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BLOOD IMMUNITY

AND

BLOOD RELATIONSHIP

HonOon: C. J. CLAY and SONS,

CAMBBIDGE UNIVEKSITY PEESS WAEEHOUSE,

AVE MAEIA LANE,

AND

H. K. LEWIS,
136, GOWER STREET, W.C.

®laagoto: 50, WKLLINGTON STREET.

Itipjis: F. A. BROCKHAUS.

ftrt) gotk: THE MACMILLAN COMPANY.

BombaB airt Ealculta; MACMILLAN AND CO., Ltd.

lAU Rights reseiTed.]

BLOOD IMMUNITY

AND

BLOOD RELATIONSHIP

A DEMONSTRATION OK CERTAIN BLOOD-RELATIONSHIPS
AMONGST ANIMALS BY MEANS OF

THE PRECIPITIN TEST FOR BLOOD

by
GEORGE H. F. NUTTALL, M.A., M.D., Ph.D.

University Lecturer in Bacteriology and Preventive Medicine, Cambridge.

Including
Original Researches by

G. S. Graham-Smith, M.A., M.B., D.P.H. (Camb.)

and
T. S. P. Strangeways, M.A., M.R.C.S.

Cambridge :

at the University Press

1904

ffiambrtfige :

PBINTED BY J. & C. F. CLAV,
AT THE CNIVEBSITY PRESS.

"■»«• MUt**

'QT91
]904

THIS VOLUME IS DEDICATED

TO
PAUL EHRLICH and ^LIE METCHNIKOFF

WHOSE GENIUS AND INFLUENCE

HAVE SO GREATLY ADVANCED AND STIMULATED

THE SEARCH AFTER TRUTH AMID

THE COMPLEX PROBLEMS OF IMMUNITY.

258;r^

"Blut ist ein ganz besondrer Saft."

Goethe's Famt, Part i

PREFACE.

r I THE investigations recorded in this volume were carried out in the
-*- Pathological Laboratory of the University of Cambridge, chiefly
during the year 1902.

My original intention was to publish a volume on the demonstration
of certain blood -relationships amongst animals as indicated by 16,000
tests made by myself with precipitating antisera upon 900 specimens
of blood obtained from various sources, followed by a critical review
of the literature on antibodies in blood.

Subsequently it was deemed advisable to add a section dealing with
the practical applications of the precipitin test for blood in legal
medicine, together with some investigations carried out at my sug-
gestion by Messrs G. S. Graham-Smith and F. Sanger. The latter were
published in the Journal of Hygiene, vol. in., 1903, but are added for
the reason that they bear directly on the subjects treated in this volume.
Finally two new sections were added dealing with further original
researches conducted in this Laboratory. The latter are entitled " The
Results of 500 Quantitative Precipitin-Tests upon the Bloods of Primates,
Insectivora, Camivora, Ungulata, Cetacea, Marsupialia and Aves" by
myself and Mr T. S. P. Strangeways, and " Blood-Relationship amongst
the Lower Vertebrata and Arthropoda, etc." by Mr G. S. Graham-Smith.

The results recorded in these papers should be of interest not only
to zoologists, physiologists, and those engaged in practical medico-legal
work, but also to those interested in the complex problems of immunity.

Although the source from which each specimen of blood was obtained
has been fully acknowledged in the text, I here wish to place on record

viii Preface

my indebtedness to the many gentlemen who, often at much incon-
venience to themselves, collected and forwarded these specimens, and
especially my appreciation of the scientific spirit which led them to
give, wherever possible, the accurate nomenclature of the species,
without which the value of the work would have been considerably
impaired.

The publication of these researches has been rendered possible
through a gi-ant made for the purpose by the Royal Society, and the
generous interest of the Syndics of the University Press, Cambridge,
to whom I am greatly indebted.

G. H. F. N.

Pathological Laboratory, Cambridge.
Januari/, 1904.

ERRATA.

Beginning page 298 (Tables) the following names of animals should be corrected
the numbers referring to those printed in black letters on the left-hand margin :

797 read Euplocamus 846 read Bitjs

827 „ Python sebae 857 „ Xenopus laevis

836 „ Zamenis i-avergieri 860 „ Megalobatrachus.
844 „ Naja Aaje

p. 260, top of first column, delete "3. Suborder Hyracoidea, Fam. Hyracidae"
and substitute

1. Suborder
MYSTACOCETI.

2. Suboi-der
ODONTOCETI.

CONTENTS.

PAOE

Introduction 1

PART I.

RELATING TO ANTIBODIES IN GENERAL OTHER
THAN THE PRECIPITINS.

Section I. Ehrlich's Theory regarding the Formation of Antibodies,

TOGETHER WITH A BRIEF CONSIDERATION OP ToXINS AND ANTITOXINS 5

Section II. Ferments and Antiferments 16

Section III. The Gytotoxins of Blood Serum 18

The mode of action of haemolysins and baoteriolysins . . . .19

The intermediary body, immune body, complement, complementoid.

Anti-cytotoxins, anti-complements 21

Special cytotoxins : leucotoxin, spermotoxin, neurotoxin, trichotoxin, etc. . 22

Source of cytolysin.s 26

Influence of the species of animal upon the character of cytolysins . . 27

Influence of animal's age upon susceptibility of corpuscles to haemolysis . 27

Bacterial haemolysins and anti-haeniolysins 28

The serum complements and their importance in physiological processes . 28

The treatment of animals for the production of specific haemolysins . . 31

Section IV. The Action of different Sera upon the Blood Corpuscles

OF certain Animals in Vitro and in Corpore 33

The effects of normal and artificial haemolysins upon the red blood corpuscles

of different animals in vitro and in corpore 40

Isolysins and autolysins 42

The relation of haemolysins to agglutinins .43

Section V. Agglutinins and Anti-agglutinins, etc 45

Differences between agglutinins and precipitins.

X Contents

PART II.

THE PRECIPITINS.
Section I. Methods : page

Treating rabbits for the production of precipitating antiscra . . . b\
Modes of injecting (p. 57), marking rabbits (p. 58), weighing the
animals (p. 58), when to kill the treated animals (p. 59).

Killing the animals and storing the antisera 59

The choice of animals for immunization 61

Collecting bloods and testing them with antisera 62

Sources of error in precipitin tests 72

Opalescent anti.sera (p. 72), over-powerful and weak antisera (p. 74),
suitable dilutions of bloods tested (p. 74), effects of preservatives on
antisera (p. 76), acid or alkaline reaction of the medium (p. 79), in-
jurious effects of acids and strong alkalies and salts (p. 81), precautions
with regard to quantitative tests (p. 86), bacterial development (p. 86).

Section II. The Nature op Precipitin Reactions 88

Inactivated haematosera, " precipitoids " — non-reactivatability of haema-

tosera — immune-bodies in haematosera 90

The nature of precipitins and precipitable .substances . . . . 96
The precipitum (p. 99), macroscopic and microscopic api)earance8
during reaction (p. 100), the supernatant fluid (p. 101), the influence
of salts upon the reaction (p. 102), regarding the claim that pre-
cipitins permit of distinguishing the different albumins of the same
animal (p. 104), the effect of tryptic and peptic digestion upon the
interacting bodies (p. Ill), regarding the supposed precipitins for
peptones (p. 112), influence of temperature upon the reaction (p. 113),
influence of heat upon the interacting bodies (p. 1 1 4), ett'ect of ttltra-
tion on normal sera (p. 118), effect of putrefaction and desiccation
(p. 119), stability of antisera and sera in vitro (p. 123).

Section III. Observations upon Precipitins and Precipitable Sub-
stances in Corpore 126

Section IV. The Specificity of Precipitins 134

The selective action of jirecipitins in blood-mixtures (p. 140),
measurement of degrees of reaction (p. 141), the delicacy of the test
(p. 144), the strength of antisera (p. 144), differences in degrees of
reaction of individual bloods (p. 145), character of precipitins formed
in difterent species of animals treated with the same blood (p. 146).

Section V. Isoprecipitins, Antipreuipitin.s, Normal Precipitins, Auto-

precipitins 148

Section VI. Qualitative Tests with Precipitins, including Summary
OF Literature and Personal Results :

1. Phytoprecipitins 152

Bacterioprecipitins (p. 152), Yeast precipitins (p. 154), Precipitins
for albumins of higher plants (wheat, roborat, ricin, crotin) (p. 155).

Contents

XI

PAOE

2. Zooprecipitins 166

Ijactoscra.

Haematosera, list of those used 160

Hacmatosera for the bloods of :

Primates: Man (p.' 161), Chimimnzee (p. 168), Ourang (p. 169),

Cercopithecus (p. 169).
Insectivora: Erinaceus (p. 172).

Carnivora: Cat (p. 173), Hyaena (p. 174), Dog (p. 176), Seal (p. 177).
Rodentiar Rabbit (p. 178).

Ungiilata: Pig (p. 179), Llama (p. 182), Mexican Deer (p. 183),
Reindeer (p. 185), Hog- Deer (p. 186), Antelojie (p. 187),
Ox (p. 188), Sheep (p. 190), Horse (p. 192), Donkey
(p. 195), Zebra (p. 197).
Cetacea: Balaenoptera (p. 198).
Marsupialia : Wallaby (p. 199)
Aves : Fowl (p. 200), Ostrich (p. 202), Fowl egg-white (p. 204),

Emu egg-white (p. 206).
Reptilia : Turtle (p. 207), Snake (p. 208), Alligator (p. 209).
Amphibia : Frog (p. 210).
Pisces: Eel (p. 212).
Crustacea : Lobster (p. 212).

Number of species of blood tested 213

General summary of the residts of 16,000 tests 214

Key to signs used in tables which follow 217

Tables recording 16,000 precipitin-tests on 900 blood specimens . 220-311

Section VII. Quantitative Tests with Precipitins for Mammalian and

Avian Bloods, by G. H. F. Nuttall and T. S. P. Strangewats.

General considerations 312

List of antisera used 316

Method of measuring the reactions , 315

Tests with Precipitating Antisera for the bloods of:

Primates: Man (p. 319), Ourang (p. 320).

Insectivora: Erinaceus (p. 321).

Carnivora : Cat (p. 322), Dog (p. 323).

Ungulata : Sheep (p. 323), Ox (p. 325), Antelope (p. 325), Mexican Deer
(p. 326), Hog-Deer (p. 327), Reindeer (p. 327), Pig (p. 328),
Horse (pp. 331, 332), Donkey and Zebra (p. 332).

Marsupialia : Wallaby (p. 333).

Aves: Ostrich (p. 334), Fowl (p. 334).
Conclusions relating to Quantitative Tests 335

Section VIII. Blood-relationship amongst the Lower Vertbbrata and
Arthropoda, etc. as indicated by 2500 Tests with Precipitating

Antisera, by G. S. Graham-Smith 336

Methods : Production of Antisera 336

Table of Antisera 337

Methods of Testing 338

Materials used in Experiments 339

xii Contents

Tests with Antisera for : page
Aves : Qualitative Tests with Antisera for egg-whites of Emu, Duck,

Fowl, Crane 340

Qualitative Tests with Antiserum for Fowl's blood . . . 343

Summary of the foregoing 343

Quantitative Tests with Antisera for egg-whitea of Emu, Duck,

Fowl, Crane 344

Reptilia : Qualitative Tests with Antisera for :

Chelonia : Tortoises (2), Turtle 347

Crocodilia : Alligator 349

Lacertilia : Lizards (2) 350

Ophidia: Snake sera and egg-white 351

Summary of the foregoing 353

Quantitative Tests with Antisera for

Chelonia : Tortoises (2), Turtle 354

Qualitative Tests with Antisera for

Amphibia: Frog 356

Pisces : Ammocoetes . 356

Ascidia 357

Arthropoda : Decapoda ; Lobster, Crab 357

Xiphosura; Limulus 358

Summary of foregoing 359

General Conclusions 361

Tables recording the foregoing Qualitative Tests .... 364-380

Section IX. On the Practical Application of the Precipitin Reactions
IN Legal Medicine, etc.

1. Antisera in the Examination and Identification of Bloods and Blood-

stains 381

2. Antisera in the Examination of Meats 404

3. Antisera in the Examination of Bones 406

4. Antisera in the Examination of Commercial albuminous Preparations

containing egg-white, as also in the examination of Honey for

adulteration 406

5. Antisera in the Study of Urine 407

Plate. Showing Test-tube Reactions .... hetween pp. 402 and 403

Conclusions 4O7

Acknowledgments 411

Bibliography 414

Appendix 43O

Index to Authors 431

Index to Subjects 433

INTRODUCTION.

In the absenoe of palaeontological evidence the question of the
interrelationship amongst animals is based upon similarities of structure
in existing forms. In judging of these similarities the subjective
element may largely enter, in evidence of which we need but look
at the history of the classification of the Primates.

Linnaeus placed Man, the Apes, Lemurs and Bats in the division
Primates. Blumenbach fii-st placed Man in a special order, the Bimana,
including the Apes and Semi- Apes under Quadrumana, this classi-
fication being retained by Cuvier and others. Huxley (1863) showed
that all true Apes are as genuinely "Bimana" as Man, and gave
comparative anatomical proof that the differences between Man and
the higher Apes are le.ss than between these and the lowest Apes.
Huxley therefore separated Primates into Anthropoidae (Man), Simiidae
(Apes), and Lemuridae (Semi- Apes). Haeckel (1866)' did not think
that Man should be placed in a separate order. Zoologists to-day agree
in placing Man and Apes in one order, the Anthropoidea.

The question as to the degree of relationship between the Anthro-
poidea is one upon which there is some disagreement. HaeckeP (1899)
has recently brought together all the evidence speaking for the descent
of man from Old World apes, whose recent ancestors belonged to the
tailless Anthropoids, whose older ancestors belonged to Cynopithecidae.
Years ago', he pointed out that the African man-like apes, the Gorilla
and Chimpanzee, are black in colour, and " like their countrymen the
Negroes, have the head long from back to front (dolichocephalic). The
Asiatic man-apes are on the contrary mostly of a brown, or yellowish-
brown colour and have the head short from back to front (brachy-
cephalic), like their countrymen, the Malays and Mongols." A closer

■ Haeckel (1879), The Evolution of Man, vol. n. (New York, D. Appleton & Co.).
' Haeckel, " Ueber unserc gcgenwartige Keuntniss vom Ursprang des Mensohen,"
(Bonn, 1899).

N. 1

2 Man's Place in Nature

relationship was claimed to exist between man and the Old World
apes than between man and the New World apes, by Darwin •.
Selenkd^ has pointed out that the placenta in Simiidae possesses a
similar structure (Placenta discoidaiis capsularis) to that in man, as
distinguished from Old World monkeys, where the placenta bidiscoidalis
prevails. The very interesting observations upon the eye made by
Johnson (1901)' also demonstrated the close relationship between the
Old World forms and man, the macula lutea tending to disappear as
we descend in the scale of New World monkeys, and being absent
in the Lemurs. The results which I published upon my tests with
precipitins directly supported this evidence, for the reactions obtained
with the bloods of Simiidae closely resembled tho.se obtained with
human blood, the bloods of Cercopithecidae came next, followed by
those of the Cebidae and Hapalidae, which gave but slight reactions
with antihuman serum, whilst the blood of Lemuroidea gave no indica-
tion of blood-relationship.

According to Dubois (1896)* the relationships amongst the Anthro-
poidea are represented by the accompanying genealogical tree, based
upon that of Haeckel (1895). In the paper by Dubois, the bones of
Pithecanthropus erectus are described as those of a probably ancestral
form of man, these having been found in early pliocene deposits in Java.
He places Dryopithecus between the Cercopithecidae and Simiidae,
after Gaudry (1890), and considers Frothy lobates to represent a general-
ized hypothetical form to the common ancestor of all the man-like apes.
He regards Palaeopithecus as the direct ancestor of Pithecanthropus.
However this may be, the fact remains, that the degrees of reaction
obtained by me in my blood tests are in strict accord with this
genealogy, as pointing to the more remote relationship of the Cercopi-
thecidae, but especially of the New World monkeys, as indicated in
the tree. And we shall see that the study of the haemolysins has
given results in accord with what has been observed for the precipitins.

A perusal of the pages relating to the tests made upon the many
bloods I have examined by means of precipitating antisera, will very
clearly show that this method of investigation permits of our drawing

^ See Darwin, The Descent of Man.

» Selenkfi,, cited by Friedentlial (i. 1900).

' Johnson, G.L. (1901), "Contributions to the Comparative Anatomy of the Mammalian
Eye, etc." Philos. Trans, of the Royal Society, B. vol. cxciv. pp. 1—82.

* Dubois, E. (14, iv. 1896), " Pitliecanthropus erectus, eiue Stammform des Menschen."
Anatom. Aiizeiger, Bd. xii. pp. 1—22. 3 figures.

Genealogy of Anthropoidea 3

certain definite conclusions. It is a remarkable fact, as I stated on
a former occasion (16, XII. 1901) with regard to my results with the
Anthropoidea, and this applies as well to other groups of animals, that

CERCOPITHECIDAE

HVLOBATES SIMtA HOMO ANTHR0P0PITHEGU8 OORILkA

HOLOCENE

AND

PLEISTOCENE

PLIOCENE

AROHIPITHEOUS

GENEALOGY OF ANTHROPOIDEA

AFTER DUBOIS T896

Fig. 1.

1—2

4 Blood- Relationships

a common property has persisted in the blooils of certain groups of
animals " throughout the ages which have elapsed during their evolu-
tion from a common ancestor, and this in spite of differences of food
and habits of life." The persistence of the chemical blood-relationship
between the various groups of animals serves to carry us back into
geological times, and I believe we have but begun the work along
these lines, and that it will lead to valuable results in the study of
various problems of evolution.

In the following pages I have given but a brief and fragmentary
summary of the immense amount of work which has been done on
antibodies other than the precipitins, my object in mentioning them
at all is to bring out the many points of resemblance between the
different antibodies. On the other hand, I believe that this con-
stitutes the first attempt to treat the subject from a general and
more especially zoological point of view. The literature relating to
the precipitins has been gone into as fully as possible. In view of
its scattered nature, a summary and criticism of our knowledge brought
up to date should prove of value, more especially to English readers.

Comparative studies upon the constitution of the bloods of different
animals by means of ordinary chemical methods have not demonstrated
diflferences such as can be proved to exist by means of what we may
well call the biological methods, the use namely of the precipitins and
haemolysins. It is true that Abderhalden and others have demonstrated
that chemical differences of a coarser nature exist between the bloods
of Carnivora and Herbivora, that a similarity exists between the blood
of the sheep and ox, etc., but the investigations afford no means of
demonstrating delicate differences such as we have been able to study
by means of biological tests.

In view of the crudity of our methods, it is not surprising if certain
discrepancies may be encountered in the course of investigations con-
ducted by biological methods, the body of evidence is however perfectly
conclusive. The object of my investigation has been to determine
certain broad facts with regard to blood-affinities, consequently my
studies must be regarded in the light of a preliminary investigation,
which will have to be continued along special lines by many workers
in the future.

PAET I.

RELATING TO ANTIBODIES IN GENERAL, AND ANTI-
BODIES OTHER THAN THE PRECIPITINS.

SECTION I.

EHRLICH'S THEORY REGARDING THE FORMATION OF ANTIBODIES,
TOGETHER WITH A BRIEF CONSIDERATION OF TOXINS AND
ANTITOXINS.

In 1890 Behring discovered that the serum of an animal immunified
against diphtheria was capable, when injected into a fresh animal, of
conferring immunity upon the latter, which, failing the use of the
immune serum, died from the effects of the diphtherial toxin it
received. Soon after, Behring and Kitasato obtained similar results
with tetanus. Ehrlicii (1891) next treated animals with increasing
doses of ricin and abrin (the toxic substances contained in the castor-
oil bean and the seeds of jequirity) and found that the toxin was
neutralized in vitro when added to the treated animal's serum, proof
of neutralization being afforded by the fact that when certain propor-
tions of toxin and immune serum were mixed in vitro, these mixtures
were innocuous when injected into animals. He proved that the
neutralizing action of immune serum upon each of these toxins was
specific, that is, the antiserum for abrin did not neutralize ricin, and
vice versa. Immunization against the toxins of snake venom had
already been practised by Sewall (1887), and subsequently Calmette
(1894) and Frazer (1895), but it was reserved for Ehrlich to throw
more light upon the nature of the acquired immunity to toxins through
the formation of antitoxins in the bodies of toxin-treated animals. He
and Madsen showed that toxin and antitoxin combined iu definitely

6 Ehrliclis Theory

measurable quantities in vitro, more rapidly in concentrated solutions,
the union being retarded by cold, hastened by heat. In other words,
the union took place in a manner similar to that observed with regard
to known cliemical bodies.

Ehrlich concluded that " the power of to.xins to combine with
antibodies must depend upon a specific atom-group in the toxin-
complex possessing a maximal specific relation to definite atom-groups
of the antitoxin-complex, so that it rapidly unites therewith, like a
lock and key," a figure borrowed from Emil Fischer in describing the
action of specific ferments.

Ehrlich's theory (1897) had its origin in an hypothesis advanced
by Weigert in 1896, to the effect that a condition of physiological
equilibrium is maintained in the body by virtue of mutually restraining
influences exerted upon each other by the cells which compose it.
Assuming that a cell or a group of cells are destroyed through some
agency, the equilibrium is upset, an element of restraint will be
removed, and there will be an overproduction, comparable to the
compensating hyperplasia observed when an organ is removed, say a
kidney, a double function being thrown upon the one which remains.
According to Weigert, there is always hypercompensation where there
has been destruction of cells. The above conception is narrowed down
by Ehrlich to what takes place in a single cell.

The proces.ses which lead to the formation of antibodies such as
antitoxins are, according to Ehrlich, essentially similar to those taking
place normally in the process of assimilation. We now know that
normal serum contains a number of antibodies having similar actions
to those artificially produced as a result of immunization with this or
that substance, we know of normal agglutinins, haemolysins, bacterio-
lysins, antitoxins, antiferments, etc., all of which go to prove the
correctness of Ehrlich's views in this respect. In normal processes of
assimilation food substances are taken up by the cells with whose
substance they enter into chemical combination. That a toxin also
enters into chemical union with certain cells of the body was
indicated by the experiments of Wassermann, who found that when
he mixed tetanus toxin with the brain substance of the susceptible
guinea-pig, that the mixture was no longer toxic for the guinea-pig.
There was evidently a special affinity between the brain substance and
the toxin, for the reason that emulsions of other guinea-pig organs
when brought in contact with tetanus toxin exerted no such effect.
It would appear from this experiment that a toxin may have a special

Toxins and Antitoxins 7

affinity for certain tissue cells, and this appeared to explain the neuro-
toxic character of the symptoms which are observed in tetanus.

Antitoxins are not formed when any of the chemically defined
poisons are introduced into an animal. The effect of toxins is also
different from these with respect to the period of incubation which
precedes their apparent action in corpore whatever the dose may be,
if we except snake venom and eel-serum, where we however have
antitoxins formed. According to Ehrlich (1901) toxins enter into
specific chemical combination with the protoplasm of certain cell
groups, other poisons like alkaloids do not. Substances which enter
into chemical combination are assimilable, it being immaterial whether
they belong to the class of substances we regard as foods or as toxins.
The toxins of vegetable and animal origin possess the same characters
as the albumens or their derivatives. Antibodies are formed not only
for toxins, but also for food substances, such as milk, serum, etc., which
exert no injurious action.

The food-stuff or toxin enters into combination with the cell or
antibody by means of its haptoptiorous group (see below), and the
protoplasm wiiich is capable of combining with these bodies, which
in other woi'ds, is receptive, possesses corresponding "receptors," which
unite with the haptophorous groups.

Diphtheria antitoxin was found to enter into chemical combination
with diphtheria toxin, combining in definite proportions according to
what is known as the law of multiples. When a culture-filtrate of the
diphtheria bacillus is allowed to stand for some time it is found to lose
its toxicity as far as its immediate effects upon experimental animals
are concerned. A larger dose of old filtrate is required to kill an
animal in 24 — 48 hours than when a fresh filtrate is used. Neverthe-
less the filtrate continues to combine with antitoxin in the same
manner as before. In other words, the combining power remains, the
toxic power is lowered. This is due to the conversion of toxin into
" toxoid." Ehrlich concluded from the above observation that the
toxin-molecule contains two independent atom-complexes, the one
haptophorous (which persists), which combines with the antitoxin or
the corresponding cell receptors, the other toxophorons (labile), being
the cause of the specific toxic effect. The haptophorous group serves
as an intermediary in binding the toxophorous group to the cell.

When an animal is treated with culture-filtrate containing toxoid
it is rendered imnnine to toxin, and its serum is found to contain
antitoxin. According to Ehrlich, the haptophorous (non-toxic) group,

8 EhrlicKs Theory

combines with the cell receptors, or " side chains," because of a special
affinity between the groups. At first the combination may be effected
within the cell substance, but owing to an increased demand upon the
cell for receptors fitted to the haptophors, receptors are produced in
increasing quantity, and are finally thrown off as free receptors into
the circulation. It is the free receptors which, circulating in the blood,
lead to its antitoxic character. The serum of an animal treated with
toxoid is antitoxic, for the reason that the receptors disarm the toxin
of its haptophorous group, and consequently do not permit the toxo-
phorous group to combine with the cell protoplasm.

Toxoid is therefore to be regarded as a toxin molecule, the toxo-
phorous group of which has been destroyed. Similar or analogous
observations have been made upon other antibodies, such as tetano-
lysin (Madsen), snake venom (Myers), milk-curdling ferment (Morgen-
roth), etc.

Immunity to the effects of a toxin would therefore appear to depend
upon the absence of certain haptophorous groups in the toxin, for failing
these the toxin does not become anchored to the cell, and the latter
remains uninjured. Whereas the haptophorous group inunediately enters
into combination with its corresponding receptor, the action of the
' toxophorous group may be delayed for weeks. If tetanus toxin is
injected into frogs, the animals being maintained at 20° C. (Courmont),
large amounts may be injected with impunity. Morgenroth has shown
that the haptophorous group of the tetanus toxin enters into combination
with the nervous sub.stance of the frog, but that the toxophorous group
does not do so unless the temperature is raised.

Antitoxins are not of themselves toxic. As stated, the evidence of
their combining with toxins is based upon animal experiment, a mixture
of suitable proportions of homologous toxin and antitoxin being without
effect upon an animal which is susceptible to the toxin alone. Much
more favourable are the conditions of experiments with other antibodies,
such as haemolysins, agglutinins, and precipitins for the reason that the
interaction of the substances can be observed in vitro.

The study of the specific bacteriolysins', begun by Behring and
Nissen (1890) on animals immnnified with Vihno Metchnikovi, but
brought especially into prominence by Pfeiffer (1894) in bis studies upon

' That a difference between the bacteriolytic power of iiornial and immune serum
might exist was already indicated by me (1888, p. 388). The serum of a sheep immunified
against anthrax was found to be more bacteriolytic for anthrax bacilli than normal sheep's
scrum. The desirability of further experiments in this direction was indicated by me at
the time.

Antibodies in General 9

immunity to cholera, led to results of great importance. It is however
especially the studies upon the specific haemolysins which have helped
to further our knowledge concerning immunity. The results with the
haemolysins show that these and the bacteriolysins are both strictly
comparable in their mode of production and constitution. In both the
injection of cellular elements is followed by the appeai-ance of specific
lysins in the serum of the treated animal. The cytolytic serum is
rendered inactive by heat (.56° C.) and can be reactivated by fresh serum
which of itself is without effect. The action between cytolysin and cell
has been compared by Ehrlich to that which takes place between toxin
and antitoxin. In the case of the cytolysins we have to deal with a
stable immune-body and a labile complement, which cannot act of itself
but requires the intermediation of the immune or intermediary body.
When this is secured, the complement produces changes of a digestive
character in the cells subject to its action, and for this reason it may be
safely considered to possess the character of a digestive ferment. The
similarities observed in these antibodies led to the extension of the
theory of Ehrlich to other antibodies such as the agglutinins and
precipitins.

It appears therefore that all antibodies are formed on the same
general principles, although they may possess different properties.
Wherever they are formed the substance must be assimilable which
gives rise to their formation. Toxins are relatively simple bodies, they
are highly soluble and readily enter into combination with the proto-
plasmic molecule through the intermediation of the haptopliorous group.
Food-stuffs cm the other hand are more complicated, they require to be
simplified for assimilation, and in consequence the antibodies to which
they give rise must necessarily be more complicated. In the latter
case the albuminous molecule anchors the food-stuff by means of
receptors, which act as intermediaries, permitting a digestive, ferment-
like group (complement or the like) to attack the complicated food
molecule.

Before proceeding to consider antibodies in general I will add a few
facts regarding the antitoxins in particular. When an animal is being
immunified against a toxin, say tetanus or diphtheria toxin, the antitoxin
content of its serum and milk gradually rises, as periodic injections of
increasing doses of toxin are being practised. Brieger and Ehrlich
(1893, p. 341) noted however that the curve of antitoxin content
pursued a wavy course (wellenfdrmiger Verlauf) in animals treated
with tetanus toxin, the milk containing antitoxin in les.ser quantity

10 Ehrlich's Theonj

after each toxin injection, the fall in antitoxin being followed in due
course by a rise to a point higher than before. Salomonsen and Madsen
(IV. 1897, p. 326) also observed this in horses treated with diphtheria
toxin. The antitoxin content of the serum or milk always fell after a
fresh toxin injection, gradually rising afterwards, the maximum amount
being usually reached 9 — -10 days after the last toxin injection. A
similar observation has been made with regard to the precipitins, and
the phenomenon will pi'obably be observable in relation to all antibodies.
(See Appendix, Note 1.) The decrease of antitoxin appears to denote
that the toxin and antitoxin are combining in corpore.

The question of the regeneration of antitoxins in corpore has
been the subject of some investigation. Thus Roux and Vaillard
(ll. 1893, p. 82) found that they could remove (by repeated bleedings
in a few days) as much blood from a tetanus-immune rabbit as the
animal originally possessed, but the newly-formed blood was apparently
as antitoxic as that removed at first. Salomonsen and Madsen (xi.
1898, p. 763) made similar observations with regard to diphtherial
antitoxin in the serum of goats and horses, the animals being in a
condition of antitoxic equilibrium. These observations, as also the fact
recorded by the last autliors mentioned {Gomjjt. rend. Acad, des Sc,
Paris, 1898) that the administration of pilocarpine increased the amount
of antitoxin present in the serum of immunified animals, have been
brought forward in evidence as to the secretive nature of the anti-
bodies.

It has been claimed that normal antitoxins are present in the serum
of animals. Thus Meade Bolton, in Philadelphia, and Cobbett, in Cam-
bridge, found a certain percentage of normal horse sera to possess slight
but distinct antitoxic properties for diphtheria toxin. Cobbett has
however recently had occasion to observe diphtheria in the horsed Con-
sequently a doubt arises as to the former observation bringing strict
evidence as to the existence of normal antitoxins. On the other hand
their existence can scarcely be doubted for the reason that substances
fulfilling the function of different antibodies have frequently been
noticed in normal sera. Quite recently, moreover, von Dungern (1902,
p. 37) has observed that rabbit serum contained a " normal antitoxin "
for the toxin contained in the eggs of starfish {Astei'ias glacialis, and
Astropecten aurantiacus), the toxin in question acting upon the sperma-
tozoa of sea-urchins.

' Cobbett, C. L. (1900), Diphtheria occurring spontaneously in the horse, Lancet, toI. ii.
p. 573.

Antibodies in General 11

The chemical nature of antitoxins has been the subject of con-
siderable investigation without much light being thrown on the subject.
Brieger and Ehrlich (1893, p. 345) precipitated tetanus antitoxin
from the milk of immuiiified animals by means of ammonium sulphate,
finding tiiat the antitoxin was included in the first precipitate obtained
by adding 27 — 30Vo of the salt. The remaining filtrate still contained
much albumin, but very little antitoxin. Freund and Sternberg
(1899, p. 432) precipitated diphtheria antitoxin from antidiphtherial
horse serum by means of oOVa ammonium sulphate. Seng used
magnesium sulphate to saturation. Jacoby (cited by Michaelis 1902,
p. 41) found antiricin in rabbit serum to be present in the fraction
precipitated through the addition of 25 — 33% ammonium sulphate.
The antitoxins are therefore precipitated under the same conditions as
the globulins, but not the albumins. We shall see that other antibodies
behave similarly in this respect. Some authors claim that the antitoxins
are identical with the globulins, others that they are only entangled
with the globulins, being precipitated under the same conditions.
Pick (1902, p. 5) refers the disagreement to differences in the chemical
metliods used by different authors, and discusses the subject at length.
Pick found diphtheria antitoxin in horse serum to be present in the
pseudoglobulin fraction, whereas in the goat it was present in the
euglobulin fraction.

Ehrlich (1901, Schlussbetrachtungen, etc.) has sought by means
of schematic figures to render our conception clearer regarding the way
in which the various antibodies enter into chemical union with different
substances, by means of what he terms receptors. Before proceeding,
the reader is referred to the figures and description on the succeeding
page.

In normal assimilative processes, as also in the earlier stages
of immunization (either artificial or in consequence of disease), the
various receptors are attached to the molecule of the cell which is
receptive either for the foodstuff, toxin or the like. When the receptors
are produced in excess they are thrown off into the cumulation.
Accepting the above diagrammatic representation of the receptors
attached to the cell-molecules as a base, then the freed receptors would
be represented similarly, only detached at their base. Such freed
receptoi-s have been termed " haptins "; they possess the same structure
as the attached receptors. The haptins corresponding to receptors of
the Orders I. and II. have been styled " uniceptors," those correspond-
ing to receptors of the III. Order as " amboceptors/' by Ehrlich.

12

Ehrlich's Theory

Fig. 2.

Receptors of the First Order, are represented in Fig. 2, I., at a, being attached to the
cell-moleeule beneath. The portion e represents the haptophorous complex, whilst b
represents a toxin molecule, which possesses a haptophorous c and toxophorous d group.
This represents the union of toxin and antitoxin, or ferment and antifennent, the latter
possessing but one haptophorous group, the union between antibody and the toxin or
ferment being direct.

Receptors of the Second Order, are represented at c, in Fig. 2, II., in which e represents
the haptophorous, and d the siymophorous group of the complex, / being the food-molecule
with which it enters into combination. Receptors of this order are possessed by agglutinins
and precipitins. The digestive cell, or the antibody present in the serum in this case
possesses one haptophorous and one zymophorous group.

Receptors of the Third Order, are represented in Fig. 2, III., e being the haptophorous
group, g the complcmentophile group of the complex, k the complement with its hapto-
phorous h and zymotoxic z group ; whilst / represents the food-molecule which has become
linked to the receptor. Such receptors are found in haemolysius, bacteriolysins and other
cytolysins, the union with these cellular elements being effected by means of the immune-
body, this permitting the complement to act as a digestive ferment.

Antibodies in General 13

It appears from the foregoing that it is immaterial whether the
receptors or haptins and the haptophorous groups are attached to
cellular elements or in solution, the diagrammatic representation
indicates the mode of chemical union. A molecule provided with
haptophoric groups is as we have seen styled a haptin, the other
groups with which it is provided being named in accordance with the
action produced by the antibody. Thus, a toxic haptin possesses
haptophorous and to^ophorous groups ; a ferment haptin possesses
haptophorous and ^^mophorous groups; precipitins a haptophorous
and e?"(7ophorous group, the latter term being suggested by Michaelis
and Oppenheimer (1902, p. 360).

Antibodies resemble each other in a number of points. Ana-
logous bodies to toxoid (Ehrlich), are ccjmplementoid (Ehrlich and
Morgenroth), agglutinoid (Eisenberg and Volk), precipitoid (Kraus
and Pirquet). The chemical examination of various antibodies by
fractional precipitation (summarized by Pick, 1902, p. 34) shows that
all observers agree in their not being bound up with fibrino-globulin
and serum-albumin. This has been found to be the case with diphtheria
and tetanus antitoxins, with cholera-lysin, with cholera and typhoid
agglutinins, with precipitins. Anti-antibodies for anti-spermotoxin,
anti-hiBmolysin, and precipitins have been obtained, but not anti-
antitoxins and anti-bacterio-agglutinins. As Kraus and Eisenberg
(27. II. 1902, p. 211) point out, this is explainable by Ehrlich's theory.
The diphtheria antitoxins and typhoid agglutinins only possess affinities
for diphtheria toxin and typhoid bacilli respectively, and consequently
if they are introduced into an animal the immune-bodies they contain
find no substances to which they can become anchored, and it follows
that they will not lead to the formation of new substances such as
anti-antitoxin and anti-agglutinin. On the other hand, other immune-
substances introduced into animals of a corresponding species, will lead
to the formation of anti-immune bodies. All the antibodies have the
property of entering into chemical union with the bodies to which they
owe their origin, specific affinities existing between them. The names
which have been applied to various antibodies are in accordance with
their action in corpore or in vitro.

I do not think it expedient here to enter further into the theories
of Ehrlich with regard to the more intimate nature of toxins, the
so-called "toxin-spectra" and the like, regarding which the reader
is referred to the papers by Ehrlich. A useful summary on the subject
will be found in the collective review by Aschoff (1902). A number

14

Antibodies in General

of points of a critical nature, directed against the acceptance of some
of Ehrlich's views, have been brought forward by Baumgarten, Gruber,
and by Emmerich and Loew. It is difficult to enter upon these
questions without considerable detail which would be out of place
here, the reader is therefore referred to these authors in the
bibliography.

That a number of different antibodies may coexist in a serum
has been amply proved, for example, Bordet (1900) after injecting
fowl's blood into rabbits, observed the formation of agglutinins, haemo-
lysins, and precipitins in the animals' serum, this having also been seen
by Tchistovitch (1899) in animals treated with horse blood. Similarly
agglutinins may coexist with antitoxins and with antiferments etc.

Normal Sera, as we shall see, may contain Antitoxin,

Antiferments,
Cytotoxins,
Agglutinins,
Precipitins,

the amount of antibody present being usually very slight as compared to what is
observable in sera which contain specific antibodies.

^Antitoxin

An immunizing
substance may
produce

The Antibodies :

iCytotoxins

Antiferment

Gytotoxin including Haemolysins,^ . ,. (Complement

Spormotoxius, Nephrotoxins, etc.v „ -llmmune-body or

Bacteriolysins ) (Amboceptor

Agglutinins

Precipitins

/Anti-complement
-^ (Anti-cytotoxins — consisting of ^Anti-inmuuie-

\ Antiferment Igwurifie Xo a,nii-\Anti-antiferiHent ( body

1 Agglutinins
Precipitins

antibodies

j Anti-agglvXinins
\Anti-precipitins

Synonymous Terms.

Gytotoxin is used for any substance in serum, venom or bacterial cultures or of
plant origin, which destroys cellular elements, either animal or vegetable. The
haomolysins and other toxic substances which kill but do not dissolve cellular
elements are included under Cytotoxins, also the bacteriolysins (bactericidal sub-
stance, alexin).

Terminology 15

Precipitin is used to include the antibodies contained in lactosera, called
"coagulins" by some authors. The term "serotoxin" (corresponding to cytotoxin)
suggested by Ncdriagailoff (U. viii. 1901) will scarcely receive acceptance. Sera
containing precipitins for blood are referred to frequently as haematosera.

Complement is used synonymously with addiment (Ehrlich), alexine, cytase (of
French authors).

Immune-body is used synonymously with Amboceptor, Immunkorpcr, Pra-
parator, Copula, De-smon, Hilfskorpcr, Zwischenkorper (of Gemian authors),
Fixateur, substance sensibilisatricc or preventive specifique (of French authors),
Fixer, and Go-between (of English authors).

Iiitermediary-hody is confined in its use to the coiTesponding substance to the
foregoing, which is found in normal sera, and corresponds to Ehrlich's " Zwischen-
korper."

SECTION II.

ANTIFERMENTS AND FERMENTS.

The discovery of antiferinents dates from Hildebrand (1893, p. 32) who found
an anti-emulsin in the serum of rabbits subjected to repeated emulsin injections.
Fermi and Pernossi (1894) found that when trypsin was injected into guinea-pigs
it disappeared within 24 hours, its presence being determined by the action of the
animals' blood and organs upon gelatin. The experiments, both in corj^ore and in
vitro, showed that the trypsin was destroyed (see Achalme, 1902). Von Dungern
(1898) obtained an antidiastatic ferment by treating animals with the proteolytic
ferment contained in certain bacterial cultures. Morgenroth (1899 and 1900),
experimenting with goats, obtained anti-rennet ferment through injecting animals
with rennet. He next obtained anti-cynarase by injecting cynara.se ferment (from
the blos.soms of Cynara cardunculus), and proved that this and the preceding
antiferment were distinct. Briot (1900) working independently to Morgenro^'..,
discovered that he could obtain anti-reimet in tlie serum of rabbits treated with
rennet. Delezenne (cited by Metchnikofl", 1901, p. 115) whilst finding that the
normal senmi of animals exerted scarcely any effect on gelatin, that of animals
treated with gelatin did. He injected animals with fluid gelatin, and observed that
their serum .soon acquired the power of rapidly dis.solving gelatin. We may there-
fore speak of this as a gelatin ferment. The ferment, or anti-gelatin serum, resembles
the precipitins in that it resists heating to 56" C. Wliei'eas Landsteiner (23. ill.
1900) stcitcs that the action of anti-trypsin is non-specific, being bound up with
serum albumin, Glassner (unpublished research, cited by his colleague Rostoski,
1902 b., p. 60) came to the opposite conclusion, exixirimenting with anti-trypsin
normally present in the serum of the horse and ox. The actions of these anti-
trypsins were most marked against their homologous trypsins. The anti-trypsin
is bound up with pseudoglobulin. Achalme (1902, ji. 744) cites several authors who
have observed the presence of anti-trypsin in normal serums. Metchnikoff (1901,
p. 117) cites Roden as having foimd normal horse serum to retard or prevent rennet
action, and states that still otliers have found normal sera to more or less impede
the digestion of albuminoids through trypsin". Moll (1902) immunified rabbits

' See also S. Korsehun, " Ueber Lab und Antilab," Zeitschr. f. physiol. Chem. xxxvi.
p. 141, who studied the anti-rennet in normal horse serum, and by injecting horse serum
into goats obtained anti-anti-rennet. He considers anti-rennet to act on rennet as does
antitoxin on toxin. He also found what appears to be a pseudo-anti-rennet. The reader
is referred to the original.

Antiferments and Ferments 17

against the urea-splitting ferment of Micrococcus ureae, demonstrating the presence
of anti-urease in their serum. Although normal serum and urine antagonize urease,
the normal antagonistic substance is distinct from anti-urea.se. I will further
mention that Sachs (1902) has in a similar manner succeeded in obtaining aiui-
pepsin, and Gessard (1902, cited above) anti-tyrosin.

Anti-coagulins have been discovered by Bordet and Gengou (ni. 1901). These
investigators injected rabbit serum, or plasma, into guinea-pigs and found that the
serum of the guinea-pigs acquired the property of preventing the coagulation
of rabbits' blood. They attribute the greater part of the anti-coagulating action
to the neutralization of the rabbit's hbrin-ferment. The anti-coagulin is specific,
or nearly so, in its action, and the experiments made therewith indicate that the
fibrin-ferments of different animals j)ossess a different constitution, although
capable, all of them, of prod\icing coagulation of the same fibrinogen. The anti-
coag>ilin resists heating to 58-5° 0. The authors cite Camus (1901) as having also
worked upon anti-coagulins.

The anti-coagulating action of leech-extract studied by Haycroft, then by
Dickinson (1870) and others, can be counteracted by a coagulin. Thus Wendelstadt
(1901) found that if he injected leech-extract into rabbits he obtained an anti-
body which impeded the anti-coagulating action of the leech-extract. According
to this author, the coagulin is chiefly formed in the pancreas, then in the liver and
kidneys. The source of these different anti-ferments will doubtless vary consider-
ably, depending on their nature.

SECTION III.

THE CYTOTOXINS OF BLOOD SERUM.

The discovery of the cytotoxins dates back to the time when
blood-transfusion was first practised, it being noticed that the bloods
of different animals transfused into man were more or less directly
injurious, and not capable of replacing human blood for this purpose.
The transfusion of foreign blood led to the formation of clots, thrombi,
serous exudation, and more or less haemolysis. A rdsume of the
earlier work on this subject will be found in Ziemssen's Klinische
Vortrage, 1887, wherein the observations of Panum, Ponfick, Hayem,
Landois and others are recorded.

Especially important for our subject are the investigations of
Landois (1875) on blood-transfusion. He found that the transfusion
of foreign blood might prove fatal to an animal. The transfusion was
followed by haemoglobinuria due to the haemoglobin derived from the
injected blood corpuscles, but in addition to the dissolution of the
treated animal's corpuscles. Where the blood transfused emanated
from a closely related species Landois observed no ill effects to follow
its transfusion, this being the case for instance when transfusion was
practised between the dog and wolf, horse and donkey, hare and rabbit.
He concluded that large transfusions could only be practised between
closely allied species. The first to study the phenomena of haemolysis
in heterologous blood serum was Creite (1869), whose description of the
appearances observed leaves little to be desired.

The term cytotoxin has been proposed by Metchnikoff (vi. 1900,
p. 369) for such animal poisons as affect cellular elements, whether they
be animal or vegetable. For the cytotoxins which dissolve the blood
cells the term haemolysin has gained general usage, and for this reason
I have retained the term in the following pages. Bordet (v. 1900, p.
257) prefers the term haemotoxin to haemolysin, but I see no

Hfiemolysins and Bacteriolysins 19

advantage in abandoning the term lysins, which certainly suggests
something more as to their effects. Corresponding terms are spermo-
toxin for toxin in serum which acts more especially on spermatozoa,
leucotoxin for serum which destroys leucocytes. Fiexner and Noguchi
(II. 1902) .separate the haemolysins into erythrolysins (dissolve red
corpuscles) and leukolysins (dissolve leucocytes).

Besides the serum of animals the secretion of certain glands may
be cytotoxic, as is seen with snake venoms. Cytotoxins may also be
of vegetable origin, being either derived from bacteria or higher plants.
We shall refer to these elsewhere (see p. 28). The haemolysins act by
separating the haemoglobin from the stroma of the blood corpuscles,
causing the blood with which they come in contact to "lake."
Haemolysis may be effected also by hypotonic salt solutions, or by
destructive chemicals, but this apparently purely physical phenomenon
does not concern us.

It is true thcat Baumgarten (16, xil. 1901 ; and 27, x. 1902) would explain
haemolysis as due to altered physical coudition.s brought about by haemolytic
.sera, as seen in plasmolysis due to non-isotonio solutions. He however found that
this did not offer a full explanation, for a haemolytic serum acted more rapidly than
saline solution of equal specific gravity. He therefore suppases that the agglutinin
present in haemolytic serum causes this increased action, claiming moreover that
the agglutinin is identical with Ehrlich's immune-body. It appears to him a priori
probable that the serum of different animals may contain different quantities of
osmotic substance. He does not lielieve that the action of complement is similar
to that of digestive ferment, a-s considered by Buchner, Bordet, and Ehrlich and his
school.

Haemolysins may be present in certain normal sera, or they may
be artificially produced by injections of the blood of other animals than
that treated, if we except the observations upon isolysius. In tlieir
fundamental characters the haemolysins, both natural and artificial,
correspond to the bacteriolysins; they offer the advantage over bacterio-
ly.sins, that it is possible to experiment with them in the test-tube.
The discovery of the artificial haemolysins dates from Belfanti and
Carbone (1898).

The Mode of Action of Haemolyshis and Bactenobjdns.

Bordet and Ehrlich have shown that there are at least two bodies concerned in
the haemolytic or iKvcteriolytic action of a serum. The one is the specific inmmne-
body, which is thermostable ; the other is the thermolabile complement, which is
present in normal sera. The artificially produced haemolysins, namely those

2—2

20 The Cytotoxins of Blood Serum

produced in animals by the injection of foreign blood cori:)uscles, appear to have
the same constitution as the normal haemolysins.

Bordet (1895 — 1900) demonstrated the existence of the two bodies referred to
above by heating immune-serum to 55° C, or allowing the serum to stand for some
hours. Such serum was no longer cytolytic, but if he added fresh normal serum
to it, the normal serum of itself being inactive, the heated immune-serum regained
its haemolytic power, that is, it was reactivated. The immune-body persists
in stored sera, and withstands a temperature of 65 — 70° C. ; the thermolabile body
as we have seen is destroyed at 55° C. and is soon lost. According to Bordet
the immune-body or " matifere sensibilisatrice," as he terms it, becomes anchored
to a red blood cell or bacterium, rendering it highly susceptible to the influence
of the complement, to the action of which it wa.s previously insusceptible. It is
the complement, or " alexine " as he terms it, which reactivates the immune-body.
Ehrlich and Morgenroth (1899) showed by most ingenious experiments that the
immune-body became fixed to the susceptible cells.

By injecting complement, Bordet (v. 1900) produced anti-complement, which
prevented the action of the immune-body by neutralizing the complement, the
anti-complement not having any specific affinity for the immune-body. Bordet
and Gruber consider the complement or "alexine" identical in all cases, the
former and Gengou (1902, p. 738) found fresh dog serum added to fresh rabbit
corpuscles lost its bactericidal properties. Ehrlich and Morgenroth consider that
normal sera contain a mrdtiplicity of complements. Metchnikoff (1901, p. 123)
points out that there is agreement as to the action of anti-complement upon
complement being direct. Bordet found that heating a mixture of these bodies
did not liberate the anti-complement, for the heated mixture does not regain its
anti-haemolytic power, as we might expect it to do if anti-complement were freed
by heating. Heating to 55°, as we have seen, aS'ects complement, but not anti-
complement. The conclusion is therefore jiistified, that complement and anti-
complement enter into chemical combination. Ehrlich and Morgenroth have found
moreover that anti-complement only prevents the action of complement, that it
exerts no action either on the immune-body or susceptible cell.

Ehrlich (1900) and Morgenroth, by the method of "elective absorption," found
that a normally haemolytic serum contains a variety of intermediary bodies
(analogous to the immune-bodies in artificially haemolytic sera). They found
that a serum which had been treated with one sjjecies of corpascles until it
ceased to act on these still had the power to haemolyse other species of corpuscles.
In the same way a multiplicity of complements appropriately adapted to the
various intermediary bodies have been found by them to exist in normal sera.
The union of cellular elements and the intermediary body is efi'ected, according to
Ehrlich, by means of combining or haptophoric groups, common to both cell and
intermediary body, the complement being similarly linked to the intermediary
body. In other words, the intermediary body possesses two haptophoric groups,
the one (complementophilic) combining with the complement, the other with the
cell. The complement has also two haptophores, the one combining with the
intermediary body, the other possessing a fermentative action (zymotoxic, or toxo-
phoric group) which dissolves the cell.

Flexner and Noguchi (1902) find that there are a multiplicity of intermediary

Immune-Body and Complement 21

bodies in snake venoms, for venom solution treated with the washed blood corpuscles
of the dog, rabbit, and guinea-pig in 8ucce«sion was found to give up to each a part
of its intermediary bodies, no one kind of corpuscle being capable of fixing the
entire content of intermediary bodies. It therefore appears as if there might be
an indefinite number of intermediary bodies in venom. When a complement
foreign to the corpuscles is used it never causes complete haemolysis. It is evident
that these results with venom are very similar to those of Ehrlich with haemo-
lytic sera.

That different immune-bodies and complements may coexist in a serum has been
shown by Wendclstadt (16, iv. 1902) who treated animals with different bloods
simultaneously, for instance with the blood of the ox, sheep, and pig, finding three
immune-bodies and three complements in their serum; the complement for the
pig being more resistant to heat than the others.

Ehrlich and Morgenroth found that they could not obtain anti-complement when
they treated an animal with complement derived from a dosely related species, but
they obtained it readily in many cases where the relationship was distant. The
injection of goat serum into the sheep and vice versa did not give rise to anti-
complement. This observation is quite in accord with what has been observed with
regard to the formation of precipitins.

An excess of immune-body may impede haemolysis upon the addition of com-
plement, as Nolf observed in haemolysing fowl corpuscles with the immune serum
of the rabbit ; moreover Neisser and Doring are cited by Eisenberg (v. 1902,
p. 303) as having made an analogous observation when haemolysing rabbit cor-
puscles with human serum from a case of clironic nephritis. Ide (27, vii. 1902,
p. 273) has confirmed this. For an explanation of this interesting phenomenon
of " Komplementablenkung " see Neisser and Wechsberg (1901), who observed that
an excess of immune-body prevented the action of a bacteriolytic serum ; as did
also Lipstein (16, iv. 1902), and Walker (i. 1903).

That the normal haemolytic action of eel serum upon rabbit corpuscles can be
prevented by specific anti-haemolyiic serum has been shown by Camus and Gley
(29, I. 1898) and Kossel (14, ii. 1898) experimenting with the serum of rabbits
immunified against the serum of the eel. The rabbits were treated with increasing
doses of eel serum, for which they gradually dcveloiied increasing quantities of
antitoxin (othei-wise anti-haemolysin), this being confirmed by Tchistovitch (1899).
Bordet (1900) also found that the serum of a guinefi-pig immunified with rabbit
corpuscles, when injected in carefully graded doses into a rabbit (being highly
haemolytic), caused the formation of anti-haemolysin in the rabbit, the anti-
body preventing haemolysi.s. The experiment was reversed by Schutze (5, vii.
1901) with similar result. Schlitze thought he could trace the formation of the
anti-haemolysin to the action of the immune-body, for he treated the guinea-pigs
with complement-free (heated) rabbit serum. Miiller (21, ii. 1901) also treated
rabbits with heated fowl serum, the antiserum obtained being anti-haemolytic.
Ehrlich and Morgenroth (1901), however, consider that Schutze, and MuUer, by
heating their sera, converted the complement into complementoid (analogous to
toxoid) and that the anti-haemolytic action they observed depended upon anti-
complement ; for the complementoids, although incapable of acting upon corpuscles,
give rise, when injected, to anti-complements. Miiller (21, il. 1901, p. 185) failed

22 The Cytotoxins of Blood Serum

to produce anti-haemolysin in guinea-pigs treated with normal rabbit senun which
had been heated. Ehi'lich and Morgenroth (iii. 1900) obtained anti-kolysin by
injecting isolysin (see later) into a goat, noting that there were individual differ-
ences in the action of both isolysin and anti-isolysin depending upon the animal
from which they were derived.

Waasermann (1901, Zeitsckr. f. Hyg., Bd 37) injected washed rabbit leucocytes
into guinea-pigs, obtaining weak but evident "anti-complement" to the complements
of rabbit serum, the haemolysis of goat blood by rabbit serum being prevented.
Donath and Landsteiner (25, vii. 1901) obtained a similar result in rabbits by
injecting dog leucocytes, crushed lymph-glands, red blood corpuscles and milk
severally, but they do not conclude that the antisera obtained in these different
ways are identical in character.

Miiller (24, vi. 1901) finds that a numlier of normal sei-a possess anti-haemolytic
properties, several for instance protected rabbit's corpuscles against duck haemnlysin.
This action appears to be due to anti-comiilement. When he treated (12, viii. 1902)
animals with such anti-haemolytic sera, these being inactivated, their serum acquired
the property of haemolysiug the corpuscles of the animal which possessed the anti-
haemolytic serum.

Besredka (25, x. 1901) also claims to have observed the e.xistence of anti-
haemolysins in the normal sera of man, rabbit, guinea-pig, fowl, and goosa Ho
considers that they represent anti-auto-cytotoxins, whose formation is brought aliout
by the constantly occurring death of certain cells in the body (I'ed blood cor|)uscle8
for example), the assimilation of these cells bringing about the formation of this
antibody. This view receives support from what has been observed with regard to
isolysins in disease. The presence of anti-haemolysin is to be considered as evidence
of a reaction on the part of the body, its object being to combat the continual
destruction of bodily cells, although at the same time haemolysins ' are produced.
We know that fresh rabbit serum, for instance, haemolyses guinea-pig corpuscles.
When heated to 55^0. it ceases to be haemolytic, whereas this heated serum when
added to fresh rabbit serum in certain proportions will prevent its exerting a
haemolytic action on guinea-pig corpuscles. This does not happen when the heated
sera of other animals (man, ox, fowl, goose, etc.) are added instead of rabbit serum.
There would therefore appear to be evidence as to the existence of both anti-
haemolysin and haemolysin in the one serum.

The action of normal haemolysin is not confined to red blood corpuscles. For
instance, Delezenne (x. 1900) found that it aftected other cells of the same animal
as well, to a gi-eater or less extent. Haemolysins act both on the red blood corpuscl&s
and leucocytes, as can be seen in experiments conducted in vitro. To see the
effects on other cells the exijeriments nnust ha conducted to a large extent in
corpore. Taking eel serum ivs a type of a haemolytic .serum, we find that it is
destructive to red blood coi'])uscl6s (Mos.so, Camus and Gley, Kossel, etc.), to
leucocytes (Delezenne, 1898), nerve cells (Kossel and Wostphal), renal epitheliimi
(Pettit, 1898). The effects on the nervous system were already observed by Mosso
(1888) and Delezenne (x. 1900, p. 693), the latter finding eel serum 200 times as toxic
when introduced into the nerve centres as when administered suboutaneously to
the same animal.

Metchnikoff (x. 1899, p. 762) produced " ieucotoxins" by injecting an emulsion

Leucotoxhu and Spermofoxins 23

of rat spleen into the guinea-pig. After 47 days the treated animal's serum was
found to agghitinate and dissolve rat leucocytes suspended in rat's abdominal
lymph. The mononuclears were finst attacked, then the polynuclejirs, and finally
the Mastzellen of Ehrlich. He also obtained leucotoxin by injecting an emulsion of
rabbits' mesenteric glands into guinea-pigs. Although these glands only contained
mononuclears, the serum was destructive both to rabbit mononuclears and polynu-
clears. The leucotoxins were markedly specific, that for the rat scarcely affe<;ted
mouse leucocytes, that for the rabbit had no efiect on rat leucocytes and vice vertd.
Funck (26, v. 1900), and Besredka (vi. 1900, p. 391) have experimented also upon
these lines, the latter preparing leucotoxins for different animals (horse, ox, sheep,
goat, dog) by injecting their lymph glands into other animals. He found that none of
the leucotoxins affected human leucocytes, but this was an exception, for ox leuco-
toxin, obtained from the guinea-pig, was leucotoxic for rabbits ; and human leuco-
toxin was leucotoxic for guinea-pigs. Be.sredka found leucotoxic sera less stable than
haemolytic sera, and no leucotoxin was formed when lymph glands heated to 55° C.
were injected. As with other toxins, the leucotoxin.?, when injected into animals,
l)roduce illness and death ; he found for instance (p. 393) that an intraperitoneal
injection of 3 c.c. of leucotoxic serum for the guinea-pig, obtained from a rabbit,
killed a guinea-pig in 3—4 hours. Besredka (p. 397) obtained anti-lettcotoxin in the
serum of guinea-pigs treated with guinea-pig leucotoxin (from rabbit), the antibody
(ireventing leucolysis when mixed with leucolytic serum.

In their studies on snake venom, Flexner and Noguchi (1902) found the venoms
to dissolve and agglutinate rabbit's leucocytes. Whereas the agglutinating principle
seemed to be identical for both leucocytes and red corpuscles, the lysin for each was
distinct. Venomized leucocytes required a complement for their destruction just
!is do the red blood corpuscles. The difiei-ent varieties of leucocytes were found
to react differently to venom.

The presence of leucotoxins in fresh normal dog serum was already observed
by Buchner (1893, p. 120), who stated that the fresh serum killed but did not
dissolve the leucocytes of man and rabbit. When dog serum was heated to 55° C.
it was no longer leucotoxic.

Tlie spermotoxins discovered by Landsteiner (29, iv. 1899, p. 549) have proved
of considerable interest. He obtained spermotoxin by injecting ox spermatozoa
into guinea-pigs. Whereas ox spermatozoa introduced into the peritoneum of the
normal guinea-pig remained motile for a considerable time, those introduced into
the peritoneum of a guinea-pig which had received several injections of spermatozoa
were immobilized very quickly. This was confirmed by Metchnikoff (x. 1899,
1). 739) who injected ox and human spermatozoa into guinea-pigs. Whereas the
sj)ermatozoa remained alive for 30 hours in normal guinea-pig's serum, they were
immobilized in a few minutes in that of a treated guinea-pig. The antiserum only
immobilized the spermatozoa, it did not dissolve them. Moxter (25, i. 1900) found
that spermotoxic serum for the sheep also haemolysed sheep's blood corpuscles,
and that the immune-body combined with sheep spermatozoa. He denied the
specificity of spermotoxin, considering, however, that it did possess a special affinity
for Si)ermatozoa. The blood cells appeared to him to be more susceptible to
a variety of antibodies than are the other cells. Metchnikoff (vii. 1900, p. 373)
considers that the haemolytic action of Moxter's spermotoxin may have been due

24 The Cytotoxins of Blood Serum

to haemolysin produced in his treated animals through injecting blood contained
in the testicular emulsion he used. He argues that if the action of these antibodies
is not specific, then an artificial haemolytic serum should destroy the spermatozoa,
whereas it does not. He found that a rabbit serum which was haemolytic for sheep
corpuscles did not exert any more action upon sheep sjiennatozoa than did normal
rabbit senim, ho claims in fact that they lived longer in the haemolytic serum. He
concludes therefrom that the antisera may be s|)ecific for certain cells as distinct
from others belonging to the same animal. To further test this point, Metalnikott'
(ix. 1900, p. 581) treated a rabbit with a mixture of blood and spermatozoa fixjm
sheep, obtaining an antiserum which haemolysed corpuscles and immobilised
spermatozoa. In confirmation of Metchnikoft"s results, he found that he could
remove the haemolysin by adding corpuscles to the senim, the remaiin'ng flviid
remaining spermotoxic. On the other hand spermatozoa fixed Ixith the intermediary
bodies of spermotoxiu and haemolysin, and he concludes that this must have been
what misled Moxter. Metalnikofl' (p. 578) obtained spermotoxin from rabbits
treated with guinea-pig sj:)ermatozoa. He noted that normal rabbit serum was
scarcely less spermotoxic than that of treated rabbits, but there was an essential
diflerence, namely, that the normal spermotoxin heated to 56° C. could not be
reactivated, whereas the artificial spermotoxin coidd be reactivated by adding fresh
normal guinea-pig serum thereto, the latter having no influence of itself. Con-
sequently artificial spermotoxins, as is the case with haemolysins, are composed
of an immune or intermediary body and of complement.

The existence of noi-mal spermotoxins, as observed by Metalnikofl', has also been
proved by Weichardt (25, xi. 1901, p. 835). Two out of three normal rat sera
were markedly spermotoxic, the third feebly so, for rabbit sijermatozoa. Pigeon
serum was found to be spermotoxic for rabbit, dog, and goose spermatozoa. He
observed the normal spermotoxic action of a goose's serum to vary widely in one
and the same animal at various times.

Following along the lines of Ehrlich and Morgenroth's work with haemolysins,
Metalnikoff (p. 585) succeeded in producing auto-spennotoxiii by injecting guinea-
pig spermatozoa into guinea-pigs. The auto-spermotoxin, which was active in vitro,
could be reactivated by fresh normal guinea-pig serum after having been inactivated
by heat.

Halban and Landsteiner (25, ill. 1902, p. 475) observed that spermotoxic .serum
obtained by injecting rabbits with ox spermatozoa agglutinated ox corpuscles much
more powerfiUly than did normal rabbit serum.

The reaction of different guinea-pigs to foreign spermatozoa injections was
found to vary considerably by Weichardt (p. 833), some producing a highly
spermotoxic serum, others little or no spermotoxin.

Anti-spermotoxin, comparable to anti-haemolysin, etc., was produced by Metal-
nikoff (ix. 1900, p. 583) by injecting guinea-pig si)ermotoxin (from rabbit) into
normal guinea-pigs. Seriun, containing anti-spermotoxin, exerted but a slight
effect when fresh, but its effect was marked when it was inactivated, this being due
to the fresh serum containing a preponderance of anti-complement. Consequently
auti-spermotoxic serum consists of anti-intermediary body and anti-complement.
Weichardt (25, xi. 1901) reacihes the same conclusion, adding that anti-spermotoxin
also contains an auti-agglutiniu.

Neurotoxins, Trichotoxins, etc. 25

Neurotoxins are another class of antibodies obtained by injecting the brain-
substance of one species into another. Metchnikoflf succeeded in obtaining
a neurotoxin for pigeons in the scrum of rats treated with pigeon-brain emulsion.
His pupil Dolezenne (x. 1900, p. 696), who cites the unpublished experiment,
sub.sequently obtained dog neurotoxin by treating a duck with dog-brain substance.
He (p. 703) tested this neurotoxin on rabbits with negative result, whereas
it affected cats, though less than dogs. This indicates a relationship between these
Carnivores, such as I have been able in a measure to demonstrate by means
of precipitins. Again as with the precipitins (see later) he was unable tf) obtain
positive results by rei)efitedly injecting rabbit-brain substance into guinea-pigs
(p. 695). Boeri (28, x. 1902) on the other hand states that he injected rabbit-
brain emulsion intraijcritoneally into guinea-pigs, and found their serum when
injecteil subdurally into rabbits to produce prolonged nervous excitation. When
heated to bo" C. the serum had no such effect.

Nevu'otoxins are normally present in venoms of serpents, as found by Flexner
and Noguchi (1902). The nem'otoxin is distinct from the haemolytic substance
in venom, for a venom, robbed of its baemolysin by the addition of corpuscles, was
neurotoxic, and vice versd. This agrees with Ehrlich's view, tis suj^ported, amongst
others, by Wassermann and Takaki's experiments upon the fixation of tetanus
toxin in certain centres, etc.

Trichotoxins were first obtained by von Dungern (1899), the antibody, as the
name implies, exerting a special effect upon ciliated epithelium. He injected
rabbits and guinea-pigs with the tracheal epithelium of the ox, and found their
serum to immobilize the ox ciliated epithelium. He however found that the
antiserum also haeiuolysed ox corpuscles, but it had more affinity for the epithelium
than did an antiserum obtained by corresponding blood injections. Rabbits
injected with cows' milk gave an anti.serum which also immobilized the epithelium,
and it was also haemolytic to a considerable degree. The three antisera (for milk,
corpuscles, epithelium) were subjected to comparative tests on S^/j, blood corpuscle
suspensions, the antisera being inactivated and then reactivated by the addition
of complement. The residts showed differences with regard to the haemolytic
properties, those of the antisera for milk and epithelium being distinct from the
third, but indistinguishable from esich other.

Other authors have finally claimed to have produced a variety of toxic substances
in animals treated with ennilsions of different glands, this l)eing at present the
subject of much active investigation. It is only neces.sary to mention a few of
these so as to indicate the general drift of the work. Lindenmann (ll. 1900, p. T)?)
treated guinea-pigs with rabbit kidney-emulsion, obtaining, he claims, a highly
potent " nephrotoxin " which caused necrosis and profomid disintegration of the
epithelium of the convoluted tubules in the rabbit's kidney, the glomeruli remaining
unaffected. Nefedieff (25, i. 1901, p. 18) repeated the experiment, and reversed
it, confirming the residt. He found the nephrotoxin to be also haemolytic.

Delezenue (viii. 1900) obtained antisera for liver cells, or " Aepatotoxins," and
these have also been studied by Deutsch (cited by Nefedieff). Mankowski (1902)
treated cats with intraperitoneal injections of dog thyroid gland emidsion, obtaining
a serum having toxic properties for the gland in dogs, the action being in the main
thyreolytic. The serum contiiining the " thyreotoxin" could be inactivated and

26 The Cytotoxins of Blood Serum

reactivated, consequcntlj' it contained an intermediary bo(iy and complement.
GoutHcharnkow (1901) also observed toxic effects in dogs injected with a similar
antiserum obtained from sheep. He however found that the thyroids of sheep
were afiected by injecting these animals with dogs' thyroids, general systemic
disturbance being jiroduced.

Von Dungern (1902, p. .51) treated rabbits with emulsions of the eggs of starfish
and sea-urchins, and found theantisera to agglutinate the corre.spouding sjjermatozoa
of these animals.

From the work of Metchnikoff it would appear as if the antibodies for special
cells of one animal exerted no influence on others. Delezenne (x. 1900, p. 704)
also found that his artificial neurotoxin for the dog did not haemolyse dog
corpuscles, and that his artificial haemoly.sin exerted no neurotoxic action. We
have seen that other workers hiive noted sjjecial eft'ects on the cells, emulsions
of which had been used for the production of antibodies, but that they acted
besides on other elements. I would add that this has been the case in the investiga-
tions of Boeri (28, x. 1902) who found a neurotoxic serum to be also haemolytic,
and a haemolytic serum to be slightly neurotoxic. The evidence is certainly
unanimous with regard to the different antibodies exerting a special action.

Regarding the Source of Haemolysins and Baderioli/sitis.

Gengou (1901, Ann. de VInst. Pasteur, xv.) concluded that the haemolysins are
derived from leucocytes, for the reason that plasma separated from fresh blood, kept
cold throughout, by centrifugalization, was less haemolytic than serum. G. Ascoli
(9, X. 1902, J). 736) came to the opposite conclusion. Ascoli obtained plasma in the
same manner as Gengou, from a dog which had been immunified with rabbit
corpuscles. When 2 — 5 c.c. of this dog's plasma were injected into a normal rabbit
the latter develoj)ed haemoglobinuria lasting 24 — 48 houre. Ascoli concludes
that plasma possesses the same haemolytic properties as serum both in corpore and
in vitro. Whereas Nolf claimed that .serum injections did not lead to the formation
of haemolysins, the contrary has been found to be the case by von Dungern (1899)
and Morgenroth (1902). Sohatteufroh (1901) wiis unable to obtain haemolytic sera
by goat serum injections, but did so by injecting goat and human urine, the latter
observation being confirmed for human urine by Ruffer and Crcndiropoulo (24, i.
1903). Ide (27, vii. 1902, p. 269) considers that such results are to be explained
by the presence of haemoglobin in serum and urine.

Ide produced anti-pseudoglobulin and anti-serum-albumin and found the first
not to haemolyse corresponding corpuscles, whereas the second did so, although
its action was but one-tenth as strong as that of anti-haemoglobulin. He concludes
therefrom that intact serum and serum albumin possess receptors in common with
those of the red blood corpuscles, and these receptors, Ide think.s, are to be found
in the haemoglobulin which remains present in .serum and serum albumin.

Shibayama (5, xil. 1901) found haemolysins for dog corpuscles in the spleen
and lymphatic glands of normal guinea-pigs, not elsewhere. He considers that
when dog blood is injected into such animals these organs are stimulated to
increased production of haemolysin in the sense of Ehrlich's theor)'.

Bacteriolysins 27

The seat of origin of bacteriolysins in corpore has been studied by a number
of observers, and has been the sulyect of considerable discussion, for which reason
I shall not enter u{)on it hei-e (see Metchnikotf, 1901 ; Pfeiffer and Marx, 1898 ;
Deutsch, 1899 ; Wassermann, 1899 ; Romer, 1902 ; and the reviews of Ritchie,
1902 and Aschoff, 1902). According to Pick (1902, pp. 16—21) bacteriolysins arc
contained in the euglobulin fraction of serum, this being contrary to Pfeiffer and
Proskauer (1896).

That there is a difference in the specific bacteriolytic sera obtained from different
animals a])pears to be the ca.se from the investigations of Sobemheim (1899), who
found that anti-anthrax serum olitainod from immunified sheep was able to protect
sheep but not rabbits. In this case we are dealing, not with an antitoxic serum but
with one whose action is antibacterial. As Elirlich (Croonian Lect. 1900) notes,
Kitt had a precisely similar o.xi>erience with .symptomatic anthrax. It will be noted
that an analogous observation has been made with regard to the precipitin.s.
This would appear to be due to the complexity of bacteriolysins and precipitins as
compared with antitoxins, where no .such differences have been noted. It may be
supposed that in reacting to a highly complex body the organism impresses
more of its own character upon the antib<xly which it evolves.

Depending upon the age of an animal, its blood corpuscles will behave differently
both to natural and artificial haemolysins. Thus Camus and Gley (1899, p. 779)
found the normal haemolysin of eel serum to be markedly resisted by the corpuscles
of young rabbits, and Delezenne (.\. 1900, p. 702) found the corpuscles of young
dogs to be much more resistant to the artificial haemolysins (for dog) than those
of adult animals. We shall see that the precipitins also give different reactions
with foetal and adult blood.

The bacteriolysins to which reference has been made in the preceding i>ages
constitute a class of cytotoxins essentially similar to the haemolysins. They act
upon bacteria. Their pr&sence in normal blood and various body fluids was
demonstrated by Nuttall (1888) ; whereas artificial or specific bacteriolysins were
first demon.strated by Pfeiffer in animals immmiitied with the cholera vibrio.
The work done on the bacteriolysins has been very extensive, but it is impossible
to enter into the subject hei-e at all in detail. It will suffice to say that the
sjiecific bacteriolysins Viehave essentially iis do the specific haemolysins, and that
tliey possess the .same constitution. They are composed of a labile complement
(destroyed at 55° C, as I first showed for normal bacteriolysins) and of a stable
imnuuie-body. The latter is capable of conferring inmuuiity, as was first .shown
by Fraenkol and Sobehiheim (1894, p. 154), on heated anti-cholera .serum which had
lost its bactericidal properties. Wassermann (3, i. 1901) was able to neutralize the
complement by meiins of an ti -complement, the latter being specific and only capable
of binding one sort of complement. As in haemolytic sera, agglutinins may be
present together with lysins. Pfeiffer observed the agglutinin to disapi>ear in
stoi-ed immune-sera (of goat and dog treated with B. typhosm) and the immuno-
body to iwrsist therein. Mertens (13, vi. 1901), who cites this unpublished
observation of Pfeiffer, wiis altle to confirm it on cholera immune-serum 5 years old.
The existence of immune-bodies in anti-microbic sera has been also proved by
Bonlet and Gengou (v. 1901) in exiicrimonts upon the blood of animals treated with

28 The Cytotoxins of Blood Serum

the germs of plague, typhoid, anthrax, rouget du pore, and with Proteus vulgaris.
Immune-bodies were found in the serum of typhoid convalescents.

The occurrence of substances corresponding to immune-bodies in nonnal sera
has been denied, although Malvoz (25, viu. 1902) has found normal adult dog
serum to act like immune-serum towards B. anthracig, that is, when inactivated
by heat its "immune-body" combined with the bacilli, and rendered them susceptible
to the bactericidal action of the complement in the senim of other animals, such a-s
the rabbit, guinea-pig and rat. It is worthy of note in this connection, that the
adult dog is most refractory to anthrax infection. The immuue-body is absent in
young dogs, as also in the guinea-pig, ox, aud rat, all of which are susceptible,
whereas it may or may not be present in the relatively resistant rabbit.

The rapid decomposition of bodies of animals which have died from the effects of
snake venom is due to the loss of bactericidal power of the blood, as has been
shown by Welch and Ewing, and by Flexner and Noguchi (1902), the venom
combining with the serum complement of bacteriolytic serum. Antiveuin (Calmette)
neutralized both the bacteriolytic and haemolytic action of venom in vitro.

Eisenberg (v. 1902) cites Kraus and Clairmont as finding that heated bacteria are
not disisolved, although, as Bail and Wilde found, they are capable of absorbing the
bacteriolysin.

Bacterial Haemolysins.

The bacterial haemolysins have received a considerable amount of attention.
Ehrlich showed that tetanus toxiu is haemolytic, Madsen finding that it contained
a toxin which produced convulsions, a tetanospasmin, as distinguished from the
tetanolysin. Kraus and Clairmont (17, -x. 1901) found tetanus toxin, as also the
products ai Staphylococcus pyogenes, Streptococcus, Vibrios, and putrefactive bacteria,
to be haemolytic. Neisser and Wochsberg, as also Bulloch and Hunter, have
studied the haemolytic action of Bacillus pyocyaneus and of the Staphylococcus ;
Levy, Castellani, and Lubenau of B. typhosus, B. dysenteriae, and Mierococciu
tetracfeniis. Madsen fo\uid tetanolysin very unstable ; Kraus and Clairmont found
the haemolysin to V)e destroyed after 15 minutes' exposure at 60' C. Neisser and
Wechsberg found staphylolysin to be injured at 48°, and destroyed at 58" C. in
20 minutes. That the bacteriohaemolysins jkisscss a constitution similar to toxins
was indicated by Ehrlich and Madsen, who found that they could neutralize them by
means of anti haemolysin. They moreover found that normal horse serum 2)osses.sed
anti-haemolytic action. They were unable to establish any relation lietween the
antitoxic value of a serum and its haemolytic power. The conclusion reached as
the result of comparative experiments is that the bacterio-haernolysins and anti-
bacterio-haemolysins are specific in character.

The Complement.

As I have stated elsewhere, cytolytic sera are inactivated by being heated
to 55° C, this being due to the destruction of the complement. A normal serum
thus trciited cannot be reactivated, whereas an ivniruuie-serum can. The ferment-

Serum Complement 29

like body, or complement, is not specific, for the reason that complement can be
supplied from another animal which has not l)een treated. (The toxophorous group
of the toxin molecule is also regarded as non-specific by Ehrlich.) Some authors
consider that there is but one complement in cytolytic sera, this being the alexin
of some writers, whereas Ehrlich and his school claim that any serum contains a
multiplicity of complements. A number of points in connection with complements
have been considered in the preceding iiages, and it is not my object to enter
at all fully into a consideration of them here. It will suffice to mention a few facts
indicative of their importance.

The amount of complement in serum appears to vary considerably. Thus
Weichardt (25, xi. 1901, p. 834), using normal rabbit sera to reactivate spermotoxic
guinea-pig serum, found that only 5 out of 11 rabbits' sera contained complement
suitable for reiictivation. Serum obtained from a man contained complement at
one time, none when tested after a period of 8 weeks. Sweet (xii. 1902) has
collected the data contained in the literature relating to the variation in the
complement-content of cytolytic sera. A decrease in complement was observed by
Abbott and Bergey (1902) in the serum of rabbits treated with ox corpuscles, the
animals also receiving alcohol. Ehrlich and Morgenroth (1900) found a decrease
in complement in rabbits which had received a do.se of phosphorus sufficient to
kill them after the lapse of 3 days ; the haemolytic power of their serum had
disappeared on the second day in a manner corresponding to what is seen in sera
inactivated by heat. Metalnikofi" (1900) observed a decrease of complement in a
rabbit treated with guinea-pig spermatozoa, an enormous abscess having formed,
which subsequently burst and healed. Wlien fresh guinea-pig .serum was added to
this rabbit's serum it was spermotoxic, not otherwise. On the other hand, Nolf
(1900) claimed to observe an increase in complement in animals treated with fowl
serum ; Mtiller (1901) made observations which show that the complement-content
may be increased by injecting peptone solution, bouillon, aleuronat .solution, in
other words indifferent fluids. Sweet, from whom I have made these citations,
studied the question in rabbits imnmnified with ox coiijuscles. He found that he
could iucreiise the complement-content by the injection of substances having a
positive chemotactic action on leucocytes, using Staphylococcus pyogenes aureus,
sterile oil of turpentine, sterile aleuronat suspension. He found the haemolytic
complement in a fre^ state in the blood pla.sma, and in the serous part of an
exudate, not being contained in leucocytes, nor being set free by the process of
coagulation.

The relation of complement in a serum coutivining blood corpuscles subject
to the action of snake venom has been very clearly brought out by Flexner and
Noguchi (1902). These authors experimented with the haemolysins contained in
the venoms of Crota/us acUimanteus (rattlesnake), Ancislrodon piscivorus (water-
moccasin), iVa;a tripudiaiis (cobra), and Ancistrodon contortrix (copperhead). They
found that when they had thoroughly washed the blood corpuscles of the dog,
sheep, ox, pig, rabbit, and guinea-pig, that the corpuscles were unaflected by
contact with these venoms. On the other hand if the corpuscles wei-e bathed
in their respective complement-containing sera, haemolysis took place promptly
upon the addition of the venoms. An excess of serum increased the haemolytic
power. They found :

30 The Cytotoxins of Blood Serum

Venom intermediary body, added to blood corpuscles, had no eflfect

„ „ „ „ „ „ „ „ and serum complement gave

haemolysis.
Serimi complement alone „ „ „ „ had no effect.

This result is comparable to what has been observed with regard to the non-
haemolytic action of blood serum from normal animals upon the washed corpuscles
of certain other species, and directly supports a contention of Friedenthal's (men-
tioned later), that tests made with normal sera upon the washed corjniscles of other
animals do not afford a means of studying blood relationships with haemolysins.

Tlie importance of a further study of the complement is well shown by the
investigations of Moro (31, x. 1901) upon serum, and its bactericidal properties
under different conditions. Using the methods devised by me (1888), but express-
ing the bactericidal effect in terms of per cent, of bacteria destroyed by contact
with the serum, he foimd that fresh

Placental blood serum killed 58-9 "/o

Older children's „ „ 463%

Bottle-fed infant's „ „ 33-4%

Breast-fed „ „ „ 77-0 %

An experiment conducted upon a single infant gave :

Placental blood of mother killed 56-0%

Breast-fed infant 2 weeks old, its serum killed 72-9 "/o

Serum of the .same child after bottle-feeding for 2 weeks killed 40-7 %•

The blood serum of a breast-fed child had more haemolytic action on rabbit
corpuscles than did that of a little-fed infant's ; even the sickliest breast-fed infant
had more bactericidal substance in its serum than the healthiest bottle-fed infant.
The serum of a new-born infant only killed 59 % of the bacteria.

That a fundamentiil interest attaches to the ferment-like complements in
relation to normal physiological processes is moreover very clear from a sug-
gestive paper by Wassermann (1, I. 1903), whose investigations were stimulated
by those of Moro just quoted. It is a well-established fact that breast-fed infants
thrive as a rule better than those fed vipon cows' milk Heubner, using Rubner's
method, has studied the process of assimilation in such children comparatively, and
found that, reckoned in calorie.s, the child receiving mother's milk showed the mo.st
growth ; in other words, that homologous mother's milk achieves as much with a
few calories as does cow's milk with a very large number of calories. The infant fed
on cow's milk has to exix^nd energy in the form of glandular and digestive activity
to assimilate the heterologous milk. That there is such an increased activity is
evident from the following.

If heterologous food-stufia, such as goat serum, etc., are injected, say into a
guinea-pig's peritoneum, the peritoneal exudation is capable, soon after, of destroying
and dissolving large numbers of bacteria {B. typhosus), whereas the contrary is
the case in a normal animal. The peritoneal exudation in the serum-treated
guinefi-pig is rich in digestive complements which have appeared for the pui-pose
of acting upon the serum injected, and it is these digestive substances (complements)
which destroy the bacteria.

Specific Haemolysins 31

If wo introduce an homologous serum, instead of the foregoing, that is guinea-
Itig serum into the peritoneum of another guine<a-pig, the bacteria subsequently
introduced into the peritoneum are not destroyed, no complements or ferments
lieing required for the assimilation of the homologous senmi.

We have noted above that Moro found the serum of bottle-fed infants markedly
loss bactericidal than that of breast-fed infants. He was unable to find complements
in milk. The conclusion api)ears therefore justified, that the reason for there being
less complement in the .serum of the bottle-fed infant is to be traced to the comple-
ment being used up in the process of assimilating the cow's milk.

The conclusion reached above api)ears all the more justified when we view the
results obtained in the study of other antibodies in corpore, such as the fall in the
amount of antitoxin or of precipitin in a semm when a substance capable of being
acted upon by the antibodies is introduced into the economy.

The treatment of animals for the production of Specific Haemolysiiis.

As it is not my object to enter at all fully into this subject, it will
suffice to cite the methods pursued by but a few observers. Bordet (x.
1898, p. 692) injected 10 c.c. of defibrinated rabbit blood intraperitoneally
into guinea-pigs and after 5-6 injections noted the presence of haemolysins.
Deutsch (15, v. 1901, p. 662) defibrinates the blood (.say human) he wishes
to inject, allows it to settle 24 hours in the ice chest, then removes the
serum and injects 10 c.c. of corpuscles subcutaneously into rabbits,
a second and third injection being made at intervals of 7 days.
He bleeds the rabbits for anti-serum 21 days after the first injection.
He says that haemolysins, agglutinins and even precipitins (?) appear
one week after the first injection, as with the antibodies for B. typhosus
(Deutsch, 1899).

That injections of urine may lead to the formation of haemolysins
was discovered by Schattenfroh (30, i. 1901). He injected rabbits sub-
cutaneously with human, goat, and horse urine, the animals receiving
120 — 150 c.c. intervals of 2-3 days elapsing between injections. The
serum of the rabbits treated with human and goat urine acquired
powerfully haemolytic and agglutinating properties for the red blood
corpuscles of the animals which had yielded the urine. This special
action was very marked in the serum of the human-urine-treated
rabbits ; normal rabbit sera, or those of rabbits treated with other urines,
possessing little or no globulicidal effect on human corpuscles. The
serum of a rabbit treated with horse urine gave no marked result.
The serum of the rabbits treated with goat urine did not contain
precipitin, nor anti-complement for goat serum, this being in marked

32 The Cytotoocins of Blood Serum

contrast to the serum of rabbits treated either with active or inactive
goat serum. Schattenfroh suggests that the method of urine injection
may be of use in the production of specific haemolysins for blood-testing
by the method recommended by Deutsch.

Ruffer and Crendiropoulo (24, i. 1903) have confirmed this observa-
tion, finding that the injection of human urine two or three times into
rabbits produced a haemolytic serum for human corpuscles, the
haemolysin acting however slightly also upon the corpuscles of the
guinea-pig.

As has been found for the precipitins, Metalnikoff (18, iv. 1901,
pp. 532, 533) has observed the formation of haemolysins in the serum
of rats fed for 1 to 8 weeks on horse blood. An exposure to 55° C.
inactivated their serum, it being reactivatable through fresh normal
serum ; it consequently contained immune-body and complement.
Similarly rats fed on rabbit blood, and rabbits fed on horse blood,
yielded specific haemolysins.

Again, as has been found for the precipitins, Calmette and Breton
(1, XII. 1902) observed a decrease in the amount of haemolysin in
animals treated for longer periods. They found, however, that when
such over-treated animals are allowed to rest for some months they
produce a much more active antiserum after but two injections.

SECTION IV.

THE ACTION OF DIFFERENT SERA UPON THE BLOOD CORPUSCLES
OF CERTAIN ANIMALS IN VITRO AND IN CORPORE.

I. The Effects of Normal Haemolysins.
Primates.

Human Serum : There is no evidence that the sera of any human
races are haemolytic for the blood corpuscles of other races of man,
in proof of which we have a large experience with regard to trans-
fusion. Transfusion of the blood from a member of one race into that
of another has not been followed by any ill effects.

Fricdenthal (1900, p. .505) transfused 25 c.c. of defibrinated human
blood into a chimpanzee, the animal remaining healthy, its urine
showing no trace of haemoglobin. He also transfused 10 to 20 c.c. of
defibrinated human blood into Macacus sinicus and M. cynomolgus
without ill effects, a slight transitory haemoglobinuria being observed
in consequence, which was attributed to the haemoglobin in the
defibrinated blood injected, the blood corpuscles contained therein
having undergone haemolysis.

The effects of human serum in vitro have been studied by
Friedenthal, upon the blood corjjuscles of other Primates, these cor-
puscles not being washed (see p. 34). He found the corpuscles of the
Oiuaiig-iiutaug and (Jibbon to remain unaffected, whereas the corpuscles
of Macacus sinicus, M. cynomolgus. Rhesus nemestrinus (Old World
Monkeys), Pithesciuriis sdureiis, and Ateles ater were haemolysed.
Human serum also haemolysed the corpuscles of Lemur varius. He
does not state that he observed any differences with regard to the rate
at which haemolysis took place, a matter worth noting when we
consider the results I have obtained with precipitins.

In his Huxley Lecture, Welch (11, x. 1902, p. 1108) refers to an
unpublished investigation by H. T. Marshall (working under Ehrlich
N. 3

34 Action of formal Haemolydns

and Morgenroth) upon the receptors of the red blood corpuscles
of man and of two species of monkey. It appears that Marshall
has found human and monkey corpuscles to have a large number
of receptors in common, but also ones peculiar to each. Welch
points out that this is in harmony with my published results upon
the precipitins in relation to the phylogenetic relationships between
animals.

The action of human serum upon the blood of other animals
than Primates has been considerably studied ; it will suffice however
if I cite but a few data. CI. Bernard ^ stated that 10 c.c. of human
blood was toxic for the rabbit. Friedenthal, experimenting with
unwashed corpuscles of the eel, frog, snake, pigeon, fowl, night heron,
horse, pig, ox, rabbit, guinea-pig, dog, cat, hedgehog, in vitro, has found
all of these to be haemolysed by human serum.

Macacus Servm was found to haemolyse some human blood cor-
puscles, not others, there being evidently differences in the resisting
power of the corpuscles of different individuals (Friedenthal).

Few as these observations are, they are certainly in remarkable
accord with what I have observed upon the precipitin reactions.

Insectivora.

Erinaceus Serum haemolyses the blood corpuscles of the cat and
rabbit (Friedenthal).

Camivora.

Canine Sera : The older experiments demonstrated that transfusion
could be practised with impunity between different Canidae : the dog,
wolf, and fox. On the other hand CI. Bernard (loc. cit.) found rabbits
to be killed by the injection of 10 c.c. of dog serum. Friedenthal and
Lewandowsky (1899, p. 532) killed rabbits of 1500 grammes weight
usually in a few minutes by injecting 7 — 14 c.c. of dog's serum ; the
lethal dose required being larger when the rabbits received subcutaneous
injections instead of intraperitoneal injections.

Experiments with dog serum in vitro, have been mostly conducted
upon washed corpuscles, that is corpuscles from which the serum
had been removed, if we except those of older date. Ehrlich and
Morgenroth (1899, ii. p. 13 repr.) found dog serum at times to powerfully

' "LeQons sur les propria t^s physiologiques et les alterations pathologiques des liquides
de I'organisme," ii. p. 459 ; cited by Friedenthal and Lewandowsky (1899, p. 532).

An Index of Bloocl-relatlonship 3o

haemolyse cat corpuscles, at other times it exerted no effect, whilst
London (1902, p. 56) states they are not haemolysed. Daremberg
(1891) found dog serum to haemolyse human corpuscles in 9 minutes.
The coi-puscles of the rahhit and guinea-pig are haemolysed in
2J minutes (Daremberg); Buchner (1893, p. 122), Ehrlich and
Morgenroth (ill. 1900), Flexner and Noguchi (1902) state that rabbit
coi-puscles are rapidly haemolysed. Buchner stated that the corpuscles
of the guinea-pig were more slowly haemolysed than those of the
rabbit, but this does not apjjear to have been noted by Ehrlich and
Morgenroth, and Flexner and Noguchi. Rat corpuscles are haemolysed
(Ehrlich and Morgenroth).

The blood corpuscles of the Ungulata are haemolysed by dog serum,
the bloods tested being those of the horse, pig, and sheep (haemolysed
in 1 minute, Daremberg), ox (haemolysed in 9 minutes, Daremberg),
sheep and goat (Ehrlich and Morgenroth).

Feline Sera: Friedenthal (1900) cites an experiment in which
he transfused the blood of the domestic Angora cat into the vessels
of Felis ocelot, and from the latter into the cat. He chose animals
of similar size, put their blood circulations into communication,
and allowed their bloods to mix until about one-half of their blood
volume had been exchanged. The cat remained perfectly well after the
operation, the ocelot unfortunately died from the effects of prolonged
narcosis. He pertinently remarks to these experiments " Also getrennte
Familien, gesondertes Blut." Friedenthal and Lewandowsky found
the lethal dose of cat serum for rabbits to be the same as that of the
dog (see above).

Experiments with cat serum in vitro have shown that it does not
haemolyse the blood corpuscles of Felis ocelot and Felis jaguarundi, and
vice versa (Friedenthal, 1900), nor of other cats (Friedenthal and
Lewandowsky, 1899). Cat sennn was found to haemolyse the corpuscles
of the rabbit (F. and L. 1899, London, 1902, p. 56), hedgehog, and frog
(Friedenthal, 1900).

Rodent ia.

Rabbit Serum: Transfusion effected between two rabbits or be-
tween the rabbit and hare is not followed by ill effects (Friedenthal,
1900, and Friedenthal and Lewandowsky, 1899).

The experiments with rabbit serum in vitro are not concordant,
apparently for the reason that some observers experimented with
washed, others with unwashed coi-puscles. Working with unwashed

3—2

36 Action, of Normal Haemolysins

corpuscles, Friedenthal found rabbit serum to rapidly haemolyse the
corpuscles of the horse, pig, ox, monkey and man, whereas it exerted no
effect on hare's corpuscles. Other observers, experimenting with washed
corpuscles, state that rabbit serum does not haemolyse the coqjuscles of
the cat (London, 1902, p. 56), that its action is very variable upon the
corpuscles of the guinea-pig (Ehrlich and Morgenroth, 1899, ii, p. 13
repr.), and only slight upon dog's corpuscles (Flexner and Noguchi, 1902).

Ouinea-pig Serum: Experiments in vitro, made apparently under
comparable conditions with washed corpuscles, have shown guinea-pig
serum to exert practically no haemolytic action on rabbit corpuscles
(Bordet, x. 1898, p. 692 ; Flexner and Noguchi, 1902); to rapidly haemo-
lyse those of the rat and mouse (Bordet, 1898); to haemolyse those of the
hedgehog (Friedenthal, 1900); and to exert a feeble action on the
amoeboid cells of the crayfish (Szczawinski, 28, XI. 1902), the artificial
haemolysin acted more powerfully.

Rat Serum : Injections of rat blood into mice and vice versd are not
injurious (Friedenthal, 1900). Metalnikoff (18, iv. 1901, p. 532) ex-
perimenting with the serum of the white rat found that it did not
haemolyse nor agglutinate the corpuscles of the horse. I have not
found further data with regard to the action of these sera.

Ungulata.

Pig Serum: The haemolytic action of pig serum upon human
corpuscles was already observed by Landois in transfusion experiments
upon man. Friedenthal (1900) also found it to haemolyse the corpuscles
of the ox, horse, dog, cat and rabbit, besides those of man.

Uhlenhuth (1897) found 12 c.c. of pig serum to be fatal to 1 kilo of
rabbit when injected intravenously; he cite.s Weiss (1896) as having
found 35 c.c. to be the fatal dose for rabbits (subcutaneous injection?).
Pig serum produced infiltration and necrosis when subcutaneouslj'
injected into guinea-pigs.

Ox Serum was found to be injurious to man in the transfusion
experiments of Landois, because it haemolysed human corpuscles.
Friedenthal found it to haemolyse the corpuscles of the pig, horse,
rabbit, dog, cat, and also man. The lethal dose by intravenous injection
in i-abbits was found to be 6 c.c. per kilo of rabbit by Uhlenhuth.
According to Rumno and Bordoni, and Weiss (1896), the lethal dose of
ox serum is 8 c.c, whilst Guinard and Dunarest give 9 c.c. as fatal for
rabbits, these authors being cited by Uhlenhuth. Friedenthal and Lewan-

An Index of Blood-relationship 37

dowsky found the lethal dose of calf serum for rabbits to be the same
as that of dog serum (see above, p. 34). Subcutaneously injected into
guinea-pigs it produced the same effects as pig serum.

Gruber (3, xii. 1901) dwells on the importance of concentration in
hacmolytic experiments, stating that ox serum, even when highly
diluted, acts on rabbit and guinea-pig corpuscles, whilst it irtdy acts on
sheep corpuscles when concentrated. This observation, we see, shows the
converse of what has been observed with the precipitins, which act on
higher dilutions of related bloods.

Sheep Semm was also found injurious to man in Landois' transfusion
experiments. According to Uhlenhuth (1897) the lethal dose per kilo
of rabbit is 11 c.c, Rumno and Bordoni give it at 12 c.c, Weiss at
20 c.c. It produces the same effects as pig serum when subcutaneously
injected into guinea-pigs, according to Uhlenhuth, who cites the authors
named.

Ooat Serum was found to exert a very slight haemolytic action or
no action upon the corpuscles of the sheep by Ehrlich and Morgenroth
(1899).

Equine Serum : The transfusion of horse serum into man was found
to be injurious to man in the experiments of Landois. On the other
hand he found transfusions between the horse and donkey not to be
injurious. Friedenthal (1900, p. 503) found the sera of the horse and
donkey without action on the corpuscles of these species, whereas they
haemolysed those of man, ox, sheep, guinea-pig, and rabbit. According
to Morgenroth and Sachs (7, vii. 1902, p. 633) the haemolytic action
of horse serum varies greatly, compared to the constant haemolytic
power of other sera, such as those of the dog and goat. The haemolysin
of the horse appears to be very labile, for they often observed it to
be active for the corpuscles of the rabbit and guinea-pig when fresh,
but inactive, or but slightly active, after being kept 24 hours on ice.
Uhlenhuth cites Weiss as giving the lethal dose of horse serum at 44 c.c,
and Guinard and Dunarest at 324 c.c, for rabbits, this great variation
being in accord with the statement of Morgenroth and Sachs just cited.
Friedenthal and Lewandowsky (1899) found it necessary to inject 70 c.c.
of horse serum intravenously into a rabbit of 1300 g. to kill it. Uhlen-
huth (1897, p. 391) found horse serum not to exert effects such as
do the sera of man, sheep, pig, rabbit, and ox, when injected subcu-
taneously into guinea-pigs. (See under pig serum, p. 36.)

88 Action of Normal Haemolysins

Aves.

Experiments conducted in vitro by Friedenthal (1900) showed the
serum of the fowl to haemolyso the corpuscles of several vertebrates in-
cluding those of some birds, namely those of Falco tinnunadus ( Accipitres)
and Nyctocorax (Ciconiiformes). The serum of the night heron haemo-
lysed the corpuscles of the fowl. The serum of the gull {Larus argen-
tatus) haemolysed the corpuscles of the frog.

Reptilia.

The toxicity of snake sera for Mammalia is well-marked. Frieden-
thal found the intravenous injection of 2 c.c. of the serum of the
"Kreuzotter" (presumably Pelias beriis Merr.) to be fatal to rabbits
of average size. Dr Graham-Smith in trying to produce precipitins in
rabbits by injecting the serum of Tropidotiotus natriw, found a similar
dose to be lethal for guinea-pigs by intraperitoneal injection. Frieden-
thal found "5 c.c. of his snake serum to kill frogs when injected into
the posterior lymph-sac. He also found the serum of Tropidonotus
(" Ringelnatter") to be highly haemolytic.

The agreement between Aves and Reptilia in possessing a highly
toxic serum, may be added to the other points of similarity, as to
anatomical structure (skeleton^ urinary apparatus, scales, eggs, etc.) and
the microscopic appearances of their blood, which have led systematists
to group both classes under Sauropsida. It will be seen, however, that
more striking proof has been obtained by means of the precipitins with
regard to the relationship between these classes.

Amphibia.

Frog Serum possesses haemolytic properties for the corpuscles of
the dog, guinea-pig and rabbit, although it acts less rapidly than the
serum of Necturus, whose corpuscles it slightly haemolyses (Flexner and
Noguchi, 1902). Friedenthal states that the haemolytic power decreases
in starved frogs.

Necturus Serum : The serum of this amphibian quickly haemolyses
the corpuscles of the dog, guinea-pig, and rabbit, but slightly affecting
those of the frog (Flexner and Noguchi).

An Index of Blood-relationship 39

Pisces.

Eel Serum : The highly toxic properties of the serum of Anguilla
were discovered by Mosso (1888, p. 144), who found that -5 c.c. thereof
injected intravenously into a dog weighing 15,206 g., produced death
in 7 minutes, even a dose of '02 c.c. per kilo being fatal. He referred to
the toxic body as "ichthiotoxin." The serum of Muraena proved also
highly toxic, '66 c.c. killing a dog of 6,160 g. in 5 minutes; "Sec. killing
a rabbit of 1,030 g. in 2^ minutes. A toxic serum is also possessed by
Conger myrm and C vulgaris. Pigeons are susceptible to large doses
according to Camus and Gley (1899, p. 785). These authors (24,
VII. and X. 1899, p. 786) found the blood corpuscles of new-born rabbits
to be highly resistant to the haemolytic action of eel serum. They con-
sidered this due to a resisting power inherent in the cell, not to the
existence of normal anti-haemolysin. (See p. 22.)

The study of the haemolytic effects of eel serum in vitro, begun by
Mosso, were extended by others. Tchistovitch (25, v. 1899, p. 407) and
Camus and Gley (x. 1899, p. 784) found the corpuscles of the fowl very
resistant. The latter found the corpuscles of the pigeon very resistant,
whilst Tchistovitch states they are susceptible, though almost insoluble.
Friedenthal, working with unwashed hedgehog corpuscles, found them
haemolysed by eel serum ; whereas Canms and Gley state that they resist
haemolysis. Camus and Gley state that the corpuscles of the guinea-pig
and rabbit are haemolysed by eel serum, whereas those of the Bat, Bufo
vulgaris, Rana temporaria and R. esculenta, and Testudo graeca are very
resistant. They attribute this resistance to the corpuscles, not to anti-
haemolysin. Friedenthal, working with unwashed frog corpuscles, states
that they were haemolysed by the eel serum. These somewhat contra-
dictory results may be explained by the fact observed by Ehrlich and
Morgenroth (1899, ii. p. 13 repr.) that the haemolytic power of eel serum
may vary greatly, at times haemolysing powerfully, at other times not
at all.

In Friedenthal's experiments, the blood of Acanthias vulgaris was
rapidly haemolysed by eel serum, and that of Labrus maculatus haemo-
lysed teleostian blood. The serum of A. vulgaris haemolysed the
corpuscles of a bird {Larus argentatus), a rat (Mtis decumanus), as well
as that of other Teleostea (Labrus inaculutus and Anguilla vulgaris),
and the serum of the shark was " not indifferent" to the corpuscles of
other Elasmobranchs, viz. Raja batis.

40 Artificial Haemolysins

Lower Animals.

Friedenthal was unable to demonstrate the existence of any haeuio-
lytic action on the part of the blood of Crustacea (Cancer pagurus),
Oligochaeta {Arenicola piscatorum), or Sea-urchin, when these were
brought in contact with the blood corpuscles of the gull (Larm urgen-
tatus) and rat.

//. The Effects of Artificial Haemolysins upon ilie Red Blood
Corpuscles of different Animals in cor pore and in vitro.

The demonstration of artificial haeuiol^'sins dates from the discovery
of Belfanti and Carbone ^viii. 1898) who found that they were formed
in the serum of animals treated with a foreign blood. They injected
large amounts of rabbit blood intraperitoneally into the horse and found
the serum of the horse to become highly to.xic to rabbits. Similarly, the
serum of a dog, treated with i-abbit blood, became toxic foi- rabbits.
Such sera were not toxic for the animals yielding the antisera, very
slightly so for other animals than those which yielded the blood injected.
Bordet subsequently showed that the toxic action of the serum of
treated animals corresponded with its haemolytic power in vitro upon
the particular species of corpuscles with which the animal had been
treated.

Ehrlich and Morgenroth (1899) treated a goat with sheep's corpus-
cles and found its serum to become haemolytic for the corpuscles of the
sheep, although the animals are so closely allied. They subsequently
(vi. 1901, p. 8, repr.) found that a rabbit treated with ox corpuscles
developed a serum which was haemolytic for the corpuscles of the goat
and sheep, as well iis those of the ox, although the haemolysin acted less
on the goat corpuscles than on those of the ox. This demonstrated
a similarity but not an identity between these corpuscles of Bovidae.
Similarly, a rabbit treated with goat corpuscles gave haemolysins for
goat and ox corpuscles, acting less on the latter. They explain that the
specificity of the immune-body formed in the blood-treated animal is
not to be confused with the conception of species held by systematists
in botany or zoology. All those elements will be affected by an immune-
serum which have receptors corresponding in type to that of the original
element injected, and consequently the influence will be most marked
where there are most receptors belonging to the C(jmmon tyjie. This
view also applies to the corresponding results obtained with precipitins.

Use in Legal Medicine 41

Deutsch (9, vin. 1900, and later) has suggested the use of artificial
haemolysins in legal medicine, in the identification of bloods, both
fresh and dried. He found that a powerful haemolytic serum dissolved
powdered blood completely, the latter being suspended in 9Yoo salt
solution. Dried blood to which saline is added brings the haemoglobin
of the injured coi-jniscles into solution, the uninjured corpuscles do not
however dissolve even after 24» hours at 37° C. If the dried blood
is extracted in normal rabbit serum more haemoglobin goes into
solution than with saline, when the proportion added is 1 : 2, whereas the
normal serum acts like saline when added in the proportion of 1:4.
When two samples of the same dry blood are brought into suspension
in normal and artificially haemolytic serum respectively, a little phenol
or toluol being added, the antiserum brings about complete haemolysis
after 24 hours, besides leading to the formation of a precipitum, due to
the action of precipitins formed in the blood-treated animal in conse-
quence of the serum which was injected together with the corpuscles.
When washed corpuscles alone are injected precipitins are not formed.
In view of the specificity of the reactions observed with human blood
corpuscles, he considers that the method can be put to use in a practical
way. There can, however, be no question but that the precipitins offer
many advantages over the haemolysins for such purposes.

