NationalLibraryOfScotland/medical-history-of-british-india huggingface dataset:
113
Statement No. XIV (Vital).—Showing the Sickness
and Mortality among Prisoners of all classes in
the Jails and Subsidiary Jails of Rajputana during
the year 1918.
No. I.
(NEW SERIES.)
SCIENTIFIC MEMOIRS
BY
OFFICERS OF THE MEDICAL AND SANITARY DEPARTMENTS
OF THE
GOVERNMENT OF INDIA.
STANDARDISATION OF CALMETTE 'S ANTI-VENOMOUS SERUM
WITH PURE COBRA VENOM : THE DETERIORATION OF THIS SERUM
THROUGH KEEPING IN INDIA.
BY
G. LAMB, M.B., CAPTAIN, I.M.S., AND WM. HANNA, ESQ., M.B., ETC.
(FROM THE PLAGUE RESEARCH LABORATORY, BOMBAY.)
ISSUED UNDER THE AUTHORITY OF THE GOVERNMENT OF INDIA
BY THE SANITARY COMMISSIONER WITH THE GOVERNMENT
OF INDIA, SIMLA.
CALCUTTA:
OFFICE OF THE SUPERINTENDENT OF GOVERNMENT PRINTING, INDIA.
1902.
Price 3 annas or 4d.
No. 1.
(NEW SERIES. )
SCIENTIFIC MEMOIRS
BY
OFFICERS OF THE MEDICAL AND SANITARY DEPARTMENTS
OF THE
GOVERNMENT OF INDIA.
STANDARDISATION OF CALMETTE' S ANTI-VENOMOUS SERUM
WITH, PURE COBRA VENOM: THE DETERIORATION OF THIS SERUM
THROUGH KEEPING IN INDIA.
BY
G. LAMB, M.B., CAPTAIN, I.M.S., AND WM. HANNA, ESQ., M.B., ETC.
( FROM THE PLAGUE RESEARCH LABORATORY, BOMBAY. )
ISSUED UNDER THE AUTHORITY OF THE GOVERNMENT OF INDIA
BY THE SANITARY COMMISSIONER WITH THE GOVERNMENT
OF INDIA, SIMLA.
CALCUTTA:
OFFICE OF THE SUPERINTENDENT OF GOVERNMENT PRINTING, INDIA.
1902.
IN 1884, the publication of the Scientific Memoirs by Medical
Officers of the Army of India was begun. The series
was intended to furnish a convenient medium for the circula-
tion of selected original papers on scientific subjects, which it
had been the custom to include in the Annual Report of the
Sanitary Commissioner with the Government of India. Twelve
parts of the Scientific Memoirs have been published, the last
in 1901.
The collection of scientific papers in a volume, appearing
periodically at considerable intervals of time, presents a serious
disadvantage, since considerable delay may occur between the
publication of some of the papers and the completion of the
work which they describe.
To obviate this, it has been decided that in future the
papers will be published as soon as possible after they are
received; and the opportunity afforded by this change is taken
to modify the title of the series, so that original papers on
scientific subjects by all officers of the Medical and Sanitary
Departments of the Government of India may be included.
B. FRANKLIN,
Surgeon-General, I.M.S.
OFFICE OF THE SANITARY COMMISSIONER
WITH THE GOVERNMENT OF INDIA;
Simla, June 30, 1902.
STANDARDISATION OF CALMETTE'S ANTI-VENOMOUS SERUM
WITH PURE COBRA VENOM :
The Deterioration of this Serum through keeping in India.
THE standardisation of an anti-venomous serum is of great practical as
well as theoretical importance.
It is the practical side of the question to which we propose to confine our-
-selves in this paper.
The snake venom which M. Calmette 1 employs, both for the purpose of the
immunisation of his horses and for the purpose of the standardisation of the serum
recovered from those immune horses, is a mixture of colubrine and viperine
poisons, the former making up about 80 per cent. of the mixture
. A solution of
this mixture is heated at about 73° C. for half an hour and then filtered. It is this
mixed and heated poison which is employed at Lille for both the purposes above
mentioned.
