NationalLibraryOfScotland/medical-history-of-british-india huggingface dataset:
18
destructive action in vitro on the red blood corpuscles of the rabbit. The method
which was used in these experiments was the following. The venom was
dissolved in a solution of common salt which was isotonic8 for the red cells
of the rabbit. The strength of this solution was 1 c.c.= 2 milligrammes, namely,
0.2 per cent. From this original solution a succession of two-fold dilutions,
made with salt solution of the same strength as used for the original solution
of venom, was prepared in a series of test tubes, so that each dilution con-
tained, half the amount of the poison of the one just above it in the series.
A convenient quantity, namely, 0.5 c.c., of each dilution was then measured
into a series of small test-tubes. To each tube a measured quantity, namely,
0.005 c.c., of fresh blood was added by means of a graduated capillary pipette.
A control tube of salt solution and blood alone was also prepared. The
preparations were allowed to stand at laboratory temperature (about 25° C.)
for 20 hours, when the results were recorded. At the same time observations
were made in an exactly similar manner with a heated solution of venom. A
0.2 per cent, solution was heated in a water bath for half an hour at 75° C. and
then filtered. The filtrate was treated in the same way as the original un-
heated solution. The results of these observations are detailed in the protocols,
series No. V. A glance at this table enables us to come to the conclusion
that the venom of Bungarus fasciatus has only a slight destructive action
on the red corpuscles in vitro and, further, that the heated venom has the
same hæmolysing power as the unheated venom. In consideration of this
result, and also in consideration of the observations in vivo to which attention
has been already drawn, we can conclude that the action which the venom of
Bungarus fasciatus has on the red blood corpuscles is slight and of quite minor
importance compared with its action on the nervous system.
Toxicity of the venom of Bungarus fasciatus.
In order to compare the toxicity of this poison with the toxicity of the
venoms of other species of snakes with which I have worked, several series of
experiments were undertaken. The results of these observations have now to be
put on record.
In the first place, the minimum lethal dose for rabbits of the venom
under consideration was determined both by intravenous and subcutaneous in-
jection. A reference to the protocols (series Nos. VI and VII ) will show that
it was found that 0.7 milligramme per kilo. was the smallest quantity which
killed, a rabbit when the injection was made directly into the blood stream,
but that 2.5 to 3 milligrammes per kilo. were required to cause death when
the injection was made under the skin.
In the second place, a series of experiments was undertaken to determine the
19
minimum lethal dose of this venom for rats of about 118 grammes in weight.
A glance at the protocols, series No. VIII, will show that the results following
the injection of small quantities of venom into rats were variable. Thus, it was
found that in two instances 0.5 milligramme caused a fatal result, while in an-
other case 1. 5 mi l l i grammes brought on very urgent symptoms but di d not
kill. Again, in one instance one milligramme caused death, while in another
experiment this amount failed to kill. It appears, therefore, that in order
to be certain of producing a fatal result a greater amount than 1.5 milligrammes
would have to be given.
In the third place, the minimum lethal dose of the venom of Bungarus fas-
ciatus for monkeys, when the poison is injected subcutaneously, was determined.
A reference to the protocols, series No. IX, will show that this amount was
ascertained to be about 3 milligrammes per kilo.
With a view of comparing the toxicity of this poison with that of cobra
venom, two series of experiments were made to determine the minimum lethal
dose of this latter venom for rabbits and monkeys, respectively, when the injec-
tions are made subcutaneously. It was found (vide protocols, series Nos. X
and XI) that 0.35 milligramme per kilo. for rabbits and 0.25 milligramme per
kilo. for monkeys were the smallest amounts which were able with certainty to
produce death. In previous series of experiments,9 which have been already
published, I demonstrated that the minimum lethal dose of cobra venom for rats
of about 118 grammes in weight was about 0.05 milligramme : it was found to
vary between 0.04 and 0.07 milligramme according to the sample of the venom
employed. From these data we can construct the following table, showing the
minimum lethal doses of the venoms of the cobra and Bungarus fasciatus for
three species of animals, when the poison is injected subcutaneously :—
Rabbits.
Rats.
Monkeys.
.
Cobra venom .
0. 35
milligramme
per kilo.
0.04 to 0.07 milli-
gramme.
