organism in a consolidated and
caseous lung. The condition suggested tuberculosis.
The lesion consisted mainly of an inflammatory exuda-
tion that had undergone caseation, but in addition there
were present isolated nodules that in size and general
appearance were difficult to distinguish from miliary
tubercles. Giant cells were not seen. The streptothrix
was abundant in the lung, appearing as masses of con-
voluted, branching threads. The contours of the rods
were not quite uniform, the staining was irregular, and
occasionally a thread was seen that, toward its extrem-
ity, appeared to be breaking up into short segments.
No coccus-like forms were seen. It is stained best by
the Weigert method, when deeply stained masses sepa-
rated from one another by more or less clear spaces are
to be detected. The organism was not obtained in
culture, and no eifect was produced on guinea-pigs
by subcutaneous inoculation with bits of the diseased
lung.1

JFor the literature on pathogenic streptothrices, see Flexner:
Journal of Experimental Medicine, 1898, vol. iii. p. 435 ; for a sum-
mary of cases in which streptothrices have been found, see Musser,
Pearce, and Gwyn : Transactions of the Association of American
Physicians, 1901, vol. xvi. p. 208.

CHAPTER XVIII.

Glanders — Characteristics of the disease — Histological structure of
the glanders nodule— Susceptibility of different animals to glan-
ders— The bacterium of glanders ; its morphological and cultural
peculiarities — Diagnosis of glanders.

SYNONYMS: Eotz (Ger.), Morve (Fr.).

The disease is generally known as glanders when the
mucous membrane of the nostrils is affected, and as farcy
when the subcutaneous lymphatics are the principal sites
of involvement.

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. The primary lesion may extend from its 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 initial
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-

389

390 BACTERIOLOGY.

verted into indurated, knotty cords — " farcy-buds " —
easily discernible from without.

In man it usually occurs in individuals who have
been in attendance upon animals affected with the dis-
ease. It may occur upon the mucous membrane of the
nares ; but its most frequent expressions are in the skin
and muscles, where appear abscesses, phlegmons, ery-
sipelas-like inflammations, and local necrosis closely
resembling carbuncles. Metastases to the lungs, kid-
neys, and testicles, as in the horse, may also be seen.

When occurring upon the mucous membrane glan-
ders is characterized by the presence of gray nod-
ules, about as large as a pin-head, that closely re-
semble miliary tubercles in their naked-eye appearance.
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
tendency to 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 in
detail.

The round-cell infiltration of the glanders nodule con-
sists essentially of polymorphonuclear leucocytes, while
that of the miliary tubercle partakes more of the nature
of a lymphocytic infiltration ; in the later stages of the
process the glanders nodule breaks down into a soft,
creamy matter, very analogous to ordinary pus, while
in the later stages of the miliary tubercle the tendency
is to an amalgamation of its histological constituents,

BACTERIUM MALLEI. 391

and ultimately to necrosis with caseation. The giant-
cell formation common to tuberculosis is never seen in
the glanders nodule. As Baumgarten aptly puts it :
" The pathological manifestations of glanders, from the
histological aspect, stand midway between the acute
purulent and the chronic inflammatory processes." l Evi-
dently these differences are only to be explained by differ-
ences in the nature of the causes that underlie the several
affections. We have studied the characteristics of bacte-
rium tuberculosis ; we shall now take up the bacillus of
glanders and note the striking differences between them.

BACTERIUM MALLEI (LOFFLER), MIGULA, 1900.

Synonyms : Bacillus mallei (Loffler), 1886 ; Eotz bacillus, Kranz-
feld, 1887.

In 1882 Loffler and Schiitz discovered in the dis-
eased tissues of animals suffering from glanders a bacte-
rium that, when isolated in pure culture and inoculated
into susceptible animals, possesses the property of repro-
ducing the disease with all its clinical and pathological
manifestations. It is therefore the cause of the disease.

This organism is a rod, with rounded or slightly
pointed ends. It usually stains somewhat irregularly.
(See Fig. 66.) When examined in stained prepara-
tions 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-

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 Historical Lesions of Acute Glanders
ib Man," Journal of Experimental Medicine, vol. i. p. 577.

392 BACTERIOLOGY.

sition to the opinion that it possesses this peculiarity.
Certain observers claim to have demonstrated spores in
the bacteria 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.

FIG. 66.

Bacterium mallei, from culture.

carbolic 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.

BACTERIUM MALLEI. 393

It grows readily on ordinary nutrient media at from
25° to 38° C.

Upon nutrient agar-agar, both with and without
glycerin, it appears as a moist, opaque, glazed layer,
with nothing characteristic about it. This is true both
for smear-cultures and for single colonies.

Its growth on gelatin is much less voluminous than
on media that can be kept at higher temperature, though
it does grow on this media at room-temperature without
causing liquefaction.

Its growth on blood-serum is 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
in from twenty-four to thirty-six hours at 37° C. as a
moist, amber-yellow, transparent deposit having some-
what the appearance of honey ; this becomes deeper
in color and denser in consistence as growth progresses,
and finally takes on a reddish-brown color; at the same
time 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 separates into clear whey
and a firm clot of casein.

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 if exposed to this temperature
for forty-eight hours it is destroyed. It is killed in five

394 BACTERIOLOGY.

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-
nate with points at which it either does not stain at all
or only slightly.

The animals 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 were 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 subctitaneously with a small
portion of a pure culture of bacterium mallei 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 nodules, the disease as
seen in this animal presents none of the features that

STAINING IN TISSUES. 395

it displays in the horse and ass. The clinical and path-
ological manifestations resulting from inoculation of
guinea-pigs are much more faithful reproductions. The
animal lives usually from six to eight weeks after inocu-
lation, and during this time becomes aifected 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
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 orchids 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.

STAINING IN TISSUES. — Though always present in
the diseased tissues, considerable trouble is usually
experienced in demonstrating the bacteria by staining-
methods. The difficulty is due to the fact that the bac-
teria 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 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 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

396 BA CTERIOLOG Y.

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 intensity 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,
absorb all water with blotting-paper, and stain with
two or three drops of

Carbol-fuchsin 10 c.c.

Distilled water 100 c.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 blot-
ting-paper ; and finally, at very moderate heat, or with
a small bellows (Kiihne), dry the section completely on
the slide. When dried clear in xylol and mount in
xylol balsam.

6. Transfer the 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

DIAGNOSIS BY MKTHOD OF STRAUSS. 397

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 in xylol and mount
in xylol balsam.

By method 6 the tissues are better preserved than by
method a, by which they are dried.

Very good preparations are also obtained by the use
of Loffler's alkaline methylene-blue, if care be taken
not to stain for too long a time nor to decolorize too
energetically with alcohol.

No method of contrast-stain for this organism in
tissues has been devised.

In properly stained tissues the bacteria 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
filling some of the epithelial cells, of which the nodule
contains more or less. They are always present in
these nodules in the tissues ; they are rarely present
in the blood, and, if so, in only small numbers.

DIAGNOSIS OF THE DISEASE BY THE METHOD OF
STRAUSS. — 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, a,iul
as this proceeds the skin over them becomes red and

398 BA GTE RIO LOG Y.

shining, desquamation occurs, evidences of pus-forma-
tion are seen, and, indeed, the abscess (purulent orchids)
often breaks through the skin. The diagnostic sign is
the tumefaction of the testicles.

MALLEIN. — The filtered products of growth of the
glanders bacillus in fluid media represent what is known
as inallein — a solution of compounds that bear to
glanders a relation analogous to that which tuberculin
bears to tuberculosis. It is used with considerable suc-
cess as a diagnostic aid in detecting the existence or
absence of deep-seated manifestations of the disease, the
glanderous animal reacting (manifested by elevations of
body-temperature greater than 1.5° C.) to subcutaneous
injections of mallein in from four to ten hours, while an
animal not so affected gives no such reactions.

Mallein is prepared from old glycerin-bouillon cult-
ures of the glanders bacterium by steaming them for
several hours in the sterilizer, after which they are fil-
tered through unglazed porcelain.

By some it is said that the repeated injection of mal-
lein in small doses confers immunity from infection by
bacterium mallei upon animals so treated ; an opinion
that is entirely in accord with the principles underlying
the artificial induction of immunity in general.

CHAPTEK XIX.

Bacterium (syn. Bacillus) diphtherise — Its isolation and cultivation —
Morphological and cultural peculiarities — Pathogenic properties —
Variations in virulence — Bacterium pseudodiphtheriticum — Bacte-
rium xerosis — Diphtheria antitoxin.

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 Loffler's blood-
serum mixture. (See chapter on Media.) Pass a stout
platinum needle, which has been sterilized, 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 carefully over
the surface of one of the serum-tubes ; without steriliz-
ing it pass it over the surface of the second, then the
third, fourth, and fifth tubes. Place these tubes in the
incubator. Then prepare cover-slips from scrapings
from the membrane on the fauces. If the case is one
of true diphtheria, the tubes will be ready for examina-
tion 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 the blood-serum mixture is a much more
favorable medium for the rapid development of the
diphtheria organism than of the other organisms present,

399

400 BACTERIOLOGY.

makes its isolation by this method a matter of but little
difficulty.

After twenty-four hours in the incubator the tubes
present a characteristic appearance. Their surfaces are
marked by more or less irregular patches of a white
or cream-colored growth, which is usually more dense
at the centre than at the periphery. Except now and
then, when a few orange-colored colonies may be seen,
these large irregular patches are the most conspicuous
objects on the surface of the serum. Occasionally,
almost nothing else appears.

The cover-slips made from the membrane at the time
the cultures were prepared will be found on 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 are
irregularly segmented. They are rarely or never regu-
lar in outline. If the preparation has been stained
with Loffler's alkaline methylene-blue solution, many
of these irregular rods are seen to be marked by cir-
cumscribed points in their protoplasm which stain very
intensely — they appear almost black. This irregularity
in outline is the morphological characteristic of bacillus
diphtherice of Loffler.

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, especially when they are stained only by the
usual method — i. e., with Loffler's methylene-blue.
There are other organisms present in the mouth-cavity,
particularly in the mouths of persons having decayed

BACTERIUM DIPHTHERIAS. 401

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 only the usual method
of microscopic examination ; and again, the genuine
diphtheria organism is sometimes found in the mouth-
cavities of healthy persons in attendance upon diph-
theria cases, such persons being 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 diph-
theria, a microscopic examination alone of the deposit
in the throat will serve to confirm or contradict this
opinion, and such examinations very frequently reveal
the diphtheritic nature, etiologically speaking, of mild
conditions of the throat which are not associated with
grave constitutional manifestations.

BACTERIUM DIPHTHERIA (LOFFLER), MIGULA, 1900.

Synonyms: Bacillus diphtheriae, Loffler, 1884; Klebs-Loffler bacil-
lus ; Corynebacterium diphtherise, Lehmann and Neumann, 1896.

Bacterium diphtherice, discovered microscopically by
Klebs, and isolated in pure culture and proved to
stand in causal relation to diphtheria by Loffler, 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 :

MORPHOLOGY. — As obtained directly from the diph-
theritic deposit in the throat of an individual sick of
the disease, it is sometimes comparatively regular in

26

402 BACTERIOLOGY.

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 stain irregularly ; in some of them very
deeply stained round or oval points can be detected.

When cultures are examined microscopically it is
especially characteristic to find irregular, bizarre forms,
such as rods with one or both ends swollen, and very
frequently rods broken at irregular intervals into short,
sharply defined 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 and size depend 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 Loffler'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, sometimes extremely slender, sometimes
thicker, irregularly staining threads that are usually
clubbed and frequently pointed at their extremities.
They are, as a rule, 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-

BACTERIUM DIPHTHERIA. 403

scrum, 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.
Another peculiar variation is that observed upon very
slightly acid blood -serum. Here the rods appear
swollen, and are usually contracted to oval or short,
oblong bodies, which stain very faintly, and in which
are usually located one or two very deeply staining
round or oval points. 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 diph-
theria organisms under conditions that might reasona-
bly be regarded as favorable to normal development ;
and in many thousand blood-serum cultures from cases
of diphtheria that have been examined by competent
bacteriologists at the laboratory of the Board of Health
of Philadelphia, the branching forms of this organism
have not been observed in a single instance. It is fair
to assume, therefore, that this peculiar morphological
variation of bacillus diphtherice is, under normal condi-
tions of growth, comparatively rare.

On the other hand, if the organism be grown on
media favorable to involution, such, for instance, as hard-
boiled egg, or coagulated egg of acid reaction, branching
may be seen, but with it degenerated organisms are so
conspicuous as to leave no doubt that the so-called
branching and involution are attributable to the same
cause, namely, unsuitable conditions of cultivation.

On plain nutrient agar-agar (that is, nutrient agar-
agar without glycerin) ; on a medium consisting of dried
commercial albumin dissolved in bouillon (about 10
grammes of albumin to 100 c.c. of bouillon containing

BACTERIOLOGY.

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 morphology of the organism is about in-
termediate, in both size and outline, between the forms
seen upon glycerin agar-agar and upon Loffler's blood-
serum. There will appear about an equal number of
short segmented and longer, irregularly staining forms ;
but in general the longest are rarely as long as the long
forms seen on blood-serum, and throughout they are not
so conspicuous for the irregularity of their staining.

FIG. 67.

A.

Bacterium diph'hcrise. A. Its morphology on glycerin-agar-agar. B. Its
morphology on Loffler's blood-serum, c. Its morphology on acid blood-
serum mixture.

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 other set
contains none, a noticeable difference in morphology can

BACTERIUM DIPHTHERIA. 405

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 less uniformly, vary more in
shape, and when -stained by Loffler's blue are not so
regularly 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.

GROWTH ON SERUM MIXTURE. — The medium upon
which bacillus diphtherice grows most rapidly and lux-
uriantly, and which is best adapted for determining its
presence in diphtheritic exudates, is, as has been stated,
the blood-serum mixture of Loffler. (See chapter on
Media.) On the blood-serum mixture the colonies of
bacillus diphtherice grow so much more rapidly than the
other organisms usually present in secretions and exuda-
tions 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 with coalescent or scat-
tered colonies of this organism is so characteristic that
one familiar with the appearance can anticipate with tol-
erable certainty the results of microscopic examination.

GLYCERIN AGAR-AGAR. — Upon nutrient glycerin
agar-agar the colonies likewise present an appearance

406 BACTERIOLOGY.

that may readily be recognized. They are in every
way more delicate in 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
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

Colonies of bacterium diphtherias on glycerin-agar-agar. a. Colonies located
in the depths of the medium. 6. Colonies just breaking out upon the sur-
face of the medium, c. Fully developed surface-colony.

or scalloped. (Fig. 68, c.) These colonies are always
quite dry in appearance. When deep down in the agar-
agar they are coarsely granular. (Fig. 68, a.) They
rarely exceed 3 mm. in diameter.

GELATIN. — On gelatin the colonies develop much
more slowly than on media that can be retained at a
higher temperature. They rarely present their char-
acteristic appearances on gelatin in less than seventy-
two hours. They then appear as flat, dry, translucent
points, usually round in outline.

BACTERIUM DIPHTHERIA. 407

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
exceed 1.5 mm. in diameter.

BOUILLON. — In bouillon it usually grows in fine
clumps, which fall to the bottom of the tube, or become
deposited on its sides without causing diffuse clouding
of the bouillon. Sometimes there are exceptions to
this naked-eye appearance : the bouillon may be dif-
fusely clouded ; but if one inspect it very closely, par-
ticularly if he examine it microscopically as a hanging
drop, the arrangement in clumps will always be de-
tected, but the clumps are so small as not to be dis-
cernible by the unaided eye.

In bouillon kept at a temperature of 35°-37° C. a
soft, whitish pellicle often forms upon 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
has been added. This play of reactions has been attrib-
uted to the primary fermentation of the muscle-sugar
often present in the bouillon. It does not occur when
the medium is free from carbohydrates.

POTATO. — On potato at a temperature of 35°-37° C.
its growth after several days is invisible, only a thin,
dry glaze appearing at the point at which the potato
was inoculated. Microscopic examination of scrapings

408 BACTERIOLOGY.

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 sur-
face-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° 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.

STAINING. — 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 obtained by the use
of Loffler's alkaline methylene-blue solution ; this brings
out the dark points in the protoplasmic body of the
bacilli and thus aids in their identification.

For the purpose of demonstrating the Loffler bacil-
lus in sections of diphtheritic membrane, both the Gram
method and the fibrin method of Weigert give excellent
results.

PATHOGENIC PROPERTIES. — When inoculated sub-

BACTERIUM DIPHTHERIA. 409

cutaneously into the bodies of susceptible animals the
result is not the production of septicaemia, 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 de-
velopment gives rise to changes in the tissues which
result entirely "Ironi the absorption of poisonous albu-
mins generated by the bacteria 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 bacteria in
the internal organs is in all probability accidental, and
usually unimportant.

By special methods of inoculation2 (the injection of
fluid cultures into the testicles of guinea-pigs) diph-
theria bacilli can be caused to appear in the ornentum ;
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.

1 Frosch : " Die Verbreitung des Diphtheric-bacillus in Korper des
Menschen," Zeit. fiir Hygiene und Infektionskrankheiten, 1893, Bd.
xiii. pp. 49-52. Booker : Archives of Pediatrics, Aug. 1893. Wright
and Stokes: Boston Med. and Surg. Journ., March and April, 1895.

2 Abbott and Ghriskey : " A Contribution to the Pathology of Experi-
mental Diphtheria," The Johns Hopkins Hospital Bulletin, No. 30,
April, 1893.

410 BACTERIOLOGY.

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 oedema,
with more or less hypersemia and ecchymoses 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 a pin-point to a pin-head, and may be
even larger. They are white and do not project above
the surface of the capsule.

The bacteria are always to be found at the site of
inoculation, 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 oedema-
tons 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 bacteria within leucocyt^ as well as
some outside, frequently stain very faintly and irregu-
larly, and may appear disintegrated and dead.

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 bacteria.

Microscopic examination of the tissues at the site of

BACTERIUM DIPHTHERIA. 411

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 formation
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 irregu-
larly crescentic, dumb-bell, flask-shape, whetstone-
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
in isolated masses, indicating the location of the
nucleus from the destruction of which they originated.
These particles always stain much more intensely than
do the normal nuclei of the part.1 Oertel showed long
ago that these peculiar alterations in their distribution
are characteristic of human diphtheria, and the demon-
stration of similar changes in animals inoculated with
this organism is important additional proof that diph-
theria is caused by it.

By the inoculation of certain animals an affec-
tion may be produced in all respects identical with
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

'See "The Histological Changes in Experimental Diphtheria," also
"The Histological Lesions Produced by the Toxalbumin of Diph-
theria," by Welch and Flexner, Johns Hopkins Hospital Unlit-tin.
August, 1891, and March, 1892.

412 BACTERIOLOGY.

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 oedematous. 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 bacteria 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 oedematous fluid about the skin-wound bacillus
diphtlierice 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 bac-
teria growing at the site 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 ba-
cillus diphtherice, and is found to belong, not to the
crystallizable ptomaines, but to the toxic albumins —
bodies which, in their chemical composition, are analo-
gous to the poison of certain venomous serpents. By
the introduction of this toxalbumin, 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 inoculation with the bacilli themselves, except,
perhaps, the production of false membranes.

BACTERIUM DIPHTHERIA. 413

Under certain circumstances with which we are
not acquainted bacillus diphtheria; becomes diminished
in virulence or may lose it entirely, so that it is no
longer capable of producing death of susceptible ani-
mals, and may cause only a transient local reaction
from which the animal entirely recovers. Sometimes
this reaction is so slight as to be overlooked, and
again careful search may fail to reveal evidence of
any 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 cultural and morphological peculiarities, but which
differed in their disease-producing power. Further
studies on this point have, however, shown that the
genuine bacillus diphtheria? may possess almost all
grades of virulence, and that absence of or dimi-
nution in virulence can hardly serve to distinguish
as separate species those 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 inoculation has
proved fatal.

In the course of their observations upon a large
number of cases Roux and Yersin found that it was
not difficult to detect, in the diphtheritic deposits of
the same individual, bacteria of identical cultural and
morphological peculiarities, but of very different degrees
of virulence, and that with the progress of the disease

414 BA CTERIOLOG Y.

toward recovery the less virulent varieties often became
quite frequent.1

There is, moreover, a mild form of diphtheria, etio-
logically speaking, affecting only the mucous membrane
of the nares, known as membranous rhinitis, from
which it is very common to obtain cultures in all re-
spects identical with those from typical diphtheria, save
for their inability to kill susceptible animals. On inocu-
lation these cultures produce only local reactions, but
these are characterized histologically by the same kind
of 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 bacteria.

For those organisms that are in all respects identical
with the virulent bacillus diphtheria?, 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 pseurfo-diphtheritic, as applied to an organ-
ism in all respects identical with the genuine bacterium,
except that it is not fatal to susceptible animals, is a
misnomer, and that it would be more nearly correct to
designate this organism as the attenuated or non-viru-
lent diphtheritic bacterium, reserving the term " pseudo-
diphtheritic " for that organism or group of organisms
(for there are probably several) that is enough like the

1 It must not be assumed from this that the bacteria lose their viru-
lence entirely, or that they all become attenuated with the establish-
ment, of convalescence, for this is contrary to what experience has
shown to be the case.

BACTERIUM PSEUDODIPHTHERIT1CUM. 415

diphtheria bacterium 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 bacterium pneu-
monias, micrococcus aureus, streptococcus pyogenes, and
the group of so-called " hemorrhagic septicaemia "
organisms — undergo marked variations in their patho-
genic properties ; and yet these organisms, when found
either devoid of this peculiarity, or possessing it in 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.

It must nevertheless be admitted that in the course
of microscopic examination of materials from various
sources, including the pharynx, one occasionally encoun-
ters micro-organisms whose morphology is so like that
of the genuine bacterium diphtherice as to create suspi-
cion, and yet they are at the same time sufficiently
unlike it to make one cautious in forming an opinion
as to their real nature.

BACTERIUM PSEUDODIPHTHERITICUM. — For a long
time bacterium pseudodiphtheriticum was looked upon as
being entirely harmless, and the only particular in which
it was regarded as being of importance was in the fact
that it was likely to be mistaken for bacterium diph-
therias. The wide dissemination of this class of organ-
isms and the demonstration of pathogenic effects in iso-
lated instances has led to the more systematic study of
members of this group of organisms.

Bacterium pseudodiphtheriticum, as found under dif-
ferent conditions, varies markedly in its morphologic
and biologic characters. Some of the varieties have

416 BACTERIOLOGY.

definite chromogenic properties, producing various
shades of yellow- and orange-colored pigment, while
others grow with a pink color.

The occurrence of bacterium pseudodiphtheriticum in
pure culture in superficial abrasions showing a slight
tendency to suppuration ; the fact that these organisms.,
when injected into the peritoneal cavity of guinea-pigs,
produce purulent peritonitis ; that such organisms are
frequently encountered in vaccine virus and in the pus
of vaccination wounds ; and that frequently in cases of
mastitis in cows such organisms occur in large numbers
in pure culture, has led to the supposition that this
group of organisms was probably responsible for sup-
purations occurring under certain special conditions.
With these facts in mind specimens of pus were derived
from thirty cases with suppurating wounds in the Uni-
versity of Pennsylvania Hospital, and careful bacterio-
logical examination of these specimens showed the pres-
ence of bacterium pseudodipthheriticum in 43 per cent,
of the cases. These organisms were always found in
conjunction with one or more of the group of pyogenic
organisms, and it is impossible to state how much of the
effect was due to any one of the organisms present. It
seems probable, however, in the light of what has been
said, that these bacteria were present not merely as
accidental invaders, but that in some way they contrib-
uted toward the results.

The fact that some of the organisms isolated from
the pus, when inoculated into the peritoneal cavity of
guinea-pigs, show distinct pyogenic properties gives
strong support to the opinion that this group is of greater
importance than was heretofore supposed. Repeated
passage through guinea-pigs serves to so increase the

BACTERIUM XEROSIS. 417

pathogenic properties of these organisms that they cause
the death of the animal in less than twenty-four hours
with marked inflammatory reaction affecting the perito-
neum as well as the abdominal organs.

The morphologic and biologic characters of some
members of the group of bacterium pseudodiphtheriticum
are very closely allied to those of bacterium diphtheria}.
Other members of the group, however, are readily dif-
ferentiated from bacterium diphtherias by either the
morphologic or the biologic characters, or by both. Many
of the members of the group produce very little acid
when grown in carbohydrate media, and the slight
degree of acidity produced is frequently obliterated by
a marked degree of alkali production. This fact is of
special value in the differentiation from bacterium diph-
therias.

BACTERIUM XEROSIS (NEISSER AND KUSCHBERT),
MIGULA, 1900.

Synonym : Bacillus xerosis, Neisser and Kuschbert, 1883.

Another organism which is also related in its mor-
phologic and biologic characters to bacterium diphtheria
is bacterium xerosis, first encountered by Kuschbert and
Neisser in xerosis of the conjunctiva, and which has
since been found on the conjunctiva by a number of
investigators, in various diseases as well as in health.

The xerosis bacteria are less likely to be mistaken for
bacterium diphtherias because they are somewhat smaller
and have less tendency to show multiple striations.
Usually they stain deeply at the poles with only one
clear unstained band in the centre. It is only occa-
sionally that a few striated organisms are encountered
in a culture.

27

418 BA CTERIOLOG Y.

Biologically bacterium xerosis is readily differentiated
from bacterium diphtherias because of the scant growth
that takes place on the ordinary culture-media. On
agar-agar the growth appears as small transparent colo-
nies which have little tendency to coalesce. On gelatin
the growth is slow, and frequently shows as minute, iso-
lated colonies along the needle track. In litmus-milk a
slight degree of acidity is produced. In bouillon the
growth is so slight as to leave the medium practically
unaltered. The growth on potato is slight and invisible.

Differentiation of Members of the Group. — Knapp1
reports that the serum-water media of Hiss, to which
different carbohydrates have been added, serve to differ-
entiate between bacterium diphtherice, bacterium pseudo-
diphtheriticum, and bacterium xerosis. Bacterium diph-
therias ferments dextrose, mannite, maltose, and dextrin
with formation of acid and the coagulation of the medium.
Saccharose is not fermented. Bacterium xerosis fer-
ments dextrose, mannite, maltose, and saccharose, with
formation of acid and coagulation of the medium. Dex-
trin is not fermented. Bacterium psendodiplttJieritieum
does not ferment any of these carbohydrates. Knapp
claims that a positive differentiation of the organisms
may be made by merely inoculating the Hiss media
containing dextrin and saccharose. If the dextrin is
alone fermented, the organism is bacterium diphtheria, if
only the saccharose is fermented, the organism is bacte-
rium xerosis, and if neither of these carbohydrates is fer-
mented, the organism is bacterium pseudodiphtheriticum.

Through the suggestion of Neisser2 we are fortu-

1 Knapp : Jour. Med. Research, vol. xii., p. 475, 1904.

2 Neisser: Zeitschrft fur Hygiene und Infektionskrankheiteu, 1897,
Bd. xxiv.

BACTERIUM XEROSIS. 419

nately enabled to differentiate between bacterium diph-
theria and the " pseudo " forms. He has found that
by the use of a particular staining method the
appearance of bacterium diphtheria? is strikingly
unlike that of the confusing forms. His differential
method comprehends the following manipulations :
the culture to be tested should be grown upon Loffler's
blood-serum mixture solidified at 100° C. ; it should
develop at a temperature not lower than 34° C. and not
higher than 36° C. ; and it should be not younger than nine
and not older than twenty-four hours. A cover-glass prep-
aration made from such a culture is stained as follows :

«. It is subjected to the following mixture for from
one to three seconds :

Methylene-blue (Griibler's) 1 gramme.

Alcohol (96 per cent.) 20 c.c.

When dissolved, mix with

Acetic acid 50 c.c.

Distilled water 950 c.c.

b. After thoroughly rinsing in water, it is stained for
from three to five seconds in vesuvin (Bismarck-brown),
2 grammes, dissolved in 1 litre of boiling distilled water,
filtered, and allowed to cool. It is again rinsed in water
and examined as a water-mount, or it may be dried and
mounted in balsam.

When so treated the diphtheria bacterium appears as
faintly stained brown rods, in which from one to three
dark-blue granules are always to be observed. The
dark granules are at one or both poles of the cell, are
more or less oval, and usually seem to bulge a little
beyond the contour of the bacterium in which they are

420

BACTERIOLOGY.

located. (See Fig. 69.) From Neisser's observations
and those of others,1 as well as from personal experience,
it seems safe in the vast majority of cases to regard all
bacteria that do uot stain in the way described as distinct
from bacterium diphtheriw.2

Blumenthal and Lipskerow3 compared all the known
staining methods that had been suggested for the differ-
entiation between bacterium diphtheria and bacterium
pseudodiphtheriticum. They decide that the method

FIG. 69.

Bacterium diphtheria:, stained by Neisser's method.

which yields the most satisfactory results is one whieh
had not heretofore been published, but which Dr. Lju-
binsky communicated to them. This method consists in
the fixation of the preparation for from one-half to two

1 Frankel : Berliner klin. Wochenschrift, 1897. No. 50.

2 Bergey : Publications of the University of Pennsylvania, New Series,
No. 4, 1898.

3 Blumenthal and Lipskerow: Centralblatt f. Bacteriologie, Bd.
xxxviii., p. 359.

BACTERIUM XEROSIS. 421

minutes in the following solutions : Pyoktanin (Merck)
0.25 grammes, acid acetic (5 per cent.) 100 c.c. Washing
in water and counterstaining with a 1 to 1000 solution
of vesuvin for one-half minute. By this method the
polar granules of bacterium diphtherias are stained bluish
black, are large, and may be seen in almost all of the
organisms. The contour of the darkly violet stained
bacterium diphtheria? is sharply defined, and it is very
easily differentiated from any other organisms that may
be present in the preparation.

NOTE. — 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 bacterium diph-
therise ? Wherein is the difference ?

In cultures and cover-slips made from both diph-
theritic and from innocent sore-throats are any organ-
isms almost constantly present ? Which are they, and
what are their characteristics ?

Which are the predominating organisms in the an-
ginas of scarlet fever?

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

Do the diphtheria organisms disappear from the throat
with the disappearance of the membrane? How long
do they persist? When obtained from the throats of
convalescents are they still pathogenic for guinea-pigs ?

Prepare a bouillon culture of virulent bacillus diph-
therise ; after it has been growing for thirty-six hours

422 B A CTERIOLOG Y.

at 37°-30° C. inoculate a guinea-pig subcutaneously
with about 0.1 c.c. of it. If the animal dies, note care-
fully the findings at autopsy, especially the distribution
of the bacilli. Now add to this culture sufficient pure
carbolic acid or trikresol to kill all bacteria in it, and
inject under the skin of another guinea-pig varying
amounts of the culture so treated, beginning with 0.05
c.c. ; determine the minimum fatal dose, and note in
which respects the post-mortem findings simulate and
in which they differ from those of the first animal.
Should any of the animals survive the injections of the
disinfected culture, note carefully their condition from
day to day, particularly any fluctuations in weight.
When they have quite recovered inoculate them with
living, virulent diphtheria organisms. Do the results
correspond with those obtained with guinea-pigs that
have never been treated at all ? Explain the results.

DIPHTHERIA ANTITOXIN. — As stated above, the
growth of bacterium diphtherias is accompanied by the
elaboration of a poison of remarkable toxicity that is
accountable for the constitutional symptoms and patho-
logical lesions by which the disease is characterized. If
by appropriate methods this poison (toxin) be separated
from the bacteria by which it was formed, it is capable,
when injected into susceptible animals, of causing death
and practically all the lesions that accompany the dis-
ease when due to the invasion of the living bacteria. If,
on the contrary, the dose of poison be so adjusted as to
cause only temporary inconvenience and not endanger
life, and this dose be injected repeatedly, gradually in-
creasing in size as the animal is able to bear it, after a
while a marked tolerance is established, so that the animal

DIPHTHERIA ANTITOXIN. 423

may be given many times the amount of the toxin that
would otherwise prove fatal — /. e., many times the lethal
dose for an animal that had not acquired such a tolerance.

If blood be now drawn from the animal that has
become habituated, so to speak, to the diphtheria toxin,
and the serum collected from it, we discover several
important facts, viz. :

That this serum when mixed with the previously
determined lethal dose of the toxin in a test-tube will
either neutralize its toxicity or greatly reduce it, accord-
ing to the amount of serum used.

That if we inject into an animal the determined fatal
dose of the toxin, and immediately afterward inject a
quantity of the serum, either the animal will not die or
the death will be more or less delayed, according to the
amount of serum employed.

That if a susceptible animal be inoculated with a
living culture of virulent bacterium diphtherise, its life
may be saved, or its death postponed, by the subsequent
injection of the serum ; the result depending upon the
amount of serum used and the lapse of time between
inoculation with the bacteria and injection of the serum.

And, finally, that although this serum has such a
marked effect upon the toxins of bacterium diphtherise in
a test-tube or in the animal, and so striking an influence
upon the course of infection with the living organisms
in the animal, it has little or no effect upon the living
bacteria either in a test-tube or at the site of inoculation
in the living animal body.

This serum with which we have been experimenting
is the so-called " diphtheria antitoxin " or " antidiph-
theritic serum."

For practical purposes, it is obtained from horses,

424 BACTERIOLOGY.

the animals being treated with gradually increasing
doses of diphtheria toxin until they are able to with-
stand enormous multiples of the ordinarily fatal dose.
When this point is reached, the protective body — the
antitoxin — is present in the blood in such large quan-
tities that the serum may be successfully employed in
the treatment of diphtheria in human beings — /. e., as
an antidote to the diphtheria toxin that is produced by
the growing bacteria in the throat, or elsewhere, and
distributed through the body by the circulating blood.

THE STANDARDIZATION OF DIPHTHERIA ANTI-
TOXIN.— The value of diphtheria antitoxin may be de-
termined according to several different standards. Those
that are best known have been proposed by Behring and
Ehrlich.

1 . Behring's Method. — He designates as a " normal "
poison a toxin of which 0.01 c.c. suffices to kill a guinea-
pig weighing 250 grammes in four days. Of such a
normal diphtheria toxin 1 c.c. will be sufficient to kill
100 guinea-pigs weighing 250 grammes each, or 25,000
grammes in weight of guinea-pigs.

The quantity of antitoxin that is required to just pro-
tect 25,000 grammes weight of guinea-pigs from the
minimum fatal dose of the toxin is called one immuni-
zing unit. If an immune serum contains in 1 c.c. one
immunizing unit, it represents a " normal " antitoxin.

To determine the strength of an immune serum, 1 c.c.
of normal toxin is mixed with increasing quantities of
the serum, and these mixtures are injected subcutaneously
into guinea-pigs ; the quantity of the serum which suf-
fices to neutralize that amount of normal toxin — i. e.,
that keeps the animal alive for four days or longer —
contains one immunizing unit.

DIPHTHERIA ANTITOXIN, 425

2. Ehrlich's Method. — Ehrlich has recently introduced
the use of a standard diphtheria antitoxin in a dry state
which contains 1700 immunizing units in each gramme.
This standard antitoxin is distributed by the Institute
for testing serum at Frankfort-on-the-Main, and is now
being used in a great many places for the standardiza-
tion of diphtheria antitoxin. A test toxin is prepared,
corresponding to this standard antitoxin, and with this
toxin the strength of the unknown serum is titrated.

If, for instance, the test toxin is of such a strength
that 0.003 c.c. represents the minimum fatal dose for a
guinea-pig of 250 grammes, then 0.3 c.c. would represent
100 times the minimum fatal dose of toxin, and, accord-
ing to Ehrlich's standard, an immunity unit is that
amount of antitoxic serum which will neutralize 100
times the minimum fatal dose of toxin. In performing
the test to estimate the strength of an antitoxic serum,
the antitoxin is diluted with sterile water in varying
proportions, and a series of guinea-pigs are injected with
mixtures of 100 times the minimum fatal dose of the
toxin and varying quantities of the diluted antitoxic
serum. For this purpose guinea-pigs of approximately
250 grammes weight are employed. If, for instance, a
guinea-pig receiving 100 times the minimum fatal dose
of toxin, and 0.1 c.c. of the diluted antitoxic serum, sur-
vives for four days, then 0.1 c.c. of the serum represents
an immunity unit of antitoxin.

An antitoxic serum of this strength, therefore, contains
10 times the normal amount of antitoxin, because it con-
tains the immunity unit in only 0.1 c.c. ; a normal anti-
toxin being one in which an immunity unit is contained
in one cubic centimetre. Antitoxic serums are frequently
of such high degree of potency that they contain from
800 to 1000 immunity units in each cubic centimetre.

CHAPTER XX. •

Typhoid fever — Study of the organism concerned in its production —
Its morphological, cultural, and pathogenic properties — Bacillus
coli — Bacillus paratyphosus — Its resemblance to Bacillus typhosus.

BACILLUS TYPHOSUS.

THE organism discovered in the tissues of typhoid
cadavers microscopically by Eberth (1880-81), and
subsequently isolated in pure culture and described by
Gaffky (1884), is now generally recognized as the etio-

FIG. 70. FIG. 71.

Bacillus typhosus, from culture Bacillus typhosus, showing flagella

twenty-four hours old, on agar- stained by Loffler's method,

agar.

logical factor in the production of typhoid fever. It
may be described as follows :

It is a bacillus about three times as long as broad,
with rounded ends. It may appear at one time as very
short ovals, at another time as long threads, and both

426

BACILLUS TYPHOSUS. 427

forms may occur together. Its breadth remains toler-
ably constant. Its morphology presents little that
will aid in its identification. (See Fig. 70.) 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 Loffler (see
this method in chapter on Staining) is seen to possess
very delicate locomotive organs in the form of fine,
hair-like flagella, attached in large numbers to all parts
of its surface. (See Fig. 71.) These flagella are not
seen in unstained preparations, nor are they rendered
visible by ordinary methods of staining.

In patients suffering from typhoid fever the organ-
ism has been found during life in the blood, urine, and
faeces, 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.

Colony of bacillus typhosus 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-

428 BACTERIOLOGY.

tricts. (Fig. 72.) These colonies have sometimes the
appearance of flattened pellicles of glass-wool, and
usually a pearl-like lustre.

On AGAR-AGAR the colonies present nothing typical.

STAB-CULTURES. — In stab-cultures the growth is
mostly on the surface, there being only a very limited
development down the track made by the needle. The
surface-growth has the same appearance in general as
that given for the colonies.

POTATO. — 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 so in many cases, yet it cannot
be considered as invariable, for at times this organism
develops more or less visibly on potato.

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

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

BOUILLON. — It causes uniform clouding of the bouil-
lon and brings about a slightly acid reaction.

INDOL FORMATION. — It is customary to regard this
organism as devoid of the power of forming indol ;
in fact, this has hitherto been considered one of its
important differential peculiarities. By the usual meth-
ods of cultivation and testing the indol reaction is not
observed in cultures of the typhoid bacillus. It has
recently been shown, however, by Peckham, that by
repeated transplantation, at short intervals, into either
Dunham's peptone solution, or, preferably, a freshly
prepared alkali-tryptone solution, made from trypton-

JtACILLUS TYPHOSUS. 429

i/cd beef-muscle, that the inclol-producing function
may be induced in the genuine typhoid bacillus obtained
directly from the spleens of typhoid cadavers.1

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 may become red. In the fer-
mentation-tube, in glucose or lactose bouillon, no evo-
lution 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.

It does not liquefy gelatin.

It grows both with and without oxygen.

It does not grow rapidly.

Owing to a tendency to retraction of its protoplasm
from the cell-envelope and the consequent production
of vacuoles in the bacilli, the staining of this organism
is frequently more or less irregular. At some points in
a single cell marked differences in the intensity of the

'A. W. Peck ham : "The Influence of Environment Upon the
Biological Functions of the Colon Group of Bacilli," Journal of Experi-
mental Medicine, 1897, vol. ii.

430 BACTERIOLOGY.

staining will be seen, and here and there areas quite
free from color can commonly be detected. These
colorless portions are often so sharply defined that they

FIG. 73.

Diagrammatic representation of retraction of protoplasm, with production
of pale points, in bacillus typhosus.

look as if they had been punched out with a sharp
instrument. (See Fig. 73.)

