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Library

Cornell University Library

The fundamentals of bacteriology,

Cornell University

The original of this book is in
the Cornell University Library.

There are no known copyright restrictions in
the United States on the use of the text.

http://www.archive.org/details/cu31924003205816

ANTHONY VAN LEEUWENHOEK

Who first saw bacteria

THE FUNDAMENTALS

OF

BACTERIOLOGY

BY
CHARLES BRADFIELD MORREY, B.A., M.D.

PROFESSOR OF BACTERIOLOGY AND HEAD OF THE DEPARTMENT IN THE OHIO
STATE UNIVERSITY, COLUMBUS, OHIO

ILLUSTRATED WITH 165 ENGRAVINGS AND 6 PLATES

LEA & FEBIGER
PHILADELPHIA AND NEW YORK

1917
lec

CopyRIGHT
LEA & FEBIGER
1917

TO

GRACE HAMILTON MORREY

AMERICAN PIANIST

PREFACE.

AN experience of nearly twenty years in the teaching of
Bacteriology has convinced the author that students of this
subject need a comprehensive grasp of the entire field and
special training in fundamental technic before specializing
in any particular line of work. Courses at the University
are arranged on this basis. One semester is devoted to
General Bacteriology. During the second semester the
student has a choice of special work in Pathogenic, Dairy,
Soil, Water, or Chemical Bacteriology. A second year
may be devoted to advanced work in any of the above
lines, to Immunity and Serum Therapy, or to Pathogenic
Protozoa.

This text-book is intended to cover the first or introductory
semester’s work, and requires two class-room periods per
week. Each student is compelled to take two laboratory
periods of three hours per week along with the class work.
The outline of the laboratory work is given at the end of
the text. Results attained seem to justify this plan. A
text-book is but one of many pedagogical mechanisms and is
not intended to be an encyclopedia of the subject.

The author makes no claim to originality of content, since
the facts presented are well known to every bacteriologist,
though the method of presentation is somewhat different
from texts in general. During the preparation of this work
he has made a thorough review of the literature of Bacteri-
ology, covering the standard text-books as well as works of
reference and the leading periodicals dealing with the sub-
ject. Thus the latest information has been. incorporated.

No attempt has been made to give detailed references in a
work of this character.

vi PREFACE

The photomicrographs are original except where otherwise
indicated and are all of a magnification of one thousand
diameters where no statement to the contrary appears.
These photographs were made with a Bausch & Lomb Pro-
jection Microscope fitted with a home-made camera box.
Direct current arc light was used and exposures were five
to ten seconds. Photographs of cultures are also original
with a few indicated exceptions. All temperatures are
indicated in degrees centigrade.

For use of electrotypes or for prints furnished the author
is indebted to the following: A. P. Barber Creamery Supply
Company, Chicago, IIl.; Bausch & Lomb Optical Company,
Rochester, N. Y.; Creamery Package Manufacturing Com-
pany, Chicago, Ill.; Davis Milk Machinery Company, N.
Chicago, Il.; Mr. C. B. Hoover, Superintendent of Sewage
Disposal Plant, Columbus, O.; Mr. C. P. Hoover, Super-
intendent of Water Filtration Plant, Columbus, O.; The
Hydraulic Press Manufacturing Company, Mt. Gilead, O.;
Loew Manufacturing Company, Cleveland, O.; Metric
Metal Works, Erie, Pa.; Sprague Canning Machine Company,
Chicago, Ill.; U. S. Marine Hospital Service.; Wallace and
Tiernan Company, New York City, N. Y.

For the preparation of many cultures and slides, for great
assistance in the reading of proof and in the preparation of
the index, Miss Vera M. McCoy, Instructor in Bacteriology,
deserves the author’s thanks.

The author trusts that the book will find a place in College
and University courses in Bacteriology.

C.B. M.

CotumBvs, 1917.

CONTENTS.

Historical Introduction—Spontaneous Generation—Causation of
Disease—Putrefaction and Fermentation—Study of Forms—
Chronological Table .

CHAPTER I.

Position of a a agama to Algee—Yeasts—Molds—
Protozoa a

Par: Ts
MORPHOLOGY.

CHAPTER II.

Cell Structures—Ce]l Wall—Protoplasm—Plasmolysis—Plasmop-
tysis—Nucleus—Vacuoles—Capsules—Metachromatic Granules
—Flagella—Spores

CHAPTER III.

Cell Forms—Coccus—Bacillus—Spirillum

CHAPTER IV.
Cell Groupings

CHAPTER, V.

Classification-—Migula’s

17

34

39

49

52

56

x CONTENTS

CHAPTER XxX.

Study of the Physiology of Bacteria—Temperature—Incubators—
Thermal Death Point—Oxygen Relationships—Study of Physi-
ological Activities—Appearance of Growth on Culture Media

CHAPTER XXI.

Animal Inoculation—Material for Bacteriological Examination

PART IV.
GENERAL PATHOGENIC BACTERIOLOGY.

CHAPTER XXII.

Introduction — Infection — Acute Infection — Chronic Infection
—Specific—Non-specific—Koch’s Postulates—Virulence—Sus-
ceptibility .

CHAPTER XXIII. .

Pathogenic Bacteria Outside the Body—As Saprophytes—<As

Facultative Saprophytes—Latent—Carriers
CHAPTER XXIV.

Channels of Infection—Skin—Mucose—Respiratory Tract—Ali-
mentary Tract—Dissemination in the Body—Paths of Elimi-
nation—Specificity of Location .

CHAPTER XXV.

Immunity—Natural—Artificial—Active—Passive—Production of
Immunity—Vaccine—Antiserum

CHAPTER XXVI.

Theories of Immunity — Pasteur — Chauveau — Baumgiirtner —
Metchnikoff—Ehrlich—Principles of Ehrlich’s Theory .

196

210

213

218

CONTENTS

CHAPTER XXVII.

\
Ehrlich’s Theory (Continued)—Receptors of the First Order—
Antitoxin—Antienzyme—Preparation of Antitoxins—Units

CHAPTER XXVIII.

Ehrlich’s Theory (Continued)—Receptors of the Second Order—
Agglutinins—Agglutination Reaction—Precipitins—Precipitin
Test ‘

CHAPTER XXIX.

Ehrlich’s Theory (Continued)—Receptors of the Third Order—
Cytolysins — Amboceptor — Complement — Anti-amboceptors
—Antisnake Venoms—Failure of Cytolytic Serums in Practice—
Complement-fixation Test

CHAPTER XXX.

Phagocytosis—Opsonins—Opsonic Index—Bacterial Vaccines—
Preparation of—Use of—Aggressins

CHAPTER XXXI.
Anaphylaxis—Author’s Theory—Tuberculin Test—Summary

xi

238

242

248

256

264

BACTERIOLOGY.

HISTORICAL INTRODUCTION.

BACTERIOLOGY as a science is a development of the latter
half of the nineteenth century. It may be said to have begun
with Koch’s proof that Bacteriwm anthracis is the cause
of anthrax in 1876. Nevertheless this discovery of Koch’s
was preceded by numerous observations and experiments
which led up to it. Some of this work was done in attempt-
ing to disprove the old “spontaneous generation’”’ theory as
to the origin of organisms; some in searching for the causes
of disease and some in the study of fermentation and putre-
faction.

SPONTANEOUS GENERATION.

Speculation as to the first origin of life is as old as history
and doubtless older. Every people of antiquity had its own
legends, as for example, the account in Genesis. This ques-
tion never can be definitely settled, even though living
matter should be made in the laboratory.

The doctrine of the “spontaneous origin’ of particular
animals or plants from dead material under man’s own
observation is a somewhat different proposition and may be
subjected to experimental test. ‘The old Greek philosophers
believed it. Anaximander (B.c. 610-547) taught that some
animals are derived from moisture. Even Aristotle (B.c.
384-322) said that “animals sometimes arise in soil, in
plants, or in other animals,” 7. e., spontaneously. It can be
stated that this belief was general from his day down through
the Dark and Middle Ages and later. Cardano (a.p. 1501-

1576) wrote that water gives rise to fish and animals and is
2

18 HISTORICAL INTRODUCTION

also the cause of fermentation. Van Helmont (1578-1644)
gives directions for making artificial mice. Kircher (1602-
1680) describes and figures animals proces under his own
eyes by water on plant stems.

However, many thinkers of the even century
doubted the truth of this long-established belief. Francesco
Redi (1626-1698) made a number of experiments which
tended to prove that maggots did not arise spontaneously
in meat, as was generally believed, but developed only when
flies had an opportunity to deposit their eggs on the meat.
It seems that by the latter part of this century the idea
that organisms large enough to be seen with the naked eye
could originate spontaneously was generally abandoned by
learned men.

The work of Leeuwenhoek served to suspend for a time
the subject of spontaneous generation, only to have it revived
more vigorously later on. He is usually called ‘The Father
of the Microscope,” though the compound microscope was
invented probably by Hans Zansz or his son Zacharias, of
Holland, about 1590. Leeuwenhoek used a simple lens, but
his instruments were so much more powerful that they
opened up an entirely new and unknown world.

Anthony van Leeuwenhoek (1632-1723) was apprenticed
to a linen draper, and accumulated a comfortable fortune
in this business. He became interested in the grinding of
spectacle lenses, then an important industry in Delft,
Holland, where he lived, and did a great deal of experimental
work in this line, mainly for his own enjoyment. Finally he
succeeded in making a lens so powerful that he could see
in water and various infusions very minute living bodies
never before observed. Leeuwenhoek contributed 112 papers
to the Royal Society of Great Britain, the first in 1673,
many of them accompanied by such accurate descriptions
and drawings, for example a paper submitted September 12,
1683, that there is no doubt that he really saw bacteria and
was the first to do so (Fig. 1). Rightly may he be styled
“The Father of Bacteriology,” if not of the microscope. He
says in one paper: “With the greatest astonishment I
observed that everywhere through the material: which I

SPONTANEOUS GENERATION 19

was examining were distributed animalcules of the most
microscopic dimness which moved themselves about in a
remarkably energetic way.”’ Thus he considered these liv-
ing objects to be animals, from their motion, and this belief
held sway for. nearly two hundred years.

Leeuwenhoek was a pure observer of facts and made no
attempt at speculation, but his discoveries soon started the
theorists to discussing the origin of these minute organisms.
Most observers, as was probably to be expected, believed
that they arose spontaneously. Needham, in 1749, described
the development of microédrganisms around grains of barley

Fig. 1.—The first drawings of bacteria by Leeuwenhoek. The dotted line
C-D indicates movement of the organism.

in water. Bonnet, in 1768, suggested that probably Need-
ham’s animalcules came from ova in the liquid. The Abbot
Spallanzani, in 1769, called attention to the crudeness of
Needham’s methods and later, in 1776, attempted to dis-
prove spontaneous origin by heating infusions of organic
material in flasks and then sealing them. His critics raised
the objections that heating the liquids destroyed their
ability to support life, and that sealing prevented the access
of fresh air which was also necessary. The first objection
was disproved by the accidental cracking of some of the
flasks which thereafter showed an abundant growth. This

20 HISTORICAL INTRODUCTION

accident seemed also to support the second objection, and
Spallanzani did not answer it. Though Spallanzani’s experi-
ments failed to convince his opponents, they led to important
practical results, since in Francois Appert, 1810, applied them
to the preserving of fruits, meats, etc., and in a sense started
the modern canning industry.

From Spallanzani to Schultze, there were no further
experiments to prove or disprove spontaneous generation.
Schultze, in 1836, attempted to meet the second objection to
Spallanzani’s experiment, 7. e., the exclusion of air, by draw-
ing air through his boiled infusions, first causing it to bubble

Fic. 2.—Schultze’s experiment. The set of bulbs next to the face con-
tained KOH and the other set concentrated H2SO.. Air was drawn through
at frequent intervals from May until August but no growth developed in
the boiled infusion.

through concentrated sulphuric acid to kill the “germs”
(Fig. 2). His flasks fortunately showed no growths, but
his critics claimed that the strong acid changed the proper-
ties of the air so that it would not support life. Schwann, in
1837, modified this experiment by drawing the air through
a tube heated to destroy the living germs (Fig 3). His
experiments were successful but the “spontaneous genera-
tion” theorists raised the same objection, 2. ¢., the change
in the air by heating. Similar arguments were brought
forward, also to the use of cotton plugs as filters by Schroeder

SPONTANEOUS GENERATION 21

and Dusch in 1859 (Fig. 4). It remained for Chevreuil and
Pasteur to overcome this objection in 1861 by the use of

HHT TG aTE ETE

PEL

Fic. 3.—Schwann’s experiment. After boiling, as shown in the diagram,
and cooling, air was drawn into the flask by aspiration while the coiled tube
was kept hot with the flame.

Fic. 4.—Schroeder and Dusch’s experiment. The aspirating bottle drew
the air through the flask after it had been filtered by the cotton in the
tube

22 HISTORICAL INTRODUCTION

Fic. 5.—Pasteur’s flask.

Fic. 6.—Tyndall’s box. One side is removed to show the construction.
The bent tubes at the top are to permit a free circulation of air into the
interior. The window at the back has one corresponding in the front
(removed). Through these the beam of light sent through from the lamp
at the side was observed. The three tubes received the infusion and were
then boiled in an oil bath. The pipette was for filling the tubes. (Popular
Science Monthly, April, 1877.)

CAUSATION OF DISEASE 23

flasks with long necks drawn out to a point and bent over.
These permitted a full access of air by diffusion but kept
out living germs, since these cannot fly but are carried
mechanically by air currents or fall of their own weight
(Fig. 5). Hoffman, the year before (1860), had made similar
experiments but these remained unnoticed. The Pasteur
flasks convinced most scientists that “spontaneous genera-
tion” has never been observed by man, though some few,
notably Dr. Charlton Bastian, of England, vigorously sup-
ported the theory from the early seventies until his death
in November, 1915.

John Tyndall, in combating Bastian’s views, showed that
boiled infusions left open to the air in a closed box through
which air circulated did not show any growth of organisms
provided the air was so free of particles that the path of a
ray of light sent through it from side to side could not be
seen (Fig. 6). Or if such sterilized infusions were exposed
to dust-free air, as in the high Alps, the majority showed no
growth, while all infusions in dusty air did show an abun-
dance of organisms. Tyndall’s experiments confirmed those
of Pasteur and his predecessors and showed that the organ-
isms developed from “germs” present in the air falling into
the liquids and not spontaneously. :

CAUSATION OF DISEASE.

Apparently the first writer on this subject was Varo; about
B.c. 70, who suggested that fevers in swampy places were
due to invisible organisms. Fracastorius (1484-1553), in
a work published in 1546, elaborated a theory of “disease
germs” and “direct and indirect contagion” very similar to
modern views, though based on no direct pathological knowl-
edge. Nevertheless Kircher (mentioned already) is usually
given undeserved credit for the “contagium vivum”’ theory.
In 1657 by the use of simple lenses he observed “worms” in

decaying substances, in blood and in the pus from bubonic
plague patients (probably rouleaux of corpuscles in the
blood, certainly not bacteria in any case). Based on these
observations and possibly also on reading the work of

24 HISTORICAL INTRODUCTION

Fracastorius, his theory of a “living cause” for various dis-
eases was published in 1671, but received little support.

The discoveries of Leeuwenhoek which proved the exist-
ence of microscopic organisms soon revived the “contagium
vivum”’ idea of Kircher. Nicolas Andry in a work pub-
lished in 1701 held such views. Lancisi in 1718 advanced
the idea that “animalcules” were responsible for malaria, a
view not proved until Laveran discovered the malarial para-
site in 1880.!. Physicians ascribed the plague which visited
Southern France in 1721 to the same cause, and many even
went so far as to attribute all disease to animalcules, which
brought the theory into ridicule. Nevertheless, the ‘“con-
tagium vivum” theory survived, and even Linnaeus in his
Systema Nature (1753-6) recognized it. by placing the
organisms of Leeuwenhoek, the contagia of diseases and the
causes of putrefaction and fermentation in one class called
“Chaos.”

Plenciz, a prominent physician and professor in the Vienna
Medical School, published in 1762 a work in which he gave
strong arguments for the “living cause” theory for trans-
missable diseases. He taught that the agent is evidently
transmitted through the air and that there is a certain
period of incubation pointing to a multiplication within the
body. He also believed that there was a specific agent
for each disease. His writings attracted little attention at
the time and the “contagium vivum”’ theory seems to have
been almost lost sight of for more than fifty years. Indeed,
Oznam, in 1820, said it was no use to waste time in refuting
hypotheses as to the animal nature of contagium.

Isolated observers were, however, keeping the idea alive,
each in his own locality. In 1787 Wollstein, of Vienna,
showed that the pus from horses with glanders could infect
other horses if inoculated into the skin. Abilgaard, of
Copenhagen, made similar experiments at about the same
time. In 1797 Eric Viborg, a pupil of Abilgaard’s, published
experiments in which he showed the infectious nature not

1Sir H. A. Blake has called attention to the fact that the ‘‘mosquito
theory”’ of malaria is mentioned in a Sanscrit manuscript of about the 6th
century A.D.

CAUSATION OF DISEASE 95

only of the pus but also of the nasal discharges, saliva,
urine, etc., of glandered horses. Prevost’s discovery of the
cause of grain rust (Puccinia graminis) in 1807 was the first
instance of an infectious disease of plants shown to be due
to a microscopic plant organism, though not a bacterium in
this case.

In 1822 Gaspard showed the poisonous nature of material
from infected wounds by injecting it into animals and caus-
ing their death. Bearing on the “contagium vivum”’ theory
was the rediscovery of the “itch mite” (Sarcoptes scabiet) by
Renucci (1834), an Italian medical student. This had been
declared several hundred years before but had been lost
sight of. Chevreuil and Pasteur, in 1836, showed that putre-
faction did not occur in meat protected from contamination,
and suggested that wound infection probably resulted from
entrance of germs from without. Bassi, investigating a dis-
ease of silkworms in Italy, demonstrated that a certain
mold-like fungus (Botrytis basstana) was the cause in 1837.
This was the first instance of a microscopic vegetable organ-
ism proved to be capable of causing disease in an animal.

Boehm, in 1838, observed minute organisms in the stools
of cholera patients and conjectured that they might have a
causal connection with the disease. The fungous nature of
Favus, a scalp disease, was recognized by Schonlein in 1839,
and the organism was afterward called ‘‘ Achorion schoen-
leinit.”” Berg, in 1839-41, showed that Thrush is likewise due
to a fungus, “ Oidiuwm albicans.”

These discoveries led Henle, in 1840, to publish a work in
which he maintained that all contagious diseases must be
due to living organisms, and to propound certain postulates
(afterward restated by Koch and now known as “ Koch’s
postulates’) which must be demonstrated before one can be
sure that a given organism is the specific cause of a given
disease. The methods then in vogue and the instruments of
that period did not enable Henle to prove his claims, but he
must be given the credit for establishing the “contagium
vivum”’ theory on a good basis and pointing the way for

+ men better equipped to prove its soundness in after years.

In 1842-43 Gruby showed that Herpes tonsurans, a form

26 HISTORICAL INTRODUCTION

of ringworm, is due to the fungus Trichophyton tonsurans.
Klencke, in 1843, produced generalized tuberculosis in a rab-
bit by injecting tuberculous material into a vein in the ear,
but did not carry his researches further. Liebert identified
the Peronospora infestans as the cause of one type of potato
rot in 1845. The skin disease pityriasis (tinea) versicolor
was shown to be due to the Microsporon furfur by Eichstedt:
in 1846.

Pollender, in 1849, and Davaine and Rayer, in 1850, inde-
pendently observed small rod-like bodies in the blood of
sheep and cattle which had died of splenic fever (anthrax).
That Egyptian chlorosis, afterward identified with Old
World “hookworm disease,” is caused by the Ankylosto-
mum duodenale was shown by Griesinger in 1851. In the
same year the Schistosomum hematobium was shown to be
the cause of the “Bilharzia disease” by Bilharz. Kiichen-
meister discovered the tapeworm, Tenia soliwm, in 1852,
Cohn, an infectious disease of flies due to a parasitic fungus
(Empusa musce) in 1855, and Zenker the Trichinella spiralis
in trichinosis of pork (‘measly pork”) in 1860. The organ-
isms just mentioned are, of course, not bacteria, but these
discoveries proved conclusively that living things of one kind
or another, some large, most of them microscopic, could cause
disease in other organisms and stimulated the search for
other “living contagiums.”” In 1863 Davaine, already men-
tioned, showed that anthrax could be transmitted from
animal to animal by inoculation of blood, but only ifthe
blood contained the minute rods which he believed to be the
cause. In 1865 Villemin repeatedly caused tuberculosis in
rabbits by subcutaneous injection of tuberculous material
and showed that this disease must be infectious also. In
this same year Lord Lister introduced antiseptic methods
in surgery. He believed that wound infections were due
to microdrganisms getting in from the air, the surgeon's
fingers, etc., and without proving this, he used carbolic acid
to kill these germs and prevent the infection. His pioneer
experiments made modern surgery possible. In this year
also, Pasteur was sent to investigate a disease, Pebrine,
which was destroying the silkworms in Southern France.

SIR JOSEPH LISTER

ROBERT KOCH

CAUSATION OF DISEASE 27°

He showed the cause to be a protozoan which had been seen
previously by Cornalia and described by Niigeli under the
name Nosema bombycis and devised preventive measures.
This was the first infectious disease shown to be due to a proto-
zoan. In 1866 Rindfleisch observed small pin-point-like
bodies in the heart muscle of persons who had died of wound
infection. _Klebs, in 1870-71, published descriptions and
names of organisms he had found in the material from simi-
lar wounds, though he did not establish their causal rela-
tion. Bollinger, in 1872, discovered the spores of anthrax
and explained the persistence of the disease in certain dis-
tricts as due to the resistant spores. In 1873 Obermeier
observed in the blood of patients suffering from recurrent
fever long, flexible spiral organisms which have been named
Spirocheta obermeiert. Lésch ascribed tropical dysentery
to an ameba, named by him Ameba coli, in 1875. Finally,
Koch, in 1876, isolated the anthrax bacillus by means of gelatin
plates (first used by Emil Chr. Hansen in his studies on yeast),
worked out the life history of the organism and reproduced
the disease by the injection of pure cultures and recovered
the organism from the inoculated animals, thus establish-
ing beyond reasonable doubt its causal relationship to the
diseasé. This was the first instance of a bacterium proved
to be the cause of a disease in animals. Pasteur, working on
the disease at the same time, confirmed all of Koch’s find-
ings, though his results were published the next year, 1877.
Bollinger determined that the Actinomyces bovis (Strepto-
thria bovis) is the cause of actinomycosis in cattle in 1877.
Woronin in the same year discovered a protozoan (Plasmo-
diophora brassice) to be the cause of a disease in cabbage
plants, the first proved instance of a unicellular animal caus-
ing a disease in a plant. In 1878 Koch published his
researches on wound infection in which he showed beyond
question that microdrganisms are the cause of this condi-
tion, though Pasteur, in 1837, had suggested the same thing
and Lister had acted on the theory in preventing infection.

These discoveries, especially those of Koch, immediately
attracted world-wide attention and stimulated a host of
workers, so that within the next ten years most of the bac-

28 HISTORICAL INTRODUCTION

teria which produce disease in men and animals were iso-
lated and described. It is well to remember that the first
specific disease of man proved to be caused by a bacterium
was tuberculosis, by Noch in 1882.

Progress was greatly assisted by the introduction of anilin
dyes as suitable stains for organisms by Weigert in 1877, by
Koch’s application of special technic and solid cultures for
isolation and study, and the great improvements in the
microscope by Prof. Abbé, of Jena.

Laveran’s discovery of the malarial parasite in 1880 turned
attention to protozoa as the causes of disease and led to the
discovery of the various piroplasmoses and trypanosomiases
in man and the lower animals.

Pasteur’s protective inoculations in chicken cholera and
anthrax directed attention to the possibility of using bac-
teria or their products as a specific protective or curative
means against particular diseases. This finally led to the dis-
covery of diphtheria antitoxin by Behring, and independently
by Roux, in 1890, a discovery which opened up the wide
field of immunity which is so persistently cultivated at the
present time.

While the causation of disease by bacteria has probably
attracted most attention, especially in the popular mind, it
should not be forgotten that this is but one of the numerous
ways in which these organisms manifest their activities, and
in a sense it is one of their least-important ways, since other
kinds are essential in many industries (dairying, agriculture)
and processes (sewage purification) and are even indispen-
sable for the very existence of all green plants and hence of
animals, including man himself.

PUTREFACTION AND FERMENTATION.

The idea that there is a certain resemblance between
some infectious diseases and the processes of putrefaction
and fermentation seems to have originated during the dis-
cussion on spontaneous generation and the “contagium
vivum”’ theory which followed Leeuwenhoek’s discoveries.
Plenciz (1762) appears to have first formulated this belief

LOUIS PASTEUR

PUTREFACTION AND FERMENTATION 29
in writing. He considered putrefaction to'be due to the
“animalcules” and said that it occurred only when there
was a coat of organisms on the material and only when
they increased and multiplied. Spallanzani’s experiments
tended to support this view since his infusions did not
“spoil” when boiled and sealed. Appert’s practical appli-
cation of this idea has been mentioned.

Thaer, in his Principles of Rational Agriculture, pub-
lished in the first quarter of the nineteenth century, expressed
the belief that the “blue milk fermentation” was probably
due to a kind of fungus that gets in from the air, and stated
that he had prevented it by treating the milk cellars and
vessels with sulphur fumes or with “oxygenated hydrochloric
acid” (hypochlorous acid).

In 1836 Chevreuil and Pasteur showed that putrefaction
did not occur in meat protected from contamination. In
1837 Caignard-Latour, in France, and Schwann, in Germany,
independently showed that alcoholic fermentation in beer
and wine is due to the growth of a microscopic plant, the
yeast, in the fermenting wort. C.J. Fuchs described the
organism which is commonly called the “blue milk bacillus”
in 1841 and conjectured that the souring of milk was prob-
ably bacterial in origin. It remained for Pasteur to prove
this in 1857. During the following six or seven years Pas-
teur also proved that acetic acid fermentation, as in vinegar
making; butyric acid fermentation (odor of rancid butter
and old cheese) and the ammoniacal fermentation of urea,
so noticeable around stables, were each due to different species
of bacteria. Pasteur also, during the progress of this work,
discovered the class of organisms which can grow in the
absence of free oxygen—the anaérobic bacteria. There is
no question that Pasteur from 1857 on did more to lay the
foundations of the science of bacteriology than any other
one man. Influenced by Pasteur’s work von Hesseling, in
1866, stated his belief that the process of cheese ripening, like
the souring of milk, was associated with the growth of fungi,
and Martin also, in 1867, stated that cheese ripening was
a process which was akin to alcoholic, lactic and butyric
fermentations. Kette, in 1869, asserted the probability of

30 HISTORICAL INTRODUCTION

Pasteur’s researches furnishing a scientific basis for many
processes of change in the soil. In 1873 Schlésing and
Miintz showed that nitrification must be due to the action
of microérganisms, though the discovery of the particular
ones remained for Winogradsky in 1889. Thus the belief
that fermentation and putrefaction are due to microérgan-
isms was as well established by the early eighties of the last
century as that similar organisms are the causes of infectious
diseases.

STUDY OF FORMS.

An important part of the scientific knowledge of living
organisms is dependent on a study of their forms and rela-
tionships. As has been stated, Leeuwenhoek considered
bacteria to be “animalcules” because they showed inde-
pendent movement. But little attention was paid to the
natural history of these animalcules for nearly a hundred
years after Leeuwenhoek. During the last quarter of the
eighteenth century, however, workers busied themselves
chiefly with the discovery and description of new forms.
Among these students were Baron Gleichen, Jablot, Lesser,
Reaumur, Hill and others. Miller, of Copenhagen, in 1786
published the first attempt at classification, a most impor-
tant step in the study of these organisms. Miller intro-
duced the terms Monas, Proteus and Vibrio which are
still in use. Ehrenberg, in his work on Infusoria, or the
organisms found in infusions, published in 1838, introduced
many generic names in use at present, but still classed the
bacteria with protozoa. Joseph Leidy, the American geol-
ogist, who took great interest in natural history in general,
considered that the ‘‘vibrios”’ of previous writers were plants
and not ‘‘animalcules.” He seems to have been the first to
have made this distinction (1849). Perty (1852) recognized
the presence of spores in some of his organisms. Ferdinand
Cohn (1854) classed the bacteria among plants. Néageli (1857)
proposed the name “Schizomycetes” or “fission fungi,”
which is still retained for the entire class of bacteria. Cohn
in the years 1872-1875 established classification on a mod-
ern basis and added greatly to the knowledge of morphology

STUDY OF FORMS 3l

and natural history of bacteria. He described spore forma-
tion and the development of spores into active bacteria,
and showed the close relationships as well as differences
between the bacteria and the lower alge. Robert Koch
was a pupil of Cohn.

An examination of the accompanying chronological table
will show how the investigations and discoveries in con-
nection with “spontaneous generation,” the “contagium
vivum” theory and putrefaction and fermentation must
have been mutually suggestive:

1546. Fracastorius, disease germs theory and direct and
indirect contagion.

1671. Kircher, “contagium vivum” theory.

-1675. Leeuwenhoek, first saw bacteria, “animalcules.”’

1701. Andry, “animalcules” cause of diseases.

1718. Lancisi, “animalcules” cause of malaria.

1749. Needham, described development of organisms in
water around barley grains.

1762. Plenciz, arguments for “living cause” theory and
that “animalcules” cause putrefaction.

1768. Bonnet, suggested that probably Needham’s organ-
isms came from germs in the liquid.

~~1776. Spallanzani, boiled and sealed infusions.

1786. Miiller, first classified “animalcules.”
1787. Wollstein, glanders pus infectious.
1797. Viborg, transmitted glanders repeatedly.

—~1807. Prevost, grain rust, Puccinia graminis.

1810. Appert, directions for “canning.”

1822. Gaspard, infectiousness of material from wounds.
1834. Renucci, itch—itch mite (Sarcoptes scabiet).
1836. Schultze, air through acid to kill “germs.”

—~1837. Chevreuil and Pasteur, protected meat did not
putrefy; suggested wound infection due to entrance
of germs from without.

1837. Caignard-Latour, Schwann, alcoholic fermentation
—yeast.

1837. Schwann, air through heated tubes to kill germs.

1837. Bassi, muscardine of silkworms, Botrytis bassvana.

32

1838.

~~ 1838.
1839.

4

HISTORICAL INTRODUCTION

Boehm, cholera, saw organisms in stools (not the
cause).

Ehrenberg, study of forms.

Schonlein, Favus, Achorion schoenleinit.

1839-41. Berg, Thrush, Oidiwm albicans.

~~ 1840.
1841.

Henle, theory of contagious diseases.
Fuchs, bacterial cause of blue milk.

1842-43. Gruby, Herpes tonsurans, Trichophyton ton-

1843.

1845.
1846.
1849.

1849.
1850.
1851.

1851.
1852.
1852.
- 1854.
1855.
1857.
1857.
1860.
1861.
1863.
1865.
1865.

~ 1865.
1866.
1866.
1867.

~1869.

surans.

Kklencke, inoculations of tuberculous material into
rabbit.

Liebert, a potato rot, Peronospora infestans.

Eichstedt, Pityriasis versicolor, Microsporon furfur.

Leidy, Joseph (American geologist), considered
“vibrios”’ to be plants.

Pollender, Anthrax, saw rods in blood.

Davaine and Rayer, Anthrax , saw rods in blood.

Griesinger, Egyptian chlorosis, Ankylostoma duo-
denatle.

Bilharz, Bilharzia disease, Schistosomum hematobium.

Kiickenmeister, tapeworm, Tenia soliwm.

Perty, saw spores in bacteria.

Cohn, classed bacteria as plants.

Cohn, Disease of Flies, Empusa musce.

Nageli, named bacteria, Schizomycetes.

Pasteur, lactic, acetic, butyric acid fermentation.

Zenker, Trichinosis, Trichinella spiralis.

Pasteur, disproof of spontaneous generation.

Davaine, transmitted anthrax by blood injections.

Pasteur, Pebrine of silkworms, Nosema bombycis.

Villemin, repeatedly transmitted tuberculosis to
rabbits.

Lister, introduced antisepsis in surgery.

Rindfleisch, Pyemia, organisms in the pus.

Von Hesseling, cheese ripening.

De Martin, cheese ripening akin to alcoholic fer-
mentation.

Kette, Pasteur’s researches scientific basis for many
processes in the soil.

STUDY OF FORMS 33

1871. Klebs, Pyemia, organisms in the pus.
1872. Bollinger, spores in anthrax.
—1872-75. Cohn, definite classification.
1873. Obermeier, Recurrent fever, Spirocheta obermevert.
1873. Schlésing and Miinz, nitrification due to organisms.
1875. Lésch, Amebic dysentery, Ameba coli.
1875-76. Tyndall, germs in the air.
—1876. Robert Koch, Anthrax, Bacteriwm anthracis.
1877. Bollinger, Actinomycosis, Actinomyces bovis (Strep-
tothrix bovis).
1877. Weigert, used anilin dyes for staining.
1877. Woronin, Cabbage disease, Plasmodiophora brassice.
-1878. Koch, wound infections, bacterial in origin.
_-1881. Koch, gelatin plate cultures. Abbé, improvements
196 in the microscope. ee
fy \. NA oid

CHAPTER I.
POSITION—RELATIONSHIPS.

BacTERIA are considered to belong to the plant kingdom
not because of any one character they possess, but because
they most nearly resemble organisms which are generally
recognized as plants. While it is not difficult to distinguish
between the higher plants and higher animals, it becomes
almost, if not quite, impossible to separate the lowest forms
of life. It is only by the method of resemblances above
mentioned that a decision is finally reached. It has even
been proposed to make a third class of organisms neither
plants nor animals but midway between in which the bac-
teria are included, but such a classification has not as yet
been adopted.

In many respects the bacteria are most ficarly related to
the lowest alge, since both are unicellular organisms, both
reproduce by transverse division and the forms of the cell
are strikingly similar. The bacteria differ in one important
respect, that is, they do not contain chlorophyl, the green
coloring matter which enables all plants possessing it to
absorb and break up carbon dioxide in the light, and hence
belong among the fungi. Bacteria average much smaller
than even the smallest alge. Bacteria are closely con-
nected with the fission yeasts and the yeasts and torule.
All are unicellular and without chlorophyl. The bacteria,
as has been stated, reproduce by division but the others
characteristically by budding or gemmation, though the fission
yeasts also by division.

There is a certain resemblance to the molds in their
absence of chlorophyl. But the molds grow as branching
threads and also have special fruiting organs for producing
spores as a means of reproduction, neither of which charac-
teristics is found among the true bacteria. The higher

POSITION—RELATIONSHIPS 35

thread bacteria do show true branching and rudimen-
tary fruiting bodies (Streptothrix) and appear to be a link
connecting the true bacteria and the molds.

\

Fic. 7.—A thread of blue-green Fic. 8.—A thread of a small blue-
alge. green alge.

Fic. 9.—A thread of bacteria. Com- Fic. 10.—A chain of spherical blue-
pare with Figs. 7 and 8. green alge.

Fic. 11.—A chain of spherical Fic. 12.—A pair of spherical blue-
bacteria. green alge,

36 . POSITION—RELATIONSHIPS

Further, the chemical composition of bacteria is more like
that of other fungous plants than of any of the forms
classed as animals.

Fic. 13. —Spherical bacteria. Fic. 14.—Yeast cells. Some show
Several pairs are shown. typical budding.

The food of bacteria is always taken up in solution by
diffusion through the outer covering of the cell as it is in all
he flan s ps ever surround and engulf particles of
¢@ ood omc c.cz: them within the cell as many single-

Fig. 15.—A portion of the mycelium of a mold. Note the large size and
the branching.

celled animals do, and as the leukocytes and similar ame-
boid cells in practically all multicelled animals do.!

One of the most marked differences between animals and
plants is with respect to their energy relationships. Plants

1 Myxomycetes excepted, and they are probably to be regarded as animals
—Mycetozoa,

POSITION—RELATIONSHIPS 37

are characteristically storers of energy while animals are
liberators of it. Some bacteria which have the power of
swimming in a liquid certainly liberate relatively large
amounts of energy, and in the changes which bacteria bring
about in the material which they use as food considerable
heat is evolved (“heating’”’ of manure, etc.). Nevertheless
the evidence is good that the bacteria as a class store much
more of the energy contained in the substances actually
taken into the body cell as food than is liberated in any
form.

Bacteria do show some resemblance to the protozoa, or
single-celled animal forms, in that the individuals of each
group consist of one cell only and some bacteria have the
power of independent motion from place to place in a liquid
as most “infusoria”’ do, but here the resemblance ceases.

Bacteria are among the smallest of organisms, so small
that it requires the highest powers of the microscope for
their successful study, and the use of a special unit for their
measurement. This unit is the one-thousandth part of a
millimeter and is called the micro-millimeter or micron.
Its symbol is the Greek letter mu ().

The size varies widely among different kinds but is fairly
constant in the same kind. The smallest described form is
said to be only 0.184 long by 0.06y thick and is just visible
with the highest power of the microscope, though it is pos-
sible and even probable that there are forms still smaller
which cannot be seen. Some large rare forms may measure
40u in length, but the vast majority are from 1p to 4y or 5u
long, and from one-third to one-half as wide.

From the above description a bacterium might be said to
be a microscopic, unicellular plant, without chlorophyl, which
reproduces by dinding transversely.

PART I.

MORPHOLOGY.

CHAPTER II.
CELL STRUCTURES.

THE essential structures which may by appropriate means
be distinguished in the bacterial cell are cell wall and cell
contents, technically termed protoplasm, cytoplasm. The
cell wall is not so dense, relatively, as that of green plants,
but is thicker than the outer covering of protozoa. It is
very similar to the cell wall of other lower fungi. Diffusion
takes place readily through it with very little selective action
on substances absorbed as judged by the comparative com-
position of bacteria and their surrounding medium.

Cytoplasm.—The cytoplasm according to Biitschli and
others is somewhat different and slightly denser in its outer
portion next to the cell wall. This layer is designated the
ectoplasm, as distinguished from the remainder of the cell
contents, the endoplasm. When bacteria are suddenly trans-
ferred from a given medium into one of decidedly greater
density, there sometimes results a contraction of the endo-
plasm, due to the rapid diffusion of water. This phenomenon
is designated plasmolysis (Fig. 16), and is similar to what
occurs in the cells of higher plants when subjected to the
same treatment. This is one of the methods which may be
used to show the different parts of the cell just described.

If bacteria are suddenly transferred from a, relatively
dense medium to one which is of decidedly less density, it

40 CELL STRUCTURES

occasionally happens that water diffuses into the cell and
swells up the endoplasm so much more rapidly than the cell
wall that the latter ruptures and some of the endoplasm
exudes in the form of droplets on the surface of the cell
wall. This phenomenon is called plasmoptysis. Students will
seldom observe the distinction between cell wall and cell
contents, except that in examining living bacteria the outer
portion appears more highly refractive. This is chiefly due
to the presence of a cell wall, but is not a proof of its
existence.

Fig. 16.—Cells of bacteria show- Fia. 17.—Vacuoles in the bac-
ing plasmolysis. The cell substance terial cell. The lighter areas are
of three of the cells in the middle of vacuoles.

the chain has shrunk until it appears
as a round black mass. The cell wall
shows as the lighter area.

Nucleus.—A nucleus as such is not present in bacterial
cells, except in a few large rare forms. Nuclein, the char-
acteristic chemical substance in nuclei, which when aggre-
gated forms the nucleus, is scattered throughout the cell con-
tents and thus intimately mingled with the protoplasm, and
cannot be differentiated by staining as in most cells. This
close association of nuclein and protoplasm may explain
the rapid rate of division of bacteria (Chapter VIII, p. 80).

The chemical composition of the bacterial cell is discussed
in Chapter VII.

In addition to the essential parts just described the bac-
terial cell may show some of the following accidental struc-
tures: vacuoles, capsules, metachromatic granules, flagella, spores.

CAPSULE 41

Vacuoles.—J”acuoles appear as clear spaces in the proto-
plasm when the organism is examined in the living condition
or when stained very slightly (Fig. 17). During life these are
_ filled with liquid or gaseous material which is sometimes
waste, sometimes reserve food, sometimes digestive fluids.
Students are apt to confuse vacuoles with spores (p. 44).
Staining is the surest way to differentiate (Chapter XIX,
p. 192). If vacuoles have any special function, it is an
unimportant one.

Capsule.—The capsule is a second covering outside the cell
wall and probably developed from it (Fig. 18). It is usually
gelatinous, so that bacteria which form capsules frequently

“3s —-eeteria seen within Fig. 19.—Metachromatic gran-
capsules, ules in bacteria. The dark round

spots are the granules. The cells

of the bacteria are scarcely visible.

stick together when growing in a fluid, so that the whole
mass has a jelly-like consistency. The term zodglea was
formerly applied to such masses, but is a poor term and
misleading (zo6n=an animal) and should be dropped. The
masses of jelly-like material frequently found on decaying
wood, especially in rainy weather, are in some cases masses
of capsule-forming bacteria, though a part of the jelly is a
product of bacterial activity, a gum-like substance which
lies among the capsulated organisms. When these masses
dry out, they become tough and leathery, but it is not to be
presumed that capsules are of this consistency. On the
contrary, they are soft and delicate, though they certainly

42 CELL STRUCTURES

serve as an additional protection to the organism, doubtless
more by selective absorption than mechanically. The
presence of capsules around an organism can be proved only
by staining the capsule. Many bacteria when stained in
albuminous fluids show a clear space around them which
appears like a capsule. It is due to the contraction of the
fluid away from the organism during drying.

Metachromatic Granules—The term “metachromatic” is
applied to granules which in stained preparations take a color
different from the protoplasm as a whole (Fig. 19). They
vary widely in chemical composition. Some of them are
glycogen, some fat droplets. Others are so-called “granu-
‘lose” closely related to starch but probably not true starch.
Others are probably nuclein. Of many the chemical com-
positionis unknown. They are also called “ Babes-Ernst cor-
puscles”’ in certain bacteria (typhoid bacillus). Since they
frequently occur in the ends of cells the term “ polar granules”
is also applied. Their presence is of value in the recognition
of but few bacteria (“Neisser granules”’in diphtheria).

Flagellum.—A flagellum is a very minute thread-like pro-
cess growing out from the cell wall, probably filled with a
strand of protoplasm. The vibrations of the flagella move
the organism through the liquid medium. Bacteria which
are thus capable of independent movement are spoken of
as “motile bacteria.” The actual rate of movement is very
slight, though in proportion to the size of the organism it
may be considered rapid. Thus Alfred Fischer determined
that some organisms have a speed for short periods of about
40 cm. per hour. This is equivalent to a man moving more
than 200 miles in the same time.

It is obvious that bacteria which can move about in a
liquid have an advantage in obtaining food, since they do
not need to wait for it to be brought to them. This advan-
tage is probably slight.

An organism may have only one flagellum at the end. It
is then said to be monotrichic (Fig. 20) (uévos=alone, single;
tpcxos = hair). This is most commonly at the front end, so
that the bacterium is drawn through the liquid by its motion.
Rarely it is at the rear end. Other bacteria may possess a

FLAGELLUM 43

bundle of flagella at one end and are called lophotrichic (Fig.
21) (Aopos = tuft). Sometimes at approaching division the
flagella may be at both ends and are then amphitrichic (Fig.
22) (aude = both). It is probable that this condition does not

Fig. 20.—A bacterium showing a Fig. 21.—A bacterium showing
single flagellum at the end—mono- a bundle-of four flagella at the
trichic. end—lophotrichic.

. persist long, but represents the development of flagella at
one end of each of a pair resulting from division of an organ-
iem which hag flagella at one end only. In many bacteria

_Fic. 22.—A bacterium showing Fie. 23.—A_ bacterium showing
flagella at each end—amphitrichic. flagella all around—peritrichic.

the flagella arise from all parts of the surface of the cell.
Such bacteria are peritrichic (Fig. 23) (reo. = around). The
position and even the number of the flagella are very con-
stant for each kind and are of decided value in identification.

44 CELL STRUCTURES

Flagella are too fine and delicate to be seen on the living
organism, or even on bacteria which have been colored by
the ordinary stains. They are rendered visible only by
certain methods which cause a precipitate on both bacteria
and flagella which are thereby made thick enough to be
seen (Chapter XIX, p. 194). The movement of liquid
around a bacterium caused by vibrations of flagella can
sometimes be observed with large forms and the use of
“dark-field” illumination.

Flagella are very delicate and easily broken off from the
cell body. Slight changes in the density or reaction of the
medium frequently cause this breaking off, so that prepara-
tions made from actively motile bacteria frequently show
no flagella. For this reason and also on account of their
fineness the demonstration of flagella is not easy, and it is
not safe to say that a non-motile bacterium has no flagella
except after very careful study.

The motion of bacteria is characteristic and a little prac-
tice in observing will enable the student to recognize it and
distinguish between motility and “ Brownian’ or molecular
motion. Dead and non-motile bacteria show the latter.
In fact, any finely divided particles suspended in a liquid
which is not too viscous and in which the particles are not
soluble show Brownian motion or “pedesis.”” This latter is
a dancing motion of the particle within a very small area
and without change of place, while motile bacteria move
from place to place or even out of the field of the microscope
with greater or less speed. There is a marked difference in
the character of the motion of different kinds of bacteria.
Some rotate around the long axis when moving, others
vibrate from side to side.

Among the higher thread bacteria there are some which
show motility without possessing flagella. Just how they
move is little understood.

Spores—Under certain conditions some bacterial cells
undergo transformations which result in the formation of
so-called spores. If the process is followed under the micro-
_ scope, the changes observed are approximately these: A very
minute point appears in the protoplasm which seems to act

SPORES 45

somewhat like the’ chromosome of higher cells as a “center of
attraction” so that the protoplasm gradually collects around
it. The spot disappears or is enclosed in the collected proto-
plasm. This has evidently become denser as it is more
highly refractive than before. In time all or nearly all of
the protoplasm is collected. A new cell wall is developed
around it which is thicker than the cell wall of the bacterium.
This thickened cell wall is called the “spore capsule.”
Gradually the remnants of the former cell contents and the

old cell wall disappear or dissolve and the spore becomes
“free” (Fig. 24).

_. ...aller oval bodies in the middle of the field are free spores.

a1 uue opuse is placed in favorable conditions the proto-
plasm absorbs water, swells, the capsule bursts at some
point, a cell wall is formed and the bacterium grows to
normal size and divides, that is, it is an active growing cell
again. This process is called “germination” of the spore.
The point at which the spore capsule bursts to permit the
new cell to emerge is characteristic for each kind of bac-
terium. It may be at the end when the germination is said
to be polar (Fig. 25). It may be from the middle of one
side which gives equatorial germination (Fig. 26). Rarely it
is diagonally from a point between the equator and the pole,
which type may be styled oblique germination. In one or
two instances the entire spore swells up, lengthens and
becomes a rod without any special germination unless this
type might be designated br-polar.

Spores are most commonly oval or ‘elliptical in shape,

46 CELL STRUCTURES

though sometimes spherical. A spore may be formed in the
middle of the organism without (Fig. 27) or with (Fig. 28)

Fic. 25.—Spores showing polar
germination. The lighter part of the
two organisms just below A and B is
the developing bacterium. In the
original slide the spore was stained
red and the: developing bacterium a
faint blue.

Fic. 27.—Spores in the middle of

the rod without enlargement of the
rod. The lighter areas in the rods
are spores.

the cell around it.
-: nereased, then the cell with the con-
spindle-shaped. Such a cell is termed

Fig. 26.—A spore showing equa-
torial germination. The spore in
the center of the field shows a rod
growing out of it laterally. In the
original slide the spore was stained
red and the developing bacterium
blue.

If the diameter

Fic. 28.—Spores in the middle
of the rod with enlargement of the
rod around them. The lighter areas
in the rods are spores.

a “clostridium.” Sometimes the spore develops in the end
of the cell either without (Fig. 29) or with enlarging it

SPORES 47

(Fig. 30). In a few forms the spore is placed at the end of
the rod and shows a marked enlargement. This is spoken
of as the “plectridium” or more commonly the “drumstick
spore” (Fig. 31). The position and shape of the spore are

Fig. 29.—Spores at the end of the Fic. 30.—Spores at the end of the
rod with no enlargement of the rod rod with enlargement of the rod,
around them. The lighter areas in A,A,A,A.
the rods are spores.

constant for each kind of bacteria. In one or two instances
only, two spores have been observed in a single organism.
_ >i. “nat the protoplasm is denser and the spore cap-

v-~- (the percentage of water in each is decidedly

Fic. 31.—Drumstick spores at the end of the rod.

less than in the growing cell) gives the spore the property
of much greater resistance to all destructive agencies than
the active bacterium has. For example, all actively growing
cells are destroyed by boiling in a very few minutes, while

48 CELL STRUCTURES

some spores require several hours’ boiling. The same rela-
tion holds with regard to drying, the action of chemicals,
light, etc. This resistance explains why it happens that
food materials boiled and sealed in cans to prevent the
entrance of organisms sometimes spoil. The spores have
not been killed by the boiling. It explains also in part the
persistence of some diseases like anthrax and black leg in
pastures for years.