In the experiments of Bordet (x. 1898, p. 692), it was found that
the serum of a guinea-pig treated with rabbit corpuscles exerted
no effect on the corpuscles of the normal guinea-pig, nor upon those
of the pigeon. Both normal and treated guinea-pig sera act strongly
on the corpuscles of the rat and mouse, the normal serum is however
less haemolytic. If 2 c.c. of defibrinated rabbit blood is injected
intraperitoneally into the blood-immunified guinea-pig, the rabbit
corpuscles are destroyed in 10 minutes. This does not happen
in the peritoneum of a normal guinea-pig unless inactivated specific
serum (heated to 55" C.) is injected together with the corpuscles. The
serum of the treated guinea-pig, injected intravenously into rabbits
kills them when 2 c.c. are given. This is in substantial agreement with
what has been observed with regard to the specific bacteriolysins, for
instance with regard to the cholera germ. Bordet considers the
haemolysin (alexine) in the treated guinea-pig serum to be probably
identical with the specific bactericidal substance of PfeiflFer.

The study of the relationships amongst animals by means of artificial
haemolysins has scarcely been begun, as we see, if we except the work
of Ehrlich and Morgenroth above cited, which gives corresponding

42 Isolysin and Autolysin

results to those which have been observed with the precipitins. It
remains to be seen whether the haemolysins offer greater advantages
in this respect than do the precipitins.

Isolysin and Autolysin.

Ehrlich and Morgenroth (Ueber Hdmolysine, lil. 1900) term the
haemolysins formed in the bodies of animals treated with the corpuscles
of another species " heterolysins," so as to distinguish them from the
" isolysins," which are formed for the coqauscles of the same species.
They injected 920 c.c. of goats' blood, obtained from three animals, into
a goat. No haemolysis occurred, but after five days or more the treate<i
goat's semm was foimd to be markedly haemolytic for the blood
corpuscles of nine different goats, the tests being conducted in vitro.

An " autolysin " is a haemolysin which acts upon the blood corpuscles
of the animal which yields the serum. Ehrlich (1901, Schlussbetracht-
ungen, p. 16, repr.) only succeeded once with Morgenroth in obtaining
autolysin, this being formed under the same conditions as the
isolysin. He injected the blood of a goat (a) into another goat (6),
and found the serum of goat {b) to haemolyse the corpuscles of (a).
The existence of isolysins and autolysins point therefore to individual
differences in animals belonging to the same species.

Besredka (2.5, X. 1901, p. 788) considers the immune-body in autocy-
totoxins to be probably identical with the cytotoxins of another animal
when we exclude the complement which is peculiar to each species.

The existence of isolysins and isoagghitinins in disease does not bear upon tlie
immediate subject of this paper. It is however well to mention that Ciriinbauni
{Brit. Med. Juurn. i. p. 1089, 1900) found the serum of typhoid and scarlatina
patients to agglutinate the corpuscles of normal pei-sons, or those affected not with
the same but with other diseases. Shattock {Joiirn. Pathol, and Biccteriol. vi. p. 303,
1900) confirmed this in cases of croupous pneumonia, typhoid, erysipelas, acute
articular rheumatism. Panichi has observed isoagglutinins in the .scrum of persons
experimentally infected with malaria, after .six days, when no parasites could be
discovered in their blood. ELsenberg (17, x. 1901) as also Ascoli have found
isolysins and isoagglutinins in the blood after crisis from croupous pneumonia,
their appearance corresiwnding with the absorption of the hemorrhagic infiltration.
It would therefore seem as if the appearance of these antibodies in the blood
depended upon the resorption of blood corpuscles or their constituents. In seven
out of eight cases of scarlatina with slight clinical signs of blood destruction,
Eisenberg observed the.se antibodies. He denies that the isolysins and isoagglutinins
are specific for the diseases named, but that they simply indicate that corpuscles
are being de.stroyed, or reabsorbed, and consequently that their presence is not of
diagnostic iinpoi'tance.

Cytolysin and Agglutinin 43

The relation of Gytolysins to Agglutinins.

The evidence in favour of the haemolysins and agglutinins being
distinct seems fairly conclusive. It is trae that Emmerich, Loew, and
Baumgarten consider that agglutination always precedes haemolysis.
Baumgarten's view receives some support from the observation of
Shibayama, who states that very powerful antisera may haemolyse
without agglutinating, Baumgarten claiming that rapid haemolysis may
mask the phenomenon of agglutination.

Most observers agree with Ehrlich and Morgenroth in considering
the antibodies distinct. These authors, as also Bordet and others, have
found the haemolytic action to be prevented by heating haemolytic
serum to 55" C, the agglutinins remaining unaffected. Mertens, as we
have seen (p. 27), has shown the non-identity of agglutinin and immune-
body. In the following pages evidence i.s given as to the non-identity
of agglutinins and precipitins. Jacoby determined that ricin-toxin and
agglutinin are different. We see that there is every evidence as to
a multitude and diversity of antibodies in sera. Further, whereas
lysins pass through a coUodium film by osmosis, Gengou claims that
agglutinins do not. The bacteriolysins in a serum may be increased
by sodium carbonate injection, but this does not affect the amount
of agglutinin present. It has been repeatedly noted that no definite
relation exists between the agglutinative and bacteriolytic power of
typhoid sera.

The studies of Mitchell and Stewart (1897) have shown that the
phenomenon of agglutination precedes haemolysis by snake venoms,
this being comparable to what we see in PfeifFer's reaction, where the
agglutination of cholera spirilla precedes lysis by the immune serum.
In snake venom, Flexner and Noguchi (1902) have also found that they
are able to separate the lysin from the agglutinin. Agglutination by
venom did not affect haemolysis, but rapid haemolysis limited or
prevented agglutination. On adding ricin, which agglutinates blood
corpuscles powerfully, and allowing it to act for less than 30 minutes
on the corpuscles, it was found that a further addition of venom effected
haemolysis within the usual time. On the other hand, when ricin had
acted for two or more hours, solution took place on the addition of
venom, but the corpuscular stroma remained at the bottom of the tube
in the form of a white conglutinated mass. It appears therefore that
the stroma undergoes a form of coagulation under the influence of
agglutinins, the venom releasing the haemoglobin. No interaction

44 Ci/toh/sin and Agglutinin

took place between venom and ricin. The haemolysin in venom resisted
an exposure of 30 minutes at 75 — 80° C, being slightly reduced in
power after 15 minutes at 100° C. Venom agglutinins exposed for
30 minutes at 75—80° C. were destroyed.

Dubois (25, ix. 1902) considers the agglutinins and immune-bodies
of artificially haemolytic sera to be distinct, although they behave
similarly in their resistance to heat, putrefaction, light, etc. He states
that he was able to separate them by repeatedly injecting rabbits,
(a) with intact washed fowl's corpuscles, and {h) with the same corpuscles
previously heated to 116° C. for 15 minutes. The fresh seriim of the
rabbit receiving treatment (a), agglutinated and haemolysed fowl's
corpuscles, whereas the second {h) did not haemolyse, although it
contained some agglutinin. Dubois cites Defalle as having found that
certain germs {B. typhosus, B. mycoides, B. mesenteric us, etc.) when heated
to 115° C, and injected into animals, produced an agglutinative serum
not containing immune-body, whereas the latter is readily obtained by
treating animals with unheated germs.

SECTION V.

AGGLUTININS AND ANTIAGGLUTININS.

It is not my wish to dwell at any length upon the agglutinins and
antiagglutinins. They are frequent in haemolytic sera, the serum of
one animal generally agglutinating the blood corpuscles of another ; thus
fowl serum has a powerful agglutinating action upon the corpuscles
of the rat and rabbit (as observed by Bordet, X. 1898), this action
depending upon the presence of normal agglutinins in the serum of the
fowl. Powerful agglutinins are also present in the venoms of serpents.
The agglutinins may or may not be specific. Where artificially produced
they belong to the latter category, as also when they develope in conse-
quence of disease. If an animal is immunified against the blood of
another, it developes agglutinins which act more or less powerfully
on the foreign corpuscles. The agglutinins may coexist with other
antibodies in a given serum, the complexity of serum in this respect
appearing to be unlimited. There may moreover be several agglutinins
present at once in a serum. Thus Myers (14, VII. 1900) found that
a precipitating antiserum for sheep's globulin, obtained from a rabbit,
agglutinated the washed blood corpuscles of the sheep as well as those
of the fowl. That the agglutinins which acted on the two kinds of
corpuscles were different was shown by adding one kind of corpuscles
to the antiserum until it ceased to agglutinate them, it being then
found that it was still capable of agglutinating the other kind; the
same result following a reversal of the order, in which the corpuscles
were subjected to the action of the serum.

The agglutinins have however received chief attention from bacterio-
logists, their presence in the serum in certain infective diseases, and
after recovei-y, being as is well known a valuable aid in diagnosis. The
agglutination test is applicable moreover to a large number of germs
against which animals may be immunified. As in the case of the

46 Agghitmins and Antiagglutinins

haemo-agglutinins, so with the bacterio-agglutinins, they may be natural
or artificial, the latter are more or less markedly specific. Since the
discovery of the bacterio-agglutinins, the principles of whose action
were worked out by Gruber and Durham, and practically applied by
Griinbaum, as also by Widal, they have been used both for the identi-
fication of micro-organisms and diagnosis of disease. In the first case,
a particular genn, B. typhosus, Micrococcus melitensis, B. coli, etc., has
been subjected to the action of graded dilutions of a specific antiserum :
in the second case, a well-identified germ has been exposed to the
action of the serum from a patient suffering from supposed typhoid
fever, and the like. At first it was thought that the reaction of agglu-
tination was strictly specific, as in the case of the precipitins, but time
has proved that they are but relatively specific. Their mode of action
has been the subject of much controversy, the literature relating thereto
being well-nigh inexhaustible. My object in mentioning agglutinins at
such length is to show that they possess many properties in common
with other antibodies, but more especially with those which are the
immediate subject of this paper, the precijjitins and haemolysins. For
the reason that the agglutinins have been most studied in their relation
to bacteria I have drawn my material chiefly from this source, for there
can be no question but that the haemo-agglutinins and bacterio-
agglutinins are essentially similar. They differ as to their value in one
respect, the agglutinins are <>{ great practical use in the identification of
bacteria, but of little use in the comparative study of blood, zoologically
or medico-legally.

Agglutinins of some kind are alwaj-s present in normal blood, and it would
appeiir that they reside chiefly in the hlood, there being less present in the organ.s.
Almo.st all observers, who have studied the question, have found agglutinins in the
lymphoid and blood-foi-ming organs. Gruber (1896), observing that the polynuclear
leucocytes took up injured micro-organisms, when cholera and typhoid gemis were
injected intraporitoncally into animals, concluded that the agglutinins were formed
within the leucocytes. ( 'oitrmoiit (1897) tested the blood and organs of typhoid
cadavers, and almost invariably foimd the blood to contiiin most agglutinin.
Similar results were obtained by Arloing (1898) with animals infected with
P^ieumobacillus bovia. Fodor and Rigler (1898) found agglutinins made their
first api>earance in the senmi of guinea-pigs rendered immune to typhoid bacilli.
Bath (29, IV. 1899), experimenting on rabbits with the .same germ.s, concluded that
the spleen (which had been extirjmted in some animals), lymph gland?j and bone-
marrow exert no demonstrable efl'ect upon ngglutinin-formation. van Emden (1899),
experimenting with B. aerogenes on rabbits, found the agglutinins chiefly in the
lymphoid tissue, less being present in the liver and kidneys. Deutsch (ix. 1899,
p. 720) found only traces of typhoid agglutinins in the liver, kidney.s, suprarenal.s,

Origin and Action 47

variable quantities in the spleen, bone-marrow and lymph glands, but never as
much as in the serum. He considers that they originate in the blood, and this
view is supported by a number of observers (Deutsch, 25, vii. 1900). Ruffer and
Crendiropoulo (5, iv. 1902), studying the normal agglutinins in the rabbit and guinea-
pig, observed (what was already known) that the serum jjos-sessed agglutinating
l)ower. A solution of red blood corjmscles, i)reviou8ly washed, did not agglutinate,
neither did similar solutions of formed elements from immunified animals exert an
agglutinating action. They on the other hand bring forward evidence to show
that agglutinins may lie formed in the leucocytes, for an extract of leucocytes
from an immune animal had greater agglutinating i)Ower than did the same
animal's serum. These observations are certainly suggestive. Agglutinins are
transmitted to the foetus in utero. They have also been found in humor aqueus.

The formation of agglutinins in animals subject to imnumiziition, follows the
same laws as does that of other antibodies. As Deutsch (p. 720) sjiys, they
gradually appear (3— 4th day), increase in quantity for a time (10—13 days) and
then gradually decroa.se in amount, there being of course individual differences
amongst animals in this respect.

Pick (1902, pp. 30—34) found that when he mixed typhoid (from
horse) and cholera (from goat) antisera, they acted independently
of one another when they were brought in contact with these germs.
This shows, as in Myers's experiment above noted, that there is no
interference exerted between the antibodies. The same thing has been
observed by others who have immunified animals with two species of
micro-organisms. Exactly comparable are the experiments made by
Uhlenhuth (11, IX. 1902) with different precipitating antisera, and (in a
measure) my earlier experiments (1, VII. 1901), in which I however reversed
the order, adding one antiserum to a mixture of bloods in solution.

The influence of salts upon agglutination is in a sense comparable
to their action upon the precipitins. Bordet (ill. 1899, p. 236),
who first studied the question, found that after cholera germs had
been agglutinated in the ordinary way and were resuspended in
saline solution, he could reagglutinate them, but that they were not
reagglutinatcd if resuspended in water alone. Joos (1901) found that
antityphoid serum did not agglutinate B. typhosus in the absence of
.salts. For agglutination to take place he considers salts as necessary
as the agglutinin and agglutinatable substance. He believes that
salts play an active part in the process, combining with both the other
substances, a conception which is contrary to Bordet's, that the
absence of salts offers only a physical impediment to agglutination. Joos
(p. 429) found that but a trace of salt added to washed bacilli which had
been impregnated with typhoid agglutinin immediately led to agglu-

48 Agglutinins and Antiagglutinins

tination'. These results have been confirmed in the main by Friedberger
(ix. 1901), who does not however consider that salts act chemically, for
he found agglutination to take place in the presence of grape-sugar,
asparagin, etc.

The agreement is fairly general as to the chemical nature of the
reaction, both components being used up in the process, as was originally
observed by Gruber. In this they are similar to the other antibodies".
How stable the union is has not been made clear. Landsteiner (1901)
finds that when red corpuscles have been agglutinated with serum or
abrin, the agglutinin may be again obtained, after they have been
washed, by simply warming them in salt solution. This would scarcely
point to a well-established union.

Widal {Semaine MMicale, 1897, No. .5, cited by Kraus) found that
typhoid bacilli which had been killed by exposure to a temperature (jf
56° C. were still subject to agglutination; a fact which Kraus (12, Vlll.
1897) confirmed both for the typhoid bacillus and that of cholera. The
reaction does not therefore depend upon the germs being alive.

Agglutinins are more resistant to heat than are haemolysins or
bacteriolysins, withstanding a temperature of 55°C. and over; for instance,
Laveran and Mesnil (25, IX. 1901, p. 695) found specific Trypanosoma-
agglutinin to be inactivated at 63 — 65° C. {\ hour), and Bordet (in. 1899,
p. 243) found haemagglutinins (of rabbit treated with fowl's coi-puscles)
to be destroyed at 70° C. in half an hour. Like the precipitins, they are
stable bodies, retaining their agglutinating power at times for many
months in vitro. Pick (1902, p. 21) cites Widal and Sicard (1897) and
Winterberg (1899) as having found the agglutinins to be precipitated
with the globulin fraction from serum. He has confirmed this, working
with typhoid-agglutinin. The latter was present in the pseudoglobulin
fraction, only traces (presumably impurities) being found with euglo-
bulin, when horse serum was examined. On the other hand in immune
goat, rabbit, and guinea-pig serum, the agglutinin was almost entirely
confined to the cuglobulin fraction. Similarly cholera-agglutinin was
almost entirely confined to the euglobulin fraction, both in iminuni'
horse and goat serum. Here again we find a similai-ity with other
antibodies (see precipitins, antitoxins, etc.).

The iignificance of the agglutinins is but imperfectly understood. They may
persist in the serum of i^ersons who have had typhoid fever for months and even
years. In some cases this would appear to be referable to a retention of these

' See further Bordet (in. 1899).

- See the investigation of Eisenberg and Volk (i!, v. 1902).

THoagglutinms and Agglutinoids 49

bacilli in some part of the body. Although anti-typhoid sera, to confine myself
to these, usually show marked agglutinating power for these germs, when potent,
it has at times been observed that such sera were strongly protective and but
slightly agglutinative. It has l)een claimed that such agglutinin.s may be ac-
cepted as an index of immunity, but this seems scarcely justified in view of the
fiict tliat strongly agglutinative .sera have been obtained from persons dying from
typhoid fever. Van Emden (1899, p. 31) has made a similar observation upon
rabbits dying of infection with B. aerogenes. I am inclined to believe that the
agglutinins represent but a group of the many antibodies with which the
organism reacts to infection or the like, that they are immune substances directed
against some of many injurious factors. It follows from what we know of the
individual differences which exist in animals that the reaction may take place
more along some lines than upon others, and thi.s would explain the presence of
an excess of one immune substance, another, perhaps more vital one, being deficient.
The use to the economy of the agglutinins under normal and pathological conditions
is certainly obscure as yet.

The occuiTence of Isoagglutinins together with isolysins in human
blood in disease hsis already been referred to on p. 42.

The existence of Agglutinoids, analogous to toxoids and precipitoids,
is indicated by the observations of Eisenberg and Volk (6, viii. 1902),
there being apparently a stable combining group, and a labile precipi-
tating group, in agglutinating sera. They found inactivated, as also old
antisera, to retard the action of fresh antiserum. Bail (Arch. f. Hyg.
1902, cited by Kraus and Pirquet) also believes in the existence of two
groups in such antisera. (Compare with precipitins.)

Antiagglutinins have been obtained through the treatment of animals
with haemagglutinins. Venom-agglutinins may be neutralized by anti-
venene (Kanthack), ricin-agglutinins by anti-ricin serum (Ehrlich). Ford
(3, VII. 1902, p. 367), taking either the normal agglutinin of rabbits, or
the artificial agglutinin developed in rabbits through fowl-blood injection,
injected it into fowls, and obtained antiagglutinin. It seems however
premature for him to conclude that the immune-agglutinins are only
quantitatively different from normal agglutinins. He states that
Wassermann has been unable to obtain an antiagglutinin for the
agglutinin of B. pyocyaneus (formed in pyocyaneus-treated goats), and
that Kraus and Eisenberg had been equally unsuccessful in obtaining
bacterial antiagglutinins.

Differences between Agglutinins and Precipitins, etc.

Tchistovitch (v, 1899, p. 418) has already drawn attention to the
fact that precipitins are distinct from agglutinins, on the ground that

50 Agfilutimns and Precipitins

anti-tetanus sennn agglutinates B. tetani, but does not produce a pre-
cipitum in culture fluids of this micro-organism. Nolf (v. 1900) found
that a serum may contain a precipitin but no agglutinin, and concluded
therefrom that they are distinct. Radziewsky (1 900, p. 434) also considers
the antibodies distinct. Bail (1901) found that after all the precipit-
able substance had been i-emoved, by adding antisennn to a diluted
agglutinating serum, the latter retained its power to agglutinate
unimpaired, as tested on B. typhosus. Both Eisenberg (5, v. 1902) and
Beljajew (1902) note the parallelism between the power of agglutination
and precipitation possessed by antisera. The latter observed that
precipitins appear much more slowly during immunization than agglu-
tinins, and consequently they nnist be distinct. In both cases we
have to deal with substances which appear to enter into chemical
combination in definite proportions; both are stable bodies, and resist heat
more than do others. There appeai-s to bo a difference though, in this
resistance to heat: thus Pick (1902, III. p. 81), working with antityphoid
serum, which he heated to 58 — 60° C, found that the bacterio-precipitins
were inactivated ; not so the agglutinins, for which reason he considei-s
them distinct antibodies. Kraus and ELscnberg (27, ii. 1902) found that
the precipitins acting upon their homologous blood did not carry down
diphtheria antitoxin, or the tj-]3hoid agglutinin they contained. They
were unable to obtain diphtheria anti-antitoxin, or t}i3hoid anti-
agglutinin, by treating rabbits with the respective antitoxic and
agglutinative sera, whereas they obtained anti-lactosenim (see under
Lact<isera), viz. antiprecipitins. The resistance of precipitins to heat,
as proved by a number of observers, will be referred to presently.
Ford (3, VII. 1902, p. 371) does not consider that the non-identity of these
antibodies is ])roved. See also Wassermann (1903).

PABT TI.
THE PRECIPITINS.

SECTION I.

METHODS.
Methods of treating rabbits fm- the pvdvction of jyrecipitating antisera.

Different methods of injecting blood, serum, etc. have been u.sed
by varioiLS aiithors. Tchistovitch and Bordet injected rabbits intra-
peritoneally, a method followed by Myers, Uhlenhuth, Nuttall, and
many othei-s since. The subcutaneous method has been used by
Wassermann, Stem, Dieudonne, Zuelzer; the intravenous by Mertens,
Leclainche and Valine, Strube. The subcutaneous method has been
claimed to have the advantage of offering less chance of infection,
a poor reason if proper aseptic precautions are employed. In my
experience it has been difficult to inject quantities of fluid at all
comparable to what may be introduced intraperitoneally, sloughing
being produced when larger quantities are injected beneath the skin.
The intravenous method has recently been advocated by Stnibc, and
Kister and Wolff, on the ground that it is possible thereby to shorten
the time of treatment needed by the intraperitoneal method, a smaller
amount of blood being required to be injected.

Rabbits when properly treated by any of the above methods will
yield effective antisei-a. The blood injected should be sterile, if necessarj-
filtered as serum through porcelain, as reconunended by myself, Uhlen-
huth and others. Otherwise freshly defibrinated blood may be used; some
claim it yields more powerful antisera. Serum is best for intravenous
injections. Pleuritic and peritoneal exudation, hydrocele fluid, albu-
minous urine, have been used in place of serum by a number of different
workers, the impression prevailing, however, that these yield less potent

4—2

52 The Precipitins : Methods

antisera. I have frequently used sera from cadavers, both human and
animal, the latter more especially from animals dying at the Zoological
Gardens, London.

As a rule no difficulties are encountered, the rabbits bearing the
treatment well, a slight loss of weight being noticeable after the first
injections. My experience suggests that it is better to grade the
dosage of serum, as is after all usual in immunization experiments,
beginning with a small dose and gi-adually raising the amount admi-
nistered, being guided by the animal's body-weight. It is especially
necessary when treating an animal with a serum whose possibly toxic
effects are unknown to begin with a small dose. Occasionally accidents
will happen, well-immunified rabbits dying, perhaps after the last injec-
tion, apparently from intoxication ; this has however happened rarely in
my experience. Of the sera injected, I found rabbits to tolerate larger
quantities emanating from the horse than from other animals. It
therefore seems strange to me that Rostoski (1902, b, p. 26) should
have experienced any difficulty when treating rabbits with hoi-se serum,
most of his animals dying, and he concludes, "so .schcint es mir sehr
schwcr bezw. unmbglich zu sein, Kaninchcn gegen die specifische Gift-
wirkung dcs Pferdesenims zu immunisiren." This is so contrary to the
experience of others that I am inclined to attribute it to his simply
having used excessive dosage. Even under identical conditions of
treatment rabbits of similar weight will yield antisera of different
strengths, so that the individual element entei-s also into the
problem.

Attempts made by myself, and othere (Whitney, 1902, etc.), to
obtain antisemm after a single injection of blood, or the like, have failed.
Michaelis (9, x. 1902, p. 734) states that he only once observed a
powerful antiserum to have developed in a rabbit bled 14 days after
a single injection of 20 c.c. of ox serum, the rabbit's blood having
contained no precipitin three days after the injection, and all traces
thereof having disapjjeared three weeks after its presence was observed.
Otherwise all workers have subjected their animals to repeated injections
of serum, a varying number of days intervening between each.

Minovici (12, VI. 1902) states that ox blood produces precipitin more
rapidly than does human blood, an observation which I have not as yet
been able to confirm. There is no reason why an animal should not
react more rapidly to one blood than to another, but to establish this
requires not a few, but many carefully conducted experiments where
the conditions are equal.

Production of Antisera

53

Uhlenhuth (15, xi. 1900) was able to obtain antiserum in a rabbit
fed on egg-white, the solution being introduced daily by means of a
sound, and the precipitin appearing after the feeding had been con-
tinued 24 days. Similarly Michaelis and Oppenheimcr (1902, p. 355)
fed rabbits with ox scrum in large quantities, and obtained antiserum
for ox blood. They attribute the formation of antiserum, through
excessive feeding with heterologous albumen, to the escape of some of it
from the action of the peptic digestion, for precipitins for milk, etc. are
not found in the serum of normally fed man.

In the following table I have summarized the methods of treatment
of rabbits adopted by difierent workers, the data in some cases being
rather incomplete.

Antiserum
for blood
or milk of

Mode of
injection

Amount in-
jected at a
time in c.c.

Total
amount in-
jected in c.c.

Duration of

treatment

in days

last injection: witli date

Man

subcut.

10

50-60

14

6?

Wassermann and
Schiitze, 18. ii. 01

ti

perit.

5—10

14—21

Stern, 28. ii. 01

fi

„

10

80—200

16—40

Kister and Wolff,
18. xi. 02

»

venous

1

6

8

7 Strube, 12. vi. 02

Ox

perit.

10

50-60

30—40

Uhlenhutb, 7. i. 01

Cow-milk
etc.

subcut.

10—50

21

6 ; Wassermann and

' Schutze, 1900-1901

)t

perit.

10

30

7

7

Moro, 31. X. 01

„ +

venous

7

56

28

Uhlenhuth, 6. xii. 02

Human al-
buminous

J)

20

150—200

90

15

Leolainche and
Valine, 25. i. 01

urine t

* Gave reaction with 1 : 1000 blood dilutions.

t Milk sterilized 90 minutes at 65'' C, injected every 4 days.

J Contained 1 — 2 g. of albumen per litre.

Uhlenhuth made injections every 5 — 6 days. Stem cveiy two days,
Wassermann and Schutze every 3 — 4 days, others at intervals which
are generally not stated. In Bordet's experiments (iii. 1899, p. 240,
not cited in the table), the milk was partially sterilised at G5° C. prior
to injection intraperitoneally. Wassermann and Schiitze sterilized it
by means of chloroform.

Without making an attempt at completeness, the above table very
clearly shows a great divergence in the mode of treatment, more especially
with regard to the duration of treatment and the amounts of substance

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56 The Precipitins: Methods

injected. Most workers followed the original method laid down by
Tchistovitch and Bordet, injecting what are evidently too large amounts ;
others have pursued a course of treatment contrary to what is usual in
other immunizations, the dose at the start being as large as that at the
finish. Very few appear to have kept accurate records of the weights
of their animals during treatment, as given in my paper of Vii. 1901,
although some state that they were guided to some extent by the
variations in weight. It is evident that a more scientific method of
treatment has to he worked out, it being, certainly for practical reasons,
desirable to discover the least amount of substance which gives a
maximum of precipitin, as also the shortest period of treatment
necessary. In my protocols (jj. 54) the duration of treatment is stated
in terms of the number of days intervening between the first and last
injection.

In the preceding table I have summarized the method pursued by
me in treating a number of rabbits with different bloods and .sera,
excluding all the older antisera which were not standardized by my
quantitative method, so as to give some conception of the amount of
precipitum yielded by antisera obtained in different ways. Owing to
other work I have been unable to study this question as I should have
desired. The results of Strube, using the infravenous method, had
already indicated, what I have also found, that the duration of treat-
ment and dosage required are much smaller than when other modes of
treatment are used. It is true that my most poweiful antisera have not
been obtained by the intravenous method, but this is doubtless due to my
not continuing the treatment thus begun longer, there being no necessity
for doing so, as the antisera obtained were as strong as was desirable
for my purjDoses, quite as strong in fact as some obtained by the other
methods. / am certainly inclined, therefore, to accept the intravenous
method as being the most advantageous^.

In the experiments by Graham-Smith and Sanger (1903, p. 2(jO),
the anti-human serum, and some of the others, were prepared by
intravenous injection, but much smaller quantities than those usually
employed were found to sufficed " For example 18 c.c. of human serum
injected in doses of 5, 5, 5 and 3 c.c. at intervals of 2, 3 and 4 days
produced a powerful anti-himiau serum. The animal was bled 14 days

' See further a similar table to mine in the chapter dealing with the experiments
of Graham-Smith.

^ Powerful anti-ox and anti-sheep sera were made by injections of 9'2 and 12 c.c. in
doses of 1, 2, 1, 3-2 and 1-5, 2, 2, 3-5, and 3 c.o. respectively. The intervals between the
injections were 4, 4, 2 and 5 days in each case, and the animals were bled 7 and 10 days
after the last injections.

Production of Antisera 57

after the last injection. Continental workers have used quantities
ranging to hundreds of c.cs., and have frequently found that their
animals stood the operation badly, whereas the above animal, and most
of the othei-s we have treatied, continued to gain weight, and appeared
to be healthy. Some of our control antisera was prepared by the intra-
peritoneal method and quantities ranging from 30 to 60 c.c. were used.
The animals stood the treatment very well."

The fact that long -continued treatment of rabbits leads to the dis-
appearance of precipitins from the blood has been already noted by
Tchistovitch (1899) in rabbits rendered immune to eel serum. I was
able to confirm this (16, XII. 1901) in rabbits treated with ox and sheep
serum, the treatment having been too prolonged. As I stated in my
paper, just cited, "There is therefore a point in the treatment of
animals, when, for purposes of obtaining an antiserum, a maximum
of reaction is reached and the animal should be bled; this can be
determined by periodic bleedings from the ear-vein."

Mode of Injection.

If the serum to be injected has been preserved by means of
chloroform, as is frequently the case, it is desirable to remove as much
of this as possible, as the injection may prove fatal from the chloroform
contained in the serum. This is best accomplished by pouring the
serum into Petri dishes, these being exposed, half covered with their
lids, in the thermostat at 87° C. for about half-an-hour, or until no
perceptible odour of chloroform emanates from the serum. For intra-
peritoneal injections I have used an all-metal 10 c.c. syringe, one of
smaller capacity (5 c.c.) being used for intravenous injections.

Intraperitoneal Injections.

The x-abbit being held by an assistant, belly up, is lathered over the
seat of operation by means of liquid soap containing lysol. A small
area about 5 cm. across is then shaved in the lower abdominal region on
the left side. After disinfecting the skin with lysol solution, applied
with cotton pledgets, a small puncture is made through the skin with
a fine scalpel, this facilitating the penetration of the needle of the
syringe, the point of which it is better to render somewhat blunt, for
the reason that this lessens the chance of penetrating the intestine.
Penetrate the abdominal wall, holding the needle at right angles to its
surface. Having gently bored the needle through the abdominal wall,
through which it passes not infrequently with a slight jerk, immediately

58 The Precipitins: Methods

proceed to empty the syringe, slightly retracting the point if there
appears to be any impediment to the outflow. After the injection has
been made, and the needle withdrawn, the parts are dried, and tincture
of benzoin is applied, sealing the small opening made by the scalj)el
(usually but 1"5 — 2 mm. long). The wound usually heals by first
intention, the animal experiencing no discomfort. In this way I have
repeatedly injected animals in the same region, all the punctures made
during the treatment being included within the area shaved as stated
above. It will be seen that the operation is exceedingly simjjlc, and
I should not describe it but for the fact that many do not know how to
perform it.

Intraven o us Injections.

Intravenous injection is practised by introducing a fine-needled
syringe into the marginal ear-vein, and injecting smaller quantities
than in the preceding method, for the reason, not only that they suffice,
but that much smaller doses exert a toxic action when introduced
intravenously as against intraperitoneally. Intravenous injections are
very easily practised after a little experience. Before introducing the
needle the skin of the car should be shaved and disinfected as in the
preceding case.

Subcutaneous injections are not to be recommended from my
experience. Absorption of injected material takes place more slowly
this way than through the peritoneum, and the mechanical eS'ects of
large injections are liable to lead to sloughing.

Marking the Rabbits.

It is absolutely essential that there should be no confusion amongst
the animals treated, especially where these arc at all numerous. The
best way to secure this is to tattoo the rabbits inside the ear. This is
Ciisily done by dipping a fairly large needle into India-ink and pricking
it through the skin, a proceeding which the rabbit does not appear to
mind. Various letters and simple signs can be made in this way, and
there is then no possibility of confusing the animals.

Weighing the Animals.

It is desirable to weigh the animals during treatment. I have
always done this before each injection, stopping treatment as a rule
when a notable fall in weight occurred. I have had less loss in weight

Production of Antisera 69

in the few animals I have treated by the intravenous method. Of
the 26 animals noted in the preceding table (j). 54) 11 show a loss of
weight, in the figures I have given, whereas the others regained their
weight or even greatly incre.ised in weight. Several of those which
had lost weight, as recorded, had regained it when killed. Of the
11 that lost in weight, 3 only lost 50—70 g., 5 lost 130—220 g., a loss
of no great moment. On the other hand 3 lost heavily, viz. Nos. 3, 6,
and 14. Of these. No. 3 was treated with pig serum ; at the third
injection it exceeded its original weight, by the last it had fallen
400 g., 60 g. more being lost by the time it was killed. A greater
loss of weight is noticeable in Nos. 6 and 14 (treated by Mr Strangeways)
where the treatment was continued longer, the intervals between in-
jections being shorter, and in No. 6 the dosage very high. This animal
however gave a powerful antiserum.

When to kill the treated Animals,

When the animals have received usually 5 to 6 injections, and some
days have elapsed, it is well to draw off samples of blood and to test
these for precipitins. This is accomplished by shaving the ear, making
a small cut into a vein, and allowing the drops of blood which flow out
to fall into a Petri dish, which is then covered and set aside, being
slightly tilted. After an hour or two the senim will have separated
and can be tested. If it prove insufficiently powerful treatment may
be continued, whereas if it shows power the animal may be killed, the
time for killing being preferably 7 to 12 days after the last injection.

Killing the Animals and Collecting the Antiserum.

The animals may be killed in a variety of ways. Uhlenhuth
(25, vii. 1901) chloroforms them, opens the thonxcic cavity under aseptic
precautions; and, cutting through the beating heart, the blood is allowed
to flow into the thoi-acic cavity, whence it is removed by means of sterile
pipettes to suitable vessels. Othcra isolate the carotid, insert a sterile
cannula, and allow the blood to flow through it into suitable sterilized
vessels.

The method I have pureued is very much simpler, and usually gives
quite as good results, and certainly more blood. Having shaved the
neck, and disinfected the skin with lysol solution, the hair on the head
and fore-part of the body being moistened with lysol to prevent hair
flying about, the animal's head is bent backward, putting the skin of

60 The Precipitins: Methods

the neck on the stretch. The animal being held in this position by an
assistant, the operator's left hand is placed upon the back of the rabbit's
neck, which is uppermost, the fingei's and thumb encircling it. These,
being drawn upward, put the skin on the stretch transversely to the
long axis of the animal. By pressing downward, the neck arches ven-
trally. A sharp sterilized knife is now allowed to make a clean sweep
through the tense skin to and through the vessels. The skin retracts,
and the blood spurts out into a large sterile dish which is immediately
covered when the main flow has ceased. Drippings are caught in a
similar manner in Petri dishes, the thorax etc. being compressed to
expel as much blood as possible from the body.

The dishes are placed horizontally until a clot has formed ; they are
then slightly tilted, and as soon as serum enough has been expressed,
this is pipetted off into sterile test-tubes, and thence transferred to
sealed bulbs of small size. The serum which comes off later is similarly
collected, but it may be necessary to add chloroform thereto or to pass
it through a Chamberland filter to exclude bacterial development'.
Even when a hair or two falls in, the first serum will generally be
sterile, the clot retaining the microorganisms, the bacteria being more-
over in part destroyed through the bactericidal properties of the fresh
serum. After serum has been drawn off once or twice, a further amount
may be gained by slashing the clot with a sterilized knife. This last
serum is bloody, and has to be centrifugalized or placed in the refrige-
rator to sediment.

Storing Antisera.

The most suitable form of bulb in which to store antisera is repre-
sented in figure 3 B (p. 65), being drawn out at both ends. The bulb
is sterilized in the process of drawing out the ends ; the latter are cut
off with a diamond. After cooling, a number of bulbs are grasped in
the hand, the ends being sterilized in the flame, after which they are
allowed to cool, resting upon sterilized wire-gauze or otherwise. The
antiserum is now drawn up into the bulbs until these arc filled, as little
air as possible being left in the portion where it narrows. By tilting
the tube a little serum flows into the opposite drawn-out portion. The
capillaries are now sealed near to the serum by means of the small
flame, such as serves to relight the large flame of certain Bun.sen
burnei-s. A large flame is unsuitable. The serum must not be heated
in the process of sealing. The tubes are now placed vertically in tin
> The effects of filtration are noted on p. 118.

ProdiicHon of Antisera 61

boxes, containing wool at the bottom, and suitably labelled. The object
in placing the tubes vertically and not horizontally, is to ensure the
gravitation of any suspended matter into the lower drawn-oxit end,
where it accumulates, and can be readily disposed of by cutting the
tube above the layer of deposit, by means of a diamond, when about to
use the serum for testing. (Regarding the use of preservatives see
p. 64.)

The Choice of Animals for Immunization.

Rabbits appear to be the most suitable animals for the production
of antisera. 32 kinds have been produced by me through injecting
rabbits with mammalian (non-rodent), avian, reptilian, amphibian, and
crustacean bloods. Most observers have used the rabbit in their
work, doubtless because of the greater convenience and moderate
expense, the guinea-jtig not being so suitable, for the reason that
it yields less antiserum. Guinea-pigs have been used by but a few
observers, including myself The rabbit is therefor'e to be selected,
apparently in all cases where antisera are required for bloods other than
those of rodents. We have sufficient evidence to show that precipitins
are not formed in the serum of closely related animals. Thus Boi-det
and Hamburger obtained no precipitins for rabbit blood by treating
guinea-pigs with rabbit serum, and all that Gengou could claim was
that the serum of such guinea-pigs produced a slight oi)alescence in
rabbit blood dilutions, never a precipitum. Nolf was unable to produce
antisera in pigeons treated with fowl serum, the reason being apparent
from my extensive investigation of avian bloods, which, throughout,
have been found to react to antifowl and antiostrich .sera. In other
words, birds appear to be too closely allied for the purpose of inter-
avian antiserum-production.

In Tchistovitch's classical experiments precipitins for eel serum
were obtained by treating goats, dogs, rabbits and guinea-pigs. Anti-
rodent serum (for rabbit) has been produced by Nolf by injecting rabbit
blood into the fowl. Hamburger (G, XI. 02, p. 1190) was unable to
obtain antiseriim for egg-white from a dog treated therewith. Corin
hits on the other hand used the dog for the production of antisera,
according to Strube (1902). TThlenhuth (18, ix. 02, p. 680) has not
had encouraging results with other animals than rabbits. For instance,
he treated a lamb and a goat with human blowl and ascitic fluid during
2 months, injecting respectively a total of 1"5 and 2 litres thereof into
the animals ; the antiseium was too weak for practical use. De Lisle

62 The Precipifiiis : Methods

claims to have obtained antisera from eels treated with rabbit serum,
and Hamburger obtained effective antisera for rabbit blood by treating
goats therewith. Wassermann and Schiitze have apparently also failed
to obtain antihuman serum from the goat.

Judging from the blood reactions obtained, I am inclined to the
theory that an antirodent serum will act upon the rodent group, as I
have found with other antisera in other groups. The rodents form such
a well-defined order among the mammalia that it appears theoretically
probable that they will produce antisera for practically any animal
outside a rodent. The rabbit is therefore the animal jmit excellence for
the purposes of such experiments.

Collecting Bloods and testinff them with Antisera.

The bloods or sera tested were collected in two ways, viz. fluid and dry.
The fluid sera were relatively few in number because of the practical
difficulty of obtaining sera in this way. The fluid sera were obtained
by collecting blood in covered vessels where it was allowed to clot, the
serum being subsequently removed to clean or sterilized well-stojipered
bottles and a small amount of chlorofonn added for purposes of preser-
vation. Dilutions of fluid sera were made in salt-solution, the dilutions
being usually of 1 : 100 or thereabouts. A number of fluid sera were filtered
through sterilised Chamberland and Berkefeld filters and subsequently
preserved in sealed tubes. In other cases the dilutions were preserved
by the addition of small quantities of chloroform, and it was foimd that
such dilutions gave good reactions after over a year, the bottles having
been kept at room temperature in the dark. Although long-stored
bloods usually give somewhat less reaction than do fresh ones, the
reverse may be the case. It is a remarkable fact, which will be
brought out more clearly in describing the results of the quanti-
tative tests, that sera or serum-dilutions preserved for six months
or more will at times give more precipitum on the addition of an
anti-serum than do the fresh sera. This applies not only to the amount
of precipitum obtained by the addition of an homologous anti-serum to
a serum dilution, but also to what I have termed the " mammalian
reaction," namely one taking place between a powerful non-homologous
antiserum and a more distantly related blood. Whereas this increased
amount of precipitation obtained with older sera (the increase may
amount to 50 "/») will naturally greatly aff"ect the quantitative tests, it
can be practically ignored in the qualitative tests here described. On
what this change in stored blood depends is a matter for future

Collecting and Testing Bloods 63

invcst.igiition. It appeai-s also to take place in stored antiaera at times.
It is not duo to evaporation in stored dilutions, nor to the prolonged
action of the chloroform added for purposes of preservation, nor to
bacterial development, for pure stei'ilc sera preserved in sealed tubes
have repeatedly given greater reactions. This observation has not as
j^et been recorded. Although it appears at first sight improbable that
a dried serum ov blood may (as long as it remains soluble) give an
increased reaction after a time, the matter will have to be submitted to
e.xperiment.

In a few instances the sera were dried on glass plates at 37° C, the
scales being preserved in bottles with slightly vaselined stoppers. In
the majority of cases the bloods or .sera were collected on strips of
filter-paper of fairly uniform size (3 by 5 inches), as noted in the paper
by Nuttall and Dinkelspiel (July 1901, p. 378) and in a circular letter
which I sent out to those who have kindly aided me in collecting bloods
in various parts of the world. The filter-paper is immersed in the
serum or defibrinated blood, clots being avoided, after which it is hung
up to dry. One end of the strip is left clean, so that the name of the
animal (both common and scientific), the date, place of collection or
natural habitat, and the name of the collector, can be noted thereon in
lead-pencil. The notes can be written clearly if the paper is rested on
a hard surface. The strips of paper dry rapidly when suspended in the
air, it being convenient to pin them against the edge of a table or shelf
or upon the branch of a tree. Collectors were particularly warned not
to allow strips of paper saturated with different bloods to come in
contact with one another, especially in a moist state. Where it was
impossible to wait for the strips to dry, as when out shooting, collectors
were requested to place each sample separately in the paraffined paper
covers which I supplied, these being of the pattern usually used by photo-
graphers. The outfit for collecting which I sent out consisted, then,
simply of pure filter-paper of fairly uniform thickness, accompanied by
paraffined envelopes and outer envelopes of ordinary stiff paper. On re-
turning from shooting, the moist strips were removed from the jmraffined
covei-s and hung up to diy. The drying may be facilitated in our climate
by exposing the strips to the sun, but this proceeding does not appear
to do in the tropics, for I have found several samples sent from hot
countries to have become insoluble. The fact that blood-stains may
frequently become insoluble in hot countries was noted in a letter to me
from Mr E. H. Hankin, of Agra, India, dated 23 August, 1901. He wrote,
"There is a practical difiiculty in canying out the test here in the

64 The Precipitins: Mefhodft

frequent insolubility of blood stains, which in this climate may be dried
at a temperature of 115° Fahr. or even more. Blood stains usually don't
give the spectroscope test when extracted. If more dried or older, they
successively refuse to give the haemin and, I believe, the guaiacum tests."
It will be seen from my tabulated results that bloods sent to me from
hot countries dried on filter-paper not infrequently refused to go into
solution in saline. Where I had to deal with such bloods I allowed
them to remain some hours in salt solution at 37° C. Some bloods
however refused to go into solution even after 24 hours under these
conditions. Such bloods natural!}- gave no reactions. Other bloods
from the same source gave full or feeble reactions. This difference in
behaviour may depend upon the different temperatures at which the}'
were dried. These observations are of importance fi'om a medico-legal
standpoint. I did not find it advisable to use a weak soda-solution
(0-1 to l7o) as recommended by Uhlcnhuth (17 Oct. 1901, and by
Ziemke, p. 732), for the reason that it frequently yielded solutions
which gave rise to a cloudiness on the addition of any antiserum', the
cloudiness being in some cases quite marked, and sufficient to obscure
the mammalian reaction to which I first drew attention, and which will
be presently referred to. In the examination of the large number of
blood samples investigated it seemed scarcely worth while to try the
method recommended by Ziemke (17 Oct. 1901) of extracting with
cyanide of potiish solution. He does not state the strength of the
cyanide solution used as a solvent, but claims that it has even a gi-eater
solvent action than the soda solution for bloods which will not go into
solution in .saline. Ziemke adds a few crj'stals of t<artaric acid to the
cyanide-blood solution, shakes the fluid and tests continuously with
red and blue litmus paper, until the reaction is almost neutral. The
solution is now decanted and filtered. If the solution is to remain
clear it must be slightly alkaline. The solution is now diluted to the
degree desired and tested with antiserum in the usual way. The
method of Ziemke was too complicated for my pui-poses, although it
may be of use medico-legally. Uhlenhuth has also recommended the
use of saline chloroform-water for extracting difficultly soluble blood
«tains. In my investigations I however preferred to pass over the
insoluble bloods, that is bloods insoluble in salt solution, and this for
the reason that, in the case of chloroform, slight cloudings frequently
occurred in solutions where all the chlorofomi was not driven off,
upon the addition of antiserum of any kind. These cloudings due to
' See farther in the section relating to the medico-legal application of the test.

Qualitative Tests

65

chloroform or to the presence of soda should not give cloudings which
would be mistaken for a full reaction with an homologous antiserum,
but as stated they at times obscured the delicate cloudings which in my
tests gave indications of blood relationships
between different animals, and consequently
if used would have vitiated the purposes
of my investigation.

This leads me to a brief mention of the
method of preserving antisera. In the paper
by Nuttall and Dinkelspiel (1, vii. 1901, p.
378), it was stated that neither the addition
of chlorofonn, nor of trikresol to an antiserum
prevented the reaction taking place with
an homologous blood. It appeared there-
from that the addition of such reagents to
antiserum might serve a useful purpose in
their preservation. I however soon found
that there were disadvantages in adding any
preservative; that it was much better to
either collect the serum under aseptic con-
ditions, or, failing this, to pass it through
Chamberland filters with a view to excluding
putrefactive organisms from it'. Antisera to
which trikresol had been added frequently
gave marked cloudings with non-homologous
bloods. Antiserum to which chloroform has
been added behaves in the same manner
unless the chloroform has been removed by
evaporation. In evaporating the chloroform
one concentrates the antiserum and alters its
power. I was led to use trikresol through the
statement by Uhlenhuth (25 April, 1901)
that he had obtained reactions with an anti-
serum preserved three months with O'S'/o
carbolic acid. My experience certainly points
to the inadvisability of adding any preserva-
tive to an antiserum, for the reason that it
may produce pseudo-reactions, and I have no

m

A.

Fig. 3.
Test-tube containing
square of filter-paper which
has been soaked in saline.
The remaining blood-dilution
has been drawn up into the
pipette.

B. Antiserum contained
in bulb which has been sealed
at both ends, without the
serum being heated.

C. Small test-tube such as
is used for testing, being figured
in the racks in fig. 4.

(The figures are reduced to
^ natural size.)

' Note the effects of filtration on serum, p. 118.

N.

66 The Precipitins: Methods

doubt but that in certain cases such reactions have led investigators
astray. (See further pp. 76 — 79.)

The tests here recorded were carried out with pure antisera, upon
bloods which proved soluble in salt solution. In some cases, through
misadventure, microorganisms developed in the sealed tubes of antiserum.
Where this occurred the serum was centrifugalised, being cleared in
this manner in some cases, not in others. The microorganisms collected
in the drawn-out lower portion of the tube (Fig. 3), and when the latter
had to be opened the glass was cut above the part including the
deposit. It is a remarkable fact that even putrid antisera continued
to produce their special reactions, in some cases at any rate, without
any apparent decrease. The microorganisms present included bacilli,
micrococci and moulds of various kinds, the species not having been
determined from lack of time. It appeared to me to be a matter
of some interest to study the effects of different germs by cultivating
these in antisera^. In the paper by Nuttall and Dinkelspiel (11, V.
1901) it was noted that human blood which had undergone stinking
putrefaction for two months still gave a full reaction on the addition
of anti-human serum, this blood not reacting to other antisera.
This observation has been confirmed by the independent investiga-
tions of Uhlenhuth (25 April, 1901) and Ziemke (27 June, 1901)
on putrid bloods. Certain putrefactive processes do not therefore seem
to affect the bodies in antiserum and serum in so far as the amount of
precipitum they produce by their interaction is concerned.

In making the tests described below square-cut pieces of filter-paper,
of fairly uniform size, saturated with blood and dried, were placed in
a definite quantity of normal salt solution. As I have noted elsewhere
(July 1901, p. 378) blood and serum which is dried on glass plates only
dissolves slowly and frequently gives clouded solutions. On the other
hand, the filter-paper method, as I have shown, rapidly yields clear
serum solutions, the fibres of the filter-paper retaining the fine particles
which would otherwise go into suspension. To clear such clouded
solutions Uhlenhuth has resorted to the tedious method of filtering
them through Chamberland filters. I have found this to be usually
unnecessary. Where I have to deal with a clouded blood, I drop some
of it on to filter-paper and place the paper for half-an-hour or so in the
thermostat to dry, after which I place it in saline and usually obtain
a clear solution without more ado.

Where serum only is present on the filter-paper the solution may be

1 See the experiments by Graham-Smith and Sanger (1903) given on pp. 119 — 122.

Testing dried bloods 67

colourless, whereas when haemoglobin is present the solution may be
more or less coloured. I have attached no particular importance to the
amount of colouring matter which has gone into solution, but have
throughout judged a blood sample to have been soluble or not by means
of the Joam-test. In earlier papers by Uhlenhuth, as also in those of
Ziemke, stress is laid upon a trace of colour in solution as an index
of the blood being in solution. In a later paper by Uhlenhuth (18, ix.
p. 679, 1902) that author for the first time mentions the foam-test,
which I have throughout my work considered essential in this respect.
I judge the solubility by blowing air gently through the pipette which
is used for transferring the solution into the test-tubes (Fig. 3 a).
If air is blown violently through the fluid containing the filter-paper,
particles thereof, as also the particulate matter in the blood attached to
the paper, are loosened and the solution is rendered cloudy. Solutions
of blood or serum of 1 : 1000 and over still foam well, the limit being
apparently reached with solutions of about 1 : 5000, when, in several
quantitative experiments, it was seen that the minute bubbles formed
" held " an instant longer than they did when air was bubbled simply
through normal salt solution. I find it possible, moreover, to tell
approximately the degree of concentration of a solution by the length
of time the bubbles remain at the surface after air has been bubbled
through the fluid. Even from a medico-legal point of view the colour
test of solubility as noted above may prove fallacious, for stains due to
serum, which we know contains the acting substance, might well be
overlooked in the absence of the foam-test to which I unqualifiedly
attach more importance.

When the filter-paper squares are placed in salt solution, the serum
contained in the meshes of the paper usually goes into solution very
promptly, often in a few minutes. At times the solution is delayed
for half-an-hour or more. Once solution has taken place, it is inad-
visable to keep the paper soaking too long as the solid particles may in
turn become detached. Solution may be entirely prevented through
the paper having become soiled with fat, this having been the case
apparently with a few of the samples collected for me from animals.
In such cases the immersed paper tended to float, and remained covered
by minute silvery bubbles of attached air.

As soon as solution has taken place, as evidenced by the foam-test,
the mixed solution is transferred to small test-tubes (Fig. 3 c) placed in
suitable racks. The test-tubes I used usually had a capacity of about
1 C.C. and were kept scrupulously clean, and were always well dried.

5—2

68

The Precipitins: Methods

The test-tube racks (Fig. 4) were made in the laboratory, and consisted
of a flat strip of wood upon which three equidistant blocks of wood were
nailed to support a strip of cardboard perforated with holes of suitable
size 2 cm. apart. The holes were bored by means of a red-hot iron rod
pointed at one end. Each rack contained holes for 20 tubes, the strip
below being indented by means of a nail-pimch with holes correspond-
ing to those above in the cardboard ; these indentations received the
convex bottoms of the small tubes and fixed them in place. Each rack
was lettered according to the alphabet at one end, and numbers 1 to 20
were written in ink upon a strip of white celluloid which was tacked

^r r t t 1 t t iTTiP-H, n T. T> ■■■ r. t. t. ^ -Xl^

Fig. 4.
Showing the test-tube racks on the stand, and a single rack in the foreground. The
black riband is shown at the top, forming a background for the top row of tubes. For a
further description see the text, p. 70. Every part of the stand and racks, except the
celluloid strip bearing the numbers, is blackened.

(The figure is reduced to ^^ natural size.)

Qimlitative Tests 69

along one side of the upper surface of the base. When a number of
blood samples, my usual limit being 80, were to be tested by different
antisera, the racks were placed in rows of suitable length upon the
table. Where 80 bloods were being tested, the racks were ordered as
follows :

Backs
Row 1. A B C D

„ 2. E F G H

„ 3. I J K L

„ 4. M N O P
the number of rows corresponding to the number of antisera to be
used in testing the series of 80. In rows of 20 bloods the number of
antisera used might number 12; in sets of 80, the number of antisera
used was usually 4 to 6.

Each blood, whether on filter-jjaper or in bottles, received a number
on arrival. This number is given in the following tables (in the second
column) in small figures, and corresponds to the order in which the
bloods were tested. My plan was to test every blood received by means
of every antiserum produced. In this way a " network " was, so to
speak, formed through which all the bloods were passed in testing. In
some cases this scheme was departed from. For instance there appeared
to be no object in continuing to test avian bloods with mammalian
antisera after ten or more such antisera had continually given negative
results. Moreover, in some cases the supply of antiserum or of
blood to be tested ceased, and the series of tests was necessarily
interrupted. In other cases again the general results obtained with
one antiserum sufificed, so that it appeared only necessary to test a given
group with a particular antiseiaim, this for instance being the case with
the tests made with anti-chimpanzee and anti-ourang serum upon the
bloods of Primates, etc. The bloods received were not tested in their
zoological order, but in the order in which they were received. For

example :

696. Sus scrofa: Pig,

697. Ateles vellerosus : Spider Monkey,

698. Balaenoptera rostrata: Rorqual,

699. Ornithorhynchus paradoxus: Platypus,

700. Podiceps : Diver,

were actually tested in succession. Similarly the tests with antisera
were made in the order of production of the antisera, which depended
entirely upon the time when the bloods for injecting rabbits were
received. For reasons of convenience the order of these antisera, as

70 The Precipitins: Methods

produced, is incidentally given in connection with the tests on the bloods
of Pisces. It will be seen that no general picture, other than a sub-
jective one, could be gained of the results until the conclusion of the
work, when the tables had been completely rearranged, the bloods being
placed in their zoological order and the antisera in theirs.

On receipt, the samples on filter-paper were cut in two after
numbering, the one half being placed in order according to system, the
other half in order according to number. The systematic collection
aided materially in the final arrangement of the tables, and permitted
of small series of tests being made upon isolated groups, the bloods
being readily found. In both collections the samples were pinned upon
clean sheets of paper which prevented their coming in contact with one
another. The slips of the collection by numbers were ordered in groups
of 20 in paraffined envelopes enclosed within envelopes of stiff paper.
All the samples on filter-paper were kept at room temperature.

When a new antiserum had been produced I began by testing
down from blood No. 1 to the end of the list.

The test-tubes containing the blood solutions were numbered and
ordered in a manner corresponding to the numbers on the racks. The
solutions were drawn up by means of the pipette (Fig. 3 A, p. 65) and
transferred to tube 1 of racks A, E, I, M (for a series of 80), then the
second blood dilution was allowed to flow into tube 2 of the same
racks, and so on to the end of the series. This system rendered any
confusion practically impossible.

When the tubes had all been filled, the racks were transferred to
a stand (Fig. 4) which I constructed at small cost and have found
essential in the work. The stand consists of narrow shelves of deal
attached to wooden uprights by means of galvanized brackets. Metal
T-pieces are attached to both ends of the uprights, the one in each case
serving for the attachment of a strip of wood which serves as a base,
the other for the attachment (at one extremity of the T), of a brass
wire cord which runs down vertically to its attachment, a screw eyelet,
screwed into the basal strip of wood. A piece of dull black silk riband
sewed to two strips of bent zinc at both ends slides up and down
along the wires, wear being prevented by the zinc, within the bend
of which the wires run. Small tacks are placed on the posterior upper
margin of the shelves to prevent the racks being pushed backward
off the shelf The whole stand, and the racks, are painted black.
Each stand carries two raicks side by side. Where a series of 80 bloods
is being tested, it is necessary to place two stands side by side to
accommodate the racks in their proper order.

Qualitative Tests 71

The racks having been ordered, the solutions are viewed by trans-
mitted light to see if any are slightly cloudy. Where the cloudiness is
likely to obscure even a slight reaction the solution must be filtered or
discarded. The contents of the sealed tube containing antiserum
(Fig. 3 b) are now allowed to flow into a small test-tube such as is used
for testing; sediment in the sealed tube being excluded. By means
of a small tube of similar shape to that used in filling the solution into
the small test-tubes in the racks, the serum is now added in equal
quantity to all the solutions in the series. A drop or two suffices (equal
to "05 or even less of a c.c.)'. Where the antiserum does not flow down
to the bottom of the blood solution it is made to do so by gently tapping
the test-tube or by inserting a clean sealed capillary. On reaching the
end of the row, a slip bearing the name of the antiserum used, the time,
temperature, etc. is attached to the last rack in the row. In this way
a different antiserum is added to each row of bloods, until all have
received the test serum. The manipulation is exceedingly simple and
rapid, drops of antiserum of very uniform size being allowed to fall
from the pipette into each tube in the row.

Where a powerful antiserum is added to its homologous blood
dilution, the reaction is almost instantaneous, in other cases it takes
place more slowly. In the case of a strong antiserum, the reaction
takes place as a rule rapidly in related bloods, more slowly in distantly
related bloods. The rate at which the reaction takes place may
depend also upon the concentration of the blood dilution, the more
concentrated dilutions, within limits, reacting earlier than higher
dilutions. A weak antiserum will act more slowly than a powerful one.
Consequently a " time limit " (Nuttall, 16, xii. '01) may have to be put
on each antiserum when testing for a particular blood. In my earlier
tests I placed an arbitrary time limit of five to fifteen minutes upon
the reactions. Subsequently I lengthened the period of observation,
periodically noting the reactions which took place up to several hours,
indeed up to 24 hours at room temperature (varied between 12 and
20° C. in winter and summer), the deposits in the tubes being noted at
the end of the time mentioned. The black riband noted above, in
describing the stand, has proved of considerable value in studying the
slight reactions which are observable in more distantly related bloods,
or when testing more nearly related bloods by means of weak antisera,
where the reactions take place slowly and faintly. Many of the

^ It is unnecessary to operate with larger quantities as other authors have done, as this
only entails a waste of antiserum.

72 The Precipitins: Methods

cloudings or feeble reactions observed, for instance in the generalized
mammalian reaction already noted, have certainly been overlooked by
other observers because of the mode of illumination employed. For the
same reason it is of advantage to have the stands and racks painted
black. By moving the riband up and down, the obliquity of the rays
of light falling across it from the window and illuminating the test-
tubes can be regulated to suit each row of racks, and render slight
reactions quite evident. I can but recommend the use of this simple
apparatus to those engaged in similar work.

Sources of Error in Precipitin Tests.
1. Opalescent Antisera.
In the absence of more obvious sources of error, such as I shall
mention below, the use of what may be described as " opalescent
antisera " constitutes one of the gravest in using the test. A little
familiarity in the process of testing, and the making of one or two
control tests on heterologous sera, will however suffice to exclude the
error which might otherwise be made by using the antisera about to be
described. Uhlenhuth (11 — 18, ix. 1902, p. 680), Miessner, and Rostoski
(1902, b. p. 29) have observed these. It was due to the fact that my
anti-bear serum (referred to in an earlier paper, 5, iv. 1902) belonged
to this category, that I never published any tests made with it.
I found that it clouded evei-y blood dilution, mammalian and otherwise,
to which I added it. Although it gave a great reaction with its
homologous serum, it gave such marked reactions with all other sera,
that I immediately discarded it, without being able to explain why
it behaved so peculiarly. Since then I have fortunately rarely
encountered such antisera. Uhlenhuth has been the first to sound
a warning against their use, medico-legally. As he says, a slight
opalescence is usually perceptible when any serum or antiserum is
added to blood dilutions, the tube being viewed by strong transmitted
light, but the clouding here referred to is totally different and much
more marked, and takes place even in salt solution. I tried to clear my
anti-bear serum by filtration through porcelain, but was unsuccessful.
Uhlenhuth has had the same result. We are still in the dark as to
the cause of this peculiar milkiness or opalescence of certain antisera.
Uhlenhuth thought it might depend upon the stage of digestion at
which the animal was killed, but this seems to me unlikely for the reason
that I have killed my animals at no fixed time with regard to meals,
and on the whole have encountered the condition but rarely. Moreover

Sources of Error 73

in a rabbit recently treated by Dr Graham-Smith, the opalescence was
seen to continue for days, samples of antiserum being taken from the
ear- vein so as to test its strength'. It would therefore appear desirable
to take samples of blood from the rabbits' ear- veins to see if the senim
is clear before proceeding to kill the animals. Uhlenhuth states that
he has encountered the condition much more frequently in intravenously
treated animals, but this has not been our experience here. Uhlenhuth
suggests that some of the results of Kister and Wolff, as also of Strube,
on non-homologous bloods with different antisera, may receive an
explanation from their having used opalescent antisera, their results
being contrary to those of about 40 other authors. I am certainly
inclined to Uhlenhuth's view on this point.

Rostoski (1902 6. p. 29) treated rabbits with different constituents
of horse serum, and seems to have encountered the condition rather
frequently. This is unfortunate as it necessarily detracts from the
value of his results. One rabbit (his No. Ii.) was treated with the
crystallized serum albumen of the horse, yielding an antiserum which
was " stark milchig getriibt," and he goes on to say, " doch habe ich
gelegentlich auch schon andere milchig getrlibte Sera beobachtet, die
nicht eine besondere starke pracipitirende Kraft besassen." Linossier
(25, III. 1902) states that he has found anti-human serum to precipitate
not only human, but also ox, horse, dog, sheep, guinea-pig, and fowl (!)
sera, the reaction, he adds, being however incomparably greater with
human blood. He does not state with what concentrations he worked,
but says the difficulty can be surmounted by the use of higher dilutions^
This is certainly not the case with opalescent antisera. Kister and
Wolff (18, XI. 1902) experimenting with anti-horse serum, which had
" surprised " them because of the non-specific character of the reactions
it gave, tried to see if there were any law with regard to its action on
different bloods. These authors expressly state (p. 412) that they only
used clear sera, but perhaps they overlooked opalescence which may
not be very marked, and which in my experience might deceive one
into accepting such antisera. In any case adding it to salt solution
would have cleared the matter up. I have referred to their results
elsewhere (under tests with anti-horse serum), and it will be seen that
they are not in accord with those of the majority of observers, as they
obtained quite marked reactions with ox and sheep serum.

> Dr Graham-Smith has found several rabbits yielding opalescent antisera to be affected
with Cysticerci. This may explain the condition,
a See p. 74.

74 The Precipitins: Methods

2. Overpower/ul and Weak Antisera.

Powerful antisera have been of considerable use to me in my work,
and have led to my being able to establish more remote relationships,
especially amongst the mammalia, as I have shown in speaking of the
"mammalian reaction." They may however be a source of error in
medico-legal work, as has been pointed out by Uhlenhuth. When an
antiserum is so powerful as to produce a reaction in non-related bloods,
its action should be weakened by dilution with salt solution as suggested
by Uhlenhuth, Kister and Wolff, and Ewing. This lessens the amount
of antibody they contain, and by suitable dilution their strength may be
graded to suit requirements. Weak antisera may also be a source of
error through their reaction being delayed and bacterial develop-
ment (see below, § 7) occurring in the mixture of antiserum and blood
dilution.

3. Suitable Dilution of Bloods tested.

In view of the fact that concentrated dilutions of different bloods
may give reactions with non-homologous antisera, it is desirable to
dilute bloods as far as possible when testing them. This remark applies
more especially to medico-legal tests. Tests in which concentrated
sera are brought together and allowed to interact, as in the experiments
of Friedenthal and others^, are liable to lead to false conclusions,
especially when reactions with non-homologous bloods are to be excluded^.
This is also true of tests carried out with strong solutions.

Linossier and Lemoine (25, i. '02), working with anti-human serum,
having, they state, gi'eat power, found it necessary to add it in the
proportion of 25 : 1 of human blood in dilution, to remove all the
precipitin; in other words, 25 volumes of antiserum were neutralized by
one of blood. The proportion of antiserum added to a given blood for
this purpose will naturally vary according to its strength. As is stated
on p. 89, an excess of blood leads to precipitum solution. This has also
been observed by L. Camus, and Linossier and Lemoine (21, iii. '02),
who dwell upon the importance of not using an excess of serum in
solutions to which antiserum is added. Eisenberg (v. '02, p. 293) finds
that dilution per se affects the interaction of equivalent proportions
of precipitin and precipitable substance. Whereas equivalent propor-
tions lead to precipitation in small quantities of fluid, he states that no

' See also under Antipreeipitina, p. 149, and Normal Precipitins, p. 150.
2 See p. 73, Linossier.

Sources of Error 75

precipitation occurs when the same proportions act upon each other in
greater dilution, although the bodies combine. A precipitum which
had been resuspended in a very large volume of fluid did not become
redeposited, whereas it did become redeposited in a smaller volume
of fluid. He states further (p. 294) that antiserum added to different
concentrations of albumen gave a greater reaction with higher dilutions
than with more' concentrated ones. As has already been noted above,
he also finds that an excess of precipitable substance checks the
reaction, he thinks mechanically, for if the precipitum is collected and
resuspended in saline and in concentrated albuminous solutions respec-
tively, the precipitum is rapidly redeposited in the first case, not at all
in the second. He appears to be somewhat confused as to the inter-
pretation of this result. It is scarcely due to viscidity alone, an excess
of albuminous substance causes re-solution of the precipitated particles.
(See Antiprecipitins, p. 149).

Rostoski (1902 h, p. 53) found that more precipitation occurred in
0-5 "/o and 1 "/o than in 6 "/o serum albumin solutions, under otherwise
similar conditions. This was still clearer in corresponding globulin
solutions. Strong dilutions of serum pseudoglobulin hindered precipi-
tation more markedly than serum albumin dilutions, and pseudoglobulin
hindered the precipitation of serum albumin. He concludes that the
globulin exerts an antiprecipitating action, besides having an effect
through concentration. He compares the antiprecipitin to antitrypsin
and antirennet in blood (Fuld and Spiro, 1900 — 01), and to the normal
antihaemolysin of pseudoglobulin (Pick, 1901).

Rostoski (p. 58) also found higher dilutions to favour precipitation
of egg albumin by its antiserum, for precipitation took place in
l'5°/o solutions, whereas it was impeded to some extent in 3"5°/o
solutions.

In medico-legal work it will be desirable to progressively dilute
a suspected blood sample, and to reach a conclusion upon the highest
dilution (within limits) which reacts to a given antiserum. In routine
work, as I have stated, I have worked with dilutions of usually 1 : 100
to 1 : 200. As the dilution increases, the reaction narrows down more
and more, the reactions with the highest dilutions being practically
specific. Of course, in medico-legal work, the possibility of blood stains
being due not to one blood, but to two or more, has to be taken into
consideration. On one or two occasions I have found an avian blood
react to an anti-mammalian serum. For instance, the blood of a
swan (reported in my paper of vii. 1901), gave a very slight clouding

76 The Precijntins: Methods

with anti-sheep serum. In such quite exceptional cases I have
attributed the result to mammalian bloods having come in contact
with avian when the samples were collected, the damp filter-paper
strips having been placed side by side, with the result that the one
contaminated the other. (See tests with blood mixtures, p. 140.)

Reverting to what I have said with regard to the grade of dilutions
tested, I would add that Aschoff (1902, p. 192), who" cites an earlier
paper of mine in this connection, pertinently adds, " Man muss stets den
Verdiinnungsgrad der Sera und die Menge des zugesetzten Pracipitins
beriicksichtigen, um die specifische Wirkung richtig zu beurtheilen."

4. Effect of Preservatives on Antisera and Bloods.

In my paper of vii. 1901 I stated that the addition of chloroform
to an antiserum did not impede its giving a reaction. Its use for
purposes of preservation has since been recommended by Uhlenhuth,
Stockis, Biondi, Robin, Rostoski, and Strube. Ziemke (17, x. 1901)
also used chloroform, but seems to find antisera thus preserved to lose
in potency, adding, however, that they also did so when stored dry. He
goes too far however when he adds, " Gegenwartig ist keine Conservir-
ungsmethode im Stande, uns das Serum in frischem Zustande zu
ersetzen," for as I have shown elsewhere (p. 123) antisera may at times
be preserved for a considerable length of time. As I noted in my paper
of 16, XII. 1901 (and the observation still holds), none of these authors
"state the fact which I have observed, namely, that it is necessary,
before using it, to drive off the chloroform by placing the test-tube in
the thermostat. A small quantity of chloroform in a test-serum will
frequently cause it to produce a considerable clouding, which may be
a source of error, as the clouding is produced in almost any serum to
which the test-serum is added." It is therefore necessary to guard
against such a possibility of error due to the preservative being present
except perhaps in minimal amount.

For the same reason I have discarded the use of trikresol, and would
advise caution in the use of carbolic acid, as recommended by Uhlenhuth.
Strube and others.

In further support of the above statement I will refer to the
experiments made in our Laboratory by Graham-Smith and Sanger
(1903, p. 285) upon the effects of various volatile antiseptics, as well as
such agents as formalin, mercuric perchloride, and copper sulphate, etc.

Sources of Error

77

"As an example of the important volatile antiseptics chloroform may
be taken. In solutions containing much of this reagent on the
addition of serum a white cloud, and later a deposit, occur. More
dilute solutions give rise to slight cloudings. The results of experiments
with a series of such volatile antiseptics are given below. When only
present in small quantities in preserved sera the possible error due to
their presence can be eliminated by placing them in the incubator for
half-an-hour to evaporate off the reagent. When present to a greater
extent it was found that the supernatant serum above the deposit
caused by them still retained its specific properties. This is in accord
with what Nuttall has found.

1—10

1—100

1—1000

1—10,000

1—100,000

1

1

1-

1

a
1

3

S 3

0*

5

s

£

^

1—25

30 mina.

c

c

+

24 hrs.

D

c

D

X

c

*

*

d

s

*

*

*

c

*

D

*

+

;

+
*

•

d

See Footnote.

c

D

*
*

*

*

*

*

*

*

*

*

.

*

*

*

Corrosive sublimate and ferrous and copper sulphates were found
to produce very marked effects. They apparently destroy the serum
in contact with them and except when present in very small quantities
it was found impossible to carry out the test. The effects of dilutions
of corrosive sublimate and copper sulphate are given below.

In this and other tables by these authors the following symbols have been nsed :

C = coagalation.

+ = marked cloud — full reaction.

+ = less marked cloud.

X = medium cloud.

# = slight cloud.

#? = very slight oloud.

D = large deposit after 24 hours.
T> = smaller ,, „ »

d = smaller „ „ „

tr = trace of deposit.
• = no result.
— = no reaction.

78

The Precipitins: Methods

Corrosive sublimate and anti-ox eerum

Ck>pper sulphate and anti-ox eerum

Dilutions

1 hour

24 hours

1 hour

24 hours

1—25

immediate coagu-
lation

large deposit

immediate coagu-
lation

large deposit
and cloud

1—100

dense cloud

,, ,,

») »»

,, »i

1—500

cloud

cloud

coagulation and
cloud

deposit & cloud

1—1000

»)

slight cloud

)> )»

»» »»

1—10,000

slight cloud

nil

marked cloud

»> »»

1—100,000

nil

nU

slight cloud

small deposit

Silver nitrate causes an opaque white cloud on dilution with salt
solution up to 1 in 10,000. Dilutions below this do not aflfect serum
when added to them.

Formalin in 1 in 10 dilutions causes marked clouding, which
increases till the whole contents of the tube are opaque white.
Dilutions below 1 in 100 do not cause sufficient clouding to interfere
with the specific reaction. Solutions of thymol of 1 in 100 cause slight
cloudings, but lower dilutions do not apparently affect sera.

Lysol and lysoforiu both cause great turbidity when added to salt
solution even in low dilutions, and moreover even in very low clear
dilutions the addition of serum causes clouding. No method has been
devised for getting rid of these effects ; consequently the presence of
these substances except in very small quantities would render the test
of doubtful value.

The effects of the reagents, which for the sake of convenience we have
grouped under the heading of antiseptics, are very marked except in
the case of the volatile class. Some of the latter when added in full
strength to liquid sera produce heavy deposits, but the supernatant
fluid retains its properties. Formalin and corrosive sublimate in
strong solutions, as well as the sulphates of copper and iron and nitrate
of silver in much weaker dilutions, completely destroy the precipitum-
forming property. Lysol, lysoform, and similar antiseptics, owing to
their property of forming cloudings with salt solution, render the
application of the test of doubtful value in their presence."

In order to determine quantitatively ^ the effects of the prolonged
action of antiseptics on fluid sera, the following experiments have been
can-ied out by Graham-Smith (29, vii. '03, p. 358).

" Antiseptics in the proportions given below were added to fluid ox
and sheep sera, and allowed to act in sealed bulbs for 4 months. None
completely checked bacterial gi'owth. After this period dilutions of 1 — 21

* For description of my quantitative method, see Section VII.

Sovrces of Error

79

in salt solution were made, and all were allowed to stand in open
dishes for 2 hours in order that the volatile antiseptics should evaporate
off. The results were compared with serum kept under the same
conditions but without the addition of any antiseptic. The following
table shows that in nearly all cases the precipitum-forming power was
slightly reduced, but in a few completely destroyed.

The effects of the presence of these substances in fluids to be tested
have been given above.

Ol

aenim

Sheep 1
Precipitum

)eniin

Precipitum

Percentage

Percentage

Normal ox and sheep sera

•0356

100

•0140

100

Chloroform

f 1—1000
1—500
1—100

•0338
■0328
•0187

95
89
55

•0103
•0093

73
65

Xylol

1—500

• 1—100

1—25

•0300
•0300
•0281

84
84
79

•0140

100

Benzol

(1—1000
] 1—500
( 1—100

•0281
•0187
•0225

79
55
63

•0112

80

Toluol

1—100

•0140

100

Ether

(1—500
1—100

•0347

•0244

97
68

•0084

60

Formalin

1-10,000

1—1000

1-500

•0262

•
•

73
0
0

Alcohol

1—1000
1—100

•0075
•0103

53

73

Lysol

ri— 600

\ 1—100
(l— 25

•0187
•0169

•

55

47
0

Lysoform

(1—1000
1—100

•
•

0
0

Chinosol

(1—500
11—200

■0262
•0262

73
73

•0112

80"

The results obtained by Graham-Smith and Sanger with carbolic
acid and chinosol are considered on page 82, those obtained with lime
in Section IX.

5. The acid or alkaline reaction of the medium.

Normal blood dilutions possess an alkaline reaction. Tchistovitch
(v. 1899) found that precipitation only took place when the reaction
was alkaline ; when it was neutral, a slight opalescence was observable ;
no reaction took place when the reaction was acid. Linossier and
Lemoine (21, III. '02) found that sulphuric acid in the proportion of
•49 : 1000 lessened reaction, that it was reduced to a minimum when

80 The Precipitins: Methods

2'45 : 1000 was present, reaction being entirely prevented by acid
4"9 : 1000. Sodium carbonate, added in the proportion '66 : 1000 had
no influence, stronger dilutions thereof checked reaction in proportion
to their strength. Nevertheless precipitation may occur in dilutions
containing 5'3 : 1000, and 10 : 1000 does not quite prevent reaction.
In other words, the reaction takes place best in neutral or slightly
alkaline fluids, an excess of acid being more inhibitive than an excess
of alkali.

Rostoski (1902, a and h, p. 52) comes to diametrically opposite
results. He states that precipitation is impeded by an alkaline, aided
by an acid reaction. He acidified with acetic acid or an acid salt
(Mononatrium phosphate) or very small amounts of HCl, as little acid
is required to prevent reaction. Reaction, he states, takes place to
a lesser degree in neutral than in acid fluids. Slightly alkaline solutions
(degree of alkalinity 5'0) also react well, but as the alkalinity is
increased precipitation is impeded. It is probable that the discordant
results of Rostoski are in part attributable to his having worked with
opalescent antisera (see page 73).

It is not impossible therefore that the changes in the alkalinity of
the blood in health, but more especially in disease (see von Rigler, 1901)
may to a certain extent affect the degree of reaction obtained with
precipitins. We propose to pursue the question by means of quanti-
tative tests, as purely qualitative tests would scarcely show any marked
difference.

I have referred elsewhere to the abnormally high figures obtained
with anti-human serum upon the (concentrated ?) bloods of diseased
monkeys. In this connection it is of interest to note an observation by
Graham-Smith and Sanger (1903, p. 285) published since the above
was written.

" Our experiments on this subject are only three in number but
suggest that important differences may be found in diseased blood
by means of this test. The following observations were made on
sera from tuberculous cattle. The first required 2 c.c. of decinormal
caustic soda per 100 c.c. of serum to give a pink tint with phenol-
phthalein, and the others l"2o c.c. and "8 c.c. respectively. Also the
former required 4"25 c.c. of decinormal caustic soda per 100 c.c.
to produce a condition in which the serum was liquid when hot and
solid when cold, and the latter 2 c.c. and 1"2 c.c. respectively. As a
mean of three estimations in each case these sera produced "OSTo cc,
"0328 c.c. and '0244 c.c. of precipitum.

Sources of Error 81

NaOH ^, ,„ . , — ifl— required per 1000 cc. to

in per 100 cc. to give i" , T, .^ .

s... ! i. ..I. u ,,»,., '"*''* serum liquid wlien

hera pink with phenolplithalein hot, aolid when cold Preclpitum

1- 2 0 0.0. 4-25 cc. '0375 cc.

2- 1"25 „ 20 „ -0328 „
8- 8 .. 1-2 „ -0244 „

Strangeways has made numerous observations (unpublished) on the
differences in precipitum-forming power of the sera in disease."

We have found that an acid-reacting blood dilution will because
of the acidity alone produce precipitation (see below and Section IX.).
The observation has considerable practical importance medico-legally, for
the reason that blood stains may occur on acid fabrics, or acid-reacting
leather, the acidity in the substratum impeding the test by the anti-
serum in the blood solution made by steeping the stained article in
saline solution. The subject has been carefully gone into by Graham-
Smith and Sanger (p. 281), who report as follows :

" Observations on the reaction to litmus of extracts of coarse cloths
and leather, as well as the possibility of the treatment of blood-stains in
forensic practice with chemical reagents, made it desirable to investigate
the action of such reagents on blood.

Acids.

Several experiments were made with dilutions of both organic and
inorganic acids in distilled water and salt solution. Dilutions from
1 in 10 to 1 in 100,000 were tested by dropping in serum and noting
the effects up to 2 hours and after standing for 24 hours. In these
observations one drop of antiserum was added to about "6 cc. of the
dilution since this has been the quantity uniformly used in qualitative
work.

The addition of larger quantities produced slightly different results,
probably owing to the alkalinity of the serum itself, and moreover,
perhaps for the same reason, the effects of different sera were noticed to
vary slightly. This remark applies to all the following experiments of
a similar nature.

With the inorganic acids a noteworthy phenomenon was observed.
Strong solutions (1 in 10) in salt solution caused coagulation of the
serum and destruction of the precipitating substance, whereas weak
solutions (1 in 100) produced no result. Dilution between 1 — .500 and
1 in 10,000 caused more or les.s clouding, in the latter case taking place
half-way up the tube. These cloudings probably resulted from the
N. 6

82

The Precipitins: Methods

precipitation of the albumen by the dilute acid and were observable
within a few minutes. With greater dilutions nothing occurred. It
was also found that neutralisation previously with sodium carbonate
prevented these cloudings and in some cases even dissolved them after
they had been formed.

One example is given below in detail.

Dilutions of Nitric Acid
in nonnal salt solution

Anti-human serum

Anti-human serum afte
neutralisation

1 hour

24 hours

1 hour 24 hours

1-10

coagulation

coagulated mass

— —

1—100

very faint cloud

cloud

—

1—1000

marked cloud

cloud

— —

1—10,000

slight cloud half-
way up tube

—

— —

1—100,000 — — — —

The precise degrees of clouding caused by the various dilutions

differ with different acids.

Some organic acids behaved differently, acetic, oxalic, and tartaric

causing little clouding in dilutions of 1 in 10, but marked cloudings in

1 in 100 and in 1—1000.

Details of the action of tartaric acid : —

Dilutions of Tartaric Add
in salt solution

1—10

1—100

1—1000

1—10,000

1—100,000

Anti-human serum

Anti-human serum after
neutralisation

1 hour
very slight cloud

ft If ))
medium cloud

24 hours
slight cloud
medium cloud
marked cloud
slight cloud half-
way up tube

1 hour

24 hours

The accompanying table shows the chief actions of acid dilutions in
salt solution on serum. These effects are more marked when the
dilutions are made in distilled water.

I

•|

S

1

f

1

<o

P 1
'1 fi

(A

»

w

•«!

o

H

A<

O

S U

sat. sol.

1—10

SOmins.
24hrs.

c

c

+

.

*

*

•

0

•

-1-

D

D

B

•

*

*

•

D

•

D

1—100

■

•

*
*

•

*
X

*
*

*
*

C
D

*

d

*
*

* *
#

1—1000

■

*
*

X
X

X
X

#

X

X
X

X
X

+
d

X

*

*
*

1—10,000

*

•

*

*

•

*

.

*

*

• •

1— 100,00C

*

•

*

•
•

•

.

•

•

• •

Sources of Error

83

Strong alkalis and salts.

Experiments with the more powerful alkalis showed that in strong
solutious cloudings were also produced in them on the addition of
serum. Ziemke (17, viii. 1901) has recommended the use of -1 7,
caustic soda in distilled water for extracting blood-stains under certain
conditions. Our observations show that in such dilutions cloudings are
apt to occur on the addition of any antiserum, and render it thus an
unsuitable agent for the process^ These cloudings are better marked in
dilutions in distilled water than with those in salt solution.

The effects of dilutions of caustic soda are given in detail below : —

Solutions 0

caustic soda
solution

-10

-100

-1000

-10,000

-100,000

Anti-human

serum

in salt

1 hour
slight cloud
medium cloud
slight cloud

24 hours
slight cloud
medium cloud

The following table shows the aJctions of dilutions of alkalis and
salts on serum : —

30 mins.
24 hrs.

*
*

*
*

X X

X X

* *

a

a

*
*

& a

0. V

a«

*
*

1—20

*

*

*
*

■9 ® («

-5 C o

I i

1-20

-10

-100
1—1000

-10,000
1—100,000

Owing to the absence of any bad results from the addition of serum
to sodium carbonate dilutions, we chose this reagent as being the most
suitable for neutralising the effects of acids.

We next made some experiments to ascertain to what extent the
acidity or alkalinity of the medium influenced the specific reaction.
For this purpose both quantitative and qualitative experiments were
conducted. The solutions in each case were made up in the following

' Confirmation of my statement on p. 64.

6—2

84

The Precipitins: Methods

way. Three series of 11 tubes were prepared, each containing "o c.c. of
a 1 in 21 dilution of human serum in salt solution. To the first tube
were added 5 drops of a solution of acid, to numbers 2, 3, 4 and 5 were
added 4, 2, 3 and 1 drops of acid respectively. The sixth tube was not
treated in any way. Numbers 7 to 11 received 1 to 5 drops of alkali
respectively.

In the first series very small drops of a 1 in 100 dilution of
hydrochloric acid was used, in the second series large drops of the same
solution, and in the third series large drops of 1 in 10 solution of the
same acid. Large drops of corresponding dilutions of sodium carbonate
were used in series two and three.

In the quantitative experiments "1 c.c. of anti-human serum was run
into each tube, and in the qualitative one drop.

Series I.

Series

II.

Series III.

No. of tube Drops Small drops »/„

Large drops «/o

1. / 5 -0309 (61)

•0009

(2)

( nil

(0%)

2. 1 in 100

4 -0319 (63)

•0018

(4)

1 in 10

,,

,,

3. Hydrochloric

3 -0431 (85)

•0140

(27)

Hydrochloric ■

M

1»

4. Acid

2 -0431 (85)

•0187

(37)

Acid

1»

»J

6.

. 1 -0478 (93)

•0422

(83)

,,

»»

6. Normal dilution of serum -0507 (100)

•0510

(100)

•0516

(100»/„)

7.

'

■0422

(83)

[ -0169

(31 „)

8. 1 in 100

•0441

(86)

1 in 10

•0150

(29 „)

9. Sodium \

•0422

(83)

Sodium

tr

?

10. Carbonate

•0469

9

Carbonate

nil

(07o)

U.

L

•0591

?

. 1>

«•

In series II the acidity and alkalinity varied from about 1 — 1000 to
1—5000 and in series III from about 1—100 to 1—500.

These experiments show that the presence of even small quantities
of acid or alkali rapidly reduce the quantity of precipitum formed
(see Plate, fig. 5). The apparent exceptions of numbers 10 and 11 of
series II are due to the fact that the precipita produced were more
flocculent and occupied more space than the more compact precipita
elsewhere obtained. They also indicate that the presence of small
quantities of acid or alkali do not alter the specificity of the reaction,
for controls with anti-sheep serum were all negative.

Qualitative experiments undertaken on the same lines showed that
with 1 in 100 solutions of acid and alkali, cloudings first occurred in the
normal tube, next in the alkaline series, the times of their appearance
increasing from No. 6 to 11. The last two showed faint traces of the
specific reaction and also general opacity. On the acid side cloudings
due to the acid rapidly appeared, but later specific cloudings were

Sotirces of Error

85

superadded in Nos. 3 to 5. Control tubes tested with anti-ox serum
showed general opacity in the last of the alkaline series and slight
clouds in the acid series. Similar experiments with 1 in 10 solutions
showed cloudings in the normal serum and first three specimens of the
alkaline series only.

In the light of these observations it becomes necessary to test the
reaction to litmus of all solutions which are to be examined and, if
found decidedly acid or alkaline, to neutralize them.

It must also be remembered that the addition of strong acid or
alkali to fluid or dried blood completely destroys it."

The effects of the prolonged action of Acids and Alkalis on Fluid Sera.

These experiments were continued by Graham-Smith (1903, p. 359)
as follows :

" Ox, and sheep, sera with acids and alkalis added in the proportions
given below were kept under conditions similar to those just mentioned.

Ox

serum

Sheep

Bemm

Precipitum

Percentage

Precipitum

Percentage

Normal ox and sheep sera

•0356

100

•0140

100

1—1000

•0093

65

Hydrochloric acid

1—500

trace

? •

1—100

•

0

1—1000

•0103

73

Nitric acid

1—500

•0046

13

1—100

•

0

•

0

, 1 1000

•0112

80

Sulphuric acid

1 1—100

0

1—1000

•0112

80

Acetic acid

1—500
1—100

•0244
•0300

68
84

•0112

80

1—10

trace

f

1—1000

•0225

63

•0140

100

Oxalic acid

1-500

•0244

68

1—10

•0028

20

1 1000

•0131

93

Carbolic acid

1-100

•0112

80

1—1000
1—100

•0131

93

Citric acid

•

0

1—1000

•0122

87

Caustic potash

\ 1—500

•0150

42

0

1 1—100

•

0

•

1 1000

•0122

87

Caustic soda

1—100

■

0

1—1000

•0103

73

1 500

•0244

68

Sodium carbonate h

1—100

•0103

73

1—10

•0244

68

1—1000

•0140

100

1—500

•0309

84

Ammonia

1-100

•0206

57

•0131

93

1—10

•0065

18

86 The Precipitins: Methods

After dilution, the solutions were neutralized, and then tested quanti-
tatively. Control antisera were also used in all cases, but gave no
reactions.

The effects of the presence of unneutralized acids and alkalis have
been given previously (p. 82).

The preceding table (p. 85) shows that, except in very small quan-
tities, the prolonged action of inorganic acids completely destroys the
precipital)le substance, but that organic acids do not exert so dele-
terious an influence. Strong alkalis act in the same way as inorganic
acids."

6. Precautions with regard to Quantitative Tests by my Method.

It is obvious that if either antiserum or the serum to be tested
undergo concentration by evaporation the figures obtained will be
fallacious. The possibility of serum becoming concentrated in corpore,
in consequence of disease, has been referred to on page 80. It is
naturally essential that there shall be no matter in suspension, or
bacterial multiplication occurring in either the test serum or blood
dilution.

7. Bacterial Multiplication.

I have referred to an objection to weak and slowly-acting antisera
being the opportunity which is given to bacteria to develope, when
tubes are left standing as long as 24 hours, especially when they are
placed at higher temperatures as has been done in experiments of
some investigators. In my experiments I have on several occasions
had my later readings vitiated by bacterial development, especially
during summer, the readings being made after 24 hours and the tubes
kept at room temperature. The object of thus prolonging the experi-
ment was to note the deposits formed in the solutions overnight.
I sought to guard against this by noting the deposits earlier or later,
depending upon the temperature of the room in which the experiments
were made; in summer the deposits were noted after 12 — 16 hours, in
winter after 24 or more hours.

Where bacteria develope, provided that the readings are made
sufficiently early, there will be little chance of confusing the clouding
produced by microorganisms, with what I wished to note, namely the
deposit at the bottom of the tube. A little experience renders it easy
to as a rule recognize early bacterial cloudings, for they generally arise
from the lower strata of the fluid, gradually float upward, the bacteria

Sources of Error 87

seeking oxygen. The bacterial cloud at times remains stationary, or
moves gradually upward in the fluid, so that clear fluid may be seen
above and below. The behaviour of various bacteria under somewhat
similar conditions, has been the subject of an interesting investiga-
tion by Beyerinck (Ueber Athmungsfiguren beweglicher Bakterien.
Centralbl. f. Bakteriol. 1893, xiv. pp. 827—845), who refers to the
" Bakterienniveau " as the point in the fluid where the bacterial cloud
may hover at times for days.

See further under medico-legal applications of the precipitin method,
Section IX., also p. 63, regarding insoluble bloods.

SECTION II.

THE NATURE OF PRECIPITIN REACTIONS.

That the precipitins are used up in the process of precipitation, was
already observed by Myers (14, vii. 1900) who took it as evidence of
the action being chemical in nature. Michaelis and Oppenheimer
(1902, p. 357) consider that the precipitins cannot be regarded as a
special form of coagulative ferment for the reason that they are used
up quantitatively in considerable amount. Whereas, it is true, that
ferments are also ultimately used up, this occurs but slowly and in a
very slight degree as compared to the amount of substratum affected.
Precipitins are used up in a manner analogous to what takes place when
toxin is added to antitoxin, or acid to base.

Eisenberg (5, v. 1902) following Ehrlich's absorption method, deter-
mined the amounts of the mixed interacting substances, before and after
reaction had taken place. In this way he was able to find that both
substances combine quantitatively, for measureable amounts of both
had disappeared from the mixture. The results of Miiller (1902) and
of Leblanc (1901) with lactoprecipitins and haemoglobinprecipitins gave
similar results, pointing to the fact that both substances are present in
the precipitum. Eisenberg therefore adds that Halban and Landsteiner
(1902) are scarcely justified in denying the chemical nature of the
reaction. These authors claim in proof of this that salt-free serum
albumin heated to 100° C. partly loses its precipitability, although it
contains as much albumin as before. It may well be said in rejoinder
that this was no longer native albumen. I have stated elsewhere
that the precipitins act independently of the amount of albumen, the
latter must therefore possess certain specific properties if they are to
combine with the precipitins.

In this connection it is of interest to cite some very careful analyses
and tests made here by Mr F. G. Hopkins, to whom I am exceedingly

Observations by F. G. HopTdns 89

indebted for the loan of his notes, the experiments not having as yet
been published. The animals were treated by me with substances
supplied by Mr Hopkins.

Obsei-vation I. Two rabbits were treated with intraperitoneal in-
jections of crystalline egg albumen, receiving 5 injections, the total
volume injected representing 45 c.c. The animals were bled 11 days
after the last injection (22, vii. 1901).

The two antisera were mixed. The mixture remained clear on
standing.

35 c.c. of antiserum were fully precipitated by crystalline egg albumen
solution, the amount of which was not determined.

The dried precipitum weighed only '0246 g.

Observation IF. Three rabbits similarly treated to the above, except
that one received 35 c.c, and that the animals were bled 7 days after
the last injection (27, vii. 1901).

The three antisera remained clear when mixed.

50 c.c. of antiserum were fully precipitated and the dried precipitum
weighed -0325 g.

Observation III. Two rabbits were treated with ox-serum, receiving
5 injections of a total amount of 45 c.c. intraperitoneally. Bled 23, v.
1901.

The two antisera remained clear when mixed.

When the antiserum was added to undiluted ox-serum, drop by
drop, the instantaneous precipitum produced was redissolved on stii~ring.
Ten c.c. of ox-serum requiring 5 — 6 drops of antiserum before permanent
clouding was obtained. Evidently then a precipitum is soluble in excess
of serum. (See Antiprecipitins, p. 149.)

The ox -serum was next added to the antiserum. When 6"5 c.c. of
ox-serum had been added to 30 c.c. of antiserum no further precipitation
occurred, and there was no further clouding subsequently when more
antiserum was added to the clear supernatant fluid. The total pre-
cipitum obtained from the above mixture of 30 c.c. antiserum and 6'5 c.c.
ox-serum, weighed when dried at 110° C. '553 g. Prior to drying, it
should be added, the precipitum first obtained with the aid of the
centrifuge had been thoroughly washed with 5 "/o NaCl solution, being
resuspended and recentrifugalized three times in saline, this being fol-
lowed by repeated washings with water, the washing being similarly
conducted, until the washings gave no trace of biuret or xanthoproteic

90 The Nature of Precipitin Reactions

reaction. The tests conducted upon this precipitum will be considered
presently.

Observation IV. A rabbit treated in the usual way with sheep-
serum, yielded an antiserum of which 25 c.c. took 4'5 c.c. of sheep-serum
to fully precipitate it. The washed precipitum weighed, when dry, -ISS g.

Observation V. Two rabbits were treated with horse-serum, receiving
6 and 5 injections each (55 and 45 c.c. total). Bled 3 days after last
injection. The mixed sera remained clear, and 40 c.c. thereof took about
5 c.c. of horse-serum to fully precipitate it. The washed precipitum
weighed when dry "366 g.

Strube (12, vii. 1902) states that his results contradict those of
Biondi. Strube, like others, has found a quantitative relation to exist
between the interacting substances, for on adding 5 c.c. of a blood solu-
tion to different proportions of its homologous antiserum (1:10 to
1 : 5000) he obtained, as I have done, decreasing quantities of pre-
cipitum. In very dilute solutions, as Biondi is also stated to have
observed, large quantities of precipitum may apparently be formed, this
being attributed by Strube to the looser character of the precipitum
making it appear more than it actually represents. He also notes, that
by repeatedly adding an antiserum to blood dilution, and filtering each
time after precipitation takes place, fresh precipitations occur every
time antiserum is added, the amounts of antiserum of course being but
fractions of the total amount required to induce complete precipitation.
This also indicates very clearly that the reaction is quantitative and not
due to enzyme action.

Inactivated Antisera.

Mtiller (18, II. 1902) found a lactoserum which had been inactivated
at 70° C. to have acquired the power of neutralizing precipitins, in the
sense that it prevented precipitation when active serum was added to
inactivated, the latter having been previously mixed with a given milk.
Lactoserum from which the precipitin had been removed by the addition
of casein is incapable of neutralizing active lactoserum. This indicates
that the i^eutralizing substances are not present in fresh lactoserum,
but that they are formed in consequence of heating. Inactivated normal
rabbit serum possesses no neutralizing power. The neutralizing sub-
stances are not affected by the presence of lime salts, and may be

Inactivated Antisera: Precipitoids 91

precipitated from inactivated lactoserum by means of dilute acetic acid,
for they are absent in the neutralized filtrate. Mliller finds that pre-
cipitins do not combine with the neutralizing substance in the presence
of (acetic) acid, on the other hand, under similar conditions, the pre-
cipitins combine with casein, precipitation occurring although reaction
is retarded. Inactivated lactoserum dissolved the precipitum after some
hours, whereas inactivated normal rabbit serum did not. Lactoserum
robbed of its precipitin by the addition of milk, acquires no neutralizing
properties even when heated to 75° C. Miiller therefore concludes that
the neutralizing substances are derivatives of precipitins, originating
from these tvhen they are heated.

Whereas normal inactivated rabbit serum possesses no anti-rennet
action, inactivated lactoserum does prevent coagulation through rennet
ferment just as it prevents precipitation through fresh lactoserum. He
concludes therefore that the neutralizing action depends upon the pre-
cipitable (casein) aiid neutralizing substances combining, thus protecting
the casein from the action of the coagulating agents.

Miiller considers the neutralizing substances "precipitoids," analogous
to the " agglutinoids " recently described by Eisenberg and Volk (see
p. 49). According to Ehrlich's theory, the precipitoids would be re-
ceptors whose "zymophoric" group has been destroyed by heat, but
whose " haptophoric " group is retained (see p. 12).

Two views may be held with regard to the nature of the neutralizing
or combining body in inactivated serum, according to Eisenberg (v. 1902,
p. 301). Either, (a) the combining body preexists in unheated serum,
its action being obscured by that of the precipitin, or (6) it originates
from another substance in precipitin through the action of heat. The
latter view, as we have seen, is held by Miiller, and apparently also by
Eisenberg. Supposition (a) appears unlikely to Eisenberg, for the reason
that if it is correct an excess of precipitatable substance should have no
effect in preventing the reaction of the bacterioprecipitin with which
Eisenberg experimented. He found, on the contrary, that an excess
thereof increased such action^. Moreover active serum robbed of its
precipitin through the addition of precipitable substatice no longer
antagonizes fresh precipitin. Neither does deprecipitinated antiserum,
as Miiller showed, when heated, prevent the action of precipitins.

' See " Observation in " p. 89 where I have stated that an excess may prevent pre-
cipitation bj haematosera. This statement of Eisenberg's may therefore have to be
modified. He was working, however, with bacterioprecipitins which possibly behave
differently.

92 The Nature of Precipitin Reactions

Eisenberg therefore concludes that precipitins contain two bodies,
(a) which is labile and produces the visible reaction of precipitation,
(6) which is stable and represents the haptophoric or combining group
of the precipitin, the part which possesses special affinity for the pre-
cipitable substance in the homologous serum. On theoretical grounds
Ehrlich {Croon. Lect, 1900) assumed the precipitins to possess such a
constitution, defining them as receptors of the second order (see p. 12),
and we find that Miiller and Eisenberg reach the same conclusion in
consequence of their experiments.

Eisenberg (p. 297) found inactivated antiserum to prevent pre-
cipitation by fresh antiserum both when it was added to precipitable
substance before, or when added simultaneously with, fresh antiserum.
In such a reaction there are three components, a. precipitable sub-
stances, b. heated antiserum, c. fresh antiserum. On adding a constant
amount of a mixture of a. and b. to a varying quantity of c, it will be
seen that the antagonism is overcome as the amount of c. added is
increased. Eisenberg notes that small amounts of b. are more anta-
gonistic than large amounts, which is not ea.sy to understand, and
requires confirmation. He does not consider the antagonism due to the
action of b. on c, for the inactivated substance antagonizes best when
added to the precipitable substance before the active antiserum. In
other words, it does not antagonize as completely when added together
with fresh precipitin. From the fact that precipitation may be pre-
vented when b. and c. are added simultaneously, it seems clear that h.
has greater affinity for a. than has c. It is only when c. is in excess
that b. exerts a limited influence, this being due to the prepon-
derating amount of c. giving it a greater chance of being the first to
enter into combination with the precipitable substance. Where a
reaction is brought about by adding an excess of c, we have possibly to
deal with a " Massenwirkung " such as has been observed by Donitz, who
found that an excess of antitoxin apparently removed diphtheria toxin
which had already become anchored to body cells.

Similar observations with regard to the bacterioprecipitins have
been made by Kraus and von Pirquet (5, vii. 1902, p. 68). They found
that the bacterioprecipitins for typhoid and cholera were inactivated by
heat (58° C), and that such inactivated sera when added to fresh culture
filtrates prevented precipitation upon the addition of active bacterio-
precipitin. A fact which the authors state has been independently
observed by Pick. Inactivated precipitin was allowed to act for 10 hours
at 37° C. before adding fresh antisenim. They do not think that the

Inactivated Antisera: Precipitoids 93

"antiprecipitin'" in inactivated serum acts upon the precipitins as Pick
supposed, for mixed active and inactive serum allowed to stand for
10 hours at 37° C. gave a precipitum on being added to culture filtrate.
They conclude that heated serum retains its combining, but loses its
precipitating power, for the reason that it consists of (stable) combining
and (labile) precipitating groups. In other words, they agree with
Miiller and Eisenberg, as stated above.

It was further found by Kraus and von Pirquet (p. 71) that an
excess of old cholera antiserum prevented precipitation in culture fil-
trates, as did the inactivated fresh antiserum. The combining groups
would therefore appear to outlast the precipitating groups in such
antisera when stored. It also appears evident that the combining
groups, or, as we shall term them precipitoids, acquire a greater affinity
for the precipitable substance than the remaining intact precipitin.
This is completely in accord with what has been observed with agglu-
tinins and agglutinoids by Eisenberg and Volk (see p. 49). The
breaking down of precipitin to precipitoid is comparable to the changes
undergone by toxin into toxoid as observed by Ehrlich.

Michaelis (25, ix. 1902) inactivated antiserum for ox-serum globulin
by exposure to a temperature of 68" C. for 15 minutes. He does not
think the precipitin is converted into precipitoid, for the reason that
when the heated antiserum is added to precipitable substance it
retards but does not prevent the action of fresh antiserum. He con-
siders the preventive action as non-specific, but depending upon physical
causes, viz. the addition of neutral colloidal substance, for any other
dilution of albuminous substance will exert the same effect. This is
contradictory to the views previously expressed, where the authors
agreed that the action was specific, for inactivated normal rabbit serum
added under similar conditions to inactivated antiserum (also from
rabbits) did not prevent precipitation. Michaelis (p. 459) moreover
claims that heated antiserum markedly reinforces the action of a
minimal quantity of fresh antiserum, as much as if one had added the
corresponding amount of fresh antiserum. These results require con-
firmation and suggest, it seems to me, that he may have worked with
incompletely inactivated antisera. Michaelis believes that precipitation
is not due to a single substance, the "precipitin," but to a complex
similar to that possessed by the haemolysins. With precipitins we
have the conjoint action of two substances, both absent in normal sera,

^ Evidently a misnomer, precipitoid is a more suitable term.

94 The Nature of Precipitin Reactions

in contrast to what is the case with the haemolysins. In the haemato-
sera then there are two substances, the thermolabile being predominant,
the thermostable being small in amount.

Referring to the neutralizing action of inactivated precipitating
antisera as observed by Muller, Eisenberg, Kraus and von Pirquet,
above described, I would add that an analogous phenomenon has also
been observed with regard to specific bacteriolytic sera by Neisser and
Wechsberg, Lipstein, and Walker (see Bacteriolysins, p. 21), the inacti-
vated bacteriolytic serum, containing an immune body, which antagonizes
the action of fresh antiserum, when it is added to the latter in excess.

The Non-reactivatahility of Heated Precipitating Antisera.

In the paper by Myers (14, vii. 1900) that author stated that he
had succeeded in obtaining an antiserum for Witte's pepton, that he
had been able to inactivate it by heat (see p. 112) and to reactivate it
with normal rabbit serum. His observations in this regard have not
been confirmed, although he was the first to refer to inactivated pre-
cipitin as precipitoid, drawing an analogy between the change observed
in precipitin with that which takes place when toxin is converted into
toxoid. If Myers' observations were correct, then precipitins would
constitute receptors of the third order (see p. 12) according to Ehrlich.

No observer has been able to reactivate precipitating antisera since
Myers. Attempts have been made by Eisenberg (v. 1902, p. 302),
Kraus and von Pirquet (5, VII. 1902, p. 68), Michaelis (9, X. 1902,
p. 734), and myself Since the addition of a complement to inactivated
antisera does not reactivate them, we must conclude in the light of
Ehrlich 's theory that the precipitins constitute receptors of the second
order (see fig. 2, p. 12), as is assumed to be the case with toxins and
agglutinins. In Eisenberg's experiments (p. 297) the antiserum was
inactivated by an exposure of 1 hour to 72° C, reactivation being
attempted both with homologous and heterologous sera, and also by
adding quantities of active antiserum, too small to produce by them-
selves any reaction.

