v&n^&JfosCont&famJ

V

I'TOM. VINES, LEUCOM VINES,

AND

BACTK1IIAL PKOTEIDS:

UK

THK CHKMICAL FACTORS IN THK CAUSATION OK DISKASE.

/'

< BY

\*
VICTOR C. Y^UGHAN, PH.D., M.D.,

1-RUKKlWOIl <ir IIViilENK AM) I'll YSKll.niilCAI. CHEMISTRY IN THK •' N I V Kltsi I 1 OP MICHIGAN,
AND DIKECTOB or THE HYUIENIC LABORATORY .

FREDERICK G, NOVY, Sc.D., M.D.,

kNr iMtorRssmi or IIYOIKNK. >M> HIYS|'PI,O«ICAL CHEMISTRY IN THE UNIVERSITY
or MICHIGAN.

SECOND EDITION, REVISED AND ENLARGED.

PHILADELPHIA:

LEA BROTHERS & CO.,
1891.

filtered according to Act of Congress in the year 1891, by

LEA BROTHERS & CO.,
In the Office of the Librarian of Congress at Washington, D. C.

DOBNAN, PRINTER,
PHILADELPHIA.

TO

A U5ERT B. PRESCOTT, PH.D., M.D., F.C.S.,

I'lliK' T.iR • •( TlIK CHEMICAL I.AB'lBATORY IN THB UNIVCB8ITY OF MICHKiAN,

THIS LITTLE WORK
18 RESPECTFULLY DEDICATED

AS A SLIGHT TOKEN OF THE HIGH ESTEEM IN WHICH
HE IS HELD BY HIS FORMER STUDENTS,

THE AUTHORS.

K TO SKCOND EDITION.

TIIK preparation of this edition has Urn made a work
of pleasure on account of the many kind words which have
l»een said concerning our first effort to collect the smttered
facts pertaining to the chemical factors in the causation of
di-'ase. \\Y mii-t !•<• allowed to express our gratification
at the "Jeii'Tal acceptance accorde<l to the statements which
we first made three yen's ago, and wliieh were then re-
isinled l»y many as extremely radi<td. At that time
many of the leading bacteriologistB held to the " mechan-
ical interference" theory, and regarded the chemical pro-
duets of IMTIII- a- of some interest, hut in no direct way
concerned in the causation of disease. Now the fact that
a -crin is pathogenic is considered to l>e sufficient evidence
that it elaliorates poisonmi- products, and the study of tin .-••
pro<luets is regarded a- of the greatest importance in the
investigation of the ^crm and the disease which it cau-
The interest in this sulijcct is not eonfine<l to a study of
the causation «•(' di-:a-r, hut efforts are In-ill^ made to
ire immunity from diseasi- and even to elli-et cures l>y
the employment of the li;i<-terial produets. This line of

VI PREFACE TO SECOND EDITION.

study has certainly become one of great interest to all
scientific students of medicine.

In the preparation of the present edition we have
endeavored to utilize the latest and best information, and
we can only express our thanks for the encouragement
which we have received from so many sources and hope
that the present effort will justify no censure.

UNIVERSITY OF MICHIGAN, SEPTEMBER, 1891.

TO FIRST EDITION.

WITHIN the past ten years much has Ixvn said and
written concerning tin1 basic substances forim-d during the
putrefaction of organic matter, and those which are pro-
duced by the normal tissue-changes in the living organism.
Many investigators have given their whole time and atten-
tion to the study of these substances, and important discov-
eries have been made and much light has been thrown upon
what have heretofore been considered problems in medical
science. To collect, arrange, and systematixe the facts
concern inn; j Romanies and leiiconia'ines has been our first
olijcct. Although many short essays, some of them of
-iv. -it value, have been written with the above-mentioned
object in view, the present work may he regarded as tin-
first attempt to make this collation embrace everything of
importance on this subject. In endeavoring to accomplish
this object we have met with many difficulties. The original
reports of the various investigators arc scattered through
the pages of medical and Scientific journals, trail-actions of
societies, monograph-, -o\ernment reports, etc. However,
with few exception- we have been able to obtain the original

viii PREFACE TO FIRST EDITION.

reports, and we think that we have included everything of
importance published up to the present year (18XX).

To the physician the facts which have been made known
concerning the putrefactive and physiological alkaloids
must be of great value, and if this little work furnishes
the means by which members of the profession may become
better acquainted with the nature of those poisons which
are introduced from without, and those which are gener-
ated within the body of man, the object of its authors will
be accomplished.

UNIVERSITY OF MICHIGAN, JULY, 1888.

CONTENTS.

MM

I \ I INDUCTION 13

CHAPTER I.

DEFINITION AND CLASSIFICATION OF THE BACTERIAL
POISONS 15

CHAPTER II.
HISTORICAL SKETCH OF THE BACTERIAL POISONS . . 22

CHAPTER III.

FOODS CONTAINING BACTERIAL POISONS : Poisonous Mus-
sels,. Oysters and Eels, Fish, Sausage, Ham, Canned
Meats, Cheese, Milk, Ice-cream, Meal and Bread . . 36

CHAPTER IV.

GENERAL CONSIDERATIONS OF THE RELATION OF BAC-
TERIAL POISONS TO INFECTIOUS DISEASES : Classifica-
tion of Diseases, How Germs Produce Disease, Definition
of Infectious Disease, Koch's Rules .... 84

CHAPTER V.

THE BACTERIAL POISONS OF SOME OF THE INFECTIOUS
DISEASES: Anthrax, Asiatic Cholera, Tetanus, Tuber-
culosis, Diphtheria, Suppuration, The Summer Diar-
rhoeas of Infancy, Typhoid Fever, Swine-plague (Hog-
cholera), Rabbit Septicaemia, Pneumonia, Malignant
(Edema, Puerperal Fever 101

CHAPTER VI.

THE NATURE OF IMMUNITY-GIVING SUBSTANCES: Methods
of securing Immunity; Bacterial Products which Favor
the Development of Infectious Diseases . . .14(5

X CONTENTS.

CHAPTER VII.

PACK

THE GERMICIDAL PROTEIDS OF THE BLOOD . . . ].r>^

CHAPTER VIII.

METHODS OF EXTRACTING PTOMAINES. Basic Impurities in
Reagents. The Stas-Otto Method, Dragendorff's Method,
Brieger's Method, The Methods of Gautier and Etard.
Remarks upon the Methods 157

CHAPTER IX.
METHODS OF ISOLATING THE BACTERIAL PROTEIDS . . 171

CHAPTER X.

THE IMPORTANCE OF PTOMAINES TO THE TOXICOLOGIST.
Coniine-like Substances, Nicotine, Strychnine, Mor-
phine, Atropine, Digitaline, Veratrine, Delphinine, Col-
chicine. Effect of Ptomaines on Alkaloidal Reactions' . 174

CHAPTER XI.

CHEMISTRY OF THE PTOMAINES: Primary Amines, Dia-
mines, etc., the Choline Group, other Oxygen-containing
Bases, Undetermined Ptomaines. Tables . . . 187

CHAPTER XII.

CHEMISTRY OF THE LEUCOMAINES : Uric Acid Group,

Creatiniue Group, Undetermined Leucomaines. Tables 280

CHAPTER XIII.
THE AUTOGENOUS DISEASES 352

CHAPTER XIV.

BIBLIOGRAPHY : Ptomaines, Leucomaiues, Bacterial Pro-

teids, Miscellaneous 364

PTOMAINES, LEUCOMAl'NES, AND BACTERIAL
PROTEIDS.

INTRODUCTION.

IT is customary to divide bacteria into the parasitic and
the saprophytic. The obligate parasite can live only on
living matter; the obligate saprophyte can live only on
dead i natter. Since all attempts to grow the bacilli of
syphilis and leprosy on artificial media have failed, they are
probably obligate parasites. True parasitic germs do not
prove speedily fatal to their hosts, because their continued
existence depends upon the continued existence of their host,
or on their transference to another host. Leaving out of
consideration the obligate bacterial parasites, about which
very little is known at best, the above classification becomes
of but little importance to us in a study of the causal rela-
tion of germs to disease, l>ecause a given bacterium may
grow and multiply in one part of the body, while it is
unable to do so in another ; or it may thrive in one species
of animal, while it finds the conditions unfavorable in an-
other species ; or similar differences may exist in individual
members of the same species. Thus, the white rat is ordi-
narily and naturally immune against the bacillus of anthrax,
l»ul if the rat be exhausted by being kept on a small tread-
mill for some hours it becomes susceptible to anthrax.
Recognizing these facts, we propose that bacteria be divided
into the toxieogenic and the non-toxicogenic. Since we
know of no infectious disease in which poisons are not
formed, the toxieogenic germs only are of interest to us.

2

14 INTRODUCTION.

Iii the study of these we must not only ascertain the nature
of the poisons which they produce, but must know the con-
ditions under which they can multiply and elaborate these
poisons. To these points the following pages, in so far as
they treat of the infectious diseases, will be devoted.

However, all diseases are not infectious; all poisons
formed within the body do not owe their existence to bac-
teria. Some originate in the altered metabolism of the
various tissues, and these will be discussed under the auto-
genous diseases.

CHAPTER I.

I'l.l'INITION AND CLASSIFICATION OF THE BACTERIAL

I '(• I SONS.

PTOMA'fNES. — All exact classification of the chemical
factors in the causation of the infectious diseases can prob-
al»lv not l>e made at present. We know of two chemically
distinct classes, one of which contains substances which
combine with acids, forming chemical salts, and which in
this nsjxvt at least correspond with the inorganic and
vegetable basis. The members of this class are designated
a- ptoma'ines, a name suggested by the Italian toxicologist,
SKLMI, and derive<l from the (Jreek word -rufia, meaning a
cadaver: A ptoma'ine may l>e defined as a chemical com-
pound which is basic in character and which is formed by
the action of bacteria on organic matter. On account of
their basic properties, in which they resemble the vegetable
alkaloids, ptomaines may !>e called putrefactive alkaloids.
Thev have also been called animal alkaloids, but this is :i
misnomer, because, in the first place, some of them are
formed in the putrefaction of vegetable matter; and, in
the second place, the term "animal alkaloid " is more prop-
erlv re-tricted to the leucomaines — -those basic substances
which result from tissue metabolism in the body. While
some «.f the ptomaines are highly poisonous, this is not an
utial property, and others are wholly inert. Indeed,
the greater number of those which have Ixvu isolated up
to the present time do not, when employed in single doses,
produce any apparently harmful effects. BRIEGER restricts
the term ptomaine to the non-poisonous basic products, and
designates the poisonous ones as " toxines." This is a
classification, however, which seems to be of questionable
utility. It is not always easy to say just what I unties are
poisonous and what are not. The poisonous action of a

16 PTOMAINES.

substance depends upon the conditions under which, and
the time during which, it is administered. Thirty grains
of quinine may be token by a healthy man during twenty-
four hours without any appreciably ill effect, yet few of us
would be willing to admit that the administration of this
amount daily for three months would lx> wise or altogether
free from injury. In the same manner the administration
of a given quantity of a putrefactive alkaloid to a dog or
guinea-pig in a single dose may do no harm, while the
daily production of the same substance in the intestine of
a man and its absorption continued through weeks and pos-
sibly months may be of marked detriment to the health.
We do not as yet know enough about the physiological or
toxicological action of the putrefactive alkaloids to render
the classification proposed by BRIEGER worthy of general
adoption.

All ptomaines contain nitrogen as an essential part of
their basic character. In this they resemble the vegetable
alkaloids. Some of them contain oxygen, while others do
not. The latter correspond to the volatile vegetable alka-
loids, nicotine and coniine, and the former correspond to
the fixed alkaloids.

Since all putrefaction is due to the action of bacteria, it
follows that all ptomaines result from the growth of these
microorganisms. The kind of ptomaine formed will de-
pend upon the individual bacterium engaged in its produc-
tion, the nature of the material being acted upon, and the
conditions under which the putrefaction goes on, such as the
temperature, amount of oxygen present, and the duration
of the process.

BRIEGER found that, although the Eberth bacillus grew
well in solutions of peptone, it did not produce any pto-
maine; while from cultures of the same bacillus in beef-tea
he obtained a poisonous alkaloid. FITZ found that whilst
the bacillus butyricus produces by its action on carbohy-
drates butyric acid, in glycerin it produces propylic alcohol,
and MoRIN has found amyl alcohol among the product- ..('
this germ. BROWN has shown that while the mycoderma
aceti converts ethylic alcohol into acetic acid, it converts

I'KKINITION. 17

propylic alcohol illl(» prOpioniG acid, ;nnl is without effect
upon mcili\lie alcohol, primary isobutylic alcohol, :iml
amvlic alcohol. Some bacteria will not multiply In-low :i
given temperature. Thus, tin- bacillus hutyrieus will not
grow ;it a temperature In-low 24°. ' The lower temperature
i|«i - tint destroy the organism, hut it lies dormant until the
conditions nre more favorable tor its growth. PASTEIU
divided the bacteria into two classes — the aerobic and the
anaerobic. As the name implies, the fornu-r grow and
thrive in the presence of air, while the latter find their
conditions of life improved by the exclusion of air. There-
fore, different ptomaines will In- formed in decomposing
matter freely exposed to the air, and in that which is huried
iM'iieath the soil or from which the air is largely excluded.
Kven when the same ferment is present the products of the
putrefaction will vary, within certain limits, according to
the extent to which the putrefying material is supplied with
air. The kind of ptomaine found in a given putrid sub-
stance will depend also upon the stage of the putrefaction.
I'toma'ines are transition products in the process of putre-
faction. They are temporary forms through which matter
pa— e- while it is being transformed, by the activity of bac-
terial life, from the organic to the inorganic state. Com-
plex organic substances, as muscle and brain, are broken
up into less complex molecules, and so the process of
chemical division goes on until the simple and well-known
final products, carbonic acid gas, ammonia, and water,
result; but the variety of combinations into which an
individual atom of carbon may enter during this long-
series of changes is almost unlimited, and with each change
in combination there is more or less change in nature. In
one combination the atom of carbon may exist as a con-
stituent of u highly poisonous substance, while the next
combination into which it enters may be wholly inert.

It was formerly supposed that putrefaction was simply
oxidation, but the researches of I'ASTF.ru and others have
demonstrated the fact that countless myriads of minute

1 All temperatures given in this work are Centigrade, unless otherwise
specified.

18 BACTERIAL PROTEIDS.

organisms are engaged constantly in transforming matter
from the organic to the inorganic form. Lock up the i>it
of flesh so that these little workers cannot reach it, and it
will remain unchanged indefinitely.

It may be asked if any of the change's occurring during
putrefaction are to be regarded as purely chemical. Without
doubt, many of the secondary products of putrefaction arise
from reactions between antecedent and more complex prod-
ucts or by the action of oxygen, water, and reducing agents
upon primary products. Ptomaines formed in this way
may be regarded as the indirect results of bacterial life.

BACTERIAL PROTEIDS. — These substances have been
known for so short a time and are at present so imperfectly
known that many difficulties arise in discussing them. In
the first place, we may divide the bacterial proteids into
two classes : (1) those which constitute an integral part of
the bacterial cells, and (2) those which have not been
assimilated by the cells, but which have been formed by
the fermentative or cleavage action of the bacteria on the
proteid bodies in which they are growing. Even this
classification is of questionable value. We allow bacteria
to grow for a number of days in a nutrient solution. We
then separate the soluble constituents from the formed cells
by filtration through porous tile ; we wash the latter and
then study their proteid contents, which constitute the first
class, as given above ; but the filtrate contains, or may e.m-
tain, any one or more of the following proteid bodies : (1)
Those portions of the proteid substances which were us«l
in the preparation of the nutrient solution and which have
escaped the action of the bateria; (2) proteids which have
been at one time integral parts of the cells, but which
have passed into solution ou the death and dissolution of
the bacteria ; and (3) proteids which have been formed by
the fermentative action of the bacteria, or those which are
defined as constituting the second class, as given above.
We know at present of no means by which one of these
proteids can with certainty be isolated from the others.
However, the above classification is a convenient one, and

BACTERIAL PROTEIDS. 19

\\illi a dear understanding that it is not free from criticism
we may employ it until a more thorough and scientific study

of these bodies has Urn made.

The difficulty in discussing these substances lies not only
in the classification, hut in the name which shall be em-
ployed to designate them. BRIEGER and FRANKEI, have
proposed the term " toxalbumius ;" but, while it is true
that some belong to the albumins, others are more truly
albumoses ; others are most closely related to the peptones ;
and still others differ in some important respects from all
of these. In view of the above facts, we have decided upon
the term " bacterial proteids " to designate those formed by
the fermentative action of germs, while those which consti-
tute an integral' part of the cell will IHJ known as "the
bacterial cellular proteids."

The Bacterial Cellular Proteids. — NENCKI first prepared
one of these substances from putrefactive bacteria. These
were obtained by dccantation, freed from fat with ether,
dissolved in fifty parts of a potash solution of 0.5 percent.,
heated for some hours at 100° and filtered. The filtrate
was acidified with dilute hydrochloric acid and precipitated
by the addition of rock salt. The precipitate was washed
with a saturated salt solution, dried at 100°, and washed
free from salt with water. NENCKI designates this sub-
stance as " myeoproteiu," and finds that it has the formula,
C25H42N6O9. Freshly precipitated mycoprotein forms in
amorphous flakes, which are soluble in water, alkalies, and
acids. The aqueous solution is acid in reaction. After
In'ing dried at 100° it is no longer wholly soluble in water.
NENCKI found that it is not precipitated from aqueous solu-
tion by alcohol, but by picric acid, tannic acid, and mercuric
chloride; that it does not give the xanthoproteid, but
docs give the Mi lion and the bin ret reactions. According
to SCHAFFER it is changed by acids into peptone, and on
Ix-ing fused with five parts of potash it breaks up into am-
monia, amylamin, phenol (0.15 per cent, of its weight), vale-
rianic acid (.'>X per cent.), leucine, and traces of indol and
sUatol. A proteid obtained from the yeast plant has the
formula, C12H21N3O3.

20 BACTERIAL PROTEIJ)S.

The purified pyogenetic agent obtained from the pneu-
monia bacillus of FJUEDKANDER was found by I>i c HNER
to give the following reactions: It is soluble in water and
the concentrated mineral acids, very soluble in dilute alka-
lies, from which it is precipitated on the addition of an acid.
From its aqueous solution, it is not precipitated by heat,
nor by saturation with sodium chloride, but is precipitated
by magnesium sulphate, copper sulphate, platinum chloride,
gold chloride, lead salts, picric acid, tannic acid, and abso-
lute alcohol. It gives the xanthoproteid, Millon, and
biuret reactions.

The Bacterial Proteids. — BRIEGER and FRANKEL ob-
tained the proteid poison of diphtheria by precipitating
the filtrate from a Chamberland filter after concentration
to one-third its volume at 30°, with absolute alcohol after
feebly acidifying with acetic acid. The precipitate was puri-
fied by repeated solution in water and repreeipitation with
alcohol. Dried in a vacuum at 40°, it forms a snow-white,
amorphous, very light mass. From its aqueous solution it
is not precipitated by heat or dilute nitric acid, singly or
combined, nor by sodium sulphate, sodium chloride, mag-
nesium sulphate, or lead salts. It is precipitated by car-
bonic acid (to saturation), concentrated mineral acids,
potassium ferrocyanide and acetic acid, phenol, organic
acids (soluble in excess), copper sulphate, silver nitrate,
and mercuric chloride. The so-called alkaloidal reagents
phosphomolybdic acid, potassio-mecuric iodide, potass io-
bismuthic iodide, platinum chloride, gold chloride, and
picric acid also cause precipitation. The xanthoproteid,
Millon, and biuret reactions give positive results. An ulti-
mate analysis furnishes the following figures computed
from the ash-free substance : C 45.35, H 7 J3, N 16.33, S
1.39, O 29.80. From these facts BRIEGER and FRANK KL
conclude that this substance is allied to serum-albumin.
Their bouillon cultures contain serum-albumin, and they
suppose that the bacteria convert this into the poison by
causing a rearrangement in the atoms; but the same poison
was formed when nutrient solutions containing no proteid

BACTERIAL P K < > T K I 1)S. J I

BRVe peptone were employed. In this ca-e they -iipp
that the bacteria reconvert (lie peptone into :m ;ill»iiiiiii).

The poisonous proteids obtained by BRIEQEB MIK!
Ki: \NKKL from cultures of the Kin-rill germ, the comma
bacillus, and the staphyloeocciis ann-ns art- practieally in-
soluble in \valrr, and more nearly related to (he globulins
than (he albumins, although (hey dill'er from the former
in their tardy and difficult solubility in dilute solutions of
sodium chloride.

The poisonous protcids isolated by VAUGHAN from cul-
tures of two species of toxico^enic licrins found in drinking
water, supposed (o IM- (he cause of (yphoid fever, an> solu-
ble in water, from which they an- not precipitated by lx>il-
iiiLi, or by concentrated nitric acid, or by both. Potassium
(errocvanide and acetic acid, sodium sulphate, magnesium
sulphate, and carbonic acid also liiil to precipitate them.
They are precipitated by the general alkaloidal reagents,
and respond to the xanthoproteid, Millon, and binret (cs(s.
They are precipitated by ammonium sulphate when added
to saturation, and for this reason cannot be classed amon<;
the peptones. Neither l>en/oyl chloride nor phenyl-hydni-
/in chloride precipitate them. Their poisonous properties
are destroyed by prolonged boiling or l>\- bein^r hea(<'<l to
S()° for some hours, though they remain active after an
exposure of (<-n minutes to the last mentioned temperature.

Of the three bacterial proteids obtained by the same ex-
perimenter from the bacilli x, a and A of Booker's list of
summer diarrlnea .irerms, the first two are soluble in water,
while the other is not. So far as their behavior with pre-
cipitating agents is concerned, the first two agree with the
proteids of the water ^erms.

TI/XOM and CATTANI find that the proteid of cultures
of their tetanus germ is rendered inert by precipitation with
absolute alcohol. It is obtained by saturation with am-
monium sulphate, and the removal of the silt by dial\ >i-.

Further description of the individual proteids will l»e
in subsequent chapter-.

2*

CHAPTER II.

HISTORICAL SKETCH OF THE BACTERIAL POISONS.

IT must have l>een known to primitive man that the
eating of putrid flesh was liable to afttvt the health more
or less seriously; and when he began his endeavor.- t«>
preserve his food for further use, instances of poisoning
from putrefaction must have multiplied. However, the
distinguished physiologist, ALBERT VON HALLER, seems
to have been the first to make any scientific experiments
concerning the effects of putrid matter upon animals. He
injected aqueous extracts of putrid material into the veins
and found that death resulted. Later in the eighteenth
century MORAND gave an account of the symptoms in-
duced by eating poisonous meat. In the early part of
the present century (1808 to 1814) GASPARD carried on
similar experiments. He use as material the putrid flesh
of both carnivorous and herbivorous animals. With these
he induced marked nervous disturbance^., MS stiffness of the
limbs, opisthotonos, and tetanus. GASPARD concluded from
the symptoms that the poisonous effects were not due to
carbonic acid gas or hydrogen sulphide, but thought it
possible that ammonia might have part in their produc-
tion. In 1820 KERNER published his first CSSMV on poi-
sonous sausage, which was followed by a second in 1 >'_'•_'.
At first he thought that the poisonous properties were din-
to a fatty acid, similar to the sebacie of THENARD, and
which originated during putrefaction. Later he modified
these views, and believed the poison to be a compound con-
sisting of the sebacic acid and a volatile principle. This may
be regarded as the first suggestion as to the probability of
the development of a poisonous substance with basic prop-
erties in decomposing matter. In 1822, Drn;i: observed
a peculiar disease among the soldiers under his care, who,

11 ISToKir AL SKETCH. 28

during tin- \v:inii Mini dry summer of that year.
<• ..... pelled to drink very foil I water. Later M.v;KNDIE,
induced by the investigations of (lAspAKD an<l the obser-
vation< of I>iri;i::. made many experiments, in which do<rs
and other animals were OOBuned over ve~- eU containing
j)Utrid animal matter and compelled constantly to breathe
the emanations therefrom. The effects varied markedly
with tin- species of animal and the nature of the putrid
material, l>nt in some instances symptoms were indn<-ed
which resembled closely those of typhoid fever in man.
l.i.i KKT directs! his attention to the chemical changes
produced in 1)1 ..... 1 by putrefaction, but accomplished noth-
ing of special value. Hri'UY injected putrid material into
the jugular vein of a horse, and with TROUSSEAU studied
alterations produced in the blood by these injections.

During the third decide of the present century there
were main investi«r«itors in addition to those mentioned
alx»ve, who endeavored to ascertain the active agent in
|M.i-onon- foods. DANN, WEISS, Bi'riiNER, SCHUMANN,
CADET DE GASSICOURT, and ORFILA studied jwisonous
sausage, but made no advance upon the work done by
KEUNKH. HENNEMAN, HUNNEFELD, WESTRUMB, and
SKRTURNER nuide contributions concerning poisonous
i-hee-c, but all In'lievcd the caseic acid of KERNER to
IM- the poi-oiious princi])lc.

In 1850 SCHMIDT, of Dorpat, made some investijrations
on the deoompomtion prodoctB and volatile substanco
found in cholera st<M>ls ; and, two years later, MEYKI:, of
licrlin, injecte<l the blcxnl and stools of cholera patients
into lower animals. In 1858 STICH made an important
contribution on the <• fleets of acute poisoning with putrid
material. He ascertained that, when -riven in sufficient
ijuantity, putrid matter produces an int4-stinal eatarrh, with
choler.iic stools. Nervous symptoms, trembling, nn-teady
trait, and, finallv, convulsions were also observed. STICK
made careful po>t-niortcm <-xaminations, and was unable
to find any characteristic or important lesions. Theo-
retically, he concluded that the putrid material contained
a ferment which pnxluced rapid decomposition of the blood.

24 BACTERIAL POISONS.

In 1856 PANUM published a most important contribu-
tion to the knowledge of the nature of the poison present
in putrid flesh. He first demonstrated positively the
chemical character of the poison, inasmuch as he shown I
that the aqueous extract of the putrid material retained
its poisonous properties after treatment which would insure
the destruction of all organisms. His conclusions were as
follows :

(1) "The putrid poison contained in the decomposed
flesh of the dog, and which is obtained by extraction with
distilled water and repeated filtration, is not volatile, but
fixed. It does not pass over on distillation, but remains in
the retort.

(2) " The putrid poison is not destroyed by boiling, nor
by evaporation. It preserves its poisonous properties even
after the boiling has been continued for eleven hours, and
after the evaporation has been carried to complete desicca-
tion at 100°.

(3) "The putrid poison is insoluble in absolute alcohol,
but is soluble in water, and is contained in the aqueous ex-
tract which is formed by treating with distilled water the
putrid material which has previously been dried by heat
and washed with alcohol.

(4) " The albuminoid substances which frequently are
found in putrid fluids are not in themselves poisonous only
so far as they contain the putrid poison fixed and condensed
upon their surfaces, from which it can be removed by
repeated and careful washing.

(5) "The intensity of the putrid poison is comparable
to that of the venom of serpents, of curare, and of certain
vegetable alkaloids, inasmuch as 0.012 of a gramme of the
poison, obtained by extracting with distilled water putrid
material which had been previously boiled for a long time,
dried at 100°, and submitted to the action of absolute
alcohol, was sufficient almost to kill a small dog."

PANUM made intravenous injections with this poison, and
with ammonium carbonate, ammonium butyrate, ammo-
nium valerianate, tyrosine, and leucine, and found that the
symptoms induced by the putrid poison differed from those

HISTORICAL SKETCH. 25

caused by the other agents. Moreover, lie (bund (he symp-
toms (oditl'er from those of typhoid (ever, cholera, pyaemia,

anthrax, and sausage poisoning. He was also in doubt a> to
whether the poison acted directly upon the nervous system,
or whether it :ie<ed as a ferment upon tlie blood, causing

decomposition, the products of which affected the nerve-
eentres; but he \vassure that it could not correspond to
the ordinary ferments, inasmuch as it was not, decomposed
by prolonged boiling nor by treatment with absolute alcohol.
Certainly, the putrid poison could not consist of a living
organism.

The symptoms observed by PANUM varied greatly with
the quantity of the poison used and the strength of the
animal. After the intravenous injection of large doses,
death followed in a very short time. In these cases there
were violent cramps, and involuntary evacuations of the
urine and feces ; the respirations were labored, the pallor
was marked, sometimes followed by cyanosis, the pulse
fi-cblc, the pupils widely dilated, and the eyes projecting.
In these cases the autopsy did not reveal any lesion, save
that the blood was dark, imperfectly coagulated and slightly
infiltrated through the tissue. Post-mortem putrefaction
came on with extraordinary rapidity.

When smaller doses or mor«% vigorous animals were used,
the symptoms did not appear l>efore from a quarter of an
hour to two hours, and sometimes even later. In the-e
cases the symptoms were less violent, and the animal gen-
erally recovered. In all instances, however, the disturbances
were more or less marked.

In addition to the " putrid poison," PANUM obtained a
narcotic substance, the two being separated by t lie solubility
of the narcotic in alcohol. The alcoholic extract was evap-
orated todryness, the residue dissolved in water and injected
into the jugular vein of a dog. The animal fell into a deep
sleep, which remained unbroken for twenty-four hours,
when it awoke apparently in perfect health.

PANUM'S first contributions, which were published in
Danish, did not attract the attention which they deserved,
until after the lapse of several years. Now, however, their

26 BACTERIAL POISONS.

importance is fully appreciate!, and the distinguished inves-
tigator lived to receive the credit and honor due him.

WEBER, in 1864, and HEMMER and SCHWENNJV.I.I;
in 1866, confirmed the results obtained by PANUM ; and
SCHWENNINGER announced that in the various stages of
putrefaction different products are formed, and that these
vary in their effects upon animals. In 1866, BKNVK
JONES and DUPRE obtained from the liver a substance
which in solutions of dilute sulphuric acid gives the blue
fluorescence observed in similar solutions of quinine. To
this substance they gave the name " animal chinoidine."
Subsequently, the same investigators found this substance
in all organs and tissues of the body, but most abundantly
in the nerves. Its feebly acid solutions give precipitates
with iodine, potassio-mercuric iodide, phospho-molybdic
acid, gold chloride, and platinum chloride. From three
pounds of sheep's liver, they obtained three grammes of a
solution in which, after slight acidulation with sulphuric
acid, the intensity of the fluorescence was about the saun-
as that of a similarly acidulated solution of quinine sulphate
which contained 0.2 gramme of quinine per litre. Still
later, this base was obtained by MARINO-ZUCO.

In 1868, BERGMANN and SCHMIEDEBERG separated,
first from putrid yeast, and subsequently from decomposed
blood, in the form of a sulphate, a poisonous substance
which they named sepsine. The sulphate of sepsine forms
in needle-shaped crystals. Small doses (0.01 gramme) of
this substance were dissolved in water and injected into the
veins of two dogs. In a short time it produced vomiting,
and later diarrho3a, which, in one of the animals, after a
time, became bloody. Post-mortem examination showed,
in the stomach and intestines, bloody ecchymoses. It was
now believed that the " putrid poison " of PANUM had been
isolated, and that it was identical with sepsine, but further
investigations showed that this was not true. There are
marked differences in their effects upon animals, and sepsiue
has not been found to be generally present in putrid ma-
terial. It is only rarely found in blood, and the clo-i-t
search has failed to show its presence in pus. BERGMANN.

HISTORICAL SKETCH. 27

following the s;mn' method which he had used in extracting
tin's j)ois()ii from yeast, lias been unable to obtain it from
other putrid material. Moreover, he was not always SIH--
- I'M I in obtaining the poison from veast. Sepsine was
not obtained in quantity sufficient to serve for an ultimate
analysis, In nee, its composition remains unknown.

In ]s<;«) /I'LXKR and SONXKNSCHKIX prepared from
decomposed meat extracts a nitrogenous base, which in its
chemical reactions and physiological ('flirts resembled a tro-
phic and hyoseyamine. When injected under the skin of
animals it produced dilatation of the pupils, paralysis of
the muscles of the intestines, and acceleration of the heart-
beat ; but it is uncertain and inconstant in its action. This
probably results from rapid decomposition taking place in
it, or to variations in its composition at different stages of
putrefaction. This suhstamr lias also been obtained from
the bodies of those who have died from typhoid fever, and
it may be possible that the lx>lladonna-like delirium which
frequently characteri/es the later stages of this disease is
due to the ante-mortem generation of this poison within
the Uxly.

Since 1X70 many chemists have Urn en<ra«red in making
investigations on the products of putrefaction. We can
only mention a few names at present, while others will IK-
referred to subsequently in di<cussin«r the individual pto-
ma'iiic-.

First of all stands the Italian SELMI, who su<r*;csted the
name ptomaine, and whose researches furnished us with
much information of' value, and, what is probably of more
importance, <rave an impetus to the study of the chemistry
of putrefaction, which has already Urn productive of much
jrood and «rives promise of much more in the future. SKI. MI
showed that ptomaines could l>e obtained (1) by extracting
acidified solutions of putrid material with ether; (2) by
extract in::- alkaline solutions with ether ; (.'?) by extracting
alkaline solution- \\ith chloroform ; (4) by extracting with
amylic alcohol ; and(">) that there yet remained in the solu-
tions of putrid matter ptomaines which were not extracted
by anv of the above-mentioned reagents. In this way he

28 BACTERIAL POISONS.

gave some idea of the great number of alkaloidal
which might be formed among the products of putrefaction,
and the promising field thus discovered and outlined was
soon occupied by a busy host of chemists. In (he second
place, he demonstrated the fact that many of the ptomaines
give reactions similar to those given by the vegetable alka-
loids. This led the toxicologist into investigations, the
results of some of which we will ascertain further on.

SEI.MI, however, did not succeed in isolating completely
a single putrefactive alkaloid. All his work was done with
extracts. He remained ignorant, except in a general way,
of the composition of these bodies. NENCKI, in 1*7<;,
made the first ultimate analysis and determined the first
formula of a ptomaine. This was an isomer of col Iodine,
which will be described later.

RORSCH and FASSBENDER, in a case of suspected poison-
ing, obtained by the STAS-()TTO method a liquid which
could be extracted from acid as well as alkaline solutions
by ether, and which gave all the general alkaloidal reac-
tions. They were unable to crystallize either extract by
taking it up with alcohol and evaporating. The colorless
aqueous solution was not at all bitter to the taste. The
precipitate formed with phospho-molvbdic acid dissolved on
the application of heat, giving a green solution, which
became blue on the addition of ammonia. They believed
that this substance was derived from the liver, since fresh
ox-liver, treated in the same manner, gave them an alkaloid
which could be extracted with ether from acid as well as
from alkaline solutions. GUNNING found this same alka-
loid in liver-sausage from which poisoning had occurred.
RORSCH and FASSBENDER state that while in some of its
reactions this substance resembles digitaline, it is distin-
guished from this vegetable alkaloid by the failure of the
ptomaine to give the characteristic bitter taste.

SCHWANERT, whilst examining the de< i posing intes-
tines, liver, and spleen of a child which had died suddenly,
perceived a peculiar odor and obtained by the STAS-Orro
method (ether extract from an alkaline solution) small
quantities of a base, which was distinguished from nicotine

HISTORICAL SK K l( II . 29

and confine l»y il< greater volatility ami its peculiar odor,
llr -upposed tliat tliis sabstHloe WM produced by decom-
po-ition, ami, in order to ascertain the truth of hi- -nppo-
sition, lie took the organs of a cadaver that had lain for
>ixtecn d:i\> at a temperature of .".(I0 and \vas well decom-
|>osed. These were treated with tartarie acid and aleohol.
The acid solution was first ext raetnl with ether, and yielded
no result; it was then rendered alkaline and extracted
with ether. The latter extract gave, on evaporation, the
same suhstamv which he had found in the organs of the
child. The residue was a yellowish oil, having an odor
somewhat similar to propylamiiie. It was repulsive, hut
not hitter to the taste, and alkaline in reaction. On the
addition of hydrochloric acid, it crystallized in white needles,
which were freely soluhle in water, hut soluhle with diffi-
culty in alcohol. On the addition of ammonium hydrate
to this crystalline substance, a white vapor of unpleasant
odor was given off'. The crystals dissolved in sulphuric acid,
forming a solution which was at first colorless, hut which
gradually became dirty hrownish -yellow, and .gravish-
hrowu on the application of heat. On Ix'ing warmed with
sodium molybdate, a splendid blue color, becoming gradu-
ally gray, was produced. Potassium bichromate and sul-
phuric acid gave a reddish-brown, then a grass-green color.
Nitric acid gave a yellow color. A tartaric acid solution
of the crystals pnxlnced, on the addition of platinum chlo-
ride, a dirty yellow precipitate of small six-sided stars,
which contained 31.55 per cent, of platinum. Gold chlo-
ride gave a pale yellow, amorphous jirecipitatc; mercuric
chloride yielded white crystals; pota.ssio-mereuric iodide a
dirty-white precipitate; and potaflSiO-Oadmio iodide yielded
no result. Tannic acid produced only a turbidity. Sodium
phospho-molybdate gave a yellow, flooonleot precipitate,
which Ix-eame blue on the addition of ammonium hydrate.
This base has a slight reducing power, and in this it
iv-cinhles a substance obtained by SKLMI, but it differs
from SKI. MI'-* extract inasmuch as it does not give a violet
coloration on being warmed with sulphuric acid. In its
amorphous character, its Miavior to the general alkaloidal

30 BACTERIAL POISONS.

reagents, and its lack of bitter taste, it resembles (lie bast-
obtained by RORSCH and FASSBENDER, but, unlike that
alkaloid, it is extractable from alkaline solutions only.

SELMI, in commenting upon the base studied by RORSCH
and FASSBENDER, SCHWANERT, and himself, believing
that all were dealing with the same body, states that it
does not contain phosphorus, and that it is separated with
extreme difficulty from the vegetable alkaloids.

LIEBERMANN, in examining the somewhat decomposed
stomach and intestines in a case of suspected poisoning,
found an alkaloids! body which was unlike that studied by
the chemists mentioned above, inasmuch as it was not vola-
tile. The STAS-OTTO method was employed. The ether
extract from alkaline solution left, on evaporation, a brown-
ish, resinous mass, which dissolved in water to a turbid
solution, the cloudiness increasing on heating. This reac-
tion agrees with coniine, but the odor differed from that of
the vegetable alkaloid. The aqueous, strongly alkaline
solution gave the following reactions :

(1) With tannic acid, a white precipitate.

(2) With potassium iodide, a yellowish-brown, turning
to dark-brown precipitate.

(3) With chlorine water, a marked white cloudiness.

(4) With phospho-molybdic acid, a yellow precipitate.

(5) With potassio-mercuric iodide, a white precipitate.

(6) With mercuric chloride, a white cloudiness.

(7) With concentrated sulphuric acid, after a while, a
reddish-violet coloration.

(8) With concentrated nitric acid, after evaporation, a
yellowish spot.

These reactions exclude all vegetable alkaloids save
coniine. The putrefactive alkaloid does not distil when
heated on the oil-bath to 200°, while coniine distils at
135°. The former is with certainty distinguished from
coniine by its non-poisonous properties.

This substance is extracted by ether from acid, as well
as from alkaline solutions. The yellow, oily drops ob-
tained after the evaporation of the ether are soluble in
alcohol. The taste is slightly burning.

II ISTORICAL SKETCH . 31

SKI. MI obtained from lx>th putrefying and fresh inte—
tin-- ;i -iihstance which <r.ive the general alkaloidal reac-
tion- with pota-siuin iodide, p>ld chloride, |)latinuin chlo-
ride. potaaBlO-mercorio iodide, and phospho-molyl)dic acid.
It has strong reducing power, and when wanned with
sulphuric acid uives a violet coloration. These reactions
are not due to leucine, tyrosine, en -a tine, or creatinine.
This is the substance which, as has Urn state<l, SfiLMI con-
sidered identical with that observed by RoitSCH and FASS-
I',KM»KI; and ScHWANERT. The minor differentia observe* 1
by the different chemists may have been due to the varying
decrees of purity in which the substance was obtained by
them.

From In i ma n Ixxlies which had l>een dead from one to
ten months, SKLMI remove<l many alkaline bases. From
an ether solution of a unmlxr of these, one was removed
by treatment with carbonic acid uas. One base which was
insoluble in ether, but readily soluble in amylie alcohol,
was tbnnd to IM> a violent poison, producing in rabbits
tetanus, marked dilatation of the pupils, paralysis, and
death.

Parts of a human body preserved in alcohol were found
by SKI. MI to yield an easily volatile, phosphorus-containing
siib-tance, which is soluble in ether and carbon disulphide,
and jrives a brown precipitate with silver nitrate. It is
not the phosphide of hydrogen. A similar substance is
produced by the slow decomposition of the yolks of <^jrs.
With potassium hydrate it jjivcs otf ammonia and yields a
substance having an intense coniine odor. It is volatile
and reduces phosphomolvbdic acid.

SKI, MI also obtained from decomposing egg-albumin a
body, whose chloride forms in net-dies, and which has a
curare-like action on fro">. From one arsenical body which
had Ixvn buried for fourteen days, he obtained, by extract-
ing from an alkaline (made alkaline with baryta) solution
with ether, a substance which formed in net -dies and which
•rave crystalline salt- with acids. With sulphuric acid it
jrave a red color; with iodic acid and sulphuric acid it
liberated free iodine and irave a violet coloration; with

32 BACTERIAL POISONS.

nitric acid it gave a beautiful yellow, which deepened on
the addition of caustic potash. Platinum chloride gave no
precipitate save in highly concentrated solutions. From a
second arsenical body, SELMI obtained by the same method
a substance which gave, with tannic acid, a white precipi-
tate ; with iodine in hydriodic acid a kermes-brown ; with
gold chloride a yellow, which was soon reduced ; with
mercuric chloride a white; with picric acid, a yellow,
which gradually formed in crystalline tablets. This sub-
stance did not contain any arsenic, but was highly poi-
sonous. From the stomach of a* hog, which had been pre-
served in a solution of arsenious acid, SELMI separated an
arsenical organic base. The fluid was distilled in a current
of hydrogen. The distillate/ which was found to be strongly
alkaline, was neutralized with hydrochloric acid and evapo-
rated to dryness, when cross-shaped crystals, giving an odor
similar to that of trimethylamine, were obtained. This sub-
stance was found by CIACCIA to be highly poisonous, pro-
ducing strychnia-like symptoms. With iodine in hydriodic
acid it is said to give a gray, crystalline precipitate.

From the liquid which remained in the retort, a non-
volatile arsenical ptomaine was extracted with ether. An
aqueous solution of this gave with tannic acid a slowly
forming, yellowish precipitate, and similarly colored pre-
cipitates with iodine in hydriodic acid, platinum chloride,
auric chloride, mercuric chloride, potassio-mercuric iodide,
potassio-bismuthic iodide, picric acid, and potassium bi-
chromate. The physiological action of this substance as
demonstrated on frogs was unlike that of the arsines, but
consisted of torpor and paralysis.

MORIGGIA and BATTISTINI experimented with alkaloids,
obtained from decomposing bodies, upon guinea-pigs and
frogs, but did not attempt their isolation because of the
rapid decomposition which they undergo when exposed to
the air and by which they lose their poisonous properties.
These alkaloids they found to be easily soluble in ainylie
alcohol, less soluble in ether.

In 1871 LOMBROSO showed that the extract from mouldy
corn-meal produced tetanic convulsions in animals. This

II ISTORIC^ SKETCH. 35

threw sonic light upon the cases of '4«1 P^i'iaines which
had long Ixrn known to occur among the j«.
banly, who eat fermented and mouldy corn-meal. *Vl<<tlV('
BRUQNATELLI and ZENONI obtained by the SrAS-OYV.
method from this mouldy meal an alkaloidal substance
which was white, non-crystalline, unstable, and insoluble
in water, but readily soluble in alcohol and ether. With
sulphuric acid and bichromate of potassium it yields a
color reaction very similar to that of strychnine.

The action of the ether extracts from decomposed brain
iv-einbles that of curare", but is less marked and more
transitory. The beats of the frog's heart were decreased in
numU-r and strengthened in force; the nerves and the
muscles lost their irritability, and the animal passed into
a condition of complete torpor. The pupils were dilated.
GUARESCHI and Mosso, using the STAS-OTTO method,
obtained from human brains which had been allowed to
decompose at a temperature of from 10° to 15° for from
one to two months, both volatile and non-volatile bases.
Among the former only ammonia and trimethylamine were
in sufficient quantity for identification. With these, how-
ever, were minute traces of ptomaines.

They obtained non-volatile bases from lx)th acid and
alkaline solutions. From the former they separated a sub-
stance which gave precipitates "with gold chloride, phospho-
tunvistic acid, phospho-molybdic acid, MAYER'S reagent,
palladium chloride, picric acid, iodine in potassium iodide,
and slightly with tannic acid. This substance was not
precipitated with platinum or mercury.

1'Yom the alkaline extract there was obtained a substance
which in dilute hydrochloric acid solutions gave with gold
chloride a heavy yellow precipitate with reduction, also
pwipitates with phospho-molybdic acid, platinum chloride,
MAYER'S reagent, picric acid, phospho-tungstic acid,
M AKMK'S reagent, iodine in potassium iodide, tannin, bi-
chromate of potassium, palladium chloride, and mercuric
chloride. It reduces ferric salts. From decomposed fibrin
the -nine i n ye.- ti gators obtained one well-defined ptoina'ine.
Analyses of the platinum compound of this substance

32 BACTERIA^ POISONS.

nitric acid it gave^ ' This substance will be discussed in
the addition of-/; ""

precipijft* fresh brain substance they separated ammonia,
Sffniethylamine, and an undetermined base. These, how-
ever, are not to be regarded as products of putrefaction,
but as resulting from the action of the reagents upon the
brain substance. The trimethylamine probably arises from
the splitting up of lecithin, while the undetermined base
is most likely choline, which also results from the breaking
up of the lecithin molecule.

They also show that when DRAGENDORFF'S method is
used basic substances can be obtained from fresh meat, and
these are shown to be produced by the action of the sul-
phuric acid on the flesh.

To BRIEGER, of Berlin, is due the credit of isolating
and determining the composition of a number of ptomaines.
From putrid flesh he obtained neuridine, C6H14N2, and
neurine, C5H13NO. The former is inert, while the latter is
poisonous. From decomposed fish he separated a poisonous
base, C2H4(NH2)2, which is an isomerideof ethylenediamine,
muscarine, C6H15NO3, and an inert substance, C7H17NO2,
gadinine. Rotten cheese yielded neuridine and trimethyla-
mine. Decomposed glue gave neuridine, dimethylamine,
and a muscarine-like base. In the cadaver, he has found
in different stages of decomposition, choline, neuridine, tri-
methylamine, cadaverine, C5H14N2, putrescine, C4H,2N2, and
saprine, C5H16N2. These are all inert. After fourteen
days of decomposition he found a poisonous substance,
mydaleine. From a cadaver which had been kept at
from — 9° to + 5° for four months, BRIEGER obtained
mydine, C8HnNO, the poisonous substance mydatoxine,
C6H13N02, also the poison methyl-guanidine. From
poisonous mussel he separated mytilotoxine, C6H15NO2.
From pure cultures of the typhoid bacillus of KOCH and
EBERTH, BRIEGER obtained a poison, typhotoxinc, and,
from like cultures of the tetanus germ of ROSENBACH,
tetanine. All of these bases will be discussed in detail in
a subsequent chapter.

HISTORICAL SKETCH. 35

GAUTIEU and KTAUD have also isolate! ptomaines which
will be described Inter.

In iss."), VAUGHAN succ^-ded in isolating an active
nt from poisonous chccsr, to which he gave the name
t v rot o\ icon. Tliis discovery has l>een confirmed by NEW-
ION. WALLACE, SCHAFFER, STANTON, FIRTH, LADD,
WOLFF, KIMURA, DAVIS, and KINNICUTT.

NICATI and RIETSCH, KOCH, and others, have shown
the presence of a poisonous substance in cultures of the
cholera bacillus. SALMON and SMITH have done the same
with cultures of the swine-plague germ ; HoFFA, with those
of the anthrax bacillus; and BRIEGER with those of the
tetanus germ.

In 1888, CHRISTMAS obtained from cultures of the
-t.iphylococcus pyogene- aureus a proteid which, when in-
jected into the anterior chamber of the eye or under the
skin, causes suppuration.

In 1889, HANKIN isolated from cultures of the bacillus
anthracis a poisonous albnmosc, which, when employed in
large doses, proves fatal, and in small doses gives immunity.

In 1888, Roux and YERSIN showed that the chemical
poison of LOFFLER'S diphtheria bacillus is a proteid body
which they l>elieved to be of the nature of a ferment. In
1X!K), this work was continued by BRIEGER and FRANKEL
in their memorable contributi«-ui on bacterial poisons, in
which they detail the methods by which they isolate their
"toxalbumins" from cultures of the LoFFLER bacillus,
the anthrax bacillus, EBERTH'S germ, the cholera vibrio,
and the staphylococeus pyogencs aureus. MARTIN made a
more detailed study of the albumoses of anthrax. VAUGHAN
reports! poisonous proteids in cultures of two toxicogenic
uerms found in drinking-water, also in cultures of three of
r>«M»K KR'S Bummer diarrhoea germs and in poisonous cheese.
Now and SnnvEiNlTZ found lx>th basic and proteid
poisons in cultures of the swine-plague bacillus.

Many other contributions have Ix-en made, many of
which will IK- mentioned in subsequent chapter-.

CHAPTER III.

FOODS CONTAINING BACTERIAL POISONS.

POISONOUS MUSSELS. — Judging from the symptoms
produced, there seem to be three different kinds of poison-
ous mussel. In one class, the symptoms resemble those of
a true gastro-intestinal irritant. FODERE reports the case of
a sailor, who, after eating a large dish of mussels, suffered
from nausea, vomiting, pain in the stomach, tenesmus, and
rapid pulse. After death, which occurred within two days,
the stomach and intestines were found inflamed and filled
with a tenacious mucus. COMBE and others also report
cases of the choleraic form of poisoning from mussel.

However, the symptoms which most frequently manifest
themselves after the eating of poisonous mussels are more
purely nervous. A sensation of heat and itching appears
usually in the eyelids, and soon involves the whole face,
and perhaps a large portion of the body. An eruption,
usually called nettle-rash, though it may be papular or
vesicular, covers the parts. The itching is most annoying,
and may be accompanied by marked swelling. There
follows a distressing asthmatic breathing, which is relieved
by ether. In some cases reported by MOHRING, dyspnea
preceded the eruption, the patients became insensible, the
face livid, and convulsive movements of the extremities
were noticed. BURROW reports similar cases with delirium,
convulsions, coma, and death within three days.

In a third class of cases, there may be a kind of intoxi-
cation resembling somewhat that of alcohol, then paralysis,
coma, and death.

In 1827, COMBE observed thirty persons poisoned, two
of them fatally, with mussels. He describes the symptoms
as follows : "None, so far as I know, complained of any-
thing peculiar in the smell or taste of the animals, and

POISONOUS MUSSELS. 37

none suffered immediately after taking them. In general,
an hour or two elapsed, sometimes more; and the bad
rtfrrts cmiMsted rather in uneasy feelings and debility than
in any distress referable to the stomach. Some children
su til -red from eating only two or three; and it will be re-
membered that Robertson, a young and healthy man, only
look live or six. In two or three hours they complained
of a slight tension at the stomach. One or two had cardi-
algia, nausea, and vomiting; but these were not general or
last in^ symptoms. They then complained of a prickly feel-
ing in their hands, heat and constriction of the mouth and
throat; difficulty of swallowing and speaking freely; numb-
ness about the mouth, gradually extending to the arms, with
great debility of the limbs. The degree of muscular de-
bility varied a good deal, but was an invariable symptom.
In some it merely prevented them from walking firmly,
but in most of them it amounted to perfect inability to
stand. While in bed they could move their limbs with
tolerable freedom, but on being raised to the perpendicular
posture they felt their limbs sink under them. Some com-
plained of a bad, coppery taste in the mouth, but in general
this was in answer to what lawyers call a leading question.
There was slight pain of the abdomen, increased on pres-
sure, particularly in the region of the bladder, which organ
suffered variously in its functions. In some the secretion
of urine was suspended, in others it was free, but passed
with pain and great effort. The action of the heart was
feeble ; the breathing unaffected ; the face pale, expressive
of much anxiety ; the surface rather cold ; the mental
faculties unimpaired. Unluckily, the two fatal cases were
not seen by any medical person ; and we are, therefore,
unable to state minutely the train of symptoms. We ascer-
tained that the woman, in whose house were five sufferers,
went away as in a gentle sleep, and that a few moments
before death she had spoken and swallowed."

The woman died within three hours, and the other death
was that of a watchman, who was found dead in his box
si x or seven hours after he had eaten the mussels. Post-

38 BACTERIAL POISONS.

mortem examination in these showed no abnormality. The
stomach contained some of the food partially digested.

The explorer VANCOUVER reports four cases similar to
those observed by COMBE. One of the sailors died in five
and a half hours after eating the mussels.

In some recent cases reported by SCHMIDTMANN, as
quoted by BRIEGER, the symptoms were as follows : Some
dock hands and their families ate of cooked blue mussels
which had been taken near a newly built dock. The
symptoms appeared, according to the amount eaten, from
soon after eating to several hours later. There was a sen-
sation of constriction in the throat, mouth, and lips ; the
teeth were set on edge, as though sour apples had been
eaten. There was dizziness, no headache ; a sensation of
flying, and an intoxication similar to that produced by
alcohol. The pulse was hard, rapid (eighty to ninety), no
elevation of temperature, the pupils dilated and reaction-
less. Speech was difficult, broken, and jerky. The limbs
felt heavy ; the hands grasped spasmodically at objects and
missed their aim. The legs were no longer able to support
the body, and the knees knocked together. There was
nausea, vomiting, no abdominal pain, no diarrhoea. The
hands became numb and the feet cold. The sensation of
cold soon extended over the entire body, and in some the
perspiration flowed freely. There was a feeling of suffoca-
tion, then a restful and dreamless sleep. One person died
in one and three-quarters of an hour, another in three and
one-half hours, and a third in five hours, after eating of
the mussels.

In one of these fatal cases rigor mortis was marked and
remained for twenty-four hours. The vessels of all the
organs were distended, only the heart was empty. ViR-
CHOW concluded from the conditions observed that the
blood had absorbed oxygen with great avidity. There was
marked hyperiemia and swelling of the mucous membrane
of the stomach and intestines, which VIRCHOW pronounced
an enteritis. The spleen was enormously enlarged and the
liver showed numerous hemorrhagic infarctions.

Many theories have been advanced to account for poison-

POISONOUS MUSSKI.S 39

OIH mussels. It was formerly believed that the effects were
due to copper which the animate obtained from the bottoms
of vessels ; but, as CIIKISTISON remarks, copper does not
produce these symptoms. Moreover, CHRISTISON made
analysis of the mussels which produced the symptoms ob-
served by (IOMBE, and was unable to detect any copper.
BOUCHAHDAT found copper in some poisonous mussels, but
he does not state the amount of the copper nor the source
of the animals.

EDWARDS advanced the theory that the symptoms were
wholly due to idiosyncrasy in the consumer. This may be
true in some instances where only one or two of those par-
taking of the food are affected, but it certainly is not a
tenable hypothesis in such instances as those reported by
COMBE and SCHMIDTMANN, where a large number or all
those who partook of the food were affected.

COLDSTREAM found the livers of the Leith mussels, as
he thought, larger, darker, and more brittle than normal,
and to this diseased condition he attributed the ill effects.

LAMOROUX, MOHRING, DE BEUME, CHENU, and DU
RONDEAU have supposed that the poisonous effects were
due to a particular species of medusa upon which the mus-
sels feed. DE BEUME found in the vomited matter of one
I>erson, suffering from mussel poisoning, some medusae, and
he states that these are most abundant during the summer,
when mussels are most frequently found to be poisonous.

The theory of BURROW that the animal is always poison-
ous during the period of reproduction has been received
with considerable credit. However, cases of poisoning have
occurred at different seasons of the year.

CRUMPE, in 1872, suggested that there is a species of
mussel which is in and of itself poisonous, and this species
is often mixed with the edible variety. SCHMIDTMANN and
V i \u iiou support this idea. They state that the poisonous
species has a brighter shell, a sweeter, more penetrating,
bouillon-like odor than the edible kind, also that the flesh
of the former is yellow and that the water in which they
are cooked is bluish. LOHMEYER also champions this
opinion. This theory, however, is opposed by the majority

40 BACTERIAL POISONS.

of zoologists. MOBIUS states that the peculiarities of the
supposed poisonous variety pointed out by VIRCHOW and
SCHMIDTMANN are really due to the conditions under which
tie animal lives, the amount of salt in the water, the tem-
perature of the water, whether it is moving or still water,
the nature of the bottom, etc. Finally, MOBIUS states that
the sexual glands, which form the greater part of the
mantle, are white in the male aud yellow in the female.
However, it has been shown later by SCHMIDTMANN and
VIRCHOW that edible mussels may become poisonous if left
in filthy water for fourteen days or longer, and, on the
other hand, poisonous ones may become fit for food if kept
for four weeks in good water.

Cats and dogs which have eaten voluntarily of poisonous
mussels have suffered from symptoms similar to those ob-
served in man ; and rabbits have been poisoned by the
administration of the water in which the food has been
cooked. A rabbit which was treated in this manner by
SCHMIDTMANN died within one minute. From these
mussels BRIEGER extracted the ptomaine mytilotoxine,
which will be discussed in a subsequent chapter. This
poison has a curare-like action. Whether or not those
mussels which produce other symptoms also contain pto-
maines, remains for future investigations to determine.

In 1887 three other cases of mussel poisoning, one fatal
case, occurred at Wilhelmshaven, the place which supplied
BRIEGER with the mussels from which he obtained mytilo-
toxine. SCHMIDTMANN has found that non-poisouons
mussels placed in the waters of this bay soon become poi-
sonous, and that the poisonous mussels from the bay placed
in the open sea soon lose their poisonous properties. LIN-
DER has found in the water of the bay and in the mussels
living in it a great variety of protozoa, amoeba, bacteria,
and other lower organisms, which are not found in the
water of the open sea nor in the non-poisonous mussel. He
has also found that, if the water of the bay be filtered, non-
poisonous mussels in it do not become poisonous. He
therefore concludes that poisonous mussels are those which
are suffering from disease due to residence in filthy water.

POISONOUS FISH. 1 1

has tested dead and decomposed mussels taken
IV.. in the (tpen sea for mytilotoxine, with negative results.

s OYSTERS AND EELS. — PAS^U-IKI: reported
< :i-es of poisoning at Havre from the eating of oysters
taken from an artificial bed which had been established
near the outlet of a drain from a public water-closet.
CHRLSTISON says that an " unusual prevalence of colic,
diarrlnea, and cholera" at Dunkirk was believed to have
been traced to an importation of unwholesome oysters from
the Normandy coast. VAUGHAN and Now obtained
tests for tyrotoxicon in the liquor of some decomposed
oysters which had caused illness in many people at a church
festival.

YIUKY states that many persons were attacked with
violent pain and diarrlm'a a few hours after eating a pate
made of eels from a stagnant cattle-ditch near Orleans,
also that similar cases have occurred in various parts of
France, and that domestic animals have been killed by
eating the remains of the poisonous dish.

POISONOUS FISH. — While many species offish are popu-
larly regarded as poisonous, but little scientific work has
been done in this line, and we are not prepared to say to
what extent this popular idea is correct. MruRA and
T \KKSAKI find that the ripe ovaries of tctrodon mhrlfn-x
contain a substance which induces in rabbits acceleration
of the respiratory movements, paralysis of the skeletal
muscles, mydriasis, increased peristalsis of the intestines,
and arrest of the heart.

The disease known as " kakke," which prevails from
May to October in Tokio is, according to MIUIJA and
others, an intoxication due to the eating of fish, which be-
long to the 8conibri<i(c. The affection is generally chronic
or subacute, seldom acute. The most characteristic symp-
tom is paralysis of the diaphragm with consequent dysp-
nn-a and disturbance of the action of the heart. Kleetri-
eal stimulation of the diaphragm ha-; proven to l>e the
most successful treatment.

42 BACTERIAL I'OISONS.

SAUSAGE POISONING. — This is also known as botulis-
mus and allantiasis. While considerable diversity has
been observed in symptoms of sausage poisoning, we can-
not divide the cases into classes from their symptoma-
tology as has been done in mussel poisoning. The first
effects may manifest themselves at any time from one hour
to twenty-four hours after eating of the sausage, and cases
are recorded in which it is stated, no symptoms appeared
until several days had passed. However, we must re-
member that trichinosis was frequently, in former times,
classed as sausage poisoning, and it is highly probable that
these cases of long delay in the appearance of the symp-
toms were really not due to putrefaction, but to the pres-
ence of parasites in the meat. A large majority of the one
hundred and twenty-four cases more recently reported by
MULLER sickened within twenty-four hours, and out of
the forty-eight of these which were fatal, six died within
the first twenty-four hours. At first there is dryness of
the mouth, constriction of the throat, uneasiness in the
stomach, nausea, vomiting, vertigo, indistinctness of vision,
dilatation of the pupils, difficulty in swallowing, and
usually diarrhoea, though obstinate constipation may exist
from the first. There is, as a rule, a sensation of suffoca-
tion, and the breathing becomes labored. The pulse is
small, thready, and rapid. In some cases the radial pulse
may be imperceptible. Marked nervous prostration and
muscular debility follow. These symptoms vary greatly
in prominence in individual cases. The rechting and vom-
iting, which may be most distressing and persistent in
some instances, in others are trivial at the beginning and
soon cease altogether. The same is true of the diarrhoea.
As a rule, the functions of the brain proceed normally, but
there may be delirium, then coma and death. In some
there are marked convulsive movements, especially of the
limbs, in others paralysis may be an early and marked
symptom. The pupils may dilate, then become normal
and again dilate. There is frequently ptosis, and paralysis
of the muscles of accommodation is not rare. Complete
blindness has followed in a few instances.

SAUSAGE POISONING. 43

The fatality varies greatly in different outbreaks. In
1820 KKIINEK collected reports of seventy-six cases, of
which thirty-seven were fatal. In his next publication
(1822) he increased the number to one hundred and fifty-
five cases, with eighty-four fatal results. This gave a
mortality of over fifty per cent., while in one outbreak
reported by MI'LLKU the mortality was less than two per
cent.

A large proportion of the cases of sausage poisoning
have occurred in Wiirtemberg and the immediately adja-
cent portions of Baden. This fact has, without doubt,
been correctly ascribed to the methods there practised of
preparing and curing the sausage. It is said to be com-
mon for the people to use the blood of the sheep, ox, and

at in the preparation of this article of diet. Moreover,
the blood is kept sometimes for days in woodeu boxes and
at a high temperature before it is used. In these cases it
is altogether likely that putrefaction progresses to the poi-
sonous stage before the process of curing is began. How-
ever, cases of poisoning have occurred from beef and pork
sausages as well.

Moreover, the method of curing employed in Wiirteni-
berg favors putrefaction. A kind of sausage known as
- "blunzeu" is made by filling the stomachs of hogs with
the meat. In curing, the interior of this great mass is not
acted upon, and putrefaction sets in. The curing is usually
done by hanging the sausage in the chimney. At night
the fire often goes out and the meat freezes. The alternate
freezing and thawing render decomposition more easy.
The interior of the sausage is generally the most poison-
ous. Indeed, in many instances those who have eaten of
the outer portion have been unharmed, while those who
have eaten of the interior of the same sausage have been
most seriously affected.

Many German writers state that when a poisonous saus-
age is cut, the putrid portion has a dirty, grayish-green
color, anil a soft, smeary consistency. A disagreeable
odor, resembling that of putrid cheese, is perceptible. The
ta-te is unpleasant, and sometimes a smarting of the mouth

44 BACTERIAL POISONS.

aud throat is produced. Post-mortem examination alter
sausage poisoning shows no characteristic lesion. It is
generally stated that putrefaction sets in very tardily, but
MULL.ER shows that no reliance can be placed upon this
point, and states that out of forty-eight recorded autop>i< •-.
it was especially stated in eleven that putrefaction rapidly
developed. In some instances there has been noticed
hypersemia of the stomach and intestinal canal, but this is
by no means constant. The liver aud brain have been re-
ported as congested, but this would result from the failure
of the heart, and would, by no means, be characteristic of
poisoning with sausage.

YON FABER, in 1821, observed sixteen persons who
were made sick by eating fresh, uusmoked sausage made
from the flesh of a pig which had suffered from an abscess
on the neck. Five of the patients died. The symptoms
were as follows: There was constriction of the throat,
difficulty in swallowing, retching, vomiting, colic-like
pains, verti'go, hoarseness, dimness of vision, and headache.
Later and in severer cases, there was complete exhaustion,
and, finally, paralysis. The eyeballs were retracted, the
pupils were sometimes dilated, then contracted; they did
not respond to light ; there was paralysis of the upper lids.
The tonsils were swollen, but not as in tonsillitis. Liquids
which were not irritating could be carried as far as the
oesophagus, when they were then ejected from the mouth
and nose with coughing. Solid foods could not be swal-
lowed. On the back of the tongue aud in the pharynx
there was observed a pur i form exudate.

Obstinate constipation existed in all, while the sphincter
ani was paralyzed. The breathing was easy, but all had
a croupous cough. The skin was dry. There was incon-
tinence of urine. There was no delirium and the mind
remained clear to the last.

Post-mortem examinations were held on four. The
skin was roughs— " goose-ski u." The abdomen was re-
tracted. The large vessels in the upper part of the stom-
ach were filled with black blood. The contents of the
stomach consisted'of a reddish-brown, semi-fluid substance,

SAUSACK I'll | S«»N '1 N(J. \:>

which gave ofl' a repugnant, acid odor. In one case the
omentuin was found greatly congested. The large intes-
tine was very pale, and the right ventricle of the heart
was filled with dark fluid blood.

Sen r/ cites thirteen cases of poisoning from liver saus-
age in which the symptoms differed from the foregoing in
the following respects:

(1) In only one out of the thirteen was there const ipa-
tinn; all the others had numerous watery, typhoid-like

stools.

(2) Symptoms involving the sense of sight were present
in only three; in all the pupils were unchanged.

(3) The croupons cough was wholly wanting ; though
in many there was complete loss of voice. Difficulty of
swallowing was complained of by only one.

(4) Delirium was marked in all ; and in one the dis-
turbance of the mental faculties was prominent for several
weeks.

ft>) There were no deaths.

(6) The time between eating the sausage and the appear-
ance of the symptoms varied from eighteen to twenty-four
hours, and the duration of sickness from one to four
weeks ; though in one case complete recovery did not occur
until after two and one-half months.

The sausages were not smok'ed, and all observed a garlic
odor, though no garlic had been added to the meat.

TRIPE reports sixty -four cases. The symptoms came
on from three and one-half to thirty-six hours after eating.
The stools were frequent, watery, and of offensive odor.
In some there was delirium. One died. In the fatal case
the hands and face were cold and swollen. The pulse was
rapid and weak. The pupils were contracted, but re-
sponded to light. The small intestine was found inflamed.

HEDINGER reports the case of a man and a woman with
the usual symptoms, but during recovery the dilatation of
the pupils was followed by contraction. Birds ate of this
sausage, and were not affected.

UO-KU reports ease-, in which there were found, after
death, abscess,..., of the tonsils, a dark, bluish appearance

3*

46 BACTERIAL POISONS.

of the mucous membrane of the pharynx, larynx, and
bronchial tubes, dark redness of the fundus of the stom-
ach, and circumscribed, gray, red, and black spots on the
mucous membrane of the intestine. The liver was brittle
and the spleen enlarged.

Many theories concerning the nature of the active prin-
ciple of poisonous sausage have been advanced. It was
once believed to consist of pyroligneous acid, which was
supposed to be absorbed by the meat from the smoke used
in curing it; but it was soon found that unsmoked sausage
might be poisonous also. EMMEBT believed that the active
agent was hydrocyanic acid, and JAGER'S theory supposed
the presence of picric acid. But these acids are not found
in poisonous sausage, and, moreover, their toxicological
effects are wholly unlike those observed in sausage poison-
ing. As we have elsewhere seen, KERNER believed that
he had found the poisonous principle in a fatty acid. This
theory was supported by DANN, BUCHNER, and SCHU-
MANN. KERNER believed the poison to consist of either
caseic or sebacic acid, or both, while BUOHNER named it
acidum botuliuicum ; but the acids of the former proved
to be inert, and that of the latter to have no existence.
SCHLOSSBERGER first suggested that the poisonous sub-
stance is most probably basic in character, and he found
an odoriferous, ammoniacal base which could not be found
in good sausage, and which did not correspond to any
known amides, imides, or nitril bases. However, this
substance has not been obtained by anyone else, nor has
it been demonstrated to be poisonous.

LIEBIG, DUFLAS, HiRSCH, and SIMON believed in the
presence of a poisonous ferment. VAN DEN CORPUT de-
scribed sarcina botulina, which was believed to constitute
the active agent. MULLER, HOPPE-SEYLER, and others
have found various microorganisms, and VIRCHOW, EICH-
ENBERG, and others have examined microscopically the
blood of persons poisoned with sausage. Recently, EHR-
ENBERG has attempted to isolate the poisonous substance
by employing BRIEGER'S method, but he obtained only
inert substances.

POISONOUS HAM. 47

GAFFKY and PAAK have made a thorougli study of
Mum- sausage which poisoned a large number of people,
among whom one, a strong man, died. The sausage was
made of horse-Hesh and liver. In the majority of the
jKirsons the symptoms came on within six hours and in
one instance within half an hour. Many had a severe
chill ; some did not. The most prominent symptoms were
headache, loss of appetite, pain in the bowels, vomiting
and purging. In the fatal case, however, there was no
vomiting. From the sausage (}AFFKY and PAAK isolated
a >hort bacillus, which when given by the mouth, sub-
c utaneously or intravenously produced the above symptoms,
with a fatal termination in most instances, in rabbits,
guinea-pigs, mice, and apes. GAFFKY and PAAK were
unable to isolate the chemical poison.

POISONOUS HAM. — Under this head we shall not discuss
cases of poisoning from trichina or other parasites, but shall
refer only to those instances in which the toxic agent has
originated in putrefactive changes. A number of such
eases have been observed within the past ten years, but
only a few of them have been investigated scientifically.
The best known of these, as well as the most thoroughly
studied, is the Well beck poisoning, which BALLARD in-
vestigated successfully. In June, 1880, a large number of
persons attended a sale of timber and machinery on the
estate of the Duke of Portland at Wellbeck. The sale
continued four days, and lunches were served by the pro-
prietress of a neighboring hotel. The refreshments con-
sisted of cold boiled ham, cold, boiled, or roasted beef,
cold beefsteak pie, mustard and salt, bread and cheese,
j)i< -kles and Chutney sauce. The drinks were bottle and
draught beer, spirits, ginger beer, lemonade, and water.
Many were poisoned, and BALLARD obtained the particu-
lars of seventy-two cases, among which there were four
deaths. The symptoms are given by BALLAUD as follows:

" I propose to speak of the attacks under the name of
'diarrlueal illness/ because diarrhoea was the most constant
of all the symptoms observed, and the other symptoms

4H BACTEUIAL POISONS.

were in some respects so peculiar that I am indisposed to
give to the disease any name otherwise generally recogni /<•<!.
As might have been anticipated from our experience of
diseases in general, there were varieties in severity among
the cases investigated ; and symptoms strongly marked in
some, were slightly marked or altogether wanting in others.
Perhaps I shall do the best service by giving first a general
sketch of the course of the illness, subsequently illustrating
it by a description of a few well-marked cases.

"A period of incubation preceded the illness. In fifty-
one cases where this could be accurately determined, it
was twelve hours or less in five cases; between twelve and
thirty-six hours in thirty-four cases ; between thirty-six
and forty-eight hours in eight cases; and later than this in
only four cases. In many cases the first definite symptoms
occurred suddenly, and evidently unexpectedly, but in some
cases there were observed during the incubation more or
less feeling of languor and ill health, loss of appetite,
nausea, or fugitive, griping pains in the belly. In about
a third of the cases the first definite symptom was a sense
of chilliness, usually with rigors, of trembling, in one case
accompanied by dyspnoea ; in a few cases it was giddiness
with faintness, sometimes accompanied by a cold sweat and
tottering; in others, the first symptom was headache or
pain somewhere in the trunk of the body, e. g., in the
chest, back, between the shoulders, or in the abdomen, to
which part the pain, wherever it might have commenced,
subsequently extended. In one case the first symptom
noticed was a difficulty in swallowing. In two cases it was
intense thirst. But however the attack may have com-
menced, it was usually not long before pain in the abdomen,
diarrhoea, and vomiting came on, diarrhoea being of more
certain occurrence than vomiting. The pain in several
cases commenced in the chest or between the shoulders, and
extended first to the upper and then to the lower part of
the abdomen. It was usually very severe indeed, quickly
producing prostration or faintness, with cold sweats. It
was variously described as crampy, burning, tearing, etc.
The diarrhceal discharges were in some cases quite uure-

ii A M . 49

strai nable, and (where a description of them could be ob-
tained) were said to have been exceedingly offensive and
usually of a dark color. Muscular weakness was an early
and very remarkable symptom in nearly all the cases, and
in many it was so great that the patient could only stand
by holding on to something. Headache, sometimes severe,
was a common and early symptom ; and in most cases there
was thirst, often intense and most distressing. The tongue,
when observed, was described usually as thickly coated
with a brown, velvety fur, but red at the tip and edges.
In the early stage the skin was often cold to the touch, but
afterward fever set in, the temperature rising in some
cases to 101°, 103°, and 104° F. In a few severe cases
where the skin was actually cold, the patient complained of
heat, insisted on throwing off the bedclothes, and was very
restless. The pulse in the height of the illness became
quick, counting in some cases 100 to 128. The above
were the symptoms most frequently noted. Other symp-
toms occurred, however, some in a few cases, and some only
in solitary cases. These I now proceed to enumerate.
Excessive sweating, cramps in the legs, or in both legs and
arms, convulsive flexion of the hands or fingers, muscular
twitchings of the face, shoulders, or hands, aching pain in
the shoulders, joints, or extremities, a sense of stiffness of
the joints, prickling or tingling or numbness of the hands
lasting far into convalescence in some cases, a sense of
general compression of the skin, drowsiness, hallucinations,
imperfection of vision, and intolerance of light. In three
cases (one, that of a medical man) there was observed yel-
lowness of the skin, either general or confined to the face
and eyes. In one case, at a late stage of the illness, there
was some pulmonary congestion, and an attack of what was
regarded as gout. In the fatal cases, death was preceded
by collapse like that of cholera, coldness of the surface,
pinched, features and blueuess of the fingers and toes and
around the sunken eyes. The debility of convalescence
was in nearly all cases protracted to several weeks.

"The mildest cases were characterized usually by little
remarkable beyond the following symptoms, vi/., abdominal

50 BACTERIAL POISONS.

pains, vomiting, diarrhoea, thirst, headache, and muscular
weakness ; any one or two of which might be absent."

The cause of this illness was traced conclusively to the
hams eaten. KLEIN found in the meat a bacillus, cultures
of which were used for inoculating animals. These inocu-
lations were found generally to be followed by pneumonia.
No attempt was made to isolate a ptomaine.

Later, BALLARD reported fifteen cases with symptoms
similar to the above, and with one death, from eating baked
pork. Not all of those who ate of this pork were made
sick. This might have been due to inequality in the putre-
factive changes in different portions of the meat, or it may
have been due to differences in temperature in various por-
tions of the meat during the cooking. In the blood, peri-
cardial fluid, and lungs of the fatal case, KLEIN observed
bacilli similar to those discovered in the Wellbeck inquiry.
Pneumonia was produced by inoculating guinea-pigs and
mice with these bacilli.

In meat which poisoned a large number of persons,
GARTNER found his bacillus euteritidis. The meat was
from a cow which had a severe diarrho3a for two days be-
fore she was killed. Of twelve persons who ate the flesh
raw, all were sick ; while of those who ate of the cooked
food a large per cent, were also affected. In the meat and
in the spleen of a person who died from the effects of the
poison, GARTNER found the bacillus, which proved fatal
to animals. Good beef, inoculated with this bacillus and
cooked some hours later, killed rabbits, guinea-pigs, and
mice. The skin of the people who were poisoned and re-
covered peeled off. The period of incubation varied from
two to thirty hours.

August 29, 1887, 256 soldiers and 36 citizens at Middle-
burg, Holland, were taken sick after eating meat from a
cow which had been killed while suffering from puerperal
fever. The symptoms were nausea, vomiting, purging,
elevation of temperature, and prostration. In some there
were observed dizziness, sleepiness, and dilatation of the
pupil. After a few days these symptoms gradually disap-
peared, and in many an eczematous eruption of the lips

POISONOUS MEAT. ;~» I

c annoyance. Pigs, cats, and dogs which ate of the
oll'al of this animal were also made sick. Thorough
cooking did not destroy the poison, and those who took
soup and houllion made from the meat were affected like
those who ate of the muscular fibre. In most of the cases
the symptoms came on within twelve bom's after eating
the meat.

On a fete-day at Zurich, in 1839, 600 persons who were
fed upon cold veal and ham were taken ill, with shivering,
giddiness, vomiting, and diarrlura. Some were delirious
and others were salivated, the saliva being extremely fetid.
In the worst cases there were involuntary stools, collapse,
and death. The cause was traced to putrefactive changes
in i he meat.

Si KDLER reports an instance of four persons having been
made sick by eating decomposed goose-grease. There were
giddiness, prostration, and violent vomiting. No metallic
poison could be found. The grease was rancid, of repul-
sive odor, and three ounces of it given to a dog produced
the same symptoms which had been observed in the
persons.

CIIKISTISON reports a number of cases in which |>ersons
were seriously, a few fatally, affected by eating various
kinds of meat which had undergone partial putrefaction.

OLLIVIKI; found six persons poisoned, four of them
fatally, by eating of decomposed mutton. He also men-
tions the poisoning of a family of three with ham pie.
Chemical analysis failed to reveal the presence of any
poison.

BOUTIQNY, having failed to find any poison in the meat
furnished at a festival, and to which the serious illness of
many was attributed, made a meal of stuffed turkey fur-
nished by the same dealer, but after a short time his coun-
tenance became livid, his pulse small and feeble, a cold
sweat bathed his body, and violent vomiting and purging
followed. His recovery was slow.

(iKisKLKR observed nausea, vomiting, purging, and
delirium after eating of bacon which was imperfectly cured.

52 BACTERIAL POISONS.

POISONOUS CANNED MEATS. — Cases of poisoning from
eating canned meats have become quite frequent. Although
it may be possible that in some instances the untoward
effects result from metallic poisoning, in the great majority
of cases the poisonous principles are formed by putrefactive
changes. In many instances it is probable that decomposi-
tion begins after the can is opened by the consumer. In
others, the canning is carelessly done and putrefaction is
far advanced before the food reaches the consumer. In
still other instances, the meat may be taken from diseased
animals, or it may undergo putrefactive changes before
the canning. What is true of canned meats is also true of
canned fruits and vegetables.

Dr. ASHWORTH, of Smithland, Iowa, has reported to us
three fatal cases of poisoning from canned apricots. An
infant, which was only eight days old, and which must
have received the poison from its mother's breasts, died
within a few hours. The mother died forty-three hours
after eating the apricots, and the father on the sixth day.
The symptoms corresponded with those of poisoning by
tyrotoxicou. However, it seems that no analysis was
made, and these may have been cases of mineral poisoning.

POISONOUS CHEESE. — In 1827 HUNNEFELD made some
analyses of poisonous cheese, and experimented with ex-
tracts upon the lower animals. He accepted the ideas of
KEENER in regard to poisonous sausage in a somewhat
modified form, and thought the active agents to be sebacic
and caseic acids. About the same time, SERTURNER,
making analyses of poisonous cheese for WESTRUMB, also
traced the poisonous principles, as he supposed, to these
fatty acids. We see from this that during the first part of
the present century the fatty acid theory, as it may be
called, was generally accepted.

In 1848, CHRISTISON, after referring to the work of
HUNNEFELD and SERTURNER, made the following state-
ment : " His (Hiinuefeld's) experiments, however, are not
quite conclusive of the fact that these fatty acids are really
the poisonous principles, as he has not extended his experi-

POISONOUS CHEESE 53

mental researches to the caseic ami sehacic acids prepared
in tin' ordinary way. His views will probably be altered
an<l simplified if future experiments should confirm the
late inquiries of BRAOONNOT, who has stated that PROUST'S
caseic acid is a modification of acetic acid combined with
an acrid oil."

In 1852 SCHLOSSBERGER made experiments with the
pure fatty acids and demonstrated their freedom from poi-
sonous properties. These experiments have been verified
repeatedly, so that now it is well known that all the fatty
acids obtainable from cheese are devoid of poisonous
properties.

It may be remarked here, that there is every probability
that the poisonous substance was present in the extracts
obtained by the older chemists. Indeed, we may say that
this is a certainty, since the administration of these extracts
to cats was, in some instances at least, followed by fatal
result. The great mass of these extracts consisted of fatty
acids, and as the chemists could find nothing else present,
they very naturally concluded that the fatty acids them-
selves constituted the poisonous substance.

Since the overthrow of the fatty acid theory, various
conjectures have been made, but none worthy of considera-
tion.

We make the following quotations from some of the best
authorities who wrote during the first half of the past
decade upon this subject :

HILLER says : " Nothing definite is known of the nature
of cheese poison. Its solubility seems established from an
observation by HUSEMANN, a case in which the poison was
transmitted from a nursing mother to her child."

Ih SKMANN wrote as follows: " The older investigations
of the chemical nature of cheese poison, which led to the
belief of putrefactive cheese acids and other problematic
substances, are void of all trustworthiness, and the dis-
covery of the active principle of poisonous cheese may not
be looked for in the near future, on account of the proper
animals for controlling the experiments with the extracts,

54 BACTERIAL POISONS.

as dogs can eat large quantities of poisonous cheese without
its producing any effect."

BRIEGER stated in 1885 : " All kinds of conjectures con-
cerning the nature of this poison have been formed, but all
are even devoid of historical interest; because they are not
based upon experimental investigations. My own experi-
ments toward solving this question have not progressed
very far."

In the above quotation we think that BRIEGER has
hardly done justice to the work of HUNNEFELD and SER-
TURNER. Their labors can hardly be said to be wholly
devoid of historical interest, and they certainly did employ
the experimental method of inquiry.

In the years 1883 and 1884 there were reported to the
Michigan State Board of Health about three hundred cases
of cheese poisoning. As a rule, the first symptoms ap-
peared within from two to four hours after eating the
cheese. In a few the symptoms were delayed from eight
to ten hours and were very slight. The attending physi-
cians reported that the gravity of the symptoms varied with
the amount of cheese eaten, but no one who ate of the
poisonous cheese wholly escaped. One physician reported
the following symptoms : " Everyone who ate of the
cheese was taken with vomiting, at first of a thin, watery,
later a more consistent reddish-colored substance. At the
same time the patient suffered from diarrhoea with watery
stools. Some complained of pain in the region of the
stomach. At first the tongue was white, but later it be-
came red and dry, the pulse was feeble and irregular ;
countenance pale, with marked cyanosis. One small boy,
whose condition seemed very critical, was covered all over
the body with bluish spots."

Dryness and constriction of the throat were complained
of by all. In a few cases the vomiting and diarrhosa were
followed by marked nervous prostration, and in some dila-
tation of the pupils was observed.

Notwithstanding the severity of the symptoms in many,
there was no fatal termination among these cases, though
several deaths from cheese poisoning in other outbreaks

POISONOUS CHKKSK. 55

have occurred. Many of the physicians at first diagnosed
the cases from the symptoms as due to arsenical poisoning,
and on this supposition some administered ferric hydrate.
Others gave alcohol and other stimulants and treated upon
the expectant plan.

VAUGHAN, to whom the cheese was sent for analysis,
made the following report: "All of these three hundred
cases were caused by eating of twelve different cheeses. Of
these, nine were made at one factory, and one each at three
other factories. Of each of the twelve I received smaller
or larger pieces. Of each of ten I received only small
amounts. Of each of the other two I received about
eighteen kilogrammes. The cheese was in good condition
and there was nothing in the taste or odor to excite sus-
picion. However, from a freshly cut surface there exuded
numerous drops of a slightly opalescent fluid which red-
dened litmus paper instantly and intensely. Although, as
1 have stated, I could discern nothing peculiar in the odor,
if two samples, one of good, the other of poisonous cheese,
were placed before a dog or cat, the animal would invari-
ably select the good cheese. But if only poisonous cheese
was offered, and the animal was hungry, it would partake
freely. A cat was kept seven days and furnished only
poisonous cheese and water. It ate freely of the cheese
and manifested no untoward symptoms. After the seven
days the animal was etherized and abdominal section was
made. Nothing abnormal could be found. I predicted,
however, in one of my first articles on poisonous cheese,
that the isolated poison would affect the lower animals.
As to the truth of this prediction we will see later.