0.25 milligramme per
kilo.
Venom of Bungarus fasciatus . 2.5 to 3 milligrammes
per kilo.
1.5 milligrammes.
3
milligrammes per
kilo.
From this table it will be seen that cobra venom is at least ten times as
toxic as the venom of Bungarus fasciatus.
It is more difficult to compare the degree of toxicity. of the venom of
Bungarus fasciatus with that of the venom of Daboia Russellii. We have seen,
however, that it requires about 10 milligrammes per kilo. of the venom of
Bungarus fasciatus, injected intravenously into a rabbit, to cause intravascular
clotting, while as small a quantity as 0.1 milligramme per kilo. of daboia
poison injected in the same way kills a rabbit from thrombosis. As regards this
D 2
20
particular physiological action daboia venom is about 100 times more potent
than the poison of Bungarus fasciatus. Injected subcutaneously into a rabbit
daboia venom varies greatly as regards the smallest amount which can cause
death. As a result of a great number of experiments on rabbits I have found
t hat a dose of from 1 t o 2 mi l l i grammes per ki l o i s requi red i n most cases. Thus,
when given subcutaneously the venom of the daboia is still somewhat more
toxic than the poison of Bungarus fasciatus, for we have seen that from 2.5 to
3 milligrammes per kilo of this latter venom is required to kill a rabbit.
The neutralising power of two antivenomous sera for the venom
of Bungarus fasciatus.
I have now to place on record some observations which were made to test
the neutralising power of two antivenomous sera for the poison of Bungarus
fasciatus. The details of these observations have been already incorporated
in a paper on the ' Specificity of Antivenomous Sera '. 10 It is, however,
thought that the present observations would not be complete unless some
reference was made to the results obtained.
In the first place, two series of observations were made with Calmette's
serum, an antivenomous serum which this observer claims to be efficacious
against the venom of all species of snakes. 11 In both series of experiments rab-
-bits were the animals employed, and the injections were made directly into the
blood stream. We have already seen (vide protocols, series No. VI) that the mini-
-mum lethal dose of Bungarus fasciatus venom for rabbits by intravenous injec--
tion is 0.7 milligramme per kilo. In the first series of experiments ( vide proto-
-cols, series No. XII A) four milligrammes, i.e., about six lethal doses, were
mixed in vitro with different quantities of serum. The mixture was allowed
to stand at laboratory temperature (about 25° C.) for half an hour. It was then
injected into the marginal vein of the rabbit's ear. A glance at the table appended
will show that even 5 c.c. of serum were of no avail to save the life of the animal,
the rabbit which received this amount of serum along with the test dose dying after
practically the same interval of time as the control animal. In the second series
of experiments with Calmette's serum and the venom of Bungarus fasciatus ( vide
protocols, series No. XII B) two milligrammes of venom, i.e., about three lethal
doses, were used as the test dose. With this exception the same technique
was employed as in the previous series of experiments. It will be seen by refer--
ence to the protocols that even with this test dose, small multiple as it was of the
minimum lethal dose, 5 c.c. of serum were not able to preserve the life of the
animal. The rabbits, however, which received 4 c.c. and 5 c.c. of serum, respec-
21
tively, lived somewhat longer than the control animal. Inasmuch as the test
dose in these cases was such a small multiple of the minimum lethal dose, this
result does not appear to me to influence the conclusion to which we are led by
these two series of observations, viz ., that Calmette's serum has practically no
neutralising power for the venom of Bungarus fasciatus.
In the second place, a series of experiments was made with a serum prepared
by Dr. Tidswell of Sydney with the venom of the Australian tiger-snake (Hoplo-
-cephalus curtus). This snake belongs to the same sub-family, v i z
E l a p i n æ , o f
Colubridæ, as Bungarus fasciatus. The details of the preparation of this venom
and the results which he obtained with it are to be found in a paper by
Dr. Tidswell in the Australasian Medical Gazette. 12 It need only be mentioned
here, that Tidswell found this serum to be strongly antitoxic to the venom with
which it was prepared, but to be quite inactive towards the venom of three
other Australian snakes. For this series of experiments rabbits again were the
animals employed. The test dose of venom used was 4 milligrammes per
kilo., namely, about six lethal doses. This test dose and different quantities of
serum were mixed in vitro. The mixture was allowed to stand at laboratory
temperature for half an hour. It was then injected into the marginal vein of the
ear of a rabbit. It will be seen from the protocols that the rabbit which received
5 c.c. of serum along with the test dose died in even a shorter time than the
control which received no serum. We have, therefore, to conclude that the
serum had failed to neutralise even a modicum of the venom. The bearing of
the results of these observations has already been entered into in full in the paper
from which they are now summarised. It need not further be discussed here.
