野口英世著

Journal of Experimental Medicine

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S N A K E VENOI~ I N R E L A T I O N TO H2EMOLYSIS,BACTERIOLYSIS, AND TOXICITY.
BY S I M O N F L E X N E R ,M.D.,AND H I D E Y ONOGUCHI,

M.D.

(From the -Pathological Laboratory of the University of Pennsylvania.)
CONTENTS.
PA.GE.
INTRODUCTION.

GENERAL CONSIDERATIONS CONCERNING H ~ M O L Y S I S AND BAC-TERIOLYSIS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

277

VENOM-.AGGLUTINATIO~ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

283

VENOM-H-~EMOLYSIS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

284

Defibrinated blood ..................................................
285
Effect o f h e a t u p o n h m m o l y t i c p o w e r o f v e n o m s . . . . . . . . . . . . . . . . . . . . . . . .
286
Effect o f v e n o m s u p o n ~ v a s h c d b l o o d - c o r p u s c l e s . . . . . . . . . . . . . . . . . . . . . . . .
286
Combined action of venom and ricin.
Relation of agglutination andh~emolysis ........................................................
VENOM-LEUCOLYSIS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

289~. . . . . . . . . . . . . .

A r e t h e h~emolysins ( e r y t h r o l y s i n s ) i d e n t i c a l w i t h l e u c o l y s i n s ? . . . . . . . . . .

289
291

VENoM-ToxXcXTY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

291

Relation of neurotoxie to h~emolytic principle .........................
EFFECTS OF VENOM UPON BACTERICIDAL PROPERTIES OF BLOOD SERUM . . . . . .
Serum venomized in vivo .............................................
Blood mixed with venom in vitro ......................................
The mechanism of the action of venom upon serum ....................
EFFECTS OF ANTIVENIN ON H~EMOLYSIS AND BACTERIOLYSIS . . . . . . . . . . . . . . . . .

292

INTRODUCTION.

294
294
295
298
300

G E N E R A L CONSIDER~.TIONS CONCERNING H~]~MOLYSIS A N D
BACTERIOLYSIS.

[I have long desired that the action of venoms upon blood should
be further examined. I finally indicated in a series of propositions
the direction I wis]~ed the inquiry to take. Starting from these thefollowing very satisfactory study has been made by Professor Flexnerand Dr. NoguehL ~-~y own share in it, although so limited, I mention with satisfaction.--S. WEIR ~[ITCHELL.]
The fol]owing research, which is presented at this time in abstract,
was conducted under a grant from the Bache fund of the National
Academy of Sciences. It forms the first instalment of a new study
20

278

SnaIce Venom in Relation to Heemolysis

of venoms upon which we have been engaged during a year past andwhich, it is hoped, wi]l be continued during anoCher year or longer.
While these studies are still incomplete, the data here given havebeen worked out in detail and may therefore be accepted as final.
On account of the large number of tables which will be given in the
final publication, and the many drawings necessary to illustrate properly the text, more or less delay in bringing out the full work will be
inevitable. But inasmuch as the results of the studies form an integral part of the work on hmmolysis and baeteriolysis, which is now
attracting so much attention among bacteriologists and pathologists,
and as they contain certain facts of fundamental importance bearing
on the theory of these phenomena~ it seems best not to delay publication until the entire series of researches shall have been completed.
At present we shall not give the full bibliogTaphy. Since the fundamental studies of Weir ~itche]l and his collaborators,* the effects
of venom upon the blood and the nervous system of animals havebeen generally recognized. The rapid putrefaction which sets inafter poisoning with venom was also explained by Welch andEwing's ' observations on the loss of bactericidal power of the serumof such poisoned animals. The close relationship of the poison withcertain toxins of bacteria and of higher plants was shown by the discovery of Sewa]l,' of Calmette' and of Fraser ~ that animals could
be immunized from the effects of venom and that they yielded an
active antitoxin. That the poison of venom is not simple, but thatit consists o,f a complex of constituents of a proteid nature was proven
by Mitchell and Reiehert. The time, therefore, seemed ripe for afurther study of the physiological effects of venom upon the blood,
upon bacterial life, and upon tissues~ in the light of the recent studies
upon various kinds of immunity.
For the purpose of these studies dried venom has been employed.
1 S m i t h s o n i a n Contrib. to Knowledge, 1860, xii, and 1886, To. 647.
~Lancet, 1894, i, p. 1236.
a J o u r n a l o f _Physiology, 1887, viii~ p. 203.
4 A n n . de l ' I n s t i t u t Pasteur~ 1894, viii, p. 275.
5 B r i t i s h Med. Journ., 1895, i, p. 1309.

Simon Flexner and ]:Iideyo Noguchi

279

Fortunately several kinds were available~through the kindness ofProf. Reichert~ that of the rattlesnake (Crotalus adamanteus); of
Dr. Joseph ~V[cFarland, that of the water mocassin (Ancistrodon
piscivorus) and of the cobra (Na]a tripudians), and of 5Iessrs. ~V[ulford & C~., tha£ of the copperhead (Ancistrodon contortrix). We
wish to express to these gentlemen our great appreciation of theirkindness.
Before presenting the matter of our studies, it seems best to preface
what we have to say with a brief statement of some of the facts andviews relating to the phenomena of htemolysis and bacteriolysis. In
this preface we shall not distribute credits for the work or the views
here embodied, inasmuch as this will be done in the complete publication. Only so much will be said as is necessary for an understanding of the experimental data relating to venom which are to follow.
By h~emolysis is meant solution of the blood-corpuscles. The term
is usually applied to solution of the red corpuscles, but the white cells
are also subject to a similar solution. It is, therefore, correct to speakof hsemolysis when both kinds of cells, erythrolysis when the red cells
only, and leucolysis when the white cells alone are dissolved. In thissolution of the red cells, which is the type of hsemolysis, the hmmoglobin is separated from the stroma of the corpuscles. The separationof hmmoglobin by hypotonic solutions and through the action of destructive chemical substances is not considered in this article. H~emo]ysis as here employed refers to such separation through the action ofcomplex agents derived from living plants or animals. This form isdistinguished as biologic hmmolysis. Of all such agents the most active
are found in the blood plasma or serum of alien animal species. Others
are the products of cellular activity, such as venom, certain toxic products of bacterial growth, as tetanolysin, staphylotoxin, etc., and stillothers are yielded by some of the higher plants, as crotin from Croton

tiglium.
The most familiar examples of hzemolysis are supplied by the effectsof the transfusion of animal blood into man. It was early discoveredthat the practice was dangerous for the reason that the red corpusclesof the host were dissolved by the foreign blood. This effect wasquickly seen to be due to the serum of the Mien blood and it was observed to take place with equal readiness in vitro. The blood of animals

