BIOLOGY
R
G
ANTONI VAN LEEUWENHOEK
1682-1723
THE PRINCIPLES
OF
BACTEEIOLOGT
A PRACTICAL MANUAL FOR STUDENTS
AND PHYSICIANS
BY
A. C. ABBOTT, M.D.
PROFESSOR OF HYGIENE AND BACTERIOLOGY, AND DIRECTOR OF THE SCHOOL OF
HYGIENE AND PUBLIC HEALTH, UNIVERSITY OF PENNSYLVANIA
TENTH EDITION, THOROUGHLY REVISED
WITH 121 ILLUSTRATIONS, 31 OF WHICH ARE COLORED
LEA & FEBIGER
PHILADELPHIA AND NEW YORK
BIOLOGY
LIBRARY
G
COPYRIGHT
LEA & FEBIGER
1921
PRINTED IN U. S. A.
PREFACE TO THE TENTH EDITION.
As in previous editions, we have kept before us the adapta-
tion of this book to the needs of the beginner. There have
been incorporated all advances that we regard as fundamental
to an understanding of the accepted principles and practices
of modern bacteriology.
Opinion is now well stabilized upon the matter of variations
in bacterial species and the subject has been treated in appro-
priate places in both a general and particular manner.
Since the last edition of this book there have been devel-
oped methods for the estimation of the exact acid or alkaline
strengths of solutions. These methods are based upon the
theory of dissociation of electrolytes under different condi-
tions of solution. They are now so simplified as to make
them adaptable to the routine estimation and correction of
reactions of culture media. In this connection an effort has
been made to explain the phenomenon of dissociation in such
manner as to make it comprehensible to those not versed in
the physics of solutions, and to clarify the steps, and reasons
therefor, taken in efforts to determine the reactions of
solutions, especially of culture media, by the estimation of
their hydrogen-ion content. The significance of pH values
as symbols of reaction and the use of indicators for different
pH values is explained.
689G30
iv PREFACE TO THE TENTH EDITION
Recently work upon that ill-defined group of micro-
organisms, the Spirochsetaceae, has at least begun to be
systematized, and already our knowledge of their nature,
their relation to disease and the possible distribution of this
group have been greatly extended. Sufficient of these
studies has been incorporated in this edition to give to the
student an idea of what has been accomplished and the
methods used. New illustrations accompany this section.
Since nothing has transpired to shake the foundations of
the science of bacteriology, the fundamental features of the
book, except for minor changes, remain as in the last edition.
A. C. A.
UNIVERSITY OP PENNSYLVANIA,
1921.
CONTENTS.
INTRODUCTION.
"Omne Vivum ex Vivo"— The Overthrow of the Doctrine of
Spontaneous Generation — Earlier Bacteriological Studies —
The Birth of Modern Bacteriology 17
CHAPTER I.
Definition of Bacteria — Differences Between Parasites and
Saprophytes — Their Place in Nature — Bacterial Enzymes
— Products of Bacteria — Nutrition of Bacteria — Their Rela-
tion to Oxygen — Influence of Temperature Upon Their
Growth — Chemotaxis 33
CHAPTER II.
Morphology of Bacteria — Chemical Composition of Bacteria —
Mode of Multiplication — Spore-formation — Motility . . 63
CHAPTER III.
^Principles of Sterilization by Heat— Methods Employed — Dis-
continued Sterilization — Fractional Sterilization — Apparatus
Employed — Sterilization under Pressure — Sterilization by
Hot Air — Thermal Death-point of Bacteria — Chemical Dis-
infection and Sterilization — Mode of Action of Disinfectants
— Practical Disinfection 76
CHAPTER IV.
Principles Involved in the Methods of Isolation of Bacteria in
Pure Culture by the Plate Method of Koch — Materials
Employed 104
(v)
vi CONTENTS
CHAPTER V.
Reactions, Methods for Adjustment — Titration — Hydrogen ion
Concentration — Preparation of Media — Bouillon, Gelatin,
Agar-agar, Potato, Blood Serum, Blood Serum from Small
Animals, Milk, Litmus-whey Milk, Dm ham's Peptone Solu-
tion, Lactose Litmus-agar, Loffler's Blood Serum Mixture,
the Serum Water Media of Hiss, Guarniari's Gelatin-agar
Mixture Ill
CHAPTER VI.
Preparation of the Tubes, Flasks, etc., in which the Media are to
be Preserved 145
CHAPTER VII.
Technique of Isolating Bacteria in Pure Culture by the Plate and
the Tube Method 149
CHAPTER VIII.
The Incubating Oven — The Safety Burner Employed in Heating
the Incubator — Thermo-regulator — Gas-pressure Regulator 157
CHAPTER IX.
The Study of Colonies — Their Naked-eye Peculiarities and Their
Appearance Under Different Conditions — Differences in the
Structure of Colonies from Different Species of Bacteria —
Stab Cultures— Slant Cultures 165
CHAPTER X.
Methods of Staining — Cover-slip Preparations — Impression
Cover-slip Preparations — Solutions Employed — Preparation
and Staining of Cover-slips — Staining Solutions — Special
Staining Methods 171
CHAPTER XL
Systematic Study of an Organism — Points to be Considered in
Determining the Morphologic and Biologic Characters of a
Culture — Methods by which the Various Biologic and Chem-
ical Characters of a Culture may be Ascertained — Variation
and Varieties — Dark Field Illumination — Facts Necessary
to Permit the Identification of an Organism as a Definite
Species 191
CONTENTS vn
CHAPTER XII.
Inoculation of Animals — Subcutaneous Inoculation — Intravenous
Injection — Inoculation into the Lymphatic Circulation —
Inoculation into the Great Serous Cavities and into the
Anterior Chamber of the Eye — Observation of Animals
after Inoculation . 232
CHAPTER XIII.
Post-mortem Examination of Animals — Bacteriological Examina-
tion of the Tissues — Disposal of Tissues and Disinfection of
Instruments after the Examination — Study of Tissues and
Exudates During Life 255
CHAPTER XIV.
Infection and Immunity — Mechanism — Defenses of the Body —
Specific Bodies and Reactions — Doctrines in Explanation . 266
CHAPTER XV.
Hemolysis — The Hemolytic System — Identification of Specific
Immune Bodies and Specific Antigens by their Ability to
Fix Complement — The Wassermann Reaction — Schematic
Representation of Reactions 312
APPLICATION OF THE METHODS OF
BACTERIOLOGY.
CHAPTER XVI.
To Obtain Material with Which to Begin Work 323
CHAPTER XVII.
Various Experiments in Sterilization by Steam and by Hot Air . 327
viii CONTENTS
CHAPTER XVIII.
Methods of Testing Disinfectants and Antiseptics — Experiments
Illustrating the Precautions to be Taken — Experiments in
Skin-disinfection 331
CHAPTER XIX.
Micrococcus Aureus — Micrococcus Pyogenes and Citreus —
Staphylococcus Epidermidis Albus — Streptococcus Pyogenes,
Types and Typing — Micrococcus Gonorrhcese — Micrococcus
Intracellularis, Varieties — Pseudomonas ^Eruginosa —
Bacillus of Bubonic Plague, Protective Inoculation and Anti-
serum 345
CHAPTER XX.
Some of the Pathogenic Organisms Encountered in the Mouth
Cavity in Health and Disease — Micrococcus Lanceolatus,
(Pneumococcus) Types and Typing — Micrococcus Tetrag-
enus, Bacterium Influenzae, Bacillus Tuberculosis, etc. . . 401
CHAPTER XXI.
Tuberculosis — Microscopic Appearance of Miliary Tubercles —
Diffuse Caseation — Cavity-formation — Encapsulation of
Tuberculous Foci — Primary Infection — Modes of Infection,
Vaccination Against — The Bacterium Tuberculosis — Loca-
tion of the Bacilli in the Tissues — Microscopic Appearance of
Bacterium Tuberculosis — Staining Peculiarities — Organisms
with Which Bacterium Tuberculosis may be Confounded:
Bacterium Leprse; Bacterium Smegmatis — Acid-proof Bac-
teria— Bacterium Tuberculosis Avium — Variations — Pseudo-
tuberculosis — Susceptibility of Animals — Tuberculin — Vac-
cination Against Tuberculosis — Actinomyces Bovis — Actino-
myces Israeli, Actinomyces Madurse, Actinomyces Farcinicus,
Actinomyces Eppingeri, Actinomyces Pseudo tuberculosis . 430
CHAPTER XXII.
Glanders— Characteristics of the Disease — Histological Structure
of the Glanders Nodule — Susceptibility of Different Animals
to Glanders — The Bacterium of Glanders; Its Morpholog-
ical and Cultural Peculiarities — Diagnosis of Glanders —
Mallein . 470
CONTENTS IX
CHAPTER XXIII.
Bacterium (Syn. Bacillus) Diphtherise — Its Isolation and Cultiva-
tion—Morphological and Cultural Peculiarities— Pathogenic
Properties — Variations in Virulence — Varieties — Bacterium
Pseudodiphtheriticum — Bacterium Xerosis — Diphtheria
Antitoxin 480
CHAPTER XXIV.
Typhoid Fever — Study of the Organism Concerned in its Produc-
tion— Its Morphological, Cultural, and Pathogenic Properties
— Prophylactic Inoculations — Bacillus Coli — Bacillus Para-
typhosus — Its Resemblance to Bacillus Typhosus . . . 508
CHAPTER XXV.
The Group of Bacilli Found in Cases of Epidemic, Endemic, and
Sporadic Dysentery — The Morphological, Biological, and
Pathogenic Characters of the Several Members of the Group
—The Differentiation of the Different Types of Bacilli . .541
CHAPTER XXVI.
The Spirillum (Comma Bacillus) of Asiatic Cholera — Its Mor-
phological and Cultural Peculiarities — Pathogenic Properties
— The Bacteriological Diagnosis of Asiatic Cholera — Micro-
spira Metchnikovi — Microspira ("Vibrio") Schuylkilliensis
— Its Morphological, Cultural, and Pathogenic Characters . 549
CHAPTER XXVII.
Study of Bacterium Anthracis, and of the Effects Produced by Its
Inoculation into Animals — Peculiarities of the Organism
Under Varying Conditions of Surroundings — Anthrax Vac-
cines— Anthrax Immune Serum 583
CHAPTER XXVIII.
The Nitrifying Bacteria— The Bacillus of Tetanus— The Bacillus
of Malignant Edema — The Bacillus of Symptomatic Anthrax
— Bacterium Welchii — Bacillus Sporogenes — The Spirochae-
tacese, Spironema, Treponema, Leptospira — Relapsing
Fever, Yellow Fever, Syphilis 600
CONTENTS
CHAPTER XXIX.
Bacteriological Study of Water — Methods Employed — Precau-
tions to be Observed — Apparatus Employed, and Methods of
Using It — Methods of Investigating Air and Soil — Bacterio-
logical Study of Milk — Methods Employed . . . . . 637
APPENDIX 669
BACTERIOLOGY.
INTRODUCTION:', >,„ " ; .- '; } ; , ; ;
"Omne Vivum ex Vivo" — The Overthrow of the Doctrine of Spontaneous
Generation — Earlier Bacteriological Studies — The Birth of Modern
Bacteriology.
BACTERIOLOGY may be said to have had its beginning
with the observations -of Leeuwenhoek in the latter part
of the seventeenth century. Though its most rapid and
important development has taken place since about 1880,
still, a review of the various evolutionary phases through
which it has passed in the course of more than two hundred
years reveals an entertaining and instructive history. From
the very outset its history is inseparably connected with
that of medicine, and from the outcome of bacteriological
research preventive medicine, in its modern conception,
received its primary impulse. Through a more intimate
acquaintance with the biological activities of the unicellular
vegetable microorganisms modern hygiene has attained
almost the dignity of an exact science, and properly merits
the importance and prominence now generally accorded to
it. From studies in the domain of bacteriology our knowl-
edge of the causation, course, and prevention of infectious
diseases is daily, becoming more accurate, and it is needless
to emphasize the relation of such knowledge to the manifold
problems that present themselves to the student of modern
2 (17)
18 BACTERIOLOGY
medicine. Though the contributions which have done most
to place bacteriology on the footing of a science are those
of recent years, still, during the earlier stages of its devel-
opment, many observations were made which formed the
foundation-work for much that was to follow. Before
regularly beginning our studies, therefore, it may be of
advantage to. acquaint ourselves with the more prominent
of those mves$igutions.
; Antony van Leeuwenhoek, the first to describe the bodies
now recognized as bacteria, was born at Delft, in Holland,
in 1632. He was not considered a man of liberal education,
having been during his early years an apprentice to a linen-
draper. During his apprenticeship he learned the art of
lens-grinding, in which he became so proficient that he
eventually perfected a simple lens by, means of which he was
enabled to see objects of much smaller dimensions than
any hitherto seen with the best compound microscopes in
existence at that date. At the time of his discoveries he
was following the trade of linendraper in Amsterdam.
In 1675 he published the fact that he had succeeded in
perfecting a lens by means of which he could detect in a
drop of rain-water living, motile "animalcules" of the most
minute dimensions — smaller than anything that had hitherto
been seen. Encouraged by this discovery, he continued to
examine various substances for the presence of what he
considered animal life in its most minute form. He found
in sea-water, in well-water, in the intestinal canal of frogs
and birds, and in his own diarrheal evacuations, objects
that differentiated themselves the one from , the other,
not only by their shape and size, but also by the peculiarity
of motility which some of them were seen to possess. In the
year 1683 he discovered in the tartar scraped from between
INTRODUCTION 19
the teeth a form of microorganism upon which he laid
special stress. This observation he embodied in the form
of a contribution to the Royal Society of London on Sep-
tember 14, 1683. This paper is of peculiar importance,
not only because of the careful, objective nature of the
description given of the bodies seen by him, but also for
the illustrations which accompany it. From a perusal of
the text and an inspection of the plates there remains little
room for doubt that Leeuwenhoek saw with his primitive
lens the bodies now recognized as bacteria.1
Upon seeing these bodies he was apparently very much
impressed, for he writes: "With the greatest astonishment
I observed that everywhere throughout the material which
I was examining were distributed animalcules of the most
microscopic dimensions, which moved themselves about in
a remarkably energetic way."
This discovery was shortly followed by others of an
equally important nature. His field of observation appears
to have increased rapidly, for after a time he speaks of bodies
of much smaller dimensions than those at first described by
him.
Throughout all of Leeuwenhoek's work there is a con-
spicuous absence of the speculative. His contributions are
remarkable for their purely objective nature.
After the presence of these organisms in water, in the
mouth, and in the intestinal evacuations was made known
to the world, it is not surprising that they were immediately
seized upon as the explanation of the origin of many obscure
diseases. So universal became the belief in a causal relation
between the "animalcules" and disease that it amounted
1 See Arcana Naturae detecta ab ANTONIO VAN LEEUWENHOEK; Delphis
Batavorum, 1695.
20 BACTERIOLOGY
almost to a germ-mania. It became the fashion to suspect
the presence of these organisms in all forms and kinds of
disease, simply because they had been demonstrated in
the mouth, intestinal evacuations, and water.
Though nothing of value at the time had been done in
the way of classification, and even less in separating and
identifying the members of this large group, still the fore-
most men of the day did not hesitate to ascribe to them not
only the property of producing pathological conditions,
but some even went so far as to hold that variations in the
symptoms of disease were the result of differences in the
behavior of the microorganisms in the tissues.
Marcus Antonius Plenciz, a physician of Vienna in 1762,
declared himself a firm believer in the work of Leeuwenhoek,
and based the doctrine which he taught upon the discoveries
of the Dutch observer and upon observations of a confirma-
tory nature which he himself had made. The doctrine of
Plenciz assumed a causal relation between the microorgan-
isms discovered and described by Leeuwenhoek and all
infectious diseases. He maintained that the material of
infection could be nothing else than a living substance,
and endeavored on these grounds to explain the variations
in the period of incubation of the different infectious diseases.
He likewise believed the living contagium to be capable of
multiplication within the body, and spoke of the possibility
of its transmission through the air. He believed in the
existence of a special germ for each disease, holding that
just as from a given cereal only one kind of grain can grow,
so by the special germ for each disease only that disease
can be produced.
He found in all decomposing matters innumerable minute
"animalculse," and was so firmly convinced of their etio-
INTRODUCTION 21
logical relation to the process that he formulated the law:
that decomposition can only take place when the decompos-
able material becomes coated with a layer of the organisms,
and can proceed only when they increase and multiply.
However convincing the arguments of Plenciz may appear,
they seem to have been lost sight of in the course of subse-
quent events, and by a few were even regarded as the pro-
ductions of an unbalanced mind. For example, as late as
1820 we find Ozanam expressing himself on the subject as
follows: "Many authors have written concerning the
animal nature of the contagion of disease; many have
indeed assumed it to be developed from animal substances,
and that it is itself animal and possesses the property of
life; I shall not waste time in effort to refute these absurd
hypotheses."
Similar expressions of opinion were heard from many
other investigators of the time, all tending in the same
direction, all doubting the possibility of these microscopic
creatures belonging to the world of living things.
It was not until between the fourth and fifth decades of
the nineteenth century that by the fortunate coincidence
of a number of important discoveries the true relation of
the lower organisms to infectious diseases was scientifically
pointed out. With the fundamental investigations of
Pasteur upon the souring and putrefaction of beer and wine;
with the discovery by Pollender and Davaine of the presence
of rod-shaped organisms in the blood of animals dead of
splenic fever, and with the progress of knowledge upon the
parasitic nature of certain diseases of plants, the old question
of " contagium animatum" again began to receive attention.
It was taken up by Henle, and it was he who first logically
taught this doctrine of infection.
22 BACTERIOLOGY
The main point, however, that had occupied the attention
of scientific men from time to time for a period of about
two hundred years subsequent to Leeuwenhoek's discoveries
was the origin of the "animalcules." Do they generate
spontaneously, or are they the descendants of preexisting
creatures of the same kind? was the all-important question.
Among the earlier participants in this discussion were many
of the most distinguished men of the day.
In 1749 Needham, who held firmly to the opinion that
the bodies which were attracting such general attention
developed spontaneously as the result of vegetative changes
in the substances in which they were found, attempted to
demonstrate by experiment his reasons for holding this view.
He maintained that the bacteria which appeared about a
grain of barley germinating in a carefully covered watch-
crystal of water were the result of changes going on in the
barley-grain itself, incidental to its germination.
Spallanzani, in 1769, drew attention to the laxity of
Needham's experimental methods, and demonstrated that
if infusions of decomposable vegetable matter be placed in
flasks, which, alter being hermetically sealed, were heated
for a time in boiling water, no living organisms would be
detected in them, nor would decomposition appear in the
infusions so treated. The objection raised by Treviranus,
viz., that the high temperature to which the infusions had
been subjected had so altered them and the air about them
that the conditions favorable to spontaneous generation
no longer existed, was promptly met by Spallanzani when
he gently tapped one of the flasks that had been boiled
against a hard object until a minute crack was produced;
invariably organisms and decomposition appeared in the
flask thus treated.
INTRODUCTION 23
From the time of the experiments of Spallanzani until
as late as 1836 but little advance was made in the elucida-
tion of this, at that time, obscure problem.
In 1836 Schulze attracted attention to the subject by
the convincing nature of his investigations. He showed that
if the air which gained access to boiled infusions be robbed
of its living organisms by first passing it through strong
acid or alkaline solutions no decomposition occurred, and
living organisms could not be detected in the infusions.
Following quickly upon this contribution came Schwann,
in 1837, and somewhat later (1854) Schroder and Dusch,
with similar results obtained by somewhat different means.
Schwann deprived the air which passed to his infusions of
its living particles by conducting it through highly heated
tubes; whereas Schroder and Dusch, by means of cotton-
wool interposed between the boiled infusions and the outside
air, robbed the air passing to the infusions of its organisms
by the simple process of filtration. In 1860 Hoffmann and
in 1861 Chevreul and Pasteur demonstrated that the pre-
cautions taken by preceding investigators for rendering
the air which entered these flasks free from bacteria were
not necessary; that all that was required to prevent the
access of bacteria to the infusions in the flasks was to draw
out the neck of the flask into a fine tube, bend it down along
the side of the flask, and then bend it up again a few cen-
timeters from its extremity, and leave the mouth open. The
infusion was then to be boiled in the flask thus prepared
and the mouth of the tube left open. The organisms which
now fell into the open end of the tube were arrested by the
drop of water of condensation which collected at its lowest
angle, and none could enter the flask.
While, from our modern standpoint, the results of these
24 BACTERIOLOGY
investigations seem to be of a most convincing nature, yet
there were many at the time who required additional proof
that "spontaneous generation" was not the explanation
for the mysterious appearance of these minute living crea-
tures. The majority, if not all, of such doubts were sub-
sequently dissipated through the well-known investigations
of Tyndall upon the floating matters of the air. In these
studies he demonstrated by numerous ingenious and instruc-
tive experiments that the presence of living organisms in
decomposing fluids was always to be explained either by
the preexistence of similar living forms in the infusion
or upon the walls of the vessel containing it, or by the
infusion having been exposed to^air which had not been
deprived of its viable organisms.
Throughout all the work bearing upon this subject, from
the time of Spallanzani to that of Tyndall, certain irregu-
larities were constantly appearing. It was found that par-
ticular substances required to be heated for a much longer
time than was needed to render other substances free from
living organisms, and even after heating under the most
careful precautions decomposition would occasionally occur.
In 1762 Bonnet, who was deeply interested in this sub-
ject, suggested, in reference to the results obtained by
Needham, the possibility of the existence of "germs or
their eggs," which had the power to resist the temperature
to which some of the infusions employed in Needham's
experiments had been subjected.
More than a hundred years after Bonnet had indulged
in this pure speculation it became the happy privilege of
Ferdinand Cohn, of Breslau, to demonstrate its accuracy
and importance.
Cohn repeated the foregoing experiments with like results.
INTRODUCTION 25
He concluded that the irregularities could only be due to
either the existence of more resistant species of bacteria
or to more resistant stages into which certain bacteria have
the property of passing. He demonstrated that some of
the rod-shaped organisms possess the power of passing into
a resting- or spore-stage in the course of their life-cycle,
analogous to the seeding stage of higher plants, and when in
this stage they are much less susceptible to the deleterious
action of high temperatures than when they are growing
as normal vegetative forms. With the discovery of these
more resistant spores the doctrine of spontaneous generation
received its death-blow. It was no longer difficult to explain
the inconsistencies in the results of former investigations,
nor was it any longer to be doubted that putrefaction and
fermentation were the result of bacterial life and not the
cause of it, and that these bacteria were the offspring of
preexisting similar forms. In other words, the law of
Harvey, Omne vivum ex ovo, or its modification, Omne
vivum ex vivo, was shown to apply not only to the more
highly organized members of the animal and vegetable
kingdoms, but to the most microscopic, unicellular creatures
as well.
The establishment of this point gave an impetus to
further investigations, and as the all-important question
was that concerning the relation of the microscopic organ-
isms to disease, attention naturally turned into this channel
of study. Even before the hypothesis of spontaneous
generation had received its final refutation a number of
observations of a most important nature had been made
by investigators who had long since ceased to consider
spontaneous generation as a tenable explanation of the
origin of the microscopic living particles.
26 BACTERIOLOGY
In the main, these studies had been conducted upon
wounds and the infections to which they are liable; in
fact, the evolution of our knowledge of bacteriology to its
present development is so intimately associated with this
particular line of investigation that a few historical facts
in connection with it may not be without interest.
The observations of Rindfleisch, in 1866, in which he
describes the presence of small, pin-head points in the
myocardium and general musculature of individuals that
had died as a result of infected wounds, represent, probably,
the first reliable contribution to this subject. He studied
the tissue-changes round about these points up to the
stage of miliary abscess-formation. He refers to the organ-
isms as "vibrios." Almost simultaneously von Reckling-
hausen and Waldeyer described similar changes that they
had observed in pyemia and, occasionally, secondary to
typhoid fever. Von Recklinghausen believed the granules
seen in the abscess-points to be micrococci and not tissue-
detritus, and gave as the reason that they were regular in
size and shape, and gave specific reactions with particular
straining-fluids. Birch-Hirschfeld was able to trace bacteria
found in the blood and organs to the wound as the point
of entrance, and believed both the local and the constitu-
tional conditions to stand in direct ratio to the number of
spherical bacteria present in the wound. He observed also
that as the organisms increased in number they could often
be found within the bodies of pus corpuscles. His studies
of pyemia led him to the important conclusion that in this
condition microorganisms were always present in the
blood.
Of immense importance to the subject were the investiga-
tions of Klebs, made at the Military Hospital at Karlsruhe
INTRODUCTION 27
in 1870-71. He not only saw, as others before him had
seen, that bacteria were present in diseases following infec-
tion of wounds, but described the manner in which the
organisms had gained entrance from the point of injury
to the internal organs and blood. He expressed the opinion
that the spherical and rod-shaped bodies which he saw in
the secretions of wounds were closely allied, and he gave to
them the designation " microsporon septicum." He believed
that the organisms gained access to the tissues round about
the point of injury both by the aid of the wandering leuko-
cytes and by being forced through the connective-tissue
lymph-spaces by the mechanical pressure of muscular
contraction.
On erysipelatous inflammations secondary to injury
important investigations were also being made, Wilde,
Orth, von Recklinghausen, Lukomsky, Billroth, Ehrlich,
Fehleisen, and others agreeing that in these conditions
microorganisms could always be detected in the lymph
channels of the subcutaneous tissues; and through the
work of Oertel, Nassiloff, Classen, Letzerich, Klebs, and
Eberth the constant presence of bacteria in the diphtheritic
deposits at times seen on open wounds was established.
. We see that the conception of a living, invisible some-
thing— a contagium vivum — was old, but by the use of the
rapidly improving compound microscope a host of investi-
gators was making this "something" more tangible; they
were describing various minute bodies seen in diseased
conditions that they believed to be living things, and to be
the cause of the conditions in which they were observed.
Yet no convincing demonstration of the relationship between
these supposed living foreign bodies and the diseases in which
they were present had been made. In 1855 Pollender
28 BACTERIOLOGY
announced the constant presence in the blood of animals
dead of anthrax of rod-shaped bodies, and in 1863 Davaine
showed the blood of such animals to be infective for normal
animals. This distinct step in advance so attracted the
attention of Pasteur that he soon became closely identified
not only with studies of this particular disease but with
other diseases of domestic animals and fowls. Pasteur was
already known in the fields of physical and natural sciences
through his basic work on the organic isomers and his dis-
coveries in the field of fermentation. His conception of
fermentation as a function of living cells was so opposed to
the views generally held at the time, and especially those
held by von Liebig — perhaps the most distinguished chem-
ist of his day— that endless discussion, amounting at times
almost to polemic, took place. Pasteur's opinions finally
triumphed. His manifold investigations at this time were
so novel, so progressive; his interests so versatile and so in
accord with the new thought that was beginning to develop,
that he was soon regarded as the most suggestive and impres-
sive contributor of his time. His subsequent studies upon
vaccination with living attenuated viruses were of such
fundamental nature both scientifically and practically as
to justify the opinion that the science of immunology began
with his observations and investigations. The circumstances
surrounding his public demonstration that sheep can be
protected from anthrax infection by the process of vaccina-
tion with a living, attenuated virus constitutes one of the
most dramatic incidents in the history of applied science.
The catholicity of Pasteur's interests in all matters con-
cerning the world of living, microscopic things, the benefits
that accrued from the application of his keen analytic men-
tality to the solution of problems of domestic, agricultural
LOUIS PASTEUR
1822-1893
INTRODUCTION 29
and commercial moment, and his contributions to our
understanding of infection, transmission and induced
immunity from disease, justify the statement that he was
easily our broadest minded and most comprehensive con-
tributor to the field of microbiology.1
Simple and natural as all this may seem to us now, the
stage to which the subject had developed when these obser-
vations were recorded did not admit of their meeting with
unconditional acceptance. The only strong argument in
favor of the etiological relation of the organisms that had
been seen to the diseases with which they were associated
was the constancy of this association and the occasional
transmission of the disease from a sick to a well animal by
the use of body fluids or bits of diseased tissue. No efforts
had been made to isolate them, and but few to reproduce the
pathological conditions by inoculation. Moreover, not a
small number of investigators were skeptical as to the
importance of such demonstrations; many claimed that
microorganisms were normally present in the blood and
tissues of the body; and some even urged that the organisms
seen in diseased conditions were the result rather than the
cause of the maladies. It is hardly necessary to do more
than say that both of these views were purely speculative,
and have never had a single reliable experimental argument
in their favor. Billroth and Tiegel, who held to the former
opinion, did endeavor to prove their position through experi-
mental means; but the methods employed by them were of
such an untrustworthy nature that the fallacy of deductions
drawn from them was very quickly made manifest by subse-
quent investigators. Their method for demonstrating the
1 See Life of Pasteur, by Vallery-Radot.
30 BACTERIOLOGY
presence of microorganisms in normal tissues was to remove
bits of organs from the healthy animal body with heated
instruments and drop them into hot melted paraffin. They
held that all living organisms on the surface of the tissues
would be destroyed by the high temperature, and that if
decomposition should subsequently occur it would prove that
it was the result of the growth of bacteria in the depths of
the tissues to which the heat had not penetrated. Decom-
position did usually set in, and they accepted this as proof of
the accuracy of their view. Attention was, however, shortly
called to the fact that in cooling there was contraction of
paraffin, resulting usually in the production of small rents
and cracks in which dust, and bacteria lodged upon it,
could accumulate and finally gain access to the tissues,
with the occurrence of decomposition as a consequence.
Their results were thus explained after a manner analogous
to that employed by Spallanzani, in 1769, in demonstrating
to Treviranus the fallacy of the opinion held by him and the
accuracy of his own views, viz., that it was always through
the access of organisms from without that decomposition
primarily originated. (See page 22.)
Under careful precautions, to which no objection could
be raised, the experiments of Billroth and Tiegel w^re
repeated by Pasteur, Burdon-Sanderson, and Klebs, but
with failure in every instance to demonstrate the presence
of bacteria in the healthy living tissues.
The fundamental researches of Koch (1881) upon patho-
genic bacteria and their relation to the infectious diseases
of animals differed from those of preceding investigators
in many important respects. The scientific methods of
analysis with which each and every obscure problem was
met as it arose served at once to distinguish him as a pioneer
ROBERT KOCH
1843-191O
INTRODUCTION 31
in this hitherto but imperfectly cultivated domain. The
outcome of these investigations was the establishment of
a foundation upon which practical bacteriology of the future
was to rest. He, for the first time, demonstrated that dis-
tinct varieties of infection, as evidenced by anatomical
changes are due in many cases to the activities of specific
microorganisms, and that by proper methods it is possible
to isolate these organisms in pure culture, to cultivate them
indefinitely under artificial conditions, to reproduce the
lesions by inoculation of these pure cultures into susceptible
animals, and to continue the disease at will by continuous
inoculation from an infected to a healthy animal.
By the methods that he employed he demonstrated a
series of separate and distinct diseases that can be produced
in mice and rabbits by the injection of putrid substances
into their tissues. The disease known as septicemia of
mice; likewise a disease characterized by progressive abscess
formation, and pyemia and septicemia of rabbits, were among
the affections first produced by him in this way. It was in
the course of this work that the Abbe system of substange
condensing apparatus was first used in bacteriology; that
the aniline dyes suggested by Weigert were brought into
general use; that the isolation and cultivation of bacteria in
pure culture on solid media were shown to be possible; and
that animals were employed as a means of obtaining from
mixtures pure cultures of pathogenic bacteria.
With the bounteous harvest of original and important
suggestions that was reaped from Koch's classical series
of investigations bacteriology reached an epoch in its devel-
opment, and at this period practical bacteriology, as we
know it today, may justly be said to have had its birth.
32 BACTERIOLOGY-
NOTE. — I have presented only the most prominent inves-
tigations that will serve to indicate the lines along which
the subject has developed. For a more detailed account
of the historical development of the work the reader is
referred to Loffler's instructive and entertaining Vorlesungen
uber die geschichtliche Entwickelung der Lehre von den Bac-
terien, upon which I have drawn freely in preparing the
foregoing sketch.
CHAPTER I.
Definition of Bacteria — Differences Between Parasites and Saprophytes
— Their Place in Nature — Bacterial Enzymes — Products of Bacteria
— Nutrition of Bacteria — Their Relation to Oxygen — Influence of
Temperature Upon Their Growth — Chemotaxis.
BACTEKIA (more properly bacteriacese or schizomycetes)
were regarded by the older writers as infusoria. This was
because of their capacity for developing in infusions, their
property of spore formation, their resistance to drying,
their power of independent motion, and the absence of
chlorophyl from their tissues. In the modern conception,
however, this classification is untenable, and bacteria, by
virtue of their distinguishing peculiarities, are now treated
as a group by themselves that may briefly be defined as
comprising microscopic, unicellular, vegetable organisms
that multiply by the process of transverse division.
Inasmuch as bacteria are not possessed of chlorophyl,
their metabolic processes are fundamentally different from
those of the higher plants in which it is present. They
cannot, as in the case of the green plants, obtain carbon
and nitrogen from such simple bodies as carbon dioxide and
ammonia, but are forced to secure these essential elements
from organic matter as such. This power to decompose and
assimilate organic matters is signally different in different
species of bacteria, and, singular to say, there is a small
group (to be described later) from which this function is
apparently absent, in spite of the fact that no compensatory
chlorophyl is discernible in their tissues.
3 (33)
34 BACTERIOLOGY
SAPROPHYTES AND PARASITES. — In the case of certain
bacteria, in fact, the majority, the source of food supply
must of necessity be dead organic matters of either animal
or vegetable origin. They cannot exist in the presence of
living tissues. To the members of this group the designa-
tion saprophytic or metatrophic (A. Fischer) is given. To
that group that can exist only upon living organic matters,
and herein belong many (not all) of the disease-producing
bacteria, the appellation parasitic or paratrophic (A. Fischer)
is applied; while for the few species that either do not
require organic matters, or do not, so far as is known, have
the faculty of decomposing and assimilating proteid stuffs
at all, the name prototrophic is suggested by Fischer. In
the strict sense of the word, a parasite can exist only in the
body of a living host, and a saprophyte only upon lifeless
organic matters, and such obligate parasites and saphrophytes
are known, but in the majority of cases such nutritive con-
ditions are not obligatory, many of both parasites and
saprophytes having the power to adapt themselves to
conditions other than those for which they are by nature
best fitted. For instance, certain species that exhibit their
most important properties under conditions of parasitism
may, nevertheless, lead a saprophytic existence when cir-
cumstances demand it, and, on the other hand, particular
species usually saprophytic by nature may find conditions
favorable to their development in a living host. To such
adaptable species the designation "facultative" is given,
and, when employed, signifies that the species in question
has the faculty of adapting itself to environments other
than those in which it is usually encountered. In this sense
all of the disease-producing bacteria that can be cultivated
artificially are manifestly facultative saprophytes.
DEFINITION OF BACTERIA 35
The life-processes of bacteria are so rapid, complex, and
energetic that they result in the most profound alterations
in the structure and composition of the materials in and
upon which they are developing.
Disintegrations and decompositions result from the acti-
vities of the saprophytic bacteria; while the changes brought
about in the tissues of their living host by the purely parasitic
forms find expression in disease-processes not infrequently
leading to complete death.
THEIR PLACE IN NATURE. — The role played in nature by
the saprophytes is a very important one. Through their
functional activities the highly complicated tissues of dead
animals and vegetables are resolved into the simpler com-
pounds, carbonic acid, water, and ammonia, in which form
they may be taken up and appropriated as nourishment by
the more highly organized members of the vegetable king-
dom. It is through this ultimate production of carbonic
acid, ammonia, and water by bacteria, as end-products in
the processes of decomposition and fermentation of dead
animal and vegetable tissues, that the demands of growing
vegetation for these compounds are supplied.
The more highly organized chlorophyl plants do not
possess the power of obtaining their carbon and nitrogen
from such complicated organic substances as serve for the
nutrition of bacteria, and as the production of the simpler
compounds, carbon dioxide and ammonia, by the animal
world is not sufficient to meet the demands of the chloro-
phyl plants, the importance of the part played by bacteria
in making up this deficit is obvious and cannot be overesti-
mated. Were it not for the activity of these microscopic
living creatures all life upon the surface of the earth would
cease. Deprive higher vegetation of the carbon and nitrogen
36 BACTERIOLOGY
supplied to it as a result of bacterial activity, and its develop-
ment comes rapidly to an end; rob the animal kingdom of
the food-stuffs supplied to it by the vegetable world, and
life is no longer possible. It is plain, therefore, that in this
cycle of life phenomenon the saprophytes, which represent
the large majority of all bacteria, must be looked upon in
the light of benefactors, without which existence would be
impossible.
With the parasites, on the other hand, the conditions are
far from analagous. Through their metabolic activities
there is constantly a loss, rather than a gain, to both the
animal and vegetable kingdoms. Their host must always
be a living body in which exist conditions favorable to their
development, and from which they appropriate substances
that are necessary to the health and life of the organism on
which they are preying; at the same time they elaborate
substances as products of their nutrition that are directly
poisonous to the tissues in which they are growing.
In their relations to terrestrial life, therefore, the posi-
tions occupied by the two functionally different groups, the
saprophytes on the one hand, and the parasites on the
other, are diametrically opposed.
SPECIFIC FUNCTIONS OF SAPROPHYTIC BACTERIA.
Appropriate investigation of the saprophytic group of
bacteria has shed important light upon certain specific
characteristics with which many of the species are endowed.
We know that numerous common phenomena are the results
of their activities. The souring of milk, the ripening of
cheese; certain of the fermentations resulting in the forma-
tion of various acids of the fatty series; the elaboration of
FUNCTIONS OF SAPROPHYTIC BACTERIA 37
other aromatic bodies of organic character and origin; the
spoiling of wine; the disintegrations incidental to the
manufacture of hemp products; the old method of making
indigo; the natural and artificial methods for the destruction
of the organic waste encountered in polluted waters and
sewage and the transformations of dead organic matter in
the soil are all illustrations of these well-known phenomena.
In a number of commercial lines constant use is made of
these bacterial activities. This is conspicuously seen in
the manufacture of butter and cheese where the excellence
of the products is due to the peculiar flavors caused by
bacterial growth in the raw materials. Before synthetic
methods became so generally in use bacterial activities were
largely employed in the manufacture of the organic acids.
In addition to the foregoing a number of saprophytes
have the specific property of producing beautiful pigments,
red, yellow, orange, pink, violet, green, etc. This group of
" chromogens" as they are called have doubtless other func-
tions in the great laboratory of nature, the soil, where they
are commonly found, but color production is the most
obvious.
Another group — the "photogens" or photogenic species
have the remarkable ability to produce luminosity in the
substances in or on which they exist. It is to the activity
of this group that the phosphorescence sometimes seen in
decayed wood, in rotten fish and other flesh is attributable.
How it is done is a mystery, just as is the means by which
the fire-fly an,d the glow-worm emit their tiny sparks of light.
Another group have as the end products of their activities
those evil smelling bodies by which putrefaction is charac-
terized, these are the so-called "saprogenic" species.
Others have as their most interesting functions the power
38 BACTERIOLOGY
to carry hydrogen sulphide to higher sulphur compounds,
the so-called "thiogenic" species.
Those saprophytes that are concerned in such well-known
fermentations as result in the production of the various
acids of the fatty series are known as "zymogens."
But of all the so-called saprophytic group none are more
interesting and none by any means so important as those
concerned in the various transformations through which
nitrogen passes in being prepared as food for higher vege-
tation. This group, or rather those groups, for there are
apparently several operating on nitrogen and its compounds
in various ways, are known as the "nitrifying" and the
"denitrifying" and the " nitrogen fixing" bacteria.
NITRIFYING BACTERIA. — They carry ammonia, resulting
from the decomposition of dead animals and plants; by
a process of oxidation first to nitrous acid, then by further
oxidation the nitrous acid is carried to nitric acid. These
two steps in the process are taken by two totally distinct
groups of bacteria of a most interesting nature. The func-
tion of one group is strictly limited to the nitrite process;
that of the other to the nitrate; the latter taking up the
work at the point where the former leaves it. The former
cannot carry its operation beyond the nitrite point, nor can
the latter begin with ammonia and carry it to complete
nitrifaction. A most singular peculiarity of this group is
the inability to develop on the nutrient media commonly
used for the cultivation of bacteria. Organic matter as
such seems to be unfavorable to their viability. To grow
them one is obliged to use a silicate jelly, a sort of water
glass of about the consistency of ordinary gelatin, to which
are added certain salts that these particular species are
able to decompose in order to secure the elements necessary
FUNCTIONS OF SAPROPHYTIC BACTERIA 39
to provide energy. In so far as life upon the earth's surface
is concerned the nitrification going on in the soil as a result
of the activities of this group is one of the most important
phenomena in operation. It is to a large extent responsible
for supplying higher vegetation with nitrogen in a form
available for food.
Denitrifieation, i. e., the reverse of nitrif action, the reduc-
tion of nitrates and nitrites to ammonia is a function peculiar
to many bacteria, particularly many species found in the
soil. Often it does not appear to be a specific function and
is frequently accomplished under conditions where organic
matter is present and is utilized. In many cases the denitrifi-
cation seems to be less a phenomenon due to the specific
activities of the bacteria themselves than to the reducing
action of the products of their growth. In the case of the
few species that have been called "true denitrifiers," the
reduction appears to be due to the respiratory demands of
those species for oxygen; this robbing of the oxides of
nitrogen of their oxygen by the bacteria resulting, mani-
festly, in reduction.
Nitrogen Fixation. — Another phenomenon having to do
with nitrogen, and resulting from the activity of saprophytes,
is the so-called "nitrogen fixation" by bacteria. For many
years we were taught that the nitrogen of the air, constitut-
ing about 80 per cent, of the entire atmosphere, was of no
biological significance and was put there by nature merely to
dilute the excessively active oxygen to a point compatible
with respiration by man and animals. This extraordinary
conception was always looked upon with suspicion by thought-
ful students. It was not, however, until about 1886 that
the real significance of atmospheric nitrogen was made
clear. Hellrigel and Wilfarth at that time demonstrated
40 BACTERIOLOGY
that the nodules found on the roots of the leguminous
plants (clover, peas, beans, etc.), might properly be re-
garded as communities of bacteria which were beneficently
cooperating with the plants in the performance of their
fundamental life processes, i. e., they were in "symbiotic"
relationship. The result of this cooperation they showed
to be the power of the legumens to fix and store the free
atmospheric nitrogen. When one realizes how inexhaustible
is the supply of free atmospheric nitrogen it is difficult
to exaggerate the importance of this function. It also sheds
interesting light upon certain practices of the agriculturalist
that have been in empirical operation since the cultivation
of the soil began. It has always been known that the
rotation of crops is essential to the successful tillage of the
soil and we find that in such rotation one or another of the
legumens was always used. The reason is evident, they do
not impoverish, but though their ability to fix nitrogen
through the aid of the "nodule bacteria" on their roots,
they actually enrich the soil.
From the foregoing it is obvious that the expression
"nature's laboratory" is properly applied to the soil. It
is here that all the saprophytes are sooner or later found.
Among her various analytic and synthetic performances
nature concerns herself with none so important to life as
those having to do with the several transformations of nitro-
gen to which allusion has just been made.
SPECIFIC FUNCTIONS OF THE PARASITIC BACTERIA.
As already intimated the parasitic bacteria are not charac^
terized by such beneficent activities as are possessed by
the saprophytic group; they exist at the expense of living
FUNCTIONS OF THE PARASITIC BACTERIA 41
hosts and usually excite detrimental changes in those hosts.
It is to the parasitic group that the pathogenic or disease-
exciting bacteria belong.
Strictly speaking none of the pathogenic bacteria with
which we are acquainted are obligate parasites, that is,
none of them grow and multiply only in the body of a living
host; for all have been cultivated under artificial conditions
on dead, nutrient cultural materials. They are nevertheless
properly classified as parasites for it is only under conditions
of parasitism that they exhibit those activities that make
them the objects of special interest.
When circumstances admit of the various members of
this group getting access to the living hosts in which they
find conditions favorable to their growth and multiplica-
tion there results the state known as "disease." In some
cases the disease is local, i. e., it involves only the tissues in
the immediate vicinity of the invading bacteria; in others
it is general; involves the entire body and eventuates in
the death of the host.
As we study the peculiarities of the disease-producing
bacteria more closely we find that in inducing disease they
do not all operate in the same way, though the ultimate
forces used by them in the destruction of living tissue are
throughout analogous, i. e., they are poisons.
In some cases the parasite finds the circulating fluids of
the host the most favorable place for its growth and develop-
ment. Under such circumstances it is not uncommon for
the blood- and lymphvessels of an infected animal to be
almost filled with the parasites within a short tune after
the invasion. To such a state the designation "septicemia"
is given, that is, there is a septic condition of the blood,
"blood poisoning" as it is commonly called.
42 BACTERIOLOGY
In other instances a parasitic species may manifest its
activities in a very insignificant way, insofar as the welfare
of the host is concerned. The causation of simple boils,
pimples and unimportant local inflammations serves to
illustrate this. In other examples we find the activities
of the parasite more or less confined to special vital organs
of the body, the restriction, practically speaking, of the
cholera and dysentery germs to the mucosa of the intestinal
canal; of the gonorrheal germ to the mucous surfaces of
the genito-urinary tract; of the typhoid bacillus to the
lymphatic structures of the abdominal cavity may serve
as illustrations.
Again we know of parasitic species that do not disseminate
beyond their portal of entry. They grow at that point and
manufacture deadly poisons which are disseminated through-
out the body by way of the circulating fluids. The germs
of diphtheria and of tetanus are striking illustrations of
this type of parasite.
In practically all cases fever is an accompaniment of the
activities of the parasitic bacteria in the body though in
certain particular instances an initial rise in temperature
may be followed by marked depressions of it, due to the
action of the poisons elaborated by the bacteria.
In considering the activities of the parasitic group of
bacteria we encounter at the beginning one factor in par-
ticular with which we are not called upon to reckon in speak-
ing of the saprophytic group. The saprophytes work upon
inert, dead matter; the parasites on active, living matter,
all of which is by nature endowed with some degree of
resistance to the inroads and activities of invading para-
sites. It is through this "vital resistance" possessed by the
living host that many of the irregularities seen in the opera-
FUNCTIONS OF THE PARASITIC BACTERIA 43
tion of the parasitic group may be explained. It is indeed
so active in certain individual cases as to give to its possessor
almost complete immunity from particular types of parasitic
invasion.
To illustrate: Diphtheria is caused by a well-known
parasite. It has many interesting properties but the most
significant of its physiological activities is its power to
elaborate a poison that causes the group of symptoms and
tissue changes which we know as diphtheria. No other
organism has this property. This same diphtheria bacillus
may invade one person and cause his death, while in another
the results of its activities may be comparatively trifling.
Similar extremes of variation are constantly seen in the case
of all the known infective diseases. It is to be explained in
but one way "the soil," i. e., the living host, in which the
disease exciting " seed," the germ, finds itself is not that
which is best suited to its active growth, or in other words
one individual possess higher natural powers of resistance
than does another, and in a large group of individuals such
differences in the degree of resistance are marked. We see
nothing like this in the action of saprophytes upon dead
matter. It is true we see their growth restrained at times.
In some cases such restraint is exercised by other species
the products of whose growth are antagonistic; and in a
number of cases the growth of saprophytes is often for a
time checked by the accumulated products of their own
activities. For instance the growth of those saprophytes
concerned in acid fermentations comes very quickly to an
end unless special provisions be made to neutralize and fix
the acids as fast as they are manufactured, for no bacteria
develop indefinitely in the presence of free acids. This is,
however, a very different kind of inhibition from that
44 BACTERIOLOGY
exercised by living tissues in repelling the invasion of
parasites.
In the foregoing brief sketch of the manifold transfor-
mation resulting from bacterial activity there is no dis-
cussion of the mechanism through which such changes are
wrought. In the case of the saprophytes the various analyses
and syntheses that accompany their growth are in general
believed to be manifestations of fermentations; while the
activities of the parasites in producing disease are referred
to poisons elaborated by these that have a destructive action
upon the tissues in which the parasites are operating. In
the following paragraphs an effort will be made to elucidate
this phase of the subject.
FERMENTS, ENZYMES, TOXINS, BACTERIAL PROTEINS,
AND PTOMAINS.
There is perhaps no department of either biology or physics
that relates to more important phenomena, more widespread
phenomena or more inexplicable phenomena than that
having to do with fermentation and the agencies that cause
it.
The phenomenon has attracted the attention of the ablest
investigators for years and we are scarcely nearer to an
understanding of its intimate nature today than we were
at the beginning.
In its older sense, the word fermentation related to all
reactions that are accompanied by the evolution of gas and,
indeed, it is probable that the word originated with the word
fervere, meaning to seethe, to boil, to bubble. In its modern
usage, however, the word comprehends many reactions,
believed to be caused by ferments, but during which no
FERMENTS, ENZYMES, TOXINS AND PTOMAINS 45
gas as such is evolved: The fermentation best and longest
known to man is that through which sugar is converted
into alcohol, seen in the making of wine from grapes. In
so far as bacteria are concerned we are aware of a multipli-
city of reactions which are believed to be manifestations
of fermentation, though opinion on these points is far from
being in agreement. However, as ferments have never
been isolated in a pure state and as the real nature of their
activities cannot with the present means at our disposal,
be finally determined, there is as much justification for
regarding such reactions as excited by ferments as not.
We shall therefore assume that both the normal metabolism
of the bacterial cell and its peculiar power to excite specific
reactions in various substances are made possible through
the agency of ferments. In some cases such ferments are
firmly bound up as integral parts of the cell protoplasm.
To such cells with their peculiar ferments the term "organized
ferments" is often applied. The common yeast cell serves
as an example. In other cases the cells throw off in the
course of their living activities, as by-products so to speak,
bodies which, when completely separated from the cells by
which they were formed, are still capable of bringing about
fermentation reactions when mixed with appropriate sub-
stances, without themselves undergoing any demonstrable
change. These are denominated "unorganized ferments"
or "enzymes."
In the case of the disease-producing bacteria we have an
analogous state of affairs. We find that the tissue changes
characterizing disease are due to poisons elaborated by the
living pathogens. These poisons are generically known as
toxins, and it is possible, though not certain, that in causing
disease their activities may be in some instances likened to
46 BACTERIOLOGY
those of the enzymes of the non-disease producing group,
while in others this is not the case. In the case of certain
pathogens, as with the yeasts and certain saprophytic bacteria,
these toxins — poisons — are so bound up with the protoplasmic
bodies of the bacteria that they become effective as poisons
only on the disintegration of the cells containing them; these
are the "endotoxins." In other instances the poisons are
diffused through the surrounding medium in which the
bacteria are growing and may readily be separated from
the cells forming them by the simple process of filtration.
These are the free or "true toxins."
At one time there was believed to be an essential difference
between the "organized" and "unorganized" ferments, but
when in 1897 E. Buchner expressed the active ferment from
the yeast cell, and demonstrated that this active principle,
"zymase," without the aid of the living cell, is capable of
transforming sugar into aclohol, just as is done by the
intact living yeast cells, it became manifest that the old
distinction between "organized" and "unorganized" fer-
ments is after all not important. The "enzyme" is the
active agent and in so far as the result is concerned it matters
not if it be tied up in the body of a cell or diffused freely in
the medium surrounding the cell.
The same may be said with regard to the analogous
"endotoxins" and "toxins" elaborated by the pathogenic
species, though it must not be assumed that the toxins act
in the same way as do the ferments or enzymes. Such
knowledge as we have of the mechanism of certain toxic
activities justifies the statement that the poisons of some
pathogenic bacteria enter into a destructive combination
with body cells for which they have a specific affinity and
that there and then their activity ceases; the result being
FERMENTS, ENZYMES, TOXINS AND PTOMAINS 47
that the physiological activities of both the poison and the
cells are destroyed. Not so with the enzymes; they are
characterized by the ability to bring. about profound altera-
tions in the substances on which they are acting without
they themselves being appreciably altered or diminished in
quantity; just as is seen with many of the inorganic catalysers
which, after having, by their mere presence, promoted con-
spicuous changes in the substances surrounding them, are
found at the end to have undergone little or no loss in
amount and to be of identically the same composition as
at the beginning. As to the way in which enzymes act
nothing definite can be said. The problem has for years
engaged the attention of competent investigators but up to
the present there is no final opinion. That they differ in
nature and mode of operation the one from the other
seems certain; the results of their activities are manifestly
different.
Neither enzymes nor toxins have ever been isolated in
a pure state. Both are assumed to be amorphous matters
of a protein nature and all are recognized by that which
they do; i. e., by the reactions which they originate. They
are characterized for their instability, particularly is this
the case with the enzymes. All have many of the essential
characteristics of living matter; they are destroyed by heat,
varying in amount and mode of application. The same
chernicals that are hurtful to living cells are likewise, in the
main, destructive of enzymes and toxins; they are soluble
(or appear to be) in water, dilute acids, alkalies and neutral
salines; they are to a slight extent dyalizable; some are
precipitated from their solutions by alcohol readily, others
less so; they may be thrown down from their solutions by
mechanically enmeshing them with certain inorganic
48 BACTERIOLOGY
precipitates. Their powers of fermentation (enzymes) and
of intoxication (toxins) are apparently specific.
The enzymes of bacterial origin with which we are best
acquainted may be defined as amorphous constituents of
living protoplasm that are able through catalytic activity
to split up complex organic substances into simpler, more
soluble and diffusible combinations. They may be classified
as proteolytic, diastatic, inverting, coagulating, sugar
splitting, fat splitting, etc. It is important to note that
such enzymes may and do originate in both the animal
and vegetable world. Those obtained from bacteria are,
in so far as it is possible to say, identical with those found
in the cells of animals.
The proteolytic or albumin-dissolving enzymes are formed
by a great many bacteria. The most familiar indications of
the formation of a proteolytic enzyme are seen in the lique-
faction of gelatin, in the digestipn of coagulated blood serum,
and of casein. Most frequently the proteolytic enzyme is
allied to trypsin, since the liquefaction, hydrolysis or
digestion induced by it proceeds only under an alkaline reac-
tion.1 Some bacteria, however, produce a proteolytic enzyme
analogous to pepsin, and this enzyme is active under an
acid reaction. The proteolytic enzymes of different bacteria
vary considerably with regard to their resistance to heat,
some being destroyed in a few minutes when heated to 60°
or 70° C.,. while others may be exposed to 100° C. for a short
time without suffering marked deterioration.2 The proteo-
lytic enzymes also differ in respect to their susceptibility to
the action of acids and other chemicals.
The formation of proteolytic enzymes is one of the func-
1 See Abbott and Gildersleeve, Journ. of Med. Research, 1903, vol. v.
2 Loc. cit.
FERMENTS, ENZYMES, TOXINS AND PTOMAINS 49
tions of bacteria that is easily disturbed by external condi-
tions, for instance, long-continued cultivation on media
in which the exercise of this function is not required may
lead to its marked deterioration, while prolonged cultivation
under conditions demanding it may result in its accentua-
tion.
The addition of carbohydrates and of glycerine to culture
media interferes with production of the proteolytic enzyme
by many species of bacteria, as shown by Auerbach.1
Diastatic enzymes convert starch into sugar. This func-
tion is best studied on media containing starch, as potato
infusion or solutions of starch. By appropriate tests the
intermediate steps in the conversion of the starch into
sugar may be traced by testing a portion of the culture
medium from time to time. Fermi2 found this function
in a large number of bacteria studied, especially in organisms
of the subtilis group and in the microspira of the cholera
group.
Inverting enzymes convert saccharose into dextrose and
levulose. These enzymes are produced by comparatively
few bacteria. Fermi found this function manifested by
bacillus megatherium, pseudoinonas fluorescens, bacillus
vulgaris, microspira comma, microspira Metchnikovi, and
others.
Coagulating enzymes are those which coagulate milk.
Rennet may be taken as the typical form. This alteration
is quite common in association with an acid reaction, but
in such instances it is not always certain that the coagulation
has not been induced by the acid formed. Gorini3 found
1 Archiv fiir Hygiene, Ed. xxxi, p. 311.
2 Ibid., Bd. xi, and Centralblatt fiir Bacteriologie, Bd. xii.
3 Centralblatt fur Bacteriologie, Bd. xii, p. 666.
4
50 BACTERIOLOGY
that cultures of bacillus prodigiosus, sterilized by heating
to 60° C., caused a solid coagulation of sterile milk in a few
days.
A small number of bacteria have also been encountered
that bring about coagulation of milk with a distinctly
alkaline reaction. This function has been noticed in bac-
teria isolated from milk, and especially in bacterium pseudo-
diphtheriticum isolated from cows' milk (Bergey).
Sugar-splitting enzymes are . very common in bacteria.
This function varies in different species as seen in the dif-
ferent end-products that are formed. Buchner succeeded
in isolating the sugar-splitting enzyme (zymase) of yeast-
cells, and when thus isolated it still possesses the power
of inducing active fermentation of sugar. It is believed that
the sugar-splitting enzymes of bacteria are similar in charac-
ter to the zymase of yeast cells. The splitting up of carbo-
hydrates appears to be brought about by the bacteria for
the purpose of obtaining oxygen, as indicated by the nature
of the end-products formed, and also by the conditions
under which it may be carried out — i. e., the absence of
atmospheric oxygen.
The splitting of the carbohydrate molecule may be illus-
trated as follows:
= 2C2H6O + 2CO2
Grape sugar = 2 alcohol + 2 carbon dioxide
or C6Hi2O6 = 2C3H6O3
Grape sugar = 2 lactic acid ,
or CsHizOe = 3C2H4p2
Grape sugar = 3 acetic acid
According to Theobald Smith1 all facultative anaerobic
bacteria2 form acids from carbohydrates, while the strictly
1 Centralblatt fur Bacteriologie, Bd., xviii.
2 See "aerobic" and "anaerobic" bacteria.
FERMENTS, ENZYMES, TOXINS AND PTOMAINS 51
aerobic bacteria do not have this function, or bring about
the alteration so slowly that it is concealed by the simul-
taneous production 'of alkali. Among the acids formed by
bacteria, besides carbon dioxide, we have lactic, acetic,
butyric, propionic, and formic; and frequently there is
also produced ethyl alcohol, aldehyde, and acetone.
The lactic acid formed by the action of different bacteria
on carbohydrates may be either dextrorotatory or levoro-
tatory, or almost equal quantities of both forms may be
present and the mixture be optically inactive.
Bacterial Proteins. — The proteid matter making up the
bodies of many species of bacteria, even those not conspicu-
ously pathogenic, was shown by H. Buchner to induce dis-
ease when isolated and injected into the tissues of animals;
in some cases causing only slight fever, in others acute
inflammation with suppuration. For such compounds he
suggested the name "bacterial proteins."
Ptomains. — Ptomains, or as they are sometimes called
"putrefactive alkaloids" or "cadaveric alkaloids," are
crystallizable, nitrogenous bodies that are the results of
bacterial action upon dead organic matter. They differ
from enzymes in that they are the occasional results of
defective bacterial metabolism and from both toxins and
enzymes in that they are crystallizable and of definite
chemical composition. Some of them are poisonous, many
are not. The conditions favorable to the elaboration of
ptomains by bacteria vary, but in the main the most
poisonous of the ptomains appear to be the result of bac-
terial activity under a limited supply of oxygen. Poisonous
ptomains are sometimes formed within the intestinal canal
of man either as a result of malf ermentation or of interruption
of normal oxidation. We have no reason for believing that
52 BACTERIOLOGY
ptomains play any part in either the causation or course
of the definite, infective diseases.
Ptomains have been isolated from decomposing cadavers,
from putrid meat, milk, cheese, and from a number of
bacterial cultures. Poisonous ptomains occasionally develop
in improperly preserved food. True toxins and dangerous
bacteria have also been found in such substances.
Nutrition of Bacteria. — :We have said that through the
agency of chlorophyl, in the presence of sunlight, the green
plants are enabled to obtain the amount of nitrogen and
carbon which is necessary to their growth from such simple
bodies as carbon dioxide and ammonia, which they decom-
pose into their elementary constituents. The bacteria, on
the other hand, owing to the absence of chlorophyl from
their tissues, do not possess this power. They must, there-
fore, have their carbon and nitrogen presented as such, in
the form of decomposable organic substances.
In general, bacteria obtain their nitrogen most readily
from soluble proteins, and to a certain extent, but by no
means so easily, from salts of ammonium. In some of
Nageli's experiments it appeared probable that they could
obtain the necessary amount of nitrogen from inorganic
nitrates. At all events, he was able in certain cases to
demonstrate a reduction of nitric to nitrous acid and ulti-
mately to ammonia. Nevertheless, in all of these experi-
ments circumstances point to the probability that the
nitrogen obtained by the bacteria for building up their
tissues in the course of their development was derived from
some source other than the nitric acid or the nitrates, and
that the reduction of this acid was most probably a secondary
phenomenon. We must bear in mind, however, the specific
group, the. nitrifying bacteria, which increase and mul-
FERMENTS, ENZYMES, TOXINS AND PTOMAINS 53
tiply without appropriating proteid nutrition. They are, as
stated above, concerned in the particular form of fermenta-
tion that results in the oxidation of ammonia to nitrous and
nitric acids, a process everywhere in progress in the super-
ficial layers of the soil.
For the supply of carbon many of the carbon compounds
serve as sources upon which the bacteria can draw. The
carbon deficit, for example, can be obtained from sugar and
bodies of like composition; from glycerin and many of the
fatty acids; and from the alkaline salts of tartaric, citric,
malic, lactic, and acetic acids. In some instances carbon
compounds, which when present in concentrated form
inhibit the growth of bacteria, may, when highly diluted,
serve as nutrition for them. Salicylic acid and ethyl alcohol
are of this class.
In addition- to carbon and nitrogen, water is essential
to the life and development of bacteria; without it no
development occurs, and in many cases drying kills them.
Certain species and developmental forms, on the contrary,
though incapable of multiplying when in the dry state,
may be dried without causing them to lose the power of
reproduction when again placed under favorable conditions.
Closer study of bacteria, and a more intimate acquain-
tance with their nutritive changes, demonstrate an appre-
ciable variability in the character of the substances best
suited for the nutrition of different species, as well as in the
end products of such nutrition, for instance: one species
may require a tolerably concentrated form of nutrition,
while another needs but a very limited amount of proteid
substance for its development; some bring about profound
alterations in the media in which they are growing, while
others produce but little apparent change; for certain species
54 BACTERIOLOGY
free oxygen is essential, for others it is harmful. In one case
alterations in the reaction of the media will be conspicuous,
while in another no such variation can be detected. As
shown above the growth of some species is accompanied by
evidence of specific fermentations; of others by the appear-
ance of poisonous; of others by putrefactive changes.
In considering the normal development of bacteria we
must not lose sight of the fact that this is influenced both
by the quality and the quantity of the nutritive materials
to which they have access, and by the character of the
metabolic products that accumulate in these materials as
a result of their vital processes. Nitrogen and carbon
compounds may be present in amount and kind entirely
suitable to normal bacterial growth, and yet this may be
checked, after a comparatively short time, by the accumu-
lated products of bacterial metabolism, some of which
possess the property of inhibiting growth and ultimately
of even destroying the bacteria that produced them. The
most common and conspicuous examples of such inhibiting
conditions is alteration in the chemical reaction of the media
in which the bacteria are developing.
In the case of a number of species there begins, coincidently
with retardation of normal development, a process of dis-
solution, self-digestion or "autolysis," which may continue
until the cells are unrecognizable as bacteria. This pheno-
menon is the result of the action of enzymes located within
the cells which, under normal conditions of growth, are
concerned in the life processes of the cell, but which, on the
advent of conditions unfavorable to the growth and mul-
tiplication of the cells, .react upon them and cause their
actual solution. An analogous "autolysis" is often to be
seen with animal cells. If bits of living tissue be removed
FERMENTS, ENZYMES, TOXINS AND PTOMAINS 55
from the body, under aseptic precaution, and kept at suit-
able conditions of moisture and temperature they may
ultimately become completely liquefied as a result of the
digestive action of hydrolysing enzymes contained within
them.
Their Relation to Oxygen. — Of primary importance and
interest in the study of the nutritive changes of bacteria
is the difference in their relation to oxygen. For certain
species free oxygen is essential to the proper performance
of their functions; in another group no evidence of life can
be detected under its access; while in a third group free
oxygen appears to play but an unimportant role, for develop-
ment occurs as well with as without it. It was Pasteur who
first demonstrated the existence of particular species of
bacteria which not only grow and multiply and perform
definite physiological functions without the aid of free
oxygen, but to the existence of which it is positively harmful.
To these he gave the name anaerobic bacteria, in contra-
distinction to the aerobic group, for the proper performance
of whose functions free oxygen is essential. In addition to
these there is a third group, for the maintenance of whose
existence the absence or presence of uncombined oxygen is
apparently of no moment — development progressing as well
with as without it; the members of this group comprise the
class known as facultative in their relation to this gas. It is
'to this third group, the facultative, that- the majority of
bacteria belong.
It is also well to remember that many of the so-called
obligate anaerobes may, by a gradual process of adaptation,
adjust themselves to atmospheres containing oxygen. A
few observations have shown that even so anaerobic a
species as the bacillus of tetanus may be brought gradually
56 BACTERIOLOGY
to grow and perform all its important functions in ordinary
atmospheric air.
Since all growing bacteria, anaerobic as well as aerobic,
generate carbonic acid in the course of their development,
it is evident that oxygen must in reality be obtained by
them from some source, and must be regarded as essential
to their life processes; but the manner in which it is
appropriated by them varies, the aerobic species taking
it from the air as free oxygen, while the anaerobic species,
not possessed of this ability, obtain it through the decom-
position of more or less stable oxygen-containing com-
pounds.
Though the multiplication of the facultative varieties is
not interfered with by either the presence or absence of free
oxygen, yet experiments demonstrate that the products of
their growth are different under the varying conditions of
absence or presence of this gas. For example: in the case'
of certain of the chromogenic forms the presence or absence
of oxygen has a very decided effect upon the production of
the pigments by which they are characterized.
NOTE. — Observe the difference between the intensity of
color produced upon the surface of the medium and that
along the track of the needle in stab-cultures of bacillus
prodigiosus and of spirillum rubrum. In the former the red
color is apparently a product dependent upon the presence
of oxygen, while in the latter the greatest intensity of color
occurs at the point furthest removed from the action of
oxygen.
Influence of Temperature upon the Growth. — Another
factor which plays a highly important part in the biological
FERMENTS, ENZYMES, TOXINS AND PTOMAINS 57
functions of these organisms is the temperature under which
they exist. The extremes of temperature between which
the majority of bacteria are known to grow range from 5.5°
to 43° C. At the former temperature development is hardly
appreciable; it becomes more and more active until 38° C.
is reached, when it is at its optimum, and, as a rule, ceases
at 43° C.; though species exist that multiply at as high a
temperature as 70° C. and others at as low as 0° C. The
investigations of Globig,1 Miquel,2 and Macfayden and
Bloxall3 have revealed the existence in the soil, in water,
in feces, in sewage, in dust, and, in fact, practically every-
where, of bacteria that under artificial cultivation show no
evidence of life at a temperature lower than 60° to 65° C.,
and will even grow at such high temperatures as 70° and
75° C., a state of affairs almost paradoxical, inasmuch as
these are temperatures that suffice for the coagulation of
albumin, and, in consequence, are generally incompatible
with life. Rabinowitsch4 has likewise described a number
of species of these thermophilic bacteria, as they are called;
but states that it was possible in her experiments to obtain
evidence of their growth at the lower temperature (34° to
44° C.), as well as at the higher temperature mentioned by
preceding investigators. It is possible that this peculiarity
is but a manifestation of adaptation to environment and
not an essential to the life processes of these species.
The most favorable temperature for the development of
.pathogenic bacteria is that of the human body, viz., 37.5° C.
There are a number of bacteria commonly present in water,
1 Zeitschrift fur Hygiene, Bd. iii, S. 294.
2 Annales de Micrographie, 1888, pp. 4 to 10.
3 Journal of Path, and Bact., vol. iii, Part I.
4 Zeitschrift fur Hygiene u. Infecktionskrankheiten, Bd. xx, Heft. 1,
S. 154 to 164.
58 BACTERIOLOGY
the so-called normal water bacteria, that grow best at about
20° C.
Reaction. — The majority of bacteria require an approx-
imately neutral medium in which to multiply and function.
Certain species may develop in weak acid materials, others
in weak alkaline — but none can live and grow in media
either strongly acid or alkaline. Even those species whose
most conspicuous function is the conversion of sugars into
acids have their activities checked by the accumulation of
free acid beyond a very limited amount.
Cooperating Bacteria. — Under natural conditions it fre-
quently occurs that the development of one species or group
of species of bacteria is directly dependent upon the func-
tional activities of another totally distinct species, the
growth of one group resulting in conditions that are of vital
importance to the existence of the other. Such interdepen-
dence is observed, for instance, in complete nitrification,
as already noted; in the course of putrefaction, where,
through exhaustion of free oxygen by the actively germinat-
ing aerobic varieties, the conditions are supplied that enable
the anaerobic species to develop and exercise their biological
activities. Again, through the proteolytic activity of
enzymes produced by certain species of bacteria, other
species are supplied with nutrition that would otherwise be
unassimilable or only imperfectly so. Similar cooperative
or symbiotic relations between bacteria and higher plants
are also noticed, notably that between certain bacteria of
the soil and the group of leguminous plants, whereby the
latter are enabled, through the assistance of the former, to
make up their nitrogen deficit in large part from the free
nitrogen of the atmosphere. This latter relationship is
probably an example of true symbiosis.1
1 See Nitrogen fixing bacteria.
FERMENTS, ENZYMES, TOXINS AND PTOMAINS 59
Influence' of Light. — Light is not only unnecessary to the
performance of functions by bacteria but appears to be
in varying degrees inhibitory.
• Direct sunlight is destructive to many species. It is a
matter of common experience that cultures of particularly
important species retain their type characteristics better
and longer if cultivated in the dark than in diffuse daylight.
Electric light has likewise a depressing influence upon
the viability of bacteria. Beyond the fact that bacteria in
vacuo are unaffected by light we have no knowledge of the
mechanism of its action. Presumably it has something to
do with oxidation processes.
The germicidal action of the direct rays of the sun may
be easily demonstrated by preparing a plate of colon bacillus,
shading a portion and allowing the sun to shine upon it for
a time, varying with the intensity of its light. Growth will
occur in the shaded part, none or only relatively little in
the illuminated part of the plate.
Influence of Pressure. — The influence of pneumatic pres-
sure on the viability of bacteria appears to depend upon the
character of the gas used. Ordinary air, or its constituents,
oxygen and nitrogen, whenever pressed heavily (600 to
2000 atmospheres) upon cultures of bacteria, have a slight
inhibitory effect; Carbon dioxide under five to ten atmos-
pheres pressure is shown by Park and his associates to
destroy almost all of the typhoid, dysentery, diphtheria
and colon bacilli exposed to it within twenty-four
hours.
Effect of Moisture. — As is the case with all living plants
a degree of moisture is essential to life. Certain species of
bacteria are killed by ordinary drying, and many of them by
absolute drying. The spores (to be described later) of bac-
60 BACTERIOLOGY
teria are not so effected, a few species retaining their power
to germinate after having been dried, as the word is ordinarily
understood, for a comparatively long time, and spores have
been kept in a dry state for years without losing their power
to germinate.
Influence of Electricity. — The methods employed for
deciding this point have led to results that are inconclusive
and not easy of interpretation.
It is true that when bacteria are exposed to the electric
current they are often inhibited and sometimes killed.
This result may be interpreted in several ways, viz. : The
elevation of temperature caused by the current may explain
the destruction; the electrolytic action of the current on
matters in which the bacteria are located may, by dissocia-
tion, liberate agents that are destructive to bacteria, or a
similar destructive dissociation within the bacteria them-
selves may result from the action of the current.
The evidence at hand does not permit of the acceptance
of either of these suggestions as the correct interpretation of
the results.
Chemotaxis. — Another interesting biological peculiarity of
bacteria is that discovered by Engelmann and by Pfeffer,
known as chemotaxis. This term applies to the peculiar
phenomena of attraction and of repulsion that are exhibited
by motile bacteria when in the presence of solutions of bodies
of various chemical composition. It was demonstrated
that the bacteria in decomposing infusions accumulate in
great numbers in the neighborhood of the sources of oxygen.
In a hanging-drop of such an infusion the bacteria will be
seen to a'ccumulate in a dense mass along the margin or
around the edge of small bubbles of air in the fluid. Even
FERMENTS, ENZYMES, TOXINS AND PTOMAINS 61
plant cells in the infusion, whose chlorophyl sets free oxygen
in the light, are surrounded by large numbers of bacteria.
The positive chemotactic affinity between oxygen and
bacteria was employed by Engelmann as a basis for the
demonstration of small quantities of oxygen in studying the
influence of various kinds of light upon the assimilation of
green plant-cell. Pfeffer showed that when a neutral fluid
(a drop of water) containing motile bacteria is brought in
contact with a weak solution of either peptone, sodium
chloride, or dextrin, the bacteria are at once attracted
toward the solution; this reaction is designated "positive
chemotaxis." On the other hand, if brought in contact with
an acid, an alkaline, or an alcoholic solution, the bacteria
are repelled or driven from the point at which the two fluids
are diffusing; that is, they exhibit "negative chemotactic"
affinities. The significance of these reactions is not under-
stood, but it has been aptly suggested that they may be
fundamentally analogous to the specific positive and negative
affinities exhibited by the ions resulting from dissociation
of electrolytes, and that they may "have their explanation
in the forces of ionic attraction and repulsion."1 In this
connection it is important to note that the wandering
cells of the animal body, the leukocytes, exhibit also these
chemotactic phenomena; and it is- especially necessary to
a complete comprehension of the process of suppuration to
bear in mind that among the substances which have the
greatest attraction for these wandering cells, are the products
of growth of certain bacteria in some cases, and the protein
constituents of the bacteria themselves in others.
1 Read Sewall on Some Relations of Osmosis and Ionic Action in Clinical
Medicine, International Clinics, vol. xi, Eleventh Series.
62 / BACTERIOLOGY
To summarize briefly the foregoing it may be said, in
general, that for the growth and development of bacteria
nitrogenous organic matter of a neutral or slightly alkaline
reaction, in the presence of moisture and at a suitable tem-
perature, is all that is necessary. From this can be formed
some idea of the omnipresence in nature of these minute
vegetables. Bacteria are found wherever these conditions
obtain.
CHAPTER II.
Morphology1 of Bacteria — Chemical Composition of Bacteria — Mode of
Multiplication — Spore-formation — Motility.
IN structure the bacteria are unicellular, always develop-
ing from preexisting cells of the same character and never
appearing spontaneously. They are seen to occur as spher-
ical, rod- and spiral-shaped bodies that multiply by the
simple process of transverse division, belonging, therefore, to
the schizomycetes or fission fungi.
In size the bacteria are among the smallest living crea-
tures with which we are acquainted, being visible only
when very highly magnified. In order that some conception
of their microscopic dimensions may be formed, it has been
computed that of the average size bacteria about thirty
billion would be required to weigh a gram, and that about
one billion seven hundred million of the small spherical forms
might readily be suspended in a drop of water.
Under what we are accustomed to regard as normal con-
ditions of development, and by the ordinary methods of
examination, bacteria appear very simple in form and
structure. They are cells consisting of a protoplasmic mass
within a membranous hull that is discernible with more or
less difficulty. The protoplasmic body is of material closely
allied, chemically speaking, to ordinary vegetable protein.
It is often homogenous, but in particular species and under
various conditions of growth the central mass in stained
1 Morphology, pertaining to shape, outline, structure.
(63)
64 BACTERIOLOGY
specimens is commonly marked by the presence of very
dark granules, the so-called metachromatic granulations.
Again, in other species paraplastic granules giving the
microchemical reactions of fat, starch, sulphur, etc., are to
be seen. Under certain physical conditions the protoplasmic
body presents irregular rents or retractions, the result of
proteolytic or of osmotic disturbances dependent upon the
character of the fluid in which the bacteria are located; in
fact, the deeply staining granules, other than those of fat,
starch, and sulphur, that are often observed, are regarded
by some writers (especially A. Fischer) as but altered or
condensed protoplasm due to the same influences.
In certain species the protoplasmic body is always more
dense at the poles of the cells than at the middle, so that
when stained the ends are much darker than the intervening
portion. In other species the reverse is the case.
By some investigators the protoplasmic central mass is
regarded as a nucleus, and, functionally speaking, possibly
it is to all intents and purposes, but this cannot be certainly
decided. In the great majority of cases, however, with the
ordinary methods of examination, it is not seen to possess
any of the structural peculiarities that we are accustomed
to regard as the distinguishing attributes of cell-nuclei.
The enveloping hull or membrane is in some cases ap-
parently only a modification of the protoplasmic central
mass, at times being only a condensation of that protoplasm;
again, it seems to be, chemically different from it. In a few
instances it appears to be allied to cellulose in its chemical
composition. Sometimes it is so thick as to be readily seen,
while again it is discernible only by special methods of
examination. In particular species it may, by appropriate
methods, be seen as a sharply defined capsule inclosing a
CHEMICAL COMPOSITION OF BACTERIA 65
clear zone in which lies the deeply stained central mass.
Occasionally the central protoplasmic mass is surrounded
by an ill-defined slimy material that causes the individual
cells to adhere to one another in more or less compact masses
or pellicles (zooglea, Fig. 1).
Chemical Composition of Bacteria. — The bodies of bacteria
consist of water, salts, and albuminous substances, with
smaller proportions of various extractives soluble in alcohol
or ether, such as triolein, tripalmitin, tristearin, lecithin,
and cholesterin. In many varieties substances giving the
reaction of starch have been found, while others give the
true reactions of cellulose (B. subtilis). Nuclein has not
*
FIG. 1
/ ^>" '¥•--«&*>. ?£H. \
!*S£5*3B?. ,.
Zooglea of bacilli.
been found in any of the bacteria, though the nuclein bases,
xanthin, guanin, adenin, have been found.
The relative amounts of water in bacteria are influenced
to a large extent by the nature of the medium on which
they have been grown. In like manner the content in
albumin, extractive substances, and salts varies with the
conditions under which the bacteria have been cultivated.
E. Cramer1 has studied the chemical composition of bacteria
in great detail. As the result of his studies of microspira
comma, he found its composition to be as follows: water
1 Archiv fur Hygiene, Bd. xiii, xvi, xxii, and xxviii.
66 BACTERIOLOGY
88.3 per cent., albumin 7.6 per cent., ash 3.6 per cent. The
dry substance of the bacteria contains the following : albumin
65 per cent., ash 31 per cent. From 76 to 80 per cent, of
the ash consists of sodium chloride and phosphate.
Morphology of Bacteria. — For the purposes of this book it
will suffice to classify the bacteria roughly into three mor-
phological groups with their subdivisions, the members of
each group being identified by their individual outline, viz.,
that of a sphere, a rod, or a spiral.1 To these three grand
FIG. 2
c d e
a, staphylococci; b, streptococci; c, diplococci; d, tetrads; e, sarcinse.
divisions are given the names cocci or micrococci, bacilli,
and spirilla.
Mode of Multiplication. — In the group micrococci belong
all spherical forms — i. e., all those forms the isolated individ-
ual members of which are practically of the same diameter
in all directions. (See Fig. 2, a, 6, c, d, e.)
The bacilli comprise all oval or rod-formed bacteria. (See
Fig. 3.)
1 For complete data on classification see Reports of Committee of Am.
Bact. Soc., Jour. Bact., 1917, vol. ii; 1920, vol. iii.
MODE OF MULTIPLICATION 67
To the spirilla belong the bacteria that are curved when
seen in short segments and that appear as undulating
threads when such segments are of greater length or when
several short segments are joined end to end. (See Fig. 4.)
FIG. 3
d e f
a, bacilli in pairs; b, single bacilli; c and d, bacilli in threads; e and /,
bacilli of variable morphology.
The micrococci are subdivided according to their pre-
vailing mode of grouping, as seen in growing cultures, into
staphylococci — those growing in masses like clusters of grapes
FIG. 4
a b c
a and c, spirilla in short segments and longer threads — the so-called comma
forms and spirals; 6, the thick spirals, known as vibrios.
(see Fig. 2, a); streptococci, those growing in chains con-
sisting of a number of individuals strung together like
beads upon a string (see Fig. 2, b) ; diplococci — those growing
68 BACTERIOLOGY
in pairs (Fig. 2, c); tetrads — those developing as fours (Fig.
2, d); and sarcince — those dividing into fours, eights, etc.,
as cubes — that is, in contradistinction to all other forms, the
segmentation, which is rarely complete, takes place regularly
in three directions of space, so that when growing the bundle
of segmenting cells presents somewhat the appearance of a
bale of cotton (Fig. 2, e).
To the bacilli belong all straight, oval and rod-shaped
bacteria — L e., those in which one diameter is always greater
than the other. In this group are found those organisms the
life-cycle of many of which presents deviations from the
simple rod shape. Many of them in the course of development
increase in length into long threads, along which traces of
segmentation may usually be found. Again, under certain
conditions, many of them possess the property of forming
within the body of the rods oval, glistening spores (see Fig. 6),
and, if the conditions are not altered, the rods may entirely
disappear and nothing be left in the culture but these oval
spores. In some of them this phenomenon of spore-formation
is accompanied by an enlargement or swelling of the bacillus
at the point at which the spore is located (see Fig. 6, c and d) .
Again, many of them, from unfavorable conditions of nutri-
tion, aeration, or temperature, undergo pathological changes
that are probably autolytic in nature — that is, the individ-
uals themselves experience degeneration of their proto-
plasm with coincident distortion of their outline; they are
then usually referred to as "involution-forms" (see Fig. 5,
a and &). In all of these conditions, however, so long as
death has not occurred, it is possible to cause these forms
to revert to the typical rods from which they originated,
by the renewal of conditions favorable to their normajj
vegetation.
MODE OF MULTIPLICATION 69
It must be borne in mind, though, that it is never possible
by any means to bring about changes in these organisms
that will result in the permanent conversion of the mor-
phology of the members of one group into that of another —
that is, one can never produce bacilli from micrococci, nor
vice versa; and any evidence which may be presented to
the contrary is based upon untrustworthy methods of
experimentation.
Very short oval bacilli may sometimes be mistaken for
micrococci, and at times micrococci in the stage of segmenta-
tion into diplococci may be mistaken for short bacilli; but
FIG. 5
tf \\) '|/|
*//»
. a, spirillum of Asiatic cholera (comma bacillus) ; normal appearance
in fresh cultures; b, involution-forms of this organism as seen in old cul-
tures.
by careful inspection it will always be possible to detect a
continuous outline along the sides of the former, and a
slight transverse indentation or partition-formation between
the segments of the latter. The high index of refraction of
spdres, the property which gives to them their glistening
appearance, will always serve to distinguish them from
micrococci. This difference in refraction is especially notice-
able if the illumination of the microscope be reduced to the
smallest possible bundle of light-rays. . The spores, more-
over, take up staining-reagents much less readily than
do the micrococci. The most reliable differential points,
70 BACTERIOLOGY
however, are the infallible properties possessed4 by the
spores of developing into bacilli, and by the spherical
organism with which they may have been confounded of
always producing other micrococci of the same spherical
form.
We have less knowledge of the life-history of the spiral
forms. Efforts toward their cultivation under artificial
conditions have thus far been successful in only a compara-
tively limited number of cases. Morphologically, they are
thread- or rod-like bodies which are twisted into the form
of spirals. In some of them the turns of the spiral are long,
in others quite short. In some the threads appear rigid, in
others flexible. They are motile and multiply apparently by
the simple process of fission.1
Mode of Multiplication. — The micrococci multiply by
simple fission. When development is in progress a single
cell will be seen to elongate slightly in one of its diameters.
Over the center of the long axis thus formed will appear a
slight indentation in the outer envelope of the cell; this
indentation will increase in extent until there exist even-
tually two individuals which are distinctly spherical, as was
the parent from which they sprang, or they will remain
together for a time as diplococci; the surfaces now in juxta-
position are flattened against one another, and not infre-
quently a fine, pale dividing-line may be seen between the
two cells. (See Fig. 2, c and d.) A similar division in the
other direction will now result in the formation of fours as
tetrads.
In the formation of staphylococci such division occur
irregularly in all directions, resulting in the production of
1 Dividing into two transversely.
MODE OF MULTIPLICATION 71
the clusters in which these organisms are commonly seen.
(See Fig. 2, a.) With the streptococci, however, the ten-
dency is for the segmentation to continue in one direction
only, resulting in the production of long chains of 4, 8, and
12 individuals. (See Fig. 2, 6.)
The sarcinse divide more or less regularly in three direc-
tions of space; but instead of becoming separated the one
from the other as single cells, the tendency is for the seg-
mentation to be incomplete, the cells remaining together
in masses. The indentations upon these masses or cubes,
which indicate the point of incomplete fission, give to the
bundles of cells the appearance commonly ascribed to them,
viz., that of a bale of cotton or a packet of rags. (See Fig.
2>e.)
The mode of multiplication of bacilli is similar to that
of the micrococci — i. e., a dividing cell elongates slightly in
the direction of its long axis; an indentation appears about
midway between its poles, and this becomes deeper and
deeper, until eventually two daughter-cells have formed.
This process may occur in such a way that the two young
bacilli adhere together by their adjacent ends in much the
same way that sausages are seen to be held together in
strings (Fig. 3, /), or the segmentation may take place more
at right angles to the long axis, so that the proximal ends
of the young cells are flattened, while the distal extremities
may be rounded or slightly pointed (Fig. 3, e). The segmen-
tation of the anthrax bacillus, with which we are to become
acquainted later, results, when completed, in an indenta-
tion of the adjacent extremities of the young segments, so
that by the aid of high magnifying powers these surfaces
are seen to be actually concave. Bacilli never divide longi-
tudinally.
72 BACTERIOLOGY
Spore Formation. — With the spore-forming bacilli, under
favorable conditions of nutrition and temperature, the
same mode of segmentation is seen to occur during vege-
tation; but as soon as these conditions become altered by
the exhaustion of nourishment, the presence of detrimental
substances, unfavorable temperatures, etc., they enter, in
their life-cycle, the stage to which we have referred as
spore-formation. This is the process by which the organisms
are enabled to enter a state in which they resist deleterious
influences to a much higher degree than is possible for them
when in the growing or vegetative condition.
In the spore, dormant, or permanent state, as it is variously
called, no evidence of life whatever is given by the spores;
though as soon as the conditions which favor their germina-
tion have been renewed these spores develop again into the
same kind of cells as those from which they originated, and
the appearances observed in the vegetative or growing stage
of their history are repeated.
Multiplication of spores, as such, does not occur ;~ they
possess only the power of developing into individual rods
of the same nature as those from which they were formed,
but not of giving rise to a direct reproduction of spores.
When the conditions which favor spore-formation present,
the protoplasm of the vegetative cells is seen to undergo a
change. It loses its normal homogeneous appearance and
becomes marked by granular, refractive points of irregular
shape and size. These eventually coalesce, leaving the
remainder of the cell clear and transparent. When this
coalescence of highly refractive particles is complete the
spore is perfected. In appearance the spore is oval or round,
and very highly refractive — glistening. It is easily differen-
tiated from the remainder of the cell, which now consists
SPORE FORMATION 73
only of a cell-membrane and a transparent, clear space
which surrounds the spore. Eventually both the cell-
membrane and its fluid contents disappear, leaving the oval
spore free; it then gives the impression of being surrounded
by a dark, sharply defined border. When thus perfectly
developed, the spore may be regarded as analogous to the
seeds of higher plants. Like the seed, it evinces no evidence
of life until placed under conditions favorable to germina-
tion, when there develops from it a cell identical in all
respects with that from which it originated. Its tenacity
of life, as in the case of seeds, is almost unlimited. It may
FIG. 6
a, Bacillus subtilis with spores; 6, bacillus anthracis with spores; c, clos-
tridium form with spores; d, bacillus of tetanus with end spores.
be kept in a dry state, and this has actually been done, for
years without loss of viability.
The glistening, enveloping spore-membrane is not of
uniform thickness throughout, and in consequence when
germination occurs the growing bacillus, the so-called vege-
tative form of the organism, protrudes through the thinnest
part of the spore-membrane — that is, through the point of
least resistance. This may be either the end or the side of
the spore, according to the species under observation. In
certain cases such a protrusion is not observed, but in its
place the spore in toto appears to be gradually absorbed or
74 BACTERIOLOGY
in some way converted directly into a vegetating cell. It
evinces no motion other than the mechanical tremor common
to all insoluble microscopic particles suspended in fluids,
and it remains quiescent until there appear conditions favor-
able to its subsequent development.
By the ordinary methods of staining, spores do not become
colored, so that they appear in the stained cells as pale,
transparent, oval bodies, surrounded by the remainder of
the cell, which has taken up the dye.
A single cell produces but one spore. This may be located
either at an extremity or in the center of the cell (Fig. 6).
Occasionally spore-formation is accompanied by an en-
largement of the cell at the point at which the process is
in progress. As a result, the cell loses its regular rod
shape and becomes that of a club, a drum-stick, or a loz-
enge, depending upon whether the location of the spore
is to be at the pole or in the center of the cell. (See
Fig. 6, c and d.)
Motility. — In addition to the property of spore-formation
there is another striking difference between various species
of bacteria, namely, the property of motility, by which some
of them are distinguished. This power of motion is due to
very delicate, hair-like appendages or flagella, by the lashing
motions of which the cells possessing them are propelled
through the fluid. In some cases the flagella are located at
but one end of the organism, either singly (monotrichic) or
in a tuft (lophotrichic) ; and in some cases, especially of
the bacillus of typhoid fever, they are given off from the
whole surface of the rod (peritrichic). (See Fig. 7.)
For a long time this property of independent motion could
only be assumed to be due to the possession of some such
form of locomotive apparatus, because similar appendages
MOTILITY
75
had been seen upon some of the large motile spirilla found
in stagnant water, but it was not until a few years ago that
the accuracy of this assumption was actually demonstrated.
By a special method of staining Loffler1 rendered visible
these hair-like appendages. His method, as well the several
FIG. 7
a, spiral forms with a flagellum at only one end; 6, bacillus of typhoid
fever with flagella given off from all sides; c, large spirals from stagnant
water with wisps of flagella at their ends (spirillum undula).
modifications that have been made of it, depends for success
upon the use of mordants, through the agency of which
the stains employed are caused to adhere with increased
tenacity to the objects under treatment.
1 Loffler's method of staining will be found in the chapter devoted to
this part of the technique. v
CHAPTER III.
Principles of Sterilization by Heat — Methods Employed — Discontinued
Sterilization — Fractional Sterilization — Apparatus Employed — Sterili-
zation under Pressure — Sterilization by Hot Air — Thermal Death-
point of Bacteria — Chemical Disinfection and Sterilization — Mode
of Action of Disinfectants — Practical Disinfection.
OF fundamental importance to successful bacteriological
manipulations are acquaintance with the principles under-
lying the methods of sterilization and disinfection, and
familiarity with the approved methods of applying these
principles in practice.
In many laboratories it is customary to employ the term
sterilization for the destruction of bacteria by heat, and the
term disinfection for the accomplishment of the same end
through the use of chemical agents. Such distinction in the
use of the terms is obviously incorrect, as we shall endeavor
to explain.
The laboratory application of the word sterilization for
the destruction of bacteria by high temperatures probably
arose from the circumstance that culture-media, and certain
other articles that it is desirable to render free from bacterial
life, are not treated by chemical agents for this purpose,
but are exposed to the influence of heat in various forms of
apparatus known as sterilizers; and the process is, therefore,
known as sterilization. On the other hand, cultures no
longer useful, bits of infected tissue, and apparatus generally
that it is desirable to render free from danger, are com-
monly subjected for a time to the action of chemical com-
pounds possessing germicidal properties — i. e., to the action
(76)
PRINCIPLES OF STERILIZATION 77
of disinfectants; and the process is, consequently, known as
disinfection, though the same end can also be reached by
the application of heat to these articles. Strictly speaking,
sterilization implies the complete destruction of the vitality
of all microorganisms that may be present in or upon the
substance to be sterilized, and can be accomplished by the
proper application of both thermal and chemical agents;
while disinfection, though it may insure the destruction of
all living forms that are present, need not of necessity do
so, but may be limited in its action to those only that possess
the power of infecting; it may or may not, therefore, be
complete in the sense of sterilization. -From this we see it
is possible to accomplish both sterilization and disinfection
as well by chemical as by thermal means.
In practice the employment of these means is governed
by circumstances. In the laboratory it is essential that
all culture media with which work is to be conducted should
be free from living bacteria or their spores — they must be
sterile; and it is equally important that their original
chemical composition should remain unchanged. It is
evident, therefore, that sterilization of these substances
by means of chemicals is out of the question, for, while the
media could be thus sterilized, it would be necessary, in
order to accomplish this, to add to them substances cap-
able not only of destroying all microorganisms present, but
whose presence would at the same time prevent the growth
of bacteria that are to be subsequently cultivated in these
media — that is to say, after performing their sterilizing
or germicidal function the chemical disinfectants would,
by their further presence, exhibit their antiseptic properties
and thus render the material useless as a culture medium.
Exceptions to this are seen, however, in the case of certain
78 BACTERIOLOGY
volatile substances possessing disinfectant powers — chloro-
form and ether, for instance; these bodies, after exhibiting
their germicidal activities, may be driven off by gentle heat,
leaving the media quite suitable for purposes of cultivation.
They are not, however, in general use in this capacity.
The circumstances under which chemical sterilization or
disinfection is practised in the laboratory are, ordinarily,
either those in which it is desirable to render materials free
from danger that are not affected by the chemical action
of the agents used, such as glass apparatus, etc., or where
destructive changes in the composition of the substances
to be treated, as in-the case of old cultures, infected tissues,
pathological exudates, feces, etc., are a matter of no conse-
quence. On the other hand, for the sterilization of all
materials to be used as culture media heat only is employed.1
The two processes will be explained in this chapter,
beginning with
STERILIZATION BY HEAT.
Sterilization by means of high temperature is accom-
plished in several ways, viz., by subjecting the articles to
be treated to a high temperature in a properly constructed
oven — this is known as dry sterilization; by subjecting
them to the action of streaming or live steam at the tem-
perature of 100° C.; and by subjecting them to the action
of steam under pressure, under which circumstance the
temperature to which they are exposed becomes more and
more elevated as the pressure increases.
Experience has taught us that the process of sterilization
by dry heat is of limited application because of its many
1 An occasional exception to this is the use of chloroform, mentioned above.
STERILIZATION BY HEAT 79
disadvantages. For successful sterilization by the method
of dry heat, not only is a relatively high temperature needed,
but the substances under treatment must be exposed to this
temperature for a comparatively long time. The penetra-
tion of dry heat into materials which are to be sterilized is,
moreover, much less thorough than that of steam. Many
substances of vegetable and animal origin are rendered
valueless by subjection to the dry method of sterilization.
For these reasons comparatively few materials can be
sterilized in this way without seriously impairing their
further usefulness.
Successful sterilization by dry heat cannot usually be
accomplished at a temperature lower than 150° C., and to
this degree of heat the objects should be subjected for not
less than one hour. For the sterilization, therefore, of the
organic materials of which the media employed in bacterio-
logical work are composed, and of domestic articles, such
as cotton, woollen, wooden, and leather articles, this method
is wholly unsuitable. In bacteriological work its application
is limited to the sterilization of glassware principally — such,
for example, as flasks, plates, small dishes, test-tubes,
pipettes — and such metal instruments as are not seriously
injured by the high temperature.
Methods Employed. — Sterilization by moist heat — steam —
offers conditions much more favorable. The penetrating
power of the steam is not only more energetic, but the tem-
perature at which sterilization is ordinarily accomplished is,
as a rule, not destructive to the objects under treatment.
This is conspicuously seen in the work of the laboratory;
the culture media, composed in the main of decomposable
organic materials that would be rendered entirely worthless
if exposed to the dry method of sterilization, sustain no
80 BACTERIOLOGY
injury whatever when intelligently subjected to an equally
effective sterilization with steam. The same may be said
of cotton and woollen fabrics, bedding, clothing, etc.
• Aside from the relations of the two methods to the mate-
rials to be sterilized, their action toward the organisms to
be destroyed is quite different. The penetrating power of
steam renders it by far the more efficient agent of the two.
The spores of several organisms which are killed by an
exposure of but a few moments to the action of steam, resist
the destructive action of dry heat at a higher temperature
for a much greater length of time.
These differences will be strikingly brought out in the
experimental work on this subject. For our purposes it
is necessary to remember that the two methods have the
following applications :
The dry method, at a temperature of 150°-180° C. for
one hour, is employed for the sterilization of glassware such
as flasks, test-tubes, culture-dishes, pipettes, plates, etc.
Sterilization by steam is practised with all culture media,
whether' fluid or solid. Bouillon, milk, gelatin, agar-agar,
potato, etc., are under no circumstances to be subjected to
dry heat.
Discontinued Sterilization. — The manner in which heat is
employed in processes of sterilization varies with circum-
stances. When used in the dry form its application is always
continuous — i. e., the objects to be sterilized are simply
exposed to the proper temperature for the length of time
necessary to destroy all living organisms which may be upon
them. With the use of steam, on the other hand, the articles
to be sterilized are frequently of such a nature that a pro-
longed application of heat might materially injure them.
For this and other reasons steam is usually applied inter-
STERILIZATION BY HEAT 81
mittently and for short periods of time. The principles
involved in the intermittent method of sterilization depend
upon differences of resistance to heat which the organisms
to be destroyed are known to possess at different stages
of their development. During the life cycle of many of the
bacilli there is a stage in which the resistance of the organism
to the action of both chemical and thermal agents is much
greater than at other stages of their development. This
increased power of resistance appears when these organisms
are in the spore- or resting-stage, to which reference has
already been made. When in the vegetative or growing
stage most bacteria are killed in a short time by a relatively
low temperature; whereas, under conditions which favor the
production of spores, the spores are seen to be capable of
resisting very much higher temperatures for an appreciably
longer time; indeed, spores of certain bacilli have been
encountered that retain the power of germinating after an
exposure of from five to six hours to the temperature of
boiling water. Such powers of resistance have never been
observed in the vegetative stage of development. These
differences in resistance to heat which the spore-forming
organisms possess at their different stages of development is
taken advantage of in the process of sterilization by steam
known as the discontinuous, fractional, or intermittent
method, and are the essential feature of the principles on
which the method is based.
As culture media are dependent for their usefulness upon
the presence of more or less unstable organic compounds,
the object aimed at in this method is to destroy the organ-
isms in the shortest time and with the least amount of heat.
It is accomplished by subjecting them to the elevated
temperature at a time when the bacteria are in the vegetat-
6
82 BACTERIOLOGY
ing or growing stage — i. e., the stage at which they are most
susceptible to detrimental influences. In order to accom-
plish this it is necessary that there should exist conditions
of temperature, nutrition, and moisture which favor the
vegetation of the bacilli and the germination of any spores
that may be present. When, as in freshly prepared nutrient
media, this combination is found, the spore-forming organ-
isms are not only less likely to enter the spore-stage than
when their environment is less favorable to their vegetation,
but spores which may already exist develop very quickly
into mature cells.
It is plain, then, that with the first application of steam
to the substance to be sterilized the mature vegetative forms
are destroyed; while certain spores that may be present
resist this treatment, providing the sterilization is not con-
tinued for too long a time. If now the sterilization be
discontinued, and the material which presents conditions
favorable to the germination of the spores be allowed to
stand for a time, usually for about twenty-four hours, at
a temperature of from 20° to 22° C., those spores which
resisted the action of the steam will, in the course of this
interval, germinate into the less resistant vegetative cells.
A second short exposure to the steam kills these forms in
turn, and by a repetition of this process all bacteria that
were present may be destroyed without the application of
the steam having been of long duration at any time. It
should be remembered that while spores which may be
present are not directly killed by such an exposure to heat
as they experience in the intermittent method of sterili-
zation, still their power of germination is somewhat inhibited
by this treatment. In this method, therefore, if the tem-
perature of 100° C. be employed for too long a time, it is
STERILIZATION BY HEAT 83
possible so to retard the germination of the spores as to
render it impossible for them to develop into the vegetative
stage during the interval between the heatings. By exces-
sively long exposures to high temperature, but not long
enough to destroy the spores directly, the object aimed at
in the method may be defeated, and in the end the substance
undergoing sterilization be found still to contain living
bacteria. In this process the plan that has given most satis-
factory results is to subject the materials to be sterilized
to the action of steam, under the ordinary conditions of
atmospheric pressure, for fifteen minutes on each of three
successive days, and during the intervals to maintain them
at a temperature of about 25°-30° C. At the end of this
time all living organisms which were present will, as a general
rule, have been destroyed, and, unless opportunity is given
for the access of new organisms from without, the substances
thus treated remain sterile. As an exception to this, certain
species of spore-forming bacteria are occasionally encountered
that are not readily destroyed by this mode of treatment.
These species are found so uniformly in the soil that the
customary designation for them is that of "the soil bacteria."
This group includes a number of species that are endowed
with remarkable resistance to heat. Some of them are
probably thermophilic by nature, which would account not
only for the failure to destroy their spores by the ordinary
exposures to steam, but also for their slow and incomplete
development from the spore to the less resistant vegetative
stage during the intervals between the heatings, for, as a
rule, the materials containing them are kept at a temperature
during these intervals that is too low to favor the rapid
germination of the species having thermophilic tendencies.
As a result of the presence of these species, media that
84 BACTERIOLOGY
have been subjected to the customary discontinuous method
of sterilization may, after having been kept for a time,
reveal the presence of isolated colonies of bacteria distrib-
uted through them in such a way as to preclude all likelihood
of their having fallen upon it from the air after sterilization
was supposedly complete.
Theobald Smith1 has called attention to an instructive
personal experience. He finds that when media are present
in vessels in only thin layers the spores of anaerobic specie's
do not develop into the vegetative forms during the interval
between the heatings, for the reason that the shallow layer
of medium does not sufficiently exclude free oxygen to per-
mit it; and by subjecting such materials, apparently steril-
ized by the intermittent method, to strictly anaerobic
conditions a development of anaerobic species will often
occur. On the other hand, if the vessels be nearly filled with
media, and especially if the area of the surface be small, the
conditions are much more favorable to the germination of
anaerobic spores, and sterilization by this process after such
precautions is usually perfect.
Fortunately, these undesirable experiences are rare, but
that they do occur, and result in no small degree of annoy-
ance, will be admitted by most bacteriologists.
It must be borne in mind that this method of sterilization
is only applicable in those cases which present conditions
favorable to the germination of the spores into mature
vegetative cells. Dry substances, such as instruments,
bandages, apparatus, etc., or organic materials in which
decomposition is far advanced, where conditions of nutrition
favorable to the germination of spores are not present, do
not offer the conditions requisite for the successful operation
1 Journal of Experimental Medicine, iii, No. 6, p. 647.
STERILIZATION BY HEAT 85
of the principles underlying the intermittent method of
sterilization.
Discontinued Sterilization at Low Temperatures. — The pro-
cess of discontinued sterilization at low temperatures is
based upon exactly the same principle, but differs in two
respects from the foregoing in the manner by which it is
practised, viz., it requires a greater number of exposures
for its accomplishment, and the temperature at which it
is conducted is not above 68°-70° C. It is employed for
the sterilization of easily decomposable materials, which
would be rendered useless by steam, but which are unal-
tered by the temperature employed, and for certain albu-
minous culture media that it is desirable to retain in a
fluid condition during sterilization, but which would be
coagulated if exposed to higher temperatures. This pro-
cess requires that the material to be sterilized should be
subjected to a temperature of 68°-70° C. for one hour on
each of six successive days, the interval of twenty-four
hours between the exposures admitting of the germination
of spores into mature cells. During this interval the sub-
stances under treatment are kept at about 25°-30° C. The
temperature employed in this process suffices to destroy,
in about one hour, the vitality of almost all organisms in
the vegetative stage. Formerly blood serum was always
sterilized by the intermittent method at a low temperature.
Direct Sterilization. — Sterilization by steam is also prac-
tised by what may be called the direct method — that is to
say, both the mature organisms and the spores which may
be present in the material to be sterilized are destroyed
by a single exposure to the steam. In this method steam
at its ordinary temperature and pressure — live steam or
streaming steam, as it is called — is employed just as in the
86 BACTERIOLOGY
first method described; but it is allowed to act for a much
longer time, usually for not less than an hour; or steam under
pressure, and consequently of a higher temperature, is now
frequently employed. By the latter procedure a single
exposure of fifteen minutes is sufficient for the destruction
of practically all bacilli and their spores, providing the
pressure of the steam is not less than one atmosphere over
and above that of normal; this is approximately equivalent
to a temperature of 122° C. to which the organisms are
exposed.
The objection that has been urged to both of these
methods, particularly that in which steam under pressure
is employed, is that the properties of the media are altered.
Gelatin is said to become cloudy and lose the property of
solidifying; in bouillon and agar-agar fine precipitates are
said to result, and some believe the reaction undergoes a
change. In the experience of those who have used steam
under pressure not exceeding one atmosphere for ten to
fifteen minutes these obstacles have rarely been encoun-
tered. There is one point to be borne in mind, however, in
using steam under pressure, viz., it is not possible to regulate
the time of exposure to the same degree of nicety as where
ordinary live steam is used. The reason for this is that if
the apparatus be opened to remove the objects being steril-
ized while the steam within it is under pressure, the escape
of steam will be so rapid that all fluids within the chamber,
thus suddenly relieved of pressure, will begin to boil violently,
and, as a rule, will boil quite out of the tubes, flasks, etc.,
containing them. For this reason the apparatus must be
kept closed until cool, or until the gauge indicates that
pressure no longer exists within the chamber, and even
then it should be opened very cautiously. It is patent from
STERILIZATION BY HEAT 87
this that the temperature and time of exposure of articles
sterilized by this process cannot usually be controlled with
accuracy. It requires some time to reach a given pressure
after the apparatus is closed, and it also requires time for
cooling after the desired exposure to such pressure before
the apparatus can be opened.
It is manifest that during these three periods, viz., (a)
reaching the pressure desired, (6) time during which the
pressure is maintained, and (c) time for fall of pressure
before the chamber can be opened, it is difficult to say
certainly to what temperature and pressure the articles in
the apparatus have, on the whole, been subjected. Clearly,
if the desired pressure and temperature have been maintained
for ten minutes, one cannot say that that is all the heat to
which the articles have been subjected during their stay
in the chamber. In this light, while steam under pressure
may answer very well for routine sterilization, still it pre-
sents insurmountable obstacles to its use in more delicate
experiments where time-exposure to definite temperature is
of importance. Nevertheless, for general laboratory pur-
poses, sterilization by steam under pressure has so much
to recommend it in the way of economy of time and cerr
tainty of accomplishment that it has practically superseded
the older methods of sterilization by streaming or live steam;
and in most laboratories the original styles of steam steril-
izers are rapidly giving way to some one or another of the
modern forms of autoclave.
For sterilization by live steam the apparatus in common
use was for a long time the cylindrical boiler recommended
by Koch. (See Fig. 8.) Its construction is very simple,
essentially that of the ordinary domestic potato-steamer.
It consists of a copper cylinder, the lower fifth, approximately,
88 BACTERIOLOGY
of which is somewhat larger in circumference than the
remaining four-fifths and serves as a reservoir for the water
from which the steam is to be generated. Covering this
section of the cylinder is a wire rack or grating, through
which the steam passes, and which supports the articles to
be sterilized. Above this, comprising the remaining four-
FIG. 8
Steam sterilizer, pattern of Koch.
fifths of the cylinder, is the chamber for the reception of
the materials over and through which the steam is to pass.
The cylinder is closed by a snugly fitting cover, through
which are usually two perforations, into which a thermo-
meter and a manometer may be inserted. The whole
of the outer surface of the apparatus is encased in a non-
conducting mantle of asbestos or felt.
STERILIZATION BY HEAT 89
The water is heated by a gas-flame placed in an enclosed
chamber, upon which the apparatus rests, which serves to
diminish the loss of heat and deflection of the flame through
the action of draughts. The apparatus is simple in con-
struction, and the only point which is to be observed while
using it is the level of the water in the reservoir. On the
reservoir is a water-gauge which indicates at all times the
FIG. 9
Arnold steam sterilizer.
amount of water in the apparatus. The amount of water
should never be too small to be indicated by the gauge;
otherwise there is danger of the reservoir becoming dry and
the bottom of the apparatus being destroyed by the direct
action of the flame.
A sterilizer that has come into very general use in bac-
teriological laboratories is one originally intended for use
90 BACTERIOLOGY
in the kitchen. It is called the "Arnold steam sterilizer."
It is very ingenious in its construction as well as economical
in its employment.
The difference between this apparatus and that just
described is that it provides for the condensation of the
steam after its escape from the sterilizing chamber, and
returns the water of condensation automatically to the
reservoir, so that in practice the apparatus requires but
little attention, as with ordinary care there is no likelihood
of the water in the reservoir becoming exhausted, with the
consequent destruction of the sterilizer. Fig. 9 shows a
section through this apparatus. »
STERILIZATION UNDER PRESSURE.
The advantages of the use of steam under pressure for
the purposes of sterilization have received such general
recognition that almost everywhere this method is sup-
planting the older one of intermittent sterilization with
streaming or live steam. By this plan one is able to accom-
plish, by a single exposure of fifteen minutes to steam under
a pressure of one atmosphere, the same end that would,
with streaming steam, require three exposures of fifteen
minutes on each of three successive days.
For sterilization by steam under pressure several special
forms of apparatus exist. The principles involved in them
all are, however, the same. They provide for the generation
of steam in a chamber from which it cannot escape when
the apparatus is closed. Upon the cover of this chamber
is a safety-valve, which can be regulated so that any degree
of pressure (and coincidently of temperature) that is desir-
able may be maintained within the sterilizing chamber.
STERILIZATION BY HOT AIR
91
These sterilizers are known as "digesters" and as "auto-
claves." Their construction can best be understood by
reference to Figs. 10 and 11.
FIG. 10
A B
Autoclave. A, external appearance; B, section.
STERILIZATION BY HOT AIR.
The hot-air sterilizers used in laboratories are simply
double-walled boxes of Russian or Swedish iron (Fig. 12),
having a double-walled door, which closes tightly, and a
heavy copper bottom. They are provided with openings
for the escape of the contained air and the entrance of the
heated air. The flame, usually from a rose-burner (Fig. 13),
92 BACTERIOLOGY
is applied directly to the bottom. The heat circulates from
the lower surface around about the apparatus through the
space between its walls.
FIG. 11
Autoclave or digester for sterilizing by steam under pressure.
The construction of the copper bottom of the apparatus
upon which the flame impinges is designed to prevent the
direct action of the flame upon the sheet-iron bottom of the
chamber. It consists of several copper plates placed one
STERILIZATION BY HOT AIR
93
above the other, but with a space of about 4 to 5 mm.
between the plates. These copper bottoms after a time
become burned out, and unless they are replaced the appara-
tus is useless. The older forms of hot-air sterilizers are so
constructed that their repair is a matter involving some time
and expense. To meet this objection I had constructed
FIG. 12
FIG. 13
Laboratory hot-air sterilizer.
Rose-burner.
some years ago a sterilizer in all respects similar to the old
form except in the arrangement of the copper bottom. This
latter is made in such a way that it can easily be removed,
so that by keeping several sets of copper plates on hand
a new plate can readily be inserted when the old one is
burned out.
In the employment of the hot-air sterilizer care should
94 BACTERIOLOGY
always be given to the condition of the copper bottom; for
the direct application of heat to the sheet-iron plate upon
which the substances to be sterilized stand results not only
in destruction of the apparatus, but frequently in destruc-
tion of the substances undergoing sterilization.
Since the temperature at which this form of sterilization
is usually accomplished is high, from 150° to 180° C., it is
well to have the apparatus encased in asbestos boards, to
diminish the radiation of heat from its surfaces. This not
only confines the heat to the apparatus, but guards against
the destructive action of the radiated heat on woodwork,
furniture, etc., that may be in the neighborhood.
Thermal Death-point of Bacteria. — By "thermal death-
point of bacteria" is meant the temperature necessary to
kill them in a given time. As this varies with different
species, it is an aid to identification. For the practical pur-
poses of the sanitarian the knowledge is of fundamental
importance. The thermal death-point of an organism is
ascertained by subjecting it to varying degrees of tempera-
ture for five or ten minutes until the point is reached where
it is killed. The test is best carried out by means of small
glass bulbs, the so-called Sternberg bulbs, or through the
use of capillary tubes containing a small amount of fluid
inoculated with the organism to be studied. The bulb,
or tube, is sealed in the gas flame and placed in a water-
bath kept at 50° C. for five minutes. Sub-cultures are now
made to learn whether the bacteria have been killed or not.
If the organism survives the test is repeated at 55°, 60°, 65 °,
and 70° C. Finally, the test is repeated for each degree of
temperature between the points where growth is still apparent
and where the organisms have been killed. If the bacteria
were killed when heated to 60° C. for five minutes, but sur-
CHEMICAL STERILIZATION AND DISINFECTION 95
vived when heated to 55° C., then similar tests are made for
the same length of time for each degree of temperature
between 55° and 60° C. It will usually be found that heating
for ten minutes suffices to kill the bacteria at a temperature
one or two degrees lower than that required when heated
for only five minutes. All such tests should be made at
least in duplicate, and the mean of the results taken.
CHEMICAL STERILIZATION AND DISINFECTION.
As has been stated, it is possible by means of certain
chemical substances to destroy all bacteria and their spores
that may be within or upon various materials and objects —
i. e., to sterilize them; and it is also possible by the same
means to rob objects of their dangerous infective properties
without at the same time sterilizing them — i. e., to disinfect
them. This latter process depends upon the fact that the
vitality of many of the less resistant pathogenic organisms
is easily destroyed by an exposure to particular chemical
substances that may be without effect upon the more resis-
tant saprophytes and their spores that are present.
In general, the use of chemicals for sterilization is not to
be considered in connection with substances that are to be
employed as culture media, and their employment is re-
stricted in the laboratory to materials that are of no further
value, and to infected articles that are not injured by the
action of the agents used, though exceptionally such vola-
tile germicides as chloroform and ether are employed for
the sterilization of special culture-media. (See Preservation
of Blood-serum with Chloroform.) In short, they are mainly
of value in rendering infected waste-material innocuous.
For the successful performance of this form of disinfection
96 BACTERIOLOGY
there is one fundamental rule always to be borne in mind,
viz., it is essential to success that the disinfectant used
should come in direct contact with the bacteria to be de-
stroyed, otherwise there is no disinfection.
For this reason one should always remember, in selecting
the disinfecting agent, the nature of the materials containing
the bacteria upon which it is to act, for the majority of
disinfectants, and particularly those of an inorganic nature,
vary in the degree of their potency with the chemical nature
of the mass to which they are applied. Often the materials
containing the bacteria to be destroyed are of such a character
that they combine with the disinfecting agent to form insol-
uble, more or less inert precipitates; these so interfere with
the penetration of the disinfectant that many bacteria may
escape its destructive action entirely and no disinfection
be accomplished, although an agent may have been employed
that would, under other circumstances, have given entirely
satisfactory results.
An antiseptic is a body which, by its presence, prevents
the growth of bacteria without of necessity killing them.
A body may be an antiseptic without possessing disinfecting
properties to any very high degree, but a disinfectant is
always an antiseptic as well.
A germicide is a body possessing the property of killing
bacteria.
Mode of Action of Disinfectants. — In the destruction of
bacteria by means of chemical substances there occurs,
most probably, a definite chemical reaction— that is to
say, the characteristics both of the bacteria and the agent
employed in their destruction are lost in the production of
an inert third body, the result of their combination. It is
impossible to state with certainty, as yet, that this is in
CHEMICAL STERILIZATION AND DISINFECTION 97
general the case; but the evidence that is rapidly accruing
from studies upon disinfectants and their mode of action
points strongly to the accuracy of this belief. This reaction,
in which the typical structures of both bodies concerned are
lost, takes place between the agent employed for disinfection
and the protoplasm of the bacteria. For example, in the
reaction that is seen to take place between the salts of mer-
cury and albuminous bodies there results a third compound,
which has neither all the characteristics of mercury nor of
albumin, but partakes of some of the peculiarities of both;
it is a combination of albumin and mercury, commonly
known by the indefinite term "albuminate of mercury."