Myers 2 has correctly pointed out that as we have in all cases clinically to
deal with intoxication by a pure venom, either colubrine or viperine, and also by
an unheated venom, it is preferable, if the true value of this serum as a curative
agent against snake-bite is to be determined, to use a pure unheated poison for
its standardisation. The object then of our investigation was to ascertain, as
exactly as possible, the neutralising power of Calmette's anti-venomous serum
when tested with pure, fresh and carefully dried cobra poison. Further, in con-
sequence of the observations made by us in a recent case of cobra intoxication, 3"
we have extended our investigations so as to ascertain if any deterioration of the
serum takes place through keeping in this country.
In another communication we hope to be able to record the results of similar
experiments made with the venom of Russell's Viper (Daboia Russellii).
The method of standardisation of the anti-venomous serum which we
adopted in our experiments was the method now recognised as giving the most
accurate results. It is based on the almost generally acknowledged fact, that
snake poison toxin and its anti-toxin interact directly, and that this neutralisation
is, so far as our knowledge goes, independent of vital activity. The evolution of
this method is, briefly stated, as follows. The method which M. Calmette4
recommends and still practises is the following :—The dose of venom (mixed and
* Reported to Government' on 24th and 25th January 1901, vide Plague Research Laboratory's No.
130, to the address of the Secretary to the Government of India, Home Department.
2
heated), which will kill a rabbit of 2 kilos in weight in from 15 to 20 minutes when
injected into the marginal vein of the ear, is determined; then the minimum
amount of serum, which will prevent death when injected intravenously five
minutes before this dose, is ascertained: this is done by experimenting with
varying quantities of serum. If one cubic centimetre is able to accomplish this,
M. Calmette says that the serum contains 2,000 immunity units per cubic centi
-metre or 20,000 per 10 cubic centimetres.
Martin of Melbourne 5 was the first to use the method of mixing the venom
and serum in vitro before injection. In some early experiments with the
poison of the Australian tiger snake (Hoplocephalus curtus) he used only a single
lethal dose in estimating the curative value of Calmette's serum. However, in
some later researches he recognised that this method was fallacious, inasmuch
as if the amount of serum was able to neutralise even a mere fraction of the
single lethal dose the animal would recover, even though the larger part of the
poison remained unneutralised. He therefore recommended the employment of
at least ten lethal doses.
Along with one of us, Major D. Semple, R.A.M.C.,6 has pointed out that
the following objections could be taken to Calmette's method:—
(1) that there is no direct estimation of the amount of venom which
a given quantity of serum will neutralise *;
(2) that there is no estimation made of the amount of venom, the
injection of which an untreated animal is capable of surviving.
Further, we showed that Calmette's test for a dose for the rabbit amounted to
about three lethal doses for that animal. We thus gave a value for the serum in
terms of its neutralising power and not, as in the method used at Lille, in terms
of its preventive power. With such a small test dose as three lethal doses,
unless careful attention be paid to the second objection mentioned above, it is
evident that a much enhanced neutralising power would be given to the serum.
The next step in advance was made by Myers.7 A s p r e v i o u s l y m e n t i o n e d ,
this observer pointed out that in order to ascertain the strength of an anti-venom
-ous serum for practical purposes, it was à priori preferable to test it against a
pure venom, such as pure cobra poison or pure daboia poison. He further showed
that it was preferable also to use unheated venom, inasmuch as heating at the
temperature employed by M. Calmette causes important changes in cobra venom,
altering it considerably from the natural poison as injected by a snake. Further,
Myers recommended that a cheap, small and susceptible animal be employed.
He showed that mice of about 15 grammes weight answer all requirements, and
recommended that these animals should be used. He further designated
* This is evident from the description of Calmette's method already stated. From this it will be seen
that the standard unit which he adopts is the weight in grammes of a rabbit which 1 C. Cm. of serum can
protect against a quantity of poison which would kill a control animal in 20 minutes.
3
Calmette's method as unscientific, in as much as by injecting the serum before
the venom the completion of the reaction between these two bodies does not
take place before they enter the blood stream. He showed that much more
accurate results are attained when the poison and serum are mixed in vitro than
by Calmette's method. Finally, he recognised the necessity of using as the test
dose a multiple, and, if possible, a large multiple of the minimum lethal dose.
In the series of experiments which we have now to present, these various
points were kept carefully in view.
We may preface the protocols by giving a short account of the method
employed and of the general results obtained.
The animals we used were rats from 115 to 120 grammes in weight.
