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POTHO<ji£NlC

MY HONORED AND RELOYBD Q^AND^THER
Mr. J^TTO H

WHOSE PARENTAL LOVE AND LIBF.I^LJ^f HAVE ENABLED ME TO. PURSUE
MY MEDICAL EDUCATION

THIS BOOK IS AFFECTIONATELY DEDICATED

PREFACE TO THE SECOND EDITION.

In preparing this second edition of “Pathogenic Bac¬
teria” I have endeavored to bring the work up to date
in all departments of the subject by introducing brief
mention of all recent work accomplished in bacteriology.
In order to aid the student whose particular interest
might make him desire to refer to the original papers,
I have thought well to depart from the plan of the orig¬
inal work and give the references in the form of foot¬
notes.

Without departing too much from the primary descrip¬
tive purpose of the book, I have made it a special point
to add considerably to the amount of technique it con¬
tains, and so make it fulfill the double purpose of a sys¬
tematic work upon bacteria and a laboratory guide.

New chapters have been added dealing with the bac¬
teriology of Whooping-cough, Mumps, Yellow Fever,
Hog-cholera, and Swine-plague ; describing the Bacillus
aerogenes capsulatus and the Proteus vulgaris ; and
describing the Methods of Determining the Value of
Antiseptics and Germicides, and of Determining the
Thermal Death-point.

To a number of friendly readers whose suggestions
have been helpful in improving the work, I desire to
extend my sincere thanks.

Joseph McFarland
r

PREFACE.

The following pages are intended to convey to the
reader a concise account of the technical procedures
necessary in the study of bacteriology, a brief descrip¬
tion of the life-history of the important pathogenic
bacteria, and sufficient description of the pathological
lesions accompanying the niicro-organismal invasions
to give an idea of the origin of symptoms and the
causes of death.

The work being upon Pathogenic Bacteria, it does
not cover the whole scope of parasitology, and the
parasites of higher orders are all omitted. Malaria and
amebic dysentery are omitted as logically as tape-worms
and pediculi. The higher fungi are also omitted, both
because they are not bacteria and because their proper
consideration would make a small book in itself.

In leaving out the non-pathogenic bacteria of course
a stumbling-block was encountered. The Sarcina ven-
triculi, for instance, may be a cause of dyspepsia, yet
can scarcely be regarded as pathogenic, and, together
with other similar bacteria of questionable deleterious
operation, has been omitted ; on the other hand, it
has been thought advisable to include and describe
somewhat -at length a long list of spirilla similar to,
and probably closely allied with, the spirillum of
cholera, yet not the cause of any particular diseased
condition.

PREFACE.

The aim has been to describe only such bacteria' rts'
can be proven pathogenic by the lesions . or .toxins
which they engender, and, while considering them, to
mention as fully as is necessary the species with which
they may be confounded.

The book, of course, will find its proper sphere of
usefulness in the hands of medical students ; its pages,
however, will be found to contain much that will be
of interest and profit to those practitioners of medicine
who graduated before modern science had thrown its
light upon the etiology of disease.

In writing this work the popular text-books have

« r

been drawn upon. Hiippe, Fliigge, Sternberg, Frankel,
Gunther, Thoinot and Masselin, and others have been
freely consulted.

The illustrations are mainly reproductions of the best
the world affords, and, being taken from the great stand¬
ards, are surely superior to anything new covering the
same ground. Credit has carefully been given for each
illustration.

Philadelphia, Feb. i, 1896.

J. McF.

CONTENTS

PART I. GENERAL CONSIDERATIONS.

PAGE

Introduction . 17

CHAPTER I.

Bacteria . ; . 30

CHAPTER II.

The Biology of Bacteria . 43

CHAPTER III.

Immunity and Susceptibility . 65

CHAPTER IV.

Methods of Observing Bacteria . 'S6

CHAPTER V.

Sterilization and Disinfection . 105

CHAPTER VI.

The Cultivation of Bacteria ; Culture-media . 124

CHAPTER VII.

Cultures, and their Study . 139

CHAPTER VIII.

The Cultivation of Anaerobic Bacteria . *53

CHAPTER IX.

Experimentation upon Animals . . 158

CONTENTS.

CHAPTER X.

' PAGR

The Recognition of Bacteria . i63

CHAPTER XI.

The Bacteriologic Examination of Air . 164 -

CHAPTER XII.

The Bacteriologic Examination of Water . 1(^9

CHAPTER XIII.

The Bacteriologic Examination of Soel . 174

CHAPTER XIV.

The Thermal Death-point and the Value of Germi¬
cides . 176

PART II. SPECIFIC DISEASES AND THEIR
BACTERIA.

A. THE PHLOGISTIC DISEASES.

I. THE ACUTE INFLAMMATORY DISEASES.
CHAPTER I.

Suppuration ; Gonorrhea ; Mumps . 182

II. THE CHRONIC INFLAMMATORY DISEASES.
CHAPTER I.

Tuberculosis . . . .208

CHAPTER II.

Leprosy . .

CHAPTER III.

Glanders . .

W TENTS.

CHAPTER IV.

PAGE

Syphilis . 255

CHAPTER V.

Actinomycosis . 260

CHAPTER VI.

Mycetoma, or Madura- foot . . . 266

CHAPTER VII.

Farcin du Bceuf . 270

CHAPTER VIII.

Rhinoscleroma . 273

B. THE TOXIC DISEASES.

CHAPTER I.

Tetanus . 274

CHAPTER II.

Diphtheria . 284

CHAPTER III.

Hydrophobia, or Rabies . 306

CHAPTER IV.

Cholera and Spirilla resembling the Cholera Spi¬
rillum . 311

CHAPTER V.

Pneumonia . . , . 345

CONTENTS.

C. THE SEPTIC DISEASES.

CHAPTER I.

PAGE

Anthrax . 356

CHAPTER II.

Typhoid Fever . 366

CHAPTER III.

The Bacieeus Coei Communis . 389

CHAPTER IV.

•Yeeeow Fever . 399

CHAPTER V.

Chicken-choeera . 409

CHAPTER VI.

Hog-choeera . 413

CHAPTER VII.

Swine-peague . 420

CHAPTER VIII.

Typhus Murium . 423

CHAPTER IX.

Mouse-septicemia . 426

CHAPTER X,

Relapsing Fever . 431

CHAPTER XI.

Bubonic Peague . 433

CHAPTER XII.

Tetragenus . .. 443

CONTENTS. 15

CHAPTER XIII.

PAGE

INFLUENZA . . 446

CHAPTER XIV.

Measles . . . .451

D. MISCELLANEOUS.

CHAPTER I.

Symptomatic Anthrax . 453

CHAPTER II.

Malignant Edema . , . . . . 459

CHAPTER III.

The Bacillus Aerogenes Capsulatus . 463

CHAPTER IV.

The Bacillus Proteus Vulgaris . 472

CHAPTER V.

Whooping-cough . 476

INDEX . 4S1

PATHOGENIC BACTERIA.

PART I. GENERAL CONSIDERATIONS.

INTRODUCTION.

It is incorrect to begin the consideration of bacteriol¬
ogy, as is so often done, with the probable discoverer of
bacteria, Leeuwenhoek, or with the so-called u Father of
bacteriology, n Henle. The controversies and ideas which
stimulated the investigations and researches which have
brought us to our present state of knowledge were begun
hundreds of years before the beginning of the Christian era.

Excepting such as taught and believed that u in six
days the Lord made heaven and earth, the sea and all
that in them is,1’ or a kindred theory of the origin of
things, the thinkers of antiquity never seem to have
doubted that under favorable conditions life, both animal
and vegetable, might arise spontaneously.

Among the early Greeks we find that Anaximander
(43d Olympiad, 610 B. c.) of Miletus held the theory that
animals were formed from moisture. Empedocles of
Agrigentum (450 B. c.) attributed to spontaneous genera¬
tion all the living beings which he found peopling the
earth. Aristotle (b. c. 384) is not so general in his view
of the subject, but asserts that u sometimes animals are
formed in putrefying soil, sometimes in plants, and some¬
times in the fluids of other animals'.” He also formulated
a principle that u every dry substance which becomes
moist, and every moist body which becomes dried, pro¬
duces living creatures, provided it is fit to nourish them.”

2 17

PATHOGENIC BACTERIA.

Three centuries later, in liis disquisition upon the
Pythagorean philosophy, we find Ovid defending the
same doctrine:1

“By this sure experiment we know
That living creatures from corruption grow :

Hide in a hollow pit a slaughter’d steer,

Bees from his putrid bowels will appear,

Who, like their parents, haunt the fields and bring
Their honey-harvest home, and hope another spring
The warlike steed is multiplied, we find,

To wasps and hornets of the warrior kind.

Cut from a crab his crooked claws, and hide
The rest in earth, a scorpion thence will glide,

And shoot his sting ; his tail in circles toss’ d
Refers the limbs his backward father lost ;

And worms that stretch on leaves their filmy loom
Crawl from their bags and butterflies become.

The slime begets the frog’s loquacious race ;

Short of their feet at first, in little space,

With arms and legs endued, long leaps they take,

Raised on their hinder part, and swim the lake,

And waves repel ; for nature gives their kind,

To that intent, a length of legs behind.”

"Not only was the doctrine of spontaneous generation
of life current among the ancients, but we find it persist¬
ing through the Middle Ages, and descending to our own
generation to be an accidental but important factor in
the development of a new branch of science. In 1542,
in his treatise called De Snbtilitate , we find Cardan as¬
serting that water engenders fishes, and that many ani¬
mals spring from fermentation. Van Helmotit gives
special instructions for the artificial production of mice,
and Kircher in his Muudus Subterraneus (chapter “ De
Panspermia Rerum n) describes and actually figures cer¬
tain animals which were produced under his own eyes
by the transforming influence of water on fragments of
stems from different plants.2

1 Ovid’s Metamorphoses , translated by Mr. Dryden, published by Sir Samuel
Garth, London, 1794.

2 See Tyndall : Floating Matter in the Air.

INTRODUCTION.

About 1668, Francesco Redi seems to have been the
first to doubt that the maggots familiar in putrid meat
arose de novo: tc Watching meat in its passage from
freshness to decay, prior to the appearance of maggots,
he invariably observed flies buzzing around the meat and
frequently alighting- on it. The maggots, he thought,
might be the half-developed progeny of these flies.
Placing fresh meat in a jar covered with paper, he found
that although the meat putrefied in the ordinary way,
it never bred maggots, while meat in open jars soon
swarmed with these organisms. For the paper he sub¬
stituted fine wire gauze, through which the odor of the
meat could rise. Over it the flies buzzed, and on it they
laid their eggs, but the meshes being too small to per¬
mit the eggs to fall through, no maggots generated in
the meat; they were, on the contrary, hatched on the
gauze. By a series of such experiments Redi destroyed .
the belief in the spontaneous generation of maggots in
meat, and with it many related beliefs.”

It was not long before Leeuwenhoek, Vallismeri,
Swammerdan, and others, following the trend of Redi’s
work, contributed additional facts in favor of his view,
and it may safely be asserted that ever since the time
of this eminent man the tide of scientific opinion has
turned more and more strongly against the idea that
life is spontaneously generated.

About this time (1675) one whose name has been
already mentioned, Anthony van Leeuwenhoek, and who
is justly called the u Father of microscopy,” came into
prominence. An optician by trade, Leeuwenhoek devoted
much time to the perfection of the compound micro¬
scope, which was just coming into use. The science of
optics, however, was not sufficiently developed to enable
him to overcome the errors of refraction, and after the
loss of much time he turned to the simple lens, using it
in so careful and remarkable a manner as to be able
to record his observations in one hundred and twelve
contributions to the Philosophical Transactions . Leeu-

PATHOGENIC BACTERIA.

wenhoek, among other things, demonstrated the conti¬
nuity of arteries and veins through intervening capil¬
laries, thus affording ocular proof of Harvey’s discovery
of the circulation of the blood; discovered the rotifers,
and also the bacteria, seeing them first in saliva.

Although one of those who contributed to the support
of Redi’s arguments against the spontaneous generation
of maggots, Leeuwenhoek involuntarily reopened the old
controversy about spontaneous generation by bringing
forward a new world, peopled by creatures of such ex¬
treme minuteness as to suggest not only a close relation¬
ship to the ultimate molecules of matter, but an easy
transition from them. Interested in Leeuwenhoek’s
work, Plencig of Vienna became convinced that there
was an undoubted connection between the microscopic
animals exhibited by the microscope and the origin of
disease, and advanced this opinion as early as 1762.
Unfortunately, the opinions of Plencig seem not to have
been accepted by others, and were soon forgotten.

In succeeding years the development of the compound
microscope showed these minute organisms to exist in
such numbers that putrescent infusions, both animal and
vegetable, literally teemed with them, one drop of such
a liquid furnishing a banquet for millions.

Much hostility arose in the scientific world as years
went on until two schools attained prominence — one
headed by Buffon, whose doctrine was that of u organic
molecules;” the other championed by Needham, whose
doctrine was the existence of a u vegetative force” which
. drew the molecules together.

Experimentation was begun and attracted much atten¬
tion. Among the pioneers was Abbe Lazzaro Spallan¬
zani (1777), who filled flasks with organic infusions,
sealed their necks, and, after subjecting their contents
to the temperature of boiling water, placed them under
conditions favorable for the development of life, without,
however, being able to produce it. Spallanzani’s critics,
however, objected to his experiment on the ground that

INTRODUCTION

air is essential to life, and that in hikfljj^ks' the air was
excluded by the hermetically-sealed necks.

Schulze (1836) set the objection aside by filling a flask
only half full of distilled water, to which animal and
'‘vegetable matters were added, boiling the contents to
destroy the vitality of any organisms which might al¬
ready exist in them, then sucking daily into the flask a
certain amount of air which had passed through a series
of bulbs containing concentrated sulphuric acid, in which
it was supposed that whatever germs of life the air might
contain would be destroyed. This flask was kept from
May to August; air was passed through it daily, yet with¬
out the development of any infusorial life.

The term “ infusorial life” having been used, here it
is well to observe that during all the early part of their
recognized existence the bacteria were regarded as ani¬
mal organisms and classed among the infusoria.

Cagniard Latour and Schwann in the year 1837 suc¬
ceeded in proving that the minute oval bodies which had
been observed in yeast since the the time of Leeuwenhoek
were living organisms — vegetable forms — capable of
growth; and when Boehm succeeded a year later in de¬
monstrating their occurrence in the stools of cholera, and
conjectured that the process of fermentation was con¬
cerned in the causation of that disease, the study of these
low forms of life received an immense impetus from the
important position which they began to assume in rela¬
tion to medical science.

The experiments of Schwann, by proving that the
free admission of calcined air to closed vessels contain¬
ing putrescible infusions was without effect, while the
admission of ordinary air brought about decomposition,
suggested that the causes of putrefaction which were in
the air were living entities.

In 1862, Pasteur published a paper lCOn the Organized
Corpuscles existing in the Atmosphere,” in which he
showed that many of the floating particles which he
had been able to collect from the atmosphere of his

PATHOGENIC BACTERIA .

laboratory were organized bodies. If these were planted
in sterile infusions, abundant crops of micro-organisms
were obtainable. By the use of more refined methods
he repeated the experiments of Schwann and others, and
showed clearly that ccthe cause which communicated life*
to his infusions came from the air, but was not evenly dis¬
tributed through it.”

Three years later he showed that the organized cor¬
puscles which he had found in the air were the spores or
seeds of minute plants, and that many of them possessed
the property of withstanding the temperature of boiling
water — a property which explained the peculiar results
of many previous experimenters, who failed to prevent
the development of life in boiled liquids enclosed in
hermetically-sealed flasks.

Chevreul and Pasteur (about 1836) proved that animal
solids did not putrefy or decompose if kept free from
the access of germs, and thus suggested to surgeons that
the putrefaction which occurred in wounds was due rather
to the entrance of something from without than to some
change within. The deadly nature of the discharges
from these wounds had been shown in a rough manner
by Gaspard as early as 1822 by injecting some of the
material into the veins of animals.

Examinations of the blood of diseased animals were
now begun, and Pollender (1849) and Davaine (1850)
succeeded in demonstrating the presence of the anthrax
bacillus in that disease. Several years later (1863) Da¬
vaine, having made numerous inoculation-experiments,
demonstrated that this bacillus was the materies morbi
of the disease.

Tyndall enlarged upon the experiments of Pasteur,
and very conclusively proved that the micro-organismal
germs were in the dust suspended in the atmosphere, not
ubiquitous in their distribution. His experiments were
very ingenious and are of interest to medical men. First
preparing light wooden chambers, with one large glass
window in the front and one smaller window in each

INTRODUCTION .

side, he arranged a series of empty test-tubes in the
bottom and a pipette in the top, so that when desired
the tubes, one by one, could be filled through it. The
chamber was first submitted to an optical test to deter¬
mine the purity of its atmosphere, and was allowed to
stand undisturbed and unused until a powerful ray of
light passed through the side windows failed to reflect
rays from suspended particles of dust when viewed from
the front. When the dust had settled so as to allow the
optical test of its purity, the tubes were filled with urine,
beef-broth, and a variety of animal and vegetable broths,
boiled by submergence in a pan of hot brine; the tubes
were then allowed to remain undisturbed for days, weeks,
or months. In nearly every case life failed to develop
after the purity of the atmosphere was established.

In 1873, Obermeier observed that actively motile, flex¬
ible spiral organisms were present in large numbers in
the blood of patients in the febrile stages of relapsing
fever.

Thus evidence slowly accumulated to establish the
theory for which Henle had labored as early as 1821, that
for many diseases at least there was a distinct and specific
contagium vivum , and the “ GERM theory” was pro¬
pounded.

Is it not strange that the very idea which was to be the
outcome of all this investigation and discussion — an idea
which would form a new era in scientific medicine and
become a fundamental principle of pathology — was one
which had been conceived and taught by a philosopher
who lived nearly two thousand years ago? Among the
numerous works of Varro 1 is one entitled Rerum Rusti-
carwn libri ires , from wThich the following is quoted :
“Animadvertendum etiam, si qua erunt loca palustria —
quod crescunt animalia quaedam minuta, quae non pos-
sunt oculi consequi et per aera intus in corpus per os ac
nares perveniunt atque efficiunt difficilis morbus” (I.,
xii. 2). — “ It is also to be noticed, if there be any marshy
1 Univ. Med. Mag., vol. iii., No. 3, Dec., 1890, p. 152.

PATHOGENIC BACTERIA .

places, that certain minute animals breed [there] which
are invisible to the eye, and yet, getting into the sys¬
tem through mouth and nostrils, cause serious disor¬
ders (diseases which are difficult to treat)” — a doctrine
which, as Prof. Lamberton, to whom the writer is in- *
deb ted for the extract, points out, is handed down to us
from “the days of Cicero and Csesar,” yet corresponds
closely to the ideas of malaria which we entertain at
present.

Pasteur had long before suggested that for the different
kinds of fermentation there must be specific ferments,
and by fractional cultures had succeeded in roughly sepa¬
rating them.

Klebs, who was one of the pioneers of the germ
theory, published in 1872 his work upon septicemia and
pyemia, in which he expressed himself convinced that
the causes of these diseases must come from without the
body. Billroth strongly opposed such an idea, asserting
that fungi had no especial importance either in the pro¬
cesses of disease or in those of decomposition, but that,
existing everywhere in the air, they rapidly developed in
the body as soon as through putrefaction a “Faulniss-
zymoid,” or through inflammation a u phlogistische-
zymoid,” supplying the necessary feeding-grounds, was
produced.

Klebs was not alone in the opposition aroused. Da-
vaine no sooner announced the contagium of anthrax
than critics declared that inasmuch as he introduced
blood from the diseased animal into the other animal
to whom the disease was to be communicated, it was
altogether unreasonable to believe the bacilli which were
in all probability accidentally present in that blood were
the cause of the disease.

In 1875 the number of scientific men who had embraced
the germ theory of disease was small, and most of those
who accepted it were experimenters. A great majority
of medical men either believed, like Billroth, that the
presence of fungi where decomposition was in progress

INTRODUCTION \

was an accidental result of their universal distribution,
or, being still more conservative, retained the old un¬
questioning faith that the bacteria, whose presence in
putrescent wounds as well as in artificially prepared
^media was unquestionable, were spontaneously generated
there.

The following extracts from Tyndall’s work1 will illus¬
trate the slow growth of the germ theory even among
men of eminence :

u At a meeting of the Pathological Society of London,
held April 6, 1875, the ‘ germ theory ’ of disease was
formally introduced as a subject for discussion, the debate
being continued with great ability and earnestness at sub¬
sequent meetings. The conference was attended by
manv distinguished medical men, some of whom were
profoundly influenced by the arguments, and none of
whom disputed the facts brought forward against the
theory on that occasion.”

“The leader of the debate, and the most prominent
speaker, was Dr. Bastian, to whom also fell the task of
replying on all the questions raised.”

“The coexistence of bacteria and contagious disease
was admitted ; but, instead of considering these organisms
as probably the essence, or an inseparable part of the es¬
sence, of the contagium, Dr. Bastian contended that they
were pathological products spontaneously generated in the
body after it. had been rendered diseased by the real con¬
tagium. ’ ’

“The grouping of the ultimate particles of matter to
form living organisms Dr. Bastian considered to be an
operation as little requiring the action of antecedent life
as their grouping to form any of the less complex chem¬
ical compounds.” “Such a position must, of course,
stand or fall by the evidence which its supporter is able
to produce, and accordingly Dr. Bastian appeals to the
law and testimony of experiment as demonstrating the
soundness of his view.” “ He seems quite aware of the

1 Op. cit.

PATHOGENIC BACTERIA .

gravity of the matter at hand ; this is his deliberate and
almost solemn appeal : 4 With the view of settling these
questions, therefore, we may carefully prepare an infusion
from some animal tissue, be it muscle, kidney, or liver ;
we may place it in a flask whose neck is drawn out'
and narrowed in the blowpipe flame ; we may boil the
fluid, seal the vessel during ebullition, and, keeping it
in a warm place, may await the result, as I have often

done . After a variable time the previously heated

fluid within the hermetically-sealed flask swarms more
or less plentifully with bacteria and allied organisms,
even though the fluids have been so much degraded in
quality by exposure to the temperature of 2120 F., and
have in all probability been rendered far less prone to
engender independent living units than the unheated
fluids in the tissues would be.’ ”

These somewhat lengthy quotations are of great in¬
terest, for they show exactly the state of the scientific
mind at a period as recent as twenty years ago.

In 1877 the introduction of the anilin dyes by Weigert
made possible a much more thorough investigation of
the bacteria by enabling the observers to color them
intensely, and thus detect their presence in tissues and
organs where their transparency had caused them to ’ be
overlooked.

Rapid strides # were immediately made, and before
another decade had passed discoveries were so numerous
and convincing that it was impossible to doubt that bac¬
teria were causes of disease.

Before the publication of the discoveries of which we
speak, however, there was suggested a practical applica¬
tion of the little known about bacteria which produced
greater agitation and incited more observation and ex¬
perimentation than anything suggested in surgery since
the introduction of anesthetics — namely, antisepsis.

u The seminal thought of antiseptic surgery may per¬
haps be traced to John Colbach, a member of the College
of Physicians, England, whose collection of tracts, printed

INTRODUCTION-.

1704, contained a description of a new and secret method
of treating wounds, by which healing took place quickly
without inflammation or suppuration; but it is to one of
old Scotia’s sons, Sir Joseph Lister, that the everlasting
* gratitude of the world is due for the knowledge we pos¬
sess in regard to the relation existing between micro¬
organisms and inflammation and suppuration, and the
power to render wounds aseptic through the action of
germicidal substances. ’ ’ 1

Lister was not the discoverer of carbolic acid nor of
the fact that it would kill bacteria; but, convinced that
inflammation and suppuration were due to the entrance
of germs from the air, instruments, fingers, etc. into
wounds, he suggested the antisepsis which would insist
upon the use of sterile instruments and clean hands and
towels; which would keep the surface of the wound
moist with a germicidal solution to kill such germs as
accidentally entered; and which would conclude an ope¬
ration by a protective dressing to exclude the entrance of
germs at a subsequent period.

Listerism, originated (1875) a few years before Koch
published his famous work on the Wundinfectionskrank -
heiten (traumatic infectious diseases) (1878), spread slowly
at first, but surely in the end, to all departments of sur¬
gery and obstetrics.

The discovery of the yeast-plant by Latour and
Schwann as the cause of fermentation, and the later dis¬
covery by Bassi of the veast-like plant causing the mias¬
matic contagious disease of silkworms, had led Henle
(1840) to believe that the cause of miasmatic, infective,
and contagious diseases must be looked for in fungi or
in other minute living organisms. Unfortunately, the
methods of study employed in Henle’ s time prevented
him from demonstrating the accuracy of his belief.

“ It would indeed have been difficult at that period to
satisfy every condition that he required to be fulfilled:
the methods now in use were then unknown, and have

1 Agnew’s Surgery , vol. i. chap. ii.

PATHOGENIC BACTERIA .

only been perfected by workers as it has been found nec¬
essary from time to time to comply in the most minute
detail with Henle’s conditions, and as, one point being
carried, it was found necessary to advance on others.
The first of these was that a specific organism should
always be associated with the disease under consideration.
As such presence, however, might be accidental, these
organisms were not only to be found in pus, etc., but actu¬
ally in the living body. As they might be, even then,
merely parasitic, and not associated directly with the
causation of the disease, it would be necessary to isolate
the germs, the contagium organisms, and the contagium
fluids, and to experiment with these separately with
special reference to their power of producing similar
diseases in other animals. We now know that it has
only been by strict compliance with all these conditions,
again postulated by Koch, that the most brilliant scien¬
tific observers and experimentalists in Germany, France,
England, [and America] have been able to determine
the causal connection between micro-organisms and
disease. ” 1

The refined methods of Pasteur, but more especially
of Koch, by making possible the fulfilment of the pos¬
tulates of Henle caused an enormous increase in the
rapidity with which data upon disease-germs were gath¬
ered. Almost within a decade the causes of the most
important specific diseases were isolated and cultivated.

In 1879, Hausen announced the discovery of bacilli in
the cells of leprous nodules. The same year Neisser
discovered the gonococcus to be specific for gonorrhea.

In 1880 the bacillus of typhoid fever was first observed
by Eberth, and independently by Koch.

In 1880, Pasteur published his -work upon u chicken-
cholera.” In the same year Sternberg described the
pneumococcus, calling it the micj'ococcus Pasteziri.

In 1882, Koch made himself immortal by his discov¬
ery of and work upon the tubercle bacillus. The same

1 Woodhead : Bacteria ami their Products , p. 65.

INTRODUCTION.

year Pasteur published a work upon Rouget du Pore , and
Lbffler and Schiitz reported the discovery of the bacillus
of glanders.

In 1884, Koch reported the discovery of the “comma
0 bacillus,” the cause of cholera, and in the same year
Loffler discovered the diphtheria bacillus, and Nicolaier
the tetanus bacillus.

In 1892, Canon and Pfeiffer discovered the bacillus of
influenza.

In 1892, Canon and Pielicke first found the bacillus
now thought to be specific for measles.

In 1894, Yersin and Kitasato independently isolated
the bacillus causing the bubonic plague then prevalent
at Hong-Kong.

A new era in bacteriology, and probably the most
triumphant result of the modern scientific study of dis¬
ease, was inaugurated in 1890 by Behring, who presented
to the world the u Blood-serum therapy,” and showed as
the result of prolonged, elaborate, and profound study of
the subject of immunity that in the blood of animals
with acquired immunity to certain diseases (diphtheria
and tetanus) a substance was held in solution which was
potent to save the lives of other animals suffering from
the same diseases.

CHAPTER I.

BACTERIA.

A BACTERIUM is a minute vegetable organism consist¬
ing of a single cell principally composed of an albumin¬
ous substance, which Nencki has called my coprotein .
Nencki found the chemical analysis of bacteria in the
active state to consist of 82.42 per cent, of water. In
100 parts of the dried constituents he found 84.20 parts
of inycoprotein; 6.04 of fat; 4.72 of ash; 5.04 of unde¬
termined substances.

Mycoprotein, which has the composition C 52.32, H
7.55, N 14.75, a perfectly transparent, generally ho¬
mogeneous body, which probably varies somewhat ac¬
cording to the species from which it is obtained, the
culture-medium in which it is grown, and the vital
products which the organism produces by its growth.
Sometimes the mycoprotein is granular, as in bacillus
megatherium ; sometimes it contains fine granules of
chlorophyl, sulphur, fat, or pigment. Each cell is sur¬
rounded by a cell-wall, which in some species shows the
cellulose reaction with iodin.

When subjected to the influence of nuclear stains the
bacteria not only take the stain faintly, but in «uch a
manner as to show the existence of a large nucleus situ¬
ated in the centre of the cell and constituting its great
bulk. The cell-wall generally is not stained, but when
it does tinge, a delicate line of unstained material can
sometimes be made out between the nucleus and the cell-
wall, showing the existence of a protoplasm.

The anilin dyes, which possess a great penetrating
power, color the organisms so intensely as to preclude
the differentiation of the cellular constituents. Under

BACTERIA.

these® conditions the bacteria appear as solidly-colored
spheres, rods, or spirals, as the case may be.

The cell-walls of some of the bacteria seem at times to
undergo a peculiar gelatinous change or to allow the ex¬
udation of gelatinous material from the protoplasm, so
that the individuals appear surrounded by a distinct halo
or capsule. This is not only a peculiarity of certain indi¬
viduals, but one which only takes place when they develop
under certain conditions; thus, Friedlander points out
that the capsule of his pneumonia bacillus, when it was
found in the lung or in the u prune-juice ” sputum, was
very distinct, while it could not be demonstrated at all
when the organisms grew in gelatin.

From the cell-walls of many bacteria numerous deli¬
cate straight or wavy filaments project. These are called
cilia or flagella , and seem to be organs of locomotion.
Sometimes they are only observed projecting from the
ends or from one end; sometimes they are so numerous
and so regular in their distribution as to give the organ¬
isms a woolly appearance.

Many of the bacteria which are thus supplied with
flagella are actively motile and swim about like mi¬
croscopic serpents. In all probability the locomotory
powers of the bacteria are not entirely dependent upon
the presence of the flagella, but may sometimes be due
to contractility of the protoplasm within an elastic cell-
wall. The micro-organisms most plentifully supplied
with them are those of the rod and spiral shape. Only
one of the spherical forms, Micrococcus agilis of Ali-
Colien, has been shown to have flagella. This and one
other species are probably the only motile cocci. Ob¬
serving that the organisms known to be most active are
those best supplied with flagella, it is reasonable to con¬
clude that the motility is dependent upon the flagella.

The presence of flagella, however, does not necessarily
imply motility, for some of the bacilli amply provided
with these appendages are not motile. The flagella may
not only serve as organs of locomotion, and be of use to

PATHOGENIC BACTERIA.

the organism by conveying it from an area where the
nutrition is less to one where it is greater, but, as Wood-
head points out, may, in the noil-motile species, serve to
stimulate the passage of currents of nutrient material
past the organism, so as to increase the food-supply. -
The flagellate bacteria have a greater number of repre¬
sentatives among those whose lives are spent in water
and in fermenting and decaying materials than among
those inhabiting the bodies of animals. This is an
additional fact in favor of the view that locomotion and
flagella are provisions favorable to the maintenance of
the species by keeping the individuals supplied with
food.

It may be added that such parasitic disease-producing
bacteria as do not habitually gain access to the tissues,
but inhabit the intestine, as the bacillus of typhoid fever
and the spirillum of cholera, are actively motile, like
the saprophytes, while those habitually entering the tis¬
sues and multiplying there are motionless and without
flagella. Of course this example is open to criticism,
because the spirillum of relapsing fever, which has never
been found elsewhere than in the blood and spleen of
affected animals, is actively motile.

One of the linear organisms, known as the Bacillus
megatherium, has a distinct but limited ameboid move¬
ment.

The commonly observed dancing movement of the
spherical forms seems to be the well-known Brownian
movement, which is simply a physical phenomenon. It
is sometimes difficult to determine whether an organism
is really motile or whether it is only vibrating. In the
latter case it does not change its relative position to
surrounding objects.

The bacteria are so minute that a special unit of meas¬
urement has been adopted by bacteriologists for their
estimation. This is the micro-millimeter (/*), or one-
thousandth part of a millimeter, and about equivalent
to the one-twenty-five-thousandth of an inch.

BACTERIA .

As a rule, the spherical organisms are the smallest and
the spiral organisms the longest, except the chains of
bacilli called leptothrix . Their measurements vary from
0.15 fjt (micrococcus of progressive abscess-formation in
rabbits) to 2.8 fi (Diplococcus albicans am plus) for cocci,
and from 1 X 0.2 fi (bacillus of mouse-septicemia) to
5 X 1.5 p (anthrax bacillus) for bacilli. Some of the
spirilla are very long, that of relapsing fever measuring
40 /jl at times.

This estimation of size almost prepares one for the
estimation of weight given by Nageli, who found that
an average bacterium under ordinary conditions weighed

i-fi-tnreoTnnnnr of a milligram.

The bacteria multiply in two ways : by direct division
(fission) and by the development of spores, seeds, or eggs
(sporulation). The more common mode is by binary
division. The bacterium which is about to divide ap¬
pears a little larger than normal, and, if a spherical
organism, more or less ovoid. No karyokinetic changes
have been observed in the nuclei, though they may occur.
When the conditions of nutrition are good, the process of
fission progresses with astonishing rapidity. Buchner
and others have determined the length of a generation
to be from fifteen to forty minutes.

The results of binary division, if rapidly repeated, are
almost appalling. l< Cohn calculated that a single germ
could produce by simple fission two of its kind in an
hour ; in the second hour these would be multiplied to
four ; and in three days they would, if their surroundings
were ideally favorable, form a mass which can scarcely be
reckoned in numbers, or, if reckoned, could scarcely be
imagined — four thousand seven hundred and seventy-two
billions. If we reduce this number to weight, we find
that the mass arising from this single germ would in
three days weigh no less than seventy-five hundred
tons.7’ “ Fortunately for us,’ 7 says Woodhead, “they
can seldom get food enough to carry on this appalling
rate of development, and a great number die both for
3

PATHOGENIC BACTERIA .

want of food and because of the presence of other con¬
ditions unfavorable to their existence.”

When the conditions for rapid multiplication are no
longer good, the organism assumes a protective attitude
and develops in its interior small oval eggs, seeds, or, as
they are more correctly called, spores (Fig. i). Such

a b c d e f

Fig. i. — Diagram illustrating speculation : a , bacillus enclosing a small oval
spore ; drumstick bacillus, with the spore at the end ; c , Clostridium ; I, free
spores ; e and f bacilli escaping from spores.

spores developed within the bacteria are called endospores.
When the formation of such a spore is about to com¬
mence, a small bright point appears in the protoplasm,
and increases in size until its diameter is nearly or quite
as great as that of the bacterium. As it nears perfection
a dark, highly-refracting capsule is formed about it. As
soon as the spore arrives at perfection the bacterium
.seems to die, as if its vitality were exhausted in the
^development of the permanent form.

Endospores are generally formed in the elongate bac¬
teria — bacillus and spirillum — but Zopf has described
similar bodies as occurring in micrococci. Escherich '
also claims to have found undoubted spores in a form
of sarcina.

The spores found in the bacilli are either round or
oval. As a rule, each bacillus produces a single spore,
which is situated either at its centre or at its end. When,
as sometimes happens, the diameter of the spore is greater
than the diameter of the bacillus, it causes a bulging- of
the organism, with a peculiar appearance described as
Clostridium. When the distending spore is in the centre
of the bacillus, it produces a barrel-shaped organism;
when situated at the end, a “ Trommelschlager, ” or drum¬
stick-shaped one. As the degeneration of the protoplasm
of the bacillus sets the spore free, it appears as a clear,

BACTERIA. 35

highly-refracting sphere or ovoid situated in a little col¬
lection of granular matter.

Spores differ from the bacteria in that their capsules
seem to prevent evaporation and to enable them to with¬
stand drying and the application of a considerable amount
of heat. Ordinarily, bacteria are unable to resist a tem¬
perature above 6o° C. for any considerable length of
time, only a few resistant forms tolerating a temperature
of 70° C. The spores, however, are uninjured by such
temperatures, and can even successfully resist that of
boiling water (ioo° C.) for a short time. The extreme
desiccation caused by a protracted exposure to a tem¬
perature of 150° C. will, however, destroy them. Not only
can the spores resist a considerable degree of heat, but
they are also unaffected by cold of almost any intensity.

While the cell-wall of the bacterium is easily pene¬
trated by solutions of the anilin dyes, it is a matter of
much difficulty to accomplish the staining of spores, so
that we see they are probably more resistant to the
action of chemical agents than the bacteria themselves.

When a spore is accidentally dropped into some nu¬
trient medium a change is shortly observed. The proto¬
plasm, which has been clear, becomes somewhat granu¬
lar, the capsule a little less distinct; the body increases
slightly in size, and in the course of time splits open to
allow the escape of the young organism. The direction
in which the escape of the young bacillus takes place is
of interest, as varying in the different species. The
Bacillus subtilis escapes from the end of the spore, where
a longitudinal fissure occurs; the bacillus of anthrax
escapes from the side, sometimes leaving the capsule of
the spore in the shape of two small cups.

As soon as the young bacillus escapes it begins to in¬
crease in size, develops around its soft protoplasm a cha¬
racteristic capsule, and, having once established itself,
presently begins the propagation of its species by fission.

In addition to the endospores, of which we have just
been speaking, there are arthrospores . The formation

PATHOGENIC BACTERIA.

of these is much less clear. It seems to be an effort to
convert the entire microbe into a permanent form. This
process is observed particularly in the micrococci, where
the substance of a cell is said to break up into segments,
each of which becomes a resisting body fruitful in prop¬
agating its species. Of the arthrospores little has, so
far, been learned. It is not improbable that among the
micrococci, and also among some of the smaller bacilli
in whom no spores have been observed, the maintenance
of the species when conditions of life become unfavor¬
able is due to the assumption of a permanent form by
some of the individuals, without the formation of any
spore-like bodies. This is at present largely a matter of
conjecture, but the indications pointing in that direction
are numerous.

It is believed by Frankel and others that sporulation
in the bacteria is not a sign of the exhaustion of nutri¬
tion, but a sign of the vital perfection of the organism.
These observers regard spore-formation as analogous to
the flowering of higher plants, which takes place only
when the conditions and development are best.

Morphology. — The morphology of the bacteria is quite
varied. Three principal forms, however, exist, from which
the others seein to be but variations.

The most simple appear as minute spheres, and from

4? \

qO <£

Fig. 2. — Diagram illustrating the morphology of the cocci : a, coccus or
micrococcus; b, diplococcus ; c, d , streptococci; e, f telragenococci or meris-
mopedia; g , /i, modes of division of cocci; i, sarcina; j, coccus with flagella;
k , staphylococci.

their fancied resemblance to little berries are called cocci
or micrococci (Fig. 2, a\ When the bacteria of this form

BACTERIA .

multiply by fission the resulting two organisms not
infrequently remain attached to each other, producing
what is called a diplococctts (Fig. 2, b). The diplococci
sometimes consist of two perfect spheres, but more often
^show a flattening of the contiguous surfaces, which are
not in absolute apposition (Fig. 2, g). In a few cases, as
the gonococcus, the approximated surfaces are slightly
concave, causing the organism to somewhat resemble the
German biscuit called a “semmel,” hence biscuit- or
semmel-cocci (Fig. 2, h). Frequently a second binary di¬
vision occurs, causing four individuals to remain closely
approximated, without disturbing the arrangement of the
first two. When division of this kind produces a distinct
tetrad, the organism is described as a tetragenococcus ,
while to the entire class of cocci dividing so as to pro¬
duce fours, eights, twelves, etc. on the same plane the
name merismopedia is given (Fig. 2, e and f).

If, as sometimes happens, the divisions take place in
three directions, so as to produce cubical masses or u pack¬
ages” of cocci, the resulting aggregation is described as
a sar cina (Fig. 2, z). This form slightly resembles a dice
or a bale of cotton in miniature.

If the divisions always take place in the same direc¬
tion, so as to produce a chain or string of beads, the
organism is described as streptococcus (Fig. 2, d). When
there are diplococci joined in this manner a strepto-diplo-
cocciis is of course formed.

More common than any of the forms already described
is one in which, without any definite arrangement, the
cocci occur in irregular groups having a fancied resem¬
blance to bunches of grapes. These are called staphylo-
cofccz, and, as it is very unusual to find cocci habitually
occurring isolated, most cocci not classified under one of
the above heads are called staphylococci.

When cocci are associated in globular or lobulated
clusters encased in a resisting glutinous, homogeneous
mass, the name ascococcus has been iised in describing
them. A modified form of this, in which the cocci are

PATHOGENIC BACTERIA .

in chains or solitary and are surrounded by an encase¬
ment almost cartilaginous in consistence, has been called
leuconostoc .

Certain bacteria, constituting a better-known if not
more important group, are not spherical, but elongate*
or u rod-shaped, ” and bear the name bacillus (Fig. 3).

ah c d e

Fig. 3. — Diagram illustrating the morphology of the bacilli: a, b, c , various
forms of bacilli ; d , e, bacilli with flagella ; f chain of bacilli, individuals dis¬
tinct; chain of bacilli, individuals not separated.

I would remark that the absence of a standard by
which to separate the cocci from the bacilli is the cause
of much confusion. In the judgment of the author, it
would be well to place all individuals having one diam¬
eter greater than the other among the bacilli. This
would prevent the error of describing one species as
“oval cocci” and another as “nearly round bacilli,”
and by giving a definite standard would greatly aid in
the formation of a differential table.

The bacilli present a considerable variety of forms.
Some are quite short, with rounded ends, so as to ap¬
pear elliptical ; some are long and delicate. Some have
rounded ends, as subtilis ; others have square ends, as
anthrax. Some are enormously large, some exceedingly
small. Some are always isolated, never forming threads
or chains ; others nearly always occur in these forms.

The bacilli always divide by transverse fission, so that
the only peculiarity of arrangement is the formation of
threads or chains.

In the older writings the short, stout bacilli were all
described under the generic term bacterium. This genus,
like some of the species it comprehended, has now passed

BACTERIA.

out of use. Some of the flexile bacilli, whose movements
are sinuous, much resembling the swimming of a snake
or an eel, were described as vibrio , but this name also has
passed into disuse.

o The long filaments formed by the division of bacilli
without their distinct separation are sometimes called
leptothrix) and when these long threads form distinct
masses surrounded by a jelly-like material, the name
myconostoc is sometimes applied to them.

Certain forms much resembling bacilli in their isolated
state, characterized by the formation of long filaments
with a peculiar grouping which gives the appearance
of a false branching, are described as cladothrix ; others
in which true branchings are seen, as streptothrix. One
other bacillus-like form, consisting of long, thick, not
distinctly segmented, straight threads, is called beggiatoa.
The only important difference between it and leptothrix
is that its filaments are thick and coarse, while those of
leptothrix are very delicate.

Some of the elongate bacteria have a remarkably
twisted form and bear some resemblance to a cork¬
screw. These are called spirilla (Fig. 4). A subdivision

a be

Fig. 4. — Diagram illustrating the morphology of the spirilla : a , b, c , spirilla ;
d, e , spirochseta.

of them, whose individuals are not only twisted but are
also very flexible, is called spirochceta. Though not
formerly differentiated from vibrio, these forms are quite
distinct.

A spiral organism of a ribbon shape is called spiro -

PATHOGENIC BACTERIA .

monas , while a similar organism of spindle shape is
called a spirulina . One species of spiral bacteria in
whose protoplasm sulphur-grounds have been detected
has been called ophidiomonas.

Some of the spirilla are exceedingly long and deli-,
cate, as the spirochaeta of relapsing fever ; others which
are stouter, like the spirillum of cholera, habitually occur
in such short individuals as to be easily mistaken for
slightly-bent bacilli.

Classification. — Leeuwenhoek, when he first saw the
bacteria — and his successors even to so recent a date as
to include Ehrenberg and Dujardin — did not doubt that
they belonged to the infusoria.

It was not until biologists had concluded that organ¬
isms which take into their bodies particles of solid or
semi-solid material, digest that which is useful, and
extrude the remainder, are animals, and that those which
live purely by osmosis and exosmosis are vegetables, that
the bacteria, which we have seen provided with a resist¬
ant cell-wall, allowing of no possibility of nutrition
except by osmosis and exosmosis, could be finally and
correctly classed among the members of the vegetable
kingdom.

The extremely simple organization of bacteria naturally
places them among the lowest members of the vegetable
kingdom, in that class of the Cryptogamia known as
Thallophytae, comprising the algae, lichens, and fungi.

The algae are mostly water-plants, containing chloro-
phyl and obtaining their nourishment from inorganic
substances.

The lichens are plants, some of which contain chloro-
phyl. They live upon inorganic matter, which they
generally absorb from the air. According to the new
view of the subject, some, if not all, of these plants are
regarded as fungi growing parasitically upon algae.

The fungi, the lowest group of .all, are minute or large
plants, mostly devoid of chlorophvl, living upon organic
matter, which they obtain as saprophytes from decom-

BACTERIA .

posing animal and vegetable matters, or as parasites
upon the tissues or juices of living animals or plants.

This lowest family, the fungi, are divisible into the —

Hyphomycetes or Mucorini, or moulds;

J Saccharomycetes, or yeasts; and

Schizomycetes, or bacteria.

Cohn divided the bacteria, according to their mor¬
phology, into —

Sphero-bacteria, or cocci ;

Micro-bacteria — short rods ;

Desmo-bacteria — bacilli ;

Spiro-bacteria — spirilla.

Davaine suggested a classification based upon motility,
making four classes — Bacterium, Vibrio, Bacteridium,
and Spirillum, neglecting to provide for the cocci.

Zopf arranged them, according to his theory of
pleomorphism, into the Coccace^, comprising those
known only in the coccus form, and comprehending
the streptococci , merismopedia , sarcina , niicrococais , and
ascococcus; the Bacteriace^E, comprehending the genera
bacterium , spirillum , vibrio , leuconostoc , bacillus , and
Clostridium (chiefly coccus, rod, and thread forms ; the
former may be absent ; in the latter there is no distinction
between base and apex ; threads straight or screw-like) ;
and the Leptothriche^E, comprehending crenothrix ,
beggiatoa , phragmidiothrix , and leptotJudx (coccus, rod,
and thread forms ; the latter show a distinction between
base and apex ; threads straight or screw-like ; spore-
formation not demonstrated).

This classification is, however, based upon what is
probably an erroneous principle, the pleomorphism of
the bacteria.

Van Tieghem, DeBary, and Hiippe formed classifica¬
tions the main feature of which was the formation of
endospores or arthrospores, but, as the sporulation of
many species is as yet unknown, they cannot be properly
placed in it.

PA THO GENIC BA CTERIA .

It has even been suggested to classify the bacteria by
the size and number of their flagella, of which so little
is known.

The most convenient classification, though it cannot
be purely scientific, seems to be the morphological one «
given by Cohn. Baumgarten, recognizing the relative
pleomorphism of certain of the species, has modified it
as follows, and thus made it answer all the needs of the
pathologist at least:

I. Cocci, •)

II. Bacilli, l species relatively monomorphous.

III. Spirilla, J

IV. Spirulina, 'j

V. Leptothrix, [ species relatively pleomorphous.

VI. Cladothrix, J

The members of the first group, the cocci, bacilli, and
spirilla, are practically the only ones which are of patho¬
logical significance.

CHAPTER II.

BIOLOGY OF BACTERIA.

The distribution of bacteria is wellnigh universal.
They and their spores float in the atmosphere we breathe,
swim in the water we drink, grow upon the food we eat,
and luxuriate in the soil beneath our feet. Nor is this
all, for, entering the palpebral fissures, they develop upon
the conjunctiva ; entering the nares, they establish them¬
selves in the nose ; the mouth is always replete with
them ; and, as many are swallowed, the digestive appa¬
ratus always contains them. The surface of the body
never escapes their establishment, and so deeply are
some individuals situated beneath the epithelial cells
that the most careful washing and scrubbing and the use
of the most powerful germicides are required to rid the
surgeon’s hands of what may prove to be dangerous
hindrances to the healing of wounds. The ear is not
without its microscopic flora ; special varieties live be¬
neath the finger-nails, and especially the toe-nails, in
the vagina, and beneath the prepuce.

While so general, however, they are not ubiquitous.
Tyndall succeeded in proving that the atmosphere of
high Alpine altitudes was free from them, and likewise
that the glacier ice contained none. Wherever man, ani¬
mals, or even plants, live, die, and decompose, bacteria
are sure to be present.

Notwithstanding their extreme familiarity with the
animal body, there are certain parts of it into which
bacteria do not enter, or, entering, remain vital for a
very short time, for the body-juices and tissues of normal
animals are free from them , and their occurre7ice there
may almost always be accepted as a sign of disease.

The presence of bacteria in the air is generally de-

PA THOGENIC BA CTERIA .

pendent upon their previous existence in the soil, its pul¬
verization, and its distribution by currents of the atmo¬
sphere. Koch has shown that the upper stratum of the
soil is exceedingly rich in bacteria, but that their num¬
bers decrease as the soil is penetrated, until below a«
depth of one meter there are very few. Remembering
that bacteria can live only upon organic matter, this is
readily understandable. Most of the organic matter is
upon the surface of the soil. Where, as in the case of
porous soil or the presence of cesspools and dung-heaps,
the decomposing materials are allowed to penetrate to a
considerable depth, the bacteria may occur much farther
from the surface, yet they are rarely found at any great
depth, because the majority of the known species require
oxygen.

The water of stagnant pools always teems with bacte¬
ria, but that of deep wells rarely contains many unless
it is polluted from the surface of the earth.

Being generally present in the soil, which the feet of
men and animals grind to powder, the bacteria, together
with the pulverized earth, are blown from place to place
into every nook and cranny, until it is impossible to es¬
cape them. It has been suggested by Soyka that the
currents of air passing over the surface of liquids might
take up bacteria, but, although he seemed to show it ex¬
perimentally, it is not generally believed. Where bac¬
teria are growing in colonies they seem to remain un¬
disturbed by currents of air unless the surface becomes
roughened or broken.

Most of the bacteria which are carried about by the air
are what are called saprophytes, and are perfectly harm¬
less to the human being ; but not all belong to this class,
nor will they do so while tuberculous patients are al¬
lowed to expectorate upon the sidewalks, and typhoid
patients’ wash to dry upon the clothes-line, and their
dejecta to be spread upon the ground.

The growth of bacteria is profoundly influenced by
environment, so that a consideration of the conditions

BIOLOGY OF BACTERIA . 45

favorable or detrimental to their existence becomes a
necessity.

Conditions influencing the Growth of Bacteria. —
(a) Oxygen . — The majority of bacteria grow best when
’exposed to the air. Some develop better when the air is
withheld; some will not grow at all where the least
amount of oxygen is present. Because of these pecu¬
liarities bacteria are divisible into the

Aerobic bacteria^ those growing in oxygen.

Anaerobic bacteria , those not growing in the presence
of oxygen.

As, however, some of the aerobic forms will grow
almost as well without as with oxygen, the term optio?ial
(facultative) anaerobics has been applied to the special
class made to include them.

As examples of strictly aerobic bacteria the Bacillus
subtilis and the Bacillus aerophilus may be given. These
forms will not grow if oxygen is denied them. The
staphylococci of suppuration and the bacilli of typhoid
fever, pneumonia, and anthrax, as well as the spirillum
of cholera, will grow almost equally well with or with¬
out oxygen, and hence belong to the optional anaerobics.
The bacillus of tetanus and of malignant edema, and the
noii-pathogenic forms, the Bacillus butyricus, Bacillus
muscoides, and Bacillus polypiformis, will not develop
at all where any oxygen is present, and hence are
strictly anaerobic.

(b) Nutriment . — The bacteria do not seem able to derive
their nourishment from purely inorganic matter. Pros-
kauer and Beck, however, have succeeded in growing the
tubercle bacillus in a mixture containing ammonium
carbonate 0.35 per cent., potassium phosphate 0.15 per
cent., magnesium sulphate 0.25 per cent., glycerin 1.5
per cent. They grow best where diffusible albumins are
present. The ammonium salts are rather less fitted to
support them than their organic compounds. The in¬
dividual bacterium varies very widely in the nutriment
which it requires. Some of the water-microbes can live

PATHOGENIC BACTERIA .

in distilled water to which the smallest amount of organic
matter has been added; others require so concentrated a
medium that only blood-serum can be used for their
cultivation. Sometimes a species with a preference for a
particular culture-medium can gradually be accustomed"
to another, though immediate transplantation causes the
death of the transplanted organism. Sometimes the ad¬
dition of such substances as glucose and glycerin has a
peculiarly favorable influence upon bacteria, causing, for
example, the tubercle bacillus to grow upon agar-agar.

(c) Moisture . — A certain amount of water is always
necessary for the growth of bacteria. The amount can
be exceedingly small, however, so that the Bacillus pro-
digiosus is able to develop successfully upon crackers and
dried bread. Materials used as culture-media should not
be too concentrated; at least 80 per cent of water should
be present. Most bacteria grow best in liquid media;
that is, they form the longest threads, and diffuse them¬
selves throughout the liquid so as to be present in far
greater numbers than when on solid media.

The statement that certain forms of bacteria can flour¬
ish in clean distilled water seems to be untrue. When
transferred to such a medium the organisms soon die and
undergo a granular degeneration of their substance. If,
however, in their introduction a good-sized drop of cul¬
ture-material is carried with them, the distilled water
ceases to be such, and becomes a dilute bouillon fitted to
support life for a time.

( d ) Reaction . — Should the pabulum supplied to bacte¬
ria contain an excess of either alkali or acid, the growth
of the organisms is inhibited. Most true bacteria grow
best in a neutral or feebly alkaline medium. There are
exceptions to this rule, for the Bacillus butyricus and the
Sarcina ventriculi can grow well in strong acids, and the
Micrococcus urea can tolerate excessive alkalinity. Acid
media are excellent for the cultivation of moulds.

(e) Light. — Most species of bacteria are not influenced
in their growth by the presence or absence of light. The

BIOLOGY OF BACTERIA.

direct rays of the sun, and to a less degree the intense
rays of the electric arc-light, retard and in numerous in¬
stances kill bacteria. Some colors are distinctly inhibi¬
tory to their growth, blue being especially prejudicial.
•Some of the chromogenic forms will only produce their
colors when exposed to the ordinary light of the room.
The Bacillus mycoides roseus will not produce its red
pigment except in the absence of light. The pathogenic
bacteria have . their virulence gradually attenuated if
grown in the light.

(f) Electricity. — Very little is known about the action
of electric currents upon bacteria. Very powerful dis¬
charges of electricity through culture-media are said to
kill the organisms, to change the reaction of the culture,
and the rapidly reversed currents of high intensity to
destroy the pathogenesis of the bacteria and change their
toxic products into neutralizing protective (antitoxin?)
bodies. Much attention has recently been devoted to
this subject by Smirnow, Arsonval and Charin, Bolton
and Pease, Bonome and Viola, and others.

(. g ) Movement. — When bacteria are growing in a liquid
medium perfect rest seems to be the condition best
adapted for their development. A slow-flowing move¬
ment does not have much inhibitory action, but violent
agitation, as by shaking a culture in a machine, greatly
hinders or prevents their growth. The practical appli¬
cation of this will show that rapidly flowing streams,
whose currents are interrupted by falls and rapids, will,
other things being equal, furnish a better drinking-water
than a deep, still-flowing river.

* (Ji) Association. — It occasionally happens that bacteria
grow better when associated with other species, or have
their pathogenic powers augmented when grown in com¬
bination. Coley found the streptococcus toxin more
active when combined with Bacillus prodigiosus.

Occasionally the reverse is true, and Pawlowski found
that mixtures of anthrax and bacillus prodigiosus were
less virulent than cultures of anthrax alone.

PATHOGENIC BACTERIA .

Rarely, the presence of one species of microorganism
entirely eradicates another species. Hankin found that
the Micrococcus Ghadialli destroyed the typhoid and colon
bacilli, and suggested the use of this coccus to purify
waters polluted with typhoid.1

(z) Temperature. — The question of temperature is of
importance from its bearing upon sterilization. Accord¬
ing to Frankel, bacteria will scarcely grow at all below
i6° and above 40° C.

* The researches of Fliigge show that the Bacillus sub-
tilis will grow very slowly at 6° C. , and as the tempera¬
ture is elevated it is said that until 12. 50 C. is reached
fission does not occur oftener than every four or five
hours. When 250 C. is reached the fission occurs every
three-quarters of an hour, and at 30° C. about every half
hour.

Most bacteria die at a higher temperature than 60-
750 C. The spores can resist boiling water, but are
killed by dry heat if exposed to 150° C. for an hour or to
1750 C. for five to ten minutes. Freezing kills many, but
not all bacteria, but does not affect the spores at all.

Most bacteria grow best at the ordinary temperature of
a comfortably heated room, and are not affected by its
occasional slight changes. Some, chiefly the pathogenic
forms, are not cultivable except at the temperature of
the animal body (370 C.) ; others, like the tubercle bacil¬
lus, grow best at a temperature a little above that of the
body — 40° C.

Variations in the amount of oxygen, temperature, moist¬
ure, etc., beyond what have been described, are prej¬
udicial to the growth and development of bacteria, first
inhibiting their growth, thus tending toward their de¬
struction. In the practical application of our knowledge
of the biology of the bacteria we constantly make use of
such precautions as removing from surgical dressings,
sponges, etc., every substance that can possibly afford
nutriment to bacteria, and heating such materials, as well

1 Brit. Med. Jour.} Aug. 14, 1897, p. 418.

BIOLOGY OF BACTERIA.

as culture-media and a variety of other substances, to a
temperature beyond that known to be the extreme limit
of bacterial endurance.

The presence of certain substances — especially some
of the mineral salts — in an otherwise perfectly suitable
medium will prevent the development of bacteria, and
when added to grown cultures of bacteria will destroy
them. Carbolic acid and bichlorid of mercury are the
best known examples.

It is interesting to mention in this connection the
results of the experiments of Trambusti, who found it
possible to produce a tolerance to a certain amount of
bichlorid of mercury by cultivating Priedlanderks bacillus
upon culture-media, containing gradually increasing
amounts of the salt, until from 1-15,000, which inhibited
ordinary cultures, it could accommodate itself to 1-2000.

( j) .r-A 'ays. — The action of the .r-rays upon bacteria
has been investigated by Bonome and (iros and others.
When the cultures are exposed to their action for pro¬
longed periods, their vitality and virulence seem to be
slightly diminished. They are not killed by the .r-ravs.

Some forms of the bacteria are never found except in
the tissues of diseased animals. Such organisms are
called parasites. The parasitic group really is divisible
into the purely parasitic and the occasionally parasitic
bacteria. Of the first division the tubercle bacillus may
be used as an illustration, for, so far as is known, it is
never found in other places than the bodies and dejecta
of diseased animals. The cholera spirillum illustrates
the second group, for, while it produces the disease
known as Asiatic cholera when admitted to the digestive
tract, it is a constant inhabitant of certain waters, where
it multiplies with luxuriance.

Bacteria which do not enter the animal economy, or if
accidentally admitted do no harm, but live upon decaying
animal and vegetable materials, are called saprophytes*

According to their products of metabolism, bacteria
are often described as —

5°

PATHOGENIC BACTERIA.

Zymogenic, or bacteria of fermentation.

Saprogenic, or bacteria of putrefaction.

Chromogenic, or color producers.

Photogenic, or phosphorescent bacteria.

Aerogenic, or gas producers.

Pathogenic, or disease producers.

The parasitic organisms alone possess much interest to
the physician, but as in their growth the saprophytes ex¬
hibit many interesting vital manifestations, it is not well
to exclude them or their products from the following
consideration of the

Results of Vital Activity in Bacteria. — i. Fermenta-
tion. — The alcoholic fermentation, which is a familiar phe¬
nomenon to the layman as well as to the brewer and the
chemist, is not the work of a bacterium, but of a yeast-
plant, one of the saccharomyces fungi. The acetic-acid,
lactic-acid, and butyric-acid fermentations are, however,
caused by bacilli. A considerable number of bacilli seem
capable of converting milk-sugar into lactic acid, some¬
times associating this with coagulation of milk, some¬
times not. The production of coagulation in milk is not
always associated with acid-production, but with the pro¬
duction of a curdling ferment similar to that belonging
to the gastric juice. There seems to be no real specific
micro-organism for the lactic-acid fermentation, although
the Bacillus acidi lactici seems to be the most powerful
generator of the acid. There may also be several bac¬
teria which produce the acetic fermentation, though it is
generally attributed to a special common form, the Myco-
derma aceti or Bacillus aceticus. The butyric fermenta¬
tion is generally due to the Bacillus butyricus, though it
also may be caused by other bacilli, the one named sim¬
ply being the most common. (For .an exact description
of the chemistry of the fermentations reference must be
made to text-books upon that subject, as their considera¬
tion here would occupy too much space.)

2. Putrefaction. — This process is in many respects sim-

IUOLOitY OF FACTFR/A .

ilar to the preceding, except that instead of occurring in
carbohydrates it takes place in nitrogenous bodies. The
first step seems to be the transformation of the albumins
into peptones, then the splitting* up of the peptones into
a large number of gases, acids, bases, and salts. In the
process the innocuous albumins are frequently changed to
toxalbumins, and sometimes to distinct animal alkaloids
known as ptomaines. Vaughan and Now declare the
term u animal alkaloid ” to be a misnomer, as ptomaines
are sometimes produced from vegetable substances in
the process of decomposition ; they suggest the term
u putrefactive alkaloids’' as preferable. The definition
of a ptomaine given by these observers is ua chemical'
compound, basic in character, formed by the action of
bacteria on organic matter.” The chemistry of these
bodies is very complex, and for a satisfactory description
of them Vaughan and Novy’s book1 is brief and excel¬
lent. Among the ptomaines, the following appear to
be important: Methylamin (CH.^NIL), the simplest or¬
ganic base formed in the process of putrefaction; dime-
thylamin ((CM;t)2NH) ; trimethylamin (C;|II«,N (CII;i);iN) ;
ethylamiu (C2H5.NH,); diethylamin (C,,IInN (CdL.h-
NH); triethylamiu (CnIIi:>N (C2IIft)aN); ]>ropylamin
(C,H7.NII,); butylamin C,llnN); iso-amylamin ; caproyl-
amin ; tetanotoxin ; spasmotoxin frlihydrolutidin ; putres-
cin ; cadaverin ; ueuridin ; saprin ; pyoevanin ; and tyro-
toxicon. It is supposed that the cases of ice-cream and
cheese-poisoning that sometimes occur are due to tyro-
toxicon produced by the putrefaction of the proteid sub¬
stances of the milk before it is frozen into ice cream or
made into cheese. The safeguard is to freeze the milk
only when perfectly fresh and avoid adding the sugar and
flavoring substances, allowing the whole to stand some
time, and then freezing. Numerous others have been
described, some toxic, others harmless.

It is to compounds of this kind that the occasional
cases of 14 Fleishvergiftung” or 41 meat-poisoning ” are

1 PtoHlilhli'S tVItf

PATHOGENIC BACTERIA.

cine, the growth of various bacteria in stale meat bring¬
ing about in its proteid substances the development of
toxic ptomaines. Kaensche1 carefully investigated the
subject, and gives a synoptical table containing all the
bacteria of this class described. His researches show
that there are at least three different bacilli whose growth
causes the development of poisonous ptomaines in meat.

Toxins and toxalbumins are also very common.

3. Chromogenesis. — Those bacteria which produce col¬
ored colonies or impart color to the medium in which
they grow are called chromogenic ; those with which no
color is associated, non-cliromogenic. Most chromogenic
bacteria are saprophytic and uon-pathogenic. Some of
the pathogenic forms, as the Staphylococcus pyogenes
aureus and citreus, are, however, color-producers. It
seems likely that the bacteria do not form the actual
pigments, but certain chroniogenetic substances which,
uniting with substances in the culture-medium, pro¬
duce the colors.

Galleotti has described two kinds of pigment, one of
which, being soluble, readily penetrates all neighboring
portions of the culture-medium, like the colors of Bacillus

pyocyaneus, and an insoluble pigment which does not
tinge the solid culture-media at all, but is constantly
iound associated with the colonies, like the pigment of
Bacillus prodigiosus. The pigments are found in their

greatest intensity near the surface of the colony. The
coloring matter never occupies the protoplasm of the
bacteria (except the Bacillus prodigiosus, in whose cells
occasional pigment-granules may be seen), but occurs in
an intercellular excrementitious substance.

The pigments are so varied as to give almost everv
known color. It sometimes happens that a bacterium

i'i icufthnf T °T “0re COlOTS' The BacilI"s PVC

produces pyocyanin and ftaorescm, both

bemg soluble plgrae„ts-,0M blue, the other Wee
JZsrZ ‘hat When a' Bacin"S P^faneus

Zatst hrijt fur Hym etc., Bd. xxii., Heft I, June 2S, 1896.

BIOLOGY OF BACTERIA.

is cultivated upon white of egg, it produces only the
green fluorescent pigment, while in pure peptone solu¬
tion it grows with the production of blue pyoevanin
alone. His experiments prove a very interesting fact,
that for the production of iluorescin it is necessary that
the culture-medium contain a definite amount of a
pliosphatic salt. Sometimes one pigment is soluble,
the other insoluble, so that the colony will appear one
color, the medium upon which it grows another. Some
organisms will only produce their colors in the light *,
others, as the Bacillus mycoides roscus, only in the dark.
Some produce them only at the room-temperature, but,
though growing luxuriantly in the incubator, refuse to
produce pigments at so high a temperature. Thus,
Bacillus prodigiosus produces a brilliant red color when
growing at the temperature of the room, but is colorless
when grown in the incubator. Colored lights seem to
have no modifying influence upon the pigment-produc¬
tion. 13 ven if for successive generations the bacterium
be grown so as to be colorless, it speedily recovers its
primitive color when restored to its old environment, no
matter what the color of the light thrown upon it. Bac¬
teria which have been robbed of their color by incuba¬
tion, when placed in the normal environment produce
the original color, no matter what color the light they
receive. Some of the pigments — perhaps most of them —
are formed only in the presence of oxygen.

4. Liquefaction of (ic/atiu. — When certain forms of
bacteria are grown in gelatin the culture-medium is
partly or entirely liquefied. This characteristic is en¬
tirely independent of any other properly of the bacte¬
rium, and is one manifested alike by pathogenic and
non-pathogcnic individuals. Sternberg and Bitter have
shown that if from a culture in which liquefaction has
taken place the bacteria be removed by filtration, the
filtrate will retain the power of liquefying gelatin, show¬
ing that the property is not resident in the bacteria, but
in some substance in solution in their excreted products.

PATHOGENIC BACTERIA.

These products are described as “ tryptic enzymes n by
Fermi, who found that heat destroyed them. Mineral
acids seem to check their power to act upon gelatin.
Formalin renders the gelatin insoluble. As some of
the bacteria not only liquefy the gelatin, but do so in a
peculiar and constantly similar manner, the presence or
absence of the change becomes extremely useful for the
separation of different species.

5. Production of Acids and Alkalies. — Under the head
of u Fermentation ’ ? the formation of acetic, lactic, and
butyric acids has been discussed. These, however, are
by no means all the acids resulting from microbic me¬
tabolism. Ziegler mentions formic, propionic, baldrianic,
palmitic, and margaric as being among those produced,
and even this list may not comprehend them all. As
the acidity due to the microbic metabolism progresses, it
impedes, and ultimately completely inhibits, the develop¬
ment of the bacteria. The addition of phenolphthalein
and litmus to the culture-medium is one of the best
methods for detecting the acids. Milk, to which litmus
is added, is particularly convenient. Rosalie acid may
also be used, the acid converting its red into an orange
color. The same tests will also determine the alkali-
production, which occurs rather less frequently than acid-
formation and depends chiefly upon the salts of ammo¬
nium.

6. Production of Gases. — This seems, in reality, to be
a part of the process of decomposition and fermentation.
Among the gases due to bacterial action, C02, H3S, NH4,
CH4, and others have been described. If the bacterium
be anaerobic and develop at the lower part of a tube of
gelatin, not infrequently a bubble of gas will be formed
about the colonies. This is almost constant in tetanus
and malignant edema. Ordinarily, the production or
liberation of gases passes undetected, the vapors escaping
from the surface of the culture-medium.

To determine the gas production where it is suspected
ut not apparent, the ordinary fermentation-tubes can be

BIOLOGY OF BACTERIA.

employed. They are filled with glucose bouillon, steril¬
ized as usual, inoculated and allowed to grow. If gases
are formed, the bubbles ascend and the
gas accumulates at the top of the tube.

In estimating quantitatively, one must
be careful that the tube is not so con¬
structed as to allow the gas to escape as
well as to ascend in the main reservoir.

For the determination of the nature
of the gases ordinarily produced, some
of which are inflammable and some not,

rn;. — Smith s firr-

Theobald Smith has recommended the mcntutiun-tubc.
following methods:

“The bulb is completely filled with a a per cent, so¬
lution of sodium hydroxid (NaOII) and tightly closed
with the thumb. The fluid is shaken thoroughly with
the gas and allowed to flow back and forth from the bulb
to closed branch, and the reverse several times to insure
intimate contact of the C(k with the alkali. Lastly,
before removing the thumb all the gas is allowed to col¬
lect in the closed branch so that none may escape when
the thumb is removed. If CO* be present, a partial
vacuum in the closed branch causes the fluid to rise sud¬
denly when the thumb is removed. After allowing the
layer of foam to subside somewhat the space occupied by
gas is again measured, and the difference between this
amount and that measured before shaking with the
sodium hydroxid solution gives the proportion of C()2
absorbed. The explosive character of the residue is
determined as follows: “The cotton plug is replaced and
the gas from the closed branch is allowed to flow into the
bull) 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.”

7. Production of Odors. — Of course, such gases as H2S
and NFL are sufficiently characteristic to be described as
odors. There are, however, a considerable number of

PATHOGENIC BACTERIA.

pungent oders which seem dependent purely upon odor¬
iferous principles dissociated from gases. Many of them
are extremely unpleasant, as the onion-like odor of the
tetanus bacillus. The odor does not have any direct rela¬
tion to decomposition, but, like the colors and acids,
seems to be a peculiar individual characteristic of the
metabolism of the organism.

8. Production of Phosphorescence. — A Bacillus phos-
phorescens and numerous other organisms have a dis¬
tinct phosphorescence associated with their growth. It
is said that so much illumination is sometimes caused by
a gelatin culture of some of these as to enable one to tell
the time by a watch. Most of them are found in sea¬
water, and are best grown in sea-water gelatin.

9. Production of Aromatics. — The most important of
these is indol, which was at one time thought to be pecu¬
liar to the cholera spirillum. For the method of deter¬
mining its presence, see “Dunham’s Solution. ” At pres¬
ent we know that a variety of organisms produce it, and
that it and phenol, kresol, hydrochinon, hydroparacuinaric
acid, and paroxy-phenylic-acetic acid are by no means
uncommon.

10. Reduction of Nitrites. — A considerable number of
bacteria are able to reduce nitrites present in the soil or
m culture-media prepared for them into ammonia and
nitrogen. To the horticulturist this is a matter of much
interest. Winogradsky has found a specific nitrifying
bacillus m soil, and asserts that the presence of ordinary
bacteria in the soil causes the reduction of no nitrites so
long as his special bacillus is withheld

BIOLOGY OF BACTERIA .

Milk usually contains bacteria, entering it from the
dust of the dairy, possessing this power. In the process
of peptonization the milk may become bitter, but need
not change its original reaction. As the peptonization
progresses the milk very often becomes poisonous, espe¬
cially to individuals under two years of age, and may
bring about a fatal enterocolitis or “summer complaint. ”
The disease does not only occur in consequence of toxic
substances formed from the split-up albumins, or from
the presence of metabolic products of the bacteria, but,
as Liibbert has shown,1 from the presence of the bacteria
themselves. One reason that the enterocolitis caused in
this way comes on in summer is that it is only in un¬
usually warm weather that these bacteria are able to
grow luxuriantly.

Sometimes the properties of coagulation and digestion
of milk are valuable aids in the separation of different
species of bacteria.

12. Production of Disease. — Bacteria which produce
diseases are known as pathogenic ; those which do not,
as non-pathoge7iic. Between the two groups there is no
sharp line of separation, for true pathogens may be culti¬
vated under such adverse conditions that their virulence
will be entirely lost, while at times bacteria ordinarily
harmless may be made toxic by certain manipulations or
by introducing them into animals in certain combina¬
tions. The diseases produced are the result of the sum
of numerous activities exhibited by the bacteria. For
example, it may be that a microbe, having effected its
entrance into an animal, grows with great rapidity,
completely blocking up the blood- and lymph-channels,
so that the proper circulation of these fluids is stopped
and disease and death must result. Perhaps more com¬
mon than this is a local establishment of the organisms,
with a resulting inflammation, due partly to the presence
of the foreign organisms, and partly to their toxic me¬
tabolic products. More often, however, the pathogenic

1 Zeitschrift fur Hygiene , xxii., Heft 2, 1896, p. 1.

PATHOGNEIC BACTERIA .

bacteria produce powerful metabolic poisons — toxins*
ptomaines, etc. — which either cause widespread destruc¬
tion of the tissues immediately acted upon, or, circulating
throughout the organism, produce fever, nervous excita¬
tion, and a general overthrow of the normal physiological
equilibrium. These peculiarities serve to divide the bac¬
teria into

Phlogistic bacteria,

Toxic bacteria,

Septic bacteria.

The bacteria of suppuration probably act in several
ways. Their products may be of a violently chemotactic
nature, or their virulence, exerted upon the surrounding
tissue, may destroy large numbers of the cells, whose
dead bodies may be chemotactic. When the suppura¬
tion is violent the toxic product of the bacterium is itself
most probably strongly chemotactic.

The great majority of suppurations depend upon bac¬
teria, but there are sterile suppurations which sometimes
follow the use of croton oil, turpentine, etc. The differ¬
ence between infectious and sterile pus is marked, for
the former, containing the virulent germs, tends to invade
new tissue or distribute its disease-producers to new parts
of the body, while the latter remains local.

There are few purely toxic bacteria, the tetanus and
diphtheria bacilli serving as typical examples. By sep¬
tic bacteria, I mean those whose habitual tendency is to
grow in the blood and lymph and distribute to all the
organs. Anthrax is a type of the class.

How the disease-producing bacteria effect their en¬
trance into the tissues is an interesting question. The
channels^ naturally open to them are those leading into
the interior of the organism, and must be separately con¬
sidered.

(a) The Digestive Tract. — Attention has already been
called to the facility with which the bacteria enter the
digestive tract in foods and drinks. Once their metabo¬
lism is m active progress, the poisons which they produce

BIOLOGY OF BACTERIA .

are ready for absorption. It seems probable that the
absorption of the toxic substances by reducing the vital¬
ity of the individual predisposes to the formation of local
lesions through which the bacteria may enter the intes¬
tinal walls to continue their existence and produce
greater damage than before. Some such theory may
explain the activity of such organisms as those of
typhoid, cholera, and meat-poisoning, but it is not true
that all bacteria can be admitted into the intestinal struc¬
ture in this way, for the experiments of Max Neisser,1 who
fed mice, guinea-pigs, and rabbits upon a variety of
pathogenic and non-pathogenic bacteria, both before and
after injuries to the intestine caused by the ingestion of
powdered glass, chemical agents, and irritating bacteria,
failed to show that with the exception of those bacteria
whose particular tendency is toward the production of
intestinal disease, none entered either the chyliferous
system, the blood-vessels, or the organs.

The occurrence of the staphylococcus aureus and other
bacteria in osteomyelitis, and of tubercle bacilli in deep-
seated diseases of the bones and internal organs, has led
many to believe that the intestine is a point of easy
entrance. There is, however, no reason to believe that
penetration of the digestive mucous membrane is any
easier than that of the respiratory or other similarly deli¬
cate tissues.

On the other hand, Beco2 is of the opinion that, with¬
out any apparent lesion of the intestine, bacteria — ba¬
cillus coli — escape from it into the blood during life.
His experiments showed that immediately after death the
colon bacillus could be found in small numbers in the
spleeu, in many cases. After twenty-four hours, in
three cases, they were present in immense numbers.
When, however, they were absent from the organ im¬
mediately after death, they were also absent after twenty-
four hours.

1 Zdtschrift fur Hygiene, June 25, 1S96, Ikl. xxii., Heft x.

1 Ann. tie I'Inst. Pasteur, 1S95, No. 3.

PATHOGENIC BACTERIA.

Achard1 studied 49 cases to determine whether or not
the intestinal bacteria entered the organism during the
death agonv. In 14 bacteria were found zntra vitciwi in
the liver and in the blood. In 24 no bacteria were found
during life, but after death. In 11 no bacteria were
found either during life or after death — before twenty-two
to twenty-seven hours, when his autopsies were made.
The passage of bacteria into the blood during agony was
unusual. The bacteria most commonly found during life
were the streptococci and staphylococci. In the dead body
the one most frequently encountered was the bacillus coli
coinmunis. Before reaching the intestine the bacteria
pass through the stomach, and must resist the deleterious
action of the acid gastric juice, which few are able to do.

(b) The Respiratory Tract . — Notwithstanding the moist
interiors of the mouth and nose and the lashing cilia of
the pharyngeal and tracheal mucous membrane, numbers
of bacteria enter the smaller bronchioles, and occasionally
penetrate as deeply as the air-cells. It is usual to find a
few bacteria in a section of healthy lung.

Thomson and Hewlett2 estimate that from 1500 to
14,000 bacteria are inspired every hour. As expired air
is usually sterile, they sought to determine what became
of these organisms, and agree wfith Lister and with
Hildebrandt that the organisms are arrested before they
reach the air-cells. They found by killing a number of
animals and examining the tracheal surface that it was
sterile, and conclude that the great majority of bacteria
are stopped in the nose against the moist surfaces of its
vestibules, where they found great numbers in the crusts.
No doubt the ciliated cells of the nose have something to
do with getting rid of the bacteria.

An ingenious experiment was performed by placing
some bacilli prodigiosus upon the septum naris, and
making a culture from the spot at intervals during two

1 Archives de medecine experimentale et d’antomie pathologique, 1895, No.
1, p. 25.

2 British Med. Jour., Jan. 18, 1896, p. 137.

BIOLOGY OF BACTERIA.

hours. Cultures made within five minutes showed con¬
fluent colonies of the bacilli, which became fewer and
fewer in number, until after two hours not a trace of a
bacillus prodigiosus could be found.

Wurtz and Lermoyez assert that the nasal mucus exerts
a germicidal action, but this is not substantiated. These
writers conclude that the bacteria were carried away by
the action of the cilia and trickling mucus.

It seems to have been proven by Buchner that micro-
organismal infection may take place through the lungs
without definite breach of continuity of the alveolar
walls. He mixed anthrax spores and lycopodium powder
together, and caused mice and guinea-pigs to inhale them.
Out of the 66 animals used in his experiments, 50 died of
anthrax and 9 of pneumonia. Our knowledge of the dis¬
position of foreign particles in the lung probably explains
such infection by assuming that the presence of the lyco¬
podium attracted numerous leucocytes to the affected air-
cells; that these took up the powder, and with it the
spores; and that the leucocytes, being cells of very sus¬
ceptible animals, were unable to resist the growth into
bacilli of the spores which they had carried into the
lymph-channels.

On the other hand, it has been shown that when
the entering spores are unaccompanied by a mechanical
irritant like the lycopodium powder, but are inspired
in a pulverized liquid, infection takes place much less
readily.

Tuberculosis and pneumonia are in all probability
generally the result of the inspiration of the specific
organisms.

(f) The Skin and the Superficial Mucous Membranes. —
The entrance of bacteria into the tissues by way of the
skin is probably extremely rare if the skin is sound.
Numerous experimenters have caused infection by rub¬
bing bacteria or their spores upon the skin. It would
seem probable that in these cases there must have
been some microscopic lesions into which the bacteria

62 PATHOGENIC BACTERIA:

were forced. My own investigations have shown viru¬
lent staphylococci of suppuration upon the conjunctive
in health. It is very improbable that the bacteria habit¬
ually present upon the skin, and ready to enter the least
abrasion, can penetrate the outer coverings of the body,
except when disease or accident • has rendered them
abnormally thin or macerated.

Turro seems to have shown that the gonococcus can
enter the tissues without any pre-existing lesion, for he
asserts that if a virulent culture simply be touched to
the meatus urinarius, the disease will be established.

(d) Wounds.— The results of the entrance of bacteria
into unprotected wounds are now so familiar that no
one deserving of the name of surgeon dares to allow a
wound to go undressed.

(e) The Placenta.— Very frequently the occurrence of
specific diseases during pregnancy causes abortion of
the product of conception. In certain cases the specific
contagion passes through the placenta and infects the
fetus. This has been pretty clearly demonstrated for
variola, malaria, syphilis, measles, anthrax, symptomatic
anthrax, glanders, relapsing fever, typhoid, and in rare
cases for tuberculosis.

Anche found streptococci and staphylococci in the tis¬
sues of aborted fceti in cases of variola.1 Except in the
case of wounds, it must be observed that, although the
bacteria are in the body — i. e ., respiratory, digestive, or
sexual apparatus, etc.— they are still not in the blood, and
really not in, but only upon the surfaces of the tissues.

For their actual entrance into the circulation, Kruse2
gives the following possible modes:

1. Passive entrance of the bacteria through the sto-
mata of the vessels where the pressure of the inflammatory
exudate is greater than the intravascular pressure.

2. Entrance of the bacteria into the vessel in the body
of leucocytes that have incorporated them.

1 La Semaine Med 1892, No. 61.

2 Fliigge’s Mikrodrganismcn.

BIOLOGY OF BACTERIA. 63

3. Actual penetration of the vessel-wall by the growth
of the microorganism.

4. Entrance into the vessels via the lymphatics, either
passively or in leucocytes.

Seeing that the channels by which bacteria can enter
the body are so numerous, and that there is scarce a
moment when some part of us is not in contact with
them, how is it that we are not constantly subject to
disease? The consideration of this question, together
with the closely related questions why we should be
subject to certain diseases only, and to these diseases
at certain times only, must be reserved for another chap¬
ter, in which the subjects Immunity and Susceptibility can
be taken up at length. Before passing on to it, however,
some attention must be paid to the subject of the

Elimination of Bacteria from the Body. — There is every
reason to think that nou-pathogenic bacteria entering the
body ordinarily, or being experimentally injected into it,
follow the same course as inert, non-vital particles; con¬
cerning which, the experiments of Siebel have shown
that they accumulate in the finest capillaries, especially
in the lung, liver, spleen, and bone-marrow, and are
slowly transferred to the surrounding tissues, either to be
collected in the connective-tissues, carried to the lym¬
phatic nodes, or to be subsequently excreted with the
bile, succus entericus, etc., or to be discharged from the
surface of the mucous membranes, pulmonary alveoli,
tonsils, etc. They also escape from suppurating wounds
to which they may be carried by leucocytes. They are
not excreted by the kidneys.

The experiments of Wyssokowitsch are in accord with
the results of SiebePs work, and show that the kidney
rarely eliminates bacteria. Cavazzani found that the
kidney had the power to retain bacteria in the blood,
unless the epithelium was injured.

The principal avenues of escape for the bacteria are,
therefore, for the non-patliogenic forms, the mucous mem¬
branes, the bile, and the sweat. For the pathogenic

PATHOGENIC BACTERIA .

forms, the mucous membranes, the intestine in particular
in such diseases as anthrax, typhoid, and cholera; the bile
almost always; the sweat generally; the kidney when
damaged; the mammae in tuberculosis and septicemia
particularly, and, of course, such of the pathological
products of the disease-process as pus from abscesses,
dejecta of typhoid and cholera, expectoration in diph¬
theria and tuberculosis, etc.

The bacteria that are not excreted, but retained in such
organs as the spleen, bone-marrow, and lymphatic nodes,
are probably slowly devitalized and dissolved.

CHAPTER III.

IMMUNITY AND SUSCEPTIBILITY.

One of the most interesting tilings observed in physi¬
ology and pathology is the resistance which certain ani¬
mals show to the invasion of their bodies by the germs
of disease.

Tims, man suffers from typhoid fever, cholera, and
other infectious diseases which are never observed in the
domestic animals; cattle are subject to a pleuro-pneunio-
nia which does not affect their attendants; man, the cow,
and the guinea-pig are peculiarly susceptible to tubercu¬
losis, which the cat, dog*, and horse resist; yellow lever
is a highly contagious, infectious disease which is almost
certain to attack all new arrivals of the human species
when epidemic, but which rarely, if ever, attacks animals.

The popular mind accepts the statement of such facts
as these without any other explanation than that the
animals are different, and so of course their diseases are
different; but the more the scientific man contemplates
them, the more complicated the matter becomes; for,
while it might be admitted that a difference in the body-
temperature and chemistry might explain why a frog-
will resist anthrax, which readily kills a white mouse, it
will not explain why a house-mouse, whose chemistry
must be almost identical with that of the white mouse,
can successfully combat the disease. Nor is this all.
That one attack of yellow fever, of typhoid fever, or
of scarlet fever renders a second attack almost impos¬
sible is not the less interesting because of its every-day
observation. The mouse that has recovered from teta¬
nus will not take tetanus again, and most interesting and

5 ID

PATHOGENIC BACTERIA .

extraordinary is the fact that a few drops of blood from
the recovered mouse injected into another will protect it
from tetanus.

Immunity is the condition in which the body of an
animal resists the entrance of disease-producing germs,
or, having been compelled to allow them to enter, resists
their growth and pathogenesis. The resistance so mani¬
fested is a distinct, potential vital phenomenon.

Susceptibility is the opposite condition, in which, in¬
stead of resistance, there is a passive inertia which allows
the disease-producing organisms to develop without oppo¬
sition. Susceptibility is accordingly the absence of im¬
munity.

Immunity is either natural or acquired.

Natural Immunity. — By this term is meant the natural
and constant resistance which certain healthy animals
exhibit toward certain diseases.

The white rat is peculiar in resisting anthrax. It is
almost impossible to develop anthrax in a healthy white
Tat, but Roger found that such an animal would easily
:succutnb to the disease if compelled to turn a revolving
wheel until exhausted. Susceptibility which follows such
an exhaustion of the vital powers cannot be regarded as
other than accidental, and makes no exception to the
statement that the white rat is immune to anthrax.
Animals such as man, sheep, cows, rabbits, and white
mice are susceptible to anthrax, while birds and reptiles
are generally immune. The great difference in the morph¬
ology between mammals and birds and reptiles, together
with the fact that their temperature, blood, and tissues
all differ, makes this immunity reasonably intelligible.
Morphological differences, however, will not suffice to
explain all cases, for the Caucasian nearly always suc¬
cumbs to yellow fever, while the negro is rarely affected ;
and scarlatina, which is one of our commonest and most
dangerous diseases of childhood, is said to be unknown
among the Japanese. Nor is this all, for, close as is their
resemblance in all respects except color, the house-mouse,

IMMUNITY ANI) SUSCEPTIBILITY. * 67

field-mouse, and white mouse differ very much in their
susceptibility to various diseases.

Acquired immunity is resistance which is the result
of accidental circumstances. It may result —

A. By recovery from a mild attack of the disease.
Most adults have suffered from rubeola, scarlatina, and
varicella in childhood, and in consequence of the attacks
are now immune to these diseases — /. e. will not become
affected again. One attack of yellow fever is always a
complete guard against another. Typhoid fever is rarely
followed by a second attack.

B. By recovery from an attack of a slightly different
disease. Sometimes the immunity is experimentally pro¬
duced, as when by vaccination we produce the vaccine
disease and afterward resist variola. Acquired immunity
is a little less complete and not so permanent as natural
immunity, for in the latter it is only when the functions
of the individual are disturbed or his vitality depressed
that the resistance is lost, while in the former time seems
to lessen the power of resistance, so that rubeola and
scarlatina may return in a few months or years, and for
complete protection vaccination may need to be done as
often as every seven years.

C\ By the injection of antitoxic substances. At
present there is much agitation over the newlv-dis-
covered antitoxin of diphtheria, the injection of about
500 units of which will give complete protection against
the disease for a period lasting from a month to six
weeks.

Immunity may be destroyed in numerous ways:

(a) By variation from the normal temperature of the
animal under observation. Pasteur observed that chick¬
ens would not take anthrax, and suspected that this
immunity might be due to their high body-temperature.
After inoculation he plunged the birds into a cold bath,
reduced their temperature, and succeeded in destroying
their immunity. The experiment was a success, but the
reasoning seems to have been faulty, as the sparrow,

PATHOGENIC BACTERIA.

with a temperature equally high, readily falls a victim
to anthrax without a cold bath.

(b) By altering the chemistry of the blood by changing
the diet or bv hypodermic injection. Leo found that
when white rats were injected with or fed upon phlorid-
zin an artificial odvcosuria resulted which destroyed their

O - '

natural resistance to anthrax. Hankin found that rats,
which possess considerable immunity to anthrax, could
be made susceptible by a diet of bread. Platania suc¬
ceeded in producing anthrax in dogs, frogs, and pigeons,
naturally immune, by subjecting them to the influence
of curare, chloral, and alcohol.

(c) By diminishing the strength of the animal. Roger
by compelling white rats to turn a revolving wheel until
exhausted destroyed their immunity to anthrax.

( d ) By removing the spleen (?). A large number of
experiments have been performed by various investi¬
gators to show that the removal of the spleen does or
does not affect immunity. From their work it seems
proper to conclude that the spleen has little, if any, in¬
fluence upon the vital resistance to disease.

I. Bardach,1 Righi,2and Montuori3 seem to have shown
that the removal of the spleen lessens the ability of the
organism to combat the infections.

II. Blumenreich and Jacoby,4 on the contrary, found
that the removal of the spleen was followed by a hvper-
leucocytosis, an increase in the bactericidal power of the
blood, and consequent increase of immunity.

III. Milkinow-Raswedenow5 found that the removal of
the spleen was a weakening factor in the immunization
of animals. The spleen itself, however, was of little
importance in combating the micro-organismal infections.

Kurlow 6 concluded from his experiments that the in-

1 Ann. de l' Inst. Pasteur , 1889, No. 2, p. 577, and 1891, No. I, p. 40.

2 To Riforma JMedica , 1893, PP* 170, 17 1.

3 Ibid., Feb., 1893, 17, 18. 4 Berlin, klin. Wochenschrift , May 24, 1807.

0 Zeitschrift fur Hygiene, 1896, xxi., 3.

6 Archtv fiir Hvg., 1889, Bd. ix., p. 450.

IMMUNITY AND SUSCEPTIBILITY 69

fluence of the spleen was not greater than that of any
other organ in overcoming bacterial infections.

Kanthack1 found that the removal of the spleen had
practically no influence upon the natural immunity of
animals to pyocyaneus infection.

(c) By combining Deo different species of bacteria, either
of which, when injected alone, would be harmless or of
-slight effect. Roger found that when animals immune
to malignant edema were simultaneously injected with
1 to 2 c.cm. of a culture of Bacillus prodigiosus and the
bacillus of malignant edema, they would contract the
disease. Pawlowski found that when rabbits, which
are very susceptible to anthrax, were simultaneously in¬
jected with anthrax and prodigiosus, they recovered
from the anthrax, as if the harmless microbe possessed
the power of neutralizing the products of the patho¬
genic form.

Giarre found that if an adult guinea-pig, which is refrac¬
tory to infection by pneumococci, were simultaneously in¬
oculated with diphtheria, it readily died of septicemia.

Sometimes an apparent immunity depends upon the
attenuation of the culture used for inoculation, and the
erroneous results to which such a mistake may lead arc
obvious. Should a culture become attenuated, its viru¬
lence may sometimes be increased by inoculating it into
the most susceptible animal, then from this to a less
susceptible, and then to an immune animal. The viru¬
lence ot anthrax is increased by inoculation into pigeons,
and also by cultivation in an infusion of the tissues of
an animal similar to the one to be inoculated.

It must be understood that the term u immunity 11 is
a relative one, and that while ua white rat is immune
against anthrax in amounts sufficiently large to kill a
rabbit, it is perhaps not immune against a quantity
sufficiently large to kill an elephant.”

It is not to be expected that such intricate phenomena
as these which have been mentioned could be observed

1 Cvntralbl. f. Balt, u. TanuiU'uk., 1S92, xii., p. 227.

jo PA TH0GEN1C BA CTERIA .

and suffered to go unexplained. We have explanations,
but, unfortunately, they areas intricate as the phenomena,
and, though each may possess its grain of truth, not one
will satisfy the demands of the thoughtful student. In
brief review, the theories of immunity are the following :

1. The Exhaustion Theory.— This hypothesis was
advanced by Pasteur in 1880, and suggests that by its
growth in the body the micro-organism uses up some
substance essential to its life, and that when this sub¬
stance is exhausted the microbe can no longer thrive.
The removal of the necessary material, if complete, will
cause permanent immunity.

As Sternberg points out, were this theory true we must
have within us a material of small-pox, a material of
measles, a material of scarlet fever, etc., to be exhausted
by its appropriate organism. It would necessitate an
almost inconceivably complex body-chemistry and a
rather stable condition of the same.

2. The Retention Theory. — In the same year
Chauveau suggested that the growth of the bacteria
in the body might originate some substance prejudicial
to their further and future development. There seems
to be a large kernel of truth in this, but were it always
the case we would have added to our blood a material
of small-pox, a material of measles, a material of scarlet
fever, etc., so that we would become saturated with the
excrementitious products of the bacteria, instead of hav¬
ing so many substances subtracted from our chemistry.

3. The Theory of Phagocytosis. — In 1881, Carl
Roser suggested a relation between immunity and the
already familiar phenomenon of phagocytosis. Stern¬
berg in the United States and Koch in Germany observed
the same thing, but little real attention was paid to the
subject until 1884, when Metschnikoff appeared, with his
careful observations upon the daphnia, as the great cham¬
pion of the theory which is now known as “ Metschni¬
koff ’s theory of phagocytosis. n

Phagocytosis is the swallowing or incorporating of

IMMUNITY AND SUSCEPTIBILITY. 7 1

particles by certain of the body-cells which are called
phagocytes. This activity of the cells toward inert
particles had been observed by Virchow as early as 1840,
and toward living bacteria by Koch in 1878, but was not
carefully studied until 1884. Metsclmikoff divides the
phagocytes into fixed phagocytes, comprising the fixed
connective-tissue cells, endothelium, etc., and the free
phagocytes , which are the leucocytes. The terms u phag¬
ocyte” and u leucocyte” are not to be regarded as synon¬
ymous in this connection ; all leucocytes are not phag¬
ocytic, the lymphocyte having never been observed to
take up bacteria.

It is obvious that only those cells can be phagocytic
which are without a resisting cell-wall and possess
ameboid movement. When an ameba, in a liquid con¬
taining numerous diatoms and bacteria, is watched
through the microscope, an interesting phenomenon is
observed. The ameba will approach one of the vege¬
table cells, even though it may be at a distance, will
apprehend and surround it, and within the animal cell
the vegetable cell will be digested and assimilated. The
ameba has no eyes, no nose, no volition, and, so far as
we can determine, no nervous apparatus which gives
it tactile sense, yet it will approach the particle fitted
for its use and swallow it. The attraction which draws
the cells together has been called by Peffer chemotaxis,
chemiotaxis , or chemotropism.

Chemotaxis is the exhibition of an attractive force
between cells and their nutriment, ameboid cells and
food-particles, and sometimes between ameboid cells and
inert particles. This attractive force, when operating so
as to draw the ameba to the particle it will devour, is
further named positive chemotaxis in order to distinguish
it from a repulsive force sometimes exerted causing the
ameboid cells to fly from an enemy, as it were, and which
is called negative chemotaxis.

The force that operates and guides the ameba in its
movements is exactly the same as that which governs the

/ ~

PATHOGENIC BACTERIA .

movement of the phagocytic cells of the human body,
and observation of these phenomena is not difficult. If
a small capillary tube be filled with sweet oil and placed
beneath the skin, only a short time need pass before it
will be found full of leucocytes — positive cliemotaxis.
If, instead of sweet oil, oil of turpentine be used, not
a leucocyte will be found — negative cliemotaxis.

Phagocytosis is almost universal in the micro-or-
ganismal diseases at some stage or another. If the
blood of a patient suffering from relapsing fever be
studied beneath the microscope, it will be found to
contain numerous active mobile spirilla, all free in the
liquid portion of the blood. As soon as the apyretic
stage comes on not a single free spirillum can be found.
Every one is seen to be enclosed in the leucocytes.

At the edge of an erysipelatous patch a most active
warfare is waged between the streptococci and the cells.
Near the centre of the patch there are many free strep¬
tococci and a few cells. At the margin there are free
streptococci, and also a great many streptococci en¬
closed in cells (leucocytes) which are, for the most part,
dead. In the newly-invaded tissue we find hosts of
active living cells engaged in eating up the enemies
as fast as they can. The phagocytologists tell us that at
the centre the bacteria are fortified, actively growing*, and
\ indent ; in the next zone the leucocytes which have
feasted upon the bacteria are poisoned by them ; outside,
the cells, which are more powerful and which are con¬
stantly being reinforced, are waging successful warfare
against the streptococci. In this manner the battle con¬
tinues, the cells now being obliged to yield to the bacteria
and the patch spreading, while the cells subsequently re-
inforce and destroy the bacteria, so that the disease comes

MetschnikofF introduced fragments of tissue from ani¬
mals dead of anthrax under the skin of the back of a fro-
and found it surrounded and penetrated by leucocvtes con¬
taining many of the bacilli. '

IMMUNITY AND SUSCEPTIBILITY.

It need scarcely be pointed out that a loophole of doubt
exists in all these illustrations: the bacteria may have been
dead before the cells ingested them, and the phenomena of
digestion and destruction which have gone on in their in¬
teriors may have been exerted upon dead bacteria. To the
relapsing-fever illustration we may take exceptions, and
state that the apyrexia may have marked the death of
the spirilla, which were taken up by the leucocytes only
when dead. In the erysipelas illustration the streptococci
remote from the centre of the lesion may have met from
the body-juices or some other cause a more speedy death
than that from the digestive juices of the leucocyte.