On Immune Bodies in Precipitating Antisera.

Until 1902 immune bodies had only been observed and studied in
sera which act on formed elements, bacteria or different cells. Gengou
(25, X. 1902, p. 739) has sought for them in precipitating antisera (from
rabbits) for milk, egg-albumen, horse fibrinogen, and also in the antiserum

Immune Bodies in Antisera 95

from guinea-pigs treated with heated rabbit and dog senim. He found
that all these antisera contained immune body, which for instance
in lactoserum attached itself to milk, and thus allowed the precipitin to
act, just as is the case with the anti-microbic and other sera above
referred to. He tested this as follows:
He prepared

LS. Fresh lactoserum heated to 56° C. for 30 minutes.
NS. Normal rabbit serum heated to 56° C. for 30 minutes.
A. Normal rabbit serum 24 hours old, containing alexin.
C. Washed fowl's corpuscles suspended in saline after having been
subjected to action of heated immune-serum of a rabbit which had
received injections of fowl's corpuscles.
L. Cows' milk.
NaCl. Normal salt solution.

He mixed these in different ways as follows (I have omitted the
quantities intentionally) :

\ result : precipitation ; fluid added to C don't haemolyse, acts

Test I. mixed L ^^ ;|. ^^ alexin were present ; alexin must be attached to

[ milk as it was not anchored to LS in Test III., nor by

j milk alone in Test II.

II. mixed h^ 1^ ^^^j^ _ ^^ precipitation, C haemoHsed as in Tests III. and
IV., alexin being free.

NS
A

III. mixed NaCl \

LS
A

IV. mixed NaCI

NS
A

result : haemolised C (added 24 hrs. later) in 30 minutes,
completely in IJ hrs. In this and Test IV. the
milk being absent, the alexin remained free to act
on the sensitized corpuscles.

V. mixed L

LS
NaCl

VI. mixed L [ P"^"*-

NS
NaCl

result: no haemolysis of C, there being no alexin

Gengou concludes from the above ingenious experiment that lacto-
serum contains immune body (" sensibilisatrice ") besides the precipitin.

96 The Nature of Precipitin Reactions

On the Nature of Precipitins and Precipitable Substances.

The power of precipitation possessed by antisera does not appear to
depend upon any alteration in the usual physical properties of a serum,
for Beljajew (1902) has found no difference in the specific gravity, nor
in the alkalinity between these and normal sera. Whitney (1902)
claims that a powerful antiserum clotted more slowly than a weak one,
a statement which requires confirmation.

The nature of precipitins has been the subject of considerable
investigation, as will appear from what follows. Nolf (v. 1900, p. 300)
treated rabbits with (a) washed blood corpuscles, and (b) the serum of
the fowl and dog, and found precipitins only in the blood of the rabbits
treated with serum, or with "plasma^." He ne.xt found that he could
produce precipitins in animals treated with globulin solutions, those
treated with albumin solution giving a negative result. Artificial
globulin solutions gave as good results in this respect as did normal
sera. Subsequently Myers (14. VII. 1900) produced antisera for sheep
and ox bloods by injecting their serum globulins into rabbits, and Stockis
(v. 1901) obtained antisera by globulin injections. Leblanc (31. V. 1901,
p. 359) injected different components of o.x blood (serum albumin,
pseudoglobulin, haemoglobin) into rabbits and found the precipitins in
the pseudoglobulin fraction of the immune serum. According to
Leblanc the precipitins are pseudoglobulins, or bodies precipitated
together with these. Eisenberg (v. 1902, p. 308) on the other hand
found the precipitins in the euglobulin fraction of the serum of rabbits
treated with horse serum. Strube (12. VI. 1902) cites the results of
Corin, Modici, and Biondi as pointing to the precipitins being bound to
the globulins. It appears doubtful however whether the globulins them-
selves exert the precipitating action (Biondi) if we rely on analogous
studies upon the antitoxins, in which Dieudonne (Arb. a. d. kaiserL
Gesundheitsamte, xiil) found in the case of diphtheria antitoxin that
precipitation with COa yielded antitoxin-free globulins, whereas precipi-
tation with magnesium sulphate, as practised by the above observers
for the precipitins, caused antitoxins to be carried down with globulins
mechanically. Biondi considers the precipitins possess a fermentative
action for the reason that he found different amounts of antiserum added
to equal amounts of serum to yield no markedly different quantities of
precipitum. We have however seen that there is every evidence in

• He (1902), and Dubois (1902, p. 692) state they have since obtained precipitins
by injecting haemoglobin solutions and intact blood corpuscles respectively.

Precipitins and Precipitable Substances 97

favour of the action not being fermentative, and I do not doubt but that
Biondi did not work with a sufficiently large series of dilutions, for if he
had he would have convinced himself to the contrary, as can be readily
done'. Oppenheimer and Michaijlis (18, vii. 1902) consider the compo-
nents in serum which lead to precipitation to be constituents of albumin,
not merely attached thereto. In a later paper Michaelis (9, x. 1902,
p. 734) states that he found precipitins in that fraction of serum which
is precipitated by half saturation thereof with ammonium sulphate,
namely, in the globulin fraction. He attempted to further isolate the
precipitin by repeating the fractional precipitation with ammonium
sulphate and found that most of the precipitin came down with the first
fraction (0 and 30 »/„ saturation), the second fraction (precipitated by
30 and 50 7o saturation) only containing traces of precipitin. The
precipitable substance behaved similarly. He finds that anti-ox
globulins do not precipitate serum-albumin. Corin (1902) considers the
active principle which leads to the formation of precipitin to be para-
globulin, and that therefore it would be best to treat animals with
paraglobulin solutions to obtain antisera, such as he prepared for the
dog. He finds the precipitin bound up with the paraglobulin, and this
can be separated and dried, the dried powder being brought into clear
watery solution and used directly for testing.

According to Obermayer and Pick (1902) the very rapid appearance
(within 15 minutes) of precipitins in the circulation of animals which
have received an intraperitoneal injection of precipitating antiserum,
indicates that the precipitin is non-albuminous. They found the
precipitins to be contained in the euglobulin fraction, both in rabbit
and horse immune serum, obtained from the goat and rabbit respect-
ively. In the hope of determining which of the constituents of egg-
albumin are concerned in causing the formation of precipitin in corpore,
they treated rabbits with subcutaneous injections of egg-albumin and
tested the action of the antiserum thus obtained upon egg-white and its
constituents. They state that normal rabbit serum produces an appre-
ciable precipitum in egg-albumin solutions*, this being due especially
to the action of dysglobulin on the serum, the action of other constitu-
ents being much less marked. They consider that the egg-white
precipitins do not constitute an integral part of the albuminous sub-

' An extensive series of tests made by Mr Strangeways at my suggestion during the
past year, which have not been hitherto published, distinctly show Biondi to have been
wrong.

^ Strength not stated ; see normal precipitins, p. 150,

N. 7

98 The Nature of Precipitin Reactions

stance, but that they adhere in different proportions to its constituents.
In support of this assumption they state that trypsin-treated albumin
gives a reaction with precipitins when all the albumin has been broken
up as indicated by the biuret reaction. They conclude that the
precipitin-formation, due to immunization with albumins of egg-white,
is independent of albumins, and that it depends upon a substance which
is difficult to separate from albumins in the process of chemical cleaning,
consequently it cannot be a specific process due to the action of albumins
of egg-white. The immunifying substance, which they wrongly style "pre-
cipitogenS" as well as the precipitin they conclude are not albuminous,
and consequently the biological test is of no use in the determination of
albumins. They found anti-egg serum to resist the action of hot 0'5 °/o
nitric acid. These observers and others have found the precipitins in
different antisera to be precipitated by alcohol.

The rapidity of the reaction between precipitin and precipitable
substance certainly points to the existence of great affinity between the
interacting substances.

In relation to the pTecipitable substance, Halban and Landsteiner
(25, III. 1902, p. 476) have found that senim-albumin, rendered salt-free
by dialysis and subsequently heated to 100° C, reacted but slightly to
precipitins, though when boiled 15 minutes it contained scarcely less
albumin (as understood by chemists and physiologists) than before.
It would therefore appear that precipitation constitutes a reaction with
definite chemical groups (within the albuminous molecule ?) like the
agglutination reaction, etc. Linossier and Lemoine (18, IV. 1902) and
Leclainche and Vallee (25, i. 1901) find that the action of antiserum is
not comparable to that of other reagents such as heat or nitric acid.
This is proved by the fact that the precipitin does not always give
reactions proportional to the amount of albumin present. Antisera are
more active towards globulins than nitric acid, less so towards serum-
albumin. With urine which contains much of the latter substance an
antiserum may give no reaction.

Eisenberg (v. 1902, p. 306) notes that heated albumin is more
alkaline than native, and that alkalized albumin is not precipitable.
He found (p. 307) albumin to be modified by contact with concentrated
solution of urea, added in the proportion of 4 : 1 to albumin solution, and

' A misnomer, as pointed out by Miohaelis and Oppenheimer (1902, p. 341) for the
reason that it suggests a relation between precipitin and ("precipitogen") precipitable
substance such as exists, for instance, between pepsin and pepsinogen, in other words that
the "precipitogen" is a forerunner of precipitin, which it is not.

The Precqntum 99

also by weak formalin, both of which are known to coagulate albumin,
the solutions thus treated being no longer precipitable. Nevertheless
the albumin retained its affinity for precipitin. Eisenberg and Volk
have made analogous observations upon agglutinins. Michaelis and
Oppenheimer (1902, p. 347) refer to the earlier observations on toxins
as possibly throwing some light upon the nature of the precipitable
substance. The toxins were at first considered albuminous, and for this
reason called toxalbumins. Further investigation showed that they
might be freed from albumin. Thus Brieger (1895) and others prepared
tetanus toxin which gave no biuret reaction, Jacoby (1900-1) did the
same with ricin, and Hausmann (1902) with abrin. Nevertheless
Michaelis and Oppenheimer consider that both in the case of toxin and
precipitable substance these substances may be simply torn away from
the large albuminous molecule to which they naturally belong.

The Predpitum.

According to Tchistovitch (v. 1899) the precipitum is soluble in
dilute acids and alkalis; insoluble in water, solutions of neutral salts and
alkaline carbonates. Nolf (v. 1900) considers the precipitum a globulin,
giving as evidence thereof that a precipitin added to globulin and
albumin solutions separately or mixed, yields a precipitum. Tchisto-
vitch states a precipitum is only formed in alkaline solutions, although
there may be some opalescence in neutral solutions, whereas acid
solutions remain clear. The precipitum (from eel and anti-eel serum)
proved non-toxic to rabbits when administered intravenously, although
the clear supernatant fluid proved toxic when it contained unneutral-
ized ichthiotoxin^

Bordet (ill. 1899) found different precipita soluble in dilute alkali.
Leclainche and Vallee (25, l. 1901) state that washed precipitum
gives all the albumin reactions. Leblanc (31, V. 1901, p. 362) states
that a repeatedly washed precipitum, obtained by adding anti-ox
globulin (from rabbit) to ox globulin solution, had a pink tint, due to
haemoglobin which had doubtless entered into union with the precipitin.
Nitrogen estimations made before and after precipitation lead to the
same conclusion regarding the occurrence of a union. Moro (31, x. 1901)
found that a lacto-precipitum was almost entirely dissolved in warmed
salt solution. Eisenberg (v. 1902, pp. 307-8) has also found lacto-
precipitum to be soluble at higher temperatures, being different in

' Ichthiotoxin, see p. 39.

7—2

100 Tlie Nature of Precipitin Reactions

this respect to the sero-precipita studied by other authors, and it also
differed in being soluble in concentrated urea solutions, and in saturated
solution of magnesium chloride. The egg-white precipitum, on the
other hand, he found to be soluble in dilute acids and alkalis^, whereas
he was unable to confirm the observation of Myers (1900) to the effect
that this precipitum is soluble in 2 "/„ saline. It proved insoluble in
solutions of alkaline carbonates, and various solutions, even up to
saturation point, of sodium chloride. Acid solutions of precipitum were
reprecipitated' on being neutralized, behaving like an acid albumin.
When washed in saline and heated, the precipitum coagulates, and
is then insoluble in weak acids, behaving therefore like coagulated
albumins which have undergone clotting through corresponding fer-
ments and have then been coagulated by heat.

The precipitum, obtained by adding anti-ox serum (from rabbit) to
ox serum as described under " Observation III." (p. 89), examined by
Mr Hopkins last year, was found to be soluble in very dilute NaHO,
much less easily soluble in Na^COj. It was precipitated from alkaline
solution on neutralization, but was soluble in excess of acid, even acetic.
The precipitum contained abundant phosphorus in organic combination.
It gave the biuret, xanthoproteic, and glyoxylic reactions, and was found
to contain " loosely bound " sulphur.

Macroscopic and Microscopic Appearances during Reaction.

When antiserum is added to blood dilution, it sinks to the bottom
of the tube. If it does not mix with the dilution, but flows down the
walls of the vessel, the reaction taking place almost instantaneously at
the zone of contact, assuming that the antiserum is powerful, and
reacting with its homologous blood dilution. The reaction consists in
the formation of a milky layer at the point where antiserum and blood
dilution come in contact. The milkiness extends gradually upward,
until the whole fluid is clouded. Where the fluids have been partially
mixed this generalized clouding occurs more rapidly, being perfectly
uniform when the bloods are thoroughly mixed by shaking. Where the
fluids have been mixed by shaking the diffuse clouding undergoes
a change ; after 10 to 20 minutes, or later, very fine granules of pre-
cipitum begin to appear, and the upper layers of fluid begin to clear, due
to the sedimentation of the particles of precipitum. The fine particles
soon become aggregated into coarser ones, and these into flocculi, which

1 Also observed by Michaelia (9, x. 1902, p. 734).

Appearances during Heaction 101

gradually sinking to the bottom of the tube give rise to more or less
deposit, having, when pure serum is used, a whitish appearance. Particles
may adhere to the walls of the tube, from which they are readily de-
tached by rotating it. With blood dilutions of, say 1 : 40 to 1 : 200 and
over, the deposit formed is usually sharply defined, where more con-
centrated dilutions are used the deposit may form an irregular mass at
the bottom of the tube. The increased viscidity of such dilutions may
retard sedimentation. The supernatant fluid remains clear. In some
few cases it may remain slightly clouded, more especially in non-homo-
logous blood dilutions. In rare instances where non-homologous, but
related bloods are tested, a cloud may form, without, however, leading
to a deposit.

Similar appearances are noted when anti-human serum is added to
albuminous urine, as described by Leclainche and Valine (25, i. '01),
who made their tests by bringing equal volumes of antiserum and urine
in contact with each other in the manner described above. In testing
with lactosera the opacity of milk diluted to 1 : 40, as recommended by
Wassermann and Schiitze, prevents one following the finer details of
the reaction. In this case the antiserum may be added in the pro-
portion of 1 : 1 or 1 : 5 as recommended by the last-named author.
Here the casein is precipitated by the antiserum.

The reaction may be followed microscopically, as recommended by
Tarchetti (1901), and Modica, who used the "hanging-drop" method,
familiar in bacteriological work. Griinbaum (18, I. 1902) used this
method also, comparing the appearances to what is seen when bacteria
are agglutinated, and observed that the particles appeared and were
more rapidly aggregated when anti-human serum was added to human
than to simian blood-dilution and vice versa. Robin (20, xii. '02) also,
apparently unaware of Tarchetti's method, has found it useful, for he
reports that he could observe the formation of gi-anules within 10 to 15
minutes microscopically, when a reaction visible to the naked eye was
only observable after two houre. He considers that the aggregation of
granules is possibly brought about by a process analogous to that of
bacterial agglutination.

The Supernatant Fluid.
If antiserum is added in insufficient amount to a blood dilution the
supernatant fluid, after removal of the first deposit, still contains pre-
cipitable substance, as may easily be proved by adding more antiserum,
and vice versd.

102 The Nature of Precipitin Reactions

Linossier and Lemoine, Eisenberg (5, v. '02, p. 291), and Ascoli
(26, VIII. '02, p. 1410), agree in finding that the supernatant fluid may
contain an excess of both interacting bodies in sokition after precipitation
has taken place. This is proved by adding either the one or the other
component to the clear supernatant fluid, which leads in either case
to fresh precipitation.

The Influence of Salts upon the Reaction.

That concentrated solutions of certain salts impede analogous re-
actions, such as those with bacteriolysins (Lingelsheim) and agglutinins
(Eisenberg and Volk) has already been observed. Linossier and Lemoine
(21, III. '02) taking a 1 : 20 serum dilution and adding increasing amounts
of NaCl thereto, found that even the presence of 1 »/o salt impeded the
precipitin reaction, and that 5 "/o salt prevented precipitation even after
a mixture had stood as long as 24 hours. They found the reaction to take
place in the presence of small amounts (1 "/„) of ammonium sulphate,
magnesium sulphate, and sodium fluoride, as they state was previously
noted by Arthus. Eisenberg's (v. '02, p. 307) results are in flat contra-
diction; he states that concentrated NaCl solutions (up to close on
18°/o, at which point albumins are thrown down) had no influence on
the reaction. The reaction was prevented by 0'25 normal magnesium
sulphate, slowed by 0'5 magnesium chloride, prevented by a 2-normal
solution of the latter salt. Moreover, Rostoski (1902, b. p. 42) observed
no noticeable difference in the precipitations which took place with
haematosera in the presence of 10 "/» NaCl, and 2 »/„ ammonium sulphate
and ammonium chloride. On the other hand, Miiller (18, ii. '02)
found the action of lactosera depends upon the presence of lime salts,
although these may be replaced by barium salts. Lactoserum only
occasionally precipitated boiled milk after the addition of lime salts.
Michaelis (9, X. '02, p. 734) removed all trace of lime salts both from
haematoserum aud its homologous blood by means of oxalate of
potassium and found the contrary, namely, that precipitation occurred
as well as before. Rostoski (1902, b. p. 42) on the other hand, also
experimenting with haematosera, obtained the same result as Miiller.
He states that precipitation does not take place in the absence of salts,
small quantities thereof are sufficient, large amounts of NaCl do not
impede precipitation. It was evident therefore that these experiments
required to be repeated.

Graham-Smith and Sanger (1903, p. 266) undertook to solve this

Influence of Salts

103

problem at my suggestion. Their experiments agree more closely with
those of Eisenberg and Rostoski. They report thereon as follows : —
"We have quantitatively estimated the influence of salt in the following
way'. Tubes containing 1 in 21 dilutions of human serum with
gradually increasing percentages of salt were arranged in a rack, and to
each •! c.c. of anti-human serum was added. We found that the
precipita in the tubes containing the most salt were more flocculent,
and owing to the increased specific gravity of the medium took longer
to settle (see Plate, fig. 2).

Results of measurements showed a slight decrease to 7 "/o and later
an increase, probably due to the fact that the more flocculent precipitum,
though really less in amount, occupies a greater volume.

Results of increasing quantities of salt in huma/n serum dilutions.

Percentage
ot salt

•6''/o

1 .,

2 „

3 „
i „

5 „

6 „

7 „

Precipitum
c.a

■0643

•0571

•0638

•0554

•0639

•0618

•0635

•0630

Percentage

of precipitum

as compared with

■6 % salt solution

100 »/o

88^8 „

99-2 „

86-1 „

99^2 „

96-1 „

98^7 „

97^9 „

Percentage
of salt

8«/o

9„

10 „

12 „

14 „

16 „

18 „

Saturated ]

solntion f

Precipitum
cc,

•0693

•0673

•0770

•0686

•0787

•0730

•0821

•0854

Percentage

of precipitum

as compared with

*6 °Iq salt solution

107-7 «/„

104-6 „

119-7 „

106^6 ,,

114-6 „

113-5 „

127-6 „

132-8 „

Experiments with sheep and anti-sheep sera, which formed more
compact precipita, show the diminution in volume plainly. In this
experiment the tubes were all centrifugalized for the same length of
time in order to diminish the error due to the increasing specific gravity
of the solutions.

Similar experiments to above with sheep serum.

Salt

l«/o

2 „

4 „

10 „

Saturated)

solution i

Precipitum
cc.

-0199

-0203

-0201

•0140

•0122

Percentage of
precipitum

100%

102 „

101 „

703 „

61 8 „

Qualitative estimations showed that when the quantity of salt was
increased above o'j, the antiserum did not sink to the bottom, and
1 The quantitative method of testing is described in Section VII. which follows.

104 The Nature of Precipitin Reactions

that clouding occurred at the top of the tubes, and also took longer in
forming."

Graham-Smith and Sanger (p. 266) moreover studied the behaviour
of aqueous blood dilutions to antisera, and report thereon as follows : —

" It has been noticed by many observers that solutions of fluid, or
dried, sera in distilled water become cloudy, and that after 24 hours a
precipitate occurs. In "5 c.c. of a 1 in 21 dilution of human serum in
distilled water this precipitate amounts to about '001 c.c. We have,
however, found that including this precipitate '1 c.c. of human
antiserum produces a smaller quantity of precipitum with blood diluted
with distilled water than with the same specimen diluted with normal
salt solution. The mean of three experiments in each case gave
■0384 c.c. of precipitum in salt solution dilutions and 0328 c.c. iu watery
dilutions."

Regarding the Claim that Precipitins permit of Distinguishing
different Albumins of the same Animal.

The investigations of Nolf (v. 1900) indicated that precipitins are
formed for serum-globulin and not for serum-albumin and solutions
of blood corpuscles. Leclainche and Vallee (25, i. 1901) however state
that antiserum for serum-albumin is very active for its homologous
substance, but almost indifferent towards globulins contained in the
albuminous urine with which they experimented. The urine from three
cases of interstitial nephritis gave marked reactions, whereas the urine
from a case of parenchymatous nephritis, which contained much globulin,
gave but slight precipitation. Albuminous horse and cow urine gave
no reaction. They add that human pleuritic exudate gave a reaction,
but not human blood serum when antiserum for human albuminous
urine was added thereto. Mertens (14, iii. 1901) treated rabbits with
human serum and found the antiserum to precipitate albumin in urine,
concluding therefrom that the latter must be derived from the blood.
The urine reacted both to this antiserum and to one obtained by treating
the rabbits with human albuminous urine. Dieudonne (2, iv. 1901)
found that peritoneal exudate and blood of man reacted similarly to
anti-human serum. Zuelzer (4, iv. 1901) treated rabbits with urine con-
taining 1 — 9°/oo albumin, and obtained antisera as did Mertens, to whose
generalization regarding the origin of albumin in the urine from the
blood he objects, although he considere that his results warrant the
conclusion that at least one albuminous body in urine is derived from

Effects on different Albumins 105

the blood'. Ide (1, iv.) and his pupil Leblanc (31, v. 1901, p. 355) state
that rabbits treated with chemically pure albumin derived from cows'
milk fonned antisera which possessed a different character, depending
upon which albumin of the same species had been used for the treat-
ment of the animals. They go so far as to consider that there are
specific antibodies not only for each cell-species but also for as many
albumins as the cell may contain. According to Ide the agglutinins
represent easily precipitated albuminous bodies, the antitoxins being
less precipitable. Leblanc {loc. dt.) treated rabbits with ox serum,
pseudoglobulin, and albumin. He states that the antisera produced
a reaction with ox, but not with sheep, horse, pig, guinea-pig and pigeon
sera. Anti-pseudoglobulin for ox, precipitated ox pseudoglobulin solu-
tions but not those of euglobulin and serum albumin. Antiserum for
serum-albumin precipitated its homologous substance but not pseudo-
globulin. Anti-ox serum had no effect on lactalbumin. Anti-ox haemo-
globin precipitated the haemoglobin of the ox. In other words, the
different antisera possessed a high degree of specificity for the different
albumins of the same species of animal.

Nuttall (v. and vii. 1901) showed that anti-human serum caused
reactions with human pleuritic exudate, the fluid from blisters, and to
a slight extent with nasal, and lachrymal secretions' and a faint clouding
even with normal urine. Anti-human serum was produced in rabbits not
only by serum but also by old pleuritic exudate of man, preserved as
long as 5 — 6 months with chloroform. Schiitze (22, XI. 1901, p. 492)
treated rabbits with human muscle albumin producing an antiserum
which he states precipitated homologous substance, but not human
albuminous urine. Levene (21, Xii. 1901) immunified rabbits with
milk and found the antiserum to precipitate milk, casein, milk albumin,
and ox serum, a result contrary to Leblanc's. This same antiserum
failed to act on fowl egg-white, egg-albumin, egg-globulin, fowl serum
and sheep haemoglobulin. Linossier and Lemoine (25, i. 1902) found
they could not distinguish albumin in urine by precipitins, as claimed
by Leclainche and Valine {loc. dt). Halban and Landsteiner (25, iii. 1902,
p. 475) treated rabbits with injections of ox spermatozoa, obtaining
(spemiotoxic) antiserum, which precipitated not only saline extract of
spermatozoa but also ox serum dilutions. Linossier and Lemoine
(28, III. 1902) found haematosera to also act on other albuminous fluids

' See a short paper by Asehoff (6, ix. 1902) on tliis subject.

" Confirmed by Biondi (1902, p. 21), who also obtained reactions with milk, vaginal
secretion, etc.

106 The Nature of Precipitin Reactions

from the same animal, viz., saliva, albuminous urine, spermatic secretion,
milk serum, muscle-extract. Moreover, blood serum was found to be
precipitated by lactoserum, as well as by antiserum for albuminous urine.
Repeating the experiment of Nolf (cited above, see p. 104) they obtained
a contrary result. Antiserum produced by globulin injections acted not
only on globulin but also on solutions of serum-albumin. Moreover they
found antiserum for serum-albumin to precipitate serum-albumin, and
also globulin. To make sure that this was not due to impurity of the
serum-albumin, which might contain globulin, they prepared as pure a
substance as was possible (see method in original, p. 370), but obtained
an identical result. They conclude that anti-globulin acts most on
globulin solutions, less on those of albumin. The antiserum for albumin
on the other hand actually produced more reaction with globulin solu-
tions than with its homologous substance. They conclude that for the
present the precipitins do not permit us to demonstrate chemical
differences between the different albumins of one animal. Strube
(12, VI. 1902) injected rabbits intraperitoneal ly with human spermatozoa
and human testicular extract (from cadaver), obtaining in both cases
a weak antiserum which acted on spermatozoa solutions. Anti-human
haematoserum had however the same effect, quantitatively and quali-
tatively, on spermatozoa solutions, and vice versa. Meyer and Aschoff
(7, VII. 1902) found that injections of blood, spennatozoa, and tracheal
epithelium led to the formation of antisera which coagulated milk
solutions (1 : 40).

Obermayer and Pick (1902) studied the different constituents of
egg-white, finding the albumin to contain : a globulin, crystallizable
albumin, a non-crystallizable albumin-constituent (conalbumin), and
ovimucoid, also other bodies upon which they will report later. The
egg-globulin of other authors they claim to have separated into four
different constituents : ovimucin, dysglobulin (both insoluble in water),
euglobulin and pseudoglobulin (both soluble in water). These bodies,
purified by washing, were injected severally into rabbits and the various
antisera tested upon the different constituents of egg-white. Re-
peatedly crystallized egg-albumin (method of Hoffmeister) did not lead
to the formation of antiserum. The result of the tests was that all the
constituents of egg-white gave a precipitum, proving that the antisera
were not specific for the different constituents. They were surprised to
find that a substance might not lead to the formation of a precipitin
which acted upon that substance, but to a precipitin which acted on
some other body in egg-albumin.

Effects on different Albnmins 107

Hamburger (cited by Aschoff, 1902, p. 193) obtained precipitins for
casein and albumin of cows' milk, stating that he was able to differentiate
the one from the other, although both antisera precipitated ox serum,
whereas ox haematoserum did not affect milk.

Michaelis and Oppenheimer (1902, p. 343), working with anti-ox
serum, euglobulin and pseudoglobulin, found these antisera to act upon
globulins but not upon albumin. Both acted more upon pseudoglobulin
than upon euglobulin, the action of anti-euglobulin being markedly stronger
with euglobulin than that of anti-pseudoglobulin. Anti-ox haematoserum
acted on ox serum-globulin, pseudoglobulin, somewhat less on euglobulin,
slightly or not at all on serum-albumin. They consider (p. 345) these
antisera not " specific " for each albumin of the same animal, although
they certainly react with some more than with others. They conclude
therefore that the specific " combining groups " are not possessed by
one form of albuminous molecule, but that they are common to
chemically related albumins. The precipitin reactions are therefore
of no use for the qualitative chemical separation of the different
albumins of the same animal.

Rostoski (1902, a.) treated rabbits with horse serum, as also with
horse serum-globulin, euglobulin, pseudoglobulin and serum-albumin,
but found that all the antisera reacted with the different serum
constituents above named. As I have stated elsewhere, this might
be ascribed to his using milky antisera (see p. 73) which unfortunately
robs his results of their value. A rabbit treated with Bence-Jones
albumins gave an antiserum which acted upon these, but also upon
human serum, serum-albumin and globulin, but not with the serum or
serum derivatives of other animals. Like the preceding authors he
concludes that precipitins afford no aid in distinguishing the albumins
of the same animal. In a subsequent paper Rostoski (1902, b., p. 21)
sums up the results of Leblanc and of Hamburger and points out their
obvious contradictions. These are evident from what I have stated
above.

Umber (14, vii. 1902) on the whole confinns the results of
Obermayer and Pick, Michaelis, and Rostoski. He treated rabbits
with egg-albumin and globulin solutions and tested egg-white and
the homologous substances with the antisera produced. Separating
fibrinogen, globulin and albumin from the antisera, he dissolved them
in saline and tested them upon the egg-white constituents named.
Anti-globulin and anti-albumin serum precipitated dilutions of egg-
white, of globulins, but not of crystallized egg-albumin. The separated

108 The Nature of Precipitin Reactions

constituents of the antisera were next tested, and it was found that a
solution of the fibrinogen fraction, and still more of the globulin fraction,
precipitated the same constituents as the preceding, but no precipitin
was contained in the albumin fraction. The precipitin was therefore
precipitated with globulin, but it is not certain whether it was simply
carried down, or whether it is a globulin itself Umber also concludes
that the precipitins do not afford a means of differentiating the albumins
of the same animal.

Oppenheimer and Michaelis (18, VII. 1902) treated rabbits with ox
serum-albumin and found their antiserum to precipitate serum-albumin,
having less effect upon pseudoglobulin, none on euglobulin, nor on
horse serum-globulin. Globulin-treated rabbits formed precipitin for
globulin alone. Landsteiner and Calvo (18, vii. 1902, p. 782) treated
rabbits with different components of horse serum, the fractions containing
a, fibrinoglobulin and euglobulin, \>, pseudoglobulin, c, albumin. The
antisera for fraction a precipitated dilutions of all three fractions, the
intensity of reaction being in the order «, h, c, being weak in the last.
The antisera for fraction h acted similarly, but more on dilutions of h
than of a. The antisera for fraction c had no effect in a first experiment,
but did have in a second, precipitating both solutions of c and of
globulins where the fraction had been reprecipitated. The behaviour
of globulins to precipitins was inconstant. The authors conclude that
the precipitable substances in serum represent several bodies with
different reactions as regards precipitation. The precipitable sub-
stances do not act in accordance with known albuminous bodies in
serum, in other words, there is no reason to identify the precipitable
substance with globulin. Ide (27, vii. 1902, p. 266) injected ox
haemoglobin into rabbits, and in confirmation of his puf)il Leblanc
found the antiserum to precipitate ox haemoglobin solutions, the anti-
serum also haemolyzing ox corpuscles. Ascoli (26, viil. 1902) treated
rabbits with fibrino-, eu-, and pseudoglobulin, sei-um-albumin, and
normal serum. Following a recommendation of Arthus he used S'/o
sodium fluoride solution with which to prevent bacterial development
in his serum dilutions, which had to be kept at 37° C. during his
experiments. He reaches the conclusion that there are qualitative
differences in eu- and pseudoglobulin, and serum-albumin solutions
made evident by the biological test, and that consequently an anti-
serum produced through injection of normal serum contains different
precipitins, each of which seizes upon different components in normal
serum when it acts thereon.

Effects on different Albumins 109

Michaelis (9, x. 1902, p. 735) injected horse and ox serum-albumin
into rabbits, and found the antisera to precipitate both serum-albumin
and globulin. Contrary to Nolf he found, possibly owing to his having
used different methods, that serum-albumin injections lead to the
formation of precipitins which acted on serum-albumin. Gengou
(25, X. 1902, p. 746), experimenting in another manner (see Immune
Bodies in precipitating Antisera, p. 94) and using antisera obtained
from rabbits treated with dog serum, found that anti-dog serum acting
on dog euglobulin and serum-albumin led to precipitation and fixation
of complement. Lactoserum, acting on cow casein and lactoglobulin
did likewise, whereas lactalbumin was not precipitated and complement
remained free. This experiment, often repeated, he considers contradicts
the statement of Hamburger (see p. 107). Schiitze (6, xi. 1902, p. 805)
found haematosera to also precipitate dilutions of the homologous sper-
matozoa of different animals, the spermatozoa tested being those of
the horse, ox, sheep, pig, and man. Uhlenhuth (1901, p. 501) had
previously noted that haematosera also clouded spermatozoa solutions
of the same animal. Ziemke (15, ix. 1902) appears to have obtained
results which do not point to the specificity of the reaction.

Falloise (25, xi. 1902, p. 836) refere to the discrepancies in the
results of other workers as being possibly due to their different method
of treating their animals. Working with the sera of the ox and horse,
Falloise separated the globulin and albumin after the methods of
Leblanc and Nolf, whose results, as I have noted above, are not in
accord. The results obtained with anti-globulin and anti-albumin for
ox and horse confirmed each other, for Falloise found that. A, anti-horse
globulin precipitated normal horse serum and globulin solution, albumin
solutions being very slightly affected. It exerted a very slight action
on ox serum, and globulin, none on ox albumin. (Anti-ox serum
behaved in a corresponding manner upon ox and horse serum etc.)
B, Anti-horse serum-albumin produced slight reactions and deposit
in horee serum, in albumin and globulin solutions. With ox serum
and globulin solutions there was only a very slight trace of deposit
in 24 hrs., no effect on ox serum albumin solutions. (Anti-ox serum-
albumin behaved in a corresponding manner towards its homologous
serum, etc.)

Falloise would explain the failure of albumin to produce precipitin
in larger amount to the presence of but little globulin therein, attributing
the precipitin formation entirely to the action of globulin which it
contains in consequence of our imperfect method of purification. He

110 The Nature of Precipitin Reactions

agrees therefore with Nolf that serum-albumins do not lead to the
formation of precipitins.

Liepmann (18, xii. 1902) treated rabbits in the usual way with
placenta emulsion, obtaining antiserum which caused precipitation in
the presence of bits of placenta, and possessed slight haemolytic power.
Pieces of organs, including pieces of the uterus, and also blood, gave
no precipitation even after :| to 1 hour. This author's technique
is certainly open to criticism. Liepmann (29, i. '03, p. 81) further
reports that his antiserum for human placenta gives reactions with
foetal serum (obtained from the cord), reacting more with placental
substance, but giving no reaction with blood serum of a man and non-
gravid woman. He proposes to test the serum of gravid women, to
see if by means of the test he is able to demonstrate the presence of
placental substance (Veit) in their circulation. Notel (13, III. 1902)
following the method suggested by Uhlenhuth for the identification
of meats (see Section IX.) states that an antiserum for muscle-albumin
gave a greater reaction with muscle extract than did the corresponding
haematoserum. The statement requires confirmation, no other experi-
ments of a comparative character having as yet apparently been made.

Uhlenhuth (6, XII. '02) obtained an antiserum for the yolk of egg,
which precipitated yolk of egg dilutions, but did not act upon egg-
white dilutions, except when these were fairly concentrated, and even
then but slight action was observable after a considerable time. This
antiserum also clouded avian serum slightly. He concludes therefore
that the albuminous constituents of egg-white and yolk are different.
Egg-white, as we know, contains albumin and globulin, egg-yolk
containing vitelline, lecithine, and nuclein. He recommends this
antiserum for the detection of egg-yolk in the examination of foods.

Graham-Smith and Sanger (1903, p. 268) working in our laboratory
with my quantitative method {q. v.) obtained the following differences
in the amount of precipitum when anti-human serum was added to
different body fluids in 1 : 21 dilutions. Anti-human serum No. I. was
much more powerful than No. II:, which moreover had undergone
putrefaction.

Material Anti-human Anti-human from means Normal

Na I. No. II. of these two Anti-ox rabbit

1. Fresh human serum (2 days)

2. Old „ „ (8 months)

3. Placental serum (8 months)

4. Pleuritic exudate (2 weeks)

5. Hydrocele fluid (9 months)

6. Fluid from ovarian cyst )

(9 months) |

7. Amniotic fluid (9 months)

Anti-human
No. I.

Anti-human
No. II.

Percentages
from means
of these two

•0291 c.c.

•0197 C.c.

100 »/„

•0272 „

•0187 ,,

93-8 „

•0150 ,,

•0112 „

5i-5 ,,

•0065 ,,

•0084 „

30^3 „

•0046 „

•0037 „

16-7 „

•0018 „

trace

6^1 „

•0009? „

trace

3-0 „ ?

Effects of Digestion on Antibodies 111

Effect of Tryptic and Peptic Digestion on Antibodies,
especially the Precipitins.

The resistance to tryptic digestion of the precipitin and agglutinin
of abrin, after the destruction of the albumen as indicated by chemical
reactions, has been observed by Hausmann', an observation which had
previously been made upon ricin by Jacoby. Landsteiner and Calvo
(18, VII. 1902, p. 786) found the precipi table substance in horse
serum-globulin solutions to give somewhat less precipitum after tryptic
digestion than before. They however succeeded in obtaining a pre-
cipitating antiserum from rabbits treated with ox serum which had
previously undergone tryptic digestion, after having been coagulated.
According to Obermayer and Pick, this resistance to tryptic digestion
contraindicates the active substance being of an albuminous nature.
Rostoski (1902, b. p. 60) found precipitins to resist tryptic digestion,
and he adds that Ringer (1902) has found the globulin molecule to
also resist. We have referred to the opinions regarding the connection
between the globulins and precipitins. Michaelis and Oppenheimer
(1902, p. 34), on the other hand, state that blood serum is only digested
with difficulty by trypsin, and that large quantities of it are required
to exert an action, this action being exerted slowly. They find that
a serum reacts to precipitins as long as it remains coagulable, but
that it does not react when trypsin has been in contact for a sufficient
length of time. Serum subjected sufficiently to the action of trypsin
is incapable of causing the formation of precipitins in animals treated
with such serum. The question evidently requires further investigation.
Destruction through peptic digestion. Leblanc (31, V. 1901, p. 361)
found precipitin, as also precipitable substances in sera, to be destroyed
by peptic digestion, as Dziergowski (1899) had previously done in the
case of diphtheria antitoxin. Obermayer and Pick (1902)'-' found the
precipitin-generating power of a serum destroyed by peptic digestion,
although large amounts of albumoses and peptones were present in
the treated solution. This agrees with what is stated below with
regard to the very doubtful properties of immunization possessed by
peptones. According to Jacoby (loc. ait.) peptic digestion destroys the
precipitable substance in egg-white, ricin, and in abrin. Michaelis
and Oppenheimer (1902, p. 34) also found that immunizing properties

' Hofmeister's Beitrdrif, ii. p. 131, cited by Landsteiner and Calvo, p. 783.
' See also Oppenheimer and Michaelis (18, vii. 1902).

112 The Nature of Precipitin Reactions

were lost in a serum subjected to peptic digestion, and the precipi table
substance in a serum, which during the first stages of digestion reacts
slightly, after a time gives no reaction to precipitins. They found a
stage in the action of pepsin when the serum remained coagulable but
not precipitable. Rostoski (1902, h. p. 60) found the precipitins to
be destroyed by peptic digestion. Michaelis (9, X. 1902, p. 735) found
that pepsin and normal HCl, taken separately, exerted no action, but
when they acted in conjunction for 1 hour, all the precipitin or pre-
cipitable substance in a serum had disappeared. The evidence there-
fore with regard to the action of peptic digestion is unanimous.

Regarding the supposed Precipitins for Peptones.

Contrary to Tchistovitch (v. 1899), whose experiments will be
referred to presently, Myers (14, vii. 1900) claimed to have produced
precipitins for peptone. He treated rabbits with solutions of Witte's
peptone and obtained what appeared to be an antiserum which pre-
cipitated homologous peptone solutions. A curious statement of Myers
is that the antiserum lost some of its precipitating power after being
heated to 56° C, this being contrary to what has been observed by
others regarding the resistance of precipitins to heat (see p. 114).
He moreover stated that the heated serum could be markedly reactivated
through the addition thereto of fresh normal rabbit serum, although
the latter alone had no such effect. Other observers, however, are
unanimous in finding that precipitins cannot be reactivated in this
manner (see p. 94). Myers found that heating antisera for ox and
.sheep globulin did not have the same efi"ect as upon the " antipeptone "
serum. The only author who appears to have confirmed Myers' observa-
tion is Schiitze (von Leyden's Festschrift, 1902, cited by this author in
his paper of 6, XI. 1902, p. 804) who claims to have been able to
distinguish different peptones, for instance, those obtained from the
muscles of man and ox. By treating a rabbit with human peptone
(from muscle) he states that he obtained an antiserum which gave a
reaction with human peptone-containing urine, derived from a patient
suffering from carcinoma of the peritoneum, no reaction being given
with normal rabbit serum, nor with anti-ox peptone. The latter, however,
gave a reaction with the stomach-washings of the same patient above
referred to after a meal of beef

As stated above, Tchistovitch was unable to obtain precipitins for
peptone, after injecting 10 "/o solutions in doses of 5 c.c. repeatedly into

Influence of Temperature 113

rabbits. Buchner and Geret (16, Vll. 1901) stimulated by the results
of Myers, treated rabbits with peptone prepared according to KUhne.
Although the peptone possessed a considerable toxicity, they succeeded
in immunifying the rabbits, obtaining an "antiserum" which caused
a precipitum in peptone solutions, the precipitum being composed of
crystalline bodies or " globulites." On further investigation (8, Vill. 1901)
they were surprised to find that these globulites consisted of barium
sulphate, the barium being derived from the peptone they had used.
When barium was excluded no reactions occurred. Briefly, they
obtained no antibody for peptone. Michaelis (9, X. 1902, p. 736)
repeated Myers' experiment, using Merck's egg-peptone, and " peptonum
siccum Riedel," both barium-free. He also obtained no antibodies for
these. On the other hand, I have noted elsewhere (under Phyto-
precipitins. Section VI.) that Kowarski has claimed to obtain precipitins
for plant albumose. Michaelis found but few of his rabbits to survive
the toxic effects of the peptones he injected. He concludes that egg-
peptone no longer possesses the side-chains which are present in the
egg-white molecule, giving rise in the latter case to the formation of
precipitins. The side-chains are destroyed through peptic digestion.
Obermayer and Pick (1902) also repeated Myers' experiment, treating
rabbits with Witte's peptone, finding that it produced little or no
precipitin when injected. The effects of peptic digestion upon pre-
cipitins and precipitable substances has been considered on p. 111.

It would appear from the foregoing, that Myers and Schtitze are
mistaken with regard to the nature of the "precipitins" they found.

I might add here that Klein (17, vii. 1902) treated rabbits
intraperitoneally with injections of starch, glycogen (from fowl and
rabbit), grape-sugar, gum, and gelatin but did not observe the formation
of precipitins.

The Influence of Temperature upon the Reaction.

The influence of temperature upon the precipitin reaction is well-
marked. Myers (14, Vil. 1900) stated that it took place more rapidly
at 37° than at room temperature. This was subsequently confirmed by
Wassermann and Schutze (18, ii. 1901), Michaelis (9, x. 1902, p. 734),
and Stockis (v. 1901), the latter stating that 40— 42°C. are most
favourable. Biondi (1902, p. 16) found the temperature to exert a
distinct influence. Linnossier and Lemoine (1902) found reactions to
occur at temperatures ranging from 0 to 58° C, the amount of pre-

K. 8

114 The Nature of Precipitm Reactions

cipitum not being materially altered, although deposits formed badly
at 0°. Reactions appeared to occur most rapidly at 85°. Working with
bacterioprecipitins Kraus (1897) kept his tubes at 37°, and Nicolle
(1898, p. 162) and Radziewsky (1900, p. 434) found this the best
temperature for the reaction. Kister and Wolff (18, xi. 1902) state
however that there is no special difference in the reaction at room
temperature and at 37° C, this being contrary to the experience of
all other workers.

Observations made in this laboratory by Mr Strangeways have
finally disposed of the question. Blood dilutions and antisera were
placed at 37°, at about 12°, and in the ice-chest at about 5°C. prior
to being mixed. After having attained the temperature of their
surroundings, the antisera were added to the blood solutions, and the
time noted when reaction took place, and it was found that a low
temperature markedly retarded the reaction, although it had no influence
upon the amount of precipitum as measured by my volumetric method.

The statement therefore of Kister and Wolff is wrong. It can
doubtless be explained in a measure by the fact that in routine work
with powerful antisera, the reaction begins almost immediately even at
low temperatures.

The effect of Heat upon Precipitating Antisera.

The following table contains the results of experiments by different
observers with regard to the effects of various temperatures upon
antisera obtained from rabbits. The antisera were tested, after heating,
upon their homologous bloods with which they had previously given
reactions. Some authors state the time of exposure, others not.

Linossier and Lemoine (21, ill. 1902) state that their anti-horse
serum, which had acted on horse blood in the dilution 3 : 100 before
heating, was exposed for 48 hours at a temperature of 60° C. At the
end of that time a coagulum had formed, and on separating the clear
fluid therefrom it was found to cloud a 10 : 100 horse blood dilution.
Diluted five times in saline, the antiserum showed a still greater
resistance to heat, for after 48 hours at 60° it showed no loss of strength.
After four days at 60° it still precipitated a 10 : 100 solution.

It will be seen that the Bacterioprecipitins are much more readily
destroyed by heat than are the others, a fact which has been brought
forward as a reason for considering them antibodies of another nature.

Effect of Heat on Precipitins

115

Temperature centiurade

to which it was

exposed

Kind of Antiserum

Kemarks

Authority

Destroyed at

Besisted

Haematoserum

70°

_„

Tohistovitch, 1899

If

70°

65°

though weakened in
power

Bordet, 1899

91

—

60°

no effect apparent

Rostoski, 1902 (6)

l>

—

60°

J hr., still effective

Obermaver and
Pick, 1902

»l

65° in 24 hrs.

60°

48 hrs., weakened

Linossier and Le-
moine, 1902

t>

72°

—

—

Eisenberg, 1902

»»

68°, 2 hrs.*

52°

no effect

Miehaelis, 1902

Anti-e?g albumin

—

60°

1 hr. , scarcely aff eated

Uhlenhuth, 1900

Anti-urinet

—

58°

2 hrs., still effective

Leelainche and
Valine, 1901

IiBotoserum

—

56°

" resisted "

Moro, 1901

Bacterioserum

58°

—

Krans and v. Pir-
quet, 1902

H

58—60°

—

destroyed in J to | hr.

Pick, m. 1902

* " Almost quite destroyed."

t Babbit treated with human albuminous urine.

It will be seen from the above that all observers agree in finding
that a temperature of 60° C. does not destroy the efficacy of haematosera.
In the absence of detailed observations regarding the effects of tem-
perature, Dr Graham-Smith (29, vii. '03, p. 354) carried out some
experiments at my suggestion by means of my quantitative method, his
results being as follows :

" In order to determine quantitatively the effects of heating on the
precipitum-forming property, specimens of antisera were heated iu
small sealed capillar}' tubes attached to the side of a thermometer in
a water-bath.

Specimens of anti-ox serum were heated for 5 minutes each, and of
anti-sheep serum for 1"5 minutes, at the temperatures given in the
following table. Suljsequently "1 c.c. of each sample was added to "5 c.c.
of a 1 : 21 dilution of its homologous blood, and the resulting precipitum
measured quantitatively.

After the process of heating, no visible change was noticed in the
anti-ox serum till a temperature of 65° C. was reached, when the fluid
became slightly opalescent. At 70" C. this opalescence was very marked,
and at 75° C. the serun» became gray, opaque, aud solid. In the case of

a-2

116

The Nature of Precipitin Reactions

the anti-sheep serum slight opalescence was noticed at 66° C, which
became more pronounced at 68° C. The following table shows that a
marked reduction in the precipitum-forming power coincided with the
visible change.

When the slightly opalescent antiserum was added to a serum
dilution a slight cloudiness appeared throughout the fluid. The more
markedly opalescent serum differentiated itself as it settled to the
bottom of the tube as a very definite cloud. After shaking the tube
the fluid appeared cloudy throughout, but remained in this condition, no
precipitum settling to the bottom.

The precipitum settled most quickly in the unheated specimens, and
the rate of formation of precipitum decreased as the temperature, to
which the antiserum had been exposed, increased.

Up to 60° C. no change in the precipitum-forming power was found
in either the anti-ox or anti-sheep sera, and both gave no trace of
precipitum when heated beyond 67° C. Between 60° 0. and 67° C. the
quantity produced in each case was diminished. The figures given are
the mean of two estimations in each case."

Anti-ox (heated for 5 minutes)

Anti-sheep (heated for 1-5 minutes)

Temp.

Precipitum

Percentagt

Remarks

Precipitum

Percentage

Remarks

87° C.

•0234

100

•0075

100

40

•0234

J,

4S

•0234

»1

60

•0234

JJ

SS

•0234

tt

•0075

100

ee

•0075

67

•0075

08

•0075

69

•0075

60

•0284

100

•0075

61

•0056

74

62

•0056

,,

68
64

•0065
•0037

83
49

66

•0187

79

Slight opalescence

trace

?

66

tt

?

Slight opalescence

67

•0108

42

It

If

0

tt tt

68

0

Marked „

69

0

tt ti

70

•

0

Marked

opalescence

0

tt It

76

4

0

Opaque,

solid

Effect of Heat on Precipitable Svhstance

The effect of Heat upon the Precipitable Substance.

117

Temperature centigrade to
which it was exposed

Substance heated

Remarlcs

Authority

Destroyed at

Resisted

Eel Berum

80°

58°

but gave less reaction

Tchistovitch, 1899

Fowl egg-white

—

56°

J hr., not appreciably
affected

Myers, 1900

Ox and Sheep serum
globulin sols.

—

1>

)l *f

»»

Fowl serum

—

70°

4hr.

Bordet, 1899

Human albuminous

—

58°

2 hrs.

Leclainche and

urine

Valine, 1901

Milk

100°, i hr.

—

no reaction

Schiitze

,,

—

100°

i hr., still reacted

Miiller

Egg-white dil.

78°, 1— li hr.

—

no reaction

Eisenberg, 1902

Serum dil. 1 : 100

100°, 5 min.

55°

4 hr., no effect

Nuttall, 1901

Sernm dil. 1 : 10

65°, 24 hrs.

60°

4 days, no effect

Linossier and Le-
moine, 190a

Linossier and Lemoine (21, iii. 1902) state that a serum dilution
containing so little albumin that it will not coagulate on boiling, has to
be boiled several minutes to destroy its precipitable substance.

The few observations noted in the preceding table made it appear
that the precipitable substances in normal serum possess about the
same resisting power as the precipitins. The results with milk are in
flat contradiction, and probably due to error on the one or the other
side (see under Lactosera). Whereas some observers exposed undiluted
serum, others exposed diluted serum to the different temperatures, con-
sequently the results cannot be compared.

The following experiments were carried out at my suggestion by
Dr Graham-Smith (29, vii. '03, p. 355).

" The heating of specimens of undiluted ox serum (1 c.e. for 3
minutes), was carried out in the same manner as described for antisera.
Subsequently 1 : 21 dilutions in salt solution were made, and tested
with anti-ox serum.

No visible change in the serum was noticed till a temperature of
56" C. was reached, when the serum became slightly opalescent. This
opalescence increased between 63 — 67° C, and was still further marked
at6S°C. All these specimens gave slightly cloudy solutions. At70°C.
the serum became very opaque, and at 75° C. white and solid.

118

The Nature of Precipitin Reactions

The quantity of precipitum formed remained constant up to 50° C,
but from 55° C. to 62' C. a marked diminution was noticed. At 63' C.
a further reduction occurred, and at higher temperatures the formation
of precipitum ceased. All solutions gave a good foam-test

The figures given below are the mean of two estimations in each
case.

Normal undiluted ox serum heated /or 3 minutes.

Temp.

Predpitum

PercenUge

nheated

■0262

100

40° C.

•0262

>*

46

•0262

• t

to

•0263

n

U

•0225

85

»

■0225

^^

n

•0215

^^

S8

•0215

82

M

•0206

74

M

•0187

71

61

•0187

W

U

•0187

II

«3

•0122

46

M

•

0

«

0

6S

0

tn

0

68

0

6»

0

70

0

75

0

Slight opalescence

Increaaed opalescence

Marked

Opaqoe

and solid

These experiments, as far as they go, appear to indicate that an
anti-serum can be exposed to a greater degree of heat than its
corresponding serum without injury, and that the precipitum-producing
property is completely destroyed in the latter at a lower temperature."

The effects of filtration of Normal Sera through " Htone " filters.

" It has been already indicated that the substance of " stone " filters
when allowed to act on serum exerts some influence on the serum
exposed to it. In order to further test this point ox serum was filtered
through a new Berkefeld filter, and through a new clean (Jhamberland
filter. After a certain quantity of serum had filtered through it was
removed, and specimens from it diluted and tested. It was found that
in the former case the precipitum-forniing power was at first diminished.

Effects of Filtration, Putrefaction, etc. 119

but returned to the normal after 110 c.c. had been filtered. No change
was noticed during the passage of a further 300 c.c. through the filter.

In the latter case the precipitum-forming property diminished
rapidly and fairly uniformly as the filter became choked." (Graham-
Smith, 1903, p. 357.)

Ox serum.

Quantity altered
in C.C.

New BeAefeld alter

New clean Cha
Precipitum

mberland alter

Precipitum

Percentage

Peiventage

Unfiltered

•0281

100

•0281

100

10

•0187

66

90

•0210

74

SO

•0229

82

•0272

97

40

•0225

80

60

•0229

83

•0272

97

60

•0229

83

70

•0216

76

80

•0286

80

90

•0844

86

100

•0245

87

110

•0226

80

136

■0881

100

140

•0872

96

160

•0168

56

166

•0881

100

aoo

•0881

100

•0158

S6

260

•0881

100

•0114

40

S60

•0114

40

400

■0881

100

27)6 Precipitins and Precipitable Substances resist
Putrefaction and Desiccation.

I have already noted elsewhere that putrid antisera might
retain unimpaired precipitating power for their homologous bloods.
I have found this to be the case repeiitedly, having found a variety
of microorg;\nisms, moulds'. Bacilli and Cocci, present in such sera,
the latter at times emitting a very putrid odour. Similarly, putrid
bloods have been found to react to their homologous antisera both
by Uhlonhuth, NutUll (1901), and Biondi (1902), the latter incidentally
mentioning that he obtained positive reactions with human blood
ingested by fie^vs, bugs, and mosquitos. In my paper of 1, vii. 1901,
I stated that human blood which had undergone putrefaction for two

> Conlirmeii by Bioudi (1902, p. 17).

120 The Nature of Precipitin Reactions

months still gave excellent reactions with its antiserum but not with
other antisera, a fact independently observed by Uhlenhuth (25, vi. 1901).

In the same paper I stated that I had preserved antiserum 42 days
dried upon filter paper, a fact which has been also noted by Corin, and
Stockis (V. 1901) as cited by Ziemke (17, x. 1901, p. 732). These
authors have kept dried antisera (globulin) for two months and found
it still active. Ziemke states that he kept dried antiserum for three
months but found that it had then lost in power. Biondi (1902) has
also preserved dried antisera for several months.

The fact that dried bloods give reactions after the lapse of a
considerable time, months or even years, has been fully established by
Uhlenhuth, and confirmed by others. It is true nevertheless that the
serum becomes insoluble after a time, the length of which appears to
vary considerably. It is doubtless for this reason that old dried bloods
not infrequently give very feeble reactions.

Ziemke (1901) obtained negative results with blood stains 25 years
old. Biondi (1902) obtained reactions with human blood stains which
had been dried 10 — 15, but not with those dried 20, years; Modica (1901)
on the other hand claims to have obtained a reaction with blood
dried 25 years. Uhlenhuth (5, vi. and 25, vii. 1901) obtained reactions
within 1 minute with bloods dried 6 to 12 years. I found (1901) that
blood dried and kept at 37° C. in the dark for 42 days and blood
hung for 6 months exposed to the air in the laboratory still gave
reactions. Dried blood exposed for 30 minutes to a temperature of
100° C. remained unaffected. Ferrai (1901) and also Biondi (1902, p. 30)
have found dried blood acted in the way that I have stated. They
found however that reactions did not take place after it had been
exposed to 130° C. for 1 hour, to 150° for 10 minutes, or to 160° for
5 — 10 minutes.

Graham-Smith and Sanger (1903, p. 274) have also studied the
influence of putrefaction on sera and antisera.

"Following a suggestion of Dr Nuttall's, in order to determine
the influence of specific bacteria on serum, 1 in 21 dilutions in salt
solution of ox and horse serum were inoculated with a series of
organisms. Undiluted human pleuritic exudate was similarly treated.
All were incubated for 5 days at 37° C. and then left at room
temperature for 36, 50, and 40 days respectively ; but the horse serum
was allowed to undergo natural putrefaction also for the last 10 days.
With the exception of the putrefactive bacteria none gave rise to very
considerable growth, and in nearly all cases by the time of examination

Putrid Antisera and Sera 121

the organisms had sunk to the bottom, leaving the supernatant fluid
clear. When necessary the fluids were filtered through filter-paper.
All were slightly alkaline or neutral in reaction.

Human pleuritic exudate Oi aerum (1 : 21) Horse Berum {1 : 21)

(1— '1) (contaminated)

... „ . , Control Control

Anti- Control anti- Antl- normal

human % antiox Anti-ox % human bone % rabbit

cc. C.C. cc.

Control. No organisms -0234 100 — -0173 100 — -0572 100 —

Putrefactive ) j^^ J -0280 119-6 - -0140 80-9 - -0713 124-4 -

organism

No. II. -0280 119-6 — -0112 647 — -0525 91-7

No. III. 0280 119-6 — _ _ _ _ _

Streptococcus — — — -01 G3 94-2

^ organism^ J No- IV. -0215 91-8 - -0163 94-2 - -0666 116-4

B. anthracis -0-206 87-8 — -0150 86-7 — -0591 103-3

Hofmaim's bacillus -0187 87-8 — _ _ _ _ _

B. subtilis -0187 80-0 — _ _ _ _ _

B. typhosus -0187 80 — -0140 80 9 — -0657 114-8

B. diphtheriae -0187 80 — — — — -0670 117-1

^organism' 1 N°- '^- '^^^'^ ^ - '^^^ *8-7 - -0582 101-1

Staphylococcus albus -0187 80 — -0169 97-6 — -0754 111-5

B. coli _ _ _ -0131 75-7 — — —

V. of cholera _ _ _ -0112 64-7 — -0670 117'1

In considering the above table in detail it is seen that the effects of
various organisms on ox and human serum agree fairly closely with a
few exceptions. The most striking are the putrefactive organisms
I, II, and V. These differences may be due to the fact that growth in
nearly all cases was less marked in the undiluted human, than in the
diluted ox serum, the latter moreover was a year old and had been
preserved in sealed tubes after filtration through porcelain. The
effects on horse serum of the action of specific organisms combined
with general putrefaction for 10 days agree with those of putrefactive
organisms I, II, and III, on ox serum, in that the capacity for forming
precipitum is increased.

It appears then from the few quantitative experiments we have
made that the results of bacterial growth on sera differ, some reducing
the quantity of precipitum produced and others i-aising it, neither
action being however very marked. Such slight changes as do occur
do not alter the specific character of the reaction.

Experiments were also made on human and other sera which had
undergone natural putrefaction. Most of the materials had been

122

The Nature of Preclpitm Reactions

kept for some time and consequently show the combined results of age
and putrefaction.

Material
Fresh human serum (2 days)

Old
Putrid
Putrid
Putrid „
Putrid placental

(9 months)
(5 months)
(8 months)
(9 months)
(9 months)

Anti-human
No. I.

■0291 C.C.

•0272 „

•0262 „

•0150 „

■0131 ,,

•0150 „

Anti-human
No. II.

•0197 e.c.

•0187 „

■0169 „

■0140 „

■0150 „

■0112 ,,

Anti-ox

7. Ox serum (1 year old) mean of 8 exps. ■0233 c.c.

8. Ox serum, putrid ( „ „ ) mean of 3 ,, ■0233 ,,

Per-
centage

100

93^8

88 ■!

59^4

57-4

o3^7

100
100

Normal
Anti-ox rabbit

Anti-human

The above table shows that in some cases advanced natural
putrefaction seems to exert little influence, for although the precipitum
is decreased considerably in Nos. 4, 5, and 6, yet this is not the case in
Nos. 3 and 8. All the specimens had been putrefying for the time
given in each case. Though time may have influenced Nos. 4, 5, and 6,
it is more probable that organisms whose growth deleteriously affected
the serum were present.

Finally, from the few experiments we have done we are of the
opinion that putrefaction to almost any extent does not affect the
specific precipitum-formiug body.

Since blood dried in small quantities does not undergo putrefaction
to any appreciable extent this factor may be neglected in ordinary
medico-legal work.

Experiments quoted on p. 124 with contaminated anti-human and
anti- fowl's albumin sera likewise demonstrated that putrefaction in
sealed tubes does not affect the antibody in them, as has also been
found by Nuttall.

An experiment conducted on the same blood dilution with a normal
and a contaminated sample of the same antiserum gave as a mean of
four estimations in each case '0433 c.c. and '0436 c.c. of precipitum
respectively.

Moreover putrid (filtered) sera when injected produce, as several of
us have found, powerful and specific antisera, and Strangeways has
shown that the power of antisera made with similar doses of fresh and
putrid filtered sera is nearly identical."

Stability of Antisera and Sera 123

The Stability of Haematosera and Sera sealed in vitro.

In my paper of 21, xi. 1901, I stated that some of my antisera
had given good reactions after being sealed in a pure state for seven
months in vitro. Wassermann and Schiitze (18, II. 1901) had only kept
antisera on ice up to two weeks. They state that fresh antisera give
greater reactions. I have some antisera which are eflfective after
14 months of storage. I have preferred to keep them on ice, being under
the impression that they remain potent longer at low temperatures. In
the majority of cases antisera deteriorate markedly after 3 — 4 months.
My observations in this respect have been confirmed by Uhlenhuth
(25, IV. 1901), Rostoski (1902, b, p. 17)', and Linossier and Lemoine
(21, III. 1902) who have kept antisera for three months. Uhlenhuth how-
ever added 0'5 % carbolic acid to them, which I consider disadvantageous,
for the reason that carbolized antisera tend to cloud blood solutions to
which they are added, irrespective of their being homologous. Strube
(12, VI. 1902) preserved antisera, both pure and with 0"2 7o carbolic acid
for three months. Moro (31, x. 1901) found sealed antisera to give
reactions after several months, a fact also observed by Biondi (1902,
p. 17). Robin (20, xii. 1902) found antiserum preserved four weeks
to give a reaction in two hours whereas it gave a reaction in 30 minutes
at first ^.

The precipitable substance appears to be even more stable. In my
paper of v. 1901 1 stated that I had successfully immunified rabbits with
old antidiphtherial horse serum preserved two years and seven months in
the laboratory at room temperature. This serum was exposed through-
out that time to diffuse light and room temperature, preservation being
secured through trikresol. The serum has given reactions with anti-
horse serum after more than four years. Similarly I immunified rabbits
with human pleuritic exudate preserved for six months with chloroform
at room temperature, and this has also given reactions after being
preserved for over two years. Further observations with regai-d to
the durability of sealed fluid sera in this respect have since been made
in this laboratory by Graham-Smith and Sanger (1903, p. 273) who
report as follows :

" A few quantitative experiments quoted below made on fluid sera,
preserved by sealing in glass bulbs, indicate that such sera lose their

' Preserved with chloroform.

2 Eobin's antiseram was evidently weak at the start.

124

The Nature of Precipitin Reactions

strength to some extent, though differences exist in the rate at which
this occurs.

Anti-hnman
No. I.

Anti-human
No. II.

Per-
centage

Normal
Anti-ojt rabbit

Human sertim (1 week)

•0291 C.C.

•0197 C.C.

ioo»/<,

—

„ „ (9 months)

■0272 „
Anti-ox

•0187 „

93 „

Ox serum (mean of 8 exps., p. 264)

•0233 C.C.

100%

—

sealed 1 year

„ „ (mean of 3 exps.) sealed

•0239 „

102,,

—

2 years

Anti-Jowrs
egg. No. I.

Anti-fowl's
egg, No. 11.

Fowl's egg albumin (2 days)

•0254 C.C.

•0162 C.C.

lOOo/o

—

„ ,, (9 months) ...

•0160 „

•0112 „

67,.

—

(U „ ) ...

•0225 „

•0144 „

88,.

—

Antidiphtherial horse serum four years and six months old preserved
with trikresol was found to produce a good but somewhat flocculent
specific precipitum amounting to '0572 c.c.

In the above experiments anti-human serum No. I. was only a few
days old, whereas No. II. was three and a half months old, and was
moreover contaminated by bacterial growths. The first anti-fowl's egg
serum was quite fresh and the second three months old.

All sera of the same kind do not give with the same antiserum
identical precipita, nor even the sera of the same individual at different
times in some cases, consequently an accurate determination of the
influence of age is not possible. Our experiments however seem to
point to a slight decrease in strength ;is the result of age, the human
serum and fowl's albumin experiments showing a decrease of
precipitum of 7% and 12''/o after 9 and 14 months respectively. The
fowl's albumin kept for 9 months shows a decrease of 33 "/o^ It is,
however, by no means easy to get accurate dilutions of egg albumin,
and the relative weakness of the specimen may be due to this cause.

The two experiments just quoted also indicate that antisera lose
some of their power, but not to the extent that some observers have
stated. Some undoubtedly preserve their power of producing specific
reactions after the lapse of 12 months. Others lose this property more
rapidly, whilst some, as Nuttall has also found, become untrustworthy
after a time, giving cloudings with all sera.

In considering the general results of these tables it appears that in
the case of dried bloods time per se does not destroy their capacity

Stability of Antisera and Sera 125

for reacting with their own antisera. Judging from the control
experiments with recently dried bloods we should think that the period
between the addition of the antiserum and the formation of the cloud
was increased, and the magnitude of the cloud diminished.

Fluid sera appear to deteriorate at any rate in some cases by
keeping. It has been occasionally observed, however, in qualitative
tests that old sera appear to react better than fresh ones."

SECTION III.

OBSERVATIONS UPON THE PRECIPITIN AND PRECIPITABLE
SUBSTANCE IN CORPORE.

We have seen that precipitins appear in the serum of suitable
animals after a longer or shorter treatment with non-homologous
albuminous substances, serum, milk, bacterial filtrates etc., administered
by intraperitoneal, subcutaneous, or intravenous injections or by excessive
feeding. The increase in the amount of precipitin is gradual, as is the
case with other immune substances, a fact that can be readily deter-
mined by occasionally bleeding the animal and testing its serum upon
the blood with which it has been treated. Obermayer and Pick (1902)
noted occasional differences in this respect in animals treated with
different blood components, some of which produced no effect at first
but great effects in the later stages of treatment, whereas in other
cases the increase was gradual. In accordance with what has been
observed with regard to the antitoxin of diphtheria in the horse and
the goat, by Salomonsen and Madsen (iv. 1897) (see p. 9) and others,
the amount of precipitin present in the serum of the animal during
immunization falls after each injection of the precipitin-producing
substance, the fall being succeeded in due course by a rise. Curves
made by roughly estimating at frequent intervals the amount of
precipitin present show that during successful treatment precipitin is
gradually formed within the animal's body. No measurements of the
amount of precipitin during the growth of immunization have as yet
been made, which would correspond to those made upon antitoxin, but
it is safe to say that a corresponding undulation would be observed.
Numerous observers, besides myself have noted that it is best to wait
for a minimum of five days, usually a week or more, before bleeding
an animal, after the last injection it receives. The object of this is
to obtain the maximum amount of precipitin, by allowing a sufficient

Behaviour of Precipitins in the Body 127

time for an increase of precipitin after the fall which succeeded the
last injection. The maximum of the precipitin content usually appears
to be reached about eight days after the final injection.

Following upon a period which may last two weeks or longer, the
precipitin content gradually begins to fall, in animals which have
ceased to receive treatment. Thus Stnibe (12, vi. 1902), who studied
eight animals in this respect, found that they furnished for about a month
sufficient precipitin to produce a reaction in a blood dilution of 1 : 1000,
but that after eight weeks the precipitin had disappeared, and that
then it was possible to again treat the animals with the same blood,
and again obtain precipitin. Rostoski (1902, b, p. 39) found scarcely
a trace of precipitin in the sera of animals 13 weeks after the last
injection. The precipitins do not therefore appear to persist as long
in the body as do, for instance, the agglutinins.

In animals subjected to long-continued treatment, the precipitins
may be seen to gradually disappear, as was noted by Tchistovitch
(v. 1899). I have not found a similar observation recorded in publica-
tions by other writers, although I was able to confirm it a year ago
(Nuttall, 16, XII. 1901, p. 407) in some rabbits which I treated with
human serum, in the false hope of increasing the strength of the
antiserum they possessed. As I wrote at the time, " There is therefore
a point in the treatment of animals, for purposes of obtaining an
antiserum, when a maximum of power is reached, and the animal
should be bled."

That the precipitins are present in other body-fluids besides the
serum is indicated by an observation of Eisenberg's (v. 1902, p. 308)
who twice found precipitins in the humor aqueus of rabbits treated
with fowl-egg injections. As far back as 1888 I noted the existence of
normal bacteriolytic substances in the aqueous humour, and I might
add that other antibodies, e.g. typhoid agglutinins, have also been found
in this situation by Levy and Giesler. Eisenberg, and Moro, (31, X. 1901
in milk-treated rabbits), were unable to find any precipitins in the urine
of their immune animals. It would however be a matter of interest
to see if they appear in the urine of rabbits suffering fi'om albuminuria
following upon injection wth foreign albumins, even though there may
be but a moderate amount of precipitin in their blood. I propose to
investigate this.

The precipitins are transmitted to the offspring in utero, as was first
shown by Mertens (14, III. 1901) who examined the serum of one out of
three newborn rabbits for precipitins, the mother having been treated

128 Function and Regeneration of Pt'ecijntins

during pregnancy. This observation was subsequently confirmed by
Moro (31, X. 1901) with lactosera, and Biondi (1902, p. 15) with
haematosera.

The function of the precipitins in corpore is not as yet cleared up.
Presumably they serve to protect the body against the injurious effects
of corresponding foreign albumins, and more probably, as is suggested
by Michaelis and Oppenheimer (1902, p. 363) to neutralize the
specifically foreign character of the albumin introduced, thereby making
it forthwith assimilable. This may explain the physiological signifi-
cance of the phenomenon, and I might add, is in substantial agreement
with Ehrlich's theory as to the function of antibodies in general.

The regeneration of precipitins in the body after large bleedings
would appear improbable from the investigations of Rostoski. It will
be remembered that a regeneration of diphtheria antitoxin has been
observed in animals subjected to large and repeated bleedings (see
p. 10). Rostoski (1902, b. p. 35), in a similar manner, subjected his
rabbits to one large bleeding (drawing 55 to 64 c.c. from animals
weighing 2000 to 2300 g.), and tested their serum for precipitins nine days
or so later. He observed a marked decrease in the precipitins and con-
cludes that they are not regenerated. It appears to me that these
experiments should be repeated with the aid of my quantitative method.
Rostoski notes, however, that the relatively rapid disappearance of
precipitins from the bloods, after treatment has ceased, indicates that
precipitin production soon ceases, owing possibly to the elimination of
the foreign substance which has stimulated their production. He
nevertheless considers it possible that the anaemia following large
bleedings may also affect the activity of the sources of precipitin
formation.

The systemic reaction of treated animals is slight, if we except the
slight loss of weight which follows the first injections in properly treated
animals, by which I understand animals treated with graded doses
which do not injure or destroy them by intoxication. It is easy to
observe that a leucocytosis follows the injection of foreign substance,
which is usually most marked in immune animals (note immunity to
bacteria). As stated by Michaelis and Oppenheimer (1902, p. 356),
rabbits treated by intraperitoneal injection of serum (they used ox
serum) develope a leucocytosis due to multi-nuclear granular, and
mono-nuclear non-granular leucocytes. On examining the peritoneum of
animals receiving an injection of serum for the first time, nothing
abnormal is observed after absorption has taken place. On the other

Coexistence tvith Precipitable Substances 129

hand, Michaelis and Ojjpenhcimer note that immunified animals present
a different appearance if examined two to three days after an injection.
Here solid masses of albumin arc found, either free or attached to the
peritoneal surface, the masses, when viewed in section, showing an
outer zone, due to leucocytic infiltration (chiefly microphages, with
macrophages at periphery). The parietal and intestinal peritoneum
is covered by small tubercle-like nodules made up of nests of leucocytes'.
Intravenous injections, they found, produced a leucocytosis, due almost
exclusively to mono-nuclear elements, the leucocytosis being more marked
in immunified animals.

Considerable interest attaches to the observation that a precipitin
and precipitable substance may coexist in the serum of immunified
animals. It was purely by accident that Mr Hopkins (Reader in
Physiological Chemistry, Cambridge) and I had occasion to observe
this in 1901, in connection with rabbits which had been treated with
crystallized horse albumin, and I herewith append the protocol of the
experiment.

Three rabbits bled 29, vil. 1901.

Treatment : Intraperitoneal injection of crystallized horso albumin. First in-
jection 22, VI., last 20, vii. 1901, all lilcd nine days after last injection. Total amount
injected 50, 50 and 53 c.c. of solution (strength undetermined) respectively, in graded
doses of 5 c.c, rising to 10 c.c. for last three injections, the day intervals between
injections being 5, 4, 5, 5, 9. The weights of the rabbits were (in g.) :

At let injection

At last injection

Wlien kUled

I.

1G90

1730

1580

II.

1940

1980

1600

III.

1910

1780

1520

Rabbit I. bled from the ear vein four days after injection 5, showed presence
of precipitin for the solution with which treatment had been administered.

It is noticeable that the weights of all the animals had fallen very
considerably during the days preceding death, this being somewhat
unusual and the reason not clear. The animals were bled to death and
their sera collected in the usual way. When we came to test their
precipitating power, we found Sera I. and III. to give a large precipita-
tion, whereas Serum II. gave no reaction at all. Before this fact
was discovered, in order that a test en gros might be made. Serum II.

1 The authors found that injection of albumoses, cither subcutaneonsly or iutraperi-
toneally, only produced sterile abscesses at the point of inoculation, or strong peritoneal
adhesions. See further on the effects of blood injections in Metchnikoff, VImmuniU,
Paris, 1901.

9

130 Behaviour of Precipitins in the Body

and one of the others were poured together, when to our astonishment
a massive precipitation occurred. Fortunately small separate samples
of these sera had been preserved which enabled us to make further tests
to see if we could find a reason for this remarkable behaviour of the
antisera. On mixing the samples we found that

Sera I. and III. gave no reaction,
„ II. and I. gave marked reaction,
„ II. and III. gave powerful reaction.

Consequently it was due to some peculiarity in Serum II., and it
seemed to us that the only reasonable explanation to be found was
that some of the precipitable substance, viz. crystallized horse albumin,
was actually present in the rabbits' serum. The matter was not pursued
farther at the time, but subsequently I directed the attention of
Mr Strangeways in our Laboratory to this observation, and asked
him to mix various antisera for human blood which he had made,
especially those taken from rabbits which, through prolonged treatment,
had shown a decrease in the amount of precipitin. On adding a
certain anti-human serum to another, he also obtained a precipi-
tation, and this was interpreted in the same manner as before. In
the latter case, the antiserum which contained precipitin likewise
contained precipitable substance. It appears somewhat remarkable
that precipitation does not occur in such antisera on standing in sealed
tubes ; perhaps the explanation of the reaction which takes place with
a similar antiserum from another animal of the same species will be
found to depend upon an individual difference in the constitution of
the precipitin. It is however premature to draw further conclusions.
The matter certainly deserves further enquiry. Owing to stress of
other work, it has been impo.ssible for me hitherto to pursue the
question, but I hope to do so shortly. I have not published this
observation before for the reason that I hoped to have had more data
before doing so. The reason that I do so now is that similar observa-
tions have been made by others, as follows.

Obermayer and Pick (1902) find that when egg-white is injected
intraperitoneally into a rabbit which has precipitins for this substance
in its serum, both precipitin and egg-white may be present in its
blood, and, although this is the case, no precipitation occurs. Ascoli
(26, VIII. 1902) also found that precipitin and precipitable substance
may coexist in the serum of immunified animals. He thinks the
condition is similar to that which some claim to have observed in vitro.

Coexistence with Precipitable Substances 131

namely the coexistence of the interacting bodies in the supernatant
fluid after a precipitum hiis formed. Hamburger (6, XI. 1902, p. 1190)
has also found this to be the case; the egg albumin may, however,
be found ;is readily in rabbits which have received their first injection
as in those which are immune. The egg albumin appeared in their
sera two hours after injection, and disappeared after four days in both
immune and non-immune rabbits, there being at no time any apparent
difference in the quantity of egg albumin present in these animals.
Hamburger (p. 1191) considers that this observation makes it all the
more difficult to explain the disappearance of the albuminuria in
immune animals, although this may possibly be due to immunity
having been acquired by the kidney cells. The probabilities are
that the reaction between the precipitin and precipitable substance
constitutes but one of several reactions which are taking place in the
body, as to the nature of which we are ignorant. As Rostoski, and
Michaelis, and Opponheimer (1902, p. 363) state, all we know is that
precipitation does not appear to occur in corpore.

By analogy with what takes place in vitro upon the mixture
of precipitin and jj^ccipitable substance, precipitation should take
place in the body of an animal whose serum contains precipitin when
precipitable substance enters its circulation. If such a reaction took
place with anything like the rapidity with which it does in vitro, it
is needless to say that it would be fatal to the animal, leading to the
formation of thrombi, etc. The remarkable thing is that the animal
remains well. Rostoski (1902, b, p. 40) thinks that the absence of
precipitation in corpore may be due to three causes, (a) the strongly
alkaline reaction of its blood, (6) the large amount of albumin contained
in its serum (about 7 "/o), (c) and possibly to the presence of antipre-
cipitins.

Michaelis and Oppenheimer (1902, p. 363) draw attention to the
possible significance of the enormous leucocijtosis observable in immune
animals upon the injection of fresh precipitable substance, and they
consider that the leucocytes may possibly take up the precipitum the
moment it is formed. This would be scarcely demonstrable. They
do not think the leucocytes would take up the foreign albumin as such,
but they might take it up after it has been acted upon by the precipitin
which circulates in the plasma.

Hamburger (6, xi. 1902, p. 1191) believes that a combination is
effected in corpore, for the reason that the amount of precipitin
decreases after fresh egg-white injections. In support of the statement

9—3

132 Seat of Origin of Precipitins

I have made (p. 126) he found that a rabbit whose serum gave a dense
precipitum with a 1 : 500 dihition of egg-white before a fresh injection
of egg-white, only gave a faint clouding with a 1 : 200 dilution 24 hours
after another injection of egg-white. No deposit takes place in such
a serum when it is removed from the body. The solubility of a
precipitum in an excess of precipitable serum has been noted on
p. 89.

The seat of oi-igin of the precipitins is unknown. Michaelis and
Oppenheimer (1902, p. 356) followed up the clue which seemed to
be presented by the leucocytosis, (if one adopts the view of Metchni-
koff and his followers,) basing an analogy upon the origin they claim for
the cytolitic complements in the breaking up of leucocytes. Michaelis,
however, found precipitins circulating freely in the plasma, a fact which
does not lend support to the view that precipitins originate from
leucocytes.

Biondi (1902, p. 15) sought to determine in which organs of
humanized rabbits the precipitins were formed. Having bled the animals
to death, he washed out the vessels with salt solution, cut up various
organs (liver, spleen, kidney, lung, brain, lymph glands, bone-marrow,
thyroid gland) and extracted these in saline. All the organ-extracts
contained precipitin, which he attributes to the possibility of blood
having been retained in the washed organs ; nevertheless the retroperi-
toneal lymph glands appeared to contain more precipitin than did the
other organs.

Referring to the experiment in which Uhlenhuth succeeded in
obtaining precipitins for egg-white in the serum of a rabbit to which
egg-white had been administered per os, (an experiment which has
been confirmed for precipitins which act on blood, sec p. 53,) Michaelis
(9, X. 1902, p. 734) notes that Uhlenhuth's antiserum only became rich
in precipitin after the rabbit had received many eggs. This points
to the probability of the precipitin being formed in consequence of the
excess of egg-white introduced, leading to quantities of it escaping
digestion. In other words, he thinks it is the non-assimilated egg-white
which stimulates the formation of precipitin. He pertinently adds
that if this were not the case, the serum of human beings, for instance,
those using milk as a part of their ordinary diet, would contain pre-
cipitins for milk, whereas as a matter of fact he has not found this
to be the case.

I might add here that Hamburger (6, XI. 1902, p. 1190) observed
the rapid appearance of egg-white in the serum of a dog fed therewith.

Albuminuria in Rabbits 133

althougli no precipitins were formed in this animal. M. Ascoli (1903)
has in a similar manner been able to demonstrate the presence of
egg-white, and of substances derived from roast fowl in the lymph
of dogs fed therewith. He was unable to note a parallelism between
the amount of precipitable substance contained in the dog's lymph
and blood serum. The serum of human subjects fed on roast beef also
contained substances which were precipitated by an homologous anti-
serum for ox meat. Where precipitins were contained in the serum
of an animal thus fed, the amount of precipitin therein underwent
considerable oscillations when a corresponding food was given. The
experiments will be reported upon in extenso in a future paper.

The presence of albuminuria in rabbits treated with foreign albumin
is noted by Hamburger (G, xi. 1902). This is however an old observa-
tion, which has been made not only on animals but also on man, by
physiologists and others, in connection with the so-called physiological
albuminuria as the result of food rich in albumin. Hamburger, however,
has made very important observations with respect to the albuminuria
which is observed in the course of immunization with egg-white. He
found that the albuminuria disappeared during the process of successful
immunization. Moreover the earlier the albuminuria disappeared, Hie
sooner did the animal form a powerful antiserum.

In a rabbit in which the albuminuria disappeared after the third
injection of egg-white, an antiserum was obtained which reacted with
egg-white dilution of 1 : 200,000, whereas in the case of another rabbit
in which the albuminuria only disappeared after the sixth injection,
the antiserum only reacted with an egg-white dilution of 1 : 40,000.
The practical bearing of this observation is clear for those engaged
in the preparation of precipitating antisera, and without doubt Ham-
burger's discovery is of general interest, not only from its physiological
aspect, but also from the standpoint of immunity. The degree of
albuminuria may very well serve as a guide as to the grading of dosage
during such immunizations. And I do not doubt but that the method
would prove the need of grading the dosage much more carefully than
some observers have done. Some indeed have made no attempt at
gradation, but have injected 10 c.c. for example of a foreign serum,
I might say blindly, every time, during the whole course of treatment.

That the albuminuria in rabbits is not simply due to an escape
of egg-white, was proved by Hamburger through the use of two antisera,
anti-rabbit and anti-egg sera. These antisera gave reactions with both
albumins, when the coarser tests of boiling and nitric acid ceased

134 Precipitins injected into Rabbits

to act. Finally the traces of albumin demonstrated by means of
antisera disappeared, nevertheless egg-white persisted in the serum
of the immune rabbit.

The behaviour of precipitins injected into normal animals has been
the subject of investigation by Obermayer and Pick (1902). They
injected anti-egg serum intraperitoneally into rabbits, and noted its
appearance after 15 minutes in the rabbits' circulation, the quantity
undergoing a rapid increase, a fact which could be observed by removing
samples of blood from the animals' ear veins at stated intervals. Control
experiments with subcutaneous injections showed that the precipitins
appeared much more slowly in the blood when this mode of administra-
tion was employed. The authore conclude that the rapid diffusibility of
these substances argues against their being of an albuminoiis nature.

SECTION IV.

ON THE SPECIFICITY OF PRECIPITINS.

That no special attention had been paid to the reactions which may
take place in non-homologous bloods upon the addition of powerful
antisera, prior to my publications, would seem clear from a comment
upon one of my papers by Rostoski (1902, b, p. 18), who \vrites: "Die
Angaben von Nuttall, dass das Serum eines mit Menschenblut be-
handelten Kaninchcns auch cine schwache Trlibung in Blutlosungen
des Pferdes, des Ochsen und des Schafes hervorrufe, wiedersjiricht alien
andern Beobachtungen direkt" ! We shall see, however, that my
observations have since been confirmed by several investigators.

Ehrlich (22, in. 1900) appears to have been the first to attribute
"a rigidly sjjecific" character to the precipitins, basing his statement
upon the investigations of Bordet and Myers, and speaking of the
pi'ecipitins as " specific coagulines — which act only in a specific manner,
i.e. precipitate only the albuminous body injected." It was natural
that he was led to this conclusion in view of the results of the investi-
gations he cited, which, however, were based on but a very limited
number of tests upon non-homologous material. Michaelis (9, X. 1902,
p. 733) refers to Wassermann (1900, p. 501), saying he "fiihrte zuerst
grundlegend aus, dass die Reaktionen strong speeifisch fur die injicirte
Eiweissart sind," although on the next page (p. 734) he contradicts this
by saying that the specificity of the reaction is not " absolutely strict."
Wassermann and Schlitzc (2, VII. 1900), it will be remembered, stated
that ox-lactosenim, for instance, did not act on goat milk and vice versd,
whereas other observers since, notably Moro (31, x. 1901, see further
under Lactosera, p. 157), have found the contrary to be the case, doubt-
less because they used stronger antisera, or made their observations at
a later i)eriod, after milk and antiserum had been brought in contact.
Uhlenhuth (7, II. 1901) was apparently the firet to consider that it

136 On the Specificity of Precipitins

might be worth while to study the reaction given by the bloods of
related species, mentioning that he intended to see if there were
any similarity of reaction between the bloods of horse and donkey,
man and monkey. In his paper of 25, iv. 1901, he first demonstrated
reactions amongst a variety of species of egg-albumins which were
brought about by an antiserum for the egg-albumin of one species (see
further under Anti-egg Sera, Section VI), a fact already indiciited earlier
by Myers (14, vii. 1900), who found that the antiserum for fowl egg-white
acted upon both fowl, and to a lesser degree upon duck egg-white, and
that antisera for ox acted on sheep globulin solutions and vice versd.
I was able (11, v. 1901) to confirm this with corresponding antisera for
ox and sheep blood, showing that slighter reactions were produced
by anti-ox serum on sheep blood dilutions and vice versa. On the
other hand Wassermann and Schiitze (18, II. 1901) stated that the
action of anti-human serum was "strong specifisch," except with the
blood of a monkey, a fact independently established by Stern (see tests
with anti-human serum. Section VI). Uhlenhuth (25, VI. 1901) found
anti-sheep serum to precipitate sheep, goat and ox blood, anti-horse to pre-
cipitate horse and donkej' blood, anti-human to precipitate human and
monkey blood. In my paper of 1, vii. 1901, in view of the results then
obtained, I stated that the " precipitins are specific, although they may
produce a slight reaction with the sera of allied animals." //( view
of the very limited number of bloods examined by vwst authors, it seemed
to me altogether premature, as some had done, to make any broad
generalizations. For this reason, I began early in 1901 to collect
as many bloods as I could from all classes of animals. It was
as important to do this from the medico-legal as from the zoological
standpoint, and in presenting the results given in this book I am safe
in saying that they constitute the first scientific demonstration, on
general lines, of the specificity or relative specificity of precipitins.

Although I have been unable to consult the original paper by
Schirokich (21, VII. 1901), it would appear that he also noted the action
of antisera on certain non-homologous bloods. He put a time limit upOn
the reaction for the reason that he observed an opalescence, as he terms
it, after 4 — 5 houi-s, when he added anti-human serum to dilutions
of ox, goat, hog, horse, camel, cat, guinea-pig and rabbit sera, a
precipitum being formed after 24 hours. I do not know with what
dilutions he worked, but should think that they were concentrated, for
it does not seem impossible to me that some of the deposits may have
been due to matter suspended in the antiserum itself, which in the

Blood Relationship 137

interval of time had been able to settle. I have observed such deposits
when antisera were not clear, although not of the opalescent kind (see
p. 72). That oven rabbit blood gave a precipitum indicates possible
errors in his technique.

Uhlenhuth (25, vii. 1901) agrees with me in finding that the
zoological relationships between animals are best demonstrated by means
of powerful antisera. He judged from reactions with such antisera,
that the ox is not so closely allied to the sheep, as the sheep is to the
goat. He found that weak anti-sheep serum produced no reaction
in ox blood. In my paper of 21, xi. 1901, 1 wrote "The more powerful
the antiserum obtained the greater is its sphere of action upon the bloods
of related species. For instance, a weak anti-human serum' produced
no reaction with the blood of the Hapalidae, whereas a powerful anti-
serum did produce a reaction, and proved what I may be permitted
to call the ' blood relationship ' in the absence of a better expression."
This generalization was based upon data, previously published, with
regard to the general action apparently possessed by anti-ungulate sera
upon certain ungulate bloods (see anti-ox and anti-sheep blood tests, etc.),
as well as on the bloods of Canidae and Primates. I also noted that
reactions took place "to a lesser extent, in the bloods of allied animals,
than in the homologous bloodl" This was of paramount importance,
the statement being based upon the examination, not of one or two
bloods, but of over 200. In a subsequent paper (16, XII. 1901, p. 408)
speaking of the reactions amongst the Primates, I wrote "If we accept
the degree of blood reaction as an index of the degree of blood-relationship
within the Anthropoidea, then we find that the Old World apes are
more closely allied to man than are the New World apes, and this is
exactly in accordance with the opinion expressed by Darwin." I cite
these earlier papers for the reason that I wish to make it clear that
my results have materially contributed to a modification of the views
held with regard to the specificity of these reactions.

I have already shown that a reaction may take place with even the
distantly related blood of the horse, very slight it is true, upon the
addition of anti-human serum. This was confirmed by Griinbaum
(18, 1. 1902, p. 143) working with another anti-primate serum, that for the

' Wlien a powerful antiserum is diluted, corresponding results may be obtained. See
pp. 71 and 142.

" Exceptions, notably in the case of ungulate blood (q. v.) have since been noted by
me and are recorded in this book. They may howcTer be due to the effects of disease on
the animals yielding the blood tested.

138 On the Specificity of Precijntins

chimpanzee, which he states " gave a slight but distinct tm-bidity after
a few hours with horse blood." In a paper which I published almost
simultaneously (20, i. 1902) I stated that when testing bloods it is
necessary " to put a time limit upon them. This may appear to be a
rather ai-bitrary proceeding. My time limit has usually been 5 minutes
at average temperatures in the laboratory. A powerful antiserum will
certainly have acted within that time upon its homologous blood-
dilution ; with powerful fresh antisera the reaction takes place almost
instantaneously. On the other hand, if u>e allow mixtures of antisera
and bloods to stand, a reaction takes place with non-homolof/ous bloods.
The results I have hitherto obtained tend however to prove that anti-
mammalian sera only produce these later reactions in mammalian bloods,
anti-avian sera similarly in avian sera alone."

Furthermore Linossier and Lemoine (8, ill. 1902) state that in view
of the unanimity amongst authors regarding the strictly specific
character of these reactions, they were surprised to find antisera not so
specific as had been supposed. They found that a precijntin may act on
a number of different bloods, although the degree of reaction vanes.
As a rule, they state that the precipitum obtained is least voluminous in
bloods of distantly related animals to the one whose blood has been used
for the production of the antiserum. Their method of testing with
concentrated sera is different from mine and is subject to criticism, as
will be seen by reference to pages 74 and 89. And this will account
for their obtaining a reaction even with fowl serum, as stated
below. They added anti-human, anti-hoi-se, and anti-ox serum in the
proportion of 10 volumes to 1 of the different sera tested, viz. those of
man, dog, ox, sheep, horse, pig, guinea-pig, fowl, and obtained a
precipitum in all of these bloods. With the exception of the fowl's
blood in which the reaction was minimal, and the guinea-pig's in
which it was feeble, all gave well-marked reactions. None of the
antisera acted on rabbit serum. They noted what appeared to be a
faint clouding in the serum of the eel. Judging from these results,
which are anomalous in some respects, it would appear desirable to
make a series of tests with a largo number of sera under the same
conditions. I have not as yet had an opportunity of doing so. Halban
and Landsteiner (25, iii. 1902, p. 475) state in a footnote, that
their haematosera (presumably anti-human) also acted to a very slight
degree on substances (serum and milk probably meant) of other not
closely related animals. Anti-human serum produced "spurweise
Pracipitation," that is, traces of precipitation, with horse, but not with

Blood Relationship 139

ox serum. Here again my observation was confirmed. Referring to
the specificity of the reaction, Lino.ssier and Lemoine {loc. cit ) remark
"lik oh. on a era voir une action sp^cifique, un examen attentif ne
permit de voir qu'ime action particulierement intense," which sums up
the question in few words.

Passing over my paper of 5, IV. 1902 (see below) in which I described
my method for measuring the degrees of reaction, by volumetric
estimations of the prccipitum formed, I shall mention some more
papers referring to the subject of these generalized reactions. Strube
(12, VI. 1902) confirms my observation that the stronger an antiserum
is the more powerfully does it act upon non-homologous bloods. He
thinks that the quantitative differences of reaction may be explained by
assuming that different species of blood do not possess an identical
constitution (which is a self-evident proposition) but a closely related
constitution, witli the result that the antiserum for one reacts but to a
limited extent upon the other, as in the case of the agglutinins.
Second!}', let us assume that the serum is composed of different
albumins (and there appears to be evidence of this) which we shall
style a, b, and c. Substance a is present in other animals, but h and c
are not. With the homologous antiseram a, b, and c are precipitated,
whereas in the non-homologous bloods only a is precipitated;. He offers
this in explanation of my "mammalian reaction," and we have no other at
present. Strube, I might add, makes no mention of a time limit, saying
only that " several hours " may be necessary for reaction to take place.
Test sera which take several hours to act would however be scarcely
desirable in practice. Whereas Strube found anti-human serum to
produce an equal, though slight, reaction in the bloods of the ox, sheep
and pig, Kister and Wolff (18, xi. 1902) claimed to have noted that
human blood is more strongly acted upon by anti-horee and anti-ox than
by anti-sheep or anti-pig sera, this observation being evidently due to
some experimental erroi-. They also note that differences are not well
observed in concentrated solutions, or when a too large proportion of
antiserum is added (note in this connection what I have said on
page 74). They note, as I have also observed, that clouds may
form in concentrated solutions, whereas flocculent precipitates will
form in more dilute solutions. Oppenheimer and Michaelis (18, VII.
1902), working with but a limited number of bloods, conclude that
precipitins are specific for a "bestinmite Thicrart." They evidently
overlooked the generalized reactions.

Ascoli (26, VIII. 1902) has also observed generalized reactions. He

140 On the Specificity of Precipitins

treated rabbits with the serum-globulins of man, horse, mule and sheep,
and found that the antisera acted on solutions of all these globulins,
only to a different degree. On the other hand he observed " scarcely any
quantitative differences" upon adding anti-horse or anti-mule sera to
both of the con-esponding globulin solutions. Much less, and quantita-
tively decreasing in the order named, were the reactions obtained with
similar solutions from the ox, sheep, and man, this again being in accord
with my results. That different substances are acted upon in homologous
and non-homologous sera (see Strube, quoted above) is clear, for as
Ascoli found, if non-homologous serum is added in excess after precipi-
tation has occurred, no more precipitum fomis, but on the addition of
homologous antiserum one obtains further precipitation. According to
Ascoli, for example, when anti-horse serum produces a large reaction with
horse blood and a small one with human blood, there are two possibilities
to be considered regarding the differences of reaction. Either the anti-
serum contains several (in this case two) precipitins acting on different
constituents of each sennn, the one being common to both horse and
man, and the other present only in the horse ; or the precipitin is simple,
there being less precipitable substance in the human serum. The latter
supposition is not borne out by experiment, for even when we add an
excess of anti-horse, the human serum continues to give less precipitum.

Uhlenhuth (11-18, ix. 1902, p. 661) dwells upon the fact that the
zoological relationship of animals is brought out by an antiserum ; he
however does not bring new facts to bear on the question.

Further details regarding the specificity of the precipitin reactions
are to be found under Tests with different antisera, in Section VI
which follows. As will be seen, my most generalized reactions amongst
the mammalia were obtained by a powerful anti-pig serum.

It is evident from the foregoing that there are many points of
similarity between the precipitins and the haemolysins. Ehrlich has
pointed out that the haemolysins of different species may possess
receptors which are identical but quantitatively different.

The Selective Action of Precipitins in Blood Mixtures.

In my paper of 1, VII. 1901, p. 384 I described some tests made
with a view of determining whether or no a mixture of several kinds of
blood in solution woukl prevent a reaction taking place upon the
addition of an antiserum which was effective when added to one of these
bloods when alone in solution. It was found that when two to six

Degrees of Reaction

141

different bloods were brought together into solution, so that each blood
in the mixture was diluted to about 1 : 500 or 1 : 600, the presence
of other bloods did not impede a reaction taking place between an
antiserum and its homologous blood in the mixture. The antisera only
acted however when a suitable blood was present in such a mixture.
(For details see original.) Ziemke (17, X. 1901) subsequently repeated
my experiment ' and confirmed it.

The measurement of Degrees of Reaction.

In my paper of 21, xi. 1901, p. 152, read before the Royal Society,
I mentioned that I had undertaken to make quantitative measurements
of the degrees of reaction obtained with precipitating antisera upon
different bloods. Owing to the labour involved in the qualitative,
and approximately quantitative tests here recorded, a report upon the
results obtained had to be deferred. I however described my method
in the following year (5, iv. 1902). I shall not dwell upon the method
here, but refer the reader to Section VII which follows. As stated in
the last paper referred to, the amount of precipitum, say 0"03 c.c,
obtained by adding 01 c.c. of an antiserum to 0'5 c.c. of its homologous
blood dilution (1 : 100 or 1 : 200) is taken as 100, and the reactions
given by non-homologous bloods are stated in percentages of that figure.
At the time I reported two sets of tests, which were, however, not to be
taken as final, as follows :

Anti-sheep

serum

Sheep

100

Ox

80

Antelope

50

Hog-deer

47

Eeindeer

30

Pig

20

Horse

16

Cat

13

Dog

7

Wallaby

5

Anti-pig serum

Pig

100

Horse

16

Hog-deer

14

Cat

U

Dog

13

Sheep

13

Wallaby

5

I only cite these results to show that there are measurable differences
in the degrees of reaction, as calculated upon the precipitum obtained
with different bloods, the precipitum being actually metisured volumet-
rically. The method I described has not as yet been tried by workers
outside our laboratory.

' Ziemke cites my paper but does not state this as well as some other particulars.

142 On the SpeciJicUij of Precipitins

All observers, including myself, who have worked with different
blood dilutions, have noted the fact that an antiserum will act upon
a higher dilution of homologous than of non-homologous blood. In view
of the labour involved, but very few figures have been obtained by this
method, employed comparatively. Linossier and Lemoine (8, in. 1902)
thus sought to express the reactions with various dilutions, as follows :

Antisera added in the proportion of 15 : 100 of dilutions.

Anti-ox serum gave reactions with Ox serum diluted 1 : 5000
„ „ Horse „ 1 : 300

„ „ Man „ 1 : 50

Reactions of "apparently equal intensity" were obtained when anti-
human serum was added to human serum diluted 1 : 1000, and to ox
serum diluted 1 : 20. In another similarly conducted test, anti-human
serum acted on human serum diluted 1 : 2500, but only on horse serum
diluted 1 : 20. As the authors state, these figures have no absolute
value, as they naturally will vary according to the antisera used being
different. Strube (12, vi. 1902) testing with anti-human serum,
obtained a reaction with human blood diluted 1 : 20,000, other bloods
reacting in dilutions of 1 : 100. He said the reactions might take place
after some hours, as already mentioned above, and he does not mention
anything with regard to the rate at which different bloods react in
a series, an omission made by most authors. I find the rate at which
a reaction takes jilace is a very fair index of the degree of relationship.
This rate would appear to be due chiefly to the differences in the
amount of matter precipitated; where this is slight, a reaction would
only be registered when the particles of precipitum form agglomerations
which are visible to the naked eye. On the other hand there may
be a slower reaction actually taking place, due to a lesser degree
of avidity between the combining substances, but to prove this will
be a matter of some difficulty. Experiments made by progressively
diluting the antiserum which is added to a blood dilution, have further-
more been reported by Ewing (ill. 1902, p. 14), following a suggestion
made by Uhlenhuth, and Kister and Wolff Ho does not state the
blood dilution used, but I presume it was 1 : 100. He found

Anti-ox serum diluted 1 : 5 to act on dilutions of "several bloods."

„ „ „ 1 : 30 „ „ „ „ bloods of ox and goat,

„ „ „ 1 : 50 „ „ „ „ ox blood alone.

Similar observations were made with anti-human serum tested upon
the bloods of man, and of the baboon, rhesus and Java monkeys. When

Degrees of Reaction 143

added to these bloods in solutions of equal strengths, the anti-human
seram in its highest dilution, only acted upon human blood dilutions.

The dilution-method has not been adopted in the study of blood-
relationships, and these are the only figures obtained by the method
which I have come across. The importance of making a series of
dilutions of a suspected blood in medico-legal work, is made parti-
cularly clear by the figures of Linossier and Lemoine, given above.

It is evident that one observer will record a reaction and another
not, when using the dilution-method without reference to time, and
undoubtedly faint cloudings will be completely overlooked if the fluids
are examined in an unsuitable light (see description of my apparatus,
p. 70 and Fig. 4). There is consequently a considerable subjective element
in the tests conducted by the dilution-method, which is absent from my
method of actual measurement.

Eisenberg (5, v. 1902, p. 290) considers a "unit of precipitablo
substance" to be that minimal quantity, which, when contained in
a given volume of fluid, suffices to produce a specific reaction. Thus,
when a given albuminous solution (dilution 1 : 1000) yields a precipitum,
then 1 c.c. thereof contains 1000 units of precipitable substance. The
"unit of precipitin," is that minimal quantity which just suffices to
produce a reaction in any albuminous solution. For example when an
antiserum still produces a precipitation in a 1 : 100 albumin dilution, he
would say it contained 100 precipitin units per c.c. It seems to me
until we know more about the possible existence of normal anti-
precipitins in sera, it is somewhat premature to attempt an exact
standardization either of the precij)itin or precipitable substance.
Nevertheless the method suggested may have its use, unless my method
of volumetric measurement is preferred. I have standardized my
antisera, simply by stating the amount of precipitum produced as may
be seen by my table of measurements quoted on p. 145. This is again
stated in most cases for the antisera with which my tests were made in
the following tables. It will be seen I express the " strength " of my
antisera simply in terms of jjrecipitum amounts.

To study the quantitative relations of the interacting bodies,
Eisenberg {loc. cit. p. 291) adds a constant amount of one substance to
a variable amount of the other, and, after reaction has taken place,
he determines the absolute as well as the relative absorption of precipitin.
As in agglutination experiments, the amount of absolute absorption
is defined in terms of the diff'erence between the number of precipitin-
units present in the fluid per unit of volume, before and afler reaction

144 On the Sjiecificity of Precipitins

has taken place. He expresses the proportion of absorbed substance
to the amount originally present in the form of a fraction.

The Delicacy of the Precipitin Test.

Whereas the ordinary chemical tests cease to give reactions in blood
dilutions of about 1 : 1000, powerful antisera greatly exceed this limit,
as the reported results of independent observers have shown. The tests
in each case were conducted with antisera added to their homologous
blood dilutions of the strengths indicated :

Strube (12, vi. 1902) anti-human serum gave reactions with a dilution of 1 : 20,000
Stern (1901) „ „ „ 1 : 50,000

Uhlciihuth (15, XI. 1900) anti-egg scrum „ „ 1 : 100,000

Ascoli (26, vni. 1902) „ „ „ 1 : 1,01X),000

Some tests conducted at my suggestion by Mr Strangeways, with
a view to determining the limit, have shown that reactions mjiy take
place even in dilutions of over 1 : 1,000,000, for on adding a constant
amount of antiserum to progressive dilutions, differences in the amoimt
of deposit, measurable to the eye, were observable even in these highest
dilutions, when the precipitum had been collected in fine capillary
tubes.

The Strength of Antisera.