From the above description it follows that the spore is to
be considered as a condensation of the bacterial protoplasm
surrounded by an especially thick cell wall. Its function ts
the preservation of the organism under adverse conditions. It
corresponds most closely to the encystment of certain
protozoa—the ameba for example. Possibly the spore
represents a very rudimentary beginning of a reproductive
function such as is gradually evolved in the higher thread
bacteria, the fission yeasts, the yeasts, the molds, etc. Its
characteristics are so markedly different, however, that the
function of preservation is certainly the main one.

It must not be supposed that spores are formed under
adverse conditions only, because bacteria showing vigorous
growth frequently form spores rapidly. Special conditions are
necessary for their formation just as they are for the growth
and other functions of bacteria (Chapters VI and VII).

CHAPTER III.
CELL FORMS.

Tuoues there is apparently a wide variation in the shapes
of different bacterial cells, yet these may all be reduced to
three typical cell forms. These are: first and simplest, the
round or spherical, typified by a ball and called the coccus
form, or coccus, plural cocci! (Fig. 32). The coccus may be
large, that is, from 1.54 to 2u in diameter. The term
macrococcus is sometimes applied to these large cocci. If
the coccus is less than 1 in diameter, it is sometimes spoken
of as a micrococcus; in fact, this term is very commonly
applied to any coccus. When cocci are growing together,
many of the cells do not appear as true spheres but are more
or less distorted ftom pressure of their neighbors or from
failure to grow to full size after recent division. Most cocci
divide into hemispheres and then each half grows to full
size. A few cocci elongate before division and then appear
oval or elliptical.

The second cell form is that of a cylinder or rod typi-
fied by a section of a lead-pencil. The name bacillus, plural
bacilli, is applied to this type (Fig. 33). The bacillus may
be short (Fig. 34), 1» or less in length, or long, up to 40u
in rare cases. Most bacilli are from 24 to 5y or 6p long.
The ends of the rod are usually rounded, occasionally square
and very rarely pointed. It is evident that a very short
rod with rounded ends approaches a coccus in form and it
is not always easy to differentiate in such cases. Most
bacilli are straight, but some are slightly curved (Fig. 35).

1 The pronunciation of this word according to English standards is
’ kék-si; the continental pronunciation is k6k-kee; the commonest American
seems to be k6Ok-ki. We prefer the latter since it is easier and more
natural and should like to see it adopted. (Author.)

4

50 CELL FORMS

The third cell form is the spiral, typified by a section of a
cork-screw and named spirillum, plural spirilla (Fig. 36). A
very short spiral consisting of only a portion of a turn is some-
times called vibrio (Fig. 37). Vibrios when seen under the

Fig. 32.—Cocci. Fic. 33.—Bacilli.

microscope look like short curved rods. The distinction
between the two can be made only by examining the organ-
ism alive and moving in a liquid. The vibrio shows a char-
acterisiie sprrai cwiseing motion. Very long flexible spirals are

Fic. 34.—Short bacilli. Fic. 35.—Curved bacilli. Only
the one in the center of the field is
in focus. The others curve out of
focus.

usually named spirochetes (Fig. 38). The spirochetes are

motile but flagella have not been shown to be present.
Besides the three typical cell forms bacteria frequently

show very great irregularities in shape. They may be

CELL FORMS 51

pointed, bulged, club-shaped or even slightly branched.
These peculiar and bizarre forms practically always occur
when some of the necessary conditions for normal growth,

© €

Fig. 36.—Spirilla. Fic. 37.—Vibrio forms of spirilla.
Compare with Fig. 35.

discussed in Chapters VI and VII, are not fulfilled. They
are best regarded as involution or degeneration forms for this
reason (Fig. 39). In a very few cases it is not possible to
obtain the organism without these forms (the diphtheria

Fic. 38.—Spirochetes. Fic. 39.—Involution forms. The

organisms are taperingand branched
at one end.

group). It is probable that these cell forms are normal in

such cases, or else conditions suitable for the normal growth
have not been’ obtained.

CHAPTER IV.
CELL GROUPINGS.

Ir has been stated that bacteria reproduce by trans-
verse division, that is, division across the long axis. Fol-
' lowing repeated divisions the new cells may or may not
remain attached. In the latter case the bacteria occur as
separate isolated individuals. In the former, arrangements
characteristic of the particular organism almost invariably
result. These arrangements are best described as cell
groupings, or growth forms.

Fic. 40.—Streptospirillum grouping. Fic. 41.—Diplobacillus grouping.

In the case of spiral forms it is obvious that there is only
one possible grouping, that is, in chains of two or more
individuals adherent end to end. A chain of two spirilla
might be called a diplospirillum (dirdés = double); of
_three or more, a streptospirillum (orpertés = necklace, chain)
(Fig. 40). These terms are rarely used, since spirilla do
not ordinarily remain attached. Likewise the bacillus can
grow only in chains of two or moré, and the terms diplo-
bacillus (Fig. 41), bacilli in groups of two, and streptobacillus
(Fig. 42), bacilli in chains are frequently used. Still the terms
thread, filament, or chain are more common for streptobacillus.

CELL GROUPINGS 53

Since the coccus is spherical, transverse division may occur
in any direction, though in three planes only at right angles
to each other. Division might occur in one plane only as in
spirilla and bacilli, or in two planes only or in all three planes.

Fig. 42.—Streptobacillus grouping. Fig. 43.—Typical diplococcus
grouping. Note that the individual
cocci are flattened on the appos-
ing sides.

As a matter of fact these three methods of ‘division are
found among the cocci, but only one method for each par-
ticular kind of coccus. As a result there may be a variety
: ----ags among the cocci. When division occurs in

Fic. 44.—Long streptococcus . Fie. 45.—Short streptococcus
grouping. grouping.

one plane only, the possible groupings are the same as among
the spirilla or bacilli. The eocci may occur in groups of two—
diplococcus grouping (Fig. 43), or in chains—streptococcus
grouping (Figs. 44 and 45). When the grouping is in diplococci,

54 CELL GROUPINGS

the individual cocci most commonly appear as hemispheres
with the plane surfaces apposed (Iig.43). Sometimes they ap-
pear as spheres, and occasionally are even somewhat elongated.
The individuals in a streptococcus grouping are most com-
monly elongated, either in the same direction as the length
of the chain, or at right angles to it. The latter appearance
is probably due to failure to enlarge completely after division.
Streptococci frequently appear as chains of diplococci, that is,
the pair resulting from the division of a single coccus remain
a little closer to each other than to neighboring cells, as a
close inspection of Fig. 44 will show.

If division occurs in two planes only, there may result the
above groupings and several others in addition. The four
cocci which result from a single division may remain together,
giving the tetracoccus or tetrad grouping. Very rarely all the
cocci divide evenly and the result is a regular rectangular
flat mass of cells, the total number of which is a multiple of
four. The term merismopedia (from a genus of algee which
grows the same way) is applied to such a grouping. If the
cells within a group after a few divisions do not reproduce
so rapidly (lack of food), as usually happens, the number of
cells becomes uneven or at least not necessarily a multiple
of four and the resultant flat mass has an irregular, uneven
outline. This grouping is termed staphylococcus (otadvdos =
a bunch of grapes) (Fig. 46). It is the most common group-
ing among the cocci.

When division occurs in all three planes, there is in addi-
tion to all the groupings possible to one- and two-plane divi-
sion a third grouping in which the cells are in solid packets,
multeples of erght. The name sarcina is applied to this growth
form (Fig. 47). The individual cells in a sarcina packet
never show the typical coccus form so long as they remain
together, but are always flattened on two or more sides.

The above descriptions indicate how the method of divi-
sion may be determined. If in examining a preparation the
sarcina grouping appears, that shows three-plane division.
If there are no sarcina, but tetrads or staphylococci (rarely
merismopedia), then the division is in two planes. If none
of the foregoing is observed but only diplo- or strepto-

CELL GROUPINGS 55

cocci, these indicate one-plane division only. Cocci show
their characteristic groupings only when grown in a liquid
medium, and such should always be used before deciding on
the plane of division.

Fic. 46.—Staphylococcus group- Fig. 47.—Sarcina grouping.
ing. The large flat masses are
staphylococcus grouping. Diplo-
coccus grouping, tetrads and short
streptococci are also evident.

ms the above description shows, these terms which are

~ ucieetives describing the cell grouping, are quite
'y wsed as nouns. Thus the terms a diplococcus, a
a, & streptococcus, etc., are common, meaning a bac-
terium of the cell form and cell grouping indicated.

Crit Form. CELL GROUPING.
diplococcus—in 2’s.

streptococcus—in chains.

tetracoccus, tetrads—in 4’s.
staphylococcus—irregular flat. masses.
sarcina—regular, solid~ packets, multiples

coccus—round or spherical.

of 8.
bacillus—rod-shaped or diplobacillus—in 2’s.
cylindrical. streptobacillus—in chains.

diplospirillum—in 2’s, little used.

spirillum—spiral-shaped. streptospirillum—in chains, little used.

CHAPTER V.

CLASSIFICATION.

THE arrangement of living organisms in groups accord-
ing to their resemblances and the adoption of fixed names
is of the greatest advantage in their scientific study. For
animal forms and for the higher plants this classification is
gradually becoming fixed through the International Congress
of Zodlogists and of Botanists respectively. Unfortunately,
the naming of the bacteria has not as yet been taken up by
the latter body, though announced as one of the subjects for
the Congress of 1916 (postponed on account of the war).
Hence there is at present no system which can be regarded
as either fixed or official.

Since Miiller’s first classification of “animalcules” in 1786
numerous attempts have been made to solve the problem.
Only those beginning with Ferdinand Cohn (1872-75) are
of any real value. As long as bacteria are regarded as
plants it appears that the logical method is to follow the
well-established botanical principles in any system for nam-
ing them. Botanists depend on morphological features
almost entirely in making their distinctions. The preceding
chapters have shown that the minute plants which are
discussed have very few such features. They are, to
recapitulate, cell wall, protoplasm, vacuoles, metachromatic
granules, capsules, flagella, spores, cell forms, and cell group-
ings. Most bacteria show not more than three or four of
these features, so that it is impossible by the aid of morphol-
ogy alone to distinguish from each other the large number
of different kinds which certainly exist. In the various
systems which are conceded to be the best these charac-
teristics do serve to classify them down to genera, leaving
‘the “species” to be determined from their physiological

CLASSIFICATION 57

activities. One of these systems has been adopted by the
laboratory section of the American Public Health Associa-
tion and by the Society of American Bacteriologists and is

Fic. 48.—Illustrates the genus Fic. 49.—Illustrates the genus

Streptococcus. Typical chains, no Micrococcus. Diplococcus, tetrads,
staphylococcus grouping, no sarcina short chains and staphylococcus;
grouping, no flagella. no sarcina, no flagella.

practically the standard in this country. It is that of the
German Bacteriologist Migula and is the only one which
will be considered here and used throughout this book.

Fic. 50.—Illustrates the genus Fie. 51.—Illustrates the genus
Sarcina. Sarcina grouping, no Bacillus. A bacillus with peritri-
flagella. chic flagella. (Student prepara-

tion.)

Personally, the author would prefer some slight modifications,
but as he is not a botanist, will not presume to make them.
Since practically the entire discussion in this book is con-

58 CLASSIFICATION

Fie. 52.—Illustrates the genus Fic. 53.—Illustrates the genus
Pseudomonas. A bacillus with flag- Microspira. It is (though the
ella at the end only. photograph does not prove it) a

short spiral with one flagellum at
the end.

Fic. 54.—Illustrates the genus Fic. 55.—Illustrates the genus
Spirillum. Spiral bacteria with Spirocheta.
more than three, in this case four,
flagella at the end.

Fig. 56.—TIllustrates the genus Fig. 57.—Illustrates the genus
Chlamydothrix. Fine threads with a Crenothrix. The thickness of the
delicate sheath. cell walls is due to deposits of iron

hydroxide. (After Lafar.)

CLASSIFICATION 59

cerned with the first three families, the generic character-
istics in these only will be given. The full classification as

Fic. 58.—Illustrates the genus Beggiatoa. The filament A is so full of
sulphur granules that the individual cells are not visible. B has fewer
sulphur granules. In C the granules are nearly absent and the separate
cells of the filament are seen. (After Winogradsky, from Lafar.)

well as a thorough discussion of, this subject is given in
Lafar’s Handbuch, whence the following is adopted:

ORDER I. EUBACTERIA.

out nuclei, free from sulphur granules and from
Suuvemupee wpavin (p. 100); colorless, or slightly colored.

1. Family: Coccacra (Zopf) Migula, all cocci.

Genus 1. Streptococcus Billroth: division in one plane only

(Fig. 48).
Non-flagellated, “ 2. Micrococcus (Hallier) Cohn:: division in two
non-motile planes only (Fig. 49).
«3. Sarcina Goodsir: division in three planes only ~
(Fig. 50).
Flagellated, “ 4, Planococcus Migula: division in two planes only.
motile “ 5. Planosarcina Migula: division in three planes only.

2. Family: Bacreriace# Migula, all bacilli.

Genus 1. Bactertum (Ehrenberg) Migula: no flagella;
non-motile.
“ 2. Bacillus (Cohn) Migula: flagella peritrichic
(Fig. 51).
3. Pseudomonas Migula: flagella at the end: mono-
trichic, lophotrichic, amphitrichic (Fig. 52).

«

60 CLASSIFICATION

3. Family: Sprrittacra# Migula, all spirilla.

Genus 1. Spirosoma Migula: non-flagellated; non-motile.
“2. Microspira (Schréter) Migula: flagella one to
Cells stiff three at the end (Fig. 53).
“3. Spirillum (Ehrenberg) Migula: flagella more fas
three at the end (Fig. 54).
4. Spirocheta Ehrenberg: motile; no flagella (Fig.
55).

“

Cell flexible

4. Family: CHLAMYDOBACTERIACES.

Cells cylindrical in long threads, and surrounded by a
sheath. Reproduction also by gonidia formed from an
entire cell.

Genus 1. Chlamydothrix Migula (Fig. 56).
“2. Crenothriz Cohn (Fig. 57).
“3. Pragmidiothriz Engler.
“ 4. Spherotilus (including Cladothrix).

ORDER II. THIOBACTERIA: SULPHUR BACTERIA.

Cells without a nucleus, but containing sulphur granules,
may be colorless or contain bacteriopurpurin and be colored
reddish or violet.

1. Family Breeratoaces.

Genus 1. Thiothrix Winogradsky.
“2. Beggiatoa Trevisan. ‘Of interest since it is with-
out a sheath, is motile, but without flagella
(Fig. 58).

2. Family RHODOBACTERIACES.

This has five subfamilies and twelve genera, most of which
are due to the Russian bacteriologist Winogradsky who did
more work than anyone else with the sulphur bacteria.

PART II.

PHYSIOLOGY.

CHAPTER VI.
GENERAL CONDITIONS FOR GROWTH.

OCCURRENCE.

BacrTeria are probably the most widely’ distributed of
living organisms. They are found practically everywhere
on the surface of the earth. Likewise in all surface waters,
in streams, lakes and the sea. They occur in the air imme-
diately above the surface, since they are carried up mechan-
ically by air currents. They cannot fly of themselves. There
is no reason to believe that any increase in numbers occurs
to an appreciable extent in the air. The upper air, for
example, on high mountains, is nearly free from them. So
also is the air over midocean, and in high latitudes. As
a rule, the greater the amount of dust in the air, the more ~
numerous are the bacteria. Hence they are found more
abundantly in the air in cities and towns than in the open
country. The soil is especially rich in numbers in the upper
few feet, but they diminish rapidly below and almost disap-
pear at depths of about six feet unless the soil is very porous
and open, when they may be carried farther down. Hence
the waters from deep wells and springs are usually devoid
of these organisms. In the sea they occur at all levels and
have been found in bottom ooze dredged from depths of
several miles. It is perhaps needless to add that they are
found on the bodies and in the alimentary tract of human

62 GENERAL CONDITIONS FOR GROWTH '

beings and animals; on clothing, utensils; in dwellings,
stables, outhouses, etc. From one-fourth to one-half of the
dry weight of the feces of animals and men is due to the
bacteria present. The urine is practically free from them
in health.

While bacteria are thus found nearly everywhere, it is an
entirely mistaken idea to suppose that all are injurious to
man. Asa matter of fact, those which are dangerous are
relatively few and are for the most part found only in close
association with man. Most bacteria are harmless and the
vast majority are beneficial or even essential to man’s
existence on the earth. These facts must be constantly
borne in mind, and it is hoped that the pages which follow
will make them clear.

In order that any organism may thrive there are a number
of general environmental conditions which must be fulfilled.
These conditions vary more or less for each kind of organ-
ism. Bacteria are no exception to this general rule. These
conditions may be conveniently considered under the gen-
eral heads of movtsture; temperature; light; oxygen supply;
osmotic pressure; action of electricity; of Réntgen and radium
rays; pressure; mechanical vibration; and chemical environ-
ment, including the reaction of the medium, the effect of
injurious chemicals, and especially the food requirements of
bacteria. For each of these conditions there is a maximum,
meaning the greatest amount of the given condition which
* the organism can withstand, a minimum, or the least amount,
and an optimum or that amount which is most favorable for
development. Further, there might be distinguished a maxi-
mum for mere existence and a lower maximum for develop-
ment; also a minimum for mere existence and a higher mini-
mum for development. These maxima, minima, and optima
for bacteria have been determined with exactness for only
a very few of the general conditions and for comparatively
few kinds.

MOISTURE 63

MOISTURE.

The maximum moisture is absolutely pure water, and no
‘organism can thrive in this alone owing to the factor of too
low osmotic pressure and to the further factor of absence of
food material. There are many bacteria which thrive in
water containing only traces of mineral salts and a large
class whose natural habitat is surface water. These “water
bacteria’”’ are of great benefit in the purification of streams.
They are as a class harmless to men and animals. Some
of the disease-producing bacteria like Bacillus typhosus (of
typhoid fever) and Microspira comma (of Asiatic cholera)
were undoubtedly originally water bacteria, and it is rather
striking that in these diseases conditions are induced in
the intestine (diarrheas) which simulate the original watery
environment. The minimum moisture condition is abso-
lute dryness, and no organism can even exist, not to say
develop, in such a condition since water is an essential con-
stituent of living matter.. Some bacteria and especially
most spores may live when dried in the air or by artificial
means for months and even years, while some are destroyed
in a few hours or days when dried (typhoid, cholera, etc.).
The optimum amount of moisture has not been determined
with any great accuracy and certainly a rather wide range
in percentage of water is permissable with many, though
a liquid medium is usually most favorable for artificial
growth. The “water bacteria” have been mentioned. In
the soil a water content of 5 to 15 per cent. seems to be
most suitable for many of the organisms which aid in plant
growth. In animals and man the organisms infecting the
intestinal tract prefer a high percentage of moisture as a
rule, especially those causing disease here. Those found on
the surface of the body (pus cocci) need a less amount of
water, while those invading the tissues (tuberculosis, black-
leg, etc.) seem to be intermediate in this respect. In arti-
ficial culture media a water content:of less than 30 per cent.
inhibits the growth of most bacteria.

As a general rule those bacteria which require the largest,
percentage of water are most susceptible to its loss and are

64 GENERAL CONDITIONS OF GROWTH

most readily killed by drying. The typhoid and cholera
organisms die in a few hours when dried, while pus cocci
and tubercle bacilli live much longer.

TEMPERATURE.

The temperature conditions for bacterial existence and
growth have been determined more accurately than any of
the other general conditions. The maximum for existence
must be placed at or near 100° since it is known that all
bacteria including spores may be killed by boiling in time.
Nevertheless, certain forms have been reported as thriving
in hot springs where the water temperature was 93°. This
is the highest known temperature for development. The
minimum for existence lies at or near the absolute zero
(—273°) since certain organisms have been subjected to the
temperature produced by the sudden evaporation of liquid
hydrogen (— 256° to— 265°) and have remained alive. Whether
they could withstand such temperatures indefinitely is not
known. The minimum for development is near the freez-
ing-point of water, since reproduction by division has been
observed in the water from melting sea-ice at a temperature
of —1.5°. Thus bacteria as a class have a range for existence
of about 373° (—273° to +100°) and for development of
94.5° (—1.5° to +93°) certainly much wider ranges than
any other group of organisms.!

The optimum temperature for development varies within
rather wide limits for different organisms. In general it
may be stated that the optimum temperature is approxi-
mately that of the natural habitat of the organism, though
there are exceptions. The optimum of the “hot spring”
bacteria just mentioned is apparently that of the springs
(93° in this case). Many ‘soil organisms are known
whose optimum is near 70° (a temperature rarely, if ever,
attained in the soil), but only when grown in air or oxygen;

1 With the possible exception of blue green algze which have been found
with bacteria in the above-mentioned hot springs. Seeds of many plants
have been subjected to as low temperatures as those above-mentioned with-
out apparent injury.

LIGHT 65

but is very much lower when grown in the absence of oxygen.
Many other soil organisms exhibit very little difference in
rate or amount of growth when grown at temperatures
which may vary as much as 10° or 15°, apparently an
adaptation to their normal environment. The disease-pro-
ducing organisms show much narrower limits for growth,
especially those which are difficult to cultivate outside the
body. For example, the bacterium of tuberculosis in man
scarcely develops beyond the limits of 2° or 3° from the
normal body temperature of man (37°), while the bacterium
of tuberculosis in birds grows best at 41° to 45°, the normal
for birds, and the bacterium of so-called tuberculosis of cold-
blooded animals at 14° to 18°.

Those bacteria whose optimum temperature is above 40°
are sometimes spoken of as the “thermophil” bacteria. The
fixing of the “thermal death-point,” that is, the tem-
perature at which bacteria are killed is a matter of great
practical importance in many ways and numerous deter-
minations of this have been made with a great many organ-
isms and by different observers. The factors which enter
into such determinations are so many and so varied that
unless all the conditions of the experiment are given, the
mere statements are worthless. It may be stated that all
young, actively growing (non-spore-containing) disease-pro-
ducing bacteria, when exposed in watery liquids and wm small
quantities are killed at a temperature of 60° within half an
hour. It is evident that this fact has very little practical
application, since the conditions stated are rarely, if ever,
fulfilled except in laboratory experiments. (See Sterilization
and Pasteurization, Chapter XIII.)

LIGHT.

Speaking generally, it can be said that light is destructive
to bacteria. Many growing forms are killed in a few hours
when properly exposed to direct sunlight and die out in
several days in the diffuse daylight of a well-lighted room.
Even spores are destroyed in a similar manner, though the
exposure must be considerably longer. Certain bacteria

5

66 GENERAL CONDITIONS FOR GROWTH

which produce colors may grow in the light, since the pig-
ments protect them. Some few kinds, like the sulphur bac-
teria, which contain a purplish-red pigment that serves
them to break up H.S, need light for their growth. Since
disease-producing bacteria are all injuriously affected by
light, the advantage of well-lighted habitations both for
men and animals is obvious.

OXYGEN SUPPLY.

Oxygen is one of the constituents of protoplasm and is
therefore necessary for all organisms. This does not mean
that all organisms must obtain their supply from free oxygen,
however, as animals and plants generally do. This fact
is well illustrated by the differences among bacteria in this
respect. Some bacteria require free oxygen for. their growth
and are therefore called aérobic bacteria or aérobes (sometimes
strict aérobes, though the adjective is unnecessary). Others
cannot grow in the presence of free oxygen and are therefore
named anaérobic bacteria or anaérobes (strict is unnecessary).
There are still other kinds which may grow either in the
presence of free oxygen or in its absence, hence the term
facultative anaérobes (usually) is applied to them. The dis-
tinction between facultative aérobe and facultative anaérobe
might be made. The former means those which grow best
in the absence of free oxygen, though capable of growing in
its presence, while the latter term means those which grow
best in the presence of free oxygen, but are capable of grow-
ing in its absence. The amount of oxygen in the atmos-
phere in which an organism grows may be conveniently
expresssed in terms of the oxygen pressure, 7. ¢., in milli-
meters of mercury. It is evident that the maximum, mini-
mum and optimum oxygen pressures for anaérobic bacteria
are the same, namely, 0 mm. Hg. This is true only for nat-
ural conditions, since a number of anaérobic organisms have
been gradually accustomed to increasing amounts of O, so
that by this process of training they finally grew in ordinary
air, that is, at an oxygen pressure of about 150 mm. Hg.
(Normal air pressure is 760 mm. Hg. and oxygen makes up

OXYGEN SUPPLY 67

one-fifth of the air.) The minimum O pressure for faculta-
tive anaérobes is also 0 mm. Hg. Some experiments have
been made to determine the limits for aérobes, but on a few
organisms only, so that no general conclusions can be
drawn from them. To illustrate: Bacillus subtilis (a com-
mon “hay bacillus’) will grow at 10 mm. Hg. pressure but
not at 5 mm. Hg. It will also grow in compressed oxygen
at a pressure of three atmospheres (2280 mm. Hg.), but not
at four atmospheres (3040 mm. Hg.), though it is not
destroyed.

Parodko has determined the oxygen limits for five com-
mon organisms as follows:

Minimum.

Maximum. Vol. Mm.

In atmospheres. Mm. Hg. per cent. Hg.

Bacillus fluorescens 1.94 to 2.51 1474 to 1908 0.00016 =0.0012

Sarcina lutea . » 2.51 to3.18 1908 to 2417 0.00015 =0.0011
Bacillus vulgaris . 38.63 to 4.35 2749 to 3306 0 0
Bacillus coli - 4.09 to 4.84 3108 to 3478 0 0
Bacillus prodigiosus . 5.45 to 6.32 3152 to 4800 0 0

These few instances do not disclose any general principles
which may be applied either for the growth or for the dis-
tinction of aérobes or facultative anaérobes.

It has been shown that compressed oxygen will kill some
bacteria but this method of destroying them has little or :
no practical value. Oxygen in the form of ozone, Os, is
rapidly destructive to bacteria, and this fact is applied prac-
tically in the purification of water supplies for certain cities
where the ozone is generated by electricity obtained cheaply
from water power. The same is true of oxygen in the
“nascent state” as illustrated by the use of hypochlorites
for the same purpose.

It was stated (p. 64) that certain thermophil bacteria in
the soil have an optimum temperature for growth in the air
which is much higher than is ever reached in their natural
habitat, and that they grow at a moderate temperature under
anaérobic conditions. It has been shown that if these organ-
isms are grown with aérobes or facultative anaérobes they
thrive at ordinary room temperature. These latter organ-

68 GENERAL CONDITIONS FOR GROWTH

isms by using up the oxygen apparently keep the tension
low, and this explains how such organisms grow in the soil.?

OSMOTIC PRESSURE.

Like all living cells bacteria are very susceptible to changes
in the density of the surrounding medium. If placed in a
medium less concentrated than their own protoplasm water
is absorbed and they “swell up;” while if placed in a denser
medium, water is given off and they shrink (plasmoptysis
or plasmolysis). Should these differences be marked or
the transition be sudden, the cell walls may even burst and
the organisms be destroyed. If the differences are not
too great or if the transition is made gradually, the organ-
isms may not be destroyed, but will either cease to grow and
slowly die out, or will show very much retarded growth, or
will produce abnormal cell forms. This is illustrated in the
laboratory in attempting to grow bacteria on food material
which has dried out. A practical application of osmotic
effects is in the use of a high percentage of sugar in preserv-
ing fruits, etc., and in the salting of meats. Neither the
cane-sugar nor the common salt themselves injure the bac-
teria chemically, but by the high concentration prevent their
development.

ELECTRICITY.

Careful experimenters have shown that the electric cur-
rent, either direct or alternating, has. no direct destructive
effect on bacteria. In a liquid medium the organisms may
be attracted to or repelled from one or the other pole or
may arrange themselves in definite ways between the poles
(galvanotaxis), but are not injured. However, electricity
through the secondary effects produced, may be used to
destroy bacteria. If the passage of the electric current
ancreases the temperature of the medium sufficiently, the bac-
teria will be killed, or if ¢njurious chemical substances are

1Tt is popularly supposed that in canning fruit, vegetables, meats, etc.,
all the air must be removed, since the organisms which cause ‘‘spoiling”’
cannot grow in a vacuum. The existence of anaérobic and facultative
anaérobic bacteria shows the fallacy of such beliefs.

MECHANICAL VIBRATION 69

formed (ozone, chlorine, acids, bases,’ etc.), the same result
will follow (see Ozone, pp. 67, 138 and 145).

RADIATIONS.

_ Réntgen or x-rays and radium emanations when prop-
erly applied to bacteria will destroy them. The practical
use of these agents for the direct destruction of bacteria in
diseases of man or animals is restricted to those cases where
they may be applied directly to the diseased area, since they
are just as injurious to the animal cell as they are to the
bacteria, and even more so. Their skilful use as stimuli to
the body cells to enable them to resist and overcome bacteria
and other injurious organisms or cell growths is an entirely
different function and will not be considered here.

PRESSURE.

Hydrostatic pressure up to about 10,000 pounds per
square inch is without appreciable effect on bacteria as has
been shown by several experimenters and also by finding
living bacteria in the ooze dredged from thé bottom of the
ocean at depths of several miles.

Préssures from 10,000 to 100,000 pounds show variable
effects. Some bacteria are readily killed and others, even
non-spore formers, are only slightly affected. The time
factor is important in this connection. The presence of
acids, even COs, or organic acids, results in the destruction
of most non-spore formers.

MECHANICAL VIBRATION.

Vibrations transmitted to bacteria in a liquid may be
injurious to them under certain circumstances. Some of
the larger forms like Bacillus subtis may be completely
destroyed by shaking in a rapidly moving shaking machine
in a few hours. Bacteria in liquids placed on portions of
machinery where only a slight trembling is felt have been
found to be killed after several days. Reinke has shown
that the passing of strong sound waves through bacterial
growths markedly inhibits their development.

CHAPTER VII.
CHEMICAL ENVIRONMENT.

REACTION OF MEDIUM.

Most bacteria are very susceptible to changes in the
degree of acidity or alkalinity of the medium in which they
grow. Some kinds prefer a slightly acid reaction, some a
slightly alkaline, and some a neutral (with reference to
litmus as indicator). The organism which is the commonest
cause of the souring of milk thrives so well in the acid
medium it produces that it crowds out practically all other
kinds, though its own growth is eventually stopped by too
much acid. Acid soils are usually low in numbers of bac-
teria and as a consequence produce poor crops. The disease-
producing bacteria as a class grow best in a medium which
is slightly alkaline.

Accurate determination, of limits have been made on but
few organisms. The reaction is a most important factor in
growing bacteria on artificial media (see Making of Media,
Chapter XVI).

INJURIOUS CHEMICAL SUBSTANCES.
(SEE DISINFECTION AND DISINFECTANTS, Chapter XIII.)

CHEMICAL COMPOSITION.

The chemical composition is subject to wide variation
chiefly for two reasons: First, the cell wall in most instances
seems to exert only a slight selective action in the absorption
of mineral salts so that their concentration within the cell
is very nearly that of the surrounding medium. Second,
the chief organic constituents vary remarkably with the
kind and amount of food material available—a rich protein

CHEMICAL COMPOSITION 71

pabulum increases the protein, a plentiful supply of carbo-
hydrates or of fat results in the storing of more fat, especially,
and vice versa. These facts must be borne in mind in con-
sidering the chemistry of bacteria.

Of the chemical elements known, only the following

seem to be essential in the structure of bacteria: carbon,
vhydrogen, oxygen, nitrogen, sulphur, phosphorus, chlorine,
potassium, calcium, magnesium, iron, manganese. Other
elements, as sodium, iodine, silicon, aluminum, lithium,
copper, etc., have been reported by different analysts, but
none of them can be regarded as essential, except possibly
in isolated instances.

These elements exist in the bacterial cell in a great variety
of combinations of which the most abundant is water. The
amount of water varies in different species from 75 to 90
per cent. of the total weight in growing cells, and is less in
spores. The amount of ash has been shown by different
observers to vary from less than 2 per cent. to as much as
30 per cent. of the dry weight. The following table com-
piled from various sources will give an idea of the relative
abundance of the different elements in the ash:

Sas SOs 7.64 per cent. (much more in sulphur bacteria)
Pas P2Os 18.14 * to 73.94 per cent.

cl Sy ee Ge od 2.29 e

K as K:0 11.1 es to 25.59 fe

Ca as CaO 12.64 ss to 14.0 S

Mgas MgO. . 0.77 es to 11.55 ss

Fe as Fe:Os 4 1.0 ss to 8.15 s (iron bacteria)
Mn. ‘ traces

As to the form in which the last six elements in the table
exist in the cell, little is known. The sulphur and phos-
phorus are essential constituents of various proteins. The
high percentage of phosphorus points to nuclein compounds
as its probable source.

The carbon and nitrogen, together with most of the
hydrogen and oxygen not united as water, make up the
‘great variety of organic compounds which compose the main
substances in the bacterial cell.

It has already been stated that the essential structures in
the bacterial cell are cell wall and protoplasm, including

72 CHEMICAL ENVIRONMENT

the nuclein. These differ markedly in chemical composition.
It is well known that the cell walls of green plants consist
largely of cellulose and closely related substances.! True
cellulose has been recognized in but very few bacteria.
(Sarcina ventriculi, Migula; Bacterium tuberculosis, Ham-
merschlag, Dreyfuss, Nishimura; Bacillus subtilis, Drey-
fuss; Bacterium xylinum, Brown; Bacterium acidi oxalic,
Banning and a few others.) It is certainly not an important
constituent of the cell wall in many. On the other hand,
hemicellulose and gum-like substances have been identified
in numerous organisms of this class as important constit-
uents of the cell wall and of the capsule which is probably
an outgrowth from the latter. Practically always associated
with these substances are compounds containing nitrogen.
One of these has been certainly identified as chitin or a
closely similar substance. Chitin is the nitrogenous sub-
stance which enters largely into the composition of the hard
part of insects, spiders and crustaceans. It is an interesting
fact to find this substance characteristic of these animals in
bacteria, as well as other fungi.

Though it is extremely difficult to separate the cell wall
of bacteria from the cell contents, in the light of our present
knowledge it can be stated that the cell walls are composed
of a carbohydrate body closely related to cellulose, though
not true cellulose, probably in close combination with
chitin.

Of the organic constituents of the cell contents the most
abundant are various proteins which ordinarily make up
about one-half of the dry weight of the entire cell. The
“Mycoproteid” of Nencki, 1879, and other earlier workers
is deserving of little more than historical interest, since these
substances were certainly very impure and probably con-
sisted of mixtures of several “proteins” in the more recent
sense.

1“By cellulose is understood a carbohydrate of the general formula
(CeHi00s) not soluble in water, alcohol, ether, or dilute acids but soluble
in an ammoniacal solution of copper oxide. It gives with iodine and sul-
phuric acid a blue color and with iodine zine chloride a violet and yields
dextrose on hydrolysis.’—H. Fischer.

CHEMICAL COMPOSITION 73

From later studies it seems probable that substances
resembling the albumin of higher forms do not occur in
bacteria, at least in appreciable quantities. Globulin has
been reported by Hellmich in an undetermined bacterium,
but is certainly not commonly found. The larger portion
of the protein is of a comparatively simple type, in fact,
consists of protamins most of which are in combination
with nucleic acid as nucleoprotamins. Practically all recent
workers find a high percentage of nuclein, both actually
isolated and as indicated by the amounts of purin bases—
xanthin, guanin, adenin—obtained, as well as by the abun-
dance of phosphorus in the ash, already mentioned. Some
of these nucleins have been shown to have poisonous
properties. ;

Closely related to but not identical with the proteins are
the enzymes and toxins which are formed in the cell and
exist there as endo-enzymes or endotoxins respectively.
These substances will be discussed later under the heading
“Physiological Activities of Bacteria” (Chapter XII).

Carbohydrates are not commonly present in the cell con-
tents, though glycogen has been observed in a few and a
substance staining blue with iodine in one or two others.
This latter substance was at first considered to be starch
“oranulose,”’ but is probably more closely related to glycogen.

Fats seem to be very generally present. The commoner
fats—tri-olein, tri-palmitin, tri-stearin have been found by
many analysts. The “acid-fast bacteria” are particularly
rich in fatty substances, especially the higher wax-like fats.
Lecithins (phosphorized fats) and cholesterins (not fats but
alcohols) have been repeatedly observed and probably occur
in all bacteria as products of katabolism.

Organic acids and esters occur as cell constituents but
will be discussed in connection with their more character-
istic oceurrences as products of bacterial activity, as will
also pigments which may likewise be intracellular in some
instances.

The following analysis of tubercle bacilli, from de
Schweinitz and Dorset, while not intended as typical for
all bacteria, still illustrates the high percentage of protein

74 CHEMICAL ENVIRONMENT

compounds which undoubtedly occurs in most, as well as
showing the large amount of fatty substance in a typical

“acid-fast” organism:

per cent. tuberculinic acid

8.5
24.5 *
In the dried } 23.0 st
organisms 8.3
26.5
9.2

nucleoprotamin

neucleoprotein ; 55.8 per cent. protein.
proteinoid

fat and wax

ash

CHAPTER VIII.
CHEMICAL ENVIRONMENT (ContinveEp).

GENERAL FOOD RELATIONSHIPS. METABOLISM.

Tue foregoing brief review of the chemical composition
of the bacterial cell illustrates the variety of compounds
which necessarily occurs, but affords no definite clue as to
the source of the elements which enter into these compounds.
These elements come from the material which the organism
uses as food. Under this term are included elements or
compounds which serve as building material, either for new
cell substance or to repair waste, or as sources of energy.

An organism which is capable of making use of an element
in the free state is said to be prototrophic for that particular
element. Thus aérobes and facultative anaérobes are proto-
trophic for O. The “root-tubercle bacteria” of leguminous
and other plants and certain free living soil organisms are
prototrophic for N.1

On the other hand, if the element must be secured from
compounds, then the organism is metatrophic in respect to
the element in question. Should the compound be inorganic,
the term autotrophic is applied to the organism, and hetero-
trophic if the compound is organic. It is very probable that
anaérobes, exclusive of a few nitrogen absorbers, are meta-
trophic for all the elements they utilize. With the excep-
tion of the anaérobes it seems that all bacteria are mizo-
trophic, that is, prototrophic for one or two elements and
auto- or heterotrophic for the others.?

1 The sulphur bacteria are partially prototrophic for 8; probably the iron
bacteria also for Fe. Some few soil bacteria have been shown to be capable
of utilizing free H, and it seems certain that the bacteria associated with
the spontaneous heating of coal may oxidize free C. So far as known no
elements other than these six are directly available to bacteria.

2 Only a few kinds of bacteria so far as known are proto-autotrophic.
The nitrous and nitric organisms of Winogradsky, which are so essential

in the soil, and which might have been the first of all organisms so far as
their food is concerned, and some of the sulphur bacteria are examples.

76 CHEMICAL ENVIRONMENT

Those bacteria whose food consists of dead material are
spoken of as saprophytes, while those whose natural habitat,
without reference to their food, is in or on other living
organisms are called parasites. The host is the organism in
or on which the parasite lives. Parasites may be of several
kinds. Those which neither do injury nor are of benefit to
the host are called non-pathogenic parasites or commensals;
many of the bacteria in the intestines of man and other ani-
mals are of this class. Those which do injury to the host
are called pathogenic or disease-producing, as the organisms
causing the transmissible diseases of animals and plants.
Finally, we have those parasites which are of benefit to and
receive benefit from the host. These are called symbionts
or symbiotic parasites and the mutual relationship symbiosis.
Certain of the intestinal bacteria in man and especially in
herbivorous animals are undoubted symbionts, as are also
the “root-tubercle bacteria’’ already mentioned.

It is also evident that all parasites that may be cultivated
outside the body are for the time saprophytic, hence the terms
strict parasites and facultative parasites, which should require
no further explanation.

The changes which the above-mentioned types of food
material undergo in the various anabolic and katabolic pro-
cesses within the cell are as yet but very slightly known.
Nevertheless there are a number of reactions brought about
by bacteria acting on various food materials, partly within
but largely without the cell which are usually described as
“physiological activities” or ‘biochemical reactions.’’ Some
of these changes are to be ascribed to the utilization of
certain of the elements and compounds in these materials
as tissue builders, some as energy-yielding reactions and still
others as giving rise to substances that are of direct benefit
to the organism concerned in its competition with other
organisms.

Though all of the twelve elements already mentioned are

1 The term pathogenic is also applied to certain non-parasitic saprophytic
bacteria whose products cause disease conditions, as one of the organisms
causing a type of meat-poisoning in man (Bacillus botulinus) and certain
fungi found on spoiled fodder or grain.

GENERAL FOOD RELATIONSHIPS—METABOLISM 77

essential for the growth of every bacterium, two of them
are of especial importance for the reason that most of the
“physiological activities” to be described in the next chap-
ters are centered around their acquisition and utilization.
These elements are carbon and nitrogen. Some few of the
special activities of certain groups have to do with one or
the other of the remaining nine, as will be shown later.
But generally speaking, when a bactertwm under natural con-
ditions secures an adequate supply of carbon and nitrogen, the
other elements are readily available in sufficient amount.

Carbon is necessary not only because it is an essential
constituent of protoplasm but because its oxidation is the
chief source of the energy necessary for the internal life of
the cell, though nitrogen and sulphur replace it in this
function with a few forms. This latter use of carbon con-
stitutes what may be called its respiratory function. Bac-
teria like other organisms in their respiration utilize oxygen
and give off carbon dioxide. The amount of the latter given
off from the cell in this way is very small as compared with
that which is frequently produced as an accompaniment of
other reactions (see Fermentation, next chapter). But there
is no doubt of its formation and it has been determined by a
few investigators. On account of this use of carbon, bacteria
require relatively large amounts of this element. One group
of bacteria concerned in the spontaneous heating of coal
seems to be able to use free carbon from this material.
Another group is said to be able to oxidize marsh gas, CH,,
and use this as its source of carbon. The nitrite, nitrate and
sulphur bacteria mentioned later utilize carbon dioxide
and carbonates as their carbon supply, and one kind has
been described which uses carbon monoxide. With these
few exceptions bacteria are dependent on organic compounds
for their carbon and cannot use CO, as green plants do.

The oxygen requirement is also high partly for the same
reason that carbon is, 2. ¢., respiration. Oxygen is alos
one of the constituents of protoplasm, and combined with
hydrogen forms water which makes up such a large part of
the living cell. Anaérobic bacteria are dependent on so-
called “molecular respiration” for their energy. That. is,

78 CHEMICAL ENVIRONMENT

through a shifting or rearrangement of the atoms in the
compounds used as food the oxidation of carbon is brought
about. Enzymes are probably responsible for this action.
Carbon dioxide is produced by anaérobes as well as aérobes,
and frequently in amounts readily collected. A carbohy-
drate is usually though not always essential for the growth
of anaérobes and serves them as the best source of energy.

Nitrogen is the characteristic element of living material.
Protoplasm is a chemical substance in unstable equilibrium
and nitrogen is responsible for this instability. No other
of the commoner elements is brought into combination with
such difficulty, nor is so readily liberated when combined
(all commercial explosives are nitrogen compounds). Bac-
teria, like other forms of protoplasm, require nitrogen. More
marked peculiarities are shown by bacteria with reference
to the sources from which they derive their nitrogen than
for carbon. Some can even combine the free nitrogen of the
air and furnish the only natural means of any importance
for this reaction. Some few forms (the nitrite and nitrate
formers, Chapter XI) obtain their energy from the oxida-
tion of inorganic nitrogen compounds, ammonia and nitrites
respectively, and not from carbon. These latter bacteria
use carbon from carbon dioxide and carbonates. A great
many bacteria can secure their nitrogen from nitrates but
some are restricted to organic nitrogen. Many bacteria
obtain their carbon from the same organic compounds from
which their nitrogen is derived.

Sulphur serves mainly to supply this element in the
protoplasmic structure. In some of the sulphur bacteria it
is a source of energy, since either free sulphur or H2S is
oxidized by them. Some of these bacteria can obtain their
carbon from CO, or carbonates, and their nitrogen from
nitrates or ammonium salts.

Whether the tron bacteria, belonging to the genus Creno-
thriz of the higher, thread bacteria, use this element or its
compounds as sources of energy is still a disputed question.
The evidence is largely in favor of this view.

Free hydrogen has been shown to be oxidized by some
forms which obtain their energy in this way.

GENERAL FOOD RELATIONSHIPS—METABOLISM 79

Whether there is a special class of phosphorus bacteria
remains to be discovered. That phosphorus is oxidized dur-
ing the activity of many bacteria is undoubted, but whether
this represents a source of energy or is the accidental
by-product of other activities is undetermined.

Practically nothing is known about the metabolism of the
other elements as such.

From the preceding brief review of the relation of certain
bacteria to some of the elements in the free state and from
the further fact that there is scarcely a known natural
organic compound which cannot be utilized by some kind
of bacterium, it is evident that this class of organisms has
a far wider range of adaptability than any other class, and
helps to explain their seemingly universal distribution.

As to the metabolism within the cell, no more is known than
is the case with other cells, nor even as much. The materials
used for growth and as sources of energy are taken into the
cell, built up into various compounds some of which have
been enumerated and in part broken down again. Carbon
dioxide and water are formed in the latter process. What
other katabolic products occur it is not easy to determine.
Certainly some of the substances mentioned in the next
chapters are such products but it is not, always possible to
separate those formed inside the cell from those formed
outside. Perhaps most of the latter should be considered
true metabolic products. It would seem that on account
of the simplicity of structure of the bacterial cell and of the
compounds which they may use as food they would serve
as excellent objects for the study of the fundamental prob-
lems of cell metabolism. Their minuteness and the nearly
impossible task of separating them completely from the
medium in or on which they are grown makes the solution
of these problems one of great difficulty.

When all of the environmental conditions necessary for
the best development of a given bacterium are fulfilled, it
will then develop to the limit of its capacity. This develop-
ment is characterized essentially by its reproduction, which
occurs by transverse division. The rate of this division
varies much with the kind even under good conditions.

80 CHEMICAL ENVIRONMENT

The most rapid rate so far observed is a division in eighteen
minutes. A great many reproduce every half-hour and this
may be taken as a good average rate. If such division could
proceed without interruption, a little calculation will show
that in about sixty-five hours a mass as large as the earth
would be produced.

Starting with 1 coccus, lu in diameter,
its volume =0.0000000000005 c.c.

} hour = 2

lhour = 4

2 hours = 16

4 hours = 256

5 hours = 1024 = 103 +
15 hours = 1,000,000,000 = 108 = 0.5 cc.
35 hours = 102+ = 500.0 cu.m.

About 65 hours = 2 x 10+ = 5x 10 cu.m. = a mass as large

as the earth.

Such a rate of increase evidently cannot be kept up long
on account of many limiting factors, chief of which is the
food supply.

The foregoing calculation is based on the assumption that
the organism divides in one plane only. If it divides in 2 or
3 planes, the rate is much faster, as is shown by the following
formule, which indicate the theoretical rate of division.

s
a

number of bacteria after a given number of divisions
number at the beginning, and n = number of divisions.

1 plane division S = 2"a
Dos « § = 2"
3 «8 = 23%

With two-plane or three-plane division, assuming that each
organism attains full size, as was assumed in the first calcu-
lation, the “mass as large as the earth” would be attained
in about 32 hours and 22 hours respectively.

This extraordinary rate of increase explains in large
measure why bacteria are able to bring about such great
chemical changes in so short a time as is seen in the rapid
“spoiling” of food materials, especially liquids. The reac-
tions brought about by bacteria on substances which are
soluble and diffusible are essentially “surface reactions.”

GENERAL FOOD RELATIONSHIPS—METABOLISM 81

The material diffuses into the cell over its entire surface
with little hindrance. The bacteria are usually distributed
throughout the medium, so that there is very intimate con-
tact in all parts of the mass which favors rapid chemical
action. The following calculation illustrates this:

The volume of a coccus ly in diameter is 0.5236 x 1078 c.c.
The surface of a coccus 1p in diameter is x 10-8 sq. cm.

It is not uncommon to find in milk on the point of sour-
ing 1,000,000,000 bacteria per c.c.

Assuming these to be cocci of 1u diameter the volume of
these bacteria in a liter is only 0.05-c.c. or in the liter there
would be 19999 parts of milk and only 1 part bacteria. ‘The
surface area of these bacteria is 3141.6 sq. cm. With this
large surface exposed, it is not strange that the change from
“on the point of souring” to “sour” occurs within an hour
or less.

Although large numbers of bacteria can and do cause
great chemical changes the amount of material actually
utilized for maintenance of the cell is very slight, infinites-
imal almost, and yet is fairly comparable to that required
for man, as is illustrated by the following computations:

E. Kohn has shown that certain water bacteria grew well
in water to which there was added per liter 0.000002 mg.
dextrose, 0.00000007 mg. (NH4)2SO, and 0.0000000007 mg.
(NHa)eHPO;. The bacteria numbered about 1000 per c.c.
Taking the specific gravity at 1 (a little too low) the mass
of the bacteria in the liter was about 0.001 mg. Hence the
bacteria used 0.002 of their weight of carbohydrate and
0.00007 of ammonium sulphate. A 150-pound (75-kilo) man
can live on 375 g. of sugar (0.005 of his weight) and 52.5 g. of
protein (0.0007 of his weight).

CHAPTER IX.
PHYSIOLOGICAL ACTIVITIES.