These animals fulfil all the conditions indicated by Myers. They are cheap and
the supply is abundant, while with care no difficulty is experienced in handling
them. At the same time they are extremely susceptible to cobra poison, as
susceptible as, if not more so than, any of the other laboratory animals. Thus for a
mouse of 15 grammes Myers 8 states the minimum lethal dose of cobra venom to
be 0·012 mgrm. or in other words 0·8 mgrm. per kilo. For rabbits Elliot 9 has
found that a certain lethal dose of pure cobra poison injected subcutaneously is
0·7 mgrm, per kilo. In our experiments we found the minimum lethal dose of
this poison for a rat of about 120
grammes was 0·04 or at the rate of 0·33 mgrm.
per kilo.
Another reason for selecting rats was that horses' serum, even in large doses
has no lethal action on them as it has on mice. As in the course of our experi-
ments large doses of serum had to be given, this was an important point.
Great care was taken in choosing rats of one size for all experiments, viz., of
about the weight mentioned above.
The venom used was pure, fresh, unheated cobra poison. This was collected
from a number of living, full-sized, healthy cobras by "milking" the glands after
the method practised by M. Calmette. The poison was then thoroughly dried
over lime and kept, till used, in an air-tight tube away from light. Before weigh
ing it was pounded in a mortar and then again dried over sulphuric acid.
A solution of this powdered venom in normal salt solution was made. The
strength of this solution was 0·1 per cent. When necessary a tenfold dilution of
this material was employed. All the solutions were freshly made as required, so
that no deterioration of the poison was possible through keeping in solution.
All injections were made in the inner side of the thigh.
Series No. I was undertaken to determine the minimum lethal dose for a rat
of 115 to 120 grammes in weight.
A certain lethal dose was found to be 0 ·04 mgrm., and the maximum non
- l e t h a l d o s e w a s f o u n d t o b e 0·
0 3 5 m g r m .
The remaining series of experiments were undertaken with a view of
4
determining the neutralising power of various samples of anti-venomous serum.
These samples were of different ages and had been in India for varying periods of
time. They had been stored in different places and had therefore been exposed
to different degrees of heat.
The test dose of venom which was used in all cases consisted of ten times
the minimum lethal dose, i.e., 0∙4 mgrm. This quantity was mixed in vitro with
varying amounts of the serum to be tested. The mixture was allowed to stand at
laboratory temperature (about 25° C.) for at least half an hour. All injections
were made, as in the experiments detailed in series No. I, subcutaneously in the
inner side of the thigh.
In calculating the amount of venom which the serum had been able to
neutralise, or had failed to neutralise, the maximum non-lethal dose— i. e., 0∙ 0 3 5
mgrm.—was deducted from the test dose in all cases.
In series No. II a serum which had recently been received from Lille was
used. We take this opportunity of thanking M. Calmette for his courtesy in
placing this serum at our disposal.
This sample was dated 17th September 1900. It arrived in Bombay on the
14th October of the same year. Our experiments with it were made between the
10th November and the 5th December. We are justified, therefore, in considering
this as perfectly fresh serum, as fresh as it would be possible to get in this
country.
A reference to the protocols will show that 0 ∙5 C. Cm. was able to prevent
death, while 0∙45 C. Cm. did not accomplish this. In other words 0∙5 C. Cm.
of this serum was able to neutralise 0 ∙4 — 0∙035 mgrm.=0∙365 mgrm.; namely,
1 C. Cm. could neutralise 0∙73 mgrm.
Working with a mixed and heated venom, Semple and one of us 10 found that
1 C. Cm. of fresh serum was able to neutralise 1 ∙5 mgrm. of this mixed poison.
This is the same value which M. Calmette attributes to his serum, when this is
expressed in terms of the neutralising power of the serum. Comparing these
three results, we see at once, therefore, the importance of testing the serum
against a pure unheated venom and not with a mixed and heated poison.*
The serum employed to perform the experiments detailed in series No. III
was obtained by purchase from Messrs. Phillips & Co., Chemists, Bombay. It
bore the date 8th May 1900. The vendors informed us that they had received
it from Europe about the end of August 1900, and that it had not been out of
Bombay. Our experiments were made between the 5th and 12th December 1900.