~80

Snalse Venom in Relation to Hcemolysis

Mso is hsemolysed by foreign sera--the red corpuscles of the rabbit, forexample, being dissolved readily by dog's serum. Some sera have very
high dissolving power, the most active thus far known being that obtained from the eel, which is correspondingly toxic. After admixtureof the corpuscles and foreign serum, solution does not occur immediately: The corpuscles first run together, become clumped, or, as we
now denominate the effect, agglutinated. The dissolving effect ofvenom upon corpuscles is also preceded by a similar agglutination, as
was first shown by Mitchell and Stewart. °There is much similarity in the phenomena of agglutination and oflysis as observed in blood-corpuscles with appearances seen in connection with bacteria. The Gruber-Widal reaction of agglutination,
which has served so well in the diagnosis of typhoid fever and otherbacterial infectious diseases, is of a similar nature. ~oreover, undercertain conditions solution of the agglutinated bacteria may also occur,when bacteriolysis more or less analogous to hsemolysis results. The
well-known Pfeiffer phenomenon, in which cholera spirilla undergo disintegration and solution in the peritoneal cavity of the immunizedguinea-pig is the classical example of baeteriolysis.
The studies of the past two or three years upon the allied phenomenaof bacteriolysis and of hsemolysis have not only demonstrated theirfundamental similarity, but provided chemical explanations of the processes involved. Pfeiffer observed that the serum of immune animals
caused agglutination only of the bacterial species used for immunization; he believed that for complete solution of the bacteria the mixture must be brought into the living body, and fo.r this purpose he chosethe peritoneum of the guinea-pig. Somewhat later Metchnikoff and Border discovered that the same effect could be produced in vitro by theaddition to the immune serum of a small quantity of peritoneal exudate,or even of the fresh serum of an animal. From this experiment it could
be inferred that for agglutination of bacteria certain bodies were required which were in the immune serum; but for solution still othersubstances contained in part in fresh sermn were requisite. That thissecond substance might also be present in the original immune serum
could now be shown; and it was also demonstrated that it is of a very
labile nature and quickly disappears spontaneously. The addition of
fresh serum or exudate restores it. I t may be destroyed by raising thetemperature of the fluid to 56 ° C.
A great advance in our knowledge of cytolysis was made when it was
6 Trans. College of Physicians of Philadelphia, 1897, 3. s., xix, p. 105.

Simon Flexner and Hideyo Noguehi

281

discovered that immunization to blood and body cells gives rise to the
production of lysins. Just as in bacterial immunization lysins for the
special bacteria employed are yielded, so also are evolved analogous
substances for red and white blood cells, for epithelial cells, spermatozoa, etc. Indeed the number and variety of lysins that can be produced
experimentally are limited only by the number and variety of animalcells available. Blood cells of one animal may be used to produce alysin in the body of another animal of the same species--isolysin; inanother species--heterolysin; and success has in rare instances followed
the re-injection of withdrawn blood through which autolysins havebeen produced. Nor is the production of lysins the only result of theinjection of cellular structures. Preceding the solution, clumping of
the cells takes place from which it may be concluded that agglutinins
are also formed.
The factors required for producing solution of cells are similar to
those for causing solution of bacteria under like conditions. Only
when the lyric serum is very fresh will solution be effected; the addition, however, of peritoneal exudate or fresh normal serum to immune
serum which has lost the solvent property, suffices to restore it.
A consideration of the preceding facts shows that the agglutinating
principle is distinct from the dissolving one. This consideration also
indicates that more than one body is necessary to bring about solution
either of bacteria or of animal cells.
An analysis of the phenomena suggests that at least two substances
are requisite. One is stable and contained in the immune sera (whether
for bacteria or animal cells); the other is labile, and while originallycontained in the immune sera, it is lost spontaneously. This latter
substance is a normal constituent of the lymph and blood plasma, forit can be restored by the addition of these fluids.
Experiments conducted in a very convincing way by Ehrlich andMorgenroth indicate: (1) that a special principle is concerned in agglut i n a t i o n - t h e so-called agglutinin; and (2) that two principles areconcerned in lysis. These principles are different in origin. One-that which is stable--is the product of immunization, and, on accountof certain combining properties possessed by it, they call it the ' inter.
mediary body." The other is normally present in the body juices but
has recently suggested the name ,ceptor,' in place of 'intermediary
According to the manner of action he distinguishes , u n i c e p t o r s ' and ~amboceptors.'
Bordercalls this body ,substance s e n s i b i l i s a t r i c e ; ' Metchnikoff
f i x a t o r ; ' P . M u e l l e r , , copula.'
7Ehrlichbody.'