Tue objects in view in the discussion of the “ physiological
activities” of bacteria in this and subsequent chapters are
to familiarize the student to some extent with the great
range of chemical changes brought about by these minute
organisms, to show their usefulness, even their necessity,
and to impress the fact that it is chiefly by a careful study
of these “activities” that individual kinds of bacteria are
identified. It should always be borne in mind that the bac-
teria, in bringing about these changes which are so charac-
teristic in many instances, are simply engaged in their own
life struggle, in securing the elements which they need for
growth, in liberating energy for vital processes, or occa-
sionally in providing conditions which favor their own
development and hinder that of their competitors.

FERMENTATION OF CARBOHYDRATES.

By this is meant the changes which different carbohy-
drates undergo when subjected to bacterial action.

These changes are marked chiefly by the production of
gas or of acid.’ The former is called “ gaseous fermentation”
the latter “acid fermentation.” The gases commonly pro-

1The term “fermentation’’ was originally used to denote the process
which goes on in fruit juices or grain extracts when alcohol and gas are formed.
Later it was extended to apply to the decomposition of almost -any organic
substance. In recent years the attempt has been made to give a chemical
definition to the word by restricting its use to those changes in which, by
virtue of a ‘‘wandering” or rearrangement of the carbon atoms “‘new sub-
stances are formed which are not constituents of the original molecule.”
It may be doubted whether this restriction is justified or necessary. <A
definition is at present scarcely possible except when the qualifying adjective
is included as ‘‘alcoholic fermentation,’’ ‘‘ammoniacal fermentation,” ‘lactic
acid fermentation,” etc.

FERMENTATION OF CARBOHYDRATES 83

duced are carbon dioxide (CO,) hydrogen and marsh gas
(CH,). Other gases of the paraffin series may also be formed
as ethane (C2H¢), acetylene (C2Hs), etc. CO2 and H are the
ones usually formed from sugars by the few gas-forming

‘

Fig. 59.—Cylinder to show Fig. 60.—A burning natural gas
the formation of gas by bacteria. well at night. From a photograph
The gauge shows 265 pounds. colored.

It went beyond 500 pounds.

84 PHYSIOLOGICAL ACTIVITIES

bacteria which produce disease, though even here some CH,
is present. The common Bacillus coli forms all three, though
the CH, is in smallest quantity.

In the fermentation of the polysaccharids—starch and
especially cellulose and woody material—large amounts of
CH occur, particularly when the changes are due to anaé-
robic bacteria. This phenomenon may be readily observed
in sluggish streams, ponds and swamps where vegetable
matter accumulates on the bottom. The bubbles of gas
which arise when the mass is disturbed explode if a lighted
match is applied to them.

The author has conducted a number of experiments to
demonstrate this action as follows: Material taken from the
bottom of a pond in the fall after vegetation had died out
was packed into a cylinder five feet long and six inches in
diameter, water was added to within about 2 inches of the
top. After leaving them open for a few days to permit all
the dissolved oxygen to be used up by the aérobes, the cylin-
ders were tightly capped and allowed to stand undisturbed.
Pressure gauges reading to 500 lbs. were attached (Fig. 59).
At the end of six months the gauge showed a pressure beyond
the limits of the readings on it. Most of the gas was col-
lected and measured 146 liters. An analysis of portions
collected when about one-half had been allowed to escape
showed the following composition, according to analysis by
Prof. D. J. Demorest of the Department of Metallurgy:

CO2 3 +8 . 18.6 per cent.
CHa oe a o 10k a

H F 1.0

N ¥ il x : - oe) 4.8 3

In the author’s opinion natural gas and petroleum have
been formed in-this way! (Figs. 60 and 61).

One of the very few practical uses of the gaseous fermen-
tation of carbohydrates is in making “salt rising” bread.
The “rising” of the material is due not to veasts but to the
formation of gas by certain bacteria which are present on
the corn meal or flour used in the process (Fig. 62).

1 See “Oil and Gas in Ohio,’ Bownocker: Geological Survey of Ohio, Fourth
Series, Bull. I, pp. 313-314.

FERMENTATION OF CARBOHYDRATES 85

Another is in the formation of the “holes” or “eyes” so
characteristic of Swiss and other types of cheese (Fig. 63).

A great many organic acids are formed during the “acid
fermentation” of carbohydrates by bacteria. Each kind of

Fic. 61.—A “flowing” oil well.

bacterium, as a rule, forms several different acids as well as
other substances, though usually one is produced in much
larger amounts, and the kind of fermentation is named from
this acid. One of the commonest of these acids is lactic.

86 PHYSIOLOGICAL ACTIVITIES

The “lactic acid bacteria” form a very large and important
roup and are indispensable in many commercial processes.
p P

Fia. 62.—A loaf of ‘salt rising’’ bread. The porous structure is due to the
gas formed by bacilli and not by yeasts.

In the making of butter the cream is first “ripened,” as is
the milk from which many kinds of cheese are made (Fig. 64).

Fig. 63.—Ohio Swiss cheese. The “eyes” are due to gas formed by bacteria
during the ripening of the cheese.

The chief feature of this “ripening” is the formation of lac-
tic acid from the milk-sugar by the action of bacteria, and a

FERMENTATION OF CARBOHYDRATES 87

similar change occurs in the popular “Bulgarian fermented
milk.” The reaction is usually represented by the equation:

Milk-sugar. Lactic acid.
CrH2On + H:O + (bacteria) = 4C3H6Os

It is not probable that the change occurs quantitatively as
indicated, because a number of other substances are also
formed. Some of these are acetic and succinic acids and
alcohol. Another industrial use of this acid fermentation is

Fig. 64.—A cream ripener. In this apparatus cream is ‘“'ripened,” 7. e.,
undergoes lactic acid fermeftation, preparatory to making it into butter.

in the preparation of “sauer kraut.’ These bacteria are
chiefly anaérobic and grow best in a relatively high salt -
concentration. They occur naturally on the cabbage leaves.

In the formation of ensilage (Fig. 65) the lactic acid bac-
teria play a very important part, as they do also in “sour
mash’’ distilling, and in many kinds of natural “pickling.”
_ In fact, whenever green vegetable material “sours” spon-
taneously, lactic acid bacteria are always present and
account for a large part of the acid. This property of lactic

88 PHYSIOLOGICAL ACTIVITIES

acid formation is also taken advantage of in the preparation
of lactic acid on a commercial scale in at least one plant in
this country.

Acetic acid is another common product of acid fermenta-
tion. However, in vinegar making the acetic acid is not |
formed directly from the sugar in the fruit juice by bacteria.
The sugar is first converted into alcohol by yeasts, then the

Fie. 65.—Filling a silo on the University farm.

alcohol is oxidized to acid by the bacteria (Fig. 66). The
reaction may be represented as follows:

Dextrose. Ethyl] alcohol. Acetic acid.
CeHOs = 2C2H,OH + 2CO2
C:H;OH + O2 + (bacteria) = CHsCOOH + H:O0

Butyric acid is generally produced where fermentation of
carbohydrate occurs under anaérobic conditions. Some of
the “strong” odor of certain kinds of cheese is due to this
acid which is formed partly from the milk-sugar remaining
in the cheese. Most of it under these conditions comes from
the proteins of the cheese and especially from the fat
(see page 90).

FERMENTATION OF CARBOHYDRATES 89

As has been indicated alcohol is a common accom-
paniment of most acid fermentations, as are the esters of
acids other than the chief product. Bacteria are not used
in a commercial way to produce alcohol, however, as the
yield is too small. There are some few bacteria in which
the amount of alcohol is prominent enough to call the pro-

LTR

Wipe OA LLL
dl Vie

Nigel,

iH ul 1

| :
Hh i
i i l
il i
Mi

|

Fig. 66.—A vinegar ripener. The tank shown opened at the side is filled
with a special type of beech shavings which thus provide a very large surface.
The apple juice which has been previously fermented with yeast, which
converts the sugar into alcohol, is allowed to trickle through the openings
at the top over the shavings. The acetic acid bacteria on the shavings
rapidly oxidize the alcohol to acetic acid. The vinegar is drawn off below.

cess an “alcoholic fermentation” rather than an acid one.
In brewing and distilling industries, yeasts are used to make
the alcohol, though molds replace them in some countries
(“sake” and “arrak” from rice).

Under ordinary conditions the carbohydrate is never com-
pletely fermented, since the accumulation of the product—

90 PHYSIOLOGICAL ACTIVITIES

acid—stops the reaction. If the acid is neutralized by the
addition of an alkali—calcium or magnesium carbonate is
best—then the sugar may all be split up. Where such fer-
mentation occurs under natural conditions, the products
are further split up, partly by molds and partly by acid-
destroying bacteria into simpler acids and eventually to
carbon dioxide and water, so that the end-products of the
complete fermentation of carbohydrate material in nature
are carbon dioxide, hydrogen, marsh gas, and water.

In all of these fermentations the bacteria are utilizing the
carbon both as building material and for oxidation and the
fermentations are incidental to this use. As a rule, the acid-
forming bacteria can withstand a higher concentration of
acid than the other bacteria that would utilize the same
material, and in a short time crowd out their competitors
or inhibit their growth, and thus have better conditions for
their own existence, though finally their growth is also
checked by the acid.

SPLITTING OF FATS.

The splitting of fats into glycerin and the particular acid
or acids involved may be brought about by bacteria. An
illustration is the development of rancidity in butter at
times and the “strong” odor of animal fats on long keeping
and of many kinds of cheese—“ Limburger”’’ in this country.
Generally speaking, however, fats are not vigorously attacked,
as is illustrated by the difficulties due to accumulation of
fats in certain types of sewage-disposal works. The chemi-
cal change is represented by the equation:

Fat. Glycerin. Fatty acid.
CaHs (CaHen-102)3 + 3 HO = CsHs (OH)s + 3 (CrHen Ov).

CHAPTER X.
PHYSIOLOGICAL ACTIVITIES (ContInvEp).

PUTREFACTION OF PROTEINS.

TueE word “ putrefaction” is now restricted to the action of
bacteria on the complex nitrogen-containing substances, pro-
teins, and their immediate derivatives. The process is
usually accompanied by the development of foul odors.

Bacteria make use of proteins chiefly as a source of nitro-
gen, but also as a source of carbon and other elements.
Proteins contain nitrogen, carbon, hydrogen, oxygen, sul-
phur and frequently phosphorus. Some of the metals—
potassium, sodium, calcium, magnesium, iron and manganese
and the non-metal chlorine—are nearly always associated
with them more or less intimately. Since these bodies
are the most complex of natural chemical substances it
follows that the breaking up of the molecule to secure a
part of the nitrogen gives rise to a great variety of products.

There are marked differences among bacteria in their
ability to attack this class of compounds. Some can break
up the most complex natural proteins such as albumins,
globulins, glyco-, chromo-, and nucleoproteins, nucleins and
albuminoid derivatives like gelatin. The term saprogenic
(campos = rotten) is sometimes applied to bacteria which
have this power. These proteins are large, moleculed and
not diffusable, so that the first splitting up that they undergo
must occur outside the bacterial cell. The products of this
first splitting may diffuse into the cell and be utilized there.
The bacteria of this class attack not only these proteins in
the natural state or in solution, but also in the coagulated
state. The coagulum becomes softened and finally changed
into a liquid condition. The process when applied to the
casein of milk is usually called “digestion,” also when coagu-
lated blood serum is acted on. In the latter case the serum

92 PHYSIOLOGICAL ACTIVITIES

is more commonly said to be “liquefied” as is the case when
gelatin is the substance changed. Most of these bacteria
have also the property of coagulating or curdling milk in an
alkaline medium, and then digesting the curd.

A second class of bacteria has no effect on the complex
proteins just mentioned but readily attacks the products of
their first splitting, 7. ¢., the proteoses, peptones, polypep-
tids and amino-acids. They are sometimes called saprophilic
bacteria.

Other bacteria derive their nitrogen from some of the
products of the first two groups, and still further break down
the complex protein molecule. Under normal conditions
these various kinds of bacteria all occur together and thus
mutually assist one another in what is equivalent to a sym-
biosis or rather a metabiosis, a “successive existence,’ one
set living on the products of the other. The result is the
complete splitting up of the complex protein molecule. A
part of the nitrogen is built up into the bodies of the bacteria
which are using it as food. A part is finally liberated as
free nitrogen or as ammonia after having undergone a series
of transformations many of which are still undetermined.

One class of compounds formed received at one time
much attention because they were supposed to be respon-
sible for a great deal of illness. These are the “ptomaines,”
basic nitrogen compounds of definite composition—amines—
some few of which are poisonous, most of them not. The
basic character of ptomaines may be understood if they be
regarded as made up of one or more molecules of ammonia
in which the hydrogen has been replaced by alkyl or other

radicals. Thus ammonia (NH;) may be represented as
/ ; : : /CHs 7 .
piven the simplest ptomaine is es , methylamine, in

which one H is replaced by methyl, a gaseous ptomaine;
CH:

with two hydrogens replaced by methyl, NCH, dimethyl-

amine, a gas at ordinary temperature also; and trimethyl-

CH;
amine, NEC, a liquid. All three of these occur in her-
3

PUTREFACTION OF PROTEINS 93

ring brine and are responsible for the characteristic odor
of this material. Putrescin and cadaverin—tetramethylene
diamine, and pentamethylenediamine respectively —
occur generally in decomposing flesh, hence the names.
They are only slightly poisonous. One of the highly poi-
sonous ptomaines is neurin C;Hi;NO or C2H3;3N(CH3);0H
= trimethyl-oxyethyl ammonium hydroxide. This is a
stronger base than ammonia, liberating it from its salts.
Numerous other ptomaines have been isolated and described.
These bodies were considered for a long time to be the cause
of various kinds of “meat poisoning,” “ice cream poison-
ing,” “cheese poisoning,” etc. It is true that they may
sometimes cause these conditions, but they are very much
rarer than the laity generally believe. Most of the “meat
poisonings” in America are due, not to ptomaines, but to
infections with certain bacilli of the Bacillus enteritidis
group. Occasionally a case of poisoning by the true toxin
(see Chapter XII) of Bacillus botulinus occurs.

As ptomaines result from the putrefaction of proteins, so
they are still further decomposed by bacteria and event-
ually the nitrogen is liberated either as free nitrogen or
as ammonia.

Another series of products are the so-called aromatic
compounds—phenol (carbolic acid) various cresols, also
indol and skatol or methyl indol (these two are largely
responsible for the characteristic odor of human feces). All
of these nitrogen compounds are attacked by bacteria and
the nitrogen is eventually liberated, so far as it is not locked
up in the bodies of the bacteria, as free nitrogen or as
ammonia.

The carbon which occurs in proteins accompanies the
nitrogen in many of the above products, but also appears in
nitrogen-free organic acids, aldehydes and alcohols which
are all eventually split up, so that the carbon is changed to
carbon dioxide or in the absence of oxygen partly to marsh
gas.

The intermediate changes which the sulphur in proteins
undergoes are not known, but it is liberated as sulphuretted
hydrogen (HS) or as various mercaptans (all foul-smelling),

94 PHYSIOLOGICAL ACTIVITIES

or is partially oxidized to sulphuric acid. Some of the H.S
and the sulphur of the mercaptans are oxidized by the
sulphur bacteria to free sulphur and finally to sulphuric
acid.

Phosphorus is present especially in the nucleoproteins
and nucleins. Just what the intermediate stages are, or
whether there are any, so far as the phosphorus is concerned,
in the splitting up of nucleic acid by bacterial action is not
determined. The phosphorus may occur as phosphoric acid
in such decompositions, or when the conditions are anaérobic,
as phosphine (PHs), which burns spontaneously in the air
to phosphorus pentoxide (P,O;), and water.!

The hydrogen in proteins appears in the forms above
indicated: H,C, H3N, H3P, H.S, H.O and as free H. The
oxygen as CO, and HO.

In the breaking down of the complex protein molecule
even by a single kind of bacterium there is not a perfect
descending scale of complexity as might be supposed from
the statement that there result proteoses, peptones, poly-
peptids, amino-acids. These substances do result, but at
the time of their formation simpler ones are formed also,
even CO2, NH; and HS. It appears that the entire mole-
cule is shattered in such a way that less complex proteins
are formed from the major part, while a minor portion breaks
up completely to the simplest combinations possible. A
more complete knowledge of these decompositions will aid
in the further unravelling of the structure of proteins. The
presence or absence of free oxygen makes a difference in the
end-products, as has been indicated. There are bacteria
which oxidize the ammonia to nitric acid and the H.S to
sulphuric acid. (See Oxidation, Chapter XI.) Bacteria
which directly oxidize phosphorus compounds to phosphoric
acid have not been described. It does not seem that such
are necessary since this is either split off from nucleic acid

1 It is probable that this is the way ‘Jack o’lanterns” or ‘Will
o’ the wisps’’ are ignited. Marsh gas is produced as above outlined from
the vegetable and animal matter decomposing in swampy places under
anaérobic conditions and likewise phosphine. These escape into the air
and the “spontaneous combustion”’ of the phosphine ignites the marsh gas.

PUTREFACTION OF PROTEINS 95

or results from the spontaneous oxidation of phosphine when
this is formed under anaérobic conditions.

Not only are proteins decomposed as above outlined, but
also their waste products, that is, the form in which their
nitrogen leaves the animal body. This is largely urea in
mammals, with much hippuric acid in herbivorous animals
and uric acid in birds and reptiles. These substances yield
NH, CO, and H.O with a variety of organic acids as inter-
mediate products in some cases. The strong odor of
ammonia in stables and about manure piles is the every-day
evidence of this decomposition.

Where the putrefaction of proteins occurs in the soil with
moderate amounts of moisture and free access of air a large
part of the products is retained in the soil. Thus the
ammonia and carbon dioxide {in the presence of water form
ammonium carbonate; the nitric, sulphuric and phosphoric
acids unite with some of the metals which are always present
to form salts. Some of the gases do escape and most where
the oxygen supply is least, since they are not oxidized.

The protein-splitting reactions afford valuable tests in
aiding in the recognition of bacteria. In the study of patho-
genic bacteria the coagulation and digestion of milk, the
digestion or liquefaction of blood serum, the liquefaction of
gelatin and the production of indol and H2S are those usually
tested for. In dairy bacteriology the coagulation of milk
and the digestion of the casein are common phenomena.
Most bacteria which liquefy gelatin also digest blood serum
and coagulate and digest milk, though there are exceptions.
In soil bacteriology the whole range of protein changes is of
the greatest importance.

The three physiological activities already discussed explain
how bacteria break down the chief complex, energy-rich
substances—carbohydrates, fats and proteins which consti-
tute the bulk of the organic material in the bodies of
plants and animals, as well as the waste products of the
latter, into energy-free compounds like carbon dioxide, water,
ammonia, nitric, sulphuric and phosphoric acids—mineralize
them, as is frequently said. By so doing the bacteria act
as the great scavengers of nature removing the dead animal

96 PHYSIOLOGICAL ACTIVITIES

Nuclein Maa.

Decomposition
ce 7 bacteria
oe
Animals a Unknown PH,
wt oxidizes
ee compo nat Pon aneoUslt
Nuclein of plant ao
ells Phosphoric
agiad

Green plants

Phosphates in the soit

Fig. 67.—Diagram to illustrate the circulation of phosphorus through the
agency of bacteria.

various CLompounds Decomposition
a bacteria

Various
’
Animal )~ Cc
respitation compounds
ie eventually
to

other C chnpounds

Green
BES NG

in”,
the air

Fic. 68.—Diagram to illustrate the circulation of carbon through the
agency of bacteria.

Dead aniyrGl protein position bacteria

_ at
: _-—nttial waste,
Aninlals — urea, eter” NH. tompounds
in soil

Free N taken
up by Sree living
N absorbers and

5 ., root tubercle bacteri
rite bacteria octeree

as a
Dead; plant protein

Plant protein - Ni

Nifrites

Gréen plants Nitrate bacteria

Nitrates in soil

Fig. 69.—Diagram to illustrate the circulation of nitrogen through the
agency of bacteria.

PUTREFACTION OF PROTEINS 97

and vegetable matter of all kinds which but for this action
would accumulate to such an extent that all life, both on
land and in the water, must cease. It is further to be noted
that not only is all this dead organic matter removed, but
it is converted into forms which are again available for
plant growth. Carbon dioxide forms the source of the car-
bon in all green plants, hence in all animals; the sulphates
and phosphates are likewise taken up by green plants and
built up again into protein compounds; the ammonia is not
directly available to green plants to any large extent but
is converted by the nitrifying bacteria (Chapter XI) into

Dead anjinal protein Decompasttwon bacteria
ea

_
-
os
=

Animals Dead plant 4,
protein

Plant protein Sulphur bacteria

. ae
Greemplants ‘ae bacteria

Sulphates in the soil

Fic. 70.—Diagram to illustrate the circulation of sulphur through the
agency of bacteria.

nitrates which is the form in which nitrogen is assimilated
by these higher types. Even the free nitrogen of the air is
taken up by several kinds of bacteria, the symbiotic “root-
tubercle bacteria” of leguminous and other plants, and some
free-living forms, and made available. Hence bacteria are
indispensable in nature, especially in keeping up the circu-
lation of nitrogen. They are also of great service in the
circulation of carbon, sulphur, and phosphorus. Though
some few kinds cause disease in man and animals, if it were
not for the saprophytic bacteria above outlined, there could
be no animals and higher plants to acquire these diseases.

CHAPTER XI.
PHYSIOLOGICAL ACTIVITIES (ContINvED).

PRODUCTION OF ACIDS.

THE production of organic acids has been sufficiently dis-
cussed in preceding chapters. It should be noted that not
only these in great variety are produced by bacteria but that
under certain conditions mineral acids, such as nitric, sul-
phuric and phosphoric may be formed (see Oxidation, p. 102).
Acid production is of great value in the identification of
bacteria in dairy and soil work and in connection with
certain types of pathogenic bacteria.

GAS PRODUCTION.

It will be sufficient merely to enumerate collectively the
various gases mentioned in preceding paragraphs and to
state that those commonly observed in the study of patho-
genic bacteria are the first six mentioned. Most of them
come in in dairy work either in the study of bacteria causing
milk and cheese “failures” or as affecting the flavors of
butter or cheese. In the study of soil organisms, any or all
of them are liable to be of importance. The gases are:
CO:, H, CH, N, NHs, HS, gaseous mercaptans, gaseous
ptomaines, volatile fatty acids, ethereal salts or esters and
others, both of pleasant and of foul odor, but of unknown
composition.

PRODUCTION OF ESTERS.

The production of esters, as mentioned in Chapters IX
and X, of various alcohols and aldehydes are activities
which are sometimes of value in the study of bacteria, but
need not be further discussed.

*

PHOSPHORESCENCE OR PHOTOGENESIS 99

PRODUCTION OF “AROMATIC” COMPOUNDS.

These have been mentioned in discussing the putrefac-
tion of proteins, as indol, skatol, phenol and various cresols.
Of these only the first is ordinarily tested for in the study
of bacteria, though others of the group become of value in
certain special cases.

Fig. 71.—Culture of phosphorescent bacteria in an Ehrlenmeyer flask
photographed by their own light. Time of exposure twelve hours. (Molisch,
from Lafar.)

PHOSPHORESCENCE OR PHOTOGENESIS.

This is a most interesting phenomenon associated with
the growth of some bacteria. The “fox fire” frequently seen
on decaying wood which is covered with a slimy deposit is
most commonly due to bacteria, though also to other fungi.
Phosphorescent bacteria are very common in sea water,

100 PHYSIOLOGICAL ACTIVITIES

hence they are frequently found on various sea foods, espe-
cially when these are allowed to decompose, such as fish,
oysters, clams, etc. The light is due to the conversion of the
energy of unknown easily oxidizable compounds directly into
visible radiant energy through oxidation without appreciable
quantities of heat. The light produced may be sufficient
to tell the time on a watch in absolute darkness, and also
to photograph the growths with their own light, but only
after several hours’ exposure (Fig. 71). None of the phos-
phorescent bacteria so far discovered produce disease in the
higher animals or man.

PRODUCTION OF PIGMENT OR CHROMOGENESIS.

One of the most striking results of bacterial activity is
this phenomenon. The particular color which results may
be almost any one throughout the range of the spectrum,
though shades of yellow and of red are of more frequent
occurrence.

In the red sulphur bacteria the “bacteriopurpurin” which
they contain appears to serve as a true respiratory pigment
in a manner similar to the chlorophyl in green plants, except
that these bacteria oxidize HS in the light as a source of
energy instead of splitting up CO.. The red pigment pro-
duced by certain bacteria has been shown to have a capacity
for combining with O resembling that of hemoglobin,’ and
some investigators have believed that such bacteria do
store O in this way for use when the supply is diminished.
With these few exceptions the pigments seem to be merely
by-products of cell activity which are colored and have no
known function.

The red sulphur bacteria above mentioned and one or
two other kinds retain the pigments formed within the cell.
Such bacteria are called chromophoric as distinguished from
the chromoparic bacteria whose pigment lies outside the
cell.

The chemical composition of no bacterial pigment has
been determined up to the present. Some are soluble in
water, as shown by the discoloration of the substances on

REDUCING ACTIONS 101

which they grow. Others are not soluble in water but are
in alcohol, or in some of the fat solvents as ether, chloro-
form, benzol, etc. These latter are probably closely related
to the lipochromes or “fat colors” of higher plants and
animals. Attempts have been made to render the produc-
tion of pigments a still more reliable means of identification
of species of bacteria through a careful examination of the
spectra of their solutions, but such study has not as yet led
to any valuable practical results.

The production of pigment depends on the same general
factors which determine the growth of the organism but does
not necessarily run parallel with these. It is especially influ-
enced by the oxygen supply (only a very few organisms
are known which produce pigment anaérobically—S pirillum
rubrum is one); by the presence of certain food substances
(starch, as in potato, for many bacteria producing yellow and
red colors; certain mineral salts, as phosphates and sulphates,
for others); by the temperature (many bacteria cease to
produce color at all if grown at body temperature, 37°—
Bacillus prodigiosus—or if grown for a longer time at tem-
peratures a few degrees higher).

REDUCING ACTIONS.

Reduction of nitrates to nitrites or to ammonia or even
to free nitrogen is brought about by a great many different
kinds of bacteria. In many instances this phenomenon is
due to a lack of free oxygen, which is obtained by the
bacteria from these easily reducible salts. In other cases a
portion of the nitrogen is removed to be used as food material
in the building up of new protein in the bacterial cell. This
latter use of the nitrogen of nitrates by bacteria might
theoretically result in considerable loss of “available nitro-
gen” in the soil as has actually been shown in a few experi-
ments. The reduction of nitrates as above mentioned would
also diminish this supply, but probably neither of these
results has any very great practical effect on soil fertility.
The building up of protein from these mineral salts by bac-
teria in the intestines of herbivorous animals has been sug-

102 PHYSIOLOGICAL ACTIVITIES

gested by Armsby as a considerable source of nitrogenous
food, and this suggestion appears possible.

The liberation of nitrogen from nitrates or nitrites, either
as free nitrogen or as ammonia, is spoken of as “denitrifica-
tion,” though this term was formerly applied to such libera-
tions from compounds of nitrogen génerally even from
proteins.

Certain bacteria may also reduce sulphates and other
sulphur compounds to HS, a phenomenon frequently
observed in sewage and likewise of importance in the soil.
It is possible that phosphates may be similarly reduced.
Further and more careful study of the reducing actions of
bacteria is needed.

OXIDATION.

As has been stated in discussing the respiration of bac-
teria (Chapter VIII) most of these organisms gain their
energy through the oxidation of carbon in various forms,
chiefly organic, so that CO, is a product of the activity of
nearly all bacteria. Some few oxidize CO to COs, others
CH, and other paraffins to CO2for this purpose. One class of
bacteria even oxidizes H in small amounts for its energy and
uses the carbon dioxide of the air or traces of organic carbon
in the air as a source of carbon for “building” purposes.

One of the familiar oxidations of organic carbon is that
of the acetic acid bacteria in the making of vinegar. These
oxidize the alcohol which results from the action of yeast
to acetic acid according to the formula CH;CH,OH+0O,=
CH;COOH+H.O (see Fig. 66).

Of the various phenomena of oxidation due to bacteria,
the formation of nitrites and nitrates has the greatest prac-
tical importance, since it is by this means that the ammonia
which results from the decomposition of animal and vege-
table tissue and waste products is again rendered available
to green plants as food in the form of nitrates. Practically
all the nitrates found in nature, sometimes in: large quanti-
ties, are formed in this way. There are two distinct kinds
of bacteria involved. One, the nitrous bacteria, oxidizes
the ammonia to nitrous acid which forms nitrites with bases,

OXIDATION 103

and the other, the nitric bacteria, oxidizes the nitrous to
nitric acid, giving nitrates with bases. A striking peculiarity
of these two classes of organisms is that they may live entirely
on inorganic food materials, are proto-autotrophic, proto-
trophic for oxygen (aérobic) and autotrophic for the other
elements. Their carbon is derived from CO, or carbonates.
The importance of such organisms in keeping up the supply
of nitrates in the soil can scarcely be overestimated.

Fig. 72.—Sprinkling filters of the Columbus sewage-disposal plant—
devices which provide a good supply of oxygen for the bacteria that
oxidize the organic matter in the sewage.

The oxidation of the H2S, which is formed in the putre-
faction of proteins, to free S by the sulphur bacteria and the
further oxidation of this free S to sulphuric acid, and of the
phosphorus, so characteristic of the nucleins, to phosphoric
acid have been referred to. These activities of bacteria are
of great value in the soil. Doubtless the commercial “ phos-
phate rock” owes its origin to similar bacterial action in
ages past.

104 PHYSIOLOGICAL ACTIVITIES

The oxidation of H2S to free S may be an explanation of
the origin of the great deposits of sulphur which occur in
Louisiana and along the Gulf coast. These deposits occur
in the same general regions in which natural gas and oil
occur. The sulphur might have been derived from the same
organic material carried down by the Mississippi which
yielded the oil and gas.

A purposeful utilization of the oxidizing power of bacteria
is in “contact beds” and “sprinkling filters” in sewage dis-
posal works. In these instances the sewage is thoroughly
mixed with air and brought in contact with large amounts
of porous material so as to expose an extensive surface for
oxidation (Fig. 72).

sigeiath ate 2

One of the University hot beds.

Fie. 73.

PRODUCTION OF HEAT.

A direct result of the oxidizing action of bacteria is the
production of heat. Under most conditions of bacterial
growth this heat is not appreciable. It may become well
marked. The “heating” of manure is one of the commonest
illustrations. The temperature in such cases may reach
70°. The heating of hay and other green materials is due
chiefly to bacterial action. This heating may lead to “spon-
taneous combustion.” The high temperatures (60° to 70°)
favor the growth of thermophil bacteria which cause a still
further rise. The heat dries out the material, portions of

ABSORPTION OF FREE NITROGEN 105

which are in a state of very fine division due to the disin-
tegrating action of the organisms. The hot, dry, finely
divided material oxidizes so rapidly on contact with the air
that it ignites.

A practical use of heat production by bacteria is in the
making of “hot beds” for forcing vegetables (Fig. 73).

ABSORPTION OF FREE NITROGEN.

This is likewise one of the most important practical
activities of certain types of bacteria present in the soil.

Fic. 74.—Root tubercles on soy bean. X 3.

The ability of plants of the legume family to enrich the
soil has been known and taken advantage of for centuries,
but it is only about thirty years since it was demonstrated
that this property is due to bacteria. These plants, and
several other kinds as well, have on their roots larger or smaller
nodules (Fig. 74) spoken of as “root tubercles” which are at
certain stages filled with bacteria. When conditions are
favorable, these bacteria live in symbiotic relationship with

106 PHYSIOLOGICAL ACTIVITIES

the plant tissues, receiving carbonaceous and other food
material from them and in return furnishing nitrogenous
material to the plant. This nitrogenous material is built up
from free nitrogen absorbed from the air by the bacteria.
The utilization of this peculiar property through the proper
cultivation of clover, alfalfa, soy beans and other legumes
is one of the best ways of building up and maintaining soil
fertility insofar as the nitrogen is concerned. The technical
name of these bacteria is Pseudomonas radicicola.

There are also types of “free-living,” as distinguished
from these symbiotic, bacteria which absorb the free nitro-
gen of the air and aid materially in keeping up this supply

Fig. 75.—Free-living nitrogen absorbing bacteria ‘“‘Azotobacter.” Note
their large size as compared with other bacteria shown in this book.

under natural conditions. One of the most important of
these types is the aérobic “Azotobacter” (Fig. 75), while
another is the anaérobic Bacillus (Clostridium) pastorianus.
The nitrogen which is absorbed is built up into the protein
material of the cell body and this latter must in all proba-
bility be “worked over” by various types of decomposition
bacteria and by the nitrous and nitric organisms and be
converted into utilizable nitrates just as other protein
material is, as has been discussed in Chapter X. At any
rate there is as yet no definite knowledge of any other
method of transformation. Up to the present no intentional
practical utilization of this valuable property of these free-
living forms has been made.

ABSORPTION OF FREE NITROGEN 107

Nitrogen Nutrition of Green Plants.—It is the belief of
botanists that green plants obtain their nitrogen chiefly
in the form of nitrates, though ammonium salts may be
utilized to some extent by certain plants at least. Excep-
tions to this general rule are those plants provided with
root tubercles (and the bog plants and others which have
mycorrhiza?). These plants obtain their nitrogen in the form
of organic compounds made for them by the bacteria grow-
ing in the tubercles. That nitrogen circulates throughout
the structure of plants in organic combination is certain.
There does not appear to be any reason why similar com-
pounds which are soluble and diffusible (amino-acids?) should
not be taken up through the roots of plants and. utilized
as such. It seems to the author that this is very probably
the case. Arguments in favor of this view are: (1) The
nitrogen nutrition of leguminous and other plants with root
nodules. (2) The close symbiosis between “Azotobacter”’
and similar nitrogen-absorbing bacteria and many species
of algee in sea water at least. (8) The vigorous growth of
plants in soils very rich in organic matter, which inhibits
the production of nitrates by the nitrous-nitric bacteria
when grown in culture, and possibly (?) in the soil, so that
nitrates may not account for the vigorous growth. (4) The
effect of nitrate fertilizers is to add an amount of nitro-
gen to the crop much in excess of the amount added as
nitrate. (5) The most fertile soils contain the largest num-
bers of bacteria. . The doctrine that nitrates furnish the only
nitrogen to plants was established before the activities of
bacteria in the soil were suspected, and, so far as the author
is aware, has not been supported by experiments under
conditions rigidly controlled as to sterility.

It would seem that one of the chief functions of soil bac-
teria is to prepare soluble organic compounds of nitrogen
for the use of green plants and thus to make a “short cut”
in the nitrogen cycle (p. 96), as now believed in, direct
from the “decomposition bacteria” to green plants.

Experiments have been made by different observers in
growing seedling plants of various kinds in water culture
with one or in some cases several of the amino-acids as

108 PHYSIOLOGICAL ACTIVITIES

sources of nitrogen. Most of these experiments were disap-
pointing. Plant proteins are not so different from animal
proteins, or plant protoplasm (apart from the chlorophyl
portions of plants) from animal protoplasm as to lead one to
suppose that it could be built up from one or two amino-
acids any more than animal protoplasm can. The author
is strongly convinced that this subject should be thoroughly
investigated. It will require careful experimentation and
perhaps rather large funds to provide the amounts of amino-
acids that would probably be needed, but might result in a
decided change in our ideas of soil fertility, and especially
in the use of nitrogen fertilizers.

CHAPTER XII.
PHYSIOLOGICAL ACTIVITIES (Continvep).

PRODUCTION OF ENZYMES.

Most of the physiological activities of bacteria which
have been discussed are due to the action of these peculiar
substances, so that a knowledge of their properties is essen-
tial. This knowledge cannot as yet be exact because no
enzyme has, up to the present, been obtained in a “pure
state,” though it must be admitted that there are no certain
criteria which will enable this “pure state’ to be recognized. |
It was formerly thought that they were protein in nature,
but very “pure’’ and active enzymes have been prepared
which did not give the characteristic protein reactions, so
this idea must be abandoned. That they are large mole-
culed colloidal substances closely related to the proteins in
many respects must still be maintained. There are certain
characteristics which belong to enzymes, though no one of |
them exclusively. These may be enumerated as follows:

1. Enzymes are dead organic chemical substances.

Dead is used in the sense of non-living, never having
lived, not in the sense of “ceased to be alive.”

2. They are always produced by lzving cells:

Sometimes as active enzymes, sometimes as pro-enzymes
or zymogens which are converted into enzymes outside the
cell by acids, other inorganic substances or other enzymes.

3. They produce very great chemical changes without
themselves being appreciably affected.

Enzymes will not continue to act indefinitely, but are
used up in the process (combination with products?). The
amount of change is so great in proportion to the amount of
enzyme that the above statement is justified in the rela-
tive sense. Thus a milk-curdling enzyme has been prepared

110 PHYSIOLOGICAL ACTIVITIES

that would precipitate 100,000,000 times its own weight of
caseinogen.

4, Their action is specific in that each enzyme acts on
one kind of chemical substance only, and the products »
are always the same.

The substance may be combined with a variety of other
chemical substances so that the action appears to be on
several, but in reality it is on a definite group of molecules
in each instance. For example, emulsin attacks several
different glucosides but always sets free dextrose from them.

5. The action is inhibited and eventually stopped, and in
some cases the enzyme is destroyed by an accumulation of
the products of the action. If the products are removed, the
action will continue, if the enzyme is not destroyed. This
effect is explained partly because the enzyme probably
combines with some of the products, since it does not act
indefinitely, and partly because of the reversibility of the
reaction.

6. Like many chemical reactions those of enzymes are
reversible, that is, the substance broken up may be reformed
by it from the products produced in many instances. Thus:

maltose + maltase 5 glucose + glucose + maltase
‘fat + lipase 7 glycerin + fatty acid + lipase.

7. The presence of certain mineral salts seems to be
essential for their action. These and other substances which
are necessary are sometimes called co-enzymes. A salt of
calcium is most favorable for a great many.

8. They may be adsorbed like other colloids by “shaking
out” with finely divided suspensions like charcoal or kaolin,
or by other colloids like aluminum hydroxide or proteins.

9. When properly introduced into the tissues or blood of
an animal, they cause the body cells to form anti-enzymes
which will prevent the action of the enzyme (see Chapter
XXVID.

10. Though inert, they show many of the characteristics
of living organisms, that is

(a) Each enzyme has an optimum, a maximum and a
minimum temperature for its action,

PRODUCTION OF ENZYMES 111

All chemical reactions have such temperature limits, the
distinction is that for enzymes as for living substance the
range is relatively narrow.

(b) High temperatures destroy enzymes. All in water
are destroyed by boiling in time and most at temperatures
considerably below the boiling-point. When dry, many will
withstand a higher degree of heat than 100° before they are
destroyed.

(c) Temperatures below the minimum stop their action,
though they are not destroyed by cold. ‘

(d) Many poisons and chemical disinfectants (Chapter
XIV) which kill living organisms will also stop the action of
enzymes, though generally more of the substance is required,
so that it is possible to destroy the living cells by such means
and yet the action of the enzyme will continue.

(e). Most enzymes have an optimum reaction of medium
either acid, alkaline or neutral, depending on the particular
enzyme, though some few seem to act equally well in a
considerable range on either side of the neutral point.

The final test for an enzyme is the chemical change it brings
about in the specific substance acted on.

The most prominent characteristic of enzymes is that
they bring about very great chemical changes without them-
selves being appreciably affected. This property is also
shown by many inorganic substances which are spoken of
as “catalytic agents” or “catalyzers’”’ so that enzymes are
sometimes called “organic catalyzers.” The function of
catalytic agents seems to be to hasten the rate of a reaction
which would occur spontaneously, though in a great many
cases with. extreme slowness.

Just how enzymes act is not certain and probably will not
be until their composition and constitution are known.
Most probably they form a combination with the substance
acted on (the substrate) as a result of which there is a rear-
rangement of the atoms in such a way that new compounds
are formed, nearly always at least two, and the enzyme is
at the same time set free. It is rather remarkable that
chiefly optically active substances are split up by en-
zymes and where two modifications exist it is usually the

112 PHYSIOLOGICAL ACTIVITIES

dextrorotatory one which is attacked. No single enzyme
attacks both. This probably means that the structure of
the enzyme corresponds to that of the substrate, “fits it as
a key fits a lock,” as Emil Fischer says.

The production of enzymes is by no means restricted to
bacteria since all kinds of living cells that have been inves-
tigated have been shown to produce them and presumably
all living cells do. Hence the number of different kinds of
enzymes and of substances acted upon is practically unlim-
ited. Nevertheless they may be grouped into a compara-
tively few classes based on the general character of the
change brought about by them.

I. Class 1 is the so-called “splitting” enzymes whose
action is for the most part hydrolytic, that is, the substance
takes up water and then splits into compounds that were ap-
parently constituents of the original molecule. As examples
may be mentioned diastase, the enzyme first discovered, which
changes starch into a malt-sugar, hence is more commonly
called amylase! (starch-splitting enzyme); invertase,’ which
splits cane-sugar into dextrose and levulose: CyH»Ou +
HO = = CeHO¢ + CgH Os. Inpase! or a fat-splitting

enzyme, | pch decomposes fat into glycerin and fatty acid:
Glycerin Fatty acid

C;H,(0C, "Han-iO)s + 3H,0 = C3H,(OH)s + 3CrHenO>.
Proteases, which split up proteins into proteoses and pep-
tones.

Other classes of “splitting enzymes” break up the prod-
ucts of complex protein decomposition, such as proteoses,
peptones and amino-acids. A variety of the “splitting
enzymes” is the group of

“Coagulases’’ or coagulating enzymes as the rennet (lab,
chymosin) which curdles milk; fibrin ferment (thrombin,
thrombase) which causes the coagulation of blood. These
apparently act by splitting up a substance in the fluids
mentioned, after which splitting one of the new products
formed combines with other compounds present (usually a

1Tt will be noted that the names of enzymes (except some of those first
discovered) terminate in ase which is usually added to the stem of the name

of the substance acted on, though sometimes to a word which indicates the
substance formed by the action, as lactacidase, alcoholase.

PRODUCTION OF ENZYMES 113

mineral salt, and in the cases mentioned a calcium salt) to
form an insoluble compound, the curd or coagulum.

Another variety is the “activating” enzymes or “kinases”
such as the enterokinase of the intestine. The action here
is a splitting of the zymogen or mother substance, or form in
which the enzyme is built up by the cell so as to liberate the
active enzyme.

Of a character quite distinct from the splitting enzymes
are

II. The zymases. Their action seems to be to cause a
“shifting or rearrangement of the carbon atoms” so that
new compounds are formed which are not assumed to have
been constituents of the original molecule. Most commonly
there is a closer combination of the carbon and oxygen
atoms, frequently even the formation of CO, so that con-
siderable energy is thus liberated. Examples are the zymase
or alcoholase of yeast which converts sugar into alcohol and
carbon dioxide; CeH»O, = 2C2H,O + 2COkz: also urease,
which causes the change of urea into ammonia and carbon
dioxide. Another common zymase is the lactacidase in lactic
acid fermentation.

III. Oxtdizing enzymes also play an important part in
many of the activities of higher plants and animals. Among
the bacteria this action is illustrated by the formation of
nitrites, nitrates and sulphates and the oxidation of alcohol
to acetic acid as already described.

IV. Reducing enzymes occur in many of the denitrifying
bacteria and in those which liberate H.S from sulphates. A.
very widely distributed reducing enzyme is “catalase” which
decomposes hydrogen peroxide.

As previously stated, most of the physiological activities of
bacteria are due to the enzymes that they produce. It is
evident that for action to occur on substances which do
not diffuse into the bacterial cell—starches, cellulose, com-
plex proteins, gelatin—the enzymes must pass out of the
bacterium and consequently may be found in the surround-
ing medium. Substances like sugars, peptones, alcohol,
which are readily diffusible, may be acted on by enzymes
retained within the cell body. In the former case the

8

114 PHYSIOLOGICAL ACTIVITIES

enzymes are spoken of as extra-cellular or “exo-enzymes,”
and in the latter as intra-cellular or “endo-enzymes.” The
endo-enzymes and doubtless also the exo-enzymes may after
the death of the cell digest the contents to a greater or less
extent and thus furnish substances that are not otherwise
obtainable. This process of “self-digestion” is known tech-
nically as “aztolysis.”

A distinction was formerly made between “organized”
and “unorganized ferments.” The former term was applied
to the minute living organisms, bacteria, yeasts, molds, etc.,
which bring about characteristic fermentative changes,
while the latter term was restricted to enzymes as just
described. Since investigation has shown that the changes
aseribed to the “organized ferments” are really due to their
enzymes, and that enzymes are probably formed by all liv-
ing cells, the distinction is scarcely necessary at present.

PRODUCTION OF TOXINS.

The injurious effects of pathogenic bacteria are due in
large part to the action of these substances, which in many
respects bear a close relationship to enzymes. The chemical
composition is unknown since no toxin has been prepared
“pure” as yet. It was formerly thought that they were
protein in character, but very pure toxins have been pre-
pared which failed to show the characteristic protein reac-
tions. It is well established that they are complex sub-
stances, of rather large molecule and are precipitated by
many of the reagents which precipitate proteins. Toxins
will be further discussed in Chapter XXVII. It will be
sufficient at this point to enumerate their chief peculiarities ‘
in order to show their marked resemblance to enzymes.

1. Toxins are dead organic chemical substances.

2. They are always produced by living cells.

3. They are active poisons in very small quantities!

4, Their action is specific in that each toxin acts on a
particular kind of cell. The fact that a so-called toxin acts

1 Tetanus toxin is about 120 times as poisonous as strychnin, both of
which act on the same kind of nerve cells.

PRODUCTION OF TOXINS 115

on several different kinds of cells, possibly indicates a mix-
ture of several toxins, or action on the same substance in the
cells.

5. Toxins are very sensitive to the action of injurious
agencies such as heat, light, etc., and in about the same
measure that enzymes are, though as a rule they are some-
what more sensitive or “labile.”

6. Toxins apparently have maxima, optima, and minima
of temperature for their action, as shown by the destructive
effect of heat and by the fact that a frog injected with
tetanus toxin and kept at 20° shows no indication of poison,
but if the temperature is raised to 37,° symptoms of poisoning
are soon apparent. Cold, however, does not destroy a toxin.

7. When properly introduced into the tissues of animals
they cause the body cells to form antitoxins (Chapter
XXVIJ) which are capable of preventing the action of the
toxin in question.

8. The determining test for a toxin rs its action on a living
cell.

It is true that enzymes are toxic, as are also various
foreign proteins, when injected into an animal, but in much
larger doses than are toxins.

A marked difference between enzymes and toxins is that
the former may bring about a very great chemical change
and still may be recovered from the mixture of substances
acted on and produced, while the toxin seems to be perma-
nently used up in its toxic action and cannot be so recovered.
Toxins seem very much like enzymes whose action is restricted
to ling cells.

Just as enzymes are probably produced by all kinds of
cells and not by bacteria alone, so toxins are produced
by other organisms. Among toxins which have been care-
fully studied are ricin, the poison of the castor oil plant
(Ricinus communis); abrin of the jequirity bean (Abrus pre-
catorius); robin of the common locust (Robinia pseudacacia) ;
poisons of spiders, scorpions, bees, fish, snakes and sala-
manders.

It has been stated that. some enzymes are thrown out
from the cell and others are retained within the cell. The

116 PHYSIOLOGICAL ACTIVITIES

same is true of toxins, hence we speak of exo-toxins or toxins
excreted from, and endo-toxins or toxins retained within the
cell. Among the pathogenic bacteria there are very few
which secrete toxins when growing outside the body.
Bacillus tetant or lockjaw bacillus, Bactertwm diphtherie or
the diphtheria bacillus, Bacillus botulinus or a bacillus caus-
ing a type of meat poisoning, Pseudomonas pyocyanea or the
blue pus bacillus are the most important. Other pathogenic
bacteria do not secrete their toxins under the above condi-
tions, but only give them up when the cell is disintegrated
either within or outside the body. For the reason that
endotoxins are therefore difficult to obtain their character-
istics have not been much studied. The description of
toxins as above given is intended to apply to the exotorins
of bacteria, sometimes spoken of as true toxins, and to the
vegetable toxins (phytotoxins) which resemble them.

The snake venoms and probably most of the animal toxins

(zoétoxins) are very different substances. (See Chapter
XXIX.)

CAUSATION OF DISEASE.

This subject belongs properly in special pathogenic bac-
teriology. It will be sufficient to indicate that bacteria may
cause disease in one or more of the following ways: (a)
blocking circulatory vessels, either blood or lymph, directly
or indirectly; (b) destruction of tissue; (c) production of
non-specific poisons (ptomaines, bases, nitrites, acids, gases,
etc.); (d) production of specific poisons (toxins).

ANTIBODY FORMATION.

Bacteria cause the formation of specific “antibodies” when
properly introduced into animals. This must be considered as
a physiological activity since it is by means of substances
produced within the bacterial cell that the body cells of
animals are stimulated to form antibodies. (See Chapters
XXVI-XXIX.)

CULTURAL CHARACTERISTICS 117

STAINING.

The reaction of bacteria to various stains is dependent on
their physicochemical structure and hence is a result of
physiological processes, but is best discussed separately

(Chapter XIX).
CULTURAL CHARACTERISTICS.

The same is true of the appearance and growth on different
culture media. (Chapter XX.)

CHAPTER XIII.

DISINFECTION—STERILIZATION—
DISINFECTANTS.