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
stain, 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 Mashed
in absolute alcohol, and -cleared in xylol and mounted
in balsam, the bacilli (particularly if the tissue be the
liver and spleen) can readily be detected, massed to-
gether 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 this
peculiar deposition in clumps must always be borne in
mind, otherwise much labor will be expended in vain.
In tissues the typhoid bacilli do not lie scattered about
in the same way as do the organisms in tissues from
certain other conditions — septicaBmia, for instance ; they

BACILLUS TYPHOSUS. 431

are not of necessity distributed along the course of the
capillaries, but are localized in small clumps through
the organs, and it is for these clumps, which are easily
detected under a low-power objective, that one should
search. This peculiar clumping of the typhoid bacilli
in the tissues cannot be satisfactorily explained. It
may possibly be due to the specific clumping or agglu-
tinating influence that typhoid blood has been shown to
have upon the typhoid bacillus, a phenomenon that is
readily demonstrable in the test-tube or under the
microscope. In other words, may it not be simply the
result of an intracapillary "Widal reaction"? (See
Widal Reaction.)

When the section is prepared for examination, if it
be gone over with a low-power objective, one will
notice at irregular intervals little masses that look in
every respect like particles of staining-matter which
have been precipitated upon the section at that point.
When these masses are examined with a higher power
objective they will be found to consist of small ovals or
short rods so closely packed that the individuals com-
posing the clump can often be seen only at the extreme
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.

RESULT OF INOCULATION INTO LOWER ANIMALS. —
A great many experiments have been made in a variety
of ways with the view of reproducing the pathological
conditions of this disease, as seen in man, in the tis-
sues of lower animals, but with practically no success.
From the time of its discovery up to within a compara-
tively recent date there was an almost continuous con-
troversy concerning the infective properties of bacillus

432 BA CTERIOL OG Y.

typhoiius for animals. By some it was held that the
effects of its introduction into animals were manifestly
of toxic l origin, while others regarded them as evidences
of genuine infection.2 These diversities of opinion are
hardly surprising when we remember that animals
never suffer naturally from a disease similar to typhoid
fever, and therefore offer many obstacles to its faithful
reproduction, and that the vigor of this organism when
cultivated from various sources is liable to a wide range
of fluctuation. For a time there seemed to be good
grounds for the opinion that under exceptional circum-
stances bacillus typhosus did exhibit truly infective
properties, and the reported experiments of Cygnaeus3
in particular, as well as a single observation by the
writer,4 in no wise weakened this opinion. By a variety
of methods Cygnseus demonstrated that this organism
possessed the property of multiplying within the in-
ternal organs of animals and of causing constitutional
symptoms and pathological lesions that very closely
simulated those of typhoid fever as seen in man. In
1890 the writer called attention to the lesions found in
one of a number of rabbits that had succumbed to
intravenous injection of large amounts of fluid cultures
of this organism.

In this case there was an ulcer in the ileum which
was macroscopically and microscopically identical with
those found at autopsy in the small intestine of human

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

2 Infective or septic — poisoning of the tissues as a result of the growth
of bacteria within them.

'Cygnseus: Ziegler's Beitrage zur Anat. und Path., 1890, Bd. vii.
Heft 3, S. 377.
4 Bulletin of the Johns Hopkins Hospital, 1890, vol. i. p. 63.

BACILLUS TYPJIUSUS. 433

subjects dead of this disease. The typhoid bacilli were
not only obtained from the spleen of the animal by
culture method, but the characteristic clumps were also
demonstrated microscopically in sections of the organ.

It must be said, however, that such results are ex-
tremely rare. As a rule, the only eifects that follow
the introduction of this organism into animals are refer-
able to the intoxicating action of the materials used.
In fact, the results of modern investigations have placed
bacillus typhosus in the category of toxin-producers,
and through the use of the toxins produced by it ani-
mals have .been rendered immune from otherwise fatal
doses. The serum of such animals has also been shown
to possess a certain degree of immunizing power.1

In connection with the inoculation of animals with
bacillus typhosus observations of a 'most important
nature have been made by Sanarelli 2 upon the arti-
ficial induction of susceptibility to its pathogenic ac-
tion. 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 — bacillus vulgaris,
bacillus prodigiosus, and bacillus coli; and that by
whatever means the animal was subsequently inocu-
lated 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 pathological alterations most conspicuous in the
digestive tract, and particularly in the small intestine.
In these cases the infection is general, and the organisms

1 Pfeiffer and Kolle : Zeitschrift fur Hygiene und Infektionskrank-
heiten, 1896, Bd. xxi. S. 208.
* Sanarelli : Annales de 1'Institut Pasteur, 1892, tome vi.

28

434. BA CTERIOLOG Y.

may be recovered from the blood and internal organs.
A It is the opinion of Sauarelli that the toxic conditions
produced by the preliminary injections of the products
of growth of the saprophytic organisms may be consid-
ered 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-
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.

Alessi1 reports 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, gradually became susceptible
to infection by the typhoid bacillus ; but, unfortunately
for the value 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

1 Alessi : Centralblatt fur Bakteriologie un. Parasitenkunde 1894,
Bd. xv. No. 7, p. 228.

BACILLUS TYPHOSUS. 435

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

Because of the variations in the morphology and cult-
ural peculiarities of this organism, and because of the
difficulty experienced in efforts to reproduce in lower
an i nials the conditions found in the human subject,
typhoid fever is bacteriologically one of the most unsat-
isfactory of the infectious diseases.

A number of other organisms appear botanically to
be closely related to the typhoid bacillus, and with our
present methods for studying them they so closely simu-
late it that the difficulty of identifying this organism
is sometimes very great. In addition 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 ba-
cillus is conspicuously inconstant ; its growth on potato,
which was formerly described as characteristic, may,
with the same stock, at one time be 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 de-
monstrable by ordinary methods, and frequently by
special methods of cultivation. (Peckham, I. c.) The

1 See paper by the author : " The Effects of the Gaseous Products of
Decomposition upon the Health, and Resistance to Infection, of Certain
Animals that are Forced to Respire Them," Transactions of the Asso-
ciation of American Physicians, 1895, vol. x. pp. 16-44.

436 BA CTERIOLOG Y.

only properties possessed by it that may be said to be
constant are its motility; its inability to cause gasnn is
fermentation of glucose, lactose, or saccharose ; its inca-
pacity for coagulating milk; and its growth on gelatin
plates; but there are other bacilli which possess these
same characteristics to a degree that renders their differ-
entiation from the typhoid organism often a matter that
requires the careful application of all the different tests.

THE AGGLUTINATION EEACTION. — An interesting
reaction of the typhoid bacillus is 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 organ-
ism. 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 bacilli appear as single, act-
ively motile cells ; when to such a drop a drop of di-
lute serum from a case of typhoid fever is added the
motility of the organism gradually becomes lessened,
and finally ceases, and the bacteria congregate in
larger and smaller clumps. The reaction may also be
produced 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 clear and contains within it flocculent
masses of the bacteria that have agglutinated together
as a result of the specific action of the serum used.

For the hanging-drop test, sufficient serum may be
obtained from a needle-prick in the finger, while for

BACILLUS TYPHOSUS. 437

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.

It is proper to state, however, that occasionally cult-
ures of genuine typhoid bacilli are encountered that do
not respond to this peculiar influence of typhoid blood,
even though the blood be tested at different stages of
the disease, and even though it causes the characteristic
cessation of motion and clumping with other cultures of
this organism upon which it may be tried.

When employed conversely — i. e., for deciding if
the serum used is from a case of typhoid fever or not —
the reaction constitutes " Widal's serum diagnosis of
typhoid fever." For this purpose it is often necessary
to test several cultures of genuine typhoid bacilli, from
different sources and of varying degrees of vitality, be-
fore a culture is procured that gives the reaction most
conspicuously 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.

WIDAL'S REACTION WITH DRIED BLOOD. — For clin-
ical purposes it is of importance to know that this reac-
tion can be obtained from dried blood — i. e., by the
method suggested by Wyatt Johnston, of Montreal.
In this method a drop of the blood to be tested, ob-
tained by a needle-prick in the cleansed finger or lobe
of the ear, is collected on a bit of clean, unglazed
paper and allowed to dry. The paper is then folded,
kept free from contamination, and taken to the labora-
tory. With a medium size platinum-wire loop a drop
of sterile bouillon, water, or physiological salt solution

438 BACTERIOLOGY.

is gently rubbed upon the drop of dried blood until the
contents of the loop are of a dark amber color; this is
then mixed with a drop of a bouillon culture of typhoid
bacilli on a cover-glass, which is mounted upon the
hollow-ground slide as a hanging drop, when the effect
of the diluted blood upon the culture can be observed
with the microscope. The reaction, if positive, should
occur within a half hour. Many object to this method
because it is impossible accurately to dilute the blood
by the plan used. A number of tests have shown us
that preparations made in this way correspond roughly
with a fresh-blood dilution of from 1 : 1 5 to 1 : 20, as
determined by the hsemoglobinometer. In a small
number of cases in which parallel tests were made
with this and with fresh fluid serum the results were
concordant. We are inclined to the opinion, however,
that in doubtful cases, in which all the available clin-
ical evidence is opposed to either the positive or nega-
tive results of the test, the difficulty is much more
certainly cleared away by the use of highly diluted
and exactly diluted fresh serum than by this method.
Competent observers are of the opinion that in all
such cases the quantity of serum in the hanging drop
should be decreased until it is present in the proportion
of from (not less than) 1 : 50 to 1 : 60, and that, if after
exposure to this dilution for two hours the bacilli are
still motile and not clumped together, or the reaction is
deficient in only one or the other of these peculiarities,
the case from which the serum was obtained may be safely
regarded as not typhoid fever, or if typhoid the exami-
nation was not made at a time when agglutinin was pres-
ent in demonstrable quantities in the circulating blood.
Experience with the dry-blood method at the Mu-

BACILLUS TYPHOSUS. 439

nicipal Laboratory of Philadelphia in more than
12,000 examinations from about 10,000 febrile con-
ditions, leads us to regard the culture used as one of
the most important factors in the test. After deciding
upon the most suitable culture for the reaction — and it
is often necessary to try a great number from various
sources — we have adopted the plan of daily trans-
planting the culture into fresh bouillon and keep-
ing it at a temperature rarely above 20°— 22° C. The
bacilli grown under these circumstances are usually
somewhat longer than when cultivated at higher tem-
perature, and they exhibit a regular, gliding motility
that renders it more easy to follow the individual cells
under the microscope than when they possess the usual
active, darting motion.

In the group of cases examined by us by the dry-
blood method, including typhoid and other febrile con-
ditions, there is a discrepancy between the clinical and
the laboratory diagnosis in from 2 to 3 per cent, of the
cases examined.

In the hands of all who have carefully employed
the Widal reaction for the diagnosis of typhoid fever
the results are reported to have been almost uniformly
satisfactory. In the great majority of cases the reac-
tion is, so far as experience indicates, specific — i. e., a
typical reaction does not occur between typhoid serum
and organisms other than the typhoid bacillus, nor be-
tween the typhoid bacillus and serums other than those
of typhoid fever. There are, however, confusing reac-
tions— so-called pseudo-reactions — in which more or
less clumping of the bacilli and a diminution of motion,
without complete cessation, are observed. These reac-
tions have been seen to occur with normal blood and

440 BA CTERIOLOO Y.

with blood from other febrile conditions. It is said by
Johnston and McTaggart l that they can be prevented
if cultures of just the proper degree of vitality are em-
ployed ; and this corresponds with the results of a fairly
wide personal experience with the test.

The blood of certain animals, as well as a number of
chemical substances, such as corrosive sublimate, alco-
hol, salicylic acid, resorcin, and safranin in high dilution,
cause agglutination of the typhoid bacilli ; but the reac-
tion is not specific, for in most cases they have the same
effect on other motile bacilli.

The method is still in the experimental stage, and
there are numerous features not entirely clear. In
the light of present experience, however, it is fair
presumptive evidence that the serum is from a case
of typhoid fever when unmistakable agglutination and
cessation of motion are seen in from fifteen to twenty
minutes after typhoid bacilli are mixed with the serum
of a suspicious febrile condition.

All the points with regard to morphologic and biologic
characters of bacillus typhosus, and of the organisms
closely resembling it, should be borne in mind in the
examination of drinking-water supposed to be con-
taminated by typhoid dejections, for the organisms
which most closely 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.
1 Johnston and McTaggart: Montreal Medical Journal, March, 1897.

ISOLATING THE TYPHOID BACILLUS. 441

METHODS OF ISOLATING THE TYPHOID BACILLUS.
— Bacillus typhosus is so variable in many of its bio-
logical peculiarities, and is so closely simulated in cer-
tain respects by a group of other organisms to which it
appears to be botanically related, that its identification,
especially outside the infected body, is usually a matter
of considerable difficulty and uncertainty. For these
reasons many efforts have been made to discover specific
reactions for the organism, and with this end in view
many methods have been devised for its isolation from
water, faeces, sewage, and other matters believed to con-
tain it. None of them, however, has given general satis-
faction, and many have proved wholly untrustworthy.
Those worthy of some degree of confidence are as follows :

HLw's Method. — In this method advantage is taken
of the fact that in semisolid nutrient media the greater
motility of the typhoid bacillus enables it to diffuse more
readily through the medium than can the less active
colon bacillus. The endeavor of Hiss was to discover
a method whereby this peculiarity would be favored,
or at least not checked, in the typhoid, and more or
less suppressed in the colon bacillus. A series of experi-
ments "demonstrated that if peptone be omitted and glu-
cose be added to the semi-solid medium, the absence of
the former important nutritive substance and the excess
of acidity resulting from the fermentation of the latter
had only slight effect upon the characteristic develop-
ment of the typhoid bacillus (a diffuse clouding of the
medium), while the influence upon the growth of the
colon bacilli was to prevent, in many cases, their ten-
dency to cloud the medium by sharply restricting their
growth to the point at which they were deposited.1

1 Hiss : Journal of Experimental Medicine, 1897, vol. ii. No. 6, p. 677.

442 BA CTERIOLOG Y.

The composition of the medium used is :

Agar-agar 5 grammes.

Gelatin 80 "

Liebig's beef-extract 5 "

Sodium chloride 5 "

Glucose 5-10

Water 1000 c.c.

The gelatin should be added after the agar-agar and
other ingredients are dissolved ; the volume of the mass
is then brought to 1000 c.c., and finally the reaction is
corrected. This should be equivalent to a degree of
acidity that would require 15 c.c. of a normal sodium
hydroxide solution to the litre to bring it to the phenol-
phtalein neutral point.

When planted as stab-cultures in this medium and
kept at body-temperature the growth of bacillus typho-
sus appears simply as a diffuse cloud, with little or no
tendency to appear more concentrated along the track of
the needle ; while the development of the colon bacillus
is confined to the neighborhood of the needle-track, is
moderately dense, is accompanied by the formation of
gas-bubbles, and the surrounding gelatin is more or
less clear.

These distinctions were found by Hiss to be much
more marked with the slowly or feebly motile speci-
mens of the colon bacillus than when the actively motile
varieties were used. Many of these latter, except for
their power to ferment glucose with liberation of gas,
were almost indistinguishable from the typhoid bacillus
in so far as their power to wander through and cloud
the medium was concerned. For the isolation and dif-
ferentiation of colonies of the two organisms by the
plate method the following medium was employed :

ISOLATING THE TYPHOID BACILLUS. 443

Agar-agar 10 grammes.

Gelatin 25 "

Liebig's beef-extract 5

Sodium chloride 5 "

Glucose 10 "

Water 1000 c.c.

The reaction of this medium is equivalent to 2 per cent,
of normal acid to the litre — i. e., an acidity that would
require 20 c.c. of normal sodium hydroxide solution to
the litre to bring it to the phenolphtalein neutral point.
In plates made from this medium the deep colonies of
bacillus typhosus are small, more or less spherical, and
have a rough, irregular outline. Their most character-
istic feature " consists of well-defined, filamentous out-
growths, ranging from a single thread to a complete
fringe around the colony. The young colonies are at
times composed solely of threads." The fringing
threads grow almost straight out from the colonies.
The surface colonies are small and have usually a
dense centre that is surrounded by an almost trans-
parent zone or by a fringe of threads somewhat similar
to those seen about the deeper colonies.

The deep colonies of the colon bacillus are, as a rule,
larger, denser, of an oval or lens-shape, and are more
sharply circumscribed than those of bacillus typhosus.
'On the surface they are also larger, and, as a rule,
spread out as a moderately thick layer from a denser
centre. The younger the colonies of the typhoid bacil-
lus the more characteristic their appearance. They are
seen at their best after from 16 to 18 hours' growth at
37.5° C.1

1 The reader is referred to the original article for many important
details that are not included here.

444 BACTERIOLOGY.

Method of Capaldi and Proskauer.1 — As a result of
an elaborate series of experiments, these authors recom-
mend the use of two special culture-media for the dif-
ferentiation of the typhoid and colon bacilli.

Medium No. 1 consists of:

Asparagine 0.2 per cent, in distilled water.

Mannite 0.2

Sodium chloride 0.02

Magnesium sulphate .... 0.01

Calcium chloride 0.02

Mono-potassium phosphate . 0.2

Medium No. 2 consists of:

Witte's peptone 2.0 per cent, in distilled water.

Mannite 0.1 "

Both media are to be sterilized, the reaction brought
to the litmus neutral point with caustic potash solu-
tion, and enough litmus tincture then added to them to
cause a distinct, though not too intense, purple color.
After this they are to be again sterilized, when they
are ready for use.

After 20 hours at 37°-38°C. typical colon bacilli
and the varieties of this organism develop in both solu-
tions, but produce add only in medium No. 1.

The typhoid bacillus grows only in medium No. 2,
and its growth is accompanied Jjy the production of
an acid reaction.

The growth of bacillus coli in medium No. 2 is ac-
companied by a slight alkaline reaction.

The alterations in reaction are indicated by the cor-
responding changes in the color of the litmus tincture
in the media.

1 Capaldi and Proskauer: Zeitschrift fur Hygiene und Infektions-
krankheiten, 1896, Bd. xxiii. S. 452.

ISOLATING THE TYPHOID JLK'ILLUS. 445

It is interesting to note that in this test the usual
reactions produced by these organisms in peptone media
containing the ordinary fermentable carbohydrates, such
as glucose and lactose, are reversed.

The authors state that this method has thus far shown
itself to be infallible for the differentiation of cultures
of typhoid and colon bacilli obtained by them from
every available source.

Hunter1 recommends the use of neutral red as a dif-
ferential test. He employs it in the proportion of 0.5
to 1.0 c.c. of a saturated watery solution to 10 c.c. of
nutrient agar-agar. The reducing action of the colon
bacillus causes the color to become yellow, while the
normal red color is not affected by the typhoid bacillus.

METHOD OF v. DRIGALSKI AND CONRADI. — v. Dri-
galski and Conradi2 published a method for the detec-
tion of bacillus typhosus in water. In this method
they sought to bring about a separation of bacillus
typhosus and bacillus coli on the basis of their ferment-
ing properties. This they sought to do in such a man-
ner as not to hinder the growth of bacillus typhosus,
but rather to make the conditions of growth as favor-
able as possible. Their studies of the fermentative
properties of bacillus typhosus and bacillus coli were
carried out upon the following carbohydrates :

1. Monosaccharides : Of hexoses : glucose, fructose,
gaiactofle, mannite and dulcit. Of pentoses : arabinose,
xylose, and rhamnose.

2. Disaccharides : Saccharose, maltose, lactose.

3. Polysaccharides : Amylum, inulin, and dextrin.

1 Hunter: The Lancet, March 2, 1901.

1 v. Drigalski and Couradi : Zeitschrift fur Hygiene, Bd. 39, 1902,
p. 283.

446 BA GTE RIO LOG Y.

These substances were added to sterile litmus agar in
the proportion of 1 to 100, and sterilization was again
carried out for five to ten minutes in streaming steam.

In their studies with these culture media they found
that different organisms behaved differently with the
lactose. While colon cultures produce a red colora-
tion of the litmus when grown on the surface of
the medium, the typhoid cultures cause no change.
They also found that a culture medium of greater
density was of distinct value, and consequently they
employed 3 per cent, agar-agar medium, and in order
to overcome the marked acid production by the colon
organism they added small quantities of sodium car-
bonate. This increase in the salt concentration of the
culture medium did not bring about plasmolysis of the
typhoid organism. In order to make the conditions
as favorable as possible for the growth of bacillus
typhosus they experimented with a large number of
artificially prepared albuminous substances. Besides
peptone they experimented with tropon and nutrose.
The addition of nutrose brought about a more intense
blue coloration by the typhoid organism, which they
attribute to the alkali albuminate nature of the sodium
casein. There was also a more voluminous growth of
the typhoid organism when they employed meat infu-
sion in the preparation of the medium.

Still another difficulty was encountered in the identi-
fication of bacillus typhosus in stools because of colo-
nies of varieties of micrococci also present, which,
through their marked acid production, colored the whole
surface of the medium. After a large number of nega-
tive experiments they succeeded in finding an elective
bactericidal aniline dye against the majority of these

ISOLATING THE TYPHOID BACILLUS. 447

disturbing organisms, which, however, was not injurious
to the typhoid and colon organisms. In the selection
of an antiseptic coloring substance they had to be cer-
tain that the fermentative properties of the typhoid
bacteria were not influenced thereby. They experi-
mented with the following analine dyes : Malachite
green, 1 to 1,000,000; brilliant green, 1 to 1,000,000;
medicinal methylene-blue, 1 to 100,000 ; methyl violet,
1 to 100,000, and crystal violet, 1 to 100,000. Of
these the crystal violet gave the most satisfactory result.

v. Drigalski and Conradi give the following direc-
tions for the preparation of their culture medium :

a. Preparation of agar : 3 pounds of finely chopped
beef are placed in two litres of water and set aside for
twenty-four hours. The meat infusion is boiled for one
hour, filtered, and 20 grammes of Witte's peptone, 20
grammes of nutrose, and 10 grammes of sodium chloride
are added and again boiled for an hour, filtered, and 60
grammes of agar-agar are added, boiled for three hours
(or one hour in the autoclave), rendered slightly alka-
line to litmus-paper, filtered, and boiled for one-half
hour.

6. Litmus solution : Litmus solution (according to
Kubel and Tiemann) 260 c.c. boiled ten minutes, add
30 grammes chemically pure lactose, boil fifteen min-
utes.

c. The hot litmus-lactose solution is added to the hot
nutritive agar, thoroughly mixed, and the alkaline reac-
tion is again restored. To this medium is then added
4 c.c. of a hot sterile solution of 10 per cent, water-free
soda, 20 o c. of freshly prepared solution of 0.1 gramme
crystal violet (Hochst) in 100 c.c. of warm sterile dis-
tilled water.

448 BA CTERIOL OG Y.

One now has a meat-infusion-peptone-nutrose-agar
with 13 per cent, of litmus solution and 0.01 per thou-
sand crystal violet, which becomes very hard on solidi-
fying, without becoming too dry. Plates are poured
with this material which can be held in readiness for
some time, and the remainder of the medium is placed
in flasks in portions of 200 c.c. each.

If the lactose is boiled for a longer time than directed
it is reduced with an acid reaction of the culture medium
and the content in lactose falls below the required quan-
tity, and the alteration in the color of the colon colonies
appears too early. For this reason it is also necessary
to liquefy the agar as quickly as possible in pouring
plates from the agar medium stored in flasks.

In employing this culture medium it is necessary to
have a uniform suspension of a portion of the material to
be examined and to make a series of plate inoculations
from this suspension. These plate inoculations are best
made by means of a sterile glass spatula.

After fourteen to sixteen hours at 37° C., and still
better after twenty to twenty-four hours, the cultures
are readily differentiated :

a. Bacillus coll: All cultures of true colon that have
been examined form colonies of 2 to 6 or more millimetres
in diameter, of reddish color and translucent. In each intes-
tinal evacuation there are usually several varieties of colon
colonies which differ according to their size and texture,
translucency, and the intensity of the alteration of the
color which they bring about. Many colon colonies are
bright red, some are cloudy, and others are quite opaque,
dark-wine red in color, while still others form large col-
onies which are surrounded by a red halo.

b. Bacillus typhosus: The .colonies have a diameter

ISOLATING THE TYPHOID BACILLUS. 449

of 1 to 3 millimetres, rarely larger. Their color is
blue, with a tendency toward violet. In structure they
are glistening, with a single contour, somewhat of the
nature of a dew drop. Only in isolated instances is the
colony larger and more cloudy in appearance.

METHOD OF HOFFMANN AND FICKER. — Hoffmann
and Ficker1 have published a new method for the isola-
tion of bacillus typhosus from infected waters, which
consists in the addition of increasing quantities of caf-
fein, crystal violet, and nutrose to large quantities of the
water. They add 1 per cent, of nutrose, 0.5 per cent,
of caffein, and 1 per cent, of a 0.1 to 100 solution of
crystal violet to the water, and incubate at 37° C. for
twelve to thirteen hours. In this manner they reduce
the number of water bacteria, while bacillus typhosus
increases in numbers. The three solutions to be added
to the water are prepared as follows :

1. A solution of 10 grammes nutrose in 80 c.c. of
distilled water. The solution is placed in a water-bath
for several hours and is not filtered.

2. A solution of 5 grammes of caffein in 20 c.c. of
warm (80° C.), sterile distilled water. The solution is
to be freshly prepared and should not be shaken.

3. A solution of 0.1 gramme of crystal violet in 100
c.c. of sterile' distilled water. This must be freshly
prepared each time.

900 c.c. of the water to be examined are placed in a
flask and the three solutions are added, and the mixture
thoroughly shaken. After incubation, the water is exam-
ined according to several well-known methods. For
instance, some of the supernatant portion of the fluid
is removed and spread out in a thin layer upon plates

1 Hoffmaiiu and Ficker : Hygienische Ruudschau, Bd. 14, 1904, p. 1.
29

450 BACTERIOLOGY.

intuit1 with Drigalski-Conradi agar, or 500 c.c. of the
water may be precipitated with typhoid immune .serum
according to the method of Altschuler and incubated
attain for three hours and then plated, or 500 c.c. may
be precipitated by the chemical-mechanical method of
Ficker.1 The sediment in the portion of water which
has been precipitated in this manner is distributed over
a series of three or four plates of the Drigalski-Conradi
medium. Subsequently the sediment is diluted three-
or fourfold and distributed over another series of plates.
By these methods Hoffmann and Ficker succeeded in
isolating bacillus typhosus when present in a mixture
of 1 : 51,867 water bacteria.

v. Jaksch and Rau l employed the method of Hoff-
mann and Ficker for the isolation of bacillus typhosus
from the water supply of the city of Prague, and suc-
ceeded in finding bacillus typhosus in three out of five
samples examined, one of which was taken from a tap
in the hospital, while the others were taken from the
Moldau, at different points along the city front. The
two negative samples were derived from points along
the upper portion of the stream where there was less
opportunity for the water to become polluted. The
demonstration of the presence of bacillus typhosus in
these samples of water was substantiated by all the
known cultural methods as well as by the agglutination
test, using for the latter purpose the serum of a highly
immunized rabbit which agglutinated the bacteria iso-
lated from the water in the proportion of 1 : 10,000.
Higher dilutions were not made.

1 Ficker: Hygienische Ruudschau, 1904, Bd. xiv., S. 7.

2 v. Jaksch and Eau : Centralblatt fur Bacteriologie, 1904, Bd. xxxvi.,

S. 584.

ISOLATING THE TYPHOID BACILLUS. 451

PRECIPITATION METHOD OF FiCKER.1 — The method
first proposed by Vallet,2 and modified by Schiider,3 for
the demonstration of bacillus typhosus in water consists
in the precipitation with sodium hyposulphite and nitrate
of lead, when the precipitate is dissolved with sodium
hyposulphite. This method was studied by Ficker, in
the laboratory, by adding to sterile river water definite
quantities of bacillus typhosus, but the results were not
satisfactory. The experiment showed that a portion of
the bacilli were not carried down with the precipitate,
while another portion were killed. These negative re-
sults led him to employ, at the suggestion of Hoffmann,
sulphate of iron as a precipitating substance, and the
sediment was dissolved with neutral potassium tartrate.

The method employed is as follows : Two litres of
the water to be examined are placed into a narrow
sterile glass cylinder and rendered alkaline with 8 c.c.
of 10 per cent, soda solution, and afterward 7 c.c. of a
10 per cent, sulphate of iron solution are added and
mixed with the water by means of a sterile glass rod.
The cylinder is then placed in the ice chest. Precipi-
tation is complete in two to three hours. The over-
standing water is syphoned off, and the precipitate or
portions thereof are poured into sterile test-tubes. To
this precipitate is now added about a half volume of a
25 per cent, solution of neutral potassium tartrate. The
test-tube is closed with a sterile rubber cork and the
mixture thoroughly agitated, whereby the precipitate is
completely dissolved. With a sterile pipette one part
of the mixture is mixed ' in a test-tube with two parts

1 Ficker: Hygienische Rundschau, 1904, Bd. xiv., S. 7.
* Vallet: Arch, de med. exp. et d'anat. path., 1901.
8 Schuder: Zeitschr. fur Hygiene, Bd. xlii., S. 317.

452 BACTERIOLOGY.

of sterile bouillon, and this mixture is distributed over
a series of Drigalski-Conradi plates. Ficker advises
when possible the use of a centrifuge for the separation
of the precipitate, as he believes the results are likely
to be more satisfactory.

ISOLATION OF BACILLUS TYPHOSUS FROM CADA-
VERS.— The spleen of a patient dead of typhoid fever is
the most reliable source from which to obtain cultures
of the typhoid bacillus for study. But it must always be
remembered that the same channels through which the
typhoid bacillus gains access to this viscus are likewise
open to other organisms present in the intestines, and
for this reason bacillus coli, a normal inhabitant of the
colon, may also be found in this locality.

NOTE. — Obtain a pure culture of typhoid bacilli, and
from this make inoculations upon a series of potatoes
of different 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.
Are any differences in the growths noticeable ?

Make a series of twelve tubes of peptone solution to
which rosolic acid has been added. Inoculate them all
with as nearly 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

BACILLUS COLL 453

marked. Examine this culture unstained. Do the
organisms look as if they contained spores? How
would you demonstrate that the vacuolations are not
spores ? What is the crucial test for spores ?

Obtain from normal faeces a pure culture of the com-
monest organism present. Write a full description of
it. Now make parallel cultures of this organism and
of the typhoid bacillus on all the different media ? How
do they differ ? In what respects are they similar ?

BACILLUS COLI (ESCHERICH), MIGULA, 1900.

Synonyms: Neapeler bacillus, Emmerich, 1884 ; Bacillus pyogenes
fcetidus, Passet, 1885 ; Emmerich's bacillus, Eisenberg, 1886 ; Bac-
terium coli commune, Escherich, 1886.

This organism was discovered by Escherich, in 1886,
in the intestinal discharges of milk-fed infants. It has
since been demonstrated to be a normal inhabitant of the
intestines of man and of certain domestic animals
(bovines, 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
subject for study. Some have even gone so far as to
regard them as but varieties of one and the same
species, though in the present state of our knowledge
this is an assumption for which as yet there are
not sufficient grounds. That they possess in common
certain general points of resemblance and often ap-
proach one another in some of their biological peculiar-
ities is true ; but, as we shall learn, they each possess
peculiarities which, when considered together, render

454 BACTERIOLOGY.

their differentiation from one another a matter of but
little difficulty.

With the wider application of bacteriological methods
to the study of pathological processes it was occasion-
ally observed that, under favorable circumstances,
bacillus coli disseminated from its normal habitat
and appeared in remote organs, often associated witn
diseased conditions. This was at first considered
of but little importance, and its presence in these
localities was usually regarded as accidental. Its
repeated appearance, however, in different parts of
the body outside of the intestines, and the frequency
of its association with pathological conditions, ultimately
attracted attention to it, and in consequence during the
past few years a great deal has been written concerning
the possible pathogenic nature of this organism.

The fact that it is a commensal species, always inti-
mately associated 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 a pathogenic organ-
ism, there is, nevertheless, sufficient evidence to war-
rant the statement that under favorable conditions of
reduced vitality on the part of the animal tissues, this
organism may assume pathogenic properties, so that its
presence in diseased conditions is not always to be con-
sidered as accidental, though this is frequently the case.

The morphological and cultural peculiarities of bacillus
coli are as follows :

BACILLUS COLL 455

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 are associated in the same culture.
It may occur as single cells, or as pairs joined end to
end.

It has no peculiar morphological features 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 at time so sluggish that a
positive opinion is often difficult.

By Loffler's method of staining, flagella can be de-
monstrated, though usually not in such numbers as are
seen to occur on the typhoid fever bacillus.

It does not form spores.

It grows both with and without free oxygen.

ON GELATIN. — On the surface its colonies appear as
small, dry, irregular, flat, blue-white points that are
commonly somewhat dentated or notched 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 ex-
amined with a low-power lens, they are at first seen to
be finely granular and of a very pale greenish-yellow
color; later they become denser, darker, and much
more markedly granular; in shape they are round,
oval, and lozenge-like. When the surface colonies are
viewed under a low power of the microscope they pre-
sent essentially the same appearance as that given for
the colonies of the bacillus of typhoid fever, viz., they

456 BACTERIOLOGY.

resemble flattened pellicles of glass-wool, or patches of
finely ground colorless glass. Colonies of this organ-
ism on gelatin are frequently encountered that cannot
be distinguished from those resulting from the growth
of bacillus typhosus ; although, 'as a rule, their growth is
a little more luxuriant.

In stab- and smear-cultures on gelatin the surface-
growth is flat, dry, and blue-white or pearl color.
Limited growth occurs along the track of the needle in
the depths of the gelatin. As the culture becomes
older the gelatin round about the surface-growth may
gradually lose its transparency and become cloudy,
often quite opaque. In still older cultures small root-
or branch-like projections from the surface-growth into
the gelatin are sometimes seen. At times these may be
of a distinctly crystalline appearance.

It does not cause liquefaction of gelatin.

Its growth on nutrient agar-agar and on blood-serum
is luxuriant, but not characteristic.

In bouillon it causes diffuse clouding with sedimen-
tation. In some bouillon cultures an attempt at pel-
licle-formation on the surface may be seen, but this is ex-
ceptional. In old bouillon cultures the reaction becomes
alkaline and a decided facal odor may be detected.

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 firm coagulation of the
casein in about thirty-six hours, though frequently this

BACILLUS COL/. 457

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
ferraentation-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.

It produces indol in both bouillon and peptone solu-
tion.

In Dunham's peptone solution it produces indol in
from forty-eight to seventy-two hours.

It stains with the ordinary aniline dyes. It is decol-
orized when treated by the method of Gram.

By comparing what has been said of bacillus typho-
ms and of bacillus coli it will be seen that, while they
simulate each other in certain respects, they nevertheless
possess individual characteristics by which they may
readily be differentiated. The least variable of the dif-
ferential points are :

1. Motility of bacillus typhosus is much more con-
spicuous, as a rule, than is that of bacillus coli.

2. On gelatin, colonies of the typhoid bacillus de-
velop 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

458 BA CTKRIOLOG Y.

milk with acid reaction The colon bacillus does this
in from thirty-six to foi y-eight liours in the incubator.

5. The typhoid bac .lus never causes fermentation,
with liberation of gas, .n media containing glucose, lac-
tose, or saccharose. The colon bacillus is conspicuous
for its power of causing gaseous 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 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-
panied by the production of indol in from forty-eight
to seventy-two hours at 37° to 38° C.

ANIMAL , INOCULATIONS. — As with the bacillus of
typhoid 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 effects that do appear are in
most instances to be attributed to the toxic rather than
to the infective properties of the culture used.

When introduced into the subcutaneous tissues of
mice it has no effect, while similar inoculations of guinea-
pigs are sometimes (not always) followed by abscess-
formation at the point of operation, 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 hyperaemia
and ecchvmoses of the small intestine, together with

BACILLUS COLL 459

swelling of Peyer's patches. The caecum 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-
rhoea before death, which may, in the acute cases, result
in from three to forty hours. In another group of
cases acute fatal intoxication does not result, and the
animal lives for weeks or months, dying ultimately of
what appears to be the effects of a slow or chronic form
of infection. For a few hours after inoculation these
animals present no marked symptoms ; exceptionally,
somnolence and diarrhoea have been observed at this
period, indicating acute intoxication from which the
animal has recovered. The affection is unattended by
fever. The most marked symptom is loss of weight.
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 an excess of serum. The viscera are
anaamic. The spleen is small, thin, and pale. Ex-
ceptionally ulcers and ecchymoses are observed in the
cfficum, 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 ; in some cases the quantity of bile may not exceed
the normal, but in others the gall-bladder may be ab-
normally distended with bile. The bile is nearly color-

460 BACTERIOLOGY.

less or has a pale yellowish or brownish tint, with little
or no 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 micro-
scopically of bile-stained, apparently necrotic, epithelial
cells ; leucocytes in small numbers ; amorphous masses
of bile-pigment, and bacteria often in zoogloea-like
clumps. Similar material is found in the larger bile-
ducts.

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 cm. in
extent. By microscopic examination they are found to
represent localities where the liver-cells have undergone
necrosis accompanied by emigration of leucocytes, and
the cells about them are in a condition of fatty degenera-
tion. In sections of the liver masses of the bacilli may
be discovered in and about the necrotic foci just de-
scribed.

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

BACILLUS PARATYPHOSUS.

During the past five years the careful bacteriological
examination of cases of continued fever in which the

1 Consult paper by Blachstein on this subject, Johns Hopkins Hos-
pital Bulletin, 1891, vol. ii., p. 96.

BACILLUS PARATYPHOSUS. 461

agglutination reaction with the typhoid bacillus was
absent, has revealed a group of bacilli which differ from
bacillus typhosus in certain important particulars. These
bacteria possess characters which are intermediate be-
tween those of bacillus typhosus and bacillus coli, some
resembling more closely bacillus typhosus, and others
bacillus coli, and for these reasons they have sometimes
been classed as the intermediate group. Some of the
organisms isolated from such cases of continued fever
resemble very closely bacillus enteriditis, which Gaertner
found in cases of meat poisoning.

The general opinion to-day is that these organisms
produce a form of infection sometimes resembling in
many of its characters that produced by bacillus ty-
phosus. The infection, however, is usually of a milder
type and only a comparatively small number* of cases
have terminated fatally, so that the pathology of the
disease is not well known. Moreover, the biological char-
acters of the different organisms isolated from cases of
paratyphoid fever show such wide variations that it is
probable that the pathology of different cases also
varies with the particular type of organism causing the
infection.

Buxton l was one of the first to make a careful com-
parative study of the morphology and biology of this
group of organisms. He classifies the intermediary
group of organisms in the following manner:

" Paracolons : those which do not cause typhoidal
symptoms in man. A group containing numerous dif-
ferent members, but culturally alike.

" Paratyphoids : those which cause typhoidal symp-
toms.

1 Buxton: Journal of Medical Research, 1902, vol. viii., p. 201.

462 BA CTERIOL OG Y.

" («) A distinct species culturally unlike the para-
colons.

" (6) A distinct species culturally resembling the
paracolons."

Buxton acknowledges, as has also been found by
others, that some of those producing typhoidal symp-
toms cannot be distinguished culturally from some
members of the paracolon group. Morphologically, the
intermediates cannot be distinguished with certainty from
each other, nor from bacillus typhosus or bacillus coli.
All the organisms of the intermediate group have the
morphological characters of the colon-typhoid group of
organisms.

The biological differences on agar-agar, blood serum,
gelatin, and bouillon, between the members of the inter-
mediate group, and between bacillus typhosus and bacil-
lus coli are too insignificant and uncertain to be of any
assistance in a differentiation between members of the
group. In litmus milk certain well-marked differences
between different members of the group are noticed.
None of the organisms of the intermediate group pro-
duce coagulation. Some produce a slight initial acidity,
which is later followed by an alkaline reaction. Still
other members of the group produce an acidity amount-
ing to 1 per cent.

Buxton states that the intermediates can be distin-
guished from bacillus typhosus by their power of fer-
menting the disaccharid maltose and all the monosac-
charids with gas formation. On the other hand they
can be distinguished from bacillus coli by their inability
to form acid and gas in lactose media.

The agglutination reaction of members of the inter-
mediate group with the serum of an animal immunized

B A (JILL US PARATYPHOSUS.. 463

with one of the organisms varies with the different
organisms. The more closely a member of the group
resembles culturally the organism employed in immuni-
zing the animal the more readily is it agglutinated. In
attempts to diagnose paratyphoid infection it is well to
bear this fact in mind and make agglutination tests with
different members of the group and the blood of the
patient.