Precipitating antisera have been termed powerful, or weak, in pro-
portion to their action upon various dilutions of their homologous bloods.
Most authors express the power of the antiserum by stating the blood
dilution upon which it will act. It is evident that the time and the
temperature at which reaction takes place, especially in high blood
dilutions, must be taken into account, but this has not been done as yet
with sufficient care to pennit of accurate standardization by this
method. My quantitative method would appear therefore to give more
accurate results, consequently I have used it throughout the greater
part of this investigation. The following figures give an idea of the
" power " of some of my antisera, which have all been standardized by
stating the amount of precipitum, measured volumetrically, obtained
by adding 0"1 c.c. of antiserum to 0"5 c.c. of a 1 : 100 dilution of its
homologous blood.

Differences in Individual Sera

145

Amo
Antiserum for

unt of pre
in c.c.

cipituni

Amoi
Antiserum for

int of preclpitum
in O.C.

Fowl's egg
Ostrich

•OG ^
•042

most

Dog and Mex. Deer
Zebra

•015
•012

Pig and Antelope
Fowl

•055
•035

powerful

Ox

Whale

•Oil
•009

- moderate

Hyaena
Turtle
Hedgehog
Sheep

•031
•03
•022
•02

■ powerful

Ourang and Horse

Wallaby

Seal

Llama

Reindeer

•008 J

•007

•006

•005

■004.

- weak

Measurements of -002 to '001 can still be made.
Keindeer were not used or standarJized.

Weaker antisora than that for the

On Differences in the Reactions of Individual Sera belonging
to the same Species of Animal.

In the investigations which I have made I have not studied the
question of individual differences in the reaction of normal sera to their
homologous precipitins. Together with Mr Strangeways, I have made
a number of measurements on normal sheep sera with anti-sheep serum,
and have obtained remarkably uniform results by means of my quanti-
tative method. These results will be published later. On the other
hand Mr Strangeways has observed a considerable amount of variation
especially in human subjects affected with various diseases. It is not
impossible that disease has in some cases been the cause of my obtaining
greater reactions than would be expected according to theory. I shall not
at present lay stress upon this point, but shall only, as an example, draw
attention to the very great reactions I obtained with two Cercopithecus
bloods (see Section VI) when testing with anti-human serum. Linossier
and Leinoine (8, iii. 1902) were unable to observe any individual differ-
ences in two human sera, the one being obtained from a case of pneumonia,
the other from a case of uraemia. One of these sera had been used for
the treatment of the rabbit which yielded the antiserum.

That there may be individual differences in health is indiaited by
the investigation of More (31, x. 1901) on lactosera (see p. 159), who found
that different women's milks reacted especially to the antisera to which
they had given rise. His results will however require confirmation. On
the other hand we have evidence of differences between matei-nal and
foetal blood in this respect'. Thus Halban and Landsteiner (25, in. 1902,

> Halban and Landsteiner (p. 473) cite Kriiger (1886) as having found foetal blood to
contain more solids but less fibrin than maternal blood. Scherenziss (1888) found the
specific gravity of foetal markedly lower than that of maternal blood, although it con-

N. 10

146 On the Sjiecificiti/ of Precijntins

p. 475) report that anti-human scrum gave reactions with higher dihitions
of adult serum than of foetal serum. On the other hand, foetal serum
gave more clouding in strong dilutions than did adult blood. Evidently
an excess of normal serum checks precipitation, and this seems to me to
be due possibly to normal antiprecipitins (see p. 149). It would appear
that normal maternal serum contains more precipitable substance, and
more antiprecipitin, than foetal serum. These differences however by
no means indicate that maternal serum contains more globulins, for
the precipitin reaction as we have seen (p. 98) does not constitute a
quantitative test for albumins.

Uhlenhuth (5, vii. 1902), using antiserum for fowl's egg-white (from
rabbit) found it to precipitate the serum of the hen much more than
that of the cock, the sera of sexually mature animals being used in both
cases for comparison. The reactions of the sera were so markedly
different, that he found it easy to distinguish the one from the other.

He therefore refers to a sexual reaction (Geschlechtreaktion), and
proposes to see if it is possible to distinguish between the mammalian
sexes in a similar manner.

On the Character of the Precipitins in different Species of Animals
treated luith the same Blood.

It has been accepted for the bacterial antitoxins obtained from
different animals that they are similarly constituted, whatever their
source. This would appear to be different for the precipitins, judging
from results obtained by Ascoli (26, viii. 1902), who, to begin with,
remarks that because two individuals, even of one species, yield sera
which react equally to their homologous precipitin, we have no right
to conclude that the sera of the two individuals are chemically identi-
cal, the reaction being perhaps insufficiently fine to demonstrate differ-
ences which actually exist. That he is right in saying this receives
confirmation from what has been observed with regard to the isopreci-

tained more salts. Doldris and Quiuquand (1893) made similar observations. Halban
(1900) found foetal blood corpuscles to be agglutinated by maternal serum, and vice versa,
whilst the sera showed differences in haemolytic and bacteriolytic power. Schumacher also
found maternal berum to agglutinate B. typhosus better than foetal serum. According to
Halban and Landsteiner (loc. cit.) maternal serum is more haemolytic than foetal, it ag-
glutinates red blood corpuscles more powerfully, it is more powerfully bacteriolytic (tested
on the cholera vibrio), it is more anti-fermentative (antitryptic), more antitoxic (as against
the haemagglutinina of abrin and ricin), and finally it yields moie precipitum with preci-
pitating antisera,

Precijntins from dif event Species 147

pitins, which would scarcely be formed unless there were chemical
differences between individuals (see p. 148).

Working with rabbits, Ascoli (p. 1411) found it impossible to
demonstrate any differences in the properties of the antisera they
produced, as tested against egg-white solutions with which they had
been immnnified. On the other hand, when he immiinified both rabbits
and guinea-pigs with the defibrinated blood of the same dog, he was
able to find a difference in the antisera they produced, for on adding
the antiserum, say from the rabbit, to dog blood dilutions, until no
more precipitation occurred, he found that he obtained a further
precipitation on adding antiserum from the guinea-pig. As an ad-
ditional proof of the antisera from the rabbit and guinea-pig being
different qualitatively, he states that

Anti-dog serum from rabbit gave a precipitum with normal guinea-pig'

but not with normal rabbit.
Anti-dog serum from guinea-pig gave a precipitum with normal rabbit

but not with normal guinea-pig.

Assuming that Ascoli is correct in this observation, then it is patent
that a multiplicity of precipitins are formed in the blood of an animal
treated with normal serum of another species. And, moi'eover, different
animals form different precijjitins when treated with the blood of the
same animal. According to Ascoli, the conclusion seems reasonable
(p. 1412) that different precipitins attach themselves to different side-
chains of the immensely complex " Rieseneiweissmolekiil," in the sense
of Ehrlich.

' Concentratiou not stated.

10-3

SECTION V.

ISOPRECIPITINS.

The occurrence of isoprecipitins analogous to isohaemolysins dis-
covered by Ehrlich and Morgenroth (see p. 42) appears to have been
observed by Schiitze (12, xii. 1901, and 6, xi. 1902, p. 804), who obtained
them by injecting animals with the scrum of other individuals belonging to
the same species. Schiitze treated a rabbit every two to three days with
5 to 10 c.c. of the serum of other rabbits, administered subcutaneously,
until a total of 60 c.c. had been given. The treated rabbit was bled
six days after the last injection, and its serum tested for isoprecipitin upon
the sera of 32 different rabbits. Of these 32 sera only two reacted,
precipitations occurring in 30 minutes at 37 ° C. The reaction was
" strictly specific," for, ui)on the addition of 2 to 4 c.c. of the treated
rabbit's serum to 3 c.c. of normal guinea-pig or human serum, no reaction
was observable after two hours under the same conditions of temperature.
Granted that the reaction took place as stated, it seems somewhat
premature to conclude as to the strictly specific character of the reaction
after only testing two other non-homologous bloods, even that of the
guinea-pig being but distantly related, not to mention the human blood.

Schiitze (p. 804) states that he found it more convenient to obtain
isoprecipitins from goats treated with goat's milk. To do this, he
injected 40 — 50 c.c. of the milk every 4 — 5 days, until a total of 400 c.c.
had been administered in the course of a month. Bled eight days after
the last injection, the goat yielded a serum which, when added in the
proportion of 0-.5 to 1 c.c. — 5 c.c. of a 1 : 40 dilution of goat's milk,
gave an immediate reaction.

Evidently then, there would appear to be individual differences in
the chemical constitution of different individuals, demonstrable by means
of isohaemolysins and isoprecipitins.

Antiprecipitins : Normal and Artificial 149

ANTIPBECIPITINS.

Normal Antiprecipitins. I have noted elsewhere (p. 145) that Halban
and Landsteiner (25, in. 1902, p. 475) observed a difference between
maternal and foetal serum with regard to their reactions to precipitins.
This difference appears to me to depend upon the possible existence of
noiTnal antiprecipitins, analogous to the normal antihaemolysins observed
by Besredka (1901) (see p. 22). Halban and Landsteiner found that
the addition of large quantities of human serum to its antiserum
prevented precipitation (but this might be explained in the light of
what I have stated elsewhere, viz., that a precipitum is soluble in an
excess of its homologous blood), whereas, additions of large quantities of
ox or horse serum were less effective, and dog, fowl, and fresh rabbit
serum did not prevent precipitation. Similarly, the authors I have cited
found that anti-ox serum acted least upon the addition of much ox
serum, more markedly upon adding the serum of the horse, most when
the sera of the dog, man, rabbit, and fowl were added. That is to say,
the latter did not impede the reaction. It is possible, it seems to me,
that this appearance may be due to normal antiprecipitin, or precipito-
lysin if I may so term it, which is present only in small quantity in
normal serum, and therefore only exerts an obvious effect when large
amounts of a serum are added to an homologous antiserum. (See
Observation III. p. 89.)

Artificial Antijjrecipitivs have been produced by Kraus and
Eisenberg ( If lereer klin. Wochenschr., 1901, p. 1191 ; see also 27, ii. 1902,
p. 212 — 213) tlirough treating goats with goat-lactoserum obtained from
rabbits which had been treated with goat's milk. They found that
antilactoserum prevented the precipitation of milk by lactoserum. In
addition, the antilactoserum contained antihaemolysin, which was
natural, for the goat lactoserum (obtained from rabbits) was haemolytic
for goat's red blood corpuscles.

Schlitze (2, xir. 1901) has also succeeded in obtaining antilactosera,
which, as stated above, are analogous to the antihaemolysins (see p. 21).
Schiitze's antiprecipitin retarded or prevented the precipitation of milk
by its homologous antiserum.

It remains to be determined if injections with precipitoids, which
have been compared to toxoids, will lead to the formation of antipreci-
pitins, as toxoids do of antitoxins, as discovered by Ehrlich (1900
Cro&nian Lect.). I propose to see if this is possible.

150 Normal Precqntlns

NOBMAL PBECIPITINS.

Noguchi (xi. 1902) and M. Ascoli (1903), report having found preci-
pitins in certain normal sera. I have noted elsewhere that normal sera
may contain antibodies, which are usually present in small amounts only.
This appears also to be the case with the normal precipitins. Ascoli was
stimulated to search for normal precipitins by a statement of Obermayer
and Pick (see p. 97), that they had found normal rabbit serum to preci-
pitate egg-white. Ascoli found ox serum to contain precipitins for the
sera of man, dog, pig, goat, rabbit, guinea-pig, fowl ; dog serum to contain
precipitins for the serum of the fowl, and for egg-white; goat serum
contained precipitins for fowl and guinea-pig serum. He judged of
the occurrence of a reaction by observing if any clouding occurred at
the zone of contact between a serum and serum dilution, made in
0"85 7o saline. He used small tubes 4 cm. long by 3 mm. wide, and
observed the reactions after the tubes had stood 30 — 60 minutes
at 38"^ C. By diluting the one or the other of the normal sera he
was able to note which contained normal precipitin, and in some
cases found both sera to contain it. As has been observed for other
normal antibodies, the amount of normal precipitin present varies
considerably in different animals of the same species.

Ascoli also states that he has found " autoprecipitins " in normal
human and animal sera, comparable to the auto-agglutinins found by
him and other observers.

The occurrence of the precipitins in normal sera may account for
some aberrant results obtained by various authors, but they do not
appear to affect my results in the slightest. Although Ascoli does
not state with what concentrations he worked, any more than do
Obermayer and Pick, it amounts to a certainty that but low dilutions,
and, in the latter case, even concentrated sera were used. That the
use of concentrated sera or dilutions constitutes a grave source of error
when making tests with specific precipitating antisera has already
been pointed out, the source of error being apparently in part
the normal precipitins. The stronger an antiserum the greater will
be the dilution of homologous blood with which it will react, and it
appeal's now all the more necessary to work with the highest possible
dilutions when attempting to identify a blood. This being in accordance
with what Wassermann (10, II. 1903) has just found in the case of the
agglutinins for bacteria.

The foregoing remarks apply also to the observations of Noguchi

Normal Precipitins 151

(xi. 02) who tested the interaction of the blood of cold-blooded animals
with reference to haemolysis, agglutination, and precipitation He says
that " the results do not lend themselves readily to classification, and
from them no systematic conclusions can be drawn."

In regard to precipitins he states that his " studies show conclusively
that normal precipitins are not uncommon, and m.iy be compared with
the occurrence of normal hacmolysins and agglutinins. The technique
of the experiments is simple : blood or body-cavity sera are obtained
after coagulation has taken place, and are rendered clear by filtration-
The clear products are mixed in given proportions, and the resulting
fluids are compared with controls of the mixed sera."

No conclusions as to the relationship of these animals can be
obtained from these experiments.

SECTION VI.

TESTS WITH PRECIPITINS.

I. PHYTOPRECIPITINS.
Bacterioprecipitins.

Under the name of Bacterioprecipitins I include the precipitins
discovered by Kraus in the serum of animals treated with certain
bacteria, which act upon culture-filtrates of the corresponding germs,
or upon a solution of the substance of such micro-organisms. It will be
remembered that these were the first precipitins to be discovered.

Kraus (30, iv.^ and 12, viii. 1897) found that if he added cholera,
plague or typhoid antisera to filtrates of the con-esponding cultures,
that a precipitation took place. The antisera were obtained from
animals which had been immunified. The culture-filtrates were prepared
by passing the culture-fluid through porcelain filtei-s. A filtrate of
crushed germs gave the same result as the filtered culture-fluid, proving
that the precipitable substance was present within the bacterial cell.
The antisera only acted upon their homologous cultures. Filtrates of
B. diphthenae to which antitoxic horse-serum was added, on the other
hand, did not give a precipitum. Kraus allowed the reaction to take
place at 37° C, observing the precipitum after 24 houi-s. He states
that the latter appeared to be composed of alkali-albuminate and
peptone, and notes that it is necessary to add more antiserum in
making these tests than is the case when adding agglutinating sera.
Corresponding to the precipitable substance in culture-filtrates of
B. typhosus and the Cholera genn, there must be a substance therein
corresponding to the agglutinin-producing substance in these bacteria,
for it may be noted that Widal, Levy and Bruns succeeded in rendering

' Kiaiis's earlier jiaper (30, iv. 1897 — A'. GeseUschafl drr Aerzte in ir/cii) is cited by
liordet (1899, p. 228). 1 have beeu unable to gain access to the origiual.

Bacteriojyrecijntins 153

animals immune by means of such culture-filtrates, the immune serum
agglutinating specifically.

NicoUe (ill. 1898, p. 162) confirmed the observations of Kraus with
regard to B. typhosus, B. pestis, and B. diphtlteriae. He in addition
carried out corresponding experiments with B. coli and Vibrio massauah,
obtaining most precipitum from old cultures of the corresponding germs.
The reactions were studied in the thermostat and noted after 15 — 20
hours. Nicolle considered the reaction specific, and found that it was
not impeded by antiseptics which were added.

According to Tchistovitch (v. 1899, p. 414) Mannorek subsequently
obtained .similar results with antistreptococcic serum when this was
added to Streptococcus culture-filtrates.

Radziewsky (1900, p. 434) repeated and confirmed the observations
of Kraus and of Nicolle upon B. coli. He, however, found that the
reaction was not hastened at 37°, but that it took place as well at room
temperature. Bail (1901) found that both typhoid immune serum and
the peritoneal exudate of a guinea-pig which had received an intra-
peritoneal injection of B. typhosus, gave a precipitum upon being added
to a culture-filtrate of this organism. In the latter case the peritoneal
exudate doubtless contained typhoid bacilli substance in solution.
Both Radziewsky and Bail consider bacterioprecipitins and bacterio-
agglutinins to be separate bodies, for, the latter states that after
all precipitable substance has been removed from a culture-filtrate
through repeated addition of precipitin, the deprecipitinated fluid
showed unimpaired agglutinating power. Markl (13, VI. 1901, p. 812)
has also found that anti-plague serum precipitated culture-filtrates
oi B. pestis. Kraus (18, VII. 1901) states that Whidinuvoff {Pete7-sburg.
med. Wochenschr., 1900) has applied the precipitation method in the
diagnosis of glandeis, finding a precipitum formed in a glycerine-free
culture-filtrate of B. mallei (aged 46 days) upon the addition of the
serinn of a provedly glandercd horee. Neufeld (2, v. 1902, p. 65) found
that anti-pneumococcic serum precipitated a solution of Pneumococci.
Castellani (28, vi. 1902, p. 1828) injected living cultures of B. typhosiis,
Staphylococcus aureus, and B. coli (two varieties) into rabbits and
obtained antisera wliich produced a precipitum in old culture-filtrates
of the corresponding germs. A rabbit received combined treatment
with culture-filtrates of B. typhosus and B. coli, its serum subsequently
precipitating the two filtrates. One of the cultures of B. coli was
agglutinated, and its filtrate precipitated by typhoid-precipitin'. When

' The existence in a serum of common receptors for certain races of /}. typhosus and
B. coli has also been observed with "specific" bacteriolysins.

154 Tests ivith Predjntins

animals were treated with culture-filtrates, both dialyzed and non-
dialyzed, precipitating and agglutinating antisera were also obtained.
In confirmation of Kraus (see above), he was unable to obtain precipitins
for B. diphtheriae.

There is fairly general agreement that the bacterioprecipitins and
agglutinins are distinct chemically. I have noted above that both may
coexist in a serum, and will refer the reader to p. 50, where the differ-
ences between agglutinins and precipitins are more especially discussed.
Kraus and von Pirquet (5, VII. 1902) found that bacterial culture-
filtrates contain not only a precipitable substance, but also specific anti-
agglutinin, the latter being probably identical, they think, with the
agglutinatable substance of bacteria. Pick (1902, p. 54) considers
the precipitable substances in old and fresh culture-filtrates to be
differently constituted, as they combine differently with bacterio-
precipitins. The precipitable substance in old typhoid cultures,
according to Pick, is not albumose, nor probably peptone, nor nucleo-
proteid, and it is not an albuminous body. The reader is referred to
Pick's third communication (1902) for details regarding the effects of
various chemical agents upon bacterioprecipitins.

According to Pick (1902, p. 81), typhoid bacterioprecipitins are
inactivated by an exposure of 30 — 45 minutes to 58 — 60° C, but not
the agglutinins. The inactivated serum prevents the action of fresh
antiserum. He considers (p. 92) that the inactivated serum acts upon
the precipitin, not upon the precipitable substance (compare p. 91).
As Rostoski (1902, h, p. 23) points out, the temperature at which
bacterioprecipitins are inactivated is lower than that at which
haematoprecipitins etc. are inactivated, and this indicates that they
may be of a different nature. He suggests that the name " coagulin "
should perhaps be retained for bacterioprecipitins so as to distinguish
them from the precipitins which act upon blood and the like. This
term, coagulin, is rarely used. It was applied by Ehrlich (1900, Croonian
Led.) and is used by Pick and a few others, but in my opinion it should
be dropjjed, as it will only lead to a confusion with the coagulins for
blood (see anticoagulins, p. 17) to which the term properly applies.

Yeast-Precipitins.

Schiitzc (6, XI. 1902, p. 805) sought to discover if it were possible
by the use of precipitins to distinguish between different yeasts, which
were severally used for the immunization of rabbits. Ho cites an

Yeast- Precipitins 155

earlier paper {Sitzung. der Gesellschaft dei' Charitedrzte, 12, xii. 1901)
wherein he reported success in obtaining yeast-precipitins. He ex-
tracted yeast-albumin by rubbing up yeast in a mortar in sterile 25 "/o
soda solution, fixcilitating the rupture of the cell membranes by the
addition of powdered glass and sand. The thick paste at first formed
soon became fluid and thin, and a clear fluid was obtained by centri-
fugalizing off the sand and cellular detritus. The clear fluid was
injected in doses of 5 — 10 c.c. every 3 — 4 days, during two months, into
rabbits, a total of 100 c.c. being administered.

In this wiiy he treated rabbits with yeast-albumin of four kinds:
Potato yeast. Baker's yeast. Top and Bottom Beer yeast. When he
came to test the antisera he found that all the yeasts reacted to the
various antisera, in other words that it was not possible to distinguish
the species of yeasts by means of these precipitins.

Precipitins for Albumins of Higher Plants.

Kowarski (4, vii. 1901) injected wheat albumose' into rabbits,
obtaining precipitins, which acted promptly on wheat albumose in
solution, less markedly on rye and barley, but not on corresponding
solutions from oats ; peas giving a very weak reaction (faint clouding).
Normal rabbit serum had no such effect.

Schiitze (22, xi. 1901, p. 493) treated rabbits with a vegetable
proteid " Roborat," obtaining an antiserum which precipitated roborat
solutions, but not muscle albumin, the antiserum for the latter having in
its turn no effect on roborat solutions. Castellani (28, vi. 1902, p. 1828)
confirmed this observation, finding that anti-roborat did not act on
somatose solutions. He also appears to have produced a weak anti-
somatose, which had no action on roborat solutions.

Jacoby (1901, cited by Bashford) found that animals which had
been rendered immune to ricin yielded a serum, which, whilst anti-
toxic, gave a precipitum on being added to ricin solutions. Bashfoi-d
(1902, Joiirn. of Pathol, and Bacteriol. Vlll. p. 59) states that he has
recently made a similar obsei-vation in rabbits treated with crotin. The
rabbits' serum gave a dense precipitum with a solution of crotin, normal
rabbit serum not exerting this effect'''.

' These results are to be received with cautiou, see p. 111.

^ Note analogous observations on auake venom, see Section VI.

156 Tests tvith Precipitins

II. ZOOPRECIPITINS.

I. Lactosera.

The term "lactosera" was applied by Bordet (ill. 1899, pp. 240 —
241), who discovered them, to antisera which precipitated milk casein.
Having partially sterilized milk at a temperature of 65° C. he injected
it intraperitoneally into rabbits at stated intervals. After a time he
bled the rabbits, and found their serum to possess the property of
causing precipitation in the milk with which the animals had been
treated. He performed the test by placing about 3 c.c. each of the
antiserum and of normal serum in tubes, to which he subsequently
added 6 — 15 drops of the milk. Whereas the mixture with noraial
serum retained its opaque white appearance, the mixture with anti-
sennn soon underwent a change; fine granules appeared, and these
rapidly growing in size led to the foraaation of flocculent masses. Soon
the fluid became limpid above, the flocculi sinking to the bottom. This
was best observed in milk which had been deprived of an excess of fat
by being passed two or three times through filter-paper. When milk rich
in fat is used, the flocculi are carried upward by the fat globules.
In studying the reaction, it is therefore best to first filter the milk.
The supernatant fluid remains clear, but may at times again show
a cloud upon the addition of fresh antiserum.

Fich (II. 1900, cited by Schiitze, 29, i. 1901, p. 7, footnote) soon
afterwards found that an emulsion of udder-cells, as well as milk, gave
rise to antisera which precipitated milk, when rabbits were treated
with these substances. The observation of Bordet lias been since
amply confirmed by a number of investigatoi-s. The first of these to
suggest a practical application of the method in the differentiation
of milks were Wassermann and Schutze (2, VII. 1900 and 29, I. 1901),
who treated rabbits with three different milks, namely those of the
cow, goat, and human subject. They claimed tliat these milks could
be distinguished from one another, the corresponding lactoserum acting
only upon its homologous milk. Their method of treating rabbits will
be found described under Methods (p. 53). Schutze (29, I. 1901, p. 5)
subsequently described their investigations in detail, and drew attention
to the possibility of using the lactosera in the examination for milk
adulteration.

Uhlenhuth (6, Xii. 1902), in testing with lactosera, adds 5 c.c. of
antiserum to 3 c.c. of a 1 : CO milk dilution in salt solution. In his tests.

Specificity of Reactions 157

lactoscruin for cow's milk produced a thick flocculent precipitate in cow
milk dilutions within a few minutes, whereas it left human and donkey
milk dilutions unaffected.

The specific character of the reaction was dwelt upon by Wassemiann
and Schiitze, who stated that no reaction took place, for instance
between the lactoserum for cow's milk and goat's milk, and vice versd.
This result may have been due to their using weak lactosera, for the
results of other workers have been different with the milks of animals
so closely related. Thus More (31, X. 1901) found the lactoserum for
cow's milk to precipitate goat's milk. This is in perfect agreement
with what I found with regard to the antisera for the corresponding
bloods, and is also in accordance with the observations of Ehrlich and
Morgcnroth, who found that injections of the blood corpuscles of the
ox into experimental animals led to the formation of an haemolysin
which acted not only on the corpuscles of the ox, but also on those
of the goat.

The question of specificity was next studied by Gcngou (25, X. 1902,
p. 749) with lactosera from rabbits treated with cow's milk. He states
that the reactions which took place in the milks of the cow, sheep and
goat were indistinguishable when he made the following tests. He
prepared two tubes containing :

I. Milk (of cow, goat, or sheep, etc.) II. Milk (a.s in I.)

Lactoserum (heated to 56° C.) Normal rabbit serum (heated to 56" C.)

Normal rabbit serum, fresh Normal rabbit serum, fresh

He found that the complement or " alexine " (of the fresh normal rabbit
serum) was absorbed in I. and not in II., throughout the series, when a
lactoserum was added to its homologous milk. It was in this that
the three milks above named were indistinguishable. The immune
substance acted to a lesser extent upon human and mare's milk.
The lactoserum, he adds (p. 754), did not act upon ox semm (see
contrary below). Schutze (6, xi. 1902, p. 804) found that a powerful
isoprecipitin (see p. 148) for goat's milk did not produce a reaction
in human milk dilution even after a considerable time, though he does
not state how long.

Milk boiled for half-an-hour, according to Schutze (loc. cit.) still
gives a slight reaction upon the addition of antiserum. More
(31, x. 1901, p. 1076) obtained an antisennn from rabbits treated
with milk which had been boiled for half-an-hour or upwards. He
found that milk which had been boiled for 30 minutes reacted im-

158 Tests with Precipitins

mediately to its antiserum. Miiller (18, II. 1902) dissolved lacto-
precipitum in boiling saline, and found that it could bo reprecipitated
by fresh lactoserum, and also by rennet ferment. He considers that the
precipitiim consists of unaltered casein combined with the precipitin.
Finally Fuld (1902) also found lactoscra to act upon boiled milk. He
considers that the casein is chiefly concerned in the reaction. He
found the presence of lime salts essential. Contrary to Moro, he
was unable to obtain an antiserum from rabbits treated with boiled
milk. This difference, it appears to me, may very well be due to the
milk being " boiled " in a different manner by the two workers. The
experiment should be repeated, the temperature of the milk within
the receptacle being accurately determined. Uhlenhuth (6, Xii. 1902)
obtained reactions when antiserum was added to milk which had been
boiled, and even with milk which had been exposed to a temperatiu'o
of 114" C. for 30 minutes in an autoclave.

Lactosera do not act on milk only, for Halban and Landsteiner
(25, III. 1902, p. 475), following Hamburger, found that human lacto-
serum acted also upon human blood serum. They also found that
human haematoserum precipitated human milk, just as Schutze (2, xii.
1901, cited in this author's paper of 6, XI. 1902, p. 805) had found
antiserum for ox blood to also precipitate cow's milk.

Meyer and Aschoff (7, vii. 1902) found ox lactoserum to be haemo-
lytic for ox blood corpuscles, and that it immobilized ox spermatozoa.
The milk which they injected into their rabbits had been previously
centrifugalized to rid it of cellular elements which of themselves might
have led to the formation of cytolysins. They also found lactoserum
to precipitate ox serum. Injections of blood, spermatozoa, and tracheal
epithelium, led to the formation of antisera which also precijjitated 1 : 40
milk dilutions. They found that lactosera (which were also haemolytic)
could be neutralized by homologous blood corpuscles or spermatozoa.

Schutze (6, XI. 1902, p. 804) found human lactoserum to jjre-
cipitate solutions of human spermatozoa, and von Dungem (1899 —
1900 Miinchener med. Wochenschr.) found lactosera to agglutinate red
blood corpuscles. (See further Hamburger and others, under the Action
of Precipitins upon different Albumins of the same animal, p. 104.)

Uhlenhuth (6, XII. 1902) found lactoserum for cows' milk to produce
clouding in ox blood dilutions. He cites an observation of von Dungem
to the effect that lactoserum for human milk exerts a toxic action on
human milk-gland epithelium.

Although perhap? not identical with the haematosera, the lactosera

Indimdual Differences 159

for the same animal would appear to show but a very slight diflference,
if any. We see that lactoscra and haematosera both act similarly.
Although there may be quantitative differences there are none
apparently of a qualitative character. Comparative quantitative ex-
periments have still to be made.

As in haematosera, the precipitin contained in lactosera is very
stable. Moro (31, x. 1901) found it still gave reactions with its
homologous milk after being sealed for months in capillaries, thus
confirming what I have found for some haematosera. Lactosera with-
stand a temperature of 56° C, in this resembling the haematosera,
and the agglutinins. On warming lactoprecipitum in normal saline
it almost comj)letely redissolves. Moro obtained lactosera by treating
animals with pure powdered human or cow's casein.

Milks of the same species show individual differences according to
Moro {loc. cit.), in their reaction to lactosera. On the other hand, the
reaction of each individual milk to lactoscrum, as tested on the milk of
wet-nurses, remained constant. Individual differences were less marked
in cow's milk. The individual difference w;is shown by the reaction of
each milk to its homologous antiserum taking place with higher dilutions
than with a non-homologous antiserum. Personally I am inclined to be
somewhat sceptical on this point in consequence of the results which
I have obtained with Mr Strangeways with haematosera. To prove
the presence of individual differences, and I do not question but that
there may be such, it is necessary to examine a large series of antisera
and homologous milks or sera, and this does not seem to have been
done by Moro. His results have therefore to be accepted with caution.
I will however state the reasons which led him to the conclusion that
there arc individual as well as specific groups in a given milk.

1. Antiserum for milk A added to milk a gives rciictioii iu a high dilutioo,

and vice vend.

2. Antiserum for milk A added to milk b gives no reaction in a high dilution,

and vice versd.

The reaction is due in the first instance to the specific and individual
groups entering into combination. In the second instance, using a
stronger dilution, only the specific, not the individual groups combine.
The deficit in the reaction A plus b, cannot be made good by adding
more of milk b. He concludes, therefore, that the specific groups are
neutralized, or destroyed, only the individual ones which are least
numerous being left. These being unable to combine account for

160

Tests with Precijntins

the precipitum-deficit. He represents this schematically by a figure
analogous to the well-known diagrams of Ehrlich, the lactoprecipitin
molecule being represented by a circle from which protrude an assumed
number (6) of receptors, the majority of which (4) represent specific
groups. Whereas the latter arc alike, the individual groups (2) are
different, and the various combinations of the two lactosera (A and B)
and milks (a and b) are thus shown, the precipitable substance in milk
being fixed, or not, as the case may be, to receptors. See further under
"Individual Differences," p. 145.

II. Haematosera.

In the following pages the results of precipitin tests with haemato-
sera are given in the zoological order of the antisera, the tests made by
other observers being summarized in each case, the results of my tests
following.

The number of tests, made by me with 30 antisera produced, is
given in the following table, the total number of tests being 16,000.

No. of teste

No. of tests

Antiserum for

therewith

Antiserum for

therewith

Man

825

Ox

790

Chimpanzee

47

Sheep

701

Ourang

81

Horse

790

Cercopitheous

733

Donkey

94

Hedgehog

383

Zebra

94

Cat

785

Whale

94

Hyaena

378

Wallaby

691

Dog

777

Fowl

792

Seal

358

Ostrich

649

Pig

818

Fowl-egg

789

Llama

363

Emu-egg

630

Mexican Deei"

749

Turtle

666

Reindeer

69

Alligator

468

Hog Deer

6U9

Frog

551

Antelojie

686

Lobster

450

7751

8249

7751

Total number o

8249

tests 16,000

In the short tables which folloiv, I have briefly summarized the general
results of the tests given in the experimental tables at tlw end, the object
of this being to render the conclusions clearer. The number of sera

Anti- Human Serum 161

reacting in each case is given, the intensity of reaction being indicated
by signs, as follows : —

• No reaction
« Faint clouding
X Medium clouding

+ Marked clouding
4- Full reaction

The number of full reactions obtained is indicated by the numbers
printed in black type. Although in some cases the number of bloods
examined has been small, it has been thought best to introduce the
percentages of reactions under each class, as the numbers alone convey
no clear impression to the mind as to the relative frequency of reactions.
Percentages arc freqtiently given alongside each class of reaction. The
percentages of positive reactions " all told," including the faintest, are
given on the right-hand margin of the tables'.

I. Antisera for bloods of Primates.
(1) Tests ivith Anti- Human Serum.

Before proceeding to give the results of my own tests with this
antiserum I shall briefly summai'ize what has been done by different
workers who have used anti-human serum in making various tests. It
may seem somewhat pedantic to give the exact day on which the
authors, cited in the following pages, published their papers ; my object
in doing so is to dispose of certain claims to priority which have been
made in various quarters. I wish to accentuate the fact that no new
principle was discovered when an antiserum for human blood was found.
The antisera for eel, fowl, horse, etc. had been found by Tchistovitch and
Bordet, and the whole impulse given to work in this line undoubtedly
emanates from them. Their papers were most suggestive, and it was
quite natural, in reading them, that many should be seized by the same
idea as to the j)ossible applications of the methods they had pointed out.
This was the more natural in view of the knowledge previously gained
with regard to other classes of antibodies.

Leclainche and Valine (25, i. 1901) injecting human albuminous
urine into rabbits, found that they obtained an antiserum which pre-
cipitated albuminous urine, and pleuritic exudation, but not serum
dilutions of man. This antiserum did not produce a reaction with
albuminous urines of the horse and cow.

' I am indebted to Dr Graham-Smith for kindly calculating the percentages given in
the succeeding tables. The percentages are given in round numbere, fractions of percent-
ages being only included in the totals at the right-hand margins of the tables.

N. 11

162

Tests with Precipitins

Uhlenhuth (7, ii. ]901) was however the first to publish tests of
a kind that awakened general interest, more especially regarding the
medico-legal aspects of the test and its possibilities. He injected
rabbits with human blood, and tested the antiserum on 19 bloods (1),
finding only human blood to react. Normal rabbit serum had no such
effect. He made the practically important observation that human,
horse, or ox blood which had been dried 4 weeks on a board, could
readily be distinguished by the antiserum when brought into solution.

A day later than Uhlenhuth, Wa.sserinann (8, II. 1901) demonstrated
experiments similar to Uhlenhuth 's at the Meeting of the Physiological
Society, Berlin. Outside of human blood only that of a " monkey" gave
the reaction. A report of the experiments appeared under the names
of Wassermann and Schiitze (18, II. 1901) shortly after, it being stated
therein that they had tested 23 bloods with this antiserum (2). I have
ordered the bloods they tested here, and it will be seen that many of
those examined were the same as in Uhlenhuth's list.

(1) Uhlenhuth's List
Man Reacted

Dog, cat No reaction

Pig

Ox, sheep, deer, fallow-deer
Horse, donkey
Hare, rabbit, guinea-pig, rat,

mouse
Fowl, turkey, pigeon, goose

(2) Wassermann and Schutze's List
Man, baboon Reacted

Dog, cat No reaction

Pig

Ox, sheep, goat „

Horso, donkey „

Babbit, guinea-pig, rat, mouse ,,

Fowl, duck, goose, sparrow ,,

Eel, pike, " Schlei " ,,

The latter authors also found that blood could be perfectly well
tested when brought into solution after being dried for 3 months on
various objects. The baboon's blood reacted much more slowly and
less markedly than did human blood. This finding possessed peculiar
interest to me, and, together with the similar observation of Stem,
led me to plan an investigation of the bloods of Anthropoidea. Stern
(28, II. 1901) also obtained anti-human serum from rabbits and found it
to act on blood which had been dried, and also on albuminous urine.
His antiserum did not act on horse, ox, sheep and pig bloods, whereas,
it gave a feeble but distinct clouding with the blood of 3 species of
monkeys "Meerkatze, Java-Aife, Kronen- Affe," the first being I suppose
a Cercopithecus, the second Macacus cynomolgus L. He does not give
their scientific names, so that, as in the case of Wassermann and Schiitze,
it is difficult to identify them.

Dieudonn^ (2, IV. 1901) obtained anti-human serum by treating

Anti- Human Serum

163

rabbits with human albuminous urine, pleuritic exudation, and blood
serum, injecting quantities of 10 c.c. every 3 to 4 days. He tested
his antiserum on only four other bloods (rabbit, guinea-pig, pigeon,
goose), finding that normal rabbit serum had not the precipitating
effect of the antiserum on human blood, albuminous urine, pleuritic
and peritoneal exudate.

At this stage I reported (11, v. 1901) having tested 24 bloods; only
human, and to a lesser degree, 2 monkey bloods reacting. Soon after
(1, VII. 1901 ; 36 bloods tested) I reported the blood of four species of
monkey as having given a slight but distinct reaction, a very faint
clouding appearing in solutions of the bloods of the horse, ox, and sheep,"
all the others remaining perfectly clear.

Ziemke (27, vi. 1901) obtained antiserum from Wassermann and
Schutze, and tested it upon 12 bloods (1). A reaction in 15 minutes,
and a precipitum after 24 hours only being found in human blood
dilutions.

Schirokich (21, vii. 1901) injected rabbits with blood from the
placenta and cord. He found that blood-stains 2 years old gave the
reaction when dissolved and brought into contact with his anti-human
serum. He tested 9 bloods (2).

(1) Ziemke's List

Man Reacted

Dog, cat No reaction

Pig

Ox, calf, sheep, lamb ,,

Horse ,,

Babbit, guinea-pig, mouse „

(2) Scbiboeicb's List
Man Reacted

Cat No reaction

Pig »

Ox, goat „

Horse „

Camel „

Rabbit, guinea-pig ,,

In the meantime my collection of bloods had reached 140, including
the bloods of 12 species of monkeys (some in duplicate) including
10 Cercopithecidae, and 2 Gehidne. I reported on these tests in my paper
of 14, IX. 1901 : "All the bloods of the Old World monkeys gave a very
marked reaction, less powerful however than that of human blood.
They also reacted to a weak antiserum. On the other hand the South
American monkeys (Cebidae) gave but a slight reaction with human
antiserum as compared to that of the other monkeys, and a weak anti-
serum produced no precipitation in the blood of Mycetes senicidus."
I reported further upon this interesting observation in subsequent
papers (21, xi. and 16, xii. 1901, p. 408), having in the meantime also
tested the bloods of the Ourang-Outang and Chimpanzee with positive

11—2

164 Tests with Precipitins

results. These bloods appeared to give about as much reaction as human
blood to anti-human serum. Grlinbaum (18, I. 1902, p. 143) also
obtained this result with anti-human serum tested upon these bloods
and that of the Gorilla. I shall refer to his results again further on.
In my paper of 20, I. .1902, I remarked that the amount of reaction
would appear to correspond with the degree of relationship amongst the
Anthropoidea, the Lemurs, as already noted, having given negative
results. Uhlenhuth seems to have tested a monkey blood of some kind
lately, for he says (11 — 18, ix. 1902, pp. 661) that the weak reaction
occurring in monkey blood upon the addition of anti-human serum
cannot be confounded with the marked reaction occun-ing in human
blood dilutions. In the remarks which follow the table below we see that
even monkey blood may give a high degree of reaction under certain
conditions. Schiitze (6, xi. 1902, p. 805) was able to obtain precipita-
tion with anti-human serum in solutions of human spermatozoa made
from stains which had been dried 6 months.

Confirming what has gone before, Whittier (18, i. 1902) obtained
negative results in testing anti-human serum on four bloods : of hoi-se,
cow, rabbit, guinea-pig. Butza (18, iv. 1902) tested anti-human serum
upon 14 bloods (man, dog, cat, pig, ox, sheep, rabbit, guinea-pig, fowl,
pigeon, turkey, duck, goose, and a fish) with negative result except the
first. Biondi (1902) found anti-human serum to precipitate the bloods
of Gercopithecus flavus vindis and Macacos radiatus. Anti-human serum
precipitated human milk but not that of the cow, goat and donkey ;
human saliva, but not that of the dog, cat, hoi-se and donkey. Human
bloods in health and disease gave similar reactions.

Biondi found furthermore that human serum injected into a monkey
did not lead to the formation of precipitins, as might be expected
theoretically in consequence of the close relationshiji between man and
monkey.

Lastly we find that Ewing (ill. 1903) tested four specimens of
monkey blood (baboon, rhesus, and two Java monkeys, species not
stated) with anti-human sera obtained from rabbits and a fowl. He
considers it possible to distinguish these bloods from human blood by
means of higher dilutions of the test sera'. He omits to mention that
the same thing can be accomplished by using higher dilutions of the
bloods tested, as others have found. It must always be remembered that
the precipitin should be present in excess.

> See page 142.

Anti- Human Serum

165

Liayton (1903, pp. 219, 220) has treated rabbits according to Uhlen-
huth's method and produced anti-human serum. He tested 28 bloods
(species not stated) and was able to identify human and exclude monkey
blood'.

825 Tests with Anti-Human Serum.

The following tests were carried out by means of 5 different anti-
human sera obtained by treating rabbits with the blood of Europeans.
All the sera were powerful, being freshly prepared in succession, so that
when the one began to weaken another was substituted'.

*

Eeactiona

97 Primates

«

X

f

+

7.

Antliropoidea

34 Hninan (4 races)

•

•

3(8''/„)

?(2l7o)

a* (71 "/o)

100

8 Simiidae (3 species)

•

•

•

8

100

36 Cercopithecidae
(26 species)

3

,

26(72,,) 3(8 „)

4 (10 „ )

92

13 Cebidae (9 species)

3

2(l5»/o)

5 (38 „ )

M23.>)

78

4 Hapalidae (3 spec.)

2

1

1

SO

Lemuroidea

2 Lemuridac (2 spec.)

2

•

•

0

29 ClIIROPTERA

26
13
70
S3
40

3(10,,)

2(13,,)
13(13,,)

7(11,,)
19(27,,)

14(14,,)

5(7 „)

11(10,,)

10

15 Inskctivora

13

97 Carnivora

27

65 EODENTIA

18

70 Unqulata

43

3 Cetacea

12

3(ioo"/„)
1(7 ..)

100

13 Edentata

7

26 Marsupialxa

25
1

319

1(4 ■.)
1(3 -)

4

1 MoNOTREMATA

0

320 AvES

0 3

49 Keptilia

49

0

14 AMPniBiA

14
19

7

0

19 Pisces

0

7 Crbstacea

0

•24«/o

•S'/o

The preceding table shows maximum reactions only amongst
the Hominidae, Simiidae, and Cercopithecidae. In the case of the
last only 4 bloods gave a maximum reaction, in two the precipitnm
was moderate in quantity, in two it was voluminous, in fjict almost
equal in quantity to that observed in the reactions with human

' Layton added -5 "/„ carbolic acid to his antiserum.
'^ See p. 144 regarding the strength of antisera.

166 Tests loith Precijyitins

and ape bloods. This great reaction with the blood of the two
Cercopithecidae appears to find its explanation in the cause of death
which was given for these two animals (Nos. 49 and 58) viz., intus-
susception and dysentery. In both of these affections the blood
is liable to become concentrated, and therefore, taken volume for
volume, the serum of such an animal will contain more of the
reacting body than does normal serum. Within limits, moreover, the
more concentrated a blood dilution, the more precipitum will it yield
upon the addition of an antiserum. In point of fact then, the bloods of
Cercopithecidae give under ordinary conditions a very moderate reaction
as compared to those of Hominidae and Simiidae. Three negative
results amongst the Cercopithecidae (Nos. 49, 59, 75) are due to some
common cause, which I am not at present able to fully explain. All
three bloods were sent to me by Dr Langmann from the New York
Zoological Gardens. None of them went well into solution, a fact which
was possibly due to their having been exposed too long in the sun, or
affected in some other way. The bloods of the Cebidae and Hapalidae
show a still further reduction in the amount of reaction.

If we include even the faint and medium cloudings ( * x)
amongst the positive reactions, we see that the percentage' of positive
reactions falls as we read doivn the column of the reactions with the
bloods of Primates, from man to the Hapalidae. It must however not
be lost sight of, that the great reactions in the two last columns are
those which more clearly show the relationship. When we come to
consider the other Mammalia, we see that there is a notable absence
of larger reactions, the largest reactions noted being a few medium
cloudings, and these occurred most frequently amongst the Carnivora
and Ungulata. This will be better seen by reference to the percentages
bracketed alongside the figures. The percentage of faint and medium
cloudings in all the mammalian bloods examined, outside the Primates,
amounts but to about a half of that noted for the Hapalidae, viz. 24°/o-
Amongst the Primates we see that the result of the test with two
lemurs was negative. I demonstrated a small series of primate bloods
before the Royal Society (21 Nov. 1901), which showed this progressive
decrease of reactions from the Hominidae downward, and no reaction
with the lemur bloods. I stated at the time, that a weaker antiserum
included the Hominidae, Simiidae and Cercopithecidae within its
sphere of action. When the antiserum was stronger it included the
Cebidae and Hapalidae, these reactions being comparatively very slight.
On the other hand, if we use a very powerful anti-human serum, faint

Anti-Human Serum 167

cloudings may also be observed in the dilutions of other mammalian
bloods, and such an antiserum will also cloud the dilution of lemur
blood. This is however a feeble reaction to which I have already
given the name of mammalian reaction, for I have not found it to occur
amongst non-mammalian bloods. This is very well seen by reference to
the preceding table, where only one avian blood, out of 320 tested,
is recorded as showing a faint clouding. On what such faint cloudings
may depend, seems fairly clear. To begin with, they are such great
exceptions that this alone suggests their being due to some error. At
times they may be due to clouded solutions, or to clouded antiserum,
at other times they may be due to the blood samples having been
brought together by collectors. In an earlier paper I first showed
that this reaction took place in blood mixtures. Consequently, if a
blood sample, say from a bird, were brought in contact with one from
a mammal, a positive reaction might take place with anti-avian as well
as with anti-mammalian serum upon these being added to a dilution
made from a sample of mixed bloods. Also, supposing that samples
of dried bloods are stored in contact with one another, and are allowed
to rub against each other, we may have the same result. This especially
might occur where blood had been allowed to clot upon the paper, for
blood-scales might break off and become attached to a paper saturated
with another specimen of blood. Finally, a certain number of these
results may be reasonably ascribed to the fact that in cutting out
squares of filter-paper in succession from a number of different samples,
a trace of a foreign blood may at times adhere to the scissors used
and thus find its way into a solution to which it does not belong. It is
remarkable however how few tests have given other than the result
which experience taught one to expect. It is nevertheless well to
remember these sources of error so as not to attach any special
importance to exceptional reactions.

The anti-human sera used in the foregoing tests were usually
powerful, and therefore we find more "mammalian reactions" noted.
In contrast to tins, we see on referring to the tests with a much
weaker antiserum (anti-monkey, p. 171) that the action of the antiserum
was practically limited to the Primates, for, oven when we include the
faint cloudings, reactions only occurred in 1 "/o of all the other mam-
malian bloods examined.

168 Tests ivith Precipitins

(2 — 3) Tests with Antisera for Simiidae.

The first to obtain antisera for the Simiidae was Griinbaum (18, i.
1902, p. 143), who treated rabbits with the blood of the gorilla, ourang-
outang, and chimpanzee, obtaining as many antisera, all of which
acted upon their homologous bloods, as well as upon those of the
related species, and man. Griinbaum was unable to "assert that
there is any difference of reaction " amongst these bloods to the several
antisera above-named. Dr Griinbaum, knowing of my investigations,
very kindly sent me some of his anti-gorilla and anti-chimpanzee serum,
so that I might test them upon the bloods of Anthropoidea which I had
collected. Unfortunately his anti-gorilla serum had gi-own so weak
that no results of value could be recorded for it. On the other hand,
the following table gives the results of tests made with the anti-
chimpanzee serum.

47 Tests luith Anti-Chimpanzee Serum.

The anti-chimpanzee serum used in these tests was kindly sent to
me by Dr A. S. F. Griinbaum of Liverpool. It bore the date 24, 1. 1902,
and the tests were made on its receipt four days later. The antiserum
was weak and limited in quantity, and consequently but few bloods
could be tested with it, the tests being confined to bloods of Primates.
Although the amount of precipitum obtained was small, considerable
differences could be noted.

Iteactioua

+

•/.

8

100

3

100

.

es

3 HoMiNiDAE (European)

3 Simiidae (2 spec.) •

23 CERcopiTnEciDAE (19 spec.) 8 9{397o) 6(26»/„)

12 Cebidae (9 spec.) 11 1

4 Hapalidae (3 spec. ) 13

2 Lemuridak 2 • •

It will bo seen that the results are in accord with those obtained
with anti-human serum. The proportion of negative results with the
bloods of Cercopithecidae and Cebidae was naturally greater because
a weaker antiserum was used. The results with Cebidae and Hapalidae
do not agree with those obtained with the preceding and two
succeeding series, where the Hapalidae gave lower values than the
Cebidae.

Anti-Simian Sera 169

81 Tests with Anti-Ourang Serum.

The antiserum used in these tests was produced by injecting a
rabbit with the serum of Simia satyriis, which died at the Zoological
Society's Gardens (No. 38). The antiserum was of moderate power.
Here again the tests were limited to those upon the bloods of Primates,
the bulk of the antiserum being reserved for studies by the quantitative
method reported in Section VII. Standardized 6 weeks after these
tests were made, this antiserum gave a precipitum of '008 c.c.

23 HoMiNiDAB (4 races)

8 SiMiiDAE (3 spec.)

32 CKHCoriTiiEciDAE (23 spec.)
12 Cemdae (8 spec.)

4 HArALiDAE (3 spec.)

2 Lemdridae (2 spec. )

Reactions

•

* X

+

"U

a

1

5

7
4
2

7(30»/„) 13(56»/„)

2(25,,)

2 ( e „ ) 23 (71 „ )

2 (17 „ ) 3 (25 „ )

• •

6(62 0/„)
2(6")

•

86
87

43
0
»

We see here that the results are again mainly in accord with the
preceding. It is possibly due to the fact of the human blood
dilutions being somewhat weaker (old dried bloods mostly) that the
human bloods gave lower values here. The quantitative tests made
with fluid sera gave results similar to those in the two preceding
tables. Owing to the fiict that this antiserum was more powerful
than the anti-chimpanzee serum the greater number of the bloods
of Cercopithecidae gave medium reactions. Of the 5 that gave
negative results 3 were samples from India, Borneo, and China
respectively, 1 came from New York (gave a * with anti-monkey),
and the fifth sample had been dried one year. Similarly the 2 giving
feeble reactions ( * ) came respectively from India and New York.
The 3 negative results with human blood were with 3 samples
(Negro, Nos. 33 — 35) sent from W. Africa, these samjiles also showing
less than full reactions even with anti-human scrum. The negative
result noted among Simiidae is due to the blood of a gorilla (No. 43)
which Dr Griinbaum informed me a year ago gave but feeble reactions
in his tests with anti-human and anti-chimpanzee serum.

(4) Tests with Anti-Cercopithecus Sera.

From my results with other anti-primate sera, it seemed of interest
to repeat a series of tests with an antiserum for Cercopithecidae, and

170 Tests with Precipitins

in due course for other families of Primates. The tests I have made
with anti-monkey serum are given in the short table which follows.
Before coming to these, I shall refer to a paper by Friedenthal
(10, VII. 1902, p. 831). Pursuing my line of investigation, he sought
to obtain an antiserum for Cynocephalus hamadryas, the blood of
which animal he injected subcutaneously into rabbits in the huge
doses of 26 — 51 c.c, which appears to have killed most of his rabbits
outright, as might naturally be expected. It is almost impossible to
attach any value to his results, because of the faulty manner in which
he proceeded to immunify his animals, and especially because of the
date at which he bled them after their last injection. In a protocol,
which he gives, he states that he obtained an antiserum from a rabbit
as follows: A rabbit received monkey serum subcutaneously in doses
of 2, 2, 3, 5, 5, 5 c.c, the day intervals between inoculations being
3, 2, 5, 5, 4. Bled 3 days after the last injection, the rabbit's serum
contained no precipitin for its homologous blood. Having waited
seven days, he injected 25 c.c. (!) subcutaneously, and 48 hours (!)
later, found precipitin (?) in the rabbit's serum, the serum being then
used for tests. He made the tests by adding 0'2 c.c. of the serum
to be tested to 5 c.c. of the "antiserum'."

Although Friedenthal's method was evidently bad, the results he
claims to have obtained are not opposed to mine. His antiserum
acted on its homologous blood, and also upon that of three other
Cercopithecidae {Cynocephalus " dschedala," Cvlubus guereza, Macacus
cynomolgus) to an equal degree. His antiserum, when fii"st used,
was certainly very weak (as might be expected from the very
early date at which he bled his rabbit after its last injection),
for he states that it failed to produce any effect upon human or
anthropomorphic ape blood even after 24 hours. He adds, later (p. 833),
that the rabbits which survived his (mal-) treatment, gave an anti-
serum which did also produce reactions, though weaker ones, with
both human and chimpanzee blood. In this he confirms my results
with the anti-primate sera described on the preceding pages, and also
agrees with what follows. It is unfortunate that Friedenthal used
such methods, for, as stated, they tend very much to vitiate his results.

• See further under "Sources of Error," p. 74.

Anti-Moiikey Serum

171

733 Tests with Anti-Monkey (Cercopithecits) Serum.

The antisenmi used in these tests was procured through the
injection of a rabbit with the blood of Cercopithecus (Papio) hamadryas.
The blood was kindly sent to me by Dr Liihe, the animal having died
at the Zoological Gardens, Konigsberg (No. 65, 14, ii. 1902). The
antiserum was feeble.

85 Primates

Anthropoidea

23 Hominidae (4 races) ... 3

8 SimiiJae (3 spec.) 2

35Cercopithecidae(2-tspec.)* •

13 Cebidae (9 spec.) 7

4 Hapalidae (3 spec.) 3

Lemuroidea

2 Lemuridae (2 spec.) 2

29 Chiroptera 29

12 Insectivora 12

95 Carnivora 93

62 Kodentia 62

67 Ungulata 66

3 Cetacea 3

13 Edentata 13

26 Marsupialia 24

1 Monotremata 1

271 AvEs (incl. 3 eggs) 271

45 Reptilia 45

9 Amphibia 9

14 Pisces 14

1 Crdstacea 1

Beactions

16(70%

2 (25 „
11(31 „

3 (23 „
1 (25 „

4(17%)

4 (50 „ )

21 (60 „ )

3 (23 „ )

3 («»/»)

87
75
100
46

as

0^
0

u

2
0
1
0
0
8
0
V
0
0
0
0

M'/o

• No. 59 Cercopithecus is not included here, as it gave negative results throughout.
See note to No. 49 in tables.

The results will be seen to con'espond to those obtained with
the preceding antisera, only that the reactions amongst the Cercopithe-
cidae form the majority of the marked reactions. The bloods of
Cebidae and Hapalidae again give less marked reactions than do those
of Hominidae and Simiidae. The Lemuridae give a negiitive result.
Owing to the weakness of this antiserum the mammalian reaction
occurring in bloods not closely related is scarcely perceptible, for only
1 "/o of faint reactions (slight clouds) were observed amongst all the
other mammalian bloods examined outside of the Primates. The action

172

Tests with Precipitins

of this antiserum very well demonstrates a fact to which I have already
drawn attention, namely that a weak antiserum is more limited in its
action.

II. Antisera for bloods of Insectivora.

Uhlenhuth (25, Vli. 1901) treated rabbits with the serum of the
hedgehog {Ennacem europaeus) and obtained an antiserum which only
acted on the blood of the hedgehog, he tested 24 bloods as follows : —

Man, hedgehog, bat, dog, fox, cat, pig, deer, ox, sheep, goat, horse,
donkey, rabbit, guinea-pig, rat, mouse. Fowl, pigeon, owl, crow, sparrow,
duck, goose.

383 Tests with Anti-Hedgehog Sei-um.

The following tests were made with a powerful antiserum (Preci-
pitum = '022 c.c.) obtained by injecting a rabbit with the mixed serum
of four Erinaceus europaeiis (No. 135). In view of the continued
negative results with different anti-mammalian sera upon non-mannna-
lian bloods, these tests were limited to mammalia.

Eeaetions

85 PmMATKS 83

27 CniRoi'TEBA 26

15 Insectivora (4 families)

6 Erinaceus europaeus •

1 Crocidura coerulea 1

6 Talpa europaea 6

2 Ceiitetes ecaudatua 1

92 Carnivora 87

58 rodentia 58

C5 Ungdlata 60

3 Cetacea 3

12 Edentata 12

25 Marsdpialia 25

1 monotbemata 1

The foregoing table shows a very limited action on the part of this
antiserum, only the blood of Erinaceits eitrojmeus giving a full reaction.
The tests with other Insectivora were negative, with the exception of
one sample from Centetes ecaudatus which only gave a faint reaction
such as was observed in a few instances amongst the other mammalia.

* X -

V 7o

2

2

1

3

<

3 100
0
0

With other than its

homulogous serum

3-5 7o

1

60

5

S

•

0

5

7

•

0

•

0

■

0

.

0

Anti-Cat Serum

173

III. Antisera for bloods of Camivora.

(1) Tests tuith Anti-Cat Serum.

In my pipor of 1, vii. 1901 (p. 381) I reported having failed to
obtain an anti-cat serum by treating rabbits in the usual manner by
intraperitoneal injections with cat serum. The rabbits were treated
until they had received as many as 8 and 9 injections respectively, but
there was no appearance of precipitin. Two further attempts were
made which failed for the reason that a fresh cat serum was used for
continuing the treatment begun with old serum, the fresh serum
proving toxic. Discouraged by these results, I made no further attempts
in this direction. Uhlenhuth (25, Vli. 1901) was more successful, how-
ever. He does not state that he had any particular difficulty in
obtaining this antiserum. He tested it on the 24 bloods cited on
page 172, with the result that only cat serum dilutions were found to
react, the antiserum being obtained, as usual, from rabbits. Stimu-
lated by Uhlenhuth's success, I again made an effort to obtain anti-cat
serum, finally succeeding, although I only obtained a weak antiserum.
My results are summarized in the following table :

Eeactiona

74 Primates 67

27 cuiropteka 27

13 Insectivoka 13

92 Cabnivora

19Felidae (8 spec.) 2

14 Viverridae (11 spec.) 12

1 Proteleidae 1

2 Hyaeuidae (1 spec.)

20 Canidae (14 spec. & ? races) 17

4 Ursidae (3 spec.) 3

7 Procyonidae (5 spec.) 5

19Mustelidac (10 spec.) 16

4 Pinnipedia (3 spec.) 4

63 bodeniia 61

73 Unoclata 72

2 Cetacea 2

13 Edentata 12

24 Maksupialla 24

1 mosotremata 1

324 Aves (incl. 4 eggs) 319

41 Eeptilia 41

10 Amphibia 10

22 Pisces 22

6 Cbustacea 6

6(8-'/o) Ml"/.)

5(20'>/„)
1 ( 7 „ )

10{52«/„)
1(7,,)
1

1 1

3
1

1(14,,) 1(U„)
3

1 1*

1

+ "/o

9

n

0

a(IO»/„) 89^
14
50
100
15 J-31-5''/„
25
28
16

0

3

1

0

7

0

0
0-J

0

0

0

0

other mam-
mals than
Carnivora
4%

174 Tests with Precipitins

785 Tests with Anti-Cat Serum.

This antiserum was obtained by injecting a rabbit with the serum
of Felis domesticus. The antiserum was weak, giving a precipitum
of 005 c.c.

It will be seen from the preceding table that this antiserum only
produced full reactions amongst the Fclidae. Many of the bloods had
been dried for over a year when brought into solution and tested.
This, but especially the weakness of the antiserum, accounts for most
of the reactions being so slight. The two negative results obtained
with Felidae were with one blood of F. domesticus (No. 155) dried
19 months, and one of a leopard (147) sent to me by Mr E. H. Hankin
from Agra, India. The blood-relationship amongst the Camivora is
however fairly well shown, excepting in the case of the Pinnipodia.
The other mammalia showed a much smaller number of faint reactions.
One rodent blood (marked *, No. 288) gave a medium reaction, this
was from an agouti and it behaved curiously with other antisera, as
will be seen by reference to its number in the tables. The agouti
blood, which had been preserved with chloroform, showed slight
reactions with anti-wallaby and anti-Mexican-deer and also gave a
medium reaction with anti-horse serum. I cannot as yet explain this
result, as this blood did not react to 16 other antisera.

(2) 378 Tests with Anti-Hyaena Serum.

This antiserum was obtained by treating a rabbit with the serum
of Hyaena stnata, the animal having died at the Zoological Gardens,
London. This antiserum was much more powerful than the preceding.
It gave a precipitum measuring 'OSl c.c. when "1 c.c. of the antiserum
was added to 'Sec. of a 1 : 100 dilution of hyaena scrum.

A glance over the following table immediately shows that a con-
siderable number of reactions took place with the bloods of mammalia
other than those belonging to the Camivora. When we however confine
our attention to the largest reactions, we find that they occur solely
amongst tlw Hyaenidae and. Felidae. If we include all the reactions,
faint and otherwise, as positive ones, we find on reading the percentages
opposite each family of Carnivora, that they are higher in each of these
families than in any other mammalia not belonging to the order
Camivora. The percentage of reactions amongst the Camivora, taken
as a whole, is also higher. The anti-hyaena serum is sufficiently

Anti- Hyaena Serum

175

powerful to include the Pinnipedia. On referring to the percentages
of reactions all told, occurring between anti-cat serum and the bloods
of Felidae and Hyaenidae, we find that even the weak anti-cat serum
proved the close relationship of these two families, in a manner corre-
sponding to what we have found with anti-hyaena serum. Moreover,
in the case of both antisera, the lowest percentages of reactions all told
are given by the bloods of Viverridae, Canidae, Ursidae and Mustelidae.

Beactions

82PRIMATES 41 35(42<'/„) 12 (l4 »/„)

27 Chiropteka 22 6

13 Insectivoka 6 7 •

90 Carnivora

18 Felidae (8 spec.) • 1(5,,) 11 (Bl „ )

14 Viverridae (11 spec.) 2 3 (21 „ ) 9(64,,)

8 Proteleidae • • 1

2 Hyaenidae (1 spec.) •

22 Canidae (13 spec. & ? var.) 4 4 (i8 „ ) 14 (63 „ )

4 Ursidae (3 spec.) 1 1(25,,) 2 (50 „ )

6 Procyonidae (4 spec.) • 2 4

18 Mustelidae (10 spec.) 4 6(33,,) 8 (44 „ )

5 Pinnipedia (3 spec.) • 1(20,,) 4(80,,)

59 Eodentia 52 5(8„) 2*(3„)

65 Ungclata 31 26(40,,) 9 (14 „ )

3 Cetacea 2 1

12 Edentata 8 3(25,,) 1 (8 „ )

25 Marsupiai.ia 22 3

1 monotremaia 1

• Rodent blood 257 (see text).

4- Vo

56
18
S4

e(3S»/„) 100 >^

85
100
100
81
75
100
77

100 J
12
54
33
33
12
0

87%

Other mam-
mals than
Carnivora

Of the 11 bloods of Carnivora giving negative results in the pre-
ceding table, there were two of Viverridae (168 from Borneo, 169 dried
18 months), four of Canidae (187, 189, 196, 204, the three first from
India, the last from Paraguay), one of Ursidae (206 dried 17 months).
These bloods have evidently been the cause of lowering the percentage
of positive results with anti-hyaena serum, but they do not account
entirely for the lower percentages obtained with these families. On
the other hand, the four bloods of Mustelidae giving a negative
reaction, had all been collected in England, and presumably had not
been subjected to any conditions which would alter the bloods so that
they would not give good reactions with a suitable antiserum.

One of the two rodent bloods(*) giving a medium clouding was No. 257

176 Tests with Precipitins

(see tables), which acted peculiarly with other antisera. The other
rodent blood giving this slight reaction was No. 283, and no explanation
can be offered for the result.

(3) Tests ivith Anti-Dog Sera.

Nolf (v. 1900) first produced anti-dog serum by treating rabbits
with dog serum. Ho was unsuccessful when he treated them
with dog blood corpuscles. The antiserum did not precipitate some
other bloods tested. In ray note of 11, v. 1901 I reported having
obtained negative results with 23 bloods other than dog's. Similar
results have been obtained by Uhlenhuth, who found the anti-serum to
react with the blood of the fox. In my paper of 21, XI. 1901 I reported
having only obtained reactions with the bloods of six species of
Canidae, 196 non-canine bloods having given a negative result. By
reference to the tables, it will be seen that a considerably larger
number of canine bloods has since been examined. Qengou (25, x. 1902,
p. 751) tested this antiserum upon four bloods (dog, horse, ox, guinea-
pig) and found only that of the dog to react.

I will add here that Uhlenhuth (25, vii. 1901) has produced an
anti-fox serum, which, tested on 24 bloods (see his list, p. 172) only
reacted with the blood of the fox and dog, less with the latter.

Farnum (28, xii. 1901) prepared anti-dog scrum and found it to
precipitate solutions of serum of the dog, but not of man and bull.

777 Tests with Anti-Dog Serum.

These tests were carried out in the couree of two years and necessi-
tated the use of five different antisera obtained from as many rabbits
which were treated with the serum of Ganis familiaris (different
breeds). Four of the sera were powerful, one weak. The weak anti-
serum was scarcely used. One of the powerful sera (the last used)
was standardized, being found to give a precipitum of "015 c.c.

We see from the following table that the dogs form a detached group
amongst the Camivora, for with the exception of one of the Mustelidae,
no other bloods gave a full reaction. Three canine bloods gave
negative or faint reactions, the two giving negative results were sent
from India (196) and South America (204), the one giving a faint
clouding only, from India (187). It may therefore be assumed that
these bloods had become relatively insoluble, The proportion of medium.

Anti-Dog Serum 177

but especially of weak, reactions amongst the Carnivora is obviously
much higher (note the percentages) than in the other mammalia when
the number of bloods examined is taken into account.

Reactions

84 Pbimates 66 16 (19"/,,)

25 Chibopteba 23 1(4 „)

14 Inseotivoba 12 1(7,,)

91 Caenivoea

18 Felidae (8 spec.) 12 3(8,

14 Viverridae (U spec.) 11 3

1 Proteleidae 1

2 Hyaenidae ( 1 spec. ) 1 1

22 Canidae (12 spec. * ? races) 2 1(7,

3 Ursidae (3 spec.) 1

7 Procyonidae (? 5 spec.) ... 4 2 (l9 ,

20 Mustelidae (9 spec. ) 16 2(10,

4 Pinnipedia (3 spec.) 2 2

61 RoDENTIA 55 4(6,

65 U.NQULATA 63 2

2 Cetacea 2

13 Edentata 12 1

22 Maesupiama 21 1

1 monotremata 1

322 AvEs (incl. 4 eggs) 321 1

40 Keptilia 40

13 Amphibia 13

18 Pisces 18

6 Cecstacea 6

2(2%)
1 H -. )
1(7 ■■ )

) 3 (8 „ )

2

) 1 (U „ )
) 1(5„)

) 2('.. )

16 \
21
0
50

l«(727o) 91

/o
SI
8
U

1*6%

(5,.)

43

20
50/

9

3

0

a

4
0
03
0
0
0
0

Other mam-
mals than
Carnivora

It is worthy of note, that the tests made with the three preceding
antisera for Carnivora agree in causing a larger proportion of reactions
amongst the Primates than amongst any of the other mammalia,
excepting the results with anti-hyaena serum which show a large
proportion of faint reactions amongst the Ungulata.

One of the two rodent bloods giving a x reaction again proved to
be No. 257 referred to under anti-hyaena serum.

(4) 358 Tests with Anti-Seal Serum.

The antiserum used in the following tests was obtained by treating
a rabbit with the serum of Phoca vitulina L., the Common Seal (No. 242).
The antiserum was weak, only giving a prccipitum measuring '006 c.c.
when •! c.c. of antiserum was added to "5 c.c. of a 1 : 100 dilution of
homologous serum.

N. 12

178

Tests with Preciintins

Reactions

7S Primates 63

24 Chiroptera 21

13 Inseciitora 12

88 Carnivora

18 Felidae (8 spec.) 14

13 Viverridae (10 spec.) 10

1 Proteleidae

2 Hyaenidae (1 spec.) 2

22 Canidae (13 spec. &? races) 11

4 Ursidae (3 spec.) 2

6 Procyonidae (? 4 spec.) ... 4

18 Mustelidae (10 spec.) 10

53

4 Pinnipedia (3 spec.)

56 eodentia 54

63 Ungulata 61

3 Cetacea 3

12 Edentata 10

23 Marscpialia .. 22

1 Monotremata 1

o(i2 7o)

3
1

1 ( 5 „ )

2 (15 „ )
1

7(31,
1
1
4

17

1 (25,

1

2

2
1

)

X "/o

3 ( 4 »/„) 16

12

7

3 (17 „ ) 22 1
1 ( 7 „ ) 23

(18 „ )

14

3 (75 „ )
1

37 '/o

Other mam-
mals than
Carnivora
s'Vo

It is owing doubtless to a slight cloudiness and also to the feebleness
of this antiserum that the results tabulated above appear at first sight
less in accord with the preceding. It is obvious that it is more
difficult to estimate different degrees of reaction when at most moderate
cloudings occur and the precipitum observed after 24 hours is so small
in quantity. It is evident nevertheless that the bloods of the Pinni-
pedia react more than do the others, numerically if not apparently
quantitatively, and it would appear also that there is a preponderance
of reactions amongst the other Carnivora. It is notable again that the
Primates stand out amongst the other mammalia, in a manner corre-
sponding (but less marked) to what has been stated with regard to the
preceding three antisera for Carnivora.

IV. Antisera for Bloods of Rodents.

Antisera for rabbit blood have been obtained by only a few observers,
the first of these being Nolf (v. 1900) who produced it by treating
a fowl with rabbit serum. Bordct (ill. 1899) failed to find any precipitin
in guinea-pigs treated with rabbit serum, whereas Gongou (25, X. 1902,
p. 743) claims that the serum of such guinea-pigs produced a " distinct
opalescence" in rabbit serum dilutions, although it did not produce

Anti-Rodent Sera 179

a precipitum. Two years ago I sought to discover if isoprecipitins
were formed in rabbits treated with the serum of other rabbits, but the
result wa« negative, and I have not mentioned it hitherto. Hamburger
(6, XI. 1902, p. 1189, footnote) states that he obtained negative results
in treating guinea-pigs with rabbit senim, consequently Gengou's state-
ment requires further confirmation before it can be accepted. On the
other hand Hamburger reports that he has succeeded in obtaining
anti-rabbit serum by treating a goat with rabbit serum. De Lisle
(XI. 1902, p. 399) states that he has obtained anti-rabbit serum by
injecting rabbit blood into eels. Haemolysins were also formed in the
eel, so that its serum after treatment was four times as toxic for rabbits
as before. He does not state the strength of the blood dilutions he
tested, nor is there mention of control tests on other sera, consequently
his precipitin results are not as valuable as they might be.

I have only had time to make two attempts to obtain anti-rabbit
sera. Following Nolf, I used a fowl and duck for treatment, but in
neither case was I successful. Working with birds is not satisfactory in
any case, as compared to mammals. The goat would appear to be the
most likely animal for such purposes. I regret not to be able to give
a series of tests with an anti-rodent serum at present, but shall perhaps
have an opportunity of doing so later. It will be very interesting to
see how the collection of rodent bloods will behave to such an anti-
serum. On glancing through the tables, we see that the results are
throughout practically negative with rodent bloods. Here and there a
rodent blood reacts, but this can be put down in some cjiscs to experi-
mental error. Also where the most powerful anti-mammalian sera were
used, we see a number of slight cloudings noted (anti-pig, anti-human,
anti-hyaena) but they appear to have no .significance excepting as slight
indications of mammalian relationship. I would surmise that a powerful
anti-rodent serum might possess a very generalized action in this group.

V. Antisera for Bloods of Ungulata.

a. Suidae.

(1) Tests with Anti-Pig Serum.

The first to produce anti-pig scrum was Uhlenhuth (25, vii. 1901)
who tested 24 bloods therewith (see list on p. 172); the only bloods ho
found to react were those of the pig and wild-boar. I subsequently
(20, 1. 1902) tested 250 bloods with this antiserum, with the result that

12—2

180 Tests tvith Precipitins

I found it to produce marked clouding in a number of mammalian
bloods: that of man, several species of monkey, bear, dog, opossum,
raccoon, cat, coati, genet, stoat, rat, mouse. It did not however produce
a full reaction with any blood except that of the pig, no other suilline
bloods being tested. Only once did the antiserum produce a slight
clouding in an avian blood, this being attributed to experimental error,
the sample in question having probably been in contact with some
mammalian blood, through carelessness in collection. Still later,
(5, IV. 1902), I observed faint cloudings to occur upon the addition of
this antiserum to the bloods of the antelope and deer, and after 30 to
120 minutes in the bloods of bats (6 species), 3 species of Edentates, and
8 species of Australian Marsupials. I also drew attention to the
interesting fact that the blood of the porpoise immediately gave a slight
clouding with this antiserum, a well-marked though slight deposit being
formed after 24 hours. I wrote at the time that "The more general
action of this particular anti-pig serum on other mammalian bloods may
at first appear to contradict what has hitherto been claimed with regard
to the relatively specific character of these antisera; but this is
actually not the case. I have already noted elsewhere the occasional
occurrence of clouding in non-homologous bloods upon the addition of an
antiserum." I have attributed these results to the great power of my
antisera, giving what I have described elsewhere as a " mammalian
reaction."

Besides Uhlenhuth and myself, only Kister and Wolfi" (18, xi. 1902,
p. 422) are known to me as having experimented with anti-pig serum.
They state that their antiserum did not act on human blood in any
concentration, and this I attribute to their antiserum not being as
powerful as mine. Under the same conditions the antiserum gave a
reaction in 5 minutes with pig blood (followed by a large deposit in
1 — 2 hours), a cloud in the sera of ox and sheep after 20 minutes, and
in the horse after 60 minutes.

Finally, Schiitze (6, xi. 1902, p. 805) found pig haematoserum to
precipitate solutions of pig spermatozoa, but not those of man.

818 Tests with Anti-Pig Serum.

Three different antisera were used in the following tests, the three
having been obtained by injecting as many rabbits with the serum of
the domestic pig. All three antisera were exceedingly powerful, two of

Anti-Pig Serum

181

«

X

+

+ »/«

. 16(18%)

19(217„)

18(20»/„)

. eo

» 2(6 „)

8(27,,)

1 (3 „ )

• 58

3 3(2i„)

2(14,,)

1(7.,)

. 4J

i 20(21,,)

20(27,, )

5(5 „)

• 53

i 16(«„)

4(6..)

l'('»/u)

32

1 1

them on being standardized giving a precipitum of -045 and '055 c.c.
respectively.

Reactions

89 Primates 36

29 Chikoptera 18

14 Insectivora

96 Carnivoba 45

65 RoDENTIA 44

69 Unoci.ata

a. Artiodactyta
Suina

4 Suidae

1 Dieotylidae

Tylopoda
4 Caraelidae (2 spec.) .
Tragulina

1 Tragulidae

Pecora
17 Cervidae (12 spec.) .
34 Bovidae (22 spec.) .
Perissodactyla

1 Tapiridae •

6 Equidae (3 spec.) 3

c. Hyracoidea

1 Hyracidae

3 Cetacea (2 spec.)

13 Edentata 3

26 Marsupialia 10

1 monotremata 1

322 AvES (incl. 4 eggs) 321

47 Reptii.ia 47

13 Amphibia 13

24 Pisces 24

7 Crustacea 7

8
23

b.

4(23,,
6(17,,

1
1

) •

) 5(15,,)

5(18,,)
12(35,,)

5 (38 „ )
5(19,,)

2

5 (38 „ )
11(42,,)

* Rodent No. 257 which gave curious resalta with other antisera,

100
100

75

0
41

67;

66%

lUO

so

j 57%

100

100 Other
77 mammals

61

than Un-

gulata

51»/o

The antisera employed for these tests were amongst the most powerful
used throughout the investigation. The general mammalian reaction is
consequently well marked. Nevertheless we see that fall reactions only
occurred amongst the Suidae. The four bloods of Suidae tested included
the blood of the domestic pig, of the wild boar (Europe), and of a
wild boar, whose blood was collected at Singapore. Moderate reactions
were observed with the blt)ods of Bovidae, Cervidae, Camelidae. Taking
the Artiodactyla as a whole we find that G6 "/o of the bloods gave some
sort of reaction. The Perissodactyla and Hyracoidea (1 sample only of

182 Tests loith Precipitms

last) gave but slight reactions. Whether it is due or not to the
fact that the pig is a " generalized mammal " I do not know, but
certainly the results, especially with the Primates, seem somewhat
anomalous. We find in fact that nearly all the mammalia are more or
less connected, the only quite negative result being with the blood of a
Monotreme, which has shown no trace of reaction with any of my
antisera, although it went perfectly into solution. These results seem
to be confirmed by our quantitative tests as far as they have gone.

This antiserum brings out the reaction with the bloods of Cetacea in
a very striking manner. It will be seen further that these bloods also
reacted to other anti-ungulate sera. The quantitative tests (see Section
VII), very clearly show a remarkable tendency of cetacean blood to
react with anti-ungulate sera, giving large perfectly measurable quan-
tities of precipitum.

The following passage from Flower and Lydekker (p. 233)' would
seem to offer the clearest explanation of this action of anti-ungulate sera
upon cetacean bloods. They write, " But the structure of the Cetacea is,
in so many essential characters, so unlike that of the Camivora that the
probabilities are against these orders being nearly related. Even in the
skull of the Zeuglodon, which has been cited as presenting a great
resemblance to that of a seal, quite as many likenesses may be traced to
one of the primitive Pig-like Ungulates (except in the purely adaptive
character of the form of the teeth), while the elongated larynx, complex
stomach, simple liver, reproductive organs both male and female, and
foetal membranes of the existing Cetacea are far more like those of that
group than of the Camivora. Indeed it appears probable that the old
popular idea which affixed the name of "Sea-Hog" to the Porpoise
contains a larger element of truth than the speculations of many
accomplished zoologists of modem times "

b. Camelidae.

(2) 363 Tests with Anti-Llama Serum.

This antiserum was produced by injecting a rabbit with the serum of
Auchenia huanacos Molina. The antiserum was weak, giving a
precipitum of '005 c.c. when standardized, six weeks later.

» Flower, W. H., and Lydekker, E. (1891), An Introduction to the Study of Mammals
Living and Extinct, London, Adam and Charles Black.

Anti-Llama Serum

183

Eeactions

#

X +

+

7o

2

4

•

0

•

0

3

9

'

0

1

. .

• "

25

•

1

•

100

•

a

a

100

.607«

•

•

•

0

4
9

23) «5°/»
29 J

,

(1

1

SO

1

0
0
0
3
0

Other mam-
mals than
Ungnlata
2-6 «/„

75 Primates 72

25 Chiroptera 25

13 Insectivora 13

89 Carnivora 85

57 rodentia 57

62 Ungulata

a. Artiodactyla
Suina

4 Suidae 3

1 Dicotylidae

Tylopoda
4 Camelidae (3 spec.) ...

Tragulina
1 Tragulidae 1

Peoora
17 Cervidae (12 spec.) ... 13
31 Bovidae (22 spec.) ... 22

b. Perissodactyla

1 Tapiridae 1

2 Equidae (1 spec.) 1

c. Hyracoidea
1 Hyracidae 1

2 Cetacea 2

12 Edentata 12

27 Marsupialia 26

1 monotbemata 1

The preceding table shows that large reactions only occurred with
two samples of the blood of two animals of the species Auchenia
huanacos, the blood of one of which was used for the treatment of the
rabbit yielding the antiserum. The blood of Auchenia glama and
Camelus dromedanus gave x reactions. The percentage of reactions all
told, is larger amongst the Ungulata than among the other mammalia.
Again the Primates and Carnivora stand out among these.

c. Cervidae.

(3) 749 Tests with Anti-Mexdcan-Beer Serum.

The following tests were made with one antiserum obtained by
injecting a rabbit with the serum of Cariacus mexicanus, H. Smith
(No. 338, belonging to the Family Cervidae). This antiserum was
powerful, giving a precipitum of '015 when standardized.

184

Tests with Precijntms

73 Primates 40

25 Chiroptera 15

11 Insectivoba 7

84 Carnivoea 62

61 bodentia 57

61 Unoclata

a. Artiodactyla
Saina

3 Suidae

1 Dicotylidae •

Tylopoda

2 Camelidae (2 sp.)
Tragulina

1 Tragulidae

Pecora
15 Cervidae (11 sp.) If
34 Bovidae (22 sp.) 2^

b. Perissodactyla

1 Tapiridae

3 Equidae (1 spec.) 2

c. IJyracoidea

1 Hyracidae 1

2 Cetacea 1

13 Edentata 10

23 Marsupialu 21

1 monotremata 1

314 AvES (incl. 4 eggs) 314

40 Heptilia 40

11 Amphibia 11

23 Pisces 23

7 Cbostacea 7

Reactions

^

*

X

+

+

7o

27 (37»/„)

4 (5 7«)

2 (2 7o)

47

10

.

.

40

3 (27 „ )

1*(9„)

•

3S

15 (18 „ )

7 (8 „ )

.

27

3

1

.

«

1(6,,)

(53 „ )

7 (20„ ) 17 (50 „ )

2 (13 „
5(14,,

100 '
100

100

3(20%)
3(8„)

• JOO %

9*7o

100
33

SO Other
23 mammals
than
Ungulata

» 27«/„

0

0

0

0

0

* Fluid serum No. 143.

t No. 332 test not repeated, result doubtful as gave good reactions with other antisera.
X No. 347 from Central Africa, 369 dried 15 months when tested.
§ No. 380 a fluid serum 17 days on ice tested with antiserum 19 days old also on ice.
I! No. 385 Balaenoptera rostrata. This and another sample of fluid blood showed
reactions with other anti-ungulate sera.

It will be noted in the preceding table that large reactions occur
only amongst the Pecora, forming 20 °/(, of such reactions amongst the
Cervidae, 8 "/a among the Bovidae. The bloods of both of these families
all reacted if we except the three giving a negative result (about which
see footnotes). Second-class reactions occurred chiefly amongst the Pecora,
as also in 1 blood each of Suidae, Equidae, Cetacea, and in only 2 (2 7o)
of the Primates. All bloods of Artiodactyla, outside the Pecora, reacted.

Anti- Reindeer Serum 185

though weakly. Of 3 equine bloods two gave no reaction, one gave a
moderate reaction ; the blood of Hyrax gave a negative result. Of the
non-ungulate bloods those of the Primates gave the highest percentage
of reactions all told.

(4) 69 Tests with Anti- Reindeer Serum.

This antiserum was obtained by injecting a guinea-pig with the
blood and clouded serum of Rangifer tarandus, kindly sent me by
Privatdocent Dr Max Luhe from the Zoological Gardens at Konigsberg.
The antiserum was unfortunately very weak, yielding only a precipitum
of -004 when standardized with its homologous blood. Owing to its
limited quantity it was impossible to carry out more than a very small
number of tests, especially as some antiserum was used for the quan-
titative tests reported in Section VII.

Beactions

4 CnraopTEBA 4

6 Carnivoba G

1 RoDENTIA 1

9 Ungulata

a. Artiodactyla
Suina

1 Suidae

Pecora

4 Cervidae (4 spec.) 1

4 Bovidae (4 spec.) •

b. Perissodacti/la

1 Equidae

1 Cetacea

1 Maksupialia 1

41 AvEs 41

5 Reptilia 6

3

a

7o

0

0
0

75
100

100 y

100
0

90»/,

Other mammalian bloods than £hose noted were not tested for the
reason that the antiserum had become exhausted. Here, as in the
preceding table, the Pecora only show greater reactions, the Cervidae
giving more of the mai-ked reactions than do the Bovidae. Of the other
manmialian sera tested those of a pig, horse, Ronjual (385 Balae-
noptera rostrata) gave a faint clouding. All other bloods showed no
trace of reaction.

186

Tests ivitti Precipitins

(5) 699 Tests with Anti-Hog-Deer Serum.

This antiserum was obtained by treating a rabbit with the serum of
Gervus porcinus Zimm. (No. 326> The antiserum gave a precipitum of
•0063 c.c. when standardized.

Beactions

74 PatMATES 70 3

22 Chibopteba 22

H Insectivoea 11

86 Cabnivoba 80 3

57 BoDENTiA 56 1

62 Unoulata

a. Artiodactyla
Saina

3 Suidae (3 spec. ?) . 3

1 Dicotylidae 1

Tylopoda

2 Camelidae (2 sp.) 1 1
Traguliua

1 Tragulidae 1

Pecora

17 Cervidae (12 sp.) 1
34 Bovidae (22 sp.,

4 races) 2

b. Perissodactyla

1 Tapiridae 1

2 Equidae (1 spec.) 2

c. Hyracoidea

1 Hyracidae 1

2 Cetacea 1 1

12 Edentata 11 1

17 Mabsupialu 17

1 mosotiiemata 1

290 AvEs (incl. 4 eggs) ... 290

33BEPTIUA 33

11 Amphibia 11

15 Pisces 15

6 Cbustacea 6

l(B7o) 7(41%) 2(l2"/„)
9(26,,) 15(44,,) 2(6„)

0/

/o
5
0
0
7
1

100 \

()

50

6(35%) 94
e (17 ,, ) 94

0
0

0

50
8
0
0

57 Vo

Other
mammals

than
Ungnlata

It will be seen from the above table that the bloods of the Pecora
only give large reactions, and that the percentage of large and moderate
reactions amongst the Cervidae is higher than amongst the Bovidae.
In some cases the bloods from both of these families appear to
react equally. One sample of cervine blood failed to react : 322, from
India. Two bovine bloods failed to react : 347 from Central Africa, and

Anti-Antelope Senim

187

369, dried over a year when tested ; if we exclude these then 100 "/o of the
bloods of Pecora reacted. Next in order come the Suidae and Caraelidae
and the Cetacea.

d. Bovidae.
(6) 686 Tests tvith Anti-Antelope Serum.
This antiserum was obtained by injecting a rabbit with the serum
of Cobus nnctuosus Lorrill., or Sing-Sing antelope (340) which died at the

68 Primates 62

23 Chiroptera 23

10 Insectivora 10

82 Carnivora 77

58 KoDENTiA 56

60 Unoulata

a. Artiodactyla
Suina

3 Suidae 1

1 Dicotylidae .

Tylopoda

2 Camelidae (2 sp.) 1
Tragulina

1 Tragulidae

Pecora
16 Cervidae (11 sp.) If
33 Bovidae (22 sp.,

?var.) 1 +

b. Perissodactyla

8 Equidae (1 sp.) ... 3
0. Hyracoidea

1 Hyracidae

2 Cetacea 2§

11 Edentata 11

16 Marscpialia 16

1 Monotremata 1

292 AvES (incl. 4 eggs) ... 292

33 Eeptilia 83

10 Amphibia 10

15 Pisces 15

5 Ckdstacea 5

Keactions

X

+

+

Vo

2

9

•

0
0

1

G

1»

3

3(18%) 7(43»/„) 5(31»/„)
3 » ) 19 (57 „ ) 7 (21 „ )

<8.

100

so

•n7o

96»/„

6(l5»/„) 97.

100
0
0
0
0

other
mammaU

than

Uniittlata

5 7o

Collected properly ?

• 257, see tables, this sample reacted most peculiarly,
t 336, roebuck, gave feebler reactions than another sample from same species when
tested with other anti-ungulate sera.
X 347 from Central Africa.
§ 385-6 showed trace of deposit after 24 hours.

188

Tests with Preelpitins

Zoological Gardens, London. The antiserum was very powerful, giving
a precipitum of "055 when standardized.

We see from the above table that the large reactions are limited to
the Bovidae, the second-class reactions to Bovidae (21 "/o) find Cervidae
(31 "Jq of their totals). Two negative results amongst the Pecora are
referred to in footnotes. Other Artiodactyla than the Pecora gave 71 "/j
total reactions. Hyrax gave a faint clouding, the Equidae negative
results. The Cetacea also gave negative results, although it is stated in
the tables that a trace of deposit was noted in the tubes after 24 hours.
Of the non-ungulate bloods those of the Primates and Camivora give
most reaction.

(7) Tests with Anti-Ox Serum.

The first to produce anti-ox serum was the late Dr Walter Myers
(14, VII. 1900) who treated rabbits with ox serum-globulin, obtaining
an antiserum which precipitated solutions of ox globulin, besides having
a slight action on a similar solution from sheep. Uhlenhuth (1, li. 1901)
produced it next by subjecting rabbits to intraperitoneal injections of
defibrinated ox blood, doses of about 10 c.c. being administered every
6 — 8 days until 5 injections had been made. He tested 19 different
bloods therewith in 1 : 200 dilutions. The bloods tested were the
following, which I have grouped in order:

Ox

Man

Fowl

Sheep

Turkey

Deer

Dog

Pigeon

Fallow Deer

Cat

Goose

Pig

Hare
Rabbit

Horse

Guinea-pig

Donkey

Rat
Mouse

Presumably his anti-ox serum was weak, for he states that he
obtained a reaction only with ox blood. In my preliminary note of
11, V. 1901, in which tests on 24 bloods were reported, I noted that
anti-ox serum also produced a weak reaction with the blood of the sheep.
As the number of bloods tested increastsd, I found that those of the
gazelle and axis-deer gave distinct reactions (1, vii. 1901), as did also
those of the goat, roebuck, and Burrhel sheep (14, IX. 1901), and in

Anti-Ox Serum 189

a later paper (21, xi. 1901) I stated that the tests conducted both
with anti-ox and anti-sheep sera had "given reactions, which indicate
the existence of a 'blood relationship' between certain of the true
ruminants," positive reactions having further been obtained with the
bloods of the deer, antelope and gnu. The bloods of the Tragulidae and
Camclidao (20, i. 1902) gave no indication of relationship with the true
ruminants. Subsequently I found what appeared to be differences in
the degree of the reaction, the antisera for ox and sheep acting " to
a greater degree upon the bloods of more closely allied species"
(5, IV. 1902).

In the meantime Uhlenhuth (25, vii. 1901) tested 24 bloods (see
his list on page 172) with this antiserum, and found it to give much
precipitum with ox blood, and a small prccipitum with the bloods of
the goat and sheep. Farnum (28, xii. 1901), in America, treated
rabbits with the semen of the bull and found the antiserum thus ob-
tained to act upon homologous solutions, not upon those of goat semen.

Michaelis and Oppenheimer (1902, p. 342) found anti-ox serum to
act on ox and on sheep blood, not on that of the horse. Michaelis
(9, X. 1902, p. 734), states that this antiserum acts very much less upon
sheep blood than upon that of the ox. Kister and Wolff (18, xi. 1902,
p. 422) publish results which certainly suggest some experimental error,
probably, as Uhlenhuth suggests, the use of " milky antisera " (see p. 72).
Correctly enough, according to the results above cited, deposits were
found to occur upon the addition of this antiserum to ox and sheep
blood, but they found a greater reaction with human blood than with
that of the pig and horse.

790 Tests tuilh Anti-Ox Serum.

Four different antisera were used in the following tests, all being
obtained from rabbits injected with the serum of the ox. The antisera
were fairly powerful, one of them, when standardized, giving a precipitum
of -Oil c.c.

The larger reactions are limited here to the bloods of the Bovidae,
the second-class and weaker reactions being about equally divided
amongst the Bovidae and Cervidae, medium cloudings occurred in
a horse, a pig, and a whale blood and in a few Primates (ll'/o) *°d
Camivora (4 "/„). All three of the cetacean bloods gave some reaction.
Two of the four cervine bloods sent from India, which gave no reaction,
should be excluded.

190

Tests with Precipitins

Reactions

94 Primates 84

29 Chiboptera 29

12 Insectivora 12

91 Carnitora 87

63 EODENTIA 62

67 Unoclata

a. Artiodactyla
Suina

4 Suidae 3

1 Dicotylidae 1

Tylopoda
4 Camelidae 4

Tragulina
1 Tragulidae 1

Pecora
17 Cervidae (12 sp.) 4
35 Bovidae (22 sp.,
etc.)

b. Perissodactyla

1 Tapiridae 1

3 Equidae 2

0. Hyracoidea

1 Hyracidae 1

3 Cetacea (2 spec.)

13 Edentata 13

26 Mabsopialia 26

1 monotremata 1

301 Aves (incl. 4 eggs) ... 299

51 Eeptilia 51

13 Ampuibu 13

19 Pisces 19

7 Crustacea 7

6(6«/o) 4(4«/„)

4» 2(n<'/„) 3(l7«/o) 8(47%)
2t 4(11,,) 6(17,,) 17(48,,)

01
10

1
0
0
4
1

85 \

0

^10%

0

n

im

0
0

n
o-«

0
0
n

0

Other
mammals

than
Ungulata

5»/o

• Two of these, 322 and 323, from India.

t?

(8) Tests with Anti-Sheep Serum.

The first to prepare this antiserum was Myers (14, viz. 1900). He
treated rabbits with sheep serum-globulin, and found that their serum
contained a " specific " precipitin for sheep globulin solutions, and also
exerted a slight precipitating action upon ox globulin. Nuttall (17, V.
1901) treated rabbits with sheep serum and obtained a similar result,
when testing 24 bloods. Subsequently he found (1, vn. 1901) the
blood of the gazelle and axis-deer to give "distinct but less marked"

Anti-Sheep Serum 191

reactions than did the blood of the sheep. Uhlenhuth (25, Vll. 1901)
tested 24 bloods (see list p. 172) with this antiserum, finding that it
gave almost as much reaction with the blood of the goat as with that of
the sheep, less with that of the ox. He observed no reaction in the
blood of a deer, nor in any of the other bloods tested. With an in-
creasing number of bloods tested, I noted more and more the tendency
of this antiserum to include other Bovidae in its positive reactions, and
not infrequently Cervidae. In several short papers these findings were
briefly dwelt upon as in the preceding tests -with anti-ox serum.

Recently Schiitze (6, xi. 1902, p. 805) has found anti-sheep serum
to act less upon ox spermatozoa solutions than upon corresponding ones
of the sheep. The results of Kister and Wolff (18, xi. 1902, p. 421)
with this antiserum are in accord with mine, and of themselves indicate
that an experimental error lay at the bottom of those they obtained
with anti-ox serum referred to above. Anti-sheep serum clouded both
sheep and ox serum dilutions in 5 minutes, leading to a large deposit
in the former after 2 hours. After 20 to 30 minutes slight clouds
appeared in pig and horse serum. No effect was exerted on human
blood after 2 hours. As the proportion of antiserum added to the
dilution decreased, the blood of the pig and horse ceased to react,
reactions still being obtained with the bloods of sheep and ox.

701 Tests with Anti-Sheep Sei'um.

Six different antisera, obtained from as many rabbits, were used for
these tests. The rabbits received injections of the serum of the domesti-
cated sheep. One of the antisera had moderate power, whereas the
others were powerful. One of the latter gave a precipitum of '02 when
standardized.

Here again the large reactions are limited to the bloods of Bovidae
(23 "jo), the second-class reactions occur amongst Bovidae and Cervidae,
forming a higher percentage of the total reactions in the latter group.
Of the bloods of other Ungulata, those of Suina and Equidae gave faint
and medium cloudings. One of two cetacean bloods tested gave a
medium clouding. Outside the Ungulata, those of Primates and
Carnivora gave most of the " mammalian reactions " recorded, viz.
5 and 10 "/o respectively. Two of the four bloods of Pecora giving
negative results were from India.

192

Tests with Precijntins

Bcactiong

75 Primates 71

24 Chikoptera 24

9 Insectivora 9

78 Cabnivoba 70

58 BoDENTU 67

61 Ungulata

a. Artiodactyla
Suina

3 Suidae 2

1 Dicotylidae

Tylopoda

2 Camelidae (2 sp.) 2
Traguliua

1 Tragulidae 1

Pecora
16 Cervidae (11 sp.) 2*
34 Bovidae (21 sp.,

4 races) 2t

b. Perissodactyla

3 Equidae 1

c. Uyracoidea

1 Hyracidae 1

2 Cetacea 1

12 Edentata 12

22 Marsdpialia 22

1 monotbemata 1

289 AvES (incl. 4 eggs) ... 287

33EEPTILIA 33

1 3 Amphibia 13

17 Pisces 17

7 Ceustacea 7

l(6"/o) 0(56%) 4(25%)

2(6„) 12(35,,) 10(23,,)

* One from India (H23), the of.
t One from China (393), the ol

10

6

n

0
10
2

88

8(23%) 94
66
0

i»7o

92"/„

50 Other
n mammals
than
Ungulata

» 5 7o

ler (329) from Germany,
her (342) from London Zoo.

e. Equidae.

(9) Tests with Anti-Horse Serum.

Anti-horse serum was first obtained by Tchistovitch (v. 1899) by
treating rabbits with horse serum, injections of 3 c.c. being made
5 — 6 times, at intervals. We may conclude that his antiserum was
very weak indeed, for the reason that he states it produced a reaction
■with the serum of the horse, but not with that of the donkey. Tests
upon normal rabbit serum also proved negative. Nolf (v. 1900), and

Anti-Horse Serum 193

several observers since, have prepared this antiserum. In a series of
24 bloods tested by me and reported in my preliminary note of 11, v. 1901,
only horse serum was found to react to its antiserum. Numbers of
tests were reported on in my subsequent papers, wherein it was
stated that the only other blood to react was that of the donkey.
Uhlenhuth (25, vii. 1901) reported having tested 24 bloods with anti-
horse serum (see list p. 172) and he found no other bloods outside those
of the horse and donkey to react. Schiitze (6, xi. 1902, p. 805) found
this antiserum to precipitate spermatozoa solutions of the horse, but not
those of man, and ox. Uhlenhuth (7, xi. 1901) suggests the use of this
antiserum for the detection of horse-meat in sausages (see under practical
applications), and, using the test on meats, Notel (13, iii. 1902) found
horse and donkey meat indistinguishable. Kister and Wolff(18, xi. 1902,
pp. 414 — 416) published seven tables of tests made by them with anti-
horse serum upon 5 bloods. They do not state whether they were all
conducted with one, or with several antisera, so it is to be presumed
that they used one, which they say "surprised" them by the general
reactions which it gave. I have no doubt, in view of my own results,
that Uhlenhuth is right in stating that these authors probably had
to deal with what is well described as a " milky antiserum " (see p. 72),
for their results are somewhat anomalous. Possibly their antisera were
preserved with chloroform (see p. 76). They tested the blood of the
horse, pig, ox, sheep, and man. On adding antiserum in the proportion
1 : 5 blood-dilution (1 : 10 to 1 : 320) all the bloods clouded, the greatest
clouding occurred with horse blood, where a large deposit soon formed.
Deposits did not occur in the other bloods (except man, tests of 1 : 10
and 1 : 40). When the amount of antiserum added was less than 1 : 10,
1 : 30, 1 : 50, the reactions amongst non-homologous bloods took place
more and more slowly, all the bloods outside that of the horse practically
ceasing to react with tests of 1 : 100. Beginning with their tests of
1 : 5, and following the series of increasing amount of dilution to which
antiserum was added to 1 : 100, we find that the blood of the pig first
ceased to react, then that of man and ox. Strong sheep blood dilutions
still gave clouds with antiserum added in the proportion of 1 : 100, the
clouding only appearing after two hours ; though the homologous horse
serum reacted in five minutes. Next to horse, most positive reactions
were obtained with man. These results are entirely in disacconl with
mine and others.

N.

13

194

Tests with Precipitins

Reactions

97 Primates 93

28 Chiropteba 29

13 Insectivoha 13

88 Caknivora 86

63 Rodentia 61

71 XjNOnLATA

a. Artiodactyla
Saida

4 Saidae 3

1 Dicotylidae 1

Tylopoda

4 Camelidae (3 sp.) 4

Tragulina

1 Tragulidae 1

Pecora

17Cervidae (I2sp.) 15

36 Bovidae (22 sp.) 36

b. Perissoddctyla

1 Tapiridae 1

6 Equidae (3 sp.) ...

c. Hyracoidea

1 Hyracidae 1

3 Cetacea 2

13 Edentata 13

26 Mabscpialia 26

1 Monotremata 1

296 AvES (incl. 4 eggs) ... 296

51 Reptilia 51

13 Amphibia 13

20 Pisces 20

6 Ckustacea 6

2t

2:

tt

+ 7o

»i

6 ino

* The 3 bloods giving « reactions were Nos. 38, 45, 58, all fluid. No. 45 reacted
peculiarly with other antisera. The other two were not tested with many antisera. .

+ One, a fluid serum No. 162, gave fuint clouding with a number of other antisera.
See tables. The other. No. 168, a sample from Borneo, also gave « with anti-human, and
X with anti-hog-deer.

X One, a clouded, chloroform-preserved serum (No. 288) rejected in some of the
tests because it gave clouded dilutions. Gave x with anti-wallaby and anti cat, both weak
antisera. Also » with anti-Mexican-deer. The other. No. 292, was a dried sample from
S. America, and this also reacted, with faint clouding, with 4 different antisera. Owing
to the peculiar behaviour of these two bloods they should be excluded.

§ No. 313, fluid serum. The negative results were with samples dried on filter-paper,
but perfectly soluble.

II Anti-diphtberial serum .5 years old, fluid, bottled (No. 378). Two sets of tests on
Nos. 37G and 377 (anti-diphtherial horse serum, dried in scales, bottled since 1895 and
1897 respectively) gave negative results with this antiserum, doubtless because of their
insolubility ; these are excluded from the above table as also from the other tables.

t+ No. 41), fluid serum. The value of this reaction is open to doubt because of the
behaviour of this blood to other non homologous antisera, notably, weak anti-cat and
anti-wallaby,

Anti-Horse and Donkey Sera 195

790 Tests with Anti-Horse Serum.

Four antisem were used in the tests noted on p. 1 94, the antisera having
been obtained from as many rabbits treated with the serum of horses
slaughtered at Cambridge. Three of the antisera were powerful, one of
moderate strength, giving, when standardized, a precipitum of -008 c.c.

The reactions with anti-horse serum, and with the antisera for
the donkey and zebra, are practically in accord. The large reactions
are in all three cases confined to equine bloods. In the case of the
last two antisera, a time limit was put upon the reactions recorded,
those only being included which took place within 40 minutes. For
this re:Tson the effects of these antisera seem more limited, although
the difference is trivial.

Although the reactions with the bloods of Equidae are put down as
equal, there is a very distinct difference both with regard to the time
in which they take place, and the amount of precipitum formed.
Anti-horse serum acts more rapidly on horse blood than upon the
othei-s, anti-donkey more rapidly upon that of the donkey, anti-zebra
more rapidly upon that of the zebra. In the quantitative tests to be
reported in Section VII. these differences arc stated in figures, and
arc dwelt upon more in detail.

(10) Tests with Anti-Donkey Serum.

This antiserum was first obtained by Uhlenhuth (25, vii. 1901) who
tested 24 bloods therewith (see list p. 172) and found it only to react
with 2 bloods, those of the donkey and horse, less with the latter.

94 Tests with Anti-Donkey Serum.

The following tests were made on one day, and with one antiserum
obtained from a rabbit which had been treated with the serum of Equus
asiinis. Only the reactions occurring within 40 minutes are recorded.
The antiserum was fairly powerful, giving a precipitum of about O'Ol c.c.
when standardized.

The tests on the bloods of Ungulata and Cetacea are included in
the extended tables at the end, but not the others, as they were made
in such limited ninnbers. Only one test on Ungulata is not included in
the tables at the end, the test having been made on a fluid ox-serum
(1, 1. 1902) not included amongst the numbered samples The following

13—3

196

Tests with Precipitins

numbers, referring to the bloods tested, are to be found in the first
column of the tables in question.