" My friend, Dr. STERNBERG, the eminent bacteriologist,
found in the opalescent drops above referred to numerous
micrococci. But inoculations of rabbits with these failed
to produce any results.

" At first I made an alcoholic extract of the cheese. After
the alcohol was evaporated in vacuo at a low temperature
a residue consisting mainly of fatty acids remained. I ate
a small bit of this residue, and found that it produced dry-
ness of the throat, nausea, vomiting, and diarrhiea. The

56 BACTERIAL POISONS.

mass of this extract consisted of fats and fatty acids, and
for some weeks I endeavored to extract the poison from
these fats, but all attempts were unsuccessful. I then made
an aqueous extract of the cheese, filtered this, and drinking
some of it, found that it also was poisonous. But after
evaporating the aqueous extract to dry ness on the water-
bath at 100°, the residue thus obtained was not poisonous.
From this I ascertained that the poison was decomposed or
volatilized at or below the boiling-point of water. I then
tried distillation at a low temperature, but by this the
poison seemed to be decomposed.

" Finally, I made the clear, filtered aqueous extract,
which was highly acid, alkaline with sodium hydrate, agi-
tated this with ether, removed the ether, and allowed it to
evaporate spontaneously. The residue was highly poison-
ous. By re-solution in water and extraction with ether, the
poison was separated from foreign substances. As the ether
took up some water, this residue consisted of an aqueous
solution of the poison. After this was allowed to stand for
some hours in vacuo over sulphuric acid, the poison sepa-
rated in needle-shaped crystals. From some samples the
poisoned crystallized from the first evaporation of the ether,
and without standing in vacuo. This happened only when
the cheese contained a comparatively large amount of the
poison. Ordinarily, the microscope was necessary to detect
the crystalline shape. From sixteen kilogrammes of one
cheese, I obtained about 0.5 gramme of the poison, and in
this case the individual crystals were plainly visible to the
unaided eye. From the same amount of another cheese I
obtained only about 0.1 gramme, and the crystals in this
case were not so large. 1 have no idea, however, that by
the method used all the poison was separated from the
cheese."

To this ptomaine VAUGHAN has given the name tyr<>-
toxicon (rvpos, cheese, and TO&KOV, poison). Its chemistry
will be discussed in a subsequent chapter.

During 1887, WALLACE found tyrotoxicon in two
samples of cheese which had caused serious illness. The
first of these came from Jeanesville, Pa., and the symptoms

POISONOUS CHEESE. 57

as reported to WALLACE by DOOLITTLE, who had
of (lie discs, were as follows: " There were at least fifty
persons poi-mied by this cheese. There were also eight
others who ate of the cheese, but felt no unpleasant effects ;
whether this was due to personal idiosyncrasy, or to an
uneven distribution of the poison throughout the cheese, I
am unable to say.

"The majority, however, comprising fifty or sixty per-
sons, were seized, in from two to four hours after eating the
cheese, with vertigo, nausea, vomiting, and severe rigors,
though varying in their order of appearance and in severity
in different cases. The vomiting and chills were the most
constant and severe symptoms in all the cases, and were
soon followed by severe pain in the epigastric region,
cramps in the feet and lower limbs, purging and griping
pain in the bowels, a sensation of numbness or pins and
needles, especially in the limbs, and lastly, very marked
prostration, amounting almost to collapse in a few cases.

"The vomit at first consisted of the contents of the
stomach, and had a strong odor of cheese ; afterward it
consisted of mucus, bile, and in three or four of the severer
cases blood was mixed with the mucus in small quantities.
Microscopic examination of the same was not made, but to
the eye it appeared as such. The vomiting and diarrhea
lasted from two to twelve hours ; the rigors and muscular
cramps, one to two hours. The diarrhosal discharges, at
first fecal, became later watery and light colored. No
deaths occurred, and for the most part the effects were
transient, and all that remained on the following day were
the prostration and numbness ; the latter occurred in about
one-half the cases, and disappeared in from one to three
days.

" Children, as a rule, seemed to suffer less than adults,
and, of course, it was not possible to elicit as definite symp-
toms from them. The suddenness of the attack was
remarked by all, some feeling perfectly well until the
moment of attack. Nor did the symptoms seem to be in
proportion to the amount of cheese taken ; some of the
severest cases declared they had not eaten more than a cubic

58 BACTERIAL POISONS.

inch of it. One of the severest cases was about six and
one-half mouths pregnant, but no interference with preg-
nancy occurred. All the cheese which caused the sickness
came from the same piece."

The second sample of cheese examined by WALLACE
came from Riverton, N. J. This outbreak included a
smaller number of persons, all of whom recovered.

WOLFF has detected tyrotoxicon in cheese which poisoned
several persons at Shamokin, Pa. The pores of this cheese
were found filled with a grayish-green fungoid growth,
though it is not supposed that this fungus was connected in
any way with the poisonous nature of the cheese. Tests
were made for mineral poison with negative results, after
which tyrotoxicou was recognized both by chemical and
physiological tests. " A few drops of the liquid (extract),
placed on the tongue of a young kitten, produced prompt
emesis and numerous watery dejections with evident depres-
sion and malaise of the animal. A larger cat was similarly
affected by it, though the depression and malaise were not
so marked nor so long continued."

Cheese poisoning caused the death of several children in
the neighborhood of Heiligenstadt, in 1879, and there were
many fatal cases from the same cause in Pyrmont, in 1878.
Unfortunately we have not been able to find any detailed
account of either the symptoms or the post-mortem appear-
ances in these cases.

EHRHART has published the history of some cases of
poisoning from cheese, of which the following is an abstract :
The family of a workman, consisting of eight persons, ate for
supper 600 grammes (about eighteen ounces) of Limburger
cheese. The rind was covered with a heavy mould, while
the interior had become fluid from putrefaction, and was of
bitter taste. Three ate only of the mouldy rind, and these
remained well. The next morning, the five who had eaten
of the inner portion suffered from vertigo, nausea, vomiting,
and abdominal pains ; no stool. The father had convulsive
movements of all the extremities. The pupils were dilated,
and did not respond to light ; there were double vision,
cold sweat, skin cyanotic, abdomen distended, difficulty in

I'oISONOUS ril KKSE. 59

swallowing, delirium, mild trismus, and temperature 40° C.
(104° F.). The temj)erature of the mother, on account of
the great collapse, was subnormal. She had no convulsive
movements, but there was prolonged loss of consciousness.
The pulse was small and thready, and threatened paralysis
of the heart. Recovery was very slow. The others suf-
fered only from gastro-enteric symptoms. EHRHARDT
discusses the question as to whether these symptoms were
due to tyrotoxicon, or to infection with microorganisms ;
but as we have not had access to his original paper, we do
not know what his conclusions are. However, there cannot
be much doubt that in those cases in which the organism
is taken into the alimentary canal, it continues the elabora-
tion of its poisonous products.

In 18!»0 VAUGHAN made the following additional report
on poisonous cheese :

" During the past two or three years we have received
at the Hygienic Laboratory of Michigan University a
number of samples of cheese which, it was claimed, had
caused nausea and vomiting in those eating of them, and
in which we were unable to detect tyrotoxicon. Some of
these samples produced vomiting and purging in cats and
dogs to which the cheese was fed directly. The evidence
that these samples had been the actual cause of the sickness
among the people who had eaten of them was thus con-
firmed by the experiments upon the animals ; but inasmuch
as we were unable to detect the poison, we were compelled
to report as follows :

u ' The poisonous character of the cheese has been proven
l>\ experiments upon animals, but .we have failed to demon-
strate the nature of the poison. Tyrotoxicon could not be
detected '

" One sample of this class was found by Now to be very
poisonous. Some of this cheese was covered with absolute
alcohol, and after standing in a dish for some weeks the
alcohol was allowed to evaporate, then 100 grammes of the
cheese was fed to a young dog and caused its death within
a few hours. Sterili/ed milk to which a small bit of the
cheese was added, after standing in the incubator at 35°.

60 BACTERIAL POISONS.

for twenty-four hours, became so poisonous that 100 c. c. of
it introduced into the stomach of a full-grown cat caused
death. Now made plate cultures from the cheese and
from the spleen and liver of the dead animals, and suc-
ceeded in identifying one germ as common to both. Ster-
ilized milk inoculated with a pure culture of this germ, and
kept in the incubator, proved fatal to cats. But with the
advent of cold weather the germ lost its toxicogenic prop-
erties, which were not restored by subsequent cultivation in
the incubator.

" In a second class of samples, the poisonous character
of the cheese was not confirmed by direct feeding. Cats,
rats, and dogs were fed with the same quantities as above,
without any appreciable effect. The report made upon the
samples was as follows :

" ' Animals fed upon the cheese were not affected. Tyro-
toxicon could not be found. The sickness in the people
was probably due to some other cause.'

" The last sentence of this report was probably wrong, as
will be shown from the following experiment. Two kilo-
grammes of a cheese of this class was extracted repeatedly
with absolute alcohol. The part insoluble in alcohol was
then extracted with water. The aqueous extract, after
filtration, was allowed to fall slowly into three times its
volume of absolute alcohol. A voluminous, flocculent
precipitate resulted. After twenty-four hours the super-
natant fluid was decanted, and the precipitate was dissolved
in water and re-precipitated with absolute alcohol ; then it
was collected and speedily dried on porous plates. A small
bit of this precipitate was dissolved in water ; and forty
drops of this solution, injected under the skin on the back
of cats, produced invariably within one hour vomiting and
purging. After the partial collapse which followed the
vomiting and purging, and which was evidenced by the
animal sitting with its chin resting on the floor, recovery
gradually followed. The same amount of the solution
injected into the abdominal cavity of white rats rendered
the animals within ten or fifteen minutes perfectly limp,
and the only evidence of life observed was rapid respiratory

POISONOUS CHEESE. 61

movements. The rats lay upon their sides, and could be
handled without manifesting any attempt at movement.
In this condition some died after three or four hours, while
others, after lying in this position for from eighteen to
twenty-four hours, gradually improved, and alter some
days seemed to be wholly recovered.

" This substance belongs to the so-called poisonous albu-
mins. From its aqueous solutions it is not precipitated by
heat or nitric acid, singly or combined. Its solutions
respond to the biuret test. It is not precipitated by satura-
tion with sodium sulphate, nor by a current of carbonic
acid gas ; therefore, it is not a globulin. It is precipitated
by saturation with ammonium sulphate; and this fact
removes it from the peptones.

"That animals were not affected when fed with the
whole cheese may be explained by the supposition that they
did not in this manner get enough of the poison to affect
them. It, cannot be said positively that the samples of
cheese of the first class mentioned above owe their poison-
ous properties to this substance. We have not had the
opportunity of testing samples of this class since the
recognition of the poisonous proteid in those of the
second class. Four samples of the latter have been tested
for the poisonous albumin with positive results.

" It may be found that traces of this poison exist in all
samples of green cheese. This point will be investigated.

" It is highly probable that the poisonous effects of some
samples of sausage and meat are due to similar products of
bacterial activity."

In reference to the poisonous proteids in cheese and other
articles of food the following interesting questions arise :
How is the poisoning explained ? Is it not generally sup-
posed that poisonous proteids are not absorbable from
mucous membranes? MITCHELL and REICHERT showed
that the venom of serpents may be absorbed from mucous
membranes; especially did they find this to be true of the
poisonous peptone of the cobra. It may be, however, that
the bacteria, which are in the cheese and to which the
formation of the poisonous proteids is due, find their way

62 BACTERIAL POISONS.

through the intestinal walls and form their poisonous pro-
ducts within the spleen and other organs. The fact that
NOVY found the bacteria in the spleen and liver of the
animals experimented upon confirms this view.

POISONOUS MILK. — In 1885 VAUGHAN found tyrotoxi-
con in milk which had stood in a well-stoppered bottle for
about six months. It was presumed that this milk was,
when first obtained, normal in composition, but since this
was not known with certainty, the following experiments
were made : Several gallon bottles were filled with normal
milk, tightly closed with glass stoppers, aud allowed to
stand at the ordinary temperature of the room. From time
to time a bottle was opened and the test for tyrotoxicon was
made. These tests were followed by negative results until
about three months after the experiment was begun. Then
the poison was obtained from one of the bottles. The coagu-
lated milk was filtered through paper. The filtrate, which
was colorless and decidedly acid in reaction, was rendered
feebly alkaline by the addition of potassium hydrate and
agitated with ether. After separation, the ethereal layer
was removed with a pipette, passed through a dry filter-
paper in order to remove a flocculent, white substance which
floated in it, and then allowed to evaporate spontaneously.
If necessary, this residue was dissolved in water and again
extracted with ether. As the ether takes up some water,
there is usually enough of the latter left after the sponta-
neous evaporation of the ether to hold the poison in solu-
tion, and in order to obtain the crystals this aqueous solu-
tion must be allowed to stand for some hours in vacuo
over sulphuric acid.

From one-half gallon of the milk there was obtained
quite a concentrated aqueous solution of the poison after
the spontaneous evaporation of the ether. Ten drops of
this solution placed in the mouth of a small dog, three
weeks old, caused within a few minutes frothing at the
mouth, retching, the vomiting of frothy fluid, muscular
spasms over the abdomen, and after some hours watery
stools. The next day the dog seemed to have partially

POISONOUS MILK. 63

recovered, but was unable to retain any food. This condi-
tion continuing for two or three days the animal was killed
with chloroform. No examination of the stomach was
made.

In 1886 NEWTON and WALLACE obtained tyrotoxicon
from milk and studied the conditions under which it forms.
Their report is of so much value that the greater part of it
is herewith inserted.

" On August 7th twenty-four persons, at one of the hotels
at Long Branch, were taken ill soon after supper. At
another hotel, on the same evening, nineteen persons were
sei/ed with the same form of sickness. From one to four
hours elapsed between the meal and the first symptoms.
The symptoms noticed were those of gastro-intestinal irri-
tation, similar to poisoning by any irritating material —
that is, nausea, vomiting, cramps, and collapse ; a few had
diarrhoea. Dryuess of the throat and burning sensation
in the oasophagus were prominent symptoms.

" While the cause of the sickness was being sought for,
and one week after the first series of oases, thirty persons
at another hotel were taken ill with precisely the same
symptoms as noticed in the first outbreak.

" When the news of the outbreak was published one of
us immediately set to work, under the authority of the State
Board of Health, to ascertain the cause of the illness. The
course of the investigation was about as follows :

" The character of the illness indicated, of course, that
some article of food was the cause, and the first part of our
task was to single out the one substance that seemed at
fault. The cooking utensils were also suspected, because
unclean copper vessels have often caused irritant poisoning.
Articles of food, such as lobsters, crabs, blue fish, and
Spanish mackerel, all of which at times, and with some
persons very susceptible to gastric irritation have produced
toxic symptoms, were looked for, but it was found that
none of these had been eaten at the time of the outbreak.
The cooking vessels were examined, and all were found
clean and bright, and no evidence of corrosion was pre-
sented.

64 BACTERIAL POISONS.

" Further inquiry revealed the fact that all who had
been taken ill had used milk in greater or less quantities,
aud that persons who had not partaken of milk escaped
entirely ; corroborative of this, it was ascertained that
those who had used milk to the exclusion of all other food
were violently ill. This was prominently noticed in the
cases of infants fed from the bottle, when nothing but un-
cooked milk was used. In one case an adult drank about
a quart of the milk, and was almost immediately seized
with violent vomiting followed by diarrhoea, aud this by
collapse. Suffice it to say, that we were able to eliminate
all other articles of food and to decide that the milk was the
sole cause of the outbreak.

" Having been able to determine this, the next step was
to discover why that article should, in these cases, cause so
serious a form of sickness.

" The probable causes which we were to investigate were
outlined as follows : (1) Some chemical substance, such as
borax, boric acid, salicylic acid, sodium bicarbonate, sodium
sulphate, added to preserve the milk or to correct acidity.
(2) The use of polluted water as an adulterant. (3) Some
poisonous material accidentally present in the milk. (4) The
use of milk from diseased cattle. (5) Improper feeding of
the cattle. (6) The improper care of the milk. (7) The
development in the milk of some ferment or ptomaine,
such as tyrotoxicon.

"At the time of the first outbreak we were unable, un-
fortunately, to obtain any of the noxious milk, as that uu-
consumed had been destroyed; but at the second outbreak
a liberal quantity was procured.

" It was soon ascertained that one dealer had supplied
all the milk used at the three hotels where the cases of
sickness had occurred. His name and address having been
obtained, the next step in the investigation was to inspect
all the farms, and the cattle thereon, from which the milk
was taken. We also learned that two deliveries at the
hotels were made daily, one in the morning and one in the
evening; that the milk supplied at night was the sole
cause of the sickness, and that the milk from but one of

POISONOUS MILK. 65

the farms was at fault. The cows on this farm were found
to l>e in good health, and, besides being at pasture, were
well li-d with bran, middlings, and corn-meal.

" So far we had been able to eliminate as causes diseased
cattle and improper feeding, and we were then compelled
to consider the other possible sources of the toxic material.

" While the inspection of the farms was being made, the
analysis of the milk was in progress. The results of this
showed that no chemical substance had been added to the
milk, that it was of average composition, that no polluted
water had been used as a diluent, and that no poisonous
metals were present. This result left us nothing to con-
sider but two probable causes : improper care of the milk,
and the presence of a ferment.

"As to the former, we soon learned much. The cows
were milked at the unusual and abnormal hours of mid-
night and noon, and the noon's milking — that which alone
was followed by illness — was placed, while hot, in the cans,
and then, without any attempt at cooling, carted eight miles
during the warmest part of the day in a very hot mouth.

"This practice seemed to us sufficient to make the milk
unpalatable, if not injurious, for it is well known that when
fresh milk is closed up in a tight vessel and then deposited
in a warm place, a very disagreeable odor and taste are
developed. Old dairymen speak of the animal heat as an
entity, the removal of which is necessary in order that the
milk shall keep well and have a pleasant taste. While we
do not give this thing a name, we are fully convinced that
milk should be thoroughly cured by proper chilling and
aeration before it is transported any distance or sold for
consumption in towns or cities.

" This opinion is based on a study of the methods prev-
alent among exj>erienced dairymen, who ship large quanti-
ties of milk to our great cities. The usual practice is to
allow the milk to stand in open vessels, .surrounded by ice
or cold water, for from eight to twelve hours before trans-
portation, and when placed on the cars it has a temperature
of from 50° to GO0 P., and is delivered to consumers in a
perfectly sweet condition. The city of New York receives

66 BACTERIAL POISONS.

about 200,000 gallons each day from the surrounding
country, and much of it brought in by the railroads has
been on the cars for a time varying from six to twelve
hours, yet we seldom hear of any of this milk undergoing
the peculiar form of fermentation set up in the Long
Branch milk. We may account for this by assuming that
the proper care of the milk after it was taken from the
cow, and the low temperature at which it was kept, have
prevented the formation of any ferment ; this opinion
seems to be endorsed by all dairymen and managers of
large creameries with whom we have consulted. They all
agree in stating that milk maintained at a low temperature
can be kept sweet and in good condition for many days.

u We have dwelt on this branch of our topic somewhat
extensively, because we are fully persuaded that the im-
proper care of the milk had much to do with the illness it
produced.

" The results of our inquiry having revealed so much,
we next attempted to isolate some substance from the
poisonous milk, in order that the proof might be more
evident. A quantity of the milk that had caused sickness
in the second outbreak was allowed to coagulate, was then
thrown on a coarse filter, and the filtrate collected. This
latter was highly acid, and was made slightly alkaline by
the addition of potassium hydrate. This alkaline filtrate
was now agitated with an equal volume of pure, dry ether,
and allowed to stand for several hours, when the ethereal
layer was drawn off by means of a pipette. Fresh ether
was added to the residuum, then agitated, and, when sepa-
rated, was drawn oif and added to the first ethereal
extract. This was now allowed to evaporate spontane-
ously, and the residue, which seemed to contain a small
amount of fat, was treated with distilled water and filtered,
the filtrate treated with ether, the ethereal solution drawn
oif and allowed to evaporate, when we obtained a mass of
needle-shaped crystals. This crystalline substance gave a
blue color with potassium ferricyanide and ferric chloride,
and reduced iodic acid. The crystals, when placed on the
tongue, gave a burning sensation. A portion of the crys-

POISONOUS MILK. 67

l:il- was mixed with milk and fed to a cat, when, in the
course of half an hour, the animal was seized with retching
and vomiting, and was soon in a condition of collapse, from
which it recovered in a few hours.

" \Vc are justified in assuming, after weighing well all
the facts ascertained in the investigation, that the sickness
at Long Branch was caused by poisonous milk, and that
the toxic material was tyrotoxicon.

" The production of this substance was no doubt due to
the improper management of the milk — that is, too long a
time was allowed to elapse between the milking and the
cooling of the milk, the latter not being attended to until
the milk was delivered to the hotel ; whereas, if the milk
had been cooled immediately after it was drawn from the
cows, fermentation would not have ensued, and the result-
ing material, tyrotoxicon, would not have been produced."

In the same year, SCHEARER found the same poison in
the milk used by, and the vomited matter of, persons made
sick at a hotel at Corning, Iowa.

In 1887, FIRTH, an English army surgeon stationed in
India, reported an outbreak of milk poisoning among the
soldiers of his garrison. From the milk he separated, by
VAUGHAN'S method, tyrotoxicon. He also obtained tyro-
toxicon from milk which had been kept for some months
in stoppered bottles, as had been previously done by
Vaughau. (See page 62.)

In 1887, MESIC and VAUOHAN observed four cases of
milk poisoning, three of which terminated fatally, and
Now and VAUCJHAN obtained tyrotoxicou from the milk,
and from the contents of the intestine in one of the fatal
cases. VAUGHAN reports these cases as follows :

" September 23, 1887, 1 was visited by Dr. A. G. Mesic,
of Milan, Michigan, who informed me that he had four
members of a family under his charge, all of whom were
seriously ill with peculiar symptoms which he believed to
be caused by tyrotoxicon. Since Dr. Mesic has written
out for me the history of these cases, I will insert his report
in full, as follows :

" ' Saturday, September 17, while passing the residence

68 BACTERIAL POISONS.

of S. H. Evans, a respectable farmer, I was called in to see
him. I found him — a man of about fifty years, spare
and muscular — vomiting severely, with flushed face, but
with a temperature of 96° F. There was marked throb-
bing of the abdominal aorta ; the tongue had a white,
heavy coating, and the breathing was very labored. I set
to work with the ordinary remedies to allay the vomiting,
which had already continued for some hours. The vomited
matters were colored with bile. Pupils were dilated, and
a rash resembling that of scarlatina, but coarser, covered
the chest, forearms, and legs below the knees, while the
abdomen and thighs remained unaffected. As the bowels
had not been moved since the beginning of the attack, I
administered a purgative dose of calomel with a little podo-
phyllin and rhubarb. On Sunday a small stool resulted.
During that day and night, and the following day, the
retching and vomiting continued. Small doses of carbolic
acid seemed to give the most relief. After the movement
of the bowels the symptoms were somewhat more prom-
ising ; but a heavy and unfavorable stupor was observable
and persistent.

" ' On Sunday the coating of the tongue remained very
thick, and had changed to a dark brown color. At first I
thought that his symptoms indicated a depressed condition,
which I had known in one instance to precede typhoid
fever. However, after a few days, I concluded that I
must look for the cause of the condition among the poi-
sons ; but I could think of no one poison which would
be likely to produce all the symptoms observed. During
Monday, Tuesday, and Wednesday, there was but little
change, and the treatment was continued.

" ' On Thursday morning I found the son Arthur, a lad
of eighteen years, strong and vigorous, suffering with the
same symptoms, only in a more violent form. After
supper on Wednesday evening he was taken with nausea
and vomiting. He had no rash, but the symptoms were
otherwise identical with those of the father, except in being
more severe. I gave a cathartic, which acted only slightly.

" * At^ my evening visit I found Mrs. Evans, a lady of

POISONOUS MILK. 09

about forty-five, previously in good health, with the same
symptoms. In this case the stupor was more marked from
the first. I was unable at any time to obtain any cathartic
action in this case. Copious enemata of warm water were
used, but succeeded only in washing some hardened lumps
from the rectum. By this time I had concluded that the
poison was most likely tyrotoxicon.

"'On Friday morning the only remaining member of
the family at home, Miss Alma, sixteen years of age, was
affected in the same way as the others. On that day I
went to Ann Arbor, and gave a history of the cases so far
to Dr. Vaughan, who, from the symptoms, thought that
my diagnosis was most probably correct, and he advised
with me as to treatment, which I carried out. I gave two
grains of sodium sal icy late every four hours, and used small
doses of the tonics and stimulants, quinine, mix vomica,
digitalis, whiskey, and the aromatic spirits of ammonia.
On Saturday the symptoms in all remained unimproved,
and in the mother and son the stupor and labored breath-
ing grew more marked.

" ' On Sunday, I again went to Ann Arbor, and brought
Dr. Vaughan with me to see the patients. The tempera-
ture of the mother on Sunday was as low as 94° F., and
that of the son 95° F. Dr. Vaughan agreed with me as to
diagnosis and treatment. Sunday evening the patients
were all removed to the house of a neighbor, about forty
rods distant (the reasons for this will be given later). Dr.
Vaughan and I both expressed the fear that the mother,
ami j>ossibly the son, would not live through the night.
Ik>th of these rapidly grew worse, and the son died at 7.45
A.M. and the mother at 4 P.M., Monday.

" ' During Monday the daughter rapidly grew worse, and
at the time of her mother's death could not be aroused, and
practically she remained unconscious from that time on.
The father was very weak, but retained his consciousness
all the time. Convulsive movements of the limbs had
been noticed in the son, but not in the mother. These
now became more marked in the daughter, who remained

4*

70 BACTERIAL POISONS.

in the heavy stupor, with labored breathing, until 5 P.M.
Thursday, when she died.

" ' Mr. Evans has slowly improved, and now, October
18th, is able to walk about the room. The sodium sali-
cylate, even in the small doses used, seemed to cause severe
headache ; so apparent was this that the drug was discon-
tinued, and drop doses of amyl nitrite, given every hour,
seemed to relieve the pain in the head. His temperature
remained below the normal until Thursday, October 14th,
when it reached the normal. After this it was found once
as high as 99.5° F.,theu 99° F.,then again normal, where
it remains.

" 'All complained of a burning constriction in the throat,
and difficulty in swallowing, and all, as long as they were
conscious, frequently called for ice. In all the pulse was
rapid and feeble, and death seemed to result from failure
of the heart. Those who died voided urine involuntarily,
while Mr. Evans passed small quantities frequently, and
for this buchu and uva ursa were given. During his con-
valescence small doses of morphine were given, as he was
unable to sleep, and became very restless. He is now
taking teaspoonful doses of the elixir of calisaya and iron
every four hours.'

" As stated above by Dr. Mesic, I first saw these patients
Sunday, September 25th. On a sofa in the room we found
the daughter, Alma. She had been vomiting during the
day, and seemed much exhausted. She was not inclined to
talk, and seemed to be in a stupor, though when spoken to
she responded rationally. Her pupils were slightly dilated,
her tongue coated, her pulse 120 and weak, her face flushed,
and a violent throbbing could be felt over the abdomen,
which was retracted. Her temperature was 96° F.

" In another room were the father, mother, and son, two
of them dying. The father was rational, and talked with
some freedom when I asked as to the kind of food they
had been eating, etc. His pupils were normal. His face
could not be said to present any peculiar feature. His
pulse was rapid, breathing somewhat labored, and the
throbbing of the abdominal aorta was plainly felt. The

POISONOUS MILK. 71

abdomen \v;is retracted, and there was no pain on pressure.
He complained of a Iwrning constriction of the throat,
swallowed with difficulty, and said that his throat and
stomach felt as though they were on fire.

"The mother lay perfectly still with eyelids closed, as if
in a deep sleep. Her pulse was rapid, her face had a livid
flush, her breathing was about 35 per minute, and labored.
The skin was cool, but neither abnormally moist nor
specially dry and harsh. She could not be aroused. In
lad. she was comatose.

" The son rolled uneasily from one side of the bed to the
other. His breathing, also, was very labored. His eyelids
were closed, and the pupils were markedly dilated — did not
respond to light. He could not be aroused. In mother
and son, as well as in father and daughter, the abdomen
was retracted, and the throbbing of the abdominal aorta
\\as easily felt.

" Now, to what were these symptoms due ? They were
certainly those of some poison. Dr. Mesic had brought
me some of the vomited matter, which I tested thoroughly
for mineral poisons, with negative results. The symptoms
certainly were not those of morphine, strychnine, digitalis,
or aconite. They did have some resemblance to those of
belladonna, but yet they were not the symptoms of bella-
donna. The pupils were not • as widely dilated as they
would l>e in belladonna poisoning. There was in none of
these persons the active delirium of belladonna poisoning.
There was no picking at the clothing, no grasping of imag-
inary objects in the air, no hallucinations of vision. Surely
it could not be any vegetable alkaloid with which I was
familiar.

" On the other hand, we know that nausea, vomiting,
headache, dilatation of the pupil, rapid pulse, heavy breath-
ing, constipation, and great prostration, with stupor, do
occur in ( -a ses of poisoning with certain ptomaines. There-
fore we began to look for conditions which would be favor-
able for the production of putrefactive alkaloids. These
conditions we were not long in finding.

" The family, which consisted of the four persons sick, and

72 BACTERIAL POISONS.

of a daughter about twenty years of age, who was away
from home at the time when the others were taken ill,
and for some months before that time, was evidently a
tidy one. This was shown by their personal appear-
ance, and by the clothing and bedding. But the house
in which they lived was very old, and very much
decayed. Mr. Evans had purchased the farm six years
ago ; and for some three years past, at least, they had been
troubled every now and then, one or more of the family,
with nausea and vomiting, followed by more or less prostra-
tion. But in no instance, up to the present illness, had the
symptoms been sufficient to cause them to summon a physi-
cian. The family had worked hard in order to pay for the
farm, and had determined to make the old house do until
they were out of debt. Even before this family had moved
to the farm, the house had been known among the neigh-
bors as an unhealthy one, and there had been much sick-
ness and a number of deaths among its former tenants.

" The house is a frame one, and one of the neighbors said
to me that it was an old house when he came to the neigh-
borhood thirty-seven years ago. It consists of two rooms
on the ground-floor, with attic rooms above. The frame
rests upon four large logs or sills, which lie directly upon
the ground, and are thoroughly rotten. There is no cellar
under any part of the house. From the front, at least, the
surface slopes toward the house, and the rain-water runs
under it. In the floor of one room a trap-door had been
placed, and directly under this a small excavation had been
made for the purpose of collecting the rain-water when it
accumulated under the house. Although this pit was dry
at the time of our examination, its sides and bottom were
marked with cray-fish holes, showing that water had stood
in it. The floor was laid of unjointed boards, and every
time that it was swept much of the filth fell through the
cracks, and every time that the tidy housewife scoured and
mopped the floor, the water, carrying with it the filth, ran
through the crevices, and thus the conditions most favorable
for putrefactive changes were brought into existence and
maintained.

POISONOUS MILK. 73

" One corner of one of the rooms had Ixniii transformed
into a small room, or buttery, as it was called, and in tin's,
on shelves, the food was kept. On account of the more
frequent scouring demanded by that part of the floor
enclosed in this buttery, the boards had rotted away, and a
Mvnnd layer of boards had been placed over the original
floor. Between these two floors we found a great mass of
moist, decomposing matter, the accumulations of years,
which the broom could not reach. When this floor was
taken up, a peculiar, nauseating odor was observable, and
was sufficient to produce nausea and vomiting in one of the
persons engaged in the examination. Some of the dirt
from beneath the floor, ami some of that which hud accumu-
lated InMieath the boards in the buttery, were taken for
further study.

" The condition of the house was supposed to be unfavor-
able to the patients, and for this reason they were moved,
as Dr. Mesic has stated, to the house of a neighbor. Of
course, thorough examination of the house was not made
until the patients had been removed.

"Special inquiry was now made concerning the food used
by this family. They had been living very simply. They
lived upon bread, butter, milk, and potatoes, with coffee
and ripe fruit. They had eaten no canned foods for months.
They ate but little meat. Occasionally a chicken was killed
and served, and rarely, some fresh meat was obtained from
the village. During the week in which they were taken ill,
all the meat used consisted of slices from a piece of bacon,
the only meat which was kept in the house, and a chicken.
None of the latter remained, but the bacon was examined.
It seemed in perfect condition, and contained no trichina?.
Moreover, as has been seen from the history of the cases,
all the members of the family were not made sick by any
one meal, but the opportunity of obtaining the poison must
have been present for some time. Moreover, the fact that
previous similar, but less severe, attacks had occurred at
intervals for the past three years, convinced us that the
poison must owe its origin to some long-existing condition.

" The drinking-water supply was also investigated. The

74 BACTERIAL POISONS.

water was obtained from a shallow well, and some of it was
taken for analysis. But several families had for years used
water from this well, and had remained healthy.

" The milk used by the family was studied. Of course,
we could get none of that which had been used before the
members of the family were stricken down. As soon as he
made the diagnosis of tyrotoxicon poisoning, Dr. Mesic
ordered the discontinuance of the use of milk, not only with
the sick, but he forbade the daughter, who had returned,
and any of the visitors using it. Mr. Evans owned four
milch cows, and they were supplied with fair pasturage and
abundant water. The greater part of the milk was placed
in tin cans which were set in a wooden trough in the yard,
and surrounded by cold water. The covers to the cans
were arranged so that the air could have free access to the
milk, and were left in this position until the milk was
thoroughly cooled. Indeed, the cans were furnished by a
creamery company, which followed the directions which I
have previously given for the care of milk. On his first
visit to me, Dr. Mesic brought some of the milk from one
of these cans. This I examined, but failed to find tyro-
toxicon in it.

" However, the family did not drink any of the milk from
the cans. That which they did use was kept in the buttery
which I have described. Here it stood upon a shelf, and
some members of the family, at least, were in the habit of
drinking from it between meals. This was especially true,
it is said, of the son. He would frequently come from his
work in the fields, go into the buttery and drink a glass or
more of the milk. Mr. Evans states that he frequently
observed that the taste of the milk was not pleasant. On
my first visit to the premises I advised that some of the
milk should be taken from the cans, allowed to stand in the
buttery over night, and be sent to me the next day. This
was done, and in this milk we found tyrotoxicou, not only
by the employment of chemical tests, but by poisoning a
kitten with it.

"On the death of the mother and son, Dr. Mesic asked for
a post-mortem, but the friends objected, and the undertaker

POISONOUS MII-K. 75

an arsenical embalming fluid, so that, although consent
was subsequently obtained, it was decided that the cxami-
nation would be so vitiated as to be worthless. On the
death of (he daughter, the coroner summoned a jury and
held an iiKjiiest. The post-mortem was conducted by I>r.
George A. Hendricks, in the presence of the jury and
several physicians who had been invited. Dr. Hendricks
has kindly furnished me with his report, which I present
here in full :

" The autopsy was held fifteen hours after death. The
alxlominal viscera were first examined. The great omen-
turn was small, in normal position, covering the small
intestine. The small intestine was moderately distended
with Hat us. The jejunum was ashy-green in color; the
ileiim purplish-green. About eighteen inches from the ter-
mination of theileum was found a diverticulum two inches
in length. The small intestine contained very little ali-
mentary matter. The vermiform appendix was free, con-
tained some small fecal lumps, and snowed no evidence of
inflammation. The ca?cum, ascending, transverse, and
descending colon were empty and their circular fibres were
tightly constricted, except at intervals where the intestine
was distended with gas. The sigmoid flexure was moder-
ately distended with gas, and the rectum contained small
bits of fecal matter. The stomach was somewhat contracted
and lay wholly upon the left side of the median line. It
contained a few ounces of fluid. Its extremities were ligated
and the organ removed. The mucous membrane of the
stomach and intestine were not examined until they reached
the chemist. The duodenum was distended with flatus.
The liver was normal in size and appearance. The gall-
bladder contained about one ounce of bile. The spleen was
normal. One-half ounce of fluid deeply stained with blood
was found in Douglas's cul-de-sac. The uterus, Fallopian
tuhcs, and ovaries were deeply congested. The left ovary
was enlarged and presented on its posterior surface a hemor-
rhagie spot, oval, about one-half line in length, and several
other less distinct ones. The right ovary was normal in
size and showed numerous Graalian scars. The ureters

76 BACTERIAL POISONS.

and bladder were normal ; the latter contained a small
amount of urine. The peritoneum, pancreas, and kidneys
were perfectly normal.

"The thoracic cavity was next opened. The lungs were
normal ; there was about one-half ounce of free serum in
the left pleural cavity ; none in the right. Pericardium
normal ; right auricle in diastole ; left auricle and both
ventricles in systole.

" The dura mater showed venous congestion ; the arach-
noid, normal ; the pia mater, congested. On the surface
of the centrum ovale, small drops of blood oozed from the
divided vessels. The large veins of the velum interposi-
tum were distended. Third and fourth ventricles were
slightly distended with serous fluid, but the walls were
normal. There seemed to be slight softening of the optic
thalami. The sub-arachnoid fluid was about twice the nor-
mal quantity.

" On examination of the mucous membrane of the
stomach and intestine in the presence of the chemist, Prof.
A. B. Prescott," nothing abnormal could be found. The
membrane was stained with bile, but there was not the
slightest redness. The solitary glands were distinct, but
not at all inflamed. Peyer's patches were normal.

"It will be seen that there existed no lesion which would
account for the death. The venous congestion observed in
the brain would follow from failure of the heart.

" Some of the post-mortem appearances bore a striking
resemblance to those which I had observed in cats poi-
soned with tyrotoxicon. This was especially noticeable in
the condition of the mucous membrane of the stomach and
intestine. Tyrotoxicon produces the symptoms of a gas-
tro-iutestinal irritant, but not the lesions. The contraction
of the circular fibres of the intestine, which undoubtedly
caused the constipation, I had also observed in cats that
died from tyrotoxicon poisoning without either vomiting
or stool.1 The action of this poison upon the stomach and

1 Marsh reports a case in which the symptoms resembled very closely
those of rapidly perforating typhlitis, but the post-mortem examination
showed absolutely no evidence of this disease or of peritonitis. In fact the

POISONOUS MILK. 77

intestine must be through the nervous system. Small
ilixcs cause both vomiting and purging, while after large
doses vomiting may be impossible, and obstinate constipa-
tion may exist. Both the vomiting and purging after
small doses are undoubtedly due in part to increased
activity of the circular fibres of the muscular coats, induced
through the nerves ; and the inability to vomit, and the
constipation, one or both of which may be observed after
large doses of the poison, are due to spasm of the same
muscles, induced in the same manner.

" Prof. A. B. Prescott was requested by the coroner to
analyze the material for mineral and vegetable poisons.
He made analyses of the stomach and part of its contents,
and a portion of the liver. His results were wholly nega-
tive.

" Novy tested a cold-water extract of the finely divided
intestine for ptomaines. The fluid, which was acid in
reaction, was filtered, then neutralized with sodium bi-
carbonate, and shaken with ether. The ether, after
separation, was removed, and allowed to evaporate spon-
taneously. The residue was dissolved in water, and
extracted again with ether. This ether residue gave the
chemical reactions for tyrotoxicou, and a portion of it was
administered to a kitten about two months old. Within
half an hour after the administration the kitten began to
retch, and soon it vomited. Within the next three hours
it was noticed to vomit as many as five times. The breath-
ing became rapid and labored. The animal sat with its
head down, and seemed greatly prostrated. The pupils
were examined, but could not be said to be dilated. There
was no purging. The retching and heavy breathing, with
evidences of prostration, continued more or less marked
for two days, after which the animal slowly improved.

" A quantity of fresh milk was divided into five por-
tions of one quart each, placed in quart bottles which had

only abnormality found in the intestines consisted of the contraction ot the
circular fibres of the transverse and descending colon. Marsh believes
that this was a case of ptomaine poisoning.

78 BACTERIAL POISONS.

been thoroughly cleansed, and treated in the following
manner :

" No. 1 consisted of the milk only, and was employed
as a control test.

"No. 2 was mixed with a drachm of vomited matter.

(l No. 3 was treated with a portion of the contents of
the stomach.

" No. 4 was treated with an aqueous extract of the in-
testine.

" No. 5 was treated with a small portion of the soil
which had been taken from the floor of the buttery, stirred
up with water.

" These bottles were placed in an air-bath, and kept at a
temperature of from 25° to 30° C. for twenty-four hours.
Then each was tested for ptomaines. No. 1 yielded no
tyrotoxicon, while all of the others contained this poison.
The tests were both chemical and physiological. All of
the samples yielded a non-poisonous base when treated
according to Brieger's method, and the same substance was
obtained from perfectly fresh milk. It is most probably
formed by the action of the heat and reagents employed in
this method. This base was obtained in crystalline form,
and several portions of it were administered to kittens
without any effect. The further study of this body will
be of interest to toxicologists, because it gives many of the
general alkaloidal reactions. At first we supposed it to be
Brieger's neuridiue, and this supposition may still be cor-
rect, but, as we obtained it, it gave some reactions which
are not given by neuridine. Further investigations will
be made on this point.

"Tyrotoxicon was obtained from the filtered milk by
two methods: (1) The one which we have previously used,
and which consists in neutralizing the filtered milk with
sodium bicarbonate, and extracting with ether. That por-
tion of the poison employed in the physiological tests was
obtained in this way, and in order to be sure that no poison
came from the ether, the extract from the milk to which
nothing had been added was given to a kitten, and was
found to produce no effect. (2) The filtrate from the milk

POISONOUS ICE-CKKAM. 79

heated to 70° C. (158° F.) (tyrotoxicon decomposes at
91° C. (195.8° F.)) for some minutes, ami filtered. Tliis
filtrate, which was perfectly clear, was treated with a small
quantity of nitric acid in order to convert the tyrotoxicon
into a nitrate, then pure potassium hydrate in the solid
form was added until the solution was strongly alkaline.
This solution was concentrated w far as it could be on the
water-bath. (The potassium compound of tyrotoxicon is
not decomposed below 130° C. (234° F.).) The dark
brown residue, after cooling, was examined with the micro-
scope and found to contain the crystalline plates of tyro-
t o\ icon-potassium hydrate, along with the prisms of potas-
sium nitrate. The former was separated from the latter
by extraction with absolute alcohol and filtration. The
alcohol was evaporated to dry ness on the water-bath, and
the residue again extracted with absolute alcohol. From
this alcoholic solution tyrotoxicon was precipitated with
ether. The precipitate was decomposed by adding acetic
acid and heating, the tyrotoxicon being broken up into
nitrogen and phenol. The phenol was recognized by pre-
cipitation with bromine water, and by other well-known
tests.

" On October 8th, the coroner's inquest, which had been
adjourned after the post-mortem in order to await the re-
sults of the analysis, was resumed, and after hearing the
testimony in accordance with the above stated facts, the
jury returned a verdict of death from poisoning with tyro-
toxicon."

CAMMAN reports twenty-three cases of milk poisoning
which he attributes to tyrotoxicon, although this poison
could not l>e found in the milk. It may be that the active
agent present belongs to the bacterial proteids.

KINNICUTT has isolated tyrotoxicon from milk which
had l>een kept for some hours in an unclean vessel.

POISONOUS ICK-CRKAM. — In 1886, V.msiiAX and
Now obtained tyrotoxicon from a cream which had
seriously affected many person at Lawton, Michigan.
Vanilla had been used for flavoring, and it was supposed

80 BACTERIAL POISONS.

that the ill-effects were due to the flavoring. This belief
was strengthened by the fact that a portion of the custard
was flavored with lemon, and the lemon cream did not
affect any one unpleasantly. Fortunately some of the
vanilla extract remained in the bottle from which the fla-
voring for the ice-cream had been taken, and this was for-
warded to the chemists. Each of the experimenters took
at first thirty drops of the vanilla extract, and no ill- effects
following this, one of them took two teaspoonfuls more,
with no results. This proved the non-poisonous nature of
the vanilla more satisfactorily than could have been done
by a chemical analysis.

Later, it was found that that portion of the custard
which had been flavored with lemon was frozen immedi-
ately ; while that portion which was flavored with vanilla
and which proved to be poisonous, was allowed to stand
for some hours in a building, which is described as follows
by a resident of the village :

" The cream was frozen in the back end of an old
wooden building on Main Street. It is surrounded by
shade, has no underpinning, and the sills have settled into
the ground. There are no eve-troughs, and all the water
falling from the roof runs under the building, the streets
on two sides having been raised since the construction of
the house. The building had been unoccupied for a num-
ber of months, consequently had had no ventilation, and
what is worse, the back end (where the cream was frozen)
was last used as a meat market. The cream which was
affected was that portion last frozen ; consequently it stood
in an atmosphere like that of a privy vault for upward of
an hour and a half or two hours before being frozen."

The symptoms observed in these cases are given by Dr.
MOFITT as follows :

"About two hours after eating the cream every one was
taken with severe vomiting, and after from one to six
hours later with purging. The vomit was of a soapy char-
acter, and the stools watery and frothy. There was some
griping of the stomach and abdomen, with severe occipital
headache, excruciating backache, and boue pains all over,

POISONOUS ICE-CREAM. 81

especially marked in the extremities. The vomiting lasted
from two to three hours, then gradually subsided, and
everybody felt stretchy, and yawned in spite of all resist-
ance. The throats of all were oedematous. One or two
were stupefied ; others were cold and experienced some
muscular spasms. A numb feeling, with dizziness and
momentary loss of consciousness, was complained of by
some. Temperature was normal, and pulse from 90 to
120. Tongue dry and chapped. All were thirsty after
the vomiting subsided, and called for cold water, which
was allowed in small quantities, with no bad results.
After getting out no one of the victims was able to be in
the hot sun for several days, and even yet (about ten days
after the poisoning) the heat affects myself. I attended
twelve persons, besides being sick myself, and all were
affected in pretty much the same way. Several complain
yet of inability to retain food on the stomach without dis-
tressing them. The man who made the cream took a tea-
spoonful of it, and he vomited the same as those who took
a whole dish, but not so often or for so long a time. All
are affected with an irresistible desire to sleep, which can
scarcely be overcome. Even yet, some of us feel that
drowsy condition, with occasional occipital headache."

The tyrotoxicon obtained from this cream was adminis-
tered to a kitten about two months old. Within ten
minutes the cat began to retch and soon it vomited. This
retching and vomiting continued for two hours, during
which the animal was under observation, and the next
morning it was observed that the animal had passed several
watery stools. After this, although the kitten could walk
about the room, it was unable to retain any food. Several
times it was observed to lap a little milk, but on doing so
it would immediately begin to retch and vomit. Even cold
water produced this effect. This condition continuing,
after three days the animal was placed under ether and its
abdominal organs examined. Marked inflammation of the
stomach was supposed to be indicated by the symptoms,
but the examination revealed the stomach and small intes-
tine filled with a frothy, serous fluid, such as had formed

82 BACTERIAL POISONS.

a portion of the vomited matter, and the mucous membrane
very white and soft. There was not the slightest reduess
anywhere. The liver and other abdotniual organs seemed
normal.

A bit of the solid portion of this cream was added to
some normal milk, which, by the addition of eggs and
sugar, was made iuto a custard. The custard was allowed
to stand for three hours in a warm room, after which it
was kept in an ice-box until submitted to chemical analysis.
In this tyrotoxicon was also found.

Tyrotoxicon has since been found in some chocolate
cream which poisoned persons at Geneva, N. Y., and in
lemon cream from Amboy, Ohio.

SCHEARER reports the finding of tyrotoxicon in both
vanilla and lemon ice-cream which made many sick at
Nugent, Iowa.

ALLABEN reports poisoning with lemon cream, and
makes the following interesting statements concerning it :

" I would first say July 4, 5, and 6 were very warm.
Monday evening, July 5, the custards were cooked, made
from Monday morning's cream and Monday night's milk,
boiled in a tin pan that had the bright tin worn off. It
was noticed that one pan of cream was not sweet, but
thinking it would make no difference, it was used; the
freezers were thoroughly cleaned and scalded, and the
custards put in the same evening while hot; the cream was
frozen Tuesday afternoon, having stood in the freezers
since the night before, when the weather was very warm."

No analysis of this cream was made, but the symptoms
agree with those of tyrotoxicou poisoning.

WELFORD observed several cases of poisoning from
custard flavored with lemon. These custards were tested
for mineral poisons, with negative results.

MORROW has put forth the claim that ice-cream poison-
ing is solely due to artificially prepared vanillin, which is,
according to his statement, used instead of vanilla extract,
but the facts stated above concerning poisoning with creams
in which other flavors had been' used contradict this claim.
Moreover, GIBSON has shown the utter absurdity of the

POISONOUS MEAL AND BREAD. 83

claim, inasmuch as he calculates from the amount of flavor-
ing ordinarily used in ice-cream, that in order to produce
the toxic symptoms observed, the flavoring must be ten
times as poisonous as pure strychnine.

BA.BTLEY suggests that poisonous cream sometimes
results from the use in its manufacture of poor or putrid
gelatin. This is highly probable, and with the gelatin
the germs of putrefaction may be added to the milk.

POISONOUS MEAL AND BREAD. — Reference has already
been made to the fact that the peasants in certain parts of
Italy are frequently poisoned by eating mouldy corn-meal.
As lias also been stated, LOMBROSO and others have ob-
tained from this meal ptomaines, some of which give the
same color reaction as strychnine. In 1886, LADD suc-
ceeded in isolating from " heated " corn-meal a ptomaine
which forms in urea-like crystals. The quantity was not
sufficient for an ultimate analysis, and the physiological
action has not been studied. Poisoning from decomposed
and mouldy bread is not unknown.

CHAPTER IV.

GENERAL CONSIDERATIONS OF THE RELATION OF
BACTERIAL POISONS TO INFECTIOUS DISEASES.

THE majority of diseases may be grouped from an etio-
Jogical standpoint into the following classes : (1) Trau-
matic ; (2) infectious ; (3) autogenous ; and (4) neurotic. It
must be understood, however, that in many diseases the
cause is not single, but multiple, and for this reason sharp
lines of classification cannot be drawn. For instance, the
greatest danger in those traumatic affections in which the
traumatism itself does not cause death, lies in infection.
The wound has simply provided a suitable point of en-
trance for the infecting agent. Indeed, the break in the
continuity of tissue may be so slight that it is of import
and danger only on account of the coincident infection.
This is true in many cases of tetanus. Furthermore, an
infectious disease, whether it originates in a traumatism or
not, is markedly influenced by what we are pleased to call
the idiosyncrasy of the patient, and by which we mean the
peculiarities of tissue metabolism taking place in the indi-
vidual at the time. A dozen men may be exposed alike to
the same infection, and the infecting agent may find a suit-
able soil for its growth and development in two of these,
while in the other ten this same agent meets with such
adverse influences that it dies without producing any appre-
ciable effects ; or all may be infected, but with difference in
degree, as is evidenced by variation in symptoms, in the
length of time that this infecting agent continues to grow
and develop in the body and in the ultimate result. Every
physician who has had experience in the treatment of
typhoid fever, diphtheria, scarlet fever, or, in short, of any
of the infectious diseases, will appreciate the importance of
the personal equation in his patients.

RELATION TO INFECTIOUS DISEASES. 85

CHARRIN and ROGER have shown that white rats,
which are naturally immune to anthrax, IM-COIUC susceptible
when fatigued by Ix-ing kept on a small tread-mill. Klcve-n
rats were inoculated with an anthrax culture; five of these
which were allowed to rest in the cage manifested no symp-
toms of the disease, while six which were placed on the
tread-mill developed the disease and died within from
twenty-four to thirty hours. The bacilli were found in
the liver and spleen of those which died; and guinea-pigs
inoculated with these germs died. The influence of the
condition of health on susceptibility to the infw-tious dis-
eases has also been shown by LEO, who found that mice
which arc- naturally insusceptible to glanders, become highly
<:i-!-cptible when they are rendered diabetic by the adminis-
tration of phloridzin.

That some- neurotic art'e-ctions originate from traumatism
we know. That others of this class are largely due to mal-
nutrition accompanied by improper metabolism or insuffi-
cient elimination, or, in other words, are to some extent
autogenous, all l>elieve. Understanding, then, that the
al»ove classification docs not attempt a sharp and marked
differentiation of the causes of disease, we will now give
our attention to a consideration of the chemical factors in
the causation of the infectious diseases, and of the trau-
matic, autogenous and neurotic, in so far as these are influ-
enced by infection.

Recognizing the fact that germs do l>ear a e-ausal relation
to some diseases, the question arises, How do these organ-
isms product- disease? In what way does the bacillus
anthracis, for instance, induce the symptoms of the disease
and death? Many answers to this question have been
oH'ere-d. Sinie of the most important of these are as fol-
lows :

1. It was first suggested by BOLLINGER that apoplecti-
form anthrax is due- to deoxidation of the blood by the
bacilli. These germs are aerobic-, and were supposed to
deprive the rc.nl blood-corpuscles of their oxygen. This
theory was suggested most probably by the resemblance of
the symptoms to those of carbonic acid pcrsoning. The

86 BACTERIAL POISONS.

most prominent of these symptoms are dyspnoea, cyanosis,
convulsions, dilated pupils, subnormal temperature, and,
in general, the phenomena of asphyxia. Moreover, post-
mortem examination reveals conditions similar to those
observed after death by deprivation of oxygen. The veins
are distended, the blood is dark and thick, the parenchy-
matous organs are eyanotic, and the lungs hypertemic.
BOLLINGER compared this form of anthrax to poisoning
with hydrocyanic acid, which was then believed to prodixv
fatal results by robbing the blood of its oxygen.

This theory was supported by the observations of SZITL-
MANN, who found that while the putrefactive bacteria are
destroyed by ozone, the bacillus anthracis thrives and mul-
tiplies in this gas.

This theory pre-supposed a large number of bacilli in the
blood, and this accorded with the estimate of DAVAINE,
which placed the number at from eight to ten million in a
single drop. But more extended and careful observation
showed that the blood of animals dead from anthrax is
often very poor in bacilli. VIROHOW reported cases of
this kind. BOLLINGER himself found the bacilli often
confined to certain organs and not abundant in the blood.
Then SIEDAMGROTZKY counted the organisms in the blood
in various cases and found not only that the estimate made
by DAVAINE is too large, but that in many instances the
number present in the blood is small. JOFFROY found in
some of his inoculation experiments that the animals died
before any bacilli appeared in the blood. These and other
investigations of similar character began to cause workers
in this field of research to doubt the truth of the theory of
BOLLINGER, and these doubts were soon converted into
positive evidence against it. PASTEUR, in support of the
theory, reported that birds were not susceptible to anthrax.
and he accounted for this by supposing that the blood
corpuscles in birds do not part with their oxygen readily.
However, it was shown by OEMLER and FESER that the
learned Frenchman had generalized from limited data, and
that many birds are especially susceptible to the disease.
OEMLER found that the blood even when rich in bacilli

RELATION TO INFECTIOUS DISKASES. 87

still possesses the bright-fed color of oxy-hannoglobin.
TOEPPEB and I\oi,oFi-' reported cases of apoplectifbrm
anthrax in which there was no difficulty in respiration.
To USSAINT cans'. -d animals which had Ix-en inoculated with
the anthrax bacillus to breathe air containing a large
volume of oxygen, and found that this did not modify the
symptoms or retard death. Finally, NENCKI determined
the amount of physiological oxidation going on in the IxKlies
of animals sick with anthrax by estimating the amount of
phenol excreted after the administration of one gramme of
benzol, and found that the oxidation of the In-n/ol was not
diminished by the disease. Thus, the theory that germs
destroy lite by depriving the blood of its oxygen has been
found not to be true for anthrax, and if not true for
anthrax, certainly it cannot l>e for any other known disease.
The bacillus anthracis is, as has l>een stilted, aerobic, while
most of the pathogenic bacteria are anaerobic — that is, they
live in the absence of oxygen. This element is not neces-
sary to their existence, and, indeed, when present in large
amount, it is fatal to them. Moreover, in many diseases,
the bacteria an- not found in the blood at all. Lastly, the
symptoms of these diseases are not those of asphyxia. These
facts have caused all bacteriologists to acknowlege that this
theory is not the right one.

2. If a properly stained sex then of a kidney taken from
a guinea-pig, which has been inoculated with the bacillus
anthracis, be examined under a microscope, the bacilli will
be found to be present in such large numbers that they form
emboli, which not only close, but actually distend the capil-
laries and larger blood vessels, and interfere with the normal
functions of the organ. A similar condition is sometimes
found on microscopical examination of the liver, spleen,
and lungs. From these appearances, it was inferred by
BoLLINOEB that the bacilli produce the diseased condition
simply by accumulating in large numbers in these impor-
tant organs, and mechanically interrupting their functions.
This is known as the mechanical interference theorv.

KLKI-.S and TOUSSAINT were formerly ardent advocat< s
of this theory in its application to anthrax, and the latter

88 BACTERIAL POISONS.

thought that the symptoms and death are due to stoppage
of the pulmonary escalation by means of emboli. How-
ever, HOFFA studied this point by making numerous post-
mortem examinations, and was unable to confirm it. A
like result followed the work of YIRCHOW, COLIN, and
SIEDAMGROTZKY, and the mechanical-interference theory
has been abandoned.

In the majority of germ diseases this theory never had
any support. There is not found any great accumulation
of bacteria in any organ, and the number and distribution
of the germs are such that the theory of mechanical inter-
ference cannot be held.

3. Another answer given to the question, How do germs
cause disease? is, that they do so by consuming the proteids
of the body and thus deprive it of its sustenance. The
proteids are known to be necessary for the building up of
cells, and it is also known that microorganisms feed upon
proteids. But this theory is untenable for several reasons.
In the first place, many of the infectious diseases destroy
life so quickly that the fatal eifect cannot be supposed to
be due to the consumption of any very large amount of
proteids. In the second place, the distribution of the micro-
organisms is such in many diseases that they do not come
in contact with any large proportion of the proteids of the
body. In the third place, the symptoms of the majority of
these diseases are not those which would be produced by
withdrawing from the various organs their food. The
symptoms are not those of general starvation.

4. Still another theory, which has been offered, is that
the bacteria destroy the blood corpuscles, or lead to their
rapid disintegration. But in many of the infectious dis-
eases, as has been stated, the microorganisms, although very
abundant in some organs, are not present in the blood.
Moreover, the disintegration of the blood corpuscles is not-
confirmed by microscopical examination.

5. Seeing the vital deficiencies in the above theories, and
being impressed by the results obtained by the chemical
study of putrefaction, bacteriologists have been led to in-
quire into the possibility of the symptoms of the infectious

RELATION TO INFECTIOUS DISEASES. 89

diseases In-iii-; due to chemical poisons. In investigating
tin's theory, three possibilities suggest, themselves:

(a) The microorganisms themselves may ta poisonous,
or the poison may IK- ;in integral part of thorn. NEELSEN,
at one time an advocate of this theory, thus accounted for
the appearance and increase in violence of the symptoms as
the germ-. increase in numlxT. In order for the conditions
of this theory to l>e fulfilled the microorganisms must be
present in the blood before any of the symptoms appear.
But in anthrax the most thoroughly studied of all the in-
flrtions diseases, and the one to which all these theories
have Ixrn applied, the bacilli first appear in the blood, as a
rule, only a few hours before death, and long after the
appearance of the first symptoms; while in many other
diseases the germs are never found in the blood. More-
over, as HOFFA has shown, if this theory l>e true, the in-
jeetion of a large quantity of anthrax bacilli directly into
the blood should be followed immediately by symptoms of
tli«' disease, and death should l>e speedy. But he found, on
making experiments of this kind, that the symptoms did
not appear until from twenty-four to seventy-two hours.
NENCKI found by analysis that the substance of the an-
thrax bacilli resembles Vegetable easein in some respects,
and animal mncin in others. This "anthrax protein " is
freely soluble in alkalies, is insoluble in water, acetic acid,
and the dilute mineral acids. It contains no sulphur and
was believed by NENCKI to be inert; but the recent re-
searches of BUCHNER has shown that this belief is not well
founded. It has IxH'ii stated by a numlxT of investigators
that suppuration might lx; induced by the injection of cer-
tain sterilized cultures, but the dictum of WEIGERT, "no
suppuration without bacteria," has been generally accepted ;
and statements to the contrary, although some of them have
Ix-en made by men of excellent reputation, have until recently
received but little credence. However, BUCHNER has shown
conclusively that the albuminate of the bacterial cell in as
many as sevenh-en different species jxxssessos well-marked
pyogenetic properties, and that the pus formed is free from
germs. BUCHNER separated the microorganisms from the

90 BACTERIAL POISONS.

soluble substances accompanying them by sedimentation and
decantation, washed the cells, dissolved them in a ().;"> per
cent, solution of potash by the aid of heat, precipitated the
albumin with dilute mineral acid, and, after repeated re-
solution in alkali and reprecipitation with acid, employed
the purified proteid in his experiments. Introduced with
antiseptic precautions under the skin, this substance invari-
ably causes suppuration. This demonstrates that the sub-
stance of the bacterial cell is not altogether inert. It is
impossible at present to say to what extent the course of
an infectious disease may be influenced by the breaking
down of a large number of bacterial cells and the intro-
duction of their substance into the blood.

(6) The microorganisms may be intimately associated
with or may produce a soluble, chemical ferment, which,
by its action on the body, produces the symptoms of the
disease and death. This theory formerly had a number of
ardent supporters, among whom might be mentioned the
eminent scientist, DE BARY. But PASTEUR proved the
theory false when he filtered anthrax blood through earthen
cylinders, inoculated animals with the filtrate, and failed to
produce any effect. NENCKI made a similar demonstration
when he inoculated a two per cent, gelatin preparation
with the anthrax bacillus, which liquefied the preparation,
and on standing the ba/'illi settled to the bottom. The
supernatant fluid, which was clear, alkaline in reaction, and
contained dissolved " anthraxprotein," was filtered and
injected into animals without producing any effect.1

It must not be inferred from the above statements that
bacteria do not produce any ferments. Many of them do
form both diastatic and peptic ferments, which may retain
their activity after the bacteria have l>een destroyed ; but
there is no proof that in any case these ferments have any
causal relation to the disease. After the diseased process
has been inaugurated some of these ferments probably play

1 We now know that if the supernatant fluid used in this experiment
had been injected in sufficient quantity death would have been produced
by the soluble chemical poisons.

RELATION TO INFECTIOUS DISEASES. 91

an important part in the production of morphological
chants, the nature of which will be indicated when these
diseases are discussed.

(e) The germ may produce chemical poisons by splitting
up preexisting complex compoiiiids in the Ixxly. This
theory finds, in the lirst place, strong support in the well-
known fact that many of the putrefactive germs produce
highly poisonous bodies; and, in the second plaee, the for-
mation of chemical poisons will account for the appearance
of the symptoms of the disease when the microorganisms
never find their way into the blood. The correctness of
this theory has been tested by a large nninl)er of investi-
gators, and with the result that its truth has been firmly
established. It was soon found that pathogenic germs
grown in meat broth and other culture media elaborate
chemical poisons which, when injected into the lower animals,
induce in an acute form one or more of the symptoms char-
acteristic of the d:s-jase caused in man by the microorgan-
ism. It is true that until quite recently this theory has
been opposed by some, and it is altogether possible that
at present there may be those who are not altogether con-
vinced of its truth. However, we are not acquainted with
any argument against it which remains unanswered. For
a while U.M'Mc AK-TEX claimed that the formation of chem-
ical poisons in the dead matter of meat broth and other
media by the germ does not prove that the same agent is
capable of forming the same or similar products within the
living body ; but the isolation of tctanine from the ampu-
tated arm of a man with tetanus, by HRIEGER, furnished
the first positive answer to this criticism, and since that
time a number of bacterial poisons have been obtained from
the bodies of men and the lower animals. We now expect
to find each sps-citic, pathogenic microorganism producing
its characteristic po:son or poisons. The evidence on
this point will l>e given further on in a brief sketch of the
chemical factors in the causation of some of the best-known
inflations diseases.

Uel'orc taking up the individual diseases, we will give

92 BACTERIAL POISONS.

what appears to us, in the present state of our knowledge,
a correct definition of an infectious disease.

An infectious disease arises when a specific, pathogenic
microorganism, having gained admittance to the body, and
having found the conditions favorable, grows and multi-
plies, and in so doing elaborates a chemical poison which
induces its characteristic effects.

In the systemic infectious diseases, such as anthrax,
typhoid fever, and cholera, this poison is undoubtedly taken
into the general circulation, and affects the central nervous
system. In the local infectious diseases, such as gonorrhoea,
and infectious ophthalmia, the principal action of the poison
seems to be confined to the place of its formation. Though
even in these, when of a specially virulent type, the effects
may extend to the general health. It may be that in some
diseases the chemical poison has both a local and a systemic
effect. Thus, it is by no means certain that the ulceration
of typhoid fever is due directly to the bacillus. On the
other hand, it is altogether probable that the anatomical
changes in the intestine result from the irritating effects of
the poison at the place of its formation.

With the proof, that the deleterious effects wrought by
germs are due to chemical poisons elaborated by them
during their growth, admitted, let us inquire what proper-
ties a microorganism must possess before it can l)e said to
be the specific cause of a disease. The four rules of KOCH
have been generally conceded to be sufficient to show that
a given germ is the sole and sufficient cause of the disease
with which that germ is associated. Briefly, these rules are
as follows :

1. The germ must be present in all cases of that disease.

2. The germ must be isolated from other organisms
and from all other matter found with it in the diseased
animal.

3. The germ thus freed from all foreign matter must,
when properly introduced, produce the disease in healthy
animals.

4. The microorganism must be found properly dis-

UK I. A TH»X TO INKKCTIOUS DISEASES. 93

tributed in the animal in which tin- disease has Ix-en
induced.

Let us give <>nr special attention to the first of these
rules for a few moments. What is meant by the state-
ment that the special germ must be found in every case of
the disease? How will A, pursuing his studios on the
bacteriology of a given disease in America, decide whether
or not a bacillus which he finds is identical with one which
\\:\< Keen reported as invariably present in the same disease
by B, who has investigated an epidemic in Germany?
What means are relic* I upon to prove the identity of these
two organisms? The means which have lxxin relied upon
wholly are the form, si/e, reaction with staining reagents,
manner of growth on various nutrient media, and, in ex-
ceptional instances, correspondence in their effects upon the
lower animals. In other words, with the exception of those
instances in which the effects upon animals are tried, the
characteristic property by which the germ causes the disease
is left wholly out of consideration. It is admitted that any
causal relation which the germ may have to the disease is
due to its capability of forming one or more chemical poi-
sons, and yet no attempt is made to ascertain whether or
not it possesses this property. Indeed, some of the most
eminent bacteriologists have taught that in the identifica-
tion of germs the reactions with staining reagents and the
appearance of the growths on the various nutritive media
are of more importance than the observation of the effects
upon animals. Thus, FLUGGE says :

" Inoculation experiments with both typhoid dejections
and pure cultuns of the Elx-rth bacillus have universally
Ix-en without success. 'Flic few experiments in which a
typhoid disease has followed inoculation or feeding have
Ixvn made with impure material containing other active
bacteria. It is known that a group of widely distributed
organisms, which, however, a/v wholly different from the
typhoid bacillus, have the power, when injected snlx-n-
taneonsly or intravenously, of producing in animals death
with marked swelling and nlccration of Peycr's patches.
To these organisms undoubtedly arc due the apparently

6*

94 BACTERIAL POISONS.

positive results which some authors have supposed to be
due to inoculation with the typhoid bacillus."

In other words, this eminent author teaches that although
other germs may cause the essential symptoms and lesions
of typhoid fever in the lower animals, they are not related
to the germ found in the spleen of man after death from
typhoid fever; because they do not react in the same man-
ner with the auilin stains, and present a different appear-
ance in their growths on potatoes.

We will suppose that in an epidemic of diphtheria, A
examines the membrane from a hundred, or we might as
well suppose a thousand, children, and finds a characteristic,
well-marked, easily recognized bacillus in all. He isolates
this organism, and obtains it in pure culture. He inocu-
lates animals, and these manifest all the signs, together with
the appearance of the characteristic membrane of diphtlu ria,
and in these animals he finds his bacillus growing as in the
throats of the children. All the rules of KOCH have been
complied with. Has A demonstrated that his bacillus is the
sole cause of diphtheria ? No. He has shown that his
bacillus is a cause of diphtheria; but he has not proven
that there may not be other germs, wholly different from
his in form and size, which may also cause diphtheria.
The most which can be proven by KOCH'S rules is that a
given germ is a cause of a certain disease. They do not
show, as most bacteriologists would have us believe, that
the given germ is the sole cause of the disease.

To illustrate, we will suppose that a botanist in visiting
Arabia should find a tree producing a berry, the coffee
berry, which, when properly prepared and taken into the
system, produces certain effects which are due to the alka-
loid, caff'ein, and which invariably follow the drinking of
a decoction of these berries; would our supposed discoverer
be justified in concluding that the coffee tree is the only
plant in the world capable of producing these supposed
characteristic effects ? Should he reach such a conclusion,
the fact that it is not warranted would be shown by a study
of the tea plant of China and the gnarana of South America.

The moment that it is granted that the real poison of the

KKI.ATloN To INFECTIOUS IHSKASKS. !».')

is chemical in character, it becomes evident that no
one is jn.-tilied in saying that one germ is the sole source of
that poison. Surh a statement would be as unwarranted
a- one that the oilier tnr is the solo source of call'ein, or
that the strvchnos Ignatii is the only species of the nat-
ural order Loganiacea- which produces a convulsive poison.
In other words, the specific cause of a given disease is not
to lie determined wholly by the morphology of the germ,
but I iv the character of the chemical poison which is the true
materics morbi.

Bacteria cannot be classified, so far as their causal rela-
tionship to disease is conecrned (and this is the most im-
portant knowledge to be gained from them), until we know
the nature of their chemical products, for it is by virtue of
these that the germs have any causal relationship to dis-
ease.

It is possible that two germs maybe unlike in form, and
yet they may produce poisons which are identical or those
which are very similar in their effects upon man. One
germ may lie stained by GRAM'S method and another fail
to be acted upon when so treated ; but this does not prove
that their chemical products are totally unlike. This is
not only a possibility, it is a fact which has Ix-en demon-
strated repeatedly, both with pathogenic and non-pathogenic
organisms. A few illustration* may IK' given here: The
yeast plant is not the only microorganism which will pro-
duce alcohol in saccharine solutions. The same prodtiet
results from the growth of the bacterium Bisehleri, bac-
terium coli commune, bacterium ilei, bacterium ovale ilei,
bacterium laetis aerogenes, and others (NENCKi). Mor-
phologically, there are marked differences lietween the yca.-t
plant and these bacteria, but they alike produce alcohol.
More than a do/en germs, including lx)th mierococci and
bacilli, are capable of generating lactic acid. Some of these
prodiur an acid which is optically inactive; others, one
which is dextro-rotatory; and others still, one which is hevo-
rotatory. The tetanus germ of KlTASATo and that of
TDBBONI and GAOTAHI are known to be different. Cultures
of the former in bouillon are virulent, while those of the

96 BACTERIAL POISONS.

latter in the same medium are inert. Not only are those
two organisms morphologically and biologically distinct,
but their poisons are chemically unlike. BRIEGER and
FRANKEL precipitated the poisonous albumin of the germ
of KITASATO with alcohol, but this reagent renders the
poison of the Italian germ inert. Notwithstanding this
difference, however, both microorganisms and their chemical
products produce tetanic convulsions and death in the lower
animals. We must, therefore, admit that there are at least
two distinct germs, each of which is capable of causing
tetanus ; and how many other bacteria with like properties
there may be no one can tell. All attempts to find a mor-
phologically specific germ in the summer diarrhoeas of
infancy have failed. The labors of BOOKER in this coun-
try and of ESCHERICH in Germany have shown that no
one species or variety is constantly present. No less than
thirty distinct germs have been obtained from the bowels
and feces of children suffering from these diarrhoeas. A
germ which is frequently present one season may not be
found at all the next. Are we to conclude from this fail-
ure to comply with the first of KOCH'S rules, that the sum-
mer diarrhoeas of infancy are not due to microorganisms?
Certainly not ; especially in view of the fact that BAGINSKY
and STADTHAGEN have obtained from pure cultures of a
saprophytic germ found in the stools of cholera infantum a
poisonous base and a poisonous proteid ; and VAUGHAN
has shown that at least three of BOOKER'S bacteria pro-
duce chemical poisons which cause in kittens retching,
vomiting, purging, collapse, and death. To the contrary
we are justified in concluding that these diarrhoeas may be
due to any one or more of a number of germs which differ
from one another sufficiently morphologic-ally to be classified
as distinct species. The similarity among these bacteria
will not be discovered by a study of their size, form, and
reactions with staining agents, but by a study of their
chemical products, the agents by virtue of which they cause
the disease.

We think that we are justified in concluding that in
those diseases in which the four rules of KOCH have been

KKI-AT10N TO INFECTIOUS DISEASKS. '.»7

complied with, the germ is '/ cause of the disease, l>ut our
ran^e of observation must IK- much wider than it now is
before we can say that the given germ is the only cause of
tin- disease.

We believe that those few infectious diseases, such as
anthrax and tuberculosis, which have such well-marked,
typical, clinical histories, are duo to equally well-marked
ami morphologically distinct microorganisms which can IM>
recognized by microscopical study alone; but we do not
Ix-lieve that this is true in diseases showing such wide vari-
ations in symptoms as is the case in typhoid fever and
cholera infantum.

In all cases, we insist that the true test of the specific
character of a germ is to l>e made with its chemical pro-
ducts. A given bacterium may not multiply in the circu-
lating Mood of a dog, and failure to do so is by no means
piHxif that the same organism might not cause disease in
man ; but every germ which rouses disease in man does so
by virtue of its chemical products, and if these be isolated
and injects! into the dog in sufficient quantity a poisonous
effect will l>e produced. In the study of the bacteriology
of the infections diseases, the third and fourth of KOCH'S
rules have not been complied with in many diseases on
account of the insusceptibility of the lower animals. The
majority of investigators, meetihg with this difficulty, have
IxH-n inclined to rest content with the first two rules, and
to conclude that when a given germ is constantly present
in a given disease, and not found in other diseases, that it
is the cause of the disease with which it is associated. In-
deed, we find so g<x>d an authority as WELCH stating that
the successful inoculation of animals is not necessary in
order to prove the causal relationship of a germ to a disease.
In 1889, VAUQHAN suggested that in those instances in
which the third and fourth of KOCH'S rulescannot be
complied with on account of the insusceptibility of the
lower animals, it must be shown that the germ can pro-
duce chemical poisons which will induce in the lower ani-
mals in an acute form the characteristic symptoms of the

98 BACTERIAL POISONS.

disease, before the proof that the given germ is the cause
of the disease be accepted as positive.

Heretofore, the science of bacteriology has been largely
founded upon morphological studies. Bacteriologists have
given their time and attention to the discovery of bacterial
forms in the diseased organism and to observations of char-
acteristics in structure and growth of different species of
bacterial life. We must now study the physiology and
chemistry of the germs, and until this is done we must
remain ignorant of the true cause of disease, and so long
as we remain ignorant of the cause, it cannot l>e expected
that we shall discover scientific and successful methods of
treatment. Suppose that our knowledge of the yeast plant
was limited to its form and method of growth ; of how little
practical importance this knowledge would be. That the
yeast plant requires a saccharine soil before it can grow,
that given such a soil it produces carbonic acid gas and
alcohol, are the most important and practical facts which
have been ascertained in its study. Likewise, the condi-
tions under which pathogenic germs multiply and the pro-
ducts which they elaborate in their multiplification must
be ascertained before their true relationship to disease can
be understood.

In saying that the morphological work upon which the
science of bacteriology rests almost wholly is inadequate,
we wish that it may be plainly understood that we are not
offering any hostile criticism upon the great men who have
done this work and who have formulated conclusions there-
from. The development of bacteriology has been in accord-
ance with the natural law governing the growth of all the
biological sciences. The study of form naturally and neces-
sarily precedes the study of function. The ornithologist
finds a new species of bird. He first studies its shape and
size, the color of its plumage, the form of its beak, the
number and arrangement of the feathers of the tail and
wing, the color of the eyes, etc. All this he can do with a
single specimen, recognizing the fact, however, that varia-
tions more or less marked are likely to be found in other
individuals. More time and wider opportunities of ob-

i: I:L. \TION TO i N KKCTIO rs IUSKASKS. 99

s 'Tx-ation will IM- nn-ded before he can tell where ami when
this hinl is a<vustome<l to build its DCS), upon what insects,
grains, and berries it feeds, with what other species of birds
it lives in peace and with what it is at war. A much
•greater range of observation and study is necessary Wore
the naturalist can tell how his newly discovered species
would thrive if carried to a new climate, where it woidd
he compelled to live upon unaccustomed food, to build its
nest of strange material, ami to encounter new foes.

We repeat that it is no discredit to the science nor to the
men who have developed it to say that the study of bac-
teriology has hitherto l>een almost wholly morphological.
Without the morphologist the physiologist and the physio-
logical chemist could not exist. The science having had
for its support only morphological studies, the deductions
and formulated statements arrived at by its students have
Urn reached in am)rdance with the Knowledge obtained
from this source. But now, it being admitted that the
causal relation between a given germ and a certain disease
is dependent upon the chemical products of the growth of
the germ, the fundamental lines of work must IK; altered in
order to correspond with this new knowledge.

The study of the chemical factors in the causation of the
infectious diseases opens up for us a field in which much
work must l>e done. Let us attempt a statement of the
nature of some of the researches that must be carried out
along this line.

In the first place, we must ascertain what germs are toxi-
eogenie. This would necessitate a chemical study of all kinds
of bacteria, l>oth the pathogenic and the non-pathogenic.
Kvery fact ascertained in this investigation will not have
its practical application in medicine, but will have its
scientific value, and many will most probably l>e of more
or less direct service to man.

Secondly, it must be determined under what conditions
these germs are toxicogenic. It is not at all probable that all
those bacteria which are capable of producing poisons when
grown on dead material outside of the body are also capable
of multiplication and the production of the same substances

100 BACTERIAL POISONS.

when under the influence of the various secretions of the
body. Some bacteria are destroyed by a normal gastric
juice within a short time, while others are not. The con-
ditions of life and growth are different when the infecting
agent is introduced into the blood from what they are when
infection occurs by the way of the alimentary canal. This
is well recognized in the two forms of anthrax, one of which
arises from inoculation through a wound and the other by
way of the intestines. A preventive treatment which is
efficient in one is of no service in the other. Then, again,
we are to study those conditions of the blood and other
fluids of the body which are especially unfavorable to the
successful implantation or the continued existence of an
infectious disease.

Thirdly, the chemical properties and the physiological
action of these poisons will demand careful attention. Some
are especially depressing in their action upon the heart,
others seem to manifest their chief energy upon the central
nervous system, while others still act like true gastro-
intestinal irritants. In the study of the toxicological effects
of these bacterial poisons every method of investigation
known in the most advanced physiological work must be
employed. The action of these agents on the heart, the
brain, the spinal cord, etc., must be thoroughly studied.

CHAPTER V.

THE BACTERIAL POISONS OF SOME OF THE INFECTIOUS
DISEASES.

WE will now give our attention to the chemical poisons,
both the ptomaines and the proteids, of some of the infec-
tious diseases, and in doing this we will illustrate and sub-
stantiate the statements made in the preceding chapter.

ANTHRAX. — The definition of an infectious disease, as
we have given it, is well illustrated by the facts which have
been learned concerning the causation of anthrax, which
lias probably been more thoroughly studied than any other
infectious disease. KAUSCH taught that this disease has its
origin in paralysis of the nerves of respiration. As to the
cause of this paralysis he gave us no information. DELAFOND
thought that anthrax has its origin in the influence of the
chemical composition of the soil affecting the food of ani-
mals and leading to abnormal mftrition. The investigations
of GERLACII in 1845 demonstrated the contagious nature
of the disease, which was emphasized by HKUSINGER in
1.S50 and accepted by VlRCHOW in 1855. However, as
early as 1849, PoLLENDER found numerous rod-like micro-
organisms in the blood of animals with the disease. This
observation was confirmed by BRAUELL, who produced
the disease in hcaltliy animals by inoculations with matter
taken from a pustule on a sick horse. Attempts were made
to ridicule the idea that these germs might l)e the cause of
the disease, and it was said that the bodies seen were only
fine shreds of fibrin or blood crystals. Some claimed that
the rod-like organisms reported were due to defects in the
glass, while others claimed that the defects existed in the
eye of the observer, and others still suggested that the de-

102 BACTERIAL POISONS.

fects might be found hack of the eye and in the brain. But
in 1863, DAVAINE showed that these little bodies must
have some causal relation to the disease, inasmuch as his
experiments proved that inoculation of healthy animals
with the blood of those sick with anthrax produced the
disease only when taken at a time when the blood con-
tained these organisms. He also demonstrated beyond any
question that these rod-like bodies are bacteria, capable of
growth 'and multiplication. The conclusions of this investi-
gator were combated by many ; but PASTEUR, KOCH,
BOLLINGER, DE BARRY, and others, studied the morph-
ology and life-history of these organisms, and then came
the brilliant results of PASTEUR and KOCH in securing
protection against inoculation anthrax by the vaccination
of healthy animals with the modified germ and subsequent
inoculation with the virulent form. Now, the bacillus
anthracis is known in every bacteriological laboratory, and
by inoculation with it the disease is communicated at will
to susceptible animals. But here the question arose, How
do these bacilli produce anthrax? and in answer to this
question the various theories which we have mentioned
were proposed.

The first successful attempt to study the chemical poisons
of anthrax was made by HOFFA, who obtained from pure
cultures of the bacillus small quantities of a ptomaine,
which, when injected under the skin of animals, products
the symptoms of the disease and death. This substance
causes at first increased respiration and action of the heart,
then the respirations become deep, slow, and irregular ;
the temperature falls below the normal; the pupils are
dilated, and a bloody diarrhoea sets in. On section the
heart is found contracted, the blood dark, and ecchymoses
are observed on the pericardium and peritoneum. HOFFA
names his poison anthracin. Recently HOFFA has isolated
this poison from the bodies of animals dead from anthrax.

It has been said that HOFFA 's work was the first suc-
wssful attempt to study the chemical poisons of anthrax.
However, his results cannot l>e considered altogether satis-
factory. The small amount of the basic substance which

ANTHRAX, 103

lie obtained rendered it highly probable that in the case of
;i germ so virulent MS that of anthrax there must ho other
chemical poisons produced. This supposition lias IxH'ii eon-
liniied l»\ the labors of HANKIN, who, in 1889, while at
work in KOCH'S laboratory, prepared from cultures of
the bacillus anthracis an albumose which, when employed
in comparatively large amount, proved fatal to animals,
but when used in very small quantity gave immunity
against sul)sc<[uent inoculations with the living germ.
Unfortunately, HANKIN does not mention the symptoms
induced by toxieal doses of this substance. Whether or
not the albumose of HANKIN contains in statu nascendi
the base of HoFFA, and owes its poisonous properties to
the same, has not been determined.

BfilBQEB and FRANKEL obtained the poisonous protcid
of anthrax from animals in which the disease had been
induced by inoculation with the bacillus. The liver, spleen,
lungs, and kidneys of these animals were finely divided
and rubbed up with water. After this had stood in a
refrigerator for twelve hours it was passed through a
Chamberland filter and the proteid precipitated from the
filtrate with absolute alcohol.

MARTIN, by growing the anthrax bacillus for from ten
to fifteen days in an alkaline albuminate from blood serum
and filtration through porcelain, obtains the following
metabolic products :

1. Protoalbumose and deuteroalbumose and a trace of
peptone. All of these react chemically like similar sub-
staiices prepared by peptic digestion.

2. An alkaloid.

.">. Small quantities of leucin and tyrosin.

The most characteristic property of the albumoses is that
their solutions are strongly alkaline, and the alkalinity is
not removed by treatment with alcohol, lx'ii/ol, chloroform,
or ether, or by dialysis.

The alkaloid is soluble in water, alcohol, and amylic
alcohol ; insoluble in chloroform, ether, and ben/ol. Its
solutions are strongly alkaline and the alkaloid forms crys-
talline -alt- with acids. It is precipitated by the general

104 BACTERIAL POISONS.

alkaloidal reagents, with the exception of potassio-mercnric
iodide. It is somewhat volatile and loses its poisonous
properties on exposure to the air.