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Snal~e Venom in Relation to Heemolysis

is easily destroyed by heat and tends to disappear spontaneously when
the fluids are removed from the body. This latter principle, on account of the complemental nature of its action, they propose to call the' complement.' 8
There is conclusive experimental evidence that, although the intermediary body unites first with the cells--bacteriM, blood cells, etc.-this substance by itself cannot bring about solution. But after theunion of this intermediary body with the cells the complement is capable of being brought into action, through this intermediation, so thatsolution takes place. The union of intermediate body and cells is conceived to take place through certain combining (haptophore) groups
present in the cells and in the intermediary substance; while the complement is linked through similar combining (haptophore) groups possessed by the intermediary body and itself. The intermediary body,therefore, carries two sets of combining or haptophore groups: one forthe cells and the other for the complement (complementophilie group).
The complement possesses in addition to such a corresponding haptophore group, another group which exhibits fermentative properties(zymotoxic or toxophore group), through the action of which solutionof cells ~akes place.
This conception of lysis applies not solely to tha.t produced by iramunization, but the same factors are believed to be operative in thesolution of blood cells or of bacteria by normal blood sera. Here also
an intermediary body and a complement are brought into action; theonly difference being that, in the one case, the intermediary body is
produced through artificial immunization, and in the other, it is present normally, but whether because of some insidious and unperceived
change, similar to but slighter than artificial immunization, is notknown.
It would carry us too far afield ~o give in detail the elaborate viewsof Ehr]ich and his co-workers as to the origin of the intermediarybodies. Suffice it to say that they are the products of immunization
by bacterial or other cel!s, and are believed by them to be yielded bycertain constituents of cellular protoplasm within the body, desigmatedas ' lateral chains,' which through their haptophore groups are capableof combining with the haptophore groups of protoplasmic constituentsof the bacteria or the cells used for the immunization. When dealing
with the toxic constitution of venom more will be said concerning this
aspect of the subject.
8 T h i s b o d y is c a l l e d ~ a l e x i n ~ b y B o r d e r , a n d p r o b a b l y a g r e e s i n p a r t w i t h t h e b o d yof the same name described by Buchner
Metchnikoff calls it ' cytase.'

Simon Flexner and Hideyo, :Noguchi

383

VENOM-AGGLUTINATION.

For the study of agglutination all the available varieties of venom
were emplo~,ed. Several kinds o.f animal blood--from the dog, rabbit, guinea-pig, sheep, ox, pig, Necturus, and frog--wer e tested.
Either t h e blood was defibrinated or the specimen consisted of the
corpuscles separated by centrifugalization and washed six times, as arule, in 0.8% normal saline solution (washed corpuscles). The different venoms showed slight differences only in the degree of agglutination, with the exceptions of the action on the blood of Necturus andthe frog, which are not affected in weak solutions but show agglutination in stronger solutions (3%).
The usual method consisted in dissolving in normal saline solution
dried venom in strengths ranging from 0.01% to 10%--the last thatof Crotalus adaraanteus. The phenomena of agglutination appear
rapidly in favorable solutions, while in very weak solutions a delay ofsome minutes up to one hour may be noted. The corpuscles whichcome together thus slowly do not show the great modification of shapethat is characteristic of those that fuse more completely and quickly.
I n a general way it may be said that the several varieties of dried
venom with which we experimented gave, when employed in thestrength of 0.5%, wha£ are to be regarded as maximal agglutinations
for mammalian corpuscles. Active agglutinatio.n still takes place in
0.2% solutions, while weaker ones either produce no change at all orshow an imperfect fusion.
The morphological changes need not be described here as thesehave been fully dealt with by Mitchell and Stewart.'
The value of the use of washed corpuscles comes especially fromthe fact that the successio.n of lytic phenomena is eliminated.
Agglutination, therefore, may be studied purely. For this purpose a
5% solution of the corpuscles in normal salt solution was employed.
That complete agglutination has no effect upon subsequent solution(]ysis) of the corpuscles will be shown when treating of the latterphenomena. On the other hand, distinct differences in susceptibility
9 Loc. cir.

~84

Snake Venom in Relation to Heemolysis

to agglutination have been observed. Of the mammalian blood thus
far employed the red corpuscles of the rabbit may be said to be highly
susceptible, while those of the guinea-pig, dog, sheep, swine, and ox
were less responsive in about the order given.
The use of defibrinated Mood permitted observation upon the succession of phenomena of agglutination and heemolysis. In general
it may be said that the first effect of the venom is the production of
agglutination to be fo]lowed by solution after a variable interval,
depending on the kind and strength of the venom and on the temperature. There are, however, notable exceptions in that the rangeof lytic activity of venoms is greater than that of the agglutinating
property. Very weak solutions o.~ venom which no longer cause
agglutination may still be capable of producing solution.
~{oreover, on account of the action of two sets of factors in defibrinated blood--one tending to produce agglutination, and the othersolution of the red corpuscles--the degree of agglutinatio.n is here
less marked than in the washed cells where no lysis occurs. Thisdifference is explained by the fact that a part of the corpuscles go
into solution hero.re agglutination can take place; and hence the extent of precipitation and fusion varies inversely with the susceptibility to lysius. As a consequence, dog's corpuscles which are moreeasily hsemolpsed by venom than any others of the animal bloods
tested by us show the least degree of agglutination. The rapidityof agglutination in any ease is not affected by ordinary temperatures.
Hence a low temperature (0 ° C.) permits, even in defibrinated blood,the separation of the phenomena of lysis and those of agglutination.
At this temperature defibrinated dog's blood behaves as do the washedcorpuscles, the amount of precipitation being, therefore, greater thanat the temperature of the room or of the thermostat.
The agglutinating power of venoms is destroyed by temperaturesof 75 ° to 80 ° C. maintained for thirty minutes.
V~O:~-~OLYSIS.
Unless mention is especially made the same group of animals was
employed in these studies as in the foregoing. The venom solutions

Simon Flexner, and Hideyo Noguchi

285

varied from 5% to 0.0001%, depending upon the source of the corpuscles and the variety of venom. The venoms differ in hsemolytic
power as follows: cobra most active; water mocassin, copperhead,rattlesnake, in less degree in the order named. A similar variationin susceptibility to the reaction could be distinguished in the different
mammalian bloods employed. Thus dog's blood was most quickly andeasily hsemolysed and it responded to the greatest dilutions, wl/fle
the corpuscles of the ox were the least susceptible. The intermediate
animals were in about the following order: sheep, g~inea-pig, pig,
and rabbit. On the other hand, the blood of ]Vecturus is acted uponslightly after longer l~riods, and frog's blood almost not at all after
equally long periods.
The extent of variation in the response of the different bloods is
considerable. Very s~ong solutions of venom (5%) are needed tocause heemolysis ~f corpuscles of the ox, but such great strengths arewithout action upon rabbit's corpuscles, although they are still capableof producing rapid solution of dog's or sheep's corpuscles. Taken
altogether solutions averaging 0.2 per cent of venom have proven themost favorable for bringing out the h~emolytic effect upon btood
generally.
Defibrinated Blood.--In making the tests with defibrinated blood
uniform mixtures were employed. In all experiments 5 per cent ofblood was added to the venom solutions, and the mixtures were kept
at temperatures varying from 36 ° to 37 ° C.
The differences in activity of venoms are shown by the following
series in which minimal active solutions are given for several kindsof blood.
Dog's blood hsemolysed by solutions of Crotalus venom 0.001%; copperhead 0.0005%; water-mocassin 0.0002~; cobra 0.0001%
Sheep's blood hsemolysed by solutions of Crotalus venom 0.002%; copperhead 0.0005%.
Guinea-pig's blood h~emolysed by solutions of Crotalus venom 0.002%;
copperhead 0.001%.
Swine's blood h~emolysed by solutions o~ Crotalus venom 0.002%;
copperhead 0.001%.