Some such reaction as this apparently occurs when the
soluble salts of mercury are brought in contact with
bacteria. This view has been strengthened by the experi-
ments of Geppert, in which the reaction was caused to take
place between the spores of the anthrax bacillus and a
solution of mercuric chloride, the result being the apparent
destruction of the vitality of the spores by the formation of
this third, inert compound. In these experiments it was
shown that though this combination had taken place, still
it did not of necessity imply the death of the spores, for if
by proper means the combination of mercury with their
protoplasm was broken up, many of the spores resumed
their vitality, with all their previous disease producing and
cultural peculiarities. Geppert employed a solution of am-
monium sulphide for the purpose of destroying the combi-
nation of spore protoplasm and mercury; the mercury was
precipitated from the protoplasm as an insoluble sulphide,
and the protoplasm of the spores returned to its original
condition. These and other somewhat similar experiments
have given a new impulse to the study of disinfectants, and
7
98 BACTERIOLOGY
in the light shed by them many of our previously formed
ideas concerning the action of disinfecting agents have
been modified.
The process of disinfection is not a catalytic one — 'i. e.,
occurring simply as xa result of the presence of the disin-
fecting body, which is not itself decomposed during its
process of destruction — but is, as said, a definite chemical
reaction occurring within more or less fixed limits; that is
to say, with a given amount of the disinfectant just so much
work, expressed in terms of destruction of bacteria can be
accomplished.
Another point in favor of this view is the increased
energy of the reaction with elevation of temperature. Just
as in other chemical phenomena the intensity and
rapidity of the reaction become greater under the influence
of heat, so in the process of disinfection the combination
between the disinfectant and the organisms to be destroyed
is much more energetic at a temperature of 37°-39° C.
than it is at 12°-15° C.
A number of important and novel suggestions with regard
to the modus operandi of disinfection were brought out
through the work of Kronig and Paul,1 who took up the
subject from its physico-chemical standpoint. The compre-
hensive nature of this elaborate investigation precludes
more than a brief mention of some of the conclusions reached,
and in order that these may be intelligible, certain beliefs
(working hypotheses) of the physical chemists should be
borne in mind. In 1887 Arrhenius proposed the theory
that when an electrolyte (a compound decomposable by an
electric current) is dissolved in water its molecules break
down, not simply into their component atoms, but into
1 Zeitschrift fur Hygiene und Infektionskrankheiten, 1897, xxv, 1-112.
CHEMICAL STERILIZATION AND DISINFECTION, 99
ions, which are atoms or groups of atoms having electro-
positive and electro-negative characteristics. According
to this theory, salts, when dissolved in water, undergo
electrolytic dissociation into metallic and acidic ions, the
former being the electro-positive cation, the latter the
electro-negative anion; sodium chloride, for example, re-
solving itself, under these conditions, into its sodium,
or metal ion, and its chlorine, or acidic ion. The electro-
positive cations, according to Ostwald, comprise the metals
and metal-like radicals, such as ammonium (NH4) and hydro-
gen (H) ; while the electro-negative anions include the halo-
gens, the acidic radicals (such as NO3 and SO4), and hydrosyl.1
Using this theory as the basis of their investigations, Kronig
and Paul reached the following conclusions with regard to
the action of chemical disinfectants:
The germicidal value of a metallic salt depends not only
upon its specific character, but also upon that of its anion.
Solutions of metallic salts in which the metallic part is
represented by a complex ion and in which the concentra-
tion of the metal ion is very slight, have but feeble disin-
fecting activity.
The halogen compounds of mercury act according to the
degree of their dissociation.
The disinfecting power of the halogens — chlorine, bromine,
iodine — (as well as their compounds) is in inverse ratio to
their atomic weights.
The disinfecting activity of watery solutions of mercuric
chloride is diminished by the addition to them of other
1 Consult Ostwald's Lehrbuch der Allg. Chemie; or Muir's transla-
tion of Ostwald's Solutions, p. 189, published by Longmans, Green &
Co., London and New York, 1891. Also The Rise of the Theory of Elec-
trolytic Dissociation, etc., by H. C. Jones, Ph.D., Johns Hopkins Hospital
Bulletin, No. 87, June, 1898, p. 136.
100 BACTERIOLOGY
halogen compounds of metals and of hydrochloric acid. It
appears probable that this is due to obstruction offered to
electrolytic dissociation.
The disinfecting activities of watery solutions of mer-
curic nitrate, mercuric sulphate, and mercuric acetate are
increased by the moderate addition of sodium chloride.
In general, acids disinfect according to the degree of their
dissociation — i. e., according to the concentration of their
hydrogen ions in the solution.
The bases, potassium, sodium, lithium, and ammonium
hydroxide, disinfect according to the degree of their dis-
sociation— i. e., corresponding to the concentration of their
hydroxyl ions in the solution.
The disinfecting activity of metallic salts is, in general,
less in albuminous fluids than in water. It is probable that
this is due to a diminution in the concentration of metallic
ions in the solution.
The reaction between the inorganic salts and albuminous
bodies is not selective; they combine in most instances with
any or all protoplasmic bodies present. For this reason
the employment of many of the commoner disinfectants
in general practice is a matter of doubtful advantage. For
example, the disinfection of excreta, sputum, or blood,
containing pathogenic organisms, by means of corrosive
sublimate, is a procedure of questionable success. The
amount of sublimate employed may be entirely used up
and rendered inactive as a disinfectant by the ordinary
protoplasmic substances present, without having any
appreciable effect upon the bacteria which may be in the
mass.
These remarks are introduced in order to guard against
the implicit confidence so often placed in the disinfecting
CHEMICAL STERILIZATION AND DISINFECTION 101
value of corrosive sublimate. In many bacteriological
laboratories it is the custom to keep at hand vessels con-
taining solutions of corrosive sublimate, into which i;nfec-
tious materials may be placed. The value of this procedure,
as we have just learned, may be more or less questionable,
especially in those cases in which the substance to be disin-
fected is of a proteid nature and where the solution used is
not freshly prepared and frequently replenished." £)n the
introduction of such substances into the sublimate solution
the mercury is quickly precipitated by "the* alSuravri,1 '.a toil* it*
disinfecting properties may be in large part or entirely
destroyed; we may in a very short time have little else
than water containing an inactive precipitate of albumin
and mercury, in so far as its value as a disinfectant is con-
cerned.
Though the other inorganic salts have not been so
thoroughly studied in this connection, it is nevertheless
probable that the same precautions should be taken in
their employment as we now know to be necessary in the
use of the salts of mercury.
The modes of action of other germicides have not been so
carefully investigated as has that of the metallic salts.
From the nature of many of them, however, we may infer
that some act through oxidation, as in the case of strong
acids and other active oxidizers; others by coagulation or
by dehydration, as in the case of strong aldehydes and
alcohols; and others by penetrating the cell wall and fatally
poisoning the bacterial protoplasm, as in the case of hydro-
cyanic acid and its compounds.
Practical Disinfection. — Where, it is desirable to use chemi-
cal disinfectants in the laboratory, much more satisfactory
results can usually be obtained from the employment of
102 BACTERIOLOGY
carbolic acid in solution. A 3 or 4 per cent, solution of
commercial carbolic acid in water requires longer for disin-
fection; but it is, at the same time, open to fewer objections
than are solutions of the inorganic salts; though here, too,
we find a somewhat analogous reaction between the car-
bolic acid and proteid matters. Under ordinary circum-
stances its action is compilete in from twenty minutes to
a, .half-hour,, . :It is not reliable for the disinfection of
resistant spores; such, for instance, as those of bacillus
All tissues containing infectious organisms should be
burned, and all cloths, test-tubes, flasks, and dishes should
be bolied in 2 per cent, soda (ordinary washing-soda) solu-
tion for fifteen to twenty minutes, or placed in the steam
sterilizer for half an hour.
Intestinal evacuations may best be disinfected with
boiling water or with milk of lime, a mixture composed of
lime in solution and in suspension — ordinary fluid "white
wash." This should be thoroughly mixed with the evacua-
tions until the mass contains a considerable excess of the
lime, and should remain in contact with them for one or
two hours. Excreta may also be easily disinfected by
thoroughly mixing them with two or three times their
volume of boiling water, after which they are kept covered
until cool.
Sputum in which tubercle bacilli are present, as well as
the vessel containing it, must be boiled in 2 per cent, soda
for fifteen minutes, or steamed in the sterilizer for at least
a half-hour.
On the whole, in the laboratory we should rely more upon
the destructive properties of heat than upon those of chemical
agents.
CHEMICAL STERILIZATION AND DISINFECTION 103
From what has been said, the absurdity of sprinkling
here and there a little carbolic acid, or of placing vessels
of carbolic acid about apartments in which infectious
diseases are in progress, must be plain. Treatment of water-
closets and cesspools by allowing now and then a few cubic
centimeters of some so-called disinfectant to trickle through
the pipes is ridiculous. A disinfectant must be applied to
the bacteria, and must be in contact with them for a long enough
time to insure the destruction of their life.
In the light of the latest experiments upon disinfectants,
the place formerly occupied by many agents in the list of
substances employed for the purpose will most likely be
changed as they are studied more closely. The agents,
then, which will prove of greatest value in the laboratory
for the purpose of rendering infectious materials harmless
are: heat, either by burning, by steaming for from half an
hour to an hour, or by boiling in a 2 per cent, sodium car-
bonate solution for fifteen minutes; 3 to 4 per cent, solution
of commercial carbolic acid; milk of lime, and a solution of
chlorinated lime ("chloride of lime") containing not less
than 0.25 per cent, of free chlorine. The chloride of lime
from which such a solution is to be made should be fresh
and of good quality. Good chlorinated lime, as purchased
in the shops, should contain not less than 25 to 30 per cent,
of available chlorine. The materials to be disinfected in
either of the lime solutions should remain in them for about
two hours. The solutions should be freshly prepared when
needed, as they rapidly decompose upon standing.
CHAPTER IV.
Principles Involved in the Methods of Isolation of Bacteria in Pure Culture
by the Plate Method of Koch — Materials Employed.
As was stated in the introductory chapter, the isolation
in pure cultures of the different species that may be present
in mixtures of bacteria was rendered possible only through
the methods suggested by Koch. Since the adoption of
these methods they have undergone many modifications,
but the fundamental principle remains the same. The
observation that lead to their development is of almost
daily occurrence. When bread, cooked potatoes or old bits
of leather are left in moist, damp surroundings they invari-
ably become "moldy" as we call it; that is to say, they
become more or less covered or spotted with deposits that
are known to be composed of living microorganisms. .
If one watches the evolution of this condition from day
to day it will be seen that the moldy deposit begins as a
number of small isolated points which, as they get larger,
may finally coalesce into a confluent mass that eventually
covers the surface. If one examine these points, however,
before they begin to run together, it is found that they are
composed of microorganisms of several different kinds,
some being molds, some yeasts, and some bacteria. The
isolated growths of these various species present different
naked-eye appearances, so that even at a glance we are
justified in suspecting that they are of a 'different nature.
They develop from single cells that have fallen upon the
(104)
PRINCIPLES IN METHODS OF ISOLATION 105
moist objects from the air, and as the cell grows and mul-
tiplies it forms these circumscribed patches or "colonies"
as they are called.
* The question that then presented itself was: If from a
mixture of organisms floating in the air it is possible in this
way to obtain in pure cultures the component individuals,
what means can be employed for obtaining the same results
at mil from mixture of different species of bacteria when
found together under other conditions? It was plain that
the organisms were to be distinguished primarily, the one
from the other, only by the structure and general appear-
ance of the colonies growing from them, for by their mor-
phology alone this is impossible. What means might be
devised, then, fpr separating the individual members of a
mixture in such a way that they would remain in a fixed
position, and be so widely separated, the one from the other,
as not to interfere with the production of colonies of charac-
teristic appearance, which would, under favorable condi-
tions, develop from each individual cell?
If one take in the hand a mixture of several kinds of
flower seeds and attempt to separate the mass into its con-
stituents by picking out the different grains, the task is
tedious, to say the least of it; but if the handful of seeds
be thrown upon a large flat surface, as upon a table, the
grains become widely separated and the matter is con-
siderably simplified; or, if sown upon proper soil, the various
grains germinate and develop into plants of entirely different
characteristics, by which they can readily be recognized
as distinct species. Similarly, if a test-tube of decomposed
bouillon be poured upon a large, flat surface, the individual
bacteria in the mass are much more widely separated, the
one from the other, than they were when the bouillon wfts
106 BACTERIOLOGY
in the tube; but they are in a fluid medium, and there is
no possibility of their either remaining separated or of
their colonizing under these conditions, so that it is impos-
sible by this means to pick out the individuals from the
mixture.
FIG. 14
Showing certain macroscopic characteristics of colonies. Natural size.
If, however, some substance can be found which possesses
the property of being at one time fluid and at another time
solid, and which can be added to this bouillon without in
any way interfering with the life-functions of the bacteria,
then, as solidification set in, the organisms would be fixed
PRINCIPLES IN METHODS OF ISOLATION 107
in their positions, and the conditions would be analogous
to those seen on the bits of potato, bread or leather.
Gelatin possesses this property, and it was, therefore,
used. At a temperature which does not interfere with the
life of the bacteria it is quite fluid, whereas when subjected
to a lower temperature it solidifies. When once solid it
may be kept at a temperature favorable to the growth of
the bacteria and will remain in its solid state.
Gelatin was added to the fluids containing mixtures of
bacteria, and the whole was then poured upon a large, flat
surface, allowed to solidify, and the results noted. It was
found that the conditions seen on the slice of moldy potato
could be reproduced; that the indivduals in the mixture
of bacteria grew well in the gelatin, and, as on the potato,
grew in colonies of typical macroscopic peculiarities, so
that they could easily be distinguished the one from the
other by their naked-eye appearances. (See Fig. 14.) It
was necessary, however, to use a more dilute mixture of
bacteria than the original decomposed bouillon. The
number of individuals in the tube was so enormous that on
the gelatin plate they were so closely packed together that
it was impossible to pick them out, not only because of
their proximity the one to the other, but also because this
packing together materially interfered with the production
of those characteristic differences visible to the naked eye.
The numbers of the organisms were then diminished by a
process of dilution, consisting of transferring a small portion
of the original mixture into a second tube of sterilized bouillon
to which gelatin had been added and liquefied; from this
a portion was added to a third gelatin-bouillon tube, and
so on. These were then poured upon large, cold surfaces
and allowed to solidify. The result was entirely satisfactory.
108 BACTERIOLOGY
On the gelatin plates from the original tube, as was expected,
the colonies were too numerous to be of use; on the plates
made from the first dilution they were much fewer in number,
but usually they were still too numerous and too closely
packed to permit of characteristic growth; on the second
dilution they were, as a rule, fewer in number and widely
separated, so that the individuals of each species were in
no way prevented by the proximity of their neighbors from
growing each in its typical way. (See Fig. 15.) There
FIG. 15
Series of plates showing the results of dilution upon the number of
colonies: A, Plate No. 1, or "original;" B, first dilution, or Plate No. 2;
C, second dilution, or Plate No. 3. About one-fourth natural size.
was then no difficulty in picking out the colonies resulting
from the growth of the different individual bacteria. This,
then, is the principle underlying Koch's method for the
isolation of bacteria by means of solid media.
The fundamental constituent of the media employed is
the bouillon, which contains all the elements necessary for
the nutrition of most bacteria, the gelatin being employed
simply for the purpose of rendering the bouillon solid. The
medium on which the organisms are growing is, therefore,
simply solidified bouillon, or beef -tea.
PRINCIPLES IN METHODS OF ISOLATION 109
In practice two gelatinous substances are employed —
the one an animal or bone gelatin, the ordinary table gelatin
of good quality; the other a vegetable gum, known as
agar-agar, the native name for Ceylon moss or Bengal
isinglass, which is obtained from a group of marine algse
found along the coast of Japan, China, and many parts
of the East, where it is employed as an article of diet by the
natives.
The behavior of the two gelatinous substances under the
influence of heat and of bacterial growth renders them of
different application in bacteriological work. The animal
gelatin liquefies at a much lower temperature, and also re-
quires a lower temperature for its solidification, than does the
agar-agar. Ordinary gelatin, in the proportion commonly used
in this work, liquefies at about 24°-26° C., and becomes solid
at from 8°-10° C. It may be employed for those organisms
which do not require a higher temperature for their develop-
ment than 22°-24° C. Agar-agar, on the other hand, does
not liquefy until the temperature has reached about 98°-99°
C. It remains fluid ordinarily until the temperature has
fallen to 38°-39° C., when it rapidly solidifies. For our
purposes, only that form of agar-agar can be used which
remains fluid at from 38°-40° C. Agar-agar which remains
fluid only at a temperature above this point would be too
hot, when in a fluid state, for use; many of the organisms
introduced into it would either be destroyed or checked in
their development by so high a temperature. Agar-agar
is employed in those cases in which the cultivation must be
conducted at a temperature above the melting-point of
gelatin.
In addition to their thermal reactions, these two gelati-
nous substances are affected very differently by different
110 BACTERIOLOGY
species of bacteria. As was said above, and as we shall soon
see for ourselves, certain bacteria elaborate in the course of
their growth digestive enzymes or ferments that in their
action upon proteid matters are strikingly like pepsin in
some and trypsin in other instances. When bacteria en-
dowed with this physiological property are cultivated upon
bone gelatin their growth is accompanied by the progressive
digestion (liquefaction) of the gelatin, which liquefied
gelatin cannot again be brought to a solid condition. We
know of no bacteria capable of producing a similar lique-
faction of agar-agar or vegetable gum.
As a rule, the colony-formations seen upon gelatin are
much more characteristic than those which develop on agar-
agar, and for this reason gelatin is to be preferred when
circumstances will permit. Both gelatin and agar-agar may
be used for the isolation of species from mixtures.
CHAPTER V.
Reactions — Methods for Adjustment — Titration — Hydrogen-ion Concentra-
tion— Preparation of Media — Bouillon, Gelatin, Agar-agar, Potato,
Blood Serum, Blood Serum from Small Animals, Milk, Litmus- whey
Milk, Durham's Peptone Solution, Lactose Litmus-agar, Loffler's
Blood-serum Mixture, the Serum-water Media of Hiss, Guarniari's
Gelatin-agar Mixture.
REACTION:— METHODS OF ADJUSTING.
OF fundamental importance to the successful cultivation
and study of bacteria upon artificial media is the reaction
of the media used. For most purposes this should be at
or about neutral.
Reaction may be roughly determined by the use of litmus
papers: Acids causing the blue paper to turn red and alka-
lies turning the red paper blue.
It may also be determined by titration, the indicator used
being a substance that announces by changes in color slight
deviations to the acid or alkaline side of neutral.
It may also be determined by estimating the total acidity
as indicated by the hydrogen-ion content resulting from
the dissociation of various electrolytes dissolved in the
medium.
For a long time the simple method of adjusting the reac-
tion of culture media by the use of litmus papers was thought
all that was necessary. Closer study of the matter, however,
revealed, among other facts, that litmus is inconstant in its
composition and that it also undergoes changes in color
resulting from influences other than those of acids and
alkalies. In consequence, for more exact work other methods
have been developed.
(ill)
112 BACTERIOLOGY
The first of these was the titration of a known volume
of the culture medium with alkaline solutions of known
strength and the use of indicators believed at the time to
be trustworthy in revealing very minute deviations from
the neutral point. From the amount of alkali used in estab-
lishing the neutral point of a small fraction of the mass
under consideration it was easily possible to calculate the
amount needed for the whole.
The indicator commonly employed in such titrations is
phenolphthalein, a compound which in neutral or faintly
acid solutions is practically colorless but which shades from
a delicate pink into deep magenta as alkalinity gradually
increases.
The adoption of still more refined physical methods to
the study of solutions, together with the fact laid down
by Arrhenius that many substances when dissolved in
water no longer retain their molecular structure but in
part or whole are dissociated into atomic modifications
that he terms ions, some of which carry positive and others
negative electrical charges, have led to a complete revision
of our views on the questions of "the reactions of solutions"
as the expression had hitherto been employed.
As a result of all this two general plans for determining
acidity or alkalinity of culture media are now in use — the
one the titration method, the other the estimation of the
aggregate electro-positive ions present in the solution, i. e.,
the hydrogen-ion concentration of the solution. As there is
every reason for regarding the latter method as the more
exact, it is likely that in the near future it will supersede all
others, particularly as the mode of its application is becom-
ing constantly more and more simplified and adapted to the
routine needs of the bacteriologist.
REACTION AS DETERMINED BY TITRATION 113
REACTION AS DETERMINED BY TITRATION.
In the development of this method, it is of prime impor-
tance that the indicator used shall announce as nearly as
possible the true neutral point, i. e., neutral as represented
by distilled water. Experience with three well-known indi-
cators will illustrate the importance of settling this point:
If, for instance, we decide to establish the reaction of a
volume of meat infusion agar-agar, with the view of ulti-
mately adjusting that reaction to neutral, we shall first
find (as with practically all other artificial media) that the
mass is acid. If we now undertake to determine the volume
of T KOH solution that is necessary to neutralize the acid
present, we shall find that volume to vary considerably
according to the indicator employed. If phenolphthalein
be selected as the indicator, about 47 c.c. of the alkaline
solution per liter of culture medium will be required; whereas,
if litmus be used instead of phenolphthalein, only about
28 c.c. of the alkali will be needed, while if rosolic acid be
substituted for litmus the figure for the alkali required falls
to about 5 c.c. Obviously, if our information ceased here
we would be at a loss to know just which of the three titra-
tions represented exact neutralization of the mass.
Fortunately, the application of precise physical methods
to the study of indicators has established their relative
values and revealed the limits of their usefulness under
various circumstances. As a result of such studies we are
informed that the neutral point (the reaction of pure dis-
tilled water) as announced by phenolphthalein is a little
high, while that given by rosolic acid is so low as to be
altogether misleading. On the other hand, the indications
afforded by litmus may be exact or nearly so, but as litmus
114 BACTERIOLOGY
is not a stable compound, as its quality and sensitiveness
is subject to variations, it is deemed wisest to rely, in this
method upon phenolphthalein, its high neutral point being
easily adjusted by the necessary correction.
The method of titration as recommended by Fuller1 is
essentially as follows: 5 c.c. of the culture medium are to
be mixed with 45 c.c. of distilled water in a porcelain evapo-
rating dish or casserole and boiled for three minutes, after
which 1 c.c. of a phenolphthalein solution (0.5 per cent, in
50 per cent, alcohol) is added and titration with •$-$• KOH is
quickly made. The neutral point, slightly to the alkaline
side, is announced by the assumption of a permanent, pale
pink color, the effect of the free alkali on the indicator.
From the volume of ^ KOH needed to neutralize the 5 c.c.
of culture medium, one can readily calculate the amount
needed for the whole mass, the volume of which must, of
course, be knowrn. For the neutralization of the entire mass
one uses not •$$ KOH but y KOH, that is, an alkaline solu-
tion twenty times as strong as that with which the titration
is made and therefore of only ^V the volume.
To illustrate : If to neutralize 5 c.c. of a nutrient gelatin
5.5 c.c. of ^r KOH are required, and the original mass
represented by the 5 c.c. is a liter in volume, manifestly
the whole mass would require two hundred times 5.5 c.c.
or 1100 c.c. of the alkaline solution, a volume much too
great to add to the gelatin because of the extreme dilution
that would result, we therefore substitute for the YTT KOH
in the final correction the normal KOH solution ( f KOH) ;
which being twenty times as strong will necessitate the
addition of only -^V of the volume, that is ^y|p = 55 c.c.
of f KOH for the liter.
1 Jour. Am. Pub. Health Assn., Quarterly Series, 1895, p. 38L
REACTION BY HYDROGEN-ION CONCENTRATION 115
On the average the neutral point as established by this
method requires for a liter of nutrient, meat infusion agar-
agar the addition of 47 c.c. T KOH and for a liter of meat
infusion gelatin 56 c.c. T KOH. Experience shows that
media neutral to phenolphthalein are somewhat too alkaline
for the best development of most bacteria. It is desirable
therefore to make certain corrections. In Fuller's experi-
ence the degree of deviation from phenolphthalein neutral-
ity that insures in general the best results is represented by
from 15 to 20 of his scale — that is, to say there should
remain enough uncombined acid in a liter of the finished
media to require a further addition of from 15 to 20 c.c.
T KOH. Thus for instance if, as in our foregoing calcula-
tion 55 c.c. T KOH were needed to bring the mass to
phenolphthalein neutrality, we would actually add from 35
to 40 c.c., i. e., from 15 to 20 c.c. less than was indicated by
the titration.
REACTION AS DETERMINED BY HYDROGEN-ION
CONCENTRATION.
This is based on Arrhenius's demonstration that when
acids, bases and salts are dissolved in water their molecules
are dissociated into electropositive and electronegative
ions. Not all the molecules of such electrolytes are always
so dissociated, the proportion being dependent upon a
number of circumstances, such as degree of dilution,
temperature, character of solvent, etc. By appropriate
electrical methods the degree of such dissociation may be
accurately discovered. Analyses made in this manner have
afforded results of fundamental importance to an understand-
ing of solutions, of acidity, of alkalinity and of relative
combining values of dissolved substances.
'116 BACTERIOLOGY
Of prime importance to the bacteriologist is the fact that
while the degree of dissociation undergone by an electro-
lyte when dissolved in pure water may be accurately deter-
mined and is constant under fixed conditions, if such electro-
lyte be dissolved in organic fluids the estimation of the
amount of dissociation is interfered with by a number of
organic bodies present. Such interfering matters are known
as "buffers" and their influence must always be taken into
account in this method of estimating reaction of a fluid,
i. e., its hydrogen-ion concentration.
The meaning of the term "hydrogen-ion concentration"
can best be understood after the statement of several funda-
mental facts: If an acid, such as HC1, for instance, be dis-
solved in water, some of it retains its characteristic mole-
cular form HC1, but a much larger proportion is broken
down, "dissociated," into its component elements H and Cl,
which are conceived as atoms or groups of atoms denomi-
nated electrically charged "ions;" those carry ing the positive
charge being the hydrogen (H) ions, those carrying the
negative charge being the chlorine (Cl) ions, or "cations"
and "anions" respectively. According to this theory the
acidity of a fluid (the strength of the acid dissolved) is pro-
portionate to the amount of dissociated hydrogen ions
present, and not to the amount of alkali required to neutra-
lize the acid. "Thus, the percentage dissociation or the
amount of H ions set free in YT HC1 solution was found
(Talbot, 1908) to be 90 per cent., while that of A acetic
acid is 1.4 per cent. Hydrochloric acid according to these
findings is therefore sixty-four times stronger in acidity
that acetic acid, although 10 c.c. of yV of either acid will
require a similar 10 c.c. portion of YTT NaOH to neutralize
it. Accordingly the only correct method of measuring the
REACTION BY HYDROGEN-ION CONCENTRATION 117
acid strength of a solution is to determine the amount of
free H ions or the hydrogen-ion concentration (H.I.C.) of
that solution, and not to determine the amount of y^ NaOH
necessary to neutralize that solution."1
From the foregoing it is obviously not possible to deter-
mine by titration with an alkaline solution if the acidity of
the fluid under consideration is due to the presence of a
strong or a weak acid or, to put it in other words, to the
presence of acids readily dissociated with the liberation of
large amounts of H ions or to weak acids in which the reverse
is the case. Total acidity can, therefore, only be deter-
mined by estimating the H ion concentration of the fluid
under consideration.
By the use of appropriate electrical devices the H.I.C. of
a fluid may be accurately determined and its variations, as
the fluid is diluted, may also be exactly detected, even
when reduced by dilution to such minute traces as would
be expressed by unwieldy decimals. To obviate the use of
such unwieldy decimals the symbol pH is employed, and by
suffixing to it a numeral representing the successive dilu-
tions by 10 that a normal solution may have undergone, we
have a brief and handy way of expressing H.I.C. Thus,
for instance, pH5 would symbolize the H.I.C. of a 0.00001
N (the fifth decimal point) solution, while pH7 (neutrality
as represented by distilled water) would symbolize the
H.I.C. of an acid the dilution of which is expressed by
0.0000001 N, or the seventh decimal point. We see, there-
fore, that in the use of the short, convenient symbol pH, we
have a means of noting the degree of dilution of an acid
which corresponds to a determined H.I.C. for that acid in that
1 Medalia, Jour. Bacteriol., 1920, No. 5, vol. v, p. 442.
118 BACTERIOLOGY
dilution; pH, therefore, increases as H.I.C. diminishes. Thus:
Tj-i N acid TTO N acid TTO N acid TTI
PH1 = ~To^; PH2 = Too~; PH3 = ToTocr» etc., or pHl =
0.1 N acid; pH2 = 0.01 N acid; pH3 = 0.001 N acid, etc.
Estimation of H.I.C. —This may be done by the employ-
ment of either electrolytic or colorimetric methods. The
former is the more exact, but for a variety of reasons is less
well adapted to routine bacteriological work than is the
latter.
By the former, the exact electrical method, the true
acidity, i. e., the H.I.C. of a number of substances in known
dilutions has been accurately determined. These "stand-
ard solutions" are so adjusted that in known dilutions
they can be made to represent a fairly regular range of
H.I.C. from extreme concentration to almost infinite dilu-
tion. When thus prepared they serve as objects on which
to establish the value of various indicators for various
H.I.C. ranges, for no single indicator will function through-
out the entire series of dilutions; that which gives its most
trustworthy indication at pH3, for instance, would be
valueless when mixed with a solution the H.I.C. of which is
symbolyzed by pH7.
By arranging, therefore, a regular succession of known
dilutions of an electrolyte whose pH under dilution is known,
from the strongest point down to the weakest, i. e., by
having each step in the range only & the strength of the
preceding, it is found that several indicators each function-
ing best at some fixed point will be required to cover the
range embraced between concentration and neutrality. And
also that as we pass from the maximum point of efficiency
for such indicators to either the acid side of the scale, that
is to the lessor pH, or to the alkaline, or greater pH, the
color of the indicator is so modified as to serve as a guide for
fairly accurate interpolations between the periods.
INDICATORS AND THEIR EMPLOYMENT 119
If, for instance, we so dilute any N acid with distilled
water that each successive dilution will be weakened by a
tenfold dilution we can by comparison with a known stand-
ard solution arrange a scale extending, say from pHl to pH8,
and by trying various indicators at various points on the
range, determine at which point each indicator functions
best. That, in fact, is what has been done and it is in that
manner that the colorimetric estimation of H.I.C. as symbo-
lyzed by pH is determined.
INDICATORS AND THEIR EMPLOYMENT.
If one prepare a normal solution of an electrolyte in pure
water and from such solution prepare successive dilutions
by negative multiples of ten, it is possible, as said above,
by appropriate electrical methods to detect the ions result-
ing from dissociation in .dilutions reaching the millionth or
the billionth part, as expressed by the appropriate decimals.
If, for instance, such dilutions be prepared for a NHC1
solution, we shall not find that the hydrogen-ion content
of each tenth strength of solution is precisely one-tenth of
that of the next stronger dilution, for the higher the dilution
of an electrolyte, the greater the ionization, i. e., the greater
the dissociation. It is necessary, therefore, to establish for
each successive tenth dilution the H.I.C., as symbolized by
pH for that dilution, and it is found that in the interval
between such dilutions the pH gradually increases from the
low to the high dilutions so that such increase may also
be expressed in decimals — thus a substance whose pH is
between 5 and 6 may be expressed as pH5.5, or pH5.8 as
the case may be.
When a solution has been so standardized by the electrical
120 BACTERIOLOGY
method that the gradations of its pH are known, it is the
"standard" solution with which solutions of unknown pH
(or H.I.C.) are to be compared when the estimations are
made by the colorimetric method.
Thus, if to a standard solution of pH6 we add the indi-
cator which exhibits its most trustworthy color changes at
or about that point, changing to one color or shade as we
approach the alkaline side, i. e., as its pH ascends, and to
another color or shade as we approach the acid side, i. e.,
as pH descends, it is observed that if such indicator behave
in an identical manner with an unknown solution, the pH
of that solution is probably the same or approximately the
same as that of the standard solution.
fV
By a creful study of indicators it is found, as said, that
for various pH values different indicators must be chosen,
as none act equally well throughout all dilutions.
According to Clark and Lubs, Medalia and others :
Thymol blue, acid range, operates best between pH1.2
and 2.8.
Brom phenol blue, between pH3 and 4.6.
Methyl red, between pH4.4 and 6.
Brom cresol purple, between pH5.2 and 6.8.
Brom thymol blue, between pH6 and 7.6.
Phenol red-, between pH6.8 and 84.
Cresol red, between pH7.2 and 8.8.
Thymol blue, alkaline range, between pH8 and 9.6.
Method of Barnett and Chapman. — As it is desirable for
routine bacteriological work to have culture media at
or about the neutrality of distilled water, pH7, it is
obvious that only that indicator which operates best at
or about that point is the one of most immediate inter-
est. Viewing the question from this standpoint Barnett
INDICATORS AND THEIR EMPLOYMENT 121
and Chapman have introduced a very simple method that
appears to meet all the requirements. The following are
the essentials of that method: For the preparation of cul-
ture media, which are to be neutral or nearly so, the bacteri-
ologist is concerned only with reactions falling between pH7
and pH8, and as phenolsulphonephthalein (phenol red) indi-
cates best at about these points it is the indicator used.
In this method use is made of the principle of superimposing
the two extreme colors of the indicators in determining the
so-called "half transformation" point. Within the range
of its transition from red to yellow -we may regard the
observed color of a phenol red solution as composed of a
definite amount of red plus a definite amount of yellow, and
such a color may be exactly duplicated by superimposing
the extreme red and the extreme yellow of the indicator
in proper concentrations. Thus, if to one test-tube we add
5 c.c. of dilute acid and to another 5 c.c. of dilute alkali and
to each add 5 drops of phenol red solution, a bright yellow
will be produced in the acid tube and a bright red in the
alkaline. But if we look toward the light through both
tubes, a color will be observed that is half-way between
yellow and red. In fact, it will be identical with the color
produced by 10 drops of the indicator solution in 5 c.c. of a
standard solution having a pH value of 7.9. This is the
''half transformation" point, and is a definite constant for
this indicator. But if instead of using equal amounts of
indicator in each of the two tubes we vary the partition
of the 10 drops of indicator between them, then by super-
imposing each pair and viewing them by transmitted light,
a series of colors will be observed which will cover the range
of usefulness of this particular indicator. Once such a
series is standardized ("calibrated") against solutions of
122 BACTERIOLOGY
known hydrogen-ion concentration, it may be used as a
standard for the determination of unknown reactions.
Results obtained by such a procedure, the phosphate solu-
tion of Sorensen being employed as the standard of com-
parison, are as follows:
RESULTS WITH PHENOL RED.
Acid tubes. Alkaline tubes.
Drops of phenol Drops of phenol
red solution. red solution. pH.
9 1 = 6.9
8 2 = 7.2
7 3 7.5.
6 4 =7.7
5 5 = 7.9
4 6 = 8.1
Outline of procedure -and equipment needed: Apparatus
and chemicals:
(a) Clean test-tubes of approximately the same diameter.
(b) A 5 c.c. and a 1 c.c. volume pipette.
(c) A medicine dropper drawn out to a fine point.
(d) A burette.
(e) Indicator solution: 0.01 per cent, phenol red in
distilled water.
; (/) /TT KOH. '
(g) HC1 or H2SO4.
(h) Test-tube rack, double row of holes.
Preparation of Standard Color Series. — Twelve test-tubes
are placed in two rows of six. Into each tube of one of
the rows five (5) c.c. of dilute alkali are placed (the ^
KOH solution may be used). Into each tube of the other
row five (5) c.c. of very dilute acid are placed (1 drop of
concentrated HC1 or H2SO4 in 100 c.c. distilled water).
Into the six acid tubes 9, 8, 7, 6, 5, 4 drops, respectively,
of the indicator solution are placed. Into the six correspond-
INDICATORS AND THEIR EMPLOYMENT 123
ing alkali tubes, 1, 2, 3, 4, 5, 6 drops of the indicator solution
are placed. (If the dropper be held vertically all drops will
be of practically the same volume.)
Each pair of tubes thus contain 10 drops of the indicator
solution between them, and the series of six pairs, when
viewed by transmitted light, will correspond to pH values
shown in the foregoing table.
In order to determine the hydrogen-ion concentration of
an unknown solution whose reaction lies within the ranges
pH6.9 and pHS.l, 5 c.c. of it are placed in a test-tube,
10 drops of the indicator solution are added, and its color
is compared with those of the six pairs of tubes; its H.I.C.
being the same as that pair with which its color corresponds.
When this unknown solution is a bacterial culture medium
the procedure is as follows: 1 c.c. of the medium to be
titrated is added to 4 c.c. of distilled water in a test-tube.
Ten drops of the indicator solution are then added and
the initial reaction is determined by comparing the color of
the mixture with each of the six pairs of standard colors
already prepared. From this initial reaction, usually too
acid, the desired reaction is obtained by titration with the
|^ NaOH solution. When the desired reaction for 1 c.c. of
the medium is thus obtained, fifty times that figure in y
NaOH (not <nr NaOH) will be needed for a liter of the
medium.
If in making the titrations the volume of fluid in the
unknown solution is greatly increased by the titrate, then
the volumes in the standard solutions must be correspond-
ingly increased by the addition of distilled water.1
1 For details see, Clark and Lubs, Jour. Bacteriol., 1917, No. 2, vol. ii, p.
109. Barnett and Chapman, Jour. Am. Med. Assn., 1918, No. 15, vol. Ixx, p.
1062. Report of Committee on Descriptive Chart, Soc. Am. Bacteriologists,
Jour. Bacteriol., 1919, No. 2, vol. iv, p. 119. Medalia, Jour. Bacteriol.,
1920, No. 5, vol. v, p. 441.
124 BACTERIOLOGY
Bouillon. — As has been stated, the fundamental con-
stituent of culture media is beef-tea or bouillon. The
directions of Koch for the preparation of this medium
have undergone many modifications to meet special cases;
but for general use the formula now employed is as follows:
500 grams of finely chopped lean beef free from fat and
tendons, are to be soaked in 1 liter of water for twenty-
four hours, during which time the mixture is to remain in
an ice-chest or to be otherwise kept at a low temperature.
At the end of twenty-four hours it is to be strained through
a coarse towel and pressed until a liter of fluid is obtained.
To this are to be added 10 grams (1 per cent.) of dried
peptone and 5 grams (0.5 per cent.) of common salt
(NaCl). It is then to be rendered neutral or very slightly
alkaline by one of the foregoing methods. The mixture is
then placed in an agateware or porcelain-lined saucepan
over a free flame, and kept at the boiling-point until all
the albumin is coagulated and the fluid portion is of a
clear, pale straw color. It is then filtered through a folded
paper filter and sterilized by steam.
Not infrequently the filtered bouillon, neutralized and
sterilized, will be seen to contain a fine, flocculent precipi-
tate. This may be due either to excess of alkalinity or to
incomplete precipitation of the albumin. The former may
be corrected with dilute acetic or hydrochloric acid, and the
bouillon again boiled, filtered, and sterilized; or, if due
to the latter cause, subsequent boiling and filtration usually
result in ridding the bouillon of the precipitate.
Another modification now generally employed is in the
substitution of meat extracts for chopped meat in making
the bouillon. Almost any of the meat extracts of com-
merce answer the purpose, though we usually employ
PREPARATION OF GELATIN 125
Liebig's. It is used in the strength of from two to four
grams to the liter of water. Peptone and sodium chloride
are added as in the bouillon made from meat-infusion.
The advantages of meat extract are: it takes less time;
affords a solution of more uniform composition if used in
fixed proportions'; and in general use gives results that are
equally as satisfactory as those obtained from the employ-
ment of infusion of meat. The disadvantage is the possible
presence of antiseptics or preservatives.
Nutrient Gelatin. — For the preparation of gelatin the
bouillon is first made in the way given, except that its
reaction is corrected after the gelatin has been completely
dissolved, which occurs very rapidly in hot bouillon. The
reaction of the gelatin of commerce is frequently more or
less acid, so that a much larger amount of alkali is needed
for its neutralization than for other media. It is possible,
however, to obtain from the makers an excellent grade
of gelatin from which practically all free acid has been
carefully washed. The gelatin is added in the proportion
of 10 to 12 per cent. Its complete solution may be accom-
plished either over a water-bath, in the steam sterilizer, or
over a free flame. If the latter method be practised, care
must be taken that the mixture is constantly stirred to
prevent burning at the bottom.
It is now almost the universal practice to use enamelled
iron saucepans, instead of glass vessels for the purpose of
making both gelatin and agar-agar; by this means the
free flame may be employed without danger of breaking the
vessel, and, with a little care, without burning the media.
Under any conditions it is better to protect the bottom of
the vessel from the direct action of the flame by the inter-
position of several layers of wire gauze, a thin sheet of asbes-
tos-board, or an ordinary cast-iron stove-plate.
126
BACTERIOLOGY
When the gelatin is completely melted it may be filtered
through a folded paper filter supported on an ordinary
funnel; if solution is complete, this should be very quickly
accomplished.
To Fold a Filter. — For the filtration of such substances
as gelatin and agar-agar it is of mportance to have a
properly folded filter. • Inability to fold a filter properly is
so common with beginners that a detailed description of
the steps may not be out of place. To fold a filter cor-
rectly, proceed as follows: A circular piece of filter paper
is folded exactly through its center, forming the fold 1, 1'
(Fig. 16); the end 1 is then folded over to 1', forming the
fold 5; 1 and 1' are each then brought to 5, thus forming
the folds 3 and 7; 1 is then carried to the point 7, and the
fold 4 is formed, and by carrying V to 3 the fold 6 is pro-
duced; and by bringing 1 to 3 and 1' to 7 the folds 2 and 8
result.
Thus far the ridges of all folds are on the side of the paper
next to the table on which we are folding. The paper is
now taken up and each space between the seams just pro-
duced is to be subdivided by a crease or fold through its
center, as indicated by the dotted lines in Fig. 16, but with
FOLDING A FILTER 127
the creases on the side opposite to that occupied by creases
1, 2, 3, 4, etc., first made. As each of these folds is made
the paper is gradually brought into a wedge-shaped bundle
(Fig. 17, a), which when opened assumes the form of a
properly folded filter (seen in 6, Fig. 17). Before placing
it upon the funnel it is well to go over each crease and see
that it is as closely folded as possible, care being taken not
to tear it. The advantage of the folded filter is that by its
use a much greater filtering, surface is obtained, as it is in
contact with the funnel only at the points formed by the
ridges, leaving the greater part of the flat surface free for
filtration.
FIG. 17
The employment of the hot-water funnel, so often recom-
mended, has been dispensed with in this work to a very
large extent, for the reason that if solution of the gelatin
is complete, filtration is so rapid as not to necessitate the
use of an apparatus for maintaining a high temperature.
The temperature at which the hot-water funnel retains the
gelatin is so high that evaporation and concentration rapidly
occur, and in consequence filtration is, as a rule, retarded.
The filtration is frequently done in the steam sterilizer;
but this, too, is unnecessary if the gelatin is quite dissolved.
At the ordinary temperature of the room, and by the means
commonly employed for the filtration of other substances,
128 BACTERIOLOGY
both gelatin and agar-agar may be rapidly filtered if they
are completely dissolved.
It not infrequently occurs that, even under the most
'careful treatment, the filtered gelatin is not quite trans-
parent, and clarification becomes necessary. For this
purpose the mass must be redissolved, and when at a tem-
perature between 60° and 70° C. an egg, which has been
beaten up with about 50 c.c. of water, is added. The whole
is then thoroughly mixed together and again brought to the
boiling-point, and kept there until coagulation of the
albumin occurs. The egg albumin coagulates as large floccu-
lent masses, and it is better not to break them up, as when
broken up into fine flakes they clog the filter and materially
retard filtration.
The practice sometimes recommended of removing these
albuminous coagula by first filtering the gelatin through a
cloth, and then through paper, is not only superfluous, but
in most instances renders the process of filtration much more
difficult, because of the disintegration of the masses into
finer particles, which have the effect just mentioned, viz.,
of clogging the filter.
Under no circumstances should a filter be used without
first having been moistened with water. If this is not done
the pores of the paper, which are relatively large when in a
dry state, when moistened by the gelatin not only diminish
in size, but in contracting are often entirely occluded by the
finer albuminous flakes which become fixed within them,
and filtration practically ceases. The preliminary moisten-
ing with water causes diminution of the size of the pores to
such an extent that the finer particles of the precipitate rest
on the surface of the paper, instead of becoming fixed in its
meshes.
PREPARATION OF NUTRIENT AGAR-AGAR 129
During boiling it is well to filter, from time to time, a
'' few cubic centimeters of the gelatin into a test-tube and boil
it over a free flame for a minute or so; in this way one can
detect if all the albumin has been coagulated — i. e., if the
solution is ready for filtration.
Gelatin should not, as a rule, be boiled more than ten or
fifteen minutes at one time, or be left in the steam sterilizer
for more than thirty minutes ; otherwise its property of
solidifying may be impaired.
As soon as the preparation of the gelatin is complete,
whether it is retained in the flask into which it has been
filtered or decanted into sterilized test-tubes, it should be
sterilized, the mouth of the flask or the test-tubes containing
it having been previously closed with cotton plugs. It may
be sterilized by either the intermittent method with stream-
ing steam or by a single application of steam under pressure
in the autoclave. If the latter method be selected, the
pressure should not exceed one atmosphere and the time
of exposure be not over fifteen minutes.
Nutrient Agar-agar. — The preparation of nutrient agar-
agar by the beginner is far too frequently a tedious and
time-consuming operation. This is due mainly to lack of
patience and to deviation from the rules laid down for
the preparation of this medium. If the directions given
below for the preparation of nutrient agar-agar be strictly
observed, no difficulty whatever should be encountered.
Many methods are recommended for its preparation, almost
every worker having some slight modification of his own.
The methods that have given us the best results, and from
which we have no good grounds for departing, are as follows :
Prepare the bouillon in the usual way. Agar-agar reacts
neutral or Very slightly alkaline, so that the bouillon may
9
130 BACTERIOLOGY
be neutralized before the agar-agar is added. Then add
finely chopped or powdered agar-agar in the proportion of
1 to 1.5 per cent. Place the mixture in a porcelain-lined
iron vessel, and on the side of the vessel make a mark at
the height at which the level of the fluid stands; if a liter
of medium is being made, add about 250 to 300 c.c. more of
water and allow the mass to boil slowly, occasionally stirring,
over a free flame, from one and a half to two hours; or until
the excess of water — i. e., the 250 or 300 c.c. that were
added — has evaporated. Care must be taken that the
mixture does not boil over the sides of the vessel. From time
to time observe if the fluid has fallen below its original
level; if it has, add hot water until its volume of 1 liter is
restored. At the end of the time given remove the flame
and place the vessel containing the mixture in a large dish
of cold water; stir the agar-agar continuously until it has
cooled to about 68°-70° C., and then add the white of one
egg which has been beaten up on about 50 c.c. of water;
or the ordinary dried albumin of commerce may be dissolved
in cold water in the proportion of about 10 per cent, and used;
the results are equally as good as when eggs are employed.
Mix this carefully throughout the agar-agar and allow the
mass to boil slowly for about another half-hour, observing
all the while the level of the fluid, which should not fall
below the liter mark. It is necessary to reduce the tempera-
ture of the mass to the point given, 68°-70° C., otherwise
the coagulation of the albumin will occur suddenly in lumps
and masses as soon as it is 'added, and its clarifying action
will not be uniform. The process of clarification with the
egg is purely mechanical; the finer particles, which would
otherwise pass through the pores of the filter, being taken
up by the albumin as it coagulates and retained in the
coagula.
FILTERING AGAR-AGAR 131
At the end of a half-hour the boiling mass may be easily
and quickly filtered through a heavy, folded paper filter
at the room temperature; as a rule the filtrate is as clear and
transparent as agar-agar usually appears.
It may be well to emphasize the fact that for the filtration
of agar-agar no special device for maintaining the tem-
perature of the mass, is necessary. Agar-agar prepared
after the methods just given should pass through a properly
folded paper filter at the rate of a liter in from twelve to
fifteen minutes.
Another plan that insures complete solution of the agar-
agar without causing the precipitates often seen when all
the ingredients are added at once and boiled for a long time
is to weigh out the necessary amount of agar-agar, 10 or 15
grams, and place this in 1300 or 1400 c.c. of water and boil
down over a free flame to 1000 c.c. The peptone, salt, and
beef extract are then added and the boiling continued until
they are dissolved. The clarification with egg-albumen may
then be done, and usually the mass filters quite clear and
does not show the presence of precipitates upon cooling.
If the mixture is positively alkaline, it is not only cloudy,
but it filters with difficulty; if it is acid, it is usually quite
clear, and filters more quickly, but, as Schultz has pointed
out, it loses at the same time some of its gelatinizing
properties.
Another method by which agar-agar can be easily and
quickly melted is by steam under pressure. If the flask
containing the mixture of bouillon and agar-agar be kept
in the digester or autoclave for ten minutes with the steam
under a pressure of about one atmosphere, as shown by the
gauge, the agar-agar will be found at the end of this time
completely melted, and filtration may then be accomplished
with but little difficulty.
132
BACTERIOLOGY
FIG. 18
If glycerin is to be added to the agar-agar, it is done after
filtration and before sterilization. The nutritive properties
of the media for certain organisms, particularly the tubercle
bacillus, are increased by the addition of glycerin in the
proportion of 5 to 7 per cent.
If after filtration a fine flocculent precipitate is seen, look
to the reaction of the medium. If it is quite alkaline, boilj
neutralize, and filter again. If the reac-
tion is neutral or only very slightly acid,
dissolve and again clarify with egg-albu-
men by the method given.
The most important feature of all the
media, aside from the correct proportion
of the ingredients, is their reaction. It
must be neutral or very slightly alkaline
to litmus. (See remarks on Neutralization
of Media.) Only a few organisms develop
well on media of an acid reaction.
* Preparation of Potatoes. — With an ordi-
nary cork borer punch out from sound
potatoes cylindrical bits that will slip
easily into the test-tubes to be used. Cut
away all particles of the skin. Then cut
on each cylinder a slanting surface extend-
Potato in test-tube, ing from about the middle diagonally to
the end. Leave the cylinders in running
water over night to prevent them from becoming dis-
colored when they are sterilized.
One potato cylinder thus prepared is then to be placed
in each of the already cleaned, plugged and sterilized test-
tubes, after which they are sterilized by either the inter-
mittent method with streaming steam or by steam under
POTATOES AND BLOOD SERUM 133
pressure in the autoclave. In the latter event one atmos-
phere of pressure should be continued for twenty minutes.
(See Fig. 18.)
For some purposes potatoes may be advantageously
peeled, sliced into disks of about 1 cm. in thickness, and
placed in small glass dishes provided with covers, similar to
the ordinary crystallizing dishes. The dish and its con-
tents are then sterilized by steam in the usual way. By
this plan a relatively large area for cultivation is obtained.
Potatoes may also be boiled, or steamed, and mashed,
and the mass placed in covered dishes, test-tubes, or flasks,
and sterilized. By this method one obtains in the mass a
mean of the composition of the several potatoes, or bits of
potatoes, used in making it, an advantage where uniformity
is desired.
Care must be given to the sterilization of potatoes, because
they always have adhering to them the organisms commonly
found in the ground, the spores of which are among the
most resistant known.
Blood Serum. — For ordinary routine work blood serum
may be obtained from either the slaughter houses or the
antitoxin manufacturers. When from the former the
blood that streams from the severed vessels of the throat
of the slaughtered animal is collected under as clean con-
ditions as possible in large, clean glass museum jars. These
are then, with the covers placed upon them, set aside in an
ice-chest until coagulation is complete. The serum may then
be decanted or pipetted off into flasks and thus transported
to the laboratory to be sterilized by the method given
below.
In many localities it is possible to purchase at a small
cost normal horse serum in bulk from firms engaged in the
134 BACTERIOLOGY
manufacture of antitoxins and other biological products.
This serum, obtained under aseptic precautions, has ob-
viously an advantage, and has in our hands proven entirely
satisfactory for routine work.
In either case the serum is to be decanted into clean,
sterile test-tubes provided with cotton plugs, after which
it must be immediately sterilized. For this purpose the
method suggested by Councilman and Mallory is now
FIG. 19
Chamber for sterilizing and solidifying blood serum. (Koch.)
generally used. It is as follows: Place the test-tubes con-
taining the serum in a slanting position in a dry air sterilizer
and heat them to from 80°-90° C. for a time necessary
to solidify the serum. After this they are kept for twenty
minutes on three successive days in the steam sterilizer at
100° C. They should be kept at room temperature between
the exposures to the steam. After this treatment the serum
should be sterile.
BLOOD SERUM 135
Serum thus prepared may be kept from drying by burning
off in the gas flame the excess of cotton protruding from the
ends of the tubes and then forcing down upon the cotton
plugs clean, new, corks that have been sterilized by steam
under pressure. (Ghriskey.)
To secure satisfactory results by this method several
precautions should be noted, viz.: The solidification of the
serum in the dry air sterilizers must be complete, else its
surface will be rough and broken by bubbles; the same
results if the temperature in the dry air sterilizer is brought
up too rapidly.
Serum prepared in this way is neither clear nor colorless.
This is ordinarily not a disadvantage. The popularity of
the method is due to its simplicity, the rapidity with which
a satisfactory serum may be prepared and especially to
the fact that the rigid precautions against contamination
observed in the older methods, where sterilization at low
temperature was practised, are not essential to success,
since even though such contaminations occur they are
eliminated by the high temperatures used in this procedure.
Blood Serum from Small Animals. — For special purposes
it is often desirable to secure blood serum under strictly
aseptic precautions from particular species of animals,
many of them being small. To this end there have been
devised a number of handy methods. That which in our
hands has proven the simplest and generally most useful
is the Rivas modification1 of Latapie's method. It is as
follows:
The Rivas apparatus is constructed from two test-tubes
about 15 x 180 mm. in size. The mouth of one test-tube is
drawn out into a long narrow neck 1 cm. in diameter and
1 University of Pennsylvania, Medical Bulletin, 1904, vol. xvii, p. 295.
136
BACTERIOLOGY
about 5 cm. in length. Three or four points on the side of
the tube are softened in the flame of a blowpipe, and the
FIG. 20
Rivas apparatus for collecting blood serum: A, long narrow neck on
first tube; B, constriction on tubes near mouth; C, invaginations on first
tube; D, small cannula drawn out on extremity of first tube; E, blood-
clot, and F, blood serum collected in bottom of second tube.
softened glass driven inward by means of a piece of pointed >
wood. This gives supports on the interior of the tube to
BLOOD SERUM 137
hold the coagulated blood in place. Between the long
narrow neck and the body of the tube a constriction is
formed by drawing out the tube while heated. The second
tube also has a similar constriction about 20 cm. from its
mouth.
The two tubes are now fitted together by inserting the
one with the long narrow neck into the second tube ; a small
amount of cotton being first carefully folded around the
neck of the first tube, so as to prevent the entrance of dust.
The two tubes are then fastened together by means of a
wire twisted around the constriction at the neck of each
tube, and the apparatus is then wrapped in cotton and
sterilized in a hot-air sterilizer.
Before using the apparatus the extremity of the first tube
is heated in the gas-flame, and by touching this point with
a piece of pointed glass rod it is gently drawn out into a
fine cannula. When the animal has been prepared for the
operation and a vessel exposed, the point of the cannula is
snipped off with a sterile scissors, when the point of the
cannula is inserted into the vessel. The pressure of blood
is sufficient to fill the first tube. The point of the cannula
is now removed from the vessel and sealed in a gas-flame.
The apparatus is laid aside in an almost horizontal position
until the blood has become completely coagulated. It is
then inverted and set aside for the serum to separate and
trickle down through the narrow neck of the first tube and
collect in the second tube. When this has occurred, the wire
holding the two tubes together is unwound, and the first
tube is removed and the second plugged with a well-fitting
sterile cotton plug, when the serum may be preserved in
the tube for several days without danger of contamination.
138 BACTERIOLOGY
Preservation of Blood Serum. — It is sometimes desirable
to preserve blood serum in a fluid state. This can be done
by the fractional method of sterilization at low tempera-
tures, already described, or with much less effort, and with-
out the use of heat, by a method that we have found very
satisfactory. In the course of Kirschner's investigations
chloroform was shown to possess decided disinfectant
properties; as it is quite volatile, it is easily got rid of when
its disinfectant or antiseptic properties are no longer required.
If, therefore, the serum to be preserved be placed in a closely
stoppered flask and enough chloroform added to form a
thin layer, about 2 mm., on the bottom, the serum may
be kept indefinitely without contamination, so long as the
chloroform is not permitted to evaporate. This latter pro-
vision is one on which success depends. If the vessel con-
taining the mixture of chloroform and serum be not tightly
corked, the chloroform vapor escapes pretty rapidly and
exerts no preservative action. In fact, bacteria will grow
uninterruptedly in a cotton-stoppered test-tube containing
bouillon to which chloroform has been added. When re-
quired for use, the serum is decanted into test-tubes, which
are then placed in a water-bath at about 50° C. until all
the chloroform has been driven off; this can be determined
by the absence of its characteristic odor. The serum may
then be solidified, sterilized by heat, and employed for
culture purposes. We have found serum so preserved to
answer all requirements as a culture medium.
Milk. — Fresh milk should be allowed to stand overnight in
an ice-chest, the cream then removed, and the remainder of
the milk pipetted into test-tubes, about 8 c.c. to each tube,
and sterilized by the intermittent process, at the tem-
perature of steam, for three successive days.
MILK 139
The separation of the cream may be accelerated and
rendered more complete if the cylinder containing the milk
be placed in the steam sterilizer for fifteen minutes before it
is placed in the ice-chest.
The cream is best separated from the milk by the use of
a cylindrical vessel with a stopcock at the bottom, by
means of which the milk, devoid of cream, may be drawn
off. A Chevalier creamometer with a stopcock at the bottom
serves the purpose very well. It should be covered while
standing.1
Milk may be used as a culture medium without any addi-
tion to it, or, before sterilizing, a few drops of litmus tinc-
ture may be added, just enough to give it a pale-blue color.
By this means it will be seen that different organisms bring
about different reactions in the medium: some producing
alkalies, which cause the blue color to be intensified; others
producing acids, which change it to red; while others
bring about neither of these changes. Similarly litmus
solution is often added to gelatin and agar-agar for the
same purpose.
Milk may also be employed as a solid culture medium
by the addition to it of gelatin or agar-agar in the propor-
tions given for the preparation of ordinary nutrient gelatin
or agar-agar. It has, however, in this form the disadvan-
tage of not being transparent, and can therefore best be
used for the study of those organisms which grow upon the
surface of the medium without causing liquefaction.
Nutrient gelatin and agar-agar can also be prepared from
neutral milk-whey, obtained from milk after precipitation
of the casein.
1 For some time past we have been using what is technically known
as "separator milk" — i. e., the fluid left after milk has been deprived of
its fat (cream) by centrifugal force.
140 BACTERIOLOGY
Litmus-whey Milk. — An important differential medium
is milk-whey to which litmus tincture has been added.
A number of methods for its preparation are in use, but the
one employed by Durham seems to be the most satisfactory.
Briefly it is as follows: fresh milk, free from antiseptic
adulterations, is gently warmed and clotted with essence
of rennet. The whey is strained off and the clot hung up
to drain in a piece of muslin. The whey, which is somewhat
turbid and yellow, is then cautiously neutralized with a 4
per cent, citric acid solution, neutral litmus solution being
used as the indicator. It is then heated upon a water-bath
to 100° C. for about half an hour; thereby nearly the whole
of the proteid is coagulated. It is then filtered clear and
neutral litmus solution is added until it is of a distinct purple
color. If the filtered whey is cloudy, let it stand in a cold
place for a day or two and decant off the clear supernatant
portion or pass it through a Berkefeld filter. The whey should
never be heated above 100° C. or neutralized with mineral
acids, otherwise there is a danger of so modifying the milk-
sugar present as seriously to impair the usefulness of the
medium. When properly prepared, the medium is free from
proteid, and contains only water, lactose, the salts of the
milk, and a small quantity of a body suggestive of dextrose
or galactose. The medium is of great utility in detecting
the power of bacteria to cause acid fermentation in a non-
proteid medium containing a fermentable sugar; and for
observing the variations of this power in closely allied though
not identical species.
Dunham's Peptone Solution. — The hiedium known as
Dunham's solution is prepared according to the following
formula :
Dried peptone 1.0 part
Sodium chloride . . . . . . . . . 0.5 part
Distilled water 100.0 parts
DUNHAM'S PEPTONE SOLUTION 141
It is usually of a neutral or slightly alkaline reaction
and neutralization is not, therefore, necessary. It is filtered,
decanted into tubes or flasks, and sterilized in the steam
sterilizer in the ordinary way. The most common use to
which this solution is put is in determining if the organism
under consideration possesses the property of producing
indol as one of its metabolic products. It is essential for
accuracy that the preparation of dried peptone employed
should be as nearly chemically pure as is possible, and indeed
the other ingredients should be correspondingly free from
impurities. Gorini1 calls attention to the fact that impurities
in the peptone, particularly the, presence of carbohydrates,
so interfere with the production of indol by certain bacteria
that otherwise produce it, that it is ofttimes impossible,
under such circumstances, to obtain the characteristic
color-reaction of this body, and where it is obtained it is
always after a much longer time than is the case where pep-
tone free from these substances has been used.
Peckham has also demonstrated that where bacteria
have the property of forming indol and also of fermenting
carbohydrates, their proteolytic function, as evidenced by
the appearance of indol as a product of metabolism, may be
completely suppressed by the addition of such fermentable
carbohydrates as glucose, saccharose, and lactose to the
proteid solution in which they are developing.2
Gorini suggests the advisability of testing the purity of
all peptone preparations before using them, by means of
the reaction that they exhibit with Fehling's alkaline copper
solution. Under the influence of this reagent pure peptoen
in solution gives a violet color (the biuret reaction), which
1 Centralblatt fur Bakteriologie und Parasitenkunde, 1893, vol. xiii, p. 790.
2 See Journal of Experimental Medicine, 1897, vol. ii, p. 559.
142 BACTERIOLOGY
remains permanent even after boiling for five minutes. If,
instead of a violet color, there appears a red or reddish-
yellow precipitate, the peptone should be discarded, as in
his experience no indol is produced from peptone giving
this reaction. Both the peptone solution and that of the
copper (particularly the latter) should be relatively dilute
in order for the reaction to be successful.
Lactose Litmus-agar, or Litmus-gelatin of Wurtz. — A
medium of much use in the differentiation of bacteria is
that recommended by Wurtz, consisting of slightly alkaline
nutrient agar-agar, to which from 2 to 3 per cent, of lactose
and sufficient litmus tincture to give it a pale-blue color have
been added. Bacteria capable of causing fermentation
of lactose when grown on this medium develop into colonies
of a pale-pink color and cause, likewise, a reddening of the
surrounding medium, owing to the production of acid as
a result of their action upon the lactose ; while other bacteria,
incapable of such fermentative activities, grow as pale-blue
colonies and cause no reddening of the surrounding medium.
It is especially useful in the differentiation of the bacillus
of typhoid fever, which does not possess the property of
bringing about fermentation of lactose, from other organ-
isms that simulate it in many other respects, but which do
possess this property.
Its preparation is as follows: to nutrient agar-agar or
gelatin, the alkalinity of which is such that 1 c.c. will require
0.1 c.c. of a 1 : 20 normal sulphuric-acid solution to neu-
tralize it, lactose is added in the proportion of 2 or 3 per
cent.; it is then decanted into test-tubes and sterilized in
the usual way. When sterilization is complete enough
sterilized litmus tincture should be added to each tube to
give a decided, though not very intense, blue color. This
SERUM WATER MEDIUM OF HISS 143
must be done carefully, to avoid contamination of the tubes
during manipulation. It is better not to add the litmus
tincture before sterilizing the tubes, as its color-character-
istics are altered by contact with organic matters under the
influence of heat. This medium is used for both test-tube
and plate cultivation, just as is ordinary agar-agar and
gelatin.
Lbffler's Blood-serum Mixture. — Loffler's blood-serum mix-
ture consists of 1 part of neutral meat-infusion bouillon,
containing 1 per cent, of grape sugar, and 3 parts of blood
serum. This mixture is placed in test-tubes, sterilized, and
solidified in exactly the way given for blood serum. It
requires for its solidification a somewhat higher tempera-
ture and a longer exposure to this temperature than does
blood serum to which no bouillon has been added. (See
also the Councilman-Mallory method.)
The Serum Water Medium of Hiss. — A medium which
has been found very serviceable in the differentiation
between closely related bacteria is prepared by mixing 1
part of blood serum (either horse or bovine) and 3 parts of
distilled water. This is neutralized, and heated in a water-
bath or an Arnold steam sterilizer until it becomes opales-
cent. A 5 per cent, aqueous solution of litmus is then
added in the proportion of 1 per cent. Any one of the
carbohydrates, as dextrose, lactose, saccharose, levulose,
mannite, etc., is then added in the proportion of 1 per cent.
The finished medium is then placed in test-tubes. The
medium must be sterilized in an Arnold steam sterilizer, and
it is advisable to allow the sterilizer to remain uncovered
during the process of sterilization to avoid excessive heating
of the medium.
The relative degree of acidity produced, with or without
coagulation, with or without gas-production, and with or
144 BACTERIOLOGY
without reduction of the litmus, in a series of tubes of this
medium containing the different carbohydrates serves to
differentiate between related species of bacteria. For
instance, the colon bacillus produces an acid reaction with
coagulation and gas-formation with some of the carbohy-
drates, while the typhoid bacillus produces a lower degree
of acidity with coagulation, but without gas-production.
Similarly, the different types of the dysentery bacillus may
be differentiated by means of their effects on the different
carbohydrates in this medium.
Guarniari's Gelatin-agar Mixture. — For special work,
particularly with the organism of pneumonia (bacterium
pneumonise) the gelatin-agar mixture recommended by
Guarniari is of very great service. It should be exactly
neutral in reaction, and should possess the following ingredi-
ients :
Meat infusion 950 c.c.
Sodium chloride 5 grams
Peptone . v' . . . . . . . . . 25 to 30 grams
Gelatin . ....••. . 40 to 60 grams
Agar-agar 3 to 4 grams
Water 50 c.c.
The agar-agar should be completely dissolved separately
in about 100 c.c. of water in the autoclave while the other
ingredients are being prepared. The latter should be filtered
and the dissolved agar-agar added to the filtrate.
A complete list of the special media would be too volu-
minous for a book of this size. For their description the
reader is referred to the current literature. Those that have
been given above will suffice for obtaining a clear under-
standing of the principles of the subject. In the chapters
upon the Pathogenic Bacteria such special media as have
proved of use for purposes of identification and differentiation
are described in detail.
CHAPTER VI.
Preparation of the Tubes, Flasks, etc., in which the Media are to be
Preserved.
WHILE the media are in course of preparation it is well
to get the test-tubes and flasks ready for their reception,
and it is essential that they should be as clean as it is pos-
sible to make them. For this purpose it is advisable that
both new tubes and those which have previously been used
should be boiled for about thirty to forty-five minutes in
a 2 to 3 per cent, solution of common soda ; it is not necessary
to be exact as to strength, but it should not be weaker than
this. At the end of this time they are to be carefully swabbed
out with a cylindrical bristle brush, preferably one with a
reed handle (Fig. 21, a), as those with wire handles are apt
to break through the bottoms of the tubes, though Messrs.
Lentz & Sons, of Philadelphia, have in large part eliminated
this objection from the wire-handle brush depicted in Fig.
21, 6. All traces of adherent material should be carefully
removed. When the tubes are quite clean they may be
rinsed in a warm solution of commercial hydrochloric acid
of the strength of about 1 per cent. This is to remove the
alkali. They are then to be thoroughly rinsed in clear,
running water, and stood top down until the water has
drained from them. When dry they are to be plugged with
raw cotton; this requires a little practice before it can be
properly done. The cotton should be introduced into the
mouths of the tubes in such a way that no cracks or creases
10 (145)
146
BACTERIOLOGY
exist. The plug should fit neither too tightly nor too loosely,
but should be just firmly enough in position to sustain the
weight of the tube into which it is placed when held up by
the portion which projects from and overhangs the mouth
of the tube. The tubes thus plugged are now to be placed
upright in a wire basket and heated for one hour in the hot-
air sterilizer at a temperature of about 150° C. A very good
guide for this process of sterilization is to observe the tubes
from time to time, and as soon as the cotton has become a
very light-brown color, not deeper than a dark-cream tint,
to consider sterilization complete. The tubes are then
removed and allowed to cool.
FIG. 21
Brushes for cleaning test-tubes.
The cotton used for this purpose should be the ordinary
cotton batting of. the shops, and not absorbent cotton;
the latter becomes too tightly packed, and is, moreover,
much too expensive for this purpose.
Care should be taken not to burn the cotton, otherwise
the tubes will become coated with a dark-colored, empyreu-
matic, oily deposit, which necessitates recleansing.
Filling the Tubes. — When the tubes are cold they may be
filled. This is best accomplished by the use of a separating
funnel, such as is shown in Fig. 22. The liquefied medium
is poured into this funnel, which has been carefully washed,
FILLING THE TUBES 147
and by pressing the pinchcock with which the funnel is
provided the desired amount of material (5-10 c.c.) may
be allowed to flow into the tubes held under its opening.
It is not necessary to sterilize the funnel,, for the medium
is to be subjected to this process as soon as it is in the test-
tubes.
FIG. 22
Funnel for filling tubes with culture media.
Care should be taken that none of the medium is dropped
upon the mouth of the test-tube, otherwise the cotton plug
becomes adherent to it, and is not only difficult to remove,
but presents a very untidy appearance and interferes mate-
rially with the manipulations.
As soon as the tubes have been filled they are to be steril-
ized either in the steam sterilizer at 100° C. for fifteen
148 BACTERIOLOGY
minutes on each of three successive days, being kept during
the intervals at room temperature, or they may be sterilized
by a single exposure of fifteen minutes in the autoclave to a
temperature equivalent to steam under about one atmosphere
of pressure.
When sterilization is complete and the medium in the
tubes is still liquid, some of them may be placed in a slant-
ing position, at an angle of about ten degrees with the sur-
face on which they rest, and the medium allowed to solidify
in this position. These are for the so-called slant-cultures.
The remainder may solidify in the erect position; these
serve for making plates.
CHAPTER VII.
Technique of Isolating Bacteria in Pure Culture by the Plate and the
Tube Method.
PLATES.
THE plate method can be employed with both agar-agar
and gelatin. It cannot be practised with blood serum,
because the serum when once solidified cannot again be
liquefied.
Plates are usually referred to as "a set." This term
implies three individual plates, each representing a mixture
of organisms in a higher state of dilution. The first plate
is known usually as "the original," or "plate No. 1," the
first dilution from this as "plate No. 2," and the second as
"plate No. 3."
In the preparation of a set of plates the following are the
steps to be observed:
Three tubes, each containing from 7 to 9 c.c. of gelatin
or agar-agar, are placed in a warm water-bath until the
medium has become liquid. If agar-agar is employed, this
is accomplished at the boiling-point of water; if gelatin is
used, a much lower temperature suffices (35°-40° C.). When
liquefaction is complete the temperature of the water, in
the case of agar-agar, must be reduced to 41°-42° C., at
which temperature the agar-agar remains liquid, and the
organisms may be introduced into it without fear of de-
stroying their vitality. The medium being now liquid and
(149)
150 . BACTERIOLOGY
of the proper temperature, a very small portion of the
mixture of organisms to be studied is taken up with a steril-
ized platinum wire (Fig. 23, a) about 5 cm. long, twisted
into a small loop at one end and fused into a bit of glass
rod, which serves as a handle, at the other extremity. This
loop is one of the most useful of bacteriological instruments,
as there is hardly a manipulation into which it does not
enter. Under no circumstances is it to be employed without
having been passed through a gas-flame until quite hot,
for the purpose of sterilization. One should form a habit
of never taking up one of these platinum-wire needles, as
FIG. 23
a
6
Looped and straight platinum wires in glass handles.
they are called, for they are curved and straight (Fig. 23, 6)
as well as looped, without passing it through a flame; and
the sooner the beginner learns to do this as a reflex action,
the sooner does he eliminate one of the possible sources of
error in his work. It must be remembered, though, that it
should not be used when hot, otherwise the organisms taken
upon it will be killed by the high temperature; after steril-
ization in the flame one waits for a few seconds until it
is cool before using.
A minute portion of the material under consideration is
transferred with the sterilized loop into tube No. 1, "the
original," where it is thoroughly disintegrated by gently
PLATES 151
rubbing it against the sides of the tube. The more carefully
this is done the more uniform will be the distribution of
the organisms and the better the results. The loop is then
again sterilized and three of its loopfuls are passed, without
touching the sides of the tube, from ''the original" into tube
No. 2, where they are carefully mixed. Again the loop is
sterilized, and again three dips are made from tube No. 2
into tube No. 3. This completes the dilution. The loop is
now sterilized before laying it aside.
FIG. 24
Levelling tripod with glass cooling chamber for plates.
During this manipulation, which must be done quickly
if agar-agar be employed, the temperature of the water in
the bath in which the tubes stand should never be lower than
39° C., and never higher than 43° C. If it falls below 38°
C., the agar-agar solidifies, and can only be redissolved at
a temperature that would be destructive to the organisms
which may have been introduced into the tubes. This is
152
BACTERIOLOGY
not of so much moment with gelatin, since it may readily be
redissolved at a temperature not detrimental to the organ-
isms with which the tubes may have been inoculated. When
completed the dilutions are poured into sterilized Petri
dishes to cool and solidify, thereby fixing the bacteria so
that the individuals may develop into their characteristic
colonies and be so separated from one another as to permit
of easy isolation in pure culture.
FIG. 25
Petri double dish, now generally used instead of plates.
The Petri dish (Fig. 25) is of glass ; round in form, about
8 or 9 cm. in diameter and 1.5 to 2 cm. deep, with a loosely
fitting cover. To hasten the solidification of the medium
FIG. 26
Metal cooling stage
the dishes may be cooled by placing them upon a cold
surface, such as is provided by the glass cooling stage (Fig.
TUBE METHODS 153
24), when packed with ice, or on the metal cooler, shown in
Fig. 26, through which cold water circulates. The plates
are labeled to correspond with their respective dilutions
and are then set aside, protected from dust and light until
colony development begins. In the case of gelatin the
plates must not be maintained at a temperature higher
than that of an ordinary living room, about 20° to 22° C. being
the most favorable. In the case of agar-agar the plates may
be maintained at the temperature of the animal body, i. e.,
between 37° and 38° C.
TUBE METHODS.
Esmarch Tubes. — A useful modification of the plating
method just described is that suggested by von Esmarch.
It insures the greatest security from contamination by
extraneous organisms and requires the least amount of
apparatus. It differs from the other methods thus: the
dilutions having been prepared in tubes contain a smaller
amount of medium than usual — as a rule, not more than
5 to 6 c.c. — are, instead of being poured upon plates or into
dishes, spread over the inner surf ace of the tubes containing
them, and, without removing the cotton plugs, solidified
in this position. The tubes then present a thin cylindrical
lining of gelatin or agar-agar, upon which the colonies
develop. In all other respects the conditions for the growth
of the organisms are the same as in flat plates.
The solidification of the media on the inner sides of the
tubes is best accomplished by rolling them upon a block
of ice (Fig. 27), after the plan devised by Booker in 1887 in
the Pathological Laboratory of the Johns Hopkins Univer-
sity. In this method a small block of ice only is needed.
154 BACTERIOLOGY
It is levelled and held in position by being placed upon a
towel in a dish. A horizontal groove is melted in the upper
surface of the ice with a test-tube of hot water. The tubes
to be rolled are then held in an almost — not quite — hori-
zontal position and twisted between the fingers until the
sides are moistened by the contents to within about 1 cm.
of the cotton plug, care being taken that the gelatin does
not touch the cotton, otherwise the latter becomes adherent
to the sides of the tube and is difficult to remove. The tube
FIG. 27
Demonstrating Booker's method of rolling Esmarch tubes on a block of ice.
is then placed in the groove in the ice and rolled until its
contents are solid.
There is an erroneous impression that Esmarch tubes are
not a success when made from ordinary nutrient agar-agar
because of the tendency of this medium to shrink and slip
to the bottom of the tube. This slipping down of the agar-
agar is due to the water, which is squeezed from it during
solidification, getting between the medium and the walls
of the tube. .This can easily be overcome by allowing the
TUBE METHODS 155
rolled tubes to remain in a nearly horizontal position for
twenty-four hours after rolling them, the mouth of the tube
being about 1 cm. higher than the bottom. During this
time the margin of the agar-agar nearest the cotton plug
dries and becomes adherent to the walls of the tube, while
the water collects at the most dependent point — i. e., the
bottom of the tubes. After this they may be retained in
the upright position without danger of the agar-agar slipping
down.
In both the plates and tubes, if the dilutions of the number
of organisms have been properly conducted, the results
will be the same. The original plate or tube, as a rule, will
be of no use because of the great number of colonies in it;
plate or tube No. 2 may be of service; but plate or tube No.
3 will usually contain the organisms in such small numbers
that there will be nothing to prevent the characteristic
development of the colonies originating from them.
For reasons of economy the "original," tube No. 1, is
sometimes substituted by a tube containing normal salt-
solution (0.6 to 0.7 per cent, of sodium chloride in water),
which is thrown aside as soon as the dilutions are completed,
and only plates or tubes Nos. 2 and 3 are made.
The Serial Tube Method of Separation. — Another method
for the separation of bacteria and their isolation as single
colonies consists in the making of dilutions upon the surface
of solid media, such as potato, coagulated blood serum,
agar-agar, and gelatin. In pursuance of this method one
selects a number of tubes containing the medium set in a
slanting position. With a platinum needle a bit of the sub-
stance to be studied is smeared upon tube No. 1; without
sterilizing the needle it is passed in succession over the surface
of the medium in tubes Nos. 2, 3, 4, etc. When develop-
156 BACTERIOLOGY
ment has occurred essentially the same conditions as regards
separation of the colonies will be found as when plates are
poured. If a slanted medium be employed, about the most
dependent angle of which water of condensation has accu-
mulated, as blood serum, agar-agar, and potato, the dilu-
tions may be made in this fluid, and this is then to be carefully
smeared over the solid surface of the medium. The tubes
thus treated should be kept in an upright position to pre-
vent the fluid flowing over the surface. When sufficiently
developed, single colonies may be isolated with comparative
ease from tubes prepared in this manner. (See also method
for the isolation of bacillus diphtherise on blood serum.)
CHAPTER VIII.
The Incubating Oven — The Safety Burner Employed in Heating the
Incubator — Thermo-regulator — Gas-pressure Regulator.
THE INCUBATOR.
WHEN the plates have been made it must be borne in
mind that for the development of certain forms of bacteria
a higher temperature is necessary than for the growth of
others. The pathogenic or disease-producing organisms grow
more luxuriantly at the temperature of the human body
(37.5° C.) than at lower temperatures; whereas for the
ordinary saprophytic forms almost any temperature be-
tween 18° and 37° C. is suitable. It therefore becomes
necessary to provide a place in which a constant tempera-
ture favorable to the growth of the pathogenic organisms
can be maintained. For this purpose a number of different
forms of apparatus have been devised.' They are all based
upon the same principles, however, and a general description
of the essential points involved in their construction will
be all that is needed here.
The apparatus known as the incubator, or brooding-oven,
is a copper chamber (Fig. 28) with double walls, the space
between which is filled with water. The incubating-chamber
has a closely-fitting double door, inside of which is a door
of glass through which the contents of the chamber may be
inspected without actually opening it. The whole apparatus
is encased in either asbestos boards or thick felt, to prevent
radiation of heat and consequent fluctuations in teinpera-
(157)
158
BACTERIOLOGY
ture. In the top of the chamber is a small opening through
which a thermometer projects into its interior. At either
corner, leading into the space containing the water, are
FIG. 28
Incubator used in bacteriological work.
other openings for the reception of another thermometer
and a thermo-regulator, and for refilling the apparatus as
the water evaporates. On the side is a water-gauge for-
showing the level of the water between the walls. The
SAFETY BURNER
159
object of the water-chamber, which is formed by the double-
wall arrangement, is to insure, by means of the warmed
water, an equable temperature in all parts of the apparatus —
at the top as well as at the sides, back, and bottom; the
apparatus should be kept filled with water, otherwise the
purpose for which it is constructed will not be served.
When the chamber between the walls is filled with water
heat is supplied by a gas-flame placed beneath it.
FIG. 29
Koch's safety burner.
The burner employed in heating the incubator was orig-
inally devised by Koch, and is known as *' Koch's safety
burner" (Fig. 291). It is a Bunsen burner provided with
an arrangement for automatically turning off the gas-supply
1 There are now many modifications of the original form.
160 BACTERIOLOGY
and thus preventing accidents should the flame become
extinguished at a time when no one is near. The gas-cock
by which the gas is turned on and off is provided with a
long arm which is weighted, and which, when the gas is
turned on and burning, rests upon an arm attached to the
side of a revolving, horizontal disk that is connected with
the free ends of two metal spirals which are fixed by their
other ends in opposite directions on either side of the flame
and heated by it. If by draughts or any other accident
the flame becomes extinguished, the metal spirals cool, and
in cooling contract, twist the horizontal disk in the opposite
direction, and by thus removing the support allow the
weighted arm of the gas-cock to fall. By its falling the gas-
supply is turned off.
Thermo-regulators. — The regulation and maintenance of
the proper temperature within the incubator are accom-
plished by the employment of an automatic thermo-regulator
or thermostat.
The common form of thermo-regulator used for this
purpose is constructed uj>on principles involving the expan-
sion and contraction of fluids under the influence of heat
and cold. By means of this expansion and contraction the
amount of gas passing from the source of supply to the burner
may be either diminished or increased as the temperature
of the substance in which the regulator is placed either rises
or falls.
The simplest form of thermo-regulator which serves to
illustrate the principles is seen in Fig. 30. It consists of a
glass cylinder, e, having a communicating branch tube 6,
and rubber stopper /, through which projects the bent tube
a. The tube a is ground to a slanting point at the extremity
which projects into the tube e, and is provided a short dis-
THERMO-REG ULA TORS
161
FIG. 30
/
tance above this point with a capillary opening, g, in one
of its sides.
When ready for use the cylinder e is filled with mercury
up to about the level shown in the figure. It is then allowed
to stand, or is suspended, in the
bath the temperature of which it
is to regulate. The rubber tubing
coming from the gas-supply is at-
tached to the outer end of the
glass tube a, and the tube going to
the burner is slipped over the
branch tube b. The gas is turned
on and the burner lighted : and
placed under the bath. The gas
now streams through the tube a
into the cylinder e and out at b
to the burner; but as the tem-
perature of the bath rises the
mercury contained in the cylinder
e, under the influence of the ele-
vated temperature, begins to ex-
pand, and, as a continuous rise
in temperature proceeds, the expan-
sion of the mercury accompanies
it and gradually closes the slanting
opening h of tube a. In this way
the supply of gas becomes dimin-
ished and the rise in temperature of the bath will be less
rapid, until finally the opening at h will be closed entirely,
when the supply of gas to the burner will now be limited
to that passing through the capillary opening g. This is
not sufficient to maintain the highest temperature reached,
11
Mercurial thermo-regulator.
162 BACTERIOLOGY
and as cooling begins a gradual contraction of the mercury
occurs until there is again an outflow of gas from the opening
h, when the temperature again rises. This contraction and
expansion of the mercury in the regulator continues until
eventually a point is reached at which its position in the
cylinder e allows of the passage of just enough gas from the
opening h to maintain a constant temperature and, there-
fore, a constant degree of expansion of the mercury in the
tube e. This, in short, is the principle on which thermo-
regulators are constructed; but it must be borne in mind
that a great deal of detail exists in the construction of an
accurate instrument. The number of different forms of
this apparatus is comparatively large, and each form has
its special merits.
The value — that is, the delicacy — of the thermo-regulator
depends upon a number of factors, all of which it would be
useless to describe in a book of this kind; but in general
it may be said that the essential points to be observed in
selecting a thermo-regulator depend in the main upon
the temperatures at which it is to be used. For low tem-
peratures, regulators containing such fluids as ether, alcohol,
and calcium chloride solution, which expand and contract
rapidly and regularly under slight variations in temperature,
are commonly employed; whereas for temperatures approach-
ing the boiling-point of water mercury is most frequently used.
Other types of regulators operate on the principle of the
unequal expansion of different metals. Thus, if two strips
of metal having different coefficients of expansion be fixed
together, when expansion occurs, it obviously will not be
in a right line, but rather at an angle to such line. Con-
sequently if one end of such a composite rod be fixed and
the other left free, the higher the temperature the greater
THERMO-REG ULA TORS
163
will be the deflection of the free end of the rod from the
right line; the lower the temperature the less of such deflec-
tion. If now the free tip of the rod be so connected with
the gas supply that with increase of temperature the supply
is decreased and with fall of temperature increased, it is
plain that by proper adjustment the gas opening can be
FIG. 31
Moitessier's gas-pressure regulator.
brought to a point that will supply just the amount of gas
needed to maintain an approximately constant temperature.
The temperature of the incubator is to be regulated,
then, by the use of some such form of apparatus as those
just described. The regulator should be of sufficient deli-
cacy to prevent a fluctuation of more than 0.2° C. in the
temperature of the air within the chamber of the apparatus.
164 BACTERIOLOGY
Gas-pressure Regulators. — A gas-pressure regulator is some-
times intervened between the gas-supply and the thermo-
regulator. This apparatus has for its object the maintenance
of a constant pressure of the gas going to the thermo-regu-
lator. There are several forms of regulator in use, but they
do not accomplish the object for which they are designed.
The instrument most commonly employed, the apparatus
of Moitessier (Fig. 31), is based on somewhat the same
principles as the large regulators seen at the manufactories
of illuminating-gas. Such apparatus act very well when
employed on the large scale, as one sees them at the gas-
works; but when applied to the limited and sudden fluctua-
tions seen in the gas coming from an ordinary gas-cock
are practically useless. They are too gross in their con-
struction, and act only under comparatively great and
gradual fluctuations in pressure. If a good form of thermo-
regulator be employed, there is no necessity for the use of
any of the pressure-regulators thus far introduced.
CHAPTER IX.
The Study of Colonies — Their Naked-eye Peculiarities and Their Appear-
ance Under Different Conditions — Differences in the Structure of
Colonies from Different Species of Bacteria — Stab-cultures — Slant-
cultures.
THE plates of agar-agar which have been prepared from
a mixture of organisms and have been placed in the incuba-
tor, and those of gelatin which have been maintained at
the ordinary temperature of the room, are usually ready for
examination after from twenty-four to forty-eight hours.
They will be found marked here and there by small points
or little islands of more or less opaque appearance. In some
instances these will be so transparent that it is with diffi-
culty one can see them with the naked eye. Again, they
may be of a dense, opaque appearance; at one time sharply
circumscribed and round, again irregular in their outline;
here a point will present one color, there perhaps another.
On gelatin some of the points will be seen to be lying on the
surface of the medium, others will have sunk into little
depressions, while at still other points the clear gelatin will
be marked by more or less saucer-shaped pits containing
opaque fluid;
Place the plate containing these points upon the stage of
a microscope and examine them with a low-power objec-
tive, and again differences will be observed. Some of these
minute points will be finely granular, others coarsely so;
some will present a radiated appearance, while a neighbor
may be concentrically arranged; here nothing particularly
characteristic will present, there the point may resolve
itself into a mass having somewhat the appearance of a
( 165)
166 BACTERIOLOGY
little pellicle of raw cotton. All these differences, and many
more, aid us in saying that these objects must be different
in their constitution. With a pointed platinum needle take
up a bit of one of these small islands, prepare it for micro-
scopic examination (see chapter on Stained Cover-slip
Preparations), and examine it under the high-power oil-
immersion objective, with access of the greatest amount of
light afforded by the illuminator of the microscope. The
preparation will be seen to be made up entirely of bodies
of the same shape; they will all be spheres, or ovals, or
rods, but not a mixture of these forms, if proper care in
the manipulation had been taken. Examine in the same
way a neighboring spot which possesses different naked-eye
appearances, and often it will be found to consist of bodies
of an entirely different appearance from those seen in the
first preparation.
These spots or islands on the surface of the plates are
colonies of bacteria, differing severally, not only in their
gross appearances, the one from the other, but, as our cover-
slip preparations show, in the morphological characteristics
of the individual organisms composing them.
If from one of these colonies a second set of plates be
prepared, the peculiarities which were first observed in it
will be reproduced in all of the new colonies which develop;
each will be found to consist of the same organisms as the
colony from which the plates were made. In other words,
these peculiarities are constant under uniform conditions.
The appearance of the colonies developing from all organ-
isms is regulated by their location in the medium in which
they are growing. When deep down in the medium they are
usually round, oval, or lozenge-shaped; whereas when on
the surface of the gelatin or agar they may take quite a
TEST-TUBE, STAB AND SMEAR CULTURES 167
different form. This is purely a mechanical effect due to
the pressure of, or resistance offered by, the medium sur-
rounding them, and is always to be borne in mind, other-
wise false interpretations may be made.
Pure Cultures. — If from one of these small colonies a bit
be taken upon the point of a sterilized platinum needle and
introduced into a tube of sterilized gelatin or agar-agar, the
growth that results will be what is known as a "pure culture,"
the condition to which all organisms must be brought before
a systematic study of their many peculiarities is begun.
Sometimes several series of plates are necessary before the
organisms can be obtained pure, but by patiently following
this plan the results will ultimately be satisfactory.
Test-tube Cultures; Stab Cultures; Smear Cultures. — After
separating the organisms the one from the other by the
plate method just described, they must be isolated from the
plates as pure stab or smear cultures.
This is done in the following way : decide upon the colony
from which the pure culture is to be made. Select preferably
a small colony and one as widely separated from other
colonies as possible. Sterilize in a gas-flame a straight
platinum-wire needle. The glass handle of the needle should
be drawn through the flame as well as the needle itself,
otherwise contamination from this source may occur. When
it is cool, which is in five or ten seconds, take up carefully
a portion of the colony. Guard against touching anything
but the colony. If during manipulation the needle touches
anything else whatever than the colony from which the cul-
ture is to be made, it must be sterilized again. This holds
not only for the time before touching the colony, but also
during its passage into the test-tube from the colony;
otherwise there is no guarantee that the growth resulting
168 BACTERIOLOGY
from the inoculation of this bit of colony into a fresh sterile
medium will be pure.
In the meantime have in the other hand a test-tube of
sterile medium: gelatin, agar-agar, or potato. This tube is
held across the palm of the hand in an almost horizontal
position with its mouth pointing out between the thumb
and index-finger and its contents toward the body of the
worker. With the disengaged fingers of the other hand
holding the needle the cotton plug is removed from the
tube by a twisting motion and placed between the index
and second fingers of the hand holding the tube, in such a
way that the portion of the plug which fits into the mouth
of the test-tube looks toward the dorsal surface of the hand
and does not touch any portion of the hand; this is accom-
plished by placing only the overhanging portion of the plug
between the fingers. The needle containing the bit of
colony is now to be thrust into the medium in the tube if
a stab culture is desired, or rubbed gently over its surface
if a smear or stroke culture is to be made. The needle is
then withdrawn, the cotton plug replaced, and the needle
sterilized before it is laid down. Neither the needle nor its
handle should touch the inner sides of the test-tube if it
can be avoided. The tube is then labelled and set aside for
observation. The growth which appears in the tube after
twenty-four to thirty-six hours should be a pure culture of
the organisms of which the colony was composed.
Cultures of this form are not only useful as a means of
preserving the different organisms with which we may be
working, but serve also to bring out certain characteristics
of different organisms when grown in this way.
If gelatin be employed and the organism which has been
introduced into it possesses the power of bringing about
TEST-TUBE, STAB AND SMEAR CULTURES 169
liquefaction — i. e., of digesting it — it will soon be discovered
that the mode of liquefaction differs with different organ-
isms and is practically constant for the same organism.
FIG. 32
Series of stab cultures in gelatin, showing modes of growth of different
species of bacteria.
Some bacteria cause a liquefaction which spreads across the
whole upper surface of the gelatin and continues gradually
downward; with others it occurs in a funnel-shape, the
broad end of the funnel being uppermost and the point down-
170 BACTERIOLOGY
ward, corresponding to the track of the needle; at times a
stocking- or sac-like liquefaction may be noticed. (See
Fig. 32.)
NOTE. — Obtain a number of- organisms from different
sources in pure cultures by the method given. Plant them
as pure cultures, all at the same time, in gelatin — preferably
gelatin of the same making — retain them under the same
conditions of temperature, and sketch the finer differences
in the way in which liquefaction occurs.
Select from your collection a non-spore-bearing, actively
liquefying species. Cultivate it as a pure culture in nutrient
bouillon for three days. Then heat this bouillon culture to
68° C. on a water-bath for ten minutes. In the meantime
prepare several tubes containing each about 10 c.c. of:
Gelatin 7 . 00 grams
Phenol 0 . 25 gram
Water 100. 00 c.c.
Let the carbolized gelatin in one tube remain solid, and
bring that in another to a liquid state by gentle heat. On
the surface of the gelatin in the first tube place 0.5 c.c.
of the heated (and cooled) culture, and mark on the side of
the tube the point of contact between the fluid culture and
the solid gelatin. To the tube of liquefied gelatin add like-
wise 0.5 c.c. of the heated culture, mix it thoroughly with
the gelatin, and place the tube containing the mixture
in cold water until the mass becomes solid. Set both tubes
aside at a temperature not above 20° C. Note what occurs
at the end of an hour, by next day, and after three days.
Alter the experiment by filtering the three-day-old bouillon
culture through a porcelain- or a Berkefeld filter, instead of
heating it as directed above. Are the results modified?
How do you interpret these results?
CHAPTER X.
Methods of Staining — Cover-slip Preparations — Impression Cover-slip
Preparations — Solutions Employed — Preparation and Staining of
Cover-slips — Staining Solutions — Special Staining Methods.
A COMPLETE list of solutions and methods that are
recommended for the staining of bacteria is not essential
to the work of the beginner, so that only those which are of
the most common application will be given in this book.
In general, it suffices to say that bacteria stain best with
watery solutions of the basic aniline dyes, and of these,
fuchsin, gentian-violet, and methylene-blue are those most
frequently employed.
In practical work bacteria are either dried upon cover-
slips and then stained, or stained in sections of tissues in
which they have been deposited during the course of disease.
In both processes the essential point to be borne in mind
is that the bacteria, because of their microscopic dimen-
sions, require to be more conspicuously stained than the
surrounding materials upon the cover-slips or in the sec-
tions, otherwise their recognition is a matter of the greatest
difficulty, if not of impossibility. For this reason, especially
in section-staining, it frequently becomes necessary to
decolorize the tissues after removing them from the staining-
solutions, in order to render the bacteria more prominent,
and for this purpose special methods, which provide for
decolorization of the tissues without robbing the bacteria
of their color, are employed. The ordinary method of
cover-slip examination of bacteria, constantly in use in these
studies, is performed in the following way:
(171)
172 BACTERIOLOGY
COVER-SLIP PREPARATIONS.
In order that the distribution of the organisms upon the
cover-slips may be uniform and in as thin a layer as possible
it is essential that the slips should be clean and free from
grease. For cleansing the slips several methods may be
employed.
The simplest plan with new cover-slips is to immerse
them for a few hours in strong nitric acid, after which they
are rinsed in water, then in alcohol, then ether, and, finally,
they may be kept in alcohol to which a little ammonia has
been added. When about to be used they should be wiped
dry with a clean cotton or silk handkerchief.
A method commonly employed is to remove all coarse
adherent matter from slips and slides by allowing them to
remain for a time in strong nitric or sulphuric acid. They
are removed from the acid after several days, rinsed in water,
and treated as above. Knauer suggests the boiling of soiled
cover-slips and slides for from twenty to thirty minutes in
a 10 per cent, watery solution of lysol, after which they are
to be rinsed carefully in water until all trace of the lysol has
disappeared. They are then to be wiped dry with a clean
handkerchief.
Loffler's method, which provides for the complete removal
of all grease, is to warm the cover-slips in concentrated
sulphuric acid for a time and then rinse them in water,
after which they are kept in a mixture of equal parts of
alcohol and ammonia. They are to be dried on a cloth
from which all fat has been extracted.
Steps in Making the Preparations. — Place upon the center
of one of the clean dry cover-slips a very small drop of water
or physiological salt-solution. With a platinum needle,
COVER-SLIP PREPARATIONS 173
which has been sterilized in a gas-flame just before using and
allowed to cool, take up a very small portion of the colony
to be examined and mix it carefully with the drop on the
slip until there exists a very thin homogeneous film over
the larger part of the surface. This is to be dried upon the
slip by either allowing it to remain upon the table in the
horizontal position under a cover, to protect it from dust,
or by holding it between the fingers (not with forceps} , at some
distance above a gas-flame, until it is quite dry. If held
with the forceps over the flame at this stage, too much heat
may be unconsciously applied, and the morphology of the
organisms in the preparation distorted. When held between
the fingers with the thin layer of bacteria away from the flame
no such accident is likely to occur. When the whole pellicle
is completely dried the slip is to be taken up with forceps,
and, holding the side upon which the bacteria are deposited
away from the direct action of the flame, it is to be passed
through the flame three times, about a second being allowed
for each transit. Unless the preliminary drying at the low
temperature has been complete, the preparation will be
rendered worthless by the subsequent "fixing" at the higher
temperature, for the reason that the protoplasm of bacteria
when moist coagulates at these temperatures, and in doing
so the normal outline of the cells is distorted. If carefully
dried before fixing, this does not occur and the morphology
of the organism remains unchanged.
A better plan for the process of fixing is to employ a
copper plate about 35 cm. long by 10 cm. wide by 0.3 cm.
thick. This plate is laid upon an iron tripod and a small
gas-flame is placed beneath one of its extremities. By this
arrangement one can get a graduated temperature, beginning
at the part of the plate above the gas-flame where it is
174 BACTERIOLOGY
hottest, and becoming gradually cooler toward the other end
of the plate, which may be of a very low temperature. By
dropping water upon the plate, beginning at the hottest
point and proceeding toward the cooler end, it is easy to
determine the point at which the water just boils; it is at a
little below this point that the cover-slips are to be placed,
bacteria side up, and allowed to remain about ten minutes,
when the fixing will be complete. In very particular com-
parative studies this plan is to be preferred to the process
of passing the cover-slips through a flame, as the organisms
are always subjected to the same degree of heat, and the
distortions which sometimes occur from too great and
irregular application of high temperatures may be elimi-
nated. The fixing consists in drying or coagulating the
gelatinous envelope surrounding the organisms, by which
means they are caused to adhere to the surface of the cover-
slip. It is sometimes desirable to fix the preparations with-
out the use of heat, as in the case of pus or other exudates.
In this event, after drying the thinly spread material care-
fully m the air, the cover-slip on which it is placed is im-
mersed in a mixture of equal parts of absolute alcohol and
ether for about fifteen minutes. At the end of this time it
may be removed and stained. The advantage of this method
is that there is less distortion and, as a rule, less precipitation
(or, perhaps better, no charring) of extraneous matter.
The majority of bacteria with which the beginner will
have to deal stain readily with watery solutions of any of
the basic aniline dyes, such, for instance, as fuchsin, methyl-
ene-blue, or gentian-violet.
To stain the fixed cover-slip preparation, it is taken by
one of its edges between forceps, and a few drops of a watery
solution of either of the dyes named are placed upon the
COVER-SLIP PREPARATIONS 175
film and allowed to remain twenty to thirty seconds. The
slip is then carefully rinsed in water, and without drying
is placed bacteria down upon a slide; the excess of water is
taken up by covering it with blotting-paper and gently
pressing upon it, after which the preparation is ready for
examination.
Another plan sometimes used is to bring the slip upon
the slide, bacteria down, without rinsing off the staining-
fluid; the excess of fluid is removed with blotting-paper and
the preparation is ready for examination with the micro-
scope. This method is satisfactory and time-saving, but
must always be practiced with care. The staining-fluid
should always be filtered before using, to rid it of insoluble
particles which might be taken for bacteria.
If upon examination the preparation prove of particular
interest, so that it is desirable to preserve it, then it may be
mounted permanently. The drop of immersion oil is to
be removed from the surface of the slip with blotting-paper,
and the slip loosened, or rather floated, from the slide by
allowing water to flow around its edges. It is then taken
up with forceps, carefully deprived of the water adhering to
it by means of blotting-paper, and allowed to dry. When
dry it is mounted in xylol-Canada-balsam by placing a
small drop of the balsam upon the surface of the film, and
then inverting the slip upon a clean glass slide. It is some-
times desirable to have the balsam harden quickly, and a
method that is commonly employed to induce this is as
follows : the slide, held by one of its ends between the fingers,
is warmed over a gas-flame until quite hot; a drop of balsam
is then placed on the center of it, and it is again warmed;
the cover-slip is then placed in position, and when the bal-
sam is evenly distributed the temperature is rapidly reduced
176 BACTERIOLOGY
by rubbing the bottom of the slide with a towel wet with
cold water. Usually the preparation is firmly fixed after
this treatment; a little practice is necessary, however, in
order not to overheat and crack the slide. The method is
applicable only to cover-slip preparations, and cannot be
safely used with tissues.
Impression Cover-slip Preparations. — Impression prepara-
tions differ from ordinary cover-slip preparations in only
one respect: they present an impression of the organisms
as they were arranged in the colony from which the prep-
aration is made. They are made by gently covering the
colony with a thin, clean cover-slip, lightly pressing upon it,
and, without moving the slip laterally, lifting it by one of
its edges. The organisms adhere to the slip in the same
relation to one another that they had in the colony. The
subsequent steps of drying, fixing, staining, and mounting
are the same as those just given for ordinary cover-slip
preparations.
By this method constancies in the arrangement and group-
ing of the individuals in a colony can often be made out.
Some will always appear irregularly massed, others show
growth in parallel bundles, while others, again, will be seen
as lorjg, twisted threads.
NOTE. — From a colony of bacillus subtilis make a cover-
slip preparation in the ordinary way; now make an impres-
sion cover-slip preparation of another colony of the same
organism. Compare the results.
ORDINARY STAINING SOLUTIONS.
The solutions commonly employed in staining cover-slip
preparations are, as has been stated, watery solutions of
ORDINARY STAINING SOLUTIONS 177
the basic aniline dyes — fuchsin, gentian-violet, and methyl-
ene-blue. These solutions ' may be made either by directly
dissolving the dyes in substance in water until the proper
degree of concentration has been reached, or by using con-
centrated watery or alcoholic solutions of the dyes which
may be kept on hand as stock. The latter method is the
one commonly practised.
The solutions of the colors which are in constant use in
staining are prepared as follows :
Prepare as stock, saturated alcoholic or watery solutions
of fuchsin, gentian-violet, and methylene-blue. These
solutions are best made by pouring into clean bottles enough
of the dyes in substance to fill them to about one-fourth of
their capacity. Each bottle should then be filled with
alcohol or with water, tightly corked, well shaken, and
allowed to stand for twenty-four hours. If by then all the
staining material has been dissolved, more should be added,
the bottle being again shaken and allowed to stand for
another twenty-four hours; this must be repeated until
a permanent sediment of undissolved coloring matter is
seen upon the bottom of the bottle. The bottles are then
to be labelled "saturated alcoholic" or "watery" solution
of fuchsin, gentian-violet, or methylene-blue, as the case
may be. These alcoholic solutions are not directly employed
for staining-purposes.
The solutions with which staining is accomplished are
made from the stock solutions by adding 5 c.c. of the latter
to 95 c.c. of distilled water. These represent the staining
solutions in every-day use. They may be kept in bottles
supplied with stoppers and pipettes (Fig. 33), and when
used are dropped upon the preparation to be stained.
For certain bacteria which stain only imperfectly with
12
178 BACTERIOLOGY
these simple solutions it is necessary to employ agents
that will increase the penetrating action of the dyes. Ex-
perience has taught us that this can be accomplished by the
addition to the solutions of small quantities of alkaline
substances, or by dissolving the staining materials in strong
watery solutions of either aniline or carbolic acid, instead
of water— in other words, by employing special solvents
and mordants with the stains.
FIG. 33
Rack of bottles for staining solutions.
Of the solutions thus prepared which may always be
employed upon bacteria that show a tendency to stain
imperfectly, there are three in common use — Loffler's
alkaline methylene-blue solution; the Koch-Ehrlich ani-
line-water solution of either fuchsin, gentian-violet, or
methylene-blue; and Ziehl's solution of fuchsin in carbolic
acid. These solutions are as follows:
Lqffler's alkaline methylene-blue solution:
Concentrated alcoholic solution of methylene-blue 30 c.c.
Caustic potash in 1 : 10,000 solution 100 c.c.
Koch-Ehrlich aniline water solution. To about 100 c.c.
of distilled water aniline oil is slowly added, a few drops
ORDINARY STAINING SOLUTIONS 179
at the time, until the solution has an opaque appearance,
the vessel containing the solution being thoroughly shaken
after each addition. It is then filtered through moistened
filter-paper until the filtrate is clear. To 100 c.c. of the
clear filtrate add 10 c.c. of absolute alcohol and 11 c.c. of
the concentrated alcoholic solution of either fuchsin, methyl-
ene-blue, or gentian-violet, preferably fuchsin or gentian-
violet.
Ziehl's carbol-fuchsin solution:
Distilled water 100 c.c.
Carbolic acid (crystallized) ...... 5 grams
Alcohol 10 c.c.
Fuchsin in substance 1 gram
Or it may be prepared by adding to a 5 per cent, watery
solution of carbolic acid the saturated alcoholic solution of
fuchsin until a metallic luster appears on the surface of the
fluid.
The Koch-Ehrlich solution decomposes after a time, so
that it is better to prepare it fresh in small quantities when
needed than to employ old solutions. Solutions older than
fourteen days should not be used.
The three solutions just given may be used for cover-
glass preparations in the ordinary way.
In some manipulations it becomes necessary to stain the
bacteria very intensely, so that they may retain their color
when exposed to the action of decolorizing agents. These
methods are usually employed when it is desirable to deprive
surrounding objects or tissues of their color, in order that
the stained bacteria may stand out in greater contrast. It
is in these cases that the staining-solution with which the
bacteria are being treated is to be warmed, and in some
cases boiled, so as further to increase its penetrating action.
180 BACTERIOLOGY
When so treated, certain of the bacteria will retain their
color, even when exposed to very strong decolorizers. The
tubercle bacillus is distinguished from the great majority
of other bacteria by the tenacity with which it retains the
color when treated in this way; it is an organism difficult
to stain, but when once stained is equally difficult to rob of
its color.
DECOLORIZING SOLUTIONS. — As regards the employment
of decolorizing agents, it must always be borne in mind that
objects which are easily stained are also easily decolorized,
and those that can be made to take up the staining-material
only with difficulty are also very difficult to rob of their
color. The most common decolorizer in use is probably
alcohol — not absolute alcohol, but alcohol containing more
or less of water. Water alone has this property, but in a
much less degree than dilute alcohol. On the other hand,
a much more energetic decolorization than that possessed
by either alone can be obtained by alternate exposures to
alcohol and water. More energetic in their decolorizing
action than either water or alcohol are solutions of the acids.
They appear, particularly when they are alcoholic solutions,
to diffuse rapidly into tissues and bacteria and very quickly
extract the staining materials which have been deposited
there. For this reason these solutions should be employed
with much care.
Very dilute acetic acid robs tissues and bacteria of their
stain with remarkable activity; still more energetic are
solutions of the mineral acids, and particularly, as has been
said, when this action is accompanied by the decolorizing
properties of alcohol.
The acid solutions commonly employed are:
Acetic acid in from 0.1 to 5 per cent, watery solution.
STAINING OF TUBERCLE BACILLI 181
Nitric acid in from 20 to 30 per cent, watery solution.
Sulphuric acid in from 5 to 10 per cent, solution in water.
Hydrochloric acid in from 1 to 3 per cent, solution in
alcohol.
NOTE. — For details as to the technique of hardening and
cutting sections and staining bacteria in tissues, the student
is referred to Mallory and Wright's Pathological Technique.
Method of Staining the Tubercle Bacillus. — Select from the
sputum of a tuberculous subject one of the small, white,
cheesy masses which it is seen to contain. Spread this
upon a cover-slip, dry and fix it in the usual way. The slip
is now to be taken by its edge with forceps and the film
covered with a few drops of either the solution of Koch-
Ehrlich or that of Ziehl. It is then held over a gas-flame,
at first some distance away, gradually being brought nearer
until the fluid begins to boil. After it has bubbled once or
twice it is removed from the flame, the excess of stain washed
away in a stream of water, then immersed in a 30 per cent,
solution of nitric acid in water, and allowed to remain until
all color has disappeared. This takes longer in some cases
than in others. One can always determine if decolorization
is complete by washing off the acid in a stream of water.
If the preparation is still distinctly colored,, it should be
immersed again in the acid; if of only a very faint color,
it may be dipped in alcohol, again washed in water, and
stained with some contrast-color. If, for example, the
tubercle bacilli have been stained with fuchsin, methylene-
blue forms a good contrast-stain. In making the contrast-
stain the steps in the process are exactly those followed in
the ordinary staining of cover-slip preparations in general:
the slip containing the stained tubercle bacilli is carefully
rinsed in water, and a few drops of the methylene-blue
182 BACTERIOLOGY
solution placed upon it and allowed to remain for thirty
or forty seconds, when it is again rinsed in water and
examined microscopically. For this purpose of observing
the difference in behavior of the tubercle bacilli and the
other organisms present in the preparation toward this
method of staining, it is well to examine the preparation
microscopically before the contrast-stain is made; then
give it the contrast-color, and again examine. It will be
seen that before the contrast-color has been given to the
preparation the tubercle bacilli are the only stained objects
to be made out, and the preparation appears devoid of
other organisms; but upon examining it after it has received
the contrast-color a great many other organisms will appear;
these take on the second color employed, while the tubercle
bacilli retain their original color. Before decolorization
all organisms in the preparation were of the same color,
bnt during the application of the decolorizing solution all
except the tubercle bacilli gave up their color. This micro-
chemical characteristic, together with other reactions to
be described, serves to differentiate the tubercle bacillus
from other organisms with which it might be confounded.
A number of different methods have been suggested for the
staining of tubercle bacilli, but the original method as
employed by Koch is so satisfactory in its results that it
is not advisable to substitute others for it. The above differs
from the original Koch-Ehrlich method for the staining of
tubercle bacilli in sputum only in the occasional employ-
ment of Ziehl's carbol-fuchsin solution and in the method
of heating the preparation with the staining fluid upon it.
As Nuttall has pointed out, however, the strong acid
decolorizer used in this method can, with advantage, be
replaced by much more dilute solutions, as a number of the
DECOLORIZING SOLUTIONS 183
bacilli are entirely decolorized by the too energetic action
of the strong acids. He recommends the following method
of decolorization : after staining the slip or section in the
usual way, pass it through three alcohols; it is then to be
washed in a solution composed of
Water ..150 c.c.
Alcohol 50 c.c.
Concentrated sulphuric acid 20 to 30 drops
From this it is removed to water and carefully rinsed.
The remaining steps in the process are the same as those
given in the other methods.
GABBETT'S METHOD for the staining of tubercle bacilli
recommends itself because of its simplicity and the rapidity
with which it can be performed. By many it is considered
the best method for routine employment. It consists in
staining the cover-slips, prepared in the manner given, for
from two to five minutes in a cold carbol-fuchsin solution,
after which they are subjected to the action of Gabbett's
methylene-blue sulphuric acid solution. This latter con-
sists of
Sulphuric acid (strength 25 per cent.) . . 100 c.c.
Methylene-blue, in substance 1 to 2 grams
The. cover-slips are then rinsed in water and are ready
for examination. The tubercle bacilli will be stained red
by the fuchsin, while all other bacteria, cell-nuclei, etc.,
will be tinted blue.
Pappenheim's Decolorizer and Counter Stain. — As with the
Gabbett method, the cover-slips are stained for from 5 to
10 minutes in cold carbol-fuchsin. They are then rinsed
in water and kept, until they are of a pale blue color, in a
decolorizing and counter-staining fluid made as follows:
184 BACTERIOLOGY
To 100 c.c. of a saturated alcoholic solution of methylene
blue add 1 gram of rosolic acid and 20 c.c. of glycerine.
The bacilli are stained red, the balance of the field blue.
Gram's Method. — Another important differential method
of staining which is very commonly employed is that recom-
mended by Gram. In this method the objects are treated
with an aniline-water solution of gentian-violet made after
the formula of Koch-Ehrlich. After remaining in this for
two or three minutes they are immersed in a solution com-
posed of
Iodine 1 gram
Potassium iodide . . . . . . . . . 2 grams
Distilled water . . . . . ..... . 300 c.c.
In this they remain for about five minutes; they are then
transferred to 95 per cent, alcohol and thoroughly rinsed.
This method is particularly useful in demonstrating the
capsule which is seen to surround some bacteria, especially
micrococcus lanceolatus of pneumonia.
After such treatment certain species of bacteria are found
to be of a very dark purple color, while all else in the prepa-
ration is decolorized; other species lose their color entirely
in the process. Those that retain the dark stain are com-
monly denominated as " Gram-positive" while those that
lose their color are known as "Gram-negative." While the
majority of bacteria are either definitely positive or negative
to this reaction, there are a few species that are indeter-
minate in this particular, that is to say, they become partly
decolorized and one cannot say certainly that they are
either positive or negative. Under certain conditions of
cultivation, and especially under conditions favorable to
degenerative changes, some species that are normally
SPECIAL STAINING 185
"Gram-positive" may in part or wholly lose their "Gram-
positive" properties.
Two theories, one chemical the other physical, have been
offered in explanation of the mechanism of the Gram method
of staining. In the chemical theory it is believed that,
through the intervention of the iodine, the gentian-violet
is linked inseparably to the protoplasm of "Gram-positive"
bacteria and is not so linked in the "Gram-negative" species.
The physical theory assumes differences in permeability
of either the bacterial envelope or the bacterial protoplasm.
In those species that are highly permeable the precipitation
resulting from the interaction between the iodine and the
gentian-violet occurs so deeply within the bacterial structure
that it is not readily washed out by the final alcohol bath,
this would be the case with the "Gram-positive" species;
while in the case of the "Gram-negative" species, assumed
to be less permeable, the precipitation is upon their sur-
faces and is readily removed by the final rinsing in alcohol.
Glacial Acetic Acid Method. — Another method that may
be employed for demonstrating the presence of the cap-
sule surrounding certain organisms is to prepare the
cover-slips in the ordinary way, then cover the layer
of bacteria upon them with glacial acetic acid, which
is instantly poured off (not washed off with water), and
the aniline-water gentian-violet solution dropped upon
them; this is allowed to remain three or four minutes, is
poured off, and a few drops more are added, and lastly the
slip is washed in a solution of sodium chloride of from 0.6
to 0.7 per cent, in strength; but at times it must be stronger,
occasionally as concentrated as 1.5 to 2 per cent. The reason
for this is that if the slips be washed in water, or in salt-
solution that is too weak, the mucin capsule that has been
186 BACTERIOLOGY
coagulated by the acetic acid is redissolved and rendered
invisible. This does not occur when the salt-solution is
of the proper strength — a point that can be determined only
after a few trials with solutions of different strengths.
(Welch.) A very clear, sharply cut picture usually follows
this method of procedure.
Ribbert also recommends for the staining of capsulated
bacteria the momentary immersion of the cover-slips in
a saturated solution of dahlia in a mixture of 100 parts of
water, 50 parts of alcohol, and 12 J parts of glacial acetic
acid; after which the excess of color is removed by washing
in water.
Staining of Spores. — We have learned that one of the
points by which spores may be recognized is their refusal
to take up staining substances when applied in the ordinary
way. They may, however, be stained by special methods;
of these, one that has given fairly satisfactory results in
our hands is as follows: the cover-slip is to be prepared from
the material containing the spores in the ordinary way,
dried, and fixed. It is then to be held by its edge with
forceps, and its surface covered with Loffler's alkaline
methylene-blue solution. It is then held over the Bunsen
flame until the fluid boils; it is then removed, and after a
few seconds is heated again. This is continued for about
one minute, after which it is washed in water and then
decolorized in
Alcohol (80 per cent.) 98 c.c.
Nitric acid 2 c.c.
until all visible blue color has disappeared. It is then rinsed
in water and dipped for from 3 to 5 seconds in
Saturated alcoholic solution of eosin 10 c.c.
