Full text of "Bacteriology, general, pathological and intestinal"

BIOLOGY
UBffeRY

BACTERIOLOGY

GENERAL, PATHOLOGICAL

AND

INTESTINAL

ARTHUR ISAAC KENDALL, B.S., PH.D., DR.P.H.

PROFESSOR OF BACTERIOLOGY IN THE NORTHWESTERN UNIVERSITY MEDICAL SCHOOL,

CHICAGO, ILLINOIS

ILLUSTRATED WITH 98 ENGRAVINGS AND 9 PLATES

LEA & FEBIGER

PHILADELPHIA AND NEW YORK
1916

BIOLOGY
RA

Entered according to the Act of Congress, in the year 1916, by

LEA & FEBIGER,
in the Office of the Librarian of Congress. All rights reserved.

THEOBALD SMITH, M.D., LL.D.

PREFACE.

"!N the study of the microscopic forms known as bacteria we have
what might be fitly called the focal points of the various branches of
biological science. Though their investigation may require careful
morphological researches, yet the unmistakable monotony of form
combined with a considerable variation of physiological activity has
compelled the bacteriologist to pay much attention to means by which
such physiological variations may be more or less accurately registered
in order that they may serve as a supplementary basis for classification.
Again, with unicellular organisms the manifestations of cell activity
become the most important phenomena for study. These manifesta-
tions bring together the fields of physiology and chemistry and make
bacteriology in one sense a branch of physiological chemistry." 1

" There is no ulterior interest in the study of bacteria as such, which
is a strong impulse in many other departments of biological science. It
is what bacteria do, rather than what they are, that commands atten-
tion, since our interest centers in the host rather than the parasite." 2

The development of bacteriology has followed very closely the
gradual improvement of the optical parts of the compound microscope,
and to a lesser degree, the perfection of other instruments of precision
on the one hand, and the production of anilin dyes and a great expan-
sion of the fields of organic and physical chemistry on the other hand.
Naturally the greatest advances in bacteriology have been made along
the lines of morphology, staining and diagnosis, because the application
of the microscope, anilin dyes, and the preparation and use of cultural
media to bacterial problems is relatively simple and direct. The final
chapters of bacteriology, in which the problems of immunology are
of paramount interest, will be intimately associated with an unfolding
of the chemistry of cellular activity, as Theobald Smith has so clearly
pointed out in the opening paragraphs of this discussion.

1 Theobald Smith. The Fermentation Tube, Wilder Quarter Century Book, 1893,
p. 187.

2 Theobald Smith. Some Problems in the Life History of Pathogenic Microorgan-
isms, Amer. Med., 19Q4, viii, 711.

vi PREFACE

The chemistry of bacterial activity is not thoroughly studied at
the present time and many of its problems must await the develop-
ment of new methods of chemistry and physics, as well as a refine-
ment of existing methods. Nevertheless, sufficient information exists
to warrant its presentation in concrete form, partly to emphasize its
deficiencies, chiefly to indicate its relation to the biology of the bacteria,
which are potentially "living chemical reagents," as Professor Folin
has so aptly termed them.

In the last analysis, the interest and importance of bacteria centers
around "what they do rather than what they are," and the elucida-
tion of this aspect of bacteriology lies largely within the field of
biochemistry.

The relation of the chemistry of bacterial nutrition to the study
of intestinal bacteriology in health and disease is self-evident; some
of the more general aspects of this subject are briefly set forth in the
chapter relating to intestinal bacteria.

It is with great pleasure that the writer acknowledges his indebted-
ness to his colleagues in the Northwestern Univeristy Medical School
for many valuable suggestions, to Doctors Noguchi and -Amoss, of
the Rockefeller Institute, for the privilege of using the original plates
illustrating the Treponemata and Poliomyelitis, and to Mrs. N. M.
Frain for the line drawings in the text. Finally, the writer would
acknowledge his deep obligation to Miss Bertha J. Schwarz, Secretary
of the Department of Bacteriology, for her invaluable assistance in
the preparation of the manuscript and in reading the proof of the
book. A. I. K.

CHICAGO, 1916.

CONTENTS.

SECTION I.-GENERAL BACTERIOLOGY.

INTRODUCTION. THE DEVELOPMENT AND SCOPE OF
BACTERIOLOGY.

CHAPTER I.
THE MORPHOLOGY OF BACTERIA.

PAGE

Normal and Abnormal Forms Size and Weight Structure and Con-
stituents of the Bacterial Cell Reproduction and Cell Division
Cell Grouping, Classification, and Mutation 17-35

CHAPTER II.

THE PHYSIOLOGY OF BACTERIA AND THE EFFECT OF ENVIRONMENTAL

INFLUENCES.

Rate of Reproduction Motility Germination of Spores Longevity
Effects of Moisture, Oxygen, Temperature, Light and Electricity,
Gravity, Osmotic Pressure Production of Enzymes, "Toxins, Pto-
maines and Pigments Symbiosis, Antibiosis, Commensalism . . 36-55

CHAPTER III.
THE CHEMISTRY OF BACTERIA.

Chemical Constitution of Bacteria and Composition of Morphological
Components of Bacterial Cell Food Relationships of Bacteria,
Bacterial Nutrition 56-67

CHAPTER IV.
BACTERIAL METABOLISM.

The Nature of Bacterial Metabolism Nitrogen Metabolism, Carbon
Metabolism Reactions of Bacterial Metabolism Significance of
Bacterial Metabolism Putrefaction and Fermentation . 68-83

viii CONTENTS

CHAPTER V.
SAPROPHYTISM, PARASITISM AND PATHOGENISM.

PAGE

The Cycle of Parasitism The Cycle of Pathogenism Distribution of
Parasitic and Pathogenic Bacteria in Nature How Parasitic and
Pathogenic Bacteria Reach Man How they Reach the Body,
Portals of Entry, Where They Multiply in the Body, Where and
How They Escape from the Body Balanced Pathogenism and
Epidemiology 84-110

CHAPTER VI.

INFECTION AND IMMUNITY.

Classification of Immunity Infection Theories of Immunity . . . 111-131

CHAPTER VII.
Anaphylaxis, Allergy or Hypersensitiveness 132-141

CHAPTER VIII.
ANTIGENS AND THE TECHNIQUE OF SERUM REACTIONS.

Nature of Antigens and Antibodies Agglutinins and Precipitins
Lysins, Hemolysis and Complement Fixation Aggressins Opson-
ins, Tropins Bacterial Vaccines . .....".. . . 142-174

CHAPTER IX.
BACTERIOLOGICAL TECHNIQUE.

Methods for the Microscopic Study of Bacteria Staining Methods-
Media Cultivation of Bacteria, Study of Bacterial Cultures . . 175-223

CHAPTER X.
BACTERIOLOGICAL EXAMINATION OF MATERIAL FROM PATIENT AND CADAVER.

Autopsy Procedure Blood Cultures Cerebrospinal Fluid Peritoneal,
Pleural and Pericardial Fluids Pus Examination of Urine,
Feces, Sputum, Buccal and Pharyngeal Material Bacteriological
Examination of the Eye, Ear and Nose The Utilization of Animals
for Bacterial Diagnosis and Experimentation ....... 224-240

CHAPTER XI.
PRACTICAL STERILIZATION, ANTISEPSIS AND DISINFECTION.

Laboratory Sterilization Physical Agents, Chemical Solutions, Test-
ing and Standardizing Liquid Disinfectants Gaseous Disinfectants
Disinfection of Sputum, Vomitus, Feces and Urine, Fomites, Skin
and Hands, Instruments . . ... -V 241-254

CONTENTS IX

SECTION II.-PATHOGENIC BACTERIA.

CHAPTER XII.

PAGE

The Pyogenic Cocci 255-268

CHAPTER XIII.
The Streptococcus-Pneumococcus Group 269-291

CHAPTER XIV.
The Meningococcus-Gonococcus Group 292-309

CHAPTER XV.
Micrococcus Melitensis 310-312

CHAPTER XVI.
The Alcaligenes Dysentery Typhoid Paratyphoid Group . . . 313-352

CHAPTER XVII.
The Coli Cloacae Proteus Group 353-362

CHAPTER XVIII.
The Mucosus Capsulatus Group 363-366

CHAPTER XIX.

Glanders, Anthrax, Pyocyaneus, Infectious Abortion, Aciduric Bacteria 367-387

CHAPTER XX.

Diphtheria Group . . . 388-406

CHAPTER XXI.
Hemorrhagic Septicemia Group 407-416

CHAPTER XXII.

HEMOGLOBINOPHILIC BACILLI.
Influenza, Pertussis, Koch- Weeks, Morax-Axenfeld and Ducrey Bacilli . 417-427

CHAPTER XXIII.

TUBERCLE BACILLUS GROUP.
Human, Bovine and Avian ... 428-462

x CONTENTS

CHAPTER XXIV.

PAGE

Leprosy and Acid-fast Bacteria other than the Tubercle Group . 463-471

CHAPTER XXV.

ANAEROBIC BACTERIA.

Tetanus, Botulinus, Aerogenes Capsulatus, Malignant Edema and

Symptomatic Anthrax . . 472-498

CHAPTER XXVI.
Cholera Group 499-513

CHAPTER XXVII.
Treponemata and Spirocheta 514-532

SECTION III.-HIGHER BACTERIA, MOLDS,
YEASTS, FILTERABLE VIRUSES, AND
DISEASES OF UNKNOWN ETIOLOGY.

CHAPTER XXVIII.

Trichomycetes, Actinomycetes, Hyphomycetes and Saccharomycetes . 533-554

CHAPTER XXIX.
Filterable Viruses Diseases of Unknown Etiology 555-578

SECTION IV.-GASTRO-INTESTINAL
BACTERIOLOGY.

CHAPTER XXX.
Gastro-intestinal Bacteriology 579-600

SECTION V.-APPLIED BACTERIOLOGY.

CHAPTER XXXI.
Bacteriology of Milk 601-613

CHAPTER XXXII.

Bacteriology of the Soil, Water and Air '^ . . . . 614-625

SECTION I.

GENERAL BACTERIOLOGY.

INTRODUCTION THE DEVELOPMENT AND SCOPE OF

BACTERIOLOGY.

BACTERIOLOGY is that branch of Natural Science which treats of
the structure, functions and chemistry of bacteria. Bacteria are
intimately related to many fields of human activity, therefore bacterio-
logy is inseparably associated with a number of the arts and sciences.
In those branches of science which treat of the diseases of plants,
of animals and of man, bacteria enter into complex reciprocal relations
with their hosts as parasites or pathogens, relations which are neither
purely bacterial, animal nor vegetal in their limitation. A new science,
Immunology, is rapidly developing which is concerned chiefly with
the elucidation of these relationships between host and parasite.

Bacteria are the smallest in size and simplest in structure of known
visible living organisms. They are rigid unicellular organisms devoid
of chlorophyll or other photodynamic pigment; they possess no
morphologically demonstrable nucleus and reproduce by simple
transverse fission, the resulting individuals being of approximately
equal size.

Bacteria are ubiquitous in their distribution; they are found in all
climates in association with animal and vegetable life. Some thrive
at temperatures but slightly above the freezing point of water; the
majority flourish between 15 and 40 Centigrade; some even develop
in thermal springs at a temperature of 70 Centigrade. Free or atmos-
pheric oxygen is essential for most types of bacteria, but to a few it
is actually a poison.

Bacteria are ordinarily classed as plants, but they exhibit several
prominent characteristics which suggest a relationship with the lowest

animals. The most important of these is the absence of photodynamic
2

18 /. ',' ". ; . : INTRODUCTION

pigment (chlorophyll), which implies an analytical or destructive
function in the economy of Nature.

The great majority of bacteria are saprophytic, living upon dead
organic matter, which they transform into simple compounds suitable
for plant use. These bacteria are Nature's analysts. Some are para-
sitic on living plants and animals; a few are progressively pathogenic
for man and animals. It is this last group, few in numbers, but for-
midable in that their activities are in partial opposition to those of
man and animals, that has given to bacteria all the notoriety which
they possess.

Anton von Leeuwenhoek, a Dutch spectacle maker, appears to have
been the first to see bacteria: in 1675, with lenses of his own grinding,
he examined various putrescent fluids, drops of water, scrapings from
his teeth, and his own diarrheal discharges. He says in his writings,
collected and edited by Robert Hooke, 1 "With great astonishment
I observed everywhere through the material which I was examining,
animalcules of the most minute size, which moved themselves about
very energetically." It is possible to recognize cocci, bacilli and
spirilla in his drawings, and it is almost certain that he actually observed
motility among his organisms. The learned monk, Athanasius Kircher,
observed and described "minute living worms" as early as 1659, but
his optical equipment was inferior to that of von Leeuwenhoek and it
is doubtful if he actually saw bacteria.

Improvements in the microscope opened a new world for investiga-
tion and speculations concerning the doctrine of the Spontaneous
Generation of Life led to numerous experiments of increasing refine-
ment that finally resulted in the brilliant researches of Pasteur, and
Tyndall, who showed by numerous ingenious and carefully executed
experiments that the phenomena in putrescible fluids erroneously
interpreted as spontaneous generation did not take place when proper
precautions in manipulation were observed. About 1835 achromatic
lenses for the microscope reached a state of perfection compatible
with the examination of minute objects and the microscope was almost
immediately applied to the study of various morbid processes, with
remarkable success. Bassi (1837) discovered a fungus which caused
a contagious disease of silk worms known as muscardine; Cagniard
de Latour and Schwann observed and described the yeast plant in
liquids undergoing alcoholic fermentation.

1 Collected Memoirs of Anton v. Leeuwenhoek, Royal Society of London, 1675, 1683.

INTRODUCTION 19

Ehrenberg (1838) began his classification of animalcules and in his
group of Vibrionia described several "species" of organisms, as follows:

1. Bacterium rigid and filamentous organisms.

2. Vibrio flexuous and filamentous organisms.

3. Spirillum rigid spiral filamentous organisms.

4. Spirocheta flexuous spiral filamentous organisms.

This classification, which contains terms widely used in bacterial
nomenclature today, was followed in 1872 by the important contribu-
tions of Cohn upon "Bacteria," the starting-point of modern bacterial
classification.

The diseases of man naturally attracted much attention and in 1839
Schoenlein examined the crusts of that disease of the scalp known as
favus with the microscope and found the mycelia of the fungus now
known in his honor as Achorion schoenleinii.

The extensive studies of Pasteur upon yeasts and the "diseases"
of beer and wine, upon the diseases of the silk worm (pebrine and
flacherie), upon furunculosis and puerperal sepsis, 1 upon anthrax
and anthrax immunization (attenuated viruses) chicken cholera, and
somewhat later, rabies laid broad foundations for the development
of the science of bacteriology.

Among the most important technical discoveries which have con-
tributed to the development of bacteriology are: The improvement
in the achromatic lens (about 1835) and the perfection of the sub-
stage condenser (Abbe) ; the use of cotton for air filters in flasks and
test-tubes by Schroeder and von Dusch (1854), the sterilization of
culture media by heat (Pasteur, Tyndall, Koch and others), the
introduction of anilin dyes as staining reagents by Weigert and Ehrlich
(1877), and finally, the use of solid culture media and the plate method
for pure cultures by Koch in 1881.

Sir Joseph Lister (1867) published an epoch-making contribution
entitled, "On the Antiseptic Principle of the Practice of Surgery,"
in which is clearly set forth the importance of bacteria in surgery
and the principles of surgical asepsis that have revolutionized this
branch of medicine.

About 1878 Koch isolated the anthrax bacillus in pure culture from
the blood of infected animals, grew the organisms for several generations
in the clear aqueous humor of the eye of the ox, and then reinjected
the organisms into experimental animals and reproduced the disease.
For the first time a specific microbe was clearly and convincingly

1 Compt. rend. Acad. d. Sci., 1880, xc, 1033.

20 INTRODUCTION .

shown to be the etiological factor of a bacterial disease. Koch also
found that the anthrax bacillus formed spores.

From this time bacteriology developed with amazing rapidity. In
1882 Koch startled the world with the announcement of the dis-
covery of the tubercle bacillus; and in rapid succession, typhoid,
diphtheria, cholera, tetanus and other well-known pathogenic bacteria
were isolated and studied in pure culture.

In 1882 Metchnikoff published the first of his highly important
contributions to immunity and phagocytosis, and a decade later von
Behring and Kitasato announced the discovery of diphtheria antitoxin.

The last three decades have not only witnessed the rise and develop-
ment of those most brilliant chapters of medicine, infection and im-
munity; but sanitation, agriculture, many industries and other fields
of human activity have benefited largely by the development of
bacteriology.

In medicine the diagnosis of bacterial disease has reached a high
degree of precision, and bacteriological diagnosis is an important
branch of medical science. The most important problem for the future
is to create a system of Bacterial Therapeutics of equal efficiency.

CHAPTER I.

THE MORPHOLOGY OF BACTERIA.

A. MORPHOLOGY NORMAL FORMS:

Coccus, BACILLUS, SPIRILLUM.

B. MORPHOLOGY ATYPICAL AND AB-

NORMAL FORMS.

1. Variation.

2. Degeneration and Involution.

3. Pleiomorphism.

4. Branching.

C. SIZE OF BACTERIA: WEIGHT OF

BACTERIA.

D. STRUCTURE AND CONSTITUENTS OF

THE BACTERIAL CELL.
1. Cell Membrane, Ectoplasm,
Capsule, Zooglea.

2. Cell Substance, Cytoplasm,
Nucleus, Metachromatic and
Polar Granules, Flagella,
Spores, Germination of Spores,
Arthrospores.

E. REPRODUCTION AND CELL DIVISION

IN BACTERIA.

F. CELL GROUPING.

G. CLASSIFICATION OF BACTERIA.

1. Relation of Bacteria to Higher

Plants.

2. Classification.

H. MUTATION. CONSTANCY OF TYPES.

A. NORMAL FORMS: COCCI, BACILLI, SPIRILLA.

THE normal forms of the true bacteria are very simple, and are
included in three fundamental types: the sphere (coccus, plural cocci),
the straight rod (bacillus, plural bacilli), and the curved rod (spirillum,
plural spirilla) . There is in addition a group of organisms intermediate
between the true bacteria and the molds, which is characterized by
a filamentous type of growth. The members comprising this group
of filamentous organisms are commonly known as the higher bacteria
or Chlamydobacteriacese. An organism belonging to one of these
groups always reproduces its kind under normal conditions; that is,
a coccus always reproduces a coccus, a bacillus always reproduces a
bacillus, and a spirillum always reproduces a spirillum.

Cocci. A single coccus is typically spherical, although those
organisms in which division is taking place may be temporarily some-
what elongated in one diameter, thus appearing oval in outline at this
stage of their development. They may even resemble very short
bacilli in extreme instances. The habitual occurrence of cocci in pairs,
frequently with their proximate surfaces flattened, is a noteworthy
morphological characteristic of certain members of this group. They
are referred to as diplococci. The flattening of the proximated sur-
faces may be associated with an elongation of the axes of the organisms
parallel to the plane of apposition, which leads to "coffee bean"

22 THE MORPHOLOGY OF BACTERIA

shaped diplococci, exemplified in the meningococcus and gonococcus,
or to an elongation of the axes perpendicular to the plane of apposition,
in which event the organisms are "lance-shaped" diplococci, as for
example the pneumococcus.

Bacilli. Bacilli are rod-shaped, cylindrical organisms in which a
longer and a shorter dimension may be recognized. They are typi-
cally circular in cross-section. When division is taking place the shorter
bacilli may be temporarily oval or even circular in outline. The dimen-
sions of bacilli vary considerably: some are habitually long, some are
short, some are thick, some are thin. The ends may be convex, less
commonly flat or even concave. A few bacilli are not typically isodia-
metric, but appear in outline as club-shaped, spindle-shaped, or even
more or less conical (cuneate) rods. Less commonly, slightly curved
rods are met with; the curvature takes place along the longer dimension.

6 ooooooo

FIG. 1. The normal types of bacteria. 1-6, cocci; 7-13, bacilli; 14-16, spirilla;
1, micrococcus; 2 and 3, diplococci; 4, tetracoccus; 5, sarcina; 6, streptococcus (the
lower chain includes an arthrospore) ; 7 and 8, bacilli; 9, 10, 12, and 13, bacilli with
various granules; 11, strep tobacillus ; 14, vibrio; 15, spirillum; 16, Spirocheta trepo-

Spirilla. Spiral bacteria, like the bacilli, exhibit a longer and
a shorter dimension; unlike the bacilli, the longer axis is curved in
three planes of space. The curvature may be slight, less than a com-
plete turn, in which event the organism is "comma-shaped" when
viewed under the microscope; it may be a series of open curves, giving
the organism a sinuous outline; or it may be very much curved, so
that the organism resembles a somewhat closely coiled spring in out-
line. As a rule, the curvature is symmetrical and uniform in each
instance.

The cocci, through almost imperceptible morphological gradations,
merge into the bacilli, and the bacilli, through the slightly curved
forms, merge into the spirilla. Even in the spirilla slight differences
in curvature are usually discernible. Thus, a culture of the cholera
vibrio may contain many straight, uncurved organisms in addition

ABNORMAL FORMS 23

to the slightly curved rods which are the characteristic morphologic
forms. There are a few bacteria in which the morphology is still a
subject of controversy. For example, Micrococcus melitensis is called
Bacillus melitensis by some observers. The vast majority of bacteria,
however, are easily referable to their proper morphological type by
simple inspection under the microscope.

B. ABNORMAL FORMS: VARIATION, DEGENERATION AND IN-
VOLUTION, PLEIOMORPHISM AND BRANCHING.

Variation. The composition of the medium in which bacteria are
growing, the age of the culture, and to a limited degree even the tem-
perature of incubation influence somewhat the average size of bacteria.
Given constant conditions, however, bacteria growing in a favorable
environment exhibit constancy of form and size, although a few organ-
isms in every culture are somewhat larger or smaller than their fellows,
appearing as occasional giants or dwarfs. These occasional giant and
dwarf forms represent normal variations in size from the average or
mean.

Degeneration and Involution. Bacteria growing in an unfavorable
environment, brought about by the accumulation of waste products,
by undue changes in reaction resulting in excessive acidity or alka-
linity, by the presence of harmful chemicals, or by specific antago-
nistic substances, may gradually assume atypical shapes, probably the
direct result of these harmful influences. These atypical organisms
may exhibit little or no resemblance to the normal organism, either in
form or size; they may or may not develop into normal organisms when
they are placed again in a favorable environment. If the change is
a morphological one, the atypical organisms are designated involution
forms : thus, plague bacilli grown in nutrient agar containing 3 per cent,
common salt appear as swollen, balloon-like bodies, notably unlike
the typical short rod-shaped bacillus. If, on the contrary, the
organisms permanently lose some morphological or chemical charac-
teristic, they are spoken of as degeneration forms. Thus, anthrax
bacilli heated for several hours at 43 to 44 C. lose their ability to
form mature spores.

Pleiomorphism. By pleiomorphism is meant a permanent or semi-
permanent change in the normal form of the organism. A pleio-
morphic organism would be one which might at one time resemble
a bacillus, again a coccus, or even a spirillum, depending upon the age

24 THE MORPHOLOGY OF BACTERIA

and growth of the organism or the fitness of the culture medium. This
phenomenon is rarely or never met with among the pathogenic bacteria.

Branching. Among the individual organisms comprising a culture
in artificial media of tubercle, diphtheria or glanders bacilli, and to a
lesser extent of other bacilli, a certain number appear as definitely
branched rods: the typical organism in each instance does not exhibit
branching. Branching has also been demonstrated in the spirilla. 1
Bacillus bifidus appears habitually as a rod-shaped organism with
bifurcated ends in artificial media, although it is an unbranched
bacillus in its normal habitat, the intestinal tract of nurslings. Occa-
sionally, bacteria, as the tubercle bacillus, may exhibit branching in
the animal body as well as in cultures, although less commonly.

The cause of this branching is unknown, and at least two theories
have been advanced in explanation of it: each theory has a certain
amount of evidence in its favor. One theory assumes that branching
is the result of unfavorable environmental conditions, and it has been
shown that old broth cultures of diphtheria bacilli contain branched
organisms; young cultures contain few or no branched forms. The
assumption is that old cultures contain harmful products of metab-
olism which cause the diphtheria bacillus to assume branched forms.
The second theory asserts that the appearance of branched forms
among bacteria demonstrates a relationship between them and higher
organisms, which are habitually branched. Bacteria, according to
this theory, exhibit branching as a part of their normal development.

Branching does not necessarily take place under conditions which
would appear to be unfavorable or partially inimical to their growth,
and, on the other hand, it may be observed occasionally when environ-
mental conditions should be favorable for development. It appears
to be reasonable to assume that branching may be a normal develop-
mental process in the life history of the organism, although the phylo-
genetic significance of branching is as yet undetermined.

C. SIZE AND WEIGHT OF BACTERIA.

Size. The unit of measurement for microscopic objects is the
micron (/*), which is 0.001 of a millimeter, or approximately ^m of
an inch, in length. Bacteria are the smallest known living organisms
which have been seen with the microscope. Measured with this unit,
they exhibit considerable differences in size. The average sized pus-

1 Reichenbach, Centralbl. f. Bakteriol., 1901, xxix ,553.

STRUCTURE AND CONSTITUENTS OF BACTERIAL CELL 25

producing coccus is 0.8 micron in diameter; Micrococcus melitensis,
the smallest of the Coccacese, varies in diameter from 0.3 to 0.5 micron.
The largest known bacillus, B. biitschlii 1 is 3 to 6 microns in diameter
and from 40 to 60 microns in length. The smallest known bacillus,
B. influenzse, is but 0.2 by 0.5 micron in diameter; an average sized
bacillus would measure about 2 microns in length and 1 micron in
diameter. Spirillum colossum 2 is from 2.5 to 3.5 microns in diameter.
The cholera vibrio is about 2.5 microns long and 1 micron in diameter.
There are certain living viruses of unknown morphology, so-called
ultramicroscopic or filtrable viruses, which are either somewhat
smaller than any known bacteria or more plastic. Viruses belonging
to this group derive their name from the fact that they retain their
viability even after passage through the pores of standard, unglazed
porcelain filters, which will hold back even the smallest bacteria.

Weight of Bacterial Cell. The weight of a bacterial cell is depend-
ent upon its size and its specific gravity. According to Rubner, 3 the
specific gravity of common bacteria varies between 1.038 and 1.065. 4
B. coli is an average sized cylindrical rod (bacillus), measuring 1
micron in diameter and 2 microns in length. The volume of a cylinder
is the product of the diameter squared, multiplied by 0.7854, multiplied
by the length of the cylinder. The volume of a single colon bacillus
consequently would be (0.001) 2 X 0.7854 X 0.002, or 0.00000000157
c.mm. The weight of a single colon bacillus would be the volume
multiplied by the specific gravity, which is approximately 1.040 or
0.00000000163 mg.; that is to say, sixteen hundred million colon bacilli
would weight approximately one milligram. For purposes of com-
parison it may be stated that a single red blood corpuscle (human)
weighs about 0.00008 mg., about fifty thousand times the weight
of a single colon bacillus.

D. STRUCTURE AND CONSTITUENTS OF THE BACTERIAL CELL.

The typical bacterial cell consists essentially of protoplasmic cell
substance, endoplasm, enclosed by a rigid cell membrane, ectoplasm.

1. Cell Membrane. Ectoplasm. Bacteria appear to possess a
special external boundary layer, cell membrane, or ectoplasm, which is

1 Schaudinn, Arch. f. Protistenk., 1902, i, 306.

2 Errera, Recueil de 1'Instit. botanique (Universite de Bruxelles), 1901, v, 347.

3 Arch. f. Hyg., 1903, xlvi, 41; 1890, xi, 385.

4 Stigell (Cent. f. Bakt., 1908, xlv, 487) finds that the specific gravity of the same
organism varies somewhat with the medium in which it is grown. The specific gravity
of ordinary bacteria varies commonly between 1.120 and 1.35, older cultures being as
a rule of less specific gravity than younger cultures of the same kind.

26 THE MORPHOLOGY OF BACTERIA

rigid and maintains the shape of the organism. Generally speaking,
this cell membrane is intermediate in character between that char-
acteristic of animal and of plant cells respectively, being somewhat
more developed than the former, less highly specialized as a rule than
the latter. Some authorities consider the cell membrane of bacteria
to be merely a concentrated external layer of endoplasm.

The thickness of the cell membrane varies among different varieties
of bacteria, and it appears to be somewhat -thinner in young organisms
of a given variety than in the older individuals of the same kind.
Ordinarily it is not seen, and special stains are required to demonstrate
it clearly. In certain spore-forming bacteria, however, the cell mem-
brane is occasionally seen after the spore has matured within the cell,
as a thin, feebly staining shadow, outlining the original contour of the
organism. Bacteria which plasmolyze easily also show the cell wall
clearly after the cell contents have shrunken away from it.

Capsule. A considerable number of bacteria are surrounded by
mucin-like envelopes, particularly when they are observed in the animal
body or grown in albuminous fluids. This envelope or capsule fre-
quently disappears when the organisms are grown in ordinary media.
This has led to the theory that a capsule represents an hypertrophy
of the ectoplasm. The significance of capsules is still a matter of
controversy. Two principal theories have been advanced to explain
the significance of capsules : according to one theory, bacterial capsules
are purely degenerative phenomena; the more widely accepted theory,
which has much evidence in its favor, maintains that capsule formation
is closely related to the virulence of the organisms. 1 The demonstration
of capsules may be an important factor in the identification of certain
bacteria, for example, the pneumococcus.

Zooglea. A very few bacteria exhibit a slimy intracellular substance
which causes cohesion between considerable numbers of bacterial
cells. This intracellular substance, zooglea, is colored lightly by
ordinary staining methods. It is not found in any of the pathogenic
bacteria.

2. Cell Substance. Cytoplasm. The cytoplasm or endoplasm of
living bacteria (particularly in young cultures) is usually a clear,
colorless, highly refractile, homogeneous appearing substance, although
at times various granules may be seen within it. Vacuoles also are
met with, usually in older bacteria. The cytoplasm usually stains
readily with basic anilin dyes. A few bacteria, notably B. viride and

1 Eisenberg, Centrabl. f. Bakteriol., 1908, xlv, 148.

STRUCTURE AND CONSTITUENTS OF BACTERIAL CELL 27

B. chlorinum, contain a yellowish pigment in the cytoplasm suggesting
chlorophyll, and the so-called purple bacteria similarly possess a
purple colored pigment, bacteriopurpurin.

Nucleus. The occurrence of a demonstrable morphological nucleus
in bacteria is by no means definitely settled: the typical bacterial
cell can not be separated chromoscopically into a nucleus and cyto-
plasm. Those who have thoroughly studied the question by staining
methods, notably Nakanishi, 1 believe that the whole bacterial cell,
as it is ordinarily seen, is potentially a nucleus surrounded by a very
thin film of cytoplasm. Others believe the nucleus substance is dis-
tributed throughout the cell in very finely divided granules : Zettnow 2
is the champion of the latter theory. He believes that the bacterial
cell, as it is viewed following the usual staining processes, is endoplasm
in which the nuclear substance is finely divided and uniformly dis-
tributed. Some observers deny that a nucleus exists at all. Chemical
analyses show beyond doubt that bacteria contain a relatively high
percentage of substances usually regarded as essentially of nuclear
origin. It is quite certain, therefore, that, although there may be no
morphologic nucleus demonstrable by ordinary staining methods,
nuclear material is present in abundance in the organism.

Metachromatic Granules. Certain types of bacteria, notably mem-
bers of the diphtheria and hemorrhagic septicemia groups, exhibit
one or more highly refractile granules in an otherwise homogeneous
endoplasm when they are examined unstained with the higher powers
of the microscope. These granules are few in number in the diphtheria
bacillus group and are distributed somewhat irregularly throughout
the cell, one or more granules usually being greater in diameter than the
cell itself, thus giving the rod a swollen appearance. In the hemor-
rhagic septicemia group these granules are arranged symmetrically,
one at each end of the organism, polar granules. Such granules are
called Ernst-Babes or metachromatic granules. They color differently
from the rest of the cell when they are stained with methylene blue,
appearing as mahogany-red spots in the deep blue endoplasm. They
retain the stain rather tenaciously. Many theories have been advanced
to explain their significance, but nothing definite is known about them,
except that these granules appear to differ widely in chemical composi-
tion. Some are colored brown with iodine, suggesting that they may
be related to glycogen. 3 Some stain black with osmic acid, suggesting

1 Centralbl. f. Bakteriol., 1901, xxx, 97, 145, 193, 225.

2 Ztschr. f. Hyg., 1899, xxx, 1; Festschr. z. 60 Geburtstage vonR. Koch, 1903, p. 383.

3 A. Meyer, Flora, 1899, Ixxxvi, 428.

28 THE MORPHOLOGY OF BACTERIA

that they may be fatty or lipoidal in composition, while others are
probably complex phosphorus-containing compounds. 1 Not all of these
varieties of granules are met with in the same organism. 2 Among
the higher bacteria granules of sulphur or of iron are demonstrable
respectively in the sulphur and the iron bacteria.

Flagella. All minute particles suspended in water or other fluids
of low viscosity are in constant motion. This motion, which is irregular
and tremulous, was first described by Brown: 3 it is variously termed
Brownian movement, pedesis, or molecular movement. Brownian
movement may be rapid or slow, extensive or circumscribed, depending
upon the nature of the particles and the composition and temperature
of the fluid in which they are suspended. This is not true motility,
even though each individual particle moves independently of the other
particles in an irregular orbit, for the particles as a whole do not
permanently change their relative positions. Dead bacteria and many

FIG. 2. Flagella. 1'and 6, peritrichic flagella; 2 and 4, monotrichic flagella; 3 and 5,

lophotrichic flagella.

living bacteria, notably the cocci, exhibit the Brownian movement.
Many bacilli and spirilla, on the contrary, possess the power of
independent motility, that is, they can progressively and permanently
change their relative positions in space. Motile bacteria are provided
with one or more long, delicate, contractile filaments flagella which
are probably the organs of locomotion. These flagella cannot be
demonstrated on living bacteria, except possibly by dark-ground illu-
mination, and ordinary staining reactions usually fail to reveal them.
Special staining methods show them clearly. They appear to arise
from the cell membrane. 4 Their arrangement and number is varied
among bacteria in general, but relatively constant for a particular
variety of bacterium: they are thinner as a rule on younger bacterial
cells, thicker on older organisms. 5 A cholera vibrio has a single

1 Grimme, Centralbl. f. Bakteriol., 1904, xxxvi, 952.

2 For literature see Marx and Woithe, Centralbl. f. Bakteriol., 1900, xxviii, 1, 33, 65,
97; Krompecher, ibid., 1901, xxx, 385, 425; Gauss, ibid., 1902, xxxi, 92.

3 Edinburgh Phil. Jour., 1828, v, 358; 1830, viii, 41.

4 Schaudinn, Arch. f. Protistenk., 1903, i, 421.

6 De Grandi, Centralbl. f. Bakteriol., 1903, xxxiv, 97.

STRUCTURE AND CONSTITUENTS OF BACTERIAL CELL 29

flagellum at one or both ends of the organism; in the typhoid bacillus
they are distributed around the sides of the organism but do not occur
at the ends.

Spores. Endospores. Many bacteria die when their environment
becomes unsuited for further growth. Death may result from the
presence of inimical substances, the absence of essential foods, or the
intervention of unsuitable physical conditions. Death is manifested
by a cessation of chemical interchange between the bacterial cell and
its environment. There is a group of bacteria, however, usually of
saprophytic origin, which is able to survive even prolonged exposure
to unfavorable environmental conditions by passing into a latent stage
during which chemical interchange with the environment is at an
extremely low ebb. This latent stage or hibernation has been known
to last for more than two decades in certain instances, and yet the
organisms have resumed their original luxuriant growth when placed

<*>

FIG. 3. Types of bacterial spores.

under favorable conditions. The bacteria which exhibit this latent
state produce within their substance highly refractile, spherical or oval
bodies called spores. Spores are not found in very young, actively
growing cultures, as a rule. Spore formation is ushered in by a clouding
of the endoplasm of the bacterial cell, which gradually becomes granular.
The granules coalesce, eventually appearing as the mature spore which
is surrounded by a dense membrane, frequently exhibiting a double
contour when stained by dilute carbol fuchsin. 1 The spore membrane
(ectoplasm) is relatively impermeable to heat and disinfectants and
confers the resistance to physical agents which spores exhibit upon
them. But one spore is formed in an individual bacterium, except
under most unusual conditions. It is to be emphasized, consequently,
that spore formation is not a reproductive process. The mature spore
may form in the center of the bacterium, at, or near one end. The
spore may be round or oval, and greater or lesser in diameter than
the parent cell. If the spore is greater in diameter it distends the
cell membrane, producing a spindle-shaped organism if the spore is

1 Meyer, A., Practicum der botanischen Bakterienkunde, Jena, 1903.

30 THE MORPHOLOGY OF BACTERIA

in the center of the rod : if the spore is at one end, a drumstick-shaped
organism results. Usually the size and position of the spore is fairly
constant in a given type of bacteria. Spore formation is most common
among the anaerobes, fairly common among the saprophytic bacteria,
practically absent in the pathogenic bacteria, and practically never
takes place spontaneously in the human or animal body. The spiral
organisms rarely produce spores, and, with the exception of Sarcina
pulmonum, spore formation is practically never observed in the cocci.

It has never been satisfactorily determined whether spore formation
is a regular definite stage in the life history of bacteria which produce
them or whether spores are produced rather under the stress of unfavor-
able evironmental conditions.

Germination of Spores. When bacterial spores are placed in an
environment favorable to the vegetative activity of the cell, they
germinate: the dense membrane which constitutes the ectoplasm of
the spore softens, usually at the pole or the equator, and the vegeta-
tive rod emerges, at first as a small bud, then rapidly assumes the
typical size and shape of the fully mature cell. The development of
the anthrax bacillus from the spore is usually in the line of the longer
axis, polar germination: B. subtilis, on the contrary, usually emerges
at right angles to the larger axis of the spore, equatorial germination.
Many spores are circular in outline, and in such cases the relation of the
developing vegetative cell to the axis of the spore is unknown. Fre-
quently the remnants of the spore membrane remain attached to one
end of the newly formed vegetative cell, appearing as a cap, as it were.
Some spores do not appear to rupture as germination takes place.
the newly forming organism appears to absorb the entire spore and
its ectoplasm, incorporating the entire structure by solution in the
vegetative cell.

Arthrospores. Certain organisms belonging to the coccal group,
more particularly the streptococci, exhibit from time to time cells
which are decidedly larger than their fellows. These cells are more
highly refractile, they usually possess a granular cytoplasm, and fre-
quently stain somewhat irregularly. They have been designated by
Hueppe as arthrospores. These arthrospores appear to have no
unusual resisting powers, and they are in no sense to be regarded as
true spores. It is very probable that they are involution forms.

REPRODUCTION AND CELL DIVISION 31

E. REPRODUCTION AND CELL DIVISION.

Bacteria are structurally the simplest known organisms which main-
tain an independent existence: all their vital functions are exhibited
in a single asexual cell devoid of a morphologically definable nucleus.
The absence of sexual characters and of a morphologic nucleus makes
bacterial reproduction mechanically a simple process, and doubtless
the rapid sequence of generations observed in various bacteria depends
in part upon this simplicity of structure.

Reproduction takes place in the following manner: A bacterial
cell placed in a favorable environment increases in size until it reaches
a maximum which is relatively constant for each variety; then a slight
equatorial constriction occurs, which deepens until a distinct septum
is produced by invagination, which divides the original cell into two
morphologically complete, fully mature individuals of approximately
equal size. It is obvious that this septum consists ordinarily of at
least two layers, since one layer is required to complete each of the
dividing individuals. Successive generations may be produced at
intervals which may be as frequent as every fifteen minutes in the
more rapidly growing types. Septation usually takes place deliberately ;
that is to say, the septum forms relatively slowly. Diptheria bacilli
and possibly related bacteria divide somewhat differently; the parental
cell appears to be under tension when the septum becomes visible,
and the daughter cells spring apart suddenly when septation is com-
pleted. So forcible is this separation that the daughter cells lie at an
angle with each other: Nakanishi 1 has observed that the septum in
this group of organisms frequently forms at a metachromatic granule.
Septation in the Bacillacese and Spirillacese normally takes place
at right angles to the long axis of the organism, and midway between
the ends, thus effecting the separation into two individuals with the
minimal expenditure of material; in the Coccacese, which are usually
isodiametric, no economy of material in septation is apparent, and no
known force determines the initial plane of septation: subsequent
fission may be definitely related to the initial plane. Noguchi 2 has
brought forward striking evidence and photographic illustrations in
favor of the view that the Spirocheta (Treponemata) may reproduce
by longitudinal fission rather than by transverse fission. If this view
be generally adopted, it would contradict the "minimal requirement

1 Centralbl. f. Bakteriol., 1900, xxvii, 641.

2 Jour. Exper. Med., 1912, xv, 201.

32 THE MORPHOLOGY OF BACTERIA

theory," which assumes that transverse fission, the more economical

process both with respect to amount of material and expenditure

of energy, holds universally for bacteria, as has previously been
maintained.

F. CELL GROUPING.

In Bacilli and Spirilla, where septation typically occurs at right
angles to the long axis of the organism, it is obvious that no geometrical
arrangement of cells is possible other than the formation of chains of
rods or of spirals if the individual organisms remain adherent. The
cocci, on the other hand, are spherical and have no longer or shorter
axis, consequently a definite sequence of septation in one, two or
three planes of space can give rise to (1) chains of cocci, if the plane
of septation is always in one plane of space; (2) groups of four cocci,
if septation takes place alternately in two planes of space; or (3) in
packets of cocci, if septation is alternate in three planes of space.
Many cocci do not exhibit a definite sequence of planes of septation.

G. CLASSIFICATION OF BACTERIA.

Relation to Higher Plants. The position of Bacteria in the Plant
Kingdom is indicated in the following table :

Plant Kingdom.

Cryptogamia
Thallophyta

Phanerogamia

Algae

Schizomycetes
(Bacteriacese)

Fungi

Saccharomycetes
Blastomycetes

(yeasts)

i
Lichens
1
Hyphomycetes
(molds)

Eubacteriaceae
Coccacese
Bacillacese
Spirillacese

Chlamydobacteriaceae
Streptothrix
Phragmidothrix
Crenothrix
Cladothrix
Actinomycetes

A complete natural classification of bacteria is impossible at the
present time. The monotony of form observed in this group of
organisms merely suffices to classify them into three great divisions:
Cocci, Bacilli, and Spirilla. Further subdivision into groups which
are potentially families, genera and species is accomplished by arrang-

CLASSIFICATION OF BACTERIA 33

ing them according to their physiological and chemical activities.
Even this artificial procedure is unsatisfactory, for bacteriological
diagnosis is a subject which has developed under the stress of practical
needs, and as bacteria play a part in many fields of activity, it has
inevitably followed that the criteria whereby they are recognized
vary greatly according to the art or science in which they are contem-
plated. Even the same species may be identified by wholly different
characteristics. Notwithstanding the difficulties which surround the
grouping of bacteria, Migula 1 has worked out a system of classification
based upon purely morphological characteristics, which effects a
primary separation of bacteria into smaller subdivisions, which is
moderately satisfactory so far as it goes, and it is the one commonly
adopted.
With certain additions it is as follows:

THE TRUE BACTERIA: EUBACTERIACE.E.

1. Coccacece. Cells in the free state spherical.

(a) Micrococcus. Cells spherical. No definite sequence of planes of septa-

tion.
(6) Diplococcus. Organisms habitually occur in pairs.

chains.

(d) Tetracoccus. Planes of septation alternate, and at right angles in two

planes of space. Form groups of four or tetrads.

(e) Sarcina. Planes of septation alternate, at right angles, in three planes

of space. Form packets.

(/) Planococcus. Motile cocci, provided with flagella.
(g) Planosarcina. Motile sarcina, provided with flagella.

2. Bacillaceoe. Cells elongated and cylindrical; straight.

(a) Bacterium. Non-motile. No flagella.
(6) Bacillus. Cells motile. Peritrichic flagellation.

(c) Pseudomonas. Cells motile. Polar flagellation. Single flagellum or
tufts of flagella at one or both poles of the organism.

3. Spirillacece. Cells elongated and cylindrical; spirally twisted about the long

axis.

(a) Spirasoma. Cells rigid and slightly curved; without flagella.
(6) Microspira. Cells rigid and slightly curved; with one, rarely several,

polar flagella.

(d) Spirocheta. Cells flexuous, closely coiled; flagellation unknown.

THE HIGHER BACTERIA.

4. Chlamydobacteriacece. Cells enclosed in a sheath.

(a) Streptothrix. Cell division always in one plane.

(6) Phragmidothrix. Cell division in three planes of space; very delicate
sheath.

(d) Cladothrix. Cells more or less branched.

5. Beggiatoacece (Thiothrix). Cells contain sulphur granules.

1 System d. Bakterien, Jena, 1907.

34 THE MORPHOLOGY OF BACTERIA

H. MUTATION: CONSTANCY OF TYPES. 1

True mutation or discontinuous variation is rarely observed among
bacteria, although a few instances are on record which have been sub-
jected to satisfactory scrutiny. Mutation must be carefully differen-
tiated from the loss of one or more characteristics of bacteria during
cultivation; the loss or suppression of one or more characteristics is
fairly commonly observed among bacteria. Pigment production, and
proteolytic activity as for example the ability to liquefy gelatin-
are frequently lost to cultures of bacteria during prolonged cultiva-
tion, but these properties may be regained when the organisms are
placed once more in a suitable environment. Similarly, strains of
fermenting bacteria may temporarily, or even permanently, become
unable to decompose certain carbohydrates. Change in virulence,
or loss of virulence is rather commonly noticed among pathogenic
bacteria grown outside the animal body. It is even possible to so
parasitize organisms by prolonged cultivation upon one medium that
they will develop not at all, or slowly at best, on other media. Thus,
a strain of B. proteus has been grown continuously upon agar with
frequent transfers for four years, and the organism will no longer
grow in broth. Similarly, B. bulgaricus is an obligate milk parasite.
Exposure to unfavorable environmental conditions may also suppress
important characters: Pasteur's celebrated experiment of growing
anthrax bacilli at 43 C. for some hours and establishing an asporeless
variety is a familiar example. The suppression of characters as out-
lined above is frequently important as the starting point for new
adjustments between pathogenic bacteria and their hosts.

Turning to the production of disease in man, it is certain that at
least some organisms produce the same reaction today they did years
ago : tuberculosis appears to be the same disease today it was centuries
ago, as is evidenced by the lesions found in Egyptian mummies. Clini-
cally, the observations of Hippocrates would be a fair exposition of the
phenomena seen in tuberculous patients at the present time. Leprosy
also appears to be the same entity now it was during the middle ages,
although the geographical distribution is much more restricted. With
respect to more acute diseases, which require more careful examination
to differentiate them, the evidence is less certain, although typhoid
bacilli do not appear to have changed since they were first isolated

1 Eisenberg, Ueber Mutationen bei Bakterien und anderen Mikroorganismen in
Ergebnisse d. Immunitatsforsch. experimentellen Therapie, Bakteriologie und Hygiene,
Berlin, 1914, pp. 28-142, for summary.

MUTATION: CONSTANCY OF TYPES 35

by Gaffky three decades ago. It appears to be reasonably certain
from what is known of bacteria and the manifestations of disease
they induce that mutation is an infrequent phenomenon: attenuation
and the partial suppresion of characteristics, on the contrary, appear
to be quite common. The available evidence indicates that bacterial
types are stabile under natural conditions : there is no definite evidence
in favor of the view that bacteria change slowly or abruptly either in
their morphology or in the changes they induce in their environment
in the sense that entirely new, unrelated types are developed de now
from preexisting types. This does not preclude the possibility that
such changes have taken place in the past, rather that such changes,
if they have taken place, have not been definitely established.

CHAPTER II.

GENERAL PHYSIOLOGY OF BACTERIA THE EFFECT
OF ENVIRONMENT ON BACTERIA.

A. RATE OF REPRODUCTION.

B. MOTILITY: RATE OF MOTION.

C. SPORULATION: GERMINATION OF

SPORES.

D. LONGEVITY.

E. MOISTURE: DESICCATION.

F. OXYGEN. AEROBIOSIS AND ANAERO-

BIOSIS.

G. TEMPERATURE.

1. General.

2. Cold.

3. Heat.

H. HEAT PRODUCTION.

I. LIGHT AND ELECTRICITY.

J. GRAVITY, OSMOTIC PRESSURE, AGI-
TATION AND CHEMOTAXIS.
K. ENZYMES, TOXINS. PTOMAINS.
L. PIGMENTS.

1. Photodynamic.

2. Phosphorescent.

3. Fluorescent.

4. Chromogenic.

M. SYMBIOSIS, ANTIBIOSIS, COMMENSAL-
ISM.

N. MEDIA COMPOSITION AND REAC-
TION.

O. GROWTH IN ANIMAL BODY.

A. RATE OF REPRODUCTION.

ONE of the striking characteristics of the Bacteriacese is their rapidity
of reproduction. Among the most actively growing types of bacteria,
as, for example, the cholera vibrio, successive generations may appear
at intervals as frequent as every fifteen minutes when the environ-
mental conditions are most favorable : that is to say, ninety-six genera-
tions are theoretically possible in twenty-four hours. If this rate of
reproduction could be maintained for three days, the progeny of a
single organism would occupy a space not less than that of the com-
bined waters of the earth. Fortunately, nature imposes many restraints
which limit the numbers of bacteria. The rapid accumulation of waste
products, the exhaustion of nutrient material, and the enormous
death rate in culture media even after a comparatively few hours'
growth, together with other factors restrict development to such a
degree that the actual number of living descendants of bacteria in
cultures or in nature falls far short of the theoretical number. Many
bacteria develop more slowy than this, however. They may require
hours or even days to arrive at maturity. The tubercle bacillus, for
example, grows comparatively slowy in artificial media (where such
observations are of necessity made), and the frequency of septation,
even in the most rapidly growing bacteria, is greatly affected by
environmental factors.

SPORULATION: GERMINATION OF SPORES 37

Generally speaking, when nutritional conditions are favorable, the
rate of reproduction is influenced by temperature, growth being most
rapid when the temperature is optimum for the organism, less rapid
when the temperature exceeds or falls below this point.

B. MOTILITY: RATE OF MOTION.

The rhythmic contractions of the flagella, with which practically
all motile bacteria are provided, drive the organisms through fluid
media in which they may be suspended, some slowly, some rapidly.
Not all bacteria even in the same culture exhibit motility. The char-
acter of the motion may be direct, serpentine, oscillatory, or irregular.
Rarely, the flagella appear to produce local currents in the medium
which immediately surrounds the organism. Various environmental
factors incite or inhibit motility. Chemotactic substances may attract
bacteria, thus in a sense directing their line of movement. Other
substances, as protoplasmic poisons, paralyze bacterial movements.
Oxygen appears to increase the motility of aerobic bacteria, and it
inhibits motility in the anaerobes. Generally speaking, in favorable
media motility increases with the rise in temperature to the optimum.
If this temperature is exceeded, even by a very few degrees, motion
ceases.

The rate at which bacteria progress through a fluid is a variable
one, although with a given organism under favorable conditions it
appears to be fairly constant. It must be remembered that the
apparent rate of motion observed under the microscope is increased
proportionately to the increase in magnification. Lehmann and
Fried 1 have measured the average speed of certain bacteria in fluid
media in millimeters per second. They find that of the -cholera vibrio
to be 0.03, typhoid bacillus 0.018, B. subtilis 0.01, B. megatherium
0.0075. If a man traveled at a rate of speed in proportion to his size
as great as that of the cholera vibrio, he would average more than a
mile a minute.

C. SPORULATION: GERMINATION OF SPORES.

Many saprophytic bacteria form within themselves spores which
appear apparently under the stimulus of the stress of conditions unfa-
vorable for the continued vegetative growth of the organism. Sporula-
tion, in other words, appears to be a specialized mechanism for the

1 Arch. f. Hyg., 1903, xlvi, 314.

38 GENERAL PHYSIOLOGY OF BACTERIA

perpetuation of the organism during periods of environmental unfit-
ness. Whether spore formation is a definite phase in the life-history of
spore-forming bacteria is not definitely settled. Sporulation is rarely
observed when the temperature of the environment falls much below
15 C., although considerable latitude is observed among the spore-
forming bacteria in this respect. Spores are rarely, if ever, produced
within the tissues of the animal body: if the tissues are exposed to
the air, however, particularly postmortem, spore formation may take
place. No bacteria progressively pathogenic for man are known to
form spores.

The unusual resistance of mature spores to desiccation, to exposure
to dry and moist heat, and to disinfectants may be due either to
their low content of water, for spores contain less than half of the
water contained in the normal vegetative cell, to the relatively thick

FIG. 4. Germination of bacterial spores. 1, by absorption of spore membrane;
2, equatorial germination; 3, polar germination.

refractile spore membrane, or to unusual concentrations of fatty and
lipoidal substances. Experiments by Lewith 1 would suggest that the
relative desiccation of the contents of spores as compared with the mois-
ture content of the vegetative organism would be the most plausible
explanation of their resistance to heat without apparent injury. He
found that egg albumen (dried) suspended in 5 per cent, of water coagu-
lated at 145 C.; suspended in 18 per cent, of water, coagulation took
place at 90 C.; with 25 per cent, of water, at 80 C.; and in a consider-
able volume of water (amount not stated) coagulation occurred when
the temperature reached 56 C.

The resistance of spores to physical conditions varies somewhat
according to the organism in which they are formed. Generally
speaking, however, several minutes' exposure to the temperature
of boiling water (100 C.) may fail to kill them. Dry heat is less
effective than moist heat, for an exposure of 160 C. for one and one-
half hours is required to certainly sterilize glassware containing spores.
Ten to fifteen pounds live steam pressure for fifteen minutes is required

1 Arch. f. exp. Path. u. Pharmakol., 1890, xxvi.

MOISTURE AND DESICCATION 39

to effect sterilization of liquids and organic matter in general. Direct
sunlight will kill spores after days of exposure.

Germination of bacterial spores takes place when they are placed
in a suitable nutritive environment in which the temperature, moisture
and oxygen relations are favorable. The vegetative cell breaks through
the spore membrane apparently after the latter has lost its refractility,
and reproduction by fission proceeds anew, and persists until environ-
mental conditions again lead to sporulation.

D. LONGEVITY.

The duration of life in the individual non-spore-forming bacterium
is unknown, but it is greatest apparently when the organism is quiescent
or nearly so. This condition is realized most commonly when bacteria
are exposed to temperatures slightly above freezing in a dark place.
This question has been studied recently under unusual conditions.
A mastodon was discovered in Siberia which had been uncovered by
an unusual recession of the ice. This animal was found to be practi-
cally intact, and cultures made with proper precautions from the
center of the proboscis contained bacteria indistinguishable from
Sarcina lutea and other well known air organisms. 1 If these cultures
are authentic, a most unexpected instance of bacterial longevity has
been unearthed, for this animal has undoubtedly been frozen for
hundreds of years.

Spores have been dried and kept in a cool dry place for more than
two decades, and yet developed with their usual luxuriance when placed
in a favorable environment. Dried anthrax spores thus retain not only
their viability but their virulence unimpaired for years. Practically,
the. average duration of life among bacteria is comparatively brief.

