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MICROBES AND TOXINS

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PASTEUR
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MICROBES & TOXINS

By Dr. ETIENNE BURNET

OF. THE PASTEUR INSTITUTE OF PARIS
WITH A PREFACE BY

ELIE METCHNIKOFF

Translated from the French

BY

Dr. CHARLES BROQUET and W. M. SCOTT, M.D.

ILLUSTRATED

LONDON:
WILLIAM HEINEMANN

Ig12

Copyright 1912

Photo) (/enri Manuel, Paris.
Eig METCHNIKOFF

TO
“Dr. EMILE -ROUX

Director of the Institut Pasteur

INTRODUCTION

THE publication in the Bibfothique de Philosophie Scientifique
of a volume dealing with micro-organisms was entirely indicated,
for microbiology is taking every day a larger and larger place
in the realm of knowledge and philosophy. Although dis-
covered more than two hundred years ago, microbes were long
neglected, and it was only during the second half of last
century that their true r6le was ascertained.

Ehrenberg in the middle of the nineteenth century had
already perceived the importance of microscopic organisms in
the evolution and life of our planet. The discovery of the
fossil remains of Diatoms and Foraminifera led him to appre-
ciate the great part these minute creatures have played in the
building up of the earth’s crust.

There were not lacking men of science disposed to attribute
to micro-organisms an important action in the phenomena of
fermentations and of disease, but it was only after the labours
of Pasteur that this truth was definitely established and became
part of our common heritage of knowledge.

Relations had long been perceived between the animal
kingdom and the vegetable, animals furnishing carbonic acid
and nourishment to plants, while these, on their side, nourish
the animals with their organic matter and provide them with
oxygen. Later it was recognised that between these two
kingdoms there lies the domain of the microbes. It is the

x INTRODUCTION

microbe which transforms the animal material supplied by
dead bodies and dejecta into simpler substances, nitrates, and
salts of ammonia, capable of assimilation by those plants which
supply us with food. Further, it is the microbe which renders
pleasant to the taste certain animal and vegetable food-
products, as, for example, the juice of the grape, the extract
of malt, cabbages, apples, and milk, transforming these
respectively into wine, beer, sauerkraut, cider, kephir, various
kinds of cheese, etc.

Thanks to Pasteur the activity of micro-organisms was
established in every case of putrefaction and fermentation ;
and, with this fact to start from, it became more easy to tackle
the problem of infectious diseases.

Putrefaction and suppuration have been recognised for cen-
turies as being phenomena of the same order. Decomposing
pus, faecal matter smelling of putrefaction, urine issuing from
the bladder in a state of decomposition, all indicated that an
illness, a state of suffering, was equivalent to an infection of the
body.

Although certain microbes, suchas the bacteridium of anthrax,
had been observed before Pasteur’s great discoveries, it was only
as a consequence of these discoveries that the fundamental réle
of microscopic organisms in disease was understood. The
labours of Lister in surgery, and of Davaine and Koch on the
“black blood of anthrax” first authorised the application of
Pasteur’s doctrine to surgery and medicine.

Pasteur himself with his pleiad of disciples was in the midst
of this activity, an activity which in a very few years revolu-
tionised medical science and endowed medicine with more
than one preventive vaccine such as those against anthrax and
rabies.

The Pasteur schoo! in France and the school of Koch in
Germany have succeeded in elucidating many medical problems
of the highest importance and have drawn valuable practical
conclusions from these.

Thanks to all this work, work which has increased during
these last years in extraordinary fashion, a universe of micro-

INTRODUCTION xi

organisms, beneficent and mischievous, has been revealed to
humanity ; and it is this new knowledge which has so largely
contributed to the diminution in disease and death at the
present day and which holds out to man the hope of a more
happy future.

The micro-organisms inhabiting our bodies have set going
there a poison factory, which cuts short our existence, and by
secreting poisons which penetrate all our tissues, injures our
most precious organs, our arteries, brain, liver, and kidneys.

Man balked of his full term of life feels himself unhappy and
is ready to accept any solution to the problem of gaining
happiness. And the progress of microbiology leads us to hope
that this science will one day liberate man from his fear of the
grave and permit -him to attain the true object, the true
conclusion of life.

It is time for bacteriological science to leave the laboratory
and the lecture theatre, and to take its place before the great
public, in order that its benefits may receive the widest and
readiest application.

It was very natural for the creator of this “ Library of
Scientific Philosophy” to apply to the Pasteur Institute for an
account of the actual position of science with regard to microbes
and toxins. Not only was the movement started from Pasteur’s
laboratory and continued in the Institute bearing his name,
and still sheltering one of his most illustrious collaborators in
the person of Dr. Roux, but it.is in this Institute that every
branch of microbiology is undergoing active study. To take
colloids and the physico-chemical laws which govern their
activity, we have at the Zystitut Pasteur studies on ferments
and fermentations as well as on the chemical processes which
lie at the root of life and of recovery from disease. In this
Institute also there are zealous workers in the field of infective
microbes and the means of combating them.

Several laboratories are specially set apart for researches on
tropical diseases, and finally the Pasteur Hospital has been
created for patients suffering from all sorts of infectious maladies.

If Pasteur were to see his Institute again, he would be

xii ‘ INTRODUCTION

astonished at the great changes which have taken place in it
and in the science of microbiology in general. It would take
him some time to overtake and realise the progress attained.
And yet in spite of all that has been done there remains still
much work for the future. Many scourges still await a remedy.
In the case of tuberculosis, although extraordinary advances
have been made in its study, the final solution of the problem
is still reserved for the future. The great question of cancer,
so important and so difficult, has been hardly more than opened.
There remain to be discovered the microbes of many diseases,
e.g., hydrophobia, scarlatina, and measles, which are perhaps
filtrable micro-organisms, invisible with the best microscopes.

The field of infectious diseases is extending wider and wider
with the progress of microbiology. We find that certain dis-
eases thought to be diseases of metabolism are beginning to be
classed in this group. Arterio-sclerosis, an affection so wide-
spread and so apt to cut short our existence, results from the
activity of our intestinal flora. Perhaps before long it will be
possible to explain diabetes, gout, and rheumatism by the
injurious activity of some variety of microbe.

Even in those problems of hygiene which affect society in
general microbiology is taking the predominant réle. The
grand problem of a rational food supply, which used to be
thought capable of solution by inventions of chemistry and
physics, will necessarily have to be studied by microbiological
methods, in view of the fact that the intestinal bacteria play
one of the most important parts in everything that concerns
nutrition. It is not sufficient to state the nutritive value of a
food in terms of the calories which it contains ; it will still be
necessary to define precisely its relations to the intestinal flora
from the point of view of the production of microbic poisons.

M. Gustave le Bon asked me to put together in a little
volume for his Library a summary of what is known about
microbes and toxins. I advised him to apply rather to one of
my young colleagues, and I indicated in particular Dr. Burnet.
I am happy to find that I could not have chosen better. In
spite of the great difficulties there are in attempting to describe

INTRODUCTION xiii

within a limited space the result of the innumerable labours
which have accumulated on microbes and their poisons and
which have been pursued in the most varied directions,
M. Burnet has succeeded in accomplishing the task in a
remarkable fashion. I am sure that those who read will share
my opinion, and I wish this book the most widespread
popularity.

ELIE METCHNIKOFF.

PaRIS, IQII.

CONTENTS

CHAPTER I

THE GENERAL FUNCTIONS OF MICROBES—THE TRANSFORMATION
CYCLES OF CARBON AND NITROGEN. 3 . fs o I

PAGE

CHAPTER II

MICROBES IN THE HUMAN BODY—LIFE WITHOUT MICROBES —
THE INTESTINAL FLORA . . « . . . e é 25

CHAPTER III

FORM AND STRUCTURE OF MICROBES. . . ~« « + ~~ 50
CHAPTER IV

PHYSIOLOGY OF THE MICROBES . Co ee er ee oe ee)
CHAPTER V

PATHOGENIC MICROBES—INFECTION .« . « «© «© « « 107
CHAPTER VI

INFLAMMATION AND PHAGOCYTOSIS. . . + 8 « 130

CHAPTER VII

THE PATHOGENIC PROTOZOA; FILTER-VASSING VIRUSES * . 141

Xvi CONTENTS

CHAPTER VIII

THE TOXINS

CHAPTER IX

TUBERCULIN AND MALLEIN—ANIMAL TOXINS—VENOMS

CHAPTER X
IMMUNITY .

CHAPTER XI
IMMUNITY

CHAPTER XII
ANAPHYLAXIS .

CHAPTER XIII

APPLICATIONS OF BACTERIOLOGY

CHAPTER XIV

VACCINES AND SERA

CHAPTER XV

CHEMICAL REMEDIES

PAGE

- 157

173

187

210

233

250

265

285

CHAPTER I

THE GENERAL FUNCTIONS OF MICROBES—THE TRANSFORMATION
CYCLES OF CARBON AND NITROGEN

The circulation of matter ; anabolism and catabolism—Views of Lavoisier—
The transformation cycles of carbon and nitrogen—The réle of micro-
organisms : I. Maturation of the soil and formation of arable land.
II. Fermentation of vegetable matter--Decomposition of starch and
manure— Hypotheses on the formation of coal. III. Putrefaction of
albuminous materials. IV. Nitrification and denitrification: in agricul-
ture ; in the biological purification of sewage. V. Fixation of atmos-
pheric nitrogen in the soil: by bacteria alone; by bacteria in
association with alge; by the nodule bacteria of Leguminose—
Some ideas about the useful micro-organisms: Fermentations in con-
nection with food production and in various industries.

A.Lmost all living matter is made up of water, that is to say,
of oxygen and hydrogen, and of compounds of carbon and
nitrogen. Other elements may enter into the tissues of
animals and vegetables, for example, sulphur, iron, arsenic,
boron; but by following the circulation of carbon and
nitrogen, it is possible to have a general view of the movements
of exchange between matter and life.

The living creature restores to nature what it has absorbed,
and eventually its own body, by its excretions during life and by
its decomposition after death. The elements set free are
recombined into organic bodies, and these exchanges and this
circulation form the essence of life. It is an abuse of our
subjective attitude to consider as two opposites life and death.
At most, life is the opposite of inertia. Death is a special kind
of accident, life being the all-embracing phenomenon. The

first of the work ers in the great cycle of life are the microbes,
B

2 MICROBES AND TOXINS

and the decompositions and re-combinations of living matter
depend entirely upon them.

Life without microbes is not conceivable at the present
day. That does not mean that they were the first living beings
to appear on the surface of the earth. It is the more difficult
to get an idea of their origin, since in all probability they have
undergone evolution, and have not always had the appearance
they have at present. It is possible that under forms that we
can hardly guess at, life appeared long before the existence of
microbial forms ; but microbes have been the chief agents in
the spread and extension of life throughout the world.

In the world of to-day, the building up and breaking down
of organic substances are functions of cells, conducted by an
infinite number of diastases. These cells include the cells
built up into animal and vegetable tissues, and the isolated
cells, the microbes. It would not be right to contrast too
much the microbial cells with the cells of tissues, merely
because the former are separate individuals, whereas the latter
are arranged in systems. In nature, micro-organisms can
exist as solitary individuals, but are seldom actively at work in
this condition. For example, in the soil, the bacteria which
fix nitrogen act rather like a wide-spread tissue. The active
agents are in any case invariably cells.

To describe in a few words the cycle which organic matter
follows, one cannot do better than quote the celebrated page
of Lavoisier, ‘Plants draw from the circumambient air, from
water and in general from the mineral kingdom, the substances
necessary for their own organisation. Animals feed either on
plants or on other animals, which have themselves fed on
plants, so that eventually the matter building them up is
always derived either from the air or from the mineral
kingdom.

“Finally, fermentation, putrefaction and combustion are
continually restoring to the atmosphere and to the mineral
kingdom the elements which plants and animals have
borrowed.

“By what rocesses does nature effect this wonderful

GENERAL FUNCTIONS OF MICROBES 3

circulation between the two kingdoms? How does nature
succeed in producing substances which are combustible,
putrescible and capable of fermentation from combinations
which have none of these properties? Here are impenetrable
mysteries. One may perceive, however, that since combustion
and putrefaction are the means which nature employs to return
to the mineral kingdom what has been drawn from it in the
building up of plants and animals, the latter process must be
the converse of the former.”

Green plants get their carbon from the carbonic acid of the
air. In virtue of their chlorophyll activity they build up this
carbon into starch, cellulose, sugars and fats. This
carbohydrate synthesis then represents the accumulation of
energy. A green plant kept in the dark burns up its
hydrocarbons, returning them to the atmosphere. A dead
plant returns its hydrocarbons after a series of decompositions,
in the form of carbonic acid and water. A plant which has
been eaten by an animal supplies the animal with glycogen and
fats which are consumed in the course of muscular work and
respiration. Dying animals like dead plants return their
hydrocarbons to nature. Plants without chlorophyll and all
animals spend and dissipate the energy accumulated by the
chlorophyllous plants, energy derived entirely from the rays
of the sun.

Nitrogen exists in the atmosphere, from which it enters the soil.
It exists in the dejecta of living animals and in the bodies of
plants and animals rotting on the surface of the earth. It
accumulates there in the mould or humus: plants take up the
nitrogenous matter of the soil in the form of salts of ammonia
and nitrates, and build up from them the vegetable proteins.
Animals which eat plants produce from these the animal
proteins. Animals and plants in decomposition, and the
excretions of animals in general, return their nitrogen to
fertilize the soil.

Animals are in a certain sense parasites of plants, since they
are unable to build up by themselves, starting from mineral
elements, their hydrocarbons and proteins. Even in plants

B 2

4

4 MICROBES AND TOXINS

the formation of the protein compounds is dependent on the
chlorophyll activity; without this it would be impossible
for them to build up the carbohydrates, and these are
indispensable in the employment and elaboration of the
nitrogenous elements of the soil, The chlorophyll activity
is itself dependent on the sunlight, so that life is one great
pean to the sun.

In the transformation of carbon and nitrogen all the
operations are not carried out by microbes, for these latter do
not count at all in chlorophyll assimilation nor in animal
digestion. But it is the bacteria which keep up the supply of
organic matter which forms the source of animal and
vegetable life ; it is the bacteria which restore to circulation
those elements which were for a moment arrested in the bodies
of animals and plants: they restore to life matter which had
ceased to live. It is they also which prepare the soil for
vegetation and cultivation: they accumulate in the soil the
nitrogen which we are to eat in the form of cereals.

It is they which carry on the impenetrable mystery referred
to by Lavoisier.

They prepare the primary material of life. Their activity
is as universal as that of water, as that of light.

The discoveries of Pasteur have not only revolutionised
medicine, they have filled with new life the science and
practice of agriculture and husbandry.

1.—WMaturation of the soil and formation of arable land.

It was not the bacteria which shattered, splintered and
powdered the rocks which formed the first crust of the cooling
globe. But as soon as in this chaos there appeared water and
alkaline phosphates, alge and bacteria could establish
themselves and with the help of carbonic acid continue the
first formation of soil.

In those rocks known as “rotten rock,” Miintz found
nitrifying microbes: the nitrifying ferment has also attacked
the Faulhorn,a mountain of the Bernese Oberland near

GENERAL FUNCTIONS OF MICROBES 5

Grindelwald, which is composed of a calcareous schist, black,
friable, and crumbling down. There is therefore a sort of
putrefaction of rocks (alkalis and alkaline earths). According
to Fausto Sestini the carbonic acid produced in the respira-
tion of plant roots hastens the breaking down of feldspars.

Mosses, lichens, algze and bacteria prepare the way for the
highest plants. The more there is growth of plants, the more
there is decomposition. In this way, soil or humus has little
by little spread over all the earth.

Il.—Fermentation of Carbohydrates.

These fermentations restore to circulation the hydrocarbons
of animals and plants.

For the decomposition of sugars, starches, fats, glucosides,
and celluloses, several series of fermentations are necessary,
each ‘supplementing another in the work. The anaerobic
organisms break down the large organic molecules, whereas
the aerobes carry out, in particular, oxidations. When the
bacteria are insufficient, the moulds continue the work.
Finally, nothing is left but carbonic acid and water. All the
operations which in our laboratory analyses we distinguish and
conceive in terms of abstract formule, are combined and
intermingled in nature, each succeeding and limiting the other.
The yeasts transform sugars into carbonic acid and alcohol.
Alcohol attacked by the acetic ferments is turned into acetic
acid. Finally, the Bacterium acetd can split the acetic acid
of vinegar into carbonic acid and water. The A/ycoderma vini
destroys and oxidises both alcohol and acetic acid, producing
again carbonic acid and water. The sugar contained in
Raulin’s fluid (v. zzfra), for example, is attacked by Aspergillus
niger, the products including oxalic acid. Various moulds and
bacteria turn starch into sugar; it is the moulds in particular
which complete the oxidations, producing carbonic acid and
water.

Milk left to itself ferments, ¢.¢., produces lactic acid, which,
meeting with an alkaline carbonate, furnishes calcium lactate.

6 MICROBES AND TOXINS

Pasteur discovered the transformation of the lactate into the
butyrate by the butyric vibrio ; the butyrate can be completely
consumed in its turn by the moulds.

The glucosides (compounds of sugar with an organic body,
an alcohol or phenol) are decomposed into their two elements.
A diastase, tannase, secreted by the Aspergi//us, splits tannin
into two molecules of gallic acid, from which other moulds
produce again carbonic acid and water.

Just as there is not one starch but several, so there are
several celluloses, which resemble starches but are more
stable: (CsH,,O;)a. They form the walls of vegetable cells,
and make up one-third of the weight of the straw, which is
the principal component of farmyard manure. If the celluloses

id
we

——
a

OE

Fic. 1.—The microbe which ferments cellulose, described by Omeliansky :
bacilli with spores.

S&

were not decomposed and restored to circulation, the earth
would soon be cumbered with useless refuse material. But from
the beginning moulds establish themselves on the outer skin of
the living plant; when it dies they invade its tissues, attacking
first the sugar and then the cellulose, the latter being hydrolysed,
transformed into sugars and consumed. The &. Amylobacter
of Van Tieghem, an anaerobe, produces from cellulose
hydrogen, carbonic acid and butyric acid. Omeliansky has
demonstrated two methods of anaerobic fermentation in
cellulose, one with production of hydrogen, the other with
production of methane. Those ferments which in a tube in
the laboratory decompose the cellulose of Berzelius’ paper,
act in precisely the same way in manure heaps. The aerobic
fermentation of cellulose is carried on by moulds, by fungi

GENERAL FUNCTIONS OF MICROBES 7

more highly organised, and by the nitrifying and denitrifying
bacteria.

Pectose is a hydrocarbon associated with cellulose in the
membranes and interstices of plant cells; the rust which
attacks hemp and linen is a fermentation of the pectose,
transforming it into pectic acid, then to sugar, by the BZ.
amylobacter and by the Granulobacter of Fribes and Winograd-
sky. In the manure heap, aerobic fermentation proceeds at
the surface and raises the temperature up to nearly 80° C. In
the depths of the heap the temperature is low, and there the
anaerobic ferments attack the cellulose. The bubbles which
rise and burst on the surfaces of ponds are signs of the
anaerobic fermentation which is decomposing organic debris at
the bottom. Manure kept in a latticed box with free access of
air heats up without there being destruction of the cellulose ;
loss of nitrogen takes place. Manure heaped in a closed box
or kept corked in a large carboy, liberates methane from the
decomposition of the cellulose. It is possible to collect the
gas and by means of an exit tube to make it furnish a light.

It was a natural step to attribute to the ferments of
cellulose the formation of peat, lignite and coal. Microbes
certainly are at work in the-decomposition of vegetable matter
in the peat-bogs, and coal is supposed to be the product of more
complete fermentations, two varieties at least of bacteria being
in activity in succession; the first dissolves the central
membranes of the cell walls, the others attacking the cellulose,
more or less pure, which constitutes the thick parts of the wall.
“The bacterial activity,” says the most convinced defender of
this theory, ‘produced a de-hydrogenation and de-oxidation,
the final result being the production of carbon. We do not
know if the final limit of this process has ever been reached,
but figures show that the more geologically ancient the fuel, the
more carbon it contains” (Bernard Renault). Fuel of less
age, lignite, and peat, for example, contains besides bacteria,
amoebze and infusoria. In the lignites, the cannel coals, and the
boghead coals, we find fungi and alge, which do not occur in
coal proper. Bacteria alone exist in all the fuels; they are

8 MICROBES AND TOXINS

much less altered than the structures which surround them:
therefore it is supposed that they must have survived tissues
which they destroyed.

The presence of bacteria in coals derived from organic
matter should not astonish us. But it appears difficult to
believe that bacteria have been the only agents in the formation
of coal. The study of their form is extremely difficult ; fine
particles of iron pyrites and little crystals often imitate bacterial
forms in the thin sections of coal, so difficult to prepare and
examine by transmitted light under the microscope. On the
other hand the fermentation of cellulose does not explain the
frequent impregnation of the debris by a blackish, bituminous
material, nor perhaps does it really explain the enrichment in
carbon of the deposits ; further, there have been produced from
the fermentation of fatty bodies, resins and similar volatile
substances, phenol bodies like those found in coal (E. Duclaux).

There is nothing surprising in the presence of algee in the
Bogheads, for these are precisely the coals derived from algz
and often named algal coals. Their formation was due to
luxuriant alge growths rapidly developed on the surface of
stagnant pools, which then sank to the bottom carried down
by a coagulum in the peaty water; this mass contained
bituminous substanceswhich must have come from elsewhere, for
there is no indication that they took their origin on the spot by
an alteration of thealge. It is these bitumens which have
produced the carbon enrichment of the mass, so that on the
whole instead of destruction a preservation process has taken
place. In other cases, instead of this enormous growth of
algee, clouds of pollen and spores from the primeval forests
have been deposited : these “rains of sulphur” also sank in
the peaty water and became saturated with bitumen, hence
the so-called spore and pollen coals.

We ought not to be too ready to reject the bacterial theories
of the formation of the various coals, especially should it turn
out to be impossible to explain without microbes the formation
of the bitumen which impregnates them. The small lower
algee are rich in fats and capable of yielding petroleum bodies

GENERAL FUNCTIONS OF MICROBES 9

by distillation. The problem of the origin of coal has therefore
some relation to the problem of the origin of petroleum. The
decomposition of fatty substances commences with their
saponification, which splits them into fatty acids and glycerine,
and glycerine is a good food medium for various bacteria.

The moulds decompose fatty acids into carbonic acid and
water. Fats resist decomposition longer than other carbo-
hydrates, and longer than nitrogenous substances; it is
from this cause that the proportion of fat increases in a cheese
which has been kept, or in a dead body which is decomposing.
The ‘adipocere’ is the final stage in an animal body left entirely
to nature.

III. Putrefaction of albuminous materials.

Putrefaction returns to the soil the nitrogen which
makes up 15 per cent. of the proteins of animal tissues.

Since plants draw their nitrogen from the soil in the form of
nitrates and salts of ammonia, it is necessary for the complex
protein molecule to be broken down so as to supply finally

tg a AIAN

a BIW

Fic. 2.—Ammoniacal fermentation Fig. 3. —Urobactllus of
of urine: Urocococcus of Pasteur. Duclaux.

nitrogen in the form of ammonia and nitric acid. Later
green plants and finally animals, raise this mineral nitrogen to
the level of organic nitrogen.

Already during life animals discharge nitrogen with their
excretions in the form of urea, uric acid and hippuric acid.
The Urobacteria (there are about a hundred species known)

10 MICROBES AND TOXINS

transform the urea, by means of a urease which they secrete,
into carbonate of ammonia. Hippuric acid is transformed
into benzoic acid and glycocoll, and finally into ammonia.

The putrefactions of albuminous materials in nature are
never simple, that is to say carbohydrates almost always
accompany the proteins ; even meat contains a little sugar.
Putrefactions therefore are almost always mixed fermentations.

Pasteur thought that all putrefaction was the work of
anaerobes. He discovered the wibrion septique,an anaerobic
bacillus capable of decomposing proteins. Later, the
anaerobes were neglected and it was thought that various
aerobes, in particular Proteus, were the principal agents of
putrefaction. But after the study of fetid suppurations had
drawn attention to the presence of anaerobes (Veillon), the
idea rose again that they too must play a part in putrefaction,
and the methodical study of this subject was recommenced,
chemical analysis going hand in hand with bacteriological
examination.

In their experiments, Tissier and Martelly followed
for months the events which took place in flasks into which
meat had been put and allowed to putrefy either with or
without access of air. Meat taken from the slaughter-house as
fresh as possible contains all the germs necessary and
sufficient for putrefaction, and, as it contains carbohydrates, it
is a mixed putrefaction which occurs.

There are two phases. In the first the sugar and the
proteins are attacked by mixed ferments, that is to say, by
microbes which decompose at the same time both proteins and
sugar, proteolytic and saccharolytic—(peptolytic is the term
applied to the ferments which attack protein only after
its reduction to peptone). In the second phase the protein
and its products are attacked by ferments which are
proteolytic or peptolytic pure and simple, not saccharolytic.

But between these two phases a critical turning-point occurs:
the decomposition of the sugars produces an acidity sufficient
to stop putrefaction. The “antagonistic force” which’
Bienstock observed in his investigations on a putrefactive

GENERAL FUNCTIONS OF MICROBES 11

bacterium the ZB. putrificus, is nothing but this acidity. A piece
of meat, as is well known, keeps excellently in unboiled milk,
because the milk on fermenting produces lactic acid, which
protects the proteins of the meat from putrefaction. That: is
why the housewife when she salts down meat, does not forget
to add some vinegar, z.e., acid. It would seem then that
putrefaction ought to cease after the action of the mixed
ferments.

If it does not stop, it is because there is not sufficient sugar.
The ¢imit of acidity for the pure proteolytic ferments has not
been reached. Further, owing to the decomposition of the
albumins already begun, there appears a base, ammonia, which
neutralises the acidity, and the pure proteolytic ferments can
begin their action. It is the anaerobes which break down the
protein molecule and produce putrefaction; but they cannot
do without the auxiliary aerobes ; thanks to the aerobes which
hasten the production of ammonia and the alkalinisation of the
medium, putrefaction passes successfully through the crisis of
the antagonistic acidity.

In the putrefaction of milk, two analogous phases succeed
each other. Milk being richer in sugar than meat, the acidity
developed in the first phase is greater and the crisis more
difficult to surmount. To surmount it, more powerful fer-
ments are required than the aerobes which produce the
ammonia in the putrefaction of meat: these are the fungi
(Otdium lactis, Rhizopus nigricans) and the yeasts, which destroy
the acids, consume the milk-sugar, and attack the casein.
The fungi prepare the way for the pure ferments, which then
carry out the second phase of putrefaction.

When an animal dies, all the microbes necessary for its
putrefaction are present in its intestine. They invade the
tissues and carry out on a larger scale what we see on a small
scale in the experimental flasks. The worm of the grave is an
old poetical image long out of date. The real destroyer is the
microbe.

It is a fact well established to-day, that in a normal intestine,
the bacteria of putrefaction are capable of vegetative existence,

12 MICROBES AND TOXINS

and that our digestion is accompanied by a commencement of
putrefaction,—and of intoxication. The aerobic bacteria of
the Proteus group can produce putrefaction there, but it is
chiefly the anaerobes, as was Pasteur’s opinion, which do this.
There have been found in the human intestine all the most
important of these, the B. putrificus, the B. sporogenes, and
the B. perfringens, The anaerobic flora of the intestine does
not differ much from the anaerobic flora of the putrefying meat
in Tissier’s experiments. These microbes produce poisons
which are the true source of auto-intoxications (Metchnikoff).
For life in general, putrefaction is necesssary to permit of the
circulation of nitrogen in nature: but our own particular
interest demands that putrefaction should not begin too soon,
Z.é.,1n our intestine, and mask itself under an appearance of
perfect health.

To combat the intestinal putrefaction, it is necessary to
adopt a ‘diet capable of producing in us that Amit of acidity -
which induces the crisis separating the two phases of putre-
faction of meat or milk, and of arresting decomposition in our
bodies at the end of the first phase. Therefore, we ought to
eat carbohydrates and sugars, and so alter the conditions
‘in our intestine as to favour the lactic ferments.

IV.—Mitrification and Denitrification.

Nitrates represent the form of nitrogen preferred by the
plants, and it has long been known that the ammonia set free
in putrefaction becomes oxidised in the soil, the ammonium
salts being transformed into nitrates. This is the process
of nitrification and is carried on by bacteria.

It was long thought that the ammoniacal salts were oxidised
in contact with the soil by the direct action of atmospheric
oxygen, in the same way that certain chemical combinations
can take place on contact with porous substances. But chalk
and sand, which ought to act as porous substances, cannot
take the place of earth. Pasteur perceived with his peculiar
intuition that the lower plant must play a part in nitrification.

GENERAL FUNCTIONS OF MICROBES = 13

That nitrification is the work of living creatures has been
proved by the celebrated experiment of Schloesing and Miintz.
If a cylinder is filled with cultivated soil, ammonia poured on
the top appears at the bottom as nitrate of lime. But this
transformation no longer occurs if the earth is previously heated
to 100° C., or if it is impregnated with the vapour of chloroform
or of carbon bisulphide, z.e., if the living organisms that it
contains are killed or paralysed. The soil recovers its activity
when the paralysing vapours have been removed by passing
through a current of air.

Nitrification is a process of two stages, and is carried out by
two species of bacteria, each with its own function. In the
first phase, the ammoniacal salts are transformed into nitrites
by the microbes known as the nitrous ferment, ¢.g. Vitroso-
monas of Europe, Witrosomonas of Java, and the JVitrosococcus
of America (Mexico and Brazil). In the second phase the
nitrites are turned into nitrates by the nitro-bacterium or nitric
ferment, the Witrobacter of Winogradsky (figs. 4-6).

Neither the nitrous nor the nitric ferments develop in
presence of organic matter. The activity of the nitrous
ferment is arrested by 0°3 per cent. of glucose, peptone or
asparagin. The nitric ferment, less sensitive, is stopped by
0°3 per cent. of glucose, 1°25 per cent. of peptone or 1 per
cent. of asparagin. The former is inhibited by 1'5 per cent.,
the latter by 3 per cent. of sodium acetate.

Now we are accustomed to the idea that bacteria live on
organic matter. Yet the bacteria of nitrification must, as we
have seen, get their nourishment elsewhere. They differ from
other bacteria in being capable of taking up carbon by decom-
posing carbonates, in being aerobic, and in behaving like chloro-
phyllous plants. They are not saprophytes in the same sense
as the majority of bacteria.

In the laboratory, the two phases of nitrification can be
demonstrated separately by pure culture. But in nature they
are simultaneous. Under laboratory conditions ammonia
exerts an inhibiting action on the nitric ferment, whereas in
nature both the actions can occur in presence of quantities of

14 MICROBES AND TOXINS

ammonia frequently very considerable. Schloesing has shown
that in the soil ammonia does not prevent the nitric ferment
from acting. These apparent contradictions between theory
and practice are capable of explanation. Ammonia prevents
the development of the nitric ferment but scarcely acts at all on
the same ferment in the adult state. The soil in nature being
populated by the adult ferment, the inhibiting action of
ammonia is much more limited than in an experimental
flask (Boullanger and Massol). .

The nitrous ferment will stand neither an excess of ammonia
nor an excess of its own product, the nitrite of magnesium.
The nitric ferment ceases to act when too much nitrite has

Fic. 4.—Nitrous ferment Fic. 5.—Nitric ferment : the nitro-bacte-
from Gennevilliers. rium from Quito (after Kayser).

been produced around it and when it has itself developed a
certain quantity of nitrates. Thus each demands suitable
proportions both of the primordial material and of the products.
It is, no doubt, to maintain these favourable conditions that
we have the denitrifying microbes which partially undo the
labour of the nitrifying organisms. They return the nitrates
and nitrites to the condition of ammonia, liberating protoxide
of nitrogen, dioxide of nitrogen, or simply nitrogen. Wheat
straw, the straw of maize and of lucerne and oilcakes contain
denitrifying bacteria. Animal excrement also contains them,
for soil to which cow dung is added loses part of its nitrates.
When soil is treated with nitrate of soda too soon after
receiving farmyard manure it loses nitrogen. The denitrifying
organisms act best-in presence of the excess of organic matter

GENERAL FUNCTIONS OF MICROBES 15

which is so prejudicial to nitrification. Probably the two
functions balance and regulate each other. The nitrogen
liberated by the denitrifying bacteria is, however, not lost ; it
may be taken up by the bacteria which fix nitrogen. It is
nevertheless true that farmers ought to be on their guard
against the denitrifying organisms and avoid putting on the
soil farmyard manure, especially if fresh, along with nitrates.
Formerly in the manufacture of saltpetre cultivated soil was

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Fic. 6.—Nitrifying bacteria (after Winogradsky).

A. Nitrous ferment from Ziirich.

B. The same in the motile form with flagella.

C. Nitrous ferment from Kazan.

D. Nitrous ferment from Java, motile cells and groups of cells.

freely sprinkled with urine. If the deposits of nitrate in Chili
and Peru should some day be exhausted, it would not be
impossible to make it with the help of bacteria (not to speak of
the electrical methods which have already been employed).
Saltpetre is produced everywhere when suitable conditions of
moisture and organic matter are given. The saltpetre of

16 MICROBES AND TOXINS

cellars is nitrate of lime which has risen from the soil through
the walls by capillarity and has undergone evaporation.

Long ago Miintz prepared artificial nitre beds which furnished
eight grams of nitrate daily; in recent experiments he and
Lainé have obtained such a yield that they find themselves
capable of preparing by means of nitrifying organisms all the
saltpetre for the powder necessary to defend the nation in war.
Distributed over beds of peat of two metres thickness on the
top of a layer of clinker, the nitrifying bacteria are capable of
producing, for each 25 acres of area, 1,500 tons of nitrate per
day, after a starting period of one month at most to get the
beds into working order; in five days, that is, 7,500 tons, or
the annual requirement of powder for the army. It is easy to
calculate what 750,000 acres of peat bogs in France could
produce if necessary.

The purification of sewage is one of the greatest tasks which
burden the hygienists of large towns. Broad irrigation followed
by cultivation demands much land and is not quite safe except
when employed solely for the growth of forage, not for market-
gardening. Hence it is gradually giving place to the biological.
method of purification, an intensive process carried on in
small space, and here again by bacteria.

We shall not enter here upon the details of its application.
In principle, complete biological purification goes on in two
phases: a phase of anaerobic fermentation in the septic tank
and a phase ot aerobic fermentation in the bacterial beds.

In the septic tank, into which the sewage must be run
gently-—-so as not to carry in the air which would favour
aerobic fermentation—in which it must be allowed to circulate
quietly, and to stay at least twenty-four hours, the disintegration
takes place both of hydrocarbons and proteins under the
agency of legions of bacteria which secrete all sorts of diastases.
The sludge dissolves and the resultant product can be sub-
mitted to the action of the nitrifying agents.

The experimental control of the ferment activity in the
septic tank can be carried out by comparing the action on
coagulated egg-white, meat, raw or cooked, fats, paper, &c.

GENERAL FUNCTIONS OF MICROBES 17

In six weeks roo grams of egg-white leave only one gram of
residue, whereas in stagnant sewage there remain 76 grams,
and in running water 83 grams. In three weeks meat loses
almost 50 per cent. of its weight, and in six weeks 96 per cent.
The body of an animal, immersed in it is for long protected
by the layer of subcutaneous fat; but cleaned cartilage and
tendon lose in five weeks 65 to 99 per cent. of their weight ;
even wool and feathers decompose.

As regards hydrocarbons, the fats are slowly split into fatty
acids and glycerine. Cabbages and potatoes are almost
completely destroyed in six weeks. A hempen rope which,
after five weeks of immersion in stagnant sewage or running
water, could still bear the weight of 12 kilograms, broke under
15 grams after the same period of immersion in the septic
tank. After three weeks newspaper begins to dissolve libera-
ting bubbles of gas. It is quite wrong to consider the septic
tank as operating like a simple settlement tank : it is rather a
sort of crucible in which the powerful microbes melt and
disintegrate the most resistant organic matter. The septic
tank liberates various gases, methane, hydrogen, nitrogen and
carbonic acid, a cubic metre of sewage furnishing from 40 to
70 litres of gas. ;

The aerobic phase of the purification is accomplished by
bacterial beds, into which the organic matter, already dissolved -
and transformed into ammoniacal compounds is discharged.
What takes place in them is an intense mtrification carried
on by the same microbes which in arable soil transform the
ammonia into mineral salts, nitrites and nitrates.

The bacterial bed consists of a thick layer of clinker or slag,
and is filled with sewage for periods of an hour and a half to
two hours separated by intervals of four to six hours. Contact
may be renewed if necessary two or three times by passing the
effluent through a second or third bacterial bed. During
contact the organic matter fixes itself on the clinker, while
during the aeration period it is oxidised by the ferments which
take up the necessary oxygen from the air.

The nitrification is balanced in the bacterial beds by a

Cc

18 MICROBES AND TOXINS

denitrification as in cultivated soil. Nitrifying and denitrifying
ferments can live side by side; the latter only interfere with
the object desired when there is present an excess of hydro-
carbons.

A town of 500,000 inhabitants, furnishing 6 million gallons
of waste water, could replace the 682 acres required for broad
irrigation, or the 15 acres required for the bacterial beds with
slags and clinker, by 2°5 acres of bacterial bed built on peat
(Calmette).

The biological purification returns the nitrogen to nature in
the form of nitrate, although a certain quantity is lost in the
form of gas. There are other bacteria, however, capable of
taking up nitrogen from the air and re-introducing it into the “
cycle of animal and vegetable life.

V.—Ffixation of Atmospheric Nitrogen in the Soil.

The life of both animal and vegetable species depends on the
stock of nitrogen retained by the soil. Although the earth
acquires nitrogen from putrefying processes it loses nitrogen
also, discharged in the gaseous condition; some is lost also
during denitrification, and in percolating water which robs
uncultivated soil of as much as 4o kilos. of nitrogen per acre
perannum. Floods also carry off the nitrates, and after the
floods of 1896, Schloesing calculated that the Seine carried off
about 5 milligrams of nitric acid per litre, at the-rate of
800,000 litres per second ; the total nitric acid lost amounted
to 350,000 kilos. per twenty-four hours, equal to 650,000 kilos,
of saltpetre. The rivers pour this nitrogen into the sea.

And yet in spite of these losses the soil retains its nitrogen.
Nay more, it accumulates it. The soil of forests is never
manured and the woodcutters carry off a great quantity of
nitrogen with the wood ; yet the soil there remains fertile. In
the hill pastures, flocks are feeding all the summer and
furnishing us with nitrogen in the form of milk, cheese and
meat: and yet the soil of these natural fields contains quantities
of nitrogen greater than is found in soil ploughed and copiously

GENERAL FUNCTIONS OF MICROBES 19

manured, 7.¢., from 5 to g milligrams of combined nitrogen
per kilo. of soil. Finally, the crops remove from the soil
much more nitrogen than the manuring supplies: the difference
varying with the rotation of the crops from 1°5 to 400 kilos.
per acre. Where does this nitrogen come from?

It can only come from the inexhaustible reservoir of the
atmosphere.

Rain water carries into the earth the ammonia which has
evaporated from it and the oxidised compounds of nitrogen
which form during thunderstorms. But these gains—about
1'°5 kilos. per acre per annum—are quite insufficient to com-
pensate for the losses occasioned by drainage and cultivation.
Plants must take up not only the ammonia and the nitrates
of the atmosphere supplied by rain, but also uncombined
nitrogen, the free nitrogen of the air. -

Cultivated soil, kept moist and exposed to the air, fixes
atmospheric nitrogen (Berthelot). This fixation does not occur
if the earth has been sterilised by heating to 120°C. Living
creatures must therefore be at work in this.

These workers are the Jacterta of the soil which are found
to a depth of one foot. They are also found in the sea,
especially in the neighbourhood of alge. They are more
abundant in soil, the better it is aerated and cultivated. They
include anaerobes (Clostridium pasteurianum) and aerobes
(genus Azotobacter: Pseudomonas leuconttrophilus). Their
organic food in soil as in laboratory cultures is carbohydrate,
glucose, saccharose, levulose, dextrine, mannite and other
sugars ; butyrates, lactates and acetates; their mineral food
consists of salts of lime and phosphates. The <Azoto-
bacter chroococcum will only develop in soil containing at least
o'r per cent. of lime.

It is not exactly known how bacteria fix nitrogen. They
build it up into Their substance and liberate it when they are
destroyed. Doubtless also they build up nitrogenous com-
pounds which are taken up by the nitrifying bacteria.

Nitrogen can only be thus combined on condition that the
bacteria are supplied with energy in the form of carbohydrate

C2

20 MICROBES AND ‘TOXINS

food. No hydrocarbons, no bacterial activity, no fixation of
nitrogen. To fixa gram of nitrogen, experiments show, 100
to 200 grams of glucose are required.

Just as we do not know all about'a disease when we know
its microbe, so it is not sufficient to have simply the bacteria
which fix free nitrogen in order to enrich at will a soil with
nitrogen. Nature is not the laboratory. he inoculation of
poor soil with these beneficent bacteria produces scarcely any
increase in the crops. To excite bacterial multiplication it
would be necessary to distribute sugars over the soil—14
kilos., it is calculated, to fix the nitrogen to which corresponds
1 kilo. of nitrate. Nitrogen at sucha price is far from cheap.
Besides, the hydrocarbons favour the denitrifying action of the
microbes which results in a loss of nitrogen. The useful
germs are already present in good soil; it is literally the soil
itself which must be altered, by adding marl, by tilling, drainage,
and all the operations which change its physical properties.

There exist certain moulds which fix the nitrogen of the air,
for example the Penida and the Sterigmatocystes. The alge
of the xostoc group, Chlorella, Stichococcus and Cystococcus, fix
nitrogen only when in symbiosis with the fixing bacteria : it is
then the bacteria which fix nitrogen ; the algze, as green plants,
nourish their associated bacteria by means of the hydrocarbons
which they synthesize in virtue of their chlorophyll activity.
According to Beijerinck’s experiments the Cyanophyceae are
capable of taking nitrogen into their tissues independently just
as they do carbon. But if all the algze possessed this double
function they would be so powerfully adapted for life that they
must have long ago invaded the whole universe.

There is a third group of microbes which fix nitrogen ; but
in this case they do not live free in the soil, but are
confined to the roots of Leguminosz.

The soils which are found to be most rich in nitrogen are
those in which there have been longest grown crops of this
family. Georges Ville in 1852 thought that the leguminose
took up oxygen from the air. He sowed leguminosz in sand
which had been washed and calcined, thus being sterile and

GENERAL FUNCTIONS OF MICROBES 21

freed from nitrogen compounds ; he kept them in an atmosphere
similarly freed from all compounds of nitrogen ; when the young
plants had overcome these rather unfavourable conditions they
contained at the end of the experiment more nitrogen than
was present in the seeds.

Malpighi observed long ago—in 1687—little nodules
on the roots of Leguminosz whose function has only in
modern times been discovered thanks to Pasteur. These little
nodules are in fact crammed with bacteria the function of which
is to fix the atmospheric nitrogen. The plant suffers from a
malady which is actually beneficent (Hellriegel and Wilfarth).

Grown in a sterile soil and protected from the germs of the

ZX

Fic. 7.—Nodules on the roots of Leguminosz (Vicéa faba).

air and of the soil, the roots of Leguminosz never present
these nodosities. If this soilis watered with a suspension of the
nodules or of earth in which Leguminosz have grown, or is
simply sprinkled with garden soil, the Leguminosz grow in
it possessing nodules, but if these suspensions are boiled for a
sufficient time they lose this property.

It is possible to inoculate the plant with the infection by
simply pricking with a needle first a nodule on the root of a
normal pea, then the root of a pea which has been grown
aseptically without nodules ; under such conditions the roots
which were free from nodules develop them.

The bacteria of the nodules have an irregular and peculiar
shape, and have been given the name of bacteroidia. The
best known is the &, radicicola of Beijerinck. Two groups

22 MICROBES AND TOXINS

have been made: those of the vetches, clovers, and peas, and
those of the beans and lupines. Mazé distinguishes those
adapted to calcareous soil, the ‘ calcicolz ’ from those adapted to
acid soils, the ‘ calcifuge.’ They are aerobic, and pure cultures
can be obtained. They require carbohydrate food, and are
not fond of nitrates. Grown in pots, the nodules are
encouraged by the addition of chromium, manganese, nickel,
and cobalt. About 100 grams of sugar are consumed for each
gram of nitrogen fixed. The bacteroidia only act well when
their food supply is regulated in quantity as well as in quality.
The broth for the cultures should contain 1 per 10,000 at

.

Fic. 8.—Bacteria and Bacterioidia from the nodules
of Leguminosee (after Beijerinck).

least and 1 per 3,000 at most of combined nitrogen; the
proportions of saccharose should lie between 2 and 6 per
cent. :

The virulence of the bacteroidia varies ; those which have
undergone several passages in the roots attack the new roots
more easily. It seems that the weaker bacteria induce a
certain immunity in the rootlets towards the more virulent.
A root already bearing nodules does not produce new ones
except when inoculated with very virulent bacteroidia. On the
same plant the recent nodules of the lateral roots contain
bacteria much more virulent than those of the main root.

GENERAL FUNCTIONS OF MICROBES) 23

The bacteroidia scattered through the soil are attracted
towards the young roots by the vegetable carbohydrates : there
is a positive chemiotaxis.

The bacteroidia and the plant are mutually beneficial.
The plant supplies carbohydrate food to the bacteroidia, which
n their turn supply the plant with nitrogen. In culture a
viscous glairy jelly appears, which is present also in the
nodules, but only in their early stage. The sap rapidly carries
it into the body of the plant, and it is this jelly probably which
contains the nitrogenous food product. Some diastase
probably of the root liberates the nitrogen compound from the
cells of the bacteroidia.

Nitrogenous manures are expensive, and hence the idea
has arisen to improve leguminous crops by planting them in
prepared soil, or by adding soil which has already grown
leguminous plants. Afterwards the attempt was made to
inoculate the soil with artificial cultures of the nodule
bacteria. The process is analogous to the injection into an
animal of a food substance or a drug: it is agricultural
bacteriotherapy.

In observing nature, not from the point of view of any
single living species, man or animal, but throughout the whole
of her operations, one sees that the pathogenic bacteria are
less prominent than the useful ones—one may say even that
all bacteria are useful and are only injurious by accident.
They all have their réle in the cycle of matter and only destroy
one existence to prepare for another.

To perceive the function of the useful bacteria, it has been
necessary to study all the fermentations in relation to both
industry and food supply: wine, beer, cider, vinegar, cheese,
bread, sauer-kraut, tobacco, and the leather of our shoes are
all more or less the result of bacterial operations. Life as a
whole could not continue without bacteria ; they do not create
life, but they supply it with the necessary material.

We may ask ourselves now if, with nature thus provided
with nutritive material, the life of a particular organism might
be possible although it did not contain microbes in itself.

24 MICROBES AND TOXINS

Logically it is possible. An animal without microbes, an
aseptic creature, might take advantage of the general activity
of microbes without itself being open to these peculiar fermen-
tations which we call diseases.

This then would be at once the first principle of hygiene,
and, as it were, its paradox.

CHAPTER II

MICROBES IN THE HUMAN BODY —LIFE WITHOUT MICROBES—
THE INTESTINAL FLORA

Microbes in and on the bodies of animals and man: skin, mucous mem-
branes, mouth, stomach, intestine.

Life without microbes —Pasteur’s ideas—Aseptic breeding—The example
of the bat, Pteropus.

The intestinal flora of man: quantity, species, and variations of the
microbes.

Intestinal putrefactions—The flora and diet—Products of putrefaction and

auto-intoxication.

Associations and antagonisms—Cholera—Experiments of Metchnikoff,
Bienstock, Tissier—Principles of intestinal bacteriotherapy—Sour
milk ; culture, pure and mixed, of the lactic ferments.

Is the large intestine useless ?—Is the intestine permeable to microbes ?—
Investigations on anthracosis and tuberculosis—The defensive powers
of the mucous membrane.

The Microbial Flora in Man and Animals.—We
have seen what innumerable legions of bacteria are at work in
the universe. Let us now proceed from the contemplation of
the macrocosm to the microcosm, as represented by our own
bodies.

There is scarcely a living being which is not crowded with
microbes. Living as we do in a universe where they swarm,
how could we avoid having them both within and without ?

Normally, we enter the world free from microbes. But from
the first moment after birth they begin to settle on our skin
and on our mucous membranes. Our mother’s first touch
communicates them to us, even before she has given us the first

drops of her milk. They penetrate the nose, the mouth, and
25

26 MICROBES AND TOXINS

the lungs with our first respiratory movements and our first
cries ; they are deposited on our skin from our first bath and
our first swaddling-clothes. Even four hours after birth - and
invariably between the tenth and the seventeenth hour of life
—they have already reached the intestine.

Our skin is inhabited chiefly by round bacteria, such as
streptococci, and especially staphylococci; these latter are most
abundant, and there are several species of them, some
commoner than others. They do not inhabit so much the
smooth surface of the epidermis as the hair follicles and their
adnexa, the sebaceous glands, which are regular dens of
bacteria. Even the cleanest persons, however little oily their
skins, have only to press between the fingers a little fold of skin
on the end or at the side of the nose, to squeeze out what
looks like alittle worm, but which is nothing else thana colony
of staphylococci.

As a matter of fact, however healthy the skin, it is more or
less inhabited by bacteria, which, however, in general, remain
harmless ; the skin is not zz/ected. But given at some point a
lesion, a boil or pustule, where bacteria have multiplied, the
microbes of these foci spread themselves over all the cutaneous
surface, even to the most distant parts. ‘On the surface of
healthy skin, the ‘diffusion of germs takes place around the
original lesion for a great distance, and with an abundance and
continuity such as is only paralleled by the law of conservation
of widely distributed species. An analogous case is that of
plants covering square miles of country round with their
pollen or their winged seeds” (Sabouraud). Hence, the best
way to avoid cutaneous infections is to preserve intact the
epidermis, and not to weaken by the abuse of antiseptics its
protecting cells, which endeavour to maintain the integrity of
the skin against bacteria.

The mucous membranes, warmer and moister than the skin,
form a better soil for microbes. There is in the conjunctiva
of the eye, among others, a little bacillus, resembling the
bacillus of diphtheria, which is found from the first hours of life
(Morax). In the cavities of the nose and pharynx, there are

MICROBES IN THE HUMAN BODY Q7

not only the common bacteria, the streptococci and staphylo-
cocci, but also pathogenic bacteria, which take shelter there
and remain latent until an opportunity offers to multiply ;
examples are the diphtheria bacillus, the microbe of pneumonia,
and that of cerebro-spinal meningitis.

In the deeper respiratory passages, there are few microbes ;
there they may be numbered by units. But it is proved that
the tubercle bacillus can penetrate with the inspired air right
to the bottom of the pulmonary alveoli.

The mouth, the vestibule of the digestive tube, contains
already a large proportion of the microbial species which
populate the stomach and intestine, ¢.g., staphylococci and
streptococci, resembling those of the skin, bacilli, aerobic
and anaerobic, resembling those found both in the healthy
intestine and in the intestine and appendix in disease, and finally
a whole flora peculiar to the mouth which plays a part in dental
caries.

The stomach being acid suits moulds and yeasts better than
bacteria. However, about thirty species of bacteria have been
described in it (Coyon), several of which have attracted special
attention because of the idea that they might favour or inhibit
the penetration of certain pathogenic bacteria into the intestine.

All the cavities and recesses of the human body deserve
study from the point of view of their flora. Many species have
been seen but are not yet well known, because we do not yet
know how to cultivate them artificially.

The flora hitherto most studied is that of the intestine. It
isalso the most important. The intestine is the great laboratory
of digestion, and at the same time, unfortunately, of putrefac-
tions, the products of which are absorbed by the body.
Hence one may say that man, like other animals, is dependent
on his belly, and no system of therapy is justified in neglect-
ing it. Just as the soil outside of us is, in nature, the great
microbial reservoir, so in us the great reservoir of microbes is
our intestine.

Under the impulse of Metchnikoff, the intestine has with
justice become the great field of study and experiment in those

28 MICROBES AND TOXINS

problems of nutrition which affect most of all the general
health, the individual development, and the evolution of man.
That is why we pay special attention here to the intestinal
microbes as being the most important of all those which
inhabit the bodies of man and animals.

The first problem in this question is the possibility of life
without microbes.

Life without Microbes.—Life in general, as we know
it on the surface of this earth of ours, is impossible without
the action of the micro-organisms. As some philosophers
have well said, “life is but a little mould growing on the
surface of a rather moist planet.” But is it possible for certain
individuals to live free from bacteria, although of course
depending on the conditions produced by bacteria, and paying
tribute to bacteria by reason of the fermentations to which
they owe their origin and nourishment and by which one day
they are doomed to be dissolved ?

Plants have bacteria both around their roots and in their
tissues. Animals have thousands of millions in their digestive
tubes. The microbial world not only surrounds them but
is in them. In our intestine there proceed fermentations
from which we can absorb the products. Is this to our
advantage or is it not?

It seems that plants derive only benefit. The bacteria prepare
for them their food. A sterile seed made to sprout in a sterile
nutritive fluid, such as milk, can use neither casein nor sugar
nor starch ; it secretes neither rennin nor casease, nor sucrase,
nor amylase. There is amylase, it is true, in the cotyledons of
the germinating seed, but its action does not extend into the
surrounding medium. Plants thus prosper by the help of
bacteria, and the aim of cultivators is to provide for each plant
the microbes most favourable for the yield desired by mankind.
But it is to be noted that these useful microbes are not zithin
the plant.

The problem is quite otherwise in the case of animals. As
in so many other cases, the question was first stated by Pasteur
in connection with Duclaux’s experiments on the nourishment

MICROBES IN THE HUMAN BODY 29

of plants under sterile conditions and their rational culture
with the help of selected microbes. Pasteur expressed an
opinion which he had not time to submit to experiment, but
which has been taken up ‘by others, the idea namely that an
animal cannot dispense with the bacteria living within it. He
suggested that a young animal should be fed from birth with
pure sterile food.

“TI do not conceal the fact that, had I time to undertake
this study, I should do so with the preconceived opinion that
life under such conditions would become impossible. Given
that such experiments are capable of being gradually simplified,
it might be possible to study digestion by adding systematically
to the sterile food of which I speak various individual bacteria
or different bacteria together, each of definitely known species.

“The hen’s ‘egg lends itself without serious difficulty to
experiments of this kind. Previously freed externally from
every sort of living impurity just before hatching, the chick
should be immediately put into a chamber free from every
sort of bacterium, so that it could be supplied with pure air
and with sterile food, easily introduced from without in the
shape of water, milk, and corn.

‘Whether the result were positive, confirming the precon-
ceived idea which I am putting forward, or negative, or even
absolutely the opposite, #.¢., that life without bacteria is easier
and more vigorous, in any case the experiment would be full
of interest.” :

The experiment /as been tried and the programme is still a
long way short of completion. One thing only has to be
altered in Pasteur’s statement. The experiments are far from
being without “ serious difficulty.”

Two currents of opinion have developed. The first, following
literally Pasteur’s idea, maintains that animals are like plants
and cannot nourish themselves with the sole help of their own
digestive juices but require also the intestinal microbes. We
do not know, say its supporters, if in the beginning of life an
animal organism has ever developed free from micro-organisms ;
it is not very probable ; what is certain is that for centuries and

30 MICROBES AND TOXINS

centuries there have been microbes within living beings, and
there must have become established a sort of understanding, or
adaptation between the intestinal flora and the intestine. Just
as plant roots absorb. from the soil the fluids elaborated by
the bacteria, so the intestinal villi absorb the juices prepared
by the microbes of the intestine. These microbes accumulate
and their presence in masses stimulates the intestinal muscles.
Further, the normal intestinal flora opposes an invasion by
foreign bacteria which might be or might become pathogenic.
Consequently the intestinal bacteria both nourish and protect ;
they are useful, salutary, and providential.

The opposite opinion is maintained by Metchnikoff. The
digestive ferments prepare the nutritive materials without any of
this problematical assistance from the microbes. The intestinal
flora is injurious (taking of course the point of view which
interests us most, that of mankind), because long before the
food stuffs leave the body, the bacteria induce in them fermenta-
tions the products of which, absorbed by the mucous membrane,
are for the most part poisonous.

From the point of view of nature, it is quite normal for the
albuminous excreta of our food to putrefy and thus return into
the general circulation of matter ; but it is regrettable when this
takes place in our bodies, for phenols, skatol, and indol, among .
other products, penetrate into our circulation and affect the cells
of our arteries and brain. It would be to our advantage if the
food-stuffs were expelled immediately after useful digestion, and
before the terminal phase, the putrefaction, begins in the waste
products. And since in the part of the intestine which pro-
perly speaking is the digesting part, the small ‘intestine, there
are practically no bacteria, and since on the other hand they
swarm in the large intestine where there are scarcely any diges.
tive ferments, it is evident that, on the whole, the intestina]
flora is injurious. The ideal condition would be to live free
from bacteria while the world remained populated by them.
Is a life of such purzty possible ?

Aseptic breeding.—There are, it has been said, certain
Arctic animals, both birds and mammals, without intestinal

MICROBES IN THE HUMAN BODY 31

microbes. But the recent polar expeditions failed to find a
single animal of this kind.

Nevertheless, such have been found among the burrowing
larvee which excavate galleries in the thickness of leaves and
live shut off from the external world by a transparent wall of
epidermal cells. The truth of this can be demonstrated by
extracting them with a sterile needle through a litttle perforation
made in the epidermis after sterilisation with peroxide of hydro-
gen. According to Portier’s experiments, the Zzthocolletis
caterpillars are aseptic in about a third of the cases ; those of
the JVepticula of rose-trees always. These burrowing larve
dispose of their excreta by stowing them aseptically in their
closed tunnels, whereas Z¢scheria, which discharges its dejections
externally, through a little hole made in the leaf, is always
contaminated.

Finding experiments on these small invertebrates easy,
Bogdanoff introduced the previously disinfected eggs of flies
into sterile meat and found that the larvee hatched and kept
under these conditions develop less well than the control larvae
fed on putrefying meat rich in bacteria. When he added to
the sterile breeding ground a digestive ferment capable of
attacking meat, 7 ¢., trypsin, the sterile larve flourished equally
with the others. Bogdanoff would have concluded that the
bacteria play the necessary part which in the latter case was
performed by the ferment, had he not found some sterile broods,
without addition of ferment, quite as vigorous as in ordinary
breeding. Larvee can thus develop without the aid of microbes.
Wollmann using an excellent technique has finally proved this :
he succeeded in breeding from the egg sterile flies. “ During
the first days of life the sterile larvae develop more slowly than
the contaminated controls, probably because the digestive
glands are not yet in full activity and find the sterilised meat
difficult to attack. Later these differences disappear and the
sterile larvee attain the weight and size of normal adults.”

Experiments on aseptic breeding of vertebrates, such as
Pasteur desired, have been carried out, with a patience
worthy of all praise, by Nuttall and Thierfelder, who took young

°

32 MICROBES AND TOXINS

guinea-pigs from their mothers by Czsarean section, by
Schottelius and Cohendy on the chick, by Mme. Metchnikoff
and by Moro on tadpoles.

The little guinea-pigs appeared to develop and increase in
weight in normal fashion, but Schottelius has questioned the
interpretation of these experiments; he maintains that the
increase in weight was due to food swallowed but not digested.
The tadpoles developed badly without microbes. The
chickens of Schottelius were ‘ weak/ings’ and lived seventeen
days at most, becoming increasingly thin and feeble. Their
digestive juices were apparently insufficient for digestion, and
they only recovered when there was added to the sterile food
certain bacteria, among which was the bacillus coli.

But it is impossible to conclude with certainty from these
experiments that life is impossible without microbes ; these
new-born animals were placed under conditions too different
from the natural ; their intestine was not yet secreting enough
ferment, and the food they were getting, sterilised as it was at
a high temperature, was not adapted to the hereditary disposi-
tion of their alimentary canal.

It would therefore be necessary to study adult animals
free from microbes, but we know that this condition hardly
exists.1

There are, however, animals, which approach it somewhat,
namely, the large fruit-eating bats of the tropics (Pteropus
medius) recently studied by Metchnikoff. These animals
have a large intestine very limited in size; there is no
reservoir in which to accumulate the waste products of their
food, and they begin to evacuate feeces one hour after the
ingestion of the food from which these are derived. They are
obliged to eat a great deal: and to evacuate in proportion.
They have, properly speaking, no intestinal flora, simply a
few bacteria conveyed by the food and changing with the diet.

1 The intestine of the scorpion is almost always free from microbes;
and the same is the case with the intestine of certain maggots provided
with digestive juices sufficiently powerful to digest seeds, wool, and even
such resistant microbes as the bacillus of tubercle.

MICROBES IN THE HUMAN BODY 33

The juices of their small intestine have no power of killing
bacteria such as has been alleged in the dog and the cat, so
the poverty of their intestinal flora cannot be thus explained.
The digestive tube being almost free from bacteria in its
whole length, the digestion of the food cannot be attributed to
microbes.

The fruit-bat above-mentioned digests the cellulose of
bananas, yet it is precisely for the digestion of cellulose
among the herbivora that the necessity of auxiliary microbes
has been maintained ; the reason is that cellulose-digesting
microbes are known, whereas no ferment has been separated
from the digestive tube capable of doing this. But the
bacterial ferment has not been isolated either: the question
of cellulose digestion is one on which our ignorance is great
and which demands fresh investigations. Finally, in the
dejecta of the fruit-eating bat the putrefactive poisons phenol,
skatol, and indol do not occur, although these are constant
among all other mammals including the herbivora. This
absence of putrefaction corresponds with the extreme short-
ness of the large intestine and the poverty of the intestinal
flora. We have then in this animal a good example of an
adult animal capable of digesting its food, under normal
conditions, by its own digestive juices without the help of
bacteria.

The Intestinal Flora of Man.—Man differs from the
bat. He harbours a very abundant intestinal flora in his large
intestine which is highly developed and in which the food-stuffs
begin to putrefy.

It is almost impossible to calculate the number of bacteria
in our intestine. Gilbert and Dominici have calculated that
we discharge daily 11,725 millions, without counting the
anaerobes, of which there is a prodigious number. Klein
speaks of 9 billions of bacteria discharged every twenty-four
hours, of which 100,000 millions are still alive. Strassburger,
using a different method of counting, calculates the quantity
of bacteria at one-third of the normal dried feeces ; in twenty-
four hours a man evacuates 120 billions of bacteria. It would

D

34 MICROBES AND TOXINS

be too much to say that these figures are imaginary, but they
are certainly only approximate. It is sufficient to state that
the real figure is enormous and certainly approaches the
magnitude stated.

The small intestine is less populous, so much so that a
bactericidal action even has been ascribed to it. Yet the
pancreatic juice which is poured into the small intestine is
invaded by microbes when collected and left outside the body.
The auto-sterilisation of the small intestine seems to be due to
the combined action of the two principal digestive juices, the
pancreatic and the intestinal. The flora of the small intestine
like that of the intestine in general varies with the diet.

It is more important to know the nature of the microbes
than their quantity. In 1898 Mannaberg counted only
twenty-seven species in the intestine ; but there are now many
more described and the improvement in our methods of
cultivation is leading daily to the discovery of new ones.
MacFadyean, Nencki and Mme. Sieber have isolated fourteen
from the small intestine alone.

A child is born with a sterile alimentary canal, but from the
tenth to the twentieth hour of life it begins to be inhabited.

Fic. 9.—Bacillus bifidus (Tissier) : Fic. 10.—Baczllus bifidus
10 day culture. (Tissier): 15 day culture.

From the first to the third day in the dveast-fed child there
appear Streptococci, Bacillus coli, B. perfringens (= Welch’s
Bacillus), B. III of Rodella, B. /actis aerogenes, Sarcinae,

MICROBES IN THE HUMAN BODY 35

enterococci, mesentericus and an acidophilus. After the third
day the flora becomes more simple, being dominated by one
of the last to appear, the anaerobe ZB. difdus of Tissier, which
is characteristic of the flora of the healthy breast-fed infant ;
along with it persists the Bacillus coli, the enterococcus and
the B. lactis aerogenes. Thanks to the presence of B. difidus
and the lactic bacilli, which produce acids from the carbo-
hydrates (human milk is rich in sugar especially after the
tenth day), the putrefying bacteria (B. perfringens, for example)
are kept in check or even entirely eliminated. Proceeding
from the stomach to the rectum the species which predominate
in turn are the B. coli, the B. /actzs aerogenes, the enterococcus,

Kj K
Fic. 11.—Bacteria in the Fic. 12.—Bacillus sporogenes
feces of a normal child of (Metchnikoff).
19 months.

.

the B. exilis, the B. acidophilus and lastly the B. bifidus. The
distribution and proportions may be altered in the case of the
infant not breast-fed. The chemical surroundings being
different, the fermentations also differ.

From the age of one to five years and in particular after
weaning, the flora has added to it several new species, while
still retaining the species found in the infant, which may be
regarded as the fundamental flora. ‘Tissier calculates the pro-
portion of this fundamental flora in the child brought up on a
vegetable diet at go per cent. of all the bacteria and 4 of

D2

36 MICROBES AND TOXINS

this are made up of B. bifidus. The proportion is less (70%)
in the child receiving a fair quantity of animal food.

In the human race three microbial species seem to be chiefly
capable of provoking the putrefaction of albuminous material
in the intestine ; namely the three anaerobes: B. putrificus of
Bienstock, B. sporogenes and the B. of Welch also called
B. perfringens.

Not only are these three anaerobes putrefying organisms but
they seem also to be true pathogenic organisms. Putrificus
has been found in peritoneal suppuration, in appendicitis and in
various intestinal disorders. 3B. sforogenes has been found in
many cases of diarrhoea ; B. perfringens is common in acute
and chronic suppurations, and in infantile diarrhcea : it is also
the cause of crepitating gangrene or gaseous phlegmon. The

= F k
Fic. 13.—Bactdlis Fic. 14.—Welch’s bacillus
putroficus (Bienstock). (B. perfringens).

study of their virulence and toxicity by experiments on small
laboratory animals and monkeys has hardly been begun. They
indicate well how delicate is the distinction between a simple
saprophyte of wide prevalence and a pathogenic organism
properly speaking.

The B. coli, the commonest and most abundant bacterium
in the intestine, though it was considered by Schottelius, in his
experiments on the aseptic chick, as an indispensable adjunct
to nutrition and though it has even been employed asa remedy
for constipation, is nevertheless prejudicial to health.

It does not, it is true, attack the genuine albumins but it

MICROBES IN THE HUMAN BODY 37

breaks down the peptones, producing indol, phenol, mercaptan
and sulphuretted hydrogen. It is precisely these bodies,
putrefaction products, which enter the blood and produce the
chronic poisoning which induces premature old age! Among
the bacteria of the intestine, B. dactis aerogenes, B. perfringens,
B. sporogenes, Staphylococcus pyogenes and Proteus produce
indol. .B. coli produces both indol and phenol, and there is ex-
perimental proof that indol, phenol, and their sulpho-compounds
can produce auto-intoxications of the body. With indol and
potassium phenyl-sulphate, aortic atheroma has been produced
in rabbits in about 60 per cent. of those tried, whereas
spontaneous atheroma does not occur in more’than 6 to ro
per cent. Monkeys treated with paracresol presented arterial
lesions in the brain and kidney. By combining the phenols
with sulphur, the body manufactures ‘ sulpho-compounds ”
which are less injurious than the pure phenols, but which are
still chronic poisons (Metchnikoff).

Intestinal Putrefaction.—The study of the putrefactive
bacteria being in its infancy and beset with great difficulties
besides, it is not surprising that certain authorities maintain that
intestinal putrefaction is harmless to man.