Water . .... 90 c.c.
FLAGELLAR STAINING 187
after which it is again rinsed in water and finally mounted
for examination. If the decolorization in the acid alcohol
be not carried too far, the preparation will show the spores
stained blue and the bodies of the cells to have taken on
the rose color characteristic of eosin.
By another process the cover-slip is floated, bacteria
down, upon the surface of freshly prepared Koch-Ehrlich
solution of fuchsin contained in a watch-crystal. This is
then held by its edge with forceps and moved up and down
over a small Bunsen flame until the fluid boils gently. This
is continued for 2 or 3 minutes. When the fluid has stood
for about five minutes after boiling the preparation is trans-
ferred, without washing in water, to a second watch-crystal
containing the following decolorizing solution :
Absolute alcohol 100 c.c.
Hydrochloric acid 3 c.c.
In this solution it is placed, bacteria up, and the vessel is
tilted from side to side for about one minute. It is then
removed, washed in water, and stained with the cold
methylene-blue solution. The spores will be stained red
and the body of the cells blue.
It must be remembered that there are conspicuous dif-
ferences in the behavior of spores of different bacteria to
staining methods and of the spores of a single species in
different stages of development. Some stain readily by either
of the methods especially devised for this purpose, while
others can hardly be stained at all, or only with the greatest
difficulty, by any of the known processes; some stain
readily when fully developed, but with difficulty when
only partly developed; others have this peculiarity reversed.
Lbffler's Method for Staining Flagella. — For the demon-
stration of the locomotive apparatus possessed by motile
188 BACTERIOLOGY
bacteria we are indebted to Loffler. By a special method
of staining, in which the use of mordants played the essen-
tial part, he has shown that these organisms possess very
delicate, hair-like appendages, by the lashing movements
of which they propel themselves through the fluid in which,
they are growing. The method as given by Loffler is as
follows:
It is essential that the bacteria be evenly and not too
numerously distributed upon the cover-slip. The slips must
therefore be perfectly clean. (See Loffler1 s method of clean-
ing cover-slips.) Five or six of the carefully cleansed cover-
slips are to be placed in a line on a table, and on the center
of each slip a very small drop of tap-water is placed. From
the culture to be examined a minute portion is transferred
to the first slip and carefully mixed with the drop of water;
from this mixture a small portion is transferred to the
second, and from the second to the third slip, and so on,
in this way insuring a dilution of the number of organisms
present in the preparations. These slips are then dried and
fixed in the ordinary way. They are next to be warmed in
the following solution:
Tannic acid solution in water (20 acid, 80 water) . 10 c.c.
Cold saturated solution of ferrous sulphate ... 5 c.c.
Saturated watery or alcoholic solution of fuchsin . 1 c.c.
This solution represents the mordant. A few drops of
it are to be placed upon the film of bacteria on the cover-
slip, which is then to be held over a flame until the solution
begins to steam. It should not be boiled. After steaming,
the mordant is washed off in water and finally in alcohol.
The bacteria are then to be stained in a saturated aniline-
water-fuchsin solution.
There are several points and slight modifications in con-
FLAGELLAR STAINING 189
nection with this method that require to be emphasized in
order to insure success: the culture to be employed should
be young, not over 18-20 hours old; it should have developed
for this time on fresh agar-agar at 37° to 38° C. ; the mordant
should not be perfectly fresh, as the best results are obtained
from the use of old solutions that have stood exposed to the
air and that have been filtered just before using; when
placed on the cover-slip and held over the flame never heat
the mordant to the boiling-point; 'indeed, the best results are
obtained when the preparation is held high above the flame
and removed from it at the first evidence of vaporization, or,
better still, a little before this point is reached.1
Duck-wall's Method2 is a modification of the Loffler method,
and the results obtained thereby are very satisfactory.
Preparation of the Staining Agents. — The fixing agent is
mordant, and the stain is carbol-gentian-violet or, prefer-
ably, carbol-fuchsin.
The Mordant.
Desiccated tannic acid 2 grams
Cold saturated solution ferrous sulphate (aqueous) 5 grams
Distilled water 12 c.c.
Saturated alcoholic solution of fuchsin .... 1 c.c.
The tannic acid is dissolved in the water first by the
application of gentle heat, then the ferrous sulphate, and
then the alcoholic solution of fuchsin are added. To these
ingredients it is advisable to add from 0.5 to 1 c.c. of a
1 per cent, sodium hydroxide solution. The best grade of
filter-paper is used for filtering the mordant, and there
should be left a heavy precipitate. After filtering, the color
1 1 am indebted to Dr. James Homer Wright, Thomas Scott Fellow in
Hygiene, 1892-1893, University of Pennsylvania, for some of the suggestions
in connection with the modification of this method.
2 The Canner, vol. xx, p. 23.
190 BACTERIOLOGY
of this mordant should be of a reddish-brown hue, not clear,
but somewhat cloudy, and this mordant must be used within
five hours after it is made. After that time it loses its fixing
power. This is indicated by its gradual clarification and
darkened color. It gives the best results when strictly
fresh, and accomplishes its work in a much shorter time, so
that very little if any heating is required when it is placed
on the cover-glass preparation.
The Stain. — To prepare the dye for this method take
about 1 gram of ordinary granulated fuchsin, put it in a
bottle, and pour over it about 25 c.c. of warm, absolute
alcohol. Shake vigorously and let it stand for several
hours before using. The carbol-fuchsin is made by diluting
the saturated alcoholic solution four or five times with a
5 per cent, solution of carbolic acid. Carbol-fuchsin should
be freshly made, heated, and filtered before using.
The application of this method of demonstrating the
flagella varies with different organisms with regard to the
length of time the mordant and stain are allowed to act,
and the amount of sodium hydroxide solution used. Usually,
it is well to heat the mordant on the cover-slip to steaming,
and allow it to act from one-half to one minute. It is then
washed off with water and a small quantity of alcohol
poured over the surface and washed off instantly. The
water on the cover-slip is now absorbed from the edge of
the cover-slip with clean filter-paper. The carbol-fuchsin
stain is now applied and heated just enough to generate a
thin vapor. The stain should not act for more than from
one-half to one minute. The cover-slip is now dried, then
xylol is poured over the surface, the excess being removed
with filter-paper. The cover-slip is now mounted in xylol
balsam.
CHAPTER XL
Systematic Study of an Organism — Points to be Considered in Determin-
ing the Morphologic and Biologic Characters of a Culture — Methods
by Which the Various Biologic and Chemical Characters of a Culture
may be Ascertained — Dark Field Illumination — Facts Necessary to
Permit the Identification of an Organism as a Definite Species.
AFTER isolating an organism in pure culture by the plate
method, considerable work is necessary in order to estab-
lish its identity. Small portions of the pure culture are
taken upon the point of a sterile platinum wire and trans-
planted into the various culture media. These sub-cultures
of the organism are then placed under suitable conditions
of temperature and environment, and examined from day
to day to note the alterations that occur in the different
media. In the systematic study of an organism no one
character can be relied upon to the exclusion of others.
It is necessary to note the microscopic appearance of the
individual organism and its behavior toward different
staining solutions and other reagents; in addition it is
necessary to note the gross appearance of the culture of
the different media as shown by naked-eye (macroscopic)
examination as well as under a lens of low magnifying
power (microscopic); while equal importance must be given
to the chemical alterations produced by the bacteria in the
different media, and the influence of different reagents,
when added to the media, to show the presence of certain
metabolic products. In this manner the entire life history
of an organism, outside the animal body, may be ascertained.
(191)
192 BACTERIOLOGY
The different characters of an organism may be grouped
as: (a) morphologic, those ascertained by examination of
the individual organism under a lens of high magnifying
power; (b) biologic, those ascertained by macroscopic and
microscopic study of the gross appearance of the culture
in the different media; (c) biochemic, the alterations pro-
duced in the different media as shown by direct examination
or by the use of different reagents; and (d) pathogenic,
the effects of the inoculation of the culture into susceptible
animals.
SCHEME or STUDY. — Record the source whence the
organism was derived. Was this the normal habitat of the
organism, or was it present accidentally?
MORPHOLOGIC CHARACTERS.
Note the shape, size, and grouping of the organism as it
occurs in the different media. Observe the nature of the
ends of the individual organism. Determine the presence
or absence of motility in very young cultures. If motility
is observed, apply one of the special methods for demon-
strating flagella to note their relative number and location
and do not be discouraged if your first attempts fail. Stain
your cultures by means of the different staining solutions,
and note the effect of each. Do the organisms stain deeply
and uniformly, or are they stained in a peculiar manner?
Apply the Gram method of staining, and note whether or
not the organisms are decolorized by the alcohol. Stain
the organisms deeply with carbol-fuchsin staining solution,
and note the effect of different decolorizing agents; and
ascertain whether the organisms are capable of resisting the
decolorizing effects of dilute acids. Do the organisms show
BIOLOGIC CHARACTERS 193
the presence of a capsule when taken from the blood or
tissues of an animal, or when taken from cultures in milk
or blood-serum ? Examine cultures that are several days old,
and note whether spores are being formed. Note particularly
the position of the spore within the cell. Is the spore of
smaller or greater diameter than the cell in which it is
forming? Examine cultures that are a week or more old,
and note whether the organisms have undergone any definite
alterations in form (involution forms), or whether they
present evidences of fragmentation or granulation of their
protoplasm (degeneration forms) .
BIOLOGIC CHARACTERS.
Colony Formation. — Observe the character of the colonies
formed in gelatin and agar-agar plates. Describe a typical
surface colony and a typical deep colony, both as to their
macroscopic and microscopic appearance. What is the
relative size of the colonies formed on each of these media
when they are sufficiently separated from one another to
permit unhindered development? Note the color and inter-
nal structure of the colonies as well as their relative density.
What is the nature of the surface contour and arrangement
of the colonies? Note their general character, as to whether
they are moist or dry, compact or loosely constructed,
sharply circumscribed or spreading over the surface of the
medium. Do the gelatin colonies show evidences of lique-
faction?
Agar-slant Inoculations.— Observe the nature of the growth
on the surface of an agar-agar-slant inoculation. Describe
the color, texture, and optical characters of the growth. r Is
the growth confined to the line of inoculation, or has it a
13
194 BACTERIOLOGY
tendency to spread over the surface of the medium? Is
it smooth or rough, moist or dry, glistening or dull in
character? If the organism forms pigment, note whether
the pigment is confined to the area of growth or whether it
extends into the medium itself. Record the manner in
which the culture changes in its appearance on successive days.
Agar-stab Inoculations. — Observe the nature of the growth
in an agar-agar-stab inoculation. Note whether the growth
is most voluminous at or near the surface or in the depth
of the stab. If the organism produces pigment, note whether
the pigment formation is most marked at or near the surface
or at the bottom of the stab. Record the alterations that
are observed on several successive days.
Gelatin-stab Inoculations. — Observe the nature of the
growth in a gelatin-stab inoculation. Is the growth most
voluminous at or near the surface or at the bottom of the
stab? Note the general character of the growth on the
surface, especially as to its contour, extent, and color. Note
the character of the growth in the stab. Is it continuous
along the whole line of inoculation, or is it confined to
isolated areas? If the organism has the property of liquefy-
ing gelatin, note carefully the manner in which the lique-
faction proceeds. How soon does liquefaction begin, and
in what length of time is a tube of gelatin completely
liquefied?
Potato Culture. — Observe the nature of the growth on
potato. This is an important differential medium, since
some organisms grow upon it very sparingly or indeed
almost invisibly. Other organisms grow very character-
istically. Some organisms have the property of breaking
up the starch of the potato into simpler compounds. This
is sometimes accompanied by the evolution of gas. Many
BIOLOGIC CHARACTERS 195
of the chromogenic bacteria find the potato a most suitable
pabulum on which to form their pigment, the pigment
formed on this medium having at times an especial bril-
liancy. Note in detail all the changes that occur in the growth
on successive days.
Growth in Bouillon. — Observe whether the fluid shows
turbidity or not, as well as the extent and distribution of
this alteration. Note whether any sediment is being formed,
as well as the nature and amount of such sediment. Does
the organism form a definite growth (pellicle or scum) on
the surface of the bouillon? What is the character of the
pellicle? Is it readily dislodged, and, when dislodged, is
it replaced by a new pellicle? Note whether the color of
the medium has become altered. Note the manner in which
the appearance of the culture changes on several successive
days.
Growth in Litmus-milk. — Observe the nature of the growth
in litmus-milk. Has the reaction of the medium become
altered? To what is such alteration attributable? Note
whether there is precipitation of casein. Record the extent
and rapidity with which this alteration takes placej as well
as the reaction of the fluid while the change is being pro-
duced. Is there any evidence of the subsequent liquefaction
of the precipitated casein? Has the litmus been altered in
any manner except as shown by altered reaction of the
medium? In what part of the tube has such alteration of
the litmus commenced? If the litmus has been decolorized,
is it possible to restore its color by the admixture of air with
the fluid? Note the order in which the appearance of the
medium ch'anges on successive days.
Growth in Special Media. — The special culture media may
be employed to ascertain additional biologic characters of
196 BACTERIOLOGY
an organism, such as the production of indol, reduction of
nitrates to nitrites, the formation of ammonia, production
of gas in media containing different carbohydrates, or the
reducing power of the organism on aniline dyes, etc.
Influence of External Agencies. — Note the vitality of the
organism under the influence of various physical and
chemical agents. Determine the temperature at which it
thrives best, as well as the lowest and highest temperatures
at which growth is possible. Determine the thermal death-
point of the organism by subjecting it to various degrees
of temperature from 55° to 75° C. for ten minutes. Deter-
mine its resistance to drying; to the influence of light; to
the influence of germicidal substances. Determine the
influence of different gases upon the growth of the organisms,
such as hydrogen, nitrogen, or carbon dioxide. Determine
the chemical reaction of the culture media best adapted for
its growth.1
BIOCHEMIC CHARACTERS.
If the organism exhibits chromogenic properties, ascertain
whether the pigment is intra- or extracellular. Ascertain
under what conditions of temperature, reaction, and con-
stitution of media, or under what atmospheric conditions
this function is best exhibited. Note the influence of dif-
ferent reagents upon the pigment, such as chloroform, ether,
alcohol, water, acids, or alkalies. Note whether the organism
exhibits photogenic properties, and if so, ascertain what
conditions are most suitable for the manifestation of this
phenomenon.
1 For more detailed description of the variations in the character of
the macroscopic and microscopic appearance of the cultures in the different
media, and for commonly employed terminology the student is referred to
Chester's Determinative Bacteriology and Eyre's Bacteriologic Technique,
PATHOGENIC PROPERTIES 197
Ascertain whether the organism produces enzymes. Does
it manifest a proteolytic function, as shown by the
liquefaction of gelatin, casein, or blood serum? Note
whether this function is manifested in alkaline or in acid
condition of the medium. Does it manifest a precipitating
effect (rennet ferment?) upon casein? Note whether this
is manifested in alkaline or in acid condition of the
medium. Does the organism have the property of breaking
up any of the carbohydrates into simpler compounds? Is
this alteration accompanied or not by the liberation of gas?
If so, ascertain the relative amount of gas formed from a
given quantity of carbohydrate. Analyze the gas formed,
and state the relative proportion of carbon dioxide and
residual (explosive) gas formed.
Ascertain whether the organism produces indol. Is this
substance formed with the simultaneous reduction of
nitrates to nitrites? Are the nitrites reduced further into
ammonia ?
PATHOGENIC PROPERTIES.
Ascertain whether any of the animals used for experi-
mental purposes are susceptible when inoculated with the
organism. Are all species of laboratory animals equally
susceptible, or are some immune? Note the size of the
dose and the manner of inoculation that gives the most
constant and characteristic results. What are the symp-
toms and postmortem appearances produced? What is the
location of the organisms in the body of the dead animal ?
Are they confined to the seat of inoculation, or are they
distributed more or less generally throughout the body?
Note whether the virulence of the organism is maintained
198 BACTERIOLOGY
when grown for several generations on artificial media,
or whether it soon becomes attenuated. Which culture-
medium is best suited to conserve the virulence of the
organism? In what manner does its environment influence
the virulence? If the virulence is readily lost, may it be
regained by any of the known methods?
Ascertain whether the organism forms a soluble toxin
when grown in fluid media, as sugar-free bouillon. If
toxin is formed, ascertain whether the antitoxic state is
readily induced in susceptible animals.
If no soluble toxin is formed, ascertain whether animals
may be immunized by the injection of sub-lethal doses
of dead or living cultures. Is a bactericidal immunity
induced by this means? Does the serum of immune animals
possess protective and curative properties when adminis-
tered to susceptible animals before or after inoculation
with the living organism? Does the serum of immune
animals possess the property of agglutinating the organ-
isms in relatively higher dilutions than the serum of normal
animals of the same species?
The majority of the bacteria may be identified without
resorting to such a detailed study of the biochemic and
pathogenic properties as given in the foregoing outline, but
for some of the pathogenic bacteria it has been necessary
to apply all the known tests in order to definitely establish
their identity. By means of such detailed studies on related
organisms, it. has been possible to differentiate varieties
whose characters are constant, yet in general they are so
closely related that it is impossible from the clinical mani-
festations produced to state definitely which particular
variety of organism is responsible for the conditions.
VARIATIONS AND VARIETIES 199
VARIATIONS AND VARIETIES.
As in the case of all other living things, bacteria are modi-
fied by their environment. Such modifications manifest
themselves in various ways. In some instances they are
degenerative, involving alterations in form and function
that are easily detected by appropriate methods of exami-
nation. Often such changes are but transitory and are
referable to the influences of well-known causes, the removal
of which permits the bacteria to resume their normal state.
(See Involution Forms.) In other instances more or less
prolonged environmental influences, of which we know but
little, appear to have brought about alterations in function
with no appreciable changes in form. Sometimes the one
or the other of such modifications may be brought about
at will by appropriate experimental methods.
From the early days of modern bacteriology confusion
has arisen at times in connection with the establishment of
definite species.
It was frequently found that among the species, as deter-
mined by methods then available, individual members of
a species would exhibit variations in particular functions
that differentiated them from the accepted type. Some-
times these differences were morphological, more often they
were physiological. Occasionally they could be detected by
crude culture methods then in vogue — more often — as the
studies progressed, they were demonstrable only by more
refined special methods. To recall this confusion one need
but mention the marked functional variations of Bacillus
coli communis and the striking morphological differences
seen in Bacillus diphtherise.
By the discovery of methods better suited to bring out
200 BACTERIOLOGY
finer differences — notably, those that took into considera-
tion the zymogenic powers of bacteria — it was soon possible
to speak of groups, strains, or types among the species, one
strain or group differing from another in its ability to ferment
certain carbohydrates, with recognizable end-products, while
other strains were devoid of this power, though in all other
particulars the two strains may have been identical. By
the application of such tests many species have been sepa-
rated into groups and some groups into sub-groups; some
fermenting all sugars, others fermenting only particular
sugars. Some fermenting with free gases as an end-product
others with no gas but only acids as end-products. These
functions are subject to quantitative variations and in a
few cases they may temporarily disappear, and occasionally
by experimental methods, may be made to disappear, but
as yet it has not been possible by any known method to
endow a species, by nature devoid of the power to ferment
sugar, with such power.
The ability of a pathogenic species to cause in animals
pathological lesion identical to those from which the species
was obtained was held for a long time as the test par excel-
lence for the identification of pathogenic species. Accord-
ing to the standards then in common use two cultures from
different cases of the same disease may have been identical
in all other particulars, yet if one was capable of reproducing
in animals lesions similar to those in man from which that
culture was obtained and the other was devoid of that
power, they were generally regarded as two distinct species.
To illustrate we have only to recall the confusion that
existed in connection with the diphtheria bacillus and the
several "pseudo" diphtheria bacilli that were described.
We now know that variations in pathogenic power is
VARIATIONS AND VARIETIES 201
one of the commonest phenomena noticed among disease-
producing bacteria. And we also know that by artificial
procedures many of the highly; pathogenic organisms may
be in part or wholly deprived of their powers to cause the
lesions peculiar to the activities of the normal organisms,
retaining at the same time all other peculiarities common to
the species.
The manifold studies upon infection and immunity have
placed at our disposal methods by which it is possible to
detect differences among closely related types of the same
species that cannot be revealed in any other way.
Such differences appear to be idioplasmic, if the word is
appropriate to bacteria, and though slight quantitative
fluctuations may be noted, the strains characterized by them
have them as fixed, inherent peculiarities.
We may regard them therefore not as indicating modi-
fications of a component common to the species, but rather
as specific components possessed by some varieties of a
species and not by others; not necessarily as new characters
evolving from environmental influences, insofar as can be
determined, but as natural, fundamental components revealed
only by newer adequate methods of investigation.
Through the use of certain immunologic methods or tests
it is now possible to subdivide most of the pathogenic species
into distinct sub-groups — the one group differing from the
other only in the presence or absence of those components
necessary to complete the differential reactions.
To make this clear: If one immunize an animal from
Bacillus typhosus the blood serum of such animal if brought
together with Bacillus typhosus causes the bacilli to gather
in distinct clumps, whereas, if such serum be brought in
contact with Bacillus coli communis, or with Bacillus dysen-
202 BACTERIOLOGY
terise, both closely related to Bacillus typhosus, no such
clumping will occur.
Again, if we mix with that serum typhoid bacilli derived
from any number of cases of typhoid fever the serum will
cause clumping of some cultures and not others, even though
all cultures came from individuals having the same disease
and by the common tests all are alike.
We note here a high degree of specificity — not only a
specificity peculiar to certain species but likewise peculiar
to certain individual members of the same species. We are
justified then in concluding that, from the standpoint of
this test, all the cultures of typhoid bacilli that were clumped
by the serum used were of the same strain or type as that
used in immunizing the animal; while all those that did not
clump with the same serum were of different strains or
types. If we now immunize an animal from anyone of
this latter group of typhoid bacilli we shall find that the
serum will cause clumping of the bacilli in the culture used
for immunization and will probably react in the same
manner with some of the other cultures — but not with all,
nor with the cultures embraced by our first group. Thus,
we will have established at least two groups, or types that
have distinct, specific serologic reactions, and so we may
go on and perhaps establish additional groups.
This reaction commonly known as the "agglutination
reaction" is invaluable in the efforts to assemble species into
groups and subgroups or types specifically different — the
one from the other insofar as the reaction goes.
If by this procedure we find that two groups — A and B of an
infective species — can be established and with the members of
Type A we render an animal so highly immune that its serum
may be expected to possess curative properties for the disease
VARIATIONS AND VARIETIES . 203
caused by the bacterial species under consideration, we may
find it to be curative or preventive for all Type A infections and
not at all so or only lowly so for Type B infections, and
vice versa. In other words those specific components of the
members of Type A which are revealed by the agglutination
test may indicate the possession by Type A organisms of com-
ponents that call forth the elaboration by the tissues of the
immunized animal of bodies that neutralize only the poisons
of the bacteria of Type A — that is, of the particular type
from which the animal was rendered immune.
This is a point of fundamental value in connection with
the use of antisera for the cure of infections. For the best
results such antisera must be homologously related to the
"type" organisms concerned in the infection for which it
is to be employed.
As the result of all this, bacteriologists are today concern-
ing themselves more with groups and group reactions than
with individual species and their peculiarities; that is to say
the "typing" of bacteria has become one of the routine
operations in bacteriological laboratories, in consequence we
speak of the "colon type," the "dysenteric type," the
" pneumococcus type," etc., meaning that the particular
organism or culture with which we are dealing is a species
belonging to one or the other "type" as determined by its
agglutinability, and it may or may not conform to all the
other reactions by which the so-called typical species is
identified. •
Working from such a standpoint we now know that
many of the important disease-producing organisms lend
themselves to such grouping, and by the adoption of this
plan of work it has been possible to make advances and to
interpret phenomena otherwise impossible.
204 BACTERIOLOGY
By using the agglutination test we now know that the
organisms causing pneumonia may be definitely subdivided
into at least four groups; that the pathogenic streptococci,
fall into at least two, possibly four groups; that several
strains of dysenteric bacilli and at least two strains of
meningococci have been demonstrated. And throughout we
find a constant specific relationship between strains and
their homologous antibodies. To repeat: This is a matter
of the greatest practical moment, for the serum from an
animal that is immune from the members of one group, while
it may possess high potency if used against infections caused
by members of that group, may be of but little or no value
if used in the treatment of infections caused by members of
another closely related group. (See " Pneumococcus " and
"Streptococcus" paragraphs on variations.)
MICROSCOPIC EXAMINATION OF PREPARATIONS.
The Different Parts of the Microscope. — Before describing
the method of examining preparations microscopically, a
few definitions of the terms used in connection with the
microscope may not be out of place. (The different parts
of the microscope referred to below are indicated by letters
in Fig. 34.)
The ocular or eye-piece (A) is the lens at which the eye is
placed when looking through the instrument. It serves to
magnify the image projected through the objective.
The objective (B) is the lens which is at the distal end of
the barrel of the instrument, and which serves to magnify
the object to be examined.
The stage (c) is the shelf or platform of the microscope on
which the object to be examined rests.
MICROSCOPIC EXAMINATION OF PREPARATIONS 205
The diaphragms are the perforated stops that fit in the
center of the stage. They vary in size, so that different
FIG. 34
— G
amounts of light may be admitted to the object by using
diaphragms with larger or smaller openings.
206 BACTERIOLOGY
The "iris" diaphragm (D) opens and closes like the iris
of the eye. It is so arranged that its opening for admission
of light can be increased or diminished by moving a small
lever in one or another direction.
The reflector (E) is the mirror placed beneath the stage,
which serves to illuminate the object to be examined.
The coarse adjustment (F) is the rack-and-pinion arrange-
ment by which the barrel of the microscope can be quickly
raised or lowered.
The fine adjustment (G) serves to raise and lower the
barrel of the instrument very slowly and gradually.
For the microscopic study of bacteria it is essential that
the microscope be provided with an oil-immersion system
and a sub-stage condensing apparatus.
The oil-immersion or homogeneous system consists of an
objective so constructed that it can only be used when the
transparent media through which the light passes in enter-
ing it are all of the same index of refraction — i. e., are
homogeneous. This is accomplished by interposing between
the face of the lens and the cover-slip covering the object
to be examined a body which refracts the light in the same
way as do the glass slide, the cover-slip, and the glass of
which the objective is made. For this purpose, a drop of
oil of the same index of refraction as the glass is placed upon
the face of the lens, and the examinations are made through
this oil. There is thus little or no loss of light from deflec-
tion, as is the case in the dry system.
The sub-stage condensing apparatus (H) is a system of
lenses situated beneath the central opening of the stage.
They serve to condense the light passing from the reflector
to the object in such a way that it is focussed upon the
object, thus furnishing the greatest amount of illumination.
MICROSCOPIC EXAMINATION OF PREPARATIONS 207
Between the condenser and reflector is placed the "iris"
diaphragm, the aperature of which can be regulated, as
circumstances require, to permit of either a very small or
a very large amount of light passing to the object.
The nose-piece (i) consists of a collar, or group of collars
joined together (two or more), that is attached to the distal
end of the tube of the microscope. It enables one to attach
several objectives to the instrument in such a way that by
simply rotating the nose-piece the various lenses of different
power may be conveniently used in succession.
Dark-field Illumination. — This refers to a result obtained
through the use of an apparatus that so deflects and reflects
the light's rays that the field is dark and the objects in it
brilliantly light. It is used only for the examination of
unstained living objects and is capable of revealing the
most minute particles and microorganisms. It is especially
useful for the study of the normal morphology and move-
ment of spirochete and trypanosomes and for the detection
of bodies so small or otherwise so constituted as not to be
visible by the ordinary methods of microscopic examination.
Two forms of the illuminator are in use — one that slips into
the collar ordinarily carrying the sub-stage condensing
apparatus, the other is made in the form of a slide and is
placed on the stage directly over the opening for illumina-
tion. Both provide for the complete cutting off of direct
central rays of light, allowing only the lateral rays to reach
the objects and be reflected by them to the eye. Both require
very intense illumination for the best results. This may be
obtained from a Welsbach burner, or a small arc or incan-
descent light. In both cases the light's rays must be
condensed upon the reflector of the microscope by means
of a condensing lens.
208 BACTERIOLOGY
Microscopic Examination of Cover-slips. — The stained cover-
slip is to be examined with the oil-immersion objective,
and with the diaphragm of the sub-stage condensing appara-
tus open to its full extent. The object gained by allowing
the light to enter in such a large volume is that the contrast
produced by the colored bacteria in the brightly illuminated
field is much more conspicuous than when a smaller amount
of light is thrown upon them. This is true not only for
stained bacteria on cover-slips, but likewise for their differen-
tiation from surrounding objects when they are located in
tissues. With unstained bacteria and tissues, on the con-
trary, the structure can best be made out by reducing the
bundle of light-rays to the smallest amount compatible with
distinct vision, and in this way favoring, not color-contrast,
but contrasts which appear as lights and shadows, due to the
differences in permeability to light of the various parts of
the material under examination.
Steps in Examining Stained Preparations with the Oil-immer-
sion System. — Place upon the center of the cover-slip which
covers the preparation a small drop of immersion oil. Place
the slide upon the center of the stage of the microscope.
With the coarse adjustment lower the oil-immersion objec-
tive until it just touches the drop of oil. Open the illumi-
nating apparatus to its full extent. Then, with the eye
to the ocular and the hand on the fine adjustment, turn the
adjusting screw toward the right until the field becomes
somewhat colored in appearance. When this is seen pro-
ceed more slowly in the same direction, and, after one or
two turns, the object will be in focus. Do not remove the
eye from the instrument until this has been accomplished.
Then, with one hand upon the fine adjustment and the
thumb and index finger of the other hand holding the slide
MICROSCOPIC EXAMINATION OF PREPARATIONS 209
lightly by its end, it may be moved about under the objec-
tive. At the same time the screw of the fine adjustment
must be turned back and forth, so that the different planes
of the preparation may be brought into focus one after the
other. In this way the whole section or preparation may
be inspected. When the examination is finished raise the
objective from the preparation by turning the screw of the
coarse adjustment toward you. Remove the preparation
from the stage, and, with a fine silk cloth or handkerchief,
wipe very gently and carefully the oil from the face of the lens.
During work, of course, the lens need not be cleaned and
put away after each examination; but when the work for
the day is over an immersion lens is best protected in this
way. Under no circumstances should it be allowed to remain
in the immersion oil or vexposed to dust for any length of
time.
Examination of Unstained Preparations. — "Hanging drops."
It frequently becomes necessary to examine bacteria in the
unstained condition. The circumstances calling for this
arise while studying the multiplication of cells, the germina-
tion of spores, and the absence or presence of motility.
In this method the organisms to be studied are suspended
in a drop of physiological salt solution or of bouillon, or a
tiny drop of either agar-agar or gelatin, inoculated with
the organism, may be employed. The drop is placed in the
center of a clean cover-slip which has been sterilized in the
flame and which is then inverted over the depression in
a sterilized so-called "hollow-ground" slide to which it is
sealed with vaseline. A convenient and quick method of
making the preparation is, after placing the drop in the
center of the cover-slip, to invert over it the slide, around
the depression in which a ring of vaseline has been painted.
14
210 BACTERIOLOGY
The slip adheres and the preparation may then be handled
without fear of disturbing the drop or the position of the
slip over the depression. When completed it has the appear-
ance shown in Fig. 35. The drop hangs in an air-tight
chamber so that both evaporation and contamination are
prevented.
This is known as the "hanging-drop" method of exami-
nation or cultivation. It is indispensable for the purposes
mentioned, and at the same time requires considerable care
in its manipulation. The fluid is so transparent that the
cover-slip may be broken by the objective being brought
down upon the preparation before one is aware that the
focal distance has been reached. This may be avoided by
FIG. 35
Longitudinal section of hollow-ground glass slide for observing bacteria in
hanging drops.
bringing the edge of the drop into the center of the field with
one of the higher power dry lenses. When this is accomplished
substitute the immersion for the dry system, when the edge
of the drop is readily detected with the higher power lens
somewhere near the centre of the field.
In examining bacteria by this method there is a possibility
of error that must be guarded against. All microscopic
insoluble particles in suspension in fluids possess a peculiar
tremor or vibratory motion, the so-called "Brownian
motion." This is very apt to give the impression that the
organisms under examination are motile, when in truth they
are not so, their movement in the fluid being only this
molecular tremor.
MICROSCOPIC EXAMINATION OF PREPARATIONS 211
The difference between the motion of bodies undergoing
this molecular tremor and that possessed by certain living
bacteria is that the former particles never move from their
place in the field, while living motile bacteria alter their
position in relation to the surrounding organisms, and may
dart from one position in the field to another. In some cases
the true movement of bacteria is very slow and undulating,
while in others it is rapid and darting. The molecular
tremor may be seen with non-motile and with dead organisms.
NOTE. — Prepare three hanging-drop preparations — one
from a drop of dilute India-ink, a second from a culture of
micrococci, and a third from a culture of the bacillus of
typhoid fever. In what way do they differ?
Study of Spore Formation. — The hanging-drop method just
mentioned is not only employed for detecting the motility
of an organism, but also for the study of its mode of spore-
formation.
Since with aerobic organisms spore formation occurs, as
a rule, only in the presence of oxygen, and is induced more
by limitation of the nutrition of the organisms than by any
other factor, it is essential that these two points should be
borne in mind in preparing the drop cultures in which the
process is to be studied. For this reason the drop of bouillon
should be small and the air chamber relatively large.
A very thin drop of sterilized agar-agar may be substi-
tuted for the bouillon. » It serves to retain the organisms in
a fixed position, and the process may be more easily followed.
. As soon as finished the preparation is to be examined
microscopically and the condition of the organisms noted.
It is then to be retained in a warm chamber, and kept under
212 BACTERIOLOGY
continuous observation. The form of chamber best adapted
to the purpose is one which envelops the whole microscope.
It is provided with a window through which the light enters,
and an arrangement by which the slide may be moved from
the outside. The formation of spores requires a much
longer time than the germination of spores into bacilli, but
with patience both processes may be satisfactorily observed.
It will be noticed that the description of this process is
very much like that which immediately precedes, but differs
from it in one respect, viz., that in this manipulation we
are not making a preparation which is simply to be ex-
amined and then thrown aside, but it is an actual pure
culture, and must be kept as such, otherwise the observa-
tion will be worthless. For this reason the greatest care
must be observed in the sterilization of all objects employed.
Studies upon spore formation by this method frequently
continue over hours, and sometimes days, and contamina-
tion must, therefore, be carefully guarded against. The
study should be begun with the vegetative form of the
organisms; the hanging-drop preparation should, for this
reason, always be made from a perfectly fresh culture of
the organism under consideration before time has elapsed
for spores to form.
The simple detection of the presence or absence of spore
formation can in many cases be made by other methods.
For example, many species of bacteria which possess this
property form spores most readily upon media from which
it is somewhat difficult for them to obtain the necessary
nourishment; potatoes and agar-agar that have become
a little dry offer very favorable conditions, because of the
limited area from which the growing bacteria can draw their
nutritive supplies, and because of the free access which they
MICROSCOPIC EXAMINATION OF PREPARATIONS 213
have to oxygen, for, their growth being on the surface, they
are surrounded by this gas unless means are taken to prevent
it. By the hanging-drop method, however, more than -this
specific property may be determined. It is possible not only
to detect the stages and steps in the formation of endogenous
spores, but when the spores are completely formed their
germination into mature rods may be seen by transferring
them to a fresh bouillon-drop or drop of agar-agar preserved
in the same way. The word rods is used because we have as
yet no evidence that, endogenous spore-formation occurs
in any of the other morphological groups or bacteria.
Hanging-block Cultures. — Hill1 has devised a method for
observing the development of individual bacteria, which
consists in the substitution for the ordinary "hanging drop"
of liquid or jelly a cube of solidified agar-agar, on the surface
of which the bacteria are distributed.
The "hanging block" i's prepared as follows: "Pour
melted nutrient agar into a Petri dish to the depth of one-
eighth to one-quarter inch. Cool this agar and cut from it
a block about one-quarter to one-third inch square and of
the thickness of the layer of agar in the dish. This block has
a smooth upper and under surface. Place it, under surface
down, on a slide and protect it from dust. Prepare an
emulsion in sterile water of the organism to be examined if
it has been grown on a solid medium, or use a broth culture;
spread the emulsion or broth upon the upper surface of the
block, as if making an ordinary cover-slip preparation.
Keep the slide and block in an incubator at 37° C. for five
to ten minutes to dry slightly. Then lay a clean sterile
cover-slip on the inoculated surface of the block in close
contact with it, avoiding, if possible, the formation of air-
1 Journal of Medical Research, 1902, vol. vii, p. 202.
214 BACTERIOLOGY
bubbles. Remove the slide from the lower surface of the
block, and invert the cover-slip so that the agar-block is
uppermost. With a platinum loop run a drop or two of
melted agar along each side of the agar block where it is
in contact with the cover-slip. This seal hardens at once,
preventing slipping of the block. Place the preparation in
the incubator again for five or ten minutes to dry the agar
seal. Invert this preparation over a moist chamber and seal
the cover-slip in place with white wax or paraffin. Vaseline
softens too readily at 37° C., allowing shifting of the cover-
slip. The preparation may then be examined at leisure."
Aerobic bacteria receive sufficient oxygen by diffusion,
and for anaerobic bacteria it will suffice to hang the block
in a chamber containing a little alkaline pyrogallic acid solu-
tion. This absorbs all oxygen.
Study of <Jelatin Cultures. — As has been previously stated,
the behavior of bacteria toward gelatin differs — some of
them producing apparently no alteration in the medium,
while the growth of others is accompanied by an enzymotic
action that results in liquefaction of the gelatin at and
around the place at which the colonies are growing. In
some instances this liquefaction spreads laterally and down-
ward, causing a saucer-shaped excavation; while in others
the colony sinks almost vertically into the gelatin and may
be seen lying at the bottom of a funnel-shaped depression.
These differences are constantly employed as one of the
means of differentiating otherwise closely allied species and
varieties. (See Fig. 32.) Studies upon the spirillum of
Asiatic cholera and a number of kindred species, for
example, reveal decided differences in the form of lique-
faction produced by these various organisms. The minutest
detail in this respect must be noted, and its frequency or
constancy under varying conditions determined.
CHANGES IN THE REACTION OF MEDIA 215
Cultures on Potato. — A useful factor in the identification
of an organism is its growth on sterilized potato. Many
organisms present appearances under this method of cul-
tivation which alone can almost be considered characteristic.
In some cases coarsely lobulated, elevated, dry or moist
patches of development occur after a few hours; again, the
growth may be finely granular and but slightly elevated
above the surface of the potato; at one time it will be dry
and dull in appearance, again it may be moist and glisten-
ing. Sometimes bubbles, due to the fermentative action of
the growing bacteria on the carbohydrates of the potato,
are produced.
A most striking form of development on potato is that
often exhibited by the bacillus of typhoid fever and the
bacillus of diphtheria. After inoculation of a potato with
either of these organisms there is usually no naked-eye
evidence of growth, though microscopic examination of
scrapings from the surface of the potato reveals an active
multiplication of the organisms which had been planted
there. The potato is one of the first of the differential media.
CHANGES IN THE REACTION OF MEDIA AS A RESULT
OF BACTERIAL ACTIVITY.
For purposes of differentiation, much stress is laid upon
the reaction assumed by media as a result of bacterial
growth. Under the influence of certain species the medium
will become acid, under that of others it is alkaline, while
some cause little or no change. In media of particular
composition — i. e., those containing traces of fermentable
carbohydrates, notably muscle-sugar, as seen in infusions
of fresh meat — the reaction may become acid with the begin-
216 BACTERIOLOGY
ning of growth and subsequently change to alkaline after
the supply of fermentable sugar is exhausted. These changes
of reaction are most conveniently observed through the use
of indicators — bodies that either lose or change their usual
color as the reaction of the medium to which they are added
changes.
Such substances as litmus, in the form of the so-called
"litmus tincture," and coralline (rosolic acid) in alcoholic
solution, have been commonly employed for this purpose,
though many other indicators having definite ranges of
usefulness are now being employed. (See paragraphs on
Reaction.) They may be added to the media in the pro-
portions given in the chapter on Media, and the changes in
their colors studied with different bacteria. Milk and litmus
tincture or peptone solution to which rosolic acid has been
added are excellent media for this experiment.
Fermentation. — The production of gas as an indication of
fermentation is an accompaniment of the growth of certain
bacteria. This is best studied in media to which 1 to 2 per
cent, of grape-sugar (glucose) has been added. A convenient
method of demonstrating this property is to employ a tube
about half full of agar-agar containing the necessary amount
of grape-sugar. The medium is to be liquefied on a water-
bath, and then cooled to about 42° C., when a small quantity
of a pure culture of the organism under consideration should
carefully be distributed through it. The tube is then placed
in ice-water and rapidly solidified in the vertical position.
When solid it is placed in the incubator. After twenty-four
to thirty-six hours, if the organism possesses the property
of causing fermentation of glucose, the medium will be
dotted everywhere with very small cavities containing the
gas that has resulted.
CHANGES IN THE REACTION OF MEDIA 217
This property of fermentation with evolution of gas is of
such importance as a differential characteristic that con-
siderable attention has been given to it, and those who have
been most intimately concerned in the development of our
knowledge on the subject do not consider it sufficient to
say that the growth of an organism "is accompanied by the
production of gas-bubbles/' but that under given condi-
tions we should determine not only the amount of gas or
gases produced by the organism under consideration, but
also their nature. For this purpose, Smith1 recommends the
employment of the fermentation tube. This is a tube bent
at an acute angle, closed at one end and enlarged with a bulb
at the other. At the bend the tube is constricted. To it
a glass foot is attached so that the tube may stand upright.
(See Fig. 36.) To fill the tube, the fluid (it is used only with
fluid media) is poured into the bulb until this is about half
full. The tube is then tilted until the closed arm is nearly
horizontal, so that the air may flow out into the bulb and the
fluid flow into the closed arm to take its place. When this
has been completely filled sufficient fluid should be added
to bring its level within the bulb just beyond the bend, and
the opening of the bulb plugged with cotton. The tubes thus
filled are then to be sterilized. During sterilization they
are to be maintained in the upright position. Under the
influence of heat the tension of the water-vapor in the closed
arm forces most of the fluid into the bulb. As the tube cools,
the fluid returns to its place in the closed arm and fills it
again, with the exception of a small space at the top, which
is occupied by the air originally dissolved in the liquid and
1 An excellent and exhaustive contribution to this subject has been
made by Theobald Smith in the Wilder Quarter-Century Book, Ithaca,
N. Y., 1803.
218 BACTERIOLOGY
which has been driven out by the heat. The air-bubble
should be tilted out after each sterilization; and finally,
after the third exposure to steam, this arm of the tube will
be free from air. The medium employed is bouillon con-
taining some fermentable carbohydrate, as glucose, lactose,
or saccharose. After inoculation the flasks are placed in the
incubator, and the amount of gas that collects in the closed
FIG. 36
Fermentation tube.
arm is noted from day to day. From studies that have been
made this gas is found to consist usually of about one part
by volume of carbonic acid and two parts by volume of an
explosive gas consisting largely of hydrogen. For deter-
mining the nature and quantitative relations of these gases
Smith1 recommends the following procedure: "The bulb
1 Loc. cit., p. 196.
CHANGES IN- THE REACTION OF MEDIA 219
is completely filled with a 2 per cent, solution of sodium
hydroxide (NaOH) and closed tightly with the thumb.
The fluid is shaken thoroughly with the gas and allowed to
flow back and forth from bulb to closed branch and the
reverse several times, to insure intimate contact of the CO2
with the alkali. Lastly, before removing the thumb all the
gas is allowed to collect in the closed branch, so that none may
escape when the thumb is removed. If C02 be present,
a partial vacuum in the closed branch causes the fluid to
rise suddenly when the thumb is removed. After allowing
the layer of foam to subside somewhat the space occupied
by gas is again measured, and the difference between this
amount and that measured before shaking with the sodium
hydroxide solution gives the proportion of CO2 absorbed.
The explosive character of the residue is determined as
follows: the thumb is placed again over the mouth of
the bulb and the gas from the closed branch is allowed
to flow into the bulb and mix with the air there present.
The plug is then removed and a lighted match inserted
into the mouth of the bulb. The intensity of the explosion
varies with the amount of air present in the bulb."
Durham's Fermentation Tube. — Durham employs a con-
venient modification of the ordinary fermentation tube,
which is constructed in the following manner: test-tubes
of about 10 or 12 c.c. capacity are placed in an inverted
position within a larger test-tube, and the latter plugged
with cotton in the usual way and sterilized. (See Fig.
37.) The small tube should fit loosely within the larger
one. The medium to be used is run into the larger tube
until there is present about 50 per cent, more than the
volume of the smaller tube. The whole is then steri-
lized in streaming steam by the fractional method. After
220
BACTERIOLOGY
FIG. 37
the first sterilization the small tube will be found almost
filled with fluid, over which a small air-bubble lies. After
the second or third sterilization this
air-bubble is completely expelled, and
the small tube contains nothing but
the liquid.
The medium that Durham employs
for the fermentation test is a 1 per
cent, solution of Witte's peptone in
distilled water, to which have been
added known amounts of some such
fermentable sugar as glucose, saccha-
rose, lactose, mannite, etc., as the case
may demand. He prefers peptone to
meat-infusion bouillon for the reason
that the latter often contains traces
of muscle-sugar, and is thereby likely
to complicate the results. He prefers
neutralization with organic acids rather
than mineral acids, and uses citric acid
by preference, the reason for this being
that where sugars such as those men-
tioned are acted upon by mineral
acids under the influence of heat their
composition is apt to be altered.
Durham's fermentation
tube.
NOTE. — Prepare two fermentation
tubes as follows: Fill one with 1 per
cent, watery solution of peptone to
which 2 per cent, of glucose has been
added; fill the other with a similar peptone solution, but
to which only 0.3 per cent, of glucose has been added.
CHANGES IN THE REACTION OF MEDIA 221
Sterilize and inoculate with Bacillus coli communis. How
do the two tubes differ from one another after eighteen
to twenty-four hours in the incubator? First, as regards
the reaction of the fluid in the open arms of the tubes.
Second, as to accumulation of gas in closed arms of the
tubes. Third, as to the capacity of each solution for
reducing copper in Fehling's solution. What differences
are observed, and how may they be explained?
Indol Production. — The detection of products other than
those that give rise to alterations in the reaction of the
media, and whose presence may be demonstrated by chemical
reactions, is a routine step in the identification of different
species of bacteria. Among these bodies is one that is pro-
duced by a number of organisms, and whose presence may
easily be detected by its characteristic behavior when
treated with certain substances. I refer to nitroso-indol,
the reactions of which were described by Beyer in 1869,
and the presence of which as a product of the growth of
certain bacteria has since furnished a topic for considerable
discussion.
Indol, the name by which this body is generally known,
when acted upon by reducing agents becomes of a more
or less decided rose color. This body was recognized as
one of the products of growth of the spirillum of Asiatic
cholera first by Poel, and a short time subsequently by
Bujwid and by Dunham, and for a time was believed to be
peculiarly characteristic of the growth of this organism.
It has since been found that there are many other bacteria
which also possess the property of producing indol in the
course of their development. It is constantly present in
putrefying matters, and is one of the aromatic compounds
that give to feces their characteristic odor.
222 BACTERIOLOGY
The methods employed for its detection are as follows:
cultivate the organism for twenty-four to forty-eight hours
at a temperature of 37° C., in the simple peptone solution
known as "Dunham's solution" (see formula for this
medium). This solution is preferred because its pale color
does not mask the rose color of the reaction when the
amount of indol present is very small.
Four tubes should always be inoculated and kept under
exactly the same conditions for the same length of .time.
At the end of twenty-four or forty-eight hours the test
may be made. Proceed as follows: to a tube containing
7 c.c. of the peptone solution, but which has not been inocu-
lated, add 10 drops of concentrated sulphuric acid. To
another similar tube add 1 c.c. of a 0.01 per cent, solution
of sodium nitrite, and afterward 10 drops of concentrated
sulphuric acid. Observe the tubes for five to ten minutes.
No alteration in their color appears, or at least there is no
production of a rose color. They contain no indol.
Treat in the same way, with the acid alone, two of the
tubes which have been inoculated. If no rose color appears
after five or ten minutes, add 1 c.c. of the sodium nitrite
solution. If now no rose color is produced, the indol reac-
tion may be considered as negative — i. e., no indol has been
formed as a product of the growth of the bacteria.
If indol is present, and the rose color appears after the
addition of the acid alone, it is plain that not only indol
has been formed, but coincidently a reducing-body. This is
found, by proper means, to be nitrous acid. The sulphuric
acid liberates this acid from its salts and permits of its
reducing action being brought into play.
If the rose color appears only after the addition of both
the acid and the nitrite solution, then indol has been formed
during the growth of the organisms, but no nitrites.
CHANGES IN THE REACTION OF MEDIA 223
Control the results obtained by treating the two remaining
cultures in the same way.
The test is sometimes made by allowing concentrated
sulphuric acid to flow down the sides and collect at the
bottom of the tube; the reaction is then seen as a rose-
colored zone overlying the line of contact of the acid and
culture medium. This method is open to the objection that,
if indol is present in only a very small amount, the faint rose
tint produced by it is apt to be masked by a brown color
that results from the charring action of the concentrated
acid on the other organic matters in the culture medium,
so that its presence may in this way escape detection. In
view of this, Petri recommends the use of dilute sulphuric
acid. He states that when indol is present the characteristic
rose color appears a little more slowly with the dilute acid,
but it is more permanent, and there is never any likelihood of
its presence being masked by other color reactions.
Muir and Ritchie recommend the use of ordinary fuming
or yellow nitric acid for this test. In this method two or
three drops of the acid are added to the culture under con-
sideration. If indol be present, the red color appears as a
result of the reducing action of the nitrous acid upon it.
The defect in this method is that it reveals only the presence
of indol, and fails to indicate whether or not reducing bodies
were coincidently formed with the indol. As a test for indol
alone it is convenient and entirely trustworthy.
Reducing Power of Bacteria. — The power to reduce chemical
compounds from a higher to a lower state may be said to
be common to all bacteria. In some bacteria, perhaps the
majority, it is most conspicuously manifested in connection
with substances containing sulphur, hydrogen sulphide being
formed. In other bacteria it is best seen in connection with
224 BACTERIOLOGY
the alterations produced in certain pigments, as litmus,
methylene-blue, indigo, etc., the normal color disappearing
in part or entirely according to the nature and activity of
the process. Other bacteria have the property of reducing
certain salts, as in the reduction of nitrates to nitrites, or
even to ammonia by the denitrifying bacteria. In some
instances these reductions result from the fact that the
bacteria liberate hydrogen from the compounds, in others
it results from the fact that the bacteria abstract oxygen
from such compounds, while in still other instances the
reduction is of a more complex nature. Each of these
changes, therefore, indicates the nature of some of the
metabolic activities manifested by the bacteria in question.
Test for Hydrogen Sulphide. — The reduction of sulphur
compounds may be determined by growing the bacteria in
peptone solution containing ferric tartrate, when the presence
of hydrogen sulphide will be indicated by the brownish-
black or jet-black color of the precipitated iron-sulphide.
Reduction of Nitrates. — The complete reduction of nitrates
is brought about by many bacteria. Other bacteria are
capable of carrying the reducing action as far as the for-
mation of ammonia, while still others merely reduce the
nitrates to nitrites. These reducing functions are encour-
aged and may be demonstrated by cultivating the bacteria
in peptone solution containing potassium nitrate.
Test for Nitrites. — The method of Griess, as modified by
Ilosvay, is quite satisfactory. These reagents are required:
(a) Naphthylamine 0.1 gram
Distilled water 20.0c.c.
Acetic acid (25 per cent, solution) . . . 150 . 0 c.c.
(6) Sulfanilic acid 0.5 gram
Acetic acid (25 per cent, solution) . . . 150.0 c.c.
CHANGES IN THE REACTION OF MEDIA 225
In preparing solution a the naphthylamine is dissolved in
20 c.c. of boiling water, filtered, allowed to cool, and mixed
with the dilute acetic acid. Solutions a and b are then mixed.
It is best prepared as needed, though it may be preserved
for some time in a glass-stoppered bottle.
In testing for nitrites the reagent is added in the proportion
of one volume of reagent to five volumes of culture. When
nitrites have been formed a deep-red color appears in a few
seconds. If no nitrites have been formed the culture remains
colorless. In testing cultures it is always necessary to control
the results by blank tests on a portion of the same medium
that had not been inoculated, as some of the ingredients of
the medium may have contained nitrites.
Another test for the formation of nitrites is a mixture of
starch and potassium iodide, as follows:
Starch 2.0 grams
Potassium iodide, 0.5 gram
Water 100.0 c.c.
Warm the mixture until the starch is completely dis-
solved.
In testing for nitrites add 0.5 c.c. of the reagent to a tube
of culture, and follow this by the addition of 2 or 3 drops
of pure sulphuric acid. If nitrites have been formed, a
dark-blue or purple color will appear. Control-tubes of the
medium show no color reaction, or merely a trace of blue
coloration.
Test for Ammonia. — The formation of ammonia may be
detected by testing with Nessler's reagent. The most satis-
factory results are obtained by cultivating the organisms
in a liter of culture fluid and then distilling off portions of
the culture, collecting in Nessler tubes, and applying 1 c.c.
of the reagent to each 50 c.c. of the distillate. The presence
15
226 BACTERIOLOGY
of ammonia in the distillate is shown by the yellow coloration
resulting from the addition of the reagent.
The direct application of the reagent to the culture will
give satisfactory results if a great deal of ammonia has been
formed. In this instance the mercury in the reagent will
be precipitated as mercurous oxide. Another rough test for
the formation of ammonia is to place a strip of filter-paper —
moistened with the Nessler reagent — over the mouth of a
test-tube containing the culture, and then gently heating
the culture. As the ammonia is driven off by the heat, it
will react on the reagent on the strip of paper.
Examination of Cultures for Bacterial Toxins.— In the sys-
tematic study of a pathogenic organism it is necessary to
know whether it is capable of producing a soluble toxin
when growing in culture media. This is done by filtering
cultures of various ages and testing the effect of the filtrate
upon susceptible animals.
FILTRATION OF CULTURES. — A variety of filters have been
devised for the purpose of filtering liquid cultures and other
fluids to obtain sterile filtrates. These filters are usually
constructed of unglazed porcelain or of infusorial earth, and
are made in the form of hollow cylinders or bulbs. The best-
known forms of bacterial filters are the Chamberland and
the Berkefeld. All the filters used for this purpose require
some motive power to force the fluid through the filter.
Compressed air may be employed to force the fluid through
the filter, or atmospheric pressure may be utilized by creating
a negative pressure on the distal side of the filter by the use
of an air-pump.
It is always necessary to test the sterility of the filtrate
by making cultures from it into nutritive media and noting
whether growth takes place or not.
ANAEROBIC METHODS 227
Cultivation without Oxygen. — As we have already learned,
there is a group of bacteria to which the designation "anae-
robic" has been given, which are characterized by inability
to grow in the presence of free oxygen. For the cultivation
of the members of this group, a number of devices are
employed for the exclusion of free oxygen from the cultures.
Method of Buchner. The plan suggested by Buchner, of
allowing the cultures to develop in an atmosphere robbed of
its oxygen by pyrogallic acid, gives very good results. In
this method the culture, which is either a slant- or stab-
culture in a test-tube, is placed — tube, cotton plug, and all —
into a larger tube, in the bottom of which have been deposited
1 gram of pyrogallic acid and 10 c.c. of yV normal caustic-
potash solution. The larger tube is then tightly plugged
with a rubber stopper. The oxygen is quickly absorbed
by the pyrogallic acid, and the organisms develop in the
remaining constituents of the atmosphere, viz., nitrogen, a
small amount of CO2, and a trace of ammonia.
Method of C. Frdnkel. Carl Frankel suggested the fol-
lowing : the tube is first inoculated as if it were to be poured
as a plate or rolled as an ordinary Esmarch tube. The cotton
plug is then replaced by a rubber stopper, through which
pass two glass tubes. These must all have been sterilized
in the steam sterilizer before using. On the outer side of
the stopper these two tubes are bent at right angles to the
long axis of the test-tube into which they are to be placed,
and both are slightly drawn out in a gas-flame. Both of
these tubes must be provided, before sterilization, with
a plug of cotton; this is to prevent the access of foreign
organisms to the medium during manipulations. At the
inner side of the rubber stopper — that is, the end which is
to be inserted into the test-tube — the glass tubes are of
228
BACTERIOLOGY
different lengths: one reaches to within 0.5 cm. of the bottom
of the test-tube, the other is cut off flush with the under
surface of the stopper. The outer end of the longer glass
tube is then connected with a hydrogen generator and
hydrogen is allowed to bubble through the gelatin (Fig. 38, A)
FIG. 38
Franke!' s method for the cultivation of anaerobic bacteria.
in the tube until all contained air has been expelled and
.its place taken by the hydrogen.1 When the hydrogen has
1 Before beginning the experiment it is always wise to test the hydro-
gen— i. e., to see that it is free from oxygen and that there is no danger
of an explosion, for unless this be done the entire apparatus may be blown
to pieces and a serious accident occur. The agents used should be pure
zinc and pure sulphuric acid of about 25 to 30 per cent, strength. With the
primary evolution of the gas the outlet of the generator should be closed
and kept closed until the gas reservoir is quite filled with hydrogen. The
ANAEROBIC METHODS 229
been bubbling through the gelatin for about five minutes
(at least) one can be reasonably sure that all oxygen has
been expelled. The drawn-out portions of the tubes can
then be sealed in the gas-flame without fear of an explosion.
The protruding end of the rubber stopper is then painted
around with melted paraffin and the tube rolled in the way
given for ordinary Esmarch tubes. A tube thus prepared
and containing growing colonies is shown in Fig. 38, B.
The development that now occurs is in an atmosphere of
hydrogen, all oxygen having been expelled. During the
operation the tube containing the liquefied gelatin should
be kept in a water-bath at a temperature sufficiently high
to prevent its solidifying, and at the same time not high
enough to kill the organisms with which it has been inocu-
lated.
One of the obstacles to the successful performance of this
method is the bubbling of the gelatin, the foam from which
will often fill the exit-tube and sometimes be forced from it.
This may be obviated by reversing the order of proceeding,
viz.: roll the Esmarch tube in the ordinary way with the
organisms to be studied, using a relatively small amount of
gelatin, so as to have as thin a layer as possible when it is
rolled. Then replace the cotton plug with the sterilized
outlet should then be opened and the entire volume of gas allowed to escape,
care being taken that no flame is in the neighborhood. This should be
repeated, after which a sample of the hydrogen generated should be collected
in an inverted test-tube in the ordinary way for collecting gases over water, ,
viz., by filling a test-tube with water, closing its mouth with the thumb,
inverting it, and placing its mouth under water, when, after removing the
thumb, the water will be kept in it by atmospheric pressure. The hydrogen
which is flowing from the open generator may be conducted to the test-
tube by rubber tubing. When the water has been replaced test the gas
by holding a flame near the open mouth of the test-tube. If no explo-
sion occurs, the hydrogen is safe to use. Should there be an explosion, the
generation of hydrogen must be continued in the apparatus until it burns
with a colorless flame when tested in a test-tube.
230 BACTERIOLOGY
rubber stopper carrying the glass tubes through which the
hydrogen is to be passed, and allow the hydrogen to flow
through as in the method first given. The gas now passes
over the gelatin instead of through it, and consequently no
bubbling results. In all other respects the procedure is the
same as that given by Frankel.
Method of Kitasato and Weil. — For favoring anaerobic
conditions Kitasato and Weil have suggested the addition
to the culture media of some strong reducing-agent. They
recommend formic acid or sodium formate, in 0.3 to 0.5 per
cent.; glucose in 1.5 to 2 per cent.; or blue litmus tincture
in 5 per cent, by volume. This is, of course, in addition to
an atmosphere from which all oxygen has been expelled.
As a reducing agent for this purpose, Theobald Smith regards
a weaker solution of glucose, 0.3 to 0.5 per cent., as more
advantageous; and Wright obtains better results when
glucose is added if the primary reaction of the media is about
neutral to phenolphthalein.
Method of Park. A very simple, convenient, and effi-
cient method is employed by Park. It consists in covering
the medium in which the anaerobic species are to be cul-
tivated with liquid paraffin (albolene). The best results
are obtained when the amount of paraffin added is about
half that of the liquid in the tube or flask. The liquid paraffin
has the advantage over the solid paraffin in not retracting
from the walls of the vessel on cooling. All air is expelled
from flasks or tubes prepared in this way, by heating them
in the autoclave. The layer of paraffin prevents the reab-
sorption of oxygen driven off by the heat. After cooling, the
inoculation is made by passing the needle through the paraffin
well down into the media.
Many other methods are employed for this special purpose,
but for the beginner those given will suffice.
ANAEROBIC METHODS 231
From what has been said, it may be inferred that the cul-
tivation of anaerobic bacteria is a simple matter attended
with but little difficulty. Such an opinion will, however,
be quickly abandoned when the beginner attempts this part
of his work for the first time, and particularly when his
efforts are directed toward the separation of these forms from
other organisms with which they are associated. The
presence of spore-forming, facultative anaerobes in mixed
cultures is always to be suspected, and it is this group that
renders the task so difficult. At best the work requires undi-
vided attention and no small degree of skill in bacteriological
technique.
CHAPTER XII.
Inoculation of Animals — Subcutaneous Inoculation — Intravenous Injec-
tion— Inoculation into the Lymphatic Circulation — Inoculation into
the Great Serous Cavities, and into the Anterior Chamber of the Eye-
Observation of Animals after Inoculation.
AFTER subjecting an organism to the methods of study
that we have thus far reviewed there remains to be tested
its action on animals — i. e., to determine if it possesses the
property of producing disease or not; and, if so, what are
the pathological results of its growth in the tissues of animals,
and in what way must it gain entrance to the tissues in
order to produce those results? The mode of deciding these
points is by inoculation, which is practised in different ways
according to circumstances. Most commonly a bit of the
culture to be tested is simply deposited beneath the skin of
the animal; but in other cases it may be necessary to intro-
duce it directly into the vascular or lymphatic circulation,
or into one or the other of the great serous cavities; or,
for still other purposes of observation, into the anterior
chamber of the eye, upon the iris or within the skull cavity,
upon the dura or brain substance.
SUBCUTANEOUS INOCULATION OF ANIMALS.
The animals usually employed in the laboratory for pur-
poses of inoculation are white mice, gray house-mice, guinea-
pigs, rabbits, and pigeons.
For simple subcutaneous inoculation the steps in the
(232)
SUBCUTANEOUS INOCULATION OF ANIMALS 233
process are practically the same in all cases. The hair or
feathers are to be carefully removed. If the skin is very
dirty, it may be scrubbed with soap and water. Sterili-
zation of the skin is practically impossible, so it need not be
attempted. If the inoculation is to be made by means of a
hypodermic syringe, then a fold of the skin may be lifted
up and the needle inserted in the usual way. If a solid
culture is to be inoculated, a fold of skin may be taken up
with forceps and a tiny pocket cut into it with scissors
which have previously been sterilized. This pocket must
be large enough to admit the end of the needle without its
touching the sides of the opening as it is inserted. Beneath
the skin will be found the superficial and deep connective-
tissue fascise. These must be taken up with sterilized
forceps, and with sterilized scissors incised in a way corre-
sponding to the opening in the skin. The pocket is then to
be held open with the forceps and the substance to be
inserted is introduced as far under the skin and fasciae as
possible, care being taken not to touch the edges of the
wound if it can be avoided. The edges of the wound may
then be simply pulled together and allowed to remain. No
stitching or efforts at closing it are necessary, though a drop
of collodion over the point of operation may serve to lessen
contamination.
As the subcutaneous inoculation is very simple and takes
only a few moments, guinea-pigs, rabbits, and pigeons may
be held by an assistant. The front legs in the one hand
and the hind legs in the other, with the animal stretched
upon its back on a table, is the usual position for the opera-
tion when practised upon guinea-pigs and rabbits. The
point at which the inoculations are commonly made is in
the abdominal wall, either to the right or left of the median
234 BACTERIOLOGY
line and about 3 cm. distant. When pigeons are used they
are held with the legs, tail, and ends of the wings in the one
hand, and the head and anterior portion of the body in the
other, leaving the area occupied by the pectoral muscles,
over which the inoculation is to be made, free for manipu-
lation. In the case of fur-bearing animals the hair over the
point selected for the inoculation should be closely cut with
scissors, and from a small area the feathers should be plucked
in the case of birds.
FIG. 39
Kitasato's mouse-holder.
It is at times, however, more convenient to dispense with
an assistant; one of several forms of apparatus that have
been devised for holding mice, guinea-pigs, rats, rabbits,
etc., may then be used. For small animals, such as mice and
rats, the holder suggested by Kitasato is very useful. It
is simply a metal plate attached to a stand by a clamped
ball-and-socket joint, so that it can be fixed in any position.
It is provided with a spring-clip at one end that holds the
SUBCUTANEOUS INOCULATION OF ANIMALS 235
animal by the skin of the neck, and at the other end with
another clamp that holds the tail of the animal. This
holder is shown in Fig. 39. For larger animals the form of
holder shown in Fig. 40 is commonly used.
The holder devised by Sweet,1 which can be made of any
size, from that suitable to a guinea-pig up to that large
enough to secure a dog, is in every way the most convenient
that we have encountered and, from the standpoint of the
animal, is the most humane. It consists of four pieces of
heavy round wire so bent that two engage the animal
FIG. 40
Holder for larger animals.
immediately behind the lower jaw while the remaining two
close over the muzzle. All are held in position by a single
clamp controlled Jby a single thumb-screw. When the screw
is reversed and the clamp opened the anterior and posterior
wire of each pair falls away from the median line, thereby
liberating the animal. To secure the animal it is placed
upon its back, the head laid in the cradle formed by the
bent wires, the latter are adjusted to the proper position,
1A Simple, Humane Holder for Small Animals under Experiment,
University of Penna. Med. Bull., 1903, No. 2, p. 78.
236
BACTERIOLOGY
and all secured by the turn of the single set-screw. Of
course, the extremities of the animal are to be secured. This
is done by means of cords securely held by a patent fastener
made by the Tie Co., of Unadilla, N. Y. These fasteners
are in every way more convenient than the cleats in common
use. An idea of the apparatus is given in Fig. 41.
FIG. 41
A very simple and useful holder for guinea-pigs consists
of a metal cylinder of about 5 cm. diameter and about 13
cm. long, closed at one end by a perforated cap of either tin
or wire netting. Along the side of this box is a longitudinal
slit 12 mm. wide that runs for 9.5 cm. from within 0.5 cm.
of the open extremity of the cylinder. The animal is placed
SUBCUTANEOUS INOCULATION OF ANIMALS 237
in such a cylinder with its head toward the perforated
bottom. It is then easily possible to make subcutaneous
inoculation by taking up a bit of skin through the slit in the
FIG. 42
The Voges holder for guinea-pigs.
side of the box, or to make intraperitoneal injection by draw-
ing the posterior extremities slightly from the box and hold-
ing them steady between the index and second finger, as
seen in Fig. 42. It is also very convenient for use when the
238 BACTERIOLOGY
rectal temperature of these small animals is to be taken.
The manipulation can easily be made without the aid of
an assistant. Its construction is seen in Fig. 42. 1
For ordinary subcutaneous inoculations at the root of the
tail in mice a simple apparatus consists of a piece of board
about 7 x 10 cm. and 2 cm. thick, upon which is tacked a
hollow truncated cone of wire gauze, about 6 cm. long and
about 1.5 cm. in diameter at one end and 2 cm. at its other
end. This is tacked upon the board in such a position that
its long axis is in the long axis of the board, being equidistant
from its sides. Its small end is placed at the edge of the
FIG. 43
Mouse-holder, with mouse in proper position.
board. .The mouse is taken up by the tail by means of a
pair of tongs and allowed to crawl into the smaller end of
the wire cone. When so far in that only the root of the
tail projects the animal is fixed in this position by a clamp
and thumb-screw, with which the apparatus (Fig. 43) is
provided. The animal usually remains perfectly quiet and
may be handled without difficulty.
The hair over the root of the tail is to be carefully cut
away with scissors and a pocket cut through the skin at
this point. The inoculation is then made into the loose
1 Centralblatt fur Bacteriologie and Parasitenkunde, 1895, vol. xviii, p. 530.
•SUBCUTANEQUS INOCULATION OF ANIMALS 239
tissue under the skin over this part of the back in the way
that has just been described. It is always best to insert the
needle some distance along the spinal column, and thus
deposit the material as far from the surface-wound as
possible.
Injection into the Circulation. — It is not infrequently
desirable to inject the material under consideration directly
into the circulation of an animal. If a rabbit is employed
for the purpose, the operation is usually done upon one of
the veins in the ear. To those who have had no practice
with this procedure it offers a great many difficulties; but
if the directions which will be given are strictly observed
the greatest of these obstacles to the successful performance
of the operation may be overcome.
When viewing the circulation in the ear of the rabbit by
transmitted light three conspicuous branches of the main
vessel (vena auricularis posterior) will be seen. One runs
about centrally in the long axis of the ear, one runs along
its anterior margin, and one along its posterior margin.
The central branch (ramus anterior of the vena auricularis
posterior) is the largest and most conspicuous vessel of the
ear, and is, therefore, believed by the inexperienced to be
the branch into which it would appear easiest to insert a
hypodermic needle. This, however, is fallacious. This
vessel lies very loosely imbedded in connective tissue, and,
in efforts to introduce a needle into it, rolls about to such
an extent that only after a great deal of difficulty does the
experiment succeed. On the other hand, the posterior
branch (ramus lateralis posterior of the vena auricularis
posterior) is a very fine, delicate vessel which runs along the
posterior margin of the ear, and is so firmly fixed in the dense
tissues which surround it that it is prevented from rolling
240 BACTERIOLOGY
about under the point of the needle. The further away from
the mouth of the vessel — that is, the nearer we approach
its capillary extremity — the more favorable become the
conditions for the success of the operation.
After shaving the ear and carefully washing it with clean
water select the very delicate vessel lying quite close to the
posterior margin of the ear, and make the injection as near
to the apex of the ear as possible. At times the vessels of
the ear will be found to contain so little blood that they are
hardly distinguishable, making it very difficult to introduce
the needle into them. This is sometimes overcome by pres-
sure at the root of the ear, causing stasis of the blood and
distention of the vessels. A very satisfactory method of
causing the veins to become prominent is to press lightly or
prick gently with the point of a needle the skin over the
vessel to be used. In a few seconds, as a result of this irri-
tation, the vessel will have become distended with blood,
and readily distinguishable from the surrounding tissue;
it may then be easily punctured by the needle of the syringe.
A sharp flick with the finger will often produce the same
result. The injection is always to be made from the dorsal,
surface of the ear.
Of no less importance than the selection of the proper
vessel is the shape of the point of the needle employed.
The hypodermic needles as they come from the makers
are not suited at all for this operation, because of the manner
in which their points are ground. If one examine carefully
the point of a new hypodermic needle, it will be seen that
the long point, instead of presenting a flat, slanting surface
when viewed from the side, has a more or less curved surface.
Now, in efforts to introduce such a needle into a vessel of
very small caliber it is- usually seen that the point of the
SUBCUTANEOUS INOCULATION OF ANIMALS 241
needle, instead of remaining in the vessel, as it would do
were it straight (or "chisel pointed"), very commonly pro-
jects into the opposite wall; and as the needle is inserted
further and further it is usually pushed through the vessel-
walls into the loose tissues beyond, and the material to be
injected is deposited in these tissues, instead of into the
circulation. If, on the contrary, the slanting point of the
needle be ground until its surface is perfectly flat when
viewed from the side, and no curvature exists, then when
once inserted it usually remains within the vessel, and there
FIG. 44
a
Hypodermic needles, magnified, a, improper point; 6, proper shape of
point.
is no tendency to penetrate the opposite wall. We never
use a new hypodermic needle until its point is carefully
ground to a perfectly flat, slanting surface with no curvature
whatever.
These differences may perhaps be more easily understood
if represented diagrammatically. In Fig. 44, a, the needle
has the point usually seen when new. In Fig. 44, b, the
point has been ground to the shape best suited for this
operation. The needles need not be returned to the maker.
One can grind them to the shape desired in a few minutes
upon an oilstone. The size of the needle is that commonly
16
242 BACTERIOLOGY
employed by physicians for subcutaneous injections in
human beings.
When the operation is to be performed an assistant holds
the animal gently but firmly in the crouching position upon
a table. If the animal does not remain quiet, it is best to
wrap it in a towel, so that only its head protrudes; though
in most cases we have not found this necessary, particularly
if the animal has not been excited prior to beginning the
operation.
The ear in which the injection is to be made should be
shaved clean of hair by means of a razor and soap and then
washed with water. It is unnecessary to attempt disin-
fection of the skin.
The animal should be placed so that the prepared ear
comes between the operator and the source of light. This
renders visible by transmitted light not only the coarser
vessels of the ear, but also their finer branches.
The filled hypodermic syringe is. taken in one hand and
with the other hand the ear is held firmly. The point of the
needle is then inserted through the skin and into the finest
part of the ramus posterior, the part nearest the apex of the
ear, where the course of the vessel is nearly straight. When
the point of the needle is in this vessel it gives to the hand
a sensation quite different from that felt when it is in the
midst of connective tissue. As soon as one supposes the
point of the needle is in the vessel a drop or two of the fluid
may be injected from the syringe, and, if his suspicions
are correct, the circulation in the small ramifications and
their anastomoses will rapidly alter in appearance — i. e.,
the circulating blood will be displaced very quickly by the
clear, transparent fluid that is being injected. At this stage
one must proceed very carefully, for sometimes when the
SUBCUTANEOUS INOCULATION OF ANIMALS 243
needle-point is not actually in the vessel, but is in the lymph
spaces surrounding it, an appearance somewhat similar is
seen. This may always be differentiated, however, by con-
tinuing the injection, when the flow of clear fluid through
the vessels will not only fail to take the place of the cir-
culating blood, but at the same time a localized swelling,
due to an accumulation of the fluid injected, will appear
under the skin about the point of the needle. The needle
must then be withdrawn and inserted into the vessel at a
point a little nearer its proximal end.
Care must be taken that no air is injected.
The hypodermic syringe and needle must, previous to
operation, have been carefully sterilized in the steam steril-
izer or in boiling water. The animal must be kept under
close observation for about an hour after injection.
The operation is one that cannot be learned from verbal
description. It can only be successfully performed after
actual practice. If the precautions which have been men-
tioned are observed, but little difficulty in performing the
operation will be experienced.
Its greater convenience and simplicity, as compared with
other methods for the introduction of substances into the
circulation, commend it as a technical procedure with which
to make one's self familiar. The animals sustain practically
no wound, they experience no suffering — at least they give
no evidence of pain — and no anesthetic is required.
The form of syringe best suited for this operation is of
the ordinary design, but one that permits of thorough
sterilization by steam. It should be made of glass and metal,
with packings that may be sterilized by steam without
injury. The syringes commonly employed are those shown
in Fig. 45.
244
BACTERIOLOGY
For operations requiring exact dosage experience has led
me to prefer a syringe after the pattern of C, in Fig. 45 — i. e.,
the form commonly used by physicians. The reason for
this is as follows: in making injections,, either into the cir-
culation or under the skin, there is a certain amount of
resistance to the passage of fluid from the needle. If one
overcomes this resistance by means of a cushion of com-
pressed air, as is the case in syringes A and B, Fig. 45, the
sudden expansion of the air in the body of the syringe when
FIG. 45
Forms of hypodermic syringe. A, Koch's syringe; B, syringe of Strohschein;
C, Overlack's form.
resistance is overcome frequently causes a larger amount
of fluid to be injected than is desired. No such accident
is likely to occur when the fluid is forced from the barrel
of the syringe by the head of a close-fitting piston, with no
air intervening between the fluid and the head of the piston.
With such an instrument, properly manipulated, the dose
can always be controlled with accuracy.
Inoculation into the Lymphatic Circulation. — Fluid cultures
or suspensions of bacteria may be injected into the lym-
SUBCUTANEOUS INOCULATION OF ANIMALS 245
phatics by way of the testicles. The operation is in no wise
complicated. One simply plunges the point of the hypo-
dermic needle directly into the substance of the testicle and
then injects the amount desired. Injections made in this
manner are usually followed by instructive pathological
lesions of the lymphatic apparatus of the abdomen.
Inoculation into the Great Serous Cavities. — Inoculation into
the peritoneum presents no difficulties if fluids are to be
introduced. In this case one makes, with a pair of sterilized
scissors, a small nick through the skin down to the under-
lying fasciae, and, taking a fold of the abdominal wall between
the fingers, plunges the hypodermic needle through the
opening just made directly into the peritoneal cavity. There
is little or no danger of penetrating the intestines or other
internal viscera if the puncture be made along the median
line at about midway between the end of the sternum
and the symphysis pubis. Though this may seem a rude
method it is rare that the intestines are penetrated or other-
wise injured. The object of the primary incision is to lessen
the chances of contamination by bacteria located in the skin,
some of which might adhere to the needle if it were plunged
directly through the skin, and thus complicate the results.
If solid substances, bits of tissue, etc., are to be intro-
duced into the peritoneum, it becomes necessary to conduct
the operation under an anesthetic and upon the lines of a
laparotomy. The hair should be shaved from a small area
over the median line, after which the skin is to be thoroughly
washed.. A short longitudinal incision (about 2 cm. long)
is then to be made in the median line through the skin and
down to the fasciae. Two subcutaneous sutures, as em-
ployed by Halsted, are then to be introduced transversely
to the line of incision about 1 cm. apart, and their ends left
246
BACTERIOLOGY
loose. This particular sort of suture does not pass through
the skin, but, instead, the needle is introduced into the
subcutaneous tissues along the edge of the incision. In this
case they are to pass into the abdominal cavity and out
again, entering at one side of the line of incision and leaving
at the other, as indicated by the solid and dotted lines in
Fig. 46. (The figure indicates the primary opening through
the skin. The longitudinal dotted line shows the opening
FIG. 46
Diagram of skin incision and sutures in laparotomy on animals.
to be made into the abdomen; the transverse dotted lines,
with their loose ends, represent the sutures as placed in
position before the abdomen is opened; it will be seen that
these sutures in all cases pass through the subcutaneous
tissues only and do not penetrate the skin proper.)
The opening through the remaining layers may now be
completed; the bit of tissue is deposited in the peritoneal
cavity, under precautions that will exclude all else, the
SUBCUTANEOUS INOCULATION OF ANIMALS 247
edges of the wound drawn evenly and gently together by
tying the sutures, and the lines of incision dressed with
collodion. It should be needless to say that this operation
must be conducted under the strictest precautions, to
avoid complications. All instruments, sutures, ligatures,
etc., must be carefully sterilized either in the steam sterilizer
for twenty minutes, or by boiling in 2 per cent, sodium
carbonate solution for ten minutes; the hands of the opera-
tor, though they should not touch the wound, must be
carefully cleansed, and the material to be introduced into
the abdomen should be handled with only sterilized instru-
ments.
Inoculation into the pleural cavity is much less frequently
required — in fact, it is not a routine method. It is not easy
to enter the pleural cavity with a hypodermic needle without
injuring the lung, and it is rare that conditions call for the
introduction of solid particles into this locality.
Inoculation into the anterior chamber of the eye is per-
formed by making a puncture through the cornea just in
front of its junction with the sclerotic, the knife being
passed into the anterior chamber in a plane parallel to the
plane of the iris. By the aid of a fine pair of forceps the bit
of tissue is passed through the opening thus made and is
deposited upon the iris, where it is allowed to remain, and
where its pathogenic activities upon the iris can be con-
veniently studied. It is a mode of inoculation of very
limited application, and is therefore but rarely practised.
It was employed in the classical experiments of Cohnheim
in demonstrating the infectious nature of tuberculous tissues,
tuberculosis of the iris being the constant result of the
introduction of tuberculous tissue into the anterior chamber
of the eye of rabbits.
248 BACTERIOLOGY
OBSERVATION OF ANIMALS AFTER INOCULATION. — After
either of these methods of inoculation, particularly when
unknown species of bacteria are being tested, the animal is to
be kept under constant observation and all deviations from
the normal are to be carefully noted — as, for instance, eleva-
tion of temperature; loss of weight; peculiar position in
the cage; loss of appetite; roughening of the hair; excessive
secretions, from either the air-passages, conjunctiva, or
kidneys; looseness of or hemorrhage from the bowels;
tumefaction or reaction at site of inoculation, etc. If death
ensue in from two to four days, it may reasonably be expected
that at autopsy evidence of either acute septic or toxic
processes will be found. It sometimes occurs, however,
that inoculation results in the production of chronic con-
ditions, and the animal must be kept under observation
often for weeks. In these cases it is important to note the
progress of the disease by its effect upon the physical condi-
tion of the animal, viz., upon the nutritive processes, as
evidenced by fluctuation in weight, and upon the body-
temperature. For this purpose the animal is to be weighed
daily, always at about the same hour and always about mid-
way between the hours of feeding; at the same time its
temperature, as indicated by a thermometer placed in the
rectum, is to be recorded.1 By comparison of these daily
observations the observer is aided in determining the course
the infection is taking.
Too much stress must not, however, be laid upon moderate
and sudden daily fluctuations in either temperature or
weight, as it is a common observation that presumably
1 The thermometer must be inserted into the rectum beyond the grasp
of the sphincter, otherwise pressure upon its bulb by contraction of this
muscle may force up the mercurial column to a point higher than that
resulting from the actual body-temperature.
SUBCUTANEOUS INOCULATION OF ANIMALS 249
normal animals when confined in cages and fed regularly
often present very striking temporary gains and losses in
weight, often amounting to 50 or 100 grams in twenty-four
hours, even in animals whose total weight may not exceed
500 or 600 grams; similarly unexplainable rises and falls
of temperature, often as much as a degree from one day to
another, are seen. Such fluctuations have apparently no
bearing upon the general condition of the animal, but are
probably due to transient causes, such as overfeeding or
scarcity of food, improper feeding, lack of exercise, excite-
ment, fright, etc.
The accompanying charts (Figs. 47, 48, 49, 50) will serve
to illustrate some of these points. The animals, two rabbits
and two guinea-pigs, were taken at random from among
stock animals and placed each in a clean cage, the kind used
for animals under experiment, and kept under as good
general conditions as possible. For the first week the rabbits
received each 100 grams of green food (cabbage and turnips)
daily, and the guinea-pigs 30 grams each of the same food.
During the second week this daily amount of food was
doubled; during the third week it was quadrupled; and for
the fourth and fifth weeks they each received an excess of
food daily, consisting of green vegetables and grains (oats
and corn). By reference to the charts sudden diurnal
fluctuations in weight will be observed that do not corre-
spond in all instances with scarcity or sufficiency of food.
With the rabbits there is a gradual loss of weight with the
smaller amounts of food, which losses are not totally re-
covered as the food is increased. With the guinea-pigs there
is likewise at first a loss; but after a short time the weight
remains tolerably constant, and is not so conspicuously
affected by the increase in food as one might expect. From
250
BACTERIOLOGY
the recorded temperatures one sees the peculiar fluctuations
mentioned. j_To just what they are due it is impossible to
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SUBCUTANEOUS INOCULATION OF ANIMALS 251
presenting such fluctuations, is about a degree or more,
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from which these charts were made were not inoculated,
nor were they subjected to any operative procedures what-
252
BACTERIOLOGY
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SUBCUTANEOUS INOCULATION OF ANIMALS 253
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reduced to from 50 to 60 per cent, of its original weight.
In other cases, after inoculations to which the animal is not
254 BACTERIOLOGY
susceptible, rabbits in particular, if properly fed, will fre-
quently gain steadily in weight. The condition of progressive
emaciation just mentioned is conspicuously seen after
intravenous inoculation of rabbits with cultures of bacillus
typhosus and of bacillus coli, referred to in the chapter on the
latter organism, and if looked for will doubtless be seen to
follow inoculation with other organisms capable of producing
chronic forms of infection, but which are frequently con-
sidered non-pathogenic because of their inability to induce
acute conditions. Not infrequently in chronic infections
there may be hardly any marked and constant temperature-
variations until just before death, when sometimes there
will be a rise and at other times a fall of temperature. In
the majority of cases, however, one must be very cautious
as to the amount of stress laid upon changes in weight and
temperature, for unless they are progressive or continuous
in one or another direction they may have little significance
as indicating the existence or absence of disease.
CHAPTER XIII.
Post-mortem Examination of Animals — Bacteriological Examination of the
Tissues — Disposal of Tissues and Disinfection of Instruments after
the Examination — Study of Tissues and Exudates During Life.
DURING bacteriological examination of the tissues of dead
animals certain precautions must be rigidly observed in
order to arrive at correct conclusions.
The autopsy should be made as soon as possible after
death. If delay cannot be avoided, the animal should be
kept on ice until the examination can be made, otherwise
decomposition sets in, and the saprophytic bacteria now
present may interfere with the accuracy of results. When
the autopsy is to be made the animal is first inspected
externally, and all visible lesions noted. It is then to be
fixed upon its back upon a board with nails or tacks. The
four legs and the end of the nose, through which the tacks
are driven, are to be moderately extended. Plates are now
to be made from the site of inoculation, if this is subcuta-
neous. The surfaces of the thorax and abdomen are then
to be moistened to prevent the fine hairs, dust, etc., from
floating about in the air and interfering with the work. An
incision is then made through the skin from the chin to the
symphysis pubis. This is only a skin incision, and does not
reach deeper than the fascise. It is best done by first making
with a scalpel an incision just large enough to permit of the
introduction of one blade of a blunt-pointed scissors. It is
then completed with the scissors. The whole of the skin is
(255)
256 BACTERIOLOGY
now to be carefully dissected away, not only from the
abdomen and thorax, but from the axillary, inguinal, and
cervical regions, and the fore and hind legs as well. It is
then pinned flat upon the board so as to keep it as far from
the abdomen and thorax as possible, for it is from the skin
that the chances of contamination are greatest.
It now becomes necessary to proceed very carefully.
All incisions from this time on are to be made only through
surfaces that have been sterilized. The sterilization is best
accomplished by the use of a broad-bladed table-knife that
has been heated in a gas-flame. The blade, made quite hot,
is to be held upon the region of the linea alba until the tissues
of that region begin to burn; it is then held transversely
to this line over about the center of the abdomen, thus
making two sterilized tracks, through which the abdomen
may be opened by a crucial incision. The sterilization thus
accomplished is, of course, directed only against organisms
that may have fallen upon the surface from without, and
therefore, it need not extend deep down through the tissues.
In the same way two burned lines may be made from either
extremity of the transverse line up to the top of the thorax.
With hot scissors the central longitudinal incision extend-
ing from the point of the sternum to the genitalia is to be
made without touching the internal viscera. The abdominal
wall must therefore be held up during the operation with
sterilized forceps or hooks. The cross-incision is made in
the same way. When this is completed an incision through
the ribs with a pair of heavy, sterilized scissors is made
along the scorched tracks on either side of the thorax. After
this the whole anterior wall of the thorax may easily be
lifted up, and by severing the connections with the dia-
phragm it may be completely removed. When this is done
POST-MORTEM EXAMINATION OF ANIMALS 257
and the abdominal flaps laid back, the contents of both
cavities are to be inspected and their condition noted without
disturbing them.
After this the first steps to be taken are to prepare plates
or Esmarch tubes from the blood, liver, spleen, kidneys,
and any exudates that may exist. This is best done as
follows: Heat a scalpel quite hot and apply it to a small
surface of the organ from which cultures are to be made.
Hold it upon the organ until the surface directly beneath
is visibly scorched. Then remove it, heat it again, and
while quite hot insert its point through the capsule of the
organ. Into the opening thus made insert a sterilized
platinum loop, made of wire a little heavier than that
FIG. 51
Nuttall's platinum spear for use at autopsies.
commonly employed. Project this deeply into the tissues
of the organ; by twisting it about enough material from
the center of the organ can be obtained for making the
cultures.
As the resistance offered by the tissue is sometimes too
great to permit of puncture with the ordinary wire loop,
Nuttall1 devised for the purpose a platinum-wire spear
which possesses great advantages over the loop. It has
the form seen in Fig. 51. It is easily made by beating a
piece of heavy platinum wire into a spear-head at one end,
and perforating this with a small drill, as seen in the cut.
It is attached by the other end to either a metal or glass
1 Centralblatt fur Bakteriologie und Parasitenkunde, 1892, Bd. xi, p. 538.
17
258 BACTERIOLOGY
handle, preferably the former. It can readily be thrust into
the densest of the soft tissues, and by twisting it about
after its introduction particles of the tissue sufficient for
examination are withdrawn in the eye of the spear-head.
Cultures from the blood are usually made from one of the
cavities of the heart, which is always punctured at a point
which has been burned in the way given.
In addition to cultures, cover-slips from the site of inocu-
lation, from each organ, and from any exudates that may
be present must be made. These, however, are prepared
after the materials for the cultures have been obtained.
They need not be examined immediately, but may be
placed aside, under cover, on bits of paper upon which the
name of the organ from which they were prepared is written.
When the autopsy is complete and the gross appearances
have been carefully noted, small portions of each organ are
to be preserved in 95 per cent, alcohol for subsequent
examination. Throughout the entire autopsy it must be
borne in mind that all cultures, cover-slips, and tissues must
be carefully labelled, not only with the name of the organ
from which they originate, but with the date, designation
of the animal, etc., so that an account of their condition
after closer study may be subsequently inserted in the
protocol.
The cover-slips are now to be stained, mounted, and
examined microscopically, and the results carefully noted.
The same care with regard to noting, labelling, etc.,
should be exercised in the subsequent study of the cultures
and the hardened tissues, which are to be stained and sub-
jected to microscopic examination. The results of micro-
scopic study of the cover-slip preparations and of those
obtained by cultures should in most cases correspond,
POST-MORTEM EXAMINATION OF ANIMALS 259
though it not rarely occurs that bacteria are present in
such small numbers in the tissues that their presence may
be overlooked microscopically, and still they appear in the
cultures.
If the autopsy has been performed in the proper way,
with the precautions given, and sufficiently soon after death,
the results of the bacteriological examination should be
either negative or the organisms which are isolated should
be in pure cultures. This is particluarly the case with cul-
tures made from the internal viscera.
Both the cover-slips and cultures made from the point
of inoculation are apt to contain a variety of organisms.
If the organism obtained in pure culture from the internal
viscera, or those predominating at the point of inoculation
of the animal, have caused its death, then subsequent
inoculation of pure cultures of this organism into the tissues
of a second animal should produce similar results.
When the autopsy is quite finished the remains of the
animal should be burned; all instruments subjected to
either sterilization by steam or boiling for fifteen minutes
in a 1 to 2 per cent, soda solution; and the board upon which
the animal was tacked, as well as the tacks, towels, dishes,
and all other implements used at the autopsy, be sterilized
by steam. All cultures, cover-slips, and, indeed, all articles
likely to have infectious material upon them, must be
sterilized as soon as they are of no further service.
What has been said with regard to the study of dead
tissues obtained at autopsy applies equally well to the
bacteriological study of tissues and exudates obtained during
life. In the latter case, however, certain .precautions are
always to be observed. In the first place, it is desirable to
260 BACTERIOLOGY
obtain the materials under aseptic precautions, care being
taken that no disinfectant fluids are mixed with them.
They should be subjected to study as soon as possible after
removal from the body. In the case of tissues that cannot
be examined on the spot, they should be placed in a sterile
Petri dish or in a stoppered, sterile, wide-mouthed bottle
and taken at once to the laboratory. The surface should
then be seared with a hot knife and an incision through the
seared area into the center made with a knife that has been
sterilized and allowed to cool. From the depths of this
incision enough material may be obtained for microscopic
examination and for the preparation of cultures. Fluid
exudates that must be taken to the laboratory should be
collected in either a sterile test-tube, or, better, in a sterile
capillary tube that is subsequently sealed at both ends in
a gas-flame. When bacteriological examination of the
blood during life is required, it is customary to obtain the
necessary sample of blood by pricking the skin. It must
be remembered, in this connection, that the skin usually
contains a number of species of bacteria that are of no
pathological significance and have nothing to do with the
disease from which the individual may be suffering. It is
manifestly essential to exclude these. It is not possible to
exclude them certainly and completely under all circum-
stances, without a more or less elaborate procedure; but
an effort to do so should always be made. As a rule, the
greater number of them may be removed from the skin by
careful washing with warm water and soap and a sterile
brush, after which the skin should be rinsed with alcohol
and allowed to dry spontaneously. The drop of blood may
then be obtained from the skin thus cleaned by a prick
with a sharp, sterilized lancet. The presence in the cultures
of a staphylococcus, growing slowly, with white colonies,
ULTRA-MICROSCOPIC OR FILTERABLE VIRUSES 261
is a frequent experience, and does not necessarily imply
that this organism bears an etiological relation to the disease
from which the individual may be suffering (see Staphylococcus
Epidermis Albus).
When more than a few drops of blood are needed, as may
be the case in deciding the general nature of an infection
process, it is customary to withdraw it from one of the super-
ficial veins of the forearm by means of an hypodermic
syringe. The operation should be done under strictly
asceptic conditions, i. e., the skin should be thoroughly
cleaned with soap, water, and alcohol; the hands of the
operator should be surgically cleaii; the syringe must have
been sterilized immediately before using, and great care
should be taken that no air bubbles be injected into the
veins during the operation.
In interpreting the results of cultures made from blood
drawn in this manner, the possibility of contamination by
skin bacteria should not be forgotten. The success of the
operation depends upon attention to the most minute
details of aseptic practice. It requires for its safe practice
skill in manipulation, experience and judgment in the inter-
pretation of the results. It is not, therefore, an operation
to be commended to the beginner.
"ULTRA"-MICROSCOPIC OR "FILTERABLE" VIRUSES.
These terms relate to particular substances capable of
causing disease, that are so small as to be beyond the visual
range of the microscopes used in bacteriological work, which
do not respond to the usual methods for the cultivation of
bacteria and which are able, because of their minute dimen-
sions, to pass through the pores of the finer grades of earth-
enware filters.
262 BACTERIOLOGY
Their existence has been suspected for a number of years
but it is only comparatively recently that sufficient became
known of them to justify our speaking confidently of them;
and even now little more than their etiological potentialities
and some of their physiological reactions can be considered.
For a long while it has been a puzzle that such character-
istic contagious diseases as certain of the acute exanthemata
in man and a number of typical transmissible diseases in
animals should have eluded all efforts to discover their
causes. By the customary methods of bacteriological
analysis nothing of a positive character is learned and yet
by the introduction into susceptible animals of bits of tissue
from the diseased animal, or small quantities of blood or
tissue juices or even of filtrates of emulsions of such tissues
or juices, it is possible in a number of instances to reproduce
the disease. It is such evidence as this that serves as the
basis for the belief in the existence of invisible or elusive
viruses for a number of diseases of man and animals and
a few for plants.
The existence of such viruses has been demonstrated
in smallpox vaccine, measles, typhus fever, dengue fever,
poliomyelitis, and trachoma, among the diseases of man
and in foot and mouth disease, contagious pleuro-pneu-
monia, sheep-pox, rabies, cattle plague, chicken sarcoma,
and distemper of dogs among those of animals, and in the
mosaic disease of the tobacco plant. Sometimes such fil-
trates when placed under special methods of cultivation
show evidence of multiplication by clouding of the media
but with no development of recognizable morphological
structures — in a few instances such cultures have shown the
development of minute spiral forms of organisms. (See
Leptospira icteroides.
Though little or nothing that is convincing can be said
ULTRA-MICROSCOPIC OR FILTERABLE VIRUSES 263
of the morphology of this group of ultra-microscopic par-
ticles, still in their reactions to a variety of physical agents
they are obviously living matter, having many analogies to
the more highly developed microorganisms with which we
are familiar. Practically all are killed at temperatures
ranging from 55° to 70° C. Some resist drying for com-
paratively long periods of time, others are quickly killed by
it. Practically all are resistant to the action of glycerin.
This is not the case as a rule with bacteria. They vary
considerably in their resistance to such germicidal substances
as formalin, boric acid, corrosive sublimate and menthol.
Practically all animals that survive their invasion have
acquired immunity from a second attack of the disease.
There is little evidence that the growth is accompanied by
the production of toxins as such. A survey of such data as
are available justifies the suspicion that these bodies are
more closely allied to the protozoa than to the bacteria.
Efforts at cultivation under artificial circumstances have
succeeded in only a few instances. In their studies upon the
contagious pleuro-pneumonia of cattle Nocard and Roux
by the use of special methods, both optical and cultural,
claim to have demonstrated the causative factor of that
disease. The method employed by them for the cultivation
of the virus is that suggested by Metchnikoff, Roux and
Salambini in 1896. It consists in placing bits of tissue or
secretions from the infected animals in small, sterilized
collodion sacs, which are finally hermetically sealed with
sterile collodion. These little sacs with their contents are
then placed in the peritoneal cavity of an animal; a rabbit,
chicken, guinea-pig, calf, dog, or sheep as the case may be,
and left there for a time. The idea on which this method is
based is that the collodion sacs are impermeable for the
specific virus but are permeable to the normal juices of the
264 BACTERIOLOGY
peritoneal cavity of the animal in which they are placed.
Under these circumstances the specific virus was expected to
develop within the sacs and receive its food supply by dif-
fusion from the surrounding peritoneum; the body tem-
perature of the animal in which they were placed being most
favorable to incubation.
The investigators found that by the use of a special
system of illumination and very high magnification, about
2000 diameters, there were to be detected within the col-
lodion sacs, in from a few days to several weeks, numerous
motile points or dots of such minute dimensions that it was
often impossible to decide as to their exact form. No such
bodies were seen in control collodion sacs placed similarly
in the peritoneum of animals but in which sacs none of the
tissue or juices from a diseased animal had been inclosed.
Nocard and Roux are disposed to regard these bodies as
the exciting cause of the disease under consideration.
Flexner and Noguchi announce that by the use of Nogu-
chi's method for cultivating spirochetse (see Spirochetacese)
they have isolated from the central nervous tissues of both
man and monkeys dead of poliomyelitis, minute coccus-like
bodies that they believe to be the cause of the disease.
The culture medium consists of human ascetic fluid to which
a fragment of sterile fresh rabbit kidney has been added.
The cultivation is conducted at first under anaerobic condi-
tions but later subcultures do not demand complete absence
of free oxygen. When ready the tubes are inoculated with
small bits of the diseased cerebrum or cord after which a
thick layer of sterile paraffin oil is placed upon the surface
of the ascetic fluid. This suffices for the exclusion of free
oxygen.
After from seven to twelve days at body temperature a dif-
fuse clouding or opalescence appears about the bit of nervous
ULTRA-MICROSCOPIC OR FILTERABLE VIRUSES 265
tissue in the tube. Microscopic examination of this opalescent
matter, especially by dark-field illumination, reveals the pres-
ence of coccoid bodies conspicuous for their variation in size.
Their true nature has not been determined. The disease
can be reproduced in monkeys by inoculation with the cul-
tures, but not with regularity.1
By an analogous method Noguchi has cultivated from
both rabies and trachoma bodies that he regards as etiolog-
ically related to the diseases from which they were obtained.
It is not possible as yet to be either certain as to the accuracy
of his suspicions or to satisfactorily classify the bodies found
in his cultures. In some respects they suggest bacteria, in
some protozoa and taking them in conjunction with the
tissue findings in the diseases it seems fair to suspect that
they may be developmental forms of the Negri bodies con-
stantly present in rabies in the one case or the singular cell
inclusions common to trachoma, the so-called "trachoma
bodies" in the other.2
In the study of many of the common diseases, notably
the exanthemata, both at autopsy and during life, by the
methods above outlined, the investigation often yields
negative results, and yet there is every reason for believing
these diseases to be dependent for their existence upon
invasion of the body by some form or another of living
microorganisms, capable of growth in the tissues and sus-
ceptible of being transmitted from individual to individual,
either directly or indirectly. It is possible that the applica-
tion of one or another of the foregoing methods to the
study of these diseases may demonstrate that some of them
at least are due to the presence of so-called filterable viruses.
1 For details see Flexner and Noguchi, Jour. Exp. Med., 1913, vol. xviii,
No. 4.
2 For particulars see Noguchi, Jour. Exp. Med., 1913, No. 4; ibid., 1913,
No. 5.
CHAPTER XIV.
Infection and Immunity — Mechanism — Specific Bodies and Reactions —
Doctrines in Explanation.
INFECTION. .
IF one examine in detail the lesions resulting from the
invasion of the body by the different types of infective
bacteria, justification is found for -the conclusion that the
physical manifestations of infection, that is, the sites of
activity and the characteristic lesions, vary with the nature
of the different invading parasites.
To a certain extent this is true; that is to say, the type
of lesion characterizing a specific disease is peculiar to that
disease and is produced only by the particular microorganism
having the power to excite the disease. But if we take up
the various lesions of specific diseases in intimate detail we
shall see, as will be shown later, that fundamentally the
essential factor in the mechanism of infection is of the same
general nature for all* diseases, be the characteristic lesions
and clinical manifestations what they may; the apparent
differences being referable to dissimilarities of structure and
function of the various species of bacteria that excite the
several phenomena on the one hand, and to the parts of
the body of the host that are attacked on the other. Thus,
by way of illustration, if we select a group of clinically and
pathologically distinct infections, such as anthrax, miliary
tuberculosis, and diphtheria, and compare the conditions
recorded at autopsy, little of a macroscopic nature will be
(266)
INFECTION 267
discovered to suggest anything that is common to all, and
even if the tissues be examined microscopically such marked •
divergencies are seen that we are still in doubt as to the
existence of a common factor. In the case of anthrax, a
true septicemia, the blood current is the seat of activity of
the exciting bacteria, and beyond congestion, enormous
numbers of bacteria in the bloodvessels and the escape of
serum into the tissues (edema), little else is to be seen to
account for death. On the other hand, in the case of miliary
tuberculosis, even though the involvement of the organs may
be general, there is no similar invasion of the blood stream.
The tubercles are circumscribed, are often surrounded by
healthy tissue and, though obviously distributed throughout
the body from a primary focus through the agency of the
circulating fluids, each tubercle may nevertheless be regarded
as a distinct local infection. There is, however, a conspicuous
difference between the lesions found here and those seen in
anthrax. The lesion of tuberculosis, the tubercle, is always
characterized by tissue death at and about its center, i. e.,
where the bacilli are located, even in the .earliest stages of
its development.
On postmortem examination of an animal dead of diph-
theria we observe conditions that are unlike those noted in
both anthrax and tuberculosis. There is neither an invasion
of the vascular system nor a distribution of conspicuous
pathological foci throughout the body. The bacteria are
confined to the primary site of invasion and when found in
distal organs are there only in small numbers and give no
evidence of an effect upon the tissues immediately surround-
ing them.
Thus far, as a result of this review, we have two points
in common to the three distinct diseases, viz.: they are all
268 BACTERIOLOGY
caused by bacteria, and they all may terminate fatally.
On the other hand the clinical symptoms and the pathological
lesions are such as to characterize each as a pathological
entity. But, as has been intimated, there is a fundamental
factor common to all, and the discovery of this factor gives
the clue to the true mechanism of all infections. Light upon
this phase of the subject can best be secured through experi-
mental methods.
Observation and experiment have taught us that some-
times highly pathogenic bacteria may lose in part or in
whole their disease producing properties without at the same
time losing their vitality. If such "attenuated" bacteria
be injected into susceptible animals the result may be
nothing; or it may be a modified lesion totally dissimilar
to that following injection of the fully virulent organism.
This is often the case with the bacteria that excite septicemia,
and the bacillus causing anthrax serves as a useful illustra-
tion. When normal, as it is usual to regard it, it is fully
virulent and causes fatal blood poisoning in suceptible
animals, but if subjected to certain chemical or physical
influences the virulence may gradually be lessened until
finally we may have a living anthrax bacillus that has been
deprived of almost all its disease producing power. If
animals be inoculated with such attenuated anthrax bacilli
the conditions found may be in striking contrast to those
produced by the normal germ. Instead of the bloodvessels
being almost packed with bacteria, they may contain few
or none, and the only bacteria to be found in the body in
numbers are at and immediately about their point of deposit.
Yet these animals exhibit clinical symptoms and occasionally
die.
Similarly, in other varieties of septicemia, the so-called
INFECTION 269
" hemorrhagic group" we see as a rule typical, fatal septice-
mias resulting from the invasion of the body by the organisms
causing them; but at times, through influences not fully
known, these organisms become modified in their physio-
logical functions so that instead of the customary general
invasion of the circulating fluids there may be only a very
slight invasion and the results of their inoculation are prin-
cipally-evidenced as local destruction of tissue, sometimes
with fatal results. Obviously then these organisms have the
power of causing constitutional disturbances, tissue changes
and even fatal results without the necessity of their being
themselves disseminated throughout the body by way of
the circulating fluids.
As said above the characteristic lesion of tuberculosis is
the tubercle, and the peculiarity of the tubercle is necrosis,
observable almost from the moment it begins to develop.
If tuberculosis be induced through the intravenous injection
of rabbits with carefully prepared suspensions of living viru-
lent tubercle bacilli the resulting miliary tubercles are always
marked by more or less death of tissue at and about their
center, which tissue death progresses as the disease progresses,
until it reaches a point easily seen with the naked eye and
finally incompatible with life. If on the other hand a similar
injection be made with a suspension of tubercle bacilli that
have been killed, by heat or otherwise, disseminated nodules,
tubercles, will also be found in the internal organs. These
may be, histologically, strikingly like those following the use
of the living organism; they are marked by the characteristic
tissue death, but it is less in evidence and it is not progres-
sive beyond certain limits and the injection does not neces-
sarily prove fatal to the animal. As a result of this experi-
ment we see that dead bacteria may produce a result differing
270 BACTERIOLOGY
only in degree from that caused by the same species when
living and fully virulent.
A similar property may be demonstrated in a number of
other pathogenic species in no way related to bacillus tuber-
culosis. Obviously, there is something within or associated
with these bacteria that may act upon the tissues even
though the bacteria themselves may be dead.
In our autopsy on the animal dead of diphtheria we saw
that the bacilli were not distributed throughout the body, but
were confined to the site of inoculation. We saw at the
site of inoculation a tissue reaction scarcely sufficient to
account for the fatal result, yet that result occurred within
a comparatively short time after inoculation.
When diphtheria occurs in human beings the same holds
true as a rule, and while occasionally the local reaction in
the throat is such as gravely to imperil life through obstruc-
tion to respiration, the real danger in most cases is not local
but remote, and the clinical observations on the living subject
affected with this disease point to the far-reaching influence
of a local phenomenon, that, of itself, may often seem to be
of but slight significance.
If the internal organs of either animals or human beings
that have died of diphtheria be examined microscopically,
changes are easily to be discovered that are incompatible
with life and that at once account for many of the clinical
manifestations of the disease, yet these changes are not
accompanied by the presence of bacteria nor by any other
agent that can be detected by the eye.
It is plain, then, that the serious influence of the local
infection of diphtheria is referable to a something that
originates at the point where the bacteria are growing and is
from that point distributed to the distant organs.
INFECTION 271
Has the specific germ of diphtheria any property to war-
rant such a view? If a fluid culture of bacillus diphtherise
be filtered through a porcelain filter, the filtrate will contain
none of the bacteria. If this filtrate, free of all bacteria,
be injected into animals, death ensues; and if the tissues of
these animals be examined, all of the most important lesions
that characterized the tissues of the animal dead after
inoculation with the living germ are to be found.
If a parallel experiment be made with the bacillus of
tetanus analogous results will be obtained.
It is clear, then, that here are two species of bacteria that
excite the characteristic results through the instrumentality
of a something that they manufacture in the course of their
growth; that may be separated from them by the simple
process of filtration, and that when so separated possesses
all the properties of specific intoxicants.
In anthrax and other septicemias we saw that, normally,
the infection was characterized by the distribution of the
bacteria throughout the body, but that modified results,
differing only in degree, might still be obtained with the
attenuated organisms without such general distribution.
These latter conditions must, therefore, have been caused
by a poison elaborated by or escaping from the locally
deposited organisms and carried to distant parts of the body
by the circulating fluids. In tuberculosis the nodules result-
ing from inoculation with the dead bacteria must have been
the result of a poison associated with the bodies of those
dead bacteria and liberated with their disintegration in the
tissues; while in diphtheria it is plain that its characteristic
manifestations are the outcome of a poison produced locally
by the growing bacteria and carried thence by the circulating
fluids to distant organs, there to exhibit its destructive
properties.
272 BACTERIOLOGY
Thus far, then, infection must be viewed as a conflict
between bacteria on the one hand and tissues on the other;
the former having as their weapons of offence destructive
poisons; the latter, vital defensive provisions that enable
them to resist infection with greater or less degree of success,
according to circumstances. It makes no difference, there-
fore, whether, in infection, the bacteria be generally or only
locally present, the mechanism of infection is at bottom a
destructive intoxication.
Bacterial Toxins. — The term "toxins," as used in bac-
teriology, refers to a group of soluable, nitrogenous, non-
crystallizable poisons that are elaborated by certain bacteria
in the course of their growth, both in the tissues of the living
host and under conditions of artificial cultivation. They
are assumed to be by-products of metabolism and they may
be separated easily from the living bacteria by which they
are manufactured by the simple process of filtration, through
fine-pore earthenware filters. As they have not been ob-
tained in a pure state their chemical composition cannot be
stated precisely but it is probable that they are allied to the
globulins, nucleo-albumens, peptones, albumoses, or the
enzymes.
The toxins are identified, not by their chemical structure,
but rather by their harmful action upon the tissues of living
animals, i. e., by their physiological reactions. It is this
property that renders them of such significance in the
phenomena designated as disease.
By the injection of either of these bacteria-free, true
toxins into the tissues of susceptible animals, lesions are
produced that are in all essential respects identical with
those occurring in the course of infection by the living bac-
teria. By varying the dose of toxin injected into the animal
INFECTION 273
one may produce either prompt death or only slight con-
stitutional reaction. In the latter event repeating the
injection of a non-fatal dose may have no apparent effect
upon the animal. In such a case the animal has acquired,
loosely speaking, a tolerance to the poison and this tolerance
is due to a newly formed, antidotal substance now circulating
in the blood of the tolerant or immune animal. For example :
If a measured quantity of the toxin under consideration be
mixed in test-tubes with varying amounts of the serum of
the tolerant animal and each of these mixtures be injected
into fresh, normal animals of the same species, it will be
seen that in some instances the toxicity of the poison is
only lessened, while in others it may be completely neutral-
ized; in other words, we have demonstrated by such an
experiment the presence in the blood of an antidote, and
"antitoxin'' as it is called. This antidote is specific, that
is, it can neutralize only the poison used in the experiment;
it is inactive when used against other toxins.
This union between toxin and its antidote is conceived to
occur according to the laws governing ordinary chemical
reactions, i. e., there is a definite numerical relationship;
a certain fixed quantity of toxin being neutralized by a
certain fixed amount of antitoxin, variations in either factor
resulting in failure to accurately neutralize. The union
between the two factors is made possible, according to
Ehrlich's conception, through the possession by the toxin
molecule and by the antitoxin molecule of constituents
having the combining function, " haptophore" side chains,
as he calls them. In addition the toxin molecule possesses
another constitutent having the poisonous destructive func-
tion, the "toxiphoric," side chains, while the antidotal or
antitoxic molecule possesses a constitutent having the neu-
18
274 BACTERIOLOGY
tralizing function. Of the functions of these side chains,
that of combination is the more permanent.
Toxoids and Toxones. — Bearing this matter of permanency
in mind we find that when toxins are allowed to stand,
acted upon by heat, light and air, for a time, they may
still combine, as may be determined numerically, with the
appropriate antidotes or antitoxins, but may show evidence
of diminution of their intoxicating principle. When in this
degenerated state they are designated as "toxoids" and
"toxones."
A point of peculiar interest in connection with the true
bacterial toxins is the extraordinary toxicity of thqse with
which we are more or less fully acquainted. Experiment
leads to the belief that the toxins of diphtheria and of tetanus
are more highly poisonous than any other known poisons.
Thus, for instance, diphtheria toxin is capable of causing
fatal intoxication in a guinea-pig weighing 400 grams when
injected subcutaneously in so small a dose as 0.05 milli-
gram,1 while typical tetanus is produced in a mouse by the
injection of 0.0001 milligram of tetanus toxin.2
The number of bacteria capable of elaborating true toxins
is very small; indeed, in so far as those of significance to
animal pathology is concerned, we are certain of only two
species having this property, viz., the bacillus of diphtheria
and the bacillus of tetanus. For most of the other pathogenic
species their toxic action is referable, not to toxins, as defined
above, but rather to toxic components of the bacterial cells,
the endotoxins or intracellular toxins.
The Endotoxins or Intracellular Toxins. — The term Endo-
toxin is generically used to designate a toxic, protein com-
ponent of the bacterial cells, i. e., it is part and parcel of the
1 Roux and Yersin, Annals de 1'Inst. Pasteur, 1889, iii, p. 287.
2 Brieger and Cohn, Zeit. f. Hyg. u. Infekt., 1893, Bd. xv, Heft 1,
INFECTION 275
cell and becomes active, presumably, only when the cells
are disintegrated. Such disintegration may occur as a
result of autolysis or self-digestion of the bacteria under
special conditions of artificial cultivation, or it may be seen
as the outcome of the lytic or solvent action of the resisting
body cells or fluids, either those of the infected animal or,
as in the case of the toxins and antitoxins, those of the
animal that has become tolerant in one way or another to
the activities of the bacteria in question. Endotoxins are
not liberated from the bacterial cells as a secretion or excre-
tion or manufactured as an extracellular by-product, as is
the case of the toxins, but are peculiarities of the protoplasm
of which the bacteria are composed.
The escape of endotoxin from bacterial cells as a result
of autolysis is seen occasionally in old cultures that have been
kept for a time under more or less constant conditions.
It is probable that it occurs to a limited degree in all cul-
tures of endotoxic bacteria as a result of the death and final
dissolution of a smaller or larger number of individual
bacteria in such cultures. For want of a better interpre-
tation this liberation is supposed to be the result of a sort
of self-digestion by enzymes that are within the bacteria
as normal components. It is most conspicuously to be seen
in cultures of those endotoxic species that most readily under-
go those morphological changes commonly denominated as
involution or degeneration; the spirillum of Asiatic cholera
and the meningococcus may be cited as conspicuous illus-
trations. The fundamental mechanisms of this phenomenon
cannot be discussed with profit as little or nothing is known
of it.
As in the case of toxins, the definite chemical nature of
endotoxins cannot be stated. Nevertheless Buchner isolated
276 BACTERIOLOGY
from a number of bacterial species protein constituents,
"bacterio-proteins," as he denominated them, having the
common properties of soluability in alkalies, relative resist-
ance to the boiling temperature, attraction for leukocytes
(positive chemotaxis), and pyogenic powers.
The liberation of endotoxins from the bacterial cells by
strictly bacteriolytic processes going on in the living body
is not a simple phenomenon. It is conceived as resulting
from a solution of the bacteria by a certain ferment-like
body in the blood. This ferment-like body can act only
when it is bound to the bacterial cell by a 4 specific inter-
mediary body. This latter is supposed to be portions of
cells that have been thrown off from fixed cells in the course
of immunization, i. e., in the course of acquiring tolerance
to the action of certain endotoxic bacteria.
In numerous instances bacteria are disintegrated by
normal blood. Here it is believed that the ferment-like body
is brought into action through the agency of intermediary
bodies of a non-specific nature, i. e., of bodies normally
present that may have the power to bind any or all bacteria
to the ferment-like body and thus lead to their destruction.
While the blood of all animals possesses some destructive
solvent or disintegrating action for most bacteria, this is
never so great as is that of the blood of immunized animals
upon the particular bacteria from which they are immune.
Endotoxins Distinct in their Action from Toxins. — Like
toxins, endotoxins, i. e., dead endotoxic bacteria, may cause
disease and death of the animal tissues. Similarly, when
endotoxic bacteria are repeatedly injected in sublethal
doses, immunity of varying degrees develops. The immunity
resulting from the use of non-fatal doses of endotoxic bac-
teria, is not, however, an antitoxic or an anti-endotoxic
THE DEFENSES OF THE BODY 277
immunity, but the substance appearing in the blood of
animals so immunized is rather bacteriolytic, and the blood
of such animals may contain little or no true antitoxic
components.