E. MOISTURE AND DESICCATION.

Bacteria normally contain at least 80 per cent, of moisture in their
substance, and they develop typically only in media containing con-
siderable amounts of moisture. Bacteria do not vegetate normally
in desiccated media, but many varieties resist drying for considerable
periods. Advantage is taken of the restriction of bacterial develop-
ment in the absence of suitable amounts of moisture in various pro-
cesses of drying meats and other foodstuffs; desiccated foods will
keep for weeks under the proper conditions. Bacterial spores pro-

1 Russian Academy of Science, 1911-1912.

40 GENERAL PHYSIOLOGY OF BACTERIA

tected from direct sunlight are extremely resistant to drying, but
they develop with characteristic vigor when environmental conditions
become suitable. Even non-sporogenic bacteria may develop after
days or weeks of desiccation. Many pathogenic bacteria are eliminated
from the body enveloped in albuminous material, as in sputum. These
organisms thus protected may resist drying for many days, provided
they are not exposed to direct light. The following table indicates
the relative viability of various bacteria pathogenic for man to air
drying. 1

1. Gonococcus, few hours.

2. Cholera vibrio, few hours to two days.

3. Plague bacillus, one to eight days.

4. Diphtheria bacillus, twenty to thirty days.

5. Streptococcus pyogenes, fourteen to thirty-six days.

6. Pneumococcus, nineteen to fifty-five days.

7. Staphylococcus pyogenes, fifty-five to one hundred days.

8. Typhoid bacillus, up to seventy days.

9. Tubercle bacillus, two to three months.

F. OXYGEN: AEROBIOSIS AND ANAEROBIOSIS.

Oxygen, either in the free state or combined, is essential to the
growth of all known bacteria. The majority of bacteria grow best in
the presence of free (atmospheric) oxygen, although the percentage
of this gas necessary to support bacterial life may be considerably
less than that occurring normally in the air. Some bacteria appear
to be wholly dependent upon free oxygen, and they are called obligate
aerobes. A small group of bacteria, on the contrary, grow only in the
absence of free oxygen, and more than minimal concentrations of this
gas are actually poisonous to them. Those bacteria which grow only
in the absence of free oxygen are called obligate anaerobes. The vast
majority of bacteria are facultative with respect to their oxygen
requirement, growing best in the presence of atmospheric oxygen but
able to develop either in the presence of small amounts of free oxygen,
as in. the tissue of the body and certain parts of the intestinal tract,
or they are able to obtain their oxygen from chemical compounds, as
certain simple sugars, if free oxygen is not available. These organisms
are called facultative anaerobes. The maximum tolerance of bacteria
for oxygen varies very considerably, as the following table indicates:

Oxygen content of the air is taken as 100 per cent.

1 Fischer, Vorlesungen iiber Bakterien 1903, II Aufl., 110.

TEMPERATURE 41

MAXIMUM OXYGEN TOLERANCE.

Atmospheric oxygen,
Per cent.

B. (clostridium) butyricus ........... 1 . 35

B. chauvei ................ 5.00

B. edematis maligni ........... . . 3.25

Purple bacteria (Molisch) ......... about 90.00

Thiosulphate bacteria (Nathansson) ........ 400.00

B. prodigiosus .............. 3000.00

G. TEMPERATURE.

1. General. The extreme temperature limits of bacterial growth
are very slightly above C. to 80 C. inclusive. Some bacteria,
notably those found in the Arctic regions, appear to develop even at
C.; others, chiefly those found in soil, feces, and certain thermal
springs, grow even at 80 C., a degree of heat considerably above that
at which the protoplasm of most animals and plants is coagulated.
The vast majority of bacteria, however, develop best within a range
of temperature from 15 C. as a minimum to 40-43 C. as a maximum.
All bacteria exhibit three cardinal thermic points : a minimum tempera-
ture, below which growth ceases; an optimum temperature, at which
growth is most luxuriant and rapid; and a maximum temperature,
above which growth ceases, and the organisms die. Fischer 1 has
classified bacteria according to their thermic relations as follows:

Minimum. Optimum. Maximum.

1. Psychrophilic bacteria . 15-20 30 Many water bacteria.

2. Mesophilic bacteria . 15-25 37 43 Pathogenic bacteria and

others.

3. Thermophilic bacteria . 25-45 50-55 85 Spore-forming bacteria

from soil, feces, and
thermal springs.

Bacteria which are progressively pathogenic for man and warm-
blooded animals develop within a much narrower range of tempera-
ture than the saprophytic bacteria which are found chiefly in nature,
as the following table, also taken from Fischer, 2 indicates:

Difference between

minimum and

Minimum.

Optimum.

Maximum.

maximum.

B. phosphorescen

B. fluorescens

. 5

20-25

B. subtilis

. 6

Vibrio cholerae

. 10

B. anthracis .

. 12

B. diphtherise

. 18

33-37

Mic. gonorrhese

. 25

B. tuberculosis

. 30

B. thermophilus .

. 40

1 Vorlesungen iiber Bakterien, 1903, II Aufl.

2 Loc. cit., 106.

42 GENERAL PHYSIOLOGY OF BACTERIA

The saprophytic bacteria, as for example B. subtilis, which develop
through a relatively wide range of temperature are also called
Eurythermic bacteria. The pathogenic bacteria, as for example the
tubercle bacillus, which exhibit but little latitude in this respect, are
called Stenothermic bacteria.

2. Cold. All bacteria grow best and most rapidly in an environ-
ment which is maintained at the optimum temperature for the organism.
If this temperature is lowered even a few degrees, the rate of reproduc-
tion is proportionately reduced. As the temperature approaches C.,
there is complete or nearly complete cessation of growth with a corre-
sponding complete or nearly complete restriction of chemical inter-
change between the organism and its environment. The viability,
and in the pathogenic bacteria the virulence, is not seriously impaired
even by exposure to these low temperatures for considerable periods
of time. Practical advantage is taken of this restriction of bacterial
development by cold in the preservation of food by refrigeration or
by cold storage, and also for the preservation of laboratory cultures
of many non-spore-forming bacteria by placing them in the ice-box
at 5-10 C. So resistant are bacteria to low temperatures that they
may be actually frozen solid and kept in this state for days and even
weeks without killing all the individuals of the cultures. Alternate
freezing and thawing is much more disastrous to them than simple
freezing. Thus, typhoid bacilli may be suspended in water and
exposed to a freezing mixture of ice and salt at 18 C. for several
weeks without killing all the organisms, although the majority of them
are killed within a few hours. At the end of a week fully 90 per cent,
are dead; over 95 per cent, succumb by the end of four weeks' continual
freezing; but from four to six months' continuous freezing is required
to kill all of the typhoid bacilli. The survivors appear to be no more
resistant to subsequent freezing than similar organisms which have
not been frozen. It is a noteworthy fact that bacteria suspended in
colloidal substances, as egg albumen, are much more resistant to freez-
ing than similar organisms frozen in water. Alternate freezing and
thawing in colloids is much less disastrous to bacteria, in other words,
than the same freezing in aqueous solutions. It is probable that the
mechanical factor of crystallization which takes place when water is
frozen actually crushes many of the bacteria, thus accounting, in part
at least, for the greater death rate in aqueous solutions than that
observed in colloids. When bacteria are once frozen, further lowering
of the temperature has surprisingly little influence upon the death
rate. Typhoid and colon bacilli will survive freezing, in moderate

HEAT PRODUCTION 43

numbers at least, in liquid air ( 176 C.) or even liquid hydrogen
(252 C.) for several hours, and develop vigorously when they are
again placed in a suitable environment at the optimum temperature.

3. Heat. Bacteria are distinctly injured by exposure to even slight
increases of temperature above that optimum for their growth, although
there are considerable differences met with among different kinds of
organisms in this respect. Generally speaking, the saprophytic bac-
teria exhibit greater latitude than the pathogenic bacteria. If the
maximum temperature of growth be exceeded by even a very few
degrees, the death of the organisms follows rather promptly. The
greater the degree of heat, the shorter the time required to kill them.
Therefore, the thermal death point of bacteria, that temperature at
which specific organisms die, is dependent not only upon the actual
temperature to which they are exposed, but also to the length of time
of exposure. A standard exposure of ten minutes has been proposed,
so that the thermal death point of the bacterium may be defined as
the lowest temperature to which it must be exposed for ten minutes
under constant conditions to ensure the sterility of the culture. The
determination of the thermal death point is influenced by many factors
besides the kind of organism under observation and the temperature.
Older cultures are usually less resistant than younger cultures of the
same kind. The reaction of the medium (acids particularly decrease
thermal resistance), the presence of extraneous substances as mucin
and other non-conductors of heat, all play a part. Certain modifica-
tions in the characteristics of bacteria are observed when they are
exposed for several hours at the maximum temperature of growth
or a degree or ,two above this point. For example, anthrax bacilli,
which habituallyjorm spores, lose this property when they are exposed
to 44 C. for several hours.

Dry Heat, Moist Heat Dry heat is less effective in killing bacteria
than moist heat. This is shown by the high temperature to which
glassware and other apparatus must be exposed in order to kill spores,
a temperature of 160 C. for one and one-half hours being required
to ensure sterility. Moist heat, which is best obtained by dry steam
under pressure, will kill even the most resistant spores in fifteen
minutes at fifteen pounds pressure.

H. HEAT PRODUCTION. .

The energy liberated by bacteria during the decomposition of organic
substances by bacterial growth is partly utilized by them for their

44 GENERAL PHYSIOLOGY OF BACTERIA

anabolic requirements. A larger part, however, is dissipated as heat.
The heat generated in actively growing cultures of bacteria can be
detected with sensitive thermometers, provided losses due to radiation
and evaporation are guarded against. The heat production is not
great as a rule, although in certain fermentations it may rise as high
as 12-15 above the uninoculated controls. The decomposition of
protein and protein derivatives (putrefaction) usually gives rise to less
heat than the decomposition of carbohydrates (fermentation) under
the same conditions.

I. LIGHT AND ELECTRICITY.

The vast majority of plants possess a photodynamic pigment,
chlorophyll. This pigment can synthesize inorganic substances, as
CO2 and water, together with nitrates, into complex organic compounds
through the energy of the sun's rays acting upon it. Plants possessed
of this pigment, therefore, are the synthetic agents of nature. Usually
this pigment is green; it may, however, be brown or red, the latter
pigment being characteristic of certain algaB. A group of the higher
bacteria, the Rhodobacteriaceae, possess a photodynamic pigment,
bacteriopurpurin, which appears to be analogous to chlorophyll of
the green plants. These sulphur bacteria prefer light and move toward
it. 1 The action of sunlight on this bacteriopurpurin enables them to
decompose CO 2 and to utilize the oxygen thus obtained to oxidize
H 2 S.

All other known bacteria have no photodynamic pigment. Light
is not a source of energy to them, and they are distinctly harmed by
it; they grow best in darkness. Direct daylight kills them rapidly,
and even prolonged exposure to diffuse light may be fatal. Bacteria
are more rapidly killed by exposure to the sun's rays in June, July
and August 2 than exposure of the same time in November, December
and January. Expressed differently, many bacteria which are killed
after an exposure of from one to two hours' direct sunlight in summer
require an exposure of from two to three hours in winter to accomplish
the same result.

Of the spectral rays, the red and infra-red rays, aside from the heating
effect, are without noteworthy action on bacteria. The blue, violet,
and ultraviolet rays, on the contrary, are distinctly bactericidal.

1 Yost, Plant Physiology, 223.

2 In the Northern Hemisphere.

LIGHT AND ELECTRICITY 45

These rays are chemodynamic and it is very probable that the death
of bacteria exposed to them in organic media results from the formation
of H 2 O 2 or other germicidal substances from the substrate. Bacteria
are also killed in non-decomposable media when they are exposed to
the ultraviolet rays. It should be remembered that one of the most
important characteristics of ultra spectral emanations is their very
short wave length. Glass is opaque to them where quartz is trans-
parent.

Electricity. It is difficult to differentiate sharply between purely
electrical effects and chemical changes which are induced in media
of various kinds by the action of electric currents. Generally speaking,
strong electrical currents sterilize media in which bacteria are growing,
but it is by no means certain that the electric current per se is the
important factor. Zeit 1 has made a careful, extensive and accurate
study of the action of various kinds of electric currents on bacterial
growth, and his conclusions are as follows:

"LA continuous current of 260 to 320 milliamperes passed through
bouillon cultures kills bacteria of low thermal death points, in ten
minutes by the production of heat 98.5 C. The antiseptics produced
by electrolysis during this time are not sufficient to prevent growth of
even non-spore-bearing bacteria. The effect is a purely physical one.

" 2. A continuous current of 48 milliamperes passed through bouillon
cultures for from two to three hours does not kill even non-resistant
forms of bacteria. The temperature produced by such a current does
not rise above 37 C. and the electrolytic products are antiseptic but
not germicidal.

" 3. A continuous current of 100 milliamperes passed through bouillon
cultures for seventy-five minutes kills all non-resistant forms of bac-
teria even if the temperature is artificially kept below 37 C. The
effect is due to the formation of germicidal electrolytic products in the
culture. Anthrax spores are killed in two hours. Subtilis spores were
still alive after the current was passed for three hours.

"4. A continuous current passed through bouillon cultures of bacteria
produces a strongly acid reaction at the positive pole, due to the libera-
tion of chlorin which combines with oxygen to form hypochlorous acid.
The strongly alkaline reaction of the bouillon culture at the negative
pole is due to the formation of sodium hydroxid and the liberation of
hydrogen in gas bubbles. With a current of 100 milliamperes for two
hours it required 8.82 milligrams of H 2 SO 4 to neutralize 1 c.c. of the

1 Jour. Am. Med. Assn., November, 1901.

46 GENERAL PHYSIOLOGY OF BACTERIA

culture fluid at the negative pole, and all the most resistant forms of
bacteria were destroyed at the positive pole, including anthrax and
subtilis spores. At the negative pole anthrax spores were killed also,
but subtilis spores remained alive for four hours.

" 5. The continuous current alone, by means of DuBois-Reymond's
method of non-polarizing electrodes and exclusion of chemical effects
by ions in Kruger's sense, is neither bactericidal nor antiseptic. The
apparent antiseptic effect on suspensions of bacteria is due to electric
osmose. The continuous electric current has no bactericidal nor
antiseptic properties, but can destroy bacteria only by its physical
effects heat or chemical effects, the production of bactericidal
substances by electrolysis.

" 6. A magnetic field, either within a helix of wire or between the
poles of a powerful electro-magnet, has no antiseptic or bactericidal
effects whatever.

"7. Alternating currents of a three-inch Ruhmkorff coil passed
through bouillon cultures for ten hours favor growth and pigment
production.

"8. High frequency, high potential currents Tesla currents have
neither antiseptic nor bactericidal properties when passed around a
bacterial suspension within a solenoid. When exposed to the brush
discharges, ozone is produced and kills the bacteria.

"9. Bouillon and hydrocele-fluid cultures in test-tubes of non-
resistant forms of bacteria could not be killed by Rontgen rays after
forty-eight hours' exposure at a distance of 20 mm. from the tube.

" 10. Suspensions of bacteria in agar plates and exposed for four
hours to the rays, according to Rieder's plan, were not killed.

"11. Tubercular sputum exposeu to the Rontgen rays for six hours
at a distance of 20 mm. from the tube, caused acute miliary tubercu-
losis of all the guinea-pigs inoculated with it.

" 12. Rontgen rays have no direct bactericidal properties. The
clinical results must be explained by other factors, possibly the pro-
duction of ozone, hypochlorous acid, extensive necrosis of the deeper
layers of the skin, and phagocytosis."

J. GRAVITY, OSMOTIC PRESSURE, AGITATION, CHEMOTAXIS.

1. Gravity. The majority of bacteria suspended in liquids are
not killed even by four hours' exposure to direct pressure of from 2000
to 3000 atmospheres (one atmosphere of pressure is equal to approxi-

GRAVITY, OSMOTIC PRESSURE, AGITATION, CHEMOTAXIS 47

mately 15 pounds to the square inch, or one kilogram per square
centimeter of surface). Bacteria are weakened, however, by these
great pressures, as is evidenced by a diminution in virulence, decreased
pigment production, and the partial or complete inability to multiply.
It is a curious fact that motile bacteria may retain their motility after
an exposure of several hours to 2000 atmospheres from the pressure
liquids, even although their powers of reproduction are quite lost.

Liquids are practically non-compressible, consequently direct pres-
sure does not affect the volume of the liquid in which bacteria are
suspended, nor does this pressure affect the amount of gas dissolved
in the liquid. If, however, bacteria are exposed in liquids to gas
pressure in the place of direct pressure, the germicidal action of the
gas plays the prominent part in the final result. The amount of gas
dissolved in the liquid increases with increase of pressure, consequently
feebly germicidal gases may become powerfully germicidal as the
pressure is increased. Thus, bacteria suspended in water overlaid
by CO2, which is feebly germicidal at ordinary pressures, are rapidly
killed if the pressure is gradually increased; that is, CO 2 under these
conditions becomes strongly bactericidal. According to Certes, 1
600 atmospheres pressure of an inert gas, as nitrogen, will not kill
anthrax bacilli.

Diminished Pressure. Diminished pressure, aside from lowering
the oxygen tension to a point below that necessary for the growth of
aerobic bacteria, does not interfere seriously with bacterial growth.

2. Osmotic Pressure. The boundary layer, ectoplasm, of every
bacterial cell reacts like a semi-permeable or osmotic membrane.
Through this membrane must pass all the elements necessary to the
nutrition of the organism. A normal bacterial cell always tends to
maintain a greater concentration of solutes within its substance than
exists in the surrounding medium; hence the pressure from within
upon the cell membrane is somewhat greater than the pressure from
without upon the cell membrane, and the cell is consequently in a
state of continual turgor. The osmotic pressure exerted by dissolved
substances varies very greatly. Those of high molecular weight, as
albuminoses or peptones, exert little or no osmotic pressure. Crys-
talloids, on the contrary, may exert very considerable pressure. Thus,
a 30 per cent, solution of dextrose exerts a pressure of about 22 atmos-
pheres. A bacterial cell placed in such a solution is under a great
strain. If bacteria which are in a state of equilibrium with reference

1 Compt. rend. Acad. de sc., 1884, 99, 385.

48 GENERAL PHYSIOLOGY OF BACTERIA

to the osmotic pressure of a solution are suddenly introduced into
media containing a greater concentration of solutes, the contents of
the cell diminish somewhat in amount, due to the rapid withdrawal of
water leaving the rigid cell membrane visible. This shrinkage of the
cell contents is spoken of as plasmolysis. 1 This shrinkage of the cell
contents would indicate that the cell membrane is differentially more
rapidly permeable to water than to crystalloids. All bacteria are not
plasmolyzed when they are suddenly introduced into hypertonic
solutions, and some organisms exhibit the phenomenon of plasmo-
lysis to a much greater extent than others. Plasmolysis does not neces-
sarily result in the death of the organism. It appears to be a fact that
older bacteria are frequently more readily plasmolyzed than younger
individuals of the same kind. The observations of Nicolle and Auclaire 2
would indicate that bacteria which retain the Gram stain are less
readily plasmolyzed than Gram-negative bacteria. Whether Gram-
positive bacteria which have become Gram-negative due to prolonged
cultivation in artificial media invariably follow the same rule is not
known.

If bacteria are gradually subjected to solutions of greater or lesser
osmotic pressure, they usually accommodate themselves to these
changes without visible effect. If bacteria are introduced abruptly
into solutions of low osmotic pressure or distilled water, water rapidly
passes through the cell membrane of the bacteria faster than the solutes
within the cell can pass out, thus rapidly increasing the intracellular
pressure until frequently the cell membrane ruptures, permitting the
escape of some of the cell contents. This phenomenon is called
plasmoptysis. 3 Most bacteria do not plasmoptyze readily, and it is
problematical how much importance should be attached to either
plasmolysis or plasmoptysis in practical bacteriology.

3. Agitation. Bacteria grow best in quiet surroundings, although
a slight amount of agitation is usually harmless and may be even
beneficial if it tends to dislodge waste products from the immediate
surroundings of sedimented organisms. Rapid agitation frequently
retards the multiplication of bacteria in fluid cultures, and Meltzer 4
and Horvath 5 have shown that violent shaking gradually kills bacteria;
not, however, by rupturing the cell membrane. The organisms undergo

1 Fischer, loc. cit., p. 23.

2 Ann. de 1'Inst. Pasteur, 1909, xxiii, 547.

3 Fischer, loc. cit., p. 48.

4 Ztschr. f. Biol., 1894, xxx.
6 Pfliiger's Arch., 1887, xvii.

ENZYMES, TOXINS, PTOMAINS 49

a gradual disintegration, and the injurious effects observed are said
by these observers to be not purely mechanical.

4. Chemotaxis. Bacteria respond to various chemical stimuli.
Substances which can be used by them for nutritional purposes, as
various constituents of laboratory media, appear to attract bacteria.
Harmful substances, as acids or alkalis, may act in the reverse manner.
Oxygen is a powerful chemotactic agent for many aerobic bacteria,
while many anaerobes are repelled by it. The mutual chemotactic
relations of bacteria and leukocytes, and the well-defined tendency
of certain invasive bacteria to localize in definite tissues or organs
of the animal body .are interesting fields for speculation. Nothing
conclusive is known about these relations.

K. ENZYMES, TOXINS, PTOMAINS.

Enzymes. The phenomena of chemical interchange between
bacteria and their environment indicate that enzyme activity plays
an important part in bacterial metabolism.

Enzymes may be defined as substances of unknown composition
produced by living cells which incite specific chemical reactions with-
out permanently combining with the products of reactien. A small
amount of enzyme acting under favorable conditions will cause a
relatively extensive transformation of substance without itself being
used up or inactivated. There is, however, a limit to the amount of
transformation which a given amount of enzyme can accomplish,
for the accumulation of reaction products tends to restrict enzyme
action; the removal of reaction products appears to extend enzyme
action somewhat. All bacterial cells appear to produce or to possess
enzymes, probably several, which may be divided somewhat arbi-
trarily into two classes, the extracellular or exo-enzymes, and the
intracellular or endo-enzymes.

Exo-enzymes. Exo-enzymes are those which are excreted from the
organism and appear as soluble, filterable and, frequently, diffusible
enzymes, which may be obtained in an active state from filtrates of
cultures of bacteria. Their diffusion from the bacterial cell and their
filterability suggests that they may be relatively simple in molecular
aggregation. Their function is essentially a "preparatory" one, for
they transform potential nutritional substances, as proteins, carbo-
hydrates or fats, to simpler compounds which are assimilable by the
bacteria. It is very probable that the exe-enzymes work uneconem-

50 GENERAL PHYSIOLOGY OF BACTERIA

ically in the sense that they transform more material than the
organisms require : this phenomenon is exhibited in the extensive lique-
faction of gelatin by proteolytic bacteria, as B. proteus. The organism^
which elaborates such an exo-enzyme probably derives but little energy
from its activity, and, conversely, probably expends comparatively
little energy in the elaboration and secretion of the exo-enzyme.

Endo-enzymes. Comparatively little is known of the endo-enzymes :
it is -generally believed that they are comparatively non-diffusible, at
least in an active state, and that they are non- or but slightly filter-
able. This suggests that they are relatively complex in their mole-
cular aggregation. Their function is probably to act upon the nutrient
substances which the cell has assimilated, partly to liberate energy
from them, and partly to participate in the organization of the cell
constituents. These endo-enzymes work economically in contra-
distinction to the exo-enzymes in the sense that the substrate is appar-
ently changed by them in proportion to the requirements of the cell.
Endo-enzymes may be obtained from bacterial cells when the latter
disintegrate, provided the rupture of the cells is not accomplished by
violent chemical means. Probably the phenomena of autolysis which
many bacteria exhibit when they are placed in an environment free
from food may be due, in part at least, to the autodigestion of the
organisms by their endo-enzymes.

Classification of Enzymes. Enzymes are usually classified according
to the substrate they act upon : thus, proteolytic enzymes, or proteases,
split proteins or protein derivatives into simpler compounds; carbo-
hydrolytic enzymes split starches or polysaccharides into simpler
carbohydrates; fat-splitting ferments, lipases, split fats into glycerin
and fatty acids. The above enzymes are -hydrolytic in character,
that is, they effect cleavage of protein or carbohydrate or fat or of
glucosides by splitting the molecule into simpler molecules which
simultaneously take up hydrogen and oxygen in the proportions to

form water, thus:

CH 2 NH 2 CO-NHCH 2 COOH + H 2 O (+ enzyme) = CH 2 NH 2 COOH + CH 2 NH 2 COOH.
Glycyl-glycine. Glycine. Glycine.

Ci 2 H 22 On + H 2 O ( + lactase) = C 6 Hi 2 O 6 + C 6 Hi 2 O 6 .
Lactose. Dextrose. Galactose.

CH 2 O-CO-CH 3 CH 2 OH

I I

CHO-CO-CHs + 3 H 2 O (+ lipase) = CHOH + 3 CH 3 COOH

Acetic acid.

CH 2 O T CO 7 CH 3 CH 2 OH

Triacetin, Glycerin.

ENZYMES, TOXINS, PTOMAINS 51

The question of specificity of action of bacterial enzymes is not
definitely settled. There is some evidence in favor of the view that
exo-proteolytic enzymes produced by various bacteria act upon a
variety of proteins: thus, the cholera vibrio produces a soluble pro-
teolytic enzyme which will digest casein, coagulated blood serum, egg
albumen, fibrin and gelatin. Other organisms, as the staphylococcus,
produce an exo-enzyme which will hydrolyze casein, coagulated blood
serum and gelatin: its action upon other proteins is not definitely
established. The important question are the products of hydrolysis
of the same protein by proteolytic enzymes from different bacteria
the same is not definitely settled; it is probable, however, that the
products differ. This suggests that the proteolytic enzymes of bacteria
are not mere "catalyzers" which accelerate reactions in relatively
unstable substances that would take place spontaneously but much
more slowly; these enzymes (proteolytic enzymes) may not only incite
reaction, they may guide it, as it were, along lines of cleavage which
would not be followed in the absence of this enzyme. The carbo-
hydrate and the fat-splitting enzymes have much less latitude in
splitting the carbohydrates and fats respectively than the proteolytic
enzymes, for these substances are less complex in structure and com-
position than the proteins and protein derivatives.

Fuhrmann 1 has classified enzymes of bacterial origin into four types
as follows:

A. SCHIZASES (HYDROLYTIC) CLEAVAGE ENZYMES.

1. Proteases, protein-splitting enzymes. Pepsin, Trypsin (Lysins, Coagu-

2. Carbohydrate-splitting enzymes. Amylase, Cellulase, Pectinase, Gelase,

Invertase, Lactase.

3. Glucoside-splitting enzymes. Emulsin (Synaptase).

4. Fat-splitting enzymes. Lipases (esterases).

B. OXIDIZING ENZYMES.

Tyrosinase, Acetic bacteria, Oxydase.

C. REDUCING ENZYMES.

Reductases.

D. FERMENTATION ENZYMES.

Zymase, Urease, Lactic acid enzyme.

The bacteriolysins are of particular importance in bacteriology:
of the bacteriolysins, those which liberate unchanged hemoglobin
from red blood cells (hemolysins) and those which digest hemoglobin
(hemodigestins 2 ) are intermediary in their general properties between
enzymes and toxins, if indeed there is any tangible distinction between

1 Vorlesungen uber Bakterienenzyme, Jena, 1907.

2 Van Loghem, Centralbl. f. Bakteriol., 1912-1913, Ixvii, 410.

52 GENERAL PHYSIOLOGY OF BACTERIA

them. Vaughan 1 has studied both enzymes and toxins extensively,
and has summarized admirably the points of resemblance between
exo-enzymes and exo-toxins as follows:

"1. Both are destroyed by heat. 2

"2. They act in very dilute solution.

"3. When repeatedly injected into animals in non-fatal doses they
cause the body cells to elaborate antibodies which neutralize the toxin
(or the enzyme) both in viw and in vitro.

"4. In the development of their effects a period of incubation is
required.

"5. It has been shown (by Abderhalden) by optical methods that
they have a cleavage effect upon proteins they split complex proteins
into simpler bodies; in other words, they have a proteolytic action.

"6. They are specific in two senses: (a) they are specific according
to the cell which produces them; (6) they are specific in the antibody
elaborated in the animal body after repeated injections of non-fatal
doses."

Bacterial toxins are usually classified as exo- or soluble (extra-
cellular) toxins, and endo- (intracellular) toxins. The former are
soluble and diffuse out from the bacterial cell into the surrounding
medium. Very few bacteria produce exo-toxins: the best known are
those of the diphtheria, tetanus, and botulismus bacilli. To these
specific antitoxins are known. Endo-toxins are non-diffusible and are
locked up in the bacterial cell; they are liberated only when the
cell disintegrates. No specific antitoxin has been produced for an
endo-toxin.

Ptomains. Ptomains are soluble, basic, nitrogen-containing sub-
stances formed from proteins or protein derivatives by the action of
microorganisms. They are non-specific, relatively poor in oxygen
content, and probably simpler in composition than either exo- or endo-
toxins. No antibodies have been produced against them. Some are

poisonous, many are not.

\
\

L. PIGMENTS.

With the exception of bacteriopurpurin, which occurs in the sulphur
bacteria and is supposed to be photodynamic and, therefore, somewhat
analogous to the chlorophyll of the higher plants, the significance of

1 Protein Split Products, Lea & Febiger, Philadelphia and New York, 1913.

2 Although they are somewhat more resistant to heat than the cells which produce
them.

SYMBIOSIS, ANTIBIOSIS AND COMMENSALISM 53

pigment formation, which is a striking cultural characteristic of many
bacteria, is wholly unknown. The pigment they produce does not
protect them 'against strong Light, and achromogenic strains may be
cultivated from the chromogenic varieties without apparent loss in the
cultural or chemical characters of the organisms. It is very probable
that these pigments are chiefly waste products of metabolic origin.

Pigments are produced in darkness and sunlight rapidly destroys
many of them. Oxygen is not necessary for their production, for the
non-colored leukobase is the form in which the pigment is excreted
by bacteria, but oxygen is necessary for the development of color from
this leukobase.

Pigment-producing bacteria may be grouped into four classes :

1. Bacteria producing photodynamic pigment. Certain sulphur
bacteria which produce bacteriopurpurin.

2. Phosphorogenic bacteria which produce a luminous substance
somewhat analogous to that of glow-worms. These organisms are
chiefly marine forms, as B. phosphorescens.

3. Fluorogenic bacteria which produce a pigment soluble in water
and culture media; this usually exhibits complementary colors as it
is viewed by reflected and transverse light respectively.

4. Chromogenic bacteria. The pigment produced is usually insol-
uble in water and soluble in organic solvents. The color varies accord-
ing to the organism producing it. The more common colors are red,
orange, yellow, green, blue, violet, brown, and black pigment. These
colored pigments are usually referred to as lipochromes because of
their solubility in organic solvents and their general relationship to
fats. Many of them give well-defined and constant absorption when
they are viewed spectroscopically in solutions. 1

M. SYMBIOSIS, ANTIBIOSIS AND COMMENSALISM.

The biological relations of bacteria are of the greatest importance
in the economy of nature and in the production of disease. Bacteria
do not grow in pure culture in nature, although they may do so in the
tissues of man or animals, as disease-producing bacteria (pathogenic
bacteria). In nature, where the reduction of dead complex organic
material to mineralized salts is the striking function of bacteria, the
successive steps in the degradation of organic matter are carried on
by different kinds of microbes. The various steps appear to vary

1 Sullivan, Jour. Med. Research, 1905, xiv, 109.

54 GENERAL PHYSIOLOGY OF BACTERIA

somewhat, but the process is on the whole an orderly and definite one.
The association of various kinds of bacteria in this process, where
each succeeding kind profits by the activities of the preceding kind,*
is a symbiotic one; that is, the several types of organisms mutually
profit by their combined activities.

It frequently happens that the products of symbiotic activity may
be greater than the sum of the products of the separate activities of
the organisms. 1 On the contrary, many instances are known in which
one kind of organism by its activity actually crowds out a preexisting
organism, as for example, the lactic acid bacteria which sour milk.
They produce sufficient lactic acid from the fermentation of the lactose
to kill the proteolytic forms. This substitution of one type of organism
by another is known as antibiosis: the latter organism profits wholly
at the expense of the first organism.

It not infrequently happens that one type of bacterium profits by
the activity of another type of organism without benefiting the former
in return. If two types of bacteria are concerned, the process is known
as metabiosis; if the bacterium is living on a host, the relationship is
spoken of as parasitism.

N. MEDIA COMPOSITION AND REACTION.

Most bacteria grow best in a medium containing a large percentage
of moisture in which diffusible proteins or protein derivatives are
present as sources of nitrogen: these substances are better adapted to
the dietary needs of the majority of bacteria than are ammonium
salts or even simple amino acids. A very few bacteria (nitrifying
bacteria) cannot grow in media containing organic nitrogen compounds :
a few strictly pathogenic bacteria appear to require nitrogen as it
exists in the highly complex tissues of man or animals for their growth.
Many bacteria can utilize carbohydrates for their carbon, hydrogen,
and oxygen requirements. Some bacteria appear to be able to utilize
fats for their carbon requirement.

A neutral or feebly alkaline reaction is best adapted to the develop-
ment of the vast majority of bacteria; a few types develop best in a
medium which is distinctly acid the aciduric bacteria. 2 Mineral
acids are germicidal; organic acids may be utilized by bacteria for
foods.

1 Kendall, Jour. Am. Med. Assn.. 1911, Ivi, 1084.

2 Kendall, Jour. Med. Research, 1910, xxii, 153.

GROWTH OF BACTERIA IN THE ANIMAL BODY 55

O. GROWTH OF BACTERIA IN THE ANIMAL BODY.

The vast majority of bacteria do not grow in the tissues of the body,
although a small number of organisms, the parasitic bacteria, live
habitually on the surface of the body or on mucous membranes, usually
without producing noticeable effects. A small, formidable group of
bacteria, the progressively pathogenic bacteria, actually invade the
tissues; they may produce within the host inhibition of function or
anatomical changes incompatible with health.

CHAPTER III.

THE CHEMISTRY OF BACTERIA. THE EFFECT OF
BACTERIA ON THEIR ENVIRONMENT.

A. GENEEAL.

B. CHEMICAL CONSTITUTION OF BAC-

TERIA.

1. Elementary Composition.

2. Chemical Constitution.

3. Chemical Composition.

C. COMPOSITION OF THE MORPHOLOGI-

CAL COMPONENTS OF THE
BACTERIAL CELL.

1. Cell membrane.

2. Capsule.

3. Cytoplasm.

4. Spores.

D. FOOD RELATIONSHIPS OF BACTERIA.

1. General.

2. Sources of Food.

(a) Nitrogen.
(&) Carbon.

(d) Oxygen.

(e) Inorganic Salts.

A. GENERAL CHEMISTRY OF BACTERIA.

THE practical significance of bacteria is summed up in the nature
and extent of the chemical changes which they induce in their environ-
ment, the result of their multiplication and vegetative activity. These
changes are essentially analytical, for the function of bacteria in
nature is to transform dead organic matter from complex unstable
combinations of carbon, hydrogen, nitrogen, and oxygen, which are
worthjess in the economy of nature, to fully mineralized, stable inor-
ganic compounds of these elements, which may be resynthesized by
plants.

A small but formidable group of bacteria, chiefly those pathogenic
for plants, animals and man, act directly upon the living plant or
animal organism, producing changes in them which may be tempor-
arily incompatible with their well-being, and not infrequently lead to
their death and eventually to their mineralization. The pathogenic
bacteria, therefore, are also analytical in their activities and do not
differ essentially in this respect from the saprophytic types.

It is necessary to consider briefly the method of the interchange
of material between the vegetable and animal kingdoms in order to
understand the full significance of bacterial action in the economy of
nature. All animals require preformed organic compounds for their
sustenance. They are unable to build up these compounds of which
their tissues are composed from chemical elements or from simple
inorganic salts. They are, therefore, dependent directly or indirectly
upon the synthetic activities of green plants for their foodstuffs. The
green plants by virtue of the chlorophyll contained within their leaves

GENERAL CHEMISTRY OF BACTERIA 57

and stems possess the power of combining CO 2 , water and nitrogenous
salts under the influence of sunlight directly into the highly complex
proteins and carbohydrates essential for animal food. These products
of the synthetic activity of the plants are utilized by the animal
kingdom for food; directly by the herbivora, indirectly by the carni-
vora. These substances are either broken down within the digestive
tract of the animal body and reconstructed to form the tissues and
supply energy to the animal, or eliminated as excreta. The excreta
of animals are not sufficiently simple in composition, as a rule, to be
used directly by plants, and the tissues of dead animals and plants
are of little value in their complex state for plant foods. Further
cleavage, both of the excreta of animals and the dead bodies of plants
and animals, is necessary to make the elements contained within them
utilizable by plants, and this cleavage is brought about by bacterial
activity. Various saprophytic bacteria act successively upon these
complex organic compounds, changing them, chiefly by hydrolytic
cleavage, into stable, fully mineralized salts, which are directly utiliz-
able in this state by the chlorophyll-bearing plants. There is, there-
fore, a constant rotation of the various elements which enter into the
composition of animal and plant tissues between the plant and animal
kingdoms respectively by means of an anabolic or constructive process
in the one (plants), and a catabolic or destructive process in the other
(animals). The cycle as outlined, however, is not a continuous one,
for there are important gaps in the process of cleavage and in the pro-
cess of synthesis which if left unbridged by the bacteria would eventu-
ally arrest all vital activity both of plants and animals, and all life
would then inevitably cease on this planet. These gaps between the
animal and vegetable kingdoms are filled by the analytical activity
of bacteria.

A small group of bacteria, on the other hand, is also important
from the synthetical point of view. A certain amount of nitrogen is
lost in the animal and vegetable kingdoms by various natural agencies,
and this supply of nitrogen must be made good from sources which
are not directly available either to plants or to animals. Approxi-
mately 80 per cent, of the atmosphere is made up of nitrogen, and a
certain group of bacteria, "the nitrogen-fixation" bacteria so-called,
which are found chiefly on the nodules or roots of leguminous plants,
are able to draw upon this great reservoir of atmospheric nitrogen
and synthesize it into nitrogen-containing compounds which plants
can utilize directly.

58 THE CHEMISTRY OF BACTERIA

Another type of bacterial activity of importance is the oxidation
of ammonia, the final step in the degradation of protein, into nitrites
and nitrates. This is carried on by the nitrifying bacteria of the soil.
Contrary to the generally accepted idea, therefore, the activities of
the majority of bacteria are not in opposition to the activities of man,
animals, and plants; bacteria are indispensable agents in the economy
of nature.

B. CHEMICAL COMPOSITION OF BACTERIA.

1. Elementary Composition. Bacteria normally contain the same
elements in their substance that the higher plants and animals contain,
viz., carbon, nitrogen, hydrogen, oxygen and phosphorus, together
with smaller amounts of sodium, chlorine, sulphur, potassium, calcium,
magnesium, and traces of iron.

2. Chemical Constitution. The elements carbon, hydrogen, nitro-
gen and oxygen, and to a certain extent phosphorus, and perhaps
sulphur are united to form proteins, nucleoproteins, carbohydrates,
and fats. The inorganic substance of bacteria is made up of the other
elements mentioned above in variable proportions. Of these elements,
carbon, hydrogen, nitrogen, oxygen and phosphorus are the most
important. 1

TABLES ILLUSTRATING THE CHEMICAL COMPOSITION OF
BACTERIA.

1. .PERCENTAGE OF THE ELEMENTS IN ASH-FREE " MYCOPROTEiN." 2

C H N

per cent. per cent. per cent.

52.1-52.6 7.3-7.38 14.5-14.9

2. PERCENTAGE COMPOSITION WITH RESPECT TO ORGANIC AND INORGANIC
CONSTITUENTS.

Putrefactive Bacillus Tubercle

bacteria. 3 prodigiosus. 4 bacilli. 5

Water 83.42 85.45 85.00

Protein 13.96 10.33 8.50

Extractive 1.00 0.70 4.00

Ash 0.78 1.75 1.40

Residue 0.84 1.77 1.10

1 Certain acid-fast bacteria can be grown in media containing theoretically but five
elements: carbon, hydrogen, nitrogen, oxygen, and phosphorus. Lowenstein, Centralbl.
f. BakterioL, Original, 1913, Ixviii, 591. Wherry, Centralbl. f. Bakteriol., 1913, Ixx,
115. Kendall, Day and Walker, Jour. Inf. Dis., 1914, xv, 428.

2 Kruse, Allgemein. Microbiol., p. 62.

3 Nencki and Scheffer, Ueber die chemische Zusammensetzung der Faulnisbakterien,
Beitr. z. Biol. d. Spaltpilze. Nencki, Leipzig, 1880, Jour. f. prakt. Chemie, N. F., xix,
u. xx.

4 Kappes, Analyze d. Massen Kulturen einiger Spaltpilze u. d. Soorhefe, Leipzig, Diss.,
1889.

5 Ruppel, Die Proteine, 1900, Heft 4, Beitr. z. exp. Therapie., Ztschr. f. physiol.
Chemie, xxvi.

CHEMICAL COMPOSITION OF BACTERIA 59

COMPOSITION OF BACTERIA. 1

Water.

In per cent, dry residue.
Acetone CHCla

Phosphorus,
per cent.

per cent.

extract.

extract. 3

in fat. 4

Glanders

. . . 76.5

10.5

11.7

8.6

2.5

Chicken cholera .

. . . 79.3

10.8

7.5

6.3

2.4

Cholera

73 4

9.8

8.7

6.8

2.4

Dysentery (Shiga)

. . . 78.2

8.9

12.8

10.6

1.6

Proteus vulgaris

. . . 80.0

10.7

10.9

7.1

1.6

Typhoid . . .

. . . 78.9

8.3

15.4

10.6

1.2

Anthrax 2

. . , 81.7

9.2

6.3

1.5

0.9

Pseudotuberculosis

. . . 78.8

10.4

15.6

10.3

0.8

B. pneumonias

. . . 85.5

10.4

15.4

10.3

0.8

B. coli ....

. . . 73.3

8.3

15.2

11.8

0.8

B. prodigiosus

. . . 78.0

10.5

9.0

6.6

0.5

B. psittacosis .

. . . 78.0

9.5

11.1

7.0

0.5

B. diphtherias

. . . 84.5

7.0

5.2

0.2

B. pyocyaneus

. . . 75.0

9.8

15.8

10.7

0.2

It will be seen that from 75 to 86 per cent, of the bacterial celt is
water. The remainder of the cell consists chiefly of protein, carbo-
hydrate-like bodies, extractives (fats, fatty acids, waxes and lipoids),
and inorganic salts. Of these, the nitrogenous substances vary greatly
in amount, depending upon the composition of the medium in which
the organisms are grown. _ The extractives (fats, waxes, lipoids, and
fatty acids) are most prominent in the tubercle bacillus and the acid-
fast group. Some extractives, however, are found in all bacteria,
they being greater in amount on a medium containing carbohydrate
and protein than on one containing protein alone. The chemical
determination of the extractives is very unsatisfactory, partly because
of the difficulty in breaking up the cell sufficiently to facilitate the
entrance of the solvent.

3. Chemical Composition of Bacteria. The percentages of the ele-
ments and various constituents of bacteria, as indicated in the above
tables, is at best only approximate. Other factors very markedly
influence the composition of the organisms.

Of these, the age of the culture, the temperature at which it is
grown, and the composition of the medium in which the organisms
are grown are the most important. Generally speaking, young cul-
tures appear to contain rather more dry residue than older cultures,
and bacteria grown at 37 C. contain more dry residue than those
grown at 20 C. 5 The inorganic constituents of the broth influence

1 Nicolle and Alilaire, Ann. 1'Inst. Past., 1909, xxiii, 547.

2 Asporeless. 3 From acetone extract. < From CHCls extract.

5 The decrease in dry residue observed in old cultures is partly attributable to auto-
lysis of bacteria ; this is usually observed earlier in cultures maintained at 37 C. than in
corresponding cultures kept at 20 C. Growth is more rapid at this higher temperature,
and recessive changes due partly to the accumulation of waste products are seen earlier.

THE CHEMISTRY OF BACTERIA

the composition of bacteria markedly. Cramer 1 has found that the
percentage composition of the ash of the cholera vibrio varies within
very considerable limits as the organism is grown under different
conditions. The following table indicates in a general way the influ-
ence of these factors:

=4
II-

9.30

22.30

25.90

1.34

2.75

3.73

1.25

2.50

4.12

28.70

34.80

10.90

7.90
16.90

39.80
7.97

2.10
46.70

23.00

11.40

49.20

Ash content of bacteria in dry substance
Ash content of moist mass ....
Ash content of medium in moist mass
Phosphoric acid in bacterial ash .
Phosphoric acid in media ash
Chlorine in bacterial ash ....
Chlorine in media ash

The phosphorus content of the medium in these experiments, as
shown in the above table, was varied almost twenty times, but in the
bacterial organisms it varied scarcely three times. The variation in
chlorine content was somewhat greater.

Even as important an element as nitrogen is subject to rather wide
variations in bacteria, as Cramer 2 and Lyons 3 have shown. The fol-
lowing tables summarize Cramer's and Lyons's results. They were
obtained by growing certain bacteria mentioned specifically below on
a medium consisting fundamentally of 1.5 per cent, agar, to which were
added various substances, as indicated in the tables, respectively
Media A, B, and C. The general procedure was to grow the bacteria
at 37 C. for several days, to wash them off with salt solution, to free
them from adherent media by centrifugalization and washing, to dry
the washed organisms in vacua to constant weight, and to analyze
the dry residue for extractives and ash.

CRAMER.

Nitrogen substance.

Ether-alcohol
extractives.

Ash.

* !

Organism.

Pfeiff er bacillus . .

66.6

70.0

53.7

17.7

14.63

24.0

12.56

9.10

9.13

Bacillus H-28 4 . .

73.1

79.6

59.0

16.9

17.83

18.4

11.42

7.79

9.20

Pneumonia bacillus .

71.7

79.8

63.6

10.3

11.40

22:7

13.94

10.36

7.88

Rhinoscleroma bacillus

68.4

76.2

62.1

11.1

9.06

20.0

13.45

9.33

9.44

1 Quoted by Kruse, Allgemeine Mikrobiologie, p. 88.

2 Arch. f. Hyg., 1893, 151. 3 Ibid., 1897, xxiii, 30.

4 From water.

COMPOSITION OF THE BACTERIAL CELL

LYONS.

Medium.

Nitrogen-
containing
substance.

Ether
extractives.

Alcohol
extractives.

Ash.

Organism.

Pfeiffer bacillus .

62.75

58.88

45.88

1.68

3.502.67

12.17

17.30

29.60

7.16

2.79

3.09

Bacillus No. 28 1 . .

71.8159.12

46.25

3.32

3.842.84

11.39

15.19

22.78

6.51

3.66

4.18

"Thread bacillus." .

61.06

44.31

33.25

1.74

2.24

1.87

18.40

21.80

27.50

8.09

4.50

3.02

Medium A agar, 1.5 per cent.
Medium B agar, 1.5 per cent.
Medium C agar, 1.5 per cent.

peptone, 1 per cent,
peptone, 5 per cent,
peptone 1 per cent.; dextrose, 5 per cent.

It will be seen that the nitrogen content of the bacteria grown in
a medium containing nitrogen plus carbohydrate is almost 25 per cent,
less than the nitrogen content in the same organisms grown in the same
nitrogen medium but with no carbohydrate. The nitrogen content is
greatest in the carbohydrate-free medium, the extractives are greater
in the carbohydrate-containing medium. This decrease in the nitrogen
content in pathological bacteria grown in sugar media may be of
considerable importance, particularly in the preparation of vaccines
and other antigens. Nothing is known definitely of the distribution
of nitrogen in bacteria, but this reduction of 25 per cent, in the
nitrogen content may well influence somewhat the immunizing value
of vaccines.

C. COMPOSITION OF THE MORPHOLOGICAL COMPONENTS OF
THE BACTERIAL CELL.

1. Cell Membrane. Typical cells of higher plants contain cellulose,
and bacteria were formerly differentiated sharply from the plant
kingdom because cellulose could not be found in them. Later observa-
tions would suggest that cellulose or substances chemically closely
related to it are demonstrable in certain bacteria. Dreyfuss 2 appears
to have identified cellulose in bacteria from pus and in B. subtilis;
Hammerschlag 3 claims to have isolated cellulose from tubercle bacilli,
Dzierzgowski and Rekowski 4 appear to have found cellulose in diph-
theria bacilli; more recently Tamura 5 has demonstrated a hemi-cellulose

1 From water.

2 Ztschr. f. phys. Chemie, 1893, xviii, 375.

3 Sitzber. Akad. Wiss., Wien, xiii, 12.

4 Arch. Soc. Biol., St. Petersburg, 1892.

6 Ztschr. f. phys. Chem., 1914, Ixxxix, 289.

62 THE CHEMISTRY OF BACTERIA

in the same organism. So that the ability of at least certain bacteria
to elaborate cellulose can hardly be doubted.

Emmerling 1 identified chitin in Bacterium xylinum, and Irvanoff 2
gives the following percentage composition of the cell membranes of
B. pyocyaneus, B. megatherium and B. anthracis: C, 46 per cent.;
H, 6.7-7 per cent. ; N, 8.4-8.8 per cent. ; which is empirically very similar
to chitin. Chitin is chemically a polymer of glucoseamine, CH 2 OH.-
(CHOH) 3 .CHNH 2 .CHO, which in turn is an amino hexose very similar
to dextrose, except that it has an amino group adjacent to the aldehyde
gro'up. Chitins are typically animal in origin, and are rarely, if ever,
found in typical plants, hence the distribution between cellulose and
chitin in bacteria is important as suggesting relationships to the
vegetable or animal kingdoms.

Many bacteria stain brown with iodin, and the assumption is that
the cell membrane of such organisms, or the cell substance contains
substances similar to glycogen. According to Arthur Meyer, 3 many
bacteria color blue with very small amounts of iodin; brown or
red-brown with an excess of iodin; indicating that there is a very
small amount of starch and a relatively large amount of glycogen
or amylodextrin in the substance. Similar observations have been
made by Heinze 4 and Levene, 5 who have isolated a substance from
tubercle bacilli which reacts chemically like glycogen.

2. Capsule. The capsules of the capsule-forming bacteria contain
considerable amounts of a mucinous substance apparently a glyco-
protein. Cultures of bacteria which do not ordinarily exhibit capsules
occasionally produce spontaneously viscid, mucinous substances in
artificial media; thus, strains of rabbit septicemia bacilli and glanders
bacilli may become viscid after repeated transfers. 6 Broth cultures
of tubercle bacilli may similarly become mucinous. 7 Rettger's observa-
tions 8 make it very probable that these viscid substances are true
mucins.

3. Cytoplasm. The cytoplasm of bacteria consists chiefly of the
bacterial protein, which appears to be specific in character for any

1 Berichte d. chem. Gesell., 1899, 541.

2 Hofmeister's Beitrage, 1902, i, 524.

3 Flora, 1899.

4 Centralbl. f. Bakteriol., 2te Abt., 1903, xii; 1904, xiv.
6 Jour. Med. Research, 1901, vi, 135.

6 Theobald Smith, Transactions of First Annual Meeting of National Association
for the Study and Prevention of Tuberculosis.

7 Weleminsky, Berl. klin. Wchnschr., 1912, xlix, 1320; Kendall, Walker and Day,
Jour. Infec. Dis., 1914, No. 11.

8 Jour. Med. Research, 1903, x, 101.

COMPOSITION OF THE BACTERIAL CELL 63

given organism, together with enzymes and at least minimal quantities
of all the products of its metabolism.

Regarding the nature of the protein substance in bacteria, but little
is known, although 50-80 per cent, of the dried substance of the
bacterial cell consists of protein and protein derivatives. Conspicuous
among these protein derivatives are the nuclein constituents, nucleins,
nucleoproteins, and nucleic acids; they occur constantly in bacteria
and apparently the greater part of the protein of the bacterial cell
consists of these nuclear constituents. Nucleins and nucleoproteins
have been isolated from many bacteria: from B. subtilis by Van de
Velde; 1 from the plague bacillus by Lustig and Galeotti; 2 from the
typhoid bacillus by Paladino-Blandini; 3 from the tubercle bacillus
by Von Ruck 4 and Ruppel; 5 from the diphtheria bacillus by Aronson; 6
and Carapelle 7 has identified a glyco-nucleo-protein in B. prodigiosus.

Numerous observations indicate that nuclein bases (xanthin bases)
are found in bacterial cells; thus, Lustig and Galeotti 8 identified
xanthin in plague bacilli. Nashimura 9 obtained xanthin bases in the
dried residue of a water bacillus in the following amounts: xanthin 0.07
per cent.; guanin, 0.14 per cent.; adenin, 0.08 per cent. No hypox-
anthin was found.

The amino-acids of bacterial protein have not been thoroughly
studied. The variable nitrogen content even of the same organism
as it is grown in different media and under different conditions would
suggest that quantitative determinations of nitrogenous substances
would be somewhat unsatisfactory. Qualitatively, so far as available
data show, many amino-acids found in protein of higher animals and
plants have been isolated or identified in bacterial cells. These amino-
acids appear to differ in amount in different organisms, and several
have not been isolated at all up to the present time. Vaughan, Wheeler,
and Leach 10 conclude that the bacterial substance contains carbo-
hydrates, nuclein bodies and polymers of mono- and diamino-acids.
They are glyco-nucleo-proteins. Kruse 11 and Vaughan 12 have arrived at

1 Ztschr. f. phys. Chem., viii.

2 Deutsch. med. Wchnschr., 1897, 225.

5 Baumgarten's Jahresberichte, 1901, 228, ref.

4 Prophylactic Immunization against Tuberculosis, Report No. 1, Asheville", 1912, 3.

6 Ztschr. f. phys. Chem., 1898, xxvi.

6 Arch. f. Kinderheilkunde, vol. xxx.

7 Centralbl. f. Bakteriol., 1907, xliv, 440.

8 Loc. cit.

9 Arch. f. Hyg., xviii, 325.

10 Tr. Assn. Am. Phys., 1902, p. 243.

11 Allgemeine Microbiologie, p. 65.

12 Protein Split Products, p. 437.

64 THE CHEMISTRY OF BACTERIA

the same conclusion. The analysis of one hundred grams of dried
tubercle bacilli by Ruppel 1 indicates the importance of the nucleins in
bacterial proteins.

Grams.

Nucleic acid (tuberculinic acid) 8.5

Nucleoprotamin 25.5

Nucleoproteid 23.0

Albuminoids (keratin, etc.) 8.3

Fat and wax 26 . 5

Ash 9.2

Carbohydrates. Glycogen or some similar carbohydrate, which is
readily detected by the mahogany color it gives with iodine, is found
in many bacteria, as has been stated previously, but it is extremely
difficult to decide definitely whether it is limited exclusively to the cell
membrane or scattered somewhat diffusely through the cytoplasm
as well.

Fats and Fatty Derivatives. Fats, fatty acids, lipoids and waxes,
which may be demonstrated by staining bacteria with Sudan III,
Scharlach R, and osmic acid, occur in variable amounts in the tubercle
bacillus and other acid-fast bacilli. The amount of these extractives
may be very great in the acid-fast group, varying from 26 to 40 per cent,
of the total dry residue. Considerable discussion has centred around
the distribution of these substances, many authorities claiming that
the fats and waxes are contained in the cell wall of the organism, while
others maintain that these substances are scattered throughout the
cell substance as well. In the acid-fast bacilli it is probable that these
fats are both intra- and extracellular, for analyses show that a certain
amount of them can be extracted from intact bacilli, while still more
can be extracted when the organisms are broken up. The following
table from Kresling 2 illustrates the distribution of the fatty substance
of the tubercle bacillus:

I. CONTENTS OF THE DRIED TUBERCLE BACILLI IN THE
PREPARATION OF TUBERCULIN.

Per cent.

Moisture (dried at 100 -l 10 C.) 3.9375

Moisture (dried in desiccator) 3 . 08

Ash 2.55

Nitrogen - 8.575

Nitrogen-containing substances (albumin) reckoned by multiply-
ing the amount of N by the factor 6.25 (the N of lecithin and
other substances soluble in chloroform, benzol, ether, and

alcohol were not reckoned) 53 . 59

Fatty substances in medium after the first four determinations 38.95
Other N-free substances, reckoned as the difference . . . . . 9725

1 Loc. cit.

2 Centralbl. f. Bakteriol., 1901, xxx, 909,

FOOD RELATIONSHIPS OF BACTERIA 65

II. FATTY SUBSTANCE OBTAINED BY EXTRACTION WITH

CHLOROFORM, POSSESSES THE FOLLOWING

CHARACTERISTICS :

Melting point 46 C.

Acid number ' 23.08

Reichert-Meissl number 2.007

Hehner number 74 . 236

Saponification number 60.70

Ether number 36 . 62

Iodine number (according to Hubl) 9 . 92

III. THE FATTY SUBSTANCE OBTAINED BY EXTRACTION WITH
CHLOROFORM CONTAINS:

Per cent.

Free fatty acids 14.38

Neutral fats and esters of fatty acids 77.25

Alcohols separated from the fatty acid esters (with melting point

43.5-44 C.) 39.10

Lecithin 0.16

Cholesterin Not determined

Substances directly soluble in water 0.73

Substances soluble in water which are formed by the complete

saponification of the fatty substances 25 . 764

Inorganic Constituents. The most conspicuous inorganic element
found in the ash of bacteria is phosphorus, and the content of phos-
phorus, recovered as phosphoric acid, frequently reaches as high as
half the total ash weight. It is probable that a considerable part of
this phosphorus is combined with nucleic acid to form nucleo-protein.