Metschnikoff, however, is prepared to show us that the
leucocytes do take up living pathogenic organisms. He
succeeded in isolating two leucocytes, each containing an
anthrax spore, and conveying them to artificial culture-
media, where he watched them. The new environment
being better adapted to the growth of the spore than for
the nourishment of the leucocyte, the latter died, and
the spore developed under his eyes into a healthy bacillus.
Seeing that the animal cells take up bacteria, and seeing
that the ameba can ingest and digest u threads of lepto-
thrix ten times as long as itself,” we need only put two
and two together to see that MetschnikofF’s theory rests
upon a very substantial foundation. The more virulent
the bacteria, the less ready the leucocytes are to seize
them. The more immune the animal, the greater is the
affinity of the leucocyte for the bacteria.

The organisms which are seized upon by the leucocytes
do not remain in the blood, but are collected in the spleen
and the lymphatic glands; and not the least important
fact in favor of phagocytosis is that observed by Bardach,
that excision of the spleen diminishes the resistance to
infectious disease.

Ouiniu also furnishes a therapeutic support to the
theory. It is known that quinin increases the destruc¬
tion of leucocytes. Woodliead inoculated a number of
rabbits with anthrax, giving quinin to some of them.

PATHOGENIC BACTERIA.

Those which had received the drug died earliest — a
result probably dependent upon the destruction of part
of the phagocytic army.

Ruffer found that the “ phagocytes evince a distinct
selective tendency between various kinds of organisms.
They will leave the bacillus of tetanus in order to seize
upon the Bacillus prodigiosus if simultaneously intro¬
duced ; also the streptococci in diphtheria for the Klebs-
Lcffler bacilli. This is illustrated in the diphtheritic
membrane, where at the surface one can see leucocytes
taking in numbers of the bacilli, but leaving the strepto¬
cocci almost untouched, with the immediate result that
streptococci are often found in the deeper parts of the
membrane, and with the remote result that secondary
abscesses occurring in the course of diphtheria are never
due to the bacillus of diphtheria, but to some other or¬
ganism. ’ ’

Hankin and Hardy found that the three varieties of
leucocytes in the frog’s blood play important parts in the
destruction of anthrax bacilli, this destructive process
being accomplished thus :

1. The eosinophile cells are first to approach and swal¬
low the bacteria. As this takes place the eosinophile
granules are seen to dissolve and act upon the bacteria.

2. The hyaline cells take up the remains of the bac¬
teria destroyed by the eosinophile leucocytes.

3. The basophile cells come to the field loaded with
basophilic granules, supposed to be antidotal to the
poisons of the bacteria, surround the combatants, neu¬
tralize the bacterial poisons, and liberate the contesting
cells.

Wyssokowitsch found that saprophytic micro-organ,
isms are quickly eliminated from the blood when in¬
jected into the circulation. This elimination is not
by excretion through organs nor by destruction in the
streaming blood, but by collection in the small capil¬
laries, where the blood-stream is slow and where the
micro-organisms are taken up by the endothelial cells.

IMMUNITY AND SUSCEPTIBILITY.

Wyssokowitsch found them most numerous in the liver,
spleen, and bone-marrow, and found that in these situa¬
tions they were destroyed in a short time — saprophytic
in a few hours, pathogenic in from twenty-four to forty-
eight hours. Spores of Bacillus subtilis remained as
living entities in the spleen for three months.

An interesting communication upon phagocytosis is
that of Bordet, whose experiments seem to show that the
lack of disposition to take up bacteria on the part of the
leucocytes may depend upon negative chemotaxis . He
found that when a guinea-pig became very ill after the
intraperitoneal introduction of a streptococcus of mild
virulence, if an injection of a culture of Proteus vulgaris
was given, the leucocytes, which had steadily refused to
take up the streptococci, seized upon the bacilli with
avidity. This seems to show that a chemical, or other
negative, or inhibitory influence felt by the leucocyte,
prevents it from taking up all the bacteria that come
within reach.

4. The Humoral Theory. — It was observed that if
anthrax bacilli were introduced into a few drops of
rabbit’s blood, they were instantly killed. This obser¬
vation was one of immense importance, and from it and
similar observations Buchner deduced the principles of
his theory, which teaches that the destruction of patho¬
genic bacteria in the body is due to the bactericidal
action of the blood-plas?7ia , not to phagocytosis, which
phenomenon amounts to nothing more than the burial
of the dead bacteria in “cellular charnel-houses.” The
experiments of Buchner and his followers, conspicuous
among whom is Nuttall, have shown that freshly drawn
blood, blood-plasma, defibrinated blood, aqueous humor,
tears, milk, urine, and saliva possess marked destructive
influence upon the organisms brought in contact with
them — an influence easily destroyed by heat.

The apparent paradox of rapid multiplication of an¬
thrax bacilli in the rabbit’s blood enclosed in the rabbit’s
body, and the reversed action in the test-tube, caused im-

PATHOGEJV/C BACTERIA .

mediate and prolonged opposition to the theory. Each side
of the question seemed well supported. The phagocytolo-
gists, however, showed that bacteria were often injured
and their vegetative powers destroyed by sudden changes
from one culture-medium to another, this being proved
bv Haffkine, who in experimenting with aqueous humor
has shown that its germicidal actions are largely imagin¬
ary, and due to the dispersion of the organisms in a large
amount of watery liquid. When the micro-organisms
are introduced into it in such a manner as to remain

together, they grow well. If the tube be shaken, so as
to distribute them, they die. Again, Adami has shown
that when blood is shed there is almost always a pro¬
nounced destruction of corpuscles, and suggests that the
antibiotic property of the shed blood may be due to
solution of the nucleins formerly in the substance of the
leucocytes. Jetter endeavored to prove the germicidal
action of the serum to be due to certain salts which it
contained. His experiments, which consisted in observ¬
ing the action of solutions of various salts in mixtures
ot water, glycerin, and gelatin, were justly condemned
by Buchner on the ground that such mixtures, though
they might contain constituents of blood-serum, were far
from approximating the normal serum in composition.

Wyssokowitsch, however, surely argued against hu¬
moral germicide when he showed that the spores of Ba¬
cillus subtilis could reside in the spleen for three months
uninjured.

In supporting their theory the humoralists experimented
\ placing beneath the skin micro-organisms enclosed in
iiUle b.ap of pith, collodium, etc. These bags allowed
the fluids of the body free access to the bacteria, but
would shut out the phagocytes. By these means Hiippe
and Lubarsch have repeatedly seen the bacteria grow
well while the attempts of Baumgarten have failed
Such experiments are by no means conclusive, for we
on remember that the operation necessary and the
presence of the foreign body in which the bacteria are

IMMUNITY AND SUSCEPTIBILITY .

encased produce an inflammatory transudate which may
have properties very different from those of the normal
juices.

How much of the immunity which animals enjoy de¬
pends upon the antibacterieidal action of their body-
juices must remain an open question. In some cases the
germicidal action of the blood seems to be unquestion¬
able. Buchner has shown that the blood-serum of ani¬
mals only possesses this germicidal power when freshly
drawn, and that exposure of the serum to sunlight, its
mixture with the serum from another species of animal,
its mixture with distilled water or with dissolved cor¬
puscles, and heating it to 55 0 C\, check the bactericidal
power. Buchner also points out that the bactericidal
and globulicidal actions of the blood are simultaneously
extinguished. Meltzer and Norris1 found that lymph
taken from the thoracic duct of the dog possessed marked
bactericidal powers upon the typhoid bacillus.

The experiments of Pfeiffer seem to add additional
support to the humoral theory of immunity. He found
that when guinea-pigs were given experimental choleraic
peritonitis, they could be saved from death from the affec¬
tion by intraperitoneal injection of serum from an
immunized animal. He also showed that when the cul¬
ture of cholera, or a culture of typhoid bacilli, was in¬
jected into the peritoneum of a guinea-pig, the multipli¬
cation of the bacteria was rapid. If, however, a lew
drops of the immunized scrum were introduced, a marked
effect was observed, for the serum seemed to exert a
germicidal effect upon the bacteria, and transform them
from living entities into inanimate little granular masses.

Hankin is of the opinion that the germicidal sub¬
stances of the blood-serum are derived from the eosiuophile
cells, and resides in the matter forming the eosin-granules.

Lowit,2 in investigating the bactericidal power of the

1 Journal of Experimental Medicine , vol. ii„ No. 6, p. 701, Nov., 1S07.

2 Bietr&gc zur Pathol. Anatomic uml zur AHgcm. Pathologic, Pel. xxih, II .
1, p. 173-

PATHOGENIC BACTERIA .

/ ^

blood in relation to its leucocytes, found that when a
marked experimental livpoleucocytosis was produced, the
bactericidal power of the blood was markedly diminished.
The most interesting feature of his work was the discov¬
ery that bactericidal matter could be extracted from
crushed leucocytes, and that it could be subjected to a
temperature of 6o° C. without change, thus differing
markedly from the alexins.

Much discussion has arisen as to exactly what the pro¬
tective substances are. Buchner has applied the term
a/txin to the protective proteid substances found in the
blood of naturally immune animals. Hankin has given
us, together with an extension of Buchner’s idea, a con¬
siderable nomenclature of somewhat questionable utility.
He divides the protective substances (alexins) into sozhts,
which occur in the blood of animals with natural immu¬
nity, and phylaxins , which occur in the blood of animals
with acquired immunity. Both sozins and phylaxins are
divisible into two groups — thus: a sozin which acts de¬
structively upon bacteria is called a myco-sozin ; one.
which neutralizes bacterial poisons, a toxo-sozin. A phy-
laxin which acts destructively upon bacteria is called a
myl o-pZiylcixin ; one which neutralizes bacterial toxins,
a toxo-phylaxin .

The anti-microbic serums obtained by Pfeiffer, Kollo,
Ivoffler, and Abel from dogs and other animals immunized
to typhoid fever belong in the group of myco-phylaxins.
The toxo-phylaxins are the antitoxins.

5. The Theory of Antitoxins.— It is a well-known
fact that individuals can accustom themselves to the use
oi certain poisons, as tobacco, opium, and arsenic, so as
o experience no inconvenience from what would be poi¬
sonous doses for other individuals. This is purely a
matter of tolerance, but is of interest in connection with
the observations winch are to follow.

Khrlich has shown that animals can tolerate gradually
increasing doses of ricin and abrin, provided that up o
a certain pomt „!e iMrease of do£ge veryt

IMMUNITY AND SUSCEPTIBILITY. 79

When this point is, however, safely passed, the increase
in dosage can be very rapid, yet without signs of poison¬
ing, seemingly because the drug is no longer simply tol¬
erated, but tolerated and simultaneously neutralized. By
experimentation Ehrlich has shown that during the
period of simple tolerance the blood of the animal is
unaltered, but that as .soon as the tolerance becomes
unlimited the blood contains a new substance, capable
not only of protecting the animal by which it is pro¬
duced, but also other animals into whose blood it is in¬
troduced. In the ricin experiments this substance was
described as antiricin ; in the experiments with abriu, as
an tiab riii.

These investigations of Ehrlich with the poisons of
higher plants succeeded, but threw much light upon, the
extraordinary work of Behring, Wernicke, and Kitasato,
who experimented with the toxins of diphtheria and
tetanus, and showed that in the blood of animals accus¬
tomed to these poisons, new substances — antitoxins, found
by Brieger to be proteid in nature — were produced.

The antitoxic theory of immunity, being, in the cases
cited at least, a fact capable of demonstration, has estab¬
lished itself at present as the most important hypothesis.
According to it, acquired immunity, at least, depends upon
the development in the blood of a neutralizing substance
probably related to the nucleins.

It is of prime importance to remember that the anti¬
toxin is an entirely new substance which does not occur
in the blood of normal animals, even when they possess
a high degree of natural immunity, except in rare in¬
stances, and then only in minute amounts not propor¬
tional to the degree of immunity. Calmette has called
special attention to this fact, and points out that while
fowls and tortoises resist abriu, their blood contains no
auti-abrin; Vaillard has shown that, although the fowl
resists tetanus, its blood contains no protective substance
destructive to tetanus-toxin. Calmette finds that the
blood of the ichneumon and hedgehog, which are ini-

PATHOGENIC BACTERIA.

mnne to serpent’s venom, contains some normal antitoxin,
but onlv in small amount.1 Fischl and v. Wunscliheini
found a small amount of a protecting substance in the
blood of newborn infants, which prevented the opera¬
tion of a fatal dose of diphtheria toxin upon guiuea-
pigs.2 •

Bolton "and the author have found some anti toxicity to
diphtheria present in the blood of normal (not experi¬
mentally immunized) horses.

The origin of the antitoxin is a very important and
interesting question. Is it in the blood, or in all the
body juices? Does it come from the leucocytes ? D/.erj-
gowskv ' has estimated the quantity of antitoxin con¬
tained in the blood and organs of horses immunized
against diphtheria. Of the constituents of the blood he
found (i) the fibrin has no antitoxic power; (2) serum
obtained normally and that got by expression from the
clot, from the plasma of the same blood, have an equal
antitoxic power; (3) the clot from the plasma, therefore,
does not retain the active principle; (4) the plasma and
the serum have an equal antitoxic power; (5) the red cor¬
puscles, compared with the plasma, contain traces only
of antitoxin; (6) serum containing the juice of the leuco¬
cytes is less rich in antitoxin than the plasma; (7) the
extract of the leucocytes contains relatively little anti¬
toxin, and the leucocytes themselves traces or none at all.
Hence the white blood-corpuscles cannot be the place
where the antitoxin is formed. The serous liquids con¬
tained in organs, such as the Graafian follicles, etc. con¬
tain as much antitoxin as the blood-serum— none of the

itself S C°ntam aS mnch of the autitoxin as the blood

Dzerjgowsky is of the opinion, held probably by a

Ann. de T I fist. Pasteur , x., 12
= Znuchriftflr Heilkunde, 1S95, *vi„ 429-4S2.

' Jour, of Experimental Medicine, vol. i No 7 Tulv

' ** **r. * /'/„»» „,l. 2*. , a „ ,

> tome V., Xos. 2 and 3, 1S97. * ' St' Peters'

IMMUNITY AND SUSCEPTIBILITY.

minority of scientists, that the antitoxin is the toxin in
a modified (oxidized ?) form, and supports his view by
the fact that the antitoxins are specific for their respec¬
tive toxins only, and by quoting the experiments of
Kondrevitsky, who, killing animals two hours after
an injection of toxin, found in the blood toxin alone ;
killing later, found some antitoxin, and still later much
antitoxin.

The difference between this theory of neutralization
by antitoxins and Cliaveaubs retention-hypothesis is quite
marked. The retention-theory teaches that a bacterium
leaves behind it a substance prejudicial to its future
growth in the economy — a distinct metabolic product.
The antitoxic theory shows the protective substance to
be a product not of bacterial growth, but of tissue-energy,
not depending upon the presence of the bacteria, but
upon the presence of a poison.

The antitoxins do not usually act harmfully upon the
bacteria, or preclude tlieir growth in the animal body, but
prevent their pathogenesis by annulling their toxicity —
z. <?., enabling the body-cells to endure the injury — and
placing them in a position exactly parallel with non-
pathogenic bacteria.

Closely related to the antitoxins, if not^identical with
them, are certain substances of an anti-infections nature
that can be generated in the blood of animals to which,
in the process of immunization, the bacteria, instead of
their poisons, have been administered. The anti-infec¬
tious serums are protective against the bacterial infections,
but powerless against the toxins. They are the only
results of immunization against cholera and typhoid
fever. When antitoxic serums can be secured they are
of far greater importance, and should always be selected
for purposes of therapeutics.

The diseases which are at present controllable by anti-
. toxins are toxic diseases, caused by the entrance of toxin-
producing bacteria into the body. The growth of these
toxin-producers probably depends upon the inability of
6

PATHOGENIC BACTERIA.

the body-cells or bactericidal body-juices to properly cope
with them, so that they develop and engender the poison¬
ous substances which are the essential factors of disease-
production. The more the body and its component ele¬
ments are injured, the more successful the inroads of the
bacteria, the more prolific the toxin-production, and the
more severe the affection.

The presence of the antitoxin annuls the poison, main¬
tains the vitality of the organism as a whole, sustains
the integrity of its tissues, and so places the pathogenic
bacterium on a very different footing in relation to the
organism.

An antitoxin is a neutralizing or annulling agent, not
a regenerating one, and therefore in therapeutics finds
its proper sphere only in the beginning of the disease
combated. Up to a certain point the symptoms of diph¬
theria and tetanus are due to the circulation of toxins in
the blood, and can be successfully combated by antitoxic
neutralization. Later in both diseases we have symp¬
toms resulting from disorganization of the nervous sys¬
tem, degeneration of the heart-muscle, destruction of the
kidneys, etc. , and the neutralization of the poison can be
of no avail because the lesions are irreparable, and the
patient must succumb.

I have used the term “ neutralization,” in speaking of
the antitoxins, in a rather free and scarcely warranted
manner, and must call attention to the fact that their
operation is probably not exactly analogous to chemical
neutralization. From mixtures of toxin and antitoxin
the unchanged poison has been recovered. The effect of
an antitoxin may be a biologic one, by which the tissues
are so stimulated as to endue the action of a substance
ordinarily disorganizing in effect.

Buchner and Roux have both pointed out that when the
toxins and antitoxins are mixed and introduced into ani¬
mals of greater susceptibility than are ordinarily used, the
presence of an unaltered toxin can easily be demonstrated.

This proof is, however, of very little value, for let the

IMMUNITY AND SUSCEPTIBILITY. 83

amount of toxin endurance of a resistent animal be repre¬
sented by x, and any addition to this as y. Then xy
would certainly be fatal. If the least quantity of anti¬
toxin that will protect the animal be expressed by z, then
xy + 2 is harmless. It is evident, however, that z does
not necessarily have any influence upon nr, but only need
neutralize y in order to save the animal, and therefore
it is obvious that the remaining x in such a mixture
could readily destroy another more susceptible animal
into which it might be injected.

I am of the opinion that the effect of the antitoxin
really partakes of the nature of chemic neutralization
from the following experiment: let a* represent the least
certainly fatal dose of diphtheria toxin for a guinea-pig,
and y the least quantity of antitoxin that will protect
against it; then

x + y is harmless. That

10 x + 10 y is also harmless is known to every one
accustomed to test antitoxins. I have con¬
tinued this and have found that
50 x + 50 y

100 + 100 y are also harmless.

According to Buchner, the antitoxins differ from the
alexins in being new substances in the blood, in being
without germicidal or chemical neutralizing power against
the toxins, and in being stable compounds which can
resist heat to 750 C., can resist a reasonable amount of
exposure to light, and which are not altered by decompo¬
sition of the substances containing them.

The antitoxins are specific for one poison only. Ehrlich
found that antiricin was powerless against abrin, and vice
versd. Diphtheria antitoxin is of no avail against tetanus,
and vice versd.

The immunity which the antitoxins produce is fuga¬
cious, varying considerably according to the particular
substance employed. As a rule, it is limited to a few
months — at least in the case of such antitoxins as we can
produce experimentally.

PATHOGENIC BACTERIA .

A new principle discovered by Pfeiffer, and bearing
directly upon the theories of immunity, is that the se¬
rum of animals immunized to certain diseases (cholera
and typhoid) contains a germicidal substance. Metchni-
koff has tried to show that the action of this body depends
upon solution of the leucocytes, but Pfeiffer has disproved
this by showing that the liquor puris from abscesses oc¬
curring in the experiment-animals did not contain the
active substance.1

The work of van de Velde2 is very interesting. An
animal immunized by progressively increasing doses of
strong filtered toxin produced a serum possessed of pow¬
erful anti-infectious and antitoxic powers; one immu¬
nized by the introduction into its body of the washed,
precipitated bodies of diphtheria bacilli collected by fil¬
tration furnished a serum of appreciable anti-infectious,
but no antitoxic properties; one immunized by the use
of bacillus cultures developed antitoxic and anti-infectious
serum identical with the first described; one immunized
to weak toxin furnished serum of considerable anti-infec¬
tious, but slight antitoxic power, and still another ani¬
mal that received toxin that had been heated developed
neither anti-infectious nor antitoxic serum.

Seeing that the serums commercially manufactured are
made by the use of strong filtered toxin, van de Velde
examined a number of samples purchased in the market,
and found that they were all possessed of both antitoxic
and anti-infectious properties. It is important to remem¬
ber the presence of both of these properties in the serum,
as the successful use of the agent for immunizing depends
upon the presence of the one, and the use in treatment
upon the presence of the other.

Immunity and antitoxins stand in unknown relation¬
ship to one another. That an animal has considerable
antitoxin in its blood is no guarantee that it is immune.
I have seen a horse in each c. cm. of whose blood there

1 Centralbl. f Bakt. u. Parasitenk ., Bd. xix., Nos. 14 and 15.

2 Ibid., Nov. 24, 1897, Bd. xxii.. Nos. 18 and 19.

IMMUNITY AND SUSCEPTJ HI UTY. 85

were 300 immunizing units of diphtheria antitoxin, die
of typical symptoms of diphtheria-poisoning after the ad¬
ministration of a comparatively small dose of the toxin.

From all that has gone before it must be clear to the
reader that no single theory thus far advanced can ex¬
plain immunity. Acquired immunity may depend in
the great majority of cases upon antitoxins, but as yet
we have no satisfactory explanation of natural immunity.
The humoral theory may be applicable in some cases ; in
others one cannot deny the importance of the role played
by the phagocytes.

CHAPTER IV.

METHODS OF OBSERVING BACTERIA.

Whoever would study bacteria must be equipped witli
a good microscope. The instruments generally provided
for the use of medical students in college laboratories, as
well as those seldom-employed u show microscopes ” seen
in physicians’ offices, are ill adapted for the purpose.
The essential features of a bacteriological instrument
are lenses giving a clear magnification extending as
high as one thousand diameters, and a good condenser
for intensifying the lights thrown upon the objects. It
naturally follows that the best work requires the best
lenses. The cheapest good microscope which is at pres¬
ent offered to the public is the BB. Continental stand,
made by Bausch and Tomb. This stand is provided with
everything necessary, is fitted with very creditable objec¬
tives, including an excellent TV' oil-immersion lens, and
seems capable of doing very good work. I do not
recommend this as the best instrument obtainable, but
as one that is both good and cheap. For those who desire
the ver}r best the rather costly outfits made by Carl Zeiss
of Jena are unexcelled.

For those who may begin the use of the Abbe con¬
denser and oil-immersion lenses without the advantage
of personal instruction a few hints will not be out of
place :

Always employ good slides without bubbles, and thin
cover-glasses; No. i are best.

Place a drop of oil of cedar upon the cover-glass of
the specimen to be examined ; rack the body of the instru¬
ment down until the oil-immersion lens touches the oil ;

METHODS OF OBSERVING BACTERIA. 87

keep on until it almost touches the glass, then look into
the microscope and find the object by slowly and firmly
racking up. As soon as the object comes into view
leave the rack and pinion and focus with the fine adjust¬
ment.

Always select the light from a white cloud if possible ;
if there are no white clouds, choose the clearest whitest
light possible. Never under any circumstances employ
sunlight \ which is ruinous to the eyes and useful only
for photomicrography.

In using low-power lenses the Abbe condenser must be
moved away from the object and the light modified by
the iris-diapliragm. The distance between condenser and
object should correspond more or less closely with the
distance between objective and object.

In using high powers the Abbe condenser must be
brought near the object and the light modified by the
iris-diaphragm.

If the oil-immersion lens is used, it is perhaps best to
employ the plane side of the mirror. When with this
lens a section of tissue is examined for details, the light
must be modified by the iris-diaphragm, opening and
closing it alternately until the best effect of illumina¬
tion is achieved. If tissue be searched for stained bac¬
teria, and no cellular detail is required, the diaphragm
should be wide open to admit a great flood of light
and extinguish everything except the deeply-colored
bacteria.

When unstained bacteria are to be examined with the
oil-immersion lens, the diaphragm should be closed so
as to leave only a small opening through which the
light can pass.

Bacteria may be examined either stained or unstained.
The former condition would always be preferable if the
process of coloring the organisms did not injure them.
Unfortunately, it is generally the case that the drying,
heating, boiling, macerating, and acidulating to which
we expose the organisms in the process of staining alter

■88

PATHOGENIC BACTERIA.

their shape, make their outlines less distinct, break up
their arrangement, and disturb them in a variety of other
ways. Because of the possible errors of appearance re¬
sulting from these causes, as well as because it must be
determined whether or not the individual is motile, in
making a careful study of a bacterium it must always be
examined in the living, unstained condition.

The simplest method of making such an examination
would be to take a drop of the liquid, place it upon a
slide, put on a cover, and examine.

While this method is simple, it cannot be recommended,
for if the specimen should need to be kept for a time
much evaporation takes place at the edges of the cover-
glass, and in the course of an hour or two has changed it
too much for further use. The immediate occurrence of
evaporation at the edges also causes currents of liquid to
flow to and fro beneath the cover, carrying the bacteria
with them and making it almost impossible to determine
whether the organisms under examination are motile or
not.

The best way to examine living micro-organisms is in
what is called the hanging drop (Fig. 6). A hollow-

Fig. 6. — The “ hanging drop ,J seen from above and in profile.

ground slide is used, and with the aid of a small camel’ s-
hair pencil a ring of vaselin is drawn on the slide about,
not in, the concavity at its centre. A drop of the mate¬
rial to be examined is placed in the centre of a large
clean cover-glass, and then placed upon the slide so

METHODS OF OBSERVING BACTERIA . 89

that the drop hangs in, but does not touch, the concavity.
The micro-organisms are now hermetically sealed in an
air-cliamber, and appear under almost the same con¬
ditions as in the cul¬
ture. Such a speci¬
men may be kept
from day to day and
examined, the bac¬
teria continuing to
live until the oxygen
or nutriment is ex¬
hausted. By means
of a special appara¬
tus (Fig. 7), in which
the microscope is
stood, the growing
bacteria may be
watched at any tem¬
perature, and very
exact observations
made.

The hanging drop
should always be ex¬
amined at the edge,
as the centre is too
thick.

In such a specimen
it is possible to de- Fig. 7. — Apparatus for keeping objects under
termilie the shape, microscopic examination at constant tempera
size, grouping, divis- tures-

ion, sporulation, and motility of the organism under
observation.

Care should be exercised to use a rather small drop,
especially for the detection of motility, as a large one
vibrates very readily and masks the motility of the
sluggish forms.

When the bacteria to be observed are in solid or semi¬
solid culture, a small quantity of the culture should be

PATHOGENIC BACTERIA .

mixed up in a drop of sterile bouillon or water and ex¬
amined.

Iii the early days of study efforts were made to facili¬
tate the observation of bacteria by the use of carmin and
hematoxylon. Both of these reagents tinge the proto¬
plasm of the organisms a little, but so unsatisfactorily
that since Weigert introduced the anilin dyes for the
purpose both of these tissue-stains have been rejected.
The affinity between the bacteria and the anilin dyes is
peculiar, and many times is so certain a reaction as to-
become an essential factor in the differentiation of
species.

For the study of bacteria in the stained condition we
now employ the anilin dyes only. These wonderful
colors, as numerous as the rainbow hues, are coal-tar
products. Hiippe classifies them as follows :

A. Dyes prepared from anilin oil.

1. Oxidation-products of pure anilin :

Methylene blue,

Chlorhydrin blue (basic indulin).

2. Oxidation-products of pure toluol :

Safranin.

3. Oxidation-products of mixed anilin and toluol :*

{a) Rosanilin. When pure this is triamido-
diplienyl-toluyl-karbinol.

Fuchsin — rosanilin hydrochlorate. It is
often mixed with the acetate and the
pararosanilin acetate and hydrochlo¬
rate. The pure rosanilin hydrochlorate*
should always be chosen for purposes of
staining.

Azalein is rosanilin nitrite.

Methylized and ethylized rosanilin :

Iodin violet,

Dahlia,

Iodin green.

(b) Pararosanilin. The colorless pure para¬
rosanilin is triamido-triphenyl-karbinol.

METHODS OF OBSERVING BACTERIA . 91

Rubi n-pararosan i 1 in hy drocli 1 orate.
Methylized, ethylized, and benzylized
pararosanilid :

Crystal violet,

Gentian violet,

Victoria blue,

Methyl green,

Auramin.

The rosanilins are more difficult to prepare
than the pararosanilins, and are generally
mixed with them. The pararosanilins
color more sharply than the rosanilins.

4. Amido-azo combinations :

Bismarck brown,

Phenylene brown,

Vesuvin.

5. Chiuolin derivatives :

Cyanin.

B. Naphthalin group. — Magdala red.

The best anilin dyes made at the present time, and
those which have become the standard for all bacterio¬
logical work, are made in Germany by Dr. Grubler. In
ordering the stain the name of this manufacturer should
always be specified.

A whole volume could easily be devoted to scientific
staining. Indeed, the technical difficulties encountered
are so great that no explanations can be too thorough to
be useful. The special methods essential for such bac¬
teria as have peculiar staining reactions will be given
with the description of the organism. General methods
only will be discussed in this chapter.

Cover-glass Preparations for General Examination.
— The material to be examined must be spread in the
thinnest possible layer upon the surface of a perfectly
clean cover-glass, and dried. Here it may be remarked
that for bacteriological purposes thin covers (No. 1) are
generally required, because thick glasses interfere with
the focussing of the oil-immersion lenses, and that cover-

PATHOGENIC BACTERIA .

o-lasses can never be too clean. It is best to immerse
them first in a strong mineral acid, then to wash them in
water, then in alcohol, then in ether, and keep them in
ether until they are to be used. Except that it some¬
times cracks, bends, or fuses the edges of the glasses, a
better and more convenient method of cleaning them is to
wipe them as clean as possible, seize them in fine-pointed
forceps, pass them repeatedly through a small Bunsen
flame until it becomes greenish yellow, then slowly ele¬
vate the glasses above the flame, so as to allow them to
anneal. This maneuvre removes the organic matter by
combustion. It is not expedient to use covers twice for
bacteriological work, though if well cleaned they may
subsequently be employed for ordinary microscopic ob¬
jects.

To return : After the material spread upon the cover
has dried, it must be fixed to the glass by immersion for
twenty-four hours in equal parts of absolute alcohol and
ether, or, as is much easier and more rapid, be passed
three times through a fla?ne. Three is not a magic
number, but experience has shown that when drawn
through the flame three times the desired effect seems
best accomplished. The Germans recommend that a
Bunsen burner or a large alcohol lamp be used, that the
arm holding the forceps containing the cover-glass in¬
scribe a circle a foot in diameter, and that, as each revo¬
lution occupies a second of time, the glass be made to pass
through the flame from apex to base three times. This
is supposed to be exactly the requisite amount of heating.
The rule is a good one for the inexperienced.

After fixing, the material is ready for the stain. Every
laboratory should be provided with several stoc solutions
of the more ordinary dyes. These stock-solutions are
saturated alcoholic solutions made by adding 25 grams
of the dye to 100 c.cin, of alcohol. Of these it is well to
have fuclisin, gentian violet, and methylene blue always
made up. The stock-solutions will not stain, but are the
standards for the manufacture of the working stains.

METHODS OF OBSERVING BACTERIA . 93

For ordinary staining an aqueous solution made in a
simple manner is employed. A small bottle is nearly
filled with distilled water, and the stock-solution is added,
drop by drop, until the color becomes just sufficiently in¬
tense to prevent the ready recognition of objects through
it. Such a watery solution possesses the power of readily
penetrating the dried protoplasm of the bacterium, taking
the stain with it. Alcohol does not have this power.

As in the process of staining the cover is apt to slip
from the fingers and spill the stain, it is well to be pro¬
vided with cover-glass forceps (Fig. 8), which hold the

Fig. 8. — Stewart’s cover-glass forceps.

glass in a firm grip and allow of all manipulations with¬
out danger to the fingers or clothes. The ordinary in¬
struments are entirely unfitted for the purpose, as capil¬
lary attraction draws the stain between the blades and
makes certain the soiling of the fingers. Sufficient stain
is allowed to run from a pipette upon the smeared side
of the cover-glass to flood it, but not overflow, and is
allowed to remain for a moment or two, after which it
is thoroughly washed off with water. If the specimen
is one prepared for temporary use, it can be examined at
once, mounted in a drop of water, but under these con¬
ditions will not appear as advantageously as if dried and
then mounted in Canada balsam.

Sometimes the material to be examined is too solid to
spread upon the glass conveniently. Under such circum¬
stances a drop of distilled water can be added and a minute
portion of the material be mixed in it upon the glass.

The entire process is, in brief :

i. Spread the material upon the cover ; 2. Dry — do not
heat ; 3. Pass three times through the flame ; 4. Stain

PA THO GENIC BA CTERIA .

two to three minutes; 5. Wash thoroughly in water;
6. Dry; 7. Mount in Canada balsam.

This simple process suffices to stain most bacteria.

Ohlmacher1 deserves credit for his observation that
when the u fixed ” preparation is immersed for a moment
or two in a 2-4 per cent, solution of formalin, the brill¬
iancy of the stain is considerably increased.

Staining Bacteria in Sections of Tissue. — It not
infrequently happens that the bacteria to be examined
are scattered among or enclosed in the cells of tissues.
Their demonstration is then a matter of some difficulty,
and the method employed is one which must be modified
according to the kind of organism to be stained. Very
much, too, depends upon the preservation of the tissue
to be studied. As bacteria disintegrate rapidly in dead
tissue, the specimen for examination should be secured
as fresh as possible, cut into small fragments, and im¬
mersed in absolute alcohol from six to twenty-four hours
to kill the cells and bacteria. Afterward they are re¬
moved from the absolute alcohol and kept in 80-90
per cent., which does not shrink the tissue. Bichlorid
of mercury may also be used, but absolute alcohol seems
to answer every purpose.

The ordinary ?nethods of imbedding suffice. The sim¬
pler of these are probably as follows:

I. Celloidin . — From the hardening reagent (if other
than absolute alcohol) —

12-24 hours in 95 per cent, alcohol,

6-12 u u absolute alcohol,

12-24 u u thin celloidin (consistence of oil),

6-12 u u thick celloidin (consistence of molasses).

The solutions of celloidin are made in equal parts of
absolute alcohol and ether.

Place upon a block of dry wood, allow to evaporate
until the block can be overturned without dislodging- the
specimen ; then place in 70-80 per cent, alcohol until

1 Medical News, Feb. 16, 1896.

METHODS OF OBSERVING BACTERIA .

ready to cut. The knife must be kept flooded with alco¬
hol while cutting.

II. Paraffin —

12-24 hours in 95 per cent, alcohol,

6-12 u u absolute alcohol,

4 u u chloroform, benzole, or xylol,

4-8 u “ a saturated solution of paraffin in one of
the above reagents. ^

Place in melted paraffin in an oven or paraffin water-
bath, at 40°-45° C., until the volatile reagent is all evap¬
orated, and the tissue impregnated with paraffin. Im¬
bed in freshly melted paraffin in any convenient mould.
In cutting, the knife must be perfectly dry.

When it is necessary, subsequently, to remove the im¬
bedding material, dissolve the paraffin in chloroform,
benzole, xylol, oil of turpentine, etc., which in turn can
be removed with 95 per cent, alcohol.

Celloidin is soluble in absolute alcohol, ether, and oil of
cloves. It is very convenient to fasten the cut sections upon
the slide — paraffin sections by oil of cloves and collodion
or gum arabic solution, celloidin sections by firmly pressing
filter paper upon them and rubbing hard, then allowing
a little vapor of ether to run upon them. ^

III. Glycerin-Gelatin . — As the penetration of the tissue
by celloidin is attended with lessened stainiug-qualities of
the tubercle bacillus, it has been recommended by Kolle 1
that the tissue be saturated with a mixture of glycerin, r
part; gelatin, 2 parts; and water, 3 parts; cemented to a
cork or block of wood, hardened in absolute alcohol and
cut as usual for celloidin with a knife wet with alcohol.

For staining bacteria (other than the tubercle and
lepra bacilli) in tissue, two universal methods can be
recommended:

Loffler’s Method. — The cut sections of tissue are
stained for a few minutes in Loffler’s alkaline methylene-
blue solution (q. v.)} and then differentiated in a 1 per

1 Fliigge’s Mikroorganisjnen.

PATHOGENIC BACTERIA .

cent, solution of hydrochloric acid for a few seconds.
The section is subsequently dehydrated in alcohol, cleared
up in xylol, and mounted in balsam, L- —

Pfeiffer’s Method. — The sections are stained for one-
lialf hour in diluted Ziehl’s carbol-fuchsin (q. v.\ then
transferred to absolute alcohol made feebly acid with
acetic acid. The sections must be carefully watched,
and as soon as the original, almost black-red color gives
place to a red violet color the section is removed to
xylol, where it is cleared preparatory to mounting in
balsam.

For ordinary work the following simple method is
recommended: After the sections are cut the paraffin
must be, and the celloidin had better be, removed.
From water the sections are placed in the same watery
stain used for cover-glasses and allowed to remain five
to eight minutes. They are next washed in water for
several minutes, then decolorized in 0.5-1 per cent,
acetic-acid solution. The acid removes the stain from
the tissues, and ultimately from the bacteria as well,
so that one must watch carefully, and as soon as the
color almost disappears from the sections remove them
to absolute alcohol. At this point the process may be
interrupted to allow the tissue-elements to be counter-
stained with alum carmin or any stain not requiring
acid for differentiation, after which the sections are
dehydrated in absolute alcohol, cleared in xylol, and
mounted in Canada balsam.

As will be mentioned hereafter, certain of the bacteria
which occur in tissue do not allow of the ready penetra¬
tion of the color. For such forms a more intense stain
must be employed. One of the best of these stains,
which can be employed by the given method both for
cover-glasses and tissues, is Loffler’s alkaline methylene
blue :

Saturated alcoholic solution of methylene blue, 30 ;

1 : 10,000 aqueous solution of caustic potash, 100.

METHODS OF OBSERVING BACTERIA.

Some bacteria, as the typlioid-fever bacillus, decolorize
so rapidly as to contraindicate the use of acid for the dif¬
ferentiation, washing in water or alcohol being sufficient.

Gram’s Method of Staining Bacteria in Tissue. —
Gram was the fortunate discoverer of a method of stain¬
ing bacteria in such a manner as to saturate them with
an insoluble color. It will be seen at a glance what a
marked improvement this is on the method given above,
for now the stained tissue can be washed thoroughly in
either water or alcohol until its cells are colorless, with¬
out fear that the bacteria will be decolorized. Its prose¬
cution is as follows : The section is stained from five to
ten minutes in a solution of a basic aniliu dye — pure
anilin (aniliu oil) and water. This solution, first devised
by Ehrlich, is known as Ehrlich’s solution. The ordinary
method of preparing it is to mix the following :

Pure anilin, 4 ;

Saturated alcoholic solution of gentian violet, 11 ;

Water, 100.

Instead of gentian violet, methyl violet, fuchsin, or any
basic anilin color may be used. The mixture does not
keep well — in fact, seldom longer than six to eight weeks,
sometimes not more than two or three ; therefore it is
best to prepare it in very small quantity by pouring
about 1 c.cm. of pure anilin into a test-tube, filling
the tube about one-lialf with distilled water, shaking
the mixture well, then filtering as much as is desired
into a small dish. To this the saturated alcoholic solu¬
tion of the basic dye is added until the surface becomes
distinctly metallic in appearance.

Friedlander recommends that the section remain from
fifteen to thirty minutes in warm stain, and in many cases
the prolonged process gives better results.

From the stain the section is given a rather hasty wash¬
ing in water, and then immersed from two to three min¬
utes in Gram’s solution (a dilute Lugol’s solution) :

PATHOGENIC BACTERIA.

Iodin crystals,

1 ;

Potassium iodid,

2 ;

Water,

3°°.

While the specimen is in the

Gram’s solution

appears to turn a dark blackish-brown color. When
removed from the solution it is carefully washed in 95
per cent, alcohol until no more color is given off and
the tissue assumes a grayish color. If it is simply
desired to find the bacteria, the section is dehydrated
in absolute alcohol for a moment, cleared up in xylol,
and mounted in Canada balsam. If it is necessary to
study the relation between the bacteria and the tissue-
elements, a nuclear stain, such as alum carmin or Bis¬
marck brown, may be subsequently used. Should a
nuclear stain requiring acid for its differentiation be
desirable, the process of staining must precede the Gram
method altogether, so that the acid shall not act upon
the stained bacteria.

The success of Gram’s method rests upon the fact that
the combination of my coprotein , basic anilin , and the
iodids forms a compound insoluble in alcohol .

The process described may be summed up as follows

Stain in Ehrlich’s anilin-water gentian violet five
to thirty minutes ;

Wash momentarily in water ;

Immerse two to three minutes in Gram’s solution ;

Wash in 95 per cent, alcohol until no more color
comes out ;

Dehydrate in absolute alcohol ;

Clear up in xylol ;

Mount in Canada balsam.

This method stains a large variety of bacteria very
beautifully, but, unfortunately, does not stain them all,
and as some of those which do not stain are important,
it seems well to mention the —

METHODS OF OBSERVING BACTERIA. 99

Spirillum of cholera and of chicken-cholera ;

Bacillus mallei (of glanders) ;

Bacillus of malignant edema ;

Bacillus pneumoniae of Friedlander ;

Micrococcus gonorrhoeae of Neisser;

Spirochaete Obermeieri of relapsing fever ;

Bacillus of typhoid fever ;

Bacillus of rabbit-septicemia.

. Gram’s method is a method of staining bacteria in
tissues, but the fact that the method colors some but not
all bacteria is one of considerable importance from a dif¬
ferential point of view ; and as the difficulty of separating
the species of bacteria is so great that every such point
must be eagerly seized for assistance, this method be¬
comes one much employed for cover-glass preparations>
where it is more easily performed than for sections.

Gram’s Method for Cover-glass Preparations. — A
thin layer of the bacteria to be examined is spread upon
the cover-glass, dried, and fixed. The cover, held in the
grip of a cover-glass forceps, is flooded with Ehrlich’s
solution. By holding the cover flooded with stain over
a small flame for a moment or two the solution is kept
warm, and the process of staining is continued from two
to five minutes. If the heating causes the stain to
evaporate, more of it must be dropped upon the glass,
so that it does not dry up and incrust.

The stain is poured off, and the cover placed in a small
dish of Gram’s solution and allowed to remain one-half
to two minutes, the solution being agitated. It is pos¬
sible to apply the Gram solution in the same manner
in which the stain is used, but as a relatively larger
quantity should be employed, the dish seems preferable.

The cover is next washed in 95 per cent, alcohol until
the blue color is wholly or almost lost, after which it can
be counter-stained wdth eosin, Bismarck brown, vesuvin,
etc., washed, dried, and mounted in Canada balsam.
Given briefly, the method is :

IOO

PA THOGENIC BA CTERIA .

Stain with Ehrlich’s solution two to five minutes ;

Gram’s solution for one-half to two minutes ;

Wash in 95 per cent, alcohol until decolorized ;

Counter-stain if desired ; wash the counter-stain
off with water ;

Dry;

Mount in Canada balsam.

Method of Staining Spores. — It has already been
remarked that the peculiar quality of the spore-capsules
protects them from the influence of stains and disinfect¬
ants to a certain extent. On this account they are much
more difficult to color than the adult bacteria. Several
methods are recommended, the one generally employed
being as follows : Spread the thinnest possible layer of
material upon a cover-glass, dry, and fix. Have ready
a watch-crystalful of Ehrlich’s solution, preferably made
of fuchsin, and drop the cover-glass, prepared side down,
upon the surface, where it should float. Heat the stain
until it begins to steam, and allow the specimen to
remain in the hot stain for five to fifteen minutes. The
•cover is now transferred to a 3 per cent, solution of hydro¬
chloric acid in absolute alcohol for about one minute.
Abbott recommends that the cover-glass be submerged,
prepared side up, in a dish of this solution and gently
agitated for exactly one minute, then removed, washed
in water, and counter-stained with an aqueous solution
of methyl or methylene blue.

In such a specimen the spores should appear red, the
bacilli blue.

I have not generally found that spores color so easily,
and for many species the best method seems to be to
place the prepared cover-glass in a test-tube half full of
carbol-fuchsin :

Fuchsin, T .

Alcohol, I0 .

5 Per cen-t. aqueous solution of phenol crystals, 100,

METHODS OF O BSE RUNG BACTERIA .

xoi

and boil it for at least fifteen minutes, after which it is
decolorized, either with 3 percent, hydrochloric or 2-5 per
cent, acetic acid, washed in water, and counter-stained blue.

Fiocca suggests the following rapid method: “About
20 c.cm. of a 10 per cent, .solution of ammonium are
poured into a watch-glass, and 10-20 drops of a saturated
solution of gentian violet, fuchsiu, methyl blue, or saf-
ranin added. The solution is warmed until vapor begins
to rise, then is ready for use. A very thinly-spread cover-
glass, carefully dried and fixed, is immersed for three to
five minutes (sometimes ten to twenty minutes), washed
in water,, washed momentarily in a 20 per cent, solution
of nitric or sulphuric acid, washed again in water, then
counter-stained with a watery solution of vesuvin, chrys-
oidin, methyl blue, malachite green, or safraniu, according
to the color of the preceding stain. This whole process
is said to take only from eight to ten minutes, and to give
remarkably clear and beautiful pictures. n

Method of Staining- Flagella. — This is much more
difficult than the staining of either the bacteria or their
spores, because each species seems to behave differently
in its relation to the stain, so that the chemistry of the
micro-organismal products must be taken into considera¬
tion.

The best method introduced is that of Loffler. In it
three solutions are used :

A. A 20 per cent, solution of tannic acid, 10 ;

Cold saturated aqueous solution of ferrous sulphate, 5 ;

Alcoholic solution of fuchsiu or methyl violet, 1;

B. A 1 per cent, solution of caustic soda.

C. An aqueous solution of sulphuric acid of such strength

that 1 c.cm. will exactly neutralize an equal quan¬
tity of Solution B.

Some of the bacteria to be stained are mixed upon a
cover-glass with a drop of distilled water. This is the
first dilution, but is too rich in bacteria to allow the

102

PATHOGENIC BACTERIA.

flagella to show well, so that it is recommended to prepare
a second dilution by placing a small drop of distilled
water upon a cover and taking a small portion from the
first cover to the second, spreading it over the entire sur¬
face. The material is allowed to dry, and is then fixed
by passing it three times through the flame. When this
is done with forceps there is some danger of the prepara¬
tion becoming too hot, so Loffler recommends that the
glass be held in the fingers while the passes through the
flame are made.

The cover-glass is now held in forceps, and the mordant,
Solution A, is dropped upon it until it is well covered.
The cover is warmed until it begins to steam, and the
stain replaced as it evaporates. It must not be heated too
strongly ; above all things, must not boil. This solution
is allowed to act from one-half to one minute, is then
washed in distilled water, then in absolute alcohol until all
traces of the solution have been removed. The real stain
— LSffier recommends an anilin- water fuchsin (Ehrlich’s
solution) — which should have a neutral reaction, is now
dropped on so as to cover the specimen, and heated for a
minute until vapor begins to arise; it is then washed off
carefully, dried, and mounted in Canada balsam. To
obtain this neutral reaction enough of the i per cent,
sodium-hydrate solution is added to an amount of the
anilin-water-fuchsin solution having a thickness of sev¬
eral centimeters to begin to change the transparent into
an opaque solution. Such a specimen may or may not
show the flagella. If not, before proceeding farther it is
necessary to study the products of the bacterium in cul¬
ture-media. If by its growth the organism elaborates
alkalies, Solution C, in proportion from i drop to i c. cm.
in 16 c. cm. of the mordant A, must be added, and the
process repeated again and again until the proper amount
is determined. On the other hand, if the organism by
its growth produces acid, Solution B must be added,
drop by drop, until i in 16 cm. have been attained, and
numerous experiments made to see when the flagella

METHODS OF OBSERVING BACTERIA . 1 03

will appear. Doffler lias fortunately worked out the
amounts required for some of the species, and of the
more important ones the following amounts of Solutions
B and C must be added to 16 c.cm. of Solution A to
attain the desired effect :

Cholera spirillum, ]/>- 1 drop of Solution C ;

Typhoid fever, 1 c.cm. of Solution B ;

Bacillus subtilis, 28-30 drops of Solution B ;

Bacillus of malignant edema, 36-37 drops of Solution B.

Part of the success of the staining depends upon
having the bacteria thinly spread upon the glass, and as
free from albuminous and gelatinous materials as possi¬
ble. The cover-glass must be cleaned most painstakingly :
too much heating in fixing must be avoided. After using
and washing off the mordant, the preparation should be
dried before the application of the anilin-water-fuchsin
solution.

Pitfield1 has devised a simple and good method of
staining flagella. A single solution at once mordant and
stain is employed. It is made in two parts, which are
filtered and mixed.

A. Saturated aqueous .solution of alum, 10 c.cm. ;

Saturated alcoholic solution of gentian-violet, 1 c.cm.

B. Tannic acid, 1 gr. ;

Distilled water, xo c.cm.

The solutions should be made with cold water, and
immediately after mixing the stain is ready for use. The
cover-slip is carefully cleaned, the grease being burned
off in a flame. After it lias cooled the bacteria are
spread upon it, well diluted with water. After drying
thoroughly in the air, the stain is gradually poured on
and by gentle heating brought almost to a boil ; the slip

1 Med. News , Sept. 7, 1895.

104 PATHOGENIC BACTERIA.

covered with the hot stain is laid aside for a minute, then
washed in water and mounted. In such preparations I
have always been able to see the flagella well, but usually
find that while the flagella are very distinct, the bodies
of the bacteria are scarcely visible.

Bunge suggests a mordant consisting of a concentrated
aqueous tannin solution and a i : 20 solution of liq. ferri
sesquichloridi in water. The best mixture seems to be
3 parts of the tannin solution to 1 part of the diluted
iron solution. To 10 c.cm. of this mixture 1 c.cin. of a
concentrated aqueous fuchsin solution is added. It is
not necessary to prepare this mordant fresh for each
staining, as it seems to improve with age. The use of
acid and alkaline solutions added to the mordant is dis¬
pensed with.

The bacteria are carefully fixed to the glass, stained
with the mordant for five minutes, warming a little to¬
ward the end, washed, dried, and subsequently colored
wTith carbol-fuchsin warmed a little.

Bacteria can best be measured by an eye-piece microm¬
eter. As these instruments vary somewhat in con¬
struction, the unit of measurement for each objective
magnification or the method of manipulating the adjusta¬
ble instruments must be learned from dealers’ catalogues.

Photographing bacteria requires special apparatus and
methods, which are fully described in text-books upon
the subject.

CHAPTER V.

STERILIZATION AND DISINFECTION.

Before considering- the cultivation of bacteria and
the preparation of media for that purpose it is necessary
to discuss methods of destroying bacteria whose acci¬
dental presence might ruin our experiments.

The dust of the atmosphere, as has already been shown,
is almost constant in its micro-organismal contamination,
so that the spores of moulds and bacilli, together with
yeasts and micrococci, constantly settle from it upon our
glassware, enter our pots, kettles, funnels, etc., and would
ruin every culture-medium with which we operate did
we not take measures for their destruction.

Micro-organisms may be killed by heat or by the action
of chemicals, the processes being generically termed
sterilization. The term sterilization is usually employed
to denote the destruction of bacteria by heat, in contra¬
distinction to disinfection, which usually means the
destruction of the bacteria by the use of chemical
agents. A chemical agent causing the death of bacteria
is called a germicide. An object which is entirely free
from bacteria and their spores is described as sterile.
Certain substances whose action is detrimental to the
vitality of bacteria and prevents their growth amid other¬
wise suitable surroundings are termed antiseptics.

The study of sterilization, disinfection, and antisepsis
will naturally lead us through the following subdivisions :

I. The sterilization and protection of instruments and
glassware used in experimentation.

II. The sterilization and protection of culture-media.

III. The disinfection of the instruments, ligatures, etc.
and the hands of the surgeon, and the use of antiseptics.

IV. The disinfection of sick-cliambers and their con¬
tents, as well as the dejecta and discharges of patients
suffering from contagious and infectious diseases.

106 PATHOGENIC BACTERIA .

The Sterilization and Protection of Instruments
and Glassware Used in Experimentation. — Steriliza¬
tion may be accomplished by either moist or dry heat.
For the perfect sterilization of objects capable of with¬
standing it dry heat is preferable, because more certain
in its action. If we knew just what organisms we had
to deal with, we might be able in many cases to save
time and gas, but while some simple non-spore-producing
forms are killed at a temperature of 6o° C., others can
withstand boiling for an hour ; it is therefore best to
employ a temperature high enough to kill all with cer¬
tainty. Platinum wires used for inoculation are held in
the direct flame until they become incandescent. In
sterilizing such wires attention must be bestowed upon
the glass handle, which should be held in the flame for
at least half its length for a few moments when used for
the first time each day. Carelessness in this respect may
cause the loss of much time by contaminating cultures.

Knives, scissors, and forceps may be exposed for a very
brief time to the direct flame, but this affects the temper
of the steel when continued too long. They may also
be boiled, steamed, or carbolized.

All glassware is sterilized by exposure to a sufficiently
high temperature, 150° C. or 302° F., for one hour in the
well-known hot-air closet (Fig. 9). A temperature of
150° C. is sufficient to kill all known bacteria and their
spores if continued for an hour.

Rubber stoppers, corks, wooden apparatus, and other
objects which are warped, cracked, charred, or melted
by so high a temperature must be sterilized by moist
heat in the steam apparatus for at least an hour before
they can be pronounced sterile.

It must always be borne in mind that after sterilization
has been accomplished the same sources of contamination
that originally existed are still present, and begin to
operate as soon as the objects are removed from the
sterilizing apparatus.

To Schroder and Van Dusch belong the credit of

STERILIZATION AND DISINFECTION 107

havingr first shown that when the months of flasks and
tubes are closed with plugs of sterile cotton no genus
can filter through. This observation has been of ines¬
timable value to every bacteriologist. Before sterilizing

Fig. 9. — Hot-air sterilizer.

flasks and, tubes we plug them with ordinary raw cotton,
and are sure that afterward their interiors will remain
free from the access of germs until opened. Instruments
may be sterilized wrapped in cotton, to be opened only
when ready for use ; or instruments and rubber goods
sterilized by steam can subsequently be wrapped in
sterile cotton and kept for use. It is of the utmost
importance to carefully protect every sterilized object,
and to allow as little dust to collect upon it as possible,
in order that the object of the sterilization be not de¬
feated. As the spores of moulds falling upon cotton
sometimes grow and allow their mycelia to work their
way through and drop into a culture-medium, Roux

io8

PATHOGEN/C BACTERIA .

has introduced little paper caps with which the cotton
stoppers are protected from the dust. These are easily
made by curling a small square of paper into a “ cornu¬
copia, ’ ’ fastening by turning up the edge or putting in a pin.
The paper is placed over the stopper before the sterilization,
after which no contamination of the cotton can occur.

Sterilization of Culture-media. — As almost all of the

culture-media contain about 80 per cent, of water, which
would be evaporated in the hot-air closet, so that the
material would be destroyed, hot-air sterilization is not
appropriate for them. Sterilization by streaming steam
is the best and surest method. The prepared media are

placed in flasks or tubes care¬

fully plugged with cotton and
previously sterilized with dry
heat, and then sterilized in what
is known as Koch’s steam appa¬
ratus (Fig. io) or in Arnold’s

Fig. io.— Koch’s steam sterilizer. Fig. ii.— Arnold’s steam sterilizer.

steam sterilizer (Fig. ii), which is more convenient and
more generally useful.

The temperature of boiling water, xoo° C., does not

STERILIZATION AND DISINFECTION. 109

kill many spores, so that the exposure of culture-media
to streaming steam is of little use unless applied in
a systematic manner — intermittent sterilization — based
upon a knowledge of sporulation.

In carrying out the intermittent sterilization the cul¬
ture-medium is exposed for fifteen minutes to the passage
of streaming steam in the apparatus or to some tem¬
perature judged to be sufficiently high, so that the bac¬
teria contained in it are killed. As the spores remain
uninjured, the medium is stood aside in a cool place for
twenty-four hours, and the spores allowed to develop into
perfect bacteria.

When the twenty-four hours have passed the culture-
medium is again placed in the apparatus and exposed to

Fig. 12. — Autoclave for rapid sterilization Fig. 13. — Kny-Spraguc steam sterilizer,
by superheated steam under pressure.

the same temperature, until these newly-developed bac¬
teria are also killed. Eventually, the process is repeated

PATHOGENIC BACTERIA.

a third time, lest a few spores remain alive and capable
of spoiling the material. When properly sterilized in
this way, culture-media will remain free from contamina¬
tion until time and evaporation cause them to dry up.

Fig. 14. Pasteur-Chamberland filter arranged to filter under pressure.

If hermetically sealed, a sterile medium will keep indef¬
initely.

If it should be necessary to sterilize culture-media at
once, not waiting the three days required by the inter¬
mittent method, it may be done by superheated steam in

STERILIZATION AND DISINFECTION

III

an autoclave (Fig. 12). Here under a pressure of two or
three atmospheres sufficient heat is generated to destroy
the spores. The objections to this method are that it
sometimes turns the agar-agar dark, and that it is likely
to destroy the gelatinizing power of the gelatin, which
after sterilization remains liquid.

Liquids may also be sterilized by filtration — i. e. by
passing them through unglazed porcelain or some other
material whose interstices are sufficiently fine to resist the
passage of the bacteria. This method is largely employed

Fig. 15. — Kitasato’s filter : a, por- Fig. 16. — Reichel’s bacteriologic filter
celain bougie ; b, attachment for sue- of unglazed porcelain: A, sterile re-
tion-pump; c, reservoir; d, sterile ceiver; J5, porcelain filter ; c, d, attach-
receiver. ments for pump.

for the sterilization of the unstable toxins and anti¬
toxins, which are destroyed by heat. Various substances
have* been used for filtration, as stone, sand, powdered
glass, etc., but experimentation has shown porcelain to
be the only reliable filter against bacteria. Even this
material, whose interstices are so small as to allow the
liquid to pass through with great slowness, is only cer¬
tain in its action for a time, for after it has been used
considerably the bacteria seem able to work their way

II 2

PATHOGENIC BACTERIA.

through. To be certain of the efficacy of such a filter
the fluid first passed through must be tested by cultiva¬
tion methods. The complicated Pasteur-Cliamberland
and the simple Kitasato and Reich el filters are shown in
Figures 14, 15, and 16.

After having been used a porcelain filter must be dis¬
infected, scrubbed, dried thoroughly , and then heated in
a Bunsen burner or blowpipe flame until all the organic
matter is consumed. In this firing process the filter first
turns black as the organic matter chars, then becomes
white as it is consumed. The greatest care must be
exercised in cleansing, and especially must care be taken
that the porcelain is dry before entering the fire, as it
will certainly crack if moist.

Before using a new filter it should be sterilized by dry
heat, then connected with receivers and tubes, also care¬
fully sterilized. It should not be forgotten that the fil¬
tered material is still a good culture-medium and must be
handled with the greatest care.

While the filtration of water, peptone solution, and
bouillon is comparatively easy, gelatin and blood-serum
pass through with great difficulty, and speedily gum the
filter, so that it is useless until fired.

A convenient apparatus used by the author for the rapid
filtration of large quantities is shown in the accompany¬
ing illustration (Fig. 17).

The Disinfection of Instruments, Ligatures, Sutures,
the Hands, etc. — There are certain objects used by the

STERILIZATION AND DISINFECTION

i*3

surgeon which cannot well be rendered incandescent,
exposed to dry heat at 150° C. , steamed, or intermittently
heated without injury. For these objects disinfection
must be practised. Ever since Sir Joseph Lister intro¬
duced antisepsis, or disinfection, into surgery there has
been a struggle for the supremacy of this or that highly-
recommended germicidal substance, with two results —
viz. that a great number of feeble germicides have been
discovered, and that belief in the efficacy of all germi¬
cides has been somewhat shaken; hence the origin of the
successful aseptic surgery of the present day, which
strives to prevent the entrance of germs into, rather than
their destruction after admission to, the wound.

For a complete discussion of the subject of antiseptics
in relation to surgery the reader must be referred to the
large text-books of surgery, where much space is thus
occupied. A short list of useful germicides of which
the respective values are given, and a brief discussion
of some of the more important measures, can alone find
space in these pages. The antiseptic value of some of
the principal substances used may be expressed as fol¬
lows, the figures indicating the strength of the solution
necessary to prevent the development of bacteria :

Pyoktanin (methyl violet) . 1 : 2,000,000 — 1 : 5000.

Formalin . 1 125,000 — 1 15000.

Bichlorid of mercury . . . 1 : 14,300.

Hydrogen peroxid . 1 : 20,000.

Formalin . 1 : 20,000.

Nitrate of silver . 1 : 12,500.

Creolin . 1 : 5000 to 1 : 200 (does not kill

anthrax).

Chromic acid . 1 : 5000.

Thymol . 1 : 1340.

Salicylic acid . 1 : 1000.

Potassium bichromate . . . 1 : 909.

Trikresol . 1 : 1000 — 1 : 500.

Zinc chlorid . 1 : 526.

Potassium permanganate . 1 : 285 ; not prompt in action.

Nitrate of lead . 1 : 277.

Izal . . : 200.

PATHOGENIC BACTERIA .

114

Boracic acid . 1 : 143.

Chloral hydrate . 1 : 107.

Ferrous sulphate . . . . . 1 : 90—1 : 200, Sternberg.

Calcium chlorid . 1 : 25.

Creosote . . 1:20.

Carbolic acid . 1 : 20 : : 1 : 50.

Alcohol . . 1:10.

Ether. Pure ether will not kill anthrax spores immersed
in it for eight days.

The value of antiseptics, like that of disinfectants, is
always relative, the destructive as well as the inhibitory
power of the solution varying with the micro-organism
upon which it acts. The following table, from Boer,
will illustrate this :

Methyl Violet ( Pyoktanin ).

Restrains.

Kills.

Anthrax bacillus . . .

. . . 1 : 70,000

i : 5000

Diphtheria .

. . . 1 : 10,000

1 : 2000

Glanders .

. 1 : 2500

1 : 150

Typhoid .

. . . 1 : 2500

1 : 150

(Cholera spirillum . .

. . . 1 : 30,000

1 : 1000

Targe numbers of both strongly and feebly antiseptic
substances have purposely been omitted from the above
lists, compiled from Sternberg and Micquel, as either in-
appropriate for ordinary use or as having been replaced
by better agents.

The newest, and one of the best germicides for all pur¬
poses is formaldehyde. Its use as a vapor for the sterili¬
zation of infected rooms was first suggested by Trillat in
1895, but it did not make much stir in the medical world
until a year or more had passed and a 40 per cent, solu¬
tion of the gas, under the name of “Formalin,” had
been placed upon the market. The original method con¬
sisted of the evolution of the gas from methyl alcohol by
volatilizing it in a steam apparatus, and passing the vapor
over a heated metal plate. At present the original auto¬
clave has been replaced by the apparatus shown in Fig.
19, in which a solution of formochloral is volatilized by
heating under a pressure of three atmospheres.

STERILIZATION AND DISINFECTION. 115

The gas is very penetrating, easily diffusing itself, and
is said to have enormous bactericidal powers. In experi¬
ments conducted by Prof.

Robinson, of Bowdoin Col¬
lege, the gas penetrated mat¬
tresses and killed cultures in
tubes wrapped up in them.

There seems to be little
doubt of the ability of the

Fig. 19. —Sanitary formaldehyde* re-

Fig. 18. — The Trillat autoclave. generator.

formaldehyde gas to disinfect, but there are few apparatus
upon the market at present that seem capable of discharg¬
ing a sufficient volume of the gas with sufficient rapidity
to do the work. The physician, therefore, who desires
to disinfect with confidence should choose an apparatus
that has been shown by competent experiments to fill the
requirements.

The 11 formalin,” or 40 per cent, solution of the gas,
when fresh and tightly corked, is fatal to most bacteria in
dilutions of from 1: 5000 to 1 : 25,000. It can be employed
with great advantage to spray the walls and floors of
rooms. It cannot be employed upon the skin or mucous
membranes, because of its marked irritating effect.

The disinfection of the skin, both the hands of the
surgeon and the part about to be incised, is a matter of
importance. It is almost impossible to secure absolute
sterility of the hands, so deeply do the skin-cocci pene¬
trate between the layers of the scarf-skin. The method at

1 1 6 PA THO GENIC BA CTERIA .

present generally employed, and recommended by Welch
and Hunter Robb, is as follows: The nails must be
trimmed short and perfectly cleansed. The hands are
washed thoroughly for ten minutes in water of as high a
temperature as can comfortably be borne, soap and a brush
previously sterilized being freely used, and afterward the
excess of soap washed off in clean hot water. The hands
are then immersed for from one to two minutes in a
warm saturated solution of permanganate of potassium,
then in a warm • saturated solution of oxalic acid, until
complete decolorization of the permanganate occurs, after
which they are washed free from the acid in clean warm
water or salt-solution. Finally, they are soaked for two
minutes in a i : 500 solution of bichlorid of mercury,
after which they are ready for use.

Lockwood,1 of St. Bartholomew’s Hospital, recommends
after the use of the scissors and penknife, scrubbing the
hands and arms for three minutes in hot water and soap
to remove all grease and dirt. The scrubbing brush
ought to be steamed or boiled before use, and kept in
1 : 1000 biniodid of mercury solution. When the soap¬
suds have been thoroughly washed away with plenty of
clean water, the hands and arms are thoroughly washed
and soaked for not less than two minutes in a solution of
biniodid of mercury in methylated spirit; 1 part of the
biniodid in 500 of the spirit. Hands that cannot bear
1 : 1000 bichlorid and 5 per cent, carbolic solutions, bear
frequent treatment with the biniodid. After the spirit
and biniodid have been used for not less than two min¬
utes, the solution is washed off in 1 : 2000 or 1 : 4000
biniodid of mercury solution.

Catgut cannot be sterilized by boiling without deterio¬
ration. The present method of preparing it is to dry it
in a hot-air chamber and then boil it in cumol, which is
afterward evaporated and the skeins preserved in sterile
test-tubes or special receptacles plugged with sterile cot¬
ton. Cumol was first introduced for this purpose by

1 Brit . Med. your., July II, 1896.

STERILIZATION AND DISINFECTION.

ii 7

Kronig, as its boiling-point is i68°-i78° C., and thus
sufficiently high to kill spores. The use of cmnol for the
sterilization of catgut has been carefully investigated by
Clarke and Miller.1

Ligatures of silk and silkworm-gut are boiled in
water immediately before using, or are steamed with the
dressings, or placed in test-tubes plugged with cotton and
steamed in the steam sterilizer.

At present, in most hospitals, instruments are boiled
before using in a 1-2 per cent, soda solution. Plain
water has the disadvantage of rusting the instruments,
and during the operation they are either kept in the boiled
water or in carbolic solution. Andrews makes special
mention of the fact that the instruments must be com¬
pletely immersed to prevent rusting.

During the operation the wound is frequently washed
with normal salt solution, applied by sterile marine or
gauze sponges.

The water and the salt solution used for surgical pur¬
poses are to be sterilized before using, either by steaming
for a prolonged period, or by the intermittent method.
Large hospitals are generally furnished with special appa¬
ratus for supplying sterile distilled water in large quantity.

To La Place belongs the credit of observing that the
efficacy of bichlorid of mercury is greatly increased by
the addition of a small amount of acid, by which the
penetration is increased and the formation of insoluble
albuminates lessened.

The knowledge that the action of germicides is chem¬
ical, and that the destruction of the bacteria is due to the
combination of the germicide with the mycoprotein, is
apt to lessen our confidence in the permanence of their
action. Geppert has shown of bichlorid of mercury that
in the reaction between it and anthrax spores the vitality
of the latter seems lost, but that the precipitation of the
bichlorid from this combination by the action of ammo¬
nium sulpliid restores the vitality of the spore.

1 Bull, of the Johns Hopkins Hospital Feb. and March, 1S96.

PATHOGENIC BACTERIA.

Again, the fact that some of tlie antiseptics, as nitrate
of silver and biclilorid of mercury, are at once precipi¬
tated by albumins, and thus lose their germicidal and
antiseptic powers, limits the scope of their employment.

I think it may* be safely said that carbolic acid is the
most reliable and most generally useful of all the germi¬
cides and antiseptics.

The Disinfection of Sick-chambers, Dejecta, etc. —
What has just been remarked concerning the unreliability
of many of the germicidal substances is eminently a
firopos of the disinfection of dejecta. It is useless to
mix biclilorid of mercury with typhoid stools or tubercu¬
lar sputum rich in albumin, and imagine these substances
rendered harmless in consequence. It should not be for¬
gotten that the sick patient is less the means of convey¬
ing the contagium than the objects with which he is in
contact, which can be carried to other rooms or houses
during or after the progress of the disease. A careful
consideration of the condition of the sick-room will
lead us to a clear understanding of its bacteriological
condition.

The Air of the Sick-room. — It is impossible to sterilize
or disinfect the atmosphere of a room during its occu¬
pancy by the patient. The disinfecting capacity of the
solutions given above must make obvious the concentra¬
tion of their useful solutions, and show the foolishness
of placing beneath the bed or in the corners of a room
small receptacles filled with carbolic acid or chlorinated
lime. These can serve no purpose for good, and may be
potent for harm by obscuring the disagreeable odors
emanating from materials which should be removed from
the room by the still more disagreeable odors of the dis¬
infectants. The practice of such a custom is only com¬
parable to the old faith in the virtue of asafetida tied
in a corner of the handkerchief as a preventive of cholera
and smallpox.

During the period of illness a chamber in which the
patient is confined should be freely ventilated, so that its

STERILIZATION AND DISINFECTION 1 19

atmosphere is constantly changing and replacing the
closeness so universally an accompaniment of fever by
fresh, pure air — a comfort to the patient and a protection
to the doctors and nurses.

After recovery or death one- should rely less upon fu¬
migation than upon the disinfection of the walls and
floor, the similar disinfection of the wooden part of the
furniture, and the sterilization of all else. The fumes
of sulphur may do some good — when combined with
steam, much good — but are greatly overestimated, and
the sulphurous vapors- are rapidly giving way to the more
penetrating and germicidal formaldehyde vapor. To
apply this, the room to be sterilized is carefully closed,
the carefully selected apparatus set in action, and the
discharged vapor allowed to act undisturbed for some
hours, after which the windows and doors are all thrown
open to fresh air and sunlight.

Instead of the gas, a 40 per cent, solution, which can
be sprayed upon the ceiling, walls, floor, and contents of
the room from a large atomizer, is sometimes used. Ex¬
perience has not shown, however, that this possesses any
distinct advantages.

So far as is at present known, the disinfection by form¬
aldehyde is complete and leaves nothing to be desired.
Only one point is to be considered, already often men¬
tioned — that is, the apparatus. Of those experimented
with by the author, few have given satisfaction.

The Dejecta. — A little thought will direct attention to
those of the dejections which are dangerous to the com¬
munity and promote efforts for their complete steriliza¬
tion. In cases of diphtheria the vomit, expectorations,
and nasal discharges are most important. They should
be received in old rags or in Japanese paper napkins —
not handkerchiefs or towels — and should be burned. The
sputum of tuberculous patients should either be collected
in a glazed earthen vessel which can be subjected to boil¬
ing and disinfection, or, as is an excellent plan, should be
received in Japanese rice-paper napkins, which can at

120

PATHOGENIC BACTERIA .

once be burned. These napkins are not quite as good
as the small pasteboard boxes (Kig. 20) recommended by

Fig. 20. — Pasteboard cup for receiving infectious sputum. When used the
pasteboard can be removed from the iron frame and burned.

some city boards of health, because, being highly absorb¬
ent, the sputum is apt to soak through and soil the fin¬
gers, etc. Tuberculous patients should be provided with
rice-paper instead of handkerchiefs, and should have their
towels, knives, forks, spoons, plates, etc. kept strictly
apart from the others of the household (though the pa¬
tients, whose mental acuity makes their sensibilities very
pronounced, need never be told of their isolation), and
frequently boiled for considerable lengths of time.

The excreta from typhoid-fever and cholera cases re¬
quire particular attention. These, and indeed all alvine
matter possibly the source of infection or contagion,
should be received in glazed earthen vessels and imme¬
diately intimately mixed with a 5 per cent, solution
of chlorinated lime (containing 25 per cent, of chlorin)
if semi-solid, or with the powder if liquid, and allowed
to stand for an hour before being thrown into the
drain.

The Clothing , etc. — All bed-clothing which has been
used in the sick-room, all towels, napkins, handkerchiefs,
night-robes, underclothes, etc. which have been used by
the sick, and all towels, napkins, handkerchiefs, caps,
aprons, and outside dresses worn by the nurse, should be
regarded as infected and subjected to sterilization. The
only satisfactory method of doing this is by prolonged
subjection to steam in a special apparatus ; but, as this

STERILIZATION AND DISINFECTION . 12 1

is only possible in hospitals, the next best thing- is boiling
for some time in the ordinary wash-boiler. When pos¬
sible, the clothes should be soaked in 1 : 2000 bichlorid
solution before or after boiling, and in drying should
hang in the sun and wind. Woollen underwear can be
treated exactly as if of cotton. The woollen clothing of
the patient, if infected, requires special treatment. For¬
tunately, the infection of the outer woollen garments is
unusual. The only reliable method for their purification
is prolonged exposure to hot air at no0 C. In private
practice it becomes a grave question what shall be done
with these articles. Prolonged exposure to fresh air and
sunlight will aid in rendering them harmless ; when it
is certain that articles of wool are infected, they may be
sent to the city hospital or to certain of the moth-destroy¬
ing and fumigating establishments which can be found
in all large cities, and be baked.

The Furniture , etc . — The wholesale destruction of fur¬
niture practised in earlier times has at present become
unnecessary. The doctor, if he properly performs his
functions, will save much trouble and money for his
patient by ordering the immediate isolation of his charge
in an uncarpeted, scantily- and cheaply-furnished room
the moment an infectious disease is suspected , before
much infection can have occurred. However, if before
his removal the patient has occupied another bed, its
clothing should be promptly handled in the above-
described manner.

After the illness the walls of the rooms, including the
ceiling .should be sprayed with formalin, or, where it can¬
not be obtained, may be rubbed with fresh bread, which
Loffler has shown to be efficacious, though scarcely prac¬
ticable, in collecting the bacteria, or, if possible, should
be whitewashed. If the walls are hung with paper, they
may be dampened with 1 : 1000 bichlorid-of-mercury so¬
lution before new paper is hung.

Aronson1 says: uFor the disinfection of living-rooms

2 Verein fur Ofcntliche C csundheitsffleg e, Berlin, April 26, 1897.

122

PATHOGENIC BACTERIA .

there is no method that can compare in the remotest
degree, as regards certainty and simplicity, with that by
means of formaldehyde gas. For example, any one who
has seen the process of cleansing walls by rubbing them
down with bread, as carried out by the disinfecting corps,
will agree with me that, however effective it may be
from a theoretical point of view, it is absolutely inefficient
in practice. The possibility of disinfecting rooms and all
their contents with certainty, by means of a simple,
cheap, harmless, and easily managed method must be
hailed as a great advance.”

The floor should be scoured with 5 per cent, carbolic-
acid solution or 1 : 1000 bichlorid of mercury, and all the
wooden articles wiped off two or three times with the
same solution employed for the floor. In this scouring
no soap can be used, as it destroys the virtue of the
germicide. If a straw mattress was used, it should be
burned and the cover boiled. If a hair mattress was
used, it can be steamed or baked by the manufacturers,
who generally have ovens for the purpose. Curtains,
shades, etc., should receive proper attention; but, of course,
the greater the precautions exercised in the beginning,
the fewer the articles which will need attention in the
end. They should be removed before the case lias-
developed.

Strehl has succeeded in demonstrating that when 10 per
cent, formalin solution is sponged upon artificially infected
curtains, etc., the bacteria are killed by the action of the
disinfectant. This knowledge will be an important ad¬
junct to our means for disinfecting the furniture of the
sick-chamber.

The patient , whether he lives or dies, may also be
a means of spreading the disease unless specially cared
for. After convalescence the body should be bathed witli
a weak bichlorid-of-mercury solution or with a 2 per
cent, carbolic-acid solution, or with 25-50 per cent, alco¬
hol, before the patient is allowed to mingle with society,
and the hair should either be cut off or carefully washed

STERILIZATION AND DISINFECTION 123

with the above solution. In desquamative diseases it
seems best to have the entire body anointed with cos-
molin once daily, the unguent being well rubbed in, in
order to prevent the particles of epidermis being distrib¬
uted through the atmosphere. Carbolated cosmolin may
be better than the plain, not because of the very slight
antiseptic value it possesses, but because it helps to allay
the itching which may be part of the desquamative
process.

After the patient is about the room again, common
sense will prevent the admission of strangers until all
the disinfective measures have been adopted, and after
this, touching, and especially kissing him, should be
omitted for some time.

The dead who die of infectious diseases should be
washed in a strong disinfectant solution, and should be
given a private funeral in which the body, if exposed,
should not be touched. In my judgment, the body
is best disposed of by cremation.

It seems, however, to be an error to suppose that a
dead body can remain for an indefinite period a source of
infection. Esmarch 1 has made a series of laboratory ex¬
periments to determine what the fate of pathogenic bac¬
teria in the dead body really is. From his results it seems
clear that in septicemia, cholera, anthrax, malignant
edema, tuberculosis, tetanus, and typhoid the pathogenic
bacteria all die sooner or later, generally more rapidly in
conditions of decomposition than in good preservation of
the tissues. Lack of oxygen may be a cause of their
disappearance.

1 Zeitschrift fur Hygiene, 1893.

CHAPTER VI.

CULTIVATION OF BACTERIA; CULTURE-MEDIA.

Accuracy of observation requires that the bacteria be
separated from their natural surroundings and artificially
grown upon certain prepared media of standard compo¬
sition, iu such a manner that only organisms of the same
kind are together.

One after another various organic and inorganic mix¬
tures have been suggested, but, although almost any
compound containing organic matter, even in small
amounts, will suffice for the nourishment of bacteria,
a certain few have met with particular favor as being
most valuable.

Rather than give a complete review of the work which
has already been done, in the following pages the most
useful preparations only will be considered.

Our knowledge of the biology of the bacteria has
shown that they grow best in a mixture containing at
least 80 per cent, of water, of a neutral or feebly alka¬
line reaction, and of a composition which, for the patho¬
genic forms at least, should approximate the juices of
the animal body. It might be added that transparency
is a very desirable quality, and that the most gener¬
ally useful culture-media are those that can be readily
liquefied and solidified.

Bouillon is one of the most useful and most simple of
the media. Its preparation is as follows : To 500 grams
of finely-chopped lean, boneless beef, 1000 c.cm. of clean
water are added and allowed to stand for about twelve
hours on ice. At the end of this time the liquor is de¬
canted, that remaining on the meat expressed through a
cloth, and then, as the entire quantity is seldom regained,

124

CULTIVATION OF BACTERIA.

125

enough water added to bring the total amount up to 1000
c.cm. This liquid is called the meat-infusion. To it io
grains of Witte’s or Fairchild’s dried beef-peptone and 5
grams of sodium chlorid are added, and the whole boiled
until the albumins coagulate. The reaction is then care¬
fully tested, in order that whatever sarcolactic acid may
have been present in the meat may be neutralized by the
addition of a few drops of a saturated aqueous solution of
sodium carbonate. The solution is added drop by drop,
and the reaction frequently tested with litmus-paper.
When a neutral reaction, or, better, a faint alkaline re¬
action, is attained, the mixture is well stirred, boiled
again for about half an hour to precipitate the alkaline
albumins formed, and filtered. The use of phenolphtha-
lein to determine the reaction of the culture-media is much
more reliable than litmus, and in many laboratories has
replaced it. The method of using it suggested by Timpe
is to continue the addition of the carbonate of sodium
solution until a drop of it produces a red spot upon phe-
nolplithalein-paper. Such a paper can easily be made by
using a solution of 5 grams of phenolphtlialein to 1 liter
of 50 per cent, alcohol. The bibulous paper is cut into
strips, moistened with the solution, and then hung up to
dry. It keeps quite well. Acids do not change the
appearance of the paper, but small traces of alkali turn
it red.

If it is necessary to be extremely accurate concerning
the acidity or alkalinity of the culture-medium, the
method of titration with phenolphthalein can be em¬
ployed. For this purpose a small quantity of the culture-
fluid — say 10 c.cm. — receives an addition of a drop or
two of a weak alcoholic solution of phenolphthalein
(1 : 300), and then drop by drop from a burette a dilute
soda solution is added until a faint rose color occurs, when
a simple calculation will show that if so much is required
to bring about the required color in the 10 c.cm., so much
more will be required for the total amount. The occur¬
rence of the rose color marks the change from a neutral

126

PATHOGENIC BACTERIA .

to a faintly alkaline reaction. Definite varying degrees
of alkalinity can be secured by adding measured quan¬
tities of the soda solution beyond that necessary to bring
about the beginning alkalinity. In using phenolphtha-
lein one should remember that the first sign of rose color
marks a change to alkalinity, and that this is a higher
degree of alkalinity than that required to turn red litmus
blue.

The bouillon thus prepared is a clear fluid of a straw
color, much resembling normal urine in appearance. It

Fig. 21. — Funnel for filling tubes with culture-media (Warren).

is dispensed in previously sterilized tubes with cotton
plugs — about io c.cm. to each — and is then sterilized by
steam three successive days for fifteen to twenty minutes
each, according to the directions already given for frac¬
tional sterilization. (See p. 109.)

CULTIVATION OF BACTERIA .

127

For the preparation of bouillon, as well as gelatin,
agar-agar, aud glycerin agar still to be described, beef-
extract (Liebig’s) may be employed, but for the most
delicate work this is rather undesirable, because of its
unstable composition and because of the precipitation of
meat-salts, which can scarcely be filtered out of the agar-
agar, owing to the fact that they only crystallize when
the solution cools. When it is desirable to prepare the
bouillon from beef-extract, the method is very simple.
To 1000 c.cm. of clean water 10 grams of Whitte’s dried
beef-peptone, 5 grams of sodium chlorid, and about 2
grams of beef-extract are added. The solution is boiled
until the constituents are dissolved, neutralized, if neces¬
sary, and filtered when cold. If it is filtered while hot,
there is always a subsequent precipitation of meat-salts,
which clouds it.

Bouillon and other liquid culture-media are best dis¬
pensed and kept in small receptacles — test-tubes or flasks
— in order that a single contaminating organism, should
it enter, may not spoil the entire bulk. A very con¬
venient simple apparatus used by bacteriologists for fill¬
ing tubes with liquid media is shown in Figure 21. It
need not be sterilized before using, as the culture-medium
will be sterilized by the intermittent method after the
tubes are filled. The test-tubes and flasks into which
the culture-medium is filled must, however, be previously
sterilized by dry heat. The dry-heat sterilization is done,
of course, after the cotton plugs are in place.

Bouillon is the basis of most of the culture-media.
The addition of 10 per cent, of gelatin makes it u gela¬
tin;” that of 1 per cent, of agar-agar makes it u agar-
agar. n The preparation of these media, however, varies
somewhat from that of the plain bouillon.

Gelatin. — The culture-medium known as gelatin has de¬
cided advantages over the bouillon, not only because it is
an excellent food for bacteria, and, like the bouillon, trans¬
parent, but because it is also solid. Nor is this all : it is
a transparent solid which can be made liquid or solid at

128

PA TJ10GENIC BACTERIA .

will. It is prepared as follows : To 1000 c.cm. of meat-
infusion or to 1000 e.cm. of water containing 2 grams of
beef-extract in solution, 10 grams of peptone, 5 grams of
salt, and 100 grams of gelatin (“Gold label” is the best
commercial article) are added, and boiled for about an
hour over a moderately hot flame. Double boilers are
very slow, and if proper care is exercised there is little
danger of the gelatin burning. It must be stirred occa¬
sionally, and the flame should be so distributed by wire
gauze as not to act upon a single point of the bottom of
the kettle. At the end of the hour the albumins of
the meat-infusion will be coagulated and the gelatin
thoroughly dissolved. Giinther has shown that the
gelatin congeals better if allowed to dissolve slowly in
warm water before boiling. The liquid is now cooled
to 6o° C. and neutralized — /. c. alkalinized. As the gela¬
tin is itself acid, a relatively larger amount of the sodium-
carbonate solution will be needed than was required for
the bouillon. When the proper reaction is attained, as.
much water as has been lost by vaporization during the
process of boiling, intimately mixed with the white of
an egg, is added, well stirred in, and the whole boiled
for half an hour, then filtered.

If the filter-paper be of good quality and properly
folded (pharmaceutical filter), and if the gelatin be prop¬
erly dissolved, the whole quantity should pass through
before cooling too much. Should only half go through
before cooling, the remainder must be returned to the
pot, heated to boiling once more, and then passed through
a new filter-paper. As a matter of fact, gelatin generally
filters readily. A wise precaution is to catch the first few
centimeters in a test-tube and boil them, so that if a
cloudiness shows the presence of uncoagulated albumin,
the whole mass can be boiled again. The finished gel¬
atin is at once distributed into sterilized tubes, and then
sterilized like the bouillon by the fractional method.

Of course, the gelatin or any other culture-medium can
be kept en masse indefinitely, but should a contaminating

CUL 77 VA TION OF BA CTERIA. 1 29

micro-organism accidentally enter, the whole quantity
will be spoiled ; if, on the other hand, it is kept in tubes,
several of them may be lost without much inconvenience.
Under proper precautions of sterilization and protection
it should all keep well.

Agar-agar. — Agar-agar is the commercial name of a
Japanese sea-weed which dissolves in boiling water with
resulting thick jelly when cold. The jelly, which solidi¬
fies between 30° and 40° C., cannot again be melted ex¬
cept by the elevation of its temperature to the boiling-
point, so that this culture-medium, which is nearly trans¬
parent, is almost as useful as gelatin. In addition to its
readiness to liquefy and solidify, it is sufficiently firm
to allow of the incubation-temperature — i. e. 370 C. — at
which gelatin is always liquid, and no better than bouillon.

The preparation of this medium is generally described
in the text-books as one l( requiring considerable patience
and much waste of filter-paper.” In reality, it is not dif¬
ficult if a good heavy filter-paper be obtained and no
attempt be made to filter the solution until the agar-agar
is perfectly dissolved. It is prepared as follows : To 1000
c.cm. of bouillon made as described above, preferably of
meat instead of beef-extract, 10 grams of agar-agar are
added. The mixture is boiled for an hour, or, if possible,
two. At the end of the first hour it is cooled to about
6o° C., and after neutralization, which may not be neces¬
sary if the bouillon was neutral, an egg beaten up in
water is added, and the liquid is boiled again until the
egg is entirely coagulated. The reaction of the agar-agar
should be neutral rather than alkaline, as, for an un¬
known reason, alkalinity seems to interfere slightly with
filtration.

After the boiling, which should be brisk, has caused
the thorough solution of the agar-agar, it is filtered, just
as the gelatin was, through a carefully-folded pharmaceu¬
tical filter wet with boiling water. It may expedite mat¬
ters to pour in about one-half of the solution, keep the
remainder hot, and subsequently add it when necessary.

130

PATHOGENIC BACTERIA.

Experience shows that 1000 c. cm. of agar-agar rarely go
through one paper, and I always expect when beginning
the filtration to be compelled to boil the material which
remains on the paper again, and pour it through a new
filter.

The formerly much-employed hot-water and gas-jet
filters seem unnecessary. If properly prepared, the whole
quantity will filter in from fifteen to thirty minutes.

If made from beef-extract, the agar-agar almost always
precipitates a considerable amount of meat-salts as it
cools. This should be anticipated, but, so far as I can
determine, cannot always be prevented. The amount is
certainly lessened by making the bouillon first, filtering
it cold , then adding the agar-agar, and dissolving and
filtering it.

The difficulty of filtering the agar-agar has led Fliigge
and others to adopt a method of sedimentation. An in¬
genious apparatus for this purpose has lately been devised
by Bleisch. The methods can be simplified by using a
small pharmaceutical percolator, the bottom of which is
closed by a rubber cork containing a tube which extends
nearly to the top of the percolator and is attached to
.a rubber tube with a pinchcock below. The melted agar-
agar is poured into this, and kept in the steam apparatus
mntil the sedimentation is sufficient to allow clear fluid to
be drawn from the top. As the clear agar-agar is drawn
off the tube is pulled down through the rubber cork, and
more drawn off until only the sediment is left.

Agar-agar is dispensed in tubes like the gelatin and
bouillon, sterilized by steam by the intermittent process,
and after the last sterilization, before cooling, each tube
is inclined against a slight elevation, so as to offer an ex¬
tensive flat surface for the culture.

After the agar-agar jelly solidifies its contraction causes
some water to collect at the lower part of the tube. This
should not be removed, as it keeps the material moist,
and also because it has a distinct influence upon the cha¬
racter of the growth of the bacteria.

CUL TJ VA TION OF BA CTERIA . 1 3 1

Glycerin Agar-agar. — For an unknown reason certain
of the bacteria which will not grow upon the agar-agar
as prepared above will do so if 3-7 per cent, of glycerin
be added. Among these is the tubercle bacillus, which,
not growing at all upon plain agar-agar, will grow well
when glycerin is added — a fact discovered by Roux and
Nocard. The glycerin may also be added to gelatin or
any other medium.

Blood Agar-agar was recommended by R. Pfeiffer for
the cultivation of the influenza bacillus. It is ordinary
agar-agar whose surface is coated with a little blood
secured under antiseptic precautions from the finger-tip,
ear-lobule, etc., of man, or the veins of one of the lower
animals. Some bacteriologists prepare a hemoglobin
agar-agar by spreading a little powdered hemoglobin
upon the surface of the agar-agar. This has the disad¬
vantage that powdered hemoglobin is not sterile, and the
medium must be sterilized after its addition.

The blood agar-agar should be kept in the incubator a
day or two before use so as to insure perfect sterility.

Blood-serum. — The great advantage possessed by this
medium is that it is itself a constituent of the body, and
hence offers opportunities for the development of the
parasitic forms of bacteria under the most natural con¬
ditions possible. It is the most difficult of all the media
to prepare. The blood must be obtained from a slaughter¬
house in an appropriate receptacle, the best things for the
purpose being tall narrow jars of about 1 liter capacity,
wTith a tightly-fitting lid. The jars are sterilized by heat
or by washing with alcohol and ether, are carefully dried,
closed, and carried to the slaughter-house where the blood
is to be obtained. As the blood flows from the severed
vessels of the animal the jars are filled one by one. It
seems advisable to allow the first blood to escape, as it is
likely to become contaminated from the hair. By waiting
until a coagulum forms upon the hair the danger of con¬
tamination is obviated. The jars when full are allowed
to stand undisturbed until quite firm coagula form within

132

PA THOGENIC BA CTERIA .

them. If these have any tendency to cling to the glass,
each one should be given a few violent twists, so as to
break away the fibrinous attachments. After this the
jars are carried to the laboratory' and stood upon ice for
forty-eight hours, by which time the clots will have re¬
tracted considerably, and a moderate amount of clear"
serum can be removed by sterile pipettes and placed i tr

Fig. 22. — Koch’s apparatus for coagulating and sterilizing blood-serum.

sterile tubes. If the serum obtained is red and clouded
from the presence of corpuscles, it may be pipetted into
sterile cylinders and allowed to sediment for twelve hours,
then repipetted into tubes. It is evident that such com¬
plicated maneuvring will offer many possible chances of
infection; hence the sterilization of the serum is of the
greatest importance.

If it is desirable to use the serum as a liquid medium, it
is exposed to a temperature of 6o° to $5° C. for one hoar
upon each of five consecutive days. If it is thought best
to coagulate the serum and make a solid culture-medium ,
it may be exposed twice, for an hour each time — or three
times if there is distinct reason to think it contam¬
inated— to a temperature just short of the boiling-point.
During the process of coagulation the tubes should be
inclined, so as to offer a large surface for the growth of

CULTIVATION OF BACTERIA .

133

the culture. The serum thus prepared may be white, or
have a reddish-gray color if many corpuscles are pres¬
ent, and is opaque. It cannot be melted, but once solid
remains so.

Koch devised a very good apparatus (Fig. 22) for coag¬
ulating blood-serum. The bottom should be covered
with cotton, a single layer of tubes placed upon it, and
the temperature elevated until coagulation occurs. The
repeated sterilizations may be conducted in this apparatus,
or may be done equally well in the steam apparatus, the
cover of which is not completely closed, for if the tem¬
perature of the serum is raised too high it is certain to
bubble.

Loffler’s blood-serum mixture, which seems rather
better for the cultivation of some species than the blood-
serum itself, consists of 1 part of a beef-infusion bouillon
containing 1 per cent, of glucose and 3 parts of liquid
blood-serum. After being well mixed this is distributed
in tubes, and sterilized and coagulated like the blood-
serum itself. Most organisms grow more luxuriantly
upon it than upon either plain blood-serum or other
culture-media. Its special usefulness is for the Bacillus
diphtlierise, which grows upon it with rapidity and with
quite a characteristic appearance.

Alkaline Blood-serum. — According to Lorrain Smith,
a very useful culture-medium can be prepared as follows:
To each 100 c.cm. of blood-serum add i-r.5 c.cm. of a 10
per cent, solution of sodium hydrate and shake it gently.
Put sufficient of the mixture into each of a series of test-
tubes, and, laying them upon their sides, sterilize like
blood-serum, taking care that their contents are not
heated too quickly, as then bubbles are apt to form.
The result should be a clear, solid medium consisting
chiefly of alkali-albumins. It is especially useful for
the bacillus diplitlierise.

Deycke’s Alkali-albuminate. — 1000 grams of meat are
macerated twenty-four hours with 1200 c.cm. of a 3 per
cent, solution of potassium hydrate. The clear brown fluid

134

PATHOGENIC BACTERIA .

is filtered off and pure hydrochloric acid carefully added
while a precipitate forms. The precipitated albuminate
is collected upon a cloth filter, mixed with a small quan¬
tity of liquid, and made distinctly alkaline. To make
solutions of it of definite strength it can be dried, pul¬
verized, and redissolved.

The most useful formula used by Deycke was a 2}^ per
cent, solution of the alkali-albuminate with 1 per cent, of
peptone, 1 per cent, of NaCl, and gelatin or agar-agar
enough to make it solid.

Potatoes. — Without taking time to review the old
method of boiling potatoes, opening them with sterile
knives, and protecting them in the moist chamber, or
the much more easily conducted method of Esmarch in
which the slices of potato are sterilized in the small
dishes in which they are afterward kept and used, we
will at once pass to what seems the most simple and
satisfactory method of using this valuable medium — that
of Bolton and Globig :l

With the aid of a cork-borer a little smaller in diam¬
eter than the test-tube ordinarily used a number of cyl¬
inders are cut from potatoes. Rather large potatoes
should be used, the cylinders being cut transversely, so
that a number, each about an inch and a half in length,
can be cut from one potato. The skin is removed from
the cylinders by cutting off the ends, after which each
cylinder is cut in two by an oblique incision, so as to
leave a broad, flat surface. The half-cylinders are placed
each in a test-tube previously sterilized, and then are
exposed three times, for half an hour each, to the pass¬
ing steam of the sterilizer. This steaming cooks the
potato and also sterilizes it. Such cultures are apt to
deteriorate rapidly, first by turning very dark ; second,
by drying so as to be useless. Abbott has shown that
if the cut cylinders be allowed to stand for twelve hours
in running water before being dispensed in the tubes,
they do not turn dark. Drying may be prevented by

1 The Medical News , vol. 1., 1887, p. 138.

CULTIVATION OF BACTERIA.

135

adding a few drops of clean water to each tube before
sterilizing. It is not necessary to have a special small
chamber blown in the tube to contain this water ; only
a small quantity need be added, and this will not touch
the potato, which does not reach the bottom of the
rounded tube.

A potato-juice has also been suggested, and is of some
value. It is made thus : To 300 c.cm. of water 100 grams
of grated potato are added, and allowed to stand on ice
over night. Of the pulp 300 c.cm. are expressed through
a cloth and cooked for an hour on a water-bath. After
cooking, the liquid is filtered and receives 4 per cent, of
glycerin. It may or may not need neutralization. Upon
this medium the tubercle bacillus grows well, especially
when the reaction of the medium is acid, but loses its
virulence.

Milk. — Milk is useful as a culture-medium. As when
the milk stands the cream which rises to the top is a
source of inconvenience, it is best to secure from a dairy
fresh milk from which the cream has been removed by
a centrifugal machine. It is placed in sterile tubes and
sterilized by steam by the intermittent method. The
opaque nature of this culture-medium often permits the
undetected development of contaminating organisms.
A careful watch should therefore be kept upon it lest it
spoil.

Litmus Milk. — This is milk to which just enough of
a saturated -watery solution of pulverized litmus is added
to give a distinct blue color. Cow’s milk is inclined to
be acid in reaction, and a small ' amount of sodium car¬
bonate may be necessary to give it a distinct blue. The
use of litmus is probably the best method of determining
whether bacteria by their growth produce acids or alka¬
lies.

The watery solution of litmus, being a vegetable in¬
fusion, is likely to spoil; hence it should always be treated
like the culture-media and sterilized by steam every time
the receptacle in which it is kept is opened.

136 PA THOGENIC BA CTERIA .

Petruschky’s Whey. — In order to differentiate be¬
tween acid and alkaline producers among the bacteria,
Petruschky has recommended a neutral whey colored
with litmus. It is made as follows:

To a liter of fresh skimmed milk i liter of water is
added. The mixture is violently shaken. About io c.ctn.
are now taken out as a sample to determine how much
hydrochloric acid must be added to produce coagulation
of the milk, and, having determined the least quantity
required for the whole bulk, it is added. After coagulation
the whey is filtered off, exactly neutralized and boiled.
After boiling it is generally found clouded and acid in
reaction. It is therefore filtered again, and again neu¬
tralized. Litmus is finally added to the neutral liquid, so
that it has a violet color, which can readily be changed to
blue or red by alkalies or acids.

The medium is a very useful aid in differentiating
the typhoid and colon bacilli, showing well the alkaline
formation of the typhoid bacillus.

Peptone Solution, or Dunham’s solution, is very use¬
ful for the detection of certain faint colors. It is a per¬
fectly clear, colorless solution, made as follows:

Sodium chlorid, 0.5^ Boil until the ingredients

Witte’s dried peptone, 1. > dissolve; then filter, fill

Water, 100. ) into tubes, and sterilize.