The mixed alhumoses are poisonous only in considerable
doses, 0.3 gramme being required to kill a mouse of 22
grammes weight when injected subcutaneously. Smaller
doses cause a local oedema and a somnolent condition, from
which the animal recovers. The larger doses produce a more
extensive oedema and the somnolence deepens into coma,
terminating in death. In some cases the spleen is enlarged.
The absence of germs was demonstrated by plate cultures.
The alkaloid causes similar symptoms. It is, however,
more poisonous and acts more rapidly than the albumoses.
The animal is affected immediately after the injection, and
the gradually increasing coma terminates in death. The
alkaloid also produces oedema, and in many cases throm-
bosis of the small veins. Extravasation into the peritoneal
cavity is occasionally seen, and the spleen is ordinarily
enlarged and filial with blood. The fatal dose for a mouse
is from 0.1 to 0.15 gramme, death resulting within three;
hours.

This alkaloid does not appear to be identical in its action
with the anthracin of HOFFA.

ASIATIC CHOLERA. — There are good reasons, apart from
experimental evidence, for believing that the comma bacillus
of KOCH produces its ill effects by the elaboration of chemi-
cal poisons. This germ is not a blood parasite. It grows
only in the intestine, and the symptoms of the disease and
death must result from the absorption of its poisonous
products. In confirmation of this statement experiment
has shown that this is one of the most active, chemically,
of all known pathogenic germs.

In the first place, BITTER has shown that the comma
bacillus produces in meat-peptone cultures a peptonizing
ferment, which remains active after the organism has been
destroyed. Like similar chemical ferments, it converts an
indefinite amount of coagulated albumin into peptone. It
is more active in alkaline than in acid solutions, thus

ASIATIC CIIOLKKA. 105

milling panel-cat in more than pepsin. This resem-
blaiuv to panereat in is further demonstrated by the fact
that its activity is increased by the presence of certain
clicinicals, such as s<xlinm carbonate and sodium salicylate.
That a diastatic ferment is also produced l>y the growth of
the bacillus was indicated in the experiments of BITTER by
the development of an acid in nutrient solutions contain-
ing starch paste. However, all attempts to isolate the
dia>tatie ferment were unsuccessful. A temperature of 00°
destroys or greatly decreases the activity of ptyalin, and
this seems to l>c true also of the diastatic ferment produced
by the comma bacillus. But the formation of an ax-id from
the starch pre-supposes that the starch is first converted into
a soluble form.

FERMI has succeeded in isolating the peptonizing ferment
of the cholera germ in the following manner : 65 per cent.
alcohol added to gelatin which has been liquefied by the
bacillus precipitates the proteid, but not the ferment. After
1 \\entv-four hours the precipitate is removed by filtration
and the ferment precipitated from the filtrate by the addi-
tion of absolute alcohol. After being collected on a filter
and dried the ferment is dissolved in an aqueous solution
of thymol and its peptoni/ing properties demonstrated on
gelatin tulx's.

RrETscii Ix'licvcs that the destructive changes observed
in the intestines in cholera are due to the action of the
peptoni/ing ferment.

CANTANI injected sterili/ed cultures of the comma bacil-
lus into the peritoneal cavities of small dogs and observed
after from one-quarter to one-half hour the following symp-
toms : (Jreat weakness, tremor of the muscles, drooping of
the head, prostration, convulsive contractions of the pos-
terior extremities, repeated vomiting, and cold head and ex-
tremities. After two hours these symptoms l>egan to abate,
and alter twenty-four hours recovery seemed complete.
Control experiments with the same amounts of uninferted
beef-tea were made with negative results. The cultures
used were three days old when sterili/ed. Older culture-
~ en id I |r-s poisonous and a high or prolonged heat in

106 BACTERIAL POISONS.

sterilization decreased the toxicity of the fluid. From these
facts CANTANI concluded that the poisonous principle is
volatile, but the effect of high or prolonged heat in dimin-
ishing the toxicity was more probably due to its destructive
effect on the poisonous proteids.

CANTANI also observed that the blood of those sick with
cholera is acid : this has been confirmed by STRAUSS by
the examination of the blood directly after death ; and
AHREND found lactic acid in the strongly acid urine of a
cholera patient.

NICATI and RIETSCII produced fatal effects in dogs by
injecting cultures, from which all germs had been removed
by filtration, into the bloodvessels. Later, the same inves-
tigators obtained from old bouillon cultures containing
peptone a poisonous base. ERMENGEN also showed that
cultures after filtration through a Chamberland filter are
poisonous.

KLEBS has attemped to answer experi mental ly the ques-
tion, In what way does the cholera germ prove harmful ?
Cultures of the bacillus in fish preparations were acidified,
filtered, the filtrate evaporated on the water-bath, the residue
taken up with alcohol and precipitated with platinum chlo-
ride. The platinum was removed with hydrogen sulphide,
and the crystalline residue obtained on evaporation was
dissolved in water and injected intravenously into rabbits.
Muscular contractions were induced. Death followed in
one animal, which, in addition to the above treatment,
received an injection of a non-sterilized culture. In this
case there was observed an extensive calcification of the
epithelium of the uriniferous tubules. KLEBS believes this
change in the kidney to be induced by the chemical poison,
and from this standpoint he explains the symptoms of
cholera as follows : The cyanosis is a consequence of arte-
rial contraction, the first effect of the poison. The mus-
cular contractions also result from the action of the poison.
The serous exudate into the intestines follows upon epithe-
lial necrosis. Anuria and the subsequent symptoms appear
when the formation and absorption of the poison become
greatest.

ASIATIC CHOLERA. 107

HiKiM'K states tliiit the severe symptoms of cholera c:m
IM- explained only on tin- supposition tliat the bacilli produce
a chemical poison, and that this poison resembles mnscarine
in its action.

VILLIEHS isolateil by the STAS-OTTO method from two
l>odies dead from cholera, a poisonous base which was liquid,
pungent to the taste, and presetted the odor of hawthorn.
It \\;is strongly alkaline, and ^ave precipitates with the
general alkaloidal reagents. From one to two milligrammes
of this substance, injected into lro«*s, caused decreased
activity of the heart, violent trembling, and death. The
heart was found in diastole, and full of blood, and the
brain slightly congested. However, the presence of this
substance in the bodies of persons who have died of cholera
does not prove that its production is due to the cholera
bacillus.

P0UCHET extracted from cholera stools, with chloroform,
an oily base l>elon«rin<r to the pyridine series. It readily
reduces ferric as well MS «;old and platinum salts, and forms
an easily decomposable hydroehlonde. It is a violent poison,
irritating the stomach, ami retarding the action of the he-art.
Subsequently, he obtained an apparently identical substance
from cultures of Kuril's comma bacillus.

In 1887, BKIKCKK made a report of his studies on the
chemistry of the cholera bacillus: He used pure cultures on
beef-broth (Mcisehbrei), which was rendered! alkaline by the
addition of a .'} per cent, soda solution. These were kept
at from .'57" to .'58°. A fter twenty-four hours, cadaverine
was found to IM- present. Older cultures furnished very
small quantities of putreseine, but cultures on blo<xl-serum
yielded much larger amounts of this base. While cada-
verine and putrcs"ine cannot be said to be poisonous, they
do cause necrosis of tissue into which they are injected, and
their formation by the cholera bacillus may account for the
necrotic tissue in the intestine in the disease. The lecithin
of the beef-broth was slowlv acted UJMHI by the therms, but
with aii'c the amount of choline increased, reachinj; its
maximum durin«r the Jburth week.

<'iv:itinr prove* I still more resistant to the action of the

108 BACTERIAL POISONS.

germs ; but, after six weeks, a considerable quantity of
creatinine was isolated, and a smaller amount of methyl-
guanidine. The latter is very poisonous, causing muscular
tremors and dyspnoea. The presence of methyl-guanidine
indicates that the comma bacillus acts as an oxidizing agent,
since creatine yields methyl-guanidine only by oxidation.

BRIEGER succeeded in finding, in addition to the above-
mentioned ptomaines, which are common products of putre-
faction, two poisons which he considers as specific products
of the comma bacillus. One of these, found in the mer-
curic chloride precipitate, is a diamine, resembling trime-
thylenediamine. It produced muscular tremor and heavy
cramps. In the mercury filtrate was found another poison,
which, in mice, produced a lethargic condition ; the respira-
tion and heart's action became slow, and the temperature
sank, so that the animal felt cold. Sometimes there was
bloody diarrhoea.

BRIEGER and FRANKEL found that the insoluble proteid
which they obtained from cultures of the cholera bacillus,
when suspended in water and injected subcutaneously in
guinea-pigs, caused death after from two to three days.
Section showed inflammatory swelling and redness of the
subcutaneous tissue, extending into the muscles for some
distance about the point of injection, but no necrosis.
There was no change in the intestines and no effusion into
the peritoneum. In some instances there were evidences
of beginning fatty degeneration of the liver. Upon rab-
bits this substance, even in large doses, was without effect.

In endeavoring to obtain immunity in guinea-pigs against
cholera, GAMALEIA employs cultures which have been ster-
ilized at 120°. Subcutaneous injections of these cause
transient O3dema, and the animals soon recover. The high
temperature destroys not only the bacillus, but renders inert
certain " ferment-like " products. However, if the cultures
be sterilized at 60°, large doses (10 c.c. per kilogramme, body
weight) cause death, injected intravenously in rabbits, and
a less amount produces marked symptoms. The animals
refiise food, and a diarrhoea, which may continue for hours,
appears. Often there is cloudiness of the cornea and ivtm-

ASIATIC CHOLERA. 109

tion of urine, which is albuminous. The ;miin;ils recover
very slowly. In this connection BOUCHARD remarks that
in 1884 he obtained l>y the intravenous injection of the
urine of a cholera patient in rabbits muscular tremor, cyan-
osis, albuminuria, and diarrh<ea, but that he has never suc-
ceeded in inducing these symptoms with the cholera vibrio.

PETRI finds that the comma bacillus produces in solu-
tions of peptone large amounts of tyros in and leticin, a
small quantity of inclol, fatty acids, poisonous bases, and a
poisonous proteid. The proteid resembles peptone in its
behavior toward heat and chemical reagents, and is desig-
nated by PETRI as " toxopeptone." It is not precipitated
by I lent or concentrated nitric acid, nor by potassium ferro-
cyanidc and acetic acid, nor by ammonium sulphate added
to saturation, \\ith sodium phospho-tungstate it gives a
precipitate which clears up on the application of heat. The
precipitate with tannic acid is insoluble in an excess of the
precipitant. It gives the biuret reaction perfectly, but
responds to Millon's test but feebly.

In quantities of 0.36 of a gramme per kilogramme and
more it is fatal to guinea-pigs within eighteen hours. It
produces muscular tremor and paralysis. Post-mortem
shows an effusion into the peritoneal cavity, marked injec-
tion of the bloodvessels of tbe intestines, and isolated
hemorrhagic spots.

This proteid is not rendered inert by a temperature of
100°. PETRI does not claim that he has obtained a chemi-
cally pure l>ody, but supposes that it is contaminated with
more or less unchanged peptone.

ScHOLL has studied the chemical products of the cholera
bacillus when grown under anaerobic conditions. Fresh
BggB were stcrili/cd and inoculated in the usual way. The
eggs, after being kept for eighteen days at 36°, were opened.
The contents smelled intensely of hydrogen sulphide, but
not of amines. The albumin was completely fluid, while
the yolk was more solid and of a dark color.

Five cc. of the fluid contents were injected into the
abdomen of a guinea-pig. Soon the posterior extremi-
ties were paraly/ed, and after ten minutes the paralysis

6

110 BACTERIAL POISONS.

became general, the animal lying on the side. After five
minutes more convulsive movements of the extremities
began, and forty minutes after the injection the animal was
dead. Section showed the vessels of the small intestines
and stomach highly injected, a colorless effusion in the
peritoneal cavity, and the heart in diastole.

The albuminous content of the egg was poured into ten
times its volume of absolute alcohol. The precipitate was
collected and washed with alcohol until a colorless filtrate
was obtained. The precipitate was then digested for fif-
teen minutes with 200 c.c. of water and filtered. Eight
c.c. of the filtrate was injected into the abdomen of^a
guinea-pig. Paralysis resulted immediately, and within
one and one-fourth minutes the animal was dead. Section
showed marked injection of the vessels of the small intes-
tines, a bloody transudate in the peritoneal cavity and the
heart in diastole.

The poisonous proteid Avas rendered inert by a tempera-
ture of 100°; it was not altered by short exposure to 75°,
but attempts to evaporate the solution at 40° in vacuo over
calcium chloride destroyed the poisonous properties. The
proteid was finally precipitated from its aqueous solution
by a mixture of alcohol and ether. It was washed with
ether and the ether allowed to evaporate spontaneously. A
small bit of this proteid proved fatal to guinea-pigs, and
the same post-mortem changes were found as given above.
SCROLL classes this proteid among the peptones. It is not
precipitated by heat or concentrated nitric acid, singly or
combined, nor by ammonium sulphate. It gives the xantho-
proteid and biuret reactions. SCHOLL regards this as the
true poison of cholera, and points out its difference from
the proteid of BRIEGER and FRANKEL and that of
PETRI.

BUJWID found that on the addition of from five to ten
per cent, of hydrochloric acid to bouillon cultures of the
cholera bacillus there was developed after a few minutes a
rose-violet coloration which increased during the next half
hour and in a bright light showed a brownish shade. The
coloration is more marked if the culture is kept at about

ASIATIC CHOLERA. Ill

37°. In impure cultures this reaction does not o<vur.
The FlNKLER-PllIOR bacillus cultures give after a longer
time a similar, but more of a brownish coloration. Cul-
tui-K of many other bacilli were trie<l and failed to give
this reaction.1

HIMEGER found that this color is due to an indol deriva-
tive. In cholera cultures on albumins he obtained indol
by distillation with acetic acid.

Bu.nvn> has made a further contribution to our knowl-
edge of the " cholera-reaction." His conclusions are as
follows :

(1) Five to ten per cent, of hydrochloric acid added to
cholera cultures produce a rose-violet coloration, which is
characteristic of the comma bacillus.

(2) No other bacterium gives the same coloration under
the same conditions.

(3) The coloration appears in such cultures which are
from ten to twelve hours old, so that this test can l>e used
lor diagnostic purposes, and will give results before they
ean be obtained by plate cultures.

(4) Impure cultures do not give this reaction.

DI.MIAM finds the In-st medium for the " cholera-reac-
tion " to be a one per cent, alkaline peptone solution with
one-half per cent, of common salt. BUJWID prefers a two
percent, tlrbly alkaline peptone solution with salt. JADAS-
SOHN finds that gelatin cultures give the reaction both
In-tiire and alter the liquefaction of the gelatin. The un-
dissolved gelatin, after the addition of hydrochloric or
sulphuric acid, Ix-comes rose-violet.

OoHEH claims that cultures of other bacilli give a similar
coloration, but BUJWID explains that the results obtained
by Co i IK N were due to the use of impure acids, which con-
tained nitrous acid. SALKOWHKI agrees with Bu.iwin, and
-t:itis that, when acids wholly free from nitrous acid arc
used, the reaction is characteristic of the comma bacillus.
He explains the reaction by supposing that the germ pro-

1 Poehl deserves the credit of being the first to call attention to this
reaction, though his work was evidently unknown to Bujwid at the time
when the latter published his report.

112 BACTERIAL POISONS.

duces nitrous acid, which exists in the culture as a nitrite.
On the addition of an acid the nitrous acid is set free, and
acting upon the indol, which is also present, gives the
coloration.

From a very exhaustive research on the importance of
this test PETRI comes to the following conclusions :

(1) Seven pure cultures of the cholera germ from as many
sources gave the reaction with equal distinctness.

(2) Of one hundred other bacteria tested in the same
way twenty gave a red coloration. In nineteen of these
the coloration is due to the nitroso-indol reaction of
Baeyer. The twentieth (anthrax) gave a color which is not
due to indol.

(3) In case of the cholera germ and the others as well,
the reaction is due to the reducing action of the bacteria on
nitrates. The reaction is most marked at blood -tempera-
ture and with the cholera bacillus ; it is least distinct with
the bacilli of FINKLER and MILLER.

(4) None of these bacteria convert ammonia into nitrite.

(5) The simple addition of sulphuric acid is sufficient to
give the test, which, however, is most marked when the
nutritive solution contains 0.01 per cent, of nitrate.

(6) The reaction is most marked if the sulphuric acid be
added after the addition of a very dilute nitrite solution.

SCHUCHARDT calls attention to the fact that VIRCHOW
observed a red coloration on the addition of nitric acid to
filtered cholera stools in 1846. GRIESINGER, in 1885,
also made mention of the production of a red coloration in
rice-water stools on the addition of nitric acid.

A " cholera-blue " has also been observed by BRIEGER
in cultures in meat extract containing peptone and gelatin.
This substance, which is yellow by reflected, and blue l>y
transmitted light, is developed by the addition of concen-
trated sulphuric acid to the culture. It may be separated
from the "cholera-red" as follows : Treat the culture with
sulphuric acid, then render alkaline with sodium hydrate,
and extract with ether. Evaporate the ether, and remove
the " cholera-red " with benzol, then again dissolve the
" cholera-blue " in ether. The characteristic absorption

TETANUS. 113

bands for this coloring matter beirin ill tin- first third of the
spectrum, between ]-] and F, and darken all of tin- zone
lyini: beyond.

WINTKU and LKSA<.I: treat a bouillon culture of the
cholera licrni with sulphuric acid, dissolve the precipitate
in an alkaline medium, reprecipitate with aeid, and rcdis-
solve in ether, which on evaporation leaves oily drops,
which, on cooling, form a yellow mass of the appearance
of a fat This substance is insoluble in water and acids,
soluble in alkalies and ether. It melts at 50°, and does
not lose its virulence on heinjj boiled with alcohol rendered
feebly alkaline. The virulence of a culture and the amount
of this substance contained therein are in direct proportion
to each other.

Small dos s of this substance (1 milligramme to 100
grammes of body weight of the animal) in feebly alkaline
solution introduced into the stomachs of guinea-pigs cause,
a- a rule, within from four to six hours, a chill, and death
alter twenty-four hours. With larger doses the tempera-
ture falls after from one-half to one hour, and death results
within from twelve to twenty hours. Smaller doses cause
a less marked reaction and the animal recovers within
twenty-four hours. If killed within this time the animal
shows a choleraic condition. Rabbits succumb only after
repeated subcutaneous injections. The substance can be
extracted from the muscles, liver, kidneys, and urine of
the poisoned animals. It can also U> obtained from cultures
of a cholera infimtuin germ. The fact that this poison l>c-
l<>ii_:~ neither to the ptomames nor albumins is of interest.

CUNNINGHAM describee ten species of the common ba-
cillus, one of which does not liquefy gelatin, and fails to
respond to the cholera reaction. lie also states that there
are cases of undoubted cholera in Calcutta in which Un-
common bacillus is wholly wanting.

TETANUS.— In 1884, NICOLA IKIJ, by inoculating 140
animals with earth taken from different places, produced
symptoms of tetanus in <>!> of them. In the pus which
formed at the point of inoculation he found microeocci and

114 BACTERIAL POISONS.

bacilli. Among the latter was one which was somewhat
longer and slightly thicker than the bacillus of mouse-
septicaemia. In the subcutaneous cellular tissue he found
this bacillus alone, but could not detect it in the blood,
muscles, or nerves. Heating the soil for an hour rendered
the inoculations with it harmless. In cultures, NICOLAIER
was unable to separate this bacillus from other germs, but
inoculations with mixed cultures produced tetanus. In the
same year, CARLE and KATONE induced tetanus in lower
animals by inoculations with matter taken from a pustule
on a man just dead from tetanus. In 1886, ROSENBACH
made successful inoculations on animals with matter taken
from a man who had diet! from tetanus consequent upon
gangrene from frozen feet. With bits of skin taken from
near the line of demarcation, he inoculated two guinea-pigs
on the thigh ; tetanic symptoms set in within twelve hours,
and one animal died within eighteen, and the other within
twenty-four hours. The symptoms corresponded exactly
with those observed in the " earth tetanus " of NICOLAIER,
and the same bacillus was found. With mixed cultures of
this, ROSENBACH was also able to cause death by tetanus
in animals. BEUMER had under observation a man who
died from lockjaw following the sticking of a splinter of
wood under his finger-nail. Inoculations of mice and
rabbits with some of the dirt found on the wood led to
tetanus. The same observer saw a boy die from this dis-
ease following an injury to the foot from a sharp piece of
stone. White mice inoculated with matter from the wound,
and those inoculated with dirt taken from the boy's play-
ground, died of tetanus. The bacillus of NICOLAIER was
again detected. GIORDANO reports the case of a man who
fell and sustained a complicated fracture of the arm. He
remained on the ground for some hours, and when assist-
ance came the muscles and skin were found torn and the
wounds filled with dirt. On the fifth day he showed symp-
toms of tetanus, from which he died on the eighth day.
Inoculations and examinations for the bacillus were again
successful. FERRARI also made successful inoctilat'ons
with the blood taken during life from a woman with

TETANUS. 115

i' 1:11111- after :ni ovariotomy. HoCKSIXGER has confirmed
(lie above-mentioned observations by carefully conduct* •< I
e\p •riiiients, tin- mat-rial for which was furnished by a case
of tetanus arising from a very slight injury to the hand,
the wound brin ir fillr<l with dirt. SHAKESPEARE has suc-
renled in inducing: tetanus in rabbits by inoculating them
with matter taken from the medulla of a horse and of a
mule, l>oth of which had died from traumatie tetanus.
These uniform observations leave no room to doubt that
tetanus is often, at least, due to a germ which exists in
many places in the soil, and that the disease is transmissible
by inoculation.

BONOME observed nine eases of tetanus among seventy
I Arsons injured by the falling of a church from the
earthquake at Bajardo. The bacillus of NlCOT-AiER was
detected in the wounds, and animals inoculated with the
lime-dust of the fallen building died of tetanus. Of many
persons Injured by the falling of another church at the
same time, none had tetanus, and animals inoculated with
the lime from this church suffered no inconvenience.

The same experimenter found the bacillus in the wound
of a sheep which died from tetanus after castration.

BEUMER found the tetanus bacillus in the sloughing
tissue of the umbilical cord of a child which was taken ill
on the sixth day after birth, ami died four days later from
tetanus. From this he concludes that tetanus neonatoruin
and " earth tetanus " are identical, and advises that the cord
should be dressed antiseptically.

KITASATO has succeeded in isolating the bacillus of
Nieolaier by growing the mixed cultures, from the pus of a
wound on a man who died from tetanus, at a high tempera-
ture (80°), and subsequently developing the germ under
hydrogen. The bacillus grows only in the absence of air,
and not. in earl ionic acid. It develops on agar, blood-serum,
and gelatin, the last of which it gradually liquefies with the
formation of gas. The growth is more vigorous when the
nutritive medium contains from 1.5 to 2 per cent of grape-
sugar.

In 1888 BELFANTI and PESCAROLO found in the pus of

116 BACTERIAL POISONS.

a wound, which was followed by tetanus, a bacillus which
they l>elieved to differ morphologically from that of Nico-
laier and Rosenbach, and which in pure cultures induces
tetanus in animals. The number of animals experimented
upon was great and included mice, guinea-pigs, frogs,
rabbits, pigeons, geese, sparrows, a chicken, and a dog.
The pigeons, chicken, geese, and frogs proved immune.
After subcutaneous injections a bloody oedema appeared at
the place of inoculation and pus formed in small quantity.
Paralysis first appeared and was followed by convulsions
and opisthotonos. Later studies lead BELFANTI and I'KS-
CAROLO to conclude that their bacillus is really that of
NICOLAIER, but differing somewhat from that of KITA-
SATO. KITASATO states positively that the germ which he
has isolated is absolutely anaerobic, while the Italians find
that theirs will not only grow aerobic-ally, but when so
grown will induce a classical tetanus.

LAMPIASI found in the blood from various organs of a
man who died from so-called spontaneous tetanus, and in
two cases of tetanus in mules, a spore-forming bacillus,
which in pure cultures induced tetanus in animals. This
bacillus is wholly different morphologically from that of
Nicolaier.

WIDENMANN reports a very interesting case of a boy
who fell from a wall and wounded his face on a piece of
vine-stake in the earth. The boy died of tetanus, and the
splinters extracted from the face and the earth about the
stake were examined. The splinter was introduced under
the skin of a mouse, which died thirty hours later of tetanus.
In the pus formed about the splinter numerous microorgan-
isms, among which a micrococcus and a short, thick bacillus
abounded, were found, but in none of the many animals ex-
perimented upon could the bacillus of NICOLAIER be de-
tected. In animals inoculated with the earth, however, the
NICOLAIER germ was found. WIDENMANN concludes that
the so-called tetanus bacillus is found in most cases on ac-
count of its very wide distribution in the soil and not as a
result of its causal relation to the disease.

FLUGGE has produced tetanus in animals without being

TETANUS. 117

able to find the bacillus of NIOOLAIBB, and WYSSOKOW-

ir-< il has examined an earth which did not induce tetanus,
Itnt which caused suppuration, and in the pus the NlCO-
I.AIKI: l)acillus was found to lxi abundant. With the pus
obtained from thnv cases of tetanus neonatonun due to
omphalitis KISCIIENSKY induced tetanus in animals. The
pus contained pyogenetk) micrococd and a short bacillus,
but the germ of \i< OLAIEU could not l>e detected.

Although KiTT claims that his tetanus had 11 us is iden-
tical with that of KITASATO (which is now regarded as a
pure culture of the germ of NICOLAIEK), the former lique-
fies solid blood-serum and the latter does not. Bacteriolo-
gists generally agree that the NicOLAlER bacillus is found
only at the place of inoculation and that it is never present
in the blood or internal organs, yet SHAKESPEARE, as we
have seen, induced tetanus in rabbits by inoculating them
with matter taken from the medulla of a horse and that of
a mule, l>oth of which had died of tetanus. The bacillus
which has l>een so well studied by TI/ZONI and CATTANI
has certain constant biological differences from that of
K ITASATO.

I'LA has studied eight cases of traumatic tetanus both by
cultures and by inoculation of animals. In none has he
found the germ of NieX)LAiEit. Moreover, since tetanus
was induced in animals by bits of matter taken from the
spinal cord, the NICOLAIER germ could not have been the
cause, if, as bacteriologists now teach, this germ is never
found save at the place of inoculation.

P>I:II:<;I:I; has obtained in the mixed cultures of the germ
of NICOLA I EU and ROSKNUA< H four jmisonous substances.
The first, tetanine, which rapidly decomposes in acid solu-
tions, but is stable in alkaline solutions, prodiuvs tetanus
in mice when inject*1*! in quantities of only a few milli-
grammes. The second, tctanotoxine, produces i\r>i tremor,
then paralysis followed by severe convulsions. The third,
to which no name has l>een given, causes tetanus accom-
panied by Ire*1 flow of the saliva and tears. The fourth,
spasmotoxine, induces heavy clonic and tonic convulsions.

BRIEGER has also isolate*! tetanine from the amputated

6*

118 BACTERIAL POISONS.

arm of a man with tetanus, thus showing that this chemical
poison is formed in the Ixxly as well as in the artificial
cultures.

BuiEGERand FRANK EL obtained a " toxalbumin " from
a culture of KITASATO'S germ in bouillon containing grape-
sugar. This substance is soluble in water, and when in-
jected in small amounts subcutaneously in guinea-pigs,
tetanus appears in about four days, and soon terminates
fatally. On the other hand, cultures of the bacillus of
TIZZONI and CATTANI in bouillon with sugar fail to pro-
duce any chemical poison, but the cultures in gelatin are
highly poisonous after nitration through porcelain. Even
one-half cubic centimetre of the latter induces the disease
and death in rabbits weighing from one and a half to two
kilogrammes. Death results never later than three days,
while, as has been seen above, the first symptoms induced
by the poison from the bacillus of KITASATO usually
appear on the fourth day. BRIEGER and FRANKEL ob-
tained their proteid by precipitation with absolute alcohol,
but the addition of this agent to cultures of the germ of
TIZZONI and CATTANI Destroys its poisonous properties.
The active substance of the Italian germ was obtained
either (1) by dialysis, solution in water, and evaporation in
a vacuum ; or (2) by precipitation with ammonium sul-
phate, separation by dialysis, and drying in a vacuum.
This poisonous body is soluble in water, non-dialyzable,
destructible by a temperature above 60°, and by treatment
with concentrated mineral acids, and is unaffected by alka-
lies or by prolonged treatment with carbonic acid gas. It
contains a ferment which liquefies gelatin and digests fibrin.
This peptonizing ferment is active only in alkaline solu-
tion, and is present in the bouillon cultures which are not
poisonous; therefore, the poison and the peptoni/ing fer-
ment must be two distinct bodies. However, on account
of the properties which we have mentioned, TIZZONI and
CATTANI conclude that the poison also belongs to the
soluble ferments or enzymes.

BUSCHETTINI has studied the distribution of this poison

TETANUS. 119

through the |NH!V mid its elimination in the following
manner :

Animals were poisoned by injections of the substance
prepared by TI//OM and CATTANI, and just before death
they were killed and bits of various organs rnblx-d up with
sterili/ed water were injected into other animals. Emul-
sions from the liver and supra-renal capsules were invariably
without etfi-ct, while those from the kidney were constantly
poisonous. This is supposed to prove that the poison is
eliminated by the kidney. The blood taken from the vena
cava was found to be poisonous in three out of four experi-
ments. When the injections were made under the skin
the lumbar cord was active in four out of eight eases, and
in all, when the injections were made directly into the
sciatic nerve. On the other hand, when the inoculations
were made under the dura mater, the brain was found to
be active while the lumbar cord remained inactive. From
these experimental it is concluded that the poison not only
circulates in the blood, but is deposited in the central
nervous system.

A. BABKS prepared, from cultures made by V. BABES
and IVscARlA in agar containing no peptone, an albumose
which causes tetanus in animals.

FA HER finds in a mixed culture a poisonous proteid
body which resembles closely, so far as it has l>een studied,
that of TIZ/OXI and CATTANI. FA BE R lays much stress
upon the arguments in favor of this substance being a
soluble ferment. With this proteid, convulsive movements
first ap|>ear and I x -come very distinct in the muscles about
the |*)iut of injection. In ease very small amounts are
employed, the convulsive movements do not lx»come general
and the animal finally recovers.

I'KYKAUD claims to have secured immunity in animals
against "earth tetanus" by giving to them strychnia in
gradually increased closes. NoCARD could not confirm this
claim.

According to LEDANTES, the poisonous arrows of the
natives of the New Hebrides are prepared as follows : The
points, which are usually made from human bones, are first

120 BACTERIAL POISONS.

covered with a vegetable resin, then smeared with the slime
of swampy places.

LIERMANN found that material taken from the arm of a
man who had died from tetanus, and who had l)een buried
for two and one-half years, induced tetanus in animals.
This would seem to show that the poison retains its viru-
lence for a long time. In this material there were found
nine kinds of bacteria, but none of these in pure culture,
or in mixed culture, induced the disease. This is explained
by the supposition that non-pathogenic bacteria may receive
toxicogenic properties from the media in which they grow.

TUBERCULOSIS. — Whatever may be the ultimate verdict
concerning the curative properties of KOCH'S tuberculin, its
employment has made us familiar with the action of the
chemical products of the bacillus tuberculosis on man. Un-
fortunately, KOCH has given us but little information con-
cerning the nature of his tuberculin, and the little which he
has given us has been to some extent misleading. We
would not imply that he has intentionally been misleading.
Indeed, we believe that such was not his intention. He
speaks of the agent as an extract of a pure culture of the
bacillus tuberculosis with 50 per cent, glycerin. One would
infer from KOCH'S statements that tuberculin is prepared
by extracting the bacterial cells with 50 per cent, glycerin,
and that the bacterial products are not present. But,
as has been shown by HUEPPE and SCROLL, the proteids
of the cells of the bacillus tuberculosis cannot be extracted
with 50 per cent, glycerin. Moreover, the same investiga-
tors have prepared a fluid identical in physical properties,
in chemical reactions, and in its effects on animals, with
KOCH'S fluid, by each of the three following methods :

1. Cultures of the bacillus are filtered, sterilized by heat,
and concentrated.

2. The supernatant, fluid portion of the culture is de-
canted from the mass of germs at the bottom of the flask,
and then concentrated.

3. The culture is freed from germs by filtration through
a Chamberland filter, and concentrated.

TUBERCULOSIS. 121

Tins*; fluids contain : 1, tin; constituents of the nutritive
medium which have not been altered by the growth of the
germ, such as ulyccrin, albumins, albumoses, and peptones;
2, the bacteria] products, which may possibly belong to the
ptomaines, the bacterial albumins or albumoses and bacterial
ferments ; and ."., any constituents of dead, broken-down
bacilli which may have passed into solution. To which of
these constituents the action of the fluid is due hat not been
positively determined. However, from the similarity in
the action of this fluid with that of the bacterial products
of other germs, we seem justified in assuming that these
constitute the active principle.

As early as 1888, HAMMERSCHLAG found a poisonous
proteid among the products of the growth of this germ.
More recently he finds that as much as 27 per cent, of the
cellular substance of the bacillus tuberculosis is soluble in
alcohol and ether. In this extract there is, in addition to
fat and lecithin, a poison which induces in rabbits and
guinea-pigs convulsions followed by death. The part insol-
uble in alcohol and ether consists of cellulose and proteids.
HAMMKUSCHLAG has also prepared from cultures of this
bacillus a " toxalbumin " which, when injected sulx'iita-
neously in rabbits, causes an elevation of temperature of
from 1° to 2°, which continues for a day or longer.

ZUKLXKI: has reported the isolation of a poisonous pto-
maine from agar cultures of the bacillus tuberculosis. He
-avs that the injection of 1 centigramme or less of this
substance sulx-utancously in rabbits or guinea-pigs causes,
alter from three to five minutes, increased frequency of
respiration (to 180 per minute?) and an elevation of tem-
perature of from 0.5° to 1°. He also reports marked pro-
trusio bulbi as a constant symptom ; the eyes become very
bright and the pupils are dilated. From two to three
centigrammes suffice to kill rabbits, death occurring in from
two to four days. The place of injection is reddened, and
hemorrhagic spots are formed in the mucous membrane of
the stomach and small intestines. In two instances from
15 to 20 cubic" centimetres of clear fluid were found in the
peritoneal cavity.

122 BACTERIAL POISONS.

BAUMGARTEN draws the following conclusions from his
experiments with tuberculin on rabbits with inoculation
tuberculosis :

It causes an exudative inflammation in the vascular
tissue about the tubercle, and in this way the tuberculous
tissue may be isolated and, when situated superficially, re-
moved. In some cases, however, after the prolonged employ-
ment of the agent, the tuberculous tissue itself may, under
the influence of the exudative fluid and the polynuclear
leucocytes, break down and form abscesses. The bacilli
themselves are in no way harmed by the use of tuber-
culin, and, after its constant employment for months, they
retain their original form and lose none of their virulence.
Some preparations seem to show that the bacilli multiply
more rapidly when the injections are made, but a positive
statement on this point is reserved until further studies
have been made. It is certain, however, that the non-
tubercular tissue of animals acquires no immunity against
the disease from the injections. This is shown by the
appearance of metastatic foci in animals in which from
seven to twelve grammes of the original lymph (an amount
which would be equivalent to from seventy to one hundred
and eighty grammes in man) has been injected. It is further
shown by the fact that in some animals treated subcutane-
ously, tubercles have appeared at the point of injection.

PRUDDEN and HODENPYL, summarize the results which
they have obtained by the inoculation of animals with dead
tubercle bacilli as follows : " These dead tubercle bacilli
are markedly chemotactic. When introduced in consider-
able amount into the subcutaneous tissue or into the pleural
or abdominal cavities, they are distinctly pyogenetic, caus-
ing aseptic localized suppuration. Under these conditions
they are capable, moreover, of stimulating the tissues about
the suppurative foci to the development of a uew tissue,
closely resembling the diffuse tubercle tissue induced by
the living germ. We have found that dead tubercle bacilli
introduced in small numbers into the bloodvessels of the
rabbit largely disappear within a few hours or days, but
that scattering individuals and clusters may remain here

TUBERCULOSIS. 123

and there in the lungs and liver, clinging to the vessel walls
for many days without inducing any marked changes in the
latter. After a time, however — earliest in the lung, later,
as a rule, in the liver — a cell proliferation occurs in the
vicinity of these dead germs, which leads to the formation
of new multiple nodular structures bearing a striking mor-
phological resemblance to miliary tubercles. There is in
them, however, no tendency to cheesy degeneration and no
evidence of proliferation of the bacilli, but rather a steady
diminution in their number. It seems to us that the new
structures originate in a proliferation of the vascular endo-
thelium under the stimulus of the dead and disintegrating
germs."

MAFFUCCI finds that cultures of the tul>erclc bacillus
(from a mammal), when grown from one to six months on
gly(vrin, blood-serum, or liquid blood-serum, and then
sterili/ed by Ix'ing repeatedly heated to from 65° to 70°,
produces in guinea-pigs, when employed sulxnitaneously, a
progressive marasmus, which terminates fatally within from
fourteen days to five or six months. He also finds that
i-^o-s inoculated with sterilized cultures of the chicken tuber-
culosis bacillus produce chickens which are feeble and soon
die of emaciation. In neither the guinea-pigs nor chickens
could he find any tubercles. This author, unfortunately,
docs not state positively whetlier the bacilli employed in his
< -\p riments on guinea-pigs TON obtained from man or some
other mammal.

CROOKSHANK and HERROUN report the isolation of a
ptomaine and an albumose not only from artificial cultures
of the l>aeillus, but also from bovine tul>erculous tissue.
The ptomaine is reported as causing an elevation of tem-
perature in tul>ereulous, and a depression in healthy, ani-
mals. "The albumose, whether obtained from pure culti-
vations of the bacillus, or from tuberculous tissue, produced
a marked rise of temperature in tulicrculous guinea-pigs.
On the other hand, in an experiment tried on a healthy
guinea-pig, there was an equally well-marked tall of tem-
perature."

124 BACTERIAL POISONS.

DIPHTHERIA. — That the LOFFLER bacillus is a cause
of diphtheria no one can now deny. The fact that this
germ, although found only at the seat of inoculation, c;iu<i s
marked systemic disturbances, indicates that its action must
be due to its soluble products. This was early recognized
by LOFFLER, who in 1887 attempted to ascertain the
nature of the poison. A flask of bouillon containing pep-
tone and grape-sugar was, three days after it had Urn
inoculated with the bacillus, evaporated to 10 c.c., and this
was injected into an animal, but was without effect. A
second flask of the same material was extracted with ether,
but this extract was also found to be inert. Next, some
neutral beef broth was extracted with glycerin some four
or five days after it had l>een inoculated with the bacillus.
The glycerin extract, when treated with five times its
volume of absolute alcohol, deposited a voluminous, floe-
culent precipitate, which was collected, washed with alcohol,
dried, and dissolved in a little water. A further precipita-
tion with alcohol and a current of carbonic acid gas secured
a white substance, and the injection of from 0.1 to 0.2
gramme of this, dissolved in water, subcutancously in
guinea-pigs, caused marked pain followed by a fibrous
swelling with hemorrhage into the muscles and oedema,
terminating in necrosis. From these studies LOFFLER
concluded that the poison belongs to the enzymes.

Roux and YERSIN found that bouillon cultures from
which the bacillus had been removed by filtration through
a Chamberland filter are poisonous, especially cultures
which are four or five weeks old. The results obtained
varied with the amount of the fluid, the species of animal,
and the method of administration. The effects observed
were a serous exudation into the pleural cavity, a marked,
acute inflammation of the kidney, fatty degeneration of the
liver, especially after injection into a bloodvessel, and cede-
matous swelling in the surrounding tissue after sulM-u-
tancous inoculation. In some instances, in dogs, rabbits,
and guinea-pigs, paralysis, generally in the posterior extre-
mities, followed. The action of the poison was found to
be very slow, and; as a rule, death occurred days, and in

HI I'lITIl K IUA. 125

instances weeks utter tin- inoculation, and WAS pre-

<-edcd by marked emaciation.

The cultures first employed were seven days old ; older
cultures (six weeks) contain more of the poison, and the
symptoms appear within a lew hours. In cultures espe-
cially rich in the poison, a small amount (from 0.2 to '1 c.c. )
injected under the skin in guinea-pins sulliees to induce the
symj)toms. .Mice and rats are markedly insusceptible, but
succumb to large doses.

Heating to 100° for twenty minutes renders the poison
inert, and a temperature of 58° maintained for two hours
markedly lessens its virulence.

The poisonous substance is precipitated by al>solute
alcohol, and is carried down mechanically on the addi-
tion of calcium chloride to the filtered cultures. These
investigators agnv with LOFFLER that the poison belongs
to the en/ymes. The great toxicity of this substance is
indicatnl by the statement of Itoux and YERSIN that 0.4
milligramme suffices to kill eight guinea-pigs or two rab-
bits, and that 2 centigrammes of the calcium chloride
precipitate, containing aboutO.2 milligramme of the pure
poison, will kill a guinea-pig within four days.

BRIEGER and FRANKEL have made a very complete
study of the chemical products of the LOFFLER bacillus.
They employed cultures of bouijlon and peptone containing
from five to six per cent, of glycerin, and others containing
ten per cent, of sterile, fluid blood-serum. The latter were
found to IM- most suitable. In these the bacilli grow most
abundantly. In all cases they confirmed the statement of
Itoux and YERSIN that the cultures, at first alkaline, be-
come strongly acid, and finally again alkaline, with the
exception that the glycerin cultures remained acid.

For the removal of the bacteria two methods were em-
ployed. First the bacilli were destroyed by heat \Vhen
a temperature of 100° was employed the cultures were
rendered inert, but it was found that exposure for from
three to four hours to a temperature of 50° was sufficient
t<> destroy the germs, while the virulence of the chemical
products was not affected. The second method of removing

126 BACTERIAL POISONS.

the bacteria consisted of filtration through a Chamberland
filter. The germ-free filtrate could be heated to 50° with-
out loss of toxicity, while a temperature of 60° rendered
it inert. In the majority of the experiments the filtration
method was used and in this way a large quantity of a
poisonous fluid of uniform strength was obtained.

Varying amounts of this fluid were used upon animals,
mostly guinea-pigs and rabbits, and it was found that the
effects varied with the quantities employed and the methods
of administration. The symptoms appeared most promptly
when the injections were made directly into a bloodvessel.
Of four rabbits which were given subcutaneously respec-
tively 1, 2|, 5, and 10 c.c. of the filtrate on December the
28th, the first died January 4th ; the second, January 2d ;
the third, December 31st ; and the fourth, December 30th.
In all cases in which death did not occur too early, paralysis
appeared. The limbs were first paralyzed, and this was
true whether the fluid was administered intravenously or
subcutaneously. The post-mortem appearances were iden-
tical with those observed after inoculation with the bacillus,
with the exception of the absence of the pseudo-membrane.
After subcutaneous injection there was a gelatinous, grayish-
white, sometimes reddish, oedematous fluid formed at the
point of injection ; and, after larger doses, necrosis. In
cases in which death was delayed, there were effusions in
the pleura, fatty degeneration of the liver, and inflamma-
tion of the kidneys. Especially marked were these cellular
changes in rabbits which were treated with small amounts
intravenously.

BRIEGER and FRANKEL, conclude this part of their
report with the following statement : " We have shown
that the LOFFLER diphtheria bacillus produces in its cul-
tures a poisonous, soluble substance, separable from the
bacteria, which causes in susceptible animals the same
phenomena which are induced by inoculation with the
living microorganism. We have further shown that this
substance is destroyed by a temperature over 60°, but that
it can be heated to 50°, even in the presence of an c.wss
of hydrochloric acid, without being destroyed. This last

DIPHTHERIA. 127

fact is contrary to the assumption that the chemical poison
of the diphtheria hacillns is a ferment or en/yme."

The fluid was tested for basic products, I nit with wholly
negative results, except that small amounts of kreatinin and
cholin were found. It was also distilled at from 20° to
3~)° in a vacuum, and the distillate was found to be inert.

The poisonous substance was found to be insoluble in
alcohol, soluble in water, and non-dial v/able. It was pre-
cipitated by saturation with ammonium sulphate.

The substance; was obtained by allowing the germ-free
filtrate, after being rendered feebly acid with acetic acid, to
fall into a large volume of absolute alcohol. It was puri-
fied by repeated solution in water and precipitation with
alcohol. It contains a large amount of sulphur, and re-
sponds to the biuret and Millon tests. It is, therefore,
classified among the albumins. Since it is not precipitated
by saturation with magnesium sulphate at 30°, it cannot
belong to the globulins. The fact that it is precipitated by
saturation with ammonium sulphate, and that it does not
dialyze, shows that it is not peptone. It is, therefore,
classified by BRIEGER and FRANKEL among the albumins,
and is designated as a " toxalbumin."

The special reactions and the results of an ultimate
analysis of this substance have already been given (page 20).

This proteid induces in animals all the symptoms and
post-mortem appearances which have been mentioned as
following the administration of the filtered cultures. It is
to be noted that the injection of small quantities of this
proteid (2£ milligrammes per 1 kilogramme of the body-
weight of the animal) does not produce its effects until after
the lapse of weeks, and possibly months. This peculiarity
in action distinguishes this class of substances from all other
chemical poisons, and it has received as yet no satisfactory
explanation. There is no reason for l>elieving that the body
obtained by BRIEGER and FRANKEL is chemically pure,
and until it has been obtained in this condition we can
only sp:-eulate concerning its true nature.

It should be remarked that the LoFFLER bacillus shows
not only marked morphological variations, but that it is

128 BACTERIAL POISONS.

very variable in its virulence, some cultures having been
obtained which are wholly without effect upon animals.
From cultures of this kind BRIEGER and FRANKEL pre-
pared a non-poisonous albumin differing in its ultimate
composition and in many of its chemical reactions from the
poisonous one.

FRANKEL has been unable to secure immunity in ani-
mals against diphtheria by the employment of small doses
of the " toxalbumin." If the dose is large enough the
animal dies. If it is smaller, the animal seems to become
more susceptible and succumbs more readily to inoculations
with the germ. While this is true of the filtered culture, it
is not the case with that which has been sterilized by heat.
FRANKEL finds that if from 10 to 20 c.c. of a cul-
ture of the bacillus three weeks old, which has been
heated for one hour at from 65° to 70°, be injected under
the skin of the abdomen of guinea-pigs, immunity against
subsequent inoculation with the virulent germ is secured,
provided that the inoculation is not made earlier than the
fourteenth day after the treatment with the sterilized
culture. He thinks that the culture contains two specific
albumins, one of which is poisonous, while the other gives
immunity. The former is destroyed by a temperature of
from 65° to 70°, while the other retains its characteristic
properties. He admits the possibility that the poisonous
albumin may be converted into the other form by the high
temperature. He finds that the modified culture, which
gives immunity, is of no service for therapeutic purposes,
and that if an animal be treated with it directly after inocu-
lation with the germ, death is not retarded, but is hastened.
From these experiments he concludes that the vaccination
albumin at first lessens, and subsequently increases the
resistance of the animal.

SPROUCK and his students have confirmed the alx>ve
statements concerning the toxicity of the germ-free cultures
of this bacillus. They have also called attention to the
albuminuria following the employment of this poison. In
the urine they find casts, white, and sometimes red, blood-
corpuscles. Microscopic examination of the kidney after

SUPPURATION. 129

death shows the same changes which are observed in the
diphtheritic nephritis of children. BAHKS also finds that
the germ-free cultures product- the parenchymatons degener-
atious of the internal organs which arc found in the human
body.

TANGL has shown that the chemical poison is formed in
the INMIV as well as in culture-Masks. A large piece of
pseudo-membrane was macerated in water in an ice-chest
for twenty-four hours, and then filtered through porcelain.
The filtrate, injected into animals, produced all the symp-
toms which have Urn obtained by a similar employment of
artificial cultures. TANUI, also observed that in some cases
in which the animals were inoculated with the sterilized
culture through the mucous membrane a pseudo-membrane
formed at the point of injection.

SUPPURATION. — As early as 1879, LEBER concluded
from his observation on infective keratitis that the asper-
gillus must produce certain soluble products which diffuse
through the cornea and set up an inflammatory action in
the adjacent vascular tissue. In 1882, he showed that sup-
puration could IK- induced by the introduction of sterilized
mercury and copper, and that the pus formed is free from
germs. In 1884, he induced suppuration by the injection
of cultures of the staphylocomis pyogenes anrens which
had Urn sterilised by being l>oiled for hours. In 1888,
the same investigator reported that he had found an alco-
holic extract of the dried Btaphjloooocas to U> highly pyo-
genetic. From this extract he has prepared a crystalline
body which he calls phlogosin. This substance is readily
soluble in alcohol and ether, sparingly soluble in water,
and it crystallizes in needles. The crystals can U- sub-
limed, leaving no residue, and the sublimate, which fi»rms
in rosettes, still possesses the pyogeiu 'tic properties. Alkalies
pnripitate this substance from its solution in amorphous
granules, which dissolve in acids, forming crystalline salts.
LKHKU refers to the observation of the botanist PFEFFER,
who found that vegetable cells are attracted by certain
chemical substances, and adopts the term cheinotactic action

130 BACTERIAL POISONS.

(chemotactische Wirkung) to indicate the property of certain
chemical agents of attracting leucocytes.

As has been stated, BUCHNER has found that the cells of
many bacteria contain pyogenetic proteids. The amount
of these substances in the cells varies with the kind of
germ, and some species (the bacillus prodigiosus, for in-
stance) seem to contain no such bodies. The bacillus pyo-
cyaneus contains a large quantity of the proteid, and is
suitable for lecture demonstration. The germs are taken
from potato cultures and rubbed up with water. Then
they are treated with about fifty volumes of a 0.5 per cent,
solution of caustic potash. This forms in the cold a muci-
laginous mass which dissolves at the temperature of the
water-bath. After being heated for some hours the fluid
is filtered through a number of small filters ; the first por-
tions should be refiltered. The filtrate is a greenish fluid
(pyocyanin) which by the careful addition of acetic or
hydrochloric acid (an excess is to be avoided) forms a
voluminous precipitate (pyocyaneus proteid). This pre-
cipitate should be collected on a filter, washed with water,
then suspended in water and a few drops of a soda solution
added, when a dark-brown fluid, with a tendency to gela-
tinize in the cold, containing about 10 per cent, of the pro-
teid, is obtained.

13.254 grammes of the moist bacteria yield 1.44 gramme
of dry bacterial substance, and this after the treatment
given above furnishes 0.2739 gramme of dry proteid =
19.3 per cent. This proteid leaves 11.52 per cent, of ash,
which contains phosphoric acid, but consists principally of
sodium chloride.

Much smaller amounts of proteid were obtained from
other germs, but the Eberth germ, bacillus subtilis, lactic
acid bacillus, red bacillus from potato, and staphylococcus
pyogenes aureus furnished considerable quantities.

* The chemotactic properties of these proteids were tested
in the following manner : The dissolved proteid was placed
in a spindle-shaped glass tube, and the tubes, sterilized by
prolonged boiling, were introduced under the skin on the

SUPPURATION. 131

backs of rabbits with antiseptic precautions, aud the ends
of the tiil»es broken off subcutaneoiisly.

Alter I'roin two to three days tlie tubes were removed
and tbnnd to contain, in addition to some of the proteid,
several millimetres of tibrinous pus, which was examined
microscopically and by the preparations of cultures, which
invariably remained sterile. The proteid of the EBERTH
bacillus was found to have specially marked pyogenetic
properties.

Similar experiments were made with the following crys-
talline substances : the butyrate and valerianate of ammo-
nia (each 1 per cent, solution), trimethylamin (2 per cent.),
ammonia ('2 per cent.), Icucin, tyrosin and glycocol (1 per
edit.), urea (/> per cent.), and urate of ammonia and skatol
(1 per cent.). Glycocol and leucin only were found to have
the chemotactic action, and with these this action was but
slight compared with that of the bacterial proteids.

The next experiments were made with the object of
ascertaining whether or not proteids similar to those derived
from the bacteria would cause a like effect. The bacterial
cellular proteids resemble very closely vegetable casein
some of which was prepared from wheat gluten and tested
as above. This proteid was found to be. possessed of
marked chemotactic properties. The subcutaneous injec-
tion of sterilized preparations -of wheat-flour and ground
peas were also found to cause suppuration. Negative
results were obtained with starch and solutions of disodium
hydric phosphate. From this it is concluded that the active
agent in the flour is its casein.

Peptone was employed without effect, while gelatin was
found to act energetically. Alkaline albuminates were
prepared from muscle, liver, lungs, and kidney by treating
finely divided portions of these organs with potash and pro-
ceeding as in the preparation of the bacterial protends. All
of these caused the formation of pus, and the preparations
from the liver were found to be specially potent.

Similar preparations from blood and egg-yolk were
active, while those from fibrin and the white of egg had no
effect. Heini-albumose was also found to be active, and

132 BACTERIAL POISONS.

this fact is placed in contrast with the negative result
obtained with peptone.

One of the most interesting results was obtained by the
daily injection of a chemotactic proteid directly into the
blood. Before the first injection the proportion of white to
red corpuscles was 1 : 318 ; on the second day, 1 : 126 ; on
the third, 1 : 102 ; on the morning of the fourth, 1 : 73 ; on
the afternoon of the fourth, 1 : 38. After this there was
no further increase. The absolute number of red corpuscles
remained unchanged, while the absolute numl>er of the
white multiplied sevenfold. The white corpuscles were on
the first days often found in groups of from two to four,
and later, of from ten to twenty. This seems to demon-
strate that these substances cause an increased production
of leucocytes. General leucocytosis was induced by the
similar employment of vegetable casein and an alkaline
albuminate prepared from the muscles of a calf.

Finally, BUCHNER tested the action of this proteid upon
himself. One cubic centimetre of a very dilute solution,
containing 3.5 milligrammes of the solid proteid, was
injected under the skin of the forearm with antiseptic pre-
cautions. Two hours later there was marked pain along
the lymphatics, especially localized in the elbow and axilla.
The temperature showed no marked elevation (only 37.8°).
On the following day there were marked erysipelatous
redness and swelling extending for some inches about the
place of injection, and accompanied by severe pain. The
inflamed area felt hot, and projected distinctly above the
surrounding surface. The lymphatics of the arm appeared
like red cords. On the third day the swelling and redness
were more marked, and extended from the wrist to the
elbow. On the fourth day the symptoms began to recede.
Here we have clinically a perfectly typical erysipelas with
lymphangitis, and BUCHNER claims that all the cardinal
symptoms of inflammation — rubor, calor, dolor — could not
be produced without involvement of the solid tissues.

Similar, but less marked, symptoms were induced by the
injection of a dilute solution of vegetable casein.

BUCHNER states that bacteria will not cause inflamma-

SUMMER DIARRHCEAS OF INFANCY. 133

lion unless they !><• broken down. The pyogenetic substance
contained within the l>:ieteri;il cell (".in have no chemotactic
action until the eell disintegrates. Thus, the anthrax
liaeillns contains a pyogenetie substance, but no pus is
forme*! in mice with anthrax, because there is no destruction
of the bacilli. This pyogenetie proteid of the anthrax
bacillus, however, manifests its action in malignant pustule.
These experiments are of the greatest interest. We must
say, however, that it is possible that the bacterial cellular
proteid may l>e modified by the treatment to which it has
Ixvn subjected in these experiments. We do not as yet
know enough alnnit the nature of this proteid to say that
its nature and its action are not altered by being heated for
hours with an alkali. However, accepting BUCHNER'S
work, it throws much light upon processes which have
heretofore been but imperfectly understood.

THE SUMMER DIARRHCEAS OF INFANCY. — In a paper
published in 1888, VAUGHAN stated that the microorgan-
isms which produce the catarrhal or mucous diarrheas of
infancy are probably only putrefactive or saprophytic in
character, and that they prove harmful by splitting up
complex molecules and forming chemical poisons. At that
time it was generally Ix'lieved that a specific germ would
!>;• found, but the truth of the above statement is being
made more manifest with every experimental study of the
subject. Able and diligent bacteriologists, among whom
BOOKER, in this country, and ESCHERICH, in Germany,
deserve special mention, have made a careful study of the
bacteria found in the intestines and stools in these diseases,
and all agree that no specific organism has l>eeu found.
HOOKER has reports! the isolation of more than thirty
kinds. In true cholera infantum the protons group of bac-
teria was found in fifteen out of nineteen eases, but in the
ordinary duurfaceaa there is no constancy in the species
piv-rnt. (lerms which are frequently found one year are
rardy seen in the cases observed the next summer. This
lias been the cxperien<« of all who have studied the bacteria
of the summer diarrhteas of infancy. VAUGHAN has studied

7

134 BACTERIAL POISONS.

the chemical products of the germs x, a, and A of Booker's
list in the following manner and with the results as stun •<!
below.

Of these germs, BOOKER makes the following statements :

" x was found almost as a pure culture in the feces of a
fatal case of diarrhoea, a was strongly pathogenic when
tested last winter. A was isolated last summer ; liquefies
gelatin, and belongs to the proteus group."

Beef-tea cultures of each of these germs were made and
kept in an incubator at 37° for forty-eight hours. At the ex-
piration of this time these cultures were used for inoculating
flasks of sterilized beef-broth. Eight flasks, each contain-
ing about ten ounces, were employed for each germ. These
cultures were kept in the incubator at 37° for ten days.
They were then twice filtered through heavy Swedish filter-
paper. The second filtrate was allowed to fall into a large
volume of absolute alcohol feebly acidified with acetic acid.
A voluminous, flocculent precipitate resulted in each case.
After the precipitates had subsided the supernatant fluid
was decanted. The precipitates were then treated with dis-
tilled water, in which those from x and a were soluble,
while that from A proved insoluble. A large volume of
absolute alcohol was again added, and the mixture allowed
to stand for four days. The precipitates from x and a com-
pletely subsided, leaving the supernatant fluids perfectly
clear; but in the case of A the subsidence was not com-
plete. The precipitates were collected, by decantation and
filtration, on porous plates, and dried over sulphuric acid.
These substances are proteid in composition, but differ from
known proteids and from one another That from x is
slightly yellow, as seen deposited in the alcohol, but be-
comes grayish on exposure to the air. It is readily soluble
in water, from which it is not precipitated by heat or nitric
acid, singly or combined.

It gives the biuret and xantho-proteid reactions. It is
precipitated by saturating its aqueous solution with ammo-
nium sulphate, and therefore cannot be classed with the
peptones. Sodium sulphate and carbonic acid fail to throw

SUMMKIt hlAKIUKKAS OF INFANCY. 135

it down from its aqueous solution, consequently we must
-ay that it is not u globulin.

This leaves us with no other choice than to place it
among the albumins, but we must admit that it possesses
properties which do not belong to the known albumins.

The proteid prepared from cultures of the germ a is, as
seen under the alcohol, very light, flocculent, and perfectly
white, but so soon as it is brought in contact with the air
it begins to blacken, and finally dries down on the porous
plate in black scales.

It possesses the same general properties in regard to the
action of solvents and other reagents which were found to
be possessed by the proteid obtained from cultures of x.

The proteid of A is peculiar, inasmuch as it is practically
insoluble in water.

These three proteids are highly poisonous. When in-
jected under the skin of kittens or dogs they cause vomit-
ing and purging, and, when employed in sufficient quantity,
collapse and death. Post-mortem examination shows the
small intestine pale throughout and constricted in places.
The heart has been in variably, so far, found in diastole and
filled with blood. The following brief notes from the
record of experiments will illustrate the nature of the
symptoms and the post-mortem appearances.

A small amount of proteid frem bacillus x, dissolved in
water, was injected under the skin on the back of a kitten
about eight weeks old. Within one- half hour the animal
began to vomit and purge, and death resulted within eigh-
teen hours. The small intestines were pale, contracted in
places, and contained a frothy mucus. The stomach was
distended with gas and contained yellowish mucus. The
liver was normal, the spleen and kidneys congested, and
the heart distended.

Another kitten was treated with the proteid from bacil-
lus a, dissolved in water. The vomited and fecal matters
in this case were green. The animal died after fifteen
hours, and presented appearances practically identical with
those mentioned above.

A third kitten was treated with some of the proteid of

136 BACTERIAL POISONS.

bacillus A, suspended in water, aud presented substantially
the same symptoms and post-mortem appearances.

A fourth animal was treated in the same manner as the
above with a proteid prepared from some canned meat.
This was done as a control on the above experiments, and
the kitten remained unaffected. This experiment demon-
strates the fact that the poisonous properties are peculiar to
the bacterial proteids.

Concerning the amount of one of these proteids neces-
sary to produce a fatal result in the animals experimented
upon a few experiments have been made.

Under the skin on the back of a guinea-pig, VAUGHAN
injected ten milligrammes of the dry-scale proteid from
bacillus a. This caused death within twelve hours. Of
two kittens treated with fifteen milligrammes each of the
a albumin, one died after forty eight hours and the other
recovered after two days of purging and vomiting. Two
dogs, of about five pounds' weight, had each forty milli-
grammes, and, after serious illness of two days' duration,
speedily recovered.

During these two days of vomiting aud purging the dogs
were constantly shivering, as with cold, but the rectal tem-
perature stood at from 102.5° to 103.5° F.

There was in no case any sign of inflammation at the
point of injection.

Plate cultures have been made from the proteids them-
selves and from the blood, liver, spleen, and kidneys of
some of the animals killed with the proteid, and these plates
have remained sterile, thus demonstrating that no germ
has been introduced into the animal along with the chem-
ical poison.

What conclusions may we draw from these facts when
considered in connection with the results of the labors of
BOOKER and ESCHERICH ? We will formulate our ideas
in the following propositions :

(1) There are many germs, any one of which, when in-
troduced into the intestines of the infant, under certain
favorable conditions, may produce diarrhoaa.

As has been stated, many different germs have been

SIMMKR 1)1 ARK IKK AS OF INFANCY. 137

found iii the intestines of infants raftering from summer
diarrhoea, and we now find that three species of tiiese are
capable of producing chemical poisons, which induce effects
substantially identical with the symptoms observed in the
infants, and it is not unreasonable to suppose that many
other of these germs produce similar poisons.

(2) Many of these germs are probably truly saprophytic.

A germ growing in the intestine does not necessarily
feet! upon living tissue. The food in the duodenum before
absorption has no more vitality than the same material in
the flask. Moreover, the excretions poured into the intes-
tines from the body are not supposed to be possessed of
vitality. A germ which will grow upon a certain medium
in the flask and produce a poison will grow on the same
medium in the intestine and produce the same poison, pro-
vided it is not destroyed by some secretion of the body.

(8) The only digestive secretion which is known to have
any decided germicidal effect is the gastric juice; therefore,
if the secretion be impaired there is at least the possibility
that the living germ will pass on to the intestine, will there
multiply, and will, if it be capable of so doing, elaborate a
chemical poison which may be absorbed.

There is no longer any doubt that the acid of the gastric
juice has a marked germicidal effect upon many of the
microorganisms.

VAUGHAX has found that an exposure to a two-tenths
percent, solution of hydrochloric acid for half an hour will
destroy Eberth's germ and two poison-producing bacilli
which he has isolated from drinking-water which was
believed to have caused typhoid fever. Although the ger-
micidal effect of this acid has not been tried on the bacteria
under consideration, doubtless it will be found to be con-
siderable.

The chief reason why the breast-fed child has a better
chance for life than the one fed upon cow's milk lies in the
fact that the former gets its food germ-free ; but a second
reason is to be found in the larger amount of acid required
to neutralize the cow's milk, as has been pointed out by

138 BACTERIAL POISONS.

ESCHERICH. The gastric juice is the physiological guard
against infection by way of the intestines.1

It is also possible that some of the secretions poured into
the intestines have germicidal properties, or that the cells,
in absorbing the poisonous proteids, may to a limited ex-
tent so alter them that they are no longer poisonous, or
that in a perfectly normal condition the liver may be able
to prevent these poisons from entering the general circula-
tion without change. These are all possibilities, which
science at some time in the future will investigate.

(4) Any germ which is capable of growing and produc-
ing an absorbable poison in the intestine is a pathogenic
germ.

It is not necessary that a germ be capable of growing
and causing disease and death when injected under the skin
or into the blood in order to establish its right to rank with
the pathogenic germs. In the blood the organism is acted
upon by a wholly different fluid from that with which it
is surrounded in the intestine, and the germicidal properties
of the blood have been unquestionably demonstrated.

(5) The proper classification of germs in regard to their
relation to disease cannot be made from their morphology
alone, but must depend largely upon the products of their
growth.

As has been stated, three microorganisms, differing suf-
ficiently to be recognized as of different species, produce
poisons, all of which induce vomiting and purging, and,
when used in sufficient quantity, death. Morphologically
these bacilli may not be closely related, but physiologically
they are near akin.

If these deductions be true, we will try to avoid the
introduction into the alimentary canal, not only of the so-
called specific pathogenic germs, but of all toxicogenic
microorganisms.

1 It has been said that this statement cannot be true, because there are
other acids which are more powerful germicides than hydrochloric acid,
but there is no force in this argument. The question is not whether the
stomach is supplied with the very best germicide, but whether it is sup-
plied with any at a'l. The human eye may not be a perfect mechanism,
but it is man's only organ of vision.

TYPHOID FEVKR. 189

BAGINSKY and STADTHAGEN have obtained from cul-
tures of the " white liquefying bacterium " of the former a
poisonous proteid which produces in mice, after about five
hours, slight dyspnoea. The coat becomes rough, the ani-
mal sits with drooping head, and when forced to move does
so sluggishly, but without any evidence of paralysis. The
narked apathy increases, and death results after two or
three days. Section shows an infiltration about the place
of injection, congestion of the spleen, liver, and peritoneum.
The intestine is hypera3inic throughout its entire length,
and its upper portion contains a reddish-brown fluid.

From cultures of the same bacterium BAGINSKY and
STADTHAGEN have also obtained a poisonous ptomaine,
which is probably identical with one found by BRIEGER
in putrid horseflesh, and which has the formula C7H,7NO2.

That tyrotoxicon is one of the causes of the violent
choleraic diarrhoea of children there can scarcely be a
doubt. The symptoms induced by the poison cannot be
distinguished from those of the disease. The post-mortem
appearances are very much alike, if not identical, and the
poison has been found in a sample of milk a part of which
had been given to a child not more than two hours before
the first symptoms of a violent attack of the disease made
themselves manifest.

TYPHOID FEVER. — In 1880, EBERTH discovered a
bacillus which he believed to be the cause of typhoid fever,
and this belief has been quite generally accepted. In the
first edition of this work it was stated that the fever and
the characteristic lesions of the disease had been produced
in animals by inoculation with this germ. This is now
known to be erroneous. As has been stated (page J).'i), the
essential lesions of typhoid fever may be produced in ani-
mals with a number of microorganisms, among which,
however, the EBERTH bacillus is not included. The results
obtained by FRANKET, and SIMMONDS, and SEITZ have been
shown by BEUMER and PEIPER to be fallacious, and the
germ with which the experiments were made by VAUGHAN
and NOVY, and mentioned in the first edition, is known

140 BACTERIAL POISONS.

not to be identical with that of EHERTH. It is true that
this germ induced in dogs a continued fever of from
twenty-eight to thirty-five days in duration, terminating
in some instances fatally and revealing ulceration and per-
foration of the small intestines, but for this reason it is
known to be different from EBERTH'S bacillus, because the
latter never induces these effects. Notwithstanding this
failure to affect the lower animals, the majority of bacteri-
ologists believe, as has been stated, that the EHERTH
bacillus is the sole and only cause of typhoid fever. In
this believe VAUGHAN refuses to concur, and claims that
the EBERTH bacillus as found in the spleen after death is
an involution form of any one of a number of germs
which are found in certain waters. As this is not the
place for an extended discussion of purely morphological
questions, the reader is referred to the literature of the
subject, and we will content ourselves with giving the
following summary of what is known concerning the
chemical products of the EBERTH bacillus and of the
germs studied by VAUGHAN.

In 1885, BRIEGER obtained from pure cultures of the
EBERTH bacillus a poisonous ptomaine, which produced in
guinea-pigs a slight flow of saliva, frequency of respira-
tion, dilatation of the pupils, profuse diarrhoea, paralysis,
and death within from twenty-four to forty-eight hours.
Post-mortem examination showed the heart in systole, the
lungs hypersemic, and the intestines contracted and pale.
At first BRIEGER was inclined to regard this as the specific
poison of typhoid fever and named it typhotoxine. How-
ever, he has more recently modified his opinion and is
inclined to regard typhoid fever as due to a mixed in-
fection.

BRIEGER and FRANKEL have found in cultures of the
EBERTH bacillus a proteid which causes death in rabbits
after from eight to ten days. They say nothing about the
symptoms.

In 1889, VAUGHAN isolated from mixed cultures from
typhoid stools a base, forming crystalline salts and capable
of inducing in cats and dogs a marked elevation of tern-

TV I' MO ID KKVKK. I 11

pe rat ii re accompanied l»y seven- purging. The following
is the record of one experiment with this substance:
"An aqueous solution of the crystals was given to a dog
by tin- month at -I I'.M. The rectal temperature before the
administration \V;LS 101° F. At 3.15, retching and vomit-
ing set in and continued at intervals for more than two

I is. At 8.80, the temperature waa 108° F. At 3.55,

the animal l>egan to purge. The first discharges contained
much fecal matter, but subsequently they were watery
and contained mucus plainly stained with blood. At 4,
the temperature was 103.5° F. and remained the same at
4.30. The animal was not seen again until 10 A.M. the
next day, when its temperature was 100.5°, and recovery
-ermed complete."

This base was not obtained in quantity sufficient for an
ultimate analysis. The platino-chloride crystallizes in fine
rhombic prisms and the hydroehloride in long, delicate,
red needles. The red color seems to l>e inherent to the
substance and not due to impurities. The mercury and
platinum compounds are insoluble in alcohol, soluble in
water. The hydroehloride is soluble in both water and
alcohol.

In 1890, VAUGHAN reported the isolation, from water
supposed to cause typhoid fever, of a number of toxi-
cogenic germs. The chemical, products of two of these
have been studied. They Ixilong to the proteids, and an
analysis of one of them by FIM.KI: shows it to belong to the
nucleins. These poisons are soluble in water, the opales-
cent solution showing a distinctly acid reaction. They are
not precipitated by heat or nitric acid singly or combined.
They dissolve in nitric acid, forming a colorless solution,
which becomes yellow on the addition of ammonia. They
dissolve in caustic alkalies and the solution becomes purple
on the addition of a dilute solution of copper sulphate.

On white rats these poisons produce symptoms which are
identical with those which follow inoculations with the
living germs. The rat seems to shiver with cold and gives
evidence of al>dominal pain. It lies with its limbs Hexed
and head drawn down for a few seconds, then stretcho out

7*

142 BACTERIAL POISONS.

the limbs. It lies on the side for a short time, then sits
with the head drawn under the body.

Dogs shiver as with cold, but at the same time the rectal
temperature is from one to four degrees above the normal.
In some instances vomiting and purging have been induced.

The following experiments seem to show that the poison
accumulates in the nerve-centres :

Two guinea-pigs were treated with hypodermic injec-
tions of one of these poisons, the amount used being about
ten times the dose which ordinarily proves fatal to these
animals. Within twelve hours both were dead. Plate
cultures made from the liver, spleen, blood, brain, and
spinal cord remained sterile. Small quantities of the brain
and cord were rubbed up in a sterilized dish with sterilized
water, and two c. c. of the emulsion were injected under
the skin of each of four guinea-pigs. These animals seemed
to be very excitable the next day, throwing themselves
about violently in the cages when slight noises were made
near them. Within a period of from sixteen to twenty-
four days all died. This experiment needs repetition, and
it will be necessary to prepare and inject similar emulsions
made from other organs before any positive conclusions can
be drawn.

In a study of fatal cases of typhoid fever at Bucharest
BABES finds that the typical germ differs markedly from
that of EBERTH.

SWINE-PLAGUE, OR HOG-CHOLERA. — The researches of
LOFFLER, SCHUTZ, LYDTiN, and SCHOTTELIUS in Europe,
and of BILLINGS and SALMON in this country, have demon-
strated the existence among swine of at least three infectious
diseases. These are —

(1) Hog-erysipelas, or rouget of France, or Schweine-
rothlauf of Germany.

(2) German swine-plague, or Schweineseuche.

(3) American swine-plague (BILLINGS), or hog-cholera
(SALMON).

The first two of these are exclusively European diseases,
and their chemical poisons have not been studied.

S\\ I N IK-PL AOT K . 143

Tin- American swine-plague is preeminently a disease of
the digestive tract involving most markedly the large in-
testine. It is the great swine disease of this country, and
is probably present in England, where it is associated with
other diseases under the name of swine-fever. A disease
which was observed in Denmark and Sweden for the first
time in 1888-89 and known as swine-pest or swine-diph-
theria, has been shown by SELANDER, FROSCH, and others
to be identical with our swine-plague. In the summer of
18b9 France was visited by a swine disease, which is con-
sidered by CORNIL and CHANTEMESSE to be identical with
the (irernian swine-plague, but which RIETSCH and JOBERT,
after a comparative study of the microorganisms, pronounce
as the American disease. In this country we have at pres-
ent no positive demonstration of the existence of any other
infectious swine disease. The swine-plague of SALMON
has been the subject of considerable discussion, but its ex-
istence can hardly be said to be established.

The following statements concerning the chemical poisons
refer to the swine-plague of BILLINGS or the hog-cholera
of SALMON, which are only two names for one disease.

In pure cultures of this bacillus Now has found a poi-
sonous base, which probably has the composition C10H26N,,
and to which he has provisionally given the name, suso-
toxine. One hundred milligrammes of the hydrochloride
of this base causes in white rats convulsive tremors and
death within one and one-half hours. Post-mortem exam-
ination shows the heart in diastole, lungs pale, stomach
contracted, a serous effusion in the thoracic cavity, and the
subcutaneous tissue pale and oedematous.

Now has also obtained a poisonous proteid from cul-
tures of this germ. The following experiments illustrate
the effects obtained with this body : 100, 50, and 25 milli-
grammes, respectively, were injected into three young rats
from the same litter. The animal which received 100 mg.
soon began to crawl about on its belly, being unable to rise.
The eyes were soon filled with a thick secretion and the
toes became red. Finally it became quiet, lying on its
belly, with feet extended. The respirations became deeper,

144 BACTERIAL POISONS.

aud a coma-like coudition set iu. The animal died, with-
out convulsions, within about three hours. The rat which
received 60 mg. went through the same course of symptoms,
but these were less intense. Death resulted four hours
after the injection. The one which received 25 mg. be-
came very sick, but finally recovered, aud one week later
it was given another injection of 30 mg., which produced
scarcely any effect. Then it was treated at intervals of
five, three, five, two, and four days, respectively, to 40, 50,
75, 100, and 125 mg. without effect. Three days after the
last injection the animal was inoculated with one c. c. of a
bouillon culture of the highly virulent germ. Only a
slight temporary effect was observed during the first day,
after which recovery was complete and permanent. A
control rat which was given the same quantity of the cul-
ture sickened the next day and died one week later. From
this it will be seen that the animal was rendered immune
against the disease.

SCHWEINITZ also reports the detection of a slightly
poisonous base, which he designates as sucholotoxin, and a
poisonous proteid, and with these he has been able to secure
immunity in guinea-pigs against the virulent germ. The
proteid body is classed among the albumoses, and is said
to crystallize in white, translucent plates when dried in
vacuo over sulphuric acid and to form needle-like crystals
with platinum chloride. No one else has reported a crys-
talline bacterial proteid, and this body is deserving of a
more extended study.

RABBIT SEPTICAEMIA. — HOFFA has killed rabbits by
inoculation with pure cultures of the bacillus of this disease,
and has isolated from the bodies of these animals methyl-
guanidin, while in the bodies of healthy rabbits this poison
could not be found. The fatal dose of methylguauidin for
rabbits was found to be 0.2 gramme when given subcu-
taneously. Since HUEPPE has suggested that the bacte-
rium of chicken-cholera is identical with that of rabbit
septicaemia, chickens were poisoned with methylguanidin,

I1 I K i: I'K i; A I, FKYKK. 145

and the symptoms were observed to be analogous to those

of (lie disease.

PNEUMONIA. — BONARDI has made a chemical study of
the diplococcus of FRAXKKL. He finds certain poisons —
ptomaines — which he has been unable as yet to obtain in
quantity sufficient for ultimate analysis. He also claims
to have secured immunity against the germ by treating
rabbits with small quantities of the chemical poisons.

M ALKJNANT (EDEMA. — KERRY finds that the bacillus
of this disease decomposes albumin with the formation of
fatty acids, leucin, hydro-paracu marie acid, and a foul-
Mnelliug oil of the composition C8H1604. This oil is in-
soluble in water, alkalies, and acids, easily soluble in ether,
benzol, bisulphide of carbon, and alcohol. It is optically
inactive, and on being oxidized furnishes valerianic acid.
Nothing is said concerning its action upon animals.
Among the gaseous products are carbonic acid, hydrogen,
and marsh gas. The author was unable to determine
whether or not free nitrogen is formed.

PUERPERAL FEVER. — BOURGET claims to have isolated
several ptomaines from the urine of women with puerperal
fever. His conclusions are as follows: (1) In puerperal
fever the urine contains highly poisonous bases. (2) The
toxicity of the urine is most marked when the symptoms
of the disease are most grave, and diminishes as the symp-
toms abate. (3) The ptomaines obtained from the urine
prove fatal when injected into frogs and guinea-pigs. (4)
Toxic bases, resembling those obtained from the urine, were
extracted from the viscera of a woman who had died of
puerperal fever.

CHAPTER VI.

THE NATURE OF IMMUNITY-GIVING SUBSTANCES.

OGATA and JASUHARA find that anthrax bacilli grown
in the blood-serum of animals naturally immune to the
disease will not on subsequent inoculation induce the dis-
ease in animals naturally susceptible. Thus, anthrax
germs grown in frog-blood make mice sick, but do not
prove fatal to them, and those grown on the blood-serum
of white rats or dogs have a similar effect upon rabbits;
but germs grown in the blood of animals not immune kill
both mice and rabbits. They also find that the injection
of one drop of frog blood-serum or one-half drop of serum
from a dog into a mouse, any time within seventy-two
hours before to five hours after inoculation with anthrax,
protects this animal from the disease. A guinea-pig weigh-
ing 400 grammes was given twenty drops of frog's blood
diluted with the 0.6 percent, salt solution and immediately
thereafter inoculated with virulent anthrax ; the animal
became slightly sick, but soon recovered. The same was
true of a rabbit weighing 1500 grammes which was treated
with 8 c.c. of defibriuated dog's blood. The experimenters
conclude that one-fourth of a drop of the serum of the dog
diluted to three times its volume with the salt solution is
the smallest amount which will give immunity against
anthrax to a mouse of 10 grammes.

KITASATO and BEHRING have secured immunity in
some animals against tetanus and diphtheria by the follow-
ing methods :

(1) By the method of FRANKEL (for diphtheria), which
has been given. (See page 128.)

(2) By the addition of iodine trichloride to cultures four
weeks old, in the proportion of 1 : 500 ; allow to stand for
sixteen hours ; inject 2 c.c. into the abdominal cavity of a

I M Ml NIT V GIN" INi; Sl'HSTANCKS. 147

guinea-pig; three weeks later inject 0.2 c.c. of a culture'
in bouillon containing iodine trichloride in the proportion
of 1 : 5500.

(3) By the metabolic products of the diphtheria bacillus
in the living Ixxly. In the pleural cavities of guinea-pigs
killed by inoculation with the germ there is often a reddish,
germ-free transudate; 1" c.c. to 15 c.c. of this kills guinea-
pigs; small amounts give immunity.

(4) By inoculating with the virulent germ and arresting
the growth of the same with iodine trichloride, gold-sodium
chloride, naphthylamine, or carbolic acid. ( )f eight guinea-
pigs, each of which was inoculated with 0.3 c.c. of a viru-
lent culture, two, which were not treated, died within
twenty-four hours ; four, which had — two each — a 1 per
• •cut. and a 2 percent, solution of iodine trichloride injected
immediately and at the place of inoculation, recovered ; of
two which had the same treatment six hours after the
inoculation, one died after four days.

(5) By peroxide of hydrogen in diluted sulphuric acid.
Guinea-pigs bear from 1 :4000 to 1 : 2500 ; mice, 1 : 2000
to 1 : 800; rabbits, less than 1 : 1500 of this substance
per body-weight. Injections of this solution before inocu-
lation give more or less immunity, or, rather, increase the
resistance to the disease; given after inoculation it hastens
death.

None of these methods are applicable to the prevention
or treatment of the disease in man.

TI//OXI and CATTANI have reviewed the above state-
ments in so liir as they refer to tetanus. These experi-
menters find that the addition of an equal volume of
either a 2 per cent, solution of fresh chlorine water or
iodine trichloride, or a 5 percent, solution of phenylic acid
to the poisonous, filtered tetanus culture destroys the tox-
icity of the same; but they state that the injection of these
substances into animals either before or after inoculation
with the germ has no effect upon the development or course
of the disease.

However, they do find that the blood-serum of an ani-
mal which is immune will protect against cither the

148 BACTERIAL POISONS.

living germ or the germ-free culture. Pigeons and dogs
are but slightly susceptible to tetanus and they are made
still less so by being treated for a number of times with
small quantities of the virulent culture. After each re-
covery these animals are found to be less susceptible, and
finally they acquire a high degree of immunity, and then
their blood is employed in securing immunity in other
animals much after the manner already detailed for
anthrax.

TI/ZONI and CATTANI have attempted to ascertain the
nature of that constituent of the blood-serum which gives
immunity. In these experiments serum from a dog which
had been rendered immune against tetanus was employed.
In the first place, a filtered culture of the tetanus germ
was concentrated in vacuo at 40° until one-half c.c. of
it would kill a rabbit within thirty-six hours. To this
amount of the culture, the blood-serum was added after
having been subjected to varied treatment, and the whole
was subsequently injected into a rabbit. The blood-serum
retains its antitoxic properties when kept in the dark at
15° for some days, and it may be heated to 60° without
injury. A temperature of 65° weakens, and one of 68°
(the temperature at which the serum coagulates) completely
destroys the antitoxic properties of the serum. The
"tetanus-antitoxine" is non-diffusible. It is precipitated
from the blood-serum on the addition of absolute alcohol
and from the dried percipitate it may be extracted either
with water or glycerin, though very slowly with the
latter. From these facts it is concluded that the antitoxin
is a proteid with the characteristics of an enzyme.

HANKIN gives the following argument in favor of the
theory that immunity is not due to ptomaines, but to pro-
teids : " It is generally admitted that in acquired immunity
against a disease we are dealing (for the most part, at least)
with a phenomenon of the nature of acquired tolerance of
a poison. If we consider what this theory really implies,
and, further, suppose that the poison involved is a pto-
maine or other body of an alkaloidal nature, numerous
difficulties immediately present themselves, For, in the

1MMT MTY-<;i VING SUBSTANCES. 149

first place, if acquired immunity is of this nature, we are
dealing with an acquired tolerance of a poison, which
tolerance is conferred by administering a single dose, or at
most a very limited number of doses. Further, this ac-
quired tolerance, thus easily obtained, is very permanent,
la-ting for mouths, or even years. Now, though acquired
tolerance of alkaloids is constantly observed, it is but
limited in degree, and only obtained as the result of a
long-continued succession of doses.1 Secondly, since ac-
quired tolerance of this hypothetical poison results in the
microbe being no longer capable of living in the body,
this theory implies that the poison in question is one that
is produced by the microbe in order to live there. In
other words, that it is a poison capable of lowering the
bacteria-killing power possessed by every living animal
body.1

" Of course, it is conceivable that a ptomaine might be
concerned in doing this, but, so far as I know, no parallel
to such action can be found among bodies of an alkaloidal
nature.

lt When, however, we turn to what is known of poison-
ous proteids, we at once find that they have properties
analogous to those of the hypothetical immunity-giving
poison.

" First, as regards the question of tolerance : Two poisons
are known, which, in the nature of the tolerance they pro-
duce, resemble the hypothetical poison in question. Both
of them are albumoses. The first is the ordinary hemi-
albumose of proteid digestion. It is known that the injec-
tion of a single minute dose confers immunity against a

1 CARBONE claims to have obtained immunity in rabbits against the action
of the proteus vulgaris by means of not more than two previous injections
of small quantities of neurin obtained from cultures of the proteus. He
still further states that immunity against the same germ is obtained by
muscarin, which produces physiological effects practically identical with
those of this ueuriii.