286

Snalce Venom in 1?elation to Hcemolys£s

Rabbit's blood h~emolysed by solutions of Crotalus venom 0.005%;
copperhead 0.002%.
Rabbit's blood h~emolysed by solutions of water-mocassin venom
0.002%; cobra 0.001¢.
Ox's blood h~emolysed by solutions of Crotalus venom 0.05%; copperhead 0.02%.

Effect of Heat upon Hoemolytic Power of Venoms.--Temperaturesof 75 ° to 80 ° C. for thirty minutes have no effect upon the h~emolytic
action of any kind of venom. From 90 ° to 96 ° C. Crotalus venom
in solution suffers a moderate reduction in hvemolytic power, while
the remaining venoms are entirely unaffected at these temperatures.
After heating te 100 C. fo,r fifteen minutes the dissolving power ofcobra, moeassin, and copperhead venom in solution is slightly reduced.
Effect of Venoms upon Washed Bloo&Corpuscles.--In no instancewere the washed Mood-corpuscles h~emolysed by venom. Agglutination occurs as already described. :But if the separated serum is restored to each of the several kinds of blood-corpuscles treaied withvenom, lysis takes place.
Certain important differences may be noted. Thus if the quantityof serum added to rabbit's corpuscles exceeds the normal, quicker
and more complete solution occurs than is noted in deflbrinated blood.
A similar but less striking action may be observed with the blood of
the guinea-pig. This action depends upon the union first effeeted
between the red corpuscles and the intermediary body o.f venom, thelatter later combining with the introduced complement of the serum
so that solution takes place. That this is the mechanism of theaction the succeeding experiments prove. But before taking them up
another set of phenomena must be briefly considered.
Rabbit's and guinea-pig's washed blood-corpuscles (hereaftertermed washed corpuscles) are. quickly dissolved by fresh dog's serum.
Dog's corpuscles are but little acted upon by fresh rabbit's serumand not at all by guinea-pig's serum. Rabbit's and guinea-pig's sera
are about equally h~emolytic for each other's corpuscles. Necturus'
serum is highly h~emo.lytic for rabbit's, dog's, and guinea-plg's corpuscles. Frog's serum is less destructive for the mammalian cor-

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287

t,uscles mentioned than that of Necturus, but is still very active.
_N'ecturus' serum and frog's serum are slightly and equally active oneach other's corpuscles.
This dissolving actSon is net noted at freezing temperatures. If,
therefore, washed corpuscles which have been treated with serum forthirty minutes at zero temperature are separated by centrifugalizatian or precipitation, the complement in the serum is unaffected, while
the intermediary body will be found to have been removed from theserum by the corpuscles. In this way the complement for a particular species of corpuscles, free frem the intermediary body, can beobtained. The addition of such complement-containing serum tovenomized washed corpuscles of the same species brings abo.ut hmmolysis, while the addition of fresh washed corpuscles to the treatedserum, from which the intermediary body for them has been remeved,is unattended by solution.
The action of complements, freed from any intermediary body bythis means, upon venomized co.rpuscles o.f different species has also
been studied. The results are of interest. The procedure is as follows: Let us suppose that it is desired to test the effects o.f rabbit's
serum upon venomized dog's corpuscles. The rabbit's serum is firsttreated with washed dog's corpuscles in the cold to withdraw all combining intermediary bodies; the clear serum having been separated is
now added te the venomiied corpuscles, when solution of a slow andlimited nature occurs, thus showing that there exists in the dog's
corpuscles a limited number of receptors capable when venomized of
uniting with rabbit's complement.
Controls for these experiments were made in the following manner: Any serum treated with alien corpuscles at zero tempera±ure
and then separated from the corpuscles by centrifugaHzation has become inactive for this kind of fresh washed corpuscles (tested for
dog's, guinea-pi~s, rabbit's, and Necturus' corpuscles). However,
if to the inactive mixture the same variety of serum heated te 58 ° C.
is added (t~is serum containing intermediary body but without complement) solution ta]~es place.
T~ae degree of interaction of different species of sera minus inter-288

Snake Venom in Relation to Hzemolysis

mediary bodies upon different species of venomized washed corpuscles
is shown by the following series, in which copperhead venom is usedthroughout. 1 cc. of 0.2% solution of venom is mixed with 0.05 cc.
of washed corpuscles and 0.5 cc. of complement, with these results:
Dog's corpuscles and rabbit's c o m p l e m e n t = s l o w and imperfecthemolysis.
Dog's corpuscles and guinea-pig's c o m p l e m e n t = slight hemolysis,
more marked than preceding.
Rabbit's corpuscles and dog's e o m p l e m e n t ~ r a p i d and imperfecthemolysis.
Rabbit's corpuscles and guinea-pig's complement ~-~ weak and imperfect hemolysis.
Guinea-pig's corpuscles and dog's complement ~ rapid and imperfecthemolysis.
Guinea-pig's corpuscles and rabbit's complement ~ slow and imperfect h~emolysis.
Dog's corpuscles and Neeturus' complement ~ slight and imperfecthemolysis.
Guinea-pig's corpuscles and Neeturus' complement ~---no action.
Rabbit's corpuscles and Necturus' complement-~-no action.
Venom solution treated with dog's, rabbit's, aad guinea-pig's
washed corpuscles in succession gives up to each a part of its intermediary bodies. No one kind of corpuscle is capable of fixing theentire content of intermediary bodies. The supernatant fluid probably contains still other intermediary bodies capable of fixation by
still other corpuscles. I f to the several kinds of venomized corpuscles
here mentioned different complements are added, then, as shown in
the foregoing series, lysis will or will not take place, depending on
the nature of the complement employed; but so long as the complement is foreign to the corpuscles it never causes complete solution.
From these results the following conclusions are warranted: (1)Venom contains several or many intermediary bodies. (2) Thesebodies show specific affinities for certain complements. I n addition
to this there is evidence that the many susceptible corpuscles contain,
besides specific haptophore groups for intermediary bodies, certain
common haptophore groups, which are shared, perhaps, by all vulnerable corpuscles.