It is one of the best media for the detection of indol.
In it the bacillus pyocyaneus produces its blue color. A
very important fact in regard to peptone has been pointed
out by Garini,1 who found that many of the peptones
upon the market were impure, and on this account failed
to show the indol reaction for bacteria known to produce
indol. He recommends the use of the biuret reaction
for testing the peptone to be employed. The reagent
_used is Fehling’s copper solution, with which pure pep¬
tone strikes a violet color not destroyed upon boiling,

1 Centralbl. f Bakt. u. Parasitenk., 1893, xiii., p. 790.

CULTIVATION OF BACTERIA .

137

while impure peptone gives a red or reddish-yellow pre¬
cipitate. Both the peptone and copper solution should
be in a dilute form to make successful tests. The
addition of 4 c.cm. of the following solution —

Rosalie acid, 0.5,

80 per cent, alcohol, 100.

makes it become an excellent reagent for the detection
of acids and alkalies. The solution is pale rose in color.
If the bacterium produces acids, the color fades; if alka¬
lies, it intensifies. As the color of rosalic acid is destroyed
by glucose, it cannot be used in culture-media contain¬
ing it.

Theobald Smith calls attention to the fact that Dun¬
ham’s solution is unsuited to the growth of many bac¬
teria, some failing altogether to grow in it, and recom¬
mends that, instead, bouillon free of dextrose shall be used.
All bacteria grow well in it, and the indol-reaction is
pronounced in sixteen-hour-old cultures. His method of
preparation1 is as follows: beef-infusion, prepared either
by extracting in the cold or at 6o° C. , is inoculated in
the evening with a rich fluid culture of some acid-pro¬
ducing bacterium (Bacillus coli), and placed in the ther¬
mostat. Early next morning the infusion, covered with
a thin layer of froth, is boiled, filtered, peptone and salt
added and the neutralization and sterilization carried on
as usual.

To test for the presence of indol, the bacterium is
planted in the culture-medium, allowed to grow for
upward of twelve hours, and then subjected to the com¬
bined action of a nitrite and chemically pure sulphuric
acid. In making the test, Smith adds to each tube 1 c. cm.
of a 0.01 per cent, solution of KN02, freshly prepared,
and 10 drops of chemically pure H2S04. The presence
of indol is characterized by the production of a red
color.

1 Journal of Exp. Medicine , vi., Sept. 5, 1897, p. 546.

138 PATHOGENIC BACTERIA.

It is not intended that the student shall infer that
there are no culture-media other than these, which have
been selected because of their usefulness and popularity.
Many other compounds and as many simple substances
are employed ; for example, eggs, white of egg, urine,
bread, sputum, sugar solutions, hydrocele fluid, and
aqueous humor.

CHAPTER VII.

CULTURES, AND THEIR STUDY.

The objects which we have had before us in the prep¬
aration of the culture-media were numerous. We have
prepared them so as to allow us to separate — or, rather,
to isolate- — bacteria, to keep them in healthy growth for
considerable lengths of time, to enable us to observe their
biologic peculiarities, and to introduce them without dif¬
ficulty into the bodies of animals.

The isolation of bacteria was impossible until the fluid
culture-media of the early observers were replaced by the
solid media, and was exceedingly crude until Koch gave
us the solid, transparent media and the well-known
“plate-cultures.”

A growth of artificially-planted micro-organisms in
which an immense number are massed together is called
a culture. If such a growth contains but one kind of
organism, it is known as a pure culture.

It has become the habit at present to use the term ‘ 1 cul¬
ture” rather loosely, so that it does not always signify a
growth of micro-organisms artificially planted, but may
signify a growth taking place under natural conditions ;
thus, typhoid bacilli are said to exist in the spleens of
patients dead of that disease “in pure culture,” because
no other bacteria are there ; and sometimes, when in ex¬
pectorated fragments of cheesy matter from tuberculosis
pulmonalis the tubercle bacilli are very numerous and
umnixed with other bacteria, the term “pure culture”
is again used to describe the condition.

Three principal methods are at present employed to
enable us to secure pure cultures of bacteria, biit before
beginning a description of them it is well to observe that

139

140

PATHOGENIC BACTERIA.

the peculiarities of certain pathogenic forms enable us
to use special means, taking advantage of their eccentrici¬
ties, for their isolation, and that the general methods are
in reality more useful for the non-path ogenic than for the
pathogenic forms.

All three methods depend upon the observation of
Koch, that when germs are equally distributed through¬
out some liquefied nutrient medium which can be solidi¬
fied in a thin layer, the growth of the germs takes place
in little scattered groups or families, called colonies , dis¬
tinctly separated from each other and capable of trans¬
plantation to tubes of culture-media.

Plate-cultures. — The plate-cultures, originally made
by Koch, require considerable apparatus, and of late years
have given place to the more ready methods of Petri and
Von Esmarch. So great, however, is the historic interest
attached to the plates that it would be a great omission
not to describe Koch’s method in full.

Apparatus . — Half a dozen glass plates, about 6 by 4
inches in size, free from bubbles and scratches and
ground at the edges, are carefully cleaned, placed in a
sheet-iron box made to receive them, and then put in

Fig. 23. — Complete levelling appa¬
ratus for pouring plate-cultures, as
taught by Koch.

the hot-air closet, where
they are sterilized. The box:,
which is tightly closed, al¬
lows the sterilized plates to
be kept on hand indefinitely
before using.

A moist chamber, or double
dish, about 10 inches in di¬
ameter and 3 inches deep, tlie
upper half being just enough,
larger than the lower to allow
it to close over it, is carefully

washed. A sheet of bibulous

paper is placed in the bottom, so that some moisture can
be retained, and a 1 : 1000 bichlorid solution is poured in
and brought in contact with the sides, top, and bottom

CULTURES ; AND THEIR STUDY.

141

by turning the dish in all directions. The solution is
emptied out, and the dish, which is always kept closed,
is ready for use.

A levelling apparatus is required (Fig. 19). This con¬
sists of a wooden tripod with adjustable screws, and a glass
dish covered by a flat plate of glass upon which a low
bell-jar stands. The glass dish is filled with broken ice
and water, covered with the glass plate, and then exactly
levelled by adjusting the screws under the legs of the
tripod. When level the cover is placed upon it, and it
is ready for use.

Method (Fig. 24). — A sterile platinum loop is dipped
into the material to be examined, a small quantity se-

Fig. 24. — Method of holding tubes during inoculation.

cured, and stirred about so as to distribute it evenly
through a tube of the melted gelatin. If the material
under examination is very rich in bacteria, one loopful
may contain a million individuals, which, if spread out
in a thin layer, would develop so many colonies that it
would be impossible to see any one clearly ; hence the
necessity for a dilution. From the first tube a loopful
of gelatin is carried to a second tube of melted gelatin
and stirred well, so as to distribute the organisms evenly
through it. In this tube we may have no more than ten
thousand organisms, and if the same method of dilution
be used again, the third tube may have only a few hun¬
dreds, and a fourth only a few dozen colonies.

After the tubes are prepared, one of the sterile glass
plates is caught by its edges, removed from the iron box,
and placed beneath the bell-glass upon the cold plate

142

PATHOGENIC BACTERIA .

covering the ice-water of the levelling apparatus. The
plug of cotton closing the mouth of tube No. i is re¬
moved, and to prevent contamination during the outflow
of the gelatin the mouth of the tube is held in the flame
of a Bunsen burner for a moment or two. The gelatin
is then cautiously poured out upon the plate, the mouth
of the tube, as well as the plate, being covered by the
bell-glass to prevent contamination by germs in the air.
The apparatus being level, the gelatin spreads out in an
even, thin layer, and, the plate being cold from the ice

beneath, it immediately solidi¬
fies, and in a few moments can
be removed to the moist chain-

^ ber prepared to receive it. As

Fig. 25.— Glass bench. ,

soon as plate No. 1 is prepared,
the contents of tube No. 2 are poured upon plate No. 2,
allowed to spread out and solidify, and then superimposed
on plate No. 1 in the moist chamber, being separated from
the plate already in the chamber by small glass benches
(Fig. 25) made for the purpose and sterilized. After the
contents of all the tubes are thus distributed, the moist
chamber and its contents are allowed to stand for some
hours, to permit the bacteria to grow. Where each or¬
ganism falls a colony develops, and the success of the
whole method depends upon the isolation of a colony
and its transfer to a tube of culture-medium where it
can grow unmixed and undisturbed.

The description must have made evident the fact that
only such culture-media can be used for plate-cultures as
can be melted and solidified at will — viz. gelatin, agar-
agar, and glycerin agar-agar. Blood-serum and L,offler\s
mixture are entirely inappropriate.

The great drawback to this excellent method is the
cumbersome apparatus required and the comparative im¬
possibility of making plate-cultures, as is often desirable,
in the clinic, at the bedside, or elsewhere than in the
laboratory. The method therefore soon underwent mod¬
ifications, the most important being

CULTURES , AND THEIR STUDY.

143

Petri’s Dishes. — These small dishes (Fig. 26), about
4 inches in diameter and ]/2 inch deep, with accurately
fitting lids, are about as convenient as anything that has
been devised in bacteriological technique. They dis-

Fig. 26. — Petri dish for making plate-cultures.

pense with plates and plate-boxes, with moist chambers
and benches, and usually with the levelling apparatus,
though this is still employed in connection with the
Petri dishes in some laboratories.

The method of the employment of Petri dishes is very
simple. The dishes are carefully cleaned, polished, and
sterilized by hot air, care being taken that they are placed
in the hot-air closet right side up, and after sterilization
are kept covered and in that position. The dilution of
the material under examination is made with gelatin or
agar-agar tubes in the manner described above, the plugs
are removed, the mouth of the tube is cautiously held
for a moment in the flame, then the contents of each
tube are poured into one of the sterile dishes, whose top
is elevated just sufficiently to allow the mouth of the
tube to enter. The gelatin is spread over the bottom
of the dish in an even layer, is allowed to solidify,
labelled, and then stood away for the colonies to develop.

Esmarch Tubes. — This method, devised by Esmarch,
converts the walls of the test-tube into the plate and dis¬
penses with all other apparatus. The tubes, which are
inoculated and in which the dilutions are made, should
contain less than, half the usual amount of gelatin or
agar-agar. After inoculation the cotton plugs are pushed
into the tubes until even with their mouths, and then
covered with a rubber cap, which protects them from
wetting. A groove is next cut in a block of ice, and

144

PATHOGENIC BACTERIA.

the tube, held almost horizontally, is rolled in this until
the entire surface of the glass is covered with a thin
layer of the solid medium (Fig. 27). Thus the tube
becomes the plate upon which the colonies develop.

Fig. 27. — Esmarch tube on block of ice (redrawn after Abbott).

Several little points need to be observed in carrying*
out Esmarch’s method. The tube must not contain too
much culture-medium, or it cannot be rolled into an even
layer. In rolling the contents should not touch the cotton
plug, lest it be glued to the glass and its subsequent use¬
fulness be injured. No water must be admitted from the
melted ice.

The offspring of each bacterium growing upon the
film of gelatin constituting a plate-culture form a mass
which has already been pointed out as a colony . These
small bacterial families may be seen through a micro¬
scope when still much too small for detection by the
naked eye, and because of their minuteness should always
be studied with the microscope.

The original plates of Koch are very inconvenient for
such examination, because it is impossible to remove
them from the moist chamber and lay them upon the
stage of the microscope without exposing them to the
danger of contamination by the atmosphere, so that the
advantages of Petri dishes and Esmarch tubes, where
the examination may be made through the glass tube or

CULTURES , AND THEIR STUDY. 145

through the bottom of the inverted dish, will be more
than ever apparent.

The colonies should be viewed from time to time in
their growth, drawings being made of the appearances,
so as to form a series showing the developmental cycle.
Most colonies will be found to originate as spherical, cir¬
cumscribed, slightly granular, yellowish, greenish, or
brownish dots, and later to send out offshoots or filaments
or to develop concentric rings or characteristic liquefac¬
tions. A few appear from the very first as woolly clumps
of entangled threads.

Some of the most diverse forms of colonies are repre¬
sented iu the accompanying illustrations (Figs. 28-32).

Figs. 28, 29, 30. — The various appearances of colonies of bacteria under the
microscope: a , colony of Bacillus liquefaciens parvus (Liideritz) ; b, colony
of Bacillus polypiformis (Liborius); c, colony of Bacillus radiatus (Liideritz).

A pure culture, when obtained from colonies growing
upon a plate, must always be made from a single colony Y
the transplantation being accomplished under a low power
of the microscope. The naked eye can rarely be depended!
upon to recognize the purity of a colony or its isolation.

Selecting as isolated, large, and characteristic a colony
as possible, it is brought to the centre of the field. A
platinum wire, securely fused into a glass handle about
8 inches long, is sterilized by being made incandescent
in a Bunsen flame, cooled, and then cautiously manipu¬
lated until, while it is watched through the microscope,
10

146

PATHOGENIC BACTERIA.

it is seen to touch the colony and take part of its con¬
tents away. In this mcineuvre the wire must not touch
the objective , the glass , or anything except the colony.
Having secured the adhesion of a few bacteria to the
sterile wire, the pure culture is made by introducing
them into a sterile culture-medium.

If the pure culture is to be made in bouillon, the tube
is held obliquely, so that when the cotton plug is cau¬
tiously removed no germs can fall in from the air. The
plug is removed by a twisting movement. The wire, with¬
out being allowed to touch the mouth or sides of the
tube, is plunged' into its

Figs. 31, 32. — The various appearances of colonies of bacteria under the
microscope : a , colony of Bacillus muscoides (Liborius) ; b, colony of Bacillus
anthracis (Fliigge).

moved and the plug replaced. The wire should be im¬
mediately sterilized by heating to incandescence, lest the
bacteria be pathogenic and capable of doing subsequent
harm.

If the culture is to be made in gelatin, a different
method is employed. The tube is either held horizon¬
tally, or, as is perhaps better, inverted ; the cotton plug

CULTURES , AND THEIR STUDY.

147

is removed cautiously ; the wire bearing the bacteria
from the colony is introduced until its point enters the
centre of the gelatin, and is then carefully pushed on
until a vertical puncture from the surface to the bottom
of the gelatin is made. This is the puncture-culture —
u stichcultur n of the Germans.

If the bacteria are only to be planted upon the surface
of the culture-medium, the wire is drawn over the surface
of a tube of obliquely solidified gelatin, agar-agar, blood-
serum, etc. with a steady, slow movement, so as to scatter
the germs along its path and cause the development of
the bacteria in an enormous colony or mass of colonies
in a line following the longest diameter of the exposed
surface from end to end. This is the stroke-culture —
“ strichcultur. ”

The method of holding the tubes, cotton plugs, and
platinum wire during the process of inoculation is shown
in Figure 20.

Sometimes it is desirable to preserve an entire colored
colony as a microscopic specimen. To do this a perfectly
clean cover-glass, not too large in size, is momentarily
warmed, then carefully laid upon the surface of the
gelatin or agar-agar containing the colonies. Sufficient
pressure is applied to the surface of the glass to exclude
bubbles underneath, but the pressure must not be too
great, as it may destroy the integrity of the colony.
The cover is gently raised by one edge, and if successful
the whole colony or a number of colonies, as the case
may be, will be found adhering to it. It is treated
exactly as any other cover-glass preparation, is dried,
fixed, stained, and mounted, and kept as a permanent
specimen. It is called an adhesion preparation — “ klatsch
praparat. ’ ’

Very often, when one is in a hurry, pure cultures from
single colonies may be secured by a very simple manipu¬
lation suggested by Banti.1 The inoculation is made
into the water of condensation at the bottom of an agar-

1 Centralbl. f Bakt. und Parasitenk.> 1895, xvii., No. 16.

148

PATHOGENIC BACTERIA.

agar tube, without touching the surface. The tube is
then inclined so that the water flows over the agar,
after which it is stood away in the vertical position.
Colonies will grow where bacteria have been floated upon
the agar-agar, and may be picked up later in the same
manner as from a plate.

In other cases pure cultures may best be secured by
animal inoculation. For example, when the tubercle
bacillus is to be isolated from milk or urine which con¬
tains rapidly growing bacteria that would outgrow the
slow-developing tubercle bacillus, it is better to inject
some of the fluid into the abdominal cavity of a guinea-
pig and await the development of tuberculosis, and then
seek to secure the bacillus from the unmixed material in
the softened lymphatic glands. Anthrax bacilli are also
more easily secured in pure culture by inoculating a
mouse and recovering the bacilli from a spleen or the
heart’s blood after death, than by going to the trouble of
making plates and picking out the colonies.

In many cases when it is desired to isolate the micro¬
coccus tetragenus, the pneumococcus, and others, it is
easier to inoculate the most susceptible animal and
recover the germ from the organs than to plate it out and
search for the colony among many others which may be
similar to it.

The development of bacteria in liquids is of less in¬
terest than that upon solid media. The growth generally
manifests itself by a diffused turbidity. Sometimes flocculi
float in the otherwise clear medium. Some forms grow
most rapidly at the surface of the liquid, and produce a
distinct membranous pellicle called a mycoderma. In
such a growth multitudes of degenerated bacteria and
large numbers of spores are to be observed. On the
other hand, it occasionally happens that the growth
occurs chiefly below the surface, and may produce gelat¬
inous masses which are known as zooglea.

In gelatin the bacteria exhibit a great variety of ap¬
pearances, many of which are beautiful and interesting.

CULTURES , AND THEIR STUDY.

149

Certain bacteria, as the tubercle bacillus, will not grow
at all upon gelatin. Some forms which are rigidly ae¬
robic will only grow upon or near the surface ; others,
anaerobic, only in the deeper parts. The majority, how¬
ever, grow both upon the surface and in the puncture
made by the wire. Sometimes the consistence of the
gelatin is unaltered ; sometimes it is liquefied throughout,
sometimes only at the surface. Sometimes offshoots ex¬
tend from the colonies into the gelatin, giving the culture

a b c d e f

Fig. 33. — Various forms of gelatin puncture-cultures: a, Bacillus typhi ab-
dominalis ; b, B. anthracis ; 4 B. mycoides ; d, B. mesentericus vulgatus ;
e, B. of malignant edema ; yj B. radiatis.

a bristling appearance. Figure 33 will serve to illustrate
different varieties of gelatin growth.

The growth in gelatin is generally so far removed from
the walls of the tube (a central puncture nearly always
being made in the culture-medium, in order that the
growth be symmetrical) that it is next to impossible to
make a microscopical examination of it with any power
beyond that given by a hand-lens.

Much attention has been given of late to the preparation
of microtome sections of the gelatin growth. To accom¬
plish this the glass is warmed sufficiently to allow the
gelatin to be removed and placed in Muller’s fluid (bi-

*5°

PATHOGENIC BACTERIA .

chromate of potassium 2.-2. 5, sulphate of sodium 1,
water 100), where it is hardened. When quite firm it
is washed in water, passed through alcohols ascending
in strength from 50 to 100 per cent., imbedded in cel-
loidin, cut wet, and stained like a section of tissue.

A ready method of doing this has been suggested by
Winkler,1 who bores a hole in a block of paraffin with
the smallest-size cork-borer, soaks the block in biclilorid
solution for an hour, pours liquid gelatin into the cavity,
allows it to solidify, inoculates it by the customary punc¬
ture of the platinum wire, allows it to develop sufficiently,
and when ready cuts the sections under alcohol, subse¬
quently staining them with much-diluted carbol-fuchsiu.

Very pretty museum specimens of plate- and puncture-
cultures in gelatin can be made by simultaneously killing
the micro-organisms and permanently fixing the gelatin
with formalin, which can either be sprayed upon the
gelatin or applied in dilute solution. As gelatin fixed
in formalin cannot subsequently be liquefied, such prep¬
arations will last indefinitely.

The growths which occur upon agar-agar are in many
ways less characteristic than those in gelatin, but as this
medium does not liquefy except at a high temperature
(ioo° C.), it has that great advantage over gelatin. The
colorless or almost colorless condition of the preparation
also aids in the detection of such chromogenesis as may
be the result of the micro-organismal growth.

Sometimes the growth is colored, sometimes not ; some¬
times the production of a soluble pigment colors the
agar-agar as well as the growth ; sometimes the growth
is one color and the agar-agar another. Sometimes the
growth is filamentous, sometimes a smooth, shining band.
Occasionally the bacterium does not grow upon agar-agar
unless glycerin be added (tubercle bacillus) ; sometimes
it will not grow even then (gonococcus).

Still less characteristic are the growths upon potato.
Most bacteria produce rather smooth, shining, irregu-

1 Fortschritte der Medicin , Bd. xi., 1893, No. 22.

CULTURES , AND THEIR STUDY . 151

larly-extending growths, which often show very beautiful
colors.

pIG> 34. — New model incubating-oven with electro-regulator.

In milk and litmus milk one must observe the presence
or absence of acid-production, the coagulation which may

*5*

PATHOGENIC BACTERIA .

or may not accompany it, and the subsequent gelatiniza-
tion or digestion of the coagulum.

Blood-serum is liquefied by some bacteria. The ma¬
jority of organisms are not very characteristic in their
development upon it. Others, as the bacillus of diph¬
theria, are, however, characterized by their shape, color,
and rapidity of development at given temperatures.

While most of the saprophytic bacteria will grow well
at the ordinary temperature of a well- warmed room, the
important pathogenic forms require to be kept at the
temperature of the body. To do this accurately an in¬
cubating oven becomes a necessity. Various forms, of
wood and metal, are in the market, the one shown in the
illustration (Fig. 34) being one of the newest and best.

It scarcely need be pointed out that gelatin cultures
cannot be grown in the incubating oven, as the medium
will not remain solid at temperatures above 20-22° C.

CHAPTER VIII.

THE CULTIVATION OF ANAEROBIC BACTERIA.

The cultivation of micro-organisms which, will not
grow where the least amount of oxygen is present is
always attended with much difficulty, and can seldom be
accomplished with certainty. Many methods have been
suggested, but not one can be described as satisfactory.

Koch originally cultivated anaerobic bacteria upon
plates by covering the surface of the soft gelatin with a
thin film of mica previously sterilized by incandescence.
Some anaerobic forms will grow quite well by such a
simple exclusion of the air, but the strictly anaerobic
forms will not develop at all.

Hesse originated the plan, still sometimes followed, of
making a deep puncture in recently boiled and rapidly
sterilized gelatin or agar-agar, then covering the surface
with sterilized oil, through which no oxygen was sup¬
posed to penetrate (Fig. 35).

Eiborius suggested the plan of having a tube nearly
full of gelatin or agar-agar, boiling it just before inocu¬
lation, so as to expand and drive out whatever air it
might contain, making the inoculation while the culture-
medium was still fluid, cooling rapidly in ice-water, and
sealing up the tube in a blowpipe as near the surface of
the gelatin as possible.

Esmarch used a regular “ Esmarch tube,” into the
central cavity of which melted sterile gelatin was poured
to exclude the air.

Buchner invented a method by which, by the use of
jjyrogallic acid, the oxygen was absorbed from the atmo¬
sphere in which the culture was kept, and the growth
allowed to continue in the nitrogen and carbonic acid

153

*54

PATHOGENIC BACTERIA.

which remained (Fig. 36). His method was to place the
tube which had been inoculated in a much larger outer
test-tube containing alkaline pyrogallic acid. The large

Fig. 35. — Hesse’s
method of making
anaerobic cultures.

Fig. 36. — Buchners
method of making an¬
aerobic cultures.

Fig. 37. — Frankel’ s meth¬
od of making anaerobic cul¬
tures.

tube was closed with a rubber cap, and the absorption of
the oxygen allowed to progress.

Gruber, instead of absorbing the oxygen as Buchner
does, prefers to use an air-pump and exhaust the contents
of the tube. He uses a tube having a slender neck and
a perforated rubber stopper. After the inoculation is
made the air is pumped out and the slender neck sealed
in the blowpipe. After this the tube can be warmed and
the melted gelatin or agar-agar rolled on its sides, as sug¬
gested by Esmarch, if desired.

Better than any of the preceding is the method of
Frankel, which removes the; air and replaces it by hy¬
drogen. Frankel prepares an ordinary Esmarch tube,
removes the cotton stopper, and replaces it by a carefully
sterilized rubber cork containing two tubes (Fig. 37). The

CULTIVATION OF ANAEROBIC BACTERIA . 155

tubes are connected with a hydrogen generator, and the
gas is allowed to pass through until all the oxygen is
forced out and replaced by the hydrogen, after which the
ends of the tubes are sealed in the flame (Fig. 36).

Iviborius has designed a special tube for accomplish¬
ing the same thing.

Kitasato and Weil found the addition of 0.3-0. 5 per
cent, of sodium formate to be of use in aiding the rapid¬
ity of the development of anaerobic cultures. Liborius
found that 2 per cent, of glucose added ‘to the culture-
medium also increased the rapidity of the process.

The methods now generally employed by bacteri¬
ologists for the anaerobic cultivations embrace all the
essentials of the foregoing methods. One of the best
arrangements for the purpose is that devised by Dr.
Ravenel. His inoculations are deeply made in culture-
media as free from air as possible. The tubes are
loosely plugged, and are placed in an air-tight cham¬
ber the bottom of which contains pyrogallic acid — py-
rogallic acid 1, solution of caustic potash 1, water 10.
The apparatus is connected by two tubes with an ex¬
haust-pump on one side, and with a hydrogen appara¬
tus on the other, by which means the atmosphere is ex¬
hausted, and replaced by hydrogen until only pure hydro¬
gen remains, after which the chamber is permanently
sealed and the germs allowed to grow. Such a chamber
can be constructed to hold a number of tubes or Petri
dishes, yet not be too large to be stood in an incubator.
Whatever oxygen may have escaped the exhaustion or
have entered by the process of leakage is at once absorbed
by the pyrogallic acid in the lower chamber of the ap¬
paratus.

Apparatus for plating out strictly anaerobic bacteria
that have met with great favor are those invented by
Botkin (Fig. 38) and Novy (Fig. 39). The first mentioned
combines the replacement of the air by hydrogen and the
absorption of the oxygen possibly remaining by alkaline
pyrogallic acid ; the other simply replaces the oxygen by

156

PATHOGENIC BACTERIA .

hydrogen. In using Botkin’s apparatus the uncovered
Petri dishes are placed one above the other in the rack c,
and covered with the bell-glass A. Liquid paraffin is
poured in the dish B until it is about half full. From a

Kipp’s apparatus hydrogen
gas enters the little rubber
tube a , subsequently escap¬
ing by the tube b. When
only pure hydrogen escapes
the rubber tubes a and b are
withdrawn, and the appa¬
ratus remains filled with hy¬
drogen. Lest a little oxygen
should remain, it is best to
have the dishes at the top
and bottom of the rack filled
with alkaline pyrogallic
acid. Tetanus can be cul¬
tivated in this apparatus.
The jars recently intro-

Fig. 38. — Botkin’s apparatus for mak- 1 v -vt

ing anaerobic cultures. dUCed N°Vy afe Sllllllar

in principle, depending
upon the replacement of the air by hydrogen. They are

Fig. 39. — Novy’s jars for anaerobic cultures.

so constructed that when the stopper occupies a certain
relative position to the neck the gas can enter and exit,

CULTIVATION OF ANAEROBIC BACTERIA. 157

but when the stopper is turned a little the jar is hermet¬
ically sealed. Alkaline pyrogallic acid in a test-tube, or
in the bottom of the jar, will serve to absorb any remain¬
ing oxygen. The larger jar (Fig. 39, a) is intended for
Petri dishes, the smaller one (b) for test-tube cultures.

Roux has suggested the simplest method of cultivating
anaerobic bacteria. The germs are distributed through
freshly boiled, still liquid, gelatin or agar-agar, as in
making the dilutions for plate-cultures, then drawn into
a long, slender sterile piece of glass tubing of small
calibre. When the tube is full the ends, which should
have been narrowed, are closed in a flame, and the cul¬
ture is hermetically sealed in an air-tight chamber. The
chief difficulty is in transplanting the growing colony.
To do this the tube must be opened with a file or- a dia¬
mond at the point where the colony desired is observed.

CHAPTER IX.

EXPERIMENTATION UPON ANIMAES.

Bacteriology lias to-day become a science whose
principal objects are to discover the cause, explain the
symptoms, and prepare the cure of diseases. We can¬
not hope to achieve these objects except by the intro¬
duction of bacteria into animals, where their effects and
the effects of their products can be studied.

No one should more heartily condemn wanton cruelty
to animals than the physician and the naturalist. In¬
deed, it is hard to imagine a class of men so much of
whose lives is spent in relieving pain, and who know so
much about pain, being guilty of the wholesale butchery
and torture accredited to them by a few of the laity,
whose eyes, but not whose brains, have looked over
the pages of physiological text-books.

Experimentation upon animals has given us almost
all our knowledge of physiology, most of our valuable
therapeutics, and the only scientific methods of treating
tetanus and diphtheria.

Experiments upon animals we must make, and, as
animals differ in their susceptibility to diseases, large
numbers and different kinds must be employed.

The bacteriological methods are not cruel. Two prin¬
cipal modes of introducing bacteria are employed : the
subcutaneous injection and the intravenous injection.

Subcutaneous injections into animals are made exactly
as hypodermic injections are given to man.

Any hypodermic syringe that can be conveniently
cleaned and disinfected may be employed for the purpose.
Those expressly designed for bacteriological work and
most frequently employed are shown in Fig. 40.' Those

158

EXPERIMENTATION UPON ANIMALS. 159

of Meyer and Roux resemble ordinary hypodermic
syringes; that of Koch is supposed to possess the decided
advantage of not having a piston to come into contact
with the fluid to be injected. This is, however, some¬
what disadvantageous inasmuch as the cushion of com¬
pressed air that drives out the contents is elastic, and un¬
less carefully watched will follow the injection into the
body of the animal. In making subcutaneous injections
there is no disadvantage or danger from the entrance of

Fig. 40. — 1, Roux’s bacteriological syringe; 2, Koch’s syringe; 3, Meyer’s
bacteriological syringe.

air beneath, the skin, but in intravenous injections it is
commonly supposed to be dangerous.

All syringes should be disinfected with carbolic acid
solutions before and after using, the carbolic acid being-
allowed to act for some time and then washed out
with sterile water. Syringes should not be boiled, as
'it ruins the packings, whether of asbestos, leather, or
rubber.

The intravenous injections differ only in that the needle
of the syringe is introduced into a vein. This is easy in a
large animal like a horse, but is very difficult in a small
animal, and wellnigh impossible in anything smaller than
a rabbit. Such injections when given to rabbits are gen¬
erally made into the ear-veins, as those most conspicuous
and accessible (Fig. 41). A peculiar and important fact
to remember is, that the less conspicuous posterior vein

160 PATHOGENIC BACTERIA.

is much better adapted to the purpose than the anterior.
The introduction of the needle should be made from the

hairy surface of the
ear.

If the ear is manip¬
ulated for a moment or
two before the injec¬
tion is begun, vaso¬
motor dilatation
occurs and the blood¬
vessels all become
larger and more con¬
spicuous. The vein
should be compressed
at the root of the ear
until the needle is in¬
troduced, and the in-
Fig. 41. — Method of making an intravenous jectiou made CIS near
injection into a rabbit. Observe that the needle the root as possjble.
enters the posterior vein from the hairy surface. r . .

The introduction of
bacteria into the lymphatics is only possible by injecting
liquid preparations of them into some organ with com¬
paratively few blood-vessels and large numbers of lym¬
phatics. The testicle is best adapted to this purpose, the
needle being introduced deeply into the organ.

Sometimes the inoculation can be made by the platinum
wire, a very small opening made in the skin by a snip of
the scissors being sufficient.

Sometimes intra-abdominal and intra-pleural injections
are made, and in cases where it becomes necessary to
determine the presence or absence of tuberculosis or
glanders in tissues it may be necessary to introduce small
pieces of the suspected tissue under the skin or into the
abdominal cavities. To do this is not difficult. The
hair is carefully, closely cut over the point of election,
which is generally on the abdomen near the groin, the
skin picked up with forceps, a snip made through it,
and the points of the scissors introduced for half an inch

f<;xi'i*:h'/.);KXTAZ'zo\r itox aximals. 161

or so and then separated. By this maneuver a subcuta¬
neous pocket is formed, into which the tissue is easily
forced. The opening should not be large enough to re¬
quire subsequent stitching.

vSmall animals, like rabbits and guinea-pigs, can be held
in the hand, as a rule. Rabbit-holders of various forms
can be obtained from dealers. I )< >gs, cats, sheej), and goats
can be tied and held in Boughs. A convenient form of
mouse-holder, invented by Kitasato, is shown in Fig. 42.

In all these experiments one must remember that the
amount of material introduced into the animal must be
in proportion to its size, and that injection-experiments
upon mice generally are so crude and destructive as to
warrant the comparison drawn by Frnnkel, that to inject
a few minims of liquid into the pleural cavity of a mouse
is u much the same as if one would inject through a fire¬
hose three or four quarts of some liquid into the respira¬
tory organs of a man."

The blood of animals, when it is necessary to experi¬
ment with it, is best secured from
a large vein, generally the jugu¬
lar. From small animals, such as
guinea-pigs, it may be secured by
introducing a small cannula into
the carotid artery.

Our observations of animals by
no means cease with their death.

Indeed, he cannot be a bacteriol¬
ogist who is not already a good
pathologist and expert in the recog¬
nition of diseased organs.

When an autopsy is to be made
upon a small animal, it is best to
wash it for a few moments in a
disinfecting solution, to kill the
germs present upon the hair and the skin, as well as to
moisten the hair and enable it to be kept out of the
incision.

i6a

PATHOGENIC BACTERIA.

The animal should be tacked to a board if small, or
tied, by cords fastened to the legs, to the corners of a
table if large, and should be dissected with sterile knives
and scissors. When a culture is to be made from the
interior of an organ — say the spleen — it should be incised
deeply with a sterile knife and the culture made from
its centre.

Fragments intended for subsequent microscopical ex¬
amination should be cut very small (cubes of i c.cm.),
placed in absolute alcohol for a few hours, then trans¬
ferred to weaker alcohol, 80-90 per cent., for preserva¬
tion. The technique of imbedding and staining the tis¬
sues can be found in almost any reliable text-book on
pathology or on the special subject of microscopical
technique.

CHAPTER X.

THE RECOGNITION OF BACTERIA.

The most difficult tiling in bacteriology is to be able
to recognize the bacteria which come under observation.

A certain few micro-organisms are so characteristic in
shape and grouping as to be separated by a microscopic
examination. Some, as the tubercle bacillus, are charac¬
teristic in their reaction to the anilin dyes, and can be
differentiated at once by this peculiarity. Some, as the
Bacillus mycoides, are so characteristic in their agar-agar
growth as to eliminate others. The red color of Bacillus
prodigiosus and the blue of Bacillus jantliinus will speak
almost positively for them. The potato culture of the
Bacillus mesentericus fuscus and its close relative the vul-
gatus is quite sufficient to enable us to pronounce upon
them. Unfortunately, however, there are several hun¬
dreds of described species which lack any one distinct
character that may be used for differential purposes, and
require that for their diagnosis we shall wellnigh ex¬
haust the bacteriological technique in an almost fruitless
•effort to recognize them.

A series of useful tables has been compiled by Eisen-
berg, and is now almost indispensable to the worker.
Unfortunately, in tabulating bacteria we constantly meet
species described so insufficiently as to make them worse
than useless on account of the confusion caused.

The only way to recognize a species is to study it
thoroughly and compare it, step by step, with the descrip¬
tions and tables of known species compiled by Eisenberg
and others.

163

CHAPTER XI.

THE BACTERIOLOGIC EXAMINATION OF THE AIR.

IT has been repeatedly emphasized — and indeed at the
present time almost every one knows — that micro-organ¬
isms float almost everywhere in the air, and that their
presence there is a constant source of danger, not only
of contamination in our bacteriologic researches, but
also a menace to our health.

Such micro-organisms are neither ubiquitous nor equally
disseminated, but are much more numerous where the air
is dusty than where it is pure — much more so where men
and animals are accustomed to live, than upon the ocean
or upon high mountain-tops. The purity of the atmo¬
sphere bears a distinct relation to the purity of the soil
over which its currents blow.

The micro-organisms that occur in the air are for the
most part harmless saprophytes which have been sepa¬
rated from their nutrient birthplace and carried about by
the wind. They are almost always taken up from dried
materials, experiment having shown that they arise from
the surfaces of liquids in which they grow with much dif¬
ficulty. They are by no means all bacteria, and a plate
of sterile gelatin exposed for a brief time to the air will
generally grow moulds and yeasts as well as bacteria.

The bacteria present are occasionally pathogenic, espe¬
cially in localities where the discharges of diseased animals
have been allowed to collect and dry. For this reason the
atmosphere of the wards of hospitals and of rooms in
which infectious cases are being treated is much more
apt to contain them than the air of the street. However,
the dried expectoration of cases of tuberculosis, of in-

164

BACTERIOLOGIC EXAMINATION OF AIR. 165

fluenza, and sometimes of pneumonia, causes the specific
bacteria of these diseases to be far from uncommon in
street-dust.

Gunther points out that the majority of the bacteria
which occur in the air are cocci, sarcina being very
abundant. Most of them are chromogenic and do not
liquefy gelatin. It is unusual to find a considerable
variety of bacteria at a time ; generally not more than
two or three species are found.

It is an easy matter to determine whether bacteria are
present in the air or not, all that is necessary being to
expose sterile plates or Petri dishes of gelatin to the air
for a while, close them, and observe whether or not bac¬
teria grow upon them.

To make a quantitative estimation is, however, much

Fig. 43. — Hesse’s apparatus for collecting bacteria from the air.

more difficult. Several methods have been suggested, of
which the most important may be considered.

The method suggested by Hesse is simple and good.
It consists in making a measured quantity of the air to

i66

PATHOGENIC BACTERIA .

be examined pass through a horizontal sterile tube about
70 cm. long and 3.5 cm. wide (Fig. 43), the interior of
which is coated with gelatin in the same manner as an
Esmarch tube. The tube, having been prepared, is
closed at both ends with sterile corks carrying smaller
glass tubes closed with cotton. When ready for use the
tube at one end is attached to a hand-pump, the cotton
is removed from the other end, and the air passed through
very slowly, the bacteria having time to precipitate upon
the gelatin as they pass. When the required amount has
passed the tubes are again plugged, the apparatus stood
away for a time, and subsequently, when they have
grown, the colonies are counted. The number of colo¬
nies in the tube will represent pretty accurately the
number of bacteria in the amount of air which

Fig. 44. —
Petri’s sand
filter for air-
examination.

passed through the tube.

In such a cylindrical culture it will be noted
that if the air is passed through with the
proper slowness, the colonies will be much
more numerous near the end of entrance than
that of exit. The first to fall will probably
be those of heaviest specific gravity — i. e . the
moulds and yeasts.

A still more exact method is that of Petri,
who uses small filters of sand held in place in a
wide glass tube by small wire nets (Fig. 44).
The sand used is made to pass through a
sieve whose openings are of known size, is
heated to incandescence, then arranged in
the tube so that two of the little filters, held
in place by their wire-gauze coverings, are
superimposed. One or both ends of the tube
are closed with . corks having a narrow glass
tube. The apparatus is heated and sterilized
in a hot-air sterilizer, and is then ready for

use. The method of employment is very simple. By

means of a hand-pump 100 liters of air are made to pass

through in from ten to twenty minutes. The sand from

BACTERIOLOGIC EXAMINATION OF AIR . 167

the upper filter is then carefully mixed with sterile
melted gelatin and poured into sterile Petri dishes, where
the colonies develop and can be counted. Sternberg re¬
marks that the chief objection to the method is the pres¬
ence in the gelatin of the slightly opaque sand, which
interferes with the recognition and count¬
ing of the colonies. This objection has,
however, been removed by Sedgwick and
Miquel, who use a soluble material — granu¬
lated or pulverized sugar — instead of the
sand. The apparatus used for the sugar-
experiments differs a little from the original
of Petri, but the principle is the same, and
can be modified to suit the experimenter.

Petri points out in relation to his method
that the filter catches a relatively greater
number of bacteria in proportion to moulds
than the Hesse apparatus, which depends
upon sedimentation.

A particularly useful form of apparatus
is a granulated sugar-filter suggested by
Sedgwick and Tucker, which has an ex¬
pansion above the filter, so that as soon as
the sugar is dissolved in the melted gela¬
tin it can be rolled out into a lining like
that of an Esmarch tube. This cylindrical
expansion is divided into squares which
make the counting of the colonies very easy
(Fig- 45)-

The number of germs in the atmosphere 45— Sedg-

will naturally be very variable. Roughly, ^
the number may be estimated at from ioo animation,
to 1000 per cubic meter.

In reality, the bacteriologic examination of air is
of very little value, as so many possibilities of error
may occur. Thus, when the air of a room is quiescent
there may be very few bacteria in it ; let some one walk
across the floor and dust at once rises, and the number

1 68 PATHOGENIC BACTERIA.

of bacteria is considerably increased : if the person be a
woman with skirts, more bacteria will probably be raised
from the floor than would be disturbed by a man ; if the
room be swept, the increase is enormous. From these
and similar contingencies it becomes very difficult to
know just when and how the air is to be examined,
and the value of the results is correspondingly lessened.

The most valuable examinations are those which aim
at the discovery of some definite organism or organisms
regardless of the number per cubic meter.

CHAPTER XII.

BACTERIOLOGIC EXAMINATION OF WATER.

Unless water has been specially sterilized or distilled
and received and kept in sterile vessels, it always con¬
tains some bacteria. The number will bear a very dis¬
tinct relation to the amount of organic matter in the
water, though experiment has shown that certain patho¬
genic and non-pathogenic bacteria can remain vital in
perfectly pure distilled water for a considerable length of
time. Ultimately, owing to the lack of nutriment, they
undergo a granular degeneration.

The majority of the water-bacteria are bacilli, and as a

Fig. 46. — Wolfhugers apparatus for counting colonies of bacteria upon plates.

rule they are non-pathogenic. Wright,1 in his examina¬
tion of the bacteria of the water from the Schuylkill
River, found two species of micrococci, two species of
cladothrices, and forty-six species and two varieties of
bacilli. Of course, at times the most virulent forms of
pathogenic bacteria — those of cholera and typhoid fever
— occur in polluted water, but this is the exception, not
the rule.

The method of determining quantitatively the number
1 Memoirs of the National Academy of Sciences, Third Memoir.

169

PATHOGENIC BACTERIA.

170

of bacteria in water is very simple, and can generally be
prosecuted without much apparatus. The principle de¬
pends upon the equal distribution of a given quantity of
the water to be examined through a sterile liquid medium,
and the subsequent solidification of this medium in a

Fig. 47. — Heyroth’s instrument for counting colonies of bacteria in Petri dishes.

thin layer, so that all the colonies which develop may
be counted.

The method, which originated with Koch, may be per¬
formed with the Koch plates or with Petri dishes or
with Esmarch rolls. It is always best to make a num¬
ber of these plate-cultures with different amounts of the
water to be examined, using, for example, 0.01, o. 1, 0.5,
and 1.0 c.cm. added to a tube of gelatin, agar-agar, or
glycerin agar-agar.

The exact method must depend somewhat upon the
quality of the water to be examined. If the number of
bacteria per cubic centimeter is small, large quantities
may be used, but if there are millions of bacteria in
every cubic centimeter, it may be necessary to dilute the

BACTERI O LOGIC EXAMINATION OF WATER. 171

water to be examined in the proportion of 1 : 10 or 1 : 100
with sterile water, mixing well, and making the plate-
cultures from the dilutions.

It is best to count all the colonies if possible, but when
there are hundreds or thousands scattered over the plate,
an average estimation of a number of squares ruled upon
a glass background (Fig. 46), as suggested by Wolfhiigel,
is most convenient. In his apparatus a large plate of glass
is divided into small square di¬
visions, the diagonals being spe¬
cially indicated by color. The
plate or Petri dish is stood upon
the glass, and the number of
colonies in a number of small
squares is easily counted, and
the total number of colonies es¬
timated. In counting the colo¬
nies a lens is indispensable.

Special apparatuses have been
devised for counting the colo¬
nies in Petri dishes (Fig. 47)
and in Esmarch tubes (Fig. 48).

The majority of the water-
bacteria are rapid liquefiers of gelatin, for which reason
it seems better to employ agar-agar than gelatin for
making the cultures.

In ordinary hydrant-water the bacteria number from
2-50 per cubic centimeter ; in good pump-water, 100-500 ;
in filtered water from rivers, according to Gunther, 50-200
are present ; in unfiltered river- water, 6000-20,000. Ac¬
cording to the pollution of the water the number may
reach as many as 50,000,000.

The waters of wells and springs are dependent for their
purity upon the character of the earth or rock through
which they filter, and the waters of deep wells are much
more pure than those of shallow wells, unless contamina¬
tion takes place from the surface of the ground.

Ice always contains bacteria if the water contained

Fig. 48. — Esmarch’s instrument
for counting colonies of bacteria
in tubes.

172

PATHOGENIC BACTERIA .

them before it was frozen. In Hudson-River ice Prud-
den found an average of 398 colonies in a cubic centi¬
meter.

A sample of water when collected for examination
should be placed in a clean sterile bottle or in a her¬
metically-sealed pre-sterilizecl glass bulb, and must be
examined as soon as possible, as the bacteria multiply
rapidly in water which is allowed to stand for a short
time. In determining the species of bacteria found in
the water reference must be made to the numerous mono¬
graphs upon the subject, and to tables such as those
compiled by Eisenberg.

The discovery of certain important pathogenic bacteria,
as those of cholera and typhoid, will be considered under
the specific headings.

Unfortunately, the bacteriologic examination of waters
does not throw satisfactory light upon their exact hygi¬
enic usefulness. Of course, if cholera or typhoid-fever
bacteria are present, the water is harmful, but the quality
of the water cannot be gauged by the number of bacteria
it contains.

The drinking-water furnished large cities is not infre¬
quently contaminated with sewage, and contains intes¬
tinal bacteria — Bacillus coli communis. For the ready
determination of this organism, which is an important one
as an indicator that the water is polluted, Smith1 has
made use of the fermentation-tube in addition to the
plate. His method is to add to each of the fermentation-
tubes containing 1 per cent, dextrose-bouillon a certain
quantity of water. The evolution of 50-60 per cent, of
gas by the third day is a strong indication that the colon
bacillus is present. Plates may be used to confirm the
presence of the bacillus, but are hardly necessary, as
there is scarcely another bacterium met with in water
that is capable of producing so much gas.

Filtration with sand, etc. diminishes the number of
bacteria for a time, but, as the organisms multiply in

1 American Journal of the Medical Sciences , 1895, II0> P* 301.

BXi CTERIOLOGIC BXAM/JVAT/ON OB WATER. 173

tlie filter, the benefit is not permanent. The filters must
frequently be renewed. Porcelain filters seem to be the
only positive safeguard, and even these, the best of which
seems to be the Pasteur-Chamberland, allow the bacteria
to pass through if used too long without renewal or with¬
out firing.

CHAPTER XIII.

BACTERIOLOGIC EXAMINATION OF SOIL.

Almost all soil contains bacteria in its upper layers.
Their number and character, however, depend some¬
what upon the surrounding conditions. Near the hab¬
itations of men, where the soil is cultivated, the ex¬
crement of animals, largely made up of bacteria, is
spread upon it to increase its fertility, this being a treat¬
ment which not only adds new bacteria to those already
present, but also enables those present to grow very much
more luxuriantly because of the increased
amount of organic matter thev receive.

The researches of Fliigge, C. Frankel,
and others show that the bacteria of the
soil do not penetrate very deeply — that
they gradually decrease in number until
the depth of a meter is reached, then
rapidly diminish until at a meter and a
quarter they rather abruptly cease to be
found.

Many of the soil-bacteria are anaerobic,
and for a careful consideration of them
the reader must be referred to monographs
upon the subject. The estimation of their
number seems to be devoid of any dis- ..

tmct practical importance. C. Frankel i<eps instrument for
has, however, originated a very accurate obtaining earth from
method of determining it. By means various depths for
of a special boring apparatus (Fig. 49) bactenoloslc stlldy-
earth can be secured from any depth without digging and
without danger of mixing that secured with that of the
superficial strata. With sterile liquefied gelatin a definite

174

BACTER/OLOGIC EX AM /NAT/ ON OF SOIL . 175

amount of this soil is mixed thoroughly and the mixture
solidified upon the walls of an Ksmarch tube. The col¬
onies are counted with the aid of a lens, Pliigge found
in virgin earth about 100,000 colonies in a cubic centi¬
meter.

Samples of earth, like samples of water, should be
examined as soon as possible after being secured, for,
as Gunther points out, the number of bacteria changes
because of the unusual environment, exposure to increased
amounts of oxygen, etc.

The most important bacteria of the soil are those of
tetanus and malignant edema, in addition to which, how¬
ever, there are a great variety which are pathogenic for
rabbits, guinea-pigs, and mice.

In the u Bacteriological Kxamiuatiou of the Soil of
Philadelphia,'7 Ravenel 1 came to the conclusion that —

1. Made soils, as commonly found, are rich in organic
matter and excessively damp through poor drainage.

2. They furnish conditions more suited to the multi¬
plication of bacteria than do virgin soils, unless the latter
are contaminated by sewage or offal.

3. Made soils contain large numbers of bacteria per
gram of many different species, the deeper layers being
as rich in the number and variety of organisms as the
upper ones. After some years the number in the deeper
layers probably becomes proportionally less. Made soils
are more likely than others to contain pathogenic bacteria.

I11 71 cultures that were isolated and carefully studied
by Ravenel, there were two cocci, one sarcina, and five
cladotlirices; all the others were bacilli.

1 Memoirs ol the National Academy ol Sciences, Fust Memoir, 1896.

CHAPTER XIV.

TO DETERMINE THE THERMAL DEATH-POINT.

Several methods may be employed for this purpose.
Roughly, it maybe done by keeping a bouillon-culture of
the micro-organism to be studied in a water-bath whose
temperature is gradually increased from that of the body
to 750 C.

Into a fresh bouillon-culture thus exposed to heat, the
experimenter cautiously, and at given intervals, intro¬
duces a platinum loop or a capillary pipette, and with¬
draws a drop of the culture which he inoculates into
fresh bouillon and stands aside to grow. It is economy
to make the transplantations rather infrequently at first
and frequently later on in the experiment, when the tem¬
perature is ascending. In an ordinary determination it
would be well to make a transfer at 40° C., one at 450 C.,
another at 50°, still another at 550, and then beginning at
6o° make one for every additional degree up to 750 C.
or above. The day following the experiment it will be
observed that all the cultures grow except those heated
beyond a certain point, as 6o° C. and upward, when it
can properly be concluded that 6o° C. is the thermal
death-point. If all the transplantations grow, of course
the maximum temperature that the bacteria can endure
was not reached, and the experiment must be performed
again with higher temperatures.

When more accurate information is desired, and one
wishes to know how long the micro-organism can endure
some such temperature as 6o° C. without losing its vital¬
ity, a dozen or more bouillon-tubes may be inoculated
with the germ to be studied, and stood in the water-bath
at the temperature to be investigated. The first can be

176

TO DETERMINE THERMAL DEATH-POINT. T77

removed as soon as it is certainly heated through, another
in five minutes, another in ten minutes, or at whatever
intervals the thought and experience of the experimenter
shall suggest.

In both of the described procedures one must be care¬
ful that the temperature in the test-tube is identical with
that of the water in the bath. There is no reason why a
sterile thermometer should not be placed in the heated
tube in the first case, and in the second experiment one
of the test-tubes exposed under conditions similar to the
others might contain a thermometer which would show
the temperature of the contents of the tube containing it,
and so be an index of the rest.

Another method of accomplishing the same end is to
use Sternberg's bulbs. These are small glass bulbs
blown on one end of a piece of glass tubing, which is
subsequently drawn out to capillarity at the opposite end.
If such a bulb be heated, and its capillary tube dipped
into inoculated bouillon, in cooling, the fluid is drawn in
so as to fill it one-third or one-half. A number of these
tubes are filled in this manner with freshly inoculated
culture-medium and then floated, tube upward, upon
a water-bath whose temperature is gradually elevated,
the bulbs being removed from time to time as the
required temperatures are reached. Of course, as the
bulbs are already inoculated, all that is necessary is to
stand them aside for a day or two, and observe whether
or not the bacteria grow, nidging the death-point exactly
as in the other case.

To Determine the Antiseptic and Germicidal Value
of Reagents.— There arc various methods whose modi¬
fications can be elaborated according to the extent and
thoroughness of the investigation to be made.

I. The Antiseptic 1'etlne.- — Remembering that an anti¬
septic is a substance that inhibits bacterial growth, the
method that will at once suggest itself is that of adding
varying quantities of the antiseptic to be investigated to
culture-media in which the bacteria are subsequently
12

178 PATHOGENIC BACTERIA .

planted. It is always well to use a considerable number
of tubes. Bouillon is generally employed. If the anti¬
septic is non-volatile, it may be added before sterilization,
which is to be preferred; but if it is volatile, it must be
added by means of a sterile pipette, with the greatest
precaution as regards asepsis, immediately before the test
is to be made. Control-experiments — i. e . without the
addition of the antiseptic — should always be made.

The results of antiseptic action are two: retardation of
growth and complete inhibition of growth. As the tubes
used for the study of the antiseptic are watched in their
development, it will usually be noticed that those con¬
taining very small quantities develop almost as rapidly as
the control-tubes; those containing more, a little more
slowly; those containing still more, very slowly, until at
last there comes at time when the growth is not deferred,
but prevented.

Sternberg points out that certain circumstances may
.modify the results obtained. They are:

1. The composition of the nutrient media, with which
the antiseptic may be incompatible.

2. The nature of the test-organism, no two organisms
being exactly alike in their susceptibility.

3. The temperature at which the experiment is con¬
ducted, a relatively greater amount of the antiseptic
being necessary at temperatures favorable to the organ¬
ism than at temperatures unfavorable.

4. The presence of spores which are always more
resistant than the asporogenous forms.

II. The Germicidal Valite. — Koch’s original method of
doing this was to dry the micro-organisms upon sterile
shreds of linen or silk, and then soak them for varying
lengths of time in the germicidal solution. After the
bath in the reagent the threads were washed in clean,
sterile water and then transferred to fresh culture-
media, and their growth or failure to grow observed. It
will be observed that this method is aimed at the deter¬
mination of the time in which a certain solution will kill.

TO DETERMINE THERMAL DEATH-POINT. 179

Sternberg suggested a method in which the time should
remain constant (two hours’ exposure), and the object be
the determination of the exact dilution of the reagent
required to destroy the bacteria. “ Instead of subjecting
a few of the test-organisms attached to a silk thread to
the action of the disinfecting agent, a certain quantity of
the recent culture — usually 5 c.cm. — has been mixed
with an equal quantity of a standard solution of the
germicidal agent, . . . and after two hours’ contact one
or two dse-fuls would be introduced into a suitable nutri¬
ent medium to test the question of disinfection.”

A very simple and popular method of determining the
germicidal value is to make a series of dilutions of the
reagent to be tested; add to each a couple of loopfuls of
a fresh liquid culture, and at varying intervals of time
transfer a loopful to fresh culture-media. By a little
ingenuity this method may be made to yield information
as to both time and strength.

When it is desired to secure information concerning
the progress of the germicidal action of reagents, body-
fluids, etc., especially in the unusual and interesting
cases in which the material subjected to the test may
exert a restraining action for a time only, or bring about
destruction of some or many, but not all of the germs,
the use of the Petri dish can be introduced.

For example, it is desired to determine whether a
blood-serum is germicidal or not. Into about 5 c.cm. of
the serum contained in a test-tube, two or three loopfuls
of any desired bacterium, in liquid culture, are added.
The tube is well agitated and immediately one loopful is
transferred to a tube of melted gelatin, distributed
through it, and poured into a Petri dish. After one
minute the operation is repeated, in five minutes again,
and so on as often as is desired.

The dishes are stood away until the bacteria develop
into colonies, which are then counted with a Wolfhiigel
apparatus. On the first dish there may be too colonies;
on the second, 80; on the third, 50; on the fourth, 20; ou

180 PATHOGENIC BACTERIA.

the fifth-, 30; on the sixth, 150; on the seventh, 1000,
etc. ; indicating that the serum exerted a destructive
action upon some but not all of the bacteria, and that
this power disappeared after the lapse of a certain time,
allowing the bacteria to develop ad libitum .

When the germicide to be studied is a gas, as in the
case of sulphurous acid or formaldehyd, a different
method must, of course, be adopted.

It may be sufficient simply to place a few test-tube cul¬
tures of various bacteria, some with plugs in, some with
plugs out, in a closed room in which the gas is afterward
evolved. The germicidal action is shown by the failure
of the cultures to grow upon transplantation to fresh cul¬
ture-media. This crude method may be supplemented
by an examination of the dust of the room. Pledgets
of sterile cotton are rubbed upon the floor, washboard,
or any dust-collecting surface present, and subsequently
dropped into culture-media. Failure of growth under
such circumstances is very certain evidence of good dis¬
infection. These tests are, however, very severe, for in
the cultures there are immense numbers of bacteria in
the deeper portions of the bacterial mass upon which the
gas has no opportunity to act, and in the dust there are
many sporogenous organisms of extreme resisting power.
Failure to kill all the germs exposed in such manner is
no indication that the vapor cannot destroy all the ordi¬
nary pathogenic organisms.

More refined is the method of saturating sterile sand
or fragments of blotting-paper or absorbent cotton with
cultures and exposing them, moist or dry, to the action
of the gas. Such materials are best made ready in Petr:
dishes, which are opened immediately before and closed
immediately after the experiment. A piece of cotton o*\
blotting-paper or a little sand transferred to fresh culture
media will not give any growth where the disinfection ha:
been thorough. By transplanting from different depths
the sand may be used incidently to show to what deptl
the gas is capable of penetrating.

TO DETERMINE THERMAL DEATH-POINT. l8l

Kasicr oi execution, but rather more severe, is a
method in which cover-classes are employed. A num¬
ber of them are spread with cultures of various bacteria,
allowed to dry, and then exposed to the gas as long as
required. 'Pile cover-glasses are afterward dropped into
culture-media to permit the growth of the germs not
destroyed.

Animal-experiments may also be employed to deter¬
mine whether or not a germ that has survived exposure to
the action of reagents has its pathogenic power destroyed.
An excellent example of this is seen in the ease of the
anthrax bacillus, a virulent form of which will kill rab¬
bits, but after being grown in media containing an
insufficient amount of a germicide to kill it will often
lose its rabbit-killing power, though still able to fatally
infect guinea-pigs, or may lose its virulence for both
rabbits and guinea-pigs, though still able to kill white

PART II. SPECIFIC DISEASES AND THEIR
BACTERIA.

A. THE PHLOGISTIC DISEASES.

I. THE ACUTE INFLAMMATORY DISEASES.

CHAPTER I.

SUPPURATION.

Suppuration was at one time supposed to be an
inevitable outcome of the majority of wounds, and,
although bacteria were observed in the discharges, the
old habit of thought and insufficiency of information
caused most surgeons to believe that they were sponta¬
neously developed there.

Lord Lister, whose name we cannot sufficiently honor,
conceived that Pasteur’s observations upon the germs of
life floating in the atmosphere, if they explained the con¬
tamination of his sterile infusions, might also explain
the changes in wounds, and upon this idea based that
most successful system of treatment known as cc antisep¬
tic surgery. ’ ’

The further development of antiseptic surgery, and the
extremes to which it was carried by specialists, almost
attain to the ridiculous, for not only were the hands of
the operator, his instruments, sponges, sutures, ligatures,
and dressings kept constantly saturated with irritating
germicidal solutions, but at one time the air over the
wound was carefully saturated with pulverized antiseptic
lotions during the whole operation by means of a steam
atomizer. This rather monstrous outcome of the appli¬
cation of Lister’s system to surgery was the very natural
result of the erroneous idea that the germs which cause
182

St 77 Y JI\A WON.

183

the suppurative changes in wounds entered the exposed
tissues principally from the atmosphere, and that the
hands and instruments of the operator, while certainly
means of infection, were secondary in importance to it.

The researches of more recent date, however, have
shown not only that the atmosphere cannot be disin¬
fected, but also that the air of ordinarily quiet rooms,
while containing the spores of numerous .saprophytic
organisms, very rarely contains many pathogenic bac¬
teria. We now also know that a direct stream of air,
such as is generated by an atomizer, causes more bacteria
to be conveyed into a wound than would ordinarily fall
upon it, thereby increasing instead of lessening the dan¬
ger of infection. It may therefore be stated, with a
reasonable amount of certainty, that the atmosphere is
rarely an important factor in the process of suppuration.

We have already called attention to the fact that
various micro-organisms are so intimate in their relation
to the skin that it is almost impossible to get rid of them,
and have cited in this relation the experiments of Welch,
Robb, and Ohriskey, whose method of disinfecting the
hands has been recommended as the best. The investi¬
gations of these observers have shown that, no matter
how rigid the disinfection of the patient’s skin, the
cleansing of the operator’s hands, the sterilization of
the instruments, and the precautions exercised, a certain
number of wounds in which sutures are employed will
always suppurate. The cause of the suppuration is a
matter of vast importance in surgery and in surgical bac¬
teriology, yet it is one which it is impossible to remove.
We carry it constantly with us upon our .skins.

STAHivr.ocoect’S Rpidkrmidis Albtjs.

Welch has described, under the name Staphylococcus
(pi derm id is albas, a micrococcus which seems to be habit¬
ually present upon the skin, not only upon the surface,
but also deep down in the Malpighian layer. He is of
the opinion that it is the same organism which is familiar

184

PATHOGENIC BACTERIA.

to us under the name of Staphylococcus pyogenes albus,
but in an attenuated condition. If his opinion be correct,
and we have seated deeply in our derm a coccus which
can at times cause abscess-formation, the conclusions of
Robb and Ghriskey, that sutures of catgut when tightly
drawn may be a cause of skin-abscesses by predisposing
to the development of this organism, are certainly justi¬
fiable.

Not only does the coccus occur in the attenuated form
described, but we have very commonly present upon the
skin, generally as a harmless saprophyte, the important
Staphylococcus pyogenes albus , which is a common cause
of suppuration.

Staphylococcus Pyogenes Albus.

Although, as stated, the Staphylococcus pyogenes albus
is a common cause of suppuration, it rarely occurs alone,
the studies of Passet showing that in but 4 out of 33 cases
which he investigated was this coccus found by itself.
When pure cultures of the coccus are injected subcu¬
taneously into rabbits and guinea-pigs, abscesses some¬
times result; sometimes there is no result Injected
into the circulation of these animals, the staphylococci
sometimes cause septicemia, and after death can be found
in the capillaries, especially of the kidneys. From these
illustrations it will be seen that the organism is feebly
pathogenic.

In its vegetative characteristics the Staphylococcus
albus is almost identical with the species next to be de¬
scribed, but differs from it in that there is no golden color
produced. Upon the culture-media it grows white.

Staphylococcus Pyogenes Aureus.

Generally present upon the skin, though in smaller
numbers, is the dangerous and highly virulent Staphylo¬
coccus pyogenes aureus (Fig. 50), or cl golden staphylococ¬
cus” of Rosenbach. As the morphology of this organ¬
ism, and indeed the generality of its characters, are

SUPPURA TION.

185

identical with those of the preceding species, it seems
convenient to describe them together, pointing out such

Fig. 50. — Staphylococcus pyogenes aureus, from an agar-agar culture; x 1000

(Gunther).

differences as occur step by step. In doing this, how¬
ever, it must not be forgotten that, although the Staphy¬
lococcus albus has been described first, the Staphylococcus
aureus is the more common organism of the suppurative
diseases.

Although they had been seen earlier by several ob¬
servers, the staphylococci were not isolated and care¬
fully described until 1884, when Rosenbach worked upon
them. The results of his study, followed by Passet and
a host of others, have now given us pretty accurate
information about them.

The cocci are distributed rather sparingly in nature,
seeming not to find a purely saprophytic existence a
suitable one. They occur, however, wherever man and
animals have been, and can be found in the dust of
houses, hospitals, and especially surgical wards where
proper precautions are not exercised. They are common
upon the skin, they live in the nose, mouth, eyes, and
ears of man, they are nearly always beneath the finger¬
nails, and they sometimes occur in the feces, especially
in children.

i86

PATHOGENIC BACTERIA .

The cocci are rather small, measuring about 0.7 fj. in
diameter. When examined in a delicately-stained con¬
dition the organisms may be seen to consist of hemi¬
spheres separated from each other by a narrow interval.
The contiguous surfaces are flat, thus differing from
the gonococcus, whose contiguous surfaces are concave.
The grouping is not very characteristic. In both liquid
and solid culture-media the organisms either occur in
solid masses or are evenly distributed. It is only in the
organs or tissues of a diseased animal that it is possible to
say that a true staphylococcus grouping is present.

The organism stains brilliantly with aqueous solu¬
tions of the anilin dyes. In tissues it can be beautifully
stained by Gram’s method.

The staphylococci grow well either in the presence or
absence of oxygen at a temperature above 180 C., the
most rapi.d development being at about 370 C. Upon the
surface of gelatin plates small whitish points can be
observed in forty-eight hours (Fig. 51). These rapidly

Fig. 51. — Staphylococcus pyogenes aureus : colony two days old, seen upon an
agar-agar plate ; x 40 ( Heim).

extend to the surface and cause extensive liquefaction.
Hand in hand with the liquefaction is the formation of
an orange color, which is best observed at the centre of
the colony. Under the microscope the colonies appear

SUPPURATION.

187

as round disks with circumscribed, smooth edges. They
are distinctly granular and dark-brown. When the col¬
onies are grown upon agar-agar plates the formation of
the pigment is much more distinct.

In gelatin punctures the growth occurs along the whole
length of the needle-track, and causes an extensive lique¬
faction in the form of a long, narrow, blunt-pointed,
inverted cone (Fig. 52) full of clouded liquid, at the apex

Fig. 52. — Staphylococcus pyogenes aureus : puncture- culture three days old
in gelatin (Frankel and Pfeiffer).

of which a collection of golden or orange-yellow precipi¬
tate is always present. It is this precipitate in particu¬
lar that gives the organism its name, “ golden staphylo¬
coccus.”

The most characteristic growth is upon agar-agar.
Along the whole line of inoculation an orange-yellow,
moist, shining growth occurs. When the growth takes
place rapidly, as in the incubator, it exceeds the rapidity

1 88

PATHOGENIC BACTERIA.

of color-production, so that the centre of the growth is
distinctly golden ; the edges may be white.

Upon potato the growth is luxuriant, producing an
orange-yellow coating over a large part of the surface.
The potato-cultures give off a sour odor.

When grown in bouillon the organism causes a diffuse
cloudiness.

In milk coagulation takes place, and is followed by
gradual digestion of the casein.

The Staphylococcus albus is exactly the same as the
aureus, with the exception that in all media it is con¬
stantly colorless.

Experiments have shown that the Staphylococcus
aureus, like its congener, the albus, exists in an atten¬
uated form, and there is every reason to believe that in
the majority of instances it inhabits the surface of the
body in that condition.

When virulent the golden staphylococcus is a danger¬
ous and often deadly organism. Its pathogeny among
animals is decided. When introduced subcutaneously,
abscesses almost invariably follow, except in a certain
few comparatively immune species, and not infrequently
lead to a fatal termination. In such cases the organisms
may be cultivated from the blood of the large vessels,
though by far the greater number collect in, and fre¬
quently obstruct, the capillaries. In the lungs and
spleen, and still more frequently in the kidneys, infarcts
are formed by the bacterial emboli. The Malpighian
tufts of the kidneys sometimes are full of cocci, and
become the centres of small abscesses.

The coccus is almost equally pathogenic for man,
though the fatal outcome is much more rare. It enters
the system through scratches, punctures, or abrasions,
and when virulent generally causes an abscess, as various
experimenters who inoculated themselves have discov¬
ered to their cost. Garre applied the organism in pure
culture to the uninjured skin of his arm, and in four
days developed a large carbuncle with a surrounding

SUPPURATION.

189

zone of furuncles. Bockhart suspended a small portion
of an agar-agar culture in salt-solution, and scratched it
gently into the deeper layers of the skin with his finger¬
nail ; a furuncle developed. Bumnv injected the coccus
suspended in salt-solution beneath his skin and that of
several other persons, and produced an abscess in every
case.

The Staphylococcus aureus is not only found in the
great majority of furuncles, carbuncles, abscesses, and
other inflammatory diseases of the surface of the body,
but also plays an important role in a number of deeply-
seated diseases of the internal organs. Becker and others
obtained it from the pus of osteomyelitis, demonstrating
that if, after fracturing or crushing a bone, the staphylo¬
coccus was injected into the circulation, osteomyelitis
would result. Numerous bacteriologists have demon¬
strated its presence in ulcerative endocarditis. Rodet
has been able to produce osteomyelitis without previ¬
ous injury to the bones ; Rosenbach was able to produce
ulcerative endocarditis by injecting some of the staphy¬
lococci into the circulation in animals whose cardiac
valves had been injured by a sound passed into the
carotid artery ; and Ribbert has shown that the injection
of cultures of the organism may cause the valvular lesion
without the preceding injury.

The Staphylococcus aureus is an easy organism to ob¬
tain, and can be secured by plating out a drop of pus in
gelatin or in agar-agar. Such a preparation, however,
generally does not contain the Staphylococcus aureus
alone, but shows colonies of the Staphylococcus albus as
well. In addition to these two principal forms, one
sometimes discovers an organism identical with the pre¬
ceding, except that its growth on agar-agar and potato
is of a brilliant lemon-yellow color, and its pathogeny for
animals much less. This is the Staphylococcus citreus of
Passet. It is not quite so common, and not so patho¬
genic as the others, and consequently much less im¬
portant.

PATHOGENIC BACTERIA.

190

Streptococcus Pyogenes.

Another organism whose colonies are frequently ob¬
tained from the pus containing the staphylococci is the
Streptococctis pyogenes of Rosenbach (Fig. 53). ft was
found by him in 18 of 33 cases of
suppurative lesions studied, fifteen
times alone and five times with the
Staphylococcus aureus. It is a
spherical organism of . variable size
(0.4-1 p in diameter), constantly

Fig. 54. — Streptococ¬
cus pyogenes : culture
upon agar-agar two days
old (Frankel and Pfeif¬
fer).

associated in pairs and chains of from four to twenty in¬
dividuals. A special variety of it, known as Streptococ¬
cus longus, sometimes forms chains of more than one
hundred members.

The organism stains well with ordinary aqueous solu¬
tions of the anilin dyes, and also by Gram’s method. Like
the coccus already described, it is not motile and does not
seem to form spores, though sometimes a large individual
— much larger than the others in its chain — may be ob¬
served, and may suggest the thought of arthro-sporulation.

SUPPURA TION ;

191

Upon gelatin plates very small colonies of translucent
appearance are observed. When superficial, they spread
out to form flat disks about 0.5 mm. in diameter. The
microscope shows them to be irregular and granular, to
have a slightly yellowish color, and to have numerous
irregularities around the edges, due to projecting chains
of the cocci. No liquefaction occurs.

In gelatin puncture-cultures no liquefaction is observed.
The minute spherical colonies grow along the whole
needle-track and form a slightly opaque granular line.

Upon agar-agar an exceedingly delicate transparent
growth develops slowly along the line of inoculation.
It consists of almost transparent, colorless small colonies
which do not become confluent.

The growth upon blood-serum much resembles that
upon agar-agar. . The streptococcus does not seem to
grow upon potato.

In bouillon the cocci develop rather slowly, seeming
to prefer a neutral or feebly acid reaction. The culture-
medium remains clear, while numerous small flocculi are
suspended in it. . When the flocculi-formation is very
distinct the name Streptococcus conglomerates is used
to describe the organism. These masses sometimes ad¬
here to the sides of the tube; sometimes they form a sedi¬
ment. Rarely, there is general clouding of the medium
( Streptococcus dijfusus ).

In mixtures of bouillon and blood-serum or ascitic
fluid the streptococcus grows much better, especially at
incubation temperatures, and in such mixtures the lux¬
uriant development causes the liquid to appear clouded.

The organism seems to grow well in milk, which is
coagulated and digested.

The streptococcus is not very sensitive to acids, and
can be grown quite well in media with a slightly acid
reaction.

Sternberg found that the streptococci succumb to a
temperature of 52°-54° C. continued for ten minutes.

Their vitality in culture is not great. Unless fre-

192

PATHOGENIC BACTERIA.

quently transplanted they die. In bouillon they are said
to die in five to ten days. On solid media they seem
to retain their vegetative and pathogenic powers much
longer. They resist drying well. Their growth in arti¬
ficial media is accompanied by the production of an
acid which probably acts destructively upon the bacteria
themselves.

The Streptococcus pyogenes is generally not very patho¬
genic for animals. Subcutaneous injections into mice
and rabbits are, as a rule, without either general or local
manifestations of importance. If, however, an ear of a
rabbit is carefully inoculated with a small amount of a
pure culture, a small patch resembling erysipelas usually
results. The disturbance passes away in a few days and
the animal recovers.

If, however, the streptococcus is highly virulent, the
rabbit dies in from twenty-four hours to six days from
a general septicemia. The cocci may be found in large
numbers in the heart’s blood and in the organs. In less
virulent cases minute disseminated abscesses are some¬
times found.

iVccording to Marmorek,1 the virulence can be increased
to a remarkable degree by rapid passage through rabbits,
and maintained by the use of a culture-medium consist¬
ing of three parts of human blood-serum and one of
bouillon. The blood of the ass, and ascitic and chest
fluids may also be used. By these means Marmorek suc¬
ceeded in intensifying the virulence of his culture to such
a degree that one hundred millionth of a c.cm. injected
into the ear vein was fatal to a rabbit.

Petruschky2 found the virulence of the culture to be
well retained if the culture was planted in gelatin, trans¬
planted every five days, and when grown kept on ice.

Holst3 succeeded in keeping an exceedingly virulent
Streptococcus brevis on artificial culture-media for eight

1 Ann. de V Inst. Pastettr , Tome ix., No. 7, July 25, 1895, P- 593*

2 Centralbl. fur Bakt. und Parasitenk Bd. xviii., No. 16, May 4, 1895, p,

551* 3 Ibid., Bd. xix.,No. 11, Mar. 21, 1896.

SUPPURA TION. 193

years without any particular precautions and found its
virulence unchanged.

Probably the virulence and attenuation are peculiarities
of the organism itself.

Dried streptococci are said by Frosch and Kolle to re¬
tain their energies longer than those growing on culture-
media.1

Tike the staphylococci, the streptococcus is frequently
associated with internal diseases, and has been found in
erysipelas, ulcerative endocarditis, periostitis, otitis, men¬
ingitis, emphysema, pneumonia, lymphangitis, phleg¬
mons, sepsis, and in the uterus in cases of infective puer¬
peral endometritis. I11 man the streptococci occur in the
most active forms of suppuration. Its relation to diph¬
theria is of interest, for, while, in all probability, the
great majority of cases of pseudomembranous angina are
caused by the Klebs-Loffler bacillus, yet an undoubted
number of cases are met with in which, as in Prudden’s
24 cases, no diphtheria bacilli can be found, but which
seem to be caused by a streptococcus exactly resembling
that under consideration.

There is no clinical difference in the picture of the
throat-lesion produced by the two organisms, and the
only positive method of diagnosticating the one from
the other is by means of a careful bacteriologic examina¬
tion. Such an examination should always be made, as it
has much weight in connection with the treatment. Of
course, in streptococcus angina no benefit could be ex-
pected from the diphtheria antitoxic serum.

Hirsh2 has shown that under pathological conditions
streptococci are by no means rare organisms in the in¬
testinal canal of infants, and may cause a streptococcic
enteritis. In these cases the organisms are found in large
numbers in the stomach and in the stools, and later in
the course of the disease in the blood and urine of the
living child and in the internal organs of the cadaver.

1 Fliigge’s Die Mikroorgctnismen.

2 Centralbl. fur Baht, und Parasitenk Bd. xxii., Nos. 14 and 15, p. 369.

194

PATHOGENIC BACTERIA.

Liebman1 reports two cases of streptococcic enteritis
that were cerefully studied bacteriologically.

Flexner,2 in a series of autopsies upon cases of death
from various diseases, found the bodies invaded by num¬
erous micro-organisms, causing what he has called 1 1 term¬
inal infections, ” and hastening the fatal issue. Of 793
autopsies at Johns Hopkins Hospital, 255 from chronic
heart or kidney diseases, or both, were sufficiently well
studied bacteriologically to meet the needs of a statis¬
tical inquiry. Tubercular infection was not included.
Of the 255 cases, 213 gave positive bacteriological results.
“The micro-organisms causing the infections, 38 in all,
were the Streptococcus pyogenes, 16 cases; Staphylococcus
pyogenes aureus, 4 cases; Micrococcus lanceolatus, 6 cases;
gas bacillus (B. Aerogenes capsulatus), three times alone
and twice combined with the Bacillus coli communis; the
gonococcus, anthrax bacillus, Bacillus proteus, the last
combined with the Bacillus coli, the Bacillus coli alone, a
peculiar capsulated bacillus, and an unidentified coccus. ”

It is interesting to observe how many cases were
accompanied by the streptococcus. All the streptococci
may not have been streptococcus pyogenes, but for con¬
venience in his statistics they were regarded by Flexner
as identical.

The streptococcus of Rosenbach is thought by many
to be identical with a streptococcus described by Fehleisen
as the Streptococcus erysipelatis (Fig. 55). The two or¬
ganisms have much in common, but much difference of
opinion exists upon the subject of their identity. It may
seem unwise to omit the Streptococcus erysipelatis as a
major topic for discussion, but the similarity of the or¬
ganism to that just described has caused us to consider
them in the same connection.

The streptococci of erysipelas can be obtained in almost
pure culture from the serum which oozes from a puncture
made in the margin of an erysipelatous patch. They are

1 Centralbl. fur Bakt. und Parasitenk., Bd. xxii., Nos. 14 and 15, p. 376.

2 Journal of Experimental Medicine , vol. i., No. 3, 1896.

SUPPURA TION.

195

small cocci, forming long chains — generally from six to
ten individuals, but sometimes reaching a hundred in

Fig. 55. — Streptococcus erysipelatis, seen in a section through human skin;
x 500 (Frenkel and Pfeiffer).

number. Occasionally the chains can be found collected
in tangled masses. They can be cultivated at the room-
temperature, but grow much better at 30-37° C. They
are not particularly sensitive to the absence of oxygen,
but develop a little more rapidly in its presence.

The erysipelas cocci, like the Streptococcus pyogenes,
are not motile, form no spores, and are destroyed by a
low degree of heat. They stain well with aqueous solu¬
tions of anilin dyes and also by Gram’s method.

The colonies upon gelatin and the development in
gelatin tubes, upon agar-agar, and upon blood-serum
are identical with the descriptions of the Streptococcus
pyogenes. No growth occurs on potato.

The growth in bouillon is generally luxuriant, and in
a short time causes the medium to be filled with chains
of the cocci. As the growth progresses these chains
gather in clusters and fall to the bottom as a whitish

1 96 PA THOGENIC BA CTERIA.

granular precipitate, above which the liquid remains
clear.

When injected into animals Fehleisen’s coccus behaves
exactly like the Streptococcus pyogenes.

Observation has shown that dire results may follow the
entrance of this organism into exposed wounds, and that
it causes not only local suppuration, but sometimes a
general infection.

The empiric experience that the occasional accidental
infection of malignant tumors with erysipelas cocci was
followed by sloughing and subsequent disappearance of
the tumor, suggested inoculation with the Streptococcus
erysipelatis as a therapeutic measure. The dangerous
character of the remedy, however, caused many to re¬
frain from its use, for when one inoculated the living
erysipelas germs into the tissues he never could estimate
the exact amount of disturbance that would follow. The
difficulty seems to have been overcome by Coley, who
recommends the toxin instead of the living coccus for
injection. A virulent culture is obtained, inoculated
into small flasks of slightly acid bouillon, allowed to
grow for three weeks, then reinoculated with Bacillus
prodigiosus, allowed to grow for ten or twelve days at
the room-temperature, well shaken up, poured into bottles
of about f 3ss capacity, and rendered perfectly sterile by an
exposure to from 50-60° C. for an hour. It is claimed
that the combined toxins of erysipelas and prodigiosus
are much stronger than the simple erysipelas toxin. The
best effects are found in cases of sarcoma, where the
toxin causes a rapid necrosis of the tumor tissue, which
can be scraped out with an appropriate instrument.
Numerous cases are on record in which this treatment
has been most efficacious ; but, although Coley recom¬
mends it and Czerny still upholds it, the majority of sur¬
geons have failed to secure the desired results.

Recently (1895) considerable attention has been be¬
stowed upon the anti-streptococcus serum of Marmorek,
which is said to act specifically upon cases of strepto-

SUPPURATION.

197

coccus-infection, both general and local. Numerous
cases are upon record in which the serum seemed to exert
a beneficial action.

It wrould seem as if an antiphlogistic serum should
occupy an important place in the future of medicine.

The serum is prepared upon the same plan as that of
Behring, except that living virulent streptococci instead
of the sterile toxin are injected into the horse.

Bacillus Pyocyanlus.

In some cases the pus evacuated from wounds exhibits
a peculiar bluish or greenish color, from the presence of

Fig. 56. — Bacillus pyocyaneus, from an agar-agar culture; x 1000 (Itzerott

and Niemann).

the Bacillus pyocyaneiis (Figs. 56, 57). This is a short,
delicate bacillus of small size, measuring 0.3 : 1-2 /*, ac¬
cording to Fliigge, frequently united in chains of four or
six. It has round ends, is actively motile, has one
terminal flagellum, does not form spores, and can exist
with or without oxygen, though it is an almost purely
aerobic organism.

It stains well with the ordinary solutions, but does not
retain the color by Gram’s method.

1 98 PA THOGENIC BA CTERIA .

The superficial colonies upon gelatin plates form small,
irregular, ill-defined collections, which produce a fluores¬
cence of the neighboring
gelatin. The gelatin soft¬
ens gradually, and about
five days elapse before
liquefaction is complete.

The microscope shows
the colonies to be round,
coarsely-granulated masses

Fig. 57. — Bacillus pyocyaneus: colonies upon gelatin (Abbott).

with notched or filamentous borders. They have a yel¬
low-green color. Upon the surface they form a delicate
clump with a smooth surface, finely granular, distinctly
green in the middle and pale at the edges. The colonies
sink into the gelatin as the liquefaction progresses.

In gelatin puncture-cultures most of the development
occurs at the upper part of the tube, where a deep saucer
of liquefaction forms. The growth slowly descends into
the medium, and is the point of origin of a beautiful
fluorescence. The bacterial growth sinks to the bottom
as it ages. At times a delicate mycoderma forms on the
surface.

Upon agar-agar the growth is at first bright green,
developing all along the line of inoculation. The green
pigment (fluorescin) is soluble, and soon saturates the cul¬
ture-medium and makes it very characteristic. As the
culture ages, or if' w the medium upon which it grows
contains much peptone, a second pigment (pyocyanin) is
developed, and the bright green fades to a deep blue-
green, dark-blue, or in some few cases to a deep reddish-
brown.

A well-known feature of the growth upon fresh agar-
agar, upon which much stress has recently been laid by

SUPPURATION.

199

Martin is the formation of crystals in fresh cultures.
Crystal-formation in cultures of other bacteria usually
takes place in old, partially dried agar-agar cultures. The
bacillus pyocyaneus, however, produces crystals in a few
days upon fresh media. In my experience freshly iso¬
lated bacilli manifest this capability more markedly than
those which have been for some time part of the labo¬
ratory stock of cultures, and subject to frequent trans¬
plantation.1

Upon potato a luxuriant greenish or brownish, smeary
layer is produced. Milk is coagulated and peptonized.

This bacillus is highly pathogenic for laboratory ani¬
mals. About 1 c.cm. of a fresh bouillon culture, if in¬
jected into the subcutaneous tissue of a guinea-pig or a
"rabbit, causes a rapid edema, a suppurative inflammation,
and death in a short time (twenty-four hours). Some¬
times the animal lives for a week or more, then dies.
There is a marked hemorrhagic subcutaneous edema at
the seat of inoculation. The bacilli can be found in the
blood and in most of the tissues.

When the dose is too small to prove fatal, suppuration
occurs in many cases.

When sterilized cultures are injected, the same results
follow, a relatively larger quantity, of course, being re¬
quired.

Intraperitoneal injections cause suppurative peritonitis.

The organism has been found in the human being in the
pus in cases of middle-ear disease (often in pure culture),
panophthalmia, bronchopneumonia, inflammations of the
nasal fossae, meningitis, etc. Escaping from such local
lesions into the blood it sometimes causes nephritis.

It may, however, be stated that ordinarily the bacillus
is harmless for human beings, the above-mentioned ex¬
amples of pathogenic activity being marked exceptions.

It is interesting to observe, in passing, that this path¬
ogeny can be set aside by the immunity which develops
after a few inoculations with sterilized cultures. These

1 See Centralbl. f Bnkt xxi., April 6, 1897, p. 473.

200

PATHOGENIC BACTERIA .

are easily prepared, as the thermal death-point deter¬
mined by Sternberg is 56° C.

The bacillus appears to be rather common as a sapro¬
phyte, and, as it has been found in the perspiration,
probably is not uncommon upon the skin.

Before leaving the subject of suppuration attention
must be called to several rather common bacteria which
may at times be the cause of troublesome suppuration.
Among these are the pneumococcus of Frankel and
Weichselbaum, the typhoid bacillus, and the Bacillus
coli communis (q. v.).

The pjieumococcus has not infrequently been discov¬
ered most unexpectedly in abscesses of the brain and
other deep-seated organs, and seems to have powerful
chemotactic powers. For a careful consideration of it
the reader must be referred to the chapter upon Pneumo¬
nia, where it is considered in full.

The Bacillus. coli communis , which is always present in
the intestine, seems at times to enter the blood- or lymph-
channels and stimulate suppuration, and numerous cases
are on record showing this. The points most frequently
attacked seem to be the bile-ducts and the vermiform ap¬
pendix, though the significance of the organism in appen¬
dicitis has no doubt been overrated. It has also been found
in the kidney in scarlatinal nephritis, and is thought to
be the exciting cause of some cases. It was originally
described by Passet as the Bacillus pyogenes foetidus .
For a more particular study of this organism the reader
is referred to the chapter devoted to its consideration.

The Bacillus typhosus is probably less frequently a cause
of suppuration than either of the others, yet it seems to
be the occasional cause of the purulent sequelae of typhoid
fever. A case has recently been reported by Flexner in
which metastatic abscesses were found to be caused by it.

The Micrococcus tetragenus has also been found in the
pus of acute abscesses: it is quite common in the cavities
of pulmonary tuberculosis, and may aid in the destructive
processes involved in the general phthisical infection.

SUPPURA TION.

201

Micrococcus Gonorrhoea.

All authorities now accept the “gonococcus” to be
the cause of gonorrhea. It was first observed in the
urethral and conjunctival secretions of gonorrhea and
purulent ophthalmia by Neisser in 1879. The organisms
are of hemispherical shape, arranged in pairs, so that
the inner surfaces are separated from each other by a
narrow interval. Sometimes, instead of pairs of cocci,
fours are seen, the group no doubt resulting from the
division of a pair.

Kig. 58.*— Gonococcus in urethral pus; x woo (Friinkel and Pfeiffer).

The described hemispherical shape is not exactly cor¬
rect, for a good lens generally shows the approximated
surfaces to be somewhat concave rather than flat. The
Germans see in the organism a resemblance to their pop¬
ular biscuit called a “semmel.”

The gonococcus is small, is not motile, like other cocci,
is not provided with flagella, and does not have spores.
It stains readily with all the aqueous anilin dyes — best
with rather weak solutions — but not by Gram’s method.
It can be found in the urethral discharges of gonorrhea
from the beginning until the end of the disease, though
in the later days its numbers may be outweighed by other

202

PATHOGENIC BACTERIA .

organisms. Wertlieim cultivated the gonococcus from a
case of chronic urethritis of two years’ standing, and
proved its virulence by producing with it gonorrhea in
a human being. The organisms are generally found
within the pus-cells (Fig. 58) or attached to the surface
of epithelial cells, and should always be sought for as
diagnostic of gonorrhea, especially as urethritis some¬
times is caused by other organisms, as the Bacillus coli
communis1 and the Staphylococcus pyogenes.

The cultivation of the gonococcus is not an easy task,
but one which requires considerable bacteriologic skill.
Wertheim accomplished it by diluting a drop of the pus
in a little liquid human blood-serum , then mixing this
with an equal part of melted 2 per cent, agar-agar at 40°
C., and pouring into Petri dishes. As soon as the media
became firm the dishes wrere stood in the incubator at
370 C., and in twenty-four hours the colonies could be
observed. Those upon the surface showed a dark centre,
around which a delicate granular zone could be made
out.

When one of these colonies is transferred to a tube of
human blood-serum or the above mixture obliquely co¬
agulated, isolated little gray colonies occur ; later these
become confluent and produce a delicate smeary layer
upon the medium. The main growth is surrounded by
a thin, veil-like extension which gradually fades away
into the medium. A slight growth occurs upon the
water of condensation.

Turro says that the gonococci may also be cultivated
upon acid gelatin, upon gelatin containing . acid urine,
and also in acid urine itself, in which the gonococci grow
near the surface, while the pus-cocci which may be mixed
with them sink deeper into the medium. His work has
not been confirmed by other investigators.

Heiman,2 who made an extensive series of culture-ex-

1 Van der Pluyn and Loag : Centralbl . f Bakt. u. Parasitenk Bd. xvii.,
Nos. 7, 8, Feb. 28, 1895, p. 233.

2 Med. Record , Dec. 19, 1886.

SIJPPUR A TJ ON.

203

periments, finds that the gonococcus gx*ows best in a mix¬
ture of 1 part of pleuritic fluid and 2 parts of 2 per cent,
agar. Wright1 prefers a mixture of urine, blood-serum,
peptone, and agar-agar.

It is ordinarily presumed that gonorrhea cannot be
communicated to animals, but Turro asserts that the
gonococci when grown upon acid gelatin readily com¬
municate urethritis to clogs, and that no Icesio continui is
necessary, the simple introduction of the organisms into
the meatus sufficing to produce the disease.

The injection of gonococci into the subcutaneous tissue
does not produce abscess.

There is no doubt that the gonococcus causes gonor¬
rhea, as it has on several occasions been intentionally
inoculated into the human urethra with resulting typical
gonorrhea. It is constantly present in the disease, and
very frequently also in the sequela: — endometritis, salpin¬
gitis, oophoritis, cystitis, peritonitis, arthritis, conjuncti¬
vitis, endocarditis, etc. — and, so far as can at present be
determined, is never found under normal conditions.

In the beginning of their activities the cocci grow in
the superficial epithelial cells, but soon penetrate between
the cells to the deeper layers, where they continue their
irritation as the superficial cells desquamate. Authorities
differ as to whether the gonococci can penetrate squamous
and columnar epithelium with equal facility.

The periurethral abscesses that occur in the course of
gonorrhea are generally due to the Staphylococci aureus
and albus, not directly to the gonococcus.

In certain of the remote secondary inflammations the
gonococci disappear after a time, and either the inflam¬
mation subsides or is maintained by other bacteria. In
synovitis this does not seem to be true, and the inflam¬
mation excited may last for months.

As long as the gonococci persist the patient may spread
contagion. It must be pointed out that after apparent
recovery from the disease the cocci sometimes remain

1 Jour, of the Amer. A/e<t. Assoc. , Feb., 1895.

204

PATHOGENIC BACTERIA.

latent in the urethra, and cause a relapse if the patient
partake of some substance, as alcohol, irritating to the
mucous membranes. Bearing this in mind, patients
should not too soon be discharged as cured.

The gonococci are not easily killed, but withstand dry¬
ing very well. Kratter was able to demonstrate their
presence upon washed clothing six months after the orig¬
inal soiling, and also found that they still stained well.

Bumm found cocci similar to the gonococcus in the
urethra, and points out that neither the shape nor the
position in the cells is positively characteristic, but that,
in addition, there must be refusal to stain by Grands
method before we can say with certainty that cocci found
in urethral pus are gonococci.

All of the urethral inflammations do not depend upon
the gonococcus, and in true gonorrhea all of the inflam¬
matory symptoms do not depend upon the gonococcus, as
the epithelial denudation following the disease permits
the entrance of the common pus cocci of the urethra into
the peri-uretliral tissues. The peri-urethral abscesses and
salpingitis, etc., not infrequently depend upon the ordi¬
nary pus cocci, and I have seen a case of gonorrhea with
double orchitis and general septic infection, with endo¬
carditis, in which the gonococci had no role in the sep¬
sis, which was caused by a large dumbbell-coccus that
stained beautifully by Gram’s method.

Mumps, or Epidemic Parotitis.

This epidemic, infectious disease of childhood, charac¬
terized by enlargement of the parotid and submaxillarv
glands, and rarely of the testicles, ovaries, and mamnue,
has not been proved to have a specific micro-organism.

Pasteur thought the disease due to bacilli which he
found in the blood. Capitan and Charrin1 and Olivier
found in the blood, urine, and saliva both cocci and ba¬
cilli, but their studies are too early, and hence too crude
to be of any value.

1 Coviptes Rendu Soc. de Bioc. de Paris , May 2<S, 1S81.

SUPPURA TION.

205

Bouchard, Boisnet, and Bordas also found micro-organ¬
isms in the blood and saliva.

Netter, L,averan, Catrin, Mecray, and Walsh have all
studied cases and isolated a diplococcus thought to be
specific. The organism is described as occurring in pairs
and in fours, sometimes in zooglea. It grows slowly in the
ordinary media, clouding bouillin in twenty-four hours,
and appearing on gelatin after forty-eight hours as small
white punctiform colonies which develop very slowly
and liquefy some considerable time after coalescence.
It grows on potato, and has a whitish appearance not
easy to detect. Laveran and Catrin found the organism
in 67 out of 72 cases examined. In their method a
few drops of exudate are withdrawn from the inflamed
gland with a hypodermic needle, some of the negative
results being due to the fact that the needle withdrew no
exudate. The blood gave pure cultures in 10 out of 15
trials.

Mecray and Walsh report that by disinfecting the
mouths of patients, suffering from mumps, with a satu¬
rated boric acid solution, and cleansing Stensen’s duct,
by careful massage expressing its secretion, and then
allowing a piece of cotton saturated with a boric acid
solution to remain for five minutes between the orifice of
the duct and the jaw, they were able to secure from the
interior of the duct upon a bougie of sterile catgut a
micrococcus identical with that Laveran had found. Of
tubes inoculated with the contents of Stensen’s duct 6
gave a mixed growth. All, however, showed the diplo¬
coccus. Out of 8 carefully made blood examinations, 3
gave pure cultures of the coccus and 3 mixed cultures; 2
were negative.

From Stensen’s duct in healthy children they obtained
the various oral bacteria, but not the diplococcus found
in the cases of mumps. The experimenters do not think
it possible that this diplococcus is the Staphylococcus
epidermidis albus, as its growth is slower and the lique¬
faction of gelatin is accomplished only after a longer

206 PA THO GENIC 'BA CTERIA .

time than is required by the staphylococcus. They did
not succeed in producing mumps in animals. In their
experience a dog was encountered which suffered from
swelling of the parotids, malaise, etc., after playing with
a child suffering from mumps.

Concerning the diplococcus, it appeared in twos and fours ;
rarely in larger groups. Each was regularly rounded and
about the size of the pus cocci. The colonies are small,
white, glistening, distinctly defined, regularly circular
spots, at first discrete and of slow growth, gradually coa¬
lescing. The slow growth is characteristic. In study¬
ing pure cultures, some gelatin tubes three days after in¬
oculation were set aside, no growth being noted; three
days later the small white colonies became distinctly vis¬
ible. At ordinary temperatures gelatin is not liquefied
until ten or twelve days, and the liquefaction proceeds
slowly. A faint white streak appears on potato on the
third day, and spreads as a delicate whitish film. The
growth upon blood-serum is more rapid than on other
media, but the colony is not so distinctly white in color.
Litmus milk is changed to pink on the third day and is
coagulated. Milk is thought to be an excellent nutrient
medium, and a possible ready means of spreading con¬
tagion.

In the paper of Mecray and yS alsh no mention is made
of the relation of the cocci to pus cells or other organized
constituents of the secretion from which they were
obtained; no animal inoculations were done and nothing
is said about the reaction to Gram’s method of staining
or possible motility the cocci might possess.

Michaelis and Bein,1 of Leyden’s clinic, found a diplo¬
coccus (previously observed by Leyden in the sputum),
which occurred chiefly in the pus cells. In severe cases
of the disease, which they studied by culture and micro¬
scopic section, the organism was not only secured from
Stensen’s duct, but in 2 cases from the pus of an abscess
(parotid ?) and in 1 case from the blood.

1 Deutsche med. Wochenschrift May 13, 1897.

SUPPURA TION.

207

In spite of the small number of cases studied, they
were of the opinion that their coccus is the specific one.
Its is about 1 fi in size and resembles the gonococcus,
though it is smaller. The cocci generally lie in the
cells, sometimes 8 or 10 in one pus cell, and are occasion¬
ally distributed throughout the pus in long chains or
strings. They stain readily with the usual anilin dyes,
especially with Loffler’s methylene-blue, and can be
decolorized by the Gram method. They grow slowly
upon the ordinary media, forming living, transparent,
dew-like points on agar-agar. These little drops do not
coalesce. In peptone-bouillon they form white, rather
granular than flocculent deposit, the bouillon itself re¬
maining clear. The growth is said to be more rapid in
strongly than feebly alkaline media. The cocci are said
to grow upon ascites-fluid and upon milk, the latter coag¬
ulating in the course of forty-eight hours. They are
capable of slight movement. Numerous inoculation ex¬
periments were made, only one animal, a white mouse,
succumbing. Control-experiments failed to disclose “the
same organisms in the healthy human parotid or its se¬
cretion.

All the observers agree in finding in the secretions of
the gland and in the blood diplococci that grow slowly,
produce small colonies, and coagulate milk. No one has
shown their specificity by inoculation, evidence of course
necessary before the claim of real importance can be
accepted.

II. THE CHRONIC INFLAMMATORY DISEASES.

CHAPTER I.

TUBERCULOSIS.