CHAPTER XXI.

BACILLUS DYSENTERIC.

The group of bacilli found in cases of epidemic, endemic, and sporadic
dysentery — The morphological, biological, and pathogen ical char-
acters of the several members of the group — The differentiation
of the different types of bacilli.

THE investigations of epidemic dysentery by Shiga,
Flexner, Kruse, Vedder, Duval, Basset, Park, and
many others, have demonstrated that this disease is
caused by an organism that varies somewhat in its char-
acters as encountered in different cases. So far at least
four distinct types of the organism have been found that
differ in minor particulars, though not sufficiently to war-
rant their separation from each other into distinct species.
The type of organism first encountered by Shiga in
Japan is the one that is probably very widely distrib-
uted because it has been found in practically every place
where investigations have been made. The type of
organism encountered by Flexner in his investigations
in the Philippine Islands, Has also been found very gen-
erally in the United States, especially in dysentery
occurring in infants. The tyj>e of organism isolated by
Hiss and Russell, and later by Park and his associates,
has most of the characteristics of the Flexner type of
organism, though the agglutination reaction shows that
it is not identical with it.

At first the German investigators were inclined to
regard the Flexner type of organism as having no caus-
ative relation whatever to dysentery, but the later de-

464

BACILLUS DYSENTERIC. 465

tailed studies all strengthen the assumption that the
Shiga type of the organism is not the only one concerned
in causing epidemic dysentery. In a number of cases
of dysentery two, and at times three, types of bacillus
dysenteric have been encountered. Thus far it has been
impossible to differentiate clinically between the infections
produced by the one or the other type of organism.
Both severe and mild cases have been shown to be in-
fected with either type, and the amount of blood and
mucus in the stools appears to be the same in infection
with each type of organism.

THE SHIGA TYPE OF ORGANISM. — The evidence
presented by Shiga, who discovered this organism in
1898, in Japan, and the subsequent observations of
Flexner upon dysentery in the Philippine Islands,
leaves little room for doubt that, in so far as acute
epidemic dysentery is concerned, the organism under
consideration may reasonably be regarded as the causa-
tive factor. By both Shiga and Flexner the organism
was almost uniformly encountered in the intestinal con-
tents, the intestinal walls, and the mesenteric glands
during the acute stages of the disease. Later it was
frequently missed, and tin's became more common as the
malady progressed to chronicity or recovery.

It is a bacillus of medium size, with rounded ends.
In general its morphology may properly be likened to
that of either the typhoid or colon bacillus.

It is motile and does not form spores.

It can be stained with any of the ordinary aniline
dyes. It is decolorized by the method of Gram. It
may l>e cultivated on all the ordinary media. It grows
at room-temperature, but bettor at th<> temperature of
the l>ody. It does not liquefy gelatin.

30

46t> BACTERIOLOGY.

The colonies upon agar-agar present nothing charac-
teristic ; those on gelatin are at first — i. e., just after iso-
lation from the body — like those of bacillus typhosus ;
later on, after the organism has been kept under condi-
tions of continuous sap rophy tic growth, the colonies may
be thicker, denser, moister, and less translucent, but
always suggesting the peculiar, leaf-like contour char-
acteristic of the colonies of the colon-typhoid group
under similar conditions. In gelatin stab-cultures there
is growth along the track made by the needle, and little
tendency to lateral development over the surface.

On potato, its growth may be so limited as to be
scarcely visible, or it may appear as a moderately volu-
minous grayish-brown or light-brown layer along the
track made by the needle, and spreading laterally be-
yond this. Between these extremes all gradations may
be seen according to the suitability of the potato used.

In bouillon it causes uniform clouding and a more or
less dense sediment. It does not form a pellicle.

Growth on blood-serum is not accompanied by lique-
faction (digestion).

Glycerin agar-agar appears less suited to its growth
than plain nutrient agar-agar.

It does noj_Jerment either glucose, saccharose, or lac-
tose, with liberatjpjL of^gas ; although in glucose media
there is a slight increase of acidity.

When grown in litmus-milk, the latter after twenty-
four to seventy-two hours at body-temperature becomes
a pale lilac. Later on — i. e., after six to eight days —
there is a development of alkali, and the lilac tint gives
way to a deep, distinct blue color. Coagulation is never
observed.

It is either incapable of producing indol, or has this

BACILLUS DYSENTERIC. 467

faculty to so limited a degree as to make the matter
doubtful.

"When mixed with blood-serum of individuals suffer-
ing from this form of dysentery a positive agglutination
reaction is often obtained.

It is pathogenic by both subcutaneous and intraperi-
toneal inoculation for the ordinary laboratory test-ani-
mals— i. e.} mice, guinea-pigs, and rabbits.

When injection is made beneath the skin, death results
in from two to four days, according to the dose and viru-
lence of the culture used.

The most striking lesion is that observed at and about
the site of inoculation. This consists of oedema, hem-
orrhagic exudation, and, in delayed cases, more or less
of pus formation. The subcutaneous lymph-glands are
often enlarged and reddened, and a serous exudation is
frequently encountered in the great serous cavities. Of
the animals mentioned, the rabbit is most apt to survive
the subcutaneous inoculation.

When injected into the peritoneal cavity, death takes
place in from a few hours to five or six days, according
to dose and virulence of the culture used.

At autopsy the superficial lymph-glands are enlarged
and reddened ; the peritoneum contains more or leas of
turbid fluid and small masses of leucocytes ; the pleural
and pericardial cavities may contain clear fluid ; the
spleen is swollen ; the adrenals and kidneys are con-
gested ; there may be a grayish exudate over the liver,
spleen, and intestines, the bloodvessels are injected ; the
small intestine may be filled with semifluid or fluid mat-
ter ; there may be ecchymosis of the intestinal mucosa,
and Peyer's patches may be enlarged and reddened.

The distribution of the bacilli varies : sometimes

468 BACTERIOLOGY.

there is a general invasion of the body by the bacilli ;
at others they are only to be found at the local site of
inoculation. Sometimes they can be detected in the
intestinal contents after both subcutaneous and intra-
peritoneal inoculation; at other times they cannot,

If the stomach contents be neutralized and large doses
of the bacilli be administered per os, death may occur.
Under these conditions the small intestine is hyperaemic
and contains blood-stained mucoid matter, from which
the bacilli may usually be cultivated.

If cultures be fed to cats after administration of
croton oil, a fatal diarrhea may ensue. The mucous
membrane of the large intestine is injected, its surface
covered with mucus, and its contents mucoid. From the
latter the bacilli may be recovered in culture.

A fatal diarrhoea may follow the simple feeding of
cultures to dogs. This occurs in somewhat less than
six days. The condition of the contents and walls of
the large intestine is essentially similar to that seen in
the cat.

In view of the fact that marked evidences of intoxi-
cation may follow upon the injection of suspensions of
dead cultures of this organism (solid cultures killed by
exposure to 60° C.), it is probable that the pathogen-
icity of this organism is referable to the poisonous nature
of the proteid making up the bodies of the bacilli, rather
than to a soluble intoxicant secreted or manufactured by
them in the course of their growth.

THE HISS-RUSSELL TYPE OF ORGANISM. — In the
detailed study of dysentery and summer diarrhoea in
infants, which has been in progress for several years,
a type of bacillus dysenteriae has been encountered
which has the property of fermenting mannite as well

BACILLUS DYSENTERIC. 469

as dextrose. The Shiga type ferments dextrose, but
none of the other carbohydrates.

THE STRONG TYPE OF ORGANISM. — This type of
organism has many of the characters of the Harris type,
though it ferments only mannite, dextrose, and saccha-
rose.

THE HARRIS TYPE OP ORGANISM. — This type of
bacillus dysenteric was first encountered by Strong
while working in the Philippine Islands. It has since
been encountered quite frequently in the United States,
especially in the summer diarrhoaas in infants. This
organism ferments mannite as well as dextrose, maltose,
saccharose, and dextrin.

It is only by careful observations of the reactions
with the different carbohydrates that it is possible to
differentiate between these different types of bacillus
dysenteriae, as has been shown by Hiss l and by others.

THE AGGLUTINABILITY OF BACILLUS DYSENTERIC.
— The study of the influence of the agglutinins in dys-
entery immune serum has also served to differentiate
between different types of bacillus dysenterise. Normal
serums, especially those of bovines and of goats, also
yield very instructive results. These variations in the
agglutinability of the several types of bacillus dysenteric,
especially in normal serums, were first pointed out by
Bergey,2 and have since been substantiated by many
other investigators (see especially Park and Hiss, loc. cit.).

The different types of bacillus dysenteriae can easily
be distinguished by their relative agglutinability. In
order to bring out the relative influence of the immune
serum upon each variety of bacillus dysenterise it is

1 Hiss : Journal of Medical Research, vol. viii., Dec., 1904.

2 Bergey: Journal of Medical Research, 1903, vol. v., p. 21.

470 BACTERIOLOGY.

necessary to test carefully the limits of its agglutinating
power for each variety. When this is done it will be
found that the serum of an animal immunized with the
Shiga type of organism will agglutinate that type of
organism in high dilutions, say 1 : 5000, while the
Harris type of organism will only be agglutinated in
dilutions of 1 : 200, and the Hiss-Russell type of organ-
ism in dilutions of 1 : 50. On the other hand, the serum
of an animal immunized with the Flexner type of or-
ganism will agglutinate that type of organism in high
dilutions, say 1 : 10,000, while the other two types of the
organism will be agglutinated only in dilutions of 1 : 100.
The serum of an animal immunized with the Hiss-
Russell type of organism will agglutinate that type of
organism in dilutions, say of 1 : 1000, while the Harris
type is agglutinated only in dilutions of 1 : 100, and the
Shiga type in dilutions of 1 : 20.

PROTECTIVE INOCULATION. — By the repeated inocu-
lation of animals with cultures of this organism, killed
either by heat or by chemicals, it has been found pos-
sible to protect them against otherwise fatal doses of the
living virulent organism. When treated in this way,
the goat supplies a serum that exhibits not only an
agglutinating power over the living bacilli, but pos-
sesses both protective and curative properties when in-
jected into other susceptible animals.

"During 1898-1899 Shiga1 employed a protective
serum, made after the foregoing principles, in the treat-
ment of dysentery in human beings. During the period
mentioned he treated 266 cases, and had a death-rate of

1 See " The Epidemic Dysentery of the Past Twenty Years in Japan,"
by Stuart Eldridge, M. D., U. S. Marine-Hospital Service, Public Health
Reports, 1900, vol. xv., No. 1, pp. 1-11.

BACILLUS DYSENTERIC. 471

9.6 per cent. ; while for 1736 cases occurring at the same
time and in the same locality, but not so treated, there
was a death-rate of 34.7 per cent.1

Through the studies of Vedder and Duval the
observations of Shiga, of Flexner, and of Kruse, upon
acute dysentery in Japan, in the Philippine Islands, in
Puerto Rico, and in Germany, are found to be appli-
cable to acute dysentery occurring in this country.
The micro-organism described by Shiga was found by
Vedder and Duval in 22 cases of acute dysentery
occurring in Philadelphia, Lancaster, Pa., and New
Haven, Conn.; those in Lancaster and in New Haven
having been institutional outbreaks of the disease.2

Kruse3 states that 80^00 gramme of dysentery im-
mune serum protects a guinea-pig against the minimum
lethal dose of the culture. In 100 cases treated with
the serum the mortality was 8 per cent, as against 10 to
11 per cent, in cases without serum treatment.

Holt4 summarizes the results obtained in the treat-
ment of 87 cases with dysentery immune serum. De-
cided improvement was noted in only 12 of the patients.
These were principally hospital cases, and hence rather
grave forms of the disease. Another factor which prob-
ably operated against the favorable influence of the
serum is the fact that the serum treatment was generally

1 The foregoing sketch is compiled from :

Shiga: "Ueber den Dysenteric-bacillus (Bacillus Dyseiiteriae),"
Centralblatt fur Bakteriologie und Parasitenkunde, 1898, Abt. i. Bd.
xxiv. Nos. 22, 23, 24.

Flexner: "On the Etiology of Tropical Dysentery," Philadelphia
Medical Journal, Sept. 1, 1900.

'Journal Experimental Medicine, 1902, vol. vi. p. 181.

3 Kruse: Deutsche mod. Wochenschr., Jan. 8, 1903.

4 Holt : Studies from the Rockefeller Institute for Medical Research,
1904, vol. ii.

472 BACTERIOLOGY.

preceded by a careful bacteriological analysis of the stools
in order to establish a positive diagnosis, requiring two
or three days so that the serum treatment was instituted
late in the course of the disease.

Holt points out that the conditions necessary to obtain
success in the serum treatment of cases of dysentery are :
First, the early use of the serum, before serious lesions
have developed or before the patient's general condition
has been too profoundly impaired ; second, the serum
must be administered in repeated doses, one or two doses
a day, and continued for several days in severe cases.

CHAPTER XXII.

The spirillum (comma bacillus) of Asiatic cholera — Its morphological
and cultural "peculiarities — Pathogenic properties — The bacterio-
logical diagnosis of Asiatic cholera — Microspira Metschnikovi —
Microspira ("Vibrio") Schuylkilliensis — Its morphological, cul-
tural, and pathogenical characters.

At the conference held in Berlin in 1884 for the pur-
pose of discussing Asiatic cholera from the sanitary
aspect, it was announced by Koch1 that he had dis-
covered in the intestinal evacuations of individuals suf-
fering from Asiatic cholera a micro-organism that he
believed to be the cause of the malady. The importance
of this statement necessarily attracted widespread atten-
tion to the subject, and as one of the consequences there
existed, for a short time following, some skepticism as
to the accuracy of Koch's claim. These doubts arose as
a result of a series of contributions from other observers,
who endeavored to prove that the organism found by
Koch in cholera evacuations was common to other local-
ities, and was not a specific accompaniment of this dis-
ease. It was not very long, however, before it was
evident that these objections were based upon untrust-
worthy observations, and that by reliable methods of
investigation the organism to which he had called atten-
tion could be easily diiferentiated from each of those
with which it was claimed to be identical.

This organism, known both as the spirillum of Asiatic
cholera, and, because of its morphology, as Koch's

1 Verhandlungen der Conferenz zur Erorterung der Cholerafrage,
1884, Berlin.

473

474 BACTERIOLOGY.

"comma bacillus," is identified by the following peculi-
arities :

MICROSPIRA COMMA (KOCH) SCHROTER, 1886.

Synonyms : Comma-bacillus, Koch, 1884 ; Spirillum cholerae asiatica,
Fliigge, 1886.

MORPHOLOGY. — It is a slightly curved rod, ranging
from about 0.8 to 2 //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. From 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. 74.)

It does not form spores, and we have no reliable evi-
dence that it possesses the property of entering, at any
time, a stage in which its powers of resistance to detri-
mental agencies are increased.

It is a flagellated organism, but has only a single
flagellum attached to one of its ends.

It is actively motile, especially in the comma stage ;
though the long spiral forms also possess this property.

GROUPING. — As found in the slimy flakes in the intes-
tinal discharges from cholera patients, Koch likens its
mode of grouping to that seen in a school of small fish

MICROSPIRA COMMA. 475

when swimming up stream — i. e., they all point in nearly
the same direction, and lie in irregularly parallel, linear

FIG. 74.

>!

Microspira comma. Impression cover-slip from a colony thirty-four
hours' old.

groups that are formed by one comma being behind
the other without being attached to it.

FIG. 75.

8.

Involution-forms of microspira comma, as seen in old cultures.

On cover-slip preparations made from cultures in the
ordinary way there is nothing characteristic about the
grouping ; but in impression cover-slips made from
young cultures the short commas will nearly always be
seen in small groups of three or four, lying together in
such a way as to have their long axes nearly parallel to
one another. (See Fig. 74.)

In old cultures in which development has ceased it
undergoes degenerative changes, and the characteristic

476 BACTERIOLOGY.

comma and spiral shapes may entirely disappear, their
place being taken by irregular involution-forms that
present every variety of outline. (See Fig. 75.) In
this stage they take on the stain very feebly, and often
not at all.

CULTURAL PECULIARITIES. — On plates of nutrient
gelatin that have been prepared from a pure culture of
this organism and kept at a temperature of from 20°
to 22° C., development can often be observed after as
short a period as twelve hours, but frequently not be-
fore 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 pre-
sent to the naked eye an appearance that has been
likened to a ground-glass surface, or to a surface that
has been stippled with a finely pointed needle, or one
upon which very fine dust has been sprinkled. This
appearance is due to the presence of minute colonies
closely packed together upon the surface of the gelatin.
In the depth of the gelatin can also be seen closely
packed, small points, likewise representing growing
colonies. As growth progresses liquefaction occurs
around the superficial colonies, and in consequence this
plate is usually entirely liquid after from twenty-four
to thirty hours ; the developmental phases through which
the colonies pass cannot, therefore, be studied upon it.

On plates 2 and 3, where the colonies are more widely
separated, they can be seen after twenty-four to thirty
hours as small, round or oval, white or cream-white
points, and when located superficially a narrow trans-
parent zone of liquefaction can be detected around
them. As growth continues this liquefaction extends
downward rather than laterally, and the colony ulti-

MICROSPIRA COMMA.

477

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 more
than one millimetre beyond the colony proper. On
plates containing few colonies there is little or no
tendency for them to become confluent, and they rarely
exceed 2 to 3 mm. in diameter.

FIG. 76.

Developmental phases of colonies of microspira comma 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, c. After thirty-eight to forty hours, d. After forty-eight to fifty
hours, e. After sixty-fonr to seventy hours.

When examined under a low magnifying lens the very
young colonies (sixteen to eighteen hours old) appear
as pale, translucent, granular globules of a very delicate
greenish or yellowish-green color, sharply outlined, and
not perfectly round. (See a, Fig. 76.) As growth pro-
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 b and c, Fig. TQ.) After forty-eight
hours (and frequently sooner) liquefaction of the gelatin

478 BACTERIOLOGY.

has taken place to such an extent that the appearance
of the colony is entirely altered. Under a magnify-
ing glass the colony proper is now seen to be ragged
about its edges, while here and there shreds of the
colony can be detected scattered through the liquid
into which it is sinking. These shreds evidently repre-
sent pqrtions of the colony that became detached from
its margin as it gradually sank into the liquefied area.

At d, in Fig. 76, is 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-power lens they now appear as
dense, granular masses, surrounded by an area of lique-
faction through which can be seen granular prolonga-
tions of the colony, usually extending irregularly be-
tween the periphery and the central mass. (See e, Fig.
76.) 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 the columnar epithelial cells lining the air-
passages.

These are the more marked phases through which the
colonies of this organism pass in their development on
gelatin plates. In some cultures the various phases
here given pass in succession more quickly, while in
cultures from other sources they may be somewhat re-
tarded.

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 moderately
transparent. The colonies on this medium at 37° C.

MICROSPIRA COMMA.

479

naturally grow to a larger size than do those upon
gelatin at 22° C.

In stab-cultures in gelatin there appears at the top
of the needle-track after thirty-six to forty-eight hours
at 22° C. a small, funnel-shaped depression. As the
growth progresses liquefaction occurs about this point.
In the centre of the depression can be distinguished

FIG. 77.

abed
Stab-culture of microspira comma in gelatin, at 18° to 20° C.

at 18° to 20° C.

a. After twenty-four hours. 6. After forty-eight hours, c. After seventy-
two hours, d. After ninety -six hours.

a small, dense, whitish clump, the colony itself. As
growth continues the depression increases in extent
and ultimately assumes an appearance that consists in

480 BACTERIOLOGY.

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 fun-
nel. (See a, 6, c, and d, Fig. 77.) 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.

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° to 38° C. it develops actively,
and gradually produces an acid reaction, with coagula-
tion of the casein. It retains its vitality under these
conditions for about three weeks or more. The blue
color of milk to which neutral litmus tincture has been
added is changed to pink after thirty-six or forty-eight
hours at body-temperature.

Its growth in peptone solution, either that of Dun-
ham (see Special Media) or the one preferred by Koch,
viz., 2 parts of Witte's peptone, 1 part of sodium chlo-
ride, and 100 parts of distilled water, is accompanied by

MICROSPIRA COMMA. 481

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 Reaction.)

(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 growth is usually said to
be accompanied by slow liquefaction. I have not suc-
ceeded in obtaining this result on Loffler's serum, nor
have I detected anything characteristic about its growth
on this medium.

The temperature most favorable for its growth is
between 35° and 38° C. It grows, but more slowly,
at 17° C. Below 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 aerobic, 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.

31

482 BACTERIOLOGY.

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, of hydrochloric or nitric acid is
present. (Kitasato.)

Under artificial cultivation the maximum develop-
ment of this organism is reached in a comparatively
short time ; after this it remains quiescent for a period,
and finally degeneration begins. The dying comma
bacilli become altered in appearance and assume the
condition known as " involution-forms." (See Fig. 75.)
When in this state they take up coloring-reagents very
faintly or not at all, and may lose entirely their charac-
teristic 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 might lead one
to regard the material under consideration as a pure
culture of this organism. Its conspicuous develop-
ment under these circumstances does not, however, last
longer than two or three days ; degeneration and death
begin, and the other organisms gain the ascendency.
This fact has been taken advantage of by Schottelius '
in the following method devised by him for the bac-
teriological examination of dejections from cholera
patients :

In dejections not examined immediately after being
passed it is often difficult, because of the large num-
ber of other bacteria that may be present, to detect
1 Deutsche med. Wochenschrift, 1885, No. 14.

MICROSPIRA COMMA. 483

with certainty the cholera organism by microscopic
examination. 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 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 Pfeiffer1 states that
in very young cultures, grown under access of oxy-
gen, there is present a body that possesses intensely
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-
form or 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
intact secondary poisons which possess a similar physi-
ological activity, but only when given in from ten to
1 Zeitschrift fiir Hygiene und Infektionskrankheiten, Bd. xi. S. 393,

484 BA CTERIOLOG Y.

twenty times the dose necessary to produce the same
effects with the primary poison.

EXPERIMENTS UPON ANIMALS. — As a result of ex-
periments for the purpose of determining if the disease
can be produced in any of the lower animals it has been
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 fseces of these
animals. Intravascular injections of a pure culture
into rabbits are followed by an illness, from which
the animals usually recover in from two to three days ;
intra peritoneal injections into white mice are, as a rule,
followed by death in from twenty-four to forty-eight
hours ; the conditions in both instances most probably
resulting from the toxic activities of the poisonous prod-
ucts of growth of the organisms present in the culture
employed. None of the lower animals suffer spontane-
ously from Asiatic cholera.

The failure to induce cholera in animals by feeding,
or by injection of cultures into the stomach, was shown
by Nicati and Rietsch1 to be due to the destructive
action of the acid gastric juice on the organisms. 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

1 Archiv. de Phys. norm, et path., 1885, t. vi., 3e ser. Comptes rendus,
xcix., p. 928. Eevue de Hygiene, 1885. Eevue de Medecine, 1885, v.

MICROSPIRA COMMA. 485

influence of the gastric juice, a pathological condition
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
development of the organisms at the point at which
they are deposited — that is, the portion of the intestine
having an alkaline reaction and beyond the influence of
the acid stomach-juice.

By this method Nicati and Rietsch, Van Ermengem,1
Koch,2 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.

At a subsequent conference held in Berlin in 1885
Koch3 described the following method, by means of
which he had been able to obtain a much greater 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
neutralized by injecting through a soft catheter passed
down the oesophagus into the stomach 5 c.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 1 0 c.c. of a
bouillon culture of microspira comma. For the pur-

1 Recherches sur le Microbe dn Cholera Asiatique. Paris-Bruxelles,
1885. Bull, de 1'Acad. roy. de Med. de Belgique, xviii., 3e ser.

2 Loc. cit. s Ibid., 1885.

486 BACTERIOLOGY.

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
eacli 200 grammes of its body-weight. Shortly after
this last injection deep narcosis sets in and lasts from
a half to one hour, after which the animal is as lively
as ever. Of 35 guinea-pigs inoculated in this way by
Koch, 30 died of an affection that was, in general, very
similar to Asiatic cholera as seen in man.

The condition of those animals before death is de-
scribed as follows : twenty-four hours after the opera-
tion the animal appears unwell ; there is 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 wears on, becomes more
pronounced ; the animal lies quite flat upon its abdomen
or on its side, with legs extended ; respiration is weak
and prolonged, and the pulsations of the heart are hardly
perceptible ; the head and extremities are cold, and the
body-temperature is frequently subnormal. The ani-
mal usually dies after remaining in this condition for a
few hours.

At autopsy the small intestine is found deeply in-
jected and filled with flocculent, colorless fluid. The
stomach and intestines do not contain solid masses, but
fluid ; when diarrhoea does not occur, firm scybala may
be detected in the rectum. Both by microscopic exam-
ination and by culture methods the organisms are
found present in the small intestine in practically pure
culture.

MICROSPIRA COMMA. 487

More recently Pfeiffer1 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 medium
si/e wire loop. This is then finely divided in 1 c.c.
of bouillon, and by means of a hypodermic syringe is
injected directly into the peritoneal cavity. When vir-
ulent cultures have been used this operation is quickly
followed by a fall in the temperature of the animal that
is gradual and continuous until death ensues, which usu-
ally occurs in from eighteen to twenty-four hours after
the operation, though exceptionally the animal recovers,
even after having exhibited marked symptoms of pro-
found kmemia.

Continuing his studies upon this disease, Pfeiffer2 has
demonstrated that it is possible to render an animal tol-
erant to or immune from 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 immunized possess a specific
germicidal action toward microspira comma — i. e., if
into the peritoneal cavity of an animal immunized
from Asiatic cholera living organisms 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 another animal of the same species, but not so

1 Zeitschrift fur Hygiene und Infektionskrankheiten, Bd. xi. and xiv.

2 Ibid., 1894, Bd. xvii. S. 355; 1894, Bd. xviii. S. 1 ; 1895, Bd. xx.
S. 197.

488 BACTERIOLOGY.

protected, and immediately afterward living cholera spir-
illa 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 destruction of microspira comma by the serum of an
immunized animal does not occur outside the animal
body — that is, it cannot be demonstrated in a test-tube,
unless, as Bordet has demonstrated, it be perfectly fresh
from the animal body, or, as Metschnikoff has shown,
there be added to it a small quantity of fresh serum
from a normal guinea-pig. The specificity of this reac-
tion is suggested by Pfeiffer as a means of differentiat-
ing the cholera spirillum from other suspicious species,
for no such bacteriolytic action is observed if species
other than microspira comma be introduced into the
peritoneal cavity of animals immunized from Asiatic
cholera.

Pfeiffer has further demonstrated that the serum of
animals artificially immunized from Asiatic cholera has
an agglutinating effect upon fluid cultures of microspira
comma similar to that seen when typhoid bacilli are
mixed with serum from typhoid cases, or from animals
artificially immunized from typhoid infection or intoxi-
cation. (See Agglutinin.)

GENERAL, CONSIDERATIONS. — In ail cases of Asiatic
cholera, and only in this disease, the organism just
described can be detected in the intestinal evacuations.
The more acute the case and the more promptly the
examination is made after the evacuations have passed
from the patient, the less difficulty will be experienced
in detecting the organism.

In some cases the organism can be detected in the

MICROSPIRA COMMA. 489

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 pres-
ent in the gall-bladder. Doyen and Rasstschewsky l
found them in the liver in pure culture, and Tizzoni
and Cattani2 in both the blood and the gall-bladder.

Microspira comma is a facultative saprophyte ; that
is to say, it apparently finds in certain parts 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 case 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 employed for the
cultivation of bacteria.3

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-

1 Reference to Vratch, 1885, in Allg. med. Central Zeitung, Berlin.

2 Centralblatt fur die med. Wissenschaften, 1886, No. 43.

3 Obviously all pathogenic bacteria that have been isolated under
artificial methods of cultivation AW facultative saprophytes. Were they
obligate parasites, their cultivation upon dead materials would be
impossible.

490 BACTERIOLOGY.

times resulted in demonstrating the multiplication of
the organisms introduced into it, while in other cases
they died very quickly.

On February 8, 1884, microspira were found in a tank at
Saheb-Began, in Calcutta, and it was possible to demon-
strate 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, wrhereas in the canal-water (sewage) of Berlin they
died after six or seven days; but if this latter were
mixed with faecal matters, the organisms retained their
vitality for but twenty-seven hours ; and in the undi-
luted contents of cesspools it was 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-wrater,
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 test with the water-supply of Berlin the
organism retained its vitality for 267 days, and in
another for 382 days, notwithstanding the fact that
many other organisms were present at the same time.
There is no ready explanation for these variations, for
they depend apparently upon a number of factors which
may act singly or together. For example, in general it
may be said that the higher the temperature of the water
in which these organisms are present, up to 20° C., the

MICROSPIRA COMMA. 491

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-saprophytes.

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 l found
that microspira comma was not markedly susceptible
to those deleterious influences lhat 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 regular development in cultures so placed.

As a result of experiments performed in the Imperial
Health Bureau at Berlin, it was found that the bodies of

1 Zeitschrift fur Hygiene, Bd. ii. S. 521.

492 BACTERIOLOGY.

guinea-pigs that had died of cholera induced by Koch's
method of inoculation contained no living cholera spir-
illa when exhumed after having been buried for nineteen
days in wooden boxes, or for twelve days in zinc boxes.
In a few that had been buried in moist earth, without
having been encased in boxes, when exhumed after two
or three months, the results of examinations for cholera
spirilla were likewise negative.

Kitasato,1 in his experiments with the cholera organ-
ism, found that when mixed with the intestinal evacu-
ations of human beings under ordinary conditions it
lost its 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 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 2 and by Celli 3 that
many substances commonly employed as food-stuffs
serve as favorable materials for the development of
the cholera organisms. In his experiments upon its
behavior in milk Kitasato 4 found that at a temperature
of 36° C. microspira comma developed very rapidly
during the first three or four hours, and outnumbered
the other organisms commonly found in milk. They
then diminished in number from hour to hour as the
acidity of the milk increased, until finally their vitality
was lost; at the same time the common saprophytic
bacteria increased in number. Relatively the same
process occurs at a lower temperature, from 22° to 25°

1 Zeitschrift fur Hygiene, Bd. v. S. 487.

2 Ibid., Bd. v. S. 527.

3 Bolletino della E. Acad. Med. di Roma, 1888.
* Zeitschrift fur Hygiene, Bd. v. S. 491.

MICROSPIRA COMMA. 493

C. ; but it is slower, the maximum development of the
cholera organisms being reached at about the fifteenth
hour, after which time they were outnumbered by the
ordinary saprophytes present.

From the foregoing it would seem that the vitality of
microspira comma in milk depends largely upon the
reaction ; 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 multiplication.

According to Laser,1 the cholera organism retains its
vitality in butter for about seven days; it is therefore pos-
sible 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 microspira
comma and other organisms with which it may come
in contact, the experiments of Kitasato2 led him to
conclude that no organism has been found which,
when growing in the same culture-medium with it,
possessed the power of depriving it of vitality within
a short time. On the other hand, the experiments
showed that there were quite a number of other organ-
isms the development of which was checked, and in
some cases their vitality was completely destroyed, when
growing in the same medium with the microspira comma.

From this it would appear that the disappearance of
the microspira comma from mixed cultures and from
the evacuations in the short time mentioned is due more
to unfavorable nutritive conditions than to the direct
action of the other organisms present.

1 Laser : Zeitschrift fur Hygieue, Bd. x. S. 513.
1 Kitasato : Ibid., Bd. vi. S. 1.

494 BACTERIOLOGY.

When completely dried, according to Koch, micro-
spira comma does not retain its vitality longer
than twenty-four hours, but by others its vitality is
said to be destroyed by an absolute drying of three
hours. In moist conditions, as in artificial cultures,
vitality may be retained for many months ; though re-
peated observations lead us to believe that under these
circumstances virulence is diminished. According to
Kitasato,1 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 persistency of
pathogenic micro-organisms in the dead body von
Esmarch 2 found that when the cadaver of a guinea-
pig dead after the introduction of cholera organisms
into the stomach was immersed in water until decom-
position set in, after eleven days, when decomposition
was far advanced, it was impossible to find any living
microspira comma by the ordinary plate methods.

A similar experiment resulted in their disappearance
in 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 3 has shown that an atmosphere of car-
bonic acid is directly inhibitory to the development of
microspira comma, and Percy Frankland 4 states that
in an atmosphere of this gas it dies in about eight days.

1 Kitasato : Zeitschrift fur Hygiene, Bd. v. S. 134.

2 v. Esmarch : Ibid., Bd. vii. S. 1.

s Carl Frankel: Ibid., Bd. v. S. 332.
4 Percy Frankland: Ibid., Bd. vi. S. 13,

THE DIAGNOSIS OF ASIATIC CHOLERA. 495

In an atmosphere of carbon monoxide its vitality is lost
in nine days, and in general the same may be said for it
when exposed to an atmosphere of nitrous oxide gas.

From what has been said, we see that the spirillum
of Asiatic cholera, while possessing the power of pro-
ducing in human beings one of the most rapidly fatal
diseases with which we are acquainted, is still one of the
least resistant of the pathogenic organisms known to us.
Under conditions most favorable to its growth its de-
velopment is self-limited ; it is markedly susceptible 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 in this
way the disease is often disseminated from points in
which it is epidemic or endemic into localities that are
free from it.

THE DIAGNOSIS OF ASIATIC CHOLERA BY BACTERIO-
LOGICAL HfiETHODS.

Because of the manifold channels that are open for
the dissemination of this disease it is of the utmost
importance that it should be recognized as quickly
as possible, for with every moment of delay in its
recognition opportunities for its spread multiply. It
is essential, therefore, when employing bacteriological
means for making the diagnosis, to bear in mind those
biological and morphological features of the organism
that appear most quickly under artificial methods of
cultivation, and which, at the same time, may be con-
sidered 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

496 BACTERIOLOGY.

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 coiiu-i-
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 toxaemia, char-
acterized by prostration and gradual and continuous
fall in the temperature of the animal's body.

In 1893 Koch1 called attention to a plan of pro-
cedure that comprehends the points just enumerated.
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
evacuations should be examined as soon as possible
after they have been passed.

MICROSCOPIC EXAMINATION. — 1. From one of the
small slimy particles seen in the semi-fluid evacuations
prepare a cover-slip preparation in the ordinary way
and stain it. If, upon microscopic examination, only
curved rods, or curved rods greatly in excess of all
other forms, are present, the diagnosis of Asiatic cholera
is more than likely correct ; and particularly is this true
if these organisms are arranged in irregular linear
groups with the long axes of all the rods pointing in
nearly the same direction — that is to say, somewhat as
minnows arrange themselves when swimming up stream
in schools. (Koch.)

In 1886 Weisser and Frank2 expressed their opinion

1 Zeitschrift fur Hygiene und Infiktionskleiten, 1893, Bd. xiv. S. 319.

2 Ibid., 1886, Bd. i. S. 379.

THE DIAGNOSIS OF ASIATIC CHOLERA. 497

upon the value of microscopic examination in these cases
as follows :

a. In the majority of cases microscopic examination
is sufficient for the detection of the presence of micro-
spira comma in the intestinal evacuations of cholera
patients.

6. Even in the most acute cases, running a very rapid
course, microspira comma can always be found in the
evacuations.

c. In general, the number of microspira comma pres-
ent is greater the earlier death occurs ; when death is
delayed, and the disease continues for a long period,
their number is diminished.

d. Should the patient not die of cholera, but of
some other disease, such as typhoid fever, that may be
engrafted upon it, microspira comma may disappear
entirely from the intestines.

2. From another slimy flake prepare a set of gelatin
plates. Expose them to a temperature of from 20° to
22° C., and after sixteen, twenty-two, and thirty-six
hours observe the appearance of the colonies. Usually
after about twenty-two hours the colonies of this organ-
ism can easily be identified by one familiar with them.

3. With another slimy flake start a culture in a tube
of peptone solution — either the solution of Dunham
or, as Koch proposes, a solution of double the strength
of that of Dunham (Witte's peptone is to be used, as it
gives the best and most constant results). Keep this at
from 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 local-

32

498 BACTERIOLOGY.

ity in almost pure culture. After the microscopic exami-
nation 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 10 drops of concentrated sulphuric
acid for each 10 c.c. 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, at most,
twenty-four hours after beginning the examination."
(Koch.)

In certain doubtful cases the organisms are present in
the intestinal canal in very small numbers, and micro-
scopic examination is not, therefore, of so much assist-
ance. In these cases plates of agar-agar, of gelatin,
and cultures in the peptone solution should be made.

The plates of agar-agar should not be prepared in
the usual way, but the agar-agar should be poured into
Petri dishes and allowed to solidify, after which one of
the slimy particles may be smeared over its surface.
The comma bacillus, being markedly aerobic, develops
very much more readily when its colonies are located
upon the surface then when in the depths of the med-
ium. A point to which Koch calls attention, in con-
nection with this step in manipulation, is the necessity
of having the surface of the agar-agar free from
the water squeezed from it when it solidifies, as the
presence of the water interferes with the development
of the colonies at isolated points and causes them to

THE DIAGNOSIS OF ASIATIC CHOLERA. 499

become confluent. To obviate this he recommends that
the agar-agar be poured into the plates and the water
allowed to separate from the surface at the temperature
of the incubator before they are used. It is wise, there-
fore, when one is liable to be called on for such work
as this, to keep a number of sterilized plates of agar-
agar in the incubator ready for use, just as sterilized
tubes of the media are always ready at hand. The
advantage of using the agar plates is the higher tem-
perature 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 bac-
teria having the morphology of the one we are seek-
ing, and as soon as such is detected gelatin plates
and cultures in peptone solution (for the indol reac-
tion) should be made. The peptone culture from the
original material should be examined microscopically
from hour to hour after the sixth hour that it has
been in the incubator. The material taken for ex-
amination should always come from near the surface
of the fluid, and care should be taken not to shake
the tube. As soon as comma bacilli are detected in
considerable numbers in the upper layers of the
fluid agar-agar plates and fresh peptone cultures
should be made from them. In from ten to twelve
hours at 37° C. the colonies will develop on the agar-
agar plates to a size sufficient for recognition by micro-
scopic examination, and from this examination an
opinion can usually be formed. This opinion should
always be controlled by cultures in the peptone solution
made from each of several single colonies, and finally

500 BACTERIOLOGY.

the test for the presence or absence of indol in these
cultures should be made.

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 cult-
ures should be obtained as soon as possible by the agar-
agar plate method and by the method of cultivation in
peptone solution, 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 large wire-loopful should be
scraped and broken up in about one cubic centimetre
of bouillon, and the suspension thus made injected
by means of a hypodermic syringe directly into the
peritoneal cavity of a guinea-pig^ of about 350 to
400 grammes weight. For larger animals more mate-
rial is used. If the material injected is from a
fresh culture of the cholera organism, toxic symp-
toms at once appear; these have their most pro-
nounced expression in depression of temperature, and
if one follows this decline in temperature from time to
time with the thermometer it will be seen to be gradual
and continuous from the time of injection to the death
of the animal (Pfeiffer1), which occurs in from eighteen
to twenty-four hours after the operation.

In general, this is the procedure employed in the In-
stitute for Infectious Diseases at Berlin, under Koch's
direction.

1 Loc. cit.

MICROSPIRA METCHNIKOVI. 501

MICROSPIRA METCHNIKOVI (GAMALEIA), MIGULA,
1900.

Synonym : Vibrio Metchnikovi, Gamale'ia, 1888.

A 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 l under the name of

FIG. 78.

, i4^>J-

~~

Microspira Metchnikovi from agar-agar culture, twenty-four hours' old.

microspira Metchnikovi. It was found post mortem in a
number of fowls that had died in the poultry-market of
Odessa, and the experiments of the discoverer led him
to believe that it was related etiologically to the gastro-
enteritis from which the chickens had been suffering.

Morphologically it appears as short, curved rods and as
longer, spiral-like filaments. It is usually thicker than
Koch's spirillum and is at times much longer, while
again it is seen to be shorter. It is usually more dis-
tinctly curved than the " comma bacillus." (Fig. 78.)

It is supplied with a single flagellum at one of its
extremities, and is therefore motile.

It does not form spores.

It is aerobic.