TuE discussion of the physiology of bacteria in the pre-
ceding chapters has shown that a number of environmental
factors must be properly correlated in order that a given
organism may thrive. Conversely, it can be stated that any
one of these environmental factors may be so varied that
the organism will be more or less injured, may even be
destroyed by such variation. It has been the thorough study
of the above-mentioned relationships which has led to prac-
tical methods for destroying bacteria, for removing them or
preventing their growth when such procedures become
necessary.

The process of killing all the living organisms or of remov-
ing them completely is spoken of as disinfection or as sterili-
zation, according to circumstances. Thus the latter term is
applied largely in the laboratory, while the former more
generally in practice outside the laboratory. So also disin-
fection is most commonly done with chemical agents and
sterilization by physical means, though exceptions are
numerous. The original idea of disinfection was the destruc-
tion of “infective” organisms, that is, organisms producing
disease in man or animals. A wider knowledge of bacteri-
ology has led to the application of the term to the destruc-
tion of other organisms as well. Thus the cheese-maker
“disinfects” his curing rooms to prevent: abnormal ripening
of cheese, and the dairy-worker “disinfects” his premises to
avoid bad flavors, abnormal changes in the butter or milk.
Sterilization is more commonly applied to relatively small
objects and disinfection to larger ones. Thus in the labor-
atory, instruments, glassware, apparatus, etc., are ‘‘steril-
ized” while desks, walls and floors are “disinfected.'' The

PHYSICAL AGENTS 119

surgeon “‘sterilizes” his instruments, but “disinfects’” his
operating table and room. The dairy-workers mentioned ,
above sterilize their apparatus, pails, milk bottles, ete.
Evidently the object of the two processes is the same, remov-
ing or destroying living organisms, the name to be applied:
is largely a question of usage and circumstances. Any agent
which is used to destroy microérganisms is called a “disin-
fectant.” Material freed from living organisms is “sterile.”’

The process of preventing the growth of organisms without
reference to whether they are killed or removed is spoken
of as “antisepsis,” and the agent as an antiseptic. Hence a
mildly applied “disinfectant” becomes an “antiseptic,”
though it does not necessarily follow that an “antiseptic”
may become a disinfectant when used abundantly. Thus
strong sugar solutions prevent the development of many
organisms, though they do not necessarily kill them.

Asepsis is a term which is restricted almost entirely to
surgical operations and implies the taking of such precau-
tions that foreign organisms are kept out of the field of opera-
tion. Such an operation is an aseptic one, or performed
aseptically.

A “deodorant or deodorizer”’ is used to destroy or remove
an odor and does not necessarily have either antiseptic or
disinfectant properties.

The agents which are used for the above-described pro-
cesses may be conveniently divided into physical agents and
chemical agents.

PHYSICAL AGENTS.

1. Drying.—This is doubtless the oldest method for pre-
venting the growth of organisms, and the one which is used
on the greatest amount of material at the present time. A
very large percentage of commercial products is preserved
and transported intact because the substances are kept free
from moisture. In the laboratory many materials which
are used as food for bacteria (see Chapter XVI) “keep”
because they are dry. Nevertheless, drying should be con-
sidered as an antiseptic rather than as a disinfectant process.
While it is true that the complete removal of water would

120 DISINFECTION—STERILIZATION—DISINFECTANTS

result in the death of all organisms this necessitates a high
temperature, in itself destructive, and does not occur in
practice. Further, though many pathogenic bacteria are
killed by drying, many more, including the spore formers,
are not. Hence drying alone is not a practical method of
disinfecting.

2. Heat.—The use of heat in some form is one of the
very best means for destroying bacteria. It may be made
use of by combustion, or burning, as direct exposure to the
open flame, as dry heat (hot air), or as moist heat (boiling

Fic. 76.—A small laboratory hot-air sterilizer.

water or steam). Very frequently in veterinary practice,
especially in the country, occasionally under other condi-
tions, the infected material is best burned. This method is
thoroughly effective and frequently the cheapest in the end.
Wherever there are no valid objections it should be used.
Exposure to the open flame is largely a laboratory procedure
to sterilize small metallic instruments and even small pieces
of glassware. It is an excellent procedure in postmortem
examinations to burn off the surface of the body or of an
organ when it is desired to obtain bacteria from the interior
free from contamination with surface organisms.

PHYSICAL AGENTS 121

Dry Heat—Dry heat is not nearly so effective as moist
heat as a sterilizing agent. The temperature must be higher
and continued longer to accomplish the same result. Thus
a dry heat of 150° for thirty minutes is no more efficient than
steam under pressure at 115° for fifteen minutes. Various
forms of hot-air sterilizers are made for laboratory purposes
(Fig. 76). On account of the greater length of time required
for sterilization their use is more and more restricted to
objects which must be used dry, as in blood and serum
work, for example. In practice the use of hot air in disin-
fecting plants is now largely restricted to objects which
might be injured by steam, as leather goods, furs, and certain
articles of furniture, but even here chemical agents are more
frequently used.

Moist Heat—Moist heat may be applied either by boil-
ing in water or by the use of steam at air pressure, or, for
rapid work and on substances that would not be injured,
by steam under pressure. Boiling is perhaps the best house-
hold method for disinfecting all material which can be so
treated. The method is simple, can always be made use of,
and is universally understood. It must be remembered that
all pathogenic organisms, even their spores are destroyed
by a few minutes’ boiling. The process may be applied to
more resistant organisms, such as are met with in canning
vegetables, though the boiling must be continued for several
hours, or what is better, repeated on several different days.
This latter process, known as “discontinuous sterilzation,”
must also be applied to substances which would be injured
or changed in composition by too long-continued heating,
such as gelatin, milk, and certain sugars. In the laboratory
such materials are boiled or subjected to streaming steam
for half an hour on each of three successive days. In can-
ning vegetables the boiling should be from one to two hours
each day. The principle involved is that the first boiling
destroys the growing cells, but not all spores. Some of the
latter germinate by the next day and are then killed by the
second boiling and the remainder develop and are killed on
the third day. Occasionally a fourth boiling is necessary.
It is also true that repeated heating and cooling is more

122 DISINFECTION—STERILIZATION—DISINFECTANTS

destructive to bacteria than continuous heating for the same
length of time, but the development of the spores is the most
important factor. Discontinuous heating may also be used
at temperatures below the boiling-point for the sterilization
of fluids like blood serum which would be coagulated by
boiling. In this case the material is heated at 55° to 56°
for one hour, but on each of seven to ten successive days.
The intermittent heating and cooling is of the same impor-
tance as the development of the spores in this case. (Better
results are secured with such substances by collecting them
aseptically in the first place.)

Fig. 77.—The Arnold steam sterilizer for laboratory use.

Steam.—Steam is one of the most commonly employed
agents for sterilization and disinfection. It is used either
as “streaming steam” at air pressure or confined under
pressure so that the temperature is raised. For almost all
purposes where boiling is applicable streaming steam may
be substituted. It is just as efficient and frequently more
easily applied. The principle of the numerous forms of
“steam sterilizers” (Fig. 77) is essentially the same. There

PHYSICAL AGENTS 123

is a receptacle for a relatively small quantity of water and
means for conducting the steam generated by boiling this
water to the objects to be treated, which are usually placed

il \é [ae ~
af

= al

Fic. 78.—Vertical gas-heated Fig. 79.—Horizontal gas-heated labora-
laboratory autoclave. tory autoclave.

124. DISINFECTION—STERILIZATION—DISINFECTANTS

immediately above the water. Surgical instruments may
be most conveniently sterilized by boiling or by steaming in
especially constructed instrument sterilizers. If boiled, the
addition of carbonate of soda, about 1 per cent., usually
prevents injury.

Fig. 80.—A battery of two horizontal autoclaves in one of the author's
student laboratories. Steam is furnished direct from the University central
heating plant.

Steam under pressure affords a much more rapid and cer-
tain method of destroying organisms. Fifteen to twenty
pounds’ pressure corresponding to temperatures of 121° to
125° is commonly used. Variations depend on the bulk and
nature of the material. Apparatus for this purpose may now
be obtained from sizes as.small as one or two gallons up to
huge structures which will take one or two truckloads of

PHYSICAL AGENTS 125

material (Figs. 78-90). The latter type is in common use
in canning factoties, dairy plants, hospitals, public institu-
tions, municipal and governmental disinfecting stations.
Very frequently there is an apparatus attached for pro-
ducing a vacuum both to exhaust the air before sterilizing,
so that the steam penetrates much more quickly and thor-
oughly and for removing the vapor after sterilizing, thus
hastening the drying out of the material disinfected.

Fig. $1.—A ‘‘process kettle’’ (steam-pressure sterilizer) used in canning.
Diameter, 40 inches; height, 72 inches.

The smaller types of pressure sterilizers are called “auto-
claves’ and have become indispensable in laboratory work.
Fifteen pounds’ pressure maintained for fifteen minutes is
commonly sufficient for a few small objects. For larger
masses much longer time is needed. The author found that
in an autoclave of the type shown in Fig. 79 it required ten
minutes for 500 c:c. of water at 15 pounds’ pressure to reach

126 DISINFECTION—STERILIZATION—DISINFECTANTS

a temperature of 100°, starting at room temperatures, 20° to
25°. Autoclaves may be used as simple steam sterilizers by

Fic. $2.—Horizontal steam chest used in canning. Height, 32 inches;
width, 28 inches; length, 10 feet.

leaving the escape valves open so that the steam is not
confined, hence they have largely replaced the latter.'

1In the author's laboratory in the past ten years all sterilization except
those few objects in blood and serum work which must be dry, has been
done in autoclaves of the type shown in Fig. 80 which are supplied with steam
from the University central heating plant. A very great saving of time is
thus secured.

PHYSICAL AGENTS 127

A process closely akin to sterilization by heat is pasteur-
ization. This means the heating of material at a tempera-

ture and for a time which will destroy the actively growing
bacteria but notgthe spores. The methods for doing this

Fig. 83.—A battery of horizontal rectangular steam chests in actual use in a canning factory.

128 DISINFECTION—STERILIZATION—DISINFECTANTS

vary but are essentially two in principle. 1. The material in
small quantities in suitable containers (bottles) is placed in

the apparatus; the temperature is raised to 60° to 65° and
maintained for twenty to thirty minutes and then the whole

Fic. §4.—A battery of cylindrical process kettles in actual use in a canning factory.

’

PHYSICAL AGENTS

129

is cooled (beer, wine, grape juice, bottled milk) (Figs. 91, 92,

and 93).

Fic. 85.—A steam chamber used in government disinfection work. Size,

4 feet 4 inches x 5 feet 4 inches x 9 feet.

Fig. 86.—Circular steam chamber used in government disinfection work,

54 inches in diameter.

2. Pasteurizing machines are used and the fluid flows
through continuously. In one type the temperature is raised

9

130 DISINFECTION—STERILIZATION—DISINFECTANTS

Fig. 88.—Steam chambers on deck of the U. S. quarantine station barge
“Defender.”

PHYSICAL AGENTS 131

to 60° and by “retarders” is kept at this temperature for
twenty to thirty minutes (Figs. 94 to97). In another type the

Fic. 89.—Steam chambers in hold of U. 8. quarantine station barge ‘‘ Pro-
tector.’ Disinfected space.

Fie. 90.—Municipal disinfecting station, Washington, D. ©,

Fic. 92.—A pasteurizer for

PHYSICAL AGENTS 133

temperature is raised to as high as 85° for a few seconds only,
“flash process” (Fig. 98), and then the material is rapidly

Fic. 94.—A continuous milk pasteurizer.

134 DISINFECTION—STERILIZATION—DISINFECTANTS

cooled. Itiscertain that all pathogenic microérganisms, except
the very few spore formers in that stage, are killed by proper

Fic. 95.—A pasteurizer for cream to be used in making ice-cream.

pasteurization. The process is largely employed in the
fermentation and dairy industries.

Fic. 96.—A continuous milk pasteurizer with holder; capacity, 1500 pounds
per hour. A, pasteurizer—the milk flows in tubes inside of a jacket of water
heated to the proper temperature; B, holder; C, water cooler; D, brine cooler.

Fic. 97.—A continuous pasteurizing plant in operation. Similar to Fig.
96, but larger. Capacity, 12,000 pounds per hour. A, pasteurizer; B, seven
compartment holder; C, D, coolers.

136 DISINFECTION—STERILIZATION—DISINFECTANTS

3. Cold.—That cold is an excellent antiseptic is illustrated
by the general use of refrigerators and “cold storage.”
Numerous experiments have shown that although many
pathogenic organisms of a given kind are killed by tempera-
tures below freezing, not all of the same kind are, and many
kinds are only slightly affected. Hence cold cannot be con-
sidered a practical means for disinfection.

Fig. 98.—A ‘flash process” pasteurizing outfit, with holder. A, flash
pasteurizer; B, holder; C, cooler.

4, Light.—It has been stated (p. 65) that light is destruc-
tive to bacteria, and the advisability of having well-lighted
habitations for men and animals has been mentioned. The
practice of “sunning” bedclothing, hangings and other large
articles which can scarcely be disinfected in a more con-
venient way is the usual method of employing this agent.
Drying and the action of the oxygen of the air assist the
process to some extent. Undoubtedly large numbers of
pathogenic organisms are destroyed under natural condi-
tions by the combined effects of drying, direct sunlight and

PHYSICAL AGENTS 137

oxidation, but it should not be forgotten that a very slight
protection will prevent the action of light (Figs. 99 and 100).

5. Osmotic Pressure.—Increase in the concentration of
substances in solution is in practical use as an antiseptic
procedure. Various kinds of “sugar preserves,” salt meats
and condensed milk are illustrations. It must be remem-
bered that a similar increase in concentration occurs when

Fig. 99.—Effect of light on bacteria. 7/10. The plate was inoculated
in the usual way. A letter H of black paper was pasted on the bottom.
The plate was then exposed for four hours to the sun in January outside the
window and then incubated. The black paper protected the bacteria.
Outside of it they were killed except where they happened to be in large
masses. Hence the letter shows distinctly. (Student preparation.)

many substances are dried, and is probably as valuable in
the preservative action as the loss of water. That the pro-
cess cannot be depended on to kill even pathogenic organ-
isms is shown by finding living tubercle bacilli in condensed
milk. The placing of bacteria in water or in salt solution
in order to have them die and disintegrate (greatly aided.
by vigorous shaking in a shaking machine) (“autolysis,’’

1388 DISINFECTION—STERILIZATION—DISINFECTANTS

p- 114) is a laboratory procedure to obtain cell constituents.
It is not a practical method of disinfection, however.

6. Electricity.—Electricity, though not in itself injurious
to bacteria, is used as an indirect means for destroying bac-
teria in a practical way. This is done by electrical produc-
tion of some substance which is destructive to bacteria as
in ozone water purification (Petrograd, Florence, and else-

Fie. 100.—Effect of light on bacteria. XX 7/10. This plate was treated
exactly as the plate in Fig. 99, except that the letter is ZL, and that it was
exposed inside the window and wire screen. The window was plate glass.
It is evident that few of the bacteria were killed, since the letter Z is barely
outlined. The exposure was at the same time as the plate in Fig. 99. (Stu-
dent preparation.)

where), or the use of ultra-violet rays for the same purpose
(Marseilles, Paris) and for treatment of certain disease con-
ditions. Electricity might be used as a source of heat for
disinfecting purposes should its cheapness justify it. It has
also been used in the preservation of meats to hasten the
penetration of the salt and thus reduce the time of pickling.
Electrolyzed sea water has been tried as a means of flushing

PHYSICAL AGENTS 139

Fic. 101.—An electric milk purifier (pasteurizer). The milk flowing
from cup to cup completes the circuit when the current is on. The effect
is certainly a heat effect. Sparking occurs at the lips of the cups.

Fig. 102.—One of the ten filter beds of the Columbus water filtration
plant with the filtering material removed. Sand is the filtering material.
All of the beds together have a capacity of 30,000,000 gallons daily.

140 DISINFECTION—STERILIZATION—DISINFECTANTS

and disinfecting streets, but it is very doubtful if the ‘waded
expense is justified by any increased benefit. A number of
electric devices have been put forth for various sterilizing
and disinfecting purposes and doubtless will continue to be,
but everyone should be carefully tested before money is
invested in it.

Fig. 103.—Suction filtration. A, Berkefeld filter in glass cylinder con-
taining the liquid to be filtered; B, sterile flask to receive the filtrate as it is
drawn through; C, water pump; D, manometer, convenient for detecting
leaks as well as showing pressure; FE, bottle for reflux water.

7. Filtration Filtration is a process for rendering fluids
sterile by passing them through some material which will

1 The author has tested an ‘electric milk purifier’ (Fig. 101) which was
as efficient as a first-class pasteurizer and left the milk in excellent condi-
tion both chemically and as far as ‘‘cream line” was concerned. The cost
of operation as compared with steam will depend on the price of electricity.

PHYSICAL AGENTS 141

hold back the bacteria. It is used on a large scale in the
purification of water for sanitary or manufacturing reasons
(Fig. 102). Airis also rendered “germ free” in some surgical
operating rooms, “serum laboratories’ and breweries by filtra-
tion. Inthe laboratory it isa very common method of steriliz-
ing liquids which would be injured by any other process. The

i et

)
ia

Fic. 104.—Pressure filtration. A, cylinder which contains the filter
candle; B, cylinder for the liquid to be filtered; C, sterile flask to receive the
filtrate; D, air pump to furnish pressure.

apparatus consists of a porous cylinder with proper devices
for causing the liquid to pass through either by suction
(Fig. 103), where the pressure will be only one atmosphere’
(approximately 15 pounds per square inch), or by the use of
compressed air at any desired pressure (Fig. 104). The two
main types of porous cylinders (“filter candles,”’ “bougies’’)

142 DISINFECTION—STERILIZATION—DISINFECTANTS

are the Pasteur-Chamberland (Fig. 105) and the Berkefeld.
The former are made of unglazed porcelain of different
degrees of fineness, the latter of diatomaceous earth (Fig.
106). The designs of complete apparatus are numerous.

8. Burying.—This is a time-honored method of disposing
of infected material of all kinds and at first thought might
not be considered a means of disinfection. As a matter of
fact, under favorable conditions it is an excellent method.

Fig. 105.—Pasteur-Chamberland filter candles about one-half natural size.

The infected material is removed. Pathogenic organisms
tend to die out in the soil owing to an unfavorable environ-
ment as to temperature and food supply, competition with
natural soil organisms for what food there is, and the injuri-
ous effects of the products of these organisms. Care must
be taken that the burial is done in such a way that the surface
soil is not contaminated either directly or by material brought
up from below by digging or burrowing animals, insects,

PHYSICAL AGENTS 143

worms, or movement of ground water to the surface. Also
that the underground water supply which is drawn upon
for use by men or animals is not also contaminated. Fre-

Fic. 106.—Berkefeld filter candles about one-half natural size.

quently infected material, carcasses of animals, etc., are
treated in some way so as to aid the natural process of
destruction of the organisms present, especially by the use
of certain chemical agents, as quicklime (see p. 146).

CHAPTER XIV.
DISINFECTION AND STERILIZATION (ConTINUED).

CHEMICAL AGENTS.

A very large number of chemical substances might be
used for destroying bacteria or preventing their growth
either through direct injurious action or by the effect of
concentration. Those which are practically useful are rela-
tively few, though this is one of the commonest methods of
disinfecting and the word “disinfectant” is frequently
wrongly restricted to chemical agents.

Chemical agents act on bacteria in a variety of ways.
Most commonly there is direct union of the chemical with
the protoplasm of the cell and consequent injury. Some-
timeés the chemical is first precipitated on the surface of the
cell without penetrating at once. If removed soon enough,
the organism is not destroyed. This is true of bichloride of
mercury and formaldehyde. If bacteria treated with these
agents in injurious strength be washed with ammonia or
ammonium sulphate, even after a time which would other-
wise result in their failure to grow, they will develop. Some
chemicals change the reaction of the material in a direction
unfavorable to growth, and if the change is enough, may
even kill the bacteria. Some agents remove a chemical
substance necessary to the growth of the organism and
hence inhibit it. Such actions are mainly preventive (anti-
septic) and become disinfectant only after a long time.

ELEMENTS.

Oxygen.—Oxygen as it occurs in the air is probably not
injurious to living bacteria but aids them with the excep-

ELEMENTS 145

tion of the anaérobes. In the nascent state especially as
liberated from ozone (O3) hydrogen peroxide (H,O.) and
hypochlorites (Ca(CI1O).) it is strongly bactericidal.

Fig. 107.—Apparatus for sterilizing water with liquid chlorine.

Chlorine.—Chlorine is actively disinfectant and is coming
into use for sterilizing water on a large scale in municipal
plants (Fig. 107). Jodine finds extended use in aseptic sur-
gical operations and antiseptic dressings. Bromine, mer-
cury, silver, gold, nickel, zine and copper are markedly
germicidal in the elemental state but are not practical.

10

146 DISINFECTION AND STERILIZATION

COMPOUNDS.

Calcium Oxide.—Calcium oxide (CaO), quick lume, is an
excellent disinfectant for stables, yards, outhouses, etc.,
where it is used in the freshly slaked condition as “white
wash;” also to disinfect carcasses to be buried. It is very
efficient against the typhoid bacillus in water, where it is
much used to assist in the softening.

Chloride of Lime.—Chloride of lime, bleaching powder,
which consists of calcium hypochlorite, the active agent,
and chloride and some unchanged quicklime is one of the
most useful disinfectants. It is employed to sterilize water
for drinking purposes on a large scale and to disinfect sewage
plant effluents. A 5 per cent. solution is the proper strength
for ordinary disinfection. Only a supply which is fresh or
has been kept in air-tight containers should be used, as it
rapidly loses strength on exposure to the air. The active
agent is nascent oxygen liberated from the decomposition of
the hypochlorite.

Sodium Hypochlorite—Sodium hypochlorite prepared by
the electrolysis of common salt has been used to some extent.

Bichloride of Mercury—Bichloride of mercury, mercuric
chloride, corrosive sublimate (HgClz), is the strongest of all
disinfectants under proper conditions. It is also extremely
poisonous to men and animals and great care is necessary
in its use. It is precipitated by albuminous substances and
attacks metallic objects, hence should not be used in the
presence of these classes of substances.

It is used in a strength of one part HgCl, to 1000 of water
for general disinfection. Ammonium chloride or sodium
chloride, common salt, in quantities equal to the bichloride,
or citric acid in one-half of the amount should be added in
making large quantities of solution or for use with albumin-
ous fluids to prevent precipitation of the mercury (Fig. 108).

None of the other metallic salts are of value as practical
disinfectants aside from their use in surgical practice. In
this latter class come boric acid, silver nitrate, potassium
permanganate. The strong mineral acids and alkalis are,
of course, destructive to bacteria, but their corrosive effect

ORGANIC COMPOUNDS 147

excludes them from practical use, except that “lye washes”
are of value in cleaning floors and rough wood-work, but
even here better disinfection can be done more easily and
safely.

Fie. 108.—Tanks for bichloride of mercury, government quarantine
disinfecting plant. ‘

ORGANIC COMPOUNDS.

Carbolic Acid or Phenol.—Carbolic acid or phenol (CsH;OH)
is one of the commonest agents in this class. It is used
mostly in 5 per cent. solution as a disinfectant and in 0.5
per cent. solution as an antiseptic. For use in large quanti-
ties the crude is much cheaper and, according to some experi-
menters, even more active than the pure acid, owing to the
cresols it contains. The crude acid is commonly mixed with
an equal volume of commercial sulphuric acid and the mix-
ture is added to enough water to make a 5 per cent. dilution,
which is stronger than either of the ingredients alone in 5
per cent. solution.

Cresols—The cresols (CsH,CH;OH, ortho, meta and
para), coal-tar derivatives, as phenol, are apparently more
powerful disinfectants. A great number of preparations

148 DISINFECTION AND STERILIZATION

containing them have been put on the market. Creolin is
one which is very much used in veterinary practice and forms
a milky fluid with water, while lysol forms a clear frothy
liquid. Both of these appear to be more active than carbolic
acid and are less poisonous and more agreeable to use.
They are used in 2 to 5 per cent. solution.

Alcohol Ordinary (ethyl) alcohol (C,H;OH) is largely
used as a preservative, also as a disinfectant for the body
surface, hands, and arms. Experiments show that alcohol
of 70 per cent. strength is most strongly bactericidal and
that absolute alcohol is very slightly so.

Soap.—Experimenters have obtained many conflicting
results with soaps when tested on different organisms, as
is to be expected from the great variations in this article.
Miss Vera McCoy in the author’s laboratory carried out
experiments with nine commercial soaps—Ivory, Naphtha,
Packer’s Tar, Grandpa’s Tar, Balsam Peru, A. D. 5. Car-
bolic, German Green, Dutch Cleanser, Sapolio—and_ ob-
tained abundant growth from spores of Bacterium anthracis,
from Bacillus coli and from Micrococcus pyogenes aureus in
all cases even when the organisms had been exposed twenty-
four hours in 5 per cent. solutions. From these results and
from the wide variations reported in the literature it is clear
that soap solutions alone cannot be depended on as dis-
infectants. Medicated soaps do not appear to offer any
advantages in this respect.

Formaldehyde.—Formaldehyde (HCHO) is perhaps the
most largely used chemical disinfectant at the present time.
The substance is a gas but occurs most commonly in com-
merce as a watery solution containing approximately 40
per cent. of the gas. This solution is variously known as
formalin, formol, and formaldehyde solution. The first two
names are patented and the substance under these names
usually costs more. It is used in the gaseous form for disin-
fecting closed spaces of all kinds to the exclusion of most
other means today. A great many types of formalin gen-
erators have been devised. The gas has little power of
penetration and all material to be reached should be exposed
as much as possible, The dry gas is almost ineffective, so

ORGANIC COMPOUNDS 149
that the objects must be moistened or vapor generated
along with the gas. A common method in use is to avoid
expensive generators by pouring the formaldehyde solution
on permanganate of potash crystals placed in a_ vessel
removed from inflammable objects on account of the heat
developed which occasionally, sets the gas on fire. The
formalin is used in amounts varying from 20 to 32 ounces
to 84 to 13 ounces of permanganate to each 1000 cubic feet
of space. This method is expensive since one pint (16 ounces)
of formalin is sufficient for each 1000 cubic feet, and since
the permanganate is an added expense. Dr. Dixon, Commis-
sioner of Health of Pennsylvania, recommends the following
mixture to replace the permanganate, claiming that it works
more rapidly and is less expensive and just as efficient:

1. Sodium bichromate, ten ounces.
2. Saturated solution of formaldehyde, sixteen ounces.
3. Common sulphuric acid, one and a half ounces.

Two and three are mixed together and when cool are
poured on the bichromate which is placed in an earthen-
ware jar of a volume about ten times the quantity of fluid
used. The quantities given are for each 1000 cubic feet of
space.

A very simple method is to cause the formalin, diluted
about twice with water, to furnish moisture enough to drop,
by means of a regulated “separator funnel’ on a heated
iron plate. The dropping should be so regulated that each
drop is vaporized as it falls. The plate must have raised
edges, pan-shaped, to prevent the drops rolling off when they
first strike the plate. Formaldehyde has no corrosive (except
on iron) or bleaching action, and is the most nearly ideal
closed space disinfectant today. In disinfecting stations it
is made use of in closed sterilizers such as were described
under steam disinfection, particularly in connection with
vacuum apparatus. It is also used in solution as a pre-
servative and as a disinfectant. The commonest strength is
2 or 3 per cent. of formalin or 0.8 to 1.2 per cent. of the
formaldehyde gas. As an antiseptic it is efficient in dilutions
as high as 1 to 2000 of the gas. It is very irritant to mucous
membranes of most individuals.

150 DISINFECTION AND STERILIZATION

In addition to the above-discussed disinfectants a large
number of substances, particularly organic, are used in
medicine, surgery, dentistry, etc., as more or less strong
antiseptics, and the list is a constantly lengthening one.

In the laboratory chloroform, H,O:, ether and other vola-
tile or easily decomposable substances have been used to
sterilize liquids which could not be treated by heat or by
filtration. The agent is removed either by slow evapora-
tion or by exhausting the fluid with an air pump. The
method is not very satisfactory, nor is absolute steriliza-
tion easily accomplished. It is much better to secure such
liquids aseptically where possible.

CHAPTER XV.
DISINFECTION AND STERILIZATION (ConrinueEp).

CHOICE OF AGENT.

THE choice of the above-described agents depends on the
conditions. Evidently a barn is not to be disinfected in the
same way that a test-tube in the laboratory is sterilized.
- Among the factors to be considered in making a choice are
the thing to be disinfected or sterilized, its size and nature,
that is, whether it will be injured by the process proposed,
cost of the agent, especially when a large amount of material
is to be treated. Among the conditions which affect the
action of all agents the following should be borne in mind
particularly when testing the disinfecting power of chemical
agents:

1. The kind of bacterium to be destroyed, since some are
more readily killed by a given disinfectant than others, even
though no spores are present.

2. The age of the culture. Young bacteria less than twenty-
four hours old are usually more readily killed than older ones
since the cell wall is more delicate and more easily pene-
trated, though old growths may be weakened by the accu-
mulation of their products and be more easily destroyed.

3. Presence of spores, since they are much more resistant
than the growing cells.

4, Whether the organism is a “good or bad” growth, 1. e.,
whether it has grown in a favorable environment and hence
is vigorous, or under unfavorable conditions and hence is
weak.

5. The number of bacteria present, since with chemical
agents the action is one of relative masses.

6. Nature of the substance in which the bacteria are.
Metallic salts, especially bichloride of mercury, are precipi-
tated by albuminous substances and if employed at all must

152 DISINFECTION AND STERILIZATION

be used in several times the ordinary strength. Solids
require relatively more of a given solution than liquids.

7. State of the disinfectant, whether solid, liquid or gas,
and whether it is ionized or not. Solutions penetrate best
and are therefore more quickly active and more efficient.

8. The solvent. Water is the best solvent to use. Strong
alcohol (90 per cent. +) diminishes the effect of carbolic
acid, formaldehyde and bichloride of mercury. Oil has a
similar effect. The action is probably to prevent the pene-
tration of the disinfectant.

9. Strength of solution. The stronger the solution, the
more rapid and more certain the action, for the same rea-
son as mentioned under 5. In fact, every disinfectant has
a strength below the lethal at which it stimulates bacterial
growth.

10. Addition of salts. Common salt favors the action of
bichloride of mercury and also of carbolic acid. Other salts
may hinder by precipitating the disinfectant.

11. Temperature. Chemical disinfectants, as a rule, fol-
low the general law that chemical action increases with the
temperature, up to the point where the heat of itself is
sufficient to kill.

12. Time of action. It is scarcely necessary to point out
that a certain length of time is necessary for any disinfec-
tant to act. One may touch a red hot stove and not be
burned. All the above-mentioned conditions are influenced
by the time of action.

PRACTICAL STERILIZATION AND DISINFECTION.

The methods for sterilizing in the laboratory have been
discussed and will be referred to again in the next chapter.
_ In practical disinfection it is a good plan always to pro-
ceed as though spores were present even if the organism is
known. Hence use an abundance of the agent and apply it
as long as practicable. Also it is best to secure the chemical
substances used as such and not depend on patented mixtures
purporting to contain them. As a rule the latter are more
expensive in proportion to the results secured.

PRACTICAL STERILIZATION AND DISINFECTION 153

Surgical instruments may be sterilized by boiling in water
for fifteen minutes, provided they are’ clean, as they should
be. If dried blood, pus, mucus, etc., are adherent, which
should never be the case, they should be boiled one-half
hour. The addition of sodium carbonate (0.5 to 1 per cent.)
prevents rusting. Surgeon’s sterilizers are to be had at
reasonable prices and are very convenient. Whether the
instruments are boiled or subjected to streaming steam
depends on whether the supporting tray is covered with
water or not. The author finds it a good plan to keep the
needles of hypodermic syringes in a small wire basket in
an oil bath. The oil may be heated to 150° to 200° and the
needles sterilized in a very few minutes. The oil also prevents
rusting.

Rooms, offices and all spaces which may be readily made
practically gas-tight are best disinfected by means of for-
maldehyde by any of the methods above described (Figs.
109 and 110).

Stables and Barnyards (Mohler): “A preliminary cleaning
up of all litter is advisable together with the scraping of the
floor, mangers, and walls of the stable with hoes and the
removal of all dust and filth. All this material should be
burned since it probably contains the infective agent. Heat
may be applied to the surfaces, including barnyard, by means
of a ‘cyclone oil burner.’ When such burning is impracti-
cable, the walls may be disinfected with one of the following:

“1. Whitewash 1 gallon + chloride of lime 6 ounces.

“2. Whitewash 1 gallon + crude carbolic acid 7 ounces.

“3. Whitewash 1 gallon + formalin 4 ounces.
The same may be applied with brushes or, more rapidly,
sprayed on with a pump; the surface soil of the yard and
surroundings should be removed to a depth of 5 or 6 inches,
placed in a heap and thoroughly mixed with quicklime.
The fresh surface of soil thus exposed may be sprinkled with
a solution of a chemical disinfectant as above described.

“Portions of walls and ceiling not readily accessible may
be disinfected by chlorine gas liberated from chloride of
lime by crude carbolic acid. This is accomplished by mak-
ing a cone of 5 or 6 pounds of chloride of lime in the top of

154 DISINFECTION AND STERILIZATION

which a deep crater is made for the placement of from 1
to 2 pints of crude carbolic acid. The edge of the crater is
thereupon pushed into the fluid, when a lively reaction fol-
lows. Owing to the heat generated, it is advisable to place

Fig. 109. — Formaldehyde
generator used in city work for Fig. 110. — Government formalde-
room disinfection. hyde generator.

the chloride of lime in an iron crucible (pot), and to have
nothing inflammable within a radius of two feet. The num-
ber and location of these cones of chloride of lime depend
on the size and structure of the building to be disinfected.

PRACTICAL STERILIZATION AND DISINFECTION 155

As a rule it may be stated that chlorine gas liberated from
the above sized cone will be sufficient for disinfecting 5200
cubic feet of air space.”

Inquid manure, leachings, etc., where collected are thor-
oughly disinfected by chloride of lime applied in the pro-
portion of 2 parts to 1000 of fluid.

Vehicles may be thoroughly washed with 2 per cent. for-
malin solution, or if closed space is available, subjected to
formaldehyde gas disinfection, after cushions, hangings, etc.,
have been removed and washed with the disinfectant.

Fig. 111.—Chamber used in government work for formaldehyde disinfec-
tion. The small cylinder at the side is the generator.

Harness, brushes, combs should be washed with a solution
of formalin, carbolic acid, or creolin as given under these
topics.

Washable articles should be boiled, dropped into disinfectant
solutions as soon as soiled, and then boiled or steamed.

Unwashable articles—burn all possible. Use formaldehyde
gas method in a closed receptacle (Fig. 111).

Stock cars—the method described for stables is applicable
here.

Animals, large and small, may have the coat and surface
of the body disinfected by washing with 1 to 1000 bichloride
or strong hot soapsuds to which carbolic acid has been added
to make a 5 per cent. solution; they should then be given a
good warm bath.

156 DISINFECTION AND STERILIZATION

Frequently time and money are saved by a combination
of steam and formaldehyde disinfection. This is a regular
practice in municipal and quarantine disinfection (Fig. 112).

Fig. 112.—Chamber in actual use at government quarantine station for
disinfecting baggage and dunnage with steam or formaldehyde or both.
The small cylinder at the side is the steam formaldehyde generator.

Persons engaged in disinfection work should wear rubber
boots, coats, and caps which should be washed in a disinfec-
tant solution and the change to ordinary clothing made in
a special room so that no infective material will be taken
away.

PART III.

THE STUDY OF BACTERIA.

CHAPTER XVI.
CULTURE MEDJA.

Tue study of bacteria may be taken up for the disci-
plinary and pedagogic value of the study of a science; with
the purely scientific idea of extending the limits of knowl-
edge; or for the purpose of learning their beneficial or
injurious actions with the object of taking advantage of
the former and combating or preventing the latter.

Since bacteria are classed as plants, their successful study
implies their cultivation on a suitable soil. A growth of
bacteria is called a “culture” and the “soil” or material
on which they are grown is called a “culture medium.”
Insofar as the culture medium is made up in the laboratory
it is an “artificial culture medium’’ as distinguished from a
natural medium. A culture consisting of one kind of bac-
teria only is spoken of as a “pure culture,’ and accurate
knowledge of bacteria depends on obtaining them in “pure
culture.” After getting a “pure culture” the special charac-
teristics of the organism must be ascertained in order to dis-
tinguish it from others. The discussion of the morphology
of bacteria in Chapters II, III and IV shows that the
morphological structures are too few to separate individual
kinds. They serve at best to enable groups of similarly
appearing forms to be arranged. Hence any further differ-
entiation must be based on a study of the physiology of the

158 CULTURE MEDIA

organism as discussed in the chapters on Physiological
Activities of Bacteria.

The thorough study of a bacterium involves, therefore:
its isolation in pure culture, its study with the microscope
to determine morphological features and special staining
reactions; growth on culture media for determining its
physiological activities, as well as the morphological charac-
teristics of the growths themselves; animal inoculations and
special serum reactions. Since isolation in pure culture
requires material for growing the organism, the first subject
to be considered is culture media.

A culture medium for a given bacterium must not only
contain the elements necessary for its food, insofar as these
may not be derived from the air, but must contain these
elements in a form readily available to the bacterium.
Further, the medium must not be too dry in order to furnish
sufficient moisture for growth and to prevent too high a con-
centration of the different ingredients. The reaction must
be carefully adjusted to suit the particular organism dealt
with. There must be no injurious substances present. The
above are the chief points to be borne in mind in preparing
culture media. Ordinarily, more attention must be paid to
the sources of the two elements N and C than to the others,
for in general the substances used to furnish these two and
the water contain the other elements in sufficient amount.
For very exact work on the products of bacteria, synthetic
media containing definite amounts of chemicals of known
composition have been prepared, but for most of the work
with bacteria pathogenic to animals such media are not
needed.

Culture media may be either /iguid or solid, or for certain
purposes may be liquid at higher temperatures and solid at
lower, as indicated later. Liquid media are of value for
obtaining bacteria for the study of morphology and cell
groupings and for ascertaining many of the physiological
activities of the organisms. Solid media are useful for study-
ing some few of the physiological activities and especially
for determining characteristic appearances of the isolated
growths of bacteria, These isolated growths of bacteria on

MEAT BROTH 159

solid media are technically spoken of as “colonies,’’ whether
they are microscopic in size or visible to the unaided eye.

It is clear that the kinds of culture media used for the
study of bacteria may be unlimited, but the undergraduate
student will need to use a relatively small number, which
will be discussed in this section.

Meat Broth (Bouillon).'.—This is used as a medium itself,
and as the basis for the preparation of other solid and liquid
media.

Finely ground lean beef is selected because it contains the
necessary food materials. Fat is not desired since it is a
poor food for most bacteria and in the further processes of
preparation would be melted and form an undesirable film
on the surface of the medium. The meat is placed in a
suitable container and mixed with about twice its weight of
cold water (not distilled) and allowed to soak overnight or
longer. The cold water extracts from the meat water-
soluble proteins, blood, carbohydrates in the form of dextrose
(occasionally some glycogen), nitrogenous extractives and
some of the mineral salts. The fluid is strained or pressed
free from the meat. This “meat juice’ should now be thor-
oughly boiled, which results in a coagulation of a large part
of the proteins and a precipitation of some of the mineral
salts, particularly phosphates of calcium and magnesium,
both of which must be filtered off and the water loss restored
by adding the proper amount of distilled water. The boil-
ing is done at this point because the medium must later
be heated to sterilize it and it is best to get rid of the
coagulable proteins at once. The proteins thus thrown out
deprive the medium of valuable nitrogenous food material
which is replaced by adding about 1 per cent. by weight of
commercial peptone. It is usual also (though not always
necessary) to add about 0.5 per cent. by weight of
common salt which helps to restore the proper concentra-
tion of mineral ingredients lost by the boiling. The chlorine
is also an essential element. The reaction is now deter-

1 The exact laboratory details for preparing various media are not given

in this chapter. It is the object to explain the choice of different materials
and the reasons for the various processes to which they are subjected.

160 CULTURE MEDIA

mined best by titration with twentieth-normal sodium
hydroxide solution with phenolphthalein as indicator. The
amount of normal alkali or acid as found by titration is now
added to the medium to make the desired end reaction (most
commonly 1 to 1.5 per cent. acid to phenolphthalein) and the
medium is again thoroughly boiled and filtered boiling hot.
The titration and boiling ordinarily cause a precipitate to
form which is largely phosphates of the alkaline earths with
some protein. The filtered medium is collected in suitable
containers, flasks or tubes, which are plugged with well-
fitting non-absorbent cotton plugs and sterilized, best in the
autoclave for twenty minutes at 15 pounds’ pressure, or discon-
tinuously in streaming steam at 100°. If careful attention is
paid to titration and to sufficient boiling where indicated, the
meat broth prepared as above should be clear, only faintly
yellowish in color and show no precipitate on cooling.

Broth may be prepared from Liebig’s or Armour’s meat
extract by adding 5 grams of either, 10 grams peptone and
5 grams NaCl to 1000 c.c. of water, boiling to dissolve, then
titrating and filtering as above.

The author after much experience finds meat juice prefer-
able to meat extract for broth and other media for patho-
genic bacteria, and has abandoned the use of meat extracts
for these organisms.

Glycerin Broth.—Glycerin broth is made by adding 4 to
6 per cent. of glycerin to the broth just previous to the
sterilization. The glycerin serves as a source of carbon to
certain bacteria which will not grow on the ordinary broth—
as Bacterium tuberculosis.

Sugar Broths.—Sugar broths are used for determining the
action of bacteria on these carbohydrates, since this is a
valuable means of differentiating certain forms, especially
those from the intestinal tract. Broth free from sugar must
first be made. This is done by adding to broth prepared as
already described, just previous to final filtering and steriliza-
tion, a culture of some sugar-destroying organism (Bacillus
colt is ordinarily used), and then allowing the organism to
grow in the raw broth at body temperature for twenty-four
hours. Any carbohydrate in the broth is destroyed by the

GELATIN CULTURE MEDIUM 161

Bacillus coli. This mixture is then boiled to kill the Bacillus
colt, retitrated and then 1 per cent. by weight of required
sugar is added. Dextrose, saccharose and lactose are the
most used, though many others are used for special purposes.
After the sugar is added the medium must be sterilized by
discontinuous heating at 100° for three or four successive
days, because long boiling or heating in the autoclave splits
up the di- and polysaccharids into simpler sugars and may
even convert the simple sugars (dextrose) into acid.

Various other modified broths are frequently used for spe-
cial purposes but need not be discussed here.

Dunham’s peptone solution, frequently used to determine
indol production, is a solution of 1 per cent. of peptone and
0.5 per cent. of salt in tap water. It does not need to be
titrated, but should be boiled and filtered hot into tubes or
flasks and sterilized.

Nitrate Broth.- Nitrate broth for determining nitrate
reduction is 1 per cent. of peptone, 0.2 per cent. of C. P.
potassium nitrate dissolved in distilled water and sterilized.

Milk.—Milk is a natural culture medium much used. It
should be fresh and thoroughly skimmed, best by a separator
or centrifuge to get rid of the fat. If the milk is not fresh,
it should be titrated as for broth and the reaction adjusted
to 0.8 per cent. acid. The milk should be sterilized discon-
tinuously to avoid splitting up the lactose as well as action
on the casein and calcium phosphate.

Litmus Milk—Litmus milk is milk as above to which
litmus has been added as an acid production indicator. The
milk should show blue when the litmus is added or be made
to by the addition of normal NaOH solution. It should be
sterilized discontinuously. Frequently on heating litmus
milk the blue color. disappears due to a reduction of the
litmus. This blue color will reappear on shaking with air
or on standing several days, due to absorption of O and
oxidation of the reduced litmus, provided the heating has
produced no other change in the milk, as proper heating will
not.

Gelatin Culture Medium.—Gelatin to the extent of 10 to
15 pes cent. is frequently added to broth and gives a culture

1

162 CULTURE MEDIA

medium of many advantages. It is solid at temperatures
up to about 25° and fluid above this temperature, a property
which is of great advantage in the isolation of bacteria.
(See Chapter XVIII.) Further gelatin is liquefied (that is
digested, converted into gelatin proteose and gelatin pep-
tone, which are soluble in water and do not gelatinize) by
many bacteria and not by others, a valuable diagnostic fea-
ture. The gelatin colonies of many bacteria are very charac-
teristic in appearance, as is the growth of many on gelatin
in culture tubes.

Gelatin medium may be prepared by adding the proper:
amount of gelatin (10 to 15 per cent. by weight) broken into
small pieces, to broth, gently warming until the gelatin is
dissolved, standardizing as for broth, filtering and sterilizing.
It is usually cleared before filtering by stirring into the gela-
tin solution, cooled to below 60°, the white of an egg for
each 1000 c.c., and then thoroughly boiling before filtering.
The coagulation of the egg albumen entangles the suspended
matter so that the gelatin filters perfectly clear, though
with a slight yellowish color. The filtering may be done
through filter paper if the gelatin is well boiled and filtered
boiling hot, but is more conveniently done through absorbent
cotton, wet with boiling water.

Or, the gelatin may be added to meat juice before it is
bowled, then this is heated to about body temperature (not
too hot, or the proteins will be coagulated too soon) until
the gelatin is dissolved. Then the material is standardized
and thoroughly boiled and filtered. The proteins of the
meat juice coagulate and thus clear the medium without
the addition of egg white. Commercial gelatin is markedly
acid from the method of manufacture, hence the medium
requires careful titration, even when made from a standard-
ized broth.

Gelatin should be sterilized by discontinuous heating at
100° on three successive days, because long boiling or heat-
ing above 100° tends to hydrolyze the gelatin into gelatin pro-
teose and peptone and it will not gelatinize on cooling. It
may be heated in the autoclave for ten to fifteen minutes at
10 pounds’ pressure and sometimes not be hydrolyzed, but

AGAR MEDIUM 163

the procedure is uncertain and very resistant spores may not
be killed. The medium should be put into the culture tubes
in which it is to be used as soon as filtered, and sterilized in
these, since, if put into flasks these must be sterilized, and
then when transferred to tubes for use, it must be again
sterilized unless great care is taken to have the tubes plugged
-and sterilized first, and in transferring aseptically to these
tubes. These repeated heatings are very apt to decompose
the gelatin, so it will not “set”? on cooling. The prepared
and sterilized tubes of gelatin should be kept in an ice-box
or cool room, as they will melt in overheated laboratories in
summer or winter.

Agar Medium.—Agar agar, usually called agar, is a com-
plex carbohydrate substance of unknown composition ob-
tained from certain seaweeds along the coast of Japan
and Southeastern Asia. It occurs in commerce as thin
translucent strips or as a powder. It resembles gelatin only
in the property its solutions have of gelatinizing when cooled.
Gelatin is an albuminoid closely related to the proteins, agar
a carbohydrate. Agar is much less soluble in water, 1 or
1.5 per cent. of agar giving a jelly as dense as 10 to 15 per
cent. of gelatin. It dissolves only in water heated to near
the boiling-point (98° to 99°) and only after much longer
heating. This hot solution “jells,” “sets” or gelatinizes at
about 38° and remains solid until again heated to near boil-
ing. Hence bacteria may be grown on agar at the body
temperature (37°) and above, and the agar will remain solid,
while gelatin media are fluid above about 25°. No patho-
genic bacteria and none of the saprophytes liable to be met
with in the laboratory are able to “liquefy”’ agar.

An agar medium is conveniently prepared from broth by
adding 1 or 1.5 per cent. of the finely divided agar to the
broth and boiling until dissolved, standardizing, clearing,
filtering, and sterilizing. The agar must be thoroughly
boiled, usually for ten to fifteen minutes, and the water loss
made up by the addition of distilled water before titration.
Agar is practically neutral so that there is little difference
between the titration of the dissolved agar and the original
broth. The agar solution should be kept hot from the begin-

164 CULTURE MEDIA

ning to the end except the cooling down to below 60°, when
the egg white for clearing is added. Though filtration
through paper is possible as with gelatin, if the agar solution
is thoroughly boiled and filtered boiling hot, it is more satis-
factory for beginners to use absorbent cotton wet with boil-
ing water, and to pour the hot agar through the same filter
if not clear the first time. The solidified agar medium is
never perfectly clear, but always more or less opalescent.
The agar medium may be sterilized in the autoclave for
fifteen minutes at 15 pounds’ pressure as the high temperature
does not injure the agar.

Potato Media.—Potatoes furnish a natural culture medium
which is very useful for the study of many bacteria. The
simplest, and for most purposes the best, way to use potatoes
is in culture tubes as “potato tube cultures” (No. 8, Fig.
117). These are prepared as follows: Large tubes are used.
With a cork-borer of a size to fit the tubes used, cylinders
about one and one-half inches long are cut from fairly large,
healthy potatoes. The skin is cut off square from the ends
and each cylinder cut diagonally from base to base. This
furnishes two pieces each with a circular base and an oval,
sloping surface. The pieces are then washed clean and
dropped for a minute into boiling water to destroy the oxi-
dizing enzyme on the surface which would otherwise cause
a darkening of the potato. (The darkening may also be
prevented by keeping the freshly cut potatoes covered with
clean water until ready to sterilize.) A bit of cotton one-
fourth to one-half inch in depth is put into each of the
test-tubes to retain moisture and a piece of potato dropped
in, circular base down. The tubes are then plugged with
cotton and sterilized in the autoclave at 15 pounds’ pressure
for not less than twenty-five minutes, since potatoes usually
harbor very resistant spores, and it is not unusual for a few
tubes to spoil even after this thorough heating.