Moreover, if the blood serum of an animal immune from
true toxin be injected into a normal animal, this latter at
once acquires some degree of resistance to the toxin from
which the first animal was protected, i. e., it is "passively"
immunized; on the other hand, if the bacteriolytic serum of
an animal artificially immunized from endotoxic bacteria
be similarly transferred to a normal animal, there is no
certainty of a transference of the state of immunity; there
may be a transference of the reaction or of the reacting factors
but not necessarily of a protective influence.
THE DEFENSES OF THE BODY.
When considered in the most comprehensive way we find
that the normal body is endowed with a number of natural
provisions that may fairly be regarded as defenses against
the invasion of hurtful parasites. Thus for instance: If
the skin of even the most cleanly persons be examined
bacteriologically, we find that in the majority of cases bac-
teria of several kinds, often those having the power to cause
disease, are to be detected. So long as the skin is intact
and the individual in good general health no harm results.
The reason for this is found in the structure of the skin.
The horny epidermis and the fat and sweat secretions serve
as effectual barriers against both the multiplication of germs
and their penetration into the underlying tissues. The hairs
about the orifices act to some extent as filters or screens for
bacteria laden dust; the ciliated epithelium of the upper air
278 BACTERIOLOGY
passages serves as a sweep to rid the body of foreign par-
ticles that may find lodgment upon it; and the acid reaction
of the gastric juice, low though it be, is thought sufficient
to render inert certain infective bacteria that enter the
alimentary tract by way of the mouth. Of all the defenses,
however, none are certainly of so much importance as those
to be detected within the internal structures of the normal
animal. In its warfare against the invasion of infective
bacteria and the activities of their poisonous products, the
most significant defenses possessed by the body are those
which directly aim at the destruction of the living germs of
disease and at the neutralization of their poisonous waste
products.
In so far as we now know the internal means of defense
used by the body in its warfare against infective bacteria
and their poisonous products are the phagocytic cells, such
as the leukocytes, the large mononuclear cells of the blood,
and the connective tissue and endothelial cells, and the
ill-defined vital substances in the circulating blood which
act, so to speak, as antidotes to bacterial poisons. If these
defenses are not of sufficient vigor to destroy the invading
bacteria, or to render inert the poisons produced by them,
the bacteria are victorious and infection results; on the
other hand, if there be failure to excite disease, the tissues
have been victorious, and are then said to be resistant to
or immune from this or that particular type of infection.
In some cases the protective agents possessed by the
animal organism act directly upon the invading parasites
themselves — i. e., they are germicidal; in others their
function is more that of antidotes, or neutralizes' in the
chemical sense, of the poisons produced by these parasites,
the parasites themselves, in certain instances, experiencing
only slight injury from a limited sojourn in the living tissues.
THE DEFENSES OF THE BODY 279
So far as we can learn the blood serum exhibits normally
a small amount of antitoxic, agglutinative, and bactericidal
action against a great variety of pathogenic bacteria. The
nature of the agents responsible for these activities is believed
to be identical with that of similar agents found in the
blood of artificially immunized animals, though in the latter
instance they are always present to a higher degree than
in normal animals.
To those ill-defined substances whose affinities are re-
stricted to the soluble toxins elaborated by the invading
bacteria the name "antitoxins" is now generally applied.
Contrary to what we have seen in the case of the germicidal
substances, normally present in the blood, antitoxins are
to be detected in the normal animal organism in very small
amounts. When they do exist under such conditions they
are of but comparatively feeble potency.1
In the great majority of instances antitoxic activities are
acquired peculiarities; acquired in some cases in a more or
less natural manner, as in the course of a non-fatal attack
of a specific malady; induced in others by purely artificial
means, as in the case of immunization from diphtheria
and tetanus.
Our acquaintance with the antitoxins extends little beyond
their physiological functions and some of .the means that
induce their generation. We have no satisfactory knowledge
of their intimate nature or of the primary sources of their
production. They are believed by some (Buchner2 and
Metchnikoff3) to represent, when artifically induced, bac-
1 See Bolton, Transactions of Association of American Physicians, 1896,
xi, 62. Pfeiffer, Deutsche med. Wochenschrift, 1896, No. 8. Fischl and
v. Wanschheim, Centralblatt fur Bakteriologie,' Parasitenkunde, und
Infektionskrankheiten, 1896, Abt. i, Bd. xix, S. 652. Wassermann, Berliner
klin. Wochenschrift, 1898, No. 1.
2 Miinchener med. Wochenschrift, 1893, Nos. 24 and 25.
3 Weil's Handbuch der Hygiene, Bd. ix, Lieferung 1, S. 48.
280 BACTERIOLOGY
terial toxins that have b'een modified by the vital action
of the integral cells of the body; and Roux1 and Buchner2
maintain that they exhibit their protective functions less
by direct combination with the toxins than by a specific
stimulation of the tissue-cells that enables the latter to
resist the harmful influences of the toxins. On the other
hand, Behring,3 Ehrlich,4 and their associates contend that
they are vital tissue elements, having the property of com-
bining directly with the toxins to form "physiologically
inert toxin-antitoxin" compounds that dre in a manner
analogous to the double salts of familiar chemical reactions.
Natural Immunity. — It is well known that among man
and the lower animals individuals are frequently encountered
who are, in general, less susceptible to infection than are
others of their species; and that particular species of animals
not only do not suffer naturally from certain specific diseases,
but resist all efforts to produce the diseases in them by
artificial methods; in other words, they are naturally im-
mune from them. The term "natural immunity," as here
employed, implies a congenital condition of the individual
or species, a condition peculiar to his idioplasm, which has
been transmitted to him as a tissue-characteristic through
generations of progenitors.
Acquired Immunity. — Again, it is often observed that an
individual or an animal after having recovered from certain
forms of infection has thereby acquired protection from
subsequent attacks of like character; in other words, they
are said to have acquired immunity from this disease. " Ac-
1Annales de 1'Institut Pasteur, 1894, p. 722.
2 Berliner klin. Wochenschrift, 1894, No. 4.
8 Infektion und Disinfektion, Leipzig, 1894, S. 248.
<Klinisches Jahrbuch, 1897, Bd. vi, Heft 2, S. 311. Fortschritte der
Medicin, 1897, Bd. xv, No. 2.
THE DEFENSES OF THE BODY 281
quired immunity" implies, therefore, a condition of the
tissues of an individual, not of necessity peculiar to other
members of the race or species, that has originated during
his life from the stimulation of his integral cells by one or
another of the specific infective irritants that may have
been purposely introduced, or accidentally gained access
to his body. Acquired immunity may be either . active or
passive in character.
Active Immunity. — Active immunity is that seen after
recovery from infection acquired in a natural way, or from
infection induced by the injection of dead or living organisms
or the poisons peculiar to them.
Passive Immunity. — Passive immunity is that condition
in which protective substances that have been generated in
a susceptible animal by one or the other methods of active
immunization are transferred directly from that animal to
•a normal animal by the injection of the blood serum of the
former into the tissues of the latter; the latter being as a
rule at once protected. The antitoxic serums have been
employed most frequently to bring about passive immunity.
The protective value of diphtheria antitoxin in those that
have been exposed to infection is well established. The use
of tetanus antitoxin for prophylactic purposes is also recom-
mended in cases where there is a possibility of the develop-
ment of tetanus.
Vaccination Against Bacterial Diseases. — The employment
of various prophylactic vaccinations against infectious dis-
eases has received much attention in recent years. The
measures employed in different diseases vary somewhat,
though in general the principles are similar.
The first measures of this nature that were employed on
a large scale are those of Haffkine in vaccination against
282 BACTERIOLOGY
cholera and plague by means of cultures that had been
killed by heating to a moderate temperature. Such dead
organisms when injected bring about a reaction in the body
which is manifested by a marked increase in the specific
agglutinative and bactericidal properties of the blood-
serum.
Wright introduced a similar method of vaccination against
typhoid fever. The prophylactic treatment consists of one
or more injections of dead cultures of bacillus typhosus.1
Metchinkoff and Besredka maintain that immunity is
less complete and is accompanied by more severe reactions
when induced by dead bacterial vaccines than when a small
quantity of "sensitized" living culture is employed.
With this in mind they prepare vaccines by subjecting
living cultures to the action of specific immune serum that
has been heated sufficiently to destroy its disintegrating
power. By this plan the haptophore side chains of the bac-
teria are saturated with specific immune bodies, manifested
by the agglutination of the bacteria. The agglutinated mass
is then washed to remove the serum, centrifuged and the
sediment used for vaccination. The subcutaneous injection
of vaccines so prepared is said to be followed by little or
no local pain and almost no constitutional reactions. These
advantages are attributed to the sensitization in vitro,
which would otherwise go on within the tissues and account
for the undesirable reactions.
The method of Gay differs from the foregoing in that the
sensitized living bacteria are killed by heating before they
are injected.
1 See chapter on Typhoid Fever.
THE DEFENSES OF THE BODY 283
Precipitins. — The immunization of animals with a variety
of substances other than bacteria has served further to de-
monstrate the complex mechanism of immunity. One of the
reactions that is noticed as the result of such immunization
is the precipitation observed when the serum of the immu-
nized animal is mixed with the substance with which it has
been treated. For instance, the serum of an animal that
has received repeated injections of blood, tissue juices or
certain secretions from alien species, will cause a precipi-
tate to form when mixed with either of these substances
in vitro. These "precipitins," as the newly formed bodies
in the blood of the treated animal are called, are specific
in that they form precipitates only with the materials
injected.
This precipitin reaction is so characteristic that it is
employed for the identification of blood in medico-legal
cases requiring the differentiation between human blood
and that of domestic animals; thus, the serum of a rabbit
into which human blood has been injected will cause a
precipitate with no other blood except that of the anthro-
poid ape.
In like manner, the repeated injection of milk of one
species of animal into the tissues of another will result in
the formation of specific precipitins in the blood serum of
the treated animal, that will precipitate only the milk of
that species of animal from which the milk was derived.
Agglutinins. — If the blood serum of an individual who has
recovered from a bacterial infection or who has been ren-
dered immune by bacterial vaccination be mixed with the
bacteria that caused the infection or those used in the vac-
cination— the bacteria, if motile, lose their motility and
finally clump together in masses, i. e., they are "agglu-
284 BACTERIOLOGY
tinated" by the serum; the reaction being referable to the
presence of a new body — "agglutinin" — that has appeared
in the blood as a result of the infection or the vaccination.
The relation of this newly formed antibody is specific, i. e.,
it agglutinates only those agents that called it forth. In
the normal blood agglutinating activity may often be de-
monstrated for a variety of bacteria (Bergey) but it is never
as high in potency as is that which may be artificially induced,
or that seen early in convalescence from a number of infec-
tions.
The agglutinating properties of an immune serum are not
indicative of the degree of immunity possessed by the indi-
vidual from whom the blood was drawn. There may be a
relatively high degree of agglutinating property with no
demonstrable correspondence in germicidal or protective
activity. Though no parallelism necessarily exists between
the degree of agglutinating and that of germicidal or bac-
teriolytic activities of an immune serum, it is nevertheless
true that both qualities develop as a result of an effort on
the part of the tissues to resist infection, and both may
represent a response to the same stimulus.
The specificity of the agglutinating reaction has proved
of use in the identification of infective bacteria, and con-
versely, in the recognization of diseases resulting from
bacterial invasion. For instance: given an unidentified
bacterium of the colon — typhoid — dysentery group that
is agglutinated by the serum from a case either of experi-
mentally induced or naturally acquired typhoid fever and
is not agglutinated by serum from a dysentery case or one
of colon infection — in all human probability that organism
is the typhoid bacillus; or given the serum from a patient
suffering from an undetermined febrile disease that agglu-
THE DEFENSES OF THE BODY 285
tinates Bacillus typhosus and no other organism, that patient
in all probability is suffering from typhoid fever. This
latter application of the reaction constitutes what is gener-
ally known as the Widal reaction.
Immunity: Historic Sketch. — In the course of our studies
aimed to secure light on the mechanism of infection, two
phenomena are constantly in evidence, notably — first,
that not all individuals are susceptible to infection by all
pathogenic bacteria, and next, that an individual who has
recovered from infection has undergone a change during
the course of the disease that, as a rule, renders him
insusceptible to subsequent infection by the same species
of bacteria. Individuals in either the one or the other
state are said to be immune; in the former to be immune by
nature, in the latter to have acquired immunity.
In its present development there is no more fascinating
subject, and none of broader biological significance than
that involving this riddle of immunity. For a quarter of
a century it has attracted the attention of the most brilliant
investigators in medicine and its cognate fields, and, though
much has been learned, it is as yet far from fully elucidated.
It is obviously inadvisable in a work of this character to
follow in detail the manifold lines of investigation aimed to
clear up this matter. We shall content ourselves, therefore,
with a statement of the significant results and such discus-
sion of them as may be necessary to indicate their bearings
upon the problem.
Knowing as we now do that infection is at bottom a
matter of intoxication, and believing, as we are led to
do by Ehrlich and his pupils, that intoxication is to be
interpreted as a destructive union, in the chemical sense,
between the poisons on the one hand and cells or parts
286 BACTERIOLOGY
of cells for which they have an affinity on the other,
natural resistance or immunity from one or another type
of infective organism may be interpreted in several ways,
namely — that the naturally immune animal is by nature
devoid of those cells or parts of cells for which the poison
of the infective organism, from which it is immune, has a
specific destructive affinity; or, that the animal is by nature
endowed with cells, parts of cells or products of cell life that
serve as antidotes for the poison of the infective organism
in question; or, again, that certain cells of the immune
animal have the power to actually destroy the infective
organism when it gains access to the body, thereby not only
preventing its growth and multiplication, but simultaneously
rendering inert the poisons liberated as a result of its
disintegration.
Long before the present state of our knowledge on this
subject had been reached, observers who were occupied
with the study of infection had' offered certain explanations
for the occasional failure of their efforts to cause disease by
inoculation. In the majority of cases such doctrines or
hypotheses were offered in connection with the immunity
that had been acquired. This is not surprising, since artifi-
cially induced immunity — L e., acquired immunity — is a
constitutional state that is more or less under the control
of the experimentor, while natural immunity is an heredi-
tary, idioplasmic peculiarity that can be modified little if
at all by any of the known experimental procedures.
Among the first to offer an explanation for the condition
of acquired immunity was Chauveau, who, in 1880, sug-
gested that the immunity commonly observed in animals
that had recovered from a specific infection, and likewise
immunity produced artificially by vaccination, is referable
ELIE METCHNIKOFF
1845-1916
THE DEFENSES OF THE BODY 287
to a product of the infective organisms that is retained in
the tissues, and which, by its presence serves to prevent
the development of the same species of organisms should
they subsequently gain access to the tissues. This doctrine
is usually known as Chauveau's "Retention Hypothesis of
Immunity." We shall see later that it is only in small part,
if at all, a tenable theory.
As opposed to Chauveau, Pasteur and his pupils, in the
same year (1880), expressed the opinion that acquired
immunity was to be explained in just the reverse way to
that conceived by Chauveau. They believed that in the
primary attack of infection something was extracted from
the tissues by the infecting organisms that was necessary to
support the growth of the same species should it subse-
quently invade the body. This doctrine is known as Pas-
teur's "Exhaustion Hypothesis" of Immunity, and has
apparently little claim to serious consideration.
Four years later (1884) Metchnikoff, while engaged upon
the study of certain lower forms of animal life, noticed
that particular mesodermal cells, in the course of their
wanderings through the body, had the power to actually
pick up insoluble particles that had gained access to it in
one way or another. He looked upon them as functioning,
therefore, as scavengers. These phagocytes, as they are
now generally known, are common not alone to the lower
forms of life, but to the most highly organized as well. In
the higher forms of animal life, the function of phagocytosis
is conspicuously exhibited by the wandering cells— i. e.,
the white blood corpuscles. In a lower degree the inclusion
of foreign bodies with their subsequent digestion or disin-
tegration may occasionally be seen in other cells as well.
Metchnikoff believed this phagocytic power to be the
288 BACTERIOLOGY
most important defensive mechanism possessed by the body,
and believed both natural and acquired immunity to be
referable to it; in the former case regarding it as a natural
endowment, in the latter as a function that had been excited
by the specific stimulus offered by the organisms or their
poisons that were concerned in the primary attack of disease
from which the animal recovered, or by the organisms used
in purposely exciting a modified form of the disease by one
or another of the modes of protective vaccination.
As the phenomenon of phagocytosis could easily be ob-
served under the microscope, and its observation therefore
accessible to all interested in the question, the plausibility
of the doctrine at once attracted many adherents, and
Metchnikoff s views were everywhere accepted as the prob-
able explanation of the defensive mechanism of the body
against infection.
In a little while, however, Fluegge, of Breslau, perceiving
the incompetency of both Chauveau's and Pasteur's doc-
trines, observing occasional inconsistencies in Metchnikoff s
teaching, and recalling certain significant reactions of the
blood that had appeared in the course of experiments by
Traube and Gscheidlen, by Fodor, by Rauschenbach, and
by Grohmann, determined to subject the whole question
to an experimental critical review.
To Nuttall, an American working in his laboratory, was
assigned the question of determining if the cell-free blood,
or the plasma, was, as had been suggested by Grohmann,
possessed of germ-destroying properties. Nuttall's work
resulted in a blow to MetchnikofFs doctrine that for a long
time seemed to be fatal. He demonstrated that certain
virulent bacteria were rendered incapable of development,
incapable of infecting susceptible animals, and, in short,
THE DEFENSES OF THE BODY 289
killed by exposure to the serum of animal blood free of all
cellular elements. These results naturally caused defections
from the ranks of MetchnikofFs followers, especially since
Nuttall's deductions were fully confirmed by many dis-
tinguished experimenters. In consequence, for a number of
years after Nuttall's work, the cell-free fluids of the body
were regarded as the real defenses of the body in so far as
invading bacteria were concerned.
The natural sequel of NuttalPs demonstration was a
general curiosity as to the manner in which the destruction
of 'bacteria was accomplished by the cell-free serum; the
conditions that modify the phenomenon; and the nature of
the ingredient of the serum to which the germicidal activity
might properly be referred.
Buchner demonstrated that active serum was robbed of
its germicidal power by dilution with water and by dyaliza-
tion; that it was not affected by dilution with physiological
salt solution; that it was rendered inert by an exposure of
fifty minutes to 55° C., and that it was not affected by
alternate freezing and thawing. He concluded that the
element of the blood to which the function of killing bacteria
may be ascribed is a living albumen and suggested "alexin"
as the appropriate designation. Hankin and Martin believed
the active germicidal principle to be a globulin, a view that
was to some extent -suggested by the investigations of Ogata
and of Tizzoni and Cattani; while the investigations of
Vaughan and of Kossel led them to regard nucleins as the
most important constituents of the blood in so far as germi-
cidal action is concerned. Fodor believed, as a result of his
experiments, that the antibacterial action of the blood could
be appreciably accentuated by the addition of alkalies.
While Baumgarten and certain of his pupils referred the
19
290 BACTERIOLOGY
death of the bacteria to purely physical conditions; believing
that their exposure to blood serum having an osmotic tension
different from the fluids in which they had been growing
resulted in disturbances of the bacterial protoplasm that
were inconsistent with bacterial life.
By the observations of Behring and Kitasato and of Roux
and Yersin entirely new light was thrown upon the subjects
of infection and immunity and a new field of inquiry, was
opened. Through the work of these investigators and their
pupils upon tetanus and diphtheria it was demonstrated
that immunity was, at least in certain diseases, not so much
a matter of actually destroying the invading bacteria as of
neutralizing their poisons.
The outcome of these investigations established the fact
that if the poisons of tetanus bacilli or of diphtheria bacilli,
entirely free from the germs themselves, be injected into
susceptible animals in minute sublethal doses the animals
presently acquired immunity from both poisons and living
organisms. Furthermore, that the blood serum of animals
thus immunized had the power when transferred directly
to normal animals of at once rendering them insusceptible
to infection by the living germs, and of equal importance,
that if the blood serum of an animal thus immunized be
added to the bacteria-free poison of either the tetanus or
diphtheria bacillus in a test-tube that the poison was neu-
tralized, i. e.j the serum of the animal acted as an antedote
which rendered the bacteria poison inert.
It is obvious therefore that through the injections into
the normal animals of non-fatal quantities of the specific
bacterial poisons the tissues had been stimulated to react
in a manner quite in harmony with the views of Buchner
expressed in 1883, to the effect that the immunity seen in
THE DEFENSES OF THE BODY 291
an animal that has recovered from a specific infection is
explainable by a "reactive change" that has occurred in the
tissue cells, as a result of the primary infection or intoxica-
tion, which serves to protect the animal from subsequent
attacks of a similar character.
The demonstration that the serum of an artificially
immunized animal can not only confer immunity upon
another animal but, in the case of tetanus and diphtheria
in particular, actually cure it after the disease is in progress,
is one of the most important steps that has been made in
this entire field of inquiry. The triumph resulting from the
practical application of this principle to the prevention
and cure of diphtheria in man fairly marks an epoch in
modern medicine. Though the results attendant upon the
application of that principle to the prevention and cure of
a number of other diseases — Asiatic cholera, typhoid fever,
lobar pneumonia, infection by the pyogenic cocci, rabies,
tuberculosis, plague, syphilis, and snake bites — have met
with comparatively indifferent success, still the knowledge
gained through these efforts has been of inestimable value
in stimulating researaches that have served to indicate not
only the manifold nature of this complex problem but have
led to discussions through which some of its most obscure
•phases have been illuminated.
Briefly stated, the outcome favors the conclusions that
the mechanism of immunity varies in different diseases, i. e.,
that it depends upon the specific peculiarities of the invading
bacteria. In some instances it is manifested as an effort on
the part of the tissues to neutralize bacterial poisons, the
bacteria themselves remaining unaffected; in others as an
actual destruction, disintegration or digestion of the invad-
ing bacteria together with the neutralization of such intra-
292 BACTERIOLOGY
cellular poisons as may be bound up as integral portions of
their constituent protoplasm.
Furthermore, in so far at least as induced immunity is
concerned, the bulk of the experimental testimony supports
the opinion that the reaction is specific; that is to say, be
the systemic reaction evidenced as the elaboration of an
antidote to a soluble poison or as increased facility to destroy
living bacteria, it is called forth only through the specific
stimulus afforded by the injection of the animal with the
particular poison or bacterium from which we desire to
protect it. Thus, for instance, an animal rendered immune
from tetanus toxin, is not immune from diphtheria toxin
or from the inroads of diphtheria bacilli; similarly an animal
immune from any of the pathogenic species of bacteria is
immune from that species only and not necessarily from any
others.
An observation of fundamental importance to an under-
standing of the mechanism of immunity was made by R.
Pfeiffer in 1895. While investigating Asiatic cholera he
found that animals could be immunized from the specific
endotoxin of the organism causing that disease; that the
blood serum of such immune animals when injected into
normal animals protected them from what would otherwise
be a fatal d®se of the cholera spirillum; that the peritoneal
fluids of the artificially immunized animal had an almost
instantaneous bacteriolytic, i. e., disintegrating, action upon
living cholera spirilla that were injected directly into the
peritoneal cavity; that the serum from the immune animal
had no such effect upon cholera spirilla in a test-tube, but
if virulent cholera spirilla were injected into the peritoneum
of an animal that is not immune, and that such injection
be followed immediately by an intraperitoneal injection of
THE DEFENSES OF THE BODY 293
blood-serum from an immune animal, almost instantly the
peculiar disintegration of the bacteria that was observed in
the peritoneum of the immune animal was to be seen. As
we shall learn presently this observation is of the utmost
importance and its bearing upon the course of certain sub-
sequent events will soon be manifest.
The significant features of Pfeiffer's observation are that
while the blood serum of an immune animal is capable of
conferring immunity upon a susceptible animal, yet, in a
test-tube it exhibits none of the bacteriolytic activity con-
stantly to be noted in the body of the immune animal; on
the other hand if a small quantity of it be injected into
the peritoneal cavity of a normal, susceptible animal, the
phenomenon of bacteriolysis, hitherto absent, at once makes
itself manifest. Clearly the serum requires the cooperation
of something within the body of the living animal to bring
about the disintegration of bacteria. The phenomenon must
therefore be the result of a composite function.
Though Nuttall's work materially lessened the number
of adherents to the phagocytic doctrine of Metchnikoff
there was still a group of active workers who retained their
belief in the fundamental soundness of the idea. Metch-
nikoff himself never swerved. Without entering into a
discussion of the many instructive investigations upon the
questions of phagocytosis it will suffice for our purposes to
state briefly their culmination. We now know, through
the studies notably of Bail and of Kikuchi that on the one
hand phagocytosis may be inhibited, and by the demon-
strations of Wright and Douglass, in particular, that, on
the other, it may be accentuated. Bail, believing the real
defenses of the body to be cellular, attributes the failure
of the cells to protect from infection to an inhibition of
294 BACTERIOLOGY
their defensive powers by a substance, "aggressin," elabo-
rated by the invading bacteria. While Kikuchi, accepting
the "aggressin" doctrine, restricts the action of "aggressin"
to the leukocytes and interprets it as in the nature of a
negative chemotactic phenomenon, whereby the leukocytes
are so repelled that they cannot approach and take up the
bacteria.
The efforts of Wright and Douglass have been in the way
of accentuating phagocytic activity and their results have
shed a flood of most important light upon the subject. In
1903 and 1904, in papers presented to the Royal Society
of London, they express the opinion that leukocytes alone
are incapable of taking up bacteria, and that in order for
them to exhibit this function the bacteria must first be
acted upon by a something contained in the normal
blood, a state of affairs analogous to that observed by
Pfeiffer. They conceived this preparation of bacteria
for ingestion by leukocytes to be in the nature of the pre-
paration of food for consumption. They employ the term
"Opsonin," (meaning to cater for; to prepare food) in
designation of the element in the blood having that property.
Prior to the observations of Wright and his associates it
had been known that if white blood cells be washed free of
all adhering serum they are incapable of taking up bacteria,
but the interpretation, in the light of Wright's work, seems
to be incorrect. It was believed that a something in the
blood, a "stimulin" as it was called by some, acted not on
the bacteria but on the leukocytes, stimulating them to
activity. Wright and his colleagues have clearly shown the
error of that view and have convincingly demonstrated that
it is the action of their "opsonin" on the bacteria that makes
phagocytosis possible. Thus, for instance, if bacteria and
THE DEFENSES OF THE BODY 295
washed leukocytes be brought together the bacteria are not
taken up by the cells; if on the other hand a drop of normal
serum be added, phagocytosis begins. Or, if bacteria be
immersed in normal serum and then carefully cleaned of all
adherent serum by washing .they will readily be taken up
by leukocytes, even those also freed of all serum by careful
washing. In short the action of the serum on the bacteria,
through its "opsonin," has been to make them ingestible or
digestible for the leukocytes.
This opsonizing property of the blood varies. Under
conditions depressing general health it may be diminished;
while in the course of infective diseases it is sometimes
lessened, sometimes increased. It may be increased by
immunization.
The nature of opsonin (or opsonins) is not known. It has
been suggested that they are allied to the enzymes. They
are destroyed by heat. They may be absorbed entirely
from the blood by bacteria with which they combine. They
are unstable, becoming gradually inert after withdrawal
from the body.
In consequence of these later investigations the phago-
cytes are again to the fore as one at least of the important
defenses of the body and certainly, in so far as the destruction
of invading bacteria is concerned, many have come to look
upon them as, after all, just what Metchnikoff originally
regarded them, the true scavengers of the body.
Though the destruction of bacteria by the fluids of the
body had been demonstrated; though their inclusion and
digestion by phagocytes could readily be observed; though
an antidote for certain of their poisons could be demon-
strated in the blood of immunized animals, there was still
296 . BACTERIOLOGY
wanting an explanation of the mechanism through which
these interesting phenomena were accomplished.
Omitting a group of highly suggestive observations made
by many competent investigators, we encounter the most
elaborate and at the same time the most fascinating effort
to interpret the nature of the reactions occurring in the
induction of immunity as well as those fundamentally
accountable for the natural condition.
To the genius of Ehrlich1 we owe the "side chain" or
"lateral band" theory (Seitenkettentheorie) of immunity.
Its fundamental features comprise the acceptance of
Weigert's doctrine concerning the mechanism of physiolog-
ical tissue-equilibrium and repair; and the assumption of a
specific combining relation, or affinity, between toxic sub-
stances and the cells of particular tissues.
At the meeting of German Naturalists and Physicians
held at Frankfort-on-the-Main, in 1896, Weigert2 advanced
an hypothesis the essential features of which are that
physiological structure and function depend upon the
equilibrium of the tissues maintained by virtue of mutual
restraint between its component cells; that destruction of
a single integer or group of integers of a tissue or a cell
removes a corresponding amount of restraint at the point
injured, and, therefore, destroys equilibrium and permits of
the abnormal exhibition of bioplastic energies on the part
of the remaining uninjured components, which activity may
be viewed as a compensating hyperplasia; that hyperplasia
is not therefore the direct result of external irritation, and
cannot be, since the action of the irritant is destructive and
1 Klinisches Jahrbuch, 1897, Bd. vi, Heft 2, S. 300.
2 Neue Fragestellungen in der pathologischen Anatomie, Verhandlungen
der Ges. deutschen Naturforscher und Aerate, 1896, S. 121.
PAUL EHRLICH
18S4-191S
THE DEFENSES OF THE BODY 297
is confined to the cells or integers of cells that it destroys,
but occurs rather indirectly as a function of the surround-
ing uninjured tissues that have been excited to bioplastic
activity through the removal of the restraint hitherto
exerted by the cells destroyed by the irritant; and, finally,
when such bioplastic activity is called into play there is
always fo/percompensation — i. e., there is more plastic
material generated than is necessary to compensate for the
loss. Ehrlich applies this idea to the individual cell, which
he conceives to be a complex molecule, comprising a primary
central nucleus to which are attached by side chains its
secondary atom-groups, in much the same way that our
conception of the reaction structure of complex organic
chemical compounds is represented graphically. Injury to
one or more of these physiologically essential atom-groups
results, according to the view of Weigert, in disturbance
of the cell-equilibrium and consequent effort on the part
of the surrounding atom-groups at compensatory repair.
With this liberation of bioplastic energy there is more
plastic material generated than is necessary for the repair
of the injury. The excess of this material finds its way into the
blood and, as we shall see presently, is regarded by Ehrlich
as the real antidotal, immune, or protective substance.
Assuming a specific combining relation between toxic sub-
stances and particular cells or secondary atom-groups of
cells — and there are experimental grounds for this assump-
tion1— it is evident that the combination between the intoxi-
cant and the particular atom-group for which it has a specific
1 See Wassermann und Takaki, Ueber tetanus antitoxische Eigen-
schaften des normalen Centralnervensystems, Berliner klin. Wochen-
schrift, 1898, No. 1, S. 5. Neisser und Wechsberg, Zeitschrift fur Hygiene
und Infektionskrankheiten, Bd. xxxvi, S. 299. Madsen, ibid., Bd. xxxii, S.
214.
298 . BACTERIOLOGY
affinity is indirectly the cause of compensatory bioplastic
activity on the part of similar surrounding atom-groups
that have not been destroyed. This results, as we learned
above, in hypercompensation, the excess of plastic material
being disengaged from the parent-cell and thrown free into
the circulating fluids, there to combine directly with the
same intoxicant should it subsequently gain access to the
animal. This excess of plastic material thrown into the
circulation combines, according to Ehrlich,1 directly with
the intoxicant to form physiologically inactive "toxin —
antitoxin' ' compounds, and can therefore be reasonably
regarded as the antitoxic material of animals rendered
immune from bacterial and other toxins.
Since the announcement of that doctrine many important
advances have been made in our knowledge of the subject.
We have learned that the reactions of immunity or tolerance
may be induced by the use of other intoxicants than those
elaborated by bacteria, and by the employment of other
cells and cell secretions. It has been demonstrated that
antibodies, differing in their specific actions from anti-
toxins, but originating probably in a similar manner, are
to be detected in the fluids of animals thus immunized .or
rendered tolerant. For a long time we have known of the
germicidal action of normal blood serum; since 1895 we
have been familiar with the singular bacteriolytic phenome-
non demonstrated by Pfeiffer in the peritoneum of animals
immune from cholera; later we learned that the development
of immunity from a variety of infections is accompanied
by a power on the part of the serum of the immune animal
to agglutinate the bacteria causing the infection; the work
1 Zur Kenntniss der Antitoxinwirkting, Fortschritte der Medicin, 1897,
Bd. xv, No. 2.
THE DEFENSES OF THE BODY 299
of Wright upon his opsonic doctrine has finally placed the
leukocyte among the important defenses of the body and
the profoundly interesting investigations of Bordet, Moxter,
von Dungern, Fish, and others, have shown that immunity
reactions may be induced with cells and secretions of animal
origin hitherto regarded as non-irritating and harmless. For
instance, we have long known that the blood of one animal
may cause fatal intoxication when injected into an animal
of different species; but later we learned if that blood be
repeatedly injected in non-fatal amounts, the animal receiv-
ing the injections after a while becomes tolerant, and its
serum reveals the property not only of robbing the alien
blood of its hurtful properties, but also of actually dissolv-
ing its corpuscles in a test-tube (hemolysis). In an analogous
way, if such tissue-cells as epithelium or spermatozoa be in-
jected repeatedly into the tissues of animals, the serum of
the blood of those animals acquires* the power of agglutinat-
ing and finally dissolving (digesting) such cells outside the
body; and if so inert a secretion. as milk be injected into
the tissues, the blood serum of the animal receiving the
injections after a time reacts specifically with that milk in
a test-tube — i. e., precipitates it.
From the foregoing we see that in the numerous phases
and expressions of this physiological possibility there are
produced antibodies having functions totally different from
those attributed by Ehrlich to antitoxins — i. e., we have
"lysins," " agglutinins," "precipitins," " aggressins," "op-
sonins," etc., that in their mode of action suggest ferments
with specific affinities. It is evident that when broadly
conceived the mechanism of immunity comprehends very
much more than the neutralization of a bacterial toxin
by an antitoxin; and, what is more to the point, in many
300 BACTERIOLOGY
of these conditions of immunity or tolerance above noted
antitoxins, as we know them, are not present at all.
In an important series of papers on the hemolysins pub-
lished by Ehrlich and Morgenroth1 an effort is made to
elucidate further the finer mechanism of immunity in its
broad sense and various expressions, and to adapt the side-
chain doctrine to those more complicated phenomena in
which immunity depends not only on the elaboration of
antitoxins, but also upon a power on the part of the animal
fluids to cause a complete metamorphosis or disappearance
of such particulate matters as bacterial and other irritating
or poisonous cells and substances. They believe the forces
at work in the establishment of immunity from bacteria
and from bacterial and other toxins, those operative in the
elaboration of the newly discovered lysins, antilysins,
agglutinins, precipitins, ferments, antiferments, etc., as
well as those concerned' in physiological assimilation and
nutrition, to be fundamentally identical. They believe
susceptibility to infection, as well as power to assimilate
nutrition, to be explainable through the assumption that
special molecular groups of the living protoplasm are endowed
with specific combining affinities for particular matters; and
in so far as the establishment of disease is concerned, they
regard the receptivity of the individual to be determined
entirely by the greater or less susceptibility of those pro-
toplasmic molecular groups — "receptors," as they designate
them — to disease-producing agents. In individuals that
have been artificially immunized from hurtful substances
they believe (in reiteration of Ehrlich's view expressed
1 Berliner klinische Wochenschrift, 1899, Bd. xxxvi, S. 6 and 481 ; 1900,
Bd. xxxvii, S. 458 and 681; 1901, Bd. xxxviii, S. 251, 569, 598. See also
Schlussbetrachtung: Ehrlich in Nothnagel's Speciellen Pathologic und
Therapie, Bd. vii, Theil 1, Heft 3, S. 161.
THE DEFENSES OF THE BODY 301
above) that the receptive molecules have been more or less
multiplied, according to the degree of immunity, through
bioplastic activity of similar, unimpaired atom-groups
surrounding those more directly influenced by the intoxicant
during the process of immunization; and that this excess
of such "receptors/' although physiologically useless, being
of no known service to normal function, circulates unchanged
in the blood, and serves, through specific combining affinity
for the poison against which the animal has been rendered
immune, to protect the normal tissues from its hurtful
action.
According to the nature of the irritant from which the
animal has been immunized, the " receptor' ' is conceived
to be either of simple or complex construction, and its pro-
tective function to be performed in either a comparatively
simple and direct way, or in a more or less complicated and
roundabout manner.
As a result of his studies of toxins, Ehrlich reached the
conclusion that they are composed of at least two function-
ally distinct atom-groups: the one, a "haptophore" group,
characterized by its combining tendencies; the other, a
"toxophore" group, distinguished for its intoxicating powers;
and that for the exhibition of its hurtful characteristics a
toxin molecule needs to be first anchored, so to speak, to
the susceptible tissue by the "haptophore" group, after
which its intoxicating characteristics are exhibited by the
"toxophore" group. He conceives the "receptors" to be
likewise provided with "haptophore" groups that pair with
the corresponding " haptophores" of the poison to which
the animal is susceptible or from which it has been immu-
nized. Where immunization has been induced against such
relatively simple substances as toxins, ferments, and certain
302 BACTERIOLOGY
cell secretions, the "receptors" and their functions are com-
paratively simple — i. e., the single haptophore of the simple
receptor pairs with that of the intoxicant and a physiologi-
cally inert complex results. He conceives antitoxins to be
simple receptors of this type, and believes the neutralization
of toxins by them to take place in this manner. On the
other hand, if the immunization of an animal is accompanied
by an acquired power on the part of its serum to disintegrate
bacteria, to dissolve alien erythrocytes, to digest such cel-
lular elements as epithelium and spermatozoa, to precipitate
milk, or agglutinate bacterial or blood-cells, as the studies
of Pfeiffer, Bordet, von Dungern, Moxter, Fish, Belfonte
and Carbon, Metchnikoff, Gruber, Durham, Widal, and
others have demonstrated, then the process becomes less
simple, and the atomic grouping of the receptive molecule
is correspondingly more complex. In some cases the recep-
tor is provided with both a haptophore and a ferment-like
(zymophore) group; the function of the former being to
combine with and hold in close proximity to the latter the
albumin molecule that is to be destroyed or assimilated; in
this way bringing and holding the albumin molecule directly
under the influence of the zymophore group. In other cases
the "receptor" functions symbolically, so to speak, with
a complementary something that circulates normally in the
blood, the so-called "complement" of Ehrlich and Mor-
genroth. Under these circumstances the "receptor" is
conceived to be provided with two "haptophore" groups,
and becomes an "amboceptor," therefore, the one hapto-
phore of which takes up and fixes the invading bacteria,
tissue-cell, or albumin molecule, while the other pairs with
the corresponding haptophore of the complement, fixing
the latter in close proximity to the invading body, and
Fig. S2
Receptors of the 1st order (Ehrlich)
Fig. ' — Normal cell, with
receptive molecular groups,
gen
multiplicity of
Attacking anti-
Fig. 2 — Cell attacked at its specifically recep-
tive point by toxin molecule ( 'I I )•
Fig. 3 — Cell equilibrium destroyed by com-
bination of toxin molecule with specific re-
ceptor (O-H-).
Fig. 4 — Cell in hypercompensation in effort to
repair injury indicated in 3. Specific receptive
molecules produced in excess (^—). All not
required for repair float free in the blood as
antibodies — in this ease as antitoxin* "* ,
Fig. 5 — Cell repaired. Antibodies floating
free. One combined \vith toxin.
Fig. 53
Receptors of the 2d order (Ehrlich).
Fig. 1 — Normal cell, with multiple specifi-
cally receptive molecular groups. Attacking
antigen (— ^).
Fig. 2 — Cell attacked at specifically recep-
tive point by antigen (— «4). This may be
bacteria, bacterial extractives, or proteins,
formed or amorphous. They stimulate the pro-
duction of agglutinins, eoagulins, precipitins,
etc.
Fig. 3 — Cell with equilibrium disturbed by
union of receptive cell* molecule \vith its
specific antigen / ErO\ •
Fig. 4 — Cell in hypercompensation in effort
toward repair. Excess of bioplastie matter
This represents the antibodies:
preeipitins, agglutinins, etc., of this order.
Fig. 5 — Cell restored. Excess of antibodies
float free. One seen in combination with its
antigen.
Fig. 54
Receptors of the 3d order (Ehrlich).
Fig. 1 — Normal cell, -with multiple receptive
molecular groups. Attacking antigen (~J*)-
Complement in surrounding fluids =C. Note
that no complement is fixed by the cell re-
eeptors.
O
Fig. 2 — Antigen attached to its specific affin-
ity. Complement is at once fixed
(4)
Fig. 3 — Amboeeptor with attached antigen
and complement disengaged from cell, cell
equilibrium thereby impaired.
Fig. 4. — Cell in hypercompensation in effort
toward repair. Excess of bioplastic matter
((JJM represents antibodies of this o.J?d)3^T— ^yiC^h,1
as bacteriolysins, hemolysins, cytoV/srri^, ^&tc\ •<
O
r^0JL°,o*-
Fig. 3 — Cell restored. Excess of antibodies ^'
free. No complement engaged, except in the
ease of union bet\veen antibody and antigen.
THE DEFENSES OF THE BODY 303
thereby favoring the immediate destructive activity of its
"zymotoxic" group.
It is of importance to note in connection with this hypothe-
sis, that both "receptors" and "complement" are present
in normal susceptible, as well as in immune animals, but
that during immunization only the "receptors" are multi-
plied as a result of the specific stimulation necessary to the
establishment of immunity, hence the commonly employed
synonymous designations: "immune bodies" and "anti-
bodies." As such bodies are generated during immuniza-
tion, the substance used for the purpose is designated
"antigen" — i. e., generator of antibodies.
The Origin of Complement. — The -origin of complement is
a question that is still unsolved. Some investigators are
inclined to believe that it is .derived from the leukocytes.
This is the opinion of Metchnikoff and his associates, while
others believe that it is derived from other cells and organs
as well as from the leukocytes. Again other investigators
believe that it is not derived from the leukocytes at all,
but from the cells of certain organs, for instance, the spleen
pancreas, liver, and the bone marrow. It is impossible with
the knowledge at hand to state definitely the origin of the
complement.
On the Specificity of Complement. — According to Ehrlich
and his pupils the term "complement" is to be used generi-
cally to indicate a group of closely allied bodies differing
from one another in that they possess specific relations to
particular antigens. By appropriate methods they claim
to have demonstrated the multiplicity of complement. They
state that by particular treatment one or more complement-
ary bodies may be removed from normal blood while others
remain in the blood intact; even by such mechanical pro-
304 BACTERIOLOGY
cedures as filtering through porcelain some complements are
held back while others pass through with the serum.
On the other hand, evidence afforded by the investigations,
particularly of Buchner, and Bordet and his pupils point in
the opposite direction so insistently as to justify some doubt
of the accuracy of Ehrlich's views. Probably the most
important evidence in favor of the unity of complement, as
conceived by these investigators, is afforded by the every day
tests for fixation of complement (to be described later). In
the light of these tests "complement," it seems, must be
nonspecific in its physiological activities, therefore it is a
unit.
SUMMARY. — According to the nature of the intoxicant
from which the individual is immunized, the one or the other
of the structurally and functionally different types of recep-
tors is increased — i. e., in immunity from a simple toxin the
simplest type of receptor, the antitoxic, appears in the blood
(receptors of the first order, Ehrlich); in immunity that is
associated with agglutinating or precipitating powers on
the part of the blood-serum receptors having a haptophore
and a zymophore group appear (receptors of the second
order); while in immunity from such molecular complexes
as blood-, tissue-, or bacterial cells there are produced
receptors of the third order, which act through their hapto-
phore groups as intermediate links between the body to be
destroyed and the normally present ferment-like comple-
ment that is to bring about the destruction. For all the
foreign cellular irritants from which animals have been im-
munized, be they alien blood, tissue-cells, milk, or bacteria,
there is assumed to be circulating normally in the blood
"complement" on the one hand, and specific "receptors"
on the other. This idea of plurality for the complement
THE DEFENSES OF THE BODY 305
is apparently the vulnerable point in the argument
(see above ''On the Specificity of Complement")- At all
events, it has been vigorously assailed by Bordet and
Buchner, especially, who as said above, consider the
complement a unit, and who do not regard it as pos-
sessed necessarily of specific affinities beyond those com-
mon to what may be termed proteolytic enzymes in
general; and Buchner regards it as nothing more than the
normally present "alexin," to which he called attention
years ago, while with equal warrant Wright might regard it
as the "opsonin" on which he has made such instructive
studies. Whether these objections be well taken or not,
whether the doctrine as a whole can be accepted or not, the
experimental data on which it is based justify the opinion
that it is the only satisfactory working hypothesis that has
been offered in explanation of the mechanism of what
Buchner years ago designated the "reactive tissue-changes"
underlying the establisment of acquired immunity.1 Ehr-
lich's conception may be graphically represented as follows:
The observations serving as the basis for this doctrine
have given to the blood and fluids of the body a new and
peculiar interest. According to circumstances, there may
be detected in the blood and tissue juices a number of mole-
cular complexes having totally different functions and
affinities, and therefore presumably different from one
another:
First, there is normally present in the blood serum of
practically all animals the defensive "alexins" already
mentioned.
1 Justice cannot be done to the beauty and ingenuity of this conception
in so brief a summary as is appropriate to a text-book. To be appreciated it
must be read as it came from the authors.
20
306 BACTERIOLOGY
Second, there are the antitoxins, found in the blood of
animals artificially immunized from special sorts of intox-
ication, as well as occasionally in the blood and tissues of
normal animals, the functions of which are susceptible of
demonstration outside the body as well as within the tissues
of the living animal.
Third, a body possessed of disintegrating, bacteriolytic
powers, a bacteriolysin — i. e., having the property of actually
dissolving bacteria, so that the phenomenon may be observed
under the microscope. This phenomenon, generally known
as "Pfeffer's Phenomenon/' is especially to be seen within
the peritoneum of guinea-pigs that have been rendered im-
mune from Asiatic cholera and from the typhoid and colon
infections and intoxications. It is not to be confounded
with the ordinary bactericidal function of the alexins that
is demonstrable in most normal serums.
Fo'urth, a body, the so-called "agglutinin" (Gruber),
that was considered by Widal to represent a " reaction of
infection," and not of immunity; though now its presence is
generally supposed to indicate or coincide with an effort on
the part of the body to resist infection. The presence of this
body in a serum of an animal is announced by its peculiar
influence on the activity and arrangement of the particular
species of bacteria from which the individual is immune, or
with which it is infected. In the case of typhoid fever in
man, for instance, the serum obtained during the early and
middle stages of the disease, when mixed with fluid cultures
or suspensions of the typhoid bacillus, causes the bacilli to
lose their motility and to congregate (agglutinate) in masses
and clumps, a condition never seen in normal cultures of
this organism, and practically never observed when normal
serum is employed instead of the typhoid serum. The
THE DEFENSES OF THE BODY 307
blood of animals artificially immunized from cholera, pyo-
cyaneus, typhoid, dysentery, and colon infections also show
the presence of "agglutinin." So far as experience has gone,
this agglutinating property is manifested in the great major-
ity of cases only upon the particular organisms from which
the animal supplying the serum is protected; that is to say,
the relation is specific. In view of the fact that the power
of a serum to agglutinate bacteria is regarded by many as a
concomitant of infection, the exhibition of this property by
the blood of immune animals may at first sight appear
paradoxical. We should not lose sight of the fact, however,
that agglutinin is presumably distinct from the other sub-
stance concerned in immunity, and its presence in immune
animals may, therefore, be reasonably explained as a more or
less permanent result of the "reactions of infection" that were
coincident with the primary stimulations by specific infec-
tive or intoxicating matters necessary to the establishment
of the condition of immunity; nor should we in this con-
nection lose sight of the fact that its presence is constantly
to be demonstrated in typical cases of typhoid fever, for
instance, that terminate fatally, and that have exhibited
little or no clinical signs of resistance at any time during
their course.
Fifth, there may be demonstrated in the blood of animals
that have received repeated subcutaneous injections of
milk a body — a "precipitin" — that causes a precipitation
of milk. This precipitation represents apparently a specific
reaction, for it occurs only when the blood-serum is mixed
with milk from the species of animal that supplied the milk
used for immunization.
Sixth, after the repeated injection of blood or of emulsions
of tissue-cells into the body of an animal, there appear in
308 BACTERIOLOGY
the blood of that animal certain solvents, or enzyme-like
bodies, "hemolysins," " cytolysins," etc., that react specif-
ically upon the blood or tissue-cells injected, agglutinating,
disintegrating, and finally completely dissolving them.
Here, too, the relations are specific. If a rabbit, for instance,
be rendered tolerant to or immune from beef-blood, its
serum dissolves only the red corpuscles of bovines; if from
dog's blood, then only the corpuscles of the dog are dissolved
by the serum of the rabbit. Similarly, if a rabbit be ren-
dered tolerant to injections of emulsions of epithelium cells,
then its serum dissolves epithelium and not necessarily
other cells. All these reactions may be seen in a test-tube
or under the microscope.
Seventh, if a hemolyzing serum, prepared as indicated
under the sixth observation, be heated for a short time to
54°-56° C., it at once loses the hemolytic function, but
regains it again if a few drops of serum from a normal
animal be added to it. In this phenomenon of hemolysis
Ehrlich's "receptors of the third order" are assumed to be
concerned; the heating, without injuring the receptors or
immune bodies, destroys the "complement," and thereby
checks the process; but the subsequent addition of nor-
mal serum supplies fresh "complement," and at once
restores the combination necessary to the phenomenon of
hemolysis.
Eighth, if blood containing a hemolysin or a cytolysin
be repeatedly injected into an animal, antibodies — "anti-
lysins" — are formed, and the serum of the animal has the
power of robbing a hemolytic serum of its hemolyzing func-
tion if mixed with it in a test-tube.
Ninth, if normal blood, containing complement, be
injected into the same or another species of animal, anti-
THE DEFENSES OF THE BODY 309
complement is formed, which has the property of inhibiting
the action of the complement.
Tenth, if emulsions of dead bacteria- be injected into
animals, the leukocytes of that animal may gain in power
to take up and destroy living bacteria of the same species,
a result usually attributed to an increase in the opsonizing
power of the blood.
Eleventh, there exists in the blood a body to which Wright
has given the name "opsonin," which has the function of
so acting upon bacteria that they may be taken up by
phagocytes. This preparation of the bacteria by opsonin
is regarded as a prerequisite to phagocytosis.
The foregoing sketch affords but an imperfect idea of the
vast amount of labor that has been and continues to be
expended upon this many-sided, absorbing topic. Of neces-
sity many important contributions have been omitted, but
those noted will serve to illustrate the lines along which the
solution of the problem has been approached. As a result
of such investigations, our knowledge upon infection and
immunity may be summarized as follows:
1. That infection may be considered as a contest between
bacteria and living tissues, conducted on the part of the
former by means of the poisonous products of their growth,
and resisted by the latter through the agency of phago-
cytic cells and the proteid bodies normally present in and
generated by their integral cells.
2. That when infection occurs it may be explained either
by the excess of vigor of the bacterial products over the
antidotal or protective proteids produced by the tissues, or
to some cause that has interfered with the normal activity of
of the phagocytic cells and production of the protective bodies.
310 BACTERIOLOGY
3. That in the serum of the normal circulating blood of
many animals there exists a substance that is capable, out-
side of the body, of rendering inert certain pathogenic
bacteria, but which is, however, present in such small
quantities as to be ineffective., either for the protection of
the animal or for the cure of infection when introduced into
the body of another animal already infected.
4. That immunity is most frequently seen to follow the
introduction into the body of the products of growth of
bacteria that in one way or another have been modified.
This modification may be artificially produced in the prod-
ucts of virulent organisms, and then introduced into the
tissues of the animal; or the virulent bacteria may be so
treated that they are no longer of full virulence, and when
introduced into the body of the animal will produce poisons
of a much less vigorous nature than would otherwise be
the case.
5. That immunity following the introduction of bacterial
products into the tissues is apparently due to the formation
in the tissues of another body or other bodies that act as
antidotes to the poisons, and thereby protect the tissues
from their hurtful effects. '
6. That this protecting proteid which is generated by the
cells of the tissues need not of necessity be antagonistic to
the life of the invading organisms themselves, but in some
cases must be looked upon more as an antidote to their
poisonous products.
7. That immunity, as conceived by Ehrlich, may be
either "active" or "passive." According to this interpre-
tation, it is "active'' when resulting from an ordinary non-
fatal attack of infectious disease; or from a mild attack
of infection purposely induced through the use of living
THE DEFENSES OF THE BODY 311
vaccines; or from the introduction of cultures of the bacteria
that have been killed by heat; or from the gradual intro-
duction of toxins into the tissues until a marked antitoxic
state is reached. It is "passive" when occurring as a result
of the direct transference of the perfected immunizing sub-
stance from an immune to a susceptible animal, as by the
injection of blood serum from the former into the latter.
"Passive immunity" is, in most cases, conferred at once,
without the delay incidental to the usual modes of establish-
ing "active immunity." As a rule, "active" is a more
lasting than "passive" immunity.
8. That phagocytosis is effective in warding off disease
in normal individuals only when the defenses of the body
are fully active; when the number of invading bacteria
is relatively small or when the bacteria are possessed of low
aggressive powers. It is probably a secondary process, the
bacteria being taken up by the leukocytes only after having
been modified through the opsonizing activity of the serum
of the blood and of other fluids in the body.
9. That of the hypotheses advanced in explanation of
acquired immunity, the one worthy of greatest confidence
is that which assumes immunity to be due to reactive
changes on the part of the tissues that result in the formation
in these tissues of antitoxic and other antibodies, which
circulate free in the blood, and in a variety of ways serve
to screen the tissues from the harmful effect of extraneous
intoxicants and irritants, in some cases acting principally
as antidotes to toxins, in others exhibiting more the
germicidal (bacteriolytic) than the antitoxic property.
CHAPTER XV.
Hemolysis — The Hemolytic System — Identification of Specific Immune
Bodies and Specific Antigens by Their Ability to Fix Complement — The
Wassermann Reaction — Schematic Representation of Reactions.
THE HEMOLYTIC REACTION.
THE term hemolysis relates to a phenomenon through
which hemoglobin is caused to escape in solution from red
blood corpuscles. The process is also known as "laking."
It may be brought about in a number of ways — physical,
chemical, and vital. It is with the latter that we are here
concerned.
As a result of the investigations of Landois we have known
for a long time that the blood of one species of animal often
exhibits destructive action Upon the corpuscles of the blood
of an animal of another species. He showed that grave
toxic symptoms, sometimes fatal results, follow upon the
introduction of the blood of one species into the veins of
another. The blood of the dog is a powerful solvent for the
blood corpuscles of many other animals, while that of the
horse and of the rabbit has very little solvent action. The
corpuscles of the rabbit are readily laked by the blood
serum of a number of other species while those of the cat
and the dog are much more resistent. The corpuscles of
the sheep and of the rabbit are dissolved by dog's serum in
a very few minutes.
Landois' investigations explain in a satisfactory way the
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THE HEMOLYTIC REACTION 313
fatalities so often attendant upon the earlier practices of
transfusion, when it was customary, after a severe hemor-
rhage, in certain cases of poisoning, especially by carbon
monoxide, and in certain pathological states, to transfuse
the blood of animals into the veins of man for purposes of
resuscitation. So convinced was Landois of the danger at-
tendant upon the practice that he states : the blood of animals
should never be transfused into the bloodvessels of man.
For a somewhat shorter time we have known that if such
toxic alien blood be injected into animals in non-fatal quan-
tities, that repeated injections of gradually increasing doses
may be made until a condition develops in which the
receptive animal is immune from the poisonous action of
the alien blood. When this point is reached the blood of
the immunized animal exhibits specific reactions with the
alien blood that are not only of very great theoretical interest,
but, as newer developments demonstrate, are susceptible
of application to the solution of other problems relating to
infection and resistance. Thus, for instance, if a portion of
the same blood used in immunizing the animal be repeatedly
washed in physiological salt solution until one has nothing
left but red-blood corpuscles suspended in salt solution
and to this there be added a small amount of the blood,
serum from the immune animal, and the mixture be allowed
to stand for a little while at body temperature, there will
be a more or less complete solution of hemoglobin from the
washed corpuscles and their stroma will finally collect at the
bottom of the vessel as a more or less pale or colorless mass.
If instead of using in the experiment the serum just as it
comes from the immune animal, we heat it for thirty minutes
to 55° C., and then mix it with the same volume of washed
corpuscles suspended in salt solution, we find that no solution
314 BACTERIOLOGY
of hemoglobin occurs. But, if after a reasonable interval
of time we now add to the mixture in which no hemolysis
has occurred, a small amount of unheated normal (not
immune) serum — hemolysis sets in almost at once and may
proceed to completion. Obviously in washing the corpuscles
and in heating the serum we have eliminated a factor
necessary to hemolysis, which factor is readily supplied by
a small quantity of fresh, unheated serum from a non-immune
animal.1
Equally obviously, three factors are concerned in this
reaction: blood corpuscles, a something in the serum of the
immune animal that is not affected by heating; and a
something that is destroyed by the heating.
The heat-proof body is the amboceptor of the third order
of Ehrlich or the "immune body" — or the "intermediary
body" or the "antibody" as it is severally called. The
heat-sensitive body is the "complement" of Ehrlich or the
"alexin" of Buchner and Bordet. The blood corpuscles of
the alien species represent the "antigen" — i. e., the body
which when injected into the animal being immunized stimu-
lates or generates the production of the specific antibodies,
immune bodies or amboceptors as we may choose to call them.
We have already learned that amboceptors, or antibodies
of this order are conceived by Ehrlich to possess two hapto-
phore groups; the one having the power to unite with a
corresponding haptophore of the "complement," the other
with a corresponding side chain haptophore, or combining
group, of the body to be destroyed — in this case, the alien
blood corpuscles. When this combination is complete the
complement by its ferment-like action, destroys the blood
1 Has this any resemblance to the reaction known as "Pfeiffer's phe-
nomenon?"
THE HEMOLYTIC REACTION
315
corpuscles which have been "sensitized" by their union with
the antibodies. Such destruction is not possible until the
complement is bound by means of the intermediary body with
the other object — the blood corpuscle; neither is such
destruction possible when complement is, as we just saw,
absent or rendered inert by heat or otherwise. In brief,
we have here a "system" the integers of which must all
be present and in appropriate adjustment before the desired
reaction occurs. The several factors and the reaction may be
for convenience of visualization graphically represented, thus :
Complement
Immune body
Immune body
FIG. 55
Factors present in the serum of the immune
animal.
No reaction, as antigen is absent.
FIG. 56
Factor present in heated immune serum.
No reaction, as both complement and
antigen are lacking.
Immune body
Antigen
FIG. 57
Factors present in heated immune serum
to which antigen has been added. No
reaction, as complement has -been de-
stroyed by the heating.
316
BACTERIOLOGY
FIG. 58
Complement
= Immune body
Antigen
Factors present and in combination in un-
heated immune serum to which antigen
has been added. Reaction complete.
In the hemolytic system it is obvious, in so far as two
factors are concerned, that specific relationship is essential
to the reaction. Thus, immune serum from an animal
immunized from sheep's blood possesses amboceptors specific
for the sheep's blood corpuscles and none for the corpuscles
of other animals, so that if to such immune serum blood cor-
puscles other than those of the sheep be added, no hemolysis
occurs, even though it may have been conspicuously active
for sheep's corpuscles.
The relationship of the complement to the amboceptor and
antigen is not specific. It reacts with any or all amboceptors
and antigens and is present in all mammalian blood.
It must not be forgotten, as stated above, that natural
hemolytic activity is sometimes exhibited by one blood for
another, consequently, in arranging studies in this field
this fact should be borne in mind and care exercised to con-
trol all experiments.
FIXATION OF COMPLEMENT.
From the investigations of Bordet and Gengou upon the
relations between antibodies and complement, methods have
been developed by which it is possible to detect very small
quantities of antibodies in fluids under question on the one
FIXATION OF COMPLEMENT
317
hand, and to identify, on the other hand, antigens whose
true nature may only be suspected.
The important points brought out in their fundamental
experiment are: that complement is not specific in its
affinities and that when once fixed by an antibody to an
antigen the union is not dissociable. The experimental pro-
cedures necessary to this demonstration consisted in two series
of mixtures — one in which antibody, its antigen and comple-
FIG. 59
SERIES I.
= Plague antigen.
Plague amboceptor.
= Complement.
The three factors
united after a
time.
FIG. 60
SERIES II.
= Plague antigen.
= Non-specific amboceptor
of normal serum.
= Complement.
Only two necessary fac-
tors present ; no union
possible.
Washed corpuscles and inactivated hemolytic immune serum now added to
each series.
ment were together, the other identical in its ingredients save
for the absence of antibodies specific for the antigen used.
(Figs. 59 and 60.) It is obvious that in the first mixture
(Fig. 59) all factors necessary to the saturation of the
haptophores of the amboceptor were present — therefore,
complement, being one of these factors was bound by the
amboceptor to the antigen. In the other mixture (Fig. 60)
this was not possible as there were no amboceptors specific
for the antigen in it. But to prove this "fixation" of com-
318 BACTERIOLOGY
plement in the first mixture: To this end, after the mixtures
had stood for a time, an incomplete hemolytic system was
added to each mixture— that is, an amount of normal
washed blood corpuscles and a portion of inactivated
immune serum otherwise hemolytic for those corpuscles,
was added. Before this addition, obviously, no hemolysis
could occur, because the complement of the hemolytic
serum had been destroyed by the heat used for inactivation.
But after the addition hemolysis did occur in one tube but
not in the other. It is plain that complement necessary
to the phenomenon of hemolysis must have been avail-
able in one of the tubes, If one recalls that in the second
FIG. 61 FIG. 62
= Blood -corpuscle. ( J = Blood corpuscle.
b^?| = Hemolytic amboceptor.
= Hemolytic amboceptor. K|||j
Jrjpijk = Complement.
No hemolysis. No complement Hemolysis. Free complement of
available; all fixed, as in a', original mixture now bound by
hemolytic amboceptor.
mixture no immune bodies or amboceptors specifically
related to the antigen were present it is clear that the com-
plement could not have been bound or fixed. It must have
remained free in the serum, available for complementing
the action of the hemolytic amboceptors and thereby hemo-
lyzing or destroying the normal blood corpuscles added, as
shown by the laking of such corpuscles in the tube. This,
in short, is what occurred. See Figs. 61 and 62.
For this particular test) Bordet and Gengou used plague
antigen (plague bacilli); plague amboceptors (present in
blood of animal immunized from plague) ; complement (free
in normal mammalian blood); normal serum (containing no
FIXATION OF COMPLEMENT 319
specific amboceptors) ; washed mammalian blood corpuscles
and inactivated immune serum hemolytic for such corpuscles
(such serum contains only hemolytic amboceptors, no
complement).
The application of the principles involved in this experi-
ment to the solution of a number of practical problems is
evident. For instance, we are called upon to identify the
nature of an obscure infection, latent syphilis, let us say.
We know that the blood in such cases contains specific
antibodies for the antigen (excitor) of syphilis. We know
that the* excitor of syphilis or -important extractives of it
are present in the organs of syphilitic fetuses, so that the
antigen is easy to obtain. We know that all normal mamma-
lian blood contains complement. If, therefore, a mixture be
made of syphilitic antigen, of normal guinea-pig blood serum
and of the patient's blood serum, we have, providing the
patient be syphilitic, all the factors necessary to the union
of complement to antigen by the amboceptors of the blood.
If, after it has stood for a time, we now add to such a mixture
hemolytic amboceptors and red corpuscles to which such
amboceptors are specifically related, we get no hemolysis,
if the patient be syphilitic, for there is no free complement
left for the completion of the hemolytic system — on the
other hand if the patient be not syphilitic, his blood will
contain no amboceptors capable of binding complement and
syphilitic antigen together, therefore, there will be free
complement available for the hemolytic system and hemolysis
results. The application of this principle to the diagnosis
of obscure syphilis constitutes what is generally known as
"The Wassermann Reaction," but it is plain that the
principle is susceptible of application to the identification
of other latent infective processes as well. In fact it is being
more and more used for that purpose.
320
BACTERIOLOGY
A glance at the graphic representation of this reaction at
once also suggests the means of identifying unknown but
suspected antigens. Thus, for instance, if in both series we
have the same amboceptors and complement but different
antigens, one being specifically related to the amboceptor,
the other not, plainly we will have a result similar to that
obtained in the first series after the incomplete hemolytic
system is added — that is, there will be no hemolysis in the
tube in which antigen and amboceptor are specifically
related, for here all free complement will be fixed — on the
other hand in the tube in which the antigen is not so related
to the amboceptor complement cannot be so fixed and it,
therefore, as in the first experiment, remains free to complete
the hemolytic system. The reaction may be expressed
graphically as follows:
a<
FIG. 63
SERIES I.
= Plague antigen.
= Plague amboceptor.
Complement.
Factors united.
FIG. 64
SERIES II.
= Unknown antigen.
Plague amboceptor.
: Complement.
No union possible.
Washed corpuscles and inactivated hemolytic immune serum now added
to each mixture.
In the second application of this test observe that the
unknown antigen used in Series II is not of the nature of
FIXATION OF COMPLEMENT 321
plague antigen. Had the problem involved the identifica-
tion of any other antigen — say gonococci, bacillus mallei
or others — one would substitute in the mixtures gonorrhea
antibodies or glanders antibodies or others as the case may
be, and proceed as above. In these cases, however, such
antibodies must be artificially produced in animals that
react to gonorrhea or glanders antigens.
In addition to the foregoing the principles involved in
these reactions have been employed for the differentiation
of closely allied proteins. Such for instance as the differen-
tiation of bloods. For instance, if an animal be immunized
from human blood its serum will contain amboceptors for
human blood corpuscles, the antigen. Such amboceptors
in the presence of human corpuscles or their protein extrac-
tives and complement fix the complement; on the other
hand if the blood under question be from other species than
man, no such fixation can occur as there is no specific affinity
between such blood and the amboceptors for human blood.
Consequently, in the final test for fixation, as determined
by + or — hemolysis, no hemolysis occurs after the addition
of hemolytic amboceptors and their related corpuscles to
the mixture of 'human blood, its amboceptors and comple-
ment, while hemolysis does occur in the mixture of alien
blood, human amboceptors, and complement.
In the former case all complement was fixed to the antigen
by the homologous amboceptors, while in the latter this was
not possible because of the lack of specific affinity of human
blood amboceptors for the alien blood.
While the principles involved in the practical application
of these reactions are very simple, yet there are so many
chances for error that each and every step demands the most
careful control.
21
APPLICATION OF THE METHODS OF
BACTERIOLOGY.
CHAPTER XVI.
To Obtain Material with Which to Begin Work.
EXPOSE to the air of an inhabited room a slice of freshly
steamed potato or a bit of slightly moistened bread upon a
plate for about one hour. Then cover it with an ordinary
water-glass, place it in a warm spot (temperature not to
exceed that of the human body — 37.5° C.), and allow it
to remain undisturbed. In from twenty-four to thirty-six
hours there will be seen upon the cut surface of the bread
or potato small, round, oval, or irregularly round patches
which present various appearances. These differences in
macroscopic appearance are due in some cases to the presence
or absence of color; in others to a higher or lower degree
of moisture; in some instances a patch will be glistening
and smooth, while its neighbor may be dull and rough or
wrinkled; here will appear an island regularly round in
outline, and there an area of irregular, ragged deposit. All
these gross appearances are of value in aiding us to distin-
guish between these colonies — for colonies they are, and
under the same conditions the organisms composing each of
them will always produce growth of exactly the same ap-
pearance. It was just such an observation as this that sug-
(323)
324 APPLICATION OF METHODS OF BACTERIOLOGY
gested to Koch a means of separating and isolating in pure
cultures the component individuals from mixtures of bacteria,
and from it the methods of cultivation on solid media were
evolved.
If, without molesting these objects, we continue the
observations from day to day, we shall notice changes in the
colonies, due to the growth and multiplication of the indi-
viduals composing them. In some cases the colonies will
always retain their sharply cut, round, or oval outline, and
will increase but little in size beyond that reached after
forty-eight to seventy-two hours; whereas others will
spread rapidly and quickly overrun the surface upon which
they are growing, and, indeed, grow over the smaller, less
rapidly developing colonies. In a number of instances,
if the observation be continued long enough, many of these
rapidly growing colonies will, after a time, lose their lustrous
and smooth or regular surface and will show here and there
elevations, which will continue to appear until the whole
surface becomes conspicuously wrinkled. Again, bubbles
may be seen scattered through the colonies. These are due
to the escape of gas resulting from fermentation, which the
organisms bring about in the medium upon which they are
growing. Sometimes peculiar odors due to the same cause
will be noticed.
Note carefully all these changes and appearances, as they
must be employed subsequently in identifying the individual
organisms from which each colony on the medium has
developed.
If we now examine these colonies upon the bread or potato
with a hand-lens of low magnifying power, we will be
enabled to detect differences not noticeable to the naked
eye. In a few cases we may still see nothing more than a
EXPOSURE AND CONTACT 325
smooth, non-characteristic surface; while in others minute,
sometimes regularly arranged tiny corrugations may be
observed. In one colony they may appear as tolerably
regular lines, radiating from a central spot; and again they
may appear as concentric rings; and if by the methods which
have been described we obtain from these colonies their
individual components in pure culture, we shall see that
this characteristic arrangement in folds, radii, or concentric
rings, or the production of color, is characteristic of the
growth of the organism under the conditions first observed,
and by a repetition of those conditions may be reproduced
at will.
So much for the simplest naked-eye experiment that can
be made in bacteriology, and which serves to furnish the
beginner with material upon which to commence his studies.
It is not necessary at this time for him to burden his mind
with names for these organisms; it is sufficient for him to
recognize that they are of different species, and that they
possess characteristics which will enable him to differentiate
the one from the other.
Exposure and Contact. — Make a number of plates from
bits of silk used for sutures, after treating them as follows:
Place some of the pieces (about 5 centimeters long) in a
sterilized test-tube, and sterilize them by streaming steam
for one hour or in the autoclave for fifteen minutes at one
atmosphere pressure. At the end of the sterilization remove
one piece with sterilized forceps and allow it to brush against
your clothing, then make a plate from it; draw another
piece across a dusty table and then plate it. Suspend three
or four pieces upon a sterilized wire hook and let them hang
for twenty minutes free in the air, being sure that they
touch nothing but the hook; then plate them separately.
326 APPLICATION OF METHODS OF BACTERIOLOGY
Note the results.
In what way do these experiments differ and how can
the differences be explained?
Expose to the air six Petri dishes into which either sterilized
gelatin or agar-agar has been poured and allowed to solidify;
allow them to remain exposed for five, ten, fifteen, twenty,
twenty-five, and thirty minutes in a room where no one is
at work. Treat a second set in the same way in a room where
several persons are moving about. Be careful that nothing
touches them, and that they are exposed only to the air.
Each dish should be carefully labelled with the time of its
exposure.
Do they present different results? What is the reason for
this difference?
Which predominate — colonies resulting from the growth
of bacteria, or those from common molds?
How do you account for this condition?
Sprinkle a little fine dust over the surface of a plate of
sterile gelatin or agar-agar; examine the dust-particles with
the microscope immediately after depositing them on the
medium, and again after eighteen to twenty-four hours.
What differences do you detect? What information of
sanitary importance does this give?
Under the description of each of the pathogenic bacteria
more or less detailed directions will be found for the dis-
covery and isolation of each of the pathogenic bacteria.
CHAPTER XVII.
Various Experiments in Sterilization by Steam and by Hot Air.
PLACE in one of the openings in the cover of the steam
sterilizer an accurate thermometer; when the steam has
been streaming for a minute or two the thermometer will
register 100° C. Wrap in a bundle of towels or rags or pack
tightly in cotton a maximum (self -registering) thermometer;
let this thermometer be in the center of a bundle large
enough to quite fill the chamber of the sterilizer. At the end
of a few minutes' exposure to the streaming steam remove
it; it will be found to indicate a temperature of 100° C.
Closer study of the penetration of steam has taught us,
however, that the temperature found at the center of such
a mass may sometimes be that of the air in the meshes of
the material, and not that of steam, and for this reason the
sterilization at that point may not be complete, because hot
air at 100° C. has not the sterilizing value that steam has
at the same temperature. It is necessary, therefore, that
this air should be expelled from the meshes of the material
and its place taken by the steam before sterilization is com-
plete. This is insured by allowing the steam to stream
through the substances a few minutes before beginning to
calculate the time of exposure. There is as yet no absolutely
sure means of saying that the temperature at the center of
the mass is that of hot air or of steam, so that the exact
length of time that is required for the expulsion of the air
from the meshes of the material cannot be given.
(327)
328 APPLICATION OF METHODS OF BACTERIOLOGY
Determine if the maximum thermometer indicates a
temperature of 100° C. at the center of a moist bundle in
the same way as when a dry bundle was employed.
To about 50 c.c. of bouillon add about 1 gram of chopped
hay, and allow it to stand in a warm place for twenty-four
hours. At the end of this time it will be found to contain
a great Variety of organisms. Continue the observation,
and ultimately a pellicle will be seen to form on the surface
of the fluid. This pellicle is made up of rods which grow
as long threads in parallel strands. In many of these rods
glistening spores will be seen. After thoroughly shaking,
filter the mass through a fine cloth to remove coarser
particles.
Pour into each of several test-tubes about 10 c.c. of the
filtrate. Allow one tube to remain undisturbed in a warm
place. Place another in the steam sterilizer for five minutes;
a third for ten minutes; a fourth for one-half hour; a fifth
for one hour.
At the end of each of these exposures inoculate a tube of
sterilized bouillon from each tube. Likewise make a set
of plates or Esmarch tubes upon both gelatin and agar-agar
from each tube, and note the results. At the same time
prepare a set of plates or Esmarch tubes on agar-agar and
on gelatin from the tube which has not been exposed to the
action of the steam.
. The plates or tubes from the unmolested tube will present
colonies of a variety of organisms; separate and study these.
Those from the tube which has been sterilized for five
minutes will present colonies in moderate numbers; but, as
a rule, they will represent but a single organism. Study this
organism in pure cultures.
The same may be predicted for the tube which has been
EXPERIMENTS IN STERILIZATION 329
heated for ten minutes, though the colonies will be fewer
in number.
The thirty-minute tube may or may not give one or two
colonies of the same organism.
The tube which has been heated for one hour is usually
sterile.
The bouillon tubes from the first and second tubes which
were heated will usually show the presence of only one
organism — the bacillus which gave rise to the pellicle-
formation in the original mixture. This organism is bacillus
subtilis. It is especially adapted to the study of those various
degrees of resistance to heat that spore-forming bacteria
exhibit at different stages of their development.
Inoculate about 100 c.c. of sterilized bouillon with a very
small quantity of a pure culture of this organism, and allow
it to stand in a warm place for about six hours. Now subject
this culture to the action of steam for five minutes; it will
be seen that sterilization, as a rule, is complete.
Treat in the same way a second flask of bouillon, inocu-
lated in the same way with the same organism, but after
having stood in a warm place for from forty-eight to seventy-
two hours — that is, until spores have formed — and it will
be found that sterilization is not complete : the spores of
this organism have resisted the action of steam for five
minutes.
To determine if sterilization is complete always resort
to the culture methods, as the macroscopic and microscopic
methods are deceptive; cloudiness of the media or the
presence of bacteria microscopically does not always signify
that organisms possess the property of life.
Inoculate in the same way a third flask of bouillon with
a very small drop from one of the old cultures upon which
the pellicle has formed; mix it well and subject it to the
330 APPLICATION OF METHODS OF BACTERIOLOGY
action of steam for two minutes; then place it to one side
for from twenty to twenty-four hours, and again heat for
two minutes; allow it to stand for another twenty-four
hours, and repeat the process on the third day. No pellicle
will be formed, and yet spores were present in the original
mixture, and, as we have seen, the spores of this organism
are not killed by an exposure of five minutes to steam. How
can this result be accounted for?
Saturate several pieces of cotton thread, each about 2 cm.
long, in the original decomposed bouillon, and dry them
carefully at the ordinary temperature of the room; then at
a little higher temperature — about 40° C. — to complete
the process. Regulate the temperature of the hot-air
sterilizer for about 100° C., and subject several pieces of
this infected and dried thread to this temperature for the
same lengths of time that we exposed the same organisms
in bouillon to the steam, viz., five, ten, thirty, and sixty
minutes. At the end of each of these periods remove a bit
of thread, and prepare a set of plates or Esmarch tubes
from it. Are the results analogous to those obtained when
steam was employed?
Increase the temperature of the dry sterilizer and repeat
the process. Determine the temperature and time neces-
sary for the destruction of these organisms by dry heat.
These threads should not be simply laid upon the bottom
of the sterilizer, but should be suspended from a glass rod,
which may be placed inside the oven, extending across its
top from side to side.
Place several of the infected threads in the center of a
bundle of rags. Subject this to a temperature necessary to
sterilize the threads by the dry method. Treat another
similar bundle to sterilization by steam. In what way do
the results of the two processes differ?
CHAPTER XVIII.
Methods of Testing Disinfectants and Antiseptics — Experiments Illustrating
the Precautions to be Taken — Experiments in Skin-disinfection.
DETERMINATION OF DISINFECTANT PROPERTIES.
THERE are several ways of determining the germicidal
value of chemical substances, the most common being to
expose organisms dried upon bits of silk thread to the
disinfectant for different lengths of time, and then, after
removing, and carefully washing the threads in water, to
place them in nutrient media at a favorable temperature,
and notice if any growth appears. If no growth results,
the disinfection is presumably successful. Another method
is to mix fluid cultures of bacteria with the disinfectant in
varying proportions, and, after different intervals of time,
to determine if disinfection is in progress by transferring a
portion of the mixture to nutrient media, just as in the other
methods of work.
By the first of these processes the bits of thread, usually
about 1 to 2 cm. long, are placed in a dry test-tube provided
with a cotton plug and carefully sterilized, either by the
dry method or in the steam sterilizer, before using. They
are then immersed in a pure bouillon culture or in a salt-
solution suspension of the organism upon which the disin-
fectant is to be tested. I say "pure culture," because it is
always desirable in testing a substance to determine its
germicidal value for several different resistant species of
(331)
332 APPLICATION OF METHODS OF BACTERIOLOGY
bacteria, both in the vegetating and in the spore stage, and
also because it is only by the use of pure cultures of familiar
species that it is possible to distinguish between the colonies
growing from the individuals that have not been destroyed
by the disinfectant under investigation and those of unknown
species that may appear upon the plate as contaminations
occurring during the manipulation.
After the threads have remained in the culture or suspen-
sion for from five to ten minutes they are removed under
aseptic precautions and carefully separated and spread
upon the bottom of a sterilized Petri dish, which is then
placed either in the incubator at a temperature not exceed-
ing 38° C. until the excess of fluid has evaporated, or in a
desiccator over sulphuric acid, calcium chloride, or any
other drying-agent. The threads are not left there until
absolutely dry, but only until the excess of moisture has
evaporated. When sufficiently dry they are immersed in
solutions of the disinfectant of different but known strengths
for a fixed interval of time, say one or two hours, after which
they are removed, rinsed in sterilized distilled water to
remove the excess of disinfectant adhering to them, and
placed in fresh, sterile culture-media, which are then placed
in the incubator at from 37° to 38° C. If after twenty-four
to forty-eight or seventy-two hours a growth occurs at or
about the bit of thread, and if this growth consists of the
organism with which the test was made, manifestly there
has been no disinfection; if no growth occurs after, at most,
ninety-six hours, it is safe to presume that the bacteria
have been killed, unless our efforts at rinsing off the excess of
disinfectant from the thread have not been successful, and
a small amount of disinfectant is still active in preventing
development — i. e., is acting as an antiseptic.
DETERMINATION OF DISINFECTANT PROPERTIES 333
By the process in which cultures or suspensions of the
organisms are mixed with different but known strengths of
the disinfectant a small portion of the mixture, usually a
loopful or a drop, is transferred at the end of a definite time
to the fresh medium which is to determine whether the
organisms have been killed or not. This is commonly a
tube of fluid agar-agar, which is poured into a Petri dish,
allowed to solidify, and placed in the incubator, as in the
'preceding method.
After the minimum strength of disinfectant necessary to
destroy the vitality of the organisms with which we are
Working has been determined for any fixed time, it remains
for us to decide what is the shortest time in which this strength
will have the same effect. We then work with a constant
dilution .of the disinfectant, but with varying intervals of
exposure — one, five, • ten minutes, etc. — until we have
decided not only the minimum amount of disinfectant
required for the destruction of the bacteria, but the shortest
time necessary for this under known conditions.
A factor not to be lost sight of is the temperature at
which these experiments are conducted, for it must always
be borne in mind that the action of a disinfectant is usually
more energetic at a higher than at a lower temperature.
Now in both of these methods it is easy to see that unless
special precautions are taken a minute portion of the disin-
fectant may be carried along with the thread, or drop, into
the medium which is to determine whether the organisms
do or do not possess the power of growth, and there have
a restraining or antiseptic action. For organisms in their
normal condition — that is, those which have never been
exposed to the action of a disinfectant — the amount of
certain disinfectants that is necessary to restrain growth
334 APPLICATION OF METHODS OF BACTERIOLOGY
is very small indeed; but for organisms that have already
been exposed for a time to such agents this amount is very
much less. It is plain, then, that if the test is to be an
accurate one, precautions must be taken against admitting
this minute trace of disinfectant to the medium with which
we are to determine whether the bacteria exposed to the
disinfectant were killed or not. The precautions hitherto
taken to prevent this accident have been, when the threads
were employed, washing them in sterilized distilled water
and then in alcohol; or, where fluid cultures were mixed
with the disinfectant in solution, an effort was usually
made to dilute the amount of disinfectant carried over, to
a point at which it lost its inhibiting power.
While such precautions are sufficient in many cases,
they do not answer for all. Certain chemicals have the
property of combining so firmly with the threads upon
which the bacteria are located as to require other special
means of ridding the threads of them; and in solutions in
which proteid substances are present along with the bacteria
a similar union between them and the disinfectant may
likewise take place. In both instances this amount of disin-
fectant adhering to the threads or in combination with the
proteids must be eliminated, otherwise the results of the
test may be fallacious. A partial solution of the problem
is given through studies that have been made upon corrosive
sublimate in its various applications for disinfecting pur-
poses, and in this connection it has been shown by Shaefer1
that it is impossible to rid silk threads of the corrosive
sublimate adhering to them by simple washing, as the sub-
limate acts as a mordant and forms a firm union with the
1 Berliner klin. Wochenschrift, 1890, No. 3, p. 50.
DETERMINATION OF DISINFECTANT PROPERTIES 335
tissues of the threads. Braatz1 found the same to hold good
for catgut. For example, he found that catgut which had
been immersed in solutions of corrosive sublimate gave the
characteristic reactions of the salt after having been im-
mersed for five weeks in distilled water which had been
repeatedly renewed. Braatz remarks that a similar com-
bination between sublimate and cotton will take place after
a long time; but it occurs so slowly that it cannot interfere
with disinfection experiments in the same way that silk does.
The most successful attempt at removing all traces of
sublimate from the threads or from the proteid substances
in which are located the bacteria whose vitality is to be
tested was made by Geppert, who subjected them to the
action of ammonium sulphide in solution. By this procedure
the mercury is converted into inert, insoluble sulphide, and
has no inhibiting effect upon the growth of those bacteria
that did not succumb to its action when in the form of the
bichloride.
Another plan that has been successfully used is to dry
the bacteria on small particles of sterile glass rod or on
sterile glass beads instead of on threads. The advantages
of the method are obvious, but the handling, especially the
washing, must be done carefully or all the bacteria will be
removed from the glass surfaces.
In the second method of testing disinfectants mentioned
above — that is, when cultures of bacteria and solutions
of the disinfectant are mixed, and after a time a drop of the
mixture is removed and added to sterile nutrient media —
the inhibiting amount of disinfectant can readily be got
rid of by dilution; that is to say, instead of transferring the
1 Centralblatt fur Bakteriologie und Parasitenkunde, Bd. vii, No. 1, p. 8.
336 APPLICATION OF METHODS OF BACTERIOLOGY
drop directly to the fresh medium, add it to 10 or 12 c.c.
of sterilized salt-solution (0.6-0.7 per cent, of NaCl in dis-
tilled water) or distilled water, and after thoroughly shaking
add a drop of this to the medium in which the power of
development of the bacteria is to be determined.
Another important point to be borne in mind in testing
disinfectants is the necessity of so adjusting the conditions
that each individual organism will be exposed to the action
of the agent used. When clumps of bacteria exist we are
not always assured of this, for only those on the surface
of the clump may be affected, while those in the center of
the mass may escape, being protected by those surrounding
them. These clumps and minute masses are especially
liable to be present in fluid cultures and in suspensions of
bacteria, and must be eliminated before the test is begun,
if this is to be made by mixing them with solutions of the
agent to be tested. This is best accomplished in the following
way: the organisms should be cultivated in bouillon con-
taining sand or finely divided particles of glass; after grow-
ing for a sufficient length of time they are to be shaken
thoroughly, in order that all clumps may be mechanically
broken up by the sand. The culture is then filtered through
a tube containing closely packed glass-wool.
The filtration may be accomplished without fear of con-
tamination of the culture by the employment of an Allihin
tube, which is practically a thick-walled test-tube drawn out
to a finer tube at its blunt end so as to convert it into a
sort of cylindrical funnel. The tube when ready for use
has the appearance shown in Fig. 65.
This tube, after being plugged at the bottom with glass-
wool (a, Fig. 65), and at its wide extremity with cotton-
wool, is placed vertically, small end down, into an Erlen-
DETERMINATION OF DISINFECTANT PROPERTIES 337
FIG. 65
meyer flask of about 100 c.c. capacity and sterilized in a
steam sterilizer for the proper time. It is kept in the steril-
izer until it is to be used, which should
be as soon as possible after sterilization.
The watery suspension or bouillon cul-
ture of the organisms is now to be filtered
repeatedly through the glass-wool into
sterilized flasks until a degree of trans-
parency is reached which will permit the
reading of moderately fine print through
a layer of the fluid about 2 cm. thick
— i. e., through an ordinary test-tube
full of it. This filtrate can then be sub-
jected to the action of the disinfectant.
As a rule, the results are more uniform
than when no attention is paid to the
presence of clumps. It is scarcely neces-
sary to say that in the practical employ-
ment of disinfectants outside the labora-
tory no such precautions are taken; but
in laboratory, work, where it is desired
to determine exactly the value of different
substances as germicides, all the precau-
tions mentioned will be found essential
to precision.
The disinfectant value of gases and
vapors is determined by their action Cylindrical funnel
used for filtering cul-
upon test-objects in closed chambers, tures on which dis-
The object is to determine the proportion ^fsetfdants are to be
of the gas, when mixed with air, that is
required to destroy the bacteria exposed to its action in
a given time. For this purpose the test is usually made
22
IP*
338 APPLICATION OF METHODS OF BACTERIOLOGY
as follows: under a sterilized bell-glass of known capacity
the test-objects are placed. Into the chamber is then
admitted sufficient of a mixture of air and the gas under
consideration, of known proportions, to displace com-
pletely all the air; or the pure gas itself may be intro-
duced in amount necessary to give the desired dilution
when mixed with the air in the chamber. At the expiration
of the time decided upon for the test the infected articles
are removed and the vitality of the bacteria upon them is
determined.
In the case of vapors of volatile fluids, such, for instance,
as formalin, the fluid is placed under the bell-glass in an
open dish; in another open dish the test-objects are placed.
The bell-glass is then sealed to an underlying ground-glass
plate by vaseline or paraffin, and the fluid is allowed to
vaporize at ordinary room-temperature. The point here
to be decided is the volume or weight of such a fluid that
it is necessary to expose in an air-chamber of known cubic
capacity in order that bacteria may be destroyed by its
vapor in a given time.
In determining the germicidal value of different chemical
agents for certain pathogenic bacteria susceptible animals
are sometimes inoculated with the organisms after they have
been exposed to the disinfectant. If no pathological con-
dition, results, disinfection is assumed to have been suc-
cessful; while if the condition characteristic of the activities
of the given organism in the tissues of this animal appears,
the reverse is the case. The objections to this method are:
"First. The test-organisms may be modified as regards
reproductive activity without being killed; and in this
case a modified form of the disease may result from the
inoculation, of so mild a character as to escape observation.
DETERMINATION OF ANTISEPTIC PROPERTIES 339
Second. An animal that has suffered this modified form of
the disease enjoys protection, more or less perfect, from
future attacks, and if used for a subsequent experiment may,
by its immunity from the effects of the pathogenic test-
organism, give rise to the mistaken assumption that this
had been destroyed by the action of the germicidal agent
to which it had been subjected." (Sternberg.)
DETERMINATION OF ANTISEPTIC PROPERTIES.
For this purpose sterile media are employed, and are
usually arranged in two groups: the one to remain normal
in composition and to serve as controls, while to the other the
substance to be tested is to be added in different but known
strengths. It is customary to employ test-tubes each con-
taining an exact amount of bouillon, gelatin, or agar-agar,
as the case may be. To each tube a definite amount of the
antiseptic is added, and if it is not of a volatile nature or
not injured by heat, the tubes may then be sterilized.
After this they are to be inoculated with the organism
with which the test is to be made, and at the same time one
of the " control' '-tubes (one of those to which no antiseptic
has been added) is inoculated. They are all then to be
placed in the incubator and kept under observation. If
at the end of twenty-four, forty-eight, or seventy-two hours
no growth appears in any but the "control' '-tubes, it is
evident that the antiseptic must be added in smaller
amounts, for we are to determine the point at which it is
not as well as that at which it is capable of preventing
development'. The experiment is. then repeated, using
smaller amounts of the antiseptic until we reach a point at
which growth just occurs, notwithstanding the presence
340 APPLICATION OF METHODS OF BACTERIOLOGY
of the antiseptic; the amount necessary for antisepsis is
then a trifle greater than that used in the last tube. If,
for example, there was no development in the tubes in which
the antiseptic was present in the proportion of 1 : 1000, and
growth in the one in which it was present in 1 : 1400, the
experiment should be repeated with strengths of the anti-
septic corresponding to 1:1000, 1:1100, 1:1200, 1:1300,
1 : 1400, and in this way one ultimately determines the
amount by which growth is just prevented; this represents
the antiseptic value of the substance for the organism with
which it was tested.
. i EXPERIMENTS.
To each of three tubes containing 10 c.c. — one of physio-
logical salt-solution, another of bouillon, a third of fluid
blood serum — add as much of a culture of micrococcus
aureus as can be held upon a looped platinum wire. Break
this up carefully to eliminate clumps, and then add exactly
10 c.c. of a 1 : 500 solution of corrosive sublimate. Mix
thoroughly, and at the end of three minutes transfer a drop
from each tube into tubes of liquefied agar-agar, and pour
these into Petri dishes. Label each dish carefully and place
them in the incubator. Are the results the same in all the
plates? How are the differences to be explained? To
what strength of the disinfectant were the organisms ex-
posed in the experiment?
To each of two tubes — the one containing 10 c.c. of
physiological salt-solution, the other of bouillon— add as
much of a spore-containing culture of anthrax bacilli as can
be held upon a loop of platinum wire. Distribute this uni-
formly through the medium, and then add exactly 10 c.c. of
a 1 : 500 solution of corrosive sublimate. Mix thoroughly,
EXPERIMENTS 341
and at the end of five minutes transfer a drop from each tube
to tubes of liquefied agar-agar. Pour these immediately into
Petri dishes. Label each dish carefully and place them in
the incubator. Note the results at the end of twenty-four,
forty-eight, and seventy-two hours. How do you explain
them?
Make identically the same experiment with the same spore-
containing culture of anthrax bacilli, except that the drop
from the mixture is to be transferred to 10 c.c. of a mixture
of equal parts of ammonium sulphide and sterilized distilled
water. After remaining in this for about half a minute,
a drop is to be transferred to a tube of liquefied agar-agar,
poured into Petri dishes, labelled, and placed in the incubator.
Note the results. Do they correspond with those obtained
in the preceding experiment? How are the differences
explained?
Prepare a 1 : 1000 solution of corrosive sublimate. To
each of twelve tubes containing exactly 10 c.c. of bouillon,
add one drop to the first, two drops to the second, and so
on until the last tube has had twelve drops added to it.
Mix thoroughly and then inoculate each with one wire-
loopful of a bouillon culture of micrococcus aureus. Place
them all in the incubator after carefully labelling them.
Note the order in which growth appears.
Do the same with anthrax spores, with spores of bacillus
subtilis, and with the typhoid bacillus, and compare the
results. From these experiments, what will be the strength
of corrosive sublimate necessary to antisepsis under these
conditions for the organisms employed?
Make a similar series of experiments using a 5 per cent,
solution of carbolic acid.
342 APPLICATION OF METHODS OF BACTERIOLOGY
Determine the antiseptic value of the common disinfec-
tants for the organisms with which you are working.
Determine the time necessary for the destruction of the
organisms with which you are working, by corrosive sub-
limate in 1 : 1000 solution, under different conditions— with
and without the presence of albuminous bodies other than
the bacteria, and under varying conditions of temperature.
In making these experiments be careful to guard against
the introduction of sufficient sublimate into the agar-agar
with which the Petri plate is to be made to inhibit the growth
of the organisms which may not have been destroyed by
the sublimate. This may be done by transferring two drops
from the mixture of sublimate and organism into not less
than 10 c.c. of sterilized physiological salt solution, in which
they may be thoroughly shaken for from one to two minutes,
or into the solution of ammonium sulphide of the strength
given.
To 10 c.c. of a bouillon culture of micrococcw aureus or
anthrax spores add 10 c.c. of a 1 : 500 solution of corrosive
sublimate, and allow it to remain in contact with the
organisms for only one-half the time necessary to destroy
them (use an organism for which this has been determined).
Then transfer a drop of the mixture to each of three liquefied
agar-agar tubes and pour them into Petri dishes. Place
them in the incubator and observe them for twenty-four,
forty-eight, and seventy-two hours. No growth occurs.
How is this to be accounted for?
At the end of seventy-two hours inoculate all of these
plates with a culture of the same organism which has not
been exposed to sublimate, by taking up bits of culture on
SKIN DISINFECTION 343
a needle and drawing it across the plates. A growth now
results. We have here an experiment in which organisms
which have been exposed to sublimate for a much shorter
time than necessary to destroy them, when transferred
directly to a favorable culture medium do not grow, and
yet, when the same organism which has not been exposed
to sublimate at all is planted upon the same medium it
does grow. How is this to be accounted for?
SKIN DISINFECTION.
With a sterilized knife scrape from the skin of the hands,
at the root of the nails, and under the nails, small particles
of epidermis. Prepare plates from them. Note the results.
Wash the hands carefully for ten minutes in hot water and
scrub them during this time with soap and a sterilized
brush. Rinse them in hot water. Again prepare plates
from scrapings of the skin on the fingers, at the root of the
nails, and under the nails. Note the results.
Again wash as before in hot water with soap and brush,
rinse in hot water, then soak the hands for five minutes in
1 : 1000 corrosive sublimate solution, and, as before, prepare
plates from scrapings from the same localities. Note the
results.
Repeat this latter procedure in exactly the same way,
but before taking the scrapings let some one pour ammonium
sulphide over the points from which the scrapings are to
be made. After it has been on the hands about three minutes
again scrape, and note the result upon plates made from the
scrapings.
Wash as before in hot water and soap, rinse in clean hot
water, immerse for a minute or two in alcohol, after this in
344 APPLICATION OF METHODS OF BACTERIOLOGY
1 : 1000 sublimate solution, and finally in ammonium sul-
phide, and then prepare plates from scrapings from the
points mentioned.
In what way do the results of these experiments differ
from one another?
To what are these differences due?
What have these experiments taught?
In making the above experiments it must be remembered
that the strictest care is necessary in order to prevent the
access of germs from without into our media. The hand
upon which the experiment is being performed must be
held away from the body and must not touch any object
not concerned in the experiment. The scraping should be
done with the point of a knife that has been sterilized in
a flame and allowed to cool. The scrapings may be trans-
ferred directly from the knife-point to the gelatin by means
of a sterilized platinum wire loop.
The brush used should be thoroughly cleansed and always
kept in 1 : 1000 solution of corrosive sublimate. It should
-be washed in hot water before using.
CHAPTER XIX.
Micrococcus Aureus — Micrococcus Pyogenes and Citreus — Staphylococcus
Epidermidis Albus — Streptococcus Pyogenes — Micrococcus Gonor-
rhcese — Micrococcus Intracellularis — Pseudomonas ^Eruginosa — Bacil-
lus of Bubonic Plague.
MICROCOCCUS AUREUS (ROSENBACH), MIGULA, 1900.
SYNONYMS: Staphylococcus pyogenes aureus, Rosenbach, 1884; Micro-
coccus pyogenes aureus, Migula, 1895; Micrococcus pyogenes, Lehmann and
Neumann, 1896.
PREPARE a set of plates of agar-agar from the pus of an
acute abscess or boil that has been opened under antiseptic
precautions. Care must be taken that none of the antiseptic
used gains access to the culture tubes, otherwise its restrain-
ing effect may be operative and the development of the
organisms interfered with. It is best, therefore, to take a
drop of the pus upon a platinum-wire loop after it has been
flowing for a few seconds; even then it must be taken
from the mouth of the incision and before it has run over
the surface of the skin. At the same time prepare two or
three coverslips from the pus.
Microscopic examination of these slips will reveal the
presence of a large number of pus-cells, both multinucleated
and with horseshoe-shaped nuclei, some threads of disin-
tegrated and necrotic connective tissue, and, lying here
and there throughout the preparation, small round bodies
which will sometimes appear singly, sometimes in pairs,
and frequently will be seen grouped together somewhat like
clusters of grapes. (See Fig. 66.) They stain readily and
(345)
346 APPLICATION OF METHODS OF BACTERIOLOGY
are commonly located in the material between the pus-cells;
very rarely they may be seen in the protoplasmic body of
the cell. (Compare the preparation with a similar one made
from the pus of gonorrhea. (See Fig. 69.) In what way do
the two preparations differ, the one from the other?
After twenty-four hours in the incubator the plates will
be seen to' be studded here and there with yellow or orange-
colored colonies, which are usually round, moist, and glis-
tening in their naked-eye appearances. When located in
. . \
FIG. 66
/ I A f
o
Preparation from pus, showing pus-cells, A, and micrococci, C.
the depths of the medium they are commonly seen to be
lozenge or whetstone in shape, while often they appear as
irregular stars with blunt points, and again as irregularly
lobulated dense masses. In structure they are conspicuous
for their density. Under the low objective they appear,
when on the surface, as coarsely granular, irregularly round
patches, with more or less ragged borders and a dark irreg-
ular central mass, which has somewhat the appearance of
masses of coarser clumps of the same material as that com-
MICROCOCCUS AUREUS 347
posing the rest of the colony. Microscopically, these colo-
nies are composed of small round cells, irregularly grouped
together. They are in every way of the same appearance as
those seen upon the original cover-slip preparation.
Prepare from one of these colonies a pure stab-culture in
gelatin. After thirty-six to forty-eight hours liquefaction
of the gelatin along the track of the needle, most conspicu-
ous at its upper end, will be observed. As growth continues
the liquefied portion becomes more or less of a stocking-
shape, and gradually widens at its upper end into an irregular
funnel. This will continue until the whole of the gelatin
in the tube eventually becomes fluid. There can always be
noticed at the bottom of the liquefying portion an orange-
colored or yellow mass composed of a number of the organ-
isms which have sunk to the bottom of the fluid.
On potato the growth is quite luxuriant, appearing as a
brilliant, orange-colored layer, somewhat lobulated and a
little less moist than when growing upon agar-agar.
It does not produce fermentation with gas production.
It belongs to the group of facultative anaerobes.
In .milk it causes coagulation with acid reaction. This
is, however, variable.
It is not motile, and being of the family of micrococci
does not form endogenous spores. It possesses, however,
a degree of resistance to detrimental agencies that is some-
what greater than that common to non-spore-bearing
bacteria.
In bouillon it causes a diffuse clouding, and after a time
a yellow or orange-colored sedimentation.
This organism is the commonest of the pathogenic bacteria
with which we shall meet. It is micrococcus aureus, or as
it is more commonly known, the staphylococcus aureus,
348 APPLICATION OF METHODS OF BACTERIOLOGY
and is the organism most frequently concerned in the pro-
duction of acute, circumscribed, suppurative inflammations.
As it is almost ubiquitous, it is a source of continuous
annoyance to the surgeon.
While it is the etiological factor in the production of most
of the suppurative processes in man, still it is with no little
difficulty that these conditions can be reproduced experi-
mentally in lower animals. Its subcutaneous introduction
into their tissues does not always result in abscess formation,
and when it does there is probably coincident interference
with the circulation and nutrition of these tissues which
renders them less able to resist its inroads. When intro-
duced into the great serous cavities of the lower animals
its presence is likewise not always accompanied by the
production of inflammation. If the abdominal cavity of
a dog, for example, be carefully opened so as to make as
slight a wound as possible, and no injury be done to the
intestines, large quantities of bouillon cultures or watery
suspensions of this organism may be, and repeatedly have
been introduced into the peritoneum without the slightest
injury to the animal. On the contrary, if some substance
which acts as a direct irritant to the intestines — such, for
example, as a small bit of potato upon which the organisms
are growing— be at the same time introduced, or the intes-
tines be mechanically injured, so that there is a disturbance
in their circulation, then the introduction of these organisms
is promptly followed by acute and fatal peritonitis. (Hal-
sted.1)
On the other hand, the results which follow their introduc-
tion into the circulation are practically constant. If one
1 The Johns Hopkins Hospital Reports. Report in Surgery, No. 1,
1891, ii, No. 5, 301-303.
MICROCOCCUS A U RE US 349
inject into the circulation of the rabbit through a vein of
the ear, or in any other way, from 0.1 to 0.3 c.c. of a bouillon
culture or watery suspension of a virulent variety of this
organism, a fatal pyemia always follows in from two and
one-half to three days. A few hours before death the animal
suffers frequently from severe convulsions. Now and then
excessive secretion of urine is noticed. The animal may
appear in moderately good condition until from eight to
ten hours before death. At the autopsy a typical picture
presents: the voluntary muscles are seen to be marked
here and there by yellow spots, which average the size of a
flaxseed, and are of about the same shape. They lie usu-
ally with their long axis running parallel to the muscle-
fibers. As the abdominal and thoracic cavities are opened
the diaphragm is often seen to be studded with them.
Frequently the pericardial sac is distended with a clear
gelatinous fluid, and almost constantly the yellow points
are seen in the myocardium. The kidneys are rarely with-
out them ; here they appear on the surface as isolated yellow
points, or, again, are seen as conglomerate masses of small
yellow points which occupy, as a rule, the area fed by a
single vessel. If one make a section into one of these
yellow points, it will be seen to extend deep down through
the substance of the kidney as a yellow, wedge-shaped mass,
the base of the wedge being at the surface of the organ.
It is very rare that these abscesses — for abscesses the
yellow points are, as we shall see when we come to study
them more closely — are found either in the liver, spleen,
or brain; their usual location being, as said, in the kidney,
myocardium, and voluntary muscles.
These minute abscesses have a dry, cheesy, necrotic
center, in which the micrococci are present in large numbers
350 APPLICATION OF METHODS OF BACTERIOLOGY
as may be seen upon cover-slips and in cultures prepared
for them.
Preserve in alcohol bits of all tissues in which the abscesses
are located. When these tissues are hard enough to cut
sections should be made through the abscess points and the
histological changes carefully studied.
Microscopic Study of Cover-slips and Sections. — In cover-
slip preparations this organism stains readily with the
ordinary dyes. In tissues, however, it is best to employ
some method by means of which contrast-stains may be
utilized, and the location and grouping of the organisms in
the tissues rendered more conspicuous. When stained, sec-
tions of tissues containing the small abscesses present the
following appearances:
To the naked eye will be seen here and there in the section,
if the abscesses are very numerous, small, darkly stained
areas which range in size from that of a pin-point up to
those having a diameter of from 1 to 2 mm. These points,
when in the kidney, may be round or oval in outline; or
may appear wedge-shaped, with the base of the wedge
toward the surface of the organ. The differences in shape
depend frequently upon the direction in which the section
has been made through the kidney. In the muscles they
are irregularly round or oval.
When quite small they appear, in stained sections, to the
naked eye, as simple, round or oval, darkly stained points;
but when they are in a more advanced stage a pale center
can usually be made out.
. When magnified they appear in the earliest stages as
minute aggregations of small cells, the nuclei of which stain
intensely. Almost always evidences of progressing necrosis
can be seen about the center of these cell-accumulations.
MICROCOCCUS AUREUS 351
The normal structure of the cells of the tissues is more or
less destroyed; there is seen a granular condition due to
cell-fragmentation; at different points about the center
of this area the tissue appears cloudy and the tissue-cells
do not stain readily. Round about and through this spot
are seen the nuclei of pus-cells, many of which are under-
going disintegration. In the smallest of these beginning
abscesses the micrococci are to be seen scattered about the
center of the necrotic tissue; but in a more advanced stage
they are commonly seen massed together in very large
numbers in the form commonly referred to as emboli of
micrococci, meaning, obviously, that they had. developed
within the lumen of a tiny bloodvessel.
When the process is well advanced, the different parts
of the abscess are more easily detected. They then present
in sections somewhat the following conditions: at the
center can be seen a dense, granular mass which stains readily
with the basic aniline dyes, and when highly magnified is
found to be made up of micrococci. Sometimes the shape
of this mass of micrococci corresponds to that of the capil-
lary in which the organisms became lodged and developed.
Immediately about the embolus of cocci the tissues are in an
advanced stage of necrosis. Their structure is almost com-
pletely destroyed, although the destruction is seen to be
more advanced in some of the elements of the tissues than
in others. As we approach the periphery of this faintly
stained necrotic area it becomes marked here and there
with granular bodies, irregular in size and shape, which
stain in the same way as do the nuclei of the pus-cells and
represent the result of disintegration going on in these cells.
Beyond this area we come upon a dense, deeply stained
zone, consisting of closely packed pus-cells; of granular
352 APPLICATION OF METHODS OF BACTERIOLOGY
detritus resulting from destructive processes acting upon
these cells; and of the normal cellular and connective-tissue
elements of the part. Here and there through this zone
will be seen localized areas of beginning death of the tissues.
This zone gradually fades away into the healthy surrounding
tissues. It constitutes the so-called "abscess wall."
Such is the picture presented by the miliary abscess
when produced experimentally in the rabbit, and it corre-
sponds from beginning to end with the pathological changes
which accompany the formation of larger abscesses in the
tissues of human beings.
From these small abscesses in the tissues of the rabbit
micrococcus aureus may again be obtained in pure culture,
and will present identically the same characteristics that
were possessed by the culture with which the animal was
inoculated.
A characteristic of all staphylococcus abscesses, small as
well as large, is centralized death of tissue; even in those of
microscopic dimensions this area of necrosis is always
discernible by appropriate methods of examination. It
represents the very starting-point of the destructive process,
and is referable to the combined action of the endotoxins
of the bacteria and the interference with the circulation
of the past due to proliferation of cells about the point at
which the bacteria are located.
As a result of the studies of van de Velde, Krauss, von
Lingelsheim, Neisser and Wechsberg, and others, our knowl-
edge of the poison that causes the destruction — staphylptoxin,
as it is called — has been greatly extended. Through the
work of these investigators we now know that the patho-
genic properties of micrococcus aureus are due to a definite
soluble toxin elaborated by it: that this poison is produced
MICROCOCCUS A U RE US 353.
under artificial conditions of cultivation, and may be sepa-
rated from the living organisms by filtration; that when
injected into the living animal body its effects upon the
tissues are essentially reproductions of those accompanying
the growth of the organism itself; that when this action
is tested upon particular cells, such as erythrocytes and
leukocytes, two distinct properties are exhibited, one a
hemolytic, through which the red corpuscles are dissolved,
the other a leucocidic, through which the white blood-cor-
puscles are destroyed; that the hemolytic and leucocidic
properties are distinct from one another, and are due to the
activities of two lysins, of which the staphylotoxin is (in
part?) composed, and which may be separated from one
another by appropriate methods of analysis; that the result
of the treatment of animals with gradually increasing non-
fatal doses of staphylotoxin is the appearance in the blood
of the animals of antibodies (antilysins) that inhibit the
action of the toxins (lysins) ; and, finally, that in the serum
of certain animals (man and horse) similar antilysins in
varying amounts are normally present.1
Petersen, Paltchikowsky, Proscher, and others have
recently attempted to prepare an antistaphylococcus serum
with the following results: The serum of patients recov-
ering from severe staphylococcus infections contains pro-
tective substances which serve to protect rabbits from twice
the fatal dose of a staphylococcus culture. Similarly the
serum of immunized rabbits and goats, as shown by the
experiments of Petersen, possesses about the same degree
1 See van de Velde, Annales de I'Institut Pasteur, tome x, p. 580; Krauss
Wiener klin. Wochenschrift, 1900, No. 3; Von Lingelsheim, Etiologie und
Therapie der Staphylokoken Infektion (monograph), Berlin-Wien, 1900;
Neisser and Wechsberg, Zeitschrift fur Hygiene und Infektionskrankheiten,
1901, Bd. xxxvi, S. 299.
23
354 APPLICATION OF METHODS OF BACTERIOLOGY
of protective powers. No antitoxic power could be demon-
strated in the serum of the treated animals. The extremely
limited degree of the protective power of antistaphylo-
coccus serums makes them useless for curative purposes in
human beings, as Petersen calculated that an adult would
require from 350 to 700 c.c. of the serum at a single dose,
as judged by its effects on the lower animals.
OTHER COMMON PYOGENIC ORGANISMS.
MICROCOCCUS PYOGENES (Rosenbach), Migula, 1900. Synonyms:
Staphylococcus pyogenes albus, Rosenbach, 1884; Micrococcus pyo-
genes albus, Lehmann and Neumann, 1896.
MICROCOCCUS CITREUS (Passet), Migula, 1900. Synonym: Staphy-
lococcus pyogenes citreus, Passet, 1895.
The pus of an acute abscess in the human being may
sometimes contain organisms other than micrococcus aureus.
Micrococcus pyogenes and micrococcus citreus may be found.
The colonies of the former are white, those of the latter
are lemon yellow. With these exceptions they are in all
essential cultural peculiarities similar to micrococcus aureus.
As a rule, they are not virulent for animals, and when they
do possess pathogenic properties, it is in a much lower
degree than is commonly the case with the golden micro-
coccus. Streptococcus pyogenes is also present sometimes.
The commonest of the pyogenic organisms, however, is
that just described, viz. : micrococcus aureus.
An organism that is almost universally present in the
skin, and is often concerned in producing mild forms of
inflammation, is Staphylococcus epidermidis albus (Welch),
an organism that readily may be confused with micrococcus
pyogenes. It differs from the latter by the slowness with
which it liquefies gelatin and by the comparative absence
STREPTOCOCCUS PYOGENES 355
of pathogenic properties when injected into the circulation
of rabbits. Welsh regards this organism as a variety of
micrococcus pyogenes.
STREPTOCOCCUS PYOGENES (ROSENBACH), MIGULA,
1900.
SYNONYMS: Streptococcus, Billroth, 1874; Streptococcus pyogenes,
Rosenbach, 1884.
From a spreading phlegmonous inflammation prepare
cover-slips and cultures. What is the predominating
organism? Does it appear in the form of irregular clusters
FIG. 67
»•
UV*
jgjf«I
**r»»> /
?«£»&
^ •"
Streptococcus pyogenes in pus.
like those of grapes, or have its individuals a definite,
regular arrangement? (See Fig. 67.) Are its colonies like
those of micrococcus aureus?
Isolate this organism in pure cultures. In these cul-
356 APPLICATION OF METHODS OF BACTERIOLOGY
tures it will be found on microscopic examination to present
an arrangement somewhat like a chain of beads. (Fig. 68.)
Its peculiarities should be as follows:
Upon microscopic examination a micrococcus should be
found, but differing in its arrangement from those just
described. The single cells are not scattered irregularly or
arranged in clumps similar to bunches "of grapes, but are
joined together in chains like strands of beads. These
strands are sometimes regular in the arrangement and size
of the individual cells composing them, but more commonly
certain irregular groups may be seen in them, appearing
FIG. 68
/,/
Streptococcus pyogenes.
as if two or three cells had fused together to form a link
in the chain, so to speak, that is somewhat longer than the
others; again, portions of the chain may be thinner than
the rest, or it may appear broken or ragged. Commonly
the individuals comprising this chain of cocci are not round,
but appear flattened on the sides. The chains are some-
times short, consisting of but four to six cells; or, again, they
may be much longer, and extend from a half to two-thirds
the way across the field of the microscope.
Under artificial conditions this organism sometimes grows
well, and can be cultivated through many generations, while
STREPTOCOCCUS PYOGENES - 357
at other times it rapidly loses its vitality. When isolated
from the diseased area upon artificial media it seems to
retain its vitality for a longer period if replanted upon fresh
media every day or two for a time; but if the first generation
be transplanted and allowed to remain upon the original
medium for from a week to ten days, it is not uncommon to
find the organism incapable of further cultivation.
Under no conditions is its growth very luxuriant.
On gelatin plates its colonies appear after forty-eight to
seventy-two hours as very small, flat, round points of a
bluish-white or opalescent appearance. They do not cause
liquefaction of the gelatin, and in size they rarely exceed
0.6-0.8 mm. in diameter. Under low magnifying power
they have a brownish or yellowish tinge by transmitted
light and are very finely granular. As the colonies become
older their regular borders may become slightly irregular or
notched.
In stab-cultures in gelatin they grow along the entire
needle-track as a finely granular line, the granules represent-
ing minute colonies of the organism. On the surface the
growth does not usually extend beyond the point of
puncture.
On agar-agar plates the colonies appear as minute pearly
points, which when slightly magnified are seen to be finely
granular, of a light-brownish .tinge, and regular at their
margins.
When smeared upon the surface of agar-agar or gelatin
slants the growth that results is a thin, pearly, finely granular
layer, consisting of minute colonies growing closely side by
side. Its most luxuriant growth is usually on glyceriri-agar-
agar at the temperature of the body (37.5° C.), and its
least on gelatin at from 18° to 20° C.
358 APPLICATION OF METHODS OF BACTERIOLOGY
On nutrient agar-agar to which, under aseptic precautions,
unheated, defibrinated or whole blood has been added, it
often causes hemolysis, i. e., decolorization of the blood in
the neighborhood of the growth. Some strains of the
organism when thus grown are surrounded by a greenish
zone, Streptococcus viridans; others by a perfectly clear,
colorless zone, Streptococcus hemolyticus.
On blood serum its colonies present little that is character-
istic; they appear as small, moist, whitish points, from 0.6
to 0.8 mm. in diameter, that are slightly elevated above the
surface of the serum. They do not coalesce to form a layer
over the surface, but remain as isolated colonies.
On potato no visible development appears, but after a
short time (thirty-six to seventy-two hours) there is a slight
increase of moisture about the point of inoculation, and
microscopic examination shows that multiplication of the
organisms placed at this point has occurred.
In milk its conduct is not always the same, some cultures
causing a separation of the milk into a firm clot and colorless
whey, while others do not produce this coagulation. The
latter, when cultivated in milk of a neutral or slightly
alkaline reaction, to which a few drops of litmus tincture
have been added, produce, as a rule, only a very faint pink
color after twenty-four hours at 37° C.
In bouillon it grows as tangled masses or clumps, which
upon microscopic examination are seen to consist of long
chains of cocci twisted or matted together.
It grows best at the temperature of the body (37.5° C.),
though development does occur at the ordinary room-tem-
perature.
It is not soluble in bile.
It ferments some of the carbohydrates, notably dextrose,
maltose, lactose and salicin, but not mannite or inulin.
STREPTOCOCCUS PYOGENES 359
It is a facultative anaerobe.
It stains with the ordinary aniline dyes, and is not decolor-
ized when subjected to Gram's method.
It is not motile. Under artificial conditions we have no
reason to believe that it enters a stage in which its resis-
tance to detrimental agencies is increased. In the tissues
of the body, however, it appears to possess marked vitality,
for it is not rare to observe recurrences of inflammatory
conditions due to this organism, often at a relatively long
time after the primary site of infection has healed.
Such in general will serve to identify the streptococci
concerned in the disease..
Streptococcus pyogenes is the organism most commonly
found in rapidly spreading suppurations, while micrococcus
aureus is most frequently found in circumscribed abscess
formations; they may also be found together, and these
relationships may be reversed at times.