* With this statement we must take issue. The experiments already
given in which immunity in induced in a susceptible animal hy the injection
ot the serum of the blood of an animal naturally immune show that the
iininiiiiity-giving substance is not necessarily of bacterial origin, and cer-
tainly that it is not necessarily a product of the germ against which the
immunity is secured.

150 BACTERIAL POISONS.

further dose for a period of twelve hours. The second
albumose is the poisouous principle of snake-poison.
SEWALL., in 1887, published a very interesting research on
acquired immunity against snake-poison. He showed that
it was possible, by the injection of a few minute doses, to
give pigeons such a tolerance of this substance that, three
months after the treatment, they were able to stand what
would otherwise be seven times the lethal dose. He sug-
gests in his paper that, by inoculation with the ptomaines
produced by bacteria, it may be possible to protect animals
against their disease- producing powers, although the re-
markable case of tolerance he had discovered suggested that
not ptomaines, but albumoses, were the substances con-
cerned in giving immunity against a disease;1 for I sug-
gest that this fact — that the only cases of tolerance known
which resemble the tolerance implied in disease-immunity
are cases of tolerance against albumoses — strongly suggests
that immunity against a disease is immunity against an
albumose produced by the microbe."

In conformity with the above-stated theory, HANKIN
prepared, as we have already stated, from cultures of the
anthrax bacillus a poisonous albumose, which, when em-
ployed in small doses, gives immunity ; in large doses,
proves fatal. HANKIN endeavored to separate any ferment
that might be present and to which the immunity might
possibly be due. A quantity of lime water was added to
a solution of the albumose and the lime precipitated by the
addition of phosphoric acid. Theoretically, the precipitate
should contain any ferment present, and the immunity-
giving property of the albumose would be diminished by
the amount of the ferment thus removed, in case the im-
munity be due to the ferment. However, the albumose
was found to have lost none of its immunity-producing
power. From this HANKIN concludes that the albumose
is the real immunity-producing agent. He does not iu-

1 We would suggest the fact that in 1887 it was not known that bacteria
produce albumoses, and at that time the term " ptomaine " was employed
to indicate all the bacterial poisons.

I>K V KLnl- M KNT < » F 1 N K Kt'T I () US DISKASKS. 151

form us whether or not any test of the phosphate precipi-
tate was made.

PRODUCTS WHICH FAVOR TIM: I >KVK LOP-
OP INFECTIOUS DISKASKS. — ROGER has made a
very interesting contribution on this subject, and if his
work be confirmed the question of mixed infection will
become more important than it has been supposed to be.
Rabbits are not naturally susceptible to the germ of char-
bon symptomatique ; indeed, inoculation with pure cultures
of the bacillus has no visible effect. But ROGER finds that
if the staphylococcus pyogenes aureus, proteus vulgaris, or
bacillus prodigiosus be injected into the animal at the same
time with the germ of charbon symptomatique the latter
develops and produces the disease. The same result is
obtained when a sterilized culture of the bacillus prodigi-
osus is employed. He at first supposed that the chemical
products of the bacillus prodigiosus so lowered the vitality
of the tissues that the pathogenic germ was enabled to
establish itself; but he found that the same results were
obtained when the two inoculations were made in distant
parts of the body. The most marked effects were seen
when the sterilized culture was injected into a vein and the
charbon bacillus subcutaneously. In these instances the
rabbits rapidly developed enormous tumors, and died
within twenty-four hours. One drop of the sterilized cul-
ture was found to be sufficient, when injected intravenously,
to render rabbits susceptible to the pathogenic germ.

In this connection it may be remarked that from time
to time statements have been made which would lead us to
infer that there are certain poisonous proteids which in
some way yet unknown render the body especially suscep-
tible to the invasion of bacteria. ROSSBACH injected a
poisonous albumose from the juice of the papain tree into
the bloodvessels of animals and obtained a septicaemia.
The blood was found to be filled with non-pathogenic
^erms which came from the intestines. The results of
|I«>-SHACH have, however, been questioned by others.
HANKIN makes the statement that a small dose of snake-

152 BACTERIAL POISONS.

poison, which is too small to kill the auimal outright, after
a certain time may cause death from septicaemia. He says:
" The albumose of the snake-poison has apparently so far
suppressed the germicidal power of the animal that ordinary
decay-producing bacteria can increase and multiply in the
blood. Further, it is often remarked that animals killed
by a snake-bite putrefy rapidly, as if the bacteria-killing
power of the blood-serum had been diminished." A simi-
lar statement has been made concerning the action of the
poisonous albumose of jequirity seeds. Further investiga-
tion must discover how much of truth and how little of
error lie in these claims.

CHAPTER VII.

THE GERMK'IDAL PROTEIDS OF THE BLOOD.

As early as 1872 LEWIS and CUNNINGHAM showed
that bacteria injected into the circulation rapidly disappear.
In the blood of twelve animals, which had been treated
with such injections, bacteria could be found in only seven
utter six hours. In thirty animals, bacteria were found in
the blood of only fourteen after twenty-four hours, and in
seventeen animals, bacteria were found in only two when
the examination was made from two to seven days after
the injection.

In 1874, TRAUBE and GSCHEIDLEN found that the
blood taken from a rabbit into the jugular vein of which
forty -eight hours before 1£ c.c. of a fluid rich in putre-
factive germs was injected, remained without undergoing
decomposition for months. These investigators attributed
the germicidal proj>erties of the blood to its o/onized
oxygen. Similar results were obtained by FODOR and
WYSOKOWICZ. The latter accounted for the disappearance
of the bacteria not by supposing that they were destroyed
by the blood, but that they found lodgement in the capil-
laries.

The first experiments made with extra-vascular blood
were conducted by GROHMANN under the direction of A.
SCHMIDT. It was found that anthrax bacilli, after being
kept in coagulating plasma, were less virulent, as shown by
their effects upon rabbits. GROHMANN supposed that in
some way the bacteria were influenced by the process of
coagulation.

In 1887, FODOR made a second series of experiments in
which he used blood taken from the heart, and showed the
marked gcrraicidal properties of this on anthrax bacilli.

In 1888, NUTTALL used defibrinated blood taken from

154 BACTERIAL POISONS.

various species of animals (rabbits, mice, pigeons, and
sheep) and found that this blood destroyed the bacillus
anthracis, bacillus subtilis, bacillus megaterium, and staphy-
lococcus pyogenes aureus when brought in contact with
them. NISSEN continued this work and employed blood-
serum as well as defibrinated blood. The conclusions
reached were as follows :

(1) The addition of small quantities of sterilized salt-
solution or bouillon to the blood does not destroy its
gerraicidal properties.

(2) Cholera germs and EBERTH'S bacilli are easily de-
stroyed by fresh blood

(3) For a given volume of blood there is a maximum
amount of bacilli which can be added. If too many
germs are used the destruction is incomplete.

(4) Blood whose coagulability has been destroyed by
the injection of peptone is still germicidal.

(5) Filtered blood-plasma from the horse is germicidal.
BEHRING has attributed the action of tbe blood of

white rats on anthrax bacilli to the presence of a hypo-
thetical basic body to which the decidedly alkaline reaction
of the blood is supposed by him to be due. Later, he lays
special stress upon the amount of carbonic acid gas in the
blood-serum.

BUCHNER has made a most exhaustive study of this
subject, in which he has been aided by VOIT, SITTMANN,
and ORTHENBERGER. The results of this work are stated
as follows :

(1) The germicidal action of the blood is not due to
phagocytes, because it is not influenced by freezing and
thawing the blood, by which the leucocytes of the blood
of the rabbit are destroyed.

(2) The germicidal properties of the cell-free serum must
be due to soluble constituents.

(3) Neither neutralization of the serum, nor the addition
of pepsin, nor the removal of carbonic acid, nor treatment
with oxygen have any effect upon the germicidal proper-
ties of the blood.

(4) Dialysis of the serum against water destroys its

GERM1CI DA I, 1'ROTKIDS OF THK HLOOD. 155

activity, while dialysis against 0.75 per cent, salt solution
does not. In the ditl'usate there is no germicidal substance.
The loss by dialysis with water must be due to the with-
drawal of the inorganic salts of the serum.

(5) The same is shown to l>c the case when the serum is
diluted with water and when it is diluted with the salt
solution. In the former the germicidal action is destroyed,
while in the latter it is not.

(6) The inorganic salts have in and of themselves no
germicidal action. They are active only in so far as they
affect the normal properties of thealbuminates of the serum.
The germicidal properties of the serum reside in its proteid
constituents.

(7) The difference in the effects of the active serum and
that which has been heated to 55° is due to the altered
condition of the album! nates. This difference may possibly
be a chemical one (due to changes within the molecule), or it
may be due to alterations in mycelial construction. The
album! nates act on the bacteria only when the former are
in an "active state."

HALLIBURTON has prepared from the lymphatic glands
a globulin which he designates as cell-globulin /3, and which
agrees with fibrin ferment in inducing coagulation in plasma.
HANKIN has tested the germicidal properties of this cell-
globulin. His experiments have been conducted in the
following manner : The lymphatic glands (in later experi-
ments the spleen, also) of a dog or cat are freed as much
as possible from fat and connective tissue, then finely
divided, and extracted with a dilute solution of sodium
sulphate (one part of a saturated sodium sulphate solution
+ nine parts of water). The cell-globulin passes into
solution, while the other proteids are but sparingly soluble.
After twenty-four hours the fluid is filtered and mixed
with an excess of alcohol. The voluminous precipitate
containing the cell-globulin is collected on a filter and
washed with absolute alcohol. For use, a part is dissolved
in water and a small quantity of a bouillon culture of the
anthrax bacillus added. Plate cultures are made along
with control plates from time to time, and in this way the

156 BACTERIAL POISONS.

germicidal property of the substance is demonstrated.
HANKIN closes this contribution with the following con-
clusions :

(1) HALLIBURTON'S cell-globulin /? has marked germi-
cidal properties.

(2) In this respect it differs from fibrin ferment.

(3) The germicidal property of this substance seems to
be identical with that of serum as described by BUCHNER,
NISSEN, and NUTTAI.L.

(4) The active properties of the serum are probably due
to this or to an allied body.

In a more recent contribution HANKIN designates the
germicidal agents of the body as "defensive proteids."
He thinks it probable that blood-serum owes its activity
to these bodies and that the assumption of an "active con-
dition of the serum albuminate" made by BUCHNER is
unnecessary. He also thinks that BEHRING'S supposed
alkaline base exists in the form of an albumose. We know
of three albumoses which are alkaline in reaction. These
are the protomyosinose and deuteromyosinose of KUHNE
and CHITTENDEN, prepared by the digestion of myosin,
and the anthrax albumose of MARTIN.

By a method similar to that which he had employed in
the preceding experiments, HANKIN has isolated a "defen-
sive proteid " from the blood-serum and the spleen of the
rat. This substance belongs to the globulins, and the nat-
ural immunity of the rat against anthrax is probably due
to its existence in the blood.

STERN finds that the blood taken from different men, or
from the same man at different times, varies markedly in
its germicidal properties ; also, that the germicidal proper-
ties of the blood when kept at 42° are at least as great as
at the normal temperature of the body. These statements
are substantially confirmed by ROVIGHI.

CHAPTER VIII.

METHODS OF EXTRACTING PTOMAINES.

FROM what has been given in the preceding pages, one
may gather some idea of the peculiar difficulties with which
the chemist has to contend in his endeavors to isolate the
basic products of putrefaction. He has to deal with very
complex substances, of the nature and reactions of many
of which he must l>e ignorant. Besides, the substances
which he seeks are often most prone to undergo decompo-
sition, and in this way escape detection. Many ptomaines
are volatile or decomposable at any temperature near that
of boiling water. In these cases, solutions cannot be
evaporated in the ordinary way and the poison separated
from the residue. Indeed, the investigator has frequently
been disappointed when on the evaporation of a solution,
which he has demonstrated to be poisonous, he finds that
the residue is wholly inert. Again, he may destroy the
ptomaine by the action of reagents which he uses. So
simple a procedure as the removal of a metallic base from
a solution containing a ptomaine, by precipitation with
hydrogen sulphide gas, has been known to destroy wholly
the ptomaine. Probably the most perplexing difficulty in
the isolation of these putrefactive alkaloids lies in the great
number, complexity, and diversity of the other substances
present in the decomposing mass. The same ptomaine may
be present in equal quantities in two samples of milk, and
yet it may be easily obtained from the one, while from the
other only minute traces can be secured. The difference is
due to the fact that the other constituents of the milk in
the two .simples are at different stages of the putrefactive
process, and, consequently, differ greatly in their reactions
and in their effects upon the agents employed to isolate
the poison. All chemists will appreciate these difficulties.

8

158 BACTERIAL POISON-.

One of the first things for the chemist who undertakes
to do this work is to ascertain whether or not his reagents
are pure. We have found a number of samples of German
ether, which was imported on account of its supposed
purity, to yield on spontaneous evaporation a residue which
gave several of the alkaloidal reactions, and a few drops of
which, injected under the skin of a frog, caused paralysis
and death within a few minutes. We would advise that 500
c.c. of the ether to be used should be allowed to evaporate
spontaneously, and its residue, if there be one, be examined
both chemically and physiologically. The basic substance
which exists in some samples of sulphuric ether is pyridine.

GUARESCHI and Mosso found commercial alcohol almost
invariably to contain small quantities of an alkaloidal sub-
stance, the odor of which is similar to that of nicotine and
pyridine. Its solutions are precipitated by gold chloride,
phosphowolframic acid, phosphomolybdic acid, potassium
iodide, and MAYER'S reagent, but not by platinum chloride
or taunic acid. It does not reduce, or reduces feebly, ferric
salts, From one sample of alcohol they obtained a base
which, in addition to the above reactions, did give a pre-
cipitate with platinum chloride. Alcohol may be freed
from these substances by distillation over tartaric acid.

In amylic alcohol, HAITINGER has found as much as
0.5 per cent, of pyridine. It may be purified in the same
manner as recommended for ethylic alcohol.

Chloroform, when found to leave any residue on evapora-
tion, should be washed first with distilled water, then with
distilled water rendered alkaline with potassium carbonate,
then dried over calcium chloride and distilled.

Petroleum ether sometimes contains a base which has an
odor similar to trimethylamine or pyridine, and which gives
a precipitate with platinum chloride, forming in octahedra.

Benzole may contain a similar substance.

The following methods have been used for the purpose
of extracting the putrefactive alkaloids :

THE STAS OTTO METHOD. — This method depends upon
the following facts : (1) The salts of the alkaloids are sol-

MKTIM'l.s OF EXTRACTING PTOMAINES.

uble in water and alcohol, and generally insoluble in ether,
and (-] the free alkaloids are soluble in ether, and are re-
moved from alkaline fluids by agitation with ether. These
principles are capable of great variety in their application.
The usual directions are as follows: Treat the mass under
exam inatiou with about twice its weight of pure 1)0 per
cent, alcohol, and from ten to thirty grains of tartaric or
oxalic acid ; digest the whole for some time at about 70°,
and filter. Evaporate the filtrate at a temperature not ex-
ceeding 35° either in a strong current of air or in vacuo
over sulphuric acid. Take up the residue with absolute
alcohol, filter, and again evaporate at a low temperature.
Dissolve this residue in water, render alkaline with sodium
bicarbonate, and agitate with ether. After separation re-
move the ether with a pipette, or by means of a separator,
and allow it to evaporate spontaneously. The residue may
1)0 further purified by redissolving in water and again ex-
tracting with ether.

The following modifications of this method are em-
ployed : Instead of tartaric or oxalic acid, acetic acid is
frequently used.

When the fluid suspected of containing a ptoma'ine is
already acid from the development of lactic or other organic
acid, the addition of an acid is often dispensed with.

Kther extracts are made from both acid and alkaline
solutions.

( 'liloroform, amylic alcohol, and benzine are used as sol-
vents alter extraction with ether.

The modification of this method, as carried out by SELMI
and MAKINO-/UCO is given in detail as follows:

The material is divided as minutely as possible, placed
in a large flask, and treated with twice its volume of 90 per
cent, alcohol, and acidulated with tartaric acid in the pro-
portion of 0.5 gramme to 100 c.c. of the mixture, taking
care from time to time that the reaction is permanently
acid. The flask, which is connected with a reflux condenser,
is now placed on the water-bath and kept at the constant
temperature of 70° for twenty-four hours. While yet
warm the liquid is transferred to a special apparatus for

160 BACTERIAL POISONS.

filtration by the aid of atmospheric pressure. The liquid
is poured upon a wet cloth supported upon a perforated
porcelain funnel, which is connected below with a receiver
exhausted by a water-pump or aspirator. In this way
rapid filtration is secured, and by repeated washing the
extraction is made thorough. The acid alcoholic liquid is
now transferred to a special distillation apparatus.

A large tubulated retort of ten litres capacity is con-
nected by means of a cork to a large tubulated receiver.
The tubulure of the retort is provided with a small per-
forated cork, which carries a glass tube finely drawn out
and extending to the bottom of the retort. The tubulure
of the receiver is connected with LIEBIG'S bulbs containing
dilute sulphuric acid (1 to 10), and the bulbs in turn arc
connected with a water-pump or aspirator.

In order to prevent the passage of air through the corks,
they are covered with animal membrane which has been
freed from fat. By means of the aspirator a fine current
of air is drawn through the liquid and suffices to keep it
constantly agitated. The retort is kept on the water-bath
at a temperature of from 28° to 30°. The receiver is kept
cold by a current of water In this manner the distilla-
tion of the alcohol goes on rapidly and conveniently. More-
over, decomposition is so far prevented that volatile bases
are never found in the bulbs.

The aqueous residue, after the removal of the alcohol by
distillation, is filtered and extracted with ether as long as
anything is dissolved. It is then mixed with powdered
glass and evaporated to dryness in vacuo. This residue is
repeatedly extracted with absolute alcohol. The alcohol is
distilled again in the apparatus already described. The
residue is taken up with distilled water and filtered. It is
then made alkaline with sodium bicarbonate and repeatedly
extracted with ether, benzine, and chloroform.

In order to obtain the base from the solvent, the greater
part may be evaporated on the water-bath and the re-
mainder allowed to evaporate spontaneously, or the re-
mainder may be treated with dilute hydrochloric acid and
the evaporation continued on the water-bath or in vacuo.

1!KI K<; Kli' S MKTIHHt. 161

I)I:A<;KNIX>KFF'S METHOD. — The finely divided sub-
-tance i> digested for some hours with water acidulated
with sulphuric acid at from 40° to 50°. This is repeated
t\\o or three times, and the united filtered extracts are
evaporated to a syrup. This is treated with four volumes
of alcohol and digested for twenty-four hours at 30.° After
cooling, the alcoholic extract is filtered, the residue washed
with 70 per cent, alcohol, and the united filtrates freed
from alcohol by distillation. The aqueous residue, diluted
if desirable, is filtered and submitted to the following ex-
tractions :

(1) The acid liquid is shaken with freshly rectified petro-
leum ether as long as this reagent leaves any residue on
evaporation.

(2) The acid fluid is now extracted with benzine.

(3) The next solvent used is chloroform.

(4) The liquid is now again extracted with petroleum
ether in order to remove traces of benzine and chloroform.

(5) The liquid is now made alkaline with ammonia and
successively extracted with petroleum ether, benzine, chloro-
form, and amylic alcohol.

(6) The remainder of the ammoniacal liquid is mixed
with powdered glass, evaporated to dryness, the residue
pulverized, and extracted with chloroform.

The residue obtained with, each of the above solvents
should \w examined for ptomaines.

BRIEGER'S METHOD. — The substance under examination
is divided as finely as possible, and then heated with water
slightly acidified with hydrochloric acid. During the
heating care must be taken that the feebly acid reaction is
maintained. The heating should continue for only a few
minutes. The liquid is then filtered and concentrated, at
first on a plate and then on the water-bath, to a syrup. If
• me has material which is highly odorous, as is the case
frequently both with aqueous and alcoholic extracts of
putrid material, BRIEGER recommends that a piece of
apparatus devised by BocKLiscii be used. The fluid to be
evaporated is placed in a globular flask, the rubber stopper

162

BACTERIAL POISONS.

of which carries two small glass tubes. One of these, B,
extends to the bottom of the flask, while A terminates just
above the surface of the liquid. The tube, A, is connected
with a water-pump or aspirator, which draws the vapor
through the tube. In order to prevent the return of con-
densed fluids, the end of A in the flask is curved upon
itself. The tube, u, is finely drawn out and through it a
current of air is constantly moving. This prevents the
formation of a deposit or a pellicle in the fluid. By regu-

lating the amount of air coming through this tube, more
or less of a vacuum will be formed in the flask. After
evaporation to a syrup, an extraction is made with 96 per
cent, alcohol, and the filtered extract is treated with a warm
alcoholic solution of lead acetate. The lead precipitate is
removed by filtration, the filtrate evaporated to a syrup and
again extracted with 96 per cent, alcohol. The alcohol i.s
driven off ; the residue taken up with water ; traces of
lead removed with hydrogen sulphide; and the filtrate,
acidified with hydrochloric acid, evaporated to a syrup.
This syrup is extracted with alcohol, and the filtrate pre-

MKTMODS <>F ( ; A I T I K K AND KTAIM). 103

cipitated with an alcoholic solution of mercuric chloride.
The mercury precipitate is boiled with water, and on ac-
ctiunt of differences in solubility of the double compounds
with mercury, one ptomaine may l>e separated from others
at this stage of the process. (If thought best, the lead pre-
cipitate may be freed from lead and carried through the
following steps of the process. BRIKGER has found small
amounts of ptomaines in the lead precipitate only in his
work with poisonous mussels.)

The mercury filtrate is freed from mercury, evaporated,
and the excess of hydrochloric acid carefully neutralized
with soda (the reaction is kept feebly acid), then it is again
taken up with alcohol in order to free it from inorganic
salts. The alcohol is evaporated, the residue taken up with
water, the remaining traces of hydrochloric acid neutralized
with soda ; the whole acidified with nitric acid, and
treated with phosphomolybdic acid. The phosphomolyb-
date double compound is separated by filtration, and de-
composed by neutral acetate of lead. This is hastened
by heating on the water-bath. The lead is removed by
hydrogen sulphide, the filtrate is evaporated to a syrup and
taken up with alcohol, from which many ptomaines are
deposited as chlorides, or double salts may be formed in
the alcoholic solution. BRIEGEK states that the chlorides
as deposited from the alcoholic solution are seldom pure,
and he advises for their purification, precipitation with
gold chloride, platinum chloride, or picric acid, and, on
account of differences in solubility of these double salts,
the process of purification is rendered more easy. The
chloride of the base is obtained by removing the metallic
base with hydrogen sulphide; while the picrate is taken
up with water, acidified with hydrochloric acid, and re-
peatedly extracted with ether, in order to remove the
picric acid.

Tin: MKTIIODS OF GAUTIER AND ETARD. — The putrid
matters, liquid and solid, are distilled at a low temperature
in vacuo. The distillate (A) contains a considerable quan-
tity of ammonium carbonate, some phenol, skatol, trimethyl-

164 BACTERIAL POISONS.

amine, and the volatile fatty acids. The residue after dis-
tillation is treated in succession by ether aud by alcohol.

The extraction with ether (B) separates the ptomaines
and some fatty acids. The alcoholic extract (C) removes
the remainder of the fatty acids, as well as the acid and
neutral nitrogenized bodies, almost all of which are crys-
tallizable. The insoluble residue is boiled with dilute
hydrochloric acid, with exclusion of air, finally evaporated
to dryness, and the residue again extracted with alcohol.
This new alcoholic solution (D) can be divided. by acetate
and subacetate of lead into two principal portions.

By operating in this manner the complex products of
putrefaction are readily separated into four portions.

. In his more recent work, GAUTIER has employed the
following method : The putrid liquids, after the removal
of fats, are feebly acidified with very dilute sulphuric acid,
then distilled in vacuo at a low temperature. The distillate
contains ammonia, phenol, iudol, and skatol. The syrupy
residue, separated from any crystals which may have
formed, is rendered alkaline with baryta, filtered, and ex-
tracted a great number of times with chloroform, in order
to dissolve the bases. The solution is distilled at a low
temperature, either in vacuo or in a current of carbonic
acid. The contents of the retort, on being treated with
water and tartaric acid, separate into a brown resin and a
liquid portion. The latter is removed and treated with a
dilute solution of potash, when it gives off the odor of
carbylamine, which was discovered by GAUTIER in 1866,
and which, according to CALMEL, is a constituent of the
venom of toads. The alkali also sets free the bases, which
are removed by extraction with ether, aud the ether evapo-
rated in a current of carbonic acid gas under slight pressure,
then under a bell-jar over caustic potash. The bases may
be separated by fractional precipitation with platinum
chloride, or, if present in sufficient quantity, by distillation
in vacuo.

Still later, GAUTIER has modified his method as follows :
The alkaline putrid liquid is treated with oxalic acid (in-
stead of sulphuric acid) to free acidulation and as long as

K KM A I: KS C !'<i\ Til K M KTIIODS. 1 ''..'•

the tiittv aci<]> continue to separate. The liquid is then
warmed and distilled as long as a turbid fluid passes over.
I'vrrol, skatol, phenol, imlol, volatile fatty acids, and some
of the ammonia pass over. The portion which remains in
the retort is rendered alkaline with lime-water. The pre-
cipitate which forms, and which contains the greater part
of the fixed fatty acids, is removed. The liquid portion,
which is alkaline, is distilled to dryuess, care being taken
to receive the distillate in very dilute sulphuric acid. The
bases and ammonia paas over. The distillate is neutralized
(with sulphuric acid) and evaporated almost to dryness,
then decanted from ammonium sulphate, which crystallizes.
The mother-liquor is extracted with concentrated alcohol,
which dissolves the sulphates of the ptomaines. After
driving oft' the alcohol, the residue is rendered alkaline
with caustic soda, and successively extracted with ether,
petroleum ether, and chloroform.

The lime precipitate is dried and extracted with ether
of thirty-six degrees, which removes any fixed bases that
may 1x3 present.

REMARKS UPON THE METHODS. — The fundamental
difference between the STAS-OTTO and the DRAGENDORFF
methods consists in the fact that in the former the first
extraction is made with a dilute solution of an organic acid
(tartaric usually), while in the second a similar solution of
a mineral acid (sulphuric) is employed. In their various
modified forms any solvent may be used for separating the
alkaloid from the other constituents of the original solu-
tion. Therefore, the question has been asked, Which is
the more suitable acid for use in making the first solution?
The answer to this question will also be the one to the
question, Which is the better method of extracting pto-
ma'ines, the STAS-(>ITO method or that of DRAGENDORFF?
The Italian chemists (JrAKi:s< in and Mosso have at-
tempted to answer this question experimentally, and the
evidence which they have furnished is condemnatory of
the method of DRAGENDORFF. They show that basic
bodies are formed by the action of the dilute sulphuric

8*

16(3 BACTERIAL POISONS.

acid upon albuminous substances. As this poiut is of
vital importance to the investigator in this branch of
chemical science, we will give a brief abstract of the work
of GUARESCHI and Mosso :

One kilogramme of fresh meat was treated with dilute sul-
phuric acid (in the proportion recommended in the DRA-
GENDORFF method) and alcohol. The dark solution after
filtration was made alkaline with ammonium hydrate and
extracted with ether. The ethereal solution gave on evap-
oration an oily substance which had the odor of extracts
obtained from putrid fibrin. This substance, which was
obtained in considerable quantity, was soluble in water and
strongly alkaline in reaction. After neutralization with
hydrochloric acid, its aqueous solutions gave the following
alkaloidal tests :

(1) With platinum chloride, a yellowi.sh-red precipitate,
insoluble in water, alcohol, and ether, and apparently iden-
tical with the compound obtained from putrid fibrin with
the same reagent.

(2) With gold chloride, yellow precipitate, then reduc-
tion to metallic gold.

(3) With phosphomolybdic acid, a heavy, yellow precipi-
tate, forming a blue solution on the addition of ammonium
hydrate.

(4) With phosphotungstic acid, a white precipitate.

(5) With MAYER'S reagent, a heavy, whitish precipitate.

(6) With picric acid, white precipitate, instantly.

(7) With iodine in potassium iodide solution, a heavy
kerraes-red precipitate.

(8") With tanuic acid, white precipitate.

(9) With mercuric chloride, white, amorphous precipi-
tate.

(10) With MARME'S reagent, heavy precipitate.

(11) With potassium ferricyanide, no precipitate, but a
cloudiness, with a formation of Prussian blue on the addi-
tion of ferric chloride.

The same quantity of this meat was also treated by the
SxAS-O'rro method. The alcoholic extract was evaporated
on the water- bath and not in vacuo. The acid was neu-

K KM ARKS ri'i.N THK MKTIIODS. 167

trali/e<l with sodium bicarbonate. The ether extract gave
on evaporation a faintly yellow residue, of not unpleasant
odor and feebly alkaline reaction. After neutralization
with hydrochloric acid, it was only slightly soluble in
water. The pale yellow filtrate gave no precipitate with
Nos. 1, 2, 8, 9, and 10 of the above-mentioned reagents,
but gave a slight turbidity with Nos. 3, 4, 5, 6, and 7, and
with 11 formed Prussian blue.

GUARESCIII and Mosso conclude from this and other
experiments that the DRAGENDORFF method is not suit-
able for the extraction of ptomaines, and they recommend
the employment of the SxAS-Orro method with these con-
ditions : (1) no more acid should be added than is abso-
lutely necessary to keep the reaction acid ; (2) the heat
used in evaporation should not be great, and it is better
that evaporation should be made in vacuo. In this way,
they say, no ptomaine will be obtained from fresh tissue.

The same investigators extracted fresh flesh without the
addition of any acid. Thirty kilogrammes of perfectly fresh
meat were digested for two hours at from 50° to 60° with
about one and one-half volumes of water. The fluids of the
meat contained enough acid to give to the whole of this solu-
tion an acid reaction. It was evaporated to half its volume
on the water-bath, filtered, and evaporated still further.
The small residue was taken up with about four volumes
of 96 per cent, alcohol. The reddish, alcoholic solution
left on evaporation on the water-bath a brownish residue,
which was dissolved in water and extracted with ether (A),
then the solution was made alkaline with ammonium
hydrate and again extracted with ether (B).

A gave on evaporation and cooling crystals of methyl-
hydantoin, while the mother-liquor contained acetic acid.

B also yielded crystals of methyl-hydantoin, while the
mother-liquor gave alkaloidal reactions with most of the
general alkaloidal reagents, none with platinum chloride.
Methyl-hydantoin does not give these reactions.

MARiNO-Zuoo has made many comparative tests with
these two methods. He ascertained that by treating fresh
eggs, brain, liver, spleen, kidney, lungs, heart, and blood

168 BACTERIAL 1'oISOXS.

by either of the methods, he could obtain a substance which
gave alkaloidal reactions, and which he demonstrated to be
choliue. His experiments led him to believe that choline
did not exist pre-formed in these fresh tissues, but that it
resulted from the action of the dilute acids upon lecithin.
It was found most abundantly in those tissues which are
rich in lecithin, such as the yolks of eggs, brain, liver,
and blood; while only traces could be obtained from the
whites of eggs, lungs, and heart. The method of DRAGEN-
DORFP was found to furnish much larger quantities of
choline than could be obtained by the STAS-OTTO method.

COPPOLA agrees with his countrymen, mentioned above,
in condemning the method of DRAGENDORFF.

Enough has been said to show that results obtained by
the STAS-()TTO method are much more reliable than those
secured by the method of DRAGENDORFF. However, the
former is not a perfect method, nor has a perfect one yet
been devised. The principal difficulties met with in the
STAS-OTTO method are as follows :

(1) In most instances the extraction of the base is very
incomplete. (2) The degree to which the putrefactive
alkaloid is removed by the solvent will depend very
largely upon the nature of the other substances present.
This fact in some cases aids and in others hinders the
labors of the investigator. Thus, several ptomaines, which
when pure are wholly insoluble in ether, may be removed,
in part at least, from organic mixtures by this solvent by
passing into the solution along with other substances, but
if the attempt is made to purify one of these bases by re-
peated solution and extraction with ether, the result is a
failure, because the more perfectly the alkaloid is freed
from impurities, the less soluble it is in ether. This criti-
cism, however, is equally applicable to the DRAGENDORFF
method, and to all others in so far as extractions are made.

However, we may state that whenever it is applicable
this method is the best now employed. By it the sub-
stances are submitted to the least chemical manipulation,
and the results obtained are the most reliable. Many of
the more complex putrefactive products are so easily de-

I: KM A UK'S ri>o\ TIIK MKTIfODS lot)

oompoaed or otherwise altered that the investigator should
seek to isolate them by the simplest methods possible. If
it <-:m IK- done without the addition of any acid or without
the application of heat, so much the better.

K-pecially is the modification of this method employed
by MAUINO-/I;<'<>, and already described, to be commended.

By his method, BRIEGEU has discovered a considerable
numlxir of basic bodies and has given great impetus to the
study of tie chemistry of putrefaction. The method is
capable of a great many modifications. As long ago as 1868,
I>KI:<;.M\NX and SOHMIEDEBERG employed precipitation
with metallic salts in order to obtain sepsine from putrid
yeast. The method used by them was as follows: Putrid
yeast was diffused through parchment paper; the diffusate
was acidifi«nl with hydrochloric acid, and treated with mer-
curic chloride solution until a heavy cloudiness and, after
some time, a slight precipitate formed. This was removed
by filtration ; the filtrate was rendered strongly alkaline
with sodium carbonate, and then further treated with a
solution of mercuric chloride as long as a precipitate
formed. This precipitate was collected on a filter, washed,
suspended in a little acidified water, and decomposed with
hydrogen sulphide. The precipitate was removed, the free
hydrochloric acid in the filtrate taken up with silver car-
bonate, and the excess of silver removed with hydrogen
sulphide. The filtrate was evaporated to dryness; the
residue dissolved in alcohol (a part remaining insoluble),
and acidified with sulphuric acid, when a colorless or
slightly yellow crystalline precipitate formed. The crys-
talline sepsine sulphate was purified by solution in water
and precipitation with alcohol.

BRIEFER has obtained some of his bases by a much sim-
plified modification of his complete method, which we have
given in full. For instance, in obtaining neuridine, he
treated the aqueous extract of the putrid material, after
boiling and filtration, with mercuric chloride, collected the
precipitate, decomposed it with hydrogen sulphide, evapor-
ated the filtrate on the water-bath, and extracted the base
from the residue with dilute alcohol.

170 BACTERIAL POISONS.

By this method and its modifications BRIEGER has
obtained many brilliant results, among which maybe men-
tioned his discovery of mytilotoxine, typhotoxine, and
tetauine. However, the method is not free from criticism.
The great number of chemical manipulations to which the
organic matter is subjected is liable to lead to the formation
of some basic substances and to the destruction of others.
One is justified in considering the isolated base as pre-
existing in the original material only when it produces
symptoms identical with those caused by the substance from
which it is extracted. There can be uo doubt that by this
method many ptomaines would be decomposed. With it
EHRENBERG obtained from poisonous sausage only inert
bases, and tyrotoxicon, the ptomaine of poisonous cheese,
is decomposed both by heat and the hydrogen sulphide
employed. The origin of the ptomaines possessing a mus-
carine-like action discovered by BRIEGER has been ques-
tioned by GRAM, who states that when the lactate of choliue,
an inert substance which is widely distributed both in plants
and animals, is heated, it is converted into a poison with
such an action.

CHAPTER IX.

MKTIloliS OK ISOLATING THE BACTERIAL PROTEIDS.

HANKIN employed the following proceas in preparing
his Muthrax proteid :

"The cultures are made iu 0.1 per cent. Liebig's extract
of meat solution, to which some fibrin is added. The Lie-
bi^'s extract is very difficult to sterilize, and must be heated
for two or three ho ill's in the steam sterilizer on two or
three successive days. The fibrin must be added only after
this has been done, and then the flask is re-sterilized by
repeated heating to boiling-point, for a short time only on
each occasion. If the fibrin were added at first it would
be decomposed by the prolonged boiling. By the above
method this only occurs to a slight degree, a mere trace of
peptone being present in the sterilized culture-fluid. After
sterilizing, this is inoculated with the blood of an animal
dead of anthrax, and kept at the ordinary temperature.
The anthrax forms a typical growth on the masses of fibrin,
and samples of the liquid removed on successive days show
a gradual increase in the strength of their biuret reaction.
After about a week the liquid is filtered and the albumose
extracted. The reason for not keeping the flask at a tem-
perature of 37° is that the albumose is gradually decom-
posed into peptone by the anthrax ferment present, and
this change takes place more rapidly at the higher tempera-
ture. For instance, I have found scarcely a trace of albu-
mose in a culture which had been kept at 37° for a week,
and which gave a strong biuret reaction. The albumose
is separated from the culture-liquid thus prepared by satu-
ration with ammonium sulphate. It is letter to acidulate
it slightly by adding a little acetic acid. The bulky pre-
cipitate of albumose which then appears is filtered off, and
the salt separated from it by dialysis. An excess of thymol

172 BACTERIAL POISONS.

must be added at this stage to prevent putrefaction, or the
dialysis can be carried on in a current of water which is
warmed to from 45° to 50° C., at which temperature the
growth of microorganisms is inhibited. After dialyzing
for twenty-four hours or more the greater part of the salt
will have vanished, and the albumose will be found in
solution in a considerable quantity of water which will not
have passed through the parchment. It is now necessary
merely to concentrate the solution and precipitate the
albumose by the addition of alcohol. In my earlier ex-
periments this was accomplished by evaporating in vacua
at a temperature of 45° to 48°. When at length the liquid
has been reduced to a few cubic centimetres it is poured
into alcohol, and the precipitated albumose is filtered off,
washed with the same reagent (alcohol), and dried.

" Evaporating in vacuo is a long and tedious process,
and it requires a somewhat complicated apparatus. When
it is used for pathogenic albumoses there is always a risk
of the temperature employed destroying or diminishing
their physiological properties. Further, if the albumose
is allowed to evaporate to dryness, it may be difficult to
make it pass into solution again. To avoid these difficul-
ties I have designed a method of concentrating such solu-
tions which is less objectionable. It depends on the prin-
ciple that, if alcohol and water are placed on opposite sides
of a membrane, the water rapidly dialyzes through to mix
with the alcohol, while only traces of alcohol pass through
to mix with the water. Consequently, if a watery solu-
tion of albumoses is dialyzed against alcohol, the solution
diminishes in bulk and is rapidly concentrated, owing to
the passage of the water through the membrane.

" My modus operandi is to place the dilute albumose
solution in a parchment sausage skin which is immersed in
a foot glass full of methylated spirit. The spirit can be
changed after some hours if it is desired to prolong the
process ; but this is not usually necessary. In this way I
have been able to bring 400 c.c. of albumose solution down
to 100 c.c. in the course of a single night, at the ordinary
temperature, without risk to the albumose or trouble to

ISOLATING THK H A r T K IU A L PROTEIDS. 173

mvself. The concentrated solution is then poured into
absolute alcohol, which precipitates the albumose and re-
moves aiiv impurities that might be derived from the
methylated spirit. This prolonged treatment with alcohol
will tend to remove any free ptomaines or other substances
soluble in alcohol. Peptones and salts present in the cul-
ture liquid remained for the most part in solution when
the allmmose was precipitated with (NH4)2SO4. No soluble
proteids (except traces of peptone) were present in the cul-
ture medium.

Ordinarily the bacterial proteids are isolated by preci-
pitation with absolute alcohol, re-solution in water and re-
pnvipitation with alcohol. However, as has been stated,
TI/./OM and CATTANI find that strong alcohol destroys the
activity of the poison of their tetanus germ. The method
employed in obtaining the bacterial cellular proteids has
already been given (see page 130).

CHAPTER X.

THE IMPORTANCE OF PTOMAINES TO THE TOXICOLOGIST.

THE presence in the cadaver of substances which give
not only the general alkaloidal reactions but respond to
some of the tests which have hitherto been considered
characteristic of individual vegetable alkaloids, must be
of the greatest importance to toxicologists. The possi-
bility of mistaking putrefactive for vegetable alkaloids
should always be borne in mind by the chemist in making
his medico-legal investigations. On the other hand, as we
have seen in preceding chapters, cases of poisoning by
ptomaines sometimes terminate fatally, and in such in-
stances the chemist should not be satisfied with determin-
ing the absence of mineral and vegetable poisons, but
should strive to detect in the food or in the dead body
positive evidence of the presence of the putrefactive
alkaloid.

We will give a brief account of those cases in which
putrefactive substances have been found to resemble in
their reactions the vegetable alkaloids.

CONIINE-LIKE SUBSTANCES. — The most celebrated case
in which a substance giving reactions similar to those of
coniine has been found, was the Braudes-Krebs trial,
which took place in Braunschweig in 1874. From the
uudecomposed parts of the body two chemists obtained,
in addition to arsenic, an alkaloid which they pronounced
coniine. This substance was referred to OTTO for further
examination. OTTO reported that the substance was
neither coniine nor nicotine, nor any vegetable alkaloid
with which he was acquainted. OTTO converted the sub-
stance into an oxalate, dissolved it in alcohol, evaporated
the alcohol, dissolved the residue in water, rendered this

O'NIIN'K-LIKB SUBSTANCES. 175

solution alkaline with potash, then extracted the base with
petroleum ether. On evaporation of the petroleum ether
the alkaloid appeared as a bright yellow oil, which had a
strong, unpleasant odor, quite different, however, from
that of coniine. It was strongly alkaline and had an
intensely bitter taste. At ordinary temperature it was
volatile. From its aqueous solution it was precipitated
by the chlorides of gold, platinum, and mercury. In
these reactions it resembled nicotine, from which it differed
in the double refractive and crystalline character of its
hydrochloride. With an ethereal solution of iodine this
substance did not give the Roussix test for nicotine, but
instead of the long ruby-red crystals there appeared small,
dark-green, needle-shaped crystals.

This substance was found to be highly poisonous. Seven
centigrammes injected subcutaneously into a large frog pro-
duced instantaneous death, and forty-four milligrammes
given to a pigeon caused a similar result. On account of its
poisonous properties the jury of medical experts decided
that the substance was a vegetable alkaloid. OTTO says
that this decision astounded the chemists.

BROUARDEL and BOUTMY found in the body of a woman,
who had died, after suffering, with ten other persons, from
choleraic symptoms from eating of a stuffed goose, a base
which gave the odor of coniine jaud the same reactions with
gold chloride and iodine in potassium iodide, etc., as
coniine. The same base was found in the remainder of
the goose. But it did not give a red coloration with the
vapor of hydrochloric acid, and it did not form butyric
acid on oxidation, and although it was poisonous, it did
not produce in frogs the symptoms of couiine poisoning.

SELMI repeatedly found coniine-like substances in de-
composing animal tissue. By distilling an alcoholic extract
from a cadaver, acidifying the distillate with hydrochloric
acid, evaporating, treating the residue with barium hydrate
and ether, and allowing the ether to evaporate spontane-
ously, he obtained a residue of volatile bases, the greater
part of which consisted of trimethylamine. After remov-
ing the trimethylamine, the residue had the odor of the

176 BACTERIAL POISONS.

uriue of the mouse. Later, SELMI obtained an unmistak-
able coniine odor from a chloroform extract of the viscera
of a person who had been buried six months, and in an-
other case ten months after burial. Two or three drops of
an aqueous solution of the alkaline residue of the chloro-
form extract allowed to evaporate on a glass plate gave off
such a penetrating odor that SELMI was compelled to with-
draw from close proximity to the substance. The odor
imparted to his hands in testing the substance with the
general alkaloidal reagents remained for half an hour.
This volatile base seemed to be formed by the spontane-
ous decomposition of other ptomaines.

An aqueous solution of a ptomaine obtained by SELMI
by extraction with ether according to the STAS-()TTO
method from the undecom posed parts of a cadaver had
no marked odor, but after having been kept for a long time
in a sealed tube it not only gave off a marked coniine
odor, but the vapor turned red litmus-paper blue. Again,
the sulphate of a ptomaine obtained from putrid egg-
albumin, on standing formed in two layers, one of which
was a golden-yellow liquid, which on being treated with
barium hydrate gave off ammonia, and later, the odor of
coniine. Since butyric and acetic acids were formed by
the oxidation of this base, SELMI concluded that he had
real coniine or methylcouiine, and that it was formed by
the oxidation of certain fixed ptomaines, or by the action
of different amido bases on volatile fatty acids. There-
fore SELMI believed in the spontaneous origin of couiine
or closely allied bases in putrid matter, also in the exist-
ence of a "cadaveric couiine."

The substance which was found by SONNENSCHEIN in a
criminal trial in East Prussia, and which was believed by
that chemist to be the alkaloid of the water hemlock (cicuta
virosa), is thought by OTTO, HUSEMANN, and others, to be
a cadaveric coniine. OTTO says that the symptoms re-
ported in the case were not those of either coniine or
cicuta. SONNENSCHEIN obtained the base six weeks after
the exhuming of the body, which had been buried three
months. The base had the odor of coniine, the taste of

A NICOTINE-LIKE SUBSTANCE. 177

tobacco, travc with potassium bichromate and sulphuric
acid the odor of butyric acid, and behaved with reagents
like coniine.

Hi -KM ANN states that at present it is very difficult, if
not impossible, for the chemist to state with certainty that
he has detected true coniine in the dead body. The symp-
toms and the post-mortem appearances must conform with
those induced by the vegetable alkaloid. The analysis
must be made before decomposition sets in, and the amount
of the base found must be sufficient for physiological ex-
periments to be made with it.

A NICOTINE-LIKE SUBSTANCE. — WOLCKENHAAR ob-
tainod from the decomposed intestines of a woman, who
had been dead six weeks, by extraction with ether from an
alkaline solution, a base which bore a close resemblance to
nicotine. The base was fluid, at first yellow, but on being
exposed to the air, brownish-yellow. It was strongly alka-
line in reaction and gave off' an odor resembling nicotine,
but stronger, not ethereal, but benumbing and similar to
that of fresh poppy-heads. It was soluble in all propor-
tions in water, and the solutions, which did not become
cloudy on the application of heat, did not taste bitter, but
were slightly pungent. The peculiar odor did not disap-
|>ear on saturating the base with oxalic acid. The hydro-
chloride was yellow, like varnish, had a strong odor, and
became moist on exposure to the air. Under the inicro-
scoj>e it showed no crystals, differing in this respect from
nicotine hydrochloride. It differed from nicotine also in
its reactions with potassio-bismuthic iodide, gold chloride,
iodine solution, mercuric chloride, and platinum chloride.
It also failed to give the ROUSSIN test for nicotine. More-
over, it could not be identified with trimethylamine, spar-
teine, mercurialiue, lobeline, or other fluid and volatile
bases.

The studies of KORSOH and FASSBENDER (page 28), of
St-Jiu ANKKT (page 28), of LIEBERMANN (page 30), and
of SELMI (page 31), have already been referred to in a
preceding chapter.

178 BACTERIAL POISONS.

STRYCHNINE-LIKE SUBSTANCES. — In a criminal prose-
cution at Verona, CIOTTA obtained from the exhumed, but
only slightly decomposed body, an alkaloid which gave a
crystalline precipitate with iodine in hydriodic acid, a red
coloration with hydriodic acid, and a color test similar to
that of strychnine with sulphuric acid and potassium
bichromate, and with other oxidizing agents. This sub-
stance was strongly poisonous, but did not produce the
tetanic convulsions which are characteristic of strychnine.
CIOTTA pronounced this substance as probably identical
with strychnine. Portions of the body were subsequently
submitted to SELMI for his opinion. SELMI found that
the substance which gave the color-reaction was not crys-
talline, and that there was only " the presumption of a
bitter taste to it," while one part of strychnine in 40,000
parts of water is intensely bitter. SELMI also held that
many ptomaines give reactions similar to strychnine with
iodine in hydriodic acid and with hydriodic acid. He
also held that its physiological properties were such that it
could not be strychnine. This substance could hardly
have been aspidospermine, which reacts with sulphuric
acid and potassium bichromate similarly to strychnine, be-
cause quebracho bark, in which this alkaloid is found, was
not at that time used as a medicine or known in Italy.

Ptomaines giving reactions similar to those of strych-
nine, and also causing tetanic spasms, have been found in
Italy in decomposed corn-meal. SELMI obtained one of
these substances, but found that it differed from, strychnine
inasmuch as it could not be extracted with ether.

LOMBROSO has named the poisonous substance found in
decomposed corn-meal pellagroceine, but this is really a
mixture of ptomaines, some of which produce narcosis and
paralysis, and others produce the symptoms of nicotine
poisoning instead of the spasms caused by strychnine.

A MORPHINE-LIKE SUBSTANCE. — In the Souzogna
trial, at Cremona, Italy, the experts seem to have con-
founded a ptomaine with morphine. This substance was
not removed from either alkaline or acid solutions with

UIGITALINE-LIKE SUBSTANCES. 179

ether, but could be extracted with amylic alcohol. It
r»-< lures iodic acid, but iu its other reactions, as well as in
its physiological properties, it bore no resemblance to
morphine. In frogs it arrested the heart in systole, which
is said never to happen in poisoning with morphine. It
failed to give both the ferric chloride and the Pellagri
tests for morphine.

In the same body there was found a substance which
was extracted from alkaline solutions with ether, and which
gave, with hydrochloric acid and a few drops of sulphuric
acid, on the application of heat, a reddish residue similar
to that obtained by the same reagents with codeine, but in
its other reactions it did not resemble this alkaloid.

ATROPINE-LIKE SURSTANCES. — Many investigators have
found products of putrefaction which in their mydriatic
properties resemble atropine and hyoseyamine. To this
class belongs the substance observed by /UELZER and SON-
NENSCHEIN. It was removed from alkaline solutions by
ether, and formed microscopic crystals, an aqueous solution
of which, when applied to the conjunctiva, produced a
mydriatic effect, and, when administered internally, in-
creased the action of the heart and arrested the movements
of the intestines. Moreover, with certain alkaloidal re-
agents, such as platinum chlorjde, it resembled atropine.
But when heated with sulphuric acid and oxidizing agents
it did not give the odor of blossoms (Rcuss's test). How-
ever, SELMI found ptomatropines which with sulphuric
acid and oxidizing agents did give the blossom odor as dis-
tinctly as the vegetable atropine. These putrefactive bases
also developed this odor spontaneously after standing for
two or three days, and this does not happen with atropine.
The odor was produced with the ptomatropines by nitric
and sulphuric acids, lx>th in the cold and on the applica-
tion of heat, while these acids in the cold do not produce
the odor with atropiue.

DIGITAI/INE-MKE SUBSTANCES. — Elsewhere we have
referred to the discovery of a ptoma'ine belonging to this

180 BACTERIAL POISONS.

class by RORSCH and FASSBENDER (see page 28). TROT-
TARELLI obtained a similar substance from the brain of a
man in whose abdominal viscera he could find no poison.
The sulphate of this base gave on evaporation an aromatic-
smelling and astringent-ta-ting residue. It became purple
with sulphuric acid alone, and dark red with hydrochloric
and sulphuric acids. On frogs this ptomaine showed no
toxic effect.

A VERATRINE-LIKE SUBSTANCE. — BROUARDEL and
BOUTMY obtained from a corpse which had lain in water
for eighteen months, and a large portion of which had
changed into adipocere, a ptomaine resembling veratrine.
It was removed from alkaline solutions by ether. On
being heated with sulphuric acid it became violet. With
a mixture of sulphuric acid and barium peroxide it be-
came, in the cold, brick-red ; and, on being heated, violet.
With boiling hydrochloric acid it took on a cherry-red
coloration. However, it differed from veratrine, inasmuch
as it reduced ferric salts instantly, and when injected into
frogs subcutaneously it did not induce in them the spas-
modic muscular contractions characteristic of veratrine.

BECHAMP obtained by the STAS-OTTO method from the
products of the pancreatic digestion of fibrin an alkaloid
body which gave with sulphuric acid a beautiful carmine-
red, similar to that given with veratrine. By digesting this
substance with gastric juice, and again extracting, lie
obtained a body which behaved with sulphuric acid similar
to curarine.

A DELPHININE-LIKE SUBSTANCE. — In 1870, General
Gibbone, an Italian of prominence, died suddenly. His
servant was accused of having poisoned him. Two chem-
ists of some reputation reported the presence of delphiuine
in the viscera. It seemed somewhat improbable that the
servant should know anything of so rare a substance, or
that he should have been able to obtain it. However, two
or more varieties of staphisagria grow in Southern Italy,
and it was possible that the servant had used some prepara-

A COLCHICINE-LIKE SUBSTANCE. 181

ti< »u made by himself from the plant. The supposed alka-
loid was given to SELMI, of Bologna, for further study. It
was removed from alkaline solutions by ether. When
heated with phosphoric acid it became red, and when
brought in contact with concentrated sulphuric acid, red-
dish-brown. In these tests the substance resembled dclph-
inine, but with sulphuric acid and bromine water, also
with FKOUDK'S reagent, the colorations characteristic of
the vegetable alkaloid failed to appear. Moreover, SELMI
showed that delphinine gave the following reactions, to
which the suspected substance did not respond : (1) Delph-
inine dissolved in ether, and treated with a freshly prepared
ethereal solution of platinic chloride, gives a white, floccu-
lent precipitate which is insoluble in an equal volume of
absolute alcohol. (2) Delphinine gives precipitates with
auro-stxlium hyposulphite, and with a sulphuric acid solu-
tion of cupro-sodium hyposulphite, the latter precipitate
l>eing soluble in an excess of the reagent.

Finally, CIACTIA and VELL.A showed that while delph-
inine arrests the heart of the frog in diastole, the suspected
substance arrests it in systole.

A COLCHICINE-LIKE SUBSTANCE. — BAUMERT found in

a suspected case of poisoning, twenty-two months after
death, a substance which gave many of the reactions for
colchicine. It was extracted from acid solutions with «ther,
to which it imparted a yellow color. On evaporation of
the ether a yellow, amorphous substance remained, and
this dissolved in warm water with yellow coloration. It
could be extracted from acid solutions also by chloroform,
ben/ol, and amylic alcohol, but not by petroleum ether. It
was removed with much more difficulty from alkaline
solutions.

All the extracts were yellow, and left on evaporation a
feebly alkaline, markedly bitter, sharp-tasting, amorphous,
yellow residue, which dissolved in water and dilute acids
incompletely, forming a resin. When this resin was dis-
solved in dilute sodium hydrate, and the solution rendered

182 BACTERIAL POlSOtfS.

•

acid by sulphuric acid, the same reactions were obtained as
with the original extract.

With phosphomolybdic acid, phosphotuugstic acid, potas-
sio-bismuthic iodide, potassio-mercuric iodide, iodine in
potassium iodide, tannic acid and gold chloride, this sub-
stance gave the same reactions which were obtained by
parallel experiments with genuine colchicine; thus, the
tanuic acid precipitates were both soluble in alcohol, and
the precipitates with phosphomolybdic acid in both cases
became blue on the addition of ammonium hydrate.

Concentrated sulphuric and dilute nitric and hydrochloric
acids dissolved the supposed colchicine with yellow colora-
tion. Strong nitric acid (1.4 sp. gr.) colored the substance
dirty red, scarcely to be called a violet. When the sub-
stance was purified as much as possible, this color became
a beautiful carmine-red. The addition of water changed
the red into yellow, and caustic soda produced a dark, dirty
orange.

In general, in the above-mentioned reactions, the putre-
factive product agreed with the real colchicine, but the
former gave precipitates with picric acid and platinum
chloride, while the latter gives no precipitates with these
reagents.

In 1886, ZEISEL, proposed the following test for colchi-
cine: When a hydrochloric acid solution of the alkaloid is
boiled with ferric chloride, it becomes green, sometimes
dark-green and cloudy. Now, if the fluid be agitated with
chloroform, the chloroform will sink, taking up the coloring
matter, and appearing brownish, granite-red or dark, and
the supernatant fluid clears up without becoming wholly
colorless.

BAUMERT applied this test to both colchicine and the
putrefactive product. To from two to five cubic centi-
metres of the suspected solution in a test-tube, he added
from five to ten drops of strong hydrochloric acid and
from four to six drops of a ten per cent, solution of ferric
chloride, then heated the mixture directly over a small
flame until it was evaporated to half its volume or less. In
the presence of .one milligramme of colchicine the originally

MORPHINE. 183

•

bright-yellow solution became gradually olive-green, and,
<.n furl her concentration, dark-green and cloudy. Then,
on shaking the fluid with chloroform, admitting as much
air as possible, the chloroform subsided, having a ruby-red
color if as much as two milligrammes of colchicine were
present, and a bright yellow if only one milligramme, and the
supernatant fluid became of a beautiful olive-green. When
ether, petroleum ether, benzol, carbon disulphide, or amylic
alcohol was substituted for the chloroform, the coloration
did not appear. From this BAUMERT infers that the red
coloring matter is either only soluble in chloroform, or that
it is not formed until the chloroform is added.

BAUMERT found this test of great value in deciding
whether or not the substance which he found was colchi-
cine. The putrefactive product did not respond to the
test.

Some of this substance was sent to BRIEGER, who de-
cided that it was not a base, but a peptone-like substance.
It was also found to be inert physiologically.

Before these investigations were made by BAUMERT,
LIEKERMANN had found the same or a similar eolchicine-
likc substance in the cadaver. His description differed
from that of BAUMERT only in regard to the taste of the
substance, LIEBERMANN having failed to observe any
marked taste in the substance .which he found, while, as has
lx?cn stated, BAUMERT reported a distinctly bitter taste.

A colchicine-like substance has been found in beer, and
it has been suggested that it was this which the above-
mentioned toxicologists found in the bodies which they
examined, but LIEHERMANN states that the man whose
body he examined had been a total abstainer from beer.

T \MISA coin pared the reactions of ptoma'ines obtained
from putrid sausage with similar reactions of various alka-
loids, and then ascertained the effect Upon the alkaloidal
reactions by mixing the alkaloids with the ptomaines. His
results are as follows :

MORPHINE. — Ptomaines are colored yellow with nitric
acid; reddish-yellow with concentrated sulphuric acid;

184 BACTERIAL POISONS.

•

blue, violet, then green with FROHDE'S reagent ; yellow
when evaporated with concentrated sulphuric acid, then
treated with hydrochloric acid and decomposed with sodium
bicarbonate. The ptomaines reduce ferric chloride, but not
iodic acid. With sugar and concentrated sulphuric acid,
they give a yellow coloration.

Mixtures of the ptomaines and morphine give absolutely
characteristic reactions for morphine with sugar and sul-
phuric acid, the violet coloration appearing distinctly ; and
by evaporation on the water-bath with sulphuric acid, addi-
tion of hydrochloric acid aud decomposition with sodium
bicarbonate, the violet color appearing. Iodic acid is re-
duced by morphine in the presence of ptomaines, only
when the ptomaines are present in minute quantity.

The other reactions for morphine are not applicable in
the presence of ptomaines.

STRYCHNINE. — The characteristic color reaction for this
alkaloid, with potassium bichromate and sulphuric acid, is
not affected by the presence of ptomaines.1

BRUCINE. — The nitric acid reaction for brucine is not
affected by ptomaines. On the other hand, the reaction
with sulphuric and nitric acids, in which a red coloration
is obtained, is scarcely visible in the presence of ptomaines.
The action of mercuric nitrate and heat on brucine, by which
a violet coloration is produced, is not destroyed by the
presence of ptomaines.

VERATRINE. — The characteristic coloration of veratrine
by concentrated sulphuric acid is not influenced by pto-
maines. The same is true of the cherry-red coloration with
concentrated hydrochloric acid. On the contrary, the action
of sugar and sulphuric acid on veratrine is without result
in the presence of ptomaines.

ATROPINE. — The deep violet coloration produced by
fuming nitric acid, subsequent concentration, and the addi-

1 In contradiction to this, see page 178.

DELPHININE. 185

lion of alcoholic potassium hydrate, is not aflectnl by the
presence of ptomaines. On the other haml, the character-
istic (nlor produced liy the action of sulphuric acid and
heat on atropine is scarcely recogni/able wlien ptonw'ines
arc present.

NARCEINE. — The blood-red color produced by concen-
trat<>d sulphuric acid fails in the presence of ptomaines.

COLCHICINE. — Fuming nitric acid colors the ptomaines
reddish-yellow, but the violet coloration of colchicine with
nitric acid appears in well-defined form, even in the pres-
ence of ptomaines. The other reactions for colehicine are
valueless when ptomaines are present.

CODEINE. — The blue coloration of codeine with concen-
trated sulphuric acid holds good when ptomaines arc present.
The same is true of the reaction with sulphuric acid, heat,
and the subsequent addition of nitric acid. FROHDE'S re-
agent fails with codeine when mixed with ptomaines, inas-
much sis the bluish coloration rapidly passes into a brown.

ACONITINE. — Phosphoric acid and concentrated sulphuric
acid are without reaction on the alkaloid when mixed with
ptomaines.

PICROTOXINE. — The reducing action of picrotoxine on
alkaline copper sulphate solution is seriously affected by the
presence of ptomaines. The same is true of other tests for
this poison.

DELPHININE. — The reaction of ddphininc with sulphuric
acid and bromine water, as well as the one with FROHPK'S
reagent, is so much influenced by the presence of ptomaines
that the alkaloid cannot l>e recognized.

These results are to be accepted with caution, a> it is not
reasonable to suppose that, all ptoma'ines will affect the test
for the vegetable alkaloids in the same manner or to the

186 BACTERIAL POISONS.

same degree. Moreover, there is no proof that TAMBA
worked with pure ptomaines.

TAMBA has also proposed to separate vegetable from
putrefactive alkaloids by adding to ethereal solutions of
mixtures an equal volume of a saturated ethereal solution
of oxalic acid, arid allowing to stand, when the oxalates of
the vegetable alkaloids will separate in crystalline form,
and the oxalates of the ptomaines will remain in solution.
In other words, the oxalates of the vegetable alkaloids are
insoluble in ether, while the oxalates of the putrefactive
alkaloids are soluble in ether. But, in contradiction to this,
BOCKLISCH states that the oxalate of cadaverine is insoluble
in ether.

The most important work which the toxieologist is called
upon to do at present is to isolate and identify beyond all
question the bacterial poisons. This work has become im-
portant on account of the frequent occurrence of poisoning
from articles of infected food.

CHAPTER XI.

CHEMISTRY OF THE PTOMAINES.

THE basic substances described in the following pages
arc arranged, so far as possible, in the regular natural order.
An inspection of the list of these bases will show the remark-
able fact of the predominancy of the amine type. Almost
two-thirds of the known ptomaines contain only C, H, and
N, and represent simple ammonia substitution compounds.
Of the oxygenated bases, all of those whose constitution is
known possess the trimethylamine molecule as their basic
constituent, and it is quite probable that most, if not all, of
the remaining ptomaines will l>e found to possess the same
or a siniilar basic nucleus.

It will be seen, furthermore, that a very large number of
the ptomaines descrilxxl possess little or no toxic action,
and are, therefore, physiologically inert. It would seem,
as BKIECEK lias already pointed out, that a certain quantity
of oxygen is necessary to the formation of poisonous bases.
A free supply of oxygen, on the other hand, invariably
yields non-toxic ptomaines. The poisonous bases begin to
appear on about the seventh day of putrefaction, and in
turn disappear if this is allowed to go on for a considerable
period of time.

METIIYLAMINE, CHS.NH2. — This is the simplest organic
base that is formed in the process of putrefaction. It is
ammonia in which one atom of hydrogen has IH-CII replaced
by the methyl radical. It occurs in herring-brine (ToL-
L.ENS, isiiii ; llocKLlsCH, 1885) ; in decomposing herring,
twelve days in spring (BocKLlSCH) ; in pike, six days in
summer ( HOTKLIKCH) ; in haddock, two months at a low
temperature (BocKUSCH) ; in the fermentation of choline
chloride (HASEBROEK). BRIEGER has shown it to be

188 BACTERIAL POISONS.

present in cultures of comma bacillus on beef-broth which
were kept for six weeks at 37°-38°. EHRENBERG re-
ported its possible presence in poisonous sausage, and ob-
tained it by growing a bacillus from this source on intes-
tines (1887). In BRIEGER'S method, methylamine is found
both in the mercuric chloride precipitate and filtrate. The
mercury double salt is readily soluble in water, and can
thus be separated from any accompanying cadaverine or
putrescine. Methylamine is an inflammable gas of strong
ammoniacal odor, and burning with a yellow flame. It is
readily soluble in water, and its solutions give reactions
similar to those of ammonia. Its salts are, as a rule, also
soluble in both water and alcohol.

The HYDROCHLORIDE, CH3 JSTH2.HC1, crystallizes in
large deliquescent plates. On being heated with alkali, it
gives off the odor of methylamine.

The PLATINOCHLORIDE, (CH3.NH2.HCl)2PtCl4 (Pt =
41.31 per cent.),1 yields hexagonal plates which usually
occur heaped up in several layers. It is soluble in about
fifty parts of water at ordinary temperature, and can be
readily recrystallized from hot water. It is insoluble in
absolute alcohol and in ether.

The AUROCHLORIDE, CH3.NH2.HCl.AuCl3 + H2O,
forms prisms, which are readily soluble in water. There
is also a readily soluble picrate.

Methylamine does not possess any toxic action, even when
given in fairly large doses. This physiological indifference
is shared by nearly all the monamines and diamines that
have been obtained among the products of putrefaction.

DIMETHYLAMINE, (CH3)2.NH, has been found in putre-
fying gelatin, ten days at 35° (BRIEGER, 1885) ; in yeast
decomposing in covered vessels for four weeks during sum-
mer (BRIEGER) ; in decomposing perch, six days in summer
(BOCKLISCH); and in herring-brine (BocKLiscn, 1886). It
has been found in poisonous sausage, and in cultures of a

1 The percentages given in the following pages are calculated from Au=
196.64 (Kriiss), Pt= 194.46 (Seubert), 01 = 35.37,0 = 15.96.

CHEMISTRY OF THE PTOMAINES. 189

bacillus obtained from tliis source, on liver and intestines
(KiiKKNHEKQ, 1887). It is also formed, together with
trimethylamine, when ncuridine hydnx-hloride is distille<l
with sodium hydrate (BRIEGER, I., 23). It occurs in the
men-uric chloride precipitate as well as filtrate. From
eadaverine it can !><• separated by platinum chloride, since
cadaverine platinoehloride is difficultly soluble in cold water,
and recrystallizes from hot water, whereas the dimethyl-
amine double salt remains in the mother-liquor. In like
manner it can be separated from neuridine. From choline
it can be isolated by recrystallizing the mercuric chloride
precipitate from hot water.

The free base is a gas at ordinary temperature, but can
be condensed to a liquid which boils at 8° -9°. The
hydrochloride, (CH3)2.NH.HCI, crystallizes in needles,
which deliquesce OD exposure to air and are soluble in abso-
lute alcohol (BRIEGER, I , 56). It is insoluble in absolute
alcohol (BOCKLISCH) but soluble in chloroform (BEHREND),
and can then be separated from methylamine hydrochloride,
which is insoluble in chloroform.

The PLATINOCHLORIDE, [(CH3)2.NH.HCl]2PtCl,, (Pt
= 39.00 per cent.), crystallizes in long needles, which are
easily soluble in hot water, less soluble in cold water. Some-
times it forms orange-yellow plates or prisms, or else small
needles.

The AUROCHLORIDE, (CHfs)a.NH.HCl.AuClj, forms
needles (BOCKLISCH), or large yellow monoclinic plates
(HJORTDAHL), which are insoluble in absolute alcohol.

TRIMETHYLAMINE, C3H9N = (CH3)3N, has been known
for a long time to occur in animal and vegetable tissues.
DESSAIGNES showed its presence in leaves of Chenopodium
(1851), in the blood of calves (1857), and later in human
urine. It has Ixrn obtained from ergot (Secalc cornntum)
by WALZ (18&2) and BBIBQBB(1886); from herring-brine
by WERTHEIM, WINKLES, TOLLENS, and lioc-KuscH.
In these substances, with the exception of herring-brine, it
probably does not exist pre-formed, but is rather a product
of the method employed for its isolation. In fact, BRIEGER

9*

190 BACTERIAL POISONS.

lias shown that it does not exist in ergot, but is formed at
the expanse of the choline present, which, on distillation
with potash, decomposes and yields trimethylamine and
glycol. Thus :

C2H4OH.N(CH3)3.OH = N(CH3)3 + C2H4(OH)2.

It is also formed when betaine and neuridine are distilled
with potash. It may have a similar origin in most of the
other cases, since choline is now known to be widely dis-
seminated in plants and animals, either as such or as a
constituent of the more complex lecithin. Trimethylamine
has been found in the putrefaction of yeast (HESSE, 18-~)7 ;
MiiLLER, 1858); in cheese after six weeks in midsummer
(BRIEGER) ; in human liver and spleen after from two to,
seven days (BRIEGER) ; in perch after six days in mid-
summer (BOCKLISCH); in mussel (Mytilus edulis) after six-
teen days (BRIEGER) • in putrefying brains after from one
to two months, and in fresh brains (GuARESCHi and
Mosso) ; in cultures of the Streptococcus pyogenes on beef-
broth, bouillon, meat extract, and blood-serum, and from
cultures of the comma bacillus (BRIEGER). It has also
been found in cod-liver oil. EHRENBERG (1887) reports
its presence in considerable quantity in poisonous sausage,
and in cultures of a bacillus, isolated from this, grown on
liver, intestines, and meat bouillon.

Trimethylamine is found both in the mercuric chloride
precipitate and filtrate. It remains in the mother-liquor
from which cadaverine, neuridine, and dimethylamine pla-
tinochlorides have crystallized. If an aqueous solution of
mercuric chloride is used as the precipitant, the trimethyl-
amine will be found almost entirely in the filtrate, from
which it can be obtained after removal of the mercury by
evaporating the filtrate to dryness, extracting with alcohol,
and treating the solution thus obtained with alcoholic pla-
tinum chloride.

The free base is a liquid possessing a strong, fish-like
odor. Its boiling-point is 9.3°. It is strongly alkaline in
reaction and freely soluble in water.

The HYDROCHLORIDE, (CH3)3N.HC1, is deliquescent and

CHEMISTRY OF THK 1'TOMAINKS. 191

freely soluble in water and alcohol. Heated to 285° it
< lecoi n j M^SCS. With alkalies it gives off the odor of the

free base.

The PLATINOCHLORIDE, [(CH3)3N.HCl]2PtCl4 (Pt =
•'HI. 92 per cent.), is soluble in hot water, from which, on
cooling, it recrystallizes in orange-red octahedra or needles,
which do not lose water when heated at 100°-110° (BocK-
LISCH).

The AUROCHLORIDE, (CH3)3N.HCl.AnCl3 (Au = 49.39
per cent.), is easily soluble, and hence can Ix? separated from
eholine aurochloride, which is difficultly soluble. Similarly,
this base can be separated from ammonia by the use of gold
chloride.

Trimethvlamiue is not a strong poison, since very large
doses of it must l>e given in order to bring out any physio-
logical disturbances.

ETIIYLAMINE, C2H5.NH2, is formed in putrefying yeast
(HESSE, 1857) ; in wheat flour (SULLIVAN, 1858) ; and also
in the distillation of beet-sugar residues.

It is a strongly ammoniacal liquid boiling at 18.7°, and
is miscible with water in every proportion. Like the other
amines, it is combustible. It possesses strong basic prop-
erties, and is capable of expelling ammonia from its salts in
a manner analogous to the action of the fixed alkalies.

The HYDROCHLORIDE, C2H5.NH2.HC1, forms deliques-
cent plates, which melt at 76°-80°. It is readily soluble
in water and alcohol.

The PLATIXOCHLORIDE, (C2H6.NH2.HCl)2PtCl,, forms
orange-yellow rhoml>ohedra (WELT/IEN), or hexagonal-
rhombohedral crystals (TopSOE).

The AUROCHLORIDE, C2H8.NH2.HCl.AuCl3, forms gold-
yellow monoclinic prisms, readily soluble in water.

With picric acid it forms short brown prisms, not very
soluble in water.

DIETHYLAMINE, C4HnN = (C2Ha)2NH, has l)een ob-
tained by BOCKLISCH from pike which were allowed to
putrefy for six days in summer ; and by growing a bacillus

192 BACTERIAL POISONS.

obtained from poisonous sausage on intestines and on meat
bouillon (EHRENBERG, 1887).

It is an inflammable liquid which boils at 57.5°, possesses
strong basic properties, and is soluble in water.

The HYDROCHLORIDE, (C2H5)2NH.HC1, crystallizes in
needles (BOCKLISCH) ; in long needles and prisms from
absolute alcohol ; in plates from ether-alcohol. These are
not deliquescent and are easily soluble in water and in
chloroform ; rather difficultly in absolute alcohol. Heated
with sodium hydrate it gives off alkaline vapors. From an
alcoholic solution it is precipitated by addition of alcoholic
mercuric chloride. The mercury double salt is difficultly
soluble in hot water, from which it recrystallizes on cooling.

The PLATINOCHLORIDE, [(C2H5)2.NH.HCl]2Pt014, crys-
tallizes in orange-yellow monoclinic crystals, which are
easily soluble in water.

The AUROCHLORIDE, (C2H5)2.NH.HCl.AuCl3 (Au =

47.71 per cent.), forms trimetric crystals (ToPSOE), which

are difficultly soluble (BOCKLISCH). It melts at about 165°.

With picric acid it forms an easily soluble picrate (LEA).

TRIETHYLAMINE, C6H16N = (C2H6)3N, was obtained
by BRIEGER (1885) from haddock which were exposed for
five days in an open vessel during summer. He obtained
it by distilling with potash, after removal of platinum by
hydrogen sulphide, the mother liquor from which neuridine,
the base C2H8N2, muscarine, and gadinine had successively
crystallized (see Gadinine). It has also been found by
BOCKLISCH (1886) in putrid pike, and by EHRENBERG
(1887). The latter obtained it from cultures of a bacillus,
found in poisonous sausage, and grown on meat bouillon.

The free base is oily in character and possesses an am-
moniacal odor. It is but slightly soluble in water, and
boils at 89°-89.5°.

The PLATINOCHLORIDE, [(C2Ha)sKHClJaPtCl4 (Pt =
31.84 per cent.), crystallizes in needles which are readily
soluble in water.

With mercuric chloride the aqueous solution gives no
precipitate.

CHEMISTRY OF THE PTOMAINES. 193

With picric acid it yields yellow needles which are but
slightly soluble in cold water.

PROPYLAMINE, CSH7.NH,, is isomeric with trimethyl-
ainine, and can therefore be easily confounded with that
base. There are two propylamines possible represented
by the formula CHS.CH2.CH2.NH2 and (CH3)2.CH.NH2.
The former, or the normal compound, boils at 47°-48°,
whilst the latter, or iso-propylamine, l>oils at 31.5°. Both
are liquids possessing an ammoniacal, fish-like odor. They
form crystalline salts ; the hydroehlorides melt respectively
at 1550-lo8°, and at 139.5°.

Iso-propylamine (?) has been found among the distilla-
tion products of the vinasse of beet-root molasses. Propyl-
amine has been obtained by BRIEGER (1887) from cultures
of the bacteria of human feces on gelatin. SCHWANERT
has isolated from the organs of a cadaver a basic substance
which was said to possess an odor similar to propylamine.

BUTYLAMINE, C4HUN, was obtained by GAUTIER and
MOURGUES (1888) in cod-liver oil. It forms a colorless,
mobile, alkaline liquid, the boiling-point of which they
found to be 86° at 760 mm. It absorbs carbonic acid from
the air and readily forms salts. The platinochloride forms
golden-yellow plates which are quite soluble.

In animals it produces an -increase in the function of the
skin and kidneys, and in large doses fatigue, stupor, and
vomiting.

ISO-AMYLAMINE, CSH1SN = (CH3)2.CH.CH2.CH2.NH2,

has been obtained by LIMPRICHT in the distillation of horn
with potash ; it also occurs in the putrefaction of yeast
(MiiLLER, HESSE, 1857) ; and in cod-liver oil (GAUTIER
and MOURGUES, 1888), where it constitutes nearly one-
third of the bases present.

It is a colorless, strongly alkaline liquid, possessing an
odor which is not disagreeable. At the ordinary pressure
it boils at 97°-98°.

The hydrochloride forms deliquescent crystals, which

194 BACTERIAL POISONS.

have a bitter, disagreeable taste. The platinoehloride crys-
tallizes in golden-yellow slender plates, which arc very
soluble in boiling water. The base is, according to GAU-
TIER and MOURGUES, identical with that obtained by treat-
ing iso-amylcarbimide with potash.

It is a very active poison, producing rigor, convulsions,
and death. Four milligrammes produces death in a green-
finch in three minutes.

CAPROYLAMINE (HEXYLAMINE), C6H15N, has been
found to occur by HESSE (1857) in the putrefaction of
yeast. HAGER isolated from some putrid material what he
thought to be a mixture of amylamine and caproylamine,
and named it septicine.

Hexylamine was found, in small quantity, in cod-liver
oil by GAUTIER and MOURGUES, and according to these
authors it resembles amylamine in its action, but is less
toxic.

TETANOTOXINE, CjI^N, (?) was obtained by BRIEGER
(1886) as one of the products of the growth of the tetanus
microbe on beef-broth or on brain-broth. It has also been
obtained by KITASATO and WEYL (1890) from pure cultures
of the tetanus bacillus, kept eight days at 36°. For its
isolation see Tetanine, and Ber. 19, 3120. It is tetanizing
in its action, produces first tremor, then paralysis and vio-
lent convulsions. It forms an easily soluble gold double
salt which melts at 130°. The platinoehloride is difficultly
soluble, and decomposes at 240°. The hydrochloride is
crystalline, and is readily soluble in alcohol and in water.
It melts at about 205°. From warm alcohol it crystallizes
in flat, pointed plates.

SPASMOTOXINE, a base of as yet unknown composition,
produces in animals violent clonic and tonic convulsions.
It was obtained by BRIEGER (1887) from cultures of the
tetanus germ on beef-broth.

CHEMISTRY OF THE PTOMAINES. 195

Another lo.riiic was obtained by HuiKOKU (1887) in cult-
ures of the tetanus microlx- which produced complete tetanus,
salivation, and tear-secretion. In its composition it is prob-
al»lv a diainine. The platinoehloride forms plates which
begin to decompose at 240°. The hydrochloride is very
deliquescent. (Jold chloride and picric acid form very
soluble compounds. Besides these three bases lie isolated
another toxie substance, tetanine, and a base (see under
Tetaniiie).

DIHYDROLUTIDINE, C7HUN, was found in cod-liver oil
by GAUTIER and MOURGUES (1888). It is the first known
hydrolutidine. It is a colorless, somewhat oily, very alka-
line and caustic liquid, the odor of which is sharp, but
somewhat agreeable when dilute. It absorbs carbonic acid
from the air, darkens and thickens; is feebly soluble in
water, and boils at 199° at 700 ram. pressure. The stilts
are hitter to the taste.

The. hydroc-hloride crystallizes in a confused mass of
needles or in plates. The nitrate reduces silver nitrate — a
property of all hydropyridine bases (HOFMANN). The
sulphate forms fine stellate deliquescent needles.

The platinoehloride is readily precipitated from concen-
trated solutions as a canary-yellow precipitate. From
warm solutions it crystallizes in lozenge-shaped plates which
are often imbricated. On boHing with water it loses hydro-
chloric acid and forms (C7HuNCl)2PtCl2, which possesses a
lighter color, is more soluble than the normal salt, and crys-
tallizes confusedly.

The aurochloride crystallizes in needles which form fan
or lozenge-shaped masses. It is scarcely altered even in hot
water.

The IODOMETHYLATE, C7HUN.CHSI, is obtained by mix-
ing, in the cold, the base and methyl iodide. The colorless
compound thus obtained is soluble in water and in alcohol,
and possesses a disagreeable, somewhat nauseating odor.
Treated with potash it yields a colorless, aromatic, very
alkaline oil.

The base on oxidation with boiling potassium perman-

196 BACTERIAL POISONS.

ganate yields an acid, C7H7NO2, and from this fact the
discoverers conclude that the base is a dihydro-dimethyl-
pyridine, C5H4(CH3)2NH.

Physiological Action. — It is moderately poisonous. In
small doses it diminishes the general sensibility ; in larger
doses it produces trembling, especially of the head ; pro-
found depression alternating with periods of extreme ex-
citement ; paralysis of the posterior limbs, and death.

A BASE, C8HnN, isomeric, but not identical, with alde-
hyde-collidine, was obtained by NENCKI as early as 1876,
by allowing a mixture of 200 grammes of pancreas and 600
grammes of gelatin in ten litres of water to putrefy for five
days at 40°. The method used by NENCKI for its isola-
tion is as follows : The fluid mass was distilled with sul-
phuric acid, to drive off' the volatile acids, then rendered
alkaline with barium hydrate, and again distilled. The
distillate was received in dilute hydrochloric acid, and on
evaporation gave a crystalline residue of ammonium chlo-
ride, and of a salt which formed in long rhombic plates.
The latter were separated from the ammonium salt by abso-
lute alcohol. The free base was obtained from the salt by
treating it with sodium hydrate, and extracting the solution
with ether.

This compound, as already stated, is isomeric with colli-
dine, and also with O. DE CONINCK'S base, with which it is
possibly identical. The latter, however, will be described
separately.

The free base is oily in character, and possesses a peculiar,
not unpleasant odor. It readily absorbs carbonic acid gas
from the air, forming after a time a lamellar, crystalline
mass of the carbonate. The salt of this base on heating
gives off an oil which burns with a smoky flame, and pos-
sesses an odor similar to that of xylol or cumol. NENCKI
was therefore at first of the opinion that the ptomaine was
an aromatic base, probably an isophenyl-ethvlamine of the

/C* FT
following composition: C6H5 — CH\^Tj3- He supposed

CHEMISTRY OF THE PTOMAINES. 197

that it was formed from the putrefaction of tyrosin, accord-
ing to the following equation :

C9HllN03=C8HllN + COa + 0.

We know that tyrosin does split up, on being heated to
270°, into carlxmie acid and oxyphenyl-ethylamine, thus :

C.H<82rCH.NirrCOOH =C.H4/ggs CHj NHj + OQr

In 1883 ERLENMEYKR and LIPP observed that phenyl-
>z-amido-propionie acid (phenyl-alanine), on dry distillation,
decomposed with the formation, among other products, of a
base having the composition C8HUN. This base was found
to l>e identical with phenvl-ethylamine, C6H5.CHrCH2.NH2,
and in its properties and composition it resembles NENCKI'S
base. Recently (1889), NENCKI has taken up a similar
view in regard to the nature of this base, and now regards
it as possessing the formula just given — that it is phenyl-
ethylamine. He regards phenyl-amido-propionic acid — one
of the three aromatic nuclei contained in the albumin mole-
cule— as the source of this base. From the fact that phenyl-
a-amido-propionic ax-id is a well-known putrefactive product,
it would seem that NENCKI'S base may arise either from the
putrefactive decomposition of that acid, or from the splitting
up of the acid as a consequence of the method employed in
isolating the base. The latter-would seem to lx> the most
probable explanation of the genesis of this base, inasmuch
as BRIE<;KK, by using his method for the isolation of pto-
ma'iues, has not Urn able to obtain it from putrid gelatin.

The PLATINOCHLORIDE, (C8H}lN.HCl)2PtCl4 (Pt =
29.89 IMT <vnt.), is readily soluble in hot, and but slightly
soluble in cold water, and can l>e, therefore, reerystalli/.ed
from water. It forms l>cautiful flat needles. On dry
heating it gives off' an oil which possesses an odor
resembling very much that of xylol or cumol, and burns
with a smoky flame. This distinguishes NENCKI'S base
from collidine, since the platinoehloride of the latter does
not show this U-havior.

NENCKI also obtained from putrid gelatin, under certain

198 BACTERIAL POISONS.

ill-defi&ed conditions, especially when no glycocoll was
present, a basic product which gave, with sulphuric acid,
large lamellar crystals. The free base forms a thick color-
less syrup, possessing a nauseous, bitter taste. It did not
become crystalline even after standing some time. Unlike
the base C8HUN, it is not volatile, and is, therefore, obtained
on evaporation of the acidulated solution after previous
removal of the volatile bases by distillation with baryta.

A BASE, C8HnN, isomer of collidine and of the preceding
base, with which it is possibly identical, was obtained by O.
DE CONINCK (1888) in the later stages of putrefaction of
sea-polyps (poulpes marine). It forms a yellowish, rather
mobile liquid, possessing a strong benumbing (vireuse) odor,
and is but slightly soluble in water. It is soluble in methyl
and ethyl alcohol, ether and acetone. Its density is 0.9865.
When dried over potash it boils at 202° without undergoing
decomposition. On exposure to the air it becomes brown,
hydrates rapidly, and the boiling-point is then lowered. It
has not been noticed to absorb carbonic acid from the air.
It resembles some of the bases obtained from Dippel's oil.
The salts are in general less stable than those of the pyri-
dine bases, and in this respect it approaches the dihydro-
pyridine bases.