Potatoes are sometimes used in “potato plate cultures.”
The term “plate culture” is a relic of the time when flat
glass plates were used for this and other “plate cultures.”
Now glass dishes of the general form shown in Fig. 113,
called ‘Petri dishes,” or plates are used for practically all

POTATO MEDIA 165

plate culture work. For “potato plates” slices from pota-
toes are cut as large and as thick as the relative sizes of
potato and dish permit (Fig. 114). The slices should be thin
enough not to touch the lid and thick enough to be firm.

Fic. 113.—Petri dish with the lid partly raised. X 4.

Fic. 114.—A potato plate. x 4.

It is a good plan to wrap each dish separately in paper to
retain the lid securely, then sterilize as for potato tubes,
and leave plates wrapped until wanted.

It sometimes happens that the natural acidity of potatoes

166 CULTURE MEDIA

is too great for the growth of many organisms. The acidity
is sufficiently corrected by saaking the pieces of potato in a
1 per cent. solution of sodium carbonate for an hour before
they are put into the tubes or plates.

Glycerinized potato tubes are conveniently prepared by
covering the potato in the tube with glycerin broth, steril-
izing and pouring off the excess broth immediately after
sterilizing, taking care that the tubes do not become con-
taminated which is not very probable if the work is quickly
done while the tubes are still hot.

Blood Serum Media.—Blood serum, usually from the larger,
domestic animals on account of convenience in securing it in
quantity, is used in the study of the bacteria causing disease
in man and animals. Most commonly the serum is collected
from the clotted blood after it has well separated (usually
about forty-eight hours is required for this). It is then run
into tubes which are plugged with cotton and placed in an
an apparatus for coagulating the serum by heat. A copper

* water bath with a tightly closed air compartment or the hori-
zontal autoclave (Fig. 80) is sufficient for this purpose, though
special forms of apparatus are to be had. It is important that
the temperature be raised slowly so that the blood gases escape
gradually. Three to five hours or longer should be allowed for
the temperature to reach the boiling-point. If the tubes are
heated too rapidly, the serum is filled with bubbles and badly
torn since the gases are driven off suddenly. Léffler’s serum is
made by adding one part of dextrose broth to three parts
of serum and then coagulating as above. The solid-
ified serum in either case is best sterilized discontinuously,
though with care the autoclave at 15 pounds’ pressure
may be used for a single sterilization. This is very apt to
cause a greater darkening of the serum and frequently also
a laceration of the solid mass by escaping gases.

Blood serum is also used in the liquid state. For this
purpose it is best to collect it aseptically; or it may be
sterilized discontinuously at a temperature of 55° or 56° on
seven to ten consecutive days. Novy has recently suggested
dialyzing the serum to free it from salts and thus prevent
its coagulation when heated. Whether the removal of the

BLOOD SERUM MEDIA 167

various “extractives” which diffuse out with the salts
deprives the serum of any of its advantageous properties
remains to be ascertained.

From the discussion of the physiological activities of bac-
teria in Chapters IX—XII it is apparent that a very great
variety of culture media other than those described is neces-
sary for the study of special types of bacteria, but such
media are beyond the scope of the present work.

The ideal culture media are without a doubt the synthetic
media, that is, media of definite known chemical composition,
so that the various changes due to the growth of bacteria
can be accurately determined and thus a means of sharply
differentiating closely related organisms be secured. Such
media have been prepared and every bacteriologist believes
strongly in their future usefulness when media of wider
application shall have been devised. An example of this
type of culture media is Uschinsky’s synthetic medium, of
which the following is one of the modifications:

Distilled water “ : 1000 parts
Asparagin. . ‘ tig 4 “
Ammonium lactate ‘ 6 “
Disodium phosphate . 2 a
Sodium chloride . ; 5.

A criticism of this medium is that the elements K, Ca,
Mg, Fe, Mn, and S which have been shown to be essential
are not present if chemically pure salts are used in the
preparation.

CHAPTER XVII.
METHODS OF USING CULTURE MEDIA.

THE way in which culture media shall be used depends
on the purpose in view. By far the larger part of bacterio-
logical work is done with cultures in “bacteriological cul-
ture tubes.” Various laboratories have their own special
types but all are more or less after the “Board of Health”
form. They differ from ordinary chemical test-tubes in that
they are usually longer, have no “lip” and have much thicker
walls to prevent breakage and consequent loss of the cul-
ture as well as danger from pathogenic organisms. The
author finds two sets of tubes most serviceable for student
use—one size 15 cm. long by 19 mm. outside diameter
(No. 9, Fig. 117), the other 15 em. long by 13 mm. (Nos.
1 to 7, Fig. 117). Culture tubes are conveniently used in
“wire baskets” circular or square in section and of a size
to correspond with the length and number of tubes used.
These baskets are light, do not break, and if made of good
galvanized wire netting do not readily rust (Figs. 115 and 116).

Liquid media such as broth, milk, litmus milk, indol and
nitrate broths are used in the above-mentioned tubes when
small quantities only are to be worked with. The tubes
are filled approximately one-third full, then plugged with
non-absorbent cotton and sterilized. Cotton plugs are used
so much in bacteriological work because they permit a free
circulation of air and gases and at the same time act as
filters to keep out the bacteria of the air.

Sugar broths or other media in which gas may be produced
are used in fermentation tubes (Smith’ tubes) of the type
shown in Fig..118 so that the gas may be collected in the
closed arm of the tube, measured (Fig. 119) and tested if
desired.

METHODS OF USING CULTURE MEDIA 169

One method of using gelatin and also agar is as “ puncture”
or “stab” cultures. The tubes (the narrower tubes are to be

4 oa MS,
Tune
Nite Ne 7)

mo

ay es at
Ce
ce os ‘

SASS

Fig. 115.—Round wire basket. Fie. 116.—Square wire basket.

Fig. 117.—Culture tubes with media in them. x 4. J to 7 are the
smaller tubes mentioned in the text; 9 the larger tube; 8 is extra large for
potato tubes; 1, plain broth; 2, plain milk; 3, litmus milk; 4, gelatin for
“stab” or ‘“‘puncture” culture; 5, agar for ‘‘stab” or ‘‘puncture” culture;
6, agar for ‘‘slope’’ or ‘‘slant’’ culture; 7, blood serum; 8, potato tube;
9, agar for plating. Note the transparency of the broth and gelatin and
the slight opalescence of the agar.

170 © METHODS OF USING CULTURE MEDIA

preferred for most “stab” cultures) are filled one-third full
of the medium while it is still fluid, plugged, sterilized and
allowed to cool in the vertical position. The medium is then
“inoculated” with a straight platinum needle by plunging
this into the center of the surface down to the bottom of the
tube (Fig. 117, Nos. 4 and 5).

Fic. 118.—Fermentation tubes. 1, filled ready for use; 2, shows a cloudy
growth and the development of gas in the closed arm.

Agar and blood serum are frequently used in the form of
“slope” or “slant” cultures. That is, the medium solidifies
with the tubes lying on their sides which gives a long, sloping
surface on which the bacteria are inoculated (Fig. 117,
Nos. 6 and 7).

Potato tubes are likewise used for “slant” or “slope”
cultures (Fig. 117, No. 8). Potatoes as “plate cultures”
have been referred to. Agar and gelatin are very largely

METHODS OF USING CULTURE MEDIA 171

Fie. 119.—Method of estimating percentage of gas in a fermentation tube
by means of the ‘‘gasometer.’’ The reading is 45 per cent.

Fic. 120.—A toxin flask showing a large surface growth.

172 METHODS OF USING CULTURE MEDIA

used in the form of “plate cultures” also. For this purpose
Petri dishes are first sterilized, then the melted agar
or gelatin poured in to them and allowed to “set” while
the plates are kept horizontal. The melted media may be
“inoculated” before they are poured, or a portion of the
material to be “plated” may be placed in the dish, then the
melted medium poured in and distributed over the dish by
tilting in various directions, or the medium after solidify-
ing may be inoculated by “strokes” or “streaks” over its
surface, according to the purpose in view in using the plate.
The larger sized tubes should be used for making plates in
order to have sufficient medium in the plate (No. 9, Fig. 117).

For using large quantities of medium, Florence flasks,
Ehrlenmeyer flasks, special toxin flasks (Fig. 120) or various
other devices (Vaughan and Novy’s “mass cultures,” Figs.
121 and 122) have been employed.

For growing anaérobic organisms it is evident that some
method for removing and excluding the.oxygen of the air
must be used. A very great variety of appliances have
been devised for these purposes. Some are based on the
principle of the vacuum, exhausting the air with an air
pump; some on replacing the air with a stream of hydrogen;
others on absorbing the oxygen by chemical means, as with
an alkaline solution of pyrogallic acid, or even by growing
a vigorous aérobe in the same culture or in the same con-
tainer with the anaérobe, the aérobe exhausting the oxygen
so that the anaérobe then develops, or finally by excluding
the air through the use of deep culture tubes well filled with
the medium, or in the closed arm of fermentation tubes.
For many purposes a combination of two or more of the
above methods gives good results.

In any event the culture medium should have been freshly
sterilized just before use, or should be boiled in order to drive
out the dissolved oxygen. For most anaérobes the presence
in the medium of about 1 per cent. of a carbohydrate, as
dextrose, is advisable.

A description of all the various devices is unnecessary in
this work, but the following have answered most of the
purposes of general work in the author’s laboratories.

METHODS OF USING CULTURE MEDIA 173

cl. “Vignal tubes” of the style shown (Fig. 123) are made
from glass tubes of about 6 to 8 mm. outside diameter,

Fig. 121.—Tank with raised lids. (Vaughan.)

LAs fA UA EAPC ROOT

; Fic. 122.—Tank with lids lowered. (Vaughan.)
Figs. 121 and 122.—Vaughan and Novy’s mass culture apparatus.

174 METHODS OF USING CULTURE MEDIA

sealed at the small end, plugged with cotton above the
constriction and sterilized. The medium, agar or gelatin,
which has been previously inoculated with the anaérobic
culture, is then drawn up into the tube, after breaking off
the tip, as far as the constriction. The tube is then sealed
in the flame at the small end and also at the constriction.
Since it is full of the medium and sealed, access of air is
prevented. This forms an excellent means for “isolation”
(Chapter XVIII); the tube needs merely to be cut with a
file at the point where colonies appear, then these may be
readily transferred.

Fic. 123.—Vignal tubes. xX 4. 1, the sterile tube ready for inoculation;
2, fourth dilution tube showing a few isolated colonies, one near the figure;
3, third dilution tube showing colonies isolated but numerous; 4, second
dilution tube showing colonies still more numerous; 4, first dilution tube
showing colonies so numerous and small as to give a cloudy appearance to
the tube. In use tube 2 would be filed in two at the colony and inoculations
made from it.

B. “Fermentation tubes” form a simple means for growing
liquid cultures of anaérobes, the growth occurring in the
closed arm only, while with facultative anaérobes, growth
occurs both in the closed arm and in the open bulb. A little
“paraffin oil” (a clear, heavy petroleum derivative) may be
poured on the fluid in the open bulb as a very efficient
seal, though it is not usually necessary.

C. “Deep culture tubes.”—The medium, agar, gelatin or
a liquid, is poured into tubes until they are approximately
one-half full, a little paraffin oil is poured on the surface
(not essential always), then the tubes are plugged and steril-

METHODS OF USING CULTURE MEDIA 175

‘ized. Inoculation is made to the bottom and anaérobes
grow well (Fig. 124).

D. For slope or plate, or any type of surface cultures the
Novy jar (Fig. 125) is the most practical device. It is good

wr

x

Fic. 124.—Deep tubes showing anaérobic growth. 1, shows a few small
gas bubbles; 2, shows the medium broken up by the excessive development
of gas.

practice to combine the vacuum method, the hydrogen
replacement method and the oxygen absorption method in
using these jars. In operation a solution of 20 per cent.
.NaOH is poured on the bottom of the jar to a depth
of 1 or 2 cm., the cultures are placed on glass supports |
above the alkali and a short wide tube of strong pyrogallol
is set in on the bottom in such a way that it may be easily

176 METHODS OF USING CULTURE MEDIA

upset and mixed with the alkali when it is desired to do so.
The cover is now clamped in position with ‘all joints well
vaselined. Then the outlet tube is connected with a suc-
tion pump and the air drawn out. Meanwhile the inlet
tube has been connected with a hydrogen generator, and
after the jar is exhausted hydrogen is allowed to flow in,
and this process is repeated until one is satisfied that the
air is replaced. The suction exhausts the air from the tubes
or plates so that much less time is required to replace the
air with hydrogen. Finally, the stop-cock is closed, and the
pyrogallol solution is gently shaken down and mixed with
the alkali so that any remaining oxygen will be absorbed.

=

Fic. 125.—Novy jars.

It must be remembered that facultative anaérobes as well
as anaérobes will grow under any of the above conditions,
so that cultures of organisms so obtained must be further
tested aérobically in order to determine to which group
the organisms belong.

Reference has been made above to the “inoculation” of
culture media, which means introducing into the medium
used the desired material in the proper way. For small
quantities this is most conveniently done with platinum
“needles,” that is, pieces of platinum wire inserted into the
ends of glass rods. The “straight” needle is a piece of heavy
platinum wire of about 0.022 inch in diameter (Fig. 126).
It is used most frequently to inoculate all forms of solid

METHODS OF USING CULTURE MEDIA 177

media. The platinum loop is of lighter wire, 0.018 inch.
The loop in the end is conveniently made by twisting the
wire around the lead of an ordinary lead-pencil. The “loop
needle” (Fig. 127) is most used in transferring liquid media.
On account of the high price of platinum, the author has
substituted “nichrome” wire for student use. This is stiffer,
not so easily made into loops and breaks out of the rods
more easily. The latter defect is remedied to some extent
by imbedding the wire only slightly for about one-fourth

Fic. 126.—Straight needle.

rt)

>————————SSEEE

|

Fic. 127.—Straight and loop needles.

Fig. 128.—Pasteur flask—‘'‘ ballon pipette.”

of an inch on the side of the end portion of the rod. The
low cost, less than one-twentieth of platinum, justifies
its use.

Sterile graduated pipettes varying in capacity from 1 c.c.,
graduated in hundredths, upward, permit the transfer of
definite amounts of liquids. Large quantities are conven-
iently transferred by means of Pasteur flasks (Fig. 128).
The details of inoculation are best derived from laboratory
practice.

12

CHAPTER XVIII.
ISOLATION OF BACTERIA IN PURE CULTURE.

As has been stated, the thorough study of a bacterium
depends on first getting it in pure culture. In the early
days of bacteriology supposedly pure cultures were obtained
by (1) dilution in liquid media. A series of tubes or flasks
containing sterile liquid media was prepared. Number one
was inoculated with the material to be examined and thor-
oughly mixed. A small portion of the mixture was trans-
ferred to number two, and mixed; from this to number
three, and so on until a sufficient number were inoculated,
the last three or four in the series receiving the same amounts
of a very high dilution of the original material. If one or
two of these latter showed a growth and the others not, it
was assumed that the dilution had been carried so far that
only a single organism was transferred and therefore the
culture obtained was “pure.” The method in this crude
form is too uncertain to be of value today and recourse is
had to more exact means. The procedure most widely used
is that of (2) “plating out” by means of gelatin or agar
plates. The material to be plated out is diluted by trans-
ferring to three or more tubes of melted gelatin or agar as
in the first method and then all the tubes are poured into
Petri dishes and grown under suitable conditions. By
proper mixing in the tubes the bacteria are well scattered
through the medium which holds the individual organisms
separate when it solidifies. On some of the plates a suffi-
cient dilution will be reached so that the colonies develop-
ing from the bacteria will be so few that they are separate
and pure cultures may be obtained by inoculating from one
of these a tube of the appropriate medium (Figs. 129
to 182). The chief uncertainty with this method is that
occasionally two kinds of bacteria stick together so closely

ISOLATION OF BACTERIA IN PURE CULTURE 179

that even the separate colonies contain both organisms.
This is not common, however. The plate colonies frequently

Fic. 129.—Dilution plates. 3%5. 1, shows the first dilution, the colo-
nies are so numerous and small that they are invisible (compare Fig. 130);
2, shows fewer and hence larger colonies, but too crowded to isolate (compare
Fig. 131); 3, shows the colonies, larger and well separated, so that it ‘is easy
to isolate from them (compare Fig. 132).

develop from groups of bacteria which were not separated,
but as these are of the same kind the culture is essentially
pure.

Fig. 130.—A portion of plate 1 in Fig. 129 as seen under the low-power
objective. X 100. Very small, closely crowded colonies.

Another method which is frequently applicable with
material from human or animal sources is to (3) rub the
material over the surface of a slope tube or of medium solid-
ified in a Petri dish with a sterile heavy platinum needle,

180 ISOLATION OF BACTERIA IN PURE CULTURE

glass rod, ‘or cotton swab. If the bacteria are not too numer-
ous, pure cultures may frequently be obtained. A modifi-
cation of this method is to make a series of (4) parallel
streaks on a slope tube or plate of medium with a needle
inserted but once into the material to be plated. On the first
streak a large part of the bacteria are rubbed off and a
continuous growth results, but usually on the last of a series
only isolated colonies appear, which are presumably pure.
The ideal method for securing pure cultures is to be abso-
lutely certain that the culture starts from a single organism.

So

Fig. 181.—From the thinnest part Fic. 132.—The smallest colony
of plate 2, Fig. 129,asseenunder the on plate 3, Fig. 129, as seen under
low-power objective. 100. Colo- the low-power objective. x 100.

nies much larger than on plate 1, Large single, isolated colony.
but still crowded.

This may be accomplished by means of the (5) apparatus
and pipettes devised by Professor Barber of the University of
Kansas (Figs. 133 and 134). With this instrument a single
organism is picked out under the microscope and isolated in a
drop of culture medium and observed until it is seen to divide,
thus proving its viability. Transfers are then made to the
proper media. The method requires much practice to develop
the necessary skill in the making of pipettes, determining the
proper condition of the large cover-glasses used over the
isolating box, and in manipulation, but the results fully
compensate,

ISOLATION OF BACTERIA IN PURE CULTURE 181

Professor W. A. Starin of the author’s department, a
former student of Professor Barber, has done some excellent
work with this apparatus.

Fic. 133.—Diagram of Barber's isolation apparatus. 6, moist chamber;
ms, large cover-glass over moist chamber; 7, small pipette drawn out to a
fine point; k, r, g, pipette holder; f, screw for raising and lowering k, r, g; s, ;
screw for lateral motion of k,r, g; n, screw for clamp on pipette which allows
it to be moved in or out; m, mechanical stage of microscope; t, rubber tube

‘held in the mouth and used to move the liquid culture medium in the
pipette. (Journal of Infectious Diseases, October 20, 1908, vol. 5, No.4, p.381.)

182 ISOLATION OF BACTERIA IN PURE CULTURE

A number of procedures may be used to greatly facilitate
the above methods of isolation by taking advantage of the
different physiological properties of different organisms in a
mixture, such as ability to form spores, different resistance
to antiseptics, special food requirements, and pathogenic

Fic. 134.—Photograph of microscope with Barber’s isolation apparatus
set up to use.

properties. (a) If material contains resistant spores, it may
be heated to temperatures high enough to kill all of the organ-
isms except the spores (80° for half an hour, for example)
and then plated out. Or (b) an antiseptic which restrains
the growth of some organisms and not others may be placed

ISOLATION OF BACTERIA IN PURE CULTURE 183

in the culture media (carbolic acid, various anilin dyes,
excess acid, or alkali, ox bile, etc.), when the more resistant
organisms grow on the final plates, the others not. (c)
Special food substances (various carbohydrates) from which
the organism desired forms special products (acids, alde-
hydes) that may be shown on the plates by various indica-
tors, is one of the commonest means. Or media in which
certain organisms thrive and others not, so that the former
soon “crowd out” the latter (unsterilized milk for lactic acid
bacteria, inorganic media in soil bacteriology), may be used. A
combination of the general methods (b) and (c) is much used
in the separation of the organisms of the “intestinal group”
in human practice. (d) The inoculation of a susceptible ani-
mal with a mixture suspected to contain a given pathogenic
bacterium frequently results in the development of the
latter in pure culture in the body of an animal, from which
it may be readily recovered. In all of the above methods
(except Barber’s) the first “pure culture’? obtained should
be “ purified” by replating in a series of dilution plates to
make sure that it is pure.

CHAPTER XIX.
STUDY OF INDIVIDUAL BACTERIA-STAINING.

WHEN an organism has been obtained in pure culture by
any of the methods described in the preceding chapter the
next step is the study of its morphology as discussed in
Chapters I-IV. This involves the use of the microscope,
and since bacteria are so small, objectives of higher power
than the student has presumably used will be needed.
Doubtless only the two-third inch or 16 mm. and the one-
sixth inch or 4 mm. objectives are all that have been used
in previous microscopic work, while for examining bacteria
a one-twelfth inch or 2 mm. is necessary. It will have
been observed that the higher the power of the objective
the smaller is the front lens or object glass and consequently
the less is the amount of light which enters. With the use
of the one-twelfth inch or 2 mm. objective it is necessary to
employ two devices for increasing the amount of light enter-
ing it, with which the student is probably not familiar. One
of these is to place a drop of cedar oil between the front
jens and the object and to immerse the lens in this oil—
hence the term ‘‘oil-immersion objective; the other is the
substage or Abbé condenser. The latter is a system of
lenses placed below the stage and so constructed as to bring
parallel rays of light—daylight—trom an area much larger
than the face of the front lens of the objective to a focus
on the object to be examined, thus adding very greatly to
the amount of light entering the objective. Since the con-
denser brings parallel rays to a focus on the object the flat-
mirror 1s always used with the condenser when working
with daylight. With artificial light close to the microscope,
the concave mirror may be used to make the divergent rays
more nearly parallel and thus give better illumination.

The function of immersion oil is to prevent the dispersion

HANGING DROP SLIDE 185

of considerable light that would otherwise occur owing to
refraction as the light passes up through the slide and into
the air. The accompanying diagram will help to make this
clearer (Fig. 135). A ray of light (.f. B) coming through the
slide will be refracted in the direction B C if the medium
has a lower refractive index than the slide, as air has, and
hence will not enter the objective O. If, however, there is
interposed between the objective and the slide a medium
which has the same refractive index as the slide, as immer-
sion oil has, then the ray will continue in the same direction
(B D) at the point B and hence enter the objective. Evi-

ront lens of objective

Immersion oil

a \ Slide

i
Fig. 135.—Diagram of use of immersion oil. {

dently the immersion oil causes much more light to enter
the front lens and makes the field brighter and at the same
time prevents considerable refraction and dispersion of light
from the object seen and hence this appears more distinct
and sharply defined. The Abbé condenser and the oil-
immersion objective are practically always used in the
microscopic study of bacteria (Fig. 136).

HANGING DROP SLIDE.

It is sometimes necessary to examine living bacteria and
for this purpose the device known as the “hanging drop

Fig. 136.— Diagram of paths of rays in microscope.

HANGING DROP SLIDE 187

slide” is used (Fig. 137). The slide has a slight concave
depression ground in the middle of one face. A ring of
vaseline is placed around this depression with the loop
needle. On a clean cover-glass, large enough to fit over the
ring of vaseline, several drops of a broth culture, or of material
from a solid culture suspended in broth or physiological
normal salt solution are placed. The slide is inverted on
the cover-glass in such a way that the ring of vaseline seals
the latter to the slide. When the whole preparation is
quickly turned cover side up, the drops are seen “hanging”’
to the under side of the cover over the depression in the
slide. In examining such a preparation with the micro-
scope great care is necessary in order to focus on the bac-
teria without breaking the cover. To see the organisms
distinctly the lower iris diaphragm of the condenser must be
nearly closed, so that the light coming through consists mainly

Cc SS |

Fic. 137.—Hanging drop slide.

of parallel vertical rays, otherwise the transparent bacteria
themselves refract and diffract the light and appear blurréd
and indistinct. By studying living bacteria with this device
it can be determined whether they are motile or not. The
motility should not be confounded with the familiar “ Brown-
ian movement” of all minute insoluble inert particles which
non-motile living bacteria and also dead bacteria show.
The hanging drop slide is of value in the measurement of
bacteria, since this is properly done on the living organism.
Measurement is done with a calibrated ocular micrometer
as in other kinds of measurement with the microscope with
which the student is presumably familiar. The direct effect
of various agents on living bacteria as light, electricity,
heat, etc., in the study of “tropisms” and “taxes” has been
investigated on various modifications of the above-described
hanging drop slide.

Cell forms and cell groupings may be studied in the same

188 STUDY OF INDIVIDUAL BACTERIA-STAINING

way but these features are best determined on starned
preparations.

“Dark field” illumination and the ultramicroscope are
of great value in the study of living bacteria and other
minute objects, but apparatus of this type would scarcely
be used by the elementary student, so that they will not be
discussed in the present volume.

STAINING.

The main use of the microscope in bacteriology is in the
study of stained preparations of the organisms. Staining
makes bacteria opaque and hence more easily seen than the
transparent unstained forms. Some methods of staining also
show morphological structures which are either imperfectly
recognized in the unstained cell, spores, or are not visible
at all—capsules, metachromatic granules, flagella. Finally
certain bacteria are colored by special methods of staining
which do not affect others, so that under proper conditions
these bacteria may be recognized by staining methods alone,
—tubercle bacilli in the organs of animals.

The phenomena of staining are essentially chemical,
though sometimes the chemical union is a very weak one,
even resembling an adsorption of the dye rather than true
chemical union—most watery stains. In other cases the
chemical compounds formed are decidedly stable and are
not decomposed even by strong mineral acids—staining of
tubercle bacilli and other “acid-fast” organisms. In still
other cases the principal action is a precipitation on the
surface of the object stained—methods for staining flagella.

In many methods of staining in addition to the dves used
other substances are added to the solution which assist in
fixing the dye in or on the organism stained. Such substances
are called mordants. The principal mordants used are alka-
lies, anilin, carbolic acid, iodine, metallic salts, tannic acid.

While it is true that some bacteria may be stained by that
standard histological nuclear dye, hematoxylin, it is of little
value for this purpose. Practically all bacteriological stains
are solutions of the anilin dyes. These dyes, as is well

STAINING 189

known, are of nearly every conceivable color and shade but
relatively very few are used in bacteriological work. The
elementary student will rarely use solutions of other than
the three dyes fuchsin (red), methylene blue and gentian
volet for staining bacteria, with occasionally Bismarck
brown, or eosin, or safranin as tissue contrast stains.

The bacteriological dyes are kept “in stock” as saturated
solutions in 95 per cent. alcohol which are never used as
stains, but merely for convenience in making the various
staining solutions of which the following are the most
common:

1, Aqueous (watery) gentian violet solution.

Saturated alcoholic solution of gentian violet Sree 3 a 1 part
Distilled water . 20 parts
Mix well and filter.

2. Anilin gentian violet.

Saturated alcoholic solution of gentian violet é 1 part
Anilin water (see below) ; . 10 parts
Mix well and filter.

3. Anilin Fuchsin.

Saturated alcoholic solution of fuchsin Bon 1 part
Anilin water (see below) . . 10 parts
Mix and filter.

These stains rarely keep longer than ten days in the
laboratory (unless kept in the ice-box) and must be made
fresh on the first sign of a deposit on the glass of the container.

Anilin Water.—Anilin water is made by putting 3 or 4 e.c.
of anilin “oil” in a 120 c.c. flask, adding 100 c.c. of distilled
water, shaking vigorously for a minute or so and filtering
through a wet filter, in other words, a saturated solution
of anilin in water.

4, Loffler’s (methylene) blue.

Saturated alcoholic solution of methylene blue . 3 parts
Aqueous solution of NaOH (or KOH), 1 to 10,000 . . 10 parts
Mix and filter.

5. Carbol-fuchsin (Ziehl’s solution).

Saturated alcoholic solution of fuchsin. 1 part
5 per cent. aqueous solution of carbolic acid ane 10 parts

Mix and filter.

190 STUDY OF INDIVIDUAL BACTERIA-STAINING

6. Gabbet’s (methylene) blue (solution).

Dry methylene blue 2 ‘ 4 parts
Concentrated H.SO,. . “ 25 parts
Distilled water . . . 75 parts

Dissolve the dry dye in the ‘acid and add the solution to the dis-
tilled water and filter.

Staining solutions are conveniently kept in square drop-
ping bottles inserted in a block as shown in Fig. 138. This
form of holder necessitates the use of one hand only in secur-
ing the stain and dropping it on the preparation.

LUEAM LOEFFLERS
SOLUTION L3U gs

Fig. 138.—Author’s staining set. Square bottles are set in square holes in
the block. The capacity of each bottle is 30 c.c.

The actual staining of bacteriological preparations can
be learned only by repeated laboratory practice, yet the
following methods have given such uniform results in class
work that it is felt they are not out of place in a text-book.

Preparation of the “Film.’—The author learned to stain
bacteria on the “cover-glass’’ but does not recall having
used this method in fifteen years and does not teach it to
his students. All staining is done on the slide. To prepare
a film from a solid culture medium the procedure is as
follows:

STAINING 191

First, be sure the slide is clean and free from grease. This
is accomplished most readily by scouring a few minutes with
finely ground pumice stone and a little water, then washing
and drying with a grease-free cloth, handkerchief, or piece
of cheese-cloth, With the “loop’’ needle place in the middle
of the slide a small loop of water. This is best done by
filling the loop by dipping in water, then tapping it gently
so that all that remains is the water that just fills the loop
level full, and this amount is placed on the slide by touching
the flat side of the loop to the glass. Then the straight
needle is sterilized, dipped into the culture and just touched
once into the small drop of water on the slide. The remain-
der of the culture on the straight needle is then burned off
and the needle is used to spread the drop of water contain-
ing the bacteria into a thin even film, which will result, pro-
vided the slide is free from grease. This is dried and then
“fixed” by passing three times through the Bunsen flame at
intervals of about one second, passing through slowly for
thick slides and a little more rapidly for thin ones. If the
culture is in a liquid medium, the use of the loop of water is
unnecessary; a loop of the fluid from the surface, middle or
bottom as the culture indicates is spread out to a thin film,
dried and fixed.

After the film is fixed the stain desired is dropped on,
allowed to act for the proper time, which will depend on the
stain and the preparation, washed in water, dried thor-
oughly and examined with the oil-immersion lens, without
a cover. If it is desired to preserve the preparation it may
then be mounted in balsam. This is not necessary, as they
keep just as well, provided the immersion oil is removed.
To do this, fold a piece of filter paper so that at least three
thicknesses result. Lay this on the slide and press firmly
several times, when the surplus oil will be taken up by the
paper. Slides not mounted in balsam are more apt to
become dusty than those that are. This is the only disad-
vantage. :

Gram’s Method of Staining.—It has been ascertained that
some bacteria contain a substance, possibly a protein, which
forms a compound with gentian violet and iodine, which

192. STUDY OF INDIVIDUAL -BACTERIA-STAINING

compound is insoluble in alcohol, and other bacteria do not
contain this substance. Consequently when bacteria are
stained by Gram’s method (given below), those that con-
tain this chemical remain colored, while if it is not present
the dye is washed out by the alcohol and the bacteria are
colorless and may be stained by a contrast stain. The bac-
teria which stain by this method are said to “take Gram’s”
or to be ‘Gram-positive,’ while those that decolorize are
called “ Gram-negative.’”” The method is:
. Prepare the film as above given.
. Stain with fresh anilin gentian violet 1 minute.
. Wash in tap water.
. Cover with Gram’s solution (1 minute).
. Wash in tap water.
. Wash with 95 per cent. alcohol three times or until no
more color comes out.

7. Dry and examine.

Gram’s solution is:

Oorkwwere

I 1 part
KI. 2 parts
H:.0 i 300 parts

This method is excellent for differentiating Gram-posi-
tive and Gram-negative organisms on the same slide. First
stain by this method and after washing with alcohol stain
with a counter-stain, carbol-fuchsin diluted ten to fifteen
times with water is excellent. The Gram-positive bacteria
are violet and the Gram-negative are red.

It is also of great value in staining Gram-positive bac-
teria in tissues, but the sections should be stained about five
minutes in the anilin gentian violet and be left about two
minutes in the Gram solution. Sections are to be counter-
stained in Bismarck brown, dilute eosin or safranin solutions
and cleared in oil of bergamot, lavender or origanum and
not in clove oil or carbol-xylol, as these latter dissolve out
the dye from the bacteria.

Staining of Spores in the Rod.—Prepare the films as usual.
Cover with carbol-fuchsin, using plenty of stain so that it
will not dry on the slide; heat until vapor arises, not to boil-
ing; cool until the stain becomes cloudy and heat again

STAINING 193

until the stain clears, and repeat once more; wash in tap
water and then wash in 1 per cent. H,SO, three times,
dropping on plenty of acid, tilting and running this over the
slide three times and then pour off and use fresh acid and
repeat this once. Wash thoroughly in distilled water, then
stain with Léffler’s blue one to three minutes. Wash, dry
and examine. The spores should be bright red in a blue rod.

This method will give good results if care is taken to
secure cultures of the right age. If the culture is too old
the spores will all be free outside the rods, while if too young
they will decolorize with the acid. For Bacillus subtilis and
Bactervum anthracis, cultures on agar slants forty-eight hours
in the 37° incubator are just right. For the spores of Bacillus
tetant, the culture should be three days old, but may be as
old as a week.

Staining of ‘“‘Acid-fast’ Bacilli— Bacterium tuberculosis,
Bacterium of Johne’s disease, “ grass’? and “butter bacilli,”
Bacterium lepre, Bacterium smegmatis.

Gabbet’s method:

. Prepare the film as usual.

. Stain with carbol-fuchsin as given above for spores.

. Wash with tap water.

. Decolorize and stain at the same time with Gabbet’s
blue, 2 or 3 minutes.

. Wash, dry and examine.

The sulphuric acid in Gabbet’s blue removes the carbol-
fuchsin. from everything except the “acid-fast” bacteria,
which remain red, and the blue stains the decolorized bacteria
and nuclei of any tissue cells present.

Rm whe

or

ZLiehl- Neelson method:

1, 2, 3, as in Gabbet’s method.

4. Decolorize with 10 per cent. HCl until washing with
water shows only a faint pink color left on slide.

5. Wash thoroughly.

6. Stain with Léffler’s blue 1 or 2 minutes.

7. Wash, dry and examine.

The results are the same as with Gabbet’s method.

13

194 STUDY OF INDIVIDUAL BACTERIA-STAINING

Staining of Capsules.—Rdbiger’s Method—Films of the
organism to show capsules should be freshly prepared, dried,
but not fixed. The preparation is covered with a freshly
prepared saturated solution of gentian violet in formalin and
this allowed to stain for from 30 to 90 seconds. Then wash
lightly, dry and examine. The organisms appear deeply
violet and much larger than with ordinary stains and capsules
are well stained and show well.

Welch’s Method.—Prepare films as in the above method.
Cover with glacial acetic acid for 10 to 20 seconds. Wash
off the acid with carbol-fuchsin. Wash the stain off with
physiological normal salt solution (0.85 per cent.) until all
surplus stain is removed. Dry and examine. Capsules and
bacteria are red.

Staining of Flagella—The rendering of flagella visible is
considered one of the most difficult processes in staining.
Experience of a number of years during which whole classes
numbering from one hundred to three hundred students
accomplish this result shows that it is no more difficult than
many other staining processes. The essentials are: (1)
clean slides, (2) young cultures on agar slopes, (3) freshly
prepared mordant and stain which are kept free from pre-
cipitate, (4) gentle heating. The author’s students are fur-
nished only stock materials and make their own cultures,
mordants and stains.

The slides are cleaned with pumice in the usual way. An
agar slope culture of the organism to be stained from 6 to
24 hours old is selected. A bit of the culture is removed and
placed in a watch-glass of water. The bacteria are allowed
to diffuse of themselves without stirring. After several min-
utes a loop of this water is removed and spread on the slide
with a single movement of the needle, dried and carefully
fixed. The film is then covered with an abundance of the
mordant by filtering through a small filter onto the slide so
that the mordant shows transparent on the slide. The
preparation is then gently warmed and cooled three times,
adding mordant if necessary. After mordanting for about five
minutes the excess is washed off under the tap. It is a good
plan to hold the slide level and allow the water to run into
the center of the mordant and flow it off. Inclining the slide

STAINING 195

is apt to cause the film on the surface of the mordant to
settle down on the slide and spoil the preparation. After
the mordant is washed off and all traces of it removed with
a clean cloth if necessary the stain is applied and gently
heated and cooled the same way for from three to five min-
utes. The preparation is then washed, dried and examined.

The mordant used is a modification of Léffler’s which is
somewhat simpler in preparation since the stock solution of
FeCl; is more permanent than FeSO, solution.

Mordant sufficient for one student:

5 per cent. solution of FeCl; 20.0 ec.
25 per cent: solution of tannic acid . . 20.0 ¢.c.
Anilin fuchsin : . 4.0 c.c.
Normal NaOH es 1.5 c.c.

The solution of FeCl; is made up in the cold and must be
perfectly clear. The tannic acid solution must be thor-
oughly boiled and filtered until clear. The iron and the acid
are carefully mixed, boiled and filtered clear. The anilin
fuchsin must be added slowly with constant stirring and the
mixture boiled and filtered. The NaOH is added in the
same way. The final mordant should not leave a film on a
clean slide when poured on and allowed to run off. Unless
the mordant is in this condition and perfectly clear, it should
not be used, but a new one must be made up. Time and
care in the preparation of the mordant are essential.

The stain to follow this mordant is anilin fuchsin.

By the use of the hanging drop slide and the methods of
staining just described all the various morphological fea-
tures of the bacterial cell may be ascertained.

It is necessary when cell groupings as characteristic of
definite modes of division are to be determined to make slides
from a liquid culture, as broth. Place a drop of the material,
preferably from the bottom of the tube in most instances,
from the top in case a pellicle or scum is formed on the sur-
face, on the slide and allow this to dry without spreading it
out, then stain lightly with Léffler’s blue. Such slides also
show characteristic cell forms as well. Slides should be
made from solid media to show variations in form and size
and involution forms. These latter are especially apt to
occur on potato media,

CHAPTER XxX.
STUDY OF THE PHYSIOLOGY OF BACTERIA.

Or the environmental conditions determining the growth
of bacteria the following are the chief ones ordinarily deter-
mined:

A. Temperature.—The optimum temperature for growth
is usually about the temperature of the natural environment
and ordinarily one determines merely whether the organism
grows at body temperature (37°) and at room temperature
(20°) or not. For exact work the maximum, minimum and
optimum temperature must be ascertained by growing in
“incubators” with varying temperatures.

A bacteriological incubator is an apparatus for growing
bacteria at a constant temperature. This may be any tem-
perature within the limits for bacterial growth. If tempera-
tures above that of an ordinary room are desired, some source
of. artificial heat is needed. Electricity, gas or oil may be
used. A necessary adjunct is some device for maintaining
the temperature constant, a “thermoregulator” or “ther-
mostat.” For lower temperatures a cooling arrangement
must be installed. For the great part of bacteriological work
only two temperatures are used, 20° so-called “room tem-
perature’ (this applies to European “rooms’’ not to Amer-
ican) and 37° or body temperature. Incubators for 37° of
almost any size and style desired may be secured from sup-
ply houses and need not be further described. Figs. 139
and 140 illustrate some of the types.

For use with large classes “incubator rooms” are to be
preferred. The author has one such room for 37° work with
200 compartments for student use which did not cost over
$60 to install.

The styles of incubators for lower temperatures, 20° and
below, are not so numerous nor so satisfactory. The author

Fic. 140.—Electric incubator.

198 STUDY OF THE PHYSIOLOGY OF BACTERIA

has constructed a device which answers every purpose for a
small class. The diagram, Fig. 141, explains it.

The thermal death-point is determined by exposing the
organisms in thin tubes of broth at varying temperatures
for ten-minute periods and then plating out to determine

Fig. 141.—Diagram of fittings for a cold incubator. 1, small tank for
constant head, about 1 foot in each dimension. a, inflow; b, overflow;
c, lead pipe. 2, refrigerator. a’, ice; b’, flat coil under ice; c’, outflow
to incubator. 3, incubator. a”, cold water inflow; b”, overflow; ther-
mometer and burner omitted. The diagram explains the construction.
The water cooled to about 14° with artificial ice by flowing through the
lead coil under the ice, flows into the incubator which may be heated and
regulated in the usual way. :

growth. The effect of heat may also be determined by
exposing at a given temperature, ec. g., 60°, for varying
lengths of time and plating out.

B. Oxygen relations—whether the organism is aérobic,
anaérobic, or facultative is determined by inoculation in

PHYSIOLOGICAL ACTIVITIES 199

gelatin or agar puncture or stab cultures and noting whether
the most abundant growth is at the top, the bottom or all
along the line of inoculation.

C. Reaction of the medium—acid, alkaline or neutral as
influencing the rate and amount of growth.

D. The kind of medium on which the organism grows best.

E. The effect of injurious chemicals, as various disinfec-
tants, on the growth.

F. Osmotic pressure conditions, though modifying decid-
edly the growth of bacteria are not usually studied as aids
in their recognition, nor are the effects of various forms of
energy, such as light, electricity, x-rays, etc.

Among the “Physiological Activities” discussed in Chap-
ters IX—XIJ those which, in addition to the staining reac-
tions described, are of most use in the identification of
non-pathogenic bacteria are the first ten listed below. For
pathogenic bacteria the entire thirteen are needed.

1. Liquefaction of gelatin.

2. Digestion of blood serum.

3. Coagulation and digestion of milk.

4, Acid or gaseous fermentation in milk, or both.

5. Acid or gaseous fermentation of various carbohydrates
in carbohydrate broth, or both.

6. Production of indol in “indol solution.”

_ 7. Production of pigments on various media.

8. Reduction of nitrates to nitrites, ammonia, or free
nitrogen.

9. Production of enzymes as illustrated in the above
activities.

10. Appearance of growth on different culture media.

11. Production of free toxins as determined by injection
of animals with broth cultures filtered free from bacteria.

12. Causation of disease as ascertained by the injection
of animals with the bacteria themselves, and recovery of the
organism from the animals.

13. Formation of specific antibodies as determined by the
proper injection of animals with the organism or its products
and the subsequent testing of the blood serum of the inocu-
lated animals.

200 STUDY OF THE PHYSIOLOGY OF BACTERIA

For special kinds of bacteria other activities must be
determined (oxidation, nitrate and nitrite formation, action
of sulphur and iron bacteria, etc.). :

The first nine activities are determined by inoculating
the different culture media already described and observ-
ing the phenomena indicated, making chemical tests where
necessary.

APPEARANCE OF GROWTH ON DIFFERENT CULTURE
MEDIA.

In addition to those changes that are associated with the
manifestation of different physiological activities of bacteria,
many show characteristic appearances on the various cul-
ture media which are of value in their identification.

Too much stress should not be laid on these appearances
alone, however, since slight variations, particularly in solid
media due especially to the age of the medium, may change
decidedly the appearance of a colony. This is true of
variations in the amount of moisture on agar plates.
Colonies which are ordinarily round and regular may assume
very diverse shapes, if there chance to be an excess of
moisture on the surface.

Also in slope and puncture cultures on the various solid
media much variation results from the amount of material
on the inoculation needle and just how the puncture is
made, or the needle drawn over the slope. These variations
are largely prevented by the use of standard media and by
inoculating by standard methods. The Laboratory Com-
mittee of the American Public Health Association has pro-
posed standard methods for all culture media and tests and
for methods of inoculation, and these have been generally
adopted in this country for comparative work.

Likewise the Society of American Bacteriologists has
adopted a standard “descriptive chart’’ for detailing the
characteristics of a given organism which includes the terms
to be used in describing the appearance on different culture
media. This chart is inserted in this chapter.

Missing Page

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Missing Page

GROWTH ON DIFFERENT CULTURE MEDIA 201

Among the cultural appearances the following are of most
importance:

Fic. 142.—Broth cultures X 3. 1, uninoculated transparent broth; 2,
broth cloudy from growth of organisms; 3, broth slightly cloudy with a
deposit,in bottom; 4, broth slightly cloudy with a heavy membrane at the
surface.

In broth cultures the presence or absence of growth on
the surface and ‘the amount of the same. Whether the

202 STUDY OF THE PHYSIOLOGY OF BACTERIA

broth is rendered cloudy or remains clear, and whether there
is a deposit at the bottom or not (Fig. 142). An abundant
surface growth with little or nothing below indicates a strict
aérobe, while a growth or deposit at bottom and a clear or
nearly clear medium above an anaérobe. These appear-

Fia. 143.—A _fili- Fic. 144.—A beaded Fig. 145.—A vill-
form stab or punc- stab or puncture cul- ous stab or puncture
ture culture. xX #. ture. xX #. culture. X }.

ances are for the first few days only of growth. If the broth
is disturbed, or after the culture stands for several days
many surface growths tend to sink to the bottom. So an
actively motile organism causes in general a cloudiness,
especially if the organism is a facultative anaérobe, which
tends to clear up by precipitation after several days when

GROWTH ON DIFFERENT CULTURE MEDIA 203
the organisms lose their motility. _Non-motile facultative

anaérobes usually cloud the broth also, but settle out more
rapidly than the motile ones.

Fig. 146 Fic. 147

Fie. 148 Fig. 149
Fic. 146.—Crateriform liquefaction of gelatin.

ae
Fic. 147.—Funnelform liquefaction of gelatin. xX 4.
Fig. 148.—Saccate liquefaction of gelatin. x }.
Fic. 149.—Stratiform liquefaction of gelatin. 4.

In gelatin and agar punctures the oxygen relationship is
shown by surface growth for aérobes, growth near the
bottom of the puncture for anaérobes, and a fairly uniform
growth all along the line of inoculation for facultative

204. STUDY OF THE PHYSIOLOGY OF BACTERIA

anaérobes. In the case of these last organisms, a preference
for more or less oxygen is indicated by the approach to the
aérobic or anaérobic type of growth.

Fie. 150.—Filiform Fic. 151.—Filiform, Fig. 152.—Beaded
slope culture. }. slightly spreading, slope culture. X 3.
slope culture. x 3.

Along the line of puncture the commonest types are
filiform (Fig. 143), which indicates a uniform growth; beaded
(Fig. 144), or small separate colonies; willous (Fig. 145), deli-
cate lateral outgrowths which do not branch; arborescent,
tree-like growths branching laterally from the line. In agar
these branchings are usually short and stubby, or technically,
papillate. .

GROWTH ON DIFFERENT CULTURE MEDIA 205

Further, in the gelatin puncture the liquefaction which
occurs is: frequently characteristic. It may be crateriform
(Fig. 146), a shallow saucer at the surface; or funnel-shaped

Fig. 153 Fic. 154 Fre. 155 Fic. 156

Fic. 153.—Effuse slope culture. x 3.
Fic. 154.—Rhizoid slope culture. x
Fig. 155.—Rugose slope culture.. x
Fic. 156.—Verrucose slope culture.

(Fig. 147); or it may be of uniform width all along the
puncture, 2. ¢., saccate (Fig. 148); or it may be stratiform,
(Fig. 149), 2. e., the liquefaction extends to the sides of the
tube and proceeds uniformly downward, .

206 STUDY OF THE PHYSIOLOGY OF BACTERIA

On agar, potato and blood serum slope tubes the amount
of growth, its form and elevation, the character of the sur-

Fic. 157.—Punctiform colonies on a plate. 3.

Fic. 158.—A rhizoid colony on a plate. Natural size.

GROWTH ON DIFFERENT CULTURE MEDIA 207

face, and the consistency should be carefully noted, and in
some few cases the character of the edge. Figures 150 to 156
show some of the commoner types.

Fie. 159.—Ameboid colonies on a plate. x }.

Fic. 160.—Large effuse colony on a plate. The edge is lacerated. Inci-
dentally the colony shows the rate of growth for six successive days. X 3.

208 STUDY OF THE PHYSIOLOGY OF BACTERIA

Fig. 161.—Colony with edge entire Fic. 162.—Colony with edge
as seen under the low-power objective. coarsely granular as seen under
xX 100° the low-power objective. 100.

Fig. 163.—Colony with edge Fig. 164.—Colony with edge rhiz-
curled as seen under the low-power oid as seen under the low-power
objective. > 100. objective. > 100.

\ *

j SS 5
a=

IIa. 165,—A sinall deep rhizoid colony as seen under the low-power objective.
x 100,

C

GROWTH ON DIFFERENT CULTURE MEDIA 209

On agar and gelatin plates made so that the colonies are
well isolated, the form of the latter, the rate of their growth,
the character of the edge and of the surface, the elevation
and the interna! structure as determined by a low-power
lens are often of almost diagnostic value. Also in the case
of the gelatin plates, the character of the liquefaction
is important. Figs. 157 to 165 show some of the commoner
characteristics to be noted.

14

CHAPTER XXI.
ANIMAL INOCULATION.