*
This comparison, as well as other similar comparisons to be detailed later on, is made on the assump-
tion that all specimens of anti-venomous sera sent out from Lille have, when fresh, the same neutralising
power. There can be no doubt, however, that such a desirable result cannot practically be obtained. It
is probable, therefore, that a certain proportion of the differences in the neutralising power of the sera with
which we experimented may be due to initial differences in this neutralising power.
5
It will be seen therefore that this serum had been in Bombay for about three
months. One of these months, October, is one of the hottest months of the
year.
A reference to the protocols will show that 0•6 C. Cm. was able to prevent
death, while 0•5 C• Cm. could not accomplish this. It is evident therefore that 1
C. Cm. of this serum was able to neutralise 0•61 mgrm.
This result would appear to point to a slight deterioration of the serum
having taken place through keeping in Bombay, even for the short time mentioned
above.
The serum used in series No. IV of our experiments was obtained from the
Natural History Society of Bombay. We take this opportunity of thanking
Mr. Phipson, the Honorary Secretary of this Society, for his kindness in placing it
at our disposal.
This sample of serum was not dated. Mr. Phipson, however, thought that it
had been in his possession for about four years. On reference to Lille,
M. Calmette informed us that he did not know the exact age of the serum, but
that it was at least more than three years old. From the protocols it will be seen
that although one rat, which received 0•75 C. Cm. of serum, recovered after
being very ill, the quantity of serum necessary to prevent a fatal result with
certainty was 1•25 C. Cm. It is evident therefore that 1 C. Cm. of this serum
was able to neutralise only 0•29 mgrm. of pure cobra venom
.
The fifth series of experiments detailed in the protocols was made with
serum which was sent to us by Captain Mitchell of Nasik. In view of the
results obtained, the history of this sample of serum is interesting. It was
originally the property of the Bombay Natural History Society, and was got from
Europe at the same time as the serum used in series No. IV of our experiments ;
it was therefore more than three years old. Soon after its arrival in Bombay
it was sent to Nasik. It was kept there till Captain Mitchell sent it to us in
October, 1900. Our experiments with this serum were made between the 16th
October and 1st November 1900.
A reference to the protocols will show that 0•8 C. Cm. was able to prevent
death when our test dose was employed. In other words 1 C. Cm. of this serum
was able to neutralise with certainty 0•46 mgrm. of pure cobra poison.
For series No. VI of our experiments the sample of serum was obtained from
Lieutenant-Colonel G. H. D. Gimlette, I.M.S., then Residency Surgeon, Indore.
This sample was dated 17th October 1896. Its history was as follows :—It
was imported to India by Messrs. Smith, Stanistreet & Co., Chemists, Calcutta,
arriving in this country about the end of August 1899. It was sent to Indore
on the 6th September of the same year. Colonel Gimlette despatched it to
Bombay at the end of November 1900, and our experiments were completed
6
between the 29th November and 3rd December. It will be seen, therefore, that
for over a year it lay at Indore and was thus subjected to the great heat of that
place during one hot season.
A reference to the protocols will show that 1 ·7 C. Cm. of this serum failed
to neutralise the test dose, while 2 C. Cm. were able to accomplish this. The
rat which received this latter quantity was ill for a short time. We may take it,
therefore, that 2 C. Cm. was the amount which could neutralise just over nine
lethal doses (0·365 mgrm.), or in other words that 1 C. Cm. was able to neutralise
only 0·18 mgrm. of venom.
The experiments detailed in series No. VII of the protocols were made with a
serum received from Mr. Hankin of Agra. This sample bore the date 17th
September 1895. It had been stored in Agra for over four years. It was
received in Bombay on the 17th October 1900, and our experiments with it were
completed between the 27th November and 10th December.
A glance at the protocols will show that 2 ·5 C. Cm. of this serum, i.e. , the
largest amount used, were not able to prevent death when our test dose of ten
lethal doses was employed. From this it is evident that 1 C. Cm. of this Agra
serum could not neutralise even 0 ·14 mgrm. of poison. The serum can, therefore,
be said to have lost all neutralising power for practical purposes.
The serum employed in the eighth and last series of experiments detailed in
the protocols was received from Lieutenant W. G. Liston, I.M S., Civil Surgeon,
Ellichpur, Berar. It was dated 7th November 1896.