5 Pbimates 5

5 Chiroptera 5

2 Insectivora 2

6 Caknivoba 6

6 EoDENTIA C

46 Ungolata

a. Artiodactyla
Suina

4 Suidae 2

1 Dicotylidae

Tylopoda

3 Camelidae 3

Tra(,'ulina

1 Tragulidae 1

Peoora

13 Cervidae 12

ISBovidae 15

b. Peritsodactyla

1 Tapiridae 1

4 Equidae (3 spec.)

c. Hyracoidea

1 Hyracidae 1

3 Cetacea 3

5 Edentata 5

5 m.vr8upialia 5

1 MoNOTREMATA 1

8 AvEB 8

1 Keptilia 1

1 AjiPHnjiA 1

Reactions

> '8 "/o

4 100%

The bloods tested in the above table wei"e Primates, Nos. 38, 44, 45,
48, 57, all fluid ; Chiroptera, Nos. 99, 100, 105, 114, 119, all samples dried
on filter-paper; Insectivora, Nos. 133, 141, both fluid; Carnivora, Nos.
145, 162, 205 a, 220, 242, and unnumbered dog's serum (4, in 1902), all
fluid; Kodentia, Nos. 247, 271, 280, 284, 286, 289, all dried on filter-
paper; Edentata, Nos. 388, 389, 390, 391, 395, all fluid; Marsupialia,
Nos. 406, 412, 415, 417, 425, all fluid ; Monotremata, No. 427, dry ; Aves,
Nos. 428, 448, 461, 463, 477, 502, and two unnumbered samples, fowl (12,
III. 1902) and pigeon (6, vi. 1902), all fluid; Reptilia (813), Amphibia
(862), both fluid.

Anti-Zehra Set'vm

197

(11) 94 Tests with Anti-Zebra Serum.

These tests were all made on one day with one antiserum obtained
from a rabbit treated with the serum of Ecptns greyvi Giinther, Grevy's
zebra, which died at the Zoological Society's Gardens, London. This
antiserum was moderately powerful, giving a precipitum of -012 c.c. with
its homologous blood.

Reactions

5 Primates

5

« X

5 Chiroptera

2 Insectivoba

5
2

•

6 Carnivoba

G

6 RODENTIA

6

46 Ungulata

a. Artiodactyla
Suina
4 Suidae

4

1 Diootylidae

Tjlopoda
3 Camelidae

1
3

•

Tragulina

1 Tragulidae

1

Pecora

13 Cervidae

18 Bovidae

.. 12
11

1

4 3

b. Perissodactyla

1 Tapiridae

1

4 Equidae (3 spec.)

c. Hyracoidea

1 Hyraoidae

3 Cetacea

1
3

5 Edentata

5

5 Mabscpialia

5

1 Monotremata

1

8 AvES

8

1 Reptilia

1

1 Amphibia

1

8%

4 ion"/.

Note. A number of the tests here recorded are not included in the
tables at the end. The footnote to the preceding tests with anti-donkey
serum applies also to those bloods. In addition, the three tests with
anti-zebra serum upon the 3 cetacean blood-samples are not included
in the tables.

The close relationship existing between Equus caballus and a
species of zebra (Equus burchelli) has been demonstrated by crossing.

198

Tests with Precipitins

See J. C. Ewart (1889), The Penycuik Experiments. London (Adam
and Charles Black), 177 pp.

VI. Antisera for bloods of Cetacea.

94 Tests with Anti-Whale Serum.

These tests are not included in the tables at the end, for the reason
that the antiserum h.ad not been obtained when they were written. The
tests were all made with one antiserum obtained from a rabbit treated
with the serum and blood of Balaenoptera rostrata, the Rorqual
(No. 385 a), the blood having been kindly sent me by Professor Torup
of Christiania.

When standardized, the antiserum gave a precipitum of '009 c.c.

Ecactions

5 Primates

5 CniROPTERA

2 Insectivora

6 Carnivora

6 eodentia

46 Unoulata

a. Arliodactyla

5 Suina

3 Tylopoda

1 Traguliua

31 Pecora

b. Perissodactyla
1 Tapiridae

4 Equidae

c. Hyracoidea

1 Hjracidae ....

3 Cetacea (2 spec.) ...

5 Edentata

5 Marsupialia

1 Monotkemata

8 AvEs

1 Reptilia

1 Amphibia

1

3

1

10

1

4

3 1

14 7

1* 1

80 7o

<a°/o

100 »/„
40 7«

* The weak reaction due to a test carried out upon a solution from a dried sample of
blood of Balaenoptera rostrata (No. 285) of older date.

The bloods tested were the same as in the list appended to the tests
with anti-donkey serum (see p. 196), and the same as those tested with
anti-donkey serum (Ungulata, Cetacea) given in the tables at the end.

Anti-Wallaby Serum

199

The reactions noted occurred within 40 minutes.

The reactions of the first and second class were confined to two
cetacean bloods, those of Balaenoptera rostrata, and Phocaena communis.
Some medium cloudings are noted amongst the Artiodactyla, some faint
cloudings amongst these and other mammalia.

VII. Antiserum for Marsupialia.

691 Tests with Anti-Wallaby Serum.

The antiserum used in these tests was obtained from a rabbit treated
with the serum of Onychogale unguifei-a Gould, the Nail-tailed Wallaby,
the animal having died at the Zoological Society's Gardens, London.
The antiserum possessed but moderate power, yielding a precipitum of
"007 c.c, on being standardized.

Reactions

M»/o

68«/„

66 Primates 59

24 CniuopTERA 24 • • • o

10 Insectivora 10 • • • 0

79 Cabnivoha 75 2 2 • 5

56 rodentia 55 • 1* • 2

61 Ungdlata 58 3 • • 5

2 Cetacea 2 • . • 0

13 Edentata 13 • . • o /

25 Marsdpialia
Suborder Polyprotodontia

6 Didelphyidae (3 spec.) 5

1 Daayiiridae

1 Peramelidae 1

Suborder Diprotodontia

2 Phalangeridae 2

15 Macropodidae (8 spec.) • 1 (ti7o) 8 (SS^^) « (4o"/„) lOO ,

1 Monotremata 1 ... 0

278 AvES (incl. 4 eggs) 278

38 Reptilia 38

11 Amphibia 11

21 Pisces 21

6 Crustacea 6

• No. 289 gave curious results with other antisera. See tables.

It will be seen from the above that the action of this antiserum was
limited, as flir as the large reactions go, to the Macropodidae. The
bloods of the latter gave 40 "/o of the large reactions, 53 % of the second-
class reactions, one gave a faint clouding; in other words, taking the

•»

X

6

1

,

,

2

2

.

1*

3

•

•

•

1

•

1

•

•

,

.

Mti%)

8 (53»/„)

•

•

•

•

.

,

•

•

•

•

200 Tests with Precipitins

reactions all told, 100 "/„ reacted. Two Phalangeridae bloods gave no
reactions. Of the Polyprotodontia bloods one of Dasyuridae gave x and
one out of 6 of Didelphyidae gave a faint clouding. The majority of
the last gave a negative result, perhaps for the reason that they were
not tested until my antiserum had grown still weaker than it was at
the start. It is true that some x reactions are noted amongst other
mammalia, but their number is very small if their percentage is
reckoned. The Didelphis blood which showed a faint clouding, was
the only one, outside that of other Marsupialia, which reacted at all in
a long series of bloods tested on one day, the faint clouding in this case
was therefore certainly suggestive. These tests will however have to be
repeated with stronger antisera.

VIII. Antisera for bloods and egg-whites of Aves.

A. Tests with Antisera for Avian Bloods.

(1) Anti-fowl serum.

Anti-fowl serum was fii-st obtained by Bordet (iii. 1899) by treating
a rabbit intraperitoneally with defibrinated fowl's blood. He observed
that the rabbit's serum acquired agglutinating and haemolyzing, as well
as precipitating, power for fowl and jjigeon blood. As in Tchistovitch's
experiments with eel serum, the precipitum Bordet obtained was
soluble in dilute alkaline solutions. Nolf (v. 1900) obtained this
antiserum by injecting fowl serum, but not when he injected the blood
corpuscles. Uhlenhuth (15, X. 1900, p. 735) obtained anti-fowl serum
in the same manner as Bordet. He found it to precipitate solutions of
fowl blood, but not those of the pigeon, horse, donkey, ox and sheep.
Evidently his antiserum was weak, otherwise it would have acted on
pigeon blood. In my paper of 20, i. 1902, 1 reported having tested 250
bloods with anti-fowl serum. Amongst birds the results were different
to those I had obtained with anti-mammalian sera amongst mammals,
the reactions in the latter case being mostly restricted to groups of
closely related animals. "Anti-fowl serum was found to produce a
reaction not only with solutions of fowl blood, and that of the closely
related pheasant, turkey, etc., but also with the bloods of widely
divergent species, such as the parrot, various species of duck, the wood-
cock, sheathbill, heron, eagle, owl, condor, pigeon, a number of small
Piisserines, and American rhea. A marked clouding was moreover
produced in the blood of the swallow, rook, landrail, stork, swan and

Anti-Fowl Serum 201

African ostrich. What I have termed a ' marked clouding ' is probably
to be regarded as an indication of a more remote relationship." Anti-
fowl serum produced a slight cloud in only one mammalian blood of
those tested, this being put down to some experimental error, the
sample having been sent me from abroad. It may have been collected
carelessly, viz. brought in contact with avian blood collected on the same
day when out shooting. These results are incorporated in the following
table. Subsequently (5, iv. 1902) I succeeded in obtaining an anti-
ostrich serum, the tests with which follow those with anti-fowl serum.

It is interesting to note that but a slight action is exerted by anti-
fowl serum upon fowl egg-white dilutions, compared to a very powerful
action upon fowl blood. Gengou (25, x. 1902, p. 753) notes that this
antiserum acts on fowl egg-white, but does not record the remarkable
difference in the amount of the reaction, which I have repeatedly ob-
served. A fairly powerful antiserum may in fact give no reaction with
its corresponding egg-white.

792 Tests with Anti-Fotul Serum.

The following tests were made with two antisera obtained from two
rabbits treated with the serum of Oallus domesticus. Both antisera
were very powerful, giving, when standardized, the one a precipitum
of '05 c.c. with "5 c.c. of a 1 : 40 fowl serum dilution, the other a pre-
cipitum of -035 c.c, with "5 c.c. of a 1 : 100 dilution.

The following table shows a strikingdiffercnce from all of the preceding,
and a great resemblance to the succeeding one with anti-ostrich serum.
We see here an absence of reactions outside of the bloods of Aves, all
of which react, whether they be of Ratitae or of Carinatae. Footnotes
refer to some of the bloods which gave negative results. It is evident
that not much importance can be attached to these when other bloods
belonging to the same family have given even the largest reactions.
It will be noted that the egg-white of the fowl, whose serum was used
for treating a rabbit, gave a much smaller reaction than did the fowl's
blood. Note that Galliformes gave 27Vo> Anserifonnes 26 »/«. of the
largest reactions in their respective families.

202

Tests with Precijntins

Reactions

85 Primates 85

28 CHinoPTERA 28

12 Insectivoba 12

88 Carnivoba 87

61 eodentia 61

70 Unoulata 70

3 Cetacea 3

12 Edentata 12

25 MARsnpiALiA 25

1 MONOTEEMATA 1

320 AvEs

9 Ratitae

Struthionidae, Rheidae
(5 sp.), Casuaiidae ...
311 Carinatae

8 Colymbiformes

1 Procellariformes 1

14 Ciconiiformes (14 sp.) 2*
34 Anseriformes (18 sp.) 1 +
44 Falconiformes (27 sp.) 12 J
30 Galliformes (17 sp.)... 1§

6 Gruifoimes (4 sp.) ...

53 Cliaradriiformes (36 s.) 8 1|

15 Cuculiforraes (12 sp.)

21 Coraciiformes (8 sp.) 1

85 Passeres (60 sp.) 12

4 Errs

1 Ratitae 1

3 Carinatae 2

42 Reptilia 41

13 Amphibia 13

22 Pisces 22

7 Crustacea 7

6 (6fi»/„)

1

7 (50»/„)

3(9«

/o)

13(38,.)

9 (20,

.)

12(27,,)

5(17

.)

8 (27 ,.. )

2

1

10 (19 ,

.)

24 (45 „ )

3 (20 ,

.)

5(33,,)

5(23,

.)

10 (47 „ )

24 (2a ,

.)

46 (54 „ )

Itt

2

8(23"/,)
7(18,,)

8 (27 „ )
3(50,,)
8(15,,)
7(46,,)

3(14,,)
3(4,.)

•

100
0

a

85

9

(26»/„) 97

4

(9,

,)72

8

(27

,) 97
lUO

3

(5.

,) 84
100

a

(9.

) 95
86

0

.

33

•

2
0

0

385
mam-
mals
I)

87»/o

* 449 from India. 453 smeary.

t 487 from India.

J 6 from India. 1 from Japan, rest England.

§ From India.

II 2 from China and India, rest England.

+t Fowl's egg-white.

(2) 649 Tests with Anti-Ostrich Serum.

The antiserum used in the following tests was obtained from a
rabbit by the injection of the serum of Struthio molyhdophanes Reichenow

Anti-Ostrich Serum

203

(No. 428), the bird having died at the Zoological Society's Gardens,
London. The antiserum was very powerful, giving, when standardized,
a precipitum of '042 c.c.

Beactiona

64 Pmmatkb 63

24 Chiropteba 24

10 Insectivora 10

69 Cabnivora 68

51 rodentia 51

51 Unqulata 51

2 Cetacea 2

12 Edentata 12

22 Marsupialia 22

1 monotkemata 1

276 AvES

8 Eatitae
3 Struthionidae (2 sp.)

2 Casuaridae (2 sp.)
3Kheidae (1 sp.)

268 Carinatae

6 Colymbiforme.s (5 sp.) 2

11 Ciconiiformes (11 sp.) 1
2'.( Anseriformes (16 sp.) 6
36 Falcouitormes (21 sp.) 8
24 Galliformes (15 sp.) ... 1

5 Giuiformes (3 sp.)

46 Charadriiformes (34s.) 9

13 Cuculiforraes (12 sp.) 1

22 Coraciiformes (9 sp.)... 4

76 Passerea (56 ap. ) 25

4 Eggs

1 Eatitae

3 Carinatae 2

33 Reptilia

12 Chelonia 10

2 Crooodilia 1

19 Sauria 18

10 Amphibia 10

14 Pisces 14

6 CKnSTACEA 6

3

3

12 (41 »/„)
11 (.10 „ )

13 (54 „ )
3

18 (39 „ )
3 (23 „ )
9 (40 „ )

32 (42 ,

1
1

2
1

5(62«/„)

(2*7o)

1

4

7

13 (36 „
9 (37 „
1

14 (30 „
8 (61 „
6 (27 „

)

)

) 18(24,,)

3

1(4..)

4(9..)
1 ( 8 „ )
3 (13 „ )
M'..)

+

1
0
0

1

0

u

0

u
u

0/

>-8"/o

3«(377„)10U

1(4 7o)

1

i(«7o)

«« ■(

9)
79
77
%
100
80
92
81
67

100

u

17

40

0
0

u

y 79«/„

•27o

* All 3 Struthionidae.

The general results correspond to those given in the preceding
tabic, nevertheless, we see that the bloods of Ratitae (three Struthio-
nidae) give a higher percentage of large reactions than do those of

204 Tests ivith Precipitins

Carinatae. Here we also observe faint cloudings occurring in dilutions
of egg-white both of Ratitae (Emu) and Carinatae. A slight action
would seem to be exerted upon reptilian bloods, all other non-avian
bloods giving practically negative results.

B. Tests with Antisera for Avian Egg-White.

(1) Anti-fowl's Egg.

Myers (14, vii. 1900) treated rabbits with crystallized egg-albumin
of the fowl and obtained an antiserum which precipitated the egg-
albumin of the fowl, and to a slight extent that of the duck. No
reaction was obtained when the antiserum was added to normal rabbit,
bullock, or sheep blood dilutions. Subsequently Uhlenhuth (15, XI.
and 1, XII. 1900) injected fowl egg-white in salt solution intraperitoneally
into rabbits, and obtained a powerful antiserum, which produced re-
actions with egg-white diluted to 1 : 100,000, and weaker reactions
with pigeon egg than with that of the fowl. He concluded from
this that the albuminous constituents of both species of eggs are
closely allied. Mertens (14, hi. 1901) confirmed the foregoing results.
Uhlenhuth (25, iv. 1901) reported later that he had obtained a powerful
anti-egg serum which acted on fowl blood dilutions, and produced
a slight cloud in goose blood. He obtained an immediate precipitation
upon adding the antiserum to 2'5''/o fowl blood dilution. It produced
almost as powerful reactions with egg-white dilutions of goose, duck,
guinea-fowl, though less with the pigeon's than with its homologous (fowl)
egg-white. A rabbit treated with goose egg yielded a serum which
produced a precipitum in goose blood, less clouding in fowl blood,
dilutions. It gave a great and immediate precipitation with goose and
duck, a considerable clouding with fowl, guinea-fowl, and pigeon eggs.
He concludes it will not be possible to differentiate eggs as has been
possible for bloods.

Nuttall (16, XII. 1901, p. 408) found anti-fowl egg serum, when
powerful, to produce a reaction with fowl blood, but never so intense an
action as upon its homologous substance. Levene (21, XII. 1901) treated
animals for two months with egg-white and found their serum to
precipitate egg-albumin, egg-globulin, yolk of egg and fowl and turkey

Anti-FowVs Egg- White 206

serum. Nearly all the preceding observers state that normal rabbit or
other sera produce no such efifect on the dilutions, these being used for
control purposes.

Nuttall (20, I. 1902) reported that this antiserum produced clouds
with a variety of avian bloods (parrot, swan, heron, stork, conure, crow,
emu) also with egg-white of emu, and in addition with blood dilutions
of Alligator sinensis, A. mississippiensis, Chelone midas, and Testudo
ibera, and it might therefrom appear " that the egg possesses a vestige
of reptilian character." In view of the continued negative results
with other bloods I referred to this observation (5, iv. 1902) as "very
suggestive in view of the reptilian origin of birds," and the reaction as
possessing possibly a " reptilian-avian " character. Dr Graham-Smith
has since been pursuing investigations in this direction, and reports
upon them in Section VIII.

Pursuing a different line of research (see p. 94) Gengou (25, x. 1902,
p. 750) found he was unable, according to his method, to find any
difference in the action of anti-fowl egg serum as tested upon eggs of
the fowl, turkey, pigeon, and duck. He was unable (p. 753) to deter-
mine that anti-egg serum possessed an action on fowl blood. As we
have seen, anti-egg only acts upon the corresponding blood when
powerful. Finally Obermayer and Pick (1902) remark that fowl serum
pseudoglobulin contains a body which reacts to anti-egg (from rabbit)
in a similar manner to egg-white.

789 Tests ivith Anti-Foiul's Egg.

The following tests were conducted with two antisera obtained from
two rabbits treated with the white of egg of the domestic fowl. The
one antiserum was moderately powerful, the other exceedingly powerful.
The antisera were standardized, but I should estimate the precipitum
of the stronger antiserum at '06 c.c.

In this table it will be seen that the largest reaction occurs with the
egg-white of the fowl, several eggs having been tested with the same
result. Second-class reactions were obtained with another carinate egg,
with the egg of the emu, with the blood of a heron, and the blood of
two Reptilia (Chelonia and Crocodilia). Otherwise all the medium
and slight cloudings occurred amongst the bloods of Aves and Reptilia,
if we except a single slight clouding in one out of 388 mammalian
bloods tested.

206

Teats with Precipitins

Beactious

389 Mammalia

312 AvEs

8 Batitae

3 Struthionidae (2 sp.)

4 Kheidae (1 sp.)
1 Casuaridae

304 Carinatae

7 Colymbiformes

388

4*

5

1
10
17
41
18

n
49
11
18
80

•

i§

29

10

23

5

1
2

10 (33»/„)
3(10,,)

7 (8 7o)

2
1

1 Procellariformes

13 Ciconiiformes

1

30 Anseriformes

S

42 Falconiformes

9

6 Grniformes

50 Charadriif ormes

16 Cuculiformes

3

21 Coraciiformes

88 Passeres

4 Eggs
1 Batitae

1

3 Carinatae

1

46 Beptilia

13 Chelonia

?

3 Crocodilia

1

30 Sauria

10 Amphibia

23 Pisces

5 Crustacea

•

(lO'/o)

It

* 4 Bheidae.

t Egg-white of fowl, several specimens of which gave same result.

t 8 of these dried on filter-paper. (See text, p. 207, top.)

§ No. 815 dried on paper, sent from Central Africa.

la
0 2

SO

100
100

23
66
3
0
0
0

r>7 7o

(2) 630 Tests with Anti-Emus Egg-White.

The antiserum used in the following tests was obtained by treating
a rabbit with the egg-white of Dromaeus novae-hollandiae Vieill., the
emu (No. 799), received from the Zoological Society's Gardens, London.
This antiserum was fairly powerful. Standardized by Dr Graham-Smith,
it gave a precipitum of 028 c.c. when tested after being stored 5 months
on ice, 1 c.c. of antiserum being added to '5 c.c. of 1 : 21 solution of
emu egg-white.

The preceding table shows that the antiserum for the egg-white
of the emu only produced a large reaction with the dilution of the

Anti-Emu's Egg- White

207

same egg-white with which the rabbit had been treated, viz. with that of
the emu. A less marked reaction occurred with the egg-white of one
of the Carinatae, whereas medium and faint cloudings occurred when
the antiserum was added to the bloods of a number of Aves, as well as
in two out of six bloods of Reptilia (Chelonia). The negative results
with other bloods of this class may be due to their partial insolubility
when dried.

Beaotions

309 Mammalia 309

258 Aves

8 Batitae 2 4 2

250 Carinatae

5 Colymbiformes 5

11 Ciconiiformes 7 13

27 Anseriforraes 25 2

32 Falconiformes 29 . 3

28 Galliformcs 25 3

5 Gruiformes 4 1

43 Charadriiformes 35 4 4

20 Coraoiif ormes 18 1 1

12 Cuculiformea 12

67 Passores 61 6

4 Eggs

1 Batitae

3 Carinatae . . 2

31 Eeptilia

6 Chelonia 4t • 2

2 Crocodilia 2

23 Sauria 23

7 AMpurBiA 7

16 Pisces 15

6 Crdbtacea 6

/o
0

75 ^

0
36

7

9
11
20
18
10

0

1* 100

100

m
u

0
0
0
0

»"/.

* Emu's egg-white with which rabbit was treated.

t Three out of the four were dried samples, on paper (see text above).

IX. Antisera for bloods of Reptilia.

(1) 666 Tests with Anti-Turtle Serum.

The following tests were conducted with two antisera obtained from
two rabbits treated with the serum of (No. 809) Chelone midas, the
Green Turtle, a specimen of which was killed in Cambridge. Both
antisera wore very powerful, one giving a precipitum of "03 c.c.

208

Tests ivith Precipitins

Beactions

323 Mammalia 323

262 AvES 262

4 Eggs of Birds 3

34 Beptilia

7 Subclass Chelonia 2* 1

3 Subclass Crooodilia 2t 1

24 Subclass Sauria 24

14 Amphibia 14

22 Pieces 22

T.Crcbtacea 7

* Both dried, one from Central Africa.

+ One dried.

/o
n

0
25

71

3S

0

0

n

0

The reactions of the first and second class will be seen to be confined
to bloods belonging to Clielonia, medium and faint reactions were
observed only amongst Crocodilia and avian egg-white dilutions. In my
paper of 20, l. 1902 I reported the first series of 2.50 bloods tested with
this antiserum, having found that the blood of Testudo ihera gave
a marked clouding, and of A. mississippienms a faint clouding. These
tests are included in the above table. For further bloods tested see
the large tables at the end.

(2) Tests with Anti-Ophidian Sera.

In view of the extensive work involved in the production of other
antisera, I have been unable to produce anti-snake sera. Dr Graham-
Smith has been carrying on a series of tests with these, which he
reports upon in Section VIII. The observations of Lamb (16, viii. 1902)
on precipitins for cohra venom fall naturally under this head.

Lamb treated rabbits subcutaneously for 4 — 5 months with injections
of pure unheated cobra venom, the injections being made at intervals of
about 10 days. At the time when precipitins appeared, the rabbit could
withstand four times the lethal dose of venom. Using Wright's sedi-
mentation tubes, he added 4 volumes of antiserum to 1 volume of 1 "/o
cobra venom solution, and obtained a reaction leading to a marked pre-
cipitum after 24 hours. An excess of antiserum added to weak venom
solutions gave the most precipitum. The precipitin was active for
those proteids in venom which are incoagulable by heat. Moderate
heating of the antiserum does not affect its action. A 0'2 "/o solution of
cobra venom, heated to 75° C. for 30 minutes, after which the coagulated
proteids were removed by filtration, gave as much precipitum as unheated

Anti-Reptilian Sera 209

venom solutions which were used as controls. Daboia venom gave the
same result. Cobra antiserum heated for 30 minutes at 55° remained
unaffected, as did also daboia venom. Six parts of the antiserum added
to one part of -05 "/„ of venom solution used up all the precipitin. I.^mb
considers that the method might be used for the standardization of anti-
sera for therapeutic purposes, the test being more exact than in animals.
Cobra venom antiserum was tested upon the venoms of

Daboia rmellii (Indian Chain viper) Reacted

Echis carinuta (Fhoorsa, small Indian Viper) No reaction
Bungarus fasciatus (Banded Krait, India, colubrine family) „

Hoplocephalm curtus (Australian Tiger Snake, colubrine family) „

The similar reactions obtained with cobra venom and that of daboia
would, according to Lamb, indicate that the venoms are alike, but he
and Hanna (Journ. Pathol, and Bacterial. 1902, viii. p. 1) have shown
their physiological action to be quite different, and they found Calmette's
antivenine to protect against cobra but not daboia venom. If the
proteids are identical then the venom action will not depend on these,
or else they possess the same haptophoric group. Nevertheless, Lamb
adds that, on inspection of the protocols, it is seen that cobra venom
gave a precipitum in higher dilutions than did that of daboia. These
investigations are of considerable interest in relation to my work. It
will be doubtless found that the injection of the serpents' bloods will
yield precipitins having a similar action. Such a line of investigation
appears to me indicated in view of the small amount of precipitin-pro-
ducing substance present in venom compared with its toxic action.

(3) 468 Tests with Anti-Alligator Serum.

The following tests were made with one antiserum obtained from
a rabbit treated with the serum of Alligator mississippiemis (No. 813),
the reptile having died at the Zoological Society's Gardens, London.
The antiserum was moderately powerful.

We see from the table (p. 210) that the action of anti-alligator serum
was confined, so far as large reactions go, to Crocodilia; medium reactions
took place with the bloods of a large proportion of Chelonia, a bird's
blood, and egg-white. I may add here that the interaction of anti-avian
egg sera and reptilian bloods, and vice versa, has been amply confirmed
by Dr Graham-Smith, who at my suggestion has investigated this
interesting avian-reptilian reaction, as I termed it elsewhere (p. 205).
Dr Graham-Smith reports upon his results, which are mainly quan-

U

210

Tests with Precipitins

titative, in Section VIII. In my preliminary communication of 20, i.
1902, 1 reported that I had tested 250 bloods with the result that those
of Alligator Tnississippiensis and A. sinensis gave reactions, a clouding
occurring in the blood of Ghelone midas. The tests with the alligator
and turtle sera were repeated several times with uniform results, these
being included in the tables.

Reactions

237 Primates 237

174AVE8 173 . 1

3 „ eggs 3 . 1

20 Eeptilia

6 Chelonia 2t

3 Crocodilia IJ

11 Sauria 11

12 Amphibia 12

15 Pisces 15

7 Crustacea 7

t One from Central Africa, dried. The other dried.
J From Central Africa.

/o
0
•5
2S

66
0
0

0
0

X. Antiserum for bloods of Amphibia.

551 Tests with Anti-Frog Serum.

The following tests were made with two antisera obtained from two
rabbits treated with the blood of freshly killed frogs (Rana temporaria).
Both antisera were weak. They were not standardized.

Reactions

247 Mammalia 247

239 AvES (incl. 4 eggs) 239

27 Beptilia 27

13 Amphibia

3 Order Urodela 3

1 Order Anura, Suborder Aglossa 1

Suborder Phaneroglossa

2 Bufonidae (2 spec.) 2

1 Engystomatidae 1

6 Eanidae (2 spec.)

18 Pisces 18

7 Crustacea 7

§ Rana temporaria.

7o

0
0

0

0
0

0
0
100
0
0

Anti-Frog, Fish and Lobster Sera 211

The foregoing table shows that the antiserum for Rana temporaria
only produced a large reaction in the blood dilutions of this species, and
that it also produced a reaction with the blood of Rana tigrina, the
results with all other bloods being absolutely negative.

In my paper of 5, iv. 1902 I reported upon tests made on 508
bloods with this antiserum, these bloods being now included in the
above table. The only investigator who has worked with anti-amphibian
sera is Philippson (about Sept. 1902), who confined himself to a few
tests. He cites my paper, and states that he has been able to
confirm my results. He injected 1 c.c. of defibrinated frog's blood {Rana
viridis var. esculenta) into a rabbit, making two such injections with
a week's interval. He found his antiserum to precipitate the serum of
Rana viridis var. ti/pica, and that of R. fusca, but it had no effect on
that of Hyla arborea, Bufo vulgaris and Salamandra maculosa. The
antiserum had also no effect upon the serum of the pig or calf. I would
remark that his antiserum must doubtless have been weak after only
making two injections and, what is more, introducing such small amounts
of frog blood. In my experience, I have not noted the presence of pre-
cipitins in the serum of animals prior to the third injection, and then
only in small amount. I did not however make a note as to the earliest
period at which my rabbit showed precipitin when treated with frog's
blood.

XI. Antisera for bloods of Pisces.

Tchistovitch (v. 1899) made his fundamental discovery upon the
blood precipitins by treating the rabbit, guinea-pig, goat, or dog ^vith
eel serum. Tests upon different fish bloods are being made the subject
of study by Dr Graham-Smith at my suggestion. The results should
be of considerable interest. With the exception of de Lisle (xi. 1902,
p. 399) who, following the method of Tchistovitch, also obtained anti-eel
serum from rabbits, no further work has as yet been done. De Lisle
(p. 403) obtained precipitins by injecting either eel serum or eel
corpuscles.

XII. Antiserum for bloods of Crustacea.

460 Tests with Anti-Lobster Serum.

These tests were made with one antiserum derived from a rabbit
treated with the blood of the lobster (Hoinarus vulgaris). The anti-
serum was fairly powerful.

14—2

212

Tests with Precipitins

BeactioDS

+

+ »/o

0
0
0
0
0
100

210 Mammalia 210

191 AvES (inel. 4 eggs) 191

17EEPTILIA 15 2 (?)

12 Amphibia 10 2 (?)

14 Pisces 14

6 Crustacea, all of the order Decapoda • 1

Note: One sample of crustacean blood noted in the tables is excluded (No. 897), as
the foam-teat of the dilution was doubtful.

From the above table it will be seen that the anti-lobster serum only
produced first and second-class reactions with crustacean bloods. The
faint clouding in one of the six examined was doubtless due to the
small quantity which went into solution from filter-paper. A faint
clouding was observed to take place in two reptilian and two amphibian
blood dilutions, the test in this case being unfortunately not repeated
owing to lack of antiserum. Dr Graham-Smith has extended the studies
in this relation also (see Section VIII). Otherwise these are the only
investigations made with anti-crustacean sera. I might add that I first
reported having produced such an antiserum in my paper of 20, I. 1902,
and briefly referred to the results above given in my paper of 5, ix. 1902.

Summary of Bloods Tested

213

The Number of Species of Blood tested.

Of the 900 specimens of blood collected by me, about one-third
were duplicates, the number of species represented being 586 or more,
including four races of man under the species, but not several breeds of
dog and sheep, as follows : —

No. of No. of
Samples Species
Mammalia

Priviates

Antbropoidea

Hominidae 35 4 (races)

Simiidae 8 3

Cereopithecidae 36 26 3 doubtful speoiei

Cebidae 13 9

Hapalidae 4 3

Lemuroidea

Lemuridae 2 2

Chiroptera 30 25 1 doubtful

Insectivora 15 4

Carntvora, incl. Pinnipedia 99 55 6 doubtful

Rodentia 69 39

Ungulata 73 50 3 doubtful

Getacea 3 2

Edentata 13 10

Marsupialia 26 16

Monotremata 1 1

AvEs

iiatitee, incl. 3 families 9 6 1 doubtful

Carina(a«, incl. 11 Orders 319 213

Avian Egg-white 4 4

Eeptilia

Chelonia 13 9

Crocodilia 3 3

Sauria 37 29

Amphibia

Urodela 3 3

Anura 11 6

Pisces

Elasmobranchia and Teleostei (over 14 families) 25 24
Crustacea

Decapoda (Maorura and Braohyura) 7 7

Bloods which proved insoluble have not been included in the fore-
going list.

214 Tests with Precipitins

Note regarding the Classification of Animals adopted in the Tables.

As will be seen, the bloods tested have been ordered according to
the zoological classification of the animals which yielded them. For the
Mammalia, I have followed Flower and Lydekker ; for Birds, see Evans ;
for Reptilia and Amphibia, see Gadow; for Fishes, see Giinther; for
Crustacea, see Glaus: the works referred to being as follows: —

Flower, W. H., and Lydekker, R. (1891). An Introduction to the Study of Mammals,
Living and Extinct. (London : Adam and Charles Black.)

Evans, A. H. (1899). Birds. The Cambridge Natural History, vol. ix. (London :
Macmillan and Co., Limited.)

Qadow, H. (1901). Amphibia and Reptiles. Ibid. vol. viii.

QtJNTHER, A. C. (1880). An Introduction to the Study of Fishes. (Edinburgh : Adam

and Charles Black.)
Clads, C. (1887). LehrbiKh der Zoologie. (Marburg and Leipzig : Elwert'sche

Buchhandlung. )

On several occasions, when in doubt, recourse was had to the British
Museum Catalogue.

General Summary of the Results of 16,000 Precipitin-Tests
conducted by g. h. f. nuttall.

I. Tests with Anti-Primate Sera.

These tests were conducted by means of antisera for man (825 tests),
chimpanzee (47 tests), ourang (81 tests), Cercopithecus (733 tests).
Maximum reactions were only obtained with bloods of Primates. The
degrees of reaction obtained indicate a close relationship between the
Hominidae and Simiidae, a more distant relationship with the Cercopi-
thecidae, the bloods of Cebidae and Hapalidae giving still smaller
reactions than the last, when we consider the results obtained with
the first three antisera. The tests with antiserum for Cercopithecus
gave the largest reactions with bloods of Cercopithecidae, next with
those of Hominidae and Simiidae, but slight reactions with those of
Cebidae and Hapalidae. All four antisera failed to produce reactions
with the two bloods of Lemuridae tested, except when sufficiently
powerful to also produce reactions with other mammalian bloods. From
this we may conclude that the Lemurs properly belong to an Order
separate from the other Primates.

Summary of Results 215

II. Tests with Anti-Insectivore Sera.

These tests were conducted with but a single antiserum, 383 bloods
being examined. Maximum reactions were only obtained with the
blood of Erinaceus, whilst slight reactions were only obtained with a
very few (3'5 7o) of all the other bloods examined, although the anti-
serum was very powerful.

III. Tests with Anti-Carnivore Sera,

Four antisera were used in this group : anti-cat (785 tests), anti-
hyaena (378 tests), anti-dog (777 tests), anti-seal (358 tests). The
results in all four cases agreed in showing a preponderance of larger
reactions amongst the bloods of Camivora, as distinguished from other
Mammalia, maximum reactions usually taking place amongst more
closely related forms in the sense of descriptive zoology.

IV. Tests with Anti-Ungulate Sera: (a) Artiodactyla.

1. Anti-pig serum (818 tests) only gave maximum reactions with
bloods of Suidae, moderate reactions with those of Pecora and Tylopoda.
Of the bloods of the Artiodactyla, as a whole, 66 "/o reacted in some
degree. Owing to the power of the antisera used a "mammalian
reaction " was frequently observed, 51 % of the non-ungulate bloods
giving slight reactions. All of the three cetacean bloods examined
gave a moderate or slight reaction.

2. Anti-llama serum (363 tests) only gave maximum reactions with
its homologous blood, moderate reactions with the bloods of an allied
species and that of Camelus. The percentage of slight reactions was
high only amongst Ungulata.

3. A considerable number of antisera were prepared for Pecora,
and tested on a large number of bloods :

Antiserum for No. of testa Besult showed

Close connection between Cervidae and Bovidae, next to other

Artiodactyla, Cetacea, Equidae
The same
Close connection between Cervidae and Bovidae, next to other

Artiodactyla, Cetacea
The same
Close connection between Cervidae and Bovidae, next to

Cetacea, Equidae, Suidae
Sheep 701 Close connection between Cervidae and Bovidae, next came

other Artiodactyla, Equidae, Cetacea

Mexican-deer

749

Reindeer

69

Hog-deer

699

Antelope

686

Ox

790

216 Tests with Precipitins

(b) Perissodactyla.

4. Tests with Anti-equine sera.

These tests were conducted with three antisera for the bloods of the
horse (790 tests), donkey (94 tests), and zebra (94 tests). The antisera
only gave maximum reactions with equine bloods, slight reactions only
with bloods of some AHiodactyla and Cetacea. (Time limit.)

V. Tests with Anti-Cetacean Serum.

Anti-whale serum, tested on 94 bloods, gave a maximum reaction
only with cetacean bloods, slight reactions with those of Pecora and
Suina, and some faint cloudings with those of other Mammalia.

VI. Tests with Anti-Marsupial Serum.

A weak anti-wallaby serum, tested on 691 bloods, showed a close
relationship amongst Marsupialia, exclusive of the carnivorous Thy-
lacine. Only a few other bloods gave slight reactions.

VII. Tests ivith Anti-Avian Sera.

1. Tests were made by means of antisera for the blood of the
fowl and ostrich upon 792 and 649 bloods respectively. They
demonstrated a similarity in the blood constitution of all birds, which
was in sharp contrast to what had been observed with mammalian
bloods when acted upon by anti-mammalian sera. Differences in the
degree of reaction were observed, but did not permit of drawing any
conclusions. Slight or faint reactions were observed with some
reptilian bloods ; only a very few mammalian bloods (0 "/o ^^d 0'6 %
respectively) showed faint reactions.

2. Tests made with antisera for egg-white of the fowl (789 tests)
and emu (630 tests) gave maximum reactions with the egg-white of
birds, moderate reactions with avian bloods, and distinct but slight
reactions with reptilian bloods, notably those of Crocodilia and Chelonia.

VIII. Tests with Anti-Reptilian Sera.

Two antisera were used, the one for the blood of Chelonia (666 tests),
the other for Alligator (468 tests). The first produced maximum
reactions with chelonian bloods, lesser reactions with crocodilian, and
slight reactions with avian egg-whites. The anti-alligator serum
produced maximum reactions with Crocodilia, followed by Chelonia,
avian egg-whites, and least with avian bloods. (See also Section VIII.)

Key to Signs in Tables 217

IX. Tests with Anti-Amphibian Serum.

Anti-frog serum, tested on 551 bloods, only gave reactions with the
bloods of Ranidae.

X. Tests with Anti-Crustacean Serum.

Anti-lobster serum, tested on 450 bloods, only gave reactions with
the bloods of Becapoda.

Key to Signs used in the Tables.

In the following tables, the animals are placed in their zoological
order, the blood samples tested being numbered continuously in the
first column, in the second column in the order in which they were
tested. As far as possible the scientific and common names of each
animal have been given, this being followed by its habitat. The
collector's name is placed in brackets. Where the bloods from one
source are numerous, the name is represented by an inital, thus (Z)
denotes that the blood was collected at the Zoological Society's Gardens,
London; (R) stands for de Rothschild; (N) for Nuttall; (F) for Foster;
(L. C.) for Dr Louis Cobbett ; (G.-S.) for Dr Graham-Smith ; (Lane)
and (Mitchell) are two of our laboratory attendants. Where the habitat
of the animal or the collector's name is omitted refer to the preceding
samples for particulars.

Where not otherwise stated, the bloods were all collected and dried
on filter-paper. Fluid sera are indicated by the abbreviations " fl." or
" fl. in cap." (cap. standing for capillary tubes). Some sera were dried
on glass, and the dry scales kept bottled and used for tests ; such are
described as " scales." In some cases the fluid serum had been filtered
(fl. ser. filt.), in others it is stated that the blood sample was collected
from a wound or cut, and again from what disease the animal died,
etc.

The names of the antisera used are given at the top of the columns
on the right. The antisera obtained from rabbits are named according
to the animal's blood or serum with which the rabbit had been treated.
These antisera are also ordered zoologically from left to right in a
manner corresponding to the whole series of bloods.

218

Tests ivith Precipitins

The order of the antisera at the top of the page will serve therefore as
an index to the order of the series of bloods tested. The columns
giving the reactions have been grouped in blocks in their order of
classification, so as to facilitate a comprehension of the reactions taking
place, for instance amongst the Ungulata, as an order. As long as
Mammalian bloods are under consideration, the anti-mammalian sera
are thus grouped; the order to which the antiserum belongs being
given at the foot of the page. When we come to the test upon avian
bloods on the other hand, all columns referring to the tests with anti-
mammalian sera are included in one " block."

By reading from left to right the reactions given by any one blood
with the different antisera will be gathered, whereas if the columns are
read from top to bottom, the reactions given by each antiserum with
the different bloods in succession will be gathered.

The reactions which took place between each antiserum and each
blood are included in the space formed by the intersection of the lines
which include the name of the animal whose blood was tested (on the
left), and those which include the name of the antiserum (above). The
degrees of reaction are indicated by the following signs : —

+ denoteti great reaction
+ ,, marked clouding
X „ medium clouding
• „ faint clouding

• denotes no reaction

/ „ blood insoluble

\ ,, blood gave clouded solution

The time when the reaction was noted is stated below the above
signs in minutes, anywhere from 1 to 240 (4 hrs.). In a number of my
earlier tests, the observations were not extended beyond 30 minutes,
and the deposits taking place after 16 — 24 hours were not studied.
This was not the case however in most of the tests. The amounts of
deposit noted on the following day are indicated by the letters beneath
the sign for the early or immediate reaction, noted above, as also
beneath the number of minutes, as follows : —

D denotes very large deposit

D „ moderate deposit

d „ small deposit

tr „ trace of deposit

0 „ cloudiness persisting after 16 — 24 hrs.

In the earlier tests where a reading of these deposits was omitted,

their significance not being appreciated at the time, the probable

Key to Signs in Tables 219

amount of deposit which would have been noted is indicated by the
above letters reversed.

To make the matter clearer by a few examples, I state the meaning
of the following signs.

+ )

30 \ means marked olouding in 30 minutes, (race of deposit after 16 — 24 hra.

tr]

• I (no time being noted) denotes marked clouding in S — 15 minutes, moderate

D| deposit after 16 — 24 hours

no reaction, usually np to 5 hrs, and no deposit subsequently

From a medico-legal standpoint, no reactions other than those of the
first class should be accepted, control tests being naturally made with
other antisera of corresponding power for their homologous bloods.

Mammalia

Class

MAMMALIA

4)

1

Order PRIMATES

1

be

£•

■s

g

ANTISERA FOB

a

1

a

3

o

1

1

n

5

a

1

QQ

1 . Suborder

1

14.

Homo sapiens. Woman, autopsy, scales

+

ANTHROPOIDEA

2

34.

CarabridHe (N) 15. iii. 01
„ ,, Man, autopsy

a

+

*

tr

.

•

.

Fam.

(N)2. iii. 01

a

120

Hominidae

3

35.

,, ,, putrified, dried in scales

4-

d

•

•

European

(N) 15. iii. 01
,, „ W. M., cut, fl. in cap.

a

4

36.

+

(N) 13. iv. 01

a

6

37.

,, ,, Dr D., cut, fl. in cap.
(N) 23. iv. 01

+
a

6

38.

,, ,, Dr N., cut, fl. in cap.
(N) 21. iv. 01

+
a

7

39.

„ „ W. M., cut, fl. in cap.

8

408.

,, ,, Serum from Blister,

Dr N.'s foot (N) 20. iv. 01

+
a

8

409.

,, ,, Serum from Burn-blister
Dr N. (N) 23. iv. 01

+
a

lO

410.

DrD., cut
(N) 23. iv. 01

X

15
d

120
d

180

180
tr

11

413.

W., cut
(N) 17. vii. 01

X

15
D

120
d

180
tr

'

180
tr

13

415.

,, „ Ascitic Fluid from autopsy
(N) 23. X. 01

+
D

10
d

180
tr

'

180

13

416.

W. M., cut
(N) 28. X. 01

X

15
D

+

a

120
d

*

45
d

*

180
tr

1«

417.

B. C, cut
(N) 29. X. 01

+
15
D

120
d

120
d

*

tr?

15

418.

„ „ A. C, out

(N) 4. xi. 01

+
15
D

120
d

180
tr

■

•

16

420.

A. C, cut
(N) 13. xi. 01

+
15
D

120
d

180

tr

'

•

17

422.

DrN., cut
(N) 10. i. 02
A., cut

+
D

•

•

•

18

449.

+

»

«

.

•

*

X

(N) 18. i. 02

D

120
d

45
tr

45

40

tr

19

475.

W. M., cut
(N) 30. i. 02

+
D

120
d

40

*

#

«
45

40

tr

ao

575.

A. C, cut

+

«

*

.

•

»

«

(N) 18. ii. 02

30

D

120
d

45

45

75

40

tr

21

576.

DrN., cut
(N) 18. ii. 02

+
30
D

+

120

22

609.

„ „ B. C, cut

«

«

•

,

«

X

(N) 4. ui. 02

S

120
d

200

tr

?
85

85

23

610.

C. S., cut
(N) 10. iii. 02

+
D'

240
tr

200
tr

■

•

X

20

X

85

24

702.

DrN., cut
(N) 8. V. 02

+
D

/

200
tr?

•

85
tr

*

85
tr

Primates

lO Bloods 10 — 16 were tested 8 Nov. 1902 with the 9 antisera noted.

220

Insecti-
vora

Camivora

Mammalia

Avee

Reptilia

Amphibia Crustacea

?

300

tr?

tr

120

120

tr

tr

tr

Ungulata

Marau-
pialia

la Solution less diluted than in tests 10 to 16.

18, ao Anti-pig semm very powerful.

221

Mammalia

Class MAMMALIA

1

I. Order PRIMATES

a

a

b«

s-

§■

g

ANTISERA FOR

1

s

6

B

3
O

i

n

fi.

K

1

1

O 1 l-M

1 . Suborder

25

778. Homo sapiens. G. N., cut

-1-

«

ANTHROPOIDEA

(N) 12. vi. 02

15
D

120
d

Fam.

as

779. „ „ E. G., cut

+

«

,

,

,

Hominidae

(Gardner) 13. v. 02

15

240

European

27

785. ,, „ C. C, wound
(N) 16. V. 02

D

+
30
tr

tr

/

•

•

tr?

•

28

786. „ „ B. C, cut
(N) 31. V. 02

+
30
d

240
tr

3*0
tr

•

tr?

•

Mongolian

39

ao

327. ,, „ Chinaman, beri-beri

Loudon (Daniels) 14. xii. 01

380. ,, „ Chinaman, beri-beri

Shanghai, China (Stanley)
ca. 27. xi. 01

+

a

+
a

240
tr

240

tr

4*0
40

•

*

15

tr?

tr?

•

E. Indian

31

381 Punjaub Sikh

Shanghai, China (Stanley)
20. X. 01

+
a

240
tr

tr

•

32

328 E. Indian, Punjaub

London (Daniels) 13. xii. 01

+
a

240

tr

40

*

#

15
tr?

•

Negro

33

819 Negro

+

»

X

,

X

«

Lagos, Africa (Strachan)

30

?

30

15

15

35

16. ii. 02

d

tr

tr?

tr?

tr

34

820

+

*

X

^

X

«

16. ii. 02

30
d

?
tr

30

15

tr?

15

35
tr

36

821. „

-1-

«

X

,

X

16. ii. 02

30
d

?
tr

80

15

tr?

1*5

35

tr

Fam.

36

254. Simia satyrvs L. Ourang-outang

+

+

+

«

*

*

X

Simiidae

Borneo (Z) 30. x. 01

D

45
D

D

41
d

320

90

35

37

297

(Griinbaum) 01,/. & dry

+
D

d

•

*

?
15

tr?

•

38

906.

(Z) 14. X. 02, fl.

+
S

+
1
D

180
tr

•

tr

180

tr

39

237. Anthropopithecus troglodytes. Chimpanzee

+

+

+

*

«

,

»

,

«

W. Africa (Z) 16. x. 01

D

45
D

D

15
d

320

35
tr

?
46

40

298. Anthrnpopithecui troglodytes

(Griinbaum) xi. 01, fl. and dry

+
S

*

d

+
20

•

•

*
?
15

•

•

41

329. Anthropopithecus troghdytes

+

+

X

*

.

tr

X

•

(Griinbaum) 7. xii. 01

a

45
D

15
D

40
d

15
tr?

43

496. Anthropopithecus troglodytes

+

-1-

,

,

+

,

«

(Griinbaum) 12. ii. 02

D

5
D

tr

45

tr

d

40

43

299. Gorilla savagei. Gorilla

Equatorial Africa (Griinbaum) xi. 01

+

a

tr

40

*
?
80

Primates Insecti- Carnlvor*

vora

43 Anti-pig very powerful.

222

Mammalia

Avt'H

Reptilia

Amphibia Cr

ustace

1

•?

s

1

1

w

Q

1

s

i

■a

1

1

1

(X

1

3

s

9

i

<

1

3

•

"7

.

.

.

,

•

60

tr?

tr

tr

tr

tr

tr

tr

«

•

•

•

•

•

•

•

•

60

tr

tr

tr

tr

tr

tr

»

•

•

•

.

40

tr

»

,

«

•

,

?

240

40

tr

X

*

•

•

•

*

•

•

■

■

'

■

•

•

•

•

•

•

•

•

.

•

•

•

'

/

/

•

•

•

/

•

•

•

•

X

•

65

•

•

•

•

•

•

•

•

•

X

#

.

«

40

50
tr

240
tr

X

«

•

»

40

45
tr

240
tr?

X

«

■

*

40

45
tr

240

tr?

+

•

*

,

,

,

,

.

.

.

.

.

d

•

90

•

•

•

•

•

•

•

•

•

#

tr

35

tr

«

.

,

*

.

.

.

•

"

•

•

•

■

d

60

•

•

•

60

•

•

,

•

•

•

'

X

•

•

.

'

•

•

•

•

•

•

+

•

«

X

«

,

X

.

.

D

300

d

•

•

120
d

■

•

•

•

Ungul&ta

Marsu-
pialia

223

Mammalia

Class

MAMMALIA

1. Onhr PRIMATES

ANTISEBA FOE.,

1 . Suborder
ANTHROPOIDEA

Fam.
Cercopithecidae

44
45

46
47
48
49

SO
61

sa

364.
501.
703.
287.
784.
789.

98.
224.

255.

63

99

64

59.

66

97

66

124

67

907

68

908

59

790

60

118

61

918

63

58

Cynocephalus mormon. Mandrill
W. Africa (Z) 10. xii. 01, /.

Cynocephalus tphinx Linn. Guinea Baboon
W. Africa (Z) 14. ii. 02, fi.

Cynocephalus babitin Desm. Baboon
Africa (Liihe, Konigsberg Z) 10. v. 02,/.

Cynocephalus porcarius Bodd. Chacma
Baboon

S. Africa (Z) 18. xi. 01
Cynocephalus porcarius

(Z) 4. vii. 02, d. intussusception, fl.

Gelada Baboon, d. of tuberculosis

? (New York Zoo., Langmann) 5. v. 02

Macacus cynomolgus. Macaque Monkey

India (Rogers) 1. vii. 01
Macacus cynomolgus

(Z) 21. viii. 01

Macacus ocreatus. Ashy-black Macaque
India (Z) 30. x. 01

Macacus assatuiensis

Bhootan (Rogers) 1. vii. 01
Macacus rhesus. Rhesus Monkey

India (Z) 11. v. 01

Macacus rhesus. Rhesus Monkey
(Rogers) 25. vi. 01

Macacus rhesus. Rhesus Monkey
(Z) 19. vii. 01

Macacus rhesus. Rhesus Monkey

d. in Engl. (L. C.) 02, tuberculosis,
septicemia ; Jl. ser.
Macacus rhesus. Rhesus Monkey

d. in Engl. (L. C.) 16. x. 02, dysentery;
fl. ser.
Bonneted Macaque Monkey, d. tuberculosis
? (New York Zoo., Langmann) ca.
14. vi. 02

Cercopithecus melogenys. Black-cheeked
Monkey

W. Africa (Z) 22. vii. 01
Cercopithecus spec. ? Monkey

Brit. Centr. Africa (Dodds) p. 23. viii. 02

Cercopithecus lalandii Is. Geoffr.
Monkey

S. Africa (Z) 15. v. 01.

Vervet

+
b

+

180^
tr ;

+

b I

15
d
+
1
d

15
d

«

15
d

+
1
D

120
d

120
d

-I-
60
d

180
tr

«

60
d

?
320
«?
45

30

tr

180
tr

180
tr

Primates Insecti- CarBivora

vera
44 Note time of reaction with anti-Mexican-deer.

46, 61, 62, 66, 60 Anti-pig serum very powerful.

48 Note cause of death.

49 Anti-human powerful ; note negative results with Nos. 59 and 75. Filter-paper not mine,
may be the cause of negative result? A second test with anti-monkey gave . tr ? Also possibly
over-exposed to sun.

224

Mammalia

+
45
tr

tr?

^

a

s

1

^

i

a

a

s

<

30

120
d

Ungulata

tr?

tr?

90

Marsu-
pialia

Aves

Reptilia

Amphibia Crnetacea

68 Great reaction with x man, doubtless due to dysentery causing blood concentration.

59 See note to 49.

ai Reaction retarded because of little Roing into solution.

225

16

Mammalia

Class

MAMMAL A

S

1

. Order PRIMATES

a

a

S

1

a

a

a

g

a

^

%

to

«

ANTISERA FOB

i

o

s

1

6

a

o

1

1. Suborder

63

53.

Cercopithecus campbelli Waterh. Camp-

+

*

*

+

*

.

ANTHROPOIDEA

bell's Monkey

W. Africa "(Z) 25. iv. 01

b

15
d

60
d

15

tr?

Fam.

64

75.

Cercopithecus diana. Diana Monkey

+

*

X

-1-

«?

*

.

Cercopithecidae

W. Africa (Z) 25. v. 01

b

5
d

60
d

320

30

tr

65

606.

Cercopithecus (Papio) hamadryas (L.), juv.
" Mantelpavian," liab. Arabia & Abyssinia
(Liihe) 14. ii. 02

+
D

*

45
tr

*

15
d

+
45
D

■

*
H5

"

66

286.

Cercopithecus mona. Mona Monkey
W. Africa (Z) 20. xi. 02

-i-
b

45
d

120
d

-1-
60
tr

'

tr

"

67

515.

Cercopithecus pygerythrus F. Cuv. Mo-

-t-

.

»

X

.

#

.

zambique Monkey

S

45

15

45

45

tr

E. Africa (Z) 14. ii. 02

d

d

D

tr

68

742.

Cercopithecus petaurista Schreb. Lesser
White-nosed Monkey
W. Africa (Z) 10. vi. 02 healthy, fl.

+
15
D

45
d

X

45
D

85

tr

69

52.

Cercopithecus patas. Patas Monkey
W. Africa (Z) 24. iv. 01

-1-
b

#

X

5
d

X

+
60
d

*

•

■

•

•

70

238.

Cercopithecus patas

+

*

,

X

.

(Z) 15. X. 01

b

5
d

10

35
tr

71

111.

Cercopitliecus callitrichus Is. Geoffr. Green

+

«

#

-f

»

X

.

Monkey

b

45

5

60

?

30

W. Africa (Z) 31. vii. 01

tr

d

tr

320

tr

73

895.

Cercopithecus callitrichus

(Z) 13. ix. 02, d. of pneumonia

+
30
tr

#
5
d

-1-
30
tr

tr

73

270.

Cercopithecus callitrichus
(Z) 12. xi. 01

+
b

*

*

5
d

10
tr

■

tr?

'

74

382.

" Small Chinese Monkey "

Canton, China (Stanley, Shanghai)
4. xi. 01

+
b

#

5

d

4*0

•

#
•f

15

•

76

788.

Monkey, d. of enteritis

? (New York Zoo., Langmann) 30. iv. 02

'

tr

3*0
tr

■

tr

•

76

271.

Semnopithecm melalophos. Simpae Monkey

+

,

^

a

«

,

Sumatra (Z) 10. xi. 01

b

tr

10
d

80
tr

77

747.

Seninopithecus rubicundus

Sarawak, Borneo (Hose) posted 10. iv.02

+
60
tr

tr

*

45
tr

«
•>

300

tr

tr

78

100.

Seninopithecus entellus

Sangur, India (Rogers) 1. vii. 01

+
b

tr

•f
60

•

•

79

146.

Seninopithecus entellus

India (Copenhagen Zoo., Schierbeck)
23. ix. 01

+
b

•

*

15
d

6*0
tr

*

30

tr

'

Fam.

Cebidae

80

88.

Mycetes seniculns. Red Howler
Colombia (Z) 24. vi. 01

X

*

9

45

*

15
d

X

100
tr

*
?

320

30

tr

'

81

246.

Uacaria ruhieonda. Red-faced Ouakari
Upper Amazons (Z) 21. ix. 01

X

»

tr

-1-
20
tr

■

*

25
tr

■

82

288.

Nyctipithecus trivirgatus. Three-banded
Douroucouli

+
b

'

*
15

*

?

•

*
15

'

Guiana (Z) 20. xi. 01

d

60

tr

67, 70 Anti-pig very powerful.

75 See note to No. 49.
226

Primates Insecti- Carnivora

vora

79, 81 Anti-pig powerful.

tr

85
tr

tr

Mammnlia

Aves

Kcptiliii Amphiliia Crustacea

60
tr

100

tr

tr

30
tr

60
tr

«

60

100

tr

30

90
tr?

+
30

60

tr?

90

tr?

O I tn O

I. I O E^

Marsu-

pialia

Ungulata

80 Gave . reaction with weak anti-human, which acted on Cercopithecidae.

227

15—2

Mammalia

Class MAMMALIA

i

1. Order PRIMATES

a

a

&

1

4)

g

ANTISEBA FOB

s

g

a

'.H

S

O

i

g

to

4)

w

6

1

X

8=

0

-3

1. Suborder

83

480. Chrysothrix sciurea h. Squirrel Monkey

*

,

,

,

•

1

1

,

1

ANTHROPOIDEA

Guiana (Z) 1. ii. 02

D

45
tr

d

tr

Fam.

84

584. Chrysothrix sciurea

.

.

•

.

*

.

*

Cebidae

Pari, Brazil (Hagmann) 14. ii. 02

85

85

86

697. Ateles vellerosus. Long-eared Spider-

X

«

«

•

,

«

,

Monkey

d

•>

?

tr?

tr?

Brazil (Z) 11. iv. 02 (organs healthy)/.

45

45

86

580. Ateles ater F. Cuv. Black-faced Spider-
Monkey

E. Peru (Z) 28. ii. 02

45
tr?

*

15
tr

f45
tr?

45

87

431. Ateles geoffroyiKuhl. Black-handed Spider-

X

.

*

«

•

*

.

»

Monkey

D

15

260

?

55

C. America (Z) 28. xii. 01

tr

15

88

513. Ateles geoffroyi

(Z) 11. ii. 02, /.

45
tr

tr

«

?

45
tr

tr

89

894. Lagothrix humboldti Geoffr. Humboldt's
Lagothrix

Upper Amazon (Z) 23. ix. 02, d. con-
stipation

30
tr

X

5
d

30
tr

tr?

90

285. Lagothrix humboldti
(Z) 20. xi. 01

+
b

■

1*5
tr

+
60
tr

'

-1-
60
tr

81

119. Ce6u» albifrons Geoffr. White-fronted
Capuchin

S. America (Z) 25. vii. 01

*

"

tr

6*0

tr

'

■

■

'

83

481. Cebus albifrons
(Z) 3. ii. 02

X

D

"

15
tr

"

'

X

tr

«

45

■

■

Fam.

Hapalidae

93
94

226. Hapnle pygmoea. Pigmy Marmoset
Upper Amazon (Z) 23. ix. 01

225. Midas oedipus L. Pinche Monkey
Colombia (Z) 16. ix. 01

•

X

«

tr

tr

•

•

•

tr

35

tr

•

•

96

514. Midas oedipus

•

»

.

ft

,

,

,

,

(Z) 21. ii. 02

46

tr

tr

45
tr

tr

96

296. Hapale jacchus. Marmoset
(Brazenor) 11. xi. 01

«

*

*

*

•

'

2. Suborder

LEMUROIDEA

97

227. Lemur rufifrons. Eed-fronted Lemur
Madagascar (Z) 26. ix. 01

•

'

"

•

?

320

■

X

35

tr

■

'

98

241. Lemur xanthomystax. Yellow-cheeked
Lemur

•

•

tr

•

•

•

35

•

•

Madagascar (Z) 23. x. 01

'

Primates

85 Note time of reaction with anti-Mexican-deer.
83, 86, 88, 91, 92, 93, 94, 96 Anti-pig powerful.

Insecti-
vora

228

Mammalia

Aves

Reptilia

Amphibia Crustacea

1

1

1

V

1

i

t

Q
<

t
5

H

o

m

1

j

!

1

1

a
•<

s

*

/

~"

""

~~

«

""

,

D

tr

tr

120

*

,

.

,

.

.

.

.80

d

X

90

tr?

D

X

.

.

.

.

.

.

.

a

d

tr

X

.

,

.

.

.

D

tr

•

*

?

30

•

tr?

•

6*0

•

•

•

•

•

•

'

'

•

+

.

,

,

.

•

.

.

•

•

•

•

D

tr

«

,

,

,

,

*

.

.

•

•

•

D

tr

tr

120

«

X

,

.

.

.

•

D

•

.

.

.

•

X

•

•

•

•

•

*

d

♦

,

,

,

,

,

.

.

•

D

•

•

•

•

d

•

•

•

•

•

•

+

«

X

.

D

?
240
tr?

60

tr?

*

.

.

«

•

•

•

•

•

*

d

60

Un

V

;ulata

Marsu-
pialia

•
_1__

_*U«

07, S8 Anti-pig very powerful. Very powerful anti-human had effect, but aUo on other mammalia.

229

Mammalia

Primatea Ineecti-
vora

aass MAMMALIA

2. Order CHIROPTERA

^

1

09

s

S

g

rS

S

bo

rs

ANTISERA FOE

1

S

td

s

&

o3

X

1 . Suborder

99

234. Pteropiis medms. Indian Fruit-Bat

»

#?

MEGACHIROPTERA

India (Z) 21. ix. 01

?

320

35

Fam.

lOO

236. Pteropus medius

.

.

.

.

«

.

,

Pteropodidae

India (Z) 19. x. 01

35

lOl

849. Romettus ample xicaudatus Geoff. Fruit-
Bat

Ceylon (Rothschild) 24. i. 02.

240

*

?

15
tr

240

2. Suborder

1

MICROCHIROPTERA

loa

259. Rhinolophus femtm-equinum

.

,

,

Section

103

England (Farren) x. 01.
856. Rhinolophus minor Temm.

Vespertilionina

Okenawa, Japan (E) 15. iii. 02

Fam.

104

809. Hipposideros diadema

.

.

•

•

•

.

Rhinoiophidae

Ceylon (R) 23. i. 02

Fam.

105

64. Plecotus auritus L. Long-eared Bat

.

.

#

.

,

,

Vespertilionidae

loe

Ireland (N) 7. iv. 01

394. Plecotus auritus

England (N) 12. xi. 01

•

•

•>
320

tr

*

?
90

•

■

107

759. Plecotus aiiritns

,

,

•

,

,

(Brazenor) 5. v. 02

tr

tr

tr

108

127. Scotophilus pipistrellus
England (R) 7. ix. 01

•
320

tr

X

■

109

442. Vespertilio dorianus Dob.
Paraguay (F) 9. xii. 01

*

'

*

•

•

•

no

441. Myotis nigricans Wied.

Paraguay (F) 11. xii. 01

•

\

\

s

•

N

111

923. Myotis ruber

Paraguay (F) 21. ii. 02

180

•

iia

928. Lasiurus boreulis bonariensis
Paraguay (F) 1. v. 02

tr

Fam.

113

445. Molossus rufus Geoff.

,

«

,

*

Emballonuridae

Paraguay (F) 16. xii. 01

tr

45
tr

«

40

,

114

374. Molossus cerastis Thos., nov. spec.

,

,

,

■

Paraguay (F) 2. xii. 01

15

tr?

116

446. Molossus fosteri Thos., nov. spec.
Paraguay (F) 5. xii. 01

•

tr

*

4*0

116

373. Molossus iemmincki Lund.
Paraguay (F) 27. xi. 01

•

9*5
tr?

•

•

117

440. Molossus Iemmincki
(F) 17. xii. 01

d

•

•

tr?

118

921. Molossus glaucinus (?)

Paraguay (F) 6. iii. 02

?
180

'

119

656. Nyctinmnxis laticaudatus Geoff.

.

«

•

.

,

Paraguay (F) 14. ii. 02

290

•^

CamWora

09, lOO, lOa Anti-pig very powerful.

230

Mammalia

Aves

Reptilia

Amphibia Cr

UBtace

b

£

*?

s

Hi

1

O

1

H

o

K

i

1

1

1

a

3

5

1
<

r

u.

s

1

•

*

~"

•

.

•

.

.

.

d

240

*

•

.

•

•

■

•

•

•

•

•

•

•

•

.

•

d

«

•

*

•

•

•

?

?

240

240

♦

•

d

•

•

•

•

•

•

•

•

•

•

•

•

tr

tr?

/

•

/

•

•

/

/

tr?

•

«

,

^

,

,

,

,

.

.

•

.

30

d

■»

•

\

.

.

•

•

•

•

*

'

9

60

«

•

25

'

«

»

.

,

,

.

60

tr?

d

♦

45

«

•

•

♦

•

•

•

30

d

•

"

•

65

*

■

*

.

,

«

.

.

•

•

•

•

30

tr?

•

tr?

•

•

•

*

•

•

•

•

Ungulata

Marau-
pialia

103 Foam-test doubtful, no reactions.

107 • with anti-remdeer.

231

Mammalia

Class MAMMALIA

2. Order CHIROPTERA

ANTISERA FOB.,

2. Suborder
MICROCHIROPTERA

Section

Vespertilionina

Fam.
Phyllostomatidae

Fam. t

Fam.
Erinaceidae

Fam.
Soricidae

Favi.
Talpidae

ISO

lai
laa

133
124

125

126
127

128

128

130
131

132

133
134
135

136
137

138
138

375.

657.

377.
655.

376.

443.

372.

Hemiderma brevicauda Wied.
Paraguay (F) 25. xi. 01

Glossophaga soricina Pall.
Paraguay (F) 26. xii. 01

Stumira lilium Geoff.

Paraguay (F) 27. xi. 01
Artibens literatus Licht.

Paraguay (F) 8. ii. 02
Desmodns rotundus Geoff.

Paraguay (F) 25. xi. 01

Pygoderma bilabiatum Wagn.
Paraguay (F) 19. xii. 01

Vampyrops Uneatus Geoff.
Paraguay (F) 27. xi. 01
444. Vampyrops lineatua
0. xii. 01

190. Chiroptera spec. ? " Short-eared Bat '
England (Garrood) 22. viii. 01

3. Order INSECTIVORA

133. Erinaceus eiiropaens. Hedgehog

England (Garrood) 9. viii. 01

134. Erinaceus europaeus

Denmark (Sohierbeck) ix. 01
232. Erinaceus europaeus

England (Z) 30. ix. 01

294. Erinaceus europaeus
(Bird) 19. xi. 01

783. Erinaceus europaeus
(N) 23. vi. 02,^.

846. Erinaceus europaeus
(E) 11. i. 02

900. Erinaceus europaeus (4 sera, mixed)

England (N) 19. viii. 02,/. (used for
treatment)

857. Crocidura coerulea. Shrew

Okenawa, Japan (E) 9. iii. 02

858. Crocidura coerulea

(E) 9. iii. 02

80. Talpa europea L. Mole
England (N) 10. vi. 01
460. Talpa europea

(Bird) 19. xii. 01

tr?

15

tr?

15
tr

tr?

tr

tr

30

tr

35
tr
*
?

45
tr

tr?
d

240

Primates Insecti-
vora
128 Anti-pig very powerful, as also in tests of most of the preceding.

232

CamiTOra

Mammal

a

Aves

Beptilia

Amphibia CruBtacea

s

a
s

V

S

ta

V
p

8

S

1
s

R
■<

a

o

"

j

1

1
1

1
1

t '

3 1

s
&

a
<

?

^

U

s

•

•

•

45

•

•

•

•

•

•

•

•

•

•

•

•

•

•

•

•

•

*

45

•

•

•

•

•

•

•

•

«

45

•

•

•

•

•

3*0
d

»

•

30
d

•

*

•

•

•

•

'

X

D

tr?

♦

d

•

^

.

,

'

•

•

.

.

.

.

.

*

.

•

-

•

X

d

*

9

240

.

.

.

•

•

•

■

•

•

•

•

■

•

•

40

tr?
tr?
tr

«

45
tr

tr?

tr?

•

•

•

•

•

•

•

•

•

•

60

•

«

tr ?

•

•

•

tr?

tr?

•

"

'

*

*

_

V

Un(fulata

Marsu-
pialia

130, 131

Anti-pig powerful.

2

33

Mammalia

Class

MAMMALIA

. Order INSECTIVORA

c4

a

ANTISEKA FOR

§

■g

3

%

W

1

1

CO

Fain,
Talpidae

140

701.

Talpa europea

*

,

,

,

«

,

,

Scotland (W. Evans) 10. v. 02

9

85

60

tr

141

782.

Talpa europea. From 2 specimens
England (N) 23. vi. 02,/.

«

85
tr

8*5

Fam.

143

247.

Centetes ecaudatus Schreb. Tenreo

#

#

.

♦

.

.

Centetidae

Madagascar (Z) 27. ix. 01

320

240
tr

143

433.
4

Centetes ecaudatiu
3. i. 02,/.

. Order CARNIVORA

d

«

?
260

15

1 . Suborder

144

103.

Felis tiqris L. Tiger

.

.

X

+

.

.

CARNIVORA VERA

India (Z) 13. viii. 01,/.

75
D

30
b

1. Section

145

738.

Felis tiqris

X

.

+

«

.

Aeluroidea

Amur (Luhe, Konigsberg Z.) 21. v.02,/.

120
tr

30
tr

85
D

120

tr

tr

Fam.
Felidae

146

300.

Felis caracal Schreb. Caracal

X

.

+

+

.

.

S. Abyssinia (Z) ca. 15. xi. 01,/.

90
d

5
d

147

137.

Felis spec. Leopard

Agra, India (Hankin) 12. vii. 01

•

'

tr

•

+
30

*

X

30

148

556.

Felis pardns. Indian Panther

Bombay, India (Phipson) 14. i. 02

•

•

120
tr?

*

45
D

X

45
tr

d

149

509.

Felit pardalis. Ocelot

Brazil (Hagmann) 30. i. 02

■

■

*

d

+
45
d

■

4*0

tr

160

798.

Felis pardalis

(Goeldi) 5. v. 02

■

tr?

*

45
tr

+
5
D

■

151

797.

Felis onca. Jaguar

Brazil (Goeldi) 4. v. 02

,

tr?

«

45
tr

+
5
D

153

588.

Felis spec. California Lynx

♦

.

X

+

♦

X

California (Robison) 30. xii. 01

tr

120
tr

85
D

tr

85

153

16.

Felis domesticus. Domestic Cat; scales
England (N) 29. iii. 01

*

■

3*0
tr

+
15
d

'

tr?

154

17.

Felis domesticus
31. iii. 01

■

6*0
tr

+
15
d

155

29.

Felis domesticus
29. iii. 01

*

•

•

*

5
d

*

•

*

156

30.

Felis dovusticus

,

.

«

.

.

4. iv. 01

90

5
d

*

157

31.

Felis domesticus

,

,

«

,

.

12. iii. 01

30

5
d

Primates Insecti-
voni

CamiTora

143 Anti-pig powerful.

147 Went badly into solution, which explains • reaction with weak anti-cat.
234

Mammalia

Aves

BeptUia

Amphibia Cragtaoea

60

?
300

45

tr

+

90

60

90
d

*

30

90
d

tr

cl

«

d

X

40

«

40

tr

75

45

tr

45

30
d

tr
tr

X

tr

180
tr?

180

tr?

£. S

75

90

tr

Vngulatft

155 Dried over a year and seven months when tested, dissolved with difficulty.

235

Mammalia

Class

MAMMALIA '

1
1

1

4.

Order CARNIVORA

s

o !
t

a

•

ANTISERA FOB

a

IS

a
o

bo

6

1

1

1

32

1. Suborder

168

32.

Felis domesticus

*

CARNIVORA VERA

4. iv. 01. Scales

90

159

135.

Felis domestieus

X

.

»

X

+

X

X

1. Section

Seeland (Schierbeck) ix. 01

320

60

30

30

Aeluroidea

tr

d

tr

160

317.

Felis domestieus

X

•

•

«

♦

•

•

Fam.

England (R) 2. xii. 01

5

15

tr

Felidae

tr

161

395.

Felis domestieus
(N) 28. X. 01

X

»

?

260

X

30

tr

X

15
d

«
tr

■

lea

739.

Felis domestieus
8. V. 02, fl.

+

120
d

+
30
d

+
85
D

+

120
tr

Fam.

163

493.

Viverra civetta Schreb. African Civet Cat

.

«

+

«

*

Viverridae

164

569.

Africa (Z) 8. ii. 02
" Civet Cat " or " Skunk "

Orange River, S. Africa (Parkinson)
10. xii. 01

•

•

•

45

120

tr

75

4*0
tr

165

362.

Genetta tigrina. Blotched Genet
Africa (Z) 9. xii. 01

•

•

45

•

+
15

•

•

166

612.

Genetta spec.

nr Lake Nyassa, C. Africa (Dodds)
p. 8. ii. 02

*

■

■

*

*

85

•

■

167

572.

Puradoxuriis urayi. Himalayan Paradoxure

*

.

.

.

X

«

X

India (Calcutta Zoo., Rogers) received

?

tr

45

30

40

21. ii. 02

90

d

168

748.

Paradoxurus hermaphroditus. "Munsang"
Sarawak, Borneo (Hose) posted 10. iv. 02

60
tr

tr

•

•

tr

•

•

169

86.

Paradoxurus niger Desm. Common Para-

,

,

.

.

,

doxure

Java (Z) 20. vi. 01

170

517.

Paradoxurus niger
India (Z) 13. ii. 02

*
d

•

'

X

+
45
tr

X

«

'

172

84.

Herpestes griseus Geoffr. Grey Ichneumon

.

,

,

,

,

,

India (Z) 15. vi. 01

tr

30

tr

173

89.

Herpestes pulverulentes Wagner. Dusty

,

.

,

,

,

,

Ichneumon

30

S. Africa (Z) 24. vi. 01

tr

174

159.

Herpestes pulverulentes
6. viii. 01

•

•

'

'

35
d

'

•

176

105.

Cynictis penicillata. Levaillant's Cyniotis

.

,

.

.

S. Africa (Z) 13. viii. 01

176

60.

Suricata tetradactyla Schreb. Suricate
S. Africa (Z) 6. v. 01

*

•

*

*

15
d

20

tr

177

102.

Suricata tetradactyla
10. viii. 01

•

*

*
?

320

*

+
30

"

178

905.

Suricata tetradactyla
17. X. 02 pleurisy,/.

180

+
10

tr

Fam.

179

494.

ProteUa cristatus Sparrm. Aard Wolf

+

*

Proteleidae

S. Africa (Z) 10. ii. 02

45
D

75

Primates Insecti-
vora

Carnivora

159, leo Anti-human and anti-pig powerful.
236

Mammalia

Aves

Reptilia

Amphibia Crastacea

5

1

0.

i

3]

1
1

a

3

a
<

1

H

o

a

1

1

■5

S.

1

1

1

3
a
u

■£

S

■<

1

1

.

.

.

.

.

.

d

X

*

•

«

•

•

•

•

•

•

•

•

•

D

tr

tr?

X

•

X

265

•

•

•

•

•

•

•

•

•

•

X

,

«

*

*

«

•

.

60

300
tr?

30

tr

110

110

120
tr

30

«

.

.

.

,

.

«

.

•

d

300

tr

?

«

tr

tr

X

•

•

'

•

*

.

*

'

*

*

«

*

«

*

»

180

75

120

60

180

cl

tr

,

X

.

.

.

«

tr

30

tr

60

*

•

•

•

•

•

'

X

X

»

*

♦

,

D

d

d

tr?

»

60
ol

■

•

•

»

*

*

•

»

,

•

,

•

d

tr

•

•

•

*

*
?

100

•

•

•

#

180

tr

#

.

,

,

,

,

.

.

.

.

•

•

•

d

tr

N»

-V

Ungulata

Marsu-
pi&lis

170 Anti-pig powerful.
237

Mammalia

aass MAMMALIA

4. Order CARNIVORA

ANTISEKA FOR.

1 . Suborder
CARNIVORA VERA

1. Section

Aeluroidea

Fam.
Hyaenidae

2. Section

Cynoidea

Fam.
Canidae

180

181

183

183

184

185

186

187

188
189

190

191
193

198

194

195

196
197

198
199

284. Hyaena striata Zimm. Striped Hyaena
Africa (Flower, Cairo) 13. ix. 01

855. Hyaena striata

N. Africa (Z) 31. vii. 02 diseased,/.

695. Canis familiaris. Pog

England (N) 27. iv. 02, filtered ; fl.

15. Canis familiaris. Terrier Dog; scales
6. lii. 01

780. Canis familiaris. Collie Dog ; aged
(Z) 19. vi. 02,/.

397. Canis familiaris. Mongrel
(N) 2. xii. 01

330. Canis familiaris
lO'. xii. 01

727. Canis. Pariah Dog

Bhadarwa, Kashmir (Donald) 13. ii. 02

289. Canis lupus. Common Wolf
Europe (Z) 18. xi. 01

890. Canis lupus

Chitral, India (Fulton) 28. v. 02

794. Canis spec. Wolf; young

? (New York Zoo., Langmann) posted
14. vi. 02

891. Canis lupus ?. Wolf, 1 month old

Chitral, India (Fulton) about v. 02
589. Canis latrans. Coyote

California (Kobison) 4. ii. 02

272. Caiiis anthus. Jackal

N. Africa (Z) 3. xi. 01

228. Canis aureus. Common Jackal
India (Z) 10. ix. 01

359. Canis aureus

(Z) 3. xii. 01

718. Canis aureus

Bhadarwa, Kashmir (Donald) 11. ii. 02
85. Canis mesomelas Schreb. Black-backed
Jackal

S. Africa (Z) 18. vi. 01
283. Canis rulpes L. Common Fox
England (Farren) 19. xi. 01
385. Canis vulpes
xi. 01

30

240

tr?

320

*
45

*

+

80

15

D

+

+

210

2

cl

S

240

tr

cl

240

X

85
trcl
+
15
d

300

tr

X

15
d

X

15

tr?

tr?

290
tr

X

80
tr
+
35
d
+
15

30

Primates Insecti-
vora

Carnivora
vora

180 Sample 11 months old on paper when tested with anti-hyaena.

181 Fluid serum 3 months old when tested by anti-hyaena, etc. This serum used for treating the rabbit which

yielded the anti-serum.

238

Mammalia

Aves

Reptilia

Amphibia Cru

stacei

•a

to

Id

5

i

V

be
o

a

i

£

1
S

<

1

o

a

1

"5

1
1

!

K
o

En

3

2

1

<

^

•

•

•

•

•

•

•

•

•

•

•

•

•

•

'

•

•

*

.

,

,

,

30

tr?

/

•

•

/

*
tr?

*
tr

/

•

/

•

•

•

»

■

.

•

*

•

•

•

.

•

•

•

•

30

tr

X

.

.

X

.

•

•

40

•

•

45
tr

265

•

•

•

•

tr

•

•

•

•

•

•

•

•

X

•

*

•

•

■

•

•

*

•

;

•

•

•

•

•

•

•

•

•

•

•

•

•

•

•

•

*

#

*

40

240
tr

tr

.

.

30

,

.

.

X

*

•

•

•

•

X

•

•

•

•

*

*

d

240

tr

X

•

«
?

240

'

120
tr?

■

•

•

■

•

•

•

•

•

•

•

•

•

>v

■y

"

^ — y —

Ungulata

Marsu-
pialia

lOI Foam-test doubtful, no reactions.
239

Mammalia

ci<.ss MAMMALIA

4. Order CARNIVORA

ANTISEliA FOB

s

a
o

1

be

■s

X

6

s

1

1

1. Suborder
CARNIVORA VERA

2. Section

Cynoidea

Fam.
Canidae

200

aoi
aoa
ao3

386. Canis vulpes

xii. 01
675. Canis vulpes

Mecklenburg (Kuse) 16. xii. 02

498. Canis pallidus. Pale Fennec Fox
Soudan (Z) 4. ii. 02

242. Cania cerdo. Fennec Fox
N. Africa (Z) 23. x. 01

*

+

60
d

#

1

1

1

X

85
d

X

45

tr
+
35
d

tr

X

+
D

*
d

+
b

/

8*5
tr

X

40
tr

X

30

ao4

654. Canis azarae

Paraquay (F) 26. i. 02

•

•

•

•

*

85
tr

*
45

ao6
ao6a

256. Canis procyonides

Japan (Z) 28. x. 01

934. Canis pallipes Sykea. Indian Wolf
India (Z) 23.x. 02, Jl.ser.

*

15
tr

•

*

320

,

«

35
d
+
5
d

+
b

3. Section

Arctoidea

aoe

76. Ursits americanus. Black Bear
N. America (Z) 28. v. 01

•

•

•

•

•

•

•

Fam.
Ursidae

ao7
aos

435. Ursus arctos Linn. Brown Bear
N. Europe (Z) 14. i. 02,/.

367. Ursus tibetanus F. Cuv. Himalayan Bear
E.Asia (Z) 27. xii. 01,/.

*

d

•

•

*

9

260

9*0
tr

+
55
d
+
15

+
d

«
d

5*6
tr?

ao9

885. Urstis tibetanus

Bhadarwa, Kashmir State (Donald)
4. vi. 02

•

•

•

•

15
tr

X

15
d

Fam.
Procyonidae

aio
ail

634. ,&urus fulc/ens F. Cuv. Panda

Nepal (Z) 23. iii. 02, d. of bronchitis

366. Procyon lotor Linn. Raccoon

N. America (Z) 27. xii. 01,/.

•

»

9

200

•

X

90

+
85
d

«

d

X

85
tr

aia

508. Nasua socialis. Coati

Brazil (Hagmann) 29. i. 02

•

•

•

•

45

tr
+
80
tr
+
90

300
tr

X

45
trcl

*

tr

ai3

273. Nasua rufa. Eing-tailed Coati
S. America (Z) 5. xi. 01

•

•

•

•

•

•

214

aift

360. Nasua rufa

6. xii. 01
781. Nasua rufa

17. vi. 02, killed fighting

•

*

•

•

6*0

•
tr

aie

577. Nasua spec. ? " Coati mundi "

Brazil (Tuckett) reed. 1. iii. 02

'

'

*

120

?
75

4*0
tr

Fam.
Mustelidae

317

114. Lutra vulgaris Erxl. Common Otter
England (Z) 30. vii. 01

*

i

•

\

X

30

tr

•

-1

Primates Insecti- Carnivora

vora
205 a This blood tested separately 29. xi. 02. Note degrees of reaction and time differences. Anti-man
much more powerful than anti-monkey.

240

Mammalia

Aves

Reptilia

Amphibia C

nistac

^

■^H

V

«

■a

s

1

13

a

i

1

<

5

s

i
S.

■s

5

1

1

9

a

2

<a
ho

<

•

/

•

60

6*0

•

•

•

•

•

X

,

«

.

.

X

,

,

,

^

.

^

tr

?

d

X

,

,

,

,

,

.

.

.

.

.

d

•

•

•

•

•

6*0
tr?

•

•

♦

•

>

•

•

•

.

.

.

.

d

X

,

,

15

d

•

•

100
d

•

•

•

•

•

•

•

•

•

•

X

■

*

90

*

■

■

■

■

*

'

■

■

X

•

X

90
d

•

•

•

*

?
85

•

•

•

•

•

•

•

•

•

X

X

d

X

90
tr

60

•

•

75

■

•

•

•

•

,

.

X

tr?

#

45
tr

tr

ft

?

X

■

«

«

*

#

«

■

•

•

•

•

D

?
75

20
tr

?

60

9

180

9

X

.

.

\

.

.

.

\

.

,

,

•

.

.

•

D

I

V

'^~~Y

Ungulata

Mareu-
pialia

241

16

Mammalia

Class

MAMMALIA

4

Order CARNIVORA

be

3

ANTISEllA FOR

o

n

5

1

1

1

1. Suborder

218

257.

Lutra vulgaris

«

*

.

CARNIVORA VERA

(Farren) x. 01

?

320

35
tr

3. Section

319

499.

Lutra vulgaris

,

,

*

«

+

,

Arctoidea

Ireland (Scharff) reod. 12. ii. 02

tr?

75
tr

75
tr

D

Fam.

230

579.

Lutra vulqaris

,

\

\

\

\

Mustelidae

England (Z) 4. iii. 02,/.

tr

1 V I %fl %^ ».*rf ■ ■ »* •i^^^

221

704.

Meles taxus. Badper

England (Cropper) 10. v. 02

6*0

■

3*0

X

85
tr
X

tr

322

758.

Meles taxus

»

.

,

,

(Brazenor) 25. iv. 02

60

tr

85

tr

tr

228

245.

Ictonyx zorilla Thunb. Cape Zorilla
S. Africa (Z) 24. viii. 01

320

•

35

X

80

224

361.

Ictonyx zorilla
30. xi. 01

\

•

tr

\

225

795.

"Sand Badger"

China (New York Zoo., Langraann)
p. 14. vi. 02

tr?

4
5
d

tr

226

22.

Mustela putorius. Pole-cat
England (N) 25. iv. 01

•

tr?

20

d

20
d

227

113.

Mustela putorius

Germany (Kuse) 7. viii. 01

•

/

X

350
tr

X

30
d

228

396.

Mustela putorius

England (Z) 16. v. 01

*

?
45

•

X

15
tr

tr

■|

228

630.

Mustela putorius. Ferret
(Brazenor) 4. ii. 02

•

tr

85

230

274.

Mustela martes. Pine Marten
England (Z) 10. xi. 01

\

•

\

8*0

\

231

516.

Mustela foina Erxl. Beech Marten

*

«

#

X

X

Russia (Z) 18. ii. 02

d

60

45
tr

tr

40
d

233

315.

Mustela erminea. Stoat
England (R) 9. xi. 01

•

tr?

•

•

333

354.

Mustela erminea

(Brazenor) 9. xii. 01

'

+
15
d

*

5*5
tr

234

355.

Mustela erminea

Canada (Hedley) 30. xi. 01

\

\

•

s

33S

629.

Mustela vulgaris. Common Weasel
England (Brazenor) 11. i. 02

tr

•

tr

•

85

236

847.

Mtistela vulgaris
(R) 14. iv. 02

•

*

15
d

240

tr

3*5
d

237

899.

Mustela spec. ? Weasel

Bhadarwa, Kashmir State (Donald)
4. iv. 02

tr?

Primates Insecti-
Tora

Carnivora

222, 229, 236

with anti-reindeer.

24:2

Mammalia

Aves

Reptilia

Amphibia Crantacea

30

tr?
tr

60
tr

?

300

\

45

tr

tr?

tr

240
d

tr?

tr

Ungulata

Mareu-
pialia

■a ?
5 1
s &

333

♦ ? 40 with anti-reindeer.

243

16-2

Mammalia

2. Suborder
PINNIPEDIA

Fam.
Otariidae

Fam.
Phooidae

Fam.
Sciuridae

cias. MAMMALIA

4. Order CARNIVORA

ANTISERA FOR.,

238

239

240

241

242

243

24.

244

130.

24S

131.

246

132.

247

140.

248

258.

249

356.

2SO

808

281

749

262

571

263

557

264

266

288

853. Otaria californiana. California Sea-lion
N. Pacific Ocean (Z) 24. vii. 02, maras-
mus,^.
910. Otaria californiana

(WashinRton D. C, Zoo.) 2. x. 01,
pneumonia
791. Otaria spec. Sea-lion, d. of pneumonia
? (New York Zoo., Langmann) 26. iv. 02

500. Halichoerug r/rypus. Gray Seal
Ireland (Scharff) 14. xi. 01

854. Phoca vittilina Ij. Common Seal, d.youn;;
British Seas (Z) 81. vii. 02, /., no dis.

5. Order RODENTIA

Sciurus vulciaris. Common Squirrel

England (Lane) 22. iv. 01
Sciurus vulgaris

Germany (N) 2. ix. 01
Sciuruji vulgaris

3. ix. Oi
Sciurus vulgaris

(Schlieffen) 16. ix. 01
Sciurus vulgaris

(Kuse) 7. viii. 01

Sciurus vulgaris

England (Farren) vii. 01
Sciurus vulgaris

(Brazeuor) 25. xi. 01

Sciurus tristriatus. Squirrel
Kandy, Ceylon (B) 24. i. 02

Sciurus lowii. Squirrel

Sarawak, Borneo (Hose) p. 10. iv. 02

Sciurus giganteus. Large Indian Squirrel
Calcutta, India (Rogers) 14. xi. 01

Sciurus pahnarum. Palm Scjuirrel

Ahmedabad, W. India (Mason) 27. i. 02

715. Pteromys inornatus. Red Flying Squirrel
Bhadarwa, Kashmir (Donald) 2. iii. 02

292. Sciuropterus fimhriatus. Gray Flying
Squirrel

Chitral, India (Z) 18. xi. 01

231. Cynomys ludovicianus Wagn. Prairie
Marmot
N. America (Z) 13. ix. 01

180

240
tr

75

60

tr?

tr?

?
320

Primates Insecti-
vora

110

tr

20
D

240

240

tr?

1
tr

tr?

1
tr?

?
60

Camivora

242 This serum used for treating the rabbit which yielded the anti-seal serum, which was feeble.

244

Mammalia

Aves

Reptilia

Amphibia Crustacea

a

tr?

60

tr

tr?

tr

tr?

?
tr

tr?

tr

120

tr?

60

20

tr

Ungulata

Marsu-
pialia

245

Mammalia

Class MAMMALIA

5. Order- RODENTIA

ANTISERA FOR..

Finn.
Sciuridae

Section

Myomorpha

Favi.
Myoxidae

Fam.
Muridae

257

258

259

104

260

719

261

293

262

260

263

295

264

636

266

473

266

267

268
260
270

271

272
273

274
275
276

277

518. Cynomys ludovicianus OiA. Prairie Marmot
N. America (Z) 17. ii. 02

340. Myoxits avellanarius L. Dormouse
England (B) 14. xi. 01

Gerbillus shawi

Algeria (Z) 10. viii. 01
Gerbilliis indicus. Indian Gerbille

Bombay Presidency (PhipRon) 11. ii. 02
Arvicola prateiisis. Bed Field Vole

England (Bird) 8. xi. 01
Hypudaetis glareolns Sohr. Bank Vole

England (R) 3. xi. 01
Uypudaeus glareolus

(R) 5. xi. 01

Mus mimihis (messorius). Harvest Mouse

England (Bird) 17. ii. 02
Mus minutus (messorius)

27. i. 02

Mus sylvaticm. Long-tailed Field Mouse
England (Bird) 25. xi. 01

Mas sylvaticm
(R) 4. xii. 01

Mus musculus L. Common House Mouse

England (N) 25. xi. 01
Mus musculus

1. i. 02
Mus musculus

28. X. 01

Mus musculm

29. X. 01

Mus musculus

(Mitchell) ix. 02
Mus musculus

(N) 27. 1. 02

Mus spec, (of M. musculus group). Mouse
Okenawa, Japan (It) 9. iii. 02

Mus decumanus Pall. House Rat
England (N) 18. vii. 01

Mus decumanus. Rat
England (R) 30. xi. 01

337.

357.

387.
388.
392.

393.

423.

472.

859.

95.

316.

61. Mus raltus. Black Rat
Ireland (N) 4. v. 01

tr?

tr?

45

tr

tr?

tr?

?

15
tr?

?
15

tr

«

55
tr?

?

15
tr

Primates Insecti-
vora

274 Foam test doubtful, no reactions.
246

Mammalia

Avea

Reptilia

Amphibia C

rustao

Llama
Hog-deer

1
■o

c

1

S
a
■<

M3

H
O

1

at

1

Eh

•§
•c

1

i

«

1

?

^

1
3

X

«

«

X

.

.

.

.

.

.

,

D

tr?

tr

.

tr

tr

•

•

•

•

•

•

•

•

•

•

•

•

tr?

\

\

•

•

•

\

s

•

•

«

/

•

•

•

•

•

•

•

•

■

«

«

•

•

•

•

•

•

•

d

?

300

tr?

■

•

•

•

•

•

#

•

•

•

•

•

•

•

•

•

•

•

?

265

*

?
265

•

•

•

•

•

•

■

*

?

300

tr?

■

■

■

■

«

•

•

•

•

•

•

•

•

♦

•

•

•

•

•

•

•

•

•

•

•

•

•

•

•

^-

f

^'' — ■r"^

Unguis ta

Mareu-
piaUa

247

Mammalia

Class MAMMALIA

5. Order RODENTIA

ANTISERA FOR.

>.

Section

Myomorpha

Fatn.
Muridae

Fam.
Spalacidae

Section

Hystricomorpha

Fam.
Octodontidae

Fam.
Hystnioidae

Fam.
Dasyproctidae

Fam.
Caviidae

278
270

280

281
282

283

284

285
286
287

288

289

290
291

292

293
294
295
296

48. Mus rattus. Black Rat, albino
England (N) ca. 12. iv. 01
705. Nesocia bemialcn^is. Indian Mole Rat

Khande'sh, Deecan (Millard) 27. iii. 02

519. Cricetomys gambianus Waterh. Oambian
Pouched Rat

W. Africa (Z) 19. ii. 02
581. Cricetomys gambianus

(Z) 22. ii. 02
924. Oryzomys angonya. Rat

Paraguay (Foster) 12. ii. 02

115. Myoscalops ar(ienteo-ciitereu». Silvery Mole
E. Africa (Z) 29. vii. 01

633. Capromys pilorides (Say). Fournier's
Capromys
Cuba'(Z) 26. iii. 02

621. Synetheres (Cercolabes) prehensilis. Tree

Porcupine

Parii, Brazil (Hagmann) 4. iii. 02

622. Synetheres (CercoWies) prehensilis

(Hagmann) 3. iii. 02

290. Atherura africana. African Brush-tailed
Porcupine
W. Africa (Z) 22. xi. 01

436. Dasyprocta crietata (Desm.). Agouti
W. Indies (Z) 6. i. 02,/.

230. Dasyprocta cristata
20. viii. 01

801. Dasyprocta agouti. Agouti

Trinidad, W. Indies (Tulloch) 3. v. 02
457. Caelogenys paca L. Spotted Cavy

S. America (Z) 24. i. 02

922. Cavia aperea

Paraguay (Foster)

11. Cavia cobaya Schreb. Guinea-pig, scales
England (N) 11. iii. 01

18. Cavia cobaya, fi. pleural exud. diphtheria

iv. 01

19. Cavia cobaya

11. iii. 01
27. Cavia cobaya, fi. ser. decomp., sealed
2. iii. 01

180

180

Primates Insecti-
vora

Camlvora

284 • with anti-reindeer.
248

Mammalia

Aves

Beptilia

Amphibia Crustacea

tr?

tr

180

tr

90
d

40

30
d

180

tr

90

180

u

Ungnlata

Marsu-
pialia

249

Mammalia

aass MAMIVIALIA

5. Order

RODENTIA

ANTISEllA FOB.,

Section

297

737.

Hystricomorpha

Fam.

Caviidae

298

50.

299

626.

Favi.
Leporidae

300

447.

301

136.

302

339.

303

164.

304

128.

30S

33.

3oe

890.

307

391.

308

848.

309

587.

310

565.

311

562.

DolichoHs patachonica Sliaw. Patagonian
Cavy

Patagonia (b. at Z) 29. iv. 02 (fl. co-
agulated)
Hydrochoerus capyhara. Capybara

S. America (Z) 24. iv. 01
Hydrochoerus capybara

Para, Brazil (Hagmaun) 3. iii. 02

Lepus brasiliemis. Hare

Paraguay (Foster) 19. xii. 01
Lepus europaeiis. Common Hare

Seeland (Schierbeck) ix. 01
Lepus europaeus

England (N) 26. xi. 01
Lf'pus europaeus

Germany (Kuse) 26. vii. 01
Lepus variiibilis. Scotch Hare

Scotland (K) 30. viii. 01
Lepus cuniculus. Tame Rabbit

England (N) 22. iii. 01
Lepus cuniculus. Wild Rabbit

Ireland (N) 4. v. 01
Lepus cuniculus

4. V. 01
Lepus cuniculus

England (R) 14. xii. 02
Lepus cuniculus. Tame Rabbit

Brazil (Hagmann) 16. ii. 02
"Long-eared Gray Hare"

Orange River, S. Africa (Parkinson)
17. i. 02
"Bushy-tailed Red Hare"

Orange River, S. Africa (Parkinson)
22. xii. 01

tr?

290

tr

tr?

76

75

Primates Insecti-
vora

Camlvon

250

Mammnlia

Aves

Reptilia Amphibia CruBtacea

9

300

?
180

TJngulata

251

Mammalia

Class

MAMMAL A

6

. Order UNGULATA

s

I

d

ANTISERA FOR

a

S3

1

3

>*

m

1

■3

1. Suborder

313

49.

Sua gcrofa domestica. Domestic Pig

.

,

«

,

,

ARTIODACTYLA

England (N) 24. iv. 01

15

Group

313

696.

Sue scrofa domestica.

X

.

.

X

.

.

Suina

(N) 29. iv. 02 (Jl. and dr. in sc. filtered)

d

85
tr

tr?

Fam.

314

744.

Sus scrofa. Wild Boar

#

,

,

»

.

.

Suidae

316

897.

Mecklenburg (von Oertzen) 25. v. 02

Sus spec. Wild Boar

Singapore (W. Kerr) 2. viii. 02

60
tr

•

•

?

85
tr

«
15

*

15

Fam,

316

600.

Dicotyles tajuca L. Collared peccary

*

X

.

Oicotylidae

•

America (Liihe) 18. iii. 02

tr

85
tr

Group

Tylopoda

317

151.

Camelus dromedarius Linn. Dromedary

*

*

*

Cairo Abattoir, Egypt (Littlewood)

30

9

Favi.

318

379.

9. ix. 01

Auchenia glama Linn. Llama

X

.

.

tr

*

,

45

Camelidae

319
330

892.
932.

S. America (Nat. Zool. Park, D. C,
.Salmon) ix. 01
Auchenia huaiiacos Molina. Huanaco
Bolivia (Z) 23. ix. 02, d. of rupture of
pulmonary artery ; Jl. ser.
Auchenia huanacos

b. in England, d. young (Z) 21. x. 02,
Jl. ter.

•

tr?

•

90

*

15
tr

tr?

Group

Tragulina

331

77.

Traqulm meminna Erxl. Chevrotain

.

,

^

«

,

,

India (Z) 17. v. 01

240

Fam.

tr

Tragulidae

Group

333

889.

Moschus moschiferus. Musk Deer

,

.

,

,

.

Pecora

Bhadarwa, Kashmir State (Donald)
30. v. 02

tr

(True Ruminants)

333

152.

Cervulus vaginalis. Barking Deer

.

.

•

.

.

.

Fam.

S. Sylhet, India (Dalgetty) 23. viii. 01

tr

Cervidae

334

623.

Cervus rufus

*

•

•

*

•

•

Brazil (Hagmann) 6. iii. 02

45
d

#

45

336

229.

Cervus aristotelis. Sambur Deer

.

.

•

.

.

b. in Zoo., London (Z) 16. ix. 01

9

35

tr

336

502.

Cervus porcinus Zimm. Hog Deer
India (Z) 15. ii. 02, jl. ser.

•

•

90
tr

*

45
tr

•

•

837

291.

Cervus sika. Japanese Deer

• 1 •

•

•

«

•

•

b. in Zoo., London (Z) 15. xi. 01

15
tr

Primates Insecti-
vora

Carnivora

816 Anti-pig serum weak, had no effect on over 20 other bloods except that of Cervus axis which

gave

120

reaction much later.

252

^[ammalia

Aves

Reptilia Amphibia Crustacea

Im

^^^

£

*?

iJ

1

X

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a

1

1

1

a

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o

a

a

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1

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1

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1

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1

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i

5

+

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+

•

.

.

.

"T"

«

•

,

.

.

30

60
d

60

230

+

«

»

X

«

«

«

.

X

»

«

•

•

.

D

85
tr

tr

90
D

15

tr

tr

d

90
tr

30

230
tr

+

.

«

X

«

.

.

.

«

«

•

.

•

D

60

30

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tr

tr

230

230

tr

d

30
tr

tr

+

.

+

•

»

«

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30
cl

30

230
tr

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X

X

#

•

.

.

#

,

,

,

.

,

,

.

15

85

20

tr

tr

30

tr

tr

tr

+

+

«

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#

.

.

,

,

.

.

,

,

D

40
d
+
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d

90

tr

60
30

90
tr?

•

•

•

•

•

•

•

•

•

•

•

•

«

+

•

•

•

•

'

30

20
D

X

+

•

•

'

5

D

cl

•

+
D

3*0
tr

80

3*0

tr

*
tr

•

•

•

•

•

•

•

•

•

•

•

•

•

•

■

X

45
d

+
15
d

X

30
d

«

45

d

3*0

d

tr

+

«

+

+

*

+

+

•

•

•

•

•

•

'

*

*

•

■

D

?

24
tr

d

30
d

d

+

,

X

+

«

«

+

+

+

•

•

*

•

•

'

■

•

D

tr

15
d

+

S
+

15
d

15
d

»

+

D

+

90

tr

75

tr

.

,

,

,

.

d

30

tr

a

d

^^^

I

_

Ungulata

Marsu-
pialia

253

Mammalia

Class MAMMALIA

6. Order UNGULATA

ANTISERA. FOR..

1. Suborder
ARTIODACTYLA

Group

Pecora

(True Buminants)

329
330

601
743.

Fam.
Cervidae

331

658.

383

746

333

154

334

745.

33S

129

336

163.

337

483.

Fam.
Bovidae

338

888

839

25

840

486

841

62

343

109

348

792

344

567

84S

568

346

289

347

SIO

63.

631.

Cervtts axis Erxl. Axis Deer
India (Z) 13. v. 01

Cerviis axin Erxl. neonat. Axis Deer

India (Liihe) 6. iii. 02
Cervus axis d. 1 day old

b. in Zoo., London (Z), 10. vi. 02, y/.ser.

Cervus dama. Common Fallow Deer
Mecklenburg (O. v. Oertzen) 10. ii. 02

Cervus dama

England (Brazenor) 22. iv. 02

Cervus elephaa L. Red Deer

Germany (Schlieffen) 15. ix. 01

Cervus elephas. Red Deer, calf

Germany (von Oertzen) 10. iv. 02

Cervus caprenhis L. Roebuck
Germany (N) 27. viii. 01

Cervus capreolus

(Kuse) 26. viii. 01
Rangifer tnrandus. Reindeer

N. Europe (Liihe, Konigsberg Zoo.)
4. ii. 02, ft.
Cariacus mexicanus H. Smith. Mexican
Deer

Mexico (Z) 9.iT.02,yJ.sec.(d. of phthisis
and pleurisy)

Connocliaetes gnu. White-tailed Gnu, clot
dried in scales

S. Africa (d. in England, N) 12. iv. 01
Cobus uiictuosus Laurill. Sing-Sing Ante-
lope

(Z) 8. ii. 02 also /.

GazelUi arabica. Gazelle

Arabia (Z) 13. v. 01

Gazella subgutterosa. Persian Gazelle
Central Asia (Z) 31. vii. 01

"American Antelope"

? (New York Zoo. , Laugmann) p. 14.
vi. 02
"Deuker Bok"

Orange River, S. Africa (Parkinson)
11. xii. 01
"Stein Bok"

Orange River, S. Africa (Parkinson)
20. xii. 01
Traqelaphux sylvaticus. Bosch-Bok
S. Africa (Z) 15. x. 01

jEpyceros melampus. Palla

Brit. Centr. Africa (Dodds) 1. i. 02

tr?

30

75

tr

240

320

9

320

85
tr

85

tr

290

tr

tr

290
tr

X

35

tr

tr?
»
45

85
tr

45

15
tr

Primates Insecti-
yan

35

?
120

Camirors

829 Anti-pig serum weak.
254

Mammalia

Aves

Reptilin Amphibia Crnetaoes

^

~~r

V

4
I

3

1
■?

m

1

1

a
<

1

o

X

1

j_

1

1

1

1

9

3

H

1

<

1

♦

+

+

+

+

.

,

,

,

,

,

,

tr

60
D

D

«

.

X

•

«

.

.

.

•

•

•

•

•

•

120

tr

d

d

tr

^

«

+

+

+

+

.

.

•

60

86

30

30

30

60

tr

tr

tr

tr
tr

D

3*0
D

D

+
45
d

tr

D

«

45
d

+
30
D

tr

•

•

•

•

60

•

«

,

+

•

+

«

#

.

.

60

60

tr

30

60

60

tr

D

d

tr

+

.