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289

Combined Action of -Venom and R iein. Relation of Agglutinationand Hcemolysis.--Agglutination produced by venom does not affectlysis. But on the other hand when l•sis takes place quickly, agglutination may fail or may appear imperfectly. The principles causing the two phenomena are distinct in the manner of combination andin action. That the agglutinative and the lytic principles are different is now proven; and there is evidence that they act upon different
constituents of the red cells. Thus, if ricin, a strong agglutinator, ispermitted to act upon red corpuscles for periods under thirty minutes, then upon the addition of venom lysis ensues in about the average time and proceeds normally. If, however, the riein has acted
for two or more hours, then solution by venom still takes place, but
the stroma of the corpuscles remains in the bottom of the test-tube asa white conglutinated mass. From this it appears that agglutination
brings about a kind of coagulation of the stroma, from which, throughthe action of the h~emolysin in venom, h~emoglobin has been released.
l~icin is without action upon venom itself, and conversely ricin isequally unaffected by venom.
V~NO~-L~UeOLVSIS.

I n the blood snake-venom causes destruction of the leucocytes aswell as of the red cells. I n order to ensure a more accurate study of
its action upon the white blood-cells, these were obtained in larger
quantities and without admixture of red cells by injecting positively
chemotactic substances into the pleural and peritoneal cavities of the
rabbit. For this purpose cultures of B. megatherium killed by heat
were injected into the pleural cavity and sterile bouillon into theperitoneum.
From eighteen to twenty-four hours after the introduction of B.

megatherium into the pleural cavity, fluid rich in leucocytes may be
withdrawn by means of capillary tubes without sacrificing the animal.
The same procedure employed twenty-four hours after the injectionof bouillon yielded a fluid less rich in leucocytes, and consequently
this second method was not extensively employed.
The leucocytes in the fluids thus obtained could be separated ac-

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Snalce Venom in Relation to Hoemolysis

cording to size and granulation into ]ymphocytes (20 to 95~ of thetotal leucocytes), finely granular, medillm-sized cells (60~), larger
non-granular cells (3~g), still larger irregular and coarsely granularcells (4~), and others again somewhat smaller but showing very coarse
granules (6~). The susceptibility to the destructive effects of venom
varied somewhat for the different cells. Those of the largest sizewith coarse grannles are most quickly affected; next to these comethe finely granular varieties, the lymphoeytes showing the least injury of all.
In studying the changes taking place in the leucocytes under the
influence of venom a warm stage (37 ° C.) was used, the edges of thecover glasses having first been sealed with vaseline,. The venom
solutions varied from 10~ to. 0.002~. The weakest effective solution
was that of c~)bra venom (0.002~), whereas in the case of the rattlesnake and of the moeassin, 0.009~ and 0.005~g respe3tively, caused
definite changes.
Only the granular cells showed motility. Weak active solutions
are without immediate effect on motion but begin to manifest an inhibiting action after about one hour, the controls being still me.tileat the end o.f two hours or longer. After the motility ceases, thecells in general, except the ]ymphocytes, show increased granulation
due to the appearance of coarser and more numerous granules in the
protoplasm, the nuclei coincident]y becoming more distinct. After
six hours the majority of the largest granular cells have already disintegrated, the nuclei having been liberated. After twenty-fourhours most of the medium-sized granular cells have suffered disintegration~ while the lymphocytes show but slight and inconspicuouschanges. Stronger solutions, varying from 0.2~ to 10~, cause instantcessation of motility and rapid agglutination without distinction ofvariety of cells. Within five to thirty minutes thereafter dissolution
sets in, affecting first the largest, then medium-sized cells, and finallythe small ]ymphoeytes.
There are variafions in the activities of the several venoms and in
the completeness of solution of the cells. Rattlesnake venom is far
less active than that of the cobra. Thus in 2~ solutions cobra venom

Simon Flexner and Hideyo Noguchi

291

causes complete solution in thirty minutes while that of the rattlesnake requires two hours to bring about the same result.
The effects upon washed leucocytes differ from those described inthat venom sohtions cause agglutination, but with the production of
only very slight lysis.

Are the H~emolysins (Erythrolysins) Identical with Leucolysins?
--Copperhead venom (1 mg. in 4 cc. of normal saline) was treatedwith washed rabbit's red corpuscles at. the thermostat temperature forthirty minutes, until the supernatant fluid after centrifugalization
was without action upon defibrinated rabbit's blood. This solution
when brought into contact with leucocytic fluid was without agglutinating action upon the cells while still causing their solution inabout thirty minutes. On the other hand, the parallel experiment inwhich venom solution was treated with washed leucocytes yielded a
fluid still active for defibrinated blood.
The co.nclusions from these experiments are as follows:(1) Venom contains principles which are agglutinating and dissolving for white blood-corpuscles.
(2) The agglutinating principles may be identical for both whiteand red cells.
(3) The dissolving principle for leucocytes is distinct from that for
red cells.
(4) In order that solution of venomized leucocytes shall occur acomplement-containing fluid is required.
(5) The several varieties of white cells of the rabbit's blood show
different susceptibilities to the action of venom.
VENOM-TOXICITY.