It is true, they say, that our instinct leads us to reject putre-
fying food and that common sense has always connected
putrefaction with disease. Yet we see the Indo-Chinese, the
Malays, Polynesians, and the Greenlanders regaling them-
selves with decayed fish, meat, or eggs! Meat which has gone
bad frequently determines diseases resembling acute poisoning ;
but it is not because it is putrefying ; it is because it contains
pathogenic bacteria of the family of B. fyphosus, or bacilli like
the B. of botulismus, which produces a violent toxin. Extracts
of putrefied meat have been injected into different animals with-
out result. Further, how many people exist in perfect health in
spite of the putrefaction going on normally in their intestine !
It recalls the dictum of Malvoz: ‘Tout ce qui pue ne tue

1 The intestinal flora of the dog does not differ from that of man.
Little rabbits eight days old have not a rich flora. The flora of parrots,
young and old, is also scanty (five species in the ileum). The flora of the
alligator is much less rich than that of man.

38 MICROBES AND TOXINS

pas, tout ce qui tue ne pue pas.” (What stinks does not kill,
what kills does not stink.)

A Belgian physiologist, Falloise, has shown that the contents
of the large intestine where the putrefications are going on are
much less toxic than those of the small intestine which is free
from putrefaction. The toxicity of feecal matter left in the
incubator, with all its contained bacteria, diminishes instead of
increases. Hence the toxicity is not in proportion to the
putrefaction.

What then causes those gastro-intestinal disorders, in
particular infantile diarrhoea, with symptoms resembling typhoid
fever or cholera? It must be poisons of some kind, but poisons
which are the result not of bacterial decomposition of the
intestinal contents but of abnormal metabolism of the food.

This theory of non-microbial intoxication, is opposed to that
which regards microbial putrefactionas the cause ofthe poisoning.
Finkelstein, for example, says that there is no specific infecting
organism in infantile diarrhoea and that the same symptoms
appear with very different intestinal flora; according to
Nobécourt and Rivet, the intestinal flora depends on the diet
and the disorders of digestion, its réle being secondary and
rather an effect than a cause. The principal réle is played by
those poisons which are produced directly in the digestion of
the food-stuffs.

But though it is true that the study of intestinal putrefaction
is not far advanced, the opponents of this theory must acknow-
ledge thatstill lessisknownabouttheauto-intoxications. Nothing
definite has been established in connection with the ptomaines
and toxalbumins which have been incriminated. Besides, the
argument of Falloise on the slight toxicity of the faecal matter
is not justified, for he forgets that the feeces do not represent
actually the intestinal contents, these latter having lost
before discharge precisely those poisons which have been
absorbed by the mucous membrane.

The phenols, for example, which are produced in our intestine
are absent in the feeces, but appear in the urine. The volatile
fatty acids also are in greater quantity in the urine than in the

MICROBES IN THE HUMAN BODY 39

fecal matter. As to the poisons found in the small intestine,
these are nothing but the digestive juices themselves, from the
pancreas and intestinal mucous membrane ; the fact of their
toxicity when injected into the veins of arabbit does not prove
in the least that they are noxious to the animal producing
them. It is, in fact, a very rough way of studying the toxicity
of feecal matter to inoculate extracts of it into animals. The
biological and chemical study of the intestinal bacteria in their
capacity as ferments is the necessary and proper introduction to
the problem of intestinal putrefaction.

The diminution of toxicity which Falloise discovered on
incubating feeces represents only the primary phase of the
phenomena which occur under such conditions, namely, a
preliminary fermentation which produces acid and suspends the
putrefaction ; it is only after the third day that true putrefaction
can begin, the reaction having then become alkaline,

Finkelstein’s argument was the speedy effects of change of
diet on an intestinal disorder ; but this action may equally well
be explained by the intervention of microbes; the flora also
varies with the diet. In the intestine, as in a culture flask,
different cultures can be got with the same microbes when the
media originate on the one hand from meat and on the other
from vegetables.

In studies so difficult as these, no good result can be got by
treating feecal material and injecting it in quantity. It is
necessary to proceed analytically, isolating patiently the
bacterial species of the intestines, studying them in a condition
of purity, as every ferment ought to be studied, studying
secondly their actions in association, and studying finally the
poisons they produce, phenols, indol, skatol, etc. It is by
this method that the action of B. putrificus, B. perfringens,
and 2B. sporogenes in intestinal decomposition has been
rendered certain as has been the réle of B. proéeus in infantile
diarrhcea.

Mutual Assistance and Mutual Antagonism.—These
legions of microbes do not escape the law of competition which
prevails throughout life. ‘They have not all the same demands

40 MICROBES AND TOXINS

or the same appetites; one requires more alkalinity, another
requires a certain degree of acidity. The unused residue of the
food of one may provide excellent nourishment for another.
Their actions on elementary waste products have to follow each
other in a regular and chemically definite order. Hence we get
agreements and antagonisms, one species, by reason of its
nature and the food of its host, being towards another either
a help or a hindrance.

This idea was first expressed in connection with the bacteria
of the intestine by the experiments of Metchnikoff on cholera
and its vibrio. Cholera is due to the multiplication in the
intestine of a spirillum, which rarely penetrates into the blood,
but elaborates in the intestine poisons which diffuse and kill
the patient. Cholera is an acute, toxic, specific enteritis.
Koch’s discovery of the spirillum or “comma bacillus” of
cholera had to contend with an obstinate scepticism, because
cholera could not be produced with it at will in laboratory
animals, unlike anthrax or fowl-cholera. Whether by inoculation
under the skin, or in the peritoneum, or by the mouth, the
result was the same. Even when several savants swallowed
cultures of it, the results of these experiments “7” anima nobiii”
were very inconstant. In this, typhoid fever resembles cholera,
the disease being very difficult to reproduce experimentally ;
it is only quite recently that experimental typhoid fever has
been successfully produced by employing the higher apes, the
nearest congeners of man.

It cannot be a matter of indifference to the cholera vibrio
what is the nature of the flora of the alimentary canal into
which it penetrates. By a very simple method of experiment,
namely, by inoculating together on gelatine plates vibrios and
various other microbes along lines which crossed each other,
Metchnikoff was able to prove that the association of certain
bacteria favoured the growth of cholera while others inhibited
it. Round the colonies of a favouring bacterium a swarm
of little cholera colonies developed. Various favouring and
inhibiting bacteria were discovered in the air, among animals,
and—most interesting of all—in the human stomach.

MICROBES IN THE HUMAN BODY 41

But here we were still only dealing with growth on plates,
z.é., under very artificial conditions. The importance of the ex-
periments increased when it was found possible to transmit to
an animal, with certainty, a genuine attack of cholera. Neither
the cat nor the guinea-pig nor the adult rabbit takes the
disease. Metchnikoff had the idea that their resistance might
be due to the presence in the alimentary canal of inhibiting
bacteria, and he experimented accordingly on little sucking
rabbits, which, while they are at the breast—a period of some
weeks-——present a very scanty intestinal flora. Little rabbits of
one to four days old, made to swallow a culture of the cholera
vibrio, died in about half the cases ; they were thus capable of
taking cholera. But further, when to the culture of the vibrio
other bacteria were added which had been recognised as
being favouring in the gelatine experiment, the little rabbits
took cholera without exception. They were refractory, on the
contrary, when the inhibiting bacteria were added.

The question naturally arose whether during an epidemic
man might not increase his resistance to a cholera infection
by ingesting cultures of these inhibiting microbes, and this
formed the starting point of intestinal bacteriotherapy.

Bienstock attributed the resistance which milk possesses
naturally towards putrefaction to the microbes which it contains,
and not, as the old idea maintained, to the presence in it of
casein or milk sugar; he found that the B. dactzs aerogenes, an
acid producer, and even the ZB. cof, prevent the development
of the B. putrificus, the agent of putrefaction, whether in an
experimental flask or in the intestine. Inoculated along with
the B. col, the growth of B. putrificus is inhibited, because it
can only develop in an alkaline medium, and the B. col
produces from the sugars of the food materials an abundance
of acid.

The idea of an inhibiting action exerted by an acid-producing
bacterium on the putrefying organisms is correct, but the above
example was not the best; in the first place because the
B. putrificus is rare in the human intestine, and secondly
because the &. cod, while playing a useful part in certain cases,

42 MICROBES AND TOXINS

is in others itself a putrefying organism. Kolbriigge’s opinion
can no longer be maintained that the B. cold is the beneficent
bacterium par excellence, growing in the appendix as in a sort of
hot-house put specially there by Providence !

The antagonism is chiefly between the B. perfringens of
Welch and the B. difidus discovered by Tissier in the normal
flora of the healthy breast-fed infant.

There is a general antagonism between the simple saccharo-
lytic bacteria which produce acid from starches and sugars
and the simple proteolytic bacteria which carry the digestion of
meat to the stage of indol, skatol, phenol, and ammoniacal
salts. When the bacteria which oppose each other are both
proteolytic and saccharolytic (hence called mixed ferments),
those which produce the most acid inhibit those which produce
less.

For example, the eéerococcus is inhibited by an acidity of
2°45 per 1000; the B. perfringens at 1°60 per 1000; the B.
acidt paralactic’ at 5°39; the B. bifidus at 4:90. The two
last are evidently successful antagonists of the B. perfringens.

The facts established by Bienstock and by Tissier and
Martelly may be expressed as the following law: in media
containing protein along with more than 10 per 1000 of sugar,
a “mixed ferment” can arrest the development and the
action of a simple ferment, whereas a strong mixed ferment
(¢.¢., a strong acid producer) can inhibit a weaker. These
inhibiting actions are entirely due to the quantity of acid
which the bacteria produce in the course of their consumption
of the carbohydrates.

Principles of Intestinal Bacteriotherapy.—
Metchnikoff’s experiments on microbial associations in cholera
contain the germ of a method of treatment which consists in
using inhibiting bacteria. The experiments of Bienstock
and Tissier indicate that the saccharolytic bacteria form a
natural obstacle to the action of the putrefying microbes.
The idea of a bacteriotherapy had already been formulated by
the well-known children’s physician, Escherich, practically in
the following terms: “It is necessary to employ the ‘acid

MICROBES IN THE HUMAN BODY 43

fermentations’ as antagonists of the ‘alkaline fermentations,
either by adding to the diet carbohydrates such as lactose (milk
sugar), or by giving cultures of acid-producing bacteria.”

It had long been the custom to employ as remedies for
diarrhoea, intoxications, and intestinal putrefactions, first
purgatives, then antiseptics, of which the most popular was
B-naphthol. This treatment has been given up.

Several days of @-naphthol treatment fail to diminish the
number of bacteria in the alimentary canal (Stern).

“ Since intestinal antisepsis is based on such a weak found-
ation,” said Quincke in a treatise on intestinal auto-intoxi-
cations, “it was natural to search for another method of
combating the decomposition caused by bacteria in the
intestine, and the idea came to me to try to supplant the
injurious bacteria by beneficent ones, just as weeds are
suppressed by the growth of useful crops, the starting point
being the antagonism of different microbes observed in
artificial culture media.” Quincke tried the yeast of beer.
Later there was employed in infantile diarrhcea the Bacterium
lactis aerogenes, the antagonist of B. proteus.

Intestinal bacteriotherapy has now become part of practical
medicine and is applied in two principal forms. The first is
to administer those foods which are produced by natural
fermentations and which contain great numbers of bacteria ;
the second is to give along with a suitable diet artificial cultures
of bacteria possessing antiputrefactive ,properties. In both
cases it is the antagonistic action of microbes towards the
bacteria of decomposition of which one takes advantage.

Fermented foods, rich in bacteria, were employed long
before modern researches on microbial antagonism came into
being.

Humanity has known from time immemorial the whey of
milk and the various forms of cheese. It was long before
the days of science that there were invented koumiss, kvass,
kephir, lebenraib and yoghourt. In the Bible we read that
Abraham offered to his guests sour milk as well as sweet.
Moses says that Jehovah has given his people to eat of the sour

44 MICROBES AND TOXINS

milk of cows and the milk of goats (at the same time, it is true,
the fat of lambs and of sheep and of kidney). There can be
no doubt that sour milk is more ancient even than the Bible
itself.

The artificial cultures chosen for their chemical properties
are chiefly A. difidus, B. acidi paralactic’ and the lactic ferment
which has become popular under the name of the Bulgarian
bacillus ; they are given either singly or in association. They
may be taken either in the form of clotted mik or in bouillon.
The characters of the Bulgarian bacillus are briefly the
following : it produces 25 grams of lactic acid per litre of milk;
not more than 0°50 gram of succinic and acetic acid, traces of
formic acid, no alcohol, and no acetone. It hardly attacks the
proteins at all, and has no pathogenic power (G. Bertrand and
Weisweiler). Although it does not inhibit the development of
the Z. colt in a culture containing peptone, it at least prevents
it from producing phenol and greatly reduces its indol
production.

The mechanism of the action of the lactic ferments in
general is not quite settled. They appear to diminish the
number of anaerobes and of the B. cof. A diet of sour milk
reduces the ethereal sulphates of the urine more than does a
diet of sweet milk.

The Large Intestine is a Useless Organ.—Since the
large intestine performs no digestive action and absorbs poison,
it follows that it is nat only a useless but an injurious organ.
Medicine may possibly perfect methods of treatment and diets
to prevent it from doing harm ; surgery perhaps will reach this
end more quickly. However that may be, certainly no one
has ever proposed that the large intestine should be removed
from every human being ; but it is not the less true that from
the zoological point of view it is a useless inheritance left to us
by our animal ancestors.1

1 We must consider it as a useless inheritance from our ancestors during
evolution, though they no doubt derived some benefit from it. Compara-
tive anatomy teaches us that, among all the vertebrates, it is only the

mammals which are provided with a large intestine properly speaking.
Birds, reptiles, and other lower vertebrates do not possess one... . .

MICROBES IN THE HUMAN BODY = 45

Examples of human beings who have lived after the partial
or total removal of the large intestine or after the suppression
of its function are far from being rare. A patient of Korte
survived eight operations on his intestine ; another of Wiesinger
remained in a satisfactory condition after losing both his trans-
verse and descending colon. Another of Ciechomski lived for
thirty years with a fistula which had put the large intestine out
of use ; he married and had children. Tavel’s patient (studied
by MacFadyean, Nencki, and Sieber), aged sixty-two, had to
digest her food for six months without the large intestine, yet
she remained well. A patient of Mauclaire has been living
for years in fair health with her large intestine out of use.
The histories of these patients are to be found in the most
respectable surgical journals.

An English surgeon, Lane, recently published an account of
thirty-nine removals of the large intestine, partial or complete ;
he lost nine out of the number, z.e., 23 per cent. Many of the
survivors, formerly subject to enteritis and severe neurasthenia,
had never been able to enjoy life till it had become thus
“simple.” The mortality is still too great for this operation
to be anything but an exceptional procedure. But surgical
technique is progressing every day. What surgeon would

It is probable that the large intestine acted as a reservoir for the waste
products of digestion. The mammals having to run fast to escape from
their enemies or to capture their prey, were inconvenienced by the
necessity of stopping to empty their bowels. A large intestine under
these conditions would be of the greatest use. Thus we see that those
mammals which run fastest, such as the horse and the hare, possess the
most highly-developed large intestine and cecum. It is remarkable that,
among birds, the runners such as the ostriches and cassowaries have
similarly acquired a large intestine and possess ceca more developed than
all the other feathered creatures. It is thus the demand made by the
struggle for life which has led to the formation of the large intestine
among the vertebrates. The high development of this organ as a
reservoir for the solid excreta has produced in its turn the development of a
very rich bacterial flora... . . Man having no necessity for the large
intestine and its flora either for the digestion of cellulose or for prolonged
retention of the residues of digestion derives no advantage whatever from its
possession. On the contrary he suffers very numerous inconveniences.
vie a 8 It is easy to conceive how an organ thus become useless con-
tributes to the shortening of life.” Metchnikoff, Wilde Lecture, Manchester
Memoirs, 1901.

46 MICROBES AND TOXINS

have prophesied fifty years ago that the removal of the
appendix would become as simple an operation as the
extraction of a tooth?

From the scientific point of view it is quite legitimate to
conclude that in a mammal so advanced in evolution as is
civilised man, both life and digestion are possible in the absence
of a large intestine.

Is the Intestine Permeable for Microbes ?—There
exist in the massive flora of the intestine, bacteria both actually
and potentially pathogenic. After a wound which penetrates
the intestinal wall, the bacteria, which were harmless while in
the alimentary canal, invade the general peritoneum and cause
a fatal peritonitis. Immediately after death the intestinal
bacteria invade all the tissues and begin the work of putre-
faction. To prevent this happening during the lifetime of the
animal it is obviously necessary for the intestine to be
impermeable, and this impermeability must be due to the
living healthy mucous membrane.

This opposing force is sometimes thrown out of action.
There are certain diseases which can only be explained by
supposing that the pathogenic bacterium has passed from the
intestine into the blood and into the organs. In hog-cholera,
which is caused by a virus still unknown, the lesions of the
lung contain a microbe which develops under cover of the
true virus and forms a regular complication in this disease.
This microbe is a normal inhabitant of the intestine of healthy
pigs. In all infectious diseases in which infection occurs by
ingestion, and which are not exclusively confinéd to the
alimentary canal, it is necessary to suppose that the microbe
passes from the intestine into the blood either directly by the
capillary vessels of the mucous membrane or by the more
roundabout way of the mesenteric lymphatic glands. Typhoid
fever, for example, is mainly a disease of the intestine, an
enteritis, but during the whole course of the fever the bacillus
circulates in the blood, and occasionally forms colonies in
distant organs such as the lungs and the bones.

A very simple explanation suggests itself: the intestinal

MICROBES IN THE HUMAN BODY 47

barrier has been pierced by a little wound permitting the
passage of the bacteria. This happened in the case of Pasteur’s
sheep, which took anthrax when he fed them with anthrax
spores mixed with minute splinters. But this explanation does
not hold for all cases. Numerous facts exist which establish
the permeability of the healthy mucous membrane.

The importance of the problem was perceived when the
conditions of infection with tubercle began to be discussed.
Behring thought, in agreement with Chauveau, that the bacillus
penetrates into the body of man and cattle, not only by the
respiratory passages, but also through the digestive tract. This
idea was suggested to him by the predominance of mesenteric
lesions in infantile tuberculosis, compared with the predomin-
ance of pulmonary lesions in the adult; he thought that
tuberculosis always began in childhood in the intestine and
did not invade the lung until maturity. The intestine of an
adult, he maintained, does not permit the passage of the
bacteria as does that of a child, the reason being that in the
infant) as in general among new born animals, the intestinal
is not coated with the continuous and perfect covering
evelops later; the covering is discontinuous, inter-
rupted and open to the passage of bacteria. No intestinal
lesion ¢an be found because none exists: there has been
penetration without violence.
ring’s idea has been developed to such an extent that
some observers declare that invasion by the intestine is more
fréquent than invasion by the lung ; and during the search for
roofs and comparisons they have studied closely the penetra-
tion of the intestine not only by the bacillus of tuberculosis,
but by various other bacteria, pathogenic and non-pathogenic,
and even by inert dust particles. Pulmonary anthracosis, the
disease or rather the histological condition which consists in the
impregnation of the lungs by carbon particles from the air of
mines and factories, has formed the field of battle for the
partisans and opponents of Behring’s idea, and a multitude of
experiments have resulted, not without instructive consequences.

Dust particles can reach the lung by passing through the

48 MICROBES AND ‘TOXINS

intestine, but that is not the rule and can only be produced by
administering the dust in large quantities in rapid succession :
these are artificial and exceptional, not natural, conditions. It
is the same in the case of bacteria. In an animal in perfect
health they do not penetrate the intestinal wall, but they can
be made to do so under the influence of fasting and fatigue
(experiments of Ficker). One may say that their passage is
the first and a very early sign of an agonal phenomenon, an
anticipation of that exodus of bacteria which occurs immed-
iately after death. :

We must not forget, however, that, according to the state-
ments of Porcher and Desoubry, bacteria pass from the
intestine into the blood each time the animal digests, and that
to get sterile sera it is necessary to bleed anti-diphtheria and
anti-tetanus horses only during the intervals between digestion.

The question is less simple than it looks, because the mucous
membrane is quite as active as, if not more so than, the
bacteria. Not only is it built up of active living cells, but there
are continually traversing it other living motile cells, the
leucocytes, which play an active part in the phenomena of
digestion and absorption, of defence and of resistance.

The cellular mechanism of defence does not invalidate the
conclusion of this chapter. Being useless for digestion, and a
constant cause of putrefaction, the intestinal flora is a perman-
ent source of danger for the body. The intestinal flora
represents in our interior the external world with its unregulated
fermentations only limited by their mutual competition. It
represents in the interior of our bodies the innumerable legions
of bacteria in nature, which find their pabulum and their prey
in fermentable material of every kind, and which are as eager
to attack the proteins and carbohydrates of our living tissues
as to attack the sugar and the casein of fermenting milk. The
power of resistance of the body is counterbalanced by its
sensitivencss towards the poisons which are being continually
secreted, and which are capable at least of accelerating the
approach of old age if not of directly causing death.

Researches on the intestinal flora are bound to take a

MICROBES IN THE HUMAN BODY 49

greater and greater place in experimental medicine. With the
help of their results microbiology will then attack the problem
of those chronic diseases, diseases of nutrition, such as gout,
rheumatism, and diabetes. Finally the discovery of the so-
called invisible viruses proves that the field of action of bacteria
is much wider than had been suspected and that there may
well exist legions of active bacteria in situations where none
have yet been seen.

CHAPTER IIT
FORM AND STRUCTURE OF MICROBES

Animal and vegetable microbes—Protozoa— Moulds and bacteria.

Morphology of bacteria—Membrane ; vibratile cilia, capsules, Multiplica-
tion, Spores.

Pleomorphism -of bacteria—The place of bacteria in a general classification.

The nucleus in bacteria: the diffuse nucleus.

Reproduction and the possession of sex among microbes.

Chemical composition.

“MICROBES” is the name given to those living beings which
can only be seen by the aid of the microscope. They are the
simplest organisms found in nature. Some belong to the
animal kingdom, others to the vegetable. The bacillus of
tuberculosis and the typhoid bacillus belong to the class of
bacteria, and the bacteria are plants. The trypanosoma of
sleeping sickness is an infusorian, ¢.¢., an animal.

Whether animal or vegetable, almost all microbes consist of
a single cell. When there are several cells, they are practically
identical individual cells in juxtaposition.

The tissues and organs are systems of cells; microbes
possess neither tissues nor organs.

Like every cell a microbe possesses a nucleus and proto-
plasm.

The vegetable cell is enveloped by a membrane whose
composition is not well known, and even the motile ones are
more rigid than the animal microbes.

Microbes are of all living creatures the most widespread.
They exist wherever there is organic matter.

Protozoa.—In a hay infusion—a handful of hay in a
50

FORM AND STRUCTURE OF MICROBES 51

vessel of water—one can see under the microscope motile
creatures, whose surface is furnished with cilia, whiplets or
flagella in vibration ; these are the infusoria, some with flagella,
others with cilia, Sleeping sickness is caused by a ¢rypanosoma,
a flagellate protozoon found at certain times in the blood,
in the lymphatic glands of the neck and in the cerebro-spinal
fluid. These protozoa resemble minute flattened worms in
the interior of which there are two nuclei, one large, one
small ; on one side there is an undulating membrane bordered
by the flagellum, which is continued beyond the anterior end.

Fic. 15.—Trypanosomes and blood corpuscles.

The trypanosomes multiply by longitudinal division beginning
with the nucleolus and continuing through the flagellum, ‘the
large nucleus, and the protoplasm. There is a possibility that
sexual reproduction exists.

There is often found in the intestine of rabbits an animal
parasite which passes through a complicated series of different
forms; it possesses a cycle of evolution, one portion in the
rabbit, one portion outside. This parasite is a coccidium
belonging to the class of sporozoa. ‘The microbe of malaria
is a parasite of the blood corpuscles, a heematozoon, which has
for long been regarded as related to the coccidia, but which

E 2

52 MICROBES AND TOXINS

possesses certain characters, inclining one rather to derive it from
the flagellates. It too passes through a cycle of development,
one stage being in the blood of man, the other in the body of
a mosquito of the genus Anopheles. According to the stage
of development, the hzmatozoa of Laveran have both asexual
and sexual reproduction, the latter occurring in the body of
the mosquito.

It cannot be too often insisted on that no protozoon is
properly known until all its cycle of development has been
investigated and settled.

Moulds.—A slice of bread or a piece of lemon left to
itself under suitable conditions of temperature and moisture
is soon covered with a fine growth of a whitish or
greenish tint; this is formed by the moulds, lower fungi
belonging to the vegetable kingdom. Under the microscope a
particle of this growth, teased in a drop of water, shows a
felt-work of tubes or filaments, sometimes segmented at
intervals, sometimes not ; this felt-work is the mycelium. From
the mycelium little stalks stand up vertically bearing a head
which itself carries little granules resembling fruit. In the
Mucors the head is a spherical sac stuffed full of these
granules. In Penici/um the head consists of ramifications
or digitations dividing in their turn, the last sections pinching
off little pieces, the conédia; the whole resembles a little brush.
In Aspergillus the fructification resembles the flower of an
onion.

A fragment of mycelium put into a suitable medium
reproduces the mycelium. The conidia can do the same.
In the case of the mucorines two cells can join, fuse, and
produce an “egg”; often these two cells are different, and
the reproduction may be regarded as sexual. If mucor is
grown at the bottom of a sugary fluid it no longer forms an
abundant mycelium; on the contrary there can be found
nothing but short pieces, round or ovoid, which reproduce
themselves by budding like yeasts. But any of these pieces
put in a sugar-containing medium in contact with the air,
forms a mycelium on the surface. This transformation of a

FORM AND STRUCTURE OF MICROBES 53

Fic. 17.—Aspergitlus.

Fig. 18.—d/ucor taking on the yeast form Fic. 19.—Aucor.
when grown at the bottom of a sugar solution.

54 MICROBES AND TOXINS

mycelium into a yeast-like form, which also produces fermenta-
tion like a.true yeast, was discovered by Pasteur, who thought
that in the course of ages the yeasts had been evolved
from moulds which had become adapted to the fermentative
life.

Fic. 20.—The yeast of beer: high Fic. 21.—Spindle-shaped
or tumbling yeast. yeast.

~ Fic. 22.—The yeast of Fic. 23.—The yeast of beer: low
wine. or heavy yeast.

Yeasts.—Yeasts are fungi of the Blastomycete group.
They consist of round or elliptical cells which multiply as a
rule by budding; some multiply by equal division into two
parts. Yeasts may furnish spores, which number 2 to 10,
and may remain enclosed in the mother cell until by the
bursting of this latter their germination becomes possible.

FORM AND STRUCTURE OF MICROBES 55

Bacteria.—The term “microbes” in current language
most frequently signifies bacteria. The bacteria belong to the
lower plants, are unicellular, contain no chlorophyll, and are
almost all incapable of taking up carbon from the carbonic
acid of the air. They are incapable of life except in the
presence of ready made organic matter, and are in consequence
confined either to a saprophytic existence (moulds on a fruit)
or to a parasitic (as in the case of the typhoid bacillus in the
intestine). Scattered as they are throughout nature the
bacteria are the chief agents in the decompositions and re-
combinations of organic and living matter.

The usual classification of bacteria depends on their external
form and is merely provisional. The round bacteria are called
micrococci or cocci. The
streptococci are, as Pas- 2 4 5 6
teur described them, like * © i- d. 8 A |
rosaries or necklaces ; the

staphylococci are like g y >

bunches of grapes. <Ac- ut 4! 7 3 @o

cording as the micrococci Ag 4

in their multiplication _ ane .

ditie iw two ae times Fig. Ny aaa cs bacterial forms. re
occi.—2. Diplococci.—3. Strepto-

planes in space, they ap- cocci.—4. Staphylococci.—5. ‘Sar-

pear arranged in the mul- $8. Becta, 7. Biel

berry form or in the form chzetes.—-11. Clostridium.

of a sarcina or woolpack.

The bacilli are long bacteria, the ends of which are sharply

cut; the dacterium, properly speaking, has the ends rounded

like a spindle or shuttle. The curved bacteria are known as

vibrios, spirilla, and spirocheetes.

The bacterial cell is clothed by a membrane and is not
naked like an amceba. It is this sheath, resistant and elastic
up to a certain point, which permits a corkscrew spirillum to
maintain its form in spite of its movements, and the motile
and flexible bacteria to recover their shape after distortion.
The membrane takes on colours different from the protoplasm.
It is not of the same composition in all the bacteria. Some-

56 MICROBES AND TOXINS

times it is of cellulose, sometimes it resembles chitin, some-
times it is impregnated with fats or waxes like the bacillus
tuberculosis. Inthe membrane of certain bacteria iron and
silicon have been

se / found. Sometimes
‘S ) in old cultures

A hy 4 one can see mem-
mm branes without

Typhoid Vibrio of Tetanus contents which re-
Bacillus Cholera Bacillus semble empty
Fic. 25.—Flagella of bacteria. sheaths.

Certain bacteria
possess besides Brownian movement an independent motility ;
they can be seen performing long journeys, pirouetting
on their own axis. It has been calculated that the cholera
vibrio can travel 18 centimetres in the hour, which is
equivalent to ten or fifteen times its length in a second.
This motility is due to cilia or flagella analogous to those
of the vibratile epithelial cells or the ciliated infusorians. They
are often longer than the bacterium which carries them. In
old cultures they become detached and scattered through the
medium. It is doubtful whether they are fine expansions of
cellular protoplasm which have passed through pores in the
enveloping membrane or whether they are expansions
of this membrane itself; whether they are not mere
propelling organs but rather tentacles of some sort which
increase the surface of the bacterium and thus play a réle in
nutrition, especially in the young bacteria. No definite
reply can be given to these questions. According to
G. de Rossi’s experiments on agglutination and immunisation
the cilia behave like protoplasm. Their action ceases at very
low temperatures and above 50°C. When a bacterium
provided with cilia only at one end (certain vibrios) is
attracted towards a definite substance (positive chemiotaxis),
the cilium or cilia take the front position and mark the head-
end of the bacterium—just as the flagellum does in the case of
the trypanosomes.

FORM AND STRUCTURE OF MICROBES 57

Certain bacteria such as the pneumococcus and the
pneumobacillus are surrounded by a sort of case or capsule.
One capsule may enclose several bacteria: when a large mass
of bacteria is included in one enormous capsule what is called
a zooglea is formed. Bacteria which appear encapsulated in
the albuminous fluids of the infected body do not always
possess them in artificial culture. The capsule appears to be
a defensive secretion. The anthrax bacillus defends itself
against the toxic action of the serum of the rat by surrounding
itself with a thick sheath made up of a sort of mucus which
fixes and renders harmless the toxin. In the blood of lizards,
which are very resistant to anthrax, the bacillus surrounds
itself with a thick mucous envelope. The streptococci
occasionally acquire in the body of resistant guinea-pigs a

Fic. 26.—Streptococci surrounded by a sheath of mucus.

similar defensive covering. The streptococci of chronic
infections (otitis and nasal infections) are frequently encap-
sulated. The sarcinze are not usually pathogenic but encap-
sulated sarcingee have been described which were pathogenic
for laboratory animals.

Bacteria multiply by transverse divisions, Pasteuria ramosa,
described by Metchnikoff, being an exception. One bacterium
put on a suitable medium produces after a certain time two,
these two producing four, and so on ; it is easy to imagine to
what enormous numbers this geometrical progression may lead.
In nature and in artificial culture media, multiplication is

58 MICROBES AND TOXINS

limited by the quantity of available nourishment and by the
accumulation of the products of excretions ; the cultures end
by poisoning themselves. But even in a drop of culture
medium, the energy of multiplications is enormous. One
filament of the Bacil/us ramosus studied by Marshall Ward
doubled its length in thirty-five minutes; in twelve hours a
single bacillus produced four millions, and one piece of one-
hundredth of a millimetre in length produced in twelve hours
the equivalent of a thread of 4o metres. Pasteur followed
under the, microscope the growth of the yeast of wine in grape
juice at13° C. One globule produced ro millions in twenty-
four hours when nothing intervened to limit its development.
It is easy to understand now how the infinitely minute grows
to form a mass and brings into play enormous forces.
Spores.— Certain bacteria producespores, thespore appearing
as a shining point in the filament ; the protoplasm of* the cell

sp diminishes as the
young spore increases, as
otf bacterium if sporulations

were a condensa-

20 O tion of the living

matter. Later the
remains of the sporé _ )acillus disappears

3 000 and the spore is
Of mR iN free. ee

birth of the Being en-
young bacteriam closed in a resist-
; ae ing sheath the
Fic. 27.—Various types of germination of spores.
1. The spore germinates by growth in all SPOre resembles a
dimensions. 2. Germination by « sort of seed, capable of
terminal budding. 3. The spore germinating
by a sort of lateral budding. (After De Bary prolonged DEC
and Prazmowski.) servation and of

sprouting into a
new bacillus when the conditions become favourable. It is
by means of their spores that the bacilli of tetanus and of
anthrax persist so tenaciously in nature. The anthrax bacillus
is killed by heating to 60° C., the spore not till after three
minutes’ boiling at 190° C,

FORM AND STRUCTURE OF MICROBES 59

The spores which develop in the interior of bacteria are
endospores. The name of arthrospores has been given to those
rounded granules which certain bacteria produce by segmenta-
tion, by a sort of pinching off, or perhaps by a shrinking of
their substance ; for example the cholera vibrio, which does not
possess endospores.

The properties of an anthrax bacillus are fixed, and
transmitted to the new generation, by a sort of heredity, by
means of the spore.

It is not impossible that some sexual differentiation exists in
the sporulating bacteria.

Pleomorphism of Bacteria.—The bacterial forms have
not the immutability of crystals, nor even the relative stability
of species among the higher plants and animals. They are
variable to an extent that absolutely confounds the bacteriolo-
gist. Ina pure culture such a diversity of forms may appear,
forms long and short, rounded and thread-like, that the culture

Fic. 28.—Involution forms: 1. Lactic bacilli.—2. Bacillus subtilis.—
3 Anthrax bacilli.

might be thought impure. The bacillus of diphtheria trans-
ferred from a natural to an artificial medium, or from one
artificial medium to another, appears short or long according
to circumstances and gives in old cultures forms which
resemble staphylococci. One bacterium exists so variable
that it has been called Profeus, the protean bacillus. The
Bacillus prodigiosus, the bacterium which gives red colonies

60 MICROBES AND TOXINS

and thus produces the miracle of the bleeding host, multiplies
very quickly in alkaline media, producing coccal forms ; these
cocci, reinoculated on a faintly acid medium, multiply less
rapidly and produce bacillary forms. Bacteria are then
multiform or pleomorphic organisms.

In old cultures or in cultures on unfavourable media, forms
are seen diverging from the typical: a micrococcus may give
relatively enormous globules ; a bacillus may give forms racket-
shaped, club-shaped, pear-shaped, &c. A bacillus which
ordinarily never branches may show branching forms. These
abnormal shapes are called involution forms, again examples of
pleomorphism.

In the early days of bacteriology this pleomorphism was
copiously discussed. Nageli held that there were no bacterial
species, but that the bacteria formed an immense group in
which neither species nor families nor genera were to be
distinguished. A coccus might give, under suitable conditions,
a rod form or a spirillum and the function might be as variable
as the form. He would not admit that a given fermentation
always corresponded to a definite bacterial form, but held that
the same microbe might be in its turn an acetic ferment, a
lactic ferment, or a butyric ferment. For him bacterial speci-
ficity did not exist. Hence arose the belief that it was easy to
render pathogenic an inoffensive bacterium like the B. sudtilis
of hay infusions.

The botanist Cohn exaggerated the stability of bacterial
species as much as Nageli exaggerated their pleomorphism, and
bacteriologist-physicians, in their desire to discover for each
infectious disease a specific agent, followed Cohn’s idea. It
was obviously impossible to hold precise opinions about the
pathogenic bacteria if there existed no stability in their forms.
R. Koch accused the ‘‘pleomorphists” of making their
observations on impure cultures or on infusions containing
already a very numerous flora; it was not pleomorphism that
was present, but confusion.

Another observer, Kurth, however, demonstrated the
pleomorphism of the Bacterium Zopfii in pure cultures ; there-

FORM AND STRUCTURE OF MICROBES 61

upon the partisans of Cohn and Koch declared that though the
saprophytic species might lack stability, the pathogenic species
were stable. In their eyes the bacillus of anthrax was always a
bacillus. But it had to be recognised later that pleomorphism
existed even among pathogenic species, for example the cholera
vibrio and the bacterium of fowl-cholera.

Finally the stability school maintained the necessity of
distinguishing between the constancy of the mere shape and
that of the species—a cholera vibrio may modify its form and
yet remain always the vibrio of cholera—the tadpole of the
green frog does not resemble an adult green frog, yet it belongs
without any doubt to the species green frog. But it is certain
that it is not only the form, but also the species gua
species which is variable amongst the bacteria, and this
variability is even more characteristic of their physiological
properties than of their shape. ‘The bacillus of tuberculosis,
amongst others, is susceptible of very divergent adaptation.

Bacterial species exist, but they are all of the kind called in
the language of the science of classification ‘“7d/-defined.”
“That the methodical classifier should be frequently embarrassed
in his attempts to set up his artificial barriers, is very far indeed
from being surprising. The world was not created for the
special pleasure of descriptive botanists, and it is equally inter-
esting to science to possess the demonstration that a classification
is impossible as to have established a classification, had such
been possible” (E. Duclaux).

In any case their faculty of adaptation and their power of
variation sufficiently explain the history of bacteria. The
pathogenic species must have come from saprophytic species
by adapting themselves to certain animal bodies. The same
virus, adapting itself in the course of ages to the human species
and to the cow, has produced the two diseases small-pox and
cow-pox, and this adaptation has become the principle of a
wonderful prophylactic procedure. It was also by causing
variations in the virus by physical and chemical action that
Pasteur first prepared vaccines.

More recently search has been made among bacteria for

62 MICROBES AND TOXINS

examples of mutation resembling those studied by the botanist
Hugo de Vries. In a pure culture of bacillus coli Massini
found that certain individuals suddenly acquired the power of
fermenting lactose, and that this property was transmitted to
their descendants. The attenuation of the anthrax vaccines of
Pasteur is an example of a property acquired and transmitted
hereditarily by the spore.

Precisely because of this pleomorphism and plasticity of
bacteria, one must take care not to imagine mutations too
frequently. Occasionally modifications may be got which are
difficult to fix and which do not constitute true races. The
bacillus prodigiosus has been cultivated at 37°5° C., with-
out producing its red colour, for a series of generations ; but
after the thirty-fifth passage, when put again at 22° C., it re-
produced its pigment. Besides, mutation is not to be defined
simply as a sudden variation. There must be transmission by
sexual reproduction in addition. One could not speak of
mutation except in cases such as those of the anthrax bacillus,
where there has been suspected a sort of sexuality in the spore
forms. It is none the less true that the life of bacteria presents
numerous facts in accordance with the Darwinian laws.
“ Bacteriology, like all branches of biology,’ has gained by the
application of the theory of evolution, and has made a fair
return by supplying the Darwinian theory with a striking
confirmation ” (Metchnikoff).

The Place of Bacteria in Classification.—
Leeuwenhoek, describing in 1683 in his Arcana naturae
detecta the micro-organisms of the mouth, which he observed
through lenses polished by himself, represented them as
animalcule. In 1838 Ehrenberg assigned them a place in his
work on the Infusoria, classing them with the Vibrios, which
he regarded as animals. But their evolution and their activities
indicate that the bacteria are lower plants. ,

Are they to be classed among the fungi (moulds and yeasts)
or among the alge? In the absence of chlorophyll, bacteria
resemble fungi, and they have long been called Schizomycetes,
z.¢., fungi multiplying by transverse division. There are

FORM AND STRUCTURE OF MICROBES 63

bacteria, the Streptothriceze, which, by their filamentous and
branching appearance, form a link between the bacteria and
the simplest fungi. On the other hand, there are yeasts which
resemble bacteria in producing endospores and multiplying by
division instead of by budding.

It is, however, with certain algze, the Cyanophycez or blue
algze that the relationship is most marked. The Cyanophycez
reproduce by division, presenting different shapes, long,

round, and curved, resembling cocci, coccobacilli and bacilli.
They are pleo-

morphic like the
bacteria. They
possess chloro-
phyll in contrast
to the bacteria,
but the pigment
which the Cyano-
phyceze possess
in addition to =
their chloro- Fic. 29.—Streptothrix. Branching bacteria.
phyll, the phyco-

chrome (most often greenish-blue, soluble in water and
diffused throughout the ‘cell) is not without some analogy to
the pigment of certain bacteria. Certain algee clump together
in mucilaginous sheaths resembling the zooglcea of certain
bacteria. The Cyanophycez do not form endospores but have
arthrospores like some bacteria. There are species like
Beggiatoa, in which it is far from certain whether they should
be classed with the bacteria or with the Cyanophycez. These
analogies are so striking that a family, the Bacteriacee, has been
made for the algze which border on the Cyanophycee.

But in view of the differences which exist between these two
families and the similarities which link up the bacteria and the
fungi (moulds and yeasts), certain authorities make of the
bacteria a heteroclitous group where there are brought together
representatives more or less altered or degenerated of various
lower plants. There exist no doubt bacterio-algz ; there are

64 MICROBES AND TOXINS

also bacterial moulds and bacterial yeasts; there are even
protozoal bacteria, 7.e., animal bacteria, the Spirochzetes.

The conclusion then is that bacteria are not the original
forms from which the others were derived but that on the
contrary the bacteria are derived from the more definitely
characterised fungi and alge, the specific characters having
become obliterated by the parasitic habit. The branching
forms rarely observed in the bacillus tuberculosis, the endo-
spores of the anthrax bacillus, the arthrospores of the
Streptothrices are all atavistic stigmata.

The Nucleus of Bacteria.—The usual nucleus, consisting
of a distinct mass in the body of the protoplasm, does not exist
in the bacterial cell.

A cell without a nucleus ! Several observers (A. Fischer,
Migula, Massart) do not shrink from such a paradoxical
statement. They can see no trace of a nucleus ; what others
have taken for it, large or small, is only, in their opinion, an
empty space or a vacuole, taking part in the cell-division and
capable of dispersion into smaller vacuoles. Thé scattered
granulations in the protoplasm are not grains of chromatin,
for they have none of its reactions. They are either products
of metabolism or reserve materials. They have been called
metachromatic bodies, because when stained with blue or violet
stains they take on a different, reddish tint.

Such observations are best made on certain large bacteria
found in nature. It is remarkable that the dacel/us asterosporus,
which was the one selected by Migula to demonstrate the
absence of the nucleus, has been employed by others to
demonstrate the presence of one or even several well-defined
nuclei. Able cytologists complain that the supporters of the
nucleus have been deceived by appearances ; that they have
taken for chromatic masses the transverse segmentations which
appear in certain large bacteria at the moment of division, and
that they have even chosen for their demonstration bacteria
which are not true bacteria; the Bacterium gammari of
Vejdowsky is said to be a fungus approaching the yeasts and
multiplying by division, while another bacterium studied by

FORM AND STRUCTURE OF MICROBES 65

the same author in the digestive tube of an annelid (Bryodrilus)
ought rather to be considered as a mould.

To-day one can agree neither with those who affirm that
there is no nucleus nor with those who describe a well-defined
nucleus. In reality, the bacteria possess a diffuse nucleus.
Instead of forming a mass with sharp contours, the chromatin
is scattered through the protoplasm in the form of granules
quite distinct from the metachromatic granules.

Long ago Weigert maintained that the nucleus is, as it were,
melted or dissolved in the cytoplasm, for it is precisely the
nuclear stains which stain the bacteria best; Biitschli con-
sidered that the cytoplasm of bacteria is reduced to a thin
layer lying in apposition with the membrane, and that the
bacterium, far from lacking a nucleus, is in reality almost all
nucleus. Further, what purpose would a large quantity of
cytoplasm fulfil, cytoplasm whose special function is nutritive ?
Bacteria are parasitic, and absorb their nourishment ready
prepared. Their chief business is to multiply, to increase in
numbers ; it is quite natural for them, therefore, to possess an
enormous nucleus like the spermatozoa and the embryonic
cells, so much so that one might even say that bacteria are
“ free nuclei.”

Schaudinn has furnished a brilliant support to these ideas
by means of his observations on the largest known bacillus, the
Bacillus Biitschiit, found by him in the intestine of cockroaches.
The chromatin granules are scattered through the protoplasm ;
to produce the two spores they come together, and the
chromatin condenses at the two ends. The condition of a
diffuse nucleus is no less evident in the B. maximus buccalis of
Swellengrebel.

The diffuse nucleus is not confined to bacteria. There are
several protozoa which normally possess a well-defined nucleus,
but at certain moments in development or in certain conditions
of nutrition this may be seen changing into a diffuse nucleus.
The diffused nuclei have been called by R. Hertwig chromidia.
Several varieties exist, and the nucleus of B. Biitschiiz, like the
nuclei of bacteria, in general should be considered as consisting

F

66 MICROBES AND TOXINS

of chromidia. ' The nuclei of the Cyanophycez have been the
subject of similar disputes; in these inferior alge also the
existence of a chromidial nucleus is admitted, quite distinct
from the metachromatic bodies.

Fic. 30.—Bactllus maximus buccalis. (After Swellengrebel.)

The spiral filament represents a system of chromidia in process of division
(4 and 5).

Reproduction and Sex.—A species is said to possess
sex when fertilisation occurs by the fusion of two nuclei
differentiated into male and female.

In the hematozoa of malaria the sexual act takes place
in the body of the mosquito by the conjunction of the
flagellum and the female cell, and at this point the sexual cycle
begins.

Since Schaudinn’s discovery of the trypanosome forms in
the cycle of certain haematozoa closely related to those of
malaria, one must admit the existence of male and female
‘forms in the case of trypanosomes. Besides the asexual
multiplication by longitudinal division there would thus exist
among trypanosomes true sexual reproduction.

Among the fungi the ovum, the complete reproductive form,

FORM AND STRUCTURE OF MICROBES 67

results from the fusion of protoplasm as well as nucleus of
two differentiated cells, the gametes, one male, the other
female. Heterogamy exists when the male gamete is manifestly

Fic. 31.—Successive stages of Fic. 32.—Zygusaccharomyces yeast.
conjugation in a Zygosac- (a) Vegetative cells : (4) Asci.
charomycete. (After Barker. }

different from the female—isogamy when they seem identical.
The ascus and the basidium are modes of sexual reproduction.

In the yeasts, examples of conjugation are not lacking.
In Zygosaccharomyces before the intracellular formation of the
spores, two neighbouring cells put out each a little bud ; the
‘two buds join and along the little canal thus formed fusion of
the nuclei takes place (according to Barker). The two cells

Fic. 33.—Nuclear fusions and spore formation in Schzzosaccharomyces
octosporus. (After Guilliermond.)

remain united forming a dumb-bell-shaped ascus. The sime

“process occurs in certain schizosaccharomycetes, where the

shape of the ascus also recalls its origin from two cells. Since
F 2

68 MICROBES AND TOXINS

the cells which join belong to the same strain, and to the same
generation or to two generations very near each other, this
conjugation in the yeasts is an example of isogamy.

In the bacteria it has long been observed that multiplication
by transverse division, 7.¢., asexual reproduction, appeared to
be an absolute rule; but Forster has described a species of
conjugation among the sulphur-forming bacteria; they come
together in twos or threes, put out little processes, unite and
finally separate; this is a conjugation between individuals
which are doubtless differentiated, and resembles somewhat -
the well-known conjugation of infusoria.

The observations of Schaudinn on B. Biitschid have demon-
strated the existence of conjugation also among the bacteria—
at least among those which produce endospores. The ordinary
multiplication by transverse division exists in B. Biitschliz, but
in the individual about to form spores, other peculiar
phenomena are seen: the individual begins by dividing into
two, but the septum disappears almost as soon as it is formed
and is dissolved, the two cells which had just been divided
by the septum melting again into a single cell; an exchange
of chromatin takes place between them, and finally almost all
the chromatic granules collect at the two poles to form two
spores. Short as is the time that the septum remains, there
have, nevertheless, existed, during that moment, two cells
which have conjugated: and this is, according to Schaudinn,
a rudimentary sexual act. An analogous conjugation occurs
with B. Sporenema.

It is really an auéogamy or fusion between two elements of
the same cell, or to be more precise it is a conjugation between
two daughter-cells of the same mother-cell, and is hence called
paedogamy.

Numerous cases of autogamy have been described among
the protozoa. Autogamy is the opposite of the amphigamy
which occurs when there is conjugation of two individals of
well-differentiated sex.

What relations are to be established between these two
methods of fertilisation? Is autogamy the simple primitive

FORM AND STRUCTURE OF MICROBES 69

type from which the other is derived, or is it simply a
degenerated degraded form? As far as bacteria are concerned,

Fic. 34.—Bacillus Biitschiit. (After Schaudinn.) 1 to 6. stages of trans-
verse division without sporulation.—7 to 9. Rudimentary sexual
process.—1I0 to 14. Spore formation.—15 to 16. Spore germination.

Schaudinn regarded the conjugation which exists in B
Biitschiti as an evidence of sex, though rudimentary, degraded
and residual. Without absolutely excluding the possibility of

70 MICROBES AND TOXINS

primitive autogamy the Schaudinn school considers autogamy
in general as a regressive type.

The truth is that sexual differentiation of the gametes is
universal in nature even in the lower orders. Researches on
the Protista permit the recognition of sexual dimorphism in
every nucleus, 7.e., there is in every nucleus a double substance
and a double function, one specially nutritive, the female, the
other specially reproductive, the male. Every protozoon cell
is, to some extent, hermaphrodite, but with one or other
element predominating. Even in autogamy, the elements
which fuse are differentiated.

Sex is thus universal in nature, and it is from this nant of
view that Schaudinn had to consider the rudimentary con-
jugation of B. Bitschiid as a degenerate type. As with the
structure of the nucleus, the fact of autogamy indicates again
that bacteria belong to an order degraded by the parasitic
habit.

Chemical Composition.—The chemical composition
varies with the species studied, and in the same species with
the age and the nutriment. Duclaux observed in the cells of
a fifteen-year-old yeast that the proportion of fatty material,
instead of being 5 per cent. as in young yeasts, had risen to
20-30 or even 52 per cent., the proportion of nitrogen varying
from 1 to 4.

Bacterial substances contain proteids, fats, and sugars, but
as there are so many proteids and so many sugars, the figures
can only give a general indication. The cell is a “ well stocked
laboratory,” and one which is always in activity.

The organic materials isolated from bacterial bodies of very
diverse species are the following :—

Coagulable albumins in the expressed juices ; globulins ; a protamine (from
B. tuberculosis) ; proteoses (by peptic digestion).

Glycoproteids. Phosphoproteids and their derivatives: nucleins, nucleic
acids, xanthin bases, and pyridin bases.

A substance resembling chitin or keratin (B. tuberculosis).

Products of hydrolysis of proteins : amino-acids and hexone bases.

Carbohydrates : Cellulose, hemicellulose, and sugars.

Fats and waxes, neutral fats (capsulated bacteria, B. diphtheria, B. tuber-
culosis). Free fatty acids (B. tuberculosis) ; waxes (B. tub.) ; lecithin

FORM AND STRUCTURE OF MICROBES 71

(B. tub., 16 per cent., according to Kressling) ; phosphorized fats ; in
cultures of B. tuberculosis of a certain age the content of fat and wax
varies between 20 and 39 per cent.

Ash : B. tuberculosis 8 per cent., B. coli 8°5 per cent.

Ruppel gives the composition of the Bacillus tuberculosis as
follows :

Tuberculinic acid... we eee cee ee eee = 8°5 per cent.
Nucleoprotamine ... 1. ek cee cee eee tee 245 ogy
INUiGIEOPrOteld: vcs: cage wed sy Gog Gu ae tee 2B 3
Fats@nd Waxes: 4. cs = a a an ven 26% 5
ASH) sie ke BS RD ee ae ee sO ay
Proteids (keratin) 32. ssa see wee tees eve OFZ gy

Most of these substances pass into the extract, which is
known as tuberculin.

For comparison: the human body, taken as a whole, con-
tains 65 to 70 per cent. of water, the plants used for food 60 to
80 per cent., algze go per cent.

Bacteria are specially rich in albuminoids. The moulds
contain more carbohydrate than nitrogenous material, their
outer covering containing cellulose.

Yeasts contain much nuclein; as they grow older the fat
content increases ; their outer covering also is cellulose. The
mineral matter consists of phosphoric acid, potassium, mag-
nesium, sodium, silicon, lime, sulphur, and oxide of iron.
Yeast cells are richer in glycogen than the liver cells of the
rabbit (31 and ro per cent. of the weight of dried yeast).

CHAPTER IV

PHYSIOLOGY OF THE MICROBES

Nutrition—The definition of food material—Nutrition of the Mucedineze—
Raulin’s experiments—Bearing of these experiments—Nutrition of
yeasts and bacteria—Importance of the chemical constitution of the
medium—The bacterium of sorbose—The idea of the ‘ soil’—Respira-
tion—Aerobic and anaerobic life.

The purple bacteria,

Secretion of diastases or enzymes.

Products of the cultures—Products of excretion.

Auto-intoxication of the cultures.

Production of heat—Production of light—Production of pigment.

Action of heat on microbes ; thermophil microbes.

Action of light ; ultra-violet rays.

Physiology of Protozoa: cells possessing a great abundance of diverse
functions—much_ differentiated—Nutiition ; digestion, respiration
Irritability—Reproduction—Parasitism—Adaptations and specificities
—Life cycle and secondary host—Cultures.

THE microbes act in nature as transformers of energy. The
energy which they get from oxygen and from various food
materials they transform into products of excretion, heat,
light, and work, retaining a portion used up in the building of
their own substance. Like the higher creatures they display
anabolism and catabolism. Each one is like a little vortex,
from which the organic matter emerges different from its state
on entry. It is characteristic of living beings to transform the
molecules of their food materials into more complicated
molecules. For example, from sugar they produce cellulose,
from carbohydrates along with the nitrogen of ammonia they
prepare the nitrogenous compounds.

To construct these new arrangements of the molecule, energy
72

PHYSIOLOGY OF THE MICROBES 73

or heat is necessary, and the microbe provides this by burning
up a portion of its food material. Thus, while part of the food
. is raised to a level of higher organisation, another portion, on
the other hand, is reduced to a simpler condition. « For
example, part becomes protein, part carbonic acid and water,
the latter not being incorporated in the living substance.

Similarly, a microbe, like the Aspergillus niger, burns up
with the help of the oxygen of the air the sugar which is
furnished by the food, and this combustion puts at its disposal
a certain number of calories ; one molecule of sugar weighing
160 grams furnishes 673 calories during its transformation into
CO, and H,O. The yeast of beer, which decomposes sugar
less completely, z.¢., into alcohol and carbonic acid, only yields
33 calories. If the yeast had the nutritive requirements of the
aspergillus it would have to use up nearly twenty times more
food. Nutrition of this kind is characteristic of a ferment,
and the fermentative power is greater the more of the food-
stuff the cell is obliged to break down.

Lacking chlorophyll as they do almost universally, the
microbes cannot take up carbon directly from the air as do
the green plants. They demand their food ready made so
that they can destroy it, turning it into carbonic acid and
water. The heat derived from this destruction takes for them
the place of the energy furnished by sunlight to the plants
which contain chlorophyll.

The food of a microbe may then be defined as “every
material from which a given microbe, under the conditions of
the experiment, can take the material necessary for its develop-
ment and the heat necessary to render it independent of solar
energy.” In calculating the total energy coming into play
in the protoplasmic activity a certain amount has to be
accounted for as external heat, and it is even the rule for an
excess to appear as heat, so that there is a rise of temperature
in the medium. Thus a vat at the surface of which acetic
fermentation is going on gets notably hotter, and the same is
the case in grapes undergoing vinous fermentation. But
under certain limited conditions the protoplasm can perfectly

74 MICROBES AND TOXINS

well employ, as part of its food, substances already oxidised.
and incapable in any way of furnishing heat, the conditions
being that these are made to enter into a nutritive compound
in the manufacture of which heat-furnishing transformations
occur in sufficient amount. For example, the nitric bacteria,
as Winogradsky has shown, can take their carbon from
carbonic acid on condition that at the same time they trans-
form nitrous acid into nitric. Similarly, the ferments which
fix nitrogen can take up this gas from the air on condition
that they destroy at the same time by oxidation sugar or some
other hydrocarbon capable of furnishing heat during oxidation.”
(E. Duclaux).

Cellular protoplasm elaborates food by means of the
diastases which it contains, and has peculiar wants and
preferences according to the species. It must therefore be
difficult for a microbe to find the particular nourishment which
suits it best. The artificial cultures of microbes which are
so useful in scientific research and in medicine demand
practically for each microbe those food-stuffs which nourish it
in nature.

Nutrition of the Mucedinee (Raulin’s Experi-
ments).—Raulin succeeded in composing with perfectly pure
sugar and mineral salts an artificial medium more favourable
to the growth of Aspergillus niger than any occurring in
nature. It is thus a cudfvation in the fullest sense of the

word.
Raulin’s Medium.

Water ... aes den ves ase 1500 grams.
@Candy-sugar ace eer ew wt ee Ges ae as
TartariG@acid: xs ase: ese kag hes, see veces ne sia
Ammonium nitrate 00. ee ee 4 ss
ss phosphate .. 0... 0. 0. 4 ©6060 gram.
Potassium carbonate jinn ieide keh ide, few eee CORGOR gg
Magnesium _,, we Ge ee awe Gk as O"AO! gg
Ammonium sulphate die Reus el > eeae “ake age JOP Z GI gs
Zinc 7 vier ge ae awe see O'OF 65
Ferrous 3 Ree ka ae we ee JOTOZ’ yp
Potassium silicate wo Sey See eee Soe as OTOT’ gs

This medium was prepared by a series of trials and
demanded a most admirable patience, for he had to make

.

PHYSIOLOGY OF THE MICROBES 75

modifications in the number and quantity of the constituents,
to compare the weights of the growth obtained, to determine
the temperature and the hygrometric condition of the atmos-
phere, and even to take account of the shape of the culture
flasks, which affected the oxygen supply.

If potassium is cut out from the above formula, the crop of
aspergillus is 25 times less, all the other conditions remaining
the same; if the figure 25 is taken as representing the
measure of utility of potassium, the utility of the other food-
stuff may be represented as follows : nitrogen, 153 ; phosphorus,
182; sulphur, 25; silicon, 1'°4; magnesium, 91; zinc, 10;
iron, 2°7.

If you take the ratio between the weight of one of these
food-constituents and the weight of the crop due to its
presence, you get a number which expresses its specific utility,

2. 22N5
e.g., for zinc eae 560.

This ratio was in several experiments found to be 953.
For nitrogen it is 17; for phosphorus 157; sulphur, 346 ;
potassium, 64; magnesium, 200; iron, 857. It is to be noted
that aspergillus takes up zinc from a medium in which the zinc
is diluted 1 in 50,000.

If 1 in 1,600,000 of silver nitrate is added to the fluid,
aspergillus spores no longer sprout in it: even when simply
poured into a silver vessel the fluid dissolves sufficient silver .
to prevent growth. The plant is thus an indicator so sensitive
as to mark, by its refusal to sprout, such small quantities as
1/240th of sulphate of copper, 1/8,oooth of platinum bichloride,
1/50,o00th of perchloride of mercury.

Zinc has a definite food value but iron acts differently,
namely, by neutralizing a substance which is produced by the
growth and becomes injurious to it, perhaps sulphocyanic acid.

Tartaric acid maintains the acidity of the medium and
prevents bacteria from contaminating the culture, for almost
all bacteria refuse to grow except in an alkaline medium.
Hence a culture of aspergillus in Raulin’s fluid produces its
own asepsis. The sugar is only assimilated after inversion by

76 MICROBES AND TOXINS

a diastase of the fungus ; three parts by weight of sugar furnish
about one of growth. When glucose is supplied instead of
saccharose, the culture starts off more quickly ; lactose is not a
good food-stuff. Alcohol (in quantity equivalent to the weight
of sugar) interferes with the germination of the spores, but in
the adult form the fungus gets on quite well with it ; alcohol
is thus a poison to the embryo, but good food for the adult.
Starch paste (boiled) can supply the necessary carbohydrate
nourishment, but raw starch alone prevents germination ; the
adult would, however, attack it as a sort of last resource.

The Bearing of Raulin’s Experiments.—These are
not mere laboratory fantasies but are in reality almost the first
exact experiments on the conditions of plant cultivation and
growth. For every plant in the fields, for every microbe in the
laboratory, a Raulin’s fluid is demanded, a fluid which would
represent ideal conditions for development: all the improve-
ments in the technique of cultivation are attempts in this
direction. For example, the first cultures of the tubercle
bacillus were made with great difficulty on coagulated blood
serum: good growth was only obtained by adding glycerine to
the nutritive medium (Nocard and Roux). Almost all our
culture media for bacteria are empirical: blood, ascitic fluid,
and serum are supplied to them because we know that they
live well in the animal body in these fluids. If our knowledge
of bacteria was as far advanced as is that of Aspergillus it
would be possible to make media of known and constant
composition with measured quantities of the constituents, and
these would undoubtedly be of the greatest value in the
preparation of vaccines and toxins. When we consider that
Aspergillus is sensitive to zinc in the dilution of 1 in 50,000
and to silver nitrate in the proportion of 1 in 1,600,000, it is
possible to foresee to some extent the solution of many
technical difficulties and to appreciate the extent of what
remains to be discovered in connection with the action of
manures, food-stuffs and drugs. Raulin’s experiments show to
what degree bacteriology and medicine depend on the progress
of chemistry.

PHYSIOLOGY OF THE MICROBES 77

We know how to prepare a ¢/ean vaccine fluid by culture
on the flanks of the calf, but we cannot prepare 7 vi/ro a vaccine
lymph bacteriologically pure. This would be possible if we
knew the nutritive demands of the vaccine virus. This virus
is a microbe still unknown, but probably living in the interior of
the epidermal cells ; it finds itself there in a medium in which
reducing actions predominate. Répin succeeded in preparing
a reducing medium by means of a living reducing agent put
into a culture flask (the tyrosinase extracted from the
mushrooms of the genus Russula); with this he got a
commencement of growth in the vaccine under artificial
conditions. It is obvious what problems lie in wait for those
who try to grow bacteria in the laboratory.

The Nutrition of Yeasts.—The yeasts are of such great
industrial value that every detail of their nutrition has had to
be studied. They demand phosphoric acid and potassium,
magnesium, lime, and sulphur; their nitrogenous food they take
from ammoniacal salts and they can use those albuminoid
substances which are soluble in water, dialysable, and more or.
less insoluble in alcohol, and which exist in serum ; also they
can use urea and allantoin. The carbohydrates they employ
are in the first place sugars, then various alcohols, acids and
organic salts. From its food the yeast accumulates reserve
material, z.e., glycogen. Yeast attacks the food-stuffs supplied
to it by means of its diastases ; and in doing so, while toiling
for its own purposes, it toils for ours—exactly like a hive of bees.

Alimentation of Bacteria.—The minerals employed in
nutrition are rather varied; sulphur, phosphorus, calcium,
magnesium, potassium, sodium, traces of iron, traces of chlorine.
As carbohydrate food, sugars and glycerine ; as nitrogenous,
ammoniacal salts and peptones, natural proteins like blood
serum and asparagine. The food-stuffs are supplied by meat
infusions, dou¢//ons with peptone and salt, by animal fluids such
as serum, urine, ascitic fluid, milk, and by fruit juices. The
preferences which bacteria show for certain foods are employed
for diagnosis, because bacteria are characterised not less by
their food preferences than by their shape.

78 MICROBES AND TOXINS

Sulphur is indispensable in the culture fluid of the sulphurous
or sulpho-bacteria (Beggéatoa, Lamprocystis, and other species
described by Winogradsky); these can do almost entirely
without organic food material and grow well in water which
contains only 4:8 milligrams of this per litre, but which contains
two milligrains of sulphuretted hydrogen. This they decompose,
fixing the sulphur and accumulating it in the cell in the same
way as yeasts with glycogen. Certain eggéafoa contain
80-95 per 100 of their weight of sulphur. When put for two or
three days in non-sulphurous water they oxidize their sulphur
turning it into sulphates. If the dearth of sulphur continues
they die.

The ferro-bacteria (eg., Cvrenothrix polyspora, Cladothrix
aichotoma, Leptothrix ochracea) oxidize the carbonate of
iron protoxide, FeH,(CO,),, and accumulate the hydrate of
ron oxide. Instead of iron oxide a deposit of oxide of
manganese has been observed in certain cases.

The Importance of the Chemical Constitution o1
, the Medium.—Pasteur observed the relations between the
chemical structure of the food material and the physiological
action on it of the microbe. A enici/iium uses up dextro-
tartaric acid, leaving intact the levo form until the former is
completely exhausted. Penicillium glaucum and certain
yeasts can decompose optically-inactive sugars, burning up the
dextrorotatory form while sparing the lzvo.

Following in the track of Emil Fischer, there have been
observed relations between the molecular constitution of a
sugar and its value as food or fermentable material to a yeast
or in general to any definite ferment. Among the numerous
sugars with the general formula CaH ynOn (7 being a whole
number 1, 2, 3, 4, etc.), the ordinary yeasts only ferment those
with the carbon atoms numbering 3 ora multiple of 3. Sugars
which are isomeric, but whose molecule does not possess the
same stereochemical configuration, do not behave exactly the
same towards a given yeast. In a mixture of glucose and
levulose one or other is the first to be decomposed, this varying
with the strain of yeast.

PHYSIOLOGY OF THE MICROBES te

There exist also, it is true, “fermentations by force of
example,” where a fermentation started on a certain sugar may
extend its attack to another sugar which at first was not
fermentable: galactose, for example, can be fermented when
it has been “baited” by glucose. Similar affinities, capable
of modification by custom, come into play without doubt in the
sensitiveness or resistance, natural and acquired, of an animal
body towards a pathogenic ferment. Beneath the biological
specificity there lies a chemical specificity.

One of the best examples that can be quoted is that of the
bacterium of sorbose studied by G. Bertrand.

This makes a selection among the polyatomic alcohols,
attacking only in their molecule a link of the formula CH’OH,
transforming it into CO and consequently producing always a
ketone body. Further, for this link to be attacked its hydroxyl
OH must not be on the side of the H atom of the neighbour-
ing lnk CHOH. Finally the secondary group attacked is
always next to one of the primary group CH,OH, which
terminates the chain, at least in the formule’ below C, The
stereochemical structure of the sugars thus plays an important
part in the matter: it is on it and on it alone that the possibility
of attack by the bacterium depends. Its action is so narrowly
specific that it only transforms certain chemical groups, taking
no interest in the rest of the molecule, whatever may be its
mass and structure. A fermentation of this kind has all the
value of a chemical reaction.

Food-stuffs, culture media, infected organisms, represent
for the bacteria their sor7. “If we consider,” says Bertrand,
“on the one hand the differences in chemical composition,
even qualitative, which may exist between two closely related
species, and on the other hand the extraordinary variety of
_proteins which it is possible to conceive of nowadays, it will
hardly appear unreasonable to compare animal species, or
physiological variations of the same species, to culture
media varied like those which I used in the study of the
sorbose bacterium, nor to account for their immunity or
susceptibility towards a given microbe by a chemical or even

80 MICROBES AND TOXINS

merely stereochemical difference in their composition.” In
two culture media, identical except that one contains sorbite,
the other dulcite, a different sugar, the former supports the
sorbose bacterium, the latter is refractory. To the bacillus
tuberculosis distributed everywhere, all the “soils” are not the
same. The chemical study of the soil ought to go hand in
hand with the study of the microbe.

Respiration: Anaerobic Life.—Oxygen is the primary
food of creatures which have respiration. Lavoisier showed
that oxygen is indispensable to life.

