Collection of Venom.
Venom can be extracted from the poison-glands of either freshly killed or living snakes.
In cases in which the venom of dead snakes has to be collected, the best method of extraction consists in fixing the head of the animal to a sheet of cork and carefully dissecting out the gland on each side. The reptile being placed on its back, the lower jaw is removed with a pair of scissors; two strong pins or two tacks are thrust through the skull, in the median line, in order to keep the head from moving. The poison-fangs are next drawn out of their sheaths, and, without injuring them, the two poison-ducts, which open at their bases, are isolated and tied with a thread in order to prevent the poison from running out.
The dissection of the glands is then very easy; they are lifted out and placed in a saucer. The end of the duct is cut between the gland and the ligature, and with a pair of fenestrated or polypus forceps the whole of the glandular mass is gently squeezed from behind forwards, the liquid which flows out being received in a large watch-glass.
If pressed for time, a more simple method of operating is to hold the head of the snake in the left hand, with the mouth open and the lower jaw directed downwards. A watch-glass, capsule, or receptacle of some sort, such as a cup or plate, is then introduced by an assistant between the jaws, and, with the index finger and thumb of the right hand, the whole of the region occupied by the glands on each side of the upper jaw is forcibly compressed from behind forwards; the poison flows out by the fangs.
The extraction of the venom from living snakes is effected in the same manner. The animal being firmly held by the neck, as close as possible to the head, so that it cannot turn and bite; it can be made to eject the greater portion of the liquid contained in its two glands by compressing the latter with force from behind forwards, as one would squeeze out the juice from a quarter of an orange ([fig. 85]).
It is necessary to take care that the reptile cannot coil itself round furniture or other objects in the vicinity of the operator, for if this should happen there would be the greatest difficulty in making it let go, especially if dealing with a strong animal such as a Cobra, Rattle-Snake, or Fer-de-lance.
Snakes of the last-mentioned kind are especially difficult to manage. In order to avoid the risk of being bitten, it is always wise to begin by pinning down the head of the animal in a corner of its cage by means of a stick, and to seize it with a pair of long fenestrated tongs shaped like forceps. The operator then easily draws the reptile towards him and grasps it firmly by the neck with his left hand, always as close to the head as possible, at the same time raising the body quickly in order to prevent it from taking hold of anything. In this way the most powerful snake is perfectly under control.
Fig. 85.—Collecting Venom from a Lachesis at the Serotherapeutic Institute at São Paulo (Brazil).
Fig. 86.—Chloroforming a Cobra in order to Collect Venom, at the French Settlement of Pondicherry, in India (Stage I.).
At Pondicherry, where is collected the greater portion of the venom of Naja tripudians used by me for the vaccination of the horses that produce antivenomous serum, it is customary to chloroform the snakes in order to render them easier to manipulate.
The reptile is placed in a large covered jar, containing a pad of absorbent wool impregnated with chloroform (figs. 86, 87), and in a few minutes it is stupefied. It is then grasped by the neck with the hands, and the edge of a plate is slipped between its jaws. On compressing the two poison-glands with the fingers, the venom dribbles out on to the plate.
A detailed description of this technique will be found in a note kindly drawn up for me by my friend Dr. Gouzien, late head of the Medical Staff of the French Settlements in India, and reproduced further on in the section of this book devoted to documents. The note in question was accompanied by figs. 17, 18, 19, 86, 87, and 88, which are reproduced from photographs, for which I am indebted to the kindness of M. Geracki, Engineer of the Savanna Spinning Mill at Pondicherry, Dr. Lhomme, and M. Serph, Assistant Surgeon-Dispenser.
The collection of the venom having been completed, the snake is put back into its cage again, the tail and the body being introduced first, and then the head. The lid or trap-door is half closed with the left hand, and, with a quick forward thrust, the right hand releases its grasp of the reptile and is immediately withdrawn; at the same time the left hand completes the closure of the cage. The snake is temporarily dazed, as though stunned, and it is only after the lapse of a moment that it thinks of darting open-mouthed at the walls of its prison.