Its growth upon gelatin plates is usually character-

1 Annales de 1'Institut Pasteur, 1888, tome ii., pp. 482, 552.

502 BA CTERIOLOG Y.

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-size 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. (Fig. 79.)

FIG. 79.

Colony of microspira 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 exaggerated in degree. The liquefaction
is far more rapid, and the characteristic appearance
of the growth is lost in from three to four days.
(See a, 6, c, d, Fig. 89.) Development and liquefaction
along the deeper parts of the needle-track are much
more pronounced than is the case with the " comma
bacillus."

Its growth on agar-agar is rapid ; after twenty-four
to forty-eight hours a grayish deposit appears, which has
a tendency to become yellowish with age.

On potato at 37° C. its growth is seen as a moist,
coifee-colored patch, surrounded by a much paler zone.

MICROSPIRA METCHNIKOVL

503

The whole growth is so smooth and glistening that it
has somewhat the appearance of being varnished.

In bouillon it quickly causes opacity, with the ulti-
mate production of a delicate pellicle upon the surface.

FIG. 80.

a 6 c d

Stab-culture of micronpira Metchnikwi 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.

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 addi-

504 B A CTERIOLOO Y.

tion of sulphuric acid alone. The production of indol
by this organism is usually greater than that com-
mon to the comma bacillus under the same circum-
stances.

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 in from
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.

Gamaleia states that chickens affected with the chol-
eraic gastro-enteritis of which this organism is the cause,
are usually seen sitting quietly with ruffled feathers.
They suffer from diarrhoea, but there is no elevation
of temperature. Hyperaemia of the entire gastro-intes-
tinal 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

M1CROSPIRA METCHNIKOVL 505

the spirilla are not found in the blood, but in young
ones they are usually present in small numbers.

After the introduction into the pectoral muscle of a
very small quantity of a culture of this organism
pigeons succumb in from eight to twenty hours. The
most conspicuous post-mortem lesion is found at the site
of inoculation. The muscle is marked by yellow,
necrotic stripes ; is more or less cedematous ; is swollen,
and contains the vibrios in enormous numbers. The
intestines are usually filled with fluid contents, which
may or may not be blood-stained ; the walls of the in-
testines are often injected with blood, and occasionally
markedly so. The conditions of the other internal
viscera are inconstant. In fatal cases the vibrios are
present in large numbers in the blood and internal
organs. In pigeons that survive inoculation the organ-
isms may be found only at the site of inoculation, or
very sparingly in the blood also. These animals usually
exhibit immunity from subsequent inoculations. In
certain instances the results of infection are chronic;
the inoculated pectoral muscle atrophies, the pigeon loses
in weight and finally dies after one or two weeks. In
these cases the organisms are usually absent from the
blood and internal organs, and may even be absent from
the site of inoculation, or, if present, in only very small
number.

Guinea-pigs usually die in from twenty to twenty-
four hours after subcutaneous inoculation. At autopsy
an extensive redema 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
of both pigeons and guinea-pigs contain the vibrios in

506 BA CTERIOLOG Y.

large numbers, the infection in these animals takes,
therefore, the form of acute, general septicaemia.

The blood-serum of both pigeons and guinea-pigs
that have survived inoculation with this organism — i. e.,
that have acquired immunity from it — is bactericidal
in vitro for this organism. It also possesses a certain
degree of immunity-conferring property, as may be
demonstrated by injecting it into normal pigeons and
guinea-pigs that are subsequently to be inoculated with
virulent cultures.

Very old cultures of this organism in bouillon be-
come distinctly alkaline in reaction. At this stage they
contain a toxin that is markedly active for susceptible
animals. This toxin is not dissolved in the fluid to any
extent, but is apparently in intimate association with the
proteid matters composing the bacteria.

Gastro-enteritis may be produced in both chickens
and guinea-pigs by feeding them with food with which
cultures of this organism have been mixed. (Gamaleia.)

MICROSPIRA SCHUYLKILLIENSIS, ABBOTT, 1896.
Synonym : Vibrio Schuylkilliensis, Abbott, 1896.

Abbott reports the discovery of a microspira in the
water of the Schuylkill River, at Philadelphia, and later,
Bergey1 reports the presence of the same organism,
as well as several varieties that are slightly diiferent,
in the waters of the Schnylkill and Delaware rivers,
along the entire city front, more especially in the efflu-
ents of the sewers.

Microspira Schuylkilliensis is a short, rather plump
" comma," often with a very decided curve, with
rounded or slightly pointed ends. As usually seen it

1 Bergey: Jour, of Exper. Med., vol. ii., 1897, p. 535.

MICROSPIRA SCHUYLKILLIENSIS. 507

is a little . shorter and thicker than the microspira
comma, though this feature is quite variable. It is
actively motile, having a single polar flagellum. It
does not form spores. It stains with the ordinary ani-
line stains, but is negative to Gram's method.

COLONIES ON GELATIN. — The colonies are sharply
defined, distinctly granular, and have usually fine
irregular markings, as if they were creased or folded.
Sometimes they present indistinct concentric mark-
ings. As growth progresses these markings be-
come more and more distinct and finally give to
the colony a decidedly lobulated or mulberry-like
appearance.

After about the third or fourth day, when liquefac-
tion is actively in progress, the majority of the colonies
lose their characteristic appearance. They are seen as
irregular, ragged, granular masses lying in the centre
of pits of liquefied gelatin.

GELATIN STAB CULTURES. — In stab cultures in gel-
atin the appearance of the growth is essentially that of
microspira comma, though at times it is a little more
rapid in its progress.

GROWTH ON AGAR-AGAR. — On meat-infusion agar-
agjir, neutral or slightly alkaline to phenolphthalein,
growth is very rapid at the body temperature. The
general character of the growth corresponds to that of
microspira comma.

BLOOD SERUM. — The growth on blood serum, after
twenty-four hours at body temperature appears as a line
of depression, which increases as a track of liquefaction,
and later results in the more or less complete liquefac-
tion of the medium.

BOUILLON. — Bouillon becomes uniformly clouded in

508 BACTERIOLOGY,

twenty-four hours at the body temperature. Its reac-
tion becomes more alkaline as growth progresses. A
pellicle, at first delicate, later denser always character-
izes the growth in this medium.

POTATO. — Usually no visible growth.

LITMUS MILK. — In fresh litmus milk a slight degree
of acidity is noticed after twenty-four hours at body
temperature. After forty-eight hours this acidity is
slightly greater, and at times the milk shows evidences
of coagulation, though not always.

BIOCHEMIC CHARACTERS. — Microspira Schuylkilli-
ensis is an aerobic, facultative organism. In fluid media
under an atmosphere of carbon dioxide in sealed tubes
no growth is observed.

The organism grows most luxuriantly at about 37.5°
C. Growth is hardly perceptible at 10° C. It is
destroyed by an exposure of five minutes to 50° C.

None of the carbohydrates are broken up with the
liberation of gas.

It produces indol and at the same time reduces
nitrates to nitrites.

PATHOGENESIS. — The pathogenic properties of this
organism are best seen in guinea-pigs and pigeons, both
of which are uniformly susceptible. Rabbits and
chickens resist relatively large doses. Mice are infected
with small doses injected subcutaneously.

The most characteristic lesions follow the injection of
cultures into the pectoral muscles of pigeons. At death
the inoculated muscle is swollen, necrotic, and the over-
lying tissues are oedematous. The bacteria are found in
large numbers in the vicinity of the seat of the inocu-
lation, and in relatively small numbers in the blood and
internal organs.

MICROSPIRA SCHUYLKILLIENSIS. 509

NOTE. — Since the epidemic of cholera in Hamburg
quite a number of curved or spiral organisms, somewhat
like microspira comma, have been discovered. For the
descriptions of these the reader is referred to current
bacteriological literature.

CHAPTER XXIII.

Study of bacterium anthracis, and of the effects produced its by inocula-
tion into animals — Peculiarities of the organism under varying
conditions of surroundings — Anthrax vaccines — Anthrax immune
serum.

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 group of investigations that have not only fully famil-
iarized us with the nature of this malady in particular,
but have perhaps contributed more incidentally to our
knowledge of bacteriology in general than studies upon
any other single infective process or its causative agent.

The direct outcome of these investigations is that a
rod-shaped micro-organism, now known as bacterium an-
thracis, is always present in the blood of animals suffer-
ing from this disease ; that this organ-ism can be obtained
from the tissues of these animals in pure cultures ; and
that these artificial cultures of bacterium anthracis when
introduced into the bodies of susceptible animals can
again produce a condition identical with that found in
the animal from which they were obtained. The dis-
ease is a true septicaemia, and after death the capil-
laries throughout the body are always found to contain
the typical rod -shaped organism in larger or smaller
numbers.

This organism, when isolated in pure culture, is a
bacterium which varies considerably in length, ranging
from short rods, 2 to 3 // in length, to longer threads,
20 to 25 p. in length. In breadth it is from 1 to

510

BACTERIUM ANTHRACJS. 511

1.25 fjt. 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. 81.)

FIG. 81.

^

Bacterium anlhracis, highly magnified to show swellings and concavities at
extremities of the single cells.

When cultivated artificially at the temperature of the
body the bacterium of anthrax presents a series of very
interesting stages.

The short rods develop into long threads, which
may be seen twisted or plaited together like ropes,
each thread being marked by the points of juncture
of the short rods composing it. (Fig. 82, a and 6.)
In this condition it remains until alterations in its sur-
roundings, the most conspicuous being diminution of its
nutritive supply, favor the production of spores. When
this stage begins changes in the protoplasm of the
bacteria may be noticed ; they become marked by irregu-
lar granular bodies, which eventually coalesce into
glistening oval spores, one of which lies in nearly every
segment of the long thread, and gives to the thread the

512 BACTERIOLOGY.

appearance of a string of glistening beads. (Fig. 83.)
In this stage they remain but a short time. The chains
of spores, which are held together by the remains of

FIG. 82.

^r%

o \V 6

Bacterium anthracis. Plaited and twisted threads seen in fresh growing
cultures. X about 400 diameters.

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-

FlG. 83.

Threads of bacterium anthracis containing spores. X about 1200 diameters.

ences, remain and, unless their surroundings are altered,
continue in this living, though inactive, condition for
a very long time. If again placed under favorable
conditions, each spore will germinate into a mature cell,

BACTERIUM ANTHRACIS.

513

and the same series of changes will be repeated until
the surroundings become again gradually unfavorable
to development, when spore-formation again takes place.
Spore-formation occurs only at temperatures ranging
from 18° to 43° C., 37.5° C. being the optimum.
Under 12° C. they are not formed. This organism
does not form spores 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 se'en.

The bacterium of anthrax is not motile.

GROWTH ON AGAR-AGAR. — Colonies of this organ-
ism, 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

FIG. 84.

Colony of bacterium anthmcis on agar-agar.

more or less dense, consisting of a felt-like mass of
long threads irregularly matted together, the growth
continues outward upon the surface of the agar-agar.
(Fig. 84.) It is made up of wavy bundles in which
the threads are seen to lie parallel or are twisted in
strands like those of a rope ; sometimes they have a
plaited arrangement. (See Fig. 82.) These bundles
twist and cross in all directions, and eventually dis-

33

5 1 4 BA CTERIOLOG Y.

appear 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 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 and opaque, and granular and rough on
the surface. When touched with a sterilized needle
one experiences a sensation that suggests somewhat
their matted structure. They are never moist or
creamy. The bit that is taken up with the needle 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 bacteria, which look very much
like bits of cotton- wool.

BOUILLON. — 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.

POTATO. — On this medium it develops rapidly as a
dull, dry, granular, whitish mass, which is more or less
limited to the point of inoculation. On potato, at the

BACTERIUM AKTHRACIS. 515

temperature of the incubator, spore-formation may be
easily observed.

STAB- AND SLANT-CULTURES. — Stab- and slant-cult-
ures on agar-agar present in general the appearances
given for the colonies, except that the growth is much
more extensive. The growth is always more pro-
nounced on the surface than down the track of the
needle.

On gelatin it causes liquefaction, which begins on the
surface at the point inoculated and spreads outward and
downward.

It grows best with access to oxygen, and very poorly
when the supply of that gas is interfered with.

Under favorable conditions of aeration, nutrition, and
temperature its growth is rapid.

Under 12° C. and above 45° C. no growth occurs.
Its optimum temperature is that of the human body,
viz., 37°-38° C.

The spores of bacterium anthracis are very resistant to
heat, though the degree of resistance varies with spores
of different origin. Von Esmarch found that anthrax
spores from some sources were readily killed by an ex-
posure of one minute to the temperature of steam,
whereas spores from other sources resisted this temper-
ature longer, in some cases as long as twelve minutes.

STAINING. — Anthrax bacteria stain readily with the
ordinary aniline dyes. In tissues their presence may
also be demonstrated by the ordinary aniline stain-
ing-fluids or by Gram's method. They may also be
stained in tissues with a strong watery solution of
dahlia, after which the sections are decolorized in 2 per
cent, sodium carbonate solution, washed in water, dehy-
drated in alcohol, cleared in xylol, and mounted in bal-

516 BACTERIOLOGY.

sam. This leaves the bacilli stained, while the tissues
containing them are decolorized ; or the latter may be
stained a contrast-color — with eosin, for example — after
dehydration in alcohol and before clearing in xylol. In
this case they must be washed again in alcohol before
using the xylol. In a preparation treated in this way
the rod-shaped organisms are of a purple color, and will
be seen in the capillaries of the tissues, while the tissues
themselves are 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 bacterium
anthracis. The animal usually succumbs in from thirty-
six to forty-eight hours. Little or no reaction at the
immediate point of inoculation will be noticed ; but
beyond this, extending for a long distance over the
abdomen and thorax, the tissues will be markedly
cedematous. Here and there, scattered through this
cedematous tissue, small ecchymoses will be seen. The
underlying muscles are pale in color. Inspection of the
internal viscera reveals no very marked macroscopic
changes except in the spleen. This is enlarged, dark
in color, and soft. The liver may present the appear-
.ance of cloudy swelling ; the lungs may be red or pale
red in color ; the heart is usually filled with blood. No
other changes can be seen by the naked eye.

Prepare cover-slip preparations from the blood and
other viscera. They will all be found to contain short
rods in large numbers. Nowhere can spore-formation
be detected. Upon microscopic examination of sec-
tions of the organs which have been hardened in
alcohol the capillaries are seen to be filled with the
bacteria ; in some places closely packed in large num-

BACTERIUM AXTHRACIS. 517

bers, at other points fewer in number. Usually
they are present in largest numbers in those tis-
sues having the greatest capillary distribution and at
those points at which the circulation is slowest. They
are uniformly distributed through the spleen. The
glomeruli of the kidneys and the capillaries of the

FIG. 85.

Bacterium anthracis in liver of mouse. X about 450 diameters. Bacteria
stained by Gram's method ; tissue stained with Bismarck-brown.

lungs are frequently packed with them. The capillaries
of the liver contain them in large numbers. (Fig. 85.)
Hemorrhages, probably due to rupture of capillaries
by the mechanical pressure of the bacteria which are
developing within them, not uncommonly occur.-
When these occur in the mucous membranes of the
alimentary tract the blood may escape through the
mouth or anus ; when in the kidneys, through the uri-
niferous tubules.

Cultures from the different organs or from the oedema-
tous fluid about the point of inoculation result in growth
of bacterium anthracis.

The amphibia, dogs, and the majority of birds are
not susceptible to this disease. Rats are difficult to

518 BA CTERIOLOG Y.

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
bacterium 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
bacterium anthracis, obtained directly from the blood of
a recently dead animal, is brought at once into sterile
nutrient bouillon in about twenty test-tubes, which are
immediately placed in an incubator that is carefully
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

BACTERIUM ANTHRACIS. 519

once on animals. The degree of attenuation experi-
enced by the cultures grown under these circumstances
is determined 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. Investiga-
tion of these attenuations shows them to possess all
the characteristics of enfeebled anthrax bacteria ; they
grow slowly and less vigorously when transplanted ;
they do not form spores when exposed to a high tem-
perature ; 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 septicaemia that occurs in susceptible animals
after inoculation with normal cultures of this organism.
In the practical employment of these attenuated cult-
ures for protective purposes two vaccines 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 " sec-
ond " is that which killed both mice and guinea-pigs,
but failed to kill the rabbit. When larger animals,

520 BA CTERIOLOG Y.

such as sheep or cattle, are to be protected by vaccina-
tion with these vaccines, a subcutaneous inoculation of
about 0.3 c.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.

Sobernheim 1 found that it was possible to bring about
a high degree of immunity against bacterium anthracis
by means of the vaccines 1 and 2 of Pasteur, with sub-
sequent inoculations of virulent organisms. He employed
the serum of animals thus immunized in the treatment of
sheep that had been injected with highly virulent anthrax
bacteria. Five sheep were treated in this way, and all
of them recovered with only slight rise in temperature and
more or less marked infiltration at the point of injection,
while control animals died very promptly.

Sobernheim2 reports an improvement on the method
of protective inoculation against anthrax in which he
uses a combination of anthrax vaccines and immune
serum, in which the results are far more satisfactory
than with the anthrax vaccines alone. He states that
this new method has the following advantages over the

1 Sobernheim : Berliner klin. Wochenschr., 1897.
» Ibid., 1902, p. 516.

BACTERIUM ANTHRACIS. 521

Pasteur method : (1) That the immunization can be car-
ried out without losing any of the animals ; (2) that it
can be completed in one day ; (3) that stronger and more
active cultures can be employed and therefore a more
durable immunity obtained; and (4) that the serum alone
can be employed as a curative agent.

Schlemmer employed the Sobernheim method on 39
oxen in which the results were not uniformly satis-
factory, as one of the animals became ill and most of
them showed febrile reactions. Schlemmer attributes
the unfavorable results to the small dose of immune
serum employed, namely, 10 cubic centimetres, and
believes that for large animals larger doses of the
immune serum should be used.

EXPERIMENTS*

Prepare three cultures of bacterium anthracis — one
upon gelatin, one upon agar-agar, and one upon potato.
Allow the gelatin culture to remain at the ordinary
temperature of the room, place the agar-agar culture
in the incubator, and the potato culture at a temper-
ature not above 18° to 20° C. Prepare cover-slips
from each from day to day. What differences are
observed ?

Prepare two potato cultures of bacterium anthra-
cis. Place one in the incubator and maintain the
other at a temperature of from 18° to 20° C. Ex-
amine them each day. Do they develop in the same
way?

From a fresh culture of bacterium anthracis, in which
spore-formation is not yet begun (which is the surest

522 BACTERIOLOGY.

source from which to obtain non-spore-bearing anthrax
bacteria), 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
a flame twelve to fifteen times, stained with aniline
gentian-violet, washed 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 43° C. add a very minute quan-
tity of an anthrax culture which is far advanced in the
spore-stage. Mix it thoroughly with the liquid agar-
agar and from this prepare several hanging drops under
strict antiseptic precautions, using the fluid agar-agar
for the drops instead of bouillon or salt-solution. Select
from among these preparations that one in which the
smallest number of spores are present. Under the
microscope observe the development of a spore into a
mature cell. Describe carefully the developmental
stages.

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

BACTERIUM ANTHRACIS, 523

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 tube
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 cultures compare? Was there
any difference in the time required for their develop-
ment on the plates?

From a potato culture of bacterium anthracis which
has been in the incubator for three or four days scrape

">1>4 BA CTERIOLOG Y.

away the growth and carefully break it up in 10 c.c.
of sterilized physiological salt-solution. The more
thoroughly it is broken up the more accurate will be the
results of the experiment. Place this in a bath of boil-
ing water, and at the end of one, three, five, seven, and
ten minutes make plates upon agar-agar each 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 a bouillon culture from the blood of an ani-
mal just dead of anthrax. 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.c. of the medium, from
either the blood of an animal just dead of anthrax or
from a fresh virulent culture in which no spores are
formed.

Place these flasks in the incubator at a temperature
of 42.5° C. At the end of five, ten, fifteen, twenty,
twenty -five, or more days remove a flask. Label
each flask as it is taken from the incubator with the
exact number of days that it has been at the tempera-

BACTERIUM ANTHRACIS. 525

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 cult-
ure 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 incubator act in the same way toward animals
as the organisms growing in the flasks ? Is the action
of each of these cultures the same for mice, guinea-pigs,
and rabbits?

Prepare a 2 per cent, solution of sulphuric acid in
distilled water ; suspend in this a number of anthrax
spores ; at the end of three, six, and nine days at 35° C.
inoculate both a guinea-pig and a rabbit. Prepare
cultures from this suspension on the third, sixth, and
ninth days; when the cultures have developed inoculate
a rabbit and a guinea-pig from the culture made on tiie
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 ?

526 BACTERIOLOGY.

ANTHRAX IMMUNE SERUM. — Saufelice1 experimented
with the serum of dogs that had been immunized from
anthrax bacteria. This serum possessed immunizing
and curative properties, as shown by experiments upon
animals. He had an opportunity of trying the serum,
with favorable results, upon a man who had contracted
anthrax. The total amount of serum employed was 56
cubic centimetres. There was no reaction at the point
of injection of the serum. The therapeutic effect of the
administration of serum was a general -improvement in
the symptoms, marked fall of the temperature on the
second, and complete apyrexia on the third, day. The
effect on the local anthrax lesion manifested itself in
reduction and, finally, disappearance of the oedema, fol-
lowed first by an increased swelling of the glands, which
decreased again subsequently. He states that the serum
treatment should be continued not only till the temper-
ature has fallen to normal and a diminution of the
oedema is apparent, but' also until there is marked re-
duction in the size of the swollen lymph-glands.

Sclavo 2 immunized a number of animals, principally
sheep and goats, with the two vaccines of Pasteur, fol-
lowed by repeated injections of increasing quantities of
virulent cultures. By this means he obtained an im-
mune serum which had protective as well as curative
properties when tested upon guinea-pigs and rabbits.
He found that this serum had neither bactericidal nor
antitoxic properties.

Cicognani3 employed Sclavo's immune serum on 12
persons suffering from various grades of anthrax infec-

1 Sanfelice : Centralblatt f. Bacteriologie, Originate, 1902, Bd. 33.

2 Sclavo: Bulletin de 1'Institut Pasteur. T. I., 1903, p. 305.

3 Cicognani : Centralblatt f. Bacteriologie, 190-2, ref. Bd. 31, p. 725.

ANTHRAX IMMUNE SERUM. 527

tion, some of the cases being severe infections in which
the prognosis would otherwise have been very unfavor-
able. The duration of the disease was always very
much shortened and all recovered.

Lazaretti l reports 23 cases of human infection with
bacterium anthracis in which Sclavo's immune serum
\\as employed with recovery in each case. Another
patient, suffering from chronic alcoholism and malaria,
did not recover.

1 Lazaretti : Deutsche Vierteljahrsschrift f. offeutliche Gesuudheits-
pflege, 1903, Bd. 35, Supplement, p. 253.

CHAPTER XXIV.

The most important of the organisms found in the soil — The nitrify-
ing bacteria— The bacillus of tetanus — The bacillus of malignant
oedema — The bacillus of symptomatic anthrax — Bacterium Welchii
— Bacillus sporogenes.

THE NITRIFYING BACTERIA.

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 momen-
tous biological phenomena there constantly in prog-
ress. Of these, the one that is of the greatest im-
portance comprises those changes that accompany the
widespread process of disintegration and decomposi-
tion, to which reference has already been made. (See
Chapter I.) This resolution of dead complex organic
compounds into simpler structures that are assimilable
as food by growing vegetation is dependent upon
the activities of bacteria located in the superficial
layers of the ground. It is not 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 us to
follow 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 the conspicuous end-products. It suffices

528

THE NITRIFYING BACTERIA. 529

to 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 surface of the earth. The
most conspicuous example of the functional activity of
this specific form of soil organism is seen in the im-
mense saltpetre-beds of Chili and Peru, where, by the
activities of these microscopic plants, nitrates are pro-
duced from the ammonia of the fa3cal evacuations of
sea-fowl 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 seen in the decomposition and
subsequent nitrification of the organic matters of sew-
age 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
u

530 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 deposited 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 the subject of much study, and are found to
possess peculiarities of sufficient interest to justify a
brief description. For a long time all efforts to iso-
late 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 Chester, Jordan, and Richards in this
country. From the similarity of the properties, given
by these several observers, of the nitrifying organisms
isolated by them, it seems likely that they have all been
working with either the same organism or very closely
allied species.

The organism generally known as the nitro-monas of

THE NITRIFYING BACTERIA. 531

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 zoogloaa. 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 ordinary
nutrient media, and cannot, therefore, be isolated by
the means commonly employed to separate different
species of bacteria. The most astonishing property of
this organism is its ability to grow and perform its
specific fermentative function in solutions devoid of
organic matter. It is believed to be able to obtain its
necessary carbon from carbonic acid. For its isolation
and cultivation Winogradsky recommends the following
solution :

Ammonium sulphate 1 gramme.

Potassium phosphate 1 "

Pure water 1000 c.c.

To each flask containing 100 c.c. of this fluid is added
from 0.5 to 1 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 or 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
diivs from this to a third flask, and so on. As this
medium does not offer conditions favorable to the

532 BA CTERIOLOG Y.

growth of bacteria requiring organic matter for their
development, those that were originally introduced with
the soil quickly disappear, and ultimately only the nitri-
fying organisms remain. These are seen as an almost
transparent film attached to the clumps and granules of
magnesium carbonate on the bottom of the flask.

For their cultivation upon a solid medium Winograd-
sky 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 spe-
cific gravity of 1.02, remains fluid and can be preserved
in flasks in this condition. (Kiihne.) Gelatinization oc-
curs after the addition of certain salts to such a solution,
and will be more or less complete according to the pro-
portion of salts added. The salts that have given the
best results and the method of mixing them are as fol-
lows :

( Ammonium sulphate 0.4 gramme.

a < Magnesium sulphate 0.05 "

( Calcium chloride trace.

f Potassium phosphate 0.1 gramme.

b X Sodium carbonate 0.6 to 0.9 "

( 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 ; the mixture representing the
solution of mineral salts 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 a culture-
dish. If it is desired to separate the colonies, as in an
ordinary plate, the inoculation and mixing of the mate-

THE NITRIFYING BACTERIA. 533

rial introduced must be done before gelatinization is
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 the 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 im-
perfect to permit of a complete description. What has
been said will serve to indicate the direction in which
further studies of the subject should be prosecuted.
(For further details, the reader is referred to the original
contributions and to current literature on the subject.1)

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 oedema (vibrion septique of
the French), the bacillus of tetanus, and the bacillus
of symptomatic anthrax (Rauschbrand (Ger.) ; charbon
symptmnatique (Fr.)). It is sometimes due to the pres-
ence 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 consequences.

1 Winogradsky : Annales de 1'Institut Pasteur, 1890, tome iv. ; 1891,
tome v.

Jordan and Richards : Report of State Board of Health of Massa-
chusetts, " Purification of Sewage and Water," 1890. vol. ii. p. 864.

Frankland, G. C. and P. F.: Proceedings of Royal Society, London,
1890, xlvii.

Wiuogradsky aud Omeliansky : " Ueber den Einfluss der organisaten
Substangen auf der arbeitder nitrifizierenden Mikroben," Centralblatt
fur Bakteriologie, 1899, Abt. ii. Bd. v. S. 329.

534 BA CTERIOLOG Y.

BACILLUS TETANI, NICOLAIER, 1884.

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 by inoculating
them with secretions from the wounds of individuals
affected with the disease. In 1889 Kitasato obtained
the bacillus of tetanus in pure culture, and described
his method of obtaining it and its biological peculiarities
as follows :

METHOD OF OBTAINING IT. — Inoculate several mice
subcutaneously with 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 suppuration at the seat of inoculation in the mice.
In order to separate the tetanus bacillus from the others
that are present the pus is smeared upon the surface
of several slanted blood-serum or agar-agar tubes and
placed at 37° to 38° C. After twenty-four hours all
the organisms will have 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-
quarters to one hour. At the end of this time series of

BACILLUS TETANL 535

plates or Esmarch tubes of slightly alkaline gelatin are
made with very small amounts of the culture and kept
in an atmosphere of hydrogen (see page 220). They
are then kept at from 18° to 20° C., and at the end
of about a week the tetanus bacillus begins to appear
in the form of colonies. After about ten days the
colonies should not only be examined microscopically,
but each colony that has developed in the hydrogen
atmosphere should be obtained in pure culture and
again grown under the same conditions. The colonies
that grow only without oxygen, and which are 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. Here are encountered a number
of spore-bearing organisms 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
the tetanus bacillus ; but they will usually be found to

536

BACTERIOLOGY.

differ from it in their relation to oxygen, and they are
also without disease-producing properties.

MORPHOLOGY. — In the vegetating stage it is a slen-
der 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.

FIG. 86.

Bacillus tetani. A. Vegetative stage. 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. 86.) When in the
spore-stage it is not motile.

It is stained by the ordinary aniline staining-reagents.
It retains the color when stained by Gram's method.

CULTURAL, PECULIARITIES. — It is an obligate anae-
robe, and cannot be brought to development under
access of oxygen. It thrives in an atmosphere of
pure hydrogen, but not in one of carbonic acid.

BACILLUS TETANL

537

II 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.

Under artificial conditions it
may be cultivated through nu-
merous generations without loss
of virulence.

APPEARANCE OF THE COLO-
NIES.— Colonies of bacillus tetani
on gelatin under an atmosphere
of hydrogen have, in their early
stages somewhat the appearance
of the colonies of the common
bacillus subtUis in their earliest
stages, viz., they have a dense,
felt-like centre surrounded by a
fringe of delicate radii. The
liquefaction is so slow that the
appearance is retained for a rela-
tively long time, but eventually
becomes altered. In very old

Colonies of the tetanus
bacillus four days old.made
by distributing the organ-
isms through a tube nearly
filled with glucose-gel-
atin. Cultivation in an at-
mosphere of hydrogen.
(From FRANKEL and
I'KEIFFER.)

538 BA CTERIOLOG Y.

colonies the entire mass is made up of a number of
distinct threads that give it the appearance of a com-
mon mould. (See Fig. 87.)

In stab-cultures made in tubes about three-quarters
filled with gelatin growth begins at about 1.5 to 3 cm.
below the surface, and gradually assumes the appearance
of a cloudy, linear mass, with prolongations radiating
into the gelatin from all sides. Liquefaction with coinci-
dent gas-production results, and may reach almost to
the surface of the gelatin.

RELATION TO TEMPERATURE AND TO CHEMICAL
AGENTS. — It grows best at a temperature of from
36° to 38° C. ; gelatin cultures kept at from 20° to
25° C. begin to grow after three or four days. In an
atmosphere of hydrogen at from 18° to 20° C. growth
does not usually occur before one week. No growth
occurs below 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 usually appear before a week, when the lower
part of the gelatin is quite fluid.

Spores of the tetanus bacillus when dried upon bits
of thread over sulphuric acid in the desiccator and 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, of hydrochloric acid,
they are no longer active after two hours. They are
killed when acted upon for three hours by corrosive

BACILLUS TETANI. 539

sublimate, 1 : 1000, and in thirty minutes by the same
solution plus 0.5 per cent, of hydrochloric acid.

ACTION UPON ANIMALS. — After subcutaneous inocu-
lation 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 c.c. of a well-
developed bouillon culture. The period of incubation
for rats and guinea-pigs is twenty-four to thirty hours,
and for rabbits from two to three days. Pigeons are
but slightly, if at all, susceptible.

The tetanic convulsions always appear first in the
parts nearest the seat of inoculation, and subsequently
become general.

At autopsies upon animals that have succumbed to
inoculations with pure cultures1 of bacillus tetani there
is little to be seen by either macroscopic or micro-
scopic examination, and cultures from the site of inocu-
lation are often negative in so far as finding the tetanus
bacillus is concerned. At the site of inoculation there is
usually only a hyperaBinic condition. In uncomplicated
cases there is no suppuration. The internal organs do not
present any macroscopic 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
site of inoculation or, which seems more probable, pro-

1 Animals and human beings that have become infected with this
organism in the ordinary way commonly present a condition of sup-
puration 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.

540 BA CTERIOLOG Y.

duced by the bacteria in the culture from which they are
obtained and introduced with them into the tissues of
the animal at the time of 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 a 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 typical 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 toxic peculiarities, as given by Kitasato, are as
follows : 1

"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.

" Inoculations 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 Zeitschrift fur Hygiene, 1891, Bd. x. S. 267.

BACILLVS TETANI. 541

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 tem-
perature in the desiccator over sulphuric acid, it is not
destroyed.

" Diffuse daylight diminishes the intensity of the
poison. Its intensity is preserved for a much longer
time when kept in the dark.

"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 intensity."

The chemical nature of this poison is not positively
known, but according to the observations of Brieger
and Cohn l its designation of " Toxalbumen " is a mis-
nomer, for its reactions do not warrant its classification
with the albumins 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 tempera-
tures above 70° C. Even when carefully protected from
light, moisture, and air, it gradually becomes diminished
in strength, doubtless due to the formation of "toxons"
and " toxoids," analogous to those observed by Ehrlich in
deteriorating diphtheria toxin. When freshly prepared
by the methods of the authors just cited, its potency is
almost incredible, 0.00005 milligramme being sufficient to
cause fatal tetanus in a mouse weighing fifteen grammes.

1 Zeitschrift fur Hygiene und Infektionskrankheiten, 1893, Bd. xv.
8.1.

542 BA CTERIOL OG Y.

The studies of Madsen l demonstrate it to consist of
two physiologically distinct intoxicating compounds :
the one, a solvent of erythrocytes — a " tetanolysin " ;
the other, a specific irritant which, through its influence
upon the central nervous system,2 accounts for the phe-
nomena by which tetanus is characterized ; to this latter
the designation a tetanospasmin " is given. Madsen's
observations, furthermore, confirm the deductions of
Ehrlich concerning the molecular structure of bacterial
toxins in general, to the effect that the molecule of tet-
anolysin, like that of diphtheria toxin, is a complex of
at least two physiologically unlike groups ; the one,
characterized by its marked combining tendencies (for
antitoxin), the so-called haptophore group ; the other,
distinguished for its intoxicating quality, the so-called
toxophore group.

The principles involved in the induction of the anti-
toxic state against diphtheria are likewise applicable to
tetanus ; in fact, the fundamental observations upon the
generation of antitoxin in the living animal body were
made in the course of studies on tetanus ; they were
subsequently applied to the study of diphtheria, with
the results already noted. It is needless to enter here
upon the details essential to the production of tetanus
antitoxin ; to all intents and purposes, they are identical
with those given in the section on diphtheria. Briefly
stated, animals may be rendered immune from tetanus
by the repeated injection of gradually increasing non-
fatal doses of tetanus toxin ; when immunity is estab-

1 Madsen : " Ueber Teanolysin," Zeitschrift fur Hygiene und Infek-
tionskrankheiten, 1899, Bd. xxxii. S. 214.

26See paper by Wassermann and Takaki : Berliner klinische Wocb-
enschrift, 1898, No. 1, S. 5.

BACILLUS TETANL 543

lished, the circulating blood contains a body, antitoxin,
that combines directly with tetanus toxin in a test-tube,
and thereby renders it physiologically inactive (non-in-
toxicating) ; and the serum of the immune animal is
not only capable of protecting non-immune, susceptible
animals from the poisonous action of tetanus toxin
(within limits), but also against the effects of the living
tetanus bacillus as well.

Tetanus antitoxin, though the first antitoxin discov-
ered and frequently employed in the treatment of
tetanus, has not yielded as brilliant results as those
obtained with diphtheria antitoxin. There are two
important reasons why tetanus antitoxin may never be
expected to yield such satisfactory results as does
diphtheria antitoxin. First, diphtheria infection can be
diagnosed by bacteriological methods and the antitoxin
administered long before any very marked constitutional
symptoms have developed, and consequently long before
the diphtheria toxin has had time to bring about very
serious tissue alterations. In tetanus it is impossible to
make such a definite bacteriological examination, and
very frequently the first manifestation of the disease is
the twitching of the muscles which is the antecedent
sign of the tetanic convulsions. When these clinical
manifestations have developed in tetanus there is already
very serious involvement of the central nervous system
by the action of the tetanus toxin upon the nerve cells.
The second reason why tetanus antitoxin is likely to
prove less helpful than diphtheria antitoxin is that the
tetanus toxin seems to have very great affinity for the
cells of the central nervous system, while the cells and
tissues of the body affected primarily by diphtheria toxin
are of far less vital importance.

544 B A CTERIOL OGY.

In the use of tetanus antitoxin it is advisable to
employ it as early as possible and to give repeated doses
until the symptoms are relieved. Whether the subdurul
administration of the antitoxin will be of greater value
than the subcutaneous administration is as yet unsolved.

A great deal of benefit is also likely to result from
the administration of tetanus antitoxin as a prophy-
lactic in the treatment of wounds in which infection by
the tetanus bacillus is possible. The prophylactic injec-
tion of the tetanus antitoxin in these cases, however,
should always be accompanied by the most rigid aseptic
and antiseptic treatment of the wound, and under these
conditions it is more or less doubtful which of these
measures is of the greatest value, but experience seems
to indicate that the antitoxin has a distinct prophylactic
influence in these cases.

BACILLUS CEDEMATIS, LIBORIUS, 1886.

The bacillus of malignant oedema, also known as
vibrion septique, is another pathogenic form almost
everywhere present in the soil. In certain respects it
is a little like bacterium anthracis, and was at one
time confounded with it ; but it differs in the marked
peculiarity of being a strict anaerobe. It was first
observed by Pasteur, but it was not until later that
Koch, Laborious, Kitt, and others described its pecu-
liarities in detail. It can often be obtained by
inserting under the skin of rabbits or guinea-pigs small
portions of garden-earth, street-dust, or decomposing
organic substances. There results a widespread oedema,
with more or less gas-production in the tissues. In the
cedematous fluid about the site of inoculation the organ-
ism under consideration may be detected. (Fig. 88, A.)

BA CILL US (EDEMA TIS.

545

It is a rod about 3 to 3.5 p. long and from 1 to 1.1 p
thick — i. e., it is about as long as bacterium anthracis,
but is a trifle more slender. It is usually found in pairs,
joined end to end, but may occur as longer threads;
particularly is this the case in cultures. When in pairs

Bacillus cedematis. A. ffldema-fluid, from site of inoculation of guinea-
pig, showing long and short threads. B. Spore-formation, from culture.

the ends that approximate are squarely cut, while the
distal extremities are rounded. When occurring singly
l)oth ends are rounded. (How does it differ in this
respect from bacterium anthracis?) It is slowly motile,
and its flagella are located both at the ends and along

35

BACTERIOLOGY.

FIG. 89.

the sides of the rod. It forms spores that are usually
located in or near the middle of the cells, causing fre-
quently a swelling at the points at which they are
located and giving to the cell a more
or less oval, spindle, or lozenge shape.
(Fig. 88, B.)

It is an obligate anaerobe, growing
on all the ordinary media, but not with
access of oxygen. It grows well in
an atmosphere of hydrogen. It causes
liquefaction of gelatin.

In tubes containing about 20 to 30 c.c.
of gelatin that has been liquefied, inocu-
lated 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 bottom of the
tube in from two to three days at
20° C. These colonies, when of from
0.5 to 1 mm. in diameter, appear as
spheres filled with clear liquid, and
are difficult, for this reason, to detect.
(Fig. 89.) As they gradually increase
in size the contents of the spheres be-
come cloudy and marked by fine radi-
ating stripes, easily to be detected with
the aid of a small hand-lens. In deep
stab-cultures in agar-agar and in gela-

t^

Colonies of the ba-
cillus of malignant

unTuVurf.^^After tin development occurs only along the
track of puncture, at a distance below
the surface. Growth is frequently ac-
companied by the production of gas-bubbles.

It causes rapid liquefaction of blood-serum, with pro-

FRANKEL and PFEIF-
FER.)

BACILLUS (EDEMATIS. 547

duction of gas-bubbles, and in two or three days the
entire medium may have become converted into a
yellowish, semifluid mass.

The most satisfactory results in the study of the
colonies are obtained by the use of plates of nutrient
agar-agar kept in a chamber in which all oxygen has
been replaced by hydrogen. The colonies appear as dull
whitish points, irregular in outline, and when viewed
with a low-power lens are seen to be marked by a 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
does not stain by Gram's method.