During his study of the fermentative change of calcium
lactate into butyrate, Pasteur discovered the wibrio butyricus,
and made the fundamental observation that this organism lives
without free oxygen and even dies on contact with the air:

“ Pure carbonic acid passed for however long a period through
the fluid in which they are growing has no effect whatever on
their life and multiplication. If atmospheric air is passed
through instead for one or two hours under precisely parallel
conditions they all die, and the butyric fermentation which
depends on their presence ceases immediately.” (Pasteur.)

Those micro-organisms for which free oxygen seemed to be
a poison were called by Pasteur azaerobes. There exist “strict
anaerobes,” ‘‘ strict aerobes,” which cannot exist without free
oxygen, and “ facultative” bacteria capable of living in either
condition.

In broth the aerobes form at the surface a little collar, a
ring, or a pellicle; in a drop of fluid under the microscope
they can be seen to make their way towards the periphery,
where there is the best provision of oxygen.

If a filament from a green alga, a plant containing chloro-
phyll, is put into a suspension of motile aerobes, and a small
spectrum of sunlight is allowed to fall upon it, the bacteria can
be seen collecting at the points where the chlorophyll assimila-
tion and the production of oxygen are most intense, #.e., at the
red and violet regions of the spectrum, the & and C lines and
the # line of Frauenhofer (Engelmann’s experiment).

On the contrary, the anaerobes avoid the surface of. the

PHYSIOLOGY OF THE MICROBES 81

drop of water and the neighbourhood of air bubbles. They
can be grown well under shelter of a pellicle of aerobic
bacteria which prevent the passage of air, this being the best
method of cultivating the tetanus bacillus. There is no
necessity to suppose, as does Kedrowsky, that the aerobes

abe D £b F

Fic. 35.—Engelmann’s spectrum. Bacteria seeking oxygen swarming
round an algal filament lying on a spectrum. The grains of chloro-
phyll are not represented. The lines of the spectrum mark out the
regions on which the bacteria collect, 7.e., the points where most
oxygen is being liberated.

secrete a special ferment which permits anaerobic growth ; it
is sufficient that the anaerobes are cut off from free oxygen.

The addition to a tube of ordinary broth, aerated and hence
unsuitable for the culture of anaerobes, of sterile animal or
vegetable tissue, ¢.g., a fragment of flesh or a piece of banana,
allows the anaerobes to grow, the tissue acting as a reducing
agent. It is not at all correct to say, however, that the
anaerobes live without oxygen. They only live, as Pasteur
said, without free oxygen gas. They use up the oxygen which
is present in combination in the nutrient fluid and decompose
the food-stuffs to procure oxygen from them.

1 Pasteur (1861): ‘‘ There exist, besides the living beings already known
which without exception, at least in the general opinion, live and breathe
only on condition of being able to assimilate free oxygen gas, others whose
respiration is so powerful that they can live cut off from the air by

G

82 MICROBES AND TOXINS

The decomposition is generally only partial and to procure
the quantity of oxygen and energy necessary the anaerobes
have to attack large quantities of the food-stuffs. Such
behaviour is typical of ferments and accordingly the anaerobes
usually produce powerful fermentation. “Fermentation is
life without air,” was Pasteur’s dictum. It is in particular
the study of alcoholic fermentation which supports this
statement. When a yeast grows in a shallow mass of fluid
with an extensive surface its cells multiply abundantly: there
is a great increase in yeast protoplasm but little or no alcohol.
When inoculated on the contrary at the bottom of the fluid
without access of air, the growth is feeble but produces
alcohol in quantity, varying in proportion to the completeness
of the anaerobic conditions. The differences in the form and
functions of sucor when aerated or deeply immersed have
already been mentioned. Several microscopic plants, muce-
dinez and yeasts, exhibit a whole series of transitional forms
between aerobic and anaerobic growths. Anaerobic life appears
to be an asphyxial condition against which the microbe
contends or adapts itself by changing its manner of nutrition.
Not only the mucedinez and the yeasts but all living cells,
animal and vegetable, act like ferments and produce alcohol
when forced to live cut off from the air in presence of sugar.
Such is the case in the experiment of Pasteur and J. B. Dumas
with the plums kept under a bell-jar: they use up the air and
fill the jar with carbonic acid ; their sugar diminishes and they-
become charged with alcohol. A similar case is that of ripe
fruits left to themselves in an atmosphere of limited volume,
as, for example, with the apples and pears kept in a closed
vessel in the experiments of Lechartier and Bellamy. Another
is that of seeds starting to germinate cut off from oxygen;
they produce alcohol, using up their reserve material (Maze’s

seizing upon the oxygen of certain compounds, in which there is in
consequence a slow progressive decomposition. This group of living
organisms is composed of ferments precisely similar to those of the first
group, living like them, assimilating like them carbon, nitrogen, and
phosphates, and like them requiring oxygen, but differing from them in
their power of doing without free oxygen gas and carrying on their
respiration with the oxygen derived from unstable compounds.”

PHYSIOLOGY OF THE MICROBES 83

experiment). There is even alcohol in animal tissues. There
is, therefore, nothing surprising in the presence of alcohol
throughout nature, in the soil, in water, in air, and in the sea;
if it is true that the latter contains one millionth of its weight
(one gram per cubic metre) there must be an enormous
supply.

Since the discovery by H. Buchner of zymase—the diastase
by which the yeast decomposes sugar—we know that it is on
the zymase rather than on the anaerobic conditions that the
alcoholic fermentation depends. But since the zymase only
appears when the yeast is shut off from the air, it too is
“an asphyxial function” and we return to Pasteur’s formula.

Duclaux has re-established the continuity between the two
methods of respiration by his idea of the constant operation
of the zymase in aerobic as much as in anaerobic life and by
maintaining that alcohol is produced by living tissues, not
pathologically but normally. “Alcohol is a normal and
necessary product in the digestion of the hydrocarbons of the
seed. When oxygen is present, this alcohol is burnt up and
escapes observation. To demonstrate it the plant must be
submitted to a degree
of asphyxia which just
lets it live, or rather,
which permits the action
of the zymase which it
contains. It is not the
asphyxia which pro-
duces the alcohol, it
only renders it per-
ceptible.”

Further, absolute
anaerobiosis does not
exist either in nature or
in our artificial cultures. Fic. 36.—Cochin’s experiment.

The pretty experiment

of Denys Cochin shows that yeast even under anaerobic con-

ditions the most complete possible cannot do without oxygen
G2

84 MICROBES AND TOXINS

indefinitely, 7e., in traces. Yeast cells are made to grow shut
off from air in a series of communicating flasks all carefully
sealed. The flasks are inoculated in series, the second with the
yeasts of the first and so on. To isolate each flask from the
preceding the little communicating tube is sealed in the flame.
Towards the tenth generation, fermentation is seen to stop and
only revives when a little oxygen is admitted to the confined
atmosphere.

Pasteur had already observed that an air bubble about the
size of a pin-head was sufficient to reawaken a slackening
fermentation.

All microbes require oxygen but their requirements are very
unequal. Betweeen the aerobes and the strict anaerobes there
exists every intermediate condition. Each species requires an
oxygen pressure suitable for itself just as do the higher animals,
which are of course aerobic organisms.

By exhausting the air under a bell-jar, Khoudiakow observed
that the B. dutyricus could still multiply at the pressure of five
millimetres, Clostridium butyricum at ten millimetres, the vtbrion
Septigue and the tetanus bacillus at twenty millimetres ; the
bacillus of systematic anthrax at forty millimetres, at which
pressure the latter microbe behaves like an aerobe, using up
the oxygen in oxidations.

An anaerobe like the B. dutyricus can be trained to live under
an oxygen pressure greater and greater up to fifty millimetres,
a pressure ten times greater than that which it will bear
normally. This acclimatisation of anaerobes to contact with
air can be carried out within certain limits in the laboratory, so
much so that it has even been thought useful to create the
barbarous phrase ‘ aerobisation’ of anaerobes.

Khoudiakow has made a complementary experiment by
modifying the pressure on aerobes. ZB. sudtilis, grown on
gelatin, lives fairly well under three atmospheres, but begins to
suffer at four. At the other end of the scale it still grows well
at ten millimetres of pressure, but not at five. Aspergillus niger
has for minimum and maximum five millimetres and three
atmospheres. Spores are more resistant to the action of air

PHYSIOLOGY OF THE MICROBES 85

than bacilli. The spores of Bact. butyricum are scarcely affected
by the action of air for 265 days, whereas the growth of the
bacillus is inhibited by the action of air for fifteen hours.

Oxygen is a food which bacteria take from compounds
liberating it more or less readily ; anaerobes take it from com-
pounds which retain it and resist decompositions. Although it
is not absolutely true that fermentation is life without air (there
are fermentations which go on in presence of oxygen), it is true
that anaerobiosis favours the majority of fermentations and is
the usual condition for these.

In nature, anaerobes occur wherever there is little penetra-
tion of air, or where the air is diluted or replaced by other
gases, as, for example, in the earth, in mud and slime, in sewage,
in the ooze of the sea, in dunghills, and in the intestines and
excrements of animals ; and it is in these surroundings that the
most important fermentations and putrefactions of organic
matter take place.

Respiration of the Pigmented Bacteria.—The
purple bacteria (a certain number of which are also sulpho-
bacteria) contain a pigment, dacterio-purpurine, quite distinct from
the pigment of Bacillus prodigiosus. According to Engelmann,
these pigmented bacteria absorb the infra-red rays of the
spectrum (of wave-length 08 to o’9 «) and employ them, as also
the red rays, in the decomposition of carbonic acid from the
air and the liberation of oxygen, just as plants do with chloro-
phyll; they have a “chromophyll” function analogous to the
chlorophyll function of green plants. This opinion is not
shared by all observers: according to Molisch, the purple
bacteria are not capable of decomposing CO, nor of assimila-
ting directly inorganic compounds. They certainly differ from
other bacteria in their power of using light in their nutritive
process, but their foods are still organic food-stuffs ready made
and they cannot do without these: they are not capable of the
synthetic function of the green plants. They can assimilate
organic food-material in the dark like other bacteria, but
they have advanced a step by adapting themselves to light
and by using it to increase their nutritive resources. But

86 MICROBES AND TOXINS

they have remained at that stage: they have not cast off the
necessity for ready-formed organic food, #.e., the parasitic habit,
nor can they break up the carbonic acid of the air, liberating
oxygen and absorbing carbon. The purple bacteria then occupy
a position intermediate between the saprophytic or parasitic
habit of bacteria in general and the chlorophyll property of the
higher plants. They carry on, like the latter, a sort of photosyn-
thesis, but what they synthesize with the aid of light is still
organic material like the ordinary bacteria.

Secretion of Diastases or Enzymes.—Microbes act
through their diastases; fermentations are thus diastasic
reactions. The diastases carry on the transformations of
matter both by breaking down and building up, and it is
through them that the bacteria transform energy.

The discovery of diastases and the possibility of extracting
them, of isolating them (not completely pure), and of making
them act without the presence of living cells, represents a great
acquisition to the dominion of chemistry in the field of the
study of life and fermentation.

A further step was made when Bertrand demonstrated the
prominent réle taken by the mineral elements associated with
the enzymes ; the activity of /accase depends on the proportion
of manganese present, and the whole reactign behaves as if
laccase were a salt of manganese with a weak acid. Besides,
all the diastasic reactions can be performed by chemical agents
and the part played by diastases seems to be that of amplifying
and stopping the action of the latter.

The nature of diastases is still unknown, and we shall not
dwell here upon the manner of preparing them, on the causes
of error which may creep into this technique, or on the theory
of diastasic actions in general.

One microbe is capable of secreting several diastases.
With the proteolytic enzymes have been grouped the dysins
including the hemolysins of bacteria. The solution of the
cell attacked may indeed be only the sequel to an action on
the cell membrane or protoplasm, injurious but not actually
dissolving. The best known hemolysins are those of the

PHYSIOLOGY OF THE MICROBES 87

tetanus bacillus, tetanolysin; of the staphylococci, staphylo-
lysin ; of the cholera and pseudo-cholera vibrios, vibriolysin ;
of the streptococci, streptocolysin; of bacillus pyocyaneus,
pyocyanolysin ; lysins also exist in the cultures of B. typhosus,
of fowl-cholera, of the anthrax bacillus and in the bacillus of
diphtheria (in this case doubtless in the body of the bacillus,
not excreted).

The majority of the bacterial hzmolysins are destroyed by
heating to 56° C. ; that of the bacillus of fowl-cholera, however,
is only destroyed at 70° C. ; while pyocyanolysin stands boiling
for a long time and is only destroyed in half an hour at
120° C,

Along with these enzymes should be classed the lysins
which attack the leucocytes (leucocidine of Vandevelde) and
other bacteria (pyocyanase of Emmerich).

Products of Cultures. Microbic Excretions.—A
medium in which a bacterial culture has grown contains bodies
which were not present before the inoculation. These are
products of the activity of the microbes in relation to their
food-stuffs ; they do not come exclusively from the microbe
itself, but they are products of these and bear their mark. It
is often difficult to draw the line between true secretory
products, the diastases and toxins—and the residual substances
remaining after enzymatic action, the study of which ought to
be included with that of the fermentations and putrefactions—
and the excreta, the catabolic products, properly speaking.

The distinctions which are currently drawn between these
products depend often on the purpose we have in view. We
stop a fermentation when it has reached the stage of the
products which are useful, as, for example, in the manufacture
of beer, wine, and cheese; if left to continue, the organic
matter breaks down finally to the simplest substances, water,
carbonic acid, and ammonia. In nature a ferment only ceases
when it has furnished the materials for a new fermentative
process.

‘A bacterial product is a substance incapable of being
attacked under the conditions of the experiment by the

‘

88 MICROBES AND TOXINS

bacterium which has produced it, but which can in its turn
become a food material if the conditions are altered, if the
bacterium takes on new properties, or if other microbes step
in” (Duclaux). Every living thing lives on the products of
others.

As the richness of a culture increases, its growth slackens :
the medium becomes less and less favourable, the food material
becomes exhausted and the bacterium, by no means always
capable of living on its own residues, ends by being embarrassed
by the substances it has produced. Acid-producing bacteria
cease to grow when the acidity reaches the point where
vegetation is no longer possible. Alcohol acts. like an
antiseptic towards the yeast that produced it and acetic
acid does the same for the ferment of vinegar. The bacterium
however can often fall back from the food of its real choice to
a sort of famine ration: when the acetic ferment has used up
all the alcohol it burns up the acetic acid. When a yeast has
no longer any sugar it consumes the glycerine which it has
produced at its expense.

Products of excretion exist which stop the growth of cultures
by a sort of auto-intoxication. The foulest waters are those
which are least easy to infect, because, according to Miquel, they
contain substances of bacterial origin injurious to bacterial
growth. If such foul waters are concentrated at a low
temperature and the filtered result is added to pure water this
latter becomes incapable of supporting life. Boiling destroys
these inhibitory substances, which indicates perhaps that they
are of the nature of diastases. There is said to be in fecal
matter an inhibitory substance which checks the extraordinary
multiplication of bacteria in the intestine, and this also is to be
regarded as a diastase; Conradi and Kurpjuweit compare its
energy to that of carbolic acid; without having been able to
isolate it they were able by dialysis to make it act without the
bacteria themselves. They callit an “‘ autotoxin.” Others, how-
ever, question this, not having been able to find it either in
tube cultures or in the human intestine, and explain the
inhibition by the exhaustion of the medium, in the same way

PHYSIOLOGY OF THE MICROBES 89

that Pasteur explained the immunity of the body by the
exhaustion of the food material which it supplies to the
microbe.

Exhaustion of the food-stuffs and action of the excreta may
exist together. For example, when gelatine is inoculated
with 6 millions of bacillus coli per milligram of medium
no growth takes place; the bacteria die and disintegrate
although the medium is not exhausted.

Further, an ‘‘exhausted” medium can be regenerated by
filtering it through porcelain and heating it without adding
any new food material (Eijkman). In cultures of B. coli of
five days at 37° C., there is of all the bacteria which can be
seen and counted under the microscope, only one living in
fifteen, and after a week only one in forty (Hehewerth).
The antitoxin of one bacterial species can act on other
species.

Heat Production.—The combustion of the food liberates
a quantity of energy which is not entirely used up in the
construction and support of the bacterial cells; there remains
an excess of heat which raises the temperature of the medium.
The yeast of beer undergoing anzrobic fermentation heats
up the whole mass by 3°9° C. (Eriksson). In heaps of
manure or hay, the temperature may rise to 50 or 7o° C.,
and in hay even to 96° C. Cohn found in the masses of
moist cotton a micrococcus which discharges carbonic acid
and raises the temperature to 67° C., when care is taken to
avoid loss of heat by radiation.

But it is not very certain that the heating of hay is really
due to microbes. It is only the spores which can resist
temperatures bordering on 100° C., and spores exert no
activity. Further, no bacteria are to be found in the places
where the heating begins, and finally, hay sterilized at 120° C.
can heat like normal hay.

According to Beekhout and Ott de Vries, the spontaneous
heating of hay is a chemical phenomenon, the cause of which
is still unknown.

Production of Light.—Rotting wood and the corpses

90 MICROBES AND TOXINS

of sea-beasts frequently emit light. Butcher-meat left to
itself in a cool place for two or three days half-immersed
in 3 per cent. salt solution very often becomes luminous.
Dead leaves fallen in the forests occasionally give out a
dim, steady light. Now it is not the animal or vegetable
tissue which shines; it must be microbes, moulds, or
bacteria.

Luminous microbes have been discovered in the Baltic Sea,
the North Sea and the Indian Ocean, and phosphorescent
bacteria in the Elbe. At present about fifteen moulds and
about thirty bacteria are known to be “photogenic” or
“luminous.”

A simple recipe may be given: take a fresh herring, sprinkle
it with 3 per cent. salt solution, leave it at a temperature of
about ro’, add a little sugar, glycerine, and peptone; in two
days the flesh and the juice become luminous.

The production of light depends on the temperature and the
food supply. Sometimes a temperature from 20 to 30° C.
suits best ; most often lower temperatures are more favourable.
The phenomenon has been seen to occur at +45° C., and at
—20°C. The luminous bacteria seem to like salt ; some only
require a nitrogenous medium, others require in addition
carbon. But the indispensable substance is oxygen, and when
this is exhausted the luminosity ceases. Ifa bubble of air is
made to pass, by turning upside down, through a long tube in
which there is a culture of a luminous bacterium, which has
just become extinguished for lack of oxygen, a wave of light
can be seen passing along the tube. Thereis no luminosity in
a vacuum.

Strains have been produced by natural selection so luminous
that their light can be seen in full daylight. Ifa flask coated
on the inside with gelatine is inoculated with the Bacterium
phosphoreum or the Pseudomonas lucifera, one gets a microbial
lamp which with an eye a little accustomed to darkness allows
one to read the time from a watch or to read moderately large
print. It might be possible even, it seems, to employ such
lamps in powder-magazines or mines, for they do not emit heat.

PHYSIOLOGY OF THE MICROBES 91

Certain fishermen employ as bait luminous fragments of dead
fish, a luminous bait provided by bacteria.

With the aid of the spectroscope, Molisch distinguished in
the light of Pseudomonas lucifera the colours green, blue and
violet. R. Dubois photographed colonies of these bacteria with
their own light alone. Although feeble this vegetable luminosity
exerts, like sunlight, heliotropism ; the shoots of young plants
such as vetches, peas and lentils turn towards it on germinating ;
but it is incapable of exciting the chlorophyll function.

Substances which kill the microbe abolish its luminosity.
Dubois stated that he had isolated a substance which shone on
contact with the oxygen of the air, /uciferine, but others failed
to repeat this experiment. It is possible that the- microbe
secretes a substance which as soon as it is produced is
destroyed by oxidation giving out in the process luminous rays.
Many organic substances, aldehydes, ethereal oils, carbides
of hydrogen, fats, and alcohols, when they combine with free
oxygen in an alkaline medium, can emit light closely resembling
that of the above bacteria; perhaps the bacteria give out
light thanks to the oxidation of substances in the cell, such
as lecithin, cholesterin, and ethereal fats; but there is one
difficulty : the living cell does not contain free oxygen.

To prove that the luminosity of bacteria is a chemical
phenomenon independent of the life of the cell which pro-
duces it, it would be necessary to repeat with these bacteria
the experiments which have been made with the secretions
of certain animal cells. With the photogenic substance of
Luciola italica one can write, and the writing becomes
luminous whenever it is moistened. If the luminous organs
of Lampyris noctiluca, which have been dried and preserved
in vacuo, are moistened with a drop of distilled water, the
luminosity reappears. Paper soaked in the secretion of
certain AZyriapods can shine when moistened, even after two
months. In these cases the luminosity cannot be attributed
to a living cell.

Bacteria which have never been exposed to light shine quite
as well as those grown in daylight, Their luminous property

92 MICROBES AND TOXINS

is therefore not like that of the salts of strontium and barium,
a case of re-emission of light formerly absorbed. It is the
discharge in the form of light of energy absorbed in another
form.

Production of Pigments.—Numerous bacteria exist,
whose cultures possess colour, green, violet, red, blue, black,
and fluorescent ; these colours have nothing in common with
the green colour of chlorophyll plants, for they are diffused
throughout the cells, whereas chlorophyll is agglomerated in
distinct masses. The bacteria which produce coloured cultures
without their ceils themselves containing the pigment are the
more numerous ; the pigment is therefore an excretory product
which diffuses into the medium or collects in little masses which
can be seen under the microscope at the side of the bacteria. All
the coloured bacteria might be put in this category if one
admits that the bacteria which contain a diffuse pigment ought
to be classed as alge.

Staphylococcus aureus and various sarcinz produce colonies
of a golden yellow : the pigment isa fatty substance (Apochrome)
insoluble in water but soluble in alcohol, benzine, chloroform,
ether and carbon bi-sulphide and capable of saponification ; it
turns to blue or bluish-green on the addition of sulphuric acid
and to orange or red on the addition of alkalies.

Everyone has heard of the miracle of the “ d/eeding host,”
the sacred bread which becomes covered, more by accident
than miracle, with red spots having a reddish-brown, somewhat
metallic lustre. It only means that it has become invaded
by one of the commonest bacteria, one which is present abund-
antly in air, milk and dust, especially at the end of summer and
inautumn, the B. prodigiosus. The pigment is insoluble in water,
soluble in alcohol ; sulphuric acid turns it into reddish-brown,
alkalies into yellow. Reducing agents decolorise it as does
light, though only after some time.

There appears occasionally on the surface of milk a bluish
colour, sometimes as a uniform film, sometimes in rings or
marbling ; this is due to the dactl/us cyanogenes. The colour
is soluble in water, insoluble in alcohol, ether, and chloroform.

PHYSIOLOGY OF THE MICROBES 93

Grown in pure culture in sterile milk, the colour is merely
grey ; to get the typical blue the collaboration of an acid-
producing bacterium is necessary. In nature this is provided
for by the lactic bacilli. The B. cyanogenes produces at the
" same time a green fluorescent pigment.

The Bacillus pyocvaneus (Gessard) is the colour-producing
bacterium which has been most studied. It used to be thought
that it was the cause of blue pus ; but it confined itself really—
in the days before antiseptics—to diffusing its blue pigment
through the linen of the dressings. This blue colour, “ pyo-
cyanin,” is soluble in water and chloroform, insoluble in
alcohol ; it becomes pink in acid solution, yellowish in alkaline,
and is a base closely approaching the ptomaines.

The B. pyocyaneus produces in addition a fluorescent pigment
and a green pigment not fluorescent ; and, finally, old cultures
take on a smoky brown tint. By heating, by inoculating on
special media, and by animal passages it is possible to dis-
associate or to associate these different colours in the same
microbe and to create different strains or even a non-pigmented
variety ; the green fluorescent pigment is particularly associated
with phosphatic food, but the strains thus obtained depend on
the medium and on the technique employed; they are not
fixed, and are rather transitory varieties than true strains.
The chromogenic function lending itself thus to modification
it is obvious that it is not one of the essential properties of the
bacterium.

With the microbe, as with higher creatures, habits are more
easily changed than nature.

The majority of the chromogenic bacteria produce their
pigment at moderate temperatures, 20 to 25° C.; at 37° C., the
B. prodigtosus and the sarcina grow excellently, but produce
no pigment. They prefer a slightly acid medium, but
fluorescence requires the medium to be alkaline. The
starches are excellent food materials, which explains why the
B. prodigiosus grows so well on the Sacred Host. The essential
nutritive material is oxygen, and with certain exceptions none
of them produce pigment when shut off from air.

94 MICROBES AND TOXINS

Action of Heat on Microbes.—Just as on ordinary
thermometers the temperatures are marked for taking a bath
or for keeping silk-worms, so it would be possible to mark the
points at which microbes develop best. Each species has an
optimum temperature ; below this, it grows feebly ; above it, ”
it begins to suffer and dies; heat, indeed, is the sovereign
disinfectant. Adapted as they are to the surroundings which
shelter and nourish them, the bacteria are parasites, not only
in regard to their food supply, but also for their heat
surroundings.

Although the names of different species might thus be written
on almost every degree of the thermometer, three types may be
distinguished, with intermediate individuals. The majority of
the bacteria of water and of soil and the phosphorescent
bacteria of fish grow well at 15—20° C.

The majority of pathogenic microbes demand in cultures
the same temperature as that of the body in which they lived
as parasites. The tubercle bacillus of the mammals develops
best at 38° C.; that of birds at 41-42°, and that of fishes at
15-20°C., z.e., practically like a water-bacterium.

The third group is that of the thermophilic bacteria. They
demand and support temperatures so high that other bacteria
would rapidly be killed. They have been found in rivers, in
sewage, in cheese and in the human intestine. The majority
are motile and possess spores. In the hot springs of Ischia,
and in the fumaroles of Naples there are bacteria which live at
60°C. Miquel found in the Seine a species living best at 67°
to 70°C. Ina spring at Luchon, Certes and Garrigou found
a bacterium developing at 64° C., the temperature of the water.
In the upper layers of the soil, Globig discovered species
which grow well at 65-70°.. Mlle. Tsiklinsky has studied the
thermophilic bacteria of the human intestine; they are all
aerobes. ;

It is remarkable that thermophilic species from the
surface of the soil have been found in the most varied
latitudes, from the tropics to the Hebrides and Norway
Perhaps those of the cold countries (rare) can live shut -off

PHYSIOLOGY OF THE MICROBES 95

from air at 35—-40° C., and can thus remain alive in the intestine
of animals.

The majority of non-sporulating bacteria are killed in a few
minutes at a temperature in the vicinity of 60°C. Further, the
nature of the medium in which they are heated must be
reckoned with ; they perish more quickly in acid than in alkaline
fluids, and dry heat kills them much less quickly than moist.
The spores, being resistant forms, are only killed at much
higher temperatures: 100°C., during 2-4 minutes for the
anthrax spore. In one single species, there are spores which
stand the same temperature twice as long as their companions.
At higher temperatures resistance is much shorter, for example
for certain sporulating bacteria of the soil and of hay (in
saturated steam) :

100° resistance... ee vee ee eee 6-6 hours,
115° ‘i Nii Medh a cael tits Saige 4 hour.
130° 35 ws Re day ee eS Minutes:
140 53 ses aes vee = SCarcely I minute.

The spores of moulds, studied for the first time by
Spallanzani, stand thirty minutcs of dry heat at 127-132°C.,
but in moist surroundings they die below 100° C. The
spores of Ustilago carbo in the presence of saturated water
vapour perish at about 60° C. ; dry, they stand 120°C. Spores
are more resistant than the bacilli, because they contain less
water, ¢.g., 38 per cent. instead of 62 per cent. Tyndall’s
method—discontinuous heating, at intervals, about one hour
per day for three days in succession—succeeds at a relatively
low temperature because the protoplasm in taking up water
becomes more vulnerable.

Heating coagulates the protoplasm, and this coagulation is
the more rapid and easy the more water the protoplasm
contains. Albumin dried ## vacuo over sulphuric acid can
be heated beyond 100° without losing its solubility in water
(Chevreul). Since coagulation is not an instantaneous but a
progressive phenomenon, instead of talking of the “ tempera-
ture of coagulation” and the “lethal temperature” it would

96 MICROBES AND TOXINS

be better to speak of the lethal zone and of the zone of
coagulation.

It was from the effects of heat on the bacilli that Pasteur
discovered the anthrax vaccines.

Microbes stand low temperatures very well. Long ago
Cagniard de la Tour observed that yeast kept at —9g0° C. in
a mixture of carbonic acid and ether does not lose its power
of fermentation. After twenty hours at — 130° C., 108 hours
at —70° C., the spores of B. sudtilis still germinate, and
the spores of anthrax are still virulent. According to
MacFadyen’s experiments, bacteria kept for six months at the
temperature of liquid air (about — 190° C.) or ten hours at
the temperature of liquid hydrogen (— 252° C.) remain living
and virulent.

Action of Light.
thus a disinfectant.

The active rays are the chemical rays of the spectrum which
act by oxidizing the protoplasm: the bacteria do not die when
the sunlight strikes them in a vacuum.

The anthrax spores stand sunlight for about thirty hours
in contact with air and eighty hours when shut off from air
(Roux). Even zz vacuo in pure hydrogen the bacteria do not
resist indefinitely. There is therefore something else than
simple oxidation taking place. The action of the air is
associated with an action belonging more particularly to the
light, and the oxidation affects not only the bacterium but the
medium in which it is.

The bactericidal rays are par excellence the ultra-violet
rays, as can be proved by cutting off certain parts of the
spectrum by means of various sorts of screens. Glass of a
thickness of 1°35 millimetres completely abolishes the action.
A solution of oxalic acid of 10 per cent., which limits the
spectrum up to 300pp, acts in the same way, whereas bacteria
are destroyed through a screen of sulphocyanide of potassium
of 10 per cent. which cuts down the spectrum to 265um
(experiments with an electric arc); the active portion of the
rays of such an arc must lie between these limits. A blue

Light is injurious to bacteria and is

PHYSIOLOGY OF THE MICROBES 97

specimen of rock salt cuts off all visible light without cutting
off the active ultra-violet portion, and rays which traverse it
destroy bacteria.

The action of ultra-violet rays is practically equally rapid in
the presence as in the absence of oxygen. They produce a
little peroxide of hydrogen in the medium of suspension, but
in quantities 400 times too weak to be active ; hence the action
is not due to the peroxide. By putting in the path of the
ultra-violet rays from a mercury lamp a plate of white glass ot
one millimetre thickness, all the ultra-violet spectrum is cut off
beyond the rays 3027-3022 ; the latter only penetrate the glass
very much weakened, and in this case the bactericidal action
is much diminished. By far the most powerfully bactericidal
rays are those which have a wave-length below 2800 units.
“ Protoplasm (albumin, gelatine, and serum) absorbs the ultra-
violet rays below 2‘goo units: it is therefore the rays absorbed
by the cells which exert the destructive action.”

The ultra-violet rays have been studied with a view to the
destruction of cancer cells. Exposed to the ultra-violet rays the
tubercle bacillus loses its property of taking on a stain which is
acid-fast. An exposure of ten minutes kills them. An exposure
of one minute attenuates them, and, inoculated in guinea-pigs,
they now produce a slow lingering infection; the animal lives
for months, whereas the controls die within forty days at most.
After an exposure of three minutes the bacilli no longer grow
on potatoes. The toxin of tubercle, tuberculin, which stands
heating at 134° C. for half an hour, is destroyed by five hours’
exposure to ultra-violet rays. The solutions should be exposed
na layer of two or three millimetres and kept shaken. Tuber-
culin exposed to the rays 7m vacuo is destroyed much more
slowly than tuberculin exposed in air (M. and Mme. Henri
and V. Baroni).

Certain coloured and fluorescent substances such as eosin
erythrosin, and bengal-rose, are injurious to bacteria; and still
more so to infusoria, in presence of light, but are quite or
almost harmless in the dark. These have been called photo-
dynamic substances. Several have been employed in photography

H

98 MICROBES AND TOXINS

to sensitise plates towards rays which alone are chemically
inactive. They exert the same action on ferments and on the
toxins and anti-toxins of tetanus and diphtheria. This action
is entirely due to oxidation and only takes place when oxygen
is dissolved in the fluids of the experiment. Thus in a solution
of iodide of potassium, with eosin added and exposed to light,
iodine is set free; this does not occur if the solution is freed
from oxygen. It has been thought that this oxidizing action is
due not to oxygen but to ozone (0%).

Physiology of Protozoa.—It must not be thought that
all the protozoa because they are unicellular are primitive
creatures and rudimentary ancestors of higher animals ; their
cell is adapted to all the requirements of life and possesses, at
least in some degree, all the properties of higher animals ;
it may be more independent, and richer than certain cells
of vertebrates. Both by structure and by function the ee
are complex and highly differentiated creatures.

Ehrenberg, an old scientist, who studied them very apahills,
held this belief, but in a naive and inaccurate form. Protozoa
to him were animals possessing in brief all the organs of
higher animals ; he saw in them a digestive tube, a brain, eyes,
kidneys, a heart, an ovary and vessels. But nothing of that
really exists ; the protozoa have simply nuclei, vacuoles which
digest the food, others which expel the waste products and a
protoplasm full of varied movements and currents. But
although they do not possess the miniature organs which roused
Ehrenberg’s admiration, they are none the less capable of
taking up food, digesting it, and expelling the waste, of moving,
and of reproducing. They possess all the functions of animal
life, but more simply and more purely than among the higher
animals ; what one might call the chemical and physical model
of life is in them more visible, more exposed to the eye.
That is why their study is so attractive and so fertile; it is to
it we owe our best knowledge and our best ideas on life in
general, so there is no necessity for the surprise expressed
by those who have only read their family “ Buffon” (Buffon’s
Natural History), that scientists should be passionately

PHYSIOLOGY OF THE MICROBES 99

studying infusoria and amcebe rather than sharks and
elephants.

Life means always the combustion of protoplasm, according
to Lavoisier’s dictum, and this protoplasm replaces its losses
by assimilating food. Three methods of assimilation we know:
the chlorophyllous plants with the help of sunlight decompose
the carbonic acid of the air, manufacture hydrocarbons, and
finally turn the starch into more complex substances which go
to build up their protoplasm ; the green plant thus starts with
non-organised substances for all its food-supply ; animals feed
on plants or on other animals which have already fed on
plants ; parasites absorb food which has already been prepared
ready-made for them by their hosts. All three methods
exist in the protozoa.

There are some which, possessing chlorophyll, manufacture
their food exactly like green plants: these are the plant-animals
which form the link between the two kingdoms. They
prepare by synthesis a starch or a para-starch (Biitschli) and
can satisfy their life conditions in water containing mineral
salts, provided they receive the light of the sun. Certain
species lose their chromatophore granules when ready-made
food is supplied to them, permitting them thus to dispense
with the labour of “ photo-synthesis.” Certain flagellates live
in symbiosis with green algze which supply them with starch:
in such a case the protozoon may be said to be attacked by a
useful infection: further, this infection may be conveyed from
one to another.

The other protozoa capture their prey, frequently in the form
of living creatures. Whether they have a mouth or not, whether
the food penetrates their bodies with the help of a current
produced in the water by cilia or flagella, or whether
it is the protoplasm of the protozoon itself which issues
from .its envelope, introduces itself into the body of the
prey, and thus devours it from the inside, as it were re-
versing the réles, in all cases the important point in the
nutrition is z/racel/ular digestion: the food is enclosed in a
little spherule inside the protoplasm where little by little it is

H 2

100 MICROBES AND TOXINS

oa dissolved by means of
\; i the digestive juices.
; A leucocyte which
‘ seizes and digests a
' bacterium in a higher
" animal acts in precisely
the same way. The
amoeba is the proto-
type of that phagocytic
digestion which occu-
pies so large a place’ in
both natural and ac-
quired immunity.

Of what nature is
this digestion from the
point of view of chem-
istry? In the digestive
vacuoles the reaction
is acid and from certain
myxomycetes (/uligo
varians) and rhizopods
(Pelomyxa palustris)
a ferment resembling
pepsine and acting in
an acid medium has
been isolated. On the
other hand, Mouton
and Mesnil have ex-
tracted from amoebe
and paramecia a fer-
ment which digests
gelatine and fibrin in
an alkaline medium

Fic. 37.— Gromia oviformds in the act of just like trypsin.
capturing in its pseudopodia a diatom, According to other
which being too large for ingestion is investigators the diges-

digested outside the body in this way. 3 ‘ i
(After Max Schultze * tive medium is first

PHYSIOLOGY OF THE MICROBES 101

acid, then alkaline, just as in the stomach and small intestine
of mammals.

The solid refuse of digestion is evacuated by an anus and the
liquid residue collects inside protoplasm in a little spherical
sac which from
time to time ex-
pels its contents
externally: this
latter is the con-
tractile vesicle
iene ee eae Fic. 38.—An amceba expelling the residue of its
took for the pul- food: various stages. (After Verworn.)
sating heart.

Oxygen isa primary necessity to protozoa as to bacteria: the
digestive vacuoles contain oxygen, the contractile vesicles
discharge carbonic acid, #.¢., aerobic respiration. The protozoa
which live in surroundings deprived of free oxygen have, it is
certain, a method of respiration analogous to that of anaerobic
bacteria ; they draw their oxygen from reserve materials which
they have stored within themselves, ¢g., glycogen. It is
believed, without being absolutely certain, that certain infusoria
can, like the intestinal worms (ascarides), break up glycogen
with the formation of valerianic and carbonic acids. Among
the products of excretion have been found uric acid and
phosphate of lime (Schewiakoff). Excretion is a fairly active
process since the vacuole contracts often (every four to eighteen
seconds according to temperature in Stylonychia pustulata).
According to Maupas the infusoria discharge during a space
of time which varies from two to forty-six minutes a volume of
liquid equivalent to the whole volume of the animal.
Stimulation from the exterior is always accompanied in the
protozoa by a manifestation of energy: they possess irritability.
The excitant may be a touch, a ray of light, heat, electricity or
a chemical substance, and the protozoon in its reactions to the
stimulus acquires habits such that its physiology is full of as
many problems as that of the higher animals, not excepting
problems in psychology. Although possessing neither nervous

102 MICROBES AND TOXINS

system nor sense organs, even to such a degree as the sea-
anemone, the protozoon is capable of choice and of determina-
tion, these phenomena of course remaining more or less
mechanical. Superposition and propagation of impressions
exist among protozoa, and after long and minute observations
it has been maintained that no essential difference exists
between them and the most complicated metazoa: activity is
neither more nor less mechanical in the one set than in the
other.

The chief business of all living creatures is reproduction.
Among protozoa there exist several principal’ methods for this,
each presenting numerous variations. They may divide by
nuclear division: they may divide by budding, and in this the
greatest diversity occurs in the number, size, and arrangement
of the buds. They may sporulate, z.e., their protoplasm may
break up, and each fragment consisting of a bit of protoplasm
and a bit of nuclear material can reproduce a creature similar
to the mother-cell which sporulated. When life-conditions
are difficult, certain protozoa encyst, z.e., they contract inside
a resistant shell, and under shelter of this various modes of
reproduction may take place. Between reproduction by
division and that by sporulation intermediate forms exist
(Tillina and Colpidium), and the continuity in nature can
always be detected by the imagination.

Parasitic protozoa, those which to subsist require to emigrate
from one host to another, most often reproduce themselves
by sporulation, and the reproduction is the more lavish the
greater the difficulties encountered by the species in propa-
gation.

Parasitism tends to modify the species in a retrograde
direction but the losses may be compensated for by new
acquisitions. Locomotory organs, protective envelopes and
the apparatus designed for capturing and digesting food
become simplified or disappear altogether. But parasites are
in general more prolific and they acquire other organs, hooks
or suckers, by which they can better cling to their host. As
their life conditions or their habits become narrowed down,

PHYSIOLOGY OF THE MICROBES 103

they present those phenomena of strict adaptation which are
equivalent as between the soil or the host and the parasite—to

a sporozoite invading the.
epithelial cell and becoming
adult
formation and discharge
of lhe sporozoites

yas D £0 UI aIp

past!

id

i” -

ee i

a mucrogamee “te
fortilising a macrogamele

Fic. 39.—Life cycle of a Coccidium (Cocezdium Schubergt).
(After Schaudinn.)

true specificities: they cannot endure any other habitat, food,
or host.

For example, Costia necatrix which lives attached to the

104 MICROBES AND TOXINS

surface of the bodies of fishes can no longer live even in the
same water when it becomes detached and floats free. The
infusoria from the paunch of ruminants or the caecum of
horses cannot live outside the bodies of these animals except
at body temperature, 37°C.: they are thus examples of
semi-parasitism. The parasites of mammalian blood are
habituated or even confined to life at definite temperatures ;
hence the effects of climate and season.

The same parasites attach themselves to one host only and
their presence becomes a specific character of the latter. The
parasite of malaria is peculiar to man and among the mosquitoes
it can only inhabit those of the genus Anopheles. But one
trypanosome can live in several species of host, one species
often serving as a sort of reservoir for others (it is probable, for
example, that cattle form a reservoir for the Z*ypanosoma
gambiense, which causes sleeping sickness in man).

Lamblia intestinalis is a parasite of the small intestine:
gregarines are only found in the large intestine, in ‘the
peritoneum, and in the genital organs of their hosts (inverte-
brates): coccidia inhabit the epithelial cells: the heemosporidia
of malaria only the red corpuscles of the blood: while’ the
sarcosporidia only occupy the muscle cells. But parasites
exist which infect all the organs of the host, eg., Wyxobolus
pfeiffert in the barbel disease.

Chemiotactic phenomena, positive or negative, are observed
among protozoa as among bacteria, and it is by an action of
this sort or by a choice of soil (which ‘closely resembles it)
that the affinity of the sporozoites of the malarial hzmo-
sporidia is explained for the salivary glands of the mosquito
which inoculates man. The hemosporidia sucked from the
blood of the patient gain the stomach of the mosquito and
there enter upon the sexual cycle, a cycle which cannot go on
in any other surroundings: this specific action is no doubt due
to certain physical and chemical conditions which are only
realised there and of which the following fact may give some
idea: the appearance of sexual forms in human blood is
favoured by adding a little distilled water to the blood of

PHYSIOLOGY OF THE MICROBES 105

a preparation (Manson). The ideas of specificity “of soil”
and of virulence must resolve themselves among the
protozoa as among the bacteria into physical and chemical
factors.

The parasitic protozoa of the intestine of one host pass
into another host through the external world in the state of
spores or cysts. The parasites of the blood cannot enter
the blood of a new individual (in nature) except through the
intermediation of a blood-sucking insect, and a portion of
their life-cycle takes place in this intermediary. Thus the
parasite of Laveran is inoculated from man to man by the
Anopheles mosquito. In these casgs the principal host is the
one in which the sexual phase of the life-cycle takes place ;
the host in which occurs the non-sexual reproduction is only
the secondary or ‘intermediate host.” With regard to the
parasite of malaria man is the intermediate or secondary host,
and the principal host is the mosquito. The tsetse fly is the
principal host of the Z7ypanosoma gambiense of sleeping
sickness.

Though the protozoa are frequently parasites, they are often
themselves attacked by parasites, by bacteria, by chytridiacez,
by saprolegnaceze, and by alge. The alge, however, may be
useful commensals, furnishing a food-stuff—starch ; but other
parasites may kill the protozoa which they infect, provided
the latter does not defend itself and overcome its parasite
by devouring and digesting it—again by intracellular
digestion.

To study the bacteria pure cultures can be made in which
they find their food material in solution. The protozoa have,
doubtless, methods of nutrition much more complicated, for
their culture is more difficult. Several trypanosomes, parasitic
in the blood, or, more precisely, in the blood plasma, have,
been grown in pure culture by Novy and MacNeal on media
to which blood was added. The parasites of malaria (parasites
of the red corpuscles) have not been cultivated. The amcebz
can be grown on condition that suitable prey is supplied: if
in a culture there are only amoebz and as prey a bacterium

106 MICROBES AND TOXINS

for example the B. coli in pure culture, the culture is said
to be “pure-mixed.” The prey may consist of a dead
bacterium.

In the absence of cultures experiment is difficult ; the study
of protozoa is still necessarily attached to the study of their
forms, and physiological study takes of necessity the second
place to morphological. The method par excellence consists
in following the life-cycle of protozoa in their natural
surroundings or in their hosts. The study of bacteria has
been capable of greater advances, thanks to pure cultures in
well-defined media which permit of chemical analysis. The
application of similar methods to protozoa is infinitely
desirable, not only for the sake of medicine, but also to extend
our knowledge of the phenomena of life in general.

CHAPTER V

PATHOGENIC MICROBES—-INFECTION

ORIGIN. —Specificety— Virulence.— ow virulence may have been acquired
—Evolution of microbes—The ‘para’ and the ‘pseudo’ forms—
Diminution and augmentation of virulence—Pasteur and attenuation of
the virus.

INFECTION.—The conflict between the microbe and the body—Methods
—of transmission—Latent microbism —Germ-carriers—The number of
microbes sufficient to produce infection— Microbial associations—Paths
of penetration and inoculation—The réle of the intestine—Seats of
election and susceptible cells—Incubation.

ORIGIN : SPECIFICITY : VIRULENCE.

THE idea of pathogenic microbes arose as a result of Pasteur’s
labours on fermentation.

For years the bacteridium had been seen in the blood of
anthrax animals without giving rise to the thought that these
microscopical rods were the cause of the illness. After the
discovery of the bacillus of butyric fermentation, Davaine
considered that the bacteridia were the cause of anthrax asa
sort of peculiar fermentation having for its subject the body of
an animal.

We do not yet know the pathogenic microbes though they
certainly exist in small-pox, in vaccinia, in measles, in
scarlatina, in mumps, and in hydrophobia. That of syphilis
remained unknown up to 1905. Pasteur was the first to handle
invisible microbes with sufficient confidence to discover a
method of vaccination. His discoveries in hydrophobia aroused

researches on the so-called invisible microbes. A nervous
107

108 MICROBES AND TOXINS

disease which resembles hydrophobia, the acute poliomyelitis
of children, has recently been studied by the same experimental
method.

The microbial doctrine has still opponents, more or less
masked, who accuse microbiologists of being able to see
nothing but the microbe and of imagining that this is the
whole malady. The body takes some part, there is no doubt ;
the malady is a sort of fermentation, but one taking place in a
medium capable of resisting the ferment. Pasteur recreated
medicine by introducing into it the spirit and method of the
exact sciences, but he knew as well as any that diseases do not
rage in an inert material. This, however, does not prevent the
various incidents of the disease from being at bottom physico-
chemical phenomena.

The Origin of Pathogenic Microbes.—The pathogenic
microbes are not instruments of a perfidious Providence, and
created to chastise man, animals, and plants. The pathogenic
species, are species the result of selection and adaptation. They
grew first of all as saprophytes on individuals who suffered no
damage, as is the case to-day with many bacteria growing on
animal bodies. They multiplied upon ill-nourished and
fatigued individuals and found on a definite animal species
nutritive materials and a chemical “soil” which suited them.
Certain bacteria have become strict parasites, incapable of
living even temporarily in the external world, e.g., the bacillus of
leprosy. These views of Pasteur are quite in conformity with
the spirit of Darwinism.

From the beginning there has been happening what occurs
every day, z.e., there has been a struggle between the parasite
and the body. Not only does the body defend itself against
the bacteria, but the bacteria defend themselves against the
body. Each is capable of gathering strength or immunising
itself against the other, and these are simply different aspects
of adaptation and of natural selection. “The science of
bacteria, as with all the branches of biology, has profited by
the theory of evolution and, making a just return, it has
supplied the Darwinian theory with a striking confirmation.

PATHOGENIC MICROBES—INFECTION 109

The great discovery of Pasteur of the attenuation of viruses
proves the plasticity of microbial species and the facility with
which they modify their primitive characters. The history of
bacterial diseases shows also the great réle which these in-
finitesimal creatures have played in natural selection, for is it
not they which have caused to disappear in the course of ages
certain vegetable and animal species insufficiently armed to
resist them? ... Experimental medicine has studied the
adaptation of certain pathogenic microbes which permit them
to attack the body in spite of the defences opposed to them.
Here it is probably a question of a selection of individuals
endowed with particularly stable characters.” 4

Starting with a bacterium almost non-virulent, Pasteur
succeeded in infecting with anthrax in succession, by the
method of passages, the new-born mouse, the adult mouse, the
young guinea-pig, the adult guinea-pig, the rabbit, and the
sheep. Vincent by introducing into the peritoneal cavity little
collodion sacs containing bacteria which are nourished by the
body fluids, while being protected from the cellular defences,
rendered pathogenic for the guinea-pig and the rabbit such
saprophytic microbes as B. megatherium and B. mesentericus
vulgatus, but the virulence thus acquired disappeared as soon
as the artifice which produced it was suspended. These
experiments do not permit of the conclusion that pathogenic
species can be created at will in the laboratory ; only more or
less stable variations are got. It is not with such ease that we
are likely to reproduce what Nature has taken centuries to
accomplish. It is very probable that small-pox and vaccinia
are two modifications of the same virus. Yet the production
of vaccinia with smallpox has not yet been successfully
performed. The experiments said to have proved variolo-
vaccination are still disputed. Nothing has been able to
produce from B. coli a Typhoid bacillus. There exist various
families of the Tubercle bacillus, which may be secular adapta-
tions from the same strain, adaptations to the human species

1 Metchnikoff, address read to the Cambridge festival in honour of the
Darwin centenary, June, 1909.

110 MICROBES AND TOXINS

and to the ox, to birds, reptiles, frogs, and fishes, but no one
has ever succeeded in producing a tubercle bacillus of the
human type from the tubercle bacilli (or acid fast bacilli) of
the frog. Nocard’s experiments in which he transformed the
human bacillus into the bird bacillus by repeated passages
have not been confirmed. The researches of recent years
confirm the idea of Th. Smith and R. Koch, that the human
bacillus and the bovine bacillus cannot be transformed one
into the other, at least under the time conditions of our
experiments ; the fixity of these acquired characters has even
raised the hope that it might be possible to vaccinate cattle
against bovine tuberculosis by means of bacilli of the human
type and vice-versd, perhaps, men with the bovine bacillus or
products derived from it.

The Darwinian conception of evolution in pathogenic mi-
crobes is nevertheless true, although direct proofs are lacking.
Similarly the simian origin of man would be quite as certain,
although some of the proofs were lacking, and even although
we might not be able to demonstrate forms intermediate
between man and monkey.

Specificity.—It is necessary to distinguish between the
specificity of the microbe and that of the disease.

Typhoid fever is caused by the bacillus of Eberth and by
it alone—specificity of the disease. The typhoid bacillus
remains the typhoid bacillus in cultures and in the intestine ;
it does not tend towards either the dysentery bacillus or the
coli bacillus—specificity of the microbe. These two ideas hang
together ; the same causes must produce the same effects if
there is to be any science at all, but to produce the same effect
it is absolutely necessary that the cause remain the same.

A disease appears with certain symptoms and anatomical
lesions due to a definite microbe, but these symptoms and
these lesions may be produced by others ; for example, the
tubercle is the lesion par excellence produced by the tubercle
bacillus of Koch, but tubercles are also produced by the
bacillus of glanders. There are not so many possibilities of
reaction in the body as there are bacteria; fever, effusions,

PATHOGENIC MICROBES—INFECTION 111

congestion, false membranes, tubercles, are all properties of
the body rather than of the microbes. The specificity of the
disease consists in a definite combination of symptoms along
with the invariable presence of a definite bacterium. To
render possible the study of disease it was a primary necessity
for a certain disease to be the result of a certain micro-
organism, and further for this latter to be of fairly stable
natural characters. The typhoid bacillus causes typhoid fever,
but if it were capable of changing its characters the hygiene
and prophylaxis of this disease would be without any sure
foundation.

Microbiology, medicine, and hygiene therefore can no more
do without this idea of specificity than science in general could
exist without the idea of causality.

Medicine has always been pursuing this conception, but has
only finally seized it by the help of microbiology and chemistry.
Even the beliefs in ‘‘ miasmata” and “epidemic causes” were
already attempts in this direction. Common sense has always
been a believer in the specificity of transmissible diseases. It
was for that reason, as we read in the Old Testament, that the
Israelites isolated lepers. Herodotus knew that leprosy passes
from man to man; Galen believed in the specificity of hydro-
phobia, of scabies, of granular conjunctivitis. The idea of
a specific disease was bound to suggest the idea of a specific
agent.

The fact that those who have had small-pox scarcely ever
take it a second time, but are still quite virgin soil for’ measles
and scarlatina and vice versé, spoke again in favour of the
specificity of diseases. Jenner’s discovery even furnished
a general principle of diagnosis between specific viruses.

In favus, in pityriasis versicolor, in the ring-worms, and in
thrush, there had already been observed before the days of
Pasteur the constant presence of the same microscopical fungi,
and it had even been concluded that these diseases were due
to parasites. But the opponents of this idea maintained
(there are perhaps still existing some who believe this) that
these organisms were not the cause, but in a way constant

112 MICROBES AND TOXINS

“cottnesses” of the diseases, and simply the inhabitants of
lesions which they had not produced—just as the same moulds
are usually found in pots of the same jam left open to the air.

The specificity of infectious diseases has been demonstrated
by Pasteur and Koch.

Since there is no “spontaneous generation,” at least in the
world of the present day, it is impossible for the micro-
organisms to originate in the diseased tissues, and the science
of fermentation has proved that a given cell, inoculated in a
sterile fluid of known composition, produces in it definite and
constant phenomena. These ideas were taken up by medicine
when Davaine maintained that the bacteridium was the
cause of anthrax, and when Obermeier held that recurrent
fever was caused by the spirochete found in the blood of the
patients during the fever.

To prove the specific activity of a ferment it is necessary to
isolate and make pure cultures of it and to re-inoculate it. It
was this that we learnt from Pasteur and Koch. Koch’s
memorandum on tuberculosis remains the complete model
for the discovery of fhe microbe of a disease and of the
demonstration of its specificity.

Specificity of function is the point of capital importance.
The fixity of form in bacteria is of great use in the search for
and identification of them ; but on this latter point science has
been obliged to become less exacting ; the form of the bacteria is
not always a sufficient distinguishing character. They are
really defined by their chemical and physiological actions.
Thus the tubercle bacillus is better defined by its staining
peculiarities (a physico-chemical reaction) than by its shape ;
still better by the appearance of pure cultures than by staining;
and better still than by the cultures, by its excretion of
tuberculin. Finally, the study of the reactions of the body,
z,é., of immunity, has shown that the cells of the patient respond
in a specific manner to the attack of the bacterial cells, and both
medicine and hygiene daily employ the property of specificity
in the antibodies.

The “ Para”’ and ‘ Pseudo” Bacteria.—We may now

PATHOGENIC MICROBES—INFECTION 113

go on to cite a series of facts which have modified ‘the idea of
rigorous specificity among microbes, a specificity which in the
early days of medical bacteriology was believed to be
absolute ; or at least these facts have restricted this idea (by
compelling us to create new varieties), although we may still
regard it as sufficient for the purposes of medicine and hygiene.

It was long the custom to talk simply of ¢he bacillus of
diphtheria, ‘Ze typhoid bacillus, ¢4e B. of dysentery, ¢he
meningococcus, ¢#e cholera vibrio, etc., but little by little
there have been discovered bacteria, close relatives of each of
these typical microbes, but not possessing all their characters.

From the time of the first bacteriological discoveries in
diphtheria, bacilli were isolated from the mouth exactly
similar to the pathogenic bacillus, but non-toxic; the best
known is that described under the name of Hoffmann’s
bacillus. They have been called pseudo-diphtheria bacilli, and
have been found in diphtheritic sore throat, in scarlatinous sore
throat, and in the normal conjunctiva, even sometimes in
vaccine lymphs. Some of them are pathogenic for the guinea
pig, and produce in it a septicaemia bearing no resemblance to
diphtheria. They are not affected by antidiphtheritic serum
and are incapable themselves of being used to produce such
a serum. Roux’s opinion was that it was a question of
degenerated diphtheria bacilli, or of bacilli not yet adapted,
not having yet found the conditions capable of exalting their
virulence. It must be added that the name of pseudo-
diphtheria has been incorrectly applied to bacilli which do
not deserve the name, even by their form.

It is no use playing with words. It is on the clinical facts
the problem ought to depend. There are found in the most
typical cases of diphtheritic sore throat diphtheria bacilli
possessing every degree of toxicity, and also bacilli which are
not pathogenic (z.¢, for the guinea-pig, since they cannot be
inoculated in man). All these bacilli are called diphtheria
bacilli. On the other hand, there exist non-diphtheritic
affections both of the throat and of the nose in healthy
individuals where bacilli resembling diphtheria but non-toxic

I

114 MICROBES AND TOXINS

are to be found; the name of pseudo-diphtheria has been
agreed upon for them. Are they capable of becoming toxic?
Under the conditions in which we can experiment it is
scarcely possible to give an answer. There is a possibility,
and even a probability. But once the natural selection has
occurred, it is undoubtedly the toxic bacilli which get all the
chances of passing from mouth to mouth and of maintaining
their hereditary privileges.

The specificity of the meningococcus has had to be defended
against a group of bacteria resembling it in form and cultural
characters—the pseudo-meningocout. ‘The true meningococcus
may be distinguished from these by various biological reactions,
but it is certain that the principal character from our point of
view is the property of causing meningitis, and it is difficult to
say whether or not, and under what conditions, the ‘‘ pseudo”
forms may acquire this power.

Not only have there been distinguished three pathogenic
types of the dysentery bacillus (Shiga, Flexner, and Strong),
but a whole group of pseudo-dysentery bacilli has been
admitted, and side by side with the true bacillary dysentery
there have been decribed dysenterifcrm affections caused by
these ‘‘pseudo-bacilli.” In reality the biological reactions
have proved that there is only one fundamental type of patho-
genic dysentery bacillus, but that there exist none the less
satellites of this which possess biological importance, although
of less importance from the point of view of medicine.

Hygiene cannot afford to neglect the non-pathogenic
cholera vibrios, often very difficult to distinguish from the vibrios
isolated from genuine fatal cases of cholera. Since the
prophylaxis is based on the discovery of the microbe, and
since no laboratory animal exists which readily takes cholera,
it has been necessary to employ refined diagnostic procedures,
and these do not solve the scientific question of the relations
in nature between these different vibrios. In certain maladies
closely resembling typhoid fever (but almost always benign,
rarely fatal), bacilli have been found which only differ from the
typhoid bacillus in certain cultural peculiarities, differing also

PATHOGENIC MICROBES—INFECTION 115

from the B. coli which is so abundant in the normal intestine.
Two principal types can be distinguished, A and B: the latter
is nearer to the B. coli, the former to the B. typhosus. They
are called para-typhoid or para-coli bacilli, but do not on that
account call in question the specificity of the Typhoid bacillus ;
the latter rather appears as a chosen specimen out of a
numerous family containing other well-defined pathogenic
bacteria, among which are not only these para-A and B, but
various bacilli producing meat-poisoninys, diarrhcea among
animals, and the pneumo-enteritis of pigs. This family is
even the one in which the biological reactions have been
found most suitable for establishing fairly definite degrees of
relationship. We class them by their relations to the human
species, putting at the head of the column the typhoid
bacillus. It is evident that if we were calves or pigs our point
of view might be somewhat modified.

One of the properties characterising the tubercle bacillus of
Koch is its acid-fastness (it takes on stains with difficulty, but
once stained by the suitable colour, it resists the decolorising
action of acids). There exist numerous acid-fast bacilli and
even numerous bacilli capable of producing tubercles in the
tissues. Between the human and bovine bacilli and the
bacilli found in grass, in manure, and even in smegma, there is
a long series of intermediate types. Are we to believe that
among the acid-fast and para-tubercle bacilli the ancestor of
the tubercle bacillus of men and the ox is to be found? It is
rather a philosophical question and escapes experimental
examination. But the general truth of the Darwinian ideas
compels us to this belief. According as one is physician or
veterinary surgeon, according as one is engaged in diagnosis or
in treatment, according as one is accustomed to think as a
naturalist and to class all living beings in groups, so one tends
to insist on differences on the one hand, on resemblances on
the other. The production of tuberculin and the re-inoculation
of tuberculous lesions in series in a given species are methods
of differentiation which do not invalidate the existence of a
great natural family.

116 MICROBES AND TOXINS

Small-pox and vaccination are without doubt of common
origin; Jennerian vaccination is based as much on their
relationships as on their differences. In the same way the
distinction between the human bacillus and the bovine bacillus
is not inconsistent with a common origin. The numerous
experiments which have been made to vaccinate cattle with
the human bacillus prove that it is a question of two adapta-
tions from the same type, and unfortunately they are not
sufficiently differentiated for one to be sure that the bovine
bacillus is incapable of producing in man a tuberculosis, not
merely local and benign, but general and fatal.

The specificity of bacteria is only relative, but it suffices for
the carefully performed bacteriological diagnosis which is so
useful in medicine. Instead of the solitary types which once
seemed to constitute the whole species, we know now varieties
and families of which one member only may be the constant
cause of a definite disease ; and this is of advantage to both
medicine and hygiene. In theory it is evident that the only
point of view to be accepted is that of the plasticity of
microbial species, 7.e., the Darwinian theory.

Virulence.—Virulence is in the first place the capacity in
a microbe to settle and develop in the bodies of animals;
secondly, its capacity of secreting its toxic substances. Even
in the strictly toxic diseases, such as tetanus, the intoxication is
not the whole malady ; there is a preliminary, the penetration
of the microbe, which may or may not find suitable conditions
for its growth.

Virulence is a variable property, and it was in connection
with modifications in virulence that Pasteur had the intuition
of the possibility of attenuating a virus.

Diminution of Virulence.—In cultures on artificial
media in the laboratory the virulence diminishes spontaneously ;
the media may be improved by adding animal fluids (serum,
blood, ascitic fluid). A little too much or too little acidity or
alkalinity, too much or too little peptone or salt, may cause
our nutrient broth to lower the virulence of the strain from
the change in its reaction.

The diphtheria bacillus and the streptococcus are not suited

PATHOGENIC MICROBES—INFECTION 117

by acids; the cholera vibrio finally suffers from the alkalinity
which it itself produces; the presence of fatty materials is
injurious to anthrax bacillus. There exist processes for
diminishing virulence: 1. The action of a high temperature
(Toussaint, Pasteur), ¢.g., a temperature of 41° to 43°C.,
instead of the body temperature of 36-37°C. 2. Temperature
+aeration (Pasteur’s experiments on fowl-cholera and swine-
erysipelas). 3. Desiccation (Pasteur : preparation of the spinal-
cord of rabbits in the treatment of hydrophobia). 4. Light,
pressure, oxygen under pressure. 5. Antiseptics (Roux:
carbolic acid, potassium bichromate, etc.).

Increase of Virulence—Passages.—For this purpose
one provides a microbe with the food-conditions which suit it
best ; oxygen of the air for the B. diphtheria; extracts of
putrefying meat for the B. of tetanus (Brieger and Cohn).
The virulence is augmented by accustoming the bacteria to
the body against which they are being prepared ; the feebler
individuals are destroyed by the natural defences, and a
selection of the strongest members takes place.

A bacterium may be habituated to the guinea-pig by com-
pelling it to live in the peritoneum of this animal enclosed in
a collodion sac, which permits the penetration of the nutrient
juices while keeping off the leucocytes. It is a culture in the
living body. Habituation is chiefly produced by the method of
passage, z.e., by inoculating the bacterium in an animal and
from this animal into another (generally of the same species).
In certain cases a degree of virulence is reached which cannot
be exceeded in the species of animal employed ; thus the virus
of hydrophobia becomes virus fixe after a certain number
of passages through the rabbit. Passages do not perceptibly
affect the tubercle bacillus, but have a pronounced influence
on the streptococcus. According to Marmorek the strepto-
coccus, which required at first a dose of 1 c.c. to kill, could be
brought by passage to kill with a dose of o’o0000000001 c.c.
Pasteur raised the virulence of the anthrax bacillus by passing
it through new-born animals, then through older ones, then
adults, and finally through different species.

Passage does not give the same results in all cases, and

118 MICROBES AND TOXINS

qualitative variations occur which render it necessary to take
into account the species of animal employed. Pasteur, after
exalting the virulence of the virus of rabies for the dog by
passage through rabbits, found that passage through monkeys
weakened it. The bacillus of swine-erysipelas becomes more
virulent (for the pig) after passage through pigeons, less virulent
by passage through rabbits. Passage through a foreign species
has been used to prepare vaccines ; thus the virus of vaccine
lymph—undoubtedly of the same origin as that of smallpox—
has become a “vaccine” by passage through the cow; the
pox of pigeons becomes a “vaccine” for pigeons by passage
through the fowl. In passing from fowl to fowl the spirillum
of Marchoux and Salimbeni gets weaker. And while it is true
that a feebly virulent anthrax strain can “ recover ” its virulence
by passages beginning on new-born mice, in the spirillosis
of fowls, on the contrary, according to Marchoux, it is precisely
the young of the species concerned which is the animal of
choice for weakening the virus and preparing an efficient
vaccine for the adult.

Further, the age of the “young” individual must be
reckoned precisely: it is known that the new-born child is
less susceptible to Jennerian vaccination than the child of
three or four months.

The modification of virulence may be expressed by a
difference in dose in relation to a “soil” agreed upon. The
example of Marmorek’s streptococcus gives a numerical
measure of the increase in virulence. The spirochete of
Schaudinn was inoculated from man into anthropoid apes,
from these into the lower monkeys, thence into the rabbit,
and thence into the guinea-pig; but this has chiefly been a
case of progress in the technique of inoculation, for nowadays
it is possible to inoculate directly from man into the rabbit.

Attenuation of Viruses.—There was in Pasteur’s
laboratory a culture of the bacterium of fowl cholera which
was being reinoculated daily and was of constant virulence.
It happened that a culture was taken for inoculation which
had remained untouched for several weeks in the incubator ;

PATHOGENIC MICROBES—INFECTION 119

the fowls became ill but did not die, and further, they resisted
a second inoculation of a very virulent strain which killed the
controls. This was the first demonstration of an attenua-
tion produced by keeping in contact with air at incubator
temperature. By taking cultures of different ages, a scale of
virulence could be produced —a series of “ vaccines.”

If on re-inoculating an enfeebled culture, one obtained a
virulent culture, it would not be correct to speak of attenua-
tion, but merely of enfeeblement—transitory lowering of the
virulence.

Attenuation is only applied to a permanent enfeeblement,
one which passes from one generation to the next. One
cannot say hereditary because, strictly speaking, heredity only
occurs when there is sexual reproduction. In the experiments
on fowl cholera the new cultures showed themselves to be
weakened in series ; it was thus a true attenuation.

“Tf you take each one of the cultures whose virulence has
been attenuated as the starting point of successive cultures
and without a perceptible interval in the starting of the
cultures, the whole series will reproduce the attenuated
virulence of the one serving as starting point. Similarly, a
culture of zero virulence reproduces another of the same”
(Pasteur).

A similar process of attenuation appeared at first inapplic-
able to the anthrax bacillus; as the culture grows older the
bacterium sporulates and the spores are not affected by the
conditions which act upon the bacillary form. One could only
therefore expect attenuation from an anthrax bacillus which
did not produce spores.

Now at 42°5 the anthrax bacillus does not sporulate.
Pasteur cultivated it therefore at 42°°5 in order to diminish its
virulence by the action of the heat and the air.

It was then found that sporulation, instead of being an
obstacle to attenuation, was a condition entirely favourable ;
reinoculated at 35°, a bacillus attenuated at 42°°5 produced
sporulating bacilli, but the bacilli germinating from these
spores possessed the same degree of virulence as the bacilli

120 MICROBES AND TOXINS

from which the spore was derived. Pasteur had obtained an
attenuation fixed by the resistant form, the spore; “ vaccine
viruses fixed in their germs, with all their peculiar qualities
without any possible alteration.”

It was by these modifications of virulence that Pasteur
explained the behaviour of the great epidemic diseases :

“There exist virulent diseases which appear spontaneously in
every country: such is, for example, the typhus fever of armies
in the field. Without doubt the germs of the microbes
responsible for these maladies are to be found everywhere.
Man may carry them on his body or in his alimentary canal
without suffering great harm, but they are, nevertheless, ready
to become dangerous when, under conditions of overcrowding
and successive development on the surface of wounds, in
weakened bodies or otherwise, their virulence becomes progres-
sively reinforced.