When it is desired to procure large quantities of venom, as is indispensable in laboratories where antivenomous serum is prepared, the endeavour must be made to keep the snakes alive for the longest possible time. It then becomes necessary to resort to artificial feeding in the manner previously described (see p. 17), for they very often refuse to feed themselves.
Fig. 87.—Chloroforming a Cobra in order to Collect Venom, at the French Settlement of Pondicherry, in India (Stage II.).
Except when a snake is moulting, the venom can be extracted from its glands about every fortnight; and it is better that the extraction be not performed concurrently with artificial feeding, since, owing to the fact that the venom serves the animal as digestive juice, the reptile will soon perish if deprived of the means of digesting the food that it is obliged to receive. It is best, therefore, to select one day of the week for artificial feeding, and the corresponding day of the following week for the extraction of the venom.
When the venom has been collected, it must immediately be placed in a desiccator over calcium chloride or sulphuric acid, in order to dry it rapidly. In hot countries, and where no laboratory specially equipped for the purpose exists, it will suffice to dry the venom in a current of air, or even in the sun. It then concretes in scales of a citrin colour, more or less dark, according to the concentration of the liquid. In this dry condition, placed in well-corked bottles, protected from damp air, it may be kept almost indefinitely without losing anything of its original toxic power. On the contrary, if the desiccation be imperfect it undergoes a somewhat rapid change, and assumes a disagreeable odour of meat peptone. I have kept samples of various venoms, dried as described, for fifteen years without any sensible diminution of their activity.
Fig. 88.—Collecting Cobra Venom at Pondicherry (Stage III.).
CHAPTER V.
THE CHEMICAL STUDY OF SNAKE-VENOMS.
In the condition in which they are received on issuing from the glands, venoms always present the appearance of a thick saliva, of an oily consistency and more or less tinged with yellow, according to the species of snake by which the poison has been produced. They are entirely soluble in water, the addition of which renders them opalescent. Tested with litmus they exhibit a slightly acid reaction; this acidity, which is due to the presence of a very small quantity of an indeterminate volatile acid, disappears on desiccation, so that solutions of dried venom are neutral. The taste of venoms is very bitter. Their density, which is slightly greater than that of water, varies from 1030 to 1050.
Venoms are composed of a mixture, in variable proportions, of proteid substances, mucus and epithelial débris, fatty matters and salts (chlorides and phosphates of lime, ammonia and magnesia), with from 65 to 80 per cent. of water.
The elementary analysis of Cobra-venom made by H. Armstrong[7] gave the following results:—
| Carbon | 43·04 | per cent. |
| Hydrogen | 7·00 | “ |
| Nitrogen | 12·45 | “ |
| Sulphur | 2·50 | “ |
| Residue | Small quantities. | |
Not much is to be learnt from these figures; it would be of far greater importance to know the exact constitution of the proteid substances to which venom owes its physiological properties. Unfortunately, our knowledge of the chemistry of the albuminoid matters is still too imperfect for it to be possible for us to determine their nature.
As early as 1843 it was pointed out by Lucien Bonaparte that in the venom of Vipera berus the most important principle is a proteid substance to which he gave the name of viperin or echidnin, and which he compared to the digestive ferments. Later on Weir Mitchell and Reichert, and subsequently Norris Wolfenden, Pedlar, Wall, Kanthack, C. J. Martin, and MacGarvie Smith, showed that venoms, like diastases, exhibit a great complexity in composition; that all their characteristic toxic constituents are precipitable by absolute alcohol, and that the precipitate, when redissolved in water, recovers the properties possessed by the venom before precipitation.