The HYDROCHLORIDE, C8HUN.HC1, forms white or
slightly yellowish radiate masses which are deliquescent
and very soluble in water. The hydrobromide, C8HnN.
HBr, resembles it, but is less deliquescent and a trifle less
soluble in cold water.

The PLATiNOCHLORiDE,(C8HuKHCl)2PtCl4, is a dark
orange-colored powder, which is insoluble, or almost so, in
cold water, and is a rather stable compound. Boiling
water and water at 80° decompose it into hydrochloric
acid and (C8H,,NCl)2PtCl2, which is a light-brown powder,
insoluble in cold, scarcely so in hot water.

The AUROCHLORIDE, C8HnN.HCl.AuClv forms a light
yellow precipitate. It is quite stable in cold, but very un-
stable in hot or even warm water. It cannot be modified
by withdrawal of hydrochloric acid.

CHEMISTRY OF THE PTOMAINES. 199

It forms t\v<> compounds with men-uric chloride. (C18HU
N.I K 'U.IIgClj crystalli/es in small white needles, which
arc -lightly soluble in water and in dilute alcohol, insoluble
in absolute alcohol, and on exj>osure to moist air undergo
change. The second compound, 2((18HnN.II('l)..'SHg(1l2, is
obtained by adding an excess of' concentrated mercuric
chloride to a concentrated solution of the hydrochloride.
It forms slightly yellow, somewhat longer needles which
are insoluble in the principal solvents, and are likewise
changed by atmospheric humidity.

The IODOMETHYLATE, C8HnN.CH3I, is formed by
mixing solutions of the base and methyl iodide in absolute
ether. It is deposited a.s a network of fine white needles,
which are but slowly altered in the air, and are soluble in
absolute alcohol. This solution on the addition of a little
potash assumes a dark-red color, which is heightened by
the addition of a little hydrochloric or acetic acid, and
d.-t roved by ammonia without any resultant fluorescence.
Warmed with excess of moist solid potash it becomes
garnet-red in color and gives off an odor resembling that
of the d i hy d ropy r id i nes. It thus behaves the same as the
pyridine iodomcthylates.

On oxidation with potassium permanganate it yields an
acid which melts at 2'29°-230°, and Ix'gins to sublime at
I-)'*0. It presents all the characteristics of nicotinic acid,
C6H5NO2, which is formed a6 the result of oxidation of
nicotine. With hydrochloric acid it forms the compound,
CjH5NO2.HCl. With copper acetate it forms a salt ; this,
distilled with lime, yields a substance which on boiling
with platinum chloride and water forms the compound
(CgHgNd^Ptdg. This same substance forms an iodo-
methylate, which in alcoholic solution gives, on addition of
potash, the characteristic reaction of pyridiue bases.

The base, C8HUN, therefore yields pyridine and nico-
tinic acid.

A BASE, CJI^N, was obtained by GAUTIER and ETARD

(1881) from the ehloroformic extracts (see method, pn^e 164)
from putrefying mackerel, as well as from the decomposing

200 BACTERIAL POISONS.

flesh of the horse and ox. It is regarded by these authors
as a constant and definite product of the bacterial fermenta-
tion of albuminoid substances, but this view is hardly justi-
fiable, inasmuch as the base has not been found by other
investigators. It is accompanied by the base C17HMN4
(page 228). NENCKI (1882) asserted the identity of this
base with the one which he had isolated in 1876, and to
which he had ascribed the formula 08HUN. On the other
hand GAUTIER and ETARD consider their base to be iden-
tical with the hydrocollidine obtained by CA HOURS and
ETARD by the action of selenium on nicotine.

The free base is an alkaline, almost colorless, oily liquid,
possessing a penetrating odor resembling that of syringa.
It is volatile without decomposition, and boils at about 20")°,
while hydrocollidine boils at 210°. Its density at zero is
1.0296. When exposed to the air it oxidizes slowly, be-
comes brown and viscous, and at the same time absorl^s
carbonic acid. It differs from a collidine in possessing a
strong reducing action, since both the gold and platinum
double salts become reduced on heating, and even in the
cold.

The HYDROCHLORIDE, C8H13N.HC1, is very soluble in
water and in alcohol, and usually forms fine needles re-
sembling snow crystals. It is neutral in reaction and pos-
sesses a bitter taste. In the presence of an excess of acid
it reddens and resinifies.

The PLATINOCHLORIDE, (08HI3N.HCl)2PtCl4 (Pt =
29.7 per cent.), is of a light yellow, flesh-color, crystalline,
and but slightly soluble. It dissolves on warming, and re-
crystallizes in bent needles.

The AUROCHLORIDE is rather soluble, and becomes
slowly reduced in the cold ; rapidly on warming.

Physiological Action. — This isomer of hydrocollidine
is strongly poisonous. Even so small a dose as 0.0017
gramme of the hydrochloride produced, when injected under
the skin of a bird, marked unsteadiness of gait, followed by
paralysis of the extremities, and finally death. The pupils
are normal and the heart stops in diastole. Larger doses
(0.007 gramme) cause at first vomiting and staggering,

CHEMISTRY OF THE PTOMAINES. 201

which S«M»U give way to a condition of exaltation. Toward
the end tetanic convulsions set in, followed by almost com-
plete paralysis.

A II \-r., C,(II13\, isoiiieric with parvolinc, has been ex-
traeted by (J.U'TiKR and ETARD (1K81) from decomposing
mackerel and horseflesh. The met IKK! employed by these
chemists for its isolation is given on page 164. The iden-
tity of this base with the synthetic parvoline, obtained by
\\ A.\(.I: by heating ammonia with propionic aldehyde in a
>ealed tiilx- at 'J(M)°, cannot IKI considered to l>e definitely
settle* I, although an apparent identity exists in regard to
their U>iling-|>oints. Tims, the synthetic parvoline boils at
lii.'^-lJKi0, while GAUTIKR and ETARD assign to their
Iwse a l>oiling-]>oint a little U'low 200°. Further investi-
gation is ncKvssary to decide upon the question of the iden-
tity of this base with parvoline, or of the ptomaine C8H13N
with hydrocollidinc.

The free base is an oily, amlM'iM-olored liquid, possessing
the odor of hawthorn blossoms. It is slightly soluble in
water ; very soluble in alcohol, in ether, and in chloroform.
Its boiling-point^ as stated above, is a trifle below 200°.
Like the bases ( 18H1SN and (1IOH,5N it l>ecomes brown and
soon resinifies on e.\]>osure to air.

The PLATINOCHLOUIDE, (O9H1SN.II< 1l)2PtCl4 (Pt =
2.S.»;.") per cent.), is slightly soluble, crystalline, and flesh
colored ; exposed to the air it soon In •comes pink.

The AUUOCHLORIDE is quit*' soluble.

A BASE, C10H15N, was isolated by GuARESCHl and
Mo.sso(188.'i) from ox-blood fibrin which had l>een allowed
to putrefy for five months. In. 1887 it was re-obtained from
putrid fibrin by (iiJARE&ciil, who this time ascribed to it the
formula CIOI I,3N. In 1886 OECHSNER DE CONINCK found
it among the basic products formed in the putrefaction of
t he jel ly-fish (jionlpe* marine, HUGOUNENQ, page 21). The
method used for its extraction was that of (JAUTIER and
KTARD (se<' page 164). It forms a brownish oil of strong
alkaline reaction, which soon resinifies. It possesses an

202 BACTERIAL POISONS.

unpleasant, weak pyridinc or coniine odor, and is but
slightly soluble in water; soluble in ether and in chlo-
roform.

In regard to the constitution of this ptomaine we know
nothing, but from its physical characters it would seem to
possess a pyridine nucleus. It is isomerie with corindine,
a homologue of parvolinc and collidine, which has l>een
obtained from coal-tor.

For the Ix'havior of the hydroehloride to alkaloidal re-
agents, see Table I.

The HYDROCHLORIDE, C,0H15N.HC1, crystal li/cs in
colorless cholesterinc-like plates which arc somewhat, deli-
quescent.

The PLATINOCHLORIDE, (C10H15N.HCl)2PtCl4 (Ft =
27.52 per cent.), forms a light flesh-colored, crystalline pre-
cipitate, and is insoluble in water, alcohol, and ether. It
does not resinify, and is stable at 100°.

Physiological Action. — This ptomaine resembles curara,
although it is by no means as strong. 0.012 gramme of
the free base produced in a frog dilatation of the pupil and
slowing of the respiration. The nostrils were motionless,
and within five hours complete paralysis of the muscles
took place. The reflex excitability gradually diminished
until it finally disappeared. An orange-blossom odor was
observed about the frogs which were poisoned by this
ptomaine. The same amount of ptomaine injected into a
greenfinch produced vomiting, and a condition of weakness
and decreased sensibility, followed soon, however, by re-
covery. A rat was not affected by 0.020 gramme of the
free base. The hydroehloride acts much more energetically.

A BASE, C10H18N, was isolated by 0. DE CONINCK, in
1886 (HUGOUNENQ, page Si, C. Rendus, 1888), from
sea-polyps in an advanced stage of putrefaction, together
with the base C8HUN. The method employed for its ex-
traction was that of GAUTIER and ETARD (see page 164).
It forms a slightly yellow, viscous liquid, and possesses a
pleasant odor resembling that of blooming broom. Its
density is about 1.18. It boils at about 230° (iincorrected),

CHEMISTRY OF THE PTOMAINES. 203

with initial decomposition. In water it is Init slightly
>ohihle, n-adily so in ether, alcohol, acetone, and ligro'in.
It is rapidly" oxidi/ed by the air, Ixvomes In-own, and
resiniu'<s Init does not absorb carlxmic acid.

The 1 1 YI>I:< .CHLORIDE, CIOH15X.H( '1, forms fine yel-
lowish, verv deliquescent needles, which in the presence of
a trace of air are at once colored red ; if more air is present
the red changes to a brown, and in the open air a rosin is
formed the same as from the free Iwse. It is very easily
soluble,

The HYDBOBROMTDB, C,0HlsN.HBr, eryetalluee in a

network of fine deliquescent needles, which Ixx-ome likewise
red on exposure to air. It is very soluble in water; less
so in strong alcohol, and almost insoluble in ether.

The I'LATIX^HLORIDE, (C10H15X.HCl),PtCl4, forms a
dark-red powder, which is insoluble in cold water; very
soluble in warm water. It win lx> kept in dry air ; in moist
air it loses hydrochloric acid and l>ecomcs partially oxidi/ed.
Boiling water decomposes it. (C10H15N.Cl)2PtCl2 forms
clear-brown plates, which are stable in moist air, and melt
at 200°. It is insoluble in cold water, soluble in tailing

water, but decomposes. In recrystallizing, warm previously
l>oiled water should l>c used.

The AUROCHLORIDE, C10H,5N.HCl.AuCl3, occurs as a
light-yellow precipitate; insoluble in cold water, soluble in
warm water. It is decomposed by tailing water; is stable
when kept in a moist atmosphere.

The IODOMETHYLATE, C10H15X.CHSI, in warm alcoholic
solution yields, on the addition of strong potash, a bright-
red color, which soon l>ecomes brown, and in alxmt an
hour the solution shows a greenish-bine fluorescence. This
rapidity of change is due to the extreme oxidi/ability of the
ptoma'ine.

O. DE CoNlNCK considers this base, as well as C8H,,N,
as l>elonging to the pyridine and not to the hydropyridine
series.

A BASE, (',,,11,-N, was deseril>od by GRIFFITHS (1890)
as derive* 1 from cultures on peptone-agar of the bacterium

204 BACTERIAL POISONS.

allii, a germ obtained from putrid onions. The base
(hydrochloride ?) forms colorless, prismatic, microscopic,
very deliquescent needles, which are soluble in warm water,
alcohol, ether, and chloroform. It gives off a hawthorn-like
odor, especially when warmed. With phosphomolybdic acid
it yields a white; with iodine in potassium iodide and with
tannic acid a chestnut-colored precipitate. NESSLER'S solu-
tion produces a yellow chestnut-colored precipitate. Picric
acid throws down a yellow slightly soluble deposit. The
platinochloride, (C10H17N.HCl)2PtCl4, is yellow, crystalline,
and difficultly soluble in cold water and in alcohol ; soluble
in warm water. Gold chloride produces a thick yellow
precipitate soluble in water. Dilute sulphuric acid pro-
duces a violet-red color. The base is apparently a hydro-
coridine.

A BASE, C^H^N, was obtained by DELEZINIER (1889)
and is said to be the alkaloid, isolated in 1879 by BROUAR-
DEL, which in its chemical and physiological properties was
described as similar to veratrine. It forms an almost color-
less oily fluid, which possesses a hawthorn-like odor. It
is very readily oxidizable and yields the veratrine-like re-
actions only in the presence of air. It is soluble in alcohol,
ether, toluene, and benzene; and forms well-defined salts
which are very deliquescent. It appears to be an amine,
and in its composition differs from eevadine by 9H2O.
Nothing is stated in regard to its source or method of
preparation. The analytical results given — C = 89.41,
H = 7.3, N = 3.03 — correspond more to the formula

ETHYI,IDENEDIAMINE(?), C2H8N2. — This base was con-
sidered at first by BRIEGER to be identical with ethylene-
diamine, but subsequent comparison showed this to be an
error. Thus, the former is poisonous and does not form
a gold salt, while the latter is not poisonous and does form
a rather difficultly soluble gold salt. Again, ethylcne-
diamine forms a platinochloride which is almost insoluble
in hot water, whereas the platinum double salt of the pto-

CHEMISTRY OF THE PTOMAINES. 205

ma'ine is much more easily soluble. BRIEOEH is, therefore,
iiicliii.il to think that it is identical with ethyl idenediamine,
CH3.('II. ML),, rather than with ethylenediamine, which
IIM- I lie structure, CH2.NII2.CH2.NH2. This ptomaine
was obtained hy liiMi:<;KU, in 1885 (I., 44), from decom-
po>ing haddock (see (J ad i nine).

The live l>asc can l>e obtained, without decomposition, on
distilling the hydrochloride with sodium hydrate.

The HYDROCHLORIDE, C2H8N,.2HC1, crystallizes in
long glistening needles which are readily soluble in water,
insoluble in absolute alcohol. It gives no combination with
gold chloride. For its behavior to alkaloidal reagents see
Table I.

The PLATINOCHLORIDE, C2H8N2.2HCl.PtCl4 (Pt =
41.4!> per cent.), forms small yellow plates which are
moderately ditlicultly soluble in water. It can be readily
recrystalli/cd from hot water.

Phytiologitial Action. — Frogs seem to be less suscepti-
ble to the action of this poison than mice or guinea-pigs.
In the latter, it produces a short time after injection an
abundant jx-riodic flow of secretion from the nose, mouth,
and eyes. The pupils dilate and the eyeballs project.
Violent dyspnoea then comes on and predominates until the
death of tin- animal, which does not take place for twenty-
four hours or more. The heart is stopped in diastole.

TRIM KTH YLENEDIAMINE (?^, CSII10N2 (?), is a toxic base
isolated by HitiEGER (1887) from cultures of the comma
bacillus on U-ef-broth. It may be stat<>d here that from
the .-aine source, cholera cultures, KUN/ (1888) obtained a
base which he considered to be identical with sperminc or
ethyleneimine (see next chapter). It is present, however, in
exceedingly minute quantity, and occurs in the mercuric
chloride precipitate, from which it is obtained by the fol-
lowing method : The precipitate is decomposed by hydrogen
sulphide, the filtrate evaporated to dry ness, and the residue
taken up with absolute alcohol and precipitated by an
alcoholic solution of sodium picrate. The precipitate

10

206 BACTERIAL POISONS.

thus obtained consists of the picrates of cadaverine, crea-
tinine, and of this new base. It is boiled with absolute
alcohol to remove the insoluble cadaverine picrate; the
filtrate is evaporated to expel the alcohol, and the bases
then converted into the platinum double salts, whereby the
easily soluble creatinine platinochloride can be separated
from the corresponding less soluble compound of the new
base.

Owing to the small quantity of this substance present, a
complete study of its properties has not as yet been made.
It gives difficultly soluble precipitates witli gold chloride
and with platinum chloride ; the compound with the latter
crystallizes in long needles. With picric acid it gives a
precipitate consisting of felted needles, which resemble
creatinine picrate; they melt at 198°. Phosphomolybdic
acid yields a precipitate crystallizing in plates, while potas-
sium-bismuth iodide gives dark-colored tine needles. From
its physiological action it seems to be identical with the
basic substance isolated from choleraic bodies by different
observers. It causes violent convulsions and muscle
tremor.

Besides trimethylenediamine another toxine was obtained
by BRIEGER from cholera cultures, but in quantity insuffi-
cient for analysis. It was obtained from the mercuric
chloride filtrate after elimination of methylamine, trimethyl-
amine, and traces of choline and creatinine, as an insoluble
platinum double salt. Subcutaneous injection of this base
into mice produced a paralysis-like lethargic condition,
slowing of respiration and heart's action, lowering of
temperature, and finally, death in twelve to twenty-four
hours. In some cases bloody stools were passed.

PUTRESCINE, C4H12N2, is a diamine which almost in-
variably occurs together with cadaverine, with which it is
apparently closely related. This base was also discovered
by BRIEGER in 1885 (II., 42), who has obtained it from
putrefying human internal organs (for four months at a
low temperature without access of much oxygen) ; and
from the same material, decomposing at the ordinary tern-

CHEMISTRY OF THE PTOMAINES. 207

perature of tin- room, for from three- days to tlnve we-e-ks.
It lias aUo been obtained from he-mnir, twe-lve days in
spring: from pike1, six days in summer ; from haddock,
two months ( BnrK use ll ). Also from putrid mussel, six-
tevn days (HRiE(;F,u) ; and from human as well as horse
flesh. liiUKCiEli has obtained it from cnltnns of the bac-
teria of human lives on «jelatin, and in small quantity in
rather old cultures of the comma bacillus on beet-broth ; in
larger quantity in cultures of th" same ttcrni on blood-
sennn.

I'm; \NSXKV and BAUMANN in 1888 demonstrated the
existence of putn si'ine- and cadaverine in the urine of cyst-
inuriu, the Ibriner ce instituting about one-third of the
total amount of the two bases present. In the feees of the
same patient, on the contrary, pntrescine constituted by far
the greater quantity, while cadaverine formed but 10 to 1 ">
IMT cent. Normal lives, as well as the tews of various
diseases with the possible exception of cholera stools, are
free from diamincs. It would seem, therefore, that these
bases occur in eystinuria as the result of putrefactive
changes ^eiinjr on in the intestines; becoming partly ab-
sorbed they appear in the urine. In two cases of eystin-
uria, reported by HKIEOKU and STADTHAGEN, cadaverine
was found almost solely present in the urine.

According to Mi-xn:u the diamines are proportionate to
the amount of eystin excreted, and therefore constitute a
fixed symptom, the cause of which is the same as that of
the eystinuria.

Although putres:-iiie is reco^ni/able on about the fourth
day of the putrefaction, yet it docs not occur in appreciable
quantity until about the eleventh day. The amount that
is formed increases as the putre&ction <roes on, so that a
Considerable quantity may be obtained after two or three
wevks. A very n-ood source for the preparation of putre seine,
cadaverine, and nenridine is ji-elatin which has \H'VI\ allowed
to decompose in contact with water for some1 weeks.
Neuridine is, appare-ntly, formed first, but is soon ivphuvd
by the ti>rmer two bases. In the process of extraction it
i> first obtained in the alcoholie- mercuric chloride- pivcipi-

208 BACTERIAL POISONS.

tate. For its separation from cadaverine and other aceom-
panying bases, see page 220.

From the urine of cystinuria it is best obtained by pre-
cipitation with benzoyl chloride (BAUMANN'S method).
For this purpose about 1500 c.c. of urine are treated
with 200 c.c. of sodium hydrate solution (10 per cent.),
then 20 to 25 c.c. of benzoyl chloride is added, and the
whole shaken till the odor of the latter disappears. The
yellowish-white precipitate which forms may consist of in-
soluble phosphates, carbohydrates, polyatomic alcohols, and
diamines. The cystin compound is precipitated only in
concentrated solutions. The precipitate contains from a
half to two-thirds of the diamines present ; it is filtered
off, digested with warm alcohol, and the solution filtered.
The alcoholic filtrate is concentrated and then poured into
about thirty times its volume of cold water. The diamine
compounds then crystallize out. To separate the two dia-
mines they are redissolved in just sufficient warm alcohol
to effect solution, and this is then poured into about twenty
times this volume of ether. The putrescine benzoyl com-
pound is thus thrown out of solution. The filtrate from
this on concentration yields the cadaverine compound. To
isolate that portion of the diamines which remained in the
original filtrate with benzoyl cystin, it is acidulated with sul-
phuric acid and extracted with ether. The residue obtained
on evaporating the ethereal solution is first neutralized with
a 12 per cent, sodium hydrate solution, then mixed with
three to four times its volume of the same solution. The
precipitate which forms consists of the sodium . compounds
of benzoyl cystin and the diamines. It is washed with
sodium hydrate, and the two compounds separated by their
different solubilities in water — the cystin compound is
readily soluble, that of the diamines insoluble. To purify
the benzoyl diamines they are dissolved in warm alcohol
and precipitated with excess of water.

Putrescine (from putresco, to rot, to putrefy) is ;i watcr-
clear, rather thin liquid which fumes in the air and lias a
a peculiar semen-like odor, almost undistinguishahle from
that of cadaverine, and reminding one somewhat of the

cirKMISTRY OF THE PTOMAINES. 209

pyridinc bases. It absorbs earlwnie acid energetically from
the air, without losing thereby the repulsive odor. The
boiling-point of the 1'n-e base, MS ordinarily obtained, is
about l.'}~>°. It is not decomposed by distillation with
pMta-^ium hydrate, and is rather dillieultly volatile with
steam. With acids it forms Ix'autiful crystalline salts.
Piitrcsrine unites with water, like ethylenediamine, to form
a hydrate, and this water can only be removal by distilla-
tion with metallic sodium. The perfectly anhydrous base
boils at 1 ."i(5°-l ")7°, and then solidifies to plates (BRlEGEli),
which melt at 24° (UDRANSZKY and BAUMANN). The
synthetic base boils at 158°-160°, and melts at 23°-
24° (LADENHURG). Like cadaverine it is difficultly solu-
ble in ether.

The constitution of putrcscine has been determined by
UDRANS/KY and BAUMANN (1888). They showed that
the diben/oyl compound of putrescine was identical with
that of the synthetic tetramethylenediamine and of the
l>a- which they found in the urine of cystinuria.

Putreseine, therefore, is tetramethylenediamine, a homo-
lomie of eadaverine, and its rational formula is:

CH.NH.

The same authors (Zdtschr. f. Physiol. Chem. 13, 591)
point out that diamines may possibly occur in putrefaction
as the result of oxidation of nronamines. Tims, putrescine
mi^ht arise from methylamine according to the expiation :

CH,.CH2.NH2 CH2— CII2— NH2

+ 0=1 2 + H20.

CHS.CH2.NH2 CII2— CH2— NH3

In a similar manner cadaverine mi^ht form from ethyl
and propylamine.

Putrescine can be prepared synthetically, according to
LADENBURG'S method, by converting ethylene bromide into
the cyanide and then reducing this by means of sodium
in absolute alcohol.

On heating the concentrated aqueous solution of the
hydrochloride with potassium nitrite there is produced an

210 BACTERIAL POISONS.

oil, soluble in water, from which it can be extracted with
ether. This oil, on treatment witli phenol and sulphuric
acid, gives LIEBERMANN'S nitroso-reaction, which would
seem to show that putrescine is not a primary diamine
(butylenediamine), but is rather a secondary diamine
(BRIEGER, II., 42). As a primary diamine it should take
up, on repeated treatment with methyl iodide, six methyl
radicals ; whereas, if it is a secondary diamine, only four
methyl radicals can enter the molecule. Thus, to illustrate,
methylamine, CII3.NH2 (a primary amine), combines with
three molecules of methyl iodide to form (CH3)4N.EII.
Similarly, dimethylamine (CH3)2.NH, requires only two
molecules to form (CH3)4N.HI. In the case of diamines,
double this number of methyl groups is required to effect
complete saturation. As a matter of fact, BRIEGER (III ,
101), on treating putrescine with methyl iodide, has suc-
ceeded in introducing four, and only four, methyl radicals.
From this, however, it does not follow that putrescine is
not a primary amine, since cadaverine, an unquestioned
primary diamine, yields a substitution compound contain-
ing only two methyl groups (see p. 215).

The tetra-methyl substitution-product of putrescine,
C4H8(CH3)4N2, can be distilled without decomposition. The
free base crystallizes in long prisms. The hydrochloride
forms small needles which are easily soluble ; with phos-
photungstic acid it gives a white crystalline precipitate,
with phosphomolybdic acid a yellow crystalline precipitate,
with picric acid needles. Potassium-bismuth iodide gives
a brownish-red amorphous deposit, while the potassium
mercuric iodide forms prisms. Gold chloride yields diffi-
cultly, and platinum chloride easily soluble octahedra ;
aqueous mercuric chloride forms needles.

The aurochloride has the formula C8H22N2.2AuCl4.

This tetra-methyl derivative of putrescine is enormously
poisonous as compared with putrescine. The symptoms
are the same as those produced by muscarine or neurine.
They are: abundant salivation; dyspnoea — respiration at
first increases, then decreases ; contraction of the pupils ;
paralysis of the muscles of the limbs and trunk ; increased

CHEMISTRY OF THE PTOMAINES. 211

peristaltic action of the intestines, ejaculation of semen,
dribbling of urine, and, finally, violent clonic convulsions.
In the case of mice anil guinea-pigs the convulsions are
prominent immediately after the injection of the poison.

PUTRESCINE HYDROCHLORIDE, C4H12N2.2HC1, forms
long colorless needles, which are very easily soluble in
water ; difficultly so in dilute alcohol ; entirely insoluble in
absolute alcohol, and can thus be separated from eadav-
erine hydrochloridc. To accomplish this separation it is,
perhaps, Iwtter to dissolve the mixture of the hydrochlo-
rides in hot 06 per cent, alcohol. On cooling the solution
thus obtained, the putrescine salt crystallizes out, whereas
that of cadaverine remains in solution. Putrescine hydro-
chloride differs from cadaverine hydrochloride in that it is
not hygroscopic and ran be exposed for days to the air
without suffering any change on the surface of the crystals.

For the behavior of the free base and the hydrochloride
to alkaloidal reagents, see Table I. Putrescine is not toxic,
though -it possesses some marked physiological properties
(si-e Cadaverine, page 215). According to SCHEURLEN
putresrine, like cadaverine, produces inflammation, suppu-
ration, and necrosis. It is not poisonous to dogs (UDRAN-
SZKY and BAUMANN). It is optically inactive.

The PLATINOCHLORIDE, C4H12N2.2HCl.PtCl4 (Pt =
.'>!>. 10 per cent.), often appears under the microscope in the
form of cholesterine-like plates. In the pure condition it
appears as six-aided plates, which are superposed in layers.
The crystals possess a splendid silvery lustre, and are rather
difficultly soluble in cold water ; less so in hot water.

The AUROOHLORIDE, C4H12N?.2HC1.2AuCl3 + 2H,(),
crystallizes likewise in plates, which are difficultly soluble
in cold water. It win, therefore, be readily separated from
cadaverine aurochloride, which is easily soluble in water.
The water of crystalli/ation can be driven off completely
only at 110° (BRIEGER). According to BOCKLISCH, it
loses this water on standing over sulphuric acid, or on
heating at 100°.

The PICRATE, C4H12N2.2C6H2(NO2)3OH, is difficultly
soluble, and crystallizes from a hot aqueous solution in

212 BACTEKIAL POISONS.

needles ; from hot aqueous alcohol, on cooling, in yellow
plates. It begins to brown at 230°, and on further heating
becomes darker, till finally, at 250°, it decomposes with
rapid evolution of gas (BocKLiscn).

The CARBONATE is crystalline.

The MERCURY DOUBLE SALT is easily soluble in a large
quantity of water, and can thus be separated from the
cadaverine salt, which is difficultly soluble. From hot con-
centrated aqueous solution it crystallizes in needles.

The DIBENZOYL - PUTRESCINE, C4H8(XHCOC6H5)2,
forms silky plates or long needles, which are more diffi-
cultly soluble in hot alcohol than those of the cadaverine
compound. From this solution it is reprecipitated by ad-
dition of water or ether. Its melting-point is 175°. It
sublimes without decomposition.

CADAVERINE, C5HUN2, is a diamine isomeric with sap-
rine and neuridine, and, like the latter, it occurs very fre-
quently in decomposing animal tissues. Twelve isomers of
this composition are possible. Another isomer, gerontine (see
next chapter) has been described by GRANDIS (1890). It is
a very striking fact, that in ordinary putrefaction as choline
disappears the diamines appear and increase in quantity
according as the time of putrefaction is extended. It is
also worthy of note that cadaverine appears in putrefaction
before putrescine. It has been obtained by BRIEGER (1885)
from human lungs, hearts, livers, etc. (hence the name),
which were allowed to putrefy at the ordinary temperature
for three days ; from the same organs, and from horseflesh,
after four months in a closed vessel at — 9° to -f- 5° ;
from putrid mussel after sixteen days ; from putrid egg and
blood albumin. It seems to be a constant product of the
growth of the comma bacillus, irrespective of the soil on
which it is cultivated.

BOCKLISCH has isolated it from perch and pike, six days
in midsummer ; from herring, twelve days in spring ; from
haddock, two months at a low temperature ; from cultiva-
tions of FINKLER and PRIOR'S vibrio proteus ou beef-
broth, thirty to thirty-five days at 37° to 38° (Ber. 20,

CHEMISTRY OF TlfK PTOMAIN'ES. 213

I 111). Cadaverine si-cms to be a constant product of the
activity of the genus vibrio, inasmuch as it does not occur
in cultures in which this genus is absent. Thus, it is not
present in the excrements of healthy or typhoid patients ; in
cultures <>!' KMMKIMCH'S bacillus, of EBERTH'S bacillus, and
of the mrogenk bacteria. It is said to occur in cultures of
the Icieillus of hoy;- cholera (v. ScHWEiNiT/). OECHSNER
DE CONINCK has found it in putrid jelly-fish (HuGOUNENQ,
page 23). It is present with putrescine in the urine and
feres of cystinuria (UDRANSZKY and BAUMANN, 1888).
The odor of cholera stools and the breath of cholera patients
may be possibly due to cadaverine, although the base has
not been demonstrated in such cases. It has also been ob-
tained from caviar.

Cadaverine occurs in the mercuric chloride precipitate,
from which it is isolate/! according to the methods given on
pages 206 and 221. For its isolation and separation from
putrescine by the use of benzoyl chloride, see page 208.

This, base was at first ascribed the formula C5H16N2, but
subsequent researches led BRIEGER and BOCKL.ISCH to the
adoption of the formula C5HUN2. In 1883, LADENBURG
prepared, as the first step in the synthesis of piperidine, a
base, pentamethylenediamine, possessing the same empirical
formula as cadaverine, and later (Ber. 18, 2956) he showed
the possibility of the identity of these two bases. This led
to their direct comparison ami the successful establishment
of their identity. In fact, LADENBURG, as a crucial test of
the identity, converted cadaverine into piperidine, and found
the latter base to agree entirely in its chemical and physical
properties with those of the natural alkaloid (Ber. 19, 2586).
LADENBURG, however, observed one apparent difference
between cadaverine and pentamethylenediamine, and that
\vas in the composition of the mercury double salts. That
of the former base, whether obtained from alcoholic or
aeneous solution (Boc'KLiscii, Ber. 20, 1441), was found
to combine with four molecules of mercuric chloride;
whereas the double salt of pentamethylenediamine was
found by LADENBURG to contain only three molecules of
mercuric chloride. Subsequently he found that he had

10*

214 BACTERIAL POISONS.

prepared this salt by mixing the aqueous solutions of tin-
hydroehloride of the base and of the mercuric chloride in
the molecular ratio of 1 to 4, and on using a larger exo-ss
of mercuric chloride he obtained a salt containing four
molecules of mercuric chloride (Ber. 20, 2216). The com-
plete identity of these two bases has, therefore, been estab-
lished. The constitutional formula of cadaverine is, there-
fore :

NH2— CH2— CH2-CH2— CH2— CH2— NH2.

Cadaverine can be prepared synthetically according to
LADENBURG'S method. For this purpose trimcthylcnc
bromide is converted into the cyanide, and this is then
reduced by sodium in absolute alcohol.

Cadaverine forms a somewhat thick, water-clear, syrupy
liquid, which possesses an exceedingly unpleasant odor,
resembling somewhat that of coniine (piperidine) and of
semen. When dehydrated with potassium hydrate it boils
at 115°-120° (BRIEGER). It boils at 175° (BRIEGER, III.,
98), and fumes in the air. The base eagerly absorbs car-
bonic acid from the air, and solidifies into a crystalline
mass, the carbonate. It is volatile with steam, and can l>e
distilled, without decomposition, even in presence of sodium
or barium hydrate, or soda lime. Neuridine, its isomer,
decomposes under these circumstances. When heated with
alcoholic potash and chloroform it does not give the iso-
nitril reaction, nor does it give the characteristic odor of oil
of mustard on treatment with carbon disulphide and mer-
curic chloride. The absence of these reactions at first
induced BRIEGER to conclude that cadaverine and putres-
cine were not primary amines, but LADENBURG (1885)
showed that this conclusion was not justifiable. These two
reactions are given by primary monamines, but in this case
they are not given by cadaverine, a primary diamine. It
is probable that this behavior holds true for all diamines.

Cadaverine is, undoubtedly, identical with the so-called
" animal coniine," which has been isolated at various times
from cadavers.

Cadaverine and putrescine were at first regarded as

CHKMFSTHY OF THE PTOMAINES. 215

physiologically indifferent, Imt i •»• iv<mt investigations

by SniF.riaKN, ( JKAUTP/, and others, show that bothti.
bases an- capable of producing strong inflammation and
nivrosis. According to BEHIUXI;, in large doses it is
poisonous to mice, rahl>its, and guinea-pigs ; it is not
poisonous to dogs (UDRANS/KY and BAUMAXX). Cadav-
erine is ono of those substances which can sot up suppura-
tion in the absence of bacteria. In cholera Asiatica the
necrosis of the intestinal epithelium is quite common, and it
would seem that this pathological change, as well as
the muscular spasms and algidity, are due to the pres-
ence of thes:1 l>ases. It should 1x3 noted, however, that
UDUANS/KY and BAUMANN failed to obtain any sign of
intestinal irritation on feeding dogs enormous doses of
cadaverine. Besides these local effects, they prevent, even
in small quantity, the coagulation of blood, and render it
" laky." According to GRAWITZ, cadaverine seems to
hinder the growth of bacteria. The cystitis observed in
cystinuria may possibly 1x3 due to the presence of cadaverine
and putresc'ine in the urine. Both bases are optically
inactive.

When cadaverine is treated with methyl iodide, a base
is obtained, the hydrochloride of which gives with pla-
tinum chloride a double salt, having the composition:
C6H12(CH3)2N2.2HCl.PtCl4. This new base, therefore, is
cadaverine in which two atoms of hydrogen have been
replaced by two methyl radicals. The platinochloride of
this derivative forms long, clear red needles, which, unlike
those of cadavcrin >, do not change their shape on repeated
recrystallixation. It is moderately difficultly soluble in
water (BRIEGER, II., 41). Sim* cadaverine is a primary
diamine it should combine with six molecules of methyl
iodide to form a saturated compound. This, however, has
not Ixien obtained.

The 11 v])K(K.:HLORiDE, C5HUN2.2HC1, crystallizes in
U-autiful, long deliquescent n^-dles^RiEUER). According
to BOCKLISCH, it forms long, colorless needles or prisms;
crystalli/es t 'nun alcohol in plates, and is not deliquescent
except on long standing. It is soluble in water, alcohol,

216 BACTERIAL POISONS.

alcohol-ether; but is insoluble in absolute alcohol, ether,
etc. It can readily be separated from putrescine hydro-
chloride by its solubility in 96 per cent, alcohol (BocK-
LISCH). The strictly pure base, as well as the hydro-
chloride, does not give a blue color with ferric chloride and
potassium ferricyanide. For reactions of the hydrochloride
and of the free base, see Table I.

Cadaverine hydrochloride on dry distillation decomposes
into NH3, HC1, and piperidine, CSHUN. The latter is a
well-known poisonous alkaloid which exists in the combined
state in black pepper. It is not known whether this change,
whereby the non-poisonous cadaverine is converted into a
toxic base, can take place under the influence of bacteria
during the processes of putrefaction or not. However, it
does not seem improbable that this simple chemical change
should be effected through the action of living organisms ;
for SCHMIDT has already shown that the almost physiologi-
cally indifferent choline, when subjected to the action of
the bacteria of hay-infusion, decomposes into a neurine-like
base possessing a muscarine-like action, and under certain
conditions it yields a base which in its action resembles
pilocarpine.

The SULPHATE likewise forms beautiful, well-formed
needles, and in its solubility corresponds to the hydro-
chloride.

The PLATINOCHLORIDE, C5H14N2.2HCl.PtCl4 (Pt =
38.08 per cent.), crystallizes after some time, on the addition
of platinum chloride to a not too concentrated solution of the
hydrochloride, in the form of long, beautiful orange-red
needles (BocKLiSGH). Ordinarily it is obtained at first in
long, dirty red needles, which on repeated recrystallization
become clearer and assume a form similar to that of ammo-
nium platinochloride. It forms chrome-yellow rhombic
prisms which are short and octahedra-like. In polarized
light they are strongly double refracting. It is very slightly
soluble in cold water ; can be recrystallized from hot water
(BOCKLISCH). Its solubility in water at 12° isjl to 113-
114. It decomposes at 235°-236°.

The AUROCHLORIDE, C5H14N2.2HC1.2 AuCl3|(Au=50.41

CHEMISTRY OF THE PTOMAINES. "2 1 7

per cent.), crystallizes partly in cubes, and partly in
needles \vliich at first possess a bright lustre, hut under the
desiccator soon effloresce and become opaque. The water
of crystallization is completely removed on standing over
sul|)huric acid. It is very easily soluble, and melts at 188°
(BoCKLiscir).

The PICRATE, CftH,4tf2.2C6H2(NO2)3OH, forms yellow
plates which arc difficultly soluble in cold water. From
hot water it crystallizes in long prisms, which melt at 221°
with decomposition. It is insoluble in absolute alcohol
and can be recry stall ized from hot dilute alcohol.

( 'adaveriue hydrochloride combines with mercuric chlo-
ride, when the aqueous solutions of these two salts are
mixed in the molecular ratio of 1 to 4, to form C5H14N2.
2HC1.3IIir< '1... This salt can be rccrystallized from hot
water (LADENBURG). When an excess of mercuric chlo-
ride is used the double salt has the composition C5HUN2.
2HC1.4HgCl2. This last salt melts at 216° (LADENBURG) ;
at 214° (BOTKLISCH). It is difficultly soluble in cold
water ; from hot water it crystallizes in needles or plates

The NEUTRAL OXALATE, C,H14N2.H2C2O4+2H2O, was
prepared by BOCKLISCH by adding a little less than the cal-
culated quantity of alcoholic; oxalic acid to the cadaverine.
The precipitate may be reerystallized from hot dilute alco-
hol, when it is obtaimxl in the- form of needles, which melt
at about 160° and at the same time give oft' ^as.

The Ann OXALATE, OSH14N2.2H2C2O4+H2O, is made
by hrimrmi: the neutral salt into alcoholic oxalic acid. It
is soluble in hot dilute alcohol, and nrrystallizes from it in
quadratic plates, sometimes in ^listeninjr needles. It melts
at 143° with decomposition. After it has been dried
over sulphuric acid, it loses, on beinii- heated to 105°-110°,
one" i nolecule of water (BocKLiseii, Her. 20, 1441). The
insolubility of these oxalates in absolute alcohol shows the
fallacy of TAMBA'S distinction between ptoma'ines and vege-
table alkaloids. (See pajso 186.)

The'DiBENZOYL derivative, CSH10(NHCOC6H5)2, crys-
tallizes in long needles and plates, readily soluble in

218 BACTERIAL POISONS.

alcohol, difficultly so in ether, and insoluble in water ;
hence the alcoholic solution can be precipitated by addition
of water or ether (separation from the putrescine compound,
see p. 208). It melts at 129°-130°. It is not changed by
boiling with dilute acids and alkalies; but boiling with
concentrated hydrochloric or sulphuric acids for a long
time finally breaks it up.

NEURIDINE, C5H14N2, was the first diamine isolated from
animal tissues (BRIEGER, 1883). It is one of the most
common products of putrefaction, and as such has been
obtained by BRIEGER from putrid horseflesh, beef, human
muscle, five to six days ; from haddock, five days in sum-
mer ; from cheese, six weeks in summer ; from gelatin,
ten days at 35° ; from decomposing human internal organs,
three to eleven days ; from cultures of the EBERTH bacillus,
witli mydine.

BOCKLISCH has obtained it from perch, six days in
summer ; from barbel after three days in summer.

It has also been obtained from fresh eggs in the prepa-
ration of choline by heating with baryta ; and also from
fresh brain by heating with 2 per cent, hydrochloric acid
(BRIEGER, I., 57-61). EHRENBERG (1887) found it in
poisonous sausage and obtained it by growing a bacillus
from this source on liver and meat bouillon.

Neuridine is almost invariably accompanied by choline,
and as the duration of putrefaction increases, the latter
gradually decreases in amount and yields a corresponding
increase in trimethylamine, whereas the yield of neuridine
increases from day to day. The amount of neuridine
formed depends upon the nature of the organ employed in
putrefaction. The greatest yield is obtained from gelatin-
ous tissues, such as intestines ; and especially from pure
gelatin. On the other hand, such tissues as the spleen
and liver yield but little.

Neuridine comes down in the mercuric chloride precipi-
tate (sometimes it occurs in the filtrate), and can then be
isolated from the other bases present in a number of ways.
One method is given under Gadiuine. Another convenient

CHEMISTRY OF THE PTOMAINKS. 219

method of separation is to precipitate it from alcoholic
solution hv alcoholic picric acid. The pierate thus ol>-
tain-d is, for the purpose of further purification, rcerystal-
li/ed from absolute alcohol, then decompose! hv extracting
it> acid solution with ether (to remove the picric acid) and
evaporating the aqueous solution to dryness. The residue
is now extracted with alcohol and the alcoholic solution
precipitated by alcoholic platinum chloride. The platino-
chloride can now be recrystallized from hot water.

The free base, as obtained by the treatment of the
hydrochloride with moist freshly precipitated silver oxide,
possesses an extremely repulsive odor, similar to that of
human s men. On evaporation of its aqueous solution it
yields a gelatinous-like mass, and at the same time slowly
decomposes. It does not crystallize when evaporated in a
vacuum, and decomposes even under these conditions. The
same disagnrable odor is obtained when the hydrochloride
is warmed with potassium hydrate. BRIEGER (I., 24) re-
gards this decom position-product of neuridine as an oxida-
tion product of the original substance.

The free base is very readily soluble in water, but is
insoluble in ether and absolute; alcohol ; difficultly soluble
in amyl alcohol. It gives white precipitates with mercuric
chloride, neutral and basic lead acetates. When distilled
with fixed alkali it yields di- and tri-methylamine, thus
probably showing some relation to neurine, hence the name
neuridine. It does not give HOFMANN'S iso-nitril reac-
tion, but it does not follow from this, as shown under
eadaverine, that it may not be a primary diamine. It is
isoinerie with eadaverine, saprine and gerontine.

The HYDROCHLORIDE, C5H14N2.2HC1, crystalli/es in
long needles which are extremely soluble in water and
in dilute alcohol, but are insoluble in absolute alcohol,
ether, ben/ol, chloroform, petroleum ether, bcn/ine, amyl
alcohol, etc. Its insolubility in absolute alcohol may be
used to effect a separation from choline hydrochloride. It
can be recrystalli/ed from slightly warm dilute alcohol.
Although the pure salt is insoluble in the reagents just
given, nevertheless, in the presence of other animal matter

220 BACTERIAL POISON'S.

it is dissolved in greater or less quantity, and hence can be
obtained by the STAS-()TTO as well as by the DRAGEN-
DORFF method. The crystals resemble urea in form. On
heating very cautiously the salt sublimes, and at the same
time appears to undergo a partial internal decomposition,
inasmuch as many of the groups of needles in the sublimate
are colored red or blue. For the behavior of the hydro-
chloride with the alkaloids! reagents, see Table I.

Pure neuridine is not poisonous, but as long as it is
contaminated with other putrefaction products it possesses a
toxic action similar to that of peptotoxine. Tin's holds
true for the other non-poisonous bases.

The PLATINOCHLORIDE, C5H14N2.2EICl.PtCl4, crystal-
lizes in beautiful flat needles. Recrystallized from hot
water, it forms aggregations of small, clear, yellow needles.
It is readily soluble in water, from which it is precipitated
on the addition of alcohol.

The AUROCHLORIDE, C5H14N2.2HC1.2AuCl3, is rather
difficultly soluble in cold water (BOCKLISCH), and crystal-
lizes on cooling of the hot, saturated solution in bunches of
clear, yellow, short needles.

The PICRATB, C5H14N2.2C6H2(NO2)3OH, can be recrys-
tallized from boiling water, in which it is very difficultly
soluble, in the form of needles united in plumose groups.
It is almost insoluble in cold water ; less difficultly soluble
in alcohol. It is not fusible, but begins to brown and
give off yellow vapors at 230°, and carbonizes completely
at 250°.

SAPRINE, C5H14N2, was found in human livers and
spleens after three weeks' putrefaction (BRIEGER, II., 30,
46, 58). It occurs together with cadaverine, putrescine,
and mydaleine in the mercuric chloride precipitate. To
separate these bases, BRIEGER (1885) used the following
process : The mercury salts were decomposed with hydrogen
sulphide, the filtrate evaporated to dryness, and the residue
then extracted with alcohol. The putrescine hydrochloride
is insoluble in alcohol, and is thus removed. The alcoholic
solution was treated with platinum chloride, which precipi-

CII KM ISTK Y OF TIIK PTOMAINES.

tated the greater part of the cadaverine. The- mothcr-
li<|iior, on concentration, yielded a mixture of the platino-
dilorides of cadaverine and sa|)rine. Each successive crop
eontained more of the saprine double salt. The two kinds
of crystals were now separated by means of a magnifyini:-
dass. The saprine platinoehloridc thus obtained was finally
jnirilie<l by repeated recrystalli/ation from water. The
ni(tther-lii|iior, after the removal of the saprine platino-
chloride, contains the mydaleine salt, which, on account of
its solubilitv in water, crystallizes only on concentration,
or on standing under a desiccator. The mercuric chloride
filtrate contains some mydaleine and the ptomaine, which
yields a platinochloride containing 28.40 per cent, platinum.

The free base is a diamine, and was first ascribed the
formula C5H16N2. It appears, however, to be isomerie
with cadaverine and neuridine. The term saprine is derived
from the Greek <*«-/><$?, signifying putrid. It possesses a
weak pyridine-like odor, and can be distilled with steam or
with potassium hydrate; without undergoing decomposition.
In its reactions it behaves the same as cadaverine, except
that it gives an amorphous precipitate with potassium-
bismuth iodide, whereas cadaveriue gives a crystalline pre-
cipitate. The free base gives an immediate intense blue
color with ferric chloride and potassium ferricyanide.

The HYDROCHLORIDE, C5H]4N2.2HC1, forms flat needles
which are not hygroscopic "(distinction from cadaverine
hydrochloride). Its reactions are the same as those of
cadaverine hydrochloride (see Table I.). It is, however,
tinged slightly blue by a mixture of ferric' chloride and
potassium ferricyanide, whereas the fret; base gives an
intense blue. It differs from cadaverine in that it does not
give the reddish-brown color with potassium bichromate
and sulphuric acid. Again, it forms no aurochloride ;
while, on the other hand, eadaverine hydrochloride yields
an easily soluble salt, crystallizing in splendid needles.

The PLATINOCHLORIDE, C5H,4N2.2HCl.PtCl4, forms
parallel, aggregated, pointed crystals, which are somewhat
soluble in water, and are thus distinguished from cadaverine

222 BACTERIAL POISONS.

platinochloride, which crystallizes in rhombs, and is diffi-
cultly soluble in water.

Physiologically, it is indifferent.

A BASE, C7H10N2. — Until very recently the nature of the
basic substances which are formed as products of the alco-
holic fermentation of sugar or molasses has been but little
understood. KRAMER and PINNER, in 1869, found in crude
fusel oil a small quantity of a volatile base: which they
apparently identified with a eollidine. This observation was
confirmed by ORDONNEAU and others ; and still more re-
cently (January, 1888) MORIN has contributed an elaborate
paper upon the bases formed during alcoholic fermentation.
The portion of crude fusel oil which boils above 130.5°
was extracted with slightly acidulated water, the acid
aqueous solution thus obtained was made alkaline, and the
oily bases which were thus set free were then distilled with
vapor of water. The free bases were dried over potassium
hydrate and then subjected to fractional distillation. Three
fractions were thus obtained, boiling respectivelv at 155°—
160°, 171°-172°, and 185°-190°. Only the second frac-
tion, which boils at 171°-172°, was studied, and was found
to possess the formula C7H10N2. Heated with concentrated
hydrochloric acid, it is decomposed in part with the forma-
tion of ammonia. It combines with ethyl iodide to form a
yellow crystalline compound, which is soluble in water
and alcohol, insoluble in ether. The hydroehloride crys-
tallizes in fine white needles, soluble in water and alcohol,
and but very slightly soluble in absolute ether. The free
base, as stated above, boils at 171°-172°, is very soluble in
water, alcohol, ether, etc. When pure it forms a colorless,
strongly refracting, very mobile oil, which possesses a char-
acteristic nauseating odor, but slightly resembling- that of
the pyridine bases. Its density at 12° is 0.9826 ; toward
litmus paper the base shows no decided reaction. The
platinochloride is crystalline and is very soluble in water
and alcohol, slightly soluble in ether. Potassio-mercuric
iodide does not precipitate the aqueous solution of the free
base, but in solutions of the hydroehloride it gives a yellow

I'UKMISTUY OF THK PTOMAI.VKS. 223

iooculent precipitate, which soon crystallines in long bril-
li:int yellow needles. This rejiction takes pla<v readily in
solution- of 1 to 1000, and only after sonic hours in solu-
tions of 1 to 1(),()0() ; and is not given by the bases of the
pyridic and tpiinolinic series. Mercuric chloride produces
an immediate flocciilent precipitate in solutions of the base
having a oOQOentratiOD of 1 to 1000, but re<juires some
time to appear in 1 to 10,000. Phosphotnngstic acid gives
an immediate white precipitate even in a dilution of 1 to
10,000. PhosphomoJybdlfl acid in solutions of the same
strength yields a yellow precipitate.

The physiological action of this baso has been examined
by I{. Writr/, who found the lethal dose for rabbits, etc.,
to be alnmt one gramme per kilogramme of body weight.
It produeea stupor, paralysis, which at first appears in the
rear extremities ; the sensibility becomes diminished and
the pupils are dilated and unresponsive to light; the rate
of heart-beat is lowered, and the rectal temperature falls as
low as 'Jo° ; death follows a more or less prolonged coma.

TANRKT obtained by the action of ammonia on glucose
a number of bases, to which he applied the generic name
of M-lueosines. One of these, having the formula CUH,0N2
(C = 6), corresponds in its formula and its general proper-
ties to MORIX'S base (UI^X. (C = 12), and, in fact, the
two bas"s are considered by TAXIJKT to be identical.

It is interesting to note in tkis connection that alkaloidal
have been found in petroleum by BANDKOWSKI, and
that similar basic siil»-taii.-«s have been detects! by WELLER
in paraffin oil.

Most of the solvents in common use, such as alcohol,
ether, chloroform, hen/ole, petroleum ether, amyl alcohol,
etc., have been shown at different times to contain basic
pyridine compounds, though ordinarily in very minute
quantity. On the other hand, HAITINCJKU has found in
some specimens of amyl alcohol as much as 0.5 per cent,
of pyridine.

SUSOTOXINE, C,0H26N2(?), is a base isolated by \<>VY ill
1890 from cultures of the hog-cholera bacillus of SALMON

224 BACTERIAL POISONS.

(swine-plague of BILLINGS). It is probably identical with
the base obtained by v. SCHWEINITZ from the same germ,
although the formula ascribed to it by him is CLHjjN..
The free base has not been obtained. The hydrochloride
forms a light-yellow syrup which shows no tendency to
crystallize. It is soluble in water and in absolute alcohol,
and is somewhat hygroscopic. When heated with fixed
alkali it gives off a strong amine odor, such as is perceived
on evaporating the original culture-fluid, if it happens to be
alkaline in reaction.

The platinochloride is obtained by precipitation as a light,
flesh-colored, granular precipitate. It is readibly soluble
in water, from which it can be reprecipitated by addition
of absolute alcohol. From aqueous solution, when allowed
to evaporate slowly, it crystallizes in long, thick needles.

The mercurochloride is thrown down from solutions of
the hydrochloride in absolute alcohol, by alcoholic mercuric
chloride, as a heavy, white, granular precipitate. This
readily dissolves on the addition of a small quantity of
water, and can be perfectly reprecipitated by addition of
absolute alcohol. On treatment with hydrogen sulphide it
is readily decomposed, yielding the pure hydrochloride

The aurochloride is very soluble in water and alcohol.
From the alcoholic solution it may be partially precipitated
by ether as a light-yellow, oily precipitate, which is adhe-
rent to the sides and bottom of the tube.

Physiological Action. — The base is toxic only in rela-
tively large doses, as seen from the following experiment.
About 100 milligrammes, dissolved in a little water, wen-
injected subcutaneously into a young rat. The animal
was at first quiet, apparently unwilling to move. After
some ineffectual attempts at jumping, it settled down in a
recumbent position, and when placet! on its side was unable
to rise. Respiration was at first retarded, later increased,
but toward the end was again very slow. Convulsive tre-
mors shook the body at frequent intervals. The animal
kicked vigorously. Reflexes were present almost to the
end. As death approached, the red eyes whitened and took
on a glazed, opaque appearance. Death resulted in one

CHEMISTRY OF THE PTOMAINES. 225

:iinl a half hours. The animal was on its side, the feet
extruded. 1'ost-iiiortem examination showed the heart
anv-trd in diastole, lungs rather pale, stomach contracts!,
serum in thoracic eavity, sulx-nta pair and tedematoiis.

Repeated doses of smaller quantities seem to confer a j>artial
immunity to the action of the germ.

METHYL-GUANIDINE, C2H7NS, =NH= C\^E

rl"his base lias long been known as a product of the oxi-
dation of creatmc and ereatinine, but had never been met
with in animal tissues. BRIEGER in 1886 (III., 33; ob-
tained it from horseflesh which was allowed to decompose
in a close* I vessel at a low temperature ( — 9° to -f- 5°) for
four months. BOCKLISCH (Ber. 20, 1441) isolated it from
impure cultures on beef-broth of FlNKLER and PRIOR'S
vibrio proteus, containing ordinary putrefaction bacteria,
fin- twenty to thirty days at 37°-38°. Vibrio proteus
alone seems incapable of forming this base. The comma
bacillus after some time (six weeks) partially decomposes
ereatinine with formation of a small quantity of methyl-
guanidine (BitiFXJEu). The bacillus of anthrax likewise is
capable of transforming creatine into methyl-guanidine.

It occurs in the mercuric chloride filtrate (BRIEGEK),
from which it is obtained, after the removal of the mercury
by hydrogen sulphide, by precipitation with phospho-
molyhdic acid. The precipitate is decomposed with neutral
lead acetate, and the filtrate from this, alter removal of the
lead l>v hydrogen sulphide, is concentrated and then sodium
picrate added. The resinous picrate precipitate is purified
by boiling with much water, and, finally, it is reerystallixed
from boiling absolute alcohol. According to BOCKLISCH,
it occurs in the mercuric chloride precipitate (not in the
filtrate), from which it is isolated, after removal of the mer-
cury and concentration of the clear filtrate, by precipitation
with sodium picrate. The precipitate containing eadaverine,
UK •thyl-guanidinc, and ereatinine, is boiled with absolute
alcohol (eadaverine picrate is insoluble) and the alcoholic
solution is then evaporated to drive oft the alcohol and

226 BACTERIAL POISONS.

taken up with water. From this aqueous solution, after
removal of picric acid, tnethjl-guanidine is precipitated by
gold chloride, whereas creatinine remains in solution.

This ptomaine is identical with the synthetic methyl-
guanidine (methylurarnine) which can be readily obtained
by boiling a creatine solution with mercuric oxide or with
lead dioxide and dilute sulphuric acid (DESSAIGNES). The
parent substance of methyl-guanidine as it occurs in putre-
faction is undoubtedly the creatine which exists preformed
in the muscular tissue. If such is the case, the bacteria
engaged in its production must be considered as possessing
an oxidizing action, since this base is prepared synthetically
from creatine by oxidation. That creatine does not offer
much resistance to the action of bacteria is shown in the
fact that FRIEDLANDER'S pneumonia coccus, which pos-
sesses but small chemical powers, is capable of slowly but
steadily decomposing creatine, yielding as one of the pro-
ducts acetic acid. STRECKER and ERLENMEYER, as well
as BAUMANN, have shown that creatine, although a sub-
stituted guanidine, is not poisonous, but is readily con veiled
into creatiuine, which is a relatively toxic substance. On
the other hand, guanidine and methyl-guanidine are quite
violent poisons. This is, therefore, another instance in
which a toxic substance is formed by the action of bacteria
from a previously non-poisonous base (see page 244).
According to LOSSEN, guanidine is formed, though in
small quantity, in the oxidation of albumin.

The formula of these closely related substances are here
given for comparison :

Oeatine, m =

/N(C!H3).CH
Creatinine, NH = 0x^x1

XN(CH3).CH2
Methyl-hydantoine, O = C\^jj __ Ar\

,r , , .-I. XTTT ^./^NH.C'II,

Metliyl-gunnidine, JNH = '

rilKMISTRY OK Till! 1'TOMAINKS. 227

Cnanidiue, NH^C

Methyl-guankline forms a colorless, ea.-ilv deliquescent

mass p., --.(--in- ;l strong alkaline reaction. On heating
with potassium hydrate it decomposes, and yields amnionia
and mcthylamine. It is a highly poisonous base.

The HYDROCHLORIDE, C2H7N3.HC1, am be obtained
from the pierate by dissolving the latter in water acidulated
with hydrochloric acid, and extracting the solution with
ether to remove the picric acid. The colorless aqueous
solution now, on evaporation, yields a thin syrup which
crystallize in vacuum to compact prisms. These are in-
soluble in alcohol, and give with platinum chloride a double
salt of monoclinic needles (II.u\siioi-'Ku) which are very
easily soluble (1 part in about 7 parts water, TATAKINOW i.

The AUROCHLORIDE, C2H7N3.IICI.AnCl3 (An = 47.71
per cent.) forms rhombic crystals (HAUSHOFER) which are
easily soluble in ether, more difficultly in water or alcohol ;
readily soluble (BRIEFER). It readily decomposes on heat-
ing in pure water, but may be nvrvstallized from water
acidulated with hydrochloric acid. It melts at 198°.

The PICRATE, C2H7NS.C6H2(N()2)3OH, comes down at
first as a resinous precipitate, which when boiled with much
water solidities in the form of felted needles. It is very
difficultly soluble in water, and ran be purified by repeated
recrystalli/ation from boiling absolute alcohol — distinction
from cadaver inc. It melts at 102°.

The Ox A LATE, (C2l!7X3),.II2C204 + 2H2O, fi.rms crystals
which are easily soluble in water.

Physiological Action. — Mcthyl-guanidine as obtained
from putrefy ing flesh is identical in its physiological
action with the synthetic base. It has already been stated
that the non-poisonous creatin • is readily converted into the
relatively energetic poison creatininc. The latter substance
possesses a paralysing action differing very much from its

228 BACTERIAL POISONS.

decomposition-product methyl-guanidine. This base is very
poisonous, and the symptoms arc marked by dyspnoea,
muscle tremor, and general clonic convulsions. BRIEGEB
has observed the following symptoms on injection of about
0.2 gramme of methyl-guanidine into a guinea-pig: The
respiration at once becomes more rapid, and in a lew min-
utes abundant passage of urine and stool takes place ; the
pupils dilate rapidly to the maximum and cease to react,
The animal is uneasy but motionless, though not exactly
paralyzed. Respiration becomes deeper and more labored,
the head moves from side to side, the extremities become
gradually paralyzed ; dyspnoea sets in, the animal falls on
its side and dies (twenty minutes) amid general clonic con-
vulsions of short duration. Fibrillary twitchings of the
trunk muscles are observed only in the beginning. Post-
mortem showed the heart to be stopped in diastole, the in-
testines filled with fluid, the bladder contracted, the cortex
of the kidney hyperaemic, but the papillae of the kidneys
surprisingly pale.

MORRHUINE, C19H27N3, was obtained by GAUTIER and
MOURGUES (1888) from the mother liquors of aselline on
concentration of the platinum-containing liquid. Tin's sub-
stance constitutes about one-third (0.07 per cent.) of all the
bases found in cod-liver oil, and is named from Gadus
niorrhua, the ordinary codfish. The free base is an oily,
very thick, amber-yellow liquid, the odor of which resem-
bles somewhat that of syringa. It floats on water and par-
tially dissolves ; is more soluble in ether and. in alcohol.
The base is very alkaline and is caustic to the tongue. It
absorbs carbonic acid and is non-volatile. The salts of
copper are precipitated by it, but the hydrate formed is not
redissolved

The hydrochloride is very deliquescent, The gold salt
forms a yellow precipitate which readily dissolves on
warming. The platinum salt, C19H27N3.2HCl.PtCl4 (Pt =
27.56 per cent.), crystallizes in barbed needles, which are
quite soluble. (Separation from aselline, p. 230).

CII KM IS TH V OF THK PTOMAINES. 229

/'A//.s7o/oy/<W A<-fiuii. — The Icisc pos-rssi - tlir property

of exciting the appetite; it arts MS a diaphoretic ami above
all as a diuretic. (>.(»2!) gramme given suliciitaix onsly
to a gninea-rpig produced in two and a half hours a l<>->
of 1 .">.") grammes in the weight of the animal. Tin1 same
Hlivt is prodiK-ed in liirds. Strong doses (0.1 gramme per
kilogramme) prodn*-*1 fatigue and hebetude.

A UASK, (113IT20N4. was obtained as early as 1H<>8 by
< >>I:K, who observe* I its formation during the fermentation
of pure cane-sugar by means of yeast. The hydroehloride
when drie<l in vaeiio is said to form a white, very hygro-
scopic foliacciins mass, which soon becomes brown on expo-
sure to air. At first it imparts a burning taste, which is
scum replaced by a very bitter sensation.

A P>.\si; corresponding to the formula C,7H38N4 was ol>-
t-iined by ( J.U"TIKI: and HTAIJD from the mother-liquors of
the platinochloride of the base CaH,.N. Very little is

~ tO »

known, however, in regard to the general properties of this
base, <»wiiiu to the small quantity which could IM> isolated.
This base ;md the one obtained by OSKK from the yeast-
li'nueiifatioii of sugar, C13ir20N4, an<l aselline, C^lI^jN^
are the only jitoma'ines thus liir isolatxxl which are known
to contain four atoms ol nitrogen.

Tlx1 PLATIN.K iii.diMiu:, (V7II38N4.-JHCl.PtCl4 (Pt =
-!.'}'! |HT cent.), is ivadily soluble, ami crystalli/es in
ncMlles which j>o~~, ~- ,-i light-yellow flesh color. When
heated to 100°, it slowly decomposes, giving oil' a syringa-
like odor.

A-KLUNK, C2,,H32N4, isolate*! by (IATTIKU and Mouii-
<.i ivS (1SSS), lordlier with live other bases from cod-liver
oil. (See p. 26.'}.) It is present only in small (piantity in
the oil. The name is derive* 1 from Asellns major, the great
codfish. The free base is thrown down from the solutions
of the hydroehloride by the addition of alkali, in amorphous
whit*1 llocciilcs which arc almost insoluble in water. It is
almost colorless, but on exposure to the air Ixvomcs slightly

11

230 BACTERIAL POISONS.

green. It is not hygroscopic, and possesses a density of about
1.05. On heating it molts to a viscid yellowish fluid, pos-
sessing an aromatic odor ; is non-volatile. Although almost
insoluble in water, it imparts to it an alkaline reaction and
a bitter taste. It is soluble in ether, more so in alcohol.

The salts arc crystal livable, but are partially dissociated
by the action of warm water. The hydrochloride forms
crossed or entangled needles which are quite bitter. The
gold salt is very reducible. The platinochloride, C2SII32N4.
2HCl.PtCl4 (Pt= 24.41), is orange-yellow in color; solu-
ble in warm waler, insoluble in cold water (separation
from morrhuine, p. 228), and is rapidly changed by boiling
water. The mercury salt is precipitated in the cold ; r< 'dis-
solves on heating, and then, on cooling, recrystallizes.

In large doses it produces fatigue, short and rapid respi-
ration, and stupor. Three milligrammes of the hydro-
chloride kills a greenfinch in fourteen minutes.

MYDINE, C8HHNO, is a non-poisonous base which
was obtained by BRIEGER in 1886 (III., 25) from the
putrefaction of about two hundred pounds of human in-
ternal organs; and also in cultures of the EBERTH bacillus
on peptonized blood-serum. It occurs in the mercuric
chloride filtrate, and is isolated from it after the removal
of the mercury by hydrogen sulphide, by precipitation with
phosphomolvbdic acid. The gummy precipitate which is
produced is decomposed on the water-bath with a solution
of neutral lead acetate, and the filtrate on evaporation yields
a colorless hydrochloride, crystallizing in plates. It is
purified by recrystallization of the picratc.

The free base is strongly alkaline, and possesses an am-
moniacal odor. It is characterized by its strong reducing
properties. The name mydine is derived from /wMw, to
putrefy. With platinum chloride it gives, after a time, an
extremely soluble salt; with gold chloride, a precipitate 6f
metallic gold. On distillation it is decomposed.

The HYDROCHLORIDE, C8IIUNO.HC1, crystallizes in
colorless plates. It gives a blue color with ferric chloride
and potassium ferricyanide.

C1IKMISTKY <>K THK I' T« > M AI N" KS . 231

The I'K-KATK, rjlnN<>.< ',,IL( N< >,).,< )H, is obtained in
broad prisms, which melt at 1!).")°. It is tin- only salt
suitable for manipulation-.

In describing WENCKi's collidine (page 196) it waa stated
that tyrosin might IK- looked upon as the source of that
base. It would seem, however, to be more appropriately
the parent snltstanee of mydine, inasmuch as it decomposes
on being heated to 270° into carbonic aeid and oxypheuyl-
ethvlamine, C8HUNO. The change that takes place can
IM- represented by the equation :

r TT

C.».\CH!.CHNH,.C02H = C.,.CHjNIIi + co,.

TYROSIN. OxyPlIlNYL-ETIITI.AMINE.

A BASE, (15IIUNO,, was isolated by E. and H. SAL-
KOWSKI (188.S) from deeomposinj; fibrin and meat. In its
composition it is isomeric with Ix'taine anhydride. It is
extremely soluble in water, very difficultly so in alcohol,
insoluble in ether, and possesses a semen-like odor ami
saline taste. The aqueous solution, which is not alkaline
in reaction, yields on evaporation a stellate crystalline mass,
which on standing over sulphuric acid becomes a white
powder, which melts at 15<)°. It dissolves silver oxide,
l)iit not cupric hydrate, thus apparently indicating that it
is not an amido acid. Moreover, it does not give a pre-
cipitate or blue coloration with copper acetate, or anuno-
niacal silver nitrate. It thus di tiered from the then known
amido-valerianie acids, its isomers. Recently, however
(1891), GABRIEL and A0CHA1* showed that ''-amido-vale-
rianic acid agrees with this base in its reactions to copper
and silver oxide, copper acetate, and ammoniacal silver
nitrate. The «>-old salt of the synthetic base possessed (lie
same composition as that of SALKOWSKI, ami melted at

The identity of this base with 's-amido-valerianic acid
(homopiperidinie acid) would seem to IM- established, and
as such it is regarded. Its structure, then, is represented by

NHrCII2.CIL.< I

232 BACTERIAL POISONS.

For its synthetic preparation see Ber. 24, l:>(>5 (Isiil).
The hose does not seem to possess a toxic action.

The HYDROCHLORIDE, C6HUNO2.HC1, forms colorless,
stellate crystals, which are permanent in the air, and arc
extremely soluble in water, even in absolute alcohol.

The AUROCHLORIDB, ( 15HnNO2.HCl.AuCl3 *+ H2O, is
obtained on slow evaporation, as large, well-Conned, heau-
tif'nl dark-yellow crystals. They are probably monoclinic,
contain water of crystallization, and melt at below 100°.

The PJ,ATINOCHLORIDE gave on analysis results cor-
responding to the formula (C7H18NO2.HCl)2PtCl4. This
may possibly be due to the presence of some higher homo-
logues of the base C5HUNO2. It forms fine orange-yellow
crystals, which arc very difficultly soluble in alcohol, easily
so in hot water, from which, on cooling, it crystalli/es in
beautiful plates.

CHOLJNE GROUP. — The following four bases arc closely
related, and, indeed, starting from choline, the oldest and
best-known individual, the remaining bases can be readily
prepared from it. Moreover, they can all l>e prepared
synthetically according to methods that will be subsequently
indicated. As choline is the most prominent member, we
have thought best to class these substances together as con-
stituting the choline group. It is very probable that my-
datoxine and mytilotoxine, when their constitution becomes
known, will be found to be homologues of certain members
of this group.

NEURINE, C5H13NO = C2H3.N(CH3)VOII. — This sub-
stance was obtained and named thus by LlEBREICH (1S<;.">),
who prepared it by boiling protagon lor twenty-four hours
with concentrated baryta. Previous to its discovery as a
decomposition-product of protagon from the brain it was
prepared synthetically by HOFMANN (1858) by treat in-
trimethylamine and ethyl cue bromide with potassium hy-
drate or silver oxide. BAEYER (18(56), by boiling an alco-
holic extract of the brain with baryta water, obtained on
separation by three' different methods, a base, or rather a

.'II KM 1ST It Y OF Til K I'ToM A I N I

mixture- of IKISCS, whie-h, on analysis, gave iv-ults conv-
sponding to the three formula' :

1 2 3

(aBHl4N001),PtCI4 (C5H,,NCl)aPtCI4 (CsH14NCl),PtCl4

Formula No. •"> was tl ..... lie accepted by LlKBUKK'II for

nearim, bat, according to !>AKVKH, LIKUKKICH'S ne-nrine-
salt is not simple, but is a mixture of Nos. 1 and '2. He
himself aeeepts formula No. 1 as the platinoddoriele- of
neurine, and distinctly states (A nun/, if. Chrm. it. Pluinn.,
142, •">-•">, 1W>7) that nenrine is in composition trimethyl-
oxyethyl-ammonium hydroxide. And, according to him,
eholine from bile, and sinkaline- from white, mustard, appear
to l>e identical with nenrine.

This noiiieMelatiire of JiAKYKK's was tit first adopt**! l>y
WuRT/ and others, who showed that the oxyothyl base
was identical with eholine and sinkaline. ( )n that account
Sii;i:( KKR, in 18H8 (AnnaL, 148, 70), suggested the re-
striction of the nanu! eholine to the o.xycthyl base, and to
reserve the name nenrine lor the base whos1.1 platinochloride
is represented in No. •">, as originally was done by LIEB-
IJKK-H. In 18<l!) LiKiiUKH'ii showc<l conclusively that
pure prota-oii, when heated with baryta for twenty-four
hours, yields a substance having the composition of the
vinyl base :

The platino -hloride of this bas:- ei-ystalli/e<l in live-sided
yellow plates, which, after a time, on exposure to the air,
became cloudy ; on treatment now with water a portion
dissolved, and the solution was found to contain the oxv-
etliyl base. Furthermore, he observe*! that when the alco-
holic extract of the brain, from which all the protagon had
Ixvn remove*!, is tivate*! with baryta, only the' latter, the-
oxyethyl Itase, is obta'ned. Finally, in 1870, WruT/
abandoned the use of the term neurine to designate the
oxyethyl bas;-, and returned to the name eholine, originallv
applied to the oxyethyl base by its discoverer, Sl'KKe'KKK.
Nevertheless, the confusion in the nse of the-se two terms

234 BACTERIAL POISONS.

continued to exist, and even at tlie present time it is the
eutise of no little misunderstanding. Tims, MABiNO-Zuoo
(1885), in his excellent researches on the genesis of pto-
maines, applies the term neurine, following BAEYER'S pre-
cedent, to the oxyethyl base, C5H15NO2, which is really
eholine, according to the proper nomenclature.

AVe have gone somewhat at this point in detail into the
history and the proper use of the terms ueurine and eholine
because of the confusion which is sure to arise if the dis-
tinction is iiot thoroughly borne in mind. The name
neurine, then, should be used only to denote the vinyl base
C8H18NO. It is trimethyl-vinyl-ammonium hydrate. On
the other hand, eholine is applied to the oxyethyl base
C5HI5NO.j, which is trimethyl-oxyethyl-ammonium hydrate.

Neurine lias been obtained by BRIEGP;R (1883) in the
putrefaction of horse, beef, and human flesh for five to six
days in summer. It also occurs in the commercial, so-called
" neurine," together with eholine (BmEGER, I., 34). Li i:r,
REICH obtained it in the decomposition of protagon by
baryta. And BRIEGER (I., 60) also has isolated it along
with eholine from fresh human brains, by boiling with
baryta; but has not obtained it by digesting the brains on
the water-bath with two per cent, hydrochloric acid. It
lias been found in putrid, and as result of this change
poisonous, mushrooms (BERLINERBLAU, 1888).

The genesis of neurine is still rather obscure, and it is
to l>e hoped that future investigations may shed more light
upon the mysterious production of this highly poisonous
base. Its occurrence in the brain together with eholine
would seem to indicate that it is either derived from
eholine by the removal of water, or that it exists together
with eholine, partly replacing the latter in the molecule of
protagou (lecithin), according to the hypothesis put for-
ward by LIPPMANN (page 241). The question of its
derivation from eholine by withdrawal of a molecule of
water has already been subjected to an interesting experi-
mental discussion. CH. GRAM attempted to explain the
production of neurine and other muscari lie-like ptomaines
as due to the dehydrating action of the acids employed in

CII KM 1ST HY OF THK PTOMAINKS

the methods of extraction, and, indeed, lie claimed to have
converted clioline platinochloridc, by heating with hydro-
chloric acid, into neiirine. This statement has been dis-
juited l»v I)i;iH(iKU, who showed that the platinochloridc
of choline, as well as the hydroehloride, may 1)0 heated
with fifteen or thirty per cent., or even concentrated hydro-
chloric acid, for six to eight hours on a water-bath, with-
out any conversion whatever (HI., 15). That neurine
mav In- obtained from choline, at least by chemical pro-
. was shown by I>.U:YKH, in 1800, who found that
choline chloride, when heated with several times its volume
of concentrated hydriodie acid and some red phosphorus,
gave a compound C5H13X L which, on digestion with fresh,
moist silver oxide, yielded a vinyl base identical with that
previously obtained synthetically by HOFMAMN, and now
known as neurine. BUIJ-XJKK has tried, unsuccessfully, to
bring about this dehydration by the putrefaction of pure
choline (I., 59). However, SCHMIDT and WEISS (1887)
were more successful, and they found that choline, as well
as the hydroehloride and lactate, is changed by the action
of microorganisms into the strongly poisonous neurine.
Their results are given in full under choline (see page 244.)
From what has been said it is evident that neurine can
only arise from choline, and this, as will be seen later, is
derived from lecithin.

Neurine is almost invariably accompanied by choline,
from which, however, it can be readily separated by the
dill'erence in the solubilities of the platinochlorides. It
occurs in the mercuric chloride precipitate (and in the
iiltrate), and from this it can be obtained, after removal of
the mercury, by precipitating the solution of the mixed
hydrochlorides in absolute alcohol by platinum chloride.
The platinochlorides are then separated by recrystalli/ation
from water, since the neurine is difficultly soluble, while
the choline salt is readily soluble.

The five base possesses a strong alkaline reaction, and
on contact with the fumes of hydrochloric acid it yields a
cloud. According to LIKHKKK n, the alkaline solution
cannot be neutrali/cd by passing through it carbonic acid.

236 BACTERIAL POISONS.

The CHLORIDE, CSII12N.C1, is extremely poisonous, and
crvstallizes in fine hygroscopic needles.

The PLATINOCHLORIDE, (CJLjN.Cl^PtCl, (Pt = 3:;.i;n
per cent.), is difficultly soluble in hot water, and crystallizes
in beautiful, well-formed octahedra belonging to the regular
system. No twin-crystals are observed. Sometimes the
crystals contain water of crystallization, at other times they
do not (BRIEGER, I., 33). According to LIEP.IJEICH, it
forms from an aqueous solution in five- or six-sided, heaped -
up plates resembling urea nitrate, while from an alcoholic
solution it forms needles, which on exposure to air become
opaque, and are partially converted into the oxyethyl base
— choline.

The AUROCHLORIDE, C5H12N.Cl.AuCl3 (An = 46.37
per cent.), forms flat prisms, which are difficultly soluble
in hot water (BRIEGER.) Dissolves easily, and can be
purified by crystallization (LIEIJREICH).

Physiological Action. — Neurine is exceedingly poisonous,
even in small doses, and in its action it strongly par-
takes of the characteristic stamp of poisoning by muscarine.
The injection of a few milligrammes into frogs produces
in a short time a complete paralysis of the extremities, with
deadening of reflex excitability. Respiration stops first,
while the rate of heart-beat gradually decreases till, finally.
stoppage in diastole takes place. The injection of atropine
at this point does away with the effect of neurine, so that
the heart begins to beat again. Previously atropinized
frogs, as a rule, withstand the action of the poison. Im-
mediately after the introduction of this substance there can
be observed a distinct period of exaltation, which, however,
soon gives way to the characteristic stage of depression seen
in the progressive slowing of the rate of heart-beat. Of
the warm-blooded animals, cats seem to be much more
sensitive to its action than mice, rabbits, or guinea-pigs.
The symptoms seen in rabbits arc profuse moistening of the
nasal cavities and upper lip, which is succeeded by an in-
tensely profuse salivation ; later on there is noticeable an
abundant secretion from the nasal mucous membrane and
from the eyes ; the latter, however, ceases in a short time.

I'll K M ISTK V i iF TIIK I'TUM A I N KS. 237

The movements of the In-art and of respiration are at first
quickened and st relict liencd, hut before long (lie paralytic
etleets produce a constant slowing and weakening, till
finally complete ces- -ition of both inoveinents results. The
decided dyspim-a observed gradually alters its character,
and just before death the respiration is irregular and super-
ficial. The heart, as in frogs, eontinnes to beat after the
respiratory movements have ceased, until finally it stops in
diastole. Direct application of concentrated solutions of
the poison to the eyes produces almost always a contraction
<»f the pupil, while a similar but less constant contraction
is seen when it is injected. The peristaltic action of the
intestines is heightened to such an extent that continual
evacuation takes place. Just before death, violent clonic
convulsions occur. Atropine possesses a strong antagonistic
action toward neurine, and the injection of even a small
quantity is sufficient to dispel the symptoms just de-
scribed.

, (15H13NO2 = C2H4OH.N(CH,)3.OH.— This
base is identical with the sinkalinc of V()N BAUO, the bili-
neurinc of LIEIUJKK n, and the neurine of BAEYEK,
M AIM N< >-/rco, and others. According to SOHMfBDBBBRG
and HAUNACK, it is identical with LETELLIER'S amanitine
(airaricine), to which they assign, however, the formula
(CH3)3N.(CHOH.CH3)()li. Choline was first prepared,
and so named, by STUKCKER, in 1862, by treating hog-bile
with hydrochloric acid. It was prepared synthetically bv
\\'II;T/ (iSu'S) by direct union of ethylene chlorhydrine
and trimethylamine. The reaction that takes place can In-
n-presented by the equation :

^3

' ( ^ 4- n?3 IN- ( "3
C113 .

l>.\KVEii (1S«!G) obtained it by boiling an alcoholic extract
of the bra'n with baryta water; and LIKUIIKK n, in 180!),
showed that if the alcoholic extract, from which all the

238 BACTERIAL POISONS.

protagon had IK-CM removed, he thus treated, only eholiue is
formed, whereas pure protagon, on heating with barvta,
yields neurine. It has heen obtained from the yelk of
eggs ; from bile; from fresh brains (BRIEGER) ; from fresh
eggs, blood, lungs, and hearts, and from lecithin (MARINO-
/uco); from human placenta (BoEHM) ; from the < ye ;
from commercial neurinc (BRIEGER) ; from fresh as well as
decomposing internal organs of the cadaver (BRIEGER,
1885) ; from herring-brine and decomposing pike, three
days in midsummer (BocKLiHCH). It has also been isolated
from cultures of vibrio protcus (BocKLiscn), and of comma
bacillus (BRIEGER). EHRENBERG (1887) found it in
poisonous sausage, and, by growing a bacillus obtained from
this, on liver.

Not only has clioline been met with in the animal tissues,
but it has also been observed within the last few years to be
very widely distributed in the vegetable kingdom, especi-
ally so. in fatty seeds. Thus, it has been found (!!ARNACK,
1876) accompanying muscarine, in toadstool ( Agaricus mus-
carius) ; in hops, and hence in beer (GRIESS and HARROW ;
in the seeds of Trigonella, in Indian hemp, arcca- and earth-
nuts, hemp seeds and lentils (JAHNS); in the seeds of white
mustard, as a glycoside (VON BABO); in ergot (BRIEGER);
in the germs of pumpkins and lupines (ScHULZS, Zcitxchr.
f. Physiol. (?/tem.,H, 365); in beech-nuts and morels (Hel-
vetia esculenta, Boletus lusidus, Amanita pantherina, B<">IIM) ;
in flores sambuci (elder), and extracts of belladonna, hyos-
cyarnus, ipecacuanha root and Acorus calamus (KuN/),
and Scopolia Japouica (SCHMIDT and HENSOHKE) ; in the
sprouts and cotyledons of Soja beans (SCHUL/E, 1888), in
the fat from hog's bean, vetch, peas and lupines (JACOB-
SON, 1889) ; from the lecithin of lupine seeds (SCHULZE and
STEIGER) ; and in Chekeu leaves (Myrtus cheken, WEIFS).
According to LIPPMANN (B<r. 20, 3206), it is present, to-
gether with betaiue, in the molasses from beet-root sugar.
Clioline (RITTHAUSEN) and betaine (BOHM) exist together
in cotton-seeds ; hence, clioline occurs in the press-cakes from
cotton-seeds (BdHli). According to SCIIUL/E, and also
RITTHAUSEN, jcholine occurs with betaiue and another

fllKMISTKY OF TIIK I1 To MA INKS. 239

iii (he seed of the vetch, and in peas with a l>a><- !•••-
•embling In'taine. The two bases have also been found
together in S-opolia atropo'idcs by SIKUKUT.

Choline may readily l>e prepared, after the method of
hi A K< »NOW, from the yelk of eggs. These are extracted
with ether, tlien with aleohol, and the extracts thus ob-
tained evaporated, when the resulting residues are boiled
with baryta for one hour. The filtrate, after the removal
of the barium by carbonic acid, is evaporated and the
residue is abstracted with absolute aleohol. The alcoholic
solution is now precipitated with platinum chloride.
l>i:iK<;Kit (II., 55) has presented a method which is much
simpler in its details and obviates the use of the expen-
sive platinum chloride. The tissues rich in lecithin, as
yelk of egg, brain, etc., are heated with concentrated
hydrochloric acid for some hours on the water-bath. The
insoluble residue is filtered otf, and the filtrate, after neu-
trali/ation of the excess of free acid with carbonate of
sodium, is evaporated. The residue is extracted with
alcohol, and the alcoholic solution is precipitated with
alcoholic mercuric chloride. The precipitate thus obtained
on recrystalli/ation several times from a large quantity of
boiling water, yields the pure double salt of choline.