We may summarise the main conclusions to be drawn from the above
observations as follows :
—
(1) Cases of intoxication with the venom of Bungarus fasciatus can be
divided into three classes :—
(a)Cases in which rapid death due to intravascular thrombosis follows
intravenous injection of large quantities of venom.
(b)Cases which present acute nervous symptoms and which terminate
fatally within 2 or 3 days after the injection of the poison.
These cases are indistinguishable, as far as symptoms are
con-cerned, from cases of cobra venom intoxication.
(c)Cases which run a chronic course and end fatally between the 6th
and 12th day after the injection of the poison. Such cases are
peculiar to intoxication with this venom and present marked
special symptoms. A histological examination of the nervous
system in these cases shows a well marked primary degeneration
of the cells of the central nervous system.
22
(2)The venom of Bungarus fasciatus has an action on the blood plasma
in vivo. Injected directly into the blood stream in considerable
quantity it produces an increase of the blood coagulability which
may end in a fatal intravascular thrombosis. In the more or less
chronic cases of intoxication there is a deficiency in the blood coa--
gulability, but this deficiency cannot be said to be a factor in the
ætiology of any symptoms observed. No definite results were ob-
-tained with citrate plasma in vitro.
(3)The venom of Bungarus fasciatus has only a slight destructive action
on the red blood corpuscles. This action is of little or no account
as far as the production of symptoms is concerned.
(4)The venom of Bungarus fasciatus is much less toxic than the venom
either of the cobra or of the daboia.
(5)The venom of Bungarus fasciatus is not neutralised either by the serum
prepared by Calmette of Lille or by a serum prepared by Dr.
Tidswell of Sydney with the poison of another colubrine snake,
namely, Hoplocephalus curtus.
Protocols.
SERIES No. I.— Experiment to estimate the amount of coagulable proteid
contained in the venom of Bungarus fasciatus.
A considerable quantity of the dried venom, viz., 93 milligrammes, having
been carefully weighed, was dissolved in a measured quantity of doubly-distilled
water. The strength of this solution was 1 per cent. The solution was then
heated in a water bath at 75° C, for half an hour. It was then thrown on to a
weighed filter paper, which had been previously dried in a hot water oven at
100° C. till it reached constant weight. The clear filtrate was caught in a
thoroughly cleaned and weighed platinum basin.
The filter was well washed through with small quantities of distilled water.
It was then, along with the coagulated proteid, again dried in a water oven at
100° C. until a constant weight was recorded. The filtrate in the platinum
basin was evaporated to dryness in a water bath at 60° C. After thorough
desiccation over lime the basin with the residue was again weighed.
The following were the results obtained :—
Amount of venom
=
0.093 gramme.
Strength of solution
= 1 per cent.
(a) Weight of filter paper and coagulated proteid
= 0.2982 gramme.
Weight of filter paper alone
= 0.2803"
23
[ I L L ] Weight of coagulated proteid
=
0.0179 gramme.
(b) Weight of platinum basin and dried residue
= 5 6 . 7 1 3 3 "
Weight of platinum basin alone
= 5 6 . 6 3 8 9 "
[ I L L ] Weight of dried residue
=
0 . 0 7 4 4 "
Thus, there were recovered from 93 milligrammes of venom 17.9 milli-
grammes of coagulated proteid and 74.4 milligrammes of incoagulable residue, a
total of 92.3 milligrammes. We arrive, therefore, at the following percentage
composition :—
Coagulable proteids
20 per cent .
Incoagulable residue
80 ,,
,,
SERIES No. II.—Experiments to ascertain the blood coagulation time at inter-
vals in chronic cases of intoxication with the venom of Bungarus fasciatus.