+

X

+

+

+

•

•

•

•

•

•

D

D

60

tr

d

a

*

,

+

+

+

+

X

•

•

•

•

60

300

60

30

30

60

60

tr

tr

tr

D

d

D

+

,

+

+

+

+

+

.

•

*

•

•

•

•

•

D

•

30
d

«

d

60
D

/

30
d

+

a

+
a

•

230

/

•

•

•

•

•

+

«

+

+

«

+

+

+

X

•

•

•

•

•

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*

•

D

300

*
85

15
D

+
D

15

D
+

15
d

«

d

D

+
D

D

+
D

D

+
30

90
tr

3*0

4*0

?
70

•

•

•

d

i

«

*

*

+

.

,

tr

tr

tr

d

a

+

«

X

+

«

+

+

+

.

.

»

*

.

•

D

300

d
tr

S

+
60
d

tr

+

D

+

+

a

•

•

40
tr

240

75

•

•

•

«

"

3*0

tr

+
60
d

+
30
d

■

«

.

«

X

#

•

•

•

40

30

45

tr

75

tr

+

,

+

+

+

+

*

•

•

•

•

•

D

30
d

20
d

20
d

D

tr

+

.

+

+

+

+

+

•

•

•

•

•

d

30
d

20
d

20
d

D

+

.

«

X

+

+

+

•

•

•

•

•

D

tr

tr

30
trcl

d

d

<

.

■

•

«

«

"

"

*

cl

tr?

180

360

ITngulata

255

Mammalia

Class MAMMALIA

Order UNGULATA

ANTISERA FOR..

1 . Suborder

ARTIODACTYLA

Group

Pecora

(True Eumiuants)

Fam.
Bovidae

348

349
860
351
362
353

354

365
356

357

358

358

360
361
362
363
364
865
866
887

243. Capra viegaceros, Markhoor

N. E. India (Z) 23. x. 01,/. ser.

282. Capra jemlaica. Tahr (Wild Goat)
Cliamba State, ludia (Ainsworth)
21. X. 01

149. Capra hircus Linn. Egyptian Goat
Cairo Abattoir (Littlewood) ix. 01

596. Capra hircus. Common Goat
England (Z) 3. iii. 02,/. ser.

158. Capra hircus

(Z) 8. viii. 01

542. Capra falconeri. Markhoor

Chitral, India (Leslie) 29. i. 02

7. Ovis ariei. Domestic Sheep, dr. in scales

England (N) 28. ii. 01

8. Ovis aries

19. iii. 01

9. Ovis aries

Slightly decomposed

10. Ovis aries

45. Ovis aries, defibr. bl.
19. ii. 01

96. Ovis aries. St Kilda Sheep
(Lane) 11. vii. 01

726. Ovis spec. ? Domestic Sheep

Bhadarwa, Kashmir (Donald) 13. ii. 02

150. Ovis aries L. Bedouin Breed

Cairo Abattoir (Littlewood) ix. 01

604. Ovis mxisimon Sclireb. Moufflon

Sardinia and Corsica (Liilie) 26. ii. 02

541. Ovis vignei typica. Oorial

Chitral, India (Sweet) 1. ii. 02

83. Ovis burrhel. Burrhel Sheep
Himalayas (Z) 31. v. 01

145. Ovis burrhel

b. in Zoo., London (Z) ca. 8. viii. 01

595. Ovis burrhel

(Z) 10. iii. 02,/. ser.

69B. Ovis tragelaphus Desm. Aoudad, young
N. Africa (Liihe, Konigsberg Zoo.)
26. iii. 02

tr?

tr?

320

«

85
320

320

tr

30

?
320

Primates Insecti-
vora

Carnivora

256

Mumnmlia

Avps

Ileptilia

Am]

>hibu

i Cru

Btacea

1

1
i

~T

i'

2

1

a

1

1

1

i

a
<

1

M

O j

o

g

Zebra
Wsllaby

p

1

1 s

jD

1

U4

s

2

+

#

+

+

«

+

+

+

«

.

D

?

240
tr

d

90

D

IS
d

d

b

90

•

*

*

d

+
30
trcl

+

d

+
b

+

■

■

■

+

*

+

+

+

+

+

.

.

•

•

D

240
tr

D

60
trcl

D

a

•

tr

+
D

+
D

«

15

tr

+
d

+

D

+
D

3*0

4*0

+

+

+

+

+

+

•

•

D

d

60
tr

D

a

+

«

+

+

+

+

+

D

120
tr

30
d

20
d

20
d

D

D

*

,

+

+

+

+

+

.

30

tr

d

d

d

a

tr

+
a

+

•

•

+

+

+

+

+

+

•

30

D

D

s

D

d

d

+

«

+

+

+

«

•

30

d

S

s

D

d

tr

3*0

tr

100

3*0
d

+

a

+

•

•

-

•

•

»

•

3*0

tr

*

tr

+
60
tr

3*0
tr

+
30
d

+
d

+
a

+
I)

+

X

tr

•

+

•

+

+

+

+

+

*

•

D

240
tr

D

60
trcl

D

a

"

tr

d

d

«
D

+

D

•
tr

■

+

,

+

«

•

+

+

.

.

D

*
?

240

30
d

X

tr

20
d

60
d

20
d

+
d

+

a

D

X

•

•

•

,

*

X

#

«

+

+

■

'

d

30

300
tr

tr

X

15
d

»

60

+

*

15
d

tr

X

15
D

X

+

♦

?

90

3*0

4*0

*

tr

a

30

d

D

230

^^^

Ungi

ilata

Marsu-
pialia

257

17

Mammalia

Class

MAMMALIA

6

. Order UNGULATA

1

1

S3

a

a

•q

%

CA

^

ANTISERA FOE

ta

s

g

n

5

O

o

S

VI

1 . Suborder

S68

4.

Bos taunts L. Ox, filtered ser., scales

X

.

.

"T"

«

.

.

ARTIODACTYLA

England (N) 19. ii. 01

tr?

?
15

tr?

1

Group

360

6.

Bos taurus

.

.

•

.

.

1

Pecora

23. ii. 01

(True Ruminants)

370

42.

Bos taurus
20. ii. 01

'

*

Farti,

371

147.

Bos taurus. Egyptian Cow

X

.

*

•

*

.

.

Bovidae

Cairo Abattoir (Littlewood) ix. 01

?

320
tr

9

30

tr

373

148.

Bos buhalis. Water Buffalo

Egypt, Cairo Abattoir (Littlewood)
ix. 01

X

*

?

320

#
?

30
tr

373

383.

Bos spec. ? Buffalo (agricultural)

Shanghai, China (Stanley) ca. 27.xi. 01

»

*

'

?
15

■

■

374

717.

Bos gaurus Evans. Indian Bison
Kandesh, India (Simcox) 17. iii. 02

X

60
tr

•

•

•

tr

X

85
tr

*
5
tr

290

2. Suborder

PERISSODACTYLA

376

796.

Tapirus americanus. Tapir
Brazil (Goeldi) 20. v. 02

•

tr?

•

•

tr

•

•

Fam.

Tapiridae

Fam.

376

1.

F.quus caballus. Horse, antidiphtber. ser.

,

.

,

.

Equidae

dr. in so. bottled (L. C.) 1895

377

2.

EquMS caballus. Antidiphtber. ser.

.

.

.

.

dr. in sc. bottled (L. C.) 1897

378

594.

Fquu-s caballus. Antidiphtber. ser. fl.
England (B. W. and Co.) 4. i. 1897

•

•

•

•

379

3.

Eqmi.1 caballus. Normal filtered
ser. sc. bottled (N) 4. iv. 01

X

•

•

•

*

•

380

694.

Equus caballus

England (N) 20. iv. 02, filtered ser.,/.

tr

■

*
?

90

8*5
tr

»

?

tr?

*

?

85

381

929.

Equus asinus L. Donkey

England (N) 3. xi. 02,/. ser. filt.

180
tr

tr

180
tr

383

930.

Equus asinus

(M^Fadyean) 6. xi. 02, /. ser.

"

•

■

383

903.

Equus grevyi Gilnther. Grevy's Zebra,
young
Abyssinia (Z) 13. x. 02, fi. ser.

15
d

*

1*0

tr

3. Suborder

HYRACOIDEA

384

563.

Hyrax spec. "Eock Eabbit"

*

.

.

.

.

.

.

Fam.

Orange Eiver, S. Africa (Parkinson)

75

tr?

Hypaoidae

24. xii. 01

Primates Insecti-
vora

Carnivora

376, 377 Foam-test doubtful, not included in small tables.

258

Mammalia

AveB

Beptilia Amphibia Crustacea

f

^"***

^^^^m

1

^^^

^^^"

^^^

^^~

"1

V

<o

1

1

V 1

1

M

s

1

a
"3

a
•<

1

s

K

I

1

1

1

El.

1

1

S

1

1

1

1

I

*

.

+

+

+

+

+

""

""

.

30

d

D

D

d

d

*

+

+

,

tr

tr

tr

d

tr

+
d

d

+

•

•

•

•

'

•

•

+

*

+

+

+

+

+

.

*

«

•

.

.

.

•

.

•

•

D

240

tr

U

D

D

d

230

40
d

+

^

+

+

+

+

+

,

»

^

,

,

.

.

.

.

.

D

240
tr

D

X

d

+
30
d

D

+
d

d

+

•

230

40
d

•

•

•

•

•

«

«

«

+

+

X

+

.

«

«

.

,

.

.

GO

300
tr

tr

30
d

d

d

d

?
230

40

«

#

#

«

40

*

tr

45

tr

•

tr

#

.

+

?
240

40

•

•

tr

tr?

.

tr

tr

,

cl

+

.

.

.

•

•

tr

D

«

^

*

*

»

#

+

+

+

+

.

•

80

85
tr

90
D

15

?

tr?

d

30
d

D

5
D

10

X

.

+

+

+

•

25

tr?

1

5

10

d

D

S

D

X

.

+

+

+

•

10

1
D

+

5

+

10

+

tr

tr

1
D

5

10

s

»

•

•

•

60

•

•

•

■

•

•

•

•

•

•

•

,

^■^

—

—

—

—

Ungulata

Marsu

pialia

373 Anti-sheep serum weak.
259

17—2

Mammalia

Class

MAMMALIA

7. Order CETACEA

ANTISERA FOR

s

s

§

s

1

t

1

s

1

a

1

1

3. Suborder
Hyracoidea

Favi*
Hyracidae

385
385 a
386

698.
935.
485.

Balaenoptera rostrata. Rorqual, also^.
Killed near Bergen, Norway (Brun-
chorst) 30. iv. 02
Balaenoptera rostrata

Killed near Tromsoe, Norway (Torup)
p. 6. xi. 02, fl. ser.
PJiocaeva communis. Porpoise

N. Coast of France (von Oertzen,
Havre) ca. 2. ii. 02, /.

8. Order EDENTATA

tr

30

tr

*
tr

*

*

•

tr

*

ii

9

tr?

*

?
85

Fam.
Bradypodidae

387

160.

Bradypus iridactylus. Three-toed Sloth
British Guiana (Z) 5. viii. 01

•

•

*

?

320

tr

•

•

388

505.

Bradiipus marmoratus. Sloth
Brazil (Hagmann) 29. i. 02

•

•

.?
45
tr

'

'

Fam.
Myrmecophagidae

389
390
391
392
393

233.

799.
507.
586.
625.

Myrmecophaga jubata. Great Ant-eater
S. America (Z) 25. ix. 01

Myrmecophaga jnhata

Brazil (Goeldi) 5. v. 02

Tainandiia tetradactyUi. Tamandua Ant-
eater

Brazil (Hagmann) 29. i. 02
Tamandua tetradactyla
16. ii. 02

Tamandua tetradactyla
3. iii. 02

d

•

•

tr

3*5

tr

tr

»

45
tr

85
tr

•

290
85

Fam.
Dasypodidae

394

235.

Dasypue villosus. Hairy Armadillo
La Plata (Z) 21. viii. 01

•

•

•

*

35

•

•

396
396
397

371.
506.
110.

Dagypus sexcinctus L. Armadillo, male
Paraguay (Foster) .30. xi. 01

Dasypus setoms. Armadillo
Brazil (Hagmann) 29. i. 02

Tatusia peba. Peba Armadillo
S. America (Z) 1. viii. 01

.

•

N

X

30
tr

*

\

398

585.

Tatusia novemcincta (young). Peba Arma-
dillo
Brazil (Hagmann) 15. ii. 02

*

*

*

•

■

Fam.
Orycteropodidae

399

561.

Orycteropus capensis. "Ant Bear" or
"Aard Vaark"
Orange River, S. Africa (Parkinson)
22. i. 02

Primates Inaecti-
vora

Camivora

385 * tr with anti-reindeer.
260

Mammalia

Aves

Reptilia

Am|)hibia Crustacea

1

V

be
0^

I

1
1

a

1

1

r

•J.

JK

&

M

a

i

1

1

1
9

1

E-i

2

<

1

I

#

X

«

*

•

»

""

15

tr

90

•>

60

30

90

d

D

tr

D

tr?

+

*

.

.

•

•

5

00

d

tr

*

.

*

.

•

.

»

.

.

.

.

•

•

•

•

•

•

•

D

tr

120
d

«

♦

•

d

60

*

*

d

?

«

«

«

,

,

,

.

.

.

•

d

?
240

60

tr

*

.

.

*

.

.

•

40

45
tr

*

«

•

•

■

•

•

•

•

cl

*

,

,

.

.

.

,

.

•

•

180

cl

•

•

•

•

•

•

•

•

•

*

\

\

\

\

.

•

d

tr
\

•

•

•

•

•

•

•

•

•

•

*

•

•

•

•

•

•

•

d

,

,

•

»

tr

^

,

,

,

,

,

.

.

•

•

180

«

?

cl

y

_

^^^

_^

,

;■

LjTnHIII.

Ungulata

Marsu
plalia

261

Mammalift

Class
9.

MAMMALIA

Order MAR8UPIALIA

ANTISERA FOR..

Suborder
POLYPROTODONTIA

Fam.
Didelphyidae

Fam.
Dasyurldae

Fam.
Peramelidae

Suborder
DIPROTODONTIA

Fum.
Phalangeridae

400
401

4oa
4oa

404

4oa
4oe

407

Fam.
Macro podidae

4oe

409

410

413

413
414

415

416

417

800. Didelphys marsujnalis. Opossum

Trinidad, W. Indies (Tulloch) 20.iv.02

448. Didelphys marsupialu

Paraguay (Foster) 19. xii. 01
927. Didelphys vuirsupialis

5. iv. 02
432. Didelphys lanigera. Woolly Opossum

Colombia (Z) 2. i. 02
926. Didelphys cinerea

Paraguay (Foster) 7. iii. 02
923. Metachii-us nudicaudatiis. Opossum

Paraguay (Foster) 9. iv. 02

482. Thylacinus cynocephalus. Thylacine
Tasmania (Z) 1. ii. 02,/.

116. Perameles spec. inc. Bandicoot
Australia (Z) 29. vii. 01

240. Trichosunis vulpecula Kerr. Vulpine Pha-
langer

Australia (Z) 18. x. 01
458. Trichosurus vulpecula

22. i. 02

896. Petanrus sciurem Shaw. Squirrel-like
Phalanger

Australia (born at Z.) reed. 26. ix. 02

51. Hypsiprymnus rufescens Gray. Eufoua
Rat- Kangaroo

N. S. Wales (Z) 24. iv. 01
459. Onychogale frenata Gould. Bridled Wal-
laby

Australia (Z) 23. i. 02
497. Onychoyule frenata

6. ii. 02
488. Onychogale unguifera Gould. Nail-tailed
Wallaby

N. W. Australia (Z) 3. ii. 02,/.
503. Onychogale unguifera
13. ii. 02

611. Onychogale unguifera

22. iii. 02,/. ger. filt., sealed

487. Petrogale xanthopus Gray. Yellow-footed
Rock-Kangaroo
Australia (Z) 10. ii. 02, /.

♦ ?
180

15

tr

tr

Primates Insecti-
vora

Carnivora

40a, 404, 406 Anti-wallaby serum very weak.
262

Mamm

ilia

Aves

Kept

ilia

Aiuphit

ia Crustace

u

t

V

be

Ij

1

X

i

s

s

1

<

1

M
O

n

•i

■3

1

1

1

1

1

1

2
5

3

«

.

.

#

""

"~

,

.

40

45

tr

*

•

•

•

.

•

.

.

#

.

.

•

•

•

•

•

•

30

,

,

,

,

,

X

•

•

•

•

•

•

•

.

•

•

•

•

•

*

'

«

#

«

X

60

?

?

75

d

90

90

d

«

.

,

d

•

«

30

d

,

.

•

♦

•

•

30

•

•

tr

?
30

+
CO
trcl

•

•

•

•

•

X

,

,

,

+

•

•

•

■

d

75
D

«

.

.

.

X

•

•

"

d

tr

D

^

«

+

•

■

d

300

75
1)

*

.

J_

•

•

d

•

•

•

75
D
+
75
del

•

•

*

,

•

+

•

■

d

75
D

y

^_

^_

—

Ungulata

Marsu*
pialia

263

Mammalia

c

iiass MAMMALIA

9. Order MARSUPIALIA

ANTISERA FOB

1

1

§

t

K

6

1

1

Sitborder
DIPROTODONTIA

Fam.
Macropodidae

418
419

430

421 §
423

433

434

435
436

437

74. Petrogalepenicillata. Brush-tailed Kanga-
roo

N. S. Wales (Z) 28. v. 01
484. Petrogale. penicillata

Australia (Liihe, Konigeberg Zoo.)
4. ii. 02,/.
373. Macropug giganleus. Great Kaugaroo

Australia (Calcutta Zoo. , Rogers) 1 . ix. 01

275. Macropus giqanteus
(Z) 2. xi! 01

676. Macropus rujicollis. Scrub Wallaby

Oberon, N. S. Wales (Cashman)
30. xii. 01
117. Macropus bennetti Waterh. Bennett's
Wallaby
Tasmania (Z) 12. vii. 01
512. Macropm bennetti
22. ii. 02,/.

605. Macropus bennetti
(Liihe) 13. ii. 02

10. Order MONOTREMATA

699. Omithorhijnchus paradoxus Blumenb.
Duck-billed Platypus

N. S. Wales (Palmer, Sidney) 29. iii.02

tr?

•
•

tr?

100

•

100
tr?

«

?

320

«
•>

85

«

290

3*0
tr

80

tr

.?
85

tr?

*

85
tr?

tr?

Primates Insecti-
vora

§ The omitted No. 421 is replaced by 385a in the series.

264

Maimimlia

Avea

lieptilia

Amphibia Crustacea

I

a

■2,

s

GO
d

«

180

tr?

*

60

tr?

Ungulata

Marsu-
pialia

^11

J u

265

Mammalia

Class

AVE8

I. Division RATITAE

ANTISERA FOE.

Fam.
StPuthionidae

Fam.
Rheidae

Fam.
Casuaridae

Fam.
Colymbidae

Fam.
Podicipedidae

Fam.
Procellariidae

438

430
430

431

433
433
434

435
436

437

438

430
440
441
443
443
444

44S

740.

489.
120.

602.

504.
342.
305.

Sti-uthio molybdaphanes Eeicheuow. Os-
trich

Somali-lnnd (Z) 7. vi. 02,/.
Struthio molyhilopliaites

20. xii. 01,/.
Struthio australis. S. African Ostrich

S. Africa (Z) 8. vii. 01

Struthio spec. Ostricli

Africa (Liibe) 21. iii. 02,/.

lihea americann. Rhea
Brazil (Haj^mann) 2!). i.

Rhea atitericaiia

S. America (Z) 2.5. xi. 01

lihea americana
(K) 11. xi. 01

02

277. Casuarius bicnrunculatus. Cassowary
Arroo Islands (Z) 14. xi. 01

304. Dromaeiis novae-hoUaudiae . Emu
Australia ? (K) 11. xi. 01

II. Division CARINATAE
1. Order COLYMBIFORMES

650. Colymbus septentricmalis. Red-throated
Diver

England (B) 27. ii. 02

700. Podiceps spec. Diver, (ier. "Hauben-
taucher "

Mecklenburg (von Oertzen) 23. iv. 02
253. Podiceps Lesser Grebe

England (Farren) x. 01
311. Tachybaptes JiuviatiUs. Little Grebe

England (B) 21. xi. 01

644. Tachybaptes /iui'iatilis

21. xii. Oi

645. Podiceps auritus. Eared Grebe

England (B) 3. ii. 02
221. Podiceps cristatus. Crested Grebe

Europe (Z) 26. ix. 01
301. Podiceps cristatus

18. xi. 01

2. Order PROCELLARIFORMES

842. Fulmarus glacialis. Fulmar Petrel
England (Clarke) 1. ii. 02

tr?

• ?
tr?

• ?
240

431 • with antisera for seal, hyaena, hedgehog, Uama.
266

Maniinalia

Aves

Reptilia

Amphibia Crastacea

tr?

+
45
d

S

+

a

+

+

D

+

+
B

+
S

d

+
d
+
d

+
d

60
tr

«

60
+
d

d

*

tr

tr

*
tr

t

tr

30

tr?

+
1)

60

d

+

tr?

u

267

Class AVE8

II. Division CARINATAE
3. Order CICONIIFORMES

ANTISEKA FOR

Mammalia

!?

%

M

V

J

^

^

O

.^

1 . Suborder
Steganopodes

Fam.
Phalacpocoracidae

Fam.
Fregatidae

Fam.
Pelecanidae

2. Suborder
Ardeae

Fam.
Ardeidae

3. Suborder
Ciconiae

Favi.
Ciconiidae

4. Suborder
Phoenicopteri

Fam.
Phoenicopteridae

446

447

448
448

4SO
461

4sa

483
454

4S6
466

467
468

469
460
461
463

463

646.

Phalacrocorax qraculus. Shag
England (B) 10. ii. 02

733. Fregata aquila. Frigate, or Manof-War
Bird
Trinidad, W. Indies (TuUoeh) 2. iv. 02

511. Pelecanm onocrotahts L. Wliite Pelican
N. Africa (Z) 18. ii. 02 fl.

466. PelecanuK spec. Pelican

Trinidad (Tulloch) 1. i. 02

155. Ardeu caiididissima. Snowy Egret

Brazil (Z) 6. viii. 01
608. Ardeu purpurea L. Purple Heron

S. Europe (Luhe) 27. ii. 02

183. Ardea cinerea. Common Heron

Germany (Kuse) 17. ix. 01
251. Ardea cinerea

England (Farren) viii. 01
648. Ardea cinerea

(B) 2. ii. 02
710. Ardea cinerea

Kashmir State, India (Donald) 16. iv. 02
368. Nycticorax violaceus Linn. Violaceous
Night Heron

S. America (Z) 18 xii. 01
506. " Large Black and White Heron "

Orange E., S. Africa (Parkinson) 3. i. 02
453. Botaurus stellaris. Bittern

England (Clarke) 18. i. 02

126. Ciconia nigra Bechst. Black Stork
Europe (Z) 27. vii. 01

529. Ciconia alba. White Stork
India (Inglis) 18. i. 02

852. Dissura muquari Bodd. Maquari Stork
S. America (Z) 18. vii. 02/., killed,
blind

527. Dissura episcopui. White-necked Stork
India (Inglis) 3. ii. 02

851. Phoenicopterm ruber Linn. Ruddy Fla-
mingo

N. America (Z) 22. vii. 02, /. no dis.

tr?

448, 451 • with 4 antisera for seal (tr?), hyaena, hedgehog, llama.

268

Mammalia

Aves

Itcptilia

Amphibia

Crustacea

1

1 1

1

1

1

1
<

1

H
O

a

4

1

i

Ostrich
Fowl-egg

3

a

Turtle
Alligator

1

2

.

tt

«

.

•

60

•

1

/

/

•

•

/

+

+

•

/

•

tr?

■

•

•

•

•

*

d

*

D

*

d

«

.?
90
tr

•

d

•

•

•

•

•

1
1

•

/

/
/

/

/
/

/

/

/

/

/

•

/

/

i

/

/

/

+
D

*

d

»

d

+
b

«

n

+

35
d

«

d

+
35
d

tr

*
d

1)

+
30
D

«

d

»

d

/
-f

+
D

240
tr

tr?

tr?

3*0
d

210
d

210

/

+

•

•

447,

452, 465

But slightly or n

3t soluble.

454 Insoluble.

269

aass AVE8 .

II. Division

4. Order

Mammalia

CARINATAE

ANSERIFORMES

ANTISEEA FOB...

4. Stiborder
Phoenicopteri

Fam.
Anatidae

Siihfinii.
Merginae

Siihfam.
Fullguiliiiw

Siibfam.
Anatina

464
465

466
467

468

469

461

470

317

471

341

473

549

473

263

474

262

475

649

476

477
478

478
480
481

482
483
484

485
486
487
488

220. Merflus albellus L. Smew

Europe (Z) 5. ix. 01
651. Merrjua alhi'llus

Englaud (B) 10. ii. 02

261. Mergus serrator. Eed-breasted Merganser

England (R) 5. xi. 01
312. Merqns serrator

(B) 12. xi. 01

672. Clangula glaucion. Golden-Eye
Orkney (B) iii. 02

Clangula glaxwion

Englaud (Bird) 6. xii. 01
Clanqula glaucion

(B) 30'. xi. 01
Clanqula glaucion

(it) 25. xi. 01
Netta riifina. Eed-crested Pochard

Parblianga, India (Inglis) 18. i. 02

Nyroca fcrina. Pochard
England (R) 5. xi. 01

FuUijula cristata. Tufted Duck
England (R) 5. xi. 01

Fuliqula cristata
(B) 8. i. 02

276. Deiidrocygiia arciiata. Wandering Tree-
Duck

E. Indies (Z) 12. xi. 01
590. Anas boscas. Domesticated Duek

England (N) 7. iii. 02, /. ser.
659. Anas boscas

Mecklenburg (G. von Oert/.en) 19. ii. 02

404. Anas boscas

England (N) 16. v. 01

405. Anas boscas

19. X. 01
917. Anas boscas

(Cobbett) V. 1900 {Ji. ser. sealed over
2 years)
141. Anas boscas? Wild Duck

Gennany (Kuse) 8. viii. 01
831. Anas boscas

England (R) 21. ii. 02
833. Mareca penelope. Widgeon

England (Clarke) 25. i. 02

161. Tadorna tadornoides. Sheldrake

S. Australia (Z) 9. viii. 01
548. Casarca riitila. Brahminy Duck

Tirhut, India (Inglis) 17. i. 02
712. NettioH crecca. 6reen-\vinge<l Teal

Kashmir (Donald) 19. iii. 02
881. Nettion crecca

Okenawa, Japan (R) 14. iii. 02

?

25

tr?

488 Insoluble.

270

Mammalia

Avi'R

Beptilia

Amphibia

Crustacea

265

«

«

d

tr

*

*

•

60

60

tr

tr

+

«

»

A

tr

«

«

•

d

tr

+

+

60

60

d

d

+

+

*

D

D

60

+

»?

<

a

tr?

+

+

•

a

tr

+

«

•

D

d

+

.

•

9

tr

+

•

«

d

tr

+

«

•

d

60

+

\

*

d

+

+

D

D

+

+

*

60

60

60

D

D

+

*

•

a

tr?

+

•

•

a

+

X

10

15

d

tr

»

«

d

d

•

•

15

+

•

15

a

+

+

D

d

+

«

D

d

tr?

, •>
240

tr

90

.?
90

u

271

Class AVE8

II. Division CARINATAE
4. Order ANSERIFORMES

ANTISEEA FOB

Mammalia

4. Suborder
Phoenicopteri

Fam,
Anatidae

Suhfnm.
Anatina

Sitbfam.
Anserinae

Subfam.
Flectropterlnae

Siibffim.
Cyg^ninae

1 . Suborder
Cathartae

Favi.
Cathartic! ae

2. Suborder
Accipitres

Fam.
Vultupidae

Fam.
Falconidae

4S»
490

491
492

493
494

49S

496
497
498

499
500
601

6oa

603
604

SOS

soe

S07
SOS
S09

832. Nettion creccn. Green- winged Teal
England (R) 12. ii. 02

187. Nettion crecca

Germany (Sehlieffen) ix. 01

677. Amer spec. Wild Goose

Mecklenburg (Kuse) 2. x. 01

671. Anser spec. Upland Goose
England (B) 4. iv. 02

73. Aex sponsa. Summer Duck
N. America (Z) 25. v. 01
219. Rhodonessa carynphyllacea. Pink-headed
Duck

India (Z) 14. viii. 01
492. Cairina moscliuta. Muscovy Duck

Trinidad, W.I. (Tulioch) 29. i. 02

23. Cygnm olor Gm. Mute Swan
England (Lane) 24. iv. 01
309. Cyffims olor

■ ' (B) 8. xi. 01
121. Cygnus olor

(Z) 2.5. vi. 01

5. Order FALCON I FORMES

495. Cathartes alratus Bartr. Black Vulture

S. America (Z) 10. ii. 02
520. Cathartes atiatu.i

19. ii. 02
438. Cathartes atratus ? " Corbeau "

Trinidad, B.W.Indies(Tulloch) 30.xii.01
369. Sarcorhamphiis gryphiisli. Condor Vulture

S. America (Z) 27. xii. 01, Jl. ser.

707. Gyps fub'escens. Vulture

Kashmir (Donald) 22. iii. 02

188. VuUur monachus ? Vulture

S. Sylhet, India (Dalgetty) 12. ix. 01

888. Circus macruriis. Pale Harrier

Kashmir State (Donald) 17. iv. 02
559. Milviis gofinda. Pariah Kite

Bombay, India (Mason) 3. ii. 02
716. Milviis govinda

Kashmir (Donald) 2. iii. 02
93. .iccipiter niaus. Sparrow Hawk

Ireland (Dillon) 7. v. 01
193. Accipiter nisiis

England (Beauford) 9. viii. 01

S i W

tr?

Ir?

tr?

SOS Note source and p. 63 in text.
272

I

Mammaliii

Aves

Keptilia

Amphibia

Crufltacea

tr

tr?

tr?

u

607 Also . with 4 antisera for seal, hedgehog, hyaena, Uama.

273

18

Class AVE8 .

II. Division CARINATAE
5. Order FALCON I FORMES

ANTISEEA FOB

Mammalia

2. Svhorder
Accipitres

Fam.
Falconidae

sio

511
612
613
614
515
516

517

518
519
520
621

622
523

524

525
526
527
528
529

630

531
632
533
634
536
536

249.
464.
828.
709.
162.
213.
79.

530.

91.
180.
183.
400.

775.
2.52.
830.

706.
139.
176.
177.
660.

829.

603.
714.
212.

806.
211.

278.

Accipiter nisus. Sparrow Hawk

(Farren) viii. 01
Accipiter nisus

(Bird) 27. xii. 01
Accipiter nisns

(B) 2. Ti. 02
Accipiter virqatus. Sparrow Hawk

Kashmir' (Donald) 11. iv. 02
Astur pabimbarius. Goshawk

Germany (Kuse) 27. viii. 01
Astur palmnbarius

England (Z) 14. viii. 01
Falco peregrinus. Peregrine Falcon

England (Lane) vi. 01

Falco peregrinus.
India (Inglis) 17.

02

Falco tinnuncuhis. Kestrel

Ireland (Dillon) 16. v. 01
Falco timiunculus

Denmark (Schierbeck) ix. 01
F'alco tinminculus

Germany (Schlieffen) ix. 01
Falco tinmmculus

England (Lane) vi. 01

Falco aesalon. Merlin

Orkney, N.B. (B) 21. iv. 02
Tinnuncuhis alaudarius. Kestrel Hawk

England (Farren) vi. 01
Tinnuncvlus alaudarius

(K) 5. vi. 02

Tinnuncuhis spec. Cerchner's Kestrel

Kashmir State (Donald) 22. iii. 02
Buteo vulgaris L. Buzzard

Germany (Kuse) 9. viii. 01
Buteo vulgaris

(N) 27. viii. 01
Buteo vulgaris

2. ix.'oi
Buteo vulgaris

N. Wales (B) 6. iv. 02

Buteo vulgaris

England (R) 15. ii. 02

Buteo Ingnpus (GraeX.) " Rauchfussbussard "

Germany (Liihe) 11. iii. 02
Buteo ferox. Long-legged Buzzard

Ka.shmir (Donald) 8. iii. 02
Archibuteo lagopus. Bough-legged Buzzard

Norway (Z) 14. viii. 01
Butastur indicus. Hawk

Okenawa, Japan (R) 14. iii. 02
Aquila chrysaetus. Golden Eagle

Norway (Z) 11. ix. 01
Aquila chrysaetus

Scotland (Z) 14. xi. 01

tr?

613 Note source; appeared to go slightly into solution.
625, 632 Note source and p. 63 in text.

274

Mammalia

8

?
265

tr?

Aves

d

«

»

a

tr

«

«

d

tr

.

»

tr

00

+

5

d

90

Beptilia

•210
d

Amphibia

Crnstaoea

tr?

S

3

531 . also with anti-seal, hyaena, hedgehog, llama.
634 Foam-test practically negative.

275

18—2

Class AVE8 .

II. Division CARINATAE
5. Order FALCONIFORMES

Mammalia

ANTIHERA FOR.

2. Suborder
Accipitres

Fam.
Falconidae

537
538

639

S40
541
643

643

531.

1. Suborder
Galli

Vam.
Cracidae

544

545
546
547
548

549
650
551
552
553
664
655
566
657

558

669

Haliaehia indm. Brahmiuy Kite
India (Inglis) 4. ii. 02
214. Ilaliaetus Uucogaster. White-tailed Sea
Eagle

Tasmania (Z) 7. x. 01
721. ilaliaetus Uucogaster

Chatrapur, India (Fischer) 23. ii. 02

539. Nisaetus fasciatus. Bonelli's Eagle
Deccan, India (Betham) 14. i. 02

886. Nisaetus prunatus. Booted Eagle

Kashmir State, India (Donald) 18. iv. 02
69. Spizaetus bellicosus. Martial Hawk Eagle
Brit. E. Africa (Z) 25. v. 01

708. Spilornis checla. Crested Serpent Eagle
Bhadarwa, Kashmir State (Donald)
21. iii. 02

6. Order GALLIFORMES

218. Crax globicera. Globose Curassow
Honduras (Z) 30. viii. 01

389. Meleagris gallipavo. Domestic Turkey
England (Mitchell) 24. xii. 01

178. Telrao tetrix. Blackcock

Scotland (R) 28. viii. 01
168. Lagopus mutus Leach. Ptarmigan

Scotland (K) 21. viii. 01
108. Lagopus scoticus. Grouse

Scotland (Haldane) 12. viii. 01

179. Lagopus scoticus

(B) 28. viii. 01
248. Lagopus scoticus

England (Wheeler) 3. xi. 01
528. Goturnix communis. Grey Quail

India (Inglis) 3. ii. 02

170. Perdix cinerea L. Partridge

Germany (Kuse) 13. viii. 01

171. Perdix cinerea

Seeland (Schierbeck) ix. 01

172. Perdix cinerea

Germany (Schlieffen) ix. 01

173. Perdix cinerea

England (Leighton) 11. x. 01

174. Perdix cinerea

England (Wheeler) 20. x. 01
724. Tetraogallus hinialayensis. Himalayan
Snow Cock

Kashmir (Donald) 27. ii. 02
728. Caccabis chukor. " Chukor " Partridge

Kashmir (Donald) 20. ii. 02

826. Phasianus colchicus L. Pheasant
England (R) 13. i. 02

641 No foam.

643 Solution ?, note source.

276

Mammalia

Avea

I

V

Beptilia

Ampliibia

Criiittacea

•s

g

g

1

1

1

+

«

*

D

D

d

«

«

•

.

d

d

*

,

,

5

tr

tr

+

+

.

.

B

1)

tr?

+

+

,

,

a

30
d

*

*

d

60
d

d

+

«

.

D

d

«

*

«

•

d

tr

?

*

«

•

•

d

tr

«

30

tr

«

+

X

*

.

d

d

?

+

«

.

-

D

tr

cl

+

«

.

*

D

d

•210

+

#

«

.

D

tr

d

+

«

*

•

D

d

d

+

*

»

•

D

tr

d

«

»

«

*

D

tr

d

+

«

«

•

D

tr

d

/

•

»

,

.

tr

5
tr

»

•

•

r.

d

587 Note source and text, p. 03.
277

Class AVES

II. Division CARINATAE
6. Orrfer GALLIFORMES
ANTISERA FOR

Mammalia

1. Suborder

Galli

Fam.
Cracidae

Fam.
Rallidae

Fam.
Gruidae

660
561

sea

663
664
566
666
567
668
569
670
671

673

673

674
675

576

677

678
579
6 SO

581

683.
334.
244.
181.
403.
750.
722.
729.
217.
308.
751.
731.

12.

40.

182.
401.

Phasiamis colcMcui L. Pheasant

Mecklenburg (Kuse) 8. xi. 01
Phasiaiins colchicus

England (Leighton) 26. xi. 01
Phasianus colchicus

23. X. 01
Phasianus colchicm

Denmark (Schierbeck) ix. 01
Phasianus colchicus x sinensis. Pheasant

Ireland (N) 4. v. 01
Argusianus grayi. Argus Pheasant

Sarawak, Borneo (Hose) p. 10. iv. 02
Pucrasia macrolopha. " Koklass" Pheasant

Kashmir (Donald) 21. ii. 02
Lophophorus impeyunus. Pheasant

Kashmir (Donald) 21. ii. 02
Thaumulea amherstiae. Amherst Pheasant

China (Z) 24. viii. 01
Thaumalea picla. Golden Pheasant

(B) 7. xi. 01
Euplocomus nohilis. Fireback Pheasant

Sarawak, Borneo (Hose) 10. iv. 02
Euplocomus leucomelanius ? Black-crested
Kalij Pheasant

Kashmir (Donald) 17. ii. 02
Gallus domesticus. Fowl, scales

England (N) 4. iii. 01

Gallus domesticus.
19. X. 01

Pntrid in tube

Gallus domeiticus

Denmark (Schierbeck) ix. 01
Gallus domeslieus

England (N) 19. x. 01

7. Order 6RU I FORMES

843. Rallus uquaticus L. Water Rail
England (R) 7. iv. 02

67. Crex pratensis. Land Rail
Ireland (N) 4. iv. 01

266. Fulica atra L. Coot

England (R) 5. xi. 01

463. Fulica atra

(Bird) 6. xii. 01

637. Fulica atra
31. i. 02

184. Grus communis. Crane

Germany (Schlieffen) ix. 01

tr

560 • with 4 antisera for seal, hyaena, hedgehog, llama.

278

Mammalia

Aves

liejitilia

Amphibia

d

tr?

240

tr

+

a

30

tr?

*

d

tr

+
D

+

D

60

180

tr?

Crustacea

5
2

U

665, 670 Certainly insoluble.

279

'Class AVES

II. Division CARINATAE

8. Order CHARADRIIFORMES
ANTISERA FOR

Mammalia

1. Suborder
Limicolae

Fam.

Charadriidae

Sub/am.

Charadrilnae

Subfam.
Tringinae

Subfam.
Scolopaclnae

683
683
684
686

686
687

688

689

690

681

692

693

694

695
696

697

698

699

600
601
602
603
604

107. Charadrius pliwialis L. Golden Plover

Scotland (R) 21. viii. 01
647. Aegialitis hiaticola. Ringed Plover

England (B) 4. ii. 02
882. AegiaiitisalexandrinavsiT.dealbatus. Snipe

Okenawa, Japan (R) 15. iii. 02
535. Lobivanellus vialabaricus. Yellow-wattled
Lapwing

Ahmedabad, India (Mason) 26. i. 02

551. Recurvirostra avocetta. Avocet

Tirhut, India (Inglis) 17. i. 02

552. Ilimantopus cartdidus. Stilt

Tirliut, India (Inglis) 14. i. 02

776. Tringa alpina. Dunlin
England (B) 19. v. 02

845. Tringa canutus. Knot

England (R) 11. ii. 02

524. Totanus stagnalis. Marsh Sandpiper

India (Inglis) 2. ii. 02

525. Totanm glareola. Wood Sandpiper

India (Inglis) 2. ii. 02

523. Totanus fuscus. Spotted Redshank
India (Inglis) 1. ii. 02

767. Totanus hypoleucus Teram. Sandpiper
England (B) 19. v. 02

522. Limosa belgicn. Black-tailed Godwit
India (Inglis) 14. i. 02

674. Numenhis arqiuita L. Curlew

England (B) iii. 02
766. Nuineiiius phaeopii^. European Whimbrel

England (B) 19. v. 02

836. Numenius phaeopus
(Clarke) 19. v. 02

673. Scolopax rusticola L. Woodcock
England (B) ii. 02

454. Scolopax rasticola

(Clarke) 24. xii. 01

336. Scolopax rusticola

(Bird) 28. xi. 01
526. Gallhuigo ctielestis. Fantail Snipe

India (Inglis) 2. ii. 02
837. Gallinago caelestis. Full Snipe

Engluud (R) 21. ii. 02
331. Gallinago caelestis vel media. Snipe

India (Dalgett^-) 4. xi. 01
264. Gallinago gallinula. Jack Snipe

England (R) 5. xi. 01

684 Reactions all negative, did not foam.
280

Mammalia

Aves

Keptilia

Amphibia Or

xiBiaci

M

■""

4)

lU

•O

'm

1

a

<

p.

1

CD

H

o

o

X

1

1

1

1

5

1

1

2

«

«

d

d

*

6*0

■

•

«

*

.

.

210

240

,

,

,

«

«

«

,

d

d

240

.

.

•

«

*

•

•

•

.

d

d

?

180

•

,

,

.

,

»

,

,

45

tr

*

35

tr

•

•

«

#

>

•

210

d

d

•

.

*

#

»

•

210

d

210

d

•

.

X

»

»

•

D

D

210
d

.

.

«

•

45

tr

•

2-io

+
D

+

d

45

tr

X

5
d

+
60
d

+
d

d

210
d

•

-N

«

«

?

•

60
d

+
b

«

D

I)

»

tr

*

d

»

,

•

•

•

.

.

*

•

•

tr?

<

•

180

«

?
d

HO

tr

•

•

•

•

S95 • with anti-tortoise.

699 • with ftnti-seal, hyaena, hedgehog, llama.
281

aass AVE8

II. Division CARINATAE
8. Order CHARADRI1F0RME8
ANTI8EBA FOR

Mammalia

■a

n

1. Suborder
Limicolae

Fam.

Charadriidae

Suhfam.

Scolopaclnae

Fam.
Chionididae

Fam.
Parridae

Fam.
Laridae

Fam.

622

Alcidae

2. Suborder

623

Columbae

Fa m.

624

Columbidae

625

605
606
607
608
609
610

611

612

613

614

616

616

617

618
619
620

621

265. Gallinago flallinago. Common Suipe

England (R) 5. xi. 01
730. Gallinago solitaria. Solitary Snipe

Kashmir (Donald) 21. ii. 02
384. Gallinago spec. " Winter Snipe "

Shanghai, China (Stanley) ca. 27. xi. 01

862. Arenaria interpres. Snipe

Okenawa, Japan (R) 19. iii. 02

863. Tringoidcs hypoleucus. Snipe

Okenawa, Japan (R) 14. iii. 02
873. Ochthodromus moitgolus. Snipe
Okenawa, Japan (R) 9. iii. 02

71. Chionis alba Forst. Yellow-billed Sheath-
bill
England (Z) 28. v. 01

614. Parra {africayia'!) Japana

Lake Nyassa, C. Africa (Dodds) p. 8. ii. 02

70. Lams argentatus. Herring Gull
England (Z) 28. v. 01

462. Larus argentatus
(Bird) 6. xii. 01

653. Larus ridibtindus L. Black-headed Gull
England (B) 24. i. 02

834. Larus ridibundus L. Black-headed Gull
England (Clarke) 15. ii. 02

652. Larus glaucus. Glaucous Gull
England (B) 3. i. 02

550. Larus brunneicephalus. Brown-headed Gull
Darbhanga, India (Inglis) 28. i. 02

344. Larus canus L. Common Gull
England (Clarke) 16. xii. 01

850. Larus .. .spec ^ Little Gull

England (Clarke) 10. ii. 02

546. Sterna anglica. Gull-billed Tern

Darbhanga, India (Inglis) 31. i. 02

835. Uria troilc. Ringed Guillemot
England (Clarke) 8. ii. 02

825. Culumba palumbiis L. Wood Pigeon
England (R) 1. iii. 02

398. Columba palumbus

(Bird) 28. xi. 01

399. Columba palumbus

(Wheeler) ca. 22. x. 01

15

607 Filter paper but slightly stained, diluted blood ?.
608 Foam-test doubtful, no reactions. 609, 610 Foam-test negative, no reactions.

282

Mammalia

Av

ea

[leptilia

Amphibia Cr

ustaci

8

1

a
<

aa

H

o

B

1

1

•s

■c

9

1

1

1

1

<

o

S

*

.

.

.

.

d

,

,

,

,

,

.

«

,

.

•

•

•

•

*

•

'

tr

*

•

tr?

•

•

*

"

+

«

b

d

tr
?

+

»

b

d

tr
?

,

,

,

.

,

♦

«

.

.

>

.

•

•

30

d

d

,

,

,

.

.

*

»

•

•

•

•

•

GO

60

tr

d

•

•

•

•

•

5
d

CO
tr

+
D

d

•

•

•

•

•

/

•

•

•

/

X

5
d

»

d

*

*

d

•

/

«
240

•

tr

•

.

.

•

•

•

,?
180

tr?

+

a

+

»

tr

•

•

•

■

•

•

a

tr

613 Foam-test doubtful, all reactions negative.

283

ei», 822 Sample greasy.

'2. Suborder
Columbae

Fam.
Columbidae

1 . Suborder
Cuculi

Fam.
Cuculidae

Fam.
Musophagidae

2. Suborder
Psittaci

Class AVE8

II. Division CARINATAE
8. Order CHARADRIIF0RME8
ANTISEEA FOR

Mammalia

636
627
628
620
630

631
632
633
634
63S

636

687

638
639
64 O

641
642

643

644

645

646

332.

313.

175.

138.

13.

21.

41.
491.
723.
547.

763.

533.

215.

583.

72.

Columba palumbtis L. Wood Pigeon

(Leigliton) 26. xi. 01
Columba palumbus

(B) 13. xi. 01
Columba palumbus

Germany (N) 4. ix. 01
Columba palumbus

(Kuse) 10. viii. 01
Columba livia. Domesticated Pigeon
(scales)

England (N) 11. iii. 01
Columba livia

11. iii. 01
Columba livia

9. V. 01
Columba livia

Trinidad (Tulloch) 19. i. 02
Columba intermedia. Blue Bock Pigeon

Kashmir (Donald) 15. ii. 02
Columba eversmanni. Eastern Stock Pigeon

Tirhut, India (Inglis) 17. i. 02

Columba oenas L. Stock Dove
England (B) 24. iv. 02

Turtur cambayensis. Little Brown Dove
Ahmedabad, India (Mason) 26. i. 02

Geopelia striata. Barred Dove

India (Z) 30. ix. 01
Caloenns nicobaricaTjinn. Nicobar Pigeon

Indian Archipelago (Z) 26. ii. 02
Caloenas nicobarica

28. v. 01

9. Order CUCULIF0RME8

112.

Cuculus canorus L. Cuckoo
Germany (Kuse) 7. viii. 01
702. Cuculus canorus

England (B) 23. iv. 02

893. Turacus buffoni Vieill. Buffon's Touracou
W. Africa (Z) 17. ix. 02 d. of ascites

87. Ara macaox A. militaris. Macaw Hybrid
Bred in Italy (Z) 24. vi. 01

122. Cacatua roseicapillaVieiW. lloseate Cock-

atoo
Australia (Z) 22. vii. 01

123. Cacatua leadbeateri Vig. Leadbeater's

Cockatoo
Australia (Z) 12. vii. 01

tr?

630, 643 • with anti-seal, hyaena, hedgehog, llama.

284

Mammalia

Avca

Uoptilia

Ampliil

ia Cm

fitacea

M

1

1

t

1

I

t

o

1

a

«.

1

■a

^

1

t

3S

3

2

=1

?

V

3

S

<

m

o

K

b.

o

u.

ui

H

<

b4

S

,

+

«

,

,

,

.

a

•

•

*

•

•

•

.

d

tr

•

«

•

•

•

•

.

d

tr

•

*

»

■

■

>

.

d

•

»

•

d

.

•

•

/

*

D

d

/

•

*

•

/

1

/

/

•

•

9

240

240

•

•

+

«

45
tr

210
d

•

d

+
D

+

a

»
«

45
tr

+
30

a

+
a

+

*
D

*

d

*

d
+
D

»

d

+
tr

+
30
d

d

#
d

180
tr?

•

•

•

•

tr?

•

•

•

1

•

a

+
a

d
d

•

•

•

•

•

•

285

Class AVE8

II. Division CARINATAE

Mammalia

9. Order CUCULIFORMES

ANTISEBA FOR

2. Suborder

Psittaci

1. Suborder
Coraciae

667

F,im.
Alcedinidae

858

659

660

Fam.
Meropidae

661

Fam.
Buoerotidae

662

2. Suborder

Striges

663

Fam.
StPigidae

664

647

848
640
660

851
652

663

654

666

656

665

363. Ghrysotis versicolor. Blue-faced Amazon
Parrot

St Lucia W. I. (Z) 27. xi. 01
346. Ghrysotis versicolor

S. America (B) 25. xi. 01
814. Loriculus indicus. Parrot

Ceylon (R) 22. i. 02
125. Pyocephalus senegalus Linn. Senegal
Pflrrot

W. Africa (Z) 24. vii. 01
223. Pyocephalus guliehni. Jardine's Parrot

W. Africa (Z) 20. viii. 01
370. Polytelis vielanura Vig. Black-tailed
Paroquet

Australia (Z) 23. xii. 01
560. Palaeoniis torquata. Rose-ringed Paroquet

Ahmedabad, India (Mason) 27. i. 02

302. Conurus auricapillus. Golden-Headed
Conure

S. America (Z) 18. xi. 01
741. Nestor notabilis Gould. Ka-Ka Parrot

N. Zealand (Z) 2. vi. 02 killed, para-
lyzed, Jl.
156. Nestor notabilis
8. viii. 01

10. Order CORACIIFORMES

841. Alcedo inpida. Kingfisher

England (Clarke) 22. i. 02

879. Alcedo ispida var. bengalensis. Kingfisher

Japan (R) 14. iii. 02
877. Alcedo ispida var. bengalensis

(R) 11. iii. 02
811. Halcyon smyrnensis fu.icus. Kingfisher

Ceylon (R) 27. i."02

810. Merops phillipinus. Bee Eater
Ceylon (R) 27. i. 02

216. Rhytodoceros undulatus. Malayan Wrinkled
Hornbill

Malacca (Z) 8. x. 01

540. Strix javanica. Indian Screech Owl
Bombay, India (Phipson) 23. i. 02

682.

Strix Jlammea L. Barn Owl
Mecklenburg (Kuse) 5. x. 01

521. Strix Jlammea

India (Inglis) 28. i. 02

240

668, 669 Foam-test doubtful, no reactions.

Mammalia

Aves

Reptllia

Amphibia

Crustacea

tr?

tr?

+

d

+

«

d

It

•

30

tr?

*

*

30

d

*

•

tr

+

X

d

*

,

D

tr?
tr?

/

30

/

30

d

d

d

+
D

+
00

s

»

D

«

d

»

d

240

tr?

287

Class AVE8

11. Division CARINATAE

10. Order CORACIIFORMES

ANTISEEA FOR

Mammalia

2. Suborder
Striges

Fam.
Strigidae

3. Suborder
Pici

Fam.
Capitonidae

Fam.
Rhamphastidae

Fam.
Picidae

Fam.
Alaudldae

666
667
668
669
670

671

673
673
674

676
676

677

678
679

680
681
683

683

684

68S

250. Strix flammea L. Barn Owl

England (Farren) v. 01
310. Strix flammea

(B) 7. xi. 01
345. Syrnium aluco L. Tawny Owl

England (Clarke) 13. xii. 01
638. Asio brachyotus. Long-eared Owl

England (B) 20. xii. 01
827. Asio brachyotus

(Clarke) 12. iv. 02

532. Bubo bengalensis. Rook Horned Owl

Ahmedabad, India (Mason) 26. i. 02

887. Bubo bengalensis

Kashmir State (Donald) 17. iv. 02
279. Bubo maximus. Great Eagle Owl

Europe (Z) 11. xi. 01
281. Nyctea scandiaca. Snowy Owl

Bylott Islands. Lancaster S'd. (Z)
14. xi. 01

720. Xanthohiema liematocepJiala ? Crimson-
breasted Barbet

Bombay, India (Phipson) 18. ii. 02
812. Xantholaema rubricapilla. I3ar-bit

Ceylon (R) 27. i. 02

624. Rhamphastus ariel. Toucan

Brazil (Hagmann) 4. iii. 02

639. Picus major h. Great Spotted Woodpecker

England (B) 4. ii. 02
452. Picus major

(Clarke) 21. i. 02

640. Gecinns viridis Ijinn. Green Woodpecker

England (B) 12. ii. 02
307. Gecinvs viridis

(B) 11. xi. 01
678. Picus spec. Woodpecker

Mecklenburg (Kuse) 14. xii. 01

761. Dendrocopus minor. Lesser-spotted Wood-
pecker

England (B) 12. v. 02
669. lynx torquilla L. Wryneck

England (B) 10. iv. 02

11. Order PASSERES

544. Pyrrhulauda grisea. Ashy-crowned Finch
Lark

Darbhanga, India (Inglis) 4. ii. 02

673 Foam test doubtful, reactions all negative.
676 Foam test negative or doubtful, no reactions.

288

i

Mammalia

Aves

Reptilia

Ampliibia Cn

DsUcei

1

1

a

a,

O

1

1

■g

!

3

a

V

1

1

.

.

,

«

.

d

tr

•

•

■

•

d

X

d

?

'

•

'

•

•

•

•

•

•

+

a

tr

'

tr?

•

•

•

•

•

•

•
•

•

•

•

*

X

5
D

+

+
d
+
d

CO

'+
D

«
tr

#

tr

16

210
d

*

•

•

•

i

•

•

•

•

/

CO
tr?

*

CO
D

«

3*0

*

45
d

120
d

CO

•

•

,

.

•

•

•

•

•

•

d

GO
d

«

45
tr

+
CO
d

d

tr
d

tr

«

d

«

?

•

•

•

:*.!.

-„*: — „i /

■> Q--;

\ l.T-

L hft

iL't^llOU.

07« Solution weak. 677

683 • with auti-tortoise.

289

19

aass AVE8

Mammalia

Fam.
Alaudidae

Fam.
Timeliidae

Fam.
Pycnonotidae

Fam.
Muscicapidae

Fam.
Turdidae

II. Division CARINATAE
11. Order PASSER ES
ANTISEEA FOB....

Fam.

688

871.

Motacillidae

689

349.

690

901.

691

545.

69a

867.

698

666

694

450.

696

451

686

687

696

697

698
689
700

70I

7oa

703
704
70S
706
707

534. Pyrrhulauda grisea. Ashy-crowned Finch
Lark

Ahmedahad, India (Mason) 26. i. 02
641. Alatida arvensis L. Skylark

England (B) 28. i. 02

Motacilla boarula melanope. Wagtail
Okenawa, Japan (R) 13. iii. 02

Motacilla lugubris. Pied Wagtail
England (B) xi. 01

Motacilla lugubris
(B) 25. viii. 02

Anthus maculatus. Indian Tree Pipit
Darbhanga, India (Inglis) 31. i. 02

Anthus maculatus

Okenawa, Japan (B) 14. iii. 02

Anthus trivialis. Tree Pipit
England (B) 9. iv. 02

Anthus pratensis. Meadow Pipit
England (Clarke) 19. xii. 01

Anthus obscurus. Book Pipit
England (Clarke) 18. xii. 01

537. Melacocercvs terricolor. " Sat-bhai " or
Bengal Warbler
Deccau, India (Pbipson) 10. i. 02

538. Pycnonotus liaemorrhous. Common Madras
Bulbul

Deccan, India (Phipson) i. 02
817. Kclaartia pcnicillata. Yellow Bulbul

Ceylon (B) 31. i. 02
865. Hypsipetes squamiceps pryeri

Okenawa, Japan (B) 10. iii. 02
815. Hypsipietes ganeesa

Ceylon (B) 31. i. 02

902. Muscicapa grisola. Spotted Fly-catcher
England (B) 22. viii. 02

82. Turdus musicus L. Thrush
England (N) 14. vi. 01

194. Turdus m^isicus

(Garrood) 28. viii. 01

195. Turdus mmicus

(B) 16. viii. 01

318. Turdus ynusicus

(B) 2. xii. 1901

319. Turdus viscivorous. Mistletoe Thrush

England (B) 30. xi. 01

465. Turdus pilaris. Fieldfare

England (Bird) xii. 01

688 Foam-test doubtful, all reactions negative. 690 • with anti-hyaeua and hedgehog.

290

Mamiiialia

Aves

tr?

Iteptilia

21U
d

180
tr

210
d

/

240
d

tr?

#
d

•

d

*

d

d

d

30

3*0

30

«

/

ft

tr?

*

tr?

X

ft

d

240

tr?

240

Amphibia

CruHtacea

?
2i0

603, 699 Foam-test doubtful, no reactions.

694 • with anti-seal, hyaena, hedgehog, llama.

291

19—2

aass AVE8

II. Division CARINATAE
11. Order PASSERES
ANTISERA FOR....

Mammalia

Fam.
Turdidae

708

709

326.

710

809.

711

868.

7ia

662.

713

876.

714

880.

715

864.

716

872.

717

768.

Fam.
Hipundinidae

Fam.
Ampelidae

Fam.
Laniidae

71B
710

7ao

721

723

723
724
725
726

727
728

720

730
731
732

191. Turdus merula L. Blackbird

England (Garrood) 22. viii. 01

Turdus merula L.

England (R) 1. xii. 01
Cisticola cisticola brunneiceps. Fan-Tail

Okenawa, Japan (R) 14. iii. 02
Cettia cantans

Okenawa, Japan (R) 6. iii. 02
Luscinia iuscinia L. Nightingale

England (B) 12. iv. 02
Geocichla varia. Missel Thrush

Okenawa, Japan (R) 10. iii. 02
Merula chrysolaus. Thrush

Okenawa, Japan (R) 15. iii. 02
Merula pallida. Thrush

Okenawa, Japan (R) 14. iii. 02
Merula pallida

13. iii. 02
Pratincola rubetra. Whinchat

England (B) 23. iv. 02

668. Saxicola oenanthe. Wheatear
England (B) 8. iv. 02

670. Phylloscopus ru/us. Chiffchaff

England (B) 11. iv. 02
268. Accentor modularis. Hedge Sparrow

England (R) 1. xi. 01
335. Accentor modularis

(Bird) 25. xi. 01
774. Sylvia hortemis Lath. Garden Warbler

England (B) 6. v. 02

169. Sylvia curruca L.

Germany (N) 1. ix. 01
166. Erithacus rubecula L. Robin

Germany (N) 25. viii. 01
314. Erithacus rnbecula

England (Bird) 9. xi. 01
661. Ruticilla phoetiicurus. Redstart

England (B) 14. iv. 02

65. Chelidon urbica. Swallow

Ireland (N) 4. iv. 01
773. Cotile riparia L. Bank Swallow
England (B) 23. iv. 02

456. Ampelis garrulus. Waxwing

England (Clarke) 20. xii. 01

777. Lanius {collurio ?). Shrike

England (Mitchell) 26. v. 02

752. Pityriasis gymnocephala

Borneo (Hose) p. 10. iv. 02

816. Hemipus picatus. Tomtit
Ceylon (R) 31. i. 02

tr?

710, 711, 714, 715, 716

Foam-test doubtful, no reactions.

723 Very dilute solution,

713 Foam-test negative, no reactions.

292

Mammalia

Aves

llcptilia

Amphibia

Cruxtacea

V

I

120

45

tr

+

•

liO

tr

cl

•

.

tr

tr

«

•

tr

tr?

♦

•

?

tr

«

90

tr

.

•

tr

•

«

d

tr

*

#

d

tr

tr?

30

U

739 . with antisera for seal, hyaena, hedgehog, llama. 730 Foam-test doabtful.

731 Foam-test doubtful, no reactions.

293

Class AVE8

Mammalia

Fam.
Laniidae

Fam.
Sittidae

Fam.
Paridae

Fam.
Panuridae

Fam.
Corvidae

II. Division CARINATAE

11. Order PASSERES
ANTISERA FOB

733

734

735
736
737
738
730

740

741

742
743
744

746

746

747
748
749

7 SO
751

763
753

764

107. Gymnorhina leuconota. White-backed
Piping Crow

S. Australia (Z) 13. viii. 01

320. Sitta caesia. Nuthatch

England (R) 30. xi. 01

860. Parus atriceps. Tomtit

Okenawa, Japan (R) 9. iii. 02
870. Parus atriceps

15. iii. 02
269. Parus ater. Coletit

England (R) 1. xi. 01
321. Parus caeruleus. Blue Tit

England (R) 30. xi. 01
306. Parus major. Great Tit

England (R) 9. xi. 01

839. Parnurtts biarmiciis. Bearded Tit
England (R) 11. iii. 02

536. Corvus macrorhynchus. Indian Corby or
Jungle Crow

Bombay, India (Phipson) i. 02
713. Corvus macrorhyncJnts

Bhadarwa, Kashmir (Donald) 12. iii. 02
353. Corvus cornix. Hooded Crow

England (B) 14. xi. 01
824. Corvus corone L. Carrion Crow

Wales (Clarke) 11. iv. 02

680. Corvus corone

Mecklenburg (Kuse) 17. ii. 02

765. Corvus corone

England (B) 8. v. 02

90. Corvus vwnedula. Jackdaw

England (N) 24. vi. 01
711. Corvus monrdula

Bhadarwa, Kashmir (Donald) 25. iii. 02
66. Corvus fruqilequs L. Rook

England (N) 20. v. 01

186. Corvus fruffilegiis

Germany (Schlieffen) ix. 01
679. Corvus frxLgilegus

(Ruse) 28. X. 01

303. Corvtis sph-ndens. Indian Crow

India (Z) 20. xi. 01
558. Con us splendens

Bombay, India (Mason) 4. ii. 02

764. Corvus corax L. Raven
Scotland (B) 23. iv. 02

I

736, 736, 740, 748 Foam-test doubtful, no reactions.

294

d

Mammalia

Avea

Reptilia

Amphibia

Cruataccft

?
120

30

tr?

60

+

X

a

tr

»

•

?

180

tr

+

*

60

d

d

X

45

tr

.

+

d

+

+

30

d

.

X

d

tr

+

•

60

d

d

#

■»

d

tr

+

*

A

d

«

45

tr

tr?

tr?

738 Foam-teat doubtful when tested with anti-oatrioh.

295

74S • with anti-tortoise.

Class
II.

AVE8

Division CARINATAE
11. Order PAS8ERES
ANTISERA FOR...

Mammalia

s

M

Fam.
Corvidae

Fam.
Sturnidae

Fam.
Icteridae

Fam.
Fringillidae

765
766
767
768
769
760
761

762

763

764
766

766

767

664.

768

56.

769

323.

770

844.

771

663.

773

324

773

351

774

■sr,o

776

840

776

777
778

222.
165.
333.

57.
352.
642.

822.

823.
681.

189.

Cissa venatoria. Hunting Crow

India (Z) 24. viii. 01
Garrulus glandarim L. Jay

Germany (N) 2. ix. 01
Garrulus glandarius

England (Leighton) 26. xi. 01
Nucifrafla cnryocatimtes. Nutcracker

England (Lane) 28. iv. 01
Pica rustica. Magpie

England (R) 18. xi. 01
Pica rustica

(B) 14. xii. 01
Pica rustica

(Clarke) 23. i. 02

Pica rustica
(R) 5. iii. 02

Pico ruMica

Mecklenburg (Kuse) 19. xii. 01

Sturnus vulgaris L. Starling
England (Garrood) 8. viii. 01
725. Acridotlieres tristis. Common Minah

Bhadarwa, Kashmir (Donald) 28. ii. 02

106. Icterus Jamaica. Brazilian Hangnest
Brazil (Z) 13. viii. 01

Fringilla coelebs L. Chaflineh

England (B) 8. iv. 02
Frinqilla coelebs

(N) 28. iv. 01
Fringilla coelebs

(R) 30. xi. 01
Chri/somitiis spinus. Siskin

England (R) 20. ii. 02
Ligurinns chloris. Greenfinch

England (B) 8. iv. 02
Ligurinus chloris

(R) 30. xi. 01
Fringilla chloris x Carduclis elegans. Hy-
brid Greenfinch-Goldfinch

England (B) 10. xii. 01
Fringilla montifringilla. Brambling

England (B) 25. xi. 01
Carduelis elei)aiis. Goldfinch

England "(R) 7. ii. 02

144. Paroaria cucullata. Red-crested Cardinal
S. America (Z) 9. viii. 01

455. Coccothraustes vulgaris Pall. Hawfinch
England (Clarke) 21. xii. 01

348. Coccothraustes vulgaris
(B) 30. xi. 01

30

766 Foam test doubtful, no reactions.

296

J

Mammalia

Ave

1

Reptilia

Amphibia CruRtacea

•a
a

8

1

5

to

H

o

<0

1

1

Fowl-egg
Ema-egg

®

1

1

1

«

.

.

.

.

d

tr

tr

,

•

•

*

*

• •

*

>

•

>

d

tr

•

•

+

X

tr

• •

"

•

'

■

•

■

,

+

X

tr
6*0

.

•

•

•

•

•

'

5
d

•

•

•

•

•
•

?

210

/

6*0
d

d

CO

+

35

6*0

+

d
tr

3*0

tr

•

•

•
•

•
•

•

•
•

•
•

•

•

•

+
+
tr

tr

*

t

«
r?

»

tr

•

•

•

•

X

>

•

•

\

«

d

*
d

+

d

til

?

tr

*

•
?

•
tr?

•

•

tr?

•

tr?

297

Fam.
Fringillidae

aass AVE8

II. Division CARINATAE

11. Order PASSERES
ANTISEBA roil....

Mammalia

779
7 SO
781

783

783
784

785
786
787
788
789

790

791

793

793
794
795

796

797

798

799

643. Loxia curvirostra Gm. Crossbill

England (B) 10. i. 02
192. Pyrrhula europaea. Bullfinch

England (Garrood) 28. viii. 01
772. Pyrrhula europaea

(B) 20. iv. 02

665. Linota cannahina. Linnet
England (B) 8. iv. 02

267. Passer domesticus L. House Sparrow
England (R) 1. xi. 01

769. Passer ilomesticus

(B) 3. V. 02

322. Passer domesticus

(H) 30. xi. 01
813. Passer domestictts indicun. Sparrow

Kandy, Ceylon (B) 29. i. 02
861. Passer montanus saturatus. Sparrow

Okenawa, Japan (R) 14. iii. 02
875. Passer montanus saturatus

Okenawa, Japan (R) 6. iii. 02
838. Plectrophanes nivalis L. Snow-Bunting

England (Clarke) 8. u. 02

667. Emberiza miliaria. Corn-Bunting
England (B) 9. iv. 02

771. Emberiza schoeniclus. Reed-Bunting
England (B) 30. iv. 02

770. Emberiza citrinella L. Tellow Hammer

England (B) 3. v. 02

325. Emberiza citrinella

(R) 30. xi. 01
878. Emberiza persotiata

Okenawa, Japan (R) 14. iii. 02
143. Crithayra sulphuratus. Sulphury Seed-
Eater

S. Africa (Z) 9. viii. 01

EGGS

402. Galius domesticus. Fowl

England (N) 26. x. 01. Egg-white, dried
on paper
632. Euplocomtis (Phasianus) nycthemerus L.
Silver Pheasant
China (Z) 2 eggs laid ca. 11. iv. 02.
Egg-white, fluid
593. Balearica regulorum Bennett. Cape
Crowned Crane

S. Africa (Z) 13. iii. 02. Egg-white,
fluid
439. Dromaeus novae-hollandiae Vieill. Emu
Australia (Z) rec'd. 18. i. 02. Egg-
white, fluid

787, 788 Foam-test doubtful, all reactions negative.
298

Mammnlia

Avcs

Rcptilia

Amphibia

Cniataoes

«

tr?

tr?

tr?

?
265

i s

tr

30

180
tr

30

+

«

00

GO

d

d

»

45

tr

»

9

tr

+

«

I

tr?

tr?

794 Foam-test doubtful, no reactions.

797 . with anti-aeal, hyaena, hedgehog, llama.
299

Class

REPTIUA

Mammalia

1. Subclass CHELONIA

Order THECOPHORA
ANTISEBA FOR

Fam.
Testudinldae

Fam.
Chelonidae

Fam.
Crocodilidae

Suborder
Geckones

Suborder
Lacertae

800
BOl

8oa

SOS
804
SOS
806
807
SOB

SOD
810
811
813

813

814
818

816

817
818

805. Chrysemys picta. Painted terrapin

N. America (Jordan) 22. v. 02

806. Chrysemys elegans

N. America (Jordan) 11. v. 02
804. Cistudo Carolina

N. America (Jordan) 30. iv. 02

807. Graptemys pseudogeographicua

N. America (Jordan) 17. v. 02
42-5. Testudo ibera. Tortoise

Greece (killed Cambridge, N) 14. i. 02,/.
793. Testudo elephantopus. Giant Tortoise

(N. York Zoo., Langmann) 14. vi. 02
904. 'Testudo elephantopus

GalapAgos (Z) 15. x. 02,/.
78. Testudo inepta. Clumsy Tortoise

Mauritius (Z) 20. v. 01
818. Testudo spec. Tortoise, egg-white

(Leighton) egg laid 2. vii. 02, /.

343. Chelone midas. Green Turtle

Trop. Seas (killed Cambridge) 4. xii. 01 ,/.
209. Chelone viidas

(Z) 9. X. 01
628. Chelone viridis ? Turtle

(B) 13. xii. 01
615. " Water Turtle "

Upper Shir(5 R., C. Africa (Dodds) p.
8. u. 02

2. Subclass CROCODILIA

Order EUSUCHIA

338. Alligator ntississipi>iensis

N. America (Z) 27. xi. 01,/.

101. Alligator sinensis

Cbiua (Z) 13. viii. 01
616. Crocodile

Upper Shir^ E., C. Africa (Dodds) p.
8. ii. 02

3. Subclass SAURIA
Order LACERTILIA

153. Gecko quttatus. Gecko

S. Sylhet, India (Dalgetty) 2. viii. 01

555. Calotes versicolor. Indian Tree-Lizard
W. Deecan, India (Phipson) 12. i. 02

911. Uromastix spinipes

Egypt (G. Elliot Smith) 7. vi. 02

806 + with auti- tortoise, last 3 tested by Dr Graham-Smith.
300

Mammalia

Aves

Beptilia

Amphibia Crustacea

V

tr
tr?

120
cl
/

812 Dried sample, note source and see text, p. 63
301

Class REPTILIA

3. Subclass SAURIA

1. Order LACERTILIA

ANTISEBA FOE

Maminaiia

Suborder

Lacertae

819
820

821

823
823
824
825

828

434.

827

280.

828

607.

82»

208.

830

802.

831

913.

832

760.

833

68.

834

909.

835

914.

836

915.

837

618.

838

554.

839

574.

840

883.

841

884.

842

553.

437.
627.

635.

206.

912.
205.

570.

lyiiana {tuberculata Laur. ?). Iguana

Trinidad (J. P. TuUoch) 1. i. 02
Anguis fragilu L. Slow- Worm

Herefordshire, England (Leighton)
31. iii. 02
Varamis saloator Laur. Two-banded
Monitor

E. India (Z) 6. iv. 02
Varaniis griseus. Grey Monitor

N. Africa (Z) 30. ix. 01
Lacerta agilis. Sand Lizard

England (G.-S.) 3. v. 02
Scincidae spec. ? Lizard

S. Sylhet, India (Dalgetty) 1. ix. 01
"Laud Lizard," 2 ft. 6 in. long

Orange River, S. Africa (Parkinson)
13. xu. 01

2. Order OPHIDIA

Python molurus Linn. Indian Python

India (Z) 11. i. 02
Python seboe. Python

W. Africa (Z) 8. xi. 01
Curallus caiiiniis L. "Hundskopfschlange"

Madagascar ?...(Liihe) 20. ii. 02
Boa comlrictor. Corumon Boa

S. America (Z) 7. x. 01
Boa constrictor

Trinidad, W. Indies (Tulloch) 4. vi. 02
Eryx jaculiis

Egypt (G. Elliot Smith) 19. vi. 02
Tropidonotus natrix Gesn. Green Snake

England (B) 28. iv. 02

Tropidonotus natrix

(N) 2. V. 01
Tropidonotus natrix

(Graham-Smith) 18. x. 02,/. ser.
Zamenis diadema

Egypt (G. Elliot Smith) 11. vi. 02
Zamenis varageus var. nummifer

Egypt (G. Elliot Smith) 19. vi. 02
Dcndrophis lioccrcus. Whip Snake

Trmidad, W. Indies (TuUoch) 2. iii. 02
Cerberus rkyncliops. Indian Estuary Snake

Bombay, India (Phipson) 17. i. 02
Cerberus rhynclwps

Calcutta, India (Rogers) 8. x. 01
Naja tripudians Merr. Cobra

Rajputana, India (MacWatt) 21. vi. 02
Naja tripudians

23. vi. 02
Naja tripudians

Bombay, India (Phipson) 4. ii. 02

60

822 Another sample (920) proved as insoluble as the following.

302

Mammalia

Aves

Reptilia

Amphibia

Crustacea

tr?

180

a

d?

823

Insoluble in saline after 30 hours at 37° C.

303

Class

REPTIUA

3. Subclass SAURIA

Mammalia

2. Order OPHIDIA
ANTISEBA FOB

!?

$

'O

a

1

5

1

be

1

'

•

"

•

/

/

/

•

•

•

•

•

/

/

/

843 157. Naja tripudians Merr. Cobra
India (Z) 6. viii. 01

844 916. Naja nuje. African Hooded Cobra (Aepis)
Egypt (G. Elliot Smith) 19. vi. 02

846 685. Pscudechis porphyriacus. Black Snake

Oberon, N.S.W. (Cashman) 21. xii. 01

846 142. Bites arietans. Puffadder
S. Africa (Z) 6. viii. 01

847 787. Puffadder
S. Nyassa, Africa (Dodds) 27. iii. 02

848 686. Vipera berue. Adder
N. Devon, England (Leighton) iv. 02

848 689. Vipera berus

Dumfriesshire, Scotland (Leighton)
iv. 02
860 688. Vipera heriis

Devon, England (Leighton) 18. iv. 02

851 898. Vipera {russelli ?). Himalayan Viper
Bhadarwa, Ka.shmir State (Donald)

4. iv. 02

852 378. Crotalus spec. Rattlesnake
N. America (Salmon) 6. xi. 01

853 613. " A poisonous .serpent "
nr. Lake Nyassa, C. Africa (Dodds)

p. 8. ii. 02

844 Solution foamed well.

304

Mammalia

I ' I

I ! =

O tB

Aves

tr?

/

Reptilia

Amphibia

CrusUoM

847 • with 4 antisera for seal, hyaena, hedgehog, llama.

306

30

Mammalia

Fa III.
Amphiumidae

Fam.
Salamandridae

1 . Suhordn'
AGLOSSA

2. Suborder
PHANEROGLOSSA

Fam.
Bufonidae

Fam.
Engystomatidae

Fam.
Ranidae

aass AMPHIBIA

1. Order U RODE LA

ANTISERA FOE.

864

866

866

867

868
859

860

861
863
863
864

8661
8661

867
868

599. AmphiuiiM means Garden. Amphiuum
N. America (Z) 19. iii. 02,/. vo-.

365. Amblystoma Hflrinum (larva). Axolotl

Mexico (raised in England by E. Bles
2(>. xii. 01
55. Triton cristatus. Crested Newt
England (N) 26. iv. 01

2. Order ANURA

598. Xenopua loevis Daud. Smooth-clawed Frci(?
E. Africa (Z) 19. iii. 02

81. Bufo vulgarU Laur. Common Toad

■ England (N) 4. vii. 01
430. Bufo mauritanica Schlegel. Moorish Toad
' N. W. Africa (Z) 10. i. 02

207. Magalohatrachus maxima. Gigantic Sala-
mander

Japan (Z) 23. ix. 01

54. Rana temporaria. P'rog

England (N) 28. iv. 01
582. Rana temporaria

3. iii. 02,/.
406. Rana temporaria

11. xi. 01

478. Rana tigrina Daud. Tigrine Frog

India (Z) 25. i. 02

479. Rana tigrina

3. ii. 02

597. Rana tigrina

19. iii. 02
732. iJan« Bpec. Common Trinidad Frog " Cra
paud"

Trinidad, W. Indies (TuUoch) 27. iii. 02

/ /

866

with 5 anliseva for monkey, seal, hyaena, hedgehog, llama.

306

Miimmalia

Aves

Beptilia

Amphibia

Crustacea

307

20—2

Glass

PISCES

The tests made with some of the antisera used in the preceding tables gave

entirely negative results. The antisera used were the following:

1. Anti-Man 10. Anti-Lobster 19. Anti-Cat

2. „ Dog

3. ,, Horse

4. „ Ox

5. ,, Sheep

6. „ Pig

7. „ Fowl

8. „ Fowl's egg

9. „ Frog

For convenience the antisera are ordered in this case in the order in which
they were produced, not in their zoological order. The fish bloods which proved
insoluble after some hours in salt solution are not included in the list. With the
exception of the blood of the dogfish (872) which was fluid, all of these bloods
were received dried on filter-paper. The bloods tested were the following :

11.

,, AUigiitor

20.

„ Wallaby

12.

„ Turtle

21.

„ Monkey

13.

„ Ostrich

22.

„ Seal

14.

„ Emu's egg

23.

,, Hedgehog

15.

,, Antelope

24.

„ Hyaena

16.

,, Hog-deer

25.

,, Llama

17.

„ Keindeer

18.

„ Mexican -deer

I. Subclass

PALAEICHTHYIE8 1

Order CHRONDROPTERYGII (Elasmobranchii)

Tested with Antisera

Suborder
Plagiostomata

A. Selachoidei
b'lim. Carchariidae

869
870
871

490.
199.
197.

Zygaena malUm 1 Hammerhead Shark
Trinidad, W. I. (Tulloch) 16. i. 02

Mustelus canis (vuhjaris ?). Dogfish
New Jersey, U.S.A. (Silvester) viii. 01

Squalus acanthias. Dogfish

New Jersey, U. S. A. (Silvester) 30. viii. 01

1—8, 11- 10, 18—2.5
6—12, 18—21
6—12, 18—21

Fani. Scylllidae

872

578.

Scyllium canicula. Dogfish

England (Plymouth Lab.) 27. ii. 02,/.

1_8, 12-10, 18—20

B. Batoidei

Fmn. Rajldae

873

198.

Raja ocellata. Skate

New Jersey, U.S.A. (Silvester) viii. 01

1—12, 18—21

Fam. Trlgonidae

874

200.

Dasyatus spec. Sting-ray

New Jersey, U.S.A. (Silvester) viii. 01

1—16, 18—21

Fam. Myliobatidae

87S

203.

Rhinoptera Imiiasiis. Sting-ray

New Jersey , U.S.A. (Silvester) 30. viii.Ol

6—12, 18—21

II. Subclass TELE08TEI

Cottoscomberiformes

Order ACANTHOPTERYGII

F(im. Carangidae

876

803.

Naucrates ductor. Pilot-fish

Trinidad, W. I. (Tulloch) 29. v. 02

1_4, 0, 8, 13, 18-25

Fnm. Scomberidae

877
878

619.
017.

Scomber auxin. Spanish Mackerel
Trinidad, W. I. (Tulloch) 13. ii. 02

Scomber curaiigus. Mackerel

Trinidad, W. I. (Tulloch) 28. ii. 02

1—9, 12—20
1— "1, 12—20

Mucilifopmes

Finn. Sphyraenidae

879

020,

Sphyraena barracuda. Barracouta
Trinidad (Tulloch) 7. iii. 02

1—9, 12—20

Gobiesociformes

F<im. Oobiesocldae

880

919.

Lepidogaster spec. ? (Muscle extract)
Guernsey (G.-S.) vi. 02

1, 3, 4, 6-8, 21, 23—25

308

11.

Suhdms TELEOSTEI

^^^*"

Order PHYSOSTOMI

Tested with Antigen

Fam. Cyprinidae

881

427.

Carassitts auratus. Ooldiish
England (N) 15. i. 02

1-10

Fam. Esocidae

883
883

358.
94.

Fsox liiciue. Pike

England (B) 26. xi. 01
Esox lucius

Ireland (Dillon) 6. v. 01

1—16, 18, 19, 21
1-16, 18, 19, 21

Fam. Salmonidae

884
886

210.
92.

Salmo fario. Trout

England (Leighton) 17. .\. 01
Salmo salar. Salmon

Ireland (Dillon) 9. v. 01

1—12, 18—21
1—16, 18—21

Fam. Hyodontidae

886

734.

Hijodon terijuus ? Moonfish
Trinidad (Tullocb) 1. iv. 02

Orde,- PLECTOGNATHIl

1—8, 12, 14—16, IS-

-20

Fam. Sclerodermi

887

204.

Aluton schoepjii. File-fish

New Jersey, U.S.A. (Silvester) viii. 01

IS— 21

Fam. Gymnodoutes

888

202.

Tetrodon turgidus ? Toadfish

New Jersey, U.S.A. (Silvester) viii. 01

UNCLAS8ED FISHES

1—16, 18—21

889
80O
891
892
893

201.

735.
730.
468.
467.

Spheroides maculatm

New Jersey, U.S.A. (Silvester) 20. viii.Ol
Grunt or "Cro-Cro "

Trinidail, W. I. (TuUoch) 27. iii. 02
"Green Chule"

Trinidad, W. I. (Tnlloch) 29. iii. 02
" Paona-fish "

Trinidad, W. I. (TuUoch) 6. i. 02
"Gfouper"

Trinidad, W. I. (TuUoch) 1. i. 02

6—12, 18—20, 22
1—8, 12, 14—16, 18-
3, 6,9, 12, 18-20
1—16, 18—20
1_16, 18-20

-20

None of these bloods gave even a trace of reaction with any of the autisera used.

309

Mammalia

Suborder
MACRURA

Fam.
Astacidae

Fam.
Loricata

Suborder
BRACHYURA

Fam.
Oxyrhyncha

Fam.
Cyclometopa

Class

CRUSTACEA

Order DECAPODA

ANTISEKA FOK.

894
88S

898

887
888

888
800

426. Astacus fluviatilis. Crayfish
England (N) 14. i. 02

407. Homarm vulgaris Bel. Lobster
England (N) 14. xi. 01

591. Palinurui vulgaris. Crawfish or Eock
Lobster

England (Plymouth Lab.) 26. ii. 02

592. Maia squinado. Spider Crab

England (Plymouth Lab.) 27. ii. 02

470. Carcinus maenas. Common Shore Crab
England (Plymouth Lab.) 25. i. 02

471. Porluiius puher. Velvet Fiddler Crab
England (Plymouth Lab.) 25. i. 02

469. Portunus depurator. Common Swimming
Crab
England (Plymouth Lab.) 25. i. 02

S I »

s

•?

ill

"

w

n

897 Foam-test doubtful.

310

Mammalia

Aves

lieptilia

Amphibia Crufitacea

^~"

^^

^^^

^^™

^^"

g

X

1

<

0.

s

H

1

1

1

1
1

1
1

<

1

U
4>

1

.

.

.

/

,

/

.

.

.

+

1)

•

•

•

•

•

•

•

•

•

•

•

+
D

«

tr

•

■

■

•

•

•

•

•

•

•

•

+

■

■

•

\

\

•

•

•

•

•

•

+
D
+
D

311

SECTION VII.

ON THE RESUTTS OF 500 QUANTITATIVE TE.S'J'S WITH
PRECIPITATING ANTISERA UPON THE BLOODS OF
PRIMATES, INSECTIVORA, CARNIVORA, UNGULATA,
CETACEA, MARSUPIALIA, AND AVES.

By GEORGE H. F. NUTTALL, M.A., M.D., Ph.D.,

AND T. S. P. STRANGEWAYS, M.A., M.R.C.S.,

Demonstrator in Pathology, Camhridye.

In view of the crudity of the methods employed ancl the many possi-
bilities of error, it is not a little surprising that the figures obtained in
the following quantitative tests are in such accord. The combined
qualitative and quantitative tests demonstrate certain broad facts,
namely, the persistence of blood affinities amongst groups of animals
which have descended from a common stock. The difficulties in the
way of determining /iHer differences appear to be considerable, owing to
the variations in the reacting power of the rabbits treated with a given
blood, possibly also to the nature of the animal yielding the blood in-
jected (even if it be normal), but especially to much of the blood
injected being derived from diseased animals. The bloods we have
tested, also those we have used for the treatment of rabbits, with few
exceptions, have been derived from animals dying at the Zoological
Society's Gardens, London. These animals have died from various
diseases, and in the majority of cases it has been impossible to
determine the exact cause of death. In some cases the animals died
naturally, and the blood was obtained from the cadaver at variable
periods after death, in other cases they were killed and the blood
collected immediately. In some cases it appears reasonable to suppose
that fluids derived from extravasations into the body cavities have
become mixed with the blood, although precautions were taken against

NuttaU and Strangeways 313

such accidents. We know that the concentration of the blood may vary
in disease and even in health, and it is possible that blood remaining
in the blood vessels after death may undergo changes in concentra-
tion. Apart from this there are chemical changes taking place in the
blood of which we have but slight knowledge at present, which may
materially affect the quantitative results of the precipitin test. The
tests were conducted upon bloods of very different ages, some preserved
with chloroform, others filtered through porcelain and preserved pure.
In most cases serum alone was used, in some instances clots were
present in the serum. All the bloods were kept in the ice-chest until
used. In a few cases putrefaction had already taken place when we
received the bloods. The bloods were kept in tightly stoppered
bottles, or in sealed tubes. In warm weather it is reasonable to
suppose that a certain amount of evaporation may have taken place
from the bloods which were allowed to clot in the covered vessels in
which they were placed for obtaining sei-um. Where glass stoppers
were used a small amount of vaseline was smeared upon the ground-
glass surfaces. In the bloods first collected a slight loss of water by
evaporation may well have taken place through the use of corks which
loosened or were defective. All bloods of later date were kept in sealed
tubes to prevent evaporation.

The quantitative method devised by Nuttall is also open to criticism.
It appears a priori improbable that the physical character of the pre-
cipitum will be the same in different bloods to dilutions of which a
given antiserum is added. We may assume the precipitum should
" pack " to a greater extent in tubes containing the most and to various
degrees in tubes of inconstant calibre. Finally, there is considerable
chance of error in the measurement of such small quantities as have at
times to be dealt with. A considerable number of bloods were tested to
determine the range of experimental error, the result being that we
found the error to be within 10 "/o- Thus Graham-Smith and Sanger
(1903, p. 264) obtained the following results:

" Results of measurements of /our samples of a 1 hi 21 dilution of
human blood and 8 samples of a similar dilution of ox serum.

Human serum O* '"'<'<'<'

•0281 c.c. \ "0215 0.0. -0233 c.c.

•02^1 •• Mean 0293 0.0. '^'' [J^^S .- ^ Mean -0283 o.c.

•0300 „ '02^ .. 023iJ ,

•030'J „ ) -0233 „ 02(52 .

314 Quantitative Precipitin Tests

" The fluctuations above and below the means in the human series
are '0012 c.c. or 4Vo, and in the ox series "0029 c.c. and '0018 c.c.
respectively, or 12 "/o and 7°/o-

" In order to arrive at the most trustworthy figures possible, in most
experiments two observations were made under identical conditions in
the hope that by this means the experimental eiTor might be reduced
to a minimum.

" In measurements of this kind with every precaution probably a
margin of 10°/„ must be allowed for experimental eri'or.

"Throughout these experiments the aim has been to indicate by
measurements the effects of varying conditions on the formation of the
precipitum as compared with controls. It must be stated, however,
that with every precaution the measurements of the same set of
materials on different days are not identical, although the proportions
which the various members of the set bear to each other remain fairly
constant. Hence though improvements in the technique of measurement
may result in more accurate and constant figures, yet it is improbable
that the genei-al results will be materially altered."

Measurements could be made with reasonable accuracy down to
"001 c.c. but of course it would be a mistake to attach importance to
minute reactions of 10 % and under, unless results of repeated tests are
concordant.

The results in spite of all these possible sources of fallacy certainly
speak for themselves, and show beyond question that much may be
accomplished by the use of the precipitin tests in the study of animal
relationships. We believe that we have but entered on the threshold
of a method of investigations which is bound with time to yield useful
data. This investigation must necessarily be regarded as preliminary
in character. A careful selection of material to work upon, especially
material which is readily obtained, together with care directed to
excluding the sources of error above mentioned, are indicated in the
future.

In view of the extent of the work, Dr Graham-Smith, whose paper
follows, was entrusted with similar investigations upon the bloods of
lower animals than those here considered (see p. 336).

The tests here recorded were made upon the bloods of Primates, In-
sectivora, Camivora, Ungulata, Cetacea, Marsupialia, and Aves by means
of the following antisera. The method of obtaining antisera has already
been described elsewhere by Nuttall (p. .51).

Nuttalfs Method 315

2.
3.

Primatei :

Man (2 series)

Ourang

Ivsectivora :

Hedgehog

Camivora :

Cat

Dog

Uttgulata :

Sheep (3 series)

Ox

Antelope (Cobus unctuosus)

OariacuB mezicanus

Cervus porcinus

Rangifer tarandus

Pig (3 series)

Horse ,

Donkey

Zebra (E. Grevvil

' several series

6' >> Manupialia : Onychogale ungnifera

6- „ Aves : , Striithio raolybdophanes

Gallus domesticua

Before proceeding to describe the different series of tests it appears
desirable to give briefly the method of testing quantitatively, quoting
Nuttall's description which appeared in the BHtish Medical Journal for
April 5th, 1902, since his original method has not been modified.

Method for the Measurement of the Degree of Reaction.

" Dilutions of the serums to be tested quantitatively are made with
0"6 per cent, normal salt solution to the degree desired — namely, 1 : 100,
1 : 200, etc. The solutions must be perfectly clear and free from
suspended matter. Accurately-measured quantities — usually 0"5 cm.
of the dilution — are transferred into a series of clean, dry test-tubes,
having a capacity of about 1 cm and a lumen of about 5 mm. The
dilution is allowed to run into the tubes, along a fine, freshly-drawn,
sealed capillary tube in order to avoid the formation of bubbles or of
overflow in filling the tubes. Each tube is supplied with a sealed
civpillary, which is left in place after the introduction of the dilution,
subsequently serving a similar purpose when adding the antisei-um.
An accurately-measured quantity of antiserum is now added to the
diltition, in the proportion of 20 : 1 to 200 : 1 or more of the blood in
dilution. After the antiserum has been added to the series of tubes
the capillaries are removed and the mixture of dilution and antiserum
is effected. This can be done very satisfactorily by applying the tip of
the clean finger to the uiouth of the tube and shaking it up and down.

316 Quantitative Precipitin Tests

By using the fingers of both hands in rotation eight tubes can be shaken
in succession, after which the hands must be cleansed before proceeding
to the next set of tubes. This is only mentioned because it saves time.
With a little practice it will be found easy to bring about complete
mixture without the formation of troublesome air-bubbles, and if the
fluid is shaken down to the bottom of the tube before removing the
finger the loss of fluid entailed by contact with the finger is practically
negligible, the finger-tip remaining apparently dry after some practice.
The mixture is now left standing at a desired temperature, the rate of
precipitum formation, etc. being noted.

After 24 hours have elapsed the precipitum has become deposited
upon the bottom of the tube. Should any adhere to the walls of the
vessel it may be dislodged by gently tapping or rotating the tube upon
its vertical axis. The particles thus displaced soon sink to the bottom
of the tube, provided it does not contain too much serum, which through
its viscidity impedes their gravitation. The clear supernatant fluid is
now pipetted off by means of a conveniently bent bulb capillary pipette,
the tip of which is allowed to slide down along the inner wall of the test-
tube, the precipitum not being disturbed, the clear fluid being aspirated
into the bulb as the tip of the pipette slides down a little beneath the
receding surface of the column of fluid in the tube. The bulk of pre-
cipitum and clear fluid left is so small that it can now be readily di-awn
up into the capillaries.

The capillaries used for measuring the precipitum volumetrically are
made of fairly thick glass, are about 12 cm. long, and have a lumen of
about 1 mm. The ends should be cut off' square by means of a diamond,
this being essential, as it is thus possible to practically remove all the
fluid and precipitum in the bottom of the 'small test-tube. The end of
the capillary is placed against the bottom of the tube, and the deposit
and fluid are well mixed by being drawn backwards and forwards into the
capillary. It is imperative that bubbles should be avoided. The fluid
is now drawn up a little way into the capillary, and the dry end of this
is drawn out and sealed in a very small flame. The sealed end should
be blunt to avoid possibility of breakage ; the other end of the tube,
which can now be cut down to a convenient length, is left open. After
a number of capillaries have been thus prepared they are placed verti-
cally in a rack in a cool place. After 24 hours the height of the column
of precipitum is marked upon the capillary with a glass pencil. After
48 to 72 hours it will be seen that the column of precipitum remains
constant. The amount of precipitum may now be measured.

NnttairH Method

317

The accompanying figure shows a little apparatus which I have
made in the laboratory, and have found very convenient for making these
measurements of the amoimt of precipitum. A fairly large thermometer
tube (A) is used, which is graduated in tenths of a c.c, the calibre
being chosen so that a fine steel scale of 10 cm. (E) subdivided into
0'5 mm. con'esponds to the length of column of fluid measuring O'l cm.
in the tube. The tube is filled with pure water up to the graduation
mark (not shown in the figure), the mouth being wiped dry with filter-
paper. The tube is fixed in a spring clip which is attached to the
wooden rod (D) so that it pivots in a vertical plane, thus permitting the
tube to rest in a horizontal position or at any desired angle. The rod
(D) slides horizontally between the rods C and C, which are nailed to
a solid block of wood beneath. Two small brass plates attached to
r(^d C prevent rod D from being displaced. The steel cm. measure is
attached to a metal support, which is screwed to the block beneath.
By a lucky chance I happened to have an old "peep-sight" from a
sporting rifle which served admirably for cairying the metal scale, as
it can be inclined at any angle and be screwed up or down. By
pivotting the scale on a screw passing through the eyehole of the sight,
the scale can be placed and retained at any angle on its support, thus
making it possible to closely apply the fine scale to the thermometer
tube in any position desired.

Fig. 5.

The height of the volume of precipitum having been marked upon
the capillary tube, the latter is cut square by means of a diamond at the
lower meniscus, a small (,uantity of air having been left in the lower end
of the tube when it was sealed. The contents of the tube are blown out.

318 Quantitative Precipitin Tests

the tube is drained as far as possible by blowing out the moisture it
contains on to filter-paper, or it can be washed out and dried. The
mouth of the capillary is now brought in contact with the end of the
thermometer tube, which has been slightly raised at one end (A), and
fluid is allowed to pass into the capillary up to the pencil mark previously
noted. The amount of fluid which has passed into the capillary is now
read off on the scale. The thermometer tube is moved by means of the
handle (F) so as to prevent expansion of the fluid through the heat of
the hand. Where it is desired to know the actual quantity of pre-
cipitum, it is necessary to note the temperature and calculate accordingly.
This may be disregarded when a simple comparison is being made with
different quantities of precipitum in a given series, the measurements
being made in rapid succession under similar outward conditions. If it
is desired to make simply comparative measurements a diamond scratch
on any suitable tube is sufficient, only the measurements on the steel
scale being recorded."

In the tables which follow the bloods are arranged in groups
according to the orders to which the animals yielding them belong.
In most instances the scientific names of the animals are given, this
is however omitted with the commoner domesticated or laboratory
animals. The numbers preceding the names of the animals refer to the
order in which they were collected by Nuttall and tested qualitatively,
as will be seen by reference to page 217. The date in the column
following is that on which the animal died, or on which its blood was
collected. The succeeding column gives the amount of precipitum
obtained with each blood tested. In all except the first series with
antihuman serum O'l c.c. of antiserum was added to 0'5 c.c. of 1 : 100
blood dilution. The percentages given in the last column are calcu-
lated upon the basis of the amount of precipitum obtained, the amount
given by an antiserum with its homologous blood dilution being taken
as 100 "/„. In most cases this figure has not been exceeded when the
antiserum acted upon a non-homologous blood, but notably in the case
of the anti-ungulate sera cases occur where non-homologous bloods give
higher figures. In some cases comments alongside the percentages
sufficiently account for high figures. Thus, the serum may have been
concentrated, or the precipitum loose in character, and consequently
with difficulty measureable. Brief comments in the text also serve to
explain and summarize the results.

Nuttall mid Straiujeways

319

1. Quantitative Tests with Anti- Primate Sera.

Tests with Antihuman Serum (11. III. '02). Series i.

The antiserum here used was obtained in the usual way by treating
a i-abbit with human blood serum. The 45 bloods tested had been
preserved for various lengths of time in the refiigerator with the
addition of a small amount of chloroform. The only diflference in this
series is that 1 : 40 blood-dilutions were used instead of the usual
1 : 100 dilutions, the usual amount of antiserum 0"1 c.c. being added
to 5 c.c. of the dilution.

Precipitum

Blood of

Date

amount

Percentage

Primites

Man

1. iii. 02

•031

100

298 Chimpanzee

xi. 01

•04

130 (loose precipitum)

299 Gorilla

xi. 01

•021

64

254 Ourang

30. x. 01

•013

42

364 Cynocephalus mormon

10. xii. 01

•013

42

501 „ sphinx

14. ii. 02

•009

29

431 Ateles geoffroyi

28. xii. 01

•009

29

Iiisectivora

433 Centetes eoaudatus

3. i. 02

•0

0

Carnivora

359 Canis aureus

3. xii. 01

•003

10 (loose precipitum)

„ familiaris

4. iii. 02

•001

3

579 Lutra vulgaris

4. iii. 02

•003

10 (concentrated serum)

367 Ursus tibetanus

27. xii. 01

•0025

8

362 Genetta tigrina

9. xii. 01

•001

3

Felis domesticus

3. xi. 01

•001

3

300 „ caracal

15. xi. 01

•0008

3

103 „ tigris

13. viii. 01

•0005

2

UttfltUata

Ox

3. ii. 02

•003

10

Sheep

—

•003

10

480 Cobus unctuosus

8. ii. 02

•002

7

502 Cervus poreinus

15. ii. 02

•002

7

483 Eangifer tarandus

4. ii. 02

•002

7

243 Capra negaceros

23. X. 01

•0005

2

Equus caballus

10. vi. 01

•0005

2

Sua scrofa

26. ii. 02

•0

0

Rodentia

436 Dasyprocta oristata

G. i. 02

•002

7 (concentrated serum dots)

Guinea-pig

—

•0

0

Eabbit

2. xii. 01

•0

0

Slanupiatio

Seven bloods (7 species,

see

■0

0

list on p. 331 under anti-pig

series No. 3)

320 Quantitative Precipitin Testis

Among the Primate bloods that of the Chimpanzee gave too high
a figure, owing to the precipitum being flocculent and not settling well
for some reason which could not be determined. The figure given by
the Ourang is somewhat too low, and the difference between Cyno-
cephalus sphinx and Ateles is not as marked as might have been
expected in view of the qualitative tests and the series following. The
possibilities of error must be taken into account in judging of these
figures, repeated tests shoidd be made to obtain something like a
constant. Other bloods than those of Primates give small reacti(jns or
no reactions at all. The high figures (10 °/o) obtained with two
Carnivore bloods can be explained by the fact that one gave a loose
precipitum, and the other was a somewhat concentrated serum.

Tests with Antihuman Serum (2. xi. '02). Senes ii. and iii.
Antiserum from two rabbits treated with human serum.

Date

Percentages

of precipitum

Blood of

Antiserum I.

Antiserum II.

Man

8. viii. 02

100

(■018)

100 (010)1

906 Ourang

14. X. 02

47

80

364 Cynocephalus mormon

10. xii. 01

30

50

742 Cercopitheous petaurista

10. vi. 02

30

60

697 Ateles vellerosus

11. iv. 02

22

25

784 Cynocephalus poroarius

4. vii. 02

61

' note text

908 Macacus rhesus

14. X. 02

72

90)

' The succeeding amounts of precipitum are purposely omitted from the table.

The results with the two antisera appear divergent. The first
antiserum was nearly twice as poiverful as the second, and appears to
cause less precipitation proportionately in the non-homologous bloods.
The bloods Nos. 784 and 908 were from animals which had died of
intussusception and dysentery respectively, these diseases, as we know,
tending to produce concentration of the serum during life ; and this
accounts fully for the very high percentage of precipitum obtained.
On the other hand, if we take the percentages obtained with the blood
of the Ourang as 100, the figures agree fairly well between the two
series, if we except that of Ateles, thus:

The sera of the diseased animals
considered separately gave with

With Antiserum I.

Antiserum II.

^

Antiserum I.

Antiserum II.

Ourang 100 »/„

100 "/„

No. 784 100 »/„

100%

364 Cynocephalus 63 „

64 „

No. 908 US ,,

128 „

742 Cercopitheous 63 „

64 „

697 Ateles 31 „

48 „

Nut tall ami Strangeways 321

Of other mammalian sera of approximately the same age seveml
reacted as follows, Human serum being taken to give a precipitum of
100 (Reactions with antiserum I.):

Carnivora: Cat and Dog ll"/u, Hyaena striata S"/,,, Seal but a trace.
Unyulata : Zebra H "/„. Imectivora : Hedgehog 3 ''/o , Mole bnt a trace.
Marsupialia: Petrogale O'/o.

Teats tuith Anti-Ourang Serum (7. i. '03).

The rabbit yielding the antiserum was treated with the serum
of Simia satyiits (906).

Blood of

Date

Precipitum
amount

Percentage

90G Simia satyriis

14. X. 02

•008

100 (2 samples gave same result)

Man

fresh

•000

75

907 MacacuB rhesus

2(i. ix. 02

•005

62 (4 samples from 2 monkeys
gave same result)

Owing to the weakness of this antiserum the moiisurements of the
smaller amounts of precipitum given by the serum of one other mammal
cannot be considered accurate. Thus the blood of the Ox (1. i. '02)
gave the percentage 34 °/o. The sera of all other Mammalia gave no
measurable precipitum, the bloods tested being as follows: Insectivora :
Hedgehog and Mole. — Ganiivora: Cat and Dog. — Ungulata : Sheep,
Deer, Llama, Horse, Donkey, Pig. — Cetacea: Balaenoptera (two examples)
and Phocaena communis. — Rodeutia : Rabbit.

It will be seen that the Primate bloods stand in the same relation
to viich other as in the series with anti-human serum, and what is
more that the percentages approximate closely to those obtained with
anti-human .serum No. II.

2. Quantitative Tests with Anti-Insectivore Serum.

Tests with Anti-Hedgehog Sei'um. (18. x. '02).

In view of the limited number of Insectivore bloods and the negative
results obtained by qualitative tests with this antiserum upon other
bloods than those belonging to this order, only a small series was
tested. Although very powerful, this antiserum ])rofluced practically
no effect on any other bloixl :

Blood of

Date

I'recipitum amount

Percentaj(e

783 Erinaceua ouropeua

23. vi. 02

■022

100

782 Talpa europea

23. vi. 02

•0013 •

7

Bos bovis

1. i. 02

■001

4

21

322

Qvmititative Predentin Testa

No trace of reaction was obtained with corresponding dilutions of
the bloods of Man, Cat, Hyaena, Dog, Seal, and Wallaby. Further
investigations will be required with antiseni for Insectivora.

3. Quantitative Tests with Anti-Carnivore Sera.

Tests with Anti-Gat Serum (1. vi. '02).
Antiserum from rabbit treated with the serum of Felis domesticns.

Blood of

Date

rrecipitum
amount

Percentage

739 Felis domesticns

8. V. 02

•00.5

100 another cat's senim gave an
identical result

579 Lutra vulRaris

4. iii. 02

•002

.^;:!-text

367 Ursns tibetanus

27. xii. 01

•001

738 Felis tigris

21. V. 02

■001

20 another tiger (103) gave 1(1 "/„
reaction

Canis familiaris

—

•0008

l(i

The antiserum used in this case was weak. The only bloods which
gave measurable quantities of precipitum were those fi-om Carnivora.
The blood of Felis caracal (300) gave no precipitum, this being probably
' due to an excess of chlorofijrm having been added to it, causing some
precipitation; it however reacted with a distinct cloudiness upon the
addition of antiserum. The bloods of the Jackal, Raccoon and Genetta
gave no reaction. The high figures given by the bloods of Lutra*
and Ursus* are attributable to these bloods being somewhat concen-
trated through keeping, as noted elsewhere. Owing to the very
small amount of precipitum with the bloods of Ureus, Canis, and
Felis tigris, these meiisurenients cannot be considered accurate. Of
11 bloods of Carnivora examined with this weak antiserum, seven
gave a precipitum, one a clouding only, and three a negative result.
Twenty-eight other mammalian bloods were tested at the same time
with uniformly negative results, the bloods examined being from
Primates (7), Ungulates (12), Cetacea (1), Marsupialia (8).