“Virulence thus appears under a new light to us and one
which is distinctly disquieting for mankind, unless nature during
the past centuries has already met with all the opportunities
possible of developing virulent or contagious diseases, which
is highly improbable.

* A microscopical organism, harmless for man or for a given .
animal species, is simply a creature which cannot develop in
our bodies or in the body of the given animal, but there is
nothing to prove that if this microscopical creature succeeded
in penetrating another of the many thousand species in creation,
it might not invade it and produce in it disease. Its virulence,
reinforced then by successive passages through individuals of
this species, might become powerful enough to attack such
and such an animal of higher position, man, or the domestic
animals. In this way it is possible to create new virulences
and new contagions. I am much inclined to think it is
thus that there have appeared throughout the ages small-pox,
syphilis, plague, yellow fever, etc. . . . and further that it is
by phenomena of this kind that there from time to time
appear certain great epidemics. . . .”

PATHOGENIC MICROBES—INFECTION 121

INFECTION.

Infection may be defined thus: the attack on one living
being by another which penetrates it and lives parasitically at
its expense.

It is simply one case of the universal struggle and com-
petition among the species.

The conflict between the invader and the invaded resolves
itself into a question of nourishment and digestion. ‘The
parasite attacks by secreting toxic or dissolving substances,
and defends itself by paralysing the digestive and expulsive
powers of its host. This latter exerts a noxious effect on its
aggressor by digesting it or eliminating it from its body, and it
too defends itself by means of secretions.” !

Infection exists among the amcebe. An amceba invaded
by the parasites described by Metchnikoff under the name of
Microsphera finally succumbs. Certain infusoria are infected
by Acinetians, which pierce their cuticle and invade them.
The green euglena is subject to infection by lower fungi of
the chytridian group; they lose their green chromatophores
and become literally anzemic. Infectious diseases are not the
peculiar privilege of man and the higher vertebrates.

On the great problem of the origin of microbes, their mode
of transmission, and the way in which they penetrate the body,
bacteriology and hygienic science have accumulated many
facts.

The microbes inhabit the air, water, the soil, animals, and
plants, and gain access to the patient either directly by simple
contact with another patient or thanks to more or less numerous
and various intermediate agents. Contact is sufficient for the
transmission of measles, small-pox, and scarlatina ; these are
the contagious diseases properly speaking. ‘The air may carty
the germ from one individual to another. In the air the
B. tuberculosis may float, attached to particles of dried dust
or to moist droplets, projected into the air by the patient

1 Metchnikoff, Pathologie comparée de ? Inflammation,

122 MICROBES AND TOXINS

during speech or coughing (Fliigge). Water-supplies convey
the cholera vibrio and the typhoid bacillus. Along with garden

nucleus

minute alge
(zoochlorella)

Fic. 41.—An ameeba dying full
(After Gruber.) of parasites.

Fig. 40.—Amceba Ameba viriais).

Sphaerophrya
applying itself
BY othe infiusorian

two parasitic
sphaerophrya

Fic. 42.—Green euglena enclosing iG. 43.-—Infusorian attacked by
a chytridium (lower fungus). acinetian parasites (Sphero-
(Metchnikoff.) phrya). (Metchnikoff.)

earth the tetanus spore may gain access to a wound ; the soil
may spread anthrax spores over sheep pastures. ‘The foods
which are eaten raw (milk, meat, vegetables) convey what they
have gathered from the soil or from the bodies of animals.

PATHOGENIC MICROBES—INFECTION 123

The vehicle of transmission may be a living creature instead ot
an inert object ; fleas carry plague from rat to rat and from
man to man. The intermediate inoculating agent may con-
stitute a storehouse and even a culture chamber for the virus,
eg. the tick in spirillum fever. This intermediate agent
becomes properly speaking a fost when the germ undergoes
in it, and can only undergo in it, a cycle of changes by which
it attains the stage at which it can infect us ; for example, the
mosquito for the parasite of malaria and the tse-tse fly for the
trypanosome of sleeping-sickness. The transmission of bovine
piroplasmosis from ox to ox is conducted by two individuals :
a tick becomes infected, produces larve, and these larve
inoculate another ox.

Latent microbism is said to exist when the organs and tissues
contain germs which remain for a longer or shorter time
unsuspected.

Pasteur thought that our organs and tissues were normally
aseptic: “‘The human body is completely closed to the intro-
duction of the germs of fermentation ” ; and he was still more
right in adding, ‘ Except the alimentary canal and again except
in certain pathological conditions.” The body defends itself
well against the microbes which enter it; “latent” microbes
can only be those which escape phagocytosis at least temporarily.
The best example is furnished by cases of spontaneous tetanus,
appearing under the influence of a heatstroke; this tetanus is
due, according to Vincent, to the germination of spores which
have penetrated the body by some unknown path, and have
remained there for several days or even several weeks, till the
day when the excessive heat accompanied by fatigue interrupted
the phagocytic defence! The experiments of Porcher and
Desoubry teach us that the blood is rarely aseptic during
digestion.

Auto-infection is said to exist when an individual is infected

1 Tetanus spores inoculated in the blood or under the skin of rabbits are
only eliminated after three or four weeks (subcutaneous inoculation), or
even afler three months (intravenous inoculation). They ‘wake up’ and
germinate, should favourable conditions supervene, among others necrosis
in the tissues (Tarozzi).

124 MICROBES AND TOXINS

by bacteria of which he is himself a carrier. The term of
auto-infection gained a precise signification only when germ-
carriers became known.

Germ-carriers are those individuals who harbour the microbe
of the disease yet present no symptom of this. The fact is not
entirely novel (it has been known for long that the pneumo-
coccus exists in individuals recovered from pneumonia, and
Pasteur discovered it in the saliva of a child dead of rabies),
but it is only during recent years that all its importance has
been recognised.

There exist carriers of typhoid and paratyphoid bacilli, of
diphtheria, of cholera, of dysentery and of meningococcus
There are individuals who have acquired the microbe without
yet contracting the disease—precocious carriers : others recently
cured and not yet freed from their bacteria—convalescent
carriers ; individuals cured weeks, months, or even years before—
chronic carriers: finally there are occasionally individuals who
have never had the disease—healthy carriers or “ paradoxical
carriers.” It is of great importance to recognise germ carriers
especially in communities where the general life is intimate and
confined, as in schools and barracks.

In typhoid fever in particular the germ-carriers are most
often women. According to Frosch’s statistics, there is one
woman for every five cases of typhoid, but of every five chronic
typhoid-carriers four are women. The typhoid bacilli in
chronic typhoid-carriers select as their favourite habitat the
bile-ducts.

Conditions of Infection.—These are very variable,
varying with the virulence, the resistance of the body, and the
method of inoculation.

The number of attacking bacteria can be calculated fairly
closely in experiments, but in the natural disease it is impossible
to tell how many bacilli are necessary to determine a given
infection.

The rabbit is extremely sensitive to the bacilli of fowl-
cholera. Now, according to Watson Cheyne, if 10,000 to
30,000 bacteria are injected there is only a local abcess ; above

PATHOGENIC MICROBES—INFECTION 125

this figure a general infection is practically certain ; but on the
other hand the same observer states that one bacillus is
sufficient to produce fatal anthrax in the mouse. There are
certain experimental facts as regards the smallest quantity of’
bacilli capable of producing tuberculosis but these have not
an absolute value. H. Buchner found that to spray 1ooc.c. of
a dilution of 1 per 100,000 of tuberculous sputum was
sufficient to give miliary tubercle of the lung to guinea-pigs.
The sputum employed contained approximately 80,000 bacilli
perc.c. and he calculated that each guinea-pig had been exposed
to the attack of roo bacilli (these figures are of course very
approximate). In the experiments of Preisz, zy milligram of
sputum, containing about forty bacilli, was found sufficient.
In the experiments of Findel, guinea-pigs inhaling about sixty
bacilli regularly took tuberculosis.1

Microbial Associations.—The microbes which cause
diseases are never derived from pure cultures. Their virulence
is modified not only by the chemical properties of the
medium and the “soil” on which they fall, but by the
presence of other species favourable or the reverse. There
are streptococci which aggravate diphtheritic sore throats.
The coliform bacillus, the torula, and the sarcina described by
Metchnikoff favour the production of intestinal cholera. In
tetanus the associated bacteria are helped also by other
favouring factors, such as splinters, bruising of the tissues, and
blood clots. It was formerly thought that the streptococcus of
erysipelas was antagonistic to the anthrax bacillus. Pasteur
observed an antagonism between the B. pyocyaneus and the
anthrax bacillus; if these two bacteria are inoculated on a
medium in lines which cross each other, the anthrax bacillus
grows very feebly at the points of intersection. ‘The proteolytic
enzyme of the B. pyocyaneus, the pyocyanase, is injurious to
many bacteria, and occasionally plays the part of a disinfectant
(Emmerich).

1 Tuberculous sputum (caseous material) frequently contains 50,000
bacilli per mg. In cultures half-dried on paper there are about 35 to 4o
millions per mg.—with a possible error of one million (Chaussé).

126 MICROBES AND TOXINS

In the infections due to a well characterised microbe,
capable of itself of causing the disease, the associated bacteria
most often act by turning upon themselves the phagocytic
attack ; their rdle is thus secondary.

The study has hardly commenced of. those bacterial associa-
tions which have no specific pathogenic power, but which act
nevertheless favourably or the reverse by the products of their
metabolism. Their sphere is chiefly the alimentary canal, and,
according as the dominant flora of the intestine is acid pro-
ducing or produces indols and phenols, the general health
escapes or is subject to the action of the sclerosing toxins.
These microbial associations thus constitute a certain condition
or disposition rather than a true disease. There can be
distinguished in it a fundamental flora and an accidental flora,
and these can be modified by fortifying one species at the
expense of the others; it is in this that bacteriotherapy
consists.

In the mouth, microbial associations produce a disposition,
more or less marked, towards the development of inflammations
of the throat.

The body presents a field capable of infinite variations ; we
have to reckon with species, age, and physiological conditions :
hunger, cold, and fatigue. Experiment alone could teach us
that a mammal, such as the rabbit, is more sensitive to avian
tuberculosis than to the tuberculosis of mammals, or that the
rabbit is extremely sensitive to the bacillus of fowl-cholera and
the pigeon to that of swine-erysipelas. The Algerian sheep is
more resistant to sheep-pox than the sheep of Camargue. In
general very young animals are more sensitive than adults,
yet the young pig hardly ever contracts swine-erysipelas under
three months.

Hunger, heat, or cold, and fatigue act by depressing the
phagocytic defences.

Paths of Penetration into the Body.—The mosquito
inoculates the virus which it carries either under the skin
or directly into a blood-vessel. The spirochzte produces
syphilis only when it is inoculated strictly in the subcutaneous

PATHOGENIC MICROBES—INFECTION 127

cellular tissue. ‘The virus of hydrophobia spreads from the
region of the bite to’the nerve centres along the nerve trunks.
Tuberculosis appears different according as it is inoculated
along one path or another ; in natural disease different methods
of propagation are associated. The two following ideas are of
great importance.

1. Zhe role of the intestine in those diseases in which the
infection ts not purely intestinal.—The question was long ago
put by Chauveau in relation to tuberculosis. Behring has
taken it up again, and maintains that every case of pulmonary
tuberculosis in the adult is the extension of an intestinal tuber-
culosis acquired from milk in the earliest infancy, but remain-
ing latent for years. Tuberculosis in general, according to
him, does not attack the lung until it has pierced the barrier
of the intestine. A similar origin has been claimed for other
infections which finally settle in the lung, such as the pneunio-
coccal inflammation. A comparative case was found in the
anthracosis of coal-miners (the impregnation of the lung with
coal-dust), and since it is easier to experiment with inert dust
particles than with virulent bacteria, anthracosis has become
the field of study and discussion in the question for and
against intestinal infection.

It has been settled by experiment that living bacteria can
pass through the intestinal mucosa like dust particles without
leaving any lesion as a mark of their passage. But such
passage only occurs as a rule when massive, repeated doses are
ingested, and when there are in the intestine such injuries as
favour penetration. It is therefore chiefly by inhalation and
by penetration of the lymphatics and blood-vessels in the
neighbourhood of the pharynx that the tubercle bacillus
reaches the lungs.

2. Seats of election and receptive cells—The hydrophobia
virus fixes itself on the nerve-tissue, the parasite of malaria on
the red corpuscle of the blood, other protozoa on the white
corpuscles. The dysentery bacillus,’ inoculated under the
skin, proceeds to make a home for itself in the large intestine.
The bacillus of swine-erysipelas inoculated in the pigeon

128 MICROBES AND TOXINS

is chiefly found infecting the large endothelial cells, the
“Kuppfer cells,” of the capillaries of the liver.

If the virus of small-pox or of sheep-pox is injected intra-
venously, it is simply carried by the blood and settles and
multiplies in the skin. In those diseases which Borrel has
grouped together under the name of Lfithelioses, there exists
a fairly strict cellular specificity: the virus only develops
vigorously in the interior of epidermal cells, and these cells
from the moment of infection take on a special character ;
they are therefore justly called the receptive cells. The reason
why we have failed to inoculate certain diseases is that the
proper site of inoculation has not yet been discovered, z.e., the
receptive cell, or that this receptive cell requires to undergo,
before becoming truly receptive, certain modifications which
are still unknown and which we cannot reproduce.

“In cancer our methods of inoculation in a normal indi-
vidual fail to strike the receptive cells and. to transform the
normal into cancer cells: it is this which constitutes the whole
etiological problem of cancer” (Borrel).

Between the date of penetration of the virus and that of the
appearance of the disease there is a period of zacudation, during
which the virus propagates itself, multiplies and affects the
cells on which depend the symptoms. The duration of the
incubation varies primarily with the virus, secondarily with its
quantity and the path by which it has gained access. For
example, the incubation is quite short when experimental
septiczemia is produced with a virulent streptococcus, whereas
in human leprosy it may last for years. In rabies the period
of incubation is shorter, and the disease more violent when
the bite is on the face than when it is on the leg. In tetanus
(a toxin disease), the longer the incubation the less serious
the disease.

The spirillum of recurrent fever causes a disease of the
septiceemic type, z.e., the microbe inhabits the blood. The
tubercles characteristic of tuberculosis and glanders represent a
reaction of the cells of the mesoderm, while the pustular
diseases like small-pox are typically reactions of ectodermic

PATHOGENIC MICROBES—INFECTION 129

cells. But the same disease may be in different phases
septiceemic or localised in a tissue, and in any case the blood
itself is simply a tissue whose cells are motile. It was long
thought that typhoid fever was an infection localized to the
small intestine and to the Peyer’s patches there, but in reality
it is septicaemic during the whole of the febrile period. There
are also septiczemic phases in pneumonia and tuberculosis.

Microbes are not inert particles but living cells acting
through their secretions and toxins. Cholera is an intestinal
infection, but it is fatal to its host from a general intoxication.
Every infection is to some extent an intoxication. Even the
macroscopic parasites, to which formerly only a physical
activity was ascribed, the bothriocephalus, the ankylostoma,
the trichina, secrete poisons which have been studied.

Microbes, ferments, and toxins are inseparable terms, and
for this reason the discovery of the diphtheria toxin by Roux
and Yersin in 1888 began a new era in bacteriology.

CHAPTER VI

INFLAMMATION AND PHAGOCYTOSIS

The comparative pathology point of view—Inflammation throughout the
series of natural species—defined by phagocytosis—Inflammation
among invertebrates without nerves or blood-vessels—The phagocytes
and intracellular digestion — Chemiotaxis — Phagocytes in man—
Phagocytosis in the chronic infections—The examples of the squirrel-
rat, spermophilus, and of the jerboa.

THE essential fact of inflammation is the reaction of the
phagocytes towards the injurious material (Metchnikoff).
Phagocytosis is not a theory but a doctrine, a collection of
accumulated facts. It has put the finishing touch to the work
accomplished in medicine by Darwin, Virchow and Pasteur.
From Darwin it has derived its fundamentally evolutional
character : itis founded primarily on comparative pathology and
demonstrates the persistence of the same phenomenon
throughout all the animal species. It has derived from Virchow
its foundation on cellular pathology, z.¢., on the essential réle of
the body cells in disease. From Pasteur it derives the
fundamental idea of the rédle of microbes in the production of
infections.

It required a zoologist applying the method of comparative
study to demonstrate that the only constant phenomenon in
the different forms-of inflammation is the active incorporation
of injurious elements by fixed or, more often, migratory cells
which are capable of digesting these. Jnflammation is
essentially phagocytosis and phagocytosis is summed up by

intracellular digestion.
130

INFLAMMATION AND PHAGOCYTOSIS 131

The four cardinal symptoms redness, heat, pain and swelling
represent only an external definition of inflammation. The
sum total of the facts is so complex that many observers in.
former days refused to give a simple definition, proposed to
abandon the vague term of 27/Jammation, and limited themselves
to a description of the variety of facts.

The tissues, the vessels, and the nerves of the injured part
participate in the state of inflammation: to which is the
primary rdle to be ascribed? Virchow maintained that it was
the tissues, and that these were in a state of supernutrition at
the expense of the #wzd parts of the blood; the cells multiply
at the injured point and it is from the tissues of this same
region that the numerous cells of the inflammatory exudate
are derived. Inflammation taken as a whole represents a
danger to the body.

But when Cohnheim, in his observations on the frog’s
mesentery exposed to the air, discovered diapedesis or the
escape of the white corpuscles through the walls of the
vessels, and when it was established that the pus cells instead
of developing on the spot by the proliferation of the cells of
the connective-tissue came from the motile cells of the blood,
the primary fact of inflammation seemed to be the vascular
irritation, the other appearances being secondary. Cohnheim
thought he had proved this by his well-known experiment ; a
frog’s tongue is ligatured at the base so as to stop the
circulation. On untying at the end of forty-eight hours the
circulation is re-established, but is now of the inflammatory
type with diapedesis.

But if the vascular phenomena are of primary importance
it is difficult to account for the fact that microbial or other
foreign substances introduced under the skin produce an
inflammatory reaction, but fail to do so when injected into the
vessels themselves.

Light was first thrown on the problem by the comparative
study of lower organisms.

Inflammation in the Lower Organisms.—The jelly-
like plasmodium of a mycetozoon pricked or burnt responds

K 2

132 MICROBES AND TOXINS

to the injury by movements of attraction or repulsion
(varying according to circumstances) of its protoplaam. A
foreign body introduced into it is engulfed and then rejected.

Fic. 44.—Plant filament surrounded by the phagocytes of Spongilla.
(Metchnikoff. ) i

If into the body of a sponge a little glass tube or an asbestos
fibre or any sharp foreign substance is introduced, motile
ameeboid cells from the mesoderm soon come and surround it,
the same cells which are capable of surrounding and digesting
both inert granules and living prey. This engulfing power of

Phagocytes

Fic. 45.—Mass of phagocytes Fic. 46.—Phagocytes of the
round aspike in Azpiznnaria worm collected round
asterigeria. (Metchnikoff.) a foreign body. (Metch-

nikoff. )

the mesodermic cells (and of certain endodermic cells also) is
aided by the sensitive contractile ectodermic elements.
The larvee of a certain sea-anemone (Astropecten pentacanthus),

INFLAMMATION AND PHAGOCYTOSIS 133

transparent and thus easily observed, have neither nervous
system nor vessels nor muscles: they react towards a pene-
trating foreign body by an accumulation of amceboid meso-
dermic cells. In a larger larva, Bipinnaria asterigeria, the cells
of the mesoderm can be seen engulfing particles of carmine or
of indigo and surrounding a splinter or a drop of blood with
masses of cells equivalent to a plasmodium. They also engulf
bacteria introduced under their outer skin.

In all these cases inflammation occurs without blood or
blood vessels. Among the Annelids which possess a closed

monosporae

Fic. 47.—Daphnia infected by Monospora. Fic. 48.—Different stages of
(Metchnikoff. ) Monospora. (Metchnikoff.)

vascular system, the reaction against foreign bodies takes place
in the same way without any intervention of the blood vessels.

In Zumbricus, whose male sex-glands are infected by
gregarines, there is a struggle between the two organisms, the
gregarine encysting itself and becoming surrounded by an outer
covering of protective chitin, while the amoeboid cells sur-
rounding it join together and form a sort of armour-plating
which stifles it. The blood-vessels remain inactive.

For an example of parasitism exactly parallel to an infectious
disease there is nothing better than to observe Daphnia magna
being invaded by a microscopic fungus, the AMonospora bicuspi-
data. The motile cells engulf the spores of the mould and

134 MICROBES AND TOXINS

attempt to destroy them by digestion: a struggle takes place,
sometimes the Daphnia is victorious, sometimes it succumbs.
In the young of a vertebrate, the axolotl, if the non-vascular
rudiment of the fin is pricked with a needle charged with a
little carmine or indigo powder, the migratory cells can be seen

CONG

F1G. 50.—Two leucocytes of Daphnia sur-
rounding a conidium of A/onospora.
(Metchnikoff.)

filaments

Fic. 49.—Spores of JMono- or oscillaria

spora, surrounded — by
leucocytes of Daphnia.

(Metchnikoff.) The spore ” 2 ae
is transformed into F
granules.

Fic. 51. - An amoeba (Amada verrucosa)
incorporating a filament of Oscillaria.
(After Rhumbler. )

hastening to the injured point and engulfing the particles. In
the older stages of the axolotl and in the tail of tadpoles, where
a well-developed vascular system exists, inflammation is accom
panied by a dilatation of the vessels and by diapedesis: the
reaction is more violent, but the essential process is the same
as in the non-vascular invertebrates.

“Tt is quite evident that inflammation in the vertebrate, in
which the protective phagocytes emerge from the vascular
system to attack the aggressor, differs from the analogous
phenomena in the invertebrate only from the purely quantitative
standpoint... . The morbid phenomena, properly speaking, such
as the lesion or the primary necrosis, equally with the processes
of repair which succeed the inflammation, do not belong to it
and must not be confused with it” (Metchnikoff).

The phenomena of vascular dilatation and hyperzemia are no

INFLAMMATION AND PHAGOCYTOSIS 135

more to be regarded as inflammation, says Cantacuztne with
justice, than the congestive phenomena which accompany
ovulation or precede coitus can be called fertilisation.

Phagocytes, phagocytosisand digestion.— Metchnikoff
has given the name of phagocytes to those cells which are
capable from their own activity of seizing and incorporating
solid particles. (There is no question here of the faculty of
absorbing substances in solution.)

Certain phagocytes are migratory cells : for example the white
corpuscles of the blood. Others are fixed cells—for example
many of the endothelial cells of the blood-vessels and lymphatics,
the endothelial cells of the omentum and the neuroglia
cells. Others originally motile may become fixed at a certain
period in their existence.

The fundamental property of phagocytes is intracellular

digestion.

Metchnikoff’s first observations were on the property of
intracellular digestion in the intestinal epithelium of a great
many Turbellarians. In the Ccelenterata and the Sponges
digestion is intracellular, 7.e., the nutritive particles instead of
being digested in a cavity with the help of juices poured out
by digestive cells undergo digestion in the interior of the cells
themselves.

The Metazoa have inherited this property from the Protozoa.
Originally all the cells of the inferior Metazoa are capable of
phagocyting ; there are both ectodermic and endodermic pha-
gocytes ; later the function devolves entirely on specialized
cells belonging to the mesoderm.

The phagocytic cells of man are descendants of cells whose
normal function was to digest intracellularly, and through them
we still possess this method of digestion, side by side with the
extracellular digestion which occurs in our stomach and
intestine.

Phagocytosis is thus a function very wide-spread among
living beings, and the struggle against infection is only a
particular case of it.

“ The phagocytes are those cells which have best preserved

136 MICROBES AND TOXINS

the primitive amoeboid type. ‘hey are in general the least
differentiated elements in the body, but they are also the most
independent and possess the greatest vitality. They assist in
building up the young animal during the embryonic period, and
when the tissues begin to wear out, when old age is coming on,
it is the phagocytes which consume the senile cells incapable
of recovery and take their place. The renewal of cells and
tissues which goes on slowly and continuously in a great many
animals is, like the abrupt transformation which occurs in
metamorphosis, the work of phagocytes.”

The importance of pathological phagocytosis from the medi-
cal point of view should not make us forget that a normal
phagocytosis exists. The histolysis in the larvee of insects, the
destruction of the tail in the tadpole forms of the Tunicata, the
degeneration of the tail muscles in the tadpole of the Frog and
Toad, the destruction of the myelinated nerve fibres in Wallerian
degeneration, the shrinking of the ovarian follicles, the fixation
of the ovum on the mucous membrane of the uterus, the daily
destruction of the red corpuscles of the blood which goes on
in the spleen, all are examples of normal phagocytosis.

The phagocytes are guided or directed in their choice and
perception of the bodies which they ingest, by a peculiar sense
whose manifestations are known by the name of chemiotaxis.

It has been known since the time of Pfeffer and Stahl that
cellular organisms and plasmodia are attracted by certain
substances (positive chemiotaxis), and repelled by others
(negative chemiotaxis). They become accustomed to sub-
stances which at first repelled them and finally are attracted by
these. *Massart and Ch. Bordet have systematically studied
chemiotactic actions, by introducing under the skin of the frog
capillary tubes containing chemical substances, microbes and
their products. Lactic acid, glycerine, bile, and guanin repel
the leucocytes; sterilised cultures of both saprophytic and
pathogenic microbes attract them. Positive chemiotaxis may
be considered as the appetite, which prepares for intracellular
digestion. Everything is not yet explained in this distant
action. Chemiotaxis is analogous to the sensations of higher

INFLAMMATION AND PHAGOCYTOSIS 137

animals, and the sensations of a plasmodium obey like ours
the law of Weber.

Chemiotaxis is a sort of chemical sense.

The phagocytes have also a sort of tactile sensibility. The
leucocytes in their reaction apply themselves to the exciting
body over the largest surface possible.

In defensive phagocytosis, the struggle of the body against
the parasitic invaders, it is not necessary to suppose any
purposeful cause but simply a function developed by evolution
and selection. “Those lower animals in which the motile cells
directed themselves towards the enemy, engulfing and destroying
them, survived, whereas others in which the phagocytes did not
act, were condemned to perish. All the useful characters and
among them those which are concerned in the inflammatory
reaction have become fixed and transmitted without the inter-
vention of any preconceived purpose whatever” (Metchnikoff).
Thus in the invertebrates with soft skins in which bacterial
invasion occurs easily there has been a selective process at
work in the phagocytic apparatus and the defensive measures
have become perfected. Among the invertebrates possessing
a natural protection, such as a chitinous covering, infection is
rarer, but the means of defence have not found suitable
conditions for their employment and development, so that the
infected organism succumbs. The phagocytic arrangements
are much reduced in the Insects, and in these the parasitic
fungi have great difficulty in penetrating the cuticle, but if
they are successful the insect is destroyed (for example in the
beetle Cleonus punctiventris invaded by Lsaria destructor). The
nematode worms which are protected by a thick skin do not
even possess cells capable of movement.

The phagocytes of man are both fixed and motile. Among
the fixed are the large mononuclears, the Kuppfer cells of the
liver, certain endothelial cells of the lung (the dust-cells) and
the myeloplaxes of the bone-marrow. The motile phagocytes are
the white corpuscles or leucocytes in general (except the small
lymphocytes), the polymorphs, the eosinophils, the large
mononuclear cells of the blood and of the lymphatic organs.

138 MICROBES AND TOXINS

Metchnikoff has divided the phagocytes into macrophages
and muicrophages ; the former are chiefly concerned in the
absorption of cells and cellular debris, and include the large
mononuclears, the fixed phagocytes of the spleen, of the
peritoneum and of the lymphatic glands. They digest the
blood corpuscles and other phagocytes. The microphages
are the polymorphs ; their
principal function is to
digest bacteria. There are
exceptions. In certain
cases the microphages take
up cells (red cells among
others), while in certain
cases the macrophages
take up bacteria ; the large
Fic. 52. — Different leucocytes. — 1. mononuclears surround the

Polymorph.—2. Microphage (poly- tubercle bacillus producing

morph) taking up staphylococci, sé. :

—3. Small lymphocyte.—4. Fo. the giant cell, and take up
sinophil.—5. Large mononuclear also the spirochetes of
(macrophage). — 6. Macrophage recurrent fever and of
from the peritoneum of a guinea- see
pig taking up red corpuscles.—7. syphilis.

Macrophage from the peritoneum It has sometimes been
taking up polymorphs (micro- Be ee
phages) and blood corpuscles. maintained that the pha-

gocytes only take up dead
bacteria and not living virulent ones; this is a mistake to
which it will be necessary to refer again in connection with
immunity. Under the microscope there can be seen inside
the phagocytes living and even motile bacilli, and cultures
can be obtained by inoculating into broth phagocytes full of
microbes: the leucocytes are destroyed and the liberated
bacteria multiply. They were still quite alive therefore,
although already seized by the phagocytes.

The phagocytes secrete digestive ferments. Rossbach has
demonstrated the existence of a starch-splitting ferment in the
leucocytes of the tonsils. The cells of pus can digest fibrin
and gelatine, and must thus secrete proteolytic ferments. In
cases of acute muscular atrophy the progressive digestion of

INFLAMMATION AND PHAGOCYTOSIS 139

the muscle fibres can be observed in the interior of the
phagocytes. The bacilli or cells taken up by the phagocytes
become distorted, and before disappearing lose their affinity
for stains. In the various cases of immunity the phagocytes
digest the bacteria by means of endo-enzymes.

The surface of the skin, and in particular of the mucous
membrane, is being continually besieged by bacteria, and
never a moment passes but some point in the body is in a state
of subinflammation. The phagocytes are in continual opera-
tion on the surface of the tonsils, of the mucous membrane of
the intestine, and of the alveoli of the lungs.

Phagocytosis plays a pre-eminent. part in chronic infections,
especially in tubercle, and the tubercle itself is a phagocytic
formation. In contrast to Baumgarten’s contention that the
tubercle is built up by epithelial cells, fixed cells from the
diseased tissue itself, lung, liver, or kidney, Metchnikoff
and his pupils have proved that it is really composed of
migratory mesodermic cells which have come from elsewhere
to the infected point. Borrel followed the formation of the
tubercle from the time of the first contact between the white
corpuscles and the bacilli, and found that injected bacilli were
engulfed by the polymorphs while still in the circulation. The
polymorphs perish and degenerate (in two or three days) and are
followed by the macrophages which fuse together into a sort of
little plasmodium with several nuclei, which is characteristic of
the tuberculous lesion, and is called by the anatomists the
giant cell, Later, the tubercle may soften, and there may be a
new afflux of polymorphs attracted chiefly by the bacteria of a
secondary infection.

In the squirrel-rat spermophilus, a rodent rather resistant to
tubercle, the phagocyted bacilli lose their staining properties,
degenerate, swell up and finally appear as yellowish bodies,
such as ave never observed either in cultures or outside the cells,
and can only be residues of phagocytic digestion. In
another rodent, the jerboa, there is found, especially in
tubercle of the spleen, instead of bacilli amorphous bodies
built up of concentric layers, which are encrusted with

140 MICROBES AND TOXINS

phosphate of lime and may be dissolved by an acid. Obser-
vation of these tubercles at different stages shows that the
concentric layers correspond to secretions of the bacillus which
has been defending itself against the phagocytes. Analogous
formations are known in actinomycosis (the club-forms of
the granules). There is no essential difference between the
struggle of the tubercle bacillus against the giant cell and the

Tubercle
bacillus

Fic. 54.—Giant cell enclos-

Fic. 53-—Giant cell from the ing the final stage of a
spleen of the jerboa: it con- calcareous particle.
tains a tubercle bacillus sur-
rounded by concentric layers.

(Metchnikoff.)

struggle of the gregarines and the nematodes (larvee of Gordius
or Rhabditis) against the phagocytes of the worm.
Inflammation is thus defined by phagocytosis: the vessels
and nerves have their importance, but are merely accessory.
Infection, inflammation, and immunity can all be seen in
miniature in the examples of the Bipinnaria with its splinter
surrounded by motile cells, and of the Daphnia with its
globules in the act of devouring the spores of Monospora.

CHAPTER VII
THE PATHOGENIC PROTOZOA: FILTER-PASSING VIRUSES

Protozoal diseases—Laveran’s discovery—Importance of the morphology
and the life-cycle—Intracellular protozoa—Heredity in bacterial and
in protozoal diseases—Diseases due to the so-called invisible microbes.
—The ultramicroscope—Filtration—Various types of virus capable
of passing filters—Microbes of extreme minuteness described in the
pustular diseases of the epithelium-—Lesions of the infected cells.

THE PATHOGENIC PROTOZOA

THE name of Pasteur must be inscribed at the head of this

chapter. It was by his study of pébrine, a protozoal disease

of silk-worms, that our ideas on the microbial diseases were so
much advanced.

The studies on anthrax, the labours of Koch sd the great
discovery of the attenuation of viruses led the new science in:
the direction rather of bacteriology ; the protozoa had even
been somewhat forgotten, when in 1880 Laveran discovered
among them the cause of malaria. Since that date their
importance in pathology has never ceased to grow.

The methods of research cannot be quite the same as in
bacteriology ; they have not the same simplicity as the bacteria.
In the case of the tubercle bacillus, the cholera vibrio, and the
streptococcus, we practically know only one single constant
fixed form for each, and there is no sign of a life-cycle. The
majority of the pathogenic protozoa on the contrary go through
a cycle in their existence whose successive forms may be very

«diverse and this cycle may take place, not in a single host, but

often in two different ones. The discoveries of Ross on the
141

142 MICROBES AND TOXINS

plasmodium of malaria present the best example of the labour
necessary to reconstruct the biology of a parasite common to
man and the mosquito.

PROTOZOAL DISEASES
Rhizopods
Ameebe.—Ameebic dysentery and liver abscess. A ciliated infusorian,
Balantidium coli, may produce the same disorders.
Hematozoa !
Trypanosomes.—Sleeping sickness (human Trypanosomiasis).—Trypano-
somiasis of animals: nagana, surra, dourine, mal-de-Cadeéras.
Leishmania.—‘‘ Leishmanioses”: Kala-azar (the oriental sore, Biskra
button, Aleppo button, etc., being particular cases).
Piroplasmoses.—Bovine Piroplasmoses (due to Pivoplasma bigeminum,
P. parvum, P. mutans ; canine, ovine, and equine Piroplasmoses.
Plasmodia.—Malaria with its varieties: tertian, quartan, and sestivo-
autumnal or tropical tertian.
Spirochzetes.—Relapsing fever (European, African, Asiatic, American).
The spirillosis of fowls and geese.
Human spirillosis = syphilis.

The more the study of these cycles was advanced, the more
one was compelled to acknowledge relationships between forms
which did not seem in
the least related. Es-
tablished classifications
have several times got
into difficulties from
trying to express genea-
logical relationships
between very diverse
forms. Schaudinn saw in
V. ROUSSE the life-cycle of the same

Fic. 55.—Trypanosomes of sleeping sick- parasite trypanosomes,

ness (7rypanosoma gambiense): the e
form on the extreme right is in process =p © hetes, and
of division. ameeboid forms. It has

been necessary to re-
cognise a relationship between the sporozoa (hzemo-sporidia)
and the flagellates. The affinities of the spirilla and spiro-

1 According to the recent views of Hartmann, originating in Schaudinn’s
ideas, all the haematozoa mentioned here may properly be arranged in one -
natural group.

THE PATHOGENIC PROTOZOA 143

chetes are not yet clearly determined. These questions,
which seemed of zoological and philosophical interest rather
than medical, have nevertheless come into the domain of
medicine since the discovery of the microbe of syphilis and its
treatment. From the study of the pathogenic protozoa there
have arisen many new ideas.

In the study of bacteria the methods are their isolation and
pure culture, the study of their biochemical reactions, and of
experimental inoculations. Their physiology, z.e., the study of
their functions, takes precedence of the study of their forms,
Z.¢., their morphology. In the case of the protozoa, morphology
has the first place. It is no longer a question of describing
one cell, but a cycle of very dissimilar cellular forms with repro-
ductive phases, sometimes sexual, sometimes asexual. And it
is only at this price that certainty can be attained on the
methods of transmission of these microbes and on the basic
ideas for medicine and hygiene. Cultivation has been success-
fully accomplished only in the case of certain species, the
trypanosomes of the rat, amcebe in mixed culture, and
piroplasma, and it does not give the same help as do bacterial
cultivations.

No intracellular protozoon has yet been cultivated.

It is the knowledge of the life-cycle which gives the key to
the transmission. The most complicated modes of transmis-
sion among the bacteria are very simple in comparison with
those of malaria and sleeping sickness. The living carriers of
certain bacteria, e.g., the rat-flea in the case of plague, appear
to be carriers pure and simple, z.e., possess practically the same
importance as the needle of a syringe or a lancet. The mos-
quito is more than a mere carrier of the hematozoon of
Laveran ; it is a second host and in it, and it alone, the parasite
accomplishes the sexual phase of its life-cycle.

It was originally thought that the tsetse fly (Glossina palpalis)
whose bite produces sleeping sickness was a simple carrier of
the virus and only remained infective for a few hours after
sucking it from the patient’s body. But the recent experiments
of Kleine, confirmed by Bruce, have proved that the tsetse is

144 . MICROBES AND TOXINS

really a second host. It remains infective for about 24 to 48
hours after the moment of drawing infected blood, then for a
period of about 17 days it is non-infective, again becoming
infective for a period of about 60 days’ duration. The trypano-
some undergoes in it
an evolution with
sexual reproduction.
The protoplasm of
protozoa appears to
possess faculties of
adaptation and _ varia-
tion much more exten-
sive than that of bac-
teria. Biologically
speaking it is a long
journey for a parasite
of the stomach of the
mosquito to reach the
blood of man. In the
successive phases and
habitats there are
forms and _ structures
so different that it se-

quires strict proofs to

Fic. 56.—Glossina palpalzs, the tsetse fly convince one that it is
which carries sleeping-sickness.

really the same species.
The Leishmania donovani of Kala-azar (a disease of India and
the Mediterranean) is in the intestine of a bug a flagellate
form: in man it is an intracellular form deprived of all
locomotory organs. The degradation resulting from para-
sitism has abolished in many forms the characteristic
structures and in many cases permits of only provisional
classification.

These degradation phenomena are more striking among the
protozoa because we know on the other hand their complete
cycle. The bacteria, as we have seen, have reached in their
apparent simplicity perhaps the final stage of degradation, and

THE PATHOGENIC PROTOZOA 145

they conceal their origins, of which we can with difficulty
discover a few vestiges.

The protozoa may injure their host both by mechanical
and by chemical means. The Zxtameba histolytica destroys
and strips the epithelial cells from the intestine, abolishing
at certain points the impermeability of the healthy mucosa.
Myxobolus pfeiferi produces atrophy of the muscle fibres.
Lentospora destroys the bones and cartilages of the trout.

The, toxins of the protozoa are little known. If they exist
they are difficult to isolate and demonstrate. The Savcocystine
of Laveran and Mesnil, which kills the rabbit and only the
rabbit, is a well-characterised toxin, but no similar substance
has been isolated from the cultures of trypanosomes, or from
the blood of animals with a trypanosome infection. The blood
of a malarial patient, filtered at the moment of the paroxysm, is
not quite harmless to a healthy individual. The blood of an
animal infected with ¢rypanosoma gambiense produces an
appearance of somnolence in experimental animals, but it is
difficult to say how much of this is due to products of the
parasite and how much to the host. These are researches
which will have to be continued ; there is no reason to believe
in’ advance that toxins and endotoxins do not exist in the
pathogenic protozoa.

Heredity in Protozoal Diseases,—The protozoa are
frequently intracellular parasites. Bacteria also may inhabit
cells, for example the bacillus of leprosy, the bacillus of swine-
erysipelas, and the tubercle bacillus... But in these cases it is
the cell which has taken up the bacterium, the cell being
mesodermic and naturally phagocytic ; the microbe has been
captured; no bacterium ever penetrates by its own activity
into a living cell. On the contrary, many protozoa have during
their life-cycle a motile form, amceboid or flagellate, thanks to
which they can penetrate spontaneously the cells of their
host.

This fact is of capital importance from the point of view of
heredity in disease. When one sees the young of an anthrax-
infected mother born with an anthrax pustule, one might think

L

146 MICROBES AND TOXINS

that the disease was hereditary, but in reality it is a case of
contagion or of transmission at short radius; the placental
filter has been injured (Chamberland’s experiment). Nowadays
heredity in tuberculosis is no longer believed in; what is
inherited is at most a physiological predisposition of the soil
(and even that is a vague and uncertain idea), or, alternatively,
conditions of life in which the bacillus, everywhere to be
found, can flourish. There is no hereditary infection in the
strict sense unless the fertilised ovum is infected by the

Fig. 57.—The spirochzte of Schaudinn in the liver of a congenital
. syphilitic. (From a microphotograph. Magnified 3,000 times.)

parasite (among the vertebrates either from infection of the
female cell or of the spermatozoon, or of both); in such a
case the disease is truly congenital. There is no certain
example of such a fact among the bacterial diseases, and this
is the reason why the idea of heredity among them has lost so
much ground.

From their power of penetrating cells the protozoa fre-
quently infect parasitically the ovum, thus producing hereditary
infections. The first thoroughly demonstrated example of
hereditary infection was that of the pébvine of the silk-worm,

THE PATHOGENIC PROTOZOA 147

rendered so famous by Pasteur. Under the microscope the
presence of the germ was demonstrated in the egg, and it was
recognized that the infected eggs produced caterpillars which
formed the point of departure for the infection of the following
year. In general, in this example the heredity is only of one
generation, for the silk-worm infected in the egg rarely survives
to become an adult ; it is the other silk-worms, infected late in
their larval stage, which succeed in reaching the adult con-
dition after more or less great vicissitudes, which produce the
infected eggs.

Eckhardt has found coccidia (Coceidium tenellum) in the
white of hen’s eggs, and, according to him, these parasites
produce an early infection of the chicks which in consequence
very soon die.

The higher vertebrates have an interest in the hereditary
transmission of protozoal diseases from two points of view ;
either it occurs in themselves or it is a condition of the
infection of an invertebrate host which transmits to them the
disease. Thus the piroplasmosis due to Proplasma bigeminum
is transmitted from ox to ox by a tick, RAipicephalus annulatus,
but it is not the same individual tick which carries the
piroplasma from one ox to another. One tick becomes
infected from an infected ox, and it is its progeny, a daughter
tick, which infects the healthy animal. On the inheritance of

the parasite in
the insect-carrier
depends the pro-
pagation of the
disease in the
vertebrate.

Fic. 58.—The spirocheete of syphilis: forms in . An inheritance

longitudinal division. is probablealsoin

other diseases of

vertebrates which are transmitted by ticks, e.g., the spirillosis of

fowls, African tick-fever and recurrent fever. R. Koch saw the

spirochete of African recurrent fever in the egg of the

tick which transmits it, Ovazthodorus moubata. In fowls
E22

148 MICROBES AND TOXINS

infected with the spirochete of Marchoux and Salimbeni the
parasite may penetrate the egg, particularly the yolk, and in
this case inheritance may occur from vertebrate to vertebrate.
Hygiene has to take account of these facts: to abolish a
parasite it is insufficient to destroy it in the vertebrate; the
invertebrate host also has to be abolished since it is capable of
transmitting the parasite to its descendants, the egg of the
infected insect preserving the disease in nature somewhat as the
anthrax spore keeps alive anthrax.

Schaudinn considered the spirochete which he discovered
in syphilis to be a protozoon. Now the clinicians regard
syphilis as a disease which can be inherited. The case of new-
born infants with syphilis does not alone prove an hereditary
infection, the spirochzete being very motile might be transmitted
from the mother to the foetus through some lesion of the
placenta. Congenital syphilis is by no means necessarily a
syphilis by conception. But from certain observed facts such
true inheritance is very probable. The spirochete has the
power of spontaneously penetrating cells: it has even a
predilection for epithelial cells: further, although there are
no certain observations of its presence in a spermatozoon, it has
been seen in the spermatic tubules in close relation to the
epithelial cells (in congenitally syphilitic boys), and it has been
seen (Levaditi and Sauvage) in the protoplasm of the ovarian
follicles of female children. Are infected ova in the woman
capable of fertilisation, and, if fertilised, capable of normal
development? It seems possible in view of the clinical facts and
by analogy with the case of the tick Ornithodorus, whose eggs
infected with spirilla give rise to larvee which as adults are
capable of conveying the infection.

To sum up, we know from Finger and Landsteiner’s experi-
ments that the semen of an adult syphilitic is, as a substance,
capable of producing syphilis, and we know that the congenital
syphilitic of the male sex shows the parasite developing in the
seminal gland in contact with the epithelial cells, but no one
has ever seen a spirocheete in an adult spermatozoon. In the
female subject there can be no doubt of the possibility of

THE PATHOGENIC PROTOZOA 149

transmission by the general circulation through the blood, z.e.,
through the placenta; and further, spirochetes have been seen
in the interior of the ova in female congenital syphilitics ; it is
not known with absolute certainty whether, and how, a spirochzete
passes from the general circulation of the mother, or from the
spermatic fluid of the father, into the ovum which after fertilisa-
tion is to produce an embryo infected from the start, whose
life in consequence will be more or less soon cut short. But
taking all the facts we know we have almost a complete
demonstration of true inheritance.

Protozoal disease and hereditary disease are two terms so
closely associated in our minds to-day that the protozoal nature
of the spirochzete is invoked to support the hereditary character
of syphilis, and this latter is brought forward as an argument
in favour of the protozoal nature of the spirocheete: there are
at the base of this somewhat easy-going argument facts which
are sufficiently certain.

The striking analogies between syphilis, a spirocheete infection,
and sleeping sickness, a trypanosome infection permit of the
belief that the spirocheete of Schaudinn is a protozoon. Among
the more or less late complications of syphilis are locomotor
ataxy and general paralysis. Now there is also known an
ataxic condition in dogs infected with trypanosomes
(Spielmeyer’s experiments), and there exists a general paralysis
with all the mental stigmata of that disease in men attacked by
sleeping sickness (G. Martin and Ringenbach).

There are doubtless more protozoal diseases than we think
to-day, and it may quite well be that protozoa are the cause ot
those infections whose nature is still unknown—e.g., yellow
fever, cattle-plague, and the horse-sickness of the Transvaal.
Yellow fever in particular is transmitted by a mosquito
(Stegomyia fasciata), which does not infect until after the
12th day from the time at which it was itself infected. The
individual bitten passes through a period of prostration which
lasts three to five days, and at this moment his blood becomes
infective for the mosquito, but only for a period of three days ;
these facts indicate a life-cycle in the mosquito and in man,

150 MICROBES AND TOXINS

a series of different forms (cf. malaria) which appear and
disappear in the blood. These forms are unknown and must
‘be extremely minute.

From the immunity point of view also the protozoal diseases
present characters quite different from the bacterial infections.

THE VIRUSES CAPABLE OF PASSING FILTERS.

The bacteria which we study under the microscope are
unequal in size. The Bacillus Biitschlid we mentioned in
connection with the nucleus of bacteria is a colossus in com-
parison with the bacilli of fowl-cholera, with the Micrococcus
parvulus of Veillon, or even with the little bacillus found
in influenza by Pfeiffer. There probably exist bacteria still
smaller. Our best microscopes do not allow us to dis-
tinguish a particle whose thickness is less than or pw. The
bacteria smaller than o'r pw are therefore invisible under the
microscope ; they are w/tra-microscopic. Since there are many
diseases in which the microbe remains unknown we are
tempted to ascribe to them ultra-microscopic microbic agents.
Already in 1884 Pasteur said that the virus of rabies was too
small for us to be able to see it.

The study of these extremely small microbes only commenced
in 1898 with an experiment by Lofflerand Frosch on the virus
of foot-and-mouth disease, which no one has yet made visible.
The serous fluid from an ulcer (in which no microbe can be seen)
is diluted with water and filtered through a porcelain bougie
(similar to those of the Chamberland filters) ; there results a
perfectly clear fluid free from visible microbes which is capable
of transmitting the disease to a fresh animal ; this is the first
example of a virus passing through filters, or, as it is commonly
called, a filtrable virus.

Since 1898, the existence of filtrable viruses has been proved
by experiments in about twenty diseases, the chief of which
are foot-and-mouth disease, pleuropneumonia of cattle (rinder-
‘pest), yellow fever, swine-plague, cattle-plague, small-pox, and
rabies.

THE PATHOGENIC PROTOZOA 151

The study of these viruses is far from being advanced ; one
only, that of cattle pleuropneumonia, has been seen (and even its
form is subject to discussion), obtained in pure cultures and treated
like an ordinary bacterium. In vaccinia, small-pox, sheep-pox,
hydrophobia, trachoma and molluscum contagiosum, microbes
have been described but they are still hypothetical ; the proof
is still to seek.

The expression, “invisible microbes,” originally employed to
describe these, has been abandoned as inexact, and they have
been called the “so-called invisible,” and later the “filtrable
viruses.” Invisible microbes are simply microbes which have
not yet been seen ; for example, the syphilitic virus was classed
among them till the day when Schaudinn discovered the
Spirochzete. The classic example of pleuropneumonia shows
that a microbe may traverse a porcelain filter without being
invisible. Little vibrios and even little protozoa (Aficromonas
Mesniti, of Borrel) have been found in water; these pass
through a filter and can yet be quite easily seen. A filtrable
microbe is not necessarily visible.

The name of ultra-microscopic microbes is the most correct,
because many of these micro-organisms, too small to be seen
under the microscope, can be studied with the ultra-micro-
scope. Everyone has heard of this improvement, consisting
in examining the object not as lighted from below and seen
by transmitted light, but lighted from the’side so as to appear
as a bright point or line on a dark field. In the observation
of microbes which are perfectly visible the ultra-microscope is
not the instrument of choice for studying the structure ; a well-
stained preparation is still the best. The ultra-microscope
furnishes to medical bacteriology above all an economy of
time and trouble ; it makes the finding of the microbes a
more rapid process, for example, when there are very few
trypanosomes in the blood or spirochetes in the fluid from a
lesion suspected of being syphilitic; but these are quite
visible microbes.

Are the ultra-microscopic viruses always microbes? May
it not be a question, at least in certain cases (as Beijerinck has

152 MICROBES AND TOXINS

suggested for the mosaic disease of the tobacco plant), of a
fluid, living contagium which is literally invisible? This
hypothesis of “soluble viruses” has been put forward, but
hitherto no positive proof has been given.

The essential procedure in the definition of the ultra-micro-
scopic viruses is filtration ; it is the current method of isolation.
The liquid containing the virus is passed through a filter—for
example, vaccinal pulp rubbed up in water; the filtrate
collected is virulent, and with it attempts at cultivation can be
made. The filters employed are of the well-known forms ;
the majority are hollow bougies, like those which are used for
filtering drinking water ; they are made of porcelain (Chamber-
land filter), of infusorian earth (Berkfeld filter), of asbestos,
charcoal, plaster, etc.

These filters do not act towards microbes as does a sieve used
to sift seeds of unequal size, or as the metallic grids used for
separating sand of different coarseness. It would not be right
to conceive the large microbes as being kept back because they
are bigger than the meshes, whereas the little ones pass easily
through, just as the smaller fishes pass through the meshes of
anet. Even the bacteria of average size, such as the vibrio of
cholera, are smaller than the pores of our filters, and their size
would permit them, to use the simile of Duclaux, to pass
through, as a train passes through a tunnel, without rubbing
against the walls ; what keeps them back is that they are held
against the walls by the capillary pressure.

Filtration is not a simple mechanical operation, various
factors act in it: the quantity of the virus, the motility of the
microbe, the pressure, the degree of dilution, the nature of the
liquid more or less albuminous, the temperature, the duration
of the filtration, and the texture of the filter. All these factors
have to be taken into account in these experiments. As arule,
several filtrations are performed one after another, the first,
rougher and more rapid, prepare for the final one by freeing
the liquid from particles which block up the pores. One must
especially avoid having thick albuminoid substances present ;
they soon cover over the surface of the filters. By prolonging

THE PATHOGENIC PROTOZOA 153

the time it would be possible to make microbes pass which are
not ordinarily “ filtrable,” but this would not be really a filtration
but a culture propagating itself by extension from one side of the
filter to the other. This is what happens in the filters of water
supplies which are badly kept. Bacteriologically speaking, they
are no longer filters at all ; instead they are continually infecting
the drinking water with the microbes which they are supposed
to be keeping out.

Filtration can show that some quite minute microbe exists
in a given infection : it gives no information as to its nature.
Fortunately, the labours of Jenner and Pasteur have proved that
it is possible to study a virus without seeing it. It can be
purified (precisely by filters as it happens), inoculated, and its
resistance to physical and chemical agents (heat, antiseptics,
etc.) determined, as well as the conditions of preservation and
attenuation. All the viruses enumerated at the head of the
chapter have been treated in this way and processes of immun-
ization have been discovered against certain of these viruses
which are still unknown, a paradox which has become familiar
to us through Jennerian vaccination and the antirabic treatment
of Pasteur.

To say that a virus is filtrable is to give it an external rough
definition ; there are undoubtedly in this group very different
microbes ; some may be bacteria, others protozoa. Borrel has
described a protozoon which passes the rough filters. In the
life cycle of the Haemamaba Ziemanni of the little owl,
Schaudinn has described motile forms smaller than the
microbe of peripneumonia ; it is admitted that even the most
visible protozoon may have ultra-microscopic stages.

Several groups may henceforth be distinguished among the
diseases due to filtrable viruses :

1. Pleuropneumonia of cattle: in this the microbe has been
filtered, cultivated, and finally seen. It would seem that it
ought to be easy to describe it. At first it was said to be a
bacterium, extremely fine cocci namely, which it was possible
to see under the ordinary microscope, not singly, but in
amorphous masses. Recently, Bordet has described by means

154 MICROBES AND TOXINS

of cultute in a special medium, forms resembling spirocheetes.
Borrel, using a different technique, has criticised the forms
seen by Bordet and concluded that it is not a spirochzete but a
new type lying perhaps between the protozoa and the bacteria,
and still incapable of precise definition.

2. Cases of blood infection or septiceemias such as the
horse-sickness and the catarrhal malarial fever of sheep
(studied in particular in the Transvaal), yellow fever, cattle-
plague, avian-plague, and hog-cholera appear to be of the same

ye] Rt LA®

a) ie: > Oa 9
nS Ge Fa
a ee ar Jo & oge ° = ~
S fror ¢) 4 ~ eq e
hk Q ¢ ¢$ 4 ition, Oe Or
4 : x “eve ~~, d ge
+ ? t OT of Fe ote , ) : ay.
= ey wae :

. $ 4 ° ee
4 ar ® a &
7 . es Fic. 60.—Various forms of the
microbe of bovine pleuro-
Fic. 59.—Various forms of the pneumonia according to Borrel
microbe of bovine pleuro- (higher magnification than in
pneumonia according to Bordet. Fig. 59).

nature. Horse-sickness and the sheep disease which resembles
malaria only exist in localities where there are certain definite
mosquitos ; like, malaria, too they were formerly called mias-
matic disease. Horses do not take horse-sickness even when
they are exposed to conditions of climate and altitude reputed
to be dangerous, provided they are protected from mosquitos
by means of wire-screens. The “ heart-water” of ruminants is
transmitted by a tick (the bont-tick—Amblyomma hebracum).

THE PATHOGENIC PROTOZOA 155

Yellow fever is inoculated by a mosquito, Stegomyia fasciata.
These diseases have all the behaviour of protozoal infections.
By reason of certain procedures of immunization which are
common to them all, horse-sickness, cattle-plague, and hog-
cholera present some points of similarity.

3. The diseases characterized by localization to, and lesions
in, the epithelium, for example, small-pox and vaccinia, foot-
and-mouth disease, molluscum contagiosum of birds and man,
scarlatina, the jaundice of silk-worms, and trachoma or granular
conjunctivitis are perhaps to be classed in this group.

Small-pox or vaccinia is the type-specimen of these infections ;
the characteristic lesion is the pustule and the pustule is a
collection of epithelial cells containing the virus and forming a
focus of culture zz vivo. The cells which build up the little
tumour have no longer the normal structure of the epidermic
or Malpighian cells; they have become globular, voluminous
and “dropsical”; the nucleus is altered, being swollen and
frequently out of position ; beside the nucleus there appears a
mass of abnormal material called the “cellular inclusion.”

This mass was for long regarded as a parasite, as the visible
stage of a protozoon which possessed other stages more minute
or ultra - micro-
scopic. Nowadays
it is known that it
is merely a de-
formation of the
nucleus appearing

after the invasion Fyg, 61,—Negri bodies in rabies; the hypothetical
of the cell by the microbe of rabies at different stages. (After
Calkins. )

virus. It is, as it
were, the stigma of the presence of a virus within the cell, or
even within the nucleus.

These stigmata have been described under different aspects
and names in small-pox, vaccinia (Guarnieri bodies), rabies
(Negri bodies), mod/uscum contagiosum (described by Virchow),
trachoma, and the jaundice of the silk-worm, and the similarity
of these infections can no longer be doubted.

156 MICROBES AND TOXINS

Borrel has discovered in the cells of molluscum contagiosum
minute corpuscles very equal in size and distinct from the
nucleus, from the chromatin and from the protoplasm: they
are small enough to pass through filters, and sufficiently
abundant and resistant to physical influences, as temperature
and drying, to explain
the powerful nature of
the contagium in these
diseases.

This may perhaps be
the type of microbe so
long sought for in
small-pox and granular
conjunctivitis, but it is

Fic. 62.—Mallory bodies; the hypothetical necessary to speak with

microbe of scarlatina under different leat
aspects. (After Calkins.) Teserve as cullivation

has not yet been suc-
cessful, What is certain is that there exist ultra-microscopic
bacteria and protozoa sufficiently small to traverse the pores of
filters made of asbestos, porcelain, plaster, or infusorian earth.
Great discoveries are still to be made in this domain—a domain
opened up twelve years ago by the study of foot-and-mouth
disease and pleuro-pneumonia.

The curiosity of investigators ought not to be monopolised
by the diseases occurring in man and animals. There is no
reason why there should not be invisible microbes elsewhere
in those fermentations which go on everywhere in nature.
They may also quite well play a part in the life-cycle of plants.
Just as insects produce injuries and mutilations in plants, so
the ultra-microscopic microbes may be responsible for the
variations and mutations which occur in the vegetable world.
Microbiology may hope here again to bring its support to the
Darwinian doctrine.

CHAPTER VIII
THE TOXINS

MICROBIAL AND VEGETABLE TOXINS—ENDOTOXINS

Microbial and vegetable toxins—Definition—Soluble toxins—Characters
—Toxins and diastases: resemblances and differences—Incubation
Penetration of the body—Elective fixation—Wassermann’s experi-
ment—Vegetable toxins: icin, abrin, crotin—Production of
antitoxins—Endotoxins—Definition—Toxity of microbial bodies—
Toxin and endotoxin of the cholera vibrio—Do anti-endotoxins
exist ?—Importance of intravenous inoculations.

MICROBIAL AND VEGETABLE TOXINS

We know toxins as properties, not as substances, properties
of certain broth-cultures, or properties of the bodies and
extracts of the bodies of bacteria. Their nature and their
exact chemical constitution are unknown, for they are bound
up with albuminoid substances the chemistry of which is still
in its infancy.

The science of toxins is therefore more physiological than
chemical, and the chief method of experimentation is on the
living body. The quantitative element is introduced by mea-
suring the incubation times, the temperature, or the magnitude
of the local phenomena, such as cedema and the duration of the
symptoms of intoxication.

In certain cases it has been possible to replace the animal
experiment by experiments 7” vitro, and to measure, with
exactitude, certain phenomena, easy to observe, such as the
lysis of the red corpuscles of the blood (hzemolysis).

Useful discoveries are much oftener reached by instinct than
187

4

158 MICROBES AND TOXINS

by reason. If progress went on logical lines the idea of anti-
bodies in general ought to have been the first, and from it the
existence of antitoxins ought to have been deduced. But, on
the contrary, it was the discovery ofa particular antitoxin which
led to the study of antibodies in general.

In experimental medicine, the chief business is not to build
up systems of ideas, z.e., to philosophize, but simply to search
patiently with many trials and many a re-beginning. The
inquisitive, prying, intuitive people have the advantage over
those who reason.

At the very beginning of the researches on toxins we find an
experiment of Pasteur: the filtrate of a culture of fowl cholera
produced in a fowl the symptoms of the disease in the absence
of microbes. At first it was thought that the bacterial toxins
belonged to, the group of alkaloid substances, the ptomaines,
found by Selmi in dead bodies, in certain molluscs, and in
bacterial cultures (e.g., muscarine, neurine, &c.). It is true that
bacteria can produce poisons of this type (Brieger), but these
poisons do not produce a specific intoxication like that
observed in such a well-defined disease as tetanus. Later, when
it had been observed that microbes killed by heat are not
harmless, but when inoculated produce a local suppuration,
attempts were made to isolate the “poison ” by making protein
extracts of the bacterial bodies; but the bacterial protezns
of H. Buchner are not specific poisons; from very diverse
bacteria one can extract almost the same poisons. They con-
sist of excretions or residues of nutrition of the bacteria, and
are found in particular in old cultures.

Excluding these alkaloids and proteins, the following are the
substances studied as toxins :—

1. Zhe Soluble Toxins.—The type-specimen is the diphtheria
toxin or the tetanus toxin. These are secretions of bacterial
cells, just as the pancreatic juice is a secretion of the gland
cells.

2. The Endotoxins.—Examples: typhoid endotoxin, plague
endotoxin. These are poisons which remain attached to the
cellular protoplasm, and do not diffuse at all or very little in.

THE TOXINS 159

broth-cultures. It is necessary to destroy the cell to set free
the poison. The process may be exemplified by the zymase
production of Ed. Buchner, in which the yeast cell is ground
up and expressed.

3. A special group has to be made for the poison of the
tubercle bacillus (tuberculin), and that of the bacillus of
glanders (mallein). These diffuse into the broth, but there
remains some in the substance of the bacterium ; hence the
bodies of tubercle bacilli form an active tuberculin.

Besides the specific toxins, cultures may contain non-specific
poisons, the proteins of Buchner.

Soluble toxins.—The toxins of diphtheria, of tetanus, and of
botulismus are known (the latter being produced by an
anaerobic bacillus growing in meat). They can be obtained by
cultivating the bacteria with an abundant supply of air (in the
case of the bacillus of diphtheria), or in broth deprived of
oxygen (in the case of the tetanus bacillus). The bacteria are
removed from the fluid by filtering through porcelain bougies.
Those strains of bacilli are picked out which furnish the best
toxins, for good toxins are necessary for the production of
good antitoxins.

The Characters of the Soluble Toxins.—The soluble
toxins possess very definite characters ; in the first place they
are strictly specific ; the symptoms produced by the diphtheria
toxin are quite distinct from the symptoms of tetanus. Their
action is specific in another sense, as it varies in the different
animal species. The fowl does not react to the tetanus toxin
as does the mouse or the human subject. Secondly, they are
extremely potent: a good culture of tetanus can kill the
mouse in a dose of yg¢goy C-m-. The diphtheria toxin kills
a guinea-pig of 250 grams in a dose of yay c.c.1 The

1 “1 ¢.c. of the active fluid toxin produces on evaporation 7% vacuo O°O!
gram of dry residue. Deducting the weight of ash and the portion in-
soluble in alcohol (which has no toxic activity) there remains 0°0004 gram
of organic matter. It is certain that the greater portion of this weight
consists of substances other than the diphtheria toxin. Yet this minute
quantity is sufficient to kill at least eight guinea-pigs of 600 grams each,
or two rabbits of 3 kil.” (Roux and Yersin.)—1Ic.c. of a good tetanus
filtrate dried 77 vacuo leaves 0'04 gram of dried residue of which 0'025

160 MICROBES AND TOXINS

botulismus toxin is fatal in the dose of zg55 c.c. (by sub-
cutaneous inoculation in each of these cases). Toxins are
soluble in water and in glycerine, and can be precipitated from
solution in virtue of the fact that they adhere to precipitates
and coagula; precipitation is a method of purification. They
are unstable or “labile” substances, heat, light, and oxygen
destroying them fairly quickly. Exposed for an hour and
a half to the temperature of 55° C., the tetanus toxin loses its
toxic properties ; at 60° C. it is destroyed in thirty minutes ;
at 68° C., in five minutes. ‘Toxins bound up with dried
precipitates are more resistant; the dry tetanus toxin is still
slightly active after an hour at 80° C., and even after fifteen
minutes at 120°C. Sunlight “inactivates” a solution of tetanus
toxin in fifteen to eighteen hours. When a photo-dynamic
substance (e.g., 1 per cent. eosin), is added, the toxin is
‘inactivated ” after six hours’ exposure to light; with 2°5 to
5 per cent. eosin it is “inactivated” even in the dark.
Comparison between: Toxins and Diastases.—The
chemical constitution of diastases is not much better known
than that of the toxins. In comparing toxins with soluble
ferments or diastases, although it is possible to note analogies, it
is for obvious reasons impossible to give a chemical definition.
Like diastases, toxins act in a very small dose, are soluble in
water and in glycerine, and are weakened by filtration, and are
sensitive to the action of oxygen, of heat, and of light; also
to changes in their reactions and to various chemical substances
which “poison” or destroy them. Roux and Yersin, who
pointed out these analogies at the time of their work on toxins
in 1889, did not see in them more than a suggestion: “It
seems to us that the diphtheria poison has many analogies
with the diastases. Its activity is quite comparable to these
and to the activity of venoms. We do not mean, how-
ever, that it produces phenomena of hydrolysis such as the
diastases produce. It neither inverts sugar nor digests

gram is organic matter. Supposing that the whole of this is toxin (which
is a great exaggeration), the lethal dose for a mouse would be 0°000,000,25
gram.

THE TOXINS 161

fibrin. If we compare it to the diastases, we do so without
forming any rash opinion as to its chemical action and
simply in order to sum up some of its properties.” This
reservation still holds good.

The disappearance of the toxic property by heating to 60° does
not necessarily mean the destruction of a diastase. Heating
modifies the reaction of the fluids, especially of organic fluids,
and coagulates the proteins: this coagulation may inhibit the
action of certain substances or properties without destroying
them. The toxins have a character possessed neither by
chemical poisons, ¢.g., strychnine or potassium cyanide, nor by
the diastases: the action of these chemical poisons is almost
instantaneous, and a zymase put into a solution of sugar begins to
ferment it at once. The toxins, on the other hand, when injected
into the body, only manifest themselves after a silent period of
apparent inactivity, the period of incubation. By changing
the path of introduction, e.g., by injecting intravenously instead
of subcutaneously, and by increasing the dose, the period of
incubation may be rendered shorter: it is never reduced to
zero. Further the #ntmum incubation period varies with the
species of animal inoculated.

It is admitted that the tetanus poison, to reach the nerve-
centres, has to travel along the peripheral nerves from the site
of its production, and within certain limits the period of
incubation varies in proportion to the distance of this : but even
when this delay is cut out the incubation period does not
reach zero. Meyer and Ransom inoculated the nerve-centres
of cats directly and still found a smdnimum incubation of three
to five hours.

This inevitable incubation period suggests that the toxin of
the culture is not the poison which kills the animal, but that
the toxin inoculated undergoes in the body certain transforma-
tions (fermentations?) which produce the true poison, the
action of which is direct andimmediate. This secondary poison
was said to have been demonstrated by inoculating mice with
extracts of the organs of animals actually in tetanus, but these

experiments have not given constant results as the symptoms
M

162 MICROBES AND TOXINS

produced differed from the pure tetanic symptoms, It is not
yet possible to believe in the existence of pro-toxins analogous
to the pro-diastases such as the pro-fibrin-ferment.