According to Armand Gautier,[8] venoms contain alkaloids. The latter may be obtained, in very small amounts, however, by finely pulverizing dried venom with carbonate of soda, and systematically exhausting the mixture with alcoholic ether at a temperature of 50° C. These alkaloids have yielded crystallized chloraurates and chloroplatinates, and slightly deliquescent crystallized chlorhydrates. The latter produce Prussian blue when treated with very dilute ferric salts, and mixed with a little red prussiate. They therefore represent reductive bodies analogous to ptomaines.
Norris Wolfenden did not succeed in extracting these alkaloids from Cobra-venom, whence they had nevertheless been isolated by Armand Gautier. Wolcott Gibbs, and afterwards Weir Mitchell and Reichert, likewise failed to find them in Crotalus-venom. The toxicity of these bases is, moreover, but very slight, for the totality of the alkaloids extracted by A. Gautier from 0·3 gramme of Cobra-venom did not kill a small bird.
It is therefore to the toxalbumins that the toxic properties of venoms are essentially due.
All venoms are not equally affected by heat. The venoms of Colubridæ (Naja, Bungarus, Hoplocephalus, Pseudechis) and those of the Hydrophiidæ are entirely uninjured by temperatures approaching 100° C., and even boiling for a short time. When the boiling is prolonged, or when venoms are heated beyond 100° C., their toxic power at first diminishes, and then disappears altogether. At 120° C. it is always destroyed.
The venoms of Viperidæ (Lachesis, Crotalus, Vipera) are much less resistant. By heating to the coagulating point of albumin, i.e., to about 70° C., their toxic properties become attenuated, and they are entirely suppressed between 80° and 85° C. Lachesis-venoms are the most sensitive; their toxicity is lost if they be heated beyond 65° C.
On separating the coagulable albumins of the venoms of Colubridæ, by heating to 72° C., followed by filtration, we obtain a perfectly limpid liquid, which is no longer injured by boiling, and in which the toxic substance remains wholly in solution. The albuminous precipitate, when separately collected and washed, is no longer toxic. The clear liquid, after being filtered, is again precipitated by absolute alcohol, and the precipitate, redissolved in an equal quantity of water, is just as toxic as the original filtered liquid.
The venoms of Viperidæ, when coagulated, by heating them to a temperature of 72° C., and filtered, are almost always inert. The albuminous coagula, if washed, redissolved in water, and injected into the most sensitive animals, produce no harmful effect whatever.
The results of dialysis likewise differ when we experiment with the venoms of Colubridæ and Viperidæ. The former pass slowly through vegetable membranes, and with greater difficulty through animal parchment. The latter do not dialyse.
Filtration through porcelain (Chamberland candle F) does not sensibly modify the toxicity of the venoms of Colubridæ; on the contrary, it diminishes that of the venom of Viperidæ by nearly one-half.
By using a special filter at a pressure of 50 atmospheres, C. J. Martin has succeeded in separating from the venom of an Australian Pseudechis two substances: a non-diffusible albuminoid, coagulable at 82° C., and a diffusible, non-coagulable albumose. The former produces hæmorrhages; the second attacks the nerve-cell of the respiratory centres.
All venoms exhibit most of the chemical reactions characteristic of the proteids:—
Millon’s reaction.
Xantho-proteic reaction (heating with nitric acid and subsequent addition of ammonia = orange coloration).
Biuret reaction (caustic potash and traces of sulphate of copper).
Precipitation by picric acid, disappearing on being heated, reappearing when cooled.
Precipitation by saturation with chloride of sodium.
Precipitation by saturation with sulphate of magnesium.
Precipitation by saturation with ammonium sulphate.
Precipitation by a 5 per cent. solution of sulphate of copper.
Precipitation by alcohol.