PATHOGENESIS. — The animals known to be suscepti-
ble to inoculation with this organism are man, horses,
calves, dogs, goats, sheep, pigs, chickens, pigeons, rab-
bits, guinea-pigs, and mice. Cases are recorded in
which men and horses have developed the disease after
injuries, doubtless due to the introduction into the
wound, at the time, of soil or dust containing the
organism.

If one 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-
quently dies in from twenty-four to forty-eight hours.
The most conspicuous result found at autopsy is a wide-
spread oedema at and about the site of inoculation. The
oedematous fluid is in some places clear, while at others it
may be stained with blood ; it is usually rich in bacilli
(Fig. 88, A) and contains gas-bubbles. Of the internal

548 BACTERIOLOGY.

organs only the spleen shows much damage. It is large,
dark in color, and contains numerous bacilli. If the
autopsy be made immediately after death, bacilli are
rarely 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 multi-
plication in the body after death. At the moment
of death they are present in varying numbers in all
the internal viscera and on the serous surfaces of the
organs.

Of all animals mice are probably the most susceptible
to the action of this organism, and it is not rare to find
it in the heart's blood, even immediately after death.
They die, as a result of these inoculations, in from six-
teen to twenty hours.

When a pure culture is used for inoculation a rela-
tively large amount must be employed, and this 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 oedema-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 cult-
ure, from the cadaver of an animal that has succumbed
to infection by the bacillus of malignant oedema, are
in all essential respects the same as those given
for obtaining cultures from tissues in general ; but it
must be remembered that the organism is a strict anae-
robe, and will not grow under the influence of oxygen.
(See methods of cultivating anaerobic species.)

BACILLUS CHAUVEI. 549

In certain superficial respects this bacillus suggests,
as said above, bacterium anthracis, but differs from it in
so many important details that there is no excuse for
confounding the two.

NOTE. — 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 oedema.
Compare its morphological, biological, and pathogenic
peculiarities with those of bacillus anthracis under simi-
lar circumstances ; especially its action on animals and
its appearance in the tissues and fluids.

Still another pathogenic organism that may be present
in the soil is

BACILLUS OHAUVEI, ARLOING, CORNEVIN, AND
THOMAS, 1887.

Synonyms : The bacillus of symptomatic anthrax — Bacterie du char-
bon symptomatique (Fr.) — Bacillus des rauschbrand (Ger.).

It is the organism concerned 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 certain localities dur-
ing the warm months, and which is characterized by
a peculiar emphyseraatous swelling of the muscular
and subcutaneous cellular tissues over the quarters.
The muscles and cellular tissues at the points af-
fected are seen on section to be saturated with bloody
serum, and the muscles particularly are of a dark,

550

BACTERIOLOGY.

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

FIG. 90.

Bacillus of symptomatic anthrax. A. Vegetative stage— gelatin culture.
B. Spore-forms— agar-agar culture.

description of its morphological and biological peculi-
arities is that of Kitasato.1 The following is from
Kitasato's contributions: it is an actively motile rod
about 3 to 5 // long by 0.5 to 0.6 // thick. It has
rounded ends, and, as a rule, is seen singly, though
now and then pairs joined end to end may occur. It

1 Kitasato : Zeitschrift fur Hygiene, Bd. vi. S. 105; Bd. viii. S. 55.

BACILLUS CHAUVEL

551

FIG. 91.

has no tendency to form very long threads. (Fig.
90, A.)

It forms spores, and when in this stage is seen to be
slightly swollen at or near one of its
poles, the location in which the spore
usually appears. (Fig. 90, B.) It is
markedly prone to undergo degenerative
changes, and involution-forms are com-
monly seen not only in fresh cultures,
but in the tissues of affected animals as
well.

Though actively motile when in the
vegetative stage, it, like all other motile
spore-forming bacilli, loses this property
and becomes motionless when spores are
forming.

It is strictly anaerobic and cannot be
cultivated in an atmosphere in which
free oxygen is present. It grows best
under hydrogen, 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-
sodium sulphate, sodium formate, etc.

When cultivated upon gelatin plates bacillus of symp-

i e i. j ii i tomatic anthrax,

in an atmosphere ol hydrogen the col- in dcep gclatin
onies appear as irregular, slightlv lobu- culture. (After

, , A „ , . * ,. FRANKEL and

lated masses. Alter a short time lique- pFEiFFER.)
faction of the gelatin occurs and the col-
ony presents a dark, dense, lobulated and broken centre,
surrounded by a much more delicate, fringe-like zone.

Colonies of the

552 BACTERIOLOGY.

When distributed through a deep layer of liquefied
gelatin that is subsequently solidified colonies develop
at only the lower portions of the tube. The single
colonies appear as discrete globules that cause rapid
liquefaction of the gelatin, and ultimately coalesce
into irregular, tabulated liquid areas. In some of the
larger colonies an ill-defined, concentric arrangement
of alternate clear and cloudy zones can be made out.
(Fig. 91.)

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 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, pene-
trating 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 round the
point of contact of the tube and the surface of the

BA GILL US CHA UVEL 553

bouillon. There is produced also a peculiar, penetrat-
ing, sour or rancid odor.

It grows best at the body-temperature — i. e., from 37°
to 38° C. — but can also be brought to development at
from 16° to 18° C. Below 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 dessiccator 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 for a long time the power of reproducing the
disease. The spores are tolerably resistant to the in-
fluence of heat : when subjected to a temperature of
80° C. for one hour their virulence is not affected, but
an exposure to 100° C. for five minutes 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, fre-
quently occupy a position inclining to one of the poles
of the bacillus, though they are as often seen in the
middle.

554 BACTERIOLOGY.

Bacilli containing spores are usually clubbed or spin-
dle shape.

This bacillus stains readily with the ordinary aniline
dyes. It is decolorized by Gram's method. Its spores
may be stained by the methods usually employed in
spore-staining.

PATHOGENESIS. — When susceptible animals, especi-
ally guinea-pigs, are inoculated in the deeper subcutane-
ous 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 site 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 oedema. The oedematous fluid is often blood-
stained and the muscles are of a blackish or blackish-
brown color. The lymphatic glands are markedly
hypersemic. 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 recent autopsies only vege-
tative forms of the organism may be found ; but later
(in from twenty to twenty-four hours) spore-bearing rods

BACILLUS CHAUVEL 555

may be detected. (How does this compare with bacte-
rium anthracisf) By successive inoculations of suscepti-
ble animals with serous fluid from the site of inoculation
of the dead animal the disease may be reproduced.

Cattle, sheep, goats, guinea-pigs, and mice are sus-
ceptible to infection with this organism, and present the
conditions above described ; whereas horses, asses, and
white rats present only local swelling at the site of 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
oedema 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 oedema. In their relation to ani-
mals they also differ ; for instance, cattle, while con-
spicuously susceptible to symptomatic anthrax, are prac-
tically immune from malignant oedema ; and while swine,
dogs, rabbits, chickens, and pigeons are readily infected
with malignant oedema, they are not, as a rule, suscepti-
ble to symptomatic anthrax Horses are affected only
locally, and not seriously, by the bacillus of symptomatic
anthrax ; but they are conspicuously susceptible to both
artificial inoculation and natural infection by the bacil-
lus of malignant oedema.

The distribution of the two organisms over the earth's
surface is also quite different. The oedema bacillus is
present in almost all soils, while the bacillus of symp-
tomatic anthrax appears to be confined to certain locali-
ties, especially places over which infected herds have
been pastured.

556 BACTERIOLOGY.

A single attack of symptomatic anthrax, if not fatal,
affords subsequent protection ; while infection with the
malignant oedema bacillus appears to predispose to re-
currence of the disease. (Baumgarten.)

BACTERIUM WELCHII, MIGULA, 1900.
Synonym : Bacillus aerogenes capsulatus, Welch and Nuttall, 1892.

This organism consists of straight or slightly curved
rods with rounded ends, somewhat thicker than bacte-
rium anthracis, varying in length ranging from 3 to 6
microns ; sometimes longer chains or threads are seen.
The rods are surrounded by a transparent capsule,
whether grown in artificial media or obtained from
animal bodies. It is a non-motile, spore-forming
organism, and is strictly anaerobic in character. It
stains with the ordinary aniline dyes and by the Gram
method.

Under anaerobic conditions the organism grows on
the usual culture media at room temperature, and forms
large quantities of gas in media containing carbohy-
drates. Gelatin is not liquefied. In agar-agar the col-
onies are usually from 1 to 2 millimetres in diameter,
but may be as large as 1 centimetre in diameter. They
have a grayish-white color, are flat, round or irregular
masses, with small hair-like projections from the mar-
gin. In bouillon there is a diffuse clouding and marked
white sediment. Milk is quickly coagulated. On
potato there is a grayish-white layer.

The organism grows more rapidly at 30° to 37° C.
than at 18° to 20° C. Cultures on agar-agar and bouil-
lon have a slight odor resembling old lime. Bouillon
cultures are killed after ten minutes at 58° C.

Bacterium Welchii was first described by Welch in

BACTERIUM WELCHII. 557

1891, and subsequently by Welch and Nuttall l in the
blood and internal organs of a patient with thoracic
aneurism opening externally. Autopsy was made eight
hours after death and the vessels were found to contain
large numbers of gas bubbles.

Injections of considerable quantities of cultures into
the circulation of rabbits did not kill the animals, but
if the animals were killed after being inoculated and
were then allowed to lie at room temperature for twenty-
four hours the organs and tissues were filled with gas
bubbles.

Welch, Howard, Hitschman and Lilienthal, Hirsch-
berg, and others have shown that the organism is fre-
quently present in the faeces of man and animals, as
well as in the soil and in dust. Schattenfroh and Grass-
berger also found the organism in market milk.

BACILLUS SPOROGENES (KLEIN), MIGULA, 1900.
Synonym : Bacillus enteritidis sporogenes, Klein, 1895.

Klein found this organism in the intestinal discharges
of infants and believed it had some relation to the acute
inflammatory conditions of the intestinal tract of bottle-
fed infants. The organism is very generally distributed
in nature and can be very readily isolated from sewage
by appropriate methods. It is an anaerobic, spore-form-
ing organism, 0.8 micron in width, and 1.6 to 4.8 microns
in length. It is actively motile and flagella have been
demonstrated.

In culture media containing carbohydrates this organ-
ism produces gas in large quantities. Russell analyzed
the gas and found it to be composed principally of
methane. Milk and other sugar media in which the

1 Welch and Nuttall : Bulletin Johns Hopkins Hospital, No. 24, 1892.

558 BAUTERIOLUdY.

organism has been grown have a distinct odor of butyric
acid.

When injected subcutaneously into guinea-pigs this
organism causes most marked alterations. There is
intense inflammation at the point of injection with
ffidema and necrosis and the surrounding tissues are
filled with gas. The bacteria are distributed throughout
the body of the animal and can be isolated in pure cul-
ture from the blood of the heart. All the internal
organs are intensely congested.

CHAPTER XXV.

Infection and immunity — The types of infection ; intimate nature of
infection — Septicaemia, toxaemia, variations in infectious processes
— Immunity, natural and acquired, active and passive — The
hypotheses that have been advanced in explanation 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 mechanism of this process we call infec-
tion ? Is it due to the mechanical presence of living
bacteria in the body, or does it result from the deposition
in the tissues of substances produced by these bacteria
that are either locally or generally incompatible with
life ? Or, is the group of pathological alterations and
constitutional symptoms seen in these diseases the result
of abstraction from the tissues, by the bacteria growing
in them, of substances essential to the vitality of both
bacteria and tissues ? These are some of the more
important 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 to what extent the observa-
tions 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 then; is a general dis-
tribution of the infective agents throughout the body.

559

560 BA CTERIOLOG Y.

This group comprises the " septicaemias," and of them
the disease of animals knowh~as anthrax represents a
type of the condition. If the cadaver of an animal
dead of anthrax be examined by bacteriological methods,
there will be discovered present in all the organs
and tissues an organism, a bacterium, of definite form
and biological characteristics; and if the organs, and
tissues generally, be subjected to microscopic examina-
tion this same organism will be found 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 entirely occluded
by the growing bacteria. 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 devel-
opment, 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 ultimately be noticed that
the animals that die at 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 with the normal organism. These
differences usually consist in a diminution of the num-

INFECTION AND IMMUNITY. 561

bcr of bacteria that appear upon culture plates from the
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
bacteria, 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 those particular lesions 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 a 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 are astonished to find that the organs, blood, and
tissues generally are sterile,1 in so far as the presence of
the organism with which the animal was inoculated is
concerned, and by both cultural and microscopic methods
it is possible to detect them only at the site of inocula-
tion— ?'. e., where they were deposited. It is very evident

1 In by far the greater number of cases this is true ; but there are
isolated exceptions to it.
36

562 BA CTERIOL OG Y.

that we have here a condition with which mechanical
plugging of the capillaries could have had no connec-
tion, 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 of doubtful value. 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 even at the point of injury.
Plainly, the 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 cult-
ural 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 scarcely 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 toxaemia (poison in the blood),
a condition distinctly different from septicaemia, 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 circu-
lating fluids is of little consequence. Conspicuous
among these are the infections that follow upon the
introduction into the tissues of susceptible animals of
cultures of micrococcus lanceolatus (pneumococcus), of
the bacillus of chicken cholera, and of the organisms con-

INFECTION AND IMMUNITY. .563

cerned in the production of the so-called " hemorrhagic
septicaemias." When running their normal course the
specific organisms of these diseases cause typical septi-
caemias in susceptible 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 septicaemias, be-,
cause 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 infected. From
what we have seen it is manifestly probable that,
whether these diseases be designated as septicaemias or
as toxaemias, death is produced in all instances by
poisonous substances that are generated by the infecting
bacteria. In the case of typical anthrax, and other
varieties of septicaemia, the production of this poison
is associated with the general dissemination of the
organisms throughout the body ; while in those infec-
tions often referred to as toxaemias, 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.

In still another group of infections there is neither
a general distribution of the organisms throughout the
vascular system nor an elaboration of toxins that can1
be readily separated from the organisms manufactur-
ing them. In these infections, of which typhoid fever
and Asiatic cholera may be taken as conspicuous ex-

564 BA CTERIOLOG Y.

amples, the toxicity of the invading bacteria appar-
ently depends upon the existence of iutracellular sub-
stances of a poisonous nature that have thus far eluded
all efforts to satisfactorily separate them from the
bodies of the bacteria in which they develop. The
mechanism by which this group of bacteria acts is as yet
far from clear. We only know that the presence of
members of this group in the bodies of susceptible ani-
mals is accompanied by the death of the tissues in which
they are located. Whether the poisons are eliminated
by the bacteria as secretions, or whether they are set free
through the disintegration of bacteria in the tissues can-
not be said. The main point is, however, that, both
clinically and anatomically, diseases due to this group
of bacteria are characterized by marked evidence of
intoxication.

BACTERIAL TOXINS. — Through special investigations
that have been made upon the products of growth of
certain pathogenic bacteria the opinion that infection is
a chemical process receives 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 culti-
vated 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 occur
in the course of infection by the organisms themselves^
In some instances these poisons — toxins,1 as they are

1 The term "toxins " is commonly applied to amorphous, nitrogenous
poisons produced by bacteria in both living tissues and dead substances ;
while, on the other hand, the term " ptoma'ins " relates to crystallizable,
nitrogenous poisons that are formed in dead tissue, and "leucomains"
to poisonous and non-poisonous alkaloidal bodies that occur in living
tissues as a result of physiological metabolism.

INFECTION AND IMMUNITY. 565

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. On the
other hand, as said above, certain bacteria do not possess
the power of generating or secreting such poisons ; they
have, nevertheless, intimately associated with their pro-
toplasmic bodies poisonous substances that manifest
themselves only when these organisms gain access "to
living susceptible tissues ; thus the toxins of bacterium
tuberculosis and of microspira comma are much more
conspicuously present in the protoplasm of these bacteria
than in the fluids in which they have grown.

Buchner has isolated from several species of bacteria
" bacterioproteins " having the common properties of
solubility in alkalies, resistance to the boiling tempera-
ture, 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.

Toxic ptoMuims 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 particular instances the production of poisonous
principles, even under artificial conditions of cultivation,

1 See Cheiuotaxis.

566 BACTERIOLOGY.

is most astonishing, and poisons are generated 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 bacterium diphtherice
and of bacillus tetani have been carefully determined
by experiments upon animals, and it has been found
that 0.4 milligramme of the former is capable of
killing eight guinea-pigs, each weighing 400 grammes,
or two rabbits, each weighing 3 kilogrammes (Roux and
Yersin1); and that 0.0001 milligramme of the latter
will produce tetanus in a mouse, with all the character-
istic manifestations of the disease (Brieger and Cohn 2).3
TOXOIDS AND TOXONES. — Ehrlich conceives the toxin
molecule to possess two atom-groups, a haptophore group,
by means of which it unites with certain cells of the body,
or with antitoxin ; and a toxophore group, by means of
which it produces its toxic effects. When preserved for a
time, or under the influence of various chemical and phys-
ical agents, the toxophore group of the toxin molecule de-
teriorates, so that it is no longer capable of exerting any
toxic action. The haptophore group is less easily dis-
turbed, and hence the combining power of the toxin
molecule may be unaltered, though it may have lost its
toxic properties. In this condition it is spoken of as
toxoid or toxone. A toxoid or toxone is still capable
of inducing antitoxin formation when injected into an
'animal, and it is also still capable of neutralizing anti-
toxin in the same proportions as before the alteration
had taken place.

1 Annales de 1'Institut Pasteur, 1889, tome iii. p. 287.

2 Zeitsehrift fur Hygiene und Infektionskrankheiten, 1893, Bd. xv.
Heft 1.

3 By the use of more recently devised methods we are enahled to
increase still further the toxicity of these poisons; especially is this
the case with regard to the diphtheria toxin.

INFECTION AND IMMUNITY. 567

MODE OF ACTION OF PATHOGENIC BACTERIA. —
The development of our knowledge of immunity began
with the recognition of the relation existing between the
toxins and antitoxins. It was found that on the injection
into animals of a dose of toxin certain reactions occurred
varying with the size of the dose — that is, if the dose
was a fatal one the animal died within a definite period
of time, which one may call the incubation period. This
incubation period is shorter the greater the amount of
toxin injected. If the amount of toxin injected is less
than the minimum fata1 dose the animal shows after a
time certain reactions, and after recovery from the v
effects of the injection larger doses of the toxin can be •
injected without destroying the animal. At the same
time there appears in the serum an antibody which when
injected into a normal animal will protect it against the
minimum fatal dose of the toxin. Of great importance
in the relation between the toxin and antitoxin is the law
of multiple proportions, whereby one understands a
definite relation between the two — that is, a definite ,
amount of antitoxin will protect a normal animal against^
a definite amount of toxin. A multiple dose of the
scrum will protect against multiple doses of the toxin
of the same proportion.

FORMATION OF TOXIN BY BACTERIA. — The num-
ber of bacteria which are capable of producing a true
toxin is quite small.. Of those that are concerned in
human pathology, diphtheria and tetanus organisms are
the most important. For the majority of the other bac- •
tcria toxin formation has not been definitely demon-
strated. In most of the other pathogenic bacteria it
has been ascertained that the toxic action which they
bring about rests upon the poisonous character of the

568 BACTERIOLOGY.

bacterial protoplasm, and we now regard the toxic action
of these bacteria to be due to the formation of endotoxins
or intracellular toxins.

THE ENDOTOXIXS. — In contradistinction to the re-
sults obtained in the injection of the soluble toxins the
action of the endotoxins is not nearly as well understood.
The injection of the endotoxins does not bring about the
formation of anti-endotoxic substances in the serum.
The serum of animals treated with the endotoxins has
merely a bactericidal action, and through the solution of
the bacteria protection against infection is seen, but mul-
tiple doses of serum do not act against corresponding
multiple doses of endotoxin. The treatment of animals
with endotoxin does not protect the animal body against
the action of the endotoxins.

The endotoxins are not secreted or excreted in the
culture media in which the bacteria are cultivated, but
are associated with the bacterial cells, and are only lib-
erated through the solution of the bacteria — that is,
through bacteriolysis.

Liberation of Endotoxins. — Bacteriolysis may occur,
on the one hand, as the result of the specific reac-
tion consisting in the anchoring of the amboceptors
contained in an immune serum through the receptors of
the particular organisms. After the addition of comple-
ment, which is normally present in the body, union takes
place with the receptor-amboceptor group and bacterio-
lysis follows.

Besides this specific bacteriolysis there is also a non-
specific bacteriolysis in which the endotoxins contained in
the bodies of bacteria are liberated — namely, through
autolytic actions. In old cultures of bacteria one often
finds only few individuals and these are usually in various

INFECTION AND IMMUNITY. 569

stages of degeneration. The different species of bacte-
ria vary greatly with regard to their proneness to auto-
lysis. In some of them — for instance, raicrospira comma,
bacillus typhosus, etc. — the autolysis occurs quite early.
The poisonous substances contained in the bodies of the
organisms thereby pass into solution. One finds in ni-
trates of relatively young cultures some poisonous action,
which is still greater in nitrates from old cultures. Other
bacteria, as for instance, bacterium tuberculosis, show
great resistance to autolysis, and in consequence culture
filtrates contain only a small quantity of poisonous
substances.

The poisonous substances found in culture filtrates
were at first assumed to be secretory products of the
bacteria, and were regarded as true toxin formations.
If we characterize toxins as having the property, in def-
inite doses, of bringing about the death of an animal, we
find that the endotoxins also have this property. We
have also characterized the toxins with the property of
bringing about an active immunity in animals when they
are injected in sublethal doses, and that the serum of
such animals when injected into normal animals protects
them against the minimum fatal dose of the toxin — that
is, it brings about a passive immunity. On the other
hand, the injection of the endotoxins results in the induc-
tion of bactericidal immunity with the formation of
amboceptors of a specific character.

THE POINT OF ACTION OF TOXIN AND ENDO-
TOXIN. — With regard to the point of action of the
toxins the experiments of Wasserman and others in-
dicate that as far as tetanus toxin is concerned this
poison has a direct affinity for nerve cells. With regard
to diphtheria toxin one sees, on making a postmortem

570 BA CTERIOLOG Y.

examination of an animal injected with the toxin, certain
characteristic lesions which have been brought about by
the action of the toxin. For instance, there is always
very marked hyperaemia and necrosis at the point of
injection. Certain internal organs also show almost
constantly marked hyperaemia, especially the adrenals
and the chain of hsemolymph glands lying in the retro-
peritoneal space. Histological studies of the different
tissues have revealed certain other lesions attributable
to the action of the toxin, especially areas of necrosis in
the liver and other organs. Our knowledge of the man-
ner in which sudden death is frequently brought about
during convalescence from diphtheria shows that the
fatal result in such conditions is, however, not due to
any of the alterations or reactions of the toxin that have
so far been considered. Death during the period of con-
valescence from diphtheria is due to degenerations occur-
ring in the nerve trunks of important nerves, especially
of the pneumogastric. With this fact in mind it seems
evident that we must regard this form of toxic action of
diphtheria toxin as of far greater moment. In poison-
ing with diphtheria toxin then we also see secondarily
at least, if not primarily, a direct affinity of the toxin
for nerve cells.

With regard to the action of the endotoxins the facts
at hand are far less satisfactory. According to Ehrlich's
side-chain theory the amboceptors formed in the serum
as the result of recover)' from infection have been formed
as the result of the overproduction of certain cell recep-
tors, because of the action of the bacteria themselves or
their endotoxins upon certain tissue cells. When we
come to look for evidence as to the particular organs or
cells that are aifected in different infections we find very

INFECTION AND IMMUNITY. 571

little that is conclusive, except the fact that Metclmikoff
has repeatedly claimed that in all of these infections the
leucocytes are alone the cause of the complicated appear-
ances resulting from infection. Wolff l is inclined to
believe that the antibody formation occurs in all other
organs except the one in which the endotoxin exerts its
poisonous action, because Pfeiffer and Marx, and Wasser-
inan in their work upon microspira comma and bacillus
typhosus have shown that the principal points of forma-
tion of the specific immune body are the haematopoietic
organs. Wolff is inclined to regard the principal point
of attack of the endotoxins of all pathogenic organisms
to be the central nervous system, and as the result of
certain experiments which he has made he believes that
the immune bodies are formed in the other organs. The
facts which have led him to this conclusion are the re-
sults obtained in intracercbral injections of certain poisons.
Such injections, of course, bring the poison in direct re-
lation with the point of selective action, and there is
little opportunity for the anchoring or destruction of the
poison by other tissue cells or body fluids. In injections
into other parts of the body, especially when endotoxins
or bacteria are employed, there is an opportunity for the
anchoring of these elements to the receptors of certain
cells and, in consequence, unless the amount of toxin is
very large, the formation of antibodies takes place. On
the basis of such experimental results it seems probable
that Wolff's conclusions are correct.

THE DEFENCE OF THE BODY AGAINST INFEC-
TION.— Briefly stated, the invasion of the body by infec-
tive micro-organisms may best be conceived as a contest

1 Wolff: Centralblatt f. BaotorioloRie, originate, 1904, Bd. 37, and
Berliner klin. Wochenschr., 1904.

572 BA CTERIOLOG Y.

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 poison-

/ ous products of their growth, while the means of
defence possessed by the latter are the phagocytic cells,
such as the leucocytes, the large mononuclear cells of
the blood, and the connective tissue and endothelial cells,
as well as the vital substances which act, so to speak, as
antidotes to bacterial poisons. If the leucocytes and
tissue elements are not of sufficient vigor to destroy the
invading bacteria or to render inert the poisons pro-
duced by them, the bacteria are victorious and infection,
in different degrees of severity, results ; on the other
hand, if there be failure to excite disease, the tissues are
victorious, and are then said to be resistant to or immune
from this particular type of infection.

In some cases the protective bodies possessed by the
animal act directly upon the invading organisms them-

" selves — i. e., they are germicidal ; in others their func-
tion is more that of antidotes, or neutralizes in the
chemical sense, of the poisons produced by these organ-
isms, the organisms themselves, in certain instances,
experiencing only slight injury from a limited sojourn
in the living tissues. For those constituents of the
animal body that are by nature endowed with germj-
cidal peculiarities, the designations "alexins" (Buchner)
and "defensive proteids" (Hankin) have been sug-
gested. Careful investigation has shown that the normal
bactericidal properties of the blood serum rest upon the
presence of immune substances similar in nature to those
found in the blood serum of immune animals — i. e., (1) a
specific immune body (intermediary body) for a partic-
ular organism, and (2) complement. So far as we can

INFECTION AND IMMUNITY. 573

learn the blood serum contains normally a small amount
of antitoxic, agglutinative, and bactericidal action against
a great variety of pathogenic bacteria.

To those ill-defined substances whose affinities are
restricted to the soluble toxins elaborated by the inva-
ding bacteria the name " antitoxins " is now generally
applied. Contrary to what we have seen in the case of
the " alexins " normally present, antitoxins are to be de-
tected in the normal animal organism in very small
amounts. When they do exist under such conditions v
they are of but comparatively feeble potency.1 In the
great majority of instances antitoxic activities are acquired
peculiarities ; acquired in some cases in a more or less nat-
ural manner, as in the course of a non-fatal attack of a
specific malady ; induced in others by purely artificial
means, as we have seen to be possible in the case of diph-
theria, tetanus, etc. Our acquaintance with these bodies
extends little further than their physiological functions
and some of the means that induce their generation.
We have no satisfactory knowledge of their intimate
nature or of the primary sources of their production.
They are believed by some (Buchner 2 and Metchnikoff'3)
to represent, when artificially induced, bacterial toxins
that have been modified by the vital action of the inte-
gral cells of the body ; and Roux4 and Buchner5 main-

1 See Bolton : Transactions of Association of American Physicians,
1896, vol. xi. p. 62. Pfeiffer : Deutsche ined. Wochenschrift, 1896,
No. 8. Fisehl and v. Wiinschheim: Centralhlatt fur Bakteriologie,
Parasitcnkunde, und InfektionskranklH'iten, 1896, Abt. i. Bd. xix. S.
(i.">2. Wasscrmann : Berliner klin. Wochenschrift, 1898, No. 1.

2 Buchner: Munchener med. Wochenschrift, 1893, Nos. 24 and 25.

'Metschnikoff: Weil's Handlmch der Hygiene, Bd. ix. Lieferung 1,
8.48.

*Roux : Annales de PInstitut Pasteur, 1894. p. 722.

'Buchner: Berliner klin. Wochenschrift, 1894, No. 4.

574 BA CTERIOL OG Y.

tain that they exhibit their protective functions less by
direct combination with the toxins than by a specific
stimulation of the tissue-cells that enables the latter to
resist the harmful influences of the poisonous bacterial
products. On the other hand, Behring,1 Ehrlich,2 and
their associates contend that they are vital tissue ele-
ments, having the property of combining directly with
the toxins to form " physiologically neutral " toxin-anti-
toxin compounds that are in a manner analogous to the
double salts of certain chemical reactions.

NATURAL IMMUNITY. — It is well known that among
man and the lower animals individuals are frequently
encountered who are, in general, less susceptible to infec-
tion than are others of their species ; and that particu-
lar species of animals not only do not suffer naturally
from certain specific diseases, but resist all efforts to
produce the diseases in them by artificial methods ; in
other words, they are naturally immune from them.
The term " natural immunity," as now employed,
implies a congenital condition of the individual or
species, a condition peculiar to his idioplasm, which has
been transmitted to him as a tissue-characteristic through
generations of progenitors.

ACQUIRED IMMUNITY. — Again, it is often observed
that an individual or an animal after having recovered
from certain forms of infection has thereby acquired
protection from subsequent attacks of like character ; in
other words, they are said to have acquired immunity
from this disease. "Acquired immunity" implies,
therefore, a condition of the tissues of an individual,

1 Behring : Infektion und Desinfektion, Leipzig, 1894, S. 248.

2 Ehrlich : Klinisches Jahrbuch, 1897, Bd. vi. Heft 2, S. 311. Fort-
schritte der Mediciu, 1897, Bd. xv. No. 2.

INFECTION AND IMMUNITY. 575

not of necessity peculiar to other members of the race
or species, that has originated 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.

Active Immunity. — Acquired immunity may be either
active or passive in character. Active immunity is
that form which results from recovery from infection
acquired in a natural way, or from infection induced by
the injection of dead or living organisms as a prophy-
lactic measure against infection.

Passive Immunity. — Passive immunity is that form
in which the immune bodies generated in a susceptible
animal, as the result of systematic injections of dead or
living cultures, are introduced into a human being
to protect against infection. The antitoxic serums have
been employed most frequently to bring about passive
immunity. The protective value of diphtheria anti-
toxin in those that have been exposed to infection is
well established. The use of tetanus antitoxin for pro-
phylactic purposes is also recommended in cases where
there is a possibility of the development of tetanus.

VACCINATION AGAINST BACTERIAL DISEASES. —
The employment of various prophylactic measures
against infectious diseases has received much attention
in recent years. The measures employed in different
diseases vary somewhat, though in general the principles
are similar.

The first measures of this nature that were employed
on a large scale are those of the Haff kine vaccination
against cholera and plague by means of cultures that
had been killed after heating to a moderate temperature.
The dead organisms when injected bring about a reac-

576 BACTERIOLOGY.

tion in the body as shown by a marked increase in the
/agglutinative and bactericidal properties of the blood
serum against the particular organism. The favorable
results following the use of the Haffkine fluid in pre-
venting plague have already been given (see p. 325).

Wright has introduced a similar method of vaccina-
tion against typhoid fever. The prophylactic treatment
consists of one or two injections of dead cultures of
bacillus typhosus. Caiger ! gives the results obtained
in the British regiments serving in India. Amongst
15,384 inoculated men the incidence of typhoid fever
was 0.8 percent., as against 1.5 per cent, in the uninocti-
lated. The case mortality amongst the inoculated was
15.6 per cent., as against 26.6 per cent, in the uninocu-
lated. The results obtained in the military hospitals in
South Africa show that the case mortality was 8.2 per
cent, among the inoculated, as against 15.1 per cent,
among the uninoculated ; a reduction in the mortality
of about 50 per cent. In the staffs of three of the mili-
tary hospitals the reduction in the mortality was nearly
threefold.

Bresredka 2 sought to find a method of immunization
against plague, cholera, and typhoid that would be free
from the objectionable features of the method now in
use. For this purpose he places a culture of bacil-
lus typhosus into typhoid immune serum until it is
completely agglutinated. The bacteria are then removed
from the serum by centrifuging and subsequently washed
with sterile physiological salt solution in order to
remove all trace of the serum. The bacteria are then
heated to 60° C. for several hours. When these bac-

1 Caiger : The Lancet, 1904, vol. ii., p. 1467.

2 Bresredka : Annals of the Pasteur Institute, 1902, T. 16.

INFECTION AND IMMUNITY. 577

teria are now injected they bring about a distinct immu-
nity within twenty-four hours without inducing any of
the unfavorable effects seen in the Haffkine vaccination,
such as fever, pain, weakness, etc. The immunity
induced in this manner rests upon the formation of
specific antibodies. The agglutinated bacteria can be
preserved for a long time. The immunization is not
dangerous, as no complications have been noted.

PREGIPITINS. — The immunization of animals with a
variety of substances other than bacteria has served to
shed light upon the intimate mechanism of immunity.
One of the reactions that is noticed as the result of such
immunization is the formation of precipitins when the
serum of the immunized animal is mixed with the sub-
stance with which it has been treated. For instance,
the serum of an animal that has received repeated injec-
tions of blood from an alien species, will cause a pre-
cipitate to form when mixed with the serum of the
species of animal from which the blood has been
derived. These " precipitins," as they are called, are
specific in that they form precipitates only with the
serum of the species of animal from which the blood has
been derived. If precipitates are formed with the serum
of other species of animals it is always in very much
lower dilutions.

This precipitin reaction is so characteristic that it is
now employed as the most satisfactory test for blood
of a particular species, especially in medicolegal cases
requiring the differentiation between human blood and
that of the domestic animals. Albuminous urine or
other albuminous fluids may also be employed in the
immunization of the animals and similar agglutinins are
formed which produce precipitates in the blood serum.
37

578 BACTERIOLOGY.

In like manner, the repeated injection of milk of one
species of animal into another will result in the forma-
tion of precipitins in the blood serum of the treated
animal that will precipitate the milk of that species of
animal from which the milk was derived.

AGGLUTININS. — In acquired immunity as the result
of recovery from a bacterial disease, and in induced
immunity after repeated injections of dead or living
cultures of bacteria, the blood serum acquires the prop-
erty of agglutinating the bacteria causing the infection.
This agglutination, as it is called, is brought about by
the presence in the blood serum of an antibody that has
the property of bringing about the clumping of the
bacteria and causes the cessation of motility in suspen-
sions of motile bacteria. This form of antibody is
spoken of as "agglutiuin." The specific action of the
agglutinin is of such a nature that it has for a long time
been employed as a means of diagnosing certain dis-
eases, especially typhoid fever, the reaction being known
under the name of Widal reaction.

It has been found that normal blood serum of both
man and the domestic animals contains normally agglu-
tinins for a variety of bacteria (Bergey).1 The normal
agglutinins are usually present in relatively low amounts,
though occasionally individuals are encountered that
possess this property to an unusual degree. These
" common " agglutinins, as they are called, may be
removed from the serum by saturation with related
organisms, but the specific agglutinins resulting from
immunization with a particular organism are not removed
in this manner.

The agglutination reaction is frequently employed for

1 Bergey : Journal of Medical Research, 1903, vol. v., p. 21.

INFECTION AND IMMUNITY. 579

the purpose of ascertaining the identity of bacteria, and
in applying this diagnostic test for this purpose it is
necessary to know the limits of the agglutinating power
of the serum for the organism employed in immunizing
the animal, so as not to be misled by the presence of
relatively large amounts of the common agglutinins.1

The agglutinating properties of the blood serum of
an immune animal are not always in proportion to its
protective or curative properties. There may be a rela-
tively high degree of agglutination without a corre-
sponding bactericidal action of the serum. From this
fact the agglutinating properties of a serum cannot be
taken as a basis of its value when employed therapeu-
tic-ally. The exact relation between the agglutinating
and the bactericidal properties of the blood serum of an
animal cannot be stated, though it seems probable that
they are both the result of certain reactive processes on
the part of the tissue-elements against the bacteria.

THE MECHANISM OF IMMUNITY. — The problem
involving the explanation of the interesting ideas and
observations with regard to immunity has aiforded
material for reflection and hypothesis for a long time.
It is only through investigations conducted during recent
years that it has met with anything approaching satis-
factory solution, and even now there remain a number
of important points that are veiled in obscurity.

Conspicuous among the observers who have endeav-
ored to explain the mechanism of immunity may be
mentioned Chauveau, Pasteur, Metchnikoff, Buchner,
Fliigge and his pupils (Smirnow, Sirotinin, Bitter,

1 A great deal of work has been done in recent years on the common
and specific agglutinins in dysentery immune serum by Park and his
associates, as well as by others (see Journal of Medical Research, vols.
v., vi., and vii.).

580 BACTERIOLOGY.

Nuttall), Fodor, Hankin, Pfciffer, Ehrlich, Behring,
Roux, and a host of others whose names are more or
less prominent in current literature. In the following
pages we will present and discuss certain results of
investigations that serve both to mark the evolutionary
progress of our knowledge and to elucidate the more
important features of this complicated subject.

THE RETENTION HYPOTHESIS OF CHAUVEAU. — In
1880 Chauveau1 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,2
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.y acids or alkalies produced by the bac-
teria themselves. So long as the organisms were not
actually dead from exposure to these substances correc-
tion of the abnormal reaction was followed by further
development of the organisms. Sirotinin also states

1 Comptes rendus, etc., July, 1880, No. 91.

2 Zeitechrift fur Hygiene, 1888, Bd. iv.

INFECTION AND IMMUNITY. 581

that materials containing the products of growth of
bacteria, so long as they are maintained at a neutral or
only slightly alkaline reaction, serve very well as media
upon which to cultivate again the same organism that
produced them, providing the nutritive elements have
not been entirely exhausted. He remarks that, if in such
a concentrated form as we find the life-products of bac-
teria in the medium in which they are growing, no inhib-
itory compounds other than 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
intact, 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,1 Roux and Chamberland,2 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 would, if them-
selves introduced into the animal body, produce fatal
results. Though this observation of Salmon and Smith
attracted comparatively little attention at the time it
was made, it serves, nevertheless, as we shall see sub-
sequently, as the starting-point for a line of investigation
that has furnished practically all the information of im-
portance that we possess on this complicated subject.

THE EXHAUSTION HYPOTHESIS OF PASTEUR. —
As opposed to the view of Chauveau, Pasteur3 and
certain of his pupils believed that the immunity fre-

1 Proceedings of the Biological Society, Washington, D. C., 1886,
vol. iii.

2 Annales de I'lnstitut Pasteur. 1888-'89, tomes i., ii.

3 Bulletin de 1' Academic <lr Mrdccinc, 1880.

582 BACTERIOLOGY.

quently afforded to the tissues by an attack of infection
or following upon vaccination against infection, was
due rather to an abstraction from the tissues, by the
organisms that were concerned in the primary attack,
of a something that is necessary to the growth of the
infecting organism should it gain entrance to the body
at any subsequent time. This view is known as the
" exhaustion hypothesis"

As to the exhaustion hypothesis of Pasteur, there is,
as yet, no evidence whatever for its support ; and, in
fact, in the light shed by more recent investigations this
is probably the least tenable of any of the several hy-
potheses advanced in explanation of immunity. The
work of Bitter/ which was undertaken with the view
of determining if, in the process of acquiring immunity,
there occurred this exhaustion from the tissues of mate-
rial 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.

THE PHAGOCYTOSIS THEORY OF METCHNIKOFF.
— In 1884 Metchnikoff 2 published the first of a series
of observations upon the behavior of certain of the
mesodermal cells of lower animals toward insoluble
particles that may be present in the tissues of these
animals. The outcome of these investigations was the
establishment of his well-known doctrine of phago-
cytosis, the principle of which is that the wandering

1 Zei tech rift fur Hygiene, 1888, Bd. iv.

2 Arbeitcn aus dem Zoologischen Institut der Universitat Wien,
1884, Bd. v. Fortschritte der Medicin, 1884, Bd. ii.

INFECTION AND IMMUNITY. 583

cells of the animal organism, more especially the leuco-
cytes, possess the property of taking up, rendering
inert, and digesting micro-organisms which they may
encounter in the tissues. Metchnikoif believed that 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 and the tissue cells on the other.
The success or failure of the leucocytes in protecting
the animal against infection depends, according to this
doctrine, entirely upon the efficiency of the provisions
possessed by them for destroying bacteria, and upon the
aggressive powers of the invading organisms. When
the activity of the body cells is of sufficient vigor to
bring about the death of the bacteria the tissues are
victorious ; but when the poisons generated by the bac-
teria are potent to arrest the phagocy tic action of the leu-
cocytes then the tissues succumb and infection results.