Tuberculosis is one of the most dreadful and, un¬
fortunately, most common diseases of mankind. It affects
alike the, young and the old, the rich and the poor, the
male and the female, the enlightened and the savage.
Nor do its ravages cease with human beings, for it is
common among animals, occurring with great frequency
among cattle, less frequently among goats and hogs, and
sometimes, though rarely, among sheep, horses, dogs,
and cats.

Wild animals under natural conditions seem to escape
the disease, but when caged and kept in zoological gar¬
dens even the most resistant of them — lions, tigers, etc. —
are said at times to succumb to it, while it is the most
common cause of death among captive monkeys.

The disease is not even limited to mammals, but occurs
in a somewhat modified form in birds, and, it is said,
even at times affects reptiles.

It is not a disease of modern times, but one which has
persisted through centuries ; and though, before the ad¬
vent of the microscope, not always clearly separated
from cancer, it has not only left unmistakable signs of
its existence in the early literature of medicine, but has
also imprinted itself upon the statute-books of some
countries, as Naples, where its ravages were great and
the means taken for its prevention radical.

While the great men of the early days of pathology

clearly saw that the time must come when the parasitic
208

rrziKRCULoszs.

209

nature of this disease would he proved, and some, as
Klebs, Villemin, and Colmheim, were il within an ace n
of the discovery, it remained for Robert Koch to succeed
in demonstrating and isolating the specific bacillus, now
so well known, and to write so accurate a description of
the organism and the lesions it produces as to render it
almost unparalleled in medical literature.

The tubercle bacillus (Pig. 59) is a rod-shaped organ-

Fi<;, Section <»f ,i ju tiloiicMl tuluMclr from ;i cow, '.liowin^ l hr tulu'ivle

bacilli ; * 50ft ( I'r.ltikrl and iMrilirn.

ism with rounded ends and a slight curve, measuring
from 1.5-3. 5 fL in length and from 0.2-0. 5 ft in breadth.
It very commonly occurs in pairs, which may be asso¬
ciated end to end, but generally overlap somewhat and
are not attached to each other. It. is very common to
observe a peculiar beaded appearance in organisms found
in pus and sputum (Fig. 60), due to the contraction of
fragmented protoplasm within the resisting capsule (?).
Ry sonic these fragmentations are thought to be bacilli
in the stage of sporulation (see Pig. 61). Koch origin¬
ally held this view himself, but researches have not been
able to substantiate the opinion, and at present the evi-

210

PA THO GENIC BA CTERIA .

dences pro and co7i point more strongly in the negative
than in the positive direction.

The fragments do not look like the spores of any other
organisms. When spores occur in the continuity of

Fig. 6o. — Tubercle bacillus in sputum (Frankel and Pfeiffer).

bacilli, they are generally discrete oval refracting bodies
easily recognized. The fragments seen in the tubercle
bacillus are irregular and biconcave instead of oval, have

Fig. 6i. — Tubercle bacilli: I, forms suggesting sporulation; 2, forms de¬
scribed as beaded ; the open spaces in the fragmented rods are sometimes mis¬
taken for spores.

ragged surfaces, and are without the refraction peculiar
to the ordinary spore.

The spaces between the bacillary fragments cannot be
made to stain like the spores of other species. Finally,

TUBERCULOSIS.

2ll

all known spores resist heat more strongly than the fully-
developed bacilli, but experimentation has shown that
these degenerative forms are no more capable of resist¬
ing heat than the tubercle bacilli themselves.

The organism is not motile, and does not possess
flagella.

The tubercle bacillus is peculiar in its reaction to the
anilin dyes. It is rather difficult to stain, requiring that
the dye used shall contain a mordant (Koch), but it is also
very tenacious of the color once assumed, resisting the
decolorizing power of strong mineral acids (Ehrlich).

These peculiarities delayed the discovery of the bacil¬
lus for a considerable time, but now that we are familiar
with them they give us a most valuable diagnostic cha¬
racter, for with the exception of the bacillus of lepra no
known bacillus reacts in exactly the same way.

Koch first stained the bacillus with an aqueous solu¬
tion of a basic anilin dye to which some potassium
hydrate was added, subsequently washing with water
and counter-staining with vesuvin. Ehrlich subsequently
modified Koch’s method, showing that pure anilin was
a better mordant than potassium hydrate, and that the
use of a strong mineral acid would remove the color
from everything but the tubercle bacillus. This modi¬
fication of Koch’s method given us by Ehrlich is at the
present time acknowledged to be the best method of
staining the bacillus. Many other methods have been
suggested, all of them, perhaps, more convenient than
Ehrlich’s, but none so good.

As being that most frequently performed by the
physician, we will first describe the method of seeking
the bacillus in sputum.

If one desires to be very exact in his examination,
it may be well to have the patient cleanse the mouth
thoroughly upon waking in the morning, and after the
first fit of coughing expectorate into a clean wide¬
mouthed bottle. The object of this is to avoid the
presence of fragments of food in the sputum.

212

PATHOGENIC BACTERIA .

The physician will secure a better result if the exam¬
ination be made on the same day than if he wait a num¬
ber of days, because if the bacilli are few they occur
most plentifully in the small caseous flakes to be de¬
scribed farther on, which are easily found at first, but
which break up and become part of a granular sediment
that always forms in decomposed sputum.

The fresh sputum when held over a black surface
generally shows a number of grayish-yellow, irregular,
translucent granules somewhat smaller than the head of
a pin. These consist principally of the caseous material
from tuberculous tissue, and are the most valuable part
of the sputum for examination. One of the granules is.
picked up with a pointed match-stick and spread over
the surface of a perfectly clean cover-glass. If no such
fragment can be found, the purulent part is next best for
examination. The mucus itself rarely contains bacilli
when free from scraps of tissue and pus.

In cases in which this ordinary procedure fails to reveal
bacilli whose presence is strongly indicated by the clin¬
ical signs, the exact method of searching for them is to
partially digest the sputum with caustic potash, and then
collect the solid matter with a centrifugal apparatus. If
a very few bacilli are present in the sputum, this method
will often secure them.

The material spread upon the cover-glasses should not
be too small in amount. Of course a massive, thick
layer will become opaque in staining, but should the
layer spread be, as is often advised, u as thin as possible, ”
there may be too few bacilli upon the glass to enable one
to make a satisfactory diagnosis.

As usual, the material is allowed to dry thoroughly,
and is then passed three times through the flame for
purposes of fixation.

Ehrlich's Method , or the Koch-Ehrlich Method. — The
cover-glasses thus prepared are floated, smeared side-
down, upon, or immersed, smeared side up, in, a small
dish of Ehrlich’s anilin-water gentian- violet solution:

TUBERCULOSIS .

21 3

Anilin, 4,

Saturated alcoholic solution of gentian violet, ii,
Water, ioo,

and placed in an incubator or a paraffin oven, and kept
for twenty-four hours at about the temperature of the
body. When removed from the stain they are washed
momentarily in water, and then alternately in 25-33
per cent nitric acid and 60 per cent, alcohol, until the
blue color of the gentian violet is almost entirely lost.
It must be remembered that the action of the strong acid
is a powerful one, and that too long a time must not be
allowed for its application. A total immersion of thirty
seconds is quite enough in most cases. After final thor¬
ough washing in 60 per cent, alcohol the specimen is
counter-stained in a dilute aqueous solution of Bismarck
brown or vesuvin. The excess of stain is then washed
off in water, and the specimen is dried and mounted in
balsam. The tubercle bacilli will appear of a fine dark
blue, while the pus-corpuscles, epithelial cells, and other
bacteria, having been decolorized by the acid, will be
colored brown by the counter-stain.

This method, requiring twenty-four hours for its com¬
pletion, is naturally one which has fallen into disuse for
practitioners who desire in the briefest possible time to
know simply whether bacilli are present in the sputum
or not.

Among clinicians ZiehPs method with carbol-fuchsin
has met with great favor. After having been spread,
dried, and fired, the cover-glass is held in the bite of an
appropriate forceps (cover-glass forceps), and the stain1
dropped upon it from a pipette. As soon as the entire
cover-glass is covered with stain it is held over the flame
of a spirit-lamp or a Bunsen burner until the stain begins
to volatilize a little, as indicated by a white vapor. When

1 Carbol-fuchsin (see p. 86) :

Fuchsin, I ;

Alcohol, io;

214

PATHOGENIC BACTERIA .

this is observed, the heating is sufficient, and the temper¬
ature can be subsequently maintained by intermittent
heating.

If evaporation is allowed to take place, a ring of in¬
crustation occurs at the edge of the area covered by the
stain and prevents the proper action of the acid. To
prevent this more stain should now and then be added.
The staining is complete in from three to five minutes,
after which the specimen is washed off with water, the
excess of water absorbed with paper, and 3 per cent,
hydrochloric acid in 70 per cent, alcohol, 25 per cent,
aqueous sulphuric, or 33 per cent, aqueous nitric acid
solution dropped upon it for thirty seconds, or until the
red color is just extinguished. The acid is washed off
with water, and the specimen is dried and mounted in
Canada balsam. Nothing will be colored except the tu¬
bercle bacilli, which will appear red.

Gabbett modified the staining by adding methylene
blue to the acid solution, which he makes according to
this formula:

Methyl blue,

Sulphuric acid,

Water,

75'

In Gabbett’s method, after staining with carbol-fuch-
sin the specimen is washed with water, acted upon by
the methylene-blue solution for exactly thirty seconds,
washed with water until only a very faint blue remains,
dried, and finally mounted in Canada balsam. By this
method the tubercle bacilli are colored red, and the pus-
corpuscles, epithelial cells, and the unimportant bacteria
blue.

The possible relation that the number of bacilli in the
expectoration of consumptives might bear to the progress *
or treatment of the case has been elaborately investigated
by Nuttall.1 The total quantity of sputum expectorated
in twenty-four hours was caught in covered, scrupulously

1 Bull, of the Johns Hopkins Hospital May and June, 1891, ii., 13.

TUBERCULOSIS.

215

clean conical glasses and measured therein. The- pro¬
portion of muco-purulent to fluid matter was noted.
Depending upon the degree of viscidity and number of
bacilli present in the sputum, a varying amount of 5 per
cent, caustic potash solution was added to it (from one-
sixth to an equal volume), and after the caustic potash
had rendered the sputum perfectly fluid more or less water
was added to dilute the mixture. The sputum, having
been measured, was poured into a perfectly clean wide¬
mouthed bottle containing fine sterilized gravel or broken
glass. Rinsings of a measured amount of the caustic pot¬
ash solution were used to free the conical glass from what
matter might remain and were added to the sputum.
The contents of the bottle were agitated in a shaking
machine for five minutes, and allowed to stand until the
caustic potash solution had had time to act As soon as
the sputum had become homogeneous an equal volume
of water was added, and the whole shaken again. The
sputum thus treated was of a pale-green or yellowish-
brown color, and contained only small fragments of elas¬
tic tissue. It was allowed to stand two to four hours,
and then shaken again for five to ten minutes.

By means of a burette of original design drops of ex¬
actly equal size were secured and caught upon clean
sterile cover-glasses. The drops were subsequently
spread into an even film by a very fine platinum wire,
while the cover-glass was rotated upon a “ turn-table. 5 ’
After spreading, the cover-glasses were laid upon a level
brass plate slightly warmed to facilitate drying. After
drying, the cover-glasses were coated with a serum film
by spraying, and the temperature raised to 8o°-90° C. to
coagulate the serum and retain the bacteria in place,
after which they were carefully stained with carbol-
fuchsin and decolorized with a solution of 150 parts of
water, 50 parts of alcohol, and 20-30 drops of pure sul¬
phuric acid. Prior to this the cover-glass was washed in
three alcohols and subsequently in water, and if necessary
in acid and alcohol again.

2l6

PATHOGENIC BACTERIA .

A special arrangement of the microscope was devised
for the purpose, and the number of bacilli in each drop
estimated with extreme care. The number varied from
472 to 240,000. To estimate the number of bacilli in a
given quantity the number of drops to a cubic centimeter
is multiplied by the number of bacilli in the drop, and
then by the number of cubic centimeters to be estimated.

The method is an ingenious one, but a glance down
the columns of figures in the original article will be
sufficient to show that the counting of the bacilli is
devoid of any particular value.

This is only to be expected when one considers the
pathology of the disease and remembers that accidents,
such as unusually violent cough one day, modified by
the use of sedatives the next, may cause wide variations
in the quality if not in the quantity of the sputum.

When the tubercle bacilli are to be sought for in sections
of tissue, considerable difficulty is at once encountered,
partly because of the thickness of the section and partly
because of the presence of nuclei which color intensely.

Again, Ehrlich’s method must be recommended as the
most certain and best method of staining a large number
of bacilli.

The sections of tissue, if imbedded in celloidin or par¬
affin, should be freed from the foreign substances. Like
the cover-glasses, they are placed in the stain for twelve
to twenty-four hours at a temperature of 370 C. Upon
removal they are allowed to lie in water for about ten
minutes to wash away the excess of stain and to soften
the tissue, which often shrinks and becomes brittle. The
washing in nitric acid (20 per cent.) which follows may
have to be continued for as long as two minutes. Thor¬
ough washing in 60 per cent, alcohol follows, after which
the sections can be counter-stained, washed, dehydrated
in 95 per cent, and absolute alcohol, cleared in xylol,
and mounted in Canada balsam.

A method which has attained great and deserved praise
is Unna’s. It is as follows: The sections are placed in

TUBERCULOSIS .

217

a dish of twenty-four-hours-old, newly-filtered Ehrlich’s
solution, and allowed to remain twelve to twenty-four
hours at the room-temperature or one to two hours in
the incubator. From the stain they are placed in water,
where they remain for about ten minutes to wash. They
are next immersed in acid (20 per cent, nitric acid) for
about two minutes, and become greenish-black. From
the acid they are placed in absolute alcohol, and are
gently moved to and fro until the pale-blue color returns.
They are then washed in three or four changes of clean
water until they become almost colorless, and are then
removed to the slide by means of a section-lifter. The
water is absorbed with filter-paper, and then the slide is
heated over a Bunsen burner until the section becomes
shining, when it receives a drop of xylol balsam and a
cover-glass.

It is said that sections staiued in this manner do not
fade as quickly as those stained by Ehrlich’s method.

The tubercle bacillus also stains well by Gram’s method,
but as this is a general method by which many different
bacteria are colored, it is ill adapted for purposes of differ¬
entiation, especially when the prosecution of the charac¬
teristic methods is not more difficult.

So far as is known, the tubercle bacillus is a purely
parasitic organism. It has never been found except in
the bodies and excretions of animals affected with tuber¬
culosis, and in dusts of which these are component parts.
This purely parasitic nature greatly interferes with the
isolation of the organism, which cannot be grown upon
the ordinary culture-media. Koch first achieved its arti¬
ficial cultivation by the use of blood-serum. When
planted upon this medium the bacilli are first apparent
to the naked eye in about two weeks, and occur in the
form of small dry, whitish flakes, not unlike fragments
of chalk. These slowly increase at the edges, and grad¬
ually form scale-like masses of small size, which under
the microscope are seen to consist of tangled masses of
bacilli, many of which are in a condition of involution.

2l8

PA THO GENIC BA CTERIA .

The best method of obtaining a culture is to inoculate
a guinea-pig with tuberculous material, allow an artificial
tuberculosis to develop, kill the animal after a couple of
months, and make the cultures from the centre of one of
the tuberculous glands.

Of course many technical difficulties must be over¬
come. The tuberculous material used for inoculaticm
may be sputum, injected beneath the skin by a hypo¬
dermic syringe. The animal is allowed to live for a
month or six weeks, then killed. The autopsy is per¬
formed according to directions already given. A large
lymphatic gland with softened contents or a nodule in the
spleen being selected for the culture, an incision is made
into it with a sterile knife, or a rigid sterile platinum
wire is introduced ; some of the contents are removed
and planted upon blood-serum. After receiving the in¬
oculated material the tubes are closed, either by a rub¬
ber cap placed over the cotton stopper, which is cut off
and pushed in, or by a rubber cork above the cotton,
the idea of this rubber corking being simply to prevent
evaporation. The tubes must be kept in an incubator
at the temperature of 37-38° C.

Kitasato has published a method by which Koch has
been able to secure the tubercle bacillus in pure culture
from sputum. After carefully cleansing the mouth the
patient is allowed to expectorate into a sterile Petri dish.
By this method the contaminating bacteria from the
mouth and the receptacle are excluded, and the expecto¬
rated material is made to contain only such bacteria as
were present in the lungs. The material is carefully
washed a great many times in renewed distilled sterile
water until all bacteria not enclosed in the muco-purulent
material are removed ; it is then carefully opened with
sterile instruments, and the culture-medium — glycerin
agar-agar or blood-serum — is inoculated from the centre.
Kitasato has been able by this method to demonstrate
that many of the bacilli ordinarily present in tubercular
sputum are dead, although they continue to stain well.

TUBERCULOSIS .

219

Kitasato’s method of washing the sputum has been
modified and simplified by Czaplewski and Hensel.1 In
their studies of whooping-cough, instead of washing the
flakes in water in dishes, they shook them in peptone
water in test-tubes. The shaking in the test-tube being
so much more thorough than the washing in dishes, fewer
'changes are necessary, three or four washings being
sufficient.

In 1887, Nocard and Roux gave a great impetus to
investigations upon tuberculosis by their discovery that

the addition of 4-8 per cent,
of glycerin to bouillon and
agar-agar made them suitable
for the development of the
bacillus, and that a much
more luxuriant development
could be obtained upon these
media than upon blood-se¬
rum. The growth upon such
4 4 glycerin agar-agar 5 ’ (F ig.
62) very much resembles
that upon blood-serum. The
growth upon bouillon with
4 per cent, of glycerin is
also luxuriant. As tubercle
bacilli require considerable
oxygen for their proper devel¬
opment, they grow only upon
the surface of the bouillon,

Fig. 62. — Bacillus tuberculosis on

where a rather thick myco- “glycerin agar-agar.”

derma forms. The surface-

growth is rather brittle, and after a time gradually sub¬
sides fragment by fragment.

The tubercle bacillus can be grown in gelatin to which
glycerin has been added, but as its development takes
place only at 37°-38° C., a temperature at which gelatin is
always liquid, its use for the purpose is disadvantageous.

1 Centralbl. f Bakt. u. Parasitenk., xxii., Nos. 22 and 23, p. 643.

220

PATHOGENIC BACTERIA .

Pawlowski was able to cultivate the bacillus upon
potato, but Sander, who found that it could be readily
grown upon various vegetable compounds, especially
upon acid potato mixed with glycerin, also found that
upon such compounds its virulence was constantly lost.

It has also been shown that the continued cultivation
of the tubercle bacillus upon such culture-media as
are appropriate so lessens its parasitic nature that in the

Fig. 63 — Bacillus tuberculosis : adhesive cover-glass preparation from a fourteen-
day-old blood- serum culture; x 100 (Frankel and Pfeiffer).

course of time it can be induced to grow feebly upon the
ordinary agar-agar.

It is really surprising to note the extremely simple
compounds in which the tubercle bacillus can be grown.
Instead of requiring the most concentrated albuminous
media, as was once supposed, Proskauer and Beck have
shown that the organism can grow in non-albuminous
media containing asparagin, and that it can even be in¬
duced to grow upon a mixture of commercial ammonium
carbonate, 0.35 per cent.; primary potassium phosphate,
0.15 per cent.; magnesium sulphate, 0.25 per cent.;
glycerin, 1.5 percent. It was even found that tuberculin
was produced in this inorganic mixture.

TUBERCULOSIS.

221

The tubercle bacillus seems to require a considerable
amount of oxygen for its development. It is also pecu¬
liarly sensitive to temperatures, not growing at a tem¬
perature below 290 C. or above 420 C. Temperatures
above 750 C. kill it after a short exposure.

The tubercle bacillus does not develop well in the
light, and when its virulence is to be maintained should
always' be kept in the dark. Sunlight kills it in from
a few minutes to several hours, according to the thick¬
ness of the mass exposed to its influence.

The widespread character of tuberculosis at one time
suggested the idea that tubercle bacilli were ubiquitous
in the atmosphere, that we all inhaled them, and that it
was only our vital resistance that prevented us all from
becoming its victims.

Cornet must be given the credit of having shown that
such an idea is untrue, and that tubercle bacilli only
exist in the atmospheres frequented by consumptives.
His experiments were made by collecting dusts from
numerous places — streets, sidewalks, houses, rooms, walls,
etc. Injecting them into guinea-pigs, whose constant
susceptibility to the disease makes them a very delicate
reagent for its detection, Cornet showed the bacilli to be
present only in the dust with which pulverized sputum
was mixed, and found such infectious dust to be most
common where the greatest carelessness in respect to
cleanliness prevailed.

Our present knowledge of the life-liistory of the tubercle
bacillus, by showing its indisposition to multiply outside
the bodies of animals, the deleterious influence of sun¬
light upon it, the absence of positive permanent forms,
and its sensitivity to temperatures beyond a certain range,
confirms all that Cornet has pointed out, and shows us
why the expectoration of millions of consumptives has
not rendered our atmospheres pestilential.

As long as tuberculosis exists among men or cattle, it
shows that the existing hygienic precautions are insuf-

222

PATHOGENIC BACTERIA.

ficient While not so radical as to suggest the unreason¬
able isolation of patients and destruction of property once
practised in the kingdom of Naples, the author would
favor the registration of all tuberculous cases as a means
of collecting accurate data concerning their origin, would
insist upon domestic sterilization and disinfection, and
would have special hospitals for as many, especially of
the poorer classes, among whom hygienic measures are
almost always opposed, as could be persuaded to occupy
them.

It has already been declared the duty of the physician
to use every means in his power to prevent the spread
of infection in the households in his care, and no disease
is more deserving of attention than this neglected one.
Patients should cease to kiss the members of their fam¬
ily and friends ; their individual knives, forks, spoons,
cups, etc. should be carefully kept apart — secretly if the
patient be sensitive upon the subject — from those of the
family, and scalded after each meal ; the napkins and
handkerchiefs, as well as whatever clothing or bed-cloth¬
ing is soiled by the discharges, should be kept apart from
the common wash, and boiled ; and of course the expec¬
toration should be carefully attended to, received in a
suitable receptacle, sterilized or disinfected, and never
allowed to dry, for it has been shown that the tubercle
bacillus can remain vital in dried sputum for as long as
nine months. A very neat arrangement for collecting
and disposing of the expectoration is recommended by
some boards of health. It consists of a metal case into
which a pasteboard box is fitted. When the box is to be
emptied the whole of the pasteboard portion is removed,
and, together with the expectoration, burned. The metal
part is disinfected, provided with a new pasteboard box,
and is again ready for use. (See Fig. 20, page 120.) The
physician should also give directions for disinfecting tbe
bedroom occupied by a consumptive before it becomes
the chamber of a healthy person.

Boards of health are now becoming more and more in-

Tl7I>E/\C( r LOS IS.

22 3

(crested in tuberculosis, and, though exceedingly slow
and conservative in their movements, are disseminating
literature among doctors for distribution to their patients,
with the hope of achieving by volition that which they
would otherwise regard as cruel compulsion.

The channels by which the tubercle bacillus enters the
organism are varied. A few cases are on record where
the micro-organisms have passed through the placenta ,
so that a tuberculous mother was able to infect lier
unborn child. It is not impossible that the passage of
bacilli in this manner through the placenta causes the
development of tuberculosis in infants after birth, the
disease having remained latent during fetal life, for
Hireh-I Iirschfeld has shown that fragments of a fetus,
itself showing no tubercular lesions, hut coming from a
tuberculous woman, were fatal to guinea-pigs into which
they were inoculated.

The most frequent channel of infection is the respira¬
tory tract , into which the finely-pulverized dust of rooms
and streets enters. Probably all of us at some time in
our lives inhale living virulent tubercle bacilli, yet not
all of us suffer from tuberculosis. Personal predisposi¬
tion seems of great importance, for it has been shown
that without the formation of tubercles virulent bacilli
may be present for considerable lengths of time in the
bronchial lymphatic glands — the dumping-ground of the
pulmonary phagocytes.

In order that infection shall occur it does not seem
necessary that the least abrasion or laceration shall exist
in the mucous lining of the respiratory tract. The
tubercle bacillus is a foreign body of irritating prop¬
erties, and, lodging upon a cell, is soon engulfed in its
protoplasm, or, arrested by a leucocyte, is dragged off to
some other region in whose narrow passages a most hos¬
tile strife doubtless takes place.

Infection also commonly takes place through the yas-
tro-intcstinal tract by infected food. At present an over¬
whelming weight of evidence points to the presence of

224

PATHOGENIC BACTERIA.

bacilli in the milk of cattle affected with tuberculosis. It
does not seem necessary that tuberculous ulcers shall be
present in the udders; indeed, the bacilli have been
demonstrated in cbtisiderable numbers in milk from
udders without tubercular lesions discoverable to the
naked eye.

The meat from tuberculous animals is less dangerous
than the milk, because the meat is nearly always cooked
before being eaten, while the milk is generally taken
uncooked. The bacilli enter the intestinal lymphatics,
sometimes produce lesions immediately beneath the mu¬
cous membrane, and lead later on to the formation of
ulcers ; but generally they first involve the mesenteric
lymphatic glands. The thoracic duct is sometimes af¬
fected, and from such a lesion it is easy to understand the
development of a general miliary tuberculosis. The oc¬
casional absorption of tubercle bacilli by the lacteals, and
their entrance into the systemic circulation and subse¬
quent deposition in the brain, bones, joints, etc., are sup¬
posed to explain primary lesions of these tissues.

Infection is said also to take place occasionally through
the sexual apparatus . In sexual intercourse tubercle
bacilli from tuberculous testicles may be discharged into
the female organs, with resulting tuberculous lesions.
The infection in this way generally is from the male to
the female, primary tuberculosis of the testicle being
much more common than primary tuberculosis of the
uterus or ovaries.

While most probably rare, in comparison with the
preceding, wounds also are avenues of entrance for the
tubercle bacilli. Anatomical tubercles are not uncom¬
mon upon the hands of anatomists and pathologists,
most of these growths being tuberculous in character.
An interesting fact concerning these dermal lesions
is the exceedingly small number of bacilli which they
contain.

The macroscopic lesions of tuberculosis are too familiar
to require a description of any considerable length. They

Tuberculosis of the lung : the upper lobe shows advanced cheesy consoli¬
dation with cavity-formation, bronchiectasis, and fibroid changes ; the lower
lobe retains its spongy texture, but is occupied by numerous miliary tubercles.

TUBER C UL OS/S.

225

consist in nodes, nodules, or collections of agminated
nodules, called tubercles, scattered irregularly through
the tissues, which are devitalized or disorganized by
their presence. When tubercle bacilli are introduced
beneath the slcin of a guinea-pig, the animal shows no
sign of disease for a week or two ; it then begins to lose
appetite and gradually to diminish in flesh and weight.
Examination at this time will show a nodule at the point
of injection and enlargement of the neighboring lymphatic
glands. The atrophy increases, the animal shows a febrile
reaction, and at the end of a varying period of time,
averaging about twelve weeks, dies. Post-mortem ex¬
amination shows a cluster of tubercles at the point of
inoculation, enlargement of lymphatic glands both near
and remote from the primary lesion (due to the presence
of tubercles), aud a widespread invasion of the lungs,
liver, kidneys, peritoneum, and other organs and tissues,
with tuberculous tissue in a more or less advanced con¬
dition of necrosis. Sometimes there are no tubercles
discoverable at the point of inoculation. There is no
regularity in the distribution of the disease. Tubercle
bacilli are demonstrable in immense numbers in all the
diseased tissues. The disease as seen in the guinea-pig is
more extended than in other animals because of its greater
susceptibility, and the death of the animal is more rapid
than in other species for the same reason. In rabbits the
lesion runs a longer course with similar lesions. In
bovines and sheep the infection is generally first seen
in, and is principally confined to, the alimentary appa¬
ratus and the associated organs, though pulmonary dis¬
ease also occurs. In man the disease is chiefly pulmonary,
though gastro-intestinal and general miliary forms are also
common. The development of the lesions in whatever
tissue or animal always depends upon the distribution of
the bacilli by the lymph or the blood, and is first inflam¬
matory, then degenerative, in type.

The experiments of Koch, Prudden and Hodeuphyl,
and others have shown that when dead tubercle bacilli
15

226

PATHOGENIC BACTERIA .

are injected into the subcutaneous tissues of rabbits
small local abscesses develop in the course of a couple
of weeks, showing that the tubercle bacilli are chemotac-
tically potent.

While it is extremely interesting to observe that this
chemotactic property exists, it seems to be by some other
irritant that most of the lesions of tuberculosis are caused.
When the dead tubercle bacilli, instead of being injected
en 7nasse into the areolar tissue, are so introduced into
the body — as by intravenous injection — as to disseminate
themselves or remain in small groups, the result is quite
different, and much more closely resembles that of the
action of the living organism.

Baumgarten, whose researches were made upon minute
tubercles of the iris, has shown that the first manifesta¬
tion of the irritation caused by the bacillus is not the
attraction of leucocytes, but the stimulation of the fixed
connective-tissue cells of the part affected. These cells
increase in number by karyokinesis, and form about the
irritating bacterium a minute focus which is the primitive
tubercle.

The leucocytes are of secondary advent, and are no
doubt attracted both by the substance shown by Prudden
and Hodenphyl to exist in the bodies of the dead bacilli
and by the necrotic changes which already affect the
primary cells. For reasons not understood, the amount
of chemotaxis varies greatly in different cases. Some¬
times the tubercles will be sufficiently purulent in type
almost to justify the name “tubercular abscess;” some¬
times there will be a marked absence of cellular ele¬
ments derived from the blood.

The important toxic substance produced by the bacillus
is evidently not associated with chemotaxis, for when the
leucocytes are absent the necrosis which is so characteris¬
tic persists.

The groups of cells constituting the primitive tubercle
have scarcely reached microscopic proportions before a
distinct coagulation-necrosis is observable. The proto-

TUBERCULOSIS .

22 7

plasm of the cells affected takes on a hyaline character,
and seems abnormally viscid, so that contiguous cells
have a tendency to become partially confluent. The
chromatin of their nuclei becomes dissolved in the nu¬
clear juice and gives stained nuclei a pale but homo¬
geneous appearance. Sometimes this nuclear change is
only observed very late. As the necrosis advances the
contiguous cells flow together and form large protoplas¬
mic masses — giant-cells — which contain as many nuclei
as there were component cells. It may be that these
nuclei multiply by karyokinesis after the protoplasmic
coalescence, but only one observer, Baumgarten, lias
found signs of this process in giant-cells. While these
changes are in progress in the cells of the primary focus,
the leucocytes may collect in such numbers as to obscure
them and make themselves appear to constitute the prim¬
itive cells. When the irritant substance is produced in
considerable quantities, the most delicate cells die first ;
and it is not infrequent to find a tubercle rich in leuco¬
cytes suddenly showing degeneration of these cells, with
recurring prominence of the original epithelioid cells.

It has been taught by some that the giant-cells are
produced by the union of the leucocytes, but a careful
observation of the role played by these cells will convince
one that such an origin for these monstrous cells must be
very rare.

Giant-cells arc not always produced, for sometimes the
necrotic changes are so violent and widespread as to con¬
vert the whole cellular mass into a granular detritus of
un recognizable fragments.

Tubercles are constantly avascular, as would be ex¬
pected of a process which is a combination of progressive
irritation and necrosis. The avascularity may be a fac¬
tor in the necrosis of the larger tuberculous masses, but
it plays no part in the degeneration of the smallest tuber¬
cles, which is purely toxic.

Tubercles may be developed in any tissue and in any
organ. In whatever situation they occur, space is occu-

228

PATHOGEN/C BACTERIA.

pied at tlie expense of the tissue, whose component cells
are pushed aside or else included in the nodule. In mil¬
iary tuberculosis of the kidney it is not unusual to find a
tubercle including a whole glomerule, and resolving its
component thrombosed capillaries and epithelium into
necrotic fragments.

As almost all tissues contain a supporting tissue-frame¬
work of connective-tissue fragments, some of these must
be embodied in the new growth. The fibres which pos¬
sess little vitality are more resistant than cells, aild, after
all the cells of a tubercle have been destroyed, will be
distinctly visible among the granules, so that the tubercle
has a reticulated appearance.

As a rule, tubercles steadily increase in size by the in¬
vasion of fresh tissue. The tubercle bacillus does not seem
to find the necrotic centres of the tubercles adapted to its.
growth, and completes its life-cycle with the tissue-cells.
It is unusual to find healthy-lookiug bacilli in the necrotic
areas, most of them being observed at the edges of the
tubercle, where the nutrition is good. From such edges
the bacilli are occasionally picked up by leucocytes and
transported through the lymph-spaces, until the phago¬
cyte falls a prey to its prisoner, dies, and sows the seed
of a new tubercle. However, for the spread of tubercle
bacilli from place to place phagocytes are not always
necessary, for the bacilli seem capable of transportation
by streams of lymph alone.

Notwithstanding the steady advance which takes place
in most observed cases of tuberculosis, and the thoroughly
comprehensible microscopic explanation of it, many cases
of tuberculosis make quite perfect recoveries.

The periphery of every tubercle is a zone of reaction,
with a marked tendency to granulation and organization.
If the vital condition is such that through inappro¬
priate nutriment or through unusually active phago¬
cytosis the activity of the bacilli is checked or their
death is brought about, this tendency to cicatrization is
allowed to progress unmolested, and the necrosed mass is

TUBERCULOSIS.

229

soon surrounded with a zone of newly-formed contracting’
fibrillar tissue, by which it is perfectly isolated. In such
isolated masses lime-salts are commonly deposited. Some¬
times this process is perfected without the destruction of
the bacilli, but with their incarceration and inhibition.
Such a condition is called latent tuberculosis , and may at
any time be the starting-point of a new infection and lead
to a fatal termination.

In 1890, Koch announced some observations upon toxic
products of the tubercle bacillus and their relation to the
diagnosis and treatment of tuberculosis, which at once
aroused an enormous but, unfortunately, a transitory
enthusiasm.

These observations, however, are of capital importance.
Koch observed that when guinea-pigs are inoculated
with a mixture containing tubercle bacilli the wound
ordinarily heals readily, and soon all signs of local dis¬
turbance other than enlargement of the lymphatic glands
of the neighborhood disappear. In about two weeks there
occurs at the point of inoculation a slight induration which
develops into a hard nodule, then ulcerates, and remains
until the death of the animal. If, however, in the course
of a short time the animals are reinoculated, the course
of the process is altogether changed, for, instead of heal¬
ing, the wound and the tissue surrounding it assume
a dark color and become obviously necrotic, and ulti¬
mately slough away, leaving an ulcer which rapidly and
permanently heals without enlargement of the lymph-
glands.

Having made this observation with injected cultures
of the living bacillus, Koch next observed that the same
change occurred when the secondary inoculation was
made with pure cultures of the dead bacilli.

It was also observed that if the material used for the
secondary injection was not too concentrated and not
too often repeated (only every six to forty-eight hours),
the animals thus treated improved in condition, and,
instead of dying of the tuberculosis induced by the

230 PATHOGENIC BACTERIA .

primary injection in from six to ten weeks, continued
to live, sometimes (Pfuhl) as long as nineteen weeks.

Koch also discovered that a 50 per cent, glycerin
extract of cultures of the tubercle bacillus produced the
same effect as the dead cultures originally used, and
gave this substance, tuberculin, to the scientific world
for experimental purposes, in the hope that the prolon¬
gation of life observed in the guinea-pig might be true
in the case of man.

The active substance of the “ tuberculin ” seems to be
an albuminous derivative insoluble in absolute alcohol.
It is not a toxalbumin.

The action of the tuberculin upon the animal organ¬
ism is peculiar, but readily understandable. It does not
exert the slightest influence up07i the tubercle bacillus ,
but acts upon the living tuberculous tissue. In the
description of the tissue-changes already given it has
been shown that the tubercle bacillus effects the coagu¬
lation-necrosis of the cells, but does not derive its nutri¬
ment from the dead tissue. As the cells die and are
incorporated in the necrotic mass, the bacilli find the
conditions of life unfavorable, and likewise seem to die.
The active bacilli, therefore, are always found at the mar¬
gins of the tuberculous tissues, where the cells are fairly
active. The necrosis is due to bacillary poisons. When
tuberculin is injected into the organism the result is to
double the amount of poisonous influence upon the cells
surrounding the bacilli, to destroy their vitality, to re¬
move the favorable conditions of growth from the organ¬
ism, and to leave it for a time checkmated.

Virchow, who well understood the action of the tuber¬
culin, soon showed that as a diagnostic and therapeutic
agent in man its use was attended with great danger.
The destroyed tissue was absorbed, and with it the bacilli
were likewise absorbed and transported to new areas,
where a rapid invasion occurred. Old tuberculous lesions
which had been encapsulated were softened, broken
down, and became sources of dangerous infection to the

TUBERCULOSIS .

231

individual, so that, a short time after its enthusiastic
reception as a “gift of the gods,” tuberculin was placed
upon its proper tooting as a diagnostic agent valuable in
veterinary practice, but dangerous in human medicine,
except in cases of lupus and other external forms of the
disease where the destroyed tissue could be discharged
from the surface of the body.

The method of preparation of tuberculin is rather
simple. Small flasks exposing a considerable surface of
liquid are filled with about 25 c.cm. of bouillon contain¬
ing about 4 per cent, of glycerin. The bouillon is prefer¬
ably made with calf- instead of ox-meat. When thor¬
oughly sterile the surfaces are inoculated with pure
cultures of the tubercle bacillus and are stood in an
incubator. In the course of two weeks a slight surface
growth is apparent, which in the course of time develops
into a pretty firm pellicle and gradually subsides. At the
end of four or six weeks development ceases and the
pellicle sinks. The contents of a number of flasks are
then collected in an appropriate vessel and evaporated
over a water-batli to one-tenth their volume, then filtered
through a Pasteur-Chamberland filter. This is crude
tuberculin.

When such a product is injected in doses of a fraction
of a cubic centimeter an inflammatory and febrile reac¬
tion occurs. The inflammation sometimes causes super¬
ficial tuberculous lesions (lupus) to ulcerate and slough
away, and for this reason is of some value in therapeutics,
although attended with the dangers mentioned above.
The feveT is sufficiently characteristic to be of diagnostic
value, though the tuberculin can only be used as a diag¬
nostic agent in practice upon animals.

A recent important work upon tuberculin has been
done by Koch.1

In his experience the attempts made to produce im¬
munity to the tubercle bacillus by the injection into
animals of attenuated cultures proved failures, because

1 Deutsche med . Wochenschrift \ 1897, No. 14.

232 PATHOGENIC BACTERIA .

abscesses invariably followed their introduction, whether
dead or alive, and nodular growths in the lungs were
constant sequelae of their injection into the circulation.
In such nodules the bacilli could be found unabsorbed
and unaltered. It seemed as if the fluids of the body
could not effect solution of the bacteria. The ineffectual
attempts at immunization, with the results given, probably
depend upon the inability of the tissues to take up from
the bacilli whatever immunizing substances they might
contain, first, because of the impossibility of dissolving
them, and, second, because the irritating powers they
possess interfere with the direct action of normal fluids
and uninjured body-cells, and always subject the bacteria
to semi-pathological conditions.

From these data, which he carefully studied out, Koch
concluded that it would be necessary to bring about some
artificial condition advantageous to the absorption of the
bacilli, and for the purpose tried the action of diluted
mineral acids and alkalies. The chemical change brought
about in this manner facilitated absorption, but the ab¬
sorption of bacilli in this altered condition was not fol¬
lowed by immunity, probably because the chemical com¬
position of tubercle-toxin (or whatever one may name
the poisonous products of the bacillus) was changed by
the reagents used.

Tuberculin, with which Koch performed many experi¬
ments, was found to produce immunity only to tubercu¬
lin, not to bacillary infection.

Pursuing the idea of fragmenting the bacilli, or in some
way treating them chemically in order to increase their
solubility, Koch found that a io per cent, sodium hydrate
solution yielded an alkaline extract of the bacillus, which,
when injected into animals, produced effects similar to
those following the administration of tuberculin, except
that they were briefer in duration and more constant in
result. The marked disadvantage of abscess-formation
following the injections, however, remained. This fluid,
when filtered, possessed the properties of tuberculin.

TUBERCULOSIS.

233

The mechanical fragmentation of the bacilli had been
used by Klebs in the studies of antiphthisin and tubercu-
locidin. Koch now used it with advantage in his studies,
and pulverized living, fresh, virulent, but perfectly dry
bacteria in an agate mortar, in order to liberate the ba¬
cillary substance from its protecting envelope of fatty
acid. In the trituration only a very small quantity of the
bacteria could be handled at a time, and Koch seemed
thoroughly aware of the risk incurred from inhalation of
the finely pulverized bacillary mass.

Having reduced the bacilli to fragments, they were
removed from the mortar in distilled water, and collected
by centrifugation, in a small glass tube, as a muddy re¬
siduum at the bottom of an opalescent, clear fluid. For
convenience he named the clear fluid TO, the sediment
TR. TO was found to contain tuberculin. In order
to separate the essential poison of the bacteria as perfectly
as possible from the irritating tuberculin, the TR or
fragments were dried perfectly, triturated once more,
re-collected in fresh distilled water and re-centrifugated.
After the second centrifugation microscopic examination
showed that the bacillary fragments had not been resolved
into a uniform mass, for when TO was subjected to stain¬
ing with carbol-fuclisin and methyl-blue it was found to
exhibit a blue reaction, while in TR a cloudy violet reac¬
tion was obtained.

The addition of 50 per cent, of glycerin had no effect
upon TO, but caused a cloudy white deposit to be thrown
down from TR. This last reaction showed that TR con¬
tained fragments of the bacilli which are insoluble in
glycerin.

Experiment showed that TR had decided immunizing
powers. Injected into tuberculous animals in too large
dose it produced a reaction, but its effects were entirely
independent of the reaction. Koch’s aim in using this
substance in therapeutics was to produce immunity in
the patient without reactions, by gradual but rapid in¬
crease of the dose. Ill so large a number of cases did

2 34 PA THO GENIC BA CTERIA .

Kocli produce immunity to tuberculosis by the adminis¬
tration of TR, that he thinks it proved beyond a doubt
that the observations are correct.

In making the TR preparation Koch advises the use
of a fresh, highly virulent culture not too old. It must
be perfectly dried in a vacuum exsiccator, and the tritu¬
ration, in order to be thorough, should not be done upon
more than ioo mg. of the bacilli at a time. A satisfac¬
tory separation of the TR from TO is said only to occur
when the perfectly clear TO takes up at least 50 per cent
of the solid substance, as otherwise the quantity of TO in
the final preparation is so great as to produce undesirable
reactions.

The fluid is best preserved by the addition of 20 per
cent, of glycerin, which does not injure and prevents
decomposition of the TR.

The finished fluid contains ro mg. of solid constituents
to the c.cm., and before administration should be diluted
with physiological salt solution (not solutions of carbolic
acid). When administering the remedy to man the in¬
jections are made with a hypodermic syringe into the
tissues of the back. The beginning dose is of a mg.,
rapidly increased to 20 mg., the injections being made
daily.

In speaking of the results of experiments upon guinea-
pigs, Koch says:

UI have, in general, got the impression in these ex¬
periments that full immunization sets in two or three
weeks after the use of large doses. A cure in tubercu¬
lous guinea-pigs, animals in which the disease runs, as
is well known, a very rapid course, may, therefore, take
place only when the treatment is introduced early — as
early as one or two weeks after the infection with tuber¬
culosis.

c 4 This rule avails also for tuberculous human beings,
whose treatment must not be begun too late. ... A
patient who has but a few months to live cannot ex¬
pect any value from the use of the remedy, and it will

TUBERCULOSIS.

235

be of little value to treat patients who suffer chiefly from
secondary infection, especially with the streptococcus,
and in whom the septic process has put the tuberculosis
entirely in the background.”

By proper administration of the TR Koch was able to
render guinea-pigs so completely immune that they were
able to withstand inoculations of virulent bacilli. The
point of inoculation presents no changes when the
remedy is administered, and the neighboring lymph-
glands are generally normal,, or when slightly swollen
contain no bacilli.

One very important objection found by Trudeau and
Baldwin against commercially prepared TR is that it is
possible for it to contain unpulverized, and hence live,
virulent tubercle bacilli. In the preparation of the rem¬
edy it will be remembered that no antiseptic or germicide
was added to the solutions, by which the effects of acci¬
dental failure to crush every bacillus could be overcome,
Koch having specially deprecated such additions as pro¬
ducing destructive changes in the TR. Until this objec¬
tion can be removed, and our confidence that our attempts
to cure patients will not cause their death be restored, it
becomes a question whether TR can find a place in
human medicine at all, or must remain an interesting
scientific laboratory demonstration.

Probably the most interesting use to which the TR-
tuberculin has thus far been put is found in the experi¬
ments of Fisch,1 who immunized a horse with it, hoping
to produce an antitoxin that might be useful in treating
tuberculosis. His experiment seems to have met with
remarkable success, for the serum thus secured, which
he calls “Antiphthisic Serum, TR,” is found to thor¬
oughly immunize guinea-pigs to tuberculosis, to cure
tuberculous guinea-pigs in the early stages of the dis¬
ease, and to neutralize the effects of tuberculin upon
tuberculous animals.

Upon human beings it is too early to make a positive

1 Jour, of the Amer. Med . Assoc., Oct. 30, 1897.

236

PA THOGENIC BA CTERIA .

report, but Fisch’s cases have shown remarkable improve¬
ment The subject is pregnant with interest and deserves
attention.

Hirshfelder 1 claims to have cured a large number of
cases of tuberculosis by the use of a preparation known
as oxytuberculin . It consists of a 4 per cent, glycerin-
bouillon culture of very virulent tubercle bacilli, which
after being sterilized for one hour, and filtered, receives
the addition of 8-10 volumes of hydrogen peroxid, and is
then sterilized for ninety-six hours in a steam apparatus.
During the sterilization the fluid is kept in a glass vessel,
plugged with cotton wool. The peroxid of hydrogen is
renewed every twelve hours.

From the fluid obtained in this way the excess of the
peroxid is removed by alkalinization. Before being em¬
ployed in human medicine the remedy is tested upon
guinea-pigs. The dose may gradually be increased to 20
c.cm. The theory of action is based upon a claimed
destruction of the toxic property of the tuberculin by the
oxidation of the peroxid of hydrogen, which leaves a
harmless but potent immunizing substance in the fluid.

Paterson 2 has suggested, for the production of immun¬
ity to tuberculosis, the use of gradually increasing doses
of the serum of a fowl immunized to avian tuberculosis
by gradually increased doses of sterilized, attenuated, and
virulent cultures of the bacillus of avian tuberculosis.
Curative results were observed in fowls thus treated, and
in mammals similarly treated, and the inference drawn
is that men treated in the same manner can be similarly
benefited. The dose recommended is 2 c.cm.

The theory depends upon the supposed identity or near
relationship of the bacilli of avian and mammalian tu¬
berculosis.

Klebs has claimed much advantage from the treatment
of tuberculosis by antiphthisin . According to the ex-

1 Deutsche med . Wochenschrift , 1S97, No. 19, and Jour, of the Amer. Med.
Assoc., 1897.

2 Amer. Medico- Surg. Bull., Jan. 25, 1898.

TUBERCULOSIS.

237

perimental studies of Trudeau and Baldwin, however,
antiphthisin is only much diluted tuberculin, and exerts
no demonstrable influence upon the tubercle bacillus in
vitro , does not cure tuberculosis in guinea-pigs, and
probably inhibits the growth of the tubercle bacillus
upon culture-media to1 which it has been added, only by
its acid reaction.

On the other hand, Ambler has used antiphthisin with
excellent results in the treatment of human tubercu¬
losis.

Numerous experimenters, prominent among whom are
Tizzoni, Cattani, Bernheim, and Paquin, have experi¬
mented with the tubercle bacillus and tuberculin, hoping
that the principles of serum-therapy might be applicable
to the disease. Nothing positive has, however, been
achieved. The first-named observers claim to have im¬
munized guinea-pigs, in whose blood an antitoxin formed;
the last-named thinks the serum of immunized horses
a specific for tuberculosis. The field of experimentation
is an inviting one, though the chronic course of the dis¬
ease lessens the certainty with which the results can be
estimated.

Babes and Proca, in an experimental research upon the
action of the antituberculous serum, claim for it a decided
specific action, and demonstrate experimentally that ani¬
mals inoculated with tubercle bacilli and injected with
the serum are protected from the spread of the disease.

Mafucci and diVestra found that by injecting guinea-
pigs with serum from sheep immunized by injections
first of dead, then of living cultures of tubercli bacilli,
although no cures were brought about, the vitality of
the animals was maintained longer. Unprotected animals
died in fifty to fifty-three days. Those injected after in¬
fection, seventy-four days, and those injected before infec¬
tion, ninety-one days.

The author1 made an elaborate study of the so-called
antituberculin , suggested by Viquerat, and widely praised
1 Jour, of the Amer. Med. Assoc., Aug. 21, 1897*

238

PATHOGENIC BACTERIA.

by Paquin. For a long period, donkeys were injected
with increasing doses of tuberculin, in order that an
antitoxin — antitnberculin — might be generated in their
blood. Experiments upon guinea-pigs showed that the
serum was powerless to immunize against the tubercle
bacillus, or to cure established tuberculosis. The serum,
however, had the power of annulling the effects of tuber¬
culin upon tuberculous animals. While a failure experi¬
mentally, certain clinicians claim that in practice it ex¬
erts a beneficial action upon patients. Indeed, presuming
that an anti tuberculin is formed, it is but natural that it
should do good in all cases in which it is probable that
the patient is poisoned by tuberculin or a similar product.

Rather nearer the desideratum are the experiments of
DeSchweinitz,1 who injected cows and horses with increas¬
ing quantities of bouillon cultures of a greatly attenuated
tubercle bacillus, and subsequently found that the serum
possessed the property of rendering guinea-pigs immune
to the virulent bacilli.

The Bacillus of Fowl-tuberculosis {Tuberculosis gal-
linarum). — The cases of tuberculosis which occasionally
occur spontaneously in chickens, parrots, ducks, and other
birds were originally attributed to the Bacillus tuberculo¬
sis hominis, but the recent works of Rivolta, Mafucci,
Cadio, Gilbert, Roget, and others have shown that, while
very similar in many respects to the Bacillus tuberculosis,
the organism found in the disease of birds has distinct
peculiarities which stamp it a different variety, but not a
separate species. Cadio, Gilbert, and Roger succeeded in
infecting fowls by feeding them upon food containing tu¬
bercle bacilli, and keeping them in cages in which dust
containing tubercle bacilli was placed. The infection
was aided by lowering the temperature with antipyrin
and lessening vitality by starvation. Morphologically,
the organisms are similar, the bacillus of fowl-tuber¬
culosis being a little longer and more slender than its
ally.

1 Centralbl. f Bakt. und Parasitenk Sept. 15, 1897, Bd. xxii., Nos. 8 and 9.

TUBERCULOSIS.

Upon culture-media a distinct rapidity of growth is
observable, and we find that, instead of growing only
where glycerin is present, the Bacillus tuberculosis galli-
narum will grow upon blood-serum, agar-agar, and bouil¬
lon as ordinarily prepared. It will not grow upon potato.
The bacillus will grow at 42-43° C. quite as well as at
370 C., while the growth of the tubercle bacillus ceases
at 42° C. Moreover, the temperature of 43° C. does not
attenuate its virulence. The thermal death-point is 70°
C. Upon culture-media it can retain its virulence for
two years.

The growth upon artificial culture-media is luxuriant,
and lacks the dry quality characteristic of ordinary
tubercle-bacillus cultures. As it becomes old a culture
of fowl-tuberculosis turns slightly yellow.

Birds are the most susceptible animals for experimental
inoculation, the embryos and young more so than the
adults ; guinea-pigs are quite immune. Artificial inocu¬
lation can only be made in the subcutaneous tissue, never
through the intestine. The chief seat of the disease is
the liver, where cellular nodes, lacking the central coag¬
ulation and the giant-cells of mammalian tuberculosis,
and enormously rich in bacilli, are found. The disease
never begins in the lungs, and the fowls which are dis¬
eased never show bacilli in the sputum or the dung.

Rabbits are easily infected, an abscess forming at the
seat of inoculation, and later nodules forming in the
lung*, so that the distribution is quite different from that
seen in birds.

The bacillus stains like the tubercle bacillus, but takes
the stain rather more easily. The resistance to acids is
about the same.

Pseudo-tuberculosis. — Eberth, Chantemesse, Charrin,
and Roger have reported certain cases of so-called pseudo¬
tuberculosis. The disease occurred spontaneously in
guinea-pigs, and was characterized by the formation of
cellular nodules in the liver and kidneys much resembling
miliary tubercles. Cultures made from them showed the

240

PATHOGENIC BACTERIA .

presence of a small motile bacillus which could easily be
stained by ordinary methods (Fig. 64). When introduced

Fig. 64. — Bacillus pseudo-tuberculosis from agar-agar; x 1000 (Itzerott and

Niemann).

subcutaneously into guinea-pigs the original disease was
produced.

Pseudo-tuberculosis seems to be an indefinite affection
of which we have very little knowledge, and which is
certainly in no way connected with or related to true
tuberculosis.

CHAPTER II.

LEPROSY.

Leprosy is a disease of great antiquity, and very early
received much attention and study. In giving the laws
to Israel, Moses included a large number of rules for its
recognition, the isolation of the sufferers, the determina¬
tion of recovery, and observances to be fulfilled before
the convalescent could once more mingle with his people.
The Bible is replete with accounts of miracles wrought
upon lepers, and during the times of biblical tradition it
must have been an exceedingly common and malignant
disease.

At the present time, although we in the Northern
United States hear very little about it, leprosy is still a
widespread disease. It exists in much the same form as
two thousand years ago in Palestine, Syria, Egypt, and
the adjacent countries. It is exceedingly common in
China, Siam, and parts of India. Cape Colony has many
cases. In Europe, Norway, Sweden, and parts of the
Mediterranean coast furnish a considerable number of
cases. Certain islands, especially the Sandwich Islands,
are regular hot-beds for its maintenance. The United
States is not exempt, the Gulf coast being chiefly af¬
fected.

At one time the view was prevalent that the disease
was spread only by contagion, at another that it was
miasmatic. At present the tendency is to view it as.
contagious to a degree rather less than tuberculosis.
Sometimes it is hereditary.

The cause of leprosy is now pretty certainly deter¬
mined to be the lepra bacillus (Fig. 65), which was dis-

16 241

242 PATHOGENIC BACTERIA .

covered by Hansen, and subsequently clearly described
by Neisser.

The bacillus is almost the same size as the tubercle
bacillus — perhaps a little shorter — but lacks the curve
which is so constant in the latter. It stains in very
much the same way as the tubercle bacillus, but permits
of a rather more rapid penetration of the stain, so that

Fig. 65. — Bacillus leprae, seen in a section through a subcutaneous node ;
x 500 (Franlcel and Pfeiffer).

the ordinary aqueous solutions of the anilin dyes color
it quite readily. It stains well by Grain’s method,
by which beautiful tissue specimens can be prepared.
The peculiar property of retaining the color in the
presence of the mineral acids which characterizes the
tubercle bacillus also characterizes the lepra bacillus,
and the methods of Ehrlich, Gabbett, and Unna can be
used for its detection.

Like that of the tubercle bacillus, its protoplasm often
presents open spaces or fractures, which have been re-

LEPROSY ;

garded by some as spores, but which are even less likely
to be spores than the similar appearances in the tubercle
bacillus.

The organism almost always occurs singly or in irreg¬
ular groups, filaments being unknown. It is not motile.

Many experimenters have endeavored to grow this ba¬
cillus upon artificially prepared substances, but in spite
of modern methods, improved apparatus, and refined
media, few claim to have met with success.

Bordoni-Uffredozzi was able to grow upon a blood-serum-
glycerin mixture a bacillus which partook of the staining
peculiarities of the lepra bacillus as it appears in the
tissues, but differed very much from it in its morphology.
After numerous generations this bacillus was induced to
grow upon ordinary culture-media. It commonly pre¬
sented a club-like form, which was regarded by Baum-
garten as an involution appearance. Frankel points out
that the bacillus of Bordoni is possessed of none of the
essential characters of the lepra bacillus except its stain¬
ing.

Czaplewski 1 offers a confirmation of the work of Bor¬
doni-Uffredozzi, together with a description of a bacillus
supposed to be the lepra bacillus, which he succeeded in
cultivating from the nasal secretions of a leper.

The bacillus was first isolated upon a culture-medium
consisting of glycerinized serum without the addition of
salt, pepton, or sugar. The mixture was placed in flat
dishes, coagulated by heat, and sterilized by the inter¬
mittent method.

The secretion, rich in lepra bacilli, was taken up with
a platinum wire and inoculated upon the culture-medium
by a series of linear strokes. The dishes (Petri dishes
were used for the experiment) were securely closed with
paraffin and stood in the incubating-oven at 370 C.

Upon the surface of the medium there grew numerous
colonies of staphylococcus aureus, the bacillus of Fried-

1 Centralbl. f. Bakt. und Parasitenk., Jan. 31, 1898, vol. xxiii., Nos. 3 and
4, P- 97-

244

PATHOGENIC BACTERIA .

lander and a number of colonies consisting of fine, slender,
often somewhat nodose bacilli about the size and form of
the lepra bacillus.

These colonies were grayish-yellow, humped in the
middle, 1-2 mm. in diameter, irregularly rounded, and
irregular at the edges. They could be inverted entire
with the platinum wire and were excavated on the under
side. The consistence was crumbly.

When a transfer was made from one of these colonies
to fresh media, in a few days the growth became apparent
and assumed a band-like form, with a plateau-like eleva¬
tion in the center.

The bacillus thus isolated grew with moderate rapidity
upon all the ordinary culture-media except potato. Upon
blood-serum the growth was more luxuriant and fluid
than upon the solid media. Upon coagulated serum the
growth was rather dry and elevated, and was frequently
so loosely attached to the surface of the medium as to
be readily lifted up by the platinum wire.

The growth was especially good upon sheep’s blood-
serum with the addition of 5 per cent, of glycerin. The
growth upon the Loffler-mixture was excellent.

Upon agar-agar the growth is not so good as upon
blood-serum ; it is more luxuriant upon glycerin agar-
agar than upon plain agar-agar; it is grayish and flatter
upon agar-agar than upon blood-serum. The growth
never extends to the water of condensation to form a
floating layer, as does that pf the tubercle bacillus.

The colonies that form upon agar-agar are much like
those described by Bordoni-Uffredozzi, and appear as iso¬
lated, grayish, rounded flakes, thicker in the center than
at the edges, and characterized by an irregular serrated
border from which a fine irregular network extends upon
the medium. These projections consist of bundles of the
bacilli.

Upon gelatin the bacillus develops well after it has
grown artificially for a number of generations. Upon
the surface of gelatin the growth is, in general, similar

LEPROSY.

245

to that upon agar-agar. In puncture-cultures most of the
growth is on the surface in the form of a whitish, or
grayish, or yellowish folded layer. In the depths of the
gelatin the development occurs as a granular rather thick
column. The medium is not liquefied.

Bouillon is not clouded; no superficial growth occurs.
The vegetation occurs only at the bottom of the tube in
the form of a powdery sediment.

Czaplewski found that the bacillus stained well with
Loffler’ s methylen-blue, and with the aqueous solutions
of the anilin dyes. It also stains by Gram’s method, and
has the same resisting power to the decolorizing action
of mineral acids and alcohol as the lepra bacillus as seen
in tissue. The young bacilli color homogeneously, but
older ones are invariably granular. They are usually
pointed at the ends when young, but may be rounded or
knobbed when older. The more rapidly the bacillus
grows, the longer and more slender it appears.

All attempts to infect the lower animals with leprosy,
either by the purulent matter or solid tissue from lepers,
or by inoculating them with the supposed specific bacilli
that have been isolated, have failed.

Ducrey seems to have cultivated the lepra bacillus in
grape-sugar, agar, and in bouillon “in vacuo .” His
results need confirmation. Very few instances are re¬
corded in which actual inoculation has produced leprosy
in either men or animals. Arning was able to secure
permission to experiment upon a condemned criminal in
the Sandwich Islands. The man was of a family entirely
free from disease. Arning introduced beneath his skin
fragments of tissue freshly excised from a lepra nodule,
and kept the man under observation. In the course of
some months typical lesions began to develop at the
points of inoculation and spread gradually, ending in
general lepra in the course of about five years.

Melcher and Artmann introduced fragments of lepra
nodules into the anterior chambers of the eyes of rabbits,
and observed the death of the animals after some months

246

PATHOGENIC BACTERIA .

with typical lepra lesions of all the viscera, especially
the cecum.

While the lepra bacillus has much in common with the
tubercle bacillus, there is not the slightest evidence of
any real identity. It has already been shown that lepra
bacilli do not grow upon artificial media, and that they
cannot be readily transmitted by inoculation. The fol¬
lowing description will show that the relation of the
bacilli to the lesions is entirely different from that of
the tubercle bacilli to the tubercles.

Like the Bacillus tuberculosis, the Bacillus leprae proba¬
bly only occurs in places frequented by persons suffering
from the disease. That individuals are infected by the
latter less readily than by the former bacilli probably
depends upon the fact that leprous infection seems to
take place most commonly by the entrance of the organ¬
isms into the individual through cracks or fissures in
the skin, while the tuberculous infection occurs through
the more accessible respiratory and digestive apparatus.
Once established in the body, the bacillus by its growth
produces chronic inflammatory nodes — the analogues of
tubercles.

The nodes of lepra consist of various kinds of cells
and of fibres. Unlike the tubercles, the lepra nodes are
vascular, and much of the embryonal tissue completes
its formative function by the production of fibres. The
bacilli are not distributed through the nodes like tubercle
bacilli, but are found in groups enclosed within the proto¬
plasm of certain large cells — the u lepra cells. ” These
cells seem to be overgrown and partly degenerated lym¬
phoid cells. Sometimes they are anuclear, sometimes
they contain several nuclei (giant-cells).

Lepra nodules do not degenerate like tubercles, and
the formation of ulcers, which constitutes a large part of
the disease, seems largely due to the action of external
agencies upon the feebly vital pathological tissue, which
is unable to recover itself when injured.

According to the recent studies of Johnston and Jamie-

LEPROSY ;

247

son,1 the bacteriological diagnosis of nodular leprosy can
be made by spreading the serum obtained by scraping a
leprous nodule upon a cover-glass, drying, fixing, and
staining with carbol-fuchsin and Gabbet’s solution as for
the tubercle bacillus. In such preparations the bacilli
are present in enormous numbers, thus forming a marked
contrast to the tubercular skin diseases, in which very few
can be found.

In that form known as anesthetic leprosy, nodules form
upon the peripheral nerves, and by connective-tissue
formation, as well as the entrance of the bacilli into the
nerve-sheaths, cause irritation, then degeneration, of the
nerves. The anesthesia which follows these peripheral
nervous lesions is one of the conditions predisposing to
the formation of ulcers, etc. by allowing injuries to occur
without detection and to progress without observation.
The ulcerations and occasional loss of phalanges that
follow these lesions occur, probably, in the same manner
as in syringomyelia.

The disease advances, having first manifested itself
upon the face, extensor surfaces, elbows, and knees, to the
lymphatics and the internal viscera. Death ultimately
occurs from exhaustion, if not from the frequent inter¬
current affections to which the conditions predispose.

1 Montreal Med. Journal , Jan., 1897.

CHAPTER III.

GLANDERS.

Geanders is an infectious mycotic disease which, very
fortunately, is almost confined to the lower animals. Only
occasionally does it secure a victim from hostlers, drovers,
soldiers, and bacteriologists, whose frequent association
with and experimentation upon animals bring them in
frequent contact with those which are diseased. Of all
the infectious diseases studied by scientists, none has
caused the havoc which glanders has wrought. Several
men of prominence have succumbed to accidental in¬
fection.

Glanders was first known to us as a disease of the horse
and ass characterized by the occurrence of discrete, clean¬
ly-cut ulcers upon the mucous membrane of the nose.
These ulcers are formed by the breaking down of nodules
which can be detected upon the diseased membranes, and
show no tendency to recover, but slowly spread and dis¬
charge a virulent pus. The edges of the ulcers are in¬
durated and elevated, the surfaces often smooth. The
disease does not progress to any great extent before the
submaxillary lymphatic glands begin to enlarge. Eater
on these glands form large lobulated masses, which may
soften, open, and become discharging ulcers. The lungs
may also become infected by inspiration of the infectious
material, and contain small foci not unlike tubercles in
appearance. The animals ultimately die of exhaustion.

In 1882, shortly after the discovery of the tubercle
bacillus, Eofiler and Schiitz discovered in the discharges
and tissues of this disease the specific micro-organism,
the glanders bacillus ( Bacillus mallei ; Fig. 66), which is
its cause.

GLANDERS.

249

The glanders bacillus is somewhat shorter and dis¬
tinctly thicker than the tubercle bacillus. It has rounded
ends, and it generally occurs singly, though upon blood-

Fig. 66. — Bacillus mallei, from a culture upon glycerin agar-agar; x 1000
(Frankel and Pfeiffer).

serum, and especially upon potato, several joined indi¬
viduals may be found. Long threads are never formed.

The bacillus is non-motile. Various observers have
•claimed the discovery of spores, but although in the
interior of the bacilli there have been observed irregular
spaces like the similar spaces in the continuity of the
tubercle bacillus not colored by the stains, they have
not yet been definitely proven to be spores. The ob¬
servation of Loffler that the bacilli can be cultivated
after being kept in a dry state for three months makes it
appear as if some permanent form (spore) occurs. No
flagella have been demonstrated upon the bacillus.

Like the tubercle bacillus, the glanders bacillus does
not seem to find conditions outside the animal body suit¬
able for its existence, and probably does not occur except
as a parasite.

The organism only grows between 250 and 420 C., and
generally grows very slowly, so that attempts at its isola-

250

PATHOGENIC BACTERIA.

tion and cultivation by the usual plate method are apt to
fail, because the numerous other organisms in the material
grow much more rapidly.

The best method of isolation seems to be the use of an
animal reagent. It has been said that glanders princi¬
pally affects horses and asses. Recent observations, how¬
ever, have shown the goat, cat, hog (slightly), field-mouse,
wood-mouse, marmot, rabbit, guinea-pig, and hedgehog
all to be susceptible animals. Cattle, house-mice, white
mice, and rats are immune.

The guinea-pig, being a highly susceptible as well
as a readily procurable animal, naturally becomes the
reagent for the detection and isolation of the bacillus.
When a subcutaneous inoculation of some glanders pus
is made, the disease can be observed in guinea-pigs
by a tumefaction in from four to five days. Somewhat
later this tumefaction changes to a caseous nodule, which
ruptures and leaves a chronic ulcer with irregular mar¬
gins. The lymph-glands speedily become involved, and
in a month to five weeks signs of general infection are
present. The lymph-glands suppurate, the testicles un¬
dergo the same process, and still later the joints exhibit
a suppurative arthritis containing the bacilli. The ani¬
mal finally dies of exhaustion. In guinea-pigs no nasal
ulcers form. In field-mice, which are even more suscepti¬
ble, the disease is much more rapid. No local lesions
are visible. In two or three days the animal seems un¬
well, the breathing is hurried, it sits still with closed
eyes, and without any other preliminaries tumbles over
on its side, dead.

Prom the tissues of the inoculated animals the pure
cultures are most easily made. Perhaps the best places
to secure the culture are from softened nodes which have
not ruptured or from the suppurating joints. Strauss
has, however, given us a method which is of great use,
because of the short time required. The material sus¬
pected to contain the glanders bacillus is injected into
the peritoneal cavity of a male guinea-pig. In three or

GLANDERS.

251

four days the disease becomes established. The testicles
enlarge a little ; the skin over them becomes red and
shining. The testicles themselves begin to suppurate,
and often discharge through the skin. The animal dies
in about two weeks. If such an animal be killed and its
testicles examined, the tunica vaginalis testis will be
found to contain pus, and sometimes to be partially ob¬
literated by inflammatory exudation. The bacilli are pres¬
ent in this pus, and can be secured from it in pure cultures.

The value of Strauss’s method has, however, been less¬
ened by the discovery by Kutcher,1 that a new bacillus,
which he has classified among the pseudo-tubercle ba¬
cilli, produces a similiar testicular swelling when injected
into the abdominal cavity.

The purulent discharges from the noses of horses
and from other lesions of large animals generally con¬
tain very few bacilli, so that their detection by the
use of the guinea-pig inoculation is made much more
simple.

The bacillus is an aerobic organism, and can be grown
in bouillon, upon agar-agar, better upon glycerin agar-
agar, very well upon blood-serum, and quite character¬
istically upon potato. It grows in gelatin, but this is
not an appropriate medium, because the bacillus develops
best at temperatures at which the gelatin is liquid.

Upon 4 per cent, glycerin agar-agar plates the colonies
appear upon the second day as pale-yellow or whitish,
shining round dots. Under the microscope they appear
as brownish-yellow, thick granular masses with sharp
borders.

The culture upon agar-agar and glycerin agar-agar
occurs as a moist, shining layer not possessed of distinct
peculiarities. Upon blood-serum the growth is rather
characteristic. The colonies along the line of inoculation
first develop as circumscribed, clear, transparent drops,
which later become confluent and form a transparent
layer unaccompanied by liquefaction.

1 Zeitschrift fur Hygiene , Bd. xxi., Heft i., Dec. 6, 1895.

252

PATHOGENIC BACTERIA .

The most characteristic growth is upon potato. It
first appears in about forty-eight hours as a transparent,
honey-like, yellowish layer, developing only at incuba¬
tion-temperature and soon becoming reddish-brown. As
this brown color of the colony develops, the potato for
a considerable distance around it becomes greenish-
brown. (See Frontispiece .) No other known organism
produces the same appearance upon potato.

In litmus milk the growth of the glanders bacillus is
associated with the production of an acid that reddens
the reagent, with the formation of a firm coagulum and
the subsequent separation from it of a clear reddish
whey.

The organism loses its virulence if cultivated for many
generations upon artificial media.

The bacillus is killed in five minutes by exposure to
55° C.

That this bacillus is the cause of glanders there is no
room to doubt. Loffler and Schiitz have succeeded by
the inoculation of horses and asses in producing the
well-known disease.

The organisms when in cultures can be stained with
the watery anilin-dye solutions, but are difficult to stain
in tissues. They do not stain by Gram’s method.

The chief difficulty in staining the bacillus in tissues
is the readiness with which it gives up the stain in the
presence of decolorizing agents. Loffler at first accom¬
plished the staining by allowing the sections to lie for
some time (five minutes) in the alkaline methylene-blue
solution, then transferring them to a solution of sulphuric
and oxalic acids —

Concentrated sulphuric acid, 2 drops ;

5 per cent, oxalic-acid solution, i drop ;

Distilled water, io c.cm.

for five seconds, then transferring to absolute alcohol,
xylol, etc. The bacilli appear dark blue upon a paler
ground. This method gives very good results, but has

GLANDERS .

253

been largely superseded by the use of Kiihne’s carbol-
methylene blue :

Methylene blue, 1. 5

Alcohol, 10.

5 per cent, aqueous phenol solution, 100.

Kiihne’s method of staining is to place the section in the
stain for about half an hour, wash in water, decolorize
carefully in hydrochloric acid (10 drops to 500 c.cm. of
water), immerse at once in a solution of lithium carbonate
(8 drops of a saturated solution of lithium carbonate in 10
c.cm. of water), place in a bath of distilled water for a few
minutes, dip into absolute alcohol colored with a little
methylene blue, dehydrate in anilin oil containing a
little methylene blue in solution, wash in pure anilin
oil, not colored, then in a light ethereal oil, clear in
xylol, and mount in balsam.

When stained in sections of tissue the bacilli are
found to occupy the interior of small inflammatory zones
not unlike tubercles in appearance. These nodules can
be seen with the naked eye scattered through the livers,
kidneys, and spleens of animals dead of experimental
glanders. The nodules consist principally of leucocytes,
but also contain numerous epithelioid cells. As is the case
with tubercles, the centres of the nodules are prone to
degenerate, soften, and also to suppurate. The retro¬
gressive processes upon exposed surfaces, where the break¬
ing down of the nodules allows their contents to escape,
are the sources of the typical ulcerations. At times the
process is progressive, and some of the lesions heal by
the formation of a stellate scar.

Baumgarten regarded the origin and course of the his¬
tological lesions of glanders to be much like those of the
tubercle. In his studies epithelioid cells first accumulated,
and were followed by leucocytes. Tedeschi was not able
to confirm the results of Baumgarten’ s work, but found the
primary change to be due to a necrosis of the affected
tissue followed by an invasion of leucocytes. The recent

254

PATHOGENIC BACTERIA.

researches of J. H. Wright1 are in accord with those of
Tedeschi rather than with those of Baumgarten, for
Wright observed first a marked degenerative effect upon
the tissue, and then an inflammatory exudation amount¬
ing in some cases to actual suppuration.

As has been mentioned, cultures of the bacillus lose
their virulence more or less after four or five generations
in artificial media. While this is true, attempts to atten¬
uate fresh cultures by heat, etc. have so far failed.

Leo has pointed out that white rats, which are immune
to the disease, may be made susceptible by feeding with
phloridzin and causing a glycosuria.

Kalning, Preusse, Pearson, and others have pre¬
pared a substance, “mallein,” from cultures of the
bacillus, and suggested its employment for diagnostic
purposes. It seems to be quite useful in veterinary
medicine, the reaction occasioned by its injection being
similar to that caused by the injection of tuberculin in
tuberculous patients. The manufacture of mallein is
not attended with great difficulty. The bacilli are grown
in glycerin bouillon for several weeks, killed by heat, the
culture filtered through porcelain and evaporated to one-
tenth of its volume. It has also been prepared from
potato cultures, which are said to produce a stronger
toxin. A febrile reaction of more than 1.50 C. following
the injection is said to be specific of the disease. Babes
has asserted that the injection of this toxic product into
susceptible animals will protect them from the disease.

Various experiments have been made with curative
objects in view. Certain observers claim to have seen
good results follow the injection of mallein in repeated
small doses. Others, as Chenot and Picq, find the blood-
serum from immune animals like the ox to be curative
when injected into infected guinea-pigs.

1 Jour, of Exp. Med., vol. i., No. 4, p. 577*

CHAPTER IV.

SYPHILIS.

Although syphilis is almost as well known as it is
widespread, we have not yet discovered for it a definite
specific cause. Whether it is due to a protozoan par¬
asite, or whether it is due to a bacterium, the future
must decide. Numerous claims have been made by those
whose studies have revealed organisms of one kind or
another in syphilitic tissues, but no one has yet suc¬
ceeded either in isolating, cultivating, or successfully in¬
oculating them.

In 1884 and 1885, Lustgarten published a method for
the staining of bacilli which he had found in syphilitic
tissues and assumed to be the cause of the disease. The
staining, which is very complicated, requires that the
sections of tissue be stained in Ehrlich’s anilin-water
gentian-violet solution for twelve to twenty-four hours at
the temperature of the room, or for two hours at 40° C. ;
washed for a few minutes in absolute alcohol ; then im¬
mersed for about ten seconds in a 1 ]/2 per cent, perman-
ganate-of-potassium solution, after which they are placed
in an aqueous solution of sulphurous acid for one to two
seconds, thoroughly washed in water, run through alco¬
hol and oil of cloves, and finally mounted in Canada
balsam dissolved in xylol.

If the bacilli are supposed to be present in pus or dis¬
charges from syphilitic lesions, the cover-glasses spread
with the material are stained in the same manner, except
that for the first washing distilled water instead of abso¬
lute alcohol is used.

This method undergoes a modification in the hands of
De Giacomi, who prefers to stain the cover-glasses in hot

255

256

PATHOGENIC BACTERIA .

anilin-water-fuchsin solution for a few moments, sections
in the same solution cold for twenty-four hours ; then
immerse them first in a weak, then in a strong, solution
of chlorid of iron. The cover-glasses are washed in
water, sections in alcohol, and subsequently passed
through the usual reagents for dehydration and clearing.

Fig. 67. — Bacillus of syphilis (Lustgarten), from a condyloma; x 1000 (Itzerott

and Niemann).

In some syphilitic tissues these methods suffice to de¬
fine distinct bacilli with a remarkable similarity to the
tubercle bacillus. The organism is about the same size
as the tubercle bacillus, and even more frequently curved,
but often presents a club-like enlargement of one
end (involution-form ?). The bacilli very frequently
occur singly, though more often in groups, and never lie
free, but are always enclosed in cells. These bacilli are
not always found in syphilitic lesions, nor is their dem¬
onstration easy under the most favorable circumstances.
Lustgarten emphasizes particularly that they are only
demonstrable after the most painstaking technical pro¬
cedures.

The probability of the specificity of this organism was
considerably lessened by the observation by Matterstock,
Travel, and Alvarez that in preputial smegma, and also*

SYPHILIS .

257

in vulvar smegma from healthy individuals, a similar
organism, identical both in morphology and staining
peculiarities, could be demonstrated. Of course the oc¬
currence of Iyustgarten’s bacillus in the internal organs
could not but argue against the probability of its identity
with the smegma bacillus ; but Uustgarten himself pointed
out that the bacilli of both tuberculosis and leprosy stain
by his method, and thus gave Baumgarten the right to
suggest that the few cases well adapted for the demon¬
stration of the Lustgarten bacilli might be cases of mixed
infection of tuberculosis and syphilis.

The most recent research upon the bacteriology of
syphilis is that of van Niessen,1 who claims to have cul¬
tivated a syphilis bacillus from the blood of a few cases.
Blood secured from a deep puncture at the end of a
thoroughly disinfected finger is caught in a sterile glass,
diluted with an equal quantity of distilled water and
kept for from ten to fourteen days at a temperature of
io°— 20° R. (i3°-i5° C.). Very often the blood of syphi¬
litics is found subject to accidental contamination by
various well-known bacteria. When this is not the case,
however, the serum remains almost perfectly clear and
contains a large number of bacilli — syphilis bacilli. The
bacillus can be transplanted to bouillon, in which it grows
with the production of grayish- white shreds and floating
flocculi, some of which are suspended in the liquid, while
others form a membrane upon the surface.

When transplanted to obliquely solidified gelatin and
kept at room temperature, in the course of forty-eight,
hours a very fine, grayish-white, thready mass like
cloudy streaks, and having a peculiar reflecting surface,,
can be seen. Under a lens this is seen to consist of lines,
of threads which sometimes seem to penetrate into the
depths of the gelatin. After a time a layer is formed
upon the surface of the medium. Some liquefaction of the
medium occurs and causes the growth to slide down upon.

1 Centralbl. f Bakt. und Parasitenk ., Bd. xxiii., No. 2, Jan. 19, 1898, p. 49;,
No. 344, Jan. 31, 1898, p. 97; and No. 546, Feb. 11, 1898, p. 177.

258

PATHOGENIC BACTERIA.

itself so as to assume the form of a fragment of a tape¬
worm. Upon agar-agar after the lapse of two days the
growth consists of a central pellicle along the line of in¬
oculation, with little sprouts projecting in all directions
from the edges. The growth is grayish, with an occa¬
sional yellowish tinge.

Punctures in agar-agar were unsuccessful, but in gela¬
tin the appearance of the growth is similar to that of
the cholera spirillum.

The bacillus also grows upon potato in the form of an
elevated layer of exactly the same color as the potato.
In the course of time the entire potato becomes colored
a dark gray. It also grows in milk, urine, serum, and
water.

The colonies of this bacillus are quite characteristic,
but so varied in appearance as to make one suspect that
the plate upon which which they grow is contaminated
with various other species of bacteria. In general, the
colonies may be said to appear slowly as transparent
•whitish drops, which become grayish and later yellow¬
ish, and finally brownish in color. The gelatin about
them presents concentric, wave-like rings, depending
upon the liquefaction of the medicine.

When the growth is more rapid and occurs at higher
temperatures bundles of threads, somewhat resembling
the early stages of a mould, are observed. Examining
microscopically, one finds in the slowly growing colonies
a surrounding zone of small centrifugally arranged fine
threads or hairs extending in all directions, with one or
two exceptionally long bundles extending beyond the
others and beyond the limits of the colony. The long
threads are never found to divide. Many of the colonies
are highly suggestive of those of anthrax.

The bacillus is motile in very slight degree. It forms
spores. It is, in general, about the size of the tubercle
bacillus.

The vegetation of the organism is said to be peculiar
in that the bacillary stage is of short duration and soon

SYPHILIS.

259

gives place to the formation of septate, V-shaped, and
branched forms. It seems to be normally a strepto-ba-
cillus in its early stages, but eventually becomes very
pleomorphous, varying in appearance from a chain of
oval cocci to the hypha of the moulds. There seems to
be nothing peculiar about the staining-capacity of the
bacillus. It stains with the ordinary solutions of the
aniliu dyes, retains the stain of Gram’s method, and is
decolorized by mineral acids.

Dohle 1 succeeded in staining certain protoplasmic
bodies in the tissues in syphilis, which resembled the
actively motile protoplasmic bodies which he had pre¬
viously encountered in the discharges. They were for
the most part round or oval, sometimes with irregular
outlines, and were provided with flagella. The staining
took place in a mixture of hematoxylon and carbol-fuch-
sin, subsequently treated with iodin or chromatin, and
washed in alcohol.

Convinced that these bodies were the cause of syphilis,
he excised small fragments from gummata and other
syphilitic tissues, and placed them beneath the skin of
guinea-pigs, which subsequently fell ill with a chronic
marasums which ultimately caused death.

In the inoculation experiments of van Niessen there
were observed as evidences of the specificity of the
organism discovered by him: (1) abortion in pregnant
female rabbits; (2) extra-genital primary lesions on the
ears of inoculated rabbits in the form of nodes; (3) sec¬
ondary ulcer and tumor formations, and irregular lesions,
such as occasional thrombosis and pneumonia.

1 Munch, med. Wochenschrift , 1897, No. 43.

CHAPTER V.

ACTINOMYCOSIS.

In 1845, Eangenbeck discovered that the specific dis¬
ease of cattle known as actinomycosis could be com¬
municated to man. His observations, however, were not
given to the world until 1878, one year after Bollinger
had discovered the cause of the disease in animals.

Fig. 68. — Actinomyces bovis, from the tongue of a calf; x 500 (Frankel and

Pfeiffer).

Actinomycosis is a disease almost peculiar to the bovine
animals, though sometimes occurring in hogs, horses,
men, and other animals.

The first manifestations of the disease are usually found
either about the jaw or in the tongue, in either of which

A C TIN OM Y CO SIS.

261

localities there are produced considerable enlargements
which are sometimes dense and fibrous (wooden tongue)
and sometimes suppurative. In sections of these nodular
formations small yellowish granules surrounded by some
pus can be found. These granules when viewed beneath
the microscope exhibit a peculiar rosette-like body — the
ray-fungus or actinomyces.

The fungus is of sufficient size to be detected by the
naked eye. It can be colored, in sections of tissue, by
the use of Gram’s method, or better by Weigert’s fibrin
stain. Tissues pre-stained with carmin, then stained by
Weigert’s method, give beautiful pictures.

The entire fungus-mass consists of several distinct
zones embracing entirely different elements. At the
centre of the mass there is found a granular substance
containing numerous bodies resembling micrococci. Ex¬
tending from this centre into the neighboring tissue is a
radiating, apparently branched, thickly-tangled mass of
mycelial threads. These threads seem to terminate in
a zone of conspicuous club-shaped radiating forms which
give the colonies the rosette-like appearance. The cells
of the tissues affected and a larger or smaller collection
of leucocytes form the surrounding resisting tissue-zone.

The degree of chemotactic influence exerted by the
organism seems to depend partly upon the tissue affected
and partly upon the individuality of the animal. When
the animal is but slightly susceptible, and when the
tongue is the part affected, the disease is characterized
by the production of enlargement due to the formation
of cicatricial tissue. If, on the other hand, the animal
is highly susceptible or the jaw is affected, the chief
symptom is suppuration, with the formation of cavities
communicating by sinuses.

Before the nature of the affection was understood it
was confounded with various diseases of the bones, prin¬
cipally with osteosarcoma.

From the tissues primarily affected the disease spreads
to the lymphatic glands, and not infrequently to the

262

PATHOGENIC BACTERIA.

lungs. Israel has pointed out certain cases of human
actinomycosis beginning in the peribronchial tissues,
probably from inhalation of the fungi.

The occurrence of three distinct elements as compo¬
nents of the rays served to class this organism among
the pleomorplious bacteria in the genus Cladothrix,
where it has remained undisturbed for at least a decade.
Recent researches have, however, changed the view held
by some bacteriologists in regard to the actinomyces, and
caused them to regard the organism as a bacillus. If it
be a bacillus, the central zone of granular cocci-like
elements is to be regarded as consisting of individuals
in process of rapid division and spore(?)-formation, the
mycelial zone as consisting of perfect individuals, and
the peripheral zone, with the rosette-like, club-shaped
elements, as consisting of individuals partly degener¬
ated through the activity of the cells and tissue-juices
(involution-forms).

Jones is of the opinion that the disease, if not inden-
tical with, is closely allied to, tuberculosis, and that the
occasional branched forms of tubercle bacilli prove the
tendency of the individual bacillus to form a reticulum.

When the mycelial threads are carefully examined, the
branchings, which appear distinct upon hasty inspection,
are found to be more the effect of a peculiar relation
which the threads bear to one another than actual bifur¬
cations, so that it must be regarded as very questionable
whether these threads ever so divide.

The organism may be grown upon artificial culture-
media, as has been proven by Israel and Wolff.

Upon agar-agar or glycerin agar-agar it forms trans¬
lucent colonies, about the size of a pin’s head, of firm,
almost cartilaginous, consistence. These colonies consist
of bacillary individuals, sometimes seemingly branched.
In bouillon similar dense globular organisms can be
grown. The blood-serum colonies, which grow simi¬
larly to the agar-agar colonies, are rather more luxuri¬
ant, and slowly liquefy the medium.

ACTINOM YCOSIS.

263

When the actinomyces are grown upon artificial media
their virulence is retained for a considerable length of
time. If introduced into the abdominal cavities of rab¬
bits, there are produced in the peritoneum, mesentery,
and omentum typical nodules containing* the actinomyces
rays.

The organism can also be grown in raw eggs, into
which it is carefully introduced through a small opening
made under aseptic precautions. In the egg the organism
forms peculiar long mycelial threads cpiite unlike the short
forms developing upon agar-agar.

The characteristic rosettes which are constantly found
in the tissues are never seen in artificial cultures.

The exact manner by which the organism enters the
body is unknown. In some cases it may be by direct
inoculation with pus, but there is reason to believe that
the organism occurs in nature as a saprophyte, or as
an epiphyte upon the hulls of certain grains, especially
barley.- Woodhead records a case where a primary me¬
diastinal actinomycosis in the human subject was sup¬
posed to be traced to perforation of the posterior pharyn¬
geal wall by a barley spikelet swallowed by the patient.

Cases of actinomycosis are fortunately of rare occur¬
rence in human medicine, and do not always occur in
those brought in contact with the lower animals. The
fungi may enter the organism through the mouth and
pharynx, through the respiratory tract, through the di¬
gestive tract, or through wounds.

The invasion has been known to take place at the roots
of carious teeth, and is more liable to occur in the lower
than in the upper jaw. Israel reported a case in which
the primary lesion seemed to occur external to the bone
of the lower jaw, as a tumor about the size of a cherry,
with an external opening. In two cases of the disease
observed by Murphy of Chicago both began with tooth¬
ache and swelling of the jaw.

When inhaled, the organisms gain entrance to the
deeper portions of the lung, and bring about a suppiira-

264

PA THOGENIC BA CTERIA .

tive bronchopneumonia with adhesive inflammation of
the contiguous pleura. After the formation of the pleu¬
ritic adhesions the disease may penetrate the newly
formed tissue, extend to the chest-wall, and form external

Fig. 69. — Section of liver from a case of actinomycosis in man (Crookshanlc).

sinuses. Or it may penetrate the diaphragm and invade
the abdominal organs, causing an interesting and charac¬
teristic lesion in the liver and other large viscera (see
Fig. 69).

Microscopically the lesion consists chiefly of a round-

A C TINOM Y CO SIS.

265

cell infiltration with circumscribing granulation-tissue
leading to the formation of cicatricial bands. In the
form known as “ wooden tongue” the disease runs an
essentially chronic course, with the production of consid¬
erable amounts of connective tissue.

But few cases recover, the disease terminating by death
from exhaustion or from complicating pneumonia or
other organic lesions.

CHAPTER VI.

MYCETOMA, OR MADURA-FOOT.

A curious disease of not infrequent occurrence in the
Indian province of Scinde is one known as mycetoma,
Madura-foot, or pied de Madura . It almost invariably
affects natives of the agriculturist class, and in most
cases begins in or is referred by the patient to the prick
of a thorn. It generally affects the foot, more rarely
the hand, and in one instance was seen by Boyce in the
shoulder and hip. It is more common in men than in
women, individuals between twenty and forty years of
age suffering most frequently, but persons of any age or
sex may suffer from the disease. It is insidious in its
onset, as has been said, generally following a slight
injury, such as the prick of a thorn. No symptoms are
observed in what might be called an incubation stage of
a couple of weeks’ duration, but after this time elapses a
nodular growth gradually forms, attaining in the course
of time the size of a marble. Its deep attachments are
indistinct and diffuse. The skin becomes purplish,
thickened, indurated, and adherent. The points most
frequently invaded at the onset are the ball of the great
toe and the pads under the bases of the fingers and toes.

In the course of months, although progressing slowly,,
the lesions attain very perceptible size, distinct tumors
being present. Later, sometimes not until after a year
or two, the nodes begin to soften, break down, discharge
their purulent contents, and originate ulcers and com¬
municating sinuses. The discharge at this stage is a
thin sero-pus, and is always mixed with a number of
fine round black or pink bodies, described, when black,
as resembling gunpowder ; when pink, as resembling
266

MYCETOMA , OR MADURA-FOOT 267

fish-roe. It is the detection of these particles upon
which the diagnosis rests, and upon which the divis¬
ion of the disease into the melanoid and pale varieties
depends.

The progress of the disease causes an enormous size
and a peculiar deformity of the affected foot or hand.
The malady is generally painless.

The micro-organismal nature of the disease was early
suspected. In spite of the confusion caused by some
who confounded the disease with and described it as

Guinea-worm, ” Carter held that it was due to some
indigenous fungus as early as 1874. Boyce and Surveyor
believe that the black particles of the melanoid variety
represent a curious metamorphosis of a large branching
septate fungus, and that the white particles of the other
variety are the remains of a lowly-organized fungus and
of caseous particles.

Kanthack tried to prove the identity of the fungus
with the well-known actinomyces, but there seems to
be considerable doubt about the correctness of his view.

Vincent succeeded in isolating the micro-organism
by puncturing one of the nodes with a sterile pipette,
and has cultivated it upon artificial media. Acid vege¬
table infusions seem suitable to its growth. It develops
scantily in bouillon at the room-temperature, better at
370 C. — in from four to five days. In twenty to thirty
days the colony attains the size of a little pea.

In the liquid media the colonies which cling to the
glass, and thus remain near the surface of the medium,
develop a rose- or bright-red color.

Cultures in gelatin are not very abundant, are colorless,
and are unaccompanied by liquefaction.

Upon the surface of agar-agar strikingly beautiful
rounded, glazed colonies are formed. They are at first
colorless, but later become rose-colored or bright red. The
majority of the clusters remain isolated, some of them
attaining the size of a small pea. They are generally
umbilicated like a variola pustule, and present a curious

268

PATHOGENIC BACTERIA .

appearance when the central part is pale and the periphery
red. As the colony ages the red color is lost and it be¬
comes dull white. The colonies are very adherent to the
surface of the medium , and are said to be of cartilaginous
consistence. The organism also grows in milk without
coagulation.

Upon potato the development is meagre, slow, and
with very little tendency to chromogenesis. The color-
production is more marked if the potato be acid in reac-

Fig. 70. — Streptothrix Madurae in a section of diseased tissue (Vincent).

tion. Some of the colonies upon agar-agar and potato
have a powdery surface, no doubt from the occurrence of
spores. It is, of course, an aerobic organism.

Under the microscope the organism is found by Vin¬
cent to be a streptothrix — a true branched fungus con¬
sisting of long bacillary branching threads in a tangled
mass. In many of the threads spores could be made out.

MYCETOMA , OR MADURA-FOOT. 269

Vincent was unable to communicate the disease to animals
by inoculation.

Microscopic study of the diseased tissues in cases of
mycetoma is not without interest. The healthy tissue
is said to be sharply separated from the diseased masses.
The latter appear as large degenerated tubercles, except
that they are extremely vascular. The mycelial or
filamentous fungous mass occupies the centre of the
degeneration, where its long filaments can be beautifully
demonstrated by the use of appropriate stains, Gram’s
method being excellent for the purpose. The tissue sur¬
rounding the disease-nodes is infiltrated with small round
cells. The youngest nodules are seen to consist of granu¬
lation-tissue, which in its organization is checked by the
coagulation-necrosis which is sure to overtake it Giant-
cells are few.

Not infrequently small hemorrhages occur from the
ulcers and sinuses of the diseased tissues ; the hemor¬
rhages can be explained from the abundance of small
blood-vessels in the diseased tissue.

Although the disease has been described as occurring
in Scinde, it is not limited to that province, having been
met with in Madura, Hissar, Bicanir, Dehli, Bombay,
Baratpur, Morocco, Algeria, one case by Bastini and
Campana in Italy, and one by Kempner in America.

CHAPTER VII.

FARCIN DU BCEUF.

The peculiar disease which sometimes affects numbers
of cattle in Guadeloupe, and which was described by
the older writers as farcin du bceuf has been carefully
studied by Nocard. It is a disease of cattle character¬
ized by a superficial lymphangitis and lymphadenitis,
affecting the tracheal, axillary, prescapular, and other
glands. The affected glands enlarge, suppurate, and
discharge a creamy, sometimes a grumous, pus. The
internal organs are often affected with a pseudo-tubercu¬
losis whose central areas undergo a purulent or caseous
degeneration.

In the researches of Nocard it was discovered, by
staining by Gram’s and by Kiihne’s methods, that in
the centres of the tubercles micro-organisms could be
defined. They resembled long delicate filaments rather
intricately woven, characterized by distinct ramifications
which made clear the proper classification of the organ¬
ism as a streptothrix. The organism was successfully
cultivated by Nocard upon various culture-media at the
temperature of the body. It is aerobic.

In bouillon the organism develops in the form of color¬
less masses irregular in size and shape, some of which
float upon the surface, others of which sink to the bottom
of the liquid. Sometimes the surface is covered by an
irregular fenestrated pellicle of a gray color.

Upon agar-agar the growth develops in small, rather
discrete, irregularly rounded, opaque masses of a yellow¬
ish-white color. The surfaces of the colonies are tuber-
culated, and an appearance somewhat like a lichen is
observed (see Fig. 71).

270

FARCIN DU BCEUF. 2 71

Upon potato very dry scales of a pale-yellow color
rapidly develop.

The growth upon blood-serum is less luxuriant, but
similar to that upon agar-agar.

In milk the organism produces no coagulation by its
growth, and does not alter the reaction.

Microscopic study always reveals the organism as the
same tangled mass of filaments seen in the tissues. The
old cultures are rich in spores, which are very small and

FlG. 71. — Streptotlirix of farcin du boeuf growing on glycerin agar-agar.

develop upon the most superficial portions of the growth.
These spores resist the penetration of stains to a rather
unusual extent.

Cultures retain their virulence for a long time : Nocard
found one virulent after it had been kept for four months
in an incubating oven at 40° C.

The streptothrix of farcin du bcritf is pathogenic for
guinea-pigs, cattle, and sheep ; dogs, rabbits, horses, and
asses are immune.

When the culture or some pus containing the micro-

272

PATHOGENIC BACTERIA.

organism is injected subcutaneously into a guinea-pig, a
voluminous abscess results. Not long afterward the lym¬
phatic vessels and glands of the region are the seat of swell¬
ing and induration, and extensive phlegmons form, which
rupture externally and discharge considerable pus. The
animal, of course, becomes extremely ill and seems about
to die ; instead, it slowly recovers its normal condition.

In other animals, as the cow and the sheep, the subcu¬
taneous inoculation results in an abscess relatively less
extensive. This ulcerates, then indurates, and seems to
disappear, but after the lapse of several weeks or months
opens again in the form of a new abscess.

In animals which are immune or nearly immune, like
the horse, the ass, the dog, and the rabbit, the subcuta¬
neous inoculation is followed by the formation of a small
abscess which speedily cicatrizes.

Intraperitoneal inoculation in the guinea-pig gives rise
to an appearance resembling tuberculosis. The omentum
may be extensively involved and full of softened nodes.
The liver, spleen, and kidneys appear full of tubercles,
but careful examination will satisfy the observer that
the tubercles are only upon the peritoneal surfaces, not
in the organs.

Intravenous introduction of the cultures produces a
condition much resembling general miliary tuberculosis.
All the organs contain the pseudo-tubercles in consider¬
able numbers.

CHAPTER VIII.

RHINOSCLEROMA.

In Austria, Hungary, Italy, and some parts of Ger¬
many there sometimes occurs a peculiar disease of the
anterior nares, characterized by the occurrence of circum¬
scribed tumors, known as rhinoscleroma. The tumor-
masses are somewhat flattened, isolated or coalescent,
grow with great slowness, and recur if excised. The dis¬
ease commences in the mucous membrane and the adjoin¬
ing skin, and spreads to the skin in the neighborhood by
a slow invasion, involving the upper lip, jaw, hard palate,
and sometimes the pharynx. The growths are without
evidences of inflammation, do not ulcerate, and consist
microscopically of infiltration of the papilla and corium
of the skin, with round cells which change in part to
fibrillar tissue. The tumors possess a well-developed
lymph-vascular system. Sometimes the cells undergo
hyaline degeneration.

In these little tumors the researches of Von Frisch dis¬
covered little bacilli much resembling both in morphol¬
ogy and vegetation the pneumo-bacilli of Friedlander,
and, like them, surrounded by capsules. The only
marked difference between the so-called bacillus of rhi¬
noscleroma and the Bacillus pneumoniae of Friedlander
is that the former stains well by Gram’s method, while
the latter does not, and that the former is rather more
distinctly rod-shaped than the latter, and more often
shows its capsule in culture-media.

The bacillus can easily be cultivated, and in all media
resembles the bacillus of Friedlander too closely to be
distinguished from it. Even when inoculated into animals
the bacillus behaves much like Friedlander’ s bacillus.