For the study of the toxic principles copperhead venom was chiefly
employed. The animal selected for these experiments was theg~inea-pig. The method of procedure was the following: We first
determined for the particular sample of venom to be used the minimal lethal dose. This was found to be 0.3 rag. for a guinea-pig
weighing from 250 to 300 grammes, death resulting within 24 hours.
A dose of 0.6 rag. caused death in from two to three hours, and of
0.9 rag. in from 30 to 45 minutes.

292

Snake

V e n o m i n Relat.ion to H ~ m o l y s i s

Our special purpose was the determination of the existence of neutralizing substances for venom in the tissues of the body. The foplowing tissues and organs were employed: brain, liver, spleen, kidney,
voluntary muscle, adrenal gland, and blood. For this purpose thetissues were first washed in tepid, sterile, normal saline and a weighed
quantity (2 grammes) was taken. This was triturated in a sterilemortar and mixed in test tubes with three times the minimal lethal
dose (]~. L. D.) of venom.
The mixture was now placed in the thermostat where it remainedfor one hour. I t was then centrifugalized and the, supernatant fluid
was injected into t h e guinea-pigs. I n t h e course of the. experiment
the original volume of the venom-solution suffered a loss amountingo~ an average to o~e-third of the whole volume. There should, therefore, a f t e r this subtraction remain behind at least twice the minimallethal dose (2 X ~/[. L. D.).
In the case of the blood, washed corpuscles were employed in excess, the other steps remaining the same as in previous experiments.
The results of these experiments are as follows:
Control . . . . . . . . . . . . . . .
Dead iu 45 minutes.
Brain . . . . . . . . . . . . . . . . .
'~19 hours.
Blood . . . . . . . . . . . . . . . . .
"3 hours and 50 minutes.
Adrenals . . . . . . . . . . . . . .
"2 hours and 35 minutes.
Spleen . . . . . . . . . . . . . . . .
"2 hours and 10 minutes.
Liver . . . . . . . . . . . . . . . . .
:'1 hour and 30 minutes.
Kidney . . . . . . . . . . . . . . .
"1 hour and 55 minutes.
Muscles . . . . . . . . . . . .
...
"1 hour and 30 minutes.
I n the next experiment 2 IV[. L. D. of venom were employed. The
control died in five hours. The animals receiving the venom solution
treated with the organs, etc., reacted as follows:
Brain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Blood . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Liver . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Survived.
Dead in 28 hours.
"
19 hours.

R e l a t i o n o f ~ e u r o t o x i c to Ha~molytic P r i n c i p t e . - - T h a t these two
principles are distinct is r e n d e r e d probable by the effects o.f washedred-corpuscles and of brain tissue respectively upon the toxicity of

Simon Flexner and ]=[ideyo Noguchi

293

venom. Blood-corpuscles remove little or perhaps none of the toxic
constituent that brain cells do away with in toto. The proof of difference can, however, be brought in another way: l%ur •. L. D. ofvenom were treated with an excess of red corpuscles and the supernatant fluid was injected into a guinea-pig; death ensued in 30 -minutes. The same quantity of venom having been treated with fourgrammes of brain emulsion, the supernatant fluid injected into aguinea-pig caused death in 48 hours. The experiments in which 9)£. L. D. of venom were used resulted, (1) in the case of the bloodin death in 25 minutes; and (2) in the case of the brain (7 grammesof brain having been employed) in 25 hours.
The supernatant fluid from the brain emulsion was strongly agglutinating and hsemolytic for defibrinated blood, while that from thewashed corpuscles had lost all these properties. The supernatantfluid from the brain emulsion, when treated with an excess of washedcorpuscles, re-centrifugalized and the fluid then injected, is non-toxic
for guinea-pigs.
These experiments show: (1) that the neurotoxic and the hsemolytic principles are physiologically distinct; (2) that while the chief
toxic constituent unites with the nerve cells, in multiple ~ . L. D.
from which the neuro¢oxic principle has been removed a quantity ofheemolysin may be contained sufficient to bring about fatal intoxication.
These results are in keeping with the views expressed by Ehrlichand supported by Wassermann and Takaki's experiments on the fixative power of cells for certain groups of toxic substances. They tend,
therefore, to support the hypothetical considerations of Ehrlich on
which he bases his well-known lateral-chain theory of immunity. Expressed in the terms of this hypothesis brain cells may be said tocontain the receptors for the neurotoxic constituent of venom, whereas
blood cells furnish the receptors for the h~emolytic principle; thesereceptors are dlstinct and specific, and are not contained to any considerable amount, and perhaps not at all, in the liver and kidney cells
and, if at all, in small quantity only in adrenal cells. Walter ~[yersfound that the adrenal cortex possessed a very feeble combining power
21

294

Snalce Venom in Relation to Hoemolysis

for cobra venom, most marked in mammals (sheep) in which thecortex of the organ is well developed. _~Ie also observed little effectfrom the adrenal of the guinea-pig.
E F F E C T S OF V E N O M U P O N B A C T E R I C I D A L

P R O P E I % ' r l E S OF B L O O D SEI~UM/°

The animals employed were the dog, rabbit, and N e c t u r u s ; thevenoms belonged to the cobra, moccasin, copperhead, and rattlesnake,and the bacteria were B. typhi, B. coli, and B. anthracis. The
method consisted in (1) introducing venom into the animal and drawing the blood from the femoral artery into sterile Nuttall bulbs; (2)permitting the blood from the normal animals to enter Nuttall bulbs
in which the venom solution was contained; (3) admixture of thevenom in sterile solution (heated for 4 days to 56o-60 ° C.) with separated serum.
The bactericidal effects of the normal sera were i~rst established.
Rabbit's serum is highly destructive for B. typhi and B. anthracis andleast for B. coll. Dog's serum is highly destructive for B. typhi.
Necturus' serum is also very destructive to B. typhi and B . coil.
It is without marked effect on B. anthracis.
S e r u m Venomized in vivo.--Cobra venom was most active. Bloodfrom rabbits which had received 10 rag., taken 57 minutes after the
injection, showed great loss of bactericidal properties.
ec. venomized serum employed.
B. anthracis
B. colt
Immediate
1841
4254
After 1 hour
767
3125
"
3 hours
2488
13460
"
6
"
Innumerable
Innumerable
" 24
"
"
"
Experiment L X X I . - - 1