If desirable, it can be made from pure lecithin, best pre-
pared according to GILSON'S method. Yelk of eggs is
rej>eatedly shaken up with ether until the latter is colored
only a faint yellow ; the ether solution then distilled, the
residue taken up in petroleum ether and filtered. The
filtrate, in a separatory funnel, is well shaken with 75
j)er cent, alcohol, and this is repeated several times with
fresh aleohol. The alcoholic extracts are combined, allowed
to stand for some time, then filtered and subjected to dis-
tillation to remove traces of petroleum ether. The solu-
tion is now set aside in a cool place for several days; the
precipitate which forms consists of cholesterine, etc., and
a little lecithin. The alcoholic solution is filtered by de-
cantatiou, then decolored by. boiling with bone-black ;
rapidly evaporated at 50-60° to a syrupy consistency.
This residue is extracted with ether, the solution filtered

240 11ACTEHI AL POISONS.

and evaporated. The lecithin thus obtained is almost per-
fectly pure, but contains traces of cliolcsterine. To com-
pletely purify it, it can be dissolved in as little absolute
alcohol as possible, and set aside to reprecipitate in the
cold, —5 to 15°.

In regard to the genesis of choliue the preponderance of
testimony goes to show that it is derived from the decom-
position of lecithin, which, according to the researches of
DIAKONOW and others, is one of the most widely distributed
compounds, occurring in greater or less quantity in all of
the animal tissues. Lecithin, which is a complex esther
(STRECKER, HUNDESHAGEN, GILSON), decomposes under
the action of acids and alkalies into a base (choline)
glycerin, phosphoric acid, and fatty acids (stearic, oleic,
palmitic, etc.). GILSON has shown that dilute sulphuric
acid slowly decomposes lecithin, forming choliue, which,
after a few days, disappears; on the other hand, sodium
hydrate, in even 1 per cent, solution, rapidly decomposes
it. This change is undoubtedly accomplished in a similar
manner through the agency of bacteria. BRIEGER (II.,
17) is inclined to believe that choline exists preformed in
the various tissues, inasmuch as he has been unable to ob-
tain it from the brain, which is rich in lecithin, by boiling
with 2 per cent, hydrochloric acid. (See SCHUL/E, page
242.) Prolonged heating with concentrated hydrochloric
acid was necessary in order to obtain any choline from the
brain. This result of BRIEGER'S is somewhat at variance
with that of MARiNO-Zuco (see Relazione, etc., pages 29,
30, and 38), who obtained from 25 grammes of lecithin, by
the method of STAS, a small quantity of the aurochloride
of a base, while from a similar amount he obtained more
relevant quantities by the method of DRAGENDORFF.

The occurrence of choline in the vegetable kingdom
would be inexplicable to us at present were it not that
we now know of the existence of lecithin-like bodies in
plants, from the decomposition of which substantially the
same products are obtained as from the lecithin obtained
from the animal tissues. The existence of such a body in
plants was first predicted by SCHEIBLER in 1870, who was

CM KM 1ST K V (•!•' TIIK I' T ( ) M A I N K S . 1J I 1

led to tlii- conclusion in Ins celebrated study of beet-root
sugar, becau-e of (lie presence of oleic acid, glycerin, phos-
phoric acid, and betaine, as well as choles(crin, in the !».•<•(
root extracts. This hypothesis \vas confinne(l by Hol'l'K-
SKYI.KIJ, who, in 1,S7!», found a lecithin substance in yea>(.
Snii 1,7,1 : found a similar compound in the cotyledon- of
lupine, while .1 ACOIISON observed its presence in mustard-
seeds, in fenugreek-seeds, in maize and wheat, in the fat
from beans, peas, vetch, and lupines. HKUKKL showed its
presence in globularia, and LIITMAXN has found it in beet-
root. According to Hoi'i'K-SEYLKR, this lecithin-like sub-
stance exists in all vegetable cells undergoing development.
SCIICLXK and LIKIKRMK (1891) were the first to prepare
lecithin in a pure condition from plants. It was found to
possess the same properties and yield the same decomposi-
tion-products as lecithin from animal tissues. Up to the
present time lecithin has always been supposed to contain a
radical, which gives rise to choline on saponification, as an
essential component, while on the other hand the fatty
acids entering its molecule are well known to be replaceable
by one another. Thus we may have a di-stearine lecithin
as well as a di-oleine lecithin. The existence of several
lecithins in the yelk of eggs has been recognized for some
time, and according to ScHULZK and LiKlKUXlK this is
also true of the lecithins in plants. Recent observations
of LiPi'MAXX (Ber. 20, 3200} show that the above basic
radical, hitherto regarded as constant in lecithin, may pos-
sibly be capable of replacement by other similar radicals,
lie found on saponifying with baryta two different speci-
mens of lecithin, both obtained from beet-root, that while
one of them yielded oleic acid, glycerin, phosphoric acid,
and betaine; the other lecithin gave oleic acid (and some
other fatty acids), glycerin, phosphoric acid, and choline,
with no betaine — at least not in isolable quantity. This
remarkable difference has led LIPPMANN to suggest an ex-
planation which, while it may not be the correct one, never-
thcle— . | •(•-<( 's-i s a high degree of probability. According
to him, the lecithin molecule may contain interchange-
able basic radicals in the same manner that it contains

I'll' BACTERIAL POI8OH8.

interchangeable acid radicals. This view i.s supported not
only in the case of beet-root, where choline and betaine
exist together, but the same two bases have been observed
in cotton-seeds. A similar coexistence was observed in the
toad-stool (Agaricus muscarins), in which choline and inus-
carine were found. And, lastly, the same condition holds
true probably for mytilotoxine and betaine, which were
shown to be present together in poisonous mussels.

Lecithin cannot always be regarded as the source of
choline in plants, since this base is known to occur as a
glucoside in the seeds of white mustard. The sinapin de-
composes according to the equation :

2H20 = C5H15N02 + CnHI205.

SINAPIN. CHOLINE. SINAPIC ACID.

According to SCHULZE (1891) the choline which is iso-
lated from pea- and vetch-seeds exists preformed in the
seeds, and does not result from lecithin by the process of
extraction. This is also probably true with reference to
cottonseed-cake. The condition in which betaine exists is
not determined.

The protoplasm itself is another possible source of choline
as well as of other nitrogenous bases, as xanthine, etc. We
know from DRECHSEL'S brilliant investigation (1890) that
casein on treatment with hydrochloric acid and staunous
chloride yields ammonia, amido acids, and organic bases —
fysatine, C6H13N3O2, and lysatinine, CfiHuN30 — homo-
logues of creatine, C4H9N3O2, and creatinine, C4II7N3O.
From lysatinine urea can be readily obtained by treatment
with baryta. Subsequently, SIEGFRIED (1 891) showed that
vegetable protoplasm (conglutin from lupine) when treated
in the same way yields similar products. Later, SCHUL/E
demonstrated that the base, arginine, C6II14N4O2, is formed
in lupine sprouts at the expense of the proteids present, and
he pointed out that this base is probably related to lysatine,
from which it differs only by NH (see next chapter).

Cll K M 1ST K Y <>F Til K 1'ToM A I N I

I )I:«-OM POSITIONS <>K CIIOUNI:. — I>AI:YI:I; (isiKJ) snc-
led in converting choline into nenrine by a purely
chemical pr<> '—. Tliis was accomplished by heating
choline chloride with concent rated hydriodic acid and
red pbosphonw in a scaled tube at 120°-loO°, whereby
the compound C.JI^NI, was formed. The iod-iodide of
choline thus obtained, OH treatment with moist silver oxide,
gave a base whose platinoehloride corresponded to the
formula (C;,H1,XCl)2l)tCl4 f II,(). This double salt, ac-
cording to DAETEB, is readily soluble in water, and gives
reactions similar to choline. Although BAKYKU is em-
phatic in his assertion that this is the vinyl compound
(nenrinc) formed from the oxy-ethyl base (choline), yet it
-reins that there is room for doubt in regard to the
interpretation of his results. Thus nenrine platinochloride
is dillicnltly soluble in water, contrary to the l>ehavior of
the platinochloride obtained by him. On the other hand,
choline platinochloride is easily soluble in water, and it
\\niild seem, therefore, that BAKYKH has not converted
choline into nenrine, but rather has regenerated choline
from its iod-iodide. If such were the case, we would ex-
pect that the iod-iodide of nenrine, C5II,3Nr2, which has
the same composition as the corresponding derivative of
choline, would yield, on treatment with silver oxide, the
o\v-ethyl base. BAKVKI: has apparently not been able to
effect this change, since he holds that the vinyl base may
IHJ prepared from the oxy-ethyl, but that the reverse, the
preparation of the oxy-ethyl base from the vinyl compound,
cannot be accomplished.

Whether the change descril>ed by BAKYKI: takes place or
not, it is, nevertheless, certain that choline docs m>t readily
give up a molecule of water and thus become converted
into nenrine. Cn.diiAM announced, in 1886, that choline
chloride and lactate on heating on the water-bath de-
compose, and that this conversion into the vinyl base was
complete when the aqneons hydrochloric acid solution of
choline platinoehloride \vas heated ibr five or six hours on
the water-bath. In this way <H:AM endeavored to explain
the formation as due to the action of acids upon choline,

244 BACTKK 1 A I, I'OISONS.

but BRIEQEB has shown that the platinum salt of choline,
as well as its hydrochloride, can be heated with fifteen or
thirty per cent., or even concentrated, hydrochloric acid for
six or eight hours without undergoing any change into
neurine, thus disproving the results obtained by GUAM.
E. SCHMIDT has confirmed BUIEGEK'S observations in
regard to the resistance of choliue to decomposition by
acids, but he has gone further, and has shown that what
the action of acids has failed to do is readily accomplished
through the agency of bacteria. He found that choline
chloride, when allowed to stand with hay infusion, or with
dilute blood for fourteen days at 30°-35°, it almost entirely
decomposed, yielding large quantities of trimethylamine
and a base, the platinochloride of which resembles in form
and solubility the double salt of neurine, and possesses a
similar physiological action. Choline lactate in hay infu--
sion developed an odor of trimethylamiue in twelve hours,
but at the end of fourteen days a good deal of choline was
still present. In this case no neurine was present, but
instead a homologous base was found, which can be obtained
synthetically by the action of trimethylamine on allyl
bromide. According to MEYER, of Marburg, this base
does not possess the muscariue-like action of uetirine, but
resembles more closely pilocarpine.

BUIEGER (I., 59) had unsuccessfully tried to transform
choline into neurine by putrefaction. He observed that the
choline decomposed with extreme slowness, even when the
putrefaction was carried on at a higher temperature, yield-
ing only trimethylamine. WURTZ (1868) showed that
dilute solutions of free choline can be heated to boiling
without any perceptible decomposition. Concentrated
solutions, however, decompose with the formation of tri-
methylamine and glycol, C2H4(OH)2 (see page 190). The
decomposition of choline was studied somewhat by
MAUTHNEU (1873), who confirmed WUUTZ'S observation
that choline was scarcely decomposed by boiling water, and
he showed that when exposed to the action of decomposing
blood it yielded trimethylamine. The results obtained by
K. HASEBKOEK (Zeitschrift f. Physiol Chem., 12, 151, 1888)

.' II K M ISTK V oF TI1K !' TO M A 1 N K S . '_' 1 •")

deserve special mention at this place, lie carried on the
putrefaction of very dilute solutions of the chloride of
eholine in the presence of little or no oxygen in HoiM'K-
SI.VI.KI: fermentation flasks. Sewer -lime, beeau-e of it-
strong fermentative properties, was used to induce the
putrefaction, and calcium carbonate was added to neu-
trali/e any acidity that might develop during the fermen-
tation.

The fermentation, as shown by the evolution of gases,
lasted fi»i- about three months. The total quantity of gas
given oft' was about one litre from 1.17 grammes eholine
chloride. The gases consisted almost entirely of carbonic
acid and marsh gas. No hydrogen was evolved. When
the fermentation ceased the flask was opened and several
cubic centimetres of the almost neutral clear liquid were
injected under the skin of a rabbit without producing the
least ell'ect.

This liquid distilled with alkali gave methylamine
and ammonia. What is remarkable about this experiment
was the total absence of the higher amines — as, for instance,
trimethvlamine, which has been observed so many times as
a decomposition-product of eholine. The absence of any
poisonous base, as neurine, was probably largely connected
with the absence of oxygen.

Free eholine ordinarily forms a strongly alkaline syrup
which combines readily with acids to form salts, most of
which are deliquescent, liy oxidation it is converted into
bctaine (see pa-e %J4!»), and on treatment with concentrated
nitric acid it give- ri-e to muscarine (see page 2">1). The-<-
reactions can be represented by the equations :

o

ril.OII (' ()I,

CHa +<>.. MI, +H30.

N(CH3)3.OH N(CII3)3.oil

Clll. I. INK. Ill I MNK.

246 BACTERIAL POISONS.

CH2OH CII.OH

CII2 + 0 * CHOII

N(CH3)3.OH N(CII,

Mrs< ACINI:.

By the action of dilute nitric acid clioline is converted
into a base the platinochloride of which is efflorescent and
corresponds to the formula (C4H10N.p3Cl)PtCl4 + 2II,( )

(SCHMIEDEBEBO and HARNACK).

According to MAUTIINER, choline resembles the caustic
alkalies in its action. Although putrefying blood decom-
poses it into trimethylamine, yet, when present in the pro-
portion of 1.4 per cent., it is said to arrest putrefaction.
A 1 to 2 per cent, solution is said to dissolve fibrin or
coagulated albumin on boiling.

The free base, as well as the carbonate, is dimorphous
and forms thin plates or long needles.

The CHLORIDE, C5III4NO.C1, is easily soluble in water
and in absolute alcohol (separation from ueuridine hydro-
chloride). It crystallizes over sulphuric acid to needles
which readily deliquesce in the air.

The PLATINOCHLORIDE, (C5H14NO.CI)2PtCl4 (Pt =
81.64 per cent.), presents an interesting case of trimorph-
ism. It crystallizes in monoclinic plates (RiNNE) which
are easily soluble in water, insoluble in alcohol ; also in
characteristic superposed plates, sometimes in the form of
orange-red flat prisms (BRIEGEK). From a warm saturated
solution containing 15 per cent, alcohol it crystallizes in
yellow regular octahedra containing one molecule of water
of crystallization (JAIINS); from aqueous solution on slow
evaporation it forms plates, clinorhombic prisms, or needles
(HOPPE-SEYLER) which are anhydrous. When rapidly
crystallized it forms prisms (IIuNDESHAGEN, JAII.NS,
SCHULZE) ; and if the solution is concentrated the prisms
are very thin, almost needles. According to SCIIULXE, it
sometimes forms beautiful orange-red, chiefly six-sided
plates. JAIINS maintains that the plates and prisms be-

CIIKMISTKV «>K TIIK PTOMAINES. -IT

long to the same system ; while 1 1 iM»i:sn A<;I:N holds that
they arc distinct. Instead of the salt presenting an in-
-laiiee of triinorphisni as fust stated by IhM>i:sii.\(;i:N, it
would seem that but two Conns occur — anhydrous mono-
clinic and octahedra with one molecule of water of crystal-
li/ation. ft contains always more or less water of crystal-
li/.ation which it does not give up completely over sulphuric
acid, but only at 1 10° (BuiKUKU). The natural platino-
chloride becomes strongly electric on rubbing, whereas the
synthetic choline double salt docs not become electric. It
melts at 2-2">° with ellervescciice (.I.viixs).

Tile Ari:orllI,<>mi>K, C.H^NO.Cl.AuCl, (An =44. IX
pel1 cent.), i* crystalline and is difficultly soluble in cold
water, but can be recrystalli/ed from hot water or from
boiling alcohol. It form* prisms, or gold-yellow long
needles, which are very easily soluble in hot water and
alcohol (LiiM'MANN). It can be separated from neuridine
aurochloride by its solubility in water (BBIBQBB). On
heating, the gold salt melts to a brown liquid (SciIUL/lo)
and decomposes at 264°.

The Mi;i:<Ti:nriiLoi;ii>K, CsH14NO.C1.6IIgCl2, is ex-
trcmely dilliculty soluble even in hot water. On this
account the mercury salt is very convenient for the separa-
tion of choline from accompanying bases.

The Pirn.vrK, r,H,4NO.OC6H2(NO2)s, forms long, broad
needles which are more easily soluble than neuridine picrate,
and hence can be separated by reorystalli/ation. It is more
easily soluble in alcohol than in water.

F%y§ioloffical A<-/i<>n of C/i<>lin<: — Choline was regardeil
for a long time as physiologically inert, but this belief
was set aside by ( J.\ KHT<;KNS (1870), who showed that,
when given in large quantity, it po—eoed a toxic action.
This observation of (i.\ I:H r«;i:.\s has since .been con-
firmed by ({LATSK and LUCIISIMJKI:, JiitiKCKi:, and
r>oi:iiM. The chloride of choline produces in animals the
same muscarme-like symptoms of poisoning as are devel-
oped by the vinyl base neurine, the only difference lies in
the intensity of the action. In order to bring about a
physiological disturbance, choline must be given in rela-

24S BACTERIAL i'OJSONS.

lively large doses. Thus, BUIK<;KU found it necessary to
give about 0.1 gramme of choliue chloride hypodermi-
eally to a one kilogramme rabbit in order to bring out the
same effects as are obtained by the injection of 0.005
gramme of the neurine salt. He also found that the
fatal dose for a one-kilogramme rabbit was about 0.5
gramme, which is about ten times as large as the fatal
dose of neurine chloride. BOEHM observed that doses of
0.025-0.1 gramme produced in frogs general paralysis,
which, in a short time, leads to death or recovery ; and
that in its curare-like paralyzing action, choline resembles
artificial muscarine, although the latter is about five
hundred times stronger. Atropine, as in the case of
neurine and muscarine, antagonizes the action of choline.
Thus, 0.05 gramme of the chloride produced in a frog in
one hour diastolic stoppage of the heart. This condition
was removed by the injection of 0001 gramme of atropine,
the heart-beat rising to the normal in about fourteen min-
utes; 0.05 gramme of choline chloride, given subcutaue-
ously to a rabbit (1250 grammes) produced salivation, which
lasted but a short time, and did not affect the heart-beat
and respiration ; 0.10 gramme was necessary to bring out
all the symptoms; 0.06 gramme, given to guinea-pigs, had
no effect whatever.

BETAINE (OXYNEURINE), C5H13NO3. — This base has
been well known for some time, because of its occurrence
in the vegetable kingdom. Thus, it is present in cotton-
seed (BOEHM, RiTTJiAusENand WEGEK); in beet- root juice
(Beta vulgaris), and hence in beet-root molasses (Scm:i-
HLEit, 1866). It occurs also in cattle-turnip and Lycium
barbarum ; and is found with choline and another base in
vetch-seeds; in peas a base similar to betaine exists
(ScHUL/E). With choline it occurs in Scopolia atropoides
(SIEBERT). It does not exist in these substances as such,
but is formed from a more complex substance by the action
of hydrochloric acid or baryta (LIEBREICH). In this respect
it resembles choline, neurine, and probably muscarine.
Quite recently, LIPPMANN (1887) has obtained a lecithin-

CHKMISIKY <>!•' TlIK PTOMAINES. 249

liUr hodv from su^ar-lwet, which, on heating with baryta
gave oleic acid, glycerin, and phosphoric acid (glyccrin-
phosphoric acid), and l>etaine. Kctaine, however, docs not
sec in to he a constant constituent, inasinncli as on one occa-
sion he obtained chielly cholinc, and little or no bctaine.
These two bases also occur together in cotton-seed, and
this fact has led SniKiHLKU to the conclusion that it is
no mere chance. Lecithin, as is well known, may con-
tain variable acid constituents (oleic, stearic, palmitic, etc.),
and reasoning on this fact, and on the results of his ex|>eri-
incnts, Lii'i'MANN has been led to suppose that it may also
contain different bases in variables proportions.

It has been obtained from human urine (LiKiiUKini,
!*<!!»), and from poisonous and non-poisonons mussel, but
not from putrid mussel (Huii-XSKU, 1885, III., 76). The
method for its separation from mussel is described on page
256.

Betaine may be obtained synthetically in several ways :

(1) by oxidation of choline with potassium permanganate;

(2) by the action of methyl iodide on glycocoll ; ('i) by
treating inonocliloraeetic acid with trimethylamine. The
last two methods are of value as indicating the constitution
of bctaine, and the changes which take place can l>e repre-
sented by the ei | nations:

Nil, N(CIT3):tI

CII2 + 3CII.1 Cir, + 2III.

co2n r<ui

I.I 1.. . .1 I. I'.l I MM lolHllR.

CIU'I

+ N(CIIS)S CH,

i

MuMM-ltl.nK \I-ITII- ACID.

l''rom the formulae of the salts of belaine it is evident

250 BACTERIAL POTSOXS.

that bctaine lias properly the composition ( ',.1 1,.{Nr( ).{, which
is expressed by the structural formula :

N(CH3)3<>II
CII2

CO2II.

The free base is, however, readily converted into the
anhydride, C5H,,NO2, tri methyl glycocoll ; the structural
formula of which is :

CH2-N(CII3)3

CO— O.

Betaine is ordinarily regarded as crystallizing with one
molecule of water, and the composition is expressed by the
formula: C5HUNO2+H2O (= OH.N(CII3)3.CII2.CO2H).
It loses this water of crystallization by heating at 100°, or
on standing over sulphuric acid, forming an anhydride of
the formula already given. LIKBRKICH claims that free
betaine possesses the formula C5HnNO2, because it yields a
compound having the composition (C5HuNO2)ZnCl2. The
free base separates from alcohol in large crystals which deli-
quesce on exposure to the air. As obtained by BBIEGEB
from the hydrochloride by treatment with moist silver
oxide, it possessed a sweetish taste and neutral reaction.
When distilled with potassium hydrate, it yields trimethyl-
amine and other bases, among which a base of the formula
C8II,7N05 occurs in the largest quantity.

The CHLORIDE, C5H12NO2.C1, forms beautiful crystals,
monoclinic plates, which are permanent in the air, and this
can be made use of to effect a separation from the choline
salt, which is deliquescent. It is insoluble in absolute
alcohol. This fact can be made use of in their separation
(LIPPMANN). It can, moreover, be easily separated from
other bases by its aurochloride, which is easily soluble. If
a little potassio-mercuric iodide is added to a solution of
the chloride, there forms a light-yellow or whitish oily

CIIKMISTKY <>F THE I' T < > M Al \ KS . 2.r)l

precipitate, which is soluble in 6X0618, Imt on rubbing the
sides of tin- tiilx- with a gla-ss rod it reappears as yellow
needles. This is said to IK? a characteric test (BuiK<;i:u,

Th«- An.-.K III.OIMI.K, C5II12NO,.Cl.AuC]3 (An = 4:5. IL>
per cent.), forms magnificent cholesterin-like plates, and is
easilv soluble (HuiK(JKlj). The aurochloride from sngar-
beet is said to crystallize in needles or plates, and to l>e
difficultly soluble in cold water (SCHKIHLKK, LIIM'.MANN).
The double salt of the ptomaine melts at 209°, and in this
it coincides with that obtained from beet-sugar, as well as
with that of the synthetically prepared base (BRIBGBfi).
The platinochloride is yellow and crystalline.

]Setaine is not poisonous.

MUSCAKINE, CJIISNO8 = CJIjsNO, + 11,0, the well-
known toxic principle which SOBMOEUBBBBG obtaiiunl
from poisonous mushroom (Agaricus muscarius), has been
obtained also by BKIKGER in 1885 (I., 48) from haddock
which had been allowed to decompose for five days. The
process by which its isolation was effected is described on
page 258. This base is specially interesting, because of the
relation it bears to choline, for DCHMIEDEBEBG has shown
that it is formed when choline, or, better still, the platino-
chloride, is oxidized by concentrated nitric acid. It is
barely possible that BiUKCKif's base is distinct from
BCHMIEDEBEBG'S j nevertheless, it closely resembles it and
apparently is identical.

The CHLORIDE, C5IIUNO2.C1, is obtained on the decom-
position of the platinoehloride with hydrogen sulphide, as
a syrupy residue, which, under the desiccator, shows a
tendency to gradually crystalli/e. It is deliquescent
(HAUNACK).

The PLATINOCHLOBIDB, ((\HuNO4.CI),PtCl4 (Pt =

:>().( >s IMT cent.), forms as a crystalline deposit of octahedra,
which are difficultly soluble in water. They lose their
water of crystalli/ation (2II./)) only by means of strong
heating.

The AIM:O< iu.m;ii>K, (16II1,N()..(1l.Aii('l3. erystalli/es

252 BACTERIAL POISONS.

in needles, and is difficultly soluble in water, more so than
the choline double salt (HARNACK).

Physiological Action. — Small doses of this ptoma'inc
induce in frogs total paralysis, with stoppage of the heart
in diastole, and this action is antagonized by subsequent
injection of atropine, as well as in the case of previously
atropinixed frogs. Very small doses produce in rabbits
profuse salivation and laehrymation, contraction of the
pupil, profuse diarrhoea, and passage of urine and semen;
finally, the animal dies in convulsions, which, however, are
only of short duration.

CONSTITUTION OF THE MEMBERS OF THE CHOLINE
GROUP. — The structure of choline was clearly demon-
strated by WURTX, who accomplished the synthesis of this
base by treatment of ethylene chlorhydrine with trimethyl-
amine. This same method can be applied to the synthe-is
of betaine and ueuriue by using monochloracetic acid and
vinylbromide instead of ethylene chlorhydrine. The struc-
tural formula? which can be deduced from these reactions
arc as follows :

CILPH CII2 CO2H CII2OH

I II I I

CH2 CH OH, CHOII

N(CH3)3.OII N(CH3)3.OH N(CH3)3.OH N(CH3)3.OH

CIIOUNE. NEUBINE. BETAINE. MITSCARINK.

The formula; of betaine and muscarine are ordinarily given
as the anhydrides, but there can be no doubt that the free
bases possess the structure indicated above. All these bases,
since they can be prepared from choline, may also be con-
sidered as oxidation-products of trimethyl-ethyl-ammonium
hydrate :

CII3

CH2

N(CII3)S.OII.

CII KM ISTKY «»F TIIK I' To M A I X KS .

&f YDATOXINE, CgHyNQ,, — This base was obtained by
I!I:IK<JKU iii 188G (III., 25, 32) from several hundred

pounds of human internal organs which were allowed to
stand in closed but spacious wooden l»arrels for four moutli>,
at a temperature varying from — 9° to +5°. He obtained
much larger quantities of it, however, from horseflesh which
had putrefied under the same conditions. In the process
of extraction it is found in the mercuric chloride precipitate
together with cadaverine, putrescine, and another base,
( '7H17N()r It can be isolated from this mixture by recrys-
tallizing the mercury salts, which removes the cadaverine,
because of its difficult solubility in water, and decomposing
the soluble mercury salts by hydrogen sulphide. The
filtrate freed from mercury is now evaporated to dryness
and the residue repeatedly extracted with absolute alcohol,
in order to remove putrescine hydrochloride, which is
insoluble. The alcoholic solution, after standing some time
to permit complete separation of any dissolved putrescine,
is then evaporated to dryness and taken up with water.
This solution gives, on the addition of gold chloride, a pre-
cipitate of the aurochloride of the base C7H17XO2. The
filtrate from this precipitate, containing the mydatoxine, is
treated with hydrogen sulphide to remove the gold, and
then evaporated to dryness. The colorless, syrupy hydro-
chloride thus obtained forms with platinum chloride a
double salt which is readily soluble in water, and can be
purified by repeated recrystallixations from absolute alcohol
containing some hydrochloric acid.

The name mydatoxine is derived from /mt,;w, to putrefy.
The free base w obtained from the hydrochloride by treat-
ment with moist, freshly precipitated silver oxide, as a
strongly alkaline syrup, which solidifies in vacuo to plates.
It is insoluble in alcohol, ether, etc. It does not distil
without decomposition. It is isomeric with the base,
r,H,3NO2, obtained by HKIKCKH in 1888 from tetanus
cultures.

The HVDIKK III.OUIDK, ('6II13NO2.HCI, is a colorless,
deliquescent syrup which does not form any double salt
with gold chloride. With platinum chloride it gives an

12

2f>4 BACTERIAL POISONS.

easily soluble salt. Otherwise it combines only with phos-
phomolybdic acid, with which it forms cubes. Ferric
chloride and potassium ferricyanidc yield, after a time,
Berlin-blue. It is readily soluble in alcohol.

The PLATINOCHLORIDE, (CflH13NO2.IICI)2PtCl4 (Pt -
29.00 per cent.), melts at 19.'J°, with decomposition. It
crystallizes in plates which are extremely soluble in water.
It can be readily recrystallized from absolute alcohol acidu-
lated with hydrochloric acid. The mercury salt is readily
soluble in water.

The exact formula of this base, of mytilotoxine, and
some other bases, cannot be considered to be permanently
settled, inasmuch as the formula of the hydrochloride,
C6H13NO2.IIC1, as deduced from the analysis of the
platinum double salt, may equally apply to the base
CCH14NO?.OH as to the base GVU13NO2. If the first
formula is correct, then mydatoxiuc is a homologue of
betaine, and its structure would be expressed by (1).

(2)
C^°

| MI
cn2 on

I II

OIL CH

N(CH3)3OII N(CII3)3OII.

The second formula would seem to correspond to an un-
saturated aldehyde of the choline group and its structure
may be indicated by (2).

This ptomaine, although it possesses toxic properties, is
not, however, a strong poison. Its action is the same as that
of the base C7H17NO2 (see page 262), with which it is associ-
ated, except that the symptoms of poisoning develop slower,
so that the death of a guinea-pig does not take place for
about twelve hours. White mice are very susceptible to the
action of these two poisons. A short time after the injec-
tion of even small doses they are taken with convulsions

en i: M is r i: v <>K TIN-: I>T<>MAINKS.

which r.imi' on in paroxysms. The eyeballs roll upward.
Laehnrmatioo, <liarrh<i'a, and dyspmea come on, and the
mice die within a short time.

A HASK (?), C61I,SNO2, an isomer of the preceding,
ol)(aiiKHl by BRIBQEB in 1888 from tetanus cultures. It
is not |)ois(»iKnis — distinction from mydatoxine. It proba-
l»ly is an amido-acid. The platinocldoride crystal li/cs in
plates, is easily soluble in water and in alcohol, and melts
at 1!>7° with decomposition (sec page 267).

M YTII,<»TOXINK, C6H15NO2, is the specific poison of toxic
mussel (Mytilns edulis), from which it was obtained by
HiMMiKU in 1885(111., 70). This poison is formed during
the life of the animal under certain conditions which have
been thoroughly studied by SciiMiDTMANN, Vmciiow, and
others (see p. 40). BRIBQEB obtained the poison by extract-
ing toxic mussel with acidulous water, and evaporating this
solution to a syrupy consistency. The residue was thor-
oughly extracted with alcohol, and this solution was treated
with lead acetate, in order to remove mucilaginous sub-
stances. The filtrate was then evaporated, and the residue
extracted with alcohol. Any lead that had dissolved was
removed by hydrogen sulphide. The alcohol was expelled,
and the resulting syrup was taken up with water and
decolored by boiling with animal charcoal. The clear solu-
tion was now neutralized with sodium carbonate, acidulated
with nitric acid, and precipitated with phosphomolybdic
acid. The precipitate was decomposed by warming with
neutral lead acetate, and the resulting filtrate, after the
removal of the lead by hydrogen sulphide, was acidulated
with hydrochloric acid and evaporated to dry ness. The
residue was extracted with absolute alcohol, whereby
betaine, on account of its insolubility, is removed, and the
alcoholic solution was precipitated by alcoholic mercuric
chloride. The mercury precipitate is repeatedly recrystal-
li/ed from water, and the poison is obtained as an easily
soluble double salt.

The free base as obtained by the addition of alkali to

256 BACTERIAL POISONS.

the hydrochloride possesses a disagreeable odor which dis-
appears on exposure to air, and the substance ceases to pos-
sess poisonous properties. BRIEGER has proposal the
application of this test for the recognition of poisonous
mussel ; on treatment of these with alkali the characteristic
odor is developed. Mytilotoxine is also destroyed on dis-
tillation with potassium hydrate and in the distillate there
is found an aromatic non-poisonous product and trimethyl-
amine. The free base, therefore, does not exist by itself
for any length of time, but soon becomes converted into an
inert substance. H. SALKOWSKI has also shown that it is
destroyed on boiling with potassium carbonate, whereas
its hydrochloric acid solution can be evaporated to dry-
ness and heated to 110° without destroying its poisonous
property.

The HYDROCHLORIDE, C6H15NO2.HC1, prepared from
the aurochloride, crystallizes in tetrahedra. It is extremely
poisonous and according to BRIEGER produces exactly the
same symptoms which have been observed by SCHMIDT-
MANN in persons who have partaken of poisonous mussels
(see page 38). On standing, however, the pure hydro-
chloride gradually becomes dark and decomposes with loss
of its poisonous property — a change corresponding to that
which tetaniue undergoes (p. 267). The gold salt is better
adapted for preservation. The ordinary alkaloidal reagents
produce in its solutions, if at all, only oily precipitates.

As stated under mydatoxine, the formula of the hydro-
chloride, C6H15NO2 HC1, is applicable to either one of two
bases, C6H16NO2.OH or C6H15NO2. The base correspond-
ing to the first formula is evidently a homologue of mus-
carine, and should possess a similar physiological action.
As a matter of fact, mytilotoxine does resemble muscarine
somewhat in its action, and its occurrence together with
betaine would seem to make it a decomposition -product of
lecithin, in which case this base must be looked upon as a
member of the choline group. It is interesting to know
that a compound corresponding tothe formula C6H16NO2.OII
has been known for some time, and was prepared by HAN-
RIOT in a manner analogous to WURTZ'S synthesis of

Til KM 1ST I: V Ol I II K 1'TOM A I N 1

cliulinc, by treating glycerin mooochlorhydrine with tri-
mcthylaniine. This base, triinetliyl-glyceryl-ammonium
hydrate., has this structure:

CIL01I
CIKHI

N(CII3)301I.

It would- seem that HANRIOT'S base might possibly be
identical with mytilotoxine, but u careful comparison made
by I5i:ii-:<;KU showed that it possesses no physiological action
and that its chemical relictions are entirely different.

Mytilotoxine would, therefore, seem to possess the for-
mula, ( 10II15NO.2, as originally given it by BRIEGER. From
the fact that on distillation with potassium hydrate it yields
trimethylamine, it follows that mytilotoxiue is a quarter-
nary base. He is inclined to regard it as a methyl deriva-
tive of betainc, which is so common in mussels, and repre-
sents it by formula No. 1.

(1) (2)

00,11 CH,OII

m.cir,

N(rH3),<

.011 N(cii3)roii

No. 1, however, is C6H15N03, instead of C61I15N(),, as
above. The formula No. 2, (J6II17N(),, would represent
a derivative of choline or muscarinc, with only a slightly
higher percentage of hydrogen.

The AUROCJILORIDK, C6II13NO.rnCl.AuCl3(Au- 41.6(>
l>er cent.), crystallizes in cubes. Its melting-point is 18'2°.

It is well to observe that BUIK<JKU has been unable to
obtain this base from mussels that were allowed to putrefy
for sixteen days.

Physiological Action. — According to KIMKCKIJ, mytilo-
toxine produces all the characteristic effects seen in mussel

258 BACTERIAL POISONS.

poisoning, and it is, therefore, a strong paralysis-producing
poison, and resembles curara in its action. This action is
explainable now that CLAUSE and LUCHSJNGER have
shown that all trimethyl-ammonium bases have a musca-
rine-like action. For the symptoms induced by poisonous
mussel see page 38.

GADININE, C7H,7NO2, was found in haddock (1885)
which was allowed to decompose in open iron vessels for
five days during summer. BRIEGER has also obtained it
from cultures of the bacteria of human feces on gelatin.
The decomposing mass was thoroughly stirred every day
in order to bring it into contact with atmospheric oxygen
(BRIEGER, I., 49). It was then treated with water, and
hydrochloric acid was added to acid reaction, and after being
warmed the mixture was filtered and the filtrate concen-
trated on the water-bath to a syrupy consistency. This
syrupy residue was extracted with water, and the aqueous
solution was precipitated with a solution of mercuric chlo-
ride. The mercuric chloride precipitate contained a base,
the quantity of which, however, was insufficient for a com-
plete analysis (see page 272). The mercuric chloride filtrate,
after the removal of the mercury by hydrogen sulphide,
was evaporated to a syrup, and this was then repeatedly
extracted with alcohol. The alcoholic solution thus ob-
tained contained neuridine, a base of the same composition
as ethylenediamiue, muscarine, gadinine, and tricthylamine.
These bases were separated in the following manner : The
alcoholic solution gave with platinum chloride a precipitate
of neuridine. The filtrate from this platinum precipitate
was heated on the water-bath to expel the alcohol, and then
the platinum was removed by hydrogen sulphide. The
aqueous filtrate was concentrated to a small volume which,
on addition of platinum chloride, gave a precipitate of the
isorner of ethylenediamiue. The mother-liquor from this
precipitate was concentrated on a water-bath, and on cool-
ing the platinochloride of muscarine crystallized out. From
the mother-liquor of this precipitate on standing in a des-
iccator, the gadinine double salt crystallized. The mother-

CII KM ISTKV <>F TIIK I'TOMAIXKS. 259

liquor from (lie gadininc platinoohlorkfa was treated with
hydrogen sulphide to remove the |ilatinuni, and the aque-
ous filtrate on distillation with potassium hydrate gave
triethylamine.

(iadininc (from (iadiis callarias, haddock) in small doses
does not appear to he poisonous; larger doses (0.5-1 gramme)
are decidedly toxic and may kill guinea-pigs. The formula
of the free IKISC as deduced from the analysis of the platino-
chloride may !><• either C7H!7N(), or C7II18NO, OIL

The IIvi)i;ocML<)i:ii)K, C-H^NO^.IICI, as obtained by
the decomposition of the platinochloride with hydrogen
sulphide, erystalli/es under the desiccator in thick, colorless
needles, which are easily soluble in water; insoluble in
alcohol. It forms no combination with gold chloride, but
does give crystalline precipitates with phosphomolybdie
acid, phosphotungstic acid, and picric acid.

The PLATINOCHLORIDE, (C7H17N()2.HCl),PtCl4 (Pt =
27.68 percent.), is at first quite soluble, and ou standing over
a desiccator it crystallizes in golden-yellow plates, which,
when once formed, are again difficultly soluble in water.
It can be recrystallized from hot water. It melts at 214°.

TYPHOTOXINE, C7n,7NO2. — This base was named thus
by JiiMKtJKi: in 1885 (III., 86), and is regarded by him as
the specific toxic product of the activity of KocH-E BERTH'S
typhoid bacillus. It is, however, probable that, as in the
OM6 <»f tetanus, there are basic and other products formed,
lie obtained it by cultivating the bacillus on beef-broth for
eight to fourteen days at the temperature .'>7.5— .'JS°. The
nature of the soil on which it grows has a great deal to do
with the formation of the poison. An especially important
factor is the temperature: for BI;IE<;EI: has observed that
no poison was produced in one case where the temperature
remained by accident at .'i9° for twenty-four hours. In
such cn-e- creatiiie is present in quantity, whereas otherwise
the reverse is the rule.

In the process of extraction it occurs in the mercuric
chloride precipitate, and from this it is obtained, after the
removal of the mercury by hydrogen sulphide, as an easily

260 BACTERIAL POISONS.

deliquescent hydrochloride. This for the purpose of puri-
fication is converted into the difficultly soluble aurochloride.

Typhotoxine is isotneric with gadinine and the compound
C-H17N02, which BHIEGEU obtained from putrefying horse-
flesh. In its properties it is, however, very different.
Thus, the free base is strongly alkaline and its hydrochloride
yields a difficultly soluble picrate. On the other hand, the
isomer from horseflesh possesses a slightly acid reaction,
and does not form a picrate. Again, typhotoxine gives
with EIIRLICH'S reagent (sulpho-diazobenzole) an imme-
diate yellow color, which disappears upon the addition of
alkali, whereas the isomer does* not give this reaction.
Furthermore, the two bases differ in their physiological
action and in their behavior to alkaloidal reagents (see
Table I.). Their aurochlorides, however, possess the same
melting-point.

The HYDROCHLORIDE is readily deliquescent, and unites
with platinum chloride to form an easily soluble double
salt crystallizing in needles.

The AUROCHLORIDE, C7H17NO2.HC1. Au013 (Au = 40.46
per cent.), is difficultly soluble, and crystallizes in prisms,
which melt at 176°. In its melting-point and solubility
(197°, BRIEGER, Arch. f. pathol Anat., 115, 489) it
agrees with its isomer from horseflesh. From some of his
first experiments in the cultivation of the typhoid bacillus,
BRTEGEII (II., 69) obtained a basic product differing in
some of its characters from typhotoxine. Its aurochloride,
on analysis, gave 41.91 and 41.97 per cent, of Au, 16.06
per cent, of C, and 3.66 per cent, of H. ; while typho-
toxine aurochloride gave 40.78 per cent. Au, 17.38 per
cent. C, and 3.85 per cent. H. For a comparison of the
reaction of these two substances see Table I.

In its physiological action, typhotoxine differs from its
isomer (page 262) in that the latter produces symptoms
with well-marked convulsions, whilst the former throws
the animal into more of a paralytic or lethargic condition.
The action of this base has been studied only on mice and
guinea-pigs. It produces at first slight salivation with
increased respiration ; the animals lose control over the

CIIKM1STUY OF 'I' UK PT< » M A I N K S . 261

muscles of the trunk and extremities, and fall down help-
less upon their sides. The pupils become strongly dilated,
and cease to react to light ; the salivation lx.'comes more
profuse; the rate of heart -!>eat and of respiration gradually
decreases, and death follows in from one to two days.
Throughout the course of these symptoms the animals
have frequent diarrlxeie evacuations, l>nt at no time are
convulsions present. On post-mortem, the heart is found
to be iu systole, the lungs are strongly hypenemie, the
other internal organs pale, the intestines firmly contracted,
and their walls pale.

A P>ASE(?), C7H17NO2, was obtained by BICIKUKI! in 188«
(III., '28) on working over about one hundred pounds of
horseflesh which had been allowed to undergo slow putre-
faction with limited access of air and at a low temperature
( — 9° to + 5°) for four months. It occurs in the mercuric
chloride precipitate together with cadaverinc, putrescine,
and mydatoxinc, and from these bases it can be separated
and isolated according to the method on page 233.

A similar, if not identical substance, having the com-
position ( '.I [,7NO2, was obtained by BAWNSKY and STAIXT-
IIACJKN (1800) from cultures on horseflesh, ten days at 35°,
of a bacillus, closely allied to FixivLKij-I'iMou's, and iso-
lated from stools of cholera infantnm. The gold salt in
crystalline form and properties-is the same as BUIKGKK'S,
except that it possesses a somewhat higher melting-point.

The free substance possesses, even after most careful
purification, a slightly acid reaction. This acidity is
removed from even a large quantity of the substance by
the addition of a drop of alkali. On account of the acid
character of the free substance, I>i;n:<;i:i; does not consider
it to Ixi a base (a ptomaine). It differs, however, from the
amido-aeids in its poisonous character; in the fact that,
unlike an acid, it does not unite with bases to form salts;
and in not giving the characteristic red coloration (II«»K-
MKISTKI.-'S reliction for the amido-acids) with ferric chloride.
Whatever the true nature of this snb.-tance mav lxi, it
nevertheless, in its other properties, behaves like a base.

262 BACTERIAL POISONS.

Thus, it forms simple as well as double salts. On boiling
with copper acetate, it gives amorphous floccules. Under
the desiccator it solidifies into plates which deliquesce on
exposure to the air. It does not combine either with silver
oxide or with cupric hydrate. On dry distillation it yields
a distillate possessing a strong acid reaction and a peculiar
odor. The distillate does not give any precipitate witli
platinum chloride, or with gold chloride, nor does it react
with copper acetate. With phosphomolybdic acid, how-
ever, it forms an amorphous mass ; with ferric chloride and
potassium ferricyauide it yields an immediate precipitate of
Berlin-blue, whereas the original- substance does not give
any blue coloration.

The HYDROCHLORIDE, C7H17NO2.HC1, crystallizes in
fine needles which are insoluble in absolute alcohol. When
its aqueous solution is treated with freshly precipitated
silver oxide, the resulting filtrate contains some silver oxide
in solution, from which it can be removed by hydrogen
sulphide; thus differing from an ammoniacal silver solu-
tion, which gives no precipitate on treatment with hydrogen
sulphide. In this respect it resembles SALKOWSKI'S base,
page 231. For reactions of the hydrochloride, see Table I.

The AUROCHLORIDE, C7H17NO2.HCl.AuCl3, forms
plates which are difficultly soluble in water, and melt at
176° — the melting point of the gold salt of typhotoxine.
It is dimorphous, since sometimes it is also obtained in
needles which can be changed into plates.

It does not form a picrate, nor does it give a reaction
with sulpho-diazobenzole.

Physiological Action. — This substance, when injected
into frogs, produces a curara-like action. A few minutes
after the injection the animal falls into a condition of
paralysis, and, although it can still react toward reflexes, it
cannot move from its place. At times fibrillary twitchiugs
pass over the body. The pupils dilate, the heart-action
becomes gradually weaker, and finally, after several hours
the animal dies, with the heart in diastole. Doses of 0.05 to
0.3 gramme of the hydrochloride, injected into guinea-pigs,
produce in a short time a slight tremor, gradual increase in

rilKMISTUV OF TIIK I'To M A 1 N KS.

re.spiration, and slight moistening of the lower lip. The
pupils at fir-t contract, then dilate ad in<i.r!iiiiini, and become
react ion less. The temperature remains at first normal ;
eliills of short duration follow in rapid succession. The
animal squats on the ground with its snout pressing against
the floor in exactly similar manner as is caused by the
mii-<c| poison. Violent elonic (Convulsions follow iu con-
tinually shorter intervals, and at the same time lachryma-
tion and salivation become profuse, but not as excessive
as in the case of the musearine-like ptoma'ines. The tem-
perature sinks with the decrease in the rate of respiration,
the ears previously gorged become pale and cold, and the
heart-action becomes irregular and less frequent than before.
( leneral paralysis sets in, but the head still moves upward
and backward. External stimuli induce violent elonic
convulsions, the animal repeats frequently choking move-
ments, and at the same time yields large quantities of
saliva; finally, it falls upon its side completely paralyzed,
and dies. The heart stops in diastole, the intestines are
pale and strongly contracted, and the bladder is empty and
likewise contracted.

MORRIIUIC ACID, C9II,.,NO3, was obtained by GAUTIER
and MOCRGUES (1888) from brown cod-liver oil, together
with six bases, already described — namely, butylamine,
amylamine, hexylamine, dihydrolutidine, aselline and mor-
rhuine. These bases constitute about 0.2 per cent, of the
oil. The discoverers regard them as true leucoma'ines,
dissolved from the hepatic cells by the oil. It is more
probable, however, that these compounds are the products
of initial decomposition, and for that reason they are de-
scribed under the head of ptomaines. This compound is
relatively abundant, and is basic as well as acid in charac-
ter. It is resinous in appearance, and can be crystallized
in flattened prisms, or large lance-shaped plates. When
recently precipitated it is oleaginous, viscous, then gradually
hardens. It pOBSeesea a disagreeable aromatic odor re-
sembling that of the sea-weeds, upon which the fish feed.
According to the discoverers its probable source is the

264 BACTEKIAL POISONS.

lecithin derived thus from these weeds. It is soluble in
alcohol, and but slightly in ether. It reddens turmeric,
decomposes carbonates and with acids forms salts which
precipitate lead acetate and silver nitrate, but not copper
acetate, even on warming.

The hydrochloride is crystalline, and is partially disso-
ciated by excess of water. The platinum salt is soluble,
and crystallizes in very small cross-shaped prismatic
needles. The gold salt is amorphous and is readily altered
on heating.

The properties of this compound show that it is of a
pyridine nature, and inasmuch as it does not give a pre-
cipitate with copper acetate, it would apj>ear that the carb-
oxyl is not directly united to the pyridine nucleus. This
does not necessarily follow now that we know that some
amido-acids exists which do not give a reaction with copper
acetate (see page 231). Its pyridine nature is further-
more shown on distillation with lime. An oily alkaline
base is thus obtained which forms an iodomethylate, and
this with potassium hydrate yields quite an intense red
color, resembling lees (DE CONINCK'S reaction). On
oxidation with permanganate of potassium it yields a mono-
basic acid. According to GAUTIER and MOUUGUES the
compound is pVobably identical with DE JUNGII'S gad nine,
and they ascribed to it the following constitution, which,
it should be said, lacks full confirmation :

H
C

S \
HC COH

I II

H2C C— C3II6.CO.,H.
\ /
N
H

Compare with ty rosin, C9HUNO3 (page 197).

"'I! KM ISTK V <>F TIIK 1' '! • • M A I X KS . 265

A BA>K, CsH.jN.p,, was obtained by I'nrrmyr (1884)
from the residual liquors resulting from an industrial treat-
ment of d6bris of bones, lle-h, and waste of all kinds, with
dilute snl|)liurie acid. It is accompanied by another base,
CLHjjNjOg, from whioli it can be separated by treatment
with alcohol. The base itself forms tufts of delicate needles
which alter or deeomjwse less easily than the accompanying
base. The platinoehloride, (C5H,2N/)4.HCl),PtCI4, forms
a dull yellow powder, somewhat soluble in strong alcohol,
luit insoluble in ether. The platinochloride (C7II18N./ )6.
IH'l)3PtCl4 is insoluble in ether.

The hydrochlorides of these bases form silky needles
which are altered by excess of hydrochloric acid and by
exposure to air. PorciiBT considers them to l>e closely
allied to the oxy-betaincs. The general alkaloidal reagents
precipitate these bases; the phosphomolybdic precipitate,
on addition of ammonia, gives a blue tint. Both bases are
toxic, and exert a paralyzing action upon the reflex move-
ments.

The method employed by POUCH ET for their isolation
was to precipitate them as tannates. The precipitate was
decomposed by lead hydrate in the presence of strong
alcohol, the excess of lead removed from the solution by
hydrogen sulphide, and the clear liquid thus obtained was
submitted to dialysis. The above bases occurred in the
dialysate. In the non-dialy/able portion volatile bases
were found probably identical with those described by
GAUTIKK and KTARD.

TKTAXIXK, C^H^N^ )4, was obtained in 1886 by
BllI£QEB (III., 94) by cultivating impure tetanus microbes
of RnsKM'.Arii, in an atmosphere of hydrogen on beef-
broth for eight days at 37°-38°. It likewise occurs in
cultures on brain-broth. Later (April, 1888), BUIKCJKU
succeeded in obtaining tetanine from the amputated arm
of a tetanus patient, identical in its physiological action
and chemical reactions with that isolated from cultures
of KosKxi'.Acn'rt germs on l>eef-broth. The proence of
tetanine during life in tetanus patients has thus been

266 BACTKHIAL POISONS.

demonstrated. It has not been found in the brain and
nerve tissue of persons dead from tetanus. A portion of
the jelly-like muss taken from the amputated arm was
found to contain tetanus baeilli as well as staphyloeocci
and streptococci, and when planted on beef-broth, tetanine
was formed, but no tetanotoxiue or spasmotoxine.

KITASATO and WEYL, (1890), employing pure cultures
of the tetanus bacillus, obtained from 1J kilogramme beef
used as culture medium 1.7118 gramme of tetauiue hydro-
chloride (0.137 per cent.). Tetanotoxine was also present.

For its isolation BKIEGER employed the following
method : The cultures were slightly acidulated with
hydrochloric acid, heated and filtered ; the filtrate was
then treated with lead acetate and with alcoholic mercuric
chloride in the manner described under mytilotoxine (page
255). KITASATO and WEYL digest the cultures with 0.25
per cent, hydrochloric acid for some hours at 60°, then render
slightly alkaline, filter, and distil in vacuo at 60°. The
residue in the retort is worked for tetauiue by BIUEGKU'S
method, while the distillate contains tetanotoxine, ammo-
nia, indol, hydrogen sulphide, phenol and butyric acid.
The filtrate from the above mercuric chloride precipitate
contains the greater part of the active principle, provided
the precipitate has been thoroughly washed. After the
removal of the mercury by hydrogen sulphide, it is evap-
orated and the residue is repeatedly extracted with absolute
alcohol, in which the tetanus poison readily dissolves, and
can thus be separated from the insoluble ammonium
chloride. The alcoholic solution is treated with alcoholic
platinum chloride, which precipitates the ammonium and
creatiuiue platiuochlorides, whilst the platinochloride of the
poison remains in solution. The filtrate from this precipi-
tate gives, on the addition of ether, a flocculent precipitate
possessing exceedingly deliquescent properties. The pre-
cipitate is, therefore, rapidly filtered off by means of a
pump, and dried in yacuo. It can then be recry stall! ml
from hot 96 per cent, alcohol, and the beautiful clear-yellow
plates thus obtained, if dried again in vacuo, become
rather difficultly soluble in water, from which it can then

CM KM I STK V <>K TIIK I' To MA IN I 267

he recrygtalliaed and obtained in a perfectly pure

ti(tn. [f boiled with boneblack it decomposes, yielding a

non-poisonoofl crystalline compound.

I'hosphomolvMie acid cannot he used in tlic separation
of tclanine, inasiniicli as it destroys the poison (BBIBQBfi).
!'><>• K i. is» ii has also observed that it destroys the poison
Conned in the putrefaction of fish.

Tetanine obtained by treating the hydrochloride with
freshly precipitated moist silver oxide forms a strongly
alkaline yellow syrnp. With alkaloidal reagents it gives
the same reactions as the hydroehloride, exeept that it does
not give a bine color with ferric chloride and potassium
ferrieyanide. It is easily decomposed in acid solution, but
is permanent in alkaline solution.

The IlYI'i;o« IILORIDE, C^II^N/ )4.2IICI, is deliques-
cent and is easily soluble in absolute alcohol. Beside with
platinum it combines only with phosphomolybdie acid to
form an easily soluble crystalline precipitate, which on the
addition of ammonium hydrate becomes white. If, how-
ever, the hydroehloride is impure, phosphomolybdic acid
produces a precipitate which is colored an intense blue by
ammonia. Potassium-bismuth iodide yields a precipitate
which is at first amorphous, but soon becomes crystalline.
Ferric chloride and potassium ferricyanide produce a slowly
developing blue color which probably is due to impurities.

When kept for some moutjis the highly poisonous hydro-
chloride becomes syrupy, brownish, and wholly inert.
Kxamined at this stage, the syrup was found, by means of
platinum chloride, to contain a substance the hydroehlo-
ride of which crystallixcd in plates. This is readily soluble
in water and alcohol, and melts at 197°, with total decom-
position, the same as tetanine. It combines only with phos-
phomolylxlic acid to form an easily soluble compound. The
platinum salt has the composition CfiII13NO2.2HCl.PtCI4.
This substance is non-poisonous and probably an amido-
acid. It is different, however, from leiicin and NKVKI'S
isomers of lencin, although possessing the same composi-
tion. It is also isomeric with mydatoxine, r6H13N()2, but
this is highly poisonous to mice, while the former is inert

268 BACTERIAL POISONS.

(see page 255). Tetanine resembles mytilotoxine with
respect to this loss of toxicity on standing.

The PLATIXOCHLORIDE, C.jH^O^HCl.PtCl, (Pt =
28.33 per cent.), is easily soluble in absolute alcohol from
which it is precipitated on the addition of ether. From
ninety-six per cent, alcohol it crystallizes in clear yellow
plates. After repeated recrystallization from alcohol and
drying in vacuo it becomes difficultly soluble in water so
that it can be recrystallized from the latter. It decomposes
at 197°.

This base produces the characteristic, though probably
not all the symptoms of tetanus, since we know of at least
three other toxines (pages 194, 195) which occur with tcta-
nine in cultures of the tetanus microbe. The symptoms
induced by relatively large doses in warm-blooded animals,
as mice, guinea-pigs, and rabbits, exhibit two distinct phases.
In the first, the animal is thrown into a lethargic paralytic
condition, then suddenly becomes uneasy, and the respira-
tion becomes more frequent. This is followed by the second
phase, in which tonic and clonic convulsions, especially the
former, predominate till death results. 0.5 gramme has
but slight action on guinea-pigs. Small doses do not
seem to affect guinea-pigs, while frogs seem to be much
less sensitive than mice. The characteristic convulsions
and opisthotouus seen in tetanus in man are also produced
in guinea-pigs on injection of large doses of this base. Dogs
and horses seem to be but slightly sensitive to the action of
this poison.

A BASE, C/nNjjNjO^ was isolated by GUARESCHI in
1887 from putrid fibrin. It occurs in the chloroform or
ether extracts along with the base CIOHUN, and is probably
an amido-acid (see page 201).

A BASE, C7H,,,N,O6, was isolated by PorriiKT in 1884.
It is said to form short, thick prisms which become brown
wheji exposed to light.

The PLATIXOCHLORIDE, (C7HlsN2O?.HCl)2PtCl4, crys-
tallizes in prismatic needles which are insoluble iu strong

'•II KM IS I li\ OF Til K 1'ToM A I N i

alcohol. For further details in regard to thi< base see
page 2 »')•").

TYI:<>IO\ K ..\ has U-en obtained in poisonous chet-se
( YAI<.II AN. \\ AI.I.A" i., WOLFF), in jwisonous ice-cream
(\"AIP(.M\N. Now. S HI.AUKK, LADD), in poisonous
milk (YAn.iiAN, Now, Ni:\\r«>\. WAI.I.M K, FIKTII.
S< HKAKKK), and in mam-puffs (SrAX'n>.\). The methods
of separating this poison and its (-flirt ujxni animals have
already been given with sufficient detail. Chemically, it is
very instahle. When warmed with water to about 90°, it
deoOBHMMeB. Hydrant sulphide also dec-omjxtses it, there-
fore all attempts to i>olate it by precipitation with some
l»a>«-, >ueh as mercury or lead, and then removing the Iwsc
with hydrogen sulphide, have failed. Its unstable char-
acter is illustrated by the tact that it may disappear
altogether within twenty-four hours from milk rich in the
11 which is allowed to stand iu au open beaker.

With jK)tassinm hydrate it forms a euni|>ound which
agrees in crystalline form, chemic-al reactions, and the |>er
cent, of potassium which it contains, with the compound of
dia/ol>enxole and potassium hydrate. This sul>stance is l>e>t
obtained from milk containing tyrotoxicon as follows : The
filtered milk, which is acid in reaction, is neutrali/ed with
sodium carbonate, agitated with an equal volume of ether,
alloweil to stand in a stoppered glass cylinder for twenty-
four hours, the ether removed, and alloweil to evaporate
-jxintaneonsly from an OJHMI dish. The aqueous residue is
acidified with nitric acid, then treated with an equal volume
of a saturated solution of potassium hydrate, and the whole
concentrated on the water-bath (this compound is not
d« -corniced below 130°). On Ix-ing heated the mixture
MOOflMI yellowish-brown, and emits a peculiar aromatic
odor. ( >n cooling the tyrotoxk-on eom|x>und forms iu
beautiful, six-sided plates along with the prisms of potas-
sium nitrate.

With equal parts of sulphuric and carbolic acids, pure
tyrotoxieon gives a green coloration, but in whey the color
varies from yellow to orange-red. This color reaction may

270 BACTERIAL POISONS.

be used as a preliminary test in examining milk for tyro-
toxicon. It is best carried out as follows : Place on a clean
porcelain surface two or three drops each of pure carbolic
and sulphuric acids. Then add a few drops of the aqueous
solution of the residue left after the spontaneous evapora-
tion of the ether. If tyrotoxicou be present, a yellow to
orange-red coloration will be produced. This test is to
be regarded only as a preliminary one, for the coloration
may be due to the presence of a nitrate or nitrite, or as
HUSTON has shown, to butyric acid. The tyrotoxicon
must be converted into the potassium compound and puri-
fied before the absence of nitrate or nitrite can be positively
demonstrated. Moreover, the physiological test should
always be made in testing for this poison.

With platinum chloride in alcoholic solution tyrotoxicon
forms a compound which explodes with great violence at
the temperature of the water-bath. This also corresponds
with the compound of platinum chloride and diaxobenzole.

Pure tyrotoxicon is insoluble in ether, and its extraction
from alkaline solutions by this solvent is due to the pres-
ence of foreign matter, with which the poison is taken up
by the ether.

The physiological action of this ptomaine has been suf-
ficiently discussed in a preceding chapter.

MYDALEINE (/wfa/^of, putrid) is a poisonous base ob-
tained in 1885 from putrefying cadaveric organs, liver,
spleen, etc. (BuiEGEii, II., 31, 48). Though it is appa-
rently present on about the seventh day, it is unobtainable
until about the third or fourth week. The method for its
separation from the accompanying bases is given under
Sapriue (page 220). It is liable to occur in the mercuric
chloride filtrate, as well as in the precipitate, inasmuch as
the double salt is insoluble only in perfectly absolute alco-
hol. In order to purify the platinochloride obtained as on
page 221, it is repeatedly rcerystalli/ed from a very small
quantity of lukewarm water. This base has not been ob-
tained in sufficient quantity to permit of a complete deter-
mination of its composition. It is probably a diamine,

CHEMISTRY 01 TIIK I'TOMAINKS. L'7 1

containing four or five earlxMi atoms, and hence is nearly
related to some of the diamines already descnlx-d.

Tin- 1'i.A'i INOCIII.IMMDK, on analysis, gave: Pt = 38.74
C = 10.83, II = 3.23. It crystallizes in small needles, and
i- extremely soluble in water.

The HTDBOCHLORIDE crystallixes with extreme dif-
ficulty, even nn standing fin- some time in a desiccator. On
exposure to the air it rapidly deliquesces.

riiifxiofo</i<-<tf Action. — Mydaleine has an entirely specific
action. Small (juantities injected into guinea-pigs or
rabbits produce, after a short time, a moistening of the
under lip, and an abundant flow of secretion from the nose
and eyes. The pupils dilate gradually to maximum, and
become react ion less ; the ear vessels become strongly in-
jected, and the body temperature rises 1° to 2°. The hairs
bristle, and the animal occasionally shudders. Gradually
the salivation ceases, the respiration and heart-action, which
were at first hastened, now decrease, the temperature falls,
the ears become pale, and the animal finally recovers.
During the action of the poison the animal shows a ten-
dency to sleep, and the peristaltic action of the intestines is
heightened. Larger doses (0.050 gramme) induce an ex-
ceedingly violent action, which invariably results in the
death of the animal. On post-mortem, the heart is found
to be stopped in diastole, and the intestines and bladder
contracted ; otherwise nothing abnormal is observed.

A Toxic BASK. — From human livers and spleens which
were decomposing for two weeks in thorough contact with
air there was isolated, besides cadaverine and putrescine, a
small quantity of a poisonous base (]»I:IK<;KI!, II., 251, IS).
The mercuric chloride precipitate was decomposed, and the
hydroclilorides were precipitated by gold chloride (to re-
move cadaverine, which is soluble), and the aurochloride
was then changed into the platinum salt, whereby the ill-
soluble putrescine platinochloride was removed. In the
mother-li(|uors from the putrescine salt an easily soluble
platinum compound was separated, and found to contain
41.30 per cent. Pt. It crystallized in fine needles. The

272 BACTERIAL POISONS.

hydrochloride formed small, readily deliquescent needles,
and did not produce a precipitate in alcoholic platinum
chloride. Injected into guinea-pigs and rabbits it induced
an exalted peristaltic action of the intestines, which lasted
several days, and produced in the animals, on account of
the continuous evacuations, a condition of great weakness.
No disturbance in the functions of the other organs was
observed.

A BASK was isolated from decomposing haddock which
were exposed for five days during summer in an open iron
vessel. BIUEGEH (I., 42) found in the aqueous mercuric
chloride precipitate (see page 258) a base the hydrochloride
of which crystalli/cd in well-formed, small needles. The
platinochloride likewise crystallized in beautiful needles,
and gave, on analysis, 36.03 i>er cent, of Ft; 7.81 per cent,
of N.

A substance of muscarine-like action was obtained by
BUIEGER (I., 59) from putrefying gelatin, ten days at
35°, though in insufficient quantity to permit a determina-
tion of its character. The residue containing this substance
gave, on distillation with alkali, only ammonia.

A BASE was obtained by BOCKLISCH (III., 52, 53) from
herring which had undergone putrefaction for twelve days.
It was found in the distillate, together with trimethylamine
and dimethylamine, obtained by distilling the mercuric
chloride filtrate, after the removal of the mercury, with
sodium hydrate. The platinochloride was easily soluble,
and crystallized in large thin plates. On analysis it gave :
Pt = 28.57, C = 22.34, H = 4.66. The hydrochloride is
easily soluble in water, and in absolute alcohol, and l>e-
sides with platinum gives only with phosphomolybdic acid
a yellow precipitate which is soluble in excess, and with
ammonia gives an immediate blue color. It immediately
reduces a mixture of ferric chloride and potassium ferri-
cyanide with formation of Berlin blue ; and similarly

CHKMISTKV <>F TIIK I' TOM A INKS 273

throws down metallic gold from solutions of gold
chloride.

From poisonous mussel, BIMKCKI: (III., 79) obtains! an
aurochloridc of a base crystallizing in needles. The quan-
tity isolated was insufficient for analysis. It is interesting
Ixtause of its property of inducing salivation, a symptom
which has been observed by SCIIMIDTMAN.N and by URUMPE
in some cases of mussel poisoning.

A BASE was obtained by GUA I;KS< IN and Mosso (Jouni.
fiir praktitche Chem., 28, 508) from fresh beef, in the
alkaline etlicr extract obtained by DRAGENDORFF'fl method.
It formed a yellowish alkaline fluid, of unpleasant odor,
and after a time gave a deposit of microscopic crystals.
The hydrochloride gave the following reactions : Gold chlo-
ride, yellow crystalline precipitate ; platinum chloride, pre-
cipitate ; potassium iodide and iodine in hydriodic acid,
kermes-red precipitate ; phosphotungstic acid, nothing ;
phosphomolybdic acid, an abundant yellow precipitate; tan-
nic acid, heavy, grayish precipitate, same with MAYEU'S
reagent ; picric acid, yellow precipitate; MAUMK'S reagent,
precipitate soluble in excess ; potassium bichromate, noth-
ing ; potassium permanganate and sulphuric acid, violet
color ; potassium ferricyauide and ferric chloride, Prussian
blue precipitate.

By giving a precipitate with tannin, and not with phos-
photungstic acid, it resembles neurine.

CH. ( Ji; AM has studied the decomposition of yeast under
the influence of an infusion of hay. The yeast was allowed
to putrefy for fourteen days, and was then treated with zinc
sulphate. The latter was precipitated by barium hydrate,
and the filtrate after the removal of the barium by sul-
phuric acid, was evaporated to dryness, and extracted with
al tsolute alcohol. The alcoholic solution was evaporated,
and the residue again extracted with alcohol. The extrac-
tion residue was taken up with water, and again subjected to
t he alxwc treatment with xinr sulphate, barium hydrate, etc.

274 BACTERIAL POISONS.

The filtrate was poisonous, and produced, in frogs, paral-
ysis and stoppage of the heart in diastole Addition of
platinum chloride and alcohol precipitated two bases. One
of these, although possessing a curara-like action, did not
affect the heart. When its solution was heated for twenty-
four hours on the water-bath, it caused general paralysis
and stoppage of the heart. The platinochloride contained
38.05 per cent, of platinum.

The other base also possessed a slight curara-like action,
and its platinochloride gave, on analysis, 40.92 and 39.4
per cent, of platinum.

BRIEGER found a basic substance in small quantities in
cultures of the staphylococcus pyogenes aureus on bouillon
and beef-broth (II., 74). The hydrochloride formed groups
of colorless, non-deliquescent needles. With platinum
chloride it yielded a double salt, crystallizing in needles,
and containing 32.93 per cent, of Pt. For its reactions,
see Table I.

From aqueous as well as alcoholic solutions of cultures of
staphylococcus aureus LEBP:R (1888) isolated a crystalline
substance which he named phlogosine. The composition
of this substance is not known. It does not seem to con-
tain nitrogen, and inasmuch as it blackens silver it prob-
ably contains sulphur. It crystallizes in fine needles which
are soluble in ether and in alcohol ; difficultly soluble in
water. It sublimes in needles. Alkalies precipitate it as
amorphous yellow floccules which are soluble in acid and
then can be recrystallized. With potassium ferricyanide
and ferric chloride it yields a blue color, and with potassio-
mercuric, cadmic, and bismuth iodides precipitates which
are soluble in excess. No precipitate is produced by gold
or platinum chlorides, phosphotungstic or molybdic, tan-
n ic or picric acids.

A small quantity applied to the conjunctiva produces
intense inflammation, suppuration, and necrosis. Intro-
duced into the anterior chamber it induces intense suppura-
tion and keratitis. The substance is entirely distinct from
the base obtained by BRIEGER, described above.

CIIKMISTUY ii F TIIK PTOMAIN] 'J7">

A P>\-i: — boil in;: point about L'SJ — was obtained by
BfUBQER (II., 61) irom human livers and spleens which
were put reiving lor two to three weeks. It occurs in the
men-uric chloride filtrate, as described under Saprine, jwige
•J'J<>, together with some mydaleine, trimetliylainine, and
hydrocarbons. The filtrate, after the mercury is removed
l>\ hydrogen sulphide, is evaporated to dryness, and finally
the hist traces of water are removed in a vacuum. The
residue is then treated with absolute alcohol, and from this
alcoholic solution the mydaleiue is precipitated by the addi-
tion of alcoholic mercuric chloride. The trimethylamine
is separated by distillation of the alkaline filtrate, previously
deprived of its mercury by hydrogen sulphide; while the
inother-li(juor yields an oily mixture of hydrocarbons and
bases. The latter were separated by fractional distillation,
whereby only one of the bases was obtained in sufficient
quantity for study. It boiled at about 284°, and gave
with hydrochloric acid, on evaporation, a salt crystallizing
in beautiful, long needles, which were very easily soluble
in j>erfectly absolute alcohol. With gold chloride and picric
acid it gave only oily products; with ferric chloride and
potassium ferricyanide, an intense blue ; with platinum
chloride, an extremely easily soluble double salt, which
appeared under the microscope in the form of very fine
needles, while from alcohol-ether the double salt slowly
separated in thin plates which, contained 30.36 per cent, of
platinum. The free base showed a slight fluorescence. It
is not poisonous, and, according to BRIEGER, is probably
a pyridine derivative.

Other non-poisonous bases were present in very small
quantity in the mother-liquor described above, after the
separation of the oily mixture.

I'KI-TOTOXI.VK. — By this name BIUEGKK (I., 14-19) has
designated a poisonous base which he has found in some
peptones, and hence in the digestion of fibrin ; in putre-
fying albuminous substances, such as fibrin, casein, brain,
liver, and muscles. It is a well-known fact that animal
tissues, in the early stages of putrefaction, possess strong
toxic properties, even tafore the decomposition could have

276 BACTERIAL POISONS.

advanced far enough to ellect a splitting-lip of the proteid
and carbohydrate molecules. BUIKGEH and others have
tried to seek an explanation of this toxicity by connecting
it with an early peptonization of the protcids brought about
by the action of ferments which are distributed throughout
the tissues, and which begin their activity immediately after
death. This poison has not been definitely isolated, but its
general properties and action have been studied by
BRIEGER and SALKOWSKI. The former prepared it by
digesting fibrin for twenty-four hours with gastric juice at
the temperature of the blood. The perfectly fresh peptone
thus obtained was evaporated to a syrupy residue, and this
was then extracted with boiling alcohol. The residue left
on evaporation of the alcoholic solution was digested for
some time with amyl alcohol, which on subsequent evapor-
ation gave amorphous brownish masses. This extract can
then be purified by neutral lead acetate. The filtrate, after
the removal of the lead by hydrogen sulphide, is repeat-
edly extracted with ether, then evaporated to dryuess, and
extracted as before, with amyl alcohol. This final extract
is evaporated to drive off the alcohol, taken up with water,
and filtered. The colorless aqueous solution thus obtained
contains the poisonous substance, which, however, can only
with extreme difficulty be brought to crystallization in
vacuo.

This poison, when in its purest condition, as shown by
its failure to give the biuret reaction, possesses a neutral
reaction. Its behavior to Millon's reagent is quite charac-
teristic: it gives a white precipitate, which on boiling
becomes intensely red. From this reaction, BRIEGER is
inclined to regard this substance as a hydroxyl or an
amido-derivative of benzole. The ptomaine can be ex-
tracted from acid as well as alkaline solution by amyl
alcohol — more difficult in the cold than on heating. It
is absolutely insoluble in ether, benzol, and chloroform ;
very soluble in water. It is not destroyed by boiling, by
passing hydrogen sulphide, or by strong alkalies; but is
destroyed, however, when the putrefaction lasts longer
than eight days. For its behavior to reagents, see
Table I.

CHEMISTRY OF THE PTOMAINES. 277

Various observers have shown that peptone possesses a
toxic action, and some have been led to regard this toxicity
as not due to the peptone itself, but rather to the presence
of this or some other ptomaine. At least BBEBGEB found
one specimen of dry Witte's peptone to be perfectly harm-
less; whereas, the fresh peptone formed by fibrin digestion
possessed strong toxic powers. Moreover, this non-
poisonous peptone when exposed to the action of gastric
juice was found to yield the poisonous substance. The
poisonous nature of proteids and the physiological action
of this base will be described later.

PYOCYANINE, C14HUNO2, is the coloring matter of blue
pus, and is produced by the action of bacillus pyocyaneus.
It was isolated by LEDDERHOSE (1887) and is said to be an
anthracene derivative. On contact with the air it is oxidized
to pyoxan those, a yellow substance. According to KUNZ
it contains nitrogen and sulphur. The picrate is of a dark
reddish-brown color; the platinum salt is black and some-
times is obtained as glittering fine golden needles.

13

278

BACTERIAL POISONS.

TABLE OF PTOMAINES.

Formula.

Name.

Discoverer.

Physiological action.1

C H6N

Methylamine.

Bocklisch.

Non-poisonous.

CjjHjN

Dimethylamine.

Brieger.

" "

C8H,N

Trimetbylamine.

Dessaignes.

" "

C2H6N

8permine(?).

K mi/..

" "

C.iH7N

Ethylamine.

Hesse.

•< a

C4 HUN

Diethylamine.

Bockliach.

U 11

C6 H16N

Triethylamine.

Brieger.

" "

C8H9N

Propylamine.

"

C4 HnN

Butylamine.

Gautier &, Uourgues.

Poisonous(?).

C6 HnN (?)

Tetanotoxine.

Brieger.

Poisonous.

C6 H18N

Amylamine.

Hesse.

"

C6 Hj6N

Hexylamine.

"

"

C7 HUN

Di-hydrolutidine.

Gautier & Mourgues.

M

C8 HUN

Collidine(V).

Nencki.

C8 HUN

Pyridine base(?).

0. de Coninck.

C8 H18N

Hydrocollidine(?).

Gautier and Etard.

Poisonous.

Cg Hi3N

Parvoline(?).

" " "

C10H15N

Unnamed.

Guareschiand Mosso.

Poisonous.

CioH16N

Pyridine base(?).

0. de Coninck.

GiQiljjri

Hydrocoridine(?).

Griffiths.

Cgo Qg}N

Unnamed.

Del6zinier.

C2 H8 N2

Ethylidenediamine(?).

Brieger.

Poisonous.

C3 Hg N2

Trimethylenediamine(?).

"

"

4 **12^*2

Putrescine.

11

Not very poisonous.

C5 H14N2

Cadaverine.

"

a «

C6 HHN2

Neuridine

"

Non-poisonous.

C5 HJ4N2

Suprino.

•'

'• "

C7 Hi0N2

Unnamed.

Morin.

" "

C10H26N2(?)

Susotoxine.

Novy.

Poisonous.

Cy H7 N8

Methyl-guanidine.

Brieger.

"

C^H^NS

Morrhnlne.

Gautier & Mourgues.

Diuretic, etc.

CuHjjoN^

Unnamed.

Oser.

C17H88N4

"

Gautier and Etard.

C25H32N4

Aselline.

Gautier & Mourgues.

Poisonous.

C5 Hi3N 0
C8 HUN 0

Neurine.
Mydine.

Brieger.

Non-poisonous.

C5 HUN 02

k-amido-valerianic acid.

E. and H. Salkowski.

C6 H16N 02

Choline.

Brieger.

Poisonous.

Cg H18N 02

Mydatoxjne.

"

C4H,,N02

Unnamed.

Brieger, 1888.
(tetanus cult.)

Non-poisonous.

C, H1BN 02

Mytllotoxine.

Brieger.

Poisonous.

1 Only those bases are here denoted as poisonous which possess a decided toxicity.

CHEMISTRY OF THE PTOMAINES.

279

TABLE OF PIOMAINES— Continued.

Formula.

Name.

Discoverer.

Physiological action.1

C, H,,N 0,

Oadinlne.

Brieger.

Poisonous.

C7 H,,N Oj Typhotoxine.

it

"

C| H,,X 0, ; Unnamed.

"

"

C,4HUN 0» Pyocyanlne.

Ledderhow.

Non-poisonous.

C, HUN O| Betaine.

Brieger.

"

Cs H16N 0, Muscarine.

'«

Poisonous.

C, H,,N 0, Morrhuic acid.

Gautier Sc Moargues.

C4 H,fN»04

Unnamed.

Poucbet.

Poisonous.

1 : " - ^."t

Tetanine.

Brieger.

"

C14H30N.!O4

Unnamed.

Guareschi.

CT HwN.O,

"

Poucbet.

Poisonous.

Tyrotoxicon.

Vanghan.

«

Mydaleine.

Brieger.

"

gpaimotoxine.

*

"

A diamine(?).

" (tetunus cult.)

"

Peptotoxine.

"

"

Phlogosine.

Leber.

Inflammatory.

1 Only those bases are here denoted as poisonous which possess a decided toxicity.

CHAPTER XII.

CHEMISTRY OF THE LEUCOMAlNES.

UNDER this head are classed those basic substances which
are found in the living tissues, either as the products of
fermentative changes or of retrograde metamorphosis.
Most of these substances have already been known for
many years, though their real significance as alkaloidal
bodies, and their relation to the functional activities of the
animal organism have been but little understood, or rather
they have not been brought together under the leading
conception that they are alkaloidal products of physiologi-
cal change. The first attempt at the systematic study and
generalization of these basic substances was made by
GAUTIER, who applied to them the name leucornaines, a
term derived from the Greek favxut*a, signifying white of
eggs. Under this name he includes all those basic sub-
stances which are formed in animal tissues during normal
life, in contradistinction to the ptomaines or basic products
of putrefaction. The distinction between vegetable and
animal alkaloids is not very well defined, and, in fact, there
seem to be reasons for considering their formation as due
to the same causes which bear an intimate relation to the
physiology of the cells and tissues of both kingdoms.
Thus, vegetable tissues are known to contain not only
definite ptomaines, such as choline, but also leucomaines, as
hypoxanthine, xanthine, etc. Indeed, in this latter group
must be placed, on account of their relation to xanthine,
those well-defined alkaloidal bases, caffeine and theobro-
mine. Not only are the representatives of these two
divisions of basic substances common to both kingdoms,
but their parent bodies, lecithin, nuclein, etc., are known
to occur in both, thus giving rise to the same bases on
decomposition.

CHEMISTRY OF THE LEUCOMAINES. 281

So far as the genesis of most of the leucomames is
ci>n«vrnc<l, we know very little, though GAUTIER is of the
belief that they are being formed continuously and inces-
santly in the animal tissues side by side with the forma-
tion of urea and carbonic acid and at the expense of the
nitrogenous elements. It is quite probable, as KOSSEL has
pointed out, that some of these products are in themselves
antecedents of end-products of metabolism. This is unques-
tionably true of the imido group, which exists in the ade-
nine and guanine molecules, and through vital or putre-
factive processes is split off, giving rise to ammonia, which
in turn serves to form urea and uric acid. BOUCHARD has
sought an explanation of the presence of these bases in the
urine, by supposing that they were originally formed in
the intestinal tract, from which they were absorbed into the
system, to be subsequently eliminated by the kidneys. This
vie\v has also been brought forward by SCHAR (1886), who
holds that these bases, which may be formed by putrefactive
changes in the intestinal tract, are absorbed into the circu-
latory system, whence they may be partly eliminated by
the kidneys or may be partly deposited in the tissues them-
selves.

The views of BOUCHARD and SCHAR have, to a certain
extent, been confirmed by the investigations of UDRANSZKY
and BAUMANN, who showed that the well-known ptomaines,
cadaverine and putrescine, occiir in the urine in cystinuria,
and are formed by putrefactive changes induced in the in-
testinal tract probably by specific microorganisms. Under
this same head fall the recent observations of WOLKOW
and BAUMANN, that alkapton is produced from tyrosin by
similar changes in the intestines. The origin of the true
leucomames cannot, however, be accounted for in this
manner, for they are indissolubly connected with the meta-
bolism of the cell itself, and are, therefore, formed in the
tissues and organs proper, especially those rich in nucleated
cells.

Another source of the nitrogenous bases must not be
lost sight of, and that is protoplasm itself. The researches
of DRECHSEL, SIEGFRIED, and SCHULZE have shown that

282 BACTERIAL POISONS.

nitrogenous bases do result from the decomposition of
animal and vegetable proteids (see p. 242).

The leucoma'ines proper can be divided into two distinct
and well-defined groups : (1) the Uric Acid Group, and
(2) the Creatinine Group.

The first of these divisions contains a number of well-
known bases which are closely related to uric acid. The
order in which they will be described is as follows :

Adenine, C5H5N5.

Hypoxanthine, C8H4N4O.

Guanine, C5H,NftO.

Xanthine, C5H4N4O2.

(Uric Acid, C5H4N4O3.)

Heteroxanthine, C6H6N4O2.

Paraxanthine, C7H8N402.

Carnine, C7H8N403.

Pseudoxanthine, C4H5N4O.

Gerontine, C5HMN2.

Spermine, C2H5N (?).

The members of the second group have all been dis-
covered by GAUTIER, and by him are regarded as allied
to creatine and creatinine. These two substances, especially
the latter, have been hitherto regarded as strongly basic in
character, but SALKOWSKI has recently shown that creati-
nine, when perfectly pure, possesses little or no alkaline
reaction, and, moreover, does not combine with acids. The
bases in this group are :

(Creatinine, C4H7NSO.)

(Creatine, C4H9N3O2.)

Cruso-creatinine, C5H8N4O.
Xantho-creatinine, C6H10N4O.

Amphi-creatine, C9HKN7O4.

Base, CnH24N1005-

Base, C12H25NU06.

Besides these two general classes of leucoma'ines, there
may be made a third class of undetermined leucomaines,

tllKMISTRY OF THE LEUCOMAINES. 283

embracing those bases which have been observed, but
studied more or less incompletely, in the various physio-
logical smvtions of the body.

LEUCOMAKNES OF THE URIC ACID GROUP.

ADENINE, C5H5Na, which was discovered by KOSSEL in
1885, forms the simplest member of the uric acid group of
1» ucoma'ines, and as such it deserves special attention, inas-
much as it shows most clearly the relation that exists
In-twi-en hydrocyanic acid and the members of this group.
This base is apparently formed by the polymerization of
hydrocyanic acid — a view that is confirmed, at least in
part, by the fact that on heating with potassium hydrate
to 200°, it yields a large quajitity of potassium cyanide.
Moreover, by the action of reducing agents, it is converted
into a substance similar to, if not identical with, azulmic
acid. It has not been prepared synthetically, though
GAUTIER has claimed to have synthesized two closely re-
lated bodies, xanthine and methyl-xanthine, by simple
heating of hydrocyanic acid in a sealed tube in contact with
water and a little acetic acid.