Inoculation has, so far, failed to produce the disease
either in men or in the lower animals.

273

B. THE TOXIC DISEASES.

CHAPTER I.

TETANUS.

One of the most exquisitely toxic bacteria of which
we have any knowledge is the bacillus discovered in
1884 by Nicolaier, obtained in pure culture by Kitasato
in 1889, and now universally recognized as the cause of
tetanus. It is a peculiar organism, whose striking feature
is a considerable enlargement of one end, in which a
bright round spore is seen (Fig. 72). The bacilli which

Fig. 72. — Bacillus tetani; x 1000 (Frankel and Pfeiffer).

are not sporiferous, are long, rather slender, have rounded
ends, seldom unite in chains or pairs, are motile, and
have no flagella. The bacilli stain readily with ordi¬
nary aqueous solution of the anilin dyes, and also very
readily by Gram’s method.

The tetanus bacillus is a common saprophytic organ¬
ism which can be found in most garden-earth, in dust,
274

TETANUS.

275

in manure, and sometimes in the intestinal discharges
of animals. It is extremely difficult to isolate and culti¬
vate, because it will not grow where the smallest amount
of oxygen is present.

The method now generally employed for the isolation
of this bacillus is that originated by Kitasato, and based
upon his observation that its spores can resist high temper-

Fig. 73. — Bacillus tetani : six-days- Fig. 74- — Bacillus tetani : culture
old puncture-culture in glucose-gelatin four days old in glucose-gelatin (Fran-
(Frankel and Pfeiffer). kel and Pfeiffer).

atures. After finding that the typical bacilli are present
in earth or pus, or whatever the material to be investi¬
gated was, Kitasato exposed a portion of it for an hour
to a temperature of 8o° C. By . this heating all the fully-
developed bacteria, tetanus as well as the others, and the

27 6

PATHOGENIC BACTERIA .

great majority of the spores except those of tetanus, were
destroyed, and, as little other than tetanus spores re¬
mained, their cultivation was made comparatively easy.

The resistance which the tetanus bacilli manifest toward
heat is only part of a great general resisting power of
which they are possessed. It is said that they can retain
their vitality in the dried condition for months. Stern¬
berg says they can resist 5 per cent, carbolic solutions
for ten hours, but will not grow after fifteen hours’ im¬
mersion. 5 per cent, carbolic acid, to which 0.5 per cent.

Fig. 75. — Bacillus tetani : five-days-old colony upon gelatin containing glucose ;
x 1000 (Frankel and Pfeiffer).

of hydrochloric acid has been added, destroys them in
two hours. They are also destroyed in three hours by
1 : 1000 bichlorid-of-mercury solution ; but when to such
a solution 0.5 per cent, of hydrochloric acid is added, its
activity is so increased that the spores are destroyed in
thirty minutes. The resistance to heat is only within
certain limits, for exposure to passing steam for from
five to eight minutes is certain to kill the spores.

The colonies of the tetanus bacillus, when grown in

TETANUS .

277

an atmosphere of hydrogen upon gelatin plates, somewhat
resemble those of the well-known hay bacillus. There
is a dense rather opaque central mass from which a more
transparent zone is readily separable. The margins of
this outer zone are made up of a radiating fringe of pro¬
jecting bacilli (Fig. 75). The liquefaction that occurs is
much slower than that caused by bacillus subtilis.

When grown in gelatin puncture-cultures the develop¬
ment occurs deep in the puncture, and consists of mul¬
titudes of short-pointed processes radiating from the
puncture, somewhat resembling a fir tree (Fig. 73).
Liquefaction begins in the second week and causes the
disappearance of the radiating processes. The liquefac¬
tion spreads slowly, but may involve the entire mass of
gelatin and resolve it into a grayish-white syrupy liquid,
at the bottom of which the bacilli accumulate. The
growth in gelatin containing glucose is much more rapid ;
that in agar-agar punctures is much slower, but similar
to the gelatin cultures except for the absence of liquefac¬
tion. The organism can also be grown in bouillon, and
attains its maximum development at a temperature of
370 C. Much gas is given off from the cultures.

Cultures of the tetanus bacillus in all media give off
a peculiar characteristic odor — a burnt-onion smell, with
a suggestion of putrefaction about it.

The methods for excluding the oxygen from the cul¬
tures and replacing it by hydrogen, as well as other
methods suggested for the cultivation of the strictly
anaerobic organisms, are given under the appropriate
heading (Anaerobic Cultures), and need not be repeated
here.

A very simple method of cultivating the bacillus in
bouillon for the purpose of securing a large amount of
toxin has been suggested by the author.1 An ordinary
bottle is filled with bouillon to the mouth, and closed
by a perforated rubber stopper containing a glass tube

1 Centralbl. f. Bakt. u. Parcisitenk xix.. Nos. 14 and 15, April 25, 1896, p.

55°-

278

PATHOGENIC BACTERIA .

a couple of inches long. Connected with this glass
tube, by means of a short piece of rubber tubing, is

Fig. 76. — Tetanus bottle.

the bulb of a broken pipette,
the other end of which is
plugged with cotton (Fig. 76).
When the steam sterilization
takes place the expanding fluid
ascends to the reservoir repre¬
sented by the pipette bulb, de¬
scending again as the fluid cools.
When the sterilization is com¬
pleted the reservoir is detached,
the inoculation made by passing
a very fine pipette into the bottle,
the projecting glass tube drawn
out to a fine tube, and the bottle
stood in hot water until the ex¬
panding fluid ascends to the apex
of the pointed glass tube. The

tube is now sealed in a flame and the bottle and its con¬

tents allowed to cool. In cooling the retracting fluid
leaves a vacuum which at once draws up any minute
bubbles of air remaining, and allows the tetanus bacillus
to grow in a condition of very fair anaerobiosis.

Tetanus bacilli exist in nature as widely distributed
saprophytes. They are quite common in the soil, and
the fact that they are most plentiful in manured ground
has suggested that they originate in the intestines of
horses and reach the earth from their excrement. Le

Dentu has, however, shown that the tetanus bacillus is
a common organism in New Hebrides, where there are no
horses. In these islands the natives poison their arrows
by dipping them into a clay rich in tetanus bacteria.

The work of Kitasato has given us a very exact
knowledge of the tetanus bacillus and completely estab¬
lishes its specific nature.

The organisms generally enter the animal body through
a wound caused by some implement which has been in

TETANUS.

279

contact with the soil, or enter abrasions from the soil
directly. Doubtless many of the wounds are so small
that their existence is overlooked, and this, together
with the fact that the period of incubation of the dis¬
ease, especially in man, is of considerable duration, and
at times permits the wound to heal before any symptoms
of intoxication occur, serves to explain to us at least some
of the reported cases in which no wound is said to have
existed.

It would seem that in some rare cases tetanus can occur
without the previous existence of a wound. Such a case
has been reported by Karaen, who found that the intes¬
tine of a person dead of the disease was rich in the
Bacillus tetani. Kamen is of the opinion that the
bacilli can grow in the intestine and be absorbed, espe¬
cially where there are imperfections in the mucosa. It
is not impossible, though he does not think it probable,
that the bacteria growing in the intestine could elaborate
enough toxin to produce the disease by absorption.

All animals are not alike susceptible to the disease.
Men, horses, mice, rabbits, and guinea-pigs are all sus¬
ceptible ; dogs are much less so. Most birds are scarcely
at all susceptible either to the bacilli or to the poison.
Amphibians are immune, though it is said that frogs
can be made susceptible by elevation of their body-
temperature.

When a white mouse is inoculated with an almost
infinitesimal amount of bouillon or solid culture, or is
inoculated with garden-earth containing the tetanus
bacillus, the disease is almost certain to follow, the
first symptoms coming on in from one to two days.
The mouse develops typical tetanic convulsions, which
begin first in the neighborhood of the inoculation, but
soon become general. Death follows sometimes in a
very few hours. In rabbits the period of incubation is
nearly two weeks, and in man may be three weeks.

The conditions in the animal body are not favorable
for the development of the bacilli, because of the free

28o

PATHOGEN/C BACTERIA .

supply of oxygen contained in the blood, and we find
that they grow with great slowness, remain localized at
the seat of inoculation, and never enter the blood- or
lymph-circulation. Doubtless most cases of tetanus are
cases of mixed infection in which the bacillus enters with
bacteria, which greatly aid its growth by using up the
oxygen in their neighborhood. The amount of poison
produced must be exceedingly small and its power tre¬
mendous, else so few bacilli growing under adverse con¬
ditions could not produce fatal toxemia. The poison is
produced rapidly, for Kitasato found that if mice were
inoculated at the root of the tail, and afterward the skin
and the subcutaneous tissues around the inoculation were
either excised or burned out, this treatment would not
save the animal unless the operation were performed
within an hour after the inoculation .

Some incline to the view that the toxin is a ferment,
and the experiments of Nocard, quoted before the Acad-
•emie de Medecine, October 22, 1895, might be adduced
in support of the theory. He says: “ Take three sheep
with normal tails, and insert under the skin at the end
of each tail a splinter of wood covered with the dried
spores of the tetanus bacillus; watch these animals care¬
fully for the first symptoms of tetanus, then amputate the
tails of two of them 20 cm. above the point of inocula¬
tion, . . . the three animals succumb to the disease with¬
out showing any sensible difference.’7

The circulating blood of diseased animals is fatal to
susceptible animals because of the toxin which it con¬
tains; and the fact that the urine is also toxic to mice
proves that the toxin is excreted by the kidneys.

From pure cultures of tetanus bacilli grown in various
media, and from the blood and tissues of animals affected
with the disease, Brieger succeeded in separating two
alkaloidal substances — ( ‘ tetanin ’ ’ and ( ‘ tetano-toxin, ’ ’
both very poisonous and productive of tonic convulsions;
and Brieger and Frankel later isolated an extremely poi¬
sonous toxalbumin.

TETANUS.

281

The pathology of the disease is of much interest be¬
cause of its purely toxic nature. There is generally a
small wound with a slight amount of suppuration. At
the autopsy the organs of the body are normal in appear¬
ance, except the nervous system, which bears the great¬
est insult. It, however, shows little else than congestion
either macroscopicallv or microscopically.

A11 interesting fact contributed to our knowledge of
the disease has beep presented by Vaillard and Rouget,
who found that if the tetanus bacilli were introduced
into the body freed from their poison, they were unable
to produce any signs of disease because of the prompt¬
ness with which the phagocytes took them up. If, how¬
ever, their poison was not removed, or if the body-cells
were injured by the simultaneous introduction of lactic
acid or other chemical agents, the bacilli would imme¬
diately begin to manufacture the toxin and produce
symptoms of the disease.

The toxin is easily prepared, being readily soluble in
water. The most ready method of preparation is to
grow the bacilli in bouillon, keeping the culture-medium
at a temperature of 37 0 C., and allowing it to remain un¬
disturbed for from two to four weeks, by which time it
will have attained a toxicity so great that 0.000005 c.cm.
will cause the death of a mouse. The toxin is very rapidly
destroyed by heat, and cannot bear any temperature above
6o°-65° C. It is also decomposed by light. The best
method of keeping it is to add 0.5 per cent, of phenol,
and then store it in a cool, dark place. It will not
keep its strength very long under the best conditions.

The purified toxin of Brieger and Cohn was surely
fatal to mice in doses of 0.00000005 gram. Lambert,1 in
his comprehensive review of the use of tetanus antitoxin,
points out that this is the most poisonous substance that
has ever been discovered.

By the gradual introduction of such a toxin into ani¬
mals Behring and Kitasato have been able to produce in

1 New York Med. Jour., June 5, 1897.

282

PATHOGENIC BACTERIA .

their blood a distinctly potent and valuable antitoxic
substance.

The method for the production of this tetanus anti¬
toxic serum is very much like that for the diphtheria
antitoxic serum (< q . v.), except that a much longer time
is required for its production, that the doses of toxin are
of necessity smaller because its toxicity is greater, -and
that triclilorid of iodin or Gram’s solution will probably
need to be added to the toxin to prevent too powerful a
local reaction. Horses, dogs, and goats may be used.

As tetanus cases are not very common, and the anti¬
toxic serum when produced is not very stable in its prop¬
erties, Tizzoni and Cattani have successfully prepared it
in a solid form, in which, it is claimed, it can be kept
indefinitely, shipped any distance, and used after simple
solution in water. Their method is to precipitate the
antitoxin from the blood of immunized dogs with alcohol.
Numerous cases of the beneficial action of this antitoxin
are on record.

The strength of the serum is generally expressed
i : 1,000,000, i : 10,000,000, etc., which indicates that 1
c.cm. of the serum is capable of protecting 1,000,000 or
10,000,000 grams of guinea-pig from infection.

The experiments of Alexander Lambert show that a
protective power of 1 : 800,000,000 can be attained.

As Welch has pointed out, the antitoxin of tetanus has
proved to be rather a disappointment in human medicine,
and also for the treatment of large animals, such as the
horse. The results following its injection, in combination
with the sterile toxin, into mice, guinea-pigs, and rabbits
are highly satisfactory, but the amount needed, in pro¬
portion to the body-weight, to save the animal from the
toxin being manufactured in its body by bacilli increases
so enormously with the day or hour of the disease as to
make the dosage, which increases millions of times where
that of diphtheria antitoxin increases but tenfold, a matter
of difficulty and uncertainty. Nocard also calls atten¬
tion to the fact that the existence of tetanus is unknown

TETANUS.

283

until there is sufficient toxemia to produce spasms, and
that therefore it is impossible to attack the disease in its
inception ; we are obliged to meet it upon the same
grounds as diphtheria in the later days of the disease —
a time when it is well known that the chances of im¬
provement are greatly lessened.

Of course, as there is no other remedy that combats
the disease at all, the antitoxin is one which, when ob¬
tainable, should always be employed.

An interesting observation has been recently made by
Wasserman,1 who, assuming that the destruction of
nerve-cells in the cerebrum and cord during tetanus tox¬
emia might have something to do with immunity, be¬
lieved it possible to obtain from these cells an immuniz¬
ing substance. Investigating the subject, he found that
when fresh brain or spinal cord was rubbed up in a mor¬
tar with physiological salt solution, and injected into ani¬
mals, the mixture had the power not only to confer upon
them an immunity lasting for twenty-four hours, but also
was potent enough to neutralize the effects of an injec¬
tion of tetanus toxin ten times as large as that necessary
to kill the animal in doses of 1 c.cm.

These observations may offer a possible solution of the
difficult problem laid before us by Montesano and Mon-
tesson,2 who unexpectedly found the tetanus bacillus in
pure culture in the cerebro-spinal fluid of a case of para¬
lytic dementia that died without a tetanic symptom.

1 Berlin, klin. Wochensckrift , 1898, No. 1.

2 Cenlralbl. f Bakt.^u. Parasitenk Bd. xxii., Nos. 22, 23, p. 663. Dec., 1897.

CHAPTER II.

DIPHTHERIA.

In 1883, Klebs pointed out the existence of a bacillus
in the pseudo-membranes upon the fauces of patients
suffering from diphtheria, but it was not until 1884 that
LofHer succeeded in isolating and cultivating the organ¬
ism, which is now known by both their names — the
Klebs-Loffler bacillus.

The bacillus as described by Loffler is about the length
of the tubercle bacillus, about twice its diameter, has a

Fig. 77. — Bacillus diphtheria, from a culture upon blood-serum; x 1000
(Frankel and Pfeiffer).

curve similar to that which characterizes the tubercle
bacillus, and has rounded ends (Fig. 77)* It does not
form chains, though two, three, and rarely four individ¬
uals may be found joined ; generally the individuals are
all separate from one another. The morphology of the
bacillus is peculiar in its considerable irregularity, for

284

DIPHTHERIA .

285

among the well-formed individuals which abound in
fresh cultures a large number of peculiar organisms are
to be found, some much larger than normal, some with
one end enlarged to a club-shape, some greatly elongated,
with both ends expanded into club-shaped enlargements.
These bizarre forms seem to represent an involution-form
of the organism, for, while present in perfectly fresh cul¬
tures, they are so abundant in old cultures that scarcely
a single well-formed bacillus can be found. It not infre¬
quently happens that ill unstained bacilli distinct gran¬
ules can be defined at the ends — polar granules — thus
giving the organism somewhat the appearance of a
diplococcus.

The bacillus can be readily stained by aqueous solu¬
tions of the anilin colors, but more beautifully and
characteristically with Loffler’s alkaline methylene blue:

Saturated alcoholic solution of methylene blue, 30 ;

1 : 10,000 aqueous solution of caustic potash, 100 ;

and an aqueous solution of dahlia, as recommended by
Roux.

When cover-glass preparations are stained with these
solutions, the bizarre forms already mentioned are much
more obvious than in the unstained individuals, and
the contrast between the polar granules, which color in¬
tensely, and the remainder of the bacillus, which tinges
slightly, is marked. Through good lenses the organisms
are always distinct bacilli, notwithstanding the fact that
the ends stain more deeply than the centres, and it is
only through poor lenses that the organisms can be mis¬
taken for diplococci. The bacilli stain well by Gram’s
method, this being a good method to employ for their
definition in sections of tissue, though Welch and Abbott
assert that Weigert’s fibrin method and picro-carmin give
the most beautiful results.

The diphtheria bacillus does not form spores, and is
delicate in its thermal range. Loffier found that it could
not endure a temperature of 6o° C., and Abbott has shown

*286

PATHOGENIC BACTERIA.

that a temperature of 58° C. for ten minutes is fatal to it.
Notwithstanding this susceptibility, the organism can
be kept alive for several weeks after being dried upon
shreds of silk or when surrounded by dried diphtheria
membrane.

No flagella have been demonstrated upon the bacillus.
It is non-motile.

Fernbach has shown that when the organisms are
grown in a medium exposed to a passing current of air,
the luxuriance of their development is increased, though
their life-cycle is shorter. The growth can also take
place when the air is excluded, so that the bacillus must
be classed among the optional anaerobic organisms.

The diphtheria bacillus grows readily upon all the
ordinary media, and is a very easy organism to obtain
in pure culture. Loffier has shown that the addition
of a small amount of glucose to the culture-medium
increases the rapidity of the growth, and suggests a
special medium which bears his name — Loffier’ s blood-
serum mixture:

Blood-serum, 3 ;

Ordinary bouillon + 1 per cent, of glucose, 1.

This mixture is filled into tubes, coagulated, and steril¬
ized like blood-serum, and is one of the best-known media
in connection with the study of diphtheria.

The studies of Michel 1 have shown that the develop¬
ment of the culture is much more luxuriant and rapid
when horse serum instead of beef or calves’ blood is used.
Horse’s blood can easily be secured by the introduction of
a trocar into the jugular vein ; 5 liters of it can be with¬
drawn without causing the animal any inconvenience or
producing symptoms.

The impossibility of clinically making an accurate di¬
agnosis of diphtheria without a bacteriologic examination
has caused many private physicians and many medical
societies and boards of health to equip laboratories where

1 Centralbl. f Bakt. u. Parasitenk Sept. 24, 1897, Bd. xxii., Nos. 10 and 11.

DIPHTHERIA .

287

accurate examinations can be made. The method re¬
quires some apparatus, though a competent bacteriologist
can often make shift with a bake-oven, a wash-boiler,
and other household furniture instead of the regular
sterilizers and incubators, which are expensive.

When it is desired to make a bacteriologic diagnosis
of a suspected case of diphtheria or to secure the bacillus
in pure culture, a sterile platinum wire having a small
loop at the end, or a swab made by wrapping a little
cotton around the end of a piece of wire and carefully
sterilizing in a test-tube, is introduced into the throat
and touched to the false membrane, after which it is
smeared carefully over the surface of at least three of
the blood-serum-mixture tubes, without either again
touching the throat or being sterilized. The tubes thus
inoculated are stood away in an incubating oven at the
temperature of 37 0 C. for twelve hours, then examined.
If the diphtheria bacillus is present upon the first and
second tubes, there will be a smeary yellowish-white layer,
with outlying colonies on the second tube, while the third
tube will show rather large isolated whitish or slightly
yellowish colonies, smooth in appearance, but rather ir¬
regular in outline. Very often the colonies are china-
white in appearance. These colonies, if found by micro¬
scopic examination to be made up of diphtheria bacilli ,
will confirm the diagnosis of diphtheria, and will at the
same time give pure cultures when transplanted. There
are very few other bacilli which grow so rapidly upon
LofRer’s mixture, and scarcely one other which is found
in the throat.

Ohlmacher recommends the microscopic examination
of the still invisible growth in five hours. A platinum
loop is rubbed over the inoculated surface ; the material
secured is then mixed with distilled water, dried on a
cover-glass, stained with methylene blue, and examined.
This method, if reliable, will be very valuable in making
an early diagnosis preparatory to the use of the antitoxin.

The presence of diphtheria bacilli in material taken

a88

PATHOGEJV/C BACTERIA.

from the throat does not necessarily prove the patient to
be diseased. Virulent bacilli can often be discovered in
the throats of healthy persons who have knowingly or
unknowingly come in contact with the disease. The
bacteriologic examination is only an adjunct to the
clinical diagnosis, and must never be taken as positive
in itself.

The bacillus grows similarly upon blood-serum and
Loffler’s mixture. Upon glycerin agar-agar and agar-agar
the colonies are much larger, more translucent, always

ale
Fig. 78. — Diphtheria bacilli (from photographs taken by Prof. E. K. Dun¬
ham, Carnegie Laboratory, New York): a , pseudo-bacillus; b , true bacillus;
c} pseudo-bacillus.

DIPHTHERIA.

289

appearance. It must be remarked that when sudden
transplantations are made from blood-serum to agar-
agar the growth resulting is meagre, but the oftener
this growth is transplanted to fresh agar-agar the more
luxuriant it becomes.

The growth in gelatin puncture-cultures is character¬
ized by small spherical colonies which develop along the
entire length of the needle-track. The gelatin is not
liquefied.

Upon the surface of gelatin plates the colonies that
develop do not attain anything like the size of the colo¬
nies upon Loffler’s mixture. They appear to the naked

Fig. 79. — Bacillus diphtherise, colony twenty-four hours old upon agar-agar;
x 100 (Frankel and Pfeiffer).

eye as whitish points with smooth contents and regular
though sometimes indented borders. Under the micro¬
scope they appear as granular, yellowish-brown colonies
with irregular borders (Fig. 79).

When planted in bouillon the organism causes a diffuse
cloudiness at first, but, not being motile, soon settles to
the bottom in the form of a rather flocculent precipitate:
which has a tendency to cling to the sides of the glass.
Sometimes a delicate irregular mycoderma forms upon

290 PA THOGENIC BA CTERIA.

the surface, especially when the cultivation is made by
the method of Fernbach with a passing current of air.
This mycodertna, which may appear quite regular when
the flask is undisturbed, is so brittle that it at once falls
to pieces if the flask be moved.

Spronck has recently determined that the characteris¬
tics of the growth of the diphtheria bacillus in bouillon,
as well as the amount of toxin-production, vary accord¬
ing to the amount of glucose in the bouillon. He divides
the cultures into three types :

Type A. The reaction of the bouillon becomes acid
and remains acid, the acidity increasing. The bacilli
accumulate at the bottom of the clear liquid. The
toxin-production is meagre.

Type B. There is no change from alkalinity to acidity,
but the original alkalinity of the bouillon steadily in¬
creases. The culture is very rich, the bottom of the
flask shows a considerable sediment, the liquid is cloudy,
and a delicate growth occupies the surface. The toxicity
.is very great.

Type C. In a few days the reaction of the culture
"becomes acid, and then later on changes to alkaline.
During the acid period the liquid is clear, with a white
surface-growth. When the alkalinity returns the bouillon
clouds and the surface-growth increases in thickness.
Sediment accumulates at the bottom of the flask. The
toxicity of the culture is much less than in Type B.

Spronck regards the varying reaction as due to the
fermentation of the glucose, and asserts that the most
luxuriant and toxic cultures are those in which no
glucose is present. To exclude as much of the undesir¬
able sugar as possible, he makes the bouillon from the
stalest meat obtainable, preferring it when just about to
putrefy. Of the meats experimented with, beef was
found to be the best.

In large cities meat is ordinarily kept sufficiently long
before being offered for sale to meet Spronck’ s require¬
ment.

DIPHTHERIA .

291

Upon potato the bacillus develops only when the reac¬
tion is alkaline. The potato-growth is not characteristic.
Welch and Abbott always secured a growth of the organ¬
ism when planted upon potato, but do not mention the
reaction of the medium they employed.

Milk is an excellent medium for the cultivation of the
Bacillus diplitheriae, and is possibly at times a means of
infection. Litmus milk is an excellent medium for ob¬
serving the changes of reaction brought about by the
growth of the bacillus. At first the alkalinity, which
is always favorable to the development of the bacillus,
is destroyed by the production of an acid. When the
culture is old the acid is replaced by a strong alkaline
reaction.

Palmirski and Orlowski 1 assert that the bacillus pro¬
duces indol, but only after the third week. Smith, how¬
ever, came to a contrary result, and found that when
diphtheria bacillus grew in the dextrose-free bouillon
that he recommends no indol was produced.2

Diphtheria as it occurs in man is generally a disease
characterized by the formation of a pseudomembrane
upon the fauces. It is a local infection, due to the
presence and development of the bacilli in the pseudo¬
membrane, but is accompanied by a general toxemia
resulting from the absorption of a violently poisonous
substance produced by the bacilli. The bacilli are found
only in the membranous exudation, and most plentifully
in its older portions. As a rule, they do not distribute
themselves through the circulation of the animal, though
at times they may be found in the heart’s blood.

The most malignant cases of the disease are thought
to be due to pure infection by the diphtheria bacillus,
though such cases are more rare than those in which the
Streptococcus pyogenes and Staphylococcus aureus and
albus are found in association with it.

In a series of 234 cases carefully and statistically studied

1 Centralbl. f Bakt. u. Parasitenk Mar., 1S95.

2 Jour, of Exper. Med.,v ol. ii., No. 5, Sept., 1897, p. 546.

292 PATHOGENIC BACTERIA .

by Blasi and Russo-Travali it was found that in 26 cases
of pseudomembranous angina due to streptococci staphy¬
lococci, colon bacilli, and pneumococci, 2 patients died,
the mortality being 3.84 per cent. I11 102 cases of pure
diphtheria 28 died, a mortality of 27.45 Per cent* Seventy-
six cases showed diphtheria bacilli and staphylococci; of
these, 25, or 32.89 per cent., died. Twenty cases showed
the diphtheria bacilli and Streptococcus pyogenes, wTith 6
deaths — 30 per cent. In 7 cases, of which 3, or 43 per
cent., were fatal, the diphtheria bacillus was in com¬
bination with streptococci and pneumococci. The most
dangerous forms met were 3 cases, all fatal, in which the
diphtheria bacillus was found in combination with the
Bacillus coli.

It may be well to remark that all pseudomembranous
diseases of the throat are not diphtheria, but that some
of them, exactly similar in clinical picture, result from
the activity of the pyogenic organisms alone, and are
neither diphtheria nor contagious.

Diphtheritic inflammations of the throat are not always
accompanied by the formation of the usual pseudomem¬
brane, it rarely but occasionally happening that in the
larynx a rapid inflammatory edema without a fibrinous
surface-coating causes a fatal suffocation. Only a bac¬
teriological examination will reveal the nature of the
disease in such cases.

Herman Biggs,1 in an interesting discussion of the
occurrence of the diphtheria bacillus and its relation to
diphtheria, comes to the following conclusions:

1. “ When the diphtheria bacillus is found in healthy
throats investigation almost always shows that the indi¬
viduals have been in contact with cases of diphtheria.
The presence of the bacillus in the throat, without any
lesion, does not, of course, indicate the existence of the
disease.

2. uThe simple anginas in which virulent diphtheria
bacilli are found are to be regarded from a sanitary stand-

1 Amer. Joicr. of the Med . Sciences, Oct., 1896, vol. xxii., No. 4, p. 41 1.

DIPHTHERIA. • 293

point in exactly the same way as the cases of true diph¬
theria.

3. u Cases of diphtheria present the ordinary clinical feat¬
ures of diphtheria, and show the Klebs-Loffler bacilli.

4. “ Cases of angina associated with the production
of membrane in which no diphtheria bacilli are found
might be regarded from a clinical standpoint as diph¬
theria, but bacteriological examination shows that some
other organism than the Klebs-Loffier bacillus is the
cause of the process. ”

No more convincing proof of the existence of a power¬
ful poison in diphtheria could be desired than the evi¬
dences of general toxemia resulting from the absorption
of material from a comparatively small number of bacilli
situated upon a little patch of mucous membrane.

In animals artificially inoculated the lesions produced
are not identical with those seen in the human subject,
yet they have the same general features of local infection
with general toxemia.

Guinea-pigs, kittens, and young pups are susceptible
animals. When half a cubic centimeter of a twenty-four-
hour-old bouillon culture is injected beneath the skin of
such an animal, the bacilli multiply at the point of in¬
oculation, with the production of a patch of inflamma¬
tion associated with a distinct fibrinous exudation and
the presence of an extensive edema. The animal dies in
twenty-four to thirty-six hours. The liver is enlarged,
and sometimes shows minute whitish points, which in
microscopic sections prove to be necrotic areas in which
the cells are completely degenerated and the chromatin of
their nuclei is scattered about in granular form. Similar
necrotic foci, to which attention was first called by Oertel,
are present in nearly all the organs in cases of death from
the toxin. The bacilli are constantly absent from these
lesions. Welch and Flexner1 have shown these foci to
be common to numerous irritant poisonings, and not
peculiar to diphtheria.

1 Bull, of the Johns Hopkins Hospital , Aug., 1891.

294 PATHOGENIC BACTERIA .

The lymphatic glands are usually enlarged ; the adrenals
are also enlarged, and, in cases into which the live bacilli
have been injected, are hemorrhagic.

Sometimes the bacilli themselves are present in the
internal organs, and even in the blood, but generally this
is not the case.

It might be argued, from the different clinical pictures
presented by the disease as it occurs in man and in
animals, that they were not expressions of the same
thing. A careful study, however, together with the evi¬
dences adduced by Roux and Yersin, who found that
when the bacilli were introduced into the trachea of
animals opened by operation a typical false membrane
was produced, and that diphtheritic palsy often followed,
and of hundreds of investigators, who find the bacilli
constantly present in the disease as it occurs in man,
must satisfy us that the doubt of the etiological role of
the bacillus rests on a very slight foundation.

All possible skepticism of the specificity of the bacillus
on my part was dispelled by an accidental infection that
kept me housed for three weeks during the busiest season
of the year. Without having been exposed to any known
diphtheria contagion, while experimenting in the labora¬
tory, a living virulent culture of the diphtheria bacillus
was drawn into a pipette and accidentally entered my
mouth. Through carelessness no precautions were taken
to prevent serious consequences, and as a result of the
accident, two days later, my throat was full of typical
pseudomembrane which private and Health Board bac¬
teriological examinations showed contained pure cultures
of the Klebs-Loffler bacilli.

One reason for skepticism in this particular is the
supposed existence of a pseudodiphtheria bacillus , which
has so many points in common with the real diphtheria
bacillus that it is difficult to distinguish between them.
We have, however, come to regard this pseudobacillus as
an attenuated form of the real bacillus. The chief points
of difference between the bacilli are that the pseudo-

DIPHTHERIA.

295

bacillus seems to be shorter than the diphtheria bacillus
when grown upon blood-serum; that the cultures in
bouillon seem to progress much more rapidly at a tem¬
perature of from 2o°— 22° C than those of the true bacillus;
and that the pseudobacillus is not pathogenic for ani¬
mals. These slight distinctions are all exactly what
should be expected of an organism whose virulence had
been lost, and whose vegetative powers had been altered,
by persistent manipulation or by unfavorable surround¬
ings.

Park 1 carefully studied this subject, and found that
all bacilli with the exact morphology of the diphtheria
bacillus, found in the human throat, are virulent Klebs-
Lbffier bacilli, while forms found in the throat closely
resembling them, but more uniform in size and shape,
shorter in length, and of more equal staining properties
with Lofflerbs alkaline methylene blue solution, can be,
with reasonable safety, regarded as pseudodiphtheria
bacilli, especially if it be found that they produce an
alkaline rather than an acid reaction by their growth in
bouillon. The pseudodiphtheria bacilli were found in
about 1 per cent, of throats examined in New York; they
seem to have no relationship to diphtheria. They are
never virulent.

A difference of possibly much greater importance is that
observed by Martini,2 that the diphtheria bacillus will not
grow in fluid antitoxic serum in which the pseudodiph¬
theria bacillus thrives. Both the real and the pseudoba-
cilli flourish upon coagulated antitoxic scrum. If this dif¬
ference in the behavior of the bacilli bears any relation
to the so-called u specific immunity-reaction” of cholera
and typhoid fever, it may be of great future importance.

The diphtheria bacilli are always present in the throats
of patients suffering from diphtheria, and constitute the
element of contagion by being accidentally discharged
by the nose or mouth by coughing, sneezing, vomiting,

1 Scientific Bulletin No. /, Health Department, City of New York, 1895.

2 Ccntnilbl. f Balt. u. Pamsitenk ., Ikl. xxi., No. 3, Jan. 30, 1897.

PATHOGENIC BACTERIA .

etc. Whoever comes in contact with such materials is
in danger of infection.

It is of great interest to notice the remarkable results
obtained by Biggs, Park, and Beebe in New York, where
the bacteriological examinations conducted in connection
with diphtheria show that the virulent bacilli may be
found in the throats of convalescents as long as five weeks
after the discharge of the membrane and the commence¬
ment of recovery, and that they exist not only in the
throats of the patients themselves, but also in the throats
of their care-takers, who, while not themselves infected,
may be the means of conveying the disease from the
sick-room to the outer world. Even more extraordinary
are the observations of Hewlett and Nolen,1 who found
the bacilli in the throats of patients seven, nine, and in
one case twenty -three weeks after convalescence. The
importance of this observation must be apparent to all
readers, and serves as further evidence why most thor¬
ough isolation should be practised in connection with
this dreadful disease.

Park 2 found virulent diphtheria bacilli in about i per
cent, of the healthy throats examined in New York City.
Diphtheria was, however, prevalent in the city at the
time. Most of the persons in whose throats they existed
had been in direct contact with cases of diphtheria. Very
many of those whose throats contained the virulent bacilli
did not develop diphtheria. He concludes that the mem¬
bers of a household in which a case of diphtheria exists
should be regarded as sources of danger, unless cultures
from their throats show the absence of diphtheria bacilli.

In connection with the contagiousness of diphtheria
the recent experiments of Reyes are interesting. He
has demonstrated that in absolutely dried air distributed
diphtheria bacilli die in a few hours. Under ordinary

1 Brit. Med. Jour ., Feb. i, 1896.

2 Report on Bacteriological Investigations and Diagnosis of Diphtheria,
from May 4, 1893, t0 May 4, 1894, Scientific Bulletin No. 1 , Health Depart¬
ment, City of New York.

DIPHTHERIA .

297

•conditions their vitality, when dried on paper, silk, etc.,
continues for a few days. In air that is moist the dura¬
tion of vitality is prolonged to about a week. In sand
exposed to a dry atmosphere they die in five days in the
light; in sixteen to eighteen days in the dark. When
the sand is exposed to a moist atmosphere the duration
of vitality is doubled. I11 fine earth they remained alive
seventy-five to one hundred and five days in dry air, and
•one hundred and twenty days in moist air.

From time to time reference has been made to the
toxin elaborated by the diphtheria bacillus. Roux and
Yersin first demonstrated the existence of this substance
in cultures passed through a Pasteur porcelain filter.
The toxin is intensely poisonous; it is not an albumin¬
ous substance, and can be elaborated by the bacilli
when grown in non-albuminous urine, or, as suggested
by Uschinsky, in non-albuminous solutions whose prin¬
cipal ingredient is asparagin. The toxic value of the
cultures is greatest in the second or third week.

In addition to the toxin, a toxalbumin has been isolated
by Brieger and Frankel.

Behring discovered that the blood of animals rendered
immune to diphtheria by inoculation, first with attenu¬
ated and then with virulent organisms, contained a neu¬
tralizing substance which was capable of annulling the
effects of the bacilli or the toxin when simultaneously or
subsequently inoculated into non-protected animals. This
substance, in solution in the blood-serum of the immu¬
nized animals, is the diphtheria antitoxin .

The preparation of the antitoxin for therapeutic pur¬
poses received a further elaboration in the hands of Roux.
The subject is one of great interest, but must be consid¬
ered briefly in a work of this kind.

The antitoxin is manufactured commercially at present,
the method being the immunization of large animals to
great quantities of the toxin, and the withdrawal of their
antitoxic blood when the proper degree of immunity has
been attained. The details are as follows :

298

PATHOGENIC BACTERIA.

The Preparation of the Toxin. — The method employed
by Roux and others at the present time was first sug¬
gested by Fernbach, and consists in growing the most
virulent bacilli obtainable in alkaline bouillon exposed
in a thin layer to the passage of a current of air.

The cultures are allowed to grow for three or four
weeks at a temperature of 370 C., with a stream of
moist air constantly passing over them. After the given
time has passed, it will be found that the acidity prima¬
rily produced by the bacillus gives place to a much more
intense alkalinity than originally existed. The acme of
the toxin-production seems to keep pace with this alka¬
line production. When tc ripe,” 0.4 per cent, of trikresol
is added to the cultures, which are then filtered through
porcelain. If the toxin must be kept before using, it is
best to preserve it unfiltered, as it deteriorates more rap¬
idly after filtration. Unfiltered toxin causes too much
local irritation. If the bacillus employed was virulent
and the conditions of culture were favorable, the filtered
culture should be so toxic that o. 1 c.cm. would be fatal to
a 500-gram guinea-pig in twenty-four hours (Roux). Even
under the most favorable circumstances it is difficult to
obtain a toxin which will kill in less than thirty hours.

The experience of the author with Fernbach’ s appara¬
tus has not been satisfactory. The passing current of air
is a frequent source of contamination to the culture, and
requires great care. In the end it is questionable whether
the toxin thus produced is better than that obtained from
an ordinary flask exposing a large surface to the air.

Park and Williams did an elaborate work upon the
production of diphtheria toxin.1 They found that
“toxin of sufficient strength to kill a 400-gram guinea-
pig in three days and a half in a dose of 0.025 c.cm.,
developed in suitable bouillon, contained in ordinary
Erlenmeyer flasks, within a period of twenty-four hours.
In such bouillon the toxin reached its greatest strength
in four to seven days (0.005 c*cm- killing a 500-gram

1 Jour, of Exper. Med., vol. i., No. I, Jan., 1896, p. 164.

DIPHTHERIA .

299

guinea-pig in three days). This period of time covered
that of the greatest growth of the bacilli, as shown both
by the appearance of the culture and by the number of
colonies developing on agar plates.”

“The bodies of the diphtheria bacilli did not at any
time contain toxin in considerable amounts.” “The
type of growth of the bacilli and the rapidity and extent
of the production of toxin depended more on the reaction
of the bouillon than upon any other single factor.”
“ The best results were obtained in bouillon which, after
being neutralized to litmus, had about 7 c.cm. of normal
soda solution added to each liter. An excessive amount
of either acid or alkali prevented the development of
toxin.” “Strong toxin was produced in bouillon con¬
taining peptone ranging from 1 to 10 per cent.” “The
strength of toxin averaged greater in the 2 and 4 per
cent, peptone solution than in the 1 per cent.”

“When the stage of acid reaction was brief and the
degree of acidity probably slight, strong toxin developed
while the culture bouillon was still acid; but when the
stage of acid reaction was prolonged little if any toxin
was produced until just before the fluid became alka¬
line.”

“Glucose is deleterious to the growth of the diphtheria
bacillus and to the production of toxin when it is present
in sufficient amounts to cause by its disintegration too
great a degree of acidity in the culture-fluid. When the
acid resulting from the decomposition of glucose is neu¬
tralized by the addition of an alkali the diphtheria
bacillus again grows abundantly.”

The Immunization of the Animal. — The animals chosen
to furnish the antitoxic serum should be animals which
present a distinct natural immunity to ordinary doses of
the toxin, and should be sufficiently large to furnish large
quantities of the finished serum. Behring originally
employed dogs and sheep ; Aronson at first preferred the
goat ; but Roux introduced the horse, which is more easi¬
ly immunized than the other animals mentioned, and,

300

PATHOGENIC BACTERIA .

being large enough to furnish a considerable quantity
of serum, recommends itself strongly for the purpose.

The animal chosen should be free from tuberculosis
and glanders, as tested by tuberculin and mallein, but
need not be expensive. A horse with a disabled foot
will answer well. Rheumatic horses should be rejected.
In the beginning a small dose of the toxin — about i
c.cm. — should be given hypodermically to detect indi¬
vidual susceptibility. Horses vary much in this particu¬
lar, as Roux has pointed out. The author found light-
colored horses to be distinctly more susceptible than
dark-colored ones, a fact which has some substantiation
in the clinical observation that blonde children suffer
more severely from diphtheria than dark-complexioned
ones.

If well borne, the preliminary injection is followed in
about six days by a larger dose, in six days more by
a still larger one, and the increase is continued every six
days or so, according to the condition of the animal,
until enormous quantities — 500-1,000 c.cm. — are intro¬
duced at a time.

As the expression of quantity alone is very misleading,
and to know exactly what strength the horse is receiving,
the author has devised a special nomenclature by which
to express it. Instead of stating that the animal received
10, 50, or 100 c.cm. of toxin, we now say it receives 10,
50, or 100 factors , the term factor being used to express
100 times the least certainly fatal dose of toxin per 100
grams of guinea-pig. The number of factors in a given
quantity of antitoxin naturally varies with its strength,
and it will at once be seen that it is advantageous to ex¬
press strength regardless of quantity.

The toxin causes some local reaction — at first a dis¬
tinct inflammation, later a painful edema and a febrile
reaction. The amount of local irritation is much less
marked when the injections are made slowly; and a
gravity apparatus, which is filled with the amount of
serum to be injected, suspended from the ceiling of the

DIPHTHERIA .

301

stable so that the toxin is allowed to take its own time
to enter the tissues, can be recommended. Sometimes
it takes an hour to inject 500 c.cm. in this manner.

The amount of local reaction, edema, etc., the appetite
and general condition, the temperature-curve, and the
stability of the body-weight, must all be taken into con¬
sideration, so that the administration shall not be too
rapid and the animal be thrown into a condition of
cachexia with toxic instead of antitoxic blood.

One of the principal things to be avoided is haste.
Too frequent or too large dosage is almost certain to kill
the animal.

Behring found that mixing the toxin with triclilorid
of iodin lessened the irritant effect upon susceptible ani¬
mals. I prefer not to use susceptible horses.

The suggestion of Prof. Pearson, that the large doses
of toxin might with readiness be introduced into the
trachea when the absorption is good, has been success¬
fully accomplished by the author. The absorption seems
to take place without any change in the toxin, and to be
as rapid as from the subcutaneous tissue.

As the antitoxin protects the horse perfectly against
the toxin, a preliminary dose will enable one to omit all
the small preliminary doses of toxin, and render the
horse immune at once. Thus, I have frequently adminis¬
tered r 00 c.cm. of antitoxin of about 100 units strength
to a horse one day and 500 c.cm. of strong toxin (500
factors) the next. This is just 500 times as much toxin
as has twice killed a horse in the laboratory. After the
lapse of a few days the same quantity can be administered
again, and in a week a third time. In this rapid way
antitoxin can often be secured at short notice. It is yet
a question, however, whether this method, modified from
Pawlowski, is as good and certain as the slow way sug¬
gested by Behring.

The possibility of producing serum rapidly may depend
upon the method, but the production of strong serums de¬
pends chiefly upon the horse and not upon its treatment.

302

PATHOGENIC BACTERIA.

The Preparation of the Serum for Therapeutic Pur¬
poses. — When, because of the tolerance to large quanti¬
ties of toxin, the horse seems to possess antitoxic blood,
a c ‘ twitch ’ ’ is applied to the upper lip, the eyes are
blindfolded, a small incision is made through the skin,
a trocar thrust into the jugular vein, and the blood al¬
lowed to flow through a cannulated tube into sterile
bottles. It is allowed to coagulate, and kept upon ice
for two days or so, that the clear serum may be pi¬
petted off. This serum is the antitoxic seriLm. It does
not always materialize according to the ^desires of the
experimenter, sometimes proving surprisingly strong in
a short time, sometimes very weak after months of
patient preparation.

The serums are preserved by Roux with camphor, by
Behring with carbolic acid (0.5 per cent.), and by Aron¬
son with trikresol (0.4 per cent.). I prefer to use tri-
kresol, as it is not poisonous, is a reliable antiseptic, and
has a very pronounced local anesthetic action. Formalin
has been tried, but it gelatinizes the serum and causes
much local pain when injected beneath the skin.

Dried antitoxic serum has also been placed upon the
market under the impression that it will keep longer and
bear shipment better than any other. This is not, how¬
ever, shown to be the case, and as the dried serum dis¬
solves with difficulty it is much less convenient than the
usual preparations. It is also less likely to be sterile
than the liquid forms.

The strength of the serum is expressed in what are
known as immunizing units. This denomination origin¬
ated with Behring and Ehrlich, whose 7iormal serum
was of such strength that 0.1 c.cm. of it would protect
against ten times the least certainly fatal dose of toxin
when simultaneously injected into guinea-pigs. Each
cubic centimeter of this normal serum they called an
immttnizing unit. Later it was shown that the strength
of the serum could easily be increased tenfold, so that
0.01 c.cm. of the serum would protect the guinea-pig

DIPHTHERIA.

3°3

against the ten-times fatal dose. Each cubic centimeter
of this stronger serum was described as an antitoxic unit,
and, of course, contained ten immunizing units. Still
later it was shown that the limits of strength were by
no means reached, and he succeeded in making serums
three hundred times the normal strength, each cubic
centimeter of which contained 300 immunizing units,
or 30 antitoxic units.

In the course of the development of strength in the
serum the exact meaning of “ immunizing unit ” grad¬
ually became obscured, until it is at present an expres¬
sion of strength rather than one of quantity.

While it is difficult to define an immunizing unit, it is
not at all difficult for one skilled in laboratory technique
to determine the number present in a sample of serum.
There are three rules of practice:

1. Determine accurately the least certainly fatal dose
of a sterile diphtheria toxin for a standard guinea-pig.

2. Determine accurately the least quantity of the
serum that will protect a guinea-pig against ten times
the determined least certainly fatal dose of toxin.

3. Express the required dose of antitoxic serum as a
fraction of a cubic centimeter and multiply it by ten.
There will then be as many immunizing units in 1 c. cm.
of the serum as there are parts in the resulting fraction.

Example: It is found that 0.01 c.cm. of toxin kills at
least 9 out of 10 guinea-pigs. It is then regarded as the
least certainly fatal dose. Guinea-pigs receive ten
times this dose (0.1 c.cm.) and varying quantities of the
serum, measured by dilution, say, yinnr c.cm., c.cm.,
-g-oVc c.cm. The first two live. The fraction ytwis *s
now multiplied by 10; ^ Xio = -3-^, an(^ we fi n d that
there are as many units per cubic centimeter in the serum
as there are parts in the result — i. e ., 250.

The most accurate definition of an immunizing unit is:
ten times the least amount of antitoxic serum that will
protect a standard (300-gram) guinea-pig against ten
times the least certainly fatal dose of diphtheria toxin.

304

PATHOGENIC BACTERIA .

The strongest serum ever obtained by the author con¬
tained 1400 units per cubic centimeter.

As the quantity to be injected at each dose diminishes
according to the number of units per cubic centimeter
the serum contains, it is of the highest importance that
the serums be as strong as possible. Various methods of
concentration have been suggested, such as the partial
evaporation of the serum in vacuo , but none have
proved satisfactory. The latest suggestion comes from
Bujwid,1 who finds that when an antitoxic serum is
frozen and then thawed, it separates into two layers,
an upper watery stratum and a lower yellowish one; the
antitoxic value of the yellowish layer is about three
times that of the original serum.

Ehrlich asserts that 500 units are valueless: 2000 units
are probably an average dose, and, as the remedy seems
harmless, it is better to err on the side of too much than
on that of too little. Fourteen thousand units have been
administered in one case with beneficial results.

The largest collection of statistics upon the results of
antitoxic treatment in diphtheria in the hospitals of the
world are probably those collected by Prof. Welch, who,
excluding every possible error in the calculations, u shows
an apparent reduction of case-mortality of 55.8 per cent.”

One of the most important things in the treatment is
to begin it early enough. Welch’s statistics show that
1 1 15 cases of diphtheria treated in the first three days
of the disease yielded a fatality of 8.5 per cent., whereas
546 cases in which the antitoxin was first injected after
the third day of the disease yielded a fatality of 27.8 per
cent.

After the toxin has set up destructive organic lesions'
in various organs and tissues of the body, no amount
of neutralization will restore the integrity of the parts,
so that the antitoxin must fail in these cases.

The urticaria which sometimes follows the injection

1 Centralbl. f. Bakt. u. Parastienk., Sept., 1897, Bd. xxii., Nos. 10 and 11,.
p. 287.

DIPHTHERIA. 305

of antitoxic serum seems to bear a distinct relation to
the age of the serum, fresh serums being more liable
to produce it than those which have been kept for a
month or two.

I have found that the cc keeping” qualities of the se¬
rums, when properly preserved, are of long duration.
Samples examined two years after having been exposed
for sale in the markets have been found unchanged.
The serums most prone to deteriorate seem to be those
of highest potency, but even here the good qualities are
unchanged for months.

Freezing is without effect and ordinary temperature-
changes are harmless to the serum. The antitoxic power
is destroyed at 720 C., the point at which the serum
coagulates.

The erythemata are probably in some way associated
with the globulicidal action of the blood. Keeping the
serum ( c until it is ripe ’ ’ lessens this effect. The serums
from different horses probably vary much in both their
irritant and globulicidal properties, so that antitoxins
prepared by mixing the serums from a number of horses
are probably preferable to those from single horses.

Dried serums are much less active than fresh ones.

For purposes of immunization smaller doses than those
used for treatment suffice. According to Biggs, 2 cubic
centimeters are sufficient to give complete protection.
The immunity that results from the injection is of a
month or six weeks’ duration.

The transitory nature of this immunity is probably
dependent upon the fact that the antitoxin is slowly ex¬
creted through the kidneys.

CHAPTER III.

HYDROPHOBIA, OR RABIES.

No micro-organism of hydrophobia has as yet been
discovered, yet the peculiarities of the disease are such
as to leave no doubt in the mind of a bacteriologist that
one must exist. To find it is now the desideratum.

Although many men have labored upon hydrophobia,
no name is so well known or so justly honored as that
of the great pioneer in bacteriology, Pasteur. The profes¬
sion and laity are alike familiar with his name and work,
and although at times the newspapers of our country
and certain members of the profession have opposed the
methods of treatment which he has suggested as the re¬
sult of his experimentation, we cannot but feel that this
skepticism and opposition are due either to ignorance
»of the principles upon which Pasteur reasoned or to a
culpable conservatism. The most vehement opponent
that Pasteur has in America seems to disbelieve the
existence of rabies. It is impossible to argue with him.

Hydrophobia, or rabies, is a specific toxemia to which
dogs, wolves, skunks, and cats are highly susceptible,
and which can, through their saliva, be communicated
to men, horses, cows, and other animals. The means
of communication is almost invariably a bite, hence the
inference that the specific organism is present in the
saliva.

The animals that are infected manifest no symptoms
during a varying incubation-period in which the wound
generally heals kindly. This period may last for as long
a time as twelve months, but in rare cases may be only
some days. An average duration of the period of incu¬
bation might be stated as about six weeks.

306

HYDROPHOBIA , OR RABIES .

307

As the incubation-period conies to an end there is an
observable alteration in the wound, which becomes red¬
dened, sometimes may suppurate a little, and is painful.
The victim, if a man, is much alarmed and has a sensa¬
tion of horrible dread. The period of dread passes into
one of excitement, which in many cases amounts to a
wild delirium and ends in a final stage of convulsion and
palsy. The convulsions are tonic, rarely clonic, and
subsequently cause death by interfering with the respira¬
tion, as do those of tetanus and strychnia.

During the convulsive period much difficulty is experi¬
enced in swallowing liquids, and it is supposed that the
popular term ( ‘ hydrophobia ’ ’ arose from the reluctance
of the diseased to take water because of the inconveni¬
ence and occasional spasms which the attempt causes.

This description, brief and imperfect as it is, will
illustrate the parallelism existing between hydrophobia
and tetanus. In the latter we observe the entrance of
infectious material through a wound, which, like the
bite in hydrophobia, sometimes heals, but often suppu¬
rates a little. We see in both affections an incubation-
period of varying duration, though in hydrophobia it is
much longer than in tetanus, and convulsions of tonic
character causing death by asphyxia.

It is maintained by some that the stage of excitement
argues against the specific nature of the disease, but
these subjective symptoms are like the mental con¬
dition of tuberculosis, which leads the patient to make a
hopeful prognosis of his case, and the mental condition
of anthrax, in which it is stated that no matter how dan¬
gerous his condition the patient is seldom much alarmed
about it.

Pasteur and his co-workers found that in animals that
die of rabies the salivary glands, the pancreas, and the
nervous system contain the infection, and are more
appropriate for experimental purposes than the saliva,
which is invariably contaminated with accidental patho¬
genic bacteria.

308

PATHOGENIC BACTERIA.

The introduction of a fragment of the medulla ob¬
longata of a dog dead of rabies beneath the dura mater
of a rabbit causes the development of rabies in the
rabbit in a couple of weeks. The medulla of this rabbit
introduced beneath the dura mater of a second rabbit
produced a more violent form of the disease in a shorter
time, and by frequently repeated implantations Pasteur
found that an extremely virulent material could be ob¬
tained.

Inasmuch as the toxins of diphtheria and tetanus
circulate in the blood, and not infrequently saturate
the nervous systems of animals affected, it might be
concluded that the material with which Pasteur worked
was a toxin. This is readily disproven, however, not
only by the fact that the toxin would weaken instead of
strengthen by the method of transfer from animal to
animal, it not being a vital entity, but also by the dis¬
covery that when an emulsion of the nervous system of
an affected animal is filtered through porcelain, or when
it is heated for a few moments to ioo° C., or exposed
for a considerable time to a temperature of 750 or 8o° C.,
its virulence is entirely lost. This would seem to prove
that that which is in the nervous system and communi¬
cates the disease is a living, active body — a parasite, and
in all probability a bacterium. However, all endeavors
to discover, isolate, or cultivate this organism have failed.

Pasteur noted that the virulence of the poison was less
in animals that had been dead for some time than in
the nervous systems of those just killed, and by experi¬
mentation showed that when the nervous system was
dried in a sterile atmosphere the virulence was attenu¬
ated in proportion to the length of time it had been dry.
This attenuation of virulence of course suggested to
Pasteur the idea of a protective vaccination, and by in¬
oculating a dog with much attenuated, then with less
attenuated, then with moderately strong, and finally with
strong, virus, the dog developed an immunity which
enabled it to resist the infection of an amount of viru-

HYDROPHOBIA , 0^ RABIES. 309

lent material that would certainly kill an unprotected
animal.

It is remarkable that this thought, which was a theory
based upon a broad knowledge, but experience with
comparatively few bacteria, should every day find more
and more grounds for confirmation as our knowledge
of immunity, of toxins, and of antitoxins progresses.
What Pasteur did with rabies is what we now do in
producing the antitoxin of diphtheria — i. e. gradually
accommodate the animal to the poison until its body-cells
are able to neutralize or resist it. As the poison cannot
be secured outside of the body because the bacilli, micro¬
cocci, or whatever they may be cannot be secured outside
of the body, he does what Behring originally did in diph¬
theria — introduces attenuated poison-producers — bacilli
crippled by heat or drying, and capable of producing only
a little poison — accustoms the animal to these, and then to
stronger and stronger ones until immunity is established.

The genius of Pasteur did not cease with the produc¬
tion of immunity, but, we rejoice to add, extended to the
kindred subject of therapy, and has now given us a cure
for hydrophobia.

For the production of a cure in infected cases very
much the same treatment is followed as has been de¬
scribed for the production of immunity. The patient
must come under observation early. The treatment con¬
sists of the subcutaneous injection of about 2 grams of
an emulsion of a rabbit’s spinal cord which had been
dried for from seven to ten days. This beginning dose
is not increased in size, but each day the emulsion used
is from a cord which has not been dried so long, until,
when the twenty-fifth day of treatment is reached, the
patient receives 2 grams of emulsion of spinal cord dried
only three days, and is considered immune or cured.

It will be observed that this treatment is really no
more than the immunization of the individual during the
incubation stadium, and the generation of a vital force —
shall we call it an antitoxin ? — in the blood of the animal

3io

PATHOGENIC BACTERIA.

in advance of the time when the organism is expected to
saturate the body with its toxic products.

This, in brief, is the theory and practice of Pasteur’s
system of treating hydrophobia. It is exactly in keeping
with the ideas of the present, and is most extraordinary
in its reasonings and details when we remember that the
first application of the method to human medicine was
made October 26, 1885, nearly ten years before the time
we began to understand the production and use of anti¬
toxins.

CHAPTER IV.

CHOLERA AND SPIRILLA RESEMBLING THE CHOLERA
SPIRILLUM.

Cholera is a disease from which certain parts of India
are never free. The areas in which it is endemic are
the foci from which the great epidemics of the world, as
well as the constant smaller epidemics of India, probably
spread. No one knows when cholera was first introduced
into India, and the probabilities are that it is indigenous
to that couutry, as yellow fever is to Cuba. Very early
mention of it is made in the letters of travellers, in
books and papers on medicine of a century ago, and
in the governmental statistics, yet we find that little is
said about the disease except in a general way, most
attention being directed to the effect upon the armies,
native and European, of India and adjacent countries.
The opening up of India by Great Britain in the last
half century has made possible much accurate scientific
observation of the disease and the relation which its epi¬
demics bear to the manners and customs of the people.

The filthy habits of the people of India, their poverty,
their crowded condition, and their religious customs, all
serve to aid in the distribution of the disease. We are
told that the city of Benares drains into the Ganges River
by a most imperfect system, which distributes the greater
part of the sewage immediately below the banks upon
which the city is built. It is a matter of religious ob¬
servance for every zealot who makes a pilgrimage to the
u sacred city” to take a bath in and drink a large quan¬
tity of this sacred but polluted water, and, as may be
imagined, the number of pious Hindoos who leave
Benares with comma bacilli in their intestines or upon
their clothes is great, for there are few months in the

311

312

PATHOGENIC BACTERIA.

year when there are not at least some cases of cholera
in the city.

The frequent pilgrimages and great festivals of the
Hindoos and Moslems, by bringing together an enormous
number of people who crowd in close quarters where filth
and bad diet are common, cause a rapid increase in the
number of cases during these periods and the dispersion
of the disease when the festivals break up. The disease
extends readily along the regular lines of travel, visiting
town after town, until from Asia it has frequently ex¬
tended into Europe, and by the steamships plying on
foreign waters has been several times carried to our own
continent and to the islands of the seas. Many cases are
on record which show conclusively how a single ship,
having a few cholera cases on board, may be the cause
of an outbreak of the disease in the port at which it
arrives.

It seems strange to us now, with the light of present
information illuminating the pages of the past, to observe
how the distinctly infectious nature of such a disease
could be overlooked in the search for some atmospheric
or climatic cause, some miasm, which was to account
for it.

The discovery of the organism which seems to be the
specific cause of cholera was made by Koch, who rvas
appointed one of a German cholera-commission to study
the disease in Egypt and India in 1883—84. Since his
discovery, but a decade ago, the works upon cholera and
the published investigations to which the spirillum has
been subjected have produced an immense literature,
a large part of which was stimulated by the Hamburg
epidemic of a few years ago.

The micro-organism described by Koch, and now gen¬
erally accepted to be the cause of cholera, is a short
individual about half the length of a tubercle bacillus,
considerably stouter, and distinctly curved, so that the
original name by which it was known was the “comma
bacillus” (Figs. 80, 81).

CHOLERA.

3I3

A study of the growth of the organism and the forms
which it assumes upon different culture-media soon con¬
vinces us that we have to do with an organism in no way
related to the bacilli. If the conditions of nutrition are

Fig. So. — Spirillum of Asiatic cholera, showing the flagella; x xooo (Gunther).

diminished so that the multiplication of the bacteria by
simple division does not progress with the usual rapidity,
we find a distinct tendency toward — and in some cases,
as upon potato, a luxuriant development of — long spiral
threads with numerous windings — unmistakable spirilla.
Frankel has found that the exposure of cultures to unusu¬
ally high temperatures, the addition of small amounts
of alcohol to the culture-media, etc., will so vary the
growth of the organism as to favor the production of
spirals instead of commas. One of the most common
of the numerous forms observed is that in which two
short curved individuals are so joined as to produce an
S-shaped curve.

The cholera spirilla are exceedingly active in their
movements, and in hanging-drop cultures can be seen
to swim about with great rapidity. Not only do the
comma-shaped organisms move, but when distinct spirals
exist, they, too, move with the rapid rotary motion so
common among the spirilla.

PATHOGENIC BACTERIA .

3*4

The presence of flagella upon the cholera spirillum
can be demonstrated without difficulty by L,6ffler’s
method (g. v.). Each spirillum possesses a single flagel¬
lum attached to one end.

Inoculation-forms of most bizarre appearance are very
common in old. cultures of the spirillum, and very often

Fig. 8 1.— Spirillum of Asiatic cholera, from a bouillon culture three weeks old,,
showing numbers of long spirals; x 1000 (Frankel and Pfeiffer).

there can be found in fresh cultures many individuals-
which show by granular protoplasm and irregular outline
that they are partly degenerated. Cholera spirilla from
various sources seem to differ in this particular, some
of the forms being as pronounced in their involution
as the diphtheria bacilli.

In partially degenerated cultures in which long spirals
are numerous Hiippe observed, by examination in the
“ hanging drop,” in the continuity of the elongate mem¬
bers, certain large spherical bodies which he described as
spores. These bodies were not enclosed in the organisms
like the spores of anthrax, but seemed to exemplify the
form of sporulation in which an entire individual trans¬
forms itself into a spore (arthrospore). Koch, and indeed
all other observers, failed to find signs of fructification in

CHOLERA .

315

the cholera organism, and the true nature of the bodies
described by Hiippe must be regarded as doubtful.
Most bacteriologists disagree with Hiippe in believing
that arthrospores exist at all, and the fact (which will be
pointed out later on) that there is very little permanence
about cholera cultures throws additional doubt upon the
accuracy of Hiippe’ s conclusion.

The cholera spirillum stains well with the ordinary
aqueous solutions of the anilin dyes ; fuchsin seems par¬
ticularly appropriate. At times the staining must be con¬
tinued for from five to ten minutes to secure homogeneity.
The cholera spirillum does not stain by Gram’s method.
It may be colored and examined while alive ; thus Cornil
and Babes, in demonstrating it in the rice-water dis¬
charges, u spread out one of the white mucous fragments
upon a glass slide and allow it to dry partially ; a small
quantity of an exceedingly weak solution of methyl violet
in distilled water is then flowed over it, and it is flattened
out by pressing down on it a cover-glass* over which is
placed a fragment of filter-paper, which absorbs any
excess of fluid at the margin of the cover-glass. Comma
bacilli so prepared and examined with an oil-immersion
lens (x 700-800) may then be seen : their characters are
the more readily made out because of the slight stain
which they take up, and because they still retain their
power of vigorous movement, which would be entirely
lost if the specimen were dried, stained, and mounted in
the ordinary fashion.”

The colonies of the spirillum when grown upon gel¬
atin plates are highly characteristic. They appear in
the lower strata of the gelatin as small white dots, grad¬
ually grow out to the surface, effect a gradual liquefaction
of the medium, and then appear to be situated in little
pits with sloping sides (Fig. 82). This peculiar appear¬
ance, which gives one the suggestion that the plate is
full of little holes or air-bubbles, is due to the evapora¬
tion of the liquefied gelatin.

One of the best methods of securing pure cultures of

316 PA THOGENIC BA CTERIA

the cholera spirillum, and also of making a diagnosis
of the disease in a suspected case, is probably that of
Schottelius. The method is very simple : A small quan¬
tity of the fecal matter is mixed with bouillon and stood
in an incubating oven for twenty-four hours. If the

Fig. 82. — Spirillum of Asiatic cholera: colonies two days old upon a gela'in
plate; x 35 (Heim).

cholera spirilla are present, they will grow most rapidly
at the surface of the liquid when the supply of air is
good. A pellicle will be formed, a drop from which,
diluted in melted gelatin and poured upon plates, will
show typical colonies.

Under the microscope the principal characteristics
can be made out. The colony of the cholera spirillum
scarcely resembles that of any other organism. The little
colonies which have not yet reached the surface of the
gelatin begin very soon to show a pale-yellow color and
to exhibit irregularities of contour, so that they are
almost never smooth and round. They are coarsely
granular, and have the largest granules in the centre.
As the colony increases in size the granules also increase

CHOLERA.

3*7

in size, and attain a peculiar transparent character which
is suggestive of powdered glass. The commencement
of liquefaction causes the colony to be surrounded with a
transparent halo. When this occurs the colony begins to
sink, from the digestion and evaporation of the medium,
and also to take on a peculiar rosy color.

In puncture-cultures in gelatin the growth is again so
characteristic that it is quite diagnostic (Fig. 83). The

1 IG. 83. — Spirillum cholera Asiatica ; gelatin puncture-cultures aged forty-
eight and sixty hours (Shakespeare).

growth takes place along the entire puncture, but devel¬
ops best at the surface, where it is in contact with the
atmosphere. An almost immediate liquefaction of the
medium begins, and, keeping pace with the rapidity of
the growth, is more marked at the surface than lower
down. The result of this is the occurrence of a short,
rather wide funnel at the top of the puncture. As the
growth continues evaporation of the medium takes place
slowly, so that the liquefied gelatin is lower than the
solid surrounding portions, and appears to be surmounted
by an air-bubble.

PATHOGENIC BACTERIA .

318

The luxuriant development of the spirilla in gelatin
produces considerable solid material to sediment and fill
up the lower third or lower half of the liquefied area.
This solid material consists of masses of spirilla which
have probably completed their life-cycle and become
inactive. Under the microscope they exhibit the most
varied involution-forms. The liquefaction reaches the
sides of the tube in from five to seven days. Liquefac¬
tion of the medium is not complete for several weeks.
According to Frankel, in eight weeks the organisms in
the liquefied culture all die, and cannot be transplanted.
Kitasato, however, has found them living and active on
agar-agar after ten to thirty days, and Koch was able
to demonstrate their vitality after two years.

When planted upon the surface of agar-agar the spi¬
rilla produce a white, shining, translucent growth along
the entire line of inoculation. It is in no way peculiar.
The vitality of the organism is retained much better upon
agar-agar than upon gelatin, and, according to Frankel,
the organism can be transplanted and grown when nine
months old.

The growth upon blood-serum likewise is without dis¬
tinct peculiarities, and causes gradual liquefaction of the
medium.

Upon potato the spirilla grow well, even when the
reaction of the potato is acid. In the incubator at a
temperature of 370 C. a transparent, slightly brownish
or yellowish-brown growth, somewhat resembling the
growth of glanders, is produced. It contains large
numbers of long spirals.

In bouillon and in peptone solution the cholera organ¬
isms grow well, especially upon the surface, where a
folded, wrinkled mycoderma is formed. Below the mv-
coderma the culture fluid generally remains clear. If
the glass be shaken and the mycoderma broken up,
fragments of it sink to the bottom.

In milk the development is also luxuriant, but takes
place in such a manner as not visibly to alter its appear-

CHOLERA.

3*9

ance. The existence of cholera organisms in milk is,
however, rather short-lived, for the occurrence of any
acidity at once destroys them.

Wolffhiigel and Riedel have shown that if the spirilla
are planted in sterilized water they grow with great ra¬
pidity after a short time, and can be found alive after
months have passed. Frankel points out that this ability
to grow and remain vital for long periods in sterilized
water does not guarantee the same power in unsterilized
water, for in the latter the simultaneous growth of other
bacteria in a few days serves to extinguish the cholera
germs.

One of the characteristics of the cholera spirillum is
the metabolic production of indol. The detection of this
substance is easy if the spirilla are grown in a transparent
colorless solution. As the cholera organisms also produce
nitrites, all that is necessary is to add a drop or two of
chemically pure sulphuric acid to the culture-medium
for the production of the well-known reddish color.

Several toxic products of the metabolism of the spirilla
have been isolated. Brieger, Frankel, Roux and Yersin
have isolated toxalbumins; Villiers, a toxic alkaloid fatal
to guinea-pigs; and Gamal£ia, two substances about
equally toxic.

The cholera spirilla can be found with great constancy
in the intestinal evacuations of all cholera cases, and can
often be found in the drinking-water, milk, and upon
vegetables, etc. in cholera-infected districts. There can
be little doubt that they find their way into the body
through the food and drink. Many cases are reported
in the literature upon cholera that show how the disease-
germs enter the drinking-water, and are thus distributed ;
how they are sometimes thoughtlessly sprinkled over veg¬
etables, offered for sale in the streets, with water from
polluted gutters ; how they enter milk with water used
to dilute it ; how they are carried about in clothing and
upon foodstuffs ; how they can be brought to articles of
food upon the table by flies which have preyed upon

320

PATHOGENIC BACTERIA .

cholera excrement; and how many other interesting in¬
fections are made possible. The literature upon these
subjects is so vast that in a sketch of this kind it is
scarcely possible to mention even the most instructive
examples. One physician is reported to have been in¬
fected with cholera while experimenting with the spirilla
in Koch’s laboratory.

The evidence of the specificity of the cholera spirillum
when collected shows that it is present in the choleraic
dejections with great regularity, and that it is as con¬
stantly absent from the dejecta of healthy individuals
and those suffering from other diseases ; but these facts
do not admit of satisfactory proof by experimentation
upon animals. Animals are never affected by any dis¬
ease similar to cholera during the epidemics, nor do foods
mixed with cholera discharges or with pure cultures of
the cholera spirillum affect them. This being true, we
are prepared to receive the further information that sub¬
cutaneous injections of the spirilla are often without
serious consequences, though cultures differ very much
in this respect, some always causing a fatal septicemia in
guinea-pigs, others being as constantly harmless.

Intraperitoneal injection of the virulent cultures pro¬
duces a fatal peritonitis in guinea-pigs.

One reason that animals and certain men are immune
to the disease seems to be found in the distinct acidity
of the normal gastric juice, and the destruction of the spi¬
rilla by it. Supposing that this might be the case, Nicati
and Rietsch, Von Ermengen and Koch, have suggested
methods by which the micro-organisms can be introduced
directly into the intestine. The first-named investigators
ligated the common bile-duct of guinea-pigs, and then in¬
jected the spirilla into the duodenum with a hypodermic
needle. The result was that the animals usually died, some¬
times with choleraic symptoms ; but the excessively grave
nature of the operation upon such a small and delicately
constituted animal as a guinea-pig greatly lessens the value
of the experiment. Koch’s method is much more satisfac-

CHOLERA.

321

tory. By injecting laudanum into the abdominal cavity
of guinea-pigs the peristaltic movements are checked.
The amount given for the purpose is very large, about
1 gram for each 200 grams of body-weight. It generally
narcotizes the animals for a short time, but they recover
without injury. After administering the opium the con¬
tents of the stomach are neutralized by introducing
through a pharyngeal catheter 5 c.cm. of a 5 per cent,
aqueous solution of sodium carbonate. With the gastric
contents thus alkalinized and the peristalsis paralyzed a
bouillon culture of the spirilla is introduced. The ani¬
mal recovers from the manipulation, but shows an indis¬
position to eat, is soon observed to be weak in the pos¬
terior extremities, subsequently is paralyzed, and dies
within forty-eight hours. The autopsy shows the intes¬
tine congested and filled with a watery fluid rich in spi¬
rilla — an appearance which Frankel declares to be exactly
that of cholera. In man, as well as in these artificially
injected animals, the spirilla are never found in the blood
or the tissues, but only in the intestine, where they fre¬
quently enter between the basement membrane and the
epithelial cells, and aid in the detachment of the latter.

Issaeff and Kolle found that when virulent cholera
spirilla are injected into the ear-veins of young rabbits
the animals die on the following day with symptoms re¬
sembling the algid stage of human cholera. The autopsy
in these cases showed local lesions of the small intestine
very similar to those observed in cholera in man.

Guinea-pigs are also susceptible to intraperitoneal in¬
jections of the spirillum, and speedily succumb. The
symptoms are — rapid fall of temperature, tenderness over
the abdomen, and collapse. The autopsy shows an
abundant fluid exudate containing the micro-organism,
and injection and redness of the peritoneum and viscera.

Although in reading upon cholera at the present time
we find very little skepticism in relation to Koch’s
“comma bacillus,” we do find occasional doubters who
believe with Von Pettenkoffer that the disease is mias-

322

PATHOGENIC BACTERIA.

matic. Pettenk offer’s theory is that the disease has
much to do with the ground-water and its drying zone.
He regards as the principal cause of the disease the de¬
velopment of germs in the subsoil moisture during the
warm months, and their impregnation of the atmosphere
as a miasm to be inhaled, instead of ingested with food
and drink. This idea of Pettenkoffer’s, combined with
his other idea that individual predisposition must pre¬
cede the inception of the disease, is scarcely compatible
with what has gone before, and cannot possibly be made
to explain the inarch of the disease from place to place
with caravans, or its distribution over extended areas
when fairs and religious gatherings among the Hindoos
break up, the people from an infected centre carrying
cholera with them to their homes.

While it is an organism that multiplies with great
rapidity under proper conditions, the cholera spirillum
is not possessed of much resisting power. Sternberg
found that it was killed by exposure to a temperature
of 520 C. for four minutes.' Kitasato, however, found
that ten or fifteen minutes’ exposure to a temperature
of- 550 C. was not always fatal. In the moist con¬
dition the organism may retain its vitality for months,
but it is very quickly destroyed by desiccation, as was
found by Koch, who observed that when dried in a thin
film its power to grow was destroyed in a few hours.
Kitasato found that upon silk threads the vitality might
be retained longer. Abel and Claussen have shown that
it does not live longer than twenty to thirty days in fecal
matter, and often disappears in one to three days. The
organism is very susceptible to the influence of carbolic
acid, bichlorid of mercury, and other germicides.

The organism is also destroyed by acids. Hashimoto
found that it could not live longer than fifteen minutes
in vinegar containing 2. 2-3. 2 per cent, of acetic acid.

This low vital resistance of the microbe is very fortu¬
nate, for it enables us to establish safeguards for the pre¬
vention of the spread of the disease. Excreta, soiled

CHOLERA .

323

clothing, etc. are readily rendered harmless by the proper
use of disinfectants. Water and foods are rendered in¬
nocuous by boiling or cooking. Vessels may be disin¬
fected by thorough washings with jets of boiling water
.thrown upon them through hose. Baggage can be steril¬
ized by superheated steam.

It often becomes a matter of importance to detect the
presence of cholera in drinking-water, and, as the dilu¬
tion in which the bacteria exist in such a liquid may be
very great, much difficulty is experienced in finding them
by ordinary methods. One of the most expeditious meth¬
ods that have been recommended is that of Loffler, who
adds 200 c.cm. of the water to be examined to 10 c.cm.
of bouillon, allows the mixture to stand in an incubator
for twelve to twenty-four hours, and then makes plate-
cultures from the superficial layer of the liquid, where,
if present, the development of the spirilla will be most
rapid because of the presence of air. A similar method
can be used to detect the spirilla when their presence is
suspected in feces.

Gruber and Wiener, Haffkine, Pawlowsky, and Pfeiffer
have all succeeded in immunizing animals against the
toxic substances removed from cholera cultures or against
living cultures properly injected. There seems, accord¬
ing to the researches of Pfeiffer, to be no doubt that in
the blood of the protected animals a protective substance
is present. In the peritoneal infection of guinea-pigs
the spirilla grow vigorously in the peritoneal cavity, and
can be found in immense numbers after twelve to twenty-
four hours. If, however, together with the culture used
for inoculation, a few drops of the protective serum be in¬
troduced, Pfeiffer found that instead of multiplying the
organisms underwent a peculiar granular degeneration
and disappeared, the unprotected animal dying, the pro¬
tected animal remaining well.

Pfeiffer and Vogedes1 have suggested the application
of this “ immunity-reaction” for the positive differentia-

1 CentralbL fur Bakt. unci Parasitenk March 21, 1896, Bd. xix., No. II.

324

PATHOGENIC BACTERIA .

tion of cholera spirilla in cultures. A hanging-drop of
a i : 50 mixture of powerful anti-cholera serum and a
particle of cholera culture is made and examined under
the microscope. The cholera spirilla at once become in¬
active, and are in a short time converted into little rolled-
up masses. If the culture added be a spirillum other
than the true spirillum of cholera, instead of destruc¬
tion of the micro-organisms following exposure to the
serum, they multiply and thrive in the mixture of serum
and bouillon.

The specific immunity-reaction of the cholera serum
has been carefully studied by Eoburnheim,1 and is
specific against cholera alone. The protection is not
due to the strongly bactericidal property of the serum,
but to its stimulating effect upon the body-cells. If
the serum be heated to 6o°-70° C., and its bactericidal
power thus destroyed, it is still capable of producing
immunity.

The immunity produced by the injection of the spirilla
into guinea-pigs continues in some cases as long as four
and a half months, but the power of the serum to con¬
fer immunity is lost much sooner.

Of the numerous attempts which have from time to
time been made, and are still being made, to produce
immunity against cholera in man or to cure cholera
when once established in the human organism, nothing
very favorable can at the present time be said. Experi¬
ments in this field are not new : we find Dr. Ferran ad¬
ministering hypodermic injections of pure virulent cul¬
tures of the cholera spirillum in Spain as early as 1885,
in the hope of bringing about immunity. The more mod¬
ern work of Haffkine seems to be followed by a distinct
diminution of mortality in protected individuals. Ac¬
cording to the work of this investigator, two vaccines are
used, one of which, being mild, prepares the animal (or
man) for a powerful vaccine, which, were it not preceded
by the weaker form, would bring about extensive tissue-

1 Zeitschrift fur Hygiene , xx., p. 438.

CHOLERA, 325

necrosis and perhaps death. Protection certainly seems
to follow the operation of these vaccines.

Haffkine’s studies embrace more than 40,000 inocula¬
tions performed in India. From his latest paper (Dec.,
1895) the following extract will show the results:

“1. In all those instances where cholera has made a
large number of victims, that is to say, where it has
spread sufficiently to make it probable that the whole
population, inoculated and uninoculated, wTere equally
exposed to the infection, — in all these places the results
appeared favorable to inoculation.

u 2. The treatment applied after an epidemic actually
breaks out tends to reduce the mortality even during the
time which is claimed for producing the full effect of the
operation. In the Goya Garl, where weak doses of a
relatively weak vaccine had been applied, this reduction
was to half the number of deaths ; in the coolies of the
Assam-Burinah survey-party, where, as far as I can gather
from my preliminary information, strong doses have been
applied, the number of deaths was reduced to one-seventh.
This fact would justify the application of the method in¬
dependently of the question as to the exact length of time
during which the effect of this vaccination lasts.

“3. In Lucknow, where the experiment was made on
small doses of weak vaccines, a difference in cases and
deaths was still noticeable in favor of the inoculated
fourteen to fifteen months after vaccination in an epidemic
of exceptional virulence. This makes it probable that a
protective effect could be obtained even for long periods
of time if larger doses of a stronger vaccine were used.

u 4. The best results seem to be obtained from applica¬
tion of middle doses of both anticholera vaccines, the
second one being kept at the highest possible degree of
virulence obtainable.

“5. The most prolonged observations on the effect of
middle doses were made in Calcutta, where the mortality
from the eleventh up to the four hundred and fifty-ninth
day after vaccination was, among the inoculated, 17.24

326 PATHOGENIC BACTERIA.

times smaller, and the number of cases 19.27 times
smaller than among the not inoculated.”

Pawlowsky and others have found that the dog is sus¬
ceptible to cholera, and have utilized the observation to
prepare an antitoxic serum in considerable quantities.
The dogs were first immunized with attenuated cultures,
then with more and more virulent cultures, until a serum
was obtained whose value was estimated at 1 : 130,000
upon experimental animals.

• Freymuth and others have endeavored to secure favor¬
able results from the injection of blood-serum from con¬
valescent patients into the diseased. One recovery out
of three cases treated is recorded — not a very glittering
result.

In all these preliminaries the foreshadowing of a future
therapeusis must be evident, but as yet nothing really
satisfactory has been achieved.

Spirilla resembling the Cholera Spirillum. •

The Finkler and Prior Spirillum. — Somewhat similar
to the spirillum of cholera, and in some respects closely
related to it, is the spirillum obtained from the feces of
a case of cholera nostras by Finkler and Prior in 1884.
It is a rather shorter, stouter organism, with a more pro¬
nounced curve, than the cholera spirillum, and rarely
forms the long spirals which characterize the latter.
The central portion is also somewhat thinner than the
ends, which are a little pointed and give the organism
a less uniform appearance than that of cholera (Fig. 84).
Involution-forms are very common in cultures, and occur
as spheres, spindles, clubs, etc. Tike the cholera spiril¬
lum, each organism is provided with a single flagellum
situated at its end, and is actively motile. Although at
first thought to be a variety of the cholera germ, marked
differences of growth were soon observed, and showed
the organism to be a separate species.

The growth upon gelatin plates is quite rapid, and leads
to such extensive liquefaction that four or five dilutions

SPIRILLA RESEMBLING CHOLERA.

327

must frequently be made before the growth of a single
colony can be observed. To the naked eye the colonies

Fig. 84. — Spirillum of Finkler and Prior, from an agar-agar culture ; x 1000
(Itzerott and Niemann).

appear as small white points in the depths of the gelatin
(Fig. 85). They, however, rapidly reach the surface,

Fig. 85. — Spirillum of Finkler and Prior: colony twenty-four hours old, as
seen upon a gelatin plate; x 100 (Frankel and Pfeiffer).

begin liquefaction of the gelatin, and by the second

PATHOGENIC BACTERIA.

328

day appear about the size of lentils, and are situated in
little depressions. Under the microscope they are of a
yellowish-brown color, are finely granular, and are sur¬
rounded by a zone of sharply circumscribed liquefied
gelatin. Careful examination with a high power of the
microscope shows a rapid movement of the granules of
the colony.

In gelatin punctures the growth takes place rapidly
along the whole puncture, forming a stocking-shaped
liquefaction filled with cloudy fluid which does not pre¬
cipitate rapidly ; a rather smeary, whitish mycoderma is
generally formed upon the surface. The much more ex¬
tensive and more rapid liquefaction of the medium, the
wider top to the funnel-shaped liquefaction at the surface,

Fig. 86. — Spirillum of Finkler and Prior : gelatin puncture-cultures aged
forty-eight and sixty hours (Shakespeare).

the absence of the air-bubble, and the clouded nature of
the liquefied material, all serve to differentiate it from the
cholera spirillum.

Upon agar-agar the growth is also very rapid, and in
a short time the whole surface of the culture-medium is

SPIRILLA RESEMBLING CHOLERA .

329

covered with a moist, thick, slimy coating, which may
have a slightly yellowish tinge.

The cultures upon potato are also very different from
those of cholera, for instead of a temperature of 3 70 C.
being required for a rapid development, the Finkler and
Prior spirilla grow rapidly at the room-temperature, and
produce a grayish-yellow, slimy, shining layer, which
may cover the whole of the culture-medium.

Blood-serum is rapidly liquefied by the growth of the
organism.

Buchner has shown that in media containing some
glucose an acid reaction is produced.

The spirillum does not grow well, if at all, in milk,
and speedily dies in water.

The organism does not produce indol.

The spirillum can be stained well by the ordinary
dyes, and seems, like the cholera spirillum, to have a
special affinity for the aqueous solution of fuchsin.

In connection with this bacillus the question of patho¬
genesis is a very important one. At first it was sus¬
pected that it was, if not the spirillum of cholera itself,
a very closely allied organism. Later it was regarded
as the cause of cholera nostras. At present its exact
pathological significance is a question. It was in one
•case secured by Knisl from the feces of a suicide, and
has been found in carious teeth by Muller.

When injected into the stomach of guinea-pigs treated
according the method of Koch, about 30 per cent, of the
animals die, but the intestinal lesions produced are not
the same as those produced by the cholera spirillum.
The intestines in such cases are pale and filled with
watery material having a strong putrefactive odor. This
fluid teems with the spirilla.

It seems very unlikely, from the collected evidence,
that the Finkler and Prior spirillum is associated with
pathogenesis in the human species. As Frankel points
out, it is probably a frequent and harmless inhabitant of
the human intestine.

33°

PATHOGENIC BACTERIA .

The Spirillum of Denecke. — Another organism with
a distinct resemblance to the cholera spirillum is one
described by Denecke as occurring in old cheese (Fig.
87). Its form is much the same as that of the spirillum
of cholera, the shorter individuals being of equal diameter
throughout. The spirals which are produced are longer
than those of the Finkler and Prior spirillum, and are
more tightly coiled than those of the cholera spirillum.

Like its related species, this micro-organism is actively
motile. It grows at the room- temperature, as well as at
370 C., in this respect, as in its reaction to stains, much
resembling the other two.

Upon gelatin plates the growth of the colonies is much
more rapid than that of the cholera spirillum, but slower
than that of the Finkler and Prior spirillum. The col-

Fig. 87. — Spirillum Denecke, from an agar-agar culture; x 1000 (Itzerott
and Niemann).

onies appear as small whitish, round points, which soon
reach the surface of the gelatin and commence liquefac¬
tion. By the second day they are about the size of a
pin’s head, have a yellow color, and occupy the bottom
of a conical depression. The appearance is much like
that of a plate of cholera spirilla.

The microscope shows the colonies to be of irregular

SPIRILLA RESEMBLING CHOLERA . 331

shape and coarsely granular. The color is yellow, and is
pale at the edges, gradually becoming intense toward the
centre. The colonies are surrounded at first by distinct
lines of circumscription, later by clear zones, which, ac¬
cording to the illumination, are pale or dark. From this
description it will be seen that the colonies differ from
those of cholera in the prompt liquefaction of the gelatin,
their rapid growth, yellow color, irregular form, and dis¬
tinct lines of circumscription.

In gelatin punctures the growth takes place all along
the track of the wire, and forms a cloudy liquid which
precipitates at the apex in the form of a coiled mass.
Upon the surface a delicate imperfect yellowish myco-

Fig. 88. — Spirillum Denecke : gelatin puncture- cultures aged forty-eight and
sixty hours (Shakespeare).

derma forms. Liquefaction of the entire gelatin gen¬
erally requires about two weeks.

Upon agar-agar this spirillum grows as a thin yellow¬
ish layer which does not seem inclined to spread widely.

The culture upon potato is luxuriant if grown in the
incubating oven. It appears as a distinct yellowish moist

332

PATHOGENIC BACTERIA.

film, and when examined microscopically is seen to con¬
tain long bean ti fill spirals.

The organism sometimes produces indol, but is irreg¬
ular in its action in this respect.

The spirillum of Denecke is mentioned only because
of its morphological relation to the cholera spirillum,
not because of any pathogenesis which it possesses. It
probably is not associated with any human disease. Ex¬
periments, however, have shown that when the spirilla
are introduced into the intestines of guinea-pigs whose
gastric contents are alkalinized and whose peristalsis is

Fig. 89. — Spirillum Metchnik off, from an agar-agar culture; x 1000 (Itzerott
and Niemann).

paralyzed with opium, about 20 per cent, of the animals
die from intestinal disease.

The Spirillum of GamalSia (Spirillum Metchnikoff).
— Very closely related to the cholera spirillum in its
morphology and vegetation and possibly, as has been
suggested, a descendant of the same original stock, is the
spirillum which Gamaleia cultivated from the intestines
of chickens affected with a disease similar to chicken-
cholera. This spirillum is a curved organism, a trifle
shorter and thicker than the cholera spirillum, a little
more curved, and with similar rounded ends (Fig* 89).

SPIRILLA RESEMBLING CHOLERA .

333

It forms long spirals in appropriate media, and is actively
motile. Each spirillum is provided with a terminal flagel¬
lum. No spores have been positively demonstrated.

The organism, like the cholera vibrio, is very suscep¬
tible to the influence of acids, high temperatures, and
drying, so that spores are probably not formed. It grows
well both at the temperature of the room and at that of
incubation.

The thermal death-point is 50° C. , continued for five
minutes.

The bacterium stains easily, the ends more deeply than
the center. It is not stained by Gram’s method.

Upon gelatin plates a remarkable similarity to the

Fig. 90.— Spirillum Metschnikoff; puncture- culture in gelatin forty-eight hours
old (Frankel and Pfeiffer).

colonies of the cholera spirillum is developed, yet there
is a difference, and Pfeiffer points out that uit is com¬
paratively easy to differentiate between a plate of pure
cholera spirillum and a plate of pure Spirillum Metch-
nikoff, yet it is almost impossible to pick out a few
colonies of the latter if mixed upon a plate with the
former.”

Frankel regards this bacterium as a kind of interme-

334

PATHOGEN/C BACTERIA.

diate species between the cholera and the Finkler-Prior
spirilla.

The colonies upon gelatin plates appear in about twelve
hours as small whitish points, and rapidly develop, so that
by the end of the third day large saucer-shaped areas of
liquefaction resembling colonies of the Finkler-Prior
spirilla occur. The liquefaction of the gelatin is quite
rapid, the resulting fluid being turbid. Generally there
will be upon a plate of Vibrio Metchnikoff some colo¬
nies which closely resemble cholera by occupying small
conical depressions in the gelatin. Under a high power
of the microscope the contents of the colonies, which ap¬
pear to be of a brownish color, are observed to be in rapid
motion. The edges of the bacterial mass are fringed with
radiating organisms (Fig. 90).

In gelatin tubes the culture is very much like that of
cholera, but develops more slowly.

Upon the surface of agar-agar a yellowish-brown
growth develops along the whole line of inoculation.

On potato at the room-temperature no growth occurs,
but at the temperature of the incubator a luxuriant
yellowish-brown growth takes place. Sometimes the
color is quite dark, and chocolate-colored potato cultures
are not uncommon.

In bouillon the growth which occurs at the tempera¬
ture of the incubator is quite characteristic, and very
different from that of the cholera spirillum. The entire
medium becomes clouded, of a grayish-white color, aud
opaque. A folded and wrinkled mycoderma forms upon
the surface.

When glucose is added to the bouillon no fermentation
or gas-production results.

When grown in litmus milk the original blue color is
changed to pink in a day, and at the end of another day
the color is all destroyed and the milk coagulated. Ulti¬
mately the clots of casein sediment in irregular masses,
and clear colorless whey is supernatant.

The addition of sulphuric acid to a culture grown in a

SPIRILLA RESEMBLING CHOLERA. 335

medium rich in peptone produces the same rose color
observed in cholera cultivations.

The organism is pathogenic for animals, but not for
man. Pfeiffer has shown that chickens, pigeons, and
guinea-pigs are highly susceptible animals. The birds
when inoculated under the skin generally die — pigeons
always. W. Rindfleish has pointed out that this positive
fatal outcome of the introduction of the spirillum into
pigeons makes it a valuable diagnostic point for the
differentiation of this spirillum from that of cholera.
According to his 'researches, the simple subcutaneous in¬
jection of the most virulent cholera cultures is never
fatal to pigeons. The birds only die when the injections
are made into the muscles in such, a manner that the
muscular tissue is injured and becomes a locus vimoris
resistenticz. When guinea-pigs are treated according to
the method of Koch for the inoculation of cholera, the
temperature of the animal rises for a short time,, then
abruptly falls to 330 C. or less. Death follows in twenty
to twenty-four hours. A distinct inflammation of the
intestine, with exudate and numerous spirilla, may be
found. The spirilla can also be found in the heart’s
blood and in the organs of such guinea-pigs. When the
bacilli are introduced by subcutaneous inoculation, the
autopsy shows a bloody edema and a superficial necrosis
of the tissues.

In the blood and all the organs of pigeons and young
chickens the organisms can be found in such large num¬
bers that Pfeiffer has suggested the term 11 vibrionensep-
ticsemie” for the condition. In the intestines very few
alterations are noticeable, and very few spirilla can be
found.

Gamaleia has shown that pigeons and guinea-pigs can
be made immune by inoculating them with cultures ster¬
ilized for a time at a temperature of ioo° C. Mice and
rabbits are immune except to very large doses.

Spirillum Berolinensis. — This organism (Fig. 91),
which was discovered by Neisser in the summer of 1893,

336

PATHOGENIC BACTERIA.

is of great interest in comparison with the spirillum of
cholera and its related forms. Its morphology is in every
particular exactly like that of the cholera spirillum, but
its growth is a little more rapid. It grows upon the
same culture-media and at the same temperature. The
colonies are, however, quite different.

Upon the second day, when grown upon gelatin
plates, the colonies of the Spirillum Berolinensis appear
finely granular and paler than those of cholera. The
borders are generally smooth and circular. As it be¬
comes older the colony takes on a slightly brownish
color, and may be nodulated or radiately lobulated. The
gelatin is very slowly liquefied.

Fig. 91. — Spirillum Berolinensis, from an agar-agar culture; x 1000 (Itzerott

and Niemann).

In puncture-cultures the development takes place along
the entire puncture, and causes a gradual liquefaction of
the gelatin.

Upon agar-agar the growth is generally similar to that
of the cholera spirillum, but at times is copious, dry,
and ragged, and suggests leather by its appearance.

When introduced intraperitoneally into guinea-pigs,
the animals die in from one to two days.

The indol reaction is exactly like that given by cul-

SPIRILLA RESEMBLING CHOLERA. 337

turfs of tlie cholera spirillum. The spirillum does not
stain by Gram’s method.

Spirillum Dunbar. — This organism (Fig. 92) was de-

Fig. 92. — Spirillum Dunbar, from agar-agar; x 1000 (Itzerott and Niemann).

scribed in 1893 by Dunbar and Oergel, who secured it
from the water of the Elbe River. It much resembles
the cholera spirillum, but it never exhibits sigmoid forms.
It stains poorly, the ends taking the color much better
than the central portion.

Gelatin is liquefied by the growth of this organism
more quickly than by the cholera spirillum. The colo¬
nies upon gelatin and the puncture-cultures in gelatin
are identical with those of the cholera spirillum.

. On agar-agar a luxuriant whitish-yellow layer is pro¬
duced.

In bouillon and peptone solution the addition of dilute
sulphuric acid produces the red color of nitro-indol.

It is said that cultures grown at a temperature of 22° C'.
phosphoresce in the dark.

The spirillum seems to be pathogenic for guinea-pigs
when introduced into the stomach according to Koch’s
method for cholera.

Spirillum Danubicus. — This organism (Fig. 93) also;

33§

PATHOGENIC BACTERIA.

much resembles cholera. It was first isolated by Heider
m 1892. In appearance it is rather delicate and decidedly
curved. It is often united in sigmoid and semicircular
forms, and exhibits long spirals in old cultures. It is
actively motile, each organism presenting a terminal
flagellum.

The growth upon gelatin plates is rapid. Small light-
gray colonies, resembling those of cholera, but exhibit-
ing a dentate margin, are observed. The growth in
gelatin punctures also much resembles cholera, and the
agar-agar growth can scarcely be distinguished from it.

The potato growth has a distinct yellowish-brown
color.

Milk is coagulated in three or four days.

* V

2,-y* ~ -

T: >3*-* sjp. ,
/ y J> 7\ '

-V0 X w ' V<

v oNC

Fig. 93. — Spirillum Danubicus, from an agar-agar culture; x 1000 (Itzerott and

Niemann).

This spirillum does not produce indol.

Heider found the spirillum pathogenic for guinea-pigs.

Spirillum I. of Wernicke. — This organism is about
twice as large as the cholera spirillum, liquefies gelatin
more rapidly, produces indol, and is feebly pathogenic
for guinea-pigs.

Spirillum II. of Wernicke. — This spirillum is smaller
than the cholera spirillum, liquefies gelatin more slowly,

SPIRILLA RESEMBLING CHOLERA. 339

produces indol, and is highly pathogenic for rabbits,
guinea-pigs, pigeons, and mice.

Spirillum Bonhoffi. — This organism (Fig. 94) was
found in water by Bonhoff. It has a decided resem-

Fig. 94. — Spirillum Bonhoffi, from a culture upon agar-agar ; x 1000 (Itzerott
and Niemann).

blance to the cholera spirillum, but is rather stouter
and less curved. Curved forms — i. e . semicircles, sig-
moids, and spirals — occur in old cultures especially.

These organisms are colored badly with ordinary stains,
dahlia seeming to be the most appropriate color, and ac¬
complishing the process better if warmed. The organ¬
ism is motile, and has a long flagellum attached to one
end.

The colonies develop slowly upon gelatin plates, first
appearing in forty-eight hours as little grayish points.
The margin of the colony is sharply circumscribed ; the
interior is broken up. The gelatin is not liquefied. In
gelatin punctures there is no liquefaction observable.

Upon agar-agar the development at the temperature
of the incubator, which is more rapid than that at the
temperature of the room, results in the production of a
bluish-gray layer.

The growth upon potato has a brownish color. The

340

PATHOGENIC BACTERIA.

growth in bouillon and in peptone solutions is accompa¬
nied by the production of indol.

The spirillum is pathogenic for mice, guinea-pigs, and
canary birds.

Spirillum Weibeli. — This spirillum (Fig. 95) was found
in 1S92 by Weibel in spring- water which had a long time

V»v 1 CWjtr'X

y6 * v- v

^ "«■ ■ tm * tV/ * 4

K 1 '/"'f

t ^ 4 *• ^ »«

\ s C* J ' vv' ' J£\

Fig. 95. — Spirillum Weibeli, from agar-agar; x 1000 (Itzerott and Niemann).

before been infected by cholera. It is short, rather thick,
and distinctly bent, often forming S-shaped figures.

The colonies before liquefaction sets in are described
as pale-brown, transparent, circular, and homogeneous.
Liquefaction is much more rapid than in cholera, and
causes the borders of the colonies to become irregular.
In the centre of each colony a little depression is ob¬
served.

In gelatin puncture- cultures the growth is rapid, be¬
ginning first upon the surface, where a large flat, saucer¬
shaped liquefaction, extending to the sides of the tube,
forms. Scarcely any growth takes place in the puncture,
but the superficial liquefaction, separated by a horizontal
line from the normal gelatin, descends slowly.