The method which was adopted to estimate the coagulation time of the
blood in these observations was the method which has been elaborated by Pro--
fessor A. E. Wright. As the observations were always made at a uniform tem-
-perature, namely, the day temperature of the laboratory in the months of January
and February, which in Bombay varies very little from day to day, it was found
unnecessary to make use of the device, described by the inventor, for maintaining
the tubes at a standard temperature.
The results obtained can therefore be considered as comparable amongst
themselves as is possible. Two rabbits and three monkeys were the animals
used. With one exception the venom was injected subcutaneously.
The following were the results obtained :—
ANIMAL.
Weight
in
grammes.
Amount of
venom per kilo.
in milligrammes.
BLOOD COAGULATION TIME IN MINUTES.
REMARKS.
Before
injection.
2nd
day.
3rd
day.
4th
day.
5th
day.
6th
day.
7th
day.
Rabbit 9
1 , 5 9 0
0 . 8
2
... 6
9 5½ ... ... Found dead on the
morning of 6th day.
(Intrav.)
„ 15
1,610
3
2 ¼ ... 10 10 3¾ 4 ... Found dead on the
morning of 8th day.
(Subcut.)
Monkey 3
2 , 7 6 0
3
2 ½3¼ 8 7½ 7½ ...
8 ½ Recovered.
(Subcut.)
,,
5
1,950
2.5
3
7 8½ 5¾ 4½ ... ... Died on 5th day.
(Subcut . )
,,
2
1,640
3 . 7
5
6 12
... 7½ ... ... Died on morning of 6th
day.
(Subcut.)
24
SERIES No. III.—Experiments to ascertain if the venom of Bungarus fasciatus
has the power of clotting citrate plasma in vitro, no soluble salt of lime
being added.
Two series of experiments were made, one with citrate donkey plasma and
the other with citrate horse plasma. In each instance the citrate of soda was
present in the strength of 1 in 100 of plasma. The plasma was completely free
from red cells.
Two cubic centimetres of plasma were measured into each tube, to each of
which there was then added a different amount of the venom of Bungarus
fas-ciatus. The strength of the venom solution used was 1 per cent . It was dis
-solved in normal saline solution.
The following were the results obtained :—
(a) Donkey plasma (1 per cent. citrate).
Plasma.
Bungarus fas-
ciatus venom in
milligrammes.
Result.
2 c . c .
.
.
.
.
.
.
.
.
.
5
Liquid after 24 hours.
2 , ,
.
.
.
.
.
.
.
.
.
4
, ,
, , 24 „
2 , ,
.
.
.
.
.
.
.
.
.
3
,,
,,
24 , ,
2 , ,
.
.
.
.
.
.
.
.
.
2
, ,
, , 24 , ,
2
, ,
.
.
.
.
.
. .
.
.
1
,,
, , 24 ,,
(b) Horse plasma (1 per cent. citrate).
Plasma.
Bungarus fas -
ciatus venom in
milligrammes.
Result.
2 c . c
.
.
.
. .
.
.
.
.
5
Liquid after 24 hours.
2 , ,
.
.
.
. .
.
.
.
.
4
,,
,,
24 , ,
2 „
.
.
.
.
.
.
.
.
.
3
,,
, , 24 ,,
2
, ,
.
.
.
. .
.
.
.
.
2
,,
, ,
2 4
, ,
2 ,,
.
.
.
. .
.
.
.
.
1
,,
,,
24 ,,
SERIES No. IV.—Experiments to ascertain if the venom of Bungarus fascia
- . t u s c a n p r e v e n t t h e c l o t t i n g o f c i t r a t e p l a s m a , w h i c h n o r m a l l y r e s u l t s f r o m
the addition of a soluble salt of lime.
Two series of experiments were made, one with citrate donkey plasma and
the other with citrate horse plasma. In each case the citrate of soda was
present in the strength of 1 in 100 of plasma.
Two cubic centimetres of plasma were measured into each tube, to each of
25
which there was then added a different amount of the venom of Bungarus fasci-
atus. The strengths of the venom solutions used were 0.1 per cent.a n d 1
p e r
cent. From one to two hours afterwards there was added to each tube a quantity
of, calcium chloride solution which clotted the control in less than 15 minutes.