4. Spores. The chemical composition of spores is not well deter-
mined, but the generally accepted theory is that they contain relatively
less water and consequently a greater proportion of proteins and ash.
Reinke 1 has suggested that the sporoplasm is an anhydride of the
cytoplasm of the vegetative cell. Sporulation implies that relatively
considerable amounts of water must be taken up by the spore sub-
stance in order to regain the proportion of this substance found in the
parent organism.

D. FOOD RELATIONSHIPS OF BACTERIA.

1. General. Food is any substance which a living organism may
utilize, either by making it a part of its living material or as a source
of energy. Food which is suitable for utilization by any organism must
contain all the elements necessary for the building up and maintenance
of that organism. Analyses of bacterial cells, which have been given
in preceding tables, show them to be made up of the same elements
as those of the higher plants and animals; viz., carbon, hydrogen,
oxygen, nitrogen, and phosphorus, together with smaller amounts

1 Quoted by Kruse, Allgem. MikrobioL, p. 57,

66 THE CHEMISTRY OF BACTERIA

of sodium, potassium, sulphur, calcium, and magnesjum. Foods to
be fully suitable for bacterial needs, therefore, should contain these
elements. It should be stated, however, that the food requirements
of bacteria vary within wide limits, but the above statements are
generally applicable.

2. Sources of Food. (a) Nitrogen. The nature of the compounds
in which nitrogen must be presented to bacteria as food varies greatly
among the different groups. The nodule bacteria found in the nodules
on the roots of many leguminous plants actually utilize atmospheric
nitrogen: nitrifying bacteria found chiefly in the soil derive their
nitrogen requirement chiefly from mineral salts which are oxidized
through their activities to nitrites and eventually to nitrates. From
this very simple source of nitrogen these bacteria are able to synthesize
the complex nitrogen-containing proteins of their bodies.

The majority of bacteria, including not only the saprophytic organ-
isms but most of those pathogenic for man, animals, and plants as
well, thrive in media in which nitrogen is presented to them as peptones,
albumoses, or even certain amino-acids; in other words, upon the pro-
ducts of protein digestion. The more strictly pathogenic organisms,
as the gonococcus, may require nitrogen in the form of highly specific
tissue proteins. Generally speaking, animal protein or its derivatives
is more easily utilized by bacteria than protein of vegetable origin.

(6) Carbon. The simplest carbon compound which occurs naturally,
CO 2 , cannot be used by bacteria, except certain nitrifying bacteria,
as a source of energy, for it is already fully oxidized. The carbon
of proteins and their derivatives, of carbohydrates, and of fats, on the
contrary, is readily utilizable by most bacteria. As a rule, hydro-
carbons of the aliphatic series are not attacked by the microorganisms,
but compounds containing oxygen as well as carbon and hydrogen are
better adapted for microbial food. Organic acids, as acetic acid,
aspartic, tartaric, and many oxy acids are utilizable by some bacteria.
The simpler alcohols can be used, but by very few bacteria. The
complex alcohols, like glycerin and mannite, on the other hand, are
available food materials for many.

The best nitrogen-free food compounds for microorganisms are the
carbohydrates, particularly those containing six and twelve carbon
atoms, the hexoses and bioses respectively. Carbohydrates containing
four, five, or any number of carbon atoms not a multiple of three are
usually not readily attacked by bacteria. Starches and cellulose are
not generally utilizable, although certain types of organisms, notably

FOOD RELATIONSHIPS OF BACTERIA 67

those found in the intestinal tracts of herbivora, appear to decompose
them very readily.

(c) Hydrogen. Hydrogen is readily obtained by microorganisms
from organic compounds containing available carbon, nitrogen, and
hydrogen, but not apparently from water.

(d) Oxygen. Oxygen is indispensable to the life of all living organ-
isms as a source of energy and for structural purposes. A few bacteria,
the obligately aerobic bacteria, can live only in the presence of free
oxygen; another small group, the obligately anaerobic bacteria, live
either in the absence of free oxygen or at best in the presence of minimal
amounts of it; more than minimal amounts of free oxygen act as
specific poisons to them. The majority of bacteria are facultative with
respect to their oxygen requirements; that is, they can either live
in the presence of free oxygen or derive their oxygen needs from
organic compounds, usually the carbohydrates or proteins.

(e) Inorganic Salts. Inorganic salts are used by bacteria almost
wholly for structural purposes. The requirement for mineral com-
pounds is very little, for these substances do not on the average make
up more than 7 to 10 per cent, of the solid matter of the bacterial cell.
The essential elements and the percentage of them found in the ash
of certain bacteria have been referred to previously, and it was stated
that the amount of inorganic salts found in the bodies of the bacteria
bore a rather direct relationship to the salt concentration of the media.
Of the inorganic elements, phosphorus is the most important, for it
makes up nearly 50 per cent, of the ash. Phosphorous in contra-
distinction to any other inorganic salt is absolutely indispensable to
bacterial growth. It is combined organically in nucleo-proteins,
glyconucleo-proteins, and nucleic acids, which form the greater part
of the protein of the bacterial cell.

CHAPTER IV.

BACTERIAL METABOLISM.

I. GENERAL.

II. THE NATURE OF BACTERIAL MET-
ABOLISM.

III. NITROGEN METABOLISM.

IV. CARBON METABOLISM.

V. QUALITATIVE CATABOLIC REAC-
TIONS OF BACTERIA.

A. In Media Containing Only

Utilizable Nitrogenous
Substances.

B. In Media Containing Both

Utilizable Nitrogenous
Substances and Utilizable
Carbohydrates .

VI. THE QUALITATIVE INFLUENCE OF
UTILIZABLE CARBOHYDRATES
UPON THE ELABORATION OF
PROTEOLYTIC ENZYMES.
VII. QUANTITATIVE MEASURE OF BAC-
TERIAL METABOLISM.
VIII. THE SIGNIFICANCE OF BACTERIAL
METABOLISM.

IX. FERMENTATION AND PUTREFAC-
TION.

I. GENERAL BACTERIAL METABOLISM.

Two distinct phases may be recognized in the life-history of a
bacterial cell; an anabolic or constructive phase, during which the
cell becomes morphologically complete; and a catabolic, vegetative,
or fuel phase, in which the mature organism reacts chemically upon
its environment to provide the energy (fuel) necessary for the main-
tenance of the cell. Chronologically, the anabolic phase precedes the
catabolic phase; that is to say, the bacterial cell must be morpho-
logically complete before it can bring about its characteristic energy
transformations; practically the two phases overlap somewhat.

The actual amount of material required for the anabolic phase of the
bacterial cell is very small, for the actual weight of the average
bacterium is but 0.000,000,0016 of a milligram, approximately (see
page 25). The structural phase is practically ended, aside from the
replacement of comparatively slight losses of substance incidental
to the elaboration of soluble enzymes or to additional requirements
for the formation of structural elements, such as capsules, when the
organism is morphologically complete. The waste incidental to the
utilization of material for purely anabolic needs is likewise very small
in amount, and the total environmental change attributable to the
purely constructive phase of bacterial metabolism is slight andordin-
arily disregarded. 1

Kendall, Jour. Med. Res., 1911, N. S. f xx, 140.

GENERAL BACTERIAL METABOLISM 69

The amount of material required for the catabolic (vegetative or
fuel) phase of the bacterial cell, on the contrary, is relatively large.
The energy requirement of cellular organisms varies rather with the
area of their surface than according to their actual volume; conse-
quently, very minute organisms, as bacteria, in which the surface is
relatively very great in comparison with their size, would require much
more material for energy purposes than for structural purposes. For
example, the total surface area of a million average-sized cocci (each
1 micron in diameter) would be approximately 3.1416 sq. mm.; the
weight of these organisms, assuming the specific gravity to be 1.030
(which is reasonably accurate), would be about 0.00054 mg. The
combined surface of all the cocci in an actively growing broth culture
of such organisms would be very considerable. It must be remembered,
however, that these figures do not carry any specific basis for the
measurement of bacterial activity in terms of chemical or physical
phenomena; they merely express in a very general manner the physical
basis for the apparent disproportion observed between the size of
bacteria and the amount of change they induce in their environment.

The energy phase commences theoretically when the cell is morpho-
logically complete, and it is a continuous process which ends only with
the death of the cell. It may be reduced to a minimum when the cell
enters upon a latent state of existence, as in spore formation; it is
greatest when the organism is growing in a favorable medium at the
optimum temperature, and it is restricted proportionately when
environmental conditions become unfavorable.

The life-history of a culture in which innumerable bacteria are
growing can not be sharply divided into the anabolic and catabolic
phases. During the first few hours after inoculation, however, the
anabolic aspect predominates; later the catabolic aspect predominates.
Thus, colon bacilli inoculated into dextrose broth fermentation tubes
do not produce gas in visible amounts during the first few hours of
incubation, although the medium gradually becomes turbid, due to the
rapid multiplication of bacteria. Somewhat later gas formation is
observed, and it then proceeds with considerable rapidity. The
production of gas is indicative of a period of great vegetative activity
in which large numbers of mature colon bacilli utilize the dextrose for
their energy requirements. Still later the production of gas ceases,
the activities of the organisms diminish, and the culture finally dies
out as waste products accumulate in sufficient amounts.

Those bacteria habitually pathogenic for man induce less striking
physical and chemical changes in their environment, as a rule, than

70 BACTERIAL METABOLISM

do the saprophytic types, as Theobald Smith 1 showed long ago. Thus,
typhoid bacilli are relatively inert culturally; they form no gas in
sugar media, no indol, and do not liquefy gelatin; on the contrary,
B. coli and even more strikingly B. proteus are characterized by strik-
ing cultural changes; B. coli produces deep-seated changes in protein,
resulting in the production of indol; it produces gas from sugar media,
but it does not liquefy gelatin. B. proteus behaves much like B. coli
in sugar media, but liquefies gelatin as well. These marked changes
in the composition of the medium, namely, the production of indol
from protein, the production of gas from sugar, and the liquefaction
of gelatin, are all phenomena associated with the vegetative or fuel
phase of bacteria.

H. THE NATURE OF BACTERIAL METABOLISM.

Chemically considered, the anabolic phase of bacterial activity is
one characterized by the synthesis of relatively simple substances,
chiefly nitrogen-containing, into the complex specific bacterial proto-
plasm through a series of synthetic reactions among which reductions
and condensations appear to be the more prominent. It is very
probable that many of these condensation reactions are hydrogenic
in nature; that is, two simpler molecules are united into one molecule
of greater complexity through the removal of hydrogen and oxygen
from them in the proportions to form water.

As simple illustrations: the formation of lactose from a molecule
each of dextrose and galactose,

C 6 Hi 2 O 6 + C 6 Hi 2 O 6 = Ci2H 22 Oii + H 2 O
Dextrose. Galactose. Lactose.

the formation of a polypeptid, glycyl-glycin, from two molecules of
glycocoll, 2

NH2.CH 2 .COOH + H.NH.CH 2 .COOH = NH 2 .CH 2 .CO.NH.CH 2 .COOH + H 2 O
Glycocoll. Glycocoll. Glycyl-glycin.

and the formation of the glyceride of a fatty acid from glycerin and
acetic acid may be cited,

CH 2 .OH + HOOC.CHa = CH 2 .O.O.CH 3

CH.OH + HOOC.CHs = CH.O.O.CHs + 3 H 2 O
I I

CH 2 .OH + HOOC.CHs CH 2 O.O.CH 3
Glycerin. Acetic acid. Triacetin.

1 Fermentation Tube, Wilder Quarter Century Book, 1893, p. 219. (See also Kendall,
Day and Walker, Jour. Am. Chem. Assn., 1913, xxxv, 1201-1249, for analytical data.)
Fischer, Ber. d. deutsch. chem. Gesell., 1906, xxxix, 530.

NITROGEN METABOLISM 71

The catabolic phase is essentially analytic; it is characterized
chemically by a series of reactions in which the cleavage of more
complex compounds to simpler ones with their simultaneous or sub-
sequent oxidation, involving the liberation of energy, is a noteworthy
feature. The catabolic phase is chiefly a series of oxidations of carbon
and hydrogen. (For illustrative catabolic reactions see infra, pp. 73, 76.)

m. NITROGEN METABOLISM.

Bacteria, like all known living things, contain nitrogen in their
substance, and nitrogen in some form is absolutely indispensable for
the building up of their structure. Nitrogen, in other words, is an
absolutely essential element in the constructive phase of the bacterial
cell. The form in which nitrogen must be presented to bacteria in
order to be utilizable by them varies with the kind of organism. The
nitrogen-fixing bacteria found on the roots of leguminous plants can
utilize the nitrogen of the atmosphere; some nitrifying bacteria can
utilize the nitrogen of ammonium salts. (These two groups of organ-
isms appear to be the only ones which can oxidize nitrogen.) Many
bacteria can obtain their nitrogen from amino-acids. The majority 1
of bacteria pathogenic for man and the higher animals are somewhat
more exacting in this respect and require more highly organized
nitrogen, as peptones and proteoses, while a small group of obligately
human pathogenic bacteria, as the gonococcus, grows only in media
containing nitrogen as it exists in the highly specialized protein of
human origin, at least during their first growth outside the human
body on artificial media.

he vegetative phase of bacterial metabolism is essentially a series
of oxidations of carbon and hydrogen; nitrogen can not be oxidized by
the great majority of bacteria, and consequently it appears to yield
little or no energy to them. When nitrogen-containing compounds
as amino-acids, peptones, albumoses, or proteins are utilized for the
energy requirements of these organisms, the nitrogen (amino nitrogen)
is usually eliminated from the ^mino-acid complex incidental to the
oxidation of the carbon and hydrogen; the nitrogen thus eliminated
appears in soluble form in the culture medium as ammonia. This
process is true deaminization. . Nitrates and even nitrites may be
sources of energy to many bacteria, usually, however, because of their
valuable oxygen content. To summarize, bacteria must have available
nitrogen for their structural needs,, but nitrogen, except for the nitrogen-
fixing and nitrifying bacteria, is not as a rule a source of energy to
them, because the great majority of bacteria can not oxidize it.

72 BACTERIAL METABOLISM

IV. CARBON METABOLISM.

Carbon is an important structural element for bacteria, and it is
equally indispensable as a source of energy, for the oxidation of carbon
is an important feature of the catabolic activity of the majority of
microorganisms. The reduced form in which this element is present
in amino-acids and other protein derivatives appears to be particularly
adapted for structural purposes; for fuel purposes it is less available,
possibly because of the necessity of introducing free oxygen into the
carbon complex to provide the requisite energy for the vegetative
activities of bacteria, as well as the additional amount of work required
to eliminate the nitrogen of the amino-acid molecule fdeaminization).
It is generally stated that bacteria with relatively few exceptions
fail to grow with their customary vigor in sugar-free media from which
free (atmospheric) oxygen is excluded; the relative absence of available
oxygen in such compounds would explain this phenomenon, in part
at least.

The carbohydrate molecule, which contains no nitrogen and in which
the carbon is already partially oxidized, can be utilized for fuel purposes
by most bacteria with less expenditure of energy for its preparation
than can be the case with most amino-acids, peptones, or proteins;
for this reason it is very probable that utilizable carbohydrate is
act6d upon by many bacteria in preference to protein carbon. In
this sense utilizable carbohydrate protects or shields protein or protein
derivatives from bacterial attack for their fuel requirements; it does
not protect protein from bacterial breakdown to supply their structural
requirements, however.

The net result of this selective protective action of carbohydrates
for protein is important because the amount of material required to
provkL* energy for the bacterial cell far exceeds the amount of material
required to build up the bacterial cell. The chemical transformations
incidental to the anabolic phase of bacterial metabolism are insignificant
in amount and ordinarily not noticeable; on the contrary, the chemical
transformations associated with the catabolic phase of bacterial
metabolism are relatively very considerable in amount; and the
nature and extent of those chemical reactions which are associated
with the transformation of material for energy are important not
only for the identification of bacteria, they collectively comprise the
important specific function of bacteria.

QUALITATIVE CATABOLIC REACTIONS OF BACTERIA 73

V. QUALITATIVE CATABOLIC REACTIONS OF BACTERIA.

The chemical changes observed in cultures of ordinary bacteria are
chiefly those associated with the breakdown of organic substances for
energy they are reactions of the catabolic phase of bacterial meta-
bolism. It should be again emphasized that the energy reactions the
catabolic reactions are those which are most profoundly influenced
by the composition of the nutritive substrate upon which the organisms
are grown.

A. Reactions of Bacteria in Media Containing Only Nitrogenous
Substances (Proteins or Protein Derivatives) Which are Utilized
for the Energy Requirements of Bacteria. Proteins are composed
of amino-acids, of which some seventeen are recognized. Bacteria
which decompose protein appear to act upon these amino-acids in the
last analysis, and several types of reaction are recognized at the present
time. Each kind of organism utilizes protein or protein derivatives
somewhat differently and characteristically, but in general one or more
of the following types of reactions are involved either successively or
simultaneously in the catabolism of these substances. The reactions
follow i 1

1 . R.CH 2 .CHNH 2 .COOH + H 2 = R.CH 2 .CH 2 .COQH 4- NH* Re-
ductive deaminization of amino-acid to fatty acid with the same
number of carbon atoms.

2. R.CH 2 .CHNH 2 .COOH + H 2 O = R.CH 2 .CHOH.COOH + NH 3v
Hydrolytic deaminization of amino-acid to oxy-acid with the same
number of carbon atoms.

3. R.CH 2 .CHNH 2 .COOH + O 2 = R.CH 2 .CO.COOH + NH 3 . Oxi-
dative deaminization of amino-acid to keto-acid with same number of
carbon atoms.

4. R.CH 2 .CHNH 2 .COOH-R.CH 2 .CH 2 .HN 2 + CO 2 . Carooxylic
decomposition of amino-acid to amine with one less carbon atom.

5. R.CH 2 .CH 2 .COOH -* R.CH 2 .CH 3 . + CO 2 . Carboxylic decom-
position of fatty acid.

6. R.CH 2 .CH 2 .COOH + 3O = CH 2 .COOH + CO 2 + H 2 O. Carbo-
xylic decomposition with the formation of a fatty acid with one less
carbon atom.

A few illustrations will indicate the nature of these changes in amino-
acids with the production of certain substances of clinical interest :
1. Formation of indol from tryptophan. Indol is a substance pro-

* See Kruse, Allgem. Mikrobiol., 505-536, for literature.

BACTERIAL METABOLISM

duced in the intestinal tract from tryptophan (an amino-acid found
in protein), chiefly by B. coli and B. proteus. The reactions through
which tryptophan is changed to indol by these organisms are as
follows. 1

\ CH 2 .CHNH 2 .COOH

+ H 2 =

Tryptophan.

CH 2 .CH 2 .COOH

V NH

(deaininization)
Indol propionic acid.

+ NHs Indol propionic acid
+ 3O =

\ _ CHz.COOH

+ CO 2

CHs

H 2 O->

Indol acetic acid.

C0 2

Skatol + 3 O

Skatol.

CO 2 + H 2 O

Indol.

Indol contains little or no energy for most bacteria, and it is left
as such in the culture medium or the intestinal tract. Indol is fre-
quently absorbed from the intestinal tract, but it has little or no
energy for the human body it is oxidized in the liver to indoxyl

and is excreted and appears
in the urine as indican

o
/

O - S - ONa

II
Q

B. coli, B. proteus, and other organisms which "form indol" utilize
the alanin radical of the tryptophan molecule (alpha amino propionic
acid) for energy, first eliminating the nitrogen (deaininization), then
oxidizing the carbon. The indol radical which is left is not a source
of energy; it can not be oxidized by these organisms, consequently
it' remains as such in culture media or is absorbed from the intestinal
tract.

Nencki, Sitzungsber. Wien. Akad., 1898, II Abt., xcviii, 412.

QUALITATIVE CATABOLIC REACTIONS OF BACTERIA 75
2. Production of phenolic bodies from tyrosine.

+ NH 3

Paraoxyphenyl propionic acid

+ 3 O =
CH 2 .CHNH 2 .COOH CH 2 .CH 2 .COOH

Tyrosine. Paraoxyphenylpropionic acid

(deaminization)

OH OH OH

C0 2 + H 2 -> + C0 2 + C0 2

Paracresol

CH 2 .COOH CH 3 + 3 O =

Paraoxyphenyl Paracresol. Phenol,

acetic acid.

Phenol is not oxidizable by bacteria, hence it remains as such
unchanged in the culture media. Phenol (or cresol) may be absorbed
from the intestinal tract, but it appears eventually in the urine as an
ethereal sulphate, precisely as indol appears in the urine as indican.
Indol and phenolic bodies are not found in cultures containing utiliz-
able carbohydrate the bacteria which produce indol and phenols
from tryptophane and tyrosine, respectively, can obtain their requisite
energy far more directly and economically from the sugar than from
the nitrogen-containing amino acid. Doubtless the same general
principle applies to the formation of these aromatic substances in the
intestinal tract.

3. Formation of amines from amino-acids by bacterial action.

(a) Cadaverin from lysine. 1

CH 2 .CH 2 .CH 2 .CH 2 .CH.COOH CH 2 .CH 2 .CH 2 .CH 2 .CH 2

I I - I I- +C0 2

NH 2 NH 2 NH 2 NH 2

Lysine. Cadaverin.

(6) Putrescin from ornithin. 2

CH 2 .CH 2 .CH 2 .CH.COOH CH 2 .CH 2 .CH 2 .CH 2

| | -'/ "! I +00,

NH 2 NH 2 NH 2 NH 2

Ornithin. Putrescin.

1 Ladenburg, Ztschr. f. phys. Chem., 1886, xix, 780.

2 Ellinger, Ztschr. f. phys. Chem., 1902, xxix, 334; Ber. d. deut. chem. Gesell., 1889,
xxxi, 3183; ibid., 1900, xxxii, 3542.

76 BACTERIAL METABOLISM

H C NH V H C NH V

\r*t \r | fi

II //^- II //^*

C N * = C N * + CO 2

I I

CH 2 CH 2

I I

CHNH 2 CH 2 NH 2

I
COOH

Histidine. Betaimidazoleethylamine.

According to Vaughan, 1 betaimidazoleethylamine is possibly the
active poisonous principle of the protein molecule. Recent investiga-
tions would suggest that its liberation in the intestinal tract as the
result of bacterial decomposition of protein there and its absorption
into the body may be associated with symptoms of considerable
severity. The substance is not formed as a product of bacterial
metabolism in media containing utilizable carbohydrates.

B. Reactions of Bacteria in Media Containing Both Utilizable
Nitrogenous Substances (Protein and Their Derivatives) and Carbo-
hydrates. Carbohydrates contain no nitrogen; consequently pure
carbohydrate solutions are not complete foods for bacteria, they
are important chiefly as sources of energy to them. Generally speak-
ing, carbohydrates containing two, four, five, seven or eight carbon
atoms are not readily fermentable by bacteria. Those containing
six carbon atoms, particularly dextrose, are most readily utilizable,
those with three carbon atoms, generally speaking, somewhat less
so. Bioses, containing twelve carbon atoms, and starches appear to
be hydrolyzed to sugars containing six carbon atoms before they are
finally oxidized.

The final utilization of sugars for energy by bacteria varies accord-
ing to the type of organism; the following qualitative reactions are
illustrative of some of the general types of decomposition usually met
with. It must be remembered that the exact quantitative utilization
of carbohydrates by bacteria and the nature and composition of many
of the intermediary products formed from them are still uncertain.

1. C 12 H 22 On + H 2 O = C 6 H 12 O 6 + C 6 H 12 O 6 .

Lactose. Dextrose. Galactose.

Hydrolytic cleavage of a biose to two molecules of hexose sugar.

2. C 6 Hi 2 O 6 = 3CH 3 COOH. Pure acetic acid fermentation.

3. C 6 H 12 O 6 = 2CH 3 CHOHCOOH. Pure lactic acid fermentation.

1 Protein Split Products.

QUALITATIVE CATABOLIC REACTIONS OF BACTERIA 77

4. C 6 H 12 O 6 = CH 3 .CH 2 .CH 2 .COOH + 2C0 2 + 2H 2 . Pure butyric
acid fermentation.

5. C 6 Hi 2 6 = 2CH 3 CH)H + 2C0 2 . Pure alcoholic fermentation.

6. 2C 6 H 12 O G + H 2 = 2CH 3 .CH#)H.COOH + CH 3 .COOH +
C 2 H 5 OH' + 2CO 2 + 2 + 2H 2 . The type of fermentation produced
by B. coli in dextrose broth. 1

The sugars containing six carbon atoms appear to be somewhat
more utilizable than their corresponding alcohols: thus, the Shiga
bacillus (B. dysenteriae) can not ferment mannite; it can, however,,
readily ferment dextrose. This would suggest that the aldehyde
group CHO is somewhat more readily attacked than the alcohol
group CH 2 OH , for mannite has no aldehyde group and dextrose
has an aldehyde group. The alcohols in general appear to be less
readily acted upon by bacteria than are the corresponding aldehydes or
even organic acids, provided the latter are not too greatly dissociable.

The products of fermentation of higher alcohols, as mannite, by
bacteria are somewhat different from those of the corresponding sugars
(aldoses). The chief points of difference, according to our present
knowledge, consist principally in the production of more alcohol
when the 'higher alcohols are utilized than when the corresponding
aldoses are concerned. This has been worked out satisfactorily for
certain bacteria, notably the colon and the typhoid bacilli, by Harden. 2
It is not definitely known for many other organisms. The gas-forming
bacteria, as a rule, produce more gas and more alcohol from the alcohols
of the Ce series than from their corresponding aldoses. This gas
formation appears to result from the decomposition of formic acid
by the activity of a specific enzyme, formiase, according to the equa-
tion HCOOH = CO 2 + H 2 O. 3 Thus, B. coli and related gas-forming
bacteria, according to this theory, produce the ferment, formiase,
while B. typhosus, which also produces formic acid from the decom-
position of dextrose, does not possess this ferment and consequently,
forms no gas in sugar solutions. Formic acid is, therefore, somewhat
prominently represented among the decomposition products of carbo-
hydrates by the typhoid bacillus, while formic acid is either not
present or present in small amounts in corresponding cultures of colon
bacilli. 4

The qualitative changes produced in fats and lipoidal substances
by bacteria are not well known.

1 Kruse, Allgemeine Mikrobiologie, p. 294.

2 Jour. Hygiene, 1905.

3 Franzen and Stuppuhn, Ztschr. f. physiol. Chem., 1912, Ixxvii, 129.
Clark, Science, November 7, 1913.

78 BACTERIAL METABOLISM

VI. QUALITATIVE INFLUENCE OF UTILIZABLE CARBOHYDRATES
UPON THE ELABORATION OF PROTEOLYTIC ENZYMES.

Certain bacteria, as for example B. proteus, characteristically pro-
duce proteolytic enzymes which rapidly dissolve gelatin by hydrolytic
cleavage. These enzymes are exo-enzymes; that is, they may be
obtained sterile and free from bacteria simply by passing gelatin
liquefied by their action through sterile unglazed procelain filters.
Although the bacteria which elaborated the enzymes are removed by
this filtration, the sterile filtrate still contains the active enzyme which
will liquefy considerable amounts of gelatin. The function of these
enzymes is to prepare the gelatin for assimilation by the proteus bacil-
lus: the gelatin is broken down by enzyme action to gelatin peptone
or even to polypeptids. The proteus bacillus does not produce soluble
gelatin-splitting enzymes in gelatin containing utilizable carbohy-
drate, although sugar-free gelatin contains them in considerable
amounts. These gelatinases, however, will liquefy sugar-gelatin quite
as readily as sugar-free gelatin, indicating that the enzyme itself
is not inactivated by the sugar, at least in the amount usually
employed, 1 per cent. The same phenomenon is observed in cul-
tures of the cholera vibrio and many other bacteria which liquefy
sugar-free gelatin. Extensive investigations by Auerbach, 1 and by
Kendall, Day and Walker 2 have shown that the gelatinase, which,
as has been noted, is produced only in sugar-free gelatin, although
it liquefies sterile sugar gelatin, prepares protein for utilization by
these bacteria for purely catabolic purposes; if the organisms have
access to utilizable carbohydrate the enzyme is not produced by
them, because they utilize the sugar, not the protein, under these
conditions as the source of their energy. These observations indicate
how fundamentally the metabolism of bacteria is influenced by the
nature and composition of the substrate upon which they are grown.

VH. QUANTITATIVE MEASURE OF BACTERIAL METABOLISM.

It is possible to measure the nitrogen metabolism of bacteria under
varying conditions with a very considerable degree of accuracy in
spite of the minute amounts of products involved. Such measure-
ments are not only indicative of the nature and degree of the decom-

1 Arch. f. Hyg., 1897, xxxi, 311.

2 Jour. Am. Chem. Assn., 1914, xxxvi, 1962.

QUANTITATIVE MEASURE OF BACTERIAL METABOLISM 79

position of purely nitrogenous substances by bacteria; they furnish
quantitative evidence of the extent of the utilization of carbohydrates
by bacteria in preference to nitrogenous substances for fuel (catabolic)
purposes; that is to say, such measurements evaluate the nitrogen
metabolism of bacteria in purely protein solutions, and their nitrogen
metabolism in media containing both protein and utilizable carbo-
hydrate.

Such determinations have been made for a large series of bacteria
by Kendall and Farmer, 1 and Kendall, Day and Walker. 2 The gen-
eral method followed is to measure the amount of ammonia (deamin-
ization) which appears in fluid cultures of bacteria under various
conditions of growth. The following table shows, respectively, the
change in reaction (to neutral red as an indicator in terms of T acid
or alkali per 100 c.c. media) and the increase in ammonia (milli-
grams per 100 c.c. media), as certain bacteria are grown for ten days
in plain and dextrose broth respectively. The broths are identical
in initial composition and reaction, except that the "dextrose broth"
contains in addition to the ingredients of the "plain broth" 1 per cent,
of chemically pure dextrose. All other conditions are exactly parallel.
The results are averages of several strains of the same organism in
various lots of media. It will be seen that B. alcaligenes, for example,
which ferments no sugars, produces an alkaline reaction (indicated
as " " in the table) both in plain and dextrose broth: the amounts
of ammonia in both media are nearly the same.

All the organisms which ferment dextrose produce less ammonia
in the dextrose medium than in the corresponding sugar-free medium,
although the numbers of living bacteria were found to be greater in
the former than the latter. The small amount of ammonia in the
dextrose broth appears to be largely the nitrogenous waste incidental
to the utilization of protein for structural purposes: the relatively
large amount of ammonia observed in the corresponding sugar-free
broths is the combined "structural waste" and the " deaminization"
incidental to the utilization of protein for their energy requirement.
The progressively pathogenic bacteria, as the diphtheria, typhoid
and dysentery bacilli, produce much Ies3 ammonia in sugar-free
media than do the same organisms in various lots of media. 3 (Kendall,
Day and Walker.)

1 Jour. Biol. Chem., 1912, xii, 13, 215, 219, 465; xiii, 63. Methods ^iven here.

2 Jour. Am. Chem. Soc., 1913, xxxv, 1201-1249.

3 Ibid.

80 BACTERIAL METABOLISM

Sugar-free broth. Sugar broth.

Ten-day observations. Reaction. 1 NH.s Reaction. 1 NH.s

B. alcaligenes -1.25 +3.50 -1.15 +5.30

B. dysenterise (Shiga) .... -0.30 +4.20 +2.80 0.00

B. dysenteries (Flexner) ... -0.25 +3.10 +2.45 0.00

B. typhosus -0.45 +5.40 +3.30 +0.60

B. diphtheria ...... 0.50 +3.10 +2.80 +1.05

B. of hemorrhagic septicemia . 0.20 +4.70 +2.25 +0.35

B. paratyphosus alpha and beta 0.10 +7.50 +3.90 +1.20

B. icteroides -0.10 +4.20 +3.80 +2.10

B. of hog cholera avirulent . . -1.25 +16.45 +3.70 +1.05

B. of hog cholera virulent . . -0.75 +8.40 +2.65 +1.05

B. of fowl cholera .... - .00 +13.65 +3.35 +0.70

B. of Morgan - .33 +29.50 +3.90 +29. 66 2

B. coli - .00 +24.40 +4.90 +0.35

B. cloacae - .20 +39.20 -0.30 +36. 40 2

B. proteus . ...,..- .98 +58.40 +3.55 +1.40

Sp. cholerse - .45 +62.80 +2.00 +0.70

Sp. of Finkler and Prior . . . -1.00 +27.30 +1.50 +0.70

Sp. of Metchnikoff . . . . -4.30 +41.30 +2.70 +0.70

B. pyocyaneus -1.85 +30.30 -1.33 +41.50

Streptococcus +0.70 +1.40 +5.00 +0.70

Staphylococcus -0.75 +38.70 +3.75 +0.70

Mic. tetragenus +1.00 +2.10 +3.00 +0.70

Mic. melitensis 0.10 +6.30 +3.50 +0.70

Vm. SIGNIFICANCE OF BACTERIAL METABOLISM, WITH SPECIAL

REFERENCE TO THE SPARING ACTION OF UTILIZABLE

CARBOHYDRATE FOR PROTEIN.

Considerable emphasis has been placed upon the sparing action
of utilizable carbohydrate for protein in the preceding pages. It now
remains to summarize the salient features of this aspect of bacteriology
and to indicate briefly by means of a few illustrations precisely how
a comprehension of the principles underlying bacterial metabolism
may be made use of in controlling, or at least influencing the action
of these microorganisms upon their environment. The examples
selected are chosen rather with a view of indicating the extreme range
of the subject than for completeness along any limited line of inves-
tigation.

]. The Composition of Bacteria. Experiments quoted previously
(page 60) show very clearly that the percentage of composition of the
bacterial cell varies according to the medium in which it is grown.
Particularly striking is the difference in nitrogen content when the
same bacterium is grown in media of the same nitrogenous composition
and reaction with and without the addition of utilizable carbohydrate.

1 Neutral red, = alkaline reaction, + = acid reaction.

2 These organisms can utilize 1 per cent, of dextrose without forming enough acid
to inhibit their growth; after the dextrose is used up they attack the protein for their
fuel needs hence the ammonia production in a medium containing utilizable sugar.
During the initial period when sugar is present, the ammonia value is very little, and
the reaction is acid.

THE SIGNIFICANCE OF BACTERIAL METABOLISM 81

2. The Recognition of Bacteria. The recognition of many kinds
of bacteria, as for example members of the intestinal group, depends
upon the reactions these organisms induce in various sugars. Thus,
B. alcaligenes ferments no sugars; B. dysenterise ferments dextrose
with the production of acid; B. proteus ferments dextrose and sac-
charose with the evolution of gas and the production of acid; B.
coli ferments dextrose and lactose with the evolution of gas and the
production of acid; B. coli coagulates milk, while B. proteus charac-
teristically peptonizes it. All of these reactions are explained per-
fectly upon the theory that utilizable carbohydrate protects protein
from bacterial breakdown. Thus, B. alcaligenes does not utilize
any carbohydrate; as is well known, it is carnivorous. B. dysenterise
can utilize dextrose, and consequently it produces acid in a medium
containing both protein derivatives and this sugar: similarly, B.
proteus and B. coli ferment dextrose and in addition a specific biose.
B. proteus, however, does not ferment lactose, hence it attacks the
protein of milk; while B. coli, which does ferment lactose, produces
an acid coagulation in milk: the acid resulting from the fermenta-
tion of the milk sugar (lactose) protects the proteins of the milk.
In each instance the organisms attack the utilizable carbohydrate
whenever it is present, in preference to the protein for their energy
requirements. If bacteria did -not habitually utilize carbohydrate
in preference to protein for their fuel needs, these fermentation reac 1 -
tions would be of no value whatsoever as diagnostic tests for these
various microorganisms.

3. Certain bacteria, notably B. proteus, produce active, soluble
(extracellular) enzymes when grown in sugar-free gelatin, that
bring about an energetic liquefaction of this medium, which becomes
alkaline in reaction. If the organisms are grown in dextrose gelatin
no liquefaction takes place; the bacilli produce CO 2 and H 2 as well
as acid in dextrose gelatin, using the sugar in preference to the protein
for their energy needs. The liquefied gelatin containing the soluble
gelatinase may be sterilized by passage through a Berkefeld filter,
thus removing all bacteria. The filtrate will liquefy sterile plain or
sterile dextrose gelatin, thus proving that the soluble enzyme, which
prepares gelatin for assimilation by proteus bacilli (and which is
only produced in a carbohydrate-free medium), acts specifically on
the protein irrespective of other substances which may be present.
In this instance the presence of utilizable sugar in cultures of living
proteus bacilli protects the protein (gelatin in the instance cited)

BACTERIAL METABOLISM

from bacterial attack, and inasmuch as proteus bacilli prepare gelatin
for assimilation through the action of a proteolytic ferment, the
ferment is not elaborated by them under these conditions. A pre-
cisely similar restriction of the development of gelatin-liquefying
ferments by utilizable sugars occurs in cultures of cholera vibrios and
other bacteria which habitually liquefy this medium. In each instance
the same explanation holds true.

4. Diphtheria bacilli do not produce their characteristic powerful
extracellular toxin in the presence of utilizable carbohydrate dex-
trose as Theobald Smith 1 showed several years ago. The toxin is
only formed in sugar-free media. In this case again the dextrose
shields the protein of culture media from attack by the diphtheria
bacillus, and consequently prevents the formation of toxin which
is apparently a true excretion produced incidental to the utilization
of protein for energy by these organisms. Similarly, tetanus and
Shiga bacilli fail to produce toxin in the presence of utilizable carbo-
hydrates.

5. Colon and proteus bacilli produce considerable amounts of
indol in sugar-free media, but no indol in the same media to which
utilizable sugar has been added. Here again the carbohydrate is
attacked by these organisms in preference to the protein. The fol-
lowing table summarizes briefly the salient features of the above
discussion :

Sugar- free media. 2 Dextrose media. 2

Nitrogen substance, Nitrogen substance,

Chemical composition of bacteria. per cent. per cent.

1. Pfeiffer bacillus 70.0 53.7

Pneumo bacillus 79 . 8 63 . 6

Rhinoscleroma bacillus . 76 . 2 62 . 1

2. Diphtheria bacillus

3. B. tetani ....

B. dysenterise (Shiga)

4. B. proteus

Sp. cholerse .

5. B. coli, B. proteus:

Odor ....
Reaction .
Products .

Sugar-free media.
Powerful extracellular toxin

of which on the average

0.005 c.c. kills guinea-pigs.
Powerful extracellular toxin

produced.
Toxin present.
Soluble, extracellular gela-

tinase formed.
Soluble, extracellular gela-

tinase formed.

Foul.

Strongly alkaline.
H2S, indol, phenols, am-
monia, etc.

Sugar media.

No toxin produced; several
cubic centimeters medium
fails to kill guinea-pigs.

No toxin produced.

No toxin present.
No gelatinase formed.

No gelatinase formed.

None.

Strongly acid.

H 2 , CO 2 , lactic acid.

1 Tr. Assn. Am. Phys., 1896.

2 Nitrogenous constituents and reaction precisely the same in both sugar-free and
sugar-containing media. The only difference is that the dextrose medium contains
1 per cent, of dextrose in addition. The organisms studied have, therefore, a choice
between protein and sugar for catabolic purposes.

FERMENTATION AND PUTREFACTION 83

DC. FEEMENTATION AND PUTREFACTION.

The terms "fermentation" and "putrefaction" have been confused
and even used synonymously- in bacteriological, chemical and even
legal nomenclature, but they represent essentially distinct and generic
types of bacterial activity. They indicate, or should indicate
respectively, microbic decomposition of two quite distinct types of
organic compounds, the carbohydrates and closely related nitrogen-
free compounds, on the one hand (fermentation), and nitrogenous
organic substances on the other hand, putrefaction. There are
substances intermediate in character between carbohydrates and
proteins, or fats and nitrogen-containing compounds in which it
would be difficult to predict a priori which term would be correct
glucose amine is such a substance. Glucose amine is an amino-
aldose, containing both nitrogen and carbohydrate groupings. Such
instances, however, are uncommon and do not militate against the
correctness of the general theory that fermentation and putrefaction
are distinct processes. 1

Fischer 2 has defined fermentation in the broad sense it should be
used in bacteriology, essentially in the following terms: "Fermenta-
tion is the biochemical decomposition of nitrogen-free compounds,
chiefly carbohydrates, by the action of microorganisms." Similarly,
putrefaction is defined as "The biochemical decomposition of nitro-
genous organic compounds by the action of microorganisms."

Fermentation and putrefaction are probably enzyme phenomena.

Transposing the sparing action of utilizable carbohydrate for
protein, which has been repeatedly emphasized in the preceding
pages, it may be stated that in the catabolic phase of bacterial metab-
olism "fermentation takes precedence over putrefaction," 3 meaning
by that that bacteria which can utilize carbohydrate derive their
energy requirements from the utilizable carbohydrate when they are
growing in media containing both carbohydrate and protein. The
results of this sparing action of utilizable carbohydrate for protein
have been indicated in the preceding pages, sections V-VIII, inclusive.

1 Kendall, Jour. Med. Research, 1911, N. S., xx, 140-144.

2 Vorlesungen iiber Bakterien, 1903, II Aufl., 206.

3 Kendall, Jour. Med. Research, 1911, N. S., xx, 140-144.

CHAPTER V.

SAPROPHYTISM, PARASITISM, AND PATHOGENISM.

I. DEFINITIONS AND LIMITS.
II. THE CYCLE OF PARASITISM.

III. THE CYCLE OF PATHOGENISM.

IV. DISTRIBUTION OF PARASITIC AND

PATHOGENIC BACTERIA IN NA-
TURE.

V. How PARASITIC AND PATHOGENIC
BACTERIA REACH MAN.

A. The Occurrence of Parasitic

Bacteria upon the Bodies of
Healthy Men and Animals.

B. How Pathogenic Bacteria

Reach the Body.

1. Air-borne Infection.

(a) Dust.
(6) Droplet.

2. Soil-borne Infection.

3. Water-borne Infection.

4. Food-borne Infection.

5. Animal Carriers.

(a) Direct Contact.
(6) Indirect Transfer.

Transfer.

(d) Intermediary Host.

6. Human Carriers.

7. Contact Infection.

8. Germinal and Prenatal

Infection.

C. Portal of Entry: Atria of In-

vasion.

1. Skin and Adnexa: Ear,

Eye. SubcutaneousTis-
sue, Tonsils, Salivary
Glands, Nasal Cavity,
Lungs.

2. Mucous Membranes:

Mouth, Stomach, In-
testines.

3. Geni to-urinary System:

Vagina, Uterus, Ure-
thra, Urinary Bladder
and Ureter, Kidneys.

D. Where Bacteria Multiply in

the Body.

E. Where and How Bacteria

Escape from the Body.
VI. BALANCED PATHOGENISM; EPIDEMI-
OLOGY.

I. DEFINITIONS AND LIMITS.

THE most conspicuous and important function of bacteria in the
economy of Nature is to maintain a continuity between the Animal
and Vegetable Kingdoms by restoring in utilizable form to the Plant
World the elements contained in the complex organic compounds
which comprise the dead bodies of plants, animals and their products.
Bacteria dissipate much of the energy accumulated in these dead
bodies and oxidize the elements contained in them to inorganic, fully
mineralized salts. These salts are resynthesized by the chlorophyll-
bearing plants through the energy of sunlight to carbohydrates,
proteins and fats, and in these complex combinations the elements
are again available for animal food.

The bacteria which live upon this dead organic matter, and whose
function it is to effect its degradation and ultimate mineralization,
are called saprophytic bacteria. They are specifically the most

DEFINITIONS AND LIMITS 85

numerous, chemically the most active, and economically the most
important members of the phylum Bacteriacese. They are rarely
pathogenic, that is, they rarely initiate disease in man or the lower
animals. Whenever they are found associated with morbid processes
their presence is usually to be explained on the ground that they are
secondary invaders.

A smaller group of bacteria are parasitic, that is, they exist upon
the bodies of living plants, animals or men. Many of them are rarely
met with in Nature far removed from their respective hosts. Their
activities are not usually in opposition to those of their host and their
presence is therefore unnoticed. They may become invasive, how-
ever, whenever the natural barriers, which ordinarily suffice to keep
them out, are impaired.

From the parasitic bacteria there has been gradually evolved a
small but formidable group of organisms, the pathogenic bacteria,
whose activities are in partial opposition to those of their host. The
pathogenic bacteria, like the parasitic bacteria, require a living host,
but they differ from the parasitic forms in that they actually invade
their hosts and induce progressive disease from host to host.

There are no sharply definable limits between these three groups
of bacteria, the saprophytic, parasitic, and pathogenic; the latter
appear to have arisen from the former by a process of evolution.
Certain general modifications in the general types of chemical activity
manifested by these groups are discernible, however, which are partly
the result and partly the cause of their change in environment as
they have passed from a saprophytic to a parasitic existence. Promi-
nent among these modifications and activities is a gradual decrease
in the intensity with which the parasitic and pathogenic bacteria act
upon their environment.

The essential function of the saprophytic bacteria in Nature is to
effect a rapid, deep-seated degradation of organic matter to simple
compounds; these organisms decompose a relatively large amount
of substance in a relatively short time. They are chemically active
and many of them form highly resistant spores which enable them
to survive prolonged periods of environmental vicissitude. The
habitually parasitic bacteria, on the other hand, which exist upon
the bodies of living animals, and the progressively pathogenic bacteria
which develop within the tissues of animals are not subjected to
extremes of temperature and food supply; they rarely or never form
spores. The chemical activity of these organisms is usually much

86 SAPROPHYTISM, PARASITISM, AND PATHOGENISM

less pronounced than that of the saprophytic bacteria. 1 Indeed,
intense chemical activity would be incompatible with their continued
parasitic existence, for the damage to their host would be insupport-
able. The parasitic and pathogenic bacteria do not, for example,
produce widespread liquefaction of the tissues, even when large
numbers of them are actually growing in the body of the host. The
growth of invasive organisms in the animal body is characterized by
subtle changes in the composition of the tissues of the host and the
development of these reciprocal reactions between host and parasite,
which collectively are included in the newly developed science of
Immunology.

It would appear, therefore, that in their evolution toward parasit-
ism, those bacteria which could thrive without producing deep-seated
and rapid degradation of proteins, that is to say, whose metabolism
approached more closely the intracellular metabolism of their host,
would be the more adaptable to a parasitic existence, and this is in
accord with what is known of the chemistry of these organisms.
Their metabolism approaches rather closely that of their host.

H. THE CYCLE OF PARASITISM.

The cycle of parasitism for bacteria whose life cycle is such that
but a limited excursion outside their host is possible for them and
this appears to be the case for the majority of organisms parasitic on
man consists of three separate and well-defined stages, as Theobald
Smith 2 has so clearly pointed out. They must first reach an appro-
priate host; secondly multiply at least temporarily thereon, and
thirdly escape to other suitable hosts. Each phase of this parasitic
existence must be exactly fulfilled, otherwise the cycle is broken and
that particular strain dies out. It is not surprising, therefore, that
the bacteria habitually parasitic for man are found variously upon
the surface of the body in the upper respiratory tract, the gastro-
intestinal tract, or upon the mucous surfaces which are in direct
communication with the exterior. Escape from the body of the host
to other hosts is readily accomplished from these positions. 3

Under special conditions, parasitic bacteria may actually invade
the body of the host and become, therefore, temporarily pathogenic.

1 Theobald Smith, Am. Med., October 22, 1904, viii; Kendall, Boston Med. and
Surg. Jour., 1913, clxix, 749.

2 Theobald Smith, loc. cit.

3 Theobald Smith, loc. cit.

THE CYCLE OF PATHOGEN ISM 87

Such an invasion is usually subsequent to a preexisting disease or to
local weakening of the tissues which under normal conditions suffice
to exclude these organisms. The disease produced by parasitic organ-
isms is usually non-specific in character and sporadic in distribution,
and ordinarily it does not attain epidemic proportions. The bacteria
which have penetrated into the tissues of the host are locked up there,
as it were, and their descendants cannot escape to other hosts, at least,
in numbers sufficient to perpetuate the invasive strain, for these
organisms have not perfected their pathogenic cycle. Parasitic
organisms, in other words, are " opportunists," as Theobald Smith
has admirably called them, rarely initiating disease, but usually able
to penetrate the body as secondary or terminal invaders. The colon
bacillus, for example, is an habitual parasite in the gastro-intestinal
tract of man and many animals. Under certain conditions it may
become invasive, causing cystitis, appendicitis, peritonitis, or other
inflammatory lesions, but it does not ordinarily become progressively
pathogenic for successive hosts, producing epidemics of cystitis, ap-
pendicitis or peritonitis. The staphylococcus is a common inhabitant
of the skin of healthy man. When the continuity of the epidermis is
destroyed, the organism may become invasive, causing furuncles,
osteomyelitis, or endocarditis. The pneumococcus is found in the
respiratory tract of many normal men, particularly in large cities,
where it exists as an "opportunist," ordinarily producing no harmful
effects, but frequently becoming invasive and producing a variety of
lesions when the general resistance of the host is lowered. 1 These
parasitic bacteria have not perfected their mechanism of entry into
the tissues of the host, and of escape from the tissues to the exterior,
consequently those strains which accidentally become invasive are
locked up in the body and, as a rule, either are overwhelmed by
thjeir host or perish with it. They are imperfectly pathogenic, in other
words.

m. THE CYCLE OF PATHOGENISM.

Habitually pathogenic bacteria those organisms which produce
progressive, specific disease from host to host actually invade the
living bodies of animals or man. This invasion may be direct* in which
event the microorganisms actually enter the tissues or body fluids

1 Recent studies by Cole and his associates indicate that the ordinary "mou^"
pneumococcus differs serologically from the strains found in the saliva of pneumonl^
cases. It is not improbable that similar serological differences may be demonstrated
in the group of the streptococci.

88 SAPROPHYTISM, PARASITISM, AND PATHOGENISM

and multiply there, or it may be indirect, in which instance their soluble
toxins alone are absorbed by the host. The cycle of pathogenism,
therefore, is more complex than the cycle of parasitism ; it necessitates
lodgement of the invading microbe on the body of the host, the location
and penetration of the necessary portal of entry (which involves an
initial skirmish between the organism and the non-specific natural
defences of the host), growth within the tissues of the host in the
presence of opposition there, escape from the tissues to the surfaces
of the host or to some channel in communication with the exterior
and, finally, the transmission of the organism, directly or indirectly,
to other suitable hosts. If the organism cannot force an entrance to
the tissues of the host, that is, if the natural defences of the host
suffice to keep out the prospective invader, the latter usually perishes
and no infection takes place; if the organism does penetrate the tissues
of the body, the invasion and growth of the microorganism leads to
disturbances of structure, function or composition of the host, which
are abnormal and inimical to his well-being. The production of disease,
therefore, depends ordinarily upon the ability of the microorganism
to multiply in the tissues or the body fluids of the host; bacteria which
cannot force an entrance into the tissues of the host, multiply there
and escape to the exterior and eventually to other susceptible hosts
do not produce progressive disease.

The nature and extent of the disease produced depends upon several
factors: (1) the kind of microorganism; (2) the number of micro-
organisms; (3) their ability to locate and force an entrance to the
tissues of the body (their virulence, in other words) ; (4) the location
and extent of their multiplication in the tissues of the host; (5) the
response of the tissues of the host to this invasion, and (6) the nature
and extent of the secondary, specific defense of the host in response to
the invasion.

The contagiousness of a disease depends upon the ability of the
invading organisms to escape from their host in sufficient numbers to
infect new hosts and to survive environmental vicissitudes until new
hosts are reached. A few examples will indicate the principal vari-
ants of the pathogenic cycle commonly met with among progressively
pathogenic bacteria.

The tubercle bacillus ordinarily gains entrance to the host through
the air passages. The organisms pass through the alveoli of the lungs,
set up infection there, and gradually are shut off from communication
with the exterior through the formation of the tubercle. After a

DISTRIBUTION OF PARASITIC AND PATHOGENIC BACTERIA 89

longer or shorter time, these tubercles eventually break down, typically
into the air passages and discharge there large numbers of tubercle
bacilli. These are coughed up by the patient and are eliminated from
the body, usually in enormous numbers, by droplets and in the sputum.
Pulmonary tuberculosis is typically a chronic, focal disease. The
perpetuation of the tubercle bacillus is assured through their elimina-
tion from the diseased body in enormous numbers through long periods
of time, their ability to resist desiccation, and the relative directness
with which they reach other hosts.

The typhoid bacillus gains entrance to the body through the mouth
and the intestinal tract. The organisms penetrate the intestinal
mucosa, develop in the internal organs, particularly the spleen, and
after a rather definite excursion in the tissues of the* body, enter into
the intestinal tract again, either through ulcers or the gall-bladder or,
occasionally, they appear in the urine. They are eliminated from the
body in great numbers, either with the feces, or less commonly, the
urine, and they gain access immediately to other subjects through
direct contact or more or less indirectly through water or food, in
sufficient numbers to set up infection in at least some of them.

The gonococcus is transmitted directly by contact. Occasionally
the infection may be somewhat less direct, involving the conjunctiva.

The plague bacillus may be transmitted from host to host,. either
directly in the case of pneumonic plague, where great numbers of
plague bacilli are coughed up from the lungs of one patient and trans-
mitted through inhalation to other patients, or somewhat more
indirectly, as is the case in bubonic plague. Bubonic plague appears to
be a true septicemia; the plague bacilli circulate, at least temporarily,
in the blood, and they are removed from the blood of one patient and
transmitted to another patient (either man or rat) through the agency
of the flea, which acts potentially as an hypodermic syringe, as it were,
in this instance. Plague bacilli are locked up in the tissues of the host
and were it not for the agency of a suctorial insect, as the flea, bubonic
plague would almost certainly disappear, because the organisms have
not perfected for themselves any mechanism of escape from one V)st
to the other.

IV. DISTRIBUTION OF PARASITIC AND PATHOGENIC BACTERIA

IN NATURE.

It has been shown in previous sections that comparatively few, if
indeed any, of those bacteria habitually parasitic or pathogenic for

90 SAPROPHYTISM, PARASITISM, AND PATHOGENISM

man are found in Nature far removed from rather intimate association
with their hosts. This is in accordance with the fact that few, if any,
of these organisms are provided with spores which would enable them
to survive exposure to long periods of conditions unfavorable to their
growth. It is true, however, that some, at least, of these organisms,
as for example, the typhoid bacillus, can survive for longer or shorter
periods of time in the soil, particularly if it be frozen, or in water, for
days or even weeks. There is little evidence that these bacteria multiply
extensively outside the body; on the contrary, they tend to die off
rather rapidly. In any event, their existence depends upon their
reaching a suitable host again within a comparatively brief period.

There- are a few spore-forming bacteria which occasionally infect
man when associated conditions are favorable for them. Of these
the bacillus of lockjaw, B. tetani; of botulism, B. botulinus; the
gas bacillus, B. aerogenes capsulatus; and the anthrax bacillus are
well-known. These organisms are not habitual parasites, however;
they are "saprophytic opportunists." That is, they could in all
probability exist if man were eliminated from their environment.

V. HOW PARASITIC AND PATHOGENIC BACTERIA REACH MAN.

A. The Occurrence of Parasitic Bacteria upon the Bodies of Healthy
Men and Animals. The continual exposure of the skin of man to his
environment makes it almost inevitable that microbes shall collect
there. It is quite probable, however, that the large number of micro-
organisms which reach the skin are not only non-pathogenic, they are not
even habitually parasitic. Most of them are found there only trans-
iently. Certain organisms, however, occur among these adventitious
microbes, which appear to be habitual parasites, and many of these
bacteria, under certain conditions, produce disease. Of these, Staphy-
lococcus aureus and albus and Streptococcus pyogenes are almost
invariably present not only on the skin, but on the exposed mucous
membranes, particularly those of the nose and throat. The influenza
bacillus, diphtheria bacillus, the pneumococcus, and even the tubercle
bacillus, meningococcus and other organisms may also be occasionally
found, particularly in the nose and throat of healthy men. The
occurrence of these organisms is readily explained; the secretions of
the nose and throat, as well as that of the skin are excellent culture
media for these organisms, which collect at these sites and grow upon
the various secretions and desquamated cells.

PARASITIC AND PATHOGENIC BACTERIA 91

The majority of these organisms, however, particularly the coccal
forms, as the staphylococcus, streptococcus and pneumococcus are to
be regarded as "opportunists"; they do not of themselves initiate
disease, as a rule. They are to be regarded rather, as Theobald Smith
has called them, "organisms of the diseased state/' because of their
invasion of the bojdy secondary to other, intercurrent diseases. Even
the tubercle bacillus and the diphtheria bacillus, particularly the
latter, have been found in the mouths of men who apparently have
had neither tuberculosis nor diphtheria, yet these organisms appear
to be virulent when tested in the usual manner and presumably might
be able to incite disease whenever conditions favor their entrance to
the tissues of the body. Theoretically at least, people who harbor
these organisms are potential sources of danger to others. Even the.
internal organs of healthy individuals may contain parasitic bacteria
without harm, although these organisms naturally are not present in
large numbers. Tubercle bacilli have been found occasionally in
lymph glands in normal man and in cattle. Intestinal bacteria also
occur not infrequently in the apparently healthy tissues of the body.
In rare instances, B. coli may be present in the urinary bladder without
causing noteworthy symptoms.