ANIMAL inoculation has been referred to (1) as a method
of assisting in the preparation of pure cultures of patho-
genic organisms; (2) as a means of testing the poisonous
properties of substances produced in bacterial cultures; (3)
in order to test the ability of an organism to cause a disease;
(4) for the production of various antibodies; it may be added
(5) that some bacteria produce in the smaller experimental
animals lesions which do not occur in animals naturally
infected, but which nevertheless are characteristic for the
given organism. The best illustration is the testicular reac-
tion of young male guinea-pigs to intraperitoneal injections
of glanders bacilli. Experimental animals are also inocu-
lated (6) to test the potency of various bacterial and other
biological products, as toxins, antitoxins, etc.

Guinea-pigs are the most widely used experimental
animals because they are easily kept and are susceptible to
so many diseases on artificial inoculation. Rabbits are used
very largely also, as are white mice. For special purposes
white rats, pigeons, goats and swine are necessary. For
commercial products horses (antitoxins) and cattle (small-
pox vaccine) are employed. In the study of many human
diseases the higher monkeys and even the anthropoid apes
are necessary, since none of the lower animals are susceptible.

The commonest method of animal inoculation is undoubt-
edly the subcutaneous. This is accomplished most readily
with the hypodermic needle. The skin at the point selected
(usually in guinea-pigs the lateral posterior half of the
abdominal surface, in mice the back near the root of the
tail) is pinched up to avoid entering the muscles and the
needle quickly inserted. Clipping the hairs and washing
with an antiseptic solution should precede the inoculation
as routine practice. Frequently a small “skin pocket’ is
all that is needed. The hair is clipped off, the skin pinched
up with small forceps and a slight snip with sharp scissors

ANIMAL INOCULATION 211

is made. The material may be inserted into this pocket
with a heavy platinum needle. Cutaneous inoculation is
made by shaving the skin and rubbing the material onto
the shaved surface or scratching with a scalpel or special
scarifier, but without drawing blood, and then rubbing in
the material to be inoculated.

Intravenous injections are made with larger animals. In
rabbits the posterior external auricular is a convenient vein.
In larger animals the external jugular is used.

Intraperitoneal, -thoracic, -cardiac, -ocular, -muscular injec-
tions, and injections into the parenchyma of internal organs
are accomplished with the hypodermic needle. In the case
of the first two, injury to contained organs should be care-
fully avoided. Intracardiac injection, or aspiration of the
heart to secure blood, requires considerable practice to be
successful without causing the death of the animal at once
through internal hemorrhage. In subdural injections into
the cranial cavity it is necessary to trephine the skull first,
while such injections into the spinal canal may be accom-
plished between the vertebra with needles longer and stronger
than the usual hypodermic needle. Occasionally animals are
caused to whale the organisms, or are fed cultures mixed
with the feed.

SECURING AND TRANSPORTING MATERIAL FROM
ANIMALS FOR BACTERIOLOGICAL EXAMINATION.

If the site of the lesion is readily accessible from the
exterior, material from the living animal should be collected
with sterile instruments and kept in sterile utensils until the
necessary tests can be made. Testing should be done on
material as soon after collection as possible, in all cases, to
avoid the effects of ‘“decomposition’’ bacteria.

If the blood is to be investigated it may be aspirated from
a peripheral vein with a sterile hypodermic syringe of appro-
priate size or allowed to flow through a sterile canula into
sterile receptacles. The site of the puncture should be
shaved and disinfected before the instrument is introduced.

Discharges of whatever kind should likewise be collected
in sterile receptacles and examined as soon as may be.

212 ANIMAL INOCULATION

If internal organs are to be examined it is best to kill a
moribund animal than to wait for death, since after death,
and in severe infections even sometimes before, the tissues
are rapidly invaded by saprophytic bacteria from the ali-
mentary and respiratory tracts which complicate greatly
the isolation of the specific organism. Hence the search for
specific bacteria in carcasses or on organs several hours after
death is frequently negative. Animal inoculation with such
material is very often followed by sepsis or septicemia in a
few hours, so that the specific organism has no opportunity
to manifest itself.

In securing material for cultures from internal organs it
is a good plan to burn the surface of the organ with a gas
or alcohol flame, or to sear it with a hot instrument to
kill surface organisms, then make the incision or puncture
through the burned area and secure material from the inte-
rior of the organ. Such punctures made with a stiff platinum
needle frequently give pure cultures of the organism sought.
Slides may be made from such material and culture media
inoculated at once

Since a bacteriological diagnosis depends most commonly on
growing the organisms, it is evident that material sent for
examination must never be treated with an antiseptic or pre-
servative. If decomposition is to be feared the only safe pro-
cedure is to pack the material in ice and forward in this way.

Tuberculous material from the parenchyma of internal
organs may be forwarded in a preservative (not formalin,
since this makes it very difficult to stain the bacteria) as in
this special case a very positive diagnosis may be made by
staining alone. Even here it is better to pack in ice in
order that the diagnosis by staining may be confirmed by
inoculating the living organisms into guinea-pigs.

In the case of material from a rabid animal and many
protozoal diseases the rule against preservatives is not abso-
lute, since staining is a reliable diagnostic means. Even
in these cases it is often desirable to inoculate animals,
hence, as before stated, it is best to make it a uniform
practice to pack material for examination in ice and use no
preservatives.

PART IV.

GENERAL PATHOGENIC BACTERI-
OLOGY.

CHAPTER XXII.
INTRODUCTION.

PatuoceEntc Bacteriology treats of the unicellular micro-
organisms which are responsible for disease conditions, i. e.,
pathological changes in other organisms. Hence not only
are bacteria considered, but also other low vegetable forms,
as yeasts and molds, likewise protozoa insofar as they
may be pathogenic. For this reason the term pathogenic
“Microbiology” has been introduced to include all these
organisms. It is largely for the reason that the methods
devised for the study of bacteria have been applied to the
investigation of other microérganisms that the term ‘‘bac-
teriology’’ was extended to cover the entire field. The
general discussion in this chapter is intended to include,
therefore, microérganisms of whatever kind pathogenic to
animals.

The term pathogenic as applied to an organism must be
understood in a purely relative sense, since there is no single
organism that can cause disease in all of a certain class, but
each is limited to a more or less narrow range. Some form
_ of tuberculosis attacks nearly all vertebrates, but no other
classes of animals and no plants. Lockjaw or tetanus
attacks most mammals, but not any other vertebrates
naturally. Typhoid fever affects human beings; hog cholera,
swine, etc.

Diseases which are due to unicellular pathogenic micro-
organisms are called infectious diseases, while if such diseases

214 INTRODUCTION

are transmitted under natural conditions from organism to
organism they are spoken of as contagious diseases. Most
infectious diseases are contagious but not all. Tetanus is
a good illustration of a non-contagious infectious disease.
There are very few such diseases.

When a unicellular microérganism gains entrance into the
body and brings about any pathological changes there the
result is an infection. Undoubtedly many pathogenic organ-
isms get into the body but never manifest their presence by
causing disease conditions, hence do not cause an infection.
It is the pathological conditions which result that constitute
the infection and not the mere invasion.

The time that elapses between the entrance of the organ-
ism and the appearance of symptoms is called the period of
incubation and varies greatly in different diseases.

The term infestation is used to denote pathological condi-
tions due to multicellular parasites. Thus an animal is
infested (not infected) with tapeworms, roundworms,
lice, mites, etc. Many of these conditions, probably all,
are contagious, 7. e., transmissable naturally from animal
to animal. The word contagious has been used in a variety
of ways to mean communicated by direct contact, communi-
cated by a living something (contagium) that might be car-
ried to a distance and finally communicable in any manner,
transmissible. The agency of transmission may be, very
roundabout—as through a special tick in Texas fever, a
mosquito in malaria, etc., or by direct personal contact, as
generally in venereal diseases. After all, though exactness
is necessary, it is better to learn all possible about the means
of transmission of diseases, than quibble as to the terms to
be used.

An infectious disease may be acute or chronic. An acute
infection is one which runs for a relatively short time and is
“‘self-limited,” so-called, 7. e., the organisms cease to mani-
fest their presence after a time. In some acute infections
the time is very short—German measles usually runs five
or six days. Typhoid fever may continue eight to ten
weeks, sometimes longer, yet it is an acute infectious dis-
ease. It is not so much the time as the fact of self-limita-
tion that characterizes acute infections.

INTRODUCTION 215

In chronic infections there is little or no evidence of limi-
tation of the progress of the disease which may continue for
years. Tuberculosis is usually chronic. Leprosy in man is
practically always so. Glanders in horses is most commonly
chronic; in mules and in man it is more apt to be acute.

Many infections begin acutely and later change to the
chronic type. Syphilis in man is a good illustration.

The differences between acute and chronic infections are
partly due to the nature of the organism, partly to the num-
ber of organisms introduced and the point of their intro-
duction and partly to the resistance of the animal infected.

An infectious disease is said to be specific when one kind
of organism is responsible for its manifestations—as diph-
theria due to the Bacterium diphtherie, lockjaw due to
Bacillus tetani, Texas fever due to the Piroplasma bigeminum,
etc. It-is non-specific when it may be due to a variety of
organisms, as enteritis (generally), bronchopneumonia, wound
infections.

Henle, as early as 1840, stated certain principles that
must be established before a given organism can be accepted
as the cause of a specific disease. These were afterward
restated by Koch, and have come to be known as “ Koch’s
postulates.”” They may be stated as follows:

1. The given organism must be found in all cases of the
disease in question.

2. No other organism must be found in all cases.

8. The organism must, when obtained in pure culture,
reproduce the disease in susceptible animals.

4. It must be recovered from such animals in pure cul-
ture and this culture likewise reproduce the disease.

These postulates have not been fully met with reference
to any disease, but the principles embodied have been applied
as far as possible in all those infections which we recognize
as specific, and whose causative agent is accepted. In many
recognized infectious and contagious diseases no organism
has been found which is regarded as the specific cause. In
some of these the organism appears to be too small to be
seen with the highest powers of the microscope, hence they
are called “wuléramicroscopic”’ organisms. Because these
agents pass through the finest bacterial filters, they are also

216 INTRODUCTION

frequently called “‘filterable.” The term “virus” or “ filter-
able virus” is likewise applied to these “ ultra-microscopic”
and “‘filterable”’ agents.

The term primary infection is sometimes applied to
the first manifestation of a disease, either specific or non-
specific, while secondary refers to later developments. For
example, a secondary general infection may follow a
primary wound infection, or primary lung tuberculosis
be followed by secondary generalized tuberculosis, or
primary typhoid fever by a secondary pneumonia. Where
several organisms seem to be associated simultaneously in
causing the condition then the term mixed infection is used—
in severe diphtheria, streptococci are commonly associated
with the Bacterium diphtherie. In many cases of hog-
cholera, mixed infections in the lungs and in the intestines
are common. Wound infections are usually mixed. Auto-
infection refers to those conditions in which an organism
commonly present in or on the body in a latent or harmless
condition gives rise to an infectious process. If the Bacillus
coli normal to the intestine escapes into the peritoneal cavity,
or passes into the bladder, a severe peritonitis or cystitis,
respectively, is apt to result. ‘‘Boils’’ and “pimples’’ are
frequently autoinfections. Such infections are also spoken
of as endogenous to distinguish them from those due to
the entrance of organisms from without—erogenous infec-
tions. Relapses are usually instances of autoinfection.

Those types of secondary infection where the infecting
agent is transferred from one disease focus to another or
several other points and sets up the infection there are
sometimes called metastases. Such are the transfer of
tubercle bacilli from lung to intestine, spleen, etc., the for-
mation of abscesses in internal organs following a primary
surface abscess, the appearance of glanders nodules through-
out various organs following pulmonary glanders, etc.

The characteristic of a pathogenic microérganism which
indicates its ability to cause disease is called its virulence.
If slightly virulent, the effect is slight, if highly virulent, the
effect is severe, may be fatal.

On the other hand, the characteristic of the host which
indicates its capacity for infection is called susceptibility.

INTRODUCTION 217

If slightly susceptible, infection is slight, if highly suscep-
tible, the infection is severe.

Evidently the degree of infection is dependent in large
measure on the relation between the virulence of the in-
vading organism and the susceptibility of the host. High
virulence and great susceptibility mean a severe infection;
low virulence and little susceptibility, a slight infection;
while high virulence and little susceptibility or low viru-
lence and great susceptibility might mean a moderate infec-
tion varying in either direction. Other factors influencing
the degree of infection are the number of organisms intro-
duced, the point where they are introduced and various
conditions. These will be discussed in another connection
(Chapter XXV).

The study of pathogenic bacteriology includes the thor-
ough study of the individual organisms according to the
methods already given (Chapters XVII-XXJ) as an aid to
diagnosis and subsequent treatment, bacteriological or other,
in a given disease. Of far greater importance than the
treatment, which in most infectious diseases is not specific,
is the prevention and ultimate eradication of all infectious
diseases. To accomplish these objects involves further a
study of the conditions under which pathogenic organisms
exist outside the body, the paths of entrance into and elimina-
tion from the body and those agencies within the body itself
which make it less susceptible to infection or overcome the
infective agent after its introduction. That condition of the
body itself which prevents any manifestation of a virulent
pathogenic organism after it has been once introduced is
spoken of as immunity in the modern sense. Immunity is thus
the opposite of susceptibility and may exist in varying degrees.

That scientists are and have been for some years in posses-
sion of sufficient knowledge to permit of the prevention and .
eradication of most, if not all, of our infectious diseases can
scarcely be questioned. The practical application of this
knowledge presents many difficulties, the chief of which is
the absence of a public sufficiently enlightened to permit the
expenditure of the necessary funds. Time and educative
effort alone can surmount this difficulty. It will probably
be years yet, but it will certainly be accomplished.

CHAPTER XXIII.
PATHOGENIC BACTERIA OUTSIDE THE BODY.

PATHOGENIC bacteria may exist outside the body of the

host under a variety of conditions as follows:
I. In or on inanimate objects or material.

(a) As true saprophytes.

(b) As facultative saprophytes.

(c) Though obligate parasites, they exist in a latent
state.

II. In or on other animals, or products from them:

(a) Sick themselves.

(b) Recovered from illness but carrying the organisms.

(ec) Never sick with the disease but carrying the
organisms.

(d) Serving as necessary intermediate hosts for cer-
tain stages of the parasite—this applies to
protozoal diseases only, as yet.

I. (a) The bacilli of tetanus and malignant edema are
widely distributed. There is no evidence that their entrance
into the body is at all necessary for the continuation of their
life processes, or that one case of either of these diseases
ever has any connection with any other case; they are true
saprophytes. Manifestly it would be futile to attempt to
prevent or eradicate such diseases by attacking the organ-
ism in its natural habitat. Bacillus botulinus, which causes
a type of meat poisoning in man, does not even multiply
in the body, but the disease symptoms are due to a soluble
toxin which is produced during its growth outside the body.

(b) Organisms like the bacterium of anthrax and the
bacillus of black-leg from their local occurrence seem to be
distributed from animals infected, though capable of living
a saprophytic existence outside the body for years. These
can no more be attacked during their saprophytic existence

' PATHOGENIC BACTERIA OUTSIDE THE BODY 219

than those just mentioned. Doubtless in warm seasons of
the year and in the tropics other organisms pathogenic to
animals may live and multiply in water or in damp soil
where conditions are favorable, just as the cholera organism
in India, and occasionally the typhoid bacillus in témperate
climates do.

(c) Most pathogenic organisms, however, when they are
thrown off from the bodies of animals, remain quiescent, do
not multiply, in fact always tend to die out from lack of all
that is implied in a “favorable environment,” food, moisture,
’ temperature, light, ete. Disinfection is sometimes effective
in this class of diseases in preventing new cases.

II. (a) The most common infectious diseases of animals
are transmitted more or less directly from other animals of
the same species. Human beings get nearly all their dis-
eases from other human beings who are sick; horses, from
other horses; cattle, from other cattle; swine, from swine,
ete. Occasionally transmission from one species to another
occurs. Tuberculosis of swine most frequently results from
feeding them milk of tuberculous cattle or from their eating
the droppings of such cattle. Human beings contract
anthrax from wool, hair and hides of animals dead of the
disease, or from postmortems on such animals; glanders
from horses; tuberculosis (in children) from tuberculous
milk; bubonic plague from rats, etc. The mode of limiting
this class of diseases is evidently to isolate the sick, dis-
infect their discharges and their immediate surroundings,
sterilize such products as must be handled or used, kill
dangerous animals, and disinfect, bury properly, or destroy
their carcasses.

(b) This class of “carriers” offers one of the most difficult
problems in preventing infectious diseases. A perfectly
healthy individual may give off dangerous organisms and
infect others for years. Typhoid carriers have been known
to do so for fifty-five years. Cholera, diphtheria, menin-
gitis and other carriers are well known in human practice.
The difficulty in detecting such individuals is obvious.
Carriers among animals have not been so frequently demon-
strated, but there is every reason for thinking that hog-

220 PATHOGENIC BACTERIA OUTSIDE THE BODY

cholera, distemper, roup, influenza and other carriers are
common. Carriers furnish the explanation for many of the
so-called ‘‘spontaneous” outbreaks of disease among men
and animals.

(c) In this class come the “accidental carriers” like flies,
fleas, lice, bed-bugs, ticks and other biting and blood-
-sucking insects, vultures, buzzards, foxes, rats and carrion-
eating animals generally; pet animals in the household, etc.
Here the animals are not susceptible to the given disease
but become contaminated with the organisms and then
through defilement of the food or drink, or contact with
individuals or with utensils pass the organisms on to the
susceptible. Some biting and blood-sucking insects transmit
the organisms through biting infected and non-infected ani-
mals successively. The spirilloses and trypanosomiases seem
to be transmitted in this way, though there is evidence
accumulating which may place these diseases in the next
class. Anthrax is considered in some instances to be trans-
mitted by flies and by vultures in the southern United States.
Typhoid transmission by flies is well established in man.
Why not hog-cholera from farm to farm by flies, English
sparrows, pigeons feeding, or by turkey buzzards? Though
this would not be easy to prove, it seems reasonable.

Preventing contact of such animals with the discharges
or with the carcasses of those dead of the disease, destruc-
tion of insect carriers, screening and prevention of fly breed-
ing are obvious protective measures.

(d) In this class come certain diseases for which particu-
lar insects are necessary for the parasite in question, so that
certain stages in its life history may be passed therein. The
most certain, means for eradicating such diseases is the
destruction of the insects concerned. Up to the present no
bacterial disease is known in which this condition exists, unless
Rocky Mountain spotted fever and typhus fever shall prove
to be due to bacteria. Such diseases are all due to protozoa.
Among them are Texas fever, due to Piroplasma bigeminum
in this country which has been eradicated in entire districts
by destruction of the cattle tick (Margaropus annulatus).

Piroplasmoses in South ‘Africa among cattle and horses,

PATHOGENIC BACTERIA OUTSIDE THE BODY 221

and in other countries are transmitted in similar ways. Prob-
ably many of the diseases due to spirochetes and trypano-
somes are likewise transmitted by necessary insect inter-
mediaries. In human medicine the eradication of yellow
fever from Panama and Cuba is due to successful warfare
against a certain mosquito (Stegomyia). So the freeing of
large areas in different parts of the world from malaria
follows the destruction of the mosquitoes. The campaign
against disease in animals and man from insect sources
must be considered as still in its infancy. The full utiliza-
tion of tropical lands depends largely on the solution of this
problem.

CHAPTER XXIV.

PATHS OF ENTRANCE OF PATHOGENIC
ORGANISMS,

OR
CHANNELS OF INFECTION.

A, The Skin.—If the skin is healthy there is no oppor-
tunity for bacteria to penetrate it. It is protected not
only by the stratified epithelium, but also in various animals,
by coats of hair, wool, feathers, etc. The secretion pressure
of the healthy sweat and oil glands acts as an effective bar
even to motile bacteria. Nevertheless a very slight injury
only is sufficient to give normal surface parasites and other
pathogenics, accidentally or purposely brought in contact
with it, an opportunity for more rapid growth and even
entrance for general infection. Certain diseases due to
higher fungi are characteristically “skin diseases” and rarely
become general—various forms of Favus, Trychophyton in-
fections, etc. A few disease organisms, tetanus, malignant
edema, usually get in through the skin; others, black-leg,
anthrax, quite commonly; and those diseases transmitted
by biting and blood-sucking insects, piroplasmoses, trypano-
somiases, spirilloses, scarcely in any other way. Defective
secretion in the skin glands from other causes, may permit
lodgment and growth of bacteria in them or in the hair
follicles. “Pimples” and boils in man and local abscesses occa-
sionally in animals are illustrations. Sharp-edged and freely
bleeding wounds are less liable to be infected than contu-
sions, ragged wounds, burns, etc. The flowing blood washes
out the wound and the clotting seals it, while there is less
material to be repaired by the leukocytes and they are free

THE SKIN 223

to care for invading organisms (phagocytosis). Pathogenic
organisms, especially pus cocci, frequently gain lodgment in
the malk glands and cause local (mastitis) or general infection.

B. Mucose directly continuous with the skin and lined with
stratified epithelium are commonly well protected thereby and
by the secretions.

(a) The eaternal auditory meatus is rarely the seat of even
local infection. The tympanic cavity is normally sterile,
though it may become infected by extension through the
Eustachian tube from the pharynx (otitis media).

(b) The conjunctiva is frequently the seat of localized,
very rarely the point of entrance for a generalized infection,
except after severe injury.

(c) The nasal cavity, on account of its anatomical structure
retains pathogenic organisms which give rise to local infec-
tions more frequently than other mucose of its character.
These may extend from here to middle ear, neighboring
sinuses, or along the lymph spaces of the olfactory nerve
into the cranial cavity (meningitis). Acute coryza (‘‘colds”
in man) is characteristic. Glanders, occasionally, is primary
in the nose, as is probably roup in chickens, leprosy in man.
The meningococcus and the virus of poliomyelitis pass from
the nose into the cranial cavity without local lesions in the
former.

(d) The mouth cavity is ordinarily protected by its epi-
thelium and secretions, though the injured mucosa is a
common source of actinomycosts infection, as well as thrush.
In foot-and-mouth disease no visible lesions seem necessary
to permit the localization of the unknown infective agent.

(e) The tonsils afford a ready point of entrance for ever-
present micrococct and streptococci whenever occasion offers
(follicular tonsilitis, “quinsy’’), and articular rheumatism is
not an uncommon sequel. The diphtheria bacillus charac-
teristically seeks these structures for its development.
Tubercle and anthrax organisms occasionally enter here.

(f) The pharynx is the seat of localized infection as in
macrococcal, streptococcal and diphtherial “sore throat” in
human beings, but both it and the esophagus are rarely
infected in animals except as the result of injury.

224 ENTRANCE OF PATHOGENIC ORGANISMS

(g) The external genitalia are the usual points of entrance
for the venereal organisms in man (gonococcus, T'reponema
pallidum, and Ducrey’s bacillus). The bacillus of contagious
abortion and probably the trypanosome of dourine are
commonly introduced through these channels in animals.

C. Lungs.—The varied types of pneumonia due to many
different organisms (tubercle, glanders, influenza, plague
bacilli, pneumococcus, streptococcus, micrococcus and many
others) show how frequently these organs are the seat of a
localized infection, which may or may not be general.
Whether the lungs are the actual point of entrance in these
cases is a question which is much discussed at the present
time, particularly with reference to tuberculosis. The
mucous secretion of the respiratory tract tends to catch
incoming bacteria and other small particles and the ciliary
movement along bronchial tubes and trachea tends to carry
such material out. “Foreign body pneumonia”’ shows. clini-
cally, and many observers have shown experimentally that
microdrganisms may reach the alveoli even though the
exchange of air between them and the bronchioles and larger
bronchi takes place ordinarily only by diffusion. The pres-
ence of carbon particles in the walls of the alveoli in older
animals and human beings and in those that breathe dusty
air for long periods indicates strongly, though it does not
prove absolutely, that these came in with inspired air. On
the other hand, experiment has sbown that tubercle bacilli
introduced into the intestine may appear in the lungs and
cause disease there and not in the intestine. It is probably
safe to assume that in those diseases which are transmitted
most readily through close association though not neces-
sarily actual contact, the commonest path is through the
lungs, which may or may not show lesions (smallpox, scar-
let fever, measles, chicken-pox, whooping-cough, pneumonic
plague in man, lobar and bronchopneumonias and influenza
in man and animals, some cases of glanders and tubercu-
losis). On the other hand, the fact that the Bacillus typhosus
and Bacillus coli may cause pneumonia when they evi-
dently have reached the lung from the intestinal tract, and
the experimental evidence of lung tuberculosis above men-

DISSEMINATION OF ORGANISMS 225

tioned show that this route cannot be excluded in inflam-
mations of the lung.

D. Alimentary Tract.—The alimentary tract affords the
ordinary path of entrance for the causal microbes of many
of the diseases of animals and man, since they are carried
into the body most commonly and most abundantly in the
food .and drink.

(a) The stomach is rarely the seat of local infection, even
in ruminants, except as the result of trauma. The character
of the epithelium in the rumen, reticulum and omasum in
ruminants, the hydrochloric acid in the abomasum and in
the stomachs of animals generally are usually sufficient pro-
tection. Occasionally anthrax “pustules” develop in the
gastric mucosa. (The author saw nine such pustules in a
case of anthrax in a man.)

(b) The intestines are frequently the seat of localized
infections, as various “choleras’”’ and “dysenteries” in men
and many animals, anthrax, tuberculosis, Johne’s disease.
Here doubtless enter the organisms causing “hemorrhagic
septicemias” in many classes of animals, and many others.
These various organisms must have passed through the
stomach and the question at once arises, why did the HCl
not destroy them? It must be remembered that the acid
is present only during stomach digestion, and that liquids
taken on an “empty stomach” pass through rapidly and
any organisms present are not subjected to the action of
the acid. Also spores generally resist the acid. Other
organisms may pass through the stomach within masses of
undigested food. The fact that digestion is ‘going on in the
stomach of ruminants practically all the time may explain
the relative freedom of adult animals of this class from
“choleras” and “ dysenteries.”

Dissemination of Organisms.—Dissemination of organisms
within the tissues occurs either through the lymph channels
or the bloodvessels or both. If through the lymph vessels
only it is usually much more restricted in extent, or much
more slowly disseminated, while blood dissemination is
characterized by the number of organs involved simul-
taneously.

15

226 ENTRANCE OF PATHOGENIC ORGANISMS

PATHS OF ELIMINATION OF PATHOGENIC MICRO-
ORGANISMS.

I. Directly from the point of injury. This is true in
infected wounds open to the surface, skin glanders (farcy),
black-leg, surface anthrax, exanthemata in man and animals
[scarlet fever (?), measles (?), smallpox; hog erysipelas, foot-
and-mouth disease]. Also in case of disease of mucous mem-
branes continuous with the skin—from nasal discharges
(glanders), saliva (foot-and-mouth disease), material coughed
or sneezed out (tuberculosis, influenza, pneumonias), ure-
thral and vaginal discharges (gonorrhea and syphilis in man,
contagious abortion and dourine in animals), intestinal dis-
charges (typhoid fever, “choleras,” ‘‘dysenteries,”’ anthrax,
tuberculosis, Johne’s disease). Material from nose, mouth
and lungs may be swallowed and the organisms passed out
through the intestines.

II. Indirectly through the secretions and the excretions
where the internal organs are involved. The saliva of rabid
animals contains the ultra-microscopic virus of rabies (the
sympathetic ganglia within the salivary glands, and pan-
creas also, are affected in this disease as well as the cells of
the central nervous system). The gall-bladder in man is
known to harbor colon and typhoid bacilli, as that of hog-
cholera hogs does the virus of this disease. It may harbor
analogous organisms in other animals, though such knowl-
edge is scanty. The kidneys have been shown experimen-
tally to excrete certain organisms introduced into the circu-
lation within a few minutes (micrococci, colon and typhoid
bacilli, anthrax). Typhoid bacilli occur in the urine of
typhoid-fever patients in about 25 per cent. of all cases and
the urine of hogs with hog cholera is highly virulent. Most
observers are of the opinion, however, that under natural
conditions the kidneys do not excrete bacteria unless they
themselves are infected.

The milk both of tuberculous cattle and tuberculous women
has been shown to contain tubercle bacilli even when the
mammary glands are not involved.

LOCATION OF INFECTIVE ORGANISMS 227

SPECIFICITY OF LOCATION OF INFECTIVE ORGANISMS.

It is readily apparent that certain disease organisms tend
to locate themselves in definite regions and the question
arises, Is this due to any specific relationship between organ-
ism and tissue or not? Diphtheria in man usually attacks
the tonsils first, gonorrhea and syphilis the external geni-
tals, tuberculosis the lung apex (in man most commonly),
“choleras” the small intestine, “dysenteries” the large
intestine, influenza the lungs. In these cases the explana-
tion is probably that the points attacked are the places
where the organism is most commonly carried, with no
specific relationship, since all of these organisms (Asiatic
cholera, excepted) also produce lesions in other parts of the
body when they reach them. On the other hand, the virus of
hydrophobia attacks nerve cells, leprosy frequently singles
out nerves, glanders bacilli introduced into the abdominal
cavity of a young male guinea-pig cause an inflammation
of the testicle, malarial parasites and piroplasms attack the
red blood corpuscles, etc. In these cases there is apparently
a real chemical relationship, as there is also between the
toxins of bacteria and certain tissue cells (tetanus toxin and
nerve cells). Whether “chemotherapy” will ever profit
from a knowledge of such chemical relationships remains
to be developed.

CHAPTER XXV.
IMMUNITY.

Immunity, as has already been stated, implies such a con-
dition of the body that pathogenic organisms after they
have been introduced are incapable of manifesting them-
selves, are unable to cause disease. The word has taken the
place of the earlier term, resistance, and is the opposite of
susceptibility. The term must be understood always in a
relative sense, since no animal is immune to all pathogenic
organisms, and conceivably not entirely so to anyone, since
there is no question that a sufficient number of bacteria of
any kind might be injected into the circulation to kill an
animal, even though it did it purely mechanically.

Immunity may be considered with reference to a single
individual or to entire divisions of the organic world, with
all grades between. Thus plants are immune to the diseases
affecting animals; invertebrates to vertebrate diseases; cold-
blooded animals to those of warm blood; man is immune
to most of the diseases affecting other mammals; the rat to
anthrax, which affects other rodents and most mammals;
the well-known race of Algerian sheep is likewise immune
to anthrax while other sheep are susceptible; the negro
appears more resistant to yellow fever than the white; some
few individuals in a herd of hogs always escape an epizootic
of hog cholera, ete.

Immunity within a given species is modified by a number
of factors—age, state of nutrition, extremes of heat or cold,
fatigue, excesses of any kind, in fact, anything which tends
to lower the ‘normal healthy tone” of an animal also tends
to lower its resistance. Children appear more susceptible
to scarlet fever, measles, whooping-cough, etc., than adults;
young cattle more frequently have black-leg than older ones

IMMUNITY 229

(these apparently greater susceptibilities may be due in part
to the fact that most of the older individuals have had the
diseases when young and are immune for this reason). Ani-
mals weakened by hunger or thirst succumb to infection
more readily. Frogs and chickens are immune to tetanus,
but if the former be put in water and warmed up to and
kept at about 37°, and the latter be chilled for several hours
in ice-water, then each may be infected. Pneumonia fre-
quently follows exposure to cold. The immune rat may be
given anthrax if first he is made to run in a “squirrel cage”
until exhausted. Alcoholics are‘far less resistant to infec-
tion than temperate individuals. ‘“ Worry,’ mental anguish,
tend to predispose to infection.

The following outlines summarize the’ different classifi-
cations of immunity so far as mammals are concerned for
the purposes’ of discussion:

Immunity.

f 1. Inherited through the germ cell or cells.
(a) By having the

disease in utero.
| 2. Acquired in utero. } (b) By absorption
of immune sub-
B. Acquired by having the disease. stances from the

mother. _ :

A. Congenital
I. Natural

II. Artificial—acquired through human agency by:

1. Introduction of the organism or its products.
2. Introduction of the blood serum of an immune animal.

Immunity.
I. Active—due to the introduction of the organism or due to the intro-
duction of the products of the organism.
A. Naturally by having the disease.
B. Artificially.
1. By introducing the organism:
1. Passage through another animal.
2. Drying.

(a) Alive and virulent. 3. Growing at a higher temperature.

(b) Aliveand virulence

reduced b 4, Heating the cultures.
(c) D d. ¥ 5. Treating with chemicals.
aes 6. Sensitizing.

7. Cultivation on artificial media.
2. By introducing the products of the organism.
II. Passive—due to the introduction of the blood serum of an actively
immunized animal.

230 IMMUNITY

Immunity present in an animal and not due to human
interference is to be regarded as natural immunity, while if
brought about by man’s effort it is considered artificial.
Those cases of natural immunity mentioned above which
are common to divisions, classes, orders, families, species or
races of organisms and to those few individuals where no
special cause is discoverable, must be regarded as instances
of true inheritance through the germ cell as other char-
acteristics are. All other kinds of immunity are acquired.
Occasionally young are born with every evidence that they
have had a disease in utero and are thereafter as immune as
though the attack had occurred after birth (“smallpox
babies,” “hog-cholera pigs’). Experiment has shown that
immune substances may pass from the blood of the mother
to the fetus in utero and the young be immune for a time
after birth (tetanus). It is a familiar fact that with most
infectious diseases recovery from one attack confers a more
or less lasting immunity, though there are marked excep-
tions.

Active Immunity—By active immunity is meant that
which is due to the actual introduction of the organism, or
in some cases of its products. The term active is used because
the body cells of the animal immunized perform the real
work of bringing about the immunity as will be discussed
later. In passive immunity the blood serum of an actively
immunized animal is introduced into a second animal,
which thereupon becomes immune, though its cells are not
concerned in the process. The animal is passive, just as a
test-tube, in which a reaction takes place, plays no other
part than that of a passive container for the reagents.

In active immunity the organism may be introduced in
what is to be considered a natural manner, as when an ani-
mal becomes infected, has a disease, without human inter-
ference. Or the organism may be purposely introduced to
bring about the immunity. For certain purposes the intro-
duction of the products of the organism (toxins) is used to
bring about active immunity (preparation of diphtheria and
tetanus antitoxin from the horse). The method of produc-
ing active immunity by the artificial introduction of the

ACTIVE IMMUNITY 231

organism is called vaccination, and a vaccine must therefore
contain the organism. Vaccines for bacterial diseases are
frequently called bacterins. The use of the blood serum of
an immunized animal to confer passive immunity on a
second animal is properly called serum therapy, and the
serum so used is spoken of as an antiserum, though the latter
word is also used to denote any serum containing any kind
of an antibody (Chapters XXVII-XXXI). Ina few instances
both the organism and an antiserum are used to cause both
active and passive immunity (serwm-simultanecus method in
immunizing against hog cholera).

In producing active immunity the organism may be intro-
duced (a) alive and virulent, but in very small doses, or in
combination with an immune serum, as just mentioned for
hog cholera. The introduction of the live virulent organism
alone is done only experimentally as yet, as it is obviously
too dangerous to do in practice, except under the strictest
control (introduction of a single tubercle bacillus, followed
by gradually increasing numbers—Barber and Webb). More
commonly the organisms are introduced (b) alive but with
their wrulence reduced (“attenuated’’) in one of several ways:
(1) By passing the organism through another animal as is
the case with smallpox vaccine derived from a calf or heifer.
(2) By drying the organism, as is done in the preparation of
the vaccine for the Pasteur treatment of rabies, where the
spinal cords of rabbits are dried for varying lengths of time—
one to four days, Russian method, one to three days, German
method, longer in this country. (It is probable that the
passage of the “fixed virus” through the rabbit is as impor-
tant in this procedure as the drying, since it is doubtful if the
“fixed virus” is pathogenic for man.) (3) The organism
may be attenuated by growing at a temperature above the
normal. This is the method used in preparing anthrax vac-
cine as done by Pasteur originally. (4) Instead of growing
at a higher temperature the culture may be heated in such
a way that it is not killed but merely weakened. Black-leg
vaccines are made by this method. (5) Chemicals are some-
times added to attenuate the organisms, as was formerly
done in the preparation of black-leg vaccine by Kruse’s

232 IMMUNITY

method in Germany. This method is no longer used to any
great extent. (6) Within the past few years the workers in
the Pasteur Institute in Paris have been experimenting with
vaccines prepared by treating living virulent bacteria with
antisera (“sensitizing them’’) so that they are no longer
capable of causing the disease when introduced, but do
cause the production of an active immunity. The method
has been used with typhoid fever bacilli in man and seems
to be successful. It remains to be tried out further before
its worth is demonstrated (the procedure is more compli-
cated and the chance for infection apparently much greater
than by the use of killed ¢ultures). (7) Growing on artifi-
cial culture media reduces the virulence of most organisms
after a longer or shorter time. This method has been tried
with many organisms in the laboratory, but is not now used
in practice. The difficulties are that the attenuation is very
uncertain and that the organisms tend to regain their
virulence when introduced into the body.

In producing active immunity against many bacterial
diseases the organisms are introduced (c) dead. They are
killed by heat or by chemicals, or by using both methods
(Chapter XXX).

When the products of an organism are introduced the
resulting immunity is against the products only and not
against the organism. If the organism itself is introduced
there results an immunity against it and in some cases also
against the products, though the latter does not necessarily
follow. Hence the immunity may be antzbacterial or anti-
toxic or both.

Investigation as to the causes of immunity and the various
methods by which it is produced has not resulted in the dis-
covery of specific methods of treatment for as many dis-
eases as was hoped for at one time. Just at present progress
in serum therapy appears to be at a standstill, though vac-
cines are giving good results in many instances not believed
possible a few years ago. As a consequence workers in all
parts of the world are giving more and more attention to
the search for specific chemical substances, which will destroy
invading parasites and not injure the host (Chemotherapy).

ACTIVE IMMUNITY 233

Nevertheless, in the study of immunity very much of value
in the treatment and prevention of disease has been learned.
Also much knowledge which is of the greatest use in other
lines has been accumulated. Methods of diagnosis of great
exactness have resulted, applicable in numerous diseases.
Ways of detecting adulterations in foods, particularly foods
from animal sources, and of differentiating proteins of varied
origin, as well as means of establishing biological relation-
ships and differences among groups of animals through
“immunity reactions” of blood serums have followed from
knowledge gained by application of the facts or the methods
of immunity research. Hence the study of “immunity
problems” has come to include much more than merely the
study of those factors which prevent the development of
disease in an animal or result in its spontaneous recovery.
A proper understanding of the principles of immunity neces-
sitates a study of these various features and they will be
considered in the discussion to follow.

CHAPTER XXVI.
THEORIES OF IMMUNITY.

Pasteur and the bacteriologists of his time discovered
that bacteria cease to grow in artificial culture media after
a time, because of the exhaustion of the food material in
some cases and because of the injurious action of their own
products in other instances. These facts were brought for-
ward to explain immunity shortly after bacteria were shown
to be the cause of certain diseases. Theories based on these
observations were called (1) ‘‘Exhaustion Theory” of Pasteur,
and (2) “ Noxious Retention Theory” of Chauveau respec-
tively. The fact, soon discovered, that virulent pathogenic
bacteria are not uncommonly present in perfectly healthy
animals, and the later discovery that immunity may be
conferred by the injection of dead bacteria have led to the
abandonment of both these older ideas. The (3) “ Unfavor-
able Environment” theory of Baumgartner, 1. e., bacteria
do not grow in the body and produce disease because their
surroundings are not suitable, in a sense, covers the whole
ground, though it is not true as to the first part, as was
pointed out above, and is of no value as a working basis,
since it offers no explanation as to what the factors are that
constitute the “unfavorable environment.” Metchnikoff
brought forward a rational explanation of immunity with
his (4) “Cellular or Phagocytosis Theory.” As first pro-
pounded it based immunity on the observed fact that cer-
tain white blood corpuscles, phagocytes, engulf and destroy
bacteria. Metchnikoff has since elaborated the original
theory to explain facts of later discovery. Ehrlich soon after
published his (5) “Chemical or Side-chain Theory’? which
seeks to explain immunity on the basis of chemical substances
in the body which may in part destroy pathogenic organ-
isms or in part neutralize their products; or in some instances

PAUL EHRLICH

THEORIES OF IMMUNITY 235

there may be an absence of certain chemical substances
in the body cells so that bacteria or their products cannot
unite with the cells and hence can do no damage.

At the present time it is generally accepted, in this coun-
try at least, that Ehrlich’s theory explains immunity in
many diseases as well as many of the phenomena related to
immunity, and in other diseases the phagocytes, frequently
assisted by-chemical substances, are the chief factors. Spe-
cific instances are discussed in Pathogenic Bacteriologies
which should be consulted. It is essential that the student
should be familiar with the basic ideas of the chemical theory,
not only from the standpoint of immunity, but also in order
to understand the principles of a number of valuable methods
of diagnosis.

The chemical theory rests on three fundamental physi-
ological principles: (1) the response of cells to stimuli, in this
connection specific chemical stimuli, (2) the presence within
cells of specific chemical groups which combine with chemi-
cal stimuli and thus enable them to act on the cell, which
groups Ehrlich has named receptors, and (3) the “over-
production” activity of cells as announced by Weigert.

1. That cells respond to stimuli is fundamental in physi-
ology. These stimuli may be of many kinds as mechanical,
electrical, light, chemical, etc. Chemical stimuli are well
illustrated along the digestive tract. That the chemical
stimuli in digestion may be more or less specific is shown by
the observed differences in the enzymes of the pancreatic
juice dependent on the relative amounts of carbohydrates,
fats, or proteins in the food, the specific enzyme in each case
being increased in the juice with increase of its corresponding
foodstuff. The cells of the body, or certain of them at
least, seem to respond in a specific way when proteins or
substances closely related to them are brought into direct
contact with them, that is, without having been subjected
to digestion in the alimentary tract, but injected directly
into the blood or lymph stream. Cells may be affected by
stimuli in one of three ways: if the stimulus is too weak,
there is no effect (in reality there is no “stimulus” acting);
if the stimulus is too strong, the cell is injured, may be

236 THEORIES OF IMMUNITY

destroyed; if the stimulus is of proper amount then it excites
the cell to increased activity, and in the case of specific
chemical stimuli the increased activity, as mentioned for
the pancreas, shows itself in an increased production of what-
ever is called forth by the chemical stimulus. In the case of
the proteins and related bodies, the substances produced by
the cells under their direct stimulation are markedly specific
for the particular substance introduced.

2. Since chemical action always implies at least two
bodies to react, Ehrlich assumes that in every cell which is
affected by a chemical stimulus there must therefore be a
chemical group to unite with this stimulus. He further
states that there must be as many different kinds of these
groups as there are different kinds of chemicals which stimu-
late the cell. Since these groups are present in the body
cells primarily to take up different kinds of food material,
Ehrlich calls them receptors. Since these groups must be
small as compared with the cell as a whole, and must be
more or less on the surface and unite readily with chemical
substances he further speaks of them as “side-chains’’ after
the analogy of compounds of the aromatic series especially.
The term receptors is now generally used. As was stated
above, the effect of specific chemical stimuli is to cause the
production of more of the particular substance for which it is
specific and in the class of bodies under discussion, proteins
and their allies, the particular product is these cell receptors
with which the chemical may unite.

3. Weigert first called attention to the practically con-
stant phenomenon that cells ordinarily respond by doing
more of a particular response than is ctually called for by
the stimulus, that there is always an “overproduction” of
activity. In the case of chemical stimuli this means an
increased production of the specific substance over and above
the amount actually needed. Whenever a cell accumulates
an excess of products the normal result is that it excretes
them from its own substance into the surrounding lymph,
whence they reach the blood stream to be either carried to
the true excretory organs, utilized by other cells or remain
for a longer or shorter time in the blood.

THEORIES OF IMMUNITY 237

To recapitulate, Ehrlich’s theory postulates specific chema-
cal stimuli, which react with specific chemical substances in
the body cells, named receptors, and that these receptors,
according to Weigert, are produced in excess and hence are
excreted from the cell and become free receptors in the blood
‘and lymph. These free receptors are the various kinds of
antibodies, the kind depending on the nature of the stimulus,
the substance introduced. Any substance which when
introduced into the body causes the formation of an anti-
body of any kind whatsoever is called an antigen, 2. ¢., anti
(body) former.

If the three fundamental principles just discussed are
thoroughly understood, the theory of the formation of
different kinds of antibodies should not be difficult to
comprehend.

CHAPTER XXVII.
RECEPTORS OF THE FIRST ORDER.

ANTITOXINS— ANTIENZYMES.

THE general characteristics of toxins have been described
(Chapter XII). It has been stated that they are more or
less specific in their action on cells. In order to affect a cell
it is evident that a toxin must enter into chemical combina-
tion with it. This implies that the toxin molecule possesses
a chemical group which can combine with the cell. This
group is called the haptophore or combining group. The
toxic or injurious portion of the toxin molecule is likewise
spoken of as the torophore group. When a toxin is intro-:
duced into the body its haptophore group combines with
suitable receptors in different cells of the body. If not too
much of the toxin is given, instead of injuring, it acts as a
chemical stimulus to the cell in the manner already described.
The cell in response produces more of the specific thing,
which in this instance is more receptors which can combine
with the toxin, 7. e., with its haptophore group. If the stim-
ulus is kept up, more and more of these receptors are pro-
duced until an excess for the cell accumulates, which excess
is excreted from the individual cell and becomes free in the
blood. These free receptors have, of course, the capacity
to combine with toxin through its haptophore group. When
the toxin is combined with these free receptors, it cannot
combine with any other receptors, e. g., those in another
cell and hence cannot injure another cell. These free recep-
tors constitute, in this case, antitowin, so-called because they
can combine with toxin and hence neutralize it. Antitoxins
are specific—that is, an antitoxin which will combine with
the toxin of Bacillus tetant will not combine with that of
Bacterium diphtherie, or of Bacillus botulinus, or of any
other toxin, vegetable or animal.

ANTITOXINS—ANTIENZY MES 239

When a toxin is kept in solution for some time or when it
is heated above a certain temperature (different for each
toxin) it loses its poisonous character. It may be shown,
however, that it is still capable of uniting with 'antitoxin,
and preventing the latter from uniting with a fresh toxin.
This confirms the hypothesis that a toxin molecule has at
least two groups: a combining or haptophore, and a poison-
ing or torophore group. A toxin which has lost its poisonous
property, its toxophore group, is spoken of as a toroid. The
theory of antitoxin formation is further supported by the
fact that the proper introduction of toxoid, the haptophore
group, and hence the real stimulus, can cause the production
of antitoxin to a certain extent at least.

The close relationship between toxins and enzymes has
already been pointed out. This is still further illustrated
by the fact that when enzymes are properly introduced into
the tissues of an animal there is formed in the animal an
antienzyme specific for the enzyme in question which can
prevent its action. The structure of enzymes, as composed
of a haptophore, or uniting, and a zymophore or digesting
(or other activity) group, is similar to that of toxins, and
enzymoids or enzymes which can combine with the substance
acted on but not affect it further have been demonstrated.

These free-cell receptors, antitoxins or antienzymes, which
are produced in the body by the proper introduction of
toxins or enzymes, respectively, have the function of com-
bining with these bodies but no other action. As was pointed
out above, this is sufficient to neutralize the toxin or enzyme
and prevent any injurious effect since they can unite with
nothing else. Since these receptors are the simplest type
which has been studied as yet, they are spoken of by Ehrlich
as receptors of the first order. Other antibodies which are
likewise free receptors of the first order and have the func-
tion of combining only have been prepared and will be
referred to in their proper connection. They are mainly of
theoretical interest.

Ehrlich did a large part of his work on toxins and anti-
toxins with ricin, the toxin of the castor-oil bean, abrin,
from the jequirity bean, robin from the locust tree, and with

240 RECEPTORS OF THE FIRST ORDER

the toxins and antitoxins for diphtheria and tetanus. Anti-
toxins have been prepared experimentally for a large number
of both animal and vegetable poisons, including a number
for bacterial toxins. The only ones which, as yet, are of
much practical importance are antivenin for snake poison,
(not a true toxin, however, see p. 252), antipollenin (sup-
posed to be for the toxin of hay fever) and the antitoxins
for the true bacterial toxins of Bacterium diphtherie and
Bacillus tetani.

The method of preparing antitoxins is essentially the same
in all cases, though differing in minor details. For commer-
cial purposes large animals are selected, usually horses, so
that the yield of serum may be large. The animals must, of
course, be vigorous, free from all infectious disease. The
first injection given is either a relatively small amount of a
solution of toxin or of a mixture of toxin and antitoxin.
The animal shows more or less reaction, increased temper-
ature, pulse and respiration and frequently an edema at the
point of injection, unless this is made intravenously. After
several days to a week or more, when the animal has recov-
ered from the first injection, a second stronger dose is given,
usually with less reaction. Increasingly large doses are
given at proper intervals until the animal may take several
hundred times the amount which would have been fatal if
given at first. The process of immunizing a horse for diph-
theria or tetanus toxin usually takes several months. Varia-
tions in time and in yield of antitoxin are individual and
not predictable in any given case.