The following were the results obtained :—
( a ) D o n k e y p l a s m a ( 1 p e r c e n t . c i t r a t e ) .
Plasma.
Bungarus fas-
ciatus venom in
milligrammes.
Calcium Chlo-
ride solution,
2%
R e s u l t .
2 C.C.
.
.
.
.
.
.
.
0 . 5
0. 5 c. c.
Clotted solid in 4 minutes.
2 ”
.
.
.
.
.
. .
0.4
0.5 ”
”
”
4
”
2 ”
.
.
.
.
.
.
.
0 . 3
o.5 ”
”
”
4
”
2 „
.
.
.
.
.
.
.
0.2
0.5 ,,
I
,,
,,
4 ”
2 ”
.
.
.
.
.
.
.
0 . 1
0.5 ”
”
”
4
”
(b) Horse plasma (1 per cent. citrate).
P l a s m a .
Bungarus fas-
ciatus venom in
milligrammes
Calcium Chlo-
ride solution, 1 %
Result.
2 C.C.
.
.
.
.
.
. .
5
0 . 5 c. c.
Clotted solid in 12 m i n u t e s .
2
”
.
.
.
.
.
.
.
4
0.5
”
”
”
1 2
”
2
”
.
.
.
.
.
.
. .
3
0.5
”
”
”
12
”
2
„
.
.
.
.
.
.
•
2
0.5
”
”
”
12
”
2
”
.
.
.
.
.
.
.
1
0 . 5
”
”
”
12
”
SERIES No. V.—Experiments to demonstrate the result of the hœmolytic action
of the venom of Bungarus fasciatus, unheated and heated, in vitro on the
blood of rabbits.
A sample of the venom of Bungarus fasciatus was dissolved in sterile salt
solution (0.75 per cent.). The strength of this solution was 0.2 per cent.A
portion of this solution was heated in a water bath at 75° C. for half an hour and
then filtered to remove the coagulated proteids.
An estimation of the hæmolytic power of the original unheated venom and
of the heated solution was made. Each tube in the series contained 0.5 c.c. of
venom solution, which was half the strength of the solution in the tube next
above it in the series.
The dilutions were made with salt solution (0.75 per cent.). To each tube
was then added 0.005 c.c. of fresh rabbit's blood drawn from the artery of the ear.
The preparations were kept at laboratory temperature (about 25° C.). The
observations were recorded 20 hours after the preparations were made
.
E
26
The following were the results obtained :—
Strength of venom solutions.
Unheated.
Heated.
0.5 c.c. = 1
milligramme .
.
.
.
.
.
. . Slight H.
Slight H.
0.5 ”
= 0.5
”
.
.
.
.
.
. .
.
”
Trace H.
0.5
”
= 0.25
”
.
. .
.
.
.
.
.
No H.
No H.
0.5
”
= 0.125
”
.
.
.
.
.
.
.
.
”
”
0.5 ”
= 0.0625
”
.
.
.
.
.
.
. .
”
”
0.5
”
= 0.03125
”
.
. .
. .
.
. .
”
Trace H.
0.5 ”
= 0.0156
”
.
.
. .
.
.
.
. Trace H.
Slight H.
0 . 5 ”
= 0.0078
”
.
.
.
.
.
.
.
. Slight H.
”
0.5
”
= 0.0039
”
.
. .
.
.
.
.
.
”
”
SALT SOLUTION
.
.
.
.
.
No H.
. . .
SERIES No. VI—Experiments to determine the minimum lethal dose for rabbits
by intravenous injection of the pure venom of Bungarus fasciatus.
The poison was carefully dried fresh venom of Bungarus fasciatus. A
1 per cent. solution in sterile salt solution was made. Five-fold and ten-fold
dilutions of this original solution were prepared when necessary. All the solu-
tions were kept unheated. The injections were made into the marginal vein of
the ear of the rabbit.
The following were the results obtained :—
Animal.
Weight.
Amount of venom
per kilo.
Result.
Rabbit
1 .
. 1,700 grammes
. 1
milligramme
Died in 2¾ minutes.
”
2
.
. 1,640
” . 5
”
.
”
1 8
”
”
3
.
. 1,500
”
. 3.5
”
.
”
3¼ hours.
”
4 .