Upon agar-agar a grayish-white layer is formed.

No growth has been obtained upon potato.

SPIRILLA RESEMBLING CHOLERA. 341

In alkaline peptone solution a slow but luxuriant
growth takes place.

Spirillum Milleri. — This spirillum (Fig. 96) was found
in the mouth by Miller in 1885. It resembles the cholera

Fig. 96. — Spirillum Milleri, from an agar-agar culture; x 1000 (Itzerott and

Niemann).

spirillum somewhat, but is much more like the spirillum
of Finkler and Prior, with which many bacteriologists
think it identical.

Upon gelatin the colonies are small, finely granular,
have a narrow border-zone and a pale-brown color. The
gelatin is rapidly liquefied.

Upon agar-agar a thick yellowish layer is produced.

The organism seems not to be pathogenic.

Spirillum Aquatilis. — Gunther in 1892 found this or¬
ganism (Fig. 97) in the water of the river Spree. It is
similar to the cholera spirillum in shape, has a long
terminal flagellum, and is motile.

The colonies which form upon gelatin are circular,
have smooth borders, and look very much as if bored out
with a tool. They have a brown color and are finely
granular. In gelatin puncture-cultures the growth occurs
almost exclusively at the surface.

The agar-agar cultures are similar to those of cholera.

o o

Scarcely any development occurs in bouillon. By the

342

PATHOGENIC BACTERIA.

growth of the organism sulphuretted hydrogen gas is
produced.

The spirillum does not grow at all upon potato.

Giinther did not find the organism to be pathogenic.

Spirillum Terrigenus. — This species, also discovered
by Gunther, was secured from earth. It generally occurs
in a slightly curved form, but sometimes is spiral. It is
actively motile and has a terminal flagellum.

The colonies, which appear in twenty-four hours, are
small, structureless, and transparent, and later take on a
‘ ‘ fat-drop ’ ’ appearance.

Upon agar-agar a thin white coating is formed. Milk
is coagulated by the growth of the organism. No indol
is produced.

The organism does not stain by Gram’s method, and
is said not to be pathogenic for guinea-pigs or for mice.

Fig. 9 7. -^Spirillum aquatilis, from an agar-agar culture; x 1000 (Itzerott and

Niemann).

Vibrio Schuylkiliensis. — This form, closely resembling
the cholera spirillum, was found by Abbott1 in sewage-
polluted water from the Schuylkill River at Philadelphia.
The colonies upon gelatin plates resemble very closely
those of Spirillum Metschnikovi. In gelatin puncture-
cultures the appearance is exactly like the true cholera spir-

1 Jour . of Exper. Med., vol. i., No. 3, July, 1896, p. 419.

SPIRILLA RESEMBLING CHOLERA .

343

ilium. At times the growth may be a little more rapid.
The growth on agar is very luxuriant, and gives off a
pronounced odor of indol. Loffler’s blood-serum is ap¬
parently not a perfectly adapted medium, but upon it the
organisms grow, with resulting liquefaction. Upon po¬
tato at the point of inoculation there is a thin, glazed,
more or less dirty yellow, shading to brownish deposit that
is sometimes surrounded by a flat, dry, lusterless zone.

In litmus milk a slightly reddish tinge is found after
twenty-four hours at body temperature. After forty-eight
hours this is increased and the milk is coagulated. In
peptone solutions indol is produced. No gas is pro¬
duced in glucose-containing culture-media. The organ¬
ism is a facultative anaerobic spirillum. The thermal
death-point is 50° C. for five minutes.

The organism is pathogenic for pigeons, guinea-pigs,
and mice. The pathogenesis is much like that of the
Spirillum Metschnikovi. No Pfeiffer’s phenomenon was
observed with the use of the serum of immunized ani¬
mals.

Immunity was produced in pigeons, and it was found
that their serum was protective against both the Vibrio
Schuylkiliensis and Spirillum Metschnikovi, the immun¬
ity thus produced being of about ten days’ duration.

In a second paper by Abbott and Bergy1 it was shown
that the vibrios were found in river water during all
four seasons of the year, and in all parts of the river
within the city, both at low and at high tide. They were
also found in the sewage emptying into the river. The
spirilla were also found in the water of the Delaware
River as frequently as in that from the Schuylkill.

One hundred and ten pure cultures of spirilla were iso¬
lated from the sources mentioned and subjected to routine
tests. It was found that few or none of them were iden¬
tical in all points. There seems, therefore, to be a family
of river spirilla related to each other like the different
colon bacilli are related..

1 Journal of Experimental Medicine , vol. ii., No. 5, p. 535*

344

PATHOGENIC BACTERIA .

The opinion of the writers is that “the only trust¬
worthy difference between many of these varieties and
the true cholera spirillum is the specific reaction with
serum from animals immune from cholera, or by Pfeiffer’s
method of intraperitoneal testing in such animals.”

In discussing these spirilla of the Philadelphia waters
Bergy1 says:

‘ 1 The most important point with regard to the occur¬
rence of these organisms in the river water around Phil¬
adelphia, is the fact that similar organisms have been
found in the surface-waters of the European cities in
which there had recently been an epidemic of Asiatic
cholera, notably at Hamburg and Altona. . . . The fore¬
most bacteriologists of Europe have been inclined to the
opinion that the organisms which they found in the sur¬
face-waters of the European cities were the remains of
the true cholera organism, and that the deviations in the
morphologic and biologic characters from those of the
cholera organism were brought about by their prolonged
existence in water. No such explanation of the occur¬
rence of the organisms in Philadelphia waters can be
given.”

1 Jour, of the Amer. Med. Assoc., Oct. 23, 1897.

CHAPTER V.

PNEUMONIA.

The term “ pneumonia,’ 5 while generally understood
to refer to the lobar disease particularly designated as
croupous pneumonia, is a vague one, really comprehend¬
ing a variety of inflammatory conditions of the lung
quite dissimilar in character. This being true, no one
should be surprised to find that a single organism cannot
be described as “specific55 for all. Indeed, pneumonia
must be considered as a group of diseases, and the various
microbes found associated with it must be described suc¬
cessively in connection with the peculiar phase of the
disease in which they occur.

i. Lobar or Croupous Pneumonia. — The bacterium,
which can be demonstrated in at least 75 per cent of the
cases of lobar pneumonia, which is now almost uni¬
versally accepted as the cause of the disease, and about
whose specificity very few doubts can be raised, is the
pneumococcus of Frankel and Weichselbaum.

Priority of discovery in the case of the pneumococcus
seems to be in favor of Sternberg, who as early as 1880 de¬
scribed an identical organism which he secured from his
saliva. Curiously enough, Pasteur seems to have cap¬
tured the same organism, also from saliva, in the same
year. The researches of the observers whose names are
attached to the organism were not completed until five
years later. It is to Frankel, Telamon, and particularly
to Weichselbaum, however, that we are indebted for the
discovery of the relation which the organism bears to
pneumonia.

The organism (Fig. 98) is variable in its morphology.
When grown in bouillon it is oval, has a pronounced dis-

345

346

PATHOGENIC BACTERIA.

position to occur in pairs, and not infrequently forms
chains of five or six members, so that some have been
disposed to look upon it as a streptococcus (Gamaleia).
In the fibrinous exudate from croupous pneumonia, in
the rusty sputum, and in the blood of rabbits and mice
containing them the organisms are arranged in pairs,
exhibit a distinct lanceolate shape, the pointed ends
generally approximated, and are usually surrounded by
a distinct halo or capsule of clear, colorless, homogeneous
material, thought by some to be a swollen cell-wall, by

Fig. 98. Diplococcus pneumoniae, from the heart's blood of a rabbit ; x 1000

(Frankel and Pfeiffer).

others a mucus-like secretion given off by the cells. When
grown ordinarily in culture-media, and especially upon
solid media, the capsules are absent.

The organism is without motility, has no spores, and
does not seem to be able to resist any unfavorable con¬
ditions when grown artificially. It stains well with the
ordinary solutions of the anilin dyes, and gives most

PNEUMONIA. 347

beautiful pictures in blood and tissues when stained by
Gram’s method. The capsule does not stain.

To demonstrate the capsule, the glacial acetic acid
method may be used. The cover-glass is spread with a
thin film of the material to be examined, which is dried
and fixed as usual. Glacial acetic acid is dropped upon
it for an instant, poured (not washed) off, and at once fol¬
lowed by anilin-water, gentian-violet, in which the stain¬
ing continues several minutes. Finally, the preparation
is washed in water, and may be examined at once in water
or mounted in balsam after drying. The capsules are
probably more distinct when the examination is made in
water.

The pneumococcus is no stranger to us; it may some¬
times be found in the saliva of healthy individuals, and
the inoculation of human saliva into rabbits frequently
causes a septicemia in which the bacillus is found abun¬
dantly in the blood and tissues. Because of its frequent
presence in the saliva it was described by Fliigge as the
Bacillus septicus sputigenus.

When desired for purposes of study, it may be obtained
by inoculating rabbits'with pneumonic sputum and re¬
covering the organisms from their heart’s blood, or it may
be secured from the rusty sputum of pneumonia by the
method employed by Kitasato for securing tubercle ba¬
cilli from sputum. A single mouthful of fresh sputum
is secured, washed in several changes of sterile water to
free it from bacteria of the mouth and pharynx, carefully
separated, and a central portion transferred to an appro¬
priate culture-medium.

The organism grows upon all the culture-media except
potato, but only between the temperature-extremes of
240 and 420 C. ; the best development is at 370 C. The
growth is always limited, probably because the formic
acid produced serves to check it. The addition of an
unusual amount of alkali to the culture-medium favors
the growth.

The organisms readily lose their virulence in culture-

348

PATHOGENIC BACTERIA.

media, and cease to be pathogenic after a few days. In his
experiments with antipneumococcic serum Washbourn
found, however, that a pneumococcus isolated from pneu¬
monia sputum and passed through one mouse and nine
rabbits developed a permanent virulence when kept on
agar-agar made carefully, so that it was not heated beyond
ioo° C. , and alkalinized 4 c.cm. of normal caustic soda
solution beyond the neutral point determined with rosalic
acid, to each liter. The agar-agar is first streaked with
sterile rabbit’s blood, then inoculated. The cultures are

Fig. 99. — Diplococcus pneumonise: colony twenty-four hours old upon gelatin;
x 100 (Frankel and Pfeiffer).

kept at 37. 50 C. Not only is this true, but ordinarily
they seem to be unable to accommodate themselves to a
purely saprophytic life, and unless continually trans¬
planted to new media die in a week or two, sometimes
sooner.

Kinyoun recommended to the writer that virulence
could be retained for a considerable time by keeping
blood from an infected rabbit, in a hermetically sealed
glass tube, on ice. This plan seems to work admirably
if the blood is not kept too long.

PNEUMONIA .

349

The colonies which develop at 240 C. upon 15 per
cent, gelatin plates are described as small, round, cir¬
cumscribed, finely granular white points which grow
slowly, never attain any considerable size, and do not
liquefy the gelatin (Fig. 99).

If, instead of gelatin, agar-agar be used and the plates
kept at the temperature of the body, the colonies which
develop upon the plates appear as transparent, delicate,
drop-like accumulations, scarcely visible to the naked
eye, but under the microscope distinctly granular, the
central darker portion being frequently surrounded by a
paler marginal zone.

In gelatin puncture-cultures, made with 15 instead of
the usual 10 per cent, of gelatin, the growth takes place
along the entire path of the wire in the form of little
whitish granules distinctly separated from each other.
The growth in gelatin is always very limited.

Upon agar-agar and' blood-serum the growth consists
of minute, transparent, semi-confluent, colorless, dew-
drop-like colonies, which die before attaining a size
which permits of their being seen without careful in*
spection.

In bouillon the organisms grow well, clouding the
medium very slightly.

Milk is quite well adapted as a culture-medium, its
casein being coagulated.

No growth can be secured upon potato at any tem¬
perature or by any manipulation yet known.1

When it is desired to maintain or increase the virulence
of a culture it must be very frequently passed through
the body of a rabbit. The degree to which the virulence
can be raised in this way is remarkable. C. W. Lincoln
has succeeded in reducing the fatal dose for rabbits to
1000 c (To 0"(T ^ c. cm.

If a small quantity of a pure culture of the virulent

1 Ortmann asserts that the pneumococcus can be grown on potato at 370 'C.,
but this is not generally confirmed. The usual acid reaction of the potato
would indicate that it was a very unsuitable culture-medium.

350 PATHOGENIC BACTERIA .

organism is introduced into a mouse, rabbit, or guinea-
pig, the animal dies in one or two days. Exactly the
same result can be obtained by the introduction of a
piece of the lung-tissue from croupous pneumonia, by
the introduction of some of the rusty sputum, and gener¬
ally by the introduction of saliva.

The post-mortem shows that an inflammatory change
has taken place at the point of inoculation, with a fibrin¬
ous exudate resembling somewhat that in diphtheria.
At times, and especially in dogs, there may be a little
pus formed. The other appearances are those of a
general disturbance. The spleen is much enlarged, is
firm and red brown. The blood in all the organs contains
large numbers of the bacteria, most of which exhibit a
distinct lanceolate form and have their capsules very
distinct. The disease is a pure septicemia unassociated
with pronounced tissue-changes.

In cases of the kind described the lungs show no pneu¬
monic changes. Likewise, if the hypodermic needle
used for injection be plunged through the breast- wall
into the pulmonary tissue, no pneumonia results. Mon¬
ti, however, claims to have found that a true character¬
istic pneumonia results from the injection of cultures
into the trachea of susceptible animals. This observa¬
tion lacks confirmation.

JNot all animals are susceptible. Guinea-pigs, mice,
and rabbits are highly sensitive to the operations of the
organism ; dogs are comparatively immune.

From this brief review of the peculiarities of the pneu¬
mococcus it must be obvious that its reputation in pneu¬
monia depends more upon the regularity with which it is
found in that disease than upon its capacity to produce a
similar affection in the lower animals.

As in numerous other diseases, we are unable to furnish
an absolute proof of specificity according to the postu¬
lates of Koch.

The disease is peculiar in that recovery from it is fol¬
lowed either by no immunity or by one of such brief dura-

PNEUMONIA.

35*

tion as to allow of frequent relapses ; and it is well known
that many cases show a subsequent predisposition to
fresh attacks of the disease. This brevity of immunity
lessens the probability that in the future we shall dis¬
cover an antitoxin that shall be powerful in its influ¬
ence upon the course and termination of the disease.

The experiments of G. and F. Klemperer, a few years
ago, showed that the serum of immunized rabbits pro¬
tected animals inoculated with the pneumococcus. The
principle failed, however, when applied to human medi¬
cine. The treatment of pneumonia by the injection of
blood-serum from convalescents has also been abandoned
as useless and dangerous.

Washbourn has recently prepared an aniipneuviococcic
serum which is efficacious in protecting rabbits against
ten times the fatal dose of live pneumococci. In general,
the lines upon which he operated were those of Behring,
Marmorek’s work with the streptococcus furnishing most
of the details. A pony was subjected to immunization
for a period of five .months, allowed to rest three or four
months until the live pneumococci introduced were all
destroyed, and then bled. Two cases of human pneu¬
monia seem to have received some benefit from the injec¬
tion of large doses of this serum.

The pneumococcus causes other lesions than croupous
pneumonia; thus, Foa, Bordoni-Uffreduzzi, and others
have found it in cerebrospinal meningitis; Frankel, in
pleuritis; Weichselbaum, in peritonitis; Banti, in peri¬
carditis; numerous observers have found it in acute ab¬
scesses; Gabbi has isolated it from a case of suppurative
tonsillitis; Axenfeld has observed an epidemic of con¬
junctivitis caused by it; and Zaufal, Levy, and Schrader
and Netter have been able to demonstrate its presence in
the pus of otitis media. It has also been reported as oc¬
curring in the joints in arthritis following pneumonia.

The pneumococcus is often present in the mouths of
healthy persons. The conditions under which it enters
the lung to produce pneumonia are not known.

352

PATHOGENIC BACTERIA .

In the opinion of most authorities, something more
than the simple entrance of the bacterium into the lung
is required for the production of the disease, but what
that something is, is still a matter of doubt. It would
seem to be some systemic depravity, and in support of this
view we may point out that pneumonia is very frequent,
and almost universally fatal, among drunkards. Whether,
however, any vital depression or systemic depravity will
predispose to the disease, or whether it depends for its
origin upon the presence of a certain leucomaine, time
and further study will be required to tell.

Bacillus Pneumonic? of Friedlander (Fig. ioo). — An un-

FlG. ioo. — Bacillus pneumonias of Friedlander, from the expectoration of a
pneumonia patient; x 1000 (Frankel and Pfeiffer).

fortunate accident has applied the name u pneumococcus n
to an organism very different from the one just described.
It was discovered by Friedlander in 1883 in the exudate
from the lung in croupous pneumonia, and, being thought
by its discoverer to be the cause of the disease, very natu¬
rally was called the pneumococcus, or, more correctly, the
pneztmobacillus . The grounds upon which the pathog¬
eny of the organism was supposed to depend were very in¬
sufficient, and the bacillus of Friedlander — or, as Fliigge

PNEUMONIA.

353

prefers to call it, the Bacillus pneumoniae — has ceased to
be regarded as specific, and is now looked upon as an
accidental organism whose presence in the lung is, in
most cases, unimportant.

As the two organisms are similar in more respects than
their names, Friedlander’s bacillus requires at least a
brief description.

It is distinctly a bacillus, but sometimes, when occur¬
ring in pairs, has a close resemblance to the pneumo¬
coccus of Frankel and Weichselbaum. Very frequently
it forms chains of four or more elements. It is also com¬
monly surrounded by a transparent capsule. It is non-
motile, has no spores and no flagella. It stains well
with the ordinary anilin dyes, but does not retain the
color when stained by Gram’s method.

Frankel points out that Friedlander’s error in suppos¬
ing this bacillus to be the chief parasite in pneumonia
depended upon the fact that his studies were made by
the plate method. If some of the pneumonic exudate be
mixed with gelatin and poured upon plates, the bacilli
grow into colonies at the end of twenty-four hours, and
appear as small white spheres which spread upon the
gelatin to form white masses of a considerable size.
Under the microscope these colonies are rather irregular
in outline and somewhat granular.

The bacillus grows at as low a temperature as i6° C.,
and, according to Sternberg, has a thermal death-point
of 56° C.

When a colony is transferred to a gelatin puncture-cul¬
ture, quite a massive growth occurs. Upon the surface a.
somewhat elevated, rounded white mass is formed, and
in the track of the wire innumerable little colonies,
spring up and become confluent, so that a u nail-growth ”
results. No liquefaction occurs. When old the cultures
sometimes become brown in color.

Upon the surface of agar-agar at ordinary temperatures
quite a luxuriant white or brownish-yellow, smeary, cir-
23

354

PATHOGENIC BACTERIA .

cumscribed growth occurs. The growth upon blood-
serum is the same.

Upon potato the growth is abundant, quickly covering
the entire surface with a thick yellowish-white layer,
which sometimes contains bubbles of gas. Gas is also
sometimes developed in gelatin cultures.

A most superficial comparison will suffice to show the
great difference in vegetation between these two so-called
pneumococci.

Friedlander had considerable difficulty in causing any
pathogenic changes by the injection of his bacillus into
animals. Rabbits and guinea-pigs were immune, and
the only actual pathogenic results which Friedlander ob¬
tained were in mice, into whose lungs and pleura he
injected the cultures. The remarks of Frankel upon
such mouse-operations, which do not add much weight
to experiments, have already been quoted.

In the status prcesens of bacteriologic knowledge the
bacillus of Friedlander is regarded as an organism of very
feeble pathogenic powers, generally a harmless sapro¬
phyte, but which may at times aid in producing inflam¬
matory changes when in the tissues of the human body.

2. Catarrhal Pneumonia. — This form of pulmonary
inflammation occurs in local areas, generally situated
about the distribution of a bronchiole. It cannot be
said to have a specific micro-organism, as almost any
irritant foreign materials accidentally inhaled can cause
it. The majority of the cases, however — and especially
those which are distinctly peribronchial — are caused by
the presence of the staphylococcus and streptococcus of
suppuration. Friedlander’ s bacillus may also aid in pro¬
ducing local inflammations.

3. Tubercular Pneumonia. — At times the process of
pulmonary tuberculosis is so rapid, and associated with
the production of so much semi-liquid, semi-necrotic
material, that the auto-infection of the lung is greatly
favored; the tubercle -bacilli are distributed to the entire
lung or to large parts of it, and a distinct inflammation

PNEUMONIA .

355

occurs. Such a pneumonia may be caused by the tubercle
bacillus alone, but more often it is aided by accompany¬
ing staphylococci, streptococci, tetragenococci, pneumo¬
cocci, pneumobacilli, and other organisms apt to be pres¬
ent in a lung in which tuberculosis is in progress and
ulceration and cavity-formation are advanced.

4. Mixed Pneumonias. — It frequently happens that
pneumonia occurs in the course of, or shortly after the
convalescence from, influenza. In these cases a mixed
infection is present, and there is no difficulty in deter¬
mining that both the influenza bacillus and the pneumo¬
coccus are present. Again, sometimes the pneumococci
and staphylococci operate simultaneously, and produce
a purulent pneumonia with abscesses as the conspicuous
feature. As almost any combination of the described
bacteria is possible in the lungs, and as these combi¬
nations will all produce varying inflammatory conditions,
it must be left for the student to imagine what the par¬
ticular characters of each may be.

Among these mixed pneumonias may be mentioned
those called by Klemperer and Levy “ complicating
pneumonias,” occurring in the course of typhoid, etc.

C. THE SEPTIC DISEASES.

CHAPTER I.

ANTHRAX.

The disease of cattle known as anthrax or ct splenic
fever n is of infrequent occurrence in this country and in
England. In France, Germany, Hungary, Russia, Persia,
and the East Indian countries it is a dreaded and common
malady which robs herdsmen of many of their valuable
stock. Siberia perhaps suffers most, the disease being so
exceedingly common and malignant as to deserve the
name 4 4 Siberian pest. ’ ’ Certain local areas, such as the
Tyrol and Auvergne, in which it seems to be constantly
present, serve as distributing foci from which the disease
spreads rapidly in summer, afflicting many animals, and
ceasing its depredations only with the advent of winter.
It seems to be distinctly a disease of the summer season.

The animals most frequently affected are cows and
sheep. Among our laboratory animals white mice,
guinea-pigs, and rabbits are highly susceptible ; dogs,
cats, most birds, and amphibians are almost perfectly
immune. White rats are infected with difficulty. Man
is only slightly susceptible, the manifestation of the dis¬
ease as seen in the human species being different from
the same disease in the lower animals in that it is usually
a local affection — malignant carbuncle — and only at times
gives rise to a general infection.

Anthrax was one of the first of the specific diseases
proven to be caused by a definite micro-organism. As
early as 1849, Pollender discovered small rod-shaped
bodies in the blood of animals suffering from anthrax,
but the exact relation which they bore to the disease was
not pointed out until 1863, w^en Davaine, by a series of
interesting experiments, proved to most unbiased minds
their etiological significance. The further confirmation
356

ANTHRAX.

357

of Davaine’s conclusions and actual proof of the matter
rested with Pasteur and Koch, who, observing that the
bacilli bore spores, cultivated them successfully outside
the body, and then produced the disease by the inocula¬
tion of pure cultures.

The anthrax bacilli (Fig. ioi) are large rods with a

Fig. ioi. — Bacillus anthracis: colony three days old upon a gelatin plate ; ad¬
hesive preparation; x 1000 (Frankel and Pfeiffer).

rectangular form, caused by the very slight rounding of
the corners. They measure 5-20 // in length and are
from 1 n to 1.25 fi in breadth. The pronounced tendency
is toward the formation of long threads, in which, how¬
ever, the individuals can generally be made out ; at times
isolated rods occur. In the threads the bacilli seem en¬
larged a little at the ends, and give somewhat the appear¬
ance of a bamboo cane. The formation of spores is pro¬
lific : each spore has a distinct oval shape, is transparent,
and does not alter the contour of the bacillus in which it
occurs. Spores are generally formed in the presence of
oxygen upon the surfaces of the culture-media. When a
spore is placed under favorable conditions for its devel¬
opment and is carefully watched, it may be observed to
increase in length a trifle, then to undergo a rupture at

358

PATHOGENIC BACTERIA .

one end, from which the new bacillus projects. The
spores of anthrax (Fig. 102), being large and easily ob-

and Pfeiffer).

tainable, are excellent subjects for the study of spolia¬
tion, for the action of germicides and antiseptics, and for
demonstration by stains. When dried upon threads of
silk they will retain their vitality for several years, and
are highly resistant to heat and disinfectants.

Spores of anthrax are killed by five minutes’ exposure
to a temperature of ioo° C., and are killed in five minutes
in a 5 per cent, solution of carbolic acid, or, at least, are
deprived of their vegetative property in relation to cul¬
ture-media. It is said by some that spores subjected to
5 per cent, carbolic acid can germinate when introduced
into susceptible animals. Spores are also killed by simple
wetting with 1 : 100,000 bichlorid-of-mercury solution.

The bacilli are not motile and are not provided with
flagella. They stain well with ordinary solutions of the
anilin dyes, and can be beautifully demonstrated in the
tissues by Gram’s method and by Weigert’s fibrin method.
Picro-carmin, followed by Gram’s method, gives a beauti¬
ful, clear picture. The spores can be stained with carbol-

ANTHRAX .

359

fuchsin, the bacilli decolorized with a very weak acid and
then counter-stained with a watery solution of methyl blue.

. Upon the surface of gelatin plate-cultures the bacillus
forms beautiful and highly characteristic colonies (Fig.
103). To the naked eye they appear first as minute

Pfeiffer).

round whitish dots occurring upon the surface, and caus¬
ing liquefaction of the gelatin as they increase in size.
Under the microscope they can be seen in the gelatin as
egg-shaped, slightly brownish granular bodies, not attain¬
ing their full development except upon the surface, where
they spread out into flat, irregular, transparent growths
bearing a partial resemblance to tufts of curled wool.
From a tangled centre large numbers of curls extend,
each made up of parallel threads of bacilli. As soon as
the colony attains any considerable size liquefaction be¬
gins. These colonies make beautiful adhesive prepara¬
tions. If a perfectly clean cover-glass be passed once
through a flame and laid carefully upon the gelatin, the
colonies can generally be picked up entire when the glass is
removed. Such a specimen can be dried, fixed, and stained
in the same manner as an ordinary cover-glass preparation.

360 PATHOGENIC BACTERIA .

In gelatin puncture-cultures the growth is even more
characteristic than are the colonies. The bacilli begin
to grow along the entire track of the wire, most luxuri¬
antly at the surface, where oxygen is plentiful. As the
growth progresses fine filaments like bristles, extend
from the puncture into the neighboring gelatin giving
the growth somewhat the appearance of an evergreen
tree inverted (Fig. 104).

Fig. 104. — Bacillus anthracis : gelatin puncture-culture seven days old
(Gunther).

The more superficial of these threads reach about half¬
way to the sides of the tube, while the deeper ones are
shorter and shorter, until near the apex branches cease.
When the projections are pretty well developed a distinct
surface-growth will be discerned, and if the tube be tilted,
one can observe that the gelatin beneath it has liquefied.
As the growth becomes older the liquefaction increases,
until ultimately the entire gelatin is fluid and the growth
is precipitated.

Upon agar-agar the characteristics are few. The
growth takes place all along the line of inoculation as
a slightly translucent, slightly wrinkled layer with irreg¬
ular edges, from which sufficient bacillary threads pro¬
ject to give it a ciliated appearance to the naked eye.
When the culture is old the agar-agar turns a distinct
brown. Spore-formation is luxuriant upon agar-agar.

ANTHRAX . 361

On potato the growth is white, creamy, sometimes
rather dry in appearance. Sporulation is marked.

Blood-serum cultures lack peculiarities ; the culture-
medium is slowly liquefied.

The bacillus only grows between the extremes of 20°
and 450 C., best at 37° C. The exposure of the organ¬
ism to the temperature of 42-43° C. for twenty-four hours
is sufficient to destroy its virulence.

The culture-media should always be faintly alkaline, as
anthrax bacilli will not grow in the presence of free acid.

The micro-organism under consideration is a parasitic
microbe, yet is one which, because of its spores, can, in
a latent form, exist without the animal organism until
appropriate conditions for its natural development are
presented.

Ordinarily, the infection takes place either through the
respiratory tract or through the alimentary canal .

Buchner has shown that when animals are allowed
to inhale anthrax spores they die of typical anthrax.
The spores establish themselves in the alveoli of the
lung, penetrate the epithelium, enter the vascular sys¬
tem, and soon give rise to typical lesions. Strange to
say, the appearance caused by the inhalation of the
bacilli in their perfect form is entirely different, for a
rapid multiplication occurs without sporulation, and
causes a violent irritative pneumonia with serous or sero¬
fibrinous exudate in which large numbers of the bacilli
occur. In these cases there may be no general infection.

When the bacilli are taken into the stomach in food
they meet with a rapid death because of the acidity of
the gastric juice. Should spores, however, be ingested,
they are able to endure the gastric juice, to pass into the
intestine, and, as soon as proper conditions of alkalinity
are encountered, to develop into bacilli. They develop
rather rapidly, surround the villi with thick networks
of bacillary threads, separate the epithelial cells, enter
the lymphatics, and thus find the appropriate environ¬
ment for the production of a general infection.

362

PATHOGENIC BACTERIA .

Sometimes the bacillus enters the body through a
wound, cut, scratch, or fly-bite. This is especially the
case with men who come in contact with diseased cattle.
As has already been pointed out, a malignant pustule
is apt to follow, and may cause death. Men whose
occupations bring them in contact with skins and hair
from animals dead of anthrax are not only liable to
wound-infection, but are sometimes the subjects of a pul¬
monary form of the disease — <c wool-sorter’s disease” —
caused by inspiration of the spores attached to the wool.

The disease as we see it in the laboratory is accom¬
panied by few but marked lesions. The ordinary method
of inoculation is to cut away a little of the hair from,
the abdomen of a guinea-pig or rabbit or the root of
a mouse’s tail, make a little subcutaneous pocket with
a snip of a pair of sterile scissors, and introduce the
spores or bacilli from a pure culture upon a rather heavy
platinum wire, the end of which is flattened, pointed,,
and perforated. An animal inoculated in this way gen¬
erally dies, according to the species, in from twenty-four
hours to three days. The symptoms are weakness, fever,
loss of appetite, and sometimes a bloody discharge from
nose and bowels. There is much subcutaneous edema.
At the autopsy very little change is observed at the seat
of inoculation. The subcutaneous tissue beneath it for
a considerable distance around is occupied by a peculiar
colorless gelatinous edema which contains the bacilli.
The abdominal cavity shows injection and congestion
of its viscera. The spleen is considerably enlarged, is
dark in color, and of mushy consistence. The liver is
somewhat enlarged. When the thorax is opened, the
lungs may be slightly congested, but otherwise no
changes are to be found.

When the various organs, which present no appreciable
changes to the naked eye, are subjected to a microscopic
examination, the appropriate staining methods bring out
a most remarkable and beautiful change. The capil¬
lary system is almost universally occupied by bacilli,.

ANTHRAX :

363

which extend throughout its meshworks in long threads.
Most beautiful bundles of these bacillary threads can, at
times, be found in the glomeruli of the kidney and in
the minute capillaries of the intestinal villi. In the
larger vessels, where the blood-stream is rapid, the bac¬
teria are relatively few, so that the burden of bacillary
obstruction is borne by the minute vessels. The con¬
dition is thus one of pure septicemia, and bacilli can be
secured in pure cultures from the blood and tissues.

The susceptibility of the anthrax bacillus to the influ¬
ence of heat, cold, antiseptics, etc. not only permitted
Buchner, Behring, and others to produce biological curi¬
osities in the form of bacilli unable to bear spores and
robbed of their pathogenic powers, but also suggested
to Pasteur the important practical measure of protective
vaccination. Pasteur found that the inoculation of non-
virulent bacilli into cows and sheep, and their reinocula¬
tion with slightly virulent bacilli, gave them the ability
to withstand the action of highly virulent organisms.
Loffler, Koch, and Gaffky, however, found that these
immunized animals were not absolutely protected from
intestinal anthrax.

The methods of diminishing the virulence of the
anthrax bacilli are numerous. Toussaint, who was cer¬
tainly the first to produce immunity in animals by inject¬
ing them with sterile cultures of the bacillus, found that
the addition of 1 per cent, of carbolic acid to blood of
animals dead of anthrax destroyed the virulence of the
bacilli ; Chamberland and Roux found it removed when
o. 1-0.2 per cent, of bichromate of potassium was added to
the culture-medium ; Chauveau used atmospheric pressure
to the extent of six to eight atmospheres and found the
virulence diminished ; Arloing found that direct sunlight
operated similarly ; Kubarsch found that the inoculation
of the bacilli into immune animals, such as the frog, and
their subsequent recovery from its blood, diminishes the
virulence markedly.

Protection can be afforded in still other ways. The

364

PATHOGENIC BACTERIA.

simultaneous inoculation of bacteria not at all related to
anthrax will sometimes recover the animal, as Hiippe
found. Hankin found in the cultures chemical sub¬
stances, especially an albuminose, which exerted a pro¬
tective influence. Chamberland has shown that pro¬
tective inoculation by Pasteur’s method has diminished
the death-rate from 10 per cent, for sheep and 5 per
cent, for cattle to about 0.94 per cent, for sheep and 0.34
per cent, for cattle, so that the utility of the method is
scarcely questionable. I11 1890, Ogata and Jasuhara
showed that in the convalescents from anthrax among
their experimental animals an antitoxic substance was
present in the blood in such quantities that 1 : 800 parts
per body-weight of dog’s serum containing the antitoxin
would protect a mouse. Similar results have been at¬
tained by Marchoux.

Experiments of interest have been performed to show
that the natural immunity enjoyed by many animals can
be destroyed. Behring found that if the alkalinity of the
blood of rats was diminished, they could become affected
with anthrax, and numerous observers have shown that
when anthrax bacilli and unrelated organisms, such as
the erysipelas cocci, Bacillus prodigiosus, and Bacillus
pyocyaneus, are simultaneously introduced into immune
animals, the immunity is destroyed and the animals
succumb to the disease. Frogs have been made to suc¬
cumb to the disease by exposure to a temperature of 370
C. after inoculation. Pasteur destroyed the immunity of
fowls by a cold bath after inoculation.

In the natural order of events anthrax in cattle is
probably the result of the inhalation or ingestion of the
spores of the bacilli from the pasture. At one time
much discussion arose concerning the infection of the
pasture. It was argued that, the bacilli being enclosed
in the tissues of the diseased animals, the infection of
the pasture must be due to the distribution of the germs
from the buried cadaver to all parts of the field, either
through the activity of earth-worms, which ate of the

ANTHRAX.

'36S

earth surrounding the corpse and then deposited the
spores in their excrement at remote areas (Pasteur), or to
currents of moisture in the soil. Koch seems, however,
to have demonstrated the fallacy of the theories by show¬
ing that the conditions under which the bacilli find them¬
selves in buried cadavers are exactly opposed to those
favorable to fructification or sporulation, and that in all
probability the majority of bacteria suffer the same fate
as the animal cells, and disintegrate, especially if the ani¬
mal be buried at a depth of two or three meters.

Frankel points out particularly that no infection of the
soil by the dead animal could be worse than the pollution
of its surface by the bloody stools and urine, rich in
bacilli, discharged upon it by the animal before death,
and that in all probability it is the live, and not the dead,
animals that are to be blamed as sources of infection.

As every animal affected with anthrax is a source of
danger to the community in which it lives, to the men
who handle it as well as the animals who browse beside
it, such animals, as soon as the diagnosis is made, should
be killed, and, together with the hair and skin, be burned.
When this is impracticable, Frankel recommends that
they be buried to a depth of at least if^-2 meters, so
that the sporulation of the bacilli is impossible. The
dejecta should also be carefully disinfected with 5 per
cent, carbolic-acid solution.

Of course, animals can be infected through wounds.
This mode of infection is, however, more common
among men, who suffer from the local disease mani¬
fested as the malignant carbuncle, than among animals.

Occasionally bacilli are encountered presenting all the
morphological and cultural characteristics of the anthrax
bacillus, but devoid of any dise'ase-producing power —
Bacillus anthracoides, etc. Exactly what relation they
may bear to the anthrax bacillus is uncertain. They
may be entirely different organisms, or they may be in¬
dividuals whose pathogeny has been lost through unfa¬
vorable environment.

CHAPTER II.

TYPHOID FEVER.

The bacillus of typhoid fever (Fig. 105) was discovered
by Eberth and Koch in 1880, and was first secured in

Fig. 105. — Bacillus typhi, from a twenty-four-hours-old agar-agar culture;

x 650 (Heim).

pure culture from the spleen and affected lymphatic
glands by Gaffky four years later.

The organism is a small, short bacillus about 1-3 n
(2-4 /x Chantemesse, Widal) in length and 0.5-0.8/A broad
(Sternberg). The ends are rounded, and it is rather ex¬
ceptional for the bacilli to be united in chains, though
this arrangement is common in potato cultures. The
size and morphology vary distinctly with the nature of
the culture-medium and the age of the culture. Thoinot
and Masselin in describing these morphological peculi¬
arities mention that when grown in bouillon it is a very
slender bacillus ; in milk it is a large bacillus ; upon
agar-agar and potato it is very thick and short ; and in
old gelatin cultures it forms very long filaments.

366

TYPHOID FEVER.

367

The organisms are actively motile, the motility prob¬
ably being caused by the numerous flagella with which
the bacilli are provided. The flagella stain well by
Loffler’s method, and, as they are numerous (ten to
twenty) and readily demonstrable, the typhoid bacillus is
the favorite subject for their study. The movements of

Fig. 106. — Bacillus typhi, from an agar-agar culture six hours old, showing the
flagella stained by Loffler’s method; x 1000 (FrSnkel and Pfeiffer).

the short bacilli are oscillating, those of the longer indi¬
viduals serpentine.

The organism stains quite well by the ordinary meth¬
ods, but loses the color entirely when stained by Gram’s
method. Its peculiarity of staining is the readiness with
which the bacillus gives up its color in the presence of
solvents, so that it is particularly difficult to stain it in
tissue.

When sections are to be stained the best method is to
allow the tissue to remain in Loffler’s alkaline methylene
blue for from fifteen minutes to twenty-four hours, then
wash in water, dehydrate rapidly in alcohol, clear up in
xylol, and mount in Canada balsam. Ziehl’s method
also gives good results. The sections are stained for fif¬
teen minutes in a solution of distilled water 100, fuch-

363

PATHOGENIC BACTERIA,

sin i, and phenol 5. After staining they are washed in
distilled water containing 1 per cent, of acetic acid,
dehydrated in alcohol, cleared, and mounted. In such
preparations the bacilli, may be found in little groups,
which are easily discovered, under a low power of
the microscope, as reddish specks, and readily resolved
into bacilli with the high power of the oil-immersion
lens.

In bacilli stained by this alkaline methylene-blue solu¬
tion dark-colored dots may sometimes be observed near
the ends of the rods. These dots were at first regarded
as spores, but are now denominated polar granules, and
are thought to be of no importance.

The typhoid bacillus is both saprophytic and parasitic.
It finds abundant conditions in nature for its growth and
development, and, enjoying strong resisting powers, can
accommodate itself to environment much better than the
majority of pathogenic bacteria, and can be found in
water, air, soiled clothing, dust, sewage, milk, etc. con¬
taminated directly or indirectly by the intestinal dis¬
charges of diseased persons.

The bacillus is also occasionally present upon green
vegetables sprinkled with water containing it, and epi¬
demics are reported in which the infection was traced to
oysters, from a certain place where the water was infected
through sewage. Newsholme1 found that in 56 cases
of typhoid fever about one-third was attributable to the
eating of raw shell-fish. In such cases the evidence
accumulated serves to show that the shell-fish were from
sewage-polluted beds. The bacillus probably enters milk
occasionally in water used to dilute it.

The resistant powers of the organisms have already
been described as great. They can grow well at the
room-temperature. The thermal death-point is given by
Sternberg as 6o° C. The bacilli can, according to Klem¬
perer and Levy, remain vital for three months in distilled
water, though in ordinary water the commoner and more

1 Brit . Med.. Jour., Jan., 1895.

TYPHOID FEVER.

3^9

vigorous saprophytes outgrow them and cause their dis¬
appearance in a few days. When buried in the upper
layers of the soil the bacilli retain their vitality for nearly
six months. Robertson 1 found that when planted in
soil and occasionally fed by pouring bouillon upon the
surface, the typhoid bacillus maintained its vitality for
twelve months. He suggests that it may do the same
in connection with leaky drains.

Cold has no effect upon typhoid bacilli, for freezing
and thawing several times are without injury to them.
They have been found to remain alive upon linen for
from sixty to seventy-two days, and upon buckskin for
from eighty to eighty-five days. Sternberg has succeeded
in keeping hermetically sealed bouillon cultures alive for
more than a year. In the experience of the author, un¬
less transplanted rather frequently, cultures upon agar-
agar are apt to die out. In the presence of chemical
agents the bacillus is also able to retain its vitality, o. I
to 6.2 per cent, of carbolic acid added to the culture-
media being without effect upon its growth. At one
time the tolerance to carbolic acid was thought to be
characteristic, but it is now known to be shared by other
bacteria. The bacilli seem to be killed in a short time
by thorough drying.

The bacillus is best secured in pure culture, either
from an enlarged lymphatic gland or from the splenic
pulp of a case of typhoid. To secure the bacillus in this
way the autopsy should be made as soon after death as
possible, lest the Bacillus coli invade the tissue.

Cultures of the typhoid bacillus may be obtained, but
with difficulty, from the alvine discharges of typhoid,
patients. In examining this material, however, it must,
be remembered that the bacilli are certain to be present
only in the second and third weeks.

As numerous saprophytic bacteria are present in the
feces, the resistance which the typhoid bacillus exhibits
to carbolic acid can be made use of in obtaining the pure

1 Brit. Med . Jour. , Jan. 8, 1898.

37°

PATHOGENIC BACTERIA.

culture. To each of several tubes of melted gelatin 0.05
per cent, of carbolic acid is added. This addition is most
easily calculated by supposing the average amount of
gelatin contained in a tube to be 10 c.cm. To the aver¬
age tube -j-tj- c.cm. of a 5 per cent, solution of carbolic acid
is added, and gives very nearly the desired quantity. A
minute portion of the feces is broken up 'with a platinum
loop and stirred in the tube of melted gelatin ; a drop
from this dilution is transferred to the second tube, a
drop from it to a third, and then the contents of each
tube are poured upon a sterile plate or into a Petri dish,

FiG. 107.— Bacillus typhi abdominalis: superficial colony two days old, as
seen upon the surface of a gelatin plate; x 20 (Heim).

or rolled, according to Esmarch’s plan, in the manner
already described. The carbolic acid present in these
cases prevents the great mass of saprophytes from de¬
veloping, but allows the perfect development of the
typhoid bacillus (Fig. 107) and its near congener, the
Bacillus coli communis (Fig. no).

The colonies that develop upon such gelatin plate-
cultures are seen under the microscope to be brownish-
yellow in color, spindle-shaped, and sharply circum¬
scribed. When superficial they are larger and form a
bluish iridescent layer with notched edges. These colo¬
nies are often described as resembling grape-vine leaves.

TYPHOID FEVER.

371

The center of the superficial colonies is the only portion
which shows the yellowish-brown color. The margins
of the colony appear somewhat reticulated. The gelatin
is not liquefied.

Unfortunately, the appearances of the colonies of the
Bacillus typhi and the Bacillus coli communis are iden¬
tical, and make it next to impossible to select a single
colony of either with any certainty. The only solution
of the problem is to transfer a large number of colonies
to some culture-medium in which a characteristic of one
or the other species is manifested, and then study the
growth ; or to grow the colonies upon some special
medium in which differences, such as rapidity of growth
or acid-production, etc. cause the colonies of the differ¬
ent species to assume characteristic appearances.

A method recently suggested by Eisner1 has materially
aided the separation .of these allied bacteria by using a
culture-medium upon which the two bacilli develop dif¬
ferently.

The Eisner medium can be made by allowing 1 kgm.
of grated potatoes (the small red German potato is best)
to macerate in 1 liter of water over night. The juice is
carefully pressed out, and filtered cold to get rid of as
much starch as possible. The filtrate is now boiled and
filtered again. The next step is a neutralization, in
which Eisner used litmus as an indicator, and added 2.5-
3 c.cm. of a ^ normal solution of sodium hydrate to each
10 c.cm. of the juice. Abbott prefers to use phenol-
phthalein as an indicator. The final reaction should be
slightly acid. Ten per cent, of gelatin (no peptone or
sodium chlorid) is now dissolved in the solution, which
is boiled for the purpose, and must then be again neu¬
tralized to the same point as before. After filtration, the
medium receives the addition of 1 per cent of potassium
iodid. It is filled into tubes and sterilized.

When water or feces suspected of containing the ty¬
phoid bacillus are mixed in this medium and poured

1 Zdtschrift fur Hygiene , xxii., Heft I, 1895 ; Dec. 6, 1896.

372

PATHOGENIC BACTERIA .

upon plates, no bacteria develop well except the colon
bacillus and the typhoid bacillus.

These two bacteria, however, differ very markedly in
their appearance upon the medium, for the colon bacillus
appears as usual in twenty-four hours, while at that time,
if present, the typhoid bacillus will have produced no
colonies discoverable by the microscope.

It is only after forty-eight hours, long after the colon
colonies have attained considerable size and are conspic¬
uous, that the little colonies of the typhoid bacillus
appear as small, round, shining, dew-like points, which
are finely granular and in marked contrast to their
coarsely granular predecessors. Unfortunately, many of
the small colonies that develop in Eisner’s medium sub¬
sequently prove to be those of the colon bacillus.

Kashida 1 prefers to make the differential diagnosis by
observing the marked acid production of the Bacillus coli
upon a medium consisting of bouillon containing i ]/2 per
cent, of agar, 2 per cent, of milk-sugar, 1.0 per cent, of
urea, and 30.0 per cent, of tincture of litmus. The cul¬
ture-medium should be blue. When liquefied and inocu¬
lated with the colon bacillus, poured into Petri dishes,
and stood for sixteen to eighteen hours in the incubator,
the blue color passes off and the culture-medium becomes
red. If a glass rod dipped in hydrochloric acid be held
over the dish, vapor of ammonium chlorid is given off.
The typhoid bacillus produces no acid in this medium,
and there is consequently no change in its color.

For the differentiation of the typhoid bacillus from the
allied bacillary forms, Hiss 2 recommends the use of two
special media. The first consists of 5 grams of agar-agar,
80 grams of gelatin, 5 grams of Liebig’s beef-extract, 5
grams of sodium chlorid, and 10 grams of glucose to the
liter. The agar is dissolved in the 1000 c.cm. of water, to
which have been added thebeef-extract and sodium chlorid.
When the agar is completely melted the gelatin is added

1 CentralbL f. Bakt. u. Paristenk., Bd. xxi.,Nos. 20 and 21, June 24, l§97-

2 Journal of Experimental Medicine , Nov., 1897, vol. ii., No. 6.

TYPHOID FEVER .

373

and thoroughly dissolved by a few minutes’ boiling. The
medium is then titrated to determine its reaction, phenol-
phthalein being used as the indicator, and enough HC1 or
NaOH added to bring it to the desired reaction — i. e . a re¬
action indicating i. 5 per cent, of normal acid. To the clear
medium add one or two eggs, well beaten in 25 c.cm. of
water; boil for forty-five minutes, and filter through a
thin filter of absorbent cotton. Add the glucose after
cleaning.

The medium is used in tubes, in which it is planted by
the. ordinary puncture. The typhoid bacillus alone, of
many of the allied forms studied, has the power of cloud -
ing this medium uniformly without showing streaks or
gas-bubbles.

The second medium is used for plating . It contains
10 grams of agar, 25 grams of gelatin, 5 grams of beef-
extract, 5 grams of sodium chlorid, and 10 grams of glu¬
cose. The method of preparation is the same as for the
tube-medium, care always being taken to add the gela¬
tin after the agar is thoroughly melted, so as not to alter
this ingredient by prolonged exposure to high tempera¬
ture. This preparation should never contain less than 2
per cent, of normal acid. Of all the organisms with
which Hiss experimented, the Bacillus typhosus alone
displayed the power of producing thread-forming colonies
upon this medium.

The colonies of the typhoid bacillus when deep in the
medium appear small, generally spherical, with a rough,
irregular outline, and by transmitted light are of a vitreous
greenish or yellowish-green color. The most character¬
istic feature consists of well-defined filamentous out¬
growths, ranging from a single thread to a complete
fringe about the colony. The young colonies are, at
times, composed solely of threads. The fringing threads
generally grow out nearly at right angles to the periphery
of the colony.

The colonies of the colon bacillus are, on the average,
larger than those of the typhoid bacillus; they are spher-

374

PATHOGENIC BACTERIA .

ical or of a whetstone form, and by transmitted light are
darker, more opaque, and less refractive than the typhoid
colonies. By reflected light, to the unaided eye they are
pale yellow. The surface-colonies are large, round, irreg¬
ularly spreading, and are brown or yellowish-brown in
color. Hiss claims that by the use of these reagents the
typhoid bacillus can be readily detected in typhoid stools.

When transferred to gelatin puncture-cultures the ba¬
cilli develop along the entire track of the wTire, with the
formation of minute confluent spherical colonies. A
small thin whitish layer develops upon the surface near
the center. The gelatin is not liquefied, but sometimes
is slightly clouded in the neighborhood of the growth.
The growth upon the surface of obliquely solidified gela¬
tin, agar-agar, or blood-serum is not very luxuriant. It
forms a thin, moist, translucent, non-characteristic band
with smooth edges.

Upon potato a growth formerly regarded as character¬
istic takes placd. When the potato is inoculated and
stood in the incubating-oven, no growth can be detected
at the end of the second day, unless the observer be
skilled and the examination thorough. If, however, the
medium be touched with a platinum wire, it is discovered
that its entire surface is covered with a rather thick, in¬
visible layer of a sticky vegetation which the microscope
shows to be made up of bacilli. No other bacillus gives
the same kind of growth upon potato. Unfortunately, it
is not constant, for occasionally there will be encountered
a typhoid bacillus which will show a distinct yellowish
or brownish color. The typical growth seems to take
place only when the reaction of the potato is acid.

In bouillon the only change produced by the growth of
the bacillus is a diffuse cloudiness.

In milk a slight and slow acidity is produced. The
growth in milk is not accompanied by coagulation.

The chief hindrance to the ready isolation of the
typhoid bacillus is the closely-allied Bacillus coli com¬
munis. This organism, being habitually present in the

TYPHOID FEVER.

375

intestine, exists there in typhoid fever, and adds no little
complication to the bacteriological diagnosis by respond¬
ing in exactly the same manner as the typhoid bacillus
to the action of carbolic acid, by having colonies almost
exactly like those of typhoid, by growing in exactly the
same manner upon gelatin, agar-agar, and blood-serum,
by clouding bouillon in the same way, by being of almost
exactly the same shape and size, by having flagella, by
being motile, and, in fact, by so many pronounced simi¬
larities as almost to warrant the assertion of some that it
and the typhoid bacillus are identical.

Not the least significant fact about the colon bacillus
is that it is also pathogenic and capable of exciting acute
inflammatory processes which are not infrequent, and
which sometimes serve to increase the seriousness of
typhoid fever.

At the present time we are in more or less of a quan¬
dary about this extraordinary resemblance, but base our
differentiation of the species upon certain constant, slight,
but distinct differences.

The typhoid bacillus does not produce indol.

The open lymphatics and vessels of the intestinal ulcers
of typhoid favor the absorption of the bacteria in the diges¬
tive tract, and the colon bacillus enters the blood no
longer to be a saprophyte, but now to be a virulent pus-
producer, and in many cases of typhoid we find suppura¬
tions and other milder inflammations due to this microbe.
This is also a stumbling-block, for the typhoid bacillus
when distributed through the blood may act in exactly
the same manner.

The typhoid bacillus may enter the body, at times,
through dust (Klemperer and Levy), but no doubt, in the
great majority of cases, enters the digestive tract at once
through the mouth. It may possibly enter through the
rectum at times, as illustrated by the mention which
Eichhorst makes of the infection of soldiers in military
barracks through the wearing of drawers previously worn
by comrades who had suffered from typhoid.

376 PA THOGENIC BA CTERIA .

When ingested the resisting power of the bacillus per¬
mits it to pass uninjured through the acid secretions of
the stomach and to enter the intestine, where the chief
local disturbances are set up.

The bacilli enter the solitary glands and Peyer’s patches,
and multiply slowly during the one to three weeks of the
incubation of the disease. The immediate result of their
residence in these lymphatic structures is increase in the
number of cells, and ultimately the necrosis and slough-

Fig. 108. — Intestinal perforation in typhoid fever. Observe the threads of
tissue obstructing the opening. (Museum of the Pennsylvania Hospital.)
(Keen, Surgical Complications and Sequels of Typhoid Fever.)

ing which cause the typical post-mortem lesion (Fig. 108).
From the intestinal lymphatics the bacilli pass, in all
probability, to the mesenteric glands, which become en¬
larged and softened, and finally extend to the spleen and
liver, and sometimes to the kidneys. The growth of the
bacilli in the kidneys causes the albuminuria of the dis¬
ease. Sometimes under these conditions the bacilli may

TYPHOID FEVER.

377

be found in the urine. P. Horton Smith 1 found the ba¬
cilli in the urine in three out of seven cases which he
investigated. They did not occur before the third week,
and remained in one case twenty-two days after cessation
of the fever. Sometimes they were present in immense
numbers. Their occurrence, no doubt, depends upon
their growth in the kidney and descent with the urine.
It is of importance from a sanitary point of view to
remember that the urine as well as the feces is infec¬
tious. Occasionally the bacilli succeed in entering the
general circulation, and, finding a lodgement at some
remote part of the body, set up local inflammatory pro¬
cesses sometimes terminating in suppuration.

Weichselbaum has seen general peritonitis from rup¬
ture of the spleen in typhoid fever with escape of the
bacilli. Ostitis, periostitis, and osteomyelitis are very
common results of the lodgement of the bacilli in bony
tissue, and Ohlmacher has found the bacilli in suppura¬
tions of the membranes of the brain. The bacilli are
also encountered in other local suppurations occurring
in or following typhoid fever. Flexner and Harris2 have
seen a case in which the distribution of the bacilli was
sufficiently widespread to constitute a real septicemia,
the bacillus being isolated from various organs of the
body, and shown to be the true bacillus of Eberth by
all the specific laboratory tests, but in which there were
no intestinal lesions.

The bacilli can be found in the intestinal lesions, in
the mesenteric glands, in the spleen, in the liver, in the
kidneys, and in any local lesions which may be present.
Their scattered distribution and their occurrence in
minute clumps have already been alluded to. They
should always be sought for at first with a low power
of the microscope.

Ordinarily no bacilli can be found in the blood, but
it has been shown that the blood in the roseolse some-

1 Brit. Med. Jour Feb. 13, 1897-

2 Bull, of the Johns Hopkins Hospital Dec., 1897.

37§

PATHOGENIC BACTERIA .

times contains them, so that the eruption may be regarded
as one of the local irritative manifestations of the bacillus.

The amount of local disturbance, in proportion to the
constitutional disturbance, is, in the majority of cases,
slight, and almost always partakes of a necrotic charac¬
ter, which suggests that in typhoid we have to do with a
toxic bacterium whose disease-producing capacity resides
in the elaboration of a toxic substance. This, indeed,
is true, for Brieger and Frankel have separated from
bouillon cultures a toxalbumin which they thought to be
the specific poison. Klemperer and Levy also point out
further clinical proof in certain exceptional cases dying
with the typical picture of typhoid, yet without char¬
acteristic post-mortem lesions, the only confirmation of
the diagnosis being the discovery of the bacilli in the
spleen.

Pfeiffer and Kolle found that the toxic substance resided
only in the bodies of the bacilli, and could not, like the
toxins of diphtheria and tetanus, be dissolved in the cul¬
ture-medium. This was an obstacle to their immuniza¬
tion-experiments as well as those of Loffler and Abel,
later to be described, for the only method of immuniz¬
ing animals to large quantities of the bacilli was to make
massive agar-agar cultures, scrape the bacilli from the
surface, and distribute them through nutrient bouillon.

When injected into guinea-pigs the typhotoxin of
Brieger is productive of increased secretion of saliva, in¬
creased rapidity of respiration, diarrhea, and mydriasis,
and usually causes a fatal termination in from twenty-
four to forty-eight hours.

As the discovery of the bacilli in the spleen, and espe¬
cially the securing of a pure culture of the bacilli from
the spleen, are sometimes attended with considerable dif¬
ficulty because of the dissemination of the colonies
throughout the organ, E. Frankel recommends that as
soon as the organ is removed from the body it be wrapped
in cloths wet with a solution of bichlorid of mercury and
kept for three days in a warm room, in order that a con-

TYPHOID FEVER . 379

siderable and massive development of the bacilli may
take place.

Typhoid fever is a disease which is communicable to
animals with difficulty. They are not affected by bacilli
in fecal matter or in pure culture mixed with the food,
and are not diseased by the injection into them of blood
from typhoid patients. Gaffky failed completely to pro¬
duce any symptoms suggestive of typhoid fever in rab¬
bits, guinea-pigs, white rats, mice, pigeons, chickens,
and calves, and found that Java apes could feed daily
upon food polluted with typhoid germs for a considerable
time, yet without symptoms. The introduction of pure
cultures into the abdominal cavity of most animals is
without effect. Frankel and Simon found that when
pure cultures were injected into mice, rabbits, and guinea-
pigs the animals died.

Germano and Maurea found that mice succumbed in
from one to three days after intraperitoneal injection of
1-2 c. cm. of a twenty-four-hour-old bouillon culture. Sub¬
cutaneous injections in rabbits and dogs caused abscesses.

Losener found the introduction of 3 mgr. of an agar-
agar culture into the abdominal cavity of guinea-pigs to
be fatal.

When animals are treated in the manner described in
the chapter upon Cholera — 2. e. the gastric contents ren¬
dered alkaline, a large quantity of laudanum injected
into the peritoneal cavity, and the bacilli introduced
through an esophageal catheter — Klemperer, Levy, and
others found that there was produced an intestinal con¬
dition which very much resembled typhoid as it occurs in
man. The virulence of the bacillus can be very greatly
increased by rapid passage from guinea-pig to guinea-pig.

In the experiments of Chantemesse and Widal the
symptoms following the injection of virulent culture into
guinea-pigs were briefly as follows: “Very shortly after
the inoculation there is a rise of temperature, which
continues from one to four hours, and is succeeded by a
depression of the temperature, which continues to the

PATHOGENIC BACTERIA .

38°

fatal issue. Meteorism and great tenderness of the abdo¬
men are observed. At the autopsy a sero-fibrinous or
sero-purulent peritonitis is observed — sometimes hemor¬
rhagic. There is also generally a pleurisy, either serous
or hemorrhagic. All the abdominal viscera are con¬
gested. The intestine is congested — contains an abun¬
dant mucous secretion. The Peyer patches are enlarged.
The spleen is enlarged, blackish, and often hemorrhagic.
In cases which are prolonged the liver is discolored. The
kidneys are congested, the adrenals filled with blood.

u In such cases the bacillus can be found upon the in¬
flamed serous membranes, in the inflammatory exudates,
in the spleen in large numbers, in the adrenals, the liver,
the kidneys, and sometimes in the lungs. The blood is
also infected, but to a rather less degree.

‘ c In cases described as chronic, the bacillus disappears
completely in from five to twenty-four hours, and pro¬
duces but one lesion, a small abscess at the point of inoc¬
ulation.

“Sanarelli has observed that if some of the poisonous
products of the colon bacillus or the Proteus vulgaris be
injected into the abdominal cavity of an animal recover¬
ing from a chronic case, it speedily succumbs to typical
typhoid fever.”

Petruschky 1 found that mice that recovered from .sub¬
cutaneous injections of typhoid cultures frequently suf¬
fered from a more or less widespread necrosis of the skin
at the point of injection.

I experienced great difficulty in immunizing a horse to
the disease, because every injection of virulent living
organisms was followed by a necrosis equalling in size the
distended area of subcutaneous tissue.

Targe quantities of filtered cultures produce symp¬
toms similar to those resulting from inoculation with
the bacilli. The toxic product of the bacilli is, how¬
ever, practically insoluble, and, according to the ex¬
periments of Toffler and Abel and those of Pfeiffer and

1 Zeitschrift fur Hygiene , Bd. xii., 1892, p. 261.

TYPHOID FEVER. 381

Kolle, cannot be separated from the bodies of the bacilli
producing it.

Animals can easily be immunized to this bacillus, and
then, according to Chantemesse and Widal, develop in
their blood an antitoxic substance capable of protecting
other animals. Stern 1 has also found that in the blood
of human convalescents a substance exists which has a
protective effect upon guinea-pigs. His observation is in
accordance with a previous one by Chantemesse and
Widal, and has recently been abundantly confirmed.

The immunization of dogs and goats by the introduc¬
tion of increasing doses of virulent cultures has been
achieved by Pfeiffer and Kolle2 and by Loffler and Abel.3
From these animals serums were secured not exactly an¬
titoxic, but anti-infectious or auti-microbic in operation,
and possessed of marked specific germicidal action upon
the typhoid bacilli when simultaneously introduced into
the peritoneal cavity of guinea-pigs.

The action of the typhoid serum is specific, and exerts
exactly the same action upon the typhoid bacilli as the
cholera serum exerts upon the cholera spirilla, killing
and dissolving them (Pfeiffer’s phenomenon).

So far, no serum has been produced that is efficacious
in human medicine.

The specific reaction of the serum can be used to dif¬
ferentiate cultures of the colon and typhoid bacilli, the
typhoid bacilli alone exhibiting the specific effect of the
typhoid serum.

Christophers 4 found that the serum from typhoid
patients occasionally caused agglutinations in cultures
of the colon bacillus, but concludes that this does not
lessen the specificity of the reaction, as there may be
two combined specific actions of these serums. Experi¬
ments on rabbits established that typhoid and colon
serums could be produced, each specific in its agglutin-

1 Zeitschrift fur Hygiene , xvi., 1894, p. 458. 2 Eid-> 1896.

3 Centralblf Bakt. u. Parasitenk Bd. xix.,No. 23, p. 51, Jan. 23, 1896.

4 Brit. Med. Jour .} Jan. 8, 1898.

382 PATHOGENIC BACTERIA.

ating power upon bouillon cultures of its respective
organism.

Loffler and Abel also prepared a colon serum which
exerted a like specific action upon the colon bacillus,
but was without effect upon the typhoid bacillus.

The serum of immunized animals has been found to
destroy the motility of the typhoid bacilli in a few mo¬
ments, and to cause them to group together. Widal
found that the serum of convalescents and of individuals
suffering from the acute disease possessed the same power,
and suggested that this specific action might prove a val¬
uable adjunct in diagnosis.

Wyatt Johnston1 and McTaggert worked upon the
subject, and found that a drop of blood from a typhoid
patient, dried upon paper and kept for some time, when
moistened and brought in contact with a culture of the
bacilli was still potent to bring about a characteristic
effect. When such a preparation in the 4 4 hanging drop ’ 5
is watched under the microscope the typhoid bacilli are
found to be paralyzed in from one minute to half an
hour, and subsequently to collect in masses — agglutina¬
tions. This reaction may occasionally be brought about
by normal blood if insufficiently diluted, but is charac¬
teristic enough to be very useful in the diagnosis of ob¬
scure cases. In a later paper Johnston states that to ob¬
tain a satisfactory reaction an attenuated typhoid bacillus
is more useful than a highly virulent one.

My own experiments have satisfied me of the value of
the test, both for making a diagnosis of the disease and
for confirming the species of the bacillus in doubtful
cases.

It is now the opinion of all observers that cessation of
motion and agglutination of the bacteria, resulting from
the contact of typhoid bacilli and typhoid serum, are
inconclusive for diagnostic purposes unless the reaction
follows the combination of a suitable culture and a
definite quantity of serum.

1 Montreal Med. Journal March, 1897.

TYPHOID FEVER.

383

The thorough investigations of Wyatt Johnston and
his associates in Montreal have shown that reliable reac¬
tions can only be secured when the cultures employed are
of an ordinarily virulent typhoid bacillus, and are grown
in an alkaline medium for about twenty-four hours.

I prefer fresh agar-agar cultures, distributed throughout
sterile clean water, rather than bouillon cultures, because
of the larger number of bacteria in the former, the con¬
sequently greater number of agglutinations formed, and
the readiness with which they are found upon micro¬
scopic examination. It is necessary, however, to make
a microscopic examination of the diluted culture before
adding the serum or blood, in order to be sure that there
are no natural clumps of bacteria present to simulate the
specific agglutinations. This is of great importance.
The natural clumps of bacilli are more apt to occur in
cultures grown upon fresh, moist agar-agar than upon
that kept for a short time until the surface has become
partially dried. The chief difficulty experienced in
making the test seems, at present, to reside in the prepa¬
ration of the blood in accurate dilution — i. e . securing
it in measured amounts.

The original method of Widal, to collect about 5 c.cm.
of blood in a test-tube by the introduction of a hypo¬
dermic needle into a vein, is a rather more serious and
disturbing operation than most patients care to undergo
for purposes of diagnosis.

Blood dried upon paper, as suggested by Johnston, or
upon glass, while extremely convenient for transporta¬
tion, is not susceptible of accurate dilution for quantita¬
tive estimation.

Cabot has successfully made dilutions with a medicine-
dropper, by using one drop of blood and as many drops
of culture, dropped from the same instrument, as were
necessary for the desired dilution. This method seems
to be very practical, but can only be employed at the
bedside, or where it is not necessary to keep or trans¬
port the blood.

384

PATHOGENIC BACTERIA.

In the absence of a satisfactory method of securing
definite small quantities of blood for immediate or sub¬
sequent use, I was led to make some experiments with
capillary tubes to determine their possible value for the
purpose.

It is a well-known physical phenomenon that in clean
capillary tubes fluids are attracted to a height varying
according to the diameter of the tube and the density of
the fluid. In tubes of equal diameter the height of the
column is invariably the same.

Such tubes can be made by heating a piece of ordinary
glass tubing, such as is to be found in every laboratory,
in a Bunsen flame for a few minutes until it becomes red
and soft, removing the glass from the flame, and then
pulling upon the ends steadily and slowly until the tube
is drawn out to the desired diameter. The errors to be
avoided in making the tubes will be — heating too much
and making the glass too soft, drawing out the tube
while still in the flame, and drawing too rapidly. The
result of these erroneous methods will be that the tubes
are much shorter and finer than is desired. A few mo¬
ments' practice will show just how the manipulation
should be done to secure the best results.

The fact was, however, established that tubes of about
the same diameter showed almost no variation in the
quantity of liquid contained. So little was the difference
in the length of the column and the weight of the con¬
tained blood in tubes recognized by the eye to have uni¬
form caliber that I have no hesitation in recommend¬
ing an application of the capillary tube for securing
small measured quantities of blood for the specific typhoid
tests and similar experiments.

The application of the method is simple and consists in:

1. Accurately weighing the amount of blood that
enters a capillary tube of a size arbitrarily selected as a
standard.

2. The manufacture of a large number of tubes of the

same size.

TYPHOID FEVER.

385

3. The dilution of the known quantity of blood con¬
tained in the tube with a measured quantity of the
bouillon, or diluted agar-agar, culture of the bacillus.

The standard tube that I adopted had a diameter
about equal to the E string of a violin. A larger or
smaller tube would have done quite as well. In such a
tube the column of blood rises about an inch and weighs
about 0.018 gram. As personal equation in judging
size is a marked source of error, the experimenter must
work out his own standard tube and not adopt that which
has just been given. It is important to know the length
of the column that has a certain weight, because, as each
tube is not separately measured and graduated, the two
chief means of avoiding error will be (1) to have the
tubes as nearly as possible of equal diameter, and (2) to
prove them to be so by observing that the columns of
fluid they contain when used are of the same length, re¬
jecting one after another all the tubes which seem to
the eye to have the proper caliber, but in which the
column is obviously longer or shorter than that of the
original tube.

Keeping the standard tube before him as a guide, and
using a Bunsen flame — which is better than a blowpipe,
because it does not heat the glass so rapidly and make it
so soft — the experimenter prepares one hundred or more
capillary tubes as nearly as possible of the same size as
that of the standardized tube. All the irregular sizes
are rejected, and the suitable sizes cut into portions
about three inches long. These pieces, which should
number several hundred (it is economy to make a large
number at a time), are now carefully sorted, being com¬
pared with the standard tube at both ends, and thrown
away if too large or too $mall at either. It is best to
sort the tubes twice on different days, or have several
different persons go over them all. Of course, some
tubes of quite different caliber will, in spite of all pre¬
cautions, remain in the bundle, but this is no serious
matter, because at the last moment the height of the

386 PA THOGENIC BA CTERIA .

column to which the blood rises can be taken as a proof
of actual variation. It may be true that no two of the
tubes have exactly — absolutely— the same contents, but
when the given precautions are taken the variation will
be so small as to make no significant error in the results
obtained.

The use of the tubes is extremely simple. The ordi¬
nary puncture is made in the lobule of the ear or the fin¬
ger-tip of the patient, and one end of one of the tubes
touched to the surface of the oozing drop and held there
until the blood ceases to rise in the tube. So little blood
is required that a number of tubes may be filled with the
blood from a single puncture if desired. The blood in
the tube coagulates in a few minutes, and can be allowed
to dry, or be drawn to the central portion of the tube and
sealed in by fusing the ends in a flame if it be desired to
keep it moist.

When the agglutination reaction is to be made the
blood should not be blown out of the tube, as the total
quantity contained is small and a large relative quantity
will remain in the tube. A better method is to crush the
lube in a small crucible or other diminutive vessel and
dissolve its contents directly in the culture.

The proper proportionate amount of culture is meas¬
ured with a finely graduated pipette (graduated to thous¬
andths of a cubic centimeter), the calculation according
to the standard tube of the writer’s experiments being:
dilution i : io *= 0.153 c.cm. of the culture; dilution
1 : 100 = 1.53 c.cm. of the culture; dilution 1 : 1000 =
15.3 c.cm. of the culture.

The now recognized specific reaction is supposed to
take place in dilutions of 1 : 50, which would require
0.71 4- c.cm. of the bouillon or diluted agar culture.

The culture is measured into the little crucible, the
blood-containing portion of the capillary tube broken off,
dropped in, and subsequently crushed to minute frag¬
ments and stirred about with a clean, rounded, glass rod,
and a drop of the mixture placed as a u hanging drop”

TYPHOID FEVER. 387

upon the stage of a microscope and examined for the
agglutinations.

As recent extended observations have shown that occa¬
sionally the blood of healthy men and animals has the
power of producing the agglutinations, the consensus of
opinion now seems to be in favor of the view that a cer¬
tain dilution of the blood is required for a satisfactory
diagnosis, and that all reactions with concentrations
greater than one part of blood in fifty of culture may be
questionable, while less concentrated dilutions are almost
positively diagnostic. A time-limit must be placed upon
the experiment. For the weak dilution not mpre than
two hours should be required for a perfect reaction, and
for the stronger solution correspondingly less time should
be required.

A curious fact that should not be overlooked is that the
agglutinating substance is not constantly present in the
blood, but sometimes alternates, being present for several
days and then absent for a day or two.

The agglutinating power of the blood occurs early in
the course of typhoid, and in typical cases seems to be
present in the first week of actual illness.

A point that should not be forgotten is that the agglu¬
tination of the bacilli seems to be a phenomenon quite in¬
dependent of any immunity possessed by the individual,
and therefore is not an u immunity-reaction. ” Just what
the agglutinating substance is, has not yet been determined.

The agglutinations are occasionally caused by the
serum and dried blood from other diseases than typhoid,
but in a collection of 4000 cases it was shown that the
errors from this source were only about 5 per cent.

Malvoz 1 has experimented with a number of chemicals,
and has found that formaldehyd, corrosive sublimate, per-
oxid of hydrogen, strong alcohol, and anilin colors (such
as chrysoidin, vesuvin, and safranin) have the power to
produce the typical agglutinations even in very dilute
solutions.

1 Ann. de V Inst. Pasteur , xl., 7, 1897.

388

PATHOGENIC BACTERIA .

Wright and Semple assert that dead cultures of the
typhoid bacillus may be used for the test, as bacilli killed
by a temperature of 6o° C. agglutinate perfectly. They
have the advantage of being easily kept.

Rumpf,1 and Kraus and Buswell2 report a number of
cases of typhoid which were favorably influenced by the
introduction hypodermically of small quantities of steril¬
ized cultures of Bacillus pyocyaneus. These experiments
are still too new to deserve extended mention.

Following the lines of experimentation suggested by
Haffkine’s researches upon preventive vaccination against
cholera Asiatica, Pfeiffer and Kolle, and Wright and Sem¬
ple have used the subcutaneous injection of sterilized cul¬
tures as a prophylactic measure. One c.cm. of a bouillon
culture sterilized, by heat is thought to be sufficient.
Wright and Semple report 18 cases in which it was used,
and by experiment showed the blood to be changed simi¬
larly to that of typhoid patients and convalescents. This
change consisted in the destruction of motility and agglu¬
tination of the bacilli, as seen in Widal’s reaction. It is
hoped that we can gauge the duration of the immunity
thus acquired by the frequent use of Widal’s test.

One of the most important and practical points for the
physician to grasp in relation to the subject of typhoid
fever is the highly virulent character of the discharges
from the bowels. In every case the greatest care should
be taken for a proper disinfection of the feces, a rigid
attention to all the details of cleanliness in the sick¬
room, and the careful sterilization of all articles which
are soiled by the patient. If country practitioners were
as careful in this particular as they should be, the disease
would be much less frequent in regions remote from the
filth and squalor of large cities with their unmanageable
slums, and the distribution of the bacilli to villages and
towns, by watercourses polluted in their infancy, might
be checked.

1 Deutsche vied . Wochenschrift , 1893, No. 41.

2 Wien. klin. Wochenschrift , July 12, 1894.

CHAPTER III.

BACILLUS COLI COMMUNIS.

The Bacillus coli was first isolated from human feces
in 1885 by Emmerich, who thought that it was the spe¬
cific cause of Asiatic cholera. Many investigators have
since studied its peculiarities, until at the present time
it is one of the best-known bacteria.

It is habitually present in the fecal matter of most ani¬
mals except the horse, and ill water and soil contaminated

Fig. 109. — Bacillus coli communis, from an agar-agar culture; x 1000 (Itzerott

and Niemann).

with it. With water or dust it gains entrance into the
mouth, where it can frequently be found, and occurs
accidentally in foods and drinks. During life the organ¬
ism sometimes enters wounds externally from the surface
of the body or internally from the intestine, and is a
cause of suppuration — or at least occurs in the pus. The
Bacillus pyogenes foetidus of Passet is almost certainly
identical with it.

389

390

PATHOGENIC BACTERIA.

The bacillus is rather variable culturally, and is some¬
what polymorphic. Probably both size and form depend
to a certain extent upon the culture-medium on which
it grows. On the average, it measures 1-3 X 0.4-0. 7 ji.
It usually occurs in the form of short rods, but very
short coccus-like elements and quite elongate forms are
often found in the same culture. The individual bacilli
are frequently isolated or in pairs. Chains are the ex¬
ception. They are provided with flagella, which are
very variable in number, generally from four to a dozen,
though there may be more. It forms no spores.

The bacillus stains well with the ordinary aqueous
solutions of the anilin dyes, but does not retain the stain
after immersion in Gram’s solution.

The bacillus is motile, though in this particular it is
subject to irregularity, the organisms from some cultures

Fig. no. — Bacillus coli communis: superficial colony two days old upon a
gelatin plate; x 21 (Heim).

always swimming actively, even when the culture is
some days old, others being exceedingly sluggish even
when young and actively growing, and a few cultures
seem to consist of bacilli that do not move at all. Fresh
cultures which, when grown at incubation temperature,
consist of entirely non-motile bacteria are probably Bacil¬
lus coli immobilis , not Bacillus coli communis.

The bacillus is readily cultivated upon the ordinary

BACILLUS CO LI COMMUNIS.

391

media. Upon gelatin plates the colonies develop in
twenty-four hours. Those situated below the surface
appear round, yellow-brown, and homogeneous. As they
grow older they increase in size and become opaque. The
superficial colonies are larger and spread out upon the
surface. Their edges are dentate and resemble grape-
leaves, often showing radiating ridges suggestive of the
veins of a leaf. They may have a slightly concentric
appearance. The colonies rapidly increase in size and
become more and more opaque. The gelatin is not
liquefied.

In gelatin punctures the culture, developing rapidly
upon the surface, and also in the needle’s track, causes
the formation of a nail-like growth. The head of the
nail may reach the walls of the test-tube. Not infre¬
quently gas is formed in ordinary gelatin, and when 1
per cent, of glucose is dissolved in the medium the gas-
production is often so copious and rapid as to form large
bubbles, which by their distention subsequently break it
up into irregular pieces. Sometimes the gelatin becomes
slightly clouded as the bacilli grow.

Upon agar-agar along the line of the inoculation a
grayish-white, translucent, smeary growth takes place.
It is devoid of any characteristics. The entire surface
of the culture-medium is never covered, the growth re¬
maining confined to the inoculation-line, except where
the moisture of the condensation-fluid allows it to spread
out at the bottom. Kruse says that in old cultures crys¬
tals may form. I have never seen them.

Bouillon is soon evenly clouded by the development
of the bacteria. Sometimes a delicate pellicle forms upon
the surface. There is rarely much sediment in the cul¬
ture.

Wiirtz found that the bacillus produced ammonia in
culture-media free from sugar, and thus caused an intense
alkaline reaction in the culture-media. The cultures
usually give off an odor that varies somewhat, but is, as
a rule, unpleasant.

392

PATHOGENIC BACTERIA.

Indol is formed in both bouillon and pepton solu¬
tions. Phenol is not produced. Litmus added to the
culture-media is ultimately decolorized by the bacilli.

The presence of indol is probably best determined by
Salkowski’s method. To the culture i c.cm. of a 0.02
per cent, aqueous solution of potassium nitrate and a
few drops of concentrated sulphuric acid are added. If
a rose color develops, indol is present.

Nitrates are reduced to nitrites by the growth of the
bacillus.

Upon potato the growth is luxuriant. The bacillus
forms a yellowish-brown, glistening layer spreading from
the line of inoculation over about one-half to two-thirds
of the potato. The color shown by the potato-cultures
varies considerably, sometimes being very pale, some¬
times quite brown. It cannot, therefore, be taken as a
characteristic of much importance. Sometimes the po¬
tato becomes greenish in color. Sometimes the growth
on potato is almost invisible.

In milk there are rapid coagulation and acidulation,
with the evolution of much gas.

The bacillus seems to require very little nutriment. It
grows in Uschinsky’s asparagin solution, and is frequently
found living in river and well waters.

It is quite resistant to antiseptics and germicides, and
grows in culture-media containing from o. 1-0.2 per cent,
of carbolic acid. It lives for months upon artificial
media.

The bacillus begins to penetrate the intestinal tissues
almost immediately after death, and is the most frequent
contaminating micro-organism met with in cultures made
at autopsy. Kxactly how it penetrates the tissues is not
known. It may spread by direct continuity of tissue, or
via the blood-vessels.

While under normal conditions a saprophytic bacte¬
rium, the colon bacillus is far from harmless. It not
infrequently is found in the pus of abscesses remote from
the intestine, and is almost always found in suppura-

BACILLUS CO LI COMMUNIS .

393

tions connected with the intestines, as, for example,
appendicitis.

It is a question whether the colon bacillus is always
virulent, or whether it becomes virulent under abnormal
conditions. Klencki 1 found that it was very virulent in
the ileum, and less so in the colon and jejunum, espe¬
cially in dogs. He also found that the virulence was
greatly increased in a strangulated portion of intestine.
Other observers, as Dreyfuss, found that the colon bacil¬
lus as it occurs in normal feces is non-pathogenic. Most
experimenters, however, believe that pathological con¬
ditions, such as disease of the intestine, ligation of the
intestine, etc., cause increased virulence.

Adelaide Ward Peckliatn, in an elaborate study of the
“ Influence of Environment on the Colon Bacillus,”2 con¬
cludes that while the conditions of nutrition and develop¬
ment in the intestine seem to be most favorable, the colon
bacillus is ordinarily not virulent, because uits first force
is spent upon the process of fermentation, and as long as
opportunities exist for the exercise of this function the
affinities of this organism appear to be strongest in this
direction.

“ Moreover, the contents of the intestine remain acid
until they reach the neighborhood of the colon, and by
that time the tryptic peptons have been formed and
absorbed to a great extent.

“ During the process of inflammation in the digestive
tract a very different condition may exist. The peptic and
tryptic enzymes may be partially suppressed. Fermenta¬
tion of carbohydrates and proteid foods then begins in
the stomach, and continues after the mass of food is
passed on into the intestine. The colon bacillus cannot,
therefore, spend its force upon fermentation of sugars,
because they are already broken up and an alkaline fer¬
mentation of the proteids is in progress. It also cannot
form peptons from the original proteids, for it does not

1 Ann. de V Inst, Pasteur, 1895, No. 9.

2 Journal of Experimental Medicine , Sept., 1897, vol. ii., No. 4, p. 549-

394

PATHOGENIC BACTERIA .

possess this property, and unless trypsin is present it must
be dependent upon the proteolytic activity of other bac¬
teria for a suitable form of proteid food. Perhaps these
bacteria form an albuminate molecule, which like leucin
and tyrosin cannot be broken up into indol, and thus
there might be caused an important modification of the
metabolism of the colon bacillus, which might have
either an immediate or remote influence upon its acquisi¬
tion of disease-producing properties, for our own experi¬
ments indicate that the power to form indol, and the
actual forming of it, are to some extent an indication of
the possession of pathogenesis.”

To the laboratory animals the colon bacillus is patho¬
genic in varying degree. Intraperitoneal injections into*
mice cause their death in from one to eight days if the
culture is virulent. Guinea-pigs and rabbits also suc¬
cumb to intraperitoneal and intravenous injection. Sub¬
cutaneous injections are of less effect, and in rabbits seem
to produce abscesses only.

When the bacilli are injected into the abdominal cavity
a sero-fibrinous or purulent peritonitis occurs, the bacilli
being very numerous in the abdominal fluids.

The pathogeny of the colon bacillus is due to irritating,
cheniotactic substances in its protoplasm. The experi¬
ments of Pfeiffer and Kolle and Loffler and Abel have
proved very conclusively that the poisonous principle is
in, and cannot by any means be separated from the bodies
of the bacteria.

Frequent transplantation lessens the virulence, passage
through animals increases it.

Numerous observers have found that cultures of the
bacillus obtained from cholera, cholera nostras, and other
intestinal diseases are much more pathogenic than those
obtained from normal feces or from pus.

Cumston,1 from a careful study of thirteen cases of sum¬
mer infantile diarrheas, comes to the following conclu¬
sions:

1 International Medical Magazine, Feb., 1897.

BACILLUS CO LI COMMUNIS. 395

The bacterium cob seems to be the path ogenk . age, at
of the water number of summer infantile diarrheas.
Tteorganism is the more often associated with the

^T^ie'virulenc^mom "considerable than in the intestine
of a healthy child, is almost always in direct mlatmm o

^ mobhityTthe Bacterium coli^in genera^ro-
portional to its virulen . exalted virulence

nevertheless, does ^,h the°caseTin which the mobility was
"ery° TnSrable, without presenting these jumping

““Silence of the Bacterium coli found b_ the

rrxtrx.*

11 of-lipr cases From this uniformity ol action ^

the colon bacilli m

cases are all of the same species.

The agglutinating reaction occurs only m the Y

stao-es and acute forms of the disease. _

Tt is not difficult to immunize an animal against th

CO „ baSltf LbS« -

progressively incmased — ^ 'Sough

“ injections “first produced hard swellings.

SfblooSf .^immunised animals poised an active
bactericidal influence upon the colon bacteria. It was
nnf in the correct sense antitoxic.

In intestinal diseases, such as typhoid, cholera, an
1 Semaine Medicale , Oct. 20, 1897.

396

PATHOGENIC BACTERIA .

dysenteiy, the bacillus not only seems to acquire an un¬
usual degree of virulence, but because of the existing
denudation of mucous surfaces, etc., finds it easy to enter
general system, with the result of secondary remote
suppurative lesions in which it is the essential factor.
When absorbed from the intestine it frequently enters
the kidney and is excreted with the urine, causing, inci¬
dentally, local inflammatory areas in the kidney, and
occasionally cystitis. A case of urethritis is reported to
have been caused by it.

In infants cholera infantum may not infrequently be
caused by the colon bacillus, though probably in this
disease other bacteria play a very important role.

The bile-ducts are very often invaded by the bacillus,
which may cause inflammation, obstruction, suppuration,
or calculous formation.

The bacillus has also been met in puerperal fever,
Wiuckel’s disease of the new-born, endocarditis, menin¬
gitis, liver-abscess, bronchopneumonia, pleuritis, chronic
tonsillitis, and urethritis.

For the determination of the colon bacillus the im¬
portant points are the motility, the indol reaction, the
milk-coagulation, and the active gas-production. As,
however, all of these features are shared by other bac¬
teria to a greater or less degree, the only positive differ¬
ential point upon which very great reliance can be placed
is the immunity-reaction of the serum of an immunized
animal, which not only protects susceptible animals from
the effects of inoculation, but produces with fresh cul¬
tures of the bacillus exactly the same reaction as that
observed in connection with the blood and serum of
typhoid patients, and convalescents and immunized ani¬
mals. This reaction has been considered at length in
speaking of typhoid fever.

For the few who are convinced that the colon and
typhoid bacilli are identical, the fact that the typhoid
serum is specific for the typhoid bacillus, and the colon
serum for the colon bacillus, with rare exceptions.

BACILLUS CO LI COMMUNIS. 397

should be important evidence of their separate individ¬
uality.

The author has no doubt that the Bacillus coli com¬
munis is not a single species of bacteria, but is a name
applied to a group whose individual differences are thus
far too similar to enable us to differentiate them. This
opinion seems to be shared by other bacteriologists, some
of whom have attempted to separate the bacillus into
groups, types, or families.

In order to establish a type species of the Bacillus coli
communis, Smith 1 says:

1 4 1 would suggest that those forms be regarded as true
to this species which grow on gelatin in the form of deli¬
cate, bluish, or more opaque, whitish expansions with
irregular margin, which are actively motile when exam¬
ined in the hanging drop from young surface-colonies
taken from gelatin plates which coagulate milk within
a few days; grow upon potato, either as a rich-pale or
brownish-yellow deposit, or merely as a glistening, barely
recognizable layer, and which give a distinct indol reac¬
tion. Their behavior in the fermentation-tube must
conform to the following scheme:

41 Variety a:

44 One per cent dextrose-bouillon (at 370 C.). Total
gas approximately ]/o ; HC02 approximately ~/i ; reaction
strongly acid.

44 One per cent, lactose-bouillon: as in dextrose-bouil¬
lon (with slight variations).

“ One per cent, saccharose-bouillon; gas-production
slower than the preceding, lasting from seven to four¬
teen days. Total gas about HC02 nearly %. The
final reaction in the bulb may be slightly acid or alkaline,
according to the rate of gas-production.

44 Variety j3 :

44 The same in all respects, excepting as to its behavior
in saccharose-bouillon; neither gas nor acids are formed
in it. n

1 American Journal of the Medical Sciences , 1895, no, p. 287.

398

PATHOGENIC BACTERIA.

Characteristics for Differentiation.

Typhoid Bacillus.

Bacilli usually slender.

Flagella numerous (10-20), long, and
wavy.

Growth not very rapid, not particularly
luxuriant.

Upon Eisner’s culture-medium de¬
velops slowly, the colonies remain¬
ing small.

Upon fresh acid potato the so-called
“ invisible growth ” formerly thought
to be differential.

Acid-production in whey not exceed-
ing 3 per cent. Sometimes slight
in ordinary media, and sometimes
succeeded by alkaline production.

Grows in media* containing sugars
without producing any gases.

Produces no indol.

Growth in milk unaccompanied by
coagulation.

In Maassen’s asparagin-glycerin solu¬
tion the bacillus does not grow.

Gives the Widal reaction with the
serum of typhoid blood.

Colon Bacillus.

Bacilli inclined to be a little thicker.

Flagella fewer (8-10).

Growth rapid and luxuriant. This
character is by no means constant.

Upon Eisner’s medium develops more
rapidly, the colonies being larger.
(Sometimes the colonies are small
and remain so.)

Upon potato a brownish-yellow, dis¬
tinct pellicle.

Acid-production well marked.

Gas-production well marked.

Indol-production marked.

Milk coagulated.

Grows in Maassen’s solution.

Does not react with typhoid blood.

CHAPTER IV.

YELLOW FEVER.

The bacteriology of yellow fever has been studied by
Domingos Freire, Carmona y Valle, Sternberg, Havel-
burg, and most recently by Sanarelli.

Sternberg, whose work is extensive and important,
says: u Facts relating to the endemic and epidemic prev¬
alence of yellow fever, considered in connection with
the present state of knowledge concerning the etiology
of other infectious diseases, justify the belief that yellow
fever is due to a living organism capable of development
under favorable local and meteorological conditions ex¬
ternal to the human body, and of establishing new cen¬
ters of infection when transported to distant localities.”

Sternberg, at the Tenth International Medical Con¬
gress (Berlin, 1890), reported the study of 42 yellow fever
autopsies in which aerobic and anaerobic cultures were
made from the blood, liver, kidney, urine, stomach, and
intestines, but the specific infectious agent was not found,
and the most approved bacteriological methods failed to
demonstrate the constant presence of any particular
micro-organism in the blood and tissues of vellow fever
cadavers. The micro-organism most frequent!}' encoun¬
tered was the Bacillus coli communis.

A few scattered bacilli were found in the liver and
other organs at the moment of death, but when a portion
of liver was preserved in an antiseptic wrapper and kept
for twenty-four to forty-eight hours the large number of
bacteria that developed were of many varieties, the most
common being the Bacillus coli communis and the Ba¬
cillus cadaveris.

The blood, urine, and crushed liver-tissue obtained

399

400 PATHOGENIC BACTERIA.

from a recent autopsy arc not pathogenic in moderate
amounts for rabbits or guinea-pigs. Liver-tissue pre¬
served at 28° F. in an antiseptic wrapper is very patho¬
genic for guinea-pigs when injected subcutaneously, but
Sternberg found that this pathogenesis was not true of
yellow fever livers only, as it developed also in control-
autopsies.

Extended research of the alimentary canal in yellow
fever showed the intestine to contain a great number of
bacteria, but no pure or nearly pure culture of any single
species, as in cholera. Few liquefying bacteria were
found, and the most abundant bacterium was, as in
health, the Bacterium coli communis.

The most important micro-organism met with was
Bacillus x (Sternberg), which was isolated by the culture-
method from a considerable number of cases, and may
have been present in all. It was not present in any of
the control-experiments. It was very pathogenic for rab¬
bits when injected into the abdominal cavity. Sternberg
says: u It is possible that this bacillus is concerned in the
etiology of yellow fever, but no satisfactory evidence that
this is the case has been obtained by experiments upon
the lower animals, and it has not been found in such
numbers as to warrant the inference that it is the veri¬
table infectious agent.”

The latest researches upon yellow fever are those of
Sanarelli.1 In studying the cadavers of yellow fever San-
arelli found them either entirely sterile or universally
invaded by certain microbic species, such as the Strepto¬
coccus pyogenes, the colon bacillus, the protei, etc.,
which cannot be the cause of the disease. In the second
case he examined he was fortunate enough to find what
he is satisfied is the specific microbe, the Bacillus icter -
oides. In n autopsies he never found the organism
alone, but always associated with the ordinary bacteria
mentioned above. The Bacillus icteroides must be sought
for in the blood and tissues, and not in the gastro-intes-
1 Brit. Med. Journ July 3, 1&97.

YELLOW FEVER.

401

tinal cavity. In the latter it is never found. The isola¬
tion of the specific microbe was only possible in 58 per
cent, of the cases, and in some rare instances may be ac¬
complished during life.

The bacillus, at first sight, presents nothing morpho¬
logically characteristic. It is a small bacillus with
rounded ends, generally united in pairs in the culture
and in small groups in the tissues. It is 2-4 ju in length,
and, as a rule, two or three times longer than broad (Fig.
iii). It is pleomorphous, and has flagella. By em¬
ploying suitable methods it can be found in the organs

Fig. in. — Bacillus icteroides (Sanarelli).

of yellow fever cadavers, usually united in little groups,
always situated in the small capillaries of the liver, kid¬
ney, etc. The best method of demonstration is to keep
a fragment of liver, obtained from a body soon after
death, in the incubator at 37 0 C. for twelve hours and
allow the bacteria to multiply in the fresh tissue before
examination.

The bacillus can be cultivated upon the ordinary
media. Upon gelatin plates it forms rounded, transpar¬
ent, granular colonies, which during the first three or
four days present somewhat the appearance of leukocytes.

402 PA THOGENIC BA CTERIA.

The granular appearance becomes continuously more
marked, and usually a central or peripheric nucleus,
completely opaque, is seen. In time the entire colony
becomes opaque, but does not liquefy gelatin.

Stroke-cultures on obliquely solidified gelatin exhibit
brilliant, opaque, little drops similar to drops of milk.

In bouillon it develops slowly, without either pellicle
or flocculi.

The culture upon agar-agar is said to be characteristic.

If grown at 370 C., the peculiar appearances of the
colonies do not develop; but if the culture is kept at 200-
220 C., the colonies appear rounded, whitish, opaque, and
prominent, like drops of milk. This appearance of the
colonies shows well if the cultures are kept for the first
twelve to sixteen hours at 370 C., and afterward at room-
temperature, when the colonies will show a flat central
nucleus, transparent and bluish, surrounded by a promi¬
nent and opaque zone, the whole resembling a drop of
sealing-wax. Sanarelli refers to this appearance as con¬
stituting the diagnostic feature of Bacillus icteroides. It
•can be obtained in twenty-four hours.

The growth upon potato corresponds to the classic
description of that of the bacillus of typhoid fever.

The bacillus is a facultative anaerobe. It cannot be
colored by Gram’s stain. It slowly ferments lactose,
more actively ferments glucose and saccharose, but is not
capable of coagulating milk. It strongly resists drying,
dies in water at 6o° C. , and is killed in seven hours by the
solar rays. It can live for considerable time in sea- water.

The bacterium is pathogenic for the majority of the
domestic animals. All mammals seem more or less
sensitive to the pathogenic action of the bacillus; birds
are often immune. Guinea-pigs are invariably killed by
either intraperitoneal or subcutaneous injection of o. 1
c.cm. White mice are killed in five days; guinea-pigs
in eight to twelve days; rabbits in four to five days.
The morbid changes present include splenic tumor, hy¬
pertrophy of the thymus, and adenitis. In the rabbit

YELLOW FEVER.

403

there are, in addition, nephritis, enteritis, albuminuria,
hemoglobinuria, and hemorrhages into the body-cavities.

The dog is the most susceptible animal. When it is
injected intravenously the disease-process that results is
almost immediately manifested with such violent symp¬
toms and such complex lesions as to recall the clinical
and anatomical picture of yellow fever in the human
being. The most prominent symptom in experimental
yellow fever in the dog is vomiting, which begins directly
after the penetration of the virus into the blood and con¬
tinues for a long time. Hemorrhages appear after the
vomiting, the urine is scanty and albuminous, or there is
suppression, which shortly precedes death. Once grave
jaundice was observed.

At the necropsy the lesions met are highly interesting,
and are almost identical with those observed in man.
Most conspicuous is the profound steatosis of the liver.
The liver-cells, even when examined fresh, appear com¬
pletely degenerated into fat, this appearance correspond¬
ing to that found in fatal cases of yellow fever. The
same result may be obtained by injecting the liver di¬
rectly or through the abdominal wall. The kidneys are
the seat of acute parenchymatous nephritis, sometimes
with marked fatty degeneration. The whole digestive
tract is the seat of hemorrhagic gastro-enteritis comparable
in intensity only to poisoning by cyanid of potassium.

Experiments upon monkeys were also of interest, in¬
asmuch as they demonstrated the possibility of obtaining
fatty degeneration more extensive than is observed in
man. In one case the liver was transformed into a mass
of fatty substance similar to wax.

Goats and sheep are also very sensitive to the icteroid
virus, and the lesions described also occur in them.

The death of a yellow fever victim is the result of one
of three causes:

1. It may be due to the specific infection principally,
when the Bacillus icteroides is found in the cadaver in
a certain quantity and in a state of relative purity.

404

PATHOGENIC BACTERIA.

2. It may be due to the septicemias established during
the course of the disease, the cadaver then presenting an
almost pure culture of the other microbes.

3. It maybe due in large measure to renal insufficiency,
when the cadaver is found nearly sterile.

The black vomit is due to the action of gastric acidity
upon the blood which has extravasated in the stomach in
consequence of the toxic products of the Bacillus icte-
roides.

The Bacillus icteroides produces a toxin the result of
whose action corresponds to the essential symptoms of the
disease. Animals immune to the infection, or only par¬
tially susceptible to it, are not much affected by the toxin.
Susceptible animals, such as dogs, are profoundly affected.
Ten to fifteen minutes after injecting the toxin the
animals experience a general rigor; abundant lachryma-
tion begins, followed by continued vomiting, first of food,
then of mucus. In a short time the animals lie help¬
less and extended. Hematuria frequently occurs. If the
dose be moderate, the dog recovers quickly from the
violent attack; but if the quantity of toxin be very large
or repeated on successive days, it finally succumbs, pre¬
senting the anatomical lesions already described as due to
infection.

The proofs, of the specificity of the Bacillus icteroides
are not limited to the animal experiments quoted. Sana-
relli also adduces five experimental inoculations upon
men. These inoculations were not made with the bac¬
teria — i. e . were not infection experiments — but were
made with the filtered sterile toxin, whose action could
be more easily controlled. “ The injection of the filtered
cultures in relatively small doses reproduced in man
typical yellow fever, accompanied by all its imposing
anatomical and symptomatological retinue. The fever,
congestions, hemorrhages, vomiting, steatosis of the liver,
cephalalgia, collapse — in short, all that complex of symp¬
tomatic and anatomical elements which in their combina¬
tion constitute the indivisible basis of the diagnosis of

YELLOW FEVER.