B. typhi

7152
4130
4320
17280
Innumerable

The controls for this experiment showed complete destruction of
all bacteria, or, as in a few experiments, of all except B. colt, which
10 I n o r d e r t o d e t e r m i n e w h e t h e r a n y e f f e c t is p r o d u c e d o n t h e g r o w t h o f b a c t e r i ab y t h e p r e s e n c e o f v e n o m in c u l t u r e m e d i a , v a r y i n g s m a l l q u a n t i t i e s o f v e n o m w e r e
a d d e d t o n u t r i e n t a g a r - a g a r . T h e b a c t e r i a - - B , anthracis~ B. coli~ and B. t y p h i - - g r o w nupon these tubes underwent rapid involutions and exhibited marked plasmolysis, ascompared with control tubes of the same organism.

295

SimoiL F l e x n e r and t t i d e y o :Noguchi

showed considerable diminution until after six hours when increasebegan.
Experiment L X X . - - 3 0 rag. rattlesnake venom injected; blood taken
after 45 minutes. 1 cc. venomized serum employed.
B. anthracis

Immediate
1 hour3 hours
6
"
24
"

838
756
2930
Increase
Innumerable

B. coli

9940
6240
Increase
Innumerable
Innumerable

B. typhi

5750
3654
6219
About 10000
Innumerable

Blood m i x e d with Venom in v i t r o . - - I n this series rabbits only
were employed. The veno,m solutions w e r e placed in Nuttall's bulbs
and the blood f r o m the femoral artery was permitted to stream into

them. I n each experiment 6 mg. of venom were mixed with 20 to
30 cc. of blood. Coagulation was v e r y slow or completely inhibitedand the serum was obtained when necessary by centrifugalization.
I t ]nvariably contained hsemoglobin.
Experiment L X X I I I . - - 1

Immediate
1 hour3 hours6"

B. anthracis

B. coli

B. typhi

1644
2080
18930
Innumerable

4580
10760
149740Innumerable

4085
4870
24730
Innumerable

Experiment L X X I I . - - 1

Immediate
1 hour3 hours6"
24"

cc. of venomized serum employed.

cc. venomized serum employed.

B. anthracis
736
407
860
5220
Innumerable

B. coli
3720
2340
22210
Innumerable

B. typhi
1275
920
8720
Innumerable

This series of experiments m a y be open to the criticism that theincreased nutritive value of the serum because of the h~emoglobinpresent m a y have been the cause of the effects noted; as a control,Snake Venom in _Relation to Ha~molysis

296

therefore, peptone was added to the s e r u m in the proportion of 6 rag.
of peptone to 20 cc. of serum.

Experiment L X X X . - - P e p t o n e added to rabbit's blood: 1 cc. employed.
Immediate
1 hour3 hours
6"
24"

B. anthraeis
1043
193
87
22
0

B. coli
5120
2240
578
520
Innumerable

B. typhi
7430
1534
71
262
About 20000

F r o m this e x p e r i m e n t it follows that i m p r o v e m e n t in nutritive
valuo reduces bactericidal effect b u t in f a r less a m o u n t than is no,ted
in the parallel ease of venom.
T h a t the nutritive change is u n i m p o r t a n t is shown b y the first experiments, in which the poisoning was done in vivo and also b y those
to follow in which v e n o m was added directly to the separated serum.

Experiment LXXXV.---Rabbit's serum 1 cc. with rattlesnake venom
1 rag.

B. anthracis
Immediate745
1 hour594
3 hours4486
6"About 100000
24"'
Innumerable

B. colt
3990
4667
12120
Innumerable

B. typhi
5430
3136
43430
About 200000
Innumerable

Experiment" L X X X I I . - - D o g ' s serum 1 ec. with copperhead venom.

Immediate
1 hour3 hours

624""

Venom 6 rag.

Venom i rag.

B. typhi
8860
21120
65250
Innumerable

B. typhi
3572
6525
14950
Innumerable

Control; norm. ser.

B. typhi
5808
584
184
92
0

I n order to d e t e r m i n e the least quantity of v e n o m required to remonte the bactericidal properties of the s e r u m v a r y i n g quantities ofcopperhead v e n o m were employed. D o g ' s serum was chosen with
B. typhi. I n each case 1 cc. of serum was used.

Simon Flexner and t t i d e y o •oguchi

297(a).--1 cc. dog's serum and varying amounts

Experiment L X X X V 1 1of copperhead venom.
Venom l / 2 m g .
Immediate5970
1 hour6240
3 hours
12810
6

"

24

'~

Innumerable

1/5mg.
3070
3960
10000
]000001/10mg.
4290
1830
6730
13140

1/20mg.
4940
9620
1350
172

Innumerable Innumerable About 10000

1/50mg.
3350
920
593
15
0

Fr om this it may be concluded that the specimen of venom employed by us destroys the bactericidal properties of dog's serum when
added in the proportion of 1/20 mg. of venom to 1 cc. of serum; andthat 1/50 rag. in the same quantity of serum is practically without
action.
I n view of these positive results the partial inaction of venom uponN e c t u r u s serum is both remarkable and important.
Experiment C I I . - - 1 cc. ~Vecturus serum employed.
B. anthracis

Immediate
1 hour3 hours
6
24

"
'"

4215
6950
18340
50170
Innumerable

B. coli

B. typhi

5127
453
84
4
0

8350
233
12
0

Experiment C I I I . - - S a m e as above and copperhead venom 6 rag.