The toxins act in extremely minute doses, like the diastases,
of which a very small quantity can determine a chemical
change in a very large mass of material. But there are
alkaloid poisons which also act in a very small dose: a man
dies after absorbing the five-millionth part of his weight of
aconitine or digitalin. The mere fact of possessing an action
produced by very small doses is not equivalent to being a
diastase ; it may be an ordinary chemical phenomenon ; an
artificial catalyst such as colloidal platinum is “inactivated ”
by one thousand-millionth part of hydrocyanic acid; Graham’s
solution of ferric hydrate under certain conditions is sensitive
to the presence of 1/5,000,o00th of ferrocyanide of potassium.!
Finally, the phenomena produced by toxins are phenomena
taking place in living creatures, which makes it all the more
difficult to determine whether they are diastasic in nature or
simply due to ordinary chemical reactions. It is true that the
toxins of diphtheria and tetanus can “kill” 20 to 100
million times their weight of living animal, but these figures
must not be allowed to produce this illusion: the quantitative
relationship is complicated by a qualitative element whose
importance cannot be exaggerated. When a horse is killed
by 1/80,oooth of its weight of tetanus toxin, the toxin is not
acting on the whole mass of the horse: to produce death it
is sufficient for it to act on the medullary nucleus of the
vagus nerve, a group of cells scarcely weighing two grams.
The diphtheria toxin similarly acts zz an elective fashion on a
group of cells in the medulla or in the cardiac ganglia. To
take facts of another order, the fixation of carbon monoxide
on the hemoglobin of the blood is not a diastasic phenomenon
(on the contrary it arrests a vital diastasic function of the
first importance), yet the toxic power of carbon monoxide in
proportion to the weight of the body is certainly more than
100,000.

1 J. Duclaux—La chimie de la matiére vivante, Chapitre X.

THE TOXINS 163

Penetration of the Body.—To reach the sensitive cells
the toxins do not always follow the same path. Injected
subcutaneously they pass into the lymph, then into the blood.
Injected into the blood they save time, since the passage
through the lymph is avoided. Introduced into the alimentary
canal the botulismus toxin retains its: potency, but the
diphtheria toxin and the tetanus toxin are inactive even after
being swallowed in much more than a lethal dose. This
destruction or neutralization cannot be attributed to an action
peculiar to the intestinal epithelium nor to any extent to the
influence of the bacteria and their fermentations in the
digestive tube, but chiefly to the action of the digestive
secretions, the pepsin, and above all the pancreatic juice.

According to the experiments of Meyer and Ransom and of
Marie and Morax, the tetanus toxin does not pass directly from
the site of inoculation to the nerve centres ; it penetrates the
peripheral nerves at their motor terminations, and follows these
nerves to reach the centres. All three species of nerve fibre,
motor, sensory, and sympathetic, can carry it; but the carrying
power of the nerve depends absolutely on the integrity of the
axis cylinder. By employing antitoxin it is possible to localize
the action of the toxin to certain definite territories and paths.
Dissociation experiments have shown that the antitoxin acts
by neutralising the toxin still in circulation, but is no longer
capable of neutralising toxin absorbed by the nerve trunks
(these do not absorb antitoxin). The fact that in certain
animals, man and the horse, tetanus always begins by.a con-
traction of the muscles of the jaw (“lockjaw”) only means
that even after a stab or a wound at the end of a limb sufficient
toxin passes sufficiently quickly into the circulation to affect
the centres on which the innervation of these muscles
depends.

The cells of the central nervous system are sensitive to
many poisons, whether these reach them by the nerve filaments
or through the blood. Directly introduced into the centres
the poisons act in a smaller dose, and often produce different
symptoms from poisons injected subcutaneously or into the

M 2

164 MICROBES AND TOXINS

blood stream. Cerebral tetanus is more like a mental disease,
a sort of delirium, than the systematic tetanic contractions
which follow the wound of a limb. In the rat which has
received tetanus toxin intracerebrally, the incubation is from
forty-eight hours to three days, and if the observer did not
know with certainty that he had injected tetanus toxin he
would never recognize tetanus in the disease which he observes.
Psychical manifestations predominate ; the rat is anxious and
vigilant; without apparent cause it is seized with sudden
terrors, and runs madly round its cage. . . . During the crisis
it seems to obey an internal impulse .... and on careful
observation the question forces itself whether many psychical
phenomena in man may not also be produced by the fixation
on certain nerve cells of bacterial toxins elaborated in the
intestine or in some other part of the body at some particular
moment (Roux and Borrel).+

Selective Fixation of Toxins.—For a toxin to kill in
the minimum dose it must possess a selective affinity for cells
whose function is important, and must proceed to fix itself on
these cells and not on others, when introduced into the
circulation. Thus it is necessary for a medullary nucleus to
attract to itself the few thousandths of a milligram of tetanus
toxin introduced into a human body. The intoxication
depends entirely on this fixation of the toxin, and it has long
been comparyed to a dyeing process.

Even in the inorganic world, and among dead substances
of animal or vegetable origin, numerous examples exist of
the fixation of a substance in solution more dilute even than
are the toxins in the blood.. The examples which follow are
taken from the book of J. Duclaux already quoted. Pre-

1 In the rat 7% c.c. of diphtheria toxin subcutaneously does not produce
even local cedema, but a rat receiving this dose intracerebrally is soon
completely paralysed, and after two or three days of inertia it succumbs.
A rabbit of less than 1,500 grams supports perfectly 30 centigrams of a
morphine salt injected subcutaneously, whereas I milligram of morphine
hydrochloride injected into the brain produces almost immediate effects in
a rabbit of the same weight. A tuberculous guinea-pig succumbs when
injected intracerebrally with a dose of tuberculin 200 times smaller than
when injected subcutaneously.

THE TOXINS 165

cipitated sesquioxide of iron absorbs arsenious acid (and
probably also phosphoric acid) till there remains in the fluid
less than one-thousand-millionth part (A. Gautier). A skein
of white silk dipped in a solution of eosin so dilute that the
eye can perceive no colour, z.¢., to about one in a million, is
dyed pink in the course of a few hours. Silk can take up
1°3 per cent. of picric acid from a solution containing only
o'006 per cent. The absorptive power increases the greater
the dilution. Passing to living cells, we find that a cullure of
Aspergillus niger can take up from a solution measuring
250 c.c. and containing one-half milligram of zinc, Ze.,
1/500,000, practically the whole of this metal (Javillier) ; the
proportion of zinc increases to about 1 in 10,000 in the
aspergillus cells and falls below 1 in 10,000,000 in the fluid
which remains. Certain plants can absorb copper from
solutions containing only 1 in 100,000,000. Certain marine
plants, such as the ordinary sea-wrack, fix abundantly the
iodine and silver which exist in traces only in sea-water.

The whole industry of dyeing is founded on similar fixations:
fabrics are sensitive to and fix selectively the colouring
materials. Picric acid, which stains the skin, does not dye
cotton. The microscopical preparations of histologists and
biologists depend on this principle of the selective fixation of
the stains by different anatomical elements: magenta is fixed
by the nucleus, picric acid by the protoplasm, indigo-carmine
by the connective-tissue fibres.

Ehrlich has pointed out that in jaundice the kidneys and the
liver become charged with bile-pigments, whereas the brain
remains free. When certain derivatives of paraphenylene-
diamine are administered to mice, the central part of the
diaphragm is found stained brown much more intensely than
the periphery, and the muscles of the eyes, of the larynx,
and of the tongue are much more deeply stained than other
muscles: this may be because these muscles are in continual
activity, receive more blood, and are the seat of more intense
oxidations. Methylene blue in the living animal is fixed by
the sensory fibres, by the nerve-endings for taste and smell,

166 MICROBES AND TOXINS

by the nerves of the plain muscles and the cardiac muscle, and
by certain fibres in the nerve centres: it does not stain the
motor-endings of striped muScle with the exception of those of
the eyes, of the larynx, and of the diaphragm (Ehrlich).

The majority of the staining substances fixed by the cerebral
cortex are also taken up by fatty substances: now the cortical
cells are rich in “fofds such as cholesterin, lecithin, and
cerebrin. The substances which stain “zz vivo” are soluble
in these lipoids, the zon-vifal stains are not. Narcotics,
cerebral poisons, have an activity proportional to their
co-efficient of absorption by the lipoids, and both this activity
and this co-efficient vary with the temperature. Antipyretics
also have, without doubt, a selective action on certain cells.

The famous experiment of Wassermann and Takaki showed
that a similar selective absorption takes place between the
cerebral grey matter of mammals and tetanus toxin when these
are mixed together in a test-tube; after a certain time the
tissue fixes the toxin and the liquid is no longer toxic.

The brain of cold-blooded animals, e.g., the lizard and the
tortoise, does not fix toxin at all, or very little. The brain of
the frog does not fix toxin 7” vitro (and yet the frog kept ina
fairly hot room is sensitive to tetanus toxin). The fixing
power of the brain seems to be proportional to its lipoid
content, and there is less of these substances in the brain of
cold-blooded animals. Brain material treated with ether,
which dissolves the fats, loses a great part of its power of
fixation: brain which has been boiled no longer fixes at all.
Filtration of a suspension of brain material (removing the cell
elements) also destroys the fixing property. Cholesterin,
lecithin, and cochineal, a fatty material extracted from the
cochineal insect, all fix toxin, but when heated to 60° in
presence of moisture, or after a previous maceration in an
alkaline fluid, the latter is no longer a fixative.

The fixation of toxin by sensitive cells is a phenomenon of
the order of dyeing or 7” vivo staining—a phenomenon of
molecular adhesion.

There is not in Wassermann’s experiment, as was thought at

THE TOXINS 167

first, a destruction or neutralisation of the toxins by an anti-
toxin elaborated by the brain cells. The fixation can be
modified by altering the physical conditions: brain matter
emulsified in physiological saline solution (0°8 per cent.) is a
stronger fixative than the same emulsified in distilled water,
but ten times weaker than the same emulsified in salt solution
of 10 per cent. The brain gives up the toxin more or less
quickly and restores the toxicity to water when it is allowed to
macerate, and also after drying or digestion with papaine: the
toxin liberated has all the biological properties which it had
before its intimate contact with the brain matter. This
observation negatives the hypothesis of a secondary toxin
elaborated by the cells from the toxin received and capable of
acting immediately without incubation.

It is because of this fixation property of the cells of the
body that the toxin injected disappears more or less rapidly
from the blood and cannot be recovered, or only in very small
degree, from the excretions. In the rabbit seventeen hours after
injection no free toxin is to be found either in the blood or in
the organs, and there is never any in the blood at the moment
when the tetanic symptoms commence (about forty-eight hours
after intravenous inoculation.—A. Marie).

Since the tetanus toxin may be fixed on cells other than the
nerve-cells, it is evident that the former keep back at least a
portion of the toxin, acting thus as a sort of screen to the
nerve-cells. For example, the rabbit is less sensitive to tetanus
than the mouse and the guinea-pig because its spleen fixes the
toxin and saves its brain. Hence it is not the power of
fixation in general which explains the sensitiveness of a
particular animal, but the selective fixation on certain definite
cells whose activity is indispensable to life, for example, the
cells of the medullary nuclei or of the sympathetic ganglia.

Scorpions can stand very large doses of tetanus toxin
without symptonis ; the toxin rapidly disappears from the blood
and accumulates in the liver. The alligator, which is re-
fractory to tetanus, retains in its blood for more than a month
toxin which has been injected into it. The carp, the axolotl,

168 MICROBES AND TOXINS

and the frog, kept at alow temperature, do not take tetanus and
retain the toxin intact in their blood for months. The tortoise,
which does not take tetanus either at high or low temperatures,
retains in its blood for months enough toxin to give tetanus to
mice on injecting it. The fowl also, very little sensitive to
tetanus, retains toxin in its blood for long periods.

The frog, which is refractory to tetanus in winter or when
kept at a low temperature, takes tetanus in summer or when it
is kept warm in an incubator at about 30° C. At this tempera-
ture the poison disappears from the blood and the organs much
more quickly than in the cold. The course of the tetanic
symptoms can be interrupted at will in the frog kept in the
incubator by putting it again at a low temperature. In this
way the phenomena may be suspended as long as the chilling
continues; if it is again put in the incubator the symptoms
recommence at the stage at which they were interrupted. In
the frog, fixation and response are, therefore, to a certain
extent dissociated, for in the cold the toxin is fixed by the cells
yet the disease does not appear.

Vegetable Toxins.—The bacterial toxins are vegetable
toxins since bacteria are microscopic plants. They have their
analogies in the higher plants, for example, 7iciz extracted from
the seeds of the common castor oil plant; adrviz from the
Abrus precatorius or jequirity ; crotin from the plant croton
tighum.

Ricin inoculated subcutaneously can kill a rabbit in a dose
of or mg. per kilo. body-weight ; it agglutinates into masses
and dissolves the red corpuscles of the blood. The agglutina-
tion is so rapid and powerful that it is necessary to keep
shaking the tube in order to perceive the hemolysis. The
chemical nature of ricin is not exactly known; it is not
absolutely certain that it is an albuminoid substance (Jacoby).
Abrin agglutinates the red corpuscles but is not a powerful
lysin. Crotin requires a dose of several centigrams per kilo. to
kill a rabbit.

Toxins and Antitoxins.—A fundamental difference

| exists between the poisons of known chemical composition,

Se

THE TOXINS 169

such as the alkaloids and glucosides, and the toxins properly
speaking. ‘The toxins alone produce antitoxins in the animal
treated by graduated doses, ze, the antitoxins employed in
serotherapy. Antitoxins exist against ricin, abrin, and crotin, but
there are none against solanin and saponin, which are glucosides.
The glucosides although capable of being fixed by sensitive
cells, do not produce anti-glucosides. Only those poisons
which are capable of giving rise to antitoxins can be regarded
as true toxins.

ENDOTOXINS.

In contrast to the soluble toxins, the endotoxins are defined
as the poisons contained in the bodies of bacteria and not
spontaneously set free in cultures. Whereas the toxins are
secretions of living bacteria, the endotoxins, according to the
strict definition of R. Pfeiffer, are only set free by the
destruction of the bacterium. It is the protoplasm of the
bacterium itself which acts as a poison on absorption by’
the body.

To bring the endotoxins into line with a well-known example,
one may compare them to the zymase of Buchner, the ferment
of yeast not excreted, or scarcely excreted, but set free by
grinding up with sand and expression of the juice under a
pressure of several hundred atmospheres. But this com-
parison is not to be taken to mean that the endotoxins are
diastases.

Bacterial extracts have been produced by Buchner’s process,
but nowadays endotoxins are obtained by simpler procedures.
MacFadyen grinds up the microbes at the temperature of
liquid air ; others subject the cultures to combined maceration
and shaking. Besredka takes young cultures, dries them, and
re-suspends them in saline solution.

The endotoxins are distinct from the proteins ; the latter are |
practically the same in the different species of bacteria. To
deserve their name they ought to be specific and to give rise |
to antiloxins.

170 MICROBES AND TOXINS

It is in the cholera vibrio, the plague bacillus, the typhoid
bacillus, and the bacillus of dysentery that they have been
chiefly sought for and thought to have been found. It is
necessary to employ this qualified method of expression,
because their definition and even their existence is still
disputed. This chapter in the physiology of bacteria contains
many uncertainties, and the facts observed cannot always be
made to agree.

The endotoxins obtained by different workers from the same
bacterium do not seem to possess the same properties,
especially from the point of view of the toxic dose and the
resistance to temperature ; but it is also well known that with
tetanus toxin different workers do not obtain equal specimens
either.

Between the endotoxins described as having the same
general characters there exist differences which are not to be
found between the diphtheria and tetanus toxins; thus the
plague endotoxin is destroyed by heat from about 75° C.
onwards, whereas the typhoid endotoxin resists 127° C. (accord-
ing to Besredka). But physical differences are known to exist
between the diphtheria and tetanus toxins also, which, though
less striking, are none the less real.

The dysentery endotoxin is easily obtained, the typhoid
endotoxin less easily, the cholera endotoxin with great
difficulty.

The following are the principal points disputed.

1. The fundamental phenomenon on which depends the
existence of specific endotoxin is the toxicity of the bacterial
bodies themselves. The nature of the bacterial bodies must
first be agreed upon, and no agreement will be reached unless
young bacteria are taken, avoiding as much as possible the
alterations caused by manipulations, however carefully or
cautiously conducted. Pfeiffer is inclined to regard with some
suspicion the endotoxins which accumulate spontaneously
in old cultures, considering them as products of a destruction
of the bacteria, which is accompanied by chemical alterations
(fermentative), of the details of which we are ignorant. These

THE TOXINS 171

errors may be avoided by employing young bacteria from
cultures of twelve to eighteen hours on solid media.

2. But in many cases difficulties are met with when one
attempts to determine the relations between this endotoxin and
the soluble poisons secreted by the same bacteria and
apparently true toxins. For example, the cholera vibrio cer-
tainly contains an endotoxin in Pfeiffer’s signification, but it is
none the less true that the cultures contain an excreted poison,
the poison studied by Roux, Metchnikoff, and Salimbeni, on
which the hope of a serotherapy in cholera has been founded.
It is this poison which is absorbed into the body and produces
the cramps, the chilling, and death, whereas the vibrios, however
numerous they may be, remain in the intestine and only very
rarely invade the blood and the tissues. ‘The free poison,”
says Pfeiffer, ‘‘ does not exist in cultures except when these are
already old and contain many vibrios already destroyed and
autolysed.” The cholera toxin of Roux and Metchnikoff is,
however, chiefly secreted during the first days of a culture
kept under the requisite conditions (a virulent vibrio and a
well-aerated culture). This example is not in favour of
Pfeiffer’s opinion, at least in the case of cholera, for although
he admits that the endotoxic bacteria can also secrete other
soluble poisons, he maintains that the latter are quite different
from endotoxins and do not possess their specificity.

3. Do the endotoxins give rise to antitoxins in the body of
an immunised animal, as do the soluble toxins? This is the
most disputed question of all. Without denying the existence
of anti-endotoxins in principle, Pfeiffer’s school considers that
hitherto none have been obtained which have passed satis-
factory tests, and that the sera prepared against plague and
typhoid fever have not hitherto been successful precisely
because they do not contain anti-endotoxin.

To prepare an anti-endotoxin, as in the preparation of an
antitoxin, it is necessary to inject several times into an animal,
for example, the horse, the toxic substance, in this case the
bacterial bodies, entire or broken up. Good results are not
obtained when endotoxins are injected subcutaneously ; intra-

172 MICROBES AND TOXINS

venous injection is essential. By intravenous injection of young
microbes, Besredka obtained anti-endotoxins which, both zz
vitro and in the animal, neutralise the toxic action of the
bacterial bodies. Since the production of antitoxin is the
essential character of toxins, these experiments would prove
the reality of endotoxins as specific poisons.

It is difficult to say to what degree the sera hitherto
prepared against typhoid, plague, and dysentery are anti-
microbic or antitoxic, ze. active against infection or against
intoxication.

Although inferior to antidiphtheritic or antitetanic sera, the
antidysenteric and antiplague sera have already given results
sufficiently good to encourage us to bring to perfection the
endotoxins and their anti-endotoxins.

CHAPTER IX

TUBERCULIN AND MALLEIN—ANIMAL TOXINS—VENOMS

Tuberculin and mallein—Koch’s phenomen—Action of tuberculin—Local
and general reactions—Resistance of tuberculin towards physical
agents which destroy other toxins—Specificity—No antituberculin—
Habituation to tuberculin—Cutaneous reaction of v. Pirquet—Tuber-
culin and anaphylaxis.

Animal toxins—venoms—The venoms in the animal kingdom—Snake
poisons—Physiological action of venoms—Digestive properties—
Heemolysis by venoms—RdGle of lecithin—Lecithids—Natural immunity
of certain animals towards venoms.

TUBERCULIN AND MALLEIN

THESE poisons are found in old broth cultures of the
bacillus of tubercle and the bacillus of glanders. They are
prepared by combined maceration and heat from glycerine
extracts of the cultures. ‘They exist also in the bodies of the
bacteria and are thus in a sense endotoxins. But hitherto no
antitoxins to them are known.

Tuberculin and mallein from their physiological properties
occupy a place apart. Tuberculin may be taken as the

type.
Koch’s Phenomenon.—The discovery of tuberculin
originated in the ‘“Koch’s phenomenon”: when tubercle

bacilli are inoculated subcutaneously in a guinea-pig nothing
is seen at the point of inoculation for from ten to fourteen days,
then a nodule appears which later produces an open sore,
which refuses to heal; the corresponding lymphatic glands
are swollen. If, however, a guinea-pig already tuberculous is

reinoculated after four to six weeks, there appears on the third
173

174 MICROBES AND TOXINS

day, without any nodule developing, a necrosis of the skin
over a zone of half to one centimetre; the necrotic patch
becomes detached, and the ulcer heals up and closes without
any swelling of the corresponding glands. The same process
takes place when, instead of living bacilli, bacilli killed by
boiling are injected the second time.

The tubercle bacilli therefore act in a different fashion in
the healthy organism from the organism already tuberculous.
Koch observed that a large dose of bacilli killed the
tuberculous guinea-pig, whereas a very small dose produced
an improvement in their condition and healed up the initial
ulceration. He saw in this a principle of treatment. Since
the bacilli are not readily absorbed, he replaced them by an
extract from cultures ; this was the original tuberculin.

This substance is practically harmless to non-tuberculous
animals, but is fatal to tuberculous animals in a very small
dose. In absolutely minimal doses, repeatedly administered, it
exerts a curative effect on certain tuberculous lesions. Accord-
ing to the size of the dose, it can act as a poison or as a remedy.
These are its fundamental properties.

Local and General Reactions.—Koch ascribed the
curative effect to the necrotic action which he had _ noticed
after reinoculation of bacilli in tuberculous guinea-pigs.
Tuberculin he said in 1890, does not kill the bacilli, it kills
the living tuberculous tissues ; it does not even act on tissues
already dead, such as the caseous masses. It acts on cells in
the same way as the bacillus tuberculosis itself, but the soluble
product has a much more extensive radius of activity than the
bacillus.

In what way does this necrotic action become therapeutic ?
Because in a necrotic tissue the bacillus is badly nourished
and grows feebly ; the dead tissue becomes a sort of slough
or sequestrum which the body strives to get rid of. The
action of tuberculin is, in a sense, surgical. It might be
hoped that every part affected by it might be thrown off, and
this is sometimes possible in tuberculosis of the skin and of
the lungs. In many cases, however, it is impossible, and it

TUBERCULIN AND MALLEIN 175

has always been feared that tuberculin might cause a necrosis
of the tuberculous tissue without completely killing the bacilli,
and might thus set them free and inoculate them on a tissue
till then unaffected.

When the tuberculin action does not quite reach the degree
of necrosis, it merely produces around the tuberculous focus
an active inflammatory reaction with an afflux of leucocytes,
which may build up a fibrous, cicatricial tissue in place of the
tuberculous ulceration.

Tuberculin does not produce only local reactions ; it pro-
vokes a general reaction of the body, the most obvious sign
of which is fever. In large doses the reaction occurs even in
a healthy individual according to R. Koch, who observed this
in himself; but it is very probable that he had at that time
some tuberculosis, and it has been maintained that tuberculin
is entirely inactive in subjects who have never been attacked
by the bacillus.

Three or four hours after the inoculation of }c.c., Koch
observed “twinges of pain in the limbs, a feeling of
fatigue, and a tendency to cough. The symptoms became
more pronounced, and about the fifth hour he had a violent
rigorwhich lasted awhole hour with general uneasiness, vomiting,
and fever (103°°3 F.). The symptoms began to settle about
the twelfth hour, and on the following day the temperature was
normal ; a heaviness of the limbs and stiffness were perceptible
for several days after. The point of inoculation remained red
and painful for a considerable time.”

In the treatment of a tuberculous patient with tuberculin,
doses are employed which do not produce these violent
symptoms. As far as possible no symptom, not even fever,
ought to occur. A well-conducted treatment produces an
improvement in many patients; this fact is certain, but the
mechanism of these cures is not yet well understood. The
indications and contra-indications for treatment are complex,
and cannot be settled except by a physician with a large
experience of tuberculosis and tuberculin treatment.

The febrile reaction which follows the inoculation of a dose

176 MICROBES AND TOXINS

too small to be dangerous is employed in the diagnosis of
tuberculosis, both among animals and in man.

Tuberculin is very different from other toxins. It bears
much less resemblance than they to the ferments. In the
liquid condition it stands a temperature of 120° to 150° C,
In the solid state, heated dry in sealed tubes, it stands 250° C.
“Tf it is a substance derived from albumins,” said Koch, “it
cannot be a toxalbumin in view of this resistance to heat and
of its dialysing properties.” It may be exposed to sunlight for
months without losing its activity. Heating with acids (for
example, 75th hydrochloric) and with alkalies simply weakens
it without destroying it. Perhaps it is not a simple poison:
in the condition in which we get it, it has no more claim to be
pure than our diphtheria and tetanus toxins. Maragliano
thinks that it contains, besides the poison which causes the
fever and is not a toxalbumin, a poison which, on the contrary,
lowers the temperature of the body, is destroyed by heating to
100° C., and is possibly a true toxalbumin.

Tuberculin without tubercle bacilli does not reproduce
tuberculosis. To provoke suppuration, caseation, and the
typical lesion, the tubercle, living or dead tubercle bacilli are
necessary : fluid tuberculin does not even produce the lesions
which dead bacilli can give rise to: it has nothing in common
with them but its destructive action on the cells and its power
of raising temperature.

It is eminently specific, not producing any definite effects
except in tuberculous individuals. In this it differs entirely
from the other bacterial poisons. It only acts on a prepared
soil, a soil prepared by the bacillus tuberculosis itself, e., by
an agent to which it is closely related both by origin and
constitution. This specificity, though very marked, is not
absolute. Tuberculin acts similarly, less, it is true, than in
tuberculous individuals, but more than in healthy subjects, on
patients affected by lesions resembling anatomically the
tubercle, ¢g., glanders and actinomycotic nodules. This
extension of its field of action is perhaps due to the close
biological relationship between the tubercle bacillus and the

TUBERCULIN AND MALLEIN 177

micro-organisms of these diseases, perhaps to the existence of
the same anatomical type of lesion, the tubercle, or perhaps
to similar inflammatory and phagocytic reactions.
Antituberculin ?—Does tuberculin in animals, tuber-
culous or not, treated and habituated to its effects, give rise to
the production of an antituberculin comparable to antitoxins
or even anti-endotoxins? No; an antituberculous serum
comparable to antidiphtheria or even antiplague sera does not
exist, in spite of all efforts to discover it. Tuberculin in
tuberculous subjects excites the production of certain reaction-
or anti-bodies, but no true antitoxin. No treated individual
has ever furnished a serum capable of neutralizing tuberculin
either ¢# vitro or iz vivo, but serum of this kind can produce
precipitates and clumps in a suspension of bacilli; this action
is, however, inconstant and of little value in medicine.
Wasserman and Bruck have given the name of antituberculin
to the reaction products which fix themselves on tuberculin as
anti-bodies do on antigens (v. Chap. X.). The interpretation
of these experiments is a question of some delicacy, and we
shall see how doubtful are the relations between the presence
of antibodies and the existence of an immunity, ¢g., a
resistance in the case of tuberculous individuals.
Habituation to Tuberculin.—By careful, repeated
injections the tuberculous subject may be made to protect
himself against the fatal action of tuberculin, and to enjoy a
general improvement in his condition, without ,stopping the
progress of his tuberculosis. Guinea-pigs may be made to
support fifty lethal doses, yet their lesions progress in the
ordinary way—or even more quickly than usual. It is not
yet well known what are the relations between the physiological
action of tuberculin and the progress of a chronic tuberculosis.
This habituation has been called immunity to tuberculin. It
is not, however, an immunity or even a resistance to tuberculosis.
It does not appear on repeated injections of egua/ small doses
of tuberculin ; in this case, the febrile reaction, which was
absent at first, finally appears and becomes severe. The fever
remains absent when one proceeds by zucreasing doses (always
N

178 MICROBES AND TOXINS

to be done with caution). With small equal repeated doses,
tuberculin behaves like a poison towards which the tuberculous
patient becomes more and more sensitive.

Cutaneous Reactions.—A drop of very dilute tuberculin,
applied by means a prick or very superficial scarification on the
non-tuberculous skin of a tuberculous subject, excites at the
point a reaction which may extend to the vessels and lympathic
glands of the neighbourhood—vV. Pirquet’s experiment ; this
represents a new diagnostic procedure, the cut-reaction. It has
been modified and perfected by dropping the tuberculin between
the eyelids (conjunctivo or oculo-reaction of Wolff-Eisner and of
Calmette) or by inoculating it with a very fine needle in the
depths of the skin itself (the z¢va-dermo-reaction of Mantoux).

It is a reaction of extreme interest, for it occurs at a non-
tuberculous point, ze, a point containing no bacilli, in a
tuberculous individual ; and can only be explained by suppos-
ing that the whole body has become impregnated with sub-
stances formed under the influence of the tubercle bacillus.

ANIMAL ToxinS—THE VeENoms!

In the struggle for existence, certain animal species have
acquired, as their means of attack and defence, organs which
secrete and inoculate toxic substances. These animal toxins
are the venoms, and such venoms are known at almost every
level in the animal scale.

Venoms in the Animal World.—Among the Ccelen-
terata the Actinians produce certain poisons which can be
extracted from their tentacles, ¢Aalassine and congestine, well
known as having formed the subject of Ch. Richet’s experi-
ments on anaphylaxis. These poisons are perhaps the cause of
the disease of sponge-fishers, who dive quite naked without a
diving-suit ; the disease consists in burning of the skin and
swelling, with gangrene and violent fever.

The pedicles of the Sea-Urchins (Echinoderms) contain a

1 Vide papers of Noguchi and Calmette.

TUBERCULIN AND MALLEIN 179

poison which stands boiling. This poison, in nature, is
dangerous to crabs and fishes; in the laboratory it is found
to kill the rabbit.

Among the Arthropoda, the Spiders and Scorpions (Arach-
nidz) secrete active poisons. The excretory tubes of the
poison glands of venomous spiders open at the point of the
two appendices which are furnished with claws at the end and
situated on each side of the mouth. The poison kills the
small animals on which the spiders feed, and causes in man
pain and contracture at the bitten point—a sort of miniature
tetanus. The poison of certain spiders contains a haemolysin,
z.é., it lakes blood, making the hemoglobin of the corpuscles
diffuse into the surrounding liquid (arachnolysin). The bite
of the Tarantula (Zycosa tarentula) is only dangerous for the
small animals on which they feed, and is quite harmless to
man. According to Brehm, all the stories of the effects on
man of the Tarantula bite are nothing but fables and
fantasies.

The venom of the Scorpion (Scorfio occitanus) of the South
of France can kill a guinea-pig in a dose of half a milligram of
dry extract ; for a rabbit one milligram. The scorpion is the
subject of a legend which says that when it is enclosed by a
circle of fire it commits suicide with its own poison. Now
the scorpion is in reality immune to scorpion venom, towards
which its serum acts like an antitoxin (Metchnikoff).

Among the Myriapoda the Centipedes, and among insects the
Hymenoptera, secrete venoms. The poison extracted from two
bees (by grinding up the terminal part of their bodies in 1 c.c.
of water) is sufficient to kill by asphyxia a mouse or a
sparrow. It also is a hemolytic poison.

There are many poisonous fishes. As ‘a rule their poison
glands are found at the base of the dorsal or caudal fins, or
under the spine of the gill-flap. These venoms all resemble
more or less that of the weever-fish, which has been most
studied. Locally it causes pain and swelling, with fever and
vomiting. At the time of spawning the poison is more
abundant and more active. The tropical tetrodons are most

N 2

180 MICROBES AND TOXINS

venomous at the time of greatest activity of their reproductive
glands.

The Toad (Batrachia) manufactures poison in its parotid
gland and the glands of the skin, but it has no other way of
secreting it than by contracting its skin and covering itself with
a viscous, nauseating slime, which is rather poisonous when
injected into small animals such as mice. Phisalix and
Bertrand have extracted two poisons from the toad, ‘ dufotaline’
and ‘ 4ufotenine,’ a poison of the nervous system. At spawning
time the cutaneous glands of the male toad are full of venom,
whereas those of the female are empty; but the poison
accumulates in her eggs, from which it may be extracted by
means of chloroform.

The Salamanders possess on their sides and tail poison
glands, and it is to the fluid which these secrete that they owe
their fame as animals capable of living in fire and even
of extinguishing it—pure legend, of course. Their secretion
permits, at most, of their surviving a few seconds.

One venomous animal exists among the mammals, the
Omithorhynchus. Its poison gland is situated on its thigh,
and the secretion escapes by a spur or claw on the hind feet.
The poison resembles that of the snake Zachesés, but is much
more feeble. ;

Medical men have been especially attracted to the study
of snake-venoms. The vipers of our own country have few
victims, but in India the cobra kills as many as an epidemic
disease. In 1889, in India, 22,480 human beings and 3,793
domestic animals died of snake-bite. Of those bitten 25 to 30
per cent. die within ten or twelve hours. The importance

is obvious of the researches which led to the antivenom~ °

serotherapy (Calmette).

As they issue from the glands the venoms resemble a thick,
oily saliva more or less yellow. ‘Their physical properties vary
a good deal with the genus. The venoms of the Viperidae
do not dialyse through a membrane and are destroyed entirely
at 75° to 80° C. (Lachesis even at 65°). Those of the
Colubridae pass slowly through vegetable membranes, with

TUBERCULIN AND MALLEIN 181

greater difficulty through animal parchment : they resist heating
at roo® C, and are only completely destroyed at 115-120”.

From the chemical point of view both consist of proto-
and deutero-albumoses: the albumins they contain are not
toxic. S. Faust has extracted from cobra venom an “ophio-
toxin” which contains neither nitrogen nor sulphur nor
phosphorus.

By using dried venom, which can be accurately dosed by
redissolving in a known volume, it has been possible, as with the
vegetable toxins, to determine the minimal lethal dose per
kilogram in different species of animal. As with tetanus toxin
the size of the animal bears no relation to its sensitiveness.
With one gram of dry cobra-venom it is possible to kill 1,250
kilos. weight of dog, 2,000 k. of rabbit, 2,500 k. of guinea-pig,
1,430 k. of rat, 8,333 k. of mouse, 20,000 k. of horse, and
10,000 of man, 7.¢., 165 adults of average weight. The horse
is thus the most sensitive of all these animals.

The toxicity of the venom is very variable: it is more
active (and doubtless less abundant) after the casting of the
skin and after a prolonged fast.

Physiological Action of Snake-venoms. — The
physiological action of snake-venoms is complex. They act
on the cells of the organs, on the liver, the kidney, and the
spleen: on the endothelial cells which line the interior of the
blood vessels (especially the rattlesnake poisons): on the nervous
system (according to Rogers the venoms of the Viperidae
paralyse the vasomotor centres, those of the Colubridae the
respiratory centres): on the blood, one of the oldest known
effects and one much studied recently because the solution of
the red corpuscles or Aaemolysts is a phenomenon very obvious
and easy to study zz witro. The condition of the blood at
autopsy varies according to the dose of the poison and the
time; this is why the same poison is called coagulant or
anticoagulant by different workers.

Snake-venoms have the properties of digestive ferments.
They can dissolve coagulated blood and can destroy the cells
of the vessel coats and even of the muscles. Cobra-venom

182 MICROBES AND TOXINS

digests albumins but without reaching the peptone stage. The
digestive properties are destroyed by heating to 70° C.

Pancreatic juice, as is well known, when perfectly pure
cannot alone, digest albumin ; it has to be “activated” by a
“kinase ” ferment, secreted either by the intestinal mucosa or
by the leucocytes. Now snake-venoms can take the place of
this kinase and activate a pure inactive pancreatic juice. This
is all the more curious since the pancreatic juice when
activated digests and destroys venoms, which, as a rule, have
in consequence no action when taken by the mouth. The
venom secretion is thus for the snake itself a normal
physiological secretion of great use in the digestion of the
huge meals for which snakes are famous. There is further
nothing surprising in the fact that non-venomous snakes, @e.,
those not provided with poison-fangs, still possess glands
capable of secreting venom ; it is simply used in the digestion
of their food.

Venoms thus, like toxins, resemble ferments—with the same
reservations. They are very closely allied to toxins. Their
action, like that of toxins, is not simple; just as in tetanus
toxin several different substances or functions can be dis-
tinguished, a nerve-cell poison and a poison of the red blood
corpuscles, so several physiological activities can be dis-
tinguished in the same venom. But venoms act without any
incubation period, or at any rate with a very short one. It is
only the time elapsing between inoculation and death that
varies with the dose, and that within rather narrow limits.

Like the toxins, the venoms are destroyed at relatively low
temperatures (resistance up to 100° to 110° C. does not interfere
with the analogy). They act in minute dose and deteriorate
or are destroyed by light, by photodynamic substances, by
iodine perchloride, and alkaline hypochlorites.

Finally, and most important, the venoms give rise to
antivenoms, as toxins do to antitoxins. It is practically only with
the help of the antivenoms indeed that the specificity of the
venoms can be definitely demonstrated.

Venom Hzmolysis.—The venoms are hemolytic

TUBERCULIN AND MALLEIN 183

poisons, and since hemolysis is a phenomenon much more
convenient to follow experimentally than paralyses or nervous
symptoms, they have been much studied, and from certain
points of view are better known than the microbial toxins.
The study of the toxins has profited by that of the venoms.
Thanks to some beautiful experimental results, there is reason
to believe that, with the help of the venoms, the study of
toxins in general may make the advance so much desired by
science, and from a physiological subject, studied only in the
living animal, become a part of chemistry with its definite
reactions.

Some definitions and examples must first be given.

When a rabbit is repeatedly inoculated with, for example,
defibrinated sheep’s blood, the rabbit’s serum acquires the
property of dissolving the red corpuscles of the sheep: these
latter suspended in physiological saline solution in a test-tube
with a little of this rabbit’s serum, instead of settling intact
and leaving a colourless supernatant fluid, break up, liberate
their hemoglobin, and colour the fluid red. The rabbit’s
serum has become Aaemolytic for the red corpuscles of the
sheep.

Bordet has shown that hemolysis depends on the operation
of two substances, or rather of two functions, of which we
shall have much to say in connection with immunity: one is
the alexine or complement of normal serum (destroyed by
heating to 56° C. for one hour) ; the other is the senstbilisatrice
or tmmune-body of the serum of immunised animals, such
as the rabbit above mentioned (it stands heating to 68° or
yo C.).

The latter owes its name to the fact that it prepares or
renders sezsttive the red corpuscles towards the action of the
complement. The complement completes the action of the
“sensibilisatrice,” hence its name. If we take the blood
corpuscles of a goat and add a little cobra-venom, hemolysis
occurs. But if the blood corpuscles are first carefully washed
with physiological saline so as to be entirely freed from traces
of blood serum which might adhere to them, no hemolysis

184 MICROBES AND TOXINS

occurs when the venom is added. But if now to this mixture
of carefully washed goats’ corpuscles and venom a little normal
blood serum is added, hemolysis proceeds at once. It would
seem from these facts that the venom acts like a sensibilisatrice
or immune-body, the ‘normal serum providing the. alexine or
complement.

But there are other facts which forbid this interpretation.
Normal serum activates the venom, it is true, but even when
heated to 65° C. or higher it still activates: there exist even
normal sera which cannot activate venom hemolysis until they
have been heated at 100° C. It is inconceivable that it is the
complement which is the active agent since complement is
destroyed at 56° C.

Further, washed corpuscles of certain animals are laked by
the venom without any addition of fresh serum (corpuscles of
dog, rat, guinea-pig, and man). Again, in the case of the
washed goats’ corpuscles, normal serum is not the only
substance which can activate: laked red corpuscles, ¢.g., of the
guinea-pig, can take its place quite well. Finally, in this last
example it is not the fluid which acts, but. the stromata or
bodies of the corpuscles which have shed their hemoglobin,
and these still possess their activity after heating to roo’ C.
It is thus impossible to attribute the activating action to the
complement or even to the serum as a fluid. The active
substance is not the complement nor is it an albumin, for
albumin coagulates below 100° C. It is not a ferment, for a
ferment heated in solution above 100° C. is no longer active.
It is a definite chemical substance present in the serum and
in the stroma of the blood corpuscles, namely Zcithin.
Lecithin is a well-defined chemical body, unlike albumin, for
which we are unable to write a formula, still more unlike the
complement and the immune body which, like the ferments, are
known as activities or properties, not as substances. In venom
hemolysis, therefore, our knowledge is more complete and
clear than in the hemolysis of hemolytic sera.

Lecithids.—-When blood of any species is easily laked
by. venom without addition of serum (¢.g., in man, the rat,

TUBERCULIN AND MALLEIN 185

and the guinea-pig) it means that the lecithin of the corpuscles
readily detaches itself and unites with the venom. When the
blood is laked only when serum is furnished in addition, it
means that the lecithin of the globules themselves is firmly
bound and difficult to set free. In certain cases heating to
65° C. or even higher is necessary to liberate lecithin from its
combination with hzmoglobin. The experiments of this
kind have many complications of detail, since the three factors
coming into play—the blood corpuscles, the lecithin, and the
venom—are subject to many variations. A step in advance
has been made in what may be called the mechanical or
purely chemical explanation of hemolysis by the discovery
that lecithin forms with venom a combination of a chemical
character in which neither lecithin nor venom can be
recognized. Kyes has named this combination or ‘“couplé”
lecithid, or since his experiments were on cobra-venom, cobra-
lecithid.

In its physical properties (solubility in water, alcohol, ether,
chloroform, and acetone) the lecithid differs from lecithin as
much as from venom. It can be isolated in a crystalline form
and redissolved in water. It acts on the blood of all animal
species, and that without any incubation period. The delay
observed in the action of venoms is not a period of incubation,
but merely represents the time necessary for the formation of
the lecithid compound. If the ready-made lecithin is added-
to the blood the hemolysis is more rapid than on the addition
of the two elements separately.

Strictly speaking, however, the venoms do not act without
incubation: the time taken by lecithid formation represents
the minimum incubation period. It is quite possible that in
the action of microbial toxins there may occur a slow forma-
tion (ze, with a longer incubation period) of compounds
analogous to lecithids.

Cholesterin behaves as an antagonist to lecithin ; it has no
effect on complement, but prevents the combined action of
the lecithid by affecting the lecithin; it thus forms a sort of
antihemolysin or antitoxin the composition of which is definitely

186 MICROBES AND TOXINS

known. It is one of the dreams of biological chemistry to
discover an equivalent to cholesterin for the other toxins.

Still more interesting is the fact that the peculiar anzemia
produced by injecting cobra-lecithid into an animal can be
prevented by giving cholesterin. It would seem that cholesterin
acts towards lecithin and lecithid, not only as an antagonist,
but as a remedy.

There are, of course, facts which prevent us from regarding
as entirely similar the cobra-hzemolysin, on the one hand, and
the solvent properties or heemolysins of normal and immune
sera (sera derived from animals prepared by the injection of
blood corpuscles) on the other. These, however, do not
invalidate Kyes’s conclusions in his experiments on the
lecithids. The study of snake-venoms has shown a participa-
tion of chemically-defined substances in phenomena hitherto
only known from the biological point of view. It would be
of immense interest, both theoretical and practical, to discover
an analogous mechanism in the effects of toxins.

Certain animals enjoy a natural immunity towards snake-
venom, which, however, is never absolute. It is possible that
these animals have received from generation to generation
small doses of the venom as the result of being bitten, and
that they have in consequence elaborated an antivenom.
This forms the starting-point of artificial immunisation and
antivenom serum-therapy. It is known that the blood of
certain animals ‘possesses normally a weak antitoxic action
against diphtheria and tetanus toxins; these animals similarly
must have harboured tetanus and diphtheria bacilli.

The animals possessing the most remarkable resistance
towards snake-venoms are the hedgehog and the mongoose.
To see the dramatic combats which take place when the
mongoose tackles the. cobra, one has only to read Kipling’s
marvellous tale of the war between “ Rikki-Tikki” and “ Nag”
in the Jungle Book.

CHAPTER X
IMMUNITY

PuHaGocyTosis AND HumoraL IMMUNITY

Early ideas of Pasteur on immunity—Opposition to the phagocytic
doctrine—Cellular and humoral immunity.

Antigens and Antibodies—Complement—The two substances: Bordet’s
experiments.

Phagocytosis a fact capable of direct observation—Ferments of the
leucocytes—Analogies with the digestive ferments.

Pfeiffer’s phenomenon—Opsonins and_ bacteriotropins—Antibodies not
an exact measure of the immunity.

Ir is not necessary to have studied medicine or science
in order to ask oneself what is this immunity which appears in
infectious disease. Certain bacteria are pathogenic for certain
animal species and not for others ; the guinea-pig, for example,
does not take fowl-cholera and the fowl does not take anthrax.
Among people living in one family under the same conditions
and among soldiers in the same barracks living under the
same rules we see disease attacking some while others remain
free. Finally it is a popular conviction that anthrax does
not occur twice and that, as a rule, once an individual has
had measles or small-pox he never takes it again: these
are everyday examples of acquired immunity.

“Immunity against infectious diseases ought to be
understood to mean the sum total of all the phenomena
to which is due the resistance of an animal body to the
microbes which produce these diseases” (Metchnikoff).

Immunity may be innate or acquired. Natural acquired
/ sy

188 MICROBES AND TOXINS

immunity appears when there is spontaneous recovery from an
infectious disease. Immunity the result of human interference
(vaccinations, serotherapy) is artificial acquired immunity.

After his work in collaboration with Chamberland and Roux
which established the attenuation of viruses and the value of
preventive inoculations, Pasteur, being a chemist, conceived
immunity as a chemical process. He considered that the
reason why the bacillus of fowl-cholera fails to grow in
the fowl vaccinated against this disease was that the body
of such a fowl no longer contained the necessary food-stuffs
for the development of the microbe. The muscle which has
been severely affected by disease has become, even after
complete recovery, in some way incapable of supporting the
life of the microbe, as if this latter during its previous growth
had made to disappear from the muscle some principle which
life is incapable of renewing and the absence of which prevents
the development of the micro-organism.?

He filtered a culture of the fowl-cholera bacillus and found
that a re-inoculation of the bacterium in the fluid thus freed
from the first germs always failed: when fresh nutritive
substances were added to the filtrate, growth took place. It was
not, therefore, the presence of some excretion, but the absence
of some nutritive substance which explained “the immunity of
a culture filtrate, or of the fowl considered as a natural culture
medium.”

In natural innate immunity also he refused to recognise the
presence of an inhibitory substance, basing his faith on the
celebrated experiment of the fowl refractory to anthrax but
rendered susceptible by chilling, and appealing to the “con-
stitution” or to a “vital resistance,” by which he conceived a
struggle between the parasites and the body-cells for the
oxygen and food materials available. But when it was found
that bacteria could grow perfectly in the blood of animals
possessing a complete immunity, Pasteur’s early conception
could no longer be maintained in its primitive simple form.

Even to-day immunity has still to be defined as a complex

1. R. Acad. des Sciences, 1880, p. 247.

ne

IMMUNITY 189

of biological phenomena, for in spite of the hope of men of
science some day to get beyond “ vitalistic” explanations, no
explanation in chemical terms can yet be given. Immunity is
a function of the cells. Immunity means phagocytosis. Further
research may fathom the nature of this activity and give a
chemical explanation as in the case of peptic or pancreatic
digestion, but the cellular activity is indisputable and is not a
theory but a collection of facts, a doctrine in the true sense.

The principles of this doctrine of phagocytic immunity have
already been indicated in the chapter on inflammation. It is
necessary to read in Metchnikoff’s book his “historical review
of our knowledge on immunity” (Chap. XVI) to comprehend
how much his doctrine has developed.

From the historical point of view it had to oppose the ruling
conceptions, not’ only in medicine, but in pathological anatomy
and in physiology. The few observers who had seen microbes
inside the white corpuscles had never deduced from this a pro-
tective function: quite the contrary, for authorities of the rank
of Waldeyer and Robert Koch firmly believed that the microbes
found in the leucocytes only a field of growth and a means of
dispersion throughout the body. Haeckel, also, had no idea
that the presence of foreign particles in the amceboid cells was
the result of an active engulfing process. The development of
the phagocytic doctrine brought it into opposition to the
humoral theory, which was sustained under the most varied
forms by the most celebrated supporters. As with many other
doctrines which have eventually been admitted as scientific
truths, the doctrine of phagocytosis was revolutionary in con-
ception and had to_conquer by main force.

It originated in zoology and is a result of the comparative
method. From the study of the biology of organisms low in
the scale of life, stage by stage it gained the field of medicine.
These stages we have indicated in the observations and ex-
periments already described in connection with the digestive
activity of the mesodermic cells, intracellular digestion in
general, the reaction of Bipinnaria to the introduction of
splinters, the diseases of such lower animals as are transparent

190 MICROBES AND TOXINS

and suitable for observation in the living condition, e.¢.,
Daphnia, and finally the infectious diseases of animals and man.
“T have sought to develop the conception that the intracellular
digestion found in unicellular organisms and in many
invertebrates has been transmitted by heredity to the higher
animals and in them has become fixed and preserved in the
ameeboid cells of mesodermic origin.” Phagocytosis is in
harmony with the Darwinian principles of evolution among
living beings.

The essential fact of immunity is the intracellular absorption
and digestion of microbes and probably of toxins under
precisely the same conditions as in the absorption and digestion
of cellular elements and albuminoid fluids in general when
introduced into the body.

The general laws are the same whether it is a question of the
absorption of extravasated blood after a wound or an internal
hemorrhage, or of blood corpuscles injected into the peritoneal
cavity of a guinea-pig ; whether one is dealing with cells so
diverse as spermatozoa or epithelial cells, injected into the
peritoneum of a foreign species, or with complex albuminoid
fluids such as blood-serum, milk, egg-albumin, or finally with
the bodies or toxins of bacteria. Laying aside the historical
development let us now attack the mass of facts accumulated on
the subject of immunity.

Taking a general view of the observations and interpretations
which are multiplying every day but are far from being
universally clear or certain, two points of view are continually
being opposed to each other, ¢he activity of the cells and the
activity of the body/luids ; the cellular theory and the humoral
theory of immunity.

The supporters of the ce theory do not deny the participa-
tion of the body-fluids separated, more or less artificially, from
the cells, z.e., the phagocytes ; but they maintain that the cells
are the primary and principal agents, the humoral properties
being secretions or excretions of the phagocytes, and the final
stage in the destruction of the microbes being digestion in the
interior of the phagocytes.

IMMUNITY 191

The supporters of the Awmoral theory consider that in
immunity the body-fluids (serum, exudates, &c.) possess or
acquire destructive properties independent of the cells; that
there is a non-phagocytic destruction of bacteria (and poisons),
and that even when this destruction appears to be completed
in the interior of the leucocytes, the réle of the latter is limited
to seizing and absorbing bacteria already killed.

Of course the antagonism between these two standpoints is
not irreconcilable, and intermediate theories exist. The
priority of the cells, however, as compared with the fluids

Fic. 64.—Intracellular (phagocytic) diges-
ae tion in an intestinal cell of Planaria.
Fic. 63. — Phagocytosis of (Metchnikoff. )
the red corpuscles of the
goose by the phagocytes
of the snail. (Metch-
nikoff. )

independent of the cells, is still the subject of dispute ; no
theory succeeds in explaining the facts of immunity without
acknowledging the activity of the phagocytes and the im-
portance of intracellular digestion.

The humoral theory first took the field with claims or
aspirations to be a “chemical” theory, when some at least of
the phenomena of immunity were successfully reproduced out-

192 MICROBES AND TOXINS

side the body in the test-tube. It must be carefully kept in
view that all the most plausible arguments in its favour are
supplied by “2 vitro” experiments. It is a laudable
tendency to attempt to reduce biological phenomena to a
mechanism reproducible at will, but it must not be allowed to
distort the facts of nature. The study of immunity is, above
all, the study of an infected
body defending itself. We
know neither the nature
nor the composition of
the substances concerned,
albuminoid or otherwise:
we are not even always sure
that the substances postu-
lated really exist. ‘Too
often we yield to the ten-
dency to describe as sub-
stances what we only
observe as functions ; these
Fic. 65.—Phagocytes taking up spores functions have been sym-
of the tetanus bacillus (heated). bolised by names and by
signs, and some have even
come to see in them actual things and things with an actual
shape. It cannot be denied that at present, while we are
still awaiting the advances so generally longed for, vitalism
(in the sense in which certain critics of the phagocytic theory
employ the word) represents the most realistic conception.
Metchnikoff has therefore never ceased to recall and empha-
size the differences which separate the corresponding (one
cannot say “the same”) phenomena “zz vivo” and “‘7n vitro”:
not that he disputes the importance of the latter, but to empha-
size the necessity of associating them always with the phenomena
in the living animal. xperiment ought always to deal as much
as possible with the living creature itself. The cells and the
body-fluids are hardly to be treated as substances capable of
preservation in bottles, and this fact we will have occasion to
recall more than once.

IMMUNITY 193

Antigens and Antibodies.—The cells which play a
part in immunity are known: they are the phagocytes, the
micro- and macro-phages. ‘The humoral properties correspond
to what are known as antibodies.

The antibodies are the products (substances or properties) of
a reaction of the body towards a natural or artificial introduc-
tion into it of certain foreign substances, bacteria and their
poisons, vegetable poisons of other kinds, and various albumi-
noids all known by the name of antigens. The exact definition
of an antigen is its capacity of exciting in the injected (or
infected) body the production of an antibody.

The discovery of the antibodies was so much more a splendid
biological acquisition in that its practical importance was at
least equal to its theoretical. The first antibodies studied were
the antitoxins of diphtheria and tetanus. The discovery of the
diphtheria toxin by Roux led to the discovery of its antitoxin
in the hands of Behring. The neutralisation in an ordinary
test-tube of a toxin by an antitoxin was one of the first and
most brilliant “2 wtvo” experiments in immunity. It might
certainly seem that this neutralisation could take place equally
simply in the living animal with no intervention of the cells, but
like a chemical combination.

When an animal of species A is injected with the red blood
corpuscles of an animal of species B, the serum of the former
acquires the property of dissolving the globules of the other
species: it becomes hemolytic and the prepared animal is
said to have developed a hemolysin. When the body is
vaccinated against the typhoid bacillus, the serum acquires
the property of agglutinating a homogeneous suspension of
typhoid bacilli: it is said to have produced an agglutinin.
The serum of an animal A which has been injected with
the blood or the serum of an animal B of a different species
forms a precipitate when to it there is added a little of the
serum B ; there is said to have been developed a precipitin to B.

Hemolysins, agglutinins, and precipitins are the antibodies
of which the blood corpuscles, the bacteria, and the serum-

proteins are the antigens.
co)

194 ‘MICROBES AND TOXINS

Complement or Alexine.—Before the discovery of the
antibodies, at the time when attempts were being made to
transfuse human patients with the blood of other mammals, in
particular of the sheep, it had been noticed that the normal
blood-serum of certain animals destroys the red corpuscles of
other species: Buchner attributed this property to a defensive
substance which he called a/exine. Its chemical composition
is unknown ; it is thought to be an albuminoid substance: it is
known to disappear from serum on dialysis and to act after a
period of inactivity or incubation ; it is destroyed by tempera-
tures about 56° C., and only acts in presence of salts. Buchner
classes it along with the digestive ferments.

The serum of many animal species, expressed from the blood
after coagulation, possesses the property of killing z# v7¢vo many
infective bacteria without any apparent assistance from the
body-cells: this destructive action is similarly attributed to the
alexine or complement, and it is this bactericidal property
which represents the simplest and crudest fact on which the
whole structure of the humoral theory of immunity has been
reared.

The Two Substances.—Bordet showed in 1895 that the
serum of an immunized animal contains two substances, or
rather two functions. Take a guinea-pig which has received
intraperitoneally spaced injections of cholera vibrios: its serum
now destroys these zz witro, and is said to be bacteriolytic.
Heated to 56°C., it loses this property, but on the addition of
a little fresh serum, recovers it. The bacteriolytic action thus
demands primarily a substance present in the fresh normal
serum of any animal species (non-immunized), a substance
which heating to 56° C. destroys: it is the alexine or comple-
ment. But it demands also another substance present in the
serum of treated animals and absent in normal serum, which is
not destroyed by heating under 65° C.

Bacteriolysis (the destruction of bacteria by serum) is thus
due to the collaboration of two functions, by custom regarded
as two substances. One, thermolabile, is the alexine or
complement and exists in all freshly prepared normal sera.

IMMUNITY 195

The other, thermostable, only exists (with rare exceptions) in
treated immunized animals: in the example given above it
makes the cholera vibrio susceptible to the action of the
alexine or complement, or it may be said to fix this to the
bacteria, or, finally, it may be regarded as forming the connect-
ing link between the alexine or complement and the vibrios.
It has been called by Bordet the ‘ sexstbihisatrice.’ 1

The idea of two substances is not a theory, as Bordet
remarks with great justice; there is nothing hypothetical in
it. It is simply putting in words the facts observed, in
particular those of re-activation.

Hemolysis, agglutination, precipitation, in a word, all the
reactions in which antibodies play a part, proceed in the same
fashion and Bordet’s discoveries have thus a general applica-
tion.

A fundamental experiment performed by Ehrlich and
Morgenroth completes Bordet’s observations. The immune
body becomes fixed on the antigens (bacteria or blood
corpuscles) without producing in them any visible modification,
so that eventually the fluid containing it is completely robbed
of immune body; this fixation prepares the way for the
destruction of the corpuscles or the bacteria, but this latter does
not occur till the complement is added. On the other hand,
the complement does not fix itself to the antigen when alone,
but only by the intermediation of the immune body.

This collaboration of two substances or ferments in one
complex physiological process is not the sole example in biology.
The digestive ferments of the pancreas, amylase, saponase,
and especially trypsin, fail to exert their full activity without the
collaboration of the enterokinase, a ferment secreted in the
juice of the small intestine not by the cells and glands of the
mucous membrane itself, but by the lymphoid tissue, which is
composed of white corpuscles: the importance of this fact will
be seen (Pawlow ; Chepowalnikoff ; Delezenne).

1 The alexine is called by Ehrlich the complement or complementary
substance ; the sensibilisatrice is often called the immunizing substance ; the
intermediate substance, zzmuze-body or amboceptor.

O02

196 MICROBES AND TOXINS

The antibodies quoted may themselves give rise to antibodies :
anticomplements and anti-immune bodies may be prepared.

We know now all the factors which come into play in the
phenomena of immunity: on the one hand cells, the phagocytes ;
on the other the body fluids, containing ferments the actions of
which supplement each other, the immune body and the com-
plements ; we know also that the experiments of bacteriolysis
and hemolysis 7 vitro appear to indicate that the chief
phenomena of immunity are independent of the phagocytic
cells. Let us now examine the problem more closely and see
if phagocytosis stands the test brought against it by the humoral
theories.

PuHacocytTic IMMUNITY

In every case in which the body possesses immunity the
bacteria against which immunity exists are devoured by the
phagocytes, which collect in crowds, incorporate, and digest
them. ‘ Looked at from this standpoint immunity becomes a
phenomenon much more general than a mere resistance of the
body to infectious disease.” On ultimate examination it
reduces itself to
a phenomenon of
cellular suscepti-
bility, of chemio-
tactic influences,
and ofintracellular
digestion, Lmmu-
nity 1s a phenome-
non of digestion,

Fic. 66. — Phagocy- Fic. 67. — Microphage of

tosis of anthrax the rat full of anthrax Phagoc yto sis

bacilli by the bacilli. (Metchnikoff ) can be directly

macrophages of a a

the rat’s liver. observed in many
* (Metchnikoff.) cases of natural

immunity: the
disease of Daphnia presents one of the simplest and most
typical examples, and similar ones have been observed among
other invertebrates. Among the vertebrates the frog owes

IMMUNITY 197

its resistance towards the anthrax bacillus to phagocytosis, the
same bacillus growing excellently in the body-fluids deprived
of cells.

Similarly, the anthrax bacillus grows very well in the body of
a fowl, although the fowl is very resistant to inoculation. The
effect of cold in rendering it susceptible (the famous experiment
of Pasteur) is to be ascribed to a benumbing of the phagocytes.
In the case of the dog resistant to anthrax, or of the guinea-pig to
the spirillum of relapsing fever, or to the cholera vibrio injected
in small dose in the peritoneum, the engulfment and digestion
of the microbes by the phagocytes are visible facts and the
figures appended are better than any description.

On the other hand, the bacteria cannot be said to be expelled
from the body through the various excretory organs. They
are never found in the urine, provided the kidney filtering
action is intact. They are never found in the sweat unless by
faulty technique a little infected blood gets mixed with it.

There is no digestion, even intracellular, without digestive
ferments. Under the microscope the digestion of the ingested
microbes can be seen going on in the digestive vacuoles of the
phagocytes, and by means of the dye, neutral red, the acidity
of the part in which digestion is proceeding is equally easy
to demonstrate, as in the case of the digestive vacuoles of a
myxomycete or an amoeba, or as in the intestinal cells of
Planarians or Actinians. Metchnikoff considers that there are
two varieties of leucocytic digestive ferments corresponding to
the two great groups of phagocytes, the macrophages, which
digest chiefly the cellular elements and the bacteria of chronic
infections such as the tubercle bacillus, and the microphages
which digest chiefly bacteria. They can be obtained by making
extracts of those organs which are rich in phagocytes, the
lymphatic glands, the spleen, and the bone-marrow. In natural
immunity the digestive ferment of the leucocytes is simply the
complement.

There have been, however, many disputes regarding this
point and regarding the origin of complement. Certain
observers have recently maintained that the complement has

198 MICROBES AND TOXINS

nothing to do with the white corpuscles. They have made
extracts of leucocytic exudates withdrawn from the body, and
shown that these extracts were either without bactericidal
power or that the bactericidal substance they contained
possessed properties quite different from those of complement.

It is true that in extracts of white corpuscles prepared
by maceration or freezing no complement can be detected
capable of destroying bacteria, but such experiments do not
prove that the production of complement by these is
impossible. The complement may easily be lost in the course
of the maceration and freezing, rather brutal’ processes in any
case for living cells. It is also possible that the complement
may be neutralised by some antagonistic substance contained
in the leucocyte, some sort of anti-ccomplement: we are
certainly far from knowing all the substances contained
by leucocytes. It is conceivable that they may respond to
a slight injury, received in the course of the manipulation
of the blood, by discharging into the surrounding fluid com-
plement alone, whereas when more seriously injured they may
discharge the neutralizing substance. Later it will be seen
that Pfeiffer’s phenomenon when correctly interpreted supports
this view: in the body thoroughly immunized against the
cholera vibrio but with the white corpuscles uninjured, the
vibrios are not destroyed by the body-fluids and are altered
only in the interior of the cells.

According to Metchnikoff the complement is secreted by the
phagocytes, never excreted, t.e., poured out into the serum or
the body-fluids, so long as the phagocytic process remains
normal ; it is only discharged when the phagocyte has been
injured or phagolysed, as this semi-destruction has been called.
It resembles the zymase of the yeast cells of beer, which
are only liberated by processes which break up the cell. The
fact is one of great importance, as will be found again in
the discussion of Pfeiffer’s phenomenon: the complement
action never takes place outside the bodies of the phagocytes
except when there has been phagolysis.

In acquired immunity, ze, in an animal which has

IMMUNITY 199

recovered from an infection or which has been treated in
the laboratory, other ferments develop which did not exist,
or hardly existed, before the appearance of immunity: these
are the sensibilisatrices, or immune-bodies or amboceptors
of Ehrlich. They are secreted by the macrophages to some
extent, but chiefly by the microphages, and are found in the
spleen, in the lymphatic gland, and the bone marrrow at
a stage in the immunizing process when they are still
absent from the blood. They resist a higher temperature than
the complement, and have properties resembling the ferment
enterokinase of the small intestine. Just as the enterokinase
prepares fibrin for the action of trypsin, so the immune-bodies
prepare the bacteria or other cell-elements for the action of the
complement: this is an analogy between extracellular and
intracellular digestion which ought to be emphasized.

There is not in any given animal a series of different com-
plements: the complement from the same animal performs
indifferently hemolysis and bacteriolysis, and dissolves equally
well the typhoid bacillus and the cholera vibrio. The immune-
bodies, on the contrary, are specific, being developed during the
immunization against the invading cells (by inoculation or
natural infection).

Complement is discharged into the fluids bathing the phago-
cytes only when phagolysis has occurred : the immune-bodies,
on the other hand, are readily excreted by the phagocytes ; they
resemble, not zymase, which is firmly bound to the protoplasm
of the yeast cell, but sucrase, which is easily discharged.
Recovery or inoculation does not increase the quantity of com-
plement, but greatly develops the quantity of immune-body.
In natural immunity the presence of immune-body is difficult
to demonstrate, probably because there is little of it in existence,
and what there is is contained in the phagocytes; but in
acquired immunity immune-body is abundant and is found, not
only in the plasma and serum of the blood, but in exudates and
cedematous fluids. Examples of acquired immunity exist in
which the body remains poor in immune-body and in which
the body-fluids entirely lack it; in these it is necessary to

200 MICROBES AND TOXINS

assume that the action goes on in the interior of the
phagocytes.

To refute the old opinion that the leucocytes form a good cul-
ture medium for bacteria and serve as convenient vehicles for
them, it has been necessary to show that in the phagocytes the
bacteria die and are digested. To refute the opinion that the
phagocytes simply incorporate bacteria already damaged or
killed by other (humoral) actions, it has been necessary to
show that bacteria are ingested in a living and virulent condition:
as a matter of fact living motile bacilli (B. pyocyaneus) can be
seen in the interior of a frog’s leucocyte. Fatal anthrax can be
produced in a guinea-pig by inoculating anthrax bacilli already
engulfed by the phagocytes of a frog: it is only necessary not
to wait too long, or digestion in the leucocyte may be completed.
Pasteur noted that it was perfectly easy to kill the fowl and the
rabbit by inoculating them with bacilli of fowl cholera already
incorporated by the leucocytes of the refractory guinea-pig.
The bacteria attacked by the phagocytes are therefore
thoroughly alive and virulent.

If we take an animal immunised against vibrios and inocu-
late it with the same microbes against which it is immune; if
we withdraw now a drop of the exudate provoked by the
inoculation and make of it a hanging drop in a sealed chamber
at incubator temperature, we find that the phagocytes thus
withdrawn from the body promptly die and the bacteria grow
in their interior as if in culture. But from the phagocytes
withdrawn from the animal a little later no such culture can be
obtained ; the phagocytes have had time to digest the bacteria.
No better conception can be furnished than this of the life and
death of bacteria in the phagocytes.

It has been said that the body-fluids have already attenuated
the virulence of the microbe before its capture by the phagocyte.
If such previous attenuation exists their remains still to be settled
whether it is due to a cellular or to a humoral action: in any
case it is far from being the rule. In Charrin and Roger’s
experiments the streptococcus, the pneumococcus, and the
pyocyaneus bacillus grown in the serum of an immunized

IMMUNITY 201

animal no longer killed fresh animals: but this was due to the
fact that they were saturated with the immune serum which
contained immune-bodies: deprived of these by thorough
washing, they regained their original virulence.

It has further been said that the microbes act through their
toxins and that the body-fluids of an immunized animal begin
by neutralizing this toxicity, after which the bacteria fall easy
victims to the phagocytes. But if this were the case why should
there be such profound differences between the immunity
towards the microbes and that towards their toxins? Why
should there be in animals immunized against the bacillus
pyocyaneus or the cholera vibrio a complete resistance to infec-
tion with these microbes along with a susceptibility to the
toxins equal to that of a fresh animal ?

In all these objections to phagocytic immunity it is always
the idea of a direct primitive action of the body-fluids which
appears, the idea of the humoral theory. Since it is admitted
that the immune-bodies circulate in the plasma whereas
according to Metchnikoff the complement remains in the
phagocytes, since the phagocytic theory maintains that no
excretion of complement occurs without phagolysis, it ought
certainly to have been on this point that the humoral theory
should have made its attacks. This is the central point on
which turns the whole question : if without phagolysis there is no
extracellular destruction of microbes in an immunised animal,
when destruction takes place outside the phagocytes it means
there has been an abnormal lesion, a phenomenon unlikely to
occur spontaneously in nature and probably only an experi-
mental accident ; it was the celebrated experiment of Pfeiffer
which threw the question into prominence.