40^

yellow fever. This fact is not only striking evidence in
favor of the specific nature of the Bacillus icteroides, but
it places the etiological and pathologic conception of yel¬
low fever on an altogether new basis. 5 5

The discovery of the Bacillus icteroides, and especially
of its toxin, entirely changes our view of the pathology
of the disease. Instead of being a disease of the gastro¬
intestinal tract, as one would conclude from the symp¬
toms, “ all the symptomatic phenomena, all the functional
alterations, all the anatomical lesions of yellow fever, are
only the consequence of an eminently steatogenous,
emetic, and hemolytic action of the toxic substances
manufactured by the Bacillus icteroides.”

The mode by which the Bacillus icteroides enters the
body to produce the disease has not been made out.
The digestive and respiratory tracts are the most likely
routes.

Sanarelli points out that when it happens that a mould
develops near the Bacillus icteroides, the products of
material exchange of this hyphomycete or the transfor¬
mation effected by it, are sufficient to nourish the ba¬
cillus and enable it to live and multiply, whereas it
would be otherwise condemned to a more or less early
death.

There seems to be no particular mould possessed of this
power, as of six experimented upon all were capable of
it. Sanarelli is of the opinion that in the holds of ships
and in damp places generally the presence of moulds
favors the development of the Bacillus icteroides.

About the same time that Sanarelli published his
researches, Havelburg announced1 the discovery of an
entirely different bacillus. Without entering into a long
description of Havelburg’ s bacillus, which seems to be
far less established in its specificity, the following are the
chief characteristic and differential points:

The bacillus is found in the stomach and intestine and
in the u black vomit.” It is almost the sole organ-

1 Ann. de V Inst. Pasteur , 1897.

4°6 PATHOGENIC BACTERIA .

ism found in the blood in the -stomach. There seems to
be very little toxin in the blood of patients with yellow
fever, 30-40 c.cm. of blood being necessary to kill a
guinea-pig when injected subcutaneously. The injection
of 1-2 c.cm. of blood from the stomach, however, caused
death of the guinea-pig. In its body an almost pure cul¬
ture of the bacillus of Havelburg was found. This ex¬
periment was repeated twenty-one times without a failure.

The micro-organism is an exceedingly small straight
bacillus 1 fi in length and o. 3-0. 5 fx in breadth, and may
be single or in pairs, never occurring as filaments. The
stained specimens are more deeply colored at the ends
than at the center, so that the bacillus somewhat resem¬
bles the bacillus of fowl-cholera and looks somewhat like
a diplococcus. It has no flagella, is not motile, and does
not seem to produce spores.

Upon gelatin plates the colonies appear in twenty-
four hours as small, round, white points, and increase
in size during the next twrenty-four hours. The older
colonies are yellowish, finely granular discs, with deli¬
cately serrated borders. The gelatin is not liquefied.

In gelatine puncture-cultures a 4 4 nail-growth ” is pro¬
duced, consisting of a delicate line of colonies along the
puncture and a broad surface-growth.

The growth on agar-agar is not characteristic, as is that
of Sanarellfi s bacillus. Bouillon becomes clouded by the
development of the organism. Rapid fermentation and
gas-production occur in media containing sugar. A
grayish growth occurs on potato. Milk is curdled in
twenty-four hours. The bacillus produces large quan¬
tities of indol and sets free H2S. Development in acid
media is rapid. The organism is a facultative anaerobic.
Guinea-pigs and mice are very susceptible: white rats
far less so. Dogs suffer only Jfrom local abscesses at the
point of injection. The bacillus rapidly alternates in
virulence. No toxin seems to be produced by it.

Havelburg is of the opinion that “yellow fever is a
disease of which the specific toxic agent enters the stom-

YELL OW FE VER. 407

ach and intestines, where it develops. It is only excep¬
tionally and in small numbers that it makes its way from
these positions to other organs. He thinks the toxic sub¬
stances formed in the stomach and intestine are probably
the result of the breaking down of the bodies of the ba¬
cilli by the digestive juices, and that to the absorption of
these the various tissue-changes and fatal terminations
are to be referred.

In a lengthy and interesting review and comparison of
Sanarelli’s and his own work, Sternberg1 concludes that
the Bacillus icteroides of Sanarelli is identical with the
Bacillus „r, which he had discovered in yellow fever
cadavers as early as 1888, and felt disposed to describe as
the specific cause of the disease, except for a few facts,
such as finding it in only one-half of the cases, etc.
vSternberg seems inclined to believe in Sanarelli’ s work,
and asserts his intention to further investigate Bacillus x.
Bacillus x was, however, isolated from the alimentary
canal, in which Sanarelli’s bacillus is said not to exist,
and was isolated from the liver of a case of tuberculosis,
wliicli takes away considerable of the evidence of its
specificity.

In a later paper2 Sanarelli discusses the validity of
Sternberg’s claim to priority of discovery, and points out
a sufficient number of differences in the original descrip¬
tions of the organisms to establish conclusively the in¬
dividuality of the Bacillus icteroides.

It would seem, from a careful consideration of the
recent literature, that Havelburg had very little ground
for considering his bacillus specific, and that it is not
possible for Sternberg to establish the identity of the
Bacillus x with the Bacillus icteroides, while at the same
time Sanarelli’s descriptions and arguments are convinc¬
ingly in favor of the accuracy of his own work and the
specificity of his bacillus.

1 Centralbl. fi'tr Bakt. und Parasitcnk Sept. 6, 1897, Bd. xxii., Nos. 6
and 7.

2 Ibid., Ikl. xxii., Nos. 22 and 23, p. 668.

408

PATHOGENIC BACTERIA .

Sanarelli’s labors have not ceased with his careful study
of the Bacillus icteroides, but have been carried into the
important field of serum-therapy. By careful manipula¬
tion he has succeeded in immunizing the horse and ox to
large doses of the bacillus, injecting into a vein so as to
prevent the intense local reaction, and has found that the
serum of these animals has the power to protect guinea-
pigs from lethal doses of the bacillus. He hopes that
the serum will also be efficacious in the treatment of yel¬
low fever in the human being.

CHAPTER V.

CHICKEN- CHOLERA.

The barnyards of Europe, and sometimes of America,
are occasionally visited by an epidemic disease which
affects pigeons, turkeys, chickens, ducks, and geese, and
causes almost as much destruction among them as the
occasional epidemics of cholera and small-pox produce
among men. Rabbit-warrens are also at times seriously
affected by the epidemic. When fowls are ill with the
disease, they fall into a condition of weakness and apathy
which causes them to remain quiet, seemingly almost
paralyzed, and ruffle up the feathers. The eyes are
closed shortly after the illness begins, and the birds
gradually fall into a stupor from which they do not
awaken. The disease leads to a fatal termination in
twenty -four to forty-eight hours. During its course
there is profuse diarrhea, the very frequent fluid, slimy,
grayish-white discharges containing numerous micro¬
organisms.

The bacilli which are responsible for this disease were
first observed by Perroncito in 1878, and afterward thor¬
oughly studied by Pasteur. They are short, broad bacilli
with rounded ends, sometimes united to each other,
with the production of moderately long chains (Fig. 112).
Pasteur at first regarded them as cocci, because when
stained with a penetrating anilin dye the poles stain
intensely, but a narrow space between them remains
almost uncolored. This peculiarity is very marked, and
sharp observation is required to observe the outline of
the intermediate substance. The bacillus does not form
spores, and does not stain by Gram’s method. When

409

4io

PATHOGENIC BACTERIA.

examined in the living condition it is found to be non-
motile.1

The bacillus readily succumbs to the action of heat
and dryness. The cultures upon gelatin plates after
about two days appear as irregular, small, white points.
The deep colonies reach the surface slowly, and do not
attain any considerable size. The gelatin is not lique¬
fied. The microscope shows the colonies to be irregularly

rounded disks with distinct smooth borders. The color
is yellowish-brown, and the contents are granular. Some¬
times there is a distinct concentric arrangement.

In gelatin puncture-cultures a delicate white line occurs
along the entire path of the wire. When viewed through
a lens this line is seen to consist of aggregated mi¬
nute colonies. Upon the surface the development is

1 Most authorities state that the bacillus is not motile, but Thoinot and Mas-
selin assert that it is so. Precis tie Microbie, 2d ed., 1893.

Fic;. 1x2. — Bacillus of chicken-cholera, from the heart’s blood of a pigeon 7
x 1000 (Frankel and Pfeiffer).

CHICKEN CHOLERA .

411

much more marked, so that the growth resembles a nail
with a pretty good-sized flat head. If, instead of a punc¬
ture, the inoculation be made upon the surface of ob¬
liquely solidified gelatin, a much more pronounced growth
takes place, and along the line of inoculation a dry,
granular coating is formed. This growth is quite similar
to that upon agar-agar and blood-serum, which growths
are white, shining, rather luxuriant, and devoid of char¬
acteristics. No growth occurs in the absence of oxygen.

Upon potato no growth occurs except at the incubation
temperature. It is a very insignificant, yellowish-gray,
translucent film.

The introduction of cultures of this bacillus into the
tissues of chickens, geese, pigeons, sparrows, mice, and
rabbits is sufficient to produce fatal septicemia. Feeding
chickens, pigeons, and rabbits with material infected
with the bacillus is also sufficient to produce the disease
with pronounced intestinal lesions. Guinea-pigs usually
seem immune, though they succumb to very large doses,
especially when given intraperitoneally.

The autopsy shows that when the bacilli are intro¬
duced subcutaneously a true septicemia results, with the
addition of a hemorrhagic exudate and gelatinous infil¬
tration at the seat of inoculation. The liver and spleen
are enlarged; circumscribed, hemorrhagic, and infiltrated
areas occur in the lungs ; the intestine shows an intense
inflammation with red and swollen mucosa, and oc¬
casional ulcers following small hemorrhagic spots. Peri¬
carditis is of frequent occurrence. The bacilli are found
in all the organs. If, on the other hand, the disease has
been produced by feeding, the bacilli are chiefly to be
found in the intestine. Pasteur found that when pigeons
were inoculated into the pectoral muscles, if death did
not come on rapidly, portions of the muscle ( sequestra ?)
underwent degeneration and appeared anemic, indurated,
and of a yellowish color.

The bacillus of chicken-cholera is one whose peculiar¬
ities can be made use of for protective vaccination.

412

PA THOGENIC BACTERIA .

Pasteur discovered that when cultures are allowed to
lemain undisturbed for several months, their virulence
is greatly lessened, and new cultures planted from these
are also attenuated. When chickens are inoculated with
such cultures, no other change occurs than a local in¬
flammatory reaction by which the birds are protected
against virulent bacilli. From this observation Pasteur
worked out a system of protective vaccination in which
fowls can first be inoculated with very weak, then with
stronger, and finally with highly virulent cultures, with
a resulting protection and immunity. Unfortunately,
the method is too complicated to be very practical. Use
has, however, been made of the ability of this bacillus
to kill rabbits, and in Australia, where they are pests,
they are being exterminated by the use of bouillon cul¬
ture. It is estimated that two gallons of bouillon culture
will destroy 20,000 rabbits irrespective of infection by
contagion.

The bacillus of chicken-cholera seems not only to be
specific for that disease, but seems able, when properly
introduced into various other animals, to produce several
different diseases. Indeed, no little confusion has arisen
in bacteriology by the description of what is now pretty
generally accepted to be this very bacillus under the
various names of bacillus of rabbit-septicemia (Koch),
Bacillus cuniculicida (Fliigge), bacillus of swine-plague
(Loffler and Schiitz), bacillus of Wildseuche’’ (Hiippe),
bacillus of u Biiffelseuche ” (Oriste-xArmanni), etc.

CHAPTER VI.

HOG-CHOLERA.

The bacillus of hog-cholera (Bacillus suipestifer) was
first found by Salmon and Smith,1 but was for a long time
confused with the bacillus of u swine-plague, ” which it
closely resembles and with which it frequently occurs.
It is a member of the group of which the Bacillus coli
communis may be taken as a type. Since the careful
studies of Smith,1 however, the claims of the discoverers
that the bacillus of hog-cholera is a separate and specific
organism can hardly be doubted.

Hog-cholera, or upig typhoid,1’ as the English call it,
is a common epidemic disease of swine, which at times
kills 90 per cent, of the infected animals, and thus causes
immense loss to breeders. Salmon estimates that the
annual losses from this disease in the United States
range from $10,000,000 to $25,000,000.

The disease is particularly fatal to young pigs. The
symptoms are not very characteristic, and the animals
often die suddenly without having appeared particularly
ill, or after seeming ill but a few hours. The symptoms
consist of fever (io6°-io7° F.), unwillingness to move,
and more or less loss of appetite. The animals may ap¬
pear stupid and dull, and have a tendency to hide in the
bedding and remain covered by it. The bowels may be
normal or constipated at the beginning of the attack,
but later there is generally a liquid and fetid diarrhea,
abundant, exhausting, and persisting to the end. The
eyes are congested and watery, the secretion drying and

1 Reports of the Bureau of Animal Industry , 1885—91.

2 Centralbl. fur Baht, und Parasitenk Bd. ix., Nos. 8, 9, and 10, March
2, 1897.

413

414

PATHOGENIC BACTERIA.

gluing the lids together. The breathing is rapid, and
there may be cough. Occasionally there is an eruption
with crusts or scabs of various sizes on the skin, which
is often congested. The animal becomes weak, stands
with arched back and drawn abdomen, and walks with a
weak, tottering gait.

The course of this disease varies from one or two days
to two or three weeks.

At post-mortem examination petechiae, ecchymoses, and
extravasations of blood into the tissues are found to be com¬
mon and form one of the principal changes in the acute

form of the disease. The spleen is enlarged to two or four
times its normal size, and is soft and engorged with blood.

The extravasations of blood are common in the lym¬
phatic glands, beneath the serous membranes of the
thorax and abdomen, and particularly along the intes¬
tines; on the surface of the lungs and kidneys and in
their substance. The contents of the intestine are some¬
times covered with clotted blood. In the subacute form
of the disease the principal changes are found in the
large intestine, and consist of ulcers which appear as
circular, slightly projecting masses varying in color from

HOG-CHOLERA.

415

yellowish to black. Occasionally these ulcers are slightly
depressed in outline. When cut across they are found to
consist of a firm, solid growth extending nearly through
the intestinal wall. They are most frequent in the
cecum, upper half of the colon, and on the ileocecal
valve. In the chronic form of the disease the spleen is
rarely enlarged.

‘ 1 In hog-cholera the first effect of the disease is
believed to be upon the intestines, with secondary inva¬
sion of the lungs. ’ ’

The most characteristic lesions of the disease are the
petechise and ecchymoses, the ulcerations of the large
intestine (Fig. 113), and the collapse and occasional bron-
cliopneumonic changes in the lung.

The kidneys are nearly always affected, the urine con¬
taining albumin and tube-casts.

The specific bacillus of hog-cholera was secured by
Smith from the spleens of more than 500 hogs. It
occurs in all the organs and has also been cultivated
from the urine.

The organisms appear as short rods with rounded
ends, 1. 2-1. 5 p- long and 0.6-0. 7 [J- in breadth. They are
very actively motile. No spore-production has ever been
observed. In general the bacillus resembles in appearance
that of typhoid fever. It stains readily by the ordinary
methods, but not by Gram’s method.

The bacilli possess numerous long flagella, easily
demonstrable by the usual methods of staining (Fig.
11 4)-

No trouble is experienced in cultivating the bacilli,
which grow well in all the media.

Upon gelatin plates the colonies become visible in
twenty- four to forty-eight hours; the deeper ones spher¬
ical with sharply defined borders. The surface is brown¬
ish by reflected light, and is without markings. They
are rarely larger than 0.5 mm. in diameter and are homo¬
geneous throughout. The superficial colonies have little
tendency to spread upon the gelatin. Their borders may

41 6 PATHOGENIC BACTERIA.

be circular and rounded, or irregular. They are said
rarely to reach a greater diameter than 2 mm. The gela¬
tin is not liquefied. There is nothing distinctly charac¬
teristic about the appearance of the colonies.

Upon agar-agar the superficial colonies attain a diam-

Fig. 1 14. — Ulceration of the intestine in a typical case of swine-fever
(Crookshank).

eter of 4 mm. and have a gray translucent appearance with
polished surface. They are round and slightly arched.

In gelatin punctures the growth takes the form of a
nail with a flat head. There is nothing characteristic
about it. The growth in the puncture shows it to be an
optional anaerobe.

HOG-CHOLERA.

417

Linear cultures upon agar-agar present a translucent,
rather circumscribed, grayish, smeary layer.

Upon potato a yellowish coating is formed, especially
when the culture is kept in the thermostat.

Bouillon made with or without pepton is clouded in
twenty-four hours. When the culture is allowed to stand
for a couple of weeks without being disturbed a thin
surface-growth can be observed.

Milk is an excellent culture-medium, but is not visibly
changed by the growth of these bacteria. Its reaction
remains alkaline.

The hog-cholera bacillus is a copious gas-producer,
capable of breaking up sugars into C02, H, and an acid,
which, formed late, eventually checks its further devel¬
opment. No indol and no phenol are formed in the
culture-media.

The bacillus is hardy. Smith found it vital after being
kept dry for four months. It ordinarily dies sooner, how¬
ever. The thermal death-point is 540 C., maintained for
sixty minutes.

The bacillus is markedly pathogenic for animals.
Small quantities introduced subcutaneously into rabbits
or mice kill them in from seven to twelve days. The
animal appears quite well for three or four days, then
begins to sit quietly in the cage and eat but little, or
refuses to eat at all, until death takes place.

In Smith’s experiments one-four-millionth of a cubic
centimeter of a bouillon culture injected subcutaneously
into a rabbit was sufficient to cause its death. Before
death the temperature abruptly rises 2°-3° C., and re¬
mains high until death. Larger quantities may kill in
five days. Injected intravenously in small doses the ba¬
cillus may cause death in forty-eight hours.

When the animal is subjected to a postmortem exam¬
ination the spleen is found enlarged, firm, and dark red
in color. The liver is found to contain small yellowish-
white necrotic areas which sometimes occur in one, some¬
times in several acini, and not infrequently surround the
27

4 1 8 PA THOGENIC BA CTERIA.

interlobular veins. The kidneys are acutely inflamed
and the urine is albuminous. The heart-muscle is
spotted, gray, and fatty. In the intestinal tract the pic¬
ture of the disease will be found to vary according to its
duration.

The contents of the small intestine are yellowish,
watery, and mucous; Peyer’s glands are enlarged. In
the neighborhood of the pylorus, ecchymoses and exten¬
sive extravasations of blood are common. The bacilli
are found in all of the organs.

The house mouse is very susceptible to the disease;
guinea-pigs much less so, ^ c.cm. of a virulent cul¬
ture often being required to kill them. Pigeons are still
more refractory, and Smith found that ^ c.cm. of a
bouillon culture injected into the breast-muscles was
required to kill them.

In spite of the fact that hog-cholera is a disease of
swine, and that it is from dead swine that the bacilli are
obtained, these animals are not very easily affected arti¬
ficially. They snow no symptoms when injected subcu¬
taneously, but almost invariably die after intravenous
injection of 1-2 c.cm. of a virulent culture.

Smith found that feeding with 200-300 c.cm. of a
bouillon culture after a day’s fasting, or with small quan¬
tities administered daily, would also cause death, with a
widespread diphtheritic inflammation of the stomach and
colon. Feeding with the organs of dead hogs produces
the same lesions as the administration of the culture.

As early as 1886 Salmon and Smith found it possible
to produce, in both very and partly susceptible animals,
immunity to hog-cholera by gradually accustoming them
to increasing doses of the bacteria. DeSchweinitz iso¬
lated from cultures of the bacteria two toxic substances,
a ptomain (sucholo-toxin) and an albumose (sucholo-aibu-
min), together with cadaverin and methylamin. With
these substances he seems to have been able to produce
immunity. Selander and Metschnikoff found that im¬
munity could be produced more quickly by the use of

HO G- CH OLERA . 419

blood of infected rabbits exposed to 58° C. This blood
was found to be exceedingly toxic.

DeSchweinitz 1 found that the introduction of progress-
ingly increased amounts of cultures into cows caused the
development in them of an antitoxic substance capable
of protecting guinea-pigs from the disease.

Working in my laboratory, Pitfield2 has found that
after a single injection of a sterilized bouillon culture of
the bacillus into the horse, the serum, which has origin¬
ally slight agglutinative reactive power, is so changed as
to show a decided reaction. If the horse be immunized
to large doses of such sterile cultures, the serum reaction
becomes so marked that with a dilution of 1 : 10,000 a
typical reaction occurs in sixty minutes.

According to this experiment, in doubtful cases the
use of this reaction should greatly facilitate the differen¬
tiation of the bacillus of hog-cholera from similar ba¬
cilli.

1 Cenlralbl. f Bakt. ?/. Parasitcnk xx., p. 573.

2 Microscopical Bulletin , 1897, p. 35.

CHAPTER VII.

SWINE-PEAGUE.

The bacillus of swine-plague, or the Bacillus suisepti-
cus of Loftier and Schiitz, and Salmon and Smith, so
closely resembles that of chicken-cholera that it is easily
confounded with it, and, indeed, at one time, they were
thought to be identical. The species has, however, suf¬
ficient well-marked characters to make its differentiation
clear (Fig. 115).

Swine-plague is a rather common and exceedingly

Fig. 1 15. — Bacillus of swine-plague (from photograph by E. A. de Schweinitz).

fatal disease. It occurs alone or in combination with
hog-cholera (y. z;.), and because of the lack of suffi¬
ciently well-characterized symptoms — sick hogs appear¬
ing more or less alike — it is often mistaken for that
disease. The confusion resulting from the mixed cases
makes it impossible to determine exactly how fatal swine-
plague may be in uncomplicated cases.

420

5 WINE- PL A G UE.

421

The symptoms of swine-plague, while closely resem¬
bling those of hog-cholera, may differ from them in the
existence of cough, swine-plague being prone to affect the
lungs and oppress the breathing, which becomes frequent,
labored, and painful, and associated with frequent cough,
while hog-cholera chiefly presents intestinal symptoms.

The course of the disease is usually rapid, a fatal result
often occurring in one or two days.

At autopsy the lungs are often found inflamed, and
contain numerous small, pale, necrotic areas, and some¬
times large cheesy masses one or two inches in diameter.
Inflammations of the serous membranes affecting- the
pleura, pericardium, and peritoneum, and associated with
fibrinous inflammatory deposits on the surfaces, are com¬
mon. There may be congestion of the mucous mem¬
brane of the intestines, particularly of the large intestine,
or the disease in this region may be an intense croupous
inflammation with the formation of a fibrinous exudative
deposit on the surface.

A hemorrhagic form of the disease is said to be com¬
mon in Europe, but, according to Salmon, is rare in the
United States.

The bacillus of swine-plague much resembles that of
hog-cholera, and not a little that of chicken-cholera. It
is a short organism, rather more slender than its con¬
geners, not possessed of flagella, and is incapable of move¬
ment and produces no spores. Its vitality is low, and
it is easily destroyed. Salmon says that it soon dies in
water or by drying, and that the temperature for its
growth must be more constant and every condition of
life more favorable than for the hog-cholera germ. This
germ is said to be widely distributed in nature, and is
probably present in every herd of swine, though not
pathogenic except when its virulence has been increased
or the resistance of the animals diminished by some un¬
usual conditions.

In its growth the bacillus of swine-plague is an optional
anaerobic organism.

422

PATHOGENIC BACTERIA.

In general, its appearance -in culture-media is very
similar to that of the bacillus of hog- cholera. Kruse,
however,1 points out that when the bacillus grows in
bouillon the liquid remains clear on account of the for¬
mation of a flocculent, stringy sediment. Upon ordi¬
nary acid potato the bacillus does not grow, but if the
reaction of the medium be alkaline a grayish-yellow patch
is formed. In its growth in milk slight acidity is pro¬
duced, but the milk is not coagulated and the litmus
color added to it is not decolorized.

The bacillus stains by the ordinary methods, some¬
times only at the poles, then resembling very closely the
bacillus of chicken-cholera. It is not colored by Gram’s
method.

The pathogenesis, while similar to that of the hog-
cholera bacillus, presents some marked differences, espe¬
cially in regard to the seat of the local manifestations,
to which attention has already been called, and in the
duration of the disease, which is much shorter. There
is also considerable resemblance to the bacillus of chicken-
cholera in pathogenesis, but the local reaction following
injection of the culture partakes of the nature of a hemor¬
rhagic edema, which is not present in chicken-cholera, and
the cases often exhibit fatty metamorphosis of the liver.

Rabbits, mice, and small birds are all very susceptible
to the disease, generally dying of septicemia in twenty-
four hours; guinea-pigs are less susceptible, except the
very young animals, which die without exception. Chick¬
ens are more immune, but usually succumb to large doses.
Hogs die after subcutaneous injection of the bacilli, and
suffer from marked edema at the point of injection, and
septicemia. If injected into the lung, a pleuropneumonia
with multiple necrotic areas in the lung follows. In
these cases the spleen is not much swollen, there is slight
gastro-intestinal catarrh, and the bacilli are present every¬
where in the blood.

Animals cannot be infected by feeding.

1 Fliigge’s Mikroorganismen , p. 419, 1896.

CHAPTER VIII.

TYPHUS MURIUM.

The Bacillus typhi murium (Fig. 116), which created
havoc among the mice in his laboratory, causing most
of them to die, was discovered by Loffler in 1889. It
is a short organism, somewhat resembling the bacillus
of chicken-cholera. It is rather variable in its dimen¬
sions, and often grows into long^ flexible filaments. No

Fig. 1 1 6. — Bacillus typhi murium, from agar-agar; x 1000 (Itzerott and

Niemann).

speculation has been observed. It is a motile organism,
with numerous flagella, like those of the typhoid-fever
bacillus. It stains well with the ordinary dyes, but
rather better with Loffler’ s alkaline methylene blue.

Upon gelatin plates the deep colonies are at first round,
slightly granular, transparent, and grayish. Later they
become yellowish-brown and granular. Superficial col¬
onies are similar to those of the typhoid bacillus. In

423

424

PATHOGENIC BACTERIA.

gelatin punctures there is no liquefaction. The growth
takes place upon the surface principally, where a grayish-
white mass slowly forms.

Upon agar-agar a grayish- white development devoid
of peculiarities occurs.

Upon potato a rather thin whitish growth may be
observed after a few days.

The bacillus grows well in milk, with the production
of an acid reaction, but without coagulation.

The organism is pathogenic for mice of all kinds,
which succumb in from one to two days when inoculated
subcutaneously, and in eight to ten or twelve days when
fed upon material containing the bacillus. The bacilli
multiply rapidly in the blood- and lymph-channels, and
cause death from a general septicemia.

Loffler expressed the opinion that this bacillus might
be of use in ridding infested premises of mice, and the
results of its use for this purpose have been highly satis¬
factory. He has succeeded in ridding a field so infested
as to be useless for agricultural purposes by saturating
some bread with bouillon cultures of the bacillus and
distributing it near the holes inhabited by the mice.
The bacilli that were eaten by the mice not only killed
them, but also infected others which ate the dead bodies
of the first victims, and so the extermination progressed
until scarcely a mouse remained in the field. In discuss¬
ing the practical applicability of the employment of cul¬
tures of this bacillus for the destruction of field-mice,
Brunner1 calls attention to certain conditions that are
requisite for a satisfactory result, (i) It is necessary,
first of all, to attack rather extensive areas of the invaded
territory, and not to attempt to destroy the mice of a
small field into which an indefinite number of fresh
animals may immediately come from the surrounding
fields. The country-people, who are the sufferers, should
combine their efforts so as to extend the benefits widely.
(2) The preparation of the cultures is a matter of im-

1 Centralbl.f Bakt. u Parasitenk Jan. 19, 1898, Bd. xxiii., No. 2.

TYPHUS MURIUM.

4^5

portance. Agar-agar cultures are best, as being most
readily transportable. They are broken up in water and
well stirred, and the liquid poured upon a large num¬
ber of small pieces of broken bread. These are next to
be distributed with reasonable care. Instead of being
carelessly scattered over the ground, they should be
dropped into the fresh mouse-holes, and pushed suffici¬
ently far in to escape the effects of sunlight upon the
bacilli. Attention should be paid to holes in walls,
under railway tracks, etc. and other places where mice
live in greater freedom from disturbance than in the
fields. (3) The attempted eradication of the mice should
be begun at a time of year when the natural food is not
plenty. By observing these precautions the mice can be
eradicated with certainty, usually in a period of time not
exceeding eight to twelve days. For this purpose, in the
course of two years, no less than 250,000 cultures were
distributed from the Bacteriological Laboratory of the
Tierarznei. Institut in Vienna. The bacilli are not
pathogenic for the animals, such as the fox, weasel,
ferret, etc. that feed upon the mice, do not affect man
in any way, and so seem to occupy a useful place in
agriculture by destroying the little but almost invincible
enemies of the grain.

CHAPTER IX.

MOUSE-SEPTICEMIA.

In 1878, during his investigations upon the infectious
traumatic diseases, Koch observed that when a minute
amount of putrid blood or of meat-infusion was injected
into mice the animals died of a septicemia caused by the
multiplication in their blood of a minute bacillus to
which he gave the name “Bacillus der Mausesepticamie”
(Fig. 1 1 7).

Fig. 1 17. — Bacillus of mouse-septicemia, from the blood of a mouse; x 1000
(Frankel and Pfeiffer).

In 1885 the bacillus was again brought into promi¬
nence by Iydffler and Schiitz, who found a very similar.

MOUSE-SEPTICEMIA . 427

perhaps identical, organism in the erysipelatous disease
which attacks the swine of many parts of Europe.

There seem to be certain slight morphological and
developmental differences between these two organisms,
but Baumgarten, Gunther, Sternberg, and others have
regarded them as insufficient for the formation of sepa¬
rate species, and have boldly described the organisms as
identical, while Lorenz has shown that immunity pro¬
duced in the rabbit by one bacillus protects against the
other. The described differences are, indeed, so very
small that I think it well to follow in the path of the ob¬
servers mentioned, pointing out in the description such
points of difference as may arise.

The bacilli are extremely minute, measuring about
1.0 x 0.2 jvl (Sternberg). Fliigge, Frankel, and Eisenberg
find the Bacillus erysipelas suis somewhat shorter and
stouter than that of mouse-septicemia : there seems to
be a ’division of opinion upon this point.

Sporulation has been described by some observers, but
nothing definite seems to be known upon this point.

Motility is ascribed by some (Schottelius and Frankel)
to the Bacillus erysipelas suis, and is denied to the bacillus
of mouse-septicemia by others. The truth seems to be
that the motility of both organisms is a matter of doubt.

No flagella have been demonstrated upon the bacillus.
It grows quite well both at the room-temperature and at
the temperature of incubation. It can grow well with or
without oxygen, but perhaps flourishes
a little better without than with it. It
is killed by a temperature of 520 C. in
fifteen minutes.

The colonies upon gelatin plates can
first be seen on the second or third day, Fig. 1 18.— Colony

then appearing as transparent grayish
specks with irregular borders, from
which many branched processes extend

of the bacillus of
mouse-septicemia; X
So (Fliigge).

(Fig. 1 18). Frankel describes them as resembling in

shape the familiar branched cells occupying the lacunae

428

PATHOGENIC BACTERIA.

of bone. When further developed the colonies flow
together and give the plate a cloudy gray appearance.
The gelatin is not liquefied, but is gradually softened and
its evaporation thus aided.

In gelatin puncture-cultures the growth is quite cha¬
racteristic, and the tendency of the bacillus to grow
anaerobically is well shown (Fig. 119). The develop-

Fig. 119. — Bacillus of mouse-septicemia: gelatin puncture- culture three and a
half days old (Gunther).

ment takes place all along the line of puncture, but is
more marked below than at the surface. The growth
takes place in a peculiar form, resembling superimposed
disks, each disk separate from its neighbors and consist¬
ing of an area of clouded grayish gelatin reaching almost
to the walls of the tube. This growth develops slowly,
and causes a softening rather than an actual liquefaction
of the gelatin.

Upon agar-agar and blood-serum a very delicate, trans¬
parent grayish line develops along the path of the needle.
It does not grow upon potato.

The bacillus grows at the room temperature, but much
better at the temperature of the incubator.

The disease affects quite a variety of animals, notably
hogs, rabbits, mice, white rats, pigeons, and sparrows.

MO USE-SEPTICEMIA .

429

The guinea-pig, which is-generally the victim of labora¬
tory experiments, is not susceptible to it. Field- and
wood-mice, cattle, horses asses, dogs, cats, chickens, and
geese are immune.

When inice are inoculated with a pure culture they
soon become ill, lose their appetite, mope in a corner,
and are not readily disturbed. As the disease becomes
worse they assume a sitting posture with the back much
bent; the eyelids are glued together by adhesive pus; and
when death comes to their relief, in the course of forty
to sixty hours after inoculation, they remain sitting in
the same characteristic position.

When the ears of rabbits are inoculated with the
bacillus from cases of erysipelas suis, a violent inflam¬
matory edema and distinct redness occurs, much re¬
sembling erysipelas. This lesion gradually spreads, in¬
volves the head, then the body of the animal, and ulti¬
mately causes death.

When swine are affected, they are dull and weak, and
have a kind of paralytic weakness of the hind quarters.
The temperature is elevated ; red patches appear upon
the skin and swell and become tender. Death follows in
two or three days. Sixty per cent, of the diseased
animals die.

In all animals the anatomical changes are much alike.
The disease proves to be a septicemia, and the bacilli can
be found in all the organs, especially the lungs and spleen.
They are- few in number in the streaming blood.

As the organisms stain well by Gram’s method, this
stain is of great value for their discovery in the tissues,
and can be highly recommended.

Most of the bacilli occupy the capillary blood-vessels ;
many of them are enclosed in leucocytes. The organs in
such cases do not appear distinctly abnormal, except the
spleen, which is considerably enlarged. The mesenteric
and other lymphatics are also enlarged, and the gastric
and intestinal mucous membranes are usually inflamed
and mottled. The bacilli also occupy the intestinal con-

PATHOGENIC BACTERIA .

43°

tents, and Kitt, who discovered them in this position,
points out that the infection of swine probably takes
place by the entrance, along with the food, of the fecal
matter of diseased animals into the alimentary apparatus
of others.

Pasteur, Chamberland, Roux, and others have worked
upon a protective vaccination based upon the attenuation
of the virulence of the organism by passing it through
rabbits. Two vaccinations are said to be necessary to
produce immunity. The vaccinated animals, however,
may be a source of infection to others, and should always
be isolated. Klemperer in 1892 found that the blood-
serum of immunized rabbits would save infected mice
into which it was injected.

Lorenz in 1894 found an antitoxic substance in the
blood of rabbits immunized to the disease. The effect
of its injection into other animals is, however, only a
temporary immunity. Later1 he found it possible to
protect hogs against the disease by injecting them first
with a serum obtained from a hog immunized in the
ordinary manner described by Pasteur, afterward with
a feeble culture of the bacillus, and finally with viru¬
lent cultures. The strength of the serum should be
determined by injecting varying quantities of it into
mice infected with definite amounts of a culture of
known virulence. The immunity produced by Lorenz
lasted for a year.

1 Centralbl. f Bakt. u. Parasitenk.y Jan., 1896, p. 168.

CHAPTER X.

RELAPSING FEVER.

As long ago as 1873, Oberineier discovered that a
flexible spiral organism, about 0.1 n in diameter and
from 20-40 fi in length, could be observed in the blood
of patients suffering from relapsing fever.

Although many of the best bacteriologists of our day
have occupied themselves with the study of this spiril¬
lum, we really have, at present, very little more know¬
ledge than that given us by Obermeier.

FlO. 120. — Spirochoeta febris recurrentis; x 650 (Heim).

The spirilla (Fig. 120) are generally very numerous,
are long, slender, and flexible (spirochseta), and possess
a vigorous movement by flagella. The ends are rather
pointed.

The spirillum stains well by ordinary methods, but

not by Gram’s method. It seems to be a strict parasite,

and has never been cultivated artificially.

Of the pathogenesis of the organism there can be no

doubt, as it is invariably present in relapsing fever and
' ,<‘21

43*

PATHOGENIC BACTERIA .

undergoes a peculiar cycle of changes according to the
stage of the disease. During the pyrexia the organisms
are found in the blood in active movement, swimming
both by rotation on the long axis and by undulation.
As soon as the crisis comes on they are found to be with¬
out motion, most of them enclosed in leucocytes and
seemingly dead. The recurrence of the paroxysm has
suggested to many that spores are formed in the spiril¬
lum, but no one has been successful in proving that this
is the case. Koch, Carter, and Soudakewitch have all
succeeded in giving the disease to monkeys, and Munch
and Moczutkowsky have gone further and have produced
it in men by introducing into them blood from diseased
patients.

Soudakewitch finds that the removal of the spleen
causes the disease to terminate fatally in monkeys.

CHAPTER XI.

BUBONIC PLAGUE.

The bacillus of bubonic plague (Fig. 121) seems to
have met an independent discovery at the hands of

Fig. 121. — Baoillus of bubonic plague (Yersin).

Yersin and Kitasato in the summer of 1894, during the
activity of the plague then raging at Hong-Kong. There
seems to be but little doubt that the micro-organisms
described by the two observers are identical.

In a recent study of the plague, Ogata1 states that
while Kitasato found his bacillus in the blood of cadavers,
Yersin seldom found his bacillus in the blood, but always,
in the enlarged lymphatic glands. Kitasato’ s bacillus
retains the color when stained by Gram’s method; Yer¬
sin’ s does not. Kitasato’ s bacillus is motile; Yersin’ s,
non-motile. The colonies of Kitasato’ s bacillus when
grown upon agar are round, irregular, grayish-white with

1 Centralbl. f. Bakt. u. Parasitenk., Bd. xxi., Nos. 20 and 21, June 24*
1897.

433

434

PA THO GENIC BA CTERIA .

a bluish tint, and resemble ,glass-wool when slightly
magnified; Yersin’s bacillus forms white, transparent col¬
onies with iridescent edges. Ogata, in the investigation

Fig. 122. — Bacilli of plague and phagocytes; x 800.. From human lymphatic

gland (Aoyama).

of the cases that came into his hands found a bacillus
that resembles that of Yersin, but not that of Kitasato.

The bubonic plague is an extremely fatal infectious
disease, whose ravages in the hospital in which Yersin
made his observations carried off 95 per cent, of the
cases. It affects both men and animals, and is character¬
ized by sudden onset, high fever, prostration, delirium,
and the occurrence of lymphatic swellings — buboes —
affecting chiefly the inguinal glands, though not infre¬
quently the axillary, and sometimes the cervical, glands.
Death comes on in severe cases in forty-eight hours. If
the case is of longer duration, the prognosis is said to be
better. Autopsy in fatal cases reveals the characteristic
enlargement of the lymphatic glands, whose contents are
soft and sometimes purulent.

Wyssokowitz and Zabolotmy1 describe two forms of
the disease:

1 Ann. de V Inst. Pasteur , Aug. 25, 1897, xi., 8, p. 665.

BUBONIC PLAGUE.

435

1. Plague with buboes. ~

2. Plague without buboes, but with a primary specific
pneumonia in which the bacilli occur in immense num¬
bers in the affected pulmonary tissue, but sparingly in the
blood and kidney.

The studies of Kitasato and Yersin show that in blood
drawn from the finger-tips and in the softened contents
of the glands a small bacillus is demonstrable. The
organisms are small, stain much more distinctly at the
ends than in the middle, so that they resemble diplo-
cocci, and in fresh specimens seem to be surrounded by
a capsule. Kitasato compares the organism to the well-
known bacillus of ckicken-cholera. It is feebly motile
(according to Abel, entirely non-motile), and does not
seem to form spores. Nothing is said in the original
descriptions about the presence of flagella, though it is
probable from the studies of Gordon 1 that some, at least,
of the bacilli may be possessed of them. It does not
stain by Gram’s method.

When cultures are made from the softened contents of
the buboes the bacillus may be obtained almost or quite
pure, and is found to develop upon artificial culture-
media. In bouillon a diffuse cloudiness results from
the growth, as observed by Kitasato, though in Yersin’s
observations the culture more nearly resembled erysipe¬
las cocci, and contained zooglea attached to the sides and
at the bottom of the tube of nearly clear fluid.

According to Haffkine,2 when an inoculated bouillon
culture is allowed to stand, perfectly at rest, on a solid
shelf or table a characteristic appearance results. In
from twenty-four to forty-eight hours, the liquid remain¬
ing limpid, flakes appear underneath the surface, forming
little islands of growth, which in the next twenty-four
to forty-eight hours grow down into a long stalactite-like
jungle, the liquid always remaining clear. In four to

1 Ceyitralbl. f Bakt. u. Parasitenk Sept. 6, 1897, Bd. xxii., Nos. 6 and 7,
p. 170.

2 Brit . Med. Jotir June 12, 1897, p. 1461.

436

PATHOGENIC BACTERIA.

six days the islands are still more compact and solidified.
If the vessel be disturbed, the islands fall like snow and
are deposited at the bottom.

Upon gelatin plates at 22 0 C. the colonies may be ob¬
served in twenty-four hours by the naked eye. They are
pure white or yellowish-white, spherical in the deep gela¬
tin, flat upon the surface, and are about the size of a
pin’s head. The gelatin is not liquefied. The borders
of the colonies are, upon microscopic examination, found
to be sharply defined and to become more granular as
their age increases. The superficial colonies occasionally
are surrounded by a fine, semi-transparent zone.

In gelatin puncture-cultures the development is scant.
The medium is not liquefied (?); the growth takes place
in the form of a fine duct, little points being seen on the
surface and in the line of puncture.

Upon agar-agar — glycerin agar-agar is best — the bacilli
grow freely, the colonies being whitish in color, with a
bluish tint by reflected light. Under the microscope
they appear moist, with rounded, uneven edges. The
small colonies are said to resemble little tufts of glass-
wool; the larger ones have large round centers. Micro¬
scopic examination of the bacilli grown upon agar-agar
reveals the presence of long chains resembling strepto¬
cocci.

Klein1 states that the colonies develop quite readily
upon gelatin made from beef-bouillon (not infusion),
appearing in twenty- four hours, at 20° C., as small, gray,
irregularly rounded dots. Magnification shows the col¬
onies to be serrated at the edges and made up of short,
oval, sometimes double bacilli. Some colonies contrast
markedly with their neighbors in that they are large,
round, or oval, and consist of longer or shorter, straight
or looped threads of bacilli. The appearance was much
like that of the young colonies of the Proteus vulgaris.
At first Klein regarded these as contaminations, but later
’ he was led to believe that their occurrence was character-

1 Centralbl. f Bakt. u. Paratitenk xxi., Nos. 24 and 25, July 10, 1897.

BUBONIC PLAGUE .

437

istic of the plague bacillus. The peculiarities of these
colonies cannot be recognized after forty-eight hours.

Involution-forms on partly desiccated agar-agar not con¬
taining glycerin are said by Haffkine to be characteristic.
The microbes swell up and form large, round, oval, pea-
or spindle-shaped or biscuit-like bodies, which may attain
twenty times the normal size and in growing gradually
lose the ability to take up the stain. Such involution-
forms are not seen in liquid culture.

Hankin and Ueumann 1 recommend for the differential
diagnosis of the plague bacillus the addition of 2. 5-3. 5
per cent, of salt to the agar-agar. When transplanted
from ordinary agar-agar to the salt agar-agar the involu¬
tion-forms which are so characteristic of the plague ba¬
cillus form with exceptional rapidity.

Upon blood-serum the growth at the temperature of
the incubator is luxuriant. It forms a moist layer of a
yellowish-gray color, and is unaccompanied by liquefac¬
tion of the serum.

Upon potato no growth occurs at ordinary tempera¬
tures. When the potato is stood away for a few days in
the incubator a scanty, dry, whitish layer develops.

Abel found the best culture-medium to be 2 per cent,
alkaline pepton solution with 1 or 2 per cent, of gelatin,
as recommended by Yersin and Wilson.

The bacillus develops under conditions of aerobiosis and
anaerobiosis. In glucose-containing media it does not
form gas. No indol is formed. Ordinarily the culture-
medium is acidified by the development of an acid that
persists for three weeks or more.

By frequent passage through animals of the same
species the bacillus increases very much in virulence.
Curiously enough, however, the observations of Knorr,
substantiated by Yersin, Calmette and Borrel, show that
the bacillus made virulent by frequent passage through
mice is not increased in virulence for rabbits.2

1 Centralbl. f Pakt. u. Parasitenk ., Oct., 1897* Bd. xxii., Nos. l6 and 17*
p. 438. 2 Ann. de V Inst. Pasteur , July, 1895.

438

PATHOGENIC BACTERIA.

Kitasato found tliat mice, rats, guinea-pigs, and rabbits
are all susceptible; pigeons are immune. Julian Haw¬
thorne, in his paper in the Cosmopolitan , speaks of hav¬
ing seen cats and dogs dying of the disease, but no men¬
tion is made of these animals in the scientific papers I
have. read. When blood, lymphatic pulp, or pure cul¬
tures are inoculated into them, the animals become ill in
from one to two days, according to their size. Their eyes
become watery, they begin to show disinclination to take
food or to make any bodily effort, the temperature rises
to 41. 50 C., they remain quietly in a corner of the cage,
and die with convulsive symptoms in from two to five
days.

Devell 1 has found that frogs are susceptible to the dis¬
ease.

Wyssokowitz and Zabolotny2 found monkeys to be
highly susceptible to plague, especially when inoculated
subcutaneously. When so small an inoculation was
made as a puncture with a pin dipped in a culture of the
bacillus, the puncture being made in the palm of the
hand or sole of the foot, the monkeys always died in
from three to seven days. In these cases the local edema
observed by Yersin did not occur. They point out the
interest attaching to infection through so insignificant a
wound and without local lesions.

According to Yersin, an infiltration or watery edema
can be observed in a few hours about the point of inocula¬
tion. The autopsy shows the infiltration to be made up
of a yellowish gelatinous exudation. The spleen and
liver are enlarged, the former often presenting an appear¬
ance much like an eruption of miliary tubercles. Some¬
times there is universal swelling of the lymphatic glands.
Bacilli are found in the blood and in all the internal
organs. Very often there are eruptions during life, and
upon the inner abdominal walls there are petechiae and
occasional hemorrhages. The intestine is hyperemic, the

1 Centralbl. f Bakt . u. Parasitenk Oct. 12, 1897.

2 Ann. de F Inst. Pasteur , Aug. 25, 1897, xi., 8, p. 665.

BUBONIC PLAGUE . 439

adrenals congested. There are often sero-sanguinolent
effusions into the serous cavities.

Klein 1 states that the intraperitoneal injection of the
bacillus into guinea-pigs is of diagnostic value, produc¬
ing in twenty-four to forty-eight hours a thick cloudy
peritoneal exudate rich in leukocytes and containing
characteristic chains of the plague bacillus.

Animals fed upon cultures or upon the flesh of other
animals dead of the disease became ill and died with
typical symptoms. When Klein inoculated animals with
the dust of dwelling-houses in which the disease had
occurred, some died of tetanus, one from plague. Many
rats and mice in which examination showed the charac¬
teristic bacilli died spontaneously in Hong- Kong.

Yersin showed that flies also die of the disease. Mace¬
rating and crushing a fly in bouillon, he not only suc¬
ceeded in obtaining the bacillus from the medium, but
infected an animal with it.

Nuttall,2 in reviewing Yersin’s fly-experiment, found
the statement true, and showed that flies fed with the
cadavers of plague-infected mice died in a variable
length of time. Large numbers of plague bacilli were
found in their intestines. He also found that bed-bugs
allowed to prey upon infected animals took up large
numbers of the plague bacilli and retained them for a
number of days. These bugs did not, however, infect
healthy animals when allowed, subsequently, to feed
upon them. Nuttall is not, however, satisfied that the
number of his experiments upon this point was great
enough to be conclusive.

Ogata found that the plague bacillus existed in the
bodies of fleas found upon diseased rats. One of these
he crushed between sterile object-glasses and introduced
into the subcutaneous tissues of a mouse, which died
in three days with typical lesions of the plague, a con¬
trol-animal remaining well. Some guinea-pigs taken

1 Centralbl f Bakt. u. Parasiienk xxi., No. 24, July 10, 1897, p. 849.

2 Ibid., Aug. 13, 1897.

440

PATHOGENIC BACTERIA .

for experimental purposes into a plague district, and
kept carefully isolated, died spontaneously of the disease,
presumably because of insect infection.

Yersin found that when cultivated for any length of
time upon culture-media, especially agar-agar, the viru¬
lence was rapidly lost and the bacillus eventually died.
On the other hand, when constantly inoculated from
animal to animal the virulence of the bacillus is much
increased.

The bacillus probably attenuates readily. Kitasato
found that it did not seem able to withstand desicca¬
tion longer than four days ; and Yersin found that al¬
though it could be secured from the soil beneath an
infected house at a depth of 4-5 c.cm., the virulence
of such bacilli was lost.

Kitasato found that the bacillus was killed by two
hours’ exposure to 0.5 per cent, carbolic acid, and also
by exposure to a temperature of 8o° C. Ogata found
that the bacillus was instantly killed by 5 per cent, car¬
bolic acid, and in fifteen minutes by 0.5 per cent, carbolic
acid. In o. 1 per cent, sublimate solution it is killed in
five minutes.

It seems possible to make a diagnosis of the disease in
doubtful cases by examining the blood, but it is admitted
that a good deal of bacteriologic practice is necessary for
the purpose.

Abel finds that the blood may yield fallacious results
because of the rather variable appearance of the bacilli,
which are sometimes long and easily mistaken for other
bacteria. He deems the best tests to be the inoculation
of broth-cultures and subsequent inoculation into ani¬
mals, which he advises should have been previously
vaccinated against the streptococcus. Plague bacilli
persist in the urine a week after convalescence.

Wilson, of the Hoagland Laboratory, found the thermal
death-point of the organism was one or two degrees
higher than that of the majority of pathogenic bacteria
of the non-sporulating variety, and that, unlike cholera,

BUBONIC PLAGUE. 441

the influence of sunlight and desiccation cannot be relied
upon to limit its viability.

Kitasato’s experiments first showed that it is possible
to bring about immunity to the disease, and Yersin,
working in India, and Fitzpatrick, in New York, have
successfully immunized large animals (horses, sheep,
goats). The serum of these immunized animals con¬
tains an antitoxin capable not only of preventing the dis¬
ease, but also of curing it in mice and guinea-pigs and
probably in man.

Haffkine in his experiments followed the line of pre¬
ventive inoculation as employed against cholera. Bouil¬
lon cultures were used in which floating drops of butter
were employed to make the islands of plague bacilli
float. The cultures were grown for a month or so, suc¬
cessive crops of the island-stalactite growth as it formed
having been precipitated by agitating the tube. In this
manner there was obtained an “ intense extra-cellular
toxin” containing large numbers of the bacilli. The
culture was killed by exposure to a temperature of yo°
C. for one hour, and the mixture used in doses of about
3 c.cm. as a preventive inoculation. In the Byculla
Gaol, where Haffkine’s experiments numbered over one
hundred, a decided prophylactic effect was observed in
twelve to fourteen hours in men already advanced in the
stage of incubation.

Wyssokowitz and Zabolotmy, whose studies have
already been quoted, used 96 monkeys in the study of
the value of the “plague-serums,” and found that
when the treatment is begun within two days from the
time of inoculation the animals can be saved, even
though symptoms of the disease are marked. After the
second day the treatment cannot be relied upon. The
dose necessary was 20 c.cm. of a serum having a potency
of 1 : 10. If too little serum was given, the course of
the disease was slowed, the animal improved for a time
and then suffered a relapse, and died in from thirteen to
seventeen days. The serum also produced immunity,

442

PATHOGENIC BACTERIA .

but of only ten to fourteen days’ duration. Immunity
lasting three weeks was conferred by inoculating a mon¬
key with an agar-agar culture heated to 6o° C. If too
large a dose of such a culture was given, however, the
animal was enfeebled and remained susceptible.

CHAPTER XII.

TETRA GENUS .

There can sometimes be found in the normal saliva,
more commonly in tuberculous sputum, and still more
commonly in the cavities of ' tuberculosis pulmonalis, a
large micrococcus grouped in fours and known as the
Micrococcus tetragenus (Fig. 123). It was discovered by

Fig. 123. — Micrococcus tetragenus in pus from a white mouse ; x 615 (Heim).

Gaffky, and subsequently carefully studied by Koch and
Gaffky. It sometimes occurs in the pus of acute ab¬
scesses, and may be of importance in connection with
the pulmonary abscesses which so often complicate tu¬
berculosis.

The cocci are rather large, measuring about i fx in
diameter. In cultures they show no particular arrange¬
ment among themselves, but in the blood and tissues of
animals they commonly appear arranged in groups of
four surrounded by a transparent gelatinous capsule.

The organism stains well by ordinary methods, and

443

444

PATHOGENIC BACTERIA.

most beautifully by Gram’s method, by which it can be
best demonstrated in tissues.

Upon gelatin plates small white colonies are produced
in from twenty-four to forty-eight hours. Under the
microscope they are found to be spherical or elongate
(lemon-shaped), finely granular, and lobulated like a
raspberry or a mulberry. When superficial they form
white, elevated, rather thick masses 1-2 mm. in diameter
(Fig. 124). _

In gelatin punctures a large white surface-growth

Fig. 124. — Micrococcus tetragenus: colony twenty-four hours old upon the sur¬
face of an agar-agar plate; x ioo (Heim).

rakes place, but very scant development occurs in the
puncture, where the small spherical colonies generally
remain isolated.

Upon the surface of agar-agar spherical white colonies
are produced. They may remain isolated or may become
confluent.

Upon potato a luxuriant thick, white growth occurs.

The growth upon blood-serum is also abundant, espe¬
cially at the temperature of the incubator. It has no
distinctive peculiarities.

The introduction of tuberculous sputum or of a most
minute quantity of a pure culture of this coccus into
white mice generally causes a fatal septicemia.

TE TEA GEN US .

445

The organisms are fQnnd in small numbers in the
heart’s blood, but are numerous in the spleen, lungs,
liver, and kidneys.

House-mice and field-mice are comparatively immune ;
dogs and rabbits are also highly resistant. Guinea-pigs
sometimes die from general infection, though sometimes
local abscesses may be the only result of subcutaneous
inoculation.

The tetragenococci are of no special importance in
human pathology, but probably hasten the tissue-necrosis
in tuberculosis pulmonalis, and may aid in the formation
of abscesses of the lung and contribute to the production
of the hectic fever.

CHAPTER XIII.

INFLUENZA.

Notwithstanding a large number of bacteriologic
examinations conducted for the purpose of determining
the cause of influenza, it was not until 1892, after the
great epidemic, that there was found simultaneously by
Canon and Pfeiffer a bacterium which conformed, at least
in large part, to the requirements of specificity.

The observers mentioned found the same organism —
one in the blood of influenza patients, the other in the
purulent bronchial discharges.

The specific organisms (Fig. 125) are bacilli, very small
in size, having about the same diameter as the bacillus

'%■

.f •

v &

.*\-'*M*i* ■*. ^ ,

a 1

«** K

V*/

? \ys^.

V- ,i

Fig. 125. — Bacillus influenzae, from a gelatin culture; x 1000 (Itzerott and

Niemann).

of mouse-septicemia, but only about half as long (0.2 by
0.5 fj). They are usually solitary, but may be united in
chains of three or four elements. They stain rather

446

INFLUENZA .

447

poorly, except with such concentrated penetrating stains
as carbol-fuchsin and Loftier’ s alkaline methylene blue,
and even with these the bacilli stain more deeply at the
ends than in the middle, so that they appear not a little
like diplococci.

For the demonstration of the bacilli in the blood Canon
recommends a rather complicated method. The blood is
spread upon clean cover-glasses in the usual way, thor¬
oughly dried, and then fixed by immersion in absolute
alcohol for five minutes. The stain which seems best is
Czenzynke’s :

Concentrated aqueous solution of methylene

blue, 40 ;

0.5 per cent, solution of eosin in 70 per cent.

alcohol, 20 ;

Distilled water, 40.

The cover-glasses are immersed in this solution, and kept
in the incubator for three to six hours, after which they
are washed in water, dried, and then mounted in Canada
balsam. By this method the erythrocytes are stained red,
the leucocytes blue, and the bacillus, which is also blue,
appears as a short rod or often as a dumb-bell.

Sometimes large numbers of the bacilli are present ;
sometimes very few can be found after prolonged search.
They are often enclosed within the leucocytes. It really
is not necessary to pursue so tedious a staining method
for demonstrating the bacilli, for they stain quite well by
ordinary methods. They do not stain by Gram’s method.

The bacillus is non-motile, and, so far as is known,
does not form spores. Its resisting powers are very re¬
stricted, as it speedily succumbs to drying, and is cer¬
tainly killed by an exposure to a temperature of 6o° C.
for five minutes. It will not grow at any temperature
below 28° C.

The bacillus does not grow in gelatin or upon ordinary
agar-agar. Upon glycerin agar-agar, after twenty-four
hours in the incubator, minute colorless, transparent,

443

PATHOGENIC BACTERIA .

drop-like cultures may be seen along the line of inocula¬
tion. They do not look unlike condensed moisture, and
Kitasato makes a special point of the fact that the colo¬
nies never become confluent. The colonies may at times
be so small as to require a lens for their discovery.

In bouillon a scant development occurs, small whitish
particles appearing upon the surface, subsequently sink¬
ing to the bottom and causing a woolly” deposit there.
While the growth is so delicate in these ordinary media,
the bacillus grows quite well upon culture-media contain-

Fig. 126. — Bacillus of influenza ; colonies on blood agar-agar; low magnifying

power (Pfeiffer).

ing hemoglobin or blood, and can be transferred from
culture to culture many times before it loses its vitality.

It cannot be positively proven that this bacillus is the
cause of influenza, but from the fact that the bacillus
can be found only in cases of influenza, that its presence
corresponds with the course of the disease in that it is
present as long as the purulent secretions last, and then
disappears, and that Pfeiffer was able to demonstrate its
presence in all cases of uncomplicated influenza, his con¬
clusion that the bacillus is specific is certainly justifiable.

INFLUENZA .

449

The bacillus is pathogenic for certain of the laboratory
animals, the guinea-pig in particular being subject to
fatal infection. The dose required to cause death of a
guinea-pig varies considerably, in the immunization ex¬
periments of Deline and Kole1 ^ of a 24-hour old culture
being fatal in twenty-four hours. These scholars found
that the toxicity of the culture resides not in a soluble
toxin, but in the bodies of the bacilli. The outcome of
the researches, which were made most scientifically and

Fig. 127. — Bacillus of influenza; cover-glass preparation of sputum from a case
of influenza, showing the bacilli in leukocytes; highly magnified (Pfeiffer).

painstakingly, was the total failure to produce immunity.
Increasing doses of the cultures injected into the peri¬
toneum resulted in enabling the animals to resist rather
more than a fatal dose, but never enabled them to main¬
tain vitality when large doses were administered. This
discovery is in exact harmony with the familiar clinical
observation that, instead of an individual being immune
after an attack of influenza, he is as susceptible as before*
if not more so.

1 Zeitschrift fur Hygiene > etc., Bd. xxiv., 1897, Heft. 2.

450

PATHOGENIC BACTERIA.

A . Catanni, Jr.1 trephined rabbits and injected influ¬
enza toxin into their brains, at the same time trephining
control-animals, into some of whose brains he injected
water. The results were that animals thus receiving
0.5-1 mgr. of the living culture constantly died in
twenty-four hours with all the nervous symptoms of the
disease, dyspnea, paralysis beginning in the posterior
extremities and extending over the whole body, clonic
convulsions, stiffness of the neck, etc. Control-animals
injected with a variety of pathogenic bacteria in the
same manner never manifested similar symptoms. The
virulence of the bacillus was also observed to increase
rapidly when transplanted from brain to brain.

1 Zeitschrift fitr Hygiene , etc., Bd. xxiii., 1896.

CHAPTER XIV.

MEASLES.

In 1892, Canon and Pielicke, after the investigation of
fourteen cases of measles, reported the discovery of a
specific bacillus in the blood in that disease.

The organism is quite variable in size, sometimes
being quite small and resembling a diplococcus, some¬
times larger, and occasionally quite long, so that one
bacillus may be as long as the diameter of a red blood-
corpuscle.

The discovery was made by means of a peculiar method
of staining, as follows : The blood is spread in a very
thin, even layer upon perfectly clean cover-glasses, and
fixed by five to ten minutes’ immersion in absolute alco¬
hol. These glasses are then placed in a stain consisting
of

Concentrated aqueous solution of methylene blue, 40 ;

0.25 per ct. solution of eosin in 70 per ct. alcohol, 20 ;

Distilled water, 40,

and stood in the incubator at 370 C. for from six to
twenty-four hours. The bacilli do not all stain uni¬
formly.

The discoverers of the bacillus claim to have made it
grow several times in bouillon, but failed to induce a
growth upon other media.

The bacilli do not stain by Gram’s method ; they seem
to have motility ; no spores were observed. They were
found not only in the blood, but also in the secretions
from the nose and eyes. They are said to persist through¬
out the whole course of the disease, even occasionally
being found after the fever subsides.

451

45^

PATHOGENIC BACTERIA.

Czajrowski asserts that the bacillus can be cultivated
upon various albuminous media except gelatin and agar.
On glycerin agar-agar, especially with the addition of
heinatogen, and on blood-serum, they should grow in
three or four days with an appearance like that of dew-
drops. Under the microscope the colonies are structure¬
less. Mice die of a septicemia after a subcutaneous in¬
oculation.

An interesting field for experimentation has been
opened by Behla,1 who seems to have successfully inocu¬
lated a sucking-pig with measles by introducing some of
the nasal secretion from a case of measles into the nose,
which had been prepared to receive it by scratching with
a wire.

1 Centralbl.f Bakt. ii. Parasitenk Oct. 24, 1896, Bd. xx., Nos. 16 and 17*
p. 36.

D. MISCELLANEOUS.

CHAPTER I.

SYMPTOMATIC ANTHRAX.

‘‘Symptomatic anthrax, ” charbon symptomatique ,
Rauschbrand \ “ quarter-evil, ” and u black-leg” are the
various names applied to a peculiar disease of cattle com¬
mon during the summer season in the Bavarian Alps,
Baden, Schleswig-Holstein, and some parts of the United
States, characterized by the occurrence of irregular, em¬
physematous, crepitating subcutaneous pustules. Dis¬
eased areas are also found in the muscles, and are most
common over the quarters, hence the name “quarter-
evil.” When incised the affected tissues have a dark
color and contain a dark, bloody serum.

The micro-organismal nature of the disease had been
suspected from an early date, but until the work of
Faser and Bollinger the disease was confounded with
anthrax. Still later, Arloing, Thomas, Cornevin, and
Kitasato studied the disease, and succeeded in demon¬
strating the specific micro-organism, which Kitasato
successfuly cultivated upon artificial media.

The bacillus which the results of these labors brought
to light is a rather large individual (3-5 y in length,
0.5-0. 6 At in breadth) with rounded ends. The bacilli
are occasionally united in twos, but are never united in
long chains (Fig. 128). They are actively motile (Thoinot
and Masselin say scarcely at all motile) when examined
in the hanging drop, but after a short time, perhaps
because of the exposure to the oxygen required in the
hanging-drop preparation, the movement is lost and the
bacilli die. When stained by Loffler’s method a con¬
siderable number of flagella can be demonstrated. Large

■ 453

454

PATHOGENIC BACTERIA.

oval spores are found ; by their presence they distort the
bacilli in which they occur, causing them to assume a
spindle shape (clostridium), or, when two are united and
a spore occupies one of them, a drumstick shape. In-

Fig. 12S. — Bacillus of symptomatic anthrax, containing spores, from an agar-
agar culture; x 1000 (Frankel and Pfeiffer).

volution-forms are exceedingly common in old cul¬
tures, and are of enormous size and of granular appear¬
ance.

The bacillus can be stained with the ordinary aqueous
solutions of the anilin dyes, but will not retain the color
by Gram’s method orWeigert’s fibrin method. It can
be colored in sections of tissue with Loffler’s solution,
and can be observed in the blood without staining shortly
after death.

The spores, which can be stained by ordinary methods,
are quite resistant to the action of heat and disinfect¬
ants, and withstand the effects of drying for a consider¬
able length of time.

The bacillus of symptomatic anthrax (Fig. 129) is a
strictly anaerobic, parasitic bacterium. It grows at tem¬
peratures above 180 C., but best at 37 0 C.

SYMPTOMATIC ANTHRAX.

455

The artificial cultivation which was achieved by
Kitasato is not more difficult than that of other an¬
aerobic organisms. In gelatin
containing i to 2 per cent, of
glucose or 5 per cent, of gly¬
cerin the organism develops
quite well, the exact appearance
depending somewhat upon the
method by which it was planted.

If the bacteria are dispersed
through the culture - medium,
the little colonies will appear
in the lower parts of the tube as
nearly spherical or slightly irreg¬
ular, clouded, liquefied areas con¬
taining bubbles of gas. If, on
the other hand, the inoculation
is made by a deep puncture, a
stocking - shaped liquefaction
forms along the whole lower
part of the puncture, leads to
considerable gas-production, and
finally causes the liquefaction of
all the gelatin except a thin
superficial stratum. A peculiar
acid odor is given off by the
cultures.

When the bacteria grow anaerobically in Esmarch
tubes, the colonies are irregularly club-shaped or spheri¬
cal, with a tangled mass of delicate projecting filaments
visible upon microscopic examination.

In agar-agar the development is similar to that in
gelatin. The gas-production is marked, the liquefaction
of course absent, and the same acid odor pronounced.

The bacillus also develops quite well in bouillon, the
bacillary masses sinking to the bottom in the form of
whitish flakes, while the gas-bubbles collect at the top.
In this medium the virulence is unfortunately soon lost.

Fig. 129. — Bacillus of symp¬
tomatic anthrax : four-days-old
culture in glucose-gelatin (Fran-
kel and Pfeiffer).

PATHOGENIC BACTERIA.

456

Milk also seems to be a favorable culture-medium*
The development of the bacilli is unaccompanied by
coagulation.

The virulence of the organism is soon lost in all
culture-media, but it is said that the virulence of the
culture can be much increased by the addition to it of
20 per cent of lactic acid.

When susceptible animals are inoculated with a minute
portion of a pure culture in a little subcutaneous pocket,
such as is described in connection with tetanus and
malignant edema, the bacilli proceed to grow, pro¬
duce the well-known affection, and lead to a certainly
fatal outcome. Cows seem to be the most susceptible
animals, especially those between six months and four
years old; sheep and goats are also sometimes affected.
Curiously enough, animals that are immune to malig¬
nant edema are seemingly more susceptible to Rausch-
brand. Of the laboratory animals, the guinea-pig is
most susceptible ; swine, dogs, and rabbits are very
slightly susceptible ; horses, goats, and birds are im¬
mune.

The virulence of the bacillus is capable of ready
attenuation by exposure to heat, by previous exposure
of its spores to heat, or by drying combined with ex¬
posure to increased temperature. The inoculation of
animals with the attenuated bacilli causes a very mild
affection, followed by complete immunity to the viru¬
lent organisms. Upon this principle the u protective
vaccination” is based. Kitt has, however, shown that
almost the same method as that employed by Pasteur
for vaccination against rabies may be employed against
this bacillus, and that when muscular tissue from an
animal dead of the disease is dried at a temperature of
32-35° C., and then exposed for six hours to a tempe¬
rature of ioo°-io4° C. , and a second portion is exposed
in the same manner to a temperature of 90°-95° C., an
emulsion of this tissue in distilled water, salt-solution,
or bouillon, injected into the animals to be protected, will

SYMPTOMATIC ANTHRAX.

457

act in a manner resembling the pulverized spinal cords
of the rabbits used in rabies, and give an almost per¬
fect immunity. Roux and Chamberland have found that
filtered cultures can also produce immunity when properly
introduced into animals.

The immunity to symptomatic anthrax seems, how¬
ever, to be one of degree, for Arloing, Cornevin, and
Thomas found that when the bacillus was introduced
into the animal body simultaneously with a 20 per cent,
solution of lactic acid, either the virulence of the bacil¬
lus or the resistance of the tissues was so changed that
natural immunity was destroyed and the bacteria allowed
to develop and produce the disease. Roger found also
that refractory animals, like the rabbit, mouse, pigeon,
and chicken, could be made susceptible by the combined
injection of the Rauschbrand bouillon, the Bacillus pro-
digiosus, Proteus vulgaris, and other harmless organisms.

When the guinea-pig is inoculated with the bacillus of
symptomatic anthrax, it dies in from twenty-four to
thirty-six hours. The post-mortem examination shows
a bloody serum at the point of inoculation, and the mus¬
cles are dark red or black, like those of the u black-leg n
of cattle. No changes are apparent in the internal organs.
The bacilli are at first found near the point of inocula¬
tion in the inflammatory exudations only, but soon after
death, being motile, they spread to all parts of the body.

The peculiarities of symptomatic anthrax point to the
entrance of the bacteria into the animal body through
wounds, but the occurrence of epidemics at certain geo¬
graphical points, known technically as “Rauschbrand
stations, n suggests that infection may also take place
through the respiratory and alimentary tracts.

At first thought, as Frankel points out, one might
imagine that an animal dead of quarter-evil and the dis¬
charges from its body might be harmless, as compared,
for example, with the cadavers and discharges of anthrax,
because of the purely anaerobic method of the growth, of
the bacillus of symptomatic anthrax and the rapidity of its

458

PATHOGENIC BACTERIA.

death in the presence of oxygen. This is, however, un¬
true, for the rapid development of a permanent form in
the resisting spores of the bacillus makes the pollution
of the soil exceedingly dangerous for cows who subse¬
quently browse upon it. That the spores are of great
vitality is shown by the well-known laboratory method
of keeping them on hand for experimental purposes, dried
in the muscular tissue of a diseased animal.

Every precaution should be exerted to have the affected
animals isolated, and their cadavers disinfected and de¬
stroyed or buried in such a manner that subsequent
infection is impossible.

Statistical results of Guillod and Simon, based upon
3500 protective inoculations, show a distinct reduction
of the death-rate from 5-20 per cent, in unprotected
animals to 0.5-2 per cent, in protected animals.

CHAPTER II.

MALIGNANT EDEMA.

The chief contaminating organism in the preparation
of pure cultures of the tetanus bacillus is a large slender
bacillus almost as large as that of anthrax, but with
rounded ends and an individual motility accomplished
by means of flagella attached to its ends and sides
(Fig. 130). It is a strictly anaerobic bacterium, and was

Fig. 130. — Bacillus of malignant edema, from the body-juice of a guinea-pig
inoculated with garden-earth; x 1000 (Frankel and Pfeiffer).

originally described by Pasteur (1875) as the Vibrion
septique. It grows well at the room-temperature, as well
as at the temperature of the incubator, produces oval
central spores, and, because of its association with a spe¬
cific edema in certain animals, is known as the Bacillus
oedema maligni.

45 9

460

PATHOGENIC BACTERIA.

The organism is widely distributed in nature, being
almost always present in garden-eartli. It is also found
in various dusts, in the waste water from houses, and
sometimes in the intestinal canals of animals.

When introduced beneath the skin this bacillus proves
pathogenic for a large number of animals — mice, guinea-
pigs, rabbits, horses, dogs, sheep, goats, pigs, calves,
chickens, and pigeons. Cattle seem to be immune.

Gunther points out that the simple inoculation of the
bacillus upon an abraded surface is insufficient to pro¬
duce the disease, because the oxygen which is, of course,
abundant there is detrimental to its growth. When an
experimental inoculation is performed, a small subcu¬
taneous pocket should be made, and the bacilli introduced
into it in such a manner as not to be in contact with the
air.

If the inoculated animal be a mouse, guinea-pig, or
rabbit, in about forty-eight hours it sickens and dies.
The autopsy shows a general subcutaneous edema con¬
taining immense numbers of the bacilli. In the blood
the bacilli are few or cannot be found, because of the
oxygen which it contains. The great majority of them
occupy the subcutaneous tissue, where very little oxygen
is present and the conditions of growth are therefore good.
If the animal is allowed to remain undisturbed for some
time after death, the bacilli spread to the circulatory sys¬
tem and reach all the organs.

Brieger and Ehrlich have reported two cases of malig¬
nant edema in man. Both cases were typhoid-fever
patients injected with musk, and developed the edema
in consequence of impurity of the therapeutic agent.
No case is reported, however, in which healthy men
have been infected with the disease.

Cornevin declares that the passage of the bacillus
through the white rat diminishes its virulence, and that
the animals of various species that recover from this
milder affection are subsequently immune to the virulent
organisms.

MALIGNANT EDEMA. 461

The bacillus of malignant edema stains well with ordi¬
nary cold aqueous solutions of
the anil in dyes, but not by
Gram’s method.

The organism is not a difficult
one to secure in pure culture,
as has been said, generally con¬
taminating tetanus cultures and
being much more easy to se¬
cure by itself than its congener.

It is most easily obtained from
the edematous tissues of guinea-
pigs arid rabbits inoculated with
garden-earth.

The colonies •which develop
upon the surface of gelatin kept
free of oxygen appear to the
naked eye as small shining
bodies with liquid grayish-white
contents. They gradually in¬
crease in circumference, but do
not change their appearance.

Under the microscope they ap¬
pear filled with a tangled mass
of long filaments which under a
high power exhibit individual
movement. The edges of the
colony have a fringed appearance, much like the hay or
potato bacillus.

In gelatin tube-cultures the characteristic growth can¬
not be observed in a puncture, because of the air which
remains in the path of the wire. The best preparation
is made by heating the gelatin to expel the air it may
contain, inoculating while still liquid, then replacing the
air by hydrogen, and sealing the tube. In such a tube
the bacilli develop near the bottom. The appearance of
the growth is highly typical, as globular circumscribed
areas of cloudy liquefaction result (Fig. 131), and may con-

Fig. 131. — Bacillus of malig¬
nant edema growing in glucose
gelatin (Frankel and Pfeiffer).

462

PATHOGEN/C BACTERIA.

tain a small amount of gas. In gelatin to which a little
grape-sugar has been added the gas-production is marked.
The gas is partly inflammable, partly C02. A distinct
odor accompanies the gas-production, and is especially
noticeable in agar-agar cultures.

CHAPTER III.

BACILLUS AEROGENES CAPSULATUS.

This very interesting micro-organism was first de¬
scribed by Welch, and subsequently carefully studied by
Welch and Nuttall,1 and Welch and Flexner.2 It was
first secured from the body of a man dying suddenly of
aneurysm with a peculiar condition of gaseous emphy¬
sema of the subcutaneous tissue and internal organs, and
a copious formation of gas in the veins and arteries.
The blood was thin and watery, of a lac-color, and
everywhere contained large and small gas-bubbles. The
blood-alteration was associated with a change in its
coloring-matter, which dissolved out of the corpuscles
and stained the tissues a deep red. The blood was found
to contain many bacilli, which were also obtained from
the various organs, especially in the neighborhood of the
gas-bubbles. The bacilli were in nearly pure culture.

The bacillus is a large organism, measuring 3-5 ju in
length, about the thickness of the anthrax bacillus, with
ends slightly rounded, or, when joined, square (Fig. 132).
It occurs chiefly in pairs and in irregular masses, but not
in chains, in this particular differing very markedly from
the anthrax bacillus. In culture-media the bacillus is
usually straight, with slightly rounded ends. In old
cultures the rods may be slightly bent, and involution-
forms occur. When several bacilli are joined together
the opposed ends are square-cut The bacillus varies
somewhat in size, especially in length, in different cul¬
ture-media. It usually appears thicker and more vari-

1 Bull, of the Johns Hopkins Hospital , July and Aug., 1892, vol. viii.,
No. 24.

2 Jour, of Exper. Med., vol. i., No. I, Jan., 1896.

463

464

PATHOGENIC BACTERIA.

able in length in artificial cultures than in the blood of
animals and of man. The bacilli occur singly, in pairs*
in clumps, and sometimes in short chains. When united,
an angle is often formed.

The bacillus is non-motile in both the ordinary hanging-
drops and in anaerobic culture. No mention is made of
the presence of flagella.

The organism stains well with the ordinary stains, and
retains the color well in Gram’s method. When stained
with methylene blue a granular or vacuolated appearance

Fig. 132. — Bacillus aerogenes capsulatus (from photograph by Prof. Simon

Flexner).

is sometimes observable, due to the presence of unstained
dots in the protoplasm.

Usually in the body-fluids and often in cultures the ba¬
cilli are surrounded by distinct capsules — clear, unstained
zones. To demonstrate this capsule to the best advan¬
tage, Welch and Nuttall devised the following special
stain: a cover is thinly spread with the bacilli, dried, and
fixed without over-heating. Upon the surface prepared,
glacial acetic acid is dropped for a few moments, then al¬
lowed to drain off, and at once replaced by a strong aque¬
ous solution of gentian violet, which is poured off and
renewed several times until the acid has been replaced by

BACILLUS AEROGENES CAPSULATUS. . 465

the stain. The specimen is then examined in the color¬
ing-solution, after soaking up the excess with filter paper,
the thin layer of coloring fluid not interfering with a clear
view of the bacteria and their capsules. After mounting
in Canada balsam the capsules are not nearly so distinct.
The width of the capsule varies from one-half to twice
the thickness of the bacillus. Its outer margin is stained,
leaving a clear zone immediately around the bacillus.

It was at first thought that the bacillus produced no
spores, but Dunham1 found that spores were produced
upon blood-serum, and especially upon Loffler’s blood-
serum bouillon mixture. The spores resist desiccation
and exposure to the air for ten months. They stain
readily in hot solutions of fuchsin in anilin water, and
are not decolorized by a moderate exposure to the action
of 3 per cent, solution of hydrochloric acid in absolute
alcohol. They are oval, and are usually situated near
the middle of the bacillus, which is distended because of
the large size of the spore and bulges at the sides.

The bacillus is anaerobic. It grows upon all culture-
media, both at the room -temperature and at the tempera¬
ture of incubation, best at the latter. The bacillus grows
in ordinary neutral or alkaline gelatin, but better in gela¬
tin containing glucose, in which the characteristic gas-
production is marked. Soft gelatin, made with 5 instead
of 10 per cent, of the crude gelatin, is said to be better
than the ordinary medium.

There is no distinct liquefaction, but in 5 per cent,
gelatin there is sometimes a softening that can be best
demonstrated by tilting the tube and observing that the
gas-bubbles change their position, as well as by noticing
that the growth tends to sediment.

In making agar-agar cultures careful anaerobic precau¬
tions must be observed. The tubes should contain con¬
siderable of the medium, which should be boiled and
freshly solidified before using. The implantation should
be deeply made with a long wire. The growth takes

1 Bull . of the Johns Hopkins Hospital April, 1897, p. 68.

466 PATHOGENIC BACTERIA .

place slowly unless such tubes a-re placed in a Buchner’s
jar. The deeper colonies are the
largest. Sometimes the growth only *
takes place within 10-12 mm. of the
surface, at others within 3-4 cm. of
it. After repeated cultivation the
organism seems to become somewhat
accustomed to the presence of oxy¬
gen, and will grow higher up in the
tube than when freshly secured from
animal tissue (see Fig. 133).

The colonies seen in the culture-
media are grayish-white or brownish-
white by transmitted light, and some¬
times exhibit a central dark dot. At
the end of twenty-four hours the larger
colonies do not exceed o. 5-1.0 mm.
in diameter, though they may subse¬
quently attain a diameter of 2-3 mm.
or more. Their first appearance is
as little spheres or ovals, more or less
flattened, with rather irregular con¬
tours, due to the presence of small
projecting prongs, which are quite
distinct under a lens. The colonies
may appear as little irregular masses
with projections.

After several days or weeks, single,
well-separated colonies may attain a
large size and be surrounded by pro¬
jections, either in the form of little
knobs or spikes or of fine branchings
Fig. 133. — Bacillus — hair-like or feathery. Their ap-

aerogenes capsulatus,
with gas-production (from
photograph by Prof. Si¬
mon Flexnerh

pearance has been compared to
thistle-balls or powder-puffs and to
thorn-apples. When the growth
takes place in the puncture the

feathery projections are continuous. Bubbles of gas

BACILLUS AERO GENES CAPSULATUS . 467

make their appearance in plain agar as well as in sugar-
agar, though, of course, less plentifully. They first ap¬
pear in the line of growth; afterward throughout the
agar, often at a distance from the actual growth. Any
fluid collecting about the bubbles or at the surface of the
agar-agar may be turbid from the presence of bacilli.
The gas-production is more abundant at incubation- than
at room-temperatures.

The agar-agar is not liquefied by the growth of the
bacillus, but is often broken up into fragments and forced
into the upper part of the tube by the excessive gas-pro¬
duction.

In its growth the bacillus produces acid in considerable
amount.

In bouillon growth does not occur in tubes exposed to
the air, but when the tubes are placed in Buchner’s jars,
or kept under anaerobic conditions, it occurs with abun¬
dant gas-formation, especially in glucose-bouillon, with
the formation of a frothy layer on the surface. The
growth is very rapid in its development, the bouillon
becoming clouded in two to three hours. After a few
days the bacilli sediment and the bouillon again becomes
clear. The reaction of the bouillon becomes strongly
acid.

In milk the growth is rapid and luxuriant under
anaerobic conditions, but does not take place in cul¬
tures exposed to the air. The milk is coagulated in
from twenty-four to forty-eight hours, the coagulum
being either uniform or firm, retracted, and furrowed
by gas-bubbles. When litmus has been added to the
milk it becomes decolorized when the culture is kept
without oxygen, but turns pink when it is exposed to
the air.

The bacillus will also grow upon potato when the tubes
are enclosed in an anaerobic apparatus. There is a
copious gas-development in the fluid at the bottom and
sides of the tube, so that the potato becomes surrounded
by a froth. After complete absorption of the oxygen a

468 . PATHOGENIC BACTERIA.

thin, moist, grayish-white growth takes place upon the
surface of the potato.

The vital resistance of the organism is not great. Its
thermal death-point was found to be 58° C. after ten
minutes’ exposure. Cultures made by displacing the air
with hydrogen are less vigorous than those in which the
oxygen is absorbed from the air by pyrogallic acid. It
was found that in the former class of cultures the bacillus
generally died in three days, while in the absorption ex¬
periments it was kept alive at the body-temperature for
one hundred and twenty-three days. It is said to live
longer in plain than in sugar-agar. To keep the cultures
alive it has been recommended to seal the agar-agar tube
after two or three days’ growth.

It is believed that the natural habitat of the bacterium
is the soil, but there is reason to think that it occurs in
the intestine at times, and it may occasionally be found
upon the skin.

The pathogenic powers of the bacillus are limited, and
while in some cases it seems to be the cause of a fatal
outcome in infected cases, its power to do mischief in the
body seems to depend upon the pre-existence of other
depressing and devitalizing conditions predisposing to its
growth.

Being anaerobic, the bacilli are unable to live in the
circulating blood, but they grow in old clots and in cav¬
ities, such as the uterus, etc., where but little oxygen
ever enters, and from such areas enter the blood and are
distributed.

In support of these views Welch and Nuttall cite the
result of inoculation into healthy and diseased rabbits.
When a healthy rabbit is injected with c.cin. of a
fresh sugar-bouillon into the ear-vein it generally recov¬
ers without any evident symptoms. One of their rabbits
was pregnant, and at time of injection was carrying two
dead embryos. After similar injection with but 1 c.cm.
of the culture it died in twenty-one hours. It seems that
the bacilli were first able to secure a foothold in the dead

BACILLUS AEROGENES CAPSULATUS. 469

embryos, and there multiply sufficiently to bring about
death later on.

After the death of the animal, when the blood is no
longer oxygenated, the bacilli grow rapidly with a
marked gas-production, which in some cases is said to
have caused the bodies to swell to twice their normal
size. The result of injection into guinea-pigs does not
differ very much from that observed in rabbits. Gaseous
phlegmons are sometimes produced.

Pigeons when inoculated subcutaneously in the pec¬
toral region frequently succumb. Following the injec¬
tion there is gas-production that causes the tissues of the
chest to become emphysematous. The bird generally
dies in from seven to twenty-four hours, but may live.

Intraperitoneal inoculation of animals sometimes
causes fatal purulent peritonitis.

The infection as seen in man generally occurs from
wounds into which dirt has been ground, as in the case
of a compound, comminuted fracture of the humerus,
with fatal infection, reported by Dunham, or in w7ounds
and injuries in the neighborhood of the perineum.

Among the twenty-three cases reported by Welch and
Flexner1 we find wounds of the knee, leg, hip, and fore¬
arm, ulcer of the stomach, typhoid ulcerations of the in¬
testine, strangulated hernia with operation, gastric and
duodenal ulcer, perineal section, and aneurism, as con¬
ditions in which external or gastro-intestinal infection
occurred.

Dobbin, P. Ernst, Graham Stewart and Baldwin, and
Kronig have met cases of puerperal sepsis and sepsis fol¬
lowing abortion caused by the bacillus, or in 'which it
played an important role.

The symptoms following infection are quite uniform.
There are usually redness and swelling of the wound,
with rapid elevation of temperature and rapid pulse. The
wound is usually more or less emphysematous, and dis¬
charges a thin, dirty, brownish, offensive fluid which con-

1 Jour, of Exper. Med., vol. I, No. 1, Jan., 1896.

4?o

PATHOGENIC BACTERIA.

tains gas-bubbles and is soinetiijies frothy. Occasionally
the patients recover, especially when the infected part is
susceptible of amputation, but death is a more common
outcome. After death the body begins to swell almost
immediately; it may attain twice its normal size and be
unrecognizable. Upon palpation a peculiar crepitation
can be felt in the subcutaneous tissue nearly everywhere,
and the presence of gas in the blood-vessels is easy of
demonstration. The gas is inflammable, and as the bub¬
bles ignite explosive sounds are heard.

At the autopsy the gas-bubbles are found in most of
the internal organs, sometimes so numerously as to justify
the German term “ Schaumorgane n (frothy-organs).
The liver especially is apt to show this frothy con¬
dition. When the tissues from such a case are hardened
and examined microscopically it is found that the bub¬
bles appear as open spaces in the tissue, the*borders of
which are lined with large numbers of the gas bacillus.
There are also clumps of bacilli without gas-bubbles, but
surrounded by tissue, whose nuclei show a disposition to
fragment or disappear, and whose cells and fibers show
signs of disintegration and fatty change. In discussing
these changes Ernst1 concluded that they were ante¬
mortem and due to the irritation caused by the bacillus.
The gas-production he regarded as postmortem.

In the internal organs the bacillus is usually found in
pure culture, but in the wound it is generally mixed with
other bacteria. On this account it is difficult to estimate
just how much of the damage before death is the result
of the activity of the gas bacillus. That gas-production
after death has nothing to do with pathogenesis during
life is shown by injecting into the ear-vein of a rabbit
a liquid culture of the gas bacillus, allowing about five
minutes’ time for the distribution of the bacilli through¬
out the circulation, and then killing the rabbit. In a few
hours the rabbit will swell and his organs and tissues
will be riddled with the gas-bubbles.

1 Virchow’s Archiv , Bd. 133, Heft ii.

BACILLUS AEROGENES CAPSULATUS. 471

At times, however, as in a case of Graham Stewart
and Baldwin, there is no* doubt that the bacillus produces
gas in the tissues of the entire body during life. These
observers, in a case of abortion with subsequent infection,
found the patient u emphysematous from the top of her
head to the soles of her feet” several hours before
death.

In this case, in which the bacillus was found in pure
culture, it would indeed be difficult to doubt that the
fatal issue was due to the bacillus aerogenes capsulatus.
Whether the fatal termination of the cases is due to the
presence of gas in the vessels, or partly to that and partly
to some toxic property it possesses, does not seem to have
been worked out as yet. It would seem, however, to have
a toxic property from the fact that the onset of the infec¬
tion is first shown by the occurrence of chill, pyrexia,
and rapid pulse, and from the change caused by the
clumps of bacilli upon the surrounding cells of the tis¬
sues in which they occur.

CHAPTER IV.

BACILLUS PROTEUS VULGARIS (HAUSER).

This bacillus was first found by Hauser in decompos¬
ing animal infusions, generally in company with two
closely allied forms, Proteus mirabilis and Proteus Zen-
keri, which, as the experiments and observations of San-
felice and others show, may be identical with or represent

Fig. 134. — Swarming islands of proteus bacilli on the surface of gelatin; x 650

(Hauser).

attenuated forms of it. According to Kruse, it is quite
probable that the old species called Bacterium termo was
largely made up of the proteus.

The bacilli are very variable in size and shape — pleo¬
morphic — and are named proteus from this peculiarity.
Some forms differ very little from cocci, some are more

472

BACILLUS PROTEUS VULGARIS .

473

like the colon bacteria in shape, others are found as very
long filaments, and occasionally sporulina-forms are met
with. True spirilla-forms are never found. All the
forms mentioned may be met with in cultures of the
same organism. The diameter of the bacillus is usually
about 0.6 fi, but the length varies from 1.2 f± or less to 4 ta
or more. No spores are formed. The organisms are
actively motile. The long filaments frequently form loops
and tangles. Flagella are present usually in large num¬
ber; upon one of the longer bacilli as many as one hun¬
dred have been counted. Involution-forms are frequent
in old cultures. The bacilli stain well by the ordinary
methods. Gram’s method is irregular in action, but
usually fails to color the bacteria.

Upon gelatin plates a typical phenomenon is observed
in connection with the development of the colonies, but
for the most advantageous observation the gelatin used
for making the cultures should contain only 5 per cent,
of gelatin instead 10 per cent., as ordinarily used. Kruse1
describes the phenomenon as follows: “at the temperature
of the room rounded, saucer-shaped depressions, with a
whitish central mass surrounded by a lighter zone, are
quickly formed. Under low magnification the center of
the growth is seen to be surrounded by radiations extend¬
ing in all directions into the solid gelatin, and made up
of chains of bacilli. Between the radiations and the
granular center motile bacteria are seen in active
motion. Upon the surface the colony extends as a thin
patch, consisting of a layer of bacilli arranged in threads,
sending numerous projections from the periphery. Occa¬
sionally filaments are found in the surroundings. Under
certain conditions the wandering of the processes can be
directly observed under the microscope. It depends not
only upon the culture-medium, but, in part, upon the
culture itself. Entire groups of bacilli or single threads,
by gradual extension and circular movement, detach
themselves from the colony and wander about upon the

1 Flugge’s Mikroorganisi7ien.

474

PATHOGENIC BACTERIA .

plate. Often from the radiated central part of the colony
peculiar zooglea are formed, having a sausage- or screw-
shape, or wound in spirals like a corkscrew. The
younger colonies, which have not yet reached the surface
of the gelatin, are more compact, rounded or nodular,
later covered with hair, and then becoming radiated and
like the superficial colonies.7 5

When the culture-medium is more concentrated, or the
culture one that has been frequently transplanted, the
phenomenon is much less marked and sometimes does
not take place at all.

Puncture-cultures in gelatin are not at all character¬
istic. They show a rapid stocking-like liquefaction of
the gelatin, extending so as to take in the entire gelatin
in the tube in a few days. Anaerobic cultures do not
liquefy.

Upon agar-agar the bacillus grows with the production
of a moist, thin, transparent, rapidly extending layer
which probably rarely reaches the sides of the tube.
Upon agar-agar plates the wandering of the colonies is
also said to occur.

Upon potato the growth is in the form of a dirty-look-
ing, smeary patch.

In culture-media containing either grape- or cane-sugar
fermentation occurs both in the presence and in the
absence of oxygen. Milk-sugar is not decomposed.

When grown in milk the medium is coagulated.

In its growth the bacillus usually produces a strong
alkaline reaction. Indol and phenol are formed from
the peptone of the culture-media. Nitrates are reduced
to nitrites, and then partly reduced to NH3. In most
culture-media not containing sugar the bacillus pro¬
duces a very disagreeable odor.

It is a question whether the Bacillus proteus is to be
ranked among the pathogenic bacteria. Small doses of
it are harmless for the laboratory animals; in large doses
it produces abscesses. A toxic substance undoubtedly
results from the metabolism of the organism, and is the

BACILLUS PROTEUS VULGARIS.

475

cause of death in caseshn which considerable quantities
are injected into the peritoneal cavity or blood-vessels.
The bacilli do not seem able to multiply in the animal
body in health, but can do so when there has been pre¬
vious injury to its tissues or when associated with patho¬
genic bacteria. In such cases, if it be enabled to grow
in considerable quantity, its toxin may cause pronounced
symptoms. By various observers the proteus has been
secured in culture from cases of wound and puerperal in¬
fections, purulent peritonitis, endometritis, and pleurisy.
When the local lesion in which it grows is small, as in
endometritis, the danger of toxemia is slight, but when
spread over large areas, as the peritoneum, may prove
serious.

It is quite probable that in some of the cases in which
blood-infection with the proteus has been found after
death it did not exist previously, as the researches of
Bordoni-Uffreduzzi have shown that the proteus quite
regularly enters the tissues after death.

While thus apparently unable to keep up an indepen¬
dent existence in the tissues during life, and important in
the body only in conjunction with other bacteria, the
proteus seems able to grow abundantly in urine and to
produce primary inflammation of the bladder when in¬
troduced spontaneously or experimentally into that viscus.
The inflammatory process may extend from the bladder
to the kidney, and so prove quite serious.

The Bacillus proteus has also been found in acute in¬
fectious jaundice and in acute febrile icterus, or Weil’s
disease.

CHAPTER V.

WHOOPING-COUGH.

It is only recently that the bacteriology of whooping-
cough has begun to assume definiteness, and even yet
there is no certainty that any of the various described
bacteria play any specific part in its etiology. In all
diseases of the respiratory apparatus the discharges are
almost certain to be so contaminated with the nasal and
oral bacteria as to make the isolation from them of a
single probably specific organism a matter of difficulty,
and its original recognition a matter of genius.

Of historical interest are the researches and observa¬
tions of Deichler, Kurloff, Szemetzchenko, Cohn, Neu¬
mann, Ritter and Afanassiew. Those of Kurloff and
Afanassiew are of especial importance because they opened
the way for the recent studies of Koplik 1 and those of
Czaplewski and Hensel.2 Koplik and Czaplewski and
Hensel worked entirely independently of each other, and
while the bacterium studied by the former differs in
several points from that of the latter, Czaplewski and
Hensel have claimed to see in Koplik’s work a confirma¬
tion of their own.

Koplik studied 16 cases of whooping-cough. The
sputum was collected in sterile Petri dishes, in which it
was allowed to stand for an hour or so in order that it
should break up into mucous fragments.

When the clear viscid expectoration from uncompli¬
cated cases of whooping-cough is allowed to stand for an

1 Centralbl. f. Bcikt. u . Parasitenk ., Sept. 15, 1897, xxii., Nos. 8 and 9,
p. 222.

2 Deutsche vied. Woch ., 1897, No. 57, p. 586, and Centralbl . f Bakt. u.
Parasitenk ., Dec. 22, 1897, xxii., Nos. 22, 23, p. 641.

476

WHOOPING - CO UGH.

477

hour or so it separates into a fluid portion and a mass of
whitish, opalescent, irregularly formed flakes or frag¬
ments. These were selected for study, and were trans¬
planted by means of a platinum-wire hook to the cul¬
ture-media. Czaplewski and Hensel used a rather better
technique than this, and secured purity of the bacteria
in the flakes by transferring them to a test-tube contain¬
ing pepton solution and violently agitating the tube to
wash off foreign bacteria. After washing, the flakes were
sown upon culture-media.

Hydrocele-fluid was found most useful as a culture-
fluid, but particles of sputum were planted upon all
the culture-media, and attempts to cultivate bacteria from
them were conducted both aerobically and anaerobically.
In 13 out of the 16 cases the same bacillus (x) was iso¬
lated. The organism when stained and examined micro¬
scopically appeared as a remarkably short and delicate
bacillus, shorter and more slender than the diphtheria
bacillus, measuring about o. 8-1.7 p in length and about
0.3-0. 4 ft in breadth. When stained it appeared some¬
what granular, and so resembled somewhat the diphtheria
bacillus. Old cultures presented similar involution-forms
to those seen in old cultures of the diphtheria bacillus.
In general the bacillus resembles the organism found by
Afanassiew 1 and others in cover-glass specimens of
whooping-cough sputum, but differs in that spores were
seen several times.

In pure cultures on coagulated hydrocele-fluid the ba¬
cillus forms a finely granular layer of pearl-white color.

On agar-agar the cultures are opaque, pearl-white, and
occur as a thin layer.

The colonies upon agar-agar are whitish by reflected
light, and straw-yellow or deeper olive-green by trans¬
mitted light. They are of an irregularly rounded shape
and are granular.

In gelatin puncture-cultures the growth resembles that
of the streptococcus, forming along the track of the wire

1 St. Petersburger med. Woch 1887, Nos. 39—42.

478

PA THOGENIC BACTERIA. *

a line of finely granular, non-liquefying colonies. Upon
the surface of the gelatin the growth expands so* as to
form the so-called u nail-growth. ”

The colonies upon gelatin have an irregularly circular
form, appear white or straw-yellow by reflected light and
olive-green by transmitted light, and are granular. They
do not liquefy and do not grow to large colonies.

In bouillon after twenty-four hours there was a faint
clouding of the liquid and subsequently a sedimentation
of the bacteria in small clusters. After a week or so
the surface of the medium is covered with a delicate
pellicle, which grows thicker with the passage of time.

The bacillus grows quite well anaerobically. It is
motile.

The bacillus is pathogenic for mice, but does not pro¬
duce characteristic symptoms in any of the experiment-
animals.