B. How Pathogenic Bacteria Reach the Body. The manner in
which bacteria of the "opportunist" type reach the body has been
considered above. It is now necessary to consider the manner in which
bacteria which cause progressive disease from man to man reach
the body.

1. Air-borne Infection. Bacteria which cause progressive disease,
particularly of the respiratory tract, are discharged from the diseased
body principally through the mouth and nose and find lodgment in the
environment of the patient through the medium of the air, from whence
they settle upon various substances, as food, clothing, and walls and
floors of rooms. These bacteria probably do not proliferate to any
extent outside of the body, but they resist drying and may remain
fully virulent for considerable periods of time and potentially able to
infect a certain proportion of those individuals who may be exposed
to them.

These air-borne infections are transmitted in at least two rather
distinct ways: (a) by dust, and (6) by droplet infection.

(a) Organisms which are transmissible through dust must first
of all be able to survive considerable periods of drying. The larger
particles of dust to which bacteria may become attached soon settle

92 SAPROPHYTISM, PARASITISM, AND PATHOGENISM

from the air, but smaller particles may remain suspended for some
time, depending on the velocity of air currents and the nature, size
and shape of the particles. Dusting and sweeping in rooms naturally
stir up particles which have settled from the air, and even larger par-
ticles may be resuspended in this way. Tuberculosis has frequently
been suspected to have been transmitted through the inhalation of
infected dust particles, that is, particles of dust which have dried
tubercle bacilli adhering to them. Careful investigation has shown that
houses in which careless consumptive patients have lived have been
responsible for the transmission of tuberculosis. The ward-room of
a battle ship is known to have become infected with tubercle bacilli
early in its career and at least two successive details of officers con-
tracted tuberculosis in this place. Guinea-pigs exposed on the floor
of these so-called tuberculous rooms are quite frequently successfully
infected with the tubercle bacillus.

The extent to which dust dissemination is a factor in transmitting
disease, however, is not at all definitely known. It must be emphasized
that the transmission of disease through dust is not necessarily a
very direct one, because the inciting organisms may pass a very con-
siderable period of time in dust before they reach a favorable host.
In this sense, transmission of disease by dust is a relatively latent one.

(6) Droplet Infection. Fliigge 1 and his pupils were the first to
demonstrate that minute droplets of spray may be eliminated from the
mouth during talking, sneezing and coughing. These droplets are
frequently carried through the air for some distance, even as much as
ten meters in a quiet room. Usually the more minute particles remain
suspended in the air for some time. The possibility of droplet infection
has been definitely proven in the following manner: Agar plates
containing sodium carbonate are placed at various heights and dis-
tances from the experimenter, who places in his mouth a solution of
phenolphthalein and then talks in a natural manner, expelling droplets
containing phenolphthalein during his speech. This dye is transmitted
with the droplets until they reach the agar plates, where bright red
spots are produced which are very readily observed. In like manner,
cultures of B. prodigiosus placed in the mouth will infect agar plates
at similar distances.

The transmission of disease by droplet infection may be, and fre-
quently is, a very direct one. Bacteria which are air-borne or borne
by droplets may remain alive for several weeks in indirect sunlight,

1 Ztschr. f. Hyg., 1897, xxv, 179.

PARASITIC AND PATHOGENIC BACTERIA . 93

but all of them are readily killed if they are exposed to direct sunlight.
The virus of whooping-cough, mumps, measles, influenza, cerebro-
spinal meningitis, pneumonic plague, tuberculosis, the exanthemata,
the diphtheria bacillus, and possibly the pneumococcus may be spread
in this manner. Air-borne infections probably rarely take place
in the open air where the sunlight is strong. This does not apply
to droplet infections where one individual coughs, talks or sneezes
directly into the face of another. Air-borne infections, particularly
droplet infections, are potentially common where overcrowding occurs,
as in tenements, public gatherings, railway trains, schools, and factories.

2. Soil-borne Infections. Those bacteria which are occasionally
pathogenic for man and produce sporadic disease in man, and whose
habitat is the soil, are for the most part spore-forming organisms. They
commonly enter the body through wounds. Of these the bacillus of
tetanus, malignant edema, symptomatic anthrax, of anthrax, and the
gas bacillus are the best known but, with the exception of the latter,
they are not habitually human parasites. Of those bacteria which
are habitually pathogenic for man, typhoid, cholera, paratyphoid
and probably dysentery may be soil-borne, but ordinarily infection
with these organisms does not take place through the soil.

3. Water-borne Infection. The viruses of excrementitious diseases
typhoid, paratyphoid, dysentery, and cholera are not infrequently
transmitted from man to man through contaminated water. Feces
containing these organisms get into water supplies, reach man again,
incite disease in man, again escape in the feces and reenter water
courses, thus being recirculated. The cycle may be somewhat more
complex, as for example, when typhoid dejecta are thrown upon the
ground and are eventually washed directly into water supplies and
thus reach man again.

4. Food-borne Infection. A considerable number of pathogenic
bacteria may reach man through food, although food which is infected
is usually rendered so through the handling of it by man. Milk is
probably the most common food thus to be infected and it is par-
ticularly dangerous for two reasons. In the first place, its opacity
makes it difficult to distinguish foreign substances which may be in it;
and again, it contains all the elements which are necessary for the
food of man and incidentally for the majority of bacteria. Scarlet
fever, diphtheria, tuberculosis both human and bovine, Malta fever,
epidemic sore throat or tonsillitis, typhoid, dysentery, foot-and-mouth
disease, many diarrheas of children, milk sickness, and the organisms

94 SAPROPHYTISM, PARASITISM, AND PATHOGENISM

of cholera infantum, and, rarely, Asiatic cholera as well, all may be
transmitted from milk.

Shell-fish, particularly oysters, have been known to transmit enteric
diseases. This has been due, in the past, largely to their exposure
in the estuaries of rivers where sewage flowed freely over them.
Typhoid bacilli enter the mantle cavity of the shell-fish, remain alive
there and enter the digestive tract in a viable state when the shell-
fish are consumed in an uncooked condition.

Meats, particularly from beef and swine, have been known to transmit
paratyphoid fever, botulismus (sausage poisoning) and meat-poisoning
as well. There is, in addition, a group of cases with somewhat insidious
symptoms, which are probably due to the consumption of food, par-
ticularly meat, which has been decomposed by saprophytic bacteria.

5. Animal Carriers. The microbic diseases which are transmissible
to man from animals and from man to man by animals are varied
in character. They comprise protozoan and bacterial infections and
the so-called " filterable viruses." Of these diseases, comparatively
few are common to man and animals. Microorganisms may be trans-
mitted to man by animal carriers in at least four distinct ways:

(a) By direct contact.

(b) By indirect transfer.

(d) By intermediary hosts.

(a) Direct Contact. The transfer of glanders from the horse to
man, of anthrax from cattle and sheep to man and of hydrophobia
from dogs to man represents direct transfer of the virus from the sick
animal to the well man. Other diseases are thus transmitted, but the
examples given suffice for illustration.

(b) Indirect Transfer. Insects are common carriers in the indirect
transmission of the virus of disease from man to man. Flies are known
to have carried typhoid bacilli from typhoid dejecta to milk or other
food, which in turn has been consumed by man, resulting in infection.
The same insect, doubtless, when conditions are favorable, can and
does carry other enteric bacteria paratyphoid, dysentery and even
cholera organisms. It is very probable that other insects also par-
ticipate in the indirect transmission of bacteria. Acute conjunctivitis,
particularly that form which is prevalent in Egypt, is supposed to
be spread in this manner.

(c) Mechanical Transfer. Suctorial insects are known to transmit
the viruses of certain diseases which circulate in the blood stream of

PARASITIC AND PATHOGENIC BACTERIA 95

animals to man, incidental to feeding. Thus, the flea transmits the
plague bacillus from rat to man, from man to man, and possibly from
man to the rat. The louse similarly spreads the virus of typhus from
man to man. In the instances cited the insect is probably not a true
intermediary host, for the virus does not necessarily multiply in the
insect, nor does the virus undergo any essential transformation, so
far as is known, in the insect. Nevertheless, the transmission of the
viruses of these diseases bubonic plague and septicemia, for example,
depends upon the agency of suctorial insects for their passage from
host to host. Other insects also transmit disease, but the evidence
in a majority of instances is somewhat less definite than the cases
cited.

(d) Intermediary Hosts. Certain insects, notably mosquitoes,
transmit disease from man to man only after the virus has passed
an extracorporeal cycle in the extrinsic host the mosquito in this
instance. Thus, Anopheles transmits malaria from man to man and
Stegomyia fasciata, or as it is now called, Aedes calopus, transmits in
similar manner, the virus of yellow fever. Transmission in these cases
is through the female insect and a definite interval (latent period)
must elapse between the time of biting the patient and the time when
the mosquito becomes infective to the non-immune host.

6. Human Carrriers. Individuals who are apparently healthy
occasionally harbor within their bodies (in free communication with
the exterior, however, either through the respiratory tract, the gastro-
intestinal tract, the urinary tract or the skin) bacteria which are
capable of inciting disease in others. Such individuals are known as
bacillus carriers; frequently they eliminate these pathogenic bacteria
in large numbers.

The bacillus carrier may or may not give a history indicating
recovery from an infection of the specific organism which he "carries."
Bacillus carriers may be temporary carriers, in which event they harbor
the pathogenic bacteria for but a few weeks; or they may become
habitual carriers, in which case the organism may be excreted for
considerable periods of time, even years. The excretion may be
constant or intermittent.

The typhoid bacillus is a common organism to be thus carried. It
appears to localize eventually in the gall-bladder or the bile ducts, less
commonly in the urinary bladder, and it may appear occasionally
in large numbers in the feces or urine of the carrier. Women are more
commonly found to be typhoid carriers than men. Similarly, para-

96 SAPROPHYTISM, PARASITISM, AND PATHOGENISM

typhoid, dysentery and cholera organisms may be excreted in the feces
through long periods of time, rarely or never, however, in the urine.
Slowly progressing focal diseases, as pulmonary tuberculosis are,
in a sense, spread by carriers, for the patient may survive for years,
excreting daily large numbers of tubercle bacilli. The line of demarca-
tion, in other words, between the human bacillus carrier and the
patient in whom a focal disease is chronic for long periods of time is
not sharply circumscribed.

7. Contact Infection. The direct transmission of bacteria from man
to man is well exemplified in the venereal diseases, gonorrhea and
syphilis, which are usually transmitted by direct contact. Diseases
of the respiratory tract, as tuberculosis, diphtheria and whooping-
cough, may be transmitted directly from patient to patient by kissing,
swapping chewing gum, by eating utensils, etc. Soiled fingers may
transmit the typhoid bacillus from a typhoid patient to other indi-
viduals. All of the excrementitious diseases may be spread in a
similar manner, under certain conditions.

8. Germinal and Prenatal Infection. True germinal infection implies
that a disease-producing microorganism is carried by the ovum or
spermatozoa and incorporated in the embryo prior to its development.
This method of transmission is not definitely worked out, although it
has been claimed that syphilis may be thus transmitted by the male
to the ovum in utero, the mother remaining uninfected by the disease.

In prenatal infections the organisms must pass the placental
barrier. This implies that the fetus becomes infected directly from the
maternal blood stream, or by continuity of growth of the organisms
through the placenta. The placental form of infection is not conceded
by all observers, but it is reasonably certain that congenital syphilis
may be contracted thus. Smallpox, measles, dysentery, various
pyogenic infections, and, rarely, pneumonia are occasionally said to be
prenatally transmitted to the fetus. With respect to tuberculosis,
there is difference of opinion. A very few cases are on record in which
prenatal infection seems almost certainly to have taken place, for the
newborn infant exhibited lesions which were so far advanced that no
other explanation than prenatal infection suffices to explain them.

C. Portal of Entry; Atria of Invasion. The bacteria which cause
infection in the human body may be provisionally divided into two
great groups: those of exogenous origin, which are not habitual
parasites of man; and those of endogenous origin, which are habitual
parasites of man.

PARASITIC AND PATHOGENIC BACTERIA 97

The girat majority of specific microbic diseases (in contradistinction
to non-specific inflammations) are incited by bacteria of exogenous
origin. These organisms must enter the host directly through their
respective appropriate atria to produce characteristic disease. For
example, the typhoid bacillus only causes typhoid fever when the
organism is swallowed and enters the body through the intestinal
tract. Infection of a skin- wound with typhoid bacilli will not result
in typhoid fever. Similarly, cholera vibrios do not produce the disease
cholera unless they enter the body through the gastro-intestinal tract,
although if cholera vibrios are introduced through the skin in experi-
mental animals they tend to migrate toward the intestinal tract, thus
suggesting a special affinity for the intestinal tissues. Pathogenic
bacteria of exogenous origin produce in general, progressive specific
disease from man to man. Bacteria of endogenous origin, on the
other hand those which occur habitually as " opportunists" on the
surface of the body or on mucous membranes opening to the exterior
ordinarily exist as harmless parasites. They may, however, and
occasionally do, become invasive, inciting local or generalized inflam-
matory reactions as a rule, rather than well-defined clinical syndromes
which are frequently so characteristic of infections with exogenous
pathogenic bacteria. The bacteria of the "opportunist" type do not
ordinarily gain entrance to the tissues of the body through sharply-
circumscribed atria and the disease they produce is usually not epidemic
in character.

1. Skin and Adnexa The intact skin is a natural barrier which
protects the underlying tissues of the body from bacterial invasion.
Its free exposure to the environment suggests that a great variety of
organisms find lodgment upon it from time to time; a majority of
these organisms are harmless, and probably transient saprophytes
which come and go irregularly. The moisture and excretions, how-
ever, appear to favor the limited development of a few types of bac-
teria, mainly those of the coccal group, which occur with sufficient
regularity to be regarded provisionally as habitually parasitic bacteria.
Of these, the pyogenic cocci are usually the most numerous; they
exist as "opportunists" on the surface of the skin or penetrate into
hair follicles and the ducts of the cutaneous glands, ordinarily, however,
without becoming invasive so long as the continuity of the skin is
maintained. Abrasions and cuts furnish a portal of entry to the sub-
cutaneous tissues, in which these parasitic bacteria frequently set up
inflammatory reactions. Friction may actually force them through

98 SAPROPHYTISM, PARASITISM, AND PATHOGEN ISM

the intact skin. The plague bacillus and certain types of staphylococci
are said to pass through the skin occasionally in this manner.

Streptococci and staphylococci are the more common habitual
bacterial parasites found on the skin. Staphylococcus epidermidis
albus (Welch), a variant of Staphylococcus pyogenes albus, is a particu-
larly common factor in the causation of the troublesome, but relatively
benign stitch abscesses which frequently develop where sutures are
introduced through the skin.

The damaged skin is the usual portal of entry for spore-forming
bacteria as well as the cocci mentioned above. Spores of the bacilli
of tetanus, anthrax, symptomatic anthrax, malignant edema and the
"gas bacillus," (B. aerogenes capsulatus, Welch) may pass to the
underlying tissues through abrasions of the skin and cause either
localized infections or widely distributed lesions. Even so insignifi-
cant an abrasion as an insect bite may furnish the necessary atrium
for infection. The umbilicus of the newborn furnishes a portal of
entry for certain bacteria; particularly severe is the infection of the
stump of the umbilicus with B. tetani, causing that very fatal "tetanus
neonatorum" which has been so common in the tropics in the past.
"Contused wounds and compound fractures are particularly dangerous;
the inflamed tissues furnish anaerobic conditions particularly favoring
the growth of anaerobic bacteria, as the tetanus and gas bacilli.
Clean-cut wounds are usually less liable to infection with anaerobic
bacteria. The free flow of blood with its bactericidal properties
washes out many bacteria, inhibits the growth of residual microbes,
and by virtue of the clot which soon seals the wound prevents the
entrance of other organisms.

The sebaceous secretions, particularly of the axilla and external
genitalia, are good culture-media for certain acid-fast bacteria, par-
ticularly B. smegmatis. The cerumen of the external ear is frequently
infected with Micrococcus cereus flavus, and the puncture of the
tympanic membrane may lead to direct infection of the middle ear
from the outside, with this or other organisms. Infection of the
middle ear may also take place directly through the Eustachian tube.
The blood and lymph may also deposit bacteria in the middle ear.

The conjunctiva, by virtue of its very exposed position, must
receive bacteria upon it very frequently. Its polished surface and
the mechanical cleansing by the flow of tears (which do not possess
germicidal properties) usually suffice to remove adventitious bacteria
and to prevent bacterial development under ordinary conditions. The

PARASITIC AND PATHOGENIC BACTERIA 99

conjunctival sac, which receives the washings from the conjunctiva,
is probably the recipient of many bacteria; of these B. xerosis occurs
with sufficient regularity in the conjunctival sac to be regarded as a
normal inhabitant. The pneumococcus is also found there. These
organisms are "opportunists," occasionally causing severe acute
conjunctivitis, although usually they are benign. Certain bacteria
affect the conjunctiva fairly readily. Among these organisms, the
gonococcus is particularly troublesome, causing a most severe inflam-
mation. Ophthalmia neonatorum, a gonorrheal infection of the con-
junctivse of the newborn of infected mothers, has been in the past
a most common cause of blindness. It has been claimed that the
meningococcus may occasionally pass from the eye through the tear
duct to the nasal cavity, and from there to the meninges.

Subcutaneous Tissue.- Many bacteria, particularly exogenous
pathogenic bacteria, do not develop in the subcutaneous tissues, as
for example, the majority of those organisms which induce specific pro-
gressive disease from man to man such as typhoid and cholera organ-
isms. On the other hand, many of those bacteria which are habitually
parasitic on the skin may produce infections of the subcutaneous
tissues which vary in severity from mild inflammations to severe
cellulitis. The staphylococci and streptococci are among the more
important of this type.

Tonsils. The crypts of the tonsils afford mechanical protection
to bacteria which gain access to them and the secretions and tissue
undoubtedly provide the necessary nutritive elements, consequently
it is not surprising to find many types of bacteria in them. Staphy-
lococci are almost invariably present and streptococci, particularly
non-hemolytic varieties, are very common. The tonsils, which are
in very direct communication with the lymphatic system, are impor-
tant atria of invasion, particularly for streptococci, and many cases
of low-grade infections of the body appear to have originated from
the passage of bacteria through the tonsils to the tissues of the body.
The extent to which the normal tonsils destroy bacteria their value
in the non-specific initial defense of the body against bacterial invasion
in other words is not clearly established. Generally speaking, how-
ever, the tonsils appear to bear the brunt of attack in certain diseases
and they are of undoubted importance in shielding the body from
invasion through the lymphatic tract by directly holding back these
bacteria. The promiscuous removal of tonsils, particularly in the
young, has no justification from available knowledge. The removal
of diseased tonsils is quite a different matter.

100 SAPROPHYTISM, PARASITISM, AND PATHOGENISM

Salivary Glands. The salivary glands of the mouth are sometimes
invaded by bacteria.

Nasal Cavity. Large numbers of bacteria, indeed practically all
known bacteria may at one time or another gain access to the nose
through the inhalation of air containing dust, by droplet infection,
from the tear ducts, and in other ways. The air which is inhaled is
freed from bacteria before it enters the trachea, largely during its
tortuous passage over the turbinates; the moist surface of the nasal
mucosa effectively arrests the progress of bacteria, which adhere to
it. The constant secretion of mucus encloses many of these organ-
isms, which are removed mechanically with the mucus. There is no
evidence that the nasal secretions are germicidal. The permanent
nasal flora is very limited, however. The pseudodiphtheria bacillus
is very frequently found there and pneumococci, streptococci and
staphylococci are relatively common. The true diphtheria bacillus
is found in the nasal cavity of about 1 per cent, of healthy individuals.

Lungs. The expired air in quiet, normal breathing is sterile:
also, the inhaled air is practically sterile before it reaches the bronchi,
for the moist tortuous passages of the nasal cavity mechanically
retain bacteria; the same mechanism prevents the expulsion of bac-
teria during exhalation, unless the breath is expelled forcibly either
through the nose or mouth. Bacteria leave the nose or mouth in
expired air only when the expiration is forcible enough to eject finely
divided droplets from the mouth or nose respectively.

The lungs are protected from bacterial invasion not only by the
tortuous nasal air passages, but by the ciliated epithelium which
covers the surface of the mucosa of the bronchi and bronchioles.
The rhythmic contractions of these cilia carry upward and outward
those bacteria which may have penetrated so deeply into the respira-
tory passages. Inhibition of the activity of these cilia by cold or other
environmental conditions may be a potent factor in the establishment
of infection in the respiratory tract. Occasionally bacteria succeed
in reaching the terminal bronchioles and alveoli of the lungs : they
are normally removed by the phagocytic activity of leukocytes (micro-
phages) or of certain fixed tissue cells (macrophages) . In spite of
these barriers, however, the lungs occasionally become infected. The
pneumococcus and tubercle bacillus are the most common primary
invaders of the lungs. Streptococci are more frequently secondary
invaders, although many primary lobular pneumonias are caused by
this organism.

PARASITIC AND PATHOGENIC BACTERIA 101

2. Mucous Membranes. The moist surface of mucous membranes
makes them excellent culture media for many bacteria which can
grow at the temperature of the body. The physiological secretions
which bathe these membranes, with the exception of the stomach,
are usually without germicidal properties; at best, their antiseptic
properties are w r eak. The removal of bacteria from such surfaces
is probably for the most part mechanical. The secretion of mucus,
which has been shown to enclose bacteria, may be an important factor
in their elimination.

Mouth. The mouth is a most important portal of entry for the
great majority of bacteria, both pathogenic and non-pathogenic,
which are associated with man. All of the intestinal bacteria, harmful
or benign, many of the bacteria which are associated with morbid
processes of the respiratory tract, and several which induce specific
lesions of the brain and spinal cord enter through this atrium. A
great majority of viruses which infect the respiratory tract and the
cerebrospirial axis also leave the body through the mouth or nose.

The normal flora of the mouth is quite varied, 1 including not only
bacteria which are ordinarily regarded as harmless, but also organisms
which occasionally or frequently incite disease. Thus, from 20 to 40
per cent, of healthy individuals living in large cities harbor typical
and apparently virulent pneumococci in their mouths; 2 about 2 per
cent, of school children harbor typical diphtheria bacilli in their
mouths. 3 Rarely, tubercle bacilli have been detected in the mouths
of apparently normal individuals.

It is worthy of note that an occasional abscess in the cervical region
may contain spiral organisms; frequently a careful examination will
reveal a sinus connecting the abscess with the mouth, perhaps origi-
nating at the base of a carious tooth. Dental caries is usually regarded
as a bacteriological process. The removal of bacteria from the teeth
and gums can not be satisfactorily accomplished by antiseptic mouth
washes and the saliva possesses no germicidal properties. Bacteria
are removed from the teeth mechanically by friction and are trans-
ported from the mouth to the stomach during the processes of mas-
tication and deglutition. ' The oral flora is most numerous before

1 For full literature and descriptions see Miller, Die Mikroorganismen der Mundhohle,
Leipzig, 1892, and Goadby, Mycology of the Mouth, 1903.

2 Recent observations by Cole and his associates indicate that the ordinary "mouth"
pneumococci differ in their serological reactions from pneumococci isolated directly
from pneumonia lesions.

3 Moss, Guthrie and Gelien have found a much larger proportion of diphtheria bacillus
carriers during a period when diphtheria was epidemic.

102 , ' SAPRaP-HYTISM, PARASITISM, AND PATHOGENISM

eating and almost absent immediately after eating a hearty meal.
Tubercle bacilli are swallowed thus and many of them eventually
appear in the feces.

Stomach. The acidity of the stomach during gastric digestion,
by virtue of the free hydrochloric acid of the gastric juice, is a potent
factor in the destruction of bacteria which reach the stomach both
from the mouth and the respiratory tract. Mineral acids are much
more powerful germicides than organic acids. The normal stomach,
therefore, is quite free from inflammations or irritations attributable
to the activity of bacteria. Many bacteria, however, run the gauntlet
of the stomach successfully, especially when the stomach is empty
(when the concentration of hydrochloric acid is very low) and pass
into the intestinal tract, where the conditions are much more favorable
for their growth. The passage of bacteria through the stomach prob-
ably takes place either very early in gastric digestion, when the
hydrochloric acid is not at its "digestive concentration" (about 0.2
per cent.), or after gastric digestion has ceased. When water or
other fluids are drunk, which do not call forth gastric juice, bacteria
doubtless pass through the stomach unharmed, and it is probable
that organisms included mechanically within food particles may
occasionally escape the action of the gastric acidity.

Certain aciduric bacteria 1 and even yeasts which are tolerant of
acid may be found occasionally in the normal stomach, but rarely
or never pathogenic bacteria. Abnormally, particularly when the
hydrochloric acid is deficient, many bacteria are found in the stomach
contents. Obstruction of the pylorus tends to increase the number
of bacteria in the stomach by promoting stasis of food. This con-
dition is particularly common in carcinoma of the pylorus. The
Oppler-Boas bacillus, sometimes called B. geniculatus, one of the
aciduric bacteria, is so frequently found in this pathological condition
it was at one time supposed to be an accessory factor; it is now known
to have no relationship to gastric carcinoma. B. geniculatus is also
found very commonly in cases of achlorhydria. Sarcina ventriculi
is also found in similar conditions.

The gastric acidity will destroy the toxins of B. diphtherise and
B. tetani; the toxin of B. botulinus is not inactivated by the gastric
juice. The toxins of the paratyphoid group of bacteria also appear
to be resistant to gastric digestion.

1 Kendall, Jour. Med. Research, 1910, N. S., xviii, 153.

PARASITIC AND PATHOGENIC BACTERIA 103

Intestines. The abundant intestinal contents, which vary some-
what in composition and reaction at different levels, provide conditions
which make the intestinal tract a very efficient combined incubator
and culture medium. Many kinds of bacteria may theoretically find
conditions well adapted to their rapid development there and it is
not surprising to find that bacterial proliferation is greater both in
nature and extent in the intestinal tract than in any other known
medium. It has been conservatively estimated that the average
daily fecal excretion of bacteria in a healthy adult on a normal diet
is expressed by the truly enormous number, 33 x 10 12 . About 47 per
cent, of the nitrogen of the feces is contained in the bodies of these
bacteria which, when dried, weigh nearly 0.5 gram.

The upper level of the intestinal tract, particularly the duodenum,
is relatively free from bacteria during interdigestive periods. The
duodenal bacterial population increases rapidly when food enters
this section of the alimentary canal and decreases when the food
passes to lower levels. The numbers of bacteria increase very greatly
where stasis of food becomes more marked and in the cecum and
large intestines generally there are continually present enormous
numbers of bacteria. 1

The types of bacteria found in the intestinal tract are influenced
markedly by the nature of the food of the host and by the ability of
the organisms themselves to change their metabolism to meet varia-
tions in the composition of this food. Those bacteria which can best
meet alternations in diet of the host are the ones which naturally
persist. The bacteria contained in the food itself may also play a
prominent part in determining the nature of the organisms which
are found in the intestinal tract. The colon bacillus is particularly
labile in meeting dietary alternations in the intestines and this organism
constitutes about 80 per cent, of the bacteria which can be isolated
from the feces of the adult.

At birth the intestinal tract is sterile and the embryonal feces, the
meconium, which is passed during the first eighteen hours after birth,
is sterile. Following this period of sterility there is a period lasting
about three days on the average, in which various adventitious organ-
isms are met with in the dejecta. The normal nursling flora begins
to appear by the end of the third day, following the ingestion of
breast milk. The dominant organism of this nursling flora is ordi-
narially an obligate anaerobe, Bacillus bifidus, which is one of the

1 Kendall, Jour. Med. Research, 1911, xxv, 126-130.

104 SAPROPHYTISM, PARASITISM, AND PATHOGENISM

best known examples of obligately fermentative organisms. It does
not thrive on a purely protein diet but requires carbohydrate, which
is normally supplied by the breast milk. Breast milk, it will be remem-
bered, contains on the average about 7 per cent, of lactose, 3 per cent,
fat and but 1.5 per cent, protein. The proportion of carbohydrates
to protein in the diet decreases as the infant becomes older and the
diet becomes more liberal, and this decrease in the percentage of
carbohydrate is associated with a diminution in the number of the
obligately fermentative bacteria, particularly of Bacillus bifidus, and
their gradual replacement by organisms which can thrive well on a
diet containing variable proportions of carbohydrate and protein. 1

Bacillus coli is a most labile organism with respect to its ability
to develop in the carbohydrate and protein constituents of the intes-
tinal contents at the ileocecal region and lower levels; this organism
is represented to the extent of fully 80 per cent, in the feces of healthy
men. Smaller numbers of other bacteria, as Micrococcus ovalis,
Bacillus acidophilus, B. proteus, B. mesentericus, B. aerogenes cap-
sulatus and many other varieties are found transiently or semi-per-
manently in the intestinal contents. Exogenic bacteria occasionally
invade the tissues of the body through the intestinal mucosa. Thus
typhoid, paratyphoid and dysentery bacilli and cholera vibrios may
produce severe infections. The tubercle bacillus may pass through
the apparently intact intestinal wall without leaving any evidence
of its passage. It is supposed that this organism penetrates the
intact mucosa and enters lymphatic channels suspended in fats and
eventually proliferates in deeper tissues.

3. Genito-Urinary System. Vagina. The vagina has an acid reac-
tion and it harbors very few bacteria, but immediately afterchild-
birth the reaction may become temporarily alkaline. The bacillus
of Doderlein, however, occurs so commonly, that it may be provision-
ally regarded as a normal inhabitant and a few strains of aciduric
cocci are not infrequently detected in cultures from the fundus of the
vagina. The Gonococcus and Treponema pallidum are the more
common pathogenic organisms whose portal of entry is the vagina.

Uterus. The normal uterus is sterile and the acid reaction of the
vagina and the closure of the cervix uteri tends to maintain sterility
under normal conditions. During menstruation and childbirth the
mechanical defenses of the uterus are impaired. The organ itself
appears to possess no specialized powers of resistance to infection.

1 A more detailed discussion on intestinal bacteria and their significance will be found
in Chapter xxx.

PARASITIC AND PATHOGENIC BACTERIA 105

Urethra. The urethra in health is practically free from bacteria.
The flow of urine mechanically frees it from bacteria. The external
orifice of the urethra, however, frequently contains an acid-fast organ-
ism, Bacillus smegmatis, which can be differentiated from the tubercle
bacilli only by animal inoculation, and, very frequently, Bacillus coli.
The gonococcus and Treponema pallidum may invade the tissues
through the urethra.

Urinary Bladder and Ureter. The slightly alkaline reaction of the
urine affords a good culture medium for many bacteria and infection
of the bladder by B. coli, B. proteus, B. typhosus and other micro-
organisms is by no means uncommon. It is probable that infection
occurs much more frequently through the blood or lymph than through
an ascending infection from the urethra. B. proteus appears to grow
with great luxuriance in the urinary bladder and a typical cystitis
may be readily incited in dogs by injecting virulent cultures of the
organism directly into the bladder. Occasionally a descending infec-
tion from an inflamed kidney may result in cystitis: whether a true
ascending infection through the ureter to the kidney takes place is
not definitely proven.

Kidneys. The kidneys are normally free from bacteria, but infec-
tion of one or both kidneys through the blood stream is a well-estab-
lished phenomenon. A variety of organisms may thus infect the
kidney. The cocci of suppuration frequently incite acute nephritis
and tubercle bacilli induce chronic infection. Theoretically, any
invasive organism which enters the blood stream may localize in the
kidney and establish metastatic foci there. The organ is susceptible
to specific bacterial toxins as well as to the bacteria themselves.

I). Where Bacteria Multiply in the Body. Practically no organ
or part of the body, except such structures as the nails, are free from
invasion with one or another kind of organism. The obvious com-
plexity of the subject makes it difficult or even impossible to present
in concrete form, a statement which shall indicate specifically the
types of organisms which incite infection in association with the
particular organs or tissues where they become localized. It is impor-
tant in this connection, however, to remember that a great majority
of progressively pathogenic bacteria exhibit rather marked affinities
for special tissues, and that they invade the tissues through definite
atria. The organisms which are habitually parasitic, on the contrary
the "opportunists" as Theobald Smith has so clearly pointed out,
are less exacting in this respect, as a rule, and they may invade the

106 SAPROPHYTISM, PARASITISM, AND PATHOGENISM

tissues whenever the natural barriers skin, mucous membranes, and
so on weaken and become vulnerable.

The following table indicates the more common and important
bacteria, parasitic or pathogenic, which may invade the tissues, and
the organs where they tend to localize and develop.

SKIN:

Staphylococcus and streptococcus groups.

Acid-fast group: tubercle bacilli, lepra bacilli, smegma bacilli.

Anaerobic group: tetanus, gas bacillus.

Anthrax.

"Bottle" bacillus (spore of Melassez).
NOSE, THROAT AND ADNEXA:

Staphylococcus group.

Streptococcus and pneumococcus group.

Diphtheria and pseudodiphtheria group.

Influenza and pertussis group.

Pneumobacillus, rhinoscleroma and ozena group.

Bacillus fusiformis and spirillum group.

Meningococcus and catarrhalis group.

Acid-fast group 'Chiefly tubercle bacilli and leprosy.

Blastomycetes and hyphomycetes.

Virus of poliomyelitis and unknown viruses, mumps, etc.

(Organisms of dental caries and pyorrhea not included above.)
EYE AND EAR:

Streptococcus and pneumococcus group.

Staphylococcus group.

Diphtheria and pseudodiphtheria group.

Influenza group.

Koch- Weeks and Morax-Axenfeld group.

Gonococcus.

Proteus group.

Pyocyaneus group.
LUNGS :

Streptococcus and pneumococcus group.

Pneumobacillus group.

Acid-fast group: tubercle bacillus.

Influenza and pertussis group.

Plague bacillus, anthrax bacillus and B. psittacosis.

Colon and typhoid group.

Actinomyces and hyphomycetes.
PELVIC ORGANS:

Streptococcus and Staphylococcus group.

Gonococcus and Treponema pallidum.

Tubercle bacillus and smegma bacillus.

Micrococcus melitensis.
SEROUS FLUIDS:

1. Cerebrospinal fluid:

(a) Fluid usually clear: tubercle bacillus and Treponema pallidum. Virus
of poliomyelitis.

(&) Fluid turbid: Pneumococcus, streptococcus, meningococcus, B. influ-
enza, B. typhosus, B. coli.

2. Pleural and pericardial fluids:

(a) Fluid usually clear: tubercle bacillus.

(&) Fluid turbid as a rule: pneumococcus, streptococcus, B. influenzce,
Pneumobacillus group, Bacillus typhosus, Staphylococcus.

3. Peritoneal fluid:

Streptococcus group.
Coli and typhoid group.
Tubercle bacillus (?).

BALANCED PATHOGENISM; EPIDEMIOLOGY 107

BLOOD:

Streptococcus and pneumococcus group.

Staphylococcus group.

Typhoid, paratyphoid and dysentery group.

B. coli.

Recurrent fever and treponemata.

B. pestis.

Certain filterable viruses: Yellow fever, poliomyelitis (?).

Tubercle bacillus (occasionally).
INTESTINAL CONTENTS, FECES:

B. bifidus and B. acidophilus group (chiefly in infants).

B. coli, B. lactis aerogenes, proteus and cloacae group.

Alcaligenes, paratyphoid, typhoid and dysentery group.

Streptococcus and Micrococcus ovalis groups.

Mucosus capsulatus group.

Spore-forming group: Aerobic B. mesentericus, B. subtilis, B. anthracis.

Anaerobic B. aerogenes capsulatus, B. tetani, B. botulinus.

Acid-fast group: tubercle bacilli, bovine and human; grass bacilli.

Spiral group: Vibrio cholerse, Sp. of Finkler and Prior.

E. Where and How Bacteria Escape from the Body. It appears
from foregoing considerations that those microorganisms which are
progressively pathogenic for man habitually invade the tissues through
atria characteristic for each microbe. Their escape from the tissues
through appropriate channels in direct communication with the out-
side is equally important. Bacteria of the "opportunist" type fre-
quently perish within the tissues because they lack a perfected
mechanism of escape to the outside. Progressively pathogenic bac-
teria leave the body through two principal avenues the mouth and
nose, and the feces. Less commonly, certain types may pass to the
outside in the urine. The skin is not a very important factor in the
elimination of pathogenic bacteria. The paths of pathogenic bacteria
from the tissues to the outside are varied, but very constant for each
special organism and the discussion of this phase of their activity is
reserved for the Section on Specific Organisms.

VI. BALANCED PATHOGENISM; EPIDEMIOLOGY.

It has been helpful, for clearness and discussion, to distinguish
rather sharply Setween parasitic and pathogenic bacteria and in a
majority of specific instances such a differentiation can be readily
established. There is no hard and fast line of demarcation, however,
between organisms of the "opportunist" type and those progressively
pathogenic, for it is undoubtedly true that some "opportunists" may
exhibit epidemic tendencies for limited periods if a combination of
conditions arise which favor the distribution of the organisms and
either increase the invasive powers of the microbe or decrease the
resistance of the host. The limited spread of such bacteria is far more

108 SAPROPHYTISM, PARASITISM, AND PATPIOGENISM

frequently attributable to unusually direct transfer of organisms by
a common vehicle through a series of susceptible hosts than to the
escape of the microbes from one host to another. Thus, milk-borne
epidemics of septic sore throat may be extensive and involve many
patients, but secondary transfer from man to man is relatively uncom-
mon. These bacteria have not, as a rule, perfected their mechanism
of escape from the tissues of one host to those of another. The
epidemics are usually of brief duration and it is probable that the
surviving microbes return to their original parasitic state.

Of far greater importance is a probable tendency of many progres-
sively pathogenic bacteria to act more and more on the defensive;
to gradually disembarrass themselves, on the one hand, of the offen-
sive weapons which originally conferred upon their possessors the
ability to invade their host, and, on the other hand, to perfect what-
ever defensive weapons they may have possessed the rudiments of. 1
Such a change, as Theobald Smith has pointed out, would be difficult
to detect, because an elimination of the more aggressive type and its
gradual replacement by a strain in which the defensive elements were
more prominently represented would require years for its accomplish-
ment. Such a change in the activities of the microorganisms would
probably be accompanied by reciprocal activities of the host, so that
eventually a strain of microorganisms would be evolved which had
reached a state of relative equilibrium with the host. Unusually
virulent strains of microbes would tend to perish with their hosts,
and unusually susceptible hosts would tend to perish with their
invaders. A mutual adjustment of virulence and resistance between
the surviving hosts and microbes would lead eventually to one of
three conditions:

1. Gradual extinction of the microorganism;

2. The gradual assumption^ of a parasitic or " opportunist " exist-
ence, or

3. A more perfect pathogenism in which the mechanism of invasion,
multiplication within the tissues and escape to other hosts is accom-
plished without acute damage to the host.

It might well happen that the introduction of such "balanced"
strains into new fields would lead to temporary disaster, as for
example, the highly fatal epidemic of measles when this virus first
gained a foothold in the South Sea Islands.

1 Theobald Smith (Some Problems in the Life History of Pathogenic Microorganisms,
Am. Med., 1904, viii, 711) clearly stated and discussed this hypothesis over a decade
ago, and it is surprising how little cognizance has been taken of it.

BALANCED PATHOGENISM; EPIDEMIOLOGY 109

Theobald Smith 1 has mentioned the diphtheria bacillus as an
organism which possibly exhibits a tendency toward a parasitic exis-
tence. The toxin of the diphtheria bacillus is not a poison specific
for man; many animals, as the horse and guinea-pig, are very sus-
ceptible to it. Yet the diphtheria bacillus is almost obligately a human
pathogen. The ever-increasing occurrence of avirulent, non-toxin
producing strains which are otherwise perfectly typical, and the
frequent occurrence of individuals whose serum contains small
amounts of natural antitoxin might be interpreted as an indication
that strains of this organism are becoming gradually accustomed to
a purely parasitic existence in the upper respiratory tract of man on
the one hand, and that man has acquired some specific resistance to
the microbe on the other hand.

The tubercle bacillus (typus humanus) is an excellent example of
an exquisitely balanced pathogenic microorganism. Its metabolism is
not markedly different from that of the host and the typical disease
excited by it is focal, chronic, and slow-going. Years may elapse before
the host finally succumbs. The development of the organisms within
the tissues of the host does not appear to lead to the formation of
substances which arouse the latent offensive and defensive mechanism
of the host to acute antagonism. During this long period the tubercle
bacilli establish communication with the outside and, in a majority
of cases, countless myriads of bacilli escape from the host before
death removes him as a source of infection. Occasionally tubercle
bacilli become widely disseminated in the body, causing rapidly fatal,
generalized miliary tuberculosis. These organisms perish with their
host.

It is well known that the virulence of bacteria, many kinds at least,
can be increased decidedly by passage from animal to animal by
providing an artificial portal of entry and of exit from animal to
animal. This is accomplished by injecting the organisms into a
first animal and reinjecting them, at brief intervals, into other animals.
In such instances there is a direct continuity of growth from animal
to animal, greater than is met with in naturally occurring infections.
It is worthy of note that bacteria of the "opportunist" type are, gen-
erally speaking, more successfully exalted in virulence under these
conditions than the progressively pathogenic forms.

There is yet another feature of Pathogenism which is worthy of
note. From time to time almost any bacterial disease, for example,

1 Theobald Smith, loc. cit.

110 SAPROPHYTISM, PARASITISM, AND PATHOGENISM

typhoid, plague or influenza, may leap suddenly to epidemic propor-
tions, spread rapidly and then subside again, to be succeeded by
sporadic cases which gradually diminish in numbers and in severity.
The bacteria causing these outbreaks appear to acquire somehow and
somewhere, an unusual degree of invasiveness and they spread rapidly,
especially in thickly settled areas, and as rapidly lose their unusual
activities and subside to what appears to be their usual level of viru-
lence. It is very probable that those strains of pathogenic bacteria
in general, which suddenly acquire unusual virulence are short-lived,
partly because their hosts perish before the microbes can escape to
new hosts. Not infrequently, these or similar epidemics are preceded
by mild, atypical disease, which may not be specifically recognized,
and during this initial period the bacteria may be quite widely dis-
tributed. 1

1 Kendall, Boston Med. and Surg. Jour., 1915, clxxii, 851.

CHAPTER VI.
IMMUNITY AND INFECTION.

2. Passive Immunity.

(a) Antibody Im-
munity.
(6) Chemotherapy.

3. Mixed, Active and

Passive Immunity.
II. INFECTION PRIMARY AND SEC-
munity. . ONDARY.

1. Active Immunity. A. Defenses of the Host, Non-

(a) Natural Ac- specific and Specific.

GENERAL PHENOMENA OF IMMUNITY.
I. CLASSIFICATION OF IMMUNITY.

A. Natural or Inherited Im-

munity.

1. Racial.

2. Individual.

B. Acquired or Induced Im-

quired Im-
munity.
(6) Artificial Ac-

Ill. THEORIES OF IMMUNITY.

A. The Humoral Side-chain or
Ehrlich Theory of Im-

quired Im- munity.

munity.

B. The MetchnikorT or Phago-
cytic Theory of Im-
munity.

GENERAL PHENOMENA OF IMMUNITY.

IT has long been recognized that man and animals exhibit refrac-
toriness to infection with specific bacteria or other microorganisms
which cause serious epizootics in closely related animals. Man is,
as a rule, quite free from the epizootic diseases of animals domesticated
by him, and the domestic animals are usually not infected with the
organisms which incite progressive disease in man. Thus, man does
not contract chicken cholera and domestic animals do not become
infected with the typhoid bacillus. Furthermore, closely related
animal species may exhibit striking differences in susceptibility to
the same disease; for example, field mice are readily infected with
the glanders bacillus, but house mice are quite resistant to infection
with this organism, and ordinary sheep readily succumb to anthrax
although Algerian sheep are practically immune to infection with the
anthrax bacillus.

This inherent or congenital resistance or refractoriness to infection
with a specific microorganism, when general among the individuals of
a species or group of animals or of man is termed natural or inherited
immunity. It is not necessarily absolute; lowering the natural resis-
tance of the individual may render him susceptible to infection. Thus

112 IMMUNITY AND INFECTION

hunger, experimental (phloridzin) diabetes, fatigue produced by pro-
longed exercise in treadmills, and excessive chilling by the removal
of hair have been shown to decrease resistance in experimental animals.

It is also a matter of common observation that the uniform exposure
of a number of theoretically susceptible individuals of the same species
to a virus does not lead to uniform infection; a certain small number
usually perish, a larger proportion become mildly or severely ill and
recover. The greatest number are not especially affected, as a rule.
Those individuals who escape infection in one epidemic may succumb
to infection during a subsequent epidemic of the same disease. This
phenomenon of individual variation in susceptibility is well exempli-
fied in water- and milk-borne epidemics of typhoid fever where typhoid
bacilli are widely distributed in a water or milk supply. A small
number become infected, but the greater number escape the disease.
The incidence of scarlet fever, of diphtheria or of other infectious
diseases among the members of the same family frequently illustrates
this same phenomenon. This resistance to infection exhibited by
certain individuals of a susceptible species is termed inherited
immunity.

Susceptible individuals who survive a naturally acquired or arti-
ficially induced infection as smallpox, measles, typhoid fever or
vaccinia are frequently resistant or refractory to subsequent infec-
tion with the same virus. They have developed a resistance to specific
infection, they have acquired immunity, in other words. This type
of immunity, which results from actual infection, is termed active,
acquired immunity. It is the outcome of a successful struggle between
the host and the invading microbe during which the former, through
cellular activity, produces or increases antibodies specifically inimical
to the latter. The immunity which is thus laboriously produced
is frequently fairly persistent. It is more commonly observed fol-
lowing invasion by exogenous, progressively pathogenic bacteria than
infection with endogenous microorganisms of the "opportunist" type.
Indeed, infection with the latter not infrequently results in increased
susceptibility to subsequent infection with the same species of microbe.
Thus, recovery from one attack of typhoid fever usually confers last-
ing immunity upon the individual; one attack of lobar pneumonia,
on the other hand, appears to predispose the individual to subsequent
infection with the pneumococcus.

The injection of specific immune substances or antibodies into
susceptible individuals may confer upon them transient or temporary

CLASSIFICATION OF IMMUNITY 113

immunity to the specific infection; the host is a passive recipient of
antibodies in such instances. These alien antibodies, however, soon
diminish in potency or disappear, leaving the susceptibility of the indi-
vidual to infection at its original level. Immunity induced by the
injection of specific antibodies is termed passive acquired immunity.
The transitory immunity to diphtheria or tetanus following the
injection of diphtheria or tetanus antitoxin is an example of passive
acquired immunity.

Immunity may be localized or general in the same individual, and
different individuals frequently exhibit varying degrees of resistance
or susceptibility to the same virus.

I. CLASSIFICATION OF IMMUNITY.

Both immunity and susceptibility are relative; there is probably
neither absolute immunity nor complete susceptibility to any infec-
tion. There is furthermore, no hard and sharp line of demarcation
between the various types of immunity; nevertheless, it is convenient
to assemble the prominent manifestations of immunity into several
types or classes.

A. Natural or Inherited Immunity. The inherited power of resist-
ing specific infection manifested by a large proportion of the individuals
comprising a family, genus or species is termed inherited or natural
immunity. It may be:

1. Racial. Observed in specific families, genera or species of the
animal kingdom, or

2. Individual. Observed in individuals of the same species. Indi-
vidual natural immunity may also be sexual observed in males or
females of the same species.

B. Acquired or Induced Immunity. The resistance or non-sus-
ceptibility to infection following naturally acquired or artificially
induced specific diseases, or resistance passively brought about by the
introduction of specific protective substances is termed acquired or
induced immunity.

1. Active Immunity. (a) Natural. Following naturally acquired
disease, as for example, immunity following recovery from smallpox
or typhoid fever.

(b) Artificial. Brought about by the introduction of attenuated
or killed viruses, vaccines or toxic products of bacteria into a sus-
ceptible host. The toxic products of bacteria may be either those
8

114 IMMUNITY AND INFECTION

excreted during life, or products arising from their disintegration.
Immunity to smallpox following vaccination and immunity to typhoid
fever following the injection of killed cultures of typhoid bacilli are
familiar examples of this type of immunity.

There is usually a period of increased susceptibility to infection
immediately after the introduction of the virus or its products, in
artificially acquired immunity. This period of susceptibility is fol-
lowed by an increase in resistance to the virus. If the process of
immunization is repeated several times, the initial level of resistance
to infection may be raised very materially. Thus, prophylactic vac-
cination with killed typhoid bacilli (anti-typhoid vaccination) increases
the resistance of the recipient of the vaccine to typhoid infection to
such a degree that his chances of acquiring the disease are greatly
lessened. It is also probable that in the event of infection of the
protected individual with the typhoid bacillus, both the duration and
severity of the attack will be diminished.

2. Passive Immunity. (a) Antibody Immunity. Introduction into
the host of specific products of immunity (antibodies) as diphtheria
antitoxin.

(6) Chemotherapy. The use of chemicals for preventing or modifying
infection.

Passive immunity is induced by the injection of antibodies into
the host, which have been developed in another animal. The recipient
of these antibodies is protected only so long as they remain in the
body. The immunity, however, is effective almost immediately after
injection; there is no latent period.

3. Mixed, Active and Passive Immunity. Mixed artificially acquired
immunity is induced by the simultaneous injection of specific anti-
bodies and the weakened or attenuated virus; resistance to infection
is usually increased at once (passive immunity), while at the same
time the host begins to react to the virus and to produce antibodies
thereto (artificially acquired immunity).

The factors which predispose the host to or protect him from inva-
sion by microorganisms are usually varied and complex. Relatively
simple explanations of the mechanism involved suffice to account for
the phenomenon in specific instances, however. For example, frogs
and hens are not naturally susceptible to infection with the anthrax
bacillus, whose optimum temperature of growth is 37 C., yet infection
could take place if the body temperature of either animal were brought
to this level, as Pasteur showed nearly two decades ago. A change

INFECTION 115

in environment may predispose to infection; the carnivora in their
native state are quite resistant to infection with the tubercle bacillus,
whereas in captivity they may succumb readily. Similarly, man
placed in bad hygienic surroundings appears to be distinctly more
vulnerable to many infectious diseases than he is when his environ-
ment is more sanitary. Unhygienic conditions, however, are rela-
tively complex in their reactions on man, for the attendant evils of
overcrowding, underfeeding and increased exposure to infection
undoubtedly play a part.

Heredity also appears to be an important factor in determining the
average severity of infection in certain types of endemic disease.
Measles is a common and usually fairly mild disease of childhood
among civilized people. Among aboriginal populations, as those of
the South Sea Islands where the inhabitants had not been exposed
to measles previous to the advent of Europeans, the introduction
of the virus has resulted in a veritable plague during which large
numbers of the people died. This phenomenon of hereditary acquired
tolerance for specific endemic disease may conceivably be even more
specific; for example, strains of a given organism might produce
mild disease in areas where it has been endemic for generations and
yet be rapidly fatal for alien populations who may have in turn become
partly tolerant for other strains of the same organism. If such prove
to be the case, unrestricted emigration may lead to temporary dis-
turbances in the balance between specific microorganisms on the one
hand and hosts on the other a feature which Theobald Smith called
attention to many years ago. 1

Racial differences in susceptibility are occasionally met with even
in the same species. Negroes and Indians are more susceptible to
infection with tubercle bacillus than the Caucasian race. The Jews
appear to be somewhat more resistant to infection with the tubercle
bacillus than the other branches of the Caucasian race.

H. INFECTION.

Pathogenic bacteria which reach the host do not necessarily incite
disease; they may be, and undoubtedly are, frequently overcome by
the body without inducing symptoms. This initial resistance to infec-
tion involves an initial struggle between host and microorganism which
brings into play non-specific lines of defense of the macroorganism

1 Theobald Smith, Tr. Assn. Am. Phys., 1893.

116 IMMUNITY AND INFECTION

consisting collectively of the skin, mucous membranes of the respira-
tory and gastro-intestinal tracts and other intact barriers discussed
in the preceding chapter. If this initial line of defense holds, the host
overcomes the prospective invader and the latter frequently perishes.
Repeated microbic assaults may be successful if the first fails. On
the other hand, if the microbe prevails and penetrates the initial line
of defense, invasion of the tissues of the host occurs and the micro-
organism encounters a second line of defense which is made up of
two rather distinct factors cellular and humoral. The cellular
defense of the host resides in the leukocytes which circulate in the
the body fluids and in certain fixed tissue cells in the lungs, lymph-
spaces and glands, the Kupfer cells of the liver, as well as large cells
which appear in serous cavities. These cells engulf and destroy
certain types of invading microorganisms. The humoral defense
resides in the natural, non-specific power of the blood and lymph to
destroy limited numbers of microorganisms or to so interfere with
their nutrition or other functions as to prevent their development
within the body. The humoral defense is frequently effective against
bacteria which do not succumb to the cellular defense of the body,
and vice versa.

It is recognized that certain environmental factors predispose to
infection. Thus, extreme climates, excessive humidity, or exposure
to unhygienic conditions, bad air, poor or insufficient food, lack of
exercise or fatigue may react upon the individual in ways not defi-
nitely understood and reduce his resistance to microbic invasion on
the one hand, and his ability to rally his specific, anti-microbic
mechanism on the other hand. Intracurrent disease frequently
weakens the initial lines of defense, permitting bacteria of the " oppor-
tunist" type to become invasive. Thus, furunculosis frequently is
a complication of diabetes, pneumonia not uncommonly terminates
a case of tuberculosis. Renal and cardiac disease may weaken the
normal barriers of the body, permitting a variety of infections with
endogenous bacteria.

It is a well-attested fact that certain occupations or professions
cause or promote pathological conditions which predispose to infec-
tion. Prominent among these is participation in arts or industries
which involve exposure to poisonous or irritating dust or fumes. The
incidence of tuberculosis among those frequently exposed to organic
or inorganic dust is a striking example of the relation of occupation
to infection.

THEORIES OF IMMUNITY 117

When an invading microorganism has reached a suitable atrium of
the body, overcome the initial defense of the host at that point, and
has successfully resisted the normal humoral or cellular opposition
of the host, a new phase of the struggle becomes prominent, during
which the host gradually develops a specific attack upon the invader,
bringing into action latent forces which constitute the third and last
defense of the body. The invader also may change its weapons to
some degree to meet the antimicrobic activity of the host and the
result of the struggle may be complete recovery from infection, chronic
disease, the bacillus carrier state, or death of the host.

The initial and secondary defensive powers of the host, therefore,
are both cellular and humoral in character. The intact skin and
mucous membranes of the gastro-intestinal, respiratory and genito-
urinary tracts are important initial non-specific lines of defense. The
phagocytic activity of leukocytes and certain fixed tissue cells, and
the natural, normal bactericidal substances of the blood and lymph,
which bathe the initial line of defense, are important adjuvants in
maintaining the integrity of these initial barriers to infection. In
certain infections the humoral factors are the more important, while
in others the cellular mechanism is conspicuous.

The defensive mechanism against the same bacterium may be
different in one or another animal. For example, dogs and rats are
relatively immune to infection with the anthrax bacillus. The immu-
nity observed in the dog appears to be due to phagocytic activity of
leukocytes which engulf and destroy anthrax bacilli which may have
gained entrance to the body. 1 The rat, on the contrary, enjoys immu-
nity not because its leukocytes engulf and destroy anthrax bacilli;
the blood of the rat possesses soluble, non-specific bactericidal sub-
stances which destroy anthrax bacilli. Frequently both the cellular
and humoral elements are engaged either simultaneously or succes-
sively as the struggle between host and invading organism proceeds.

m. THEORIES OF IMMUNITY.

Two distinct explanations have been advanced to account for the
mechanism of immunity as it is observed during the course of disease :
the cellular or phagocytic theory championed by Metchnikoff and
his followers, and the humoral theory developed by Ehrlich.