The results of its inoculation into the tissues of lower
animals are described by Rosenbach and Passet as pro-
tracted, progressive, erysipelatoid inflammations; and
Fehleisen, who first described a streptococcus in erysipelas
that is closely related to the Streptococcus pyogenes under
consideration, stated that it produced in the tissues of
rabbits (the base of the ear) a sharply defined, migratory
reddening without pus formation. The writer encountered
a strain of this organism that possessed the property of
inducing erysipelas when introduced into the skin of the
ear, and disseminated abscess formation when injected into
the circulation of rabbits. In one animal these conditions
appeared simultaneously. This observation has an impor-
tant bearing upon the question concerning the identity of
streptococci found in various inflammatory conditions, such
360 APPLICATION OF METHODS OF BACTERIOLOGY
for instance, as the spreading erysipelatoid manifestations
on the one hand, and the circumscribed abscess formations
on the other.
The results that follow upon the inoculation of animals
with cultures of streptococci obtained from various inflam-
matory lesions are, as a rule, inconstant. At times cultures
will be encountered that are apparently without virulence,
no matter how tested; while again cultures from other
sources exhibit the most marked pathogenic properties,
even when employed in almost infinitesimal quantities.
Between these extremes every gradation may be expected.
The virulence of a culture as exhibited upon animals under
experiment is not necessarily proportional to the intensity
of the pathological process from which it was derived.
In general it may be said that the virulence of strepto-
coccus is directly proportionate to its power to hemolyze
blood. That is to say: a culture that actively and con-
spicuously brings about the laking of blood with complete
decolorization of the hemoglobin is more apt to be virulent
than one devoid of that property. With fluctuations in
such hemolytic function there are corresponding fluctuations
in virulence.
There is never any certainty of faithfully reproducing, by
inoculation into susceptible animals, the pathological lesion
from which a culture of the organism may have been ob-
tained. The introduction into a susceptible animal of a
culture derived from either a spreading phlegmon or an
erysipelatous inflammation may result in erysipelas, general
septicemia, local abscess-formation, or, as said, may have
no effect at all. Cultures may be encountered that are
pathogenic for one susceptible species of animals and not
for another.
STREPTOCOCCUS PYOGENES 361
Under the ordinary conditions of artificial cultivation
fully virulent varieties of Streptococcus pyogenes usually
lose their virulence after a short time. This property may
sometimes be preserved by cultivation upon nutrient gela-
tin for two days at 22° C., keeping the cultures after this
time in the refrigerator, and transplanting upon fresh gela-
tin every five or six days; or by growing the organism in a
mixture of 2 parts of horse or human blood serum and 1
part of nutrient bouillon, or of 1 part of ascites fluid and
2 parts of bouillon. < „ ^
Its virulence may sometimes be increased by passage
through a series of susceptible animals.
Variations in Streptococci. — The obiquity of strepto-
cocci and their frequent relationship to pathological condi-
tions of the gravest nature combine to make them of more
than passing interest. Our knowledge of the group is as
yet far from satisfactory, yet it has been enhanced in many
important particulars during the past three or four years.
When streptococci are encountered in the various diseased
conditions of the body we cannot longer content ourselves
with the fact that they conform to the commonly accepted
morphological specifications and possess the customary
gross cultural peculiarities as outlined above. We have
known for a long time that streptococci vary considerably
in a number of particulars, and if we arbitrarily decide to
call a given streptococcus a typical example of the species
we shall find in cultures from different sources very many
deviations from such a standard.
According to the nature of these deviations or variations
numerous efforts have been made to arrange the streptococci
in more or less fixed groups. Such efforts have not been
entirely satisfactory in their results, though they have con-
362 APPLICATION OF METHODS OF BACTERIOLOGY
tributed helpful information to our general comprehension
of the subject.
The conception that streptococci forming long chains
are more often pathogenic than those forming short chains is
not always a trustworthy criterion ; and the differences in the
action of different streptococci upon particular ingredients
of special culture media has helped, but not enough for
satisfactory grouping.1
The discovery that the majority of streptococci associated
with serious pathological phenomena have the power of
hemolyzing blood, while others do not possess this function
is a great step in advance, but unfortunately for the sim-
plicity of the matter we find such differences among those
hemolyzing or hemolytic streptococci as to make further
subdivision (classification) of the group desirable.
With this in mind recent studies by a number of investi-
gators have contributed information of the greatest practical,
as well as theoretical, value.
As in the case of the pneumococci (which see) there is
now an agreement of opinion that differentiation of these
closely allied varieties of pathogenic organisms is best
accomplished through specific immunologic reactions, and
to this end the agglutination test made with the serum of
immunized animals seems to prove trustworthy; though as
yet there have not been established such definite groups
or types as has been done with the pneumococci.
If, for instance, any given group of hemolytic strepto-
cocci be obtained from different diseases and an animal
be rendered immune from one of them by appropriate
means, the serum from that animal will certainly agglu-
1 See The Use of Blood Agar for the Study of Streptococci, Monograph
No. 9, Rockefeller Inst. Med. Research, January 21, 1919.
STREPTOCOCCUS PYOGENES 363
tinate the strain of streptococci used for immunization.
It may agglutinate a certain number of the other hemo-
lytic varieties but it is unlikely that it will agglutinate
all. We would assume then that etiologically all those
agglutinated by that serum were of one type, while all
others of our group were of another type or probably types,
and so we might continue throughout the whole group
originally selected and arrange them into types or classes
the members of each of which would react specifically with
its homologous serum and not with other sera.
The object of such grouping is more far-reaching than,
that of simply identifying variations; it has a most practical
bearing on all efforts to produce antisera that may be used
in preventing infection or in curing it when once under way.
We have already enough evidence to justify the general
statement that for any immune serum to possess protective
or curative properties for a bacterial infection the animal
from which it is obtained must have been immunized with
either bacteria direct from the disease against which the
serum is to be used or with types closely allied to them in
the fundamental infective and immunologic characteristics.
(Does this hold for such infections as tetanus and diphtheria?)
This being the case the desirability of establishing groups
or types of streptococci, the members of which are closely
allied in these particulars, becomes evident: for it is not
practicable in efforts to treat infections along these lines
to always immunize animals from which the curative serum
is to be obtained with the organism specifically concerned
in the case under consideration.
Efforts have been made to overcome this difficulty by the
production of "polyvalent" antisera, i. e., serum from ani-
mals immunized not by the use of a single strain of strepto-
364 APPLICATION OF METHODS OF BACTERIOLOGY
cocci, but by many, the idea being that one or the other
of the component streptococci used in the process of immun-
ization may be identical, or sufficiently nearly so, to the one
concerned in the infection to be treated, as to play its part
in the production of the desired specific component of the
antiserum so obtained. This shot-gun-like proceedure some-
times succeeds, but even so, the element of uncertainty is
too evident to justify the adherence to it as a permanent
method. All the indications point to the substitution of a
more scientific, a more logical procedure in the near future,
a procedure closely allied to that by which the pneumo-
cocci have been grouped and from which so much light
has been shed upon the complicated problem of pneumonia.
Though the last word has not yet been said, the indi-
cations are that in erysipelas, septic sore-throat, pleurisy,
rheumatism, scarlet fever anginas, measles sore-throat,
postinfluenzal pneumonia, wound infections, etc., we have
streptococci fundamentally different from the ubiquitous
Streptococcus pyogenes as commonly described.
Not any of the many investigations of this phase of the
subject appear to illustrate more clearly the possibilities
and practical value of studies upon the streptococci than
do those of Havens, conducted in U. S. Gen. Hospital No.
12, at Biltmore, N. C.1
Havens undertook to classify the hemolytic streptococci
only, as there is now a general agreement that the non-
hemolytic varieties are of but subordinate importance
insofar as they concern infections in man.
As material for his studies 292 different strains of strepto-
cocci were used; that is to say, there were that number of
1 Jour. Infect. Dis., 1919, No. 4, vol. xxv.
STREPTOCOCCUS PYOGENES 365
cultures, each from a different individual. These indi-
viduals came from- nearly all parts of the country and
therefore fairly represented conditions to be found through-
out the population in general. The physical conditions
of the persons from whom the cultures were obtained were
sufficiently diverse to x indicate the comprehensive nature
of the investigation. They were:
Throat cultures:
Healthy carriers 80
Acute bronchitis 60
Measles 9
Sore-throat . . . .... 30
Pneumonia:
Sputum 20
Autopsy 21
Empyema -. > ., . . .- . 67
Gunshot wounds 4
Renal infections . : . . . . . ... -. . . 1
Total 292
By selecting at random from the 292 cultures three that
were characteristic in their gross peculiarities and immun-
izing animals from them, it was found: (1) That in their
agglutinating reactions these cultures were identical; and (2)
that by the same test 139 other strains, or 47 per cent, of the
whole number, proved to be like them, while the remainder,
153 strains, failed in their agglutinating reactions with that
serum and were therefore regarded as different. Animals
were then immunized from one member of this negative
group and of the 153 strains in that group 54, or 19 per
cent., of the whole number agglutinated with the serum
from this immunized animal. Again a culture of those that
failed to agglutinate in the second test was chosen and a
third animal rendered immune from it. With the serum
from that animal 79 members of the group, or 27 per cent.,
of all the cultures reacted positively.
366 APPLICATION OF METHODS OF BACTERIOLOGY
In appropriately high dilutions the members of one group
did not react with the serum homologous for either of the
other groups.
A fourth group, containing only 22 cultures, gave such
irregularities that no final attempt was made at subgrouping.
However, of the total of 292 strains examined, 93 per cent,
could be definitely arranged by specific reactions into these
groups; while 7 per cent., constituting a fourth group, was
not conclusively investigated. The four strains of hemo-
lytic streptococci thus established grouped themselves about
the various normal and pathological conditions from which
the individual cultures were obtained according to the
following table:
GROUPING OF 292 STRAINS OF HEMOLYTIC STREPTOCOCCI
(HAVENS).
Group 1.
Per cent.
Group 2.
Per cent,
Group 3.
Per cent.
Group 4.
Per cent.
Total in
per cent.
Number
of sources.
Total strains, all
sources .
47
19
27
7
100
Throat cultures:
Healthy carriers
46
20
22
12
100
80
Acute bronchitis
48
20
23
9
100
60
Measles .
55
45
0
0
100
9
Tonsillitis and
sore-throat .
30
10
57
3
100
30
Bronchopneumonia
Sputum .
40
20
30
10
100
20
Necropsy
95
0
5
0
100
21
Emphysema .
42
22
30
6
100
67
Gunshot wounds
0
0
75
25
100
4
Renal infections
100
0
0
0
1
From this, Havens suggests that healthy carriers may be
the source of supply for all hemolytic streptococci causing
disease.
STREPTOCOCCUS PYOGENES 367
The study of bronchopneumonias — though small in num-
ber— he thinks indicates a special virulence of streptococci
of Group I in this disease.
In his sore-throat and tonsillitis cases the members of
Group III predominate and it is of special interest to note
that these cases all came from one hospital ward and prob-
ably therefore had a common origin.
These studies further show that the specificity of this
grouping is not limited to the agglutinating reactions of
the members of the groups but is still further demonstrated
by the fact that in vitro the streptococci of one group are
killed by its homologous immune serum, while such serum is
without germicidal action on the members of the other groups.
By analogous proceedures Tunnicliffe1 has shown that
according to their specific serologic reactions the strepto-
cocci accountable for the angina of scarlet fever and those
present in typical cases of erysipelas represent distinct
varieties of the hemolytic group of streptococci.
The bearing of all this on efforts to produce serum for
the treatment and prevention of streptococcus infections is
evident.
We can no longer expect the serum from an animal immun-
ized from any strain of streptococcus taken at random from
whatever source to be effective. It may be, but if so it is
only by chance. Serum A may be expected to be effective
when used against infection caused by streptococcus of A
group, but not those of Group B and vice versa.
Antistreptococcus Serum. — Certain animals - - notably
horses and asses — as well as some smaller animals, may be
rendered immune from Streptococcus pyogenes. In vary-
1 Jour. Am. Med. Assn., 1920, No. 20, vol. Ixxv, p. 1339.
368 APPLICATION OF METHODS OF BACTERIOLOGY
ing degrees the blood serum of such immunized animals
has both a curative and a prophylactic influence upon the
course of streptococcus infection in human beings.
The method of producing the antiserum is, in general,
to inject gradually increasing doses of virulent Streptococcus
pyogenes (beginning with dead cultures)- into the tissues
of the animal until its blood serum is found to have an
inhibiting effect upon experimentally produced streptococcus
infection in test animals.
Reports upon the therapeutic use of antistreptococcus
serum in a variety of streptococcus infections are dis-
cordant; some authors being enthusiastic as to its curative
value, others skeptical or actually denying to it such virtues.
The reasons for these divergent opinions are now pretty
manifest from what has been said under the preceding
heading "Variations in Streptococci."
THE LESS COMMON PYOGENIC ORGANISMS.
The organisms that have just been described are com-
monly known as the "pyogenic cocci" of Ogston, Rosenbach,
and Passet, and up to as late as 1885 were believed to be the
specific factors concerned in the production of suppurative
inflammations. Since that time, however, there has been
considerable modification of this view, and while they are
still known to be the most common causes of suppuration,
they are also known to be not the only causes of this phe-
nomenon.
With the more general application of bacteriological
methods to the study of the manifold conditions coming
under the eye of the physician, the surgeon, and the patholo-
gist, observations are constantly being made that do not
LESS COMMON PYOGENIC ORGANISMS 369
accord with the earlier ideas upon the dependence of all
forms of suppuration on invasion by the pyogenic cocci.
There is an abundance of evidence to justify the opinion
that a number of organisms not commonly classed as pyo-
genic may, under certain circumstances, assume this property
or may, in fact, have pus formation as one of the common
accompaniments of their pathogenic activities. For example :
The bacillus of typhoid fever has been found in pure
culture in osteomyelitis of the ribs, in acute purulent otitis
media, in abscess of the soft parts, in the pus of empyema,
and in localized fibrino-peritonitis, either during its course or
as a sequel of typhoid fever.
Bacillus coli communis has been found in pure culture in
acute peritonitis, in liver-abscess, in purulent inflammation
of the gall-bladder and ducts, and in appendicitis. Welch1
found it in pure culture in fifteen different inflammatory
conditions.
Micrococcus lanceolatus (pneumococcus) has been found
alone in abscess of the soft parts, in purulent infiltration of
the tissues about a fracture, in purulent cerebrospinal
meningitis, in suppurative synovitis, in acute pericarditis,
and in acute inflammation of the middle ear.
Organisms simulating bacterium diphtheriticum are fre-
quently encountered in large numbers in the pus of superfi-
cial wounds, and especially in ulcerations of the skin and
mucous membranes.
Moreover, many of the less common organisms have been
detected in pure cultures in inflammatory conditions with
which they were not previously thought to be concerned,
and to which they are not usually related etiologically.
1 Conditions Underlying the Infection of Wounds, American Journal of
the Medical Sciences, November, 1891,
24
370 APPLICATION OF METHODS OF BACTERIOLOGY
In consideration of such evidence as this it is plain that
we can no longer adhere rigidly to the opinions formerly
held upon the etiology of suppuration, but must subject
them to modifications in conformity with this newer evi-
dence. We now know that there exist bacteria other than
the "pyogenic cocci," which, though not normally pyogenic,
may give rise to tissue-changes indistinguishable from those
produced by the ordinary pus-organisms.
Furthermore — of organisms not classified as of the
"pyogenic group," but where growth in the tissues is always
accompanied by pus formation — one may mention micro-
coccus gonorrhea, micrococcus intracellularis, and bacillus
pestis as conspicuous examples.
MICROCOCCUS GONORRHECE-ffi (NEISSER), 1879.
SYNONYM: Gonococcus Neisser, Bumm, 1887.
One observes upon microscopic examination of cover-slips
prepared from the pus of actue gonorrhea that many of the
pus-cells contain within their protoplasm numerous small,
stained bodies that are usually arranged in pairs. Occasion-
ally a cell is seen that contains only one or two pairs of
such bodies; again, a cell will be encountered that is packed
with them. Occasionally masses of these small bodies will
be seen lying free in the pus. (See Fig. 69.) The majority
of the pus cells may not contain them.
These small, round, or oval bodies are the so-called
"gonococci" discovered by Neisser, and more fully studied
subsequently by Bumm, to whom we are indebted for much
of our knowledge concerning them.
As the name implies, this organism is a micrococcus, and
as it is commonly arranged in pairs (flattened at the sur-
MICROCOCCUS GONORRHCE& 371
faces in juxtaposition) it is often designated as diplococcus
of gonorrhea. It is always to be found in gonorrheal pus,
and often persists in the genital discharges and secretions far
into the stage of convalescence. It is not present -in inflam-
matory conditions other than those of gonorrheal origin.
It is easily detected microscopically in the secretions of
acute gonorrhea. In secondary lesions and in very old,
chronic cases it is difficult of detection and frequently
FIG. 69
'
f *M /
ljj«
i^e
•
Pus of gonorrhea, showing diplococci in the bodies of the pus-cells.
eludes all efforts to find it. It is stained by the ordinary
methods, but perhaps most satisfactorily with the alkaline
solution of methylene-blue. Most important as a differen-
tial test is its failure to stain by the method of Gram. (How
does this compare with the behavior of the other pyogenic
cocci when treated in the same way?)
It does not grow upon ordinary nutrient media, and has
only been isolated in culture through the employment of
special methods. Its growth under artificial conditions
seems to be favored by some particular nutrient substance
that is supplied by blood or blood serum, and in many of
372 APPLICATION OF METHODS OF BACTERIOLOGY
the media that have been successfully used for its cultiva-
tion this substance is apparently an essential constituent.
It was first isolated in culture by Bumm, who used for
this purpose coagulated human blood serum obtained from
the placenta.
Wertheim improved the method of Bumm by using a
mixture of equal parts of sterile human blood serum and
ordinary sterilized nutrient agar-agar, the latter having
been liquefied and kept at 50° C. until after the mixture
was made, when it was allowed to cool and solidify.
Other investigators have substituted for human blood
serum certain pathological fluids from the human body,
such as ascites-fluid, fluid from ovarian cysts, and serous
effusions from the pleura and from the joint-cavities.
The method used by Pfeiffer for the cultivation of bac-
terium influenzse (see that method) is also said to have been
successfully employed.
A simple medium that has given satisfactory results in
our hands is that devised by Vedder. It consists of ordi-
nary beef infusion agar (1.5 per cent, agar) to which 1 per
cent, of corn starch is added. The medium contains neither
sodium chloride nor peptone and has a reaction correspond-
ing to 0.2 to 0.5 per cent, acid to phenolphthalein.
Wassermann1 calls attention to the success he has had
in cultivating this organism upon a mixture of swine serum
and nitrose, the latter being a commercial product chemically
known as casein-sodium phosphate.
The preparation of the medium and its composition are
as follows:
In an Erlenmeyer flask mix 15 c.c. of swine serum, as
1 Zeitschiift fur Hygiene und Infektionskrankheiten, Bd. xvii, p. 298.
MICROCOCCUS GONORRHCEM 373
free as possible from hemoglobin; 30 to 35 c.c. of water;
2 to 3 c.c. of glycerin; and finally 0.8 to 0.9 gram (i. e.,
about 2 per cent.) of nitrose. This is boiled, with gentle
agitation, over a free flame, until all ingredients are dissolved
and the cloudy fluid has become quite clear. After such
boiling the mixture can be sterilized by steam without
precipitating the albumen, and may then be kept indefi-
nitely ready for use.
When needed, the flask and its contents are heated to
50° C.; from six to eight tubes of 2 per cent, peptone-
agar-agar are dissolved by boiling, brought to 50° C.,
and then mixed with the solution in the flask and the mass
poured into Petri dishes. Upon the surface of this serum-
nitrose-agar the cultivation is to be conducted. Wassermann
lays particular stress upon two points that are essential to
success, viz.? the preliminary boiling of the serum-nitrose
mixture before steam sterilization, as this prevents precipi-
tation of the albumin; and the necessity of having both
the serum-nitrose mixture and the agar-agar, to be mixed
with it, at not over 50° C., for if they are at a boiling tem-
perature when mixed, or if they are brought to the boiling
temperature after mixing, the albumin will be precipitated
notwithstanding the presence of the nitrose,- which otherwise
prevents this.
Wassermann further observes that some samples of serum
require to be more highly diluted with water than in the
proportions given above; that the agar-agar should be
feebly, but distinctly, alkaline to litmus, causing no red-
dening whatever of blue litmus paper; and, finally, that
the Petri dishes containing the solidified medium on which
the cultures are growing are best kept bottom upward, so
as to prevent water of condensation collecting on the surface.
374 APPLICATION OF METHODS OF BACTERIOLOGY
By the use of the above mediflm he has cultivated the gono-
coccus from about one hundred different cases.
If micrococcus gonorrhceae be transplanted from the origi-
nal culture to either glycerin-agar-agar or to Loffler's serum
mixture, a growth is sometimes observed, more often in the
latter than in the former, but of so feeble a nature that these
substances cannot be regarded as suitable for its cultivation
and certainly not for its direct isolation from the body. As
a rule, development does not occur on glycerin-agar.
Its growth is favored by at least partial anaerobic con-
ditions.
Microscopic examination of colonies of this organism
reveals the presence of a diplococcus somewhat larger than
the ordinary pyogenic cocci. The opposed surfaces of the
individual cells that comprise the couplets are flattened and
separated by a narrow slit. At times the cocci are arranged
as tetrads.
This organism cannot be grown at a temperature lower
than that of the human body, and cultures that have been
obtained by either of the favorable methods are said to
lose their vitality when kept at ordinary room-temperature
for about two days.
It is killed in a few hours by drying.
Cultures retain their vitality under favorable conditions
of nutrition, temperature, and moisture for from three to
four weeks.
This organism is without pathogenic properties for
monkeys, dogs, and horses, as well as for the ordinary
smaller animals used for this purpose in the laboratory.
In man typical gonorrhea has been produced by the
introduction into the urethra of pure cultures of this
organism.
MICROCOCCUS GONORRHCEM 375
In addition to its causal relation to specific urethritis,
it is the cause of gonorrheal prostatitis in man, of gonorrheal
proctitis in both sexes, and of gonorrheal inflammation of
the urethra, of Bartholin's glands, of the cervix uteri, and
of the vagina in women and young girls. It is etiologically
related to the specific conjunctivitis (ophthalmia neona-
torum) of young infants, and also occasionally to ophthalmia
in adults.
Secondarily, it is concerned in specific inflammations of
the tubes and ovaries, of the lymphatics communicating
with the genitalia, of the serous surfaces of joints, and of
those of the heart, lungs, and abdominal cavity.
Other species of micrococci have from time to time been
described as occurring in the pus of acute urethritis and of
other purulent inflammations. Many of these are of no
significance. Some of them possess peculiarities that might
lead to confusion. The diplococcus described by Heiman1
has certain points of resemblance to the gonococcus, such
as its location in the ^bodies of pus-cells, its grouping as
diplococci, its size and general appearance; but it is still
readily distinguished from the gonococcus by its retention
of color when treated by Gram's method. The diplococcus
detected by Bumm in puerperal cystitis is likewise often
found within pus-cells, but it is readily differentiated from
the gonococcus by its growth upon ordinary nutrient media.
Micrococcus intracellularis of Weichselbaum, isolated
from the sero-purulent fluid of the spinal canal in cases of
epidemic cerebrospinal meningitis, is microscopically also
strikingly like the gonococcus as it is seen in pus; but,
unlike the latter organism, may be cultivated by the ordinary
1 New York Medical Record, June 22, 1895.
376 APPLICATION OF METHODS OF BACTERIOLOGY
methods. Micrococcus catarrhalis, so often seen within the
bodies of pus cells in the nasal discharges of acute catarrh
also suggests the organism under consideration, but is -easily
differentiated by its growth on the ordinary culture media.
Summary of Distinguishing Peculiarities. — Since gonorrheal
discharges may be contaminated with pyogenic cocci other
than those causing the specific inflammation, it is important
in efforts to identify the gonococcus that the differential
tests be borne in mind and put into practice. The gonococcus
is differentiated from the commoner pyogenic organisms
by the following peculiarities.
First, it is practically always seen in the form of diplococci,
the pair of individual cells having the appearance of two
hemispheres, with the diameters opposed, and separated
from one another by a narrow, colorless slit. (Is this the
case with micrococcus aureus or streptococcus pyogenes?)
Second, in gonorrheal pus it is nearly always within the
protoplasmic bodies of pus-cells. (How does this compare
with the conditions found in ordinary pus?)
Third, it stains readily with the ordinary staining reagents,
but loses its color when treated by the method of Gram. (Treat
a cover-slip from ordinary pus by this method and note
the result.)
Fourth, it does not develop upon any of the ordinary
media used in the laboratory; while the common pus-
organisms, with perhaps the exception of the streptococci,
are vigorous growers and are not markedly fastidious as to
their nutritive medium.
Fifth, when obtained in pure culture by either of the
special procedures noted above, its cultivation may be
continued upon the same medium; but growth will usually
not be observed if it is transplanted to ordinary nutrient
MICROCOCCUS INTRACELLULARIS 377
gelatin, agar-agar, bouillon, or potato; should it grow under
these circumstances its development will be very feeble. (Is
this -the case with common pus-producers?)
Sixth, it has no pathogenic properties for animals, while
several of the pyogenic cocci, notably micrococcus aureus
and streptococcus pyogenes, are usually capable of exciting
pathological conditions. (This is less commonly true of
streptococcus pyogenes than of micrococcus aureus.)
Seventh, it has the power of fixing complement, and this
method of identification is of particular service in all medico-
legal cases as well as in other obscure cases not readily
diagnosed by the microscopic and cultural methods.1
MICROCOCCUS INTRACELLULARIS (WEICHSELBAUM),
MIGULA, 1900.
SYNONYMS: Diplococcus Intracellularis Meningitidis, Weichselbaum,
1887; Streptococcus Intracellularis (Weichselbaum), Lehmann and Neu-
mann, 1896.
Of the several organisms mentioned that might be mis-
taken for the gonococcus, no one of them is as important
as that concerned in the causation of epidemic cerebrospinal
meningitis.
This organism, described by Weichselbaum in 1887 under
the name "diplococcus Intracellularis meningitidis," was
found by him in the exudations of the brain and spinal
cord in six cases of acute cerebrospinal meningitis.
As its name implies, it is a diplococcus, practically always
seen within the bodies of pus-cells (polymorphonuclear
leukocytes) in the exudations characteristic of this disease.
It is not seen within the other cells of the morbid process.
1 See "Compliment-fixation;" also Schwartz and McNiel, Am. Jour.
Mod. Sc., cxliv, p. 815.
378 APPLICATION OF METHODS OF BACTERIOLOGY
It stains readily with any of the ordinary aniline dyes,
but is decolorized by the method of Gram. It is conspicuous
for the irregular way in which it takes up the dye, some
cells in a preparation (either from the exudate or from cul-
tures) being brightly and intensely colored, others being
much less so, or, indeed, often nearly colorless. There is
also a marked variation in the size of individual cocci, some
being normal, others being apparently swollen. These
latter are often pale, with a deeply staining center, giving
the appearance of a coccus surrounded by a capsule; it
is not improbable that these are degenerated. The ir-
regularities here noted are more common in cultures
than in fresh exudates from acute cases, and more common
in old than in young cultures, a state of affairs fully explained
by the self-digestion (autolysis) that this organism is known
to experience under conditions of artificial cultivation.
As seen in cultures, it is commonly arranged in pairs with
the individuals flattened at the surfaces of juxtaposition.
Sometimes it is seen grouped as four and occasionally as
short chains of three or four cells, but never as long chains.
Its size is that of the common pyogenic micrococci, and its
outline and arrangement in the pus-cells are so like those of
the gonococcus that the figure depicting gonorrheal pus
answers equally well to illustrate the appearance of the
exudate from acute meningitis.
Though facultative, still its parasitic nature is so
dominant that it can only be cultivated with difficulty
and uncertainty. The most satisfactory medium for its
isolation in pure culture from the diseased meninges is
coagulated blood serum (LofEer's mixture), and even here
one is not successful with each attempt. So uncertain is
its growth under artificial conditions that it is always advis-
MICROCOCCUS INTRACELLULARIS 379
able to inoculate a number of tubes with relatively large
quantities of the exudate, and even then growth often occurs
in only a part of them, notwithstanding the fact that on
microscopic examination the organism may have been
readily detected in large numbers in the exudate. Illus-
trative of this difficulty, the following experience of Council- *
man, Mallory, and Wright may properly be quoted i1
"As showing the difficulty in growing the organisms in
cultures made from the meninges at the postmortem exami-
nation, ten cultures were made in one case from the exuda-
tion on the brain and six from the cord, cover-slip exami-
nations showing abundant organisms in the cells. Only
two of the cultures from the brain and one from the cord
showed a growth. As a rule, the organisms were more
easily obtained in cultures made from the acute cases than
from the chronic."
When successfully isolated in pure culture its growth is
never profuse on any medium. On the serum mixture of
Loffler the isolated colonies appear as round, viscid, smooth,
sharply defined points that may attain a diameter of 1 to
1.5 mm. There is no liquefaction of the medium. Cultures
from very acute cases occasionally present an abundant
growth of fine, transparent colonies strongly suggestive of
those of micrococcus lanceolatus.
On glycerin-agar the colonies are round, pearly, trans-
lucent, flat, and viscid in appearance. They tend to become
confluent. Under low magnifying power they are homo-
geneous, semitransparent, faintly brownish, with well-defined
smooth margins. On plain agar the growth is feeble and
uncertain.
1 See Epidemic Cerebrospinal Meningitis, etc., Report of the State Board
of Health, Mass., 1898, by Councilman, Mallory, and Wright.
380 APPLICATION OF METHODS OF 'BACTERIOLOGY
Its growth in bouillon is slow and uncertain. It does not
cause clouding of the fluid, but collects at the bottom of the
tube as a scanty grayish sediment, that when disturbed
gives the impression of having a mucoid consistency. ;> .
It does not grow on potato and causes no change in litmus-
' milk.
It grows only at the temperature of the body, and can
be kept growing only by being transplanted to fresh media
about every two days, and even then growth often ceases
after a comparatively small number of transplantations.
If from a fresh growing culture a number of tubes be inocu-
lated and kept under favorable conditions it is a common
experience to have growth on only a part of them. It is
sometimes impossible to obtain a second growth on agar-agar.
In addition to its presence in the meningeal exudation
of epidemic cerebrospinal meningitis, this organism may
appear as a secondary invader of the lung, causing more or
less extensive pneumonic exudation; of the joints; the ear;
the eye; and the nose and throat. Though rarely, its
presence in the circulating blood may sometimes be demon-
strated.
Subcutaneous inoculation with pure cultures has usually
no effect. Injections into the great serous cavities may or
may not result in serofibrinous or fibrinopurulent inflam-
mation. Positive results are oftener obtained on young
guinea-pigs weighing about 150 grams, than on larger,
more mature animals. Intravenous inoculations are equally
unsatisfactory, though the results depend upon the original
virulence, the age of the culture and the animal selected.
In horses toxic symptoms are often the conspicuous result of
this mode of inoculation.
The only successful attempts to reproduce the morbid
MICROCOCCUS INTRACELLULARIS 381
conditions from which the organism is obtained are those
in which the living cultures have been injected directly
into the meninges. Weichselbaum produced congestion
with pus formation in the meninges of dogs and rabbits by
direct injection through openings made in the skulls; Coun-
cilman, Mallory, and Wright caused the death of a goat by
the injection into the spinal canal of 1 c.c. of a bouillon
suspension of a pure culture of the organism, the autopsy
revealing intense congestion of the meninges of both brain
and cord, with slight clouding of the meninges and slight
increase of meningeal fluid, and Flexner1 succeeded, through
injections of cultures into the spinal canal of monkeys, in
causing death of the animals with inflammation of the men-
inges of the cord and brain.
While the portal of entry for this organism to the system
is not definitely known, it is still of importance to note that
it often makes its exit from the body by way of the organs
that are secondarily involved and that open to without,
as the ear, nose, eye and lungs.
It is of equal importance to note that the organism is of
very low power of resistance, being destroyed in twenty-
four hours by direct sunlight and by drying at body-tem-
perature, and in seventy-two hours by drying in the dark
at ordinary room-temperature.
For the diagnosis of epidemic cerebrospinal meningitis
by bacteriological methods it is essential that the meningeal
fluid be obtained by lumbar puncture during the most
acute stage of the disease.
Varieties. — As in the case of the pneumococci and strepto-
cocci variations are observed among the meningococci.
1 Jour. Exp. Med., 1907, ix, 168.
382 APPLICATION OF METHODS OF BACTERIOLOGY
The greatest variations are seen among cultures obtained
from healthy persons who have been associated with cases
of meningitis, i. e., "the carriers/' while the least degree
of variation is noted in cultures direct from the diseased
tissues. This is of special importance in indicating the
sources from which cultures should be derived that are to
be used in the immunization of animal whose serum is to be
employed for the treatment of the disease in man.
Thus far two main groups or types of meningococci have
been established, and there is a possibility of further sub-
division of these types.
Experience in this field shows the line of demarcation
between the two main types to be distinct, but for the pro-
posed subtypes it is less sharp than that for the other organ-
isms in which typing has succeeded, that is to say, in each
of the subgroups certain individual cultures may tend to
react in a manner suggesting characters common to members
of the other groups.
In establishing the groups of this organism the method
used is that generally employed, i. e., the agglutinating
reaction with homologous immune serum. (See paragraph
on "varieties" in articles on Pneumococci and Strepto-
cocci.)
ANTIMENINGITIS SERUM.1 Flexner has demonstrated
that the blood serum of horses and of goats that have
received repeated subcutaneous injections of cultures of
diplococcus meningitidis possesses a marked restraining
action upon the course of meningitis. This is true not only
for the experimental manifestations of the disease, but for
those occurring in man as well. The analysis of about 400
1 Flexner and Jobling, Arch, of Pediatrics, 1908, p. 747.
PSEUDOMONAS MRUGINOSA 383
cases of true epidemic cerebrospinal meningitis in man in
which the serum was used shows that the general death
rate was considerably lower than that following any other
known mode of treatment. For cases treated between the
first and third days of the disease it was as low as 16.5 per
cent., while for those treated as late as, and later than the
seventh day, it was 35 per cent. Between these figures
the rates ran from 20 to 25 per cent. For success, therefore,
early diagnosis and early administrations of the serum are
essential.
There is no agreement of opinion as to how antimeningitis
serum produces its favorable results. Several suggestions
have been offered: It may stimulate phagocytosis and
thus lead to the death and removal of the meningococci; it
may enter into destructive union with the specific endo-
toxin of the meningococci; or it may act directly germicidal
upon the organs themselves.
PSEUDOMONAS -ffiRUGINOSA (SCHROTER, 1872),
MIGULA, 1900.
SYNONYMS: Bacterium ^Eruginosum, Schroter, 1872; Bacillus
ginosus, Schroter, 1872; Bacillus Pyocyaneus, Gessard, 1882; Pseudo-
monas Pyocyanea, Migula, 1896.
Another common organism that may properly be men-
tioned at this place, though perhaps not strictly pyogenic,
is a pseudomonas frequently found in discharges from
wounds, viz., pseudomonas seruginosa, or bacillus pyocyaneus
or "bacillus of green pus," or of blue pus, or of blue-green
pus, as it is by custom variously designated. Pseudomonas
seruginosa is a delicate rod with rounded or pointed ends.
It is actively motile; does not form spores. As seen in
384 APPLICATION OF METHODS OF BACTERIOLOGY
preparations made from cultures, it is commonly clustered
in irregular masses. It does not form long filaments, there
being rarely more than four joined end to end, and most
frequently occurs as single cells.
It grows readily on all artificial media, and gives to some
of them a bright-green color that is most conspicuous where
it is in contact with the air. This green color, which becomes
more and more marked as growth advances, is not seen in
the growth itself to any extent, but is diffused through the
medium on which the organism is developing. Ultimately
this color becomes much darker, and in very old cultures
may become almost black (sometimes very dark blue-green,
at others brownish-black, at others more or less of a claret
red).
NOTE. — To a fresh agar culture of this organism, in
which the green coloration of the medium is especially
marked, add about 2 c.c. of chloroform. Shake gently, and
note that the chloroform extracts a blue coloring-matter
from the culture, leaving the latter more or less yellow.
Allow the chloroform extract to stand for several days;
note what occurs; how do you account for it?
Prepare a 100 c.c. Ehrlenmeyer flask with 75 c.c. of
sterile bouillon or peptone solution. Inoculate it with this
organism and allow it to stand, without shaking, in the
incubator at body temperature for about a week. Note
its condition on removal. Now agitate it thoroughly with
air; best by pouring it into a beaker and stirring with a
glass rod. Note what now occurs. Now abstract with 5 c.c.
of chloroform — again the blue extract of "pyoscyanin"
is obtained. The dirty yellowish or reddish-yellow color
of the supernatant fluid, somewhat fluorescent, is due to
PSEUDOMONAS &RUGINOSA 385
a yellowish pigment, soluable in alcohol and water, known
as "fluorescin."
Cultivate the organism in one or another of the synthe-
sized media — Frankel's modification of Uschinsky's medium,
for instance :
• Water distilled 1000 c.c.
Asparagin 4 grams
Ammonium lactate 6 grams
Hydrogen Sod. phosphate (Na4H Po2) . . . 2 grams
Sodium chloride 5 grams
Does it produce any color? Is chloroform extract of such
cultures colored? How do you explain the result?
Obtain from the water or the soil an organism that in
several particulars suggests B. pyocyaneus, namely, bac-
illus fluorescens liquefaciens. Repeat the foregoing cul-
tivations and tests. In what way do the results differ from
those obtained with B. pyoscyaneus?
Make two bouillon cultures of bacillus fluorescens lique-
faciens. Place one in the incubator and keep the other at
room-temperature. How do they differ at end of forty-eight
hours?
Its growth in gelatin-stab-cultures is accompanied by
liquefaction and the diffusion of a bright-green color
throughout the surrounding unliquefied medium. As
liquefaction continues, and the whole of the gelatin ulti-
mately becomes fluid, the green color is confined to the
superficial layers in contact with the air. The form taken
by the liquefying portion of the gelatin in the earliest stages
of development is somewhat that of an irregular slender
funnel. (See Fig. 70.)
On gelatin plates the colonies develop_rapidly; they
25
386 APPLICATION OF METHODS OF BACTERIOLOGY
are not sharply circumscribed, but usually present at first
a fringe of delicate filaments about their periphery. (See
Fig. 71.) As growth progresses and liquefaction becomes
more advanced the central mass of the colony sinks into
FIG. 70
FIG. 72
FIG. 70. — Stab-culture of ps, ceruginosa in gelatin after twenty-eight
hours at 22° C.
FIG. 71. — Colony of ps ceruginosa after twenty-four hours on gelatin at
20°-22° C.
FIG. 72. — Colony of ps. ceruginosa after forty-two hours on gelatin at
20°-22° C.
the liquid, while at the same time there is an extension of
the colony laterally. At this stage the colony, when slightly
magnified, may present various appearances, the most
common being that shown in Fig. 72.
The gelatin between the growing colonies takes on a
PSEUDOMONAS &RUGINOSA 387
bright yellowish-green color; but as growth is comparatively
rapid, it is quickly entirely liquefied, and one often sees the
colonies floating about in the pale-green fluid.
On agar-agar the growth is dry, sometimes with a slight
metallic luster, and is of a pale gray or greenish-gray color,
while the surrounding agar-agar is bright green. With
time this bright green becomes darker, passing into blue-
green, and finally turns almost black.
On potato the growth is brownish, dry, and slightly
elevated above the surface. In some cultures the potato
about the line of growth becomes green; in others this
change is not so noticeable. With many cultures a peculiar
phenomenon, consisting of a change of color from brown to
green, may be produced by lightly touching the growth with
a sterile platinum needle. The change occurs only at the
point touched. It is best seen in cultures that have been
kept in the incubator for from seventy-two to ninety-six
hours. It occurs in from one to three minutes after touching
with the needle, and may last for from ten minutes to a half-
hour. This is the " chameleon phenomenon" of Paul Ernst.
In bouillon the green color appears, and the growth is
seen in the form of delicate flocculi. A very delicate my co-
derma is also produced. As growth progresses, the bouillon
becomes darker and darker in color, and more or less fluores-
cent, until it finally is about comparable in this respect to
crude petroleum; at the same time it assumes a peculiar
ropiness, and very old cultures (four to six weeks in the incu-
bator) may attain about the consistency of raw egg-albumen.
This is due to the production of a substance closely allied,
chemically speaking, to mucin. Whether it is a metabolic
product or one resulting from the degeneration or the auto-
digestion, so to speak, of the bacteria, cannot now be said;
388 APPLICATION OF METHODS OF BACTERIOLOGY
at all events, in cultures presenting this peculiarity very
few bacteria of normal appearance — indeed, very few
bacteria at all — are to be seen on microscopic examination.
In milk it causes an acid reaction, with coincident coagula-
tion of the casein.
On blood serum and egg-albumen its growth is accom-
panied by liquefaction. The growth on coagulated egg-
albumen is seen as a dirty-gray deposit surrounded by a
narrow brownish zone; the remaining portion of the medium
is bright green in color. As the culture becomes older the
green may give way to a brown discoloration.
In peptone solution it causes a bluish-green color. In
one of four cultures from different sources we observed the
production of a distinct blue color. In another specimen
the fluid was of a distinct wine red color, after five days at
body temperature.
It produces indol.
It stains with the ordinary dyes, and its flagella may
readily be demonstrated by appropriate methods of staining.
It is an active producer of a proteolytic enzyme that may
readily be separated and its digestive properties observed
by the following simple method: Prepare a bouillon culture
of about 70 to 80 c.c. volume, and allow it to grow at 37°
to 38° C. for four or five days. Filter through a Berkefeld
filter into a sterile receiver. Under aseptic precautions
decant the filtrate into sterile test-tubes, about 7 c.c. to
each tube. Then under aseptic precautions make the
following tests: To one tube add a small bit of hard-boiled
egg (about one-half the size of a pea) and place in an incu-
bator. Render another tube slightly acid with dilute
hydrochloric acid, and add a bit of the white of egg to it
also. Do the results differ?
PSEUDOMONAS MRUGINOSA 389
Heat another tube to 80° C. for fifteen minutes, and
repeat the experiment. Has the heating had any effect ?
To another tube add carbolic acid to the extent of 2 or 3
per cent. Is the digestive activity of the solution modified?
To two ordinary tubes of gelatin add carbolic acid 'until
it is present to the extent of 0.25 per cent, in each tube.
Solidify the gelatin in one tube in the upright position; let
that in the other remain fluid. On the surface of the former
pour 0.5 c.c. of the pyocyaneus filtrate, and mark the point
of contact between the gelatin and filtrate. To the other
tube add a similar amount of filtrate, mix thoroughly, and
solidify in a glass of cold water.
At the end of eighteen to twenty hours note result. Is
it possible to solidify again the gelatin through which the
filtrate was mixed, by placing the tube in cold water?
Do the activities of this enzyme suggest those of any of
the enzymes encountered in the animal body? Which? and
Why?
Extract with chloroform a six days' old bouillon culture
of this organism. In which portion of the liquid so extracted
is the proteolytic ferment contained, the chloroform extract
or the supernatant fluid?
Mix slowly a two weeks' old bouillon culture of this
organism, grown at body temperature, with six times its
volume of absolute alcohol. Allow to stand over night.
Filter. Redissolve the precipitate in a few c.c. (5 or 6), of
physiological salt solution. In the meantime evaporate the
alcohol filtrate to dryness at a temperature not exceeding 40°
C., and redissolve the sedement in 5 or 6 c.c. of physio-
logical salt solution. Test both of these solutions on car-
bolized gelatin for proteolytic activity. What are the
results and how are they explained?
390 APPLICATION OF METHODS OF BACTERIOLOGY
Inoculation into Animals. — As a rule, cultures of this
organism obtained directly from the discharges of the wound
are capable, when introduced into animals, of producing
diseased conditions; but cultures kept on artificial media
for a long time may in part, or completely, lose this power.
When guinea-pigs or rabbits are inoculated subcutaneously
with 1 c.c. of virulent fluid cultures of this organism, death
usually results in from eighteen to thirty-six hours. At
the seat of inoculation there are found an extensive purulent
infiltration of the tissues and a marked zone of inflammatory
edema.
When introduced directly into the peritoneal cavity the
results are also fatal, and at autopsy a genuine fibrinous
peritonitis is found. There is usually an accumulation of
serum in both the peritoneal and pleural cavities. At
autopsies after both methods of inoculation the organisms
will be found in pure cultures in the blood and internal
viscera.
When animals are inoculated with small doses (less than
1 c.c. of a bouillon culture) of this organism death may not
ensue, and only a local inflammatory reaction (abscess
formation) may be set up. In these cases the animals are
usually protected from subsequent inoculation with doses
that would otherwise prove fatal.
Most interesting in connection with pseudomonas ceru-
ginosa is the statement of Bouchard, and of Charrin and
others, that its products possess the power of counteract-
ing the pathogenic activities of bacterium anthracis. That
is to say, if an animal be inoculated with a virulent anthrax
culture, and soon after be inoculated with a culture of pseudo-
monas oeruginosa, the fatal effects of the former inoculation
BACILLUS PEST IS 391
may be prevented. Emmerich and Low1 are inclined to
attribute this to the direct bacteriolytic action of the enzymes
upon the anthrax bacteria introduced into the tissues.
In the literature upon the green-producing organisms that
have been found in inflammatory conditions several varieties
—believed to be distinct species — have been described; but
when cultivated side by side their biological differences are
seen to be so slight as to render it probable that they are
but modifications of one and the same species.
BACILLUS PESTIS, YERSIN, 1894. THE BACILLUS OF
BUBONIC PLAGUE.
Before passing from the subject of suppuration it may
not be inappropriate to call attention to the light that
modern methods of investigation have shed upon the etiology
of bubonic plague, an epidemic disease characterized by
suppuratipn of the lymphatic glands, and accompanied by
a very high rate of mortality, especially when the infection
involves the lungs, as is sometimes the case.
This pestilence, probably endemic in certain sections of
the Orient, is one of the most conspicuous epidemic diseases
of history. Since early in the Christian era epidemics and
pandemics of plague have made their appearance in Europe
at different times. During and for a time after the Middle
Ages it was more or less frequent in India, China, Arabia,
Northern Africa, Italy, France, Germany, and Great Britian.
In history it is variously known as the "Justinian Plague"
of the sixth century, the "Black Death" of the fourteenth
century, and the "Great Plague of London" of the seven-
1 Munchener med. Wochenschrift, 1898, No. 40; Centralblatt fiir Bakter-
iologie und Parasitenkunde, 1899, Abt. i, No. 1, p. 33.
392 APPLICATION OF METHODS OF BACTERIOLOGY
teenth century, though it is difficult to say to what extent
these outbreaks were uncomplicated manifestations of
genuine bubonic plague. During the existence of the Jus-
tinian Plague 10,000 people are said to have died in Con-
stantinople in a single day, and Hecker estimates that during
the pandemic of the Black Death 25,000,000 people (a
quarter of the entire population of Europe) succumbed to
the disease. During the Great Plague of London (1664-65)
the total mortality for one year was 68,596, out of an esti-
mated population of 460,000 souls.
It is not surprising to learn that it was to guard against
the plague that quarantine regulations were first estab-
lished.
The first and certainly the most exact information con-
cerning the exciting cause and the pathology of the plague
was furnished by investigations of Yersin, of Kitasato, and
of Aoyama, conducted during the epidemic of 1894 in Hong
Kong, China; although since then numerous other inves-
tigators have made additional important contributions to
our knowledge of the subject. The results of these studies
demonstrate that bubonic plague is an infectious, not
markedly contagious disease (except in the case of the
pulmonic variety), that depends for its existence upon the
presence in the tissues of a specific microorganism — the
so-called plague or pest bacillus.
This organism is described as a short, oval bacillus, usually
seen single, sometimes joined end to end in pairs or threes,
less commonly as longer threads. It stains more readily
at its ends than at its center. It is sometimes capsulated;
is non-spore-forming; is aerobic, and is non-motile. It is
found in large numbers in suppurating glands. (Fig. 73.)
It is also to be detected in the blood, spleen, lungs, liver,
BACILLUS PESTIS
393
kidneys, walls of the stomach and intestines, urine, and
intestinal contents of fresh cadavers; and during life in the
blood, expectorations, feces, and urine of persons sick of the
disease. From these findings the infection is obviously a
septicemia.
FIG. 73
A
'<*/"' C <
^ ' " '41
Bacillus of bubonic plague: A, in pus from suppurating bubo; B, the
bacillus very much enlarged to show peculiar polar staining.
It is negative to the Gram method but stains readily with
the ordinary aniline dyes. It may be cultivated upon
ordinary nutrient media, although preference is given by
some to a neutral or slightly alkaline 2 per cent, peptone
solution containing from 1 to 2 per cent, of gelatin.
The most favorable temperature for its growth is between
394 APPLICATION OF METHODS OF BACTERIOLOGY
36° and 39° C. Its colonies on glycerin-agar-agar and on
coagulated blood-serum are described as iridescent, trans-
parent, and whitish. On gelatin at 18°-20° C. it develops
as small, sharply defined, white colonies without liquefaction
of the medium. In stab-cultures it develops both on the
surface and along the track of the needle. Its growth is
slow. It does not cause a diffuse clouding of bouillon, but
grows rather as irregular, flocculent clumps that adhere to
the sides or sink to the bottom of the vessel, leaving the fluid
clear. It shows but limited growth on potato. It does not
ferment glucose with production of gas, nor does it form
indol. It coagulates milk.
This organism is killed by drying at ordinary room-tem-
perature in four days. It is killed in three or four hours
by direct sunlight. It is destroyed in a half hour by 80°
C., and in a few minutes by 100° C. (steam). It is killed in
one hour by 1 per cent, carbolic acid and in two hours by
1 per cent, milk of lime.1
It is pathogenic for rats, mice, guinea-pigs, ground squir-
rels, rabbits, hogs, horses, monkeys, cats, chickens, and
sparrows. Pigeons, hedgehogs, and frogs are immune, and
dogs and bovines are apparently so.2
Animals succumb to subcutaneous inoculation in from two
to three days. According to Yersin, the site of subcutaneous
inoculation becomes edematous and the neighboring lym-
phatics are enlarged in a few hours. After twenty-four hours
the animal is quiet, the hair is rumpled, tears stream from the
eyes, and later convulsions set in, which last till death. The
1 See Viability of the Bacillus Pestis, by M. J. Rosenau, U. S. Marine-
Hospital Service, Bulletin No. 4, of the Hygienic Laboratory, U. S. M.-H.,
Washington, D. C., 1901.
JNuttall, Centralblatt fiir Bakteriologie und Parasitenkunde, 1897,
Abt. 1, Bd. xxii, S. 97.
BACILLUS PESTIS 395
results found at autopsy are: blood-stained edema at the
site of inoculation, reddening and swelling of the lymphatic
glands, bloody extravasation into the abdominal walls, serous
effusion into the pleural and peritoneal cavities; the intes-
tine is occasionally hyperemic, the adrenal bodies congested,
and the spleen enlarged, often being studded with grayish
points, suggestive of miliary tubercles. The plague, or pest,
bacillus is detected in large numbers in the local edema, the
lymph glands, the blood, and the internal organs.
As is the case in general with the group of hemorrhagic
septicemia bacteria, the members of which it resembles in
certain other respects, when death does not result promptly
after infection there is usually only local evidence of the
inoculation, the distribution of the microorganisms through-
out the body being considerably diminished.
Animals that survive inoculation with this organism
usually exhibit a certain degree of immunity from subsequent
infection.
Nuttall1 notes that feeding experiments have resulted
in fatal infection in gray and white rats, house- and field-
mice, guinea-pigs, rabbits, hogs, apes, cats, chickens, sparrows,
and flies. He also calls attention to the fact that flies may
live for several days after being infected with this organism,
and if at liberty to fly about may infect persons or foodstuffs
on which they alight or fall.
All opinions and investigations agree in that the flea is the
most common and important of the agents of transmission,
carrying the disease from man to animals (rodents, rats in
particular) and from animals to man.
The bacilli apparently lose their virulence after long-con-
1 Loc cit.
396 APPLICATION OF METHODS OF BACTERIOLOGY
tinued cultivation under artificial conditions, and it is said
that from slowly developing, chronic buboes non-virulent
or feebly virulent cultures are often obtained. Variations
in the degree of virulence have been observed in different
colonies from the same source. Virulence is said to be
accentuated by passing the organism through a series of
susceptible animals.
It has been observed that in the suppurating lymphatic
glands of man a variety of organisms may be present, but
among them are always the plague bacilli. Occasionally
micrococci predominate. In these cases of mixed infection
the pest bacilli are said to stain less intensely with alkaline
methylene-blue than do the streptococci, and more intensely
than do the micrococci that are present. Also, in this event,
the streptococci retain the Gram stain, while the pest bacilli
do not and the staphylococci may or may not.
It is the opinion of Aoyama that the suppuration of the
glands is not caused by the plague bacillus, but is rather the
result of the action of the pyogenic cocci with which it is
so often associated. He does not regard either the air-
passages or the alimentary tract as frequent portals of infec-
tion. Wilm, on the contrary, is inclined to regard the
alimentary tract as a frequent portal of infection;1 and there
are numerous opinions that in the pulmonic type of plague,
its most fatal manifestation, infection is always by way
of the respiratory tract.
The order in which the lymphatics manifest disease ap-
pears to depend upon the location of the primary infection.
That is to say, if it is upon the lower extremities, the super-
ficial and deep inguinal glands are the first to show signs
1 Wilm, Hyg. Rundschau, 1897, p. 217.
BACILLUS PESTIS 397
of the disease; while if infection occurs via the hands and
arms, the buboes appear first in the axillary region. As a
rule, the wound through which infection is received shows
little or no inflammatory reaction.
The blood of patients convalescing from plague has an
agglutinating action upon fluid cultures of the plague bacillus
analogous to that observed when the blood-serum of typhoid
or of cholera patients is mixed with similar cultures of the
typhoid or the cholera bacillus. (See Agglutinins).
Protective Inoculation; Vaccination. — Active immunization
from plague infection by protective inoculation has been
variously attempted; by subcutaneous or intramuscular
injection of old bouillon cultures of bacillus pestis that had
been killed by heat; by similar injections of emulsions made
from agar-agar cultures of different ages suspended in
isotonic salt solution and likewise killed by heat; by the
injection of determined amounts of extractives from plague
bacilli; by the injection of mixtures of dead plague bacilli
and plague immune serum; by injection of the filtrate from
fluid cultures of the organism; by the injections of peri-
toneal exudates and organ extracts of animals infected with
plague; and by the injection of attenuated living cultures
of the organism. For the most part these efforts have been
experimental, that is to say, they have been made upon
animals susceptible to plague infection, notably guinea-pigs
and monkeys. In the problem of protecting human beings
from plague, dead cultures have been used practically to
the exclusion of all other methods. The method of Haffkine1
has enjoyed more favor than any of the others, though it
is difficult to determine its protective value with any degree
of exactness.2 This method consists in the subcutaneous
1 British Med. Jour., 1897, No. 12.
2 Bull, de 1'Institut Pasteur, 1906, No. 4, p. 825.
398 APPLICATION OF METHODS OF BACTERIOLOGY
injection of from 0.5 c.c. to 7.0 c.c. of a six weeks' old,
specially prepared bouillon culture of bacillus pestis that
had been killed by exposure to 65° C. for one hour. Some-
times the smaller, sometimes the larger doses are indicated;
sometimes a single injection is given, sometimes several are
repeated at shorter or longer intervals according to circum-
stances. The injections are followed by both local and con-
stitutional reactions, varying in the degree of intensity and
length of duration with different individuals. The immunity
resulting is said to be established fairly promptly and to last
for six weeks and longer. The investigations of the Indian
Plague Commission justify the conclusion that both mor-
bidity and mortality for Plague is less among the inoculated
than among the uninoculated.
In so far as experiments upon animals and observations
upon human beings afford positive light on this subject,
the protective inoculations protect only against the bubonic
type of plague and are practically without influence in
preventing the pulmonary or pneumonic manifestation.
The comprehensive critical review of this subject made
by Strong1 led him to the same conclusion as that of Kolle
and Otto:2 that the most effective protection from plague
is that afforded by the injection of attenuated, living cul-
tures. Tests made upon monkeys and guinea-pigs demon-
strated this method to be, in round numbers, three times
as effective as when cultures killed by heat are used. While
the results of these investigations fully warrant the conclu-
sions drawn by the authors, it is doubtful if the method
will be generally approved as applicable to man. The pos-
sibility of accident where living cultures are used even
Philippine Journal of Science, Section B, 1907, p. 155; 1912, p. 223.
2 Zeit. f. Hyg. Infektionskr., 1903, S. 45.
BACILLUS PEST IS 399
though they be attenuated to the point of harmlessness,
as decided by animal tests, is more than likely to operate
against the routine employment of such cultures in the
protection of human beings by vaccination.
Besredka, of the Pasteur Institute,1 advocates the use
of a "sensitized vaccine" against plague. This consists of
dead pest bacilli (killed by heat) that have been mixed with
antiplague immune serum obtained from an artificially
immunized animal. It is claimed by him that the process
of sensitizing lessens the toxic action of the dead bacteria;
diminishes the risk run by injecting them and eliminates
the uncomfortable local and constitutional reactions that
so often accompany the injections; while at the same time
the protective properties of the "vaccine" are preserved.
Rowland,2 in a critical review of the subject, fails to find
any neutralization of the toxic properties of the dead bacteria
through sensitization, but states that Besredka's "vaccine"
possesses good immunizing power and users of it have
reported favorably as to the minimum of discomfort fol-
lowing its inoculation. The principle here used has been
applied by Besredka, Gay and others to the making of pro-
tective agents for other types of infection.
Antiplague Serum. — The general principles that are
involved in the induction of immunity with antibody
formation hold for plague as for a number of other types
of infection; that is to say, the repeated injection into sus-
ceptible animals of non-fatal doses of the specific organism
or the products of its growth and disintegration, results in the
elaboration in the injected animal of substances that are in
one way or another antidotal, destructive or neutralizing for
the matters injecteoT.
1 Bull, de 1'Institute Pasteur, 1910, viii, p. 241 ; 1912, x, p. 529.
2 Journal of Hygiene, Plague Supplement II, 1912, p. 344.
400 APPLICATION OF METHODS OF BACTERIOLOGY
In the effort to secure specific antiplague serum two
general plans have been followed: one, by the repeated
injection of horses with at first increasing doses of dead
pest bacilli followed by ascending doses of the living organ-
ism (Yersin's method);1 the other by the injection of the
toxic extractives from artificially treated plague bacilli (the
method of Lustig2). The former method aims to establish
an antibacterial immunity, the latter an antitoxic immunity.
By both modes of procedure sera are obtained that possess
some degree of curative value in the treatment of plague,
but in both instances this is low. When tested on human
beings sick of plague under as well controlled conditions as
are offered by a good hospital, it was concluded by the
Indian Plague Commission:3 "From the whole inquiry
therefore it appears that the administration of the available
sera is not a practicable means of bringing about any material
diminution in the mortality from plague in India. It may
well be that better results would be obtained if the treatment
could be commenced within a few hours of the onset of
the disease, this however, is in the great majority of cases,
impossible in ordinary practice."
The investigations of the Pest Commissions of Germany,
Austria, and Egypt, as well as those of the Institutes for
Infectious Diseases at Berlin, Berne, and the Pasteur Insti-
tute of Paris,4 have contributed much additional informa-
tion of importance to this subject. They confirm the orig-
inal views upon the protective or prophylactic value of the
antiplague serum, but demonstrate that as a therapeutic
agent it is of but limited usefulness.
1 Annales de 1'Institute Pasteur, 1897, p. 81.
2 Deutsche med. Wchnschr., 1897, No. 15.
3 Journal of Hyg., Plague Supplement II, Seventh Report, 1912, p, 326.
4 The important literature bearing on this subject is appended to the
report of Kolle, Hetsch, and Otto (Zeitschr. f. Hygiene, Bd. xlviii, p. 368).
CHAPTER XX.
Some of the Pathogenic Organisms Encountered in the Mouth Cavity in
Health and Disease — Micrococcus Lanceolatus, Micrococcus Tetragenous,
Bacterium Influenzas, Bacillus Tuberculosis, etc.
USUALLY in the course of certain diseases, and from time
to time in health, pathogenic bacteria are to be found in
the mouth. In the latter instance the organisms, while
often fully pathogenic, as shown by tests on animals, do
apparently no harm to their hosts, with whom they live
in a commensal relationship. Moreover, they are often not
regularly and persistently present — at times they may
disappear permanently, at other times they may be recur-
rent, with varying intervals, for longer and shorter periods.
The "pneumococcus," as it is called; the Micrococcus
tetragenous; the influenza bacillus, the Bacillus diphtherias,
and the ordinary pyogenic streptococci may be cited as
occasional guests in the normal mouth cavity. In diphtheria,
tonsillitis, influenza and tuberculosis, the specific organisms
of these diseases may usually be detected either in the ordi-
nary saliva or in the sputum brought up from the deeper
respiratory tract.
To familiarize one's self with these organisms and the
customary technique for their isolation one may proceed as
follows :
Obtain from a tuberculous patient a sample of fresh
sputum — that of the morning is preferable. Spread it in
a thin layer upon a black glass plate and select one of the
small, white, cheesy masses or dense mucous clumps scat-
26 (401)
402 APPLICATION OF METHODS OF BACTERIOLOGY
tered through it. With a pointed forceps smear this carefully
upon two or three thin cover-slips, dry and fix them in the
way given for ordinary cover-slip preparations. Stain one
with Loffler's alkaline methylene-blue solution, another by
the Gram method, and a third after the method given for
bacterium tuberculosis in fluids or sputum.
In that stained with Loffler's blue — slip No. 1 — will be
seen a great variety of organisms — round cells, ovals, short
and long rods, perhaps spiral forms. But not infrequently
will be seen diplococci having more or less of a lancet shape,
joined together by their broad ends, the points of the lancet
being away from the point of juncture of the two cells.
There may also be seen masses of cocci which are conspicuous
by their arrangement into groups of fours, the adjacent
surfaces being somewhat flattened.
In the slip stained by the Gram method the same groups
of cocci which grow as threes and fours will be seen; but
the lancet-shaped diplococci may now present an altered
appearance — they are usually surrounded by a capsule.
This capsule is very delicate in structure, and, though a
frequent accompaniment, is not constant. It can sometimes
be demonstrated by the ordinary methods of staining,
though the method of Gram is most satisfactory. (Fig. 75.)
In the third slip, which has been stained by the method
given for tubercle bacteria in sputum, if decolorization has
been properly conducted and no contrast-stain has been
employed, the field will be colorless or of only a very pale
rose color. None of the numerous organisms seen in the
first slip can now be detected; but instead there will be
seen scattered through the field very delicate, stained rods,
which present, in most instances, a conspicuous beading of
their protoplasm — that is, the staining is not homogeneous,
PATHOGENIC ORGANISMS IN MOUTH CAVITY 403
but at tolerably regular intervals along each rod are seen
alternating stained and unstained points. These rods may
be found singly, in groups of twos and threes, and sometimes
in clumps consisting of large numbers. When in twos or
threes it is not uncommon to find them describing an X or a
V in their mode of arrangment, or again they may be seen
lying parallel the one to the other.
If contrast-stains are used, these rods will be detected
and recognized by their retaining the orginal color with
FIG. 74
v vvv it'
' -fif* ^\ '&>':
^ H ^ il C '"'" '
•"' -A /* / ^ t.' //
Tuberculous sputum stained by Gabbett's method. Tubercle bacteria seen
as red rods; all else is stained blue.
which they had been stained; whereas all other bacteria
in the preparation, as well as the tissue-cells which are in
the sputum, will take up the contrast-color. (Fig. 74.)
This delicate, beaded rod is bacterium tuberculosis. The
lancet-shaped diplococcus with the capsule is bacterium pneu-
monia?. The cocci grouped in fours are sarcina tetragena.
Inoculation Experiment. — Inoculate into the subcutaneous
tissues of a guinea-pig one . of the small, white, caseous
masses, similar to that which has been examined micro-
404 APPLICATION OF METHODS OF BACTERIOLOGY
scopically. If death ensue, it will, in all probability, be the
result of one of the three following types of infection :
a. Septicemia resulting from the introduction into the
tissues of bacterium pneumonia.
b. A less active form of septicemia resulting from the
introduction of sarcina tetragena, an organism frequently
seen in the sputum.
c. Local or general tuberculosis.
SPUTUM SEPTICEMIA. BACTERIUM PNEUMONIAS
(WEICHSELBAUM), MIGULA, 1900.
SYNONYMS: Diplococcus pneumoniae, Weichselbaum, 1886; Pneumo-
coccus, Frankel, 1886; Micrococcus of sputum septicemia; Diplococcus
lanceolatus; Streptococcus lanceolatus; Streptococcus pasteuri; Micro-
coccus lanceolatus.
If at the end of twenty-four to thirty-six hours the animal
be found dead, we may reasonably predict that the result
was produced by the introduction into the tissues of the
organism of sputum septicemia above mentioned, viz.,
bacterium pneumonia, which is not uncommonly found in
the mouths of healthy individuals as well as in other con-
ditions.
Inspection of the site of inoculation usually reveals a
local reaction. " This may be of a serous, fibrinous, hemor-
rhagic, necrotic, or purulent character. Frequently we may
find combinations of these conditions, such as fibrino-puru-
lent, fibrino-serous, or sero-hemorrhagic."1 The most con-
spicuous naked-eye change undergone by the internal organs
will be enlargement of the spleen. It is usually swollen, but
may at times be normal in appearance. It is sometimes
1 Welch, Johns Hopkins Hospital Bulletin, December, 1892, vol. iii,
No. 27.
SPUTUM SEPTICEMIA 405
hard, dark red, and dry; or it may be soft and rich in blood.
Frequently there is a limited fibrinous exudation over por-
tions of the peritoneum.
Except in the exudations, the organisms are found only
in the lumen- of the bloodvessels, where they are usually
present in enormous numbers. In the blood they are prac-
tically always free, being but rarely found within the bodies
of leukocytes.
FIG. 75
Bacterium pneumonias in blood of rabbit. Stained by method of Gram
Decolorization not complete.
In stained preparations from the blood and exudates a
capsule is not infrequently seen surrounding the organisms.
(Fig. 75.) This, however, is not constant.
If a drop of blood from the dead animal be introduced
into the tissues of a second animal (mouse or rabbit), iden-
tically the same conditions will be reproduced.
If the organism be isolated in pure culture from the blood
of the animal, and a portion of this culture be introduced
into the tissues of a susceptible animal, we shall see again
the same pathological picture.
406 APPLICATION OF METHODS OF BACTERIOLOGY
It must be remembered, however, that this organism when
cultivated for a time on artificial media may lose rapidly
its pathogenic properties. If, therefore, failure to reproduce
the disease after inoculation with old cultures should occur,
it is in all probability due to such loss of virulence.
This organism was discovered by Sternberg in 1880. It
was subsequently described by A. Frankel as the etiological
factor in the production of actue fibrinous pneumonia.
It is not uncommonly present in the saliva of healthy
individuals, having been found by Sternberg in the oral
cavities of about 20 per cent, of healthy persons examined
by him, and certain Authors are of the opinion that it occurs
in the oral or nasal cavities of all individuals at one time
or another during life. It is constantly to be detected in
the rusty sputum of patients suffering from acute fibrinous
pneumonia. Its presence has been noted in the middle ear,
in the pericardial sac, in the pleura, and in the serous cavities
of the brain; and indeed it may penetrate from its usual
site of development in the mouth to any of the more distant
organs.
The organism is commonly found as a diplococcus, though
here and there short chains of four to six individuals may
be seen. (Fig. 75.) The individual cells are more or less
oval, or, more strictly speaking, lancet-shaped, for at one
end they are commonly pointed. When joined in pairs the
junction is always at the broad ends of the ovals. When
in chains only the terminal cells are pointed, and then at
their distal extremities.
As already stated, in preparations directly from the sputum
or from the blood of animals a delicate capsule may fre-
quently be seen surrounding them. Though fairly constant
in preparations directly from the blood of animals and from
SPUTUM SEPTICEMIA 407
the sputum or lungs of pneumonic patients, the capsule is
but rarely observed in artificial cultures. Occasionally in
cultures on blood-serum, in milk, and on agar-agar it can,
according to some authors, be detected; but this is by no
means constant, or even frequent.
Under the most favorable artificial conditions this organism
grows but slowly, and frequently not at all.
When successfully grown upon the different media it
presents somewhat the following appearances:
On gelatin its development is very limited and often no
growth at all occurs. This is probably due in part to the
low temperature at which gelatin cultures must be kept.
If development occurs, the growth appears as minute whitish
or blue-white points on the plates. These very small colonies
are round, finely granular, sharply circumscribed, and
slightly elevated above the surface. They do not cause
liquefaction of the gelatin.
If grown in slant- or stab-cultures, the surface develop-
ment is very limited; along the needle-track tiny whitish
or bluish-white granules appear.
On nutrient agar-agar the colonies are almost transparent,
more or less glistening, and very delicate in structure.
On blood-serum development is more marked, though
still extremely feeble, appearing as a cluster of isolated fine
points growing closely side by side.
Growth on potato is not usually observed.
When grown in milk it commonly causes an acid reaction
with coincident coagulation of the casein. Some varieties,
especially non-virulent ones, do not coagulate milk.1
It is not motile.
1 Welch, loc. cit.
408 APPLICATION OF METHODS OF BACTERIOLOGY
In media containing inulin acid is produced as a result
of its fermentive action. When suspended in bile or in a
solution of bile salts the organism is dissolved.
(NOTE. — Compare this with Streptococci and Staphylo-
cocci.)
It grows best at a temperature of from 35° to 38° C. Below
24° C. there is usually no development, but in a few cases
it has been seen to grow at as low a temperature as 18° C.
Above 42° C. development is checked.
It grows as well without as with oxygen. It is therefore
one of the facultative anaerobic forms.
Cultivation of this organism is most successful when
some one of the serum-agar or agar-gelatin mixtures is
employed. (See the medium.)
It may be stained with the ordinary aniline staining
reagents. For demonstrating the capsule the method of
Gram and the acetic-acid method give the best results.
(See Stainings.)
This organism is conspicuous for the irregularity of its
behavior when grown under artificial conditions: usually it
loses its pathogenic properties after a few generations; but
again this peculiarity may be retained for a much longer
time. Often it fails to grow after three or four trans-
plantations on artificial media, though at times it may be
carried through many generations.
Inoculation into Animals. — The results of inoculations with
pure cultures of this organism are also conspicuous for their
irregularity. When the organism is of full virulence the
form of septicemia above described is usually produced, but
at times it is found to be totally devoid of pathogenic powers :
between these extremes cultures may be obtained possess-
SPUTUM SEPTICEMIA 409
ing every variation in the intensity of their disease-produc-
ing properties. The principal pathological conditions that
may be produced by the inoculation of susceptible animals
with this organism are, according to the degree of its viru-
lence, acute septicemia, spreading inflammatory exudations,
and circumscribed abscesses. All three of these conditions
may sometimes be produced by inoculating rabbits with the
same cultures in varying amounts.
Rabbits, mice, guinea-pigs, dogs, rats, cats, and sheep are
susceptible to infection by this organism. Chickens and
pigeons are insusceptible. Young animals, as a rule, are
more easily infected than old ones. Rabbits and mice are
the most susceptible of the animals used for expermental
purposes, and in testing the virulence of a culture it is
well to inoculate one of each, for the same culture may
sometimes be virulent for mice and not for rabbits, or
vice versa.
If the culture is virulent, intravascular or intraperitoneal
injections into rabbits may produce rapid and fatal sep-
ticemia; while subcutaneous inoculation of the same material
may result in only a localized inflammatory process. On
the other hand, subcutaneous inoculation of less virulent
cultures may produce a local process, while intravenous
inoculation may be without result.
This organism is the cause of a number of pathological
conditions in human beings that are not usually consid-
ered as related to one another etiologically. It is always
present in the inflamed area of the lung in acute fibrinous
or lobar pneumonia; it is known to cause acute cerebrospinal
meningitis, endo- and pericarditis, certain forms of pleuritis,
arthritis and periarthritis, and otitis media.
410 APPLICATION OF METHODS OF BACTERIOLOGY
Varieties. — The foregoing general description of pneumo-
coccus suffices for the recognition of the organism as it is
frequently found in the normal upper air passages and in
cases of pneumonia; that is, it is a lancet-shaped, Gram-
positive, encapsulated diplococcus, having the property of
fermenting inulin, of dissolving in bile or bile salts and of
usually causing septicemia when introduced into the bodies
of mice and rabbits. But this description by no means
includes certain other important aspects of the subject
that have been revealed by special researches.
It has been shown that the variations in virulence upon
animals of those pneumococci isolated from the mouth
of normal human beings is of but small importance to
an interpretation of the role of the organism in the causa-
tion of pneumonia in man; and intimate study of pneu-
monias in man, together with the organisms associated with
them, have revealed a state of affairs not only not suspected
a few years ago, but of the utmost importance to an under-
standing of the variations in the disease; of the greater
fatality of one expression of the disease over another and
the likelihood of the transmission -of the disease from the
sick to the well.
These studies have brought out the fact that in about 80
per cent, of all cases of pneumonia pneumococci are present
that are markedly different in their specific immunologic
reactions from those often found in the normal mouth, and
that are distinguished only by such reactions. Such varie-
ties of pneumococcus are found only in cases of pneumonia,
and if more than one case of pneumonia occur in succession
among persons domiciled together the same type of pneu-
mococcus will frequently be found in all of them; suggesting
the transmission of this particular variety of the disease
from one person to another.
PNEUMOCOCCUS, VARIETIES AND VARIATIONS 411
These highly pathogenic types of pneumococcus are
rarely found in the normal mouth, except in case of persons
in close contact with cases of pneumonia; and of equal
importance is the fact that under such circumstances the
pneumococcus found in the mouth of the normal individual
("the contact") is identical to that found in the discharges
from the lungs of the particular patient suffering from
pneumonia with whom he has been in contact. Such
virulent pneumococci ultimately disappear from the air
passages of the convalescent from pneumonia, as well as
from the mouth of the healthy contact.
If one secure from the normal air passages the pneumo-
coccus commonly found there and at the same time secure a
culture of pneumococcus from a case of typical lobar pneumo-
nia, it will be found that in morphology and other biological
peculiarities the two cultures are, as a rule, indistinguishable
the one from the other. On the other hand, if animals be
immunized from each culture it will be found that the blood
serum of each of the immune animals agglutinates only its
homologous cultures; that is to say, the serum of the animal
immunized by the use of pneumococcus from the case of
pneumonia agglutinates the pneumococcus from only that
case and similar cases, but not the pneumococcus common
to the normal mouth or pharynx. While the serum from the
other animal agglutinates only the pneumococcus used in
the immunization of that animal. In other words, we have
specific agglutinations.
If we examine in the same manner all cases of pneumonia
we find pneumococci differing specifically in their agglu-
tinating reactions not only from those frequent in the
normal mouth, but with various manifestations of the
disease we find variations in the virulent pneumococci
specifically related to them.
412 APPLICATION OF METHODS OF BACTERIOLOGY
In other words, certain " types " of pneumococcus are
most common to this than to that expression of pneumonia
and are more or less identified with the varying fatalities
of the disease.
At the present time, four types of pneumococci, dis-
tinguished from one another by specific agglutinating
reactions, are recognized and no transmutation from one
type to the other has been observed, even though every
experimental effort has been made to determine if such
occurs.
Types. — Type I pneumococcus causes between 30 and 50
per cent, of all true pneumonias and results in a fatality of
almost 25 per cent, of the cases with which it is associated.
Type II pneumococcus causes from about 15 to 33 per
cent, of true pneumonias and is fatal to nearly 60 per cent,
of the cases in which it is found.
Type III pneumococcus is present in from about 8 to 12
per cent, of lobar pneumonias. Its presence is associated
with a mortality in the neighborhood of 60 per cent.
This type (III) is distinguished from the other types not
only by its specific agglutinating reactions, but by the
mucoid character of its growth under artificial conditions
and its tendency to develop into streptococcus-like chains.
Type IV pneumococcus comprises a heterogeneous group
none of which can properly be included in either of the
other groups. In this group are found those pneumococci
so often present in the saliva of normal individuals and
which were regarded at one time as the specific exciters of
pneumonia. In morphology and general biological par-
ticulars the organisms in this group are alike, but by the
agglutinations test they are found to differ from one another
as well as from the other types. Etiologically, Type IV
PNEUMOCOCCUS— TYPES AND TYPING 413
pneumococci are of less importance than those of the other
groups. They are associated with about one-fifth of all
pneumonias and cause only about 7 per cent, of fatalities.
Further, when by appropriate methods of procedure
animals have been immunized from these groups, the blood
serum of such immune animals are found to have a favor-
able action in preventing infection in normal animals, but
here too there is a specific relationship, for the serum of
animals immune from either Type I, II or III is impotent
when employed against infection by pneumococci of the
types not used for immunization. This specific relation-
ship must always be borne in mind in efforts to produce
sera possessing either prophylactic or curative properties
for the disease pneumonia.
By an interchange of result, methods and materials
between various laboratories especially identified with the
development of these ideas, it has become possible to stand-
ardize the "typing" of pneumococci in a very satisfactory
manner. Specific antisera from animals highly immun-
ized from each type group of pneumococci are now avail-
able, and by the correct use of such sera in performing the
agglutination reaction, we may easily determine to which
type any pneumococcus in question belongs, as well as
form an approximate estimate as to the probable outcome
of the case of pneumonia from which that pneumococcus
was obtained.
In consequence of all this we are obliged to modify our
views formerly held on the relation of pneumococci to
pneumonia. We are not any longer justified in believing
that the pneumococcus found in the normal mouth, under
conditions not known to us, changes from a harmless com-
mensal to a dangerous pathogenic species. All modern
414 APPLICATION OF METHODS OF BACTERIOLOGY
trustworthy evidence is to the contrary, arid we now believe
that with pneumococci, as with all other living things, there
have been established in the course of time, as a result of
environmental influences, variations of a type species result-
ing in the acquisition of essentially fixed characters of
fundamental importance.
The Mechanism of Pneumonic Infection — The most impor-
tant result of pneumococcus infection in man is pneumonia.
The mechanism of the origin, course and recovery from
pneumonia still constitutes o"ne of the obscure problems
of medicine, even though special investigations have shed
much light upon several important phases of the subject.
For a clear appreciation of the current views on the
essential features of this riddle, we must bear in mind several
fundamental facts:
1. That pneumonia is not invariably the consequence of
the presence of pneumococci upon the mucous surfaces or
in the body, for that organism is often found, fully virulent
for experimental animals in the mouth, nose or upper air
passages of persons in perfect health. ("Carriers" and
"Contacts.")
2. That pneumonia, when not terminating fatally, is a
self -limited disease, i. e., the signs and symptoms increase
from the start until a point is reached, "the crisis," when
their severity suddenly begins to lessen and may continue
to do so until recovery is established.
3. That up to, and for a time after the crisis, often far
into convalescence, living virulent pneumococci are present
in the lungs. They can be found constantly in the sputum
and often in smaller or larger numbers in the circulating
blood. Their number seems at times to be affected little,
if at all, by the forces that occasion the crisis.
PNEUMONIA 415
4. That the pathogenic activities of the pneumococcus are
not referable to an extracellular toxin, properly so called,
but rather to an endotoxic component that is liberated
in the body when the bacteria are disintegrated and that
may be liberated artificially by certain solvents and under
such conditions as favor autolysis, i. e., self-digestion of
the bacteria.
5. That in the blood of convalescents from pneumonia
specific, protective antibodies are to be found, but as they
are inconstant both as to their presence and as to their
amounts it is impossible to decide their role in the mechanism
of recovery.
6. That animals may be actively immunized from pneu-
mococcus infection with but little difficulty, but the serum
from such animals is not always of value in either preventing
infection in other animals in which it is injected or of miti-
gating or curing infections already established in animals.
It is only by keeping in mind the foregoing facts that we
are able to appreciate the difficulties surrounding the problem
of pneumonia or to properly estimate the value of certain
important experimental results having a bearing upon it;
notwithstanding the light already thrown on the subject
by the discovery of various types of the causative organism
and the development of knowledge upon their several pecu-
liarities.
Given a group of persons with either of the established
types of pneumococci in their mouths, noses and pharynges,
why is it that some may develop pneumonia and others
remain in health?
It has been customary to reply: that in those developing
the disease there has been a lessening of the general vitality
416 APPLICATION OF METHODS OF BACTERIOLOGY
(resistance) through a variety of agencies, to a point that
enables the pneumococcus, hitherto present only in a com-
mensal relationship, to exhibit its pathogenic activities.
This is plausible, but that is all. There is nothing definite
in the way of experimental evidence to support it.
The most satisfying explanation* of the beginnings of
pneumonia is that offered by the investigations of Meltzer1
and his associates. They demonstrated that if fairly large
amounts (5 or 6 c.c.) of fluid cultures of pneumococci be
insufflated into the lungs of dogs, that many of the bron-
chioles became occluded as the result of the exudation
following such insufflations. The occlusion converts the
termini of those bronchioles, with their alveoli, into tiny
cavities. In such cavities the pneumococci develop and
produce irritating substances which in time bring about
more or less extensive inflammation of the lung tissues
round about them. The characteristics of these inflamma-
tory areas are in all important details identical with those
of true pneumonia in man. This experimentally-produced
pneumonia is not, however, clinically identical with pneu-
monia in man, as it is not accompanied by the crisis, nor
does one observe the sequence of local changes leading to
resolution that are commonly noticed in the course of pneur
monia in man. Nevertheless, the results of this investiga-
tion justify the conception that pneumonia in man may
not, after all, be from the start a matter purely and simply
of the invasion of the lung by pneumococci, but rather that
for such invasion to be followed by the characteristic lesions
of the disease, there must first exist physical conditions
favorable to the massed or circumscribed development of
Uour. Exp. Med., 1912, xv, 133.
PNEUMONIA 417
the organism. In the light of Meltzer's studies one can
conceive that through one or another of many causes
exudations, non-specific in character, may occur in the
lungs, occlude terminal bronchi and, as in the experimental
cases, cause small cavities into which pneumococci, gaining
access, develop as in a closed space — and by the products
of their growth bring about progressive inflammation of
the tissues surrounding them. The experimental evidence
also suggests the view that pneumonia probably always
starts as such isolated patches which, by extension, coalesce
until finally larger areas or indeed whole lobes of the lungs
are involved. When this inflammation of the lung, with its
accompanying symptoms, have progressed for about a week,
the crisis may be expected, i. e., the distressing symptoms
become more or less suddenly relieved, fever begins to
decline, respiration is less difficult, and there are beginning
signs of changes in the diseased lung tissue, i. e., resolution
may set in.
These sudden changes for the better, so often observed
in true lobar pneumonia, and as said, denominated "the
crisis," constitute one of the dramatic phenomena of clinical
medicine. As if by magic, often within a few hours, a patient
apparently in extremis, may be found in comparative comfort
and progressing steadily to recovery with little or no return
of the distressing symptoms. It is needless to say that this
is not the history of every case, but it is so frequently seen
in non-fatal cases as to fairly characterize the course of a
case destined to recover.
What are the forces that work this remarkable change for
the better? It cannot be that the pneumococci causing the
trouble are suddenly killed off and their hurtful action in
this way terminated; for we have seen that long after the
27
418 APPLICATION OF METHODS OF BACTERIOLOGY
crisis they may be found in the sputum of the patient alive,
fully virulent and in almost countless numbers. It has been
suggested that after about a week there develops in the
tissues of the body a sufficient amount of antibodies to
neutralize the .poison of the pneumococci and that coincident
with this neutralization there is a cessation of the evil
effects, i. e., the crisis occurs. Vague as this may appear
it is probably as satisfactory as any other explanation
available at this time. There are objections or criticisms
that may, however, be offered in discussing it. If that be
the correct explanation of the crisis, one might reasonably
expect to detect in the blood of convalescents from pneumonia
protective antibodies in sufficient amount and with such
constancy as to support the view, but such is not always
the case. In some instances antibodies are found in the
blood immediately after the crisis in such amounts that a
fraction of 1 cubic centimeter of the serum will protect
a mouse from infection by a hundred fold the ordinary
fatal dose of virulent homologous pneumococci; in other
cases no such protective bodies are to be demonstrated at
all; in the majority of cases limited amounts of such pro-
tective agents are to be demonstrated. In some cases pro-
tective bodies may be detected in the blood a few hours
after the crisis, and none may be found a few days later.
It is such inconstancies as these that call into question
the explanation offered above, or at least justify the sus-
picion that the crisis may be dependent upon other forces
in addition to those having to do with the neutralization of
poison or the destruction of a certain number of the germs.
It has been suggested that such other factors may com-
prise provisions for preventing further growth of the pneu-
mococci in the tissues without actually killing them or
PNE UMONIA—IMM UNIZA TION 419
robbing them of their power to produce infection when
removed alive from the pneumonic patient.
It also has been suggested that the crisis constitutes the
advent of a refractory state on the part of the tissues— a
state having some analogies to anaphylactic shock. As
yet this can be taken only as a suggestion. Much more in
the way of experimental evidence is needed before it can
be accepted.
It is scarcely suitable to a book of this character to pursue
all the lines of argument that have been advanced in con-
nection with this subject. It suffices to say that at present
we are forced still to speculate as to the nature of at least
some of the important factors responsible for the self limi-
tation of this desease.
Immunization and Specific Antisera. — Little difficulty has
been experienced in the efforts to actively immunize animals
from pneumococcus infection. Horses have been carried
to such a high degree of immunization by repeated intra-
venous injection of pneumococcus cultures that as much as
2500 c.c. of a virulent culture has been injected into the
veins at one time.
From such highly immunized animals sera have been
obtained of remarkable potency in preventing infection;
thus Cole found that 0.2 c.c. of serum from one of his
immunized horses would protect a mouse from a million-
fold the lethal dose of virulent pneumococci, provided the
serum and the culture be injected into the animal at the
same time. But if the animal be first infected, then the
serum has practically no saving powers even though it be
injected only a few hours later and in very much larger
amounts; in fact, Cole states, it is difficult or impossible
to rescue the animal, no matter how much serum is injected.
420 APPLICATION OF METHODS OF BACTERIOLOGY
We see then that while active immunization is compara-
tively easy of accomplishment, the matter is altogether
different when the serum of animals so immunized is used
for therapeutic purposes. The failure of serum from im-
munized animals to assist in the cure of pneumonia or
other pneumococcus infection with certainty is variously
explained, but as yet none of the explanations are univer-
sally accepted. By some it is believed that immune serum
has not been used in sufficient quantities; by others it is
believed that if the intensity of the infection exceeds a
certain degree that no amount of immune serum will suffice
to rescue. This latter view is particularly applicable to
pneumonia, a disease in which one is dealing with an unusu-
ally severe type of infection associated with enormous
numbers of bacteria in the body.
Cole suggests that the failure of immune serum to exhibit
its curative powers in the cure of pneumonia may not be
due to too small amounts of serum used, but rather to an
inability on the part of the infected body to supply the
factors necessary to complement the action of the serum.
His investigations lead him to several important conclu-
sions, among which may be mentioned: Since pneumococci
may be divided into several distinct groups, it is necessary
to use for curative purposes a serum from an animal immu-
nized from a strain of pneumococci belonging to the same
group as that with which the patient is infected. In order
to be effective antipneumococcus serum must be adminis-
tered early and in large doses. With these facts in mind the
treatment of human beings suffering from pneumonia with
homologous, immune serum has resulted in very low mor-
tality. In cases so treated the bacteria in the blood are
destroyed and specific immune substances appear in the
INFECTION WITH SARCINA TETRAGENA 421
blood very promptly after the injection of the serum. A
part of the action of the immune serum seems to be anti-
toxic.1
INFECTION WITH SARCINA TETRAGENA (GAFFKY),
MIGULA, 1900.
SYNONYM: Micrococcus tetragenus, Gaffky, 1883.
Should the death of the animal not occur within the first
twenty-eight to. thirty hours after inoculation, but be post-
poned until between the fourth and eighth day, it may
result from the invasion of the tissues by the organism now
to be described, viz., sarcina tetragena.
This organism was discovered by Gaffky, and was subse-
quently described by Koch in the account of his experiments
upon tuberculosis. It is often present in the saliva of
healthy individuals and is commonly present in the sputum
of tuberculous patients. Koch found it very frequently in
the pulmonary cavities of phthisical patients. It, however,
plays no part in the etiology of tuberculosis. It is principally
of historic interest, being of little pathogenic significance.
It is a small round coccus of about IM transverse diam-
e£er. It is seen as single cells, joined in pairs, and in
threes; but its most conspicuous grouping is in fours, from
which arrangement it takes its name. In preparations made
from cultures of this organism it is not rare to find single
bodies which are much larger than the other individuals in
the field. Close inspection reveals them to be cells in the
initial stage of division into twos and fours. A peculiarity
of this organism is that the cells are bound together by a
transparent gelatinous mass.
1 Cole, Jour. Am. Med. Assoc., 1912, lix, 693 and 1913, xli, 663.
422 APPLICATION OF METHODS OF BACTERIOLOGY
When cultivated artificially it grows very slowly.
Upon gelatin plates the colonies appear as round, sharply
circumscribed, punctiform masses which are slightly elevated
above the surface of the surrounding medium. Under a
low magnifying power they are seen to be slightly granular
and to present a more or less glassy lustre.
The colonies increase but little in size after the third or
fourth day. If cultivated as stab-cultures in gelatin, there
appears upon the surface at the point of inoculation a cir-
cumscribed white point, slightly elevated above the surface
and limited to the immediate neighborhood of the point
of inoculation. Down the needle-track the growth is not
continuous, but appears in isolated, round, dense white
clumps or beads, which do not develop beyond very small
points.
It does not liquefy gelatin.
Upon plates of nutrient agar-agar the colonies appear as
small, almost transparent, round points, which have about
the same color and appearance as a drop of egg-albumen;
they are very slightly opaque. They are moist and glisten-
ing. They rarely develop to an extent exceeding 1 to 2
mm. in diameter.
Upon agar-agar as stab- or slant-cultures the surface
growth has more or less of a mucoid appearance. It is
moist, glistening, and irregularly outlined. The outline of
the growth depends upon the moisture of the agar-agar.
It is slightly elevated above the surface of the medium.
In contradistinction to the gelatin stab-cultures, the
growth in agar-agar is continuous along the track of the
needle.
The growth on potato is a thick, irregular, slimy-looking
patch.
INFECTION WITH SARCINA TETRAGENA 423
The transparent mucilaginous substance which is seen
to surround these organisms renders them coherent, so
that efforts to take up a portion of a colony from the agar-
agar or potato cultures result usually in drawing out fine,
silky threads, consisting of organisms imbedded in the
mucoid material.
The organism grows best at from 35° to 38° C., but can
be cultivated at the ordinary room-temperature — about
20° C.
The growth under all conditions is slow.
It grows both in the presence of and without oxygen.
It is not motile.
It stains readily with all the ordinary aniline dyes.
In tissues its presence is readily demonstrated by the
staining-method of Gram.
The grouping into fours is particularly well seen in sec-
tions from the organs of animals dead of this form of septi-
cemia. In such sections the organisms will always be found
within the capillaries.
INOCULATION INTO ANIMALS. — To the naked eye no altera-
tion can be seen in the organs of animals that have died as
a result of inoculation with sarcina tetragena; but micro-
scopic examination of cover-slip preparations from the blood
and viscera reveals the presence of the organisms throughout
the body — especially is this true of preparations from the
spleen. White mice and guinea-pigs are susceptible to the
disease. Gray mice, dogs, and rabbits are not susceptible
to this form of septicemia. Subsequent inoculation of
healthy animals with a drop of blood, a bit of tissue, or a
portion of a pure culture of this organism from the body of
an animal dead of this disease, results in a reproduction of
the conditions found in the dead animal from which the
tissues or cultures were obtained.
424 APPLICATION OF METHODS OF BACTERIOLOGY
It sometimes happens that in guinea-pigs which have
been inoculated with this organism local pus-formations
result, instead of a general septicemia. The organisms will
then be found in the pus-cavity.
BACTERIUM INFLUENZA (E. PFEIFFER), LEHMANN
AND NEUMANN, 1896.
SYNONYM: Influenza bacillus, R. Pfeiffer, 1892.
Influenza is one of the important historic epidemic
diseases, on the nature of which much light has been shed
through modern methods of investigation. Quoting Hirsch:
—the first trustworthy literary records that we have of
this disease date from the early part of the twelfth century.
Between 1173 and 1874 it made its epidemic or pan-
demic appearance on eighty-six different occasions. Its
first recorded appearance in this country was in Massa-
chusetts in 1672; since that time there have been twenty-
two visitations of influenza in the United States. The pan-
demic of 1889-90, to that date the most severe for a long
time, appears to have originated in Central Asia and to
have spread pretty much over the entire civilized world; that
of 1918 also seems to have had its origin somewhere in the
Orient and to have spread along lines of traffic, principally
by human contact.
The advent of influenza in a community is always remark-
able for its astonishing rate of transmission from person to
person and its dissemination over wide areas.
During the pandemic of 1889-90 investigations having
for their object the discovery of its cause, resulted in demon-
strating in the catarrhal secretions from the air passages a
BACTERIUM INFLUENZA 425
microorganism that is claimed to stand in causal relation
to influenza.
This organism, bacterium influenzse, as it is called, was
discovered, isolated, cultivated and described by R. Pfeiffer.
By appropriate methods of staining it is also frequently
possible to demonstrate the presence of bacterium influenzse
in the secretions of the nose, mouth, and throat of appar-
ently healthy persons, as well as in those from persons
suffering from such diseases as diphtheria, scarlet fever,
measles, etc.
It appears to be a widely distributed organism. These
facts are of importance and must be borne in mind in con-
nection with the contention that Bacillus influenzse is not
the cause of influenza, but is only an early invader after the
disease has been started by some other, as yet unknown,
living virus.
Between the epidemic of 1889-90 and that of 1918 opin-
ion was in general concordant in regarding this bacillus
as the exciting cause of influenza. During and since the
outbreak of 1918, however, there has been a divergence
of opinion on the subject; some still believing in the
etiological relationship of Pfeiffer's bacillus to the disease,
others regarding it as only a secondary, though early, invader
after the disease itself has been started by some other living,
transmissible agent. Just what that agent is cannot now be
said, but there is some ground for believing that it may be
one of the so-called filterable, ultramicroscopic, amorphous
viruses.
If this latter view should ultimately prove to be correct,
we shall still have not only Bacillus influenzse, but pneu-
mococci and streptococci, as very early invaders in practic-
ally cases, and as agents with which we must reckon; for
426 APPLICATION OF METHODS OF BACTERIOLOGY
there can be no doubt that singly or together they are
responsible for most of the clinical symptoms and patho-
logical changes by which influenza is characterized.
Bacterium influenzae is a very small, slender, non-spore-
forming, non-motile, aerobic bacillus, occurring singly and
in pairs, joined end to end. It stains with watery solutions
of the ordinary basic aniline dyes; somewhat better with
FIG. 76
Bacterium influenzae in sputum.
alkaline methylene-blue, but best when treated for five
minutes with a dilution of Ziehl's carbol-fuchsin in water
(the color of the solution should be pale red). (Fig. 76.)
It is decolorized by the method of Gram.
It develops only at temperatures ranging from 26° to
43° C. Its optimum temperature for growth is 37° C. It
possesses the peculiarity of developing upon only those
artificial culture media to which blood or blood-coloring-
BACTERIUM INFLUENZA 427
matter has been added. Its cultivation is best conducted
and its development most satisfactorily observed by the
following procedure : over the surface of a slanted agar tube
or over agar-agar solidified in a Petri dish smear a small
quantity of sterile blood (not blood-serum). A bit of the
mucus from the sputum of the influenza patient is then
taken up with sterilized forceps or on a sterilized wire loop,
rinsed in sterile bouillon or water and rubbed over the sur-
face of the prepared agar-agar. The plate or tube is then
placed in the incubator at 37° to 38° C. If influenza bacilli
be present, they will develop as minute, transparent, watery
colonies that are without structure, and which resemble
somewhat minute drops of dew. They are discrete and
show little or no tendency to coalesce.
If a small bit of mucus be rubbed over the surface of
ordinary nutrient agar-agar, no such colonies develop.
In making the diagnosis by this method cultures on both
agar-agar containing blood (not blood-serum) and agar-
agar containing no blood should always be made, for the
reason that growth of these peculiar colonies in the former
and no such growth in the latter are evidence that one is
dealing with the organism under consideration.
The organism may also be cultivated in bouillon to which
blood has been added, if kept at body-temperature. The
growth appears as whitish flakes. Since this organism is
a strict ae'robe, its cultivation can only be conducted on
the surface of the medium used— i. e., where it has freest
access to oxygen. It is therefore inadvisable to prepare
plates in the usual way. When its cultivation is attempted
in bouillon it is recommended, in order to favor the free
diffusion of oxygen, that the depth of fluid be very shallow.
Contrary to what might be supposed, bacterium influenzse
428 APPLICATION OF METHODS OF BACTERIOLOGY
has very little tenacity of life outside of the diseased body.
It is destroyed in from two to three hours by rapid drying,
and in from eight to twenty-four hours when dried more
slowly. Cultures retain their vitality for from two to three
weeks. The organism dies in water in a little over a day.
As a result of these observations, Pfeiffer did not believe
the disease to be disseminated by either the air or the water,
but rather by direct infection from the catarrhal secretions
of the patients. During the outbreak of 1918 this opinion
received additional confirmation, though some of the disease
spread among army troops at that time is believed to have
been referable to dirty eating utensils, infected food residue
and lack of facilities for or care in the proper conduct of
kitchens and mess-rooms.
This organism has not been found outside of the human
body. In the influenza patient it is present very early,
practically with the advent of symptoms, in the catarrhal
secretions from the upper air passages and lungs. It may
be demonstrated microscopically in the mucus by cover-
slip preparations made in the usual way and stained with
diluted carbol-fuchsin, referred to above. In the tissues
it may be' demonstrated in sections stained in the same
solution. In the sputum the bacteria are found as masses
and as scattered cells. (See Fig. 76.) They are also found
within the bodies of leukocytes, especially in the later
stages of the disease when convalescence has set in; at
this time they appear as very small, irregular, evidently
degenerated bacteria within white blood corpuscles. They
are also present in the nasal secretions.
At autopsies it is advisable to cut out pieces of the diseased
tissue about the size of a pea or a bean, break them up in
a small quantity of sterile water or bouillon, and make the
BACTERIUM INFLUENZA 429
cultures from this infusion. By this procedure two advan-
tages are gained: first, a dilution of the number of bacteria
present; and, secondly, the tissue furnishes the amount of
hemoglobin necessary for the growth of the organism. Under
these circumstances it is, of course, not necessary to make
a further addition of blood to the culture-medium.
The only animal that has been found susceptible to
inoculation with this organism is the monkey. By intra-
tracheal injection Pfeiffer succeeded in causing a toxic
condition that proved fatal. He does not regard the death
of the animals as due to general infection, but rather to
intoxication. The disease, as seen in man, has not been
reproduced in animals.
CHAPTER XXL
Tuberculosis — Microscopic Appearance of Miliary Tubercles — Diffuse
Caseation — Cavity-formation — Encapsulation of Tuberculous Foci —
Primary Infection — Modes of Infection — The Bacterium Tuberculo-
sis— Location of the Bacilli in the Tissues — Microscopic Appearance
of Bacterium Tuberculosis — Staining Peculiarities — Organisms with
which Bacterium Tuberculosis may be Confounded: Bacterium
Leprae; Bacterium Smegmatis — Acid-proof Bacteria — Bacterium Tu-
berculosis Avium — Variations — Pseudotuberculosis — Susceptibility of
Animals — Tuberculin— Vaccination Against ' Tuberculosis — Actino-
myces Bovis — Actinomyces Israeli, Actinomyces Madura, Actino-
myces Farcinicus, Actinomyces Eppingeri, Actinomyces Pseudotuber-
culosis.
BACTERIUM TUBERCULOSIS (KOCH), MIGULA,
1900.
SYNONYM: Bacillus tuberculosis, Koch, 1882.
LOCAL OR GENERAL TUBERCULOSIS.— Should the animal
succumb to neither of the infections just described, then
its death from tuberculosis may reasonably be expected.
When this disease is in progress alterations in the lym-
phatic glands nearest the site of inoculation may be detected
by the touch in from two to four weeks. They will then be
found enlarged. Though not constant, tumefaction and
subsequent ulceration at the point of inoculation may be
observed. Progressive emaciation, loss of appetite, and
difficulty in respiration point to the existence of the general
tuberculous process. Death ensues in from four to eight
weeks after inoculation. At autopsy either general or local
tuberculosis may be found. The expressions of tuberculosis
are so manifold and in different animals vary so widely the
(430)
BACTERIUM TUBERCULOSIS 431
one from the other, that no fixed law as to what will appear
at autopsy can a priori be laid down.
The guinea-pig, which is best suited for this experiment
because its susceptibility to tuberculosis is greater and more
constant than that of other animals usually found in the
laboratory, presents, in the main, changes that are charac-
terized by coagulation-necrosis and caseation. This is
particularly the case when the infection is general — i. e.,
when the process is of the acute miliary type; then the tissues
of the liver and spleen present the most favorable field for
the study of this pathological-anatomical alteration.
In general, the tubercular lesions can be divided into
those of strictly focal character — i. e., the miliary and the
conglomerate tubercles — and those which are more diffuse.
The latter lesions, although primarily of the same nature
as the miliary tubercles, are much greater in extent and not
so sharply circumscribed. These latter lesions play a more
conspicuous role in the pathology of the disease than do
the miliary nodules, although it is the miliary nodules
(tubercles) that give to the disease its name.
At autopsy the pathological manifestations of the disease
are not infrequently seen to be confined to the seat of inocu-
lation and to the neighboring lymphatic glands. These
tissues then present all the characteristics of the tuberculous
process in the stage of cheesy degeneration. When the
disease is more general the degree of its extension varies.
Sometimes the small gray nodules — miliary tubercles — are
only to be seen with the naked eye in the tissues of the liver
and spleen. Again, they may invade the lung, and frequently
they are distributed over the serous membranes of the
intestines, the lungs, the heart, and the brain. These gray
nodules, as seen by the naked eye, vary in size from that of
432 APPLICATION OF METHODS OF BACTERIOLOGY
a pin-point to that of a hempseed, and, as a rule, are, in
this stage, the result of the fusion of two or more still smaller
foci. Though the two terms "miliary" and "conglomerate"
are employed for the description of the macroscopic appear-
ances of these nodules, yet it is very rarely that any condition
other than that due to the fusion of several of these minute
foci can be detected by the naked eye.
The miliary tubercles are of a pale gray color, with a
white center, are slightly elevated above the surface of the
tissue in which they are located, and, as stated, vary con-
siderably in dimensions, usually appearing as points which
range in size from that of a pin-point to that of a pin-head.
They are not only located upon the surface of the organs,
but are distributed through the depths of the tissues. To
the touch they sometimes present nothing characteristic,
but when closely packed together in large numbers they
usually give a mealy or sandy sensation to the hand
passed over them. Stained sections of miliary tubercles
present a distinctly characteristic appearance, and the dis-
ease may be recognized by these histological changes alone,
though the crucial test in the diagnosis is the demonstra-
tion of tubercle bacilli within the nodules.
Microscopic Appearance of Miliary Tubercles. — A miliary
tubercule under a low magnifying power of the microscope
presents somewhat the following appearance: there is a
central pale area, evidently composed of necrotic tissue
because of its incapacity for taking up the nuclear stains
commonly employed. Scattered through this necrotic area
may be seen granular masses irregular in size and shape;
they take up the stains employed and are evidently frag-
ments of cell-nuclei in course of destruction. Throughout
the necrotic area may be seen irregular lines, bands, or
BACTERIUM TUBERCULOSIS 433
ridges, the remains of tissues not yet completely destroyed.
Around the periphery of this area may sometimes be noticed
large multinucleated cells, the nuclei of which are arranged
about the periphery of the cell or grouped irregularly at
its poles. The arrangement of these nuclei as observed
in sections is usually oval, or somewhat crescentic. In
tubercles from the human subject these large "giant-cells,"
as they are called, are quite common. They are much less
frequent in tubercular tissues from lower animals.
Round about the central focus of necrosis is seen a more
or less broad zone of closely packed small round and oval
bodies, which stain readily but not homogeneously. They
vary in size and shape, and are seen to be imbedded in a
delicate network of fibrinous-looking tissue. This fibrin-
like network in which these bodies lie, and which is a
common accompaniment of giant-cell formation, is in part
composed of fibrin, but is in the main, most probably, the
remains of the interstitial fibrous tissue of the part. This
zone of which we are speaking is the zone of so-called " granu-
lation-tissue/' and consists of leukocytes, granulation-cells,
fibrin, and the fibrous remains of the organ; the irregularly
oval, granular bodies which take up the stain are the nuclei
of these cells. The zone of granulation-tissue surrounds
the whole of the tuberculous process, and at its periphery
may fade gradually into the healthy surrounding tissues or
be sharply outlined or may fuse with a similar zone sur-
rounding another tubercular focus.
Diffuse Caseation. — The diffuse caseation, as said, plays
a more important role in the tuberculous lesion, both in the
human and experimental forms, than does the formation
of miliary tubercles. Here a large area of tissue undergoes
the same process of necrosis and caseation as the center of
28
434 APPLICATION OF METHODS OF BACTERIOLOGY
the miliary tubercle. In certain tissues, notably the lungs
and lymphatics, it is more marked than in others. In
rabbits, particularly, all the changes in the lung frequently
come under this head. When this is the case solid masses
are found, sometimes as large as a pea, or involving even
an entire lobe or the whole lung in some cases. They are
opaque and of a whitish-yellow color, and on section are
peculiarly dry and hard. Entire lymphatic glands may
be changed in this way. The conditions which appear to be
most favorable to the occurrence of this widespread casea-
tion of the tissues are the simultaneous deposition of masses
of tubercle bacilli in them, and the involvement of a wide
area instead of a single isolated point, as in the miliary
tubercle. Necrosis is so rapid that time does not suffice for
those reactive changes to take place in the tissues which
result in the formation of the outer zone of the miliary
tubercle. In other instances the entire caseous area is sur-
rounded by a granulation-zone similar to that around the
caseous center of the miliary tubercles. It is of special
importance to recognize the etiological connection between
this' diffuse caseation and the tubercle bacillus, because
until its nature was accurately determined caseous pneu-
monia of the lungs formed the chief obstacle which many
encountered in recognizing the specific infectiousness of
tuberculosis.
Cavity Formation. — The production of cavities, a prominent
feature in human tuberculosis, particularly of the lungs,
is due to softening of the necrotic, caseous masses or of
aggregations of miliary tubercles. The material softens,
is expelled by way of the bronchi, and a cavity results.
In the wall of this cavity the tuberculous changes still pro-
ceed, both as diffuse caseation and formation of miliary
BACTERIUM TUBERCULOSIS 435
tubercles. The whole cavity with the reactive changes in
the tissues of its walls may be properly conceived as a single
gigantic tubercle, its wall forming a tissue very analogous
to the outer zone of the single tubercle, the cavity itself
corresponding to the caseous center.
In animals used for experiment cavity formation of this
sort is very rare, owing to the greater resistance of the
caseous tissue. That it is, however, possible to produce
in rabbits conditions that eventuate in pulmonary cavities
in all physical respects similar to those seen in the human
being has been beautifully demonstrated by Prudden. He
showed that when he had injected fluid cultures of strepto-
coccus pyogenes into the trachea of rabbits already affected
with tubercular consolidation of the lungs, the result of the
mixed infection thus brought about was cavity formation
in eight out of nine lungs subjected to the conditions of the
experiment; while in only one out of eleven did cavities
form under the influence of the tubercle bacillus alone.1 The
investigations of Ayer2 not only confirm the findings of
Prudden, but reveal additional facts of very great practical
importance. He demonstrated that experimental cavity
formation is very largely dependent upon the mass, physi-
cally speaking, of tubercle bacilli used for the intratracheal
injection; that uncomplicated tubercular infection is not
usually accompanied by fever, but that if there be engrafted
upon such infection, another type of infection (in Ayer's
Experiments, Streptococcus Infection) that fever was ob-
served in something over 69 per cent, of the animals 'used
in his investigations.
1 Prudden, Experimental Phthisis in Rabbits, with the Formation of
Cavities, etc., Transactions of the Association of American Physicians,
1894, ix, 166.
2 Journal of Medical Research, November 2, 1914, xxx, 141.
436 APPLICATION OF METHODS OF BACTERIOLOGY
In the contents and in the walls of tubercular cavities
in man bacteria other than bacillus tuberculosis are found.
It is to the influence of some of these, as we have seen, that
diseases other than tuberculosis may sometimes be produced
by the inoculation of animals with the sputum from such
cases; and it is also to the absorption of their toxic products
that some of the constitutional manifestations, particularly
fever, commonly seen in cases of advanced pulmonary tuber-
culosis are attributed.
Encapsulation of Tubercular Foci. — It not uncommonly
occurs that round about a necrotic tuberculous focus there
is formed a fibrous capsule which may completely shut off
the diseased from the healthy tissue surrounding it; or a
tuberculous focus may, through the resistance of the tissue
in which it is located, be more or less completely isolated.
In this condition the diseased foci may lie dormant for a
long time and give no evidence of their existence, until
they are made to break through their envelopes by some
disturbing cause. With the passage of the bacilli from such
a focus into the vascular or lymphatic circulation the disease
may become general.
It is to some such accident as this that the sudden ap-
pearance of general tubercular infection in subjects supposed
to have recovered from the primary local manifestations
may often be attributed. The breaking-down of old caseous
lymphatic glands is a common example of this recurrence of
tuberculosis.
Primary Infection. — Primary infection occurs through
either the vascular or lymphatic circulation. Through
these channels the bacilli gain access to the tissues and
become lodged in the finer capillary ramifications or in the
more minute lymph spaces. Here they find conditions
BACTERIUM TUBERCULOSIS 437
favorable to their development, and in the course of their
life-processes produce substances of a chemical nature
which serve to bring about characteristic changes in the
immediate neighborhood. In the beginning the fixed cells,
particularly the endothelial cells of the capillaries and
lymph spaces, are stimulated to proliferation. With the
onset of this phenomenon, evidence of other cell multipli-
cation may readily be detected in and about the affected
focus. As proliferation continues and as the local circulation
becomes more and more impaired, the center of the diseased
area gradually assumes a condition of inactivity, and ulti-
mately presents all the characteristics of dead and dying
tissue. This death of tissue is one of the earliest, the most
easily recognized, and the most characteristic results of
tubercular infection, and may usually be detected, in greater
or less degree, even in the youngest and most minute tuber-
cles. With the production of this progressive necrosis — for
progressive it is, as it proceeds as long as the bacilli live and
continue to produce their poisonous products — there is
in addition a reactive change in the surrounding tissues,
which results in the formation of a granulation zone at the
outer margins of the dying and dead tissue. This zone
consists of small, round granulation cells and of leukocytes,
all of wnich are seen in the meshes of the finer fibrous tissues
of the part. At the same time alterations are produced
in the walls of the vessels of the locality; these tend to
occlude them, and thus the process of tissue-death is favored
by a diminution of the amount of nutrition brought to them.
These changes may continue until eventually conglomerate
tubercles, widespread caseation, or cavity formation results;
or from one cause or another the life-processes of the bacilli
may be checked and recovery occur.
438 APPLICATION OF METHODS OF BACTERIOLOGY
Modes of Infection. — Experimentally, tuberculosis may
be produced in susceptible animals by subcutaneous inocu-
lation, by direct injection into the circulation, by injection
into the peritoneal cavity, by feeding of tuberculous material,
by the introduction of the bacilli into the air-passages, and
by inoculation into the anterior chamber of the eye.
In the human subject the most common portals of infec-
tion are, doubtless, the air-passages, the alimentary tract,
and cutaneous wounds. When introduced subcutaneously
the resulting process finds its most pronounced expression
in the lymphatic system. The growing bacilli make their
way into the lymphatic spaces of the loose cellular tissue,
are taken up in the lymph stream and deposited in the
neighboring lymphatic glands. Here they may remain and
give rise to no alteration other than that seen in the glands
themselves; or they may pass on to neighboring glands,
and eventually be disseminated throughout the lymphatic
system, ultimately reaching the vascular system.
Having gained access to the bloodvessels the results are
the same as those following intra vascular injection of the
bacilli, namely, general tuberculosis quickly follows, with
the production of miliary tubercles most conspicuous in
the lungs and kidneys; less numerous in the spleen, liver,
and bone-marrow.
When inhaled into the lungs, if conditions are favorable,
multiplication of the bacilli quickly occurs. Coincident
with their growth they are mechanically pressed into the
tissues of the lungs. As multiplication continues some are
transported from the primary site of infection to healthy
portions of the lung-tissue, where, through their develop-
ment, the process is repeated.
In the same way infection by way of the alimentary tract
BACTERIUM TUBERCULOSIS 439
is in the main due to the bacilli being forced by mechanical
pressure into the walls of the intestines. Investigation has
shown that lesions of the intestinal coats are not necessary
for the entrance of tubercle bacilli from the lumen of the
gut into the internal organs and tissues. They may be
transported from the intestinal tract into the lymphatics
in the same way that the fat-droplets of the chyle find
entrance into the lymphatic circulation.
They may gain access to the tissues by way of the tonsils.
Unlike most pathogenic organisms, the tubercle bacillus
is resistant to drying. When thrown off from the lungs in
the sputum of tuberculous patients, unless special precau-
tions be taken to prevent it, the sputum becomes dried, is
ground into dust, and sets free in the atmosphere the
tubercle bacilli which came with it from the lungs, and which
have the property of exciting the disease in susceptible
persons who receive them into the nose and throat.
Location of the Bacilli in the Tissues. — The bacilli will be
found most numerous in those tissues in which the disease
is most active.
In the initial stage of the disease the bacilli will be fewer
in number than later; at this time only scattered bacilli
may be found; later they are more numerous; and, finally,
when the process has advanced to a stage easily recognizable
by the naked eye, ,they are distributed through the granula-
tion zones in clumps and scattered about in large numbers.
In the central necrotic masses, which consist of cell-
detritus, it is rare that the organisms can be demonstrated
microscopically. It is at the periphery' of these areas and
in the progressing granular zone that they are to be seen
most frequently.
440 APPLICATION OF METHODS OF BACTERIOLOGY
This apparent absence of the bacilli from the central
necrotic area and often from old caseous tissues must not
be taken, however, as evidence that these materials are not
infective, for with them the disease can be reproduced in
susceptible ,animals by inoculation. A conspicuous example
of this condition is seen in old scrofulous glands. These
glands usually present a slow process, are commonly caseous,
and always possess the property of producing the disease
when introduced into the tissues of susceptible animals,
but yet they are the most difficult of all tissues in which
to demonstrate microscopically the presence of tubercle
bacilli.
In tubercles containing giant-cells the bacilli can usually
be demonstrated in the granular contents of these cells.
Frequently they will be found accumulated at the pole of
the cell opposite to that occupied by the nuclei, as if there
existed an antagonism between the nuclei and the bacilli.
In some of these cells, however, the distribution of the bacilli
is seen to be irregular, and they will be found scattered
among the nuclei as well as in the necrotic center of the
cell. As the number of bacilli in the giant-cell increases
the cell itself is ultimately destroyed.
Tubercular tissues always contain the bacilli and are
always capable of reproducing the disease when introduced
into the body of a susceptible animal. From the tissues
of this animal the bacilli may be obtained and cultivated
artificially, and these cultures are capable of again produc-
ing the disease when further inoculated. Thus are fulfilled
the postulates formulated by Koch for proving the etio-
logical role of an organism in the production of a malady.
BACTERIUM TUBERCULOSIS 441
BACTERIUM TUBERCULOSIS.
Of the three pathogenic organisms liable to occur in the
sputum of a tuberculous subject, the tubercle bacillus gives
us the greatest difficulty in our efforts at cultivation.