PFEIFFER’Ss PHENOMENON AND THE HuMmMorAL THEORY

It was the following experiment of Behring and Nissen which,
after the primary investigations of Fliigge, Nuttall and Buchner,
seemed best to explain immunity by the bactericidal power of
the body-fluids ; the serum of guinea-pigs well vaccinated against

202 MICROBES AND TOXINS N

the vibrio Metchnikowii, a cholera vibrio, becomes much more
powerfully bactericidal than the serum of fresh guinea-pigs.1

It is easy to immunize guinea-pigs against lethal doses of
the cholera vibrio injected intraperitoneally. Pfeiffer took a
guinea-pig thus prepared, injected into it a certain quantity of
vibrios, and then abstracted from its peritoneum a little of the
exudate. He found that after a few minutes the vibrios had
almost entirely disappeared from the peritoneum; they had
been transformed into granules, the first stage of destruction,
the “commas” turning into “dots.” Later these granules
dissolved in the peritoneal fluid like a piece of sugar in
water. The same phenomena were observed when the vibrios
were injected along with immune guinea-pig serum into the
peritoneum of a fresh guinea-pig.

Pfeiffer’s interpretation was that in the immunized body the
bacteria are destroyed directly by the body-fluids without the
intervention of the leucocytes.

Such then is Pfeiffer’s phenomenon, ‘so long discussed and
for long a sort of touchstone in the two immunity doctrines.
Metchnikoff and his pupils have subjected it to merciless
criticism. First of all they showed that the granule formation
takes place also outside the body when the vibrios are mixed
with a little fresh serum from an immunised guinea-pig, or even
when to the same serum, which from age or heating has lost
its complement, a little fresh peritoneal fluid is added. (It
was, in fact, while repeating Pfeiffer’s experiment that Bordet
discovered the two substances in the serum of immune guinea-
pigs). In the test-tube, as in the peritoneum, the vibrios fall
victim to the action of the complement through the inter-
mediation of the immune-body.

Since the granule formation is due to the combined action
of the two substances, and since we know that the leucocytes
do not readily shed their complement, which is therefore

1 Tt must be said in this connection that the experiment was insufficient
to permit a general conclusion on the nature of acquired immunity; a
similar experiment with other bacteria gives a different result and even the

vibrio itself when injected into an immunised animal remains alive in its
body for several days.

IMMUNITY 203

rarely found in the normal body-fluids, this destructive action
ought not to take place in these nor in any position except the
peritoneum. As a matter of fact, if the immune guinea-pig is
injected under the skin, in the anterior chamber of the eye, or
in the fluid of a passive cedema, the phenomenon does not
take place: the immune-body is present, but not the comple-

Fic. 69..—Cholera vibrios
phagocyted by a

Fic. 68.—Cholera vibrios phagocyted microphage of the
by a macrophage of the guinea-pig guinea-pig and
and not yet transformed into turned into granules.
granules. (Metchnikoff.) (Metchnikoff. )

ment. When this latter is added in the form of a little fresh
serum, the transformation into granules occurs.

Why then does the phenomenon occur in the peritoneum if
the complement remains inside the leucocytes? It is because
the mere act of intraperitoneal injection produces phagolysis.
The injection of any liquid into the peritoneum, water or
nutrient broth, for example, destroys some at least of the
leucocytes which are found in it: they discharge, as is known,
one ferment under these conditions, that which produces
coagulation of the blood ; in the same way, they discharge this
other ferment, the complement, which acts upon the sensitized
vibrio.

If then this initial phagolysis could be prevented, the
phenomenon of Pfeiffer would also fail. Experiment has
proved this: by injecting into the peritoneum sterile broth,
freshly prepared and tepid, the leucocytes are rendered much
less sensitive to a succeeding injection, and in the peritoneum
thus habituated, thus “prepared,” Pfeiffer’s phenomenon does

204 MICROBES AND TOXINS

notoccur. When phagolysis is thus prevented, the vibrios in all
the variations of the experiment possible fail to be transformed
into granules but disappear by digestion in the interior of the
phagocytes. If we take a guinea-pig strongly immunized
against the vibrio of cholera and inject these bacteria directly
into its circulation by the jugular vein, we find half-an-
hour after no granule transformation in the circulating blood ;
the vibrios retain their shape and are to be seen inside the
leucocytes. No phagolysis has occurred, accordingly no
Pfeiffer’s phenomenon, and no extra-cellular destruction by the
body-fluids.

The resistance of the immunized guinea-pig depends so
much upon phagocytosis, that if the leucocytic activity is paralysed
by means of a dose of opium, the animal succumbs to a
smaller dose of vibrios than is necessary to kill the non-
narcotized animal.

The destruction after phagolysis, Pfeiffer’s phenomenon, is
not even a general fact. It is true of the cholera vibrio, a
fragile bacterium, but even with the typhoid bacillus, also
comparatively fragile, it is only a modified Pfeiffer’s phen-
omenon which occurs. With the bacillus pyocyaneus there is still
greater resistance, and greater still with the bacilli of swine-
erysipelas and anthrax. Jn those cases in which the humoral
action ts imperceptible, phagocytosis ts active and constant.

Opsonins and Bacteriotropins.—There exist more
recent theories which, while recognizing the action of the
phagocytes, attribute to the body-fluids an important part in
immunity: they are said to prepare the bacteria for phagocytic
digestion. These preparatory substances or actions are the
opsonins of Wright and the bacteriotropins of Neufeld.!

1 In their experiments these workers have pursued the same general
method ; they have studied the phagocytosis occurring with leucocytes
withdrawn from the body and suspended in glass tubes, z.¢., the phago-
cytosis zz wztro already studied in the old experiments of Denys and
Leclef. Metchnikoff himself did not fail to compare phagocytosis iz
vivo and 72 vétre, having observed the incorporation of the anthrax
bacillus by leucocytes suspended in urine and in aqueous humour which had
been boiled and thus deprived of all antibodies.

IMMUNITY 205

According to Wright the opsonins are the principal and
determining cause of phagocytosis, the act of incorporation by
the leucocytes being only the final concluding operation.
Opsonins, being the essential factor in immunity, ought not to
be present in the serum of fresh animals, 7.e., there ought not
to be any phagocytosis without opsonins, no spontaneous
phagocytosis. But spontaneous phagocytosis is incontestable,
as has been established by the experiments of Metchnikoff and
Bordet : it is only necessary in 7x witvo experiments to allow
enough time for it to take place: and from the moment that
the existence of spontaneous phagocytosis is granted opsonins
can no longer play the primary part in the phagocytic
process.

It is certain that the presence of normal serum favours
phagocytosis 7 witro (Wright and Douglas, etc.): the serum
acts on the bacteria, which are capable of fixing certain of its
elements. Are the opsonins substances or properties new and
unknown before Wright’s researches? Numerous experiments
ascribe to the opsonins of normal serum the same properties as
characterize the complement. They are products of the
leucocytes.

In the serum of immunized animals, which favours phagocytosis
much more actively than normal serum, the opsonins are not
to be distinguished from the immune-body: they can be used
for the same re-activation experiments (Levaditi) and they also
are products of the phagocytes.

The Jacteriotropins of Neufeld are considered by the
majority of workers as being equivalent to the opsonins of
immune serum and to the immune-body.

There is no reason to deny these actions which favour
phagocytosis. The work on opsonins and bacteriotropins is
simply, to use Ehrlich’s expression, a new flowering of the
phagocytic doctrine. In Wright’s and Neufeld’s experiments it
js chiefly the experimental method which Metchnikoff criticises.
Leucocytes taken from the body, washed and in a different
surrounding medium, can no longer accurately represent the
phenomena occurring in the living body. The conditions are

206 MICROBES AND 'TOXINS

abnormaland yet one knows that itis only in abnormal conditions
that the leucocytes discharge complement. ‘The least change
in the salt content of the surrounding fluid is sufficient to
modify notably the phagocytosis. The leucocytes, of patients
suffering from various diseases present a marked. diminution
in their vital activities. The destruction of bacteria is
the work of phagocytes which are living and vigorous”
(Metchnikoff, Nobel Lecture). Washing, chilling, maceration
are quite sufficient to destroy the complement of the leucocytes ;
how is it possible to conclude after such procedures that the
leucocytes do not contain the complement ?

The opponents of phagocytosis declare that it is the humoral
properties which undergo the most marked increase during
immunization. There is no doubt of such a development of
bacteriotropins, opsonins and immune-bodies—which in any
case are phagocytic products. But it can be shown experi-
mentally that the phagocytes are modified in immunity and
modified sooner than the body-fluids. Leucocytes taken from
an animal vaccinated against some microbe and injected into
a fresh animal protect the latter from several lethal doses
of the microbe, whereas the leucocytes of a normal animal fail.
(Pettersson’s experiment). The white corpuscles of the
immunized animal supply protective substances at a time when
the blood-fluids are not yet affected: and it is owing to the
leucocytes that the body remains refractory after the body
fluids have already lost their protective properties (Salimbeni).

Serum is a fluid into which have been poured the ferments
of the lencocytes, the fibrin-ferment and the complement.
Injury to the leucocytes is necessary before blood will coagulate.
By very delicate operations and with great trouble it has been
possible to separate the blood corpuscles and obtain a
plasma which remains—for a certain time—incoagulable.
Now the properties of such plasma are very different from
those of serum: the leucocytic excretions are absent. It is, how-
ever, so difficult to obtain a true plasma identical with that of
the circulating blood that such experiments have to be very
carefully analysed. When quickly prepared immediately after

IMMUNITY 207

bleeding the plasma contains no complement, but every
minute afterwards injured leucocytes pour into it little by
little the active substance.

Antibodies and Immunity.—There are innumerable
facts preventing us from regarding as a law any correspondence
between the quantity of antibodies in the serum and the
degree of immunity of the animal: this is a definite proof that
there is something else besides the humoral properties and that
the preponderating part is played by the cell elements.

The serum of guinea-pigs inoculated against anthrax was
found by Behring and Wernicke to be incapable of protecting
fresh guinea-pigs from a fatal infection. Pfeiffer immunised
guinea-pigs against the bacterium which he regards as the cause
of influenza in man, but these immunized animals did not
produce a protective serum.

In protozoal diseases such as malaria, there seems to be
immunity in certain cases, but no one has ever demonstrated
a protective property in the serum. To take an example
among the invertebrates, the larvze of the rhinoceros beetle
(Oryctes nasicornis) are immune to anthrax and in them the
phagocytic incorporation of injected anthrax bacilli can be
very well seen; yet the blood fluid of these larve forms a
culture medium equally favourable to the anthrax bacillus, to
which they are immune, as to the cholera vibrio which produces
in them a fatal infection.

Again, the dog is extremely resistant to anthrax, yet the
anthrax bacillus grows very well in dog-serum ; all these are
examples of immunity in which the bactericidal power does
not play a part. It has been known since the experiments of
Behring that rat’s serum possesses a remarkable destructive
power towards the anthrax bacillus; now the rat is not
immune to anthrax, and the degree of immunity which it does
possess is due to the phagocytes. The bactericidal substance
exists in the leucocytes, but not in the circulating plasma, nor
in plasma carefully prepared by Gengou’s method. It exists
in the serum, but only because it has been discharged into
this by the leucocytes. The rat is extremely susceptible to

208 MICROBES AND TOXINS

anthrax if it is inoculated with a very fine needle so as not to
provoke a hemorrhage at the point of inoculation.?

Immunity towards toxins could be made to furnish analogous
examples in abundance; it will be discussed in the next
chapter.

In many cases Pfeiffer has seen his guinea-pigs, which had
been thoroughly immunized against the cholera vibrio, succumb
to the injection of a moderate dose of vibrios. Yet the serum
of these guinea-pigs was capable of producing Pfeiffer’s phe-
nomenon.?

Tuberculin carefully employed exerts a favourable effect in
many tuberculous patients, and leads to the production of
antibodies in their serum. Jochmann has quite recently made
several observations of this kind under the direction of R. Koch,
and has sought for a correspondence between the appearance
and quantity of the antibodies and the resistance of the
patient. He found it was impossible to maintain that the
presence of antibodies meant recovery. Certain patients mani-
fested great clinical improvement simultaneously with the
appearance of antibodies; in others, the improvement was
quite as great without the antibodies, while in other cases the
appearance of the antibodies: coincided with marked and fatal
aggravation of the malady. It is obviously impossible to draw
any conclusion as to the immunity of these patients from such
im vitro experiments.

1 In this example of the rat there is no question of an antibody produced
by immunization, but rather of a want of agreement between the watural
immunity of the animal and the zatwra/ bactericidal power of the serum.

2 Quite recently Citron has shown that the serum of rabbits actively
immunised against the so-called bacillus of hog-cholera possesses protective
properties for guinea-pigs, whereas it has none of this for fresh rabbits.
Rabbits prepared with extracts of the bacilli and without active immunity
of their own (they succumbed to the test inoculation of living bacilli),
nevertheless furnished a protective serum for guinea pigs. Choukewitch
taking up this question again, prepared rabbits by large intravenous
inoculations of the same bacilli, but in the killed condition. One rabbit
of the lot acquired immunity towards the living microbes, but in every one
of ‘the series, not only the immune individuals but also the non-immune,
the blood contained an abundance of antibodies, immune-bodies, opsonins,
ete... .. It even contained much more than the serum of rabbits
rendered truly immune by subcutaneous inoculations of virulent bacilli.

IMMUNITY 209

Immunity is entirely a cellular function and the inherent
tincture of “ vitalism ” in the phagocytosis doctrine is unavoid-
able.

“The final phagocytic reaction is represented by the physical
or physico-chemical processes in the digestion of microbes,
conducted with the help of cytases, and favoured by the
presence of immune bodies; in the resistance to poisons the
phagocytes must also exert chemical influences. But before
these phenomena occur, the phagocytes present activities which
are purely biological; such are the chemiotactic perceptions
and movements directed towards the threatened spot, the
engulfment of bacteria, and the absorption of toxins, and
finally the secretion of substances utilized in cellular digestion ”
(Metchnikoff, Z’Limmunité, p. 590).

CHAPTER XI

IMMUNITY

Toxins and antitoxins—Chemical and physical conceptions of immunity.

paar theory—Origin of the antibodies—Theory of chemical equi-
librium.

The physical point of view : Bordet—Phenomena of absorption or molecular
adhesion—Explanation of specificity—Analogies between the reactions
of antibodies and the reactions of colloids — Lipoid actions.

Phagocytosis and toxins—The body plays an essential part—Origin of
antibodies and Wassermann’s experiments—The phagocytes in their
connection with mineral poisons and microbial poisons, toxins, and
endotoxins.

Ir was the discovery of antitoxins which inaugurated the study
of antibodies. It was the necessity of explaining the action of
antitoxins on toxins which gave rise to the theories on this
peculiar problem and on antibodies and immunity in general.

It was thought at first that in the immunized animal which
manufactures the antitoxin, as well as in the animal immunized
by the injection of the antitoxic serum, the cells play a part.
Buchner enunciated the hypothesis that the body produces
antitoxin by transforming the toxin; he quoted, as a distant
analogy, the transformation of one compound into another by
polymerization. But it is difficult to understand how there
could be such a disproportion between the toxin injected and
the antitoxin produced; the horse produces, according to
Knorr, for one unit of toxin injected 100,000 units of anti-
toxin.

Buchner performed a pretty experiment, which has lost none
of its interest, by showing that after accounting for the

differences in weight and in natural susceptibility, a mixture of
210

IMMUNITY 211

tetanus toxin and antitoxin which is neutral for the guinea-pig
is fatal to the mouse ; it is impossible to avoid the conviction
that the body, that of the guinea-pig or of the mouse in this
example, counts for something in the phenomena. The same
ideas were maintained by Roux at the time of the discovery of
serotherapy.

But, on the other hand, the action of antitoxin on toxin
seemed to be a neutralization, and to behave both zz vitro and
in vivo like a chemical reaction ; and in practice it was found
necessary for medical purposes to titrate the sera in this way
to measure their activity; the ideas of Buchner and of Roux
were then laid aside, only to be rediscovered later; the
biological phenomena were subordinated as much as possible
to quantitative studies, and the endeavour was made to
represent the action of antitoxins on toxins as a chemical
reaction.

The Side-chain Theory.—The best known chemical
theory for the action of antitoxins and antibodies in general
is that of Ehrlich, which is currently known under the name
of the “side-chain theory.” The primary idea of its author
was to find in the facts mutual relations as much as possible
fixed and independent of the body, and to eliminate all
“vitalism” in favour of exact quantitative work. To begin
with, he adopted the method of zz witvo experiment. The
nature of the tetanus and diphtheria toxins had been rendered
much clearer by the study of other toxins more easy to work
with, such as heemolysins, agglutinins, ferments, and anti-
ferments (ricin, abrin, rennin, and antirennin, etc.). Pre-
liminary experiments i” vitro showed the general applicability
of the same laws. Originally Ehrlich believed that the curative
and preventive action zz vivo was equivalent to the neutralising
action towards the diphtheria toxin zz vitro, a belief which later
experiments were to disturb.

The second step was to demonstrate that antitoxin does not
destroy toxin, but that the two bodies combine to form a com-
pound (neutral from the physiological point of view), just as an
acid and a base combine to form a salt.

P 2

212 MICROBES AND TOXINS

Antitoxin does not destroy toxin because the two reacting
bodies can be recovered. For example, the neurotoxin of
cobra venom resists heating to 68° C. ; when the neutral mixture
of venom and anti-venom is heated, the toxin can be recovered
if this isdone during the ten minutes which followthe preparations
of the mixture (Calmette’s experiment). From a neutral mixture
of cobra-hemolysin and anti-venom, it is possible to recover the
hemolysin by the action of hydrochloric acid ; the recovered
heemolysin manifests its action on the addition of the necessary
lecithin (Morgenroth’s experiment). A mixture of diphtheria
toxin and antitoxin (twenty-four hours’ contact), harmless to
the rabbit, recovers its toxicity when treated with hydrochloric
acid, the toxin being set free (Morgenroth and Willanen’s
experiment). Heat, filtration, the action of a digestive diastase,
are other methods of dissociating the toxin-antitoxin com-
bination provided the intervention takes place without too
long delay.

How then are the toxin and anti-toxin to be represented ?
Probably as albuminoid substances of large molecules capable
of being represented by stereochemical models and possessing
a nucleus on which are grafted lateral chains. To conceive of
the action of the toxin on a cell, it is only necessary to imagine
the molecules of the cell protoplasm as containing figures of
the same kind. The toxin molecules enter into relation or
combination with the cell molecules by means of these atom
groups or side-chains. Ehrlich calls them “receptors”
or “haptophorous groups.” The term “side-chains” was
borrowed directly from the chemistry of benzene. Such
stereochemical symbols were introduced into science by Emil
Fischer to represent the specific action of the ferments: one
body acts specifically on another, because an atom group of
the one is adapted to an atom group of the other, as with a
key and the corresponding lock. ‘Thus the haptophorous group
of a toxin fixes itself on the receptor of a cell, both haptophore
and receptor being “ side-chains.”

This in fact is the central idea of the theory, the same mode
of chemical action, the same relations between receptors and

IMMUNITY 213

haptophorous groups, conceived after the manner of the
reactions of organic chemistry ; and this molecular stereo-
chemistry explains all vital phenomena : the action of a toxin
on the cell, the action of antitoxin on toxin, the production of
antitoxins, and immunity in general.

Finally, since the same combinations and linkages go on in
the metabolic changes of all living matter, the conception of
side-chains becomes a general theory of nutrition; so much
so that, with all his chemical language and mechanical attitude
of thought, Ehrlich arrives at the same formula as Metchnikoff
with his ‘‘vitalistic,” or rather, biological explanation :
immunity is a function of nutrition.

This idea has been Ehrlich’s guiding principle in all his
scientific work, and it is from it that we have to start in order
to arrive at the principal laws of immunity.

Let us represent the protoplasm molecule as possessing
numerous and varied functions, the agents or the bases of
which are distinct atomic groups. This molecule consists of a
central nucleus (analogous to the nucleus of the aromatic
compounds), which maintains its continuous individuality,
and of numerous side-chains or receptors which act towards
the nucleus as organs of communication or nutrition. The
primary food-stuffs and the toxins which circulate in the blood
and body-fluids have haptophore groups which are fixed by
the cell receptors; and it is thus that all the modifications
of the protoplasm are carried on.

Take a poison like the tetanus toxin introduced into the’
body. We know by definite experiments that it is ‘“ fixed”
by various cells and in particular by the nerve-cells. The
toxin molecule is treated by certain definite receptors of the
nerve-cell as a food material ; it possesses a haptophore group
which hooks on to the receptor, but it possesses also a toxophore
group, an atom group which exerts the toxic action; it is
through the haptophore group that the toxophore group is fixed
by the cell and acts as a poison.

If the toxin has been injected in sufficient quantity,
numerous cell receptors are occupied, monopolized by the

214 MICROBES AND TOXINS

toxic molecules; the cell, deprived of the use of these
receptors, has its functional activity diminished and its nutrition
threatened. But all injured protoplasm possesses a reparative
or regenerative power ; the cell reproduces receptors ; it even
manufactures a great many more than is necessary. To follow
Weigert’s dictum the living matter overstimulated by the lesion
regenerates itself much beyond its needs; these receptors
regenerated in excess are cast off by the cell and pass into the
body-fluids ; and it is these which fix and neutralize a fresh
dose of toxin injected into the body.

Antitoxin is nothing but these Free Receptors.—
It acts like a lightning-conductor. Withdrawn from the body
which produces it and introduced into another animal, it
retains the same fixing property and is the active agent in
therapeutic sera.

Thus there is no essential difference between the receptors
which produce the antitoxin and the normal “nutrition

i
? T A
t Ah ar
2 ar. _
1 2g 4

3
Fic. 70.—Diagrams to represent Ehrlich’s theory.

1, Haptophore group 4 and toxophore group ¢.

2. Cell C injured at 7: its receptor y, The toxin is represented by 7
with its haptophore group / and its toxophore group ¢.

3. A toxin molecule: haptophore group 4: toxophore group /: anti-
body or free receptor a. 7.

4. A cell C with a fixed receptor +: a detached free receptor forming
be ae a. r.: the toxin 7 with its haptophore and toxophore groups

and 2.

receptors” of the cell. A cell susceptible to the poison pro-
duces an antidote, but the antidote may also be produced by
cells which are insusceptible, z.e., not only by the “noble”
cells, but also by the connective-tissue cells, and it is necessary

IMMUNITY 215

to add, in the light of the experiments of the Metchnikoff
school, by the leucocytes.

In bacteriolysis and hemolysis as defined by Bordet’s
experiments, also immunity phenomena, the complement and
the amboceptor (or immune-body) come into play. Com-
plement is an atom group already present in the body, whereas
amboceptor is analogous to the antitoxin: it possesses, how-
ever, two haptophore groups, one uniting with the cells (blood
corpuscles or bacteria), the cytophil group, the other linking
up the complement, the complementophil group. The anti-
bodies are receptors or amboceptors set free and detached
from the cells which form them.

It is impossible to follow here all the developments of the
side-chain theory. Ehrlich has complicated it almost to
excess in order to include in it the infinite complexity of facts
observed in experiments with the various antibodies, anti-
toxins, heemolysins, bacteriolysins, and the other cytolysins,
precipitins, and agglutinins. The dominant idea is always to
give a chemical interpretation of nutrition and, as a particular
case of this,-of immunity.

The conception was first intended to explain the physiology
of toxin and antitoxin action, and it is to this that it is best
adapted ; but it has had to undergo complication, not only to
include the largest number possible of the ascertained facts,

but to act up to its original intention as an explanation in |

terms of chemistry.
It is to explain all these facts! that the distinction between

1 For example these, that the broth culture of the diphtheria bacillus
which constitutes crude toxin is not a simple substance: nor is there any
reason to believe that the broth cultures of the tetanus bacillus is any
more so, since it contains at least two poisons, tetanolysin and tetano-
spasmin. The products of cell-life are frequently very complex : Ehrlich,
for example, quotes with justice cinchona bark with its twenty odd
alkaloids and the liver cells with their round dozen ferments.
Toxin left to itself, even protected from light and heat, rapidly becomes
modified : it feels the effect of age and not only deteriorates in activity but
undergoes qualitative changes. Toxin acts after a period of incuba-
tion. The neutralization by antitoxin no longer proceeds in the same
way when the appropriate dose of antitoxin is added in separate fractions
instead of all at once (Danysz-Dungern phenomenon).

216 MICROBES AND TOXINS

the haptophorous and toxophorous groups has been conceived,
and that in toxin the toxones and toxoids have been postu-
lated. But the complexity is also due to the necessity of
making the facts conform to the idea of the chemical nature
of these actions, and especially to the law of mu/tipla, which
demands that the same quantity of toxin should always be
neutralized by the same quantity of antitoxin. The theory
flatters itself also on its capacity to explain the specificity of
the antibodies ; for the complexity of the groups is supposed
to be sufficiently great to permit a hemolysin against goat
corpuscles to differ from a hemolysin against rabbit corpuscles.
Even for the fairly numerous cases in which specificity is not
rigorous an explanation is forthcoming: different receptors
possess certain elements in common, and it is possible that a
serum which precipitates horse serum may also precipitate the
serum of the ass.

The side-chain theory has been of great service ; it has, its
supporters say, a great “‘ heuristic” value, z.¢., it has been the
means of discovering many interesting phenomena, and has led
Ehrlich on to his chemotherapeutic studies, in which he has
gained such magnificent successes.

Nevertheless, it cannot be said that these fortunate results
prove the truth of the theory: the discovery of “606,” for
example, proves neither the existence of the side-chains nor the
truth of the chemical theory of immunity,

Theory of Chemical Equilibria.—Again to explain the
action of toxin and antitoxin, Arrhenius, Madsen and Walbum
have proposed another chemical theory. They criticize the
complexity of the theory of Ehrlich, they do not admit the com-
plex nature of the diphtheria toxin, and they attach great im-
portance to the experiment of Danysz-Dungern on partial
saturation. The facts can be explained by conceiving the
toxin + antitoxin reaction as a chemical interaction, not
between a strong acid and a strong base, but between a weak
acid and a weak base, for example, ammonia and boric acid.
The reaction toxin and antitoxin is comparable with those
reactions known as reversible and is governed by the law of |

IMMUNITY 217

mass-action of Guldberg and Waage, if one supposes a state
of unstable equilibrium to exist among the combinations.}

The combination toxin + antitoxin must therefore be dis-
sociable ; Arrhenius and Madsen are of the opinion that they
have demonstrated this by their experiments on the diffusion
of the mixture in a column of solidified gelatine and by their
distribution experiments (¢.g., distribution of agglutinin between
bacteria and the immersing fluid). Many facts which led
Ehrlich to his hypothesis on the toxins are explained by
Arrhenius in terms of these dissociations, and in general by
the fact that antibodies and antigens have only a feeble affinity
for each other.

To the diffusion experiments it has been urged in reply that
the dissociation only takes place because the mixture poured
on the gelatin has not had time to form the final combination ;
while to the hypothesis that a quantity of /vee toxin is present
in the mixture, it has been replied that if this were the case
antitoxin would never act in the body; the body would fix
the free toxin, the toxin-antitoxin equilibrium would be dis-
turbed, a new quantity of toxin would be set free, and
so on. ;

Physical chemists regard the dominant idea of Arrhenius’s
theory with great reserve ; they doubt the justice of employing
the laws of chemical equilibrium and rates of reaction in
speculations as to the reactions which go on between bodies of
which nothing is known from the chemical point of view.
Nernst has verified the use made of the laws of reversible
actions, and he denies the possibility of applying to colloidal
substances laws established only for homogeneous liquids.

This does not mean that there is anything odd in attempting

! When a substance in solution of a molecular concentration # reacts
with another substance in solution in concentration 2, the mass of the
substance formed by their combination is in a given time proportional to
the product #272.

For example, when in a given volume 3 molecules of alcohol react with
2 molecules of acetic acid, the quantity of acetic ether formed in a given
time is 3x2 =6; if 5 molecules of alcohol react with 3 molecules of
acetic acid the quantity of acetic ether formed in the same time is expressed
by 5x3 = 15.

218 MICROBES AND TOXINS

to apply physico-chemical laws to biological phenomena in
spite of the variability of living creatures and of their products.
It is impossible for biologists to refrain from seeking quanti-
tative laws and from applying physico-chemical laws to
immunity phenomena. Quantitative results have been ob-
tained in the study of diastases, and Ehrlich has discovered
facts of the greatest interest by means of his experiments
in vitro on titrations and measuring. It is only necessary to
agree upon the conventions necessary (in physics itself these
cannot be dispensed with) and not to employ such unjustifiable
expressions as “ guérison in vitro” (in vitro cure).

It is always possible to return to the biological point of
view when this becomes necessary, as Ehrlich himself did,
when considering Weigert’s ideas on the regeneration of
protoplasm.

THE PHYSICAL POINT OF VIEW.

Bordet rejected Ehrlich’s system, and compared the “anti-
gens + anti-bodies ” reactions to phenomena of absorption and
molecular adhesion, even before the closer comparison with
colloidal reactions had been arrived at.

He does not only complain that the side-chain theory is too
complicated ; he criticises its whole disposition.

Immunity is a problem not yet ripe; and the solution will
probably come from a quarter quite unexpected. There
are enormous gaps in our knowledge. Why, then, make
adventurous generalizations when the biological facts are far
from permitting this? Every theory that can be constructed
must base itself for the moment on facts not yet demonstrated.
Let us keep close to the experiments, and be content to
advance step by step. Ehrlich’s theory is dangerous, in that
it offers too readily conceptions which have the appearance of
explanations, and which, therefore, are apt to dull the appetite
for research. “For my part,” adds Bordet, “I have been
unwilling to construct a theory ; I do not adduce any general
conceptions ; the hypotheses which I have proposed are

IMMUNITY 219

scarcely worthy of the name, they differ so little from mere
critical statements of experimental results. Even at the risk
of being regarded as incapable of generalizing, I prefer to stick
to the facts without moulding them into a system.”

In immunity phenomena, we observe certain activities ; but
why materialize them and picture each by an atomic group?
In the side-chain theory we are told that the antibody is
nothing but a cell-receptor affected by the antigen. This
identity is not proved. Why should not the cell with its
power of adaptation and reaction produce some new and
original substance ?

When the “substance” which we call agglutinin clumps
cells or bacteria, does it really bring into action an atomic
group, or side-chain, which attaches itself and another group
which agglutinates? The explanation is artificial. In reality
it is not the agglutinin which agglutinates ; it is a salt. The
antigen (bacteria) and the antibody (agglutinin) form a com-
bination which produces floccules, or, as it is expressed
nowadays, is ‘‘flocculable” by electrolytes. It is this couple
or “ complex ” which agglutinates.+

An analogous coupling must be regarded as taking place in
all the reactions of antigens and antibodies, and Ehrlich’s
theory is wrong in attributing everything to the antibodies
and nothing to the antigen. There are no “ amboceptors”
in reality, there only exist ‘“uniceptors” capable of being
absorbed.

It was therefore not with the purpose of disputing details
that Bordet accumulated his experiments on the mode of
fixation of the complement on the immune body; in this
field he has discovered the principal facts which render the
side-chain theory impossible as a dogma, if not altogether
impossible as a conception of certain phenomena. The
important fact is that there is never absorption of complement
by an immune body without the presence of an antigen, so

1 The salt acts on bacteria saturated with agglutinin but it acts also on
bacteria which have absorbed various chemical substances, iron, uranium,
or aluminium.

220 MICROBES AND TOXINS

true is it that it is the antigen-antibody compound which
absorbs the complement. Although the complement of one
animal species may differ from that of another species, yet in
a given serum, in opposition to Ehrlich’s theory, there exists but
one complement or rather one complementing property (exp.
of Bordet, Gay, Muir and Browning, etc.). It is due to
Bordet’s correct attitude on these points that the Bordet-
Gengou reaction (complement fixation) has been capable of
such successful application in various bacteriological diagnostic
methods, and recently by Wassermann in the diagnosis of
syphilis.

Finally, since the complement attaches itself, not to the
immune body, but to the antigen-antibody combination, there
is no reason to suppose the existence of a haptophore group
of the ‘complementophile” kind, indispensable to the
immune body if complement is to be fixed. This question
has for some time been a sort of Zest for the side-chain
theory, and it seems to have resulted in favour of Bordet’s
ideas.?

From the beginning of his researches in 1896, Bordet has
imagined immunity reactions, not as chemical combinations,
but as physical phenomena of absorption or molecular adhesion.
He considered that in agglutination (where the bacteria are
passive, since dead bacteria also agglutinate) serum acts by
modifying the relations of molecular attraction between the
bacteria and the fluid bathing them, and that, in the first
phase of the phenomenon at least, the bacteria behave like
particles in general. Under the influence of Duclaux’s ideas,
he observed the resemblances between agglutination and
coagulation. From the point of view of their coagulating and
dissolving properties, he compared the active sera to the
digestive juices, and, like Metchnikoff and after him Ehrlich,
he also saw in immunity, though from a different point of
view, a particular case of the physiology of digestion.

At that time the results of the study of colloids had

- Experiments of Ehrlich and Sachs, of Sachs and Bauer; of Bordet
and Gay and Bordet and Streng on hemolysis by ox-serum.

IMMUNITY 221

scarcely begun to be applied to the study of immunity, and
the body-fluids, the toxins, and the antitoxins had not yet
been studied from the point of view of their colloidal con-
stitution. The phenomenon of adsorption (ad in preference
to ad as expressing the idea of attraction or adhesion) is a very
general one, and does not depend absolutely on the colloidal
state. Bordet therefore, in explaining his conception, pre-
ferred to employ the comparison with dyeing processes. ‘The
action of antitoxin on toxin appeared to him to resemble, for
example, the action of iodine on starch: the immune body
which prepares bacteria or cells for the action of complement
he regarded as acting after the manner of mordants in dyeing,
intermediary substances necessary for the fixing of certain dyes
on certain cloths. Thus in hemolysis the union of the
immune-body with the blood corpuscle (anti-body + antigen)
forms a combination possessing a greater adsorptive affinity
than the normal corpuscle: the complement tends to become
precipitated on the sensitized corpuscle, and the attraction
which the latter exerts is more powerful the more heavily it is
sensitized.?

Inorganic substances present similar phenomena. Water
runs off a watch glass coated with paraffin without sticking to
it, but if the water contains barium sulphate in suspension it
wets the paraffin and spreads over it; this depends on the fact
that the surface of the paraffin becomes coated by molecular
adhesion with a thin white film of barium sulphate, which
water can wet; this film is not removed by rinsing in water,
and can only be removed by rubbing. There are even sub-
stances which inhibit this fixation of the barium sulphate on
the paraffin, just as there are substances which inhibit the
fixation of complement by sensitized corpuscles.

On all the most important points of the question of the
toxin and antitoxin combination, the physical theory is the

1 Those sera which possess the power of inhibiting hemolysis act by
keeping the complement in a condition of greater suspension or dissemina-
tion in the fluid ; they thus render it more stable, unlike sajine solution,

which produces a condition of instability in which the complement
condenses itself or is precipitated on the attracting sensitized cells.

aN

\

\

Leek

222 MICROBES AND TOXINS

reverse of the chemical theory of Ehrlich; the one simplifies
where the other complicates.

In the chemical theory the same quantity of antitoxin ought
to combine with the same quantity of toxin, so, to account for
the irregularities in the actual facts, there have been introduced
the hypotheses of the haptophore group separate from the
toxophore, of the toxones, toxoids, &c. On the contrary,
Bordet supposes that the antitoxin really unites with the toxin
in varying proportions: the toxin can fix, can, so to speak,
dye itself with toxin in greater or less amount just as starch
can take up varying quantities of iodine and become thereby
stained a more or less dark blue. In the same way, in
heemolysis the corpuscles can absorb variable amounts of the
active substance according to the concentration of the solutions
and the duration of the contact. The distance of this idea
from the theory of chemical equivalents is apparent. When a
given quantity of toxin is mixed with a quantity of antitoxin
insufficient for complete neutralization, what occurs is not a
monopolization of the antitoxin by a portion of the toxin mole-
cules, forming a complete combination with it while the rest of
the toxin remains free (z.e., the chemical conception). On the
contrary, the antitoxin is equally distributed over all the toxin
present, so that the latter is attenuated throughout and presents
a diminished activity. To return to the same analogy, it is
faintly dyed. It can produce toxic effects qualitatively different
from those due to an intact toxin or a toxin completely
neutralized without necessitating the hypothesis of a special
chemical condition (toxones).?

The phenomenon of Danysz-Dungern (the antitoxin has a
different action on toxin when the mixture of the same
quantities is made at one instead of several additions) does not
compel the hypothesis that in toxin there are several chemically

1 The fact first observed by Ehrlich, namely, the difficulty of preparing
exactly neutral mixtures of toxin and antitoxin, can thus be easily explained.
The effects produced by a hemolysin more or less neutralized and the
experiments of Grossberger and Schattenfroth on the toxin and antitoxin of
the bacillus of quarter-evil have confirmed Bordet’s views of the nature of
the toxin + antitoxin reaction.

IMMUNITY 223

distinct components. If a large piece of filter-paper is dipped in
a rather dilute solution of a dye it is faintly stained; if the
plece is cut into small pieces and these are immersed in turn
for a certain time, we find that the first pieces take up the colour
and leave almost none for the last. Substitute for the dye the
antitoxin and for the paper the toxin ; if the mixture is made at
one blow the toxin is attenuated in its whole bulk : if, however,
the mixture is made by several additions, the first portions of
the toxin take up the antitoxin and the later portions, not being
neutralized, remain much more toxic.

Further, the fact that toxin and antitoxin mixtures (and in
general antigens and antibodies) become in time less dissolvable
and more stable is also explained by adsorption phenomena.

When a piece of cloth is placed in a dyeing vat the dye
loses its attachment to the dissolving fluid and adheres more
and more intimately to the cloth until it can no longer be
redissolved. A similar example may be quoted in connection
with the precipitates produced by alcohol in certain albuminous
fluids, precipitates which are fairly easily redissolved in water
immediately after their precipitation, but which are no longer
soluble when a certain time has been allowed to elapse for
their aggregation. When three substances exist together, two
of them may compete with each other in the fixation of the
third ; it is thus that the protective action of certain substances
is to be explained, as, for example, the albuminous substances
of the blood which protect blood corpuscles against the action
of soap (Meyer) or of eel-serum (Frouin).}

One thing that the physical theory has not yet explained is
the specificity of the reactions of immunity, but it is not
incapable of explaining even this.

It is not difficult to imagine that slight physical modifica-
tions may change the affinities on which depend the molecular
attraction ; that is certainly no more difficult to imagine than

1 Citrate of soda protects blood corpuscles against the agglutinating and
hemolytic action of sulphate of barium. The lecithin of ox-serum is held
in check as regards its action on guinea-pigs’ corpuscles by some albuminoid
material.

Q24 MICROBES AND TOXINS

the innumerable molecular groups postulated by the side-chain
theory. For example, the antiserum prepared by an animal
against a protein has not the same properties as the serum
obtained against the same substance previously subjected to
heat (exp. of Obermayer and Pick). Hens’ serum agglutinates
the lipoids extracted from the red corpuscles of the rabbit
much more vigorously than those from the ox. It is easy to
found a theory for specificity on the absorption phenomena,
‘and such theories already exist ; hitherto they have been too
philosophical, but it is satisfactory to know that experiment
has already furnished the germ of a scientific explanation.

The Colloids.—After Bordet’s explanations of agglutination
and hemolysis in terms of molecular attractions and cohesions,
and in the light of his comparison of these phenomena to
dyeing processes, Zangger, Landsteiner, and Jagic established
experimentally the first analogies between immunity phenomena
and the physics of colloids.

Reactions between colloids or between colloids and true
solutions can be reduced to phenomena of molecular attrac-
tion, of absorption, and adsorption. The bodies participate
in the reactions in variable proportions, influenced by tem-
perature and pressure. Colloids of opposite electrical charge
exert on each other a “flocking” or precipitating action,
which may be,masked when one or other is in excess in the
mixture ; one colloid may inhibit the precipitation of another
colloid by a, salt. In many cases, the law of the opposite
electrical charge may be masked by the fact that the proteins
are amphoteric colloids, and may neutralize acids and alkalies
equally well, behaving in acid solution as bases, in basic
solutions as acids.

Are the antigens and antibodies of immunity colloids?
The only ones whose chemical composition is known, namely,
the lipoids (fatty bodies typified by lecithin or cholesterin),
behave, in watery suspension, like the colloids ; the others,
which probably belong to the proteins, behave like colloids
from the point of view of diffusion in dialysis, heat, and
instability, and their principal reactions are closely analogous

IMMUNITY 2 225

«
to those of colloids. In any case, there might exist between
the antibodies and antigens on the one hand, and the colloids
on the other, considerable differences without preventing the
general laws of attraction and adsorption from being equally
applicable to the former as to the latter.

Agglutination and precipitation closely resemble the flocking
of colloids. Bacteria behave towards a precipitating serum in
the same way as they behave to certain substances quite
foreign to the body, such as gelatine or gum-arabic.

Bacteria, in presence of a solution of ferric chloride, are
protected by the colloidal ferric hydrate from agglutination by
their specific agglutinin.

The phenomena of specific hemolysis (¢.e., haemolysis by
the serum of immunized animals) have been imitated
by attacking the corpuscles by means of such systems as
silicic acid + lecithin, colloidal ferric hydrate + dog-
serum, ord saponin + taurocholate of sodium: the two kinds
of phenomena may be expressed by the same curves
(Zangger, Mlle. Cernovodeanu, and V. Henri). Comple-
ment can be fixed by the most diverse substances, by
Witte peptone, yeast cells, the cells of organs, and various
precipitates.

The toxin + antitoxin reaction has been “imitated” by the
interaction of arsenious acid and colloidal ferric hydrate. The
phenomenon of Danysz-Dungern can also be reproduced with
colloids; the final result in the precipitation of a colloidal
suspension is different according as the precipitant is added in
one large or in fractional doses.

The reactions between cholesterin and various poisons and
between cholesterin and lecithin have the appearance of
colloidal reactions. One may even observe affinities of the
character of the specific affinities. For example, cholesterin
neutralizes saponin and tetanolysin, but has no effect on ricin
or staphylolysin. Complement is absorbed, as has been seen,
by a great variety of substances ; agglutinin, however, possesses
affinity chiefly for the colloidal protein, while tetanus toxin is
chiefly fixed by the lipoids. It is legitimate to conceive of

Q

226 MICROBES AND TOXINS

theseraffinities as depending upon physical conditions, including
that of the electrical charges.

The “poid substances which are found in such abundance in
nerve-tissue and which constitute a constant component of
protoplasm, according to Overton furnish the cell with a sort
of envelope through which the food materials have to pass and
which behaves as a sort of colloidal atmosphere ; the principal
members of this group are lecithin and cholesterin. It is
inconceivable that they do not play some part in immunity
phenomena which are phenomena of nutrition. Hzemolysin,
for example, undoubtedly induces changes in the lipoid coating
of the red blood corpuscles.

The lipoid extracts of red corpuscles readily fix normal
hzemolysins, whereas the lipoid extracts of bacteria fix certain
immune-bodies. The lipoids also are capable of fixing the
complements.

We have already mentioned two series of experiments in
which lipoids play a most definite part. In the first place we
have the activation of cobra venom by lecithin (Kyes), in which
the latter appears to play the part of complement. According
to Noguchi, triolein, oleic acid, exerts the same effect and loses
its activity quite like complement when heated. Oleic acid is
said to be capable of activating specific heemolysins, and silicic
acid, which alone possesses a very feeble hemolytic power, is
said to form with lecithin a complex or “lecithid” which is
much more powerful.

In the second place lecithin plays the part of antitoxin
towards certain toxins. The bile or the soluble elements
of bile neutralize: snake venom in appropriate dose; the
cholesterin is the active constituent. Cholesterin and lecithin
neutralize the botulismus toxin. Cholesterin neutralizes
saponin, solanin, agaricin, vibriolysin, lecithids of cobra venom
and of the poison of bees: again it is its cholesterin content
which makes serum neutralize saponin. Finally in the
celebrated experiment of the fixation and neutralization of
tetanus toxin by the cerebral cortex (Wassermann and Takaki’s
experiment) the lipoids of the grey matter are the active

IMMUNITY 227

agents; the brain extracted with ether loses much of its
neutralizing power, and the dye carmine neutralizes because it
contains lipoids derived from the cochineal insect (Metchnikoff).

It is facts such as these which encourage investigators to
pursue the line opened up by Bordet’s experiments, although
there is no absolute promise that they will find in this the key
to immunity.

If we add now that phagocytosis is not incompatible either
with Bordet’s theory or with Ehrlich’s, it is true enough, but it
is not all the truth. It is not a question of reconciling
theories. There are only two “theories,” that of Ehrlich and
that of Bordet, which, with their conjectures, their uncertainty,
their attempts at explanation, and their continual state of
incompleteness, are striving to round off the positive doctrine,
the expression of undoubted facts, namely phagocytosis.
When a physiologist is studying digestion he founds his study
on facts which are essential and certain, such as the action of
trypsin and enterokinase ; this is no theory; it is only when
he proceeds to interpret this action in terms of physics or
chemistry by fixations or combinations that he enters the
domain of théory. Similarly, it is no way of recognizing the
capital importance of phagocytosis to admit that antibodies
and other humoral properties are produced by the phagocytes.
The essential fact is the destruction of the microbes by
incorporation and digestion. Extraphagocytic destruction is
so much an exceptional case that it cannot even be brought in
as opposition.

There would be no temptation to forget this fact if, instead
of limiting our attention to human pathology, we kept in view
the universality of intracellular digestion throughout the series
of living beings. Phagocytosis is quite different from a medical
theory. It is a doctrine as fundamental in general biology as
is the existence of the cell or the variation of species.

Toxins and Phagocytosis.—The immunity of an animal
towards a toxin is not to be ascribed simply to the activity of
its body-fluid. We must take account of the body also in the
reaction. There are many facts indicating a similar state of

Q2

228 MICROBES AND TOXINS

affairs, as inthe experiment of Heymans and Masoin on the
neutralization 7 vivo of hydrocyanic acid by hyposulphite of
soda.

Zn vivo the hyposulphite of soda acts as an antidote or
chemical antitoxin to hydrocyanic acid. Now no one has ever
succeeded in reproducing this experiment in the test-tube,
whereas in the body it is perfectly easy. “In consequence it
is legitimate to appeal to certain peculiar conditions in the
living animal, which, however, does not exclude the possibility
that the transformation of the toxic substance into a harmless
material may be due to a chemical reaction.”

Fresh nutrient broth possesses an antitoxic action towards
abrin intoxication (Calmette). The serum of an animal
immunized against certain toxins or venoms protects other
animals to a greater or less extent against the action of other
toxins or offer venoms ; here there can be no question of a
specific direct antitoxic action.

The fresh blood of the crayfish is capable of preventing the
fatal intoxication of mice by scorpion venom ; yet the crayfish
is killed by a dose of scorpion venom three or four times
smaller than that necessary to kill a mouse, and the blood of
the crayfish has no protective action for another crayfish.

Roux and Vaillard observed long ago that animals might die
of tetanus although possessing an abundance of antitoxin in
their blood. There are certain horses originally good furnishers
of diphtheria or tetanus antitoxin which suddenly cease to
produce this in their serum although they remain immune.

Rabbits may be immunized against tetanus by inoculating
them under the skin of the tail several times in succession
with tetanus spores along with a little lactic acid ; the animal
becomes resistant to the toxin and yet 100 volumes of its
serum fail to neutralize a single minimum lethal dose of
toxin (Vaillard). The antitoxic power of the body-fluids is
thus not sufficient to explain acquired immunity, since it is
not an invariable fact in animals rendered immune.

The actions of the body itself have again to be reckoned
with in attacking the question of the origins of antitoxins.

IMMUNITY 229

We have already mentioned the old opinions of Buchner and
Roux. Buchner considered that the antitoxin was derived
from the toxin, and Metchnikoff advanced the opinion that
certain body-cells might produce this transformation. But,
it has been protested, how could a horse react to a single unit
of toxin by producing 100,000 units of antitoxin? The toxin
may, however, be seized by certain organs which retain it for
a long period and transform it slowly. The toxin may induce
in the cells which produce antitoxin the very stimulus which
Ehrlich was among the first to appeal to. The experiment of
Roux and Vaillard on rabbits immunized against tetanus
would thus be explained: after repeated bleedings the
antitoxin power of the blood rapidly regained its former
titre. But why should the serum of healthy animals sometimes
have a certain antitoxic power? Because without having
actually suffered from diphtheria or tetanus they may have
harboured diphtheria or tetanus bacilli in their bodies: in
the intestine of the horse for example the tetanus bacillus
abounds.

Whether or not the toxin is transformed into antitoxin, it is
certain that the antitoxin is a product of the body: no other
way of producing it is known. Ehrlich formerly thought that
the cells sensitive to the toxin were its chief producers; but
if this were true, antitoxin ought to be present in these cells
and be capable of neutralizing the toxin. Wassermann and
Takaki’s experiment seemed to prove this: the brain tissue of
mammals ground up with tetanus toxin, neutralizes it, furnish-
ing a mixture which no longer gives tetanus to animals. But
Wassermann’s experiment has in reality a quite different
signification. The brain does not act as an antitoxin, for,
if injected into a guinea-pig along with a dose of toxin, but at
separate points in the body, it has no protective action what-
ever, and does not act in the least like a dose of antitoxin.
Further, if the so-called neutral mixture is injected into the
thigh of a guinea-pig, the animal becomes tetanic, whereas it
remains protected if the injection is made into the peritoneum.
The neutralizing property is in reality a property peculiar to

230 MICROBES AND TOXINS

the cerebral cortex (and the grey matter of the spinal cord) of
mammals only ; in fowls immunised against tetanus the brain
has much less neutralising power than the blood, the liver, or
the kidney. The brain of the frog does not neutralise the
toxin, although under certain conditions the frog is susceptible
to tetanus.

Cholesterin, lecithin, and even olive oil and carmine (a
substance derived from the fatty body of the cochineal insect)
are capable of neutralizing a certain quantity of toxin; now
the brain substance contains both cholesterin and lecithin.
It is, however, another lipoid in the brain, profagon, which
chiefly fixes the toxin and perhaps permits of its transport
along the nerves (Landsteiner and Botteri.) A. Marie and
Tiffeneau have recently insisted that in Wassermann’s experi-
ment it is not a destruction which takes place, but a combina-
tion from which the toxin may be recovered. Anyhow, the
neutralization by the cerebral tissue is a phenomenon of
molecular adhesion, analogous to a dyeing process.

By injecting the tetanus toxin directly into the brain it has
been shown that this very brain substance which, ground up
in a glass, neutralizes the toxin, does not neutralize in the
living animal the most minute dose. It is therefore impossible
to suppose that the brain is a source of antitoxin (Roux and
Borrel).

An immunized rabbit, rendered resistant to toxin by subcu-

taneous inoculation, succumbs when the toxin is injected into
the brain: the antitoxic action is therefore due to cells lying
between the periphery and the centre ; the poison is neutralized
en route.
- What are these cells? We find that sublethal doses of
tetanus toxin produce in the fowl a great afflux of leucocytes
into the blood ; further, in a fowl injected with tetanus toxin,
far less toxin is to be found in the blood than in (aseptic)
exudates rich in leucocytes. Metchnikoff therefore considers
that the protection of the body against toxins also depends on
the leucocytes.

The rabbit can stand large doses of atropin injected subcu-

IMMUNITY 231

taneously or into the circulation, but it is very susceptible
to intracerebral injection; is it the leucocytes which dispose
of the poison when injected into the veins? This is possible,
for the poison can be recovered from the leucocytes after it
has disappeared or is only to be found in traces in the blood
plasma (Calmette).

When guinea-pigs are injected intraperitoneally with arsenic
trisulphide, a salt which is only slightly soluble, the particles
are taken up by the macrophages. When a soluble salt like
potassium arsenite is injected, when the animal recovers it is
in the leucocytes that the arsenic is found on chemical analysis
(Besredka). The phagocytes also absorb lead salts (Carles).
The white corpuscles of the blood are thus equally capable of
resisting mineral as microbial poisons.

When a guinea-pig is inoculated with the mixture of
mammalian brain ground up with tetanus toxin the solid
particles attract the phagocytes which seize them and with
them the attached toxin. If the toxin is injected along with
particles to which it does not attach itself (e.g. the mixture of
toxin + frog’s brain) it can diffuse, and the leucocytes no
longer protect the animal.

After the discovery of the antitoxins one was apt to think
that in every infection there was an intoxication and that the
neutralization of the poisons ought to precede phagocytosis
which is only a secondary phenomenon. But experience has
shown that the phagocytes can also digest the microbial toxins.

Certain bacteria secrete substances which weaken and
destroy the phagocytes ; among these antiphagocytic microbial
poisons may be classed the agvessins of Bail. Now the
phagocytes are capable of absorbing and digesting these
substances without any outside assistance.

Certain bacterial extracts prepared outside the body and
injected in sufficient quantity are prejudicial to phagocytosis.
Yet the same bacteria which furnish these extracts are
absorbed by the leucocytes when these latter have their
activity reinforced.

When dead typhoid bacilli are injected into the peritoneum

232 MICROBES AND TOXINS

of a guinea-pig they are of course incapable of producing an
infection, but as they contain the typhoid endotoxin the
animal dies nevertheless from the intoxication. But if before
the injection the peritoneum is “prepared” so that the
bacteria at once meet with a great number of vigorous leuco-
cytes the poison is absorbed by these and the animal is saved.

A staphylococcus habituated to the rabbit by the method of
passages, secretes a poison which is very injurious to the
leucocytes of this animal ; but if along with the staphylococci
vigorous living leucocytes are injected into the pleura, the
rabbit is protected against the intoxication (Bail and Weil).

Immunity against toxin therefore can be reduced, like the
immunity against bacteria, to the simple fact of phagocytic
digestion. Metchnikoff finds himself justified in the light of
all these experiments, in thinking that it is the phagocytes
which take up poisons and which perhaps themselves elaborate
the antitoxins employed in serotherapy.

CHAPTER XII
ANAPHYLAXIS

Definition of anaphylaxis—Experiments of Richet and Portier—Anaphylaxis
to various poisons—Anaphylaxis to normal serum—Arthus’ phenomenon
—Serum sickness: observations of V. Pirquet and Schick—Serum
anaphylaxis in guinea-pigs ; phenomenon of Th. Smith—Anaphylaxis
to cells and organ extracts—Passive anaphylaxis.

Study of Richet’s poisons and serum anaphylaxis—.Anti-anaphylaxis
(Besredka) : not a vaccination—Application to serotherapy—Heating
of sera—Theories of anaphylaxis—General theory of antibodies.

ANAPHYLAXIS is the opposite of vaccination. An animal is
vaccinated when the first attack by a virus (microbe or toxin)
produces in it changes through which it is protected against
another more serious attack. If you suppose the animal to
become, after the first attack, not more resistant, but more
susceptible, you have in a word the root idea of anaphylaxis.

The paradox is even greater than this definition shows, for
anaphylaxis exists not only towards viruses against which
immunity also occurs, but also it appears towards substances
which in the normal individual are apparently quite innocuous,
for example, egg-white, milk, and serum. In this way the
first injection with these substances, instead of producing
immunity, seems to destroy the natural immunity of the normal
animal.

The supersensitiveness produced by a first inoculation and
brought to light by a second is not due to a cumulative action,
such as may be observed after several successive doses of
certain drugs ; in anaphylaxis the effect is out of all proportion

to the quantity of material ingested.
233

234 MICROBES AND TOXINS

This chapter of experimental medicine was opened up by
the experiments of Richet and Portier on the poison of
Actinians (1902).

Anaphylaxis to Poisons—Richet’s Experiments.—
From the tentacles of actinians a poison may be extracted
which has been called congestin, because it produces in the
animals injected an intense congestion of the viscera, of the
stomach, liver, kidney, and, above all, the intestine. The
fatal phenomena do not appear until after several hours of
incubation. If a dog which has recovered from a small dose is
inoculated after a certain interval of time with 54 of the
quantity of the first dose, violent symptoms suddenly appear
after a few seconds, severe vomiting, difficult respiration,
paralysis, profuse and blood-stained diarrhoea. On comparing
the size of the dose with its effect, it works out that the
dog has become, between the first injection and the second,
eighty times more susceptible.

Anaphylaxis to Normal Serum—Arthus’ Phe-
nomenon.—lIf a rabbit is injected subcutaneously every six
days with 5 c.c. of horse serum, it is found that after the fifth
injection the serum is absorbed with difficulty ; succeeding
injections produce local lesions which continually increase in
severity, from simple inflammation to actual necrosis of the
tissue. The same phenomenon may be produced with milk
instead of serum.

‘‘Serum-sickness’’: Observations of Von Pirquet
and Schick.—Therapeutic sera (antidiphtheria, antitetanus)
are almost always got from immunized /orses; they are not
invariably devoid of toxicity for man. In a small proportion
of cases, about 14 per 100, the injection is followed by various
symptoms, not very severe it is true, which only appear after
an incubation period of five to fifteen days, and consist of
urticaria, erythemas, and pains in the joints. But should the
patient fall ill again months or years later, long after the serum
has disappeared from his body, and should he be reinjected
with it, the symptoms are reproduced and are more frequent
(86 per 100, according to Weil-Hallé and Lemaire), more

ANAPHYLAXIS 235

intense, and more rapid ; they appear within an hour, or even
within a quarter of an hour, after the injection.

In the light of these observations V. Pirquet observed later
that the unknown germs of vaccine lymph produce a premature
reaction in the skin of those individuals formerly vaccinated
and now supersensitive. The same observation in its turn
suggested to him the idea of applying a droplet of tuberculin
to the skin of a tuberculous patient to test his susceptibility,
and this was the origin of the cutaneous reaction of
tuberculosis.

Through these observations the study of anaphylaxis entered
the sphere of human medicine, and the important question
arose, How are we to render harmless our therapeutic sera?

Serum Anaphylaxis in the Guinea-pig. The
Phenomenon of Th. Smith.—The question entered the
laboratories when attempts were made to elucidate the fact
observed in the American serotherapeutic institutes, that
guinea-pigs which had already been employed in the titration
of antidiphtheritic serum (injected with a mixture of toxin and
serum) became eventually supersensitive to horse serum.
If three to twelve weeks after the titration injection 5 c.c. of
horse serum is injected under the skin or especially into the
peritoneum, the animals immediately manifest anxiety and
discomfort ; respiration is rapid and laboured, the heart
becomes weaker, the temperature falls below normal and after
one hour 50 per cent. of the animals die, whereas normal
guinea-pigs support doses of the same serum five times as great
without any disturbance. The phenomenon is specific, for
guinea-pigs receiving horse serum in the first dose behave
on re-injection quite like normal guinea-pigs towards rabbit,
goat or ox serum. In the toxin+antitoxin titration mixture
the antitoxin, ze, the horse serum, is responsible for the
supersensitiveness induced. A small dose sensitizes more
certainly than a large dose; even one millionth of a c.c.
may suffice! The symptoms resemble serum-sickness in man ;
there is an incubation period of twelve days at least after the
first injection. The supersensitive condition persists for

236 MICROBES AND TOXINS

months (experiments of Otto, Rosenau and Anderson,
Besredka).

Anaphylaxis to -Cells.—Animals injected with red
corpuscles, washed (to prevent the action of the serum) or un-
washed, resist the first injection well but support a second
badly:

Similarly with bacterial cells, typhoid, paratyphoid bacilli,
etc., they present more or less severe symptoms after the
second injection. The specificity of this reaction does not
appear to be very strict. This variety of anaphylaxis is of
great practical importance for in the preparation of anti-
plague sera it is necessary to inject the cultures into the
jugular vein of horses, and severe symptoms are quite
common.

Organ Extracts.—The phenomena induced by injections
of extracts of spleen, lymphatic glands, bone marrow, or
spermatozoa are analogous to the preceding. The supersensi-
tive state can be induced towards extracts of the crystalline lens
of the eye and an animal thus prepared reacts only towards the
lens tissue, no matter from what species of animal, even when
it presents no reaction to the serum of that animal.

A tuberculous patient becomes supersensitive to the product
of the tubercle bacillus, to tuberculin. But in this case it
appears that only the disease itself can induce this excessive
susceptibility permanently: it is very doubtful if it can be
produced by the injection of the bacterial bodies or of
tuberculin. Physiologically speaking it is an anaphylactic
phenomenon.

_ Passive Anaphylaxis. _M. Nicolle has shown that if a
fresh rabbit is given a large dose of the serum of a rabbit
rendered anaphylactic to horse serum, the fresh rabbit takes on
am anaphylaxis which is therefore called passive. -It may in its
turn present the typical symptoms on injection 24 hours after the
preparatory injection. This brief delay shows that it is a true
passive anaphylaxis, and not an active anaphylaxis induced by
small quantities of horse serum which might have remained in
the serum of the sensitive rabbit. "This experiment. demon-

ANAPHYLAXIS 237

strates the existence of an antibody in the serum of the sensitive
animal ; this antibody has been transferred to the other animal
just as the diphtheria antitoxin of the horse can transfer passive
immunity to man.

Passive anaphylaxis can be produced particularly well by
injecting the serum of an anaphylactic rabbit into a normal
guinea-pig. Passive transference has also been produced in the
case of Richet’s poisons. It is therefore a general fact, although
experiments of transference between animals of the same species
do not always succeed.

From these fundamental experiments there have proceeded
several sets of researches ; especially with the poisons and with
serum considerable advances have been made. It is not at all
certain in spite of their manifest similarities that the laws of
anaphylaxis against poisons and serum (or other non-toxic pro-
tein) are really entirely the same. There is always this differ-
ence, that in the one case it is a question of substances
manifestly toxic to the normal body, whereas in the other the
substances are such that the healthy animal shows no visible
reaction.

For the practical point of view, these researches, both
on the poisons and on the sera (because of serotherapy), are
of obvious value; there is an actual disease to prevent or
cure. ,

It does not seem as if the anaphylaxis to egg-white should
interest us to the same extent. We do not take albumen or
milk by subcutaneous, intraperitoneal, or intravenous injections.
If, as we know already, our body tends to resist the introduc-
tion of foreign albumens, we have a digestive tube which trans-
forms these into our own specific protein ; that is perhaps why
so few examples exist hitherto of anaphylaxis acquired by taking
food. Further, it is probable that we are much more exposed
to the cumulative action of toxic bodies, e.g., the phenols of the
digestive tube, than to an anaphylaxis to the proteins of the ox
or of the fowl. But the digestive tube may occasionally be
defective as a defensive agent.

Poisons also may be absorbed by other routes, such as the

238 MICROBES AND TOXINS

pulmonary alveoli or the skin. We are here in a region as yet
little explored ; there are many mysteries in the action of drugs,
there are still greater mysteries in the experimental study of
diseases of nutrition. Once again anaphylaxis raises this
general problem of nutrition, towards which already the whole
study of immunity is directed, and it is probable that, did we
know more of this, we would be less ignorant of the nature of
life, old age, and death.

Let us consider then separately what knowledge has been
gained on the subject of anaphylaxis to those substances which
have been most studied, the poisons and serum.

Researches on the Poisons.—There have been studied
by Richet the congestin of Actinians, the congestin extracted
from mussels, and a vegetable toxin, analogous to abrin and
ricin (well-known from Ehrlich’s experiments), called crepitin,
and extracted from the plant Mura crepitans of the Euphor-
biaceze, known in Brazil under the name of Assaku. The
mytilocongestin (from mussels) produces vomiting, a symptom
very definite and easy to. observe, a great convenience in
experimental work. “The symptoms of anaphylaxis to
crepitin are exactly the same as with actino- or mytilo-con-
gestin, and even the most acute observer, I am sure, could not
distinguish them. . . . There is the same profound abolition
of all innervation, both motor and sensory, and above all,
vaso-constrictory: there is the same intense hemorrhagic
congestion of the intestine with an enormous fall in the
arterial pressure ” (Richet).

It is to be observed that these belong to the group of slow
poisons—resembling thus the bacterial toxins--which differ
from the crystalloid group such as strychnine, and it is probable
that on this depends their anaphylactic effect.

An animal is rendered anaphylactic because the first
injection has induced in it, after a period of incubation, the
formation of a new substance, an antibody, the product of a
reaction of the body. This antibody is not itself poisonous,
but it liberates a poison when it comes in contact with the
congestin or crepitin of the second injection.

ANAPHYLAXIS 239

The antibody is named by Richet foxogénine, and the new
poison, apotoxine. He therefore imagines anaphylaxis after the
formula: Zoxogénine + Congestine = Apotoxine.

He considers that the apotoxin results from the action of
the two substances on each other, the one, congestin, introduced
from without and non-toxic in the dose employed for the test
injection, the other, toxogenin, non-toxic, prepared by the
body itself as a result of the first small injection of congestin.
In a similar way we have emulsin and amygdalin acting
together to produce hydrocyanic acid.

At first the existence of this toxogenin was hypothetical, but
experiment has in two ways proved its real existence—1. When
a normal animal is injected with the blood of a sensitized
animal, it becomes anaphylactic without any period of incubation,
7.é., there is passive anaphylaxis. 2. Richet has several times
succeeded in demonstrating the reaction with crepitin 7” vitro.
The mixture in a test-tube of anaphylactic serum and poison,
when injected after incubation, produces immediate anaphylactic
effects: the test-tube contains both the antibody and the
antigen. 3. The toxogenin does not exist only in the blood ; it
is also to be found in the brain tissue. By mixing crepitin with
brain substance (freed from blood) or even with the alcoholic
precipitate from the brain substance of an anaphylactic animal,
immediate anaphylactic symptoms can be produced in a fresh
animal: there is thus “‘ cerebral anaphylaxis ” 7z vitro. There is
thus antibody present in the nerve-cell and capable of reacting
with the antigen at the moment of the anaphylactic shock.
It is this very reaction which Besredka regards as the
essence of the anaphylactic shock, which appears to him to be
eminently a cerebral phemonenon.

According to Richet those animals which have received
an anaphylactising toxin become from this sole fact more
sensitive to other poisons. ‘The injection of an antigen, for
example crepitin, renders an animal more sensitive to toxic
action of other kinds, for example to that of apomorphine,
although the increase of sensitiveness is particularly directed
towards the antigen itself. There exists therefore, it would

240 MICROBES AND TOXINS

seem, a sort of general anaphylaxis in addition to the
specific form of anaphylaxis. Richet is inclined to consider
that in the phenomena of anaphylaxis there is in reality only
a relative specificity, and that the apotoxin is a poison without
any specificity which attacks and paralyses the central nervous
system, affecting in this particularly the vasomotor centres.
“Tt seems to me very probable that in the study of the whole
field of the different forms of anaphylaxis produced by different
substances a great analogy if not identity would be discovered
in the symptoms of all forms of anaphylaxis, so that we may be
permitted to believe in the general analogy if not in the identity
of all the different apotoxins, the various anaphylactic poisons.
There would thus be a very simple conclusion, namely, that
there is one poison and one only, the apotoxin produced in
all the forms of anaphylaxis.”

Richet’s poisons produce in the body, besides anaphylaxis,
immunity. With mytilocongestin the anaphylaxis disappears
after about six weeks and the immunity persists. Ana-
phylaxis thus gives place to its opposite “ prophylaxis” and
toxogenin to antitoxin. The two conditions “develop side by
side from the moment of the first injection. Hence it is
necessary to distinguish closely between the immediate and the
late effects. During the anaphylactic period there is a striking
supersensitiveness as regards the immediate symptoms, but
there is already some immunity towards the late effects of the
poison. If the animal survives the immediate effects of the
second dose, it presents no further symptoms during the days
following’... Anaphylaxis ts the first stage in prophylaxis.”
These views recall those of Behring. ‘ However para-
doxical it may seem, there can be no doubt that horses which
have become strongly immune as the result of treatment with
tetanus toxin yet possess a histogenic supersensitiveness of
their tissues towards the tetanus toxin.” These properties are

1 Richet applies the same idea to serum-anaphylaxis and to anaphylaxis
in general. ‘‘The anaphylactic reaction is a defensive function and is
destined to maintain intact the chemical constitution and homology of
each animal species by preventing foreign albumins from entering the
protoplasm of the cells so as to modify their specific chemical structure.”