Immediate
1 hour3 hours
6"
24"
Control:

B. coli

B. typhi

5237
615
95
180
2950

3846
394
11
2
3105

Necturus serum 1 cc. -~ peptone 6 mg.
B. coli

B. typhi

Immediate
1 hour3 hours

9270
3810
632

4530
577
92

6
24

78

2

1850

927

"
"

Snake Venom in Relation to Hcemolysis

298

Experiment CIV.--ATecturus serum 1 co. -+- copperhead venom 1 rag.
Immediate
1 hour3 hours
6"
24"

B. coli

B. typhi

6240
1170
583
25

7360
833
96
13

0

0

I t m a y be remarked that Necturus is highly r e f r a c t o r y to venom.
A n animal weighing 250 grammes received without effect 0.05 grm.
venom, equivalent to 160 ~[. L. D. for the guinea-pig.
T h e effect of heat upon venom in relation to its action upon thebactericidal prope~ies is of interest. F o r this purpose cobra, rattlesnake, moccasin, and copperhead venoms were studied. Tempera,
tures varying f r o m 75 ° to 90 ° C. were employed, and the heatedvenoms were mixed with the streaming blood in Nuttall's bulbs andwith the separated serum. The venom was kept at the lower temperature (75 °) for 30 and the higher (90 °) for 15 minutes.
T h e heated venom acts just as the unheated except in the case of
rattlesnake venom, the effect of which is somewhat diminished at thehigher temperature, 90 ° C.

The Mechanism of the Action of Venom upon Serum.--That thebactericidal action of serum depends upon the intelnnediary body andcomplement seems established. T h a t the influence of venom uponthis p r o p e r t y does not depend upon changes in the nutritive valueof the serum the foregoing experiments prove conclusively. I t is
t h e r e f o r e possible that venom acts injuriously upon the intermediary
body or the complement or upon both bodies at the same time. T h ecomplement is destroyed by heating serum to 56o-58 ° C . - - a temperature which does not affect the i n t e r m e d i a r y body.

Experiment XCVIII.---To test effect of venom on the intermediarybody.
(1) Copperhead venom 1/20 rag.; rabbit's serum 1 co., and rabbit's
serum heated to 58 ° C., 1 co. (2) Control; rabbit's serum heated to
56 ° C.

Simon Flexner and ~ i d e y o Noguchi

Immediate
1 hour3 hours
6
"'

(1)

(2)

B. typhi
4990
5800
18840
Innumerable

B. typhi
5320
7800
12400
Innumerab]e299

Experiment X C I X . - - T o test effect on the intermediary body.
(1) Copperhead venom 1/10 rag.; dog's serum 1 cc.; dog's serum
heated to 56 °, 1 cc. (2) Control; serum heated to 56 ° C.

Immediate
1 hour3 hours
6
"

(1)

(2)

B. typhi
2270
2680
71950
Innumerable

B. typhi
3440
3950
12800
Innumerable

From these experiments the conclusion can be drawn that venom
is without action upon the intermediary body contained in dog's andrabbit's serum.
The next experiment was to determine whether any action was exerted by venom upon the complements of these sera. For the purpose of obtaining the serum-complement free from the intermediarybody, the rabbit was treated with dog's serum heated to 56 ° C.
In this way the anti-intermediary-body was obtained, which, whenheated to 56 ° C. (to remove rabbit's complement) and added to fresh
dog's serum neutralized the action of the latter upon rabbit's corpuscles. From this it could be concluded that the intermediary bodyof the dog's serum was neutralized b y the anti-intermediary-body contained in the immunized rabbit's serum, leaving behind the pure
dog's complement in the fluid.
Experiment X C I X (a).--Action on complement.
(1) Fresh dog's serum 1 cc., and copperhead venom 1/10 rag., anddog's complement 1 cc.11 (2) Control, dog's serum 1 cc., and 1/10 rag.
venom.

n To obtain these complements fresh dog's or rabbit's serum was treated with rabbit's or guinea-pig's serum containing the anti-intermediary-body which was heated800

Snake Venom in Relation to Hoemolysis

Immediate
1 hour3 hours
6
"
24
"

(1)

(2)

B. typM

B. typhi

5270
930
28
15
0

4360

5980
25410
Innumerable

A similar experiment in which anti-lntermediary-body for rabbit's
serum was produced in the guinea-pig gave practically identical results except that when 1/10 rag. of venom was employed the neutralizing effect of this quantity on the complement was also exerted upon
the second quantity of complement added.
.Experiment X C V I I I (a).--Copperhead venom; fresh rabbit's serum
1 cc., and rabbit's complement 1 cc.

Immediate
1 hour3 l~ours
6"24"

B. typhi
1/10 rag. venom
4590
3740
1850
4900
Innumerable

B. typhi
1/20 rag. venom
3280
1360
730
110
0

From the experiments under the present heading the following
conclusions are warranted:(1) All venoms when used in suitable quantities destroy the bactericidal properties of many normal blood sera.
(2) The manner of this destruction consists in the fixation of theserum-complements by the venoms.
(3) Venoms have no action upon the intermediary bodies of serum.
(4) I f the venom is incapable of uniting with the aerum-eomplements (Necturus) then the original bactericidal properties remain
unaffected by the presence of the venom.
EFFECTS OF ANTIVENIN ON S~EMOLYSlS A N D BAOTERIOLYSIS.
T h r o u g h the kindness of Dr. ~eFarlandof Calmette's antivenin.

w e secured a s m M l vial

This was used to test the restraining action

Simon Flexner and Hideyo Noguchi

301

upon venom hremolysis and venom anti-bacteriolysis. The antivenin
was first proven to be non-h~emolytic for rabbit's corpuscles and to
improve slightly the nutritive value of fresh rabbit's serum.
Erythrolysis by cobra venom on rabbit's corpuscles is prevented if
neutralization by antivenin is effected. Thus 2 rag. of venom + 1 cc.
of antivenin is still ]ytic although action is retarded; 1.5 rag. ofvenom + 1 cc. of antivenin caused slight h~emolysis after 24 hours,
while 1 mg. + 1 cc. was without action.
In the case of rattlesnake venom 1 cc. of antivenin neutralized 3
rag. of the poison.
Leucolysis was affected in approximately the same degree as in thecase of erythrolysis.
The effect on bacteriolysis is equally marked. W h e n cobra or
rattlesnake venom is treated with a neutralizing quantity of antivenin
and fresh serum is added the resulting fluid behaves in a manner similar to that of the control mixture of normal fresh serum and antivenin.
Antivenin, therefore, neutralizes venom and removes both thehmmolytie and the anti-bacteriolytic actions.