THE ALEXIN THEORY OF BUCHNER. — Attractive as
this doctrine is, plausible as were the arguments in sup-
port of it, it is nevertheless, in the light of later evi-
dence, inadvisable to accept it unconditionally in the
sense in which it was originally propounded ; in fact,
Metchnikoif himself has in recent years seen fit to adopt
certain modifications of his views as first expressed.
The later studies of a number of investigators indicate
that while the leucocytes play an important part in the
phenomenon of immunity, especially in natural immu-
nity, it is hardly likely that this always occurs through
their taking up and digesting within themselves inva-
ding bacteria, as Metchnikoff believed ; but rather that
their part in the process is to secrete protective chemical

584 BA CTERIOLOG Y.

substances that are thrown into the circulating blood,
and which, in part at least, comprise the defensive bodies
to which Buchner has given the name " alexins." 1

The first severe blow that Metchnikoff's theory of
phagocytosis received was given by Nuttall,2 in his
work upon the bactericidal property of the animal econ-
omy. In these experiments Nuttall demonstrated that
the destruction of virulent bacteria in the blood of
animals was not necessarily dependent upon the actual
presence of living leucocytes, but that the serum of the
blood when quite free from cellular elements possessed
this power to a degree equal to that of the blood
when all the constituent parts were present. In the
blood, as such, phagocytosis could be seen ; but, as a
rule, the bacteria observed within leucocytes presented
evidence of having undergone degenerative changes be-
fore they had been taken up by the wandering cells — i. e,
the bacteria had evidently been injured or killed by the
fluids before they were attacked by the phagocytes.

There is a bit of interesting history leading up to the
idea on which Nuttall worked. Contrary to the beliefs in
existence at the time, Traube and Gscheidlen,3 as far back
as 1874, demonstrated that considerable quantities of sep-
tic material could be injected into the circulation of warm-
blooded animals without apparently any effect upon the
animal. Particularly was this the case with dogs. If
they injected into the circulation of a dog as much as
1.5 c.c. of decomposing fluid, blood drawn from the ani-
mal after from twenty-four to forty-eight hours showed
no special tendency to decompose, though it was kept

1 See Hahn : Archiv fur Hygiene, 1895, Bd. xxv. S. 105.

2 Zeitschrift fur Hygiene, 1888, Bd. iv.

3 Jahresb. der Schlesischen Ges. fur Cultur., 1874 ; Jahr. iii. p. 179.

INFECTION AND IMMUNITY. 585

under observation for a long time. As a result of these
experiments, the question that naturally presented itself
was : Does the animal organism possess the power of
rendering septic organisms inert, and if so, to what
extent? They believed this power of rendering living
organisms inert to be possessed by the circulating
blood to only a limited degree, for after the injection
of much larger amounts of the putrid fluid into the
blood of the animal death usually ensued in from
twenty-four to forty-eight hours. The blood drawn
from the animal just before death contained the living
bacteria of putrefaction and promptly underwent decom-
position. They attributed the germicidal phenomenon
to the action of the " ozonized oxygen of the corpuscles
of the blood."

Similar observations were made in 1885 by Fodor,1
who remarks upon the rapidity with which living bac-
teria disappear from the circulating blood of animals ;
and Wyssokowitsch,2 who endeavored to explain this
disappearance experimentally, went wide of the mark
by concluding that they were filtered from the blood
and digested by the parenchymatous tissues.

In 1882 Rauschenbach 3 demonstrated that when in
the process of coagulation fibrin was formed, it was not
as a specific product of the action of the colorless ele-
ments 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 disintegration of the leucocytes present. In

1 Fodor: Arcliiv fiir Hygiene. I',<1. iv. S. 129.

2 Wyssokowitsrli: /citsrlirirt fiir Ilyjriene, 188(5, S. 3.

3 Uohcr die \Vcrliscl\virkiinjy savisrlion I'rotoplasniii mid lilutplasma.

|)i>siTlaliun, Dorpat, IKH'-i.

586 BACTERIOLOGY.

1884 Groth1 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,2
a pupil of Alexander Schmidt, undertook to study the ac-
tion of the circulating blood upon the vegetable proto-
plasm of bacteria. He noticed that clotting of the blood
of the horse was very much accelerated by the addition to
it of certain bacteria ; that at the same time the develop-
ment of the bacteria was checked, and in the case of the
pathogenic varieties their virulence was diminished.
This was particularly the case when the anthrax bacil-
lus was employed.

Grohmann seems to have appreciated the significance
of this observation, though he made no attempt to study
the subject more closely. He remarks that the system
probably possesses, in the plasma of the blood, a body hav-
ing disinfectant properties.3 His observations on this
particular point were, however, only incidental, and it
was not until the researches of Nuttall, directed especi-
ally toward the question of germicidal properties of
normal animal fluids, that a complete and acceptable
demonstration of this important function was forthcom-
ing, and that a general interest, commensurate with its
importance, was created in the subject.

1 Ueber die Schieksale der farblosen Elemente in kreisendem Blut.
Dissertation, Dorpat, 1884.

2 Ueber die Einwirkung des zellenfreien Blutplasma auf einige
pflauzliche Mikro-organismen. Dissertation, Dorpat, 1884.

8 Loc. cit., pp. 6 and 33.

INFECTION AND IMMUNITY. 587

Nuttall's results received confirmation from all sides.
Fodor,1 Buchner,2 Lubarsch,3 Nissen,4 Stern,5 Prudden,6
Charrin and Roger,7 and many others continued in the
same line, and all made practically the same observation.

After the demonstration by Nuttall that the serum
of the blood was directly detrimental to the vitality of
certain pathogenic bacteria, a number of investigators
undertook to determine the conditions most favorable to
the exhibition of this phenomenon, and further to decide
upon the constituent of the serum to which this property
is due, or if it is a function of the serum only as a whole.

In the course of Buchner's experiments it was de-
monstrated that the serum was robbed of this property
by 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 a reduction of its bactericidal
activity. From this he concluded that the active ele-
ment in this phenomenon is a living albumin, an essen-
tial constituent of which is sodium chloride, and which,
when robbed of this salt either by dialysis or dilution,
becomes inert in its behavior toward bacteria. For this

1 Central, fur Bakteriologie mid Parasitenkunde, 1890, Bd. vii. No. 24.

2 Archiv fur Hygiene, 1890, vol. x. parts 1 and 2.

3 Centralblatt fur Bakteriologie und Parasitenkunde, 1889, Bd. vi.
No. 18.

4 Zeitscrift fur Hygiene, 1889, Bd. vi. part 3.
5Zeitscrift fur klin. Medicin, 1890, Bd. viii. parts 1 and 2.
•New York Medical Rec;rd, 1890, vol. xxxvii. pp. 85 and 86.
7 Societe de Biologic de Paris.

588 BACTERIOLOGY.

or these germicidal constituents of the blood he sug-
gested the name " alexins."

Buchner found, moreover, that the activity of the
serum alone against bacteria was greater than when the
cellular elements of the blood were present. This he
explains by the assumption that in the serum alone the
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.1 The former isolated from the spleen and
lymphatic glands a body — a globulin — which in solu-
tion possesses germicidal properties.

Similar germicidal, ferment-like globulins have been
isolated from the blood by Ogata,2 and in their studies
upon tetanus Tizzoni and Cattani 3 found a body that
was antagonistic to the poison produced by the organism
of this disease.

In 1890 Fodor4 showed that the fatal results of
anthrax infection could be materially retarded by
alkalinization of the circulating blood ; and he further
states that it is occasionally possible by the admin-
istration of alkalies per os to rescue animals already
infected.

1 British Medical Journal, May 31, 1890.

2 Centralblatt fiir Bakteriologie mid Parasitenkunde, 1891, vol. ix,
S. 599.

3 Ibid., p. 685.

« Fodor: Centralblatt fiir Bakteriologie, 1890, Bd. vii. S. 753-7(56.

INFECTION AND IMMUNITY. 589

According to the observations of Vaughan,1 the most
important germicklal or protective agents possessed by
the body are the nuclcins ; 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 believed the globulins or " defensive pro-
teids" that he had 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 poison-
ous metabolic products of growth of the organisms.
For example, if the poisonous products of virulent
anthrax bacilli be isolated and mixed with the glob-
ulin 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 employed as vaccines was much less than that
produced by organisms possessing full virulence ; and
lie suggested that perhaps the protective influence of
vaccinations that are practised by introducing into the
animal the organisms that have been attentuated in vir-
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 Sewall2

1 Vaughan : Medical News, May 20, 1893.

2 Journal of Physiology, 1887, vol. vii. p. 203,

590 BACTERIOL OG Y.

(and more recently in the work of Fraser,1 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 investigations upon
the blood and fluids of the body are the experiments
of Behring and Kitasato 2 upon the production of im-
munity from tetanus. In their studies upon the blood
of animals subjected to these experiments it was found
that it was not only possible to render animals immune
from this disease, but that the serum of the blood of
these immunized animals afforded immunity when in-
jected into the peritoneal cavity of other animals that
had not been so protected ; and moreover, that in some
cases this serum possesses curative powers over the
disease after it has been in progress for a time. They
found, further, that the serum of animals that had been
rendered immune from tetanus, when brought in con-
tact 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.

1 One of the important results of Eraser's studies is the demonstra-
tion that the bile of normal serpents and of certain warm-blooded
animals, notably bovines, rabbits, and guinea-pigs, is antitoxic for
serpent venom. The antitoxic value of the bile in this connection
seems to be fairly comparable to that of other antitoxins obtained
from the blood of artificially immunized animals. Its action is not
only prophylactic, but also curative, Fraser having succeeded in
rescuing animals by the injection of bile-extract thirty minutes after
they had received what would otherwise have been a fatal dose of
cobra poison. (See Centralblatt fur Bakteriologie, 1897, Bd. xxii. S.
420.)

2 Behring und Kitasato : Deutsche med. Wochenschrift, 1890, Bd.
xvi. S. 1113.

INFECTION AND IMMUNITY. 591

The demonstration by Behring and Kitasato of the
fact that the serum of an immunized animal can not
only confer immunity upon another susceptible animal,
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 application of the principle
involved in this observation to the cure of diphtheria
in man has resulted in a triumph which marks an epoch
in modern scientific medicine. The same principle has
been employed for obtaining curative agents against
other forms of infection and intoxication, notably, of
Asiatic cholera, typhoid fever, lobar pneumonia, strep-
tococcus and staphylococcus infection, rabies, tubercu-
losis, bubonic plague, and serpent-venom ; but unfor-
tunately, as yet, with only indifferent success ; cer-
tainly 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
ad vanced by Buchner,1 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 has,
nevertheless, much to recommend it, and in the light
of subsequent research is regarded by many as prob-

1 Buchner : Eine neue Theorie uber Erzielung vou I m muni tat gegen
Infektionskraukueiteu. Muenchen, 1883.

592 BACTERIOLOGY,

ably the correct explanation of the establishment of im-
munity in a number of, if not in all, cases. Experiments
that bear directly upon this idea have demonstrated
that, if animals be subjected to injections of the poison-
ous products of growth of certain virulent bacteria,
they respond to this treatment by more or less pro-
nounced 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
were 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 varieties.
For instance, Emmerich and Mattel1 claim to have
rendered rabbits insusceptible to anthrax through injec-
tions into them of cultures of the streptococcus of ery-
sipelas.

This, they claimed, is not due to any antagonism be-
tween the two organisms themselves, for in culture
experiments they 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

1 Emmerich und Mattei : Fortscbritte der Medicin, 1887, S. 653,

INFECTION AND IMMUNITY. 593

a product that is detrimental to the pathogenic activity
of the anthrax bacilli. In a later paper1 Emmerich, in
a>sociation with Low, offers still another explanation for
the phenomenon. They believe that the protection
afforded an animal from a specific infective organism
by the injection into it of another organism or the
products of its growth is due to the direct bacteriolytic
action of the enzymes peculiar to the latter upon the
former species. In their opinion, the enzymes of the
latter organism enter into a more or less stable combina-
tion with the living protoplasm, and in this state are
actively destructive (digestive) for the invading patho-
genic species.

Pawlowsky,2 who obtained similar results from the
introduction into animals of cultures of bacillus pro-
diffiosus, of micrococcus aureus, and of micrococcus lan-
ceolatus, believes them to be due to the induction of
increased energy on the part of the wandering cells,
preparing them thus for the difficult task of destroying
the more virulent organisms with which the animal is
subsequently to be inoculated.

Protection afforded in this way apparently centra-
indicates a specific relation between the morbific ele-
ments 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 Mattel and of Paw-
lowsky can be interpreted in another way: in the blood
of animals there is present what may be termed a nor-

1 Emmerich und Low : Zeitschrift fur Hygiene uiid Infektiouskrank-
heiten, 1899, Bd. xxxi. S. 1-5.
* Pawlowsky: Virchow's Archiv, vol. cviii. p. 494.
38

594 BACTERIOLOGY.

mal protective substance (Buchner's alexins) having no
specific relations to any particular variety of infection, but
offering some protection, more or less complete, to the
animal 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 anti-
dote was simply temporarily accentuated through the
tissue-stimulation resultant upon the treatment that the
animals had received, for it is not possible to bring
about in this way as high or as permanent a degree of
immunity 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.

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 is possible to confer immunity upon
animals from this disease; that the blood-serum of
such animals protects susceptible animals into which
it is injected against what would otherwise be a
fatal dose of the cholera spirillum ; that the perito-
neal fluid of the artificially immunized animal has
an almost instantaneous disintegrating (bacteriolytic),
bactericidal action upon living cholera spirilla that
are injected into the peritoneal cavity ; that the
serum from the immune animal, when kept for a
time, has no such effect upon cholera spirilla in
the test-tube ; but when virulent cholera spirilla are
injected into the peritoneum of an animal that is

1 Pfeiffer: Zeitschrift fur Hygiene und Infektionskrankheiten, Bd.
xviii. S. 1 ; Bd. xx. S. 198.

INFECTION AND IMMUNITY. 595

not immune, and this is at once followed by an intra-
peritoneal injection of serum from an immune animal,
almost instantly the peculiar disintegration of the bac-
teria, as observed in the peritoneum of the immune ani-
mal, can 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 susceptible animal, of endowing its
fluids with the peculiar disintegrating, germicidal
function noted in the peritoneum of the immune ani-
mal from which the serurn originated ; quickly loses its
bacteriolytic activity outside the body, but the influ-
ence of which in the peritoneum of a susceptible ani-
mal is to call forth at once this interesting phenomenon.
Manifestly the germicidal substance in this case is
either generated by the tissues as a result of the specific
irritation by a something contained in this serum — i. e.,
in consequence of a reaction on the part of the peritoneal
tissues, or possibly those of the entire animal — or else it
is, as Ehrlich conceives it to be, a complex whose physi-
ological activity depends upon the union of at least two
essential groups — the one present in the serum of the im-
mune animal, and the other in the fluids of the normal
animal.

In more recent investigations Pfeiffer, in association
with Marx, has found that the bactericidal substances in
cholera-immune animals are much more abundant in
the blood-building organs — spleen, lymphatic glands,
and bone-marrow — than in either the blood or other
tissues.1

1 Pfeiffer and Marx : Deutsche med. Wochenschrift, 1898, No. 3.

596 BA CTERIOLOG Y.

The experiments of G. and F. Klcmperer l upon acute
fibriuous pneumonia, though too limited in extent to
be accepted as conclusive, presented nevertheless a
number of most significant suggestions, not only in
connection with several obscure features of this disease,
but in their broader bearing upon acquired tissue-resist-
ance in general .

These authors found but little difficulty in conferring
immunity upon animals that are otherwise susceptible to
the pathogenic action of the organisms concerned in the
production of this disease,2 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 transmitted to
the young, during the nursing-period, through the milk
of the mother, as Ehiiich3 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
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

1 G. and F. Klemperer : Berliner klin. Wochenschrift, 1891, Nos.
34 and 35.

2 Animals do not, as a rule, present the pneumonic changes seen in
human beings. The introduction of micrococcns lanceolatus into their
tissues results, in the case of susceptible animals, in the production of
septicaemia.

3 Ehrlich : Zeitschrift fur Hygiene und Infektionskrankheiten, 1892,
Bd. xii. S. 183.

INFECTION AND IMMUNITY. 597

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 injeetion was followed by complete immunity
in from three to four days ; whereas, if the unwarmed
material was used, immunity did not appear before
fourteen days, and then only after the employment of
relatively large amounts. Moreover, when the pre-
viously heated products are introduced into the circu-
lation of the animal the systemic reaction is of but
short duration ; but if the unwarmed substance is
employed, immunity is manifest only after the onset
of considerable elevation of temperature, which lasts
for a long time.

In explanation of these differences they suggested
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
subjected, and which is necessary before they are in a
position to bring about the condition of immunity.
They claimed 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 protects the animal. In support of this, their
argument was 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 intro-
duced into the circulation of another animal, no such
delay is seen, but instead the animal is forthwith pro-
tected. In the former case the actual protecting body
had first to be manufactured by the tissues ; whereas

598 BACTERIOLOGY.

in the second it is already prepared, and is introduced
as such into the second animal.

These authors found the serum of artificially immun-
ized animals to be not only capable of rendering other
animals immune, but to be possessed of curative powers
when the disease was already in progress. The serum
of immunized animals when injected into the circula-
tion of animals in which there was a body-temperature
of from 40.4° to 41° C. reduced this temperature to
normal (37.5° C.) in twelve consecutive experiments
during the first twenty-four hours following its employ-
ment.

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
sufficient 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 sys-
temic manifestations gradually disappear. The Klem-
perers claim to have isolated from cultures of micro-
coccus lanceolatus a proteid body that is the agent con-
cerned in producing the tissue-reaction which results in

INFECTION AND IMMUNITY. 599

the formation of the protecting substance. They like-
wise isolated from the serum of immunized animals
a proteid that possessed 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.1

In the light of these experiments the hypothesis
advanced by Buchner, that the establishment of im-
munity is to be explained by reactive changes in the
integral cells of the body, receives additional support,
and when we consider the observations of Bitter,2 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 site of inoculation,
and from this point produce, by the absorption of their
chemical products, the systemic changes through which
the animal is protected against subsequent infection by
the virulent organisms, we feel justified in concluding
that the wreight of evidence is strongly in favor of this
view.

1 Emmerich and Fowitzky : Miincheuer med. Wocliensehrift, 1891,
No. 32.
1 Bitter: Zeitschrift fur Hygiene, 1887, Bd. iv.

600 BACTERIOLOGY.

EHRLICH'S " SIDE-CHAIN " THEORY. — The most re-
cent interpretation of the phenomenon of acquired im-
munity is that proposed by Ehrlich in 1897.1 It is
generally known as his "side chain" or. "lateral bond"
(Seitenketten) theory of immunity. It is one of the most
attractive of all the hypotheses that have been advanced,
and is in many ways the most satisfactory.

Its fundamental features comprise the acceptance of
Weigert' s doctrine concerning the mechanism of physio-
logical tissue-equilibrium and repair ; and the assump-
tion of a specific combining relation, or affinity, between
toxic substances and the cells of particular tissues.

At the meeting of German Naturalists and Physicians
held at Frankfort-on-the-Main, in 1896, Weigert2 ad-
vanced an hypothesis the essential features of which
are that physiological structure and function depend
upon the equilibrium of the tissues maintained by virtue
of mutual restraint between its component cells ; that de-
struction of a single integer or group of integers of a tissue
or a cell removes a corresponding amount of restraint at
the point injured, and therefore destroys equilibrium and
permits of the abnormal exhibition of bioplastic ener-
gies on the part of the remaining uninjured components,
which activity may be viewed as a compensating hyper-
plasia ; that hyperplasia is not therefore the direct
result of external irritation, and cannot be, since the
action of the irritant is destructive and is confined to
the cells or integers of cells that it destroys, but occurs
rather indirectly as a function .of the surrounding unin-
jured tissues that have been excited to bioplastic activity

1 Ehrlich: Klinisches Jahrbuch, 1897, Bd. vi. Heft 2, S. 309.

2 Weigert, Carl : " Neue Fragestellungen in der pathologischen Anat-
omie," Verhandlungen der Ges. deutscher Naturforscher und Aerzte,
1896, S, 121.

INFECTION AND IMMUNITY. 601

through the removal of the restraint hitherto exerted by
the cells destroyed by the irritant ; and, finally, when
such bioplastic activity is called into play there is always
/i^pe/'compensation — /. e.} there is more plastic material
generated than is necessary to compensate for the loss.
Ehrlich applies this idea to the individual cell, which
he conceives to be a complex molecule, comprising a
primary central nucleus to which are attached by side
chains its secondary atom-groups, in much the same way
that our conception of the reaction-structure of complex
organic chemical compounds is represented graphically.
Injury to one or more of these physiologically essential
atom-groups results, according to the view of Weigert,
in disturbance of the cell-equilibrium and consequent
effort on the part of the surrounding atom-groups at
compensatory repair. With this liberation of bioplastic
energy there is more plastic material generated than is
necessary for the repair of the injury. The excess of
this material finds its way into the blood and, as we
shall see presently, is regarded by Ehrlich as the real
antitoxic substance. Assuming a specific combining
relation between toxic substances and particular cells or
secondary atom-groups of cells — and there are experi-
mental grounds for this assumption1 — it is evident that
the combination between the intoxicant and the partic-
ular atom-group for which it has a specific affinity is
indirectly the cause of compensatory bioplastic activity
on the part of similar surrounding atom-groups that
have not been destroyed. This results, as we learned

1 See Wassermann und Takaki : " Uebcr tetanus antitoxische Eigen-
<c hat'icii drs iM.rnialcii ( Vntralnervensysti'ins," Berliner klin. Wcx-lion-
*Hirift, 1898. No. 1. S. .">. NYissor mid \\YrlislxTtf : X.'itsclirift fiir
Hyjrirno mid Infrktionskrankhcitcii, Bd. \\xvi. S. 299. Madsen :
Il.id., Bd. xxxii.S. 214.

602 BACTERIOLOGY.

above, in hypercornpensation, the excess of plastic mate-
rial being disengaged from the parent-cell and thrown
free into the circulating fluids, there to combine directly
with the same intoxicant should it subsequently gain
access to the animal. This excess of plastic material
thrown into the circulation combines, according to Ehr-
licli,1 directly with the intoxicant to form physiologic-
ally inactive toxin — antitoxin compounds (see page 497),
and can therefore be reasonably regarded as the antitoxic
material of animals immune from bacterial and other
toxins. Since the announcement of that doctrine many
important advances have been made in our knowledge of
the subject. We have learned that immunity or tolerance
may be induced by the use of other intoxicants than those
elaborated by bacteria, and by the employment of other
cells and cell secretions. It has been demonstrated that
anti-bodies, differing in their specific actions from anti-
toxins, but originating probably in a similar manner, are
to be detected in the fluids of animals thus immunized or
rendered tolerant. For a long time we have known of
the germicidal action of normal blood-serum ; for several
years we have been familiar with the singular bacterio-
lytic phenomenon demonstrated by Pfeiffer in the peri-
toneum of animals immune of cholera ; more recently
we have learned that immunity from a variety of infec-
tions is accompanied by a power on the part of the
serum of the immune animal to agglutinate the bacteria
causing the infection ; and the profoundly interesting
investigations of Bordet, Moxter, von Dungern, Fish,
and others, have shown that immunity may be induced
from cells and secretions of animal origin hitherto re-

1 Ehrlich : " Zur Kenntniss der Antitoxinwirkttng," Fortschritte der
Mediciu, 1897, Bd. xv. No. 2.

INFECTION AND IMMUNITY. 603

garded as non-irritating and harmless. For instance,
we have learned that the blood of one animal may cause
fatal intoxication when injected into an animal of dif-
ferent species ; but if that blood be repeatedly injected
in non-fatal amounts, the animal receiving the injections
after a while becomes tolerant, and its serum reveals the
property not only of robbing the alien blood of its
hurtful properties, but also of actually dissolving its
corpuscles (haemolysis) in a test-tube. In an analogous
way, if such tissue-cells as epithelium or spermatozoa
be injected repeatedly into the tissues of animals, the
serum of the blood of those animals acquires the power
of dissolving (digesting) such cells outside the body ;
and if so inert a secretion as milk be injected into the
tissues, the blood-serum of the animal receiving the
injections after a time reacts specifically with that milk
in a test-tube — /. e., precipitates it. From the foregoing
we see that in the numerous phases and expressions of
this physiological possibility there are produced anti-
bodies having functions totally different from those
attributed by Ehrlich to antitoxins — /. e., we have
lysins, agglutinins, precipitins, etc., that in their mode
of action suggest ferments with specific affinities. It is
evident that when broadly conceived the mechanism of
immunity comprehends very much more than the neu-
tralization of a bacterial toxin by an antitoxin ; and,
what is more to the point, in many of these conditions
r,i immunity or tolerance above noted antitoxins as we
know them, are not present at all.

In an important series of papers on the hajmolysins
published by Ehrlich and Morgenroth l an effort is

und Morgeirroth: Berliuer klinische Wochenschrift, 1899,

604 BA CTERIOL OG Y.

made to elucidate further the finer mechanism of im-
munity in its broad sense and various expressions,
and to adapt the side-chain doctrine to those more
complicated phenomena in which immunity depends not
only on the elaboration of antitoxins, but also upon a
power on the part of the animal fluids to cause a com-
plete metamorphosis or disappearance of such participate
matters as bacterial and other irritating or poisonous
cells and substances. They believe the forces at work
in the establishment of immunity from bacteria and
from bacterial and other toxins, those operative in the
elaboration of the newly discovered lysins, antilysins,
agglutinins, precipitins, ferments, antiferments, etc., as
well as those concerned in physiological assimilation and
nutrition, to be fundamentally identical. They believe
susceptibility in general, as well as power to assimilate
nutrition, to be explainable through the assumption that
special molecular groups of the living protoplasm are
endowed with specific combining affinities for particular
matters; and in so far as the establishment of disease is
concerned, they regard the receptivity of the individual
to be determined entirely by the greater or less suscepti-
bility of those protoplasmic molecular groups — " recept-
tors," as they designate them — to disease-producing
agents. In individuals that have been artificially im-
munized from hurtful substances, they believe (in reit-
eration of Ehrlich's view expressed above) that the
receptive molecules have been more or less multiplied,
according to the degree of immunity, through bioplastic

Bd. xxxvi. S. 6 and 481 ; 1900, Bd. xxxvii. S. 453 and 681 ; 1901, Bd.
xxxviii. S. 251, 569, 598. See also Schlussbetrachtung : Ehrlicli, \i\
Nothnagel's Speciellen Pathologic und Therapie, Bd. viii. Theil i.
Heft 3, S. 161.

INFECTION AND IMMUNITY. 605

activity of similar, unimpaired atom-groups surrounding
those more directly influenced by the intoxicant during
the process of immunization (see page 601); and that
this excess of such "receptors," although physiologically
useless, being of no known service to normal function,
circulates unchanged in the blood, and serves, through
specific combining affinity for the poison against which
the animal has been rendered immune, to protect the
normal tissues from its hurtful action.

According to the nature of the irritant from which
the animal has been immunized, the "receptor" is con-
ceived to be either of simple or complex construction,
and its protective function to be performed in either a
comparatively simple or in a more or less complicated
and roundabout manner.

As a result of his studies of toxins, Ehrlich reached
the conclusion that they are composed of at least two
functionally distinct atom-groups : the one, a " hapto-
phore " group, characterized by its combining tendencies ;
the other, a "toxophore" group, distinguished for its
intoxicating powers ; and that for the exhibition of its
hurtful characteristics a toxin molecule needs to be first
anchored, so to speak, to the susceptible tissue by the
" haptophore " group, after which its intoxicating char-
acteristics are exhibited by the " toxophore " group. He
conceives the " receptors " to be likewise provided with
" haptophore " groups that pair with the corresponding
" haptophores" of the poison to which the animal is
susceptible or from which it has been immunized. Where
immunization has been induced against such relatively
simple substances as toxins, ferments, and certain cell
secretions, the " receptors " and their functions are com-
paratively simple — L c., the single haptophore of the

606 BACTERIOLOGY.

simple receptor pairs with that of the intoxicant and a
physiologically inert complex results. He conceives
antitoxins to be simple receptors of this type, and
believes the neutralization of toxins by them to take
place in this manner. On the other hand, if the im-
munization of an animal is accompanied by an acquired
power on the part of its serum to disintegrate bacteria,
to dissolve alien erythrocytes, to digest such cellular
elements as epithelium and spermatozoa, to precipitate
milk, or agglutinate bacterial or blood-cells, as the
studies of Pfeiffer, Bordet, von Dungern, Moxter, Fish,
Belfonte and Carbon, Metchnikoff, Gruber, Durham,
Widal, and others, have demonstrated, then the process
becomes less simple, and the atomic grouping of the
receptive molecule is correspondingly more complex.
In some cases the receptor is provided with both a hap-
tophore and a ferment-like (zymophore) group; the
function of the former being to combine with and hold
in close proximity to the latter the albumin molecule
that is to be destroyed or assimilated ; in this way
bringing and holding the albumin molecule directly
under the influence of the zymophore group. In other
cases the " receptor " functions symbolically, so to speak,
with a complementary something that circulates nor-
mally in the blood, the so-called " complement " of Ehr-
lich and Morgenroth. Under these circumstances the
" receptor " is conceived to be provided with two " hap-
tophore " groups, and becomes an " amboceptor," there-
fore, the one haptophore of which takes up and fixes
the invading bacteria, tissue-cell, or albumin molecule,
while the other pairs with the corresponding hap-
tophore of the complement, fixing the latter in close
proximity to the invading body, and thereby favoring

INFECTION AND IMMUNITY. 607

the immediate destructive activity of its "zymotoxic"
group.

It is interesting to note in connection with this
hypothesis, that both " receptors " and " complements "
are present in normal susceptible, as well as in immune
animals, but that during immunization only the "recep- N//
tors " are multiplied as a result of the specific stimula-
tion necessary to the establishment of immunity.

THE ORIGIN OF COMPLEMENT. — The origin of com-
plement is a question that is still unsolved. Some
investigators are inclined to believe that it is derived
from the leucocytes. This is the opinion of Metchnikoff
and his associates, while others believe that it is derived
from other cells and organs as well as from the leucocytes.
Again other investigators believe that it is not derived
from the leucocytes at all, but from certain other organ
cells, for instance, the spleen, pancreas, liver, and the
bone marrow. It is impossible with the knowledge at
hand at the present time to state definitely the origin of
the complement.

Multiplicity of Complement. — Ehrlich and his associates
have demonstrated that in normal serum several comple-
ments occur in association. These complements appar- \/
ently have somewhat different functions, as indicated by
the influence upon blood serum when saturated with cer-
tain elements ; in this way removing one form of com-
plement, and leaving the others intact. Again, filtering
the serum through a porcelain filter, serves to separate
some complements and allows others to remain in the
serum.

CONCLUSIONS. — According to the nature of the intoxi-
cant from which the animal is immunized, the one or the
other of the structurally and functionally different types

608 BACTERIOLOGY.

of receptors is increased — i. e., in immunity from a simple
toxin the simplest type of receptor appears in the blood
(receptors of the first order, Ehrlich) ; in immunity that is
associated with agglutinating or precipitating powers on
the part of the blood-serum receptors having a haptophore
and a zymophore group appear (receptors of the second
order) ; while in immunity from such molecular com-
plexes as blood-, tissue-, or bacterial cells there are
produced receptors of the third order, which act through
their haptophore groups as intermediate links between the
body to be destroyed and the normally present ferment-
like complement that is to bring about the destruction.
For all the foreign irritants from which animals have
been immunized, be it alien blood, tissue-cells, milk^ or
bacteria, there is assumed to be circulating normally in
the blood a "complement" specifically related to that
^rritant on the one hand, and to its " receptor " on the
other. This idea of plurality for the complement is
apparently the vulnerable point in the argument. At
all events, it has been vigorously assailed by Bordet and
Buchner, especially, who consider the complement as a
unit, and who do not regard it as possessed necessarily
of specific affinities beyond those common to what may
be termed proteolytic enzymes in general ; and Buchner
regards it as nothing more than the normally present
"alexin," to which he called attention years ago.
Whether these objections be well taken or not, whether
the doctrine as a whole can be accepted or not, the experi-
mental data on which it is based warrant the opinion
that it is the only satisfactory working hypothesis that
has been offered in explanation of the mechanism of
what Buchner years ago designated the "reactive tis-

INFECTION AM) IMMUNITY. 609

sue-changes " underlying the establishment of acquired
immunity.1

The observations serving as the basis for this doctrine
have given to the blood and fluids of the body a new
and peculiar interest. According to circumstances, there
may be detected in the blood and tissue-juices a number
of bodies having totally different functions and affinities,
and therefore presumably different from one another.
To summarize briefly : First, there is normally present
in the blood-serum of practically all animals the de-
fensive " alexins " already mentioned. Second, the
antitoxins that are found in the blood of animals arti-
ficially immunized from special sorts of infection and
intoxication, as well as occasionally in the blood and
tissues of normal animals, 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, a bac-
teriolysin — i. e., having the property of actually dissolving
bacteria, 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 ren-
dered immune from Asiatic cholera and from the typhoid
and colon infections or intoxications.2 It is not to be
confounded with the ordinary bactericidal function of
the alexins that is demonstrable in most normal serums.
Fourth, a body, the so-called " agglutinin " (Grnber),
that was considered by Widal to represent a " reaction
of infection," and not of immunity ; though at this time
its presence is generally supposed to indicate an effort

1 Justice cannot be done to the beauty and ingenuity of this con-
ception in so brief a summary as is appropriate to a text- book. To be
appreciated it must be read as it came from its authors.

* It is generally kuowu as Pfuifler's phenomenon.
39

610 BACTERIOLOGY.

on the part of the body to resist infection. The pres-
ence of this body in a serum of an animal is announced
by its peculiar influence on the activity and arrangement
of the particular species of bacteria from which the indi-
vidual is immune, or with which it is infected. In the
case of typhoid fever in man, for instance, the serum
obtained during the early and middle stages of the dis-
ease, when mixed with fluid cultures or suspensions of
the typhoid bacillus, causes the bacilli to lose their
motility and to congregate (agglutinate) in masses and
clumps, a condition never seen in normal cultures of this
organism, and practically never observed when normal
serum is employed. There are evidences of the pres-
ence of " agglutinin " in certain of the antitoxic serums
from artificially immunized animals, viz., that of ani-
mals immune from cholera, pyocyaneus, typhoid, dysen-
tery, and colon infections. So far as experience has
gone, this agglutinating property is manifested in the
great majority of cases only upon the particular organ-
isms from which the animal supplying the serum is
protected ; that is to say, the relation is specific. In
view of the fact that the power of a serum to agglutinate
bacteria is regarded by many as a concomitant of infec-
tion, the exhibition of this property by the blood of
immune animals may at first sight appear paradoxical.
We should not lose sight of the fact, however, that
agglutinin is presumably distinct from the other sub-
stances concerned in immunity, and its presence in im-
mune animals may, therefore, be reasonably explained
as a more or less permanent result of the " reactions of
infection" that were coincident with the primary stimu-
lations by specific infective or intoxicating matters nec-
essary to the establishment of the condition of immunity;

INFECTION AND IMMUNITY. 611

nor should we in this connection lose sight of the fact
that its presence is constantly to be demonstrated in
typical cases of typhoid fever, for instance, that termi-
nate fatally, and that have exhibited little or no clinical
signs of resistance at any time during their course.
Fifth, there may be demonstrated in the blood of ani-
mals that have received repeated subcutaneous injections
of milk a body — a " precipitin " — that causes a precipi- \/
tation of milk. This precipitation represents apparently
a specific reaction, for it occurs only when the blood-
serum is mixed with milk from the species of animal
that supplied the milk used for the injections. Sixth,
after the repeated injection of blood or of emulsions of
tissue-cells into the body of an animal, there appear in
the blood of that animal certain solvents, or enzyme-like
bodies, " haemolysins," " cytolysins," etc., that react
specifically upon the blood or tissue-cells injected ;
agglutinating, disintegrating, and finally completely
dissolving them. Here, too, the relations are specific.
If a rabbit, for instance, be rendered tolerant to or
immune from beef-blood, its serum dissolves only the
red corpuscles of bovines ; if from dog's blood, then
only the corpuscles of the dog are dissolved by the
serum of the rabbit. Similarly, if a rabbit be rendered
tolerant to injections of emulsions of epithelium cells,
then its serum dissolves epithelium and not other cells.
All these reactions may be seen in a test-tube or under
the microscope. Seventh, if a hsemolyzing serum, pre-
pared as indicated under the sixth observation, be heated
for a short time to 54°-56° C., it at once loses the
hsemolytic function, but regains it again if a few drops
of serum from a normal animal be added to it. In this
phenomenon of haemolysis Ehrlich's " receptors of the

612 BACTERIOLOGY.

third order " are assumed to be concerned ; the heating
destroys the " complement," and thereby checks the proc-
ess ', but the subsequent addition of the normal serum
supplies fresh " complement/' and at once restores the
activity of the hsemolyzing receptors. Eighth, if blood
containing a hsemolysin or a cytolysin be repeatedly
injected into an animal, anti-bodies — "antilysins" — are
formed, and the serum of the animal has the power of
robbing a haemolytic serum of its hsemolyzing function
if mixed with it in a test-tube.1 Ninth, if normal blood,
jj containing complement, be injected into the same or
another species of animal, anticomplement is formed,

1 It is evident, from what has been said, that the belief in a vital
germicidal function possessed by the fluids and tissues of the body
is widespread ; is based upon the best of experimental evidence ;
and has served as the starting-point for all the important investi-
gations that have been instrumental in moulding our present ideas
of immunity. Notwithstanding this, one occasionally encounters a
dissenter. Baumgarten, in an address before the German Pathological
Society at Munich (see Berliner klinische Wochenschrift, 1899, No. 41),
made a vigorous attack upon the evidence that has been presented in
favor of a vital germicidal function of the blood-serum. He believes
the destruction of bacteria observed when they are mixed with blood-
serum to be due less to vital than to physical causes. He regards the
death of the organisms in fresh serum as the result of disturbances of
assimilation and osmosis, consequent upon their sudden transference
from the culture-medium, on which they have been accustomed to
develop, to an alien medium of different physical and chemical char-
acteristics, and not as a result of vital activities exhibited by any of
the ingredients of the serum.

His opinion is based upon the investigations carried on in his labo-
ratory by Jetter in 1892 and by Walz in 1899 (see Arbeiten aus dem
Path.-Anat. Institut zu Tubingen, Bd. i. and iii.).

Revolutionary though it may be, this doctrine, coming as it does
from so distinguished an authority, must be given due considera-
tion. As yet, it has not attracted very general attention ; nor will
it, in all probability, until the evidence advanced by Baumgarten
has been subjected to careful experimental scrutiny by other com-
petent investigators. Until such is the case, the matter may be held
tubjudicc.

INFECTION AND IMMUNITY. 613

which has the property of inhibiting the action of com-
plement.

The foregoing sketch affords but an imperfect idea of
the vast amount of labor that has been and continues to
be expended upon this many-sided, fascinating topic.
Of necessity, many important contributions have been
omitted, but those noted will serve to illustrate the lines
along which the solution of the problem has been
approached. As a result of such investigations, our
knowledge upon infection and immunity may at present
be summarized 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 phagocytic cells and the proteid bodies nor-
mally present in and generated by their integral cells.

2. That when infection occurs it may be explained
either by the excess of vigor of the bacterial products
over the antidotal or protective proteids produced by
the tissues, or to some cause that has interfered with the
normal activity of the phagocytic cells and production
of the protective bodies.

3. That in the serum of the normal circulating blood
of many animals there exists a substance that is ca-
pable, outside of the body, of rendering inert certain
pathogenic bacteria, but which is, however, present in
such small quantities as to be ineffective, either for the
protection of the animal or for the cure of infection when
introduced into the body of another animal already in-
fected.