Both of these theories, the cellular and the humoral, have in com-

1 Hektoen, Jour. Am. Med. Assn., 1906, xlvi, 1407.

118 IMMUNITY AND INFECTION

mon, tacitly at least, two important features:, the specificity of the
protective substances (antibodies) formed as the result of infection,
and the principle that no new mechanism is evolved de novo to meet
the conditions existing during an infection; rather, there is an increase
in activity along definite lines in the preexistent, latent or reserve
mechanism of defense.

Neither theory affords a satisfactory explanation of all the features
of immunity following infection and it is very probable that cellular

911-

FIG. 5. Side-chains, first order (antitoxins and antif erments) . 1, side-chain attached
to cell; c,.haptophore group; 2, side-chain to which is attached a toxin molecule; 3,
a cast off side-chain of the first order: antitoxin or antif erment ; 4, a toxin or enzyme
molecule; a, toxophore group; 6, haptophore group; 5, a toxoid: the toxophore group
is destroyed, leaving the haptophore group (6) intact; 6, a toxin molecule attached to
a cast-off side-chain (antitoxin), illustrating the neutralization of toxin by antitoxin in
the blood stream.

activity and the production of specific antibodies is more important
in certain types of infection, while phagocytic activities are more
intimately concerned in other types.

A. The Humoral, Side-chain or Ehrlich Theory of Immunity.
According to Ehrlich's conception, every cell of the body has two
functions: a physiological function, which constitutes a special type
yt/bf activity of the cell secretory for a glandular cell, contractile for
Vv a muscle cell, or conductive for a nerve cell and a nutritional func-
tion, which is concerned with the removal of the necessary food sub-
stances from the general supply circulating in the blood or lymph

THEORIES OF IMMUNITY

119

channels, and the appropriation and eventual utilization of these
specific food materials by the cell. These nutritional substances
undoubtedly serve two purposes: Structural, to replace cellular
waste, and Fuel, to supply cellular energy.

The nutritional requirements of the individual cell are varied as
their activities are varied, and Ehrlich conceives that each cell
possesses a number of chemical affinities or receptors, for convenience
of discussion designated as "side-chains" or "haptines," which are

FIG. 6. Side-chains, second order (agglutinins and precipitins) . 1, side-chain attached
to cell; c, haptophore group; b, zymophore group (agglutinophore or precipitinophore
group); 2, side-chain to which is attached a bacterial cell; a, haptophore group of
bacterial cell; 3, a cast-off side-chain of the second order, agglutinin or precipitin; 4,
a side-chain attached to a bacterial cell (agglutination); 5, a bacterial cell; a, hapto-
phore group; 6, an agglutinoid; the zymophore group is destroyed, leaving the hapto-
phore group intact.

the means of attaching to the cell by chemical union, the essential
nutritive substances preparatory to their assimilation. When the
particular food attached to the cell by chemical affinity anchored
by the side-chain, to use Ehrlich's terminology has been assimilated,
more of the same kind of food is removed from the blood stream and
attached to the cell, in accordance with its normal physiological
requirement. The cell, acting through its side-chain, does not exhibit

120 IMMUNITY AND INFECTION

discrimination between nutritive substances and irritating or harmful
substances which may accidentally possess the same combining
affinity for the cell. Consequently, when poisonous substances pos-
sessing chemical affinities similar to those of the normal food sub-
stances circulate in the blood stream, they may become attached to
the cell in place of the normal physiological nutrients. The anchoring
of these poisonous substances, unlike the attachment of normal
nutrient substances, is followed by damage to the cell, or, in extreme
cases, by the death of the cell. 1 If the cell is not actually killed by the
presence of the toxic substance acting upon it through the side-chains,
it is irritated, as it were, and the toxic substance imposes a twofold
burden upon the cell loss of the side-chains to which it is attached
and which are essential to maintain the nutrition of the cell, and
greater or lesser damage to the function of the cell, due to toxic inhibi-
tion of its normal activities. A cell cannot disembarrass itself of the
poison, nor can it assimilate it. It can, however, throw off the side-
chain with the poison still firmly united to it chemically; the extruded
poison cannot enter into chemical combination with other cells pos-
sessing the same chemical affinity, for it is already attached to a side-
chain. Its combining power is saturated.

Side-chains are a necessity to the cell, however; without them
the cell would starve. Consequently the cell regenerates new side-
chains of precisely the same kind to replace those thrown off after
being bound to non-assimilable substances. If enough of the soluble
poison or toxin circulates in the blood stream, this process of union
of toxin to the cell by its side-chains and its expulsion from the cell
with the side-chains attached to it is so frequently repeated that the
cell regenerates side-chains in excess of the normal requirements, in
accordance with the Weigert theory of overproduction. This casting
off of supernumerary side-chains is important. Were they not cast
off the cell would be vulnerable to toxin in direct proportion to the
extra number of side-chains, which would furnish extra bonds for its
attachment. As the cast-off side-chains circulate in the blood stream,
however, they are an element of protection to the cell, for they retain
their original combining power for the toxin and unite with it and
neutralize it as it circulates in the blood stream; that is, before it
can reach the cell itself. It will be seen, therefore, that the same

1 If the toxic material circulates in the blood stream but does not become attached
to the body cells, it is harmless to the host, according to this theory, and the host is
naturally immune.

THEORIES OF IMMUNITY

121

mechanism of the living body which is susceptible of being poisoned
becomes the protective agent if it circulates in the blood stream. It
is obvious that the cast-off side-chains constitute antitoxin. The
body as a whole is qualitatively the same after as before these side-
chains are formed in excess of the normal cellular needs; the difference
is a quantitative one. An animal is naturally immune, according to

FIG. 7. Side-chains, third order (bacteriolysins, hemolysins and cytolysins). 1,
side-chain attached to cell; c, haptophore group; b, complementophile group; 2, side-
chain to which is attached a bacterial cell (6) and complement (5) ; 3, a cast-off side- chain
of the third order; amboceptor; 4, a cast-off side-chain to which are attached a bacterial
cell (6) and complement (5) illustrating lysis; 5, complement.

this theory, if the cells of the body do not unite with toxin, that is,
if they do not contain side-chains which fit the toxin " as a key fits a
lock," to use Emil Fischer's analogy. Toxin may circulate in the
blood stream of such animals, but it does not unite with the cells.

Side-chains of the First Order. From the standpoint of the side-
chain theory, the toxin molecules consist of two groups-^a combining

122 IMMUNITY AND INFECTION

or haptophore group, and a poisoning or toxophore group. The former
is relatively thermostabile, the latter thermolabile. If toxin is heated
to 70 C. for a few minutes, or allowed to stand for several weeks, it
will be found that the poisonous property of the toxin has disappeared,
or has been materially reduced. It still retains its original powder of
uniting with and neutralizing antitoxin, however. The thermolabile
toxophore group has been destroyed or weakened by the heating
process, or on standing. The thermostabile group the haptophore
group has not been impaired. Toxin which has lost part or all of
its original poisoning properties, but which still unites with antitoxin
is called toxoid.

The soluble toxins of the diphtheria and tetanus bacilli are not
simple substances; they contain at least two physiologically separate
poisons. Thus, the toxin of the diphtheria bacillus contains in addi-
tion to the poison which produces acute symptoms, a second poison
which acts slowly and appears to be responsible for postdiphtheritic
paralyses and emaciation. This second poison has less affinity for
antitoxin than the acute poison, and it is called a toxone. Similarly,
the tetanus toxin appears to consist of at least two distinct poisons
tetanospasmin, which has an especial affinity for nerve cells and
which elicits the acute symptoms of tetanus, and tetanolysin, which
causes hemolysis of erythrocytes. The injection of soluble or exo-
toxins produced by bacteria leads to the formation of soluble specific
antibodies which are called antitoxins. Antitoxins are supernumerary
side-chains which have been produced in excess of the physiological
needs of the cell, in response to the stimulus of a specific toxin, and
cast off into the blood stream.

It has been shown that repeated injections of solutions containing
active enzymes as, for example, rennin into animals, is followed
by the appearance in the blood stream of specific antibodies which
will prevent the activity of the homologous enzyme. These anti-
bodies, or anti-enzymes, as they are called, exhibit the specificity and
other characteristics which distinguish antitoxins.

Antitoxins and anti-enzymes are called side-chains of the first
order by Ehrlich. They possess the property of combining with and
neutralizing their respective toxins or enzymes.

Side-chains of the Second Order. If substances of greater com-
plexity than those just described are needed for the nutrition of the
cell, some preliminary treatment, probably in the nature of digestion,
may be required to prepare these substances for assimilation after

THEORIES OF IMMUNITY 123

they are bound to the cell. A side-chain of the first order, which
possesses simply a combining group, does not provide the requisite
power of digestion, according to Ehrlich, and to effect this digestion
side-chains of somewhat more complex structure are required. Side-
chains of this more complex type, side-chains of the second order,
possess not only a combining group for the foodstuff, but a digestive
group as well. This digestive or zymophore group, as it is called, acts
upon foodstuffs after they are anchored to the cell by the combining
or haptophore group. The complete side-chain of the second order,
therefore, is composed essentially of a combining or haptophore group,
and a zymophore group as well. The haptophore group of the second
order side-chain is relatively stabile, but the zymophore group is
labile and readily becomes inactive without, however, impairing the
original combining ability of the side-chain. Side-chains of the second
order are as vulnerable to pathological substances possessing the
requisite chemical affinity as side-chains of the first order, and repeated
irritation of a cell by such pathological substances leads eventually
to an overproduction of side-chains of the second order and an
elimination of the supernumerary side-chains in excess of the physio-
logical need of the cell into the blood stream. Side-chains of the
second order which are thus cast off from the cell in response to the
stimulation of bacterial or other alien protein are of importance
immunologically. If the serum of an animal containing such side-
chains is brought into contact with a suspension of the homologous
bacterium, the organisms are sooner or later clumped together or
agglutinated. If, on the contrary, the serum is brought into contact
with a clear solution of the homologous protein, a precipitate forms.
These reactions are highly specific and those side-chains which cause
agglutination of the specific bacterium pr precipitation with the
homologous protein solution are called respectively, agglutinins and
precipitins.

The relative instability of a zymophore group of a side-chain of the
second order may be inferred from the following experiment:

A serum obtained by injecting a horse with repeated graduated
doses of typhoid bacilli will clump or agglutinate the specific organism
in high dilution. If the serum is heated to 60 or 70 C. for a few
minutes, or if it has been kept for a long time, it will no longer clump
the bacilli, or, at least, it will clump them imperfectly. If such a serum
is allowed to stand in contact with typhoid bacilli for an hour or two
then removed by centrifugalization, it will be found that the bacilli

124 IMMUNITY AND INFECTION

will no longer agglutinate with a fresh, highly potent agglutinating
serum. The bacteria are saturated with the combining group of the
serum whose agglutinophore group had been inactivated by heating.
This experiment shows that the combining group is relatively stabile,
and that it is active even though the zymophore group is inactive.
A side-chain of the second order which has lost its ability to cause
agglutination with a specific organism, but which still retains its
combining power, is called an agglutinoid. It bears a striking resem-
blance to a toxoid in that the active or ergophore group is destroyed,
but the combining group remains intact.

Sera containing specific precipitins readily lose their ability to form
precipitates with the homologous protein. The precipitins have
changed to precipitinoids, due to a functional loss of their precipitino-
phore group.

The part played by side chains of the second order, agglutinins and
precipitins, in immunity is not well understood. Their relation to
immunity is less clear than the relation of antitoxin to immunity.

Side-chains of the Third Order. Nutritive substances of large mole-
cular aggregation may require considerable modification to fit them
for cellular assimilation. Such substances are removed from the
blood stream and bound to the cell by side-chains of the third order.
They are then acted upon by an enzyme (complement) which is
also present in the blood stream. It will be seen that both the nutri-
tive element and a digestive enzyme circulate in the blood, but that
no reaction occurs between them until they are both united by a side-
chain of the third order, which must therefore consist essentially of
two combining groups. One of these, the cytophilic group or hapto-
phore, unites specifically with the nutritive element. The other com-
bining or haptophore group, the complementophilic group, unites
with the enzyme or complement which is present in the blood stream.

Side-chains of the third order are called amboceptors because they
possess two combining groups. An excessive irritation of a cell by a
substance capable of uniting with the cytophilic group of a side-chain
of the third order will lead to overproduction and elimination of these
side-chains precisely as toxins lead to an overproduction of side-chains
of the first order (antitoxin formation). The side-chains of the third
order, furthermore, exhibit specificity for the substance which led to
their overproduction, just as antitoxins exhibit specificity for their
homologous toxins.

It has been shown that the zymophoric group of a side-chain of the

THEORIES OF IMMUNITY 125

second order is permanently a part of the structure. The comple-
ment, which is analogous to the zymophore group of the second order,
is not attached to a side-chain of the third order until the cytophilic
group of the latter has combined with its antigen. The zymophore
group of the second order side-chain is readily destroyed and it
cannot be replaced. The zymophoric group of the third order side-
chain is not an integral part of the structure, and it can be introduced
under appropriate conditions.

Third order side-chains or amboceptors are cytolysins. Those
specific for bacteria are called bacteriolysins; those specific for blood
are called hemolysins; and those specific for the cells of various tissues
or organs are called cytolysins.

The activity of the lysins, according to the Ehrlich theory, depends
on the union of non-specific complement and a specific antigen by the
specific amboceptor. A union of antigen and amboceptor may take
place in the absence of complement, but a union of antigen and com-
plement cannot take place in the absence of amboceptor. The
amboceptor, like other haptophore groups, is relatively thermostabile.
The non-specific complement (found in fresh blood serum from any
animal) is thermolabile and readily destroyed.

Thus far it has been assumed that the cells of the body defend
themselves against toxins, alien protein or alien cells by the formation
of specific antibodies or side-chains. Welch 1 has made the important
suggestion, which has experimental evidence in its favor, that bac-
teria may also produce side-chains which are specific for certain
cells of the host. A struggle between host and microbe, therefore,
would not be one-sided; a dual attempt at immunization is going
on during a bacterial invasion, in which the microbe attempts to
protect itself against the specific weapons of the host as the host
attempts to protect itself against the weapons of the invading micro-
organism. Thus, bacteria grown in media containing agglutinating
sera gradually lose their agglutinability, but this acquired loss of
agglutinating power is not exhibited by descendants of the inagglu-
tinable strain 'grown for some time in media not containing agglutinins.

The side-chain theory, originally formulated to explain antitoxin
immunity, but enlarged in its scope to include the phenomena of
agglutination, precipitation and cytolysis, has been subjected to much
adverse criticism. It was assumed that toxin and antitoxin, for
example, united in simple proportions as a strong acid and a strong

1 Huxley Lecture, 1902.

126 IMMUNITY AND INFECTION

base unite; the chemical analogy of toxin-antitoxin union to form
an inert mixture comparable to a salt was further accentuated by the
effect of moderate degrees of heat in hastening the reaction between
the two. A very thorough investigation of the quantitative neutraliza-
tion of toxin by antitoxin revealed the error of this supposition and
Ehrlich was led to assume a very complex structure for the toxin mole-
cule, in which there existed several fractions possessing individually,
different affinity for antitoxin.

Madsen and Arrhenius 1 studied the toxin-antitoxin union from
the standpoint of physical chemistry and found that the slightly dis-
sociated reactive substances united in conformity with the law of
mass action of Guldberg and Waage. Their conclusion was that
toxin and antitoxin react like a weak acid and weak base, and that it
is a reversible reaction, so that a mixture of toxin and antitoxin always
contains free toxin, free antitoxin and toxin-antitoxin, the relative
amounts being calculable according to the law of mass action. The
observations of Theobald Smith 2 and of many other observers that
neutral mixtures of toxin and antitoxin would induce active immunity
in experimental animals are in harmony with this-view. Biltz 3 has
advanced an hypothesis, based upon the assumption that toxin and
antitoxin are colloids, which in essence assumes that the toxin-anti-
toxin reaction is a phenomenon of adsorption, quite unlike the reaction
of a weak acid and a weak base.

The humoral theory of immunity fails to attribute to phagocytic
cells any prominent part in immunity. No theory has been advanced,
up to the present time, which explains all the phenomena of humoral
immunity; whatever the final solution may be, the side-chain theory
as developed and defended by Ehrlich must, and always will be, a
worthy monument to a great man.

B. The Cellular or Phagocytic Theory of Immunity. The cellular
theory of immunity, formulated and championed by Metchnikoff,
had its inception in observations of the nutritive activities of amebse,
which could be watched under the microscope. It was observed
that these simple, transparent protozoa, when about to feed, ap-
proached and flowed around a minute organism, as a bacterial cell.
Shortly after engulfment the contour of the ingested bacterium lying
within the substance of the ameba became less and less distinct and

1 See Arrhenius, Immunochemie, Leipzig, 1907, for full details.

2 Jour. Exp. Med., 1909, xl, 241, Active Immunity Produced by So-called Balanced
or Neutral Mixture of Diphtheria Toxin and Antitoxin.

3 Ztschr. f. physiol. Chem., 1904, 615.

THEORIES OF IMMUNITY

127

finally disappeared entirely. His attention was soon directed to a
small, transparent crustacean, daphnia, within whose body cavity
could be distinguished minute wandering cells which exhibited ameboid
movements. The physiological significance of these ameboid cells
which are potentially leukocytes was not clear until it was found
that they engulfed and digested certain yeast spores that occasionally
gained entrance to the body cavity of the crustacean. If the yeast
spores were not too numerous the wandering cells flowed around and
eventually destroyed them; if, on the contrary, the number of yeast
spores was too great, the wandering cells could not remove the entire

FIG. 8. Phagocytosis of gonococcus.

number and the residual spores germinated and killed the host. It
was evident that the phagocytic activity of the ameboid cells played
a prominent part in protecting daphnia from an infection with the
yeast.

Next Metchnikoff injected anthrax bacilli into the lymphatic sac
of frogs and found again that wandering cells leukocytes engulfed
and destroyed the bacteria, thus preventing infection and death of
the frog. This line of observation was followed through an extensive
series of lower animals, mammals, and finally in man, where the
engulfment of the meningococci, gonococci, pneumococci, and staphy-
lococci by polymorphonuclear leukocytes during the course of acute
infections with these organisms afforded a striking demonstration

128 IMMUNITY AND INFECTION

of the phagocytic activity of leukocytes which circulate normally in
the blood and lymph streams. These and many other observations
and experiments led to the formulation of the phagocytic theory of
immunity. Natural immunity, according to this theory, is leukocytic
immunity that is, the natural barriers of the body, reenforced by
the activity of leukocytes in the blood and lymph streams which
bathe the intact skin, mucous membranes, etc., suffice to protect
the body against invasion by moderate numbers of bacteria or other
microorganisms. Infection of the body, according to this view, is
attributable to a failure of the leukocytic defense, or to too large
numbers of invading organisms, or both factors combined.

Metchnikoff classified phagocytic cells of the body into two groups :

1. .Macrocytes or Macrophages. Large mononuclear cells and certain
fixed tissue cells, particularly of the spleen, liver, lungs, and lymph
nodes. Macrophages are active in the removal of necrotic tissue,
injured blood cells, and similar abnormal cellular elements of the
body, and in chronic bacterial' infections, notably in tuberculosis,
leprosy, and actinomycosis. They contain a digestive enzyme
macrocytase which dissolves or digests these abnormal cells.

2. Microcytes or Microphages. Chiefly polymorphonuclear leuko-
cytes which occur in the blood stream. They engulf bacteria and
similar cells. Microcytes contain a digestive enzyme microcytase
which dissolves or digests bacteria.

The substance which Ehrlich regards as complement is normally
present in the leukocytes as macro- and microcytase, according to
Metchnikoff. These cytases are liberated into the blood stream when
the leukocytes are destroyed (phagoly sis) .

The phenomenon of phagocytosis may be divided into three separate
and distinct phases: the method of approach of the phagocytic cell
to its prey (chemotaxis), the engulf ment, and finally the digestion or
destruction of the latter.

The Method of Approach. It was a matter of observation by Metch-
nikoff and his followers that phagocytosis was more marked in mild
bacterial infections and during recovery than in severe infections and
the early acute stages of the disease. The importance of chemotaxis
as the attractive force of leukocytes to bacteria, however, was not
clearly realized until Massart and Bordet 1 showed by ingenious
experiments that non- virulent bacteria apparently secrete substances

1 Ann. Inst. Past., 1891, v, 417.

THEORIES OF IMMUNITY 129

which draw phagocytic cells to "them. 1 Virulent organisms of the
same strain not only do not appear to attract leukocytes, but they
appear to repel them. Bordet explained the increase of virulence of
bacteria through passage in experimental animals on the ground that
the less virulent individuals were engulfed and killed; the more viru-
lent members survived and produced a thoroughly virulent strain.
Yaillard and Vincent 2 and Vaillard and Rouget 3 showed that bacterial
toxins may repel or paralyze leukocytic activity; if tetanus spores
are bathed with tetanus toxin before injection into the animal body,
the leukocytes do not collect at the point of injection, the spores ger-
minate and the animal dies of tetanus. If, however, the spores are
washed free from tetanus toxin and then injected, leukocytes appear
at the site of inoculation, engulf the spores, and either destroy them
or prevent their germination.

The mechanism of chemotaxis has been a subject of much discus-
sion. Evidence is accumulating which would suggest that chemo-
tactic stimuli of bacterial origin which reach leukocytes enter the
phagocytic cell in greater concentration on that side which is nearer
the source of the chemotactic substance, lowering the surface tension
at that point. A flow of protoplasm in this direction, in obedience
to the lowered resistance, will result in the protrusion of a pseudo-
podium, which will continue to advance until the surface tension is
equalized. 4 This generally occurs when the leukocyte has flowed
around or engulfed the organism.

Engulfment. The earlier view associated the protrusion of pseudo-
podia and the subsequent engulfment of bacteria or other cell as an
auto voluntary act of the leukocyte. The inclusion of inert particles,
as dust or other minutely comminuted granules, would appear to
discredit this hypothesis. The engulfment of living or inert bacteria
or other minute bodies is, as Wells aptly expresses it, 5 " but an exten-
sion of the phenomena of chemotaxis. When the substance toward
which the leukocyte is drawn is small enough, the leukocyte simply
continues its motion until it has flowed entirely about the particle."

Digestion. The ultimate solution of engulfed substances other
than purely inert particles is by intracellular enzymes contained within

1 Inert particles, as coal dust, are engulfed by phagocytic cells; it is difficult to explain
this phenomenon on the basis of chemotaxis.

2 La semaine medicale, 1891, xi, No. 5.

3 Ann. Inst. Past., 1892, vi, No. 6.

4 See Well's Chemical Pathology, 1914, 2d ed., pp. 230-251 (Saunders & Co.), for
an excellent resume of the literature.

6 Well's Chemical Pathology, 1914, 2d ed., p. 238 (Saunders & Co.).
9

130 IMMUNITY AND INFECTION

the phagocytic cells. These enzymes are of two kinds: macrocytase,
present in the macrophages, and microcytase, found in the micro-
phages. 1 Van de Velde, 2 Buchner, 3 Hahn, 4 and Bordet 5 have demon-
strated such endo-enzymes. The solution of bacteria engulfed in leuko-
cytes can be shown by appropriate staining methods; the organisms
gradually lose their ability to take up stain and eventually disappear.

At this stage of the development of the phagocytic theory of immu-
nity, the important part played by the blood serum in preparing bac-
teria for phagocytosis was prominently set forth in the investigations
of Wright and Douglas, 6 although foreshadowed by the excellent and
comprehensive observations of Denys and LeClef 7 and Neufeld and
Rimpau. 8 Wright and Douglas showed that leukocytes, freed care-
fully from adherent serum by washing with salt solution, would not
engulf bacteria, or, at least, but slowly. The addition of serum from
a normal or immunized animal caused active phagocytosis to take
place. The substances in the blood serum which prepare bacteria
for engulfment by leukocytes were called "opsonins" by Wright and
Douglas: the immune opsonins which are specifically increased in
immunized animals are almost certainly identical with the substances
called bacterial tropins by Neufeld and Rimpau. That the opsonic
substances of the serum act primarily upon the bacteria rather than
upon the leukocytes was clearly shown by the observations of Hektoen
and Reudiger. 9 Streptococci suspended in plasma, blood serum or
defibrinated blood were engulfed by leukocytes. Leukocytes, washed
free from serum or plasma, were without phagocytic action upon the
same bacteria. If the streptococci, however, were allowed to stand
in contact with serum, plasma, or defibrinated blood for a short time
at 37 (a much longer exposure at to 4 C. was necessary), then
washed free from adherent serum or plasma, and exposed to washed
leukocytes, active phagocytosis took place.

The present tendency is to ascribe to phagocytosis an important
part both in the destruction of many kinds of invading bacteria and
in the removal of alien or abnormal cells as well. The importance

1 For a detailed discussion of leukocytic enzymes, see Opie, Jour. Exp. Med., 1905,
viii, 410.

2 La Cellule, x, 2; Cent. f. Bakt., 1898, xxxiii, 692.

3 Miinchen. med. Wchnschr., 1894, 718.

4 Arch. f. Hyg., 1895, xxviii, 312.
8 Ann. Inst. Past., 1895, ix, 398.

6 See Wright, Studies in Immunization, 1909, Constable.

7 La Cellule, 1895, xi.

8 Deutsch. med. Wchnschr., 1904, 1458.

9 Jour. Inf. Dis., January, 1905, ii, No. 1.

THEORIES OF IMMUNITY 131

of substances contained within the plasma or blood serum, which
prepare bacteria for phagocytosis to use Wright's terminology has
modified somewhat the original conception of phagocytosis as proposed
by Metchnikoff.

The phagocytic theory and the humoral theory of immunity would
appear to be in direct opposition. Metchnikoff maintained that the
fundamental basis of immunity resides in the phagocytic activity of
macro- and microphages. He believed that the humoral immune
bodies are derived either from leukocytes or the organs in which
they are formed the bone marrow and lymphatic system. The
champions of the humoral theory, on the other hand, would attribute
the healing principle to soluble substances contained in the body
fluids. The leukocytes and other phagocytic cells, according to the
extremists who advocate this theory, would be rega/ded as scavengers
merely, whose function it is to remove the debris dead bacteria or
disabled bacteria after they are overwhelmed by the activity of the
soluble natural and immune antibodies.

A final decision of the importance of cellular and humoral factors
in immunity cannot be made at the present time. It is not unlikely
that both theories will be modified somewhat as additional evidence
accumulates.

CHAPTER VII.
ANAPHYLAXIS, ALLERGY OR HYPERSENSITIVENESS. 1

PROTEIN fed to man or animals is reduced to simple compounds,
chiefly amino-acids, by the action of gastro-intestinal enzymes before
it is absorbed from the alimentary canal. These gastro-intestinal
enzymes act rapidly under normal conditions, and without an appre-
ciable latent period. One noteworthy result of digestion is a complete
denaturization of all ingested protein before it enters the tissues of
the host; absorption of unaltered or partially-digested protein is
prevented or reduced to a minimum.

The importance of a denaturization of protein before it enters the
tissues becomes apparent when a comparison is made between the
effects of parenteral injections of the end-product^ of prnfpin
on the one hand, and of the unaltered protein jtsplf nn tVi
Repeated parenteral injections of amino-acids in moderate amounts
appear to be without serious or noteworthy effects upon experimental
animals. A single parenteral injection of an unaltered protein is also
without visible effect, as a rule. A second parenteral injection of the
same protein, after an interval of ten to fourteen days, frequently is
followed by a rather definite train of symptoms, severe in character
and wholly unlike the negative response to a corresponding treatment
with amino-acids or normal end-products of gastro-intestinal digestion.

Sensitization. The first parenteral injection of a protein 2 which
is foreign to the body, or in some instances, natural for the body but
alien for the blood, is without visible effect upon the animal, but leads
to its sensitization to the specific protein. The sensitizing agent is
variously referred to as a sensitizer, sensibilisinogen, or anaphylac-
togen, and may be effective in very small doses. Rosenau and Ander-
son 3 were able to sensitize guinea-pigs with one-millionth of a cubic
centimeter of horse serum; Wells 4 has sensitized the same animal

1 For an excellent resume of the literature of anaphylaxis complete to 1912, see
Hektoen, Jour. Am. Med. Assn., 1912, Iviii, 1081.^

2 Proteins deficient in tryptophane or tyrosin are said not to sensitize.

3 Bull. 29 and 36, Hygienic Laboratory, Washington, D. C., 1906, 1907.

4 Wells' Clinical Pathology, 1914, 2d ed., 180.

REINJECTION OF THE HOMOLOGOUS PROTEIN 133

with one twenty-millionth of a gram of crystallized egg albumen.
Usually 0.001 to 0.1 c.c. of serum is an effective sensitizing dose.

A latent period intervenes between the initial injection of the
animal with sensitizing protein and sensitization on the average
this is about ten to fourteen days. Gay and Southard 1 showed, how-
ever, that the time necessary to effect sensitization depends somewhat
upon the size of the sensitizing dose, larger amounts requiring longer
periods than smaller amounts. White and A very 2 have found that a
relation exists between the minimum sensitizing and the maximum
intoxicating dose, larger amounts of protein being required on rein-
jection to elicit a reaction when the sensitizing dose is very small, and
vice versa.

Reinjection of the Homologous Protein. Repeated injections of the
homologous protein spaced at intervals less than ten days do not,
as a rule, cause symptoms of acute anaphylaxis after a third or a
fourth injection, however, there appears at the site of the first injec-
tion a swelling, usually indurated and more or less edematous, which
may lead to extensive necrosis and sloughing. These local reactions,
the so-called Arthus 3 phenomenon, are closely related phylogenetically
to the anaphylactic symptoms described below.

If the second parenteral injection is made after sensitization is
established usually after ten to fourteen days symptoms follow
almost immediately, which vary somewhat according to dosage and
the site of inoculation. A very large dose frequently results in rapid
death, the Theobald Smith phenomenon. 4 Very broadly speaking,
it requires 200 to 2000 times as much protein to cause acute anaphy-
laxis as to effect sensitization.

Intravenous or intracerebral injections of moderate doses are fol-
lowed very soon by a period of excitement (in dogs, followed by a
period of depression), 5 the animal is restless and moves about in a
bewildered manner and shows signs of respiratory embarrassment.
It coughs (a normal guinea-pig rarely or never coughs) and scratches
the corners of its mouth. This state is followed by dyspnea, with
involvement of the diaphragm and bronchial musculature leading to

1 Jour. Med. Res., 1908, xviii,.407.

2 Jour. Inf. Dis., 1913, xiii, 103.

3 Compt. rend. Soc. Biol., 1903, Iv, 20; 1906, Ix, 1143.

4 Theobald Smith, Jour. Med. Res., 1905, xiii, 341; Otto, Leuthold-Gedenkschrift,
1096, i, 153.

5 Guinea-pigs in general react most strikingly to anaphylactic stimuli; man is less
sensitive. Rabbits, sheep, goats, horses, and birds, in the order mentioned, are less
susceptible than man. Cold-blooded animals appear to be refractory.

134 ANAPHYLAXIS, ALLERGY OR HYPERSENSITIVENESS

bronchial spasm and later to paralysis of respiration, 1 lowered blood-
pressure, frequently cyanosis, an4 jdeath. Smaller intravenous injec-
tions are followed by the saijfce sympt$|Kis of excitement and respiratory
involvement, but to a lessfc degree, j Frequent micturition and fluid,
often bloody stools togetheV^ItK^reat prostration and dyspnea are
usually observed. The animal cannot stand and may die after several
hours, or eventually recover.

Intraperitoneal injections elicit similar symptoms. Subcutaneous
injections rarely cause acute death; as a rule the animal has a febrile
reaction and repeated injections may be followed by the Arthus pheno-
menon. If the animal survives an anaphylactic reaction it is fre-
quently observed to be more refractory or even temporarily immune
to subsequent injections of the same protein. This refractory state
is called anti-anaphylaxis by Besredka and Steinhardt. 2 This period
of refractoriness is of variable duration.

The postmortem appearance of guinea-pigs which have died from
the effects of acute anaphylaxis is usually striking and characteristic.
The lungs remain fully distended when the thorax is opened, the cut
surface is rather dry, and death appears to have resulted from
asphyxiation due to a tonic spasm of the bronchial musculature. 3

Severe but non-fatal anaphylactic reactions are accompanied by a
lowering of the body temperature, lowered arterial pressure, leucopenia,
frequently with a temporary partial or complete loss of coagulability
of the blood, 4 followed by a secondary febrile rise of temperature and
a leukocytosis in which polymorphonuclear leukocytes and frequently
eosinophiles 5 are increased. Animals killed during the early acute
symptoms show but little distention of the lungs the lesions may
resemble those of an acute toxic gastro-enteritis. Ecchymoses and
ulcers may be found occasionally in the stomach and intestines,
together with parenchymatous degeneration of the liver and particu-
larly the kidneys, which may lead eventually to fatty degeneration of
these organs.

The symptoms of anaphylaxis may be masked or even prevented by
the administration of certain drugs immediately before the reinjec-
tion of these atropin, chloral hydrate and similar narcotics are con-
sidered particularly efficient.

1 Auer and Lewis, Jour. Am. Med. Assn., 1909, liii, 6; Biedl and Kraus, Wien. klin.
Wchnschr., 1910, 844.

2 Ann. Inst. Past., 1907, xxi, 117, 384.

3 Auer and Lewis, loc. cit.

4 Biedl and Kraus, Wien. klin. Wchnschr., 1909, 363; Friedberger and Grober, Zeit.
f. Immunitatsforsch., 1911, ix, 216.

B Moschowitz, New York Med. Jour., 1911, Ixxxxiii, 15.

THE NATURE OF THE POISON, ANAPHYLATOXIN 135

THE NATURE OF THE POISON, ANAPHYLATOXIN.

The anaphylactic reaction, like other serological reactions, appears
to depend upon the elaboration of a specific antibody in the sen-
sitized animal. The specificity of the reaction is very striking in the
physiological sense the serum of one animal fails to sensitize for the
serum of an unrelated animal. Egg protein of one species also fails
to sensitize an animal against the egg protein of another species.
Osborne and Wells, 1 using vegetable proteins which can be obtained
in a state of relative purity, have shown that sensitization, in the
last analysis, depends chiefly upon the chemical composition of the
sensitizer. Thus, one vegetable protein fails to sensitize against a
second, unlike protein, even though they be derived from the same
seed.

The specificity of the reaction is striking it takes place only in
response to a second injection of the_jipmplogQus protein, but the
symptomatology is essentially the same, irrespective of the sensitizer.
The promptness with which the reaction appears after the reinjection
suggests at once that the poison, is radically different from a true
bacterial toxin, which invariably requires a definite latent, period
before symptoms can be detected,.] In this respect the anaphylatoxin
resembles somewhat an alkaloidal poison. Up to the present time no
antitoxins have been prepared. The action of the poison is peripheral
rather than central, according to Auer and Lewis. 2 Schultz 3 and
others have shown that it acts powerfully upon smooth muscle fibers;
Biedl and Kraus 4 and others have shown that an injection of peptone
into dogs elicits symptoms and pathological changes indistinguishable
from those of anaphylaxis. They were inclined to regard the anaphy-
latoxin as similar to, or possibly identical with peptone. Animals
immune to anaphylactic reactions react slightly or not at all to peptone
injections.

Passive anaphylaxis may be induced in a non-sensitized animal
by an injection of the serum of a sensitized animal. Usually a few
hours elapse before the recipient of the specific antibody is reactive,
however. The experiments of Pearce and Eisenbrey, 5 of Weil, 6 Dale, 7

1 Jour. Inf. Dis., 1913, xii, 341.

2 Loc. cit.

3 Hygienic Laboratory Bulletin, 1912, No. 80.

4 Wien. klin. Wchnschr., 1901, No. 11.

5 Journ. Inf. Dis., 1910, vii, 565.

6 Jour. Med. Res., 1913, xxvii, 497; 1914, xxx, 87, 299.

7 Jour. Pharm. and Exp. Therap., 1913, iv. 167.

136 ANAPHYLAXIS, ALLERGY OR HYPERSENSITIVENESS

Schultz 1 and others indicate that the reaction occurs within the cells
of the body rather than in the blood stream. The urine of anaphy-
lactic animals is toxic and 2 c.c. is frequently sufficient to kill guinea
pigs with anaphylactic symptoms, according to Pfeiffer. 2
<( Anaphylaxis may be defined as a congenital or acquired condition
of hypersensitiveness of man or animals tojthe parenteral introduction
of proteins, which is incited byjme or more injections of Jbacterial,
plant, animal or huma_n protein^ Active acquired hypersensitiveness
can be transmitted to non-sensitized individuals by the injjectionpf
the serum of an anaphylacticized individual, inducing in the recipient
of the serum a condition of passive anaphylaxis.) Anaphylaxis, there-
fore, belongs to the group of immuhological reactions.

Theories. Vaughan 3 has shown that all proteins may be split into
two fractions if they are heated with alcoholic potassium hydroxide;
one portion, insoluble in alcohol, when injected into animals gives
symptoms indistinguishable from those of anaphylaxis, irrespective
of the protein. The alcohol-soluble fraction is not toxic. The alcohol-
insoluble fraction obtained from various animal, vegetable, and
bacterial proteins always reacts the same, not only symptomatically,
but quantitatively as well. His theory is that the protein molecule
consists of two parts: an archon or nucleus, which is poisonous and
elicits the symptoms of anaphylaxis when it is injected parenterally
into animals, and common to all proteins; and additional groups
which are non-poisonous, but confer upon a protein by their number
and arrangement, its specificity. When a protein is injected paren-
terally into an animal, the cells of the animal elaborate an enzyme
which will specifically disintegrate it. Among the products of disin-
tegration is the poisonous nucleus or archon in a more or less free
state. The liberation of this substance causes acute poisoning of the
host. This substance, for which no antibody or antitoxin has been
prepared so far, is the "endotoxin" of bacteria.

Many of the phenomena of anaphylaxis are readily explained in
the light of Vaughan's work. The latent period or pre-anaphylactic
state which intervenes between the injection of a protein and the
appearance of sensitization is the time required to mature the specific
enzyme. The specificity of the enzyme (called forth by the stimulus
of alien protein in the tissues) is determined by the arrangement and

1 Loc. cit.

2 Zeit. f. Immunitatsforsch., 1911, x, 550.

3 Protein Split Products, 1913, for full discussion.

THE NATURE OF THE POISON, ANAPHYLATOXIN 137

number of groups arrayed around the poison group of the protein;
the similarity or identity of the symptoms of anaphylaxis irrespective
of the protein depends upon the liberation of the poison nucleus
(common to all proteins) in a relatively free state. The induction
of passive sensitization depends upon the injection of this specific
enzyme, which is present in the serum of a sensitized animal, into a
non-sensitized animal. 1

Vaughan regards the formation of a specific proteolytic enzyme in
response to the injection of alien protein into the tissues as a protec-
tive mechanism to rid the body of foreign substance; the theoretical
importance of this conception as a purposeful reaction is clearly
shown in bacterial infections. The incubation period of many bac-
terial infections is about two weeks, during which clinical symptoms
are not pronounced. This is interpreted as the time required by the
cells of a host to mature a specific enzyme capable of disintegrating
the alien protein (bacterial cells). The symptomatology of bacterial
disease is caused largely by the liberation of the poisonous nucleus
of the bacterial protein in special tissues or organs. Natural immunity
to bacterial disease, according to this theory, is due to the inability
of the organism to grow in the tissues of the host; active immunity
is conferred on the host by the presence of a persistent enzyme which
will disintegrate the specific organism whenever it is reintroduced
into the body.

Chemically, the poison nucleus or endotoxin is stated by Vaughan
to resemble beta-imidazoleethylamine, described previously. 2 The
specificity of the anaphylactic reaction depends upon the cleavage of
the protein molecule by a specific proteolytic enzyme with the libera-
tion of a non-specific poisonous product of protein degradation.
Abderhalden 3 and his associates have demonstrated proteolytic
enzymes in the blood stream.

Friedberger 4 has shown that a poison may be obtained by incubating
the inactivated serum of a sensitized animal with an excess of com-
plement and homologous (sensitizing) protein, which, when injected
into guinea-pigs, elicits the symptoms of anaphylaxis. It is not
true toxin, for no antibody is produced in response to repeated, sub-
lethal injections; it appears to differ from Vaughan's poison in that

1 The importance of the degradation of protein in the alimentary tract can be appre-
ciated in the light of what has been stated about anaphylaxis.

2 See page 76.

3 Zeit. f. physiol. Chem., 1912, Ixxxii, 109; Abwehrfermente des tierischen Organismus,
Berlin, 1913.

4 Zeit. f. Immunitatsfbrsch., iv, 636; vii, 94; Ueber Anaphylaxie, Ibid., 1911, ix, 394
(in collaboration with Goldschmidt, Schmanowsky, Schiiltze, and Nathan).

138 ANAPHYLAXIS, ALLERGY OR HYPERSENSITIVENESS

it is destroyed or inactivated at a temperature above 65 C. The
poison does not form if complement is not present in solution with
the inactivated serum and antigen, which would suggest a resemblance
to other cytolytic reactions in which the specific amboceptor is acti-
vated by complement.

The essential distinction between the theory of Vaughan and that
of Friedberger would appear to rest upon the nature of the poisonous
substance liberated; Vaughan would maintain the specificity of the
enzyme and the identity of the poisonous substances formed from
various proteins. Friedberger's theory, which was developed several
years after Vaughan's first work, would emphasize the distinction
between an enzyme and the specific amboceptor, which requires com-
plement for its activation. Keysser and Wassermann 1 and more
recently, Jobling and Petersen 2 have found that serum shaken with
kaolin, chloroform, and other agents will absorb substances from
serum, leaving the remainder toxic for guinea-pigs; the reaction
induced by the injection ef small amounts of altered serum resembles
closely that of anaphylaxis. Jobling and Petersen believe that the
toxic substance originates not from bacteria necessarily, but from
serum itself. Under normal conditions, anti-enzymes prevent the
normal serums from causing auto-autolysis; kaolin, bacteria, etc.,
added to the serum, absorb and thus remove the anti-enzymes, thus
permitting the serum to digest itself. In other words, the poisonous
substance may originate in the serum rather than in the bacteria or
other alien protein. These facts do not necessarily detract from
Vaughan's theory, but until more is known of the entire subject, a
final discussion of the mechanism of anaphylaxis must be postponed.

ANAPHYLAXIS IN MAN.

Natural Hypersensitiveness. It has long been known that the
inhalation of organic substances as the pollen of various plants, or
emanations from horses or guinea-pigs, of peptone or other similar
material may excite acute coryza and that train of symptoms popu-
larly recognized as " hay-fever" or "pollen fever" in some, but by no
means all, individuals. If the specific pollen or dust is rubbed on the
nasal mucosa of these sensitized individuals a violent reaction will
take place. Other individuals develop a severe urticaria if they eat
certain proteins: the flesh of arthropods, particularly crabs and
lobsters, vegetables, eggs, milk are known to excite symptoms in indi-

1 Ztschr. f. Hyg., 1911, Ixviii, p. 535.

2 Jour. Exp. Med., June, 1914, xix, p. 480.

ANAPHYLAXIS IN MAN 139

viduals who exhibit an "idiosyncrasy" to one or another of these
substances. This idiosyncrasy to foreign protein may be either
congenital or post-natal; the protein is supposed to have passed
unchanged through the intestinal tract in the latter case. The pheno-
mena in these instances are explained on the basis of sensitization
with specific protein; a mild anaphylactic reaction occurs when
the specific dust reaches the nasal mucous membrane or the specific
protein enters the digestive tract.

The tendency at the present time is to regard certain clinical and
pathological symptoms of bacterial infections particularly fever
and the production of specific pathological lesions as manifestations
of anaphylaxis as outlined by Vaughan. 1 The body is sensitized to
the alien protein, be it organic dust, protein of the food, or invasive
bacteria; the anaphylactic reaction takes place when the homologous
protein is brought into contact with the sensitized individuals through
the proper channels. It will be remembered that the incubation period
in many bacterial infections was explained as the time elapsing between
the arrival of the alien protein (bacterial cells) in the tissues of the
host and the maturing of a specific proteolytic enzyme that would
effect their disintegration. The symptomatology of bacterial infec-
tions, according to Vaughan, is largely due to the liberation of the
anaphylatoxin incidental to the lysis of the residual organisms.

Artificial or Acquired Hypersensitiveness. The phenomena
grouped for convenience as acquired hypersensitiveness are met with
chiefly in connection with the administration of the sera of animals
immunized for therapeutic purposes. Three types of anaphylactic
reaction may be recognized :

1. Sudden Death. A very few cases are on record in which the
administration of antitoxin for therapeutic purposes, either for
immunization or curatively, has been followed within a few minutes
or hours by death. Already, in 1896, Gottstein 2 had collected 12
which followed the injection of diphtheria antitoxin, 8 of whom were
diphtheritic, 4 healthy individuals. About 1 in every 50,000 appears
to be the proportion of deaths due to an injection of therapeutic sera.
The symptoms are essentially those observed in sensitized experi-
mental animals which die shortly after the injection of the homologous
protein. Behring, Kitasato and other observers had noticed many
years ago, when antitoxin was first prepared on a large scale, that
animals immunized with large amounts of tetanus or diphtheria toxin

1 Loc. cit.

2 Therap. Monatschr., 1896, Heft 5.

140 ANAPHYLAXIS, ALLERGY OR HYPERSENSITIVENESS

occasionally succumbed to a subsequent small dose of the homologous
toxin, although the blood serum of these animals contained much
specific antitoxin.

2. Serum Sickness or Serum Disease. Attention was first directed
to serum sickness by von Pirquet and Schick, 1 who noticed that there
occasionally developed in individuals who had received an injection
of antitoxic sera, usually after seven to fourteen days, fever and a
rash which might be urticarial, scarlatinal, or, in the more severe
cases, morbilliform; enlargement of lymph glands, particularly those
near the site of inoculation; and joint pains, more frequently of the
metacarpal joints. A slight edema, frequently of the angioneurotic
type, was also occasionally observed. The fever is usually slight
and there are signs of respiratory embarrassment, not as a rule
marked, but occasionally severe. These reactions, sudden death
and serum sickness, are more common in asthmatics, and in those
individuals presenting the pathological syndrome known as status
lymphaticus.

According to Moschowitz, 2 these individuals, particularly the
asthmatics, present an eosinophilia. The exact cause of sudden death
following the administration of diphtheria antitoxin is not definitely
known, but it has been assumed that respiratory involvement is a
potent factor. The appearance of serum disease seven to fourteen
days after the administration of antitoxin is supposed to depend upon
the fact that some of the alien protein (serum) remains in the body
during the period of pre-anaphylaxis (period of sensitization), and
that this residual protein is broken down by the mature specific enzyme
or enzymes with the liberation of a poisonous substance which causes
the anaphylactic shock.

3. Arthus Phenomenon. During the course of immunization against
rabies by the Pasteur method it is frequently noticed that after three
or four injections a subsequent injection causes symptoms of inflam-
mation at the site of the first injection, and that this phenomenon is
repeated, usually, but not always, with diminishing intensity at the
site of earlier injections as the treatment progresses. This inflam-
matory reaction at the site of injection is not due to bacterial infection
ordinarily, but is rather an expression of anaphylaxis. It is comparable
to the Arthus phenomenon produced in rabbits by successive injec-
tions of serum referred to above. Also in re vaccination (vaccinia)
a so-called accelerated reaction may occur the second time the indivi-

1 Die Serumkrankheit, Leipzig, 1905.

2 New York Med. Jour., 1911, Ixxxxiii, 15.

ANAPHYLAXIS IN MAN 141

dual is vaccinated. This accelerated reaction again is a mild edition
of the Arthus phenomenon.

4. Prophylaxis. At first sight it might appear that the administra-
tion of diphtheria and tetanus antitoxin for therapeutic purposes would
be a dangerous procedure. If there is reason to suspect that the
patient would react to the injection of antitoxin it is advisable to
inject 0.1 or 0.2 c.c. subcutaneously and wait half an hour. If no
symptoms develop, the full dose may be given without danger; it
is generally believed that even if mild symptoms do follow the initial
injection, the full dose may be given with safety after half an hour;
the first injection appears to abort what otherwise might be a reaction
dangerous to the patient

The present method of concentrating diphtheria antitoxin by frac-
tional precipitation of the globulin 1 appears to reduce very materially
the incidence of serum sickness. According to German investigators,
antitoxin which has stood for one or two months has lost to a very
considerable extent the substance or substances which cause the
symptoms of serum sickness.

Practical and Theoretical Considerations. A. Advantage is taken
of the sensitization of individuals by bacterial protein during certain
bacterial infections, particularly those with the tubercle bacillus,
B. mallei, and in syphilis, for diagnostic purposes. It has been shown
almost beyond doubt that individuals suffering from these diseases are
sensitized to the bacterial protein, and it is possible to make a fairly defi-
nite clinical diagnosis by introducing extracts of the specific organisms
into the skin and inducing there an anaphylactic reaction which, if the
dose is small, is local in character, but which may be general and severe
if the dose is increased in amount. The von Pirquet, Calmet, Moro, and
Koch methods of utilizing tuberculin for diagnostic purposes are directly
dependent upon this reaction of hypersensitiveness. The diagnostic
use of mallein and luetin depend upon the same phenomenon.

B. Advantage is also taken of the specificity of the anaphylactic
reaction for the recognition of proteins. Wells and Osborn 2 and
many others have sensitized guinea-pigs with proteins and then
injected into these sensitized animals proteins which are to be iden-
tified either specifically or phylogenetically. The nature and extent
of the anaphylactic reaction in these animals furnishes the most deli-
cate test (except possibly the precipitin test) which is available for
such investigations.

1 Banzhof, Johns Hopkins Hosp. Bull., 1911, xxii, 241.

2 Loc. cit.

CHAPTER VIII.

ANTIGENS AND THE TECHNIC OF SERUM
REACTIONS.

NATURE OF ANTIGENS AND ANTIBODIES.
AGGLUTININS AND PRECIPITINS.
LYSINS.

Hemolysis and" the Complement Fixa-
tion Reaction.

AGGRESSINS.

OPSONINS, TROPINS. BACTERIAL VAC-
'CINES.

NATURE OF ANTIGENS AND ANTIBODIES.

THOSE substances which cause specific antibody formation when
they are introduced into the tissues or the body fluids of the host are
called antigens. Their chemistry is as yet unknown, but available
evidence would indicate that they are protein in nature and highly
organized chemically. Degradation products of proteins, as albu-
moses and peptones and carbohydrates and fats, are not ordinarily
antigenic, that is, they do not lead to antibody formation when they
are introduced into the animal body. 1 The antigenic properties of
lipoids are still a subject of controversy: lipoids appear to play a
prominent part in certain types of immunological reactions, but their
ability to stimulate specific antibody formation cannot be regarded
as proven at the present time. 2

The function of antibodies as specific offensive weapons of the
host against alien organisms or their products has long been recog-
nized in bacteriology, and most important laboratory diagnostic
methods have been elaborated through a study of the reactions
between specific antigens and their respective antibodies. Antibodies
are soluble and are found in various concentrations in blood serum
derived from immunized animals. Many attempts have been made
to determine changes in the chemical composition or physical proper-
ties of immune sera from those of normal serum. Atkinson, 3 Gibson,

1 The injection of carbohydrates and fats may, however, lead to specific enzyme
formation. See Rohmann, Antigene Wirkung der Kohlenhydrate, Deutsch. med.
Wchnschr., 1914, xl, 204.

2 See Pick, Kolle, and Wassermann, Handbuch der pathogenen Mikroorganismen,
2d ed., Bd. I, for discussion of the chemistry of antigens.

3 Jour. Exp. Med., 1899, iii, 649.

AGGLUTININS. AGGLUTINOIDS AND PROAGGLUTINOIDS 143

and Banzhaf 1 and others have found that the sera of horses immunized
to diphtheria toxin show a marked increase in globulin content, with
a decrease in albumin content. Beljaeff 2 could find no appreciable
change in the refractive index, specific gravity, freezing point or
reaction of the serum of an immune animal above that of a normal
animal.

The chemical nature of antibodies, aside from their apparently
close relation to globulin, has not been determined. There is evidence
that antitoxin molecules may be larger than toxin molecules, how-
ever. Martin and Cherry 3 found that toxins could be forced through
dense porcelain filters impregnated with gelatin, which would restrain
antitoxin, and Arrhenius and Madsen 4 determined that the toxin mole-
cule diffused several times as rapidly as the antitoxin molecule, from
which observation they assumed that the antitoxin molecule was
larger than the toxin molecule.

AGGLUTININS. AGGLUTINOIDS AND PROAGGLUTINOIDS.

Gruber and Durham 5 appear to have been the first to clearly demon-
strate specific clumping in broth cultures of typhoid and cholera
organisms when their respective sera were added to them. Somewhat
later Widal, 6 and independently Griinbaum, 7 utilized the principle
of the specific agglomeration of bacteria by their immune sera for
the diagnosis of typhoid fever. They found that relatively early in'
the disease, sera of typhoid patients clumped typhoid bacilli from
broth cultures. Pfaundler 8 observed that typhoid bacilli grown in
broth containing low concentrations of specific sera grew out into long,
tangled filaments, the " thread" reaction. Originally this phenome-
non was regarded as highly specific, but it has largely given way to the
macroscopic or microscopic agglutination test.

Agglutination in the bacterial sense may be defined as a clumping
or agglomeration of bacteria from a uniform suspension in a fluid
medium, brought about by the addition of specific antibodies
agglutinins. It takes place in two stages if motile bacteria are con-
cerned. First there is loss of motility "immobilization" and

1 Jour. Exp. Med., 1910, xii, 411.

2 Cent. f. Bakt., Orig., 1903, xxxiii, 293, 396.

3 Proc. Royal Soc., 1898, Ixiii.

4 Festskrift Statens Serum Institute, 1902.
6 Munchen. med. Wchnschr., 1896, No. 13.

6 La Semaine Medicale, 1896, No. 13.

7 Brit. Med. Jour., 1897, May 1, and Munchen. med. Wchnschr., 1897, 330.

8 Cent. f. Bakt., 1898, xxiii, 9, 71, 131.

144 ANTIGENS AND THE TECHNIC OF SERUM REACTIONS

eventually clumping. Smith and Reagh 1 working with a non-motile
hog cholera bacillus have demonstrated both flagella and somatic
agglutinins, the former paralyzing the activity of the flagella, the
latter agglomerating the organisms themselves. Non-motile bacteria
usually agglutinate somewhat more slowly than motile organisms.
Small amounts of neutral salts are necessary for the clumping of
bacteria, 2 although a union of the specific organism and its agglutinin
will take place even if salts are absent. The specific substance (or
substances) of the bacterial cell which reacts with the specific antibody
of the serum (agglutinin) is known as agglutinogen. Closely related
bacteria, as typhoid and paratyphoid bacilli, may possess a certain
amount of agglutinogen in common, but, as a rule, the specific
organisms are clumped in immune sera at much, greater dilution than
related organisms are clumped. Also, the specific organisms will
remove the agglutinin completely from immune sera, while closely
related bacteria only remove that portion of the agglutinating sub-
stance which is common to both organisms, leaving behind the
specific agglutinin which will then agglutinate the specific organism,
but not its closely related fellow; that is to say, closely related
bacteria will react with the common or group agglutinin, but fail to
absorb the specific agglutinin.

Experience has shown that the sera of normal adults frequently
contain agglutinin which will clump various bacteria and the potency
of these "normal" or natural agglutinins may even be sufficient to
clump moderate numbers of typhoid bacilli in dilutions as great as
1 to 30. The sera of normal nurslings contain only minimal amounts
of normal agglutinins as a rule, and the conclusion has been drawn
that normal agglutinin may be either:

(a) Group agglutinin, derived from mild infection with closely
related organisms, or

(6) True immune agglutinins resulting from mild or unrecognized
infection with the specific organism.

No definite distinction has been noted between natural and immune
agglutinins; the latter are usually present in sera, however, in much
greater concentration than the former.

The site of formation of agglutinins in the body is not definitely
known, although lymphoid tissues appear to be intimately concerned,
especially bone-marrow and the spleen. Pryzgode 3 states that

1 Jour. Med. Res., August, 1903, x, No. 1.

2 Bordet, Collected Studies in Immunity, 1909 (translation by Gay).

3 Wien. klin. Wchnschr., 1913, xxvi, 84.

AGGLUTININS AGGLUTINOIDS AND PROAGGLUTINOIDS 145

cultures of spleen tissue in vitro will form specific agglutinins for
typhoid bacilli if the virus is brought into contact with the tissue
cells. As a general rule the concentration of a specific agglutinin is
greater in the blood stream than in the tissues of the body.