It is almost an obligate parasite, and finds c6nditions
entirely favorable to its development only in the animal
body. On ordinary artificial media the bacilli taken directly
from the animal body grow only very imperfectly, or, in
many cases, not at all. From this it seems probable that
there is a difference in the nature of individual tubercle
bacilli — some appearing to be capable of growth in the
animal tissues only, while others are apparently possessed
of the power to lead a limited saprophytic existence. It
may be, therefore, that those bacilli which we obtain as
artificial cultures from the animal body are offsprings of
the more saprophytic varieties. At best, one never sees
with the tubercle bacillus a saprophytic condition in any
degree comparable to that possessed by many of the other
organisms with which we have to deal.
For the cultivation of bacillus tuberculosis directly from
the tissues of the animal, the best method is that recom-
mended by Koch, viz., cultivation upon blood serum. Its
parastitic tendencies are so pronounced that even very
slight variations in the conditions under which one endeavors
to isolate bacillus tuberculosis from the tissues may cause
total failure. It is, therefore, necessary that the injunc-
tions for obtaining it in pure culture be carefully observed.
Preparation of Cultures from Tissues. — Under strict aseptic
precautions remove from the animal a diseased organ — the
liver, spleen, or a lymphatic gland being preferable. Place
the tissue in a sterilized Petri dish, and dissect out with
442 APPLICATION OF METHODS OF BACTERIOLOGY
sterilized scissors and forceps the small tubercular nodules.
Place each nodule upon the surface of the blood serum, one
nodule in each tube, and without attempting to break it
up or smear it over the surface, leave it for four or five days
in the incubator. After this time it may be rubbed over
the surface of the serum. The object of this is to give to
bacilli in the nodule an opportunity to multiply, under
the favorable conditions of temperature and moisture,
before an effort is made to distribute them over the surface
of the medium. It is best to dissect away twenty to thirty
such tubercles and treat each in the same way. Some of
the tubes will remain sterile, others may be contaminated
by extraneous saprophytic organisms during the manipula-
tion, while a few may give the result desired, viz., a growth
of the tubercle bacillus itself.
The blood serum upon which the organism is to be cul-
tivated should be comparatively freshly prepared — that is,
should not be dry.
After inoculating the tubes they should be carefully
sealed to prevent evaporation and consequent drying.
This is done by burning off the overhanging cotton plug
in a gas-flame, and then impregnating the upper layers of
the cotton with either sealing-wax or paraffin of a high
melting-point; or by inserting over the burned end of the
cotton plug a soft, closely fitting cork that has been sterilized
in the steam sterilizer just before using (Ghriskey). This
precaution is necessary because under the most favorable
conditions tubercle bacilli directly from the animal body
show no evidence of growth for about twelve days after
inoculation upon blood serum, and, as they must be re-
tained during this time at the body-temperature — 37.5° C. —
evaporation would take place very rapidly and the medium
would become too dry for their development.
BACTERIUM TUBERCULOSIS 443
If these primary efforts result in a growth of the bacilli,
further cultivations may be made by taking up a bit of
the colony, preferably a moderately large quantity, and
transferring it to fresh serum, and this in turn is sealed
up and retained at body temperature. Once having ob-
tained the organism in pure culture, its subsequent culti-
vation may be conducted upon the glycerin-agar-agar
mixture — ordinary neutral nutrient agar-agar to which
from 4 to 6 per cent, of glycerin has been added. This is
a very favorable medium for the growth of this organism
after it has accommodated itself to its saprophytic mode
of existence, though blood serum is perhaps the best medium
to be employed in obtaining the first generation of the
organism from tuberculous tissues.
The organism may be cultivated also on neutral milk
to which 1 per cent, of agar-agar has been added, also upon
the surface of potato, and likewise in meat-infusion bouillon
containing from 4 to 6 per cent, of glycerin.
Cultures of the tubercle bacillus are characteristic in
appearance — once having seen them there is little proba-
bility of subsequent mistake. They appear as dry masses,
which may develop upon the surface of the medium either
as flat scales or as coarse, heaped up, granular nesses.
They are never moist, and frequently have the appearance
of dry meal spread upon the surface of the medium. In
the lower part of the tube in which they are growing — i. e.,
that part occupied by a few drops of fluid which has in part
been squeezed from the medium during the process of solidi-
fication, and is in part water of condensation — the colonies
may be seen to float as a thin pellicle upon the surface of the
fluid.
The individuals composing the growth adhere so tena-
444 APPLICATION OF METHODS OF BACTERIOLOGY
ciously together that it is with the greatest difficulty they
can be separated. In even the oldest and dryest cultures
pulverization is impossible. The masses can only be sepa-
rated and broken v up by grinding in a mortar with the
addition of some foreign substance, such as very fine,
sterilized sand, or ground glass, etc.
The cultures are of a dirty-drab or brownish-gray color
when seen on serum or glycerin-agar-agar.
On potato they grow in practically the same way, though
the development is much more limited. On this medium
they are of nearly the same color as the potato on which
they are growing. When cultivated for a time on potato
they are said to lose their pathogenic properties.
On milk-agar-agar they, are of so nearly the same color
as the medium that, unless they are growing as character-
istic mealy-looking masses, considerably elevated above the
surface, their presence is less conspicuous than when on
other media.
In bouillon they grow as a thin pellicle on the surface.
This may fall to the bottom of the fluid and continue to
develop, its place on the surface being taken by a second
pellicle.
The, tubercle bacillus does not develop on gelatin because
of the low temperature at which this medium must be used.
Microscopic Appearance of Bacterium Tuberculosis. — Micro-
scopically the organism itself is a delicate rod, usually
somewhat beaded in its structure, though rarely it is seen
to be homogeneous. It is either quite straight, or somewhat
curved or bent on its long axis. In some preparations
involution-forms, consisting of rods a little clubbed at one
extremity or slightly bulging at different points, may be
detected. Branching forms of this organism have been
BACTERIUM TUBERCULOSIS 445
described. It varies in length — sometimes being seen in
very short segments, again much longer, though never as
long threads. Usually its length varies from 2 to 5ju. It
is commonly described as being in length about one-fourth
to one-half the diameter of a red blood corpuscle. It is
very slender. (See Fig. 74.)
These rods usually present, as has been said, an appear-
ance of alternate stained and colorless portions. At times
these colorless portions are seen to bulge slightly beyond
the contour of the rod, and in this way give to the rods the
beaded appearance so commonly ascribed to them. These
oval colorless areas were at one time thought to be spores.
A number of competent observers have expressed the opinion
that the rods which we see in tubercular lesions and which
we call bacillus tuberculosis are not, strictly speaking,
bacilli, but are fragments or developmental phases of a more
highly organized fungus — possibly related to the strepto-
thrices or actinomyces. The point cannot now be decided.
Staining Peculiarities. — A peculiarity of this organism is
its behavior toward staining reagents, and by this means
alone it may easily be recognized. The tubercle bacillus
does not stain by the ordinary methods. It possesses a
peculiarity in its composition that renders it proof against
the simpler staining processes. It is therefore necessary
that more energetic and penetrating reagents than the
ordinary watery solutions be employed. Experience has
taught us that certain substances not only increase the
solubility of the aniline dyes, but their penetration as well.
Two of these are aniline oil and carbolic acid. They are
employed in the solutions to about the point of saturation.
(For the methods of staining B. tuberculosis see Chapter
on Staining.)
446 APPLICATION OF METHODS OF BACTERIOLOGY
Under the influence of heat these solutions are seen to
stain all bacteria very intensely — the tubercle bacilli as
well as other forms. If we subject our preparation, which
may contain a mixture of tubercle bacilli and other species,
to the action of decolorizing agents, another peculiarity
of the tubercle bacillus will be observed. While all other
organisms in the preparation give up their color and become
invisible, the tubercle bacillus retains it with- marked tenacity.
It stains with great difficulty; but once stained it retains
the color even under the action of strong decolorizing agents.
Variations of B. Tuberculosis. — Theobald Smith1 called
attention to certain very conspicuous differences that may
be observed between the bacilli obtained from human and
those from bovine tuberculosis; and in a series of inocula-
tion experiments Ravenel has shown that for a large number
of animal species tubercle bacilli of bovine origin were con-
stantly more virulent than .those from human sources ; both
of which observations have been repeatedly confirmed.
Susceptibility of Animals to Tuberculosis. — The animals
that are known to be susceptible to tuberculosis are man,
apes, cattle, horses, sheep, hogs, guinea-pigs, pigeons, rab-
bits, cats, and field-mice. White mice, dogs, and rats
possess immunity from the disease.
Tuberculin. — The filtered sterile products of growth from
old fluid cultures of the tubercle bacillus represent what is
known as tuberculin — a solution containing a group of
protein substances possessing most interesting properties.
When injected subcutaneously into healthy subjects tuber-
culin has no effect; but when introduced into the body of
a tuberculous person or animal a pronounced systemic
1 Transactions of the Association of American Physicians, 1896, xi, 275.
BACTERIUM TUBERCULOSIS 447
reaction results, consisting of sudden but temporary ele-
vation of temperature, with, at the same time, the occur-
rence of marked hyperemia about the tuberculous focus,
a change histologically analogous to that seen in the pri-
mary stages of acute inflammation. This zone of hyperemia,
with the coincident exudation and infiltration of cellular
elements, probably aids in the isolation or casting off of
the tuberculous nodule, the inflammatory zone forming,
so to speak, a line of demarcation between the diseased and
healthy tissue.
As a curative agent for tuberculosis, tuberculin has not
proved worthy of the confidence that was at first accorded
to it. Its field of usefulness is now almost limited to the
diagnosis of obscure cases.
In veterinary medicine it has proved trustworthy as a
diagnostic aid, and is practically everywhere in use for the
detection of incipient tuberculosis in cattle.
VACCINATION AGAINST TUBERCULOSIS. — Experiments by
Pearson and Gilliland, v. Behring, Calmette, and others have
shown that it is possible to partly immunize animals with
lowly virulent tubercle bacteria of human origin. After one
or two injections with such organisms the animals showed for
a time some degree of tolerance to the more highly virulent
bovine strains. The results of experiments in this direction
have been so encouraging as to justify further research
in this direction, but complete immunity has not as yet
resulted.
We have reviewed the three common pathogenic organ-
isms that may be encountered in the sputum of tuberculous
individuals. Occasionally other species may be present.
The pyogenic forms are not rarely found, and for some time
448 APPLICATION OF METHODS OF BACTERIOLOGY
after an attack of diphtheria the bacillus of Loffler is demon-
strable in the pharynx, so that it, too, may be present under
exceptional circumstances.
Organisms with which Bacterium Tuberculosis may be Con-
fused.— It is important to note that in the study of tubercu-
losis one may fall into error unless it be borne in mind that
there is a group of microorganisms whose members are in
many respects so like the genuine bacillus tuberculosis as
easily to be mistaken for it. While its peculiar micro-
chemical reaction is usually sufficient for identification, par-
ticularly in connection with human pathological lesions, it is
well to remember that the confusing organisms are not only
characterized by the same staining peculiarities as bacillus
tuberculosis, but may readily be mistaken for it on morpho-
logical grounds also. Furthermore, while not all the mem-
bers of this group are capable of causing disease, some of
them are pathogenic for the same animals that are suscep-
tible to true tubercular infection; and there may produce in
those animals lesions which are distinguishable from genuine
tubercles only by their finer histological structure. A few
words concerning some of these varieties, with a brief
summary of their more important peculiarities, may not
be out of place.
BACTERIUM LEPILE. — Between 1879 and 1881 there was
described by Hansen and by Neisser an organism, a bacillus,
that was constantly to be found in the nodules, characteristic
of leprosy. For this organism the name bacillus leprce was
suggested. Though very like bacterium tuberculosis in
both morphology and staining properties, it is, however,
a little shorter, thicker, and much less homogeneously
stained. Its presence in the tissues and secretions is demon-
strated by the same method as that employed for detecting
BACTERIUM TUBERCULOSIS 449
bacillus tuberculosis. In secretions of leprous nodules,
stained by the ordinary Koch-Ehrlich process, the bacilli,
crowded together in the large so-called "lepra cells/1 are
always to be seen in great abundance. Numerous efforts
to cultivate bacillus leprse from the diseased tissues and to
reproduce the disease fby inoculation have led to little more
than a mass of confusing results. It is possible that a recent
observation of Johnston1 may assist in clearing away some
at least of the confusion. Johnston believes the acid-proof,
so-called bacilli, seen in the lepra cells to be developmental
phases of a streptothrix which is itself not acid proof. His
opinion appears to be justified by the results of carefully
made culture and inoculation studies.2
BACTERIUM SMEGMATTS. — In 1885 Alvarez and Tavel
discovered in the fatty secretions about the genitalia an
organism that suggested the bacterium of tuberculosis.
Their observation has been abundantly confirmed by others,
and the organism to which they directed attention is now
regarded as pretty commonly present in the smegma. It
is known, therefore, as the smegma bacterium (bacterium
smegmatis). In this secretion it is found in clumps located
upon or within epithelial cells. It stains by the method
used in staining bacterium tuberculosis. It has no patho-
genic power. It is said to have been artificially cultivated
upon coagulated hydrocele fluid and in milk.
THE ACID-PROOF BACTERIA. — In addition to the species
mentioned, quite a group of other "acid-proof" bacteria,
as they are called, have been described by different inves-
tigators. They are characterised by staining, as does bac-
1 Philippine Journal of Science, June, 1914, vol. ix, No. 3, Section B,
Tropical Medicine, p. 227.
2 For a general discussion on this ubject, together with literary references
see Wolbach and Honeij, Journal of Medical Research, 1914, xxix, 367.
29
450 APPLICATION OF METHODS OF BACTERIOLOGY
terium tuberculosis, by retaining the stain to a greater or
less extent when treated with decolorizers, and by being
in many instances strikingly like bacterium tuberculosis
in their morphology. The members of this group seem
to be distributed pretty widely in nature. They have been
detected in non-tuberculous sputum, in gangrene of the
lung, in the normal intestinal contents of man and domes-
tic animals, in certain of the cold-blooded species, in the
soil, in fodder — i. e., grass, hay and seed — in manure,
and in butter. They are not regularly found under any
of these conditions, and they are rarely present in very
large numbers. Inasmuch as they are occasionally en-
countered under circumstances that might lead one to
look for true tubercle bacilli, and since they possess certain
peculiarities similar to those by which it has been the cus-
tom to identify bacillus tuberculosis — i. e., retention of the
stain when acted upon by acids or alcohol, and a more
or less delicate, beaded form — the possibility of their being
confounded with that organism is obvious. In consequence
they received a great deal of attention for a time.
Space does not permit of a description of the twenty
odd species (?) that have been described by different in-
vestigators. It will suffice to say, from personal study of
the group, that in all probability not more than three, per-
haps only two, species are really represented, and that the
remainder may fairly be regarded as varieties. As said,
the characteristic common to all the members of this group
is that they are to greater or less extent acid-proof — i. e.,
when once stained by the Koch-Ehrlich or Ziehl process
the color is not in all cases removed by the ordinary acid
decolorizers. In this particular, however, there is such a
striking difference between the degree of their resistance to
BACTERIUM TUBERCULOSIS 451
acid decolorizers and that of the tubercle bacillus as to
render this an important differential aid; for instance, the
tubercle bacillus, when stained, may be treated for several
minutes with so strong a decolorizer as 30 per cent, nitric
acid without losing its color; whereas, none of the members
of this group retain their color after a few seconds of such
treatment, and particularly if it be followed by washing
in alcohol. In morphology some of them might readily be
mistaken for bacillus tuberculosis, though even these are
usually a trifle larger and less delicately formed than that
organism; others are at once differentiated from normal
tubercle bacilli, but have somewhat their appearance when
degenerated or involuted; still others have nothing in their
general appearance to lead to confusion.
When mixed with other bacteria, as is the case in the soil,
in manure, in intestinal contents, etc., their isolation in
pure culture is a matter of difficulty, and this is by no means
lessened by the fact that under these circumstances they
are always numerically in the minority. When present
in butter, their isolation offers fewer difficulties, for by the
injection of the butter containing them into the peritoneal
cavity of guinea-pigs conditions are created that favor their
development, and from animals so treated they may usually
be recovered in pure culture.
When studied in pure culture, all of them are at once
distinguished from bacillus tuberculosis by the following
group characteristics: they are of relatively rapid growth,
there being usually an abundant development on glycerin-
agar-agar after twenty-four to forty-eight hours at body-
temperature; they grow well but less rapidly at ordinary
room-temperature — i. e., at 18° to 20° C.; they grow well
in litmus-milk, and, as a rule, produce alkali that causes
452 APPLICATION OF METHODS OF BACTERIOLOGY
the color to become a deep blue; the growth on agar-agar
is dry, shrivelled, and wrinkled in appearance, and of a soft,
mealy consistency in some cases (Holler's grass bacillus
II, Rabinowitsch's butter bacillus, for instance), while in
others it is more membranous, as in the case of Holler's
timothy bacillus. We have never seen in these cultures
the hard, coarse granules so common to cultures of bacillus
tuberculosis; on glycerin-agar-agar some of them, namely,
the timothy bacillus of Holler and its varieties, grow with
a distinct orange color, while others, the grass bacillus II
of Holler, the butter bacillus of Rabinowitsch, and their
closely allied varieties, begin as a grayish-white deposit
which may ultimately become of a pale or even distinct
salmon color.
When pure cultures of them are injected into such animals
as rabbits or guinea-pigs, some of them have no effect, and
others cause lesions of more or less importance, the result
being dependent upon the quantity employed and the mode
of inoculation. By subcutaneous or intraperitoneal injec-
tion of pure cultures the result is usually negative. Occa-
sionally the superficial lymphatic glands in the neighborhood
of the site of inoculation may be inflamed and purulent.
This we have seen only after subcutaneous inoculation.
If pure cultures be injected into the peritoneal cavity along
with some sterile, irritating substance, such as sterilized
butter, a widespread fibrinopurulent peritonitis is commonly
the result.
When injected directly into the circulation of rabbits,
the kidneys are almost uniformly affected, and in the ma-
jority of instances they are, singularly enough, the only
organs in which lesions are to be detected. If, for instance,
BACTERIUM TUBERCULOSIS 453
a cubic centimeter of a carefully prepared suspension in
bouillon of, let us say, Holler's grass bacillus II, be injected
into the circulation of a rabbit, and the animal be killed
after twelve to fourteen days, the kidneys will be found
marked by gray or yellowish points that range in size from
that of a pin-point to that of a pin-head. They are some-
times very few in number, but in other cases both kidneys
may be thickly studded with them. Often they are not
elevated above the cortex of the organ, but in as many cases
they are sharply defined, yellow in color, and stand up
prominently from the cortical surface, being at the same
time so adherent to the capsule that the removal of the
latter tears them out bodily from the substance of the organ.
In the very early stages of development these nodules are
often difficult to distinguish from young tubercles, the reac-
tion of the tissues being, as in the case of tubercles, charac-
terized by proliferation of the fixed cells with little evidence
of leukocytic invasion; later on, true giant-cell formation
is recorded by some observers. We have not seen this.
Clumps of endothelial nuclei or of lymphoid cells that
remotely suggest the arrangement seen in giant cells are
often encountered, but we have not regarded them as true
giant cells. When fully developed, the nodule may present
a mixed condition of caseation and suppuration. The
conditions, as a whole, when advanced suggest a low grade
of inflammatory reaction. Occasionally nodules are en-
countered, especially in the kidney, that cannot be dis-
tinguished from tubercles. The bacilli are always to be
found within the nodules; most frequently as single rods or
clumps of rods, occasionally as rosette-like mycelia very
suggestive of the characteristic growth of the actinomyces
454 APPLICATION OF METHODS OF BACTERIOLOGY
fungus in the tissues. We have also observed this mode
of development by Bacillus tuberculosis. (Figs. 77 and 78.)
It is important to note the difference between the results
of intravenous inoculation of rabbits with bacillus tuber-
culosis arid with the organisms under consideration. When
bacillus tuberculosis is employed, the lungs, as well as the
kidneys, are always involved, while with the grass bacillus
II, the timothy bacillus, and the butter bacillus, involve-
ment of the lungs, in our experiments, has been the exception
rather than the rule.
Another point of interest is the lack of tendency on the
part of the non-tuberculous process to progress or become
disseminated.
That the members of this group are botanically related
to bacillus tuberculosis there seems little room for doubt;
but from personal study and from available evidence from
other sources it appears unlikely that they are, except
experimentally, concerned in disease production or that they
are of importance to either human or animal pathology.1
In the microscopic examination, particularly of urine,
of secretions from about the anus, rectum, and genitalia,
and of butter, it is manifestly of importance to bear in mind
the existence of this confusing group, for it is in such secre-
tions and substances that its members are most often en-
countered. The smegma bacillus and the butter bacillus
are especially liable to lead one into error of diagnosis.
This is less apt to be the case with the comparatively rare
lepra bacillus.
1 For the literature on "acid-proof" bacilli, see Cowie, Journal of Experi-
mental Medicine, 1900, v, 205.
Fig. 77
\ ,
'<3f
Q ' S'9 '"'
I *Jf, * &
Showing Aetinomyees Development of Bacillus Tuber-
culosis in Lung of Rabbit, thirty days after intravenous
injection of suspension of the organism, o ,
Fig. 78
Showing Aetinomyees Development of Acid-resisting
Bacteria (Butter Bacillus of Rabinowitsch) in Kidney of
Rabbit, following upon intravenous injection of suspension
of the organism.
BACTERIUM TUBERCULOSIS AVIUM 455
BACTERIUM TUBERCULOSIS AVIUM (MAFFUCCI),
MIGULA, 1900.
SYNONYMS: Bacillus tuberculosis avium, Maffucci, 1891; Mycobacter-
ium tuberculosis avium, Lehmann and Neumann, 1896.
From time to time fowls are known to suffer from a form
of tuberculosis that in a number of ways suggests human
or mammalian tuberculosis. The bacillus causing the disease,
the so-called bacillus of fowl tuberculosis, bacillus tuber-
culosis avium, while simulating the genuine bacillus tuber-
culosis morphologically, differs from it both in cultural
and pathogenic peculiarities. Thus, for instance, it develops
into much longer and somewhat thinner threads; grows
rapidly on media without glycerin or glucose; does not
grow on potato; develops as well at from 42° to 43° C. as
at 37° to 38° C.;1 its virulence is not diminished by cul-
tivation at 43° C.; development on artificial media begins
in from six to eight days after inoculation; young cultures
on solid media are whitish, soft, and moist, becoming yel-
lowish and slimy with age; it is somewhat more resistant
to drying and high temperatures than the bacillus of mam-
malian tuberculosis; the results of its pathogenic activities
are almost always chronic, are rarely located in the lungs
or intestines, but are especially frequent in the liver and
spleen; the lesions are conspicuously rich in bacteria, do
not show the central necrotic area that characterize the
mammalian tubercle; the disease is transmissible from the
hen to the embryo chick; the only susceptible mammal is
the rabbit; the guinea-pig and dog are naturally immune;
it has the same micro-chemical staining reactions as mam-
1The normal body-temperature of fowls ranges between 41.5° and
42.5° C.
456 APPLICATION OF METHODS OF BACTERIOLOGY
malian bacillus tuberculosis; it has never been certainly
detected in human tuberculosis.
Some are inclined to regard this organism as but a variety
of genuine bacillus tuberculosis, and it is not unreasonable
to believe that the sojourn of that organism in the body of
a refractory animal, whose normal temperature is so high as
that of the fowl, when not fatal to the organism, might
result in striking modifications of certain of its biological
functions. In fact, Nocard1 has shown that if the genuine
bacillus tuberculosis from man be left in the peritoneal cavity
of chickens (by the collodion-sac method of Metchnikoff,
Roux, and Sallembini, which see) for from five to eight
months, they will, by the end of this time, have become so
modified in their biological peculiarities as to simulate very
closely the bacillus of fowl tuberculosis.
Moore2 reports studies on bacterium tuberculosis avium
in an epidemic occurring in California. He obtained pure
cultures by inoculating glycerin-agar or blood serum tubes
directly from tuberculous livers and spleens. In the origi-
nal cultures little difficulty was experienced in cultivating
the organism on glycerin-agar, fresh dog serum, Dorset's
egg-medium, potato, and glycerin-bouillon. The general
cultural peculiarities observed agreed with those described
by Maffucci, Nocard, Straus and Gamaleia, and others.
He states that the avian tubercle bacteria as found in the
tissues of the fowl resemble quite closely those of the bovine
and human varieties in their size and general morphology.
The average length of a large number of measurements was
2.7 microns. Moore also tested the pathogenesis of the
freshly isolated avian tubercle bacteria on fowls, rabbits,
1 Annales de 1'Institut Pasteur, 1898, p. 561.
8 Journal of Medical Research, 1904, vol. vi.
ACTINOMYCETES 457
guinea-pigs, and pigeons. The results of these inocula-
tions, however, were unsatisfactory, as were also feeding
experiments of healthy fowls with human tuberculous
sputum rich in bacteria.
Pseudotuberculosis. — Anatomical lesions very suggestive
of, though not identical with, those produced by bacillus
tuberculosis, have also from time to time been observed in
mice, rats, guinea-pigs, rabbits, cats, goats, bovines, hogs,
and man. They do not appear to be of a specific nature as
regards etiology, for the reason that different authors have
described different organisms as the causative agents.
These affections are usually classed under the name pseudo-
tuberculosis.
ACTINOMYCETES.
The term actinomycetes is restricted to a group of organ-
isms having morphological affinities with the bacteria on
the one hand and the hyphomycetes on the other. They
resemble the bacteria in that they occur as homogeneous
threads which under artificial cultivation may become
segmented into short bacillus- or coccus-like fragments.
Furthermore, they are unlike the molds in that they have
not a double wall; are not filled -with fluid containing gran-
ules, and the segments are not separated from one another
by a distinct partition. They simulate the molds in that
they develop from spores into dichotomously branching
threads, which ultimately form colonies having more or
less resemblance to true mycelia. Certain of the threads
composing such a mycelium become fruit hyphse, breaking
up into round, glistening, spore-like bodies. As a rule,
these spores are devoid of the high resistance to heat exhib-
458 APPLICATION OF METHODS OF BACTERIOLOGY
ited by bacterial spores, and are stainable by the ordinary
methods.
The limits of this group are ill defined and its recognized
components are not as a whole well understood.
The longest known and most carefully studied actinomy-
cetes are act. bovis, act. madurce, act. farcinicus, and act.
Eppingeri, although many other varieties have been en-
countered in association with important and interesting
pathological lesions.
The fact that certain bacteria, viz., B. tuberculosis, B.
mallei, B. diphtherise are, as a rule, segmented and occa-
sionally show a tendency to branch, has led to their being
classified at times with the actinomycetes. On this point,
however, there is as yet no concensus of opinion.
It is interesting to note that the pathological lesions in
which actinomycetes have been detected show in many cases
certain similarities to true tubercular processes, and in few
instances, save for the absence of tubercle bacteria, as we
usually see them, were indistinguishable from tuberculosis.
More or less imperfectly studied varieties of actino-
mycetes have been encountered in abscess of the brain,
cerebrospinal meningitis, endocarditis, bronchopneumonia,
pleuropneumonia, pustular exanthemata, abscess of the lung,
bronchiectasis, pulmonary gangrene, necrosis of the vertebrae,
subphrenic abscess, noma, and pseudotuberculosis.
In some cases the actinomycetes can be obtained in culture
from the diseased tissues; almost as often they can not.
Sometimes the inoculation of animals with bits of the
diseased tissue or with cultures results in the production
of pathological lesions referable to the organism; again
no effect follows upon such inoculation. As seen in the
tissues by microscopic examination, actinomycetes may
ACTINOMYCETES 459
appear as long, convoluted, irregularly staining, beaded,
branching threads, or as clumps of short, markedly beaded,
sometimes branched rods. At times a clump of the short
or longer threads is encountered in the tissues that gives the
distinct impression of mycelial structure.
Some of the varieties that have been described are best
demonstrated in the tissues or exudates by the Gram or
Gram-Weigert method of staining; others are decolorized
by this process, and are rendered visible only by the simpler
procedures. Some of them are to a limited extent proof
against the action of acid decolorizers. Though many
accounts of the presence of these morphological types in a
variety of conditions have been recorded, the descriptions
in the main are meagre and often insufficient for identifi-
cation. A few, however, have been found so constantly in
association with more or less definite clinical and pathological
conditions that a brief description of them may be of service.
Actinomyces Bovis (also commonly known as streptothrix
actinomyces, actinomyces fungus, ray fungus) was first
observed by von Langenbeck in a case of vertebral caries
in 1845. According to Bollinger, the fungus had been seen
by Hahn a number of years before in museum specimens,
but had been regarded by him as a penicillium. The name
actinomyces or ray fungus originated with Harz.
This fungus is constantly to be detected in the tissues and
exuda'tes of the disease of cattle known as actinomycosis,
"lumpy jaw," "wooden tongue," etc. The typical tumor
of this disease is characterized by inflammation, pus forma-
tion, excessive new formation of connective tissue, abscesses,
cavities and sinuses. Viewed as a whole, the tumor pre-
sents points of resemblance to the osteosarcomatous, to
the scrofulous or tuberculous, and to the cancerous processes.
460 APPLICATION OF METHODS OF BACTERIOLOGY
The disease occasionally occurs in man, and according to the
point of entrance of the parasite may arise in the mouth, the
pharynx, the lungs, the intestines, or the skin. In animals
the disease is characterized by an excessive new forma-
tion of connective tissue, so that tumefaction is always a
conspicuous peculiarity. In man, on the other hand,
suppuration is the most prominent feature.
If the purulent discharge from an actinomycotic tumor
be examined fresh, it will be found to contain tiny yellow
FIG. 79
Actinomycosis fungus in pus. Fresh, unstained preparation. Magnified
about 500 diameters.
(sulphur color as a rule) clumps. If these be examined,
unstained, in a drop of physiological salt solution or water
under the microscope, they will be found to be made up of
a rosette-like mass of closely interwoven threads. (See
Fig. 77.) At the center the mass may show the presence of
spherical, coccus-like bodies or granules, while at the per-
iphery the free ends of the threads are more or less distinctly
bulbous or nodular, or both, and they may show branching.
Sometimes the free ends of the threads are only slightly or
not at all swollen.
ACTINOMYCETES 461
These mycelia — the actinomyces — may be stained by
the ordinary aniline dyes, or by the Weigert or the Gram
method, though by either of these procedures their full struct-
ure is not, as a rule, brought out. The reason for this is
that the terminal bulbs are not due to enlargement of the
thread itself, but rather to a colloid degeneration of its
enveloping sheath. This colloid matter, having different
microchemical reactions from the enclosed thread, requires
different reagents to stain it. The entire structure may be
seen when the fungus is stained first by the Gram method,
and subsequently with eo.sin or saffranin. For the demon-
stration of the fungus in sections, the method of Mallory
gives satisfaction. It is as follows; Stain the section on the
slide with gentian- violet; clear and dehydrate with aniline
oil in which a little basic fuchsin has been dissolved; remove
the aniline oil-fuchsin with xylol, and mount in xylol balsam.
In sections treated in this way the coccus-like central
masses and the filamentous threads making up the mass of
the mycelium are stained blue; the club-like extremities of
the thread are red. Often the red-stained hyaline material
is seen to be penetrated to its extremity by a sharply defined
blue thread.
Cultivation of the fungus from the actinomycotic pus
presents difficulties for the following reasons: Not all the
mycelia seen by microscopic examination are living; as a
rule they grow slowly even under the best of circumstances;
and generally there are many other, more rapidly growing,
living organisms in the pus. When pure cultures are ob-
tained, it grows (according to Bostrom) on all the ordinary
artificial media. It develops at room-temperature, but
better at that of the body.
It grows both with and without oxygen.
462 APPLICATION OF METHODS OF BACTERIOLOGY
The young colonies appear as grayish points composed of
a felt-work of fine threads. As the colonies become older
they become denser and more opaque. Very old colonies
are almost leathery in consistency. On blood serum the
growth after a time assumes a salmon, an orange, or a
yellowish-red color. Growth on gelatin is accompanied by
slow liquefaction.
A yellowish-red growth, limited in extent, occurs on
potato. It causes no clouding of bouillon, but grows as
cottony clumps that sink to the bottom.
The bulbous extremities seen upon the mycelial threads
fresh from the pus are not usually seen under conditions
of artificial cultivation. They are sometimes observed in
colonies located in the depths of solid media. The white,
powdery coating seen on old colonies represents the so-called
"spores." They are not, however, resistant to heat, being
destroyed, according to Domec, by 75° C. in five minutes.
Bo vines are the animals most frequently affected. The
disease has been seen in swine, dogs, and horses.
The most common seat of the disease is the jaw, and this,
together with the fact that particles of fodder, such as bits
of grain, chaff, straw, and barley beard, have been detected
in the diseased tissues in association with the causative
fungus, has led to the belief that the parasite gains access
to the tissues with such foodstuffs. It has not, however,
been recognized outside the animal body. The disease is
apparently not transmissible from animal to animal or from
animal to man. Inoculation of animals with pure cultures
is usually negative, although nodular formations have fol-
lowed the injection of large quantities into the peritoneal
cavity of rabbits. In Bostrom's cases the nodules presented
only a few of the club-shaped extremities of the ^threads,
ACTINOMYCETES 463
and there was no evidence of multiplication of the fungus;
while in the experiments of Israel and Wolf it is said there
developed, in from four to seven weeks after intraperitoneal
inoculation, larger and smaller tumors in which typical
mycelia were present, and from which the fungus was
obtained in pure culture.
Actinomyces Madurae. — This organism is suppposed to
be concerned in the causation of mycetoma or Madura foot.
Two varieties of mycetoma are known, viz., the pale or
ochroid and the black or melanoid. Save for its occur-
rence in the foot, mycetoma is almost a counterpart of
actinomycosis; and the suspicion of their identity is by
no means lessened by the fact that the actinomyces con-
stantly associated with the ochroid variety is to all intents
and purposes identical with actinomyces bo vis. It differs
from that organism only in such minor details as to leave
little doubt that they are very closely related, if not iden-
tical, so that a description of the one serves equally to aid
in the identification of the other.
The investigations of Wright,1 conducted upon a case
encountered in Boston, point to another type of parasite as
the causative factor in the black mycetoma. Instead of an
actinomyces, Wright found a true mold. He expresses
the opinion that the pale mycetoma is, etiologically,
actinomycosis, and that the black is a hyphomycetic
infection.
The fungus encountered and isolated in pure culture by
Wright presented the following characteristics: As ob-
tained from the affected tissues, the mycelia under the
microscope appear as black or brown mulberry-like masses
1 A Case of Mycetoma (Madura Foot), Journal of Experimental Medi-
cine, 1898, iii, 421.
464 APPLICATION OF METHODS OF BACTERIOLOGY
less than one millimeter in diameter. They are hard, rather
brittle, and difficult to break up under the cover-glass. On
soaking them in a strong solution of sodium hydroxide they
become softened and the structure of the fungus-mass can
be made out. Under high magnifying power these masses
are found to consist of pigment granules, ovoid translucent
bodies, and distinctly branching separate hyphse. Some-
times these latter exhibit dilatations or varicosities of their
segments. The periphery of a fungous mass shows the pres-
ence of club-shaped hyphse, closely set and radially arranged.
From such masses growth on artificial culture-media may
be obtained. When transferred direct from the tissues to
artificial media, growth in every case starts from the granule
about four or five days after it is placed upon the culture
media.
On solid media it first appears as delicate tufts of whitish
filaments. These in the course of a few days increase in
number and length, and, in the case of the potato, form a
dense whitish or pale-brown felt-work having a tendency
to spread widely.
In pure cultivation it is seen as long, branching hyphse
with delicate transverse septa. In old forms the hyphse
may be swollen at the points marked by the septa, and may
then appear as strings of plump oval segments. The fila-
ments have a definite wall, inclosing granules and pale areas.
No spore-bearing organs are seen.
On potato, it grows as a dense, widely spreading, velvety
membrane; pale brown at the center and white at the
periphery. The potato takes on a dark-brown color and
becomes very moist and dark; coffee-colored granules
appear upon the surface of the growth.
In bouillon the growth assumes a puff-ball appearance.
ACTINOMYCETES 465
The medium assumes a deep coffee-brown color, and ulti-
mately a mycelium growth appears upon the surface and
throughout the fluid.
When grown in potato infusion (20 grams of potato
boiled in water, filtered and made up to a liter), the growth
is characterized by the appearance of black granules in the
midst of the mycelium. The black granules consist of
closely packed spherical or polyhedral cells, together with
some short, thick segmented hyphse. The walls of these
cells have a black appearance, and masses of them are black
and opaque under the microscope.
On agar-agar, growth appears as a grayish mesh-work of
widely spreading filaments. In old cultures black granules
(sclerotia) appear among the filaments. No growth occurs,
in the depth of the medium.
No results were obtained by the inoculation of animals
with either the material direct from the tissues or with pure
cultures.
The tissue from which this fungus was obtained con-
sisted, briefly, of a more or less atypical connective- tissue
new-growth, with numerous areas of suppuration marked by
the presence of the black granules just described.
On histological study of the tumor the primary effect*
produced by the parasite appears to be the development
of nodules of epithelial cells and of giant cells from the
tissues immediately about them. Later, suppuration of the
nodules and abscess formation occur. This in time gives
rise to excessive development of granulation and connective
tissue.
Actinomyces Farcinicus (bacille du farcin des bceufs
(Nocard); oospora farcinica; actinomyces bovis farcinicus).
— This organism was discovered by Nocard (1888) in a
30
466 APPLICATION OF METHODS OF BACTERIOLOGY
disease of cattle that is suggestive of farcy as seen in horses.
The lesions consist of chains of enlarged subcutaneous
lymph glands, which on examination are found to be in a
condition somewhat simulating tuberculosis. Similar nodules
are sometimes encountered in the internal organs.
By microscopic examination the organism is seen as long,
branching threads consisting of short segments.
It is non-motile. Spore-formation is questionable, Nocard
having seen it, while Lehmann and Neumann have not.
The organism may be stained by the ordinary methods, and
also by the Gram-Weigert process. It grows on all the
ordinary culture media, and at both room- and body-tem-
perature, especially well at the latter. It is aerobic.
Colonies in agar-agar reach a size of from 1 to 2 mm.;
are yellowish-white in color, irregular in outline, and have
the appearance of a glazed, membranous mass.
On gelatin, the growth is much slower, so that after ten
days the colonies appear as tiny translucent round glistening
points. Under low power of the microscope these colonies
are sharply circumscribed, grayish or greenish in color, and
are without characteristic structure.
Growth in bouillon is characterized by a tough, slimy
sediment, and at times by more or less of pellicle formation.
Pellicle formation is encouraged by the addition of glycerin.
The bouillon is not uniformly clouded by the growth.
In milk, it causes an alkaline reaction, solution of casein,
but no coagulation.
On potato, it grows slowly as a dull yellowish-white dry
membrane.
Bovines, sheep, and guinea-pigs are susceptible to inocu-
lation; rabbits, dogs, cats, horses, and asses are not.
ACTINOMYCETES 467
When pure cultures are injected into either the circulation
or the peritoneal cavity of guinea-pigs, death ensues in
from nine to twenty days. The autopsy reveals diffuse
pseudotuberculosis of the omentum. Within the pseudo-
tubercles the organism is seen as long, branching threads,
often matted together as a true mycelium.
By subcutaneous inoculation only the neighboring lymph-
glands are affected.
The disease farcin des bceufs is said to be more common
in Guadeloupe than elsewhere.
Actinomyces Eppingeri. — This organism was discovered by
Eppinger in an abscess of the brain. He regarded it as a
cladothrix, and gave to it the designation cladothrix aster-
oides. It grows well in pure culture under artificial con-
ditions, and is pathogenic for animals. In the case studied
by Eppinger the organism was present not only in the
abscess, but also in the meninges of the brain and cord and
in the bronchial and supraclavicular lymph glands. There
is no doubt of its causal relation to the conditions.
In pure culture it grows well on ordinary media. It
appears as long, branching threads, many of which are com-
posed of short quadratic segments. Spores are not formed.
Motility is doubtful; it has been observed by Eppinger,
while Lehmann and Neumann failed to detect it. It stains
both by the ordinary dyes and by the method of Gram. It
grows scarcely, if at all, under anaerobic conditions. It
grows at room-temperature, but much better at the tem-
perature of the body. The best growth is observed on
nutrient agar-agar containing 2 per cent, of glucose. The
colonies on the surface of glucose-agar-agar appear as
yellowish-white, round, finely granular, dull patches that are
468 APPLICATION OF METHODS OF BACTERIOLOGY
surrounded by a narrow paler zone. In the depths of the
medium they do not develop beyond very small points.
On gelatin the growth is very slow; there is no lique-
faction, and after a time the colonies take on an orange-red
color.
Bouillon is not uniformly clouded. Growth takes place
on the surface in the form of a whitish pellicle, in which
dense white masses may be seen. These latter increase in
size, become detached, and fall to the bottom of the vessel,
to collect as mycelium-like sediment.
On potato, growth begins as a coarsely granulated white
layer, which becomes gradually red in color. It is ultimately
covered by a fine, hair-like growth.
Both rabbits and guinea-pigs are susceptible to its patho-
genic action. When injected into either the circulation, the
peritoneal cavity, or beneath the skin, there develop in from
one to four weeks a condition closely simulating tubercu-
losis (" pseudotuberculosis cladothrica")- The organism
quickly loses its pathogenic properties under artificial
cultivation.
Actinomyces Pseudotuberculosis. — In 1897 Flexner detected
this organism in a consolidated and caseous lung. The con-
dition suggested tuberculosis. The lesion consisted mainly
of an inflammatory exudation that had undergone casea-
tion, but in addition there were present isolated nodules
that in size and general appearance were difficult to distin-
guish from miliary tubercles. Giant cells were not seen.
The streptothrix was abundant in the lung, appearing as
masses of convoluted, branching threads. The contours of
the rods were not quite uniform, the staining was irregular,
and occasionally a thread was seen that, toward its extrem-
ACTINOMYCETES 469
ity, appeared to be breaking up into short segments. No
coccus-like forms were ' seen. It is stained best by the
Weigert method, when deeply stained masses separated
from one -another by more or less clear spaces are to be
detected. The organism was not obtained in culture, and no
effect was produced on guinea-pigs by subcutaneous inocu-
lation with bits of the diseased lung.
CHAPTER XXII.
Glanders — Characteristics of the Disease — Histological Structure of the
Glanders Nodule — Susceptibility of Different Animals to Glanders —
The Bacterium of Glanders; Its Morphological and Cultural Pecu-
liarities— Diagnosis of Glanders — Mallein.
THE disease is generally known as glanders when the
mucous membrane of the nostrils is affected, and as farcy
when the subcutaneous lymphatics are the principal sites
of involvement.
Though most commonly seen in the horse and ass,
glanders is not rarely met with in other animals, and
is occasionally encountered in man. When occurring
in the horse its primary seat is usually upon the mucous
membrane of the nostrils. It appears in the form of
small gray nodules, about which the membrane is con-
gested and swollen. These nodules ultimately coalesce to
form ulcers. There is a profuse slimy discharge from the
nostrils during the course of the disease. The primary
lesion may extend from its seat in the nose to the mouth,
larynx, trachea, and ultimately to the lungs. Its secondary
manifestations are observed along the lymphatics that com-
municate with the initial focus; in the lymphatic glands, and
as metastatic foci in the internal organs.
Less frequently the disease is seen to begin beneath the
skin, particularly in the region of the neck and breast. When
in this locality the subcutaneous lymphatics become in-
volved, and are converted into indurated, knotty cords —
"farcy-buds" — easily discernible from without.
(470)
GLANDERS 471
In man it usually occurs in individuals who have been
in attendance upon animals affected with the disease. It
may occur upon the mucous membrane of the nares; but
its most frequent expressions are in the skin and muscles,
where appear abscesses, phlegmons, erysipelas-like inflam-
mations, and local necrosis closely resembling carbuncles.
Metastases to the lungs, kidneys, and testicles, as in the
horse, may also be seen.
When occurring upon the mucous membrane glanders is
characterized by the presence of gray nodules, about as
large as a pin-head, that closely resemble miliary tubercles
in their naked-eye appearance. These consist histologically
of granulation-tissue — i. e., of small round cells, very similar
to proliferating leukocytes — of some lymph cells, and, in the
earliest stages, of a small portion of necrotic tissue. As
they grow older, and the process advances, there is a tendency
to central necrosis, with the ultimate formation of a soft,
yellow, creamy, pus-like material. Though strikingly like
miliary tubercles in certain respects in the early stages,
they present, nevertheless, decided points of difference when
examined more in detail.
The round-cell infiltration of the glanders nodule consists
essentially of polymorphonuclear leukocytes, while that of
the miliary tubercle partakes more of the nature of a lym-
phocytic infiltration; in the later stages of the process the
glanders nodule breaks down into a soft, creamy matter,
very analogous to ordinary pus, while in the later stages
of the miliary tubercle the tendency is to an 'amalgamation
of its histological constituents, and ultimately to necrosis
with caseation. The giant-cell formation common to tuber-
culosis is never seen in the glanders nodule. As Baumgarten
aptly puts it: "The pathological manifestations of glanders,
472 APPLICATION OF METHODS OF BACTERIOLOGY
from the histological aspect, stand midway between the
acute purulent and the chronic inflammatory processes."1
Evidently these differences are only to be explained by dif-
ferences in the nature of the causes that underlie the several
affections. We have studied the characteristics of bacterium
tuberculosis; we shall now take up the bacillus of glanders
and note the striking differences between them.
BACTERIUM MALLEI (LOFFLER), MIGULA, 1900.
SYNONYMS: Bacillus mallei (Loffler), 1886; Rotz bacillus, Kranzfeld,
1887.
In 1882 Loffler and Schiitz discovered in the diseased
tissues of animals suffering from glanders a bacterium that,
when isolated in pure culture and inoculated into susceptible
animals, possesses the property of reproducing the disease
with all its clinical and pathological manifestations. It is
therefore the cause of the disease.
This organism is a rod, with rounded or slightly pointed
ends. It usually stains somewhat irregularly. (See Fig.
78.) When examined in stained preparations its continuity
is marked by alternating darkly and lightly stained areas.
It is usually seen as a single rod, but may occur in pairs,
and less frequently in longer filaments. ,.
The question as to its spore-forming property is still an
open one, though the weight of evidence is in opposition
to the opinion that it possesses this peculiarity. Certain
observers claim to have demonstrated spores in the bacteria
by particular methods of staining; but this statement can
1 For a further discussion of the pathology and pathogenesis of this
disease, see Lehrbuch der pathologischen Mykologie, by Baumgarten,
1890. See, also, Wright, The Histological Lesions of Acute Glanders in
Man, Journal of Experimental Medicine, i, 577.
BACTERIUM MALLEI
473
have but little weight when compared with the behavior
of the organism when subjected to more conclusive tests.
For example, it does not, at any stage of development, resist
exposure to 3 per cent, carbolic acid solution for longer than
five minutes, nor to 1 : 5000 sublimate solution for more than
two minutes. It is destroyed in ten minutes in some experi-
ments, and in five in others, by a temperature of 55° C.;
FIG. 80
Bacterium mallei, from culture.
and when dried it loses its vitality, according to different
observers, in from thirty to forty days; all of which speak
directly against this being a spore-bearing bacillus.
It is not motile, and does not therefore possess flagella.
It grows readily on ordinary nutrient media at from 25°
to 38° C.
Upon nutrient agar-agar, both with and without glycerin,
it appears as a moist, opaque, glazed layer, with nothing
474 APPLICATION OF METHODS OF BACTERIOLOGY,
characteristic about it. This is true both for smear cultures
and for single colonies.
Its growth on gelatin is much less voluminous than on
media that can be kept at higher temperature, though it
does grow on this medium at room-temperature without
causing liquefaction.
Its growth on blood serum is in the form of a moist,
opaque, slimy layer, inclining to a yellowish or dirty,
brownish-yellow tinge. It does not liquefy the serum.
On potato its growth is moderately rapid, appearing in
from twenty-four to thirty-six hours at 37° C. as a moist,
amber-yellow, transparent deposit having somewhat the
appearance of honey; this becomes deeper in color and
denser in consistence as growth progresses, and finally takes
on a reddish-brown color; at the same time the potato
about it becomes darkened.
In bouillon it causes diffuse clouding, with ultimately the
formation of a more or less tenacious or ropy sediment.
In milk to which a little litmus has been added it causes
the blue color to become red or reddish in from four to five
days, and quite red after two weeks at 37° C. At the same
time the milk separates into clear whey and a firm clot of
casein.
Its reactions to heat are very interesting. At 42° C. it
will often grow for twenty days or more. It will not grow
at 43° C., and if exposed to this temperature for forty-eight
hours it is destroyed. It is killed in five hours when exposed
to 50° C., and in five minutes by 55° C.
It grows both with and without oxygen; it is therefore
facultative as regards its relation to this gas.
On cover-slips it stains readily with all the basic aniline
dyes, and, as a rule, as stated, presents conspicuous irregu-
BACTERIUM MALLEI 475
larities in the way that it takes up the dyes, being usually
marked by deeply stained areas that alternate with points
at which it either does not stain at all or only slightly.
The animals susceptible to infection by this organism are
horses, asses, field-mice, guinea-pigs, and cats. Baumgarten
records cases of infection in lions and tigers that were fed,
in menageries, with flesh from horses affected with the
disease. Rabbits are but slightly susceptible; dogs and
sheep still less so. Man is susceptible, and infection not
rarely terminates fatally. White mice, common gray house-
mice, rats, cattle, and hogs are insusceptible.
Inoculation Experiments. — The most favorable animal
upon which to study the pathogenic properties of this
organism in the laboratory is the common field-mouse.
When inoculated subcutaneously with a small portion of a
pure culture of bacterium mallei death ensues in about
seventy-two hours. The most conspicuous tissue changes
will be enlargement of the spleen, which is at the same time,
almost constantly, studded with minute gray nodules, the
typical glanders nodule. They are rarely present in the
lungs, but may frequently be seen in the liver. From these
nodules the glanders bacillus may be obtained in pure culture.
With the exception of the characteristic nodules, the disease
as seen in this animal presents none of the features that
it displays in the horse and ass. The clinical and patho-
logical manifestations resulting from inoculation of guinea-
pigs are much more faithful reproductions. The animal
lives usually from six to eight weeks after inoculation, and
during this time becomes affected with a group of most
interesting and peculiar pathological processes. The specific
inflammatory condition of the mucous membrane of the
nostrils is almost always present. The joints become swollen
476 APPLICATION OF METHODS OF BACTERIOLOGY
and infiltrated to such an extent as often to interfere with
the use of the legs. In male animals the testicles become
enormously distended with pus, and on closer examination
a true orchitis and epididymitis are seen to be present.
The internal organs, particularly the lungs, kidneys, spleen,
and liver, are usually the seat of the nodular formations
characteristic of the disease. From all of these disease-foci
the bacillus causing them ca.n be isolated in pure culture.
Staining in Tissues. — Though always present in the diseased
tissues, considerable trouble is usually experienced in demon-
strating the bacteria by staining methods. The difficulty is
due to the fact that the bacteria are very easily decolorized,
and in tissues stained by the ordinary processes are robbed
of their color even by the alcohol with which the tissue is
rinsed and dehydrated. If we will remember not to employ
concentrated stains, and not to expose the sections to the
stains for too long a time, but little treatment with decolor-
izing agents is necessary, and very satisfactory preparations
will be obtained. A number of methods have been suggested
for staining the glanders bacilli in tissues, and if what has
been said will be borne in mind, no difficulty should be
experienced. Two satisfactory methods that we have used
for this purpose, though perhaps no better than some of the
others, are as follows :
a. Transfer the sections from alcohol to distilled water.
This lessens the intensity with which the stain subsequently
takes hold of the tissues, by diminishing the activity of the
diffusion that would occur if they were placed froin alcohol
into watery solutions of the dyes. Transfer from distilled
water to the slide, absorb all water with blotting-paper,
and stain with two or three drops of
Carbol-fuchsin 10 c.c.
Distilled water . ... 100 c.c.
BACTERIUM MALLEI 477
for thirty minutes; absorb all superfluous stain with blot-
ting-paper, and wash the section three times with 0.3 per
cent, acetic acid, not allowing the acid to act for more than
ten seconds each time. Remove all acid from the section
by carefully washing in distilled water; absorb all water
by gentle pressure with blotting-paper; and finally, at wry
moderate heat, or with a small bellows (Kiihne), dry the section
completely on the slide. When dried clear in xylol and mount
in xylol balsam.
6. Transfer the sections from alcohol to distilled water;
from water to the dilute fuchsin solution, and gently warm
(about 50° C.) for fifteen to twenty minutes. Transfer
sections from the staining-solution to the slide, absorb all
superfluous stain with blotting-paper, and then treat them
with 1 per cent, acetic acid from one-half to three-quarters
of a minute. Remove all trace of acid with distilled water,
absorb all water by gentle pressure with blotting-paper, and
then treat the sections with absolute alcohol by allowing
it to flow over them drop by drop. For small sections three
or four drops are sufficient. Under no circumstances should
the alcohol be allowed to act for more than one-quarter of
a minute. Clear in xylol and mount in xylol balsam.
By method b the tissues are better preserved than by
method a, by which they are dried.
In properly stained tissues the bacteria will be found
most numerous in the center of the nodules, becoming fewer
as we approach the periphery. They usually lie between
the cells, but at times may be seen almost filling some of
the epithelial cells, of which the nodule contains more or less.
They are always present in these nodules in the tissues; they
are rarely present in the blood, and, if so, in only small
numbers.
478 APPLICATION OF METHODS OF BACTERIOLOGY
Diagnosis of the Disease by Agglutination and Complement-
fixation. The quickest and surest method of recognizing the
disease is by the specific agglutinating effect of the serum of
the diseased animal upon the organism of the disease. Many
different plans have been recommended. That of Moore, of
Cornell University, is one of the most trustworthy. He
recommends a test emulsion made by suspending a glycerin-
agar culture of glanders bacilli in physiological salt solution.
This is then exposed to 60° C. for two hours, whereby the
bacteria are killed, and is finally preserved by the addition
of 0.5 per cent, carbolic acid. To this suspension the serum
of the suspected animal is added in varying proportions
until a distinct clumping and sedimentation of the bacteria
is observed. Whenever done in a small test-tube of about
0.5 cm. diameter this reaction manifests itself as a gradual
clarification of the milky fluid and the accumulation of a mass
on the bottom of the tube. Normal horse serum in a dilution
of 1 to 300 to 1 to 200 causes the agglutination, while that
from glanders animals does the same in from 1 to 3200 to
1 to 500 dilution.
The "complement-fixation" reaction may also be applied
both for the recognition of the condition — i. e., for detecting
the specific antibodies in the tissues or fluids, as well as for
the identification of the specific exciter of the condition —
i. e., the antigen. (See that reaction.)
Mallein. — The sterile filtered products of growth of the
glanders bacillus in fluid media represent what is known as
mallein — a solution of compounds that bear to glanders a
relation analogous to that which tuberculin bears to tuber-
culosis. It is used with considerable success as a diagnostic
aid in detecting the existence or absence of deep-seated
manifestations of the disease, the glanderous animal reacting
BACTERIUM MALLEI 479
(manifested by elevations of body-temperature greater than
1.5° C.) to subcutaneous injections of mallein in from four
to ten hours, while an animal not so affected gives no such
reactions.
Mallein is prepared from old glycerin-bouillon cultures of
the glanders bacterium by steaming them for several hours in
the sterilizer, after which they are filtered through unglazed
porcelain.
By some it is said that the repeated injection of mallein
in small doses confers immunity from infection by bacterium
mallei upon animals so treated; an opinion that is entirely
in accord with the principles underlying the artificial induc-
tion of immunity in general.
CHAPTER XXIII.
Bacterium (Syn. Bacillus) Diphtherias — Its Isolation and Cultivation —
Morphological and Cultural Peculiarities — Pathogenic Properties —
Variations in Virulence — Bacterium Pseudodiphtheriticum — Bacterium
Xerosis — Diphtheria Antitoxin.
FROM the gray-white deposit on the fauces of a diph-
theritic patient prepare a series of cultures in the following
way:
Have at hand five or six tubes of Loffler's blood-serum
mixture. (See chapter on Media.) Pass a stout platinum
needle, which has been sterilized, into the membrane and
twist it around once or twice, or brush it gently over the
surface of the membrane. Without touching it against
anything else rub it carefully over the surface of one of the
serum tubes; without sterilizing it pass it over the surface
of the second, then the third, fourth, and fifth tubes. Place
these tubes in the incubator. Then prepare cover-slips
from scrapings from the membrane on the fauces. If the
case is one of true diphtheria, the tubes will be ready for
examination on the following day.
The reason that plates are not made in the regular way
in this examination is that the bacillus of diphtheria develops
much more luxuriantly on the serum mixture, from which
plates cannot be made, than it does on the media from which
they can be made. The method employed, however, insures
a dilution in the number of organisms present, and this, in
addition to the fact that the blood serum mixture is a much
more favorable medium for the rapid development of the
diphtheria organism than of the other organisms present,
(480)
BACTERIUM DIPHTHERIA 481
makes its isolation by this method a matter of but little
difficulty.
After twenty-four hours in the incubator the tubes present
a characteristic appearance. Their surfaces are marked by
more or less irregular patches of a white or cream-colored
growth, which is usually more dense at the center than at
the periphery. Except now and then, when a few orange-
colored colonie^ may be seen, these, large irregular patches
are the conspicuous objects on the surface of the serum.
Occasionally, almost nothing else appears.
The cover-slips made from the membrane at the time the
cultures were prepared will be found on microscopic examina-
tion to present, in many cases, a great variety of organisms;
but conspicuous among them will be noticed slightly curved
bacilli of irregular size and outline. In some cases they will
be more or less clubbed at one or both ends; sometimes
they appear spindle in shape, again as curved wedges; now
and then they are irregularly segmented. They are rarely
or never regular in outline. If the preparation has been
stained with Loffler's alkaline methylene-blue solution, many
of these irregular rods are seen to be marked by circumscribed
points in their protoplasm which stain very intensely —
they appear almost black. This irregularity in outline is
the morphological characteristic of bacillus diphtherise of
Loffler, the most pleomorphic organism with which we have
to deal.
It must be remembered, however, that the diagnosis
of diphtheria should not under all circumstances be made
from the examination of cover-slip preparations alone, espe-
cially when they are stained only by the usual method —
i. e., with Loffler's methylene-blue. There are other organ-
isms present in the mouth cavity, particularly in the mouths
31
482 APPLICATION OF METHODS OF BACTERIOLOGY
of persons having decayed teeth, the morphology of which
is so like that of the bacillus of diphtheria that they might
easily be mistaken for that organism if subjected to only
the usual method of microscopic examination; and again,
the genuine diphtheria organism is sometimes found in the
mouth cavities of healthy persons in attendance upon diph-
theria cases, such persons being at the time insusceptible
to the pathogenic activities of the organism^ In the vast
majority of instances, however, where the clinical condition
of the patient justifies a suspicion of diphtheria, a micro-
scopic examination alone of the deposit in the throat, made
by an experienced person, will serve to confirm or contradict
this opinion, and such examinations very frequently reveal
the diphtheritic nature, etiologically speaking, of mild con-
ditions of the throat which are not associated with grave
constitutional manifestations.
BACTERIUM DIPHTHERIA (LOFFLER), MIGULA, 1900.
SYNONYMS: Bacillus diphtherias, Loffier, 1884; Klebs-Loffler bacillus;
Corynebacterium diphtheriae, Lehmann and Neumann, 1896.
Bacterium diphtherise, discovered microscopically by
Klebs, and isolated in pure culture and proved to stand in
causal relation to diphtheria by Loftier, can readily be
identified by its cultural peculiarities and by its pathogenic
activity when introduced into tissues of susceptible animals.
In guinea-pigs and kittens the results of its growth are his-
tologically identical with those found in the bodies of human
beings who have died of diphtheria.
When studied in pure culture its morphological and cul-
tural peculiarities are as follows :
Morphology. — As obtained directly from the diphtheritic
deposit in the throat of an individual sick of the disease,
BACTERIUM DIPHTHERIAS 483
it is sometimes comparatively regular in shape, appearing
as straight or slightly curved rods with more or less pointed
ends. More frequently, however, spindle- and club-shapes
occur, and not rarely many of these rods stain irregularly;
in some of them very deeply stained round or oval points
can be detected.
When cultures are examined microscopically it is especially
characteristic to find irregular, bizarre forms, such as rods
with one or both ends swollen, and very frequently rods
broken at irregular intervals into short, sharply defined
segments, either round, oval, or with straight sides. Some
forms stain uniformly, others in various irregular ways, the
most common being the appearance of deeply stained
granules in a lightly stained bacillus.
By a series of studies upon this organism when cultivated
under artificial conditions it has been found that its form and
size depend very largely upon the nature of its environment.
That is to say, its morphology is always more regular, and
it is smaller on glycerin-agar-agar than on other media used
for its cultivation; while upon Loffler's blood serum the
other extremes of development appear: here one sees,
instead of the very short, spindle-, lancet-, club-shaped,
always segmented and regularly staining forms as seen upon
glycerin-agar-agar, long, sometimes extremely slender, some-
times thicker, irregularly staining threads that may be either
clubbed or pointed at their extremities. They are, as a rule,
marked by areas that stain more intensely than does the
rest of the rod, and at times they may be a little swollen
at the center. These differences are so conspicuous that
microscopic preparations from cultures from the same source,
but cultivated in the one case on glycerin-agar-agar and in
the other upon blood serum, when placed side by side would
484 APPLICATION OF METHODS OF BACTERIOLOGY
hardly be recognized as of the same organism, unless its
peculiar behavior under these circumstances was already
known. Another peculiar variation is that observed upon
very slightly acid blood serum. Here the rods appear
swollen, and are usually contracted to oval or short, oblong
bodies, which stain very faintly, and in which are usually
located one or two very deeply staining round or oval points.
Various authors have called attention to branching forms
of this organism that are occasionally encountered, especially
when cultivated upon albumin. We have never seen the
branching diphtheria organisms under conditions that might
reasonably be regarded as favorable to normal development;
and in many thousand blood serum cultures from cases of
diphtheria that have been examined by competent bacteriol-
ogists at the laboratory of the Bureau of Health of Philadel-
phia, the branching forms of this organism have not been
observed in a single instance. It is fair to assume, there-
fore, that this peculiar morphological variation of bacillus
diphtheriae is, under normal conditions of growth, com-
paratively rare.
On the other hand, if the organism be grown on media
favorable to involution, such, for instance, as hard-boiled egg,
or coagulated egg of slightly acid reaction, branching may be
seen, but with it degenerated organisms are so conspicuous
as to leave no doubt that the so-called branching and involu-
tion are attributable to the same cause, namely, unsuitable
conditions of cultivation.
On plain nutrient agar-agar (that is, nutrient agar-agar
without glycerin); on a medium consisting of dried com-
mercial albumin dissolved in bouillon (about 10 grams
of albumin to 100 c.c. of bouillon containing 1 per cent, of
grape-sugar); in bouillon without glycerin, and in bouillon
BACTERIUM DIPHTHERIA
485
to which a bit of hard-boiled egg has been added, the mor-
phology of the organism is about intermediate, in both
size and outlines, between the forms seen upon glycerin-
agar-agar and upon Loffler's blood-serum. There will
appear about an equal number of short segmented and
longer, irregularly staining forms; but in general the longest
FIG. 81
A.
Bacterium diphtheria. A, its morphology on glycerin-agar-agar; B,
its morphology on Loffler's blood-serum; (7, its morphology on acid blood-
serum mixture.
are rarely as long as the long forms seen on blood-serum,
and throughout they are not so conspicuous for the irregu-
larity of their staining.
In cultures made upon two sets of nutrient agar-agar
tubes, differing only in the fact that one set contains glycerin
to the extent of 6 per cent., while the other set contains
486 APPLICATION OF METHODS OF BACTERIOLOGY
none, a noticeable difference in morphology can usually
be made out: while the forms on the glycerin-agar-agar
cultures are throughout small, and pretty** regular in size,
shape, and staining, those on the plain agar-agar are larger,
stain less uniformly, vary more in shape, and when stained
by Loffler's blue are not so regularly marked by pale trans-
verse lines that give to them the appearance of being made
up of numerous short segments.
Though the outline of this organism is more regular
under some circumstances than others, it is nevertheless
always conspicuous for its manifold variations in shape.
Growth on Serum Mixture. — The medium upon which
bacillus diphtherise grows most rapidly and luxuriantly and
which is best adapted for determining its presence in diph-
theritic exudates, is, as has been stated, the blood-serum
mixture of Loffler. (See chapter on Media.) On the blood-
serum mixture the colonies of bacillus diphtherise grow so
much more rapidly than the other organisms usually present
in secretions and exudations in the throat that at the end of
twenty-four hours they are often the only colonies that
attract attention; and if others of similar size are present,
they are generally of quite a different aspect. Its colonies
are large, round, elevated, grayish- white or yellowish, with
a center more opaque than the slightly irregular periphery.
The surface of the colony is at first moist, but after a day or
two becomes rather dry in appearance.
A blood serum tube studded with coalescent or scattered
colonies .of this organism is so characteristic that one familiar
with the appearance can anticipate with tolerable certainty
the results of microscopic examination.
Glycerin-agar-agar. — Upon nutrient glycerin-agar-agar the
colonies likewise present an appearance that readily may
BACTERIUM DIPHTHERIA 487
be recognized. They are in every way more delicate in
structure than when on the serum mixture. They appear
at first, when on the surface, as very flat, almost transparent,
dry, non-glistening, round points which are not elevated
above the surface upon which they are growing. When
slightly magnified they are seen to be granular, and to
present an irregular central marking, which is denser and
darker by transmitted light than the thin, delicate zone
which surrounds it. As the colony increases in size the thin
granular peripheral zone becomes broader, is usually marked
FIG. 82
c*
Colonies of bacterium diphtherise on glycerin-agar-agar. a, colonies
located in the depths of the medium; 6, colonies just breaking out upon
the surface of the medium; c, fully developed surface-colony.
by ridges or cracks, and its periphery is notched or scalloped.
(Fig. 80, c.) These colonies are always quite dry in ap-
pearance. When deep down in the agar-agar they are
coarsely granular. (Fig. 80, a.) They rarely exceed 3 mm.
in diameter.
Gelatin. — On gelatin the colonies develop much more
slowly than on media that can be retained at a higher tem-
perature. They rarely present their characteristic appear-
ances on gelatin in less than seventy-two hours. They then
appear as flat, dry, translucent points, usually round in
outline.
488 APPLICATION OF METHODS OF BACTERIOLOGY
When magnified slightly the center is seen to be more
dense than the surrounding zone or zones, for they are
sometimes marked by a concentric arrangement of zones.
The periphery is irregularly notched. Like the colonies
seen on agar-agar, they are granular, but are much more
granular when seen in the depths of the gelatin than when
on its surface. On gelatin the colonies rarely become very
large; usually they do not exceed 1.5 mm. in diameter.
Bouillon. — In bouillon it usually grows in fine clumps,
which fall to the bottom of the tube, or become deposited
on its sides without causing diffuse clouding of the bouillon.
Sometimes there are exceptions to this naked-eye appear-
ance; the bouillon may be diffusely clouded; but if one
inspect it very closely, particularly if he examine it micro-
scopically as a hanging drop, the arrangement in clumps will
always be detected, but the clumps are so small as not to
be discernible by the unaided eye.
In bouillon kept at a temperature of 35°-37° C. a soft,
whitish pellicle often forms upon the surface.
The reaction of the bouillon frequently becomes at first
acid, and subsequently again alkaline, changes which can
•be observed in cultivations in bouillon to which a little
rosolic acid has been added. This play of reactions has
been attributed to the primary fermentation of the muscle-
sugar often present in the bouillon. It does not occur
when the medium is free from carbohydrates.
Potato.— On potato at a temperature of 35°-37° C. its
growth after several days is invisible, only a thin, dry glaze
appearing at the point at which the potato was inoculated.
Microscopic examination of scrapings from the potato,
after twenty-four hours at 35°-37° C., reveals a decided
increase in the number of individual organisms planted.
BACTERIUM DIPHTHERIA 489
Stab- and Slant-cultures. — In stab- and slant-cultures on
both gelatin- and glycerin-agar-agar the surface-growth is
seen to predominate over that along the track of the needle
in the depths of the media.
Isolated colonies on the surface of either of the media in
this method of cultivation present the same characteristics
that have been given for the colonies on plates.
The growth in simple stab-cultures does not extend later-
ally very far beyond the point at which the needle entered
the medium.
It is a non-motile organism.
It does not form spores.
It is killed in ten minutes by a temperature of 58° C.
It grows at temperatures ranging from 22° to 37° C.,
but most luxuriantly at the latter temperature.
Its growth in the presence of oxygen is more active than
when this gas is excluded.
Staining. — In cover-slip preparations made either from
the fauces of a diphtheritic patient or from a pure culture
of the organism it is seen to stain readily with the ordinary
aniline dyes. It stains also by the method of Gram, but the
best results are obtained by the use of Loffler's alkaline
methylene-blue solution; this brings out the dark points
in the protoplasmic body of the bacilli and thus aids in their
identification. »
For the purpose of demonstrating the Loffler bacillus in
sections of diphtheritic membrane, both the Gram method
and the fibrin method of Weigert give excellent results.
Pathogenic Properties. — When inoculated subcutaneously
into the bodies of susceptible animals the result is not the
production of septicemia, as is seen to follow the introduc-
tion into animals of certain other organisms with which
490 APPLICATION OF METHODS OF BACTERIOLOGY
we shall have to deal, but the bacillus of diphtheria remains
localized at the point of inoculation, rarely disseminating
further than the nearest lymphatic glands. It develops at
the point in the tissues at which it is deposited, and during
its development gives rise to changes in the tissues which
result entirely from the absorption of poisons generated by
the bacteria in the course of their development.
Occasionally diphtheria bacilli may be found in the
blood and internal organs of individuals dead of the disease;
but all that has been learned from careful study of the
secondary manifestations of diphtheria tends to the opinion
that they are in no way dependent upon the immediate
presence of bacteria, and that the occasional appearance
of diphtheria bacteria in the internal organs is in all prob-
ability accidental, and usually unimportant.
By special methods of inoculation1 (the injection of fluid
cultures into the testicles of guinea-pigs) diphtheria bacilli
can be cawed to appear in the omentum; but this is purely
an artificial manifestation of the disease, and one that is
probably never encountered in the natural course of events.
More rarely similar results follow upon subcutaneous
inoculation.
If a very minute portion of a virulent pure culture of
this organism be introduced into the subcutaneous tissues
of a guinea-pig or kitten, death of the animal ensues in
from twenty-four hours to five days. The usual changes
are an extensive local edema, with more or less hyperemia
and ecchymoses at the site of inoculation; swollen and red-
dened lymphatic glands; increased serous fluid in the peri-
toneum, pleura, and pericardium; enlarged and hemorrhagic
1 Abbott and Ghriskey, A Contribution to the Pathology of Experi-
mental Diphtheria, The Johns Hopkins Hospital Bulletin, No. 30, April,
1893.
BACTERIUM DIPHTHERIA 491
adrenal bodies; occasionally slightly swollen spleen; and
sometimes fatty degeneration in the liver, kidney, and myo-
cardium. In guinea-pigs, especially, the liver often shows
numerous macroscopic dots and lines on the surface and
penetrating the substance of the organ. They vary in size
from a pin-point to a pin-head, and may be even larger.
They are white and do not project above the surface of the
capsule.
The bacteria are always to be found at the site of inocu-
lation, most abundant in the grayish-white, fibrino-purulent
exudate. They become fewer at a distance from this, so
that the more remote parts of the edematous tissues do
not contain them. They are found not only free, but con-
tained in large number in leukocytes, some of .which have
fragmented nuclei, or have lost their nuclei. The bacteria
within leukocytes, as well as some outside, frequently stain
very faintly and irregularly, and may appear disintegrated
and dead.
Culture tubes inoculated from the blood, spleen, liver,
kidneys, adrenal bodies, distant lymphatic glands, and
serous transudates, generally yield negative results; and
negative results are also obtained when these organs are
examined microscopically for the bacteria.
Microscopic examination of the tissues at the site of
inoculation, as well as of the liver, spleen, kidneys, lymphatic
glands, and elsewhere, reveals the presence of localized foci
of cell-death, characterized by a peculiar fragmentation of
the nuclei of the cells of these parts.
This destruction of nuclei results in the formation of
groups of irregularly shaped, deeply staining bodies, having
at times the appearance of particles of dust, while again
they may be much larger. Some of them are tolerably
492 APPLICATION OF METHODS OF BACTERIOLOGY
regular in outline, while others are irregularly crescentic,
dumb-bell, flask-shape, whetstone-shape, or bladder-like
in form. Occasionally nuclei having the appearance of
being pinched or drawn out can be seen. At some points
the fragments are grouped in isolated masses, indicating the
location of the nucleus from the destruction of which they
originated. 'These particles always stain much more in-
tensely than do the normal nuclei of the part.1 Oertel
showed long before bacillus diphtherise was discovered that
these peculiar alterations in cell nuclei, both in distribution
and appearance, are characteristic of human diphtheria,
and the demonstration of similar changes in animals inocu-
lated with this organism is important additional proof that
diphtheria is caused by it.
By the inoculation of certain animals an affection may
be produced in all respects identical with diphtheria as it
exists in man. If one open the trachea of a kitten and rub
upon the mucous membrane a small portion of a pure culture
of this organism, the death of the animal usually ensues in
from two to four days. At autopsy the wound will be found
covered with a grayish, adherent, necrotic, distinctly diph-
theritic layer. Around the wound the subcutaneous tissues
will be edematous. The lymphatic glands at the angle
of the jaws will be swollen and reddened. The mucous
membrane of the trachea at the point upon which the bac-
teria were deposited will be covered with a tolerably firm,
grayish-white, loosely attached pseudomembrane in all
respects identical with the croupous membrane observed
in the same situation in cases of human diphtheria. In the
1 See The Histological Changes in Experimental Diphtheria, also Th.e
Histological Lesions Produced by the Toxalbumin of Diphtheria, by Welch
and Flexner, Johns Hopkins Hospital Bulletin, August, 1891, and March,
1892.
BACTERIUM DIPHTHERIA 493
pseudomembrane and in the edematous fluid about the
skin-wound bacillus diptherise may be found both in cover-
slips and in cultures.
From what we have seen — the localization of the bacilli
at the point of inoculation, their absence from the internal
organs, and the changes brought about in the cellular ele-
ments of the internal organs — there is but one interpre-
tation for this process, viz., that it is due to the production
of a soluble poison by the bacteria confined to the site of
inoculation, which, gaining access to the circulation, produces
the changes that we observe in the tissues of the internal
viscera.
This poison has been isolated from cultures pf bacillus
diphtheriae, and is found to belong, not to the crystallizable
ptomains, but to the toxins — bodies which, in their chemical
composition, are analogous to the poison of certain venom-
ous serpents. By the introduction of this toxin into the
tissues of guinea-pigs and rabbits the same pathological
alterations may be produced that we have seen to follow
inoculation with the bacilli themselves, except, perhaps,
the production of false membranes.
Under certain circumstances with which we are not
acquainted bacillus diphtherias becomes diminished in
virulence or may lose it entirely, so that it is no longer
capable of producing death of susceptible animals, and may
cause only a transient local reaction from which the animal
entirely recovers. Sometimes this reaction is so slight as
to be overlooked, and again careful search may fail to reveal
evidence of any reaction at all. These exhibitions of the
extremes of its pathogenic properties, viz., death of the
animal, on the one hand, and only very slight local effects
on the other, was at one time thought to indicate the existence
494 APPLICATION OF METHODS OF BACTERIOLOGY
of two separate and distinct organisms that were alike in
cultural and morphological peculiarities, but which differed
in their disease-producing power. Further -studies on this
point have, however, shown that genuine bacillus diph-
therise may possess almost all grades of virulence, and that
absence of or diminution in virulence can hardly serve to
distinguish as separate species those varieties that are other-
wise alike; moreover, the histological conditions found at
the site of inoculation in animals that have not succumbed,
but in which only the local reaction has appeared, are in
most cases characterized by tissue changes that are identi-
cal in kind though less in degree to those seen at autopsy
in animals in which inoculation has proved fatal.
In the course of their observations upon a large number
of cases Roux and Yersin found that it was not difficult to
detect, in the diphtheritic deposits of a patient ill of diph-
theria bacteria of identical cultural and morphological
peculiarities, but of very different degrees of virulence, and
that with the progress of the disease toward recovery the
less virulent varieties often became quite frequent.1
There is, moreover, a mild form of diphtheria, etiologically
speaking, affecting only the mucous membrane of the nares,
known as membranous rhinitis, from which it is very com-
mon to obtain cultures in all respects identical with those
from typical diphtheria, save for their inability to kill sus-
ceptible animals. On inoculation these cultures produce
only local reactions, but these are characterized histologi-
cally by the same kind of tissue-changes that follow inocu-
lation with the fully virulent organism.
1 It must not be assumed from this that the bacteria lose their virulence
entirely, or that they all become attenuated with the establishment of con-
valescence, for this is contrary to what experience has shown to be the case.
BACTERIUM DIPHTHERIA 495
Clinically, membranous rhinitis is never such an alarming
disease as is laryngeal or pharyngeal diphtheria, and, as
stated, the organisms causing it are often of a low degree of
virulence, though they are, nevertheless, genuine diphtheria
bacteria.
For those organisms that are in all respects identical with
the virulent bacillus diphtherise, save for their inability to
kill guinea-pigs, the designation " pseudodiphtheritic bacil-
lus" is usually employed; but from such observations as
those just cited we are inclined to the opinion that pseudo-
diphtheritic, as applied to an organism in all respects iden-
tical with the genuine bacterium, except that it is not fatal
to susceptible animals, is a misnomer, and that it would
be more nearly correct to designate this organism as the
attenuated or non-virulent diphtheritic bacterium, reserving
the terms "pseudodiphtheritic" or " diphtheroid " for that
organism or group of organisms (for there are probably
several) that are enough like the diphtheria bacterium to
attract attention, but is distinguishable from it by certain
morphological and cultural peculiarities aside from the
question of virulence.
It is a well-known fact that many pathogenic organisms —
conspicuous among these being bacterium pneumoniae,
micrococcus aureus, streptococcus pyogenes, and the group
of so-called "hemorrhagic septicemia" organisms — undergo
marked variations in their pathogenic properties; and yet
these organisms, when found either devoid of this peculiarity,
or possessing it in a diminished degree, are not designated
as "pseudo" forms, but simply as varieties, the virulence of
which, from various causes, has been modified.
It must nevertheless be admitted that in the course of
496 APPLICATION OF METHODS OF BACTERIOLOGY
microscopic examination of materials from various sources,
including the pharynx, one occasionally encounters micro-
organisms whose morphology is so like that of the genuine
bacterium diphtheriae as to create suspicion, and yet they
are at the same time sufficiently unlike it to make one cau-
tious in forming an opinion as to their real nature.
Variations. — The pleomorphism of bacillus diphtherise,
together with its many irregularities in physiological func-
tion, render some satisfactory grouping or typing, highly
desirable. Numerous efforts in this direction have been
made but none as yet with entirely satisfactory results.
The efforts to group the varieties of this organism accord-
ing to minute peculiarities of form, structure and staining
peculiarities and to attribute to one such group pathogenic
powers and to another no such power, involves far too
much that is subjective 'to be of permanent value, in fact
it is in most instances misleading.
The grouping or typing according to certain functional
characteristics, such as zymogenesis, has been of use, but it
still leaves something to be desired.
The grouping in accordance with pathogenic potency is
surrounded by too many complications to be routinely
useful.
The efforts to group the large number of varieties of this
species, though specific' agglutinating reactions, as has
been done with pneumococci, streptococci, meningococci
and certain other organisms, though not far advanced, holds
out, nevertheless, much promise of success. Up to the
present the results of the efforts may be summarized about
as follows:
1. There is apparently no constant relationship between
morphology and antigenic power.
BACTERIUM DIPHTHERIA 497
2. By immunizing an animal from any one of a large
number of strains of genuine bacillus diphtherise a certain
number of the varieties in the group will agglutinate with
the serum of that animal in very high dilutions; while others
will either not agglutinate at all with that serum or only in
very low dilutions.
3. These agglutination reactions are specific for the several
groups, i. e., cross-reactions are not observed.
4. There is a sharp distinction between the agglutining
antigenic component and the antitoxin antigenic component
in bacillus diphtherise.
5. The antitoxin produced through the use of the several
groups are not so sharply distinguished from one another
as are the agglutinins, though they manifest specific rela-
tionship to their homologous antigens.
The bearing of all this on the recognition of bacillus
diphtheria and on the production of antitoxin is obvious.1
Bacterium Pseudodiphtheriticum. — For a long time bac-
terium pseudodiphtheriticum was looked upon as being
entirely harmless, and the only particular in which it was
regarded as being of importance was in the fact that it was
likely to be mistaken for bacterium diphtherise. The wide
dissemination of this class of organisms and the demon-
stration of pathogenic effects in isolated instances has led
to the more systematic study of members of this group of
organisms.
Bacterium pseudodiphtheriticum, as found under different
conditions, varies markedly in its morphologic and biologic
characters. Some of the varieties have definite chromogenic
1 See Langer, Die Agglutination der Diphtherie bacillen, Centralbl. f.
Bact., Abt. I, Originale, 1916, vol. Ixxviii, p. 117. Havens, Biologic Studies
of the Diphtheria Bacillus, Jour. Infect. Dis., 1920, vol. xxvi, p. 38$, Addi-
tional literature given .in these papers.
32
498 APPLICATION OF METHODS OF BACTERIOLOGY
properties, producing various shades of yellow- and orange-
colored pigment, while others grow with a pink color.
The occurrence of bacterium pseudodiphthetiticum in
pure culture in superficial abrasions showing a slight ten-
dency to suppuration; the fact that these organisms, when
injected into the peritoneal cavity of guinea-pigs, produce
purulent peritonitis; that such organisms are frequently
encountered in vaccine virus and in the pus of vaccination
wounds; and that frequently in cases of mastitis in cows
such organisms occur in large numbers in pure culture has
led to the supposition that this group of organisms was
probably responsible for suppurations occurring under
certain special conditions. With these facts in mind speci-
mens of pus were derived from thirty cases with suppurating
wounds in the University of Pennsylvania Hospital, and
careful bacteriological examination of these specimens
showed the presence of bacterium pseudodiphtheriticum in
43 per cent, of the cases. These organisms were always
found in conjunction with one or more of the group of pyo-
genic organisms, and it is impossible to state how much
of the effect was due to any one of the organisms present.
It seems probable, however, in the light of what has been
said, that these bacteria were present not merely as acci-
dental invaders, but that in some way they contributed
toward the results.
The fact that some of the organisms isolated from the
pus, when inoculated into the peritoneal cavity of guinea-
pigs, show distinct pyogenic properties gives strong sup-
port to the opinion that this group is of greater importance
than was heretofore supposed. Repeated passage through
guinea-pigs serves to so increase the pathogenic properties
of these organisms that they cause theMeath of the animal
BACTERIUM XEROSIS 499
in less than twenty-four hours with marked inflammatory
reaction affecting the peritoneum as well as the abdominal
organs.
The morphologic and biologic characters of some members
of the group of bacterium pseudodiphtheriticum are sug-
gestive of those of bacterium diphtherise. Other members
of the group, however, are readily differentiated from bac-
terium diphtherias by either the morphologic or the biologic
characters, or by both. Many of the members of the group
produce very little acid when grown in carbohydrate media,
and the slight degree of acidity produced is frequently
obliterated by a marked degree of subsequent alkali
production. This fact is of special value in the differentia-
tion from bacterium diphtherise.
BACTERIUM XEROSIS (NEISSER AND KUSCHBERT),
MIGULA, 1900.
SYNONYM: Bacillus xerosis, Neisser and Kuschbert, 1883.
Another organism which is also related in its morphologic
and biologic characters to bacterium diphtherise is bacterium
xerosis, first encountered by Kuschbert and Neisser in xerosis
of the conjunctiva, and which has since been found on the
conjunctiva by a number of investigators, in various diseases
as well as in health.
The xerosis bacteria are less likely to be mistaken for
bacterium diphtherias because they are somewhat smaller
and have less tendency to show multiple striations. Usually
they stain deeply at the poles with only one clear unstained
band in the center. It is only occasionally that a few striated
organisms are encountered in a culture.
500 APPLICATION OF METHODS QF BACTERIOLOGY
Biologically bacterium xerosis is readily differentiated
from bacterium diphtherise because of the scant growth
that takes place on the ordinary culture media. On agar-
agar the growth appears as small transparent colonies which
have little tendency to coalesce. On gelatin the growth
is slow, and frequently shows as minute, isolated colonies
along the needle track. In litmus-milk a slight degree of
acidity is produced. In bouillon the growth is so slight as
to leave the medium practically unaltered. The growth
on potato is slight and invisible.
Differentiation of Members of the Group. — Knapp1 claims
that a positive differentiation of the organisms may be
made by merely inoculating the Hiss media containing dex-
trin and saccharose. If the dextrin is alone fermented, the
organism is bacterium diphtheriae, if only the saccharose is
fermented, the organism is bacterium xerosis, and if neither
of these carbohydrates is fermented, the organism is bac-
terium pseudodiphtheriticum.
Through the suggestion of Neisser2 we are assisted in
differentiating between bacillus diphtherise and the confusing
forms. He has found that by the use of a particular staining
method the appearance of bacterium diphtherise is charac-
teristic. His differential method comprehends the following
manipulations: the culture to be tested should be grown
upon Loffler's blood serum mixture solidified at 100° C.;
it should develop at a temperature not lower than 34° C.
and not higher than 36° C.; and it should not be younger
than nine and not older than twenty-four hours. A cover-
glass preparation made from such a culture is stained as
follows :
1 Jour. Med. Research, 1904, xii, 475.
2 Zeitschrift fur Hygiene und Infektionskrankheiten, 1897, Bd. xxiv.
BACTERIUM XEROSIS 501
(a) It is subjected to the following mixture for from one
to three seconds:
Methylene-blue (Griibler's) 1 gram
Alcohol (96 per cent.) . .*»,./. • >t • • • 20 c.c.
When dissolved, mix with
Acetic acid '.'..,. \.. . . . .50 c.c.
Distilled water ..'.'. . . 950 c.c.
(6) After thoroughly rinsing in water, it is stained for
from three to five seconds in vesuvin (Bismarck-brown),
2 grams, dissolved in 1 liter of boiling distilled water,
filtered, ond allowed to cool. It is again rinsed in water
and examined as a water-mount, or it may be dried and
mounted in balsam.
When so treated the diphtheria bacterium appears as
faintly stained brown rods, in which from one to three
dark-blue granules are to be observed. The dark granules
are at one or both poles of the cell, are more or less oval,
and usually seem to bulge a little beyond the contour of the
bacterium in which they are located. (See Fig. 83.) From
Neisser's observations and those of others,1 as well as from
personal experience, it seems safe in the vast majority of
cases to regard all bacteria that do not stain in the way
described as distinct from bacterium diphtherise.
Blumenthal and Lipskerow2 decide that the differential
method which yields the most satisfactory results consists
in the fixation of the preparation for from one-half to two
minutes in the following solutions: Pyoktanin (Merck)
0.25 grams, acetic acid (5 per cent.) 100 c.c.; washing in
1 Frankel, Berliner klin. Wochenschrift, 1897, No. 50. Bergey, Publica-
tions of the University of Pennsylvania, New Series, 1898, No. 4.
2 Centralblatt f. Bacteriologie, Bd. xxxviii, p. 359.
502 APPLICATION OF METHODS OF BACTERIOLOGY
water and counterstaining with a 1 to 1000 solution of
vesuvin for one-half minute. By this method the polar
granules of bacterium diphtherias are stained bluish black,
are large, and may be seen in almost all of the organisms.
The contour of the darkly stained bacterium diphtherias
is sharply defined, and it is very easily differentiated from
any other organisms that may be present in the preparation.
FIG. 83
Bacterium diphtherias, stained by Neisser's method.
NOTE. — Prepare cover-slip preparations from the mouth-
cavities of healthy individuals and from those having decayed
teeth. Do they correspond in any way with those made
from diphtheria? Do the same with different forms of
sore-throat. Do the peculiarities of any of the organisms
suggest those of bacterium diphtherise? Wherein is the
difference ?
In cultures and cover-slips made from both diphtheritic
BACTERIUM XEROSIS 503
and from innocent sore-throats are any organisms almost
constantly present? Which are they, and what are their
characteristics?
Which are the predominating organisms in the anginas
of scarlet fever?
Do these organisms simulate, in their cultural and mor-
phological peculiarities, any of the different species with
which you have been working?
Do the diphtheria organisms disappear from the throat
with the disappearance of the membrane? How long do
they persist? When obtained from the throats of convales-
cents are they still pathogenic for guinea-pigs ?
Prepare a bouillon culture of virulent bacillus diphtherise;
after it has been growing for thirty-six hours at 37°-38° C.
inoculate a guinea-pig subcutaneously with about 0.1 c.c.
of it. If the animal dies, note carefully the findings at
autopsy, especially the distribution of the bacilli. Now add
to this culture sufficient pure carbolic acid or trikresol to
kill all bacteria in it, and inject under the skin of another
guinea-pig varying amounts of the culture so treated,
beginning with 0.05 c.c. ; determine the minimum fatal dose,
and note in which respects the postmortem findings simulate
and in which they differ from those of the first animal.
Should any of the animals survive the injections of the
disinfected culture, note carefully their condition from day
to day, particularly any fluctuations in weight. When
they have quite recovered inoculate them with living,
virulent diphtheria organisms. Do the results correspond
with those obtained with guinea-pigs that have never been
treated at all? Explain the results.
Diphtheria Antitoxin. — As stated above, the growth of
bacterium diphtherise is accompanied by the elaboration
504 APPLICATION OF METHODS OF BACTERIOLOGY
of a poison of remarkable toxicity that is accountable for
the constitutional symptoms and pathological lesions by
which the disease is characterized. If by appropriate
methods this poison (toxin) be separated from the bacteria
by which it was formed, it is capable, when injected into
susceptible animals, of causing death and practically all the
lesions that accompany the disease when due to the invasion
of the living bacteria. If, on the contrary, the dose of poison
be so adjusted as to cause only temporary inconvenience
and not endanger life, and this dose be injected repeatedly,
gradually increasing in size as the animal is able to bear
it, after a while a marked tolerance is established, so that
the animal may be given many times the amount of the
toxin that would otherwise prove fatal — i. e., many times
the lethal dose for an animal that had not acquired such a
tolerance.
If blood be now drawn from the animal that has become
habituated, so to speak, to the diphtheria toxin, and the
serum collected from it, we discover several important
facts, viz.:
That this serum when mixed with the previously deter-
mined lethal dose of the toxin in a test-tube will either
neutralize its toxicity or greatly reduce it, according to the
amount of serum used.
That if we inject into an animal the determined fatal
dose of the toxin, and immediately afterward inject a quan-
tity of the .serum, either the animal will not die or the death
will be more or less delayed, according to the amount of
serum employed.
That if a susceptible animal be inoculated with a living
culture of virulent bacterium diphtheriae, its life may be
saved, or its death postponed, by the subsequent injection
BACTERIUM XEROSIS 505
of the serum; the result depending upon the amount of
serum used and the lapse of time between inoculation with
the bacteria and injection of the serum.
And, finally, that although this serum has such a marked
effect upon the toxins of bacterium diphtherise in a test-
tube or in the animal, and so striking an influence upon
the course of infection with the living organisms in the
animal, it has little or no effect upon the living bacteria
either in a test-tube or at the site of inoculation in the
living animal body.
This serum with which we have been experimenting is
the so-called "diphtheria antitoxin" or " antidiphtheritic
serum."
For practical purposes, it is obtained from horses, the
animals being treated with gradually increasing doses of
diphtheria toxin until they are able to withstand enormous
multiples of the ordinarily fatal dose. When this point is
reached, the protective body — the antitoxin — is present in
the blood in such large quantities that the serum may be
successfully employed in the treatment of diphtheria in
human beings — i. e., as an antidote to the diphtheria toxin
that is produced by the growing bacteria in the throat, or
elsewhere, and distributed through the body by the cir-
culating fluids.
The Standardization of Diphtheria Antitoxin. — The value
of diphtheria antitoxin may be determined according to
several different standards. Those that are best known
have been proposed by Behring and by Ehrlich.
1. Behring' s Method. — He designates as a "normal" poison
a toxin of which 0.01 c.c. suffices to kill a guinea-pig weigh-
ing 250 grams in four days. Of such a normal diphtheria
toxin 1 c.c. will be sufficient to kill 100 guinea-pigs weigh-
506 APPLICATION OF METHODS OF BACTERIOLOGY
ing 250 grams each, or 25,000 grams in weight of guinea-
pigs.
The quantity of antitoxin that is required to just protect
25,000 grams weight of guinea-pigs from the minimum fatal
dose of the toxin is called one immunizing unit. If an
immune serum contains in 1 c.c. one immunizing unit, it
represents a "normal" antitoxin.
To determine the strength of an immune serum, 1 c.c. of
normal toxin is mixed with increasing quantities of the
serum, and these mixtures are injected subcutaneously into
guinea-pigs; the quantity of the serum which suffices to
neutralize that amount of normal toxin — i. e., that keeps the
animal alive for four days or longer — contains one immunizing
unit.
2. Ehrlich's Method. — Ehrlich introduced the use of a
standard diphtheria antitoxin in a dry state which contains
1700 immunizing units in each gram. This standard anti-
toxin, distributed by the Institute for testing serum at
Frankfort-on-the-Main, is now being used in a great many
places for the standardization of diphtheria antitoxin. A
test toxin is prepared, corresponding to this standard anti-
toxin, and with this toxin the strength of the unknown serum
is titrated.
If, for instance, the test toxin is of such a strength that
0.003 c.c. represents the minimum fatal dose for a guinea-
pig of 250 grams, then 0.3 c.c. would represent 100 times
the minimum fatal dose of toxin, and, according to Ehrlich's
standard, an immunity unit is that amount of antitoxic
serum which will neutralize 100 times the minimum fatal
dose of toxin. In performing the test to estimate the
strength of an antitoxic serum, the antitoxin is diluted
with sterile water in varying proportions, and a series of
BACTERIUM XEROSIS 507
guinea-pigs are injected with mixtures of 100 times the
minimum fatal dose of the toxin and varying quantities of
the diluted antitoxic serum. For this purpose guinea-pigs
of approximately 250 grams weight are employed. If, for
instance, a guinea-pig receiving 100 times the minimum fatal
dose of toxin, and 0.1 c.c. of the diluted antitoxic serum,
survives for four days, then 0.1 c.c. of the serum represents
an immunity unit of antitoxin.
An antitoxic serum of this strength, therefore, contains
10 times the normal amount of antitoxin, because it con-
tains the immunity unit in only 0.1 c.c.; a normal anti-
toxin being one in which an immunity unit is contained in
one cubic centimeter. Antitoxic serums are frequently of
such high degree of potency that they contain from 800
to 1000 immunity units in each cubic centimeter.
CHAPTER XXIV.
Typhoid Fever — Study of the Organism Concerned in its Production —
Its Morphological, Cultural, and Pathogenic Properties — Bacillus Coli
— Bacillus Paratyphosus — Its Resemblance to Bacillus Typhosus.
BACILLUS TYPHOSUS.
THE organism seen in the cadavers of typhoid subjects
by Eberth (1880-81), and subsequently isolated in pure
culture and described by Gaffky (1884), is generally recog-
nized as the exciting factor of typhoid fever. It may be
described as follows:
FIG. 84
FIG. 85
Bacillus typhosus, from cultures
twenty-four hours old, on agar-agar,
Bacillus typhosus, showing
flagella stained by Loffler's
method.
Morphology. — It -is a bacillus about three times as long
as broad, with rounded ends. It may appear at one time
as very short ovals, at another time as long threads, and
both forms may occur together. Its breadth remains toler-
(508)
BACILLUS TYPHOSUS 509
ably constant. Its morphology presents little that will
aid in its identification. (See Fig. 84.) It is actively motile,
and when stained by special methods, is seen to possess very
delicate locomotive organs in the form of fine, hair-like
flagella, attached in large numbers to all parts of its surface.
(See Fig. 85.) These flagella are not seen in unstained
preparations, nor are they rendered visible by ordinary
methods of staining. (See methods for staining flagella.)
Owing to a tendency to retraction of its protoplasm from
the cell-envelope and the consequent production of vacuoles
in the bacilli, the staining of this organism is frequently
FIG. 86
Diagrammatic representation of retraction of protoplasm, with production
of pale points, in bacillus typhosus.
more or less irregular. At some points in a single cell marked
differences in the intensity of the staining will be seen, and
here and there areas quite free from color can commonly
be detected. These colorless portions are often so sharply
defined that they look as if they had been punched out
with a sharp instrument. (See Fig. 86.)
It does not form spores.
Gelatin Plates. — Its growth, when seen in the depths of
the medium, presents nothing characteristic, appearing
simply as round or oval, finely granular points. On the
surface it develops as very superficial, blue-white colonies,
with irregular borders. They are a little denser at the
510 APPLICATION OF METHODS OF BACTERIOLOGY
center than at the periphery. When magnified, the colonies
present wrinkles or folds, which give to them, in miniature,
the appearance seen in relief maps (Fig. 87). These colonies
have sometimes the appearance of flattened pellicles of glass-
wool, and usually a pearl-like luster.
Agar-agar. — On agar-agar the colonies present nothing
typical.
Stab-cultures. — In stab-cultures the growth is mostly on
the surface, there being only a very limited development
down the track made by the needle. The surface growth
has the same appearance in general as that given for the
colonies.
FIG. 87
Colony of bacillus typhosus on gelatin,
Potato. — The growth on potato is usually described as
luxuriant but invisible, making its presence evident only
by the production of a slight increase of moisture at the
inoculated point, and by a limited resistance offered to a
needle when it is scraped across the track of growth. While
this is so in many cases, yet it cannot be considered as
invariable, for at times this organism develops more or less
visibly on potato.
Potato-gelatin. — The growth is similar to that upon
ordinary nutrient gelatin.
Milk. — It does not cause coagulation when grown in
sterilized milk.
BACILLUS TYPHOSUS 511
Bouillon. — It causes uniform clouding of the bouillon and
brings about a slightly acid reaction.
Indol Formation. — It is customary to regard this organism
as devoid of the power to form indol; in fact, this has hitherto
been considered one of its important differential peculi-
arities, and by the usual methods of cultivation and test-
ing the indol reaction is not observed in cultures. It has
been shown, however, by Peckham, that by repeated
transplantation, at short intervals, into either Dunham's
peptone solution, or, preferably, a freshly prepared alkali-
tryptone solution, made from tryptonized beef-muscle, that
the indol-producing function may be induced in the genuine
typhoid bacillus obtained directly from the spleens of
typhoid cadavers.1
It does not produce gaseous fermentation. On lactose-
litmus-agar-agar it grows as pale-blue colonies, causing no
reddening of the surrounding medium; though if glucose
be substituted for lactose, both the colonies and the sur-
rounding medium may become red. In the fermentation-
tube, in glucose or lactose bouillon, no evolution of gas
as a result of fermentation occurs.
It grows at any temperature between 20° and 38° C.,
though more favorably at the latter point. It is very sen-
sitive to high temperatures, being killed by an exposure of
ten minutes to 60° C., and in a much shorter time to slightly
higher temperatures.
It does not liquefy gelatin.
It grows both with and without oxygen.
It does not grow rapidly.
1 A. W. Peckham, The Influence of Environment Upon the Biological
Functions of the Colon Group of Bacilli, Journal of Experimental Medi-
cine, 1897, vol. ii.
512 APPLICATION OF METHODS OF BACTERIOLOGY
Presence in Tissues. — In patients suffering from typhoid
fever the organism has been found during life by the appli-
cation of appropriate culture methods in the blood, urine,
and feces, and at autopsies in the tissues of the spleen, liver,
kidneys, intestinal lymphatic glands, and intestines. It is
not easy to demonstrate this organism in tissues unless it is
present in large numbers. The manipulations to which
the sections are subjected in being mounted often rob the
bacilli of their stain, and render them invisible, or nearly so.
If, however, sections be stained in the carbol-fuchsin or the
alkaline methylene-blue solution, either at the ordinary
temperature of the room or at a higher temperature (40° to
45° C.), then washed in absolute alcohol, and cleared in
xylol1 and mounted in xylol balsam, the bacilli (particu-
larly if the tissues be the liver and spleen) can readily be
detected, massed together in clumps.
In searching for the typhoid bacilli in tissues this peculiar
disposition in clumps must always be borne in mind, other-
wise much labor will be expended in vain. In tissues the
typhoid bacilli are not scattered about as are the organ-
isms in certain other conditions — septicemia, for instance;
they are not regularly distributed along the course of the
lymphatics or capillaries, but appear in small masses through
the organs, and it is for these agglutinations that one should
search. This peculiar clumping of the typhoid bacilli in
the tissues cannot be satisfactorily explained, unless it be
due to the specific agglutinating influence that typhoid
blood has upon the typhoid bacillus, a phenomenon that
is readily demonstrable in the test-tube or under the micro-
scope. In other words, may it not be simply the result of an
intracapillary "Widal reaction"? (See Widal Reaction.)
1 Do not clarify with oil of cloves. It is too active as a decolorizer.
BACILLUS TYPHOSUS 513
When the section is prepared for examination, if it be gone
over with a low-power objective, one will notice at irregular
intervals little masses that look in every respect like par-
ticles of staining-matter which have been precipitated upon
the section at that point. When these masses are examined
with a higher power objective they will be found to consist
of small ovals or short rods so closely packed that the indi-
viduals composing the clump can often be seen only at the
extreme periphery of the mass. This is the characteristic
appearance of the typhoid organism in tissues, to which
allusion has just been made. The little masses are usually
in the neighborhood of a capillary.
Isolation of Bacillus Typhosus from Cadavers. — The spleen
of a patient dead of typhoid fever is the most reliable source
from which to obtain cultures of the typhoid bacillus for
study. But it must always be remembered that the same
channels through which the typhoid bacillus gains access
to this viscus are likewise open to other organisms present
in the intestines, and for this reason bacillus coli, a normal
inhabitant of the colon, may also be found in this locality.
Result of Inoculation into Lower Animals. — A great many
experiments have been made in a variety of ways with the
view of reproducing the pathological conditions of this
disease, as seen in man, in the tissues of lower animals, but
with practically no success. From the time of its discovery
up to within a comparatively recent date there was an almost
continuous controversy concerning the infective properties
of bacillus typhosus for animals. By some it was held that
the effects of its introduction into animals were manifestly
of toxic1 origin,' while others regarded them as evidences of
1 Toxic — poisonous results not necessarily accompanied by the growth
of organisms throughout the tissues.
33
514 APPLICATION OF METHODS OF BACTERIOLOGY
genuine infection.1 These diversities of opinion are hardly
surprising when we remember that animals never suffer
naturally from typhoid fever, and therefore offer many
obstacles to its faithful reproduction, and that the vigor of
this organism when cultivated from various sources is liable
to a wide range of fluctuation. Numerous investigations
lead to the belief that the poison peculiar to this organism
is so intimately bound up with its protoplasmic structure
as to make its separation difficult, if not impossible. How-
ever, by the use of dead cultures (i. e., cultures of well
developed organisms destroyed by heat) results are obtained
that leave no doubt that the clinical symptoms and patho-
logical changes seen in man and in animals under experiment
are referable to a specific intoxication, and, as a rule, the
only effects that follow the introduction of this organism
into animals are referable to the intoxicating action of the
materials used. In fact, the results of modern investigations
have placed bacillus typhosus in the category of endotoxin
producers, and through the use of the toxins (not pure, but
mixed with other substances in the culture media) produced
by it animals have been rendered immune from otherwise
fatal doses of highly toxic cultures. The serum of such
animals has also been shown to possess a certain degree of
immunizing power.2
Because of the variations in the morphology and physiology
of this organism, and because of the difficulty experienced
in efforts to reproduce in lower animals the condition
found in the human subject, our knowledge of typhoid
fever, though fairly accurate in many respects, is, never-
1 Infective or septic — poisoning of the tissues as a result of the growth
of bacteria within them.
2 Pfeiffer and Kolle, Zeitschrift fur Hygiene und Infektionskrankheiten
1896, Bd. xxi, S. 208.
BACILLUS TYPHOSUS 515
theless, in certain essential details relating to its causation,
very far from satisfying.
A number of other organisms appear botanically to be
closely related to the typhoid bacillus, and under the avail-
able culture methods for studying them they so closely
simulate it that the difficulty of identifying this organism
is sometimes very great. In addition the variability con-
stantly seen in pure cultures of the typhoid bacillus itself
in no way renders the task more simple.
For example, the morphology of the typhoid bacillus
is conspicuously inconstant; its growth on potato which was
formerly considered unique, may, with the same stock, at
one time be the typical invisible development, at another
it is easily to be seen with the naked eye; and the change of
reaction which it is said to produce in bouillon is sometimes
much more intense than at others. The indol-producing
function, hitherto regarded as absent from this organism,
is now known to be occasionally demonstrable by ordinary
methods, and frequently by special methods of cultivation
(Peckham, /. c.). The only properties exhibited by it under
the usual conditions of cultivation that may be said to be
constant are its motility; its inability to cause gaseous fer-
mentation of glucose, lactose, or saccharose; its incapacity
for coagulating milk; and its growth on gelatin plates
but there are other bacilli which possess these same character-
istics to a degree that renders their differentiation from
the typhoid organism often a matter that requires the careful
application of all the different tests.
The Agglutination Reaction. — The nearest approach to a
trustworthy means of identification is the specific reaction of
typhoid bacilli with the blood of typhoid subjects. When
typhoid bacilli are brought in contact with the blood serum
516 APPLICATION OF METHODS OF BACTERIOLOGY
from human beings sick of typhoid fever, or from animals
that have survived inoculation with cultures of this organ-
ism, there occurs a peculiar alteration in the relation of the
organisms to one another in the fluid. As ordinarily seen
in a hanging drop of bouillon, the typhoid bacilli appear as
single, actively motile cells; when to such a drop a little
dilute serum from a case of typhoid fever is added the motility
of the bacteria gradually lessens and finally ceases, and they
then congregate, "agglutinate" in larger and smaller clumps,
or if one add to 4 or 5 c.c. of a twenty-four-hour-old bouillon
culture of typhoid bacilli in a narrow test-tube about eight
drops of serum from a case of typhoid fever and maintain
this mixture at body temperature the normally clouded
culture will be seen after a few hours to have undergone a
change; instead of a diffuse clouding it is clear and floccu-
lent masses of the bacteria that have agglutinated together
as a result of the specific action of the serum used will be
scattered about in it.
For the hanging-drop test, sufficient serum may be
obtained from a needle-prick in the finger, while for the
test-tube reaction a larger amount is needed; this may be
obtained from blood drawn from a superficial vein by means
of a hypodermic syringe, or from the cleansed skin by a wet-
cup, or, better still, from a small cantharides or ammonia
blister.
It is proper to state, however, that occasionally cultures
of genuine typhoid bacilli are encountered that do not
respond to this peculiar influence of typhoid blood, even
though the blood be tested at different stages of the disease,
and even though it may cause the characteristic cessation
of motion and clumping with other cultures of this organism
upon which it is tried.
BACILLUS TYPHOSUS 517
"Widal's Reaction." — When employed conversely — i. e.,
for deciding if the serum used is from a case of typhoid fever
or not — the reaction constitutes " Widal's serum diagnosis
of typhoid fever." In beginning these tests it is often neces-
sary to try several cultures of genuine typhoid bacilli from
different sources and of varying degrees of vitality, before
a strain is procured that reacts conspicuously and quickly
with genuine typhoid serum.
WIDAL'S REACTION WITH DRIED BLOOD. — This reaction
can also be obtained with redissolved dried blood — i. e.,
by the Johnston method: a drop of the blood to be tested,
obtained by a needle-prick in the cleansed ringer or lobe of
the ear, is collected on a bit of clean, unglazed paper and
allowed to dry. The paper is then folded, kept free from
contamination, and taken to the laboratory. With a
medium-size platinum-wire loop a drop of sterile bouillon,
water, or physiological salt solution is gently rubbed upon
the loop of dried blood until the contents of the loop are of
a dark amber color; this is then mixed with a drop of
bouillon culture of typhoid bacilli on a cover-glass, which is
mounted upon the hollow-ground slide as a hanging drop,
when the effect of the diluted blood upon the culture can
be observed with the microscope. The reaction, if positive,
should occur within a half hour. Many object to this
method because it is impossible accurately to dilute the
blood by the plan used. A number of tests have shown us
that preparations made in this way correspond roughly
with a fresh-blood dilution of from 1 : 15 to 1 : 20, as deter-
mined by the hemoglobinometer. In a small number of
cases in which parallel tests were made with this and with
fresh fluid serum the results were concordant. We are
inclined to the opinion, however, that in doubtful cases, in
518 APPLICATION OF METHODS OF BACTERIOLOGY
which all the available clinical evidence is opposed to either
the positive or negative results of the test, the difficulty is
much more certainly cleared away by the use of highly dilute
and exactly diluted fresh serum than by this method. Com-
petent observers are of the opinion that in all such cases
the quantity of serum in the hanging drop should be
decreased until it is present in the proportion of from 1 : 50
to 1 : 60, and that, if after exposure to this dilution for two
hours the bacilli are still motile and not clumped together
or the reaction is deficient in only one or the other of these
peculiarities, the case from which the serum was obtained
may be safely regarded as not typhoid fever, or if typhoid
the examination was not made at a time when agglutinin
was present in demonstrable quantities in the circulating
blood.
Experience with both the dry-blood and the fresh serum
methods show the culture used to be one of the most impor-
tant factors in the test. After deciding upon the most
suitable culture for the reaction — and it is often necessary
to try a great number from various sources — it should be
transplanted daily into fresh bouillon and kept at a tem-
perature rarely above 20° or 22° C. The bacilli grown
under these circumstances are usually somewhat longer
than when cultivated at higher . temperature, and they
exhibit a regular, gliding motility that renders it more easy
to follow the individual cells under the microscope than
when they possess the usual active, darting motion.
In a group of cases examined by us by the dry-blood
method, including typhoid and other febrile conditions
there was a discrepancy between the clinical and the labora-
tory diagnosis in from 2 to 3 per cent, of the cases examined.
In the hands of all who have carefully employed the
BACILLUS TYPHOSUS 519
Widal reaction for the diagnosis of typhoid fever the results
are reported to have been almost uniformly satisfactory. In
the great majority of cases the reaction is, so far as experi-
ence indicates, specific — i. e., a typical reaction does not
occur between typhoid serum or blood and organisms other
than the typhoid bacillus, nor between the typhoid bacillus
and serums other than those from cases of typhoid fever.
There are, however, confusing reactions — so-called pseudo-
reactions — in which more or less clumping of the bacilli
and a diminution of motion, without complete cessation,
are observed. These reactions have been seen to occur
with normal blood and with blood from other febrile con-
ditions. It is said by Johnston and McTaggart1 that they
can be prevented if cultures of just the proper degree of vitality
are employed; and this corresponds with the results of a
fairly wide personal experience with the test.
In the light of present experience it is fair presumptive
evidence that the serum is from a case of typhoid fever
when unmistakable agglutination and cessation of motion
are seen in from fifteen to twenty minutes after typhoid
bacilli are mixed with the serum of a conspicuous febrile
condition.
The blood of certain animals, as well as a number of
chemical substances, such as corrosive sublimate, alcohol,
salicylic acid, resorcin, and safranin in high dilution, cause
agglutination of the typhoid bacilli; but the reaction is
not specific, for in most cases they have the same effect on
other motile bacilli.
Drinking Water. — All the points with regard to morpho-
logic and biologic characters of bacillus typhosus, and of
the organisms closely resembling it, should be borne in
1 Montreal Medical Journal, March, 1897.
520 APPLICATION OF METHODS OF BACTERIOLOGY
mind in the examination of drinking-water supposed to be
contaminated by typhoid dejections, for the organisms which
most closely approach the typhoid bacillus in growth and
morphology are just those organisms which would appear
in water contaminated from cesspools — i. e., the organisms
constantly found in the normal intestinal tract. Even in
the stools of typhoid-fever patients the presence of these
normal inhabitants of the intestinal tract renders the isola-
tion of the typhoid organisms somewhat troublesome.
Methods of Isolating the Typhoid Bacillus. — From the fore-
going it is obvious that bacillus typhosus is so variable in
many of its biological peculiarities, and is so closely simu-
lated in certain respects by a group of other organisms to
which it appears to be botanically related, that its identi-
fication, especially outside the infected body, is a matter of
considerable difficulty and uncertainty. For these reasons
many efforts have been made to discover specific cultural
reactions for the organism, and with this end in view many
methods have been devised for its isolation from water,
feces, sewage, and other matters believed to contain it.
None of them, however, have given general satisfaction,
and many have proved wholly untrustworthy.
In deciding upon a suitable routine these are several points
that should be borne in mind :
(a) As bacillus typhosus when present in water, feces,
soil, milk, etc., is always numerically in the minority, as
compared with other organisms, it is desirable to employ
a method that will encourage its multiplication without
at the same time favoring the same rate of multiplication
by other organisms present, that is to say, to use an " enrich-
ing medium;" (6) and to possess a method that will make
comparatively simple the isolation or separation of the
BACILLUS TYPHOSUS 521
typhoid bacilli, after "enrichment," from the other organ-
isms with which it is associated. With these objects in
mind a routine that gives very general satisfaction is as
follows :
Enriching Media. — For this purpose ox bile and " brilliant
green" have been found to favor the growth of typhoid
bacilli, and to be less favorable to the growth of other
organisms associated with it; consequently if a bit of
typhoid feces or a portion of infected water or milk be
mixed with either of these media and kept at suitable tem-
perature for a time, the result will be a more conspicuous
growth of bacillus typhosus than of the other organisms.
Two forms of ox bile may be employed:
(1) Pure fresh bile direct from the gall-bladder of a freshly-
slaughtered ox, or (2) a solution of peptone and dried ox
bile of the following proportions:
Dried ox bile . . . . . . . . . . .10 parts
Peptone 1 part
Water 100 parts
In either event convenient amounts are placed in test-
tubes and sterilized; after which they are ready for inocu-
lation with the mixture suspected of containing the typhoid
bacillus. After inoculation they are kept at body tem-
perature for about twenty-four hours, when plates may be
made with the differential media to be described below.
Instead of the ox bile the aniline dye known as "brilliant
green" may be employed. This substance suppresses to
some extent the growth of organisms other than bacillus
typhosus, particularly those of the colon group. It is used
in the following manner: To test-tubes containing a known
amount (8 to 10 c.c.) of peptone solution, "brilliant green" is
522 APPLICATION OF METHODS OF BACTERIOLOGY
added in varying amounts so as to have a series of solutions
ranging in strength from one part of the green to 500,000, to
one part to 100,000 of the peptone solution. A convenient
stock solution of the "brilliant green" is 1 : 1000 in water.
From this such amounts are added to the tubes of peptone
solution as will give the desired series of dilutions. The
tubes of peptone solution should have been sterilized before
the green is added. When ready, one adds to each of these
tubes an amount of the substance under consideration:
if it be feces — a moderate loopful may be broken up in 1 c.c.
of bouillon and one or two loopfuls of this used; if it be water
or milk from 0.1 to 0.3 c.c. The amount best suited must
be determined by experiment.
When inoculated the tubes are kept at body temperature
for from eighteen to twenty-four hours, when they are
ready for the "differential" or "selective" plating.
The enriching media should be free of sugar.
In the process of plating, specially prepared selective
media are used that aim to render evident to the naked eye
distinguishing differences between the colonies of bacillus
typhosus and those of other confusing organisms, Of a
number of special media employed for this purpose two have
proved very satisfactory — notably that recommended by
Drigalski and Conradi, and that by Endo.
METHOD OF v. DRIGALSKI AND CoNRADi.1 — This method
aims to separate bacillus typhosus from bacillus coli on the
basis of their fermenting properties, in such a manner as not
to hinder the growth of bacillus typhosus, but rather to
make the conditions for its growth as favorable as possible.