In discussing the results of Koplik’s work, and com¬
paring it with their own, which very shortly preceded it,
Czaplewski and Hensel suggest that the bacillus is better
described as a bacterium than as a bacillus. The finely
granular (“fein punktiertes ”) appearance described by
Koplik, in their observations seems to consist of a deeper
staining at the poles of the cells. The growths on
gelatin and on Loffler’s blood-serum mixture correspond
in every way. The agar-agar growths are similar,
though a slight difference in color is noted, and is attrib¬
uted to a difference in the quality of the medium used.
The bouillon culture differs, the description of Czaplewski
and Hensel being as follows: at the end of a day at 37 0
C. the bouillon is scarcely clouded. At the bottom of
the tube is a sharply defined, lentil-like sediment, which
arises in the form of slimy threads when the fluid is
whirled about, and mixes with the fluid when ener¬
getically shaken. Neither bacillus grows on potato.
Koplik’ s bacillus was also peculiar in that it was motile.
Regarding Koplik’ s bacillus as identical with their own,
Czaplewski and Hensel do not agree with him in believ-

WH O OPING- C O UGH.

479

ing it to be the same as. that described by Afanassiew,
and by comparison found the latter to be a much larger,
shorter, more elongate bacillus. Czaplewski and Hen-
sel’s studies embraced 44 cases of whooping-cough, in
which the bacillus was isolated 18 times; 5 cases of
bronchitis, which subsequently developed whooping-
cough, in all of which it was found; and r case of
rhinitis and bronchitis which developed whooping-cough,
and in which it was found on three different occasions.

From the preceding, it will be seen that many scholars
have labored to detect the specific organism of this dis¬
ease. At present several agree upon the presence of«a
certain bacillus in the expectorated matter; but none of
them have yet succeeded in producing the disease or any
modification of it in the lower animals. The specificity
is, therefore, a matter of much doubt, and rests solely
upon the constancy of the presence of the micro-organ¬
ism in the sputum.

INDEX.

Abbe condenser and oil-immersion
lenses, hints as to the use of,
86

Acid, carbolic, value of, as a germi¬
cide, 118

Acids and alkalies, production of,
by bacteria, 54
Actinomyces bo vis, 260
Actinomycosis, 260
fungus of, 261
growth of, 262
in man, 263
of human liver, 264
resemblance of, to tuberculosis,
262

Activity, vital, in bacteria, results
of, 50

Adhesion preparation, 147
Aerobic bacteria, 45
Aerogenic bacteria, 50, 54
Agar-agar as a culture-medium,
129

advantages of, over gelatin,
150

blood, 13 1
hemoglobin, 131
preparation of, 129
sedimentation of, 130
Air, bacteriologic examination of,
164

Hesse’s apparatus, 165
Petri’s filter for, 166
Sedgwick’s expanded tube,
167

value of, 167
micro-organisms in, 164
pathogenic bacteria in, 164
Alexin, 78

Alkali albuminate, Deycke’s, 133
Alkaline blood-serum, 133
31

Alkaloids, animal, 51
putrefactive, 51
Anaerobic bacteria, 45
cultivation of, 153
cultures, Novy’s jars for, 156
Anilin dyes and bacteria, affinity
between, 90
classification of, 90
employment of, in study of
bacteria, 90

for bacteriological work, 91
introduction of, in 1877, by
Weigert, 26

Animals, experimentation upon,
158

inoculation of, with bacteria, 1 59,
160

to secure cultures, 148
Anthrax, 356

animals most frequently affected

by, 356

antitoxin of, 364
bacillus of, 357
cultures of, 359
discovery of, 356
morphology of, 357
other bacilli resembling, 365
pathogeny of, 361
resistant powers of, 363
staining of, 357

susceptibility of, to heat, cold*
etc., 363

foci for the distribution of, 356
immunity to, experiments in de¬
struction of, 364
in cattle, how acquired, 364
measures to prevent the spread

of. 365

means by which infection takes
place, 361

4SI

INDEX.

482

Anthrax, means of protecting ani¬
mals against, 363, 364
microscopic examination of the
various organs in, 362
resistant powers of, 358
spores, 357, 358, 360
symptomatic, 453
bacillus of, 454
cultures of, 455
staining of, 454
precautions to be observed in,
458

protective inoculations in, 456
statistics of, 458
Antiabrin, 70

Anti-infectious substances, 81
Antiphthisin, 236
Antipneumococcic serum, 351
Antiricin, 79
Antisepsis, origin of, 26
Antiseptic action, results of, on
bacteria, 178

value of some of the principal
germicides, 113

value of reagents, determination
of, 1 77

Antistreptococcic serum, 196
Antitoxic serum, preparation of, for
therapeutic purposes, 302
of tetanus, 282
Antitoxin of anthrax, 364
of cholera, 325
of diphtheria, 297
of tetanus, 282
theory of immunity, 78
Antitoxins, 79

action of, upon bacteria, 81
origin of, 80

specific for one disease only, 83
Antituberculin, 237
Arnold’s steam sterilizer, 108
Aromatics, production of, by bac¬
teria, 56

Arthrospores, 35
Ascococcus, 37

Asiatic cholera, spirillum of, 313,
314

Association, effects of, on bacteria,
47

Atmosphere as a factor in the
causation of suppuration,’
183 * <

bacteria in, 183
germs in, number of, 167

Autoclave, in
Trillat, 115

Bacilli, division of, 38
morphology of, 38
motility of, 31

Bacillus aerogenes capsulatus, 463
colonies of, 466
cultures of, 465
gas-production by, 467
infection of man by, 469
morphology of, 465
natural habitat of, 468
origin of, 463
pathogeny of, 468
staining of, 464
symptoms produced in man
by, 470

vital resistance of, 468
anthracis, 357
colony of, 146

gelatin puncture-culture of,
149

spores of, 358

coli communis, 200, 371, 389
a cause of cholera infantum,
396

cultures of, 390, 391
determination of, 396
differentiation from typhoid
bacillus, 398

immunization against, 395
in drinking-water, deter¬
mination of, 172
in yellow fever, 399, 400
pathogeny of, 392-396
for animals, 394
penetration of intestinal
tissue, 392
staining of, 389
varieties of, 39 7
coli immobilis, 390
colon, 389. See Bacillus coli
communis .

INDEX.

Bacillus, comma, discovery 0^29
diphtherias, 284, 288, 289
cover-glass preparations of,
285

growth of, 285, 286
morphology of, 284, 285
staining of, 285
toxin elaborated by, 297
icteroides, 400

antitoxic serum of, 408
cultures of, 402
immunity to, 408
morphology of, 401
pathogenesis of, 402
specificity in man, 404
staining of, 402
toxin of, 404
influenzas, 446
Klebs-Loffler, 284
leprae, 242
growth of, 243
staining of, 242

liquefaciens parvus, colony of,
r45

mallei, 249

cultivation of, 250, 251
staining of, 252

in sections of tissue, 253
Kiihne’s method, 233
Loffler’s method, 252
mesentericus vulgatus, gelatin
puncture-culture of, 149
muscoides, colony of, 146
mycoides, gelatin puncture-cul¬
ture of, 149

oedema maligni, 459, 461
of Bordoni, 243
of bubonic plague, 433
of chicken-cholera, 409
use of, to kill rabbits, 412
of fowl-tuberculosis, 238
growth of, 239
staining of, 239
of Friedlander, 352
of Havelburg, 405
of hog-cholera, 413
of Koplik, 476-478
of malignant edema, gelatin
puncture-culture of, 149

483

Bacillus of mouse-septicemia, 426,
428

of pseudo-diphtheria, 294
of pseudo-tuberculosis, 240
of rhinoscleroma, 273 .
of Sanarelli, 406
of swine-plague, 420, 421
of symptomatic anthrax, 453
inoculation with, 456
virulence of, 456
of syphilis, 256
Van Niessen’s, 257
of tetanus, method of cultivating,
2 77

of typhoid fever, 370
of whooping-cough, 476, 477
of yellow fever, 400
pneumoniae, 352
cultures of, 353
pathogeny of, 354
polypiformis, colony of, 145
proteus vulgaris, 472

colonies of, 472, 473
cultures of, 473, 474
discovery of, 472
found in human body, 475
morphology of, 472
pathogeny of, 474
pyocyaneus, 197

crystal formation by, 199
cultures of, 198
occurrence in human being,
199

pathogeny of, 199
pyogenes fcetidus, 200
radiatus, colony of, 145

gelatin puncture-culture of,
149

suipestifer, 413
suisepticus, 420
tetani, 274

colony on gelatin, 276
cultures of, 277
distribution of, in nature, 278
isolation of, 275
means of entrance into animal
organism, 278
puncture-culture of, 275
resistant powers of, 276

INDEX.

484

Bacillus tuberculosis, blood-serum
culture of, 220

channels by which it enters
the organism, 223
chemotactic property of, 226
difficulty in staining, 210, 21 1
infection by, 222

through the gastro-intestinal
tract, 223

through the placenta, 223
through the respiratory tract,
223

through the sexual appa¬
ratus, 224

through wounds, 224
isolation of, by Koch, 204
pure cultures of, 218
relation of number of, in spu¬
tum to the progress of the
case, 214

staining of, Ehrlich’s method,
211

Koch’s method, 21 1
toxic products of, 229
typhi, 366

abdominalis, 370
gelatin puncture-culture of,
149

and bacillus coli communis,
resemblance between,
371-375

cultures of, 369-375
distribution of, in nature, 368
means of entrance into the
body, 379
morphology of, 366
murium, 423
cultures of, 423
pathogenesis of, 424, 425
staining of, 423
resistant powers of, 369, 370
staining of, 367
in sections, 367

typhoid, means of entrance into
the body, 379

typhosus, as a cause of suppura¬
tion, 200
X, 400

in whooping-cough , 477

Bacteria, absence of, from normal
body-juices and tissues,
43

action of antitoxins upon, 81
aerobic, 45
aerogenic, 50, 54
anaerobic, 45

cultivation of, 153
Botkin’s method, 156
Buchner’s method, 153
Esmarch’s method, 153
Frankel’ s method, 154
Gruber’s method, 154
Hesse’s method, 153
Liberius’ method, 153
Ravenel’s method, 155
Roux’s method, 157
facultative, 45
optional, 45

and anilin dyes, affinity between,
90

and spores, difference between,
35

biology of, 43
changes in cell- walls of, 31
changes undergone by, in process
of staining, 87
chemical analysis of, 30
chromogenesis of, 52
chromogenic, 52
classification of, 40, 50
Cohn’s morphological, 42
colonies of, appearance under
the microscope, 145
in tubes, Esmarch’s instru¬
ment for counting, 17 1
cover-glass preparations for ex¬
amination of, 91
cultivation of, 124
development of, in liquids, 147
distribution of, 43
elimination of, from the body,

entrance of, into the circulation,
62

examination of, in solid or semi¬
solid cultures, 89
growing, apparatus for examina¬
tion of, 89

INDEX. 485

Bacteria, growth of, conditions in¬
fluencing, 45

association with other bac¬
teria,, 47
electricity, 47
light, 46
moisture, 46
movement, 47
nutriment, 45
oxygen, 45
reaction, 46
temperature, 48
.r-rays, 49
in gelatin, 148, 149
in air, 43, 183

determination of, 165
number of, 167
quantitative estimation of,
165

in body-juices and tissues a sign
of disease, 43

influence of anilin dyes on, 30
of nuclear stains on, 30
in ice, 171

injections of, into animals, 158,
159, 160
in milk, 57
in soil, 44, 174

estimation of the number of,
*75

in tissue, Gram’s method of
staining, 97

introduction of, into animals, by
injection, 158-160
in water, 44

apparatus for counting, 169,
170

filtration as a means of di¬
minishing the number of,
172

quantitative determination of,
169, 170
isolation of, 139
liquefaction of gelatin by, 53
locomotory powers of, 31
measurement of, 104
methods of cultivating, 139
Esmarch tubes, 143
Petri dishes, 143

Bacteria, methods of cultivating,
plate-cultures, 140
of observing, 86

microscopic examination of, 145
morphology of, 36
multiplication of, 33
non-chromogenic, 52
non-pathogenic, 57
of specific disease, 182
organization of, 40
parasitic, 49
pathogenic, 57
in the air, 164

means of entrance into the
tissues, 58

peptonization of mik by, 56
photogenic, 50, 56
photographing of, 104
production of acids and alkalies
by, 54

of aromatics by, 56
of disease by, 57
of gases by, 54
of odors by, 55
of phosphorescence by, 56
rate of development of, 33
recognition of, 163
reduction of nitrites by, 56
results of vital activity in, 50
saprogenic, 50
size of, 32

stained or unstained, examina¬
tion of, 87

staining of, in sections of tissue,
94-

Loffler’s method, 95
Pfeiffer’s method, 96
study of, in the stained condition,
90

taken in respiration, 60
that do not stain by Gram’s
method, 99

thermal death-point of, 176
unstained, method of examining,
88

weight of, 33
zymogenic, 50

Bacteriologic examination of air,
value of, 1 67

486 INDEX.

Bacteriologic examination of soil,
174

of water, 169
Bacteriology, history of, 17
Bacterium, 38
definition of, 30

Beef-peptone in preparation of
bouillon, 125
Beggiatoa, 39

Benches, glass, for use in making
plate-cultures, 142
Binary division, 33
results of, 33

Birds, susceptibility of, to experi¬
mental inoculation with
tubercle bacilli, 239
Black-leg, 453. See Anthrax ,
symptomatic.

Black vomit of yellow fever, cause
of, 404

Blood agar-agar, 131
Blood-serum, alkaline, 133
as a culture-medium, 131
Koch’s apparatus for coagulating
aijd sterilizing, 133
mixture, Loffler's, 133
therapy, discovery of, 29
Body -juices, antibactericidal action
of, 77

Bordoni, bacillus of, 243
Botkin’s apparatus for making
anaerobic plate- cultures,
156

Bottle for cultivating tetanus-
bacillus, 278

Bouillon as a culture-medium,
124

preparation of, 124
free from dextrose as a culture-
medium, 137
Brownian movement, 32
Bubonic plague, 433
antitoxin of, 441
bacillus of, 435
cultures of, 435
discovery of, 29
pathogenesis of, 437, 438
forms of, 434
serum, 441

Buchner’s method of making an-

r 0

aerobic cultures, 153

Capillary tubes for securing
definite quantities of blood
in typhoid test, 381-390
Carbol-fuchsin, 100
Carbolic acid, value of, as a germi¬
cide, 1 18

Carmin and hematoxylon, efforts
to facilitate observation of
bacteria by means of, 90
Catgut, sterilization of, 116
Celloidin as an imbedding agent, 94
Cells, phagocytic, 71
Charbon symptomatique, 453.
See Anthrax , symptom¬
atic.

Cheese-poisoning, cause of, 51
Chemotaxis, 71
negative, 75
Chicken-cholera, 409
bacillus of, 409
cultures of, 410
pathogenesis of, 41 1
pneumococcus of, discovery of,
28

Cholera, 31 1
Asiatic, spirillum of, 312
cultures of, 315-318
staining of, 315

effect of vaccination in preven¬
tion of, 325
hog, 413

immunity to, attempts to pro¬
duce, 324
reasons for, 320
infectious nature of, 312
spirillum of, in drinking-water,
means of detecting, 323
pure cultures of, Schottelius’
method of securing, 316
toxic products of the metab¬
olism of, 319

theories as to the cause of, 321,
322

Cilia, 31

Circulation, modes of entrance of
bacteria into, 62

INDEX.

487

Cladothrix, 39 »

Closet, hot-air, 107
Clostridium, 34
Clothing, disinfection of, 120
Cocci, 36

morphology of, 36
Cohn’s classification of the bac¬
teria, 41, 42

Comma bacillus, discovery of,

Cotton, sterile, value of, in bacte¬
riological work, 107
Cover-glass forceps, 93
preparations for general exam¬
ination, 91

Gram’s method for staining,

method of fixing material for
examination, 92
Cover-glasses, cleaning of, 92
Crystal-formation by bacillus pyo-
cyaneus, 199

Cultivation of bacteria, 124
Culture-media, 124
agar-agar, 129
alkaline blood-serum, 133
blood agar-agar, 13 1
blood-serum, 131
bouillon, 124
free from dextrose, 137
Deycke’s alkali-albuminate, 133
Eisner’s, 371
Dunham’s solution, 136
gelatin, 127
glycerin agar-agar, 13 1
hemoglobin agar-agar, 131
liquid, best means of keeping,
127

development of bacteria in,
147

litmus milk, 135

Lbffier’s blood-serum mixture,
133

milk, 135

peptone solution, 136
Petruschky’s whey, 136
potato, 134
potato-juice, 135
sterilization of, 108

Cultures, anaerobic, various meth¬
ods for making, 1 53—1 57
by animal-inoculation, 148
plate-, 140
puncture-, 147

in gelatin, various appearances
of, 149
“pure,” 139
method of making, 146
solid or semi-solid, examination
of bacteria in, 89
stroke-, 147
study of, 139

Culture-tubes, method of filling,
126, 127

method of inoculating, 141
Cumol for sterilization of catgut,
1 16

Czenzynke’s staining-fluid, 447

Davaine’s classification of the
bacteria, 41

Death-point, thermal, 176
Dejecta, sterilization of, 119
Deycke’s alkali-albuminate, 133
Digestive tract, entrance of bac¬
teria into, 58

Diphtheria, antitoxic serum in
treatment of, 302-305
determination of strength
of, 303

preparation of, 302
preservation of, 302
antitoxin, 297

preparation of, 298
bacillus of, 284, 288, 289
discovery of, 29
relation of, to diphtheria, 292
susceptibility of different ani¬
mals to, 293

toxin elaborated by, 297
bacteriologic diagnosis of, 287
in man and in animals, 293,
294

pseudo-, bacillus of, 294
Diplococci, 36, 37
Diplococcus of mumps, 205
cultures of, 206
morphology of, 206

4B8 INL

Diplococcus pneumoniae, 346
cultures of, 347
morphology of, 346
pathogenesis of, 350

Disease, production of, by bacteria,

Disease-germs, isolation and culti¬
vation of, 28

Diseases, acute inflammatory, bac¬
teria of, 182

chronic inflammatory, bacteria
of, 208

specific, bacteria of, 182
toxic, 284

Dishes, Petri, 143

Disinfection of clothing, 120
of furniture, 120
of patients, 122

of the air of the sick-room, 1 1 8
of the skin, 1 15

Disposal of the bodies of persons
dead of infectious diseases,
123

Division, binary, of bacteria, 33
results of, 33

Drinking-water, means of detecting
cholera-spirilla in, 323

Dunham’s solution as a culture-
medium, 136

Dyes, anilin, classification of, 90
introduction of, in 1877, by
Weigert, 26

use of, in study of bacteria, 90

Edema, malignant, 459
bacillus of, 459
cultures of, 461
pathogenesis of, 460
staining of, 461

Ehrlich’s method of demonstrating
the presence of tubercle
bacilli in sputum, 212
of staining tubercle bacilli in
sections of tissue, 216
solution, 97

Electricity, influence of, on growth
of bacteria, 47

Elimination of bacteria from the
body, 63

Elsper’s culture-medium, 371
method of separating bacillus
typhi and bacillus coli com¬
munis, 371

Endocarditis, ulcerative, production
of, by injection of staphylo¬
coccus pyogenes aureus, 189
Endospores, 34
Enzymes, tryptic, 54
Epidemic parotitis, 204
Erysipelas, streptococcus of, 194
Esmarch tubes, 143
Esmarch’s instrument for counting
colonies of bacteria in tubes,
171

method of making anaerobic
cultures, 153

Examination, bacteriologic, of air,
164

of soil, 174
of water, 169, 170
Excreta, disinfection of, 120
Exhaustion theory of immunity,
70

Experimentation upon animals,
i58

Factoks of diphtheria toxin, 300
Farcin du boeuf, 270

streptothrix of, 270, 271
Fermentation, 50
-tube, Smith’s, 55

Fetus, infection of, through the
placenta, by the bacillus
tuberculosis, 223

Fever, relapsing, 431. See Relap¬
sing fever.

splenic, 356. See Splenic fever.
typhoid, 366. See Typhoid fever.
yellow, 399. See Yellow fever.
Filter, Kitasato’s, 1 12
Pasteur-Chamberland, 1 1 1
Petri’s, for air-examination, 166
Reich el’s, 112

Filters, porcelain, sterilization of,
90

Filtration of culture-media, no
various substances used for,

INDEX.

Filtration of toxins, apparatus for,
1 1 2

Fiocca’s method of staining spores,

IOI

Fission, 33
Flagella, 31
staining of, 101

conditions essential to success
in, 103

Pitfield’s method, 103
Forceps, cover-glass, 93
Formaldehyde as a germicide, 114
in disinfection of rooms, 199, 122
regenerator, 115
Formalin, 114

in disinfection of rooms, 122
use of, as a disinfectant, 1 1 5
Fowl-tuberculosis, bacillus of, 238
Frankel’ s instrument for obtaining
earth from various depths
for bacteriologic study, 174
method of making anaerobic
cultures, 154

Friedlander’s method of staining
bacteria in tissue, 97
pneumonia bacillus, 352
cultures of, 353
pathogeny of, 354
Funnel for filling tubes with cul¬
ture-media, 126
Furniture, disinfection of, 121

Gabbett’s method of demonstrat¬
ing the presence of tuber¬
cle bacilli in sputum, 214
■Gases, production of, by bacteria,
54

determination of, 54
Gelatin as a culture-medium, 127
growth of bacteria in, 148, 149
growth, microtome section of,
159, 160

liquefaction of, by bacteria, 53
Generation, spontaneous, doctrine
of, 18

“ Germ theory” of disease, 23
Germicidal value of gaseous re¬
agents, determination of,
180

489

Germicidal value of reagents, deter¬
mination of, 178
Koch’s method, 178
Sternberg’s method, 179
Germicides, chemical action of,

11 7

value of different, 113, 144
Glanders, 248
bacillus of, 249
cause of, 248

injections of mallein in, 254
Glassware, sterilization of, 106
Glycerin agar-agar as a culture-
medium, 131

-gelatin as an imbedding me¬
dium, 85

Gonococci, cultivation of, 202
Gonococcus, 201
in urethral pus, 201
Gonorrhea, 201

communication of, to animals,
203

Gram’s method of staining bacteria
in tissue, 97

of staining cover-glass prepa¬
rations, 99

solution for staining bacteria in
tissue, 98

Gruber’s method of making an¬
aerobic cultures, 154

Hands, disinfection of, 115
Hanging-drop method of examin¬
ing living micro-organisms,
88

Havelburgs bacillus, 405
Heat, moist, in sterilization of ap¬
paratus used in experi¬
mentation, 106

use of, ift sterilization of instru¬
ments, etc., 106
Hemoglobin agar-agar, 13 1
Hesse’s apparatus for collecting
bacteria from the air, 165
method of making anaerobic
cultures, 153

Heyroth’s instrument for counting
colonies of bacteria in Petri
dishes, 170

490 INDEX .

Hiss’s culture-media for differentia¬
tion of typhoid bacillus
from allied forms, 372
History of bacteriology, 17
Hog-cholera, 413
bacillus of, 413, 415
culture of, 415
pathogeny of, 417
vitality of, 417
immunity to, 418
lesions of, 414, 415, 417
pathology of, 414
symptoms of, 413
Hot-air closet, 107
Humoral theory of immunity, 75
Hydrant-water, number of bacteria
in, 17 1

Hydrophobia, 306

and tetanus, parallelism existing
between, 307
cure for, 309

incubation period of, 306
treatment of, Pasteur’s system,
309

Hygienic precautions recommen¬
ded for preventing the spread
of tuberculosis, 221, 222
Hypodermic syringes for introduc¬
tion of bacteria into ani¬
mals, 158, 159

Ice, bacteria in, 171
-cream poisoning, cause of, 51
Imbedding in celloidin, 94
in glycerin-gelatin, 95
in paraffin, 95
methods of, 94
Immunity, acquired, 69
and susceptibility, 65, 66
apparent, 67

means of destroying, 67, 68
natural, 66

produced bv antitoxins, length
of 83

-reaction, 323
theories of, 70-83
antitoxin, 78
exhaustion, 70
humoral, 75

Immunity, theories of, phagocy¬
tosis, 70
retention, 70

Immunizing unit, definition of, 303
Incubating oven for use in cultiva¬
tion of bacteria, 1 5 1
Indol, 11, 56, 137, 319,

Infection, bacterial, through the
digestive tract, 58
through the placenta, 62
through the respiratory tract,.

through the skin and super¬
ficial mucous membranes,.

through wounds, 62
Influenza, 446
bacillus of, 446

cultures of, 447, 448
discovery of, 29
pathogeny of, 449
staining of, 447

toxin, effects of, when injected
into animals, 450
“Infusorial life,” 21
Injection of bacteria into animals,
158, 159

Injections of tuberculin, results of,.
231

Instruments, disinfection of, 117
sterilization of, 106
Intra-abdominal and intrapleural
injections for introduction
of bacteria into animals,
160

Intravenous injections for the in¬
troduction of bacteria into
animals, 159

Kashida’s method of differentiat¬
ing between typhoid ba¬
cillus and bacillus coli com¬
munis, 372
Kitasato’s filter, 1 1 1
'* Klatsch praparat,” 147
Kny-Sprague steam sterilizer, 109
Koch-Ehrlich method of demon¬
strating the presence of tu¬
bercle bacilli in sputum, 212

INDEX.

49 1

Koch’s apparatus for coagulating j
and sterilizing blood-serum, j
T33

method of determining the germi¬
cidal value of reagents, 178
new tuberculin, 231-236

injection of, in man, 234
steam apparatus for sterilization
of culture-medium, 108
syringe, 159

Koplick’s bacillus, 476-478
Kuhne’s carbol-methylene blue,
253

Lenses, high-power, use of, 87
low-power, use of, 87
oil-immersion, use of, 87
Leprosy, 241
anesthetic, 247
bacillus of, 242
cause of, 241
discovery of, 28
nodes of, 246
Leptothrix, 33, 39
Leuconostoc, 38

Levelling apparatus for pouring
plate-cultures, 140
Liborius’ method of making anaer¬
obic cultures, 153

Life, spontaneous generation of,
doctrine of, 18

Ligatures, disinfection of, 116, 1 17
Light, influence of, on growth of
bacteria, 46

selection of, in study of bacteria
by means of the microscope,
87

Liquid culture-media, development
of bacteria in, 147
Liquids, sterilization of, 1 1 1
Listerism, 182
origin of, 27

Litmus milk as a culture-medium,
135

Loffler’s alkaline methylene-blue,

blood-serum mixture, 286

as a culture-medium, 133
method of staining flagella, 10 1

Loffler’s method of staining sec¬
tions, 95

Lugol’s solution, dilute, for stain¬
ing bacteria in tissue, 97
Lymphocytes, 71

Madura-foot, 266
cause of, 267
streptothrix of, 268
Malignant edema, 459
Mallein, 254

injections of, in glanders, 254
Measles, 451
bacillus of, 451
cultures of, 452
discovery of, 29
staining of, 451
Meat-infusion, 125
Meat-poisoning, cause of, 51
Merismopedia, 36, 37
Methods of observing bacteria, 86
Methyl- violet, antiseptic value of,
destructive and inhibitory,

1 14

Meyer’s bacteriological syringe,
159

Micrococci, 36

Micrococcus gonorrhoese, 201
tetragenus, 200, 443
cultures of, 444
pathogenesis of, 445
staining of, 444

Micro-organisms, living, hanging-
drop method of examina¬
tion, 88

methods of destroying, 105
on the skin, 183

Microscope, essential features of,
86

Microtome sections of gelatin
growths, 149

Milk as a culture-medium, 135
as a medium for the cultivation
of the bacillus diphtheriae,
291

bacteria in, 57

peptonization of, by bacteria, 56
Mineral salts, effect of, in bacterial
cultures, 49

492

INDEX .

Moisture, influence of, on growth
of bacteria, 46
Mouse-holder, 16 1
Mouse-septicemia, 426
bacillus of, 426
cultures of, 427
pathogeny of, 429
staining of, 429
Movement, Brownian, 32

influence of, on growth of bac¬
teria, 47
Mumps, 204

diplococcus of, 205
Mycetoma, 266
streptothrix of, 268
cultures of, 267
Mycoderma, 148
Myconostoc, 39
Myco-phylaxin, 78
Mycoprotein, 30
composition of, 30
Myco-sozin, 78

Nasal mucous membrane, germi¬
cidal power of, 61
Negative chemotaxis, 75
Nitrites, reduction of, by bacteria,

Novy’s jars for anaerobic cultures,
156

Nutriment, influence of, on growth
of bacteria, 45

Odors, production of, by bacteria,

Ophidiomonas, 40
Osteomyelitis, production of, by in¬
jection of the staphylococcus
pyogenes aureus, 189
Oxygen, influence of, on growth
of bacteria, 45
Oxytuberculin, 236

Paraffin as an imbedding me¬
dium, 95

Parotitis, epidemic, 204
Pasteur-Chamberland filter, 1 1 1
Pasteur’s treatment of hydropho¬
bia, 309

Patients, disinfection of, 122
Peptone solution as a culture-me¬
dium, 136

Peptonization of milk by bacteria,

Pest, Siberian, 334. See Anthrax.
Petri’s dishes, 143

filter for air-examination, 166
Petruschky's whey, 136
Pfeiffer’s method of staining sec¬
tions, 96
Phagocytes, 71

Phagocytosis theory of immunity,
70

Phenolphthalein as a test for reac¬
tion of culture-media, 125
Phosphorescence, production of, by
bacteria, 56

Photogenic bacteria, 50, 56
Phylaxins, 78
Pigment-production, 52
Pig typhoid, 413

Pitfield’s method of staining fla¬
gella, 103

Placenta, entrance of bacteria
through, 62

Plague, bubonic, 433. See Bubonic
plague.

Plate-cultures, 140
anaerobic, Botkin’s apparatus for
making, 156

apparatus for making, 140
drawbacks to, 142
method of making, 14 1
Pneumobacillus, 352
as a cause of suppuration, 200
of Frankel and Weichselbaum,
200

Pneumonia, 345
bacillus of, 352, 353
catarrhal, 354
lobar or croupous, 345
diplococcus of, 346
cultures of, 347
morphology of, 346
pathogenesis of, 350
tubercular, 354

Pneumonias, complicating, 355
mixed, 355

INDEX.

493

Potato as a culture-medium, 134
-juice as a culture-medium, 135
Preparations, cover-glass, 91, 92
staining of, Gram’s method, 99
Pseudodiphtheria, bacillus of, 294
relation of, to diphtheria, 295
Pseudotuberculosis, 239
bacillus of, 240
Ptomaines, definition of, 51
Pump-watei, number of bacteria
in, 17 1

Puncture-cultures, r 47
gelatin, various appearance of,

149

Pus, urethral, gonococcus in, 201
Putrefaction, 50

Ouarter-evil, 453. See Anthrax,
symptomatic.

Rabbits, method of making intra¬
venous injections into, 1 59
Rabies, 306. See Hydrophobia .
Rauschbrand, 453. See Anthrax ,
symptomatic .

Ravenel’s method of making an¬
aerobic cultures, 155
Ray-fungus, 261

Reaction, influence of, on growth
of bacteria, 46

Reagents, determination of anti¬
septic value of, 1 77
germicidal value of, 178
Reichel’s filter, 1 1 1
Relapsing fever, 431
spirillum of, 431

pathogenesis of, 431, 432
staining of, 431

Respiratory tract, entrance of bac¬
teria into, 60

Results of vital activity in bacteria,
50

chromogenesis, 52
fermentation, 50
liquefaction of gelatin, 53
production of acids and
alkalies, 54
of aromatics, 56
of disease, 57

Results of vital activity in bacteria,
production of gases, 54
of odors, 55

of phosphorescence, 56
putrefaction, 50
reduction of nitrites, 56
Retention theory of immunity, 70
Rhinoscleroma, 273
bacillus of, 273

River-water, number of bacteria in,

, 171

Roux’s bacteriological syringe, 159
method of cultivating anaerobic
bacteria, 157

Salkowski’s method of determin¬
ing indol in cultures of ba¬
cillus coli, 392

Salt solution as a disinfectant, 117
Sanarelli’s bacillus, 400. See Ba¬
cillus icteroides.

Saprogenic bacteria, 50
Saprophytes, 49
Sarcina, 36, 37
Schaumorgane, 470
Schottelius’ method of securing
pure cultures of the chol¬
era spirillum, 316

Sedgwick’s expanded tube for air-
examination, 167
Septic diseases, the, 431
Serum, anti-streptococcus, 176
antitoxic, of anthrax, 364
of cholera, 325, 326
of diphtheria, 302
of tetanus, 282

-test for typhoid fever, 382, 383
Siberian pest, 453. See Anthrax .
Sick-room, disinfection of, 11S
Skin and mucous membranes, en¬
trance of bacteria through,
61

disinfection of, 1 1 5
Smith’s fermentation-tube, 55
Soil, bacteria of, important, 175
bacteriologic examination of, 174
Solution, Dunham’s, 136

Ehrlich’s anilin-water gentian-
violet, 212

494

INDEX.

Solution, Ehrlich’s, for staining bac- ; Spirpchaeta febris recurrentis, 431
teria in tissue, 97 Spiromonas, 40

Solutions, disinfecting, uselessness Spirulina, 40

of, in the sick-room, 1 1 8 Spleen, influence of, on the vital
staining-, 92 resistance to disease, 68 •

Sozins, 78 Splenic fever, 356

Spirilla, 39 Spores, 34

morphology of, 39 and bacteria, difference between,

of Philadelphia waters, 344 '35

resembling the cholera spirillum, destruction of, by intermittent
326 sterilization, 109

Spirillum aquatilis, 341 in the atmosphere, 183

Berolinensis, 335 presence of, in atmospheric dust,

Bonhoffi, 339 105

cholera Asiatica, 3 13-3 19 resistant power of, 35

characteristics of, 319 staining of, 35, 100

cultures of, 315-318 Fiocca’s method, 101

differentiation of, in cultures, Sporulation, 33, 34

323. 324

distribution of, 319
in drinking-water, means of
detecting, 323
inoculation forms of, 314
production of indol by, 319
resistant powers of, 322
staining of, 315
toxic products of the metab¬
olism of, 319
Danubicus, 337
Denecke, 330
cultures of, 330
Dunbar, 337
Finkler and Prior, 326
cultures of, 327, 328
staining of, 329
Gamaleia, 332

cultures of, 333, 334
of Gamaleia, differentiation of,
from spirillum of cholera,
335

pathogenesis of, 335
Metschnikoff, 332
Milleri, 341

of Asiatic cholera, 3 1 3 — 3 1 9
terrigenus, 342
Weibeli, 340

I. of Wernicke, 338

II. of Wernicke, 338
Spirochaeta, 39

diagram illustrating, 34
Sputum, tubercle bacilli in, 210
demonstration of, 210, 21 1
Sputum-cup, sanitary, 120
Staining bacteria in sections of
tissue, 94-97
Loffler’s method, 95
Pfeiffer’s method, 96
cover-glass preparations, Gram’s
method for, 99
flagella, method of, 101
Pitfield’s method, 103
fluid, Czenzynke’s, 447
of tubercle bacilli in sections of
tissue, 216
solutions, stock, 92
spores, 100

Fiocca’s method, 101
Staphylococci, 37

Staphylococcus epidermidis albus,

183

“golden,” 184
pyogenes albus, -184

distribution of, in nature,
185

growth of, 186, 187
staining of, 186
aureus, 185
citreus, 189

Steam, sterilization of culture-media
by, 108

INDEX.

495

Steam sterilizer, Kny-Sprague, 109
superheated, for quick steriliza¬
tion of culturermedia, no
Sterilization and disinfection, 105
fractional, 108
intermittent, 109
of. air of the sick-room, 118
of blood-serum, Koch’s appa¬
ratus for, 133
of culture-media, 108
of dejecta, 119

of instruments, etc., used in ex¬
perimentation, 106
of liquids, hi
of porcelain filters, 112
of surgical dressings, ligatures,
etc., 116, 117
Sterilizer, Arnold’s, 108
hot-air, 107
Kny-Sprague, 109
Koch’s, 108
Sternberg’s milk, 177
method of determining germi¬
cidal value of reagents,
179

Stock-solutions for staining, 92
Streptococci, 36, 37
in intestinal canal of infants,

Sucholo-albumin, 418
Sucholotoxin, 418
Suppuration, 182
air as a factor in the causation
of, 183

Suppuration, causes of, 183
Surgery, antiseptic, 182
Sutures, disinfection of, 117
Swine-plague, 420
bacillus of, 420, 421
culture of, 422
pathogenesis of, 422
staining of, 422
lesions in, 421
symptoms of, 421
Symptomatic anthrax, 453
Syphilis, 255
bacillus of, 256
staining of, 255, 256
Van Niessen’s, 257
Syringes, disinfection of, 159
for subcutaneous injections of
bacteria into animals, 158,
159

Temperature, influence of, on
growth of bacteria, 48
Tetanin, 280

193

Streptococcus conglomeratus, 191
diffusus, 191
erysipelatis, 194
as a therapeutic measure in
treatment of tumors, 196
longus, 190
pyogenes, 190
growth of, 19 1
staining of, 190
virulence of, 192
vitality in culture, 19 1
Strepto-diplococcus, 3 7
Streptothrix, 39
Madurae, 268
cultures of, 267, 268
of farcin du boeuf, 270, 271
Stroke-cultures, 147
Subcutaneous injections for the in¬
troduction of bacteria into
animals, 158

Tetano-toxin, 280
Tetanus, 274

and hydrophobia, parallelism
existing between, 307
antitoxic serum of, preparation
of, 282

therapeutic value of, 282
bacillus of, 274
cultures of, 277
discovery of, 29
distribution of, in nature, 278
method of cultivating, 277
-bottle, 278
pathology of, 281
susceptibility to, of different ani¬
mals, 279

-toxin, nature of, 280
preparation of, 281
Tetragenococci, 36, 3 7, 443
Thermal death-point of bacteria,
determination of, 176

INDEX.

496

Toxin elaborated by the bacillus
diphtherias, 297

Toxins, rapid filtration of, appa¬
ratus for, 1 1 2
Toxo-phylaxin, 78
Toxo-sozin, 78
Trillat autoclave, 1 1 5
TR-tuberculin, 233

injection of, in man, 234
objection to, 235
Tubercle bacilli, 209

channels by which they enter
the organism, 223
cultivation of, 217— 221
discovery of, 28
growth of, 219

in sections of tissue, 216, 217
methods of demonstrating
the presence of, 216,
217

in sputum, demonstration of,

21 I, 213

Ehrlich’s method, 212
Gabbett’s method, 214
Koch-Ehrlich method, 2 1 2
Ziehl’s method, 213
pure cultures of, 218
toxic products of, 226, 229
Tubercles, 226, 227, 228
Tuberculin, 230
action of, 230
Koch’s new, 231-236
preparation of, 231
result of the injection of, 231
TO, 233
TR, 233

Tuberculosis, 208
bacillus of, 209-229. See Tuber¬
cle bacilli .
discovery of, 28
fowl-, 238
gallinarum, 238

hygienic precautions recom¬
mended for preventing the
spread of, 222, 223
latent, 229

macroscopic lesions of, 224
Tuberculous patients, sanitary
sputum-cup for use of, 120

TubeA Sedgwick’s, for air-examina¬
tion, 167

Tubes, Esmarch, 143

for securing definite quantities
of blood for typhoid test,
384-390

Tumors, treatment of, by inocula¬
tion with the streptococcus
erysipelatis, 196

Tyndall on the “ germ theory ” of
disease, 25

Typhoid fever, 366
bacillus of, 366
cultures of, 369
differentiation of, from bacil¬
lus coli communis, 37 1—

379> 4°2
discovery of, 28
resistant powers of, 368
staining of, 367
comparative immunity of ani¬
mals to, 379-382
inoculation experiments on
animals, 379
prophylaxis in, 379
Widal's serum-test for, 386,
387

Pig-, 4i3

serum, action of, 381

Typhotoxin, 378

Tyrotoxicon, 51

Unna’s method of staining tubercle
bacilli in sections of tissue,
216

Van Niessen’s syphilis-bacillus*
257

Vibrio, 39

Schuylkiliensis, 342
colonies of, 342
growth of, 343
pathogeny of, 343

Vital activity in bacteria, results of*

5°

Water, bacteria in, quantitative
determination of, 169
bacteriologic examination of, 169

INDEX .

497

Whey, Petruschky’s, 136 *

Whooping-cough, 476
bacillus of, 476, 477
bacillus X in, 477

Widal serum-test for typhoid fever,
382, 383

Wolf huge!’ s apparatus for count¬
ing colonies of bacteria upon
plates, 169

Wooden tongue, 265

Wounds, unprotected, entrance of
bacteria into, 62

-Y-rays, effects of, on growth of
bacteria, 49

Yeast-plant as the cause of fer¬
mentation, discovery of, 27
S2

Yellow fever, 399

antitoxic serum of, 408
bacillus of, 400. See Bacillus
icteroidcs .

coli communis in, 399, 400
bacillus X in, 400
cause of black vomit in, 404
causes of death in, 403
Havelburg’s bacillus in, 405
pathology of, 405

Ziehl’s method of demonstrating
the presence of tubercle ba¬
cilli in sputum, 213

Zooglea, 148

Zoph’s classification of the bacteria,
4i

Zymogenic bacteria, 50

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The name of the author is so well known in connection with advanced aural
surgery, that one approaches this volume with feelings of the greatest anticipation,
feelings which are truly satisfied, for there exists but one or two works on Aural
Surgery which can compare with it, and they are all of slightly older issue. This
volume is by far the most scientific work of its kind. It is complete, full of detail,
and exhibits at the same time the knowledge and skill of the writer, and his apti¬
tude in teaching the same. ...

The portion of the work devoted to Anatomy and Physiology is exceptionally
clearly rendered, the plates being excellent as well as numerous.55 — Treatment.

DEXTER. — The Anatomy of the Peritonaeum. By Franklin

Dexter, M.D., Assistant Demonstrator of Anatomy, College of Physicians
and Surgeons (Columbia University), New York. With 3S full-page illustra¬
tions in colours. Price 6s net.

DORLAND— A Manual of Obstetrics. By W. A. Newman

Dorland, A.M., M.D., Assistant Demonstrator of Obstetrics, University of
Pennsylvania ; Instructor in Gynaecology in the Philadelphia Polyclinic ; one
of the consulting Obstetricians to the South-Eastern Dispensary for Women ;
Fellow of the American Academy of Medicine. With 163 illustrations in the
text, and 6 full-page plates. 760 pages. Price 12s net.

e< Among the many recent manuals of midwifery— and truly their name is legion
— the work now under review deserves more than a passing notice. In all its parts
the book shows evidence of great care and up-to-dateness, a remark which applies
even to the somewhat recondite matters of foetal disease and deformity, often very
inadequately discussed in obstetric text-books. By the help of paragraphing,
italicising, and numbering, the information is made easy of access to the
busy practitioner, and the diagnostic tables, of which there are many, will doubt¬
less serve a useful end. The illustrations are plentiful and good.55 — Scottish Medi¬
cal and Surgical Journal.

DRUMMOND. — Diseases of Brain and Spinal Cord;

Their Diagnosis, Pathology, and Treatment. By David Drummond, M.A.,
M.D., T.C.D., et Dnnelm, Physician to the Infirmary, Newcastle-on-Tyne.
Svo, 300 pages, with 50 illustrations. 10s 6d.

FICK.— Diseases of the Eye and Ophthalmoscopy. A Hand¬
book for Physicians and Students. By Dr. Eugene Fick, University of
Zurich. Authorised Translation by A. B. Hale, M.D., Assistant to the Eye
Department, Post-Graduate Medical School, and Consulting Oculist to
Charity Hospital, Chicago ; late Vol. Assistant, Imperial Eye Clinic, Univer¬
sity of Kiel. With a Glossary and 157 illustrations, many of which are in
colours. Octavo. 21s net.

Pick represents the ambitious Zurich school, a middle ground between the
german and French schools. This book takes an entirely unoccupied place in
brerman literature. It is compact, thorough, and exhaustive, has no padding in
e way o statistics or unnecessary pathology. Its physics is clearer and more
or er y than m the majority of books, while its arrangement is far superior and
more logical. The treatment is modern, simply and plainly given. Disputed
ancl special operations do not occupy an unequal amount of space. The translator
will also assume the role, of editor, and adapt the text, when necessary, to
American and English methods, and has added sections on skiascopy, etc. Dr.
contributed some special notes for this edition.

^ vf v?^ume before us has been written by the author, because he is of opinion
that the best text-books of ophthalmology are too exhaustive, and he has en¬
deavoured to supply the student with a compact treatise, in which pathological
statements and hypotheses, as well as authorities, should be referred to, only so
far as they may be necessary to illustrate diseased conditions, and which might
prove supplementary and complementary to the clinical study of diseases. The
translation, we may say at once, is creditable to Dr. Hale of Chicago. It reads
easily, and is, as a rule, satisfactory. ...

“ The treatise is divided into two parts, the first dealing with the methods of ex¬
amination, including the means of determining the acuteness of vision and errors
of refraction, the sense of light and of colour, the field of vision, and the tests for
binocular vision and for strabismus, and giving also an account of the objective
methods of examination, such as keratoscopy, oblique illumination, and the use of
the ophthalmoscope. The second part is devoted to the diseases of the eye,
which are considered in the usual topographical order, each being preceded by a
short account of the histology of the part. The observations made by the author
are, as a rule, those of an unprejudiced mind, and although they might, in some
instances, have been extended with advantage, yet they are sufficiently intellig¬
ible. ...

“ The book is a valuable one, and represents truthfully and well the

PRESENT STATE OF OPHTHALMIC SCIENCE AND PRACTICE.” — Lancet.

FROTHINGHAM. — A Guide to the Bacteriological Labora¬
tory. By Langdon Frothingham, M.D. Illustrated. Price 4s
net.

The technical methods involved in bacteria culture, methods of staining, and
microscopical study are fully described and arranged as simply and concisely as
possible. The book is especially intended for use in laboratory work.

GARRIGUES.— Diseases of Women. By Henry J. Garrigues,

A.M., M.D., Professor of Obstetrics in the New York Post-Graduate Medical
School and Hospital ; Gynaecologist to St. Mark’s Hospital, and to the
German Dispensary, etc., New York City. In one very handsome octavo
volume of about 700 pages, illustrated by numerous wood-cuts and coloured
plates. Price, cloth, 21s net.

A practical work on Gynaecology for the use of students and practitioners,
written in a terse and concise manner. The importance of a thorough knowledge
of the anatomy of the female pelvic organs has been fully recognised by the
author, and considerable space has been devoted to the subject. The chapters on
Operations and on Treatment are thoroughly modern, and are based upon the
large hospital and private practice of the author. The text is elucidated by a
large number of illustrations and coloured plates, many of them being original,
and forming a complete atlas for studying embryology and the anatomy of the
female genitalia , besides exemplifying, -whenever needed, morbid conditions,
instruments, apparatus, and operations.

Excerpt of Contents.

Development of the Female Genitals — Anatomy of the Female Pelvic Organs —
Physiology — Puberty — Menstruation and Ovulation — Copulation — Fecundation —
The Climacteric — Etiology in General — Examinations in General — Treatment in-
General — Abnormal Menstruation and Metrorrhagia — Leucorrhea — Diseases of .
the Vulva — Diseases of the Perineum — Diseases of the Vagina — Diseases of the
Uterus — Diseases of the Fallopian Tubes — Diseases of the Ovaries — Diseases of
the Pelvis — Sterility.

The reception accorded to this work has been most flattering. In the short period
which has elapsed smce its issue, it has been adopted and recommended as a text¬
book by more than sixty of the Medical Schools and Universities of the United
States and Canada .

“ One of the best text-books for students and practitioners which has been pub¬
lished in the English language ; it is condensed, clear, and comprehensive. The
profound learning and great clinical experience of the distinguished author finds
expression in this book in a most attractive and instructive form. Young practi¬
tioners, to whom experienced consultants may not be available, will find in this
book invaluable counsel and help. ”

Thad. A. Reamy, M.D., LL.D.

Processor of Clinical Gynaecology , Medical College of Ohio ; Gynecologist
to the Good Samaritan and Cincinnati Hospitals.

GOULEY. — Diseases of the Urinary Apparatus, Phleg-masic
Affections. By John W. S. Gouley, M.D., Surgeon to Bellevue
Hospital. 355 pages. Price 7s 6d.

HARE. — Practical Diagnosis. The use of Symptoms in the
Diagnosis of Disease. By Hobart Amory Hare, M.D., Professor of Thera¬
peutics and Materia Medica in the Jefferson Medical College of Philadelphia,
Laureate of the Medical Society of London, of the Royal Academy in
Belgium, etc., etc. Second Edition revised and enlarged. In one octavo
volume of 605 pages, with 201 engravings, and 13 coloured plates. Price, 21s
n®t.

“ The rapidity with which a second edition has followed the first in little over
a year, is the best possible proof of the success of this book. Dr. Amory 'Hare has
the gift of making whatever he writes interesting, and those unacquainted with
his work could not have a better introduction than this volume. It will prove of
most value to those recently qualified, but useful and suggestive to almost every¬
one. The general arrangement is to take the various parts of the body one after
another, and describe the abnormalities of signs and symptoms associated with
each. Separate chapters are devoted to the Face and Head, Hands and Arms,
Feet and Legs, and so on, with special chapters interpolated on subjects that need
fuller treatment, such as Hemiplegia and Convulsions. The illustrations are well
chosen, and good in themselves. In the chapter on the Face and Head, there are
in close juxtaposition excellent figures of a mouth-breather with post-nasal
growths, a cretin, an acromegalic, a patient with myxcedema, syphilitic ptosis,
and exophthalmic goitre, with short, pithy descriptions of the conditions repre¬
sented. The grouping is unlike that which is ordinarily employed, and is there¬
fore striking. In the chapter on the Hands and Arms there are some good photo¬
graphs and skiagams of gout and rheumatoid arthritis, and progressive muscular
atrophy. Dr. Hare’s large clinical experience and knowledge of students come
out well in the third chapter on the Feet and Legs, where the usual difficulties of
the different forms of paralysis are made as clear as possible by good plates and
tables. . . .

“ The description of the diseases of the eye is very full and good, and the diffi¬
cult subject of diplopia is well treated. There is a long and elaborate chapter on
the skin, giving practically every abnormality met with, and good coloured

diagrams are supplied of the skin areas, corresponding to the different nerve roots
as mapped out by Thorburn, Starr, and Head. The chapter on the Thorax and
its Viscera could not be better done ; it is everywhere obvious that the state¬
ments made are the result of careful thought and experience. The latest methods
of diagnosis in abdominal disease, 4 the gastrodiaphane of Einhorn,5 and Torek’s
gyromele all find their proper place. There is a good clinical account of the
abnormalities of blood and urine, of forms of vomiting, and types of sputa. The
index, forty-six pages in length, is excellent. Dr. Hare is to be congratu¬
lated ON HAVING WRITTEN A MOST STIMULATING AND SUGGESTIVE BOOK.” — Lancet.

“No better criterion of the value of this handsome and beautifully illustrated
volume can be given than the fact that the first edition, which was published in
August, 1896, was so rapidly exhausted that a second edition had to be issued
last September. The book was written as a guide to bedside practice, and that
the profession needed such a book is proved by the welcome given to the first
edition. A striking feature of the book is the wealth of illustration, more especi¬
ally of the appearances, attitudes and deformities characteristic of certain diseases.
For example, pictures of acromegaly, exophthalmic goitre, spastic paraplegia,
paralysis agitans, pseudo-hypertrophic paralysis, hysterical spasm, the ape hand
in progressive muscular atrophy, tabetic ulcer, etc., will enable the reader to
recognise these diseases at a glance. Beautiful coloured pictures of the eye-
ground in health and in certain medical diseases are given. . . .

“ Much can be learnt at a glance from the coloured charts of localisation of
cortical centres, and from the equally beautifully executed diagram showing
course of motor fibres from cerebrum and cord to the periphery. The matter is
worthy of the illustrations, and greater praise cannot be given to it. It is made
very readily accessible by a very full index, which fills more than forty pages.” —
Quarterly Medical Journal.

“ The warm appreciation of Dr. Hare’s treatise, which we expressed in our
columns only a few months since, has been fully justified by the rapidity with
which a second edition has followed upon the first. The profession were in need
of a book dealing with diagnosis from the standpoint of the symptoms, and this
gap has now been satisfactorily filled. The second edition is no mere replica of
the first, but the author has made a substantial addition of material in every
part. We notice also several new engravings. However, it is high testimony to
the care bestowed upon the former edition that the only important alterations in
the latter are of the nature of addition and not of revision. We congratulate the
author on a book that lias been of value to a very varied class of readers, both
students and practitioners.” — Practitioner.

HARE. — A Text-book of Practical Therapeutics, with especial

reference to the Application of Remedial Measures to Disease, and their Em¬
ployment upon a Rational Basis. By Hobart Amory Hare, M.D., B.Sc.,
Professor of Therapeutics and Materia Medina in the Jefferson Medical
College of Philadelphia ; Physician to the Jefferson Medical College Hospital;
Laureate of the Royal Academy of Medicine in Belgium, of the Medical
- Society of London ; Corresponding Fellow of the Sociedad Espanola de
Higiene of Madrid ; Member of the Association of American Physicians ;
Author of “ A Text-Book of Practical Diagnosis,” etc. Sixth Edition, en¬
larged, thoroughly revised and largely rewritten, in one royal octavo volume
of 758 pages. Price, 21s net.

“ The fact that this work has passed through five editions in seven years, and
that a sixth is now called for, is sufficient evidence that not only has a want been
supplied, but that the author has been successful in his endeavours to carry out
his intention of producing a work on therapeutics which should teach a distinct
practical application of remedial agents in the treatment of disease, and their em¬
ployment upon a rational basis.

“ The book is divided into four parts. Part I. is concerned with general thera¬
peutical considerations, modes of administering drugs, dosage, strength and
reliability of drugs, classification of drugs, etc. «

“ Part II., which occupies the main part of the work, is simply headed
‘ Drugs,’ and contains a full description of the various mechanical agents included
under that term, together with their therapeutic measures. This portion is
worthy of the highest praise. After a technical description of the drug, its
source and preparation, follows its physiological action on the various systems of
the body ; then the modes of its elimination ; next any peculiar properties, such
as an antiseptic action or toxic changes from prolonged use, etc. Then we find
the symptoms of poisoning, and the measures to be adopted should such an occur¬
rence present itself. The therapeutics are very plainly and fully considered, and
conclude with ‘ untoward effects’ (if any). Then follow the methods of adminis¬
tration, the doses being given according to the two systems in vogue (apothecaries
and metric), and finally are the * contraindications ’ for the use of each drug.

“In Part III. are described remedial measures other than drugs, and a descrip¬
tion of the methods employed in preparing foods for the sick.

“ Part IV. commences with a consideration of the various diseases, purely
from a therapeutic point of view. Here also will be found a large amount of use¬
ful information. In conclusion, various tables are given — namely, ‘ doses of
medicines,’ ‘tables of relative weights and measures in the metric and apothe¬
caries’ systems’ ; index of drugs and remedial measures, and index of diseases and
remedies.

“ We can thoroughly recommend this book to practitioners and stu¬
dents.” — Lancet.

“We strongly recommend the book as a useful aid to the practical work
of the profession.” — Scottish Medical and Surgical Journal.

“ The work can be strongly recommended to English practitioners, to

WHOM, PERHAPS, IT IS NOT SO WELL KNOWN AS IT UNDOUBTEDLY DESERVES TO BE.”

— Quarterly Medical Journal.

HAYNES. — A Manual of Anatomy by Irving S. Haynes, Ph.D.,
M.D., Adjunct Professor and Demonstrator of Anatomy in the Medical De¬
partment of the New York University, visiting Surgeon to the Harlein Hos¬
pital, etc., etc. With 134 half-tone illustrations and 42 diagrams. 680
pages. Price 12s net.

HEMMETER.— Diseases of the Stomach. Their special Patho¬
logy, Diagnosis and Treatment, with sections on Anatomy, Physiology, Analysis
of Stomach contents, Dietetics, Surgery of the Stomach, etc. In three parts.
By John C. Hemmeter, M.B., M.D., Philos. D., Clinical Professor of Medi¬
cine at the Baltimore Medical College ; Consultant to the Maryland General
Hospital, etc. W'ith many illustrations, a number of which are in original
colours, and a lithograph frontispiece. 1 volume, royal Svo, 788 pages.
Price 30s net.

HIRT. — The Diseases of the Nervous System. A Text-Book for

Physicians and Students. By Dr. Ludwig Hirt, Professor at the University
of Breslau. Translated, -with permission of the Author, by August Hoch,
M.D., assisted by Frank R. Smith, A,M. (Cantab.), M.D., Assistant Physi¬
cians bo the Johns Hopkins Hospital. With an Introduction by William
Osier, M.D., F.R.C.P., Professor of Medicine in the Johns Hopkins Uni¬
versity, and Physician-in-Chief to the Johns Hopkins Hospital, Baltimore.
Svo, 671 pages. With 17S illustrations. Cloth. 21s net.

HOLT. — The Diseases of Infancy and Childhood. For the

Use of Students and Practitioners of Medicine. By L. Emmett Holt, A.M.,
M.D., Professor of Diseases of Children in the New York Polyclinic Attend¬
ing Physician to the Babies’ Hospital and to the Nursery and Child’s Hospital,
. New York ; Consulting Physician to the New York Infant Asylum and to
the Hospital for Ruptured and Crippled. 1 volume of 1134 pages, with 7
• full-page coloured plates and 203 illustrations. Half -morocco gilt. 25s net.

“This is in every way an admirable volume, and we are genuinely pleased to
congratulate Dr. Holt on his work. Its very size led us to expect something of
the nature of a dictionary— a mere book of reference— but we have found it con¬
spicuously free of the stock-in-trade of the wholesale compiler. It is a monument
of labour, and labour not of collation, but the ripe fruit of the many-sided
practical experience of the author himself. It is a book that we can confidently
recommend to every practitioner as the best we know in this department of
medicine, and full of interest and useful suggestiveness from cover to cover. And
when to excellence of matter and style are linked good printing, good paper, and
good binding, we have a most acceptable volume. To the pathologist also there
is a special attraction in the large amount of space devoted to the morbid anatomy
of infantile disease, a subject that receives sparse illustration in existing text¬
books ; lesions are fully described, and by means of numerous drawings, photo¬
graphs, and coloured plates, brought more within the range of those whose duties
withdraw them from the post-mortem room to the bedside. The coloured plate of
acute meningitis is a masterpiece of its kind, and represents most exactly what
we so often see in the deadhouse.

“ Detailed attention is very properly devoted to the question of nutrition,
with its derangements and associated diseases, and great stress laid upon
diet and hygiene, ‘ since in this rather than in drug-giving lies the secret of
success, certainly in all disorders of digestion and nutrition,5 and there is no
more promising field for therapeutic activity than the prevention of disease,
in children. The experience of our large Children’s Hospitals goes far to show
that there are two chief factors in the causation of infantile disease — bad
feeding and squalor. The former we can only hope to remedy by the better in¬
struction of ignorant mothers, and this by medical men whose therapeutic range
is not entirely limited to grey powder and circumcision. We notice with pleasure
a praiseworthy absence of the numerous formulae of food-stuffs that make most
text-books unreadable ; while the graphic chart method brings the essentials of
composition readily to recognition. We quite agree with Dr. Holt that artificial
feeding, as at present ignorantly practised, is the most fertile cause of infantile
disease, and fully endorse his experience that ‘ it is exceedingly rare to find a
healthy child who has been reared in a tenement house, and who has been arti¬
ficially fed from birth.5

“ A most instructive chapter is that on the ‘ Peculiarities of Disease in
Children,5 wdiile another of no less value is devoted to a discussion of Rickets,
with copious illustrations of the incident bony deformities. We should have
said that the antero -posterior curvature of the lower third, of the tibia was much
more frequent than the author suggests, and not necessarily associated with
how legs ; indeed, it is the common and usually the only curvature in those
children who, while kept off their legs, have been nursed on their mother’s lap,
with the leg supported in such a way as to incur the bending strain of the full
weight of the foot. The carbo-hydrate phantom, too, is relegated to the sub¬
servient position it really occupies in the setiology of the disease. The handling
of the system diseases, one and all, leaves but little to he desired. True to the
intention expressed on the title-page, the author caters at once both for the
student and the practitioner; the general principles of treatment are so explained
as to he most helpful to the uninitiated, while many practical hints of the highest
value are to be found on every page. We miss many old friends— to wit, the
fallacies and misstatements handed from author _ to author- and we welcome
many new ones that are usually conspicuous by their absence. On the whole, the
chapters on diseases of the lungs attract us most in this portion of the book.
The statistics of pneumonia and broncho-pneumonia point to a much greater fre¬
quency of pneumonia in infancy than is generally imagined to be the case. In
the first twelve months of life the highly bronchial texture of the lung favours
the peribronchial variety; but after this period, as the vesicular element becomes
relatively more abundant, we find at first a tendency to a mixed process, and after
the third year a great preponderance of the croupous type. Thus it is that

though the pneumococcus Is the infective agent in almost every case of pneumonia
and primary broncho-pneumonia, the anatomical distinction is maintained. In
secondary broncho-pneumonia, however, there is nearly always a mixed infection,
and with the familiar streptococcus are often found Friedlander’s bacillus and
staphylococci, of the specific germ of influenza, diphtheria, pneumonia, or tuber¬
culosis. Dr. Holt is certainly much to be congratulated on his discussions of the
bacterial agencies at work in the production of disease. We have said enough to
convey the very high opinion we have formed of the whole volume, and we con¬
fidently expect that it will rank in the estimation of the profession as one of the
best of many good books that have come to us from across the Atlantic.” —
Practitioner .

HYDE AND MONTGOMERY.— A Manual of Syphilis and
the Venereal Diseases. By James Nevins Hyde, M.D., Professor
of Skin and Venereal Diseases, Hush Medical College, Chicago, and Frank H.
Montgomery, M.D., Lecturer on Dermatology and Genito- Urinary Diseases,
Rush Medical College, Chicago. Profusely illustrated. Price 12s net.

This Manual is intended as a thoroughly practical guide, and represents the
latest knowledge of the Venereal Diseases which are included under the heads of
Syphilis, and Gonorrhoea and its complications, with very complete instructions
for their diagnosis and carefully prepared instructions for their treatment, cure,
and alleviation.

The illustrations (some of which are coloured) have been selected with the
greatest possible care, and with the view of elucidating the text.

“We can commend this Manual to the student as a help to him in his study of
venereal diseases.” — Liverpool Med, -Chi. Journal.

“ The work may safely be recommended, being modern in spirit and concise
and complete.” — The Physician and Surgeon.

INGALS. — Diseases of the Chest, Throat, and Nasal Cava-
ties, including Physical Diagnosis and Diseases of the Lungs, Heart,
and Aorta, Laryngology and Diseases of the Pharynx, Larynx, Nose, Thyroid
Gland, and (Esophagus. By E. Fletcher Ingals, A.M., M.D., Professor of
Laryngology and Practice of Medicine, Rush Medical College ; Professor of
Diseases of the Throat and Chest, North-Western University Woman’sMedical
School ; Professor of Laryngology and Rhinology, Chicago Polyclinic ;
Laryngologist to the St. Joseph’s Hospital and to the Presbyterian Hospital,
etc. ; Fellow of the American Laryngological Association and American
Climatological Association ; Member of the American Medical Association,
Illinois State Medical Society, Chicago Medical Society, Chicago Pathological
Society, etc., etc. Third Edition, revised. With 240 illustrations, in one
volume. Price 21s net.

KELLY. — Operative Gynecology. By Howard A. Kelly, A.B.,M.D.,

Fellow of the American Gynecological Society ; Professor of Gynecology and
Obstetrics in the Johns Hopkins University, and Gynecologist and Obste¬
trician to the Johns Hopkins Hospital, Baltimore; formerly Associate Pro¬
fessor of Obstetrics in the University of Pennsylvania ; Corresponding Mem¬
ber of the Society Obst6tricale et Gynecologique de Paris, and of the Gesell-
schaft Fiir Gebiirtshulfe Zu Leipzig. In two royal octavo volumes, with 24
plates and 590 illustrations. Handsomely bound in half-morocco. Gilt tops.
Vol. I., 580 pages ; Vol. II., 573 pages. Price £3. 3s net.

The author’s aim in preparing this book has been to place in the hands of his
friends who have followed his gynecological work, and before the medical public,
a summary of the various gynecological operations that he has found best in his
own practice. He does not undertake to present a digest of the literature of the

subject and the work is not burdened by numerous references. His claims to
originality lie m his special researches in connection with the operation for sus¬
pension of the uterus, and in the investigation of vesical and ureteral diseases.

1HE WORK COVERS QUITE FULLY THE GENERAL FIELD OF GYNECOLOGICAL SURGERY,
AND IS ENRICHED BY MORE THAN FIVE HUNDRED ORIGINAL ILLUSTRATIONS, WHICH
FROM A SCIENTIFIC AS WELL AS FROM AN ARTISTIC STANDPOINT ARE EQUALLED BY
THOSE OF NO other work extant. Expense and labour have not been spared in
the preparation of the drawings for the illustrations, or in their reproduction for
the books, of which, by their accuracy in detail and clearness in delineation, they
form a very important and valuable part. The work does not appeal to the
gynecological surgeon only, but is one which will be found of inestimable value to
the general practitioner and to the surgeon, whose practices bring them in contact
with gynecological cases. Dr. Kelly has had a long and successful career as a
gynecological surgeon, and his experience has fitted him pre-eminently for the
preparation of a work such as he has written.

KIMPTON’S POCKET MEDICAL LEXICON ; or,
Dictionary of Terms and Words used in Medicine and
Surgery. By John M. Keating, M.D., editor of “Cyclopaedia of Dis-
eases of Children,” etc., author of the “New Pronouncing Dictionary of
Medicine”; and Henry Hamilton, author of “A New Translation of
Virgil’s ^dUneid into English Verse”; co-author of a “New Pronouncing
Dictionary of Medicine.” A new and revised edition. 32mo, 282 pages.
Price, cloth, 2s 6d net.

This new and comprehensive work of reference is the outcome of a demand for a
more modern handbook of its class than those at present on the market, which,
dating as they do from. 1855 to 1884, are of but trifling use to the student by their
not containing the hundreds of new words now used in current literature,
especially those relating to Electricity and Bacteriology.

“Remarkably accurate in terminology, accentuation, and definition.” — Journal
of American Medical Association.

“ Brief, yet complete ... it contains the very latest nomenclature in even
the newest departments of medicine.” — New York Medical Record.

KING’S MANUAL OF OBSTETRICS.— New (7th) Edi¬
tion. A Manual of Obstetrics. By A. F. King, M.D., Pro¬
fessor of Obstetrics and Diseases of Women in the Medical Department of the
Columbian University, Washington, D.C., and in the University of Vermont,
etc. Seventh Edition, revised and enlarged. In one demy 8vo volume of 574
pages, with 223 illustrations. Cloth, 10s 6d net.

“ Prof. King’s Manual is now so well known that the appearance of the sixth
edition calls only for a congratulatory note from the reviewer. A large number of
additional illustrations have been introduced into the work, which is quite worthy
of the high place it has attained in the undergraduate mind.” — Edinburgh Medical
Journal.

“ For clearness of diction it is not excelled by any book of similar nature, and
by its system of captions and italics it is abundantly suited to the needs of the
medical student. The book is undoubtedly the best manual of obstetrics
extant in English.” — The Philadelphia Polyclinic.

“ Prof. King’s Manual has had a remarkably successful career, passing rapidly
from one edition to another. It is just such a work as the obstetrician turns to
in time of need with the assurance that he will in a moment refresh his memory
on the subject. A vast amount of knowledge is expressed in small space.” — The
Ohio Medical Journal.

“ This is undoubtedly the best manual of obstetrics. Six editions in thirteen
years show not only a demand for a book of this kind, but that this particular one
meets the requirements for popularity, being clear, concise, and practical. The^

present edition has been carefully revised, and a number of additions and modifi¬
cations have been introduced to bring the book to date. It is well illustrated, well
arranged ; in short, a modern manual.55 — The Chicago Medical Recorder .

“ Tliis popular manual now appears in the sixth edition. Published originally
in 1SS2, and designed particularly for the students attending the author’s lectures
on obstetrics, the work maintains much of its peculiar character. It cannot be
regarded as more than it professes to be — a manual for students and junior prac¬
titioners. There is no straining after abstruse problems, no elaborate arguments,
nor portentous bibliographies. But, so far as it goes, it is an excellent and
reliable guide to the junior student of midwifery. The language employed is
clear and simple, and there is a healthy dogmatism about the methods of practice
recommended which suggests the sort of teacher a student loves to listen to.
Chapter vi., on Fecundation, is a most valuable one. We do not know of any
work of similar size which treats of the early physiology of pregnancy with equal
lucidity. Young practitioners will derive much help from chapter viii. on the
Diseases of Pregnancy. Palpation of the abdomen for diagnosis of the position of
the fcetus is clearly described at page 193. It would be well if more attention
wTere given to this mode of examination. It is now taught as an important clinical
method in America and on the Continent, but we know that our English students
are less conversant with it than is desirable. The mechanism of labour is well de¬
scribed in accordance with generally accepted beliefs. The chapter on Symphy¬
siotomy will be welcomed by practitioners desirous of knowing the most recent
ideas from America regarding this reviving procedure. Chapter xxii. on Pelvic
Deformities is terse, but clear and practical. We notice that Dr. King adopts the
now favoured treatment of puerperal eclampsia in America by hypodermic injec¬
tions of veratrum viride. There is a useful chapter on the Jurisprudence of Mid¬
wifery, containing many valuable points of information. We welcome this new
edition, which gives a very excellent resume of the main facts of obste¬
trical THEORY AND PRACTICE, AND IS LIKELY TO PROVE AS FULLY POPULAR AS

its predecessors.55 — British Gynaecological Journal.

LOCKWOOD. — Manual of the Practice of Medicine. By

George Boe Lockwood, M.D., Professor of Practice in the Woman’s Medical
College and in the New York Infirmary ; attending Physician to the Coloured
Hospital and to the City (late Charity) Hospital ; Pathologist to the French
Hospital, etc. 935 pages, with 75 illustrations in text, and 22 coloured and
half-tone plates. Price, 12s net.

This manual presents the essential facts and Principles of the Practice of
Medicine in a concise and available form.

LONG. — A Syllabus of Gynaecology, arranged in conformity with
the American Text-Book of Gynecology. By J* W. Long, M.D., Professor of
Diseases of Women and Children, Medical College of Virginia, etc. Cloth
(interleaved). Price 4s net.

“ Based upon the teaching and methods laid down in the larger work, this will
not only be useful as a supplementary volume, but to those who do not already
possess the text-book it will also have an independent value as an aid to the
practitioner in gynecological work, and to the student as a guide in the lecture-
room, as the subject is presented in a manner at once systematic, clear, succinct,
and practical.55

McFARLAND. — Text-Book upon the Pathogenic Bac¬
teria. For Students of Medicine and Physicians. By Joseph
McFarland, M.D. Demonstrator of Pathological Histology and Lecturer on
Bacteriology in the Medical Department of the University of Pennsylvania ;
Fellow of the College of Physicians of Philadelphia ; Pathologist to the Bush
Hospital for Consumption and Allied Diseases. New edition in preparation.

1 5

‘ ‘ In a work of moderate size, the author has succeeded admirably in presenting
the essential details of bacteriological technics, together with a judiciously chosen
summary of our present knowledge of pathogenic bacteria. As indicated in the
preface, the work is intended as an elementary text-book for students of medicine,
but Part II., or Specific Diseases and their Bacteria, will readilv commend itself
to a large class of practitioners who recognise the value of acquaintance with the
behaviour of the bacterial causes of disease, even without a technical knowledge
of bacteriology. It is no unfavourable reflection on the scientific character of the
treatise, moreover, to mention the fitness of this second part for the use of the non¬
professional readers who may be interested in the science or in its bearing on
matters of vital general interest.

“In the Introduction the author has sketched briefly, but in a sufficiently com¬
plete and very interesting way, the history of bacteriology. The chapter on Im¬
munity and Susceptibility is a more than usually successful attempt to briefly out¬
line the present status of this very occult study, and in the discussion of the
various theories presented, the author has not given undue prominence to any of
the tenets. Tuberculosis is considered at comparative length, and all the more
important relations of this subject have received attention in the practical way
best adapted to the class of readers to which the book is addressed.

“Numerous photographic plates illustrate the text in the description of the
various bacterial species. Of these photographs, many are very characteristic.
The author has adhered with considerable uniformity to an easy and correct style
of diction, which is so often lacking in the treatment of very technical subjects.
The work, we think, should have a wide circulation among English-speaking
students of medicine.” — New York Medical Journal.

MAISCH’S Materia Medica. — Sixth Edition. — A Manual of Or¬
ganic Materia Medica : Being a Guide to Materia Medica of the Vegetable
and Animal Kingdoms. For the use of Students, Druggists, Pharmacists,
and Physicians. By John M. Maisch, Phar.D., Professor of M ateria Medica
and Botany of the Philadelphia College of Pharmacy. New (sixth) edition,
thoroughly revised by H. 0. C. Maisch, Ph.G. In one very handsome 12mo
volume of 509 pages, with 285 engravings. Cloth. 10s 6d net.

“ New matter has been added, and the whole work has received careful revision,
so as to conform to the New United States Pharmacopoeia. The great value of
the work is the simplicity of style and the accuracy of each description. It
considers each article of the vegetable and animal pharmacopoeia, and, where
important, sections on antidotes, etc., are added. Several useful tables are incor¬
porated.” — Virginia Medical Monthly.

“ The best hand-book upon pharmacognosy of any published in this country.
The revision brings the work up to date, and is in accord with its previous high
standard.” — The Boston Medical and Surgical Journal.

“We can add nothing to our previous commendatory notices of this standard
text-book of materia medica. It is a work of such well-tried merit that it
stands in no danger of being superseded.” — American Druggist and Pharmaceutical
Record.

OSLER. — Lectures on the Diagnosis of Abdominal Tumours.

Delivered before the Post-Graduate Class, Johns Hopkins University,
By William Osler, M.D., Professor of Medicine, Johns Hopkins University :
Physician-in-Chief to Johns Hopkins Hospital, Baltimore, M.D., Small Svo.
Illustrated. Cloth. 6s net.

“ The volume before us contains six lectures delivered before the post-graduate
course at the Johns Hopkins University, which have already appeared in the
pages of the ‘New York Medical Journal.’ The first two are devoted to
the stomach, the first dealing with tumours formed by the dilated stomach itself,
almost always associated with a nodular mass at the pylorus. Amongst the special
points to which he calls attention in reference to diagnosis may be mentioned the

two kinds of movement that are observable— -namely, a peristalsis that can be seen
in the walls of the stomach, which occurs from left to right ; and, secondly, the
development of irregular protuberances of the stomach wall, generally near the
greater curvature, and often synchronous with the above-mentioned peristalsis.
Another point of importance is the gurgling of gas through the pylorus, which can
sometimes be felt. Inflation constitutes a most valuable aid to diagnosis, and is
best effected by administering half a drachm of bicarbonate of soda in solution,
followed by a similar quantity of tartaric acid, also in solution. In a few cases a
tumour may be formed by a contracted stomach, as in oesophageal obstruction, or
from cirrhosis or diffuse cancer of the stomach walls. The second lecture is de¬
voted to nodular and massive tumours of the stomach, including thereby instances
of thickening and induration round an old ulcer. In none of his cases was a tumour
situated at the cardiac orifice or on the posterior wall. Tumours of the liver form
the subject of the third lecture, cases of abscess, syphilis, and cancer being
described, whilst dilated gall bladder and cancer of the gallbladder are considered
in the fourth. The diagnosis of the latter condition is not always easy, but the
following points would be helpful : Two-thirds of the patients are women, and in
seven-eighths of the cases there is an association with gall stones, so that a history
of colic and previous attacks of jaundice should be sought for. Rapid emaciation
and the development of cachexia within three or four months would favour cancer ;
chills and fevers would be against it ; ascites is often present, but jaundice is not
necessary till the disease spreads to the walls of the duct. The fifth lecture deals
with tumours of the intestine, omentum, and pancreas, and some miscellaneous
cases of obscure origin, whilst the last lecture is devoted to tumours of the kidney,
dealing with movable kidney, which is so common that he says they are never
without an example in the wards, intermittent hydronephrosis, sarcoma of the
kidney, including a very interesting case in which the tumour was successfully
extirpated, and tuberculosis. The lectures are entirely confined to a consideration
of cases that had been under treatment during the preceding twelve months , and we
may congratulate Dr. Osler both on the wealth of his material and on the excel¬
lent use he has made of it. The whole set constitutes a most excellent piece

OP CLTNICAL WORK, AND WE BELIEVE THAT HO PHYSICIAN COULD PAIL TO DERIVE
benefit from a careful perusal of these lectures, which, we may add, are pro¬
fusely illustrated with photographs and diagrams.” — British Medical Journal.

PELLEW. — Manual of Practical Medical and Physiological
Chemistry. By Charles E. Pellew, E.M. Demonstrator of Physics
and Chemistry in the College of Physicians and Surgeons (Medical Depart¬
ment of Columbia College), New York. Honorary Assistant in Chemistry at
the School of Mines, Columbia College, etc. With illustrations, 330 pages.
Price 15s.

PHELPS.— Traumatic Injuries of the Brain and its Mem¬
branes. With a Special Study of Pistol-Shot Wounds of the Head
in their Medico-Legal and Surgical Relations. By Charles Phelps, M.D.,
Surgeon to Bellevue and St. Vincent’s Hospitals. 8vo, 596 pages, with 49
illustrations. Cloth. £1 Is net.

RAYMOND. — A Manual of Physiology. By Joseph H. Rat-

mond, A.M., M.D., Professor of Physiology and Hygiene, and Lecturer on
Gynecology in the Long Island College Hospital; Director of Physiology in
the Hoagland Laboratory ; formerly Lecturer on Physiology and Hygiene in
the Brooklyn Normal School for Physical Education ; Ex- Vice-President of
the American Public Health Association ; Ex-Health Commissioner, City of
Brooklyn, etc. Illustrated. Cloth. Price 6s net.

In this m mual the author has endeavoured to put into a concrete and available
fqsm the results of twenty years’ experience as a teacher of physiology to medical

. u<*en^s>. iias produced a work for the student and practitioner, representing
i^^50nC1Se f°rm ^ existing state of physiology and its methods of investigation,
cased, upon comparative and pathological anatomy, clinical medicine, phvsic, and
chemistry, as well as upon experimental research. A

SAHNnDER’S POCKET MEDICAL FORMULARY. ByWni.

M. Powell, M.D., Attending Physician to the Mercer House for Invalid
Women at Atlantic City. Containing 1,750 Formulae, selected from several
hundred of the best known authorities. Forming a handsome and convenient
pocket companion of nearly 300 printed pages, with blank leaves for Addi¬
tions ; with an Appendix containing Posological Table, Formulae and Doses
for Hypodermatic Medication, Poisons and their Antidotes, Diameters of the
Female Pelvis and Foetal Head, Obstetrical Table, Diet List for Various
Diseases, Materials and Drugs used in Antiseptic Surgery, Treatment of
Asphyxia from Drowning, Surgical Remembrancer, Tables of Incompatibles,
Eruptive Fevers, Weights and Measures, etc. Fourth Edition, revised and
greatly enlarged. Handsomely bound in morocco, with side index, wallet
and flap. Price 7s 6d net.

A concise, clear, and correct record of the many hundreds of famous formulas
which are found scattered through the works of the most eminent physicians and
surgeons of the world. The work is helpful to the student and practitioner alike,
as through it they become acquainted with numerous formulae which, are not found
in text-books, but have been collected from among the rising generation of the
profession, college professors, and hospital physicians and surgeons.

“This volume contains a collection of prescriptions arranged under the head of
various diseases which they are designed to benefit. The diseases are classified
in alphabetical order, and the volume is supplied with a thumb-nail index, which
renders consultation the more easy. The prescriptions given appear to have been
selected with judgment from a large number of sources, and this handbook will
doubtless often be useful in indicating how an unfamiliar drug may best be
prescribed. It will also be of use sometimes in suggesting newlines of treatment,
for there is no doubt that we are all rather disposed to fall into habits in the
matter of drug prescribing.” — British Medical Journal.

“ Designed to be of immense help to the general practitioner in the exercise of
his daily calling.” — Boston Medical and Surgical Journal.

“ An excellent pocket companion, containing the most satisfactory and rational
formulae used by the leading medical men of Europe and America, introducing in
the many prescriptions contained therein a considerable number of the more im¬
portant recently-discovered drugs.” — Southern Practitioner.

SIMON’S CLINICAL DIAGNOSIS. A Manual of Clinical
Diagnosis by means of Microscopic and Chemical Methods.

For Students, Hospital Physicians and Practitioners. By Charles E. Simon-,
M.D., Late Assistant Resident Physician, Johns Hopkins Hospital, Baltimore.
In one very handsome octavo volume of 563 pages, with 133 Illustrations on
wood, and 14 full-page coloured plates. Second Edition, revised and en¬
larged. Cloth, price, 16s net.

“The author sets forth the methods most satisfactory and most approved for
determining pathological conditions by chemical and microscopical examinations.
Without other special training the work will be a guide to the attaining of the
essential facts which only chemistry and the microscope can reveal.” — The Morth
American Practitioner.

“ This is a very much-needed book. It tells the meaning of the clinical
chemistry and results of microscopical examination of a case, and without their
aid it is impossible to master a diagnostic study of many diseases told by the
various secretions and excretions. A most excellent arrangement consists in the
Differential Table of the More Important Diseases, or of the fluid, secretion or
excretion, under consideration — the table being at the end of each subject dis-

cussed. Another excellence of the book consists in the full detail of the technique
as to mode of securing, preparing, and examining specimens. There are so many
practical, helpful points in this book that we must add it to the library which we
regard as essential for the practitioner in his daily round of duties. — The Va.
Med. Semi-Monthly.

“There is little need in the present day to dwell on the value and importance
of the assistance given to clinical diagnosis, and therefore to treatment, by a
thorough microscopical and chemical examination of the products of disease or of
the blood and the various excretions. So important is it that within the past
decade many a work has been published devoted solely to this one branch of
clinical investigation, and there is no medical school where instruction upon it of
a systematic kind is not to some extent imparted. Nevertheless, this necessary
extension of the field of observation is in itself so wide and comprehensive that it
becomes more and more difficult for the practitioner to follow7. It needs a
special department and a staff of highly-trained experts to carry it out to the full;
and it is this class of work which is being so well undertaken in this country by
the Clinical Research Association. The author of the volume before us has en¬
joyed at the Johns Hopkins Hospital, Baltimore, ample opportunities for the
study of the subject, and his treatise is in every respect excellent. Covering

PRACTICALLY THE SAME GROUND AS THE WELL-KNOWN WORK OF PROFESSOR VON
JAKSCH, THE BOOK CONTAINS IN SOME SECTIONS EVEN MORE INFORMATION THAN

does that volume. It is evident, too, that the author has himself largely con¬
firmed the statements which he makes, and occasionally he feels bound to differ
from the somewhat too dogmatic teaching that has dominated parts of the sub¬
ject. We have, after a careful review of the contents of the book,

NO HESITATION IN COMMENDING IT AS ONE OF THE BEST AND MOST COMPENDIOUS
MANUALS FOR THE CLINICAL LABORATORY THAT HAS APPEARED. The Sllbject-
matter is arranged on a very systematic plan, the text is not burdened
by references to literature, and the descriptions of apparatus as well as the
instructions for the performance of tests are clear and concise. Perhaps the best
section is that devoted to the urine, occupying about one-half of the volume, but
the sections on the blood and on the gastric juice and gastric contents are little, if
at all, inferior in scope and fulness^ In his preface Dr. Simon pleads for a more
thorough recognition of these studies in places of instruction, and urges the younger
members of the profession to pursue them with diligence. As he says, ‘ It is incon¬
ceivable that a physician can rationally diagnose and treat diseases of the stomach,
intestines, kidneys and liver, etc., without laboratory facilities.’ Whether his
suggestion that physicians might usefully employ a laboratory assistant to enable
them to carry out this duty will ever be realised, time, with its advance of know¬
ledge, can alone show. Lancet.

“ The sciences of chemistry and microscopy, as applied to medicine, are year by
year becoming of great Importance ; and while both form part of every medical
curriculum in the preliminary stages, it is rare to find a medical school in which
they are taught purely from the point of view of their clinical application. Too
often they are learned by the student only to be forgotten as soon as he commences
the ‘ professional’ part of his studies. That the time has come when this state of
things should be altered, and a separate study made of these sciences in their ap¬
plication to diagnosis, will impress all who read Dr. Simon’s volume.

“ It has evidently been the author’s aim in this work to present to students and
practitioners not only the facts of physical science which are of practical import¬
ance, but also the reasons which have led up to that union of empirical deduction
and scientific reasoning of which the modern science of diagnosis largely consists.
Consequently, we find in the volume precise descriptions for the examination of
the various fluids, secretions, and exudates of the body, both in health and disease.
In every case a description of the normal material precedes the pathological con¬
siderations, which latter are in turn followed by a detailed account of the methods
and apparatus used in examination. Following the directions given, no worker
ought to. find any insuperable difficulty in learning to recognise, say, the presence

of tubercle bacillus in sputum, or of the diphtheria bacillus in membranous
exudate.

“ The volume is most appropriately illustrated both by coloured plates and by
woodcuts in the text. We heartily welcome the appearance of the work,

WHICH WE FEEL SURE WILL FIND A PERMANENT .PLACE IN THE WORKING LITERATURE
OF THE PROFESSION, AND WILL ADEQUATELY SUPPLY A WELL-RECOGNISED DEFICI¬
ENCY.” — British Medical Journal.

STARR. — Diets for Infants and Children in Health and in
Disease. By Louis Starr, M.D., Editor of “ An American Text-
Book of the Diseases of Children.” 230 blanks (pocket-book size), perforated
and neatly bound in flexible morocco. Price 6s net.

“The first series of blanks are prepared for the first seven months of infant life.
Each blank indicates the ingredients, but not the quantities, of the food, the
latter directions being left for the physician. After the seventh month, modi¬
fications being less necessary, the diet-lists are printed in full. Formulae for
the preparation of diluents and foods are appended.”

“ We recommend every one who has occasion to treat infants and children to
obtain a copy.” — St. Louis Med. and Surg. Journal.

“The work on the whole will commend itself highly to the practitioner.” —
Archives of Pediatrics.

STEVENS. — A Manual of Practice of Medicine. By A. A.

Stevens, A.M., M.D., Instructor of Physical Diagnosis in the University of
Pennsylvania, and Demonstrator of Pathology in the Women’s Medical
College of Philadelphia. Specially intended for students preparing for gradua¬
tion and hospital examinations, and includes the following sections : General
Diseases, Diseases of the Digestive Organs, Diseases of the Kespiratory

. System, Diseases of the Circulatory System, Diseases of the Nervous System,
Diseases of the Blood, Diseases of the Kidneys, and Diseases of the Skin.
Each section is prefaced by a chapter on General Symptomatology. Third
Edition. Post 8vo, 502 pages. Numerous illustrations and selected formulae.
Price 6s net.

“ Contributions to the science of medicine have poured in so rapidly during the
last quarter of a century that it is well-nigh impossible for the student, with the
limited time at his disposal, to master elaborate treatises or to cull from them that
knowledge which is absolutely essential. From an extended experience in teach¬
ing, the author has been enabled, by classification, to group allied symptoms, and
by the judicious elimination of theories and redundant explanations to bring
within a comparatively small compass a complete outline of the practice of
medicine.”

TAYLOR. — A Practical. Treatise on Sexual Disorders of the
Male and Female. ' By Kobert W. Taylor, A.M., M.D.,
Clinical Professor of Venereal Diseases at the College of Physicians and
Surgeons (Columbia College), New York ; Surgeon to Bellevue Hospital, and
Consulting Surgeon to the City (Charity) Hospital, New York. In one
octavo volume of 451 pages. With 73 illustrations and 8 plates in colour
and monotone. Price 12s net.

“The branch of surgery with which this work deals is one about which very
little is said in most text-books of surgery, and yet its importance is by no means
small, for whether we consider the frequency with which such cases present them¬
selves, or the amount of unhappiness which results from them, it is very obvious
that they are worthy of the most careful attention of the surgeon. Dr. Taylor
deals in the first place with the anatomy and physiology of the male sexual ap-

paratus, and it is interesting to note that he is inclined to accept the result of the
researches of Professor George S. Huntingdon, who asserts that the vesiculae
seminales never contain semen, and that they do not act as places of storage of
this fluid, but they provide a special form of mucus to dilute and carry on the
semen. Impotence and sterility in the male, are thoroughly considered, and a
chapter is devoted to the mental effects of sexual disorders. With regard to
sterility in the male, the author thinks that probably in one case in six of unfruit¬
ful marriages this is the cause. The second half of the book deals with sexual
disorders in the female. The final chapter treats of a peculiar new growth of the
vulva, three examples of which Dr. Taylor has seen. He has already written on
this condition in the American Journal of the Medical Sciences . In some respects
it resembled a tertiary syphilitic condition, but potassium iodide seemed to have
no effect upon it, and microscopically it appeared to be inflammatory. The
VOLUME IS A TRUSTWORTHY TREATISE ON A DIFFICULT SUBJECT.55 — Lancet .

THAYER. — Lectures on the Malarial Fevers. By William

Sydney Thayer, M.D. , Associate Professor of Medicine in the Johns Hopkins
University. Small Svo, 326 pages. With 19 charts, and 3 lithographic
plates showing the Parasite of Tertian, Quartan, and iEstivo-Autumnal
Fevers. Cloth, 12s net.

THOMAS. — Abortion and its Treatment: From a Standpoint
of Practical Experience. By T. Gaillard Thomas, M.D., Emeritus Prof,
of Obstetrics and Gynaecology. Crown Svo, 5s.

THOMPSON. — Practical Dietetics, with Special Reference to
Diet in Disease. By W. Gilman Thompson, M.D., Professor of Materia
Medica, Therapeutics, and Clinical Medicine in the University of the City
of Hew York ; Visiting Physician to the Presbyterian and Bellevue Hospitals,
New York. Large Svo, 830 pages, illustrated. Cloth. Price 21s net.

<c We quite agree with the author that the subjects which are so fully discussed
in this volume are frequently dismissed in brief and indefinite phrases by the
writers upon the theory and practice of medicine. . . . The fact that the author
has written a successful book is due not only to his knowledge as a chemist and
his studies as a physiologist, but as well as to the fact that he is a practical physi¬
cian. . . . On the whole, the book shows that the author has industriously col¬
lected the best opinions upon the subject, that he has drawn from the results of
his own experience, that he has endeavoured to bring the findings of the laboratory
into practical relations with the observations of the consulting- room, and, finally,
to produce a hook of value to the practising physician. W e believe that he has
succeeded admirably in presenting a useful and readable book.55 — The American
Jo urnal of the Medical Sciences.

“ The book will be of great assistance to the practitioner in the dietetic treat¬
ment of diseases that are influenced by proper feeding to the trained nurse in
hospital and private nursing, and as a guide in the administration of proper food'
to infants and invalids in the home.55 — College and Clinical Record.

“It is a great pleasure to welcome Dr. Thompson’s work on dietetics. For a
long time we have longed for a book giving detailed and accurate information as
to foods, their nutritive values, and their appropriate uses in disease. Other
books have appeared, written by English and Continental writers, but they have
not been suited to American needs. . . . The book is encyclopedic in its com¬
pleteness. . . . We recommend it most heartily. It fills a place in medicine
more important even than therapeutics, and one which has been too much
neglected.55 — University Medical Magazine.

“ Fewer subjects in medicine present greater difficulties to an author than that
of dietetics ; and Dr. Thompson has done the profession a service in collecting so
much information on this subject, and presenting it in so systematic and attrac¬
tive a manner.” — Boston Medical and Surgical Journal.

The work is so complete, and has been so systematically prepared, that it is
almost impossible to find a condition in which some benefit cannot be obtained
by suitable diet.”— 0 hio Medical Journal.

TILLMAN NS. — A Text-Book of General Surgery. By Dr.

Hermann Tillmanns, Professor in the University of Leipsig. Edited by

Lewis A. Stimson, M.D., Professor of Surgery in the New York University.

8vo. Cloth, £1 Is net, per vol.

Yol. I.— The Principles of Surgery and Surgical Pathology. General Rules
governing Operations and the Application of Dressings. Trans¬
lated from the Third German Edition by John Rogers, M.D.,
and Benjamin T. Tilton, M.D. With 447 Illustrations.

Yol. II.— Regional Surgery. Translated from the Fourth German Edition
by Benjamin T. Tilton, M.D., New York. With 417
Illustrations .

Yol. III. — Regional Surgery. With 517 Illustrations.

Dr. Hermann Tillmanps, Professor of Surgery in the University of Leipsig,
possesses as a teacher those rare qualities which enable him to instruct the student
step by step, beginning by the laying of a firm, broad foundation, upon which is
built the solid surgical structure. It was on account of these exceptional qualities
of the author that his work was selected as the best for the use of students, and
at the same time well adapted to the needs of the practitioner.

Surgery, as presented in the present volumes, is a translation of his works on
General Surgery and Surgical Pathology, and on Regional Surgery. Of the latter
there are two volumes.

Volume 1., General Surgery and Surgical Pathology, is largely devoted to the
exposition of the essential principles which underlie a solid surgical structure.
This applies not only to general surgical operations, but also to all surgical condi¬
tions. The work covers the entire field of general surgery and of surgical diseases,
dealing not so much with special operations as with the conditions which should
govern them — general directions for their performance, after-treatment, and the
etiology, pathology, and treatment of the various surgical diseases.

Volume II., Regional Surgery, is devoted to the surgery of the head, neck,
thorax, and spine and spinal cord ; including, in the first division , injuries and
diseases of the scalp, of the cranial bones, of the brain and its adnexa, of the face,
of the nose and nasal fossae, of the jaws, of the mouth, fauces, and pharynx, of the
ear, and of the salivary glands. The second division includes injuries and surgical
diseases of the neck, of the larynx and trachea, and of the oesophagus. The third
division covers injuries and diseases of the thorax and of the heart ; and the fourth
division treats of the surgery of the spine and spinal cord, including deformities,
fractures, gunshot injuries, tumours, etc.

Volume Hi., Regional Surgery, is devoted toTthe surgery of the abdomen, the
upper extremity, and the lower extremity; including in the first section injuries
and diseases of the abdominal wall, of the peritoneal cavity, the surgery of the
liver, gall bladder, pancreas, spleen, stomach, and intestinal canal (with the
exception of the rectum and anus), injuries and diseases of the rectum and anus,
hernia, surgery of the kidney and ureter, injuries and diseases of the male bladder,
of the urethra and penis, of the scrotum, testicle, epididymis, spermatic cord, and
seminal vesicles, of the prostate and Cowper’s glands, surgery of the female genito¬
urinary organs, and injuries and diseases of the pelvis. The second section in¬
cludes injuries and diseases in the region of the shoulder, of the upper arm and
the elbow joirit, of the forearm and the wrist, and of the hand and the fingers.
The third section includes injuries and diseases of the hip-joint and the thigh, of
the knee-joint and the leg, and of the ankle and the foot.

The list of subjects is so full that it includes even the great surgical rarities,
and the descriptions are sufficiently complete to save the reader from the necessity

of consulting other works to obtain the knowledge necessary to understand and to
treat.

“ The translators are to be congratulated on their selection of this work as a
medium through which to bring the current views of German surgeons before the
Englisli-reading medical public. Written by an acknowledged master of his art,
accepted in the country of its production as a standard text-book, and bearing
the imprimatur of a fourth edition within five years of its publication, it is
admirably calculated to reflect the opinions and practice of the surgeons of to-day
in Germany. . . .

‘ ‘ The first volume consists of the e Principles of Surgery and Surgical Pathology, 5
and constitutes one of the best expositions of these subjects at present available. . . .
The work before us (vol. ii.) deals with the Regional Surgery of the head, neck,
thorax, and spine, and after a careful survey of it we do not hesitate to say that
it would be difficult to find a more satisfactory presentation of the modern aspects
of scientific surgery than its pages afford. ... It is sufficient praise to the
publisher to say that the paper, type, and illustrations are worthy of the text.” —
Scottish Medical and Surgical Journal.

ARRANGED IN QUESTION AND ANSWER FORM.

Essentials of Physiology. By H. A. Hare, M.D., Prof. Thera¬
peutics in Jefferson Med. College, Philadelphia. Numerous illustrations.
Fourth edition, revised and enlarged, containing a series of handsome plate
illustrations taken from the celebrated “ Icomes Nervorum Capitis ” of
Arnold. 192 pages. Cloth, 4s net, post free.

Essentials of Diagnosis. By Solomon Solis Cohen, M.D., Prof,
of Clin. Med. in the Philadelphia Polyclinic ; and Augustus A. Eshner
M.D., Instructor in Clin. Med. in Jefferson Med. Coll. 382 pages. 55 illus¬
trations, some of which are coloured, and a frontispiece. 6s net, post free.

Essentials of Obstetrics, By Easterly Ashton, M.JD., Obstetrician
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The following Catalogues can be had Post
Free on application.

Catalogue 110:

MIDWIFERY AND DISEASES OF WOMEN AND CHILDREN

URINARY AND VENEREAL DISEASES, SYPHILIS

SKIN DISEASES

FEVERS

CHOLERA

GOUT, RHEUMATISM, LIVER, STOMACH, RECTUM, AND DROPSY
SPINAL DISEASES

Catalogue 111 :

ANATOMY, PHYSIOLOGY
MEDICINE, PATHOLOGY

SMALL POX, VACCINATION, CHOLERA, FEVERS, &c.
MIDWIFERY, DISEASES OF WOMEN
CUTANEOUS DISEASES AND VARIOUS SUBJECTS

Catalogue 112 :

DISEASES OF THE CHEST, LUNGS, HEART, BLOOD, ASTHMA,
CONSUMPTION

DISEASES OF THE BONES AND JOINTS, DEFORMITIES, CLUBFOOT,
FRACTURES

INDIGESTION, DIET, FOOD, HEALTH
CANCER, TUMOURS, ULCERS, WOUNDS

DISEASES OF INDIA, EAST & WEST INDIES & TROPICAL CLIMATES
SURGERY

VALUABLE WORKS AND SETS

Catalogue 113 :

INSANITY, DISEASES OF THE BRAIN AND NERVOUS SYSTEM
DISEASES OF THE EYE

DENTISTRY

Catalogue 114:

Catalogue 115 :

VACCINATION AND SMALL POX

Catalogue 116 :

DISEASES OF THE EAR AND THROAT

Printed, by Cowan Co., Limited , Perth.

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