This base was first prepared from pancreatic glands —
hence the term adenine, which is derived from the Greek
word <"*>/", meaning a gland. -It has since been shown to
mrur together with guanine, hypoxanthine, etc., as a
decomposition-product of nuclein, and, therefore, it may
be obtained from all tissues and organs, animal or vege-
table, rich in nucleated cells. Accordingly, it has been
found in the kidneys, spleen, pancreatic, thymus and lym-
phatic glands, in beer-yeast, in spermatic fluids, but not in
testicles of the steer ; occurs also in tea-leaves. In the latter
adenine appears to exist in a preformed condition, since it can
be extracted without the use of acid reagents. The thymus
gland, as a prototype of embryonic, highly cellular tissue,
yields a considerable amount of adenine ; that from a calf,
for instance, was found by SCHINDLER to contain 0.18 per
cent. It has also been observed in the liver and urine of

284 BACTERIAL POISONS.

leucocythsemic patients ; its occurrence in this disease will
be readily understood when it is remembered that leueocy-
thsemia is characterized by the presence in the blood of an
unusual proportion of the nucleated white blood-corpuscles,
which, owing to various unfavorable conditions, become de-
stroyed in time, and the contained nuclein, as a result,
splits up into adenine and guanine. These two bases may,
therefore, be expected in all pathological conditions where
there is an abnormal accumulation of pus. Indeed, as early
as 1865, NAUNYN extracted from pus, obtained from the
pleural cavity, a considerable quantity of a substance
which was probably either adenine or guanine, or both.
Adenine does not occur, or only in minute traces, in meat
extract ; and in this it resembles guanine, which is present
only in traces. This may be due to the fact that adenine
and guanine are readily converted into hypoxanthine and
xanthine respectively, as has been shown in the putrefaction
experiments of SCHINDLER. They may be considered as
transitional products of cell-metabolism, the imido group
contained in each readily being replaced by oxygen, and
giving rise to ammonia, and this in turn to urea. KOSSEL,
however, explains this fact on the ground that the muscle
tissue is very poor in nucleated cells, i. e., in nuclein. It
would seem that the muscle cell in losing the morphological
character of a cell has also suffered a corresponding loss in
its chemical properties. For while the decomposition-
products of nuclein — hypoxanthine, xanthine, phosphoric
acid, etc. — are found in the muscle tissue, they do not exist
in combination as they do in the nuclein molecules. This is
seen in the fact that the bases exist in the free condition,
since they can be extracted by water ; and again, the phos-
phoric acid is present in the muscle tissue, not in organic
combination, but as a salt. In the nucleated cell, adenine,
guanine, etc., do not exist in the free condition, but form,
in part at least, with albumin and phosphoric acid, a loose
combination which is readily decomposed by the action of
acids at the boiling temperature. This same change takes
place spontaneously after death.

There can be no doubt that adenine and guanine play an

CHEMISTRY OF THE LEUCOM AINES. 285

im]>nrtnnt part in the physiological function of the cell
nucleus, which, from recent ol)servations, appears to be
nnvssary to the formation and building up of organic
matter. It is now known that non-nucleated eells, though
capable of living, are not capable of reproduction. We
must look, therefore, to the nucleus as the seat of the
functional activity of the cell — indeed, of the entire organ-
ism. Nuclein, the parent substance of adeninc and guanine,
is the best known and probably most important constituent
of the nucleus, and as such it has l>een already credited
with a direct relation to the reproductive powers of the
cell. This chemical view has recently been confirmed by
ZACH ARIAS, who showed that chromatin of histologists is
identical with nuclein. LlEBERMANN has questioned nu-
clein as being the source of xanthine compounds, but in
this he js not supported by the mass of evidence.

The method employed by KOSSEL for the preparation
of adenine, is as follows : The finely divided pancreatic
glands are heated to boiling, for three or four hours, with a
large quantity of dilute sulphuric acid (0.5 per cent, by
volume of concentrated acid), and the acid solution thus
obtained is treated with a slight excess of hot concentrated
baryta water. The excess of baryta is removed by carbonic
acid, and the solution is then filtered ; the filtrate is con-
centrated to a small bulk, al>out 100 c.c., rendered alkaline
with ammonium hydrate, and finally precipitated with an
:mmioniacal solution of silver nitrate. The precipitate,
consisting of the silver compound of the xanthine bodies,
is partially dried by spreading over porous porcelain
plates ; then dissolved in warm nitric acid of specific
gravity 1.1, to which a little urea lias l>een added to pre-
vent the formation of hypoxanthine should traces of nitrous
acid IK- present. The filtered acid solution, treated with
silver nitrate, on cooling, gives a deposit of the silver
salts of hypoxanthine, guanine, and adenine, which is fil-
tered oil :intl thoroughly washed. The adenine separates
out almost quantitatively if a little silver nitrate solution
is added. The filtrate contains any xanthine silver com-
pound that may be present. The washed precipitate of the

13*

286 BACTERIAL POISONS.

silver salts is suspended in water, nitric acid added, decora-
posed with hydrogen sulphide (ammonium sulphide, or,
better, hydrochloric acid, may be used), and the clear filtrate
is concentrated on the water-bath to a small volume. It is
then saturated with ammonium hydrate and digested on the
water-bath for some time, whereby adenine and hypoxan-
thine go into solution, while the guanine remains undis-
solved (see p. 287). From the ammoniacal solution on
partial concentration and subsequent cooling, the adenine
crystallizes out first, whereas the more soluble hypoxanthine
remains in solution. If the adenine is still colored it can
be purified by dissolving in water and boiling with animal
charcoal. The hot aqueous solution is then rendered very
slightly alkaline with ammonium hydrate and allowed to
cool ; adenine crystallizes out, and can be still further puri-
fied by recry stall ization from water.

Ammonium sulphide has been employed by SCHINDLER,
in place of hydrogen sulphide, in decomposing the silver
compounds of the above bases. BRUHNS recommends in-
stead warming with very dilute hydrochloric acid, espe-
cially if guanine is present. The solution can then be
neutralized with NaliCO3, using methyl-orange as indi-
cator, and the adenine separated from hypoxanthine by the
picric acid method described below.

Another method for the separation of adenine from
hypoxanthine is based upon the behavior of the nitrates
of these bases in aqueous solution. From concentrated
aqueous solutions of the nitrates, free hypoxanthine crystal-
lizes out first, because the nitrate is decomposed ; whereas,
adenine, which is a stronger base, remains in combination
with the acid, in solution.

SCHINDLER determines adenine and hypoxauthine indi-
rectly. The ammoniacal solution which is filtered from the
insoluble guanine is evaporated to dryness on a weighed
platinum dish, dried at 110°, and weighed. A nitrogen
determination is now made of the mixed bases and from
these data the proportion of each is calculated.

By far the best method for the quantitative separation of
adenine and hypoxanthine is the picrate method of BRUHNS.

CHEMISTRY OF THE LEUCOMAINES. 287

Tin- solution of the salts of the bases, preferably as nitrates
or sulphates, must be neutral or faintly acid ; excess of
alkali or acid interlen s. Such a solution can be obtained
by evaporating the filtrate from the guanine in KOSSEL'S
method (page 286), and dissolving the residue in nitric
acid ; this is neutralized with sodium carbonate, using
methyl-orange as indicator. On the addition of excess of
sodium picrate the adenine is thrown down as a clear yel-
low floceulent precipitate. If the precipitation is made at
the boiling temperature, on cooling the adenine salt sepa-
rates in a crystalline condition and is more easily filtered
and washed. After standing fifteen minutes the precipitate
is filtered off by the aid of a suction-pump on a weighed
filter, washed with cold water, and dried at 100°. As a
correction for the solubility of the adenine picrate, 2.4 nig.
per 100 c.c. filtrate can be added to the calculated amount
of adenine.

The hypoxanthiue picrate is very soluble, and, therefore,
remains in solution. In this it is estimated according to
the method described on page 302.

Adenine, when crystallized from warm or impure solu-
tions, is obtained either as an amorphous substance, pearly
plates, or in the form of very small microscopic needles ;
from dilute cold solutions it separates in long, needle-shaped
crystals containing three molecules of water. Tin's water
of crystallization is lost on exposure to the air or on heating
to 53 , and the crystals become opaque. It is soluble in
about 1086 parts of water at the ordinary temperature ;
more easily in hot water, from which, on cooling, it recrys-
tallizes. The aqueous solution possesses a neutral reaction.
The free base is insoluble in ether, chloroform, and alcohol ;
soluble in glacial acetic acid, and somewhat in hot alcohol.
It dissolves readily in mineral acids, yielding well-crystal-
lizable salts. The fixed alkalies dissolve it with ease, but
on neutralization of the solution it is reprecipitated In
aqueous ammonium hydrate it is more readily soluble than
guanine (which is insoluble, SCHINDLER), and more diffi-
cultly soluble thaji hypoxanthine — a fact which is made use

288 BACTERIAL POISONS.

of to effect a separation from these bases. It is but slightly
soluble in sodium carbonate.

Adenine can be heated to 278° without melting ; at this
temperature it becomes slightly yellow, and yields a white
sublimate. It can be completely volatilized without decom-
position, by heating on an oil-bath to 220° ; the sublimate
consists of pure, white, plumose needles of adenine, but at
250° partial decomposition occurs, and some hydrocyanic
acid forms. When heated with potassium hydrate to 200°
on an oil-bath, it yields a considerable quantity of potas-
sium cyanide. Adenine is quite indifferent to the action of
acids, alkalies, and even oxidizing agents. Thus, it may be
boiled for hours with baryta, potash, or hydrochloric acid,
without suffering decomposition. But when heated with
dilute hydrochloric acid, or concentrated hydriodic acid,
in a sealed tube at a temperature exceeding 100°, adenine
is completely decomposed, with formation of carbonic acid
and ammonia :

C5H8N5 + 5H20 + 50 = 5CO2 + 5NH3.

The free base, as well as benzoyl-adenine, is unaffected by
the weak oxidizing action of potassium permanganate, but
on stronger oxidation it is wholly destroyed. Bromine water
produces in aqueous solutions of adenine an oily precipitate,
which, on contact with potassium hydrate or ammonia, gives
a beautiful red or violet color. Sodium amalgam and zinc
chloride appear to have no action ; but on boiling with zinc
and hydrochloric acid it yields a very unstable reduction-
product, which in the presence of oxygen first assumes a red
color, and finally throws down a reddish-brown precipitate.
This brown substance appears to be identical with azulmic
acid, which has been known for a long time as a product of
the polymerization of hydrocyanic acid.

When adenine is evaporated on the water-bath with dilute
or fuming nitric acid, it gives a white residue which fails to
give any coloration with sodium, ammonium, or barium
hydrate. Similarly, it does not give the so-called WEIDEL'S
reaction (murexide test) on evaporation with chlorine water
and exposure of the residue to an ammoniacal atmosphere.

CHEMISTRY OF THE LEUCOM AINES. "289

In tin's respect it ns.inlihs hypoxanthine, which, when
pure, «loi > IK it answer to either of these tests. Another test
for adenine, which, however, is given also by hypoxanthine
but not by gnanine and catleine, is as follows : The sub-
stance to be tested is digested for half an hour with "zinc
and hydrochloric acid in a test-tube on the water-bath. If
adcnine is present, the solution will assume on standing,
more rapidly on shaking, a ruby-red coloration, which later
on turns into a brownish-red. This reaction depends upon
the formation of a reduction-product, which, owing to its
unstable nature, is soon oxidized by the oxygen of the
atmosphere into a brownish, amorphous substance, appa-
rently identical -with azulmic acid.

On treatment with nitrous acid, it is converted into hypo-
xanthine according to the equation :

C5H5N6 + HN02 = C6H4N40 + N2 + H20.

This formation of hypoxanthine from adenine is analogous
to STACKER'S transformation of guanine into xanthine by
a similar action of nitrous acid (see Gttanine). In both
cases the change of a highly nitrogenized into a less nitro-
gen ized body is accomplished by replacing an NH group
by O, or, more exactly, of an NH2 group by OH. In fact,
the change is identical with that seen in the conversion
of primary amines into primary alcohols. Thus,

C2H5.NH2 + HN02 = C2H9OH -f N2 +H2O.

ETHTLAMINE. ETHYL ALCOHOL.

In the extraction of adenine from the mother-liquors of
tea-leaves after removal of caffeine, if urea is not added to
the nitric acid, nearly one-half of the adenine may be con-
verted into hypoxanthine. By processes of putrefaction
adenine is converted into hypoxanthine and guanine into
xanthine (SCHINDLER). The change is, therefore, some-
what analogous to that produced by nitrous acid. Adenine
undergoes this decomposition much more rapidly than the
other xanthine compounds.

The ease with which adenine and guanine are reduced
outside of the organism shows that similar changes may take

290 BACTERIAL POISONS.

place within the cell-nucleus proper. For we know that
every cell is endowed with an enormous reducing power,
and hence it is not difficult to see how the oxygen-free
adenine can be readily converted into a body or bodies
which greedily take up oxygen. We must, therefore, look
upon adenine and guanine not only as the antecedents of
hypoxanthine and xanthine, but also as intermediate pro-
ducts which, when they form in the cell, may give rise to
important chemical processes, especially those of a synthetic
nature. It is highly probable that the study of the decom-
position-products of nuclein will explain to us many of the
metabolic changes in the organism, and throw additional
light upon the migration of the amido group from the
proteid molecule to the amido acids and urea derivatives.
Thus, the formation of xanthine from guanine represents
the conversion of a guanidine residue into a urea residue.
A similar change is undoubtedly effected in the transforma-
tion of adenine into hypoxanthine.

Adenine unites with bases, acids, and salts. The salts
of adenine with -mineral acids can be recrystallized, thus
differing from the corresponding salts of guanine and hypo-
xanthine, which are dissociated by the action of water.
The solutions of the salts, however, show an acid reaction
to litmus but not to methyl-orange.

The hydrochloride, C5HSN5.HC1 + JH2O, forms color-
less, transparent, strongly refracting crystals. One part of
the anhydrous salt is soluble in 41.9 parts of cold water.
M icroscopically it is distinct from that of hypoxanthine and
adenine-hypoxanthine.

The nitrate, C?H5N5.HN03 + £H2O, crystallizes from
the aqueous solution in fine, stellate needles. One part of
the dry salt dissolves in 110.6 parts of water.

The sulphate, (C5H5N5)2.H2SO4 + 2H20, can be obtained
from the aqueous solution in two different crystalline forms.
This may possibly be due to the presence of adenine-hypo-
xanthine compound (BRUHNS). It is easily soluble in hot
water, and at the ordinary temperature it is soluble in 1 53
parts of water.

The oxalate, C6H6N6.C3H2O4 -f H2O, is obtained by dis-

CHEMISTRY OF THE LEUCOMAINES. 291

solving adenine in hot, dilute, aqueous oxalic acid, from
which solution, on cooling, it separates as a voluminous,
difficultly soluble precipitate of roundish masses which are
composed of long, delicate needles. The oxalates of guanine,
hypoxanthine, and xanthine are more easily soluble than
th.it of adenine, and exhibit, moreover, a different appear-
ance.

The pic-rate, C5H5N6.C6H2(XO,)SOH + H2O, is thrown
down as a bright yellow floeculent precipitate, when aqueous
solutions of adenine salts are treated with sodium pierate.
Recry stal li/ed from hot water it forms bright-yellow, very
voluminous bunches of long fine needles, which, on drying,
acquire a silky lustre and form a felted mass. It is diffi-
cultly soluble in cold water (1 : 3500) ; more readily in hot
water and in alcohol (96 per cent.) ; is insoluble in dilute
acids. The water of crystallization is not lost on exposure
to air but is driven off at 100° ; the salt then remains un-
changed even at 220°. A cold concentrated aqueous solu-
tion of the salt treated with one-tenth its volume of cold con-
centrated solution of sodium pierate produces a precipitate
of short fine needles consisting of most of the adenine pierate
(five-sevenths). The solubility of the pierate can thus l)c
reduced to as low as 1 : 13750, and on this fact is based the
quantitative method of BRUHNS. The salt can also be
obtained in its characteristic groups by combining cold
saturated aqueous adenine solution (1 : 1086) with picric
acid ; with sodium pierate, fiowever, adenine gives no pre-
cipitate, since the pierate is soluble in an equivalent quan-
tity of sodium hydrate. Thus is explained KOSSEL'S
statement that adenine forms an easily soluble compound
with picric acid. Heated on a platinum foil it burns slowly
and leaves considerable carbon residue. The very bright
yellow color of the salt serves to distinguish it from most
of the other picrates, especially guanine pierate.

The platinochloride, (C5H5N5.HCl)2PtCl4, crystallizes
from dilute aqueous solution in small yellow needles. The
concentrated aqueous solution of this salt, when boiled for
some time, decomposes, with the separation of a clear,

292 BACTERIAL POISONS.

yellow powder, which is but slightly soluble in cold water,
and has the composition C5H5N6.HCl.PtCl4.

The aurochloride, on evaporation, yields very charac-
teristic forms.

The silver salt of adenine, C5H4AgN8, is formed when
silver nitrate is added in molecular proportion to a boiling
ammoniacal solution of adenine. An excess of silver
nitrate produces, in the cold, the compound C5H3Ag2N6 +
H2O, which is converted slowly in the cold, immediately on
warming, into the other salt, according to the equation :

2(C9H3Ag2N5 + H20) = 2C8H4AgN5 + Ag2O + H2O.

Owing to this instability the two compounds are always
found together in varying proportion. Both are difficultly
soluble in water, and ammonia even at the boiling-point.
The precipitation of adenine by an ammonical silver solu-
tion is complete, and is therefore available for quantitative
estimation.

Adenine silver nitrate, C5H5N5. AgNO3, (Ag = 35.4 per
cent.), corresponds to the similar hypoxanthine and guanine
salts. It is obtained by dissolving the above silver com-
pounds in hot nitric acid ; and from this solution, on cool-
ing, it separates in needle-shaped crystals, which are not
permanent. This lack of stability, as compared with the
permanent hypoxanthine silver nitrate, was first pointed
out by KOSSEL, and was thought to be due to loss of nitric
acid in washing, and also .by heating at 100°. BRUHNS,
however, has shown that the acidity of the wash-water is
indicated by litmus, but not by methyl-orange, which is
not colored red by silver nitrate. The reaction is, there-
fore, due not to free nitric acid, but to silver nitrate. It
would seem that adenine, as well as hypoxanthine, and pos-
sibly xanthine, form silver compounds containing one and
two molecules of silver nitrate ; the greater the quantity of
silver nitrate used the higher is the per cent, of silver, i.e.,
the more of the latter compound is formed. These are very
unstable, and are decomposed by dilute nitric acid, more so
by water, into silver nitrate and the compound containing
one molecule of silver nitrate. We have in this behavior

CHEMISTRY OF THE LEUCOMAINES. 293

an interesting case of mass-action and chemical equilibrium
between lull-nine, silver nitrate, nitric acid and water.
Ammonium hydrate removes the nitric acid from this as
easily as from the hypoxanthine compound, and there is
titi-med, aivonlinir to the composition of the original salt, a
varying mixture of C8H4AgN8 and C5H3Ag2N5 + H2O.
The solubility in nitric acid is about the same as that of
hypoxanthine silver nitrate.

Adenine silver picrate, C8H4AgN8.C6H2(NO2)sOH +
H2O, is obtained as an amorphous voluminous yellow pre-
eipitat-- when silver nitrate is added to a cold aqueous solu-
tion of adeninc pierate. If the latter solution is previously
raised to the boiling-point the precipitate then soon becomes
crystalline and rapidly subsides. The adenine can thus be
almost wholly removed from solution. The crystalline
form loses its water of crystallization at 120°, while the
amorphous form does not appreciably decrease in weight and
its composition does not appear to be as constant as that of
the corresponding hypoxanthine compound. On treatment
with ammonium hycfrate the picric acid is removed, and
adenine silver, C8H4AgN8, is left, stained yellow by traces
of picric acid.

Adenine-mercury picrate, (C5H4N8)2Hg.2C6H2(X02)3OH,
can be prepared by treating a hot concentrated aqueous
solution of adenine picrate with an excess of sodium picrate
and then with mercuric chloride. It forms a yellow gran-
ular crystalline precipitate (microscopic needles) which rap-
idly subsides and increases in quantity as the solution cools.
Its composition apparently varies, containing one to two
molecules of water, according to the temperature of the
solution. One molecule is given off at 100°, and the second
at 105°-120°. The latter preparation, then, on exposure to
the air, rapidly absorbs one molecule of water. The ob-
ject of the sodium picrate in the precipitation is to combine
with the hydrochloric acid, which is set free. The precipi-
tate produced by mercuric; chloride in cold adenine picrate
solution shows yellow and white granules, and is not homo-
_:< neous. BRUHNS considers it to be a mixture of the aden-
iiv-niercnry picrate and the compound ( 'jH^NjHgjClj ; if

294 BACTERIAL POISONS.

sodium picrate is added, however, the pure adenine-mercury
picrate forms, since no hydrochloric acid is set free.

Adenine-mercuric chloride, C5H4N5HgCl, is thrown down
as a white, finely granular precipitate when a boiling aque-
ous adenine solution is treated gradually with concentrated
mercuric chloride solution. It is formed according to the
following reaction :

C5H5N6 + HgOl, = C8H4NaHgCl + HC1.

That free hydrochloric acid forms can be ascertained by
methyl orange. Treated with ammonium hydrate the
chlorine is removed, and there is formed apparently the
compound CfiH4N5HgOH. If dissolved in warm dilute
hydrochloric acid and allowed to crystallize, the double salt
C5H5N5.HCl.HgCl2 -f- 2H2O separates in long stellate silky
needles.

Another mercury compound, C5H4N5Hg2Cl3, is obtained
when the precipitation takes place in the cold. The
precipitate is white, flocculent, and anhydrous. In this
reaction, as above, for each adenine molecule an equivalent
of hydrochloric acid is set free. This same body is also
produced when an adenine solution is boiled with a large
excess of mercuric chloride and as little hydrochloric as
possible to effect solution. On cooling small stellate needles
separate out, which do not lose their weight at 110°. It
can also be obtained by boiling the following compounds
with water.

When adenine is boiled with a large excess of mercuric
chloride and much hydrochloric acid to completely dissolve
the precipitate that first forms, there is deposited on cooling
a crystalline product, which is variable in its composition,
and apparently consists of double salts of adenine and
mercuric chloride, such as C5H6N5.HC1.5HgCl2 aud C5H5N5.
HC1.6HgCl2. On boiling with water these rapidly de-
compose, forming the compound C6H4N5.Hg2Cl3. The
formation of a double salt, C5H5N5.HCl.Hg012 + 2H2O
is described above.

Adenine-mercury cyanide, (C6H5N5)2Hg(CN)2, separates

CHEMISTRY OF THE LEUCOMAINES. 295

as stellate n^-dles and plates when a mixture of hot solu-
tions of adenine and mercuric cyanide are allowed to eool.

Anadenine bismuth iodide, C5H,N5.H 1. 2BiI, + 2H2O,
is ol)taine<l when an aqueous adenine solution is treated
with potassium bismuth iodide containing free hydriodie
acid. The heavy precipitate, which in color resembles
earl >on monoxide haemoglobin, consists of microscopic glit-
tering red needles. On contact with much water it partly
<le< (imposes, forming light reddish-yellow amorphous floo-
cules, which l>ee<>me darkish-brown at 100°.

Adenine bromide. By treating well-dried adenine with
extvss of dried bromine a dark-red body is obtained which
appears to contain six atoms of bromine. On mere ex-
posure to the air, more rapidly on heating at 100°-120°,
it decomposes, yielding bromine and a brom-adenine,
C5H4BrN5. This compound is white, difficultly soluble in
cold water (1 : 10,000), more readily in hot water, very
easily in ammonia. It crystallizes from water or dilute
ammonia in stellate needles. It is a rather strong base and
forms well-characterised salts from which it is thrown
down as a white micro-crystalline precipitate by addition
of ammonia. It is also formed from adenine- bromide by
treatment with sodium bisulphite. The picrate resembles
that of adenine but is more voluminous ; silver compounds
are also formed resembling those of adenine. The silver
nitrate compound decomposes- on l>oiling with nitric acid
with separat'on of silver bromide. It is only difficultly
attacked by boiling alcoholic potash.

When adenine is treated with zinc and hydrochloric acid,
in the cold, it forms a difficultly soluble crystalline double
salt which has not been obtained in the pure state. This
double salt is not obtained by direct treatment of adenine
hydrochloride with zinc chloride.

One of the hydrogen atoms of adenine is capable of re-
placement by organic radicals. Thus it forms crystalline
methyl and ethyl compounds.

Acetyl-adenine, C6H4N6.CO.CH3, can be obtained by
heating the anhydrous base with an excess of acetic anhy-
dride for some time, in an oil-bath, at 130°. It crystal li/» s

296 BACTERIAL POISONS.

in small white scales which dissolve but slightly in cold
water and in alcohol ; more readily in hot water, in dilute
acids and alkalies. Heated to 260° if becomes yellow but
does not melt.

Benzoyl-adenine, C5H4N5.CO.C6H5, is obtained by the
action of benzoic anhydride, but not of ben/oyl chloride,
on adenine. It crystallizes from water in long, lustrous,
thin needles which sometimes are grouped in bundles, and
melt at 234°-235°. It is easily soluble in hot alcohol, from
which it recrystallizes on cooling ; also in dilute acids and
in ammonia. With ammoniacal silver nitrate it gives a
precipitate resembling that of adenine, but is more readily
soluble in ammonia. This compound is quite stable, since
it decomposes very slowly on boiling with hydrochloric
acid ; on protracted boiling with water it is changed into
adenine and benzoic acid.

Benzyl-adenine, C5H4N5.CH2.C6H5, was obtained by
THOISS by heating well-dried adenine with benzyl chloride
to boiling (178°) on an oil-bath. The compound forms
pure white microscopic crystals and melts at 259°. It is
easily soluble in hot water and in hot alcohol. With acids
it forms salts from which alkalies throw down the base.
The hydrochloride forms fine glossy needles which are
readily soluble in alcohol and in water, but not in ether.
The sulphate and nitrate possess similar properties. Like
adenine it yields a silver compound which is insoluble in
ammonia. On reduction with zinc and hydrochloric acid
it forms an amorphous red unstable compound. Treated
with nitrous acid, benzyl-adenine is reduced to benzyl-hypo-
xanthine, thus showing that the benzyl group replaces a
hydrogen atom in the group C5H4N4, which KOSSEL has
called adenyl (see page 307).

Benzyl-adenine picrate, C12HnN5.C6H2(NO2)3OH, is ob-
tained as fine felted yellow needles, which are fairly soluble
in water and in alcohol ; insoluble in ether.

A methyl-adenine was obtained by THOISS in an impure
state by heating dried adenine with methyl iodide in a sealed
tube at 100°. It can be crystallized from absolute alcohol.
The aqueous solution of the base is precipitated by baryta

CHEMISTRY OF THK LEUCOMAINES. 297

water ; alcoliolic zinc chloride also yields a precipitate which
is soluble in excess of ammonium hydrate. Mercuric nitrate
also gives a precipitate. Cadmium chloride yields a pre-
cipitate which dissolves on warming, reappears on cooling,
and is soluble in ammonia. Basic lead acetate lias no effect.
Nothing definite is known in regard to the physiological
action of adenine, except that when fed to dogs it appears
to be eliminated as such, in part at least, by the urine.

ADENINE-HYPOXANTHINE, CaH5N5 + C6H4N4O. The
occurrence of this compound was observed by KOSSEL, but
it was isolated and studied for the first time by BRUHNS.
It can be prepared by cooling a hot aqueous solution of
t-ijual parts of the two bases. At first it is obtained as
thick, starch-like semi-transparent masses, which later in
part l>ecome white and chalky. By spontaneous evapora-
tion of its solution in very dilute ammonia it forms pearly
aggregates of very small radially arranged needles, which
contain water of crystallization. These effloresce some-
what and lose the water at 100°. The compound is more
readily soluble in water than its components, but an exact
<l( t< naination of its solubility is impossible, inasmuch as
the separation from hot solutions is not completed for some
weeks. Any adenine present can be separated by recrys-
tallization. It forms a distinct crystalline hydrochloride,
which should be borne in mind-when examining microscopic-
ally for the two bases ; but the combination is loose, since
addition of gold chloride brings down the characteristic
gold salt of adenine. Ordinarily it does not form salts
with sulphuric or nitric acids, but more often is decomposed
by these, so that the difficultly soluble adenine crystallizes
out. Once, however, BRUHNS obtained a sulphate which
differed from the pure adenine and hypoxanthine sulphates ;
thus is perhaps explained the observation of KOSSEL that
adenine sulphate forms crystals belonging to two systems.
The compound can be decomposed into its constituents by
fractional crystallization of the sulphate or nitrate ; but
better by forming the picrates, which are very unequally
soluble in water. The existence of this compound undoubt-

298 BACTERIAL POISONS.

edly explains many of the mistakes and discrepancies con-
cerning the properties of hypoxanthine, which it resembles
more than adenine, and for the same reason, perhaps,
adenine was so often overlooked.

HYPOXANTHINE, C5HfN4O, sometimes also known as
sarcine or sarkine, was discovered by SCHERER (1850) in
splenic pulp and in the muscles of the heart, and was
named thus because it contains one atom of oxygen less
than xanthine. It has since been obtained, usually accom-
panying adenine and guanine, from nearly all of the
animal tissues and organs rich in nucleated cells, i. e., in
nuclein. It has been found in blood after death, but not
in blood when flowing through the bloodvessels. SALOMON
has recently shown it to be a normal constituent of urine,
present, however, in an exceedingly minute quantity. In
the blood and urine of leucocythsemic patients it occurs in
increased quantity owing to the abnormally large number
of nucleated white blood-corpuscles in circulation (see page
284). BENCE JONES observed in the urine of a boy, who
about three years before showed the symptoms of renal
colic, a deposit of characteristic whetstone-like crystals,
resembling uric acid, but differing from the latter by dis-
solving readily on the application of heat, while from
hydrochloric acid it crystallized in elongated six-sided
plates. These crystals he believed to be those of xan-
thine, but SCHERER and others consider them to be hypo-
xanthine. It is therefore quite possible, though very rare,
for this base to form a deposit in the urine and to be
confounded in shape with uric acid. THUDICHUM has
obtained it from the urine of persons sick with liver or
kidney diseases.

Among other places it has been found in the brain,
muscle, serum, marrow of bones, kidney, heart, spleen,
liver, peripheral muscles (sarkine of STRECKER) ; in the
spawn of salmon (PiccARD), in the testicles of the bull
(SALOMON), in the nuclein of pus and red corpuscles (Kos-
SEL), in developing eggs, and in putrefaction of albumin
(SALOMON). It has also been found in the spores of lyco-

CHEMISTRY OF THE LEUCOMAINES. 299

jxxlimn, ;m<l in the pollen of various plants, in seed of bhu-k
pepper, in grass, clover, oats, bran of wheat, larvae of ants;
in the juice of potato (ScHULZE) ; in certain wines
(KAYSER) ; in the aqueous decoction of yeast of beer
(ScHtir/ENBERGER) ; and also in the liquid in which yeast
is grown (BECHAMP). DEMANT has shown it to be rela-
tively abundant in the muscles of pigeons in a state of in-
anition, while in muscles of well-ted pigeons it is said to
be entirely absent. SALOMON found hypoxanthine and
xanthine in the cotyledons of lupine, as well as in the
sprouts of malt, while REINKE and RODEWALD observed
these two bases together with guanine in JEthalium sep-
ticum — with adenine, xanthine, and theophylline, it occurs
in tea-leaves (KosSEL).

Hypoxauthine has not been extracted from the pancreas,
where it seems to be replaced by guanine, or rather by
adenine. It seems that hypoxanthine bears a relation to
adenine similar to that which we see between glycocoll and
glycocollic acid.

Hypoxanthine occurs frequently in plants together with
the other members of this group, namely, adenine, guanine,
and xanthine. The widely distributed character of these
bases is due to the presence of a parent substance, viz.,
nuclein, the necessary constituent of all cells capable of
development, which under the influence of acids, and
probably likewise of ferments, decomposes into the above-
mentioned bases. They may, therefore, be considered as
the first steps in the retrograde metamorphosis of all
tissues, since they have their origin in nuclein, an impor-
tant proteid substance. Recent advances in biological
chemistry have shown that the undeveloped eggs of various
insects and birds yield much less quantity of xanthine
bodies (hypoxanthine, xanthine, etc.) on treatment with
dilute acid than the partially developed eggs (TICHOMIROFF,
KOSSEL). This is dependent upon the remarkable fact
observed by KOSSEL that the nuclein . of undeveloped
chicken eggs differs from the nuclein of cell nuclei and
resembles that obtained from milk. For, while the nuelein
from the cell nuclei decomposes into adenine, guanine,

300 BACTERIAL POISONS.

hypoxanthine, etc., that from undeveloped eggs and from
milk yields no nitrogenous bases on treatment with acids.
But as the egg develops, i.e., the nucleated cells increase in
number, this latter nuclein is gradually converted or gives
way to the ordinary cell nuclein, and hence it is that the
chick embryo yields guanine, hypoxanthine, and possibly
adenine.

Unquestionably, the presence of hypoxanthine, etc., in
developing cells is due to the presence of the nuclein mole-
cule, from which it is readily split off. In muscle, however,
hypoxanthine and xanthine appear to exist preformed, and
bear no relation to nuclein, since they are in the free condi-
tion, and can be extracted from the tissue by water. For
an explanation of this peculiar fact, see Adenine, page 284,
and Guanine, page 308.

According to the observations of SALOMON and CHIT-
TENDEN, hypoxanthine is formed by the digestion of blood
fibrin with gastric juice, pancreatic juice, or on heating with
water or dilute acids. Egg albumin under the same con-
ditions does not yield any hypoxanthine, except when
treated with pancreatic juice. These observations require
repetition, inasmuch as the fibrin used undoubtedly con-
tained nuclein, which, as we now know, readily decomposes
under those conditions into its characteristic nitrogenous
bases. Be that as it may, it appears, however, to be one
of the products formed by the decomposition and succes-
sive oxidation of proteid matter previous to the formation
of uric acid and urea.

When a mixture of guanine, xanthine, and hypoxanthine
is allowed to putrefy, the bases decompose and disappear in
the order named. Hypoxanthine resists bacterial action
the longest, and this corresponds with its behavior to re-
agents (BAGINSKY). Adenine during putrefaction, in the
absence of air, is converted into hypoxanthine, and guanine
is correspondingly changed into xanthine (SCHINDLER).
An imido group is, therefore, replaced by oxygen, and
probably goes to form urea. This conversion is a very
important fact, since the process of putrefaction, as HOPPE-
SEYLER has repeatedly pointed out, is analogous to the

CHEMISTRY OF THE LEUCOM A I NES. 301

vital process, and the same chemical change may take place
in the animal organs. The same change very probably
pla<v in the auto-digestion of yeast. Its formation
these conditions can be represented thus :

C5H5X5 + HaO = C6H4N40 + NHS.

Bypoxanthine can be readily obtained from a number of
closely related substances. Thus, carnine, by the action of
oxidizing agents, is converted into hypoxanthine (page 328).
For this reason WEIDEL and SCHUTZENBERGER regard
hypoxanthiue as derived from carnine, but this view is now
entirely set aside by our present knowledge of the relation
of this base to nuclein.

Again, it can be obtained from adenine (page 289) by the
action of nitrous acid. The relation that hypoxanthine
bears to uric acid is shown by the fact that the latter is
converted by nascent hydrogen first into xanthine, and
finally into hypoxanthine.

C6H4N40S + 2H2 = C6H4N40 + 2H2().

URIC ACID.

This transformation of uric acid into hypoxanthine is of
especial importance, since together with HORBAC/EWSKI'S
synthesis of uric acid, accomplished by acting on urea with
either glyeocoll or trichlorlaetamide, it constitutes the last
step in the complete synthesis of hypoxanthine from its
dements.

Hypoxanthine has l>een hitherto regarded as a step lower
than guanine in the series of nitrogenous products of
regressive metamorphosis, and consequently was considered
as derived from guanine. The investigations of KOSSEL,
however, show that it arises not from guanine but from
adenine. On the other hand, guanine is to be looked upon
as the source of xanthine. It is probable that in the
organism it is oxidized as soon as it is set free from the
nuclein, forming successively xanthiue, uric acid, urea, etc-.,
and the small quantity present in the urine is all that has
escaped oxidation. When fed to dogs, it was observed that
the amount of hypoxanthine present in the urine decreased,

14

302 BACTERIAL POISONS.

and even became less in amount than before the experiment ;
but, on the other hand, the amount of xanthine in the urine
was found to have been increased above the normal. This
shows that hypoxanthine in the body is oxidized probably
first to xanthine, then into uric acid. According to ROBERT
hypoxanthine is a true muscle stimulant.

The fact that hypoxanthine is so widely distributed in
the organism, and in much larger quantities than was
formerly supposed, shows that it constitutes, together with
the closely related bodies creatine, xanthine, guanine, etc.,
the normal antecedents of urea and uric acid. This view is
furthermore strengthened since hypoxanthine is especially
abundant in those organs which are most active in pro-
ducing metabolic changes in the body, viz., the liver and
spleen.

It may be prepared from the urine, according to the
method given under paraxanthine (page 322) ; or from
extract of meat, or from glandular organs, such as the liver,
spleen, etc., by the process on page 285. Nuclein, on de-
composition with acids, yields about one per cent, of this
base. It can be determined with adenine indirectly by
SCHINDLER'S method (page 286) ; but better still directly
by BRUHN'S picrate method (see page 286). After the
adenine has been precipitated by sodium picrate, the deter-
mination of hypoxanthine in the filtrate is not difficult if
hydrochloric and other acids, the silver salts of which do
not quite dissolve in ammonia, are absent. The filtrate
from the adenine picrate is rendered slightly alkaline with
ammonia and precipitated with silver nitrate at the boiling-
point. The slightly yellow-colored precipitate is washed
with hot water till the wash-water is colorless ; then dried
at 120° for from two to three hours, when it has the com-
position 2C5H2Ag2N4O + H2O. It contains, however, traces
of picric acid and some adenine silver, and hence the quan-
tity of hypoxanthine calculated from the weight obtained is
higher than it really is. BRUHNS, as a correction, subtracts
3.0 mg. from the calculated quantity of hypoxanthine.

A more convenient method than the preceding is to esti-
mate hypoxanthine as hypoxanthine silver picrate. The

CHEMISTRY OF THE LEUCOMAINES. 303

filtrate from the adenine pi«-rate (page 287) is raised to the
lx>iling-point and silver nitrate solution gradually added.
The precipitate is washed with cold water till the wash-
water is colorless, then dried at 100°, when its composition
is represented by the formula C5H3AgN4O.C6H2(NO2)3OH.
The calculated quantity of hypoxanthinc here is likewise
slightly higher than it should be. BRUHNS deducts 1.0
mg. from the calculated result.

in the presence of hydrochloric acid, etc., the deter-
mination of hypoxanthine is somewhat circuitous since the
precipitated silver chloride must be separated from the
!i\ pnxanthine compound. The best procedure in this case
is to saturate the filtrate from adenine picrate with am-
monia and precipitate it completely with silver nitrate.
The precipitate is washed with hot water (a thorough wash-
ing is not necessary), then it is boiled several times with
nitric acid of 1.1 specific gravity. The acid each time is
rapidly decanted on to a small filter, and finally the residue
washed on the filter with 10 c.c. of the hot acid (total 100
c.c.). To the combined acid filtrate silver nitrate is added,
and the whole set aside for twenty-four hours. The pre-
cipitate is dried at 100°. The amount of hypoxantliine
lost depends upon the quantity of silver chloride present.
The correction to be added is 3.1 mg. (BRUHNS). In
XKUBAUER-KOSSEL'S method the mixed adenine and hypo-
xanthine silver salts can be decomposed with a little hydro-
chloric acid and estimated in this way.

Hypoxanthine is a white, colorless, crystalline powder,
sometimes in part amorphous ; according to BRUHNS, pure
hypoxanthine does not form floccules and bunches of micro-
scopic needles, but usually coherent crusts, which consist of
roundish, sharp-cornered granules; some resemble quadratic
octahedra. It is soluble in about 300 parts of cold water
(STRECKER). The base separates slowly from aqueous
solutions, and when pure the solubility, even in the begin-
ning, is less than 1 : 300. At the end of four days BRUHNS
found it to l>e 1 : 1880. It is more easily soluble in boiling
water (78 parts), and, on cooling, separates in the form of
white, crystalline floccules, thus differing from xanthine,

BACTERIAL POISONS.

which is amorphous. The solubility in cold alcohol is very
slight, about 1 : 1000. It dissolves in acids and alkalies
without decomposition, and from solutions in the latter
it can be precipitated by passing carbonic acid, or by the
addition of acetic acid. The aqueous solution possesses a
neutral reaction. The free base can be heated up to 150°
without suffering decomposition, but above this temperature
it sublimes, and partially decomposes, with evolution of
hydrooyanic acid. When heated with potassium hydrate
to 200°, it yields ammonia and potassium cyanide.. Heated
with water to 200°, it decomposes into carbonic acid, formic
acid, and ammonia, and in this respect it agrees with adenine
(page 288). The properties of STRECKER'S sarkine agree
closely with those of adenine-hypoxanthine ; and, inasmuch
as the latter has been often described as hypoxanthine, it is
very desirable that the properties of hypoxanthine be re-
determined.

When evaporated with an oxidizing agent, chlorine water
or nitric acid, the residue is said to give on contact with
ammonia vapors a rose-red color (WEIDEL, murexide test).
KOSSEL, however, has shown that this is due to the presence
of xanthine, and that pure hypoxanthine does not give either
the murexide test or the xanthine reaction. According to
STRECKER, concentrated nitric acid converts hypoxanthine
into a nitro-compound, which in turn, by the action of a
reducing agent, is changed into xanthine. This statement
has not been confirmed either by FISCHER or by KOSSEL.
It does not give a green color with sodium hydrate and
chloride of lime — distinction from xanthine (page 316).

With acids it yields crystallizable compounds, and, like
the amido acids, it forms compounds with bases, and also
with metallic salts, such as silver nitrate and copper acetate.

The hydrochloride, C6H4N4O.HC1 + H2O, crystallizes in
needles, and, like the nitrate and sulphate, it is dissociate 1
on contact with water. The crystalline form is character-
istic and distinct from that of adenine, as well as adenine-
hypoxanthine. The nitrate forms thick prisms or roundish
masses, readily soluble in water and ammonia. Platinum

CHEMISTRY OF THE LEUCOMAINES. 305

chloride forms :i vellow, crystalline double salt, having the

c,,m |

The picrate f'nrnis yellow prisms easily soluble in water.
which solution is not a fleet 4 -d as that of adenine by sodium
pkmte.

Ilypoxanthine silver, C,H2Ag2N4O.H2O. All attempts
tu obtain a eorapound containing but one atom of silver
in the molecule, corresponding to the adenine compound
O5H4AgNs, have failed. The above compound was first
prepared by STRECKER, and given the formula C5H4N4O.
1 : but the former is preferable, since on heating at 120°
two and a half molecules of water are lost and

2C5H2Ag2N4O -f- H20 (Ag = 60.2 per cent.) results.

At 140°-150° it loses again in weight and becomes gradu-
ally gray ; on exposure to air it absorbs moisture. In this
tin-in li\ -poxanthinc can lie estimated quantitatively (see
I Age 302j 5 the presence of sodium picrate does not interfere,
but chlorides, etc., do. It is insoluble in hot water. The
compound, C5H2Ag2N4O.3II2O, is obtained in the form of
microscopic needles, by treating pure hypoxanthine silver
nitrate with excess of aqueous ammonia. On boiling with
ammonia-water it is but slightly dissolved, and appears to
slowly lose a part of its water of crystallization. As a
nsnlt of the decomposition one-half of the hypoxanthine
passes into solution and can be recovered on boiling with
addition of silver nitrate in the crystalline form ; or in the
cold, as the usual amorphous precipitate, CsII2Ag2N4O.H2O.
Hypoxanthine silver nitrate, C5H4X4O.AgNO3, (Ag =
.".">. •_'!» per cent.), is the best-known compound ; its formula
-talilished by STRECKER. It is obtained by dissolving
the above precipitate, produced by addition of silver
nitrate to an ammoniacal solution of the base, in hot nitric
acid, specific gravity 1.1 ; on cooling the hypoxanthine
silver nitrate crystalli/es in the form of tufts of microscopic
needles or plates. Heated at 100°-120° it remains con-
stant in weight; the quantity of silver present, when deter-
mined, is always somewhat higher than the thn>nti<al,

306 BACTERIAL POISONS.

especially if an excess of silver nitrate is employed in the
precipitation. The explanation of this fact is probably
that given under Adenine, though presence of silver chlo-
ride may partly be the cause. On treatment with am-
monia it loses not only nitric acid but also half of the
hypoxanthine, and C5H2Ag2N4O.3H2O forms. The change
takes place readily even in the cold, and if during the
digestion an excess of silver nitrate is added, the hypo-
xanthine set free is converted into this compound, which is
wholly constant in composition compared with the hypo-
xanthine silver nitrate. The conversion is quantitative.
Very dilute hydrochloric acid, as well as hydrogen sulphide,
removes the silver from this compound.
Hypoxanthine-silver picrate,

C5H3AgN4O.C6H2(NO2)3OH (Ag = 22.88 per cent.),

is gradually formed by adding silver nitrate to a boiling
solution of hypoxanthine picrate. The precipitate is granular
and of a lemon-yellow color, and consists of aggregations of
fine short needles. It is slightly soluble in hot, insoluble in
cold water. It is, therefore, applicable for a quantitative
determination of the base. Aqueous ammonia very readily
and completely removes the picric acid from the compound,
and the residue is hypoxanthine silver, which is slightly
colored yellow by a trace of picric acid ; half of the hypo-
xanthine passes into solution. Nitric acid with difficulty
converts it into hypoxanthine silver nitrate.

Hypoxanthine mercuric chloride, C5H3N4OHgCl, is ob-
tained by adding an equivalent quantity of mercuric chloride
to a boiling solution of hypoxanthine. The precipitate,
which increases on cooling, is crystalline.

A second compound, C5H3N4OHg2Cl3, is produced by
adding a strong excess of mercuric chloride, in the cold, to
an aqueous solution of hypoxanthine. It forms a heavy
granular micro-crystalline precipitate, which contains some
water of crystallization.

By boiling the preceding compound with just sufficient
hydrochloric acid to effect complete solution, there is formed
on standing a precipitate of white roundish aggregates of

CHEMISTRY OF THE LEUCOMAINE3. 307

leafy or WH-dle-shaped glittering crystals which have the
oompoflition C5H4N,< >IlgCl, + H2O.

The following table of BRUHNS illustrates the analogy
(\i-tinu between the mercury compounds of adenine and
hvp'.xantliine and similar derivatives of ammonium:

AMMONIUM. ADENINB. HYPOXANTKINI.

Mil [tjCM C.H4N5HKC1 C5HsN4OHgC1(-f H,O)

NH^Hg.Cl, aH4N5H«r,Cl, C.H.N^Hg.Cl.C+H.O)

C5H4N4OHgCl,(+H,0)

A l)rom-hyix)xanthine compound corresponding to that of
adcuinc has not been obtained.

Ken/yl-hypoxauthine, CSH,N4O.CH2.C6H5, was obtained
by THOISS by the action of nitrous acid on benzyl-adenine.
It forms a white crystalline mass which under the micro-
scope consists of thin plates. It is easily soluble in hot
water, dilute alcohol, and in acetic ether ; insoluble in ether
and chloroform. It melts at 280°. It appears, as KOSSEL
has pointed out, that adenine and hypoxanthine contain a
group, C5H4N4, which he named adenyl. The formation of
the benzyl derivatives of these two bases shows that the
hydrogen atom which is replaced occurs in the adenyl and
not in the imido group. According to this view adenine is
to be considered as adenylimide (C5H4N4.NH) and hypo-
xanthine as adenyloxide (C5H4N4O).

Phosphomolybdie acid precipitates hypoxanthine from
acid solution, and in general it gives the ordinary alkaloidal
reactions.

It is not precipitated by ammoniacal basic lead acetate.
Copprr atvtate does not precipitate it in the cold, but does
on tailing. This fact has been made use of in the isolation
of liypoxanthine. Mercuric chloride, as well as mercuric
nitrate, produces a flocculent precipitate.

Altogether, in its behavior to reagents it resembles xan-
thine to a very considerable degree. The two can be
separated. ln>\\rvcr, by the different solubilities of the
livclpK-lilorides in water, and more esp<vially of (lie silver
salt in nitric acid.

Physiological A<-/i<ni. — iM-loo mg. begin to act on Irons

308 BACTERIAL POISONS.

in from six to twenty-four hours, and produce increased re-
flex excitability and convulsive attacks ; 5-100 mg. is fatal
(FiLEHNE). When injected subcutaneously into hepato-
tomized geese or chickens a corresponding increase in uric
acid secretion is observed (v. MACH). This conversion is
analogous to that observed by STADTHAGEN in the case of
guanine (page 310), and shows that in the xanthine bodies
we have antecedents of uric acid apart from the synthesis of
the latter from ammonia in the liver. The process by which
this change is eifected is undoubtedly one of oxidation.

GUANINE, C5H5N5O, was discovered, in 1844, by UNGER,
as a constituent of guano, in which it is present in varying
quantities according to the region from which the guano
comes. Thus, the Peruvian guano is reported as containing
the largest proportion of this base, and on that account this
variety is employed when it is desired to prepare guanine.
Since its discovery by UNGER, it has been met with in a
very large number of tissues, both animal and vegetable ;
in the liver, pancreas, lungs, retina, in the thymus gland of
the calf, and in the testicle substance of the bull ; in the
scales of the bleak, and in the swimming-bladder of fish, as
well as in the excrements of birds, of insects, as the garden
spider, in which it occurs with a small quantity of uric acid
(WEINMANN), and is to be regarded as a decomposition
product of proteids formed in the tissues of the spider. It
is also found in the spawn and testicle of salmon, and
SCHULZE and others have shown it to be present in the
young leaves of the plane-tree, of vine, etc., also in grass,
clover, oats, as well as in the pollen of various plants.
SCHUTZENBERGER has isolated it, together with hypoxan-
thine, xanthine, and carnine, from yeast which had been
allowed to stand in contact with water at near the body-
temperature. Pathologically, it occurs in the muscles, liga-
ments, and joints of swine suffering from the disease known
as guanine-gout. Normally, guanine, like adenine, is
present in muscle tissue only in traces. It has never been
found in the urine, though xanthine has been mistaken for
guanine by some.

CHEMISTRY OF THE LEUCOMAINES. 309

As to the origin of th's eul stance in the- organl.-m v< i y
little has Inn kixwn up to within a i« \v yens, exe<j>t so
(in- a- it I.:-- Inn sl.e> \\ n to I c. t< Hi th( r with othe r in< mhers
of (li:s group, a (i; ii-!iiii\ j re;duet in the retrograde meta-
morphosis oi' nitn»ge nous foods and tissues. In the case of
the lower air'mals it is evidently the (iid-produet of all
change-, 'ni'smueh as it is excreted as such. Our knowledge
:;- to the immediate origin of this and the other allied l:ases
has lately hern exteneled by the brilliant researe-hcs of
K OSS EL on the decomposition products of nuelein, in whirli
he has shown that this essential constituent of all nucleated
cells whether animal or vegetable1, dee oniposrs under the
ae-tion of water or dilute aeicls into adenine, guanine, hypo-
xanthine, and xanthine. We know that the first two bases
are readily converted by the aetion of nitrous ae-id into the
other two ; that is to say, an NH group in these bases is
re-placed l>y an atom of 0 — a change which it is not at all
unlike-ly take s place in the tissues, perhaps in every cell
nucleus. That such a change is quite probable is shown by
the putrefaction experiments of SCHINDLER, whereby aden-
ine and guanine were converted respectively into hypoxan-
thine and xanthine. If this e-xplanation is correct, then
adt nine and guanine are- transition-products between the
complex proteiel molce ule on the one hand, ami hypoxan-
thine and xanthine on the other. These two, in turn, form
the connecting link te> the last -step in the- regnssive meta-
morphosis of the nitrogenous elements of the tissues, viz.,
the formation of uric acid and urea. We can thus trae'e a
sne-ee'ssion of cycles from the complex nuelein molecule,
which is apparently indispensable to the functional aetivity
of all reprodue table cells, to the physiole)gically waste pro-
ducts urea and urie- aciel.

S< HII./K and BOSSHARD recently (1886) found in young
vi teh, e-Iover, e-rge)t, < te-., a in w base, to which they have
given the name vernine. It has the formula C^H^N^Og,
and is of especial interest at this point, since on heating
with hyelroclileirie aciel it apparently yields guanine. We
have, there-lore, at ha-t t\\o \\ell-deline il sou i-e» s of guanine,
the nne-leinsjind vernine.

14*

310 BACTERIAL POISONS.

Neither adenine nor guanine occur in normal muscle
further than in mere traces, a fact which can only be
explained on the ground that the muscle tissue is poor in
nucleated cells, and hence in nuclein. Just as the muscle
cell has become morphologically differentiated from the
typical cell, it may be looked upon also as having under-
gone a concomitant chemical differentiation, inasmuch as we
no longer find the phosphoric acid, xanthine, and hypo-
xanthine in the same chemical combination as they occur in
the original cell. The phosphoric acid, instead of existing
as a part of an organic compound, is present in the muscle
tissue as a salt ; similarly the hypoxanthine and xanthine
occur in the free condition, extrattable by water, and no
longer in combination with other groups of atoms consti-
tuting a part of a more complex molecule — nuclein.

Guanine and creatine apparently mutually replace one
another. Thus, in the muscle, as just stated, guanine occurs
only in traces, whereas creatine is especially abundant.
This may find its explanation in the fact that both are sub-
stituted guanidines. Creatine is regarded by HOPPE-
SEYLER as an intermediate product in the formation of
urea, and a similar role, it will be remembered, belongs to
guanine. From STADTHAGEN'S experiments on dogs we
know that guaniue ingested, produces an increase in the
amount of uric acid and urea excreted, and the same is
also true of the nuclein derived from yeast. These results
have led him to the conclusion that in mammals uric acid
is a direct, or more or less altered cleavage product of pro-
teids, notwithstanding the fact that in birds it is the result
of synthesis in the liver.

In the decomposition of nuclein-containing substances,
such as yeast, liver, spleen, etc., by dilute acids, neither
adenine nor guanine is found alone, but they are always
accompanied by hypoxanthine, and usually by a very small
quantity of xanthine.

Guanine may be readily prepared from Peruvian guano
by boiling it repeatedly with milk of lime until the liquid
becomes colorless. The residue, consisting largely of uric
acid and guanine, is boiled with a solution of sodium car-

CIIKMISTKV OK TIIK I, K I O > M A I N ES. 311

Inmate, filtered, and tin- filtrate, after the addition of sodium
acetate, ig BtrOBgfy acidulated with hydrochloric acid. This
precipitates the guanine. together with some uric acid. The
precipitate is dissolved in boiling hydrochloric acid, and the
guanine then thrown out of solution by the addition of am-
monium hvdrate. (Juanineis also obtained in the decom-
position of mieleiu with dilute acids, and (-.in, therefore, l)e
prepared from such cellular organs as the spleen, pan-
ereas, etc., according to the method given on page 285. It
should IM- noted here that in the decomposition of the mixed
silver compounds with hydrogen sulphide or ammonium
sulphide (SCHINDLER) the guanine, often only in part, passes
into solution with adenine and hypoxanthine, and the re-
mainder is held hack in the silver sulphide precipitate. The
latter should, therefore, Ix? boiled with dilute hydrochloric
acid, and on saturating the filtrate with ammonia the guan-
ine after a while separates. That portion of the guanine
which did pass into solution with the other two bases is
separated from them by digestion with ammonia onawater-
bath. The two portions are then combined, transferred to
a filter, previously dried at 110° and weighed, washed well
with ammonia, then dried and weighed.

The free base forms a white, amorphous powder, insol-
uble in water, alcohol, ether, and ammonium hydrate;
ea-ily soluble in mineral acids, fixed alkalies, and in CM •< H
of concentrated ammonium hydrate. It can be heated to
alwve 200° without undergoing decomposition. When
evaporated with strong nitric acid it gives a yellow residue,
and this on the addition of sodium hydrate assumes a red
color, which on heating becomes purple, then indigo-blue ;
on cooling it returns to a yellow, passing through purple
and reddish-yellow shades due, according to V. BRUCKE, to
absorption of water. This is the so-called xan thine reac-
tion, and is supposed to be due to the formation of xanthine
and a uitro product. It is given l>est by guanine, then by
xanthine, and is not given by either hypoxanthine or
adenine.

Nitrons acid converts it directly into xanthine, thus:

( 'ftl I5N50 + HN02 = C6H4N402 + N2 + H2O.

312 BACTERIAL POISONS.

This reaction is identical with that of adenine, whereby
hypoxanthine is formed (see page 289). By putrefaction in
the absence of air it forms xanthine (SCHINDLER). The
change can be represented by the equation :

C5H5N50 + H20 = CSH4N402 + NH

3.

On oxidation with potassium permanganate it yields urea,
oxalic acid, and oxy-guanine. By hydrochloric acid and
potassium chlorate it is oxidized to carbonic acid, guani-
dine, and parabanic acid, according to the equation :

CO-NH HX

C5H5N50 + H20 -f 30 = | y CO + g$\ C = NH + CO2.

PARABANIC ACID. GUANIDINE.

According to STRECKER, a small amount of xanthine is
formed in this reaction, and it is quite possible that this
base is also formed on oxidation with nitric acid.

Guanine combines with acids, bases, and salts. It unites
with bases to form crystalline compounds ; and with one or
two equivalents of acid it also yields crystallizable salts.
Thus, with hydrochloric acid it forms the two salts,
C5H5NSO.(HC1)2 and C5H5N,O.HC1 + H2O. Similar com-
binations can be obtained with nitric acid. The sulphate
(C5H5N5O)2H2SO4, crystallizes in long needles, and, like
the other salts, is decomposable by water. The plat'no-
chloride, (C8H,NsO.HCl)|PtCl4+ 2H20, is readily obtained
in a crystalline condition. The silver compound is
soluble in hot nitric acid, and on cooling recrystallizes
in fine, needle-shaped crystals, having the composition
C5H5N6O.AgN03.

The solutions of the hydrochloride are precipitated by
mercuric chloride and nitrate, potassium chromate, potas-
sium ferricyanide, and by picric acid. Basic lead acetate
gives a precipitate only on addition of ammonium hydrate.
The reaction with picric acid (CAPRANICA) is said to be
very characteristic, and a means of distinguishing this base
from xanthine and hypoxanthine. It is best obtained by
adding a cold, saturated solution of picric acid to the warm

CHEMISTRY OF THE LEUCOMAINES. 313

acidulated solution of guanine, when a light, crystalline

preeipitate forms. Under the microscope it appears in

prm-il-shaped. fern-like tufts of fine, orange-yellow needles.

Physiologii -allv uuaniiir like uric acid is inert (FiLEHNE).

XANTHINE, C5H4N402, is also very widely distributed in

the organism, and has been met with in almost all the

tissues and liquids of the animal economy. Together with

hypoxanthine, guanine, and possibly adenine, it occurs in

many plants, among which may be mentioned lupine,

{ethalinm, sprouts of malt, tea-leaves (BAGINSKY), auto-

diirestion of yeast, gourd seeds, sqja beans, etc. It was

first discovered by MARCET (1819) in a urinary calculus,

and since then has been frequently found as the only or

chief constituent of many calculi. UNGER and PHIPSON

have extracted it from guano, while SALOMON has shown

it to be one of the products formed in the pancreatic diges-

tion of fibrin. SCHUTZENBERGER found it together with

carnine and hypoxanthine in the liquors from yeast. It is

a normal constituent of the urine, but is present only in

extremely minute quantities. During the use of sulphur-

baths, or after the thorough application of sulphur salves,

the quantity of xanthine in the urine is considerably in-

creased. It is likewise more abundant in the urine of leuco-

cythsemic patients, for the reasons already given on pane

283. BAGINSKI holds that the amount of xanthine nor-

mally present in the urine may be increased tenfold in the

case of acute nephritis. BENCE JONES observed in the

urine of a child sick with renal colic, a deposit of crystals

which he considered to be xanthine, but other observers

arc inclined to regard the crystals as those of hypo-

xanthine. VAUGHAN has reported the presence of xan-

thine in deposits from the urine of patients with enlarged

spleen.

Xanthine may be prepared synthetically in several ways.
Thus, it may be obtained by the reduction of nric acid by
means of sodium amalgam, according to the equation :

CftH4N403 + H2 = C5H4N402 + H2O.

T'RH- ACID. XANTHINE.

314 BACTERIAL POISONS.

Now that uric acid has been prepared synthetically, this
forms the final step in the complete synthesis of xanthinc.
By further action of nascent hydrogen the xanthine in turn
is converted into hypoxanthine. The reverse operation,
the conversion of hypoxanthine into xanthine, though re-
ported by STRECKER has not been confirmed by FISCHER
or by KOSSEL. It is, therefore, evident that these bodies
form a continuous oxidation series with uric acid as the
final product. Although this change is unquestionably the
one which goes on in the animal economy, yet all attempts
to reproduce it in the laboratory by oxidation with potas-
sium permanganate or nitric acid have apparently yielded
only negative results. Again, xanthine may be prepared
from guanine by putrefaction of the latter, or by oxidation
with nitrous acid. The change may be represented by this
equation :

C5H5N50 + HN02 = C5H4N402 + N2 + H2O.

GUANINE. XANTHINB.

This reaction, first described by STRECKER (1858), corre-
sponds exactly to the one by which KOSSEL has transformed
adeniue into hypoxanthine (see page 289).

GAUTJER, starting out on the hypothesis that xanthine
is a polymerization-product of hydrocyanic acid, has en-
deavored to prepare it directly from this compound. In-
deed, he claims to have succeeded in effecting the synthesis
of not only xanthine, but also its homologue, by simply
heating hydrocyanic acid in a sealed tube with water and a
little acetic acid, the latter being added to neutralize any
ammonia that might form. He expresses the reaction as
follows :

11HCN + 4H2O = C5H4N4O2 + C6H6N4O2 + 3NH3.

XANTHINE. METHYL-XANTHINE.

Nearly all of the methods that have been employed for
the preparation of xanthine are based upon its precipitation
as the insoluble silver compound. From the urine it can
be isolated according to the method given under paraxan-
thine, on page 322. It may also l)e obtained from the

CIIKMISTKV <>K TIIK !. K I' < '< ) M \ I N KB . 315

urine by 1 1- TM i :i>i i i: '- method. The urine, acidulated
with hydrochloric acid, ia precipitated with pho0photang0tic

acid ; the precipitate is decomposed by wanning with
liaryta, filtered, ;in<l tin filtrate Is freed from barium by the
raiitiuii.- addition of sulphuric acid. The solution is thru
made alkaline with ammonium hydrate, any traces of phos-
phates that ap|M-ar are filtered off, and finally it is precipi-
tated by addition of ammoniacal silver nitrate. The pre-
cipitate which forms consists of the silver compounds of the
\ant liine bodies, and is purified by dissolving in hot nitric
acid, as given on page 285. Xanthine has l>een shown to
IM- formed at the same time with guanine, adenine, and
hypoxanthinc, in the decomposition of nuclei n by means of
dilute acids. It may, therefore, be prepared from cellular
organs according to the metluxl given under Adenine. The
method of its preparation from tea-leaves is also given
elsewhere.

Xanthine is a white, granular, amorphous body, and is
drpo.-ited f roii i hot aqueous solution on cooling in colorless
Hoceiik's, or as a fine powder, which, under the microscope,
is seen to consist of rounded granules. When occurring in
calculi, it forms compact, moderately hard, yellow or brown
fragments, which, on being rubbed with the finger-nail,
as-unie a wax-like appearance. It is difficultly soluble in
cold water (about 14,000 parts), alcohol, and ether; some-
what more soluble in boiling water (about 1200 parts). It
is soluble in alkalies and alkali carbonates, not bicarbon-
ate, and from these solutions it is precipitated on neutral-
i/ation with acids, or by passing carbonic acid. In warm
ammonia it dissolves more readily than does uric acid or
guanine, and on cooling the ammonium compound recrys-
talli/.es. It acts as a weak ba>e, and as a weak acid ; with
salt- of the heavy metals it forms difficultly soluble or
insoluble compounds. Its basic properties, however, are
weaker than those of h\ poxanthine or guanine.

When xanthine is evaporated with nitric acid it leaves a
lemon-yellow n •sidue (hence its name), which is not changed
by ammonium hydrati — distinction from uric acid — but
with potassium hydrate I x -comes yellowish-red, on heating
purple-red. When added to a mixture of bleaching powder

316 BACTERIAL POISONS.

and sodium hydrate in a watch-glass the solution becomes
covered by a dark-green scum, which changes to a brown,
and soon disappears — distinction from hypoxauthine.

By means of a very interesting synthetic reaction, xan-
thine may be converted into thcobromine, the active con-
stituentof Theobroma cacao. Thus, the xanthine is dissolved
in a sufficient quantity of sodium hydrate, necessary to form
the neutral compound C5H2Na2^f4O2, and this product,
when treated with boiling acetate of lead, yields a white
precipitate of lead xanthine, C6H2PbN4O2. This is dried
at 130°, then heated for twelve hours at 100° with methyl
iodide, when the dimethyl derivative, C6H2(CH3)2N4O2, is
formed. This compound is identical with the natural theo-
bromine, and by a similar treatment is converted into tri-
methyl-xanthine or caffeine. The relation of xanthine to
theine (caffeine) is shown in the fact that it exists together
with hypoxanthine, adenine, and possibly guanine, in fresh
tea-leaves. It is, therefore, clear, that by starting from
guanine of guano we can produce successively xanthine,
dimethyl xanthine, and trimethyl xanthine, the last two
compounds being identical with the alkaloids of theobroma
and of coffee.

Nascent hydrogen converts this base into hypoxauthiue,
but the reverse operation, the oxidation of hypoxanthine
into xanthine, has been questioned of late by KOSSEL and
others. On heating, a small portion volatilizes ; the
greater part decomposes into ammonium carbonate, cyan-
ogen, and hydrocyanic acid. Heated to 200° with hydro-
chloric acid, it decomposes with the formation of ammonia,
carbonic acid, formic acid, and glycocoll (E. SCHMIDT).
When bromine is allowed to act on xanthine, there is
formed a substitution compound, having the formula
C6H,BrN4O2. With potassium chlorate and hydrochloric
acid it yields alloxan and urea.

Xanthine is a weak base, which dissolves in acids with
the formation of salts.

The hydrochloride, CSH4N4O2.HC1, is difficultly soluble
in water, more so than the corresponding salt of hypoxan-
thine, from which it is deposited in glistening six-sided

CHEMISTRY OF THE LEUCOMAINES. 817

plates, often forming aggregations. Its solution does not
precipitate platinum chloride. The nitrate forms fine yellow

<T\ -tak

'The sulphate, C.H.NA.HgSO, -f H2O, crystallizes in
microscopic glistening rhombic plates, decomposable l>\
water.

\\ "ith baryta water xanthine forms the difficultly soluble
compound C5H4N4O2.Ba(OH)2, which corresponds to the
hypoxanthine salt C5H4N4O.Ba(OH)2, and to that of
gnanine.

From ammoiiiacal solution, silver nitrate precipitates the
compound C,H4N4O2.Ag20, which is insoluble in ammonia,
but soluble in hot nitric acid. From the nitric acid solu-
tion, on long standing, there separates the compound
C8H4N4O2.AgNO3, which, on contact with water, decom-
poses, giving off nitric acid. The ammoniacal solution is
also precipitated by lead acetate — separation from hypo-
xan thine — also by calcium and zinc chlorides. Cupric
acetate gives a precipitate only on boiling. The aqueous
solution is not precipitated by lead acetate, but is by phos-
phomolylxlie acid, phosphotungstic acid, by mercurous and
mercuric salts. Picric acid gives an easily soluble com-
pound, which resembles that of hypoxanthine, but differs
from that of guanine.

As to the physiological relation of xanthine very little
nce<l IM> said. It l>ears the same relation to guanine that
hypoxanthine does to adenine, and, like the latter, is to be
look.-d upon as an intermediate compound, a step lower
than guanine, and nearer the limit of oxidation — uric acid.
It is quite probable that in the body it is oxidized about
as rapidly as it is formed. Like hypoxanthine, it is to
be regarded as a true muscle stimulant, especially of the
heart. (BAQINSKI). According to FILEHNE it produces
in frogs a decided muscular rigor and paralysis of the spinal
cord. The heart muscle is also affected, which is not the
ca-e with caffeine or theobromine. The fatal dose is less
than one-halt' pro mille. In its action it is stronger than
theobromine, while caffeine is weaker than either of the
two. PASOHKIS and PAL hold that the reverse is true.

318 BACTERIAL POISONS.

In closing the description of the preceding bodies it may
l>e well to present briefly our present knowledge as to thei'r
constitution. GATJTIER, starting out with the idea that
they are polymerization-products of hydrocyanic acid, has
deduced theoretically cyclic formulae, recalling the hexagon
of the benzole derivatives. These formulae, though formid-
able in appearance, are a complete failure so far as they are
expressions of chemical reactions. Thus, the formula of
guanine :

N=CH
H — CO —

HN NH

fails to show either a urea or a guanidine residue, and yet
it is a well-known fact that guanine on oxidation yields
parabanic acid and guanidine (page 312). In a similar
manner, his xanthine formula fails to show up the urea
residues which we know to be present.

HORBACZEWSKI'S synthesis of uric acid has thrown con-
siderable light upon the constitution of these bases. As a
consequence of his method of synthesis uric acid was shown
to possess the structural formula given below. E. FISCHER
has found, as a result of experimental work, the constitu-
tion of xanthine to be expressed by the subjoined formula.
We know that uric acid on treatment with nascent hydro-
gen is converted into xanthine, then into hypoxanthine.
It follows, therefore, that a relation exists between hypo-
xanthine and xanthine similar to that between xanthine
and uric acid. The formula of hypoxanthine, as deduced
from this relation, and given below, probably represents
its constitution quite closely. It is possible, however, that
the CH and CO groups will be found to occupy the
reverse position which they are given in this formula, in
which case corresponding changes must be made in the
formulae of guanine and adenine. The latter two arc b;isnl
upon the relation which. these bodies bear to xanthine and

CH KM ISTKY OF TIIK I. K I' CO M AINES.

319

hypoxanthinc, and cannot be? said to be tin- result of direct
experimental evidence.

NH— 0 — NH N==C — Nil N=C — NH

CO

CO

CH

NH-

-(

3 C

0

NH—

3 C

II
'0 N— C

J C

0

CO— NH

II 1 II
CH— NH CH— 1

^E

URIC ACID.

XANTHINI. HYPOXANTHIMB.

C,H4N403

C5H4N402 C5H4N40

N = C — NH
CO

NH— C C=NH

II I
CH— NH

GlTANINK.

C5H5N50

N=C
CH

NH

N— C C=NH

II I
CH— NH

APKNINK.

C8H5N5.

HETEROXANTHINE, C6H6N402, is a now base which was
isolated from tlie urine in 1884 by SALOMON. In its
eompontion it is niethvl-xanthine, and is intermediate

l)i 'tween xanthine and ]>araxanthine or dimethyl-xanthine.
It occurs in the urine of man and of the do£ in about the
same amount as para.xanthine, and the method for its isola-
tion will be found under the description of that base. It
is a remarkable fact that this base occurs in dog's urine
unaccompanied by parazanthine, and the same seems to
hold true for the urine of lcu< •ocytlwmic jK'rsons. SALOMON
examined the liver and muscles of a dog, but was unable
to obtain any hctcroxanthine or paraxanthine, and the total
amount of .xanthine liodics pn sent was about normal.
Hence, he is inclined to think that these two bases may
|K>"iMy have their origin in the kidney. Unlike the other
xanthine liodics, h< teroxanthinc has not as yet Ixrn isolated

320 BACTEKIAL POISONS.

from plants, meat extract, or guano. The amount of
xanthine bodies present in the urine is unaffected by phos-
phorus poisoning. Neither this base nor paraxanthine has
been found in bull's testicles ; xanthine is also absent, and
only hypoxanthine and guanine were found to be present.

Heteroxanthine forms a white amorphous powder, which
sometimes on prolonged contact with water forms micro-
scopic crystalline tufts. It is very difficultly soluble in
cold water ; much more easily in hot water, and the solu-
tion thus obtained is neutral in reaction. It is easily soluble
in ammonium hydrate, but is insoluble in alcohol and ether.
When heated it volatilizes without melting and at the same
time gives off a small quantity of hydrocyanic acid. On
evaporation with nitric acid on the water-bath (xanthine
reaction) it remains as a pure white residue, which on con-
tact with sodium hydrate develops only a trace of reddish
coloration or none at all. WEIDEL'S test (page 328) pro-
duces a splendid red color, which becomes blue on the ad-
dition of sodium hydrate. Simple evaporation with chlo-
rine water gives a similar though not so strong a color
reaction.

Silver nitrate produces in ammoniacal, as well as in
nitric acid solutions, a precipitate which readily dissolves
on warming in even very dilute nitric acid ; from this
solution, if not too concentrated, the heteroxanthine silver
nitrate compound crystallizes in well-formed plate-like
prismatic crystals. Copper acetate produces in the cold, in
solutions of heteroxanthine, a clear-green precipitate. It
is also precipitated by phosphotungstic acid, and by ammo-
niacal basic lead acetate. Picric acid does not give a yellow-
colored precipitate in solutions of the hydrochloride. Mer-
curic chloride readily precipitates heteroxanthine in the
form of a grayish-yellow compound, which on standing
twelve to twenty-four hours becomes converted into pure
white crystalline aggregations. This mercuric compound
can be converted directly into the corresponding silver
compound by the addition of silver nitrate and ammonia,
as described under paraxanthine.

The hydrochloride is characterized by its rather difficult

CHEMISTRY OF THK I, K UCOM AI NES. 321

solubility and ready crystallization (a distinction from the
paraxanthine salt). The salt forms large colorless tufts
• -I' < r\-tal-, \\liidi on contact with water soon lose their
transparency and become opaque ; gradually their crystal-
line form di. -appears, till Anally tfiey completely decom-
pose with the formation of heteroxanthine. This decora-
position is hastenetl by warming, either with or without
addition of ammonia. Platinum chloride produces in the
hydrochloric acid solution a precipitate of crystalline double
salt.

This base resembles paraxanthine in its property of
yielding a difficultly soluble precipitate with the fixed
alkali. This reaction is best brought al>out by dissolving
the heteroxanthine hydrochloride in warm dilute sodium
hydrate, when, on cooling, the corresponding sodium salt
will crystallize out in oblique-angled plates. These crystals
dissolve easily in water, and on neutralization of the
solution with an acid a dense pulverulent precipitate of
heteroxantliine forms. It can thus be distinguished from
paraxanthine, the sodium compound of which, on similar
treatment, yields the characteristic crystalline form of the
free base. This sodium reaction, therefore, distinguishes it
at once from xanthine, hypoxanthine, guanine, and para-
xanthine. It differs from the latter, as has already been
indicated, in the solubility and amorphous character of the
free base ; in the behavior of the hydrochloride and the
sodium compound, and in the not giving a precipitate with
picric acid, nor the characteristic odor given by paraxan-
thine on heating.

In its composition, hetero xanthine is, as has already
been stated, methvl-xanthhu1 and probably is related to if
not identical with an isomeric body obtained synthetically
by GAUTIEB (see page 314). The fact nevertheless re-
mains, that in the nrin • we have normally a homologous
series of xanthine bodies, namely, xanthine, hcteroxanthine,
and paraxanthine.

I'M: \ XANTHINE, C7H8N4O2, was isolated in 1883 by
SALOMON, who has since shown it to IK; a constituent of

322 BACTERIAL POISONS.

normal urine, although present in exceedingly minute
quantity. Thus from 1200 litres of urine, only 1 .2 grammes
(0.0001 per cent.) of this substance! were obtained. It has
not been found in the urine of dogs or in that of leueo-
cythsemic patients. THUDICHUM was the first to isolate
paraxanthine from the urine, and he named it urotheo-
bromine (1879).

The method employed for the isolation of this base is,
with a slight modification, that of E. SALKOWSKI, as
originally proposed for the preparation of xanthine bases
from urine. The urine in portions of 25 to 50 litres is
made alkaline with ammonium hydrate and allowed to
stand twenty-four hours. The clear supernatant fluid is
decanted from the precipitate of phosphates and treated
with silver nitrate (0.5 to 0.6 gramme per litre). The gray-
ish precipitate of xanthine compounds which forms is trans-
ferred to a filter and washed with water till free from
chloride ; it is then suspended in water and decomposed
with a current of hydrogen sulphide. The liquid is filtered
by decantatiou and the filtrate is evaporated to dryness ;
the residue is extracted with 3 per cent, sulphuric acid to
remove uric aeid ; the solution thus obtained, after it has
been rendered alkaline with ammonia, is precipitated by
silver nitrate.

A better procedure is to concentrate the filtrate directly
over the flame or on the water-bath, till the uric acid begins
to crystallize out. It is then filtered, and the filtrate, after
diluting somewhat with water, is rendered alkaline with
ammonium hydrate in order to precipitate any remaining
uric acid and phosphates. The whole is allowed to stand
one or two days, then filtered, and the filtrate again pre-
cipitated with silver nitrate. The thoroughly washed pre-
cipitate of the xanthine compounds, now free from uric
acid, is dissolved in as little as possible of hot nitric acid
of specific gravity 1.1, to which a little urea has been
added, and the clear solution is set aside for twenty-four
hours. The silver salt of hypoxanthine crystallizes from
the solution and is filtered off. It can be purified by re-
peated recrystallization from hot nitric acid, containing a

CHEMISTRY OP THE LEUCOMAINES. 323

little urea, then decomposed With hydrogen sulphide, ami
the filtrate, rendered alkaline with ammonium hydrate, is
con<vntrat<-d to a small volume. On standing, pure hypo-
xanthine crystallizes out. The filtrate from the silver salt
of hypoxanthineon being rendered alkaline with ammonium
hydrat • ^ivc- a precipitate which formerly was regarded as
consisting entirely ot' the xanthine silver compound, but
which from the investigations of SALOMON, lias, ^>een shown
to l»e a mixture of the salts of xanthine, paraxanthine,
ami heteroxanthine.

The separation of thes.- bases is efl'eeted l»y the solubility
of the free bases in ammoniuni hydrate. For this purpose
the precipitate of the mixed silver salts is decomposed with
hydrogen sulphide, and the filtrate, rendered anunoniacal to
remove traces of phosphates and oxalates, i- moderately
concentrated. After standing twenty-four hours, heteroxan-
thine crystallizes out, partly in finely formed sheaves and
tufts of needles, partly in radially striated masses. The
fluid is decanted from the crust of heteroxanthine which
forms in the bottom of the beaker, and after being concen-
trated somewhat is again allowed to stand. In tnis way a
second crop is obtained, and this is repeated till finally the
separated masses scarcely give a precipitate with sodium
hydrate. All the heteroxanthine is now united and dis-
solved in a little hot water by the aid of sodium hydrate.
After twenty-four hours the greater part of the heteroxan-
thine crystallizes out in bunches of crystals of sodium
heteroxanthine, while a small part together with any traces
of xanthine remains in solution. The crystalline ma-- i.-
dried by pressure, dissolved in a little water, and the solu-
tion neutralized by addition of hydrochloric acid, when the
heteroxanthin;1 -.-paratc- as a pulverulent precipitate. To
remove any traces of paraxan thine, dissolve in hydrochloric
acid; on standing forty-eight hours the hetcroxanthine salt
separates, while the easily soluble salt of paraxanthine
remains in solution. To obtain the pure tree hetcroxau-
t.hine, the hydrochloric salt is evaporated with ammonium
hydrate; the well-washed residue of heteroxanthine is then
di--i»lved in dilute ammonia, the solution filtered, evapor-

324 BACTERIAL POISONS.

ated slowly, and the precipitate which forms is finally washed
with alcohol and ether.

The original amraoniacal mother-liquors of heteroxan-
thine yield on further concentration amorphous floccules of
xanthine, which are remoyed by filtration ; from the filtrate,
when concentrated still more, paraxanthine crystallizes out.

Paraxanthine is obtained in colorless, glassy, generally
six-sided plates, which are arranged in tufts or rosettes;
From very concentrated aqueous solutions it crystallizes in
long, colorless, interwoven needles, which on drying exhibit
the silky lustre of tyrosin. The crystals belong to the
monoclinic system, and may crystallize with as well as
without water. If water is present on careful heating (110°)
the crystals lose their brilliancy and become whitish and
opaque, and at 120°-130° the water is completely driven
off. The conditions under which crystals containing water
are formed are not known ; probably by slow crystalliza-
tion, whereas rapid crystallization from hot concentrated
solution yields the anhydrous needles. At about 170°-180°
sublimation takes place. The melting-point is at atxnit
284° (KosSEL). It can be heated to 250° without melting
or sutfering any decomposition, but when heated more
strongly it gives oft' white vapors which possess a distinct
iso-nitril odor, at the same time it carbonizes and takes fire.
When evaporated with concentrated nitric acid, as in the
ordinary xanthine test, it gives only a slight yellow residue.
On the other hand, WEIDEL'S test, evaporation with chlo-
rine water containing a trace of nitric acid, and then placing
the dry residue into an ammoniacal atmosphere under a
bell-jar, gives a beautiful rose-red color.

It is difficultly soluble in cold water (though more easily
than xanthine); somewhat more readily soluble in hot
water, and insoluble in ether and alcohol. It is soluble in
ammonium hydrate, hydrochloric acid, and nitric acid. Its
solutions are neutral in reaction.

Silver nitrate produces in nitric acid, as well as in ammo-
niacal solutions, a flocculent or gelatinous precipitate, which
in concentrated solutions forms an almost perfect jelly-like
mass. This silver precipitate is soluble in \\ann nitric

rilKMISTRY OF THE LEUCOMAINES. 325

acid. t'r<>m which on cooling it si-parats in white crystalline
tnits possessing a silky lustre. On decomposition with
hydrogen sulphide the silver salt yields pure paraxanthine.
I'icric acid pro.lu<vs in the hydrochloric acid solution a
precipitate consisting of densely felted yellow crystalline

>pan..

It IB also precipitated bypho0phatanEBticaoid and copper
.I : mercuric chloride when added in excess gives a
precipitate composed of a mass of colorless prisms, which
are rather difficultly soluble in cold water ; easily in hot
water. The crystals of paraxanthine mercuric chloride
when moderately heated become opaque from loss of water
of crystallization; at a higher temperature they melt, under-
going at the same time partial decomposition, and on strong
heating they evolve disagreeable naussatiug vapors. The
aqueous solution of this mercuric double salt gives with
silver nitrate an abundant precipitate of silver chloride,
which disappears on the addition of ammonium hydrate
and is replaced by the floceulent gelatinous precipitate of
silver paraxanthiue. The hydrochloric acid solution of
paraxanthine crystallines with difficulty even when strongly
concentrated, and on the addition of platinum chloride it
yields a well-crystallizable orange-colored paraxanthine
platinoc-hloride. It is not precipitated by basic lead acetate
nor by mercuric nitrate.

In its behavior to the xanthme test this base ressmbles
hypoxanthin.', whereas in giving WEI D EL'S reaction it
approaches xanthine. Finally, it coincides with guanine
by yielding a precipitate with picric acid. Although it
thus agrees in some of its reactions with all three of these
xanthine bodies, it can, however, be easily distinguished
from them by its behavior with the fixed alkalies. Sodium
or potassium hydrate dissolves these basas and holds them
in solution, but when added to concentrated paraxanthine
solution the alkali proilun- a piv.-ipitate of long, glittering,
crystalline spangles, which under the micros 'op? are seen to
consist of delicate rectangular, often longitudinally striated,
plates which are either isolatea or united in tufts. Besides
tin- .-ry-tals there are also present hexagonal plates resem-

16

326 BACTERIAL POISONS.

bliug cystin. The crystals are soluble in a little water, or
ou warming, but precipitate again on cooling. Paraxan-
tliine, however, shares with heteroxanthine the property of
forming a difficultly soluble compound with the fixed alka-
lies, but can be distinguished from the latter by neutralizing
with an acid the solution of the sodium or potassium com-
pound, when, in the case of paraxanthine, there will be
obtained a precipitate of the characteristic crystals of that
base ; whereas heteroxanthine is obtained on similar treat-
ment as a dense pulverulent precipitate. This reaction is
not given by theophylline.

It is interesting to observe that paraxanthine is isomeric
with theobromiue, theophylline, and also with a body re-
cently described by FISCHER as dioxy-diinethyl-purpurine.
In its composition it is, therefore, a dimethyl-xanthine.

The physiological action of paraxanthine has been studied
by SALOMON. Injections into the muscles of 1-2 mg. pro-
duced almost at once a rigor-morlis-l'ike condition of the
muscles affected, with diminished reflex excitability without
previous increase ; 6-8 mg. introduced into the lymph sac
brings on a gradual loss of voluntary motion as well as of
reflex excitability ; the rigor is more marked in the anterior
extremities, which have a wooden or waxy consistency.
Dyspnoea is likewise an early symptom, but as soon as rigor
sets in the respirations drop far below the normal, and may
even be absent for several minutes. At times the lungs are
enormously dilated, same as in theobromine. The heart's
action is intact till the very last. In mice the reflexes are
increased almost to a tetanus. The lethal dose for frogs,
subcutaneously, was found to be 0.15-0.2 per cent, of the
body-weiglnV— somewhat lower than that of theobromine and
xanthine. The action of these three bases is very similar.
They produce in common the slow creeping movements,
followed by cessation of spontaneous muscle action, com-
plete loss of reflex excitability without a previous rise,
and the heart's action is not affected till in the latest stages.