The authors give the following directions for the prepara-
tion of their culture medium :
1 Zeitschrift fur Hygiene, 1902, Bd. xxxix, p. 288.
BACILLUS TYPHOSUS 523
a. Preparation of agar: 1500 grams of finely chopped beef
are placed in two liters of water and set aside for twenty-
four hours. This meat infusion is then boiled for one hour,
filtered, and 20 grams of Witte's peptone, 20 grams of
nutrose, and 10 grams of sodium chloride are added and
again boiled for an hour, filtered, and 60 grams of agar-agar
are added, boiled for three hours (or one hour in the auto-
clave), rendered slightly alkaline to litmus paper, filtered,
and boiled for one-half hour.
b. Litmus solution: (Litmus solution according to Kubel
and Tiemann) 260 c.c., boil ten minutes, add 30 grams
chemically pure lactose, boil fifteen minutes.
c. The hot litmus-lactose solution is added to the hot
nutritive agar, thoroughly mixed, and the alkaline reaction
is again restored. To this medium is then added 4 c.c. of
a hot sterile solution of 10 per cent, water-free sodium
carbonate, 20 c.c. of freshly prepared solution of 0.1 gram
crystal violet (Hochst) in 100 c.c. of warm sterile distilled
water.
One now has a meat-infusion-peptone-nutrose-agar with
13 per cent, of litmus solution and 0.01 per thousand crys-
tal violet. It becomes very hard on solidifying, without
becoming too dry. Plates are poured of this material and
held in readiness for some time, and the remainder of the
medium is preserved in flasks in portions of 200 c.c. each.
If the lactose is boiled for a longer time than directed
it is reduced, with an acid reaction of the culture medium,
and the content in lactose falls below the required quantity,
and the alteration in the color of the colon colonies appears
too early. For this reason it is also necessary to liquefy
the agar as quickly as possible in pouring plates from the
agar medium stored in flasks.
524 APPLICATION OF METHODS OF BACTERIOLOGY
In employing this culture medium it is necessary to have
a uniform suspension of a portion of the material to be
examined and to make a series of plate inoculations from
this suspension by smearing carefully the material under
consideration over the surface of the medium in the plates, a
sterile platinum spatula or a sterile bent glass rod being used
for the purpose.
After fourteen to sixteen hours at 37° C., and still better
after twenty to twenty-four hours, the cultures are readily
differentiated :
a. Bacillus Coli: All cultures of true colon that have been
examined form colonies of 2 to 6 or more millimeters in
diameter, of reddish color and translucent. In each intes-
tinal evacuation there are usually several varieties of
colon colonies which differ according to their size and tex-
ture, translucency, and- the intensity of the alteration of
the color which they bring about. Many colon colonies
are bright red, some are cloudy, and others are quite opaque,
dark-wine red in color, while still others form large colonies
which are surrounded by a red halo.
b. Bacillus Typhosus: The colonies have a diameter of
1 to 3 millimeters, rarely larger. Their color is blue, with
a tendency toward violet. In structure they are glistening,
with a single contour, somewhat of the nature of a dew drop.
Only in isolated instances is the colony larger and more
cloudy in appearance.
The Endo Media. — (Modification of Kendall and Day.)
Prepare the following :
(a) Water 1000 c.c.
Powdered agar-agar 15 grams
Peptone (Witte) 10 grams
Meat Extract (Liebig) 3 grams
BACILLUS TYPHOSUS 525
Heat until the agar-agar is dissolved, keeping the mass to
100 c.c. volume by addition of water. This should require
about an hour over the flame, or less if the mass be dissolved
in the autoclave. Render just alkaline to litmus by the
addition of decinormal sodium hydroxide solution. Filter
and decant to flasks containing 100 c.c. each. Sterilize.
(b) Prepare a 10 per cent, solution of fuchsin in 96 per
cent, alcohol.
(c) Prepare a 10 per cent, solution of sodium sulphite in
water.
For the making of the plates mix 1 c.c. of (b) with 10 c.c.
of (c) and heat in the steam sterilizer (100° C.) for 20 minutes.
This decolorizes the fuchsin. To each 100 c.c. of the agar
prepared as (a), add 1 per cent, of chemically pure lactose
and heat in the steam sterilizer at 100° C. until the agar-
agar is completely liquefied and the lactose dissolved. To
each 100 c.c. of this lactose-agar add 1 c.c. of the decolorized
fuchsin solution, mix thoroughly and while still fluid and
warm, pour into sterile Petri dishes; sufficient in each dish
to give a layer of from 3 to 5 mm. depth. Place these dishes,
with the covers removed, in the incubator until the agar-
agar has set; this will require about 30 minutes. They are
then ready for inoculation. The plates are now inoculated
by spreading evenly over the surface small quantities from
the primary "enriching" cultures. This is best done by
the use of a bent glass rod that has been sterilized in the
flame and allowed to cool.
If typhoid bacilli be present they develop as tiny, trans-
parent, practically colorless colonies of from 1 to 2 mm.
in diameter. Colonies of the colon or paracolon group
appear as larger, denser pink or red massses and cause
a reddening of the medium about them.
526 APPLICATION OF METHODS OF BACTERIOLOGY
All small, transparent, colorless colonies, i. e., those, sug-
gestive of bacillus typhosus are to be isolated in pure cul-
ture and identified by the usual procedures.
Precipitation Method of Ficker.1 — Two liters of the water
to be examined are placed in a narrow sterile glass cylinder
and rendered alkaline with 8 c.c. of 10 per cent, sodium
carbonate solution, and afterward 7 c.c. of a 10 per cent,
sulphate of iron solution are added and mixed with the
water by means of a sterile glass rod. The cylinder is then
placed in the ice-chest. Precipitation is complete in two
to three hours. The overstanding water is syphoned off,
and the precipitate or portions thereof are poured into
sterile test-tubes. To this precipitate is now added about
a half volume of a 25 per cent, solution of neutral potassium
tartrate. The test-tube is closed with a sterile rubber cork
and the mixture thoroughly agitated, whereby the precipi-
tate is completely dissolved. With a sterile pipette one part
of this fluid is mixed in a test-tube with two parts of sterile
bouillon, and this mixture is distributed over a series of
Drigalski-Conradi plates. Ficker advises when possible
the use of a centrifuge for the separation of the precipitate,
as he believes the results are likely to be more satisfactory.
Prophylactic Vaccination. — That typhoid fever may be
prevented by vaccination is an accomplished fact. Expe-
rience gathered during the past few years by all civilized
governments, notably those of England, France, Germany
and this country is unanimous in support of this statement.
No argument could be more convincing than the results
obtained through the vaccinations practiced in the United
States Army and Navy, where the procedure is com-
pulsory. The following abstract from one of the several
1 Hygienische Rundschau, 1904, Bd. xiv, S. 7.
BACILLUS TYPHOSUS 527
excellent reports submitted by Major Russell of the U. S.
Army Medical Corps, suffices to illustrate the protective
value of antityphoid vaccination:
In 1898, during the Spanish- American War, when no
preventive vaccination was practised, there were assembled
at Jacksonville, Florida, 10,759 troops, among whom there
were certainly 1729 cases of typhoid fever, and including
those cases that were probably typhoid fever, this figure
is increased to 2,693 cases with 248 deaths. Contrast that
with the following:
In 1911 there were assembled for maneuvers along the
Mexican frontier about 20,000 United States troops. All
were vacinnated against typhoid fever; with the result
that after four months in camps (about the same time as
the men remained in the Jacksonville camp) there developed
one case of typhoid fever. This case did not prove fatal.
It should be said that the disease was known to exist among
residents in the immediate vicinity of this camp and that
the soldiers were allowed free access to the infected
districts.
By the adoption of compulsory vaccination in the Army,
typhoid fever has been practically eliminated. For the
entire United States the typhoid mortality for the year 1913
was at the rate of 12.7 per 100,000, while for the entire
army it was 0 per 100,000.*
It is needless to pursue the argument further; though it
should be said that the vaccination is harmless to the
individual.
Major Whitmore of the Medical Corps of the United
States Army states that of 130,000 adults vaccinated, 97
per cent, gave no disagreeable reaction.
1 For a discussion of typhoid fever during the war see Annual Reports of
the Surgeon-General of the Army, 1919 and 1920.
528 APPLICATION OF METHODS OF BACTERIOLOGY
Major Russell has also shown by a very careful study that
children under five years of age may be safely vaccinated
if appropriate doses of the vaccine be employed.
The Vaccine. — The agent used in vaccination is typhoid
bacilli that have been killed by heat. In some instances
living, sensitized typhoid bacilli have been employed with
good results, but as the bulk of experience has been obtained
with the dead cultures and as this is much the more simple
procedure it is probable that it is the method that will be
generally adopted.
The vaccine is prepared as follows: A proven culture of
bacillus typhosus is grown on nutrient agar-agar at body
temperature for eighteen to twenty hours. The growth is
then carefully washed from the surface with a small quantity
of sterile physiological salt solution. This emulsion is then
heated in a water bath to 53° C. for one hour, after which
it is diluted with sterile salt solution to a point at which a
billion bacilli are contained in a cubic centimeter of the
emulsion. Finally tricresol in the proportion of 0.25 per
cent, is added as a preservative. Before using such
vaccine its safety: i. e., its freedom from objectionable
qualities, especially from the germs of tetanus, is invariably
tested, as is also its efficiency in calling forth the customary
reactions of intoxication and resistance. These tests are made
upon such sensitive reagents as mice, guinea-pigs, and rabbits.
The original vaccination consisted in the subcutaneous
injection of a volume of emulsion equivalent to 500 million
bacilli followed on the tenth and twentieth days with doses
equivalent to 1000 million bacilli; that is to say, the first
dose is 0.5 c.c. of the above-mentioned emulsion, while
the second and third doses are 1 c.c. each.
BACILLUS TYPHOSUS 529
As a rule the injections — particularly the primary one —
are followed by a red, tender, swollen area at the site of
puncture. This may be accompanied by headache, fever,
general malaise and sometimes by a chill with vomiting
or diarrhea. In the majority of individuals the reactions
are mild and disappear in from thirty-six to forty-eight
hours.
In the later use of this vaccine it was found possible to
secure the desired protection by one single injection, instead
of three. In from one to three persons out of every thousand
vaccinated the reaction may be severe, though they are
not dangerous. No ill effects of a permanent nature have
thus far been noted in the thousands of civilians and millions
of soldiers inoculated during the war, nor have the vaccina-
tions been seen to influence unfavorably the course of other
diseases from which the individual may be suffering.
It should be needless to say that strict aseptic precau-
tions are to be taken in performing the operation. The
resistance that is excited by the vaccination is an "active
immunity" — that is, it is an immunity identical in nature
with that acquired by an individual who has recovered from
an attack of typhoid fever. In so far as can be stated now,
however, the immunity is not permanent. All indications
point to its gradual diminution and possible disappearance
often in two to three years, so that revaccination after the
lapse of this time is advisable.
NOTE. — Obtain a pure culture of typhoid bacilli, and
from this make inoculations upon a series of potatoes of
different ages and from different sources. Do they all
grow alike ?
34
530 APPLICATION OF METHODS OF BACTERIOLOGY
Before sterilizing render another lot of potatoes slightly
acid with a few drops of very dilute acetic acid; render
others very slightly alkaline with dilute caustic soda. Are
any differences in the growths noticeable ?
Make a series of twelve tubes of peptone solution to which
rosolic acid has been added. Inoculate them all with as
nearly the same amount of material as possible (one loopful
from a bouillon culture into each tube) ; place them all in the
incubator. Is the color-change, as compared with that of
the control-tube, the same in all cases ?
Compare the morphology of cultures of the same age on
gelatin, agar-agar, and potato.
Select a culture in which the vacuolations are quite marked.
Examine this culture unstained. Do the organisms look
as if they contained spores? How would you demonstrate
that the vacuolations are not spores? What is the crucial
test for spores?
Obtain from normal feces a pure culture of the com-
monest organism present. Write a full description of it.
Now make parallel cultures of this organism and of the
typhoid bacillus on all the different media? How do they
differ? In what respects are they similar?
BACILLUS COLI (ESCHERICH), MIGULA, 1900.
SYNONYMS: Bacillus neapolitanus, Emmerich, 1884; Bacillus pyogenes
fcetidus, Passet, 1885; Emmerich's bacillus, Eisenberg, 1886; Bacterium
coli commune, Escherich, 1886.
This organism was discovered by Escherich, in 1886, in
the intestinal discharges of milk-fed infants. It has since
been demonstrated to be a constant inhabitant of the intes-
tines of man and domestic animals, and is, therefore, con-
sidered a commensal species.
BACILLUS COLI 531
For a time after its discovery it was considered of but
little importance and attracted attention only because of
its resemblance, in certain respects, to the bacillus of typhoid
fever, with which it was occasionally confounded. In this
particular it still serves as a subject for study. Some have
even gone so far as to regard them as but varieties of one and
the same species, though in the present state of our knowl-
edge this is* an assumption for which as yet there are not
sufficient grounds. That they possess in common certain
general points of resemblance and often approach one
another in some of their biological peculiarities is true; but,
as we shall learn, they each possess peculiarities which,
when considered together, render their differentiation from
one another a matter of but little difficulty.
With the wider application of bacteriological methods
to the study of pathological processes it was occasionally
observed that, under favorable circumstances, bacillits coli
disseminated from its normal habitat and appeared in
remote organs, often associated with diseased conditions.
This was at first considered of but little importance, and its
presence in these localities was viewed as accidental. Its
repeated appearance, however, in different organs of the
body and the frequency of its association with pathological
conditions, ultimately attracted attention to it, and in
consequence a great deal has been written concerning the
possible pathogenic nature of this organism.
The fact that it is a commensal species, always intimately
associated with certain of our life-processes, together with
the fact that it is known to appear in organs other than
that in which it is normally located, and that its occurrence
in diseased conditions is not rare, justifies the opinion that
it is one of the most important of the microorganisms with
which we have to deal.
532 APPLICATION OF METHODS OF BACTERIOLOGY
While not generally considered a pathogenic organism,
there is, nevertheless, sufficient evidence to warrant the
statement that under favorable conditions of reduced vitality
on the part of the animal tissues, this organism may assume
pathogenic properties, so that its presence in diseased con-
ditions is not always to be considered as accidental, though
this is frequently the case.
The morphological and cultural peculiarities of bacillus
coli are as follows :
Morphology. — In shape it is a rod with rounded ends,
sometimes so short as to appear almost spherical, while
again it is seen as very much longer threads. Often both
forms are associated in the same culture. It may occur as
single cells, or as pairs joined end to end.
It has no peculiar morphological features that can aid
in its identification. It is usually said to be motile, and
undoubtedly is motile in the majority of cases; but its
movements are at times so sluggish that a positive opinion
is often difficult.
By Loffler's method of staining, flagella can be demon-
strated, though usually not in such numbers as are seen to
occur on the typhoid fever bacillus.
Cultural Characteristics. — It grows both with and without
free oxygen.
On the surface of gelatin its colonies appear as small, dry,
irregular, flat, blue-white points that are commonly some-
what dentated or notched at the margin. They are a trifle
denser at the center than at the periphery, and are often
marked at or near the middle by an oval or round nucleus-
like mass — the original colony from which the layer on the
surface developed. When located in the depths of the
gelatin, and examined with a low-power lens, they are at
BACILLUS CO LI 533
first seen to be finely granular and of a very pale greenish-
yellow color; later they become denser, darker, and much
more markedly granular; in shape they are round, oval,
and lozenge-like. When the surface colonies are viewed
under a low power of the microscope they present essen-
tially the same appearance as that given for the colonies
of the bacillus of typhoid fever, viz., they resemble flattened
pellicles of glass-wool, or patches of finely ground colorless
glass. Colonies of this organism on gelatin are frequently
encountered that cannot be distinguished from those result-
ing from the growth of bacillm typhosus; although, as a rule,
their growth is a little more luxuriant.
In stab- and smear-cultures on gelatin the surface- growth
is flat, dry, and blue-white or pearl color. Limited growth
occurs along the track of the needle in the depths of the
gelatin. As the culture becomes older the gelatin round
about the surface-growth may gradually lose its trans-
parency and become cloudy, often quite opaque. In still
older cultures small root- or branch-like projections from
the surface-growth into the gelatin are sometimes seen.
At times these may be of a distinctly crystalline appear-
ance.
It does not cause liquefaction of gelatin.
Its growth on nutrient agar-agar and 'on blood-serum is
luxuriant, but not characteristic.
In bouillon it causes diffuse clouding with sedimentation.
In some bouillon cultures an attempt at pellicle formation
on the surface may be seen, but this is exceptional. In old
bouillon cultures the reaction becomes alkaline and a decided
fecal odor may be detected.
Its growth on potato is rapid and voluminous, appearing
after twenty-four to thirty-six hours in the incubator as a
534 APPLICATION OF METHODS OF BACTERIOLOGY
more or less lobulated layer of a drab, dark-cream, or
brownish-yellow color.
In neutral milk containing a little litmus tincture the
blue color is changed to red after from eighteen to twenty-
four hours in the incubator, and, in addition, the majority
of cultures cause firm coagulation of the casein in about
thirty-six hours, though frequently this takes longer. Very
rarely the litmus may indicate the production of acid and no
coagulation occur.
In media containing glucose it grows rapidly and causes
active fermentation, with liberation of carbonic acid and
hydrogen. If cultivated in solid media to which glucose
(2 per cent.) has been added, the gas-formation is recognized
by the appearance of numerous bubbles along and about
the points of growth. If cultivated in fluid media, also
containing glucose, in the fermentation-tube, evidence of
fermentation is given by the collection of gas in the closed
arm of the tube.
On lactose-litmus-agar-agar its colonies are pink and the
color of the surrounding medium is changed from blue to red.
In Dunham's peptone solution it produces indol in from
forty-eight to seventy-two hours.
It stains with the ordinary aniline dyes. It is decolorized
when treated by the method of Gram.
By comparing what has been said of bacillus typhosus
and of bacillus coll it will be seen that, while they simulate
each other in certain respects, they nevertheless possess
individual characteristics by which they may readily be
differentiated. The least variable of the differential points
are:
1. Motility of bacillus typhosus is much more conspicuous,
as a rule, than is that of bacillus coli.
BACILLUS COLI 535
2. On gelatin, colonies of the typhoid bacillus develop
more slowly than do those of the colon bacillus.
3. On potato, the growth of the typhoid bacillus is usually
invisible (though not always); while that of the colon
bacillus is rapid, luxuriant, and always visible.
4. The typhoid bacillus does not cause coagulation of
milk with acid reaction. The colon bacillus does this in
from thirty-six to forty-eight hours in the incubator.
5. The typhoid bacillus never causes fermentation, with
liberation of gas, in media containing glucose, lactose, or
saccharose. The colon bacillus is conspicuous for its power
of causing gaseous fermentation in such solutions.
6. In nutrient agar-agar or gelatin containing lactose and
litmus tincture, and of a slightly alkaline reaction, the color
of the colonies of typhoid bacillus is pale blue, and there is
no reddening of the surrounding medium; while colonies of
the colon bacillus are pink and the medium round about
them becomes red.
7. The typhoid bacillus does not, us a rule, possess the
property of producing indol in solutions of peptone; the
growth of the colon bacillus in these solutions is accompanied
by the production of indol in from forty-eight to seventy-
two hours at 37° to 38° C.
Animal Inoculations. — As with the bacillus of typhoid
fever, the results of inoculation of animals with cultures
of this organism cannot be safely predicted. According to
numerous observers the effects that do appear are in most
instances to be attributed to the toxic rather than to the
infective properties of the culture used.
When introduced into the subcutaneous tissues of mice
it has no effect, while similar inoculations of guinea-pigs
are sometimes (not always) followed by abscess formation
536 APPLICATION OF METHODS OF BACTERIOLOGY
at the point of operation, or by alterations very similar to
those produced by intra vascular inoculation, viz., death in
less than twenty-four hours, accompanied by redness of
the peritoneum and marked hyperemia and ecchymoses of
the small intestine, together with swelling of Peyer's patches.
The cecum and colon may remain unchanged or present
enlarged follicles. There may or may not be an accumula-
tion of fluid in the abdominal cavity; but peritonitis is
rarely present. The small intestine may contain bloody
mucus.
Intravenous inoculation of rabbits may be followed by
similar changes, with often the occurrence of diarrhea
before death, which may, in the acute cases, result in from
three to forty hours. In another group of cases acute fatal
intoxication does not result, and the animal lives for weeks
or months, dying ultimately of what appears to be the
effects of a slow or chronic form of infection. For a few
hours after inoculation these animals present no marked
symptoms; exceptionally, somnolence and diarrhea have
been observed at this period, indicating acute intoxication
from which the animal has recovered. The affection is
unattended by fever. The most marked symptom is loss of
weight. This is usually progressive from 'the first or second
day after inoculation, with slight fluctuations until death.
At autopsy the animal is found to be emaciated. The
subcutaneous tissues and the muscles appear pale and dry.
The serous cavities, particularly the pericardial, may con-
tain an excess of serum. The viscera are anemic. The
spleen is small, thin, and pale. Exceptionally ulcers and
ecchymoses are observed in the cecum, but generally there
are no lesions of the intestinal tract.
The most striking and constant lesions, those most
BACILLUS CO LI 537
characteristic of the affection, are in the bile and in the
liver; in some cases the quantity of bile may not exceed
the normal, but in others the gall-bladder may be abnor-
mally distended with bile. The bile is nearly colorless or
has a pale yellowish or brownish tint, with little or no
greenish color. Its consistence is much less viscid than
normal, being often thin and watery. It usually contains
small, opaque, yellowish particles or clumps which can be
seen floating in it, even through the walls of the gall-bladder.
These clumps consist microscopically of bile-stained, appar-
ently necrotic, epithelial cells; leukocytes in small numbers;
amorphous masses of bile-pigment, and bacteria often in
zooglea-like clumps. Similar material is found in the larger
bile-ducts.
The liver frequently contains opaque, whitish or yellow-
ish-white spots and streaks of irregular size and shape, which
give a peculiar mottling to the organ when present in large
number. These areas may be numerous, or only one or two
may be found. In size they range from minute points to
areas of from 2 to 3 cm. in extent. By microscopic exami-
nation they are found to represent localities where the liver-
cells have undergone necrosis accompanied by emigration
of leukocytes, and the cells about them are in a condition
of fatty degeneration. In sections of the liver masses of
the bacilli may be discovered in and about the necrotic
foci just described.
At these autopsies the colon bacillus is not found generally
distributed through the body, but is only to be detected in
the bile, liver, and occasionally in the spleen.1
1 Consult paper by Blachstein on this subject, Johns Hopkins Hospital
Bulletin, 1891, ii, 96.
538 APPLICATION OF METHODS OF BACTERIOLOGY
BACILLUS PARATYPHOSUS.
During recent years careful bacteriological examination
of cases of continued fever, the blood from which had no
agglutinating action upon typhoid bacillus, has revealed a
group of bacilli which differ from bacillus typhosus in certain
important particulars. These bacteria possess characters
which are intermediate between those of bacillus typhosus
and bacillus coli, some resembling more closely the former,
others the latter, and for these reasons they have sometimes
been denominated the intermediate, "near" or "para"
group. Some of the organisms isolated from such cases
of continued fever resemble very closely bacillus enteriditis,
which Gaertner found in cases of meat poisoning.
The general opinion is that these organisms produce a
form of infection sometimes resembling in' many of its
clinical characters that produced by bacillus typhosus.
The infection, however, is usually of a milder type and
only a comparatively small number of cases have terminated
fatally, so that the pathology of the disease is not well
known. Moreover, the biological characters of the different
organisms isolated from cases of paratyphoid fever, as the
condition is called, show such wide variations that it is
probable the pathology of different cases also varies with
the particular type of organism causing the infection.
Buxton1 was one of the first to make a careful compara-
tive study of the morphology and biology of this group of
organisms. He classifies the intermediary group of organ-
isms in the following manner:
" Paracolons : those which do not cause typhoidal symp-
1 Journal of Medical Research, viii, 201.
BACILLUS PARATYPHOSUS 539
toms in man. A group containing numerous different
members, but culturally alike.
" Paratyphoids : those which cause typhoidal symptoms.
" (a) A distinct species culturally unlike the paracolons.
"(6) A distinct species culturally resembling the para-
colons."
Buxton and others state that some of those producing
typhoidal symptoms cannot be distinguished culturally
from some members of the paracolon group. All the organ-
isms of this intermediate group have* the morphological
characters of the colon-typhoid group of organisms, and
they cannot, therefore, be distinguished from one another
by the form or size.
The biological differences on agar-agar, blood serum,
gelatin, and bouillon, between the members of the inter-
mediate group, and between bacillus typhosus and bacillus
coli are too insignificant and uncertain to be of any assist-
ance in a differentiation between members of the group.
In litmus milk certain well-marked differences between
different members of the group are noticed. None of the
organisms of the intermediate- group produce coagulation.
Some produce a slight initial acidity, which is later followed
by an alkaline reaction. Still other members of the group
produce an acidity amounting to 1 per cent.
Buxton states that the intermediates can be distinguished
from bacillus typhosus by their power of fermenting the
disaccharid maltose and all the monosaccharids with gas
formation. On the other hand they can be distinguished
from bacillus coli by their inability to form acid and gas in
lactose media.
The agglutination reaction of members of the intermediate
group with the serum of an animal immunized with one of
540 APPLICATION OF METHODS OF BACTERIOLOGY
the organisms varies with the different strains. The more
closely a member of the group resembles culturally the
organism employed in immunizing the animal the more
readily is it agglutinated. In attempts to diagnose para-
typhoid infection it is well to bear this fact in mind and make
agglutination tests upon different members of the group
with the blood of the patient.
CHAPTER XXV.
The Group of Bacilli Found in Cases of Epidemic, Endemic, and Sporadic
Dysentery — The Morphological, Biological, and Pathogenic Char-
acters of the Several Members of the Group — The Differentiation of
the Different Types of Bacilli.
BACILLUS DYSENTERIC.
THE investigations of epidemic dysentery by Shiga,
Flexner, Kruse, Vedder, Duval, Basset, Park, and many
others, have demonstrated that this disease is caused by
an organism that varies somewhat in its characters as
ericountered in different cases. So far at least four types of
organisms have been found that differ in minor particulars.
The type of organism first encountered by Shiga, in Japan,
is the one that is probably very widely distributed, because
it has been found in practically every place where search
has been made for it. The type of organism encountered
by Flexner in the Philippine Islands, and believed by him
to differ from the Shiga type, has also been found very
generally in the United States, especially in dysentery
occurring in infants. The type of organism isolated by
Hiss and Russell, and later by Park and his associates, has
most of the characteristics of the Flexner type of organism,
though the agglutination reaction shows that it is not
identical with it.
At first certain investigators were inclined to regard the
Flexner type of organism as having no causative relation
(541)
542 APPLICATION OF METHODS OF BACTERIOLOGY
whatever to dysentery, but later detailed studies all
strengthen the assumption that the Shiga type of the
organism is not the only one concerned in causing epidemic
dysentery. In a number of cases of dysentery two, and at
times three, types of bacillus dysenterise have been encoun-
tered. Thus far it has been impossible to differentiate clini-
cally between the infections produced by the one or the
other type, both severe and mild cases being caused by each.
The Shiga Type of Organism. — The evidence presented
by Shiga, who discovered this organism in 1898, in Japan,
and the subsequent observations of Flexner upon dysentery
in the Philippine Islands, leaves little room for doubt that,
in so far as acute epidemic dysentery is concerned, the
organism under consideration may reasonably be regarded as
the causative factor. By both Shiga and Flexner the
organism was almost uniformly encountered in the intestinal
contents, the intestinal walls, and the mesenteric glands
during the acute stages of the disease. Later it was fre-
quently missed, and this became more common as the
malady progressed to chronicity or recovery.
It is a bacillus of medium size, with rounded ends. In
general its morphology may properly be likened to that of
either the typhoid or colon bacillus.
It is motile and does not form spores.
. It can be stained with any of the ordinary aniline dyes.
It is decolorized by the method of Gram. It may be cul-
tivated on all the ordinary media. It grows at room-
temperature, but better at the temperature of the body.
It does not liquefy gelatin.
The colonies upon agar-agar present nothing character-
istic; those on gelatin are at first— i. e., just after isolation
from the body — like those of bacillus typhosus; later on,
BACILLUS DYSENTERIC 543
after the organism has been kept under conditions of con-
tinuous saprophytic growth, the colonies may be thicker,
denser, moister, and less translucent, but always suggesting
the peculiar, leaf-like contour characteristic of the colonies
of the colon-typhoid group under similar conditions. In
gelatin stab-cultures there is growth along the track made
by the needle, and little tendency to lateral development
over the surface.
On potato, its growth may be so limited as to be scarcely
visible, or it may appear as a moderately voluminous gray-
ish-brown or light-brown layer along the track made by
the needle, and spreading laterally beyond this. Between
these extremes all gradations 'may be seen according to
the suitability of the potato used.
In bouillon it causes uniform clouding and a more or less
dense sediment. It does not form a pellicle.
Growth on blood-serum is not accompanied by liquefac-
tion (digestion).
Glycerin-agar-agar appears less suited to its growth than
plain nutrient agar-agar.
It does not ferment either glucose, saccharose, or lactose,
with liberation of gas; although in glucose media there is
a slight increase of acidity.
When grown in litmus-milk, the latter, after twenty-four
to seventy-two hours at body-temperature, becomes a pale
lilac. Later on — i. e., after six to eight days — there is a
development of alkali, and the lilac tint gives way to a
deep, distinct blue color. Coagulation is never observed.
It is either incapable of producing indol, or has this
faculty developed to so limited a degree as to make the
matter doubtful.
When mixed with blood serum of individuals suffering
544 APPLICATION OF METHODS OF BACTERIOLOGY
from this form of dysentery a positive agglutination reaction
is often obtained.
It is pathogenic by both subcutaneous and intraperitoneal
inoculation for the ordinary laboratory test-animals— i. e.t
mice, guinea-pigs, and rabbits.
When injection is made beneath the skin, death results
in from two to four days, according to the dose and viru-
lence .of the culture used.
The most striking lesion is that observed at and about
the site of inoculation. This consists of edema, hemor-
rhagic exudation, and in delayed cases, more or less of pus
formation. The subcutaneous lymph-glands are often en-
larged and reddened, and a serous exudation is frequently
encountered in the great serous cavities. Of the animals
mentioned, the rabbit is most apt to survive the subcu-
taneous inoculation.
When injected into the peritoneal cavity, death takes
place in from a few hours to five or six days, according
to dose and virulence of the culture used.
At autopsy the superficial lymph-glands are enlarged and
reddened; the peritoneum contains more or less of turbid
fluid and small masses of leukocytes; the pleural and peri-
cardial cavities may contain clear fluid; the spleen is swollen;
the adrenals and kidneys are congested; there may be a
grayish exudate over the liver, spleen, and intestines, the
bloodvessels are injected ? the small intestine may be filled
with semifluid or fluid matter; there may be ecchymosis
in the intestinal mucosa, and Peyer's patches may be
enlarged and reddened.
The distribution of the bacilli varies : sometimes there
is a general invasion of the body by the bacilli; at others
they are only to be found at the local site of inoculation.
BACILLUS DYSENTERIC 545
Sometimes they can be detected in the intestinal contents
after both subcutaneous and intraperitoneal inoculation;
at other times they cannot.
If the stomach contents be neutralized and large doses of
the bacilli be administered per os, death may occur. Under
these conditions the small intestine is hyperemic and con-
tains blood-stained mucoid matter, from which the bacilli
may usually be cultivated.
If cultures be fed to cats after administration of croton
oil, a fatal diarrhea may ensue. The mucous membrane of
the large intestine is injected, its surface covered with
mucous, and its contents mucoid. From the latter the
bacilli may be recovered in culture.
A fatal diarrhea may follow the simple feeding of cultures
to dogs. This occurs in somewhat less than six days. The
condition of the contents and walls of the large intestine
is essentially similar to that seen in the cat.
In view of the fact that marked evidences of intoxica-
tion may follow upon the injection of suspensions of dead
cultures of this organism (solid cultures killed by exposure
to 60° C.), it is probable that the pathogenicity of this
organism is referable to its endotoxin, rather than to a
soluble intoxicant secreted or manufactured as a by product
in the course of growth.
The Hiss-Russell Type of Organism. — In the detailed study
of dysentery and summer diarrhea in infants, a type of bacil-
lus dysenterise has been encountered which has the property
of fermenting mannite as well as dextrose. The Shiga type
ferments dextrose, but none of the other carbohydrates.
The Strong Type of Organism. — This type of organism has
many of the characters of the Harris type, though it ferments
only mannito-dextrose, and saccharose.
35
546 APPLICATION OF METHODS OF BACTERIOLOGY
The Harris Type of Organism. — This type of bacillus
dysenterise was first encountered by Strong while working
in the Philippine Islands. It has since been encountered
quite frequently in the United States, especially in the
summer diarrheas in infants. This organism ferments
mannite as well as dextrose, maltose, saccharose, and
dextrin.
It is only by careful observations of the reactions with
the different carbohydrates that it is possible by culture
methods to differentiate between these different strains
of bacillus dysenterise, as has been shown by Hiss1 and by
others.
The Agglutinability of Bacillus Dysenterise. — The influence
of agglutinins in dysentery immune serum has also served
to differentiate between different types of baccillus dysen-
terise. Normal serums, especially those of bovines and
of goats, also yield very instructive results. Variations
in the agglutinability of the several types of bacillus
dysenterise, especially in normal serums, were first pointed
out by Bergey,2 and have since been noticed by other
investigators (see especially Park and Hiss, loc. cit.).
The different types of bacillus dysenterise can be distin-
guished easily by their relative agglutinability, but in order
to do so animals must be rendered immune from each
variety and the serum of such animals employed as specific
reagents. When this is done it will be found that the serum
of an animal immunized with the Shiga type of organism
will agglutinate that type of organism in high dilutions,
say 1 : 5000, while the Harris type of organism will only be
agglutinated in dilutions of 1 : 200, and the Hiss-Russell
1 Journal of Medical Research, December, 1904, viii.
2 Ibid., 1903, v, 21.
BACILLUS DYSENTERIC 547
type of organism in dilutions of 1 : 50. On the other hand,
the serum of an animal immunized with the Flexner type
of organism will agglutinate that type of organism in high
dilutions, say 1 : 10,000, while the other two types of the
organism will be agglutinated only in dilutions of 1 : 100.
The serum of an animal immunized with the Hiss-Russell
type of organism will agglutinate that type of organism
in dilutions, say of 1 : 1000, while the Harris type is agglu-
tinated only in dilutions of 1 : 100, and the Shiga type in
dilutions of 1 : 20.
Protective Inoculation. — By the repeated inoculation of
animals with cultures of this organism, killed either by heat
or by chemicals, it has been found possible to protect them
against otherwise fatal doses of the living virulent organism.
When treated in this way, the goat supplies a serum that
exhibits not only an agglutinating power over the living
bacilli, but possesses both protective and curative properties
when injected into other susceptible animals.
During 1898-1899 Shiga1 employed a protective serum,
made after the foregoing principles, in the treatment of
dysentery in human beings. During the period mentioned
he treated 266 cases, and had a death-rate of 9.6 per cent.;
while for 1736 cases occurring at the same time and in the
same locality, but not so treated, there was a death-rate
of 34.7 per cent.2
Holt3 summarizes the results obtained in the treatment
1 See The Epidemic Dysentery of the Past Twenty Years in Japan, by
Stuart Eldridge, M.D., U. S. Marine-Hospital Service, Public Health
Reports, 1900, xv, No. 1, 1-11.
2 The foregoing sketch is compiled from:
Shiga, Ueber den Dysenteric-bacillus (Bacillus dysenteriae) , Centralblatt
fur Bakteriologie und Parasitenkunde, 1898, Abt. i, Bd. xxiv, Nos. 22, 23, 24.
Flexner, On the Etiology of Tropical Dysentery, Philadelphia Medical
Journal, September 1, 1900.
3 Studies from the Rockefeller Institute for Medical Research, 1904, vol. ii.
548 APPLICATION OF METHODS OF BACTERIOLOGY
of 87 cases with dysentery immune serum. Decided im-
provement was noted in only 12 of the patients. These
were principally hospital cases, and hence rather grave
forms of the disease. Another factor which probably
operated against the favorable influence of the serum is the
fact that the serum treatment was generally preceded by
a careful bacteriological 'analysis of the stools in order to
establish a positive diagnosis, requiring two or three days
so that the serum treatment was instituted late in the course
of the disease.
Holt points out that the conditions necessary to obtain
success in the serum treatment of cases of dysentery are:
First, the early use of the serum, before serious lesions have
developed or before the patient's general condition has been
too profoundly impaired; second, the serum must be
administered in repeated doses, one or two doses a day, and
continued for several days in severe cases.
CHAPTER XXVI.
The Spirillum (Comma Bacillus) of Asiatic Cholera — Its Morphologica
and Cultural Peculiarities — Pathogenic Properties — The Bacterio-
logical Diagnosis of Asiatic Cholera — Microspira Metchnikovi — Micro-
spira ("Vibrio") Schuylkilliensis — Its Morphological, Cultural, and
Pathogenic Characters.
THE CHOLERA GROUP OF ORGANISMS.
AT the conference held in Berlin in 1884 for the purpose
of discussing Asiatic cholera from the sanitary aspect, it
was announced by Koch1 that he had discovered in the
intestinal evacuations of individuals suffering from Asiatic
cholera a microorganism that he believed to be the cause
of the malady. The importance of this statement naturally
attracted widespread attention to the subject, and as one
of the consequences there existed, for a short time following,
some skepticism as to the accuracy of Koch's claim. These
doubts arose as a result of a series of contributions from
other observers, who endeavored to prove that the organism
found by Koch in cholera evacuations was common to other
localities, and was not a specific accompaniment of this
disease. It was not very long, however, before it was
evident that these objections were based upon untrust-
worthy observations, and that by reliable methods of
investigation the organism to which he had called attention
could be easily differentiated from each of those with which
it was claimed to be identical.
1 Verhandlungen der Conferenz zur Erorterung der Cholerafrage, 1884,
Berlin.
(549)
550 APPLICATION OF METHODS OF BACTERIOLOGY
This organism, commonly known both as the spirillum
of Asiatic cholera, and, because of its morphology, as Koch's
"comma bacillus/' is identified by the following peculiarities:
MICROSPIRA COMMA (KOCH), SCHROTER, 1886.
SYNONYMS. Comma-bacillus, Koch, 1884; Spirillum cholerae Asiatica,
Fliigge, 1886.
Morphology. — It is a slightly curved rod, ranging from
about 0.8 to 2fj. in length and from 0.3 to 0.4^ in thickness
— that is to say, it is usually from about one-half to two-
thirds the length of the tubercle bacillus, but is thicker and
plumper. Its curve is frequently not more marked than
that of a comma, and, indeed, it is often almost straight;
at times, though, the curve is much more pronounced, and
may even describe a semicircle. Occasionally the curve
may be double, one comma joining another, with their
convexities pointing in opposite directions, so that a figure
similar to the letter S is produced. In cultures long spiral
or undulating threads may often be seen. From these
appearances this organism cannot be considered as a bacillus,
but rather as an intermediate type between the bacilli and
the spirilla. Koch thinks it not improbable that the short
comma forms represent segments of a true spirillum, the
normal form of the organism. (Fig. 86.)
It does not form spores, and we have no reliable evidence
that it possesses the property of entering, at any time, a
stage in which its powers of resistance to detrimental agencies
are increased.
It is a flagellated organism, but has only a single flagellum
attached to one of its ends.
MICROSPIRA COMMA 551
It is actively motile, especially in the comma stage
though the long spiral forms also possess this property.
Grouping. — As found in the slimy flakes in the intes-
tinal discharges from cholera patients, Koch likens its mode
FIG. 88
\-
Microspira comma. Impression cover-slip from a colony thirty-four
hours old.
of grouping to that seen in a school of small fish when
swimming up stream — i. e., they all point in nearly the
FIG. 89
/;/ .
^ <c<y -h
— , S / " ''*>
(>(> '<••£.
€ - ^ '< vv
Involution-forms of microspira comma, as seen in old cultures.
same direction, and lie in irregularly parallel, linear groups
that are formed by one comma being behind the other with-
out being attached to it.
On cover-slip preparations made from cultures in the
552 APPLICATION OF METHODS OF BACTERIOLOGY
ordinary way there is nothing characteristic about the
grouping; but in impression cover-slips made from young
cultures the short commas will nearly always be seen in
small groups of three or four, lying together in such a way
as to have their long axes nearly parallel to one another.
(See Fig. 88.)
In old cultures in which development has ceased it under-
goes degenerative changes, and the characteristic comma
and spiral shapes may entirely disappear, their place being
taken by irregular involution-forms that present every
variety of outline. (See Fig. 89.) In this stage they take
on the stain very feebly, and often not at all.
Cultural Peculiarities. — On plates of nutrient gelatin that
have been prepared from a pure culture of this organism
and kept at a temperature of from 20° to 22° C., develop-
ment can often be observed after as short a period as twelve
hours, but frequently not before sixteen to eighteen hours.
This is especially true of the first or "original" plate, con-
taining the largest number of colonies. At this time the
plate will present to the naked eye an appearance that has
been likened to a ground-glass surface, or to a surface that
has been stippled with a finely pointed needle, or one upon
which very fine dust has been sprinkled. This appearance
is due to the presence of minute colonies closely packed
together upon the surface of the gelatin. In the depth
of the gelatin can also be seen closely packed, small points,
likewise representing growing colonies. As growth progresses
liquefaction occurs around the superficial colonies, and in
consequence this plate is usually entirely liquid after from
twenty-four to thirty hours; the developmental phases
through which the colonies pass cannot, therefore, be studied
upon it.
MICROSPIRA COMMA 553
On plates 2 and 3, where the colonies are more widely
separated, they can be seen after twenty-four to thirty
hours as small, round or oval, white or cream-white points,
and when located superficially a narrow transparent zone
of liquefaction can be detected around them. As growth
continues this liquefaction extends downward rather than
laterally, and the colony ultimately assumes the appearance
of a dense, white mass lying at the bottom of a sharply-cut
FIG. 90
Development phases of colonies of microspira comma at 20° to 22° C. on
gelatin. X about 75 diameters, a, after sixteen to eighteen hours; 6, after
twenty-four to twenty-six hours; c, after thirty-eight to forty hours; d, after
forty-eight to fifty hours; e, after sixty-four to seventy hours.
pit or funnel containing transparent fluid. This liquefaction
is never very widespread nor rapid, and rarely extends
more than one millimeter beyond the colony proper. On
plates containing few colonies there is little or no tendency
for them to become confluent, and they rarely exceed 2
to 3 mm. in diameter.
When examined under a low magnifying lens the very
young colonies (sixteen to eighteen hours old) appear as
pale, translucent, granular globules of a very delicate
554 APPLICATION OF METHODS OF BACTERIOLOGY
greenish or yellowish-green color, sharply outlined, and
not perfectly round. (See a, Fig. 90.) As growth progresses
this homogeneous granular appearance is replaced by an
irregular lobulation, and ultimately the sharply-cut margin
of the colony becomes dentated or scalloped. (See b and c,
Fig. 90.) After forty-eight hours (and frequently sooner)
liquefaction of the gelatin has taken place to such an extent
that the appearance of the colony is entirely altered. Under
a magnifying glass the colony proper is now seen to be
ragged about its edges, while here and there shreds of the
colony can.be detected scattered through the liquid into
which it is sinking. These shreds evidently represent
portions of the colony that became detached from its margin
as it gradually sank into the liquefied area.
At d, in Fig. 90, is seen a representation of the several
appearances afforded by the colonies at this stage. At the
end of the second, or during the early part of the third day,
the sinking of the colonies into the liquefied pits resulting
from their growth is about complete, and under a low-power
lens they now appear as dense, granular masses, surrounded
by an area of liquefaction through which can be seen
granular prolongations of the colony, usually extending
irregularly between the periphery and the central mass.
(See e, Fig. 90.) If the periphery be examined, it will be seen
to be fringed with delicate, cilia-like lines that radiate from
it in much the same way that cilia radiate from the ends
of the columnar epithelial cells lining the air-passages.
These are the more marked phases through which the
colonies of this organism pass in their development on
gelatin plates. In some cultures the various phases here
given pass in succession more quickly, while in cultures
from other sources they may be somewhat retarded.
MICROSPIRA COMMA
555
On plates of nutrient agar-agar the appearance of the
colonies is not characteristic. They appear as round or
oval patches of growth that are moist and moderately
transparent. The colonies on this medium at 37° C. natu-
rally grow to a larger size than do those upon gelatin at
22° C. tl^
FIG. 91 j
a
abed
Stab-culture of microspira comma in gelatin, at 18° to 20° C. a, after
twenty-four hours; b, after forty-eight hours; c, after seventy- two hours;
d, after ninety-six hours.
In stab-cultures in gelatin there appears at the top of
of the needle-track after thirty-six to forty-eight hours
at 22° C. a small, funnel-shaped depression. As the growth
progresses liquefaction occurs about this point. In the
center of the depression can be distinguished a small, dense,
556 APPLICATION OF METHODS OF BACTERIOLOGY
whitish clump, the colony itself. As growth continues the
. depression increases in extent and ultimately assumes an
appearance that consists in the apparent sinking of the
liquefied portion in such a way as to leave a perceptible
air-space between the top of the liquid and the surface of
the solid gelatin. The growth now appears to be capped by
a small air-bubble. The impression given by it at this
stage is not only that there has been a liquefaction, but
also a coincident evaporation of the fluid from the liquefied
area and a constriction of the superficial opening of the
funnel. (See a, b, c, and d, Fig. 91.) Liquefaction is not
especially active along the deeper portions of the track
made by the needle, though in stab-cultures in gelatin the
liquefaction is much more extensive than that usually seen
around colonies on plates. It spreads laterally at the upper
portion, and after about a week a large part of the gelatin
in the tube may have become fluid, and the growth will
have lost its characteristic appearance.
Stab- and smear-cultures on agar-agar present nothing
characteristic.
Its growth in bouillon is luxuriant, causing a diffuse
clouding and the ultimate production of a delicate film
upon the surface.
In sterilized milk of a neutral or amphoteric reaction at
a temperature of 36° to 38° C. it develops actively, and
gradually produces an acid reaction, with coagulation of
the casein. It retains its vitality under these conditions
for about three weeks or more. The blue color of milk to
which neutral litmus tincture has been added is changed to
pink after thirty-six or forty-eight hours at body-temperature.
Its growth in peptone solution, either that of Dunham
(see Special Media) or the one preferred by Koch, viz.,
MICROSPIRA COMMA 557
2 parts of Witte's peptone, 1 part of sodium chloride, and
100 parts of distilled water, is accompanied by the produc-
tion of both indol and nitrites, so that after eight to twelve
hours in the incubator at 37° C. the rose color characteristic
of indol appears upon the addition of sulphuric acid alone.
(See Indol Reaction.)
(What does the presence of nitrites in these cultures
signify?)
In peptone solution to which rosolic acid has been added
the red color is very much intensified after four or five days
at 37° C.
Its growth on potato of slightly acid reaction is seen after
three or four days at 37° C. as a dull, whitish, non-glistening
patch at and about the site of inoculation. It is not elevated
above the surface of the potato, and can only be distinctly
seen when held to the light in a particular position. Growth
on acid potato occurs, however, only at or near the body-
temperature, owing probably to the acid reaction, which
is sufficient to prevent development at a lower temperature,
but does not have this effect when the temperature is more
favorable. On solidified blood-serum growth is usually
said to be accompanied by slow liquefaction. I have not
succeeded in obtaining this result on Loffler's serum, nor
have I detected anything characteristic about its growth on
this medium.
The temperature most favorable for its growth is between
35° and 38° C. It grows, but more slowly, at 17° C. Below
16° C. no growth is visible.
It is not destroyed by freezing. When exposed to 65° C.
its vitality is destroyed in five minutes.
It is strictly aerobic, its development ceasing if the supply
of oxygen be cut off.
558 APPLICATION OF METHODS OF BACTERIOLOGY
It does not grow in an atmosphere of carbonic acid, but
is not killed by a temporary exposure to this gas. It does
not grow in acid media, but flourishes best in media of
neutral or slightly alkaline reaction. It is so sensitive to
the action of acids that at 22° C. its development is arrested
when an acid reaction equivalent to 0.066 to 0.08 per cent,
of hydrochloric or nitric acid is present. (Kitasato.)
Under artificial cultivation the maximum development
of this organism is reached in a comparatively short time;
after this it remains quiescent for a period, and finally
degeneration or involution begins. When in this state
they take up coloring reagents very faintly or not at all,
and may lose entirely their characteristic shape. (See Fig.
93.)
When present with other bacteria, under conditions
favorable to growth, the comma bacillus at first grows
much more rapidly than do the others; in twenty-four
hours it will often so outnumber the other organisms present
that microscopic examination might lead one to regard the
material under consideration as a pure culture of this
organism. Its conspicuous development under these cir-
cumstances does not, however, last longer than two or
three days; degeneration and death begin, and the other
organisms gain the ascendancy. This fact has been taken
advantage of in the bacteriological diagnosis of cholera.
In connection with his experiments upon the poison
produced by the cholera organism Pfeiffer1 states that in
very young cultures, grown under access to oxygen, there
is present a body that possesses intensely toxic properties.
This primary cholera-poison stands in very close relation
to the material composing the bodies of the bacteria them-
1 Zeitschrift fur Hygiene und Infektionskrankheiten, Bd. xi, S. 393.
MICROSPIRA COMMA 559
selves, and is probably an integral constituent of them, for
the vitality of the cholera spirilla can be destroyed by means
of chloroform or thymol, or by drying, without apparently
any alteration of this poisonous body. Absolute alcohol,
concentrated solutions of neutral salts, and a temperature
of 100° C., decompose this substance, leaving intact second-
ary poisons which possess a similar physiological activity,
but only when given in from ten to twenty times the dose
necessary to produce the same effects with the primary
poison.
Experiments upon Animals. — As a result of experiments
for the purpose of determining if the disease can be pro-
duced in any of the lower animals it has been found that
white mice, monkeys, cats, dogs, poultry, and many other
animals are not susceptible to infection by the methods
usually employed in inoculation experiments. When animals
are fed on pure cultures of the comma bacillus no effect
is produced, and the organisms cannot be obtained from
the stomach or intestines. They are destroyed in the
stomach, and do not reach the intestines; they are not
demonstrable in the feces of these animals. Intra vascular
injections of a pure culture into rabbits are followed by an
illness, from which the animals usually recover in from two
to three days; intraperitoneal injections into white mice
are, as a rule, followed by death in from twenty-four to
forty-eight hours, the conditions in both instances most
probably resulting from the toxic activities of the specific
poisons contained in the cultures used.
None of the lower animals suffer spontaneously from
Asiatic cholera.
The failure to induce cholera in animals by feeding or
by injection of cultures into the stomach, was shown by
560 APPLICATION OF METHODS OF BACTERIOLOGY
Nicati and Rietsch1 to be due to the destructive action of
the acid gastric juice on the organisms. They showed that
if cultures of this organism were introduced into the aliment-
ary tract of certain animals in such a manner that they
would not be subjected to the influence of the gastric juice,
a pathological condition closely simulating cholera as it
occurs in man could be produced. For this purpose the
common bile-duct was ligated, after which the cultures
were injected directly into the duodenum. Such inter-
ference with the flow of bile lessens intestinal peristalsis,
and thus permits development of the organisms at the point
at which they are deposited — that is, the portion of the
intestine having an alkaline reaction and beyond the influence
of the acid stomach-juice.
By this method Nicati and Rietsch, Van Ermengem,2
Koch,3 and others were enabled to produce in the animals
upon which they operated a condition that was, if not
identical, at all events very similar pathologically to that
seen in the intestines of subjects dead of the disease.
At a subsequent conference held in Berlin in 1885 Koch4
described the following method, by means of which he had
been able to obtain a much greater degree of constancy
in his efforts to produce cholera in lower animals: bearing
in mind the point made by Nicati and Rietsch as to the
effect produced by the acid reaction of the gastric juice,
this reaction was first to be neutralized by injecting through
a soft catheter passed down the esophagus into the stomach
5 c.c. of a 5 per cent, solution of sodium carbonate. Ten
1 Archiv de Phys. norm, et path., 1885, t. vi. 3e ser. Comptes rendus,
xcix, p. 928; Revue de Hygiene, 1885; Revue de Medecine, 1885, v.
2 Recherches sur le Microbe du Cholera Asiatique, Paris-Bruxelles,
1885; Bull, de 1'Acad. toy. de Med. de Belgique, xviii, 3e ser.
3 Loc. cit. 4 Loc. cit.
MICROSPIRA COMMA 561
or fifteen minutes later this was to be followed by the injec-
tion into the stomach (also through a soft catheter) of
10 c.c. of a bouillon culture of microspira comma. For the
purpose of arresting peristalsis and permitting the bacteria
to remain in the stomach and upper part of the duodenum
for as long a time as possible, the animal was to receive,
immediately following the injection of the culture, an
intraperitoneal injection, by means of a hypodermic syringe,
of 1 c.c. of tincture of opium for each 200 grams of its
body-weight. Shortly after this last injection deep narcosis
sets in and lasts from a half to one hour, after which the
animal is as lively as ever. Of 35 guinea-pigs inoculated
in this way by Koch, 30 died of an affection that was,
in general, very similar to Asiatic cholera as seen in
man.
The condition of those animals before death is described
as follows: twenty-four hours after the operation the animal
appears unwell; there is loss of appetite, and the animal
remains quiet in its cage. On the following day a paralytic
condition of the hind extremities appears, which, as the
day wears on, becomes more pronounced; the animal lies
quite flat upon its abdomen or on its side, with legs extended;
respiration is weak and prolonged, and the pulsations of
the heart are hardly perceptible; the head and extremities
are cold, and the body-temperature is frequently subnormal.
The animal usually dies after remaining in this condition
for a few hours.
At autopsy the small intestine is found deeply injected
and filled with flocculent, colorless fluid. The stomach and
intestines do not contain solid masses, but fluid; when
diarrhea does not occur, firm scybala may be detected in
the rectum. Both by microscopic examination and by cul-
36
562 APPLICATION OF METHODS OF BACTERIOLOGY
ture methods the organisms are found present in the small
intestine in practically pure culture.
Later Pfeiffer1 determined that essentially similar con-
stitutional effects may be produced in guinea-pigs by the
intraperitoneal injection of relatively large numbers of this
organism. His plan is to scrape from the surface of a
fresh culture on agar-agar as much of the growth as can
be held upon a medium size wire loop. This is then finely
divided in 1 c.c. of bouillon, and by means of a hypodermic
syringe is injected directly into the peritoneal cavity.
When virulent cultures have been used this operation is
quickly followed by a fall in the temperature of the animal
that is gradual and continuous until death ensues, which
usually occurs in from eighteen to twenty-four hours after
the operation, though exceptionally the animal recovers,
even after having exhibited marked symptoms of profound
toxemia.
Continuing his studies upon this disease, Pfeiffer2 demon-
strated that it is possible to render an animal immune
from the poisonous properties of this organism by repeated
injections of non-fatal doses of dead cultures (cultures that
have been killed by the vapor of chloroform or by heat).
He also demonstrated that animals so immunized possess
a specific germicidal action toward microspira comma — i. e.,
if into the peritoneal cavity of an animal immunized from
Asiatic cholera living organisms be introduced they will all
be destroyed (disintegrated) within a relatively short time.
Furthermore, if the serum of an animal immunized from
cholera be injected into the peritoneal cavity of another
animal of the same species, but not so protected, and imme-
1 Zeitschrift fur Hygiene und Infektionskrankheiten, Bd. xi and xiv.
2 Ibid., 1894, Bd. xvii, S. 355; 1894, Bd. xviii, S. 1; 1895, Bd. xx, S. 197.
MICROSPIRA COMMA 563
diately afterward living cholera spirilla be introduced, a
similar disintegration and destruction of the bacteria will
also result. He shows that a more or less definite relation
exists between the amount of serum and the number of
organisms introduced. Such a destruction *of microspira
comma by the serum of an immunized animal does not
occur outside the animal body — that is, it cannot be demon-
strated in a test-tube, unless, as Bordet demonstrated, it be
perfectly fresh from the animal body, or, as Metchnikoff
showed, there be added to it a small quantity of fresh
serum from a normal guinea-pig. The specificity of this
reaction is suggested by Pfeiffer as a means of differentiating
the cholera spirillum from other suspicious species, for no
such bacteriolytic action is observed if other bacteria be
introduced into the peritoneal cavity of animals immunized
from Asiatic cholera.
Pfeiffer further demonstrated that the serum of animals
artificially immunized from Asiatic cholera has an agglu-
tinating effect upon fluid cultures of microspira comma
similar to that seen when typhoid bacilli are mixed with
serum from typhoid cases, or from animals artificially
immunized from typhoid infection or intoxication. (See
Agglutinin.)
General Considerations. — In all cases of Asiatic cholera,
and only in this disease, the organism just described can be
detected in the intestinal evacuations. The more acute the
case and the more promptly the examination is made after
the evacuations have passed from the patient, the less
difficulty will be experienced in detecting the organism.
In some cases the organism can be detected in the vomited
matters, though by no means so constantly as in the intes-
tinal contents.
564 APPLICATION OF METHODS OF BACTERIOLOGY
As a rule, bacteriological examination fails to reveal the
presence of the organisms in the blood and internal organs
in this disease, though exceptions have been noted.
Microspira comma is a facultative saprophyte; that is
to say, it apparently finds in certain parts of the world,
particularly in those countries in which Asiatic cholera is
endemic, conditions that are not entirely unfavorable to
its development outside of the body. This was found to
be the case not only by Koch, who detected the presence
of the organism in water-tanks in India, but by many
other observers who have succeeded in demonstrating its
growth under conditions not embraced in the ordinary
methods employed for the cultivation of bacteria.1
The results of experiments having for their object the
determination of the length of time during which the
organism may retain its vitality in water are conspicuous
for their irregularity.
Koch states that in ordinary spring-water or well-water
the organisms retained their vitality for thirty days, whereas
in the sewage of Berlin they died after six or seven days;
but if this latter were mixed with fecal matters, the organ-
isms retained their vitality for but twenty-seven hours;
and in the undiluted contents of cesspools it was impossible
to demonstrate them after twelve hours. In the experi-
ments of Nicati and Rietsch they retained their vitality
in Marseilles sewage for thirty-eight days; in sea-water,
sixty-four days; in harbor-water, eighty-one days; and in
bilge-water, thirty-two days.
In one test with the water-supply of Berlin the organism
1 Obviously all pathogenic bacteria that have been isolated under artificial
methods of cultivation are facultative saprophytes. Were they obligate
parasites, their cultivation upon dead materials would be impossible.
MICROSPIRA COMMA 565
retained its vitality for 267 days, and in another for 382 days,
notwithstanding the fact that many other organisms were
present at the same time. There is no ready explanation
for these variations, for they depend apparently upon a
number of factors which may act singly or together. For
example, in general it may be said that the higher the tem-
perature of the water in which these organisms are present,
up to 20° C., the longer do they retain their vitality; the
purer the water — that is, the poorer in organic matters — the
more quickly do the organisms die, whereas the richer it
is in organic matter the longer do they retain their vitality.
The effect of light upon growing bacteria must not be
lost sight of, for it has been shown that a surprisingly large
number of these organisms are robbed of their vitality by
a relatively short exposure to the direct rays of the sun; and
it is therefore not unlikely that the non-observance of this
fact may be, in part at least, accountable for some of the
discrepancies that appear in the results of these experiments.
In his studies upon the behavior of pathogenic and other
microorganisms in the soil Carl Frankel1 found that micro-
spira comma was not markedly susceptible to those dele-
terious influences that cause the death of a number of other
pathogenic organisms. At a depth of one and a half meters
vitality was not destroyed, and there was a regular develop-
ment in cultures so placed.
As a result of experiments performed in the Imperial
Health Bureau at Berlin, it was found that the bodies of
guinea-pigs that had died of cholera induced by Koch's
method of inoculation contained no living cjiolera spirilla
when exhumed after having been buried for nineteen days
in wooden boxes, or for twelve days in zinc boxes. In a
1 Zeitschrift fur Hygiene, Bd. ii, S. 521.
566 APPLICATION OF METHODS OF BACTERIOLOGY
few that had been buried in moist earth, without having
been encased in boxes, when exhumed after two or three
months, the results of examinations for cholera spirilla were
likewise negative.
Esmarch1 found that when the cadaver of a guinea-pig
dead after the introduction of cholera organisms into the
stomach was immersed in water until decomposition was
far advanced, it was impossible to find any living microspira
comma by the ordinary plate methods. Several experi-
ments resulted in their disappearance in five days. In one
experiment, in which decomposition was allowed to go on
without the animal being immersed in water, none could be
detected after the fifth day.
Kitasato2 found that when mixed with the normal intes-
tinal evacuations of human beings it lost its vitality in from
a day and a half to three days. If the evacuations were
sterilized before the cultures were mixed with them it
retained its vitality from twenty to twenty-five days.
Hesse3 and Celli4 demonstrated that many substances
commonly employed as food serve as favorable materials
for the development of the cholera organisms.
Kitasato5 found that at 36° C. microspira comma devel-
oped very rapidly in milk during the first three or four
hours, and outnumbered the other organisms commonly
present. It then diminished in number from hour to hour
as the acidity of the milk increased, until finally its vitality
was lost; at the same time the common saprophytic bac-
teria increased in number. Relatively the same process
1 v. Esmarch, Zeitschrift fur Hygiene, Bd. vii, S. 1.
2 Zeitschrift fur Hygiene, Bd. v, S. 487.
3 Ibid., S. 527.
* Bolletino della R. Acad. Med. di. Roma, 1888.
& Zeitschrift fur Hygiene, Bd. v, S. 491.
MICROSPIRA COMMA 567
occurs at a lower temperature, from 22° to 25° C.; but it
is slower, the maximum development of the cholera organ-
isms being reached at about the fifteenth hour, after which
time they were outnumbered by the ordinary saprophytes
present.
From the foregoing it would seem that the vitality of
microspira comma in milk depends largely upon the reac-
tion; the more quickly the milk becomes- sour the more
quickly does the organism become inert.
According to Laser,1 the cholera organism retains its
vitality in butter for about seven days; it is therefore
possible for the disease to be contracted by the use of butter
that has in any way been in contact with cholera material.
When dried microspira comma retains its vitality for
from about three to twenty-four hours, according to the
degree of desiccation. In moist conditions vitality may be
retained for many months; though repeated observations
lead us to believe that under these circumstances virulence
is diminished.
Carbon dioxide, carbon monoxide and nitrous oxide gases
kill this germ in from seven to ten days.
From what has been said, we see that the spirillum of
Asiatic cholera, while possessing the power of producing
in human beings one of the most rapidly fatal diseases with
which we are acquainted, is still one of the least resistant
of the pathogenic organisms known to us. Under conditions
most favorable to its growth its development is self -limited ;
it is markedly susceptible to acids, alkalies, other chemical
disinfectants, and heat; but when partly dried upon cloth-
ing, food, or other objects, it may retain its vitality for a
relatively long period of time, and it is more than probable
1 Zeitachrift fur Hygiene, Bd. x, S. 513.
568 APPLICATION OF METHODS OF BACTERIOLOGY
that in this way the disease is often disseminated from points
in which it is epidemic or endemic into localities that are
free from it.
THE DIAGNOSIS OF ASIATIC CHOLERA BY BACTERIO-
LOGICAL METHODS.
Because of the manifold channels that are open for the
ready dissemination of this disease it is of the utmost impor-
tance that it should be recognized as quickly as possible, for
with every moment of delay opportunities for its spread
multiply. It is essential, therefore, when employing bac-
teriological means for making the diagnosis, to bear in mind
those biological and morphological features of the organism
that appear most quickly under artificial methods of cul-
tivation, and which, at the same time, may be considered
as characteristic of it, viz., its peculiar morphology and
grouping; the much greater rapidity of its growth over that
of other bacteria with which it may be associated; the
characteristic appearance of its colonies on gelatin plates
and of its growth in stab-cultures in gelatin; its property
of producing indol and coincidently nitrites in from six to
eight hours in peptone solution at 37° to 38° C.; and its
power of causing the death of guinea-pigs in from sixteen
to twenty-four hours when introduced into the peritoneal
cavity, death being preceded by symptoms of extreme
toxemia, characterized by prostration and gradual and
continuous fall in the temperature of the animal's body.
Koch1 devised a plan of procedure that comprehends the
points just enumerated. By its employment the diagnosis
can be established in the majority of cases of Asiatic cholera
1 Zeitschrift fur Hygiene und Infektionskrankheiten, 1893, Bd. xiv, S. 319.
THE DIAGNOSIS OF ASIATIC CHOLERA 569
in from eighteen to twenty-two hours. In general, the
steps to be taken and points to be borne in mind are as
follows:
1. Microscopic Examination. — From one of the small slimy
particles seen in the semi-fluid evacuations, obtained as
soon as possible after their passage, prepare a cover-slip
preparation in the ordinary way and stain it. If, upon
microscopic examination, only curved rods, or curved rods
greatly in excess of all other forms, are present, the diagnosis
of Asiatic cholera is more than likely correct; and particularly
is this true if these organisms are arranged in irregular linear
groups with the long axes of all the rods pointing in nearly
the same direction.
2. Plate Cultures. — From another slimy flake prepare a set
of gelatin plates. Keep them at a temperature of from
20° to 22° C., and after sixteen, twenty-two, and thirty-six
hours observe the appearance of the colonies. Usually after
about twenty-two hours the colonies of this organism can
easily be identified by one familiar with them.
3. Peptone Cultures. — With another slimy flake start a
culture in a tube of peptone solution — either the solution of
Dunham or, as Koch proposes, a solution of double the
strength of that of Dunham (Witte's peptone is to be used,
as it gives the best and most constant results). Keep this
at from 37° to 38° C., and at the end of from six to eight
hours prepare cover-slips from the upper layers (without
shaking) and examine them microscopically. If comma
bacilli were present in the original material, and are capable
of multiplication, they will be found in this locality in almost
pure culture. After the microscopic examination prepare
a second peptone culture from the upper layers of the one
just examined, also a set of gelatin plates, and with what
570 APPLICATION OF METHODS OF BACTERIOLOGY
remains make the test for indol by the addition of 10 drops
of concentrated sulphuric acid for each 10 c.c. of fluid con-
tained in the tube. If comma bacilli are growing in the tube,
the rose color characteristic of the presence of indol should
appear.
By following this plan "a bacteriologist who is familiar
with the morphological and biological peculiarities of this
organism should make a more than probable diagnosis at
once by microscopic examination alone, and a positive diag-
nosis in from twenty to, at most, twenty-four hours after
beginning the examination." (Koch.)
Since the publication of the foregoing plan many other
methods have been suggested. They all comprehend the
"enrichment," by special culture methods, of the number
of cholera organisms in the original material without at
the same time encouraging the multiplication of the other
bacteria present, and the subsequent isolation of the cholera
organism by the use of selective plating media. Of these
methods, the following gives general satisfaction and can be
recommended :
1. Enrichment in the peptone solution exactly as recom-
mended above by Koch if it be intestinal contents that are
under consideration; if it be water or sewage, then add to
90 c.c. of the water or sewage in an Erlinger flask 10 c.c.
of a 10 per cent, solution. Keep at 37° to 38° C. for about
eight hours.
2. Without shaking the tube or flask, now transfer one
wire loopful from the surface of the mixture of feces, water
or sewage and peptone solution, to several tubes containing
the Benedict1 medium :
Water 1000 c.c.
Peptone 10 c.c.
Sodium chloride 5 c.c.
1 Cent, of Bact., etc., Abt. i, Bd. Ixii, S. 536.
THE DIAGNOSIS OF ASIATIC CHOLERA 571
Boil and render neutral to phenolphthalein.
Add 1 grand of anhydrous sodium carbonate; boil and
filter through double filter paper. Add :
Saccharose 5 grams
Phenolphthalein (sat. sol. in 50 per cent, alcohol) 5 c.c.
Tube and sterilize by steam at 100° C.
The phenolphthalein in this alkaline solution gives to the
tubes a bright, rose-red color.
As the vibrios ferment saccharose rapidly, with resultant
acid production, the tubes containing them are quickly
decolorized. One, therefore, discards all tubes that are
not decolorized after eight hours at 37° C. Those that are
decolorized may contain cholera vibrios or other closely
allied spirilla or any of the group of bacteria having the
power to ferment saccharose. The isolation of the cholera
spirilla from this possible mixture is now accomplished by
differential or selective plating.
3. Of the many differential plating media recommended,
that which gives uniformily satisfactory results is the
alkaline egg-medium recommended by Krumwiede, Pratt
and Grund,1 and slightly modified by Goldberg:2
(a) Alkaline Egg Solution. — Beat up a whole egg with an
equal volume of distilled water. Mix an equal volume of
this with an equal volume of 6.5 per cent, solution of anhy-
drous sodium carbonate and steam for from one-half to one
hour.
(6) Meat extract-glucose-agar —
Distilled water 1000 c.c.
Meat extract (Liebig) . 3 grams
Peptone (Witte) 10 grams
Sodium chloride (c. p.) 5 grams
Glucose 1 gram
Agar-agar 30 grams
1 Jour. Infect. Dis., 1912, x, 134.
2 U. S. Pub. Health Service, Hygiene Lab. Bull., 1913, No. 91, p. 19.
572 APPLICATION OF METHODS OF BACTERIOLOGY
Steam at 100° C., for three hours to insure complete solution
of the agar-agar.
Decant, or filter through cotton, and distribute in 100
c.c. flasks.
Sterilize by steam at 100° C. for an hour and a half.
For use mix one volume of a with 5 volumes of 6, the
latter having been completely liquefied by steam. When
thoroughly mixed pour into Petri dishes, to a depth of about
3 mm. in each dish, and allow to solidify. When the medium
is solid, the dishes may be placed in the incubator with the
covers partly removed until the condensed vapor has eva-
porated. The medium should be comparatively dry before
attempting to use it. When dry the plates so prepared may
be stored in dust proof receptacles at 15° C. The plates may
now be inoculated from the surface of the Benedict medium.
This is best done by transferring a loopful of the Benedict
culture to the surface of the solid alkaline egg-glucose-
agar and distributing it over the surface with a sterile, bent-
glass spreader. When thus inoculated the plates are placed
in the incubator at 37°-38° C. until colonies develop.
On this medium the cholera, and the cholera-like spirilla
grow luxuriantly, while the other bacteria are to some extent
restrained.
The colonies of the cholera vibrios, and of those other
vibrios that closely resemble it, when well developed on
this medium, i. e., after about twenty hours at 37°^38° C.,
are, to the naked eye, more opaque than those of other
bacteria; under the low power lens they seem as if in the
depths of the medium, are more or less hazy, are surrounded
by an indistinct halo or fringe which may be in turn
surrounded by a clear zone. All such colonies should be
examined microscopically and from all that are composed
THE DIAGNOSIS OF ASIATIC CHOLERA 573
of curved or spiral organisms pure cultures should be made
for subsequent identification.
In abortive cases of cholera the organisms may be present
in the intestinal canal in very small numbers, and micro-
scopic examination is not, therefore, of so much assistance.
In these cases the adoption of one or the other of the fore-
going methods is imperative.
In the foregoing suggested plans it will be observed that
plates are not made in the usual way. The reason for this
is the cholera spirillum being markedly aerobic develops
much more readily on the surface than in the depths of the
medium. For the same reason the material taken for
plating from the enriching media should always be from
the surfaces, without the tubes or flasks having been shaken.
It being desirable to have the colonies isolated from one
another the plates should be relatively dry, that is, there
should be no collection of moisture on their surfaces that
would cause the colonies to become confluent. After pour-
ing, the plates should always be kept in a dust-free incubator
with their lids off until all excess of moisture is evaporated.
All colonies of curved rods should be isolated in pure culture
in peptone solution, and after twenty to twenty-four hours
at 37° to 38° C. such cultures should be tested for the
presence of indol. After giving positive indol reaction
should be regarded as probably cholera spirilla.
In all doubtful cases, in which only a few curved bacilli
are present, or in which irregularities in either the rate or
mode of their development occur, pure cultures should be
obtained as soon as possible and their virulence tested upon
animals. For this purpose cultures upon agar-agar from
single colonies must be made. From the surface of one of
such cultures a large wire-loopful should be scraped and
574 APPLICATION OF METHODS OF BACTERIOLOGY
broken up in about one cubic centimeter of physiological
salt solution, and the suspension thus made injected by
means of a hypodermic syringe directly into the peritoneal
cavity of a guinea-pig of about 350 to 400 grams weight.
For larger animals more material is used. If the material
injected is from a fresh culture of the cholera organism, toxic
symptoms at once appear; these have their most pronounced
expression in depression of temperature, and if one follows
this decline in temperature from time to time with the
thermometer it will be seen to be gradual and continuous
from the time of injection to the death of the animal
(Pfeiffer1), which occurs in from eighteen to twenty-four
hours after the operation.
MICROSPIRA METCHNIKOVI (GAMALEIA), MIGULA, 1900.
SYNONYM: Vibrio Metchnikovi. Gamalela, 1888.
A spirillum that simulates very closely the comma bacillus
of cholera in its morphological and cultural peculiarities,
but which is still easily distinguished from it, is that de-
scribed by Gamalei'a2 under the name of microspira Metch-
nikovi. It was found postmortem in a number of fowls that
had died in the poultry-market of Odessa, and the experi-
ments of the discoverer led him to believe that it was
related etiologically to the gastro-enteritis from which the
chickens had been suffering.
Morphologically it appears as short, curved rods and as
longer, spiral-like filaments. It is usually thicker than
Koch's microspira and is at times much longer, while again
it is seen to be shorter. It is usually more distinctly curved
than the " comma bacillus." (Fig. 92.)
1 Loc. cit. 2 Annales de 1'Institut Pasteur, 1888, tome ii, pp. 482, 552.
MICROSPIRA METCHNIKOVI 575
It is supplied with a single flagellum at one of its extremi-
ties, and is therefore motile.
It does not form spores.
It is aerobic.
FIG. 92
" .'>•
Microspira Metchnikovi from agar-agar culture, twenty-four hours old.
Its growth upon gelatin plates is usually characterized,
according to Pfeiffer, by the appearance of two kinds of
liquefying colonies, one strikingly like those of the Finkler-
Prior organism, the other very similar to those produced
by Koch's comma bacillus, though in both cases the lique-
FIG. 93
Colony of microspira Metchnikovi in gelatin, after thirty hours at 20° to
22° C. X about 75 diameters.
faction resulting from the growth of this organism is more
energetic than that common to the spirillum of Asiatic
cholera. After from twenty-four to thirty hours the medium-
size colonies, when examined under a low power of the
576 APPLICATION OF METHODS OF BACTERIOLOGY
microscope, show a yellowish-brown, ragged central mass
surrounded by a zone of liquefaction that is marked by a
border of delicate radii. (Fig. 93.)
In gelatin stab-cultures the growth has much the same
FIG. 94
abed
Stab-culture of microspira Metchnikovi in gelatin, at 18° to 20° C. a,
after twenty-four hours; 6, after forty-eight hours; c, after seventy-two
hours; d, after ninety-six hours.
general appearance as that of the cholera spirillum, but is
exaggerated in degree. The liquefaction is far more rapid,
and the characteristic appearance of the growth is lost in
from three to four days. (See a, b, c, d, Fig. 94.) Develop-
ment and liquefaction along the deeper parts of the needle-
MICROSPIRA METCHNIKOVI 577
track are much more pronounced than is the case with the
"comma bacillus."
Its growth on agar-agar is rapid; after twenty-four to
forty-eight hours a grayish deposit appears which has a
tendency to become yellowish with age.
On potato at 37° C. its growth is seen as a moist, coffee-
colored patch, surrounded by a much paler zone. The
whole growth is so smooth and glistening that it has some-
what the appearance of being varnished.
In bouillon it quickly causes opacity, with the ultimate
production of a delicate pellicle upon the surface.
It causes liquefaction of blood-serum, the liquefied area
being covered by a dense, wrinkled pellicle.
When grown in peptone solution it produces indol and
coincidently nitrites, so that the rose-colored reaction
characteristic of indol is obtained by the addition of sul-
phuric acid alone. The production of indol by this organism
is usually greater than that common to the comma bacillus
under the same circumstances.
In milk it causes an acid reaction with coagulation of the
casein. The coagulated casein collects at the bottom of the
tube in irregular masses, above which is a layer of clear
whey. If blue litmus has been added to the milk, the color
is changed to pink in from twenty-four to thirty hours,
and after forty-eight hours decolorization and coagulation
occur. The clots of casein are not re-dissolved. After about
a week the acidity of the milk is at its maximum, and the
organisms quickly die.
It causes the red color of the rosolic-acid-peptone solution
to become very much deeper after four or five days at 37° C.
It does not cause fermentation of glucose with production
of gas.
37
578 APPLICATION OF METHODS OF BACTERIOLOGY
It is killed in five minutes by a temperature of 50° C.
(Sternberg.)
It is pathogenic for chickens, pigeons, and guinea-pigs.
Rabbits and mice are affected only by very large doses.
Gamaleia states that chickens affected with the choleraic
gastro-enteritis of which this organism is the cause, are
usually seen sitting quietly with ruffled feathers. They
suffer from diarrhea, but there is no elevation of tempera-
ture. Hyperemia of the entire gastro-intestinal tract is
seen at autopsy. The other internal organs do not, as a
rule, present anything abnormal to the naked eye. The
intestinal canal contains yellowish fluid with which blood
may be mixed. In adult chickens the spirilla are not found
in the blood, but in young ones they are usually present in
small numbers.
After the introduction of a very small quantity of a culture
of this organism directly into the pectoral muscle pigeons
succumb in from eight to twenty hours. The most con-
spicuous postmortem lesion is found at the site of inocula-
tion. The muscle is marked by yellow, necrotic stripes;
is more or less edematous; is swollen, and contains the
vibrios in enormous numbers. The intestines are usually
filled with fluid contents, which may or may not be blood-
stained; the walls of the intestines are often injected with
blood, and occasionally markedly so. The conditions of
the other internal viscera are inconstant. In fatal cases
the vibrios are present in large numbers in the blood and
internal organs. In pigeons' that survive inoculation the
organisms may be found only at the site of inoculation, or
very sparingly in the blood also. These animals usually
exhibit immunity from subsequent inoculations. In certain
instances the results of -infection are chronic; the inoculated
MICROSPIRA METCHNIKOVI 579
pectoral muscle atrophies, the pigeon loses in weight and
finally dies after one or two weeks. In these cases the
organisms are usually absent from the blood and internal
organs, and may even be absent from the site of inoculation,
or, if present, in only very small number.
Guinea-pigs usually die in from twenty to twenty-four
hours after subcutaneous inoculation. At autopsy an
extensive edema of the subcutaneous tissues about the seat
of inoculation is seen, and there is usually a necrotic condi-
tion of the tissues in the vicinity of the point of puncture.
As the blood and internal organs of both pigeons and guinea-
pigs contain the vibrios in large numbers, the infection in
these animals takes, therefore, the form of acute, general
septicemia.
The blood-serum of both pigeons and guinea-pigs that
have survived inoculation with this organism — i. e., that
have acquired immunity from it — is bactericidal in vitro
for this organism. It also possesses a certain degree of
immunity-conferring property, as may be demonstrated by
injecting it into normal pigeons and guinea-pigs that are
subsequently to be inoculated with virulent cultures.
Very old cultures of this organism in bouillon become
distinctly alkaline in reaction. At this stage they contain
a toxin that is markedly active for susceptible animals.
This toxin is not dissolved in the fluid to any extent, but
is apparently in intimate association with the proteid mat-
ters composing the bacteria.
Gastro-enteritis may be produced in both chickens and
guinea-pigs by feeding them with food with which cultures
of this organism have been mixed. (Gamalei'a.)
580 APPLICATION OF METHODS OF BACTERIOLOGY
MICROSPIRA SCHUYLKILLIENSIS, ABBOTT, 1896.
SYNONYM: Vibrio Schuylkilliensis, Abbott, 1896.
Abbott1 discovered a microspira in the water of the
Schuylkill River, at Philadelphia, and later, Bergey2 reports
the presence of the same organism, as well as several varieties
that are slightly different, in the waters of the Schuylkill
and Delaware rivers, along the entire city front, more
especially in the effluents of the sewers.
Microspira Schuylkilliensis is a short, rather plump
"comma," often with a very decided curve, with rounded
or slightly pointed ends. As usually seen it is a little shorter
and thicker than the microspira comma, though this feature
is quite variable. It is actively motile, having a single polar
flagellum. It does not form spores. It stains with the
ordinary aniline stains, but is negative to Gram's method.
The colonies on gelatin are sharply defined, distinctly
granular, and have usually fine irregular markings, as if
they were creased or folded. Sometimes they present
indistinct concentric markings. As growth progresses these
markings become more and more distinct and finally give
to the colony a decidedly lobulated or mulberry-like ap-
pearance.
After about the third or fourth day, when liquefaction is
actively in progress, the majority of the colonies lose their
characteristic appearance. They are seen as irregular,
ragged, granular masses lying in the center of pits of lique-
fied gelatin.
In stab cultures in gelatin the appearance of the growth is
essentially that of microspira comma, though at times it is
a little more rapid in progress.
1 Jour, of Exp. Med., 1896, i, 419. 2 Ibid., 1897, ii, 535.
MICROSPIRA SCHUYLKILLIENSIS 581
On meat-infusion agar-agar, neutral or slightly alkaline
to phenolphthalein, growth is very rapid at the body tem-
perature. The general character of the growth corresponds
to that of microspira comma.
The growth on blood serum, after twenty-four hours- at
body temperature, appears as a line of depression, which
increases as a track of liquefaction, and later results in the
more or less complete liquefaction of the medium.
Bouillon becomes uniformly clouded in twenty-four hours
at the body temperature. Its reaction becomes more alkaline
as growth progresses. A pellicle, at first delicate, later
denser, always characterizes the growth in this medium.
Usually no visible growth occurs on a potato.
In fresh litmus-milk a slight degree of acidity is noticed
after twenty-four hours at body temperature. After forty-
eight hours this acidity is slightly greater, and at tunes the
milk shows evidences of coagulation, though not always.
Microspira Schuylkilliensis is a facultative aerobe. In
fluid media under an atmosphere of carbon dioxide in sealed
tubes no growth is observed.
The organism grows most luxuriantly at about 37.5°
C. Growth is hardly perceptible at 10° C. It is destroyed
by an exposure of five minutes to 50° C.
None of the carbohydrates are broken up with the libera-
tion of gas.
It produces indol and at the same time reduces nitrates
to nitrites.
The pathogenic properties of this organism are best seen
in guinea-pigs and pigeons, both of which are uniformly
susceptible. Rabbits and chickens resist relatively large
doses. Mice are infected with small doses injected
subcutaneously.
582 APPLICATION OF METHODS OF BACTERIOLOGY
The most characteristic lesions follow the injection of
cultures into the pectoral muscles of pigeons. At death
the inoculated muscle is swollen, necrotic, and the overlying
tissues are edematous. The bacteria are found in large
numbers in the vicinity of the seat of the inoculation,
and in relatively small numbers in the blood and internal
organs.
CHAPTER XXVII.
Study of Bacterium Anthracis, and of the Effects Produced by Its Inocu-
lation into Animals — Peculiarities of the Organism Under Varying Con-
ditions of Surroundings — Anthrax Vaccines — Anthrax Immune Serum.
THE discovery that the blood of animals suffering from
splenic fever, or anthrax, always contains minute rod-shaped
bodies (Pollender, 1855; Davaine, 1863), led to a group of
investigations that have not only fully familiarized us with
the nature of this malady in particular, but have perhaps
contributed more, incidentally, to our knowledge of bac-
teriology in general than studies upon any other single
infective process or its causative agent.
The direct outcome of these investigations is that a rod-
shaped microorganism, now known as bacterium anthracis,
is always present in the blood of animals suffering from this
disease; that this organism can be obtained from the tissue
of those animals in pure cultures; and that such artificial
cultures of bacterium anthracis when introduced into the
bodies of susceptible animals can again produce a condition
identical with that found in the animal from which they
were obtained. The disease is a true septicemia, and after
death the capillaries throughout the body are always found
to contain the typical rod-shaped organism in larger or
smaller numbers.
This organism, when isolated in pure culture, is a bac-
terium which varies considerably in length, ranging from
short rods, 2 to 3^ in length, to longer threads, 20 to 25/x
(583)
584 APPLICATION OF METHODS OF BACTERIOLOGY
in length. In breadth it is from 1 to 1.25/z. Frequently
very long threads, made up of several rods joined end to
end, are seen.
When obtained directly from the body of an animal it
is usually in the form of short rods square at the ends. If
highly magnified, the ends are seen to be a trifle thicker
than the body of the cell and somewhat indented or concave,
suggestive of the joints of bamboo, peculiarities that help to
distinguish it from certain other organisms that are some-
what like it morphologically. (See Fig. 95.)
FIG. 95
Bacterium anthracis, highly magnified to show swellings and concavities at
extremities of the single cells.
When cultivated artificially at the temperature of the
body the bacterium of anthrax presents a series of very
interesting developmental phases.
The short rods grow into long threads, which may be
seen twisted or plaited together like ropes, each thread being
marked by the points of juncture of the segments com-
posing it. (Fig. 96, a and 6.) In this condition it remains
until alterations in its surroundings, the most conspicuous
being diminution of its nutritive supply, favor the produc-
tion of spores. When this stage begins changes in the proto-
plasm may be noticed; the bacteria become marked by
irregular granular bodies, which eventually coalesce into
BACTERIUM ANTHRACIS 585
glistening oval spores, one of which lies in nearly every
segment of the long thread, and gives to the thread the
appearance of a string of shining beads. (Fig. 97.) In
FIG. 96
§"*S«£
f «~^-
i *ft
TV
a '»* 6
Bacterium anthracis. Plainted and twisted threads seen in fresh-growing
cultures. .X about 400 diameters.
this stage they remain but a short time. The chains of
spores, which are held together by the remains of the cells
in which they formed, become broken up, and eventually
nothing but free oval spores, and here and there the remains
FIG. 97
Threads of bacterium anthracis containing spores. X about 1200 diameters.
of mature bacilli which have undergone degenerative changes,
can be found. In this condition the spores, capable of resist-
ing deleterious influences, remain and, unless their sur-
586 APPLICATION OF METHODS OF BACTERIOLOGY
roundings are altered, continue in this living, though inactive,
condition for a very long time. If again placed under favor-
able conditions, each spore will germinate into a mature cell,
and the same series of changes will be repeated until the
surroundings become again gradually unfavorable to develop-
ment, when spore-formation again takes place. Spore forma-
tion occurs only at temperatures ranging from 18° to 43° C.;
37.5° C. being the optimum. Under 12° C. they are not
formed. (Why?) This organism does not form spores in
the tissues of the living animal, its usual condition at this
FIG. 98
Colony of bacterium anthracis on agar-agar.
time being that of short rods; occasionally, however, some-
what longer forms may be seen.
The bacterium of anthrax is not motile.
Colonies of this organism, as seen upon agar-agar, present
a typical appearance, from which they have been likened
unto the head of Medusa. From a central point, which is
more or less dense/ consisting of a felt-like mass of long
threads irregularly matted together, the growth continues
outward upon the surface of the agar-agar (Fig. 98.) It
is made up of wavy bundles in which the threads are seen
to lie parallel or are twisted in strands like those of a rope;
BACTERIUM ANTHRACIS 587
sometimes they have a plaited arrangement. (See Fig. 96.)
These bundles twist and cross in all directions, and even-
tually disappear at the periphery of the colony. At the
extreme periphery of the colonies it is sometimes possible
to trace single bundles of these threads for long distances
across the surface of the agar-agar. The colony itself is
not circumscribed in appearance, but is more or less irregu-
larly fringed or ragged, or scalloped. To the naked eye
they look very much like minute pellicles of raw cotton that
have been pressed into the surface of the agar-agar.
As the colonies continue to grow they become more and
more dense and opaque, and granular and rough on the
surface. When touched with a sterilized needle one experi-
ences a sensation that suggests somewhat their matted
structure. They are never moist or creamy. The bit that
is taken up with the needle is always more or less ragged,
suggesting a tiny particle of moist blotting paper.
The colonies on gelatin at the earliest stages also present
the same wavy appearance; but this characteristic soon
becomes in part destroyed by the liquefaction of the gelatin
which is produced by the growing organisms. This allows
them to sink to the bottom of the fluid, where they lie as
irregular masses. Through the fluid portion of the gelatin
may be seen small clumps of growing bacteria, which look
very much like bits of cotton-wool.
In bouillon the growth is characterized by the formation
of flaky masses, which also have very much the appearance
of bits of raw cotton. Microscopic examination of one of
these flakes reveals the twisted and plaited arrangement of
the long threads.
On potato it develops rapidly as a dull, dry, granular,
whitish mass, which is more or less limited to the point of
588 APPLICATION OF METHODS OF BACTERIOLOGY
inoculation. On potato, at the temperature of the incubator,
spore-formation may be easily observed.
Stab- and slant-cultures on agar-agar present in general
the appearances given for the colonies, except that the
growth is much more extensive. The growth is always
more pronounced on the surface than down the track of the
needle.
On gelatin it causes liquefaction, which begins on the
surface at the point inoculated and spreads outward and
downward.
It grows best with access to oxygen, and very poorly when
the supply of that gas is interfered with.
Under favorable conditions of aeration, nutrition, and
temperature its growth is. rapid.
Under 12° C. and above 45° C. no growth occurs. Its
optimum temperature is that of the body, viz., 37°-38° C.
The spores of bacterium anthracis are very resistant to
heat, though the degree of resistance varies with spores
of different origin. It has been found repeatedly that
anthrax spores from some strains are readily killed by an
exposure of one minute to the temperature of steam, whereas
spores from others resist this temperature for as long as
twenty minutes and sometimes longer.
Staining. — Anthrax bacteria stain readily with the
ordinary aniline dyes. In tissues their presence may also
be demonstrated by the ordinary aniline staining fluid
or by Gram's method. They may also be stained in tissues
with a strong watery solution of dahlia, after which the
sections are decolorized in 2 per cent, sodium carbonate
solution, washed in water, dehydrated in alcohol, cleared
in xylol, and mounted in balsam. This leaves the bacilli
stained, while the tissues containing them are decolorized;
BACTERIUM ANTHRACIS 589
or the latter may be stained a contrast-color — with eosin,
for example — after dehydration in alcohol and before
clearing in xylol. In this case they must be washed again
in alcohol before using the xylol. In a preparation treated
in this way the rod-shaped organisms are of a purple color,
ano^ will be seen in the capillaries of the tissues, while the
tissues themselves are of a pale rose color.
Inoculation into Animals. — Introduce into the subcutaneous
tissues of the abdominal wall of a guinea-pig or rabbit a
portion of a pure culture of bacterium anthracis. The animal
usually succumbs in from thirty-six to forty-eight hours.
Little or no reaction at the immediate point of inoculation
will be noticed; but beyond this, extending for a long dis-
tance over the abdomen and thorax, the tissues will be
markedly edematous. Here and there, scattered through
this edematous tissue, small ecchymoses will be seen. The
underlying muscles are pale in color. Inspection of the
internal viscera reveals no very marked macroscopic changes
except in the spleen. This is enlarged, dark in color, and
soft. The liver may present the appearance of cloudy
swelling; the lungs may be red or pale red in color; the
heart is usually filled with blood. No other changes can
be seen by the naked eye.
Prepare cover-slip preparations from the blood and other
viscera. They will all be found to contain short rods in
large numbers. Nowhere can spore-formation be detected.
Upon microscopic examination of sections of the organs
which have been hardened in alcohol the capillaries are seen
to be filled with the bacteria; in some places closely packed
in large numbers, at other points fewer in number. Usually
they are present in largest numbers in those tissues having
the greatest capillary distribution and at those points at
590 APPLICATION OF METHODS OF BACTERIOLOGY
which the circulation is slowest. They are uniformly dis-
tributed through the spleen. The glomeruli of the kidneys
and the capillaries of the lungs are frequently packed with
them. The capillaries of the liver contain them in large
numbers. (Fig. 99.) Hemorrhages, probably due to
rupture of capillaries by the mechanical pressure of the
bacteria which are developing within them, not uncommonly
occur. When these occur in the mucous membranes of the
alimentary tract the blood may escape through the mouth
Bacterium anthracis in liver of mouse. X about 450 diameters. Bacteria
stained by Gram's method; tissue stained with Bismarck-brown.
or anus; when in the kidneys, through the uriniferous
tubules.
Cultures from the different organs or from the edematous
fluid about the point of inoculation result in growth of
bacterium anthracis.
The amphibia, dogs, and the majority of birds are not
susceptible to this disease. Rats are difficult to infect.
Rabbits, guinea-pigs, white mice, gray house-mice, sheep,
and cattle are susceptible. Infection may occur either
BACTERIUM ANTHRACIS 591
through the circulation, through the air-passages, through
the alimentary tract, or, as we have just seen, through the
subcutaneous tissues.
Protective Inoculation. — The most noteworthy application
of artificially prepared living vaccines to the protection of
animals from infection is, seen in connection with anthrax
in sheep and in bo vines.
By a variety of procedures the virulent anthrax bacterium
may be in part or totally robbed of its pathogenic properties.
It is through the very mild constitutional disturbance
caused in animals vaccinated with such weakened cultures
that protection is often afforded against the severer, fre-
quently fatal, form of the infection.
Without reviewing the various methods that have been
employed for attenuating the virulence of this organism to
a degree suitable for protective vaccination, it will suffice
to say that the most satisfactory results have been obtained
by the classical method of Pasteur. This comprehends the
long-continued cultivation, (ten to thirty days) at a tem-
perature of from 42° to 43° C. In this procedure the spore-
free, virulent bacterium anthracis, obtained directly from
the blood of a recently dead animal, is brought at once into
sterile nutrient bouillon in about twenty test-tubes, which
are immediately placed in an incubator that is carefully
regulated to maintain a temperature of 42.5° C. There
should not be a fluctuation of over 0.1° C.
After about a week a tube is removed from the incubator
on each successive day and its virulence tested at once on
animals. The degree of attenuation experienced by the
cultures grown under these circumstances is determined by
tests upon rabbits, guinea-pigs, and mice. The first culture
removed may or may not kill rabbits, the most resistant
592 APPLICATION OF METHODS OF BACTERIOLOGY
of the three animals used for the test, while it will certainly
kill the guinea-pigs and mice; after another two or three
days rabbits will no longer succumb to inoculation with the
culture last removed from the incubator, while no diminu-
tion will as yet be noticed in its pathogenesis for the other
two species. After four to seven ^days more a culture may
be encountered that kills only mice, the guinea-pigs escap-
ing; while ultimately, if the experiment be continued, a degree
of attenuation may be reached in which the organism has
not even the power of killing a mouse, though it still retains
its vitality. Investigation of these attenuations shows
them to possess all the characteristics of enfeebled anthrax
bacteria; they grow slowly and less vigorously when trans-
planted; they do not form spores when exposed to a high
temperature; and microscopically they present evidences
of degeneration. When introduced beneath the skin of
animals they disseminate but slightly beyond the site of
inoculation, and do not, as a rule, cause the general septicemia
that occurs in susceptible animals inoculated with normal
cultures of this organism. In the practical employment of
these attenuated cultures for protective purposes two
vaccines are employed. These were designated by Pasteur
as "first" and "second" vaccines. The "first" is the one
that killed only the mice in the preliminary tests; while
the " second" is that which killed both mice and guinea-pigs,
but failed to kill the rabbit. When larger animals, such as
sheep or cattle, are to be protected by vaccination with
these vaccines, a subcutaneous inoculation of about 0.3 c.c.
of the first vaccine is usually given. This should be prac-
tically without noticeable effect, causing neither rise of
body-temperature nor other constitutional or local symp-
toms. After a period of about two weeks the second vaccine
BACTERIUM ANTHRACIS 593
is injected in the same way; this may or may not cause
disturbance. In the event of its doing so the symptoms are
rarely alarming, and, if the vaccines have been properly
prepared and tested before use, all symptoms disappear
within a short time after the injection.
In the large majority of cases sheep, bovines, horses, and
mules may be safely protected against anthrax by the careful
practice of this method.
Sobernheim1 found that it was possible to bring about a
high degree of immunity against bacterium anthracis by
means of the vaccines 1 and 2 of Pasteur, with subsequent
inoculations of virulent organisms. He employed the serum
of animals thus immunized in the treatment of sheep that
had been injected with highly virulent anthrax bacteria.
Five sheep were treated in this way, and all of them recovered
with only slight rise in temperature and moderate infiltra-
tion at the point of injection, while control animals died
very promptly.
He further2 reports an improvement on the method of
protective inoculation against anthrax in which he uses a
combination of anthrax vaccines and immune serum, in
which the results are far more satisfactory than with the
anthrax vaccines alone. He states that this new method
has the following advantages over the Pasteur method:
(1) That the immunization can be carried out without
losing any of the animals; (2) that it can be completed
in one day; (3) that stronger and more active cultures
can be employed and therefore a more durable immunity
obtained; and (4) that the serum alone can be employed as
a curative agent.
1 Berliner klin. Wchnschr., 1897.
2 Ibid., 1902, p. 516.
38
594 APPLICATION OF METHODS OF BACTERIOLOGY
Anthrax Immune Serum. — Sanfelice1 experimented with
the serum of dogs that had been immunized from anthrax
bacteria. This serum possessed immunizing and curative
properties, as shown by experiments upon animals. He had
an opportunity of trying the serum, with favorable results,
upon a man who had contracted anthrax. The total amount
of serum employed was 56 cubic centimeters. There was
no reaction at the point of injection of the serum. The
therapeutic effect of the administration of serum was a
general improvement in the symptoms, marked fall of the
temperature on the second, and complete apyrexia on the
third day. The effect on the local anthrax lesion manifested
itself in reduction and, finally, disappearance of the edema,
followed first by an increased swelling of the glands, which
decreased again subsequently. He states that the serum
treatment should be continued not only till the temperature
has fallen to normal and a diminution of the edema is
apparent, but also until there is marked reduction in the
size of the swollen lymph-glands.
Sclavo2 immunized a number of animals, principally
sheep and goats, with the two vaccines of Pasteur, followed
by repeated injections of increasing quantities of virulent
cultures. By this means he obtained an immune serum
which had protective as well as curative properties when
tested upon guinea-pigs and rabbits.
Cicognani3 employed Sclavo's immune serum on 12 per-
sons suffering from various grades of anthrax infection,
some of the cases being severe infections in which the prog-
nosis would otherwise have been very unfavorable. The
1 Centralblatt f. Bacteriologie, Originate, 1902, Bd. xxxiii.
2 Bulletin de 1'Institut Pasteur, T. I., 1903, p. 305. .
3 Centralblatt f. Bacteriologie, 1902, ref. Bd. 31, p. 725,
BACTERIUM ANTHRACIS 595
duration of the disease was always very much shortened
and all recovered.
Lazaretti1 reports 23 cases of human infection with bac-
terium anthracis in which Sclavo's immune serum was
employed with recovery in each case. Another patient,
suffering from chronic alcoholism and malaria, did not
recover.
Experiments. — Prepare three cultures of bacterium an-
thracis— one upon gelatin, one upon agar-agar, and one upon
potato. Allow the gelatin culture to remain at the ordinary
temperature of the room, place the agar-agar culture in the
incubator, and the potato culture at a temperature not
above 18° to 20° C. Prepare cover-slips from each from
day to day. What differences are observed?
Prepare two potato cultures of bacterium anthracis.
Place one in the incubator and maintain the other at a
temperature of from 18° to 20° C. Examine them each day.
Do they develop in the same way?
From a fresh culture of bacterium anthracis, in which
spore-formation is not yet begun (which is the surest source
from which to obtain non-spore-bearing anthrax bacteria?),
prepare a hanging-drop preparation; also a cover-slip
preparation in the usual way and stain it with a strong
gentian- violet solution; and another cover-slip preparation
which is to be drawn through a flame twelve to fifteen times,
stained with aniline gentian-violet, washed in iodine solu-
tion and then in water. Examine these microscopically.
Do they all present the same appearance? To what are
the differences due?
1 Deutsche Viertel j ahrsschrif t f. offentliche Gesundheitspflege, 1903,
Bd. xxxv, Supplement, p. 253.
596 APPLICATION OF METHODS OF BACTERIOLOGY
Do the anthrax threads, as seen in a fresh, growing,
hanging drop, present the same morphological appearance
as when dried and stained upon a cover-slip? How do they
differ?
Liquefy a tube of agar-agar, and when it is at the tem-
perature of 40° to 43° C. add a very minute quantity of an
anthrax culture which is far advanced in the spore-stage.
Mix it thoroughly with the liquid agar-agar and from this
prepare several hanging drops under strict antiseptic pre-
cautions, using the fluid agar-agar for the drops instead
of bouillon or salt solution. Select from among these
preparations that one in which the smallest number of
spores are present. Under the microscope observe the
development of a spore into a mature cell. Describe care-
fully the developmental stages.
Prepare a 1 : 1000 solution of carbolic acid in bouillon.
Inoculate this with virulent anthrax spores. If no develop-
ment occurs after two or three days at the temperature of
the thermostat, prepare a solution of 1 : 1200, and continue
until the point is reached at which the amount of carbolic
acid present jwt permits of the development of the spores.
When the proper dilution is reached prepare a dozen of
such tubes and inoculate one of them with virulent anthrax
spores. As soon as development is well advanced transfer
a loopful from this tube into a second of the carbolic acid
tubes; when this has developed, then from this into a third,
etc. After five or six generations have been treated in this
way study the spore production of the organisms in that
tube. If it is normal, continue to inoculate from one car-
bolic acid tube to another, and see if it is possible by this
BACTERIUM ANTHRACIS 597
means to influence in any way the production of spores by
the organism with which you are working. What is the
effect, if any?
Prepare two bouillon cultures, each from one drop of
blood of an animal dead of anthrax. (Why from the blood
of an animal and not from a culture?) Allow one of them
to grow for from fourteen to eighteen hours in the incubator;
allow the other to grow at the same temperature for three
or four days. Remove the first tube after the time men-
tioned and subject it to a temperature of 80° C. for thirty
minutes. At the end of this time prepare four plates from
it. Make each plate with one drop from the heated bouillon
culture. At the end of three or four days treat the second
tube in identically the same way. How do the number of
colonies which develop from the two cultures compare?
Was there any difference in the time required for their
development on the plates?
From a potato culture of bacterium anthracis which has
been in the incubator for three or four days scrape away
the growth and carefully break it up in 10 c.c. of sterilized
physiological salt-solution. The more thoroughly it is
broken up the more accurate will be the results of the
experiment. Place this in a bath of boiling water, and at
the end of one, three, five, seven, and ten minutes make
plates upon agar-agar each with one loopful of the contents
of this tube. Are the results on the plates alike?
Determine the exact time necessary to sterilize objects,
such as silk or cotton threads, on which anthrax spores have
been dried, by the steam method and by the hot-air method.
598 APPLICATION OF METHODS OF BACTERIOLOGY
Prepare a bouillon culture from the blood of an animal
just dead of anthrax. After this has been in the incubator
for from three to four hours subject it to a temperature of
55° C. for ten minutes. At the end of this time make plates
from it and also inoculate a rabbit subcutaneously with it.
What are the results? Are the colonies on the plates in
every way characteristic?
Inoculate six Erlenmeyer flasks of sterile bouillon, each
containing about 35 c.c. of the medium, from the blood of an
animal just dead of anthrax.
Place these flasks in the incubator at a temperature of
42.5° C. At the end of five, ten, fifteen, twenty, twenty-five,
or more days remove a flask. Label each flask as it is
taken from the incubator with the exact number of days
that it has been at the temperature of 42.5 C. Study each
flask carefully, both in its culture peculiarities and in its
pathogenic properties when employed on animals.
Are these cultures identical in all respects with those that
have been kept at 37° C.?
If they differ, in what respect is the difference most con-
spicuous?
Should any of the animals survive the inoculations made
from the different cultures in the foregoing experiment,
note carefully which one it is, and after ten to twelve days
repeat the inoculation, using the same culture; if it again
survives, inoculate it with the culture preceding the one
just used in the order of removal from the incubator; if
it still survives, inoculate it with virulent anthrax. What is
the result? How is the result to be explained? Do the
cultures which were made from these flasks at the time of
their removal from the incubator act in the same way toward
BACTERIUM ANTHRACIS 599
animals as the organisms growing in the flasks? Is the
action of each of these cultures the same for mice, guinea-
pigs, and rabbits?
Prepare a 2 per cent, solution of sulphuric acid in dis-
tilled water; suspend in this a number of anthrax spores;
at the end of three, six, and nine days at 35° C. inoculate
both a guinea-pig and a rabbit. Prepare cultures from this
suspension on the third, sixth, and ninth days; when the
cultures have developed inoculate a rabbit and a guinea-
pig from the culture made on the ninth day. Should the
animals survive, inoculate them again after three or four
days with a culture made on the sixth day. Do the results
appear in any way peculiar?
CHAPTER XXVIII.
The Nitrifying Bacteria — The Bacillus of Tetanus — The Bacillus of Malig-
nant Edema — The Bacillus of Symptomatic Anthrax — Bacterium
Welchii — Bacillus Sporogenes.
THE NITRIFYING BACTERIA.
BY the employment of bacteriological methods in the
study of the soil much light has been shed upon the cause
and nature of the interesting and momentous biological
phenomena there constantly in progress. Of these, the one
of the greatest importance comprises those changes that
accompany the widespread process of disintegration and
decomposition, to which reference has already been made.
(See Chapter I.) This resolution of dead complex organic
compounds into simpler structures assimilable as food by
growing vegetation is dependent upon the activities of
bacteria located in the superficial layers of the ground. It
is not a simple process, brought about by a single, specific
species of bacteria, but represents a sequence of events each
of which probably- results from the activities of different
species or groups of species, working alone or together. Our
knowledge upon the subject does not permit of the following in
detail of the manifold alterations undergone by dead organic
material, but we do know that much of it is ultimately
converted into inorganic matters and that carbon dioxide,
ammonia and water are always conspicuous end products.
When the process of decomposition occurs in the soil it
does not cease at this point, but we find still further altera-
(600)
THE NITRIFYING BACTERIA 601
tions — alterations having to do more particularly with the
ammonia. This change in ammonia is characterized by the
products of its oxidation, viz., by the formation of nitrous
and nitric acids and their salts; this is not a result of the
direct action of atmospheric oxygen upon the ammonia,
but occurs through the instrumentality of a special group
of saprophytes known generically as the nitrifying organ-
isms. They are found in the most superficial layers of the
ground, and though more common in some places than in
others, they are, nevertheless, present over the entire surface
of the earth. The most conspicuous example of the func-
tional activity of this group of soil organisms is seen in the
immense saltpeter-beds of Chili and Peru, where, by the
activities of these microscopic plants, nitrates are produced
from the ammonia arising from the decomposing fecal
evacuations of sea-fowls and from decomposing seaweeds
in such enormous quantities as to form a source of supply
of crude saltpeter for the commercial world. A more
familiar example is seen in the decomposition and subse-
quent nitrification of the organic matters of sewage and
other fluid wastes of organic nature in the process of puri-
fication by percolation through the soil, a process in which
it is possible to follow, by chemical means, the organic
matters from their condition as such to their ultimate con-
version into inorganic forms of ammonia, nitrous and nitric
acids. In fact, the same breaking down and building up,
resulting ultimately in nitrification, occurs in all nitrogenous
matters that are deposited upon the soil and allowed to
decay. It is largely through this means that growing vege-
tation obtains the nitrogen necessary for the nutrition of its
tissues, and when Viewed from this standpoint we appre-
602 APPLICATION OF METHODS OF BACTERIOLOGY
ciate the importance of this process to all life, animal as
well as vegetable, upon the earth.
Under special circumstances there occurs in the soil a
process the reverse of nitrification, that is, a reduction of
nitrates and nitrites to lower compounds and ultimately
to free gaseous nitrogen. This so-called "denitrification,"
while the result of bacterial activity is not dependent upon
such specific varieties of bacteria as is nitrification. For
instance, true denitrification is known to be an attribute
of bacillus coli communis, of bacillus fluorescens lique-
faciens, of bacillus pyocyaneus, and of bacillus typhosus.
While this group of species ordinarily develop under
free access of oxygen they can develop without it and
secure their necessary oxygen from such oxides of nitro-
gen as nitrates and nitrites, thus reducing them. It seems
probable that certain products o'f bacterial growth have
also a reducing action on soil nitrates. Denitrification
occurs most often and most actively in soils containing an
excess of undecomposed organic matter.
In addition to nitrification and denitrification there is
seen in the soil a phenomenon resulting in "nitrogen fixa-
tion." In some instances this results from the symbiotic
activities of bacteria and higher plants,. in others it appears
to be peculiar to certain definite species of bacteria acting
alone. While a discussion of the extreme agricultural
importance of these phenomena would be of great interest,
yet this is scarcely the place to undertake it.1
The unusual nature of the nitrifying bacteria, demanding
as they do special methods for their cultivation, renders
1 See Bacteria in Relation to Country Life, by Lipman-MacMillan, 1911.
THE NITRIFYING BACTERIA 603
them of sufficient technical interest to justify — for purposes
of illustration — a more or less detailed description of one of
them.
These very important and interesting nitrifying organisms,
of which there appear to be several, evade all efforts to
isolate them from the soils and to cultivate them by the
methods commonly employed in bacteriological work.
They can be successfully studied only through the employ-
ment of special media.
The organism generally known as the nitro-monas of
Winogradsky will serve as an illustration: It is a short,
oval, and frequently almost spherical cell. It reproduces
by segmentation as usual for bacteria, but there is little
tendency for the daughter-cells to adhere together or to
form chains. In cultures they are commonly massed
together, by a gelatinous material, in the form of zooglea.
It does not form spores, and is probably not motile, though
Winogradsky believes he has occasionally detected it in
active motion. As has been stated, it does not grow upon
ordinary nutrient media, and cannot, therefore, be isolated
by the means commonly employed to separate different
species of bacteria. The most astonishing property of
this organism is its ability to grow and perform its specific
fermentative function in solutions devoid of organic matter.
It is believed to be able to obtain its necessary carbon from
carbon dioxide. For its isolation and cultivation Wino-
gradsky recommends the following solution:
Ammonium sulphate 1 gram
Potassium phosphate 1 gram
Pure water . 1000 c.c.
604 APPLICATION OF METHODS OF BACTERIOLOGY
To each flask containing 100 c.c. of this fluid is added from
0.5 to 1 gram of basic magnesium carbonate suspended in
a little distilled water and sterilized by boiling. One of the
flasks is then to be inoculated with a minute portion of the
soil under investigation, and after four or five days a small
portion is to be withdrawn, by means of a capillary pipette,
from over the surface of the layer of magnesium carbonate
and transferred to a second flask, and similarly after four
or five days from this to a third flask, and so on. As this
medium does not offer conditions favorable to the growth
of bacteria requiring organic matter for their development,
those that were originally introduced with the soil quickly
disappear, and ultimately only the nitrifying organisms
remain. These are seen as an almost transparent film
attached to the clumps and granules of magnesium carbonate
on the bottom of the flask.
For their cultivation upon a solid medium Winogradsky
employs a mineral gelatin, the gelatinizing principle of
which is silicic acid. A solution of from 3 to 4 per cent,
of silicic acid in distilled water, and having a specific gravity
of 1.02, remains fluid and can be preserved in flasks in this
condition. (Kiihne.) Gelatinization occurs after the
addition of certain salts to such a solution, and will be more
or less complete according to the proportion of salts added.
The salts that have given the best results and the method
of mixing them are as follows :
Ammonium sulphate 0.40 gram
Magnesium sulphate 0.05 gram
Calcium chloride trace.
Potassium phosphate 0.10 gram
b\ Sodium carbonate 0.6 to 0.90 gram
Distilled water 100.00 c.c.
THE NITRIFYING BACTERIA 605
The sulphates and chloride (a) are mixed in 50 c.c. of the
distilled water, and the phosphate and carbonate (6) in
the remaining 50 c.c., in separate flasks.
Each flask is then sterilized with its contents, which after
cooling are mixed; the mixture representing the solution
of mineral salts is to be added to the silicic acid, little by
little, until the proper degree of consistency is obtained (that
of ordinary nutrient gelatin). This part of the process is
best conducted in a culture-dish. If it is desired to separate
the colonies, as in an ordinary plate, the inoculation and
mixing of the material introduced must be done before
gelatinization is complete; if the material is to be distributed
over only the surface of the medium, then the mixture
must first be allowed to solidify.
By the use of the silicate-gelatin Winogradsky has isolated
from the gelatinous film in the bottom of fluids undergoing
nitrification a bacillus which he believes to be associated
with the nitro-monas in the nitrifying process.
The developments in this field of study are of such breadth
and importance that they can scarcely be comprehended
in a book of this character. For particulars the reader is
referred to the special books and journals dealing with the
subject.
In addition to the bacteria concerned in the various trans-
formation of nitrogen, there are occasionally present in the
soil microorganisms possessing disease-producing properties.
Conspicuous among these may be mentioned the bacillus
of malignant edema (vibrion septique of the French), the
bacillus of tetanus, and the bacillus of symptomatic anthrax
(Rauschbrand (Ger.); charbon symptomatique (Fr.)). It is
sometimes due to the presence of one or the other of these
606 APPLICATION OF METHODS OF BACTERIOLOGY
organisms that wounds to which soil has had access (crushed
wounds from the wheels of cars or wagons, wounds received
in agricultural work, gunshot wounds, etc.) are followed by
such grave consequences.
BACILLUS TETANI, NICOLAEER, 1884.
In 1884 Nicolaier produced tetanus in mice and rabbits
by the subcutaneous inoculation of particles of garden-
earth, and demonstrated that the pus produced at the point
of inoculation was capable of reproducing the disease in
other mice and rabbits. He did not succeed in isolating
the organism in pure culture. In 1884 Carle and Rattone,
and in 1886 Rosenbach, demonstrated the infectious nature
of tetanus as it occurs in man by producing the disease in
animals by inoculating them with secretions from the
wounds of individuals affected with the disease. In 1889
Kitasato obtained the bacillus of tetanus in pure culture,
described his method of obtaining it and detailed its bio-
logical peculiarities as follows :
Method of Obtaining. — Inoculate several mice subcu-
taneously with secretions from the wound of a case of
typical tetanus. This material usually contains not only
tetanus bacilli, but other organisms as well, so that at
autopsy, if tetanus results, there may be more or less sup-
puration at the seat of inoculation in the mice. In order
to separate the tetanus bacillus from the others that are
present the pus is smeared upon the surface of several
slanted blood serum or agar-agar tubes and placed at 37°
to 38° C. After twenty-four hours all the organisms will
have developed, and microscopic examination will usually
BACILLUS TETANI 607
reveal the presence of a few tetanus bacilli, recognizable
by their shape, viz., that of a small pin, with a spore repre-
senting the head. After forty-eight hours at 38° C. the
culture is subjected to a temperature of 80° C. in a water-
bath for from three-quarters to one hour. At the end of
this time series of plates or Esmarch tubes of slightly alkaline
gelatin are made with very small amounts of the culture
and kept in an atmosphere of hydrogen (see page 242).
They are then kept at from 18° to 20° C., and at the end of
about a week the tetanus bacillus begins to appear in the
form of colonies. After about ten days the colonies should
not only be examined microscopically, but each colony
that has developed in the hydrogen atmosphere should be
obtained in pure culture and again grown under the same
conditions. The colonies that grow only without oxygen,
and which are composed of the pin-shaped organisms,
must be tested upon mice. If they represent growth of the
tetanus bacillus, the typical clinical manifestations of the
disease will be produced in these animals.
In obtaining the organism from the soil much difficulty
is experienced. Here are encountered a number of spore-
bearing organisms that are facultative in their relation to
oxygen, and are therefore very difficult to eliminate; and
there is, moreover, one in particular that, like the tetanus
bacillus, forms a polar spore. This spore is, however,
much more oval than that of the tetanus bacillus, and gives
to the organism containing it more the shape of a javelin
(or clostridium, properly speaking) than that of a round-
headed pin, the characteristic shape of the spore-bearing
tetanus organism. It is non-pathogenic, and grows both
with and without oxygen, and should, consequently, not
608 APPLICATION OF METHODS OF BACTERIOLOGY
\
be mistaken for the latter bacillus. It must also be borne
in mind that there are occasionally present in the soil still
other bacilli which form polar spores, and which, when in
this stage, are almost identical in appearance with the tetanus
bacillus; but they will usually be found to differ from it
in their relation to oxygen, and they are also without disease-
producing properties.
FIG. 100
Bacillus teiani. A, vegetative stage; B, spore-stage, showing pin-shapes.