Lieutenant Liston had purchased it from Lille in September 1898, and had
brought it with him to India the following month. During the hot seasons of
1899 and 1900 the serum had been subjected to the heat of Berar at two stations,
viz. , Hingoli and Ellichpur. It was sent to Bombay in December 1900. The
experiments made by us were completed between the 10th and 14th December.
From the protocols it will be seen that 2 C. Cm. of this serum failed to neutralise
the test dose. We can therefore conclude that 1 C. Cm. was not able to neutra-
lise 0·18 mgrm. of venom. It is unfortunate that no more serum was available
to complete this series. It is, however, evident that, like the Agra serum, it had
lost for all therapeutical purposes its power to neutralise pure cobra poison.
We may now pass on to consider some of the practical bearings which the
data collated above and detailed in the protocols have on the standardisation of
anti-venomous serum and on the therapeutics of cobra-poisoning.
Firstly, our experiments conclusively prove Myers's statement that it is
necessary in standardising this serum to use a pure unheated poison. Both in
Calmette's method of standardisation and in the method followed by Semple and
one of us, a mixed venom containing about 80 per cent, of colubrine poison was em
ployed. The solution of this mixture was heated at 73° C. for half an hour and then.
7
filtered. The conclusion arrived at in both cases was the same, viz., that
1 C. Cm. of serum was able to neutralise 1•5 mgrm. of this venom. As this mixture
contained only a fifth part of viperine poison it appeared justifiable to conclude
t h a t 1 C . C m . o f s e r u m w o u l d b e a b l e t o n e u t r a l i s e a t l e a s t a b o u t 1 • 2 m g r m . o f p u r e
cobra poison. Our experiments, however, have shown that such a hypothesis is
not borne out by fact, and that 1 C. Cm. of serum can only neutralise about 0•7
mgrm. of pure cobra venom.*
Secondly, it is evident that any attempt to calculate the therapeutic dose of
the serum can only be made when the exact measure of its power of neutralising
a natural poison has been ascertained. Let us consider this point in some detail.
Calmette† recommends that from 10 to 20 C. Cm. be given as the dose
in all cases of bites from poisonous snakes. We shall see later that such a dose
would probably be insufficient in some cases of cobra bite.
Semple and one of us11 calculated that 15 C. Cm. were sufficient to save a
patient, if this quantity were injected into the blood before the lapse of such
period of time as might be required for the absorption of the lethal dose of venom.
This calculation, however, was made on the following assumptions :—
(1) That the minimum lethal dose of cobra venom for a man of 60 kilos
weight was, as estimated by Fraser, 12 about 31 mgrms., and that consequently
we may take 30 mgrms. as the maximum non-lethal dose for an average man.
(2) That a full-sized cobra can inject at each bite a quantity of venom
which in the dried state amounts to from 30 to 45 mgrms. These amounts have
been demonstrated experimentally by Calmette.1 3
To save a man bitten by a cobra, injecting the maximum quantity possible,
we should therefore have to give an amount of serum capable of neutralising
45—30 mgrms.=15 mgrms. of cobra poison.
( 3) That 1 C. Cm. of s er um was capabl e of neut r al i s i ng 1 mgr m. of pur e
cobra venom. This figure was arrived at by assuming that pure unheated cobra
poison was not more than a third more deadly than the mixed venom we
employed. This mixture, it will be remembered, contained 80 per cent. of
col ubri ne poi son, and 1 C. Cm. of serum was abl e t o neut ral i se 1•5 mgrm. of i t .
It is evident, therefore, that were these three assumptions correct, 15 C. Cm,
of serum would be a safe dose to employ in all cases of cobra bite.
Our experiments, however, have demonstrated that 1 C. Cm. of serum is
only capable of neutralising about 0•7 mgrms. of pure cobra venom, while
Myers 14 from his experiments on mice makes the quantity neutralised by 1 C. Cm.
even less than this, viz., 0•6 mgrms.
* The serum which we used in these experiments, although newly arrived in India, had necessarily
been subjected for a short time to the heat of the voyage out— viz ., in the Red Sea and the Indian Ocean.
A certain deterioration may thus have taken place. This, however, is likely to account for only a limited
proportion of this great difference.
† This is the dose prescribed in the printed instructions sent out with every bottle of serum.