Nnttall and Strangeioays

323

Testa with Anti-Dog Serum (25. iii. '02).
Antiserum from rabbit treated with the senim of Caniti familiarifi.

Blood of Date rrecipitum Percentage

.S97 Canis familiaris 2. xii. 01 -015 100

359 Cania aureus 3. xii. 01 -003.5 24

367 Ursus tibetaniis 27. xii. 01 •0022 15

739 Felis domesticus 8. v. 02 -0021 14

362 Oenetta tigrina 9. xii. 01 -002 18

579 Lutra vulgaris 4. iii. 02 -0018 12

103 Felis tigris 13. viii. 01 -0016 10-6)

738 „ „ • 21. V.02 0012 g ^ average 9-3%

366 Procyon lotor .27. xii. 01 -001 7

No other bloods than those of Camivora were tested. A canine
blood, that of the Jackal (8.59), gave 24 "/o reaction ; the four succeed-
ing non-canine blootls gave fairly unifonn results, a somewhat lower
figure being obtained with the last three. Owing however to the small
amoTint of precipitum obtained in these cases too much reliance cannot
be placed upon the figures.

4. Quantitative Tests with Anti-Ungulate Sera.

Tests luith Anti-Sheep Senmi (18. ii. '02). Series I.

Antiserum from rabbit (F) treated with the serum of the domes-
ticiited sheep {Ovis aries).

In this series bloods cif approximately the same age were taken
for testing. The sera of five sheep gave the following amounts of
precipitum: 0325, 028.5, 0830, 028.5, 0280, the average amount
•030 being obtained with 1 : 100 sheep semm dilution. The figures
obtained with 1 : 200 dilutions were somewhat lower, a.s might be
expected, but also remarkably unifonn: -0215, -025, 0265, •02(i, -026,
•0235, the aveiage here being '024. The results obtained were as follows.
With 1 : 100 blood dilutions taking the average amount obtained with
the blood of the five sheep (-03) as 100 :

Blood of

Date

Precipitum

Percentage

u sheep

—

•030

100

502 Cervus porcinus

15. ii. 02

•0135

45

Horse

—

•0045

15

Cat

3. X. 01

•0035

12

Dog

10. xii. 01

•002

7

488 Wallaby

3. ii. 02

•0015

5

21—2

324 Quantitatwe Precipitin Tests

Here an immediate and marked reaction only took place with sheep
blood, a marked clouding in that of Cervus, a faint clouding in that
of the dog. The figure for the cat appears too high.

Tests with Anti-Sheep Serum (15. II. '02). Seiies II.

Antiserum from another rabbit (B) treated as before.

In this series the bloods of two sheep (4. i. and 10. Ii. '02) gave
a precipitum of -QISS and "022 respectively in 1 : 100 dilution (average
•02), the corresponding figures in 1 : 200 dilution being 0135 and •Oil.
Tests conducted as before, "02 being taken as 100 gave the following
results :

Blood of

Date

Percentage

Ovis aries

—

100

Bos bovis

1. i. 02

75

Cobus unctuosus

8. ii. 02

67

Rangifer tarandus

4. ii. 02

35

dried in scales 19. iii.

01

gave in I'lOO dilution in 1-200 dilution

•017

•015 j

fluid, with chloroform ,

•025

0125 Vcc. preoipituni

,, sealed in tubes ,

t

•026

•0155)

Tests luith Anti-Sheep Serum (11. ill. '02). Series III.
Antiserum from a third rabbit treated as before.

Blood of Date Precipitum amount Percentage

Ovis aries

—

•051

100

243 Capra megaoeros

23. X. 01

•02

39

Bos bovis

3. ii. 02

•019

37

486 Cobus unctuosus

8. ii. 02

•017

33

483 Bangifer tarandus

4. ii. 02

■015

29

502 Cervus porcinus

15. ii. 02

•Oil

22

Sus scrofa

2fi. xii. 01

•002

4

EquUB caballus

10. vi. 01

■0015

3

The antisenim in this case was exceptionally powerful, and it
appears due to this fact that such a contrast is noted in related
Ungulate bloods. The results with the bloods of other Bovidae and
the Gervidae appear fairly uniform, whereas very little reaction was
obtained with the bloods of the pig and horse.

The results with 28 other mammalian bloods were as follows:

Primiites. 8 bloods examined from 7 species: Man (2 samples gave 0 and 3»/(| reaction),
Ourang and Chimpanzee (0''/u), Mandrill and Baboon (4 "/„), Gorilla and Ateles (C and
10 »/o respectively, but both these sera were thick and certainly somewhat concentrated
through keeping).

Nuttall and Stranyeways 325

Carnivora. Bloods from 9 species: Tiger, Caracal, Genetta, Dog (07„), Eaccoon (3"/„),
Cat and Bear (4%), Otter and Jackal (7 and S'/n respectively). It was noted at the
time that the last three bloods were viscid or thick, doubtless concentrated.

Rodentiu. Three bloods examined, from the Agouti, Rabbit, and Guinea-pig (O"/,).

Insectivora. One blood examined, that of the Tenrec (0 "/(,).

Martupialia. Seven bloods examined (O"/,,).

If we exclude the bloods which were regarded as concentrated wc
have 23 non-ungulate bloods of which 3 gave a 4"'/o reaction, and 2 a 3V»
reaction, all the others yielding no precipitum.

Tests with Anti-Ox Serum (15. II. '02).

Antiserum from rabbit treated with the scrum of Bos bovis, the
domesticated Ox.

Blood of Datu Trecipitum amount Percentage

Bos bovis 1. i. 02 -Oil 100

Cobus unctuosus 8. ii. 02 -005 43

Ovis aries -004 36

In the above tests three sheep sera giivc the same figure, these
having been obtained on liift'erent dates (19, III. '01, sealed in tubes,
4, I., and 10, i. '02). Two other sheep sera preserved since 19, III. '01,
the one dried in scales and brought into solution for the experiment,
the other jjreservcd with chloroform, gave a precipitum of 003 and "0035
respectively. The ox and sheep sera were from animals slaughtered in
Cambridge and presumably normal. Whereas granules formed in the
solution of ox serum soon after the addition of antiserum, a cloudiness
persisted for over three hours in the other blood.s. Compare these
percentages with those of the corresponding series with anti-antelope
serum, which follows.

Tests with A nti- Antelope Serum (6. VI. '02).
Antiserum from rabbit treated with the serum of Cobus unctuosus
(486).

I'reci|)itum

Bloort of

Date

amount

Percentage

486 Cobus unctuosus

8. ii. 02

•055

100

243 Capra megaceros

23. X. 01

■031

56

631 Cariacus mexicanu.s

9. iv. 02

•028

SI

Bos bovis

—

•025

45

596 Capra hircus

13. iii. 02

•021

38

483 Eangifer taraudus

4. ii. 02

•020

36

Ovis aries

—

•019

35

695 Ovis bnrrhel

10. iii. 02

•012

22

502 Cervus porcinus

15. ii. 02

•008

15

698 Balaenoptera rostrata

31. iv. 02

•0045

8

Horse (2 samples avei

■age)'

20. iv. 02

■002.J

5

Sus scrofa ( ,, ,,

., )'

29. iv. 02

■0015

3

' Date of but one

sample of each pair

given.

326

Qmmtitative Precipitin Tests

From the above it will be seen that the larger reactions arc all
confined to Bovidae and Cervidae, although from some cause the last
two of these (Nos. 595 and 502) gave low figures. These measurements
were unfortunately not made in duplicate consequently there is no
control. Nevertheless the result agrees fairly with those obtained with
other antisera of this class. It will be seen that the whale (098) gives
a higher figure than either the horee or pig, although the difference
is very slight.

Tests with Anti-Meadcan-Deer iieriuii (1. \\. '02).

Antiserum from rabbit treated with the serum of Canncus
meodcanm (631).

Ulood of

Date

Precipiluni
amount

Percentage

Vnijulata

631 Cariacus mexicanuij

9. iv. 02

■015

100 Died ftoiu pbthisis, pleurisy.

483 Bangifer tarandus

4. ii. 02

•022

146 ? serum with clots, probably
concentrated.

502 Cervus porcinus

1.5. ii. 02

•009

60

486 Cobus unctuosus

8. ii. 02

•018

1-20

Ovis (domestic)

—

■018

120

Bovis (domestic)

—

•016

107

596 Capra hircus

13. iii. 02

■012

77

595 Ovis burrhel

10. iii. 02

■01

67

243 Capra megaceros

23. X. 01

■0095

63

696 Sug Bciofa

29. iv. 02

■006

40

)» »'

—

•0055

37

694 Equus caballus

20. iv. 02

■006

40

)! M

—

■005

33

Cetucea

698 Balaenoptera rostrata

30. iv. 02

■006

40

Of other mammalian bloods, those of Carmvora (11) gave 15"/„
reaction, those of Primates (7) gave 18 "/o reaction, the percentage given
being the average for the gi'oups. On the other hand the bloods of
Marsupialia (8) all gave no trace of reaction.

From the above we see that the high percentages are confined to
Cervidae and Bovidae amongst the Ungulata. The high percentages
obtained with non-homologous bloods in these groups is difficult to
intei"pret, the blood-relationship appears very close in any case. The
bloods of Sus and Equus give results remarkably in accord, and both
are practically the same as with the Rorqual (Balaenoptei-a). A very
moderate reaction is observed with the bloods of Cai-nivora and Primates,
both being fairly equal, whereas the distance separating the Mai-supialia
is clear from the total absence of reaction with their bloods.

Ntittall and Stnmyctcuijs '.>'27

Testa with Aidi- Hoy- Deer Serum ((i. VI. '02).

Antiserum from rabbit treated with the serum of Cervus porcwus
(502).

Bloo.1 of

Date

Pr»d|iituiii
amount

I'ercelitage

502 Cervus porcinus

13. ii. 02

•0063

100

6.H1 Cariacus mexicanus

9. iv. 02

•01

159

Bo.s bovis

—

■009.5

151

Ovis (domestic)

2. ii. 02

•008

127

o'JG Capia hircus

13. iii. 02

•0078

124

.59.5 Ovis burrhel

10. iii. 02

•000

93

486 Cobiis unctuosus

8. ii. 02

•004

64

243 Capra megaceros

23. X. 01

•004

64

098 Balaenoptera rostrata

30. iv. 02

•0038

60

696 Sus scrofa
694 Equus caballus

29. iv. 02
20. iv. 02

I

f average of 2 observations

Tests vtith Anti- Reindeer Serum (6. vi. '02).

The antiserum u.sod in this series \Vci.s obtained by treating a rabbit
with the serum of Rauf/ifer tarandus (483). Only the bloods of Ungu-
lata and that of the whale were tested. Antisenmi weak.

Precipitum

Blood of

Date

amount

483 Bangifer taraudus

4. ii. 02

•004

590 Capra hircus

13. iii. 02

•0015

631 Cariacus mexicanus

9. iv. 02

•0014

502 Cervus porcinus

15. ii. 02

•0013

595 Ovis burrliel

10. iii. 02

•0013

243 Capra megaceros

23. X. 01

•0007

480 Cobus unctuosus

8. ii. 02

•OOWi

Ovis aries

2. ii. 02

•0006

Equus caballus

10. vi. 01

•0004

Sus scrofa

26. ii. 02

•0003

698 Balaenoptera rostrata

30. iv. 02

•0011

Percentage
100
38
35
33
33
18
15
15
10
8
28

The above figures are fairly eomparable with tho.se obtained with
other anti.scra for this group. The figures for Capra, Cobus, anil Ovis
(towai\ls the end of the series) are too low, judging fn^m what has been
obtained in other series. The whale on the other hand again gives
a remarkably high figure, this being in accord with the qualitative tests
on three samples of cetticean blood.

Blood of

Date

rrecipitum
amount

Pig (fresh)
Horse

—

•04.5
•0075

Cervus porcinus

Sheep

Cat

15. ii. 02
3. X. 01

•0065

•OOP)

•0065

Dog
Wallaby

10. xii. 01
3. ii. 02

•006
•0025

328 Quantitative Precipitin Tests

Tests with Anti-Pig Serum (18. ii. '02). Series I.

Antiserum from rabbit troated with the serum of Sus scrofa
(domesticated Pig).

The figures given here were published in part by Nuttall (5, iv. 1902)
in the British Medical Journal. Of five normal sheep sera four gave
a precipitum oi'QQQ, one of •OOSS with this antiserum, the sheep having
been slaughtered 20 hours previously. The i-eactions obtained wore as
follows :

PrRfiinitum

Percentage

100
16
14
13
14
13
5'

' It may be noted that such a reaction was only obtained with a marsupial blood with
two very powerful anti-pig sera (Series I and III) whereas in Series II less precipitum was
obtained although an apparently even more powerful antiserum was used. Series II was
tested 4 months later than Series I. The most powerful anti-sheep serum (Series I) also
gave a 5 "/o reaction with a marsupial blood.

It will be noted that the antiserum in this case was very powerful
(precipitum of '045). It produced an immediate reaction with pig
serum dilution, a deposit being fijrmed, and the supei^natant fluid clear
within 30 minutes. In the other bloods the clouding took place more
slowly as also did deposition. The Wallaby blood dilution only began
to show faint clouding after 30 minutes. This antiserum demonstrates
the " mammalian reaction," and does not show any distinct difference
between the behaviour of the other non-homologous bloods if we except
that of the Wallaby.

Tests with Anti-Pig Serum (11. vi. '02). Series II.

In this series another antiserum to the preceding was tried, being
obtained in the same way from a rabbit. In this case a larger series
of bloods was tested, with the following residts :

Nuttall and Stranyeicaija

329

Bloo.1 of
Vngulata

696 Pig

694 Horse

596 Goat

483 Iteindeer

D&te

29. iv. 02

20. iv. 02

13. iii. 02

4. ii. 02

i'reci|iHuni
amount

•055
■006

•oa5

•0045

Perrentagt:

100*

12

9

8

595 Burrhel's sheep

10. iii. 02

•004

7

Ox

—

•004

7

6.B1 Cariacus mexicanus

9. iv. 02

•0035

«

486 Cobus unctuosus

8. ii. 02

•003

6

502 Cervus porcinus
243 Capra megaceros
Cetacea

15. ii. 02
23. X. 01

•0022
•001

4
2

698 Balaenoptera rostrata
Primates

30. iv. 02

•003

6

364 Cynocephalus moniiou
501 „ sphinx

10. xii. 02
14. ii. 02

■004
•003

7
6

703 „ babuiu

10. V. 02

•002

4

Homo sapiens
254 Simia satyrua

30. X. 01

•003
•001

6
2

697 Ateles vellerosus

11. iv. 02

•002

4

431 ,, geoffroyi

28. xii. 01

•0011

2

C'dCBit'ora

739 Felis domesticus

8. V. 02

•005

9

M M

—

•004

7

Canis familiaiiii

—

•0035

6

738 Felis tigris
103 „

21. V. 02
13. viii. 01

•003
•0022

6
4

579 Lutra vulgaris

4. iii. 02

•0025

5

359 Canis aureus

3. xii. 01

•0021

4

366 I'rocyon lotor

•27. xii. 01

■0021

4

367 Ursus tibetanus

•27. xii. 01

■002

4

300 Felis caracal

15. xi. 01

■0015

3

3<)2 Uenetta tigrina

9. xii. 01

■0014

3

Uodeiilin

436 Dasyprocta ciistata

0. i. 0:i

•0005

1

Kabbit

—

•0

0

Guinea-pig

—

•0

0

Marmpialia

611 Onychogale unguifera
488

22. iii. 02
3. ii. 02

•0012
•0001

2

0-2

503

13. ii. 02

•0005

1

459 ,, frenata

23. i. 02

•0001

0 2

487 I'etrogale xanthopus
484 ,, penicillata
512 Macropus bennetti
482 Thylacinus cynocephalus

10. ii. 02
4. ii. 02

22. ii. 02
1. ii. 02

•0006
•0

•0001
•0001

11
0

0 2
0 2

* The serum of another pig of older date (26. ii. 02) gave a precipitum of -07 = 127 "/,.
Pig serum No. 696 had been used for treating the rabbit yielding the antiserum.

330

Quantitative Precipitin Tests

The results recorded hei-e will be considered at the end of the
succeeding series.

Tests with Anti-Pig Serum (11. in. '02). Senes III.

The following series is recorded for the reason that it shows how an
antiserum may at times give apparently aberrant results. The antiserum
was obtained exactly like the preceding ones by injecting a rabbit intra-
jjeritoneally with pig serum. Why the antiserum should have behaved
in this manner we cannot explain unless it be that it belonged to the
class we have described as " milky," althijugh we should not have used
it if it had shown any marked opacity. The antiserum reacted most
promptly with its homologous blootl and would be classed as very
powerful. These results should serve as a warning to always use
controls in medico-legal work with the precipitin methotl.

Blood of
Vngulala

Date

Precinitum
amount

rercentage

Pib'

26. ii. 02

•044.

100

Horse

10. vi. 01

•012

27

Ox

3. ii. 02

•009

■20

i«(j Cobus uiictuosUB

8. ii. 02

•00!)

20

Sheep

—

•00()

14

48B Kangifer tarandus

4. ii. 02

•006

14

502 Cervus porcinus

15. ii. 02

•0035

8

243 Capra megaceros

23. X. 01

•0015

3

Primates

298 Chimpanzee

xi. 01

•012

27

2'J9 GoriUa

xi. 01

•012

27

Man

1. m. 02

•Oil

25

)(

5. iii. 02

•005

11

2.54 Ourang

30. X. 01

•006

14

364 Cynocephalus iiioriuuii

10. xii. 01

•013

30

501 „ sphiux

14. ii. 02

•01

23

431 Ateles geotlroyi

28. xii. 01

•0125

28

Carnivora

362 Genctta tigiina

9. xii. 01

•Oil

25

579 Lutra vulgaris

4. iii. 02

■009

■20 (conceiitr.

serum)

Feli8 domesticiis

3. xi. 01

■0075

17

367 Ursus tibetaiuis

27. xii. 01

•006

14

359 Cauis ameus

3. xii. 01

■004

9

Canis fainilinris

4. iii. 02

•0035

8

103 Felis tigiis

13. viii. 01

•003

7

300 Felis caracal

15. xi. 01

•003

7

366 Procyon lotor

27. xii. 01

•0

0

liisci'th'oru

433 Ceutetes ecauiiatiis

3. i. 02

•Oil

25

Nuttall and Strangetvays

yai

Uluudof
Rudentia

Date

Precipitum
amount

Perantage

Guinea-pig

•002

6

436 Dasyprocta cristata

6. i. 02

•012

27 (oonoentr. clots)

Kabbit

2. xii. 02

•0

0

Martiipialiii

487 Petrogale xantliopus

10. ii. 02

•003

484 „ peuioillata

4. ii. 02

•001

459 Onycbogale frenata

23. i. 02

•0007

488 ,, nnguifera

3. iL 02

•OOOiJ

503

13. ii. 02

•0005

512 Macropus bennetti

22. ii. 02

•0

0

482 Tliylacinus cynocephalus

1. ii. 02

•0

0

Testa with Anil-Horse Serum (1. vi. '02).

The following tt'sts wore coiiductocl with anti-horse suruni obttvincd
from a rabbit killed 48 hours previously.

HlootI of

Date

Prtjcipitum
amount

Percentage

Equu8 caballas

10. vi. 01

•012

150

094

20. iv. 02

•008

lOO

502 Cervus porcinus

15. ii. 02

•003

38

483 Kangifer tarandus

4. ii. 02

•0015

1»

090 Sua scrofa

29. iv. 02

•001

13

tt ff

20. ii. 02

0

Also negative with the following Ungulate Bloods : Ox, Sheep, Antelope, Markhoor,
Burrhel Sheep, Goat, Mexican Deer.

Vetaceu : Result negative with the blood of Balaenoptera rostrata (30. iv. 02).

I'rittMtet : Kesult negative with the bloods of Man, Baboon, Ouraug, Mandrill and two

species of Ateles. The blood of a Cynocephalus (703) gave a curiously

high figure, viz. 25 "/„.
llodentia : Ilesult negative with the bloods of Agouti, Babbit, and Guinea-pig.
Ctiniiiora : Besult negative with the bloods of Otter, Genetta, Kaccoon, .Jackal, Caracal,

and one Tiger. On the other hand a curious result was obtained with

some bloods which gave tlie following percentages : two Cats (31 and

25 "/o), Dog (31 "/„), Bear (14 %), Tiger (13 «/„).
Mamupialia : Besult negative with 8 bloods recorded on p. 329.

* The blood used for treating the rabbit which yielded the antiseram.

The antiserum used in this case was of moderate power only, and
the precipitum amounts obtained were so small in most cases as to
permit of no small error in stating the percentages. A considerable
distance seimmtes the blood of the horse from that of other animals,
a relatively high figure being only obtained with the blood of CcrA-us
porcinus amongst the Ungulata, others giving less or no reaction. Of

'332 Qmintitative Frecijiitin Tests

the Primate bloods only that of a Cynocephalus gave any reaction, and
then one of 25 "/„, which we cannot explain, having been unable to repeat
the test. Possibly the result was due to eiTor. It will be noted however
that two cats and a dog, amongst the Carnivora, also gave relatively
high figures.

l^ests with Anti-Donkerj Serinn (8. I. 'O.S).
Antisenim from rabbit treated with serum of donkey (929).

Blood of

Date

X*recipituiii amount
2 tests

Vercentages

029 £(]UU8 asinus

3. x\. 02

•010—009

100—90

930 „

5. xi. 02

•008— '008

80—80

Equus oaballus

17. xii. 02

■009— -009

90-90

tf It

10. vi. 01

•007— •OO?

70—70

903 Equus grevyi

13. X. 02

•004— -004

40—40

The immediate reaction observed on the addition of anti-donkey
serum to donkey blood dilutions was very jnarked. A faint clouding
appeared in the dilution of the more recent horse-blood. After 90 minutes
gramdes and deposit had formed in these solutions, whilst the horse
serum of okler date and that of the zebra still showed but a marked
clouding. In other words, there was a well-marked .sequence in the
reaction.

Parallel Tests with Antisem for H&rse, Donkey, and Zebra.

The anti-horse serum used was taken from two different rabbits
(A and B). The anti-donkey serum was a different one from that used
in the preceding series. The anti-zebra serum as in the other cases was
obtained by treating rabbits with the corresponding serum. The order
in which the immediate reactions took place corresponded exactly to
the order of precipitum amounts, that is, the dilutions giving the largest
amounts of precipitum reacted first, those giving the smallest amounts
reacted last. Corresponding appearances were noted after 90 minutes,
the precipitum had accumulated entirely at the bottom of the tubes
giving the highest precipitum amounts, partially at the bottom and on
the walls of the tubes giving lesser reactions, whilst the solutions in the
tubes giving the least reactions sometimes only showed a clouding or
granulation when deposition was complete in the first of the series.
Consequently the rate at which reaction takes place is of value in judging
the result. Only two horse-bloods («, 10. vi. '01 and b, 10. Vii. '02), two

Nuttall and Sfrangewai/s

833

donkey-bloods (a, 3, and b, 5, xi. 02) and one zebra blorKi (13, x. '02,
No. 903, see above) were tested.

Anti-horse serum A and B

iUoo<l of Precip.

Horse ti -OVA

Donkey n 'Oil

Donkey b 012

Zebra '008

I' reel p.
•017
•010
•Oil
•005

A

B !
% ,

100

100

84

59

85

66

58

30

Anti-donkey
Blood of Precip.
Donkey a
Donkey b
Horse a
Zebra

•017
•01-2
•010
•006

%
100
70
59
35

IUoo<l of
Zebra
Donkey b 010
Donkey « -008
Horse -008

%
100
83
66
66

5. Quantitative Tests with Anti-Marsupial Serum.
Tests with Anti-Wallaby Serum (1. vi. '02).

The antiserum was obtained by treating a rabbit with the serum of
Onychogale ungtiifera (No. (ill). The antiserum was weak. Nine mar-
supial bloods were tested, of which 8 reacted as follows :

Blood of

Date

Preclpitum
amount

Percentage

611 Onychogale unguifera

22. iii- 02

•007

KM)

488

3. ii. 02

•fK)25

30

503

13. ii. 02

•002

29

459 „ frenata

23. i. 02

•002

29

512 Macropus bennetti

22. ii. 02

•005

71

487 Petrogale xanthopus

10. ii. 02

•004

57

484 , , penicillata

4. ii. 02

•0025

30

482 Thylacinus cynoceplialus 1. ii. 02 yieldiHl no precipitum, as did also the blood of
35 non-marsupial mammalia, including I'riwiitet (7), Caniivom (U), Vngultla (13),
Cetacea (1), liodeutin (3).

The serum which gave the greatest precipitum with antiserum was
the one (611) used for treating the rabbit. In the absence of reconls
reganling the disea.ses from which the animals suffered it is impossible
t(j oli'er an exi)lanation of the low tigui'es observed in the precipitum in
blo<xls 611, 488, 503. Nevertheless we see the antiserum excluding all
other bloods than those of the Mai-supialia, excepting that of the carni-
vorous Thylacine, upon which it failed to act.

334

Qumifitative Precipitin Tests

6. Quantitative Tests with Anti-Avian Sera.

Tests with Anti-Ostrich Serum (11. iii. '02).

Antiserum from rabbit treated with the serum of Stiiithin molyhdn-
phanes.

lilowl of
740 Struthio molybdophanes. Ostrich
277 Casuarius bicurunculatus. Cassowary
342 Rhea americana. Bhea
.'511 Pelecanus onocrotalus. Pelican
369 Sarcorhamphus gryphus. Condor

Gallus domesticus. Fowl
590 Anas boscas. Duck.
281 Nyctea scandiaca. Owl.

Date

Precipitum
amount

Percentage

•20. xii. 01

■042

100

14. xi. 01

•020

ti2

•25. xi. 01

•017

41

18. ii. 02

•017

41

27. xii. 01

•015

36

25. ii. 02

•007

17

7. iii. 02

•007

17

14. xi. 01

•007

17

From the above it will be seen that a considerable reaction was
obtained with all the avian bloods tested, the percentage falling in the
onler given. It must be noted that the blood of Rhea (342) was putrid
when received, and that it contained a considerable amount of deposit
due possibly to the chloroform, this may account for the low percentage
obtained in this case. Although all avian bloods react appjirently to
anti-avian sera (see qualitative tests) it is evident that there are quanti-
tative differences which will require further study.

Tests ivith Anti-Fowl Sei-ion (H. vr. '02).
Antiserum from rabbit treated with the serum of Gallus domesticus.

I'recipituni

Blood of

nate

amount

Percentage

Gallus domesticus

—

•035

100

590 Anas boscas

7. iii. 02

•012

34 (average of 2 tests

277 Casuarius

14. xi. 01

•0095

27

Columba

vi. 02

•008

•23

281 Nyctea

14. xi. 01

•0072

21

369 Sarcorhamphus

•27. xii. 01

•0065

19

342 Rhea

25. xi. 01

•0065

19

511 Pelecanus

18. ii. 02

•004

11

Note the comments in connection with the [)receding tests.

Nuttall and Strangeways 335

Conclusions relating to Quantitative Tests.

The 500 quantitative tests recorded above give results in substantial
accord with those obtained by the qualitative method, they however
exclude the personal element which necessarily must enter into records
of tests done qualitatively. The summary of conclusions following the
qualitative tests (p. 214) therefore applies equally to the quantitative
tests. The reader will be able by glancing down the column of per-
centages to rapidly gather an impression of the results obtained.

SECTION viir.

BLOOD-RELATIONSHIP AMONGST THE LOWER VERTE-
BRATA AND ARTHROPODA, ETC., AS INDICATED
BY 2,500 TESTS WITH PRECIPITATING ANTISERA.

By G. S. GRAHAM-SMITH, M.A., M.B., D.P.H. (Camb.).

In the foregoing pages Nuttall has described in detail the various
methods of collecting and preserving sera, and of preparing antisera,
together with the precautions which have to be adopted in testing,
both by the qualitative and quantitative methods.

These experiments were conducted by the methods he has described,
and it is therefore unnecessary again to describe them.

On reference to Nuttall's tables it will be seen that his work was
principally carried out on Mammalian and Avian sera, though Reptilian,
Amphibian, Fish, and Crustacean sera as well as a few Egg-albumins
were tested. In order to render these experiments more complete it
was thought desirable to further study the relationships indicated by
this test amongst the latter groups of animals, and Egg-albumins. For
this pini)ose Dr Nuttall kindly placed at my disposal all his specimens
of blood of the Reptilia, Amphibia, Pi.sces, and Crustacea, and these
together with a few others have been tested with antisera derived from
animals of their own classes.

Methods.

Production of Antisera.

In the.se experiments some of the antisera were prepared by the
intraperitoneal method, and some by the intravenous, but the injection
of much smaller quantities than those usually employed was found to
suffice (see table, p. .S.S7). Continental workers have used quantities

(

r'rrt

r/awi

->S^>

»7/*

337

bled j

last Power of

ons, Antiserum

1

is

-

2 Si

:

%

:

=

1
%

I

=

=

09
1

1^

1

^ il

1 ia

:

bliss's

»?>

«

-^

t*

^

o

to

35

'^

-*

't*

o

U3

5S

" 2

»

^-I

I.I

y, 's

00

oe

00

ts

55

t-

X

oo

00

S5

05

o

a>

■s

-t< "O

C!

ration
tr«at-
nt in
ays.

rH

o

c*

-n

i-H

o

!0

o

■*

IM

o

o

1-4

o

* s

5^

.-(

1— (

CC

f*

IM

IM

■*

C^

w

I^a'

.

Total
quantity
Injected

in C.C.

't*

o

«

wr

t>

CO

I>

00

tC

O

»«

'<5

o

00

» s

«5

I>

o

■'J'

■*

CD

rH

ss

«>

oi

<Z>

<o

(N

SS

n

00

>o

to

00

I-t

CC

115

o

1-^

§

US

'I'

tity injected on eac
on. Intervals in da
tween injections'.

cf "^

o

o

I-H lO

-CC

'-I -

•o .

.115
■rH ^

«■:«

1-H ^

«5 .

«2

00-""
oo"

o

uj « 2 -

j;^ . ■«■

^1*
US

??

LO

00

rH

M

-<"'o.r

■*

s

s

o

o

S

g

g

s

g

O

?

s

g s

^

.g « s 0 c

1— t

00 ®

- S s~ s

CI

fN

C^l

n

c-l

i-H

c^

M

M

1

1

1(5

C-l

1

.-1
(M

CO

1

1

1

o

i

i^

1
1

fH

0 >

I-H W

J

w

^

,SF

.»

^J

b<

4S

di.

■w

cu

*»

B.

-*»

il
^1

,3

=

1

^

1

-

"

•^

^

2

.s

'3
O

g
(S

'

1

5

1

1 "

"

1

c
o

.

-

.

i

1
a

1

CD
P-t

;

§

2

'3
1
%

;

:

:

s

r

i 1

§

a

%
t

"^

HI

C

Oi

a "^

— _^

.

^_^

^-.^

a

i4

s

.a

a

QD

•s-s

e

5

i

s

3
to

J"

c

g

s

a

a a

i
a

^H

>i^

1

3

33 a

1?

s

3"S

DQ

3

2|

1

g

M

H

a

« 1

:

s

s

to

r

Si

o "

E-i

en

H

i

.1-

CO -p.

I" e

c

a
■Ji

1

a

i

•<

en p

^ i

y 5

:

— s s>

338 Methods of Testing

extending to hundreds of c.c, and have frequently found their animals
stood the operation badly, whereas most of the animals treated in these
experiments continued to gain weight, and appeared to be healthy.

In the preceding table the methods of production, animals treated,
their weight before and after treatment, the number of injections, and
the quantities used in each case, together with the power of the resulting
antisera are given.

Methods of Testing.

In the Qualitative Experiments the method which has already been
fully described (p. 62 et seq.) was followed.

Throughout the subsequent tables the following signs have been
used to denote the degree of clouding, and quantity of precipitum after
24 hours.

-f = very marked cloud— full reaction. D =very large deposit after 24 hours.

+ = marked cloud. D = large „ ,, ,,

X =medium cloud. d = small ,, „ „

• = small cloud. tr = trace of a deposit.

*? =very slight, or doubtful, clouding. • =iio reaction.

The very marked cloud, followed by a very large deposit, was seldom
obtained except on the addition of an antisenun to its homologous blood
dilution. In a few instances closely allied sera also produced this con-
dition. Marked cloudings followed by large de])osits as indicated by the
second signs occurred on the addition of antisera to nearly related bloods.
In some cases the antisera only gave this amount of reaction with their
homologous sera. Medium clouds occurred under the same conditions.
Powerful antisera, however, occasionally produced reactions of this degi'ee
with distantly related sera. Small cloudings indicated in the tables by
the sign • were usually obtained when antisera reacted with distantly
related bloods. Such cloudings were occasionally followed by small
deposits (d), or traces of deposits (tr), but not infrequently after the
lapse of 24 hours no precipitum was found.

The sign * ? indicates that there was a haze at the line of junction
of the serum and antiserum. In most cases this result was probably due
to some cause other than a reaction between the serum and antiserum,
but where an antiserum produces several such reactions on the sera of
a group of animals a remote relationship is probably indicated. For
this reason in the following tables summarizing the results of the tests
with the various antisera, these very small reactions have been inserted.

Grraham-Smith 339

though they are probably only of value where the antiserum reacts in this
way with several members of a group. In every table the number of
reactions of each kind hiis been given, and the results of tests with each
antiserum either including, or omitting, these reactions of doubtful value
can be seen.

Several instances occurred in which though no clouding appeared in
two hours yet a precipitum was present aftei- 24 hours. The majority
of these are probably true reactions, and are in.serted in the tables at the
end, but are not included in the summarised results of the individual
antisera.

In Quantitative experiments the method devised by Nuttall (5. vi. '02)
and described elsewhere (p. 315) was employed.

In all cases dilutions of the strength of 1 : 21 in "6 "/o salt solution
of Egg-albumins or sera to be tested were used.

In similar observations on Human and other sera it had been found
that with every precaution probably a margin of 107„ must be allowed
for experimental error (p. 313).

In the following table the antisera are placed in order according to
their strength, as shown by the amount of precipittim produced by each
with its homologous serum when tested by the quantitative method.

Anti-Limulus (I) -0980 c.o. Anti-Fowl's-egg (I) ..-0159 0.0.

,, (II) -0500 ,, Auti-Snake serum ■0159 „

Anti-Tortoise (T. ibera) 0894 „ Anti-Crab sernm -0140 „

Anti-Diick-egg -0384 „ Anti-Fowl's egg (II) -0122 „

Anti-Turtle -0800 „ Anti-Crane's egg -0078 „

Anti-Emu-egg -0281 ,, Anti-Uromastix trace

Anti-Tortoise (T. vicina) ...0234 ,, Anti-Python ,,

The anti-ammocoetes, and anti-snake's-egg sera produced well-marked
precipita, but the strengths of the dilutions tested were unknown. These,
however, were certainly not as strong as 1 : 21.

Anti-lizard {Varanus griseus) was a powerful antisenim, but no
quantitative tests were made, as the quantity of it was very limited.

Materials used in these eooperimenta.

All the egg-albumins were kejjt in a fluid condition in 1 : 21 dilution
with -6 % salt solution. Fluid sera, reptilian, amphibian, and crustacean,
were similarly preserved. In many cases a few drops of chloroform were
added to prevent putrefaction. Before testing, small quantities of the
dilutions were allowed to stand in watch-glasses in order to allow the

22—2

340 Antisera for Aves

chloroform to evaporate off. In the case of the egg-albiimins accurate
dilutions of 1 in 21 are difficult to make, and consequently the results
of quantitative experiments are not so trustworthy as those done on sera.
The sera were of various ages, but the majority were derived from animals
which were killed for the purpose. Some, however, came from animals
which had died from disease. The great majority of the specimens
tested were bloods dried on filter-paper, and preserved in this way for
various periods of time. Some of these were apparently insoluble in
salt solution and failed to react with any antisera, but the rest gave
tinted solutions, which foamed well on shaking. Nearly all these bloods
had been tested by Dr Nuttall with various mammalian and avian anti-
sera. The results of these experiments have been tabulated by him in the
preceding pages (pp. 300 et seq.), and in the following tables the number
attached to each specimen by him is quoted, so that the results of tests
with mammalian, avian, egg-albumin, reptilian, amphibian, and crustacean
antisera can be followed.

In a few cases, in which it was found impossible to obtain blood
serum, extracts of muscle in salt solution were made. After standing the
supernatant fluid was usually clear and slightly tinted, and produced
foam on shaking. The few specimens obtained in this way are marked
' extracts ' in the tables.

It may here be stated that the specimens were not tested in their
zoological order, but in the order in which they were obtained. Con-
sequently it was only after the observations had been reduced to
zoological order that any conclusions could be an-ived at as to the
general results of the experiments.

The results of the tests with each antisenim upon all the specimens
are given in the tables at the end of this section. The following short
tables summarize the actions of the antisera upon zoological groups of
egg-albumins and bloods.

I. Antisera for Aves.

A. Qualitative Tests with Antisera to Birds' Egg-Albumins.

(1) Tests with Anti-Emu Egg Serum.

This antiserum was made by the repeated intraperitoneal injection
into a Rabbit of Emu egg-albumin {Dromaeus novae-hollandiae).

Grahain-Smitli

341

Material tented •
22 Egg-Albumins

(a) 15 Birds' egg-albumins 1 ((i »/„)

(6) 1 Reptile „ 1

(r) 1 Amphibian ,, 1

(<l) 5 Fish „ 5
69 Blood Sera

flO Chelonia 9

3 Crocodilia 3

8 Lacertilia 8

lU Ophidja 14

10 Amphibia 10

14 Pisces 14

10 Arthropoda 10

of pcMitive
reactions

1 («»/„)

33 BeptiliaJ

4(26%) 9{m<>i„)

93
0
0
0

10
0
0
0
0
0
0

This antisuriini \v;i.s made by l)r Xuttall, who has rccordcfl the results
of tests therewith upon Mainmalian and Avian sera. When the above
tests were niiide the antiserum had lost some of its power; but it,
nevertheless, gave well-marked reactions with most of the egg-albumins.
One egg-albumin, that of the chaffinch, for some unknown re<ison failed
to react with any of the anti-egg sera. A moderate reaction was
obtained also with the blood serum of one Chelonian (tortoise).

(2) Tests with Anti-Duck's-Egg Seruvu

This antiserum was made by repeated intraperitoneal injections of
the egg-albumin of the domestic Duck into a Rabbit.

Material tested

,

.?

»

X +

Percentage

of poRitive

+ reaction*

27 Egg-.Ubumim

(a) 15 Birds' egg-albumins

1

B'V,,)

3(20"/„)

• 3(20%)

8(53%) 93

(b) 2 Kcptile

1

I

50

(i) 3 Amphibian ,,

3

.

0

(d) 7 Fish

7

.

0

129 niood Sera

J 15 Chelonia

12

3(13"/„)

1(7";„) ■

20

60 Keptilia J ^ Crocodilia
1 13 Lacertilia
^28 Opbidia

4

13

0
0

28

0

18 Amphibia

18

0

39 Pisces

39

0

12 Arthropoda

12

0

This antiserum was decidedly more powerful than the i.ist. The
maximum reactions were confined to the birds' egg-albumins, but small

1(7"/,,)
1

342 Antisera for Birds' Eggs

I'eactions were obtained with the egg-albumin of a tortoise, and with
a Chelonian blood (tortoise). Three very slight cloudings were also pro-
duced with the sera of three tortoises. No sign of a reaction was seen
with any other specimen of egg-albumin or sei'um tested.

(3) Tests with Anti-Fuwl's-Egg Serum (I).

This antiserum was prepared by intraperitoneal injections of Fowl's
egg-albumin into a Rabbit.

Percentage
„ of ixwitive

Material tested ' « .' « x + + results

24 Eijij- Albumins

(«) 13 Birds' egg-albumius 1 (7",g • 3C23";„) 3C2a";„) 3(23"/,,) 3(23'7„) <J3

(b) 2Keptile ,,1.1

(c) 3 Amphibian ,, 3
\il) 6 Fish „ 6

128 Blood Sera

TlSChelonia 9 3(207,,) 2(13"/,,

.•n T. i-v 4 Crocodilia 2 1 . . ^ . ^ ,

I 13 Lacertilia 12 i . - - - 7 ( ■"-' '»

(28 Ophidia 26 1 1

18 Amphibia 17 1

38 Pisces 3 s

12 Arthropoda 12

This was one of the most powerful of the antisera to egg-albumins.
Its maximum effects were produced on the Avian egg-albumins, but
well-marked reactions occurred with the egg-albumin of the grass snake,
and with the blood sera of three Chelonians (Tortoise (2), Turtle), one
crocodile, and one snake. Very feeble reactions occurred with three
Chelonians, one lizard, one snake, and one Amjjhibian (Toad).

(4) Tests with Anti-Fovd's-Egg Serum (II).

This antiserum was prepared by intmperitoneal injections of Fowl's
egg-albumin into a Guinea-pig.

I'crcentage
,, . . , ^ , , « , of positive

Material tested • . ? « x + + reactions

8 Birds' egg-albumius 3 (377") • 3 (37%,) • 2 (257„) • 62

This was a weak serum, and only a few birds' egg-albumins were
tested with it. A few quantitative tests were also made.

Graham-Smith

343

(5) Tests with Anti-Crane' s-Egg Serum.

This antisermii was prepared by intraperitoneal injections of the
egg-albumin of the Cape Crowned Crane (Balearica reguhmm) into
a Rabbit.

Material testtd •
27 Eijg-Alhumim

(a) 15 Birds' egg-albumins 4

(6) 2 Reptile „ 2

(c) 3 Amphibian ,, 3

(d) 7 Fish 7
129 Blood Sera

(15 Chclonia 13

4 Crocodilia 4

13 Lilcertilia 13

28 Ophidia 28

18 Amphibia 18

39 Pisces 39

12 Aitluopoda 12

2(13"/,,)

+ +

(33«/„) 4(26%,)

Petcenlage

of poalUre

reacUana

2 (137,,)

73
0
0
0

18
0
0
0
0
0
0

This Wivs a rather weak antiserum and failed entirely to react with
four birds' egg-albumins, and gave only two very feeble reactions with
two tortoise bloods.

B. Qtuditative tests with Antiserum for Fowl's Blood.

This antiserum was preijared by Dr Nuttall, and with it only the
following tests on biixls' egg-albumins were made to render the experi-
ments already cjvn'ied out more complete.

Material tested * • ? • x +

13 Birds' egg-albumius 3 (23»/„) 1 (8»/„) 5 (38»/o) 2 (140/.) 2 (14»/„)

Perc«ntmge
of iK>sitive
+ reactions

77

Summari/ of results of Qiuilitative Tests witit, Antisera to Avian

Egg- A Ibumiits.

With the five antisera for Avian egg-albumins 66 tests were made
with dilutions of birds' egg-albumins and 85 */o of distinct positive
reactions were obtained, of which 62 7o were very well marked. One
egg-albumin dilution, namely that of the chaffinch, failed to react with
any of these antisera, but reacted with antiserum to fowl's blood.

344 Antisera for Birds' Eggs

Seven tests were carried out with dilutions of Reptile egg-albumins
of which 28 "/(, were j)ositive.

No positive reactions were obtained with 10, and 25 tests, respect-
ively with Amphibian, and Fish egg-albumins.

The 215 tests with Reptilian sera yielded noteworthy results. All
the antisera tested reacted to some extent with Chelonian blo(xls, but
only anti-fowl's egg serum (I) gave any positive results with the other
Reptilia.

;epUlian Sera

Tests

Positive reactions

• ;oo<l reactions

Cbelonia

55

23-5 "/„

9"/,,

Crocodilia

15

13 „

7„

Lacertilia

47

2 „

o„

Ophidia

98

2 ,,

1„

Out of 64 experiments with Amphibian sera only (jne showed any
trace of reaction, namely, a very feeble clouding when anti-fowl's-egg
serum was added to the diluted blood of the Toad.

130 tests with Fish, and 4G with Crustacean sera gave no indications
of positive reactions.

These experiments, therefore, indicate a relatitmship between the
Aves and Chelonia, and a less marked one between the Aves and
Crocodilia. As indicated by this test the relationship between the
Aves and Dicertilia and Ophidia is very remote (see pp. 200 — 207,
216, 300—305).

C. Quantitative Measurenwnts with Antisera to
Birds' Egg-albmuin.

These experiments were conducted by the mothod which has already
been described (p. 315), and the results of the measurements of precipita
in each case are stated in c.c. in the second colunni. The jiercentages
given in the last colunni are calculated on the basis of the amount
of precipituin obtained, the amount given by an antiserum with its
homologous blood being taken as 100 7o- In most cases this figure
has not been exceeded when the antisenim acted on a non-homologous
blood.

Qraham-SinUh

34.=

(1) Teats with Anti- Emu-Egg Serum.

Material tested

Amount of
precipituui

Percentage

Emu's egg-albumin -0281

100

Duck"s

■0234

80

Blackbird's

■0131

46

Crane's ,

■0122

43

Fowl's

•0122

43

Greenfinch's ,

•0122

43

Silver Pheasant's ,

•0112

40

Pheasant's ,

■0073

26

Moorhen's ,

•0054

20

Thrush's

•0054

20

Hedge- Sparrow's ,
Chaffinch's

•0018
, trace

6
1

Turtle serum

•0018

6

Tln' loUcnving sera were also tested but gave no trace of prccipituin :
Tortoise, Alligator, Frog, and Dogfish.

The egg-albtimins shown by qualitative tests to produce full reac-
tions occupy the first positions on this table. Those which produced
marked reactiijns with the e.xception of the Greenfinch cgg-albuniin
occupy the lower places.

(2) Tests with Anti-Duck' s-Egg Serum.

Material teste<l

Amount of
precipitum

Percentage

Duck's egg-albumin ■0384

100

I'heasant's ,

■03^28

85

Fowl's

■0234

61

Silver Pheasant's ,

•0140

36

Blackbird's ,

•0065

15

Crane's ,

•0051

14

Moorhen's ,

•0046

12

Thrush's ,

•0046

12

Emu's ,

•0018

5

Hedge- Sparrow's ,

, trace

?

Chaffinch's

,

0

Tortoise serum

trace

?

Turtle ,,

n

?

Alligator ,,

•

0

Frog, Amphiunia, and Dogfish sera, as well as Tortoise and Dogfish
egg-albumins, were also tested, with negative results.

346 Antisera for Birds' Eggs

In this as in the last table the results of quantitative and qualitative
experiments closely correspond. Egg-albumins from the Duck's to the
Moorhen's in the above list were shown by qualitative tests to produce
full reactions. By the latter method a medium clouding occurred with
Thrush egg-albumin, and small cloudings with the albumins of the
Emu and Hedge-sparrow.

(3) Tests with Anti-Fowl' s-Egg Serum.

Material tested

Amount of
j>reeipitxim

I'eruentajie

Fowl's

egg-i

ilbumin (old)

•0159

100

„

(fresh)

•0140

88

Silver Pheasant's

•0075

47

Pheasant's

•0075

47

Crane's

•0046

29

Blackbiid's

•0046

29

Dack'g

•0087

28

Moorhen's

•0028

18

Thrush, Emu, Greenfinch, and Hedge-sparrow egg-albumins were
tested and gave traces of precipita, as also did Tortoise and Turtle sera.
The egg-albumins of the Tortoise, Frog, Skate, and two species of Dog-
fish did not react. Alligator, Frog, Amphiuma and Dogfish sera also
yielded no results.

As before the quantitative and qualitative methods closely agree.
By the former method full reactions were recorded with Fowl and Silver
Pheasant egg-albumins, marked reactions with Pheasant, Crane and
Blackbird, medium with Duck, Thrush and Greenfinch, and small with
Moorhen, Emu and Hedge-sparrow.

(4) Tests tuith Auti-Cnine's-Egy Seriua.

Material tested

Amount of
precipitum

Tercentage

Crane's egg-albumin

•0073

100

Greenfinch's „

•0054

75

Silver Pheasant's ,,

•0018

'24

Moorhen's ,,

•0018

•24

Emu's ,,

•0018

'24

Hedge-spiirrow's ,,

•OOO'J

1'2

Turtle scrum

•0018

24

Tortoise, Alligator, Frog, Dogfish and Salmon sera were also tested
but failed to give any traces of precipita.

By the qualitative method full reactions were recorded with Crane

Graham-Smith

347

and Silver Pheasant egg-albumins, marked reactions with Greenfinch,
Moorhen, and Hedge-spari'ow, and small with Enni.

The quantitative method fails to bring out the delicate reactions
which can be determined by the qualitative method, but in the case of
the major reactions differences can be recognised which are not appirent
by the latter method. By its means closely allied sera and egg-albumins
can be differentiated, which cannot be done with certainty by the quali-
tative method unless low dilutions are made use of. The results of the
few quantitative experiments which have been made are exactly in
accord with those obtained by the qualitative method.

II. Antisera for Reptilia.

Qualitative Tests with Anti-Reptilian Sera.

A. Anti-Gltelonian Sera.

(1) Tests with Anti-ToHoise Serain.

This antiserum was prepared by intraperitoneal injections of the
serum of the Tortoise {Testudo ibem) into a Rabbit. The scrum used
was obtained from healthy tortoises.

Material tested - # . *

26 Kgy-AWiimiit"

(u) 15 Birda' eggalbumius 8 2 (13"/„) 5(33";„)

(6) 2Heptile „ -1

(c) 3 Amphibian ,, 2 1

(d) 6 Fish ,, 0 .
129 Blood Sera

ri5 Chelonia

4 Crocodilia 3

GO Eeptiha .

1

o(33»/,)
1

I 13 Lacertilia 13

I 28 Ophidia 26

18 Amphibia 17

3'J riscfs 39

12 Aithropoda 12

Percentage
of poeitive
reactions

46

lUO

33

0

(13»/„) 6(40''/„) 87
25
0
6
5
0
0

This was the most powerful anti-chelonian serum made. Large
reactions occurred with 87 "/o of the Chelonian bloods tested, but slight
reactions only with a few cither specimens of Reptilian blood, id'^/f, of
the Bii-ds' egg-albumins tested gave some indications of clouding.

Material tested

•

♦ ?

*

24 Eijij-Albumins

(a) 13 Birds' egg-albumins

10

.

2(14"/„)

(6) 2 Reptile

•2

.

•

(c) 3 Amphibian ,,

3

•

•

(d) 6 Fish

6

.

.

128 Blood Sera

rib Chelonia

5

.

5(33"/o)

_ ^ ... 14 Crocodilia

2

1

1

59 Keptilia <,„ ^

' ] 12 Lacertiha

11

1

.28 Ophidia

24

2

2

18 Amphibia

18

.

•

39 Pisces

39

.

.

12 Arthropoda

12

•

.

348 Antisera for Reptilia

(2) Tests with Anti-Tortoise Senna.

This antiserum was prepared by intravenous injections of the serum
of a giant Tortoise (Testiido vidua) into a Rabbit. The serum used was
collected after the death of the tortoise from natural causes.

Percentage
of positive
^ + "T* reactions

1 (7 "/„) • . 23

0
0
0

2(13"/,,) . 3(20»/„) 66

50
8
12
0
0
0

Only a small quantity of the tortoise scrum was available for ]ire-
paring this antiserum, which was considerably, weaker than the preceding
one. The results of the tests are, however, very much alike. In this
case large reaction.s with Chelonian sera were obtained in fiG^/o ^^
against 87 7o i" ^^^ former, and in 23 70 ^^ against 46 7o of Birds' egg-
albumins. Again, the few reactions obtained amongst the rest of the
Reptilia were feeble, and the results of tests with Reptile egg-albumins
were negative.

(3) Tests with Anti-Turtle Serum.

This antiserum was prepared by the intraperitoneal injection of the
blood of a Turtle (Ckelone 7nidas) into a Rabbit. The turtle was healthy.

Although this was a very powerful antiserum, giving well-marked
reactions with 66 7o of the Chelonia, yet only 20 7o of the Birds' egg-
albumins gave any indication of clouding, and in each case the reaction
was very feeble. Fewer positive results were obtained with the other
specimens of Reptilian bloods than with either of the anti-tortoise sera.

Omitting very feeble cloudings the anti-chelonian .sera give well-
marked reactions with the Chelonia and moderate reactions with the
Crocodilia. With Birds' egg-albumins moderate cloudings are obtained,

Graham-Smith

349

indicating a relationship with the Chelonia. The few positive results
obtained with Liicertilia and Ophidia were feebly marked.

Material tested

.?

tt

26 Eiig- Albumins

(a) 15 Birds' egg-albuming

12 2

1

(6) 2 Reptile

1

■

(«) 3 Amphibian , ,

3

•

(d) 6 Fish

6

•

129 Blood Sera

llo Chelonia

<.« Tj x-1- ^ Crocodilia
60 Rcptiha -, , .. - ^.,.
^ 1 13 LacertUia

(28 Ophidia

5

1

3

,

13

.

28

•

18 Amphibia

18

•

39 Pisces

39

•

12 Arthropoda

12

•

Fgnoitafe
of pariUra

ao

50
0
0

1(7"W l(7»/o) 3(20'V„) 5(33«/.,) 66

25
0
0
0
0
0

B. Anti-Crocodilian Sei'a.
Tests tvith Anti-Alligator Seiiim.

This antiserum was made by Dr Nuttall with the serum of the

Chinese Alligator {Alligator sinensis).

Material tested
8 Egfi-Alliuminn

{a) 4 Birds' egg-albumins
(b) 1 Reptile „
{{■) 3 Amphibian „
59 Ulood Hera

,11 Chelonia
4 Crocodilia

2 (18»/„)

26Reptilia ■< 5 Lacertiha 5

I 6 Ophidia
12 Amphibia
12 Pisces
9 Arthropoda

6
12

la
9

Percentage
of positive
reactions

26

100

0

(90/,,) 2(18%) 54
2 100

0
0
0
0
0

This was a moderately powerful antiserum. It showed maximum
reactions with the Crocodilia and Chelonia. One out of the four Birds'
egg-albmnins tested gave a slight reaction. The tests on the Lacertilia,
Ophidia, Amphibia, Pisces, and Arthropoda were carried out on the best
material obtainable, but all with negative results.

350

Anfisei'a for Reptilia

C. Anti-Lucertilian Sent.

(1) Tests ivith Anti-Lizard Serum.

Tliis antiserum was obtained by the intravenous injection into a
Rabbit of the filtered serum of a Lizard (Uromastix spinipe.s). This
serum had undergone slight putrefaction, and concentration, and had to
be filtered througli porcelain before treatment was commenced.

Percentage

Material tested

•

.?

K

X

+

of ]to6itive
+ reactions

25 Egg-Albumiiis

(a) 14 Birds' egg-albumina

14

.

.

0

(6) 2 Reptile

.

.

100

(c) 3 Amphibian „

3

.

0

((f) 6 Fish

6

.

0

128 Blood Sera

/15 Chelonia

nn T. ..1. 4 Crocodilia
GO Beptiha -,,„ ^

•^ 13 Lacertiha

28 Ophidia

14

1

7

4
11

0

15

28

0

17 Amphibia

17

0

39 Pisces

39

0

12 Arthropoda

12

0

This antiserum was very feeble, giving good reactions only with two
specimens of the blood of Uromastix, moderate with the serum of
Testudo vicina, and very feeble with the two reptile egg-albumins. All
the other tests were negative.

(2) Tests with Anti-Lizard Serum.

This anti.serum was obtained by the intraperitoneal injection into a
Guinea-pig of a solution of the dried serum of a Lizard ( Varanus griseus).

Material tested

•

23 Egg-Alhumins

(a) 15 Birds' egg-albumins

15

(h) 1 Eeptile

(c) 1 Amphibian ,,

(d) 6 Fish
84 Blood Sem

1
6

15 Chelonia

8

„„ r> ,-,■ 4 Crocodilia
60 Reptilia/,, ^

1 13 Lacertilia

128 Ophidia
18 Amphibia
6 Pisces

l(i

18

6

.?

3(20"/„) 2(13"/„) 1(7"/,,)

•2 1

1 3C23"/„) G(4(;"/„)

4(147,,) 6(21"/,,) 2(7"/o)

1(7"/,,)

(23";„)

Percentage

of positive

reactions

0

100

0

0

47

100

1(10

42

0

0

Graham-Snnth 351

This antiserum was a powerful one, but was very slightly opalescent.
As shown in the above table, however, it wiis entirely confined to the
Reptilia in its reactions. The nia.\inium reactions occurred amongst
the Lacertilia, though fairly well-marked clouding and precipita were
obtained with the Ophidia as well as the Chelonia and Crocodilia. It
is unfortunate that both the Lacertilian antisera were somewhat un-
satisfactory, the first being very weak, and the second slightly opalescent,
rendering the determination of slight reactions difficult. In the Latter
ca.se all very slight cloudings have been disregarded, and a clouding
usually described by the sign » has here been marked as • ?. The
larger reactions were however well marked and unmistakeable.

D. Anti-Ophidian Sera.

a. Qualitative Tests.

(1) Tests with Anti-Snake Serum.

This antisenim was obtained by the intraperitoneal injection into a
Rabbit of Snake serum {Tropidonot^is natri.r). The serum had been
previously heated to .5.5° C. in orfler to dostroy its toxic properties.

Percentage
of positive
X + + reactions

10

1 . 100

0

0

13

60

40

4 1 1 4(!

0

0

0

This was a moderately powerful antiserum, producing its maximum
effects amongst the Ophidia. The few reactions obtained with Chelonian,
Crocodilian and Lacertilian blootis were all feeble. One bird's egg-
albumin (Moorhen) gave a small reaction, but a well-marked clouding
followed by a deposit w;xs obtained with a dilution of snake's egg-
albumin, and a smaller clouding with tortoise egg-albumin.

Material tested

.

.?

«

22 Efig-Alhiimiiis

((/) 10 Birds' egg-albumins

9

1

(6) 2 Beptile
(c) 3 Amphibian ,,
((/) 7 Fish
127 niootl Sera

3

7

•

1

15 Chelonia

13

1

1

rn T> i-i- 1 Crocodilia
59 Reptiha {,. ^

12 Lacertuia

2

7

1

1

.28 Ophidia
17 Amphibia
39 Pisces

15
17
39

7

12 Arthropoda

12

.

352

Antisera for ReptiUa

(2) Tests with Antiserum to Snake's Egy-alhumiii.

This antiserum was obtained by the intraperitoneal injection into a
Rabbit of diluted Snake's egg-albumin (Tropidonotvs natria;).

Percentage

Material tested

•

,?

«

X

+

+

of positive
reactions

23 Egg-Albumins

(a) 11 Birds' egg-albumins

7

.

30

(h) 2 Reptile „

.

1

100

(c) 3 Amphibian „

3

0

(d) 7 Fish

7

0

127 Blood Sera

IS Chelonia

9

.J (33

'!«)

M"?"/,,)

40

,„ „ .... 4 Crocodilia
59 Bepfha - ^^ ^^^^^^.,.^

3
4

3 (15

Vo)

5(41"/,,)

25
6G

28 Ophidia

14

11 (39»/„)

(7%) •

50

17 Amphibia

17

0

39 Pisces

38

2

12 Arthropoda

12

0

This was a vei-y powerful antiserum giving an immediate dense
clouding, followed by a very large precipitiini, with a dilution of snake
serum. Moderately well-marked reactions were obtixined with specimens
from all the sub-classes of the Reptilia, especially the Ophidia, as well
as with the diluted egg-albumin of the tortoise. 36 V^ of the Biifls'
egg-albumins also reacted with this antiserum.

(3) Tests with Anti-Python Servm.

This antiserum was obtained by the intraperitoneal injection into
a Guinea-pig of Python's serum {Python mohirns). The serum was
obtained from an animal which had filed, and was very slightly cf)n-
taminated with bile.

Percentage
1^ _i_ _L ^^ positive

* • * ^ + + reactions

7
0
0
14
0
0
0

Material tested

•

18 Egg-Albumim

18

(11 Bird, 1 Reptile, 6 Fish)

127 Blood Seru

/15 Chelonia
-a r> »•!• 4 Crocodilia
•^'^^P">'^ 111 Lacertilia

Us Ophidia

14

4
11

24

18 Amphibia

18

39 Pisces

39

12 Arthropoda

12

Graham- Smith 353

This antiserum was very feeble and failed to react except with the
blood of four Snakes and one .Chelonian (Testudo ibera). Even with
its homologous serum the reaction was feeble.

Summary of results of Qualitative Tests with A nti- Reptilian Sera.

The anti-reptilian sera may be divided into two groups according to
their reactions. The first group, comprising the antisera for Chelonia
and Crocodilia, shows its maximum reactions with the sera of these two
sub-classes, and few and feeble reactions with the bloods of the Lacertilia
and Ophidia; This group also gave moderate results with the Avian
egg-albumins. These experiments confirm those done with the antisera
to Avian egg-albumins.

The second grou}), comprising the antisera for the Lacertilia and
Ophidia, exerts its maximum effects on the Lacertilia and Ophidia.
Moderate reactions were obtained with Chelonian and Crocodilian sera.
With one exception no positive results were obtained with Avian egg-
albumins. The antiserum for Snake's egg-albumin, though behaving
othei-wise like anti-snake serum, gave 36 "/o of moderately well-marked
reactions with Avian egg-albumins.

All anti-reptilian sera reacted well with dilutions of Reptilian
egg-albumins, but gave negative results with Amphibian and Fish
egg-albumins, and Amphibian, Fish and Crustacean sera.

The results of these experiments indicate a close relationship between
the Chelonia and Crocodilia and between the Lacertilia and Ophidia.
A distant relationship is shown between the Aves and the former group,
and a more distant one between them and the latter group. This last
is best seen in the tests with the antiserum to Snake's egg-albumin.
Anti-Chelonian and Crocodilian sera show very little aflSnity between
these animals and the Lacertilia and Ophidia, but antisera to the latter
indicate a relationship between them and the former.

The following table summarises the results of tests with anti-
Reptilian sera. The percentages of the better marked reactions only
are given, and the experiments with anti-Lizard I ( Uromastix spinipes)
and anti-Python are not included.

K. 23

354

Antisera for Reptilia

Marked positive results with Antisera to

Material tested

y

Chelonia

Crocodilia

Lacertilia

Ophidia

Ophidian egg-alb.

Egg-Albumi^is

Birds' egg-albumins

21%

25%

OO/o

10%

36 T

Beptile

33 „

100,,

100,,

100,,

100,,

Amphibian ,,

o„

o„

o„

o„

o„

Fish

o„

o„

o„

o„

o„

Blood Sera

/Chelonia

73 „

45 „

27 „

7.,

7„

„ ,.,. Crooodilia
Eeptiha \^ ....
Lacertilia

25 „

100,,

50 „

25 „

25 „

o„

o„

92 „

40 „

40 „

'■ Ophidia

3 „

0 „

30 „

46 „

46 „

Amphibia

0..

0 „

o„

o„

0 „

Pisces

o„

o„

o„

o„

o„

Arthropoda

. 0.,

o„

o„

o„

o„

Quantitative measurements with Anti- Reptilian Sera.

The methods used in tabulating these results are the same as those
made use of in recording similar observations with the antisera to egg-
albumins.

(1) Tests with Anti-Tortoise Serum (T. ibera).

Material tested

Amount of
precipltum

Percentage

Turtle serum

•0463

' 117

Tortoise „

•0394

100

Alligator ,,

•0018

7

Greenfinch egg-albumin

•0046

12

Silver Pheasant's ,,

■0041

10

Moorhen's ,,

•0087

9

Hedge-Sparrow ,,

•0018

5

Frog, Amphiuma, and Dogfish sera failed to react. The few quanti-
tative tests made with this antiserum are in accord with the qualitative
tests. By the latter method Tortoise and Turtle sera gave full reactions.
This is the only instance in which a non-homologous serum gave a
higher reading than the homologous serum, and the result is probably
due to some error in measurement, or dilution. By the qualitative
test Alligator serum, Greenfinch, Silver Pheasant, and Moorhen's egg-
albumins gave slight reactions, and Hedge-sparrow egg a doubtful
reaction,

Ch'aham-Smith 355

(2) Tests with Anti-Tortoise Serum (T. vidna).

Amount of
Muterial tested precipitum Percentage

Tortoise serum (T. vicina) ■03M 100

(T. ibera) -0054 24

Turtle „ -0028 12

Alligator „ • 0

Tortoise egg-albumin • 0

Duck's „ -0056 24

Traces of precipita were given by Emu's, Fowl's, Silver Pheasant's,
Pheasant's, Greenfinch's and Thrush's egg-albumins, but Crane's, Black-
bird's, Moorhen's, Hedge-sparrow's, Chaffinch's, Skate's and Dogfish
egg-albumins, as well as Frog, Amphiuma, and Dogfish sera, failed to
react.

The results of these tests again agree fairly closely with those done
by the qualitative method. In these a full reaction was indicated with
Tortoise serum (T. vicina) and small reactions with Tortoise (T. ibei-a),
and Turtle. Alligator serum showed a doubtful reaction, whereas Duck's
egg-albumin gave a medium precipitum. No signs of reaction were
obtained qualitatively with Fowl's, Pheasant's and Thrush's egg-
albumins, but quantitatively they yielded traces of precipita.

(3) Tests viith Anti-Turtle Serum.

Material tested

Amount of
precipitum

Percentage

Turtle serum

•0300

100

Tortoise ,,

•0018

6

Alligator ,,

•0009

3

Emu's egg-albumin

•0037

12

Pheasant's ,,

•0028

9

Duck's ,,

trace

?

Fowl's ,,

»»

?

Moorhen's „

»t

?

The results in this case differ slightly from the qualitative experi-
ments. In the latter, Tortoise serum showed a full reaction and the
quantity of precipitum yielded ought accordingly to be more than 6 '/o
of that given by Turtle senim. Fluid Alligator serum on the other
hand failed to react. Emu's and Pheasant's egg-albumins showed small
reactions, but Duck's, Fowl's and Moorhen's egg-albumins faile<l to react,
although by the quantitative method they showed traces of precipita.

23—2

356

Antisera for Amphibia, Pisces

These quantitative Tests, as in the case of those made with antisera
to Avian egg-albumins, are ahnost entirely in accord with the quali-
tative experiments.

III. Anti-Amphibian Serum.

Tests with Anti-Frog Serum.

This antiserum was prepared by Dr Nuttall by the intraperitoneal
injection of Frog's blood (Rana temporaria) into Rabbits.

Percentage
- j^ of positive

* • » >^ + + reactions

HaterlalB tested

•

18 Egg-Albumi)u (1 Amphibian)
70 Blood Sera

18

31 Eeptilia

IE > T,-!,- 4 Urodela
15 Amphibia , , ,

11 Anura

31
4
5

15 Pisces

15

9 Crustacea

9

4 (360/0)

2(18»/o)

0

0

54

0

0

This was a moderately powerful antiserum. Good reactions occurred
only with two specimens of the blood of the Frog (Rana temporaria)
and moderate with four examples of Frog's blood (1 Rana temporaria
and 3 R. tigrina). All other tests were negative (see p. 210).

IV. Anti-Fish Serum.
Tests vjith Anti-Ammocoetes Serum.

This antiserum was prepared by the intraperitoneal injection into a
Rabbit of Ammocoetes serum, which had been dried on filter-paper, and
dissolved out by means of salt solution.

Percentage
of positive
X + + reactions

0
0

8

8

100

0

Material tested

•

«?

23 Egg-Albumins

23

• •

131 Blood Sera

60 Reptilia

60

•

17 Amphibia

17

• •

/12

Chondropterygii

11

1

39 Pisces 26 Teleostei

24

2

1

Cyclostomata

.

• •

15 Arthropods

15

• •

Graham-Smith 357

This antiserum was only of moderate power, producing with its
homologous serum a medium clouding, followed by a well-marked pre-
cipitum. Only three doubtful cloudings were, however, obtained with
other fish sera, and other tests made with it were entirely negative.
Several tests were made with Limulus, and Arthropod sera, but with
negative results.

V. Anti-Ascidian Serum.

Tests with Anti-Ascidian Serum.

This antiserum was obtained by the intraperitoneal injection into a
Guinea-pig of Ascidian extract. The extract was made by cutting large
fixed Ascidians into small pieces, and extracting these with salt solution.
The supernatant fluid which was used in these experiments was clear
and of a light yellow tint.

Percentage
of positive
• ? ♦ X + + reactions

Material tested

•

14 Egg-AIbtimins

14

22 Blood Sera

6 Amphibia

6

3 Pisces

3

13 Arthropoda

13

2 Ascidian extracts

.

■ • • • • 0

0

0

1.1. 100

This antiserum produced a well-marked precipitum with its homo-
logous extract, and a slight reaction with an extract of a species of small
fixed Ascidian, but foiled to give any trace of reaction with any of the
blood sera or egg-albumins.

VI. Anti-Crustacean Sera.

(1) Tests with Anti-Lobster Serum.
This antiserum was made by Dr Nuttall by the injection of Lobster
{Homarus vulgaris) serum into Rabbits. Peroenta«e

of positive
Materiai tested . « ? • X + + reactions

14 Egg-Albumins

(a) 13 Birds' egg-albumina 13
(6) 1 Reptile „ 1

53 Blood Sera

20 Beptilia 18

12 Amphibia 10

12 Pisces 12

0
0

11

20
0

9 Crustacea

2.1. 5(65°/„) 1 77

358

Antisera for Arthrojwda

This was a powerful antiserum producing well-marked results with
the Crustacean sera. Slight indications of a reaction were obtained
with two specimens of Reptilian, and two of Amphibian sera, probably
due to some fault in the dilutions (see pp. 211, 217, 310).

(2) Tests with Anti-Grab Serum.

This antiserum was obtained by the intravenous injection into a
Rabbit of the blood of a Crab (see pp. 211, 217, 310).

Percentage
of poeitive
• ? « X + + reactions

0
0
0
0

0

0

0

0

100

Material testeil

•

21 Egg-Albumins

(a) 13 Birds' egg-albumins
(6) 1 Reptile

(c) 1 Amphibian ,,

(d) 6 Fish

13
1

1
6

128 Blood Sera

60 Beptilia
18 Amphibia
38 Pisces

60
18
38

12 Arthropoda / ^ Xiphosura
(11 Deoapoda

1

This was a very powerful antiserum giving well-marked reactions
with all specimens of the Crustacea. The serum of the King-crab
(Limuhcs polyphemus) failed to react. Tests with all other blood sera
and egg-albumins were negative.

VII. Anti-Xiphosura Serum.

Tests with Anti-Limulus Serum.

These tests were conducted with two different antisera for the serum
of the King-crab {Limulus polyphemus). The first was made by intra-
peritoneal, and the second by intravenous injections into Rabbits. In
the following table the tests made with both antisera are recorded.

Both these antisera were extremely powerful, the first giving more
than three times as much precipitum as any other antiserum. The
second was not quite so powerful. The first antiserum produced a small
reaction with ciub's serum and with spider serum (very dilute), and
three doubtful cloudings with fish, and two with Crustacean sera. The

Graham-Smith

359

second antiserum gave two medium reactions with spider serum. All
other tests were negative.

Material tented •
46 Egg-Albumins

(a) 26 Birds' egg-albumins 26

(6) 4 Reptile „ 4

(c) 3 Amphibian „ 3

id) 12 Fish „ 12

222 Blood Sera

120 Beptilia 120

30 Amphibia 30

46 Pisces 43

26 Arthropoda

(Serum No. I)

(Serum No. II)

C 1 Xiphosura •
■! 11 Decapoda 8
\ 3 Arachnida •
I 1 Xiphosura •
(10 Decapoda 10

Penentage
, of podtlTe
+ ractioni

0
0
0
0

0

0

6

100

27

100

100

0

Numerous experiments were made with these antisera on dilutions
of Ammocoete.s blood to determine whether this test would bring for-
ward any evidence in support of Gaskell'si hypothesis on the origin of
the Vertebrates. On one occasion a small clouding resulted on the
addition of the first antiserum to a very strong solution of Ammocoetes
blood. All subsequent experiments were negative. Anti-Ammocoetes
serum also failed on all occasions to react with dilutions of Linmlus
serum. By this means therefore no evidence can as yet be produced in
support of this hypothesis.

Unlike Mammalian and Avian sera Limulus serum when passed
through a porcelain filter loses its (blue) colour, and no longer produces
any precipitum when tested with anti-Limulus serum.

Summary of Results with Anti-Arthropod Sera.

Well-marked reactions were obtained with the anti-lobster serum
amongst the Crustacea. Limulus serum was not tested. No positive
results of any importance were produced by tests on other sera.

Anti-crab serum though pnxlucing verj' marked reactions with
specimens of the Decapoda, failed entirely to react with the blood of
Limulus, or any of the other sera, or egg albumins.

Of the extremely powerfiil anti-Limulus sera only one showed any

1 Gaskell, W. H. "On the Origin of the Vertebrates," Journal of Anatomy and
Physiology, 1898, et »eq.

360 Antisera for Arthropoda

positive results with the Decapoda and these were very slight. Good
reactions were however obtained with dilutions of spider sera.

The results of these tests indicate a fairly close relationship between
the King-crabs and the Spiders, and a very distant one between the King-
crabs and the Decapoda. These sera were much more powerful than
the anti-egg, and anti-Chelonian sera which interacted, and it might
have been expected that even with distant relationship the sera of the
Decapoda would have produced at least small reactions.

During its collection the anti-crab serum behaved in a rather curious
manner. The blood clotted rapidly, and a very milky serum separated
from the clot. The latter in a short time also coagulated, and from this
coagulum a clear serum eventually came off.

Anti-Linnilus I behaved in the same way, but only a very partial
coagulation of the serum first separated took place.

In the previous tables the reactions produced with all specimens,
whether preserved in a fluid or dried condition, have been given. Some
of the latter, owing to overheating during the process of drying in the
sun, or some other cause, failed to go into solution and consequently the
tables of percentages of positive results are probably somewhat under-
estimated. The results of the actions of the antisera to the Avian egg-
albumins and Reptilian sera with the egg-albumins, and fluid Reptilian
sera are, therefore, given below. In the other gi'oups very few fluid
sera were used, and their reactions with the various antisera may be
seen in the tables at the end.

Percentage of well-marked positive reactions with Antisera to

Fluid materials tested

Avian egg
albumins (5)

Clielonia (3)

Crocodilia (1)

Lacertilia (1)

Ophidia (2)

15 Birds' egg-albumins

85 0/,,

21%

25 "/o

0%

10«/o

2 Reptile ,,

28 „

33 „

100,,

100,,

100,,

4 Chelonian sera

16 „

100,,

75 „

25 „

0,,

1 Crocodilian ,,

o„

50 „

100,,

100,,

100,,

2 Lacertilia „

o„

o„

o„

100 „

100,,

3 Ophidian ,,

0,.

o„

o„

o„

100,,

The present experiments can only be regarded as of a preliminary
nature, and indicate the interesting results which might be obtained by
systematically following up this line of research. For more accurate
and trustworthy results both quantitative and qualitative tests would
have to be undertaken with fluid sera obtained from healthy animals
and repeated two or three times. Most of the experiments, which
are the subject of this paper, have only been done once. Had it been

Graham-Smith 361

possible to repeat them, however, the results would probably not have
been materially affected.

In the following tables the name of the collector of the serum is
given with the majority of specimens. Dr Nuttall acknowledges the
source of all specimens examined by him at the end of this book.
After all these samples the numbers given in Nuttall's tables are
quoted, so that the results obtained by him with a large number of
anti-Mammalian and anti-Avian sera may also be followed.

The Limulus and Ammocoetes sera were procured for me by
Dr W. H. Gaskell, F.R.S., to whom I am much indebted for these very
interesting bloods.

The previous work on these lines has already been fully considered
in the foregoing monograph, and for this reason no attempt has been
made here to summarize the results of other observers.

The following works have been used in the construction of the annexed tables :
Evans, A. H. "Birds," Cambridge Natural Hi»tory, Vol. ix. 1900.
Gadow, H. "Amphibia and Reptiles," Cambridge Natural Hiitory, Vol. viii. 1901.
GuNTHER, A. C. L. G. The Study of Fuhes. 1880.

General Conclusions.

(1) Powerful antisei-a to Avian egg-albumins produce very large
reactions with dilutions of Bii-ds' egg-albumins. They also give distinct
positive reactions with Chelonian and Crocodilian sera, as well as with
dilutions of Reptile egg-albumins. No reactions of any importance were
obtained with Amphibian or Fish egg-albumins or with Lacertilian,
Ophidian, Amphibian, Fish, or Crustacean sera. These tests, therefore,
show a distinct relationship between the Aves and Chelonia and
Crocodilia.

(2) Powerful anti-Ghelonian sera gave maximum reactions with the
Chelonia and Crocodilia. Smaller reactions were obtained with Reptile
egg-albumins and Avian egg-albumins, but with Lacertilian and Ophidian
sera the results were almost negative. No positive reactions occurred
with other egg-albumins or blood sera.

These results, showing a well-marked relationship between the Che-
lonia and Crocodilia, and distinct one with the Aves, confirm the above
conclusion.

(3) A moderately jwwerful anti- Crocodilian serum behaved like the
anti-Chelonian sera.

362 Conclusions

(4) A powerful anti-Lacertilian serum produced its maximum
reactions with the Lacertilia, and also reacted well with the Ophidia.
Smaller reactions were obtained with the Crocodilia and Chelonia, but
none with the Avian egg-albumins or other albumins or blood sera.

(5) A powerful anti-Ophidian serum gave well-marked results with
the Ophidia, and also reacted well with the Lacertilia. Reactions with
the Crocodilia and Chelonia were much smaller. A moderate reaction
was obtained with one Avian egg-albumin. All other tests were negative.

(6) A very powerful antiserum to Ophidian egg-albumin showed its
maximum rcfictions with the Ophidia, good reactions with the Lacertilia,
and smaller ones with the Chelonia and Crocodilia. 36 "/o of distinct
positive reactions were obtained with Avian egg-albumins. Other tests
were negative.

The results of experiments with anti-Lacertilian and anti-Ophidian
sera show a well-marked relationship between these two gnjups, a more
distant relationship between them and the Chelonia and Crocodilia, and
a still more distant one between them and the Aves. The latter is
most markedly shown by the anti-snake-egg serum.

(7) , A moderately powerful anti-Frog serum reacted only with the
Anura. No reactions were obtained with the Urodela or any other
serum or egg-albumin tested.

(8) A moderately powerful anti-Ammocoetes serum failed to show
any affinity between this animal and any other.

(9) A weak anti-Ascidian serum reacted only with its homologous
extract.

(10) Two powerful anti-Crustacean {Decapoda) sera reacted well
with the sera of Decapoda, but tailed to react with the serum of the
King-crab (Limulus polyphemus). Tests with egg-albumins and other
sera were all negative.

(11) Two extremely powerful anti-Limulus sera reacted well with
dilutions of Limulus serum, and produced moderate reactions with spider
sera. One showed a small reaction with a crab serum, but the other
did not. Other tests with Crustacean sera were negative. Experiments
with Ammocoetes and other Vertebrate sera and egg-albumins were
negative. These tests, therefore, show a distinct relationship between
Limulus and the Arachnida, and a doubtful one between Limulus and
the Decapoda.

•BLOOD-RELATIONSHIP AMONGST THE LOWER VERTE-
BRATA AND ARTHROPODA, ETC., AS INDICATED
BY 2,500 TESTS WITH PRECIPITATING ANTISERA.

By G. S. GRAHAM-SMITH, M.A, M.B., D.P.H. (Camb.).

TABLES.

EGG ALBUMINS

Division I.
Ratitae

Division II.
Carinatae

Order
Anseriformes

Order
Galliformes

Order

Gruiformes

Favi.

Rallidae

Fani.
Gruidae

Order

9

Passerifopmes. Oscines

Fam.

lO

Tuididae

11

13

Fam.

13

FiingiUldae

14

15

Class AVES

Subclass NEORNITHES

Dronmeus novae-hollandiae. Emu
Australia (Z) 18. i. 02 (No. 799)

Aims boncns. Common Duck
Cambs. 24. v. 02

Galltis domesticns. Domestic Fowl

Cambs. 24. v. 02
Gallus (lomesticus

Cambs. 30. xi. 01
Phasiamis colchicm. Pheasant

Sussex 26. v. 02
F.uplocmmis nycthemerus. Silver Pheasant

China (Z),'laid 11. i v. 02 (No. 797) •

Anti-Avian Sera

a

+

D

GalHnula chloropus.
Sussex 26. v. 02

Moorhen

Balearica reguloritm. Cape Crowned Crane
S. Africa (Z) 13. iii. 02 (No. 798)

Turdus merula. Blackbird

Cambs. 13. v. 02
Turdus musicus. Song Thrush

Cambs. 13. v. 02
Accentor modularu. Hedge Sparrow

Cambs. 12. v. 02
Sylvia atrica2iilla. Blackcap

Sussex 26. v. 02

Ligurhius chloris. Greenfinch

Sussex V. 02
Fringilla coelebs. Chaffinch

Cambs. 12. v. 02
Linota eannabina. Linnet

Cambs. 13. v. 02

+
D

+
D

+
D

+
D

+
S

+
D

+

+
D

+
d
+
d

+
d

+
D

+

+
D

+
D

+
S

+
D

+
S

+

d

+

+
d

*

d

+
D

+
D

tr

+
D

+
D

+
d

+
D

+
tr

+
d

X

tr

X

tr

+
D
+
D

tr

I

364

Anti-Reptilian Sera
Chelonia Lacertilia Ophldia

Anti-Arthropod
Sera

1

i

H

a

1

1

1

<

2

1

c

S
o

13

n

3

S

s
s

«

c

i

<

c
<

a
•<

1

SI

a
<

£1

B

•a

a
<

3

a

s
i

<

1

a

a
-<

*
*
« ?

*

tr

X

D

tr

«
tr

tr

*

.?

.?

*

d

•

•

'

*

•
*

•

•

•

•

•

•
•

•
•

•
•

•
•

365

EGG ALBUMINS

Class REPTILIA

Anti -Avian Sera

a

C4

Subclass
CHELONIA

Fam.
Testudinldae

Subclass
SAURIA

Fam.
Colubrldae

Fiim.
Salamandridae

Fam.
Ranldae

Subclass
PALAEICHTHYES

Fam.
Scylliidae

Fam.
Rajldae

Subclass
TELEOSTEI

Fam.
Scomberidae

Fam,
Cottldae

Fatit.
Gobiidae

Fam.
Salmonidae

16 I Tettudo ibera. Tortoise

(Leighton) laid 2. viii. 02 (No. 808)

17 Tropidonotus natrix. Grass Snake
Cambs. vii. 02

31
33

33

34

35

36

37

aass AMPHIBIA

18 Triton cristatus. Crested Newt

Cambs. 8. v. 02
18 ; Triton ruhjaris. Common Newt
! Cambs. 8. v. 02

30 j Rami tfmporaria. Common Frog
Cambs. v. 02

aass PISCES

Scylliitm canicula. Dog-fish

Plymouth 27. vi. 02
Scyllium catulus. Dog-fish

Plymouth 27. vi. 02

Baja bat is ? Skate
Plymouth vi. 02

Scomlier scomber. Mackerel

Coitus scorpius. Bull-head
Guernsey vi. 02

Gobius ruthenspari
Plymouth v. 02

Salmo salar. Salmon
02

^

1?

1

.*

1

S,

1

+3

a

B

<

i

a

II

366

s

S
I

Anti-Reptiliau Sera
IjKertilia

a

o

Opiiidia

C

A.
3

+
D

Anti-Arthropod
Sera

I I

s

367

Anti-Avian Sera

IS

?

M

!

SERA

i

%

*«

H 1 n

^

9

Class REPTILIA

c c

a

c

< <

<

<

Subclass

as

Chryumys picta. Painted Terrapin

,

,

,

CHELONIA

39

N. America (Jordan) 22. v. 02 (No. 800)
Chrysemys eUgans

,

,

Fam.

N. America (Jordan) 11. t. 02 (No. 801)

Testudinldae

80

31

Cistudn Carolina

N. America (Jordan) 30. iv. 02 (No. 802)
Grapteinys paeudogeoqraphica

N. America (Jordan) 17. v. 02 (No. 803)

•

•

•

33

Testudo ibera. Tortoise (fluid)

(irecce, killed Cambs. 14. i. 02 (No. 804)

'

.?

tr

♦ ?

38

Testudo ibera (fluid)
13. V. 02

•

«-

*

♦ ?

34

Testudo ibera

*

d

•

+
d

•

35

Testudo viciiui. Giant Tortoise

(N. York Zoo., Langmann) 14. vi. 02

(No. 805)

■

,?

86

Testudo vidua (fluid)

Galapagos (Z) 20. x. 02 (No. 80(i)

•

*?

•

•

87

Testudo inepta. Clumsy Tortoise
Mauritius (Z) 20. v. 02 (No. 807)

Fam.

88

Chelone midas. Green Turtle (fluid)

.

.?

.

Chelonldae

89
40
41
43

King's Coll., Camb. 14. xii. 02 (No 809)
Chelone midas

Tropical seas (Z) 9. x. 01 (No. 810)
Chelone midas

4. xii. 02
Chelone viridis ? Turtle

Brighton 13. xii. 01 (No. 811)
Chelone ? Water Turtle

L. Nyassa (Dodds) 8. ii. 02 (No. 812)

tr

tr

•

Subclass

48

Alligator mississippiensis. Alligator (fluid)

.

.

CROCODILIA

N. America (Z) 27. xi. 01 (No. 813)

44

AlUiiator missisxipplen-iis. Alligator
N. America (Z) 27. viii. 01 (dried)

•

*

.?

■

45

Allitfator sinensis. Chinese Alligator
China (Z) 13. viii. 01 (No. 814)

•

+

•

46

Crocodile

L. Nyassa (Dodds) 8. ii. 02 (No. 815)

■

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Subclass

SAURIA

Order

47

Gecko quttatus. Gecko

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Lacertilia

India (Dalgetty) 24. viii. 02 (No. 816)

Suborder

Geckones

Fam.

Geckonidae

368

i

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Anti-Arthropod

Chelonin

Lacertilia

Ophidta

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24

Subclass

SAURIA

Order

Lacertilia

Suborder
Lacertae

Fam.
Agamidae

Fnm.
Ignanidae

Fam.
Ang^ldae

Fam.
Varanidae

Finn.
Lacertldae

Fam.
Scincidae

Order
Ophidia

Fam.
Boidae

SERA

aa.ss REPTILIA

Fam.
Colubrldae

Series A
Aglypha

48
40
SO

61

63

S3

84
66

6«

67
68

68
60
61
82
63
64
66

66
67

Calotes versicolor. Indian Tree-lizard
India (Phipson) 12. i. 02 (No. 817)

Uromasti.T spinipes

Cairo (Elliot-Smith) 7. vi. 02 (No. 818)

Uromastix spinipes (fluid)

Iguana tuberculata. Iguana

Trinidad (TuUoch) 1. i. 02 (No. 819)

Anguis frag His. Slow- Worm

Hereford (Leighton) 31. iii. 02 (No. 820)

Varanus salvator. Two-banded Monitor

India (Z) 6. vi. 02 (No. 821)
Vararixis salvator (fluid)

Varamts griseus. Grey Monitor.

N. Africa (Z) 30. ix. 01 (No. 822)
Varanus griseus (fluid)

Lacerta agilis. Sand Lizard
Cambs. 3. v. 02 (No. 823)

Spec. ? Indian Lizard

India (Dalgetty) 1. ix. 02 (No. 824)
Land Lizard

Natal (Parkinson) 13. xii. 02 (No. 825)

Python molurus. Indian Python
India (Z) 11. i. 02 (No. 826)
Python tiiolurus (fluid)

Python sebae. We.st African Python

W. Africa (Z) 8. xi. 01 (No. 827)
Boa constrictor. Common Boa

S. America (Z) 7. x. 01 (No. 829)
Boa constrictor (Marajuel)

Trinidad (TuUoch) 4. vi. 02 (No. 830)
Eryx jaculus

Cairo (Elliot-Smith) 19. vi. 02 (No. 831)
Corallus caninus L. "Hundskopfschlange"

Madagascar (Luhe) 20. ii. 02 (No. 828)

Tropidonotus natrix. Grass Snake
Cambs. 28. iv. 02 (No. 832)

Tropidonotus natrix (fluid)
2. V. 02 (No. 833)

Anti-Avian Sera

^

i

1

370

Chelonia

Anti-Reptilian Sera
LacertiUa

Opbidla

Anti-Arthropod
Sera

a

3
I

a

S

I

S

5

I

s

5

-I-
d

d

+

B

+
D

+

X

tr

d

tr

»

«

d

'I

X

•

D

d

«

*

D

d

X

*

D

d

X

«

D

d

+

X

D

D

371

24—2

SERA

Class REPTILIA

Subclass

SAURIA

Order

Ophidia

Fam.

Colubridae

Series A

Aglypha

Series B
Opisthoglypha

Series C
Proteroglypha

Fam.
Vlperldae

68
69
70
71

73

73

74
75
76
77
78
79

80
81
82
88
84
85
86

Anti-Avian Sera

Tropidonotus natrix (fluid)

18. X. 02 (No. 834)
Zamenis diadema

Cairo (Elliot-Smith) 11. vi.02 (No. 835)
Zamenis ravergieri, var. nummifere

Cairo (Elliot-Smith) 19. vi. 02 (No. 836)
Dendrophis liocerus. Whip Snake

Trinidad (TuUoch) 2. iii. 02 (No. 837)

Cerberus rhynchops. Indian Estuary Snake
Bombay (Phipsou) 17. i. 02 (No. 838)

Cerberus rhynchops

Calcutta (Rogers) 8. x. 02 (No. 839)

Naja tripiidians. Cobra

India (MacWatt) 23. vi. 02 (No. 841)
Naja tripudians

21. vi. 02 (No. 840)
Naja tripudians

(Z) 6. viii. 01 (No. 843)
Naja tripudians

(Phipson) 4. ii. 02 (No. 842)
Naja haje. Hooded Cobra of Africa (Aspis)

Cairo (Elliott-Smith) 19. vi. 02 (No. 844)
Pseudechis porphyriaceus. Black Snake

N. S.Wales (Cashman)31.xi. 01 (No.845)

Bitis arietans. Puff Adder

S. Africa (Z) 6. viii. 01 (No. 846)
Bitis arietans

L. Nyassa (Dodds) 27. iii. 02 (No. 847)
Viper berus. Adder

N. Devon (Leighton) iv. 02 (No. 848)
Viper berus

iv. 02 (No. 849)
Viper Russelli. Himalayan Viper

India (Donald) 4. vi. 02 (No. 851)
Crotalus horridut. Rattlesnake

United State.s ( Salmon) 6. xi. 01 (No. 852)
Poisonous Serpent

L. Nyassa (Dodds) 8. ii. 02 (No. 853)

tr

372

Cbelonis

Anti-lieptiliiiu Sera
Ucartllis

Ophldta

Auti-Artliropod
Sen

Eh
B

■s

6-1

■s

I

Hi
■a

s

a

t
Of

s

+

D

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tr

373

SERA

Class AMPHIBIA

Anti-Avian Sera

Order
Urodela

Fam.
AmpUumldae

Fam.
Salamandrldae

Order
Anura

Suborder
Aglossa

Suborder
Phaneroglossa

Fam.
Bufonidae

Fam.
Engystomatidae

Fam.
Ranidae

87

88

8S

»o

91

»a

98

94
96
96

97

98

99

lOO

lOl

loa

103
104

Amphiuma means. Araphiuma (fluid)
N. America (Z) 19. iii. 03 (No. 854)

Amblystoma tigrinum (Axolotl)

Bless. 26. xii. 01 (No. 855)
Triton cristatus. Crested Newt

Cambs. 8. v. 02
Triton cristatus

6. v. 02
Triton cristatus

29. iv. 02 (No. 85G)
Triton vulgaris. Common Newt
Cambs. 8. v. 02

Xenopus laevis. Smooth-clawed Frog
E. Africa (Z) 19. iii. 02 (No. 857)

Bufo vulgaris. Common Toad
■ Cambs. 14. vii. 01 (No. 858)

Bufo vulgaris (fluid)
viii. 02

Bufo mauritanica. Moorish Toad
'N.W. Africa (Z) 10. i. 02 (No. 859)

Megalobatrachus maxima. Gigantic Sala-
mander
Japan (Z) 23. ix. 01 (No. 860)

Bana temporaria. Common Frog

Cambs. 29. iv. 02 (No. 861)
Sana temporaria

11. xi. 01 (No. 863)
Sana temporaria

11. iii. 02 (No. 862)
Rana tigrina. Tigrine Frog

India (Z) 19. iii. 02 (No. 867)
Rana tigrina

(Z) 3. iii. 02 (No. 865)
Rana tigrina

25. i. 02 (No. 864)
Rana (spec. ?). Trinidad Frog

Trinidad (TuUoch) 27. iii. 02

374

Chelonia

Anti-lieptilian Sera
Lacertilia

Ophidia

2
&

3

a

33
5

Auti-Arthropod
Sera

+
+

tr
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1)

375

SERA

Phylum ARTHROPODA.

Anti-Avian Sera

I I

Xiphosura
Decapoda

Arachnida

106

106
107
108
109

no

111
112
113
114
115
116

117
118

lie

Limulus polyphemttt. King Crab
OaskeU

Crangon vulgaris. Common Shrimp

Guernsey vi. 02
Homarus vulgaris. Lobster

England 14. xi. 01 (No. 895)
Astucus fluviatilis. Cray-fish

Cauibs. 14. i. 02 (No. 894)
Palinurus vulgaris. Craw-fish

Plymouth 26. ii. 02 (No. 896)
Pagurus Bernhardus. Hermit Crab

Carcinus maenas. Shore Crab
Plymouth 25. i. 02 (No. 898)

Carcinus maenas (fluid)
Guernsey vi. 02

Cancer pagurus. Crab

Portunus puber. Velvet Fidler-Crab
Plymouth 25. i. 02 (No. 899)

Portunus depurator. Swimming Crab
Plymouth 25. i. 02 (No. 900)

Maia squinado. Spider Crab
Plymouth 27. li. 02 (No. 897)

Spider Serum
10. viii. 03

Spider Serum
vi. 02

Spider Extract
vi. 02

376

Chelonia

Anti-Keptilian Sera
Lacertilia

Opbldla

Auti' Arthropod
Sera

hi

I

e

3

a
■a

tr

+
d
+
D

+
d

+
d

tr

X

d

+
D

d

X

d

+
D

X

d

tr

+
S

377

SERA

Class PISCES

Suborder Plagiostomata
A. Selachoidei
Fam. Carcharildae

Fam. Scylliidae

B. Batoidei

Fam. Prlstidae

Fam. Rajidae

Fam. Trygonldae

Fam. Myliobatidae

Suborder Holocephala

Division Perciformes
Fam. Fercidae

Division

Cotto- Scorn beriformes

Fam. Carangldae

lao

121

laa

133
134

135

136

137
138

139

130

131

133
133

134

Subclass PALAEICHTHYES
Order I. Chondropterygii

Carcliarias glaucus. Common Shark

Sarawak (Hose) 10. iv. 02
Zygaena malleus ? Hammerhead Shark

Trinidad (Tulloch) 15. i. 02 (No. 869)
Glassheaded Shark

Sarawak (Hose) 10. iv. 02
iliistelus canis. Dog-fish

New Jersey, U.S.A. (Silvester) viii. 01 (No. 870)
Squalus acanthias. Dog-fish

New Jersey, U. S. A. (SUvester) 30. yiii. 01 (No. 871)

Scyllium canicula. Dog-fish (fluid)
Plymouth 27. ii. 02 (No. 872)

Saw-fish

Sarawak (Hose) 10. iv. 02

Raja ocellata. Skate

New Jersey, U.S.A. (Silvester) viii. 01 (No. 873)
Kay

Sarawak (Hose) 10. iv. 02

Dasyatus. Sting Bay

New Jersey, U.S.A. (Silvester) vui. 01 (No. 874)

Rhinoptera bonasus. Sting Bay

New Jersey, U.S.A. (Silvester) 30. viii. 01 (No. 875)

Chimaera vionstra
Naples 6. xi. 02

Subclass TELEOSTEI
Order II. Acanthopterygii

Perca fluviatilis. Common Perch (fluid)

Horsham (S. C.) 15. viii. 02
Mesoprion

Trinidad (Tulloch) 1. i. 02 (No. 893)

Naucrates ductor. Pilot-fish

Trinidad (Tulloch) 29. v. 02 (No. 876)

378

SERA

Glass PISCES

Division
Cotto-Scomberiformes

Fam. Bcomberidae

Fam. Cottidae

Division Gobiiformes
Fam. Ooblidae

Division Muciliformes
Fam. Sphyraenidae

Fam. Hugllidae

Divisimi
Gastrosteiformes
Fam. Gaatrosteidae

Division
Gobiesciformes
Fam. Ctobiesocldae

Division Pleuronectoidei
Fam. Pleuronectidae

186
188
187

188

189

140
141

143

148

Subclass TELEOSTEI
Order II. Acanthopterygii

Scomber auxis. Spanish Mackerel

Trinidad (TuUoch) 13. ii. 02 (No. 877)

Scomber carangut

Trinidad (TuUoch) 28. ii. 02 (No. 878)

Scomber scomber. Common Mackerel (muscle extract)
02

Cottus Scorpio 1 Bull-head
vi. 02

Gobius ruthenspari (muscle extract)
Plymouth 02

Sphyraena barracuda. Barracuda

Trinidad (Tulloch) 7. iii. 02 (No. 879)

Mugil capita. Grey Mullet
Sarawak (Hose) 10. iv. 02

Gastrosteus spinachia. Stickleback (muscle extract)
Plymouth 02

144
14ft

Lepadogaster

Guernsey vi. 02

(No. 880)

Fam. Oyprlnldae

Fam. Esocldae

148

147

Order III. Anacanthini

Rhombus triacanth. Butter-fish

New Jersey, U.S.A. (Silvester) viii. 01

Pleuronectes limanda. Dab (muscle extract)
15. viii. 02

Order IV. Physostomi

Carassius auratus. Gold-fish
15. i. 02 (No. 881)

Etox lucius. Pike
26. xi. 02 (No. 882)

379

SERA

Class PISCES

Fam. Esocidae

Fam. Salmonldae

Fam. Hyodontidae

Fam. Muraenldae

Fam. Sclerodeimi

Fam. Oymnodontea

Fam. Petromyzontidae

148

149

160

161

152

163

164

ISS

168

Subdass TELEOSTEI
Order IV. Physostomi

Emx litciiis. Pike

Ireland (DUlon) 6. v. 01 (No. 883)

Salmo fario. Trout

(Leighton) 17. x. 01 (No. 884)
Salmo salar. Salmon

Ireland (Dillon) 9. v. 01 (No. 885)
Salmo salar (fluid)

V. 02

Hyodon lergigus ? Moon-fish

Trinidad (TuUoch) 1. iv. 02 (No.

Anguilla imlqaris. Common Eel
Sussex (S. C.) 16. viii. 01

Order VI. Plectognathi

Aluton schoepjii. File-fish

New Jersey, U.S.A. (SiWester) viii. 01 (No. 887)

Tetrndon turgidxis. Toad-fish

New Jersey, U.S.A. (Silvester) viii. 01 (No.

Subclass CYCLOSTOMATA

Petromyzon bratwhialis (Ammocoetes). Larval Lam-
prey
Cambs. (Gaskell) 24. x. 02 + 03

Also 4 undetermined fish-bloods, 167 — 180

Most of the above fish bloods were tested with antisera to Emu's, Duck's,
Fowl's, Crane's and Snake's egg-albumins, and Tortoise (2), Turtle, Alligator,
Lizard (2), Snake, Python, Frog, Ammocoetes, Ascidian, Lobster, Crab, and
Limulus (2) sera. No well-marked positive reactions occurred except that
between anti-Ammocoetes and Ammocoetes sei'um.

380

SECTION IX.

ON THE PRACTICAL APPLICATION OF THE PRECIPITIN
REACTIONS IN LEGAL MEDICINE, ETC.

1. Antisera in the Examination and Identification of Bloods
and Blood-Stains.

The reactions given by specific haemolysins and agglutinins possess
scarcely any value medico-legally for the reason that to make such tests
a large number of intact blood corpuscles must be in suspension. The
use of the haemolysins medico-legally was first suggested by Deutsch
(see p. 41) who claimed that they might even be used upon dried
corpuscles. There are certainly grave sources of error in the method, as
compared to what we find when using precipitins.

The specific character of precipitins was already indicated by Kraus
for bacterio-precipitins and by Bordet, Fich, and Morgenroth, in their
work on Lactosera. It was recognized by Ehrlich, who refei's in his
Croonian Lecture, to still unpublished experiments of Morgenroth upon
lactoprecipitins. Although at the time very few data had been collected
regarding the precipitins, the assumption seemed justified that they
would prove to possess similar specific characters to the haemolysins
and agglutinins. Ehrlich 's paper was read before the Royal Society, at
a meeting which I attended on the 23rd of March, 1900.

Subsequently, Wassermann (18 — 21 April, 1900)' brought the
question of specificity into greater prominence, speaking of the reaction
as " eine strong specifische Methode " which permits us " iiber die
Stellung verschiedener Eiwcisskorper Aufschluss zu geben : namlich
durch die Bildung der streng specifischen Agglutinine in Thierkorpcr
die wir seit Jahresfrist kennen, und die mir noch nicht genug gewurdigt
scheint." He refers to the work of Bordet on lactosera. Bordet, and

' I am indebted to Profegaor Wassermann for a manuscript copy of the paper cited,
the original being inaccessible to me. G. H. F. N.

382 Medico-Legal

Ehrlich spoke of precipitins as " coagulins," Wassermann termed them
" agglutinins'." Wassermann referred to experiments of his own upon
lactosera. With lactoserum for cows' milk he only obtained a pre-
cipitation with cows' milk, not with human or goat milk. " Es handelt
sich iramer um specifische Korper, je nach der Milchart, welche dem
Thier vorher injiciert wurde....Die Eiweisskorper in verschiedenen
Thiergattungen sind demnach streng different und verschieden." He
added that experiments made with fowl egg-white also gave " in der
That specifische Coaguline gegeniiber dem Hlihnereiweiss." It is there-
fore fully evident that Wassermann recognized the practical bearings of
the discoveries of Tchistovitch and Bordet, and credit is due to him for
having suggested the use of precipitins in the differentiation of al-
bumins of different animals.

It will be noticed that Wassermann refers to the precipitins, as
affording a means of distinguishing the albumins of different animals,
iiTespective of their being contained in blood or the like, and it is
therefore not correct, as he states in a personal letter to me (15. i. '03),
strictly speaking, to refer to the precipitin or biological method in hlood
diagnosis. To be certain that a reacting substance is blood, it is still
necessary, in forensic practice, to utilize the ordinary tests for blood,
such as that for haematin etc. According to Wassermann the reaction
demonstrates the presence of albumins belonging to certain animals,
without especially telling us which albuminous substance we are
dealing with.

The medico-legal use of precipitins was almost simultaneously
discovered by Uhlenhuth, Wassermann and Schiitze (see p. 162).
Uhlenhuth (7. II. 1901) considered that the method might have forensic
value in the identification of blood stains. He tested 19 kinds of
blood and only obtained a reaction with human blood upon adding
anti-human serum to the series of dilutions. He moreover found that
human blood which had been dried 4 weeks on a board, could be readily
distinguished by means of anti-human serum from the blood of the horse
and ox. Wassermann and Schiitze (18. II. '01), a few days later,
reported having examined 23 bloods, none of which reacted to anti-human
serum except human blood and that of a baboon, the reaction in the

' I prefer to refer to these antibodies as precipitins for the reason that the term
conveys a definite meaning with regard to the appearances observed in their reactions.
The term agglutinin has been used with a definite meaning hitherto, and it can
only lead to confusion to include precipitins under agglutinins even assuming that
the antibodies are identical, which is unproved. The same objection holds for the term
coagulins (see p. 17).

Tests with Precipitins 383

latter case taking place much more slowly and to a lesser degree than
in human blood. Bloods of different species of animals, dried on knives,
linen, etc., when dissolved in saline solution after 3 months, and cleared
by filtration, gave specific reactions. In making their tests they took
about 5 — 6 c.c. of salt solution for the solution of a dried blood drop of
about the size of a sixpence, adding about -5 c.c. of antiserum to 5—6 c.c.
of the blood dilution, the mixture being afterwards placed at 37° C.
Under these conditions marked reaction had taken place within
20 minutes. The lists of bloods tested by the above authors will be
found on page 162.

Uhlenhuth (25. iv. '01) next reported that his anti-human serum had
served to identify human blood which had been dried 3 months. Six
samples of blood obtained from human cadavers and from healthy
persons, were allowed to putrefy, control tests being made with the
putrid bloods of the sheep, pig, horse, donkey, ox, cat, dog, goose, fowl,
hare, rabbit, deer. Solutions of these putrid bloods gave specific
reactions (see p. 119 et seq. also Plate and explanation at the end of
this section). All the bloods smelt of sulphuretted hydrogen, the
reaction being slightly alkaline. To obtain clear solutions, the bloods
were filtered through Berkefeld filters, the sterile filtrate being diluted.
A blood which had undergone putrefaction for 3 months still reacted
specifically to its antiserum. Faintly alkaline blood solutions con-
taining a small amount of soap, menstrual urine, human blood spots
on snow for two weeks (- 10° C), blood containing CO-haemoglobin,
albuminous, and especially pus-containing human urine, all reacted to
human antiserum, other bloods not doing so.