4. That immunity is most frequently seen to follow
the introduction into the body of the products of growth

614 BACTERIOLOGY.

of bacteria that in some way or other have been
modified. This modification may be artificially pro-
duced in the products 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 apparently due to the
formation in the tissues of another body or other bodies
that act as antidotes to the poisons, and thereby protect
the tissues from their hurtful effects.

6. That this protecting proteid which is generated by
the cells of the tissues need not of necessity be antago-
nistic to the life of the invading organisms themselves,
but in most cases -must be looked upon more as an
antidote to their poisonous products.

7. That immunity, as conceived by Ehrlich, may be
either " active " or " passive." According to this inter-
pretation, it is "active" when resulting from an ordi-
nary non-fatal attack of infectious disease ; or from a
mild attack of infection purposely induced through the
use of living vaccines; or from the introduction oif
cultures of the bacteria that have been killed by heat ;
or from the gradual introduction of toxins into the tis-
sues until a marked antitoxic state is reached. It is
" passive " when occurring as a result of the direct trans-
ference of the perfected 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

INFECTION AND IMMUNITY. 615

the delay incidental to the usual modes of establishing
" active immunity." As a rule, " active " is more lasting
than " passive " immunity.

8. That phagocytosis, though frequently observed, is
effective in warding off disease in normal individuals
only when the normal defenses of the body are fully
active ; when the number of invading bacteria is rela-
tively small ; or when the bacteria are possessed of low
aggressive powers ; while in acquired immmunity it is
more probably a secondary process, the bacteria being
taken up by the leucocytes only after having been
modified in virulence through the germicidal activity
of the serum of the blood and of other fluids in the
body. It is, however, probable that the living leuco-
cytes contribute to the circulating fluids certain sub-
stances that are important to the establishment of im-
munity.

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 and other
anti-bodies, which circulate free in the blood, and in a
variety of ways serve to protect the tissues from the
harmful effect of extraneous intoxicants and irritants,
in some cases acting principally as antidotes to a toxin,
in others exhibiting more the germicidal (bacteriolvtic)
than the antitoxic property.

CHAPTER XXVI.

Bacteriological study of water — Methods employed — Precautions to be
observed — Apparatus employed, and methods of using it — Methods
of investigating air and soil — Bacteriological study of milk —
Methods employed.

BACTERIOLOGICAL STUDY OF WATER.

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 occurrence of these diseases in epidemic form through
alterations in the soil resulting from fluctuations in the
level of the soil-water ; and assign to drinking-water
either a very insignificant role, 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 outbreak of these dis-
eases ; but that, as a rule, they appear as a result of
direct infection, through the use of waters contaminated

616

BACTERIOLOGICAL STUDY OF WATER. 617

with materials containing the specific organisms known
to cause such diseases.

As a result of numerous observations by the disciples
of both schools, the evidence is now 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
liable to pollution.

The object aimed at in such investigations should be
to determine the number and kind of bacteria con-
stantly present in the water — for all waters, except
dec]) ground-water, contain bacteria ; if sudden fluctua-
tions in the number and kind of bacteria occur in
these waters, and if so, to what they are due ; and
finally, and most important, whether the water contains
constantly, or at irregular periods, bacteria that can be
traced to human excrement, not of necessity pathogenic
varieties, but bacteria that arc known to be present
normally in the intestinal canal. For if conditions
are continuously favorable to pollution of the water
by the normal constituents of the intestinal canal, the
same conditions would allow of the occasional pollution
of such water by infective matters from the b&wds of
persons suffering from specific disease of the intestines.

In considering water from a bacteriological stand-
point it must always be borne in mind that com-
parisons with fixed standards are not of much value,

618 BACTERIOLOGY.

for just as normal waters from different sources are
seen to present variations in their chemical composition,
without being unfit for use, so may the relative number
and variety of species 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 employment. For this reason systematic
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 charac-
ter of the prevailing species ; and in order to do this
the investigations must cover a long period of time
through all the seasonal variations of weather. From
data obtained in this way it may be possible without
analysis to predict approximately at any season the
bacteriological condition of the water studied. Marked
deviations from these " means," either in the quantity
or quality of the organisms present, can then be con-
sidered as indicative of the existence of some unusual,
disturbing element, the nature of which should be
investigated. It is impossible to formulate an opinion
of much value from either a single chemical or bac-
teriological analysis of a water, or from both together
in many cases; for the results thus obtained indicate
only the condition 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 waters of the immediate
neighborhood.

The interpretation of the results of both chemical
and bacteriological analyses of a sample of water ac-
quires its full value only through comparison, either
with " means " that have been determined for this

BACTERIOLOGICAL STUDY OF WATER. 619

water, or with the results of simultaneous analyses of
a number of samples from other sources of supply of
the locality.

The aid of the bacteriologist is frequently sought in
connection with investigations of waters that are sup-
posed to be concerned in the production of disease, par-
ticularly typhoid fever, either in isolated cases or in
widespread epidemic outbreaks, and in these cases both
the bacteriologist and the person employing his services
are cautioned against being too sanguine of positive
results, for in the Vast majority of instances reliable
bacteriologists fail to detect in these waters the bacillus
that is the cause of typhoid fever.

Failure to find the organism of typhoid fever in
water by the usual methods of analysis does not by
any means prove that it is not present or has not
been present. The means ordinarily employed in the
work admit of 'such a very small volume of water being
used in the test that we can readily understand how
typhoid bacilli might be present in moderate numbers
and yet none be included in the drop or two of the
water taken for study. The conditions are not those
of a W////o/(, 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 susjiension, in
every drop or volume of which the number of *»*-
pendcd particles is liable to the greatest degree of
variation. Furthermore, there arc other reasons that
would, a priori, preclude our expecting to find the
typhoid bacilli in water in which we may have reason
to believe they had been deposited, because attention is
not usually directed to the water until the disease
has become conspicuous, usually in from two to four

620 BACTERIOLOGY.

weeks after the pollution probably occurred. These
intervals of time are ordinarily sufficient for the deli-
cate, 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 influence of more hardy
saprophytic bacteria, particularly the so-called " water-
bacteria," and of more highly organized water-plants ;
the effect of precipitation and of sedimentation ; and,
of great importance, the disinfecting action of direct
sunlight.

Though the positive demonstration of typhoid bacilli
in drinking-water by bacteriological methods is of ex-
treme rarity, it must not be concluded that bacteriological
analyses 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 bacillus in the intes-
tines of typhoid-fever patients, is an organism that is
frequently found in polluted drinking-waters, and
whose presence is proof positive of pollution by either
normal or diseased intestinal contents ; and though
efforts may result in failure to detect the specific
bacillus of typhoid fever, the finding of the other
organism, bacillus coli, 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 exposed as to be liable to
such pollution should never be considered as other than
a continuous source of danger to those using them.

BACTERIOLOGICAL STUDY OF WATER. 621

Another point to be remembered is in connection with
chlorine as an indicator of contamination by human
excrement. It is commonly taught that an excessive
amount of chlorine in water points to contamination by
human excreta. This may or may not be true, accord-
ing to circumstances. A high proportion of this element
in a sample of water from a locality, the surrounding
waters of which are poor in chlorine, is unquestionably
a suspicious indication ; but in a district close to the sea
or near salt-deposits, for instance, where the proportion
of chlorine (as chlorides) in the water is generally
high, the value of the indications thus afforded is very
much diminished unless the amount found in the sample
under examination greatly exceeds the normal " mean,"
previously determined, for the amount of chlorine in
the waters of the neighborhood.

A striking example of the latter condition 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 subjected to chemical
analysis revealed such an unusually high proportion
of chlorine that, had this sample alone been con-
sidered, 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

622 BA CTER10LOQ Y.

presence of an excess of chlorine in water, while often
indicating pollution from human evacuations, may
nevertheless, sometimes arise from other sources; but
the presence in water of bacteria normally found in the
intestinal canal can manifestly admit of but one inter-
pretation, viz., that faecal matters from either man or
animals have at some time been deposited in this water,
and that while no specific disease-producing organisms
may have been detected, still waters in which such pol-
lutions are possible are a constant 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 composi-
tion of a water, calls at once for a thorough in-
spection of the supply, while at the same time the
organisms present are to be subjected to the most care-
ful study. In many instances, even after the most
thorough bacteriological and chemical study of a sus-
picious water, one is forced to admit that information
of but limited usefulness has been obtained through the
employment of such analytical methods. In these
cases too much stress cannot be laid upon the im-
portance of a systematic inspection of the supply, and
its relation to sources of pollution. Optical evidence
of more or less dangerous contamination may often be
obtained when laboratory methods fail to detect them.
The reasons for such failure, in addition to those already
given, are obvious — the polluting matters are often so
diluted by the large mass of water into which they find
their way as to be beyond recognition by the tests
usually employed in such work, and still be present in
amounts sufficient to originate disease.

THE QUALITATIVE BACTERIOLOGICAL ANALYSIS

BACTERIOLOGICAL STUDY OF WATER. 623

OF WATER. — The qualitative bacteriological analysis
of water entails much labor, as it requires not only
that all the different species of organisms found in the
water should be isolated, but that each representative
should be subjected to systematic study, and its patho-
genic 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
should be made as quickly as possible after collecting
the sample.

When circumstances permit, all water analyses should
be made on the spot where 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 for making the plates.

624 BACTERIOLOGY.

In this way one can form an idea as to the approximate
number of organisms in the water, and can, in conse-
quence, determine the amount of water best suited for
the 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 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 it is 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. As a rule, the
greater number of colonies appear upon the gelatin
plates 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 de-
velopment as do the organisms not naturally present in
water, particularly the pathogenic varieties. From these
plates the different species are to be isolated in pure
culture, the morphological and cultural characteristics
determined, and finally, by tests upon animals, it is to
be decided if any of them possess disease-producing
properties.

NOTE. — What use should be made of this observa-
tion in examining water for the presence of pathogenic
bacteria ?

The waters most frequently studied from the quali-
tative bacteriological standpoint are those suspected

BACTERIOLOGICAL STUDY OF WATER. 625

of containing specific pathogenic bacteria — i. e., waters
polluted with sewage and with human excreta that
are believed to be the source of infection of typhoid
fever, or, less frequently, of Asiatic cholera. In the
investigations of such waters there are several points of
which we should never lose sight, viz., unless the
water is under continuous study there is only a chance
of detecting the specific pathogenic species, for, as a rule,
the dangerous pollution occurs either but once or is
intermittent, so that even in the case of exposed streams
there are periods when no specifically dangerous con-
tamination may be in operation. As stated above, atten-
tion is commonly called to the water when the disease,
presumably caused by its use, is fully developed, and
this is often days or weeks after the pollution of the
stream really occurred. By an analysis made at this
time one could scarcely hope to detect the specific organ-
isms that had caused the disease. The organisms sought
for may have been present in the water and may have
infected the users, and yet have disappeared by the time
the sample taken for analysis was collected.

When present in polluted waters pathogenic bacteria
are always vastly in the minority. Were they con-
stantly present in large numbers infection among the
users of such waters would be more frequent and more
widespread than is commonly the case. They may be
present in a water-supply in small numbers ; they may
even be in the sample supplied for analysis, and yet es-
cape detection if only the ordinary direct plate method
of isolation be used.

From these considerations it is obvious that before
attempts are made to isolate the various sjK'cies directly
from a suspicious sample of water it is advisable to

40

626 BA (JTERIOLOG Y.

subject it to some method of treatment that will aid in
separating the few specific pathogenic from the numer-
ous common saprophytic species. For this purpose
numerous methods have been devised. The most use-
ful of these aim to favor the rapid multiplication of
pathogenic forms that may be present and to suppress
or check the growth of the ordinary water saprophyte.

Attention has been called to the fact that when ex-
posed to the body-temperature many of the ordinary
water-bacteria develop only slowly or not at all, while
under similar circumstances the disease-producing spe-
cies develop most luxuriantly. Advantage has been
taken of this observation in devising methods for this
particular work, of which some of the following will
prove serviceable :

Collect in a sterilized flask a sample of about 100 c.c.
of the water to be tested, and add to this about 25 c.c.
of sterilized 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 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 (see pages
454, 467) ; and by taking advantage of the effect of ele-
vated temperature upon the bacteria of water Vaughan
has succeeded in isolating from suspicious water- a
group of organisms very closely allied to the bacillus
of typhoid fever.

Theobald Smith has suggested a method by which it
is easily possible to isolate, from waters in which they
are present, certain organisms that are of the utmost

BACTERIOLOGICAL STUDY OF WATER. 627

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 an Fermenta-
tion-tube) containing bouillon to which 2 per cent, of
glucose has been added, and keeping them at the tempera-
ture of the body (37° to 38° C.), the growth-of 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 fer-
mentation-characteristics 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
intestinal bacteria • will not infrequently be found
present.

For the isolation of the typhoid bacillus, especially from
water, a host of other methods have been devised. Some
of these aim, through the addition of special chemical
reagents to the media, to retard the development of
ordinary saprophytes without interrupting the growth
of the colon and the typhoid bacillus. Most of these
methods have proved disappointing. One of them, that
of Parietti, still finds favor in the hands of some. It
consists in adding to the culture-media to be used in
the test varying amounts of the following mixture :

Phenol 5 grammes.

Hydrochloric acid 4 "

Distilled water 100 c.c.

Of this solution 0.1, 0.2, and 0.3 c.c. are added re-
spectively to each of three tubes containing 10 c.c. of
nutritive bouillon. Several such sets of tubes are to be

628 BACTERIOLOGY.

made. To each are then added from 1 to 3 c.c. of the
water, and they are placed in the incubator at body-tem-
perature. It is said that whatever development occurs
consists only of the typhoid or colon bacillus, or both, if
they were present in the original sample. They may
then be isolated and separated by the usual plate method,
or, better still, through the application of the methods
of v. Drigalski and Conradi, of Ficker, or of Hoffmann
and Ficker, or several of these methods in conjunction,
detailed in the chapter on bacillus typhosus. Personally
we have not had much success with the Parietti method.
The typhoid bacillus has been isolated from water by
passing very large quantities of water through an ordi-
nary Pasteur or Berkefeld filter, brushing off the matters
collected on the filter into a sterilized vessel and examin-
ing this by plate methods.

It has occurred to us that possibly the employment
of chemical coagulants, such as alum and iron, might
prove serviceable for this purpose. Their action would
be to mechanically drag down, in precipitating as hy-
droxides, the suspended bacteria contained in the fluid.
This precipitate could then be examined bacteriologi-
cally, instead of the water, and the recent experiments
of Ficker (loc. tit.) appear to demonstrate the value of
such a procedure.

The difficulties in this field of work are obviously
due to the suspension of a very small number of the
disease-producing organisms sought for in large volumes
of fluid, and the association with them of large numbers
of other species that offer a very great obstacle to the
successful search for the pathogenic varieties.

If by either of the above procedures bacilli that bear
any resemblance to bacillus typhosus be isolated, re-

BACTERIOLOGICAL STUDY OF WATER. 629

course must then be had to all the differential tests
detailed in the chapter on that organism.

THE QUANTITATIVE ESTIMATION OF BACTERIA IN
WATER. — Quantitative analysis requires more care in
the measurement of the exact volume of water em-
ployed, for the results are to be expressed in terms of the
number of individual organisms to a definite volume.
The necessity for making the plates at the place at
which the sample is collected is to be particularly
accentuated in this analysis, for 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.

NOTE. — 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
from this tube, and on each succeeding day determine
by counts whether there is an increase or diminu-
tion in the number of organisms — i. e., if they are
growing or dying. Represent the results graphically,
and it will be noticed that in many cases there is
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 death in from
ten to twelve days. This is not true for all organisms,
but does hold for many.

630

BACTERIOLOGY.

ill

Where it is not convenient, however, to make the

analysis on the spot, the sample of water should be

taken and packed in ice and kept on

FIG. 92.

ice until the plates can be made from
it, which should in all cases be as soon
after its collection as possible.

For the collection of samples from
the deeper portions of streams, lakes,
etc., a number of convenient devices
have been made. A very satisfactory
apparatus has been made for me by
Messrs. Charles Lentz& Sons, of Phila-
delphia. It consists of a metal frame-
work, in which is encased a bottle
provided with a ground-glass stopper.
To the stopper a spring clamp is ?t-
tached, and this in turn is operated by
a string, so that when the weighted
apparatus is allowed to sink into the
stream the stopper may be removed
from the bottle at any depth by simply
pulling upon the string. When the
bottle is filled with water the stopper
is allowed to spring back into position
by releasing the string. The whole
apparatus (depicted in Fig. 92) is pro-
vided with a weight that insures its
sinking, and a heavy cord by which it may be lowered
and raised. It should be sterilized before using. After
collecting the sample the bottle should be wiped dry
with a sterilized towel. Before removing the stopper
the mouth of the bottle should be rinsed with alcohol

Bottle for collecting
water.

BACTERIOLOGICAL STUDY OF WATER. 631

and heated with a gas-flame, to prevent contamination
of its contents by matters that may have been upon
its surface.

In beginning the quantitative analysis of water with
which one is not acquainted certain preliminary steps
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 c.c., 0.5 c.c., 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
— 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.c. being the
amounts most convenient for use.

Duplicate plates should always be made, and the

632 BACTERIOLOGY.

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, carefully mixed and
poured as a plate. When development occurs the
number of colonies is 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
in 0.25 c.c. of the original water we had 180 X 100 -
18,000 bacteria, which will be 72,000 bacteria per cubic
centimetre (0.25 c.c. = 18,000, 1 c.c. = 18,000 X 4 =
72,000). The results are always to be expressed in
terms of the number of bacteria per cubic centimetre
of the original water.

Another point of very great importance (already men-
tioned) is the effect of temperature upon the number of
colonies of bacteria that will develop on the 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

BACTERIOLOGICAL STUDY OF WATER. 633

kept for the same length of time in the incubator at
from 37° to 38° C. It will be seen from the table
that much the larger number of colonies — i. e., much
higher results — were always obtained when gelatin was
employed. The importance of this point in the quan-
titative bacteriological analysis of water is too apparent
to require further comment.

TABLE COMPARING THE RESULTS OBTAINED BY THE USE OF GEL-
ATIN AT 18°-20° C. AND AGAR-AGAR AT 37°-38° C. IN QUANTI-
TATIVE BACTERIOLOGICAL ANALYSES OF WATER. RESULTS
RECORDED ARE THE NUMBER OF COLONIES THAT DEVELOPED
FROM THE SAME AMOUNT OF VARIOUS WATERS IN EACH
SERIES.i

NUMBER OF COLONIES FROM WATER THAT DEVELOPED UPON—

Jeiatin plates at 18° to 20° C. Agar-agar plates at 37° to 38° C.

310 170

280 . 140

310) J180

340) 1160

650) 1 210

630 1 1320

380) f 290

400) • • (210

1000) flOO

890) (130

340) |280

370 J (210

490) rllO

580) llOO

Another point of equal importance in its influence upon
the number of colonies that develop is the reaction of the
gelatin. A marked excess of either alkalinity or acidity
always has a retarding effect upon many species found in
water. Fuller's experience at the Lawrence (Mass.) Ex-

1 I am indebted to James Homer Wright, Thomas Scott Fellow in
Hygiene (1892-'93), University of Pennsylvania, for the results pre-
sented in this table.

634 BACTERIOLOGY.

periraent Station has shown that gelatin of such a degree
of acidity as to require the further addition of from 15 to
20 c.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 liiver 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-
phtalein neutral point — i. 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 reference is made to plates it is not to
a set, as in 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. The
latter procedure obtains, of course, only when all the
areas are of the same size. By this method, however,
the results vary so much in different counts of the same
plate that they cannot be considered as more than rough
approximations.

NOTE. — Prepare a plate ; calculate the number of
colonies upon it by this latter method. Now repeat

BACTERIOLOGICAL STUDY OF WATER. 635

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.

WOLFFHUGEL'S COUNTING-APPARATUS. — This appa-
ratus (Fig. 93) 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

FIG. 93.

Wolffhugel's apparatus for counting colonies.

upon it. When the gelatin plate containing the colonies
luis been placed upon this background of glass it is
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
colonies, 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

636 BA CTERIOLOG Y.

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 indi-
cated, if the number of colonies be very great, a mean
may be taken of the number in several (six or eight)
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. (Fig. 94.)

FIG. 94.

Lens for counting colonies.

Several useful modifications of the apparatus of WolfiF-
hiigel have been introduced. The most important is
that of Lafar.1 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 is divided into sectors of such sizes that the
spaces between the concentric circles and the radii form-
ing the sectors are of equal size. Three of the sectors
are subdivided into smaller areas of equal size for con-
venience in counting when the colonies are very numer-
ous. The principles involved are similar to those of the

1 Lafar : Ceutralblatt fur Bakteriologie und Parasitenkunde, 1891,
Bd. xv. S. 331.

BACTERIOLOGICAL STUDY OF WATER. 637

preceding apparatus, but the circular form of the appa-
ratus admits of more exactness when counting colonies
on a circular plate.1

Pakes 2 has introduced a cheap and convenient modi-
fication of Lafar's apparatus. It consists of a sheet of
white paper on which is printed a black disk ruled

Pakes'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 Thor-
strasse, Stuttgart, who holds the patent for it. Its price is about 8
marks.

J Journal of Bacteriology and Pathology, 1896, vol. iv. No. 1.

638 BACTERIOLOGY.

relation to each other.1 To use this apparatus (Fig.
95) 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 centered over the black disk the portion lying
over one sector is exactly one-sixteenth of the whole
plate.

ESMARCH'S COUNTER. — Esmarch devised a counter
(Fig. 96) for estimating the number of colonies present

FIG. 96.

Esmarch's apparatus for counting colonies in rolled tubes.

upon a cylindrical surface, as when in rolled tubes.
The principles and methods of estimation are practically
the same as those given for Wolffhiigel's apparatus.
A simpler method than by the use of Esmarch's

1 Copies of this apparatus are to be had of Ash & Co., 42 Southwark
Street, London, or of Lentz & Sons, North Eleventh Street, Phila-
delphia, Pa. (The cost is but a few cents per copy.)

B A CTERIOL OGICA L STUDY OF WA TER. 639

apparatus may be employed for counting the colonies
in rolled tubes. It consists in dividing the tube by
lines into four or six longitudinal areas, which are sub-
divided by transverse lines about 1 or 2 cm. apart. The
lines may be drawn with pen and ink. They need not
be exactly the same distance apart nor exactly straight.
Beginning with one of these squares at one end of the
tube, which 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 difficult to
eliminate : it is assumed that each colony has grown
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 — ob-
viously this is of necessity estimated as a single organ-
ism 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.

THE SEWAGE STREPTOCOCCUS. — Houston1 reached
the conclusion that there is constantly present in sewage
a particular form of streptococcus which is really more

1 Houston : Ann. Report, Local Gov. Board, xxviii.

640 BACTERIOLOGY.

positively indicative of the contamination of water
by sewage than is bacillus coli. This opinion has
recently been under investigation by members of the
staff of the Massachusetts Institute of Technology, and
they have reached the conclusion that considerable
reliance can be placed upon the presence of this organ-
ism as an indication of sewage pollution of water.

The presence of the sewage streptococcus is most
readily shown in the sediment in fermentation tubes
inoculated with water under examination. If the sew-
age streptococcus is present it is very easy to demon-
strate it by microscopic examination of the sediment after
twenty-four to forty-eight hours. In addition to this
test it has also been demonstrated by Winslow l that the
estimation of the degree of acidity of the contents of the
fermentation tube is a safe indication of the presence of
the sewage streptococcus. When this organism is pres-
ent the acidity rises far more rapidly and to a greater
height than is the case when it is absent, so that in this
.way an additional indicator is available as to the pota-
bility of a water under examination.

BACTERIOLOGICAL ANALYSIS OF AIR.

Quite a number of methods for the bacteriological
study of the air exist. In the main they consist either
in allowing air to pass over solid nutrient media (Koch,
Hesse) and observing the colonies which develop upon
the media, or in filtering the bacteria from the air by
means of porous and liquid substances, and studying the
organisms thus obtained. (Miguel, Petri, Strauss, Wiirz,
Sedgwick-Tucker.) Because of their greater exactness,
the latter have supplanted the former methods.

1 Winslow : Jour. Med. Research, vol. iii., 1902.

BACTERIOLOGICAL ANALYSIS OF AIR. 641

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 is
done in water analysis. This is the simplest procedure.
An objection sometimes raised against it is that organisms

FIG. 97.

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.
Filtration through porous substances appears, on the
whole, to give the best results. Petri recommends as-
piration of a measured volume of air through glass
tubes into which sterilized sand is packed. (Fig. 97.)
When aspiration is finished the sand is mixed with
liquefied gelatin, plates are made, and the number of
developing colonies counted, the results giving the

41

642 BACTERIOLOGY.

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 sand ; this, when
brought into the liquefied gelatin, dissolves, and no such
error as that possible in the Petri method can be made.

SEDGWICK-TUCKER METHOD. — On the whole, the
method proposed by Sedgwick and Tucker gives such
uniform results that it is to be preferred to others. It
is as follows :

The apparatus employed by them consists essentially
of three parts :

1. A glass tube of special form, to which the name
aerobioscope has been given.

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

3. An air-pump.

The aerobioscope (Fig. 98) is about 35 cm. in its
entire length ; it is 15 cm. long and 4.5 cm. in diam-
eter at its expanded part ; one end of the expanded part
is narrowed to a neck 2.5 cm. in diameter and 2.5 cm.
long. To the other end is fused a glass tube 15 cm.
long and 0.5 cm. inside diameter, in which is to be
placed the filtering-material.

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.
When thoroughly cleaned, dried, and plugged, the

BACTERIOLOGICAL ANALYSIS OF AIR. 643

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 cm. (d) of the narrow tube above the wire gauze.
This column of sugar is the filtering-material em-
ployed 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

e da

The Sedgwick-Tucker aerobioscope.

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 the mark corresponding to the amount of air
required.1

1 Such a cylinder and air-pump arc not necessary. A pair of ordinary
aspirating-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 aerobioscope.

644 B A CTERIOL OG Y.

A sterilized aerobioscope 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 aspi rating-bottle by
means of a perforated rubber stopper. The cotton plug
is then moved from the upper end of the aerobioscope,
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 aerobioscope while held in an almost
horizontal position the sugar, and with it, the bacteria,
are brought into the large part (e) of the apparatus.
When all the sugar is thus shaken down into this part
of the apparatus about 20 c.c. of liquefied, sterilized
gelatin is poured in through the opening at the end c,
the sugar dissolves, and the whole is then rolled on ice,
just as is done in the preparation of an ordinary Esmarch
tube.

The gelatin is most easily poured into the aerobio-
scope by the use of a small, sterilized, cylindrical funnel
(Fig. 99), the stem of which is bent to an angle of about
1 10° with the long axis of the body.

The larger part of the aerobioscope is divided into
squares to facilitate the counting of the colonies.

By the employment of this apparatus one can filter
the air at any place, and can then, without fear of con-
tamination, carry the tubes to the laboratory and com-
plete the analysis. Aside from this advantage, the filter
being soluble only the insoluble bacteria are left im-
bedded in the gelatin.

BACTERIOLOGICAL ANALYSIS OF AIR. 645

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

BACTERIOLOGICAL STUDY OP THE SOIL.

Bacteriological study of the soil may be made by
either breaking up small particles of earth in liquefied
media and making 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

FIG. 99.

Bent funnel for use with aerobioscope.

the soil in sterilized water and then making plates
immediately from the water.

It must be borne in mind that many of the ground-
organisms belong to the anaerobic 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-

646 BACTERIOLOGY.

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 for
their cultivation are described in the chapter on Soil-
organisms.

BACTERIOLOGICAL STUDY OP MILK.

The possibility of milk serving as a vehicle in which
disease- producing bacteria may be disseminated through-
out a community has long been recognized, and epi-
demics of typhoid fever have been traced directly to
infected milk, while such diseases as diphtheria and
scarlet fever are also frequently regarded as being con-
veyed in the same manner.

In recent years the detailed study of the milk of
individual cows has revealed the fact that streptococcus
mastitis is not an uncommon occurrence in herds, and
it has frequently been observed that milk rich in strep-
tococci may prove dangerous when fed to infants and
convalescents.

Since milk is such a favorable medium for the growth
of a variety of bacteria it is not at all uncommon to
find market milk very rich in bacteria, especially if it
has been collected in a careless manner in dirty recep-
tacles, in unsanitary stables, and has been shipped long
distances at comparatively high temperatures.

For these various reasons the bacteriological study
of milk has gained considerable prominence during the
past few years — so much so that in some localities an
effort is being made to establish a bacterial standard for
market milk — that is, milk containing more than a cer-
tain number of bacteria is not regarded as suitable for
use. Whether such a standard can be maintained or

BACTERIOLOGICAL STUDY OF MILK. 647

not remains to be demonstrated. The several milk
commissions composed of pediatrists in various large
cities have established a bacterial standard for pediatric
milk of 10,000 bacteria to the cubic centimetre. Expe-
rience has shown that it is possible to market milk that
meets this bacterial standard sometimes with merely
ordinary precautions with regard to cleanliness. In
larger dairies it has frequently been a question of some
difficulty on account of the elaborate scale on which
everything is conducted.

QUANTITATIVE BACTERIOLOGICAL ANALYSIS. — In
the quantitative bacteriological examination of market
milk it is necessary to dilute the milk with sterile water
or sterile salt solution before plating on account of the
very large numbers of bajcteria present. The degree
of dilution that is necessary will depend upon the
nature of the dairy from which the milk is derived, the
age of the milk, and the temperature at which it has
been kept. Usually a dilution of 1 to 100, 1 to 1000,
and 1 to 10,000 is sufficient. From these dilutions
plate cultures are made with 0.1, 0.2, 0.3 cubic centi-
metre of each dilution.

QUALITATIVE BACTERIOLOGICAL ANALYSIS. —
Aside from the quantitative bacteriological analysis of
milk the qualitative analysis has received a great deal
of attention. Detailed qualitative analysis necessarily
entails an enormous amount of labor, but the detection
of certain forms of bacteria is not always very difficult,
This applies especially to the detection of streptococci.

Since milk containing streptococci in considerable
numbers is derived from the udder of a cow suffering
from some form of mastitis, it is always possible to find
pus in such milk. Consequently it is customary to

648 BACTERIOLOGY.

examine such milk for the presence of both streptococci
and pus. This is done by centrifuging a cubic centi-
metre of the milk and collecting the sediment on a
clean cover-slip and staining with Loffler's methylene-
blue. In this manner practically all the sediment derived
from one cubic centimetre can be obtained on the cover-
slip and a fairly satisfactory estimate can be made of
the relative number of pus cells in this quantity of
milk as well as at the same time an estimation of the
relative number of streptococci.

Milk that shows pus cells along with distinct chains
of streptococci, either extra- or intracellular, is usually
regarded as dangerous in character, and boards of health
usually direct that the cows from which such milk is
derived be excluded from the dairy until such time as
the milk is free from these elements.

CHAPTER XXVII.

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
(self-registering) thermometer ; let this thermometer be
in the centre of a bundle large enough to quite fill the
chamber of the sterilizer. At the end of a few minutes'
exposure to the streaming steam remove it; it will be
found to indicate a temperature of 100° C.

Closer study of the penetration of steam has taught
us, however, that the temperature 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 ster-
ilizing properties that steam has at the same temperature.
It is necessary, therefore, that this air should be ex-
pelled 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

•49

650 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 1 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 ultimately a pellicle will be
seen to form on the surface of the fluid. This pellicle
is 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 parti-
cles.

Pour into each of several test-tubes about 10 c.c. of
the filtrate. Allow one tube to remain undisturbed 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

STERILIZATION BY STEAM AND HOT AIR. 651

five minutes will present colonies in moderate numbers ;
but, as a rule, they will represent but a single organ-
ism. 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 the original mixture. This organ-
ism is bacillus subtilis. It is especially adapted to the
study of those various degrees of resistance to heat that
spore-forming bacteria exhibit at different stages of their
development.

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 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-

652 BACTERIOLOGY.

scopic methods are deceptive ; cloudiness of the media
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 steam. How can this result be ac-
counted for ?

Saturate several pieces of cotton thread, each about 2
cm. long, in the original decomposed bouillon, and dry
them carefully at the ordinary temperature of the room ;
then at a little higher temperature — about 40° C. — to
complete the process. Regulate the temperature of the
hot-air sterilizer for about 100° C., and subject several
pieces of this infected and dried thread to this 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 dry heat. These threads should not be simply

STERILIZATION BY STEAM AND HOT AIR. 653

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 side to side.

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 XXVIII.

Methods of testing disinfectants and antiseptics — Experiments illus-
trating the precautions to be taken — Experiments in skin-disin-
fection.

DETERMINATION OF DISINFECTANT PROPERTIES.

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 methods of work.

By the first of these processes the bits of thread,
usually about 1 to 2 cm. 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 substance to determine its germicidal value

654

METHODS OF TESTING DISINFECTANTS. 655

for several different resistant species of bacteria, both
in the vegetating and in the spore stage, and also be-
cause it is only by the use of pure cultures of familiar
species that it is possible to distinguish between the
colonies growing from the individuals that have not
been destroyed by the disinfectant under investigation
and those of unknown species that may appear upon
the plate as contaminations occurring during the manip-
ulation.

After the threads have remained in the culture or
suspension for from five to ten minutes they are re-
moved under antiseptic precautions and carefully sepa-
rated and spread upon the bottom of a sterilized Petri
dish, which is then placed either in the incubator at a
temperature not exceeding 38° C. until the excess of fluid
has evaporated, or in a desiccator over sulphuric acid,
calcium chloride, or any other drying-agent. The threads
are not left there until absolutely dry, but only until the
excess of moisture has evaporated. When sufficiently dry
they are immersed 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 in sterilized distilled water to remove the ex-
cess of disinfectant adhering to them, and placed in
fresh, sterile culture-media, which are then placed in
the incubator at from 37° to 38° C. If after twenty-
four to forty-eight or seventy-two hours a growth occurs
at or about the bit of thread, and if this growth consists
of the organism with 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

6 56 B A CTERIOLOG Y.

the thread have not been successful, and a small amount
of disinfectant is still active in preventing development
— i. e., is acting as an antiseptic.

By the process in which cultures or suspensions
of the organisms are mixed with different but known
strengths of the disinfectant a small portion of the
mixture, usually a loopful or a drop, is transferred
at the end of a definite time to the fresh medium
which is to determine whether the organisms have
been killed or not. This is commonly a tube of fluid
agar-agar, which is poured into a Petri.dish, allowed to
solidify, and placed in the incubator, as in the preceding
method.

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
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
varying intervals of exposure — one, five, ten minutes,
etc. — until we have decided not only the minimum
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
at which these experiments are conducted, for it
must always be borne in mind that the action of a dis-
infectant is usually more energetic at a higher than at a
lower temperature.

Now in both of these methods it is easy to see that
unless special precautions are taken a minute portion of
the disinfectant may be carried along with the thread,
or drop, into the medium which is to determine whether

METHODS OF TESTING DISINFEC1ANTS. 657

the .organisms do or do not possess the power of growth,
and there have a restraining or antiseptic action. For
organisms in their normal condition — that is, those
which have never been exposed to the action of a 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 very much less
It is plain, then, that if the test is to be an accurate
one, precautions must be taken against admitting this
minute trace of disinfectant to the medium with which
we are to determine whether the bacteria exposed to the
disinfectant were killed or not. The precautions hitherto
taken to prevent this accident have been, when the threads
were employed, 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 car-
ried over, to a point at which it lost 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 threads or in
combination with the proteids must be gotten rid of,
otherwise the results of the test may bo fallacious. A par-
tial solution of the problem is given through studies that
have been made upon corrosive sublimate in its various

42

658 BACTERIOLOGY.

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 2 found the same to hold
good for catgut. For example, he found that catgut which
had been immersed in solutions of corrosive sublimate
gave the characteristic reactions of the salt after having
been immersed for five weeks in distilled water which
had been repeatedly renewed. Braatz remarks that
a similar combination between sublimate and cotton
will take place after a long time ; but it occurs so
slowly that it cannot interfere with disinfection-experi-
ments in the same way that silk does.

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 was made by Geppert, who subjected
them to the action of ammonium sulphide in solution.
By this procedure the mercury is converted into inert,
insoluble sulphide, and has no inhibiting effect upon the
growth of those bacteria that did not succumb 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,

1 Shaefer : Berliner klin. Wochenschrift, 1890, No. 3, p. 50.

2 Braatz : Centralblatt fur Bakteriolgie und Parasitenkunde, Bd. vii.
No. 1, p. 8.

METHODS OF TESTING DISINFECTANTS. 659

instead of transferring the drop directly to the fresh
medium, add it to 10 or 12 c.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
testing disinfectants is the necessity of so adjusting the
conditions that each individual organism will be ex-
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 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 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 cultivated in bouillon
containing sand or finely divided particles of glass;
after growing for a sufficient length of time they are
to be shaken thoroughly, 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 a thick-walled test-
tube drawn out to a finer tube at its blunt end so as to
convert it into a sort of cylindrical funnel. The tube
when ready for use has the appearance shown in Fig.
111.

660

BACTERIOLOGY.

FIG. 100.

This tube, after being plugged at the bottom with
glass-wool (a, Fig. 100), and at its wide extremity
with cotton-wool, is placed vertically,
small end down, into an Erlenmeyer
flask of about 100 c.c. capacity and
sterilized in a steam sterilizer for the
proper time. It is kept in the 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 de-
gree of transparency is reached which
will permit the reading of moderately
fine print through a layer of the fluid
about 2 cm. thick — i. e., through an or-
dinary test-tube full of it. This filtrate
can then be subjected to the action of
the disinfectant. As a rule, the results
are more uniform than when no atten-
tion is paid to the presence of clumps.
It is scarcely necessary to say that in
the practical employment of disinfec-
tants outside the laboratory no such pre-
cautions are taken ; but in laboratory
work, where it is desired to determine
exactly the value of different substances
as germicides, all the precautions men-
tioned will be found essential to preci-
sion.

The disinfectant value of gases and vapors is de-
termined by their action upon test-objects in closed

Cylindrical fun-
nel used for filtering
cultures on which
disinfectants are to
be tested.

METHODS OF TESTING DISINFECTANTS. 661

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 usually 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 mixture of air and the gas under
consideration, of known proportions, to displace com-
pletely all the air ; or the pure gas itself may be intro-
duced in amount necessary to give the desired dilution
when mixed with the air in the chamber. At the
expiration of the time decided upon for the test the
infected articles are removed and the vitality of the
bacteria upon them is determined.

In the case of vapors of volatile fluids, such, for
instance, as formalin, 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 vaselin or paraffin,
and the fluid is allowed to vaporize at ordinary room-
temperature. The point here to be decided is the vol-
ume cr 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 for 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

662 BACTERIOLOGY.

reverse is the case. The objections to this method
are : " First. The test-organisms may be modified as
regards reproductive activity without being killed ;
and in this case a modified form of the disease may
result from the inoculation, of so mild a character
as to escape observation. Second. An animal that
has smTered this modified form of the disease enjoys
protection, more or less perfect, from future attacks,
and if used for a subsequent experiment may, by
its immunity from the effects of the pathogenic test-
organism, give rise to the mistaken assumption that
this had been destroyed by the action of the germicidal
agent to which it had been subjected." (Sternberg.)

DETERMINATION OP ANTISEPTIC PROPERTIES.

For this purpose sterile media are employed, and are
usually arranged in two groups : the one to remain
normal in composition and to serve as controls, while to
the other the substance to be tested is to be added 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, the
tubes may then be sterilized. After this they are to be
inoculated with the organism with which the test is to
be made, and at the same time one of the " control "-
tubes (one of those to which no antiseptic has been
added) is inoculated. They are all then to be placed
in the incubator and kept under observation. If at the
end of twenty-four, forty-eight, or seventy-two hours
no growth appears in any but the " control "-tubes, it
is evident that the antiseptic must be added in smaller

EXPERIMENTS. 663

amounts, for we are to determine the point at which it
is not as well as that at which it is capable of preventing
development. The experiment is then repeated, using
smaller amounts of the antiseptic until we reach a point
at which growth just occurs, notwithstanding the pres-
ence of the antiseptic ; the amount necessary for antisep-
sis is then a trifle greater than that used in the last tube.
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 should be repeated
with strengths of the antiseptic corresponding to
1 : 1000, 1 : 1100, 1 : 1200, 1 : 1300, 1 : 1400, and in
this way one ultimately determines the amount by which
growth is just prevented ; this represents the antiseptic
value of the substance for the organism with which it
was tested.