After several injections a few hundred cubic centimeters
of blood are withdrawn from the jugular vein and serum
from this is tested for the amount of antitoxin it contains.
When the amount is found sufficiently large (250 “units”
at least for diphtheria per c.c.) then the maximum amount
of blood is collected from the jugular with sterile trocar and
canula. The serum from this blood with the addition of an
antiseptic (0.5 per cent. phenol, tricresol, etc.) constitutes
‘“‘antidiphtheritic serum,” or “antitetanic serum,” etc. All
sera which are put on the market must conform to definite
standards of strength expressed in “units” as determined

ANTITOXINS—ANTIENZY MES 241

by the U. S. Hygienic Laboratory. In reality a “unit” of
diphtheria antitoxin in the United States is an amount
equivalent to 1 c.c. of a given solution of a standard diph-
theria antitorin which is kept at the above-mentioned
laboratory. This statement, of course, gives no definite idea
as to the amount of antitoxin actually in a “unit.” Specifi-
cally stated, a “unit” of antitoxin contains approximately
the amount which would protect a 250-gram guinea-pig from
100 minimum lethal doses of diphtheria toxin, or protect
100 guinea-pigs weighing 250 grams each from one minimum
lethal dose each. The minimum lethal dose (M. L. D.) of
diphtheria toxin is the least amount that will kill a guinea-
pig of the size mentioned within four days. Since toxins
on standing change into toxoids to a great extent, the amount
of antitoxin in a “unit,” though protecting against 100
M.L. D., in reality would protect against about 200 M. L. D.
of toxin containing no toxoid.

The official unit for tetanus antitoxin is somewhat dif-
ferent, since it is standardized against a standard toxin which
is likewise kept at the Hygienic Laboratory. The unit is
defined as ‘“‘ten times the amount of antitoxin necessary to
protect a 350 gm. guinea-pig for 96 hours against the standard
test dose’’ of the standard toxin. The standard test dose is
100 M. L. D. of toxin for a 350 gm. guinea-pig. To express
it another way, one could say that a “unit” of tetanus anti-
toxin would protect one thousand 350 gm. guinea-pigs from
1M. L. D. each of standard tetanus toxin.

Various methods have been devised for increasing the
amount of antitoxin in 1 c.c. of solution by precipitating
out portions of the blood-serum proteins and at the same
time concentrating the antitoxin in smaller volume. It is
not considered necessary in a work of this character to enter
into these details nor to discuss the process of standardizing
antitoxin so that the exact content of “units” per c.c, may
be known.

16

CHAPTER XXVIII.
RECEPTORS OF THE SECOND ORDER.

AGGLUTININS.

CHARRIN and Rogers appear to have been the first (1889)
to observe the clumping together of bacteria (Pseudomonas
pyocyanea) when mixed with the blood serum of an animal
immunized against them. Gruber and Durham (1896) first
used the term “agglutination” in this connection and called
the substance in the blood-serum “agglutinin.” Widal
(1896) showed the importance of the reaction for diagnosis
by testing the blood serum of an infected person against a
known culture (typhoid fever).

It is now a well-known phenomenon that the proper injec-
tion of cells of any kind foreign to a given animal will lead
to the accumulation in the animal’s blood of substances
which will cause a clumping together of the cells used when
suspended in a suitable liquid. The cells settle out of such
suspension much more rapidly than they would otherwise
do. This clumping is spoken of as “agglutination’’ and the
substances produced in the animal are called ‘ agglutinins.”
If blood cells are injected then “hemagglutinins”’ result; if
bacterial cells “bacterial agglutinins” for the particular
organism used as “glanders agglutinin” for Bacterium mallet,
“abortion agglutinin” for Bacterium abortus, “typhoid
agglutinin” for Bacillus typhosus, etc.

The phenomenon may be observed either under the micro-
scope or in small test-tubes, that is, either microscopically or
macroscopically.

Tn this case the cells introduced, or more properly, some
substances within the cells, probably protein in nature, act
as stimuli to the body cells of the animal injected to cause
them to produce more of the specific cell receptors which

AGGLUTININS 243

respond to the stimulus. The substance within the intro-
duced cell which acts as a stimulus (antigen) to the body
cells is called an “agglutinogen.” That ‘“‘agglutinogen’’ is
present in the cell has been shown by injecting animals exper-
imentally with extracts of cells (bacterial and other cells)
and the blood serum of the animal injected showed the
presence of agglutinin for the given cell. It will be noticed
that the receptors which become the free agglutinins have
at least two functions, hence at least two chemical groups.
They must combine with the foreign cells and also bring
about their clumping together, their agglutination. Hence
it can be stated technically that an agglutinin possesses a
haptophore group, and an agglutinating group.

The formation of agglutinin in the body for different bac-
teria doés not as yet appear to be of any special significance
in protecting the animal from-the organism, since the bac-
teria are not killed, even though they are rendered non-
motile, if of the class provided with flagella, and are clumped
together. The fact that such bodies are formed, however,
is of decided value in the diagnosis of disease, and also in
the identification of unknown bacteria.

In many bacterial diseases, agglutinins for the particular
organism are present in the blood serum of the affected
animal. Consequently if the blood serum of the animal be
mixed with a suspension of the organism supposed to be the
cause of the disease and the latter be agglutinated, one is
justified in considering it the causative agent, provided cer-
tain necessary conditions are fulfilled. In the first place it
must be remembered that the blood of normal animals fre-
quently contains agglutinins (‘normal agglutinins’) for
many different bacteria when mixed with them in full
strength. Hence the serum must always be diluted with
physiological salt solution (0.85 per cent.). Further, closely
related bacteria may be agglutinated to some extent by the
same serum. It is evident that if they are closely related,
their protoplasm must contain some substances of the same
kind to account for this relationship. Since some of these
substances may be agglutinogens, their introduction into
the animal body will give rise to agglutinins for the related

244 RECEPTORS OF THE SECOND ORDER

cells, as well as for the cell introduced. The agglu-
tinins for the cell introduced will be formed in larger quan-
tity, since a given bacterial cell must contain more of its own
agglutinogen than that of any other cell. By diluting the
blood serum from the animal to be tested the agglutinins
for the related organisms (so-called ‘‘coagglutinins’”) will
become so much diminished as to show no action, while the
agglutinin for the specific organism is still present in an
amount sufficient to cause its clumping. Agglutinins are
specific for their particular agglutinogens, but since a given
blood serum may contain many agglutinins, the serwm’s
specificity for a given bacterium can be determined only by
diluting it until this bacterium alone is agglutinated. Hence
the necessity of diluting the unknown serum in varying
amounts when testing against several known bacteria to
determine for which it is specific, 7. e., which is the cause
of the disease in the animal.

Just as an unidentified disease in an animal may be deter-
mined by testing its serum as above described against known
kinds of bacteria, so unknown bacteria isolated from an
animal, from water, etc., may be identified by testing them
against the blood sera of different animals, each of which has
been properly inoculated with a different kind of known bac-
teria. If the unknown organism is agglutinated by the blood
of one of the animals in high dilution, and not by the others,
evidently the bacterium is the same as that with which the
animal had been inoculated, or immunized, as is usually
stated. This method of identifying cultures of bacteria is
of wide application, but is used practically only in those
cases where other methods of identification are not readily
applied, and especially where other methods are not sufficient,
as in the “intestinal group” of organisms in human
practice.

The diagnosis of disease in an animal by testing its serum
is also a valuable and much used procedure. This is the
method of the “ Widal’” or “Gruber-Widal” test for typhoid
fever in man and is used in veterinary practice in testing
for glanders, contagious abortion, etc. In some cases a dilu-
tion of the serum of from 20 to 50 times is sufficient for

PRECIPITINS 245

diagnosis (Malta fever), in most cases, however, 50 times
is the lowest limit. Evidently the greater the dilution, that
is, the higher the “titer,” the more specific is the reaction.

PRECIPITINS.

Since agglutinins act on bacteria, probably through the
presence of substances protein in nature within the bacterial
cell, it is reasonable to expect that if these substances be
dissolved out of the cell, there would be some reaction
between their (colloidal) solution and the same serum. As a
matter of fact Kraus (1897) showed that broth cultures
freed from bacteria by porcelain filters do show a precipi-
tate when mixed with the serum of an animal immunized
against the particular bacterium and that the reaction is
specific under proper conditions of dilution. It was not
long after Kraus’s work until the experiments were tried
of “immunizing” an animal not against a bacterium or its
filtered culture, but against (colloidal) solutions of proteins,
such as white of egg, casein of milk, proteins of meat and
of blood serum, vegetable proteins, etc. It was ascertained
that in all these cases the animal’s serum contains a sub-
stance which causes a precipitate with solutions of the pro-
tein used for immunization. The number of such precipi-
tating serums that have been made experimentally is very
large and it appears that protein from any source when
properly introduced into the blood or tissues of an animal
will cause the formation of a precipitating substance for its
solutions. This substance is known, technically as a
“orecipitin.” The protein used as antigen to stimulate its
formation, or some part of the protein molecule (hapto-
phore group), which acts as stimulus to the cell is spoken
of as a “precipitinogen,” both terms after the analogy of
“agelutinin” and “agglutinogen.”’ In fact the specific pre-
cipitation and agglutination are strictly analogous phenom-
ena. Precipitins act on proteins in (colloidal) solution and
cause them to settle out, agglutinins act on proteins within
cells which cells are in suspension in a fluid and cause the
cells to settle out. Ehrlich’s theory of the formation of

\

246 RECEPTORS OF THE SECOND ORDER

precipitins is similar to that of agglutinins, and need not be
repeated. Substitute the corresponding words in the theory
of formation of agglutinins as above given and the theory
applies.

The precipitin reaction has not found much practical use
in bacteriology largely because the “agglutination test”’
takes its place as simpler of performance and just as ac-
curate. The reaction is, however, generally applicable to
filtrates of bacterial cultures and could be used if needed.
The so-called “mallease”’ reaction in glanders is an instance.

Precipitins find their greatest usefulness in legal medi-
cine and in food adulteration work. As was noted above,
if animals, rabbits for example, are immunized with the
blood of another animal (human beings) precipitins are
developed which are specific for the injected blood with
proper dilution. This forms an extremely valuable means
of determining the kind of blood present in a given spot
shown by chemical and spectroscopic tests to be blood, and
has been adopted as a legal test in countries where such rules
of procedure are applied. Similarly the test has been used
to identify the different kinds of meat in a sausage, and
different kinds of milk in a mixture. An extract of the
sausage is made and tested against the serum of an animal
previously treated with extract of horse meat, or hog meat,
or beef, etc., the specific precipitate occurring with the
specific serum. Such reactions have been obtained where
the protein to be tested was diluted 100,000 times and more.
Biological relationships and differences have been detected
by the reaction. Human immune serum shows no reaction
with the blood of any animals except to a slight extent with
that of various monkeys, most with the higher, very slight
with the lower Old World and scarcely any with New World
monkeys.

It is a fact of theoretical interest mainly that if agglutinins
and precipitins themselves be injected into an animal they
will act as antigens and cause the formation of antiagglu-
tinins or antiprecipitins, which are therefore receptors of
the first order since they simply combine with these immune
bodies to neutralize their action, have only a combining or

PRECIPITINS 247

haptophore group. Also if agglutinins or precipitins be
heated to the proper temperature they may retain their
combining power but cause no agglutination or precipita-
tion, 7. e., they are converted into agglutinoid or precipi-
tinoid respectively after the analogy of toxin and toxoid.

Precipitins like agglutinins possess at least two groups—
a combining or haptophore group and a precipitating (some-
‘times called zymophore) group. Hence they are somewhat
more complex than antitoxins or antienzymes which have
a combining group only. For this reason Ehrlich classes
agglutinins and precipitins as receptors of the second order.

CHAPTER XXIX.
RECEPTORS OF THE THIRD ORDER.

CYTOLYSINS.

Berore Koch definitely proved bacteria capable of caus-
ing disease several physiologists had noted that the red cor-
puscles of certain animals were destroyed by the blood of
other animals (Creite, 1869, Landois, 1875), and Traube and
Gescheidel had shown that freshly drawn blood destroys
bacteria (1874). It was not until about ten years afterward
that this action of the blood began to be investigated in
connection with the subject of immunity. Von Fodor (1885)
showed that saprophytic bacteria injected into the blood
are rapidly destroyed. Fliigge and his pupils, especially
Nuttall in combating Metchnikoff’s theory of phago-
cytosis, announced in 1883, studied the action of the blood
on bacteria and showed its destructive effect (1885-87).
Nuttall also showed that the blood lost this power if heated
to 56°. Buchner (1889) gave the name “‘alexin’”’ (from the
Greek ‘‘to ward off’) to the destroying substance and showed
that the substance was present in the blood serum as well as
in the whole blood, and that when the serum lost its power
to dissolve, this could be restored by adding fresh blood.
Pfeiffer (1894) showed that the destructive power of the blood
of animals immunized against bacteria (cholera and typhoid)
was markedly specific for the bacteria used. He introduced
a mixture of the blood and the bacteria into the abdominal
cavity of the immunized animal or of a normal one of the
same species and noted the rapid solution of the bacteria
by withdrawing portions of the peritoneal fluid and exam-
ining them (“‘Pfeiffer’s phenomenon’’). Belfanti and Car-
bone and especially Bordet (1898) showed the specific dis-
solving action of the serum of one animal on the blood cor-

CYTOLYSINS 249

puscles of another animal with which it had been injected.
Since this time the phenomenon has been observed with a
great variety of cells other than red blood corpuscles and
bacteria—leukocytes, spermatozoa, cells from liver, kidney,
brain, epithelia, etc., protozoa, and many vegetable cells.

It is therefore a well-established fact that the proper
injection of an animal with almost any cell foreign to it
will lead to the blood of the animal injected acquiring the
power to injure or destroy cells of the same kind as those
introduced. The destroying power of the blood has been
variously called its “cytotoxic” or “cytolytic” power, though
the terms are not strictly synonymous since “cytotoxic’’
means ‘cell poisoning” or “injuring,” while “cytolytic”
means “cell dissolving.” The latter term is the one gen-
erally used and there is said to be present in the blood a
specific “cytolysin.” The term is a general one and a given
cytolysin is named from the cell which is dissolved, as a
bactertolysin, a hemolysin (red-corpuscle-lysin), epitheliolysin,
nephrolysin (for kidney cells), etc. If the cell is killed but
not dissolved the suffix “cidin” or “toxin” is frequently used

s “bacteriocidin,” “spermotoxin,” “neurotoxin,” etc.

The use of the term “cytolysin” is also not strictly ‘cor-
‘rect, though convenient, for the process is more complex
than if one substance only were employed. As was stated
above, the immune serum loses its power to dissolve the cell
if it is heated to 55° to 56° for half an hour, it is inactivated.
But if there be added to the heated or inactivated serum a
small amount of normal serum (which contains only a very
little cytolytic substance, so that it has no dissolving power
when so diluted) then the mixture again becomes cytolytic.
It is evident then that in cytolysis there are two distinct
substances involved, one which is present in all serum, normal
or immune, and the other present only in the immune cytolytic
serum. Experiment has shown that it is the substance
present in all serum that is the true dissolving body, while
the immune substance serves merely to unite this body to the
cell to be destroyed, 7. e., to the antigen. Since the immune
body has therefore two uniting groups, one for the dissolv-
ing substance and one for the cell to be dissolved, Ehrlich

250 RECEPTORS OF THE THIRD ORDER

calls it the “amboceptor.” He also uses the word “comple-
ment’ to denote the dissolving substance, giving the idea
that it completes the action of dissolving after it has been
united to the cell by the amboceptor, thus replacing
Buchner’s older term “alexin” for the same dissolving body.

AMBOCEPTORS.

The theory of formation of amboceptors is similar to that
for the formation of the other types of antibodies. The
cell introduced contains some substance, probably protein,
which acts as a chemical stimulus to some of the body cells
provided with proper receptors so that more of these special
receptors are produced, and eventually in excess so that they
become free in the blood and constitute the free ambocep-
tors. It will be noticed that these free receptors differ from
either of the two groups already described in that they have
two uniting growps, one for the antigen (cell introduced)
named cytophil haptophore, the other for the complement,
complementophil haptophore. Hence amboceptors are spoken
of as receptors of the third order. They have no other func-
tion than that of this double combining power. The action
which results is due to the third body—the complement.
It will be readily seen that complement must possess at
least two groups, a combining or haptophore group which
unites with the amboceptor, and an active group which is
usually called the zymophore or toxophore group. Comple-
ments thus resemble either toxins, where the specific cell
(antigen) is injured or killed, or enzymes, in case the cell is
likewise dissolved. This action again shows the close rela-
tion between toxins and enzymes. Complement may lose
its active group in the same way that toxin does and becomes
then complementoid. Complement is readily destroyed in
blood or serum by heating it to 55° to 56° for half an hour,
and is also destroyed spontaneously when serum stands for
a day or two, less rapidly at low temperature than at room
temperature.

Amboceptors appear to be specific in the same sense that
agglutinins are. That is, if a given cell is used to immunize

COMPLEMENTS 251

an animal, the animal’s blood will contain amboceptors for
the cell used and also for others closely related to it. Immun-
ization with spermatozoa or with epithelial or liver cells gives
rise to amboceptors for these cells and also for red blood
corpuscles and other body cells. A typhoid bactericidal
serum has also some dissolving effect on colon bacilli, etc.
Hence a given serum may contain a chief amboceptor and a
variety of “coamboceptors,”’ or one amboceptor made up
of a number of “partial amboceptors” each specific for its
own antigen (“‘amboceptorogen”). Amboceptors may com-
bine with other substances than antigen and complement, as
is shown by their union with lecithin and other “‘lipoids,”
though these substances seem capable of acting as comple-
ment in causing solution of blood corpuscles. ~

COMPLEMENTS.

As to whether complements are numerous, as Ehrlich
claims, or there is only one complement, according to Buch-
ner and others, need not be discussed here. In the practi-
cal applications given later as means of diagnosis it is appar-
ent that all the complement or complements are capable of
uniting with at least two kinds of amboceptors.

If complement be injected into an animal it may act as
an antigen and give rise to the formation of anticomplement
which may combine with it and prevent its action and is
consequently analogous to antitoxin. If amboceptors as
antigen are injected into an animal there will be formed by
the animal’s cells antiamboceptors. As one would expect,
there are two kinds of antiamboceptors, one for each of its
combining groups, since it has been stated that it is always
the combining group of any given antigen that acts as the
cell stimulus. Hence we may have an ‘“‘anticytophil ambo-
ceptor” or an “anticomplementophil amboceptor.” These
antiamboceptors and the anticomplements are analogous to
antitoxin, antiagglutinin, etc., and hence are receptors of
the first order.

252 RECEPTORS OF THE THIRD ORDER

ANTISNAKE VENOMS.

A practical use of antiamboceptors is in antisnake venoms,
Snake poisons appear to contain only amboceptors for differ-
ent cells of the body. In the most deadly the amboceptor
is specific for nerve cells (cobra), in others for red corpuscles,
or for endothelial cells of the bloodvessels (rattlesnake).
The complement is furnished by the blood of the individual
bitten, that is, in a sense the individual poisons himself,
since he furnishes the destroying element. The antisera
contain antiamboceptors which unite with the amboceptor
introduced and prevent it joining to cells and thus binding
the complement to the cells and destroying them. With
this exception these antibodies are chiefly of theoretical
interest.

FAILURE OF CYTOLYTIC SERUMS.

The discovery of the possibility of producing a strongly
bactericidal serum in the manner above described aroused
the hope that such sera would prove of great value in passive
immunization and serum treatment of bacterial diseases.
Unfortunately such expectations have not been realized and
no serum of this character of much practical importance has
been developed as yet (with the possible exception of Flex-
ner’s antimeningococcus serum in human practice. What
hog cholera serum is remains to be discovered).

The reasons for the failure of such sera are not entirely
clear. The following are some that have been offered: (1)
Amboceptors do not appear to be present in very large
amount so that relatively large injections of blood are neces-
sary, which is not without risk in itself. (2) Since the com-
plement is furnished by the blood of the animal to be treated,
there may not be enough of this to unite with a sufficient
quantity of amboceptor to destroy all the bacteria present,
hence the disease is continued by those that escape. (3) Or
the complement may not be of the right kind to unite with
the amboceptor introduced, since this is derived from the
blood of a heterologous (“other kind’’) species. In hog-
cholera serum, if it is bactericidal, this difficulty is removed

COMPLEMENT-FIXATION TEST 253

by using blood of an homologous (“same kind’’) animal.
Hence Ehrlich suggested the use of apes for preparing bac-
tericidal sera for human beings. (4) The bacteria may be
localized in tissues (lymph glands), within cavities (cranial,
peritoneal), in hollow organs (alimentary tract), etc., so that
it is not possible to get at them with sufficient serum to
destroy all. This difficulty is obviated by injecting directly
into the spinal canal when Flexner’s antimeningococcus
serum is used. (5) Even if the bacteria are dissolved it does
not necessarily follow that their endotorins are destroyed.
These may be merely liberated and add to the danger of the
patient, though this does not appear to be a valid objec-
tion. (6) Complement which is present in such a large excess
of amboceptor may just as well unite with amboceptor
which is not united to the bacteria to be destroyed as with
that which is, and hence be actually prevented from dissolv-
ing the bacteria. Therefore it is difficult to standardize the
serum to get a proper amount of amboceptor for the com-
plement present.

COMPLEMENT-FIXATION TEST.

Although little practical use has been made of bactericidal
sera, the discovery of receptors of this class and the peculiar
relations between the antigen, amboceptor and complement
have resulted in developing one of the most delicate and
accurate methods for the diagnosis of disease and for the
recognition of small amounts of specific protein that is in use
today.

This method is usually spoken of as the “ complement-fixa-
tion” or the ‘“complement-deviation test” (‘“Wassermann
test” in syphilis) and is applicable in a great variety of mi-
crobial diseases, but it is of practical importance in those
diseases only where other methods are uncertain—syphilis
in man, concealed glanders in horses, contagious abortion
in cattle, etc.

The principle is the same in all cases. The method
depends, as indicated above, on the ability of complement
to combine with at least two amboceptor-antigen systems,

?

254 RECEPTORS OF THE THIRD ORDER

and on the further fact that if the combination with one
amboceptor-antigen system is once formed, it does not dis-
sociate so as to liberate the complement for union with the
second amboceptor-antigen system. If an animal is infected.
with a microérganism and a part of its defensive action con-
sists in destroying the organisms in its blood or lymph, then
it follows from the above discussion of cytolysins that there
will be developed in the blood of the animal amboceptor
specific for the organism in question. If the presence of this
specific amboceptor can be detected, the conclusion is war-.
ranted that the organism for which it is specific is the cause
of the disease. Consequently what is sought in the “com-
plement-fixation test’ is a specific amboceptor. In carrying
out the test blood serum from the suspected animal is col-
lected, heated at 56° for half an hour to destroy any comple-
ment it contains and mixed in definite proportions with the
specific antigen and with complement. The antigen is an
extract of a diseased organ (syphilitic fetal liver, in syphilis),
a suspension of the known bacteria, or an extract of these
bacteria. Complement is usually derived from a guinea-
pig, since the serum of this animal is higher in complement
than that of most animals. The blood of the gray rat con-
tains practically as much. If the specific amboceptor is
present, that is, if the animal is infected with the suspected
disease, the complement will unite with the antigen-ambo-
ceptor system and be “‘fixed,”’ that is, be no longer capable
of uniting with any other amboceptor-antigen system. No
chemical or physical means of telling whether this union has
occurred or not, except as given below, has been discovered
as yet, though doubtless will be by physicochemical tests,
nor can the combination be seen. Hence an “indicator,” as
is so frequently used in chemistry, is put into the mixture
of antigen-amboceptor complement after it has been allowed
to stand in the incubator for one hour to permit the union
to become complete. The “indicator” used is a mixture of
sheep’s corpuscles and the heated (“inactivated”) blood
serum of a rabbit which has been injected with sheep’s
blood corpuscles and therefore contains a hemolytic ambo-
ceptor specific for the corpuscles, which is capable also of

COMPLEMENT-FIXATION TEST 255

uniting with complement. The indicator is put into the
first mixture and the whole is again incubated for at least
two hours and examined. If the mixture is clear and color-
less with a deposit of red corpuscles at the bottom, that would
mean that the complement had been bound to the first
complex, since it was not free to unite with the second
sheep’s corpuscles (antigen)—rabbit serum (hemolytic ambo-
ceptor) complex—and destroy the corpuscles. Hence if the
complement is bound in the first instance, the specific ambo-
ceptor for the first antigen must have been present in the
blood, that is, the animal was infected with the organism in
question. Such a reaction is called a “positive” test.

On the other hand, if the final solution is clear but of a red
color, that would mean that complement must have united
with the corpuscles—hemolytic amboceptor system—and
destroyed the corpuscles in order to cause the clear red
solution of hemoglobin. If complement united with this
system it could not have united with the first system, hence
there was no specific amboceptor there to bind it; no specific
amboceptor in the animal’s blood, means no infection.
Hence a red solution is a ‘‘negative test.”

In practice all the different ingredients must be accurately
tested, standardized and used in exact quantities, and a test
must also be run as a control with a known normal blood

‘of an animal of the same species as the one examined.

The complement-fixation test might be applied to the
determination of unknown bacteria, using the unknown cul-
ture as antigen and trying it with the sera of different animals’
immunized against a variety of organisms, some one of which
might prove to furnish specific amboceptor for the unknown.
organism and hence indicate what it is. The test used in
this way has not been shown to be a practical necessity and
hence is rarely employed. It has been used, however, to
detect traces of unknown proteins, particularly blood-serum
proteins, in medicolegal cases in exactly the above outlined
manner and is very delicate and accurate.

CHAPTER XXX.
PHAGOCYTOSIS—OPSONINS.

Ir has been mentioned that Metchnikoff, in a publication
in 1883, attempted to explain immunity on a purely cellular
basis. It has been known since Haeckel’s first observation
in 1858 that certain of the white corpuscles do engulf solid
particles that may get into the body, and among them bac-
teria. Metchnikoff at first thought that this engulfing and
subsequent intracellular digestion of the microdrganisms
were sufficient to protect the body from infection. The
later discoveries (discussed in considering Ehrlich’s theory
of immunity) of substances present in the blood serum and
even in the blood plasma which either destroy the bacteria
or neutralize their action have caused Metchnikoff to modify
his theory to a great extent. He admitted the presence of
these substances, though giving them other names, but ascribed
their formation to the phagocytes or to the same organs
which form the leukocytes—lymphoid tissue generally, bone
marrow. It is not within the province of this work to
attempt to reconcile these theories, but it may be well to
point out that Ehrlich’s theory is one of chemical substances
and that the origin of these substances is not an essential
part of the theory, so that the two theories, except in some
minor details, are not necessarily mutually exclusive.

Sir A. E. Wright and Douglas, in 1903, showed that even in
those instances where immunity depends on phagocytosis,
as it certainly does in many cases, the phagocytes are more
or less inactive unless they are aided by chemical substances
present in the blood. These substances act on the bacteria,
not on the leukocytes, and change them in such a way that
they are more readily taken up by the phagocytes. Wright
proposed for these bodies the name opsonin, derived from a

ELIE METCHNIKOFF

PHAGOCY TOSIS—OPSONINS 257

Greek word signifying “‘to prepare a meal for.” Neufeld
and Rimpau at about the same time (1904), in studying
immune sera, observed substances of similar action in these
sera and proposed the name bactertotropins, or bacteriotropic
substances. There is scarcely a doubt that the two names
are applied to identical substances and that Wright’s name
opsonin should have preference.

The chemical nature of opsonins is not certainly deter-
mined, but they appear to be a distinct class of antibodies
and to possess two groups, a combining or haptophore and
a preparing or opsonic group and hence are similar to anti-
bodies of Ehrlich’s second order—agglutinins and_precipi-
tins. Wright also showed that opsonins are just as specific
as agglutinins are—that is, a micrococcus opsonin prepares
micrococci only for phagocytosis and not streptococci or any
other bacteria.

Wright showed that opsonins for many bacteria are present
in normal serum and that in the serum of an animal which
has been immunized against -such bacteria the opsonins
are increased in amount. Also that in a person infected
with certain bacteria the opsonins are either increased or
diminished, depending on whether the progress of the infec-
tion is favorable or unfavorable. The opsonic power of a
serum normal or otherwise is determined by mixing an
emulsion of fresh leukocytes in normal saline solution with
a suspension of the bacteria and with the serum to be tested.
The leukocytes must first be washed in several changes of
normal salt solution to free them from any adherent plasma
or serum. The mixture is incubated for about fifteen min-
utes and then slides are made, stained with a good differ-
ential blood stain, Wright’s or other, and the average num-
ber of bacteria taken up by at least fifty phagocytes taken
in order in a field is determined by counting under the
microscope. The number so obtained Wright calls the
phagocytic index of the serum tested. The phagocytic index
of a given serum divided by the phagocytic index of a nor-
mal serum gives the opsonic index of the serum tested.
Assuming the normal opsonic index to be 1, Wright asserts
that in healthy individuals the range should be not more

17

258 ? PH AGOCYTOSIS—OPSONINS

than from 0.8 to 1.2, and that an index below 0.8 may show
a great susceptibility for the organism tested, infection with
the given organism if derived from the individual, or improper
dosage in case attempts have been made to immunize by
using killed cultures, vaccines, of the organism.

On the occasion of the author’s visit to Wright’s clinic
(1911) he stated that he used the determination of the opsonic
index chiefly as a guide to the dosage in the use of vaccines.

Most workers outside the Wright school have failed to
recognize any essential value of determinations of the
opsonic index in the use of vaccines. Some of the reasons
for this are as follows: The limit of error in phagocytic
counts may be as great as 50 per cent. in different series of
fifty, hence several hundred must be counted, which adds
greatly to the tediousness and time involved; the variation
in apparently healthy individuals is frequently great, hence
the “normal” is too uncertain; finally the opsonic index
and the clinical course of the disease do not by any means
run parallel. Undoubtedly the method has decided value
in the hands of an individual who makes opsonic determina-
tions his chief work, as Wright’s assistants do, but it can
scarcely be maintained at the present time that such deter-
minations are necessary in vaccine therapy. Nevertheless
that opsonins actually exist and that they play an essential
part in phagocytosis, and hence in immunity, is now generally
recognized.

BACTERIAL VACCINES.

Whether determinations of opsonic index are useful or
not is largely a matter of individual opinion, but there is
scarcely room to doubt that Wright has conferred a lasting
benefit by his revival of the use of dead cultures of bacteria,
bacterial vaccines, both for protective inoculation and for
treatment. It is perhaps better to use the older terms
“vaccination” and “vaccine’’ (though the cow, vacca, is not
concerned) than to use Wright’s term “opsonic method”’ in
this connection, bearing in mind that the idea of a vaccine
is that it contains the causative organism of the infection as
indicated on p. 231.

BACTERIAL VACCINES 259

As early as 1880 Touissant proposed the use of dead cul-
tures of bacteria to produce immunity. But because injec-
tions of such cultures were so frequently followed by abscess
formation, doubtless due to the high temperatures used to
kill the bacteria, the method was abandoned. Further, Pas-
teur and the French school persistently denied the possi-
bility of success with such a procedure, and some of them
even maintain this attitude at the present time. The suc-
cesses of Wright and the English school which are being
repeated so generally wherever properly attempted, leave
no doubt in the unprejudiced of the very great value of the
method and have unquestionably opened a most promising
field both for preventive inoculation and for treatment in
many infectious diseases. That the practice is no more
universally applicable than are immune serums and that it
has been and is still being grossly overexploited is undoubted.

The use of a vaccine is based on two fundamental prin-
ciples. The first of these is that the cell introduced must not
be in a condition to cause serious injury to the animal by its
multiplication and consequent. elaboration of injurious sub-
stances. The second is that, on the other hand, it must con-
tain antigens in such condition that they will act as stimuli
to the body cells to produce the necessary antibodies,
whether these be opsonins, bactericidal substances, or anti-
endotoxins. In the introduction of living organisms there
is always more or less risk of the organism not being suffi-
ciently attenuated and hence of the possibility of its pro-
ducing too severe an infection. In using killed cultures,
great care must be exercised in destroying the organisms,
so that the antigens are not at the same time rendered inactive.
Hence in the preparation of bacterial vaccines by Wright’s
method the temperature and the length of time used to kill the
bacteria are most important factors. This method is in gen-
eral to grow the organisms on an agar medium, rub off the
culture and emulsify in sterile normal salt solution (0.85
per cent. NaCl). The number of bacteria per c.c. is deter-
mined by staining a slide made from a small volume of the
emulsion mixed with an equal volume of human blood drawn
from the finger and counting the relative number of bac-

260 PHAGOCYTOSIS—OPSONINS

teria and of red blood corpuscles. Since the corpuscles are
normally 5,000,000 per c.mm., a simple calculation gives
the number of bacteria. The emulsion of bacteria is then
diluted so that a certain number of millions shall be contained
in each c.c., “standardized” as it is called, then heated to
the proper temperature for the necessary time and it is
ready for use. A preservative, as 0.5 per cent. phenol, tri-
cresol, etc., is added unless the vaccine is to be used up at
once. The amounts of culture, salt solution, etc., vary with
the purpose for which the vaccine is to be used, from one
or two agar slant cultures and a few c.c. of solution, when a
single animal is to be treated, to bulk agar cultures and
liters of solution as in preparing antityphoid vaccine on a
large scale.

Agar surface cultures are used so that there will be as
little admixture of foreign protein as possible (see Anaphy-
laxis, p. 264 et seq.). Normal saline solution is isotonic with
the body cells and hence is employed as the vehicle.

Vaccines are either ‘‘ autogenous” or “stock.” An ‘‘autog-
enous” vaccine is a vaccine that is made from bacteria
derived from the individual or animal which it is desired to
vaccinate and contains not only the particular organism but
the particular strain of that organism which is responsible
for the lesion. Stock vaccines are made up from organisms
like the infective agent in a given case but derived from
some other person or animal or from laboratory cultures.
Commercial vaccines are “stock” vaccines and are usually
“polyvalent’”’ or even “mixed.” A “polyvalent” vaccine
contains several strains of the infective agent and a “ mixed”’
contains several different organisms.

Stock vaccines have shown their value when used as pre-
ventive inoculations, notably so in typhoid fever in man,
anthrax and black-leg in cattle. The author is strongly of the
opinion, not only from the extended literature on the sub-
ject, but also from his own experience in animal, and espe-
cially in human cases, that stock vaccines are much inferior
and much more uncertain in their action when used in the
treatment of an infection, than are autogenous vaccines. This
applies particularly to those instances in which streptococci,
micrococe?, and colon bacilli are the causative agents, but to

BACTERIAL VACCINES 261

others as well. The following are some of the reasons for
this opinion: The above organisms are notoriously extremely
variable in their virulence. While there is no necessarily
close connection between virulence and antigenic property,
yet since virulence is so variable, it is rational to assume
that antigenic property is also extremely variable. Indi-
viduals vary just as much in susceptibility and hence in
reactive power, and generally speaking, an individual will
react better in the production of antibodies to a stimulus to
which he has been more or less subjected, 7. ¢., to organisms
derived: from his own body.

In the preparation of a vaccine great care must be used
in heating so that the organisms are killed, but the antigens
are not destroyed. Many of the enzymes present in bac-
teria, especially the proteolytic ones, are not any more sen-
sitive to heat than are the antigens, hence are not destroyed
entirely. Therefore a vaccine kept in stock for long gradu-
ally has some of its antigens destroyed by the uninjured
enzymes present with them, and so loses in potency. There-
fore in treating a given infection it is well to make up a
vaccine from the lesion, use three or four doses and if more
are necessary make up a new vaccine.

If the above statements are borne in mind and vaccines
are made and administered accordingly, the author is well
satisfied that much better results will be secured.

In accordance with the theory on which the use of vac-
cines is based, 7. e., that they stimulate the body cells to
produce immunizing antibodies, it is clear that they are espe-
cially suitable in those infections in which the process is
localized and should not be of much value in general infec-
tions. In the latter case the cells of the body are stimulated
to produce antibodies by the circulating organisms, prob-
ably nearly to their limit, hence the introduction of more of
the same organisms, capable of stimulating though dead, is
apt to overtax the cells and do more harm than good. It is
not possible to tell accurately when this limit is reached, but
the clinical symptoms are a guide. If vaccines are used at
all in general infections they should be given in the early
stages and in small doses at first with close watch as to the
effect. In localized infections only the cells in the immedi-

262 PHAGOCYTOSIS—OPSONINS

ate neighborhood are much stimulated, hence the introduc-
tion of a vaccine calls to their aid cells in the body gener-
ally, and much more of the resulting antibodies are carried
to the lesion in question. Manifestly surgical procedures
such as incision, drainage, washing away of dead and ne-
crotic tissue with normal saline, solution, not necessarily
antiseptics, will aid the antibodies in their action and are to
be recommended where indicated.

In the practical application of any remedy the dosage is
most important. . Unfortunately there is no accurate method
of determining this with a vaccine. Wright recommended
determining the number of the organisms per c.c. as before
mentioned, and his method or some modification of it is still
in general use. From what was said with regard to varia-
tion, both in organisms and in individuals, it can be seen that
the number of organisms is at least only a very rough guide.
This is further illustrated by the doses of micrococcus
(staphylococcus) vaccines recommended by different writers,
which vary from 50,000,000 to 2,000,000,000 per c.c. The
author is decidedly of the opinion that there is no way of
determining the dosage of a vaccine in the treatment of any
given case except by the result of the first dose. Hence it is
his practice to make vaccines of a particular organism of
the same approximate strength, and to give a dose of a
measured portion of a cubic centimeter, judging the amount
‘by what the individual or animal can apparently withstand,
without too violent a reaction. If there is no local or gen-
eral reaction or if it is very slight and there is no effect on
the lesion, the dose is too small. If there is a violent local reac-
tion with severe constitutional symptoms clinically, and the
lesion appears worse, the dose is too large. There should
be some local reaction and some general, but not enough to
cause more than a slight disturbance, easy to judge in human
subjects, more difficult in animals. In cases suitable for
vaccine treatment no serious results should follow from a
properly prepared vaccine, though the process of healing
may be delayed temporarily. Wright claimed, and many
have substantiated him, that always following a vaccination
there is a period when the resistance of the animal is dimin-
ished. This is called the “negative phase,” and Wright

AGGRESSIN 263

considered this to last as long as the opsonic index remained
low, and when this latter began to increase the stage of the
“positive” or favorable phase was reached. As has been
stated the opsonic index is pretty generally regarded as of
doubtful value, though the existence of a period of lowered
resistance is theoretically probable from the fact that the
body cells are called upon suddenly to do an extra amount
of work and it takes them some time to adapt themselves.
This time, the “negative phase,” is much better determined
by the clinical symptoms, general and especially local. It
is good practice to begin with a dose relatively small. The
result of this is an indication of the proper dosage and also
prepares the patient for a larger one. The second dose
should follow the first not sooner than three or four days,
and should be five to seven days if the first reaction is
severe. These directions are not very definite, but clinical
experience to date justifies them. It is worth the time and
money to one who wishes to use vaccines to learn from one
who has had experience both in making and administering
them, and then to remember that each patient is an indi-
vidual case for the use of vaccines as well as for any other
kind of treatment.

AGGRESSIN.

Opsonins have been shown to be specific substances which
act on bacteria in such a way as to render them more read-
ily taken up by the leukocytes. By analogy one might
expect to find bacteria secreting specific substances which
would tend to counteract the destructive action of the
phagocytes and bactericidal substances. Bail and_ his
co-workers claim to have demonstrated such substances in
exudates in certain diseases and have given the distinctive
name “‘aggressins” to them. By injecting an animal with
“agegressins,” antiaggressins are produced which counteract
their effects and thus enable the bacteria to be destroyed.
The existence of such specific bodies is not generally accepted
as proved. The prevailing idea is that bacteria protect
themselves in any given case by the various toxic substances ©
that they produce, and that “aggressins” as a special class
of substances are not formed.

CHAPTER XXXI.
ANAPHYLAXIS.

DaLiera, in 1874, and a number of physiologists of that
period, observed peculiar skin eruptions following the trans-
fusion of blood, that is, the introduction of foreign proteins.
In the years subsequent to the introduction of diphtheria
antitoxin (1890) characteristic ‘‘serum rashes’? were not
infrequently reported, sometimes accompanied by more or
less severe general symptoms and occasionally death —a
train of phenomena to which the name “serum sickness”
was later applied, since it was shown that it was the horse
serum (foreign protein) that was the cause, and not the anti-
toxin itself. In 1898 Richet and Hericourt noticed that some
of the dogs which they were attempting to immunize against
toxic eel serum not only were not immunized but suffered
even more severely after the second injection. They
obtained similar results with an extract of mussels which
contain a toxin. Richet gave the name “anaphylaxis”
(“no protection’’) to this phenomenon to distinguish it from
immunity or prophylaxis (protection).

All the above-mentioned observations led to no special
investigations as to their cause. In 1903 Arthus noticed
abscess formation, necrosis and sloughing following several
injections of horse serum in immediately adjacent parts of the
skin in rabbits (“Arthus’ phenomenon”). Theobald Smith,
in 1904, observed the death of guinea-pigs following properly
spaced injections of horse serum. This subject wasinvestigated
by Otto and by Rosenau and Anderson in this country and
about the same time von Pirquet and Schick were making a
study of serum rashes mentioned above. The publications of
these men led to a widespread study of the subject of injec-
tions of foreign proteins. It is now a well-established fact that

ANAPHYLAXIS 265

the injection into an animal of a foreign protein—vegetable,
animal or bacterial, simple or complex—followed by a
second injection after a proper length of time leads to a
series of symptoms indicating poisoning, which may be so
severe as to cause the death of the animal. Richet’s term
“anaphylaxis” has been applied to the condition of the
animal following the first injection and indicates that it is
in a condition of supersensitiveness for the protein in ques-
tion. The animal is said to be “sensitized’’ for that protein.
The sensitization is specific since an animal injected with white
of chicken’s egg’ reacts to a second injection of chicken’s
egg only and not pigeon’s egg or blood serum or any
other protein. The specific poisonous substance causing the
symptoms has been called “anaphylotoxin” though what it
is, is still a matter of investigation. It is evident that some
sort of an antibody results from the first protein injected
and that it is specific for its own antigen.

A period of ten days is usually the minimum time that
must elapse between the first and second injections in guinea-
pigs in order that a reaction may result, though a large pri-
mary dose requires much longer. If the second injection is
made within less time no effect follows, and after three or
more injections at intervals of about one week the animal
fails to react at all, it has become “immune” to the protein.
Furthermore, after an animal has been sensitized by one
injection and has reacted to a second, then, if it does not
die from the reaction, it fails to react to subsequent injec-
tions. In this latter case it is said to be “antianaphylactic.”

It must be remembered that proteins do not normally get
into the circulation except by way of the alimentary tract.
Here all proteins that are absorbed are first broken down
to their constituent amino-acids, absorbed as such and these
are built up into the proteins characteristic of the animal’s
blood. Hence when protein as such gets into the blood it is
a foreign substance to be disposed of. The blood contains
proteolytic enzymes for certain proteins normally. It is also
true that the body cells possess the property of digesting
the proteins of the blood and building them up again into
those which are characteristic of the cell. Hence it appears

266 ANAPHYLAXIS

rational to assume that the foreign proteins act as stimuli
to certain cells to produce more of the enzymes necessary
to decompose them, so that they may be either built up
into cell structure or eliminated as waste. If in this process
of splitting up of protein a poison were produced, then the
phenomena of ‘anaphylaxis’ could be better understood.
As a matter of fact Vaughan and his co-workers have shown
that by artificially splitting up proteins from many different
sources—animal, vegetable, pathogenic and saprophytic bac-
teria—a, poison 1s produced which appears to be the same in
all cases and which causes the symptoms characteristic of
anaphylaxis. On the basis of these facts it is seen that
anaphylaxis is simply another variety of immunity. The
specific antibody in this case is an enzyme which decomposes
the protein instead of precipitating it. The enzyme must
be specific for the protein since these differ in constitution.
Vaughan even goes so far as to say that the poison is really
the central ring common to all proteins and that they differ
only in the lateral groups or side chains attached to this
central nucleus. The action of the enzyme in this connec-
tion would be to split off the side chains, and since these are
the specific parts of the protein, the enzyme must be spe-
cific for each protein. The pepsin of the gastric juice and
the trypsin of the pancreas split the native proteins only to
peptones. As is well known these when injected in suffi-
cient quantity give rise to poisonous symptoms, and will
also give rise to anaphylaxis under properly spaced injec-
tions. They do not poison normally because they are split
by the intestinal erepsin to amino-acids and absorbed as
such. Whether Vaughan’s theory of protein structure is the
true one or not remains to be demonstrated. It is not essen-
tial to the theory of anaphylaxis above outlined, i. ¢., a
phenomenon due to the action of specific antibodies which are
enzymes. On physiological grounds this appears the most
rational of the few explanations of anaphylaxis that have
been offered and was taught by the author before he had
read Vaughan’s theory along the same lines.

On the basis of the author’s theory the phenomena of
protein immunity and antianaphylaxis may be explained in

ANAPHYLAXIS 267

the following way which the author has not seen presented.
The enzymes necessary to decompose the injected protein
are present in certain cells and are formed in larger amount
by those cells to meet the increased demand due to injection
of an excess of protein. They are retained in the cell for a
time at least. If a second dose of protein is given before the
enzymes are excreted from the cells as waste, this is digested
within the cells in the normal manner. If a third dose is
given, the cells adapt themselves to this increased intra-
cellular digestion and it thus becomes normal to them.
Hence the immunity is due to, this increased intracellular
digestion. ;

On the other hand, if the second injection is delayed long
enough, then the eacess enzyme, but not all, is excreted from
the cells and meets the second dose of protein in the blood
stream and rapidly decomposes it there, so that more or less
intoxication from ‘the split products results. This uses up
excess enzyme, hence subsequent injections are not digested
in the blood stream but within the cells as before. So that
“antianaphylaxis” is dependent on the exhaustion of the
excess enzyme in the blood, and the condition is funda-
mentally the same as protein immunity.

As has been indicated “serum sickness’ and sudden death
following serum injections are probably due to a sensitization
of the individual to the proteins of the horse in some unknown
way. Possibly urticarial rashes and idiosyncrasies follow-
ing the ingestion of certain foods—strawherries, eggs, oysters,
etc., may be anaphylactic phenomena.

In medical practice the reaction is used as a means of
diagnosis in certain diseases, such as the tuberculin test in
tuberculosis, the mallein test in glanders. The individual
or animal with tuberculosis becomes sensitized to certain
proteins of the tubercle bacillus and when these proteins in
the form of tuberculin are introduced into the body a reac-
tion results, local or general, according to the method of
introduction. The practical facts in connection with the
tuberculin test are also in harmony with the author’s theory
of anaphylaxis as above outlined, Milder cases of tubercu-
losis give more vigorous reactions because the intracellular

268 ANAPHYLAXIS

enzymes are not used up rapidly enough since the products
of the bacillus are secreted slowly in such cases. Hence
excess of enzyme is free in the blood and the injection of
the tuberculin meets it there and a vigorous reaction results.
In old, far-advanced cases, no reaction occurs, because the
enzymes are all used in decomposing the large amount of
tuberculous protein constantly present in the blood. The
fact that an animal which has once reacted fails to do so
until several months afterward likewise depends on the fact
that the excess enzyme is used in the reaction and time must
elapse for a further excess to accumulate.

The anaphylactic reaction has been made’ use of in the
identification of various types of proteins and is of very
great value since the reaction is so delicate, particularly
when guinea-pigs are used as test animals. Wells has
detected the 0.000,001 gm. of protein by this test. It is evi-
dent that the test is applicable in medicolegal cases and in
food examination and has been so used.

SUMMARY.

The discussion of “immunity problems” in the preceding
chapters serves to show that protection from disease either
as a condition natural to the animal or as an acquired state
is dependent on certain properties of its body cells or fluids,
or both. The actual factors so far as at present known may
be summarized as follows:

1. Antitoxins which neutralize true toxins, shown to exist
for very few diseases.

2. Cytolytte substances which destroy the invading organ-
ism: in reality two substances; amboceptor, which is specific,
and complement the real dissolving enzyme.

3. Phagocytosis or the destruction of the invading organ-
isms within the leukocvtes.

4. Opsonins which render the bacteria more readily taken
up by the phagocytes.

5. Enzymes other than complement possibly play a part
in the destruction of some pathogenic organisms or their
products. This 'remains to be more definitely established.

ANAPHYLAXIS 269

6. It is possible that in natural immunity there might
be no receptors in the body cells to take up the organisms
or their products, or the receptors might be present in
certain cells but of a very low chemical affinity, so that com-
bination does not occur. It is even highly probable that
many substances formed by invading organisms which might
injure specialized cells, such as those of glandular, nervous
or muscle tissue, have a more rapid rate of reaction with, or
a stronger affinity for, lower unspecialized cells, such as con-
nective and lymphoid tissue, and unite with these so that
their effects are not noticed.

The importance of these different factors varies in differ-
ent diseases and need not be considered in this connection.

The question “which of the body cells are engaged in the
production of antibodies” is not uncommonly asked. On
physiological grounds it would not seem reasonable that the
highly specialized tissues above mentioned could take up
this work, even though they are the ones which suffer the
greatest injury in disease. Hence it is to be expected that
the lower or unspecialized cells are the source, and it has
been shown that the antibodies are produced by the phago-
cytes (though not entirely as Metchnikoff maintained), by
lymphoid tissue generally, by the bone marrow and also by
connective-tissue cells, though in varying degrees.

Since immunity depends on the activity of the body cells
it is evident that one of the very best methods for avoiding
infectious diseases is to keep these cells up to their highest
state of efficiency, to keep in “good health.’’ Hence good
health means not only freedom from disease but also protection
against disease.