Preparation of Specific Agglutinating Sera. Specific agglutinating
sera for experimental purposes are best obtained from rabbits, whose
serum normally contains no agglutinin. Several, usually three to
five intravenous injections of 1, 2, 3 and 5 loopfuls respectively of
killed cultures of typhoid bacilli at eight-day intervals, produce pow-
erful agglutinating sera. The animal is bled about two weeks after
the last injection. For large amounts of agglutinating sera horses or
asses must be used.

Properties of Agglutinins. Agglutinins are of unknown chemical
composition, but they may be separated from solution by those pre-
cipitants w r hich throw down globulins, and they may be removed
from solution by absorption in animal charcoal. Toward heat they
are moderately resistant, usually remaining active after an exposure
of twenty minutes to 55 C., a degree of heat sufficient to inactivate
complement. Agglutinins, therefore, appear to be quite distinct
from bacteriolysins. The temperature at which agglutinins are de-
stroyed depends upon their specificity, agglutinins for plague bacilli
being more sensitive than typhoid agglutinins. The reaction of the
medium also affects .their stability. Alkalis, even in dilute solution,
rapidly destroy agglutinins; acids are somewhat less harmful. Nat-
urally the duration of exposure to these various agents exercises an
important influence upon their resistance. Agglutinins do not appear
to pass through parchment membranes, but it is stated that agglu-
tinogen will slowly diffuse under similar conditions. This would sug-
gest that the agglutinin molecule is larger than the agglutinogen
molecule. Preserved in a dry state, in a cool place away from light,
agglutinins preserve their properties unimpaired for days. In
solution and upon standing agglutinins rapidly lose their property
of clumping bacteria, but they still retain their original ability to unite
firmly with bacteria. Ehrlich designates agglutinins which have
lost their ability to cause clumping but still retain their combining
power for agglutinogen, agglutinoids. In his terminology they are
side-chains of the second order which have lost their agglutinophore
(ergophore) group. Agglutinins acting in neutral salt-free media
also fail to cause clumping of bacteria, but in this case the addition
of a small amount of NaCl or even some weak acid very soon brings
10

146 ANTIGENS AND THE.TECHNIC OF SERUM REACTIONS

about a typical reaction. 1 This and similar observations have
attracted attention to the similarity between the precipitation of
bacteria to which agglutinin is anchored by neutral salts, and the
precipitation of finely suspended 'clay by the addition of neutral salts;
the inference has been drawn that the phenomenon of agglutination

one of physico-chemistry.

Specificity of Agglutination Reactions: Group Agglutinins. The
composition of the agglutinogen that constituent of the bacterium
which stimulates agglutinin formation is unknown, but it appears
to be complex and probably not a single chemical compound. Closely
related bacteria may possess in common a small amount of agglu-
tinogen a least common multiple, as it were which stimulates the
production of "group agglutinin" that reacts with related bacteria
more or less in proportion to their content of the common antigen or
agglutinogen. . The specific agglutinin produced by the entire agglu-
tinogen content of an organism is more potent and fails to react with
related bacteria. Thus, the serum of an animal immunized against
B. typhosus may agglutinate that organism in a dilution of 1 to 3000;
B. paratyphosus will be agglutinated in a dilution of 1 to 300 by the
same serum, and B. coli would agglutinate only in a dilution of 1 to
50. The group agglutinin in this example would be effective for B.
paratyphosus in a dilution of 1 to 300, but in greater dilutions it would
be ineffective. For B. coli in the instance cited, the group agglutinin
is ineffective in dilutions above 1 to 50.

The common or group agglutinin for B. paratyphosus in this typhoid
serum could be quantitatively removed by leaving it in contact with
a large number of paratyphoid bacilli for a few hours, then centrifu-
galizing to remove the organisms. The residual serum would contain
only agglutinin specific for B. typhosus. If B. typhosus were added
to the serum, all the agglutinin both "group" and specific would
be removed.

As a general rule, group agglutinins constitute a minor fraction of
the total agglutinin and in practice the degree of dilution of the serum
used in specific cases is ample to exclude error. It occasionally happens
that sera of low dilution, especially those rich in agglutinoids, fail to
clump the specific organism; as the serum is diluted more and more
the phenomena of clumping become more and more marked; finally
a degree of dilution is reached beyond which the serum again becomes
ineffective. The initial negative agglutination in concentrated serum

1 Bordet, Ann. Inst. Past., 1899, xiii, 225.

AGGLUTININS AGGLUTINOIDS AND PROAGGLUTINOIDS 147

is known as a " proagglutinoid" reaction; it is attributed by Ehrlich
to the presence of " agglutinoids" in the serum side-chains of the
second order which have lost their agglutinophore group, but still
retain their combining group (haptophore group). These " agglu-
tinoids," which are deteriorated agglutinins, have a greater affinity
for the agglutinogen of the bacteria than have the unchanged agglu-
tinins, and consequently prevent the latter from becoming attached
to the organisms. If the serum is diluted a point is reached where
the agglutinoids are numerically too few to interfere with the action
of the agglutinins, which usually far outnumber the agglutinoids. As
the serum is more and more diluted a point is eventually reached
where the content of agglutinin is insufficient to react with the bacteria.
If, however, bacteria are cultured in this dilute serum, they frequently
develop into long, thread-like, interwoven filaments, the so-called
"thread-reaction" of Pfaundler. It is obvious that the maximum
dilution at which a serum will agglutinate bacteria depends somewhat
upon the number of organisms; there is, in other words, a relation
between the amount of agglutinin in the serum and agglutinogen in
the bacteria.

Non-agglutinable Bacteria. Occasionally strains of bacteria, as
B. typhosus, freshly isolated from the body, may not agglutinate
with the specific serum. This resistance to agglutination is supposed
to result from some unknown change in the agglutinogen of the bac-
terium during its development in the body. A similar loss of agglu-
tinability may be experimentally brought about by growing the
bacteria in gradually increasing concentrations of specific agglutinat-
ing serum outside the body. This inagglutinability is usually lost
after a few days' development on artificial media; the organisms will
then clump in a characteristic manner in a serum that originally was
ineffective.

The Reaction of Agglutination. The practical value of the reaction
of agglutination depends upon the visible clumping or agglomeration
of a suspension of bacteria in a fluid medium containing some neutral
salt, when a relatively small amount of immune serum specific for
the organism is added to it. The reaction may be expressed thus:

Organism (Agglutinogen) + Specific Serum (Specific Agglutinin)
= Agglutination.

If a specific organism is added to an appropriate dilution of unknown
serum with proper precautions, and characteristic clumping takes
place, or if a known specific serum is added with suitable precautions

148 ANTIGENS AND THE TECHNIC OF SERUM REACTIONS

to a suspension of an unknown organism, and characteristic clumping
takes place, a specific diagnosis of the serum or of the organism can
be arrived at. In the first instance, a diagnosis of disease may be
made; in the second instance the identity of an organism may be
established. The laboratory diagnosis of typhoid and paratyphoid
fever, of the various types of bacillary dysentery and of other bacterial
infections is frequently made by testing the serum of the patient
for agglutination with a known culture of the organism. 1 The labora-
tory identification of specific bacteria, conversely, is frequently estab-
lished or corroborated through their agglutination with known specific
agglutinating sera.

The reaction of agglutination may be made either microscopically
or macroscopically.

1. Microscopic Method. A drop of serum from a patient, diluted to
the proper degree, is mixed with an equal amount of a broth culture
of the desired organism on a clear cover-glass, 2 and then suspended
over the cavity of a hollow ground slide, ringed with vaseline to pre-
vent evaporation, and examined under the microscope. Motile bac-
teria, as for instance B. typhosus, soon lose their motility (immo-
bilization) and gradually collect in small groups which tend to coalesce
into larger and larger clumps, leaving the field between them practically
free from organisms. The bacteria are not necessarily killed by agglu-
tination. The reaction ordinarily is complete within two hours.
Killed cultures of bacteria may be used in place of living cultures
but the reaction is usually less clear-cut.

The advantage of the microscopic method lies chiefly in the small
amount of serum required to perform the test. One of its chief disad-
vantages lies in the relative inaccuracy of the dilution of the serum.
(See chapter on B. typhosus for full discussion of technic.)

2. Macroscopic Method. Various dilutions of serum, accurately
measured by volumetric pipettes, are brought into small, sterile test-
tubes, together with suspensions or broth cultures of the bacteria.
Agglutination is manifested by the gradual accumulation of a floccu-
lent sediment of bacteria, leaving the supernatant liquid perfectly
clear. Control tubes without serum remain uniformly clouded.

The part played by agglutinins in immunity is unknown; the

1 The technic and precautions to be observed are discussed individually in the chap-
ters upon specific pathogenic bacteria.

2 For a majority of bacteria, eighteen-hour cultures in 0.1 per cent, dextrose broth
are particularly advantageous. Cultures grown in plain broth are usually much less
actively motile and agglutinate less readily.

PRECIPITINSPRECIPITINOIDS 149

concentrations of agglutinins in immune sera, as measured by present-
day methods, throws no light upon the degree of immunity or the
prognosis. Very severe typhoid infections, for example, may show
little agglutinin in their sera, and mild cases may exhibit sera com-
paratively rich in agglutinin content. Their chief value at the present
time lies in their relation to the diagnosis of disease.

PRECIPITINS. PRECIPITINOIDS.

In the preceding section it was shown that the sera of animals
immunized with various bacteria contained substances agglutinins
which agglutinated the specific organisms. Kraus 1 showed that
these immune sera would cause a precipitate when they were added
to clear filtrates of the specific organisms. During the process of
immunization, therefore, specific antibodies, termed precipitins, are
formed, which react with the specific soluble antigen, precipitinogen,
in germ-free filtrate of broth cultures of the specific organisms, to
form a precipitate. Later investigations have shown that any soluble
protein, as egg-albumen, injected into experimental animals may
stimulate the production of specific precipitins which will cause a
precipitation in clear solutions of the homologous protein. These
reactions have a marked specificity: The sera of animals immunized
against casein of cows' milk, for example, will cause precipitation in
clear solutions of this protein, but will fail to cause precipitation in
solutions of casein from human milk. The sera of closely-related ani-
mals may contain small amounts of "group" precipitins, and biological
relationships have been established, based upon the community of
these antibodies. Thus, the sera of certain anthropoid apes 2 are
said to be precipitated by the sera of animals immunized to the serum
of man; sera from the lower- monkeys fail to react with the human
serum. From these observations the inference has been drawn that
these anthropoid apes are more closely related to man than are the
lower monkeys. 3

Precipitins closely resemble agglutinins in their method of formation,
their resistance to physical agents and their reactions. Like the
agglutinins, they possess both a thermostabile haptophore or combining
group and a thermolabile ergophore group. The precipitinophore

1 Wien. klin. Wchnschr., 1897, 736.

2 Griinbaum, Lancet, January, 1902.

3 See Nuttall, Jour. Hyg., 1901, i, No. 3; Proc. Royal Acad., November, 1901, Ixix;
Proceedings Cambridge Philosophical Society, January, 1902; Brit. Med. Jour., April,
1902, i, for full details.

150 ANTIGENS AND THE TECHNIC OF SERUM REACTIONS

group is very labile and readily becomes non-functionating, but the
combining group is relatively stabile. A precipitin which has lost
its ergophore group is called a precipitinoid.

The precipitate formed by a specific serum acting upon a clear
solution of the antigen (precipitinogen) probably is derived from the
serum, because very dilute solutions of the immunizing protein will
throw down a relatively bulky precipitate, far too great in amount
to come from the antigen in the dilution used. 1

Precipitins have been extensively studied in their relation to cer-
tain aspects of Forensic Medicine, but they have little practical value
in the laboratory diagnosis of bacterial disease. They are found in
sera under the same conditions as agglutinins, but the technic for their
demonstration is more involved than that for agglutinins. Their
relation to immunity is unknown, but probably similar to that of
agglutinins.

LYSINS.

Mention has been made (see preceding section) of the bactericidal
power of fresh blood serum of a normal animal and man. This impor-
tant discovery, that normal sera contain substances that will destroy
moderate numbers of bacteria, was made by Nuttall, 2 who also
observed that there was a limit to this destructive activity and that
this property was lost upon standing, or rapidly destroyed by an
exposure of the serum to 55 C. for half an hour. Buchner 3 corrobo-
rated and extended these observations and designated the . unknown
stabile component "alexin." Pfeiffer 4 then showed that the destruc-
tive action of normal sera could be increased many fold above its
original level for a specific organism if that organism were repeatedly
injected into an animal in sublethal, but gradually increased doses.
The serum of such an animal would still destroy only moderate num-
bers of heterologous bacteria, but relatively great numbers of the
homologous bacteria. This observation opened the way for the highly
important study of active acquired immunity against bacteria.
Pfeiffer observed that heating immune sera to 50 to 56 C. for half
an hour destroyed their bactericidal properties, precisely as Nuttall
had found that natural, non-specific bactericidal properties were
destroyed under similar conditions. Bordet 5 then discovered that the

Welsh and Chapman, Ztschr. f. Immunitasforsch., 1911, ix, 517.

Ztschr. f. Hyg., 1888, iv, 353.

Cent. f. Bakt., 1889, v, 817; vi, 1, 561.

Ztschr. f. Hyg., 1894, xviii, 1 ; 1895, xix, 75-100.

Ann. Inst. Past., 1895.

LYSINS 151

addition of a small amount of unheated blood serum from a non-
immune animal would "reactivate" the heated inactive immune serum
and restore its bactericidal power to its original level. These experi-
ments collectively demonstrated clearly that :

1. Normal sera had an inherent but limited destructive action upon
a variety of bacteria.

2. That this destructive or bactericidal action could be greatly
increased for specific organisms through repeated injections of sub-
lethal doses of them. 1

3. That both normal and immune sera lost their bactericidal prop-
erties by heating them to 55 C. for half an hour.

4. That immune sera would regain their specific bactericidal power
if a small amount of fresh normal blood serum of a non-immune
animal were added to them. 2

Bordet 3 showed similarly that the red blood cells of an alien animal
were also destroyed to a limited degree by the serum of a normal
'animal, but that the destruction could be greatly increased for specific
erythrocytes if they were repeatedly injected into an experimental
animal. The blood serum becomes specifically hemolytic. Here
again Bordet 4 found that heating an immune serum to 55 C. for
thirty minutes destroyed its activity, but that a small amount of
fresh serum from a non-immune animal (whose serum per se would
not dissolve the homologous cells) would reactivate the serum. Thus,
both specific bacteriolytic sera and specific hemolytic sera must con-
tain two distinct components a thermostabile component resisting
an exposure to 55 C. for half an hour and contained only in the
immune serum, and a thermolabile component destroyed or inacti-
vated at 55 C., which is present both in active immune bacteriolytic
and hemolytic sera, and also in normal sera. To the thermolabile
substance present in unheated normal and immune sera, Bordet gave
the name "alexin;" to the thermostabile specific substance in immune
sera he gave the name "substance sensibilitrice." He regarded the
"substance sensibilitrice" as a sensitizer or mordant which made
bacteria or blood cells vulnerable to the ferment-like or digestive
action of the "alexin."

Ehrlich and Morgenroth 5 studied the phenomena of hemolysis in

1 Presumably leaving the original non-specific bactericidal power at its initial level
for all except the specific organism, and possibly for closely related forms.

2 Moxter, Cent. f. Bakt., 1899, xxvi, 344.

3 Loc. cit.

4 Ann. Inst. Past., 1898, xii, No. 10.

6 Berl. klin. Wchnschr., 1899, No. 1 and 22. See also Collected Studies on Immunity,
Ehrlich, translated by Bolduan, 1910.

152 ANTIGENS AND THE TECHNIC OF SERUM REACTIONS

great detail and demonstrated by very careful and ingenious experi-
ments that the phenomena observed by Bordet were fundamentally
correct. They showed :

1. That inactivated specific hemolytic serum (heated to 55 C.)
was absorbed by the homologous red blood cells, and that these " sen-
sitized " cells, separated from the serum after a few hours and washed
carefully, were readily hemolyzed when resuspended in salt solution
to which was added a small amount of fresh, unheated, normal guinea-
pig serum.

2. The supernatant residual fluid from which the red blood cells
had removed all the immune body was incapable of causing hemolysis
of the homologous red blood cells when fresh normal serum was added
to it. The erythrocytes, in other words, quantitatively removed the
thermostabile "substance sensibilitrice" from solution.

3. If normal sera were allowed to remain in contact with the same
red cells for an equal length of time, and these red cells were then
removed by centrifugalization and resuspended in salt solution con-
taining normal fresh serum, no hemolysis took place, leading to the
conclusion that the thermolabile substance (alexin of Bordet) is not
removed from solution by erythrocytes. Apparently alexin is not
bound or anchored directly to the red cells.

4. Finally, it was shown that inactivated immune serum, red blood
cells and fresh normal serum could be maintained at C. without
apparent hemolysis. At 37 C. the same solution soon exhibited com-
plete hemolysis. Thus, at the lower temperature, the normal serum
failed to cause hemolysis. If the mixture maintained at C. were
centrnugalized, however, after some hours, and the red blood cells
washed thoroughly and resuspended in salt solution, hemolysis
promptly occurred when a small amount of normal serum was added
to the suspension, thus showing clearly that the inactivated immune
serum was bound or anchored by the red blood cells at C., even
though activation did not take place.

Ehrlich substituted the term " amboceptor" for Bordet's term
"substance sensibilitrice" and complement for the term "alexin,"
and conceived that the immune body amboceptor consisted essen-
tially of two combining or haptophore groups one the cytophilic
group, possessing a specific combining power for the specific cell
(bacterium or erythrocyte), the other, complementophilic group,
combining with the non-specific complement. According to this
theory the union of complement to specific cell takes place through the

LYSINS 153

amboceptor; Bordet maintains that neither the specific cell (antigen)
of itself nor the substance sensibilitrice (amboceptor) of itself unites
with alexin (complement). When both are simultaneously present,
however, alexin is absorbed. In other words, amboceptors as such do
not exist, according to this view, and consequently complement can-
not be bound to the specific cell by a complementophile (haptophore)
group.

Multiplicity of Amboceptors and Complement. The researches of
Nuttall and Buchner and of Moxter 1 have shown that fresh normal
serum possesses definite but limited bactericidal powers, apparently
not specific (for a variety of bacteria may be destroyed) which are
destroyed by an exposure of thirty minutes to 55 C. Furthermore,
the "inactivated" serum appears to regain its original bactericidal
value for various organisms when it is mixed with a relatively small
amount of normal serum. In other words, normal serum and specific
immune serum (unheated) alike appear to depend upon thermostabile
amboceptor and thermolabile complement for their bacteriolytic and
hemolytic activities. They differ in the highly specific potency of
the immune serum for its homologous cell. Ehrlich and Morgenroth 2
believe that the normal or natural cytolytic activities of sera depend
upon a multiplicity of specific amboceptors, each for its specific red
blood cell or other cell, and Pfeiffer 3 has made similar observations
for the normal bactericidal powers of blood. Ehrlich and Morgen-
roth have attempted to demonstrate a multiplicity of complements in
normal sera also; heated normal sera injected into normal animals
are claimed by the Ehrlich school to give rise to anticomplementoids,
the supposition being that the heat has destroyed the ergophore
group of complement but not its combining group, giving rise to a
"complementoid," precisely as a toxin which has lost its toxophore
group becomes a toxoid. There appears to be no theoretical limit
to the anti- and anti-antibodies which may thus be produced by
various increasingly complicated investigations. Bordet and Gay 4
deny the multiplicity of complement.

Fixation of Complement. Bordet and Gengou, 5 in a series of experi-
ments, brought forth experimental evidence of the unity of com-
plement and, incidentally, developed a method of investigation now

1 Loc. cit. 2 L OC> eft.

3 Harben Lecture, Jour. Royal Inst. Public Health, 1909, xvii, 385.

4 Collected Studies in Immunity by Bordet and his associates (translated by Gay,
1909).

8 Ann. Inst. Past., 1901, xv.

154 ANTIGENS AND THE TECHNIC OF SERUM REACTIONS

extensively utilized to demonstrate the presence of various specific
immune antibodies. If a specific immune body (as for example,
the serum of an animal immunized to typhoid bacilli) .is heated to
55 C. for half an hour, then added to a suspension of typhoid bacilli
together with normal unheated serum, a union between the bacilli,
the specific antibody of the serum (amboceptor, substance sensibili-
trice) and the complement (alexin) will take place. If the proportions
of the three reactive bodies are correct, all the complement or alexin
will be bound, provided the mixture is incubated a few hours at 37
C. If, now, red blood cells and inactivated immune serum specific
for the red blood cells are added to the mixture of bacteria, immune
body and complement, no hemolysis should be noticed, because the
complement is quantitatively anchored to the bacteria-immune serum
complex. If, on the other hand, the inactivated immune serum added
to the suspension of typhoid bacilli be not typhoid immune serum,
complement will not be bound to the bacteria, for the specific ambo-
ceptor or substance sensibilitrice will not be present. The complement
or alexin, therefore, is not anchored to the bacteria, and it is free to
act when the red blood cells and their specific inactivated serum are
added to the mixture of bacteria and serum. Under this condition
hemolysis occurs, because the red blood cells, inactive immune body
and complement unite. The production of hemolysis being visible,
it acts as an indicator in such instances. Wassermann and his asso-
ciates have utilized this method of "fixation of complement" for the
serologic diagnosis of syphilis, and gradually a relatively large number
of diagnoses of clinical importance have been developed along the
same lines.

The Determination of Specific Antibodies by the Method of Complement
Fixation. Principle Invoked. When an antigen (bacteria, erythro-
cytes, tissue cells, protein, or other substance which stimulates specific
antibody formation) is mixed intimately with its specific inactivated
immune serum and fresh normal complement a firm union of the
three components takes place. 1 Jhe result of this union is an injury
or destruction of the antigen, ^f the antigen be bacterial cells or
tissue cells there is usually no visible change in the gross appearance
of the mixture, and cultural or chemical investigation must be relied
upon to demonstrate the lytic process. Erythrocytes, on the other
hand, undergo changes in the presence of inactivated specific immune
serum and complement which result in the liberation of hemoglobin,

1 Bordet and Gengou, Ann. Inst. Past., 1901, xv, 290.

LYSIN& 155

which colors the solution deep red. This change is clearly visible
and requires no additional procedure for its demonstration; the
liberation of hemoglobin is in itself an indicator of the reaction which
has taken place.

The relation between antigen, immune serum, and complement
is quantitative; consequently, if the respective amounts of the three
components are correctly proportioned, no free unattached comple-
ment will be present in a mixture of them after an appropriate incuba-
tion at body temperature is practiced to allow of their union. Usually
an hour at 37 C. suffices for this union to take place quantitatively.

These very important observations of Bordet and Gengou have
led to the development of a technic for the diagnosis of infection, and
the identification of antigens by the method of complement fixation.

The underlying principles of the reaction of complement fixation
are three:

(a) The union of specific inactivated immune serum and homologous
antigen.

(b) The quantitative activation of the antigen inactivated specific
immune serum complex by non-specific complement; and

(c) The visible hemolysis that results from the activation of an
erythrocyte inactivated specific immune serum complex by non-
specific complement.

The general plan of procedure is to incubate an antigen (as bacterial
cells) and inactivated serum and complement in proper proportions
for an hour, to permit the three components to unite. A mixture of
erythrocytes and specific inactivated hemolytic serum is now added.
If the reactive substances are properly proportioned and the inac-
tivated serum first added is specific for the antigen (bacteria), no
hemolysis will occur when the hemolytic system is added, because
all the complement present is bound by the bacteria-immune serum
complex. On the contrary, if the inactivated serum is not specific
for the bacterial antigen, no union between the two will take place,
complement will not be bound, and it is free in the mixture. It will
activate the erythrocyte-inactivated immune serum complex, and
hemolysis will occur.

It will be seen that the hemolytic system is added as an indicator;
an absence of hemolysis shows a union of bacterial antigen, inactive
specific bacterial immune serum and complement. Hemolysis shows
that the union has not been formed, the complement was free in the
mixture and it united with the hemolytic system, causing hemolysis

156 ANTIGENS AND THE TECHNIC OF SERUM REACTIONS

in the erythrocyte antigen through the specific amboceptor or
hemolysin.

The method of complement fixation may be employed to examine
sera for specific antibodies, using a known antigen, or to test suspected
antigens with sera containing specific antibodies. The most practical
application of the method in medicine is the serum diagnosis of syphilis,
glanders, and other bacterial infections. t

The Technic of Complement Fixation. The technic of complement
fixation is simple in principle, but it requires the most scrupulous
attention to details. All glassware must be neutral in reaction,
chemically clean, and bacteriologically sterile. Physiological salt
solution (0.85 to 0.90 per cent. C.P. NaCl in neutral distilled water)
used for washing red blood cells and for dilutions should be sterile
and stored in clean containers.

The Wassermann Serum Diagnosis of Syphilis. Five elements enter
into the Wassermann test for syphilis: the antigen, suspected syphilitic
serum, complement, and a hemolytic system consisting of red blood
cells and specific immune hemolytic serum (hemolysin).

Preparation and Standardization of Antigen. The antigen originally
employed by Wassermann and his collaborators was an aqueous
extract of syphilitic tissue which was prepared by suspending one part
by weight of finely comminuted liver of a syphilitic fetus 1 in five parts
of physiological salt solution containing 0.5 per cent, phenol as a
preservative. After several days' violent agitation in the dark it is
strained through several layers of cheesecloth to remove coarser par-
ticles and stored in amber bottles in the refrigerator. Sedimentation
takes place until a brownish, slightly opalescent fluid remains, which
is the luetic antigen.

Later work 2 showed that alcoholic extracts of luetic liver were more
stable than watery extracts. The specific reacting component, accord-
ing to Forges and Meier, is lipoidal in nature, and in this sense it is
not biologically specific; The fixation of complement appears to
depend upon a substance in the antigen, lipoidal in nature, which
effects a union of antigen, immune body and complement. Citron
has proposed the term "lues reagine" for this substance. Alcoholic
extracts of syphilitic liver are prepared by shaking finely comminuted
liver with ten times the weight of absolute alcohol for a few days,

1 The tissue is examined for the specific organism; if Treponemata are abundant it
is converted into antigen, otherwise it is discarded.

2 Especially by Forges and Meier, Berl. klin. Wchnschr., 1908, No. 15.

LYSINS 157

then digesting the mixture at 37 C. for a week. The extract is
filtered through filter paper and placed in the refrigerator.

Alcoholic extracts of normal organs, prepared in the same manner
as luetic livers, have been found to be quite as good as alcoholic
extracts of syphilitic livers for the diagnosis of syphilis. In practice
heart-muscle of normal guinea-pigs, freed from all fat, is used.

Noguchi's Acetone-Insoluble Lipoidal Antigen. 1 Noguchi and others
have shown that .alcoholic extracts of organs may, and frequently
do, contain sufficient amounts of neutral fats, or their hydrolytic
cleavage products, to make the antigen hemolytic or anticomplemen-
tary. These substances are for the most part soluble in acetone, while
the antigenic fraction is insoluble in acetone. One part of fat-free
heart muscle or liver from a guinea-pig is cut into very fine pieces,
mixed with ten parts of absolute alcohol, and extracted in the incu-
bator at 37 C. for a week or ten days, being thoroughly shaken every
day. The soluble substances are freed from the fragments of tissue
by filtration through fat-free filter paper, and rapidly evaporated to
dry ness. 2 Sufficient ether is then added to take up the brownish
residue, and it is then allowed to stand until a clear, slightly colored,
ethereal solution is obtained, free from suspended material. The
ethereal solution is concentrated by evaporation to a point where
separation of a sediment begins, then it is poured into several volumes
(usually ten) of pure acetone. A voluminous precipitate forms at
once, and settles out as a tenacious gummy mass. This is retained,
under acetone, as the antigen. The acetone-soluble solution is dis-
carded. The antigen thus prepared consists largely of lecithins and
related substances. It keeps well and appears to be very sensitive
and reliable. From 0.2 to 0.3 gram are dissolved in a mixture of 1
c.c. of ether (free from alcohol and having a neutral reaction) and
10 c.c. of neutral absolute methyl alcohol. This solution is kept
in an amber bottle in the refrigerator as a stock antigen. One cubic
centimeter of this stock antigen is added to 19 c.c. of physiological
salt solution; this is the antigen used for the test.

Before making a test it is necessary to standardize the antigen.
It is essential to know the anticomplementary titer, that is, that
maximum amount of antigen which will inhibit hemolysis in the
presence of syphilitic serum, but which will not inhibit hemolysis

1 Noguchi, Serum Diagnosis of Syphilis.

2 Best by exposing the nitrate in a broad shallow dish to an air current from an
electric fan.

158 ANTIGENS AND THE TECHNIC OF SERUM REACTIONS

when non-syphilitic serum is used. In addition, the following deter-
minations are sometimes desirable.

The hemolytic titer, that amount of antigen which will of itself
cause lysis of red blood cells, and the antigenic titer, the amount of
complement it will absorb or "fix" in the presence of a definite amount
of specific syphilitic serum.

The anticomplementary titration is made by mixing graded amounts
of antigen and a constant amount of complement (0.1 c.c. of a 10 per
cent, solution 1 ) with constant amounts (0.1 c.c.) of known syphilitic
serum and normal serum, both inactivated.

The various mixtures are incubated in a water-bath at 37 C. for
an hour, then 0.2 c.c. of red blood cell suspension and inactivated
hemolytic serum are added and again incubated in the water-bath at
37 C. The maximum amount of antigen which will give complete
inhibition of hemolysis with syphilitic serum and no inhibition of
hemolysis in the non-syphilitic serum is regarded as the unit.

EXAMPLE OF AN ANTICOMPLEMENTARY TITRATION OF ANTIGEN.

Normal

Comple-

Hemolytic

Tube.

Antigen.

serum
Inactive,

ment, 10
per cent.

Red blood
cells, c.c.

serum
inactivated

Result.

c.c.

units.

0.2

0.1

0.10

1.0

1.5

Complete hemolysis.

0.4

0.1

0.10

1.0

1.5

" "

0.6

0.1

0.10

1.0

1.5

" "

0.8

0.1

0.10

1.0

1.5

CO 3

" "

1.0

0.1

0.10

5 ^

1.0

1.5

Partial inhibition.

1.5

0.1

0.10

J g

1.0

1.5

Marked inhibition.

2.0

0.1

0.10

1.0

1.5

Complete inhibition.

8 2

0.1

0.10

os y>

1.0

1.5

-2

Complete hemolysis.

9 3

0.10

1.0

1.5

Complete hemolysis.

Tube 5, containing 1.0 c.c. antigen, shows beginning inhibition of
hemolysis. This is regarded as the anticomplementary titer of the
antigen.

As a general rule, the hemolytic titer is higher than the anti-
complementary titer. The test is readily made, if desired, by using
the same amounts of antigen mixed with 1 c.c. of red blood cell
suspension and sufficient salt solution to bring the volume to 4 c.c.

It is customary to use one-fourth the anticomplementary titer as
the standard amount of antigen to be used in the actual test. In the

1 Prepared by adding fresh normal guinea-pig serum to physiological salt solution
in the proportion of one part serum to nine parts salt.

2 Serum control. 3 Hemolytic control.

LYSINS 159

example cited, 1.0 c.c. of the antigen was found to be anticomple-
mentary, consequently 0.25 to 0.3 c.c. would be the proper amount
of antigen to employ in the test.

Complement. Fresh guinea-pig serum is the usual source of com-
plement for fixation reactions. The animal should be healthy and
not previously injected with protein of any nature. The serum of
pregnant pigs is not trustworthy. Blood may be obtained directly
from the heart of the living animal by aspiration through a hypo-
dermic needle, from a severed carotid artery, or, more expeditiously
by cutting the throat of the animal, avoiding the esophagus, and
collecting the blood in sterile Petri dishes. The freshly drawn blood
is allowed to stand for a few hours at a low temperature and the serum
is pipetted off. Complement must be kept cold (below 16 C.) and in
the dark. It must be used fresh, for it deteriorates rapidly. In a
frozen condition, however, it will remain active for two or three
weeks. Both the "activating" and combining properties of normal
fresh guinea-pig serum are sufficiently constant for the reaction of
complement fixation.

Hemolytic System. (a) Hemolytic Serum (Hemolysiri). Hemolytic
serum is obtained from rabbits which have been injected with 2 c.c.,
4 c.c., and finally 6 c.c. of a 50 per cent, solution of washed sheep
red blood cells 1 at intervals of two or three days. The injections
may be made intraperitoneally or intravenously, the latter being
preferable. Not less than nine days after the injection the animal
is bled to death from the carotid artery under anesthesia, the blood
being received in sterile test-tubes, which are placed in an inclined
position in the ice-box. The serum is removed, centrifugalized if not
wholly free from blood corpuscles, and placed in small amber bottles
with aseptic precautions. These are heated to 56 C. for half an hour
to effect inactivation (to destroy complement).

(6) Red Blood Cells. Erythrocytes of the sheep are used. The
blood of a sheep is collected either in small sterile flasks containing
one volume of 0.85 per cent, salt solution and 0.5 per cent, sodium
citrate, or in sterile centrifuge tubes. If the former is used, nine
volumes of blood are allowed to flow into the flask and immediately
mixed intimately with the citrate solution, which prevents clotting.
This method is applicable if the blood cannot be centrifuged imme-

1 Fresh red blood cells of the sheep are freed from serum by repeated washings with
physiological salt solution usually five washings "suffice. The corpuscles are then
suspended in a volume of salt solution twice that of the corpuscles themselves.

160 ANTIGENS AND THE TECHNIC OF SERUM REACTIONS

diately. If centrifuge tubes are used, an amount of blood not more
than one-third the capacity of the tube (about 5 c.c.) is collected and
twice the volume of sterile salt solution is added to it. The corpuscles
are sedimented, the supernatant solution is pipetted off, fresh salt
solution is poured in, and the corpuscles resuspended by careful
stirring with a clean glass rod. This process is repeated five times,
each time discarding the washings. The last time the volume occu-
pied by the erythrocytes is read off on the graduations of the tube
and they are suspended in a volume of salt solution twenty times that
occupied by the erythrocytes. This makes a 5 per cent, suspension.
Erythrocytes are obtained by centrifugalization from the citrated
blood in precisely the same manner. This suspension of red blood
cells, kept in a cool, dark place, may be used for two days, but not
longer. Beyond that time the cells deteriorate and hemolyze with
abnormal readiness, thus vitiating the value of the test.

(c) Standardization of Hemolytic System. It is very important to
know with exactness the amount of hemolytic serum (inactivated,
of course) which will effect complete hemolysis of 1 c.c. of a 5 per
cent, suspension of sheep erythrocytes in the presence of a constant
amount of complement. The determination of this factor gives the
hemolytic titer of the hemolytic serum. It is readily determined
by adding to a series of tubes, 0.1 c.c. of fresh guinea-pig serum (com-
plement), 1 c.c. of erythrocyte suspension, and varying amounts of
the inactivated hemolytic serum. The smallest amount of hemolysin
which will effect hemolysis under the conditions stated is the hemolytic
titer orunit. Thus, the following tubes incubated at 37 C. for one
hour showed :

Result.
Complete hemolysis.

Partial hemolysis.
No hemolysis.

0.0025 of this serum is one unit; the hemolytic titer is 0.0025 c.c., in other words.
It is customary to use two units in the actual test, consequently 0.005 c.c. would be
the amount used.

Tube.

Complement.

c.c.

II 1

c.c.

12 2

0.0 c.c.

5 per cent, suspension
sheep erythrocytes.

Inactivated
hemolytic serum.

1 C.C.

0.10 c.c.

1 c.c.

0.075 c.c.

1 c.c.

0.050 c.c.

1 c.c.

0.025 c.c.

1 c.c.

0.010 c.c.

1 c.c.

0.0075 c.c.

1 c.c.

0.0050 c.c.

1 c.c.

0.0025 c.c.

1 c.c.

0.0010 c.c.

1 c.c.

0.00075 c.c.

1 c.c.

0. c.c.

1 c.c.

0. c.c.

Complement control.

2 Erythrocyte control.

LYSINS 161

It must be emphasized that precision of measurement is an absolute
requirement for success; the activating power of complement for
hemolysin does not follow the law of multiple proportions it is rather
an inverse ratio, as Noguchi 1 has pointed out. Relatively less com-
plement is required to induce complete hemolysis in a system contain-
ing four units than is required for a system containing but a single
hemolytic unit.

The serum to be examined for specific antibodies by the method
of complement-fixation must be sterile and free from hemoglobin.
The products of bacterial growths in serum may be anticomplementary
and the presence of hemoglobin in serum also tends to inhibit hemolysis.
Blood, therefore, should be withdrawn with aseptic precautions from
the median basilic vein of the patient into sterile test-tubes, and either
centrifugalized at once and the serum removed from the clot, or
placed in an inclined position in a cool place until the serum has
separated. The serum must be clear 2 and free from erythrocytes or
hemoglobin. 3 It is inactivated at 54 to 55 C. for half an hour in a
water-bath. 4

The Technic of the Test. It is essential that the hemolytic system
erythrocytes, hemolysin, complement be standardized daily.
Varying amounts of hemolysin are added to constant amounts of
erythrocyte suspension and complement, as outlined above. A
known positive syphilitic serum and a known negative syphilitic
serum, together with suitable controls, must be examined along with
the unknown serum to be tested.

The following reagents are required :

1. Sterile physiological salt solution.

2. Fresh guinea-pig serum (complement) add 0.1 c.c. to each tube.

3. Five per cent, suspension of washed sheep erythrocytes in normal
salt solution use 1 c.c. to each tube.

4. Hemolysin (amboceptor) use twice the hemolytic unit (the unit
must be determined daily).

5. Known syphilitic serum inactivate and use 0.2 c.c.

6. Known normal (non-syphilitic) serum, inactivated use 0.2 c.c.

7. The serum to be tested inactivate, use 0.2 c.c.

1 Serum Diagnosis of Syphilis.

2 Blood is best obtained early in the morning, before the patient has eaten; blood
obtained at the height of digestion frequently contains fats which make the serum
turbid.

3 Small amounts of blood, yielding a few drops of serum, may be obtained from the
finger-tip or the lobe of the ear. Massage must not be practised, for there is danger of
damaging erythrocytes with the liberation of hemoglobin.

4 Noguchi, Serum Diagnosis of Syphilis, states that inactivation at 54 C. should be
practised the higher temperature weakens the reactive substance somewhat.

162 ANTIGENS AND THE TECHNIC OF SERUM REACTIONS

Unknown serum.

Known positive
syphilitic serum.

Known normal
non-syphilitic serum.

Controls.

Tube 1.
Serum, 0.2 c.c.
Complement, 0.1 c.c.
Salt solution, 2.7 c.c.

Tube 3.
Serum, 0.2 c.c.
Complement, 0.1 c.c.
Salt solution, 2.7 c.c.

Tube 5.
Serum, 0.2 c.c.
Complement, 0.1 c.c.
Salt solution, 2.7 c.c.

Tube 7.

Complement, 0.1 c.c.
Salt solution, 2.9 c.c.

Tube 2.
Serum, 0.2 c.c.
Complement, 0.1 c.c.
Antigen, 1 1 c.c.
Salt solution, 1.7 c.c.

Tube 4.
Serum, 0.2 c.c.
Complement, 0.1 c.c.
Antigen, 1 c.c.
Salt solution, 1.7 c.c.

Tube 6.
Serum, 0.2 c.c.
Complement, 0.1 c.c.
Antigen, 1 c.c.
Salt solution, 1.7 c.c.

Tube 8.

Complement, 0.1 c.c.
Antigen, 1 c.c.
Salt Solution, 1.9 c.c.

After mixing the tubes are placed in a water-bath maintained at
37 C. for one hour, to permit of the fixation of complement; 1 c.c.
of a 5 per cent, suspension of erythrocytes and two units of hemolysin
are then added to each tube, mixed and reincubated for one hour,
then read. Tubes 1, 3, 5, 7, 6 and 8 should show complete hemolysis.
Tube 4 should show complete inhibition of hemolysis (positive reac-
tion). If such be the case all the reagents are properly adjusted, and
Tube 2, containing the unknown serum, is read. If hemolysis is
absent the reaction is positive; if hemolysis is complete the reaction
is negative. 2

The Method of Noguchi. 3 A rigorous standardization of reagents
is a prerequisite for accuracy in the serum diagnosis of syphilis, and
Noguchi has pointed out that a variable inherent inaccuracy exists
in the Wassermann method. He has shown that human sera may
contain variable amounts of hemolysin specific for sheep erythrocytes.
Human sera, however, contain no hemolysin for human erythrocytes.
The Noguchi modification, therefore, substitutes human red blood
cells (obtained from placenta or at autopsies) for sheep red blood
cells. Rabbits are immunized to carefully washed human erythro-
cytes and the hemolytic unit of the rabbit serum is determined in the
usual manner. The following reagents are required to perform the
Noguchi test:

1. Complement Fresh guinea-pig serum in 40 per cent, dilution
(one part clear fresh serum to 2.5 parts sterile salt solution).

2. Hemolytic Serum Rabbit serum, immunized against human
erythrocytes, is titrated against human erythrocytes to determine the
hemolytic unit. Two units are used in the test.

1 Twice the antigen titer, determined by titration, diluted with salt solution; thus, if
the antigenic titer of the acetone insoluble extract is 0.2 c.c., and the anticomplementary
titer is found to be 1.75 c.c., 0.4 c.c. of the extract are diluted with 0.6 c.c. salt solution
and used in the diluted state. In practice, enough extract should be diluted to last one
day.

2 For a discussion of results, see section on Treponema pallidum.

3 Noguchi, Serum Diagnosis of Syphilis.

PLATE I

Wassermann Reaction. (Simon.) *

A, positive; B, partial; C, negative reaction.

Note undissolved blood corpuscles in A, partial hemolysis in B, and complete hemolysis in C.

LYSINS

163

3. Human Erythrocytes Red blood cells are obtained from a
normal individual, washed thoroughly with salt solution, and made
up as a 1 per cent, suspension in salt solution. 1 c.c. of the suspension
is used in the test.

4. Antigen The acetone-insoluble antigen is used.

5. Patient's Serum Obtained fresh, from 2 to 5 c.c. of blood. It
is used unheated.

6. Known syphilitic serum.

7. Known normal (non-syphilitic) serum.
The test is performed as follows:

Unknown serum.

1
Known positive serum.

Known negative serum.

Controls.

Tube 1.
Serum, 1 drop.
Complement, 1 0.1 c.c.
Erythrocytes, 1.0 c.c.

Tube 3.
j Serum, 1 drop
Complement, 0.1 c.c.
Erythrocytes, 1.0 c.c.

Tube 5.
Serum, 1 drop.
Complement, 0.1 c.c.
Erythrocytes, 1.0 c.c.'

Tube 7.
Complement, 0.1 c.c.

Erythrocytes, 1.0 c.c.

Tube 2.
Serum, 1 drop.
Complement, 0.1 c.c.
Antigen, 2 units.
Erythrocytes, 1.0 c.c.

Tube 4.
Serum, 1 drop.
Complement, 0.1 c.c.
i Antigen, 2 units.
Erythrocytes, 1.0 c.c.

Tube 6.
Serum, 1 drop.
Complement, 0.1 c.c.
Antigen, 2 units.
Erythrocytes, 1.0 c.c.

Mix and incubate one hour in water-bath at 37 C. Remove and add 2 units hemolysin to each
tube and incubate in water-bath for one hour. Tubes 1, 3, 5, 6 and 7 should show complete hemo-
lysis. Tube 4 should show no hemolysis (positive control). If such be the case the reagents are
correctly adjusted and a reading of Tube 2 will be positive (no hemolysis) or negative (hemolysis).

A further simplification of the method has been made by Noguchi.
The hemolysin and antigen respectively may be absorbed on squares
of filter paper, dried, and standardized. In this state they retain
their potency for several weeks. In practice the squares of paper are
added directly to the tubes, thus saving much time.

Complement-fixation in Bacterial Infections. Preparation of Antigen
from Bacteria. Experience has clearly shown that bacterial antigens
should be polyvalent prepared by mixing in equal amounts, several
strains of the same organism. The antigen may be prepared in one
of several ways.

The simplest method is to wash off bacteria from agar slants,, at
the period of maximum growth, with salt solution and shake thoroughly
to make a uniform suspension. A small amount of phenol (0.5 per
cent.) and 3 per cent, glycerin are then added and the whole sterilized
at 56 to 60 C. for one hour. Relatively more of the proteins of the
bacterial cell may be obtained in solution if the bacterial emulsion
is shaken in a shaking machine with sterile, sharp quartz-sand for
twenty-four hours: filtration through coarse Berkefeld filters removes

1 Forty per cent, solution of fresh guinea-pig serum in salt solution.

164 ANTIGENS AND THE TECHNIC OF SERUM REACTIONS

the sand and broken bacterial cells, and the filtrate is preserved with
0.5 per cent, phenol. Besredka prepares a bacterial antigen from dried
bacterial cells, which are obtained by drying bacteria scraped from
agar slants or other solid media over sulphuric acid or calcium chloride.
The dried organisms are ground in agate mortars with crystals of
NaCl to an impalpable powder, which is then gradually rubbed up
in successive portions of water until a physiological salt solution is
obtained (corresponding to 8.5 grams NaCl in a liter of distilled water).

It has been found that much of the antigenic substance of bacteria
is precipitated by an excess of alcohol; a considerable excess of alcohol
is added to a suspension of bacteria, or to an emulsion of the cell
substance prepared according to Besredka's process, outlined above.
The precipitate from the alcoholic solution is separated by filtration,
dried, and ground to an impalpable powder with NaCl crystals.
The powder is gradually brought into solution by the addition of
water in successive amounts until isotonicity is reached. An attempt
is made to create a definite concentration of antigen by starting with
a known quantity of dried bacteria and a corresponding amount of
NaCl crystals. Thus, 1 gram of dried bacterial substance, ground
in a mortar with 0.85 gram NaCl crystals and gradually brought to
a volume of 100 c.c. with distilled water, would yield, theoretically,
an antigen of 1 per cent, strength. Bacterial antigens must be kept
cold and in a dark place, preferably in sealed amber bottles. Deter-
ioration gradually occurs and all bacterial antigens suspended or
dissolved in liquids are relatively unstable.

Standardization of Bacterial Antigens. The standardization of bac-
terial antigen differs in no respect from that of a syphilitic antigen.
The anticomplementary titer and the antigenic titer are determined,
the latter by titration with a specific immune serum.

The Diagnosis of Glanders by the Method of Complement-fixation
The antigen is prepared from glycerin-agar cultures 1 of several strains
of B. mallei incubated at 37 C. for forty-eight hours. The organisms
are autolyzed in distilled water for several hours at a relatively high
temperature (70 to 80 C.), then freed from suspended particles by
filtration through coarse Berkefeld filters. The filtrate is stored in
amber bottles in the ice-box after the addition of 0.5 per cent, phenol.

The anticomplementary titer is determined from a series of tubes
containing constant amounts of complement and graduated amounts

1 Reaction 1.5 per cent, acid to phenolphthalein.

AGGRESSINS 165

of antigen (1 to 20 dilution in salt solution). 1 The total volume of
complement and antigen is brought to 3 c.c. by the addition of salt
solution. After one hour's incubation in the water bath at 37 C., 1
c.c. of sheep erythrocyte suspension and 1.5 units sheep erythrocyte
hemolysin are added and reincubated. That dilution of antigen which
shows the slightest inhibition of hemolysis is taken as the anti-
complementary titer of the antigen. Not more than one-half this
amount, and preferably one-fourth of the anticomplementary titer,
is used in the test.

The actual determination is made in the same manner as for the
Wassermann test. 2 It is well to include a known positive and known
negative glanders serum of the same animal species as the unknown,
together with suitable controls of the hemolytic system. The length
of incubation is determined by the time it takes to effect complete
hemolysis in the known negative and the hemolytic controls. Fre-
quently ten or more hours will elapse before this occurs.

AGGRESSINS.

Progressively pathogenic bacteria appear to differ from parasitic
bacteria or "opportunists" in that they are able to force an entrance
to the underlying tissues of the host through natural, non-specific
barriers which ordinarily suffice to restrain the more parasitic types
of microbes. Bail 3 has advanced an hypothesis, based upon experi-
mental evidence, which attributes the invasiveness of pathogenic
bacteria and their ability to develop in the tissues of the host to
"aggressins." These aggressins, according to Bail, are present and
may be demonstrated in exudates resulting from bacterial infection,
but they are not, as a rule, found in artificial cultures of the same
organism. To demonstrate the action of aggressins, Bail removed
bacteria from exudates by centrifugalization and injected the clear
supernatant fluids, together with a sublethal dose of the homologous
bacterium, into experimental animals. Rapidly fatal infections
developed. The aggressin-containing exudates were not inactivated
by prolonged exposure to 50 C., and it was shown, furthermore, that

1 Usually a range of antigens from 2 c.c. to 0.05 c.c. will be found sufficient.

2 For full discussion of results, see Mohler and Eichhorn, Bureau of Animal Industry
Bulletin 136, April 7, 1911.

3 See Der Problem der bakteriellen Infektion, Bail, in Bibliothek medizinischer Mono-
graphien, xi; see also Miiller in Oppenheimer's Handbuch der Biochemie, 1909, ii, 1,
681.

166 ANTIGENS AND THE TECHNIC OF SERUM REACTIONS

their injection into susceptible animals stimulated the formation
of "antiaggressin," which greatly increased the resistance of the
animal to subsequent infection. The sera of animals immunized
with aggressin-containing fluids conferred a limited degree of immunity
to specific infections in non-immune animals (passive immunity).
It has been claimed by Doerr 1 and others that the aggressins are of
the nature of bacterial endotoxins and that the immunizing properties
of aggressin fluids are due to their content of specific substances
derived from the autolysis of bacterial cells.

The aggressin theory must, for the present, be regarded as not
definitely proved.

OPSONINS. TROPINS. BACTERIAL VACCINES.

A most important contribution to the literature of immunity is
the work of Denys and his associates, 2 who showed that the sera of
rabbits immunized to Streptococcus pyogenes possessed two properties
not exhibited by the serum of a normal animal, namely, the property
of restricting the development of the organism, and the property of
stimulating phagocytosis. Their very comprehensive studies demon-
strated that the leukocytes of normal animals, suspended in the serum
of immunized animals, phagocytized streptococci energetically, but
the leukocytes of immunized animals suspended in normal serum
failed to exhibit phagocytic activity. Their conclusion was that the
immunity of rabbits to the streptococcus resides in the serum. These
observations not only added materially to the restricted field in which
they were cast they brought sharply into focus the interrelation of
the humoral and cellular aspects of immunity.

Wright and Douglas, 3 using a modification of the technic of Leish-
man, 4 were able to study phagocytosis in vitro: by an ingenious series
of experiments they showed that normal serum contains substances
opsonins which prepare bacteria for phagocytosis, as described
in a preceding section (Cellular Immunity). The technic of meas-
uring the potency of opsonins in the sera of normal and infected
individuals, as practised by Wright and his associates, consisted

1 Wien. klin. Woch., 1906, No. 25.

2 Denys and Le Clef, La Cellule, 1895, xii; Bull, de 1'Acad. roy. de Belgique, 1895;
Denys and Marchand, Ibid., 1896; Van de Velde, Ann. Inst. Past., 1886, x; Marchand
Arch, de Med. exp., 1898; Denys, Cent. f. Bakt., 1898, xxiv, 685.

3 See Studies in Immunization, Constable, 1909, for complete biography.
Brit. Med. Jour., 1902, i, 73.

OPSONINS TROPINS BACTERIAL VACCINES 167

essentially in mixing intimately equal volumes of bacterial emulsion,
serum, and leukocytes; after incubation at body temperature the
mixture was spread evenly upon microscopic slides, stained, and
examined with the microscope. The average number of bacteria per
polymorphonuclear leukocyte was determined by direct count. A
comparison, under parallel conditions, of the phagocytic activity of
leukocytes for a specific organism in the serum of a normal individual
and that of an individual infected with the specific organism, accord-
ing to the technic outlined below, was called by Wright the opsonic
index.

Procedure. 1. Leukocyte Suspension. About 0.5 c.c. of blood,
drawn from the lobe of the ear or the tip of the finger, is collected in
a centrifuge tube containing 10 c.c. of sterile physiological salt solu-

FIG. 9. Phagocytosis of streptococci.

tion in which has been dissolved 1 per cent, of sodium citrate; this
mixture is centrifuged at moderate speed until a sharp separation
of blood cells and clear supernatant fluid is obtained. The super-
natant fluid is carefully poured off and the top layer of blood cells,
which contains practically all the leukocytes, is removed to a fresh
centrifuge tube containing 10 c.c. of physiological salt solution.

A second centrifugalization is made, and again the supernatant
fluid, containing the last traces of blood serum, is discarded. The
sediment, rich in leukocytes, is used as the leukocyte suspension in
the test.

2. Suspension of Bacteria. Bacteria from a culture on solid media
are suspended in sterile salt solution and agitated until a fine opales-
cent emulsion is obtained. This is most conveniently accomplished

168 ANTIGENS AND THE TECHNIC OF SERUM REACTIONS

in a shaking machine, but repeated shaking in a stoppered test-tube
containing glass beads will usually suffice. The coarser clumps of
bacteria are removed by filtration through a coarse filter paper. The
density of the bacterial suspension should be such that not more than
ten bacteria per leukocyte will be taken up as the average.

3. Serum. (a) Blood from three or four normal individuals is
collected in capillary tubes; after the serum has separated a "pool"
or mixture is made, composed of equal volumes of each serum. Experi-
ence has shown that "pooled" serum furnishes a more reliable normal
opsonic index than that obtained from a single individual.

(6) Serum from the Patient. This is prepared in the manner
described above.

The Test. A capillary pipette of 1 to 1.5 mm. bore is made by
drawing out a piece of glass tubing previously softened in the flame.
If the tubing is heated in the center until it softens, then, after a few
seconds, drawn slowly and steadily out, the desired size and shape is
readily obtained. A close-fitting rubber bulb attached to the larger
end is a convenience.

A mark about 1 to 1.5 cm. from the capillary end is made with a
wax pencil, and a volume each of the leukocytes, pooled serum, and
bacterial suspension are drawn into the pipette. It is convenient to
separate each ingredient by a small air bubble, to insure uniformity
of volume. Mixing is accomplished by carefully expelling and drawing
back the respective elements into the pipette. Finally, the mixture
is drawn well up into the pipette, the end is sealed in the flame of a
Bunsen burner, and the charged pipette is placed in the incubator
at 37 C. This is the normal or control.

A precisely similar preparation is made, using the serum of the
patient in place of the pooled serum.

Incubation is maintained for fifteen minutes.

The ends of the pipettes are now broken off, and the contents of
each pipette mixed as before. A large drop of each respective mixture
is spread upon clean glass slides, using the same technic as that for
preparing a blood smear, and air-dried. The preparations are stained
with Loffler's methylene blue, Wright's stain, or other stain suitable
for the organism used.

The number of bacteria in fifty, one hundred, or two hundred leuko-
cytes are determined by direct count, and the average number of
bacteria per leukocyte of the normal serum compared with the average
number of bacteria per leukocyte in the pathological serum:

OPSONINS TROPINS BACTERIAL VACCINES 169

EXAMPLE.

Bacteria in Bacteria per

100 leukocytes. leukocyte.

Staphylococcus suspension + pooled serum and

leukocytes 750 7.5

Staphylococcus suspension + patient's serum and

leukocytes 250 2.5

2 5

Opsonic index, patient's serum = '-- or 0.33 per cent.

7 . 5

Numerous observers have been unable to obtain uniform results
with the technic of Wright for opsonic index determination, and this
is not surprising when the many variable factors entering into the
method are reviewed. Attempts have been made to eliminate or
limit the variable factors: Simon proposed a dilution method in
which the pooled and patient's serum are diluted 1 to 10, 1 to 100,
etc., before incubation with the bacteria and leukocytes. That dilu-
tion of serum at which phagocytosis practically ceases in the normal
and patient's serum respectively is taken as a basis for comparison.
Inasmuch as the opsonic index is rarely determined as a guide for
treatment of bacterial disease with bacterial vaccines at the present
time, however, a discussion of these modifications, which are too
involved for practical use, is left for more pretentious volumes.

The Nature of Opsonins. There appears no doubt that the hypo-
thetical substance or substances called opsonin by Wright exist in
normal sera, and it is equally certain that they may be diminished
during infection. Furthermore, opsonin may be increased either in
amount or in potency by careful immunization. The relation of
opsonins to other antibodies, normal or specific, is a subject of con-
troversy at present. The researches of Neufeld and Rimpau, 1 Hek-
toen 2 and others indicate that the normal opsonins those of normal
sera are thermolabile, but those developed during immunization
to a specific organism bacteriotropins are relatively thermostabfte.