. 1,600
” . 3
”
.
”
1 hour.
”
5
.
. 1,370
” . 2.5
”
. Found dead after 20 hours.
”
6 .
. 1,520
” . 1
”
. Died in 24 hours.
”
7
.
. 1,470
”
. 1
”
.
” 30 ”
”
8
.
. 1,590
” . 0.8
”
.
” 120 ”
”
9 .
. 1,760
” . 0.75
”
. Found dead after 66 hours.
”
10
.
. 1,280
” . 0.75
”
. Died in 48 hours.
”
1 1 .
. 1,680
”
. 0.7
”
. Found dead after 48 hours.
”
12
.
. 1,380
” . 0.6
”
. III: lost weight ; recovered.
”
13
.
. 1,830
” . 0.5
”
.
”
”
”
”
14 .
. 1,340
”
. 0.25
”
. Slightly ill ; recovered.
27
From the above table it appears that about 0.7 milligramme per kilo. of
the venom of Bungarus fasciatus is the minimum lethal dose for a rabbit when
the injection is made intravenously.
SERIES No. VII.—Experiments to determine the minimum lethal dose for
rabbits by subcutaneous injection of the pure venom of Bungarus fasciatus.
The poison was the same as used in the previous series of experiments.
The strengths of the solutions employed were 0.2 per cent. and 0.1 per cent.
These were made with normal salt solution. All injections were made in the
inner side of the thigh.
The following were the results obtained :—
Animal.
Weight.
Amount of venom
per kilo.
Result.
Rabbit 1
.
. 1,610 grammes
. 3 milligrammes . Died in 8 days.
2
.
. 1,530
”
. 2.5
”
.
”
26 hours.
”
3
.
. 1,560
”
. 2
”
.
Lost weight ; recovered.
” 4
.
. 1,300
”
. 1.5
”
.
No symptoms.
”
5
.
. 1,460
”
. 1
”
.
Lost weight; recovered.
”
6
.
. 1,430
”
.
0.75
”
.
No symptoms.
„
7
.
. 1,270
”
. 0.5
”
.
”
”
From the above table it appears that 2.5 to 3 milligrammes per kilo. of
weight is the minimum lethal dose for a rabbit when the injection is made
subcutaneously.
SERIES No. VIII.—Experiments to determine the minimum lethal dose for
rats by subcutaneous injection of the pure venom of Bungarus fasciatus.
The poison was the same as used in the experiments of series Nos. VI
and VII. The strengths of solutions employed were 0.2 per cent. and 0.1 per
cent. These were made with normal salt solution. Care was taken in choosing
rats of about 118 grammes in weight. The injections were made subcutane-
ously in the inner side of the thigh.
E 2
28
The following were the results obtained :—
Animal.
Amount of venom
in milligrammes.
Result.
Rat
1
.
. .
4
Found dead after 18 hours.
”
2
2
”
”
1 8
”
”
3 .
.
.
.
1 . 5
Very ill : recovered.
”
4 .
.
.
.
1 . 5
Found dead after 11 days.
”
5 .
.
.
.
1
”
” 36 hours.
”
6 .
.
.
.
1
Very ill : recovered.
”
7 .
.
.
.
0 . 5
Found dead after 36 hours.
”
8 .
.
.
.
0 . 5 ”
”
48
”
”
9 .
.
.
.
0 . 2 5
III : recovered.
”
1 0 .
.
.
.
0 . 1
Slightly ill : recovered.
”
1 1 .
.
.
.
0 . 0 7 5
No symptoms.
” 12 .
.
.
.
0 . 0 5 ”
”
From the above series of experiments it is difficult to determine exactly the
amount of poison which can be depended on to kill a rat. Thus 0.5 milli-
gramme killed in two cases, while three times this amount, viz., 1.5 milli-
grammes, failed to kill, although grave symptoms followed the injection. We
must, therefore, take it that to produce a certain lethal effect an amount greater
than 1.5 milligrammes must be given.
SERIES No. IX.—Experiments to determine the minimum lethal dose for
monkeys by subcutaneous injection of the pure venom of Bungarus
fasciatus.
The poison was the same as used in the experiments of series Nos. VI, VII,
and VIII. The venom was dissolved in normal saline solution. The strength