CARNINE, C7H8N4O3, was isolated in 1871 from Amer-
ican meat-extract by WEIDEL, but has not been obtained

I'HKMISTRY OF THK LK r fu M A I \ K8. 327

from mil- 'Ic-tis- IK- it-.-lf. It has also been obtained from
yeast liquors by Scui T/KM-.KRGER, and from urine by
POUCIIET. It <"iii In- separated from the meat-extract, of
which it forms aUdit one per cent., by the following method
originally employed l»y WEIDEL. The extract is dissolved
in six or seven parts of warm water, then concent rated
baryta water is added, avoiding, however, an excess. The
filtrate ie precipitated by basic lead acetate. The precipitate
is collected, thoroughly washed and pressed, and finally it
is repeatedly extracted with a large quantity of boiling
water. The caruine lead salt is thus dissolved out; the
filtrate, alter removal of the lead by hydrogen sulphide, is
evaporated to a small volume. The concentrated solution
thus obtained is treated with silver nitrate, which givi - a
precipitate of silver chloride and of the silver salt of car-
uine. \\\ treatment with ammonium hydrate the silver
chloride etui lx> completely removed from the precipitate,
whereas the silver compound of carniue is insoluble in that
nt. To obtain pure carnine the silver salt is decom-
posed with hydrogen sulphide, and the filtrate, after purifi-
cation by bone-black, is evaporated to crystallization.

Carnine forms white crystalline masses, which on drying
become loose and chalk-like. It is very difficultly soluble
in cold water, easily and completely in boiling water, and
ivcrystallizes on cooling. It is insoluble in alcohol and
ether. The taste is decidedly, bitter, and the reaction is
neutral. The base is not precipitated by neutral lead
acetate, but is precipitated by the basic salt as a Hoccnlcnt
white precipitate, soluble in boiling water. On heating,
carniue decomposes and fakes fire, and at the same time
gives otl'a peculiar odor. It crvstalli/.es with one molecule
of water, which it loses at 10()°-110°.

The hydrochloride, (UTgN/ )3.I1( 'I. is crystalline, and
decomposes on heating with concentrated hydrochloric acid.

The platinoehloride, ( U W >3.HCl.Ptrl4, f<»nns a fine,
sandy. golden-yellow powder.

With silver nitrate, caruine unites to forma white floccu-
lent pm-ipitate, insoluble in nitric acid or in ammonium liy-
• Ir.u •. It- formula corresponds to 2(C7II7Ag\4Oj)-f-AgN03.

328 BACTERIAL POISONS.

Carnine is not affected by prolonged boiling with concen-
trated barium hydrate. Bromine water decomposes it with
the evolution ot" gas and the formation of hypoxanthine.
This change takes place according to the following equa-
tion :

C7H8N4O3 + 2Br = C5H4N4O.HBr + CH3Br + CO,.

A similar decomposition into hypoxanthine is brought about
by the action of nitric acid, though in this case oxalic acid
and a yellow body are formed. When carnine is evaporated
with chlorine water containing a little nitric acid, the resi-
due, on contact with ammonia, gives a rose-red color
(murexide test). This is due, according to WEIDEL, to
the formation of hypoxanthine, but it has since been shown
that the latter base does not give this reaction, and hence it
is due to the production of xanthine, or some similar body.
The physiological action of carnine has been examined
somewhat by BRUCKE, and according to him it is not very
poisonous. The only effect observed, when taken inter-
nally, was a fluctuation in the rate of the heart beat, though
even this was by no means definite in its nature.

A BASE, C4H5N6O, was obtained by GAUTIER from
fresh musole tissue of beef, according to the method given
on page 334, and on account of a resemblance in some of
its properties with xanthine, he named it pseudoxanthine.
This name is very inappropriate, not only because it differs
so much in its empirical Ibrmula from that of xanthine,
C5H4N4O2, but also -because the term pseudoxanthine has
already been applied by SCHUL.TZEN and FILEHNE to a
body isorneric with xanthine, which was obtained by the
action of sulphuric acid on uric acid.

The free base forms a light-yellow powder, slightly
soluble in cold water, soluble in weak alkali and in hydro-
chloric acid. The hydrochloride is very soluble, and it
forms stellate prisms with curved faces, which resemble
the corresponding salt of hypoxanthine, and to some extent,
also, the whetstone-shaped crystals of uric acid.

Like xanthine, its aqueous solution is precipitated in the

CHEMISTRY OF THE LEUCOMAINES. 329

cold l)v mrmirir chloride, silver nitrate, and by ammo-
niacal lead acetate, l>ut not by normal lead acetate. .On
evaporation with nitric acid, the residue gives, on contact
with potassium hydrate, as in the case of xanthinc, a beau-
tiful orange-red coloration (xanthine reaction). It differs
from xantliinc, not only in its empirical composition, but
also in its greater solubility, and in its crystalline form.
It is possible that this base, on account of its great resem-
blance to xanthine, may have been mistaken, at different
times, for that compound.

GERONTINE, C,H14N2, is a new base which was isolated
by GRANDIS in 1890. It has been repeatedly observed in
the form of peculiar crystals found in the cell nuclei in the
liver, particularly of old dogs. The free base is an isomer
of cadaverine, etc., and resembles it somewhat. It crystal-
lizes in needles which are readily soluble in water and alco-
hol ; possesses a strongly alkaline reaction, and yields the
ordinary alkaloidal reactions.

The hydrochloride forms prismatic crystals, which are
deliquescent and easily soluble in alcohol.

The platinochloride, CsH,4N2.2HCl.PtCl4, is soluble in
water and crystallizes in spindle-shaped needles, arranged
in rosettes. It decomposes at 115°.

The gold salt forms small needles, and is easily soluble
in water and alcohol.

It combines with one molecule of mercuric chloride to
form deliquescent cubes or rectangular prisms containing
two molecules of water of crystallization. It decomposes
above 100°. This distinguishes it from eadav< riuc, which
combines with three to four molecules of men uric chloride.
The crystals observed in the liver are probably the phos-
phate.

The new base also yields a benzoyl compound which
melts at 175°-176°.

Phyriological Action. — It seems to exert a paralyzing
action upon the nerve centres, and leaves the nerves and
muscles unaffected.

330 BACTERIAL POISONS.

SPERMINE, C2H6N, or C10HMN4 (?), is the basic substance
obtained by SCHREINER (1878) from semen, calf's In-art,
calf's liver, bull's testicles, from the organs of leucocytlue-
mics, and also from the surface of anatomical specimens
kept under alcohol. In 1888 KUNZ reported the presence
of a non-poisonous base, C2H6N, spermine or ethyleneimide
in cholera cultures. In this case it occurs, then, as a pto-
maine. A confirmation of the identity of the two bases is
necessary. Previous to this, however, it had been known
for a long time under the name of "CHARCOT-NEUMANN
or LEYDEN crystals," which are the phosphate of spermine.
These peculiarly shaped crystals have been found in the
sputa of a case of emphysema with catarrh, in the bronchial
discharges in acute bronchitis, as well as in sputa of chronic
bronchitis, in the blood, spleen, etc., of leucocythaemics and
anamics, and in the normal marrow of human bones, as
well as in human semen. Altogether it seems to have a
very wide distribution, especially in certain diseases, as in
leucocythaemia.

It can be prepared from fresh human semen in the fol-
lowing manner : The semen is washed out of linen by a
little warm water, evaporated to dryness, boiled with alco-
hol, and the insoluble portion is allowed to subside by
standing some hours. The precipitate is filtered off, washed,
and dried at 100°. This residue, containing the spermine
phosphate, is triturated, and then extracted with warm
ammoniacal water. From this solution, on slow evapora-
tion, the phosphate crystallizes in its peculiar-shaped
crystals.

The free base is obtained, on decomposing the phosphate
with baryta and evaporating the filtrate, as a colorless
liquid, which, on cooling, crystallizes. From alcohol it
crystallizes in wa veil ite- shaped crystals, which readily
absorb water and carbonic acid from the atmosphere.
They are readily soluble in water and in absolute alcohol,
almost insoluble in ether, and possess a strongly alkaline
reaction. When heated with platinum it gives off thick,
white fumes, and a weak ammoniacal odor. With potas-
sium bismuth iodide it yields orange-colored crystalline

« II K \IISTR Y OF TIIK LEUCOMAINES. 331

Moeenles, which, under tlic microscope, appear :is long,
sli:u-|), pliimos.- iiccdics — distinction from diethylencdiamine.
The aqueous solution of the base is precipitate! by phos-
phomolybdic mid phosphotungstic acids, tannic acid, uold
:in<l platinum chlorides. It cannot !><• volatili/cd from
alkaline -olution by steam without undergoing decomposi-
tion (MA.IERT and SCHMIDT). It is not poisonous.

The hydroehloride, (/2H,X.1I(11 (?), crystalli/cs in six-
sided prisms, united in tufts, and is extremely soluble in
water, alnio-t in-oluhlc in absolute alcohol and ether.

The aurorhloride, ( 'JI..N.II(1l.AuCls(?), forms shining,
LLolden-yellow, irreiiiilar plates, and when freshly procipi-
tated it is easily soluble in water, alcohol, and ether, but
the dried salt is incompletely soluble in water. The aque-
ous solution, treated with magnesium, gives oft" a sperm-
like odor. The platiuochloride crystalli/es in prisms.

The phosphate, (O2H5N)2.H3PO4-f 3H2O(?), forms prisms
and slender double pyramids arranged in rosettes. It is
ditlieultly soluble in hot water, insoluble in alcohol, easily
soluble in dilute acids, alkalies, and alkali carbonates. It
melts with decomposition at about 170°. It is probable
that the above formula does not represent the salt as found,
and from theoretical considerations LADENBURO was in-
clined to think that SCHREINER'S phosphate had the com-
position (C2H5NH)4Ca(PO4)2.

LADKXHI-RC and A MEL prepared in 1888 a compound,
cthyleneimine, which was first supposed to be isomeric with
spermine. The reaction whereby it is prepared is similar
to the one by which LADENBURG effected the synthesis of
piperidine. Kthylenediamine hydrochloride is subjc-cted to
dry distillation, when it decomposes into ammonium
chloride and the hydroehloride of the new base. The re-
action was supposed to be represented by the equation :

CH2NH2.HC1 CH2N

>XH.HC1+NH4C1.
CHjNHj.IICl CH,

Sin<v then LADKMMIK; has shown that the boiling-point
of this compound did not auree with what it should be

332 BACTERIA'L POISONS.

theoretically, if represented by the above formula. A de-
termination of the vapor density showed that the molecular
weight was twice that corresponding to the formula given,
and hence was C4H)0N2. MAJERT and SCHMIDT assuming
spermine to be ethyleneimine, as was apparently shown by
LADENBURG and ABEL'S investigation, attempted to pre-
pare the latter on a manufacturing scale with the expecta-
tion that it might be used as a substitute for BROWN-
SEQUARD'S testicular fluid. They were soon able to show,
however, that ethyleneimine did not possess the composition
assigned to it, but that it was identical with HOFMANN'-
diethylenediamine (piperazine),

This was soon confirmed by HOFMANN and by LADENBURG.
Spermine was then assumed to be identical with piperazine,
but recently (1891) MAJERT and SCHMIDT compared some
spermine from SCHREINER with their own piperazine and
found the two bases to be distinct, especially with reference
to the phosphates and the potassium bismuth iodide pre-
cipitates.

About the same time (1891) POEHL announced that the
composition of spermine was more complex than what it
had been hitherto supposed to be. He ascribed to it the
formula Cj0H26N4. The formula of the platinum salt cor-
responded to C10H26N4.4HC1.2PtCl4 ; and that of the gold
salt was represented by C,0H26N4.4HC1.4AuCl3.

From this it would appear that spermine is essentially
distinct from piperazine. The composition and structure
of this interesting base must therefore be considered as not
settled.

The nuclein of the spawn of salmon has been found
by MIESCHER to exist in a salt-like combination with a
basic substance, to which he applied the name protamine.
PICARD has found it in the same source, together with
hypoxanthioe and guanine, but no xanthine. The formula
assigned to this base is quite complex, and cannot be con-
sidered as definitely settled. Analysis of the platino-

CHEMISTRY OF THE LEUCOMAINES. 333

Pt=24.64, 01=26.45, N=15.03,C=22.80,
H=4.15, O=6.93. The hydrochloride forms an amor-
phous, hygroscopic, sticky mass.

Li: i . KS OF THE CREATININE GROUP.

The knowledge of the formation of basic substances
(ptomaines) during the putrefaction of nitrogenous organic
matter, led to a series of investigations having for their
object the isolation of alkaloidal bodies, if such existed,
from the normal living tissues of the organism. A number
of compounds possessing alkaloidal properties, such as the
xanthine derivatives, already described, had been known
for a long time, although their physiological relation to
the animal economy was little, if at all, understood.
GUARESCHI and Mosso, in the course of their researches on
ptomaines, were among the first to direct their attention to
the possible presence of ptomaine-like bodies in fresh tissues.
They obtained in those cases where the extraction was
carried on without the use of acids, only very minute traces
of an alkaloidal body (possibly choline), and an inert sub-
stance, methyl-hydantoin, which, although it can scarcely
be classed as a basic compound, is closely related to creatine,
and for this reason will be described at the end of this sec-
tion. Other Italian chemists, as PATERNO and SPICA and
MARiNO-Zuco, had also shown that the normal fluids and
tissues of the body were capable of yielding substances
alkaloidal in nature, and these were regarded by them as
identical with, or similar to, the ptomaines of SELMI.

Aririnine, C6H,4N4O2, is a base obtained by SCHULZE
from the conglutin of lupine sprouts, and according to him
it is related to creatinine and possibly to the leueoma'ines of
GAUTIER. Lysatine, C6HI3N3O2, and lysatinine, C6HnNsO,
are analogous bases, obtained by DRECHSEL from casein
(page 242). These three1 bases ("an properly be looked upon
as important sources of the nitrogenous bases found in
animals and plants.

LiKiuiKifH, in 1S()!I, discovered in normal urine an
oxidation-product of eholine, probably identical with

lo*

334 BACTERIAL POISONS.

betaine (pp. 249 and 343), and POUCHET, in 1880, announced
the presence in the same secretion of all antoin, carnine (pa^r
344), and an alkaloidal base, which, however, was not
obtained at that time in sufficient quantity to permit a
determination of its character. Subsequently he succeeded
in isolating this base as well as another closely related
body, both of which will be described in their proper
place. GAUTIER has been engaged for a number of years
in the study of the leucomames occurring in fresh muscle
tissue, and he has succeeded in isolating several nc\\
compounds.

A number of these substances are credited with possess-
ing an intensely poisonous action, and if such is the case
it is very evident that any undue accumulation of such
bases in the system, resulting from an interference in the
elimination, may give rise to serious disturbances. The
amount of these substances present in the daily yield of
the urine is very small — so small, indeed, that we must
rather look upon this small quantity as having escaped
oxidation in the body. It is well known that the living
tissues possess an enormous oxidizing and reducing power,
and, according to GAUTIER, there is constantly going on
in the normal tissues of the body a cycle of changes — the
formation of leucomames and their subsequent destruction
by oxidation, before they have accumulated in sufficient
quantity to produce poisonous effects.

The following method was employed by GAUTIER in
his study of the leucomames of muscle tissue : The
finely divided fresh beef-meat or the Liebig's meat extract
is treated with twice its weight of water, containing 0.25
gramme of oxalic acid, and one to two c.c. of commercial
peroxide of hydrogen per litre. The purpose of these
precautions is to prevent fermentation. At the end of
twenty-four hours the liquid is raised to the boiling-point,
then filtered through linen, and the residue is thoroughly
squeezed. The filtrate is again raised to the boiling-point
in order to coagulate any remaining albumin, and finally
filtered through paper. The clear liquid thus obtained is
evaporated in a vacuum at a temperature not exceeding

( IIKMISTRY OF TMK LEUCOM AINES. 335

50°, and the acid syrupy residue is extracted with 99
per (-(-lit. alcohol ; the alcoholic extract is in turn
evaporated in a vacuum, and the residue taken up with
warm alcohol of the same -tn-ngth. The filtered alcoholic
solution is set aside for twenty-four hours, and any deposit
which forms is removed by filtration ; ether (65°) is then
added as Ion;: MS a prcvipitate continues to form, and the
whole is again allowed to stand for twenty-four hem's.
The ether-alcoholic filtrate from this precipitate is evapo-
rated first on the water bath, and finally in a vacuum ;
the slight residue obtained contains a small quantity of
basic substances j assessing an odor of hawthorn.

The syrupy precipitate produced by the ether partially
• Tystalli/es on standing; a little absolute ether is then
a< Met 1, and alb -r standing several days more the liquid is
separated by means of an aspirator from the deposit of
crystals (A). These are first washed with 99 per cent.
alcohol, and then extracted with boiling 95 per cent,
alcohol. The alcoholic solution, concentrated by evapora-
tion, gives, on cooling, a deposit of lemon-yellow-colored
crystals of xantho-creatinine (B), from the mother-liquor
of which there separates a crop of new crystals (C). The
residue of the crystals (A) left after treatment with the
boiling 95 per cent, alcohol is extracted with boiling water,
which afterward gives a slight deposit of yellowish- white
crystals of 'amphi-creatinc (D). The aqueous mother-liquors
on concentration yield another deposit of orange-colored
crystals of cruso-creatinine (E). GAUTIER has, further-
more, separated three other bases from the mother-liquors
of the crystals obtained as above. Thus, a base which he
named pseudo.xauthiue is stated to have been obtained by
evaporating the alcoholic mother-liquors of B, D, E(?) in
a vacuum, taking up the residue with water, and precipi-
tating the hot solution with copper acetate. The precipitate
is decomposed with hydrogen sulphide, and the aqueous
solution, filtered while boiling-hot, yields a deposit of a
sulphur-yellow powder of pseiidoxanthine. Thus, by tin-
use of al« ohol, ether, and water, ( JATTIKI:, according to his
statement, has succeeded ill obtaining a sharp separation

336 BACTERIAL POISONS.

between these bases. The importance of the subject is such
as to require not only confirmation of the results arrived at
by GAUTIER, but also a more detailed and exact study of
the chemical and physiological behavior of these bodies.

To the physiological chemist these substances are of
especial interest because of the possible relation which they
bear to the formation of creatinc and creatinine in the
muscle. It will be seen that in the leucomames of this
group, as well as in those of the uric acid group, hydro-
cyanic acid plays a very important part in the molecular
structure of these bases. Just what the function of this
cyanogen group is, so far as the vital activity of the tissues
is concerned, we know very little, though recent investiga-
tions seem to show that the seat of the cyanogen formation
lies within the nucleated cell, and is intimately connected
with the functions of the nuclein molecule.

CRUSO-CREATININK, C5H8N4O, forms orange-yellow crys-
tals which are slightly alkaline in reaction, and possess a
somewhat bitter taste. It yields a soluble, non-deliquescent
hydrochloride crystallizing in bundles of needles; also a
soluble platinochloride which forms tufts of beautiful,
slender prisms. The aurochloride is obtained as slightly
soluble, crystalline grains, and, like the platinum double
salt, is partially decomposed on heating. It is not precipi-
tated by acetate of zinc or by mercuric nitrate, but is pre-
cipitated in the cold by solutions of alum. Zinc chloride
produces in somewhat concentrated solutions a pulverulent
precipitate which dissolves on heating, and recrystalli/es
again when it cools. Like xantho-creatinine, it is not thrown
out of solution by oxalic or nitric acid, and is thus distin-
guished from urea and guanidine ; nor is it precipitated by
acetate of copper — a distinction from xanthine derivatives.
Mercuric chloride produces an abundant flocculent precipi-
tate which on heating partially dissolves, decomposing at
the same time. Sodium phosphomolybdate gives a heavy
yellow precipitate, whereas potassium mercuro-chloridc ;m<l
iodine in potassium iodide have no effect. Potassium ferri-
cyanide is not reduced. This base differs in its composition

CHEMISTRY OF TIIH I, K UCOM AI NES. 337

from ereatinine l»y IK 'N, tin- element- of hydroeyanie aeid,
hut in its er\>talline liirni ami alkaline reaction, and some
other properties, it would seem to be elosely relate* 1 to this
latter sultstance. Heeause of this apparent relationsliip and
Men-yellow color, GAUTIER named it eruso-crcatiiiine.

XAMIIO-« I;I:AIIM.NE, CBH10N4O, is the most abundant
of niiiM'le letiroina'ines. It crystalli/es in sulphur-velln\\ ,
thin span-lrs, ei.nsisting of nearly rectangular plan's \vhieli
reseinlile somewhat those of eholesterin. It is soft and
tale-like to the toueh ; possesses a slightly hitter taste, and
when dissolved in tailing; alcohol it gives off the odor of
aeetamide, though ordinarily in the fold it has a slight
cadaveric odor. When heated, the substance evolves an
odor of roast meat, carbonizes in part, and yields ammonia
and methylamine. The crystals are amphoteric in reaction,
are soluble in cold water, and can be recrystallized from
tailing 99 per cent, alcohol.

It forms a hydrochloride crystallizing in plumose needles,
and a very soluble platinochloride ; the aurochloride crys-
tallizes with difficulty. Like creatinine, it is precipitated
by zinc chloride ; the yellowish-white precipitate dissolves
with partial dissociation on warming, and on cooling sepa-
rates as isolated or stellate groups of fine needles which
possess the composition (C8H10N40)2ZnCl2. Silver nitrate
throws down, in the cold, a Hoeculent precipitate which
likewise dissolves on heating, and recrystallizes in needles.
Mercuric chloride produces a yellowish-white precipitate.
It is not precipitated bv oxalic or nitric acid, nor by potas-
taasio-mercuric chloride, or iodine in potassium iodide.
Tannin produces in time a slight turbidity, while sodium
pboephomolybdate gives a heavy yellowish precipitate.
This base is distinguished from the members of the uric
aeid group by not giving :i precipitate with copper acetate,
not even on heating.

On gentle oxidation with potassium permanganate it is
converted into a black substance insoluble in acids and
alkalies, and resembling a/ulmic acid. By treatment with
recently precipitated mercuric oxide, it yields a substance

338 BACTERIAL POISONS.

which can be recry stall ized from boiling 93 per cent,
alcohol in needles which possess a slight alkaline reaction,
and forms a slightly soluble, crystalline platinochloride.
This new substance is precipitated from alcoholic solution
by the addition of ether, as a mass of beautiful, white, silky
needles resembling caffeine. These crystals melt at 174° ;
cafVeine melts at 178°.

Xantho-creatine, given in fairly large doses, is poison-
ous, producing in animals depression, somnolence, and
extreme fatigue, accompanied by frequent defecation and
vomiting. In its general properties this base resembles
creatinine very much, and it was on account of this resem-
blance and its yellow color that it was named xantho-crea-
tinine. This relation becomes especially evident since this
base appears in the physiologically active muscle at the
same time with creatinine, constituting sometimes one-tenth
of the creatinine present. MONARI has found this base in
the aqueous extract of the muscles of an exhausted <!<>;r,
and also in the urine of soldiers tired by several hours'
march. He also demonstrated its presence in the urine of a
dog after previous injection of creatinine.

AMPHI-CREATINE, C9H,9N7O4, is slightly soluble and
crystallizes from boiling water in yellowish-white oblique
prisms, which possess, if any, a slightly bitter taste.
When heated to 100° it decrepitates somewhat, and at
110° it becomes opaque white. Potassium hydrate does
not decompose it in the cold. Although a weak base, it
combines to form salts just as the preceding members of
this group. The hydrochloride is crystalline, and is not
deliquescent; the platinochloride forms rhombic plates,
which are soluble in water, but are insoluble in absolute
alcohol ; the aurochloride crystallizes in easily soluble, very
small, microscopic crystals, which are tetrahedral to hexa-
hedral in their habit. It is not precipitated by copper
acetate or by mercuric chloride ; nor does it give the
murexidc test, or the xanthine reaction. Sodium phospho-
molybdate produces a yellow, pulverulent precipitate. In
its properties it resembles creatine, and indeed GAUTIER

CHEMISTRY OF TIIK LEUCOMAINES.

think-; it may !«• possibly a combination of creatine,
( ,1 1 ,N3O,, and a l>a-« < .II10N4O2, which, it will be seen,
differs from tin- former only by a HCN group. This
second ((impound, if it really exists, has an analogy in
criiHi-creatininc, the relation of which to creatinine may be
exjin s-cd by the equation :

C6H8N40 = C4H7N30+HCN.

CRUSO-CBBATININB. CHKATININK.

In a similar manner, amphi-ereatine may be regarded as
C9H19N7O4 = 2C4H9N302+HCN.

AMPHI-CRKATINB. CRBATINB.

A BA-K, ( 'MHj4NIOO5, was isolated by GAUTIER from
the mother-liquors of xantho-ereatinine. It crystallizes in
colorless or yellowish, thin, apparently re< tanjinlar plates,
which an- tastehss, and possess an amphoteric reaction.
The hydrochloridc forms bundles of fine needles; the sul-
phate yields a confused mass of ne«ll«s; the platinochlo-
ride is soluble, non-del iqiiescent, and erystalline. When
heated with water in a sealed tube at 180°-200°, it gives
otl ammonia and carbonic acid, and is converted into a
new base, which, however, lias not l>een studied. This
reaction may be expressed by the expiation :

CnH24N1009 = 2C8H10N402+CO(NH2)2.

UREA.

The urea which at first forms, is, in turn, decomposed,

thus :

CO(NH2)2+H30 = C()2+2NH3.

It is to !H' observed that this base ditters iu OOmpOBttioa
from the following one by HCN, the hydrocyanic acid

molecule.

A l'>\-i:, CjjHjjNnOj, was obtained from the mother-
liquors of eruso-ercatinine, and forms rectangular silky
|)lati.-. i'( .-einbling those of the preceding base and of
.xantho-ereatinine. It forms erystalli/able salts.

These complex bases will require further study in order

340 BACTERIAL POISONS.

to elucidate their physiology, and the possible connection
which they may have with the formation of urea, and of
the creatinine derivatives already described.

METHYL-HYDANTO*N, C4H6N2O2, = co\.' OCT

— This substance was obtained by GUARESCHI and Mosso
(1883), by extracting fresh meat with 1-1.5 volumes of
water (without addition of acid), for two hours at 50°-60°.
The aqueous extract was evaporated on the water-bath and
the residue was extracted with 95 per cent, alcohol. This
alcoholic solution, after the alcohol was driven off, was
taken up in water, filtered, and the aqueous solution was
first extracted with ether, then rendered alkaline with
ammonia, and again extracted with ether. The alkaline
ether extract gave on evaporation a white crystalline residue
of methyl-hydanto'in. The amount of this substance
present in flesh appears to be quite variable, since, at times,
none whatever can be extracted. ALBERTONI has isolated
it from dog's flesh. Previous to its discovery in flesh by
GUARESCHI and Mosso, it was known for a long time as a
decomposition-product of various nitrogenous bases of the
body. Thus, NEUBAUER prepared it by heating creatin-
ine with barium hydrate, while HUPPERT obtained it by
fusing together sarcosine with urea. As it occurs in muscle
it is probably derived from the creatine, though under
what conditions this splitting up takes place is not definitely
known. Acetic and lactic acids are incapable of effecting
this change. At all events, it belongs to the ureides, and
is intermediate between creatinine, sarcosine, and urea
Compare the above formula with that of creatinine, p. 226.
Methyl-hydanto'in forms prisms which are easily soluble
in water and alcohol, and but slightly soluble in cold ether.
It melts at 156° (SALKOWSKI) ; at 159°-160° (GUARESCHI
and Mosso). Its aqueous solution is slightly acid in reac-
tion. On strong heating it volatilizes. When fused with
potassium hydrate it gives off ammonia; it reduces mercuric
nitrate in the cold. Treated with mercuric oxide it assumes
an alkaline reaction, and the filtrate on heating yields

CHEMISTRY OF THE LEUCOM AINES. 341

metallic mercury. Witli silver oxide it forms pearly lanceo-
late plates having tin- composition C4H5N2O2.Ag. It does
not give any alkaloidal reactions.

UNDETERMINED LEUCOMA¥NES.

Leucoma'ines of Expired Air.

It was shown at quite an early period that exhalations
from animals contain, besides an increased amount of car-
bonic aeid, some organic matter, the nature of which, on
account of the exceedingly minute quantity in which it
occurs, has never been satisfactorily determined. Never-
theless, various observers did not hesitate to ascribe to it
the ill effects consequent upon breathing impure air, while
at the same time the carbonic acid formed during respira-
tion was considered as either entirely inert or as insignifi-
cant in its action. Thus, respired air from which moisture
and carbonic acid have been removed, but which still contains
the organic vapors, has been found to be highly poisonous.
On the other hand, if the respired air is drawn througli
a red-hot tube to destroy the organic matter, the air thus
purified is capable of sustaining life even in presence of a
large percentage of carbonic acid. While it cannot be,
then-fore, doubled that the organic matter of expired air
plays a most important part in producing the well-known
noxious effects resulting from breathing confined and vitiated
air, nevertheless it would seem from experiments made by
ANGUS SMITH that the increase of even such small quanti-
ties of carbonic acid in the air, as from 0.04, the normal
amount present, to 0.1 per cent., is capable of producing
systemic disturbances characterised by a decrease in the
pulse-rate ami an increase in the rate of respiration.

SMITH is consequently of the opinion that the constant
lowering of the pulse in impure air occasioned by the pres-
ence of carbonic acid must have a depressing effect on
the vitality. Whatever ill effects the carlnmic acid may
produce of itself, it remains certain that this gas is not the
most potent and most injurious constituent of respired air ;

342 BACTERIAL POISONS.

and the investigations of HAMMOND, NOWAK, SEEGEN,
and others, point conclusively to the organic matter as the
direct and immediate agent which produces those symp-
toms of sickness and nausea experienced in badly ventilated
closed rooms.

Of special importance to the sanitarian and physician is
the work on the nature and action of the poisonous principle
of expired air which has been done by BROWN-SEQUARD,
D'ARSONVAL, and R. WURTZ. The first two observers
found that the vapors exhaled by dogs, when condensed,
and the aqueous liquid (20-44 c. c.) thus obtained was in-
jected into other animals, death was produced, generally
within twenty- four hours. The symptoms observed were
dilatation of the pupil, increase of heart- beat to 240-280
per minute, which may last for several days or even weeks,
while the temperature remains normal ; the respiratory
movements are generally slowed, and usually there is ob-
served paralysis of the posterior members. Choleraic diar-
rhoea is invariably present. As a rule, it appears that
larger doses cause labored respiration, violent retching, and
contraction of the pupil. A rapid lowering of temperature,
0.5° to 5°, is sometimes observed. These same symptoms,
apparently in aggravated form, were obtained when the
liquid had been previously boiled for the purpose of de-
stroying any germs that might be present. The appearances
presented on post-mortem were much like those observable
in cardiac syncope.

The above work has been confirmed, in part, by R.
WURTZ, who, by passing expired air through a solution of
oxalic acid, has obtained besides ammonia a volatile organic
base which is precipitated by BOUCHARDAT'S reagent and
by potassio-mercuric iodide. It is said to form a platinum
double salt crystallizing in short needles, and a soluble
gold salt. When heated to 100° it gives off a peculiar
odor. This basic substance may properly be looked upon
as a leucoma'ine.

DASTRE and LOYE and LEHMANN and JESSEN have
repeated the above experiments with wholly uegative re-
sults. It is possible that the most highly poisonous sub-

CIIKM1STKY OF THK I, K I TO M A I \ KS. 343

stands formed in the b<»dy when there is an insufficient
air supply an1 not eliminated in the exhaled air.

r-air, according to observations made l»y ODLINO,
contains a basic substance which is probably in com position
a com pon no! aiunionia. It contains, however, more carbon
than methylamine and less than ethylamine.

It should be remarked that JACKSON has (Dee, 1887)
announced the presence in expired air of ((iiantities of car-
lion monoxide <i~as sufficient to produce the ill effects ordi-
narily attributed to the organic matter. The presence of
this poisonous gas must first be fully demonstrated l>efore
it can be taken into account in the consideration of the
toxieity of air ; certainly, even if present, it cannot explain
the results obtained by the French investigators as stated

above,

According to lLOSVA, expired air contains nitrous acid.
This may possibly be derived from that which is constantly
Ix HIM formed in the month, probably by the reduction of
nitrates ( MILLER).

Lcucoma'i'nes of the Urine.

A number of basic substances have IKVII isolated at
different times from the urine, and on that account they
may be properly classed as leiicoma'ines. Thus, LlEBREICH
(1869) found in the urine a base which apparently was an
oxidation-product of eholine, and which has since been
regarded as identical with U-taine. In 18GG DlJPRfc and
I>KN< K .IO.NK.S found, among other thing! in the urine, an
alkaloidal body which in sulphuric acid solution possessed
a bine fluorescence (see p. .%>>47). Most of the meml>ers of
the uric acid <;Tonp of leucoma'incs have Ix^en detected in
the urine and on account of their well-defined nature they
are described by themselves. In the urine and fo
cystinuria UDKANS/K v and BAUMANN discovered the well-
known ptomaines, cadaverine and pntrescine. For isola-
tion, sec j)|t. 207 and 208.

In 1x7!), THUDICHUM annoumx-d the presence in the
urine of four new alkaloids, one of which, urotheobromine,

344 BACTERIAL POISONS.

was subsequently rediscovered by SALOMON and named
paraxanthine (page 321). Another base which was ob-
tained, namely, reducine, yielded a barium salt which readily
reduced the salts of silver and mercury. Its formula prob-
ably corresponds to C12H24N6O9 or C6HUN3O4. A third
alkaloid, parareducine, formed a zinc compound having the
composition C6H9N3O.ZnO. A fourth base is said to give
a compound with platinum chloride and to contain an aro-
matic nucleus (aromine). Besides these four bases THUDI-
CHUM describes two other substances which he considers
basic. These are urochrome, the normal pigment of the
urine, and creatinine.

In 1880, POTJCHET announced the presence of carnine,
C7H8N403, and of another base which he subsequently ana-
lyzed and found to have either the composition C7H,2N4O2
or C7H14N4O2. This substance formed deliquescent fusi-
form crystals, sometimes crystallized in bundles or irregular
spheres, which possessed a slightly alkaline reaction and
combined with acids to form cry stall izable salts. It was
soluble in dilute alcohol, almost insoluble in strong alcohol,
insoluble in ether. The hydrochloride yielded double salts
with gold chloride, platinum chloride, and mercuric chlo-
ride. The platinochloride formed deliquescent golden-
yellow rhombic prisms. This base occurred in the dialysate
(see page 265). From the non-dialyzable portion, POUCHET
obtained another base corresponding to the formula
C3H5NO2, which he calls the "extractive matter of urine."
It yields precipitates with the general alkaloidal reagents,
is non-crystallizable and is altered on exposure to air and
resinified by hydrochloric acid. On the addition of plati-
num chloride it is rapidly oxidized, but does not yield a
platinochloride. The same author regards the urine as
containing very small quantities of some pyridine bases
which are analogous or identical with those obtained by
GAUTIER and ETARD from decomposing fish.

The distinguished Italian toxicologist SELMI was, per-
haps, the first to draw attention to the probable formation
of basic substances in the living body during those patho-
logical changes brought on by the presence of pathogenic

CHEMISTRY OF THE LEUCOMAINES. 845

MS; and in a memoir pre-nited to (In- Academy of
of Bologna, in I)eceml>er, 1880, he announced
that intivti) ins diseases, or those in which tin-re omirs an
internal disarrangement of some element, either plasmic or
histol<>M-ical, must !><• iooompanied or followed by an elirai-
nation (if more or less characteristic products, which would
!><• a sign of the pathological condition of the patient. To
support this theory he examined a number of pathological
urines, and succeeded in obtaining from them basic sub-
stances, some of which were poisonous, others not. Thus,
a specimen of urine from a patient with progressive paraly-
sis gave two basis strongly resembling nicotine and coniine;
from other pathological urines the bases obtained usually
had either an ammoniacal or trimethylamine odor. A
strong confirmation of SELMI'S theory is seen in the obser-
vations made by BOUCHARD, VILLIERS, LEPINE, GAU-
TIER, and others, all of whom have found basic substances
in the urine of various diseases.

It is now a well-established fact that the urine of disease,
as cholera (BOUCHARD) and septicaemia (FELTZ), etc., is i'ar
more poisonous than normal urine. That poisons which
are generat<xl within the body by the activity of bacteria
can be excreted in the urine is seen in the fact that im-
munity to the action of bacillus pyocyaneus has been con-
ferred on animals by previous injection of urine taken from
animals inoculated with that bacillus (BOUCHARD) or with
filtered cultures of the same (CiiARRix and RUFFER).

Unfortunately, none of these bases supposedly character-
istic of pathological urines have been isolated in a chemi-
cally pure condition ; nor has the study of normal urine
'been carried sufficiently far to show the positive absence of
such bodies.

VILLIERS has denied the existence of alkaloids in normal
urine, and this has been confirmed experimentally by
STADTHAGEN, who, moreover, agreed with FELTZ and
HITTER that specific organic poisons are absent from nor-
mal urine. The observed physiological action is there-
fore largely (70-80 per cent.), or wholly, due to the potas-
sium salts present.

346 BACTERIAL POISONS.

Leucoma'ines of the Saliva.

According to the statement of GAUTIER (1881), normal
human saliva contains divers toxic substances in small
quantities which differ very much in their action according
to the time of their secretion, and probably according to
the individual gland in which they are secreted. The
aqueous extract of saliva at 100° is poisonous or narcotic
in its action toward birds. To show the presence of basic
substances, the aqueous extract was slightly acidulated with
dilute hydrochloric acid, then precipitated by MAYER'S
reagent ; the precipitate was washed, then decomposed by
hydrogen sulphide, and the solution filtered. The filtrate
on evaporation gave a residue consisting of microscopic
slender needles of a soluble hydrochloridc. This salt,
purified by extraction with absolute alcohol, forms soluble
crystalline, but easily decomposable double salts with
platinum chloride and with gold chloride. The solution
of the hydrochloride produces an immediate precipitate of
Prussian blue in a mixture of potassium ferricyanide and
ferric chloride, and when injected into birds produces a
condition of stupor.

Leucoma'ines from other Tissues of the Body.

SELMI'S work upon the formation of ptomaines during
the process of putrefaction led many investigators to doubt
the production of these bases by the decomposition of the
proteid or other complex molecules. To substantiate this,
a number of chemists, especially Italian, endeavored to
show that SELMI'S bases, to a large extent at least, exist
preformed in the various tissues. PATERNO and SPICA
(1882) succeeded in extracting from fresh blood as well as
from fresh albumin of eggs substances identical, or at least
similar, to those designated under the name of ptoma'iiics.
Their observations, however, were confined to the detection
of alkaloidal reactions in the various extracts obtained by
DRAGENDORFF'S method, and at no time were they in
possession of a definite chemical individual. MARINO-

I'll KM 1ST BY OF THE I. K T . :« ' M A I N KS. 347

/!<•<> I Iss.", ) \\-;H more snooeBlftll, inasmuch as lie succeeded
in obtaining fr<>m fresh tissues and organs relevant quan-
tities of a liase identical with choline, and, in addition, he
obtained extremely minute traees of other alkaloidal bodies.
One of these, obtained by the STAS method from tlie liver
and spleen of an <»x, exhibited in hydrocliloric acid solution
a beautiful violet fluorescence resembling very much that
of the salts of quinine. A similar base, probably identical
with this one, was obtained by BENCE JONES and DUPRE
(1856) from liver, nerves, tissues, and other organs, and
was named by them " animal chinoidine." A greenish-
blue fluorescence is frequently observable in the alcoholic
extracts of decomposing glue as well as from other putrefy-
ing substances. From a number of very thorough experi-
ments, he concluded that basic substances do not preexist
in fresh organs, but that the acids employed in the process
of extraction exert a decomposing action upon the lecithin
present in the tissues, resulting in the formation of choline.
II. further showed that the method of DRAOENDORFF, on
account of the larger quantity of extractives which form,
invariably gave a larger yield of this base than did the
STAS-OTTO method. Similar observations were made by
GUARESCHI and Mosso, by COPPOLA and others. At the
present time there is no doubt that some basic substances,
among these eholine, can be formed by the action of re-
agents. Mini, on the other hand, it is equally well demon-
strated that similar bases do preexist in the physiological
condition of the tissues and fluids of the body.

Recently R. WURTZ has obtained from normal blood M
number of crystalline products of alkaline reaction, which
form well-crystallixable double salts with gold, platinum,
and mercuric chlorides. These, however, have not been
a- yet subjected to analysis, because of the minute quan-
tities which were isolated.

MORELLE (1886) showed the presence in tin- spleen of
the ox of a base, the hydrochloride of which crystallised
in deliquescent needles and likewise formed crystalline
platino- and aurochlorides. 1'Yom experiment- made by
KAI;«»I:I>K, the base would seem to possess decided toxic

348 BACTERIAL POISONS.

properties, bringing on a dyspnoeic condition with con-
vulsive movements and loss of motion. The post-mortem
examinations revealed an extended visceral oadematous
infiltration, and stoppage of the heart in systole.

A. W. BLYTH has claimed to have isolated from milk
two alkaloidal substances, namely galactine, the lead salt
of which is said to have the formula Pb2O3C64Hi8N4O2s,
and lactochrome, the mercury salt of which is represented
by the formula HgOC6H18NO6.

Leucomdines of the Venoms of Poisonous Serpents.

The study of the chemistry of the venoms of serpents
and of batrachians is fraught with so many difficulties and
with so much danger, that we cannot wonder at the present
unsatisfactory condition of our knowledge in regard to the
poisonous principles which they contain. Much of the work
that has been doue hitherto is not only inaccurate and very
contradictory, but is far from meeting the requirements of
exact toxicological research. From recent investigations
it seems, however, to be quite certain that the most active
constituent of the venom of serpents is not alkaloidal in
its nature as has been supposed by some. In 1881
GAUTIER announced the isolation of two alkaloids from
the venom of the cobra which gave precipitates with tannin,
MAYER'S reagent, NESSLER'S reagent, iodine in potas-
sium iodide, etc. They formed crystallizable platinochlo-
rides and aurochlorides, and also crystalline, neutral, some-
what deliquescent hydrochlorides. The neutral or slightly
acid solutions produced an immediate precipitate of Prus-
sian blue in a mixture of potassium ferricyanide and ferric
chloride. These substances possess a decided physiological
action, though GAUTIER himself does not consider them
to be the most dangerous constituents of the venoms. This
observation of GAUTIER as to the presence of distinct basic
substances in venoms is at variance with that of WOLCOTT
GIBBS, who has been unable to obtain an alkaloid from the
rattlesnake (Crotalus) venom. S. WEIR MITCHELL and
E. T. REICHERT likewise state that they have been utterly

CHEMISTRY OF THE LEUCOMAINES. 349

unable to suletantiate GAUTIER'S statements. Still more
ntly WOLFENDEN, in an elaborate paper on the nature
of col>ra venom, has confirmed WoLOOTT GIBBS as to the
entire absence of any ulkaloidal Ixxly.

MITCHELL and REICHERT have made a careful study of
tin- venoms of various serpents, such as cobra, rattlesnake,
moccasin, and Indian viper, and have succeeded in isolating
two proteid constituents, one belonging to the class of
globulins and the other to the peptones. The peptone is
said to be non-precipitable by alcohol. According to
them, the globulin constituent consists of at least three
distin.'t globulins. They found that boiling coagulates
and destroys the globulin as a poison, but leaves the
venom peptone toxically unchanged, so that the solution,
though still poisonous, fails to produce the characteristic
local lesions due to fresh or unboiled venom. On the other
hand, GAUTIER asserts that the venom is not sensibly
alt-red on being heated to 120°-125°, and that the toxic
action ivinains constant even when all the proteid con-
stituents aiv removed, thus showing that the toxic action
cannot be attributed to the albuminoids. The venom pep-
tone from the rattlesnake or the moccasin, however, when
injected into animals produced toxic effects which were
marked by an oedematous swelling over the site of injection ;
the tumor was filled with serum, and so also was the sub-
cutaneous cellular tissue. Furthermore, a gradual breaking
down of the tissues occurred, accompanied by rapid putre-
factive changes and a more or less extensive slough. That
peptones may possess intensely poisonous properties has been
shown to be the case by a number of authors, among whom
may be mentioned ScHMiDT-MiJLHEiM, HOFXIBISTER,
POLLITZER, and others. BRIEOER has, moreover, demon-
strated that the formation of peptones in the process of
digestion is accompanied by the development of a toxic
ptomaine which he has named paptotoxine.

The venom globulins, on the other hand, though present
in less quantity than the peptones, induced the same re-
markable local effects seen on injection of the pure venom.

16

350 BACTERIAL POISONS.

They cause local bleedings, destroy the coagulability of the
blood, and rapidly corrode the capillaries.

These results of MITCHELL and REICHERT, which are
given here somewhat in full, have been questioned by
WOLFENDEN, who, while agreeing in the main that the
poisonous property of venom is due to proteid constituents,
regards their peptone not as a true peptone, but rather as
one or more bodies of the albumose group of proteids. He
likewise regards the globulin of moccasin venom to be
some other proteid body. According to him, the cobra
venom owes its toxicity to the proteids, globulin, serum-
albumin, acid albumin. Occasionally there seem to be
present traces of peptone and of heraialbumose.

BRIEGER was at first apparently inclined to believe that
the action of venoms is due to animal alkaloids, on the ground
that these bases are extremely soluble, and hence always go
into solution along with the likewise very soluble proteid
constituents, and that the difficulty in their isolation lies in
the elimination of these proteids. Since then BRIEGER
and FRANKEL pointed out the poisonous nature of some
bacterial proteids, and also showed that cobra poison yields
with alcohol a precipitate which gives proteid reactions.

The proteids of serpents' venom should be compared
with the poisonous proteids formed by the activity of the
pathogenic bacteria, as well as with similar compounds,
the phytalbumoses of castor seeds, jequirity, etc. Possibly
similar compounds will be found in croton and other
species of ricinus, jatropha, loco-weed, etc. The poisons
secreted by certain spiders and fish may be mentioned in
this connection.

CLOEZ and GRATIOLET in 1852 examined the poison
contained in the cutaneous pustules of some batrachians,
and succeeded in extracting a substance which gave a white
precipitate with mercuric chloride and formed a platinum
double salt. Beyond this meagre information very little is
known in regard to the character of these poisons, though
ZALESKY, in 1866, announced the isolation of an alkaloid
to which he assigned the formula C34H60N20S, and which
he named salamandarine. According to DUTARTRE (1890)

i-HKMISTRY OF THE LEUCOMAINES.

351

this base is a Icucoma'ine. aud similar products, but with
dim-rent physiological action, are to be found in other
hatraehians, as the toad, triton(?), green and red frogs,
and in the epidermis of some fish. According to CALMEIL,
the poison from the toad contains methyl-carbylamine and
isocyanacetic acid.

TABLE OF LEUCOMAINES.

Formula.

Name.

Discoverer.

Source.

Physiological action.

C,H»NJ

Adenine.

Kossel.

Nucleln-contain-

Non-poisonous; muscle

ing organs.

stimulant.

C& H, N4 O Hypoxanthine.

Schorer.

N in l.-in-ci in ta iu-

Non -poisonous; muscle

ing organs.

stimulant.

C» Hg Ng 0

Guanine.

Unger.

Nuclein-contain-

Non-poisonous ; muscle

ing organs,

stimulant.

guano.

CgH4N40,

Xantbine.

Marcet.

X ur If iu-enii in! n-

Non-poisonous; muscle

ing organs, | stimulant.

cal.-.uli.

C, II. N4 0,

Heteroxanthine.

Salomon.

Urine.

C, H8 N4 0,

Paraxanthine.

Thudichnm

"

Poisonous.

Salomon.

C7 Hg N4 0, Carnine.

Weidel.

Liebig's meat

Non-poisonous ; muscle

extract.

stimulant.

C4 H6 N( 0 Pseudoxanthine(?)

Gautier.

Muscle.

Cg II ,N. Gerontine.

iJr.ui.li-.

Liver of dog*.

Poisonous.

C, Hg N ( ? ) Sperraine.

Schreiner.

Sperma, in tis-

Non-poisonous.

sues of leuco-

cytbaemics.

Cg H8 N4 0

Cruso-creatiniue.

Gautier.

Muscle.

Cg 1 1 N4 O ' Xantbo-creatiniue

"

"

Poisonous.

C, H)»Nj 04 Amphi-creatine.

"

"

CiiH«N1006

Unnamed.

"

"

C, H,,N4 Os

«

Pouchet.

Urine.

C, H6 NO,

"

"

"

CMUMN, Og Salamandarine.

Zalenky.

Salamander.

Poisonous.

CHAPTER XIII.

THE AUTOGENOUS DISEASES.

ALL living things are composed of cells. The simplest
forms of life are unicellular, and in these all the functions
of life devolve upon the single cell. Absorption, secretion,
and excretion must be carried on by the same cell. A
collection of unicellular organisms might be compared to a
community of men with every individual his own tailor,
shoemaker, carpenter, cook, farmer, gardener, blacksmith,
etc. However, only the lowest forms of life are unicellular;
all others are multicellular. In the higher animals there
is a differentiation not only in the size and structure of the
cells, but in the labor which they perform. The body of
man may be compared to a community in which labor has
been specialized. Certain groups of cells, which we desig-
nate by the term " organ," take upon themselves the task
of doing some special line of work, the well-doing of which
is essential to the health, not only of that group, but of
other groups as well, or of the body as a whole. There
is an interdependence among the various organs. Certain
groups of cells supply the fluids or juices which act as
digestants, and among these there is again a division of
labor. The salivary glands supply a fluid which partially
digests the starch of our food ; the peptic glands supply
the gastric juice which does the preliminary work in the
digestion of the proteids ; while the pancreatic juice com-
pletes the digestion of the starches begun in the mouth, of
the proteids begun in the stomach, and does the special
work of emulsifying the fats. But even some of these
products of complete digestion would be harmful should
they enter the circulation unchanged. The peptones must
be con verted into serum-albumin by the absorbing mechan-
ism of the walls of the intestines, and while 10 per cent.

IIIK AUTOGENOUS DISEASES. 353

of the fat of the food is split up into glycerin and fatty
acids by the action of the pancreatic juice, a much smaller
]>••!• n lit. enters the thoracic duct in this divided form.
The food may lie taken in proper quality and quantity ;
the digestive juices may do their work promptly and
properly, but if the absorbents fail to perform their func-
tions properly, disease results. It may happen that the
failure lies in improper or imperfect assimilation and the
result becomes equally disastrous, and with the effects of
non-elimination we are fairly conversant. Of the myriads
of cells in the healthy human body there are none which
are superfluous. It is true that among these ultimate
entities of existence, death is constantly occurring, but in
health regeneration goes on with equal rapidity and each
organ continues to do its daily and hourly task. The
micm-copc h;i> made us familiar with the size and shape
of the various cells of the body, and students of pathology
have described the alterations in form and size character-
istic of various disease states. But we must remember
that in the study of these ultimate elements of life there
are other things, besides their morphological history, to
investigate. They are endowed with life, and they, as well
as the germs, have a physiology and chemistry which we
know but slightly. They are influenced beneficially or
harmfully, as the case may be, by their environment.
They grow and perform their functions properly when
supplied with the needed pabulum. They are not immune
to poisonous agents. They are injured when the products
of their own activity accumulate about them.

The object in writing this chapter has been to collect
what evidence we may concerning those diseases which
arise from imperfect or improper activity of the cells of the
body, not due to the introduction of foreign cells. To
designate this class of diseases we have selected the word
autogenous, and we understand that in these diseases the
materies morbi is a product of some cell of the body, and
not, as in the case of the infectious diseases, of cells intro-
duced from without the body.

It is true, without exception so far as we know, that the

354 BACTERIAL POISONS.

excretions of all living things, plants and animals, contain
substances which are poisonous to the organisms which
excrete them. A man may drink only chemically pure
water, eat only that food which is free from all adultera-
tions, and breathe nothing but the purest air, free from all
organic matter, both living and dead, and yet that man's
excretions would contain poisons. Where do these poisons
originate? They are formed within the body. They
originate in the metabolic changes by which the complex
organic molecule is split up into simpler compounds. We
may suppose — indeed, we have good reasons for believing —
that the proteid molecule has certain lines of cleavage along
which it breaks when certain forces are applied, and that
the resulting fragments have also lines of cleavage along
which they break under certain influences, and so on until
the end-products, urea, ammonia, water, and carbon-dioxide
are reached ; also that some of these intermediate products
are highly poisonous has been abundantly demonstrated.
The fact that the hydrocyanic acid molecule is a frequent
constituent of the leucoma'ines is one of great significance.
We know that chemical composition is an indication of
physiological action, and the intensely poisonous character
of some of the leucomaiues conforms to this fact. It
matters not whether the proteid molecule be broken up by
organized ferments, bacteria, or by the unorganized fer-
ments of the digestive juices, by the cells of the liver or by
those still unknown agencies, which induce metabolic
changes in all the tissues — in all cases poisons may be
formed. These poisons will differ in quality and quantity
according to the proteid which is acted upon, and according
to the force which acts.

Peptones formed during digestion do not in health reach
the general circulation. When injected directly into the
blood they act as powerful poisons. They destroy the
coagulability of the blood, lower blood-pressure, and in
large quantities cause speedy death. BRUNTON attributes
the lassitude, depression, sense of weight in the limbs, and
dulness in the head occurring in the well-fed, inactive man,
after his meals, to poisoning with peptones. The remedy

TIIK AUTOGENOUS DISEASES.

which lie proposes i- Ir-s fund, especially less nitrogenous
food, and more exercise. That some substance resulting
from the proteids of the food is the cause of this trouble
P>I:UNTON thinks is evidenced by the fact that the weak-
ii« -s and languor are apparently less after meals consisting
of farinaceous foods only.

That peptone finds its way into the" general circulation
frequently is shown by its detection in the urine in many
diseased conditions, some of which are infectious and others
autogenous iu character. However, propeptonuria, or albu-
raosuria, is more common than peptonuria, and we have
already seen that many of the bacterial albumoses are among
the most highly poisonous bodies known, but the action of
th<- albumoses formed during digestion has not, so far as
we know, been studied. The valuable work of KUHXE and
CHITTENDEN on the chemical character of these bodies
should be supplemented by a thorough investigation of their
physiological effects when injected into the blood. It is more
than probable that valuable information would be secured
by such studies. That albumose is frequently found in the
urine is shown by the following list of diseases in which
it has been observed, given in the last edition of the work
of NEUBAUER and YOOEL on the urine : KOSNER has
found it in spermatorrhosa ; KOPPEX, in mental diseases
without spermatorrhoea ; KAHLER, in osteomalacia ; BEXCE
JONES, in multiple myeloma ; SENATOR and others, in
dermatitis, intestinal ulcer, liver abscess, croupous pneu-
monia, apoplexy, vitium cordis, resectio coxae, parame-
tritis, endocarditis, typhoid fever, nephritis, phthisis, etc. ;
LOEB, in measles and scarlet fever ; LEUBE, in urticaria ;
and LASSAR, after inunctions of petroleum. KOTTNITZ,
FURSTNER, and others, find albumose frequently in the
urine in mental diseases. Evidently, there is much to
learn from the study of the conditions accompanied by the
elimination of the albumoses in the urine. It is more than
probable that the acute Bright's disease following scarlet
fever, diphtheria, and the other acute infectious diseases,
owes its existence to the poisonous albumoses of these dis-
-'•-. I'KIOK has recently shown that undigested egg

356 BACTERIAL POISONS.

albumin is sometimes absorbed and produces marked dis-
turbances. A boy, after eating sixteen raw eggs, had a
high fever accompanied by the appearance of both albumin
and haemoglobin in the urine.

BRIEGER obtained by digesting fibrin with gastric juice
a substance which gives reactions with many of the general
alkaloidal reagents and to which he has given the name
" peptotoxine." A few drops of a dilute aqueous solution
of this substance sufficed to kill frogs within fifteen min-
utes. The frogs became apparently paralyzed and did not
respond to stimuli. Slight tremor was perceptible in the
muscles of the extremities. Rabbits of about one kilo-
gramme weight were given from 0.5 to 1 gramme of the
extract subcutaneously. About fifteen minutes after the
injection, paralysis beginning in the posterior extremities
set in ; the animal fell into a somnolent condition, sank
and died. In some rabbits several hours elapsed before
the above-mentioned symptoms appeared.

Peptotoxine was found by BRIEGER to be formed not
only by the digestive juice, but to be among the first
putrefactive products of proteids, as fibrin, casein, brain
substance, liver, and muscle.

It is highly probable that many of the nervous symptoms
which accompany some forms of dyspepsia are due to the
formation and absorption of poisonous substances.

In some persons the tendency to the formation of poisons
out of certain foods is very marked. Thus, there are some
to whom the smallest bit of egg is highly poisonous ; with
others, milk will not agree ; and instances of this kind are
sufficiently numerous to give rise to the adage, " What is
one man's meat is another man's poison."

BRUNTON is of the opinion that the condition which we
term " biliousness," and which is most likely to exist in
those who eat largely of proteids, is due to the formation
of poisonous alkaloids ; but of this we have no positive
proof.

Whether or not the unorganized digestive ferments ever
find their way into the blood in quantity sufficient to cause
deviations from health, we are not in a position to state

THE AUTOGENOUS DISEASES.

definitely. The older physiological chemists teach us that
pepsin and trypsin are frequent, if not constant constituents
of normal urine, but their experiments were made without
any reference to the possibility of the ferments which they
found Ixnng formed by the bacteria of the urine, and after
carefully going over the literature of the subject we are not
prepared to pass judgment on the truth of their statements.
However this may be, the fact that these ferments manifest
a marked toxicologioal effect when introduced into the
blood is of great interast, especially at this time. HILDE-
BRANDT has recently reported the results of some experi-
ments made by himself upon this subject. He finds that
a fatal dose of pepsin for dogs is from 0.1 to 0.2 gramme
per kilogramme of body weight. The subcutaneous injec-
tion of these quantities is followed by a marked elevation
of temperature, which he designates as " ferment fever."
This fever begins within an hour after the injection,
reaches its maximum after from four to six hours, and
may continue for some days. On the day preceding death,
the temperature generally falls below the normal. During
the period of "elevation there are frequent chills.

The symptoms which accompanying the fever vary
somewhat with the species of animal. Rabbits lose flesh
notwithstanding the fact that they continue for a while
to eat well, they become very weak, and death is preceded
by convulsive movements. Dogs tremble in the limbs, be-
come uncertain in gait, and vomiting, dyspnoaa and coma
are followed by death.

On section there is observed parenchymatous degenera-
tion of the muscles of the heart and similar changes in the
liver and kidney. There are abundant hemorrhages in the
intestinal canal, in PEYER'S patches, in the mesenteric
glands; and in the lungs in cats. Thrombi are frequently
found in the lungs and in some cases in the kidneys.

The effect upon the coagulability of the blood is worthy
of note. At first there is a period during which the coagu-
lability of the blood is greatly lessened, then follows a
period of greater rapidity in coagulating, and it is in this
latter stage that the thrombi are formed.

16*

358 BACTERIAL POISONS.

These experiments are interesting not only as a possible
explanation of the cause of some of the autogenous fevers,
which will be discussed later, but in view of the present
tendency to inject such complex animal solutions as
BROWN-S£QUARD'S elixir and KOCH'S lymph subcutaue-
ously, and they will probably cause us to exercise a little
more care in this direction.

That certain febrile conditions are autogenous there can
be no doubt. These, like other diseases originating within
the system, may be due to either of the following causes :

1. There may be an excessive formation of poisonous sub-
stances in the body. Thus, BOUCHARD has shown that
the urine excreted during the hours of activity is much
more poisonous than that excreted during the hours of rest.
Both physical and mental labor are accompanied by the
formation of these deleterious bodies, and if the hours of
labor are prolonged and those of rest shortened, there will
be an accumulation of effete matters within the system.

2. The accumulation of the poisonous matters may be due
to deficient elimination. 3. Some organ whose duty it is
to change harmful into harmless bodies may fail to prop-
erly perform its functions. Illustrations of diseased con-
ditions arising from these several causes will be given.

First, we may mention fatigue fever, which is by no
means uncommon, and from which the overworked physi-
cian not infrequently suffers. One works night and day
for some time ; elimination seems to proceed normally ;
but after a few days there is an elevation of temperature
of from one to three degrees, the appetite is impaired, and
then if the opportunity for rest is at hand sound and rest-
ful sleep is impossible. The tired man retires to his bed
expecting to fall asleep immediately, but he tosses from side
to side all night, or his sleep is fitful and unrefreshing.
The brain is excited and refuses to be at rest. The senses
are alert, and all efforts to sink them in repose are unavail-
ing. Fatigue fever is frequently observed in armies upon
forced marches, especially if the troops are young and un-
accustomed to service. Mosso has studied this fever in

TIIK AUTOGENOUS DISEASES. 359

the Italian army. He states that in fatigue the blood is
subjected to a process of decomposition brought about by
the infiltration into it from the tissues of poisonous sub-
stances, which, when injected into the circulation of healthy
animals, induce malaise and all the signs of excessive ex-
haustion. It is possible that in this decomposition of the
blood the fibrin-ferment, which, according to SCHMIDT, is
held in combination in the colorless corpuscles, is liberated ;
and it has been shown by EDELBERG that the injection of
small quantities of free fibrin-ferment into the blood causes
fever, while the injection of larger quantities is followed by
the formation of thrombi, as has been demonstrated by the
. xpcriments of EDELBERG, BONNE, BIRK, and KOHLAR.

Fatigue fever is often accompanied, especially during the
period of elevation, by chilly sensations, and consequently
it is pronounced malarial and quinine is administered, but
it does no good — often harm, by increasing cerebral excite-
ment. The proper treatment is prolonged rest, with proper
attention to elimination.

Then there is the fever of exhaustion, which differs from
fatigue fever only in degree. It is brought on by pro-
longed exertion without sufficient rest and often without
sufficient food. The healthy balance between the formation
and elimination of effete matter is disturbed, and it may
be weeks before it is reestablished — indeed, it may never
be regained, for some of those oases terminate fatally. The
fever of exhaustion may take on the typhus form, delirium
may apj>ear, muscular control of the bowels may be lost,
and death may result.

That the fever of exhaustion has been mistaken for
typhoid by some of the ablest clinical teachers is shown by
PETER in the following quotation. "It was in 1852," says
he, "when entering upon my clinical studies and ardent in
my attendance at the clinic of CHOMEL, I was witness of
the following instance: A young man was nveived under
the celebrated professor's charge -uttering from prostration,
muscular pain, and rhachialgia. CHOMEL made the exam-
ination with all the care and attention used by him; then

360 BACTERIAL POISONS.

— as was also usual with him in the presence of the patient
— he gave the diagnosis in Latin, which was 'Aut Jebris
peyerica, aut variola indpientis ' (either typhoid fever or
incipient smallpox). I felt rather dissatisfied at a diagnosis
so little precise by one so eminent in his art. The truth of
the matter was, though CHOMEL was not aware of it, this
young fellow in a state of destitution had walked from
Compiegne to Paris, sleeping by the wayside at night and
nourishing himself with such refuse food as chance supplied.
It was under such circumstances the patient had developed
febrile symptoms. The day after his admission, and simply
from rest in bed, he felt better, and the day following he
was altogether well."

That all cases of the fever of exhaustion do not terminate
so rapidly as that instanced above many physicians know.
We have seen at least one such case terminate fatally.

Then, again, there is the fever of non-elimination, which
all physicians of experience have observed. There is a
feeling of languor, the head aches, the tongue is coated, the
breath offensive, and the bowels constipated. The physi-
cian fears typhoid fever, but finds that a good, brisk cathar-
tic dissipates all unpleasant symptoms, and the temperature
falls to the normal. This fever is also liable to appear
among those who are confined to bed from other causes.
BRUNTON says : " No one who has watched cases of acute
diseases, such as pneumonia, can have failed to see how a
rise of temperature sometimes coincides with the occurrence
of constipation, and is removed by opening the bowels."
The surgeon and obstetrician have often had cause to rejoice
when they have found a fever, which they feared indicated
septicaBmia, disappearing after free purgation.

BOUCHARD has shown that normal feces contain a highly
poisonous substance, which may be separated from them by
dialysis, and which, when administered to rabbits, produces
violent convulsions. He estimates that the amount of
poisonous alkaloids formed in the intestines of a healthy
man each twenty-four hours would be quite sufficient to
kill, if it was all absorbed. He proposes the term "ster-

THH AUTOGENOUS DISEASES. 361

eonemia" for that condition which results from arrest of
excretion from the intestine.

It is more than probable that the poisons of the intes-
tines are due to the bacteria which are normally present;
but this would not exclude the fever of nou-elimination
from the list of autogenous diseases. The bacterial cells
which are normally present in the intestines cannot be
regarded as invaders from without.

It would seem from some recent studies that not all sur-
gical fevers are due to bacterial activity. The absorption
of aseptic blood-clots and of disintegrated tissue in cases of
complicated fractures and contusions of the joints is accom-
panied by an elevation of the temperature above normal.
A like result may follow the intravenous injection of a
sterile solution of haemoglobin or of the blood of another
animal. The causative agent in the production of these
fevers remains unknown. In the blood of twelve out of
fifteen patients with aseptic fever, at the clinic of NOTH-
NAGEL, HAMMERSCHLAG has found free fibrin -ferment,
but in five persons without fever he found the same sub-
stance in the blood. This leaves the causative agent in the
production of the aseptic, or, more properly speaking, the
non-bacterial, fevers unknown.

The chemical theory of so-called uraemia has received
support in recent researches, notwithstanding the fact that
the old idea that urea is the active poison and the theory
of FREREICHES that ammonium carbonate is the active
agent have been abandoned.

LANDOIS laid bare the surface of the brain in dogs
and rabbits, and sprinkled the motor area with creatine,
creatinine, and other constituents of the urine. Urea,
ammonium carbonate, sodium chloride, and potassium
chloride had but slight effect ; but creatine, creatinine,
and acid sodium phosphate caused clonic convulsions on
the opposite side of the body which later became bilateral.
The convulsions continued at intervals for from two to
three days, when, growing gradually weaker, they disap-
peared. LANDOIS concludes that chorea gravidarum is a

362 BACTERIAL POISONS.

forerunner of eclampsia. These experiments have been
confirmed by LEUBUSCHER and ZEICHEN.

FALCK injected into both sound and nephrotomized ani-
mals fresh urine, urine and the ferment of MUSCULUS and
LEA, and urine which had undergone spontaneous decom-
position, without producing any symptoms which were
comparable with those observed in uraemia. However, he
did find that if a few drops of an infusion of putrid flesh
were added to the urine before injection all the typical
symptoms of uraemia were induced. That the infusion of
putrid flesh alone had no effect was also demonstrated.
This would lead us to believe that some ferment in the
infusion converts some constituent of the urine into a
highly poisonous body. In this connection attention may
be called to the fact that creatine may be converted by the
action of certain germs into methyl -guauidine, which pro-
duces convulsions. Whether such conversion occurs in
uraemia or not, and if it does what the cause of it is, are
questions which must be left for future investigations to
decide. It would be well for someone to test the brain
and blood of a person who has died in ursemic convulsions
for methyl-guanidine.

That there is a marked disturbance of tissue metabolism
caused by the inhalation of vitiated air has been shown by
ARAKI. In the urine of animals rendered unconscious by
being kept in a confined space this experimenter found
albumin, sugar, and lactic acid. If the animals had been
kept without food for some days before being subjected to
this experiment albumin and lactic acid were found, but
no sugar appeared. This was undoubtedly due to the fact
that the glycogcn of the body had been exhausted by the
fasting. Identical results were observed in animals which
were poisoned with carbon monoxide. Dogs which were
poisoned with curare, and in which the respiratory move-
ments were maintained artificially, secreted very little
urine; but the blood Avas found to contain considerable
quantities of sugar and lactic acid. The urine of frogs in
which the respiration was retarded by the production of
tetanus with strychnine secreted urine containing sugar and

TIIK AUTOGENOUS DISK AS I 363

lactic acid. In the urine of three epileptics there were
found albumin and lactic acid directly after the sci/mv.
Tin1 factor common to all these cases is diminished oxygen-
ation of the blood, and to this is ascribed the appearance of
the abnormal constituents of the urine. These investiga-
tions demonstrate the influence of impure air upon the
chemistry of the living cells of the animal Ixxly.

CHAPTER XIV.

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Xchciffer. Journal f. praktische Chemie, [2] 23, 202.
Scholl. Berliner klin. Wochenschrift, 27, 933.

Medical News, 57, 158. Medical and Surgical Reporter,

68, 584. Archives of Pediatrics, 7, 863. Transactions of the

American Pediatric Society, 2, 109.
ro/t Schiodnitz. Medical Newsi 57, 237, 332.

MISCELLANEOUS.

Virchow's Archiv, 119.
Kehring. Zeitechrift f. Hygiene; 6, 117.
fielfunti e Petcarolo. Central blatt f. Bacteriologie.
BUHngt. Swine-Plague, 1888-9.
Bouchard. Compt. Rend., 110.
/{in-finer. Archiv f. Hygiene, 10, 84.
l-'ei-ini. Archiv f. Hygiene, 10, 1.
Wiiijge. Die Mikrotirganismen.
Fodor. Archiv f. Hygiene, 4, 129.
/•'/vw//. Xeitschrift f. Hygiene, 9, 225.
(i'lmnli-'in. Cinnpt. Rend., 110.
Grohmann. Ueber die Einwirkung des Zellenfreien Blutplasma

auf einige pflanzliche Mikroorganismen, 1884.
Jfiinkin. British Medical Journal.
Kitt. Centralblatt f. Bakteriologie, 7, 297.
/\i/ti.<uto. Zeitschrift f. Hygiene, 8, 41.
AV.« /(///.s/.v/. Medisjinskoje Obozrenie, 1888.

386 BIBLIOGRAPHY.

Lewis and Cunningham Eighth Annual Report of the Sanitary

Commission with the Government of India.
Liermann. Archiv. f. exper. Pathologic, 27, 241.
Loffler. Arheiten aus d. kaia Gesundheitsamte, 1, 46.
Lydtin u. Schotteliw. Der Rothlauf der Schweine, 1885.
Maffucd. Centralblatt f. Pathologic, 1, 440, 449, 825.
Nissen. Zeitschrift f. Hygiene, 6, 487.
NuttaU. Zeitschrift f. Hygiene, 4, 353.
Ogata u. Jusuhara. Centralblatt f. Bakteriologie, 9, 25.
Petri. Arbeiten aus d. kais. Gesundheitsamte, 6, 1.
Rovighi. Riforma Medica, 1890, No. 110.
Salmon. Reports of the Bureau of Animal Industry, 1884, '85,

'86, '87, and '88.

Schiifz. Arbeiten aus d. kais. Gesundheitsamte, 1, 56, 376.
Sponck. Centralblatt f. Pathologic, 1, 217.
Stern. Zeitschrift f. klin. Med., 1890, No. 18.
Tizzoni e Cattani. Centralblatt f. Bakteriologie, 9, 685.
Traube u. Gscheidlen. Schlesische Gesellschaft f. Vaterhiml.

Cultur, 1874.

Tungl. Centralblatt f. Pathologic, 1, 795.
Vaughan. Journal American Med. Assoc., 13, 831. Canadian

Practitioner, 16, 77.

Welch. Journal American Med. Assoc., 13, 836.
Widenmann. Zeitschrift f. Hygiene, 5, 522.
Wyssokowitsch. Zeitschrift f. Hygiene, 1, 1.

INDEX.

AI'KVINK. 283
Adi'uine-liy]N>xaii tlii ne, 297
Ai;rotiic bacteria, 17
Agaricine, 237
Alliiimins, ji lismi'iiis, 61
Albumoses, 35

immunity from, 150

in urine, 355
Alcohol, basic substances in, 158

dialysis into, 172

effect on bacterial proteids, 173
Alcoholic fermentation, bases in, 222
Aldehyde collidine, 196
Alkaloids, interference in reactions
of, by ptomaines, 183

separation from ptomaines, 186
Alkapton, 281
Anmnitine, 237
American swine- plague, 142
Amido-valerianic acid, 231
Amphi-creatine, 338
Ainyluiiiiiio, 193

Amylic alcohol, impurities in, 158
Anaerobic bacteria, 1 7
Alkaloids, 15
Animal chinoidine, 26, 243, 347

coniine, 214

Anthracin, 102, 104, 277
Anthrax, 101

theories of. 85 ct seq.

bacillus, products of, 101 et seq.

proteid, 103, 171

albumose, 103, 156, 171
Apricots, poisonous, 52
Arginine, 189, 242, 333
Arornine, 344
Aseliine, 229
Aseptic fever, 361
Asiatic cholera, 104

bacillus of, products of, 107
Atrnpme-like substances, 27, 179
Autogenous diseases, 14, 352
Azulinic acid, 283, 288, 337

BACILLUS butyricus, 16
enteriditis, 50
Bacon, poisonous, 51
Bacteria, classification of. 13

in summer diarrhoeas, 133,

136
Bacterial cellular proteids, 19

method of extraction,

130

poisons, definition and classifica-
tion of, 15

historical sketch of, 22
foods containing, 36
relation to infectious dis-
eases, 84
proteids, 18

method of extraction, 170,

171

Bacterium allii, 203
Batrachians, poison of, 350
Beer, colchicine-like substance in,

183

Benzol, impurities in, 158
Bergmann and Schmiedeberg's me-
thod, 169

Betaine, 248, 334, 343
Bibliography of the leucomaines,

381

of the ptomaines, 364
Bilineurine, 237
Biliousness, 356
Blood, germicidal properties, 153

leucomaines in, 347
Bocklisch's base, unnamed, 272
Botulinic acid, 46
Bread, poisonous, 83
Bricger's bases, unnamed, 195, 255,

261,271, 272,273,274,275
methods, 161

disadvantages of, 170
Brouardel's veratrine, 204
Bujwid's cholera- reaction, 110
Butylamine, 193

388

INDEX.

pADA VERIO coniine, 176, 214*»

\J Cadaverine, 34, 107, 212

Caffeine, 316

Canned meats, poisonous, 52

Caproylamine. 194

Carbon monoxide in expired air, 343

Carbonic acid, 341

Carnine, 326, 334, 344

Caseic acid, 23, 46, 52

Charcot-Neumann, crystals of, 330

Cheese, poisonous, 52

Chemotaxis, 129

Cholera, 104

Bujwid's reaction, 110

-blue, 112

-infantum, 133

-red, 110

-stools. 213
Choline, 34, 107,237

decompositions of, 243

-group, 232

constitution of, 252
Chorea gravidarum, 361
Cicuta virosa, 176
Codeine-like substances, 179
Cod-liver oil, bases from, 263
Comma bacillus, ferments produced

by, 104

products of, 107 et seq.
Colchicine-like substances, 181

Zeisel's test for, 182
Collidine, 28, 196, 198
Coniine, difficulties in detection of,
177

-like substances, 30, 174
Coridine, 204
Corindine, 202

Corn-meal, ptomaines in, 33, 83, 178
Creatine, 189, 226
Creatinine, 189, 226

-group, 333
Cruso-creatinine, 336
Cyanogen, r61e of, 336
Cystinuria, bases in, 207

DE CONINCK'S bases, 198, 202
Delezinier's base, 204
Delphinine-like substances, 180
Deutero-albumose, 103

-myosinose, 156
Dialysis, concentration by, 172
Diamines, 204
Diarrhoeas of infancy, 133
Diethylamine, 191
Digitaline-like substances, 28, 179

Dihydrolutidine, 195
Dimethylamine, 34, 188
Dimethyl-xanthine, 336
Diphtheria, 124

bacillus of, products of, 124

immunity to, 128
Dippel's oil, 198
Diseases, classification of. 84

relation of bacterial poisons to, 84
Dragendorffs method, 161, 167
Dyspepsia, 356

T? BERTH'S bacillus, 16, 139
JL products of, 140

Eclampsia, 362
Eel, poisonous, 41
Ehrlich's reagent, 260
Enzymes, 35, 105, 118
Ethylamine, 191
Ethyleneimine, 205, 330
Ethylidenediamine, 34, 204
Expired air, leucoma'ines in, 341

FECES, poisons in, 360
Ferments, 35, 105, 118
from comma bacillus, 104
in urine, 357
Fever, aseptic, 361
of exhaustion, 359
of fatigue, 358
of non-elimination, 360
Fish, poisonous, 41, 350
Foods containing bacterial poisons, 36

r\ ADININE,258

VT Gaduine, 264

Gaduinic acid, 263

Galactine, 348

Gautier's pseudo-xan thine, 328

Gautier and Etard's bases, 199, 201,

229

methods, 163, 164
extraction of leucomaiines, 334
German swine-plague, 142
Germs, relation of, to disease, 85 et seq.
Gerontine, 329
Globulins, germicidal properties of,

155

Glucosines, 223
Glycol, 190

Goose-grease, poisonous, 51
Gram's bases, 273
Griffith's base, 203

INI" K X .

889

,-. MT,

::os
< Juaiv*'hi'.- II.-IM-, 208

an- 1 Mosso's bases, 2<)l,

HAM, p.. i*, nous. 47
Hankin's method, I 71
Hoti't-oxaiithine, 319
Hex ylaiiiiiii-. I'.i I
II i-iori.-al ski-trh of the bacterial jtoi

sons, 22
Hog -i-holcra, 142

-erysipelas, 142
Homo j>i|M'riiliiiii- acid, 231
Ilydrocollidiiic', 200
Hy<lnx»ridine, 204
Hydrocyanic acid, 283, 354
Hydrolutidine, 195
ll\ ..-icyamine-like substances, 27
Hyjwxanthine, 298

ICE-CREAM, poisonous, 79
Immunity from blood serum,

146, 147

methods of securing, 146
-producing substances, nature of

of, 14(1 ct xeq.
by intoxication with ptomaines,

225

to diphtheria, 12S
to pneumonia, 145
to swine-plague, 144
to tetanus, i I'.i

Illdol, Ill

Infectious diseases, 84. |n|

how produced, 85

definition of, 92

favored by bacterial pro-
ducts, 151

Iso-amylamine. Iv::
Iso-cyanacetic acid, 351
Iso-propylamine, 193

KAKKK. 41
Koch's rules. 92

LACTIC acid. I or,
Lactocliruiiif. :; 1^

Locitliin, ill mpii.-iiioii ..(', '.'I

prcpiiraticiii «f, 239
Leucin, 19, 103, 109
Leucocythii-iniu, urine in, 284

LeuooiiniiiK's, liiMin./rapliv ol

<-licmistry ..I'. I

f.xirai-iioii of. ::::i

pathological importain-i-

tal,!,-s of. 351
Lutidiue, 195
Lysatin,-. is'.i, 241'.
Lysatinino, 189, 242, :;:;:;

MALIGN ANT <Bdem», 145
Marino-Zuco's method, 159
Menl and bread, poisonous, 83
Meat, iMiisonous, 50

carbylamine, 351

guanidine, 34, 108. 144, 225, 362

liydantoin, 22fi, 340

method of extraction, 1(57

uramine, 226

xanthine, 314,319
Milk, leucoma'ines in, 348

poisonous, 62
Monamines, 187
Mori M 's base, 222
Morphine-like substances, 178
Morrhuic acid, 263
Morrhuine. 228
Mu-c. irin", 34, 251
Mussel, poisonous, 36
Mutton, poisonous, 51
Mycoderma acet i , If,
Mycoprotein, 19
Mydatoxine, 34, 253

isomer of, 25J, 2(>7
Mydaleino, 34. L'TO
Mydine. 34, 2.",0
Mylitotoxine, 34, 40, 255

VT ARCOTIC substance of Panum, 25
11 Nencki's base, 196
Neuridine, 34. L'ls
Neurine, 34, 232
Nicotine-like substances, 177
Nicotinic acid, 199
Non-toxicogenic bacteria, 13
Nucleins, 141

OSER'S base, 229
Oxy-betainea, 265
Oxygenated bases, 230
Oysters, poisonous, 41

390

INDEX.

PANUM'S narcotic substance, 25
putrid poison, 24
Paraffin oil, bases in, 223
Parareducine, 344
Parasitic bacteria, 13
Paraxanthine, 321
Parvoline, 201
Pellagroce'ine, 178
Pentamethylenedifiinine, 213
Pepsin, action of, 357
Peptones, poisonous nature of, 354

et seq.

Peptotoxine, 275, 356
Petroleum, bases in, 223
Peptotoxine, 275, 356
Phenyl-ethylamine, 197
Phlogosiue, 274, 129
Phosphorus-containing substances,31
Phytalbumose, 350
Piperazine, 332
Piperidine, synthesis of, 213
Pneumonia, chemical products in,

145

Poisonous foods, 36
Pouchet's bases, 265, 268, 344
Propylainine, 193
Protalbumose. 103
Protamine, 332
Protomyosinose, 156
Pseudo-xanthine, 328
Ptomaines, bibliography of, 364

chemistry of, 187

definition of, 15

table of, 278, 279

separation of alkaloids from, 186

methods of extraction of, 157

remarks upon, 165
Ptomatropine, 179
Puerperal fever, 145
Putrefactive alkaloids, 15
Putrescine, 34, 107, 206
Putrid poison of Panum, 24
Pyocyanine, 277
Pyogenetic proteids, 130
Pyoxanthose, 277
Pyridine, 107, 199, 202, 203, 275, 344

RABBIT septicaemia, 144
Reagents, purity of, 158
Reducine, 344
Reus's test for atropine, 179
Rouget, 142
Roussin's test for nicotine, 177

ALAMANDARINE, 350

Saliva, leucomaines in, 346
Salkovvski's base, 231
Sjiprophytic bacteria, 13
Saprine, 34, 220
Sarcina botulina, 46
Sarcine, 2!)8
Sarcosine, 340
Sausage, poisonous, 22, 42
Schweineseuche, 142
Sebacic acid, 22, 46, 52
Selmi's method, 27, 159
Sepsine, 26

method of extraction, 1 69
Septiciemia of rabbits, 144
Septicine, 194
Sinapin, 242
Spasmotoxine, 117, 194
Spermine, 205, 330
Spleen, leucomaines in, 347
Staphisagria, 180
Staphylococcus pyog. aureus, bases

from, 274

Stas-Otto method, 158, 167
Stercoraemia, 360

Strychnine-like substances, 32, 178,
204

reactions, 33, 180
Sucholotoxine, 144
Summer diarrhoeas of infancy, 133
Suppuration, 129
Susotoxine, 143, 223
Swine-plague, American, 142

products of bacillus of, 143

German, 142

rriETANINE, 34, 117,265
_L Tetanizing substance, 32
Tetanotoxine, 117, 194
Tetanus, 113, 147

bacillus, products of, 1 17 et seq.

immunity to, 119

neon atom in, 1 15

toxines 194, 195, 255,265. 267
Tetrahydronaphthylamine, 201
Tetramethylenediamine, 209
Tetramethyl-putrescine, 210
Theine, 316

Theobromine, synthesis of, 316
Theophylline, 326
Toxalbumins. 19, 35, 118. 121, 127
Toxicogenie bacteria, 13, 99
Toxicology of ptomaines, 174
Toxines, 15
Toxopeptones, 109

INDEX.

301

Trifthyl'i'iiiinr. 192
Trimefhyhuniiif
Trimothyli'Mrdiaiiiiiif, His, 205

Toberaulin, 120

TuU'iviil.isis. 120

iinxlncls df bacillus nl'. 121
niil bacillu-*. 139
products nl", 140
fever, i::-.i

Typhotoxine. 34, 140, 259
isiuncr nf, 258, 261

Trroda, HI:;, lit'.', HIT.IM
Tyr.toxicoii, :;i,4l,56, 61, 70, 2(i'.l
in summer diarrhoea, 139

ITNDETERMINED leucomaines,
U :>. 1 1

ptomaines, 269 ct seq
Urwniic poisoning, 361
Urea, 227

I'ri.- a.-id. 318

group of leucomaines, 282
Urine. fermenU in, 357
leucomaines in, 343
toxicity of, 345

i

Urotheobromine, 343

VALEKIANICacid, 231
Vanilla, 79
Veal, poisonous, 51
Venoms of serpents, 348
Vcratriin- like substances, 180, 204
Vet-nine, 309

Vitiated nir, effects of inhalation ui.
362

WEI D EL'S reaction, 288
White liquefying bacterium,
products of, i:;;i

VANTJIINE, 313

J\. group, constitution of, 318

Xantho-creatinine, 337

EISEL'S test for colchicine, 182

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