It has been suggested that opsonins or bacteriotropins are not to
be distinguished from other immune bodies as normal and specific
amboceptors or agglutinins. The rapidity with which the opsonic
index may be increased or diminished within a few hours following
injections of bacteria, however, would suggest a possible distinction
between these antibodies and the slowly developing specific bacteri-
cidal and agglutinating antibodies.

Vaccine Therapy. The value of vaccines and of autogenous vac-
cination in bacterial prophylaxis and bacterial immunization as set

1 Deutsch. med. Wchnschr., 1904, 1458.

2 Jour. Inf. Dis., 1906, iii, 434; 1909, vi, 78; 1913, xii, 1.

170 ANTIGENS AND THE TECHNIC OF SERUM REACTIONS

forth by Wright marks a distinct epoch in bacterial therapeutics in
spite of the practical failure of his opsonic index determination as
a theoretical guide to immunization and treatment. He has used
bacterial vaccines both for prophylaxis to prevent infection with
specific bacteria and therapeuitically to arrest infection.

Prophylactic Vaccination. The object of prophylactic vaccination is
to increase the resistance of the recipient to specific infection. This
is accomplished by reinforcing the natural initial defenses of the
body with specific antibodies, generated in the host in response to
the injection of the specific microorganism as a vaccine. In prophy-
lactic vaccination the host has ample time to work over the vaccine,
and by prolonging the treatment through repeated graduated doses
the maximum degree of immunity may be expected. To attain the
maximum immunizing effect the bacteria of the vaccine should be as
near their normal state as possible, that is, they should be endowed
with all the antigenic properties they possess in the natural disease
produced by them in the host.

Following the brilliant work of Jenner with cowpox vaccine and the
epoch-making observations of Pasteur, observers are fairly agreed-
that the best results from prophylactic vaccination are obtainable
only by the use of an attenuated living virus. The action of such a
living virus is, as Theobald Smith 1 has aptly expressed it, " a multitude
of feeble blows, each of which produces an immunological response."
The dangers attending the use of attenuated viruses, however, ordi-
narily preclude their employment, due to inability to control the
virulence of attenuated cultures. The possibility of creating carriers
cannot be overlooked. For this reason killed cultures are almost
invariably selected.

It is, of course, impossible to utilize an autogenous vaccine, but
for purposes of immunization a polyvalent vaccine is indicated. The
action of a dead virus is limited practically to a single immunological
response, hence the need of repeated inoculations.

Therapeutic Vaccination. In chronic, long-drawn out focal or local
infections, the invading microbes are either holding their own or
gaining the ascendency and the object of bacterial vaccination is to
turn the tables on the invaders. The products of immunization must
be used at once, arid the organisms comprising the vaccines for this
purpose cannot ordinarily be as resistant as their originals in the host.
The underlying principle of therapeutic vaccination, according to

i Jour. Am. Med. Assn., 1913, Ix, 1591.

OPSONINS TROPINS BACTERIAL VACCINES 171

Wright, 1 is to exploit the normal tissues of the body in the interest
of the infected tissue. For this purpose, microbes similar to those
causing the infection (autogenous organisms) are inoculated into
some other part of the body. This inoculation is not, to use Wright's
phraseology, a mere replica of the original infection; there are two
important points of difference: (1) the microbes of the vaccine are
killed, so that their multiplication within the host is impossible; (2)
the dose of vaccine must be so regulated that the tissues of the host
at the site of inoculation and elsewhere must inevitably win. Victory
of the host is brought about through the elaboration of specific anti-
bodies generated in the healthy tissues on a scale more than adequate
to bring about a destruction of the organisms introduced into the
healthy tissue. The surplus of the specific antibodies will find its
way, through blood and lymph channels, to the focus of infection,
and will reinforce the partially depleted defensive forces which have
ineffectually opposed the invading organisms.

It should be borne in mind that vaccine therapy cannot be reason-
ably applied unless an exact bacteriological diagnosis has been made.
The immunizing effects of vaccines are definitely limited by the
ability of the normal tissues of the patient to produce antibodies;
to inject too frequently or in too large doses may not only be barren
of results it may result in a decrease rather than an increase of
resistance to infection.

It is essential for the best results of vaccination that the focus of
infection be so situated anatomically that the newly formed antibodies
be drawn to the infected area by the production of local hyperemia.
Infections of long standing naturally respond to treatment more
slowly than newly acquired infections.

Preparation of Vaccines. Much discussion has arisen concerning
the use of autogenous vaccines as compared with stock or polyvalent
vaccines. So little is actually known of what vaccines may accomplish
in the body that it is impossible to answer this question definitely.
It is desirable, however, to retain in the vaccine all possible anti-
genie properties which were possessed by the organism in the body.
It is a well-known fact that certain kinds of organisms rapidly lose
their ability to produce disease when they are grown for any length
of time outside the body. Others retain their virulence for some
time. This would appear to indicate that stock vaccines of the former
would be unsatisfactory, while stock vaccines of the latter might be

1 Proc. Roy. Soc. of Med., London, 1910, iii.

172 ANTIGENS AND THE TECHNIC OF SERUM REACTIONS

more successful. It is a safe general rule to state that an autogenous
vaccine is desirable.

The preparation of vaccine is carried out as follows:

1. Obtain pure cultures of the organisms from the lesion or what-
ever material is available. The details of culture vary with the type
of organism that is expected.

2. Inoculation of the pure culture, or cultures in the event of mul-
tiple infection, in suitable media to furnish the desired amount of
growth.

3. Removal of the growth, with sterile precautions, to a sterile
container, such as a test-tube containing sterile glass beads. This
is accomplished by washing the growth from the medium into sterile
saline solution : 5 to 1 c.c. of salt solution are required for an ordinary
agar slant culture. When enough growth is accumulated it is trans-
ferred to the sterile test-tube, being careful that no organisms con-
taminate the upper part, else they may escape sterilization.

4. Sterilization: Heat the bacterial suspension in a water-bath.
Usually one hour at 60 to 65 C. suffices. Care must be taken that
the level of water in the water-bath is well above that of the level
of the suspension in the test-tube.

5. Test sterility of the suspension. Inoculate suitable media and
observe the absence of growth. In skin infections it is sometimes
desirable to exclude the presence of the tetanus bacillus.

6. Shake the suspension vigorously to distribute the organisms
uniformly in it.

7. Standardize: Determine the number of bacteria in a cubic
centimeter. This is very simply accomplished by thoroughly mixing
equal volumes of freshly drawn blood and bacterial emulsion in a
pipette, spreading the mixture on a microscope slide, drying and
staining it with Wright's or Jenner's stain. Determine by actual
counting in a number of fields the proportion of bacteria to red cells.
Knowing the number of red blood cells in a cubic centimeter of blood
(5,000,000,000) and the proportion of bacteria to red blood cells, it is a
simple matter to determine the number of bacteria in the suspension.

A more accurate procedure is to draw up one volume of vaccine in
the erythrocyte pipette of a hemocytometer, dilute to the 101 mark
with a dilute solution of fuchsin or other suitable stain, mix and
transfer to the counting chamber. An enumeration of the bacteria
is made in precisely the same manner that a blood count is made.

OPSONINS TROPINS BACTERIAL VACCINES 173

8. Dilute the suspension to the required degree with phenol, so
that the finished vaccine shall contain 0.25 to 0.5 per cent, of it. This
is the finished vaccine.

9. Redetermine sterility if necessary.

Sensitized Vaccines. Killed bacteria which have been immersed
in a specific serum sensitized vaccines are said to be less liable
to produce general and local reactions. The immunity developed
in response to the injection of these sensitized vaccines is said to
appear more rapidly, and doses thirtyfold those of unsensitized vac-
cines may be injected without serious effect.

The Injection. The skin at the site of injection is cleaned with
soap and water and then with alcohol; or better, after carefully dry-
ing it is painted with tincture of iodin. The required amount of
vaccine is injected subcutaneously through this area, from a sterile
syringe.

The Dosage and Frequency of Injection. It is advisable to begin
with small doses of vaccine, quantities which past experience has
shown to do no harm so far as can be determined by clinical evidence,
and to increase the size of the dose gradually, the injections usually
being given at intervals of about a week. If no change results from
the treatment, larger doses may be tried. If the symptoms become
aggravated the doses should be diminished and given at less frequent
intervals. Generally speaking, in the more acute cases smaller doses
should be selected to begin with, larger doses being reserved for the
more chronic cases. The amounts of vaccine to be injected vary widely
according to different investigators. Generally speaking, the following
figures are fairly representative :

Minimum. 1 Maximum. 1 Average. 1

Staphylococcus ...... 5.0 1000 25

Streptococcus 2.5 100 25

Pneumococcus 2.5 100 25

Goriococcus 2.5 300 30

Coli 5.0 1000 100

Pyocyaneus ........ 5.0 1000 100

Indications for the Use of Bacterial Vaccine. Generally speaking,
bacterial vaccines are contraindicated in acute disease, but may be
employed in practically any localized infection, or an infection which
has become chronic. 2

1 Figures represent millions of organisms.

2 An excellent discussion of the present status of vaccine therapy is that of Theobald
Smith, An Attempt to Interpret the Present-day Use of Vaccines, Jour. Am. Med. Assn.,
1913, Ix, 1591.

174 ANTIGENS AND THE TECHNIC OF SERUM REACTIONS

Results. Opinions differ widely as to the value of vaccines. Accord-
ing to the theory of bacterial vaccination, subacute and chronic infec-
tions which are localized should give the best results, and such indeed
appears to be the case. For example, a streptococcus septicemia
abates and leaves a joint involvement or a heart valve vegetation.
Vaccine therapy has a better chance of producing results during this
secondary stage than during the earlier acute septicemic stage. Gon-
orrheal arthritis, pneumonias which resolve by lysis, pus sinuses, and
localized colon infections are suitable for treatment. In acute inflam-
mations of the mucous membranes of the intestines, bladder, throat,
etc., the results have been either negative or unsatisfactory.

So far as specific organisms are concerned, staphylococcus vaccines
give the most constant and satisfactory results. Furuncles, severe
carbuncles, some cases of acne, and even low-grade staphylococcus
septicemias yield rather readily to vaccine therapy with this organism.
Streptococcic and pneumococcic infections are much more resistant,
generally speaking, to vaccine treatment than are staphylococcus
infections.

CHAPTER IX.

THE MICROSCOPIC AND CULTURAL STUDY OF
BACTERIA.

2. Capsules.

3. Polar Bodies.

4. Flagella.

F. Differential Stains for Bac-
teria.

1. Gram.

2. Ziehl-Neelsen.

3. Gabbett.

4. Polychrome. Stains.

B. Preparation of Stains. 5. Smith Sputum Stain.

C. Technio of Staining Bac- III. STAINING BACTERIA IN TISSUES.

METHODS FOR THE MICROSCOPIC STUDY

OF BACTERIA.
I. LIVING BACTERIA.

A. Hanging Drop.

B. Hanging Block.

C. Dark Ground Illumination.

D. Intra Vitam Staining.
II. STAINING OF BACTERIA.

A. Chemistry of Stains.

teria.

D. Intensive Stains for Bac-
teria.

IV. METHODS AND MEDIA FOR THE

CULTIVATION OF BACTERIA.
V. CULTIVATION OF BACTERIA.

E. Stains for Special Struc- 1 Inoculation of Cultures.

tures of the Bac-
terial Cell.

1. Spores.

Isolation of Pure Cultures.
Incubation of Cultures.
VI. STUDY OF BACTERIAL CULTURES.

METHODS FOR THE MICROSCOPIC STUDY OF BACTERIA.

BACTERIA may be examined directly under the higher powers of the
microscope for their morphology, motility, arrangement, method of
reproduction, and their behavior in specific sera, or they may be
stained with various anilin dyes and chemicals to bring out details
of structure or composition, and their relation to various tissues in
pathological processes.

Glass slides and cover-glasses are conveniently used for this purpose.
Microscopic slides should be made from clear, colorless glass. Cover-
glasses should be made of thin glass. The available working distance
of oil-immersion lenses is somewhat less than 1.5 mm., consequently
cover-glasses should not measure more than 1 mm. in thickness as
a maximum limit. Number 1 cover-glasses are suitable for bacterio-
logical work.

Glass slides and cover-glasses are best cleaned in a mixture of
potassium bichromate, 1 part; water, 4 parts; sulphuric acid, 6 parts.
The bichromate is dissolved in the water with the aid of heat and
cooled; the acid is added slowly with constant stirring. Immersion
in this mixture for twenty-four hours removes dirt and grease from

176 MICROSCOPIC AND CULTURAL STUDY OF BACTERIA

both slides and cover-glasses. The cleaned glassware is removed from
the cleansing bath and washed with running water until neutral to
litmus paper. It is stored either in slightly ammoniacal alcohol, or
dried with a soft cloth, previously freed from grease by boiling in a
5 per cent, sodium carbonate solution.

I. Examination of Living Bacteria. A. Hanging Drop. The motil-
ity, shape, and size of bacteria may be studied in a "hanging-drop"
preparation. A drop of fluid from a bacterial culture in liquid media

FIG. 10. Hollow-ground slide for hanging drop.

is transferred to the center of a thin cover-glass. If the growth is
upon solid media a drop of physiological salt solution 1 is placed upon
the center of the cover-glass as before, and a very small amount of
the culture is removed with a platinum needle and emulsified in it.
Next, the rim of the concavity in a " hollow-ground slide" is ringed
with vaselin and the cover-glass is inverted over it in such a manner
that the drop is suspended in the hollow, but touches neither the
sides not the bottom. The vaselin seals the preparation, causing it
to adhere to the slide, and also prevents evaporation. The prepara-
tion is now ready for microscopic examination. The one-sixth or one-
eighth-inch objective should be used, with the diaphragm partly closed
to reduce the intensity of illumination. It is desirable to focus first
upon the edge of the drop; the edge is sharply defined and readily
located. Bacteria are usually more numerous at the edge than in the
center of the drop.

B. Hanging Block. It is desirable occasionally to follow the
development of bacteria through several generations, to study the
germination of spores, or to examine special structures within the
bodies of individual organisms. The hanging-drop method is
unsuited for this purpose, which presupposes immobilization of the
organism. Hill 2 has invented an ingenious modification of the hang-
ing-drop method, the hanging block, which fulfils this requirement.
His directions for preparing it are:

"Pour melted nutrient agar into a Petri dish to the depth of about
one-eighth or one-quarter inch. Cool this agar and cut from it a block

1 Physiological salt solution is prepared by dissolving 8.5 grams NaCl in distilled
water 1000 c.c.

2 Jour. Med. Research, March, 1902, vii, 202.

METHODS FOR THE MICROSCOPIC STUDY OF BACTERIA 177

about one-quarter inch to one-third inch square and of the thickness
of the agar layer in the dish. This block has a smooth upper and
under surface. Place it, under side 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. Place the
slide and block in a 37 C. incubator 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, carefully avoiding air-bubbles.
Remove the slide from the lower surface of the block and invert

FIG. 11. Warm stage, electrically heated, for the cultivation of bacteria.

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, to fill the angles between the sides of the block and 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-agar seal. Invert this preparation over a moist 4.
chamber and seal the cover-slip in place with white wax or paraffin.
Vaselin softens too readily at 37 C., allowing shifting of the cover-
slip. The preparation may then be examined at leisure. 1

1 A light, detachable, electrically heated warm-stage incubator, manufactured by
the Chicago Surgical and Electrical Company according to specifications furnished
by the writer is very satisfactory for this purpose. Bacteria may be maintained con-
stantly at any desired temperature between that of the room and 45 C. for several
days, and observed continuously without difficulty. If the warm-stage incubator is
attached to a graduated mechanical stage, many individual bacteria may be observed
in the same preparation by recording their respective positions as indicated on the
graduated rectilinear stage verniers.
12

178 MICROSCOPIC AND CULTURAL STUDY OF BACTERIA

C. Dark Field Illumination and Ultramicroscopic Examination. For
the study of very minute particles in suspension, the ultramicroscope
of Siedentoff and Zsigmondy 1 has been used, but the dark-ground
illumination apparatus of Reichert, 2 a much simpler device, readily
adjusted to any microscope, has largely supplanted it for bacterial
examinations. With the Reichert apparatus the flagella of bacteria
and other structures of low-refractive index may be observed. Tre-
ponema pallidum in fresh smears from lesions is readily seen with the
dark ground illuminating apparatus.

D. Intra Vitam Staining. Nakanishi 3 has applied the method of
infra vifam staining to the study of spores and granules in living bac-
terial cells. The method consists essentially in emulsifying a small
amount of bacterial growth in normal salt solution containing suffi-
cient aqueous methylene blue to impart a distinct blue color to the
solution. The preparation is viewed as in the hanging-drop slide.
The organisms absorb sufficient dye to impart to them a faint color,
and granules within their bodies frequently stain with moderate inten-
sity. The development of spores from pre-sporogenic granules may be
studied by this method.

II. Staining of Bacteria. A. Chemistry of Stains. The stains of
value for coloring bacteria are almost exclusively anilin dyes which
contain one or, more commonly, several benzene rings. Their color-
ing properties have been shown to depend upon two distinct radicals;
double-bonded atoms as C = C, C = O, C = N, N = N, known as
chromophoric groups, and auxochromic groups, which impart to or
intensify the color. Of the chromophoric groups, NH 2 and OH are
the more important. The latter form salts which may be either basic
or acid in character. Bacteria usually stain best with basic dyes, as
do nuclei of higher plant and animal cells.

The chemistry of the staining process itself is a matter of discussion.
It was formerly held that the cell protoplasm united chemically with
the stain as an acid unites with a basic salt, but later investigations,
particularly those of Michaelis, 4 are not in harmony with this view.
It is probable that the physical state of the cell membrane as well
as the composition of the cytoplasm play a part in the staining process.

B. Preparation of Stains. Stains prepared by Griibler or Merck
are commonly used for the staining of bacteria. They are conveniently

1 Zeit. f. wissenschaftl. Mikroskopie, 1909, xxvi, 391.

2 Miinchen. med. Wchnschr., 1906, 2351; Hyg. Rund., 1907, No. 18; Cent. f. Bakt.
Orig., 1909, li, 14.

3 Miinchen. med. Wchnschr., 1900, No. 6; Cent. f. Bakt., 1901, xxx, 97, 145, 193, 225.

4 Einfiihrung in die Farbstoffchemie, 1902, Berlin.

METHODS FOR THE MICROSCOPIC STUDY OF BACTERIA 179

kept in stock as saturated aqueous or alcoholic (96 per cent.) solutions.
The solubility of stains in water and in alcohol respectively varies,
but, as a rule, the solubility in alcohol is greater than that in water.
Saturated solutions of anilin dyes are unsuited for the staining of
microorganisms, but they are more stable than diluted solutions
provided they are kept in tightly-stoppered bottles away from the
light. Dilutions of saturated solutions are prepared as they are
needed for current use.

C. Technic of Staining Bacteria. 1. Preparation of a film of bac-
teria for staining: A drop of a culture of bacteria from a fluid medium
as broth is removed with a platinum loop and spread upon a clean
cover-glass or glass slide. Bacteria from a solid medium are emul-
sified in a small drop of water on the slide. 1

2. The film of bacteria is allowed to dry in the air; evaporation
may be hastened in the incubator at 37 C.

3. The air-dried film is next fixed by passing it once slowly through
the flame of a Bunsen burner, film side upward; about one-half second's
exposure to the flame suffices; a longer exposure destroys or changes
the staining properties of the organisms.

4. Staining: A 5 per cent, aqueous solution of methylene blue,
fuchsin, or gentian violet, prepared by adding 5 c.c. of filtered satu-
rated stock solution to 95 c.c. of distilled water, is used. The slide
or cover-glass is flooded with the desired stain, and after one to five
minutes, depending upon the intensity of the stain used, the excess
is poured off and the preparation is washed thoroughly with water.
The residual moisture is removed with filter paper or by air-drying,
and a small drop of Canada balsam (dissolved in xylol) is placed in
the center of the stained area. The film is finally enclosed between a
slide and a cover-glass.

D. Intensive Stains for Bacteria. Simple aqueous or alcoholic solu-
tions of anilin dyes are frequently inefficient for staining bacteria and
resort is made to intensified stains. One of the most useful of the
intensified stains is Loffler's alkaline methylene blue, prepared in the
following manner:

1 to 10,000 aqueous solution of potassium hydroxide 2 70 c.c.

Saturated alcoholic solution methylene blue 30 c.c.

1 It is essential that the emulsion shall be but faintly opalescent when viewed by
reflected light; a distinct clouding indicates that too many organisms have been added,
in which event the preparation will be found to be unsatisfactory.

2 Conveniently prepared by dissolving 1 gram of KOH in 100 c.c. distilled water
and adding 1 c.c. of this solution to 99 c.c. of distilled water.

180 MICROSCOPIC AND CULTURAL STUDY OF BACTERIA

Fixed films of bacteria are stained from one to five minutes with
this stain, or the films are flooded with the stain and heated until
steam rises (not bbiled) for one to three minutes. It is difficult to
overstain with Loffler's methylene blue unless evaporation takes
place to such a degree that the stain dries on the slide. The stain is
washed off with water, dried, and mounted.

E. Stains for Special Structures of the Bacterial Cell. 1. Spores.
(a) Flood fixed film of bacteria with carbol-fuchsin 1 and steam (not
boil) for five minutes.

(6) Wash thoroughly in running water.

(d) Wash thoroughly in running water.

(e) Flood with saturated aqueous solution methylene blue (or
Loffler's alkaline methylene blue) and allow to stain one minute.

(/) Wash in water, dry, and mount.
Spores stain red, vegetative cells blue. v

Holler's Spore Stain: 2 (a) Suspend the fixed film of bacteria in
chloroform for two minutes.
(6) Wash with water.

(d ) Wash thoroughly in running water.

(e) Flood with carbol-fuchsin and steam for five minutes.
(/) Wash thoroughly in water. . \* i

(g) Decolorize with 1 per cent, sulphuric acid until excess stain is
removed.

(h) Wash thoroughly in water.

(i) Flood with Loffler's alkaline methylene blue and allow to stain
one minute.

(j) Wash in water, dry, and mount.

Spores stain red, vegetative cells blue.

2. Capsule Stains. Welch Method* (a) Fixed films of bacteria are
flooded with glacial acetic acid for a few seconds.

(6) The acid is poured off and the preparation is washed two or
three times with anilin oil gentian violet, then flooded with the stain,
which is allowed to act for three to five minutes.

(d) Mount in salt solution and examine.
Capsules faint purple, bacterial body deep purple.

1 Saturated alcoholic solution of basic fuchsin, 10 c.c.; 5 per cent, aqueous phenol
solution, 90 c.c.

2 Cent. f. Bakt., 1891, x, 273. 3 Johns Hopkins Hosp. Bull., 1892, 128.

METHODS FOR THE MICROSCOPIC STUDY OF BACTERIA 181

Hiss's Method. 1 (a) Place a drop of sterile blood serum upon a
slide and emulsify bacteria in it.

(b) Dry in the air and fix by heat.

(d) Remove excess of stain by washing in a 20 per cent, solution
of copper sulphate.

(e) Dry with filter paper. Mount and examine.

Capsule faint pink or purple; body of organism deep red or purple.
Rosenow Method. 3 (a) Prepare the smear on perfectly clean cover-
glass.

(b) When smear is nearly dry, cover with 10 per cent, aqueous
solution of tannic acid for twenty seconds.

(d) Flood with anilin-oil gentian violet and steam gently for thirty
to sixty seconds.

(e) Wash thoroughly in water. ^

(/) Cover with Gram-iodin solution, one minute.
(g) Decolorize with 96 per cent, alcohol.

(h) Stain one or two minutes with a saturated (60 per cent.) alco-
holic solution of Griibler's eosin.

(i) Wash in water; dry and mount in balsam.
Capsules pink; bacteria blue.
3. Polar Bodies. -Neisser Stain. 4
Preparation of Stain.

Solution A Methylene blue 1 gram

Ninety-six per cent, alcohol ........ 20 c.c.

Glacial acetic acid 50 c.c.

Distilled water 950 c.c.

Solution B Bismarck brown 1 gram

Distilled water 500 c.c.

(a) The air-dried film, fixed by heat, is flooded with solution A for
three to five seconds.

(b) Wash with water.

(d) Wash with water, dry, and mount.
Polar bodies stain blue; bacterial cells brown.

1 Jour. Exp. Med., 1905, vi, 338.

2 Saturated alcoholic solution of the dye, 5 c.c.; distilled water, 95 c.c.

3 Jour. Infect. Dis., 1911, ix, 1.

4 Ztschr. f. Hyg., 1897, xxiv, 443.

182 MICROSCOPIC AND CULTURAL STUDY OP BACTERIA

4. Flagella. Preparation of Film. 1 (a) Add enough of an eighteen
to twenty-four-hour agar culture to a test-tube containing 5 c.c. of
sterile salt solution to produce a faint turbidity in the upper half of
the solution.

(6) Incubate at 37 C. for thirty to sixty minutes.

(c) Place two or three loopfuls of the suspension upon a perfectly
clean cover-glass and allow to dry spontaneously in the air or in the
incubator.

Do not attempt to spread the films with the platinum loop; agita-
tion breaks off flagella.

Staining Flagella. Pittsfield's Flagella Stain. 2

Preparation of Stain.

(a) Mordant:

Tannic acid, 10 per cent, aqueous solution 10 c.c.

Mercuric chloride, saturated aqueous solution 5 c.c.

Alum, saturated aqueous solution 5 c.c.

Carbol fuchsin 5 c.c.

(b) The Stain:

Alum, saturated aqueous solution 10 c.c.

Carbol fuchsin 5 c.c. or,

Gentian violet 2 c.c.

Flood the dried and fixed film with the mordant and steam gently
for one minute. Wash in running water, air-dry and flood with the
stain. Heat gently two minutes, wash thoroughly in water, air-dry
and mount.

F. Differential Stains for Bacteria. 1. Gram Stain. 3 A most impor-
tant differential method of staining bacteria is that, of Gram. Bacteria
may be divided into two groups: those which retain the initial stain
Gram-positive organisms and those which fail to retain the initial
stain but color with the counter stain Gram-negative bacteria.

It was believed formerly that the organisms which retained the
initial stain the Gram-positive bacteria contained within their
protoplasm, a substance of unknown composition which united
chemically with gentian violet (or other pararoseanilin dye) and
iodin to form a compound relatively insoluble in alcohol. Gram-
negative bacteria did not contain the hypothetical substance, which,
in association with the dye and iodin, was insoluble in alcohol. Treat-
ment of the latter group with alcohol, therefore, would remove the

1 Kendall, Jour. Applied Microscopy, 1901, v, 1836.

2 Medical News, September 7, 1895.

3 Gram, Fortschr. d. Med., 1894, ii,

METHODS FOR THE MICROSCOPIC STUDY OF BACTERIA 183

gentian violet, leaving them unstained. In the unstained condition
the organisms were colored with the second or counter stain. Subse-
quent investigation has largely discredited this view. It has been
shown by Kruse 1 that the cytoplasm of Gram-positive bacteria is
more resistant to autolysis, to the action of trypsin, and to solution in
dilute KOH than that of Gram-negative organisms, probably because
the cytoplasm of the former is less permeable to these various reagents
than is that of the latter. Eisenberg, 2 and Guerbet, Mayer and Schaef-
fer 3 have advanced the hypothesis that Gram-positiveness is due to
the lipoidal content of the cell membrane (ectoplasm) and specifically
to unsaturated fatty acids and phosphatids. The addition of iodin,
according to this theory, through the formation of alcohol-insoluble
combinations with the lipoids in the ectoplasm, renders the cell wall
impermeable to alcohol and thus prevents removal of the dye which
has already penetrated into the cell contents.
Preparation of Stain:

Solution A Saturated aqueous solution of anilin 4 90 c.c.

Saturated alcoholic solution of gentian violet . . 10 c.c. or,

Five per cent, aqueous solution of carbolic acid . . 90 c.c.

Saturated alcoholic solution of gentian violet . . 10 c.c.

The above solutions are unstable, but retain their tinctorial value
for two or three days if they are kept stoppered.

Solution B 5 Distilled water 300 c.c.

Potassium iodide 2 grams

Iodin crystals 1 gram

Solution C Bismarck brown, saturated aqueous solution ... 10 c.c.
Distilled water 90 c.c.

Procedure. (a) Prepare and fix film of bacteria in the usual manner.
(6) Flood with anilin-oil gentian violet (or carbolic gentian violet)
and stain five minutes.

(d) Decolorize with 96 per cent, alcohol until no more stain can be
removed.

(e) Wash thoroughly in water.

(/) Counterstain with Bismarck 1 brown 6 13r two minutes.
(g) Wash in water, dry, and mount.

1 Miinchen. med. Wchnschr., 1910, p. 685.

2 Cent. f. Bakt., 1909, xlix, 465; 1910, li, 115; liii, 481, 551; Ivi, 183.

3 Compt. rend., Soc. biol., Ixviii, 353.

4 Three c.c. of anilin oil are shaken for several minutes in 100 c.c. of distilled water.
The solution is filtered through filter paper to remove the undissolved anilin.

5 This iodin solution is variously known as Gram's iodin solution or Lugol's solution.

6 Dilute aqueous fuchsin, 1 to 10, may be used in place of Bismarck brown.

184 MICROSCOPIC AND CULTURAL STUDY OF BACTERIA

Bacteria which retain the initial stain Gram-positive bacteria
are colored dark purple or blue; those which fail to retain the initial
stain Gram-negative bacteria are brown, or bright pink if fuchsin
is used as a counterstain.

2. Stains for Acid-fast Bacteria. Ziehl-Neelsen Method. 1 (a) Stain
dried and fixed smear with carbol fuchsin, as described on page 180.

(6) Wash thoroughly with water.

(d) Wash with water.

(e) Counterstain lightly with Loffler's alkaline methylene blue.
(/) Wash, dry, and mount.

Acid-fast bacilli and spores red; all others blue.

3. Frdnkel-Gabbet Method. 3 (a) Stain with carbol fuchsin as in
the Ziehl-Neelsen method and wash in water.

(b) Decolorize and counterstain simultaneously with the following
solution :

Methylene blue 2 grams

Water . 75 c.c.

Sulphuric acid 25 c.c.

The counterstain is allowed to act for one minute.

4. Polychrome Stains. Polychrome stains are of special value for
the examination of exudates, body fluids or tissues in which the his-
tological relations of bacteria are to be investigated. These stains,
or modifications of them, are also useful in the study of treponemata,
spirochetes, and protozoa.

Wright's Stain. 4 Preparation. "To a 0.5 per cent, aqueous
solution of sodium bicarbonate add methylene blue (B.X., or ' medi-
cinally pure') in the proportion of 1 gm. of the dye to each 100 c.c.
of the solution. Heat the mixture in a steam sterilizer at 100 C. for
one full hour, counting the time after the sterilizer has become thor-
oughly heated. The mixture is to be contained in a flask, or flasks,
of such size and shape that it forms a layer not more than 6 cm. deep.
After heating the mixture is allowed to cool, placing the flask in cold
water if desired, and is then filtered to remove the precipitate which

1 Ziehl, Deutsch. med. Wchnschr., 1882, 451; Neelsen, Fort. d. med., 1885, 200.

2 Ninety per cent, alcohol containing 3 per cent, by volume of hydrochloric acid.

3 Frankel, Berl. klin. Wchnschr., 1884; Gabbet, Lancet, 1887.

4 Mallory and Wright, Pathological Technic, 1915, 6th ed., p. 382.

METHODS FOR THE MICROSCOPIC STUDY OF BACTERIA 185

has formed in it. It should when cold have a deep purple red color
when viewed in a thin layer by transmitted yellowish artificial light.
It does not show this color while it is warm.

"To each 100 c.c. of the filtered mixture add 500 c.c. of a 0.1 per
cent, aqueous solution of 'yellowish, water-soluble' eosin and mix
thoroughly. Collect on a filter the abundant precipitate which imme-
diately appears. When the precipitate is dry, dissolve it in methylic
alcohol (Merck's 'reagent') in the proportion of 0.1 gm. to 60 c.c.
of the alcohol. In order to facilitate solution the precipitate is to be
rubbed up with alcohol in a porcelain dish or mortar with a spatula
or pestle.

"This alcoholic solution of the precipitate is the staining fluid. It
should be kept in a well-stoppered bottle because of the volatility of
the alcohol. If it becomes too concentrated by evaporation and thus
stains too deeply, or forms a precipitate on the blood smear, the
addition of a suitable quantity of methylic alcohol will quickly correct
such faults. It does not undergo any other spontaneous change than
that of concentration by evaporation.

"A most important fault met with in the working of some samples
of this fluid is that it fails to stain the red blood corpuscles a yellow or
orange color, but stains them a blue color which cannot readily be
removed by washing with water. This fault is due to a defect in the
specimen of eosin employed. It can be eliminated by using a proper
'yellowish, water-soluble' eosin."

Method of Staining. (a) Unheated air-dried films 1 are covered
with the stain, which is allowed to act for one minute.

(6) Add an equal volume of distilled water to the stain and allow to
stand for three minutes.

(d) Dry rapidly with filter paper and mount in balsam. 2
Giemsa Method. 3 Preparation of Stain:

Azur II (eosin) 3.0 grams

Azur II 0.8 grams

Glycerin, C. P 250 c.c.

Neutral absolute methyl alcohol 250 c.c.

The dyes are dissolved in the glycerin at 60 C.; the alcohol, warmed
to 40 C., is then added, thoroughly mixed by shaking, and allowed
to cool slowly to room temperature, then filtered. Immediately before

1 Films more than a few hours old do not stain as well as fresh ones.

2 The balsam must be neutral in reaction.

3 Giemsa, Cent. f. Bakt., 1904, xxxvii, 308.

186 MICROSCOPIC AND CULTURAL STUDY OF BACTERIA

use, 10 c.c. of distilled water are slightly alkalinized by the addition
of two drops of a 10 per cent, solution of sodium carbonate, and
exactly ten drops of the stain are then added.

Staining with Giemsa Solutions. (a) Films are fixed by immersion
in neutral absolute methyl alcohol for one minute, air-dried, and
covered with the diluted stain, which is allowed to act for fifteen to
twenty minutes when ordinary exudates and bacteria are used; for
one to three hours if Treponemata or Negri bodies are sought for.

(b) Wash in water, dry and mount.

5. W. H. Smith's Solution Stain. (a) Stain the fixed smear with
anilin oil gentian violet for one minute.

(b) Wash with water.

(d) Decolorize with 95 per cent, alcohol.

(e) Wash with ether for a few seconds.

(/) Flood with absolute alcohol for five seconds.

(g) Stain with saturated aqueous solution eosin for one to two
minutes.

(k) Wash with absolute alcohol for a few seconds.

(i) Clear with xylol.

( j) Mount in balsam.

III. Staining Bacteria in Tissues. Paraffin sections are preferable,
partly because very thin sections may be cut; chiefly because celloidin
stains somewhat with the stains ordinarily used.

The Gram-Weigert Stain for Bacteria in Tissues. 1 (a) Stain paraffin
sections with anilin oil methyl violet for five to twenty minutes.

(6) Wash in water to remove excess of stain.

(d) Wash in water to remove excess of iodin.

(e) Decolorize with several changes of absolute alcohol until no
more color comes out.

(/) Clear section in xylol.
(g) Mount in neutral xylol balsam.

Mallory and Wright Modification for Celloidin Sections. 2 (a) Stain
sections with lithium carmine for two to five minutes.

(b) Remove excess of stain with acid alcohol.

(d) Dehydrate in 95 per cent, alcohol.

1 Mallory and Wright, Pathological Technic, 6th ed., 1915, p. 432.
* Ibid.

METHODS FOR THE MICROSCOPIC STUDY OF BACTERIA 187

(e) Expose to ether vapor to fix section to slide.
(/) Stain with anilin oil methyl violet for five to twenty minutes.
(g) Remove excess stain with normal salt solution.
(h) Gram-iodin solution for one minute.
(i) Remove excess iodin with water.

( j) Remove moisture as thoroughly as possible with filter paper.
(k) Dehydrate in several changes of anilin oil.
(/) Clear with several changes of xylol.
(m) Mount in neutral xylol balsam.

Staining Tubercle Bacilli in Tissues. (a) Paraffin sections are
covered with carbol-fuchsin and steamed gently for five minutes.

(b) The excess stain is removed with water.

(d) Remove excess of stain and acid with water.

(e) Dehydrate with absolute alcohol.
(/) Clear section in xylol.

(g) Mount in xylol balsam.

Staining Actinomyces in Tissues Mallory Method. 1 (a) Stain
paraffin sections with saturated aqueous eosin for ten minutes.
(6) Remove excess stain with water.

(d) Remove excess stain with normal salt solution.

(e) Remove excess water with filter paper.
(/) Clear in anilin oil.

(g) Remove anilin oil with several changes of xylol.

(h) Mount in neutral xylol balsam.

The clubs stain pink, the filaments blue.

IV. Methods and Media for the Cultivation of Bacteria. One of
the most important procedures in bacteriology is the preparation
of nutritive media in which the morphology, chemistry, and cultural
characteristics of the organism may be studied; furthermore, it is
possible by cultural methods to separate one type of bacterium in
pure culture from associated organisms, and to study its reactions
apart from all contaminating microorganisms. The technic of isolat-
ing and cultivating bacteria is exacting at every step of the process,
from the preparation of glassware to the selection of suitable nutritive
media, and their preparation requires not only scrupulous cleanliness;
it necessitates a most rigorous maintenance of sterility.

1 Loc. cit., p. 433.

188 MICROSCOPIC AND CULTURAL STUDY OF BACTERIA

Bacterial cultivation is usually carried out in glass vessels test-
tubes, flasks, fermentation tubes, and Petri dishes because glass is
transparent and permits an unobstructed view of the reactions taking
place within. It is obvious that glassware employed in bacterial
laboratories must be chemically and bacteriologically clean.

Preparation of Glassware. The method to be employed in the clean-
ing of glassware depends somewhat on the purpose for which it is

FIG. 12. Petri dish.

used. New glassware frequently contains alkali, which is readily
neutralized by diluted acid, hydrochloric or sulphuric. Glassware
that has contained cultures of bacteria is first sterilized in the auto-
clave to remove all danger of infection, then immersed in a strong
solution of soap-powder and soap-suds maintained at a boiling tem-
perature for half an hour. The adherent media is removed with a
brush or swab; a final thorough rinsing in clear water removes all

FIG. 13. Fermentation tubes various types.

traces of soap. Very dirty glassware or glassware in which chemical
determinations are to be made should be cleaned in chromic acid
solution, which is prepared by adding a saturated aqueous solution
of potassium bichromate to a 1 to 3 dilution of sulphuric acid. Twenty-
four hours' exposure to chromic acid removes all traces of organic
matter, as a rule. Following the acid bath the glassware is thoroughly
rinsed in clear water and dried.

METHODS FOR THE MICROSCOPIC STUDY OF BACTERIA 189

The cleaned glassware test-tubes, flasks, or fermentation tubes
is then stoppered with non-absorbent cotton cotton batting which
has a long staple or fiber. The cotton plugs must be carefully fitted
neither too loose, which would permit of the passage of adventitious
microorganisms, nor too tight, for obvious reasons. The cotton plugs
are conveniently prepared from a layer of cotton batting about two
inches square (for a test-tube of ordinary diameter, about 15 mm.),
which is laid squarely over the orifice. The center of the square is
gently pushed down into the neck of the tube for a distance of about
three-fourths to one inch; sufficient cotton protrudes from the tube
to be conveniently grasped by the fingers and removed. It is fre-
quently advisable to cover the cotton plugs with two or three layers
of filter paper, which prevents an accumulation of dust on the cotton.
Wide-mouthed containers are sealed with several layers of unglazed
paper fastened in place with a piece of twine. Flasks are frequently
not plugged with cotton; the neck is simply covered by an inverted
beaker of appropriate size.

Glassware should always be sterilized before media is placed in
it; this is readily accomplished by dry heat. A hot-air sterilizer is
used, in which a temperature of 180 C. is maintained for one hour.
A higher temperature must be avoided, to prevent charring of cotton
plugs. The heat must be increased gradually and diminished gradually,
to prevent cracking of the glass. By this process not only is the
utensil rendered sterile, the plugs of cotton retain their shape when
withdrawn, as well.

A majority of the bacteria pathogenic for man and many parasitic
and saprophytic forms as well require relatively complex organic
compounds containing carbon, hydrogen, nitrogen, and oxygen,
together with other elements for their nutrition. These foodstuffs
provide both the structural and fuel requirements of the organism,
as explained in the chapter on Bacterial Metabolism. Experience has
shown that a medium containing meat infusion, peptone, and salt is
a satisfactory one for many bacteria. This medium may be enriched
by the addition of various ingredients to meet the requirements of the
more fastidious organisms.

Meat infusion is prepared from finely comminuted lean meat 1 freed
from fat. 500 grams of meat are intimately mixed with 1000 c.c. water
and allowed to infuse over night in the refrigerator. It is then strained

1 Beef hearts make a very satisfactory meat infusion and their cost is much less than
the better cuts of meat.

190 MICROSCOPIC AND CULTURAL STUDY OF BACTERIA

through several layers of cheese-cloth, the volume recorded, then
heated to boiling. The coagulum which forms is removed by filtration
through filter paper and the clear, amber-colored fluid, after restoring
the loss due to evaporation, is run into flasks and sterilized in an
autoclave at 15 pounds' pressure for fifteen minutes. This plain meat
infusion contains but little protein; it is relatively rich, however, in
soluble meat extractives, soluble salts and muscle-sugar dextrose.
It is not suitable in itself as a complete nutritive medium for most
bacteria, but it forms the basis of many of the commonly used nutri-
tive media. Meat extract (Liebig's or other kinds) is frequently
substituted for meat infusion. Three grams of meat extract are dis-
solved in a small volume of water, filtered through a cold wet filter
paper to remove fat, and made up to a volume of a liter. The solution
contains some meat extractives, including a relatively large propor-
tion of xanthin bases and a very considerable amount of salts, par-
ticularly sodium chloride. Little or no muscle-sugar is present. It
is distinctly inferior to meat infusion, however, as a basis for cultural
media, especially for the more delicate pathogenic organisms.

The Reaction of Media. Bacteria are relatively sensitive to com-
paratively slight changes in the reaction of their nutritional environ-
ment, and it is essential to create a suitable initial degree of acidity
or alkalinity in media to favor their growth. A reaction neutral to
phenolphthalein slightly alkaline to litmus is suitable for most of
the bacteria pathogenic for man human tissues and blood are slightly
alkaline to litmus. A reaction of 1 per cent, acid (+1.0), using phenol-
phthalein as an indicator, has been recommended by the Laboratory
Section of the American Public Health Association for the routine
bacterial examination of water, ice, sewage, milk, cream, and ice-cream.
A reaction of 1 per cent, signifies that 1 c.c. of normal NaOH would
be required to neutralize the acid in 100 c.c. of the medium. Ten
c.c. of Y NaOH would be required to exactly neutralize one liter of
medium having an acidity of 1 per cent.

The reaction may be determined accurately in the following manner :
to 45 c.c. of distilled water, contained in a porcelain evaporating dish
of 100 c.c. capacity, are delivered exactly 5 c.c. of the medium from a
graduated pipette. The solution is brought to the boiling-point over
the free flame to expel CO 2 and 1 c.c. of a solution of phenolphthalein 1

1 Made by dissolving 0.5 gram phenolphthalein in 100 c.c. 50 per cent, alcohol. This
indicator is colorless in acid solution pink in an alkaline solution. CO2 interferes with
its accuracy as an indicator. It is especially sensitive to organic acids which occur in
ordinary media, hence its value in media titrations.

METHODS FOR THE MICROSCOPIC STUDY OF BACTERIA J91

is added as an indicator. The solution usually remains colorless,
because ordinary media are acid in reaction; Jg NaOH is added slowly
from a burette until a faint pink color appears and persists after one
minute's boiling. From the amount of f alkali required to neutralize
5 c.c. of medium, the reaction of the entire amount is readily com-
puted. Thus:

5 c.c. media are neutralized by 3 c.c. ^ NaOH.
100 c.c. media would be neutralized by 3 c.c. normal (y)NaOH.
1000 c.c. media would be neutralized by 30 c.c. normal (j)NaOH.

To reduce the reaction of a liter of medium whose initial reaction
is + 3.0 to + 1.0, 20 c.c. of normal NaOH would be required. It is
necessary to heat the medium after adding the alkali, in order to
promote the reaction between the acids of the medium and the neu-
tralizing solution and a redetermination of the reaction should be
made to make certain that the desired change in acidity has taken
place. Frequently a second addition of alkali is necessary to create
the desired final reaction.

A satisfactory reaction for cultural media designed for most patho-
genic bacteria may be created by adding ^ NaOH solution a few
drops at a time, to the entire volume, using filter paper dipped in
phenolphthalein solution, and dried, as an indicator. When the paper
shows a faint pink color the addition of alkali is discontinued. The
reaction is practically neutral under these conditions.

The Clarification of Media. It is desirable, in the preparation of
culture media, to remove all insoluble substances. This is accom-
plished by filtration methods, with or without preliminary treatment,
to flocculate the substances in suspension. The addition of non-heat-
coagulable proteins, as gelatin, frequently requires clarification with
a coagulable protein, as egg-albumen, to remove the finely divided
suspended matter.

Clarifying with Eggs. For each liter of medium to be clarified, two
eggs thoroughly whipped in a small amount of water are added. The
temperature of the medium should not exceed 50 C. The eggs are
thoroughly stirred in and the entire mixture is slowly heated to 100
C., either in a double boiler or in the Arnold sterilizer. A firm coagulum
forms during the heating process, which enmeshes the suspended par-
ticles it is desired to remove. The medium should never be disturbed
during the coagulating process. The clear underlying medium is
drawn off and filtered through cotton.

192 MICROSCOPIC AND CULTURAL STUDY OF BACTERIA

Filtration through Cotton. A large glass funnel is lined with a double
layer of absorbent cotton; the layers are placed at right angles,
thus laying the fibers of cotton at right angles. The cotton is moistened
with a small amount of hot water if agar or gelatin is to be filtered.
The medium is then carefully poured into the funnel, care being taken
that the cotton is not displaced by the force of the inflowing fluid. The
first portion of filtrate may not be clear and it is somewhat diluted

FIG. 14. Hot-air sterilizer. Lautenschlager form. (Park.)

with the water originally used to wet the cotton hence it should be
returned and refiltered. Agar and gelatin filter slowly, which may
lead to congelation, therefore the top of the funnel should be covered
to prevent undue loss of heat. Funnels surrounded by a hot water
jacket are sometimes used in the filtration of these media. Media
that are fluid when cold may be often advantageously clarified by
filtration through a good grade of heavy filter paper, with or without
a preliminary clarification with eggs, as occasion demands.

METHODS FOR THE MICROSCOPIC STUDY OF BACTERIA 193

The Distribution of Media. The clarified media are either stored in
flasks or transferred to smaller containers for immediate use. Then
they are sterilized. Most media are used in test-tubes. Test-tubes
are filled from a reservoir, usually a large funnel, the smaller end of
which is provided with a short length of rubber tubing, into which a
short glass tube, constricted somewhat at the outer end, is introduced.
The flow is controlled by a pinch cock, which constricts the rubber
tubing midway between the funnel and the delivery tube. The cotton
plug is removed from a test-tube and the delivery tube is introduced
into the open end of it to a depth of about two inches. The pinch

FIG. 15. Arnold steam sterilizer.
(Abbott.)

STERILIZING CHAMBER

U A 4 A

FIG. 16. Arnold steam sterilizer.
Ordinary type. (Park.)

cock is opened somewhat and the desired volume is allowed to flow in.
The pinch cock is then released to stop the flow, the delivery tube
removed, care being taken that no media touches that part of the
test-tube where the cotton fits, so that it will not adhere to the sides
of the tube, and the cotton plug is replaced. Usually about 8 to 10
c.c. of media are added to a tube.

Sterilization of Media. Media which do not contain coagulable
proteins, gelatin or carbohydrates are sterilized for fifteen minutes in
an autoclave at a live steam pressure of fifteen pounds (121.3 C.).
Media containing gelatin or carbohydrates are sterilized at a lower
temperature by discontinuous sterilization half an hour on three

194 MICROSCOPIC AND CULTURAL STUDY OF BACTERIA

successive days, in flowing steam in an Arnold sterilizer. After each
sterilization the medium is kept at room temperature to permit of the
germination of spores. Lower temperatures are occasionally employed,
particularly for the sterilization of blood serum or other native pro-
teins an exposure of to 70 C. for an hour on each of six successive
days usually suffices. Bacteria may be removed from fluid media
and from various sera and solutions containing thermolabile toxins
or similar products by filtration through sterile porous filters made of
unglazed porcelain or diatomaceous earth Pasteur or Berkefeld
filters. These filters are made with varying degrees of porosity,

FIG. 17. Arnold steam sterilizer. Boston Board of Health type. (Park.)

regulated largely by the thickness of their walls to accommodate vary-
ing needs. Usually the fluid is forced through the walls of the filter
into the center, which is hollow, by suction. The clear, bacteria-free
filtrate passes into a sterile container attached to the filter. The filters
and their necessary accessory parts are sterilized in the autoclave for
fifteen minutes at fifteen pounds live-steam pressure. Turbid fluids
should be passed through several layers of filter paper prior to filtra-
tion, to remove the grosser particles which otherwise would soon
clog the filter. A time limit, usually not exceeding two hours as a
maximum, should be set, beyond which filtration should be stopped

METHODS FOR THE MICROSCOPIC STUDY OF BACTERIA 195

bacteria may be forced through filters and contaminate the filtrate
if the process is carried much beyond this interval.

New, unused filters should be cleaned by running several liters of
clean water through them and they should invariably be tested before
use to guard against " pin-holes."

After filtration the filter is sterilized to kill whatever bacteria have
contaminated it. Then the surface is thoroughly scrubbed with a
brush and 1 per cent, alkaline permanganate solution (potassium per-
manganate 10 grams, water 1000 c.c.) is run through to remove organic
matter. Five per cent, oxalic acid is then passed through to remove

FIG. 91 FIG. 92 FIG. 93 FIG. 94

FIGS. 91 to 94. Types of unglazed porcelain filters. (Park.)

the permanganate solution and the acid finally removed by repeated
washings with water. If the filter becomes so clogged with organic
matter that it can no longer deliver a reasonable amount of filtrate,
the filter is placed in a muffle-furnace, gradually heated to about
250 C., and as gradually cooled. It is then cleaned as before with
permanganate solution, to remove the last traces of organic matter.

Storage of Media. If media are not to be used at once it is necessary
to protect them from evaporation and contamination. Flasks of media
are preserved best by tying paper caps over the cotton plugs if the
period of storage does not exceed a few days, or by pouring melted
paraffin over the plugs if longer periods of storage are contemplated.

196 MICROSCOPIC AND CULTURAL STUDY OF BACTERIA

It is necessary to burn the surface of the plug to destroy surface con-
tamination, then to push the plug into the neck of the flask for a dis-
tance of 1 cm. to make room for the paraffin. Flasks hermetically
sealed in this manner may remain visibly unchanged for weeks or even
months. It is good practice to place a lead foil cap over the paraffin

FIG. 22. Autoclave. (Park.)

plug and lead foil caps are better than paper caps as coverings for cot-
ton plugs. Media in storage should be maintained at a temperature
not exceeding 45 C., in a dry ice-box.

The Preparation of Nutrient Bouillon (Broth). M eat Infusion Broth
To 1000 c,c. of meat infusion (see page 189 for preparation), in a tared,

METHODS FOR THE MICROSCOPIC STUDY OF BACTERIA 197

agate-ware boiler, add 5 grams of common salt (NaCl) and heat to
boiling. Dust 10 grams of Witte peptone over the surface and stir
until it is thoroughly dissolved. Restore the loss by evaporation and
adjust the reaction to the desired degree of acidity. Boil for five
minutes, verify the reaction and filter through filter paper until the
filtrate is perfectly clear. Sterilize in the autoclave.

Meat Extract Broth. 1 To 1000 c.c. of meat extract (see page 190 for
preparation) in a tared agate-ware boiler, add 10 grams of Witte pep-
tone, dusting the peptone on the surface. Heat to boiling, restore
loss by evaporation and adjust the reaction. Continue the boiling for
five minutes, verify the reaction and cool to room temperature. 2 Filter
cold through filter paper until perfectly clear and sterilize.

Nutrient Sugar-free Broth. Meat infusion contains small amounts
of muscle-sugar dextrose usually about 0.1 per cent. This sugar
is present in nutrient meat infusion broth prepared as outlined above.
It is frequently desirable to prepare meat infusion broth free from all
sugars. The dextrose is readily removed by fermentation with Bacillus
coli, adding a broth culture of this organism to the meat infusion before
it is heated and maintaining the infusion at 37 C., for eighteen to
twenty-four hours. The sugar which is attacked by Bacillus coli in
preference to the protein constituents of the medium 3 is quantitatively
removed. The organism must be killed as soon as the sugar is
exhausted, otherwise the protein constituents will be attacked. The
end of the fermentation may be judged with a fair degree of certainty
if one removes some of the infusion seeded with Bacillus coli to a fer-
mentation tube, kept at the same temperature, 37 C.; when gas is
no longer evolved the sugar is exhausted. Sugar-free broth contains
lactic acid, one of the products of fermentation of dextrose by Bacillus
coli. After the sugar is removed the medium is sterilized in the usual
manner, or made directly into sugar-free nutrient meat infusion broth
as outlined above.

Nutrient Sugar Broth. One per cent, of dextrose, lactose, saccharose,
mannite, or other carbohydrate is added to nutrient sugar-free broth
immediately before filtering. Media containing sugars are best steri-
lized in the Arnold sterilizer on three successive days; the high tem-
perature of the autoclave tends to decompose carbohydrates.

1 It is unnecessary to add salt to meat extract.

2 A precipitate containing phosphates, soluble in the hot medium, settles out upon
cooling. It must be removed before the medium is used.

8 See chapter on Bacterial Metabolism.

198 MICROSCOPIC AND CULTURAL STUDY OF BACTERIA

Calcium Carbonate Nutrient Sugar Media. Bacteria grown in
sugar media frequently form acid products from the fermentation of
the sugars the amount of acid products may be sufficient to inhibit
the development of the organisms even after one or two days'
growth. The addition of insoluble carbonates as calcium carbonate
neutralizes the acid as it is formed and thus maintains automatically
a favorable reaction for prolonged development. Bolduan 1 has shown
that pieces of marble about 0.5 centimeters square in 100 c.c. of broth
not only restrain the development of free acid the marble appears to
create a somewhat more favorable medium, especially for the pneumo-
coccus and streptococcus as well. The bits of marble should be-
sterilized in the hot-air sterilizer before they are introduced into the
broth.

Nutrient Glycerin Broth. To 1 liter of sugar-free broth add 3 to
5 per cent, pure, redistilled glycerin immediately before filtering.
Sterilize in the autoclave fifteen minutes at fifteen pounds pressure.
Glycerin broth is extensively used for the cultivation of the tubercle
bacillus 2 and it is frequently employed in the culture of bacteria which
are susceptible to desiccation the glycerin conserves the moisture
and retards evaporation.

The various sugar-broths may be prepared with meat extract as
a basis; pathogenic bacteria develop less luxuriantly as a rule in
extract media than in meat infusion media, however.

Dunham's Solution. Five grams of common salt and 10 grams of
Witte peptone are added to one liter of water and heated to boiling
until the peptone is completely dissolved. Pass through filter paper
until perfectly clear, tube, using 10- c.c. to each test tube, and sterilize
in the autoclave. The reaction does not require adjustment.

This medium is frequently used to test the ability of bacteria to
form indol. Indol is formed in the absence of utilizable sugars by
Bacillus coli; members of the cholera group and other bacteria form
tryptophan by the splitting off of alanin :

CH 2 .CHNH 2 .COOH

\ /\/

\/ NH
Tryptophan

Alanin. The alanin is decom-
posed by the bacteria.

1 New York Medical Journal, May 13, 1905.

2 The reaction of glycerin broth designed for the cultivation of tubercle bacilli should
be +1.0 acid. The organism does not develop well in media neutral to phenolphthalein.

METHODS FOR THE MICROSCOPIC STUDY OF BACTERIA 199

Samples of Witte peptone occasionally do not contain tryptophan,
consequently each lot of peptone should be tested. When an especially
favorable sample is found it should be reserved for this purpose. Plain
neutral sugar-free broth is a better medium than Dunham's solution
for the indol test, and it should be employed for this purpose whenever
possible.