Nuttall (11. V. '01), working independently, found antihuman serum
to cause specific precipitation in human blood which had undergone
putrefaction for 2 months. Bloods dried for 2 months and kept at room
temperature, or at 37° G. in the dark, and bloods exposed for a week in
the sun, gave specific reactions, as did also blister-fluid, nasal secretion
and human tears, the latter however to a slight degree only. On the
other hand, old horse serum (31 months in the laboratory, preserved
with trikresol) and human pleuritic exudate (6 months in the laboratory)
yielded effective antisera when injected into rabbits. He drew attention
to the reactions occurring in the bloods of allied animals which he had
tested. The allied species whose bloods reacted to antiserum for human
blood were man and 2 species of monkey, whilst ox and sheep bloods
reacted to both anti-ox and anti-sheep sera, the most marked reactions
being obtained when an antiserum acted upon its homologous blood.

384 Medico-Legal

Ziemke (27. vi. '01) next published an extensive series of tests
possessing especial medico-legal interest. Using anti-human serum, he
tested bloods which had been dried for years. Human blood dried for
2 years, reacted in 3 hours by clouding, the cloud persisting after
24 hours. Ox blood dating from 1863, 1869, 1876 gave no reaction
with anti-human senxm either at room temperature or at 37° C. Human
bloods dried on various fabrics and dating from 1878 to 1899 (shirting,
gauze, linen) were extracted with soda solution and gave reactions after
several hours, excepting a sample of the year 1883 which did not go into
solution. None of the samples gave more than a clouding which
persisted 24 hours. Of the other bloods tested by way of control (sheep,
calf, pig, dog, ox, horse, rabbit) and which had been dried 2 months on
linen, only the rabbit blood gave a slight " Opazitat." Human blood
mixed in garden earth since 1898 and 1900, gave a slight but distinct
clouding which persisted after 24 hours. This blood was soluble in soda
solution, not soluble in saline. As controls he used the bloods of the
horse, ox, sheep, calf and pig, which had been kept in garden earth for
8 weeks, but none of these gave a reaction. Soda solution of human
blood from a case of CO-poisoning, kept 8 weeks on linen and in earth
gave a moderate reaction in one hour, the clouding persisting 24 hours ;
saline dilutions gave negative results. Human blood dried on instru-
ments such as a rusty knife (1896) and clean axe (1896), gave moderate
reactions in 1 hour, clouding persisting after 24 hours. He only used
soda dilutions, finding that rust and soda solution alone produced no
reaction. Washed human blood-stains, still possessing a pale yellow
colour (1883) gave slight clouding in one hour, control tests with rust-
spots on linen were negative. Human blood from a white-washed cellar
wall (1899), gave rather marked clouding in 3 hours, both in soda and
saline dilutions ; but he does not state that he controlled the effect of
the white-wash. Human blood dried on wooden matches for 1 year,
gave moderate reactions in soda dilutions in 3 hours, whilst blood on a
tree branch gave a slight clouding in saline solution after 3 hours.
Human blood dried 3 months on glass, gave gi-eat clouding after one
hour at room temperature. Human blood exposed for 7 months on
linen in the open, gave marked clouding in soda dilution in 3 hours
at room temperature. Human blood dried 10 years on paper, gave
slight clouding in 3 hours in soda dilution. Human blood from a
cadaver, 3 days old, gave a great clouding in a few minutes and a
flocculent precipitum after 24 hours at 37° C. Putrid human blood,
diluted in saline and tested at 37° C. reacted, whereas putrid pigeon.

Tests with Precipitins 385

fowl, goose, ox and pig blood did not. He gathered the impression that
the older the blood, the less intense is the clouding, for he never obtained
a regular precipitum with old dried blood.

I have noted elsewhere that there are drawbacks to using soda
solution, because of the pseudo-reactions it may give (see pp. 64 and 83),
also to the necessity of observing a " time limit," facts which detract
from the value of Ziemke's observations. He did not state that controls
were used in all his tests, nor that the control bloods were also diluted
with soda solution.

Uhlenhuth (25. vii. '01) next reported experiments upon blood-
stained articles. He obtained positive reactions with human blood-
stains on a stick (1900), blood-stained sand (1896), a stain on cotton
(1897), a stain on a coat and pair of trousers (1901), and on a hatchet
(1900), upon adding anti-human serum to saline solutions of these bloods.
With anti-pig serum he obtained positive reactions with pig blood-stains
on linen (anti-human, anti-sheep, anti-horse sera gave no reaction), also
with blood dried since 1897. Anti-pig serum gave a reaction with
a mixture of sheep and pig blood (dried 1889), also with solutions of the
organs of a pig, dried 18 months. I have noted my observations on
reactions in mixed bloods (1. vii. '01) on p. 140. Uhlenhuth found that
wash-water, containing carbolic acid, sublimate, and soap, gave positive
reactions with anti-human serum when it contained human blood, the
same being the case with blood containing 3 */o borax, and blood-soaked
garden earth after the expiration of 3 months.

The observations of Dr Graham-Smith, in this laboratory, do not
confirm the results of Uhlenhuth's tests in the presence of carbolic acid,
and of soap (see p. 82).

The fact that monkey bloods give similar, though less reactions than
human blood upon the addition of anti-human serum, may not be a
matter of any importance in most countries, where monkeys are not
indigenous. Nevertheless it might happen that a murderous organ-
gi-inder, or perchance a similarly inclined keeper of monkeys, backed by
a well-instructed defence, would claim that suspected blood-stains were
not human but derived from a monkey. On the other hand, Mr Hankin
of Agra informed me, as stated in my paper of 21. xi. '01, of a case in
India which had come to his notice, where it appeared essential to make
a test to determine if certain blood-stains were caused by human or
monkey blood. As I stated at the time, it would be necessary in such
cases to be provided with an antiserum for the most prevalent genera
or species of monkeys belonging to such a region. My tests with anti-

K. 25

386 Medico-Legal

monkey serum (p. 169) have clearly shown that such an antiserum
would give a greater reaction with monkey blood, so that by tests
conducted with both antisera, there should be no especial difficulty in
determining to which primate the blood belongs.

In his papers of later date, Uhlenhuth (11-18 viii. '02 and '02 a)
reports at length upon a large number of tests made for medico-legal
purposes, these proving the value of the method. As it is impossible to
give his results in detail, those especially interested will have to refer
to the original papers. The lists of tests in both papers are the
same, and include 5 tests mentioned in his paper of 25. vii. '01, already
cited, 16 further cases being given. Leaving out of consideration the
5 cases above referred to, he obtained positive results in the following :
(6) Blood stain several years on linen, tested with anti-pig serum, gave
a reaction, not so with anti-sheep, anti-horse, anti-human serum.
Professor Beumer subsequently informed him that the stain was due
to pig blood. (7) Dried blood (1897) acted similarly, also subsequently
.''tated to be pig blood by Beumer. (8) Dried blood mixture (1899).
reacted to both anti-pig and anti-sheep serum, diagnosis recognized as
correct by Beumer, who, as in the other cases, supplied the specimens with-
out letting Uhlenhuth know what they were until after he reported the
result of his tests. (9) Blood-stain on paper found in puddle of blood on a
road, reacted to anti-pig serum, a suspicion of murder being thus removed.
(10) Blood-stains on penknife and handkerchief, medico-legal case,
diagnosis human blood, subsequently confirmed by prisoner, who stabbed
a man with the knife, explaining the spots on the handkerchief as due
to his own nose having bled. (11) Blood -stains on trousers and shirt,
sent from Landgericht Munich and relating to a case of rape, diagnosis
human blood. (12) Shavings from a blood-stained box, same source
as preceding, tests negative with anti-human, anti-sheep, and anti-horse
serum, further tests not made, and subsequently discovered that the
stains were due to roebuck blood. (13) Blood-stained waistcoat and
trousers, the owner being suspected of having killed some sheep, tests
negative with anti-sheep, positive with anti-fowl, and it was subsequently
proved that he had killed a fowl a day before the sheep-killing. (14)
Blood-stained wood-shavings from a floor, sent from Braunschweig in
connection with a murder case, reaction with anti-human serum,
murderer subsequently confirmed this. (1.5) Two samples of blood-
stained cloth sent by Prof. Minovici (Medico-legal Institute, Bucharest),
and 11 other articles, all blood-stained, were correctly diagnosed as sub-
sequently reported by Minovici. (16) Blood-stained coat, tests negative

Tests with Precipitins 387

with anti-human and anti-pig sera, subsequently proved to be roebuck
blood-stains, medico-legal case at Marklissa. (17) Dried blood sent from
Luxemburg, diagnosed human, subsequently proved to come from a
suicide. The blood had been found in front of a house where the suicide
lived, the body having been thrown into the Mosel (whence it was
recovered) by his relatives who wished to keep the fact hidden that the
man had committed suicide. (18) Blood-stains on wool-fragments from
waistcoat and basket for carrying wood, diagnosed as human, confirmed
by evidence in court. (19) Blood-stained trousere, diagnosis fowl blood.
Prisoner suspected of stealing chickens, had claimed the spots to be due
to rabbit blood ; microscopic examination of the stains had however
shown the presence of elliptical corpuscles. Comparative tests made
with other avian bloods (goose, duck) showed the reaction to take place
much more slowly and feebly with these bloods. (I doubt that such tests
to distinguish avian bloods medico-legally can have much value, in view
of my results, see p. 200.) The diagnosis confirmed in the course of the
trial. (20) Three shirts and a handkerchief in connection with a
murder, human blood proved to be spattered on two of the shirts.
(21) Blood-stained trousers, shirt, stockings from a murder case at Strass-
burg Landgericht, diagnosis human blood, the prisoner having claimed
that the blood came from a cow which had knocked off a horn. (22) Blood-
stains on numerous articles of clothing were diagnosed to be human and
from sheep. It was subsequently proved in court that the man had
committed a murder, also that he had slaughtered some sheep two
weeks before the murder.

Uhlenhuth (18. IX. '02, p. 679) does not attach any particular im-
portance to the weaker or stronger reactions which occur in dilutions
of dried blood, depending upon the length of time the blood has been
dried. He makes control tests upon material as far as possible of similar
age. So as to have such material of different ages on hand for this
purpose, he dries sterile bloods of various kinds in Petri dishes, and
stores the dried scales in test-tubes, a method by no means as convenient
nor as compact as mine, where the blood is allowed to dry on filter-paper
which it has saturated (see p. 63).

The generalized reactions obtained with anti-bovine sera with different
bovine bloods, will apparently make it unnecessary in most cases to
prepare special antisera for the blood of each bovine species, for we have
seen that antibovine sera produce marked, in some cases almost equivalent
reactions in the blood dilutions of different species of Bovidae. Uhlenhuth
reached this conclusion, although he has examined but three bovine

25—2

388 Medico-Legal

bloods in this respect (ox, sheep, goat). The very large number of
bloods, both of Bovidae and Cervidae, examined by me and reported
upon in part in earlier papers (see pp. 183 — 192, 252 — 258) show
that either anti-cervine or anti-bovine sera will usually suffice for the
identification of a blood as belonging to the group Pecora. I suspect
therefore that Uhlenhuth's anti-sheep serum used in Case 12 must have
been weak, for I have obtained quite marked reactions with roebuck
blood upon the addition of anti-sheep serum, although the reaction was
naturally less intense than with the sheep blood.

In medico-legal work, Uhlenhuth states that he would proceed to
test the blood dilutions with the different antisera in succession, until
a positive reaction is obtained. I should however warn against adding
different antisera in succession to the same blood sample. The reasons
being that no time limit is observable for the reaction which one or the
other antiserum may give, the first antiserum may give a "mammalian
reaction" which will be attributed to the last antiserum added, or finally,
the increasing serum concentration due to having added several antisera,
will mask the reaction.

I have already drawn attention to the fact that over-powerful anti-
sera may be a source of error (p. 74) in medico-legal work, as is also
stated by Uhlenhuth. Reaction should take place within a few minutes
after antiserum and blood-dilution have been mixed. Uhlenhuth properly
dwells upon the necessity of every antiserum used being provedly effective,
and to insure this he considers, as does also Ziemke, that the preparation
and testing of antisera for medico-legal purposes should be under State
control. The use of weak antisera, which require a period of 24 hours,
or the like, to exert their action should certainly be condemned for
medico-legal work. A great many bloods may react to an antiserum
after such a lapse of time, and there may be bacterial development
(see p. 86). I have not infrequently observed, and this has been con-
firmed by Ziemke, that quite a marked clouding may occur which does
not necessarily lead to a deposit after 24 hours. This result is recorded
in my protocols, no deposits being noted at times under bloods which
gave even marked cloudings. In my short tables which summarise the
contents of those at the end, I found it necessary to give, for instance,
equal value to, say a marked clouding, leading to little or no deposit,
and to a faint clouding leading to a deposit.

In my investigations, of course, the main thing was to see if any
blood-i-elationship could be established, the question of identification
was of secondary importance.

Tests with Precipitins 389

Biondi (1902) obtained reactions with blood stains on rasty knives,
on leather, and on fabrics exposed to the rain. Blood stains washed 15
and 30 minutes at 70° and 80° C. respectively gave no reaction, whereas
they had to be washed some hours at low temperatures to remove all
traces of blood as evidenced by the reaction with antiserum. He found
that strong acid, 5 minutes contact with 5Vo carbolic, and 1 : 1000
sublimate or chloride of lime, caustic potash, soap, and borax destroyed
the reacting substance.

Whittier (18. I. '02) and Wood (24. iv. '02) have used the precipitin
test in medico-legal cases in America with positive result. Okamato
(x. 1902) testing various human blood-stains obtained negative results
in about | of them, all animal bloods giving negative results when
tested by anti-human serum. He found very old and putnd blood
generally to give a negative result. The piipers of several other authors,
bearing indirectly upon this subject, will be found mentioned under the
tests with various haematosera p. 161 et seq.

In some tests which I conducted together with Mr Sanger and which
are mentioned in his Thesis' (23. xii. '02) it was found in several
instances that blood spots on leather, owing to the varying acidity of
leather, at times gave pseudo-reactions, that is, such blood dilutions gave
a clouding upon the addition of any serum. Sanger found that it was
possible to neutralize this acidity and obtain positive specific reactions,
neutralization being effected by the addition of '1 "/o sodium carbonate.
The studies ujion the effect of various agents on blood were continued
by Graham-Smith, and are cited fully on pp. 76 — 86, 390 et seq.

Farnum (28. xii. 1901) obtained antisera by injecting semen intra-
peritoneally into rabbits, or using testicular emulsions, derived from
man, dog, and bull. The injections were made at intervals of 5 — 6 days,
the rabbits receiving 5 — 8 injections of 5 to 10 c.c. at a time. The
antisera were specific, when tested on the semens mentioned. Anti-
human serum produced a reaction with human serum dried as long as
34 days.

Layton (1903, p. 220) obtained positive results with anti-human
serum tested on fresh human blood-stains on cloth and filter-paper, old,
dried stains on cloth, newspapers, filter-paper, putrid blood and blood
soaked in earth. Also with human blood mixed with others as in
Nuttall's earlier experiments. He does not state the age of the blood-
stains he tested.

1 Incorporated in the Paper by Graham-Smith and Sanger, the main results of which
are reprinted in this book, having appeared in the Journal of Hygiene, toI. ni. 1903,

390 Medico-Legal

Finally Austin (12. in. '03) in a paper on "the limitations of the
Uhlenhuth test for the differentiation of human blood" showed that
" other fluids of the human body, like effusions and exudates, were of
little value " in the production of antisera. These facts, however, can
have no bearing on the test in its medico-legal aspect, and but confirm
the observations of others.

Graham-Smith and Sanger (1903, pp. 269 — 272) at my suggestion
examined a number of articles obtained through the courtesy of Mr Henry,
Chief of the Criminal Investigation Department of Scotland Yard. The
objects in question all possessed forensic interest. They consisted of
various weapons and blood-stained fabrics. These authors found that
blood which had been dried many years (see also p. 119 et seq.) could
still be tested by means of precipitins. I quote the following from
their publication which appeared in the Journal of Hygiene.

A. Blood dried on metal.

" The results of experiments on 17 samples covering a period of
30 years, are arranged according to age in the following table. The
number after each specimen refers to the catalogue of the Scotland
Yard museum, and a short description of each is given in the appendix.

The reactions of all were neutral.

The following table refers entirely to weapons which had been
preserved from rusting by the application of oil to the surface of the
metal. This process had caked the blood into black masses, making it
frequently difficult to say whether the mass consisted of blood and oil
or rust and oil. In the majority of cases however it was possible to
make certain of scraping off some blood. The material thus obtained
was extracted with distilled water, and subsequently an equal volume
of 1*2 "/o salt solution added to it. If necessary the solution was
filtered through filter-paper, and tested in the way described.

Excellent results were obtained from these materials, and showed
conclusively that the property of producing a precipitum with its
appropriate antiserum is not lost by blood dried on metal even after
30 years have elapsed.

In one case. No. 11, however, no reaction was obtained ; the negative
result was probably due to little or no blood being present on that part
of the knife which was examined. The condition of the weapon was
such that it was impossible to be certain that the material scraped from
it was blood, but it was thought better to include it in the series.

Tests with Precipitins

391

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392 Medico-Legal

so as to point out the possibility of a mistake occurring under such
circumstances.

It appears that the effect of oil on blood is to lessen the reaction.
This is probably due to the blood being coated with a film of oil, and
therefore not so easily passing into solution.

B. Blood dried on organic materials.

The experiments quoted below have been inserted here to show the
effects of age on blood dried on organic fabrics, but further experiments
(p. 394) indicate some of the fallacies which may arise from the
character of the materials. It happened, however, that in the specimens
chosen few were of such a character as to give rise to possibilities of
error.

The blood-stained materials tabulated below were all obtained from
Scotland Yard, and with them two series of tests were conducted, the
antiserum employed in the second being more powerful than that in
the first.

In the first series very small quantities were employed, but in the
second the amount in each case was slightly greater. It was, however,
not found possible on either occasion to obtain more than very small
fragments, and, moreover, none of the specimens, with the exception of
No. 11, were markedly encrusted with blood. The one exception, a
specimen of hair, 28 years old, was in some parts thickly plastered, and
gave well-marked reactions with each antiserum.

The following table shows that numbers 1, 2, 3, 4, 6, 8, and 11, or
64 °/o of the whole, gave well-marked reactions, their ages varying from
3 to 28 years. In No. 5 the paper was badly burnt and the capacity for
reacting was probably destroyed by the heat. Nos. 7 and 10 produced
alkaline solutions, and in each case the reaction with anti-human serum
was very slight. This was probably due to the retarding influence of
the alkali, which has been discussed on p. 79 et seq. At the time these
experiments were carried out we were not aware of this action of alkalis.
The negative result of No. 9 may have been due to its acidity. No. 12
failed to react, but we were unable to discover any reason for this.
The controls in all cases were negative.

As so little material was available the results may be looked upon
as most satisfactory, for it can scarcely be doubted that more distinct
reactions would have been obtained had it been possible to make more
extensive use of the specimens."

Tests with Precipitins

393

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394 Medico-Legal

Graham-Smith and Sanger (1903, p. 287) further studied the in-
fluence of various materials upon the blood with which they had been in
contact. They reported upon their results as follows :

Experiments upon the detection of blood dried on fabrics.
(See Plate and explanation p. 402.)

"In order to determine to what extent the composition of different
cloths influenced blood which had dried on them we procured a
number of samples. Human blood was dropped upon these so as to
leave some patches unafifected and others saturated. Subsequently the
specimens were allowed to dry under natural conditions and were left
undisturbed at room temperature and in the light for at least 30 days ;
some were not tested for nine months. First a series of control tests
were carried out on unstained pieces of cloth in the following way.
Small pieces 1x2 cms. were soaked overnight in 2 c.c. of distilled water.
In the morning an equal quantity of double normal salt solution was
added and the condition and reaction to litmus of the extract recorded.
The majority of samples was found to be nearly neutral, some were
distinctly alkaline, whilst most of the coarser materials were acid.
About '6 c.c. of each extract, if necessary after filtration, were placed in
small test-tubes and 1 drop of serum added. No cloudings were noticed
except in the markedly acid specimens. After neutralisation with
sodium carbonate these also produced no effect on the serum. Certain
solutions, especially the acid ones, were found to be opalescent, or slightly
cloudy, before the addition of serum, but it was noticed that neutralisa-
tion tended to make these clearer. In all our experiments we have
avoided shaking the extracts, as we frequently observed deposits and
cloudy precipitates at the bottom of the tubes, which in some cases
were very difficult to remove by filtration. After removing the
supernatant fluid in solutions containing blood the tubes were, however,
shaken to ascertain whether sufficient serum was in solution to produce
marked foaming.

In testing for blood, stained patches were treated in the way
described above and neutralised if necessary. Two small tubes of
each solution were prepared. To one was added one drop of anti-human
serum and to the other a drop of anti-ox serum. The results of some of
these experiments are given on the opposite page.

Tests with Precipitins

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396 Medico-Legal

The detection of blood-stains on leathei'^.

Several observations have been made with various samples of
leather, which have been placed under a separate heading to more fully
bring into prominence their peculiarities. It was found that nearly all
gave acid reactions on solution. The degree of acidity, however, varied
greatly, chamois leather being alkaline, suede kid glove only slightly
acid, and the coarser leathers very decidedly acid. The addition of
a drop of serum to the acid solutions produced clouding, and even
coagulation with extracts of the coarser leathers. The latter also gave
rise, especially if shaken, to bulky deposits in the original solutions.

Nearly all the solutions of leather could be neutralised and the
blood test satisfactorily employed. One class of leather was, however,
a marked exception, namely, thick polished yellow leather. Solutions
of this gave rise to extremely acid yellow fluids, whose colour deepened
on the addition of alkali. It was found impossible to obtain the
specific test for blood dried on it. At first it was thought possible that
the blood was destroyed by the acid after solution, and extracts were
made in alkaline salt solution to neutralise this effect. Even under
these conditions no positive results could be obtained. Up to the
present although many meth(jds have been tried we have been unable
to devise one which gives satisfactory results, and are forced to conclude
that the mode of preparation of such leathers produces conditions which
destroy the blood in contact with them. Under favourable conditions,
when blood has been thickly deposited on the surface, it might, however,
be possible to scrape it off and obtain a positive reaction. In the
following table all solutions when necessary were neutralised, and
filtered, before the addition of anti-human serum.

A series of experiments was also made to determine the effects of
boot-blacking and polish. Blood-stains blackened over were hard to
detect on the boot, but by neutralisation and filtration clear solutions
could be obtained, and yielded well-marked reactions. Polish also
made no difference to the test.

Experiments with saline solutions of tannin show that it has a very
deleterious action on serum, rendering the application of the test when
it is present in large quantities impossible. Solutions of 1 in 20 to

1 See also p. 79 et seq. regarding tlie effects of acids on bloods, and Plate and
explanation on p. 402.

Tests with Precipitins

397

1 in 500 produce instant coagulation of the serum, and 1 in 1000
produces marked clouding.

Con-
dition

Colour

Ee-
action

Anti-ox

Anti-human .

Material

Unneutrallsed

NeutmUnd

Ifimina

34 houn

IS mina.

34hra.

IS mina.

Mhoun

Chamois leather

clear

clear

neutral

medium

medium

Su^de kid glove

cloudy

slightly

_

_

reaction
good

deposit
large

White „ „

clear

yellowish

acid
acid

slight
cloud

Blight
cloud

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II

deposit
11

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Patent leather

Yellow leather

If

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lation

deposit

cloud

oload

cloud

cloud

Detection of blood on materials not previously mentioned.

Ten examples of wall paper of various textures and colours, red,
brown, yellow, blue, and green, were tested and gave typical reactions.
All produced neutral solutions, some of which were tinted.

Extracts of blood dried on various kinds of paper, stones, flint, slate,
coal, cork, string, straw, rubber, linoleum, as well as silver and copper
coins, yielded satisfiictory results.

Although one piece of oak on which blood had been thickly
incrusted gave a marked reaction with anti-human serum, we failed to
obtain any reaction with blood on two blocks of cedar and pine. The
quantity present on each of these was exceedingly small, and the negative
result was probably due to this cause.

These experiments demonstrate that many substances in common
use give acid solutions. In most instances the acidity is not so marked
as to be of importance, but in some, unless recognised and neutralised
might be liable to lead to grave error. Extracts of certain substances
are sufficiently alkaline to impede the reaction.

398

Medico-Legal

The detection of blood in the presence of lime, mortar, and earth.

The wide distribution of these substances rendered it necessary to
investigate their action on blood, since in medico-legal practice it might
often be necessary to test blood dried on, or mixed with, these materials.

Solutions of earthy salts, mortar, and lime of various strengths
were made in salt solution and tested qualitatively with various
antisera to determine their action on serum. These actions vary to
some extent with the quantity of serum added. In the following table
the quantity added was one drop, since this was the unit chosen for
qualitative experiments.

Dilutions

Saturated jSOmins.
solution* (24 hrs.

1 : 10 I

1 : 100 I

1 : 1000 I
1 : 10,000

o

s o

Si

OX!

I

it

JPh 3

a. <

1 :25
+
D

* Where saturated solutions are mentioned the dihitions are 1
signs used here are described on p. 338.

II

10 etc. of tliese. The

The addition of serum to strong solutions of lime resulted in a
general clouding, which later gave place to a dense cloud below, which
would be hard to distinguish from a positive reaction. Mortar gave rise
to a similar but smaller clouding. Calcium chloride and sodium and
calcium phosphates caused cloudings in very strong solutions only.
The actions of caustic soda and potash in certain solutions are very
marked, and are referred to on pp. 83, 85. They are briefly mentioned
here owing to their presence in earth.

At this point it should also be noted that strong lime and calcium
solutions give rise on standing, even after filtration, to deposits of the
salt at the bottom of the tube and a filmy layer on the surface.

It was found, however, that the difficulty in testing due to the
presence of lime in mortar, plaster, and earth, could generally be

Tests with Precipitins 399

eliminated in the following way. Solutions of all of the above
substances were allowed to stand till the excess had settled to the
bottom. The supernatant fluid was then pipetted off and filtered.
Carbon dioxide giis generated by the action of dilute hydrochloric acid
on chalk, and washed by passing through distilled water, was next
passed through the fluid and the latter again filtered to free it fix)m the
presence of the precipitated calcium carbonate. By this procedure
clear filtrates could be obtained in most cases, which remained so for
an indefinite period, and produced no cloudings on the addition of sera.
The following formula explains the reaction :

Ca (OH), + CO, = Ca CO, + H,0.

Too much of the gas must not however be passed into the solution
owing to the fact that excess of CO, rendere the insoluble carbonate
again soluble :

Ca CO, + H,0 + CO, = Ca H, (CO,),.

By quantitative experiments it was found that though this process
caused a deposition of blood pigment from blood solutions, yet the
property of producing precipitation on the addition of appropriate
antisera was not in any way affected.

The action of dry and wet lime, etc.

Lime was intimately mixed with human blood and then spread on
porcelain and exposed to the action of air for three months. The
resulting compound turned a greenish colour. Solutions of this gave
an immediate clouding on the addition of serum. After the passage of
C0„, however, and subsequent filtration, no reaction could be obtained
with anti-human or other serum. Under these conditions it seems that
unslaked lime completely destroys the reacting power of blood in
contact with it.

Quantitative experiments over a shorter period bring out the
destructive quality of lime and mortar very markedly. To ascertain
the action on semm of dry and wet lime, mortar, brick, earth, etc.
weighed quantities of one gramme of each were mixed with 1 c.c. of
human serum and allowed to act for 4 days. Similar mixtures but with
10 c.c. of water added were also prepared and allowed to stand for 4
days, to determine whether any different action was excited by these
materials in the presence of water. At the end of this period all were
made up to 1 : 21, by the addition in the former case of 20 c.c. of
normal, and in the latter of 10 c.c. of double normal siilt solution.

400

Medico- Legal

After applying the method of removing lime which has just been
described quantitative estimations (Method, p. 315) were made.

Anti-

Anti-

Material

human

Anti-ox

»/o

Material

human

Anti-ox

%

1.

Control

■0403

—

100

6.

Earth

idry

•0244

tr

61

2^

Chalk

Idry

■0367

tr

91

(wet

•0291

tr

72

(wet

•0357

tr

89

7.

White

i^

3.

RpH brick

\dry

•0281

tr

70

brick

dry

•0262

tr

65

4.

Pasteur

jwet
jdry

•0347
•0309

tr
tr

86

77

8.

Mortar

jdry
(wet

tr
•0028

tr
tr

0

7

filter

jwet

•0319

tr

79

9.

Lime

dry

tr

tr

0

5.

Berkefeld
filter

Idry
(wet

•0291
•0291

tr
tr

72
72

wet

tr

tr

0

The above table shows that all the materials used in this series
produced slight effects on the serum, but that mortar and lime
completely destroyed its power of reacting. All had been ground up
very finely before the addition of the serum.

The effects of the lime present in ordiwiry earths.

The next point of importance was to determine whether the amount
of lime present in ordinary earths was sufficient to interfere to any
serious extent with the reaction. For this purpose 9 samples of
analysed earth were obtained, five from a field of gravelly land near
Trowse, divided into five plots, and the others from different localities.
The results of the analyses of these earths dried at 100° C. are given in
the following table, arranged according to the percentage of lime
present. We are indebted to Mr T. B. Wood for these analysed earths.

I.

Trowse,
plotl

II.

Trowse.

plot 2

111.

Trowse,
plots

IV.

Trowse,
plot 4

V.

Wryde

clay

VI.

Need ham

salt

VII.
Trowse,
plots

VIII.

Bentwick

fen

IX.

Uttleport

fen

Total lime

Organic matter, loss )
by ignition J

•91
a •62

1^01
516

143
5^27

1^46
6 -52

1^48

14^48

1^57
5-40

1-97
6-31

2 95
39^35

4^39
50^82

Calcium carbonate
Total phospboric acid
Total potash
Total nitrogen

116
■16
•13
•15

1-39
■18
•14
•11

1^94
•16
•13
•14

1'98
•18
■14
•53

•37

132

•24

•19
•31
•24

3 06
•20
•13
•13

•30

•63

142

•28
•56
1^8

Strong solutions of the above after simple filtration were at first
clear but showed a white filmy deposit after standing. The quantity of
this increased with the percentage of the lime present. On the
addition of anti-human serum a thin cloud spread through the entire

Testa with Precipitins 401

solution and gradually deepened, being considerably denser in No. IX
than in No. I.

After the passage of COj and filtration every solution was clear, with
the exception of V and VIII, and produced no deposit on standing.
The two mentioned were opalescent. No clouding occurred on the
addition of anti-human serum.

Two sets of quantitative experiments were carried out with these
soils. In the first 1 c.c. of finely divided soil was placed in a test-tube
with 1 c.c. of human pleuritic exudate and 5 c.c. of water. After 4 days
5 c.c. of double normal salt solution were added to each, making a dilution
of 1 in 11 of pleuritic exudate. In the second series 1 c.c. of dry earth
was allowed to act for 4 days on 1 c.c. of pleuritic exudate. At the
end of this period the specimens were diluted to 1 in 11 with salt
solution.

These solutions were treated with COj as described, and the
precipita measured quantitatively.

Earth solution

Percentage of lime

Quantity of predpitum,

mean of tlie two

observations

Percentage

Control anti-ox

No. I.

•91

•0404

100

—

No. 11.

1-01

•0394

97^5

—

No. m.

1-48

•0319

78 •g

—

No. IV.

1-46

•0389

962

—

No. V.

1-48

•0241

59^6

—

No. VI.

1-67

•0258

63 3

—

No. VII.

1-97

•0389

96-2

—

No. vm.

a -95

•0314

11-1

—

No. IX.

4-89

•0383

94^8

—

The above table shows that the quantity of precipitum obtained did
not decrease in proportion to the increase of the lime, which was
apparently never present in sufficient quantity to materially affect the
reaction. Excess of potash probably accounts for the low figures
obtained in No. V, and possibly No. VIII (p. 83). In neither of these
could a clear solution be obtained. Whatever may be the cause of the
variations in the quantity of precipitum obtained, these experiments go
to show that blood mixed with ordinary earth can be readily detected
if present in sufficient quantity and that its specific character remains
unaltered.

From our experiments on earth and lime salts we have drawn the
following conclusions: (1) that the intimate mixture of lime with blood
completely destroys the latter; (2) that a clouding occurs in the

26

M.

402 Medico-Legal

solution of earth on the addition of serum ; (3) that this is due
principally to the presence of lime salts ; (4) that the lime can be got
rid of and the solution rendered clear, and not liable to clouding, by the
passage of CO2 and subsequent filtration ; (5) that the passage of COj
in no way interferes with the reaction ; (6) that the quantity of lime
present in ordinary earth does not materially affect blood mixed
with it.

EXPLANATION OF PLATE
(Graham-Smith and Sanger).

Fig. 1. No. 1 shows the precipitum with normal human serum (1 : 21 in salt solution)
and anti-huuian serum (•! c.c). No. 2 with putrid human serum (1 : 21) and antiserum,
and No. 3 with normal human serum (1 : 21) and putrid anti-human serum. No. 4
shows a clear solution of human serum in salt solution (1 : 21). No. 5 shows the
deposit resulting from the solution of human serum in distilled water (1 ; 21).
No. 6 the precipitum formed with human serum diluted with distilled water (1 : 21)
and anti-human serum. Nos. 7, 8 and 9 show three capillary tubes such as are
used in quantitative measurements, and containing precipitum.
Fig. 2 Shows effects of increasing quantities of NaCl on the formation of precipitum;
each tube contains '5 c.c. of a 1 in 21 dilution of human serum, and 'Ic.c. of
anti-human serum. No. 1 contains -G"/,, of salt, and those following 1 %, 2°/o, ^"lo,
^"Itx ^°lo> 10%, 16%, 18%, and No. 11 is saturated with salt. Results of
measurements are given on p. 103.
Fig. 8 Shows the specific precipitum in tests for human blood dried for a month on
various materials. The lower series shows controls with anti-ox serum. The
cloudings in the tubes are due to the opalescence of the solutions ; and the various
solid particles are portions of undescended precipitum.

No. 1 test for blood dried on silk handkerchief, No. 2 on tweed cloth, No. 3 on
black dress fabric, No. 4 on dark green cloth, No. 5 on coarse green cloth, No. 6 on
coarse red cloth. No. 7 on kid glove, No. 8 on blanket, No. 9 very coarse sack material.
No. 10 on flannel. Nos. 11 — 20 show control tests with anti-ox serum : all negative.

The solutions whenever necessary were neutralised before testing.
Fig, 4. Nos. 1 to 0 show the effects on serum of dilutions of Hydrochloric acid in salt
solutions of strengths of 1 : 10, 1 : 100, 1 : 1000, 1 : 10,000 and 1 : 100,000. No. 1 has
a dense white cloud. No. 2 a slight cloud at the bottom, No. 3 a marked cloud, and
the others are unaffected. Photographed after 6 hours.

Nos. 6 — 10 similarly illustrate the action of Tartaric acid, No. 1 (1 : 10) having
a slight cloud. No. 2 (1 : 100) a medium cloud. No. 3 (1 : 1000) a marked cloud,
Nos. 4 and 5 (1 : 10,000 and 1 : 100,000) are unaffected, the apparent deposit being
due to the light.

Nos. 11 — 15 illustrate the action of Nitric acid. No. 1 (1 : 10) shows the
coagulum, No. 2 (1 : 100) a very faint cloud. No. 3 (1 : 1000) a medium cloud, and
Nos. 4 and 5 are unaffected.

Nos. 16 — 20 show the effects of Acetic acid. Nos. 1 and 2 (1 : 10 and 1 : 100)
have slight clouds, No. 3 (1 : 1000) a medium cloud, and No. 4 (1 : 10,000) a marked
cloud. No. 5 (1 : 100,000) is not affected.

BLOOD IMMUNITY

12 3 4 5 6 7

Fig. I.

23 456 78 9 10 11

Fig. 2.

123 4567 89 10

11 12 13 14 15 16 17 18 19 20
Fig. 3-

PLATE I

12 3 4

7 8 9 10

12 34 56 78 9 10 11

{Reprinted from Journal of Hygiene, Vol. III. So 2)

Testa with Precipitins 403

Fig. 5 Illustrates the action of acids and alkalis on the formation of the specific
precipitum. All the tubes contain -Sec. of human serum dilution (1 : 21) in salt
solution. Noa. 1— S contain 5 to 1 drops of 1 in 10 Hydrochloric acid. No
precipitates have been formed. No. 6 did not receive any acid or alkali. Nos. 7—11
contain 1—5 drops of 1 in 10 sodium carbouate solution ; the quantity of precipitum
shows a decrease along the series. Nos. 12—22 have been similarly treated but
received drops of 1 in 100 acid and alkali respectively. The precipitum is seen to
increase from 12 to 16 and decrease from 18 to 22. The slight clouding above the
precipitum in each case is due to bacterial growth, the tubes having stood 48 hours.

We are indebted to Walter Mitchell, our laboratory attendant, for the
time and attention he has bestowed on the photographing of these specimens."

Graham-Smith and Sanger (p. 260), as the result of their investi-
gations, reach the following conclusion, which I can but endorse :

" These experiments have led us to the conclusion that with sufficient
materials, and due precaution to exclude the various sources of error,
there are but few conditions met with in forensic practice under which
human could not be readily differentiated from other bloods. By this,
however, we do not mean to imply that a considerable acquaintance
with the action of precipitating antisera on blood solutions is not
necessary in the successful application of this test."

That this conclusion is fully justified is proved by the official
recognition of the precipitin method in forensic practice by foreign
Governments. Prof Uhlenhuth (19. IX. '03) informs me that the
method has been recommended by the Ministers of Justice in Germany
and Austria, and that it has been officially recognized by the Govern-
ments of Egypt and Roumania.

I herewith append a copy of the Order issued by the German
Authorities, the same having appeared in the Medicinalhlatt fur Medi-
ciner und medicinische Unteri-ichtsangelegenheiten No. 17 (1. X. '03): —

Berlin, W. 64,

den 8. Sept. 1903.

WlLHELMSTRASSK 65.

Der Minuter de» geistlichen Vnterrichts
und MediciiMlangelegenheiten.
U. I, No. 12328 M.
Der Juttuminister,
I. No. I, 6283.

Von dem Stabsarzte Professor Dr. Uhlenhuth in Greifswald ist eine
Methode der Blutuntersuchung vermittelt wordeti welche es ermoglicht, die Art
des zu untersuchenden Blutes festzu.stellen und namentlich Menschenblut mit
Sicherheit von Thierblut zu unterschelden. Bei dor Behaudlung des zu unter-

404 Identification of Meats

suchenden Blutes mit Serum aus deni Blute von Kaninchen, denen zuvor Blut
anderer Thiere oder Menschenhlut eingespritzt war, ergeben sich bestimmte
Erscheinungen wenn das zu untersucheude Blut von deraelben Art ist wie das zuvor
den Kaninchen eingespritzte. Es kann desshalb jede Art von Blut, wenn das
entsprecheude Serum angewendet wird, bestimmt werden. Die wissenschaftliche
Deputation fiir das Medicinalwesen hier hat sich iiber den Werth der Methode mit
Hervorhebung von deren grossen Bodeutung vi'ie folgt geaussert :

" Die Erfahrungen iiber die Serummethode der Blutuntersuchung sind bereits in
Deutschland wie im Auslande so ausgedehnte. die Resultate der Forschungen
in weseutliohen so iibereinstimmende, dass kein Zweifel mehr dariiber bestehen
kann, das diese neue biologische Methode in der Mehrzahl der Fiille mit grosser
Sicherheit gestattet, frisches sowie angetrocknetes Blut nach seiner Herkunft zu
bestimmen, Menschenblut von Thierblut, Blut vorschiedener Tierarten zu unter-
scheiden. Es ist dalier dringend geboten, diese vortreffliche Methode, welche
natUrlich die alten bowahrten Methoden des Blutnachweisses nicht verdrangen
sondem nur erganzen u. vervollstandigen soil, fur die gerichtliche Praxis allgemein
nutzbar zu machen."

Als Institute, bei deuen diese Methode seit liingerer Zeit zur Anwenduug
gelangt, werden bezeichnet :

Das hygienische Institut der Univcrsitiit Greifswald.

Das Institut fiir Infektionskrankheiten in Berlin, Nordufer 39.

Das Institut fiir Staatsarzneikunde in Berlin.

Das Institut fiir e.xperimeutelle Therapie in Frankfurt a/M.

Diese Institute werden in erster Linie fiir die Vornahme von Untersuchungen
der in Rede stehenden Art empfohlen.

Indem ich auf diese Methode der Blutuntersuchung aufraerksam mache,
empfehle ich, in alien geeigneten Fallen die Untersuchungen nach ihr ausfiihren zu
lassen.

Abdriicke dieser Verfiigung sind zur weiteren Mitteilung an die Landgerichts-
Prasidenten und die Ersten Staatsanwiilte des dortigen Bezirks beigefiigt.

Im Auftrage,

gez. ViTSCH.
An die Herreu Vorstandsbeamten des Kammergerichts
sowie der sammtlichen Oberlandesgerichte.

2. Antisera in the Examination of Meats.

From previous observations it seemed natural to conclude that meat
extracts would react to corresponding haematosera, for the reason that
they contain blood. As was noted on page 385 Uhlenhuth (1901)
obtained a positive reaction with anti-pig serum, tested upon the organs
of a pig which had been dried for 18 months. Continuing this line of
investigation, he obtained positive results with the antisera for pig,
sheep, horse, donkey, and cat blood, when these were tested upon the
corresponding meats. He found (7. xi. '01) that anti-sheep serum gave

Tests with Precipitins 405

almost as much reaction with goat as with sheep, and less reaction with
beef extract. The method is of no especial use in the examination of
large pieces of meat, for the reason that these can be readily recognized.
Tokishige has informed Uhlenhuth that meat is usually sold in small
pieces in Japan. Minced meat is sold in most countries. Admixtures
of horse, dog, and cat meat can be detected by the precipitin method in
minced meat, in sausage, and smoked meat, these of course not having
been subjected to cooking. He obtained reactions with pig and horse
hams which had been smoked the year before, also with horse siiusage
(" Pferdemettwurst ").

The method of examination consists in scraping the meat and
extracting it with water or saline. It takes a long time to extract the
meat in some cases. An extract is suitable for testing when it foams
on being shaken. Meat can be more rapidly extracted by adding a
small amount of chloroform, extraction being then usually effected in a
few minutes. The extract is very cloudy, and has to be cleared by
repeated filtration through filter-paper or a Berkefeld filter. If extracted
with water, an equal volume of double normal salt solution has to be
added to the watery extract before testing this. In testing add
10 — 15 drops of antiserum to 3 c.c. of the saline meat extract.

Von Rigler (1902) has used this method in the study of meat-
adulteration. He prepared 20"/o watery extracts of the meat of 7
species of animals (roebuck, hare, rabbit, horse, ox, pig, cat) and
injected 5 — 10 c.c. thereof every 3 days into rabbits subcutaneously,
during one month. Whereas normal rabbit serum had no effect on the
meat extracts, the antisera obtained from the treated rabbits were
specific, acting on extracts of mixed meats, as also upon some boiled
and roasted meats of an homologous kind.

Notel (13. III. '02) treated rabbits with horse serum, muscle juice
and muscle extract (in '1 "/o soda solution) injecting subcutaneously
every 2 to 3 days, amounts of 10 c.c, until 10 to 12 doses had been
administered, after which 6 days were allowed to elapse before the
rabbits were bled. He obtained the least effective antisera from the
serum-treated rabbits *.

To obtain clear solutions of muscle, he found it best to extract by
means of 1 % soda solution, without disturbing the meat mechanically.
He found roasted meat, imderdone in the centre, as also cold-smoked
meat, to give positive reactions. Donkey meat reacted like that of the

' Vailed and Nicolas (30. vi. '03) have recently confirmed these observationB, Thev
speak of sero-precipitins in contradistinction to muBculo.precipitins.

406 Identification of Bones, etc.

horse. He noted that sausage extract alone, when placed at 40° C. for
5 minutes, often becomes clouded in a manner which might lead to
mistakes, so it is necessary to use controls. It has not occurred to this
author that the soda solution might be responsible for the clouding
noted.

3. Antisera in the Examination of Bones.

Beumer (1902) has applied the precipitin method to the medico-
legal examination of bones, in a case where bone fragments only 1 to 6 cm.
long were found in a house which had been destroyed by fire, and it was
suspected by the authorities that they might be human. Because of
their small size, the fragments could not be determined. The bones
were somewhat charred on the outside, remains of soft tissues adhering
thereto. After removal of the latter, the bone fragments were placed
in saline, where they were allowed to remain 4 days, the fluid foaming
then on being shaken. The fluid was now filtered through a Berkefeld
filter, and the clear solution thus obtained was tested with anti-human,
anti-pig and anti-ox sera, reacting only with anti-ox serum. The con-
clusion was therefore obvious. Fresh bones were readily determined by
the method, best when the marrow or spongy portions were extracted,
more slowly and less surely when cortical substance was used. The
addition of chloroform facilitated extraction. Cortical substance was
best extracted by sawing the bone through and extracting the bone-
dust thus obtained. Beumer found bones which had been exposed
for several weeks to the air, to be still determinable by the precipitin
method. Boiled bones gave a negative result, as did also roasted or
incinerated bones.

Schiitze (22. I. '03) has made similar experiments, with regard to
the identification of bone fragments by means of antisera. He placed
the fragments in 0'857o saline to which "25 "/o soda solution had been
added, thus obtaining an extract which gave reactions with an homo-
logous antiserum.

4. Antisera in the Examination of Commercial albuminous Prepara-
tions containing egg-white, and in the examination of Honey for
adulteration.

Uhlenhuth (15. xi. 1900) found that the antiserum for the egg-white
of the fowl constantly gave negative results with commercial albuminous
preparations which did not contain egg-white, the contrary being the

Conclusions 407

case where they contained egg-white. The use of such antisera is there-
fore suggested where it is desired to prove the presence of egg-white in
prepared foods.

von Rigler (1902) treated rabbits with honey, in the manner em-
ployed when they are immunified with blood. The anti-honey serum
only produced precipitation in dilutions of honey, not in those of grape-
or cane-sugar. Normal rabbit serum had no such effect.

5. Antisera in the Study of Urine.

In the part referring to precipitins and precipitable substances
in corpore, I referred to observations on the urine (see p. 133), which I
shall not recapitulate. It is obvious that the precipitins may be put
to use in the study of assimilation. I would state here that M. Ascoli
(11. III. '02) succeeded by means of antiserum for egg-white, in
demonstrating the existence of apparently unaltered egg-white in the
urine of persons showing albuminuria in consequence of excessive al-
buminous diet. Linossier and Lemoine (18. iv. '02) made observations
on a young man suffering from orthostatic albuminuria, evidently due
to malassimilation, the albumen being derived from non-assimilated
food. On giving him cow's milk, traces thereof were found in his urine
by means of lactoserum for cow's milk.

Conclusions.

In view of the mass of material treated of in this book, it is difficult
to draw any detailed conclusions. It has been shown that there are
many points of resemblance between the different antibodies. The
little work which has been done with the haemolysins bears directly upon
the immediate subject of this book, namely, the blood-relationship
amongst animals, but it is scarcely to be expected, owing to technical
difficulties, that the haemolysins will be of such general use in the study
of the problem.

With regard to the precipitins, it is evident that more scientific
methods of treating animals for the production of antisera are called for.
Powerful antisera may, however, be produced by intravenous injections
of much smaller quantities of serum than have hitherto been used. Care
should be exercised with regard to the addition of preser\'atives to anti-
sera, and in the use of solvents other than salt solution for the extraction
of dried bloods.

408 Conchisions

The precipitins and precipitable substances combine quantitatively.
The precipitum is soluble in an excess of precipitable substance.
Heated antisera cannot be reactivated. The precipitins constitute
receptors of the second order (Ehrlich). There is no evidence that the
action of precipitins is fermentative. Heated haematoserum (precipitoid)
combines with precipitin, and prevents its action upon precipitable
substance. There is evidence of the existence of immune-bodies in
precipitating antisera. There is no evidence that antisera differ in
their ordinary properties from normal sera. The precipitins and pre-
cipitable substances are intimately bound up with the globidins in
serum, and have not been separated therefrom. The precipitins do not
give reactions corresponding to the amount of albumin present in a
solution of precipitable substance ; the albumin, to be acted upon, must
possess certain specific properties. The presence of even small quantities
of acids or alkalis, markedly reduces the amount of precipitum formed,
but an increase of salt (NaCl) has little effect. The supernatant fluid
in a mixture of antiserum and precipitable substance, after precipitation
has taken place, may contain an excess of both interacting bodies.
There is evidence that the precipitins may exert a special, but not a
specific action on different albumins from the same species of animal.
It is doubtful if the precipitins and precipitable substance can withstand
tryptic digestion, whereas both are certainly destroyed by peptic
digestion. The weight of evidence is against the possibility of preci-
pitins being fonned for peptones'.

The rate at which interaction takes place between precipitins and
precipitable bodies is markedly influenced by temperature, being re-
tarded at low temperatures, hastened at higher temperatures. The
quantity of precipitum formed is not influenced by the temperature
(5 — 37° C.) at which the experiment is made. Undiluted haematosera
are but slightly affected by exposure to a temperature of 63° C, ; they
are inactivated at 68 — 70°, unaffected at 60° and under. (Bacterio-
precipitins are inactivated at 58 — 60° C.) Undiluted normal sera seem
to be rendered non-precipitable at a somewhat lower temperature than
that which inactivates haematosera. Both interacting bodies resist
desiccation. The precipitins are apparently more unstable than the
precipitable substances, which may give reactions even when dried
many years. Fluid sera, preserved in vitro, may give reactions after
being stored 4 or more years ; they appear to deteriorate slightly by

> See Appendix, note 2.

Conclusiotis 409

keeping. Fluid antisera, sealed in bulbs and kept cold and in the dark,
may at times give good reactions after 6 to 14 months. Putrefaction of
serum, or antiserum, does not affect the production of a specific
precipitum.

There is evidence that the precipitin-content of the serum in corpore
undergoes fluctuations during immunization, corresponding to those
observed in animals under toxin-treatment. The precipitins begin to
disappear about 1 month after treatment has ceased. The precipitins
disappear from the blood of animals which have undergone prolonged
treatment, the animals having become immune. The precipitins may
■ be present in the humor aqueus, and are transmitted to the offspring in
utero, whilst they are absent from the urine. The precipitin and
precipitable substance may co-exist in corpore, no precipitation ap-
parently occurring in the body. The presence of foreign precipitable
substance in an animal's serum may be readily demonstrated by means
of an antiserum for the foreign substance. No explanation can as
yet be given of the marked leucocytosis after injection of a foreign
albumin into an animal which has been rendered more or less immune
to the albumin. The seat of origin of the precipitins is unknown.

The more powerful an antiserum is, the greater is its sphere of action
on other bloods. The degree and rate of blood reaction appear to offer
an index of the degree of blood-relationship ; in other words, closely
related bloods react more powerfully (more precipitum) and more
rapidly than do distantly related bloods, provided the latter react at all.
The interaction of a precipitin with its homologous blood dilution is not
impeded through non-homologous bloods of various kinds being present
in the blood-dilution in mixture. The amount of reaction may be
expressed in terms of the volumetric measurement of the precipitum
produced by mixing known quantities of the interacting substances.
An antiserum acts upon a higher dilution of homologous than of non-
homologous substance. The strength of an antiserum may be expressed
cither in terms of precipitum-volume, or, by giving the highest dilution
of blood with which it reacts, the quantities of the interacting substances
being stated. The amount of reaction may be affected by altered blood-
concentration in diseiise, possibly also by the variation in the alkalinity
of the blood in disease, or even in health. There is evidence of iso-
precipitins, autoprecipitins, and antiprecipitins being artificially formed
in the bodies of treated animals. Certain normal sera may contain
precipitins, but these do not possess a specific character.

The bacterio-precipitins appear to be distinct from the others

410 Conclusions

hitherto studied. In addition to these, various authors have obtained
precipitins for the albumin of yeasts, of higher plants (I have grouped
these together as "Phytoprecipitins" in contradistinction to the following
" Zooprecipitins "), for the casein of different milks (lactosera), also for
different bloods, etc.

To avoid needless repetition, I will refer the reader to pages 156 to
160, where the results of other authors with lactosera are given. The
results of tests with different precipitating antisera are summarized on
pp. 214, 335, 353, 359, 361, 403. The general bearing of the investiga-
tion from a zoological point of view is considered in the Introduction.

In Part II, Section ix., pp. 381 — 407, evidence of the value of the
precipitin reaction in legal-medicine, based upon the work of various
authors, is given.

In conclusion I would add that this investigation must necessarily
be regarded as preliminary in character. The exhaustive treatment
which our present knowledge of the precipitins has received, should
prove of use to others, and I hope that the work done will stimulate
many to further investigate the many problems which present them-
selves. Like other lines of investigation, this one appeared relatively
simple at first; it is evident however now that the phenomena of
precipitation are of an exceedingly complex nature.

Acknowledgments 411

Acknowledgments .

The extensive collection of bloods whose examination forms the
main subject of this book, was only rendered possible through the
generous aid of some seventy gentlemen throughout the world. I take
this occasion to thank them very cordially for the friendly assistjince
they have given me in my work. Of the 900 blood samples collected,
nearly 200 were brought together by mo personally, some with the aid
of our laboratory attendants, 6 by purchase from the Zoological
Laboratory at Plymouth ; a few have been contributed by Dr iKtuis
Cobbett and Dr Graham-Smith of Cambridge.

My work has been especially furthered by Mr Frank E. Beddard, F.R.S.,
Prosector of the Zoological Society's Gardens, London, under whose
direction 224 blood-samples were collected in the course of about two
years by his assistant Mr E. Ockenden. Mr Ockenden has been most
painstaking and exact in his attention to my directions with regard to
the methods of collecting bloods. It is needless to say that I have
obtained an invaluable material from this source.

Especial thanks are also due to the Hon. N. Charles Rothschild,
who personally collected 89 samples of blood in Great Britain, Ceylon
and Japan, besides stimulating others to supply me with material.
Thus Messrs Brazenor Brothers ("B" in the tables), Naturalists at
Brighton, have kindly supplied me with 69 specimens, Mr W. J. Clarke,
Naturalist at Scarborough, with 19 specimens, a few being also supplied
by Mr Head, Naturalist at Scarborough. Mr Rothschild also put me
in communication with a number of gentlemen in different parts of
the world.

Mr H. M. Phipson, Honorary Secretary of the Bombay Natural
History Society, sent me 11 specimens which he had collected. He
helped me considerably by circularizing the members of that Society
with copies of my letter containing directions for the collection of blood
specimens, a tiisk which was continued by Mr W. S. Millard, who
assumed his self-imposed duties during Mr Phipson's absence from
India. Of the members of this Society, I am indebted to Mr C. H.
Donald, of Bhadarwa, Kashmir State, for 29 specimens; Mr Charles
M. Inglis, of Tirhut, for 21 specimens ; Mr J. Mason, Curator of the
Society, for 8 specimens; Captain H. T. Fulton, of Chitral, for 2;

412 Achiotvledgnients

and for one specimen each to Major R. M. Betham, of Akalkote,
Deccan; MrC. Fisher, of Chatrapur; Major G. A. Leslie, of Chitral; Mr
A. H. A. Simcox, of Akrani, Khandesh ; Captain E. H. Sweet, of Chitral.
In addition, M. Wm. Foster, of Sapucay, Paraguay, supplied me
with over 26 specimens, a large number of them from Chiroptera ;
Herr Kuse, Gamekeeper at Kittendorf in Mecklenburg-Schwerin, sent
20 specimens ; Dr John P. Tulloch, of Cronstadt, Trinidad, sent 18
specimens ; Prof. E. A. Goeldi, Director of the State Museum of Natural
History and Ethnography, at Parfl, Brazil, together with his colleague
Dr G. Hagmann of the Zoological Gardens at Pari, sent 20 specimens,
many from Edentata; Rev. M. C. H. Bird, of Stalham, Norfolk, sent 1.5;
Dr Max Lliho, Privatdocent in Zoology, at Konigsberg in Prussia, sent 14 ;
Dr Gerald Leighton, of Pontrilas, Hereford, sent 11 (and over, chiefly
Reptilian); Mr Farren, Naturalist at Cambridge, supplied me with 11;
Mr F. B. Parkinson, F.R.G.S., etc., of Baviaankrantz, Cape Colony,
sent 9 (a number of unique specimens) ; Dr H. B. Dodds, Medical
Officer at Fort Johnson, British Central Africa, sent 8; Dr G. Langmann,
of New York City, sent 8 (at the kind suggestion of Prof T. Mitchell
Prudden); Surgeon-Major Leonard Rogers, I.M.S., of Calcutta, sent 8 ;
Dr N. P. Schierbeck, Privatdocent in Hygiene at Copenhagen, sent 8
(several from the Zoological Gardens there); Count Carl Otto von
Schlieffen-Schwandt, of Mecklenburg-Schwerin, sent 7 (some collected
by his gamekeeper); Prof R. P. Bigelow, of the Massachusetts Institute
of Technology, U.S.A., sent 7 (mostly from fish, collected by Mr C. F.
Silvester); Dr Charles Hose, of Baram, Sarawak, sent 6 ; Kammerherr
G. von Oertzen, of Mirow in Mecklenburg-Schwerin, sent 5. Five
specimens each were sent by Prof F. M. Sandwith, of Cairo, Egypt
(collected by Mr Littlewood, Chief Veterinary Inspector to the Egyptian
Government); Dr J. R. Garrood, of Alconbury, Hunts., and his friend
Mr W. F. Beauford; Dr A. S. Grlinbaum, of University College, Liverpool
(Simian bloods); Dr A. B. Dalgetty, late of Madapore, S. Sylhet, India;
Prof G. Elliott-Smith, of Cairo (Reptilian). Four specimens each were
sent by the Hon. R. E. Dillon, of Clonbrock, Co. Galway, Ireland ; Prof
E. O. Jordan, Chicago University. Three each were sent by Dr D. E.
Salmon, Chief of the Bureau of Animal Industry, U.S. Department of
Agriculture, Washington (collected at the Zoological Laboratory of the
Bureau); Dr Henry Strachan, Chief Medical Officer at Lagos, W. Africa
(Negro bloods); Mr E. G. Wheeler, of Alnwick, Northumberland; Dr
Arthur Palmer, of Sydney, N. S. Wales (collected by himself and
Dr J. F. Flashman). Two specimens each were supplied by Dr Daniels

Acknowledgments 413

of the London School of Tropical Medicine (Mongolian and Indian);
Major K. C. Mac Watt, I.M.S., of Ajraur, Rajputana, India; Mr E. F.
Robison, of Evergreen, California; Dr R. J. Scharfif, of the Science and
Art Museum, Dublin. Finally single specimens were sent me by Dr H.
Ainsworth, I. M.S., Bombay ; Professor J. Brunchorst, Director of Bergens
Museum, Bergen, Norway (fluid whale blood); Mr E. J. Bles, of King's
College, Cambridge ; Dr John Cropper, of Chepstow, Monmouthshire ;
Mr William Evans, of Edinburgh ; Captain Stanley Flower, Director
of the Government Zoological Gardens, Gizeh, Egypt ; Mr E. H. Hankin,
Director of the Government Laboratory, Agra, India ; Dr J. S. Haldane,
F.R.S., of Oxford; Mr M. 0. Hedley, of Carievale, N.W.T. Canada;
Mr W. Kerr, of Singapore ; Prof John MacFadyean, Principal of the
Royal Veterinary College, London (donkey serum); Herr G. von Oertzen,
Consul-General to the German Empire at Havre, France (fluid porpoise
blood); Prof Sophus Torup, Christiania, Norway (fluid whale serum);
Mr I. LI. Tuckett, of Trinity College, Cambridge.

Some specimens sent by Dr C. Christy (23 bloods) from Kampala,
Uganda, by Mr William Foster (2) from Sapucay, Paraguay, by Dr H.
Ainsworth (3), by Captain Flower (1), by the Scottish Antarctic
Expedition (5), as well as others (11 specimens) received from the
Zoological Society's Gai-dens, London, unfortunately arrived too late
to be included in the present work. They will, however, be utilized
in due course.

My laboratory attendant. Mr Bertie Clarke, has been of great
assistance in the routine work connected with this investigation, where
80 much depended upon conscientious attention to details, and I feel
it is but fair to include him amongst those to whom I am indebted.

The expenses of this investigation have been in part defrayed
personally and through two grants, the one from the Government Grants
Committee of the Royal Society, the other from the John Lucas Walker
Trust, Cambridge.

BIBLIOGRAPHY.

Papers referring to the Precipitins are marked with a star *.

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Bihliographtf 426

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APPENDIX.

Note 1 (to p. 10). Wright {Brit. Med. Journ., vol. i., p. 1069, 9. v. 1903)
observed a decrease (" negative phase ") of the bactericidal power of blood serum
following inoculation with anti-typhoid vaccine, the antibody subsequently increasing
in amount ("positive phase"). The haemolysins and anti-rennet behave in a
similar manner.

Note 2 (to p. 112). Regarding Anti-Peptoties: Dr E. F. Bashford has sent me
his Report to the Scientific A.s.sessors of the Worshipful Company of Grocers,
London, dated 25. in. 1902, in which he writes :

" In his paper on immunisation against various proteids, the late Dr Myers
stated, that the precipitate produced by the reaction between Witte peptone and
the .serum of an animal immunised against it, did n(jt give the biuret reaction, and
therefore was a new product. By the kindness of Professor Liebreich I was enabled
to immunise two goats against Witte jjeptone (albumose), and thereby to obtain the
product referred to in large amount. It seemed that this step was likely to throw
much light on the nature of the reaction, and on that between toxine and antitoxine.
It was, however, found that the rea.son why the biuret reaction had failed was
simply the extreme insolubility of the product. Thoroughly dried in vacuo over
phosjjhoric anhydride along with a specimen of the mother albumose {i.e. which had
been boiled and filtered as for injection into the immunised animals), both were
submitted to elementary analysis, with the following mean result for two analyses : —

Product precipitated — 043.5 ^13-29 ^6.^4 per cent.
Mother substance — C53.15 N,3.j7 Hj.j per cent.

This did not afford grounds sufficient for speculation as to the nature of the
product precipitated, nor as to the mechanism of its production. Many other
difficulties, esi)ecially the fact that albiunoses in presence of other albuminous
bodies in neutral solution may yield precipitates made it doubtful if the product
analysed had any claim to be considered sufficiently free from admixtures, etc.
I have not given up this investigation, on the contrary hope that immunisation
against nucleohiston now proceeding at Dr T. H. Milroy's suggestion, may afford
means for obtaining a reaction product more distinctly difl'ering in composition
from the mother luicleohiston than was foimd to be the case for the albumoses of
Witte peptone."

Note 3. On looking through the Index Medians, a paper by W. d'E. Emery
{Barfs Hosp. Journ., London, 1902 — 1903, x. pp. 34 — 40) relating to the precipitins
was noted, but too late to be considered in this volume.

INDEX TO NAMES.

•See in additimi under Bibliography, p. 414, and under AcknowUdgmentt, p. 411.

Abbott and Bergey 29

Abderhaldeu 4

Acbalme 16

Arloing 46

Arthus 102, 108

Aschoff 13, 27, 76, 105, 107 see Meyer

and
Aseoli, G. 26, 42
Ascoli, M. 102, 108, 130, 133, 139, 140,

144, 146, 147, 150, 407
Austin 390

Bail 49, 50, 153

Bail and Wilde 28

Bashford 155, 430

Baumgarten 14, 19, 43

Uehring 5

Behring and Kitasato 5

Behring and Nissen 8

Belfanti and Carbone 19, 40

Beljajew 50, 96

Bennett See Patek and

Bernard 34

Besredka 22, 23, 42, 149

Beumer 386, 406

Beyer i nek 87

Binda 415

Biondi 76, 90, 96, 97, 105, 113, 119. 120,
123, 128, 132, 164, 389

Blumenbach 1

Boeri 25, 26

Bolton 10

Bordet 14, 18-21, 31, 36, 40, 41, 43, 45,
47, 48, 51, 53, 56, 61, 99, 115, 117, 135,
152, 156, 161, 178, 200, 381, 382

Bordet and Gengou 17, 27

Bordoni See Runino and

Breton See Caliuette and

Brieger 99

Brieger and Ehrlich 9, 11
Briot 16

Buchner 19, 23, 35
Bucbner and Geret 113
Bullocb and Hunter 28
Butza 164

Calmette 5

Calmette and Breton 32

Calvo ^V« Landsteiner and

Camus 17, 74

Camus and Gley 21, 22, 27, 39

Carbone See Belfanti and

Carrara 416

Castellan! 28, 153, 155

Chcinesso 416

Clairmont See Kraus and

Claus 214

Cobbett 10, 217

Corin 96, 97, 120

Courmont 8, 40

Creite 18

Crendiropoulo See KufFer and

Cuvier 1

Daruiuberg 36

Darwin 2, 137

Defalle 44

Delizcnne 16, 22, 25-27

Deutsch 27, 31, 41, 46, 47, 381

Dickinson 17

Dicudonn^ 51, 96, 104, 162

Dinkelspiel .SV<f Nuttall and

Doliris and Quinquaud 146

Donath and Landsteiner 22

Diinitz 92

During 21

Dubois, A. 44, 96

Dubois, E. 2, 3

432

Index to Names

Dunarest See Guinard and
V. Dungein 10, 10, 25, 26, 158
Durham 46
Dziergowski 111

Ehrlich 5-7, 13, 14, 19, 21, 27-29, 42, 49,

91-94, 128, 135, 140, 147, 149, 154, 160,

381, 382
Ehrlich aud Morgenroth 13, 20-22, 24,

34-37, 39-43, 148, 157
Ehrhch See Brieger and
Eisenberg 21, 28, 42, 50, 74, 88, 91-94, 96,

98, 99, 102, 103, 115, 117, 127, 143
Eisenberg and Volk 13, 48, 49, 91, 93, 99,

102
Eisenberg See Kraus and
V. Emden 46, 49
Emery 430

Emmerich and Loew 14, 43
Evans 214, 361
Ewing 28, 74, 142, 164

Falloise 109

Farnum 176, 189, 389

Fermi and Pernossi 16

Ferrai 120

Fich 156, 381

Flexner and Noguchi 19, 20, 23, 25, 28,

29, 35, 36, 38, 43
Flower aud Lydekker 182, 214
V. Fodor and Rigler 46
Ford 49, 50
Foster 217

Fraenkel and Sobernheim 27
Frazer 5

Freund and Sternberg 11
Friedberger 48
Friedenthal 33-40, 74, 170
Friedenthal and Lewandowsky 34-37
Fuld 158
Fuld and Spiro 75
Funck 23

Gadow 214, 361

Gaskell 359, 361

Gaudry 2

Gengou 20, 26, 43, 61, 94, 95, 109, 157,

176, 178, 201, 205
Gengou See Bordet and
Geret See Buclmer and
Gessard 17
Glassner 16
Gley See Camus and

Goutscharnkow 26

Graham-Smith 38, 78, 85, 115, 117-119,

161, 205, 206, 208, 209, 211, 212, 217,

336, 380, 385, 389
Graham-Smith and Sanger 56, 76, 80, 81,

102, 104, 110, 120-125, 390 et seq.
Gruber 14, 20, 37, 46, 48
Gruber and Durham 46
Grilnbaum 42, 46, 101, 137, 164, 168,

169
Guinard and Dunarest 36, 37
Gilnther 214, 361

Haeckel 1, 2

Halban 146

Halban and Landsteiner 24, 88, 98, 105,

138, 145, 146, 149, 158
Hamburger 61, 62, 107, 109, 131-133, 158,

179
Hankin 63, 174, 385
Hanna 209
Hausmann 99, 111
Haycroft 17
Hayem 18
Herrscher 419
Heubner 30
Hildebrandt 16
Honl 419

Hopkins 88, 89, 100, 129
Hunter See Bulloch and
Huxley 1

Ida 21, 26, 96, 105, 108

Jaeoby 11, 43, 99, 111, 155
Johnson 2
Joos 47

Kanthack 49

Kister and Wolff 51, 53, 73, 74 114, 139,

142, 181, 189, 191, 193
Kitasato See Behring and
Klein, A. 113
Korschun 16
Kossel 21, 22
Kowarski 113, 155
Kratter 420

Kraus 48, 114, 152-154, 381
Kraus and Clairmont 28
Kraus and Eisenberg 49, 50, 149
Kraus and von Pirquet 13, 49, 92-94, 115,

154
Kriiger 145

Index to Nanus

433

Lamb 208, 209

Landois 18, 36, 37

Landsteiner 16, 23, 48

Landsteiner and Calvo 108, 111

Landsteiner See Donath and

Landsteiner See Halban and

Lane 217

Langmann 166

Laveran and Mesnil 48

Layton 165, 389

Leblano 88, 96, 99, 105, 107, 109, 111

Leolainohe and Valine 51, 53, 98, 99, 101,

104, 105, 115, 117, 161
Lemoine See Linossier and
Levene 105, 204

Levy 28

Levy and Bruns 152

Levy and Giesler 127

Lewandowsky See Friedenthal and

Liepmann 110

Lindenmann 25

Lingelsheim 102

Linnaeus 1

Linossier 73

Linossier and Lemoine 74, 79, 99, 102,

105, 113-115, 117, 123, 138, 139, 142,
143, 145, 407

Lipstein 21, 94

Lisle 421

de Lisle 61, 179, 211

Loew See Emmerich and

London 35, 36

Lubenau 28

Liihe 171, 185

Madsen 5, 8, 28

Madsen See Salomonsen and

Malvoz 28

Maukowski 25

Markl 153

Marmorek 153

Marshall 33

Marx See Pfeiffer and

Mertens 27, 43, 51, 104, 127, 204

Mesnil See Laveran and

Metalnikoff 24, 29, 32, 36

Metchnikoff 16, 18, 20, 22-27, 129, 132

Meyer and Asohoff 106, 158

Miohaelis 52, 93, 94, 97, 102, 107, 109,

112, 113, 115, 132, 135, 189
Miohaelis and Oppenheimer 13, 53, 88, 98,

99, 107, 111, 128, 129, 131, 132, 189
Michaelis See Oppenheimer and
Miessner 72

N.

Minovici 52, 387

Mirto 422

Mitchell 217

Mitchell and Stewart 43

Modici 96, 101, 120

Moll 16

Morgenroth 8, 16, 26, 29, 381

Morgenroth and Sachs 37

Morgenroth See Ehrlich and

Moro 30, 31, 53, 99, 115, 123, 127, 128,

135, 145, 157-159
Mosso 22, 39
Moxter 23, 24
Mailer 21, 22, 29, 88, 90, 91, 93, 94, 102,

117, 158
Myers 8, 45, 47, 51, 88, 94, 96, 100, 112,

113, 117, 185, 136, 188, 190, 204

Nedriagailoff 15

N^Kdieff 25

Neisser 21

Neisser and Wechaberg 21, 28, 94

Neufeld 153

NicoUe 114, 153

Nissen See Behring and

Noguchi 150

Noguchi See Flexner and

Nolf 21, 26, 50, 61, 96, 99, 104, 106, 109,

110, 176, 178, 179, 192, 200
Niitel 110, 193, 405
Nuttall 27, 30, 47, 51, 63, 71, 105, 117,

119, 120, 122-127, 134, 136-141, 163, 176,

179, 190, 201, 205, 206, 217, 336, 382,

etc.
Nuttall and Dinkelspiel 65, 66
Nuttall and Strangeways 312-335

Obermayer and Pick 97, 106, 107, 111,

113, 115, 130, 134, 150, 205
Ogier 424
Okamoto 424

Oppenheimer and Michaelis 97, 108, 139
Oppenheimer See Michaelis and
Orlovsky 424

Panicbi 42

Panum 18

Patek and Bennett 424

Pemossi See Fermi and

Pettit 22

Pfeiffer 8

Pfeiffer and Marx 27

Pfeiffer and Proskauer 27

Philippson 211

28

434

Index to Names

Pick 11, 13, 27, 47, 48, 50, 75, 92, 93, 115,

154
Pick See Obermayer and
Piorkowski 425
V. Pirquet See Kraus and
Ponfick 18
Proskaner See Pfeiffer and

Eadziewsky 50, 114, 153

Rath 46

Ringer 111

V. Rigler 80, 405, 407

V. Rigler .Sec v. Fodor and

Ritchie 27

Robin 76, 101, 123

Roden 16

Romer 27

Rostoski 16, 52, 72, 73, 75, 76, 80, 102,

103, 107, 111, 112, 115, 123, 127, 128,

131, 135, 154
Roux and Vaillard 10
Ruffer and Crendiropoulo 26, 32, 47
Rumno and Bordoni 36, 37

Sachs 17

Sachs See Morgenroth and

Salomonsen and Madsen 10

Sanger 389

Schattenfroh 26, 31, 32

Scherenziss 145

Schirokich 136, 163

Schumacher 146

Schiitze 21, 105, 109, 112, 113, 117, 148,

149, 154-158, 161, 180, 191, 382, 406
Schiitze See Wassermann and
Selenka 2
Seng 11
Sewall 5
Shattock 42
Shibayama 26
Sieradzki 426
Sobernheim 27
Sobernheim See Fraenkel and
Spiro See Fuld and
Stern 51, 53, 144, 162
Sternberg See Freund and
Stewart See Mitchell and
Stookis 76, 113, 120
Strangeways 59, 81, 97, 114, 130, 144, 145
Strangeways See Nuttall and
Strube 51, 53, 56, 61, 73, 90, 96, 106, 123,
127, 139, 140, 142, 144

Sweet 29
Szczawinski 36

Takaki See Wassermann and

Tarchetti 101

Tchistovitch 14, 21, 89, 49, 51, 56, 57, 61,

79, 99, 112, 115, 117, 127, 153, 161, 192,

200, 211, 382
Torup 198

Uhlenhuth 36, 37, 47, 51, 53, 59, 61, 64-
67, 72-74, 76, 109, 110, 115, 119, 120,
123, 132, 135-137, 140, 142, 144, 146, 156,
158, 162, 164, 172, 173, 176, 179, 180, 188,
189, 191, 193, 195, 200, 204, 382, 383, 385-
388, 403-406

Umber 427

Vaillard See Roux and
Valine and Nicolas 405
Vallee See Leclainche and
Volk See Eisenberg and

Walker 21, 94

Wassermann 6, 22, 27, 30, 49, 50, 51, 135,

150, 162, 381, 382
Wassermann and Schiitze 53, 62, 101, 113,

123, 135, 136, 156, 157, 162, 163
Wassermann and Takaki 25
Wechsberg See Neisser and
Weichhardt 24, 29
Weigert 6
Weiss 36, 37
Welch 28, 33
Wendelstadt 17, 21
Westpbal 22
Whitney 52, 96
Whittier 164, 389
Widal 46, 48, 152
Widal and Sioard 48
Wilde 28
Wiuterberg 48
Wladimiroff 153
Wolff 428

Wolff See Kister and
Wood 389
Wright 430

Ziemke 64, 66, 67, 83, 109, 120, 141, 163,

384, 388
Zuelzer 51, 104

INDEX TO SUBJECTS.

To avoid overloading the Index with tjie names of the nuvieroug aninuih whose bloods
were tested with precipitins it has been deemed advisable to exclude them. The names
will, however, be readily found by reference to the Tables on pp. 220-311, 364-380, where
they have been ordered zoologically. In the following Index the reader will find references
to the different bloods tested by looking up the Class or Order to which the animal belongs,
and in addition by referring to the Tables as follows :

Mammaha :

Tables

Page

Tables

Page

Primates

220-229

*Aves

266-298,

364

Chiroptera .
Insectivora .

230-232
232-234

•Reptilia .

300-305,

366,

868-373

Carnivora
liodentia

234-244
244-251

'Amphibia

306, 366,

374

Ungulata
Cetacea .

252-259
260

*Pisce8

308-309,

366,

378-380

Edentata
Marsupialia .

260
262-264

Arthropoda

310, 376

Monotremata

264

Ascidia .

357

* Kgg albumins also tested.

Abrin, antitoxin 5

Acknowledgments 411

Acids, action on serum 81, 82, 85
effects on precipitin reaction 402

Addiment See Complement

Age, influence on susceptibility of cells to
cytotoxins 27

Age, influence on susceptibility of dogs
to anthrax, etc. 28

Age of individual, influence on bacteri-
cidal power of serum 30

Age of individual, influence on degree of
precipitin reaction 146

Agglutination, chemical nature of the re-
action 48

Agglutination, salts, influence on 47

Agglutinins, Ehrlich's theoi7 regarding 12

Agglutinins, Ehrlich's theory regarding,
for bacteria 13, 27, 44
for blood corpuscles 45
chemical nature 13, 14, 48
for cholera 13, 27
differ from precipitins 49
in humor aqueus 127
mixed, act independently 47
multiplicity in serum 45
natural and artificial 45
in normal bloods 46
relation to haemolysina 19
relation to cytolysins 43
resistance to heat 48
significance of 48
in snake venom 23
stability in vitro 48

436

Index to Subjects

Agglutinins, source in the body 46

for typhoid 13, 27

See also Iso-agglutiuins, Anti-agglutinins
Agglutinoids 13, 49
Albuminuria in blood-treated rabbits 133

See also Urine
Alexin 14, 15

See also Complement
Alkalis, effects on precipitin reactions 403

effects on sera 83, 85
Alkaloids do not give rise to antibodies 7
Amboceptor, See Immune-body
Amphibia, bloods and eggs tested with
precipitins 210, 356

sera, haemolytic effects of 38

tables 306, 366, 374
Anthrax 27, 28 See Age, Anti-Immune

bodies
Anti-antibodies 13 and below

-agglutinins 14, 45, 49

-agglutinins not obtained for bacteria 13

-anthrax serum 27

-antiferment 14

-antiferment for rennet 16

-antitoxin, not obtained 13

-bodies in general 5, 9, 12

-bodies in general, chemical constitution
13

-bodies in general, coexistence in one
serum 14

-bodies in general, formed for assimilable
substances only 7

-bodies in general See Anti-antibodies

-cholera serum 27

-coagulin 17

-complement 14, 22

-complement neutralizes complement 20

-complement how obtained 20, 21

-complement resists 55° C. 20

-cynarase 16

-cytotoxins 14

-diastase 16

-egg-albumin sera 130, 132, 136 See
Aves, Egg-white

-emulsiu 16

-epithelium 25

-ferments (artificial) See this list under
Anti-emulsin, cynarase, coagulin, dia-
stase, etc.

-ferments (artificial) 16, also Anti-anti-
ferments

•ferments (normal) for rennet, trypsin,
urease 16, 17

-haemolysins 13

Anti-haemolysins normal 22

-haemolysins, normal, coexistence with
normal haemolysins 22

-haemolysins for sera of eel and fowl 21

-haemolysins for sera of immune guinea-
pig and rabbit 21

-human serum, actions on different
bloods 136, 138, 139, 143, 161, 167,
320, 381-404

-immune bodies 13, 14

-isolysiu 22

-lactosera 149

-leucotoxins, specific 23

-pepsin 17

-peptone, so-called 94, 112, 430

-precipitins 14, 149

-pseudo- globulin 26

-rennet 16

-sera for different bloods See Precipitins,
Precipitating

-serum-albumin 26

-spermotoxin 13 reactivatable 24

-symptomatic anthrax serum 27

-toxins 5

-toxins, chemical nature of 11, 13, 14

-toxins, chemical nature simpler than of
bacteriolysins, etc. 27

-toxins not formed for chemically de-
fined poisons 7

-toxins in milk and serum, fluctuations
during immunization 9

-toxins, non-toxic 8

-toxins, union with toxin 5-12

-toxins, normal antitoxins 10

-trypsin 16

-tyrosin 17

-urease 17

-venine 5
Appendix 430

Arthropoda, precipitin tests on bloods of
211, 357-360

tables 311, 376
Ascidia, precipitins and tests on extracts

of 357
Authorities, re Classification 214
Auto-cytotoxin See Auto-lysin

-lysin 42 (defined)

-precipitins 150

■spermotoxins, inactivation and reactiva-
tion, etc. 24
Ares, precipitin-tests upon their bloods,
qualitative 201-207, 266-298 ; quan-
titative 334

precipitin-tests upon their egg-whites,

Index to Subjects

437

qualitative 201-207, 298, 340-344,
364 ; quantitative 344-347

Summaries of foregoing 216, 361

See further under Eggs

tables 266-298, 364

haemolytic effects of their sera on dif-
ferent bloods 38

Bacillus diphtheriae See Diphtheria

dysenteriae See bacterial-haemolysins

pyocyaneus See bacterial-haemolysins

tetani See bacterial-haemolysins

typhosus See bacterial-haemolysins
Bacterial haemolysins 8, 19, 28

haemolyains neutralized by anti-hae-
molysin, normal and artificial 28

growth, a cause of error in precipitin
tests See Error

growth See Putrefaction
Bactericidal effects of fresh normal serum
27, 30

destroyed at 55° C. 27

destroyed by snake venom 28

substance in serum 14
Bacterio-agglutiuins 13, 27, 44

agglutinins act on dead germs 48
Bacterio-lysins 8, 14

absorbed by heated bacteria 28

artificial and normal, similar to haemo-
lysins 19, 27

constitution 19

defined 14

Ehrlich's theory relating to 12

inactivated by heat 20

mode of action 19

presence in humor aqueus 127

specific, different in different species of
animals 27

source of, in the body 26
Bacterio-precipitins 152
Bats See Chiroptera
Bibliography 414-429
Blood, collecting for testing with precipi-
tins 62 et seq.

precipitins .Sec Precipitins

preservation dried in scales or on filter-
paper 63

preservation fluid 62, 76

relationship indicated by precipitin re-
actions etc. 4, 137

stains Sfe Medico-Legal teat, Extraction of

testing by chemical methods 4

testing with precipitins 62 See Pre-
cipitable to Precipitoid

Blood, transfusion, effects of, with different
bloods 18
See Serum, etc.
Bones, identification of by precipitins 406

Carnivara, bloods, precipitin tests on,
qualitative 173-178, 234-244 ; quanti-
tative 322

bloods, precipitin tests on, tables 234-
244

haemolytic effects of their sera on dif-
ferent bloods 34
Cetacea, bloods, precipitin tests on, qnali-
tative 198, 260

bloods, precipitin tests on, summary of
results 216

bloods, precipitin tests on, tables 260
Chemiotactic substances for leucocytes,

increase complement 29
Chiroptera, bloods tested with precipitins
(tables) 230-232

formerly classed as Primates 1
Cholera agglutinins, chemical nature 13

anti-cholera serum 27

lysin, chemical nature of 13

lysin See Immune bodies
Classification of animals adopted in this
book 70

authorities used 214

of Primates 1
Coagulins 15

anti-coagulins 17

counteract anti-ooagulins in leech-ex-
tract 17
Colour-test for blood solutions 67
Complements, amount varies in serum 29

amount may be artificially increased
29, 30

amount may be artificially diminished
by phosphorus 29

amount, effect of suppuration on 29

of cytolytic sera 28

destroyed at 55° C. 20

in different antibodies 14, 15

Ehilich's theory in relation to 12, 14

in haemolysins and bacteriolysins 19

injection of, leads to formation of anti-
complement 20

multiplicity of 20, 21

neutralized by anti-complements 20

in plasma 29

relation to haemolytic action of snake
venom 29

See Anti-complement, Chemiotactic

28—3

438

Index to Subjects

Complementoid 13, 21
Complementophile group of Ehrlich 12
Conclusions 214-217, 335, 361-362, 403,

407
Copula 15

Cretin, precipitins for 155
Crustacea, precipitin tests on bloods of
211, 310, 357, 358, 376

no haemolysins found in blood of 40

tables 310, 376
Culture filtrates, toxin converted into

toxoid 7
Cynarase See Ferments, Anti-eynarase
Cytase See Complement
Cytolysins 9

Ehrlioh's theory in relation to 12

relation to agglutinins 43
Cytolytic serum See Cytolysins
Cytotoxins of blood serum 14, 18

of snake venom 19

affecting bacteria 27

bacterial 19

term defined 14, 18

See Cytolysin, Anti- and Auto-cytotoxin,
Age

Decomposition, hastened after death from
snake venom intoxication 28

Degree of precipitin reaction an index of
blood-relationship 137

Desiccation, effects on precipitable sub-
stances in blood 119
effects on precipitins 119

Desmon 15

Detection of blood See Medico-Legal

Diastase See Ferment, Antidiastase

Digestion, tryptic and peptic, action on
precipitins 111

Dilutions of blood suited for precipitin
tests 74

Diphtheria antitoxin 5, 7, 10, 11
antitoxin, chemical nature of 13
toxin 7

Disease, effect on bloods in relation to
precipitin reactions 145

Echinoidea, no haemolysins found in

"blood" of 40
Edentata, precipitin tests on bloods of

(tables) 260
Eel serum, haemolytic action of, on dif-
ferent bloods 21, 22
anti-haemolysins for 21
See Pisces

Egg-white of birds, precipitin tests there-
on See Aves
of birds, identification of in prepared
foods 406
Egg albumins of Amphibia, Pisces, Bep-

tilia, precipitin tests on 352, 366
Ehrlich's theory (figure p. 12) 5 ct seq.
Emulsin See Ferment, Anti-emulsin
Epithelium See Anti-epithelium serum
Ergophorous group of Ehrlich 13
Error, sources of, in precipitin tests :
Bacterial multiplication 86
Dilution used 74
Evaporation of serum 86
Opalescent antisera 72
Overpowerful antisera 74
Preservatives added 76
in quantitative tests 313
Beactiou of medium (acid or alkaline)

79
Weak antisera 74
Erythrolysin 19

Extraction of blood-stains which are in-
soluble in water or saliue 63, 64,
83, 385
Eye-structure in Primates 2

Fat, preventing bloods going into solution

67
Feeding blood to animals leads to hacmo-

lysiu formation 32
blood and egg-white lead to precipitin

formation 132
Ferments and antiferments 12-17
Filter-paper method of collecting bloods

67
Filtration, effects on normal sera in relation

to precipitins 118
Fish See Pisces
Fixateur 15
Fixer 15

Foam-test for blood solutions used in pre-
cipitin tests 67
Foetal transmission of precipitins 127
Food, influence on bactericidal power of

serum 30
Fowl, Anti-haemolysins for fowl serum 21

See Aves
Frog, effect of tetanus toxin on 8

See Amphibia
Function of precipitins in the body 128

Gelatin ferment, artificial 16
Go-between 15

Index to Subjects

439

Government recognition of precipitin test
for blood in forensic medicine 403

Haematosera, defined 15

See Precipitins
Haemoagglntinins 45

See Agglutinins
Haemoglobinuria following blood • trans -

fusion 18
Haemolysins 9, 19, 26, 32, 40, etc. See
following :

artificial (specific) discovery of 40

artificial (specific) decrease after pro-
longed treatment with blood 32

artificial (specific) effects on different
bloods and animals 40

artificial (specific) use in legal medicine,
suggested 41

artificial (specific) constitution 19

artificial (specific) formed in rats fed on
blood 32

artificial (specific) immune body and
agglutinin in 44

artificial (specific) inactivated by heat 20

artificial (specific) intermediary bodies in
20

artificial (specific) presence in plasma 26

artificial (specific) source of in the body
26

artificial (specific) relation to agglutinins,
bacteriolysins 19

normally present in sera, effects on diffe-
rent bloods and animals 33

normally present in sera, effects on diffe-
rent cells of body 22

specific See artificial (above)

theories regarding mode of action, Banm-
garten's 19

theories regarding mode of action, Ehr-
lich's 12

term defined 14, 18

visible effects on blood-cells 19

See further under Age, Anti-antibodies,
Bacterial haemolysins, Complement,
Cytotoxins, Immune body. Treatment,
Urine
Haemolysis, through action of chemicals
19

hypotonic salt solution 19

consequent upon blood transfusion 18

in relation to disease 42

through snake venom 29
Haemolysis, experiments on washed cor-
puscles 30

Haemotoxin See Haemolysin (synonymous)
Haptins, defined 11, 13
Haptophorous group of Ehrlich 7, 12
Heat, effect on agglutinins 48

effect on anti-complement 20

effect on bacteriolysins 20, 27

effect on complements 20

effect on haemolysins 20

effect on precipitins 114

effect on precipitable substances 117
Hepatotoxins 25
Heterolysins 42
Hilfskorper 15

Honey adulteration discoverable by pre-
cipitins 406
Human blood, transfusion experiments
33

blood See Anti-human

serum haemolysins, tested on different
animals and bloods 33
Humor aqueus, presence therein of agglu-
tinins, bacteriolysins, precipitins 127

Identification of blood-stains by haemolysins
(Deutsch) 41
of blood-stains See Precipitins et seq.
Illumination, important in precipitin tests

72
Immune-bodies 11, 12, 14, 15, 19, 21, 27,
28, 94
to anthrax iu normal adult dog serum (?)

28
in anti -typhoid and cholera sera, etc. 27
in cytolysins (Ehrlich's figure) 12
in different antibodies 14
in haemolytic and bacteriolytic sera 21
excess of, impedes bacteriolysis and hae-
molysis 21
in precipitating antisera 94
See Anti-Immune-bodies
Immunity to toxin 8
" Immunkorper " See Immune-body
Incubation and toxin action 7
Individual differences iu blood : adult, foetal,
maternal, sexual differences 145, 146
differences in milk from different women

(precipitin tests) 145, 159
differences in guinea-pigs forming spermo-
toxin 24
Insectivora, bloods tested with precipitins
172, 232-234, 321
effects of their normal haemolysins on

different bloods 34
tables 232-234

440

Index to Subjects

" Insoluble " bloods, extraction thereof,

medico-legal importance 63, 64, 83, 385

Intermediary bodies, multiplicity of, in

haemolysins and snake venoms 20, 21

body 15
Isoagglutinins in disease 42
Isolysins (and Anti-isolyeins) 22

in disease 42

term defined 42
Isoprecipitins, for serum and milk 148

Key to signs used in tables of bloods tested

217-219, 338
" Komplementablenkung " 21

Lacto-preeipitins See Lactosera
Lactosera 15, 95, etc. as follows

action on blood serum 158

discovery of 156

haemolytic action etc. 158

for human milk 145

for ox and goat milk 135

methods of testing with 156

reactions with boiled milk 157

reactions with individual milks 145,
159

specificity of reactions 157

stability in vitro 159

See Anti-Lactosera 149
Leucocytosis in blood-treated animals 128,
131

See Cheraiotactic
Leucolysins 19

distinct from erythrolysins q.v. 23
Leucotoxins 19, 22, 23

normal 23

term defined 19

See aUo Anti-leucotoxins, Leucolysins
Lysins 9

See Bacterio-, Cyto-, Haemo-lysins

Macacus serum, haemolytic action of on
other bloods 34
See Primates
Mammalia, precipitin tests on their bloods

160-200, 220-265
Mammalian reaction 139

See Medico-Legal
Man See Primates, Anti-human serum
Marsnpialia, precipitin tests on their bloods
199
summary of results 216
tables 262-264
Meat-identification through precipitins 404

Medico-Legal : applications of precipitin
tests for bloods 64, 381-402

blood-stains on cloth, metal, leather, etc.
390 et seq.

effects of earth, lime, mortar on blood
398-402

insolubility of bloodstains 63, 64, 83,
385

See Colour-test, Error, Fat, P'oam-test,
Methods, Testing
Methods of collecting precipitating antisera
59

of killing animals 59

of storing antisera 60

of testing with precipitins 336

of testing See Medico-Legal

of treating animals for production of pre-
cipitins See Treatment
Micrococcus tetragenus See Bacterial hae-
molysins
Milk, boiled, reacts to precipitins 157

-curdling ferment 8

influence of breast- and bottle-feeding
on bacteriolysins in infant's serum 30

-precipitins See Lactosera

See Antitoxin
Monotreviata, tests on one blood of
(table) 264

Nephrotoxins 14, 25

Neurotoxins, artificial in serum 25

in snake venom 26
Normal antitoxins 10
precipitins 97

See also under Agglutinins, Antihaemo-
lysins, Bactericidal, Coagulins, Hae-
molysins, etc.

Oligochaeta, no haemolysins found in

blood of 40
Opalescent antisera (precipitating) 72

Palaeontological evidence of animal rela-
tionships 1-3

"Praparator" 15

Pepsin and antipepsin 17

Peptic digestion, effect on precipitins 111

Peptones and "antipeptones" 94, 112, 430

Peritoneum, appearances, in blood-treated
animals 129

Phosphorus poisoning reduces complement
in serum 29

Physiological processes and serum-com-
plements 30

Index to Subjects

441

Phytopreoipitins 152

Pilocarpin, effect ou antitoxin content of

Bcrum 10
Pisces, bloods, precipitin tests on 211,
308-309, 378-380

egg albumins, precipitin tests on 366

haemolytic effects of sera on different
bloods 39

tables 308-309, 366, 378-380
Placenta, structure in Primates 2
Plant albumins, precipitins for 155

albumoses, precipitins for 155
Plasma, complement contained in 29

haemolytic effects of 26
Precipitable substances,

effect of desiccation on 119

effect of heat on 117

effect of putrefaction on 119

nature of 96

observations on, in corpore 89, 126, 132

and precipitins, coexistence in immune
animals 129 et seq.
Precipitating antisera,

effect of heat on 114

effect of filtration on 65

effect of antiseptics on 65, 76-79

immune bodies in 94

preservation of 65

stability of in vitro 123

strengths of 144, 145, 337, 339

See further under Precipitin reactions.
Precipitins
Precipitin reactions,

acids and alkalis, effects on See. Acids,
Alkalis

with baoterio precipitins 92

with blood precipitins See above and
below

with blood solutions in water 104

with distantly related bloods 137

with homologous substances in high
dilutions 142

with non-homologous 135 et seq.

disease, influence of 145

inactivated antisera 90

individual differences in sera, etc. 145, 146

interacting bodies unite quantitatively 88

mammalian reaction 139

nature of 88

precipitable substance, behaviour in cor-
pore 89

precipitin used up in reaction 88

precipitoids 91, 92

precipitum 88, 89, 101

Precipitin reactions,
precipitum soluble in excess of semm 89
rate of reaction 142
reactivation of antisera not successful 94
salts, influence on reaction 102, 402
supernatant fluid, character of, after

precipitation 101
studied in hanging'drop (microscopically)

101
temperature influence upon 113
tests with concentrated sera 138
time limit 138, 139
See further under Precipitable substances,

et seq.
Precipitins, absence in urine 127
chemical nature of 13, 14, 88, 89, 96,

99-101
compared to heat or HNO., as a test

for albumins 98
defined 15

differ from agglutinins 49
disappear after prolonged treatment of

animals 57, 127
disappear from serum after stopping

treatment 127
effect of desiccation on 119
effect of heat on 114
effect of putrefaction on 66, 119, 402
Ehrlich's theory in relation to (figure) 12
formed after feeding animals with serum,

etc. 132
function of, in corpore 128
injection of, into animals, behaviour of

97, 134
measure of degrees of reaction with 141
normal 150
not formed for starch, glycogen, gelatin,

etc. 113
observations on, in corpore 126
obtained from different species of

animals 146
and precipitable substances coexisting

in corpore 129 et seq.
peptic digestion destroys 111
presence in humor aqueus 127
regeneration after bleeding immune

animal 128
seat of origin in corpore 132
selective action in blood mixtures 140
specificity of 135 et seq.
transmitted to foetus in utero 127
tryptic digestion resisted 111
"unit" of Eisenberg 143
for blood See above. Precipitin tests, etc.

442

Index to Subjects

Precipitins, for crystallized horse albumin
129
for egg albumin See Egg
for milk See Lactosera
for peptones (?) 94, 112, 430
for plant albumin and albumose 155
for serum globulins 140
for snake venom 208-209
See further under Precipitable to Pre-
cipitoid, Anti-, Auto-, Iso- and Phyto-
precipitins
Precipitin tests with Antisera for Bloods
of Mammalia :
Primates : qualitative 161-172, 220-
229; quantitative 319-321
Man, qualitative 161-167 ; quanti-
tative 319-320
Simiidae: Chimpanzee, qualitative 168
Simiidae : Ourang, qualitative 169 ;

quantitative 321
Cercopithecidae : Ceroopithecus, quali-
tative 169-172
Summary of foregoing 214
Insectivora : Hedgehog, qualitative 172 ;
quantitative 321
Summary 215
Camivora : Cat, qualitative 173 ; quan-
titative 322
Hyaena, qualitative 174
Dog, qualitative 176; quantitative 323
Seal, qualitative 177
Summary 215
Kodentia : Babbit, qualitative 170
Ungulata: Suidae, Pig, qualitative 179-
182 ; quantitative 328-331
Camelidae, Llama, qualitative 182
Cervidae, qualitative 183-187; quan-
titative 326-327
Mexican Deer, qualitative 183 ;

quantitative 326
Reindeer, qualitative 185 ; quanti-
tative 327
Hogdeer, qualitative 186; quanti-
tative 327
Bovidae : qualitative 187-192 ; quan-
titative 323-325
Antelope, qualitative 187 ; quanti-
tative 325
Ox, qualitative 136, 188 ; quanti-
tative 325
Sheep, qualitative 136, 190; quan-
titative 323
Equidae : qualitative 192-198 ; quan-
titative 331-332

Precipitin tests with Antisera for Bloods
of Mammalia :

Horse, qualitative 192; quantitative

331
Donkey, qualitative 195 ; quanti-
tative 332
Zebra, qualitative 197; quantitative

332
Summary relating to Ungulata 215
Cetacea : Balaenoptera, qualitative 198
Marsupialia: Wallaby, qualitative 199;
quantitative 333
Aves: Ratitae: Ostrich, qualitative 202;
quantitative 334
Carinatae: Fowl, qualitative 200, 343;

quantitative 334
Summaries 216, 335
lieptilia : Chelonia : Tortoises (2 spec),
qualitative 317, 368 ; quantitative 354
Chelonia : Turtle, qualitative 207, 348,

300, 368; quantitative 355
Crocodilia : Alligator, qualitative 209,

300, 349, 368
Lacertilia : Lizard, qualitative 350, 370
Ophidia : Snakes (2), qualitative 208,

351, 370-373
Summaries 216, 353, 355, 361
Amphibia: Ranidae: Frog, qualitative
210, 356, 306, 374
Summaries 217, 362
Pisces : Ammocoetes, qualitative 356

Eel, quahtative 211
Ascidia, qualitative 357
Arthropoda : Decapoda, qualitative 211,
310, 357, 358, 376
Xiphosura : Limulus, qualitative 358,

376
Summaries 217
Egg-whites of Aves :
Ratitae: Emu, qualitative 206, 340,

364 ; quantitative 345
Carinatae : Fowl, qualitative 204, 342,

364 ; quantitative 346
Carinatae: Duck, quahtative 341, 364;

quantitative 345
Carinatae: Crane, qualitative 343,364;

quantitative 346
Summaries 216 See also p. 406
Reptilia: qualitative 352, 366
Varia : Bacteria 152
Milks (See also Lactosera) 156
Plant albumins, etc. 155
Venom of snake (Cobra) 208
Yeasts 154

Index to Subjects

443

Precipitin tests:
general considerations 312
delicacy of the test 144
errors in testing See Error
testing with diluted antisera 142
quantitative method (Nuttall's) 315
testing blood dried on filter-paper 1)3

et teq.
testing method, for a large series 70

et seq.
time-limit placed on reactions 71
See further under Precipitable substances,
etc. above
Precipitoid 13
Precipitum 99-101
Preservatives, effects on blood 76
Primates, bloods See Precipitin tests on
classification and relationships 1-3
eye structure in 2
genealogical tree (Dubois) 3
haemolytic effects of their sera on different

bloods and animals 33
placental structure in 2
See further under Belationship, tables
220-229.
Putrefaction, effects on blood and serum
precipitins 66, 119, 383, 402 (plate)

Qualitative tests with precipitins See Pre-
cipitin tests

Quantitative tests with precipitins See
Precipitin tests

Reaction, ncid or alkaline, effects on pre-
cipitin reactions 79
Beactivation of haemolysins, bacteriolysins,
spermotoxius, thyreotoiins, etc. 20,
24, 25
of precipitins not obtained 94
See Bacteriolysins, Complement.
Receptors (Ehrlich) 7, 11, 12
Regeneration of antitoxins in the body 110

of precipitins in the body 128
Relationships of auimals indicated
by anatomical structure 1-3
by effect of artificial haemolysins 41
by effect of normal haemolysins 41
by effect of precipitin tests on bloods, q.v.
Rennet See Ferment
Reptilia, haemolytic effects of their sera on

dificreut animals and bloods 38
Reptilia, See precipitin tests on bloods of, and
tables, including lists of bloods and
eggs tested 300-305, 366, 368-373

Rioin antitoxin 5, 11

precipitins 155
Roborat precipitins 155
RodeiUia, haemolytic effects of their sera on
different animals and bloods 35
tables including lists of bloods tested

244-251
See Precipitin tests on bloods of

Salts, effect of (NaCl) on precipitin re-
actions 102, 402

effect of on agglutination 47

effect of on sera 83, 85
Serotoxin (synonym for Precipitin) 15
Sera, normal, bactericidal power of 30

normal, effect of filtration on 118

normal, preservation (fluid) by chloroform,
filtration 62

normal, stability in vitro with regard to
precipitins 62, 123

See Acids, Alkalis, Antitoxin, etc.
Serum precipitins See Precipitins, Precipi-
tating Antisera, etc.
Side-chain theory of Ehrlich 5, 7
Signs used in tables. Key to 217-219, 338
Snake venom 5, 8 and following :

agglutinins in 23

haemolytic effects of 19, 29, 43

intermediary bodies in 21

precipitins for 208-209

See Reptilia, Antivenine, Bactericidal,
Neurotoxin
Source of agglutinins in the body 46

of haemolysins 26

of precipitins 26, 132
Species of blood tested with precipitins,

summary 213
Specificity of precipitins 104 et seq., 381
Spermotoxins, Autospermotoxins 24

normal 24

specific, reactivation, etc. 23, 24

term defined 19

See also Anti-antibodies 14
Stability of agglutinating antisera in intra
48

of precipitating antisera and normal
sera in vitro 123
Staphylococcus pyogenes 29

See Bacterial haemolysins
Staphylolysin See Bacterial haemolysins
Streptococcus See Bacterial haemolysins
Substance sensibilisatrice 15
Summary of number of blood species tested
213

444

Index to Subjects

Summary of number of tests on different
bloods 160
of results of 16,000 precipitin tests

(Nuttall) 214-217
of results of 500 precipitin tests (Nuttall

and Strangeways) 335
of results of 2500 precipitin tests (Graham-
Smith) 361-362
of results of Medico-Legal tests 403, 407
Suppuration, reduces complement in serum

29
Symptomatic anthrax antiserum 27
Synonymous terms for bodies in serum 14
Systemic reactions in animals receiving
blood injections 128

Treatment of animals for production of
specific haemolysins 31
of animals, prolonged, leads to decrease

of haemolysin 32
of animals for production of specific
precipitins 51-58
choice of animals for 61
of animals, feeding experiments 53

effect of long-continued treatment 57
injection methods 51-58
Trichotoxins, artificial 25
Trypsin See Ferment
Tryptic digestion, effect on precipitins 111
Typhoid agglutinins, nature of 13, 27
immune bodies in serum 28

Tables to 16,000 precipitin tests by Nuttall
220-311
to precipitin tests by Graham-Smith
364-380
Temperature, effect on precipitin reactions

113 ,SVe Heat
Terminology of bodies in serum 15
Testing See Precipitin tests, Antisera,

Beactions, Medico-legal, etc.
Test-tube racks used in the investigation 68
Tetanolysin 8, 28
Tetanus antitoxin 5, 10, 11, 13
immunity in rabbits 10
toxin in frogs 8
Toxic effects of transfusion of certain

normal bloods 33 et scq.
Toxicity of foreign blood due (in part)
to haemolysins, normal and artificial
33, 40
Toxins 5

fixation in the body 6
See Antitoxins
Toxoid 7, 13

stimulates antitoxin formation in the
body 8
Toxophorous complex (Ehrliob) 7, 12, 13
Thyreotoxin 25

Transfusion experiments witii different
normal bloods 33 et eeq.

Ungnlata, bloods, precipitin tests on 179
et seq., 323
haemolytic effects of their normal sera

on bloods of different animals 36
tables 252-259
Uniceptors (Ehrlich) 11
Urease See Ferments
Urine, absence of precipitins in 127
albumin in, and the precipitin test 133,

407
injections lead to haemolysin formation
26, 31, 32

Washed blood corpuscles and haemolysis

30
Watery dilutions of dried bloods and the

precipitin test 402
Weigert's hypothesis 6

Yeast precipitins 154

Zoological relationships brought out by
powerful precipitating antisera 137,
139

Zooprecipitins 156

"Zwischenkorper" (Ehrlich) 15

Zymophorous group (Ehrlich) 12, 13

Zymotoxic group (EhrUch) 12

CAMBRIDGE : PBINTED BY J. AND C. T. CLAY, AT THE CNIVEBSITY PEE8B.

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