List or Lasporatory EXERCISES GIVEN IN CONNECTION
WITH THE CLAss Work INCLUDED IN THIS TEXT-BOOK.

Exercise
Exercise
Exercise
Exercise
Exercise
Exercise
Exercise
Exercise
Exercise

Exercise 10.
Exercise 11.

Exercise 12.
Exercise 13.
Exercise 14.
Exercise 15.

Exercise 16.
Exercise 17.

Exercise 18.
Exercise 19.
Exercise 20.
Exercise 21.
Exercise 22.
Exercise. 23.
Exercise 24.
Exercise 25.
Exercise 26.
Exercise 27.
Exercise 28,

COMBA pw

. Cleaning glassware.

Preparation of broth medium from meat juice.

. Preparation of gelatin medium from broth.
. Preparation of agar medium from broth.

Potato tubes.

. Potato plates.

. Plain milk tubes.

. Litmus milk tubes.
. Sugar broth media.

Blood-serum tubes.

Inoculation of tubes.
proteins.

Production of gas from carbohydrates.

Production of indol.

Reduction of nitrates.

Chromogenesis: Illustrates nicely the varia-
tion with environment.

Enzyme production.

Making of plate cultures; isolation in pure
culture.

Stain making and staining.

Cell forms and cell groupings.

Hanging drop slides.

Staining of spores.

Staining of acid-fast bacteria.

Staining of capsules.

Staining of flagella.

Study of individual species.

Determination of thermal death-point.

Action of disinfectants on bacteria.

Action of sunlight on bacteria.

Action on complex

INDEX.

A

Apps, 28
condenser, 184-185
ABILGAARD, 24
Abortion, contagious, 224, 226, 253
Abrin, 239
Absorption of free nitrogen, 105
Accidental carriers, 220
structures, 40
Acetic acid, 88
fermentation, 29
Achorion schoenleinii, 25
Acid, acetic, 88
fermentation, 29
butyric, 88
fermentation, 29
carbolic, first used, 26
fast bacilli, staining of, 193
bacteria, 73
fermentation, 82
hydrochloric, 225
lactic, fermentation, 85-88
production of, 98
soils, 70
Acquired immunity, 230
Actinomyces bovis, 27
Actinomycosis, 223
Actions, reducing, 101
Activating enzymes, 113
Active immunity, 230
production of, 230-232
Activities, physiological, definition

of, 76
in identification, 199

Activity, overproduction, of cells,

235, 236
Acute coryza, 223

disease, 214
Aérobe, facultative, 66
Aérobes, 66

Aérobic, 66
Agar medium, 163-164
Agent, choice of, for disinfection,
151-156
Agents, physical, for disinfection,
119-143
Agglutinating group, 243
Agglutination, 242
macroscopic, 242
microscopic, 242
Agglutinin, 242
bacterial, 242
hema-, 242
Agglutinins, 242-245
anti-, 246
co-, 244
compared with precipitins, 245
functions of, 243
normal, 243
specificity of, 244
theory of formation of, 242
use of, 243, 244
Agglutinogen, 243
Aggressin, 263
ugg 2 isolation of bacteria, 182,
Air, bacteria in, 61
filtration, 141
Albumin in bacteria, 73
Alcohol as disinfectant, 148
Alcoholase, 113
Alcoholic fermentation, 29, 89
Alexin, 248
Alge, relation to bacteria, 34
Alimentary tract as channel of
infection, 225
Amboceptor, 250
hemolytic, 254
partial, 251
specific, 254
Amboceptorogen, 251

272

Amboceptors, 250
anti-, 251, 252
co-, 251
specificity of, 250
Ameba coli, 27
Ameboid colony, 207
Ammoniacal fermentation, 29
Amphitrichic, 43
Amylase, 112
Anaérobe, facultative, 66
Anaérobes, 66
Anaérobic, 66
bacteria, first discovered, 29
organisms, cultivation of, 172-
176
Analysis, chemical, of tubercle
bacilli, 74
of ash, 71
Anaphylactic, anti-, 265, 267
phenomena, 267
reaction, uses of, 268
Anaphylatoxin, 265
Anaphylaxis, 264-268
anti-, 267
antibodies in, 266
theory of, 266
ANDERSON, 264
Awnory, Nicuouas, 24
Anilin dyes, 27, 188
fuchsin, 189
gentian violet, 189
Animal inoculation, 210
Animalcules, bacteria considered to
be, 19
Animals, disinfection of, 155
experimental, 210
Ankylostomum duodenale, 26
Anthrax, 26, 218, 222, 225, 226
spores of, 27
vaccine, 231
Antiagglutinins, 246
Antiaggressin, 263
Antiamboceptors, 251, 252
Antianaphylactic, 265
Antianaphylaxis, 267
Antibacterial immunity, 232
Antibodies, 237
in anaphylaxis, 266
place of production of, 269
Antibody formation, 116
Anticomplement, 251
Antidiphtheritic serum, 240
Antienzyme, 239
Antienzymes, 238-241

INDEX

Antigen, 237
syphilitic, 254
Antipollenin, 240
Antiprecipitins, 246
Antisepsis, 119
Antiseptic, 119
methods introduced, 26
Antisnake venoms, 252
Antitetanic serum, 240
Antitoxic immunity, 232
Antitoxin, 238
diphtheria, 28
preparation of, 240
standard, 241
unit of, 241
Antitoxins, 238-241, 268
Antivenin, 240
Apex of lung, 227
Apparatus of Barber, 180
Appearance of growth in identifi-
cation, 200
AppERT, 20
Aqueous gentian violet, 189
Arborescent growth, 204
Aromatic compounds, 93
production of, 99
ARTHUS, 264
Arthus’s phenomenon, 264
Articles, unwashable, disinfection
of, 155
washable, disinfection of, 155
Artificial immunity, 230
Ase, termination of name of
enzyme, 112
Asepsis, 119
Aseptic, 119
Ash, analysis of, 71
Asiatic cholera, 227
Attenuated, 231
Autoclave, 125
Autogenous vaccine, 260
Autoinfection, 216
Autolysis, 114
Autotrophic, 75
Azotobacter, 106

B

Bases-Ernst corpuscles, 42
Bacilli, butter, 193

colon, 226

grass, 193

tubercle, chemical analysis of, 74

INDEX

Bacillus, 49, 59

botulinus, 98, 116, 218, 238

coli, 67, 84, 148, 161, 316, 224

Ducrey! 8, 224

enteritidis, 93

fluorescens, 67

of blue milk, 29

pastorianus, 106

prodigiosus, 67

subtilis, 67, 72, 193

tetani, 116, 193, 215, 238, 240

typhosus, 63, 224, 242

vulgaris, 67

Bacteria, acid-fast, 73

aids in isolation of, 182, 183

anaérobic, first: discovered, 29

as scavengers, 95

chemical elements in, 71

classed as fungi, 34

considered to be animalcules, 19

definition of, 37

energy relationships of, 36, 37

first classed as plants, 30
seen, 18
shown to cause disease, 27

in feces, 62

in the air, 61

in the sea, 61

in the soil, 61

in-urine, 62

in water, 61

iron, 75, 78

isolation of, 178-183

measurement of, 37

metabolism of, 75-81

morphology of, 39

motile, 42

nitric, 103

nitrous, 102

occurrence, 61

pathogenic, outside the body,
218

position of, 34
rate of movement of, 42
related to protozoa, 37
relation to alge, 34

to molds, 34

to yeasts, 34
relationships of, 34
size of, 37
staining of, 188-195
study of, 157

of physiology of, 196-209
sulphur, 75, 94

18

273

Bacteriacece, 59
Bacterial agglutinin, 242
vaccines, 258
preparation of, 259, 261
Bacterin, 231
Bacteriocidin, 249
Bacteriological examination, ma-
terial for, 211
microscope, 184
Bacteriology, pathogenic, defini-
tion of, 213
reasons for study, 217
\Bacteriolysin, 249
‘Bacteriotropin, 257
Bacterium, 59
abortus, 242
acidt oxalici, 72
anthracis, 148, 193
diphtheria, 116, 215, 216, 238, 240
lepre, 193
malle?, 242
of Johne’s disease, 193
smegmatis, 193
study of, necessary steps for, 158
tuberculosis, 72, 160, 193
aylinum, 72
BarsBer and Wess, 231
apparatus, 180
Barnyards, disinfection of, 153-155
Baskets, wire, 168
Bass, 25 ;
Bastian, Dr. CHaruton, 23
BAUMGARTNER, 234
Beaded growth, 204
Bed-bugs as carriers, 220
Beds, contact, 104
Beer, pasteurization of, 129, 133
Beggiatoa, 60
Beggiatoacee, 60
BrEuRING, 28
BELFANTI, 248
Bere, 25
BERKEFELD, 142
Bichloride of mercury as disinfec-
tant, 146
BILHaRzZ, 26
Bilharzia disease, 26
Biochemie! reactions, definition of,
6

Bipolar germination of spore, 46
Black-leg, 218, 222, 226
vaccine, 231
Bleaching powder as disinfectant,
146

274

Blood, detection of, 246
serum, Loeffler’s, 166
medium, 166, 167
Blue milk bacillus, 29
fermentation, 29
Borum, 25
Boils, 216, 222
Bo.uincER, 27
Bonnet, 19
Borpet, 248
Botrytis bassiana, 25
Bottles, staining, 190
Bougie, 141
Bouillon, 159
Bread, salt rising, 84
Bronchopneumonia, 215, 224
Broth, glycerin, 160
meat, 159
nitrate, 161
sugar, 160
Brownian motion, 44
movement, 44
Bucuner, 248
Budding of yeasts, 34
Burning, 120
Burying, 142
Butter bacilli, 193
rancidity of, 90
Butyric acid, 88
fermentation, 29
Buzzards as carriers, 220

Cc

Ca1Gnarp-LatTour, 29
Calcium hypochlorite as disinfec-
tant, 146
oxide as disinfectant, 146
Candles, filter, 141
Canned goods, spoiling of, 48
Canning, 68
Capsule, 41
of the spore, 45
Capsules, staining of, 194
Carbohydrates, fermentation of, 82
in bacteria, 73
Carbol-fuchsin, 189
Carbolic acid as disinfectant, 147
first used, 26
Carbon, circulation of, 96
uses of, 77
CarBone, 248
Carriers, 219

INDEX

Carriers, accidental, 220
Cars, stock, disinfection of, 155
Catalase, 113
Catalyzer, 111 ;
Causation of disease, 23, 116
Cell contents, 39
forms, 49, 55
staining for, 195
groupings, 52-55
staining for, 195
structures, 39
wall, 39
composition of, 72

| Cells, chemical stimuli of, 235

overproduction activity of, 235,
236
specific
235 ;
Cellular theory of immunity, 234
Cellulose, 72 :
Chain, 52
Channels of infection, 222
alimentary tract, 225
conjunctiva, 223
external auditory meatus, 223
genitalia, 224
intestines, 225
lungs, 224
milk glands, 223
mouth cavity, 223
mucose, 223
nasal cavity, 223
pharynx, 223
skin, 222
stomach, 225
tonsils, 223
Chaos, 24
Characteristics of toxins, 114, 115
Cuarrin, 242
Chart, descriptive, 200
CHAUVEAU, 234
Cheese, eyes in, 85
poisoning, 93
ripening, 29
Chemical analysis of tubercle
bacilli, 74
composition, 70-74
of bacteria, 36
elements in bacteria, 71
stimuli, effect of, 236
of cells, 235
specific, 236
theory of immunity, 234
Chemotherapy, 227, 232

chemical stimuli of,

INDEX

CHEVREUIL and Pasteur, 21, 25,

29
Chicken-pox, 224
Chitin, 72
Chlamydobacteriacee, 60
Chlamydothrix, 60
Chloride of lime as disinfectant, 146
Chlorine as disinfectant, 145
Chloroform as disinfectant, 150
Chlorophyl, absent in bacteria, 34
Chlorosis, Egyptian, 26
Cholera, Asiatic, 227

hog, 220, 226, 230
Choleras, 225, 226, 227
Chromogenesis, 100
Chromoparic, 100
Chromophoric, 100
Chronic disease, 214
Chronological index, 31
Circulation of carbon, 96

of nitrogen, 96

of phosphorus, 96

of sulphur, 97
Cladothriz, 60
Classification, first attempt at, 30

Migula’s, 59, 60
Cleaning of slide, 191
Clostridium, 47, 106
Clothing for disinfection, 156
Coagglutinins, 244
Coagulases, 112
Coagulating enzymes, 112
Coamboceptors, 251
Cobra, 252
Coccacee, 59
Coccus, 49
Coenzymes, 110
Coun, 26, 30
Cold, 136

incubator, 198
‘Colds, 223
Colon bacilli, 226 »
Colonies, plate, study of, 209
Colony, 159

ameboid, 207

effuse, 207

punctiform, 206

thizoid, 206, 208
» Combustion, spontaneous, 104
Commensal, 76
Complement, 250

anti-, 251

deviation test, 253

fixation test, 253-255

275

Complement, lecithin as, 251

source of, 254
Complementoid, 250
Complementophil haptophore, 250
Complements, 251 :
Composition, chemical, 70-74

of bacteria, 36

Compounds, aromatic, 93
Compressed oxygen, 67
Condenser, Abbé, 184, 185
Conditions for formation of spores,

48
for growth, general, 62
maximum, 62
minimum, 62
optimum, 62
Conjunctiva as channel of infec-
tion, 223
Contact beds, 104
Contagion, direct and indirect, 23
Contagious abortion, 224, 226, 253
diseases, 25, 214
Contagium vivum, 23
Contents, cell, 39
Continuous pasteurization, 129
Corpuscles, Babes-Ernst, 42
red, 227
Corrosive sublimate as disinfectant,
146
Coryza, acute, 223
Cotton plugs, 20, 168
Coughing, 226
Crateriform liquefaction, 205
Cream, ripening of, 86
CrEItTE, 248
Crenothrix, 60
Creolin as disinfectant, 148
Creosols as disinfectants, 147
Cultivation of anaérobic organisms,
170-176
Cultural characteristics as physio-
logical activity, 117
Culture medium, 157
essentials of, 158
gelatin, 161-163
inoculation of, 170, 176
pure, 157
tubes, 168
Cultures, mass, 172
plate, 164, 172
puncture, 169
pure, methods of obtaining, 178-
183

slant, 170

276

Cultures, slope, 170
stab, 169
Curled edge, 208
Cutaneous inoculation, 211
Cycle, nitrogen, etc., 96, 97
Cytolysin, 249
Cytolysins, 248-255
Cytolytic, 249 ;
serums, failure of, 252
substances, 268
Cytophil haptophore, 250
Cytoplasm, 39
Cytotoxic, 249

D

Da.iera, 264
DavaineE and Rayer, 26
Death-point, thermal, 65
determination of, 198
Decomposition of urea, 95
Deep culture tubes, 174
Definition of bacteria, 37
of immunity, 217
of pathogenic bacteriology, 213
of spore, 48
of unit, 241
Degeneration forms, 51
Denitrification, 102
Deodorant, 119
Deposits of sulphur, 104
Descriptive chart, 200
Detection of blood, 246
a of oxygen relations,
of thermal death-point, 198
Deviation test, complement, 253
Diastase, 112
Diffusion, food of bacteria by, 36
Digestion of milk, 91
. Diluting blood serum, necessity
for, 244
Dilution plates, 178
Diphtheria, 223, 227
antitoxin, 28
Diplobacillus, 52
Diplococcus, 53
Diplospirillum, 52
Discharges, intestinal, 226
nasal, 226
urethral, 226
vaginal, 226
Discontinuous sterilizaticn, 121

INDEX

Disease, acute, 214
Bilharzia, 26
causation of, 23, 116
chronic, 214
contagious, 25
foot-and-mouth, 223, 226
germs, 23
hookworm, 26
Johne’s, 225, 226
non-specific, 215
of flies, 26
of silkworms, 26
silkworm, 25
Diseases, contagious, 214
infectious, 213
skin, 222
specific, 215
Dishes, Petri, 164
Disinfectant, 119
Disinfectants, factors affecting,
152-156
Disinfection, 118
and sterilization, practical, 152-
156
chemical agents, 144-150
choice of agent, 151-156
clothing for, 156
of animals, 155
of barnyards, 153-155
of harness, 155
of manure, liquid, 155
of rooms, 153
of stables, 153-155
of stock cars, 155
of surgical instruments, 153
of unwashable articles, 155
of vehicles, 155 :
of washable articles, 155
physical agents for, 119-143
steam and formaldehyde, 156
Dissemination of organisms in the
body, 225
Distilling, sour mash, 87
Division, planes of, 53
rate of, 80
Dosage of vaccines, 262
Dose, minimum lethal, 241
standard test, 241
Dovatas, 256
Dourine, 224, 226
Drumstick spore, 47
Dry heat, 121
Drying, 119
Ducrey’s bacillus, 224

INDEX

Dunham’s peptone solution, 161
Douruam, 242

Dyes, anilin, 28, 188
Dysenteries, 225, 226, 227
Dysentery, tropical, 27

; E

Ecroriasm, 39
Edge, coarsely granular, 208
curled, 208
entire, 208
lacerate, 207
rhizoid, 208
Edema, malignant, 218, 222
Effuse colony, 205
EHRENBERG, 30
Euruicu, 234
Ehrlich’s theory of formation of
precipitins, 245
of immunity, fundamental
principles of, 235
summary of, 237
EIcusTep, 26
Electric milk purifier, 139, 140
Electricity, 68, 138
Elements, chemical, in bacteria, 71
Elimination of pathogenic organ-
isms, 226
paths of, gall-bladder, 226
kidneys, 226
saliva, 226
urine, 226
Empusa muce, 26
Endo-enzymes, 114
Endogenous infection, 216
Endoplasm, 39
Endotoxins, 116
Energy relationships of bacteria,

Ensilage, 87

Enteritis, 215

Entire edge, 208

Entrance, paths of, of pathogenic
organisms, 222

Enzyme, final test for, 111

Enzymes, 109-114, 268
activating, 113
and toxins compared, 114, 115
as catalyzers, 111
chief characteristics of, 111
classification of, 112, 113
co-, 110

277

Enzymes, coagulating, 112
name of, 112
oxidizing, 113
produced by all cells, 112
production of, 109
reducing, 113
similar to living organisms, 109,
110
splitting, 112 ,
Enzymoid, 239
Epitheliolysin, 249
Equatorial germination of spore, 45
Erysipelas, hog, 226
Essential structures, 39
Essentials of a culture medium, 158
Esters, production of, 98
Ether as disinfectant, 150
Eubacteria, 59
Examination, bacteriological, mate-
rial for, 211
Exanthemata, 226
Exhaustion theory of immunity,
234
Exo-enzymes, 114 ‘
Exogenous infection, 216
Exotoxins, 116
Experimental animals, 210
Explanation of natural immunity,
216
External auditory meatus as chan-
nel of infection, 223
genitalia, 227
as channels of infection, 224
Eyes in cheese, 85

F

Factors affecting disinfectants,
151, 152
modifying immunity, 228
Facultative, 66
aérobe, 66
anaérobe, 66
parasite, 76
Failure of cytolytic serums, 252
of vaccines, 261
Fats in bacteria, 73
splitting of, 90
Favus, 25
Feces, bacteria in, 62
Fermentation, 82
acetic acid, 29, 88
acid, 82

278

Fermentation alcohols, 29, 89
ammoniacal,
and aca a 28
blue milk, 29
butyric acid, 29, 88
gaseous, 82
lactic acid, 85-88
of carbohydrates, 82
tubes, 68, 174
Ferments, organized, 114
unorganized, 114
Fever, Malta, 245
recurrent, 27
scarlet, 224, 226
Texas, 214, 215, 220
typhoid, 214, 226
yellow, 221
Filament, 52
Filiform growth, 204
Film, fixing of, 191
preparation of, 190
Filter, Berkefeld, 142
candles, 141
Pasteur-Chamberland, 142
sprinkling, 104
Filterable organisms, 216
virus, 216
Filtration, 140, 141
air, 141
water, 141
First order, receptors of 239
Fixation test, complement, 253-255
Fixed, 254
Fixing of film, 191
Flagella, position of, 42, 43
staining of, 194
Flagellum, 42
Flash process, 133
Fleas as carriers, 220
FLEXNER, 253
Flies as carriers, 220
disease of, 26
. Fitiacr, 248
Fopor, von, 248
Food taken in by diffusion, 36
uses of, 75
Foot-and-mouth disease, 223, 226
Foreign-body pneumonia, 224
Formaldehyde and steam disinfec-
tion, 156
as disinfectant, 148-150
Formation of agglutinins, theory

of, 24
of antibodies, 116

INDEX

Formation of precipitins, Ehrlich’s
theory, 245
of spores, 45
conditions for, 48
Forms, cell, 49, 55
degeneration, 51
growth, 51
involution, 51
study of, 30
Foxes as carriers, 220
FRACASTORIUS, 23
Free receptors, 237
spore, 45
Fucus, C. J., 29
Fuchsin, anilin, 189
carbol, 189
Functions of agglutinin, 243
Fungi, bacteria classed as, 34
Funnel-shaped liquefaction, 205

G

GasBsEtT’s blue, 190
method of staining, 193
Gall-bladder as a path of elimina-
tion, 226
Gas, natural, 84
production of, 98
Gaseous fermentation, 82
GasparD, 25

Gelatin culture medium, 161-163

liquefaction of, 92
plates first used, 27
General conditions for growth, 62
infections, vaccines in, 261
Generation, spontaneous, 17
Generic names introduced, 30
Genitalia, external, 227
as channels of. infection, 224
Gentian violet, anilin, 18
aqueous, 189
German measles, 214
Germination of spore, 45
bipolar, 46
eae 45
oblique, 45
polar, 45
Germs, disease, 23
GuscHEIDEL, 248

‘Glanders, 215, 223, 224, 226, 227,

253
infectious, 24
mallease, reaction in, 246

INDEX

Glands, mammary, 226
salivary, 226
GLEIcHEN, 30
Globulin in bacteria, 73
Glycerin broth, 159
Glycerinized potato, 166
Gonococcus, 224
Gonorrhea, 226, 227
Good health, 269
Grain rust, 25
Gram-negative, 192
positive, 192
Gram’s method of staining, 191
, solution, 192
Granular, coarsely, edge; 208
Granules, metachromatic, 42
Neisser, 42
.. polar, 42
Grape juice, pasteurization of, 129,
132 ‘

Grass bacilli, 193
ren plants, nitrogen nutrition of,
10
GRIESINGER, 26
Group, agglutinating, 243
haptophore, 238, 239, 243, 247,
250,
precipitating, 246
' toxophore, 250
zymophore, 239, 247, 250
Groupings, cell, 52, 55
Growth, appearance of, in identifi-
cation, 200
arborescent, 204
beaded, 204
filiform, 204
forms, 51
papillate, 204
villous, 204
GRuBER, 242
Gruber-Widal test, 244
Grusy, 25

H

HAECKEL, 256
Hanging drop slide, 185
Hansen, Emit Cur., 27
Haptophore, complementophil, 250
cytophil, 250
group, 238, 239, 243, 247, 250
Harness, etc., disinfection of, 155
Health, good, 269

279

Heat, 119-135
dry, 121
moist, 121
production of, 104
Heated serum, 248
Hemagglutinin, 242
Hemicellulose, 72
Hemolysin, 249
Hemolytic amboceptor, 254 |
Hemorrhagic septicemias, 225
HENLE, 25, 215
Hericourt, 264
Herpes tonsurans, 25
HESSELING, VON, 29
Heterologous, 252
Heterotrophic, 75
Hitt, 30
HorrMan, 23
Hog cholera, 220, 226, 230
erysipelas, 226
Homologous, 253
Hookworm disease, 26
Host, 76
Hot beds, 105
Hydrochloric acid, 225 ;
Hydrogen peroxide as disinfectant,
145, 150
uses of, 78
Hydrophobia, 227
Hydrostatic pressure, 69
Hypochlorites as disinfectant, 145

I

Icr-cREAM poisoning, 93
Identification,appearance of growth
in, 200
of blood, 246
of meat, 246
of milk, 246
physiological activities in, 199 °
Immunity, 228
acquired, 230
active, 230
production of, 230-232
antibacterial, 232
antitoxic, 232
artificial, 230
cellular theory of, 234
chemical theory of, 234
definition of, 213

Ehrlich’s theory of, fundamental |
principles of, 235 ,

280

Immunity, Ehrlich’s theory of,
summary of, 237
exhaustion theory of, 234
inherited, 230
natural, 230
explanation of, 269
noxious retention theory of, 234
outlines of, 229
passive, 230.
phagocytosis theory of, 234
relative, 228
serum-simultaneous method in,
231
side-chain theory of, 234
summary of, 268
theories of, 234
to protein, 265, 267
unfavorable environment theory
of, 234
Inactivated, 249
Incubation, period of, 214
Incubator, cold, 198
Incubators, 196
Index, chronological, 31
opsonic, 257, 258
phagocytic, 257
Indicator, 254
Indol, 93
Infection, 214
auto-, 216
channels of, 222
endogenous, 216
exogenous, 216
mixed, 216
primary, 216
secondary, 216
wound, 25, 27, 215
caused by bacteria, 27
Infections, general, vaccines in, 261
localized, vaccines in, 261
Infectious diseases, 213
Infective organisms, specificity of
location of, 227
Infestation, 214
Infested, 214
Influenza, 224, 226, 227
Infusoria, 30
Inherited immunity, 230
Inoculation, 210
by feeding, 211
by inhalation, 211
cutaneous, 211
intracardiac, 211
intramuscular, 211

INDEX

Inoculation, intra-ocular, 211
intraperitoneal, 211
intraspinal, 211
intrathoracic, 211
intravenous, 211
needles, 176
of culture medium, 170, 176
subcutaneous, 210
subdural, 211
Inoculations, protective, first, 26
Instruments, surgical, disinfection
of, 153
Intestinal discharges, 226
Intestine, large, 227
small, 227
Intestines as channel of infection,
22

Intracardiac inoculation, 211

Intramuscular inoculation, 211

Intra-ocular inoculation, 211

Intraperitoneal inoculation, 211

Intraspinal inoculation, 211

Intrathoracic inoculation, 211

Intravenous inoculation, 211

Invasion, 214

Invertase, 112

Involution forms, 51

Todine as disinfectant, 145

Iron bacteria, 75, 78

uses of, 78

Isolation of bacteria, 178-183

aids in, 182, 183

J

JAaBLoT, 30

Jack o’lanterns, 94

Jar, Novy, 175

Johne’s disease, 225, 226

K

Kerrte, 29

Kidneys as path of elimination, 226
Kinase, 113

Kircuer, 18, 23

IXLEess, 27

KLENCKE, 26

Kocn, Rosert, 17, 27, 31, 215, 248
Ixoch’s postulates, 215

Kravs, 245

Kruse, 231

KKUicHENMEISTER, 26

INDEX

L

Lacrrate edge, 207
Lactacidase, 113
Lactic acid fermentation, 85-88
Lancisi, 24
Lanpo!ls, 248
Large intestine, 227
Latour, 29
LavERAN, 24, 28
Lecithin as complement, 251
LErvwENHOEK, ANTHONY VAN, 18
Legumes, 106 :
Leivy, Joszrsu, 30
Leprosy, 215, 223, 227
Lesser, 30
Lethal dose, minimum, 241
Leukocytes, washing of, 257
Lice as carriers, 220
Li=BERT, 26
Light, 65, 136
production of, 99
LinnaEvs, 24
Lipase, 112
Liquefaction, crateriform, 205
funnel-shaped, 205
of gelatin, 92
saccate, 205
stratiform, 205
Liquid manure, disinfection of, 155
media, 158
Lister, 26
Litmus milk, 161
Lobar pneumonia, 224
a infections, vaccines in,
61
Location, specificity of, of infective
organisms, 227
Loeffler’s blood serum, 166
blue, 189
Léscu, 27
Loop needles, 177
Lopotrichic, 43
Lung, apex of, 227
Lungs, 227
as channels of infection, 224
Lysol as disinfectant, 148

M

McCoy, Vira, 148
Macrococcus, 49
Macroscopic agglutination, 242

281

Malaria, 24, 214, 220, 227
Malarial parasite, 28
Mallease reaction in glanders, 246
Mallein test, 267
Malignant edema, 218, 222
Malta fever, 245
Mammary gland, 226
Manure, liquid disinfection of, 155
Margaropus annulatus, 220
MartTINn, 29
Mass cultures, 172 -
Material for bacteriological exami-
nation, 211
Maximum conditions, 62
Measles, 224, 226
German, 214
Measly pork, 26 ,
Measurement of bacteria, 37
Meat broth, 159
identification of, 246
poisoning, 93
Meatus, external auditory, as chan-
nel of infection, 223
Mechanical vibration, 69
Media, liquid, 158
selective, 182, 183
solid, 158
synthetic, 158
Medium, agar, 163, 164
blood serum, 166, 167
culture, 157
essentials of, 158
inoculation of, 170, 176
gelatin, 161-163
large quantities of, 172
potato, 164-166
reaction of, 70
synthetic, 167
Meningitis, 223
Meningococcus, 223
Meanie chloride as disinfectant,
146
Merismopedia, 54
Metabiosis, 92
Metabolism of bacteria, 75- ‘81
relative to man, 81
Metachromatic granules, 42
Metastases, 216
Metatrophic, 75
Metcunikorr, 234, 256
Methods, antiseptic, 26
of ebieining pure cultures, 178-
Microbiology, 213

282 INDEX
Micrococci, 226 N
Micrococcus, 49, 59, 223, 224

pyogenes aureus, 148 N&cEtt, 30

Micromillimeter, 37
Micron, 37
Microscope, bacteriological, 184
improvements in, 28
invented, 18
Microscopic agglutination, 242
Microspira, 60
comma, 63
Microsporon furfur, 26
Migula’s classification, 59, 60
Milk, 161 ;
blue, bacillus of, 27
fermentation in, 29
digestion of, 91
ee as channel of infection,

identification of, 246

litmus, 161

pasteurization of, 129, 132, 133,
134, 135

purifier, electric, 139, 140
souring of, 29
tuberculous, 226
Minimum conditions, 62
lethal dose, 241
Mixed infection, 216
vaccine, 260
Mixotrophic, 75
M. L. D., 241
Moist heat, 121
Moisture, 63
Molds, relation to bacteria, 34
Monas, introduced, 30
Monotrichic, 42
Mordants, 188
Morphology of bacteria, 39-55
Mosquito theory of malaria, 24
Motile bacteria, 42
Motion, Brownian, 44
Mouth cavity as channel of infec-
tion, :223
Movement, Brownian, 44
rate of, in bacteria, 42
By, 37
Mu, 37
Mucose as channels of infection,
223
Miter, 30
Minrz, 30
Mycoproteid, 72
Myxomycetes, 36

Names, generic, introduced, 30
Nasal cavity as channel of infec-
tion, 223
discharges, 226
Natural gas, 84
immunity, 230
explanation of, 269
NrEpuaM, 19
Needles, inoculation, 176
loop, 17
platinum, 176
straight, 176
Negative phase, 262, 263
test, 255
Neisser granules, 42
Nephrolysin, 249
NEUFELD, 257
Neurotoxin, 249
Nichrome wire, 177
Nitrate broth, 161
Nitric bacteria, 103
Nitrification, 103
due to organisms, 30
Nitrifying organisms, 30
Nitrogen, absorption of, 105
circulation of, 96
nutrition of green plants, 107
uses of, 78
Nitrous bacteria, 102
Non-pathogenic, 76
Non-specific disease, 215
Normal agglutinins, 243
Nosema bombycis, 27
Novy jar, 175

‘| Noxious retention theory of im-

munity, 234

Nuclein, 40
in bacteria, 73

Nucleus, 40

Nutrition of green plants, nitrog-
enous, 107

Nurta, 248

ce)

OBERMEIER, 27

Objective, oil immersion, 184, 185
Oblique germination of spore, 45
Occurrence of bacteria, 51

| Oidium albicans, 25

INDEX

Oil immersion objective, 184, 185
Opsonic index, 257, 258
value of, 258
power, 257
Opsonin, 256
Opsonins, 256-263, 268 ‘
-antibodies of second order, 257
ispecificity of, 257
Optimum conditions, 62
Order, first, receptors of, 239
second, receptors of, 242, 247
third, receptors of, 248, 250
Organisms, anaérobic, cultivation
of, 172-176
dissemination of, in the body, 225
filterable, 216
aa specificity of location,
2

nitrifying, 30
pathogenic, elimination of, 226
paths of entrance, 222
ultramicroscopic, 215
Organized ferments, 114
Osmotic pressure, 68, 137
Otitis media, 223
Orro, 264
Outlines of immunity, 229
Overproduction activity of cells,
235, 236
Oxidation, 102
Oxidizing enzymes, 113
Oxygen, 66
as disinfectant, 144
compressed, 67
pressure, 66, 67
relations, determination of, 198
uses of, 77
Oznam, 24
Ozone, 67, 138
as disinfectant, 145

Pp.

PaNncrEAs, 226
Papillate growth, 204
Parasite, 76
facultative, 76
of malaria, 28
strict, 76
Partial amboceptor, 251
Passive immunity, 230
Pasteur, 21, 25, 26, 27, 28, 29, 231,
234

283

Pasteur flask, 22, 23
| treatment of rabies, 231
Pasteur-Chamberland filter, 142
Pasteurization, 127-135
continuous, 129
of beer, 129, 133
of grape juice, 129, 132
of milk, 129-132, 133, 134, 135
of wine, 129
Pathogenic, 76
bacteria, definition of, 213
outside body, 218
a reasons for study,
21

organisms, elimination of, 226
paths of entrance, 222
Paths of elimination, gall-bladder,
226

kidneys, 226
saliva, 226
urine, 226
of entrance of pathogenic organ-
isms, 222
Pebrine, 26
Peptone solution, Dunham’s,
161
Period of incubation, 214
Peritrichic, 43
Peronospora infestans, 26
Prrty, 30
Petri dishes, 164
Petroleum, 84
PFEIFFER, 248
phenomenon, 248
Phagocytes, 234
Phagocytic index, 257
Phagocytosis, 256-263, 268
theory of immunity, 234
ee as channel of infection,

Phase, negative, 262, 263
positive, 263

Phenol as disinfectant, 147

Phenomena, anaphylactic, 267

Phenomenon, Arthus’s, 264
Pfeiffer’s, 248

Phosphate rock, 103

Phosphine, 94

Phosphorescence, 99

Phosphorus, circulation of, 96
uses of, 79

Photogenesis, 99

Physical agents for disinfection,
119-143

284

Physiological activities, definition
of, 76
use in identification, 199
Physiology of bacteria, study of,
196-209
Pigment, production of, 100
Pimples, 216, 222
Piroplasma bigeminum, 215, 220
Piroplasmoses, 220, 222
Piroplasms, 227
von PirQvET, 264
Pityriasis versicolor, 26
Plague, 224
Planes of division, 53
determination of, 54
Planococcus, 59
Planosarcina, 59
Plasmodiophora brassice, 27
Plasmolysis, 39
Plasmoptysis, 40
Plate colonies, study of, 209
cultures, 164, 172
Plates, dilution, 178
gelatin, first used, 27
Platinum needles, 176
Plectridium, 47
PLENciIz, 24, 28
Plugs, cotton, 168
Pneumococcus, 224
Pneumonia, broncho-, 224
foreign body, 224
lobar, 224
Pneumonias, 226
Poisoning, cheese, 93
ice-cream, 93
meat, 93
Polar germination of spore, 45
granules, 42
Poliomyelitis, 223
POLLENDER, 26
Polyvalent vaccine, 260
Pork, measly, 26
Position of bacteria, 34
of flagella, 42, 43
of spore, 46, 47
Positive phase, 263
test, 255
Postulates, Koch’s, 215
Potato, glycerinized, 166
medium, 164-166
rot, 26
Power, opsonic, 257
Practical sterilization and disinfec-
tion, 152-156

INDEX

Pragmidiothriz, 60
Precipitatory group, 246
Precipitin, 245
Precipitinogen, 245

-Precipitins, 245-247

anti-, 246
compared with agglutinins, 245
formation of, Ehrlich’s theory,
245
uses of, 246
Preparation of antitoxin, 240
of bacterial vaccine, 259, 261
of film, 190
Preservative in vaccine, 260
Pressure, hydrostatic, 69
osmotic, 68, 137

oxygen, 66, 67
steam, 124

Preventatives, stock vaccines as,
260

Prevost, 25
Primary infection, 216
Production of acid, 98
of active immunity, 230-232
of antibodies, place of, 269
of aromatic compounds, 99
of enzymes, 109
of esters, 98
of gas, 98
of heat, 104
of light, 99
of pigment, 100
of toxins, 114
Prophylaxis, 264
Protamin in bacteria, 73
Protease, 112
Protective inoculations, first, 28
Protein immunity, 265, 267
split products, 266
Proteins, putrefaction of, 91-97
Proteus, introduced, 30
Protoplasm, 39
Prototrophic, 75
Protozoa related to bacteria, 37
Pseudomonas, 59
pyocyanea, 116, 242
radicicola, 106
Ptomaines, 92
Puecinia graminis, 25
Punctiform colony, 206
Puncture cultures, 169
Pure culture, 157
methods of obtaining, 178-183,
Purin bases in bacteria, 73

INDEX

Putrefaction, 25, 91
and fermentation, 28
of proteins, 91-97

Q

Quick lime as disinfectant, 146
Quinsy, 223

R

RaBiEs, 226
Pasteur tréatment of, 231
Rabiger’s method of staining, 194
Radiations, 69
Radium, 69
Rashes, serum, 264
Rate of division, 80
of movement in bacteria, 42
Rats as carriers, 220
Rattlesnake, 252
RayEr, 26
Reaction, anaphylactic, use of, 268
mallease, in glanders, 246
of medium, 70
surface, 81
Reactions, biochemical, definition
of, 76
Reavumvr, 30
Receptor, 238
Receptors, 235, 236
free, 237
of the first order, 239
of the second order, 242, 247
of the third order, 248, 250
Recurrent fever, 27”
Red corpuscles, 997
Revi, FRaNcESco, 18
Reducing actions, 101
enzymes, 113
Relapses, 216
Relationships of bacteria, 30
Renucct, 25
Resistance of spores, 47
Retarder, 131
Rheumatism, 223
Rhizoid colony, 206, 208
edge, 208
Rhodobacteriacee, 60
Ricwet, 264
Ricin, 239
Rimpav, 257

285

RInpFLEISCH, 27

Ringworm, 26

Ripening of cheese, 29
of cream, 86

Robin, 239

Rock, phosphate, 103

Rods in blood, 26

Roacerrs, 242

Rooms, disinfection of, 153

Root tubercles, 105

RosEnav, 264

Rot, potato, 26

Roup, 223

Rovx, 28

Rust, grain, 25

5

Saccate liquefaction, 205
Saliva as path of elimination, 226
Salivary glands, 226
Salt rising bread, 84
Saprogenic, 91
Saprophilic, 92
Saprophyte, 76
Sarcina, 29, 54
lutea, 67
Sarcoptes scabiei, 25
Sauer kraut, 87
Scarlet fever, 224, 226
Scavengers, bacteria as, 95
Scuick, 264
Schistosomum hematobium, 26
Schizomycetes, first used, ’30
ScHGNLEIN, 25
Scuésine and Mintz, 30
ScHROEDER and Duscu, 20
experiment, 20, 21
ScHuLtze, 20
experiment, 20
Scuwann, 20, 29
experiment, 20, 21
Sea, bacteria in, 61 ~
Sealing air-tight, 19
Second order, receptors of, 242, 247
Secondary infection, 216
Selective media, 182, 183
Sensitization, 265
Sensitized, 265
vaccine, 232
Septicemias, hemorrhagic, 225
Serum, antitetanic, 240
blood, necessity for diluting, 244

286

Serum, diphtheritic, 240
heated, 248
rashes, 264
sickness, 264, 267
Serum-simultaneous method in im-
munity, 231
Serums, cytolytic, failure of, 252
Shape of spore, 46
Sickness, serum, 264, 267
Side-chain theory of immunity, 234
Side-chains, 236
Silkworm disease, 25, 26
Size of bacteria, 37
Skatol, 93
Skin as channel of infection, 222
diseases, 222
- Slant cultures, 170
Slide, cleaning of, 191
hanging drop, 185
Slope, cultures, 170
Small intestine, 227
Smallpox, 224, 226, 230, 231
vaccine, 231
SmitH, THEOBALD, 264
Snake venoms, anti-, 252
Sneezing, 226
Soap as disinfectant, 148
Sodium hypochlorite as disinfec-
tant, 146
Soil, bacteria in, 61
Soils, acid, 70
Solid media, 158
Solution, Gram’s, 192
stock, of stains, 189
Sore throat, 223
Sound, 69
Sour mash distilling, 87
Source of complement, 254
Souring of milk, 29
SPALLANZANI, 19
Specific: amboceptor, 254
chemical stimuli of cells, 235
disease, 215
Specificity of agglutinins, 244
of amboceptors, 250
of location of infective organisms,
227
of opsonins, 257
Spermotoxin, 249
Spherottilus, 60
Split products of protein, 266
Splitting enzymes, 112
of fats, 90
Spirillacee, 60

INDEX

Spirilloses, 220, 222
Spirillum, 50, 60
‘pirosoma, 60
Spirocheta, 60
Spirocheta obermeiert, 27
Spirochete, 50
Spirochetes, 221
Spoiling of canned goods, 48
Spontaneous combustion, 104
generation, 17
Spore capsule, 45
definition of, 48
free, 45
germination of, 45
of anthrax, 27
position of, 46, 47
shape of, 46
Spores, 44
cause of spoiling of canned
goods, 48
conditions for formation of, 48
first recognized, 30
formation of, 45
resistance of, 47
staining of, 192
two, in bacterium, 47
Sprinkling filters, 104
Stab cultures, 169
Stables, disinfection of, 153-155
Stain, anilin-fuchsin, 189
gentian violet, 189
aqueous gentian violet, 189
carbol-fuchsin, 189
Gabbet’s blue, 190
Loeffler’s blue, 189
Staining bottles, 190
for cell forms, 195
groupings, 194
Gabbet’s method, 193
Gram’s method, 191
of acid-fast bacilli, 193
of bacteria, 188-195
of capsules, 194
of flagella, 19+
of spores, 192
Ribiger’s method, 194
reasons for, 188
vont of physiological activity,
11

Welch’s method, 194

Zichl-Neelson method, 193
Stains, stock solutions, 189
Standard antitoxin, 241

test. dose, 241

INDEX 287

Standard toxin, 241
Standardization of vaccines, 259,
260 |
Staphylococcus, 54
Srarin, W. A., 181
Steam, 122
ae Zpnnaldehyde disinfection,

sterilizers, 122
under pressure, 124
Stegomyta, 221
Sterile, 119
Sterilization, 118
and disinfection, practical, 152,
156

discontinuous, 121
Sterilizers, steam, 122
Stimuli, chemical, effect of, 236
of cells, 235
specific chemical, 235
Stock cars, disinfection of, 155
solutions of stains, 189
vaccine, 260
as preventatives, 260
in treatment, 260
piesa as channel of infection,
Straight needles, 176
Stratiform liquefaction, 205
Streptobacillus, 52
Streptococcus, 53, 59, 223, 224
Streptospirillum, 52
Streptothrix bovis, 27
Strict parasite, 76
Structures, accidental, 40
cell, 39
essential, 39
Study of bacteria, 157
of bacterium, necessary steps for,

of forms, 30
of pathogenic bacteriology, rea-
sons for, 217

of plate colonies, 209

of physiology of bacteria, 196-209
Subcutaneous inoculation, 210
Subdural inoculation, 211
Substances, cytolytic, 268
Substrate, 111
Sugar broth, 160
Sulphur bacteria, 94

circulation of, 97

‘deposits of, 104

uses of, 78

Summary of Ehrlich’s theory of
immunity, 237

of immunity, 268

Surface reaction, 81

Surgical instruments, disinfection
of, 153

Susceptibility, 216

Symbionts, 76

Symbiosis, 76

Synthetic media, 158
medium, 167

Syphilitic antigen, 254

Syphilis, 215, 226, 227, 253

i

T

Taprworm, 26
Temperature, 64
Tenia solium, 26
Test, complement-deviation, 253
complement-fixation, 253-255
dose, standard, 241
for enzymes, 111
for toxins, 115
Gruber-Widal, 244
mallein, 267
negative, 255
positive, 255
tuberculin, 267
Wassermann, 253
Widal, 244
Testicle, 227
Tetanus, 214, 218, 222, 227
Tetracoccus, 54
Tetrad, 54
Texas fever, 214, 215, 220
THaEr, 29
Theories of immunity, 234
Theory, cellular, of immunity, 234
chemical, of immunity, 234
contagium vivum, 23, 24
Ehrlich’s, of formation of precipi-
tins, 245
of immunity, fundamental
principles of, 235
summary of, 237
exhaustion, of immunity, 234
for use of vaccines, 261
mosquito, of malaria, 24
noxious retention, of immunity,
234
of anaphylaxis, 266
of formation of agglutinins, 242

288

Theory, phagocytosis, of immu-
nity, 234
side-chain, of immunity, 234
unfavorable environment, of im-
munity, 234
Thermal death-point, 65
determination of, 198
Thermophil, 65
Thiobacteria, 60
Thiothrix, 60
Third order, receptors of, 248-250
Thread, 52
Thrush, 25, 223
Ticks as carriers, 220
Titration, 160
Tonsillitis, 223
Tonsils, 227
as channels of infection, 223
Tovissant, 259
Toxin, 227, 238
final test for, 115
standard, 241
T one a enzymes compared, 114,
characteristics of, 114, 115
_ other than bacterial, 115
production of, 114
true, 116
Toxoid, 239
Toxophore group, 250
Tract, alimentary, as channel of
infection, 225
TRAUBE, 248
Treatment of rabies, Pasteur, 231
stock vaccines in, 260
Treponema pallidum, 224
Trichinella spiralis, 26
Trichinosis, 26
Trichophyton tonsurans, 26
Tropical dysentery, 27
True toxins, 116
Trypanosomes, 221
Trypanosomiases, 220, 222
Tubercle bacilli, chemical analysis
of, 74
Tubercles, root, 105
Tuberculin test, 267
Tuberculosis, 215, 223, 224, 225,
226, 227
due to bacteria, 28
infectious nature of, 26
Klencke’s experiment, 26
Villemin’s experiment, 26
Tuberculous milk, 226

INDEX

Tubes, culture, 168
deep, 174
fermentation, 168, 174
Vignal, 173
Two spores in bacterium, 47
TYNDALL, JOHN, 23
Tyndall’s box, 22, 23
Typhoid fever, 214, 226
transmission by flies, 220

U

ULTRAMICROSCOPIC organisms, 215
Ultra-violet rays, 138
Unfavorable environment theory
of immunity, 234
Unit, definition of, 241
of antitoxin, 241
Unorganized ferments, 114
Unwashable articles, disinfection
of, 155
Urea, decomposition of, 95
Urease, 113
Urethral discharges, 226
Urine as path of elimination, 226
bacteria in, 62
Use of agglutinins, 243, 244
of anaphylactic reaction, 268
Uses of carbon, 77
of food, 75
of hydrogen, 78
of iron, 78
of nitrogen, 78
of oxygen, 77
of phosphorus, 79
of precipitins, 246
of sulphur, 78
of vaccines, theory of, 261

Vv

VACCINATION, 231
Vaccine, 231
anthrax, 231
autogenous, 260
. bacterial preparation of, 259, 261
black-leg, 231
mixed, 260
polyvalent, 260
preservative in, 260
sensitized, 232
smallpox, 231

Vaccine, stock, 260
Vaccines, bacterial, 258
dosage of, 262
failure of, 261
in general infections, 261
in localized infections, 261
standardization of, 259, 260
stock, as preventatives, 260
in treatment, 260
theory for use of, 261
Vacuoles, 41
Vaginal discharges, 226
Value of opsonic index, 258
Varo, 23
VauGHan, 266
Vehicles, disinfection of, 155
Venoms, antisnake, 252
Visore, Eric, 24
Vibration, mechanical, 69
Vibrio, 50
introduced, 30
Vignal tubes, 173
VILLEMIN, 26
Villous growth, 204
Vinegar, 88
Virulence, 216
Virus, filterable, 216
Vultures as carriers, 220

Ww

Watt, cell, 39
composition of, 72

Washable articles, disinfection of,
155

Washing of leukocytes, 257

Wassermann test, 253

Water, bacteria in, 61
filtration, 141

Wess and Barser, 231

WBEIGERT, 28, 235

INDEX

289

Welch’s method of staining, 194

Whooping-cough, 224
Wipat, 242
test, 244
Widal-Gruber test, 244
Will o’ the wisp, 94
Wine, pasteurization of, 129
Winocrabsxy, 30, 60
Wire baskets, 168
nicrome, 177
WOLLSTEIN, 24
Woronitn, 27
Wound infection, 25, 27, 215
caused by bacteria, 27

Wriaut, 256

x
X-rays, 69

Y
Yast, 29

relation to bacteria, 34
Yellow fever, 221

Z

Zansz, Hans and Zacuarias, 18

ZENKER, 26

Ziehl-Neelson method of staining,
193

Zooglea, 41
Zootoxins, 116
Zymase, 113
Zymases, 113
Zymogen, 113

Zymophore group, 239, 247, 250

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