ANAPHYLAXIS 241

probably common to many toxins, but in serum anaphylaxis
the same characteristics do not necessarily exist.

Anaphylaxis to poisons thus means a hastening of the
reaction of the body towards microbial toxins: it is a process
adapted for rapid defence and in particular for defence against
small doses : it is a sort of alarm signal sent to the body-cells
calling its attention to minute quantities of poison which
without this would have been insufficient to induce immunity :
immunity is established thanks to the preceding anaphylaxis.
Perhaps anaphylaxis is equivalent to Ehrlich’s superexcitation
of the cells required for the production of antitoxins.

Serum Anaphylaxis.—A preparatory injection of serum
in very small dose (1/250 to 1/1,000,000 c.c.), a period of
incubation, a test injection larger than the first and producing
violent symptoms, these are the conditions of serum
anaphylaxis in the guinea-pig.

On observing the symptoms it is impossible to avoid the
idea that the guinea-pig has fallen victim to some cerebral
lesion which was latent during the period of incubation but
has been revived by the test injection. The symptoms of
this lesion ought to be still more definite if the injection
were made into the brain itself. The nerve-centres can be
more directly attacked by intracerebral or intradural in-
jections or through the circulation by the intravenous route
(Besredka).

One quarter of a c.c. injected intracerebrally produces the
same phenomena as 5 c.c. intraperitoneally and produces them
with much greater regularity, for instead of about 25 per cent.,
practically all the guinea-pigs die. The experiments are thus
on a surer footing. Should the still unknown reaction take
place in the brain cell itself, ze. the reaction between the
poison and the antibody manufactured by the body as a
result of the first injection, one might hope to modify the
anaphylactic phenomena by acting on the nerve-cells of the
supersensitive animal. And this is really the case, as is shown
by a very pretty experiment of Roux and Besredka. The
sensitive guinea-pig is put to sleep with ether or put under the

R

242 MICROBES AND TOXINS

influence of a suitable dose of alcohol; an intracerebral
injection which kills the sensitive control leaves the
narcotised guinea-pig unharmed.

In practice intracerebral injections have supplied Besredka
with a useful means of measuring the toxicity of a serum, for
example, anti-diphtheria serum, from the point of view of the
symptoms of serum sickness. Should a method be discovered
of diminishing or destroying the toxicity of sera, the improve-
ment acquired could thus be estimated. In particular, the
method will tell us whether a serum may be safely injected by
the route by which it manifests its greatest toxicity, but also its
greatest efficacy, #¢., intravenously. Experience has shown
that a serum, very toxic the day of the bleeding (minimum
lethal dose #, c.c.), rapidly loses this toxicity during the ten
days following (lethal dose 4, c.c.). It continues to diminish
slowly, for a month and a half, but after about two months it
remains almost indefinitely at the same level (lethal dose 4 c.c.),
and never becomes non-toxic. For example, a sample of
serum kept in Roux’s laboratory for thirteen years still kills
sensitive guinea-pigs in a dose of } c.c. intracerebrally
(Besredka).

Antianaphylaxis.—Anaphylaxis is a morbid state predis-
posing to the occurrence of accidents. But these may be
prevented. Antianaphylaxis is the name given by Besredka to
the condition of insensibility to which the sensitive animal can
rapidly be brought. But though in current language one talks

1 There exist other physiological shocks which are deadened by narcosis,
and it is impossible to avoid a comparison between the above experiment
and one of Jellinek’s (the director of the laboratory of electrical pathology
in Vienna) reported in a recent lecture by D’Arsonval.

“* A rabbit is fairly easily killed when the opposite poles of an alternat-
ing current of 1,500 volts are placed in its mouth and rectum, whereas a
rabbit of the same breed but so deeply chloroformed that all its vital
phenomena have ceased is at once reawakened and saved from death by
the same current.

“* At the time that this experiment was published an English engineer,
Aspinall, observed that two electrical engineers who had during sleep come
into accidental contact with an alternating current of 3,000 volts were
simply awakened by burning sensations in the back without other injury.”
(Jellinek).

ANAPHYLAXIS 243

vaguely of ‘ vaccinating ” against anaphylaxis by Besredka’s
method, axtianaphylaxis is in reality not a vaccination.

Originally it was conceived as a true vaccination, and to
produce it Rosenau and Anderson made a series of injections
of 5 c.c. of serum intraperitoneally, starting before the period
of incubation for the anaphylactic state was up: they proceeded
as one does for antitoxins.

But this idea had to be given up when it was seen that it
was sufficient to protect the guinea-pig to give it, not a series of
injections, but a single injection, not a large injection, but a
minimal one, 74g to z#5 Cc, #¢, much less than the toxic
dose ; finally, and above all, that the resistance of the guinea-
pig develops the day after, or even some hours or minutes
after, the injection, according to the route employed. After
subcutaneous injection the resistance is present in four or five
hours ; after intradural injection in about two hours; after
intraperitoneal in an hour and a half; ten to fifteen minutes
after intravenous injection. The shortness of this latter incu-
bation period may be employed to render the resistance more
and more complete: it is simply a question of repeating these
minute doses intravenously every ten or fifteen minutes: each
injection reinforces the effect of the preceding: this is the
method of “ continuous vaccination ” (vaccinations subintrantes).
It is not even necessary to inject by the veins: by taking a
little more time one may proceed by other routes and even
changing routes in successive doses. In this way a guinea-pig
may be protected from as many lethal doses of serum as is
desired (Besredka).

In this there is the germ of a method which may be applied
to man. Certain sera, in particular the anti-plague serum,
have to be injected in large quantities, and as far as possible
by the veins. The anti-diphtheria serum is 500 times more
active, other things being equal, if introduced directly into the
circulation instead of under the skin (Berghaus). Besredka’s
process promises to relieve such intensive serumtherapy from
all danger of death by anaphylaxis.

What sort of immunity then is this which develops with such

R 2

244 MICROBES AND TOXINS

small doses and so rapidly ?? A vaccinated animal departs from
the normal to acquire a new condition ; in sensitized animals
under these circumstances there is not the departure from a
normal state but there is an appearance of returning towards it.
Antianaphylactic vaccination proceeds like a disintoxication
and antianaphylatic immunity seems to be a return to the
natural immunity.

The sensitizing injection induces the formation of an antibody ;
at the moment of the test injection itis probable that the toxic
serum combines with or rather fixes itself on this antibody
and this abrupt reaction produces a shock which the nerve-cell
resents profoundly. It is “ disintoxicated” by it, but with such
brutal suddenness as to kill the animal. When by narcosis with
aneesthetics or alcohol the sensitiveness of the nerve-cell is
diminished, as in the experiment of Besredka and Roux, it
becomes disintoxicated during sleep, just as in a surgical
operation under chloroform, and wakes up free. If instead of
injecting as in the test injection a massive toxic dose, a
minute dose is introduced (3, c.c. intravenously, for example),
the disintoxication of the body, and in particular of the nerve-
cells, is carried out gradually, little by little ; in the continuous
vaccination method the disintoxication leads back the animal
by degrees to its normal condition.

To the normal condition, but not quite, for the injection
leaves as a rule a trace of serum in the body with which it
re-sensitizes itself in time ; the guinea-pig has become a fresh
animal, and like a fresh animal may be sensitized. It may lose
its virginity, regain it, and again lose it. Certain experimenters
do not believe that the body ever becomes again normal.

The digestive tube is, physiologically speaking, as much a
barrier to the invasion of the body by foreign proteins as it is

1 “The immunity to a toxin does not appear till after eight days at
least and is more pronounced the more numerous and prolonged the
successive doses: it is accompanied by the appearance of the antibody in
the serum, and finally it never extends to the nerve-centres. On the
contrary anaphylactic immunity is established after a single injection, is
practically instantaneous, is never accompanied by the appearance of
antibodies and finally always extends to the nerve-centres, the brain and
spinal cord-” (Besredka).

ANAPHYLAXIS 245

towards bacteria: by its very power of digesting and assimilat-
ing them it renders inoffensive those substances which, if
injected under the skin or into the blood stream, would perhaps
be toxic: it maintains the natural immunity.

But for the present it must be pointed out that Besredka’s
conception only applies to substances like serum whick. are
harmless in general to the normal body even when injected
direct into the veins. It is not necessarily true of the tox-
albumins, which are toxic from the first.

We have briefly indicated two procedures for rendering a
sensitive animal resistant, narcosis during the test injection and
the method of continuous vaccination with small doses. The
same result may be obtained by heating the toxic serum to
certain definite temperatures. Heating produces an effect un-
realized hitherto by any chemical means. Serum heated to
100° C. (diluted to prevent coagulation) becomes practically
harmless for both intracerebral and intravenous injections :
But this heated serum, no longer lethal, can still protect
against the anaphylactic shock by gradually disintoxicating the
sensitive cells as in the method of small doses or narcosis ; no
doubt the mechanism is the same.

Heating is thus a good means of lowering the toxicity of a
serum. But sera heated to 100° C. lose all preventive and
curative power, and it is impossible therefore to exceed 59-60° C.
Experiment shows that heating for several hours at this tem-
perature diminishes considerably the anaphylactic toxicity.
The reason for the low toxicity of the Pasteur Institute sera,
duly recorded by various laboratories, is simply that their
sterilization is effected by means of several heatings at a low
temperature (without antiseptics).

Man is not alone in profiting by laboratory research on
anaphylaxis. In certain countries the Pasteur vaccine against
anthrax is injected along with a few c.c. of an anti-anthrax
serum: the method is named sero-vaccination. Many cattle
are inoculated over again every two years, so that severe
anaphylactic effects are not uncommon. _ Recently sero-
vaccination was repeated on 180 cattle in Roumania. The

246 MICROBES AND TOXINS

animals were divided into two lots, the first receiving 1 c.c., as
an anti-anaphylactic injection, five hours before the sero-
vaccination. In the result it was found that during the twenty-
four hours following the sero-vaccination, not one of these
showed any anaphylactic symptoms, whereas ten of the other
ninety showed symptoms, such as cedema of the muzzle with
salivation, cedema of the vulvar and anal mucous membranes,
and colic (Alexandresco and Ciuca).

Theories.—No final theory for anaphylaxis yet exists, but
there are various hypotheses which keep experimental work
active. There is one point, however, which is certain, that it
is impossible to explain the phenomena without the presence of
an antibody formed by the animal as a consequence of the
first injection. This has been demonstrated by M. Nicolle in
connection with Arthus’s phenomenon.

It is probable that the anaphylactic shock is due to the union
of antibody and antigen, that this union is abrupt and affects
especially the nerve-cells, which does not mean that the nerve-
cells produce the antibody. On the contrary, everything points
to their not creating the supersensitiveness, but being passively
affected in it.

According to Vaughan and Wheeler, who studied in par-
ticular the anaphylaxis to egg-white, the sensitizing injection, as
an antibody, provokes the formation of a proferment which can
only act after the reinjection by splitting the protein molecule
into two components, one toxic, the other not. As a matter of
fact it is possible in the laboratory to produce from albumin
two such components, but the toxic one is also toxic for normal
animals quite as much as for sensitive ones: this therefore is
not an explanation of anaphylaxis, though it shows that there
exist toxic elements in substances normally non-toxic.

It was natural to attempt to identify the antibody of anaphylaxis
with some antibody already known in the reactions of immunity.
This was Friedberger’s idea : in serum anaphylaxis the antibody
is nothing but the precipitin, in anaphylaxis to blood corpus-
cles a hemolysin. The facts, however, do not confirm Fried-
berger’s theory.

ANAPHYLAXIS 247

Besredka’s theory approximates more closely. In anaphyl-
axis three activities come into play, the sensibilisinogen, the
property of serum by virtue of which it sensitizes, the
sensibtlisin, the property due to the body and corresponding
to the antibody generally admitted, and the azdisensibilisin, by
which is meant the property of normal serum in virtue of
which it combines with the sensibilisin and determines the
anaphylactic shock.

Thus serum is toxic, not because it contains a poison ready
made, but because two substances, non-toxic themselves, “enter
into abrupt combination within the nerve-cells and thus
disturb their equilibrium.”

Why should small doses sensitize so well, whereas massive
doses only do so after a long delay? The answer is that a
minute dose induces the formation of sensibilisin without
furnishing it with anything on which to fix: in consequence it.
remains avid, in the nerve-cells among others. After a large
dose the sensibilisin manufactured is neutralized as it forms
by the “anti-sensibilisin” of the serum and anaphylaxis is
therefore delayed. It seemed probable at first that by heating
the serum at suitable temperatures it might be possible to
dissociate the two activities which do not depend on the
body, ze. the sensitizing and toxic properties. But on
closer examination it was found that heating acted equally on
both ; the two in reality follow the same curve. It had also
to be recognized that the toxic function, the sensitizing
function, and the vaccinating or anti-anaphlyactic function, are
all fundamentally identical and occur in the same serum, but
that they correspond to different physical conditions of that
serum.

To sensitize, a serum must be injected highly diluted; to
produce the anaphylactic shock it must possess its normal
concentration ; to “vaccinate” or protect against the shock it
must unite very slowly with the sensibilisin of the body or
must unite with it in minute quantities at a time (eg., in the
method of “ continuous vaccination ”).

Heated at 100° C. it is no longer toxic or very slightly

248 MICROBES AND TONINS

so, because being partly coagulated its fixation is delayed.
The action of serum, sensitizing, vaccinating, or toxic, depends
thus on its physical condition. ,

There are therefore really only two activities of serum
in question, that of the antigen and that of the antibody. But
according to its physical condition, according to the manner
and time of its injection, the antigen plays the part of sensitizer,
of vaccinator or of toxin. These explanations, it must be
mentioned, are confined to the field of serum-anaphylaxis as it
is known to-day.

We always return thus to the antigen and the antibody, z.e., to
the activities which we found to exist in all the phenomena of
immunity.

General Theory of the Antibodies.—M. Nicolle has
proposed a general explanation embracing anaphylaxis as a
particular case of the general physiology of the antibodies.
Every antigen induces in the body the simultaneous formation
of antibodies of two classes, the coagulins and the lysins. The
coagulins condense albuminoids and toxins (to speak only
of these two varieties of antigen); the lysins, on the contrary,
break them up and liberate from them their real toxic
components :1 in intoxication by proteid poisons it is not these
themselves which injure the body, but secondary poisons
elaborated by it itself. The fate of the animal depends on its
species, on the nature and quantity of the antigen, on the
channel of inoculation and on the route by which the assaulting
dose is introduced.

Thus, in the phenomenon of Th. Smith, the supersensitive-
ness is explained by the development of a lysin and the
absence of all coagulin makes of it the type example of pure
supetsensitiveness. In bacterial anaphylaxis, the super-

1 When the coagulins predominate they rapidly condense the antigens,
giving the body time to attack them bit by bit without liberating enough
poison at a given moment to cause toxic or, at least, fatal symptoms. The
lysins on the contrary, when they predominate, make their appearance as
the agents of an inevitable and often fulminating intoxication, for the body
has only limited protection against the true endotoxins and the true toxins,
no more than it has against alkaloids, for example ” (Nicolle).

ANAPHYLAXIS 249

sensitiveness is due to a lysin which sets free from the
bacteria a true endotoxin.

Infection and intoxication arouse in the body a many-
sided conflict between these coagulins and lysins, which are in
general the good and evil antibodies. A lytic action may,
however, be salutary when it occurs slowly, whereas it is lethal
when it takes place abruptly: under these formule may be
arranged all the facts mentioned in the course of this chapter.
* Although diametrically opposed to each other, as inevitable
results from their definition, immunity and supersensitiveness
may co-exist in the same individual, as well as succeed each
other, often again and again.”

Nicolle’s theory is frankly inclined to the physical theory of
immunity, without overlooking the intimate relations which
exist between the physical properties of bodies, and their
chemical constitution. It also sees in immunity phenomena of
nutrition; for the body digests the antigens, and the theory
supposes simply that every digestive act is due to the successive
application of a coagulin and a lysin.

CHAPTER XIII

APPLICATIONS OF BACTERIOLOGY

DIAGNOSTIC METHODS.

Direct diagnostic methods—Direct diagnosis of the microbe—Cultures
from the blood—Examination of feeces—Indirect diagnostic methods
—Cytodiagnoses.

Biological diagnostic methods—Age/utination: specificity and group
agglutinations : variations in bacteria from the agglutination point of
view—Precipitation : employment in forensic medicine and in the
adulteration of foods— Applications to anthropology : confirmation of
the simian origin of man—Complemént-fixation : first experiment of
Bordet: clinical application—Wassermann’s reaction and the sero-
diagnosis of syphilis—The nature of the substances coming into play
in this reaction—Sufersensitive reactions : tuberculin.

THE simplest and surest way to diagnose an infectious
disease is to demonstrate the presence of the specific microbe,
z.e., direct bacteriological diagnosis. When this is impossible,
indirect diagnosis is resorted to, z.e., the lesions of the tissues
which are constant accompaniments of a virus which is
invisible are sought for. The presence in exudates of certain
cell elements is noted, or the body-fluids and bacteria are
made to react together specifically (antibodies and antigens) :
in the latter case it is more properly a case of biological
diagnosis.

e
Direct Diagnostic Methods.

The microbe is sought for wherever there is a possibility
of finding it; blood, exudates and transudates, pus, mucous

discharges, false membranes, ulcers and chancres, sputum,
250

APPLICATIONS OF BACTERIOLOGY 251

cerebro-spinal fluid, urine, feces, all may be examined. Such
observations are completed by cultivating and experimental
inoculations.

Direct Diagnosis of the Microbe.—This has to suffice
when it is a microbe which cannot be cultivated. It is
sufficient when the microbe has characteristics which cannot be
mistaken. The sight of the malarial parasite of Laveran,
of a filaria embryo, of a trypanosome (in man) is a certain
diagnosis. Medicine has profited by every step in advance
in technique: the ultra-microscope now permits us to see
trypanosomes and spirocheetes living and motile much more
easily than with the best microscope.

Pasteur’s method of “seeding” silk-worms was based on
direct diagnosis. Direct diagnosis is currently employed in
connection with sputum and false membranes ; it is completed
by culture and inoculation. Inoculation is the rule when the
pneumococcus is observed in sputum: subcutaneously inocu-
lated it kills a mouse within twenty-four hours: the mouse is
the pneumococcus reagent.

Blood Culture.—Blood, taken aseptically from an animal
not in a state of active digestion, and kept free from external
germs, may be kept indefinitely without putrefying. If
microbes develop in the blood itself or in nutrient broth
into which it has been put, it means that these microbes
existed in the blood during life. Pasteur’s observations on
the sterility of normal blood form the foundation of the
diagnosis of infectious diseases by blood culture. The strepto-
coccus was isolated by him for the first time from the blood of
women suffering from puerperal fever.

It has been proved by blood culture that gonorrhcea, which is
usually quite a local disease, may infect the blood with
gonococci and cause arthritis and endocarditis. In a frankly
acute pneumonia it is the rule to find pneumococci in the
blood.

In typhoid fever blood cultivations have given results which
have upset our conceptions of this disease. It used to be
thought an exclusively local disease of the intestine and

252 MICROBES AND TOXINS

the lymphoid organs: even when the bacillus had been found
in the urine and in the red eruption spots on the skin it was
still thought that it only exceptionally entered the blood. But
when cultures were regularly made from the blood of typhoid
patients. it was perceived that the typhoid bacillus is present
there during the whole febrile period of the disease and again
during relapses: in typhoid fever, therefore, the acute enteritis
is complicated by a blood infection, a septiceemia.

The examination of the faeces is regularly performed in the
diagnosis and prophylaxis of cholera, typhoid: fever, and
dysentery, particularly in order to detect germ-carriers. Also
in the stools are sought the eggs of worm parasites and of
ankylostoma and amoebe. The bacteriological examination
of feeces is becoming more and more common in proportion
“as our knowledge of the intestinal flora is increasing; the
composition of this flora is a guide to the state of digestion and
nutrition, and aids the physician in his choice of a diet,
especially in infants, who are so often threatened by infections
of the alimentary canal.

Indirect Diagnostic Methods.

Even when no microbe is found in it, the blood may furnish
diagnostic proofs from its leucocytic formula.

Cytodiagnosis (Widal) depends on the examination of the
cells floating in pleural effusions or cerebro-spinal fluids.
Different cells are found in a pleurisy due to the bacillus
tuberculosis and in one due to heart disease. Cytodiagnosis
of the cerebro-spinal fluid is an indirect diagnostic method in
certain tuberculous and syphilitic affections.

Biological Methods : Agglutination.

The first clinical sero-diagnostic method was discovered by
Widal.
When a drop of a broth culture of the typhoid bacillus is
looked at under the microscope, the bacteria are seen actively
motile, isolated from each other and dispersed regularly

APPLICATIONS OF BACTERIOLOGY 253

throughout the liquid ; the suspension is “homogeneous.” If
a trace of serum is added from an animal prepared by injections
of typhoid bacilli or from a patient suffering from typhoid
fever, the bacilli lose their
motility and collect into
masses: they are said to
become agglutinated by the
serum. If the serum is
added to a broth culture
floccules can be seen with
the naked eye forming and
sinking to the bottom of
the tube ; agglutination is
also a sedimentation. Nor-
mal serum never possesses
this property, certainly Fic. 71.—Agglutination of the typhoid
never to the same degree. bacillus by the serum of a typhoid

The agglutinating power lens 4, cumpef baci gs.
may be measured by try-
ing the effect on a suspension of the bacilli of various dilutions
of the serum: one may say that such and such a serum
agglutinates at 1 in 50, 1 in 100, rin 1000... .

The reaction is capable of two applications. With a bacillus
definitely known as a genuine ZB. typhosis, one can say that
the serum which agglutinates it is an antityphoid serum. If,
on the other hand, with such a definitely known serum we
find a bacillus agglutinated, we may say that it is a true
B. typhosus. Agglutination may be used to diagnose now the
bacillus, now the disease. The agglutinating power depends
on an antibody named the agglutinin. This substance keeps
much longer than the time required for its carriage to long
distances for examination, when this is necessary. Dead
bacteria also agglutinate, so that the method may be employed
even without living cultures. Seroagglutination is therefore
the simplest and most convenient of the biological methods.

In performing the test it is necessary to avoid certain
sources of error, among others the existence of bacterial strains

254 MICROBES AND TOXINS

which agglutinate spontaneously or do not agglutinate at all.
Strains can be artificially produced which refuse to agglutinate :
this resistance of the microbe to the action of the serum
is an example of the immunity of the microbe towards the
animal, the converse case of the immunity of the animal
towards the microbe.

Agglutination is applied to as a test for suspected bacteria
found during cholera scares in suspected water or choleraic
diarrhoea ; the serum employed is obtained from an animal
prepared by several immunizing injections of a definitely known
cholera vibrio.

Agglutination has also been applied to the diagnosis and
prophylaxis of bacillary dysentery and epidemic cerebro-spinal
meningitis. In tuberculosis it has only been applied as a
means of controlling the treatment by tuberculin. As a
diagnostic agent the tuberculin test is much more convenient
and certain (R. Koch). But agglutinin is no better than the
other antibodies (vide p. 208) for the estimation of the resist-
ance of the body towards tubercle.}

Precipitation.

If we take a culture of B. typhosus and filter it, we obtain
a clear fluid free from bacteria. Ifa little of a very active

1 Agglutination is.the touchstone in the study of bacterial strains and
their variations. Recently Bordet and Sleeswyk, working with the bacillus
of whooping-cough discovered by Bordet, have created in the laboratory
varieties of this analogous to the varieties of the dysentery bacillus.

What fixity do these varieties or strains possess, created as they are by
growth on different media and separated by their different reaction to
such-and-such a serum? What is of interest here is the conclusion of these
workers. They maintain, at least as far as the agglutinating action of
serum on bacteria is concerned, that sera ‘‘do not act on the fundamental
bacterial substance, which is inherent to its life and whose presence is
indissolubly bound with the nature and constitution of the species, but
that they act on substances to some degree accessory, of possible but
facultative presence, whose production is in no way one of the bundle of
hereditary, immutable characters which give to a living creature its own
physiognomy and autonomy.”

It is obvious that this interpretation of the facts is not in favour of a
chemical theory of immunity : the more one considers the agglutination as
a surface reaction, the more probable becomes the physical explanation, 7.¢.,
the explanation of Bordet.

APPLICATIONS OF BACTERIOLOGY 255

‘

antityphoid serum is added, the mixture becomes turbid and a
precipitate forms which settles at the bottom of the tube.
Instead of agglutination we have precipitation (R. Kraus).

If we inject a rabbit repeatedly with proteins of animal
origin, horse serum, eel’s serum, defibrinated fowl’s blood, its
serum thus immunized forms a precipitate when mixed with
the serum of the eel, of the horse, or of the fowl (Tchistovitch
and Bordet).

The precipitating property of sera thus prepared is ascribed
to an antibody, precipitin. There are bacterial precipitins
and protein precipitins, the former being simply a species of
the latter : all proteins, animal or vegetable, in clear solution, are
precipitated by the corresponding antisera: the action is quite
specific.

This reaction is nearly related to the agglutinating reaction.
It is scarcely ever used in the diagnosis of infections, but for
the differentiation of protein substances it has furnished a very
sensitive test which is employed in forensic medicine : nowa-
days serodiagnosis is applied to the detection of bloodstains
and to the adulteration of meat and milk.

A man is accused of murder and in his house is found a
garment stained with blood: the accused (he may be, for
example, a butcher) declares that it is ox-blood and justice
demands that serodiagnosis be applied to the stains. Some of
the stains are therefore cut out of the cloth and extracted with
physiological saline and with this liquid the precipitation test
is applied : if the spot was due to human blood, the liquid
produces a precipitate with the serum of an animal previously
treated with human blood.

A merchant is accused of selling for pork-sausages sausages
made from horseflesh. An extract is therefore made with
water from the suspected meat. If the sausage contained
horseflesh, the extract gives a precipitate with the serum of an
animal previously injected with horseflesh extract. The
method can still be applied when the meat has been smoked
and dried. By the same method may be detected cow’s,
goat’s milk, &c. The reaction is sensitive to 1 in 100,000, 7¢.,

256 MICROBES AND TOXINS

it gives a result with an extract containing only zggg00 Of
its weight of the protein to be determined.

The precipitation test has given positive results with the
organs of forty-year-old mummies. It is even said to have
been successful with mummies of 3,000 to 5,000 years, but
in this case there is some doubt. It is probable that these
latter may contain albumoses, but probably they no longer
contain coagulable albumins.

The same biological reaction has furnished an additional
proof of evolution. Just as there are group agglutinations, so
there are group precipitations. The antiserum which pre-
cipitates rabbit serum precipitates also hare serum. Anti-dog
serum precipitates with both dog and fox serum. The serum
against the horse precipitates also the serum of the ass and of
the tapir. The relationship, the dood relationship literally,
between the goat and the sheep, between the fowl and the
pigeon, and between the domestic and wild pig can thus be
demonstrated.

What interests us more particularly is that the serum of a
rabbit prepared with human serum precipitates with the serum
of monkeys, a definite proof that we are their near relations.
This relationship extends even to the lemurs, which are only
half-monkeys, and is closer in the case of the monkeys of the
Old World than with those of America. Griinbaum, who
experimented with forty-six species of monkeys, maintains that
from the point of view of the quality and quantity of the
precipitates furnished by their sera, he has been able to detect
no difference between man, the gorilla, the orang-outang, and
the chimpanzee: this new proof of the correctness of man’s
simian origin may be welcomed.

Complement Fixation.

This new sero-diagnostic took origin from an experiment of
Bordet: he was trying to show that there is in a serum,
not several, but a single complement and that it is the same
complement which acts in bacteriolysis as in hemolysis.

APPLICATIONS OF BACTERIOLOGY 257

We know that complement (z.e., fresh normal serum) is fixed
by the combination antigen-antibody. Let us prepare two
such combinations :

(1) Cholera vibrios and anticholera serum (heated to 56° C.
to destroy its complement).

(2) Blood corpuscles and hemolytic immune body
(hzemolytic immune serum heated to 56° C.).

Now on the first combination fix complement (z.¢., add fresh
serum to the mixture and put in the incubator : the complement
exerts its bacteriolytic action).

Now add the second combination ready for hemolysis and
return to the incubator. If the serum contains hemolytic
complement over and above the bacteriolytic, its presence
will be shown by the laking of the corpuscles and the
red tint spreading throughout the mixture. If, on the other
hand, no hemolysis occurs, it means that the first fixation
has used up all the complement of the fresh serum: there
is no hemolytic complement therefore distinct from the
bacteriolytic.

Given a mixture complement + immune-body (antibody)
+ antigen, we have always a means of knowing if the com-
plement has been fixed, namely, to add a mixture of blood
corpuscles and hemolytic immune-body ready for hemolysis.
Laking of the blood indicates the absence of a previous
fixation of the complement. For example, a serum is sent to
us of a man who has been suffering for some time from fever ;
typhoid fever is suspected ; if this is the case his blood ought
to contain an antibody (antityphoid immune-body) and in the
mixture of complement + serum of the patient + typhoid
bacilli, the complement will be fixed on the bacilli through the
intermediation of the serum. If after a suitable lapse of time
one adds to the same tube a mixture of hemolytic antibody
and red corpuscles, the complement being no longer free, the
corpuscles are not dissolved.

Bordet’s reaction furnishes therefore a means of recognising
by the presence or absence of hemolysis the presence or
absence of another immune-body (antibody), and in conse-

s

258 MICROBES AND TOXINS

quence of discovering the effect remaining in the animal body
from a previous infection.

It has been used like agglutination to diagnose both a past
infection and the nature of a bacterial strain. There is no
reason to use it in the diagnosis of typhoid fever, Widal’s
reaction being much more convenient. It is very useful in
those cases where agglutination cannot be employed.

The Sero-diagnosis of Syphilis: Wassermann
Reaction,—Wassermann’s reaction is simply the application
of the Bordet-Gengou reaction to discover the syphilitic
antibody in ‘the serum of patients affected by this malady.
In practice it is rather delicate, and demands an experienced
worker. In theory nowadays it is regarded as a precipitating
reaction between certain colloid substances of the serum to be
examined and the colloids of an organ extract which is
employed as “antigen.”

The reaction is not strictly specific ; in leprosy, in trypano.
soma infections in animals, perhaps in scarlatina, modifications
of the serum often occur which give a positive reaction. The
method is nevertheless capable of clinical application, since
the: physician has only to decide between a trypanosoma
infection, such as sleeping sickness, which scarcely exists in
Europe, leprosy, which produces characteristic lesions, scarlet
fever, which is easy to diagnose, and syphilis, which is
extremely common; the selection is therefore easy. <A
laboratory reaction which, taken by itself, does not give an
absolute diagnosis, becomes nevertheless an absolutely certain
sign when taken along with the sum-total of clinical and
biological indications. .

As a matter of fact, the Wassermann test, if carried out with
the best technique, is positive in about 90 per cent. of the,
cases of primary syphilis, in roo per cent. in secondary syphilis,
and in 50-60 per cent. of late tertiary cases (Citron’s figures) ;
the proportions are rather lower in the statistics hitherto collected
by the Pasteur Institute. The reaction becomes more definite
the more acute the infection and the more active the virus.

There is some doubt as to whether it indicates syphilis in

APPLICATIONS OF BACTERIOLOGY 259

full activity or rather only that the individual has been affected
at a more or less distant period, and that the infection is now
latent. According to certain observers, itis always the sign of
active infection, although in a part of the body where the
lesions are not perceptible, the proof being that specific treat-
ment makes the reaction disappear, whereas it reappears
during relapses or recurrences of the disease. The experience
hitherto with “606” does not apparently authorize such a
simple interpretation, however.

In many cases the diagnosis of syphilis is only too easy ; the
lesions visible and active are sufficient proof. But humanity
suffers from many ills which are more or less distant con-
sequences of an attack of syphilis, neglected, forgotten, or
even unsuspected. Since syphilis is a curable disease the value of
Wassermann’s reaction in the diagnosis of these obscure cases
is obvious : lesions of the liver, of the heart, of the aorta, and
failure of the sight may all by its means be assigned to their
true cause and yield to the specific treatment. An old clinical
law holds that the mother of a syphilitic infant, without being
herself infected, is immune to infection from her child, and
thus may nurse it without danger ; in reality, it turns out, she is
infected : the serum reaction proves it. A Dresden physician,
Rietschel, has thus diagnosed syphilis in 1o per cent. of wet
nurses apparently healthy, and the confirmation was obtained
when it was found that the children of these women eventually
showed signs of syphilis.

Syphilis unsuspected or without symptoms is a disease less
rare than is generally thought, and Wassermann’s reaction
furnishes us with an additional weapon against this formidable
enemy.

Clinical medicine has long maintained the syphilitic origin
of general paralysis of the insane, and of locomotor ataxy.
The sero-diagnosis brings a striking proof of this. Not only
the serum but the’ cerebro-spinal fluid of general paralytics
furnishes a positive reaction in 88 per cent. of all cases, and in
95 per cent. of advanced cases. In locomotor ataxy the per-
centage is not so high.

260 MICROBES AND TOXINS

The reaction is also of value in the distinction between
general paralysis and other mental affections which bear an
external resemblance to it, among others from dementia praecox.
Bordet’s reaction has also been applied in the diagnosis of
hydatid cysts due to the echinococcus.

Also it has been employed in the detection of blood and
foreign proteins for which it is an even more sensitive test than
precipitation: it requires, however, such delicate manipulation,
and is so extremely sensitive, that it can hardly be trusted in
certain cases; according to Friedberger it is capable of
detecting the minute trace of protein present in human per-
spiration, so that the hands of the experimenter himself may
introduce an error.

It has been employed in the differentiation of different races
of man and of monkeys: not that the distinction between man
and monkeys is so absolutely necessary as to show the sort of
hierarchy which exists. Man is as far removed from the orang
as the orang is from the macacus ; however, man is more nearly
related to the orang than the latter is to certain species of
macacus. It is even possible, it is said, to distinguish by sero-
diagnosis an individual of Mongolian race from a Malay.

The biological reactions thus are of value, not only in the
physiology of immunity and of nutrition, but even in zoology
and anthropology.

Tuberculin Tests.

A dose of tuberculin which produces no effect in a healthy
subject may determine in a tuberculous individual a definite
reaction, consisting of general and local phenomena, fever,
inflammation, and cedema round the tubercles and at the site
of injection.

It was long thought that the reaction only took place in the
neighbourhood of the tuberculous lesions themselves, and
consisted in the inflammation and destruction of a certain
number of cells, the fever being due to the absorption of the
cellular debris. Von Pirquet, however, discovered that the
skin of a tuberculous patient reacts to tuberculin at every point,

APPLICATIONS OF BACTERIOLOGY 261

and it has therefore been necessary to suppose that the body is
impregnated throughout by some substance produced by the
chronic infection.

The body responds to the attack of the tubercle bacillus by
a reaction product, an antibody, and the tuberculin reaction
appears to be produced by the coming together or combination
(not necessarily in the chemical sense) of this antibody and
tuberculin ; that is roughly how the facts are being interpreted
nowadays.

The body by producing this antibody is said to become
“sensitive” to tuberculin. The tuberculous individual behaves
differently from the healthy subject, being more sensitive, and
his excessive susceptibility acting as a warning to him of his
danger. It is in this way that the supersensitiveness which is
a phase of immunity (v. Chap. XII., page 240) has occasionally
been interpreted. There is some resemblance between the
tuberculin reaction and the anaphylactic shock ; the diagnosis
of tuberculosis by tuberculin is based on the “ supersensitive-
ness” of the indiyidual.

The reaction represents in a sense a resistance to the action
of tuberculin ; we have seen that the prototype of this reaction,
Koch’s phenomenon, consists in the expulsion of the re-
inoculated virus by the infected subject ; it is therefore also an
immunity reaction. .

Tuberculin was at first employed as a remedy, but later only
as a diagnostic agent. It was even more studied in veterinary
science than in medicine, because it was easier to make experi-
ments and control them by autopsies on animals. Even to-day
the tuberculin test is quite as important in veterinary as in
human practice.

In the diagnos's of tubercles, nowadays, smaller doses are
used for subcutaneous injections than at the time of the first
trials of tuberculin.

The temperature reaches its greatest height about eight
hours after injection. A positive reaction indicates merely
“tuberculous lesions” and nothing more. It gives no
information as to the site or the age of the lesions, nor

262 MICROBES AND TOXINS

as to the probable fate of the patient. The reaction may be
negative in exhausted, cachectic tuberculous patients.

The number of individuals who give a positive reaction
much exceeds the number of those who show clinical signs’ of
tubercle: latent dormant tuberculosis, or even healing tubercle,
gives a reaction because the body is saturated with the
products of its reaction to the tuberculous infection.!

In veterinary practice, it happens that animals once sub-
jected to tuberculin injection fail to react to a second test
performed twenty-five to thirty days after the first; this fact
has been employed in fraudulent dealing to throw out the
diagnosis, but the second test will still act if a double dose
is given, and if the temperature is taken at the fourth
hour (instead of the tenth); the fever appears prematurely
(Vallée).

Cutaneous Reactions,—In individuals affected by tuber-
culosis, if the skin is subjected to a quite superficial scarifica-
tion on which is put a drop of tuberculin, a red spot is seen to
appear after a few hours. This swells, and comes to resemble
a little vaccine pustule. It was this phenomenon which was
discovered by Von Pirquet. Several variations have been
tried. It is possible to do without scarification by dropping
into the inner corner of the eye a drop of a dilute solution of
tuberculin ; the conjunctiva reacts with more or less intense
inflammation and more or less exudate ; this is the ophthalmo-
reaction of Wolff-Eisner and of Calmette.

It was observed during subcutaneous injections of tuberculin,
that the needle-track of the syringe, being wetted with a trace
of the tuberculin, became red and swelled up; from this

1 The statistics of Franz dealing with certain regiments of the Austro-
Hungarian army are. worth reproducing, since the author had the
opportunity of observing the same men during several years.

In a regiment of four hundred apparently healthy men recruited from a
district where tuberculosis was rife, 61 per cent. gave during their first year
of service a positive reaction ; of 323 similar men in their second year of
service 68 per cent. were positive. Of 279 healthy men recruited from a
district where tuberculosis was scarce, there were 30 per cent. of positive
reaction. These 1002 individuals furnished in the course of the seven
following years sixty-four cases of manifest tubercle and twenty died of it.

APPLICATIONS OF BACTERIOLOGY 263

subordinate phenomenon an independent diagnostic test has
been established by making a simple prick in the skin (Moro),

Further, it is possible to inject into the thickness of the
skin itself a drop of the solution containing only 74, of a
milligram of tuberculin ; there is thus obtained a very definite
diagnostic reaction: the intradermo-reaction (Mantoux).

These procedures are all very convenient, especially in the
examination of children; the methods of pricking and of
intradermo-reaction are less severe than the intra-ocular
instillation.

The cutaneous reaction of V. Pirquet gives about 85-90 per
cent. of positive results in children presenting signs of tuber-
culosis, 20 per cent. in children with no clinical symptoms, and
48 percent. in doubtful cases. The percentage of positive
reactions iz children not clinically tuberculous varies with the
age. During the first 6 months of life it is practically o;
from 6 to 24 months 2 per cent.; from 2 to 4 years 13 per
cent. ; from 4 to six years 17 per cent.; from 6 to 10 years
35 per cent.; from ro to 14 years 55 per cent. ; in adults
there are at least 77 per cent. of positive reactions.

The great value of V. Pirquet’s cutaneous reaction is that
with a little prick, which is itself quite harmless and produces
no trace of fever, and with a minimal quantity of tuberculin
(down to 7/59 Of a milligram), it is possible to tell if a child is
affected with tubercle bacillus.

The younger the child the more definitely does the
reaction signify a true active infection, since tuberculosis is less
frequent—and more recent—the younger the infant. In the
adult, it indicates for the most part a tuberculous impregnation,
great or small, recent or remote. .

The cutaneous tuberculin reaction, performed as it has been
on thousands and thousands of human beings, has taught us
that man becomes infected with tubercle in childhood from his
first to his fourteenth year; so that tuberculosis, although it
may only become serious and fatal at a more advanced age, is
really a disease of children and not of adult life. It is then
during childhood that protection is necessary and since we

264 MICROBES AND TOXINS

know that the child is infected by the individuals surrounding

.it, it is in the family itself and in the home that contagion
must be prevented. The disease is kept up and spread by
infected members of the family and by servants and also by
unhealthy houses, in particular by dark, airless rooms. The
cutaneous reaction tells us that of a hundred adults harbouring
the tubercle bacillus, only twelve are destined to die of
tubercle. It is therefore very evident that many human
beings recover from the attack of the bacillus and that man is
an animal naturally resistant to tuberculosis ; in most of us a
sort of spontaneous vaccination takes place. These facts
ought not to be overlooked in the search for a method of
treatment or prevention of tubercle.

CHAPTER XIV
VACCINES AND SERA

Vaccination with unknown viruses—Small-pox : inoculation and Jennerian
vaccination—Sheep-pox—Rabies: Pasteur’s treatment—Vaccination
with microbes of attenuated virulence—Anthrax, Swine-erysipelas,
Pleuropneumonia of cattle—Vaccines against cholera, plague, typhoid
fever—Attempts at anti-tuberculous vaccination.

Bacteriotheraphy: intestinal, buccal, nasal—Applications to surgery.
Serotherapy, Diphtheria, Tetanus, Venoms, Cholera—Serum against
plague, bacillary dysentery, cerebrospinal meningitis— Anti-strepto-
coccic sera—Anti-tuberculous sera.

Sero-vaccinations : Sheep-pox—Bovine plague—Swine-plague.

Virus-vaccines sensitized— Experiments of Ehrlich and Morgenroth.
Besredka’s vaccines: Typhoid fever, Plague—Virus-serum in the
treatment of rabies.

Phagocytic therapy.

It is fairly easy to immunise laboratory animals against
bacteria by repeated doses of the living microbe: a preliminary
experiment determines the minimum lethal dose, and the first
injections are to be kept well below this. Immunization is
possible also with viruses which are incapable of killing in
any dose.

With extremely virulent bacteria, immunization is much
more difficult. It is impossible to immunize a guinea-pig
against anthrax, for example. In man, where one is not
entitled to run the slightest risk, it is impossible to begin even
with very small doses of living virulent microbes. Hence
arises the great importance of Pasteur’s discovery in the history
of active immunization, the discovery of the attenuation of a
virus. Only vaccines may be used in human immunization.

In general, a non-virulent microbe neither kills nor im-
265

266 MICROBES AND TOXINS

munises, whereas a virulent organism -kills before immunity
has time to develop. It is often necessary to find inter-
mediate stages of virulence. This is what Pasteur perceived,
and hence arose his two vaccines against anthrax.

It is possible to vaccinate with viruses which one handles
without actually knowing their nature, with cultures of bacteria
virulent or attenuated, and with bacterial products freed from
the living microbes. i

VACCINES
I. Vaccinations with Unknown Viruses.

It is not necessary actually to know the microbe of a
disease in order to immunize against it. The vaccinations
with unknown viruses are, as a matter of fact, among the most
perfect known to medicine.

Small-pox.—The microbe of small-pox is unknown, yet
preventive inoculations have long been carried on. There
exist two methods, both empirical in origin; the older of the
two has been entirely given up in favour of Jenner’s method
of vaccination. Jnoculation or variolisation consisted in
inoculating the virus of small-pox itself, so as to produce one
or more cutaneous pustules, the development of which saved
the body from a general attack. Small-pox acquired in the
natural manner invades the whole body: the virus may enter
perhaps by the tonsils or the respiratory passages, gaining the
blood stream from these and thence reaching the skin and
mucous membranes. Once the eruption has appeared it is
too late to intervene. When the natural virus is inoculated
artificially on the skin, it may give rise to a general infection
later on, but in general the first local pustules produce
rapidly sufficient immunity to prevent such a general invasion ;
but not always; occasionally inoculation results in a fatal
attack of small-pox. The virus inoculated was by no means
constant, and it was always a case of working in the dark.
A correct and witty account of the ‘history of inoculation is to
be found in the eleventh of Voltaire’s Letires Philosophiques.

VACCINES AND SERA 267

There exists among cattle a disease resembling small-pox,
and also due to an unknown microbe, the cow-pox, which
attacks also those who milk infected cows. Those individuals
who have suffered from the pustules of cow-pox are immune to
small-pox.

This idea, popular in origin, had long been current among
farmers and cattle-breeders in certain districts of England,
France, Holland, and Germany when Jenner took it up
and consecrated his life and his genius to publishing it
abroad.

He made of the subject a definite: experimental study: he
inoculated in turn the virus from animal to animal, from the
animal to man, from man to man, and finally submitted his
“ vaccinated ” subjects to experimental inoculation with small-
pox.

In all countries where public health is well organized vacci-
nation has been made compulsory ; in consequence smallpox
has almost disappeared from Germany: the rare cases recorded
there are cases of immigrants or inhabitants on the frontiers.
It has very much diminished in France and is being actively
combated in the French colonies. It may one day become
extinct.

Three improvements have been introduced in Jennerian
vaccination since Jenner’s time. In the first place, it was
recognized that in many cases vaccination does not protect for
the whole of life, and periodical revaccinations have become
customary and even in certain countries compulsory. In the
second place, instead of vaccinating from man to man the
vaccine employed is derived from animals. Jenner practised
““arm to arm” vaccination, 7.¢., transmitted from man to
man a virus whose distant origin was cowpox. This practice
had the inconvenience of occasionally transmitting human
diseases such as syphilis. From 1860 onwards vaccination in
Europe has been performed with “animal lymph” taken
from vaccine calves (in certain colonies from buffalos, rabbits,
zebras, &c.).

Finally we have learned to preserve and purify the supply of

268 MICROBES AND TOXINS

virus by mixing it with glycerine, so that it isno longer necessary
to vaccinate directly from the calf. Inflamed arms are nowa-
days quite the exception.

According to certain observers there are really only two types
of smallpox, the pox of sheep and that of man. All the others
are said to be mere modifications of these two types ; for small-
pox is very apt to become modified in adapting itself to different
animal species.

Vaccinia, in this theory, would simply be an adaptation of
human smallpox to cattle, and, starting with this idea, attempts
have been made to transform smallpox into cowpox experiment-
ally by inoculating from man to the calf. This question of
variolo-vaccinia has been very much studied and is being taken
up again to-day. The results obtained have been much dis-
cussed, and itis not yet absolutely proved that it is possible to
produce in the laboratory in a short time a change which
nature has probably required centuries to accomplish.

Rabies.—Pasteur’s treatment takes advantage of the period
of incubation, which is on the average six weeks. The patient
ought to be treated as soon as possible after the bite and before
the appearance of symptoms.

In the process there is inoculated the virus fixe, which is
kept going in the nervous system of the rabbit, is contained in
the spinal cord, and may be modified by drying the latter in
the dark at the temperature of 23°. To begin with, a cord dried
for fourteen days is injected, and at the end of the treatment a
cord dried for three days. The rate of treatment varies accord-
ing to the site and gravity of the bites, there being three
formule, the fifteen-day, eighteen-day, and twenty-one day
treatments. The mortality in rabies has been reduced to 0°3
per cent. instead of 15 to 20 per cent.

The microbe of rabies is unknown, and Pasteur treatment
is one of the crowning glories of experimental medicine.
Consider the facts which it has been necessary to determine
before arriving at its application: in the first place, the virus
of rabies exists in the brain of rabid animals ; further, it is
possible to make a pure inoculation of it; the virus of any

VACCINES AND SERA 269

rabid dog taken at random inoculated under the dura mater
of the brain of a rabbit after trephining the skull produces
rabies in the rabbit after a variable period of incubation ; but
after about twenty passages from rabbit to rabbit the incuba-
tion period falls to six to seven days; the virus has then
become the virus fixe, and the passages from rabbit to rabbit
are really cultures in the living body. The culture exposed to
dry air, but sheltered from light, gradually loses some of its
virulence. The dried cords, inoculated in the form of emulsions
under the skin of animals, produce in them a certain and
stable immunity against the strongest virus inoculated under
the dura mater. The cultivation zz vivo of the wrus fixe was
for Pasteur the key to vaccination against rabies after the bite
of the rabid animal.

There is some doubt whether the virus of the dried cords
really undergoes attenuation. Even in 1885 Pasteur said that it
was rather a rarefaction which occurred. “The delays observed
in the duration of incubation of the rabies communicated day
by day to rabbits, to estimate the virulence of our cords dried
in contact with air, result from a diminution in quantity of the
virus contained, and not from a diminution in virulence.” A
variation of Pasteur’s treatment, the treatment of Hoégyes,
consists in replacing the graduated desiccation by a graduated
dilution.

1 The nature of this vzrus fixe cultivated in the rabbit is rather a
question, Nitsch and Marx, who inoculated themselves with fresh vérus
fixe, consider that after several hundred passages in rabbits the virus
becomes harmless to man ; they consider it as a true Jennerian vaccine for
man, resulting by adaptation to a different species. But it is not right to
maintain the absolute innocuousness of wzrus fixe. It may well be harm-
less when injected into the subcutaneous tissue, but would it continue to be
so if injected in a region rich in nerve filaments such as the face or the end
of finger ?

It is probable that the virus of dried cords is destroyed by the body, that
the microbe is absorbed, and that protective substances are produced in
consequence and are found in the blood of treated individuals. But these
substances play only a secondary part, for there is no constant relation
between the properties of the serum of an animal and its resistance to
rabies. There are certain animals refractory to rabies, such as a species of
tortoise whose blood nevertheless possesses no antirabic power. It is
impossible to avoid thinking of the phagocytes in such a condition.

210 MICROBES AND TOXINS

ii. Vaccination with Attenuated Cultures.

Anthrax.—The anthrax vaccines are cultures attenuated
by contact with the air at a temperature of 42°5°C. Two
graduated vaccines are employed: the weaker kills mice and
small guinea-pigs, the stronger kills adult guinea-pigs and even
a certain percentage of rabbits. The animals treated with
these preserve their immunity for about a year, and it has been
calculated that during the twenty years which followed their
introduction at least twelve million animals were inoculated.
The method of anthrax vaccination has never required alter-
ation since its discovery by Pasteur, Chamberland, and Roux,
and the celebrated experiment of Pouilly-le-Fort.

Swine-Erysipelas.—In this the same principle is
employed, 7.¢., a virus attenuated in the laboratory. But the
bacillus of swine-erysipelas does not produce spores, and a
method of sero-vaccination has been invented instead (v. infra).

iii. Pleuropneumonia of Cattle (Rinderpest).

The discovery of the microbe dates only from 1898, but
vaccination had been practised against the disease a con-
siderable time before. Willems employed as virus the serous
fluid from the diseased lungs : he had observed that inoculation
of this in a healthy animal produced variable effects according
to the site of injection chosen: injected on the trunk or neck
the infection was fatal ; at the tip of the tail or of the ears it
produced only a limited inflammation which left the animal
immune towards the naturally occurring disease.

The first improvement was introduced by Pasteur, who
showed that the pure virus could be got by taking the abundant
exudate produced in the subcutaneous tissue of a calf after
inoculation there. Later, pure cultures were substituted.

The tip of the tail is a good site for vaccination, since the
tissue is dense and the absorption slow. A few animals lose
their tails as a result of the vaccination.

VACCINES AND SERA 271

iv. Cholera, Typhoid Fever, Plague.

Cholera.—Human cholera is a toxic disease, the toxin
being secréted by the vibrios invading the intestine. The true
remedy for cholera, therefore, should be an antitoxic serum.
It was before our present conceptions as to the toxicity of the
cholera vibrio that Ferran, in 1884, tried to vaccinate men
against cholera by injecting the living microbe subcutaneously.
This method has been taken up and modified by Haffkine,
who has tried it in India, where cholera is always present.
In vaccinated individuals he found the mortality seven times
less than in the non-vaccinated, but although the death-rate
may be lower, the severity of the symptoms is quite as great
among the former as among the latter, which is rather dis-
concerting, as in other kinds of preventive vaccination this
does not occur. The solution of the cholera problem lies
elsewhere, but it is worth while recalling the attempts of Ferran
and Haffkine, because their method has proved of more value
in other diseases. :

Typhoid Fever.—Typhoid fever is a toxic enteritis, but
it is at the same time a blood-infection ; blood-cultures show
that there is septicemia. The bacillus inhabits chiefly the
small intestine, but it also extends to the blood, the spleen,
and the bone-marrow. Hence there is much more hope of
success than with cholera for preventive injections of the
bacteria under the skin. Bacilli killed by heat are employed.
The injection produces some swelling and pain with fever and
stiffness the discomfort lasting about two days. Two, or even
three, injections ought to be made. The treatment is succeeded
by what is called a ‘negative phase,” during which the
individual may be in a condition of lowered resistance.

Anti-typhoid vaccination has been chiefly employed hitherto
in the English army in India, in the Transvaal, in Egypt, and
in Cyprus. A certain number of the inoculated individuals
have nevertheless taken the disease (in many of these the
vaccination had been insufficient), but in a form milder and

272 MICROBES AND TOXINS

with less frequent relapses. The mortality is about four times
less among the inoculated than among the non-inoculated.
As far as can be judged at present, the effect of the vaccination
lasts for about four years.

There is no necessity to submit a whole family to the dis-
comforts of inoculation when one member has taken typhoid ;
it is sufficient to isolate the patient and to disinfect strictly
the excreta, linen, etc. Vaccination is adapted only for the
members of an ambulance and hospital corps during an
epidemic, or for troops campaigning in a country where
typhoid is raging, and where it is difficult to adopt hygienic
measures.

Plague.—Haffkine’s vaccine consists of bacteria killed by
heat. The inoculations, especially the first, produce inflamma-
tion and pain with a high temperature, the illness lasting for
about three days. Two, or even three, inoculations ought to
be made. Such vaccinations are suitable in the case of plague,
as this disease is a general infection, spreading throughout the
blood and organs. In 1902-1903 about half a million persons
were inoculafed in the Punjab, and fairly accurate informa-
tion could be gathered regarding a portion of them. On
comparison we find :—

186,797 inoculatéd, with 3339 cases = 1°8 per cent.
», 814 deaths = 0°4 per cent.

639,630 non-inoculated, with 49,433 cases = 7°7 per cent.
1» 29,733 deaths = 4°7 per cent.

It appears that the inoculation continues to be of benefit for
several years after.

Anti-tuberculous Vaccination.—As far as is known,
vaccination against tubercle is possible with living bacilli only,
dead bacilli being ineffective, but there is no vaccine as yet
which is entirely safe.

Behring has sought “ Jennerian” vaccines, #.¢., he has tried
to vaccinate one species, cattle, with the virus derived from
another species, man. Innumerable experiments have already
been made on bovo-vaccination, and they prove at least that
a certain degree of immunity may be created towards tuber-

VACCINES AND SERA 273

culosis. But even among cattle the method is not yet suited
for practical application.

In man there exists an auto-vaccination, since practically all
adults have been slightly affected by this disease, and only a
small fraction succumb. Both in medical and surgical practice
it is common to find tuberculosis of the skin, of the joints, and
of the bones healing up and even apparently protecting the
patients against contracting pulmonary phthisis. It is on these
lines that is to be sought a method of vaccination against
tuberculosis. Up to now none exists.

BACTERIOTHERAPY.

Vaccines and sera are not the only remedies furnished to
medicine by bacteriology. Bacteriotherapy consists in modify-
ing a given flora by adding to it other bacteria which are
antagonistic or favourable to certain microbial species. In
discussing the intestinal flora we have indicated the principles
of the bacteriotherapy of intestinal infections. In the future
this will be developed, and health-giving bacteria will form
part of our diets.

Bacteriotherapy has also been attempted in the case of
cancer and lupus, where injections of streptococci have been
given, and in the case of chronic boils, where injections of
yeast have been tried. These attempts are still semi-empirical,
but this is no reason for despising them.

The lactic bacilli have not been utilized in intestinal bacterio-
therapy only. Surgery has employed them to combat puer-
peral infections, injuries occurring during labour, inflammations
of the cervix, and even peritonitis. They evidently act by
modifying the “soil.” It is obvious that they find their chief
success in combating infections of closed cavities, such as we
have in rhinitis, sinusitis, ethmoiditis, and otitis.

Bacteriotherapy in the mouth is not less rational than in
the intestine. The lactic flora has succeeded in curing
chronic inflammations of the gums and obstinate pyorrhea
alveolaris. All the infectious diseases which enter by the

T

Q74 MICROBES AND TOXINS

mouth, the tonsils, and the nasopharynx may be combated by
this bacteriotherapy, and already very encouraging observations
exist. For example, epidemic cerebro-spinal meningitis starts
from the nasopharynx, and there are meningococcus carriers
just as there are diphtheria carriers. It is the more important
to employ the lactic bacilli against the meningococcus from the
fact that 2” vdtro the former antagonize the latter (A. Berthelot).
The bacterium most to be recommended for the purpose is
the Bacillus bulgaricus administered in powders and derived
only from young fresh cultures in liquid media. It is not
necessary to insist upon the inconvenience of the use of anti-
septic washes and to contrast with these the biological
disinfection produced by modifying the flora.

SERA.

Serotherapy means treatment by means of the sera of
immunized animals. It may be preventive or curative, or both.
The animal furnishing the serum has produced its own
immunity, ze, it possesses active immunity. The animal
which receives such a serum acquires a passive immunity.
Serotheraphy is equivalent to the transfusion of antibodies.

The antitoxins were discovered by Behring in 1890.

Antitoxic sera are not the only therapeutic sera: by
immunizing an animal with the bodies of bacteria antibacterial
serum can be prepared: with such sera there may be
transfused bacteriolysins, agglutinins, bacteriotropins, in fact
all the antibodies they contain, but above all there is transferred
their property of exciting the reaction of the phagocytes.

Certain antibacterial sera are also anti-endotoxic in the
sense that they are capable of neutralizing endotoxins. They
are best prepared as we have seen by zu¢vavenous injections of
the antigen. Finally, sera may be prepared against viruses
only slightly known or not known at all; the preventive action
of serum in general may be disregarded.

Antidiphtheria Serum.—This is a serum of horses
immunised against the diphtheria toxin. The treatment of the

VACCINES AND SERA 275

horse begins not with pure toxin but with a mixture of toxin +
Gram’s iodine or of toxin+antitoxin. Every horse has its
own susceptibility, and the treatment requires a good deal of
skill. It is impossible to foretell the supply of antitoxin from
any particular horse.

The serum treated aseptically is put into little bottles and
“tyndallised,” z.e., sterilised by heating to a low temperature
on several successive days. It is stored in a dark, cool place,
and under these conditions it only loses about one-tenth of its
activity, so long as it remains clear, even after several years.

Sera possessing great activity per unit volume have been
sought for by selecting those horses which are active pro-
ducers, or by concentrating or “refining” the serum by
various procedures, freezing, precipitation, &c. The best
method is still to take an active serum, to keep it pure and
preserve it carefully without further treatment.

The titration of the serum is indispensable: only serum of
known activity is issued for serum therapy. Hitherto, titration
has only been possible by resorting to experimental animals,
guinea-pigs. The laboratories of all countries have agreed
to adopt the method proposed by Ehrlich. The serum is
standardized by mixing with a toxin which has itself been
estimated against a standard antitoxin. The activity of the
serum is expressed in antitoxic units which, like all units of
measure, are conventional. The standard antitoxin is preserved
with precautions similar to those with which are guarded our
standard weights and measures.

But in spite of all the precision introduced, we are still
dealing here with a biological, not a chemical process.
Experience has made clear three points on which too much
stress cannot be laid. (1) The serum ought to be inoculated
as soon as possible after the appearance of the disease. A
little serum given early is worth more than a great deal of
serum given too late. Similar guinea-pigs receiving the same
dose of toxir may be cured by a small dose at the sixth
hour, whereas at the eighth hour even a large dose fails. In
hospital, unfortunately, the children are usually brought too

T 2

276 MICROBES AND TOXINS

late, and it is on ¢his account that the mortality in diphtheria is
still 10 per cent. (or 14 per cent. in epidemics ; L. Martin).

(2). There should be no fear of giving large doses: in fact
in bad throats large doses ought to be resorted to at once.

(3). Intravenous injection is the most efficacious: it is
indicated in all serious cases. This holds good for all sera. .

There is said to exist still in certain minds a certain
scepticism as to the value of antidiphtheritic serum but it is
scarcely credible. The figures speak for themselves, The
death rate from diphtheria in the Sick Children’s Hospital in
Paris before serotherapy was about 50 per cent.; it has fallen
to 6—ro per cent., and would be much less if the children were
treated at the very beginning of the disease.

The serum is used as a prophylactic against diphtheria in
schools, barracks, créches and hospitals: a dose of 5—10 c.c.
protects for about three weeks.

Anti-tetanic Serum is an antitoxic prophylactic serum.
The serum furnished by the Pasteur Institute possesses an
activity such that zogs95 Cc. suffices to neutralize Zn uitro
r00 lethal-doses for a mouse.

In human tetanus when the incubation is long and the
progress slow, the serum may check the extension, z.e., may
have a limiting effect. According to the figures collected by
Vallas the serum has lowered the death rate from about 75
per cent. to 45 per cent. The mortality remains about 70
per cent. in those cases where the incubation period is less
than eight days.

1 When tetanus toxin and a sufficient dose of antitoxin are injected at
different points of the body a minute quantity of toxin escapes neutraliza-
tion and induces a slight local tetanus which is always recovered from.

It is possible to cure animals inoculated in the paw if the serum is
injected at the first appearance of symptoms : when the poison has reached
the nerve-centres the antitoxin is of no avail. Roux and Borrel had the
idea of bringing the antitoxin in direct contact with the brain so as to
prevent the poison reaching the centres. By intracerebral inoculation
they succeeded in curing a number of guinea-pigs which had manifested
tetanus and were therefore doomed to die. But they do not extend their
conclusions to other animals. In man the first symptoms of tetanus
affect always the medullary centres so that cerebral treatment has even less
hope of success than in the guinea-pig.

VACCINES AND SERA Q77

The employment of this serum in veterinary practice is due
to the efforts of Nocard. He collected, now a long time ago,
2,708 cases of horses injected immediately after one of those
operations frequently followed by tetanus (castration, ampuita-
tion of the tail, inguinal or umbilical hernia) ; not a single case
of tetanus occurred. A second group comprising 600 animals,
treated one to four or even more days after an accidental
wound (nail-prick, harrow-tooth wound, kicks, wounds soiled
with earth or manure, etc.), presented one single case of mild
tetanus (a horse treated five days after the accident, a nail-
prick in shoeing). The veterinary surgeons who supplied these
3,308 cases observed in their practice at the same time 314
cases of tetanus (of which 220 were horses) in animals not
treated with serum. In 705 equide, wounded or operated
on, Labat observed three cases of tetanus among the un-
treated, not a single case among the treated animals. Accord-
ing to statistics supplied by eight veterinary surgeons to
Vallée of Alfort, from 1898 to 1906, 13,126 animals were
inoculated as a preventive measure, and not one took tetanus.
During the same period, two of these surgeons alone had
139 cases of tetanus among animals not treated. At present
the Pasteur Institute supplies to veterinary surgeons more than
100,000 doses per annum.

There is no reason known to science why man should not
derive a similar advantage. There exists, however, with
regard to the prophylactic serotherapy of tetanus in man a
distrust or scepticism which finds vent from time to time in
the discussions of surgical societies or congresses. It has
been maintained that the employnient of preventive injections
has not affected the death rate from tetanus in Paris ;. that
tetanus occurs fairly often in spite of prophylactic injections ;
that the conditions which permit of the prophylactic employ-
ment in veterinary practice are unrealizable in human medicine,
and finally that in man the serum is less active than in the
horse. When examined none of these objections hold good.
The numerous unsuccessful cases depend on the conditions of
tetanus intoxication and its neutralization ; there is not oné of

278 MICROBES AND TOXINS

the objectors who, if he received a tetanus-suspected wound,
would not call for a prophylactic injection of serum for
himself.

It is necessary to avoid giving too small doses and, should
the wound fail to heal, to repeat the injection several times ;
of course, the surgical cleansing of the wound should never be
omitted.

Antivenom Sera,—These are prepared by immunizing
horses, beginning with venom + calcium hypochlorite and ending
with pure venom. Eventually they can be made to support
80 lethal doses (2 grams of dried venom) at a single injection.
The immunization is a difficult operation to bring to a satis-
factory end; frequently it is interrupted by serious accidents,
endocarditis, nephritis, and abscesses. It takes 16 months
of treatment to get a good serum from a horse (Calmette).
Antivenom serum is Jolyvalent, i.e., is prepared with several
species of venom.

The quantity of serum required to save life increases with
the susceptibility of the animal and the delay in the application
of the remedy. As a rule 10-20 c.c. suffice to save a bitten
human being.

Anticholera Serum.—Cholera is an acute intoxication
due to a poison elaborated by the vibrios in the intestine.
This toxin has been sought for and an antitoxic serum
attempted. The serum prepared against the bacteria is not
active against the poison; the principle of serotherapy is
thus quite different from the Ferran-Haffkine method of
immunization.

The cholera toxin prepared in 1896 by Metchnikoff, Roux,
and Salimbeni presented this peculiarity, that it resisted heating
to 100° C. for a quarter of an hour. Horses are prepared by
intravenous injections.

Anticholera serum is still at the trial’stage. Its only trial out-
side the laboratory was in 1909, during the epidemic of St.
Petersburg. The doses ought to be large, 150-350 c.c., given
100 c.¢. at a time intravenously. There are cases so fulminant
as to defy almost any remedy. In the above trial cases, not

VACCINES AND SERA 279

very numerous, the mortality was reduced by about a half. In
the laboratories work is still going on towards the improvement
and perfection of an anticholera serum.

Antiplague Serum.—It is derived from horses immunized
against the plague bacillus. The treatment is long and difficult.

The statistics are in need of discussion. Whereas Mayer,
for example, records numerous cases of cure, other physicians
declare that there are more deaths among the treated than the
non-treated: they allege that the serum is too weak and the
Hindu too susceptible to plague. When these unfavourable
statistics are more closely examined, it becomes apparent that
the serum has been given too late, in too small dose, by subcu-
taneous injection, and not long enough continued: the patients,
abandoned after the fall of the fever, died in many cases
fifteen to thirty days after the last serum injection. In man,
plague is all over in five to six days, and if treatment is only
carried on during the third or fourth day cure is well nigh
impossible.

The injections ought to be massive and repeated. It is no
use trying to treat plague with the doses suitable to antitetanus
serum.+

It is evident that against pneumonic plague the serum which
we possess at present is not to be relied on, but that against
bubonic plague it has already been of service. It may be of
great value in a population where the hygiene is good, to
complete the isolation of a case of plague by giving prophylactic
injections to the contact individuals, and thus to limit the
epidemic focus.

Antidysenteric serum is given in doses of 20, 80 to
100 c.c.—the latter doses in very serious cases where there are
more than roo motions of the bowel per day. Between 1905

1 Duprat, using doses of 300 c.c., succeeded in curing thirty-eight out of
forty-five plague patients treated, a mortality of only 15 per cent.

Employing intravenous injections (100 c.c. repeated after twenty-four
hours), Penna had a mortality of 14°2 per cent. in 200 cases. Ferrari at
Sao Sabastiao had only 7°2 per cent. of fatal cases among forty-four.
Ferrari’s statistics throw light on the different modes of injection: by the
skin, 38 per cent. death-rate, intraperitoneally, 18 per cent, intravenously
7°2 per cent.