EXPERIMENTS.

To each of three tubes containing 10 c.c. — one of
physiological salt-solution, another of bouillon, a third
of fluid blood-serum — add as much of a culture of
micrococcus aureus as can be held upon a looped
platinum wire. Break this up carefully to eliminate
clumps, and then add exactly 10 c.c. of a 1 : 500 solution
of corrosive sublimate. Mix thoroughly, and at the
end of three minutes transfer a drop from each tube
into tubes of liquefied agar-agar, and pour these into
Petri dishes. 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?

664 BA CTERIOL OGY.

To each of two tubes — the one containing 10 c.c.
of normal salt-solution, the other of bouillon — add as
much of a spore-containing culture of anthrax bacilli
as can be held upon a loop of platinum wire. Dis-
tribute this uniformly through the medium, and then
add exactly 10 c.c. of a 1 : 500 solution of corrosive
sublimate. Mix thoroughly, and at the end of five
minutes transfer a drop from each tube to tubes of
liquefied agar-agar. Pour these immediately into
Petri dishes. 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
micrococcus aureus. Place them all in the incubator

EXPERIMENTS. 665

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 com-
pare the results. From these experiments, what will be
the strength of corrosive sublimate necessary to anti-
sepsis under these conditions for the organisms em-
ployed ?

Make a similar series of experiments using a 5 per
cent, solution of carbolic acid.

Determine the antiseptic value 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 sufficient sublimate into the
agar-agar with 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 micrococcus
aureus or anthrax spores add 10 c.c. of a 1 : 500 solu-

666 BACTERIOLOGY.

tion of corrosive sublimate, and allow it to remain 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-
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 cult-
ure on a 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
wrater 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

EXPERIMENTS. 667

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-
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 from one 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 a 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.

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 of about 1 cm. in diameter
in centre.

Cover-slips, 15 by 15 mm. square and not more than
from 0.15 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 with glass handles. One straight,
nljniit 4 cm. long; one looped at the end, about 4 cm.
long; and one straight, about 8 cm. long. Glass

669

670 APPENDIX.

handles to be about 3 mm. in thickness and from 15
to 17 cm. long.

STAINING- AND MOUNTING-REAGENTS.

200 c.c. of saturated alcoholic solution of fuchsin.

200 c.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 and 30 grammes of potassium
iodide in substance.

100 grammes of tannic acid.
100 grammes of ferrous sulphate.
Distilled water.

FOR NUTRIENT MEDIA.

\ pound of Liebig's or Armour's beef-extract.
250 grammes of Witte's or Sargent's peptone.

2 kilogrammes of gold-label gelatin (Hesteberg's).
100 grammes of agar-agar in substance.

200 grammes of sodium chloride (ordinary table-salt).
500 grammes of pure glycerin.

APPENDIX. 671

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 of C. P. granulated zinc.

GLASSWARE.

200 best quality test-tubes, slightly heavier than those
used for chemical work, about 12 to 13 cm. long and 12
to 14 mm. inside diameter.

15 Petri double dishes about 8 or 9 cm. in diameter
and from 1 to 1.5 cm. 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
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.

072 APPENDIX.

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 cm. 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.c. capacity.

6 ordinary water tumblers for holding test-tubes.

1 ruled plate for counting colonies.

1 gas-generator, 600 c.c. capacity, pattern of Kipp
or v. Wartha.

BURNERS, TUBING, ETC.

2 Bun sen burners, single flame.
1 Rose-burner.

1 Koch safety-burner, single flame.
6 feet of white-rubber gas-tubing.
12 feet of pure red-rubber tubing, 5 to 6 mm. inside
diameter.

1 thermo-regtilator, pattern of L. Meyer or Reichert.

2 thermometers, graduated in degrees of Centigrade,
registering from 0 to 100° C., graduated on the stem,

APPENDIX. 673

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.

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.

43

674 APPENDIX.

1 set of cork-borers.
1 hand-lens.

1 wooden filter-stand.

2 iron stands with rings and clamps.

3 round, galvanized iron-wire baskets to fit loosely
into steam sterilizer.

3 square, galvanized iron-wire baskets to fit loosely
into hot-air sterilizer.

1 sheet-iron box for sterilizing pipettes, etc.

1 covered agate-ware saucepan, 1200 c.c. capacity.

2 iron tripods.

1 yard of moderately heavy wire gauze.

2 test-tube racks, each holding 24 tubes, ] 2 in a row.

1 constant-level, cast-iron water-bath.

2 potato-knives.

2 test-tube brushes with reed or wire handles.

Cotton-batting.

Copper wire, wire nippers.

Round and triangular files.

Labels.

Towels and sponges.

INDEX.

ABBE, 30
substage condensing system

of, 203

Abbott and Gildersleeve, 37
Abscess, histological study of, 270

production of, 275
Alisccss-wall, 278
Acid-proof bacteria, 365
Acids, production of, 40
Actinomyces bovis, 378
Eppingeri, 386
farcinicus, 385
madurae, 382
pseudotuberculosis, 388
Actinomycetes, 376
Aerobic bacteria, 44
Aerobioscope, 644
Agar-agar, cultures on, 195
preparation of (see Media),
properties of, 102
Agglutinins, 431, 436, 578
Air, bacteriological analysis of, 640

640

Petri's method, 641
Sedgwick-Tucker method,

642

Alexin theory of Buchner, 583
Alexins, 573, 583
Alkali, production of, 40
Alkaloids, vegetable, 41
Ammonia, test for, 232
Anaerobic bacteria, 44

methods of cultivating, 220-

226

Burner's, 222
Esmarch's, 226
Friinkel's, 223
Hesse's, 221

Kitasato and Weil's, 225
Koch's, 220
Liborius's, 221
Park's, 226

Aniline dyes for differentiating

bacteria, 214
Animals, fluctuations in weight

and temperature of, 250
inoculation of, 235
apparatus used in, 237-241
intralymphatic, 247
intraocular, 250
intraperitoueal and pleural,

248

in tra vascular, 242
subcutaneous, 235
observations of, after inocula-

lation, 250

postmortem examination of, 258
cultures f roru tissues of, 260
disinfection of implements

after, 262
disposal of remains from,

262

incision through skin, 258
Nuttall's spear for use at,

261
opening the body cavities,

259

position of animal, 258
precautions during, 258
preservation of tissues after,

262
Anthrax, 510

animals susceptible to, 517
bacterium of, 510
biology of, 513
discovery of, 510
experiments with, 521
immune serum, 526
morphology of, 510
pathogenesis of, 516
protective inoculation against,

518

spore formation of, 511
staining of, 515

675

676

INDEX.

Anthrax, bacterium of, sympto-
matic bacillus of, 549
vaccines of, 519
Antidysenteria serum, 471
Antilysin, 279
Antiplague serum, 323
Antiseptic, definition of, 89
Antiseptics, mode of action of, 89

tests of, 662

Antistaphylococcus serum, 279
Antistreptococcus serum, 287
Antitoxin, diphtheria, 422

tetanus, 542
Apparatus for bacteriological

work, 669
preparation of, 138
Appendix, list of apparatus, 669
Arloing, Cornevin and Thomas,

549

Arnold steam sterilizer, 83
Aronson, 288
Auerbach, 38
Autoclave, 84

BACILLI, 58
differentiation from spores, 64
flagella upon, 66
involution-forms of, 60
life-cycle of, 59
mode of multiplication of, 57,

59

motility of, 66
spore-formation in, 63
Bacillus, 55
anthracis, 510
butter, 367
Chanvei, 549
coli, 54, 291, 453

characteristics, cultural, of,

455

morphological, 455
pathogenic, 458
differentiation of, from bacil-
lus typhosus, 457
where found, 453
" comma," 473
diphtherise, 399
dysenterise, 464
influenza3, 340
leprse, 364
mallei (of glanders), 391

Bacillus, Holler's grass, 367
nitrifying, 528
oadematis, 544
of bubonic plague, 315
pestis, 315
psetidodiphtheriticurn, 292, 336,

415

pyocyaneus, 309
smegma, 364
sporogenes, 557

biology of, 557

morphology of, 557

pathogenesis of, 558
subtilis, 651, 665
symptomatic anthrax, 549
tetani, 534

tuberculosis, 179, 357
typhosus, 426
Bacteria, 52
acid-proof, 365
aerobic, 44
anaerobic, 44

methods of cultivating, 220
behavior of, towaixl staining

reagents, 215

capsule surrounding, 50, 182
chromogenic, 35
classification of, 52
composition of, 51
conditions necessary to growth,

31-47

constancy in morphology of, 60
definition of, 31
denitrifying, 36
discovery of, 17-19
facultative, 33, 44
fermentation by, 215

apparatus for testing, 216

gases resulting from, 218
flagellated forms of, 66
identification of, 193
involution-forms of, 60
isolation of, in pure culture, 97

on slanting media, 150

plate method, 145

principles of, 99
metatrophic, 32, 33
microscopic examination of,

194, 204

mode of multiplication, 57
morphology of, 49, 194

INDEX.

677

Bacteria, motilitv of, 66
nitrifying, 36,*528
nutrition of, 41
parasitic, 32
paratrophic, 32
pathogenic, 35, 40, 559

mode of action of, 567
photogenic, 35
place in nature, 33
points to be observed in describ-
ing, 194

prototrophic, 32
reaction produced by, 214
reducing power of, 230
relation to man, 34
to oxygen, 44
to temperature, 45
results of growth, 35
role in nature, 33
saprogenic, 36

special method of Metchnikoff,
Roux, and Selembini for
cultivation, 265
spore-formation of, 63

study of, 207
staining-reactions of, 215
structure of, 49
systematic study of, 193
thermal death-point of, 67
thermophilic, 46
thiogenic, 36
toxin formation by, 567
very minute, method of ex-
amination, 266
zymogenic, 36
Bacteriacese, 31, 54
Ifcictcriaemia, 200
Bacterial enzymes, 37
proteins, 40, 565
toxins, 564

formation of, 567
point of action of, 569
Bacteriological study of air, 640
of milk, 646
of soil, 645
of water, 616
Bacteriology, application of

methods of, 2(17
beginning of, 17
Bacteriolysis, 568
Bacterium, 54

Bacterium anthracis, 510
diphtherite, 399
influenzas, 340
leprse, 364
mallei, 391

biology of, 393
discovery of, 391
inoculation of, 394
staining of, 395
pneumonia?, 330
development of, 333
immune serum, 336
inoculation of, 335
morphology of, 333
staining of, 334
variation of virulence, 335
where found, 332
pseudodiphtheriticum, 292, 336
smegmatis, 364
tuberculosis, 179, 357
Welchii, 556
biology of, 556
morphology of, 556
pathogenesis of, 557
xerosis, 417
Baumgarten, 371, 556
Becgiatoa, 52

Behring and Kitasito, 590
Berkefeld filter, 166
Beyerinck, 35
Billroth, 27

and Tiegel, 28

Biochemic characters, 194, 198
Biologic characters, 194, 195
Birch-Hirschfeld, 26
Blood, Baumgartt.'n's views on,

612
relations to bacteria and to

toxins, 584
Blood-serum as a culture-medium

(see Media).

germicidal element of, 587
methods of obtaining, 126
Nuttall's, 126
Latapie's, 127
Rivas's, 128
preservation of, 130
Bolton's potato method, 118
Bonnet, 24, 25

Booker's modification of Ee-
march's method, 148

678

INDEX.

Bouillon (see Media).

Bresredka, 565

Brieger and Cohn, 541, 566

Brooding-over, 152

Brownian motion, 207

Bubonic plague, 315

Buchner, 39, 40, 565, 573, 583,

587

Burdon-Sanderson, 29
Burner, Koch's safety, 154

nAEBOLIC acid as disinfectant,

\J 94

Chameleon phenomenon of Ernst,

312

Characters of cultures, 194
biochemic, 194, 198
biological, 194, 195
morphologic, 194
Chauveau, 579, 580
Chemical composition of bacteria,

51

sterilization, 71, 87
Cbemotaxis, 47, 565
Chevreul and Pasteur, 23
Chlamydobacteriaceae, 56
Chlamydothrix, 56
Chlorophyll, 31, 32
Cholera Asiatica, diagnosis of, 495
method of Schottelius, 482
microspira of, 473, 474

behavior of, in butter, 493 '
in milk, 492
in soil, 491
in water, 490
characteristics of, cultural,

476

morphologic, 474
effects of drying, 494
existence outside of the

body, 494
experiments upon animals

with, 485
general considerations upon,

488

isolation of, 482, 495
location in the body, 486,

489

morphology of, 474
persistence of, in dead body,
491, 494

Cholera, microspira of, PfeifTer's

studies upon, 483, 487
poisons produced by, 483
relation to gases, 481, 494
to other bacteria, 482, 493
to putrefaction, 493
to sunlight, 491
specific reaction of immun-
ized animals to, 487
toxin of, 483

Chromogenic bacteria, 35
Classen, 27

Classification of bacteria, 52
Cleaning of tubes, etc., 138
Coagulating enzymes, 37, 38
Coccoceae, 53
Cohn, 25, 566

Collodion capsule cultures, 211
Colon bacillus (see Bacillus coli).
Colonies, appearance of, 99, 101

counting of, 634

formation of, 99, 101

study of, 160, 195
Colony formation, 195
Comma bacillus (see Cholera

Asiatica).
Complements, 607

multiplicity of, 607

origin of, 607
Cooling-stage, 145, 147
Cornet, 355

Corrosive sublimate as disinfect-
ant, 89
Cover-slips, cleaning of, 168

impression, 172

microscopic examination of,
204, 276

preparation of, 168

steps in making, 169
Crenothrix, 56
Cultures, agar, 195

bouillon, 197

collodion capsule, 211

gelatin, 196, 212

hanging-block, 210

hanging-drop, 205

litmus milk, 197

potato, 196, 213

pure, 162

reactions of, 214

stab- and smear-, 162, 195

INDEX.

679

Cultures, test-tube, 162, 195
Cygnaeus, 432

DAVAINE, 21
Death-point, thermal, 67
Decolorizing solutions, 178
Decomposition, 33
Defensive proteids, 477, 589
Denitrifying bacteria, 36
Diaphragm, iris, 201
Diastatic enzymes, 37, 38
Differential diagnosis with aniline

dyes, 214

Differentiation between members
of the bacterium
diphtheria? group,
418

Hiss's media, 418
Knapp's method, 418
Ljubinsky's method,

240 -

Neisser's method, 418
Diphtheria antitoxin, 422
standardization of, 424
Behring's method, 424
Ehrlich's method, 425
bacterium of, 399,401

cultural peculiarities of, 403
experiments upon, 421
location, in tissues, 410
method of obtaining, 399
modification in virulence, 414
morphology of, 401
pathogenesis of, 408
poison produced by, 412
principles of immunizing

against, 542
staining, 408
toxin formation, 412

potency of, 566

histological changes accompany-
ing, 410
I)ip]()cocci, 59
Diplococcus intracellularis menin-

gitidis, 330
Disinfectants and antiseptics, 89,

654

experiments with, 663
general considerations, 87
methods of testing, 654

Disinfectants, methods of testing,
precautions to be observed,
657

mode of action, 89
use of animals as test objects

for, 661

in the laboratory, 94
Disinfection, general considera-
tions, 87

influence of temperature on, 91
inorganic salts in, 92
in the laboratory, 94
investigations of Kronig and

Paul on, 91
modus operandi, 91
reliable agents for purposes of,

94
selection of agents to be used

in, 88

Dissociation, electrolytic, 48, 91
Dunham's solution, 133
Durham's fermentation tube, 219

milk-whey, 132
Dysentery, bacilli of, 463-472
agglutination of, 469
cultural peculiarities of, 466-

469

discovery of, 463
immune serum, 471
morphology of, 465
pathogenesis, 467
protective inoculation, 470
staining, 465

17 BERTH, 27

LJ Ehrlich, 27, 574

" side-chain " theory of im-
munity of, 600
and Morgenroth, 603
Electrolytes, 48, 91
Electrolytic dissociation, 48, 91
Emboli of micrococei, 277
Emmerich and Fowitzky, 599
and Low, 593
and Mattel, 592
Endotoxins, 568
liberation of, 568
point of action of. 569

Enzymes, 36, 37, 102
bacterial, 37

680

INDEX.

Enzymes, bacterial, coagulating,

37

diastatic, 37
inverting, 37
proteolytic, 37
sugar-splitting, 37
Ernst, chameleon phenomenon of,

312

Erysipelas, 285
Escherich, 432
Esmarch's counter, 638
potato method, 119
tubes, 147

Booker's method of rolling,

148

made of agar-agar, 149
Eubacteria, 52
Examinations, bacteriological,dur-

ing life, 263
Exhaustion hypothesis of Pasteur,

581

Exposure and contact-experi-
ments, 269
External agencies, influence of,

198
Eye-piece, 201

"FACULTATIVE bacteria, 33, 41
JL Families, bacterial, 52
bacteriaceae, 54
chlamydobacteriacese, 56
coccacese, 53
spirillaceae, 55
FehleSsen, 27

Fermentation, 33, 36, 39, 215
gases resulting from, 218
particular forms of, 36, 37
Fermentation-tube, 216, 219

method of using, 216
Ferments, 36
Fermi, 38
Filling tubes, 139
Filter, method of folding, 111
Filtration of cultures, 233
Fission fungi, 49
Flagella, 66

methods of staining, 186
Bunge's, 188
Duckwall's, 189
Loffler's, 186
von Ermengen's, 191

Flagellated organisms, 66
Flasks, preparation of, 138
Flexner, 388, 464
Fluids, examination of, during

life, 263
Fodor, 585, 588
Foulerton, 289
Fowl tuberculosis, 372
Frankland, G. and P. F., 530
Fungi, fission, 49
Funnel for filling aerobioscope,
645

for filling test-tubes, 140

for filtering cultures, 660

hot-water, 112

pABBETT'S method, 181
\J Gas-pressure regulator, 158
Gelatin, cultures in, 212
liquefaction of, 102

characteristics of, 164, 212
preparation of (.see Media),
properties of, 99
Geppert, 90
Germicide, 89
Genus bacillus, 55
bacterium, 54
chlamydothrix, 56
crenothrix, 56
micrococcus, 53
microspira, 55
phragmidiothrix, 57
planococcus, 54
pseudomonas, 55
sarcina, 53
sphaerotilus, 57
spirillum, 55
spirochseta, 56
spirosoma, 55
streptococcus, 53
Glanders, 389
bacterium of, 391
cultivation of, 393
inoculation with, 394
morphology of, 391
staining of, in tissues, 395
diagnosis of, by Strauss's

method, 397
by use of mallein, 398
manifestations of, 389
histology of, 390

INDEX.

681

Glanders, susceptibility of animals

to, 389

synonyms, 389, 391
Gla"ss plates, 145
Gonococcus, 292

appearance in pus, 293
cultivation of, 294
Bumm's method, 294
Lipsehtitz's method, 299
Wassermann's method, 298
Wertheim's method, 294
Wright's method, 295
distinguishing features of, 302
morphology of, 292, 300
organisms that simulate it, 301
pathogenesis of, 301
vitality of, 300
Gonorrhoea, pus of, 293
Gorini, 38

(irass bacillus of Moller, 367
Green pus bacillus (see Pseudo-
monas aeruginosa).

TJAFFKINE vaccine against
11 plague, 325, 575
Hanging-block cultures, 210
Hanging-drop, 205, 207
Hankin, 572,589

and Martin, 588
Harvey, 25
Henle, 22

Hiss's media, 136,418, 441
Hoffman, 23
Hot-water funnel, 112
Hydrogen, test for purity of, 224

sulphide, test for, 231
Hypodermic syringes and needles,

243, 247

Hypothesis, exhaustion, of Pas-
teur, 581

retention, of Chauveau, 580

IMMUNITY, 574
1 acquired, 574

blood in, 585
active, 575

conclusions concerning, 607
earlier studies on blood rela-
tive to, 585

Ehrlich and Morgenroth, 003
Ehrlich's theory of, 600

Immunity, "exhaustion" hypoth-
esis, 581
experiments of Klemperers on,

596
hypothesis of Buchner, 591

of Emmerich and Low, 593
mechanism of, 579
natural, 574
nature of protective bodies, 571,

603
observations of Behring and

Kitasato, 591
passive, 575

" retention " hypothesis, 580
theory of MetchnikoflT, 582
Impression cover-slip prepara-
tions, 172
Incubator, 152

burner for heating, 154
Indol, method of detecting, 228
production of, by bacteria,

227
Infection, 559

chemical nature of, 563
conclusions concerning, 607
defense of body against, 571
wxlits operandi, 569
poisons present in, 567
Influence of external agencies,

198

Influenza, bacterium of, 340
cultivation of, 343
dissemination of, 344
isolation of, from tissues, 344
morphology of, 342
occurrence of, in tissues, 344
staining of, 342
susceptibility of animals to,

345

vitality of, 344
Inoculation of animals, 235
apparatus used in, 237-241
intnilymphatic, 247
intraocular, 250
intraperitoneal and pleural,

248

intravascular, 242

subcutaneous, 235

Inoculations, agar slant, 195

stab, 196
gelatin stab, 196

682

INDEX.

Inverting enzymes, 37, 38

Involution forms of bacteria, 60

Ions, 48, 91

Iris diaphragm, 201

Isolation of bacteria in pure cul-
tures, 97
Esmarch's roll-tube

method, 147
Petri plate method, 145
serial-tube method, 150

JORDAN and Richards, 530
tl Justinian plague, 316

T7ITASATO, 534, 540, 550

IV Klebs, 27, 29

Klein, 557

Klempercr, F. and G., work on

^neumonia, 596
Koch, fundamental researches, 29

postulates of, 357

safety-burner, 154

steam-sterilizer, 81
Kronig and Paul, 91

T ACTOSE-LITMUS agar-agar
Jj or gelatin (see Media).
Latapie apparatus, 127
Leeuwenhoek, 17-19
Lens for counting colonies, 636
Lepra bacillus, 364
staining of, 370
Leptothrix, 56
Letzerich, 27
Levelling-tripod, 145
Liborius, 544
Lime, chloride of, 96

milk of, 95

Litmus milk (see Media).
Lender's alkaline methylene-blue,
175

blood-serum mixture, 123, 136

method of isolation of bacte-
rium diphtherise, 399

stain for flagella, 186
Loffler and Schiitz, discovery of

bacterium mallei, 391
Lophotrichic flagella, 66
Lukomsky, 27
Lumbar puncture, 307
Lysin, 279

MADSEN, 542
Malignant cedema, bacillus

of, 544
cultural peculiarities of,

546

morphology of, 545
pathogenesis of, 547
susceptibility of animals

to, 548
Mallein, 393
Meat-extracts, in culture-media,

109

Meat-infusion, 104
Media, culture-, 104
agar-agar, 114

clarification of, 115
filtration of, 115
glycerine, 116
neutralization of, 114
solution of, 115
blood-serum, 120

Councilman and Mallory

method, 125

mixture of Loftier, 123, 136
NuttalFs method, 126
original method of Koch,

120
preservation of, 130

by chloroform, 130
sterilization and solidifica-
tion of, 122
bouillon, 104

neutralization of, 104
gelatin, 109

clarification of, 112
filtration of, 110
solution of, 110
sterilization of, 113
Hiss's medium of, 136, 418,

441
lactose-litmus agar-agar and

gelatin, 135

Lipschiitz's •medium, 299
litmus-agar-agar, 131
litmus-milk, 132
litmus-whey, 132
meat infusion, 104
milk, 131
peptone solution, Dunham's,

133
potatoes, 117

INDEX.

683

Media, culture-, potatoes, Bolton's

method, 118
Esmarch's method, 119
mashed, 119
original method, 117
Proskauer and Capaldi's, 444
special, 131

growth in, 197

Meningitis, cerebrospinal, causa-
tive organism of, 303
lumbar puncture in, 307
Metatrophic bacteria, 32
Metchnikoff, 582

phagocytosis theory of, 582
Rom and Selembini, method

of, 265 .

Methods of isolating bacteria, 97
of separating bacteria, 142
Esmarch's, 147
Koch's plate, 142
Petri dish, 145
serial tube, 156
of staining bacteria, 167
Gabbett's, 181
glacial acetic acid, 182
Gram's, 182
spores, 183
tubercle bacteria, 179
of standardization of diphtheria

antitoxin, 424
Bearing's, 424
Ehrlich's, 425
Micrococci, 57
emboli of, 277

mode of multiplication of, 62
Micrococcus, 53

aureus, 271 •

citreus, 280

gonorrhoea (see Gonococcus).
intracellularis, 303
cultivation of, 304
diagnosis, by lumbar punc-
ture, 307 "
morphology of, 303
powers of resistance, 307
results of inoculations, 306
lanceolatus, 330
pyogenes, 280
tctra genus, 337
Microscope, parts of, 201
Abbe condenser, 203

Microscope^, adjustment, coarse,

202

fine, 203

condenser, Abbe, 203
diaphragm, iris, 201
eye-piece, 201
iris diaphragm, 201
nose-piece, 201
objective, 201
ocular, 201
oil immersion, 203
reflector, 201
stage, 201

substage condenser, 201
Microspira, 55
comma, 474
Metchnikovi, 501
biology of, 501
morphology of, 501
pathogenesis, 504
Schuylkilliensis, 506

biochemical characters of,

508

biology of, 507
morphology of, 506
pathogenesis of, 508
Milk (see Media),
coagulation of, 38, 39
study of, 646
Mode of multiplication of bacteria,

57

Monotrichic flagella, 66
Morphologic characters, 197
Morphology of bacteria, 49, 197
Moller's grass bacillus, 367
Morgenroth, 603
Motility of bacteria, 66
Multiplication of bacteria, mode
of, 57

NAGELI, 41
Nassiloff, 27
Needham, 22

Neisser, gonococcus of, 292
N'risser's stain, 418
Neutralization of culture-media,

104-109
Nicolaier, 534
Nitrification, 528
Nitrifying bacteria, 36, 528
Nitrites, tests for, 231

684

INDEX.

Nitromonas of Winogradsky, 530
cultural pecularities of, 531
morphology of, 531

Nocard and Roux, 264

Normal serum, 289
solution, 218

Nutrition of bacteria, 41

Nuttall, 126, 260, 320, 536, 586

Nuttall's bulb, 127

OBJECTIVE, 201
Ocular, 201
Oertel, 27
Ogata, 588

Oil-immersion system, use of, 204
Organisms, nitrifying, 528
pathogenic, 35, 40, 1 99
pyogenic, 271
less common, 280, 291
Orth, 27

Oven, incubator, 152
Oxygen, relation of bacteria to,

44
cultivation of bacteria without,

220

Buchner's method, 222
Frankel' s method, 223
Hesse's method, 221
Koch's method, 220
Liborius's method, 221
Ozanam, 21

PARASITE, 32

f Paratrophic bacteria, 32
Parietti's solution, 627
Pasteur, 21, 23, 29, 44, 544, 581

exhaustion hypothesis of, 581
Pathogenic organisms, 35, 40, 199

mode of action of, 567
Pepsin, 37

Peptone, Dunham's solution of,
133

test of purity of, 134
Peritonitis, production of, 274
Peritrichic flagella, 66
Petersen, 279
Petri dishes, 146
Pfeiffer, 340, 595, 602, 609
PfeinWs phenomenon, 594, 602,

609

Phenomenon of Pfeiffer, 594, 602,

609

of Ernst, 312
Phagocytosis theory of Metchni-

koff, 582

Photogenic bacteria, 35
Phragmidiothrix, 57
Pigments, tests with, 231
Plague, bubonic, bacillus of, 315
cultivation of, 318
curative serum, 323
Haffkine vaccine, 323
immunity from, 322
mode of infection with, 321
morphology of, 317
occurrence in tissues, 321
pathogenesis, 319
vitality of, 319, 320
Planococcus, 53
Planosarcina, 54
Plates, apparatus employed in

making, 142
Esmarch's modification,

147

Booker's modifica-
tion, 148

Koch's fundamental ob-
servations, H7
materials used in making, 142

Petri's modification, 145
principles involved in the

method, 97

technique in making, 142
Platinum needles and loops, 143
Plenciz, 20

Pleuro-pneumonia of cattle, 265
Pollander, 21

Post-mortem examination of ani-
mals, 258

cultures from tissues of, 260
disinfection of implements

after, 262
disposal of remains from,

262
incision through the skin

at, 258
Nuttall's spear for use at,

261
opening of body cavities,

259
position during, 258

INDEX.

685

Post-mortem examination of ani-
mals, precautions during,
258
preparation of cover-slips

at, 262
preservation of materials

after, 262

Postulates of Koch, 357
Potato, characteristics of cultures

on, 213

preparation of, for culture pur-
poses (see Media).
Practical disinfection, 94
Precipitins, 577

1'ivMTvation of blood-serum, 130
Products of bacteria, 40
Proskauer and Capaldi, 627
Proteins, bacterial, 40, 565
Proteolytic enzymes, 35, 37
Prototrophic bacteria, 32
Prudden, 351

Pseudodiphtheria bacterium, 415
Pseudomonas, 55
aeruginosa, 309

chameleon phenomenon of,

312

cultural characters, 309
enzymes of, 313
morphologic characters, 309
pathogenic properties, 314
protective properties of, 315
Ptomains, 41, 565
Pure culture, 102
Pus, microscopic appearance of,

271, 280

Putrefaction, 33, 36, 46
Pyaemia, production of, 274
Pyogenic organisms, 271, 280, 291

QUARTER evil or quarter ill
(.see Symptomatic anthrax ).

D ABINOVITCH, 36, 367

It Reaction of media, changes

in, 214

Receptors, 605, 608
Recklinghausen, 26, 27
Reducing power of bacteria, 230
Reflector, 201

Regulator, gas-pressure, 158
thermo-, 155

Rennet, 38

Retention hypothesis, 580

Rindfleisch, 26

Rivas apparatus, 128

Roux and Yersin, 413, 566

SAFETY burner, 154
Saprogenic bacteria, 36
Saprophyte, 32

role of, in nature, 33
Sarcina, 53, 58

mode of multiplication, 59, 62
tetragena, 337

cultural peculiarities, 338
morphology of, 338
susceptibility of animals, 340
where found, 337
Scheme of study of bacteria, 194
Schizomycetes, 31, 49, 52
Schottehus's method of examin-
ing cholera evacuations, 482
Schroder and Dusch, 23
Schrotter, 309
Schulze, 23
Schwann, 23
Sections, study of, 276
Separation of bacteria, 142
Esmarch's method, 147
Koch's plate method, 142
Petri dish method, 145
serial tube method, 150
Septicaemia, 327, 562
Septicaemias, 560, 563

hemorrhagic, 563
Serum, antidysenteriae, 471
antiplague, 323
antistreptococcus, 287
antitoxic, diphtheria, 422

tetanus, 542
blood-, methods of obtaining,

126

of preserving, 130
Serum-water media of Hiss, 136
Sewage streptococcus, • >.">'.)
Shiga's bacillus (.tee Dysentery).
"Side-chain" theory of Ehrlich,

600
Skin disinfection, experiments in,

666
Smear-cultures, 162

686

INDEX.

Smegnia bacillus, staining pecu-
liarities of, 364
Smith, Theobald, 374
Soil, bacteriological analysis of,
645

nitrifying bacteria in, 528

organisms present in, 528

phenomena in operation in, 528
Spallanzani, 22, 23, 24, 29
Special media, 131
growth in, 197
Sphaerotilus, 57
Spirilla, 58
Spirillaceae, 55
Spirillum, 55

of Asiatic cholera (see Cholera).

of Metchnikovi (see Microspira
Metchnikovi).

Schuylkilliensis (see Microspira

Schuylkilliensis).
Spirochaeta, 56
Spirosoma, 55
Spores, formation of, 61, 63
method of studying, 207

mode of development of, 63, 66

recognition of, 61, 64

staining of, 183

Sputum, inoculations with, 327,
329

microscopic examination of,
327

pathogenic properties of, 329

septicaemias, 327, 330

tuberculous, 315, 327
Stab-cultures, 162
Staining, methods and solutions
used in, 167

acetic acid, 182

Bunge's, 188

Duckwall's, 189

Gabbett's, 181

general remarks on, 177

Gram's, 182

Koch-Ehrlich's, 175

Loffler's blue, 175
flagella, 185

Molar's, 185

of spores, 183

of tubercle bacteria, 174

ordinary solutions used in, 173
bottles for holding, 174

Staining, van Ermengem's, 191

Ziehl-Nielsen's, 176
Staphylococci, 58
Staphylococcus epidermidis albus,

281

pyogenes albus, 280
aureus, 271

cultural characters of, 272

pathogenesis, 274

toxin of, 279

where to be expected, 273,

285

citreus, 280
Staphylotoxin. 278
Sterilization, chemical, 76, 87
by heat, 69, 71

principles involved, 71
by hot air or dry heat, 85
apparatus used in, 86
by steam, 72

apparatus used in, 83, 84
discontinued, 73, 75
fractional, 75, 78
under pressure, 79, 83
experiments upon, 649
intermittent, 73, 75

at low temperature, 78
methods employed in, 72
principles involved in, 71-84
use of the term, 69
Sternberg, 332
Strauss's method for diagnosis of

glanders, 397
Streptococci, 58

mode of multiplication, 59
Streptococcus, 53
pyogenes, 281, 285
biology of, 281-285
curative serum, 287
effects of, in inoculation, 285
longus and brevis, 287
morphology of, 281
where found, 281, 285
of sewage, 639

Streptothrices, pathogenic, 376
Streptothrix, 56
Subtilis bacillus, 651, 665
Sugar-splitting enzymes, 37, 39
Suppuration, 271

bacteria common to, 271, 280,
291

IXDKX.

687

Suppuration, general remavks

upon, 'J'.'l

less common causes of, 291
microscopic appearance of pus,

270, 280, 292
Symbiosis, 36, 46, 528
Symptomatic anthrax, bacillus of,

549

biology of, 551
differentiation from bacil-

cillus oedematis, 555 '
morphology of, 550
pathogenesis of, 554
susceptibility of animals to,
555

rPKCHNIQUE, novel, 226
J. Test-tubes, cleaner for, 138
cleaning of, 138
filling with media, 139
apparatus for, 140
plugging with cotton, 129
position after filling, 141
sterilization of, 141
Tests for ammonia, 232
for hydrogen sulphide, 231
for indol, 228
for nitrites, 231
for toxins, 233
with pigments, 231
Tetanolysin, 542
Tetanospasmin, 542
Tetanus antitoxin, 542
bacillus of, 534
biology of, 536
effects of, on animals, 539
method of obtaining, 534
Morphology of, 536
poison produced by, 540
toxin, composition of, 542

potency of, 541, 566
Tetrads, 58
Theory, alexin, 583
phagocytosis, 582
"side-chain," 600
Thermal death-point of bacteria,

67

Thermophilic bacteria, 46, 77
Thermo-regulator, 155
Thermostat (see Incubator).

Tliiogenic bacteria, 36
Tissues, cultures from, at au-
topsies, 260

examination during life, 263
Nuttall's spear for making,

260

Toxemia, 199, 562
Toxin molecule, Ehrlich's con-
ception of, 566
Toxins, 41, 564
formation of, 567
point of action, 567
Toxoids, 541, 566
Toxones, 541, 566
Traube and Gscheidlen, 584
Treviranus, 22, 29
Tripod for levelling plates, 144
Trypsin, 37
Tube, Esmarch, 147
Tuberculin, 375
Tuberculosis, 346-376
avian, 372
bacterium of, 357

appearance in cultures, 361
cultivation from tissues, 358
methods of staining, 179,

362

Gabbett's, 181
Koch-Ehrlich's, 175, 179
Ziehl-Nielsen's, 176, 180
microscopic appearance of,

330
organisms that simulate it,

363
differential diagnosis of,

370
staining peculiarities of, 179,

329, 362
toxin of, 565
varieties of, 374
cavity-formation in, 350
condition simulating, 375
diffuse caseation of, 375
encapsulation of tubercular

foci, 352

giant cells in. 349
location of bacteria in, 355
manifestations in experimental,

347

miliary tubercle, structure of,
348

688

INDEX.

Tuberculosis, modes of infection,

353

primary infection, 352
pseudo-, 375
sputum in, 315

inoculation of animals with,

329
microscopic appearance of,

330

staining of, 179, 329
susceptibility of animals to,

375

vaccination against, 376
Tubes, Esmarch roll, 147
fermentation, 217, 219
filling of, 139
preparation of, 138
serial, 150
Tyndall, 24
Typhoid fever, bacillus of, 426

constant properties of,

435

cultivation of, 427
differentation from bacillus

coli, 448, 457
Drigalski and Conradi's

method, 445
Fischer's method, 451
Hiss's method, 441
Hoffmann and Picker's

method, 449
Parietti's method, 627
Proskauer and Capaldi's

method, 444
difficulty in identifying,

435

experiments with, 452
inoculations with, 431
isolation from cadavers,

452

location of, in tissues, 430
morphology of, 426
reaction of, with typhoid
serum, 436

source from which to ob-
tain, 452

vaccination against, 576
water as a carrier of,

441, 616

Widal's reaction with, 431,
436, 578

T7ACCINATION against dis-
f eases, 575
Vaccines, 575

plague, Haffkine method, :!'_'•")
typhoid fever, Wright method,

576

Vaughan, 589
Vegetable alkaloids, 41
Vibrio Metchnikovi, 501

characteristics of, cultural,

501

morphological, 501
pathogenesis, 504
Schuylkilliensis, 506
biochemistry of, 508
biology of, 507
morphology of, 506
pathogenesis of, 508
Vibrion septique, 549

WALDEYER, 26
Wasserman, studies on te-
tanus toxin, 569

Water, general observations upon
bacteriological study of,

616

qualitative bacteriological an-
alysis of, 622
precautions in obtaining

sample, 623
preliminary steps in,

623
quantitative bacteriological

analysis of, 629
collection of sample,

630

counting of colonies,
634

apparatus for, 635,

639

reaction of media, 633
selection of proper me-
dium for, 632

relation of, to epidemics, 616
sewage streptococcus, 639
typhoid bacilli in, 616, 619
value of bacteriological exam-
ination of, 618

of chemical examination of,
618

INDEX. 689

Weichselbeum, 303, 330 i Wright, 295, 382, 576

Weigert, 30, 600 I Wurtz's agar-agar and gelatin, 135

doctrine of cell-equilibrium, i

600 ! YER°SIS bacterium, 417

Welch, 281, 291, 331, 556 i A

Widal's reaction, 431, 437, 578,

609 i VERSIN> 322> 566

Wilde, 27 | I

Winogradsky, nitro-monas of, 530
Wolfl hiigel's counting-apparatus, r^OOGLCEA of bacteria, 50

635 U Zymase, 39

Wound-infection, 26-29 Zymogenic bacteria, 36

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C. W. MANSELL MOULLIN, M.A., M.D. OXON., F.R.C.S. ENG.

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ILLUSTRATIONS OF THE INDUCTIVE METHOD
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WHAT TO DO IN CASES OF POISONING. Ninth
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12 New and Recent Works published by

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T

WILLIAM OSLER, M.D., F.R.C.P. LOND.

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7TQUANIMITAS : With other Essays and Addresses to
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LOUIS PARKES, M.D. LOND., D.P.H.

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INFECTIOUS DISEASES, NOTIFICATION AND

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DR. THEODOR PUSCHMANN.

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HISTORY OF MEDICAL EDUCATION FROM THE
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SAMUEL RIDEAL, D.SC. (LOND.), F.I.C., F.C.S.

Fellow of University College, London.

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PRACTICAL CHEMISTRY FOR MEDICAL STU-
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FREDERICK T. ROBERTS, M.D., B.SC., F.R.C.P.

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WILLIAM ROSE, B.S., M.B. LOND., F.R.C.S.

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14 New and Recent Works published by
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late Medical Superintendent, Royal Albert Asylum for Idiots and

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HERBERT TILLEY, M.D., B.8. LOND., F.R.C.S. ENG.

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DURULENT NASAL DISCHARGES: their Diagnosis
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HN DEAFNESS, GIDDINESS, AND NOISES IN
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16 H. K. Lewis's Publications.

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