Nitrate Broth. Add 10 grams of Witte peptone to one liter of water
and dissolve by boiling. Then add 0.2 gram chemically pure potas-
sium nitrate free from nitrites and filter. Sterilize in the autoclave.
The reaction does not require adjustment.

Nutrient Gelatin Media. Ten grams Witte peptone and 5 grams
NaCl are added to 1 liter of sugar-free meat infusion 1 and dissolved by
boiling. When the ingredients are in solution, 100 2 grams of "Gold
Label" gelatin are added, a few leaves at a time, and stirred until
dissolved. The reaction is then adjusted to the desired degree and
verified after an additional five minutes' heating. The medium is
cooled to 50 C., and clarified with eggs, using two eggs for each liter.
Filter through a double layer of absorbent cotton in a large glass funnel
until clear, and sterilize. When sterilization is accomplished, cool
quickly and store in the ice-box.

Nutrient Agar. (a) Dissolve 12 grams of powdered or shredded agar
in one liter of meat infusion by the aid of heat and add 5 grams NaCl
and 10 grams Witte peptone. Maintain a boiling temperature for at
least thirty minutes, or until the ingredients are completely dissolved;
restore the loss by evaporation, adjust the reaction, and filter through
a double layer of absorbent cotton in a large glass funnel. Pass through
filter until clear. It is frequently necessary to clarify agar with eggs.
After the reaction is adjusted, cool to 50 C. add two eggs beaten up
in water and mix thoroughly. Heat slowly to the boiling-point, boil
ten minutes, and filter through absorbent cotton; sterilize in the
autoclave.

(b) Prepare " double-strength" meat infusion; 1000 grams of finely
comminuted lean meat are suspended in one liter of water; infuse in
the ice-box for twenty-four hours; heat to boiling and filter through
filter paper. Prepare nutrient meat infusion broth with this strong
infusion as a basis and adjust the reaction to twice the desired acidity
thus, if +1.0 is to be the final reaction, make the infusion broth

1 Meat extract may be used in place of meat infusion, but the medium is not as satis-
factory for pathogenic bacteria.

2 Use 120 grams gelatin during warm weather.

200 MICROSCOPIC AND CULTURAL STUDY OF BACTERIA

+2.0. Dissolve 24 grams agar in one liter of water, boiling steadily
until complete solution is attained. Add to the meat infusion broth,
boil for ten minutes and clarify with eggs in the usual manner. Filter
and sterilize.

Meat Extract Agar. Meat extract agar is made by substituting
meat extract solution for meat infusion; otherwise the process is the
same. The medium must be clarified with eggs.

Glycerin Agar. Five per cent, of glycerin is added to meat infusion
agar immediately before filtration. The reaction for cultivation of the
tubercle bacillus should be +1.0 acid to phenolphthalein. Tubercle
bacilli do not thrive in media neutral to phenolphthalein.

Loffler's Blood Serum. Add one part by volume of 1 per cent,
nutrient dextrose broth 1 to three parts of clear, hemoglobin-free beef
or sheep serum, and distribute in test-tubes. The tubes are placed
in a Koch's serum inspissator or in specially designed racks in an auto-
clave in an inclined position to produce a slanted surface, and slowly
heated to 80 C. This temperature is maintained until the medium
is firmly coagulated. The temperature is then raised to 95 or 100
C., and maintained for an hour on each of three successive days, or to
115 in the autoclave, and maintained for one hour. The medium is
opaque and white and the surface is smooth and should be free from
a metallic lustre when viewed by reflected light. The lustre indicates
an accumulation of salts, which are inimical to the growth of many
bacteria.

Coagulated Serum. 2 Clear blood serum from the dog, sheep, cow. or
other animal, preferably sterilized by filtration through Berkefeld
filters, and with or without the addition of glycerin, is placed in test
tubes and slanted and coagulated in a serum inspissator at a tempera-
ture of 75 to 80 C. An exposure of one hour to this temperature on
each of six successive days is necessary to insure sterility. The medium
should be translucent, free from bubbles, and firm.

Hiss Serum Water Media. Hiss 3 has recommended a serum water
medium for the cultivation of pneumococci and similar organisms.
It is prepared in the following manner :

Sheep or beef serum, 4 clear and free from hemoglobin, is added to
water in the proportion of one volume of serum to three of water.

1 If the liquefaction of blood serum by bacteria is to be tested, sugar-free broth must
be used in place of dextrose-broth.

2 Theobald Smith, Tr. Am. Phys., 1898, xiii, 417.

3 Jour. Exp. Med., 1905, vii, 223.

4 It is advisable to sterilize the serum by passage through an unglazed porcelain filter.

METHODS FOR THE MICROSCOPIC STUDY OF BACTERIA 201

Ten per cent, aqueous solutions of various sugars are prepared and
sterilized, and a sufficient amount of the desired sugar to make a
final concentration of 1 per cent, is added to the sterile serum solution.
Sufficient sterile 5 per cent, litmus solution is added for an indicator.
Fermentation of the carbohydrate is shown by the development of an
acid reaction, and frequently by a well-defined coagulation of the
medium as well.

Endo Medium for the Isolation of Typhoid, Paratyphoid, and Dysentery
Bacilli. I. Preparation of Agar. (a) Prepare plain, sugar-free nutri-
ent agar as described on page 197, using 15 grams of agar per liter.

(6) Adjust the reaction to a point just alkaline to litmus.

II. Preparation of Indicator. (a) Prepare a 10 per cent, solution
of basic fuchsin in 96 per cent, alcohol. This solution is fairly stabile
if kept away from the light.

(b) Prepare a 10 per cent, aqueous solution of chemically pure
anhydrous sodium sulphite (1 gram in 10 c.c. water). This solution
does not keep.

' (c) Add 1 c.c. of "II, a" to 10 c.c. of "II, b" and heat in the Arnold
sterilizer for twenty minutes. The color of the fuchsin is nearly
discharged if the solutions are of proper strength. This solution must
be prepared each day it does not keep.

III. Preparation and Use of Endomedium. (a) Add 1 gram of
C. P. lactose (free from dextrose) to 100 c.c. of agar and place in the
autoclave until melted and the lactose is thoroughly dissolved.

(b) Add a sufficient volume of "II, c" (about 1 c.c) to impart a
faint pink color to the medium.

(c) Pour into sterile Petri dishes and allow to harden in a dark
place with the covers partly removed. When cool the medium should
be colorless when viewed from above and a very faint pink when viewed
from the edge. The medium must be kept in a dark place because
the color is restored by the action of daylight.

Those bacteria which ferment lactose as Bacillus coli form lactic
acid which restores the color of the medium in the immediate neigh-
borhood of the colony; the colony therefore is colored red. Some
aldehydes also restore the color, but it is not very probable that alde-
hyde production is commonly observed among the lactose-fermenting
organisms. Non-lactose fermenting bacteria grow as colorless colonies.

If the plates are to be incubated two or three days it may be
advisable to increase the agar to 2.5 per cent, to limit the diffusion of

202 MICROSCOPIC AND CULTURAL STUDY OF BACTERIA

color from the acid colonies. For rapid isolations the medium with
the normal percentage of agar is preferable. 1

The Technique of Inoculation of Endomedia is described on page 231.

Lactose Litmus Agar. I. Prepare 1 per cent, lactose nutrient agar
by adding 10 grams of C. P. lactose (free from dextrose) to one liter
of plain nutrient agar. Adjust the reaction to a point slightly alkaline
to litmus. Tube and sterilize in the Arnold sterilizer.

II. Prepare an aqueous solution of litmus either a 5 per cent,
solution of purified litmus (Merck) or a 1 per cent, solution of azo-
litmin (Kahlbaum) and sterilize.

To Use Lactose Litmus Agar. Add about 1 c.c. of sterile litmus
solution to a sterile Petri dish and pour over it the melted lactose
agar, previously inoculated with the desired material. For water and
milk, add 1 c.c. of water or milk (diluted to the proper degree) to the
Petri dish before adding the lactose agar. Mix intimately by rotating
gently, allow to harden, and incubate.

Those bacteria which ferment lactose with the production of acid
appear as red colonies. Non-lactose-fermenting organisms appear as
blue colonies.

Blood Agar. Blood is drawn with aseptic precautions from the
carotid or femoral artery of a dog or rabbit into a sterile flask con-
taining beads. The blood is defibrinated by prolonged agitation and
added to plain (not dextrose) nutrient agar previously melted and
cooled to 45 C., in the proportion of 2 c.c. of blood to 10 c.c. of agar.
Small amounts of blood may be withdrawn directly from the heart
of an animal without difficulty, provided a small hypodermic needle
is used. The blood may be injected directly into the melted agar
without defibrination.

Occasionally human blood is added to agar; if a series of agar slants
are prepared it is possible to convert them into blood agar with a small
amount of blood, as follows :

Withdraw 10 c.c. of blood, using aseptic -precautions, from the
median basilic vein, in a large syringe. Inject the blood at once into
four times the volume of plain nutrient agar melted and cooled to 45
C. Mix at once and run 2 c.c. over the slanted surface of each agar
slant, and allow to harden in the inclined position in such a manner
that a uniform layer of the blood-agar mixture is obtained. Incubate
to prove sterility.

1 Kendall and Day, Jour. Med. Res., 1911, xxv, 95.

METHODS FOR THE MICROSCOPIC STUDY OF BACTERIA 203

Ascitic and Hydrocele Fluid Media. Ascitic and Hydrocele Agar. 1
Collect ascitic or hydrocele fluid in a sterile bottle, using aseptic
precautions. Allow to stand in the ice-box until clear, and heat to
50 C. for half an hour to destroy enzymes. Two parts of hydrocele
or ascitic fluid to eight or ten parts of plain nutrient agar previously
melted and cooled to 45 C. make a medium especially adapted to the
growth of many of the more fastidious pathogenic bacteria. 2

Ascitic broth is prepared by adding 20 to 50 per cent, by volume of
sterile ascitic fluid to plain nutrient broth. Incubate to prove sterility.

Egg Media. Eggs are a very good substitute for blood serum in
Loffler's medium. Eggs are carefully broken into a clean beaker
stirred gently with a rod (avoiding the formation of air bubbles) until
homogeneous, and mixed with dextrose broth in the proportion of
one part by volume of broth to three volumes of egg. The medium
is coagulated in a slanted position and sterilized precisely as Loffler's
blood serum is coagulated and sterilized.

Egg Medium. No. 1. Mix four to six volumes of thoroughly homo-
genized eggs with one volume of nutrient broth, and add sufficient
glycerin to make the concentration of the latter 3 per cent, by weight.
Coagulate and sterilize in the slanted position precisely as Loffler's
blood serum is coagulated and sterilized. This medium is excellent
for the cultivation of tubercle bacilli.

No. 2. Add one volume of physiological salt solution to ten volumes
of egg which have been lightly stirred with a rod until the yolks and
whites are intimately incorporated. Coagulate and sterilize in a
slanted position in test tubes.

Milk and Litmus Milk. One liter of fresh milk is thoroughly mixed
and tubed in the ordinary manner. Litmus milk is prepared by adding
sufficient litmus solution to impart a clea'r blue color. It is tubed,
using 10 c.c. to each tube, and sterilized in the autoclave.

For some purposes it is desirable to remove the cream before tubing,
but for cultural work the color of the cream ring in litmus milk is of
some diagnostic importance. Thus, members of the paratyphoid
group of bacilli almost invariably show a blue-green cream ring; the
colon bacillus colors the cream ring red brown. It should be remem-
bered that litmus milk does not coagulate as readily or as rapidly as
plain milk.

1 Ascitic and hydrocele fluids may be sterilized by passage through an unglazed porce-
lain filter.

2 It should be remembered that ascitic and hydrocele fluids usually contain about
0.08 per cent, dextrose.

204 MICROSCOPIC AND CULTURAL STUDY OF BACTERIA

Potato. New potatoes have an acid reaction, as a rule, and old
potatoes are slightly alkaline.

Large potatoes are thoroughly scrubbed, the skin removed, and
cut into cylinders with a cork-borer. The cylinders, which should be
at least 1.5 cm. in diameter, are divided into equal parts by a diagonal
cut. The pieces are placed in running water overnight so that they
will not darken, and are inserted, base downward, in large test tubes.
It is advisable to add about 1 c.c. of water to each tube to prevent
drying. Sterilize in the autoclave.

Hiss's 1 Semisolid Medium.

FORMULAE

Water . . -. 1000 c.c.

Agar 8 grams

Peptone 10 "

Meat extract 3 "

NaCl 5 "

Gelatin 2 40 "

When all the ingredients are dissolved, adjust the reaction to +0.5
(phenolphthalein), filter, and add sufficient litmus solution to impart
a clear blue. Dissolve 1 per cent, of dextrose, lactose, saccharose,
mannite, or other carbohydrate in the medium, and fill test-tubes
with it. Sterilization of lactose and saccharose semisolid media is
preferably carried out in the Arnold sterilizer. Dextrose and mannite
media may be sterilized in the autoclave.

Semisolid media are inoculated by the stab method. A change in
reaction is indicated by the litmus; gas-forming organisms form bubbles
in the depth of the medium.

Russell Double Sugar Medium. To 1 liter of nutrient agar, slightly
alkaline to litmus, add sufficient sterile 5 per cent, litmus solution to
impart a distinct clear blue color. Add 1 per cent, of C. P. lactose
and 0.1 per cent, dextrose, and distribute in test-tubes.

Sterilize in the Arnold sterilizer for three successive days, and allow
to harden in a slanted position.

Media for the Cultivation of Aciduric Bacteria. Acid Broth. Add
sufficient glacial acetic acid to a liter of 2 per cent, dextrose broth
to make the reaction equal to 50 c.c. of normal acid. A precipitate
forms, which will settle out, leaving a clear supernatant fluid that may
be removed to sterile test tubes with a sterile 10 c.c. pipette.

Oleate Agar. The addition of 0.2 per cent, sodium oleate to dextrose
agar makes a favorable medium for the cultivation of aciduric bacteria.

1 Jour. Exp. Med., 1897, ii, 677.

2 Add after the other ingredients are in solution.

METHODS FOR THE MICROSCOPIC STUDY OF BACTERIA 205

V. The Cultivation of Bacteria. Inoculation of Culture Media.
A platinum wire of 24-gauge is generally used to transfer bacteria from
medium to medium. A piece of platinum wire 1 three inches in length
is fused into the end of a glass rod 5 mm. in diameter and about 15
cm. in length. Metal handles are preferred by many; they possess
the great advantage of not breaking, but become heated during the
process of sterilization. The straight wire or " needle" is commonly
used for the inoculation of slant and stab cultures in solid media;
for the inoculation of fluid media a loop is formed on the end of the
wire. The use of the loop permits of the transfer of a greater amount

FIG. 23. Needle sterilizer. (A. de Khotinsky.)

of material. It is occasionally necessary to transfer more material
than a drop or two obtained with a loop in order to insure growth,
and for this purpose sterile capillary pipettes are very convenient.
Many anerobic bacteria and organisms which grow poorly in artificial
media must be transferred with the pipette.

The transfer of bacteria from media to media involves the following
steps :

(a) Flame cotton plugs to destroy molds and spores of bacteria;
extinguish flame.

(6) Twist cotton plugs to ^destroy adhesion to the neck of the
tube. The plugs may then be removed intact.

(c) Sterilize platinum wire in Bunsen flame. Heat wire white hot
and pass that portion of the handle adjoining the wire through the
flame, rotating it between the fingers while doing so. Allow the wire
to cool.

(d) Grasp the tubes in the left hand and remove plugs from the
tubes, holding one between the third and fourth fingers of the right

1 A cheap and efficient substitute for platinum wire is "Nichrome" wire. It is rather
less durable than platinum, and melts at a lower temperature.

206 MICROSCOPIC AND CULTURAL STUDY OF BACTERIA

hand, the other between the second and third fingers, the plugs pro-
jecting outward. Flame the mouths of the tubes and test coolness
of the platinum wire by plunging it for a distance of about a centi-
meter into the sterile medium.

(e) Remove some material from the infected tube by dipping the
tip of the wire in it, and transfer to the sterile tube.

(/) Replace the plugs in their respective
tubes and sterilize the wire before laying it
down.

II. The Isolation of Pure Cultures of Bacteria.
A. Aerobic Organisms. It is the exception
rather than the rule that bacteria exist in nature
or in many pathological processes in pure cul-
ture, that is, that a single kind of organism
alone is present. From such mixtures of bac-
teria it is frequently necessary to isolate one or
more organisms in a pure state, uncontami-
nated by other microorganisms. A common
and efficient method of separating bacteria from
mixtures is to distribute them in melted gelatin
or agar, 1 in such a manner that individual cells
are somewhat widely separated. The medium
is then allowed to harden. The organisms are
immobilized in or upon the medium and sur-
rounded by nutrients; the descendants of each
individual organism thus develop locally and

apart from the descendants of other organisms. Under favorable
conditions the descendants of individual cells become so numerous
they may be seen with the unarmed eye as spots or colonies, each
of which is made up of the progeny of a single organism. It is a
simple matter to touch such a colony with a sterile, cool platinum
needle, and infect sterile media with the adherent bacteria. In this
manner pure cultures are obtained. The technic of the isolation of
aerobic and facultatively anaerobic bacteria is technically termed
plating, or streaking, depending upon the apparatus used.

1. Plate Method. Three tubes of nutrient agar or gelatin are melted
and cooled to 42 to 45 C. A platinum wire, previously sterilized

1 Agar melts at about 95 C. and solidifies at about 40 C. It is necessary to work
rapidly with melted and cooled agar, to carry out the technic of inoculation before
solidification takes place.

FIG. 24. Platinum needle
and platinum loop.

METHODS FOR THE MICROSCOPIC STUDY OF BACTERIA 207

and cooled, is dipped to a depth of about 0.5 c.c. in a mixture of bac-
teria in fluid media, or touched to a growth in solid media, and then
rotated two or three times in a tube of the sterile melted medium.
Without sterilizing the needle, the process is repeated in the second
and third tubes. Each tube is then rotated between the palms of the
hands, to distribute the organisms thoroughly, and poured individually
into the sterile Petri dishes. The medium is flowed uniformly over
the bottom of the dish and set aside to harden.

It will be seen that the first tube inoculated contains the greatest
number of organisms, and that the third tube would theoretically
contain but few. The colonies in one of the plates will be so widely
separated that they can be "fished" with the platinum wire without
the danger of touching other colonies, and transferred to fresh, sterile
media. The success of this procedure depends largely upon a rigorous
observance of details. The mouths of the tubes and the cotton plugs
should be flamed thoroughly before inoculation is practiced, and the
transfer of the contents of the tube to the Petri dish must be done
carefully to prevent contamination. *The cover of the Petri dish should
be raised with the left hand, but directly over the bottom, to prevent
the entrance of adventitious bacteria from the air. The mouth of the
tube should not touch the bottom or edge of the Petri dish and, finally,
the cover of the latter should be replaced at once.

After the medium has hardened the plates are incubated gelatin
plates at 20 C., agar plates at 37 C. It is customary to invert agar
plates during incubation; when agar cools and becomes solid a con-
traction takes place which squeezes out some fluid. (This is well
defined in slanted agar as the water of condensation.). If the fluid
were allowed to remain on the surface of the agar plate it would con-
vert the surface potentially into a broth culture, in which the various
organisms would mix in hopeless confusion. Inversion of the plates
prevents the accumulation of moisture on the surface to a large degree;
the water of condensation collects on the cover instead. The porous
tops recommended by Hill may advantageously be used they absorb
moisture as it is formed. Gelatin plates are not inverted; fluid is
not expressed as the medium solidifies, and liquefied gelatin formed
during the growth of actively proteolytic organisms would collect on
the cover and probably contaminate the entire plate.

2. Streak Method. The isolation of pure cultures of bacteria by the
streak method differs from the plate method in that the medium
(gelatin, agar, blood serum) is not inoculated in the fluid state; the

208 MICROSCOPIC AND CULTURAL STUDY OF BACTERIA

necessary dilution to secure isolated colonies is attained by drawing
a platinum needle infected with bacteria several times across the sur-
face of sterile, slanted gelatin, agar, blood serum, blood agar or^ other
solid medium, each time covering an area not previously touched.
Eventually a degree of dilution is reached where discrete colonies are
discernible.

The plating method and streak method possess advantages and
disadvantages. A considerable proportion of the growth in plates
inoculated in the fluid state is beneath the surface, where it
is less characteristic than surface colonies. The distribution of
organisms, however, is more uniform, and small numbers of bacteria
occurring in mixture with larger numbers of undesirable organisms
are somewhat less likely to be overlooked. It is possible, moreover,
to obtain a quantitative estimation of the numbers of bacteria in
mixtures by the plate method. The streak method is advantageous
both with respect to the economy of time necessary to inoculate the
medium, and in that the colonies are wholly upon the surface of the
medium. There is less danger of contamination when u fishing" from
streak plates than from the regular method of plating, because there
is no chance for submerged colonies to underlie those upon the surface.

The use of certain kinds of media, as that of Endo, of blood agar,
and Loffler's blood serum, requires that surface inoculation shall be
made. The possibility of missing or overlooking small numbers of
the less hardy types of bacteria is greater with the streak method of
isolation.

3. The Barber Method for the Isolation of a Single Cell. It is occa-
sionally necessary, in very refined bacteriological studies, to be abso-
lutely certain that the starting point of a pure culture is a single
organism. Theoretically, single cells are the progenitors of the colonies
observed in media inoculated by the plate or the streak method, and
such is usually the case. Undoubtedly it may happen that a chain of
streptococci may remain adherent and their descendants appear as a
single colony, and it is equally certain that two alien bacteria may
occasionally become adherent by intertwining of flagella or adhesion
of viscid capsular substance and develop into a mixed colony. The
apparatus of Barber, 1 which consists essentially of a delicate capillary
pipette mounted in the substage of the microscope, and capable
of upward and downward motion in the optical axis of the instrument,
is designed to circumvent this possibility. In practice a very thin

1 Univ. Kansas Science Bull., No. 1, March, 1907.

METHODS FOR THE MICROSCOPIC STUDY OF BACTERIA 209

emulsion of bacteria in a fluid medium is placed on the surface of a
sterile thin plate of glass in such a manner that a drop of the con-
taminated fluid hangs in the opening in the stage ordinarily occupied
by the condenser. The drop is manipulated by a mechanical stage,
guided by direct observation with a one-sixth-inch lens until a single
organism appears in the field of vision. The sterile capillary pipette
is carefully brought upward until the tip engages the dependent drop;
the organism will be seen to enter the pipette, which is then lowered and
removed from its attachments to the microscope. The single cell is
transferred to a suitable medium and incubated in the usual manner.

B. Anaerobic Bacteria. 1. Plating Methods. The cultivation of
anaerobic bacteria which do not grow in the presence of atmospheric
(free) oxygen requires special apparatus and technic. The simplest
method, and one which is successful if gas-forming bacteria are absent;
is to make dilutions in dextrose agar precisely as described under
Plating in the preceding paragraphs. The tubes should be filled to a
depth of 10 cm. with the medium, and tubes of relatively large dia-
meter 2 to 3 cm. are preferable. The tubes, previously heated to
the boiling point, and rapidly cooled to 43 to 45 C. to prevent reabsorp-
tion of oxygen, are inoculated by the dilution method, rotated between
the hands to distribute the organisms uniformly, and cooled rapidly
in an upright position.

Colonies appear within the depths of the media after incubation;
in the thinly seeded tubes these colonies are discrete, and they may
be removed without contamination, either in sterile capillary pipettes
introduced through the surface of the medium, or after breaking the
tubes from the side. It is, of course, necessary to sterilize the outside
of the tube if it is to be broken. A greater degree of anaerobiosis may
be obtained within the tubes if after solidifying they are placed neck
downward with the cotton plugs removed, in a beaker containing
freshly prepared alkaline pyrogallate solution. 1 Growth of anaerobic
bacteria upon the surface of agar or blood serum may be obtained in
this manner. 2 Those bacteria which produce gas during their growth
cannot be isolated in pure culture in deep agar tubes; the liberation
of gas bubbles fragments the medium and permits the various colonies
to coalesce.

1 Five grams of dry pyrogallic acid are placed in a beaker and covered with 15-25 c.c.
of water: when dissolved a layer of kerosene or paraffin oil about 1 cm. in depth is added,
and a 10 per cent, solution of sodium hydroxide is introduced below the oil layer with
a pipette.

2 See Rickards, Cent. f. Bakt., 1904, I Abt., xxxvi, 557.

210 MICROSCOPIC AND CULTURAL STUDY OF BACTERIA

The " bottle-plate" method of Simonds and Kendall 1 overcomes this
difficulty to a considerable degree through the use of simple appli-
ances.

Sixteen-ounce French square tincture-mouth bottles are plugged
with cotton and sterilized with dry heat. With the bottles lying on
their sides, sufficient blood agar is poured in to form a layer 5 to 10

mm. deep, and allowed to harden. Dorset's
egg medium, dextrose agar or other media
may be substituted for the blood agar if
desired.

As soon as the medium has hardened the
bottles should be turned on the opposite
side, thus bringing the medium uppermost
and preventing condensation water from
adhering to it. Inoculation is made with a
bent glass rod infected with bacteria from
a thin suspension in a liquid medium, and
rubbed over the surface of the agar within
the bottle. A partial vacuum is next cre-
ated within the bottle, and residual oxygen
dissolved in alkaline pyrogallate solution
in the following manner: A closely fitting
rubber stopper with one hole carrying a
glass tube four inches in length is inserted
in the bottle. The outer end of the glass
tube projects three-quarters of an inch
beyond the stopper and is fitted with a
rubber tube three inches in length. That
portion of the glass rod within the bottle
is bent at an angle of 45 and the stopper
is turned in such a manner that the end of
the glass tube points toward the side of the
bottle opposite the layer of agar. As much

air as possible is aspirated from the bottle, and the rubber tube
closed with a pinch-cock to prevent reentrance of air.

The bottle is now placed on its side, with the medium uppermost,
and with a pipette, 10 c.c. each of a 50 per cent, solution of pyrogallic
acid and 10 per cent, sodium hydrate are run in through the rubber
tube, avoiding the entrance of air. A few cubic centimeters of clean

1 Jour. Inf. Dis., 1912, xi, 207.

FIG. 25. Wright's method of
making anaerobic cultures in
fluid media. (Mallory and
Wright.)

METHODS FOR THE MICROSCOPIC STUDY OF BACTERIA 211

water are also run in, to wash the rubber tube free from caustic alkali.
The apparatus is working properly when the rubber tube between the
pinch-cock and the bottle is collapsed, indicating a partial vacuum
within the bottle itself. Residual oxygen is rapidly absorbed within
the pyrogallate solution, leaving an inert atmosphere of nitrogen.

The bottle is incubated, medium uppermost, for the required time.
Inspection of the surface of the medium will show the colonies. After
incubation the pinch-cock is carefully opened, admitting air very gently
to avoid spattering the medium, and the stopper is removed. The
pyrogallate solution is poured out and residual traces removed with
clean water. The bottle is drained standing upon end, mouth down,
and then the colonies are ready for fishing. The colonies which develop
are all surface growths : the isolation of gas-forming anaerobic bacteria
is as readily accomplished as the isolation of non-aerogenic types.

FIG. 26. Novy jar for anaerobic cultures. (Park.)

Pure cultures of anaerobic bacteria may be obtained in an atmos-
phere of hydrogen; plates prepared in the usual manner are placed
on a rack in a Novy jar or other similar vessel provided with a tightly
fitting stop-cock, through which hydrogen can be admitted in sufficient
volume to displace the air. The stop-cock must be hydrogen-tight.
The procedure is to place inoculated plates without covers on a rack
within the jar in an inverted position, one above the other. A few
grams of pyrogallic acid are placed on the bottom of the jar with a
small piece of solid sodium hydroxide. At the last moment, when
everything is in readiness, 20 to 30 c.c. of water are gently poured
down the side of the jar to prevent spattering, and the cover quickly
clamped down. A current of hydrogen gas, either from a cylinder or
from a Kipp generator, is passed through the jar at a fairly rapid rate.

212 MICROSCOPIC AND CULTURAL STUDY OF BACTERIA

The hydrogen should enter at the top, and the outlet for the gas should
be as near the bottom of the apparatus as possible. A sample of the
escaping gas, collected in a test-tube by downward displacement,
will ignite without an explosion when all oxygen is displaced. The
inlet tubes are closed, and incubation practiced in the usual manner.
An atmosphere of nitrogen is to be preferred to an atmosphere of
hydrogen whenever it is practicable.

b c d

FIG. 27. Koch i&fety
burner. (Park.)

FIG. 28. Dunham thermo-
regulator. (Park.)

FIG. 29. Roux Bimetallic
regulator. (Park.)

2. Anaerobic Cultures in Fluid Media. A simple method of main-
taining anerobiosis in fluid media, sufficiently effective for ordinary
usage, is to overlay a flask or test tube containing dextrose broth
with a layer of albolene about 1 cm. in depth. Immediately before
inoculation all residual oxygen in the medium should be removed
by an exposure of half an hour in the Arnold sterilizer, or ten minutes
in an autoclave. The liquid is cooled rapidly to minimize reabsorp-
tion of oxygen. Wright 1 has maintained anaerobic conditions in test-
tube cultures with alkaline pyrogallate solution. Test tubes are
prepared with absorbent cotton plugs, which are made tighter than
ordinary usage demands. After the culture medium (freed from dis-
solved oxygen by heating and rapid cooling) is inoculated, the cotton

1 Mallory and Wright, Pathological Technic, 4th ed., p. 126.

METHODS FOR THE MICROSCOPIC STUDY OF BACTERIA 213

plug is pushed into the tube until the upper end is about 15 mm.
below the top. The space above the cotton plug is filled loosely with
dry pyrogallic acid and a strong solution of sodium hydroxide, 2 to
3 c.c., is added to dissolve the acid. Immediately a tightly fitting
rubber stopper is inserted into the mouth of the tube. The alkaline
pyrogallate solution absorbs the oxygen within the tube, leaving an
atmosphere of nitrogen.

FIG. 30. Incubator. (Park.)

The addition of bits of fresh, sterile tissue, 1 fresh, sterile defibrinated
blood, or of the coagulum which is formed during the coagulation of
meat infusion adds greatly to the nutritional value of cultures for the
growth of anaerobic bacteria.

1 Theobald Smith, Cent. f. Bakt., 1890, vii, 502.

214 MICROSCOPIC AND CULTURAL STUDY OF BACTERIA

Special mention of the preparation of tissue media is made in the
sections on Specific Anaerobic Organisms.

The Incubation of Bacterial Cultures. The growth of bacteria in
artificial media is markedly influenced by the temperature to which
they are exposed. A majority of those organisms parasitic upon or
pathogenic for man develop most luxuriantly at the temperature of
the human body, 37 C. Exposure to temperatures but slightly above
37 C. leads to rapid death of these organisms, consequently incuba-
tors must be available within which cultures may be safely exposed
to a uniform and constant degree of heat equal to that of the human
body. Gelatin cultures must be maintained at temperatures not
exceeding 22 C.

Incubators are single- or double-walled chambers of various sizes,
heated directly by gas or electricity, or indirectly through a water
jacket. The latter run more uniformly, because water receives and
imparts heat more slowly than air. On the other hand, large incuba-
tors cannot be surrounded with water jackets because of mechanical
difficulties. The regulation of temperature within incubators is con-
trolled by bimetallic regulators which actuate valves or electromagnets
controlling the supply of gas or electricity which heats the chamber,
or by mercurial thermoregulators working upon the principle of the
mercury thermometer. Bimetallic regulators, in which the movement
imparted to the regulator of the source of supply of heat is due to the
differential expansion or contraction of two dissimilar metals, are more
sensitive to slight variations in heat and they possess the additional
advantage of being less fragile than mercurial regulators. Various
patterns of thermoregulators of tried efficiency are on the market and
a selection between them is largely a matter of mechanical adaptability
to local needs.

VI. The Study of Bacterial Cultures. I. Growth in Solid Media.
(a) Colonies. The macroscopic appearance of bacterial colonies upon
solid media is of considerable value for the differentiation and recog-
nition of the various types; in a similar manner their microscopic
appearance, stained or unstained, permits of some differentiation.

The aspect of a colony is influenced.

1. By the kind of organism Streptococcus colonies, for example,
are habitually small and nearly transparent; anthrax colonies are
habitually larger and opaque.

2. By the consistency of the medium in firm, dense media the
growth of bacteria is limited and relatively dry; in moist, semisolid

METHODS FOR THE MICROSCOPIC STUDY OF BACTERIA 215

media the growth of the -same organism is usually luxuriant, moist,
and spreading.

3. By the composition and reaction of the medium the addition
of specifically nutritive substances, as of fresh sterile tissue to media
for the cultivation of anaerobes; of utilizable carbohydrates to media
for the cultivation of carbohydrophilic bacteria; of fresh, defibrinated
blood to media for the cultivation of hemoglobinophilic organisms;
these may improve conditions otherwise unfavorable for bacterial
development.

The reaction of the medium, furthermore, is important; many
bacteria are extremely sensitive to slightly acid media; the aciduric
bacteria thrive in media too acid for the existence of other organisms.
Even the ordinary laboratory media, made according to a definite
formula, vary sufficiently in chemical and physical properties to
influence materially the appearance of bacterial colonies. The degree
of influence is more pronounced in the feebly growing forms, but it
may affect the appearance of colonies of the more hardy types as well.

4. The rate of growth of bacteria also affects the appearance of
colonies.

It is useless, as a scientific procedure, to attempt to recognize dif-
ferences of greater refinement than the accuracy of the method permits
of, and for this reason the descriptions of bacterial colonies should
not be carried to extremes. In general, bacterial growths on solid
media are described as solids in space the average size, form, color,
lustre and texture. This applies equally well to colonies, slant and
stab cultures. The really valuable information gleaned from a study
of bacterial growths is the recognition of types of growth. For example,
spore-forming bacteria (aerobic) produce rather heavy, opaque, floc-
culent colonies; members of the Alcaligenes dysentery, typhoid,
paratyphoid group grow characteristically as rather small, round,
transparent colonies.

(b) The Enumeration of Bacteria. A very practical application of
the plating method for the isolation of bacteria is the enumeration of
bacteria in water, milk and other similar substances. The principle
involved depends upon the development of colonies of bacteria from
single cells. If a definite volume of water, 1 c.c. for example, is dis-
tributed in melted agar, thoroughly mixed in the tube by rotation
between the hands, and poured carefully into a sterile Petri dish, the
number of colonies which develop within a definite period of incuba-
tion may be regarded as a measure of the number of living bacterial

216 MICROSCOPIC AND CULTURAL STUDY OF BACTERIA

cells in a cubic centimeter of water. Experience has shown that the
accuracy of the method is influenced somewhat by the number of
organisms in the sample. A large number of bacteria, by mutual
antagonism, will fail to develop into a proportionate number of colonies.
The most accurate results are obtained when the bacterial content of
the sample as plated lies between fifty and two hundred individual
organisms. If more than two hundred bacteria are probably present,
a dilution of the sample with sterile water is made before plating, to
reduce this source of error. It is convenient in making dilutions to
use a multiple of ten, because the subsequent calculation is much
simplified. A dilution of 1 to 10 is made by adding 1 c.c. of the sample
to 9 c.c. of sterile water, shaking thoroughly and plating 1 c.c. If
the technic is all right, each colony on the plate represents one-tenth
the number of living bacteria in the original sample. The total number
of colonies multiplied by ten gives the theoretical bacterial count of
the sample. A dilution 1 to 100 is made by adding 1 c.c. of the sample
to 99 c.c. of sterile water. The plating method is inexact, partly because
an unknown proportion of organisms in the original sample will fail
to develop for various reasons in the cultural medium; furthermore,
certain types of organisms, as streptococci, may remain adherent
in chains of greater or lesser length and develop as a single colony.
Anaerobic bacteria do not develop under aerobic conditions.

A template of paper or glass ruled in square centimeters is used
to facilitate the enumeration of colonies; for densely colonized plates,
each centimeter square of the template is subdivided into smaller
units, usually one-ninth of a square centimeter. The Petri dish con-
taining colonies is placed upon the template in such a manner that
the colonies appear superimposed upon the rulings. It is a simple
matter, with the lines as a guide, to count either the entire number of
colonies in the Petri dish, or a few representative areas, which can be
multiplied by a factor. (The average Petri dish contains about 63
square centimeters.)

Example. A sample of milk diluted 1 to 100 shows a large number
of colonies after forty-eight hours' incubation. The total count of
nine squares (each a square centimeter) is 180 colonies, an average
of twenty colonies per square centimeter. The colonies upon the
entire plate (63 square centimeters) is 63 x 20, or 1260. The number
of living bacteria in 1 c.c. of the sample of milk would be 1260 x 100
or 126,000, because the number of colonies upon the plate is T^TT the
entire number in 1 cm.

METHODS FOR THE MICROSCOPIC STUDY OF BACTERIA 217

The value of the method as a convenient means of comparison of the
bacterial content of various samples of milk, water, sewage, and the
like depends largely upon the supposition that the same types of bac-
teria present in different samples will grow quantitatively under like
conditions. The comparison of bacterial counts is therefore a com-
parison of a section of the total bacterial flora, not an absolute measure
of the number of living organisms. The method of counting bacterial
colonies has been highly developed for the regulation of water and milk
supplies of cities. (See section Water and Milk.)

(c) Growth of Bacteria in Gelatin. Gelatin is added to cultural
media both to confer upon the media the property of solidifying, and
to enrich the content in nitrogenous substances.

Pure gelatin does not contain tyrosine and it is relatively rich in
diamino acids; according to Hausmann, 1 nearly 36 per cent, of the
nitrogen in gelatin is diamino nitrogen about 63 per cent, in the form
of mono-amino acids. Chemically, gelatin media are convenient for
the demonstration of soluble, proteolytic enzymes. 2 In the absence of
utilizable carbohydrate, several types of bacteria "liquefy" gelatin,
that is, through the activity of their proteolytic enzymes the gelatin
molecule is split by hydrolytic cleavage to molecules so simple in
their state of aggregation that they can no longer produce a "gel."
The presence of utilizable carbohydrate prevents the liquefaction of
gelatin by many bacteria. 3

Formerly the morphology of the liquefied zone in gelatin stab cul-
tures was regarded as distinctive for individual organisms; thus, the
napiform liquefaction produced by cholera vibrios was supposed to
be sufficiently constant to possess diagnostic value. It is now generally
conceded that this morphological characteristic is of comparatively
little importance for the identification of the organism. On the other
hand, the liquefaction of gelatin and of coagulated blood serum and
casein as well is important from a chemical viewpoint, because it
indicates the activity of a soluble proteolytic enzyme. 4

II. Growth of Bacteria in Fluid Media. (a) Plain Broth. Plain
broth, prepared from meat infusion and peptone in the usual manner,

1 Zeit. f. physiol. Chem., 1899, xxvii, 95.

2 Kendall, Boston Med. and Surg. Jour., 1913, clxviii, 825.

3 Kendall and Walker, Jour. Inf. Dis., 1915, xvii, 442.

. 4 The enzyme may be obtained sterile and in an active state in the filtrates of liquefied
gelatin, blood serum, casein, and from plain broth cultures as well, if the bacteria are
removed by filtration through unglazed porcelain: Auerbach, Arch. f. Hyg., 1897,
xxxi, 311; Berghaus, ibid., 1906, Ixiv, 1; Kendall and Walker, Jour. Inf. Dis., 1915,
xvii, 442.

218 MICROSCOPIC AND CULTURAL STUDY OF BACTERIA

and freed from sugar with Bacillus coli, 1 contains, on the average,
about 300 milligrams of nitrogen per 100 c.c. A small but variable
amount of the nitrogen exists as free ammonia. 2 Less than 10 per
cent, of the total nitrogen, as a rule, exists as aminonitrogen (deter-
mined by the method of Van^Slyke) . 3

The visible changes in the appearance of broth cultures incidental
to the development of bacteria are not of great importance; they
consist essentially of turbidity, sediment, and occasionally a ring
or pellicle. The development of a pellicle is of importance in the pro-
duction of toxin by the diphtheria bacillus, however, because it indi-
cates the maximum oxygenation of the bacteria. The character of
the turbidity and sediment the viscidity and color may afford
some information of the character of the organism. Products of
importance are frequently detected by chemical or physiological
examination in plain broth cultures of bacteria. Thus, in the absence
of utilizable carbohydrate, diphtheria and tetanus bacilli produce
their very potent toxins; 4 proteolytic bacteria elaborate soluble
enzymes; 5 Bacillus coli, Bacillus proteus and other bacteria form indol
and phenolic bodies from tryptophan and tyrosine respectively; the
cholera vibrios form nitroso indol, 6 and in sugar-free broth containing
freshly drawn, sterile, defibrinated blood, various bacteria produce
hemolysis.

The rate of decomposition of the protein constituents of the broth
may be measured by the Folin microscopic method for ammonia; the
increase in ammonia indicates the extent of deaminization. 7 The
rate of hydrolysis of protein is conveniently estimated with the Van
Slyke 8 amino-acid apparatus, after removal of ammonia from the
medium. 9 A combination of these methods affords an approximate
analysis of plain broth media before and after bacterial growth. Un-
doubtedly the application of the Emil Fischer esterification method
of aminonitrogen determination will throw much light upon the utili-
zation of various amino acids by specific bacteria during their growth
in artificial media. Amino acids containing aromatic nuclei as tryp-

1 Theobald Smith, Cent. f. Bakt., 1897, xxii, 45.

2 Determined by the Folin Method, Jour. Biol. Chem., 1912, xi, 523.

3 Jour. Biol. Chem., 1912, xii, 275; 1913, xvi, 121.

4 Theobald Smith, Tr. Assn. Am. Phys., 1896; Jour. Exp. Med., 1899, iv, 373.

5 Kendall and Walker, Loc. cit.

c Kendall, Jour. Med. Res., 1911, xxv, 117.

7 Kendall and Farmer, Jour. Biol. Chem., 1912, xii, 13, 215, 219, 465; xiii, 63; Kendall,
Day and Walker, Jour. Am. Chem. Soc., 1913, xxxv, 1201; 1914, xxxvi, 1937.

8 Van Slyke, Loc. cit.

9 The rate of hydrolysis may also be estimated by Sorenson's formol titration method,
but this is less accurate for small amounts than Van Slyke's method.

METHODS FOR THE MICROSCOPIC STUDY OF BACTERIA 219

tophan, tyrosine, histidin give colored compounds with various
reagents because they contain the chromophoric group, C = C. The
formation of indol from tryptophan (see page 74 for chemistry) has
long been used as a diagnostic test for Bacillus coli and other bacteria.
The test depends upon the removal of alanin from the tryptophan
molecule by the activity of the organism, and the addition of an auxo-
chromic group, NO 2 in the beta position of the pyrrol ring, previously
occupied by alanin. In an acid medium the compound, betanitroso-
indol, is brownish red.

Procedure, Indol Test. To a three-day plain broth culture of Bacil-
lus coli (or other organism) add 1 c.c. of concentrated hydrochloric
acid. 1 Mix thoroughly and overlay the acid broth with 1 to 2 c.c. of a
0.1 per cent, solution of sodium nitrite. 2 At the junction of the two
solutions a brownish-red ring of nitroso indol develops.

(b) Carbohydrate Broths. The addition of sugars, as dextrose, lac-
tose, saccharose, or of alcohols, as glycerol, to plain broth media,
greatly enriches the medium in non-nitrogenous substances which
may be readily utilizable sources of energy for bacteria. It is hardly
necessary to emphasize the importance of purity in all sugars and
other carbohydrates intended for bacterial purposes, nor the fallacy of
attempting to determine the action of bacteria upon specific carbohy-
drates in media not freed from muscle sugar (dextrose). The use of
serum as a basis for fermentation media frequently introduces a source
of error, because blood serum normally contains about 0.08 per cent,
of dextrose, an amount quite sufficient to give rise to considerable
amounts of acid. 3

The observations made in carbohydrate media are usually restricted to :

(a) Change in reaction.

(b) Gas formation, and in fermentation tubes, to growth in the
closed arm as well.

1 Any strong mineral acid will answer the purpose.

2 Best accomplished by running the nitrite solution carefully down the side of the
tube held in a slanting position; a stratification of the two liquids should be obtained.

3 The significance of fermentation media for the classification and identification of
bacteria depends upon their content both of protein and carbohydrate. Bacteiia derive
their energy requirements from carbohydrate, if it is utilizable, but of course they must
obtain their "Bausteine" from the nitrogenous constituents. If the carbohydrate
cannot be utilized, both structural and energy requirements are derived from the protein
constituents. Bacteria vary greatly in their ability to ferment carbohydrates; some
types, as Bacillus alcaligenes, do not appear to ferment even dextrose. Bacillus lactis
aerogenes, on the contrary, can ferment hexoses, bioses, and even starches. The fer-
mentability of a carbohydrate depends apparently upon its stereo-isomeric configuration,
and relatively slight differences in the configuration of similar carbohydrates may
determine their value for specific organisms as sources of energy. This point is discussed
somewhat later in this section.

220 MICROSCOPIC AND CULTURAL STUDY OF BACTERIA

Bacteria which can utilize carbohydrates for their energy require-
ments usually produce acid; many types produce gas as well. The
acid, which is commonly lactic, together with small amounts of acetic
and other fatty acids may be estimated by titration with standard
alkali. A more accurate estimation is based upon the determination
of the hydrogen ion concentration. 1 The gases formed are usually
carbon dioxide and hydrogen. An approximate ratio of the proportion
H/CO 2 is conveniently made in the Smith Fermentation Tube, 2 in the
following manner:

The level of the gas in the closed arm is marked with a wax pencil.
The bulb of the fermentation tube is then completely filled with
a 1 per cent, solution of sodium solution, and the gas brought
into contact with the alkaline solution by inverting the tube several
times. The gas is then entirely run back into the closed arm, and the
volume again measured. The volume is diminished proportionately
to the absorption of CO 2 by the caustic alkali.

Smith 3 has determined the "gas ratio" for the principal aerogenic
bacteria as follows :

Organism. Dextrose. Lactose. Saccharose

H C0 2 H CO 2 H C0 2

B. coli 63 37 63 37 63 37

Hog cholera 66 34

B. lactis aerogenes .... 65 35 62 38 80 20

Friediander bacillus ... 67 33 86 14 67 33

B. edematis maligni ... 67 33 ? . . ?

B. proteus 72 28 67 33

B. cloacse 70 30 37 63 58 42

Bacteria -which ferment sugars grow in the closed arm of the fer-
mentation tube; those organisms, with very few exceptions, which
cannot utilize the carbohydrate of a fermentation medium fail to
grow in the closed arm where free oxygen is not available; growth
appears only in the open arm.

Occasionally a very slight change in the stereo-isomeric formula of
a carbohydrate, or a very small change in its terminal groups will
determine its fermentability by various organisms. Thus dextrose,
mannose, and their respective alcohols, sorbite and mannite, according
to Emil Fischer, 4 have the following stereo-isomeric formulae:

1 Clark, Jour. Inf. Dis., 1915, xvii, 109.

2 Theobald Smith, The Fermentation Tube, Wilder Quarter Century Book, 1895,
187 et seq.

3 LOG. cit.

4 Untersuchungen iiber Kohlenhydrate und Fermente, 1884-1908, Berlin, 1902.

METHODS FOR THE MICROSCOPIC STUDY OF BACTERIA 221

H H

I I

H C=O H C=O H C OH H C OH

I I I I

H C OH HO C H H C OH HO C H

I I I I

HO C H HO C H HO C H HO C H

till

H C OH H C OH H C OH H C OH

I I I I

H C OH H C OH H C OH H C OH

I I I I

H C H H C H H C H H C H

I I I I

o o o o

H H H H

D. Glucose. D. Mannose. D. Sorbite. D. Mannite

The fermentation of these hexoses and their respective alcohols
by certain bacteria is shown in the accompanying table:

Organism. D. Dextrose D. Mannose. D. Sorbite. D. Mannite

B. dysenterise Shiga + +

B. dysenteiise Flexner . + + +

B. Morgan No. 1 . . . + +

B. paratyphosus Beta + + + +

An explanation for the phenomenon set forth in the table does not
readily suggest itself. Somewhat similar selective action upon specific
amino acids by other bacteria is known, qualitatively at least. Thus,
members of the Hemorrhagic Septicemia Group, particularly those
derived from animal sources, produce indol in plain broth media.
Typhoid bacilli, diphtheria bacilli and many other pathogenic organ-
isms usually fail to produce indol in ordinary media under similar con-
ditions. It is possible that this noteworthy action of members of the
Hemorrhagic Septicemia Group upon tryptophan may be related to
the fact that this amino acid is an important constituent of the hemo-
globin, the coloring matter of the blood; the Hemorrhagic Septicemia
Bacteria are particularly likely to grow in the blood stream of infected
animals.

Fermentation reactions of bacteria in varied carbohydrate media
are of importance in their cultural identification. The table on page
316 illustrates the separation of members of the Intestinal Group of
Bacteria by their fermentation reaction in various carbohydrates.

Milk. Milk is an important natural medium for bacterial growth.
It contains protein, carbohydrate and fat, together with inorganic
salts. A variety of reactions and changes in milk are produced by
bacterial development.

222 MICROSCOPIC AND CULTURAL STUDY OF BACTERIA

(a) Change in Reaction. Milk contains, in addition to protein, two
carbohydrates, which play a prominent part in determining the reac-
tion of the medium. The principal carbohydrate is lactose, but fresh
milk contains in addition, a small amount about 0.08 per cent. of a
sugar reacting like dextrose. 1 Changes in the reaction of milk caused
by bacterial activity, therefore, may be of several types. An initial
acidity followed by an alkaline reaction, as exhibited by the dysentery
bacilli and other organisms, is probably due to the initial fermentation
of the small amount of dextrose, which results in the formation of
acid and then the production of alkaline products from the decom-
position of protein when the dextrose is exhausted. These organisms
do not ferment lactose.

A permanent acid reaction is induced either by bacteria which fer-
ment lactose, or less commonly, by the decomposition of fat with the
liberation of fatty acids. Bacillus typhosus and Bacillus paratyphosus
alpha produce a permanently acid reaction in milk, but do not ferment
lactose. The exact chemistry of their activity in the medium is not
known. An alkaline reaction in milk is usually an indication of proteo-
lytic action with the formation of basic products of protein decom-
position.

The accumulation of acid incidental to the fermentation of lactose,
as, for example, by B. coli, may be sufficient in amount to cause an
acid coagulation of the casein. 2 An acid coagulation can be distin-
guished from an enzyme (lab or rennin) coagulation; the acid coagulum
will redissolve in alkali, but an enzyme coagulum fails to redissolve
by merely neutralizing the reaction.

Some types of bacteria, as Bacillus aerogenes capsulatus, ferment
lactose energetically, liberating a large amount of gas, and forming
butyric acid as well. For some unknown reason, Bacillus coli and
allied organisms, which ferment lactose in fermentation tubes with
the liberation of considerable amounts of gas, fail to produce gas from
the lactose as it exists in milk. It has been shown, however, 3 that the
colon bacillus will liberate gas from lactose if the milk is first acted upon
by a strongly proteolytic organism, as B. mesentericus.

Proteolytic bacteria, which are unable to utilize either the small
amount of dextrose, the lactose or the fats of milk, usually produce

1 Theobald Smith, Jour. Boston Soc. Med. Sci., 1897, ii, 236; Jones, Jour. Inf. Dis.,
1914, xv, 357.

2 It must be remembered that bacteria grown in litmus milk frequently fail to cause
coagulation unless the medium is heated to boiling.

3 Kendall, Boston Med. and Surg. Jour., 1910, clxiii, 322.

METHODS FOR THE MICROSCOPIC STUDY OF BACTERIA 223

an alkaline reaction which may be a simple alkalinity without obvious
change in the appearance of the medium (as, for example, B. alkali-
genes) or a deep peptonization of the casein, as illustrated by B.
pyocyaneus. B. mesentericus peptonizes casein energetically, but
the reaction of the medium is persistently acid. The initial acidity
is probably due to the formation of acid from the dextrose of the
milk; the residual acid may be associated with the activity of an
esterase which certain strains of this organism appear to elaborate.
Fatty acids are formed by hydrolysis of the glycerides of the cream
by the soluble esterase, while the metabolic activities of the organism
appear to be largely directed to the proteins of the medium. 1

It is apparent, therefore, that the chemical and physical changes
induced in milk incidental to bacterial development in the medium
are, or may be, complex in their origin. A knowledge of the proteo-
lytic and fermentative activities of bacteria in the simpler media,
however, will frequently furnish an explanation for the more involved
reactions in the highly complex medium, milk.

1 Kendall, Day and Walker, Jour. Am. Chem. Soc., 1914, xxxvi, 1937.

CHAPTER X.

BACTERIOLOGICAL EXAMINATION OF MATERIAL FROM
THE PATIENT AND THE CADAVER.

MATERIAL FROM THE LIVING SUBJECT.
Blood Culture.

Technic of Blood Cultures.
Bacteriological Examination of Cere-

brospinal Fluid.
The Examination of Peritoneal, Pleu-

ral and Pericardial Fluids.
Pus.

Examination of Urine.
Examination of Feces.
Examination of Sputum, of Buccal

and Pharyngeal Material.

Examination by Staining.
Cultural Methods.

Bacteriological Examination of the
Eye.

Bacteriological Examination of the

Ear and Nose.

THE UTILIZATION OF ANIMALS FOR BAC-
TERIAL DIAGNOSIS AND EXPERI-
MENTATION.

The Inoculation of Animals.

THE successful outcome of a bacteriological examination of material
from a patient or a cadaver depends to a large degree upon the appli-
cation of proper technic at the time of collection. Naturally this is
varied according to the nature of the case.

Postmortem cultures are taken from organs or tissues usually
indicated by the nature of the infection, and a choice of media for the
isolation of a specific bacteria, or types of bacteria, is made with this
information in view. The value of a postmortem bacteriological
examination is frequently measured by the promptness with which
it is made after death; postmortem invasion of tissues, organs, and
even the heart and larger bloodvessels by bacteria from the mouth
and gastro-intestinal tract takes place very quickly. Even if the
cadaver is placed at once in a cold room, some time must elapse before
the internal organs are cooled sufficiently to arrest bacterial growth.

The spleen, liver, kidneys, and bloodvessels are more commonly
examined for evidence of pathogenic microorganisms. The surface
of the undisturbed organ is first seared with a hot iron, then incised
through the sterile area, and some of the contents withdrawn in a
platinum loop or with a sterile capillary pipette and introduced at once
into suitable media. (The kind of media to be used is clearly set forth
for each organism, in succeeding chapters.) Blood may be obtained
from the heart, after searing the surface of the organ, or from the
larger veins of the extremities. Exudates from the pleural, peritoneal

MATERIAL FROM THE LIVING SUBJECT 225

or pericardial cavities may be removed with sterile pipettes and trans-
ferred temporarily to sterile test-tubes or flasks. Purulent discharges
are, if small in amount aspirated directly into sterile capillary pipettes ;
if in considerable quantity, removed to test tubes or flasks, and inocu-
lated as soon as practicable into suitable media.

MATERIAL FROM THE LIVING SUBJECT.

Blood Cultures. The organisms of septicemia may be numerous
or few in number in the blood stream furthermore, they may be
associated with specific lysins and agglutinins, as occasionally hap-
pens in typhoid fever. For these various reasons, experience has shown
that from 5 to 15 c.c. of blood, drawn aseptically, should be discharged
at once with aseptic precautions, into at least 100 c.c. of 0.1 per cent,
meat infusion dextrose broth, 1 and evenly distributed by careful agita-
tion. The degree of dilution attained practically renders lytic action
and agglutination ineffective; the enrichment of the medium by the
relatively large proportion of blood creates a very favorable medium
for the development of the organisms.

Technic of Blood Cultures. 1. Apparatus. An all-glass syringe with
a platinum-iridium needle of moderately large bore is sterilized in the
autoclave, preferably enclosed in a large test tube. A syringe cannot
be sterilized for bacterial purposes by boiling in water.

As an alternate apparatus, a 250 c.c. Ehrlenmeyer flask fitted with
a rubber stopper containing two glass tubes bent at right angles may
be used. The flask contains 100 c.c. of 0.1 dextrose meat infusion
broth. One tube is connected with a platinum-iridium needle by a
short length of rubber tubing, and the needle is protected during steril-
ization by a small test tube slipped over it and extending its full
length. The test tube is removed when the blood is to be taken. The
other tube is protected by a short length of rubber tubing containing
a small filter of absorbent cotton. Suction is applied through the
latter tube. It will be seen that blood may be drawn directly into
the broth in this apparatus, and in practice it has been found con-