280 MICROBES AND TOXINS

and 1907 Vaillard and Dopter applied the serum in 512 acute
cases of all ages, of whom 32 had certainly fatal dysentery
and 170 a very severe attack. The average death rate was
only 1°3 per cent., whereas in different epidemics it has varied
from 10 to 25 or 50 per cent. ‘The serum relieves the patients
of their terrible abdominal pains, of the vomiting, and of the
hiccough ; the stools become less numerous and less painful,
the temperature falls, and a feeling of comfort succeeds that
of absolute prostration. ‘These good results occur as a rule in
the course of one day.

The antimeningococcus serum of Flexner is employed
in epidemic cerebro-spinal meningitis. It has hardly any
action when injected subcutaneously. It must be injected at
the site of the disease—z.e., into the cerebro-spinal canal by
lumbar puncture. The treatment should be begun as soon as
possible and be repeated. It has lowered the mortality from
60 to 80 per cent. to 25 to 10 per cent.

Antistreptococcic serum isa polyvalent serum—z.e., it
is prepared with several strains of streptococci. It is difficult
to get an accurate idea of its efficacy in man. The infections
in which streptococci are responsible are multiple, and the
serum is employed in the most diverse diseases, from puerperal
fever to scarlatina and acute rheumatism. Large quantities of
this serum are supplied from the Pasteur Institute both for
medical and veterinary use, but unfortunately it is rare to get
any information as to the results obtained.

Antituberculous Sera.—Maragliano’s serum is supplied
by horses treated with the soluble constituents of the tubercle
bacillus.

Marmorek’s serum is furnished by horses immunized
with “primitive bacilli,” ze, young bacilli which scarcely
possess any of the acid-fast property due to the wax and fat
constituents. The same horses are at the same time im-
munized against streptococci, since the streptococci are
frequently associated with Koch’s bacillus in tuberculous
lesions. As regards this serum opinions differ. Certain
observers report a favourable action on cases of tuberculosis of

VACCINES AND SERA 281

the bones and joints and on early cases of pulmonary tubercle
(checking the fever and improving the general condition).

SERO-VACCINATIONS.

When a vaccination fer se involves a certain risk it may be
associated with a specific serum: the combined treatment is
equivalent to an active immunization under cover of a passive
protection.

Sheep-pox.'—In France the law enjoins the vaccination
against sheep-pox of all the flocks in the affected regions,
whereas it forbids it in the regions free from the scourge. In
Algeria sheep-pox is endemic, and the Algerian sheep are
much more resistant than the French sheep. In consequence,
Algerian sheep, which are imported in great numbers, may be
landed apparently healthy, yet may induce a malignant
epidemic in a French flock. Nowadays only vaccinated sheep
may be imported.

Borrel, who perfected the method of vaccination by showing
how to prepare cheaply large quantities of pure virus, has
prepared an active anti-serum by “ charging ” with virus sheep
which had recovered from the disease. When injected into a
sheep twenty-four hours before the virus this serum prevents
the development of the disease (in a dose of 20-30 c.c.), and
even inhibits the inoculation pustule. By graduating the dose
of serum it is possible by inoculating the virus simultaneously
to produce simply an immunizing pustule without any risk of
a general infection.

The serum employed alone is sufficiently active to render
certain a prophylactic treatment of the disease by curative and
preventive injections in a flock. It permits of the importation
of Algerian sheep without danger of infecting French territory.
The sheep to be imported receive the serum three weeks
before shipment. Inspection eliminates those which were
incubating the disease at the time of inoculation, and which
might therefore be shipped while still ill. This serotherapy is

1 Non-existent in Great Britain.— Z7vanslators’ note.

282 MICROBES AND TOXINS

of advantage to the Algerian breeders, and helps in the
suppression of the disease.

Cattle plague or rinderpest is an epizootic disease which
has caused incalculable losses throughout Europe, especially
by attacking the herds of pure blood. The mortality always
exceeds 50 per cent. The microbe is unknown, but it is
known that the virus can pass through filter bougies, even
the finest: it is therefore an ultramicroscopic organism. An
animal which survives possesses an immunity which seems
quite invincible: as soon as the illness is over any quantity
of virulent blood may be injected into it without killing it.
But it is not possible to immunize by a method similar to
that in sheep-pox, for inoculation produces an attack as severe
as the naturally acquired disease. _

The animals may be made to acquire an immunity lasting
for four to six months by injecting the bile of animals dead of
the plague, but the method is not practical.

A good serum can be obtained by “charging ” with virus an
animal which has recovered. The sero-vaccination of Kolle
and Turner consists in injecting simultaneously this serum and
virulent blood: they must be injected at different spots.

It is always risky to employ a virus in therapeutics : there is
always the danger of spreading it or introducing it into a
country where it does not hitherto exist. The serum alone
suffices for prophylaxis, for treatment, and for the prophylaxis _
of the healthy animals. The prophylactic dose is one of
50c.c. Capable veterinary. surgeons have succeeded by using
the serum alone in reducing the mortality to 12 per cent.
(Nicolle). The same principles are at work in the treatment of
the horse-sickness of the Transvaal and the plague of pigs, hog-
cholera, which are also septicemic diseases due to an ultra-
microscopic virus.

SENSITIZED VACCINES.

The vaccines employed against typhoid fever, cholera and
plague may be rendered less painful, more prompt, more
powerful and more durable by impregnating the bacteria of

VACCINES AND SERA 283

which they consist with the corresponding anti-sera
(Besredka). After soaking in the serum the bacteria are
washed free from all trace of it: they must only retain what
they have been able to fix: there must be no free serum.

These qualities of such vaccines are due to the immune
body which they have fixed.!

PuHAGocytTic THERAPY.

When the leucocytes are counted to determine the
diagnosis and prognosis in certain infectious diseases it is
really a case of measuring to some extent the reactions and
natural means of defence of the body: the laws of phagocy-
tosis are being applied. It was natural not to stop at
recording these phenomena, but to proceed to active inter-
vention by attempting to modify, fortify or direct the
phagocytic apparatus.

The great danger in surgical operations, especially on the
abdomen, is an infection of the field of operation: formerly
it was the custom to flood this with antiseptics ; the bacteria
were destroyed but the tissues were injured. Nowadays not
only has antisepsis given place to pure asepsis but the attempt
is made to summon to the field of operation, particularly to
the peritoneum, legions of the cells capable of taking up
bacteria and healing wounds. For this purpose it is
customary to inject into the abdominal cavity either blood
serum (warmed to body temperature), or substances which

} The association of an antirabic serum with the injection of the
emulsions of spinal cord may be regarded as a sero-vaccination or as
treatment with a sensitized vaccine.

Such a serum cannot be relied upon to prevent rabies in animals: still
less can it furnish the basis of treatment for man. But it has been
employed with success in conjunction with the wirus fixe of the Pasteur
treatment to induce the greatest possible absorption by the body. In such
virus-serum mixtures it is only the virus which tmmunizes: the serum
merely favours absorption. An excess of serum would be actually
injurious and must be avoided.

In man the virus-serum treatment is employed i in those cases where the
treatment has to be rapid, z.¢.., when the patient presents himself long after
the bite or where the injuries have been very serious (A. Marie).

284 MICROBES AND TOXINS

exert a positive chemiotaxis on the leucocytes, e.g., nucleic
acid.

Again, in the method of Bier, which consists in cupping and
surrounding with elastic bands to retain a large volume of
blood around an abscess, a boil, or other acute infection, it is
also the phagocytes which are called upon, for the artificial
cedema which is produced is accompanied by an afflux of
leucocytes (Schimodaira’s experiment).

Wright’s method of vaccino-therapy consists in treating
infections by means of inoculations of bacterial bodies (the
bacteria of the infection itself), in order to increase the
opsonic power of the serum (v. Chap. X.); the treatment
would not be applicable without the quantitative and quali-
tative control in which the phagocytes are employed.

For some -years the study has been going on of those
chemical actions capable of stimulating phagocytosis and
reinforcing the phagocytes. Weak solutions of quinine
(o'002 per cent.) increase the phagocytic power, whereas
solutions fifty times stronger produce the opposite effect.
The peptones have been recognized to be powerful stimulants.
According to quite recent experiments, it will be necessary to
add to these iodoform and various chemical substances which
are soluble in fats, and which doubtless act in virtue of their
consequent power of penetrating protoplasm in solution in the
lipoids. These substances are true chemical sé/mulins.

It is possible that a new and most interesting chapter is
about to open in therapeutics with the discovery of Hamburger
among others: the action of certain mineral springs is said
to be due to their power of exciting phagocytosis (experiment
with the water of the Virchow spring in Wiesbaden).

Thus, chemistry and physics are far from taking possession
of medicine and relegating phagocytosis to the region of pure
speculation. On the contrary, the doctrine of phagocytosis,
which has already bloomed a second time in the purely
biological researches on opsonins and _ bacteriotropins, plays
the premier réle still as inspiration and explanation in the
study of the chemical therapeutic agents.

CHAPTER XV
CHEMICAL REMEDIES

CHEMIOTHERAPY

Ancient origin of chemical therapeutic agents—‘ Sterilization” of the
infected animal—Protozoal diseases : example of quinine—Association
of chemical research with animal experiment.

The arsenical bodies and ‘‘ 606” or Salvarsan.

Progress in the treatment of sleeping-sickness and syphilis—-Observations
on immunity in Protozoal diseases. :

Strains resistant to drugs—Strains resistant to sera—The future of chemio-
therapy.

CHEMICAL therapeutics is old as the world. It fills our
pharmacopeeias, still further enriched by organic chemistry.
But all over it furnishes only symptomatic remedies: one
induces sleep, another stimulates the heart, others deaden pain.
The drugs which really cure are easily counted, few exist
besides quinine and mercury.

Chemical therapeutics of to-day is engaged in searching
for other examples of the order of quinine and mercury, drugs
capable of destroying pathogenic microbes without injuring the
cells of the body. In theory and practice it is chemistry
applied to therapeutics.

Such remedies should resemble more or less the antiseptics
and should have as function sterilization. When the antiseptic
properties of corrosive sublimate became known, R. Koch
tried to inject it into anthrax-infected guinea-pigs to destroy
the bacteria, but the animals died. This simple experiment
puts the problem well before us with all its difficulties.

Corrosive sublimate cannot be employed to “sterilize” a
285

286 MICROBES AND TOXINS

living creature because it kills the cells of the organs at the
same time as the bacterial cells. Quinine is a good thera-
peutic agent because it destroys the parasites of the red
corpuscles without acting—at least in general—on: the
corpuscles themselves.

There are cases where the sterilization of a tissue may be
performed without the least inconvenience. Mercury is the
traditional remedy in syphilis: its internal application is
limited by its toxicity ; but after a contact possibly dangerous,
it may be applied as a prophylactic to the skin. Metchnikoff’s
experiments on the chimpanzee and on man _ have proved
that simple rubbing with calomel: ointment sterilises the skin
where the microbe of syphilis has just been inoculated and
prevents infection. This preventive treatment, which is as
efficacious as it is simple, is already practised.

Origins.—The renaissance of chemiotherapy has had
several causes. The first is the revolution due to Pasteur’s
teaching. When a disease is found to be due to a germ, to a
germ which is known and which can be made to transmit the
disease by inoculation into a laboratory animal, then there is
room for experimental therapeutics. Animal experiment
permits us to go much further than clinical observation.
Nevertheless, the progress of microbiology at first seemed about
to throw drug therapy into the background. Marvellous
biological remedies were discovered, both preventive and
curative, surpassing in specificity and efficacy all the drugs of
the Pharmacopeeia, e.g. the Pasteur vaccine against anthrax,
vaccination against rabies, and all the serotherapies. The idea
at once suggested itself that all infectious diseases might be
treated in similar ways.

But that has not proved to be the case ; there is a group of
infectious diseases against which vaccination and serotherapy
have achieved nothing or almost nothing,-the protozoal diseases.
In no case has the serum of an animal susceptible to the
trypanosome of sleeping sickness, and immunized against this
microbe, been capable of exerting a curative effect upon the
experimental disease in other animals. In malaria, all the

CHEMICAL REMEDIES 287

biological methods have failed, and quinine remains the
sovereign remedy. Hence, instead of searching for a vaccine
or a serum in protozoal diseases, the search has been directed
to finding an equivalent of quinine for each. .

When fortune favours, the history of cinchona bark and
quinine may be recapitulated. Chance or an empirical dis-
covery or a tradition of unknown origin, then enriches
humanity with a remedy on which chemists and experimenters
exercise their talents. This is the history of the recent arsenical
remedies.

The discovery of the microbes and the necessity of dis-
covering for protozoal diseases other remedies than vaccines
and serum, have been aided by the development of chemistry
itself, and especially of industrial chemistry. The great aniline-
dye factories have been the chief furnishers of the laboratories
of experimental medicine. ‘The remedies tried have been
varied as the dyes are varied, and in many cases chemiotherapy
has been really a chromotherapy.

The method of research consists in associating experiments
on the living animal with the reactions of organic chemistry.
A certain body endowed with a certain property is known ; the
molecule of this body possesses a principal nucleus on which
are grafted secondary atomic groups; it is found that the
property desired depends upon one of these groups ; this group
is then shifted about, varied or substituted by another group ;
experiment then informs us as ‘to the properties of the
new compound and as to the relations between molecular
structure and therapeutic effect.

Ehrlich has rendered great services to medicine by combining
more closely than had ever been done, the technique of the
chemist with the zz v’ve experiments of the biologist. His
guiding principle, which dominates his whole career and is to
be found in his earliest works, is the attempt to explain vital
actions by a similar mechanism as in the reactions of organic
chemistry. He has represented by stereochemical formule the
reciprocal reactions of bodies of the chemical composition of
which we are almost entirely ignorant, the toxins and antitoxins.

288 MICROBES AND TOXINS

He has disarticulated protoplasm into atomic groups, both
structural and functional, which act like the organs of the
cells; he has extended the anatomy and physiology of the
tissues and organs by imagining a sort of molecular anatomy
and physiology for the cell.

The cell is a microcosm in which multiple functions take
place by continual exchanges between the protoplasm and the
external world. Each of these functions is represented by a
group or “side-chain,” capable of entering into (chemical) com-
binations with corresponding groups in foreign substances,
food-stuffs, poisons, and also drugs. ‘ All the vital phenomena
can be reduced to a series of such exchanges, which are nutri-
tion phenomena and retain this character, whether it is a
protein which is being assimilated, or a serum producing
immunity, or quinine killing the malaria parasite.

For a substance to act on a cell, whether an organ cell or a
microbe, it must fix itself on the protoplasm. Did not a
philosopher in the middle ages declare that drugs ought to
haye points or hooks to enable them to seize upon the organs ?
There is scarcely any phenomenon which haunts the mind of
the biologist more than this fixation, which, according to some,
is a physical phenomenon, one of molecular adhesion, according
to others the chemical interplay of the side-chains.

The antitoxin injected into a patient has affinities only for
the corresponding toxin. But the arsenic which we inject into
an individual suffering from sleeping-sickness possesses affinities
both for the parasites and for the cells of certain organs. Itis
a double-edged weapon. We must suppress, or at least atten-
uate as much as possible, the dangerous affinity in favour of
the useful one, a problem in chemical substitution. The task
is chiefly met with in connection with protozoal diseases, but
it is not impossible to aim in a similar fashion at the bacterial
infections.

We already know examples of these, we may call them,
elective affinities. Ehrlich himself showed long ago, that
methylene-blue possesses a special affinity for the living nerve-
fibres ; there has even been derived from this zz vivo staining, a

CHEMICAL REMEDIES 289

valuable method for anatomical analysis as follows: the
animal is injected with methylene-blue and is then killed, and
one finds in sections the nerve-fibres as fine blue lines. By
injecting the same dye into a frog, the nervous system of the
parasites of its body-cavity can be stained in the most delicate
way. There are in cells, granules which are stained electively
by neutral-red, others by pyrrol-blue. One colour is taken up
by the nerve-cell, another by the fatty material, and these
stains are more or less specific. Drug treatment ought to be
imagined in the same way. To cure syphilis, it is necessary to
find a chemical compound which will “stain” electively the
parasite of Schaudinn without staining the cells.

It is curious that we find here again this analogy of staining,
so often brought forward by both Ehrlich and Bordet, but with
different significations. It is still too soon to ask which is true,
the chemical or the physical theory.

The great merit of a theory is its usefulness, and Ehrlich’s
theory has certainly stimulated much work. It is not necessary
to retain all the scaffolding once the house is built.

The new impulse of chemiotherapy is scarcely six years old.
Research has followed different tracks and attached itself to
different chemical substances: hence there arose several
methods in which various remedies were soon combined and
alternated.

Here is a list of the principal remedies tried against the
protozoal diseases.

1. Zrypan-red and the benzidine colours.

2. The Triphenylmethane series: malachite-green, brilliant-
green, crystal-violet, victoria-blue, parafuchsin, tryparosane.. . .

3. Antimony, tartar emetic, etc. In historical and logical
order this series was studied after arsenic and atoxyl, the
chemical relationship of antimony and arsenic suggesting this
trial. Tartar emetic acts on the parasite of sleeping sickness :
it has been employed in man. Salmon has observed that it
acts—slightly—in syphilis.

4. The arsentcal bodies and atoxyl. Arsenic is one of the
most ancient remedies. Even before Dioscorides and Pliny,

U

290 MICROBES AND TOXINS

the Chinese had employed it. As soon as it was found that
sleeping sickness had as its cause a trypanosome, the arsenical
treatment was applied to it. But arsenious acid was too toxic
to be easily employed, and the discovery of trypan-red and the
first successes reported with the chromotherapy might have led
to its abandonment, had not W. Thomas brought again into
prominence the substance afoxy/in 1905. Atoxyl, discovered
by Béchamp in 1863, hardly entered the sphere of human
medicine until 1902, when it was employed in dermatology,
both by intravenous and subcutaneous inoculation. Thomas
has the credit of employing it against sleeping sickness.

It is not, as was first thought, metarsenical aniline, which
contains 37°7 per cent. of arsenic. According to Ehrlich and
Bertheim, it is the monosodic salt of paraminophenylarsenic
acid, but it is still the compound discovered by Béchamp.

It contains 24 per cent. of arsenic. It is a white, crystalline
powder soluble in water and easily sterilized. It may also be
applied as an ointment. Although almost thirty times less
toxic than arsenious acid excessive doses produce nephritic
symptoms and above all disturbance of vision reaching even to
complete blindness.

Atoxyl has been employed in the trypanosome infections
and in several spirillum diseases, such as recurrent fever and
syphilis: it exerts both a prophylactic and a curative action.
It has been said that it no longer acts in the trypanosomes in
sleeping sickness once these have penetrated into the cerebro-
spinal fluid: but according to several observers the meninges
are in general quite permeable (L. Martin). JZ vitro it acts
neither on trypanosomes nor on spirilla, so that the body itself
must play an active part in the treatment.

Atoxyl would be a perfect remedy were it non-toxic. By
ringing the changes on the chemistry of this arsenical subject
a series of compounds have been discovered of which the last
is 1,500 times less toxic than the first.

Three compounds superior to atoxyl have been discovered
in this series: in the first place arsacetin, which is four times
less toxic than atoxyl for the mouse and distinctly less toxic

CHEMICAL REMEDIES 291

for the monkey; secondly, arsenophenylglycin (arsenophenyl
glycocollate of sodium), two to four times less toxic than
atoxyl and more active in killing the parasites and in pro-
phylactic power. Quite recently Strong and Teague in the
Philippines have been treating surra in horses with arseno-
phenylglycin on the scale of veterinary practice and not of
laboratory experiment, and have cured more than one-third of
the animals, besides checking the epidemic. In the series of
bodies studied by Ehrlich arsenophenylglycin had the number
418. Finally we reach the body numbered 606, the dzoxy-
diamidoarsenobenzol (C\,H,,O,N,AS,), of which the hydro-
chloride is employed as the celebrated remedy termed “606”
or Salvarsan.

5. The toxicity of these chemical remedies, the necessity of
treating relapses and the fear of rendering habituated to the
drug both the patient and the parasite, have suggested the
method of combined or more correctly alternated medication
(Laveran). The drugs associated may be of the same series
or of a different chemical series from the principal drug. In
spite of the good qualities of atoxyl arsenious acid still retains
the first place in the alternated treatment.

Treatment of Sleeping-Sickness.—From 1906 on-
wards atoxyl was the remedy most employed in the treatment
of sleeping-sickness. R. Koch during his sojourn in the Sese
Islands on the Victoria Nyanza, gave half a gram two days in
succession: in about eight hours the parasites disappeared
from the blood and from the swollen glands. They remained
absent for about ten days, when the injections were repeated ;
without inconvenience this double injection was continued
every six days for from four to six months.

Koch observed further that on ceasing the injections the
parasites reappeared in the glands after a minimum period of
eleven days; from the twenty-fifth day they could be found in
about 25 per cent. of the patients treated as above ; they then
disappeared and after the sixtieth day were not to be found
again. But the disappearance from the glands was not a final
cure: there were relapses. The prolonged treatment, how-

U2

292 MICROBES AND TOXINS

ever, permitted the campaign of prophylaxis; the patients
thus treated had no longer parasites in their blood and could
not therefore furnish the glossina carriers with new suppli¢s of
virus. :

Mild cases were cured after a treatment of from four to six
months. At the end of 1907 Koch calculated that the death-
rate among the treated individuals was between a tenth and a
twentieth of what it had been among the non-treated.

Relapses occur even among the patients treated from the
earliest period, and even 14 months after the treatment ceases.
The more intense the treatment the later the relapse.

Soamin (atoxyl with one molecule of water in addition) was
apparently rather less toxic than atoxyl; arsenophenylglycine
produces pain at the point of injection, but it seems to act
in patients in whom atoxyl fails.

Of the remedies supplied by other chemical groups the best
hitherto is tartar-emetic injected intravenously. It ought to be
tried when atoxyl fails and the association of atoxyl with tartar-
emetic has often seemed better than either of these drugs
alone. With one injection per week it is possible to keep the
blood and glands free from trypanosomes. The combinations
atoxyl+ mercury and atoxyl+sulphide of mercury (orpiment)
have been tried ; atoxyl remains the basis of all the remedies.

The Treatment of Syphilis——Experimental study of
syphilis is quite recent and dates from the announcement of
Metchnikoff and Roux that the disease can be inoculated with
certainty in anthropoid apes, presenting not only the primary
symptoms, but secondary symptoms as in man. By his
discovery of the specific microbe, ScMaudinn furnished us
(1905) with the best means of controlling under the microscope
both inoculation and treatment. For centuries the idea has
prevailed that syphilis is exclusively a human ailment ; since
1903 it has been transferred from man to the higher apes and
from these to the lower, and now it has been inoculated in
rabbits and guinea-pigs. Man however remains the chief.

Six years ago the medical world would have been astonished
at a prediction that syphilis and the trypanosome. diseases

CHEMICAL REMEDIES 293

would one day be treated practically by the same remedies.
Yet analogies were already in existence to act as a4 guiding
principle, for the name of horse-syphilis had long been given
to a trypanosome disease, dourine, which possesses clinical
resemblances. It was Schaudinn, again, who maintained
from his study of the facts the relationship between trypano-
somes and spirochetes ; he was convinced that a protozoon
cannot be properly known until its biological cycle has been
followed completely, and he therefore studied the hzematozoa
as a zoologist and found in the development of the same
species trypanosome forms and spirochetes. When the
spirochzte of syphilis came under his eye, he was not only
prepared to see it but to believe it and maintain it, There
was great discussion in the scientific world as to the relation-
ships of this new microbe: was it a protozoon or a bacterium ?
These researches had the happy result of drawing attention to a
problem which concerns medical practice much more closely
than was guessed, and they prepared the way for the revolution
in the treatment of syphilis.

This sprang from an old idea. In an article dated 1867 in
the Dictionnaire des Sciences médicales, one may read that arsenic
occasionally succeeds in completing a cure after mercury and
iodine: “it is especially the symptoms which have resisted the
former treatment which yield to the action of arsenic.”

The arsenical treatment of syphilis is an adaptation of the
arsenical treatment of the trypanosome infections, inspired by
the ideas of Schaudinn and the labours of Ehrlich.

Salmon was the first to undertake in Metchnikoff’s laboratory
methodical studies with atoxyl. The well-known toxicity of this
substance confined him to small doses. Arsenophenylglycine,
employed by Alt in general paralysis, was found to produce
temporary improvements. It was in December, 1909, that
Ehrlich mentioned for the first time a new substance con-
taining the same arsenobenzol group as arsenophenylglycine,
namely, the dioxydiamidoarsenobenzol, famous to-day under
the name of “606” or Sa/varsan.

All that has been published hitherto on Ehrlich’s remedy con-

294 MICROBES AND TOXINS

firms the great progress it has realized. It is to be hoped that
the future will confirm these promises and make of this drug
one of the great victories of therapeutical science.

Observations on immunity in Protozoal Diseases.
Strains resistant to Sera. The future of Chemio-
therapy.—To chemiotherapy, also, we owe much new
knowledge on the immunity to the protozoal diseases, and
on immunity in general.

The question of the dose is here of capital importance ; the
object is to begin and to finish the treatment at one blow, but
it must be a sledge-hammer stroke. Small doses cure for a
time without producing immunity, and relapses therefore occur.
In the treatment of a relapse the trypanosome is found to be
different. Sometimes it becomes more sensitive to the action
of the drug. Sometimes it gives the impression “that as the
result of the absorption of those parasites killed by the atoxyl,
the body acquires a degree of immunity which then interferes
with their normal development” (Ehrlich). Asa rule, however,
such immunity is ephemeral.

But what is most commonly observed in following the course
of a first attack on to a relapse, and again from one relapse to
another, is a diminution in the efficacy of the remedy. The
question is, is it the body or the parasite which has changed ?
We find it is the parasite, for when inoculated now into a fresh
animal it still resists the same drug. The injection of a drug
in small doses is in fact the best way to render a trypanosome
resistant to it, and further to create a strain of trypanosome which
resists the remedy and preserves this resistance even after
hundreds of passages, transmitting it as an acquired character.

Trypanosome strains have been created in the laboratories
resistant to trypanred, to benzidine dyes, atoxyl, and tartar-
emetic. The resistance is a group resistance, z.¢., it applies to
all drugs of the same series or chemical family: a trypanosome
resistant to trypanred is still sensitive to the arsenic bodies ; it
resists, however, also the benzidine dyes, which are a good deal
different from trypanred. It can be seen by testing various
drug-groups against the same resistant strain that biology agrees

CHEMICAL REMEDIES 295

with chemistry in putting tartar-emetic, 7.¢., antimony, in the
same group as arsenic.

The resistance acquired towards the bodies of one group
presents, however, different degrees; thus, in the arsenical
group a strain resistant to atoxyl is still sensitive to arseno-
phenylglycine ; if it is now rendered resistant to this latter
substance it still remains sensitive to tartar-emetic and arsenious
acid. Again subjected to arsenious acid it becomes resistant
to tartar-emetic; no strain, however, has yet been created:
resistant to arsenious acid.

These observations have important consequences for practice ;
they indicate that it may be necessary to attack the same
trypanosome by different remedies and form the reason for.
the method of combined or alternated drug treatment. The
drugs superpose their actions on the parasite, but not necessarily
on the body, because their toxicity does not bear entirely on
the same organ cells. Further, the different degrees of resis-
tance which exist towards drugs of the same group show that
it is proper to associate atoxyl and arsenious acid, although
they are both arsenical bodies. Attacking the same parasite
with several remedies is doing the same, says Ehrlich, as the
entomologist who pins out a butterfly with several pins.

Although the difficulty may be overcome by using these
combined treatments the appearance of resistant strains is
always adanger. In sleeping-sickness, for example, there is only
one good drug, atoxyl, to destroy the trypanosome, and in
presence of strains resistant to this, one is rather helpless, the.
other remedies being either too weak or too toxic. The
danger would be aggravated should the trypanosomes preserve.
hereditarily their acquired resistance through their passages in
the tsetse-fly which conveys them. It would represent the
creation of a more virulent and less curable disease, not only in
an individual, but throughout a whole country.

Things do not go on in the living body exactly as in a
test-tube. This fact, so often referred to already, must be.
insisted upon again.

There are certain chemical substances which act on the

296 MICROBES AND TOXINS

trypanosome neither 7” vitro nor iv vivo ; others are active in
both, for example, tartar-emetic.

Methylene-blue kills certain spirocheetes in the test-tube in
a dilution of 1 in 6,000,000, but has no action in the body of
a mouse even when in 500 times greater concentration.

Atoxyl does not act zz vitro but acts in the living body.
There are even substances which in the body stimulate the
multiplication of certain parasites ; one must here reckon with
the body as a factor as well as the parasite, and the problem
of the best drug to use both in the first attack and in relapses
is a new one for each species of trypanosomes, and for each
species of animal: chemical therapeutics is therefore not
likely to become more simple.

When an animal acquires immunity towards a bacterial
infection, there develop, as we have seen, aztzbodtes, as manifested
by new properties of the serum, bactericidal, preventive, and
curative. Is it the same in protozoal infections? Do the
chemical remedies induce the formation of antibodies ?

The serum of animals infected with trypanosomes possesses
microbicide and preventive properties, weak it is true, but
definite ; they appear in the course of chronic or subacute
infections. There is reason to believe in the presence of an
immune body analogous to those known in antibacterial
immunity ; it acts by inducing a phagocytosis of the parasite.

Strains of protozoa may arise resistant to the serum of
animals, infected or provisionally cured, just as with the
chemical remedies; in many cases it is a true variety forma-
tion, for the acquired character can be transmitted through
several generations (counted by mouse passage).

The antibodies in animals infected by trypanosomes originate
doubtless from trypanosomes destroyed and absorbed. At the
same time, the rapid destruction of a great number of the
parasites may flood the body of the host with substances which
act as poisons—a sort of endotoxins. In a man suffering from
sleeping sickness, a disappearance of the trypanosomes under
the influence of atoxyl is often followed by an attack of fever
(L. Martin).

CHEMICAL REMEDIES 297

A dose of a chemical remedy, itself non-toxic, sometimes
kills the mice while destroying their parasites. Ehrlich warns
against treating with “606” infants suffering from the
septicaemic form of congenital syphilis; he fears an intoxica-
tion with substances derived from the suddenly dissolved
spirocheetes,

The paradox that a patient may give a negative Wassermann
reaction which after treatment with “606” becomes positive, is to
be explained, according to Ehrlich, by the production of anti-
bodies as a consequence of a non-curative dose. And it isa
local production of endotoxin, under the influence of arsenic
or mercury, which produces the sudden revival of cutaneous
eruptions in syphilis, known as the phenomenon of Herxheimer.

These observations on strains resistant to drugs and to sera
have thrown light on the question of the virulence of
protozoa. Bacteria also acquire resistance to the defensive
powers of the higher animals; the streptococci and the
anthrax bacilli clothe themselves with a mucous capsule ;
the typhoid bacilli become insensitive to the agglutinins of
the patient whom they are infecting ; other bacteria secrete
agressins; all these are manifestations of resistance. This
“ immunization of microbes” raises the thought that the laws of
virulence may be fundamentally the same for both bacteria and
protozoa.

There is nothing to exclude the possibility of a drug-therapy
in bacterial infections also, and it is always possible that our
vaccines of to-day, and in particular our sera, marvellous
discoveries as they are, and originating in the most scientific
empiricism, may give place to physico-chemical remedies better
defined and more direct. It is from chemistry that we have
to expect advances in the healing art. The phrase of Duclaux
is very apt in this connection.

“ With Pasteur chemistry invaded the field of medicine
probably never to leave it.”

GLOSSARY

Actinomycosis—an infectious disease affecting cattle and some-
times man, characterized by tumour growths of the Jaw, the
lungs, the tongue, &c., and due to a streptothrix, q.v. : known
popularly as woody- -tongue.

Algze—cellular cryptogamaceous plants including many seaweeds.

Amylase—a ferment capable of decomposing starch.

Ankylostoma—the “‘ hook-worm,” a parasite of the small intestine,
common in the tropics and among miners, and producing
severe anzemia, the ‘“‘ miners’ anzemia.”

Annelids—a class of Vermes, or worms, including the common
earthworm.

Ascitic fluid—the fluid producing abdominal dropsy.

Ascus—an enlarged cell of a fungus in which the spores are
developed, usually the terminal end of a hypha or thread,
Auto-intoxication—the poisoning of the body by materials developed
within itself: gout, arterio-sclerosis, &c., are supposed to be

auto-intoxications.

Basidium—the spore-bearing hypha (or thread) of a fungus.

Bothriocephalus—a broad tape-worm occasionally infecting man
and derived from the ingestion of certain fish in which its
cystic stage occurs.

Botulismus—the poisoning produced by the consumption of meat,
particularly in the form of sausages, in which an anaerobic
bacillus, the B. Botulinus of Van Ermenghem, has grown and
produced a toxin, the botulismus toxin.

Brownian movement—dancing vibratile movements seen among
minute particles, even of inert substances such as charcoal,
when suspended in a fluid and examined under the microscope :
it is not to be confused with true motility.

Calories—units of heat : large and small calories distinguished by
capital and small letters ; small calory is the amount of heat
necessary to raise I gram of water 1° centigrade : large calory
the amount necessary to raise 1 kilogram 1° centigrade.

299

300 MICROBES AND TOXINS

Carbohydrates—compounds of the three elements, carbon, oxygen,
and hydrogen. the chief representatives are the sugars and
starches.

Catalysts—substances which modify the rapidity of a reaction
without forming part of its final products: such a reaction is
termed “catalysis” (Berzelius, 1835): for example peroxide of
hydrogen, H,O,, is decomposed into water, and oxygen on
contact with spongy platinum, or platinum in a state of fine
division. The platinum remains unaltered: it has acted
merely by its presence.

Chemiotaxis—a sort of “chemical sense” by means of which living
cells seem to seek the substances favourable to them, and
avoid those which are injurious. Positive and negative
‘chemiotaxis are indicated by movements of attraction and
repulsion respectively, z.e., by approach or flight.

Chromatin—the substance composing the greater part of the
nucleus of cells, so-called because it stains very deeply with
certain dyes.

Chromidia—masses or networks of chromatin distinct from the
nucleus itself.

Colloids—Graham gave the name of “colloids” to those substances
which in watery solution do not dialyse (ze, do not pass
through a parchment membrane dipping in pure water), or
dialyse extremely slowly in contrast to the “crystalloid”
substances which rapidly dialyse. Types of these two classes
are’gum and salt. Nowadays it is more usual to speak of
“substances in the colloidal state” than of ‘colloidal sub-
stances.” The “colloidal state” is a state of suspension of one
substance in another as contrasted with true solution. For
example gutta-percha forms a true solution in alcohol, a
colloidal solution in water. In the animal body the cells, the
cell-membranes, and the body-fluids all consist of colloidal
solutions, the condition and activities of which are regulated
by physico-chemical laws: the colloids are thus of extreme
interest to biologists.

Conidium—a spore of a fungus produced asexually and borne on a
special branch.

Cytoplasm—the protoplasm of the cell-body as distinguished from
the nucleus, the protoplasm of which is known as nucleo-
plasm.

Daphnia—the common water-flea.

Dialysis—wide Colloids.

Diapedesis—a phenomenon discovered by Cohnheim : the white
corpuscles of the blood (leucocytes) emigrate from the interior
of the blood-vessels by a process of active movement not
passive expulsion. They insinuate themselves between the
cells forming the walls of the capillaries, and drag themselves
through as narrow threads to recover their rounded shape
outside.

GLOSSARY 301

Diastases—also known as fermends or enzymes: they are sub-
stances which can produce fermentations in the absence of
living cells : their chemical nature is unknown and they are
defined simply by their activities: they occur in the form of
organic substances of indefinite composition which are soluble
in water. In the embryo of grain (barley, &c.) there exists a
diastase which transforms the starch of the grain into sugar in
the presence of warmth and moisture, the process of “ malting.”
(Vide Fermentation.)

Electrolytes—bodies which in solution break up into “ions”
carrying electrical charges which are equal and opposite. For
example, common salt in solution in water breaks up into a
sodium ion carrying a charge of negative electricity, the cation
and a chlorine ion carrying a charge of positive electricity the
anion. The presence of electrolytes in water renders it a good
conductor of electricity, hence the name.

Enzymes—vide Diastases.

Epizootic—infectious disease occurring widespread among animals
—contrasted with “ epidemic” in which the disease affects
man.

Fats—compounds of carbon, oxygen, and hydrogen in the form of
“esters” of glycerine, z.e., compounds of glycerine with a fatty
acid. Palmitin, stearin, and olein are fats in which glycerine
is combined with palmitic, stearic, and oleic acids. The
natural fats are mixtures in varying proportions of palmitin,
stearin, and olein. The “saponification” of fats is their
decomposition into the two elements glycerine and fatty acid,
and the combination of the latter with sodium or potassium
(“salting out” in the language of soap manufacture). Soap is
thus a salt of sodium or potassium with a fatty acid, e¢.g.,
stearate of sodium. By saponification 100 grams of fat can
furnish 90 grams of fatty acid.

Fermentation—a chemical transformation produced by the action
of living cells, ¢.g., the cells of the yeast of beer, or by the
action of the secretions of these cells (diastases), ¢.g., zymase
extracted from yeast cells. A typical fermentation is that of
sugar by yeast in which the sugar is broken up into carbonic
acid, water, and alcohol.

Fibrin—a protein (more exactly, a globulin) which forms the clot in
coagulated blood. It does not exist in circulating blood in the
living animal, but is derived from a different substance which
exists in this and is termed fidrinogen. “ Defibrinated” blood
is blood: from which the fibrin is removed as it forms by
whipping with a bunch of twigs immediately after the blood is
drawn. Such blood is no longer capable of coagulation.

Flora—the aggregate of plants growing without cultivation in a
given district or indigenous to a particular geological formation :
hence applied to the aggregate of bacterial species inhabiting
the intestine, the mouth, &c.

302 MICROBES AND TOXINS

Gametes—sexually differentiated cells which unite to form the
fertilized cell, the zygoée.

Glucosides—substances which are capable of decomposition into a
sugar (glucose), and various other organic substances, alcohol,
phenol, aldehyde, &c. For example, amygdalin the active
principle of bitter almonds breaks up into glucose benzaldehyde
(the odorous constituent) and hydrocyanic (prussic) acid.
Many poisonous plants contain glucosides as their active
principle.

Glycogen—a carbohydrate analogous to the starches, stored by the
body in the cells of the liver and muscles, and drawn upon by
the body for the supply of sugar consumed by the cells during
muscular work and other activities.

Humours—body-fluids, for example the aqueous and vitreous
humours of the eyeball.

Incubation—the time elapsing between the moment of penetration
of the body by a virus (microbe or toxin) and the moment when
the first symptoms appear.

Indol—Among the products of the digestion and putrefaction of
proteins (q.v.) there appears a substance, ¢ryptophane, from
which the indol bodies are derived, zzdol and skatol (indicated
by various colour reactions depending upon their relationship
to indigo) : also a substance, ¢yvosin, from which are derived
paracresol and phenol (carbolic acid).

Inflammation—the reaction of living tissues to injuries and
infections. The essential fact in inflammation is the activity
of cells, the phagocytes which are capable of taking up
substances and digesting them.

Isomeric—with different chemical or physical properties but
containing the same quantity of chemical elements in the
molecule: the difference is due to the different arrangement in
space of the atoms in the molecule.

Lipoids—a group of substances such as lecithin, cerebrin, protagon
cholesterin, possessing certain of the properties of fats (Aurov—
fat). The name was introduced by Overton to indicate the
possession by these bodies of powers of solution similar to fats,
in particular for anesthetics. According to Overton the outer-
most layer of protoplasm in a cell consists of lipoid substances
and the cell absorbs only those materials which are soluble in
the lipoids.

Medulla—the medulla oblongata or bulb is the continuation of the
spinal cord within the skull before its junction with the brain:
it contains the nuclei of certain cranial nerves regulating the
respiration and the heart-beat.

Molluscum contagiosum—an infectious skin disease characterized
by small teat-like protuberances.

Mucedineze—fungi of the group to which the ordinary white mould
mucor mucedo belongs.

GLOSSARY 303

Mutations—sudden variations appearing in individuals of one
species and capable of transmission to their descendants.

Mycetozoa or Myxomycetes—protozoa with naked protoplasm
occurring on damp surfaces in the form of jelly-like masses,
living on organic debris, amcebiform without mycelium but
later plant-like.

daar i multinucleated cell occurring in the marrow of

ones.

Nematodes—a class of worms with thread-like body, mouth and
intestinal canal, including the parasitic thread-worms, &c.
Neuroglia—cells in the nervous system which correspond to the

connective- tissue cells in other organs.

Nitrification—-the transformation of salts of ammonia (chiefly the
sulphate and the carbonate) into nitrites and nitrates under the
influence of the nitrifying bacteria.

Plasma—the fluid portion of the blood freed from the red and
white corpuscles which float in it: it contains fibrinogen but,
not having been allowed to coagulate, no fibrin (véde Fibrin).

Proteins—substances resembling egg-white and containing nitrogen,
carbon, oxygen, hydrogen, and sulphur.

Ptomaines—products of putrefaction, some of them poisonous,
isolated from putrefying organic matter (first discovered in
dead bodies).

Putrefaction—the decomposition of protein substances by microbes
and their ferments with the production of gas, foul smells, and
sometimes poisonous substances: as a result of putrefaction
organic matter is restored to the state of inorganic elements.

Septic—derived from oemois—corruption and applied to material
which can produce or undergo bacterial infection, ¢.g., a septic
wound, a septic instrument, a septic dressing. Hence the
words “antiseptic,” a substance acting against this action, and

« “aseptic,” the absence of sepsis. In modern surgery antisepsis
has given place to asepsis.

Serum—the clear fluid expressed from the clot of coagulated blood :
it represents the fluid portion of the blood minus the cells and
minus the fibrin which forms the clot (véde Fibrin).

Stereochemical—the chemistry of matter may be treated as
depending on arrangements of the atom in space in the
molecule (vzde isomeric).

Sucrase—sugar-splitting ferment.

Symbiosis—the living together of dissimilar organisms each
dependent on the other: the best examples are the lichens

. where a fungus and an alge live together.

Urease—urea-splitting ferment: producing ammonia from the
urea, the main nitrogenous constituent of urine.

Vagus—the nerve supplying the heart, lungs, and stomach.

Vitalism—the modern application is to a theory which postulates,
at least provisionally, some other activity in the life processes

304 MICROBES AND TOXINS

than mechanical, physical, and chemical forces. The theory
of immunity as being due to phagocytosis calls in the activity
of living cells the mechanism of which is not known. It is
certain, however, that in vital activities it is not necessary to
assume anything beyond physico-chemical phenomena which
will some day become clear. Biologists no longer talk of a
“vital principle.”
Zymase-——vzde Fermentation.

INDEX

INDEX

ABRIN, 168
Acidity, inhibiting power, 10, 42
Acidophilus, 35
Acinetians, 121
Actinians, venom of, 178, 234, 238
Actino-congestin : see Congestin.
Adulteration, detection of, 255
Aerobes, 80
in fermentation, 5
in putrefaction, 10
Agglutination, Bordet’s
tion, 221
diagnosis by, 252
Agglutinin, development of, 193
power of, 253
Agressins, 231
Albuminous materials, putrefaction

explana-

ol,
Alcohol production, 82
Alexine: see Complement.
Algze, 299
bacteria relationship, 63
coals, 7
nitrogen fixation, 20
Algal coals: see Bogheads.
Amboceptor : see Immune-body
Ameebee, culture, 105
digestion, 99
excretion, IOI
infection among, I21
inflammation in, 134
pathogenic, 142
Amylase, 299
Ancerobes, 80
in fermentation, 5
in putrefaction, 10
Anaphylaxis, 233
Ankylostoma, 299
Annelids, 299
“¢ Antagonistic force,’ 10

Anthracosis, 47, 127
Anthrax, immunity, 207
infection, 122
vaccination, 270
bacillus, antagonistic microbes,
125
encapsulation, 57
involution forms, 59
light effect on, 96
lysin, 87
phagocytosis of, 196
respiration, 84
virulence, 117
virus attenuation, 119
Antianaphylaxis, 242
Antibodies, 193, 248
immunity, 207
Antiendotoxins, 171
Antigens, 193
Bordet’s researches, 218
Antinomycosis, 299
Antisensibilism, 247
Antitoxins, 168, 210, 227
Apotoxine, 239
Arachnolysin, 179
Arsacetin, 290
Arsenic chemiotherapy, 289
Arsenophenylglycin, 291
Arrhenius immunity theory, 216
Arthraspores, 59
Arthus’ phenomenon, 234
Ascarides, respiration, IOI
Ascitic fluid, 299
Ascus, 229
Aseptic breeding, 30
Aspergillus, 52
Niger, fermentation, 5
nutrition, 73, 75
respiration, 84
zinc absorption, 165

x*

308

Atoxyl, 290
Auto-infection, 123, 88
Auto-intoxication, 299
Avian plague, 154
Axolotl, infection, 134
Azotobacter, 19

BACILLUS, 55: for bacilli of different
diseases see under disease.
acidi paralactici, 42, 44
acidophilus, 35
amylobacter, 6, 7
asterosporus, 64
bifidus, 35
chemical properties, 44
inhibiting power, 42
botulinus, 299, 37
toxin, 159
Biitschlii, nucleus, 65
reproduction, 68
_ Size, 150
coli, culture of, 89
in intestine, 34, 36, 37
mutation of, 62
para-, 115
putrefaction inhibited, 41
cyanogenes, pigment of, 92
exilis, 35
lactis erogenes, 34
putrefaction inhibited, 41
maximus buccalis, 65, 66
megatherium, 109
mesentericus, 35, 109
perfringens, inhibiting action, 42
in intestine, 34, 36
prodigiosus, pigment, 92
pleomorphism, 59
putrificus, Bienstock, 11
inhibiting action, 41
in intestine, 36
pyocyaneus, inhibiting
125
Pfeiffer’s phenomenon, 204
pigment, 93
radicicola, 21
ramosus, 58
TIL. of Rodella, 34
sporogenes, 36
sporonema, reproduction, 68
cold effect, 96
subtilis, involution forms, 59
respiration, 84

action,

INDEX

Bacteria, 55
alimentation, 77
chemical composition, 70
diseases, 107
heat effect, 94
light effect, 96
luminous, 89
nitrogen fixation, 19
organic cycles, 41
pleomorphism, 59
respiration, 80
toxins, 168
Bacteriaceze, 63
Bacterial beds, 17
Bacteriolysis, 194
Bacterio-purpurine, 85
Bacterio-therapy, 273
intestinal, 42
Bacteriotropins,
Bacterium aceti, 5
butyricum, respiration, 80, 84, 85
gammari, 64
phosphoreum, 90
sorbose, 79
zopfii, 60
Basidium, 299
Beggiatoa, 63
nutrition, 78
Behring, immunity experiment, 201
on intestinal permeability, 47
Bellamy, fermentation experiment, 82
Bengal rose, bactericidal action, 97
Bertrand, G., experiments” on
medium, 79
Besredka, anaphylaxis theory, 241,
247
endotoxins, 169
Bienstock, putrefaction experiments,
10, 41
Bier, phagocytic therapy, 284

‘Bitumen, bacterial origin, 3

Bleeding, post, 92
Blood, pathogenic microbes in, 251
Bloodstains, detection of, 255
Blue alge: see Cyanophycee.
Bogdanoff, aseptic breeding, 31
Bogheads, formation, 8
Bordet, Ch., agglutination, 254
chemiotaxis, 136
complement fixation, 256
hemolysis, 183
immunity theory, 218
pleuropneumonia, 154

INDEX

Bordet-Gengou reaction, 220
Borrel, anti-tetanic serum, 276
pleuropneumonia, 154
Bothriocephalus, 299
Botulismus, 299
Brownian movement, 299
Bruck, antituberculin of, 177
Buchner, H., antitoxin formation,
210
tubercle infection, 125
Bufotaline, 180
Bufotenine, 180
Bulgarian bacillus, 44
Biitschlii, on bacterial nucleus, 65

CAGNIARD DELA Toor, cold effect
on bacteria, 96
Calcicolee, 22
Calcifugee, 22
Calmette, antitoxin experiments,
212, 228
ophthalmo-reaction, 262
Carbohydrates,’ 300
fermentation, 5
synthesis, 3
Cattle plague : see Rinderpest.
Celluloses, fermentation, 5, 6
Centipedes, venom, 179
Cerebrin, 302
Charrin, immunity experiments, 200
Chemiotaxis, 300, 136
Chemiotherapy, 285
Cheyne, Watson, infection experi-
ments, 124
Chlorella, nitrogen fixation, 20
Chlorophyll, 3, 99
Cholera, anticholera serum, 278
infection, 122
vaccination, 271
vibrio, 40
agglutination test, 254
endotoxin, 170
flagella, 56
microbes favourable to, 125
phagocytosis, 202
pseudo-, 114
toxin, 171
virulence diminution, 117
Cholesterin, 302
lecithin reaction, 185
poisons reaction, 225, 226
Chonkewitch, immunity experi-
ments, 208

re

309

Chromatin, 300
Chromidia, 65, 300
Citron, immunity experiments, 208
Cladothrix dichotoma,
Clostridium, 55
pasteurianum, nitrogen fixation,

19
Coagulins, 248
Coal, bacterial formation, 7
Cobra venom, 181
Cocci, 55
Coccidia, life cycle, 103

parasitism, 104

Cochin, Denys, fermentation, 83
Cohen, microbial heat production,

89

Cohendry, aseptic breeding, 32
Cohn, stability of species, 60
Cohnheim, inflammation, 131
Colloids, 300

immunity analogies, 224
Comma bacillus, 40
Complement, 183

bactericidal properties, 194

Ehrlich’s theory, 215

non-specificity, 256

fixation, 256
Complementophil group, 215
Congestion, 178, 234

Richet’s researches, 238
Conidium, 300, 52
Conjunctis-reaction, 178
Conradi: inhibition, 88
Contagious diseases, 121
Costia necatrix, 103
Cow-pox, 267
Crenothrix polyspora, 78
Crepitin, 238
Crotin, 168
Cuti-reaction, 178
Cyanophycez, bacteria relationship,

63

nitrogen fixation, 20
Cystococcus, nitrogen fixation, 20
Cytodiagnosis, 252
Cytophil group, 215
Cytoplasm, 300

Danysz-DUNGERN PHENOMENON,
215, 222
Daphnia, 300
infection by monospora, 133

310

Denitrification, 12
Diagnosis, microbial, 250
Diapedesis, 300
Diarrhcea, infantile, 38, 43
Diastases, 301

secretion, 86

toxins compared, 160
Diplococci, 55
Diphtheria, antidiphtheric serum,

274
bacillus, lysin, 87
pleomorphism, 59
pseudo-, 113
toxin, 158, 159
virulence, increase and diminu-
tion of, 116, 117
Dubois, B., bacterial luminosity, 91
Duclaux, fermentation, 83
Dumas, J. B., fermentation, 82
Dysentery, antidysenteric serum,
279
bacillus, endotoxin, 170
pseudo-, 114
receptive cell, 127

EHRLICH, chemiotherapy research,
287
immunity theory, 211, 213
serum titration, 275
side-chain theory, 211
Endospores, 59
Endotoxins, 158, 168
Engelmann, respiration experiments,
80, 85
Entamoeba histolytica, 145
Enterococci, 35
inhibiting power, 42
Enterokinase, 199
Enzymes: see Diastases.
Eosin, bactericidal action, 97
Epidemics, 120
Epitheliosis virus, 128
Erythrosin, bactericidal action, 97
Evolution :
bacterial, 108
serum action, evidence of, 256

FACULTATIVE BACTERIA, 80
Falloise, intestinal putrefaction, 38
Fats, 301

fermentation, 5

resistance to decomposition, 9

INDEX

Fermentation, 5, 301
anaerobic production, 82
chemical constitution of med-
ium, 78, 79
heat production, 89
phagocytosis analogy, 195
Ferments: see Diastases.
Ferrau, cholera vaccination, 271
Ferro-bacteria, 78
Fibrin, 301
Fibrinogen, 301
Ficker, intestinal permeability, 48
Filtration, 152.
Findel, infection experiments, 125
Finkelstein, on diarrhoea, 38
Fishes, venoms of, 178
Foot-and-mouth disease virus, 155
Fowl-cholera, infection, 124
bacillus, lysin, 87
size, 150
virus attenuation, 118
Friedberger, anaphylaxis
246
Fungi, bacteria relationship, 62
coals, 7
sex, 66

theory,

GALACTOSE, fermentation, 79
Germ-carriers, 124
Giant cell, 139
Glossina palpalis : see Tsetse fly.
Glucose, fermentation, 78
Glucosides, 302

fermentation, 5, 6
Glycogen, 302
Granular conjunctivitis, see Tra-

choma.

Granulobacter, 7
Gregarines, 104
Gromia oviformis, digestion, 100
Guarnieri bodies, 155

HARMATOZOA, pathogenic, 142
Haemolysin, 179
secretion of, 86, 193
Haemolysis, Bordet’s theory, 221
specific, 225
of venoms, 182
Haffkine, cholera vaccination, 271
plague vaccination, 273
Haptophorous groups, 212
Heat-production, microbial, 89
Heredity, 145

INDEX

Herxheimer, phenomenon of, 297
Hippuric acid, fermentation, 10
Hoffmann’s bacillus, 113
Hog-cholera, 46, 154

Hogyes, rabies treatment, 269
Hlorse-sickness, 154

Humoral immunity, 191
Hymenoptera venom, 179

IMMUNE BODY, 183, 195
Ehrlich’s theory, 215
specificity, 199

Immunity, 187 e¢ seg.
anaphylaxis, 240

Incubation of disease, 128, 302

Indol, 302
toxicity, 37

Infection, 121
intestinal, 127

Inflammation, 130, 302

Infusoria, 51
excretion, IOI
infection, 121

Inoculation, 266

Intermediate substance: see Immune

body.

Intestine, flora, 11, 27, 33

Intra-dermo reaction, 178

JOCHMANN, tuberculin experiments,
208

KALA-AZAR, 144
Khondiakon, 84
Koch, R., sleeping sickness treat-
ment, 291
specificity of disease, 112
tubercle phenomenon, 173
Kurpjuweit, on inhibition, 38
Kyes, lecithids, 185

Laccask, 86
Lactic bacilli, bacteriotherapy, 273
fermentation, 43
involution forms, 59
Lainé, artificial nitre beds, 16
Lamblia intestinalis, 104
Lamprocystis, 78
Lampyris noctiluca, 91
Latent microbism, 123
Lechartier, fermentation
ment, 82
Lecithids, 184, 226

experi-

311

Lecithin, 184, 226, 302
Leguminosae bacteria, 20 ¢¢ seg.
Leishmania: pathogenic, 142
donovani, varying forms, 144
Lentospora, 145
Leprosy, incubation period, 128
bacillus, 108
Leptothrix ochracea, 78
Leucocidine, 87
Leucogytes, 48, 136, 138: see also
Phagocytes.

Levulose, fermentation, 78
Lignite, bacterial formation, 7
Lipochrome, 92
Light production, microbia’, 89
Lipoids, 166, 302

colloids, analogy with, 224, 226
Lithocolletis caterpillars, aseptic

condition, 31

Luciferine, 91
Luciola italica, 91
Lumbricus, inflammation in, 133
Lysius, 86, 248

MACFAYDEN, bacterial extract, 169
cold, effect on microbes, 96
Macrophages, 138, 197
Madsen, immunity theory, 216
Malaria, infection, 123
mosquito, 143
parasite, 51, 104
path of penetration, 126
receptive cell, 127
reproduction, 66
Mallein, 173
Manure, denitrification, 14
fermentation, 7
Marmorek’s serum, 280
Martelly, putrefaction experiments
10
Marx, virus fixe, 269
Massart, chemiotaxis, 136
Massini, bacterial mutation, 62
Maze, fermentation experiment, 82
Meat, adulteration detection, 255
putrefaction, 10
Meningitis, antimeningococcus se-
rum, 280
bacteriotherapy, 274
Meningococcus, 27
pseudo, 114
Mercaptan, poisoning, 37
Metachromatic bodies, 64

312

Metchnikoff, aseptic breeding, 32
cholera bacillus, 40
non-bacterial life, 30
phagocytic immunity, 197, 198

Micrococci, 55

Micrococcus parvulus, 150

Microphage, 138, 197

Microsphaera, amoebae infection,

121

Miguel, inhibition,. 88

Milk, fermentation, 5
putrefaction, I1, 41

Molisch, microbial luminosity, 91
respiration, 85

Molluseum contagiosum, 302
virus, 155

Monospora tricuspidata, Daphnia
infection, 133

Morgenroth, antitoxin experiments,

212
Moro, aseptic breeding experiments,
32

Moulds, 52
heat effect, 95
nitrogen fixation, 20

Mouth, microbes in, 27

Mucedinae, 302.
nutrition, 74

Mucor, 52

Mucous membranes, microbes in,
26, 46

Miintz, nitrification experiments
13, 16

Mycelium, 52
Mycetozoa, 303

inflammation, 131
Mycoderma vini, 5
Myeloplax, 303
Myriapods, 9
Mytilocongestin, 238
Myxobolus pfeifferi, 104, 145
Myxomycetes : see Mycetozoa.

NAGELI, pleomorphism, 60

Negri bodies, 155

Nepticula, aseptic condition, 31
Neufeld, bacteriotropins, 264
Nicolle, M., antibodies theory, 248
Nictric ferment, 13, 74
Nitrification, 12, 303

Nitrobacter, 13

Nitro-bacterium: see Nitric ferment.
Nitrogen fixation, 18

INDEX

Nitrosococcus, 13

Nitrosamonas, 13

Nitrous ferment, 13

Nitsch, virus fixe experiments, 269
Nobécourt, intestinal putrefaction,

Nocard, anti-tetanic serum, 277

Nose, microbes in, 26

Nucleus, bacterial, 64

Nutrition, microbial, 72

Nuttall, aseptic breeding experi-
ments, 31

OCULO-REACTION: see Conjunctivo-
reaction.

Oidium lactis, 11

Omeliansky, cellulose
tion, 6

Opsonins, 204

Ornithodorous monbata, 147

Ornithorhynchus venom, 180

fermenta-

PANCREATIC JUICE,
action, 182
Para, bacteria, 112
Para, cresol, 302
Parasitism, 102
Passages, method of, 117
Pasteur, anaerobic life, 80
fermentation experiment, 82
immunity, 188
microbial association, 125
non-bacterial life, 28
nutrition, 78
pathogenic microbes, 108
pleuro-pneumonia vaccination,
270
putrefaction, 10
rabies treatment, 268
virulence, 117
Pasteuria ramosa, 57
Pathogenic microbes, 107
Peat, bacterial formation, 7
Pébrine, 146
Pectose, fermentation, 7
Penicillium, 52
nitrogen fixation, 20
nutrition, 78
Peptolytic microbes, 10
Petroleum, bacterial origin, 9
Pettersson, immunity experiment,
206
Pfeiffer’s phenomenon, 201

venom re-

INDEX

Phagocytes, 135, 138
Phagocytosis, 130, 191 (figs.), 100
chemical stimulation, 284
immunity, 187
toxins, 227
Phagolysis, 198
Pharynx, microbes in, 26
Phenol, 302, 37
Phosphorescence (animal), 90
Photodynamic substances, 97
Phycochrome,, 63
Pigments, microbial, 92
Piroplasma, bigeminum, 147
Piroplasmosis, 142
infection, 123, 147
Pirquet, von, serum-sickness, 234
tuberculin, 178, 262
Plasmodia, 142
Plague, anti-plague serum, 279
vaccination for, 272
Plants, microbes in, 28
Plasma, 303
Pleomorphism, 59
Pleuropneumonia, microbe, 153
vaccination for, 270
Pneumococcus, germ carriers, 124
in throat, 27
Pollen coals, 18
Portier, anaphylaxis experiments,
234
Precipitation diagnosis, 254
Precipitin, 193, 255
Preisz, tubercle infection experi-
ment, 125
Prophylaxis, 240
Protagon, 230, 302
Proteins, 303
putrefaction, 9
Proteolytic microbes, 10
Proteus, intestinal, 12, 37
pleomorphism, 59
putrefaction, 10
Protozoa, 50
diseases due to, 141, 286
physiology of, 98
Pseudo-bacteria, 112
Pseudomonas lucifera, 90
Ptomaines, 303
Purple bacteria, 85
Putrefaction, 3, 303
intestinal, 37
Pyocyanase, 87
Pyocyanin, 93

313

Pyocyanolysin, 87
Pyorrhea alveolaris, bacteriotherapy,
273

RaBigs, incubation period, 128
vaccination, 268
-virus, path of penetration, 127
receptive cell, 127
virulence increase, 117, 118
Raulin, nutrition experiments, 74
Raulin’s medium, 74
Receptive cells, 127
Receptors (Ehrlich), 212
Recurrent fever, 147
Reproduction, microbial, 67, 102
Respiration, 80
Rhipicephalus annulatus, 147
Rhizopods, pathogenic, 142
Rhizopus nigricans, 11
Richet, anaphylaxis experiments,
234
Ricin, 168
Rinderpest, 154
sero-vaccination, 282
Rivet, intestinal putrefaction, 38

Roger, immunity experiments,
200

Roux, immunity experiments, 228,
276

pseudo-bacteria, 113
serum anaphylaxis, 241
toxins, 160

SACCHAROLYTIC microbes, 10
Sacred host : see Bleeding host.
Salamander venom, 180
Saltpetre, bacterial formation, 15
Salvarsan, 291, 293
Sarcine, 34
pigment production, 93
Sarcocystine, 145
Sarcosparidia, 104
Scarlatina, 155
Schaudinn, nucleus, bacterial, 65
reproduction, 68
syphilis, 118, 292
Schick, serum-sickness, 234
Schizomycetes, 62
Schizosaccharomycetes, 67
Schloesing, nitrification
ment, I
Schottelius : see Aseptic breeding
Scorpion venom, 179

experi-

314

Sea-urchin venom, 178
Sensibilisatrice : see Immune body.
Sensibilism, 247
Sensibilisinogen, 247
Septic tank (sewage), 16
Sero-agglutination, 252
Sero-diagnosis, 255
Serotherapy, 274
Serovaccination, 245
Serum, 274, 303
Sewage purification, 16
Sex, microbial, 67
Sheep-pox sero-vaccination for,
281
Side-chain theory, 211
Skatol, 302
Skin, bacteria in, 26
Sleeping sickness :
ment, 290
infection, 123
treatment, 291
trypanosome of: see Trypano-
soma gambiense.
tsetse-fly, 143
Sleeswyk, agglutination
ments, 254
Small-pox, vaccines, 266
virus, 128, 155
Smith, Th., anaphylaxis phenome-
non, 235, 248
Snake venom, 180
Soamin, 292
Sorbose, bacterium of, 79
Spallanzani: heat effect on moulds,

atoxyl treat-

experi-

95

Spider venom, 179
Spirilla, 55
Spirillosis, infection, 123, 148

virus, 118
Spirochetes, 55

diseases due to, 142
Spore coals, formation, 8
Spores, 58, 102

heat resistance, 95
Staphylococci, 55

in skin, 26
Staphylococcus aurens, 92

pyogenes, 37
Staphylolysin, 37
Starch fermentation, 5
Stegomyia fasciata, 149
Sterigmatocystes, 20
Stichococcus, 20

INDEX

Stomach, microbes in, 27
Streptococci, 34, 55
anti-streptococcic serum, 280
encapsulated, 57
in skin, 26
virulence, 116, 117
Streptocolysin, 87
Streptothriceze, 63
Stylonychia pustulata, Io1
Sucrase, 303
Sugar, fermentation, 5, 78
soil-enrichment, 20
Sulpho-bacteria, alimentation, 78
respiration, 85
Sulphuretted hydrogen poisoning,

37
Sulphurous bacteria: see Sulpho-
bacteria.
Surgery, phagocytic
283
Swine-erysipelas : vaccination, 270
-bacillus, 127
virulence increase, 118
Symbiosis, 303
Syphilis, chemio-therapeutical treat-
ment, 292
hereditary, 146
sero-diagnosis, 258
transmission, 148
spirochzte, path of penetration,
126

therapy in,

Takaki, toxicity experiments, 166
226
Tannase fermentation, 6
Tarantula venom, 179
Tartar-emetic, in sleeping sickness,
292
Tetanolysin, 87, 215
Tetanospasmin, 215
Tetanus, anti-tetanic serum, 276
favouring factors, 125
incubation period, 128
infection, 122
latent, 123
bacillus, flagella, 56
path of penetration, 163
respiration, 84
toxin, 159
virulence increase, 117
Tetrodon venom, 180
Thalassine, 178

INDEX

Thermophilic bacteria, 94
Thierfelder, aseptic breeding, 31
Thomas, W., atoxyl use, 290
Tissier, putrification experiments,
10
Toad venom, 180
Toxins, 157, 210
phagocytosis, 227
Toxogenin, 239
Toxophore group [Ehrlich], 213
Trachoma virus, 155
Trypanosoma, 51
culture, 105
diseases due to, 142, 290
parasitism, 104
sexual reproduction, 66
gambiense, 51, 105
toxin, 145
Trytophane, 302
Tsetse fly, 143
Tubercle bacillus,
position, 71
culture media, 76. 94
detection, 112
light, effect of, 97
in lung, 27
para-, 115
path of penetration, 127
phagocytosis, 138
virulence increase, 117
Tuberculin, 173
chemical composition, 71
diagnosis by, 260
immunity, 208
light, effect on, 97
Tuberculosis, antituberculous — se-
rum, 280
antituberculous vaccination, 272
cutaneous reactions, 262
heredity, 146
infection, 47, 121, 125
tuberculin treatment, 174
tuberculin diagnosis, 260
bacillus : see Tubercle-bacillus.
Typhoid bacillus, agglutination test,
2

chemical com-

2
blood infection, 251, 46
in decayed food, 37
endotoxin, 170
flagella, 56
infection, 122
lysin, 87

ip para-, 115

315

Typhoid bacillus, Pfeiffer’s pheno-
menon, 204
-fever, germ carriers, 124
vaccination, 271
Typhus fever bacillus, 120
Tyrosin, 302

ULTRA-MICROSCOPE, I5I

Urea fermentation, 9

Urease, 303

Urine fermentation, 9
toxicity, 38

Urobacteria, 9

Urtilago carbo, 95

VACCINATION, 265
anaphylaxis, 234, 242
continuous, 243
sero-, 281

Vaccines, 265
passage of, 118
sensitized, 282

Vaccinia virus, 155

Vaillard, immunity

228

Variolisation, see Inoculation

Vaughan, anaphylaxis theory, 246

Venoms, 178
anti-venom sera, 278

Vibrio, 55

Vibriolysin, 87

Vibrion septique, putrefaction, 10
respiration, 84

Villes, Georges, leguminose ex-

periments, 20

Vincent, pathogenic microbes, 109,

Virulence, 116

Virus, attenuation of, 118
filtrable, 150
fixe, 268

Vitalism, 303

experiments,

WALBRUM, immunity theory, 216

Wassermann, antituberculin, 177
toxin experiments, 166, 226

reaction, 258

Weever fish venom, 179

Weigert, 65

Welch’s bacillus: see

perfringens.

Bacillus

316 INDEX

Wheeler, anaphylactic theory, 246 Yeasts, fermentation, 5

Widal, biological diagnosis, 252 milk putrefaction, 11

Willanen, toxin experiment, 212 nutrition, 77, 73

Wollmann, aseptic breeding, 31 reproduction, 67

Wolff-Eisner, ophthalmo-reaction, Yellow fever, 149, 154
262 Yersin, toxins, 159

Wright, opsonins experiment, 204

ZOOGLAA, 57

YEASTS, 54 Zischeria, aseptic condition of, 31
chemical composition, 70 Zygosaccharomycetes, 67
cold, effect on, 96 Zymase, 83, 169

R. CLAY AND SONS, LTD., BRUNSWICK ST., STAMFORD ST., S.E., AND BUNGAY, SUFFOLKoe

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