Morphology. — In the vegetating stage it is a slender rod
with rounded ends. It may appear as single rods, or, in
cultures, as long threads. It is motile, though not actively
so. The motility is rendered somewhat more conspicuous
by examining the organism upon a warm stage.
At the temperature of the body it rapidly forms spores.
These are round, thicker than the cell, and usually occupy
one of its poles, giving to the rod the appearance of a small
pin. (Fig. 100.) When in the spore-stage it is not motile.
BACILLUS TETANI
609
FIG. 101
It is stained by the ordinary aniline staining reagents.
It retains the color when stained by Gram's method.
Cultural Peculiarities. — It is an
obligate anaerobe, and cannot be
brought to development under access
of oxygen. It thrives in an atmos-
phere of pure hydrogen, but not in
one of carbonic acid.
It grows in ordinary nutrient
gelatin and agar-agar of a slightly
alkaline reaction. Gelatin is slowly
liquefied, with the coincident, pro-
duction of a small amount of gas.
Blood serum is not liquefied by its
growth.
The addition to the media of from
1.5 to 2 per cent, of glucose, 0.1
per cent, of indigo-sodium sulphate,
or 5 per cent, by volume of blue
litmus tincture favors its growth.
It grows well in alkaline bouillon
under an atmosphere of hydrogen.
Under artificial conditions it may
be cultivated through numerous gene-
rations without loss of virulence.
Appearance of the Colonies. — Colo-
nies of bacillus tetani on gelatin under
an atmosphere of hydrogen have,
Colonies of the tetanus
bacillus four days old,
made by distributing the
in their early stages, somewhat the organisms through a tube
of the colonies of the
appearance
common bacillus subtilis in their
earliest stages, viz., they have a
39
nearly filled with glucose-
gelatin. Cultivation in
an atmosphere of hydro-
gen. (From Frankel and
Pfeiffer.)
610 APPLICATION OF METHODS OF BACTERIOLOGY
dense, felt-like center surrounded by a fringe of delicate
radii. The liquefaction is so slow that the appearance is
retained for a relatively long time, but eventually becomes
altered. In very old colonies the entire mass is made
up of a number of distinct threads that give it the ap-
pearance of a common mould. (See Fig. 101.)
In stab-cultures made in tubes about three-quarters filled
with gelatin growth begins at about 1.5 to 3 cm. below the
surface, and gradually assumes the appearance of a cloudy,
linear mass, with prolongations radiating into the gelatin
from all sides. Liquefaction with coincident gas-production
results, and may reach almost to the surface of the gelatin.
Relation to Temperature and to Chemical Agents. — It grows
best at a temperature of from 36° to 38° C.; gelatin cultures
kept at from 20° to 25° C. begin to grow after three or four
days. In an atmosphere of hydrogen at from 18° to 20° C.
growth does not usually occur before one week. No growth
occurs below 14° C. At the temperature of the body spores
are formed in cultures in about thirty hours, whereas in
gelatin cultures at from 20° to 25° C. they do not usually
appear before a week, when the lower part of the gelatin
is quite fluid.
Spores of the tetanus bacillus when dried upon bits of
thread over sulphuric acid in the desiccator and subse-
quently kept exposed to the air, retain their vitality and
virulence for a number of months. Their vitality is not
destroyed by an exposure of one hour to 80° C.; on the
other hand, an exposure of five minutes to 100° C. in the
steam sterilizer kills them. They resist the action of 5 per
cent, carbolic acid for ten hours, but succumb when exposed
to it for fifteen hours. In the same solution, plus 0.5 per
cent, of hydrochloric acid, they are no longer active after
BACILLUS TETANI 611
two hours. They are killed when acted upon for three
hours by corrosive sublimate, 1 : 1000, and in thirty minutes
by the same solution plus 0.5 per cent, of hydrochloric acid.
Action upon Animals. — After subcutaneous inoculation
of mice with minute portions of a pure culture of this
organism tetanus develops in twenty-four hours and ends
fatally in from two to three days. Rats, guinea-pigs, and
rabbits are similarly affected, but only by larger doses than
are required for mice, the fatal dose for a rabbit being from
0.3 to 0.5 c.c. of a well-developed bouillon culture. The
period of incubation for rats and guinea-pigs is twenty-four
to thirty hours, and for rabbits from two to three days.
Pigeons are but slightly, if at all, susceptible.
The tetanic convulsions always appear first in the parts
nearest the seat of inoculation, and subsequently become
general.
At autopsies upon animals that have succumbed to
inoculations with pure cultures1 of bacillus tetani there is
little to be seen by either macroscopic or microscopic exami-
nation, and cultures from the site of inoculation are often
negative in so far as finding the tetanus bacillus is concerned.
At the site of inoculation there is usually only a hyperemic
condition. In uncomplicated cases there is no suppuration.
The internal organs do not present any macroscopic change,
and culture-methods of examination show them to be free
from bacteria. The death of the animal results from the
absorption of a soluble poison, either produced by the bac-
teria at the site of inoculation or, which seems more probable,
produced by the bacteria in the culture from which they are
1 Animals and human beings that have become infected with this organism
in the ordinary way commonly present a condition of suppuration at the
site of infection; this is not due, however, to the tetanus bacillus, but
to other bacteria that gained access to the wound at the time of infection.
612 APPLICATION OF METHODS OF BACTERIOLOGY
obtained and introduced with them into the tissues of the
animal at the time of inoculation. In support of the latter
hypothesis; mice have been inoculated with pure cultures
of this organism; after one hour the point at which the
inoculation was made was excised and the tissues cauterized
with a hot iron; notwithstanding the short time during
which the organisms were in contact with the tissues and
the subsequent radical treatment, the animals died after
the usual interval and with the typical symptoms of tetanus.
The poison produced by the tetanus bacillus, and to
which the symptoms of the disease are due, has been isolated
and subjected to detailed study; some of its toxic peculiari-
ties, as given by Kitasato, are as follows:1
"When cultures of this organism are robbed of their
bacteria by filtration through porcelain the filtrate contains
the soluble poison, and is capable, when injected into animals,
of causing tetanus.
"Inoculations of other animals with bits of the organs
of the animal dead from the action of the tetanus toxin
produce no result; but similar inoculations with the blood
or with the serous exudate from the pleural cavity always
result in the appearance of tetanus. The poison is, there-
fore, largely present in the circulating fluids.
" The greatest amount of poison is produced by cultivation
in fresh neutral bouillon of a very slightly alkaline reaction.
"The activity of the poison is destroyed by an exposure
of one and one-half hours to 55° C.; of twenty minutes to
60° C.; and of five minutes to 65° C.
"By drying at the temperature of the body under access
of air the poison .is destroyed; but by drying at the ordinary
iZeitschrift fur Hygiene, 1891, Bd. x, S. 267.
BACILLUS TETANI 613
temperature of the room, or at this temperature in the desic-
cator over sulphuric acid, it is not destroyed.
"Diffuse daylight diminishes the intensity of the poison.
Its intensity is preserved when kept in the dark.
"Direct sunlight robs it of its poisonous properties in
from fifteen to eighteen hours.
" Its activity is not diminished by diluting a fixed amount
with water or nutrient bouillon.
"Mineral acids and strong alkalies lessen its intensity."
The chemical nature of this poison is not positively known,
but its designation "Toxalbumen" is probably a misnomer,
for its reactions do not warrant its classification with the
albumins in the sense in which the word is commonly used.
When obtained in a concentrated form, its toxic properties
are seen to be altered by acids, by alkalies, by sulphuretted
hydrogen, and by temperatures above 70° C. Even when
carefully protected from light, moisture and air, it gradually
becomes diminished in strength, doubtless due to the forma-
tion of "toxons" and "toxoids," analogous to those observed
by Ehrlich in deteriorating diphtheria toxin. When freshly
prepared its potency is almost incredible, 0.00005 milligrams
being sufficient to cause fatal tetanus in a mouse weighing
15 grams.
The studies of Madsen1 demonstrate it to consist of two
physiologically distinct intoxicating compounds; the one,
a solvent of erythrocytes — a "tetanolysin;" the other, a
specific irritant which, through its influence upon the central
nervous system,2 accounts for the phenomena by which
1 Ueber Teanolysin, Zeitschrift fur Hygiene und Infektionskrankheiten,
1899, Bd. xxxii, S. 214.
2 See paper by Wassermann and Takaki, Berliner klinische Wochen-
schrift, 1898, No. 1, S. 5.
614 APPLICATION OF METHODS OF BACTERIOLOGY
tetanus is characterized; to this latter the designation
" tetanospasmin" is given. Madsen's observations, further-
more, confirm the deductions of Ehrlich concerning the
molecular structure of bacterial toxins in general, to the
effect that the molecule of tetanolysin, like that of diph-
theria toxin, is a complex of at least two physiologically
unlike groups; the one, characterized by its marked com-
bining tendencies (for antitoxin), the so-called haptophore
group; the other, distinguished for its intoxicating quality,
the so-called toxophore group.
Tetanus Antitoxin. — The principles involved in the induction
of the antitoxic state against diphtheria are likewise applicable
to tetanus; in fact, the fundamental observations upon the
generation of antitoxin in the living animal body were made in
the course of studies on tetanus; they were subsequently ap-
plied to the study of diphtheria, with the results already noted.
It is needless to enter here upon the details essential to the
production of tetanus antitoxin; to all intents and purposes,
they are identical with those given in the section on diph-
theria. Briefly stated, animals may be rendered immune
from tetanus by the repeated injection of gradually increas-
ing non- fatal doses of tetanus toxin; when immunity is
established, the circulating blood contains a body, anti-
toxin, that combines directly with tetanus toxin in a test-
tube, and thereby renders it physiologically inactive (non-
intoxicating); and the serum of the immune animal is not
only capable of protecting non-immune, susceptible animals
from the poisonous action of tetanus toxin (within limits),
but also against the effects of the living tetanus bacillus as
well.
Tetanus antitoxin, though the first antitoxin discovered
BACILLUS TETANI 615
and frequently employed in the treatment of tetanus, has
not yielded as brilliant results as those obtained with diph-
theria antitoxin. There are good reasons why tetanus
antitoxin may never be expected to yield such satisfactory
results as does diphtheria antitoxin. Diphtheria infection
can be recognized by bacteriological methods and the anti-
toxin administered long before very marked constitutional
symptoms have developed, and consequently long before
the diphtheria toxin has had time to bring about serious
tissue alterations. In tetanus it is impossible to make such
a definite bacteriological examination, and very frequently
the first suggestion of the disease is the twitching of the
muscles, the antecedent sign of the tetanic convulsions.
When these clinical manifestations have developed in tetanus
there is already very serious involvement of the central
nervous system.
In the use of tetanus antitoxin it is advisable to employ
it as early as possible and to give repeated doses until the
symptoms are relieved. Whether the subdural adminis-
tration of the antitoxin will be of greater value than the
subcutaneous administration is as yet undecided.
A great deal of benefit results, from the administration
of tetanus antitoxin as a prophylactic in the treatment of
wounds in which infection by the tetanus bacillus is possible.
The prophylactic injection of the tetanus antitoxin in these
cases, however, should always be accompanied by approved
surgical treatment of the wound, and under these conditions
it is more or less doubtful which of these measures is of
the greater value, but experience seems to indicate that the
antitoxin has a distinct prophylactic influence in these cases.
616 APPLICATION OF METHODS OF BACTERIOLOGY
BACILLUS EDEMATIS, LIBORIUS, 1886.
The bacillus of malignant edema, also known as vibrion
septique, is another pathogenic form almost everywhere
present in the soil. In certain respects it is a little like
bacterium anthracis, and was at one time confounded with
it; but it differs in the marked peculiarity of being a strict
anaerobe. It was first observed by Pasteur, but it was not
until later that Koch, Laborious, Kitt, and others described
its peculiarities in detail. It can often be obtained by
inserting under the skin of rabbits or guinea-pigs small
portions of garden-earth, street-dust, or decomposing
organic substances. There results a widespread edema,
with more or less gas-production in the tissues. In the
edematous fluid about the site of inoculation the organism
under consideration may be detected. (Fig. 102, A.)
It is a rod about 3 to 3.5/z long and from 1 to 1.1 ju thick
— i. e., it is about as long as bacterium anthracis, but is a
trifle more slender. It is usually found in pairs, joined end
to end, but may occur as longer threads; particularly is
this the case in cultures. When in pairs the ends that
approximate are squarely cut, while the distal extremities
are rounded. When occurring singly both ends are rounded.
(How does it differ in this respect from bacterium anthracis?)
It is slowly motile, and its flagella are located both at the
ends and along the sides of the rod. It forms spores that
are usually located in or near the middle of the cells, causing
frequently a swelling at the points at which they are located
and giving to the cell a more or less oval, spindle, or lozenge
shape. (Fig. 102, B.)
It is an obligate anaerobe, growing on all the ordinary
BACILLUS EDEMATIS
617
media, but not with access of oxygen. It grows well in an
atmosphere of hydrogen. It causes liquefaction of gelatin.
In tubes containing about 20 to 30 c.c. of gelatin that
has been liquefied, inoculated with a small amount of the
FIG. 102
a
'*•
'•*
Bacillus edematis. A, edema-fluid, from site of inoculation of guinea-pig,
showing long and short threads; B, spore-formation, from culture.
culture, and then rapidly solidified in ice-water, growth
appears in the form of isolated colonies at or near the bottom
of the tube in from two to three days at 20° C. These
colonies, when of from 0.5 to 1 mm. in diameter, appear as
spheres filled with clear liquid, and are difficult, for this
618 APPLICATION OF METHODS OF BACTERIOLOGY
FIG. 103
reason, to detect. (Fig. 103.) As they gradually increase
in size the contents of the spheres become cloudy and
marked by fine radiating stripes, easily
to be detected with the aid of a small
hand-lens. In deep stab-cultures in agar-
agar and in gelatin development occurs
only along the track of puncture, at a
distance below the surface. Growth is
frequently accompanied by the produc-
tion of gas-bubbles.
It causes rapid liquefaction of blood
serum, with production of gas-bubbles,
and in two or three days the entire
medium may have become converted
into a yellowish semifluid mass.
The most satisfactory results in the
study of the colonies are obtained by the
use of plates of nutrient agar-agar kept
in a chamber in which all oxygen has
been replaced by hydrogen. The colo-
nies appear as dull whitish points, irreg-
ular in outline, and when viewed with a
low-power lens are seen to be marked by
a net-work of branching and interlacing
lines that radiate in an irregular way
from the center toward the periphery.
It grows well at the ordinary tempera-
ture of the room, but reaches its highest
development at the temperature of the
body.
It stains readily with the ordinary aniline dyes. It does
not stain by Gram's method.
Colonies of the ba-
cillus of malignant
edema in deep gela-
tin culture. ' (After
FrankelandPfeiffer.)
BACILLUS EDEMATIS 619
Pathogenesis. — The animals known to be susceptible to
inoculation with this organism are man, horses, calves,
dogs, goats, sheep, pigs, chickens, pigeons, rabbits, guinea-
pigs, and mice. Cases are recorded in which men and horses
have developed the disease after injuries, doubtless due to
the introduction into the wound, at the time, of soil or dust
containing the organism.
If one introduce into a pocket beneath the skin of a sus-
ceptible animal about as much garden-earth as can be held
upon the point of a penknife, the animal frequently dies in
from twenty-four to forty-eight hours. The most conspic-
uous result found at autopsy is a wide-spread edema at
and about the site of inoculation. The edematous fluid is
in some places clear, while at others it may be stained with
blood; it is usually rich in bacilli (Fig. 102, A) and contains
gas-bubbles. Of the internal organs only the spleen shows
much damage. It is large, dark in color, and contains
numerous bacilli. If the autopsy be made immediately
after death, bacilli are rarely found in the blood of the
heart; but if deferred for several hours, the organisms will
be found in this locality also, a fact that speaks for their
multiplication in the body after death. At the moment of
death they are present in varying numbers in all the internal
viscera and on the serous surfaces of the organs.
Of all animals mice are probably the most susceptible
to the action of this organism, and it is not rare to find it
in the heart's blood, even immediately after death. They
die, as a result of these inoculations, in from sixteen to
twenty hours.
When a pure culture is used for inoculation a relatively
large amount must be employed, and this should be deposited
in the subcutaneous tissues at some distance from the surface.
620 APPLICATION OF METHODS OF BACTERIOLOGY
In continuing the inoculations from animal to animal
small portions of organs or a few drops of the edema-fluid
should be used. The inoculation may also be successfully
made by introducing into a pocket in the skin bits of steril-
ized thread or paper upon which cultures. have been dried.
The methods for obtaining the organism in pure culture,
from the cadaver of an animal that has succumbed to infec-
tion by the bacillus of malignant edema, are in all essential
respects the same as those given for obtaining cultures
from tissues in general; but it must be remembered that
the organism is a strict anaerobe, and will not grow under
the influence of oxygen. (See methods of cultivating
anaerobic species.)
In certain superficial respects this bacillus suggests, as
said above, bacterium anthracis, but differs from it in so
many important details that there is no excuse for con-
founding the two.
NOTE. — From what has been said of this organism, what
are the most important differential points between it and
bacillus anthracis? Inoculate several mice with small por-
tions of garden-earth and street-dust. Isolate the organism
that agrees most nearly with the description here given for
the bacillus of malignant edema. Compare its morpholog-
ical, biological, and pathogenic peculiarities with those of
bacillus anthracis under similar circumstances; especially
its appearance in the tissues and fluids.
Still another pathogenic organism that may be present
in the soil is : —
BACILLUS CHAUVEI
621
BACILLUS CHAUVEI, ARLOING, CORNEVIN, AND
THOMAS, 1887.
SYNONYMS: The bacillus of symptomatic anthrax — Bacterie du cha'rbon
symptomatique (Fr.) — Bacillus des rauschbrand (Ger.).
It is the organism concerned in the production of the
disease of young cattle and sheep commonly known as
"black leg/' "quarter evil," and "quarter ill," a disease
FIG. 104
Bacillus of symptomatic anthrax. A, vegetative stage — gelatin culture
B, spore-forms — agar-agar culture.
that prevails in certain localities during the warm months,
and which is characterized by a peculiar emphysematous
swelling of the muscular and subcutaneous cellular tissues
over the quarters. The muscles and cellular tissues at the
points affected are seen on section to be saturated with
bloody serum, and the muscles particularly are of a dark,
almost black color. In these areas, in the bloody transu-
622 APPLICATION OF METHODS OF BACTERIOLOGY
FIG. 105
dates of the serous cavities, in the bile, and, after death,
in the internal organs, the organism to be described can
always be detected. It is manifest from
this that the soil of localities over which
infected herds are grazing may readily
become contaminated through a variety
of channels, and thus serve as a
source of further dissemination of the
disease.
The organism was first observed by
Feser, and subsequently by Bollinger
and others. The most complete de-
scription of its morphological and bio-
logical peculiarities is that of Kitasato.1
The following is from Kitasato's contri-
butions: it is an actively motile rod
about 3 to 5/z long by 0.5 to 0.6/z
thick. It has rounded ends, and, as a
rule, is seen singly, though now and then
pairs joined end to end may occur. It
has no tendency to form very long
threads. (Fig. 104, A)
It forms spores, and when in this stage
is seen to be slightly swollen at or near
Colonies of the one of its poles, the location in which
the SPMe USUally aPPearS' ^- 104,
B.) It is markedly prone to undergo
, ... , , . , ,.
degenerative changes, and involution-
forms are commonly seen not only in
fresh cultures, but in «the tissues of affected animals as
well.
iZeitschrift fur Hygiene. Bd. vi, S. 105; 13d. viii, S. 55.
deep gelatin culture.
(After Frankel and
Pfeiffer.)
BACILLUS CHAUVEI 623
Though actively motile when in the vegetative stage, it,
like all other motile spore-forming bacilli, loses this property
and becomes motionless when spores are forming.
It is strictly anaerobic and cannot be cultivated in an
atmosphere in which free oxygen is present. It grows best
under hydrogen, and does not grow under carbonic acid.
The media most favorable to its growth are those con-
taining glucose (1.5 to 2 per cent.), glycerin (4 to 5 per
cent.), or some other reducing-body, such as indigo-sodium
sulphate, sodium formate, etc.
When cultivated upon gelatin plates in an atmosphere of
hydrogen the colonies appear as irregular, slightly lobu-
lated masses. After a short time liquefaction of the gelatin
occurs and the colony presents a dark, dense, lobulated and
broken center, surrounded by a much more delicate, fringe-
like zone.
When distributed through a deep layer of liquefied gelatin
that is subsequently solidified colonies develop at only the
lower portions of the tube. The single colonies appear as
discrete globules that cause rapid liquefaction of the gelatin,
and ultimately coalesce into irregular, lobulated liquid
areas. In some of the larger- colonies an ill-defined, concen-
tric arrangement of alternate clear and cloudy zones can
be made out (Fig. 105).
In deep stab-cultures in gelatin growth begins after about
two or three days at 20° to 25° C. It begins usually at
about one or two centimeters below the surface, and causes
slow liquefaction at and around the track of its development.
During its growth gas-bubbles are produced.
In deep stab-cultures in agar-agar at 37° to 38° C. growth
begins in from twenty-four to forty-eight hours, also at
about one or two centimeters below the surface, and is
624 APPLICATION OF METHODS OF BACTERIOLOGY
accompanied by the production of gas-bubbles. There is
produced at the same time a peculiar, penetrating odor
somewhat suggestive of that of rancid butter. Under these
conditions spores are formed after about thirty hours.
It grows well in bouillon of very slightly acid reaction
under hydrogen, but does not retain its virulence for so
long a time as when cultivated upon solid media. In this
medium it develops in the form of white flocculi that sink
ultimately to the bottom of the glass and leave the super-
natant fluid quite clear. If the vessel be now gently shaken,
these delicate flakes are distributed homogeneously through
it. In bouillon cultures there is often seen a delicate ring
of gas-bubbles round the point of contact of the tube and
the surface of the bouillon. There is produced also a pecu-
liar, penetrating, sour or rancid odor.
It grows best at the body-temperature — i. e., from 37° to
38° C. — but can also be brought to development at from
16° to 18° C. Below 14° C. no growth is seen. Spore-
formation appears much sooner at the higher than at the
lower temperatures. When its spores are dried upon bits
of thread in the desiccator over sulphuric acid, and then
kept under ordinary conditions, they retain their vitality
and virulence for many months. Similarly, bits of flesh
from the affected areas of animals dead of this disease, when
completely dried, are seen to retain for a long time the power
of reproducing the disease. The spores are tolerably resist-
ant to the influence of heat: when subjected to a tempera-
ture of 80° C. for one hour their virulence is not affected,
but an exposure to 100° C. for five minutes destroys them.
They are also seen to be somewhat resistant to the action
of chemicals: when exposeo! to 5 per cent, carbolic acid
they retain their disease-producing properties for about
BACILLUS CHAUVEI 625
ten hours, tvhereas the vegetative forms are destroyed in
from three to five minutes; in corrosive sublimate solution
of the strength of 1 : 1000 the spores are killed in two hours.
When gelatin cultures are examined microscopically the
organisms are usually seen as single rods with rounded ends.
When cultivated in agar-agar at a higher temperature
spores are formed after a short time; the spores are oval,
slightly flattened on their sides, thicker than the bacilli,
and, as stated, frequently occupy a position inclining to
one of the poles of the bacillus, though they are as often
seen in the middle.
Bacilli containing spores are usually clubbed or spindle
shape.
This bacillus stains readily with the ordinary aniline
dyes. It is decolorized by Gram's method. Its spores
may be stained by the methods usually employed in spore-
staining.
Pathogenesis. — When susceptible animals, especially
guinea-pigs, are inoculated in the deeper subcutaneous
cellular tissues with pure cultures of this organism, or with
bits of tissue from the affected area of another animal dead
of the disease, death ensues in from one to two days. It
is preceded by rise of temperature, loss of appetite, and
general indisposition. The site of inoculation is swollen
and painful, and drops of bloody serum may sometimes be
seen exuding from it. At autopsy the subcutaneous cellular
tissues and underlying muscles present a condition of
emphysema and extreme edema. The edematous fluid is
often blood-stained and the muscles are of a blackish or
blackish-brown color. The lymphatic glands are markedly
hyperemic. The internal viscera present but little altera-
tion visible to the naked eye. In the blood-stained serous
40
626 APPLICATION OF METHODS OF BACTERIOLOGY
fluid about the point of inoculation short bacilli are present
in large numbers. These often present slight swellings at
the middle or near the end. They are not seen as threads,
but lie singly in the tissues. Occasionally two will be seen
joined end to end. If the autopsy be made immediately
after death, these organisms may not be detected in the
internal organs; but if not made until after a few hours,
they will be found there also. In recent autopsies only
vegetative forms of the organism may be found; but later
(in from twenty to twenty-four hours) spore-bearing rods
may be detected. (How does this compare with bacterium
anthracisf) By successive inoculations of susceptible
animals with serous fluid from the site of inoculation of the
dead animal the disease may be reproduced.
Cattle, sheep, goats, guinea-pigs, and mice are susceptible
to infection with this organism, and present the conditions
above described; whereas horses, asses, and white rats
present only local swelling at the site of inoculation. Swine,
dogs, cats, rabbits, ducks, chickens, and pigeons are, as a
rule, naturally immune from the disease.
Though closely simulating the bacillus of malignant
edema in many of its peculiarities, this organism can
nevertheless, be readily distinguished from it. It is smaller;
it does not develop into long threads in the tissues; it is
more actively motile, and forms spores more readily in the
tissues of the animal than does the bacillus of malignant
edema. In their relation to animals they also differ; for
instance, cattle, while conspicuously susceptible to symp-
tomatic anthrax, are practically immune from malignant
edema; and while swine, dogs, rabbits, chickens, and
pigeons are readily infected with malignant edema, they are
not, as a rule, susceptible to symptomatic anthrax. Horses
BACTERIUM WELCHI, MIGULA 627
are affected only locally, and not seriously, by the bacillus
of symptomatic anthrax; but they are conspicuously sus-
ceptible to both artificial inoculation and natural infection
by the bacillus of malignant edema.
The distribution of the two organisms over the earth's
surface is also quite different. The edema bacillus is present
in almost all soils, while the bacillus of symptomatic anthrax
appears to be confined to certain localities, especially places
over which infected herds have been pastured.
A single attack of symptomatic anthrax, if not fatal,
affords subsequent protection; while infection with the
malignant edema bacillus appears to predispose to recurrence
of the disease. (Baumgarten.)
BACTERIUM WELCHH, MIGULA, 1900.
SYNONYM: Bacillus aerogenes capsulatus, Welch and Nuttall, 1892.
This organism consists of straight or slightly curved rods
with rounded ends, somewhat thicker than bacterium an-
thracis, varying in length from 3 to 6/z; sometimes longer
chains or threads are seen. The rods are surrounded by a
transparent capsule, whether grown in artificial media or
obtained from animal bodies. It is a non-motile, spore-
forming organism, and is strictly anaerobic in character.
It stains with the ordinary aniline dyes and by the Gram
method.
Under anaerobic conditions the organism grows on the
usual culture media at room temperature, and forms large
quantities of gas in media containing carbohydrates. Gela-
tin is not liquefied. In agar-agar the colonies are usually
from 1 to 2 millimeters in diameter, but may be as large as
1 centimeter in diameter. They have a grayish-white color,
628 APPLICATION OF METHODS OF BACTERIOLOGY
are flat, round or irregular masses, with small hair-like pro-
jections from the margin. In bouillon there is a diffuse
clouding and marked white sediment. Milk is quickly
coagulated. On potato there is a grayish-white layer.
The organism grows more rapidly at 30° to 37° C. than
at 18° to 20° C. Cultures on agar-agar and bouillon have
a slight odor resembling old lime.
Bacterium Welchii was first described by Welch in 1891,
and subsequently by Welch and Nuttall1 in the blood and
internal organs of a patient with thoracic aneurism opening
externally. Autopsy was made eight hours after death and
the vessels were found to contain large numbers of gas
bubbles.
Injections of considerable quantities of cultures into the
circulation of rabbits did not kill the animals, but if the
animals were killed after being inoculated and were then
allowed to lie at room temperature for twenty-four hours
the organs and tissues were filled with gas bubbles.
Welch, Howard, Hitschman and Lilienthal, Hirschberg,
and others have shown that the organism is frequently
present in the feces of man and animals, as well as in the
soil and in dust. Schattenfroh and Grassberger also found
the organism in market milk.
BACILLUS SPOROGENES (KLEIN), MIGULA, 1900.
SYNONYM: Bacillus enteritidis sporogenes, Klein, 1895.
Klein found this organism in the intestinal discharges of
infants and believed it had some relation to the acute
inflammatory conditions of the intestinal tract of bottle-fed
«:; i Bulletin Johns Hopkins Hospital, No. 24, 1892.
THE SPIROCH&TACEM 629
infants. The organism is very generally distributed in
nature and can be very readily isolated from sewage by
appropriate methods. It is an anaerobic, spore-forming
organism, 0.8/JL in width, and 1.6 to 4.8/z in length. It is
actively motile and flagella have been demonstrated.
In culture media containing carbohydrates this organism
produces gas in large quantities. Russell analyzed the gas
and found it to be composed principally of methane. Milk
and other sugar media in which the organism has been
grown have a distinct odor of butyric acid.
When injected subcutaneously into guinea-pigs this
organism causes most marked alterations. There is intense
inflammation at the point of injection with edema and
necrosis and the surrounding tissues are filled with gas.
The bacteria are distributed throughout the body of the
animal and can be isolated in pure culture from the blood of
the heart. All the internal organs are intensely congested.
THE SPIROCHJETACE-ffi.
The spirochsetacese may be roughly defined as a family
of the unicellular non-nucleated, spiral organisms, of the
order spirochetales ,! which may or may not possess an undu-
lating membrane as an extension from a central filament
and which may multiply in some instances by transverse,
in others by longitudinal division.
This is scarcely the place to discuss in detail the various
opinions that have been expressed since the tune of Ehren-
berg concerning the true nature of this group of micro-
1 Preliminary Report, Committee on Classification, etc., Soc. Am. Bact.,
Jour. Bacteriol., 1917, No. 5, vol. ii. Studies in Classification, etc., by
R. E. Buchanan, Jour. Bacteriol., 1918, No. 6, vol. iii.
630 APPLICATION OF METHODS OF BACTERIOLOGY
organisms. It will suffice to say for our purposes that as
yet there is no agreement as to their exact status in the world
of living things. They appear to occupy a position some-
where between the bacteria on the one hand and the protozoa
on the other.
A few of them are susceptible of cultivation under arti-
ficial conditions, and on these we possess at least the begin-
nings of an understanding, while for many others which
cannot be cultivated (or, at least, have not been) we know
little more than their gross morphological characteristics.
A certain number of them are found in association with
particular diseases of man and of animals and are believed
to stand in causal relation to such conditions.
Many more are found living free in nature and are
regarded as of no significance, insofar as disease production
is concerned.
The definite causal relationship of a few of them to cer-
tain diseases is now generally accepted as proved.
To the student of etiology three genera in particular of
the spirochaetacese are of special interest, notably: the spi-
ronema, the treponema and leptospira.1
Genus Spironema. — To this genus belong all those species
or varieties that have been seen in the several manifesta-
tions of relapsing fever in man; such for instance as spi-
ronema carteri, spironema obermaieri, spironema novyi,
spironema duttoni; also those found in the so-called relaps-
ing fever of fowls, spironema gallinarum, spironema anserina;
also those found in the mouth, spironema buccalis, spironema
refringens, spironema vincenti and a number of others.
They are seen as wavy, irregular threads with pointed
1 See Noguchi, Jour. Exper. Med., 1918, vol. xxvii, p. 575.
Fig. 106
Spironema, Blood, Relapsing Fever, Giemsa Stain.
Fig. 1O7
Treponema Pallida, Syphilis, Silver Preparation.
THE SP1ROCH&TACE& 631
ends; varying in length from 8 to 16 /z. and in thickness
from 0.3 to 0.5 ju. They possess a delicate undulating
membrane, though this is rarely seen in stained preparations.
They are motile but not flagellated. They multiply by
transverse and occasionally by longitudinal division.
When stained by the Giemsa method they appear as
violet threads (see Fig. 106). When obtained in artificial
cultivation they are said (Noguchi) to be soluble in 10
per cent, bile salts; to be immobilized and ultimately dis-
integrated by 10 per cent, saponin.
Genus Treponema. — To this genus belong the species
treponema pallada causing syphilis and treponema pertenue
causing frambesia tropica (yaws).
They are seen in stained preparations and by dark field
illumination as tightly wound, short, rigid spirals. They
vary in length from 6 to 14 /*• and in thickness of the thread
from 0.2 to 0.3 p. No undulating membrane is observed
(see Fig. 107).
Multiply by transverse and possibly also by longitudinal
division.
The spirals stain pink by the Giemsa method.
When obtained in culture (Noguchi) they are dissolved
by 10 per cent, bile salts and more slowly by 10 per cent,
saponin.
Genus Leptospira. — Type species leptospira icerto hemor-
rhagise — found by Inado and Ido in infectious jaundice.
The genus also includes leptospira icteroides found by
Noguchi in yellow fever1 and described by him as very
small, regular, rigid spirals with hook-like prolongations
at one or both ends. They vary in length from 7 to 14 /z.
1 See Jour. Exper. Med., 1919, vol. xxx, p. 13.
632 APPLICATION OF METHODS OF BACTERIOLOGY
and in thickness from 0.2 to 0.3 /z. The whole organism
is very flexible and has highly motile end-portions. No
undulating membrane'.
Divide's transversely.
Stains reddish violet by the Giemsa method (see Fig.
108). When obtained in culture it is soluble in 10 per
cent, bile salts; insoluble in 10 per cent, saponin.
Detection by Microscopic Examination. — The methods
commonly employed in searching for these organisms are:
The microscopic examination of fresh unstained prepara-
tions by means of the dark-field illumination (see the
method), or by the examination of stained smears. In
the former method the organisms are recognized as color-
less, brightly illuminated, motile threads or spirals that
stand out in striking contrast to the almost black field
through which they are moving. If the necessary equip-
ment is available this is by far the quickest and most direct
method of getting positive indications.
By the latter method the organisms are seen as. fixed,
stained undulating threads or spirals according to their
nature. In this method the examination is of smears made
as follows: The smears are made in the routine way on
either clean cover slips or slides. They are then allowed
to dry in the air, after which they are fixed in pure methyl
alcohol for three or four minutes. They may then be stained
by the Giemsa method or by some one or another of the
silver impregnation, methods.
STAINING SOLUTION.
Azure II (eosin) 0.30 grams
Azure II . ... 0.08 u
Fig. 108
Leptospira leteroides. Blood of Experiment Guinea-pig.
(After Noguehi)
THE SPIROCHMTACEM 633
Mix with 25 c.c. of pure anhydrous glycerin at 60° C.
When dissolved add 25 c.c. of pure methyl alcohol, also at
60° C., allow mixture to stand over-night, then filter.
To stain the smears take from the above "stock" solution
1 c.c. and mix with 10 or 12 c.c. of 1 : 1000 potassium car-
bonate in distilled water. Pour this diluted stain over
the smears and allow to stand for from a quarter to half
an hour. If the smear be very thick, exposure to the stain
should be longer. Wash repeatedly in clean water until the
smear has in general a pink tinge. This clearing up may be
hastened in thick smears by immersion for an instant in
methyl alcohol, followed by repeated washings in water.
When dried, the preparations are examined in the way
common to microscopic examination of bacterial prepara-
tions.
Silver Impregnation. Stern's Method. — Prepare smear;
dry in incubator at 37° to 38° C. for several hours (do not
heat over flame).
Immerse in 10 per cent, silver nitrate solution and expose
to diffuse day-light (not direct sun-light) for from several
hours to several days. When the preparation is of a deep
brown color and shows a metallic sheen, wash thoroughly
in clean water, dry and mount for examination.
The organisms are seen as black threads or spirals in a
brown field.
India Milk Method. — Mix a drop of the suspected blood
or exudate with a drop of India milk on one end of a slide.
With the edge of another slide draw this quickly down the
length of the slide so as to make an even tolerably thin film.
Allow to dry. Examine by the usual method. Any organ-
isms present will appear as colorless objects in an almost
black field. The picture is that of a photographic negative.
634 APPLICATION OF METHODS OF BACTERIOLOGY
Levaditis Method for Tissues. — Fix bits of suspected tissue,
not over 2 m.m. thick, in 10 per cent, formalin for one or
two days. Wash out in 95 per cent, alcohol for eighteen to
twenty hours. Wash thoroughly in distilled water until the
tissues no longer float.
Impregnate in:
Nitrate of silver 1 gram
Pyridin 10 grams
Distilled water 100 c.c.
Made up fresh.
Allow to stand at room temperature for two or three
hours, followed by 50° C. for from four to six hours. Wash
rapidly in 10 per cent, pyridin, after which reduce the silver
,by immersing the tissue for several hours in:
Pyrogallic acid ...;.".,.... 4 grams
Acetone 10 "
Pyridin 15 "
Distilled water 100 c.c.
Made up fresh.
Harden in alcohol. Embed in paraffin and cut sections
about 5 /*. thick; mount in balsam. The spirochetes appear
in tissues so treated as intensely black objects.
Cultivation.1 — Certain of the spirochsetacese lend them-
selves to artificial cultivation; others have eluded all such
efforts. There are not as yet any standard methods of
artificial cultivation such as are used in the routine study
of "bacteria. Special methods have been devised by a
number of investigators and all have met with more or less
success. Practically all such methods depend for success
upon certain fundamental requirements:
The culture fluids must consist essentially of sterile animal
juices or exudates; must contain bits of fresh animal tissue;
1 See Noguchi, Jour. Exper. Med., 1918, vol. xxvii, p. 593.
THE SPIROCHMTACE& 635
must in some instances present strict, in others only partial
anaerobic conditions.
The preparation of such culture media is done under strict
aseptic conditions as the heating of the media for purposes
of final sterilization robs them of their usefulness.
Noguchi, whose investigations in this field have con-
tributed so much to our knowledge on the subject, finds
that in general a medium made up of about 12 to 15 c.c.
of sterile ascitic or hydrocele fluid to which is added a few
drops of citrated blood and a bit (about the size of a bean)
of fresh rabbit kidney, serves very well for the cultivation
of most blood spirochetes. He has also had success with
the following mixture:
Rabbit serum ; . 1.5 parts
Ringer's solution1 . . 4.5 "
Citrate plasma 1.0 "
If it be desirable to stiffen the medium, and certain
spirochetes develop better in such than in fluid media, sterile
agar (free of peptone and sugar) in the proportion of 2 per
cent, may be added.
For those species requiring partial anaerobic conditions
a little sterilized paraffin oil may be run over the surface.
For those requiring strict anaerobic conditions tubes should
be kept in an oxygen-free atmosphere (see anaerobic
methods) .
The citrated blood in the above mixture may be drawn
under aseptic conditions from the animal or person whose
1 Ringer's solution:
Sodium chloride 10.0 grams
Potassium chloride 0.2 "
Calcium chloride 0.2 "
Sodium bicarbonate 0.1 . "
Glucose 1.0 "
Water . 1000.0 c.c.
636 APPLICATION OF METHODS OF BACTERIOLOGY
blood contains the organism to be cultivated. The value
of this medium is destroyed by the addition of either bouillon
or sugar; and the bit of kidney should be taken from a
freshly killed animal. The ascitic fluid should be free
from bile and when placed in the test-tube should permit
of the formation of a loose fibrin meshwork. The culti-
vation should be conducted at 37° to 38° C. The growth
of the organisms in these media causes no appreciable
change in appearance. Multiplication of the organisms is
noted after two or three days at body temperature. Sub-
cultures should be made between the fourth and ninth
days. After about ten days the multiplication of the
organisms ceases. After several generations of artificial
cultivation the spirochetes gradually lose the power to
infect susceptible animals.
In the various manifestations of relapsing fever the
organisms are found in the circulating blood; in syphilis
they are found in the diseased tissues and in the juices
squeezed from the primary sore and from other superficial
lesions; in yellow fever the leptospira icteroides is found
in the blood.
CHAPTER XXIX.
Bacteriological Study of Water — Methods Employed — Precautions to be
Observed — Apparatus Employed, and Methods of Using It — Methods
of Investigating Air and Soil — Bacteriological Study of Milk — Methods
Employed.
BACTERIOLOGICAL STUDY OF WATER.
THE conditions that favor epidemic outbreaks of typhoid
fever, Asiatic cholera, and other maladies of which these
may be taken as types, have served as a subject for dis-
cussion by sanitarians for a long time.
Of the opinions that have 'been advanced in explanation
of the existence and dissemination of these diseases, two
should be considered: one, the ground-water doctrine of
von Pettenkofer, because of its historic interest; the other,
the belief that the diseases are disseminated by specifically
polluted waters, bacause it is the view now prevalent among
modern sanitarians.
The advocates of the "ground-water" view explained
the occurrence of these diseases in epidemic form through
alterations in the soil resulting from fluctuations in the
level of the soil- water; and assigned to drinking-water
either a very insignificant role, or ignored it entirely. On
the other hand, those who have been instrumental in
developing the drinking-water hypothesis claim that altera-
tions in the soil play little or no part in favoring epidemic
outbreaks; but that, as a rule, they appear as a result of
direct infection, through the use of waters contaminated
(637)
638 APPLICATION OF METHODS OF BACTERIOLOGY
with materials containing the specific organisms known to
cause such diseases.
As a result of evidence now known to everyone it is the
general belief that polluted water is primarily the under-
lying cause of most widespread epidemics of intestinal
infections and this too, very often, when the state of the
soil-water, in the light of the "ground-water" hypothesis, is
just the reverse of what it should be in order to render it
answerable for them. It is manifest, therefore, that the
careful bacteriological study of water intended for domestic
use is of the greatest importance, and should be a routine
procedure in all communities receiving their water-supply
from sources liable to pollution.
The object aimed at in such investigations should be to
determine the number and kind of bacteria constantly
present in the water — for all waters, except deep ground-
water, contain bacteria; if sudden fluctuations in the
number and kind of bacteria occur in these waters, and if
so, to what they are due; and finally, and most important,
whether the water contains constantly, or at irregular
periods, bacteria that can be traced to human excrement,
not of necessity pathogenic varieties, but bacteria that are
known to be present normally in the intestinal canal. For
if conditions are continuously favorable to pollution of the
water by the normal constituents of the intestinal canal,
the same conditions would allow of the occasional pollution
of such water by infective matters from the bowels of persons
suffering from specific disease of the intestines.
In considering water from a bacteriological standpoint it
must always be borne in mind that comparisons with fixed
standards are not of much value, for just as normal waters
from different sources are seen to present variations in their
BACTERIOLOGICAL STUDY OF WATER 639
chemical composition, without necessarily being unfit for use,
so may the relative number and variety of species of bacteria
in water from one source be always greater or smaller than
in that from another, and yet no difference may be seen
to result from their employment. For this reason systematic
study of any water, from this point of view, should begin
with the establishment of what may be called its normal
mean number of bacteria, as well as the character of the
prevailing species; and in order to do this the investigations
must cover a long period of time through all the seasonal
variations of weather. From data obtained in. this way it
may be possible without analysis to predict approximately
at any season the bacteriological condition of the water
studied. Marked deviations from these "means," either
in the quantity or quality of the organisms present, can
then be considered as indicative of the existence of some
unusual, disturbing element, the nature of which should be
investigated. It is impossible to formulate an opinion of
much value from either a single chemical or bacteriological
analysis of a water, or from both together in many cases;
for the results thus obtained indicate only the condition
of the water at the time the sample was procured, and give
no indication as to whether it differed at that time from its
usual condition, or from the normal condition of the waters
of the immediate neighborhood.
The interpretation of the results of both chemical and
bacteriological analyses of a sample of water acquires its
full value only through comparison, either with "means"
that have been determined for this water, or with the results
of simultaneous analyses of a number of samples from other
sources of supply of the locality.
The aid of the bacteriologist is frequently sought in con-
640 APPLICATION OF METHODS OF BACTERIOLOGY
nection with investigations of waters that are supposed to
be concerned in the production of disease, particularly
typhoid fever, either in isolated cases or in widespread
epidemic outbreaks, and in these cases both the bacteriolo-
gist and the person employing his services are cautioned
against being too sanguine of positive results, for in the
vast majority of instances reliable bacteriologists fail to
detect in these waters the bacillus that is the cause of
typhoid fever.
Failure to find the organism of typhoid fever in water by
the usual methods of analysis does not by any means prove
that it is not present or has not been present. The means
ordinarily employed in the work admit of such a very small
volume of water being used in the test that we can readily
understand how typhoid bacilli might be present in moderate
numbers and yet none be included in the drop or two of the
water taken for study. The conditions are not those of a
solution, each drop of which contains exactly as much of
the dissolved material as do all other drops of equal volume;
but are rather those of a suspension, in every drop or volume
of which the number of suspended particles is liable to the
greatest degree of variation. Furthermore, there are other
reasons that would, a priori, preclude our expecting to find
the typhoid bacilli in water in which we may have reason
to believe they had been deposited, because attention is
not usually directed to the water until the disease has become
conspicuous, usually in from two to four weeks after the
pollution probably occurred. These intervals of time are
ordinarily sufficient for the delicate, non-resistant bacillus
of typhoid fever to succumb to the unfavorable conditions
under which it finds itself in water. By unfavorable con-
ditions are meant the absence of suitable nutrition; un-
BACTERIOLOGICAL STUDY OF WATER 641
favorable temperature; probably the antagonistic influence
of more hardy saprophytic bacteria, particularly the so-
called "water-bacteria," and of more highly organized
water-plants; the effect of precipitation and of sedimenta-
tion; and, of great importance, the disinfecting action of
direct sunlight.
Though the positive demonstration of typhoid bacilli in
drinking-water by bacteriological methods is of extreme
rarity, it must not be concluded that bacteriological analyses
of suspicious waters shed no light upon the existence of
pollution and the suitability or non-suitability of the water
for drinking-purposes.
In the normal intestinal tract of human beings and
domestic animals, as well as associated with the specific
disease-producing bacillus in the intestines of typhoid-
fever patients, is an organism that is frequently found in
polluted drinking-waters, and whose presence is indicative
of pollution by either normal or diseased intestinal con-
tents; and though efforts may result in failure to detect
the specific bacillus of typhoid-fever, the finding of the
other organism, bacillus coli, justifies one in concluding
that the water under consideration has been polluted by
intestinal evacuations from either human beings or animals.
Waters so exposed as to be liable to such pollution should
never be considered as other than a continuous source of
danger to those using them.
Another point to be remembered is in connection with
chlorine as an indicator of contamination by human excre-
ment. It is commonly taught that an excessive amount
of chlorine in water points to contamination by human
excreta. This may or may not be true, according to cir-
cumstances. A high proportion of this element in a sample
41
642 APPLICATION OF METHODS OF BACTERIOLOGY
of water from a locality, the surrounding waters of which
are poor in chlorine, is unquestionably a suspicious indica-
tion; but in a district close to the sea or near salt-deposits,
for instance, where the proportion of chlorine (as chlorides)
in the water is generally high, the value of the indications
thus afforded is very much diminished unless the amount
found in the sample under examination greatly exceeds the
normal "mean," previously determined, for the amount of
chlorine in the waters of the neighborhood.
A striking example of the latter condition occurred in
the experience of the writer while inspecting a group of
water-supplies on the east coast of Florida. In each in-
stance the water was obtained from properly protected
artesian wells, ranging from 200 to 400 feet deep, and located
within a few hundred yards of the sea. The first sample
subjected to chemical analysis revealed such an unusually
high proportion of chlorine that, had this sample alone
been considered, the opinion that it was polluted by human
excreta might have been advanced. To prevent such an
error samples of water from a number of wells in the neigh-
borhood were examined, and they were all found to contain
from ten to twelve times the amount of chlorine that ordi-
narily appears in inland waters, the excess being evidently
due to leakage through the soil into the wells of water from
the sea. In short, the presence of an excess of chlorine in
water, while often indicating pollution from human evacua-
tions, may nevertheless, sometimes arise from other sources;
but the presence in water of bacteria normally found in
the intestinal canal can manifestly admit of but one inter-
pretation, viz., that fecal matters from either man or
animals have at some time been deposited in this water,
and that while no specific disease-producing organisms may
BACTERIOLOGICAL STUDY OF WATER 643
accompany them, still waters in which such pollutions are
possible are also open to other dangerous pollutions, and
must be regarded as a constant menace to the health of
those who use them for domestic purposes.
A sudden variation from the normal, mean number of
bacteria, or from the normal chemical composition of a
water, calls at once for a thorough inspection of the supply,
while at the same time the organisms present are to be sub-
jected to the most careful study. In many instances, even
after the most thorough bacteriological and chemical study
of a suspicious water, one is forced to admit that informa-
tion of but limited usefulness has been obtained through
the employment of such analytical methods. In these
cases too much stress cannot be laid upon the importance
of a systematic inspection of the supply, and its relation
to sources of pollution. Optical evidence of more or less
dangerous contamination may often be obtained when
laboratory methods fail to detect them. The reasons for
such failure, in addition to those already given, are obvious —
the polluting matters are often so diluted by the large
mass of water into which they find their way as to be beyond
recognition by the tests usually employed in such work,
and still be present in amounts sufficient to originate
disease.
The Qualitative Bacteriological Analysis of Water. — The
qualitative bacteriological analysis of water entails much
labor, as it requires not only that all the different species
of organisms found in the water should be isolated, but
that each representative should be subjected to systematic
study, and its pathogenic or non-pathogenic properties
determined.
For this purpose a knowledge of the methods for the
644 APPLICATION OF METHODS OF BACTERIOLOGY
isolation of individual species which have been described
already, and of the means of studying these species when
isolated, is indispensable.
For this analysis certain precautions essential to accuracy
are always to be observed.
The sample is to be collected under the most rigid pre-
cautions that will exclude organisms from sources other
than that under consideration. If drawn from a spigot,
it should never be collected until the water has been flowing
for fifteen to twenty minutes in a full stream. If obtained
from a stream or a spring, it should be collected, not from
the surface, but rather from about one foot beneath the
surface.
It should always be collected in vessels which have pre-
viously been thoroughly freed from all dirt and organic
particles, and then sterilized; and the plates should be
made as quickly as possible after collecting the sample.
When circumstances permit, all water analyses should be
made on the spot where the sample is taken, as it is known
that during transportation, unless the samples are kept
packed in ice, a multiplication of the organisms contained
in it always occurs.
For the purpose of qualitative analysis it is necessary
that a small portion of the water — one, two, three, five
drops — should first be employed for making the plates.
In this way one can form an idea as to the approximate
number of organisms in the water, and can, in consequence,
determine the amount of water best suited for the plates.
Duplicate plates are always to be made — one set upon
agar-agar, which are to be kept in the incubator at body-
temperature, and one set upon gelatin, to be kept at from
18° to 20° C.
BACTERIOLOGICAL STUDY OF WATER 645
As soon as colonies have developed the plates are to be
carefully compared and studied. It is to be noted if any
difference in the appearance of the organisms on correspond-
ing plates exists, and if so, to what it is due. It is to be
particularly noted which plates contain the greater number
of colonies, those kept at the higher or those at the lower
temperature. In this way the temperature best suited
for the growth of the majority of these, organisms may be
determined. As a rule, the greater number of colonies
appear upon the gelatin plates kept at 18° to 20° C.; and
from this it would seem that many of the normal water-
bacteria do not find the higher temperature so favorable
to their development as do the organisms not naturally
present in water, particularly the pathogenic varieties.
From these plates the different species are to be isolated
in pure culture, the morphological and cultural character-
istics determined, and finally, by tests upon animals, it is
to be decided if any of them possess disease-producing
properties.
NOTE. — What use should be made of this observation
in examining water for the presence of pathogenic bacteria?
The waters most frequently studied from the qualitative
bacteriological standpoint are those suspected of containing
specific pathogenic bacteria — i. e.} waters polluted with
sewage and with human excreta that are believed to be the
source of infection of typhoid fever, or, less frequently,
of Asiatic cholera. In the investigations of such water
there are several points of which we should never lose sight,
viz., unless the water is under continuous study there is
only a chance of detecting the specific pathogenic species,
for, as a rule, the dangerous pollution occurs either but
646 APPLICATION OF METHODS OF BACTERIOLOGY
once or is intermittent, so that even in the case of exposed
streams there are periods when no specifically dangerous
contamination may be in operation. As stated above
attention is commonly called to the water when the disease,
presumably caused by its use, is fully developed, and this
is often days or weeks after the pollution of the stream really
may have occurred. By an analysis made at this time one
could scarcely hope to detect the specific organisms that
had caused the disease, especially in water from flowing
streams. The organisms sought for may have been present
in the water and may have infected the users, and yet have
disappeared by the time the sample taken for analysis was
collected.
When present in polluted waters pathogenic bacteria are
always vastly in the minority. Were they constantly
present in large numbers infection among the users of such
waters would be more frequent and more widespread than
is commonly the case. They may be present in a water-
supply in small numbers; they may even be in the sample
supplied for analysis, and yet escape detection if only the
ordinary direct plate method of isolation be used.
From these considerations it is obvious that before
attempts are made to isolate the various species .directly
from a suspicious sample of water it is advisable to subject
it to some method of treatment that will aid in separating
the few specific pathogenic from the numerous common
saprophytic species. For this purpose numerous so-called
methods of "enrichment" have been devised. The most
useful of these aim to favor the rapid multiplication of
pathogenic forms that may be present and to suppress or
check the growth of the ordinary water saprophytes.
Attention has been called to the fact that when exposed
BACTERIOLOGICAL STUDY OF WATER 647
to the body-temperature many of the ordinary water-
bacteria develop only slowly or not at all, while under
similar circumstances the disease-producing species develop
most luxuriantly. Advantage has been taken of this obser-
vation in devising methods for this particular work, of
which some of the following will prove serviceable:
Collect in a sterilized flask a sample of about 100 c.c.
of the water to be tested, and add to this about 25 c.c. of
sterilized bouillon of four times the usual strength. This is
then placed in the incubator at 37° to 38° C., for thirty-six
to forty-eight hours, after which plates are to be made from
it in the^usual way; the results will often be a pure culture
of some single organism, either one of the intestinal variety
or a closely allied species. By a method analogous to the
latter the spirillum of Asiatic cholera has been isolated from
water (see article on that organism) ; and by taking advan-
tage of the effect of elevated temperature upon the bacteria
of water Vaughan has succeeded in isolating from suspicious
waters a group of organisms very closely allied to the bacillus
of typhoid fever.
Theobald Smith has suggested a method by which it is
easily possible to isolate, from waters in which they are
present, certain organisms that are of the utmost impor-
tance in influencing our judgment upon the fitness of the
water for domestic use. By the addition of small quantities
— one, two, or three drops — of the suspicious water to
fermentation-tubes (see article on Fermentation-tube) con-
taining bouillon to which 2 per cent, of glucose has been
added, and keeping them at the temperature of the body
(37° to 38° C.), the growth of intestinal bacteria that may
be present in the water is favored, while that of the water-
organisms is not; in consequence, after from thirty-six to
648 APPLICATION OF METHODS OF BACTERIOLOGY
forty-eight hours the fermentation characteristics of most
of these organisms is evidenced by the accumulation of gas
in the closed end of the tube. From these tubes the growing
bacteria can then be easily isolated by the plate method,
and intestinal bacteria will not infrequently be found present.
For the isolation of the typhoid bacillus, especially from
water, a host of other methods have been devised. Some of
these aim, through the addition of special chemical reagents
to the media, to retard the development of ordinary sapro-
phytes without interrupting the growth of the colon and
the typhoid bacillus. Most of these methods have proved
disappointing. One of them, that of Parietti, still finds
favor in the hands of some. It consists in adding to the
culture media to be used in the test varying amounts of
the following mixture :
Phenol 5 grams
Hydrochloric acid 4 grams
Distilled water 100 c.c.
Of this solution 0.1, 0.2, and 0.3 c.c. are added respectively
to each of three tubes containing 10 c.c. of nutritive bouillon.
Several such sets of tubes are to be made. To each are
then added from 1 to 3 c.c. of the water, and they are placed
in the incubator at body-temperature. It is said that
whatever development occurs consists only of the typhoid
or colon bacillus, or both, if they were present in the original
sample. They may then be isolated and separated by the
usual plate method, or, better still, through the application
of the methods of v. Drigalski and Conradi, of Ficker, or
of Hoffmann and Ficker, or several of these methods in
conjunction, detailed in the chapter on bacillus typhosus.
Personally we have not had much success with the Parietti
method. The typhoid bacillus has been isolated from water
BACTERIOLOGICAL STUDY OF WATER 649
by passing very large quantities of water through an ordinary
Pasteur or Berkefeld filter, brushing off the matters collected
on the filter into a sterilized vessel and examining this by
plate methods.
It has occurred to us that possibly the employment of
chemical coagulants, such as alum and iron, might prove
serviceable for this purpose. Their action would be to
mechanically drag down, in precipitating as hydroxides,
the suspended bacteria contained in the fluid. This preci-
pitate could then be examined bacteriologically, * instead
of the water, and the recent experiments of Ficker (loc. tit.)
appear to demonstrate' the value of such a procedure.
The difficulties in this field of work are obviously due to
the suspension of a very small number of the disease-pro-
ducing organisms sought for in large volumes of fluid, and
the association with them of large numbers of other species
that offer a very great obstacle to the successful search for
the pathogenic varieties.
If by either of the above procedures bacilli that bear
any resemblance to bacillus typhosus be isolated, recourse
must then be had to all the differential tests detailed in the
chapter on that organism.
The Quantitative Estimation of Bacteria in Water. — Quan-
titative analysis requires more care in the measurement of
the exact volume of water employed, for the results are to
be expressed in terms of the number of individual organisms
to a definite volume. The necessity for making the plates
at the place at which the sample is collected is to be particu-
larly accentuated in this analysis, for multiplication of the
organisms during transit is so great that the results of
analyses made after the water has been in a vessel for a
day or two are often very different from those that would
have been obtained on the spot.
650 APPLICATION OF METHODS OF BACTERIOLOGY
NOTE. — Inoculate a tube containing about ten cubic
centimeters of sterilized distilled or tap water with a very
small quantity of a solid culture of some one of the organ-
isms with which you have been working, taking care that
none of the culture medium is introduced into the water-
tube and that the bacteria are evenly distributed through
it. Make plates at once from this tube, and on each suc-
ceeding day determine by counts whether there is an increase
or diminution in the number of organisms — i. e., if they are
growing or dying. Represent the results graphically, and
it will be noticed that in many cases there is during the
first three or four days a multiplication, after which there
is a rapid diminution; and, if the organism does not form
spores, usually death in from ten to twelve days. This is
not true for all organisms, but does hold for many.
Where it is not convenient, however, to make the analysis
on the spot, the sample of water should be taken and packed
in ice and kept on ice until the plates can be made from it,
which should in all cases be as soon after its collection as
possible.
For the collection of samples from the deeper portions
of streams, lakes, etc., a number of convenient devices have
been made. A very satisfactory apparatus has been made
for me by Messrs. Charles Lentz & Sons, of Philadelphia.
It consists of a metal frame-work, in which is encased a
bottle provided with a ground-glass stopper. To the stopper
a spring clamp is attached, and this in turn is operated by
a string, so that when the weighted apparatus is allowed to
sink into the stream the stopper may be removed from the
bottle at any depth by simply pulling upon the string.
When the bottle is filled with water the stopper is allowed
to spring back into position by releasing the string. The
BACTERIOLOGICAL STUDY OF WATER
651
FIG. 109
whole apparatus (depicted in Fig. 109) is provided with a
weight that insures its sinking, and a heavy cord by which
it may be lowered and raised. It should be sterilized before
using. After collecting the sample the
bottle should be wiped dry with a
sterilized towel. Before removing the
stopper the mouth of the bottle should be
rinsed with alcohol and heated with a gas-
flame, to prevent contamination of its
contents by matters that may have been
upon its surface.
In beginning the quantitative analy-
sis of water with which one is not ac-
quainted certain preliminary steps are
essential.
It is necessary to know approximately
the number of organisms contained in
any fixed volume, so as to determine the
quantity of water to be employed for
the plates or tubes. This is usually done
by making preliminary plates from one
drop, two drops, 0.25 c.c., 0.5 c.c., and
1 c.c. of the water. After each plate
has been labelled with the amount of
water used in making it, it is placed
aside for development. When this has
occurred one selects the plate upon which
the colonies are only moderate in number
— about 200 to 300 colonies presenting
— and employs in the subsequent analysis the same amount
of water that was used in making this plate.
If the original water contained so many organisms that
Bottle for collecting
water.
652 APPLICATION OF METHODS OF BACTERIOLOGY
there developed on a plate or tube made with one drop too
many colonies to be easily counted, then the sample must
be diluted with one, ten, twenty-five, fifty, or one hundred
volumes, as the case may require, of sterilized distilled water.
This dilution must be accurate, and its exact extent noted,
so that subsequently the number of organisms per volume
in the original water may be calculated.
The use of a drop is not sufficiently accurate. The dilu-
tion should therefore always be to a degree that will admit
of the employment of a volume of water that may be exactly
measured, 0.25 and 0.5 c.c. being the amounts most con-
venient for use.
Duplicate plates should always be made, arid the mean
of the number of colonies that develop upon them taken
as the basis from which to calculate the number of organ-
isms per volume in the original water.
For example: from a sample of water 0.25 c.c. is added
to a tube of liquefied gelatin, carefully mixed and poured as
a plate. When development occurs the number of colonies
is too numerous to be accurately counted. One cubic cen-
timeter of the original water is then to have added to it,
under precautions that prevent contamination from with-
out, 99 c.c. of sterilized distilled water — that is, we have now
a dilution of 1 : 100. Again, 0.25 c.c. of this dilution is
plated, and we find 180 colonies on the plate. Assuming
that each colony develops from an individual bacterium,
though this is perhaps not strictly true, we had 180 organ-
isms in 0.25 c.c. of our 1 : 100 dilution; therefore in 0.25
c.c. of the original water we had 180X100 = 18,000 bacteria,
which will be 72,000 bacteria per cubic centimeter (0.25 c.c.
= 18,000, 1 c.c. = 18,000x4 = 72,000). The results are
always to be expressed in terms of the number of bacteria
per cubic centimeter of the original water.
BACTERIOLOGICAL STUDY OF WATER
653
Another point of very great importance (already men-
tioned) is the effect of temperature upon the number of
colonies of bacteria that will develop on the plates made
from water. It must always be remembered that a larger
number of colonies appear on gelatin plates made from water
and kept at 18° to 20° C. than on agar-agar plates kept in
the incubator. The following table, illustrative of this
point, gives the results of parallel analyses of the same waters,
the one series of counts having been made upon gelatin
plates at the ordinary temperature of the room, the other
upon plates of agar-agar kept for the same length of time
in the incubator at from 37° to 38° C. It will be seen from
the table that much the larger number of colonies — i. e.,
much higher results — were always obtained when gelatin was
employed. The importance of this point in the quantita-
tive bacteriological analysis of water is too apparent to
require further comment.
TABLE COMPARING THE RESULTS OBTAINED BY THE USE OF GELATIN AT
18°-20° C. AND AGAR-AGAR AT 37°-38° C. IN QUANTITATIVE BAC-
TERIOLOGICAL ANALYSES OF WATER. RESULTS RECORDED ARE THE
NUMBER OF COLONIES THAT DEVELOPED FROM THE SAME AMOUNT OF
VARIOUS WATERS IN EACH SERIES.1
NUMBER OF COLONIES FROM WATER THAT DEVELOPED UPON —
Gelatin plates at 18° to 20° C. Agar-agar plates at 37° to 38° C.
310 170
280 . 140
310 180
340 160
650 210
630 320
380 290
400 210
1000 . . 100
890 130
340 280
370 210
490 110
580 / 100
1 I am indebted to James Homer Wright, Thomas Scott Fellow in Hygiene
1892-1893), University of Pennsylvania, for the results presented in this
table.
654 APPLICATION OF METHODS OF BACTERIOLOGY
Another point of equal importance in its influence upon
the number of colonies that develop is the reaction of the
gelatin. A marked excess of either alkalinity or acidity
always has a retarding effect upon many species found in
water. Fuller's experience at the Lawrence (Mass.) Ex-
periment Station has shown that gelatin of such a degree
of acidity as to require the further addition of from 15 to
20 c.c. per liter of a normal caustic alkali solution to bring
it to the phenolphthalein neutral point gives, on the whole,
the best results. Thus, by way of illustration, Fuller found
that a sample of Merrimac River water gave 5800 colonies
per c.c. on phenolphthalein neutral gelatin, 15,000 colonies
on gelatin that would need 20 c.c. of normal alkali solution
to bring it up to the phenolphthalein neutral point — i. e.,
a feebly acid nutrient gelatin, and 500 colonies on a gelatin
so alkaline as to require 20 c.c. of a normal acid solution
to bring it back to the phenolphthalein neutral point.
Throughout this part of the work it is to be borne in
mind that when reference is made to plates it is not to a
set, as in isolation experiments, but to a single plate.
Method of Counting the Colonies on Plates. — For conven-
ience in counting colonies on plates or in tubes it is customary
to divide the whole area of the gelatin occupied by colonies
into smaller areas, and either count all the colonies in each
of these areas and add the several sums together for the
total, or to count the number of colonies in each of several
areas, ten or twelve, take the mean of the results and multiply
this by the number of areas containing colonies. The latter
procedure obtains, of course, only when all the areas are
of the same size. By this method, however, the results
vary so much in different counts of the same plate that they
cannot be considered as more than rough approximations.
BACTERIOLOGICAL STUDY OF WATER 655
NOTE. — Prepare a plate; calculate the number of colonies
upon it by this latter method. Now repeat the calcula-
tion, making the average from another set of squares. Now
actually count the entire number of colonies on the plate.
Compare the results.
For facilitating the counting of colonies several very
convenient devices exist.
Wolffhugel's Counting-apparatus.— This apparatus (Fig.
110) consists of a flat wooden stand, the centre of which is
FIG. 110
Wolffhiigel's apparatus for counting colonies.
cut out in such a way that either a black or white glass
plate may be placed in it. These form a background upon
which the colonies may more easily be seen when the plate
to be counted is placed upon it. When the gelatin plate
containing the colonies has been placed upon this back-
ground of glass it is covered by a transparent glass plate
which swings on a hinge. This plate, which is ruled in
square centimeters and subdivisions, when in position is
just above the colonies, without touching them. The gelatin
656 APPLICATION OF METHODS OF BACTERIOLOGY
plate is moved about until it rests under the center of the
area occupied by the ruled lines. The number of colonies
in each square centimeter is then counted, and the sum
total of the colonies in all these areas gives the number of
colonies on the plate; or, as has already been indicated, if
the number of colonies be very great, a mean may be taken
of the number in several (six or eight) squares; this is to
be multiplied by the total number of squares occupied by
the gelatin. The result is an approximation of the total
number of colonies.
When the colonies are quite small, as is frequently the
case, the counting may be rendered easier by the use of a
small hand lens. (Fig. 111.)
FIG. Ill
Lens for counting colonies.
Several useful modifications of the apparatus of Wolff-
hiigel have been introduced. The most important is that
of Lafar.1 Lafar's counter consists of a glass disk of the
diameter of ordinary size Petri dishes. It is supplied with
a collar or flange that fits around the bottom of the Petri
dish, and thus holds the counter in position. The disk is
ruled with concentric circles, and its area is divided into
sectors of such sizes that the spaces between the concentric
circles and the radii forming the sectors are of equal size.
1 Centralblatt fur Bakteriologie und Parasitenkunde, 1891, Bd. xv, S. 331 .
BACTERIOLOGICAL STUDY OF WATER 657
Three of the sectors are subdivided into smaller areas of
equal size for convenience in counting when the colonies
are very numerous. The principles involved are similar
to those of the preceding apparatus, but the circular form
of the apparatus admits of more exactness when . counting
colonies on a circular plate.1
8
Fakes' apparatus for counting colonies (reduced one-third).
Pakes2 has introduced a cheap and convenient modifi-
cation of Lafar's apparatus. It consists of a sheet of white
paper on which is printed a black disk ruled with white
lines, in somewhat the same fashion as is Lafar's counter,
1 Lafar's apparatus is to be obtained from F. Mollenkopf, 10 Thor-
strasse, Stuttgart, who holds the patent for it. Its price is about 8 marka.
2 Journal of Bacteriology and Pathology, 1896, iv, No. 1.
42
658 APPLICATION OF METHODS OF BACTERIOLOGY
though the areas of the smallest subdivisions are not of one
size and do not bear a constant relation to each other.1 To
use this apparatus (Fig. 112) the Petri dish is placed cen-
trally upon it, the cover of the dish is removed, and the
colonies are counted as they lie over the spaces bounded by
the white lines on the black disk beneath. When the plate
is centered over the black disk the portion lying over one
sector is exactly one-sixteenth of the whole plate.
FIG. 113
Esmarch's apparatus for counting colonies in rolled tubes.
Esmarch's Counter. — Esmarch devised a counter (Fig.
113) for estimating the number of colonies present upon
a cylindrical surface, as when in rolled tubes. The prin-
ciples and methods of estimation are practically the same
as those given for Wolffhugel's apparatus.
1 Copies of this apparatus are to be had of Ash & Co., 42 Southwark
Street, London, or of Lentz & Sons, North Eleventh Street, Philadelphia,
Pa. (The cost is but a few cents per copy.)
BACTERIOLOGICAL STUDY OF WATER ' 659
A simpler method than by the use of Esmarch's apparatus
may be employed for counting the colonies in rolled tubes.
It consists in dividing the tube by lines into four or six
longitudinal areas, which are subdivided by transverse
lines about 1 or 2 cm. apart. The lines may be drawn with
pen and ink. They need not be exactly the same distance
apart nor exactly straight. Beginning with one of these
squares at one end of the tube, which may be marked with
a cross, the tube is twisted with the fingers, always in one
direction, and the exact number of colonies in each square
as it appears in rotation is counted, care being taken not
to count a square more than once; the sums are then added
together, and the result gives the number of colonies in
the tube. This method may be facilitated by the use of a
hand-lens.
In all these methods there is one error difficult to eliminate :
it is assumed that each colony has grown from a single
organism. This is probably not always the case, as there
may exist clumps of bacteria which represent hundreds or
even thousands of individuals, but which still give rise to
but a single colony — obviously this is of necessity estimated
as a single organism in the water under analysis.
Where grounds exist for suspecting the presence of these
clumps they may in part be broken up by shaking the
original water with sterilized sand.
What has been said for the bacteriological examination
of water holds good for all fluids which are to be subjected
to this form of analysis.
The Sewage Streptococcus. — Houston1 reached the con-
clusion that there is constantly present in sewage a particular
form of streptococcus which is really more positively indica-
5Ann. Report, Local Gov. Board, xxviii.
660 APPLICATION OF METHODS OF BACTERIOLOGY
live of the contamination of water by sewage than is bacillus
coli. This opinion was under investigation by members
of the staff of the Massachusetts Institute of Technology,
who reached the conclusion that considerable reliance can
be placed upon the presence of this organism as an indication
of sewage pollution of water.
The presence of the sewage streptococcus is most readily
shown in the sediment in fermentation tubes inoculated
with water under examination. If the sewage streptococcus
is present it is very easy to demonstrate it by microscopic
examination of the sediment after twenty-four to forty-
eight hours. In addition to this test it has also been demon-
strated by Winslow1 that the estimation of the degree of
acidity of the, contents of the fermentation tube is a safe
indication of the presence of the sewage streptococcus.
When this organism is present the acidity rises far more
rapidly and to a greater height than is the case when it is
absent, so that in this way an additional indicator is avail-
able as to the potability of a water under examination.
BACTERIOLOGICAL ANALYSIS OF AIR.
Quite a number of methods for the bacteriological study
of the air exist. In the main they consist either in allowing
air to pass over solid nutrient media (Koch, Hesse) and
observing the colonies which develop upon the media, or
in filtering the bacteria from the air by means of porous and
liquid substances, and studying the organisms thus obtained.
(Miguel, Petri, Strauss, Wiirz, Sedgwick-Tucker.) Because
of their greater exactness, the latter have supplanted the
former methods.
1 Jour. Med. Research, 1902, vol. iii.
BACTERIOLOGICAL ANALYSIS OF AIR 661
In some of the methods which provide for the filtration
of bacteria from the air by means of liquid substances a
measured volume of air is aspirated through liquefied
gelatin; this is then rolled into an Esmarch tube and the
number of colonies counted, just as is done in water analysis.
This is the simplest procedure. An objection sometimes
raised against it is that organisms may be lost, and not
come into the calculation, by passing through the medium
FIG. 114
Petri's apparatus for bacteriological analysis of air. The tube packed with
sand is seen at the point a.
in the center of an air-bubble without being arrested by
the fluid — an objection that appears to have more of specu-
lative than of real value. Filtration through porous sub-
stances appears, on the whole, to give the best results.
Petri recommends aspiration of a measured volume of air
through glass tubes into which sterilized sand is packed.
(Fig. 114.) When aspiration is finished the sand is mixed
with liquefied gelatin, plates are made, and the number of
662 APPLICATION OF METHODS OF BACTERIOLOGY
developing colonies counted, the results giving the number
of organisms contained in the volume of air aspirated through
the sand.
The main objection to this method is the possibility of
mistaking a sand-granule for a colony. This objection has
been overcome by Sedgwick and Tucker, who employ
granulated sugar instead of sand; this, when brought into
the liquefied gelatin, dissolves, and no such error as that
possible in the Petri method can be made.
Sedgwick-Tucker Method. — On the whole, the method
proposed by Sedgwick and Tucker gives such uniform
results that it is to be preferred to others. It is as follows:
The apparatus employed by them consists essentially of
three parts:
1. A glass tube of special form, to which the name aerobio-
scope has been given.
2. A stout copper cylinder of about sixteen litres capacity,
provided with a vacuum-gauge.
3. An air-pump.
The aerobioscope (Fig. 115) is about 35 cm. in its entire
length; it is 15 cm. long and 4.5 cm. in diameter at its
expanded part; one end of the expanded part is narrowed
to a neck 2.5 cm. in diameter and 2.5 cm. long. To the other
end is fused a glass tube 15 cm. long and 0.5 cm. inside
diameter, in which is to be placed the filtering-material.
Upon this narrow tube, 5 cm. from the lower end, a
mark is made with a file, and up to this mark a small roll
of brass-wire gauze (a) is inserted; this serves as a stop
for the filtering-material which is to be placed over it.
Beneath the gauze (at 6), and also at the large end (c),
the apparatus is plugged with cotton. When thoroughly
cleaned, dried, and plugged, the apparatus is to be steril-
• BACTERIOLOGICAL ANALYSIS OF AIR 663
ized in the hot-air sterilizer. When cool the cotton plug is
removed from the large end (c), and thoroughly dried and
sterilized No. 50 granulated sugar is poured in until it just
fills the 10 cm. (d) of the narrow tube above the wire gauze.
This column of sugar is the filtering-material employed to
engage and retain the bacteria. After pouring in the sugar
the cotton-wool plug is replaced, and the tube is again
sterilized at 120° C. for several hours.
Taking the air sample. In order to measure the amount
of air used the value of each degree on the vacuum-gauge
is determined in terms of air by means of an air-meter, or
by calculation from the known capacity of the cylinder.
FIG. 115
c e da
The Sedgwick-Tucker aerobioscope.
This fact ascertained, the negative pressure indicated by
the needle on exhausting the cylinder shows the volume of
air which must pass into it in order to fill the vacuum. By
means of the air-pump one exhausts the cylinder until the
needle reaches the mark corresponding to the amount of
air required.1
A sterilized aerobioscope is now to be fixed in the upright
position and its small end connected by a* rubber tube
1 Such a cylinder and air-pump are not necessary. A pair of ordinary
aspirating bottles of known capacity graduated into liters and fractions
thereof answer perfectly well. Or one can determine by the weight of
water that has flowed from the aspirator the volume of air that has passed
in to take its place — i. e., the volume of air that has passed through the
aerobioscppe.
664 APPLICATION OF METHODS OF BACTERIOLOGY
with a stopcock on the cylinder, or to a glass tube tightly
fixed in the neck of an aspirat ing-bottle by means of a
perforated rubber stopper. The cotton plug is then moved
from the upper end of the aerobioscope, and the desired
amount of air is aspirated through the sugar. Dust-par-
ticles and bacteria will be held back by the sugar. During
manipulation the cotton plug is to be protected from con-
tamination.
FIG. 116
Bent funnel for use with aerobioscope.
When the required amount of air has been aspirated
through the sugar the cotton plug is replaced, and by gently
tapping the aerobioscope while held in an almost horizontal
position the sugar, and with it, the bacteria, are brought
into the large part (e) of the apparatus.. When all the sugar
is thus shaken down into this part of the apparatus about
20 c.c. of liquefied, sterilized gelatin is poured in through
the opening at the end c, the sugar dissolves, and the whole
BACTERIOLOGICAL STUDY OF THE SOIL G65
is then rolled on ice, just as is done in the preparation of
an ordinary Esmarch tube.
The gelatin is most easily poured into the aerobioscope
by the use of a small, sterilized, cylindrical funnel (Fig.
116), the stem of which is bent to an angle of about 110
degrees with the long axis of the body.
The larger part of the aerobioscope is divided into squares
to facilitate the counting of the colonies.
By the employment of this apparatus one can filter the
air at any place, and can then, without fear of contamination,
carry the tubes to the laboratory and complete the analysis.
Aside from this advantage, the filter being soluble only the
insoluble bacteria are left imbedded in the gelatin.
For general use this method is to be preferred to the
others that have been mentioned.
BACTERIOLOGICAL STUDY OF THE SOIL.
Bacteriological study of the soil may be made by either
breaking up small particles of earth in liquefied media and
making plates directly from this; or by what is perhaps
a better method, as it gets rid of insoluble particles which
may give rise to errors; breaking up the soil in sterilized
water and then making plates immediately from the water.
It must be borne in mind that many of the ground-organ-
isms belong to the anaerobic group, so that in these studies
this point should be remembered and the methods for the
cultivation of such organisms practised in connection with
the ordinary methods. It must also be remembered that
the nitrifying organisms, everywhere present in the ground,
cannot be isolated by the ordinary methods, and will not
666 APPLICATION OF METHODS OF BACTERIOLOGY
appear in plates made after either of the above plans. The
special devices for their cultivation are described in the
chapter on Soil-organisms.
BACTERIOLOGICAL STUDY OF MILK.
The possibility of milk serving as a vehicle in which
disease-producing bacteria may be disseminated through-
out a community has long been recognized, and epidemics
of typhoid fever have been traced directly to infected milk,
while such diseases as diphtheria and scarlet fever are also
frequently regarded as being conveyed in the same manner.
In recent years the detailed study of the milk of individ-
ual cows has revealed the fact that streptococcus mastitis
is not an uncommon occurrence in herds, and it has fre-
quently been observed that milk rich in streptococci may
prove dangerous when fed to infants and convalescents.
Since milk is such a favorable medium for the growth of
a variety of bacteria it is not at all uncommon to find market
milk very rich in bacteria, especially if it has been collected
in a careless manner in dirty receptacles, in unsanitary
stables, and has been shipped long distances at comparatively
high temperatures.
For these various reasons the bacteriological study of
milk has gained considerable prominence during the past
few years — ;so much so that in some localities an effort is
being made to establish a bacterial standard for market
milk — that is, milk containing more than a certain number
of bacteria is not regarded as suitable for use. Whether
such a standard can be maintained or not remains to be
demonstrated. The several milk commissions composed
of pediatrists in various large cities have established a
BACTERIOLOGICAL STUDY OF MILK 667
bacterial standard for approved milk of 10,000 bacteria to
the cubic centimeter. Experience has shown that it is
possible to market milk that meets this bacterial standard
sometimes with merely ordinary precautions with regard
to cleanliness. In larger dairies it has frequently been a
question of some difficulty on account of the elaborate
scale on which the business is conducted.
Quantitative Bacteriological Analysis. — In the quantitative
bacteriological examination of market milk it is necessary
to dilute the milk with sterile water or sterile salt solution
before plating on account of the very large numbers of
bacteria present. The degree of dilution that is necessary
will depend upon the nature of the dairy from which the
milk is derived, the age of the milk, and the temperature
at which it has been kept. Usually a dilution of 1 to 100,
1 to 1000, and 1 to 10,000 is sufficient. From these dilutions
plate cultures are made with 0.1, 0.2, 0.3 cubic centimeter
of each dilution.
Qualitative Bacteriological Analysis. — Aside from the
quantitative bacteriological analysis of milk the qualita-
tive analysis has received a great deal of attention. Detailed
qualitative analysis necessarily entails an enormous amount
of labor, but the detection of certain forms of bacteria is
not always very difficult. This applies especially to the
detection of streptococci.
Since milk containing streptococci in considerable num-
bers is derived from the udder of a cow suffering from some
form of mastitis, it is always possible to find pus in such
milk. Consequently it is customary to examine such milk
for the presence of both streptococci and pus. This is done
by centrifuging a cubic centimeter of the milk and collecting
the sediment on a clean cover-slip and staining with Loffler's
668 APPLICATION OF METHODS OF BACTERIOLOGY
methylene-blue. In this manner practically all the sediment
derived from one cubic centimeter can be obtained on the
cover-slip and a fairly satisfactory estimate can be made of
the relative number of pus cells in this quantity of milk as
well as at the same time an estimation of the relative number
of streptococci.
Milk that shows pus cells along with distinct chains of
streptococci, either extra- or intracellular, is usually regarded
as dangerous in character, and boards of health usually
direct that the cows from which such milk is derived be
excluded from the dairy until such time as the milk is free
from these elements.
APPENDIX.
LIST of apparatus and materials required in a beginner's
bacteriological laboratory :
MICROSCOPE AND ACCESSORIES.
Microscope with coarse and fine adjustment and heavy,
firm base; Abbe sub-stage condensing system, arranged
either as the "simple" or as the regular Abbe condenser,
in either case to be provided with iris diaphragm; objec-
tives equivalent, in the English nomenclature, to about
one-fourth inch and one-sixth inch dry, and one-twelfth
inch oil-immersion system; a triple revolving nose-piece;
three oculars, varying in magnifying power; and a bottle
of immersion oil.
Glass slides, English shape and size and of colorless glass.
Six slides with depressions of about 1 cm. in diameter in
centre.
Cover-slips, 15 by 15 mm. square and not more than from
0.15 to O.L8 mm. thick.
Forceps. One pair of fine-pointed forceps and one pair
of the Cornet or Stewart pattern, for holding coverslips.
Platinum needles with glass handles. One straight,
about 4 cm. long; one looped at the end, about 4 cm. long;
and one straight, about 8 cm. long. Glass handles to be
about 3 mm. in thickness and from 15 to 17 cm. long.
(669)
670 APPLICATION OF METHODS OF BACTERIOLOGY
STAINING- AND MOUNTING-REAGENTS.
200 c.c. of saturated alcoholic solution of fuchsin.
200 c.c. of saturated alcoholic solution of gentian-violet.
200 c.c. of saturated alcoholic solution of methylene-blue.
200 grams of pure aniline.
200 grams of C. P. carbolic acid.
500 grams of C. P. nitric acid.
500 grams of C. P. sulphuric acid.
200 grams of C. P. glacial acetic acid.
1 liter of ordinary 93-95 per cent, alcohol.
1 liter of absolute alcohol.
500 grams of ether.
500 grams of pure xylol.
50 grams of Canada balsam dissolved in xylol.
100 grams of Schering's celloidin.
10 grams of iodine and 30 grams of potassium iodide in
substance.
100 grams of tannic acid.
100 grams of ferrous sulphate.
Distilled water.
FOR NUTRIENT MEDIA.
J pound of beef-extract.
250 grams of peptone.
2 kilograms of first quality gelatin.
100 grams of agar-agar in substance.
200 grams of sodium chloride (ordinary table-salt).
500 grams of pure glycerin.
50 grams of pure glucose.
20 grams of pure lactose.
100 grams of caustic potash.
APPENDIX 671
200 c.c. of litmus tincture.
10 grams of rosolic acid (corallin).
Blue and red litmus-paper; curcuma paper.
5 grams of phenolphthalein in substance.
Filter-paper, the quality ordinarily used by druggists.
100 grams of pyrogallic acid.
1 kilogram of C. P. granulated zinc.
GLASSWARE.
200 best quality test-tubes, slightly heavier than those
used for chemical work, about 12 to 13 cm. long and 12 to
14 mm. inside diameter.
15 Petri double dishes about 8 or 9 cm. in diameter and
from 1 to 1.5 cm. deep.
6 Florence flasks, 1000 c.c. capacity.
6 Florence flasks, 500 c.c. capacity.
12 Erlenmeyer flasks, 100 c.c. capacity.
1 graduated measuring-cylinder, 1000 c.c. capacity.
1 graduated measuring-cylinder, 100 c.c. capacity.
25 bottles, 125 c.c. capacity, narrow necks with ground-
glass stoppers.
25 bottles, 125 c.c. capacity, wide mouths, with ground-
glass stoppers.
1 anatomical or preserving jar, with tightly fitting cover,
of about 4 liters capacity, for collecting blood-serum.
2 battery jars of about 2 liters capacity, provided with
loosely fitting, weighted, wire-net covers for mice.
10 feet of soft-glass tubing, 2 or 3 mm. inside diameter.
20 feet of soft-glass tubing, 4 mm. inside diameter.
6 glass rods, 18 to 20 cm. long and 3 or 4 mm. in diameter.
6 pipettes of 1 c.c. each, divided into tenths.
672 APPLICATION OF METHODS OF BACTERIOLOGY
2 pipettes of 10 c.c. each, divided into cubic centimeters
and fractions.
1 burette of 50 c.c. capacity, divided into cubic centimeters
and fractions.
1 separatirig-funnel of 750 c.c. capacity for filling tubes.
2 glass funnels, best quality, about 15 cm. in diameter.
2 glass funnels, best quality, about 8 cm. in diameter.
2 glass funnels, best quality, about 4 or 5 cm. in diameter.
2 porcelain dishes, 200 c.c. capacity.
6 ordinary water tumblers for holding test-tubes.
1 ruled plate for counting colonies.
1 gas-generator, 600 c.c. capacity, pattern of Kipp or v.
Wartha.
BURNERS, TUBING, ETC.
2 Bunsen burners, single flame.
1 Rose-burner.
1 Koch safety-burner, single flame.
6 feet of white-rubber gas-tubing.
12 feet of pure red-rubber tubing, 5 to 6 mm. inside
diameter.
1 thermo-regulator, pattern of L. Meyer or Reichert.
2 thermometers, graduated in degrees of Centigrade,
registering from 0° to 100° C., graduated on the stem.
1 thermometer graduated in tenths and registering from
0° to 50° C.
1 thermometer registering to 200° C.
INSTRUMENTS, ETC.
1 microtome, pattern of Schanze, with knife.
1 razor-strop.
APPENDIX 673
6 cheap-quality scalpels, assorted sizes.
2 pair heavy dissecting-forceps.
1 pair medium-size straight scissors.
1 pair small-size straight scissors.
1 hypodermic syringe that will stand steam sterilization.
2 teasing-needles.
1 pair long-handled crucible-tongs for holding mice.
1 wire mouse-holder.
2 small pine boards on which to tack animals for autopsy.
2 covered stone jars for disinfectants and for receiving
infected materials.
INCUBATORS AND STERILIZERS.
1 incubator, simple square form, either entirely of copper
or of galvanized iron with copper bottom.
1 medium-size hot-air sterilizer with double walls, asbes-
tos jacket, and movable false bottom of copper plates.
1 medium-size steam sterilizer; either the pattern of
Koch or that known as the Arnold steam sterilizer, prefer-
ably the latter.
MISCELLANEOUS.
1 pair of balances, capacity 1 kilogram; accurate to 0.2
grams.
1 set of cork-borers.
1 hand-lens.
1 wooden filter-stand.
2 iron stands with rings and clamps.
3 round, galvanized iron-wire baskets to fit loosely into
steam sterilizer.
43
674 APPLICATION OF METHODS OF BACTERIOLOGY
3 square, galvanized iron-wire baskets to fit loosely into
hot-air sterilizer.
1 sheet-iron box for sterilizing pipettes, etc.
1 covered agate-ware saucepan, 1200 c.c. capacity.
2 iron tripods.
1 yard of moderately heavy wire gauze.
2 test-tube racks, each holding 24 tubes, 12 in a row.
1 constant-level, cast-iron water-bath.
2 potato-knives.
2 test-tube brushes with reed or wire handles.
Cotton-batting.
Copper wire, wire nippers.
Round and triangular files.
Labels.
Towels and sponges.
INDEX.
A
ABSCESSES, 350-352
Acid proof bacteria, 449
actinomycotic growth, 453
cultivation of, 451
distinguished from bacillus
tuberculosis, 451
distribution of, 450
inoculation with, 452
mycelial growth, 453
relation to bacillus tuber-
culosis, 454
staining of, 449
Actinomycetes, 457
bovis, 459
Eppingeri, 467
fascinicus, 465
madurse, 463
pathogenesis of, 459, 463, 466,
467, 468
pseudotuberculosis, 468
Aerobic bacteria, 55, 227
cultivation of, Buchner's
method, 227
Frankel's method, 227
Kitasato and Weil's method,
230
Park's method, 230
Aerobioscope, 663
Agar-agar, lactose-litmus, 142
nutrient, 129
filtration of, 131
preparation of, 129
Agglutinins, 299
Aggressins, 299
Air, bacteriological analysis of, 660
apparatus for, 661-663
methods of, 661-665
Alexin, 289
Ammonia, test for, 225
Anaerobic bacteria, 55, 227
method of cultivation of,
227
Buchner's, 227
Frankel's, 227
Kitasato's, 230
Park's, 230
Animalculse, 20
Animals, cultures from, 259
blood, 260
precautions, 260
holders for, 234-238
inoculation of, 232-254
into eye, 247
into lymphatics, 244
into serous cavities, 245
intravascular, 239
subcutaneous, 252
observation of, 248-254
postmortem examination of, 255-
259
cultures at, 257
Anthrax, bacillus of, 583. See
Bacillus anthracis.
discovery of, 21
immune serum, 594
protective inoculation against,
591
Antibodies, 303
Antiseptics, determination of pro-
perties of, 339
experiments with, 340
testing of, 339
Antitoxins, 273
Apparatus for beginner's labora-
tory, 669-674
Arnold's sterilizer, 89
Autoclave, 91
Avian tuberculosis, 455
(675)
676
INDEX
B
BACILLI, 66
Bacillus aerogenes capsulatus, 627
cultivation of, 627-628
morphology of, 627
pathogenesis of, 628
anthracis, 583-599
cultivation of, 586-588
discovery of, 583
experiments with, 595-599
immune serum from, 594
inoculation experiments, 589
morphology of, 583-585
Pasteur's vaccine, 591
pathogenesis of, 589
protective inoculation against,
591
spore formation in, 584-586
staining of, 588
Chauvei, 623-627
cultivation of, 623-625
differentiation of, 626
discovery of, 622
morphology of, 622
occurrence of, 621
pathogenesis of, 625
spores of, 622
coli, 530
bacillus typhosus and, 534
cultivation of, 532-535
discovery of, 530
distribution of, 531
inoculation experiments, 535
morphology of, 532
pathogenesis of, 531
diphtherise, 482-498
bacteria simulating, 482
cultivation of, 480, 486-489
differential tests for, 497-500
stainings, 500
experiments with, 502
isolation of, 480-481
loss of virulence of, 494
morphology of, 482-486
pathogenesis of, 489-493
staining of, 489
toxin of, 493
dysenteriae, 541-548
agglutination of, 546
cultivation of, 542
discovery of, 541
Bacillus dysenterise, immune
serum, 546
inoculation experiments, 544
protective inoculations with,
547
types of, 541-546
Flexner, 541
Harris, 546
Hiss-Russell, 545
Shiga, 542
Strong, 545
edematis, 616-620
cultivation of, 616-618
discovery of, 616
how to obtain, 616, 619
inoculation experiments, 616
morphology of, 616
pathogenesis of, 619
spores of, 616
where found, 616
enteriditis sporogenes, 628
influenzas, 424
cultivation of, 426
demonstration of, 426
discovery of, 425
distribution of, 425
location of, 428
morphology of, 426
pathogenesis of, 429
vitality of, 427
leprae, 448
characteristics of, 448
location of, in tissues, 448
microchemical characters of,
449
mallei (of glanders), 472
agglutination of, 478
cultivation of, 473
inoculation experiments, 475
morphology of, 472
pathogenesis of, 475-476
resistance of, 473
staining of, 476
of symptomatic anthrax, 623
paratyphosus, 538-540
characteristics of, 538
discovery of, 538
r61e of, in disease, 538
pestis, 392
antiserum, 399
cultivation of, 393-394
discovery of, 372 .
INDEX
677
Bacillus pestis, flea as carrier of,
395
history of, 391
inoculation against, 397
morphology of, 392
pathogenesis of, 394
portals of entry of, 395-396
resistance of, 394
staining of, 393
vaccination in, 397
variations in, 395
pseudotuberculosis, 457, 468
pyocyaneus, 383. See Pseudo-
monas semginosa.
smegmatis, 449
sporogenes, 628
cultivation of, 629
distribution of, 628
pathogenesis of, 629
tetani, 606-615
antitoxin, 614
colonies of, 609
cultivation of, 609
discovery of, 606
inoculation experiments, 611
morphology of, 608
pathogenesis of, 611
poisons of, 612
relation of, to germicides, 610
resistance of, 610
spores of, 608, 610
to obtain, 606-608
tuberculosis, 441. See Tubercu-
losis.
avium, 455
general characters of, 455-457
typhosus, 508-530
agglutination of, 515
cultivation of, 509-511
in drinking water, 519
isolation from, 520
methods of, 520
Endo-media of, 524
enriching media of, 521
flagella of, 509
indol production, 511
inoculation experiments, 513.
isolation of, 513, 520
Drigolski-Conradi method,
522
Endo method, 524
precipitation method, 526
Bacillus typhosus, morphology of,
508
prophylactic vaccination, 526
staining of, 509, 512
in tissues, 512
Welchi, 627
xerosis, 499
Bacteria, acid proof, 449
aerobic, 55
anaerobic, 55, 227
autolysis of, 54
biochemic characters of, 184
chemotaxis of, 60
chromogenic, 37
classification of, 33
colonies of, 193
commensal, 401, 413, 416
composition of, 65
cooperating, 58
cultures of, 193-196
definition of, 33
denitrifying, 39, 602
discovery of, 17
electricity and, 60
enzymes of, 44
coagulating, 49
diastatic, 49
inverting, 49
properties of, 44, 51
proteolytic, 48
sugar splitting, 50
facultative, 55, 231
flagella of, 74-75
grouping of, 66
importance of, 35
involution of, 68-69
isolation of, Koch's observations
on, 104
principles involved, 105
in pure culture, 104, 110
life processes of, 35
light and, 59
metabolism of, 33
metatrophic, 34
moisture and, 59
morphology of, 63-66
motility of, 74
multiplication of, 66-70
nitrifying, 38, 600-606
nitrogen fixing, 39, 600-606
nutrition of, 52-55
natural, 33
678
INDEX
Bacteria, parasitic, 40-44
specific functions of, 40-44
paratrophic, 34
pathogenic properties of, 197
peculiarities of, 33
photogenic, 37
place in nature, 35
pressure and, 59
products of, 53-54
prototrophic, 34
reduction by, 223
relation to oxygen, 55
results of disease, 29
saprogenic, 37
saprophytic, 34-40
specific functions of, 36-40
size of, 63
spores of, 72-74
structure of, 63-65
symbiosis, 58
systematic study of, 191
bio-chemistry, 193-196
biology, 193
enzymes, 197
gas from, 197
light and, 196
morphology, 192
pathogenesis, 197
pigment and, 196
reaction, 197
temperature and, 56
thermal death-point of, 94
thermophilic, 57
thiogenic, 38
, toxins of, properties of, 44
variations and varieties, 199
of species, 199
water, 57
zymogenic, 38
Bacteriacese, 33
Bacterial diseases, vaccination
against, 281
proteins, 44
toxins, 272
examination of cultures for,
226
types, 203
Bacteriological analysis of air, 660
of milk, 666
of soil, 665
of water, 637
Bacteriology, application of, 323
Bacteriology, beginnings of, 17-32
birth of modern, 30-32
historic sketch of, 17-32
preliminary experiments, 323
Bacterium Welchii, 627-628
cultivation of, 627
discovery of, 628
morphology of, 627
pathogenesis of, 628
Bail, 293
Behring-Kitasato, 290
Billroth, 29, 30
Biology of bacteria, 193
Birsch-Hirschfeld, 26
Blood cultures, 260
reactions of coagulation, 288
Foder, 288
Grohmann, 288
immunity from, 313
Rauschenback, 288
toxicity of alexin, 312
Traube and Gscheidlen, 288
serum apparatus, Rivas', 135-
137
from small animals, 135
Loffler's, 143
preparation of, 133
preservation of, 138
serum-water mixture, 143
Body, defenses of, 277
Bonnet, 24
Bordet, 299
and Gengou, 318
Bouillon, 124
Braatz, 335
Brownian motion, 210
Brushes for cleaning test-tubes, 146
Buchner, 289
Bumm, 370
Burdon-Sanderson, 30
Burner, Koch's safety, 159
CHEMOTAXIS, 60
Chevreul, 23
Cholera, Asiatic, bacteria of, 549-
573
antagonisms of, 566
cultivation of, 552
in dead bodies, 566
INDEX
679
Cholera, Asiatic, bacteria of, dis-
covery of, 549
in food, 566
general considerations of,
563
grouping of, 551
immunity from, 562
Pfeiffer's work on, 562,
563
influences of acids on, 558
of gases on, 567
of light on, 565
inoculation experiments,
559
morphology of, 550
pathogenesis of, 561
poison of, 558, 562
resistance of, 567
in soil, 565
in water, 564
when dried, 567
diagnosis of, 568
by cultures, 569
by microscope, 569
Classen, 27
Cohn, 24
Colon bacillus, 530. See Bacillus
coli.
Colonies, macroscopic characteris-
tics of, 165
study of, 165-167
Colony-formation, 166
Commensal relations, 401, 413, 416
Complement, 302-311, 316, 319-
321
fixation of, 316
applications of, 319-321
origin of, 303
specificity of, 303
Cooling chamber for plates, 151,
152
Comma bacillus, 549
Corrosive sublimate, 334
Cover-slip preparations, 172
examination of, 208-211
hanging block, 213
drop, 209
impression, 176
staining of, 171, 632
steps in making and staining,
172-176
Culture media, 111-144
Cultures, filtration of, 226
from blood, 260
gelatin, 214
hanging block, 213
drop, 209 '
potato, 194, 215
pure, 167
smear, 167
stab, 167
test-tube, 167
DARK-FIELD illumination, 207
Davaine, 21
Decolorizing solutions, 180
Pappenheim's, 183
Defenses of body, 277
antibodies, 279
antidotes, 278
blood serum, 279
ciliated epithelium, 277
gastric juice, 277
germicidal juices, 278
orificial hairs, 277
phagocytic cells, 278
skin, 277
Denitrification, 39, 602
Dentrifying bacteria, 38, 602
Diphtheria, 480
antitoxin, 503
production of, 504
standardization of, 505
Behring, 505
Ehrlich, 506
bacillus of, 482. See Bacillus
diphtherias,
bacteriological diagnosis of,
480, 482
variations in, 483
varieties of, 493, 495, 496-497
Diplococcus, 67
intracellularis meningitidis, 377.
See Micrococcus intracellularis.
Dishes, Petri, 152
Disinfectants, determination of
properties of, 331
experiments with, 340
mode of action of, 96-109
testing of, 331
methods of precaution, 334
680
INDEX
Disinfectants, testing of, methods
of precaution, Braatz's
work, 335
Geppert's work, 335
Disinfection, 95-103
practical, 101
Dry heat, experiments, 330
DuckwalTs staining method, 189
Durham's fermentation tube, 220
solution, 140
Dysentery, 541
antiserum, 547
bacillus of, 541. See Bacillus
dysenteriae.
protective inoculation against,
547
EBERTH, 27
Ehrlich, 27, 296
and Morgenroth, 300
side chain theory of, 296
Emboli of micrococci, 351
Endotoxins, 46, 274
action of, 276
distinction of, from toxins, 276
immunity for, 277
Enzymes, 44-50
coagulating, 49
diastatic, 49
inverting, 49
proteolytic, 48
sugar splitting, 50
Erysipelas, 359
Esmarch's counting apparatus, 658
tubes, 153
Booker's method, 154
Experiments, contact, 325
exposure, 325
sterilization, 327
FACULTATIVE bacteria, 55
Fehleisen, 27
Fermentation, 216
early views on, 28
tube, 218-221
Durham's, 219
erments, 44-45
^ilter, folding of, 126
Filterable virus, 261
nature of, 265
study of, 263-265
where found, 265
Filters, bacterial, 226
Fish, 296
Fixation of complement, 316
Flagella, 74-75
staining of, 187-189
Flasks, preparation of, 145
Flexner, 382
GAS-PRESSURE regulators, 163
Gelatin, agar mixture, 144
cultures, 214
filtration of, 126
lactose-litmus, 142
nutrient, 175
preparation of; 125
Generation, spontaneous, 22-25
Geppert, 335
Glanders, 470
bacillus of, 472. See Bacillus
mallei.
diagnosis of, 478
by agglutination, 478
by mallein, 478
pathology of, 476
Glassware, preparation of, 145
Gonococcus, 370. See Micrococcus
gonorrhoeas.
Gonorrhea, cause of, 370
Gram's method of staining, 184
Guarniari's medium, 144
HANGING-BLOCK cultures, 213
Hanging-drop cultures, 209
Haptophores, 301
Harvey, 25
Havins, 364-367
Hemolysis, 312
alien blood and, 312, 313
Landois on, 312
mechanism of, 314-316
INDEX
681
Hemolytic reaction, 312
system, 315
Henle, 21
Hiss' medium, 143
Hoffman, 23
Holders for animals, 234-238
Hydrogen-ion concentration, 115
Hydrogen sulphide, test for, 224
Hypodermic needle, 241
syringes, 244
ILLUMINATION, dark-field, 207
Immune bodies, 303
Immunity, 277-311
acquired, 280
active, 281
alien proteins, 299
Behring and Kitasato on, 290
Buchner on, 289
Chauveau on, 286
conclusions on, 305-311
diverse reactions in, 302
Ehrlich's side-chain theory, 300
receptors, 300
"exhaustion" hypothesis, 287
historic sketch of, 285
Metchnikoff's doctrine, 287
natural, 280
Nuttall's work in, 288
opsonic doctrine, 294
passive, 281
Pasteur's doctrine, 287
Pfeiffer's reaction, 292
phagocytosis, 287
"retention" hypothesis, 287
Roux and Yersin on, 290
views on, 304-311
Wright and Douglass on, 278
Immunology, beginnings, 28
Incubator, 157
Indicators, 119
Indol production, 221
tests for, 222
Infection, 266
mechanism of, 272, 309
types of, 266-272
views on, 309-311
Influenza, bacillus of, 424. See
Bacillus influenzse.
Influenza, filterable virus in, 425
historic outbreaks of, 424
pneumococci in, 425
streptococci in, 425
Inoculation methods, 232-254
eye, 247
lymphatics, 244
needles and syringes for, 241,
244
serous cavities, 245
subcutaneous, 232
vessels, 239
Intracellular toxins, 46, 274
Involution, 68
Isolation of bacteria in pure cul-
ture, 104-110
of pathogenic organisms in the
sputum, 401
KLEBS, 26, 27, 30
Koch, 30
Koch-Ehrlich aniline water solu-
tion, 178
Koch's postulates, 440
safety burner, 159
sterilizer, 88
LABORATORY outfit, 669-674
Lactose litmus,-agar, 142
Landois, 312
Leeuwenhoek, 17, 19
Lens, oil-immersion, 208
Leprosy, bacillus of, 448
Leptospira, ictero-hemorrhagiae,
631
icteroides, 631
Letzerich, 27
Liebig, 28
Litmus gelatin of Wurtz, 142
milk, growth of bacteria in, 138
Litmus- whey milk, 140
Loffler, 32
Loffler's alkaline methylene-blue
solution, 178
method of staining flagella, 187
serum mixture, 143
Lukomsky, 27
Lysin, 299
682
INDEX
M
MALIGNANT edema, bacillus of, 616.
See Bacillus edema tis.
Mallein, 478
Media, culture, 111-144
agar-agar, 129
blood-serum, 133
from small animals, 132
preservation of, 138
bouillon, 124
Dunham's peptone solution,
140
gelatin, 125
-agar mixture, 144
lactose-litmus agar, 142
litmus-gelatin, 142
Loffler's serum mixture, 143
milk, 138
litmus-whey, 140
neutralization of, 111-123
potatoes, 132
' reaction of, 111-123
changes in, 215
determination of hydrogen-
ion concentration, 115-124
litmus, 110
method of Barnett and
Chapman, 120
titration, 113
serum-water, of Hiss, 143
Meningitis, cerebrospinal, 377
antiserum for, 382
coccus causing, 377
Meningococcus, 377. See Micro-
coccus intracellularis.
types, 381
Mercurial thermoregulator, 161
Metatrophic bacteria, 34
Metchnikoff, 287
Micrococci, 66
Micrococcus aureus, 345
abscesses from, 349
antiserum for, 353
characteristics of, 345-352
cultural, 346-348
pathogenic, 348
inoculation with, 348
microscopic study of, 359
source, 345
toxins, 352
citreus, 354
Micrococcus epidermidis • albus,
354
gonorrhea, 370-377
characteristics of, 376
cultivation of, 371-374-
morphology, 370
organisms simulating, 375
pathogenesis of, 374
peculiarities of, 376
staining reactions of, 371
intracellularis, 377
antiserum for, 382
cultures of, 378
discovery of, 377
inoculation with, 380
morphology of, 377
peculiarities of, 378
varieties of, 381
lanceolatus, 369
pyogenic, 345-368
tetragenus, 421
Microscope, parts of, 204
Microscopic examination, 204, 207-
211
Microspira comma, 549-573. See
Cholera.
Metchnikovi, 574-579
cultivation of, 575-578
discovery of, 574
immunity from, 579
inoculation experiments, 578
morphology of, 574
pathogenesis of, 578
resistance of, 578
source of, 574
Schuylkilliensis, 580-582
cultivation of, 580-581
morphology of, 580
pathogenesis of, 581
Miliary abscesses, 350-352
Milk as culture medium, 138
preparations of, 138
bacteriological analysis of, 666
microscopic, 666, 668
qualitative, 667
quantitative, 667
Moitessier's gas pressure regulator,
163
Molecular tremor, 210
Morphology of bacteria, 63, 192
Motility of bacteria, 74
Mouth, organisms in, 401
INDEX
683
Moxter, 299
Multiplication of bacteria, 66, 70
N
NASSILOFF, 27
Needham, 22
Neisser, 370
Neutralization of culture media.
See Reaction.
Nitrates, reduction of, 224
Nitrifying bacteria, 38, 600-606
cultivation of, 603
function of, 600-602
morphology of, 603-606
where found, 600
Nitrites, tests for, 224
Nitrogen fixation, 39, 602
Nocard and Roux, 263
Nutrition of bacteria, 52-55
NuttalPs platinum spear, 257, 293
OERTEL, 27
Ogston, 368
Oil-immersion lens, 208
system, 208
Opsonin, 294
Orth, 27
Oven incubator, 158
Oxygen, cultivation without, 227
relation of bacteria to, 55
Ozanam, 21
FAKES' counting apparatus, 657
Pappenheim's decolorizer, 183
Parasitic bacteria, 34-36
Paratrophic bacteria, 34
Paratyphoid bacillus, 538. See
Bacillus paratyphosus.
Passet, 368
Pasteur, 21, 23, 28, 30
Pathogenic properties of bacteria,
266-272
Peptone, purity of, 141
solution, 140
Pest, 391
Petri dish, 152
Phagocytosis, 287
Photogenic bacteria, 37
Plague, 391-400
antiserum, 399
bacillus, 392. See Bacillus pes-
tis.
historic outbreaks of, 391
protective inoculation, 397
reports on, 400
Plates, in isolating bacteria in pure
culture, 149-153
Plenciz, 20, 21
Pleuro-pneumonia of cattle, 263
Pneumococcus, 404
Pneumonia, 414
antiserum, 419
bacterium of, 404
cultivation of, 407
discovery of, 406
immunization, 419
inoculation of, 405, 408
morphology of, 405, 406
pathogenesis of, 409
types, 410-412 •
crisis in, 417
mechanism of infection, 414
Pollender, 21, 27
Post-mortem examination of ani-
mals, 255-259
Postulates of Koch, 440
Potato culture, 194, 215
preparation of, 132
Precipitins, 299
Proteins, bacterial, 51
Prototrophic bacteria, 34
Pseudodiphtheric bacteria, 497
Pseudomonas seruginosa, 383
color variations in, 384
cultivation of, 384-385
enzymes of, 388
experiments, 384, 388
inoculation of, 390
morphology of, 383
pathogenesis of, 390
Pseudotuberculosis, bacteria of,
457, 468
Ptomains, 44, 51
Pure cultures, 167
isolation of, 149
plates, 149
serial tubes, 155 f
684
INDEX
Pyocyaneus bacillus, 363. See
Pseudomonas seruginosa.
Pyogenic bacteria, common, 345-
368
less common, 368
REACTION, adjustment of, 111-124
Barnett and Chapman, 120
changes in, 215
hydrogen-ion concentration, 115-
124
litmus as indicator, 111
pH values, 115-124
titration method, 113-115
Receptors, 301
Recklinghausen, 27
Reduction by bacteria, 223
Regulators, pressure, 164
thermo-, 160-163
Resistance, 277
Rhinitis, membranous, bacteria of,
494
Rindfleisch, 26
Rosenbach, 268
Roux and Yersin, 290
S
SALIVA, study of, 403 .
Sanderson, 29
Saprogenic bacteria, 37
Saprophytic bacteria, 34-35.
specific functions of, 36-40
chrompgenesis, 37
denitrification, 39
nitrification, 38
nitrogen fixing, 39
photogenesis, 37
saprogenesis, 37
thiogenesis, 38
zymogenesis, 38
Sarcina, 68
tetragena, 421
cultivation of, 422
discovery of, 421
inoculation of, 423
morphology of, 421
Schaefer, 334
Schizomycetes, 33
Schroder and Dusch, 23
Schulze, 23
Sedgwick and Tucker, 663
Septicemia, sputum, 404
Serial tube method of separation,
155
Serum, blood. See Blood serum.
Serum-water media of Hiss, 143
Sewage streptococci, 659
presence of, in water, 660
Skin disinfection, 343
Smear cultures, 167
Smegma bacillus, 449
Soil, bacteria in, 665
isolation of, 665
Spallanzani, 30
Species, types of, 203
variations of, 199
Spirilla, 67-70
Spirillum of Asiatic cholera, 549-
574. See Cholera, bacteria of.
Metchnikovi, 574. See Micro-
spira Metchnikovi.
Schuylkilliensis, 580. See Micro-
spira Schuylkilliensis.
Spirochsetacese, 629-636
classification of, 629-630
definition of, 629
detection of, 632
in smears, 632
in tissues, 634
genera of, 630
leptospira, 631
ictero-hemorrhagiae, 631
icteroides, 631
spironemia, 630
in relapsing fever, 630
treponema, 631
pallada, 631
pertenue, 631
Spironema of relapsing fever, 630
Spores, discovery of, 24
formation of, 72-74, 211
methods of study, 209-213
staining of, 186
Sputum, bacteria in, 401-403
Stab cultures, 167
Staining, 176-190
cover slips, 172, 632
methods, 176
acetic acid, 185
INDEX
685
Staining methods, aniline water,
178
carbol-fuchsin, 179
Duckwall's, 189
flagella, 187
Gabbett's, 183
Gram's, 184
Loffler's, 178
Neisser's, 500
of spirocheta pallida, 632, 633
spores, 186
Stern's, 633
of tubercle bacillus, 181
solutions, 176-190
Koch-Ehrlich's, 178
Loffler's blue, 178
Ziehl's carbol-fuchsin, 179
Staphylococcus, 67
aureus, 345. See Micrococcus
aureus.
citreus, 354
epidermidis albus, 354
Staphylotoxin, 352
Steam, sterilization by, 80-90
. experiments, 327-330
Sterilization, chemical, 95
definition of, 76-78
experiments in, 327-330
heat, 78
methods, 79
direct, 85
discontinued, 80-85
fractional, 80-85
hot air, 91-94
pressure, 90
steam, 87-95
Sterilizers, 88-93
Sternberg, 339, 406
Stern's staining method, 633
Streptococci, 67
Streptococcus hemolyticus, 360
pyogenes, 354, 355-368
antiserum, 367
cultivation of, 357-359
hemolysis from, 360
inoculations, 359-361
isolation of, 355
morphology of, 356
pathogenesis of, 359-361
source of, 359
types of, 363
variations and varieties of, 361
Streptococcus pyogenes viridans,
358
virulence of, 360-361
Suppuration, causes of, 368-370
Sweet's animal holder, 236
Symbiosis, 58
Symptomatic anthrax, bacillus of,
621
Syphilis, diagnosis of, 319
treponema of, 631
TEST-TUBE cultures, 167
preparing and filling of, 146
Tests for ammonia, 225
for hydrogen sulphide, 224
for indol, 221
for nitrates, 224
for nitrites, 224
for toxins, 226
Tetanus, antitoxin for, 614
bacillus of, 606-615
Tetrads, 68
Thermal death-point, 94
Thermo regulators, 160-163
Thermophilic bacteria, 57
Thiogenic bacteria, 38
Tiegel, 29, 30
Toxins, 44, 46, 47, 272
action of, 272
antitoxins, 273
bacterial, 226, 272
formation of, 226
immunity from, 277
intracellular, 274
nature of, 272
reactions of, 272
tests for, 226
Toxoids, 274
Toxones, 274
Treponema pallada, 631
pertenue, 631
Treviranus, 30
Tubercles, miliary, 406
Tuberculin, 446
Tuberculosis, bacillus of, 441-448
cultivation of, 441
isolation of, 441
lesions produced by, 430-437
morphology of, 444
686
INDEX
Tuberculosis, bacillus of, organ-
isms simulating, 448
parasitic tendencies of, 441
peculiarities of, 441, 445
staining of, 445
variations of, 446
cassation in, 433
cavity formation in, 434
encapsulation of foci in, 436
giant cells, 440
location of bacilli in, 439
miliary tubercles, 432
modes of infection in, 438
organisms with which it may be
confused, 448
primary infection in, 436
susceptibility of animals to, 446
vaccination against, 447
Tubes, Booker, 154
Esmarch, 153
fermentation, 218-221
filling of, 146
preparation of, 145
serial, 155
Tyndall, 24
Types of bacteria, 203
Typhoid fever, agglutination test
in, 515
bacillus of, 508. See Bacillus
typhosus.
prophylactic vaccination in,
526
vaccine in, preparation of, 528
water and, 519
Widal's reaction in, 515
UNSTAINED preparations, 207, 209
VACCINATION, bacterial, 281
Van Dungeon, 296
Variation of species, 199
Variations and varieties of bacteria,
199
Vibrio Metchnikovi, 574. See
Microspira Metchnikovi.
Schuylkilliensis, 580. See Micro-
spira Schuylkilliensis.
Vibrion septique, 616. See Bacil-
lus edematis.
Viruses, filtrable, 261
ultra-microscopic, 261
W
WALDEYER, 26
Wassermann reaction, 319
Water bacteria, 641
bacteriological study of, 637-660
collection of samples for, 647
colon bacteria, 641
counting colonies in, 654
apparatus, 655-658
disease from, 638
interpretation of results,
635-643
objects of, 638
pathogenic bacteria in, 646
precautions in, 644
qualitative, 643
special methods, 648
quantitative, 649
methods, 649-654
sewage in 659
typhoid bacteria, 640
Weichselbaum, 377
Weigert, 30, 296
Weigert's doctrine, hyperplasia,
296
Welchii bacterium, 627
Widal's reaction, 517
Wilde, 27
WolffhugeFs counting apparatus,
655
Wound infections, 26-27
Wright and Douglass, 293-294
Wurtz, litmus gelatin of, 142
XEROSIS bacillus, 499
differentiation of, 500-501
ZIEHL'S carbol-fuchsin solution,
179
Zymogenic bacteria, 38
Zymophores, 302
,
I VJ
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