According to C. J. Martin and MacGarvie Smith, the albumoses of the venoms of Colubridæ are hetero-albumoses, proto-albumoses, and perhaps deutero-albumoses in small quantities. They can be separated in the following manner:—
The solution of venom is heated to 90° C., and filtered in order to separate the albumins coagulable by heat. The filtrate, saturated with sulphate of magnesium, is shaken for twelve hours. By this means there is obtained a flocculent precipitate, which is placed upon a filter and washed with a saturated solution of sulphate of magnesium. The filtrate is dialysed for twenty-four hours in a stream of distilled water, and then concentrated, likewise by dialysis, in absolute alcohol. Thus we obtain a few cubic centimetres of liquid, which contains a small quantity of proteids in solution. These proteids can be nothing but a mixture of proto- and deutero-albumoses with peptones. That there is actually no trace of the latter can easily be ascertained.
Neumeister[9] has shown that it is impossible to precipitate all the proto-albumoses of a solution by saturation with neutral salts, and, since the filtrate becomes slightly turbid when a few drops of a 5 per cent. solution of sulphate of copper are added to it, we must conclude that it contains a small proportion of these proto-albumoses.
The deposit retained upon the filter after washing with sulphate of magnesium is redissolved in distilled water, and dialysed for three days. An abundant precipitate then becomes collected in the dialyser. This is centrifuged. The clear liquid is decanted with a pipette, then concentrated by dialysis in absolute alcohol, and finally evaporated at 40° C. until completely desiccated. The solid residue is washed and centrifuged several times in distilled water, after which it is dried on chloride of sodium.
This method enables us to separate two albumoses, both precipitable by saturation with sulphate of magnesium, and belonging to the class of primary albumoses: one of these, proto-albumose, is soluble in distilled water, the other, hetero-albumose, is insoluble; but the latter can be dissolved in dilute solutions of neutral salts. These bodies are respectively identical with those obtained by the pepsic digestion of proteids.[10]
In order to study separately the local and general effects of these different albumoses, C. J. Martin and MacGarvie Smith performed the following experiment:—
They introduced beneath the skin of the belly of a guinea-pig, previously shaved and rendered aseptic, two small pieces of sterilized sponge, about 2 c.mm., one of which was impregnated with the solution of proteid, while the other served as control. The two small incisions, one on either side of the median line, were then sutured and covered with collodion. In this way the maximum of local effect and the minimum of general effects was obtained. The solutions of albumoses introduced by this method into the organism produced an enormous œdema, which, in from six to eight hours, extended along the whole side of the abdomen containing the sponge charged with poison.
To test the general toxic effects, the solutions were injected into a vein or into the peritoneal cavity. It was thus found that the proto- and hetero-albumoses killed the animals in a few hours.
It must therefore be concluded from these facts that the active principles of venom are proto- and hetero-albumoses, the albumins that it contains being devoid of all toxic power.
Many chemical substances modify or destroy venoms, and we shall see in another chapter that several of them, by reason of their properties, may be very usefully employed for the destruction, in the actual wound resulting from a venomous bite, of the venom that has not yet been absorbed in the circulation.
Among these substances the most important are:—
A 1 per cent. solution of permanganate of potash (Lacerda).
A 1 per cent. solution of chloride of gold (Calmette).
Chloride of lime or even hypochloride of calcium (Calmette), in a solution of 1 in 12, which is augmented, at the moment of use, by 5 to 6 volumes of distilled water, so as to bring it to the standard strength of about 850 cubic centimetres of active chlorine per litre of solution.
A 1 per cent. solution of chromic acid (Kaufmann).
Saturated bromized water (Calmette).
A 1 per cent. solution of trichloride of iodine (Calmette).
All these chemical bodies also modify or destroy the diastases and the microbic toxins. The venoms, although more resistant to the influence of heat, behave, therefore, like these latter, and exhibit the closest affinity with them. Moreover, like all the normal glandular juices, they possess very manifest zymotic properties, which singularly complicate their physiological action, and upon which we shall dwell later on.
Electricity, employed in the form of continuous electrolytic currents passing through a solution of venom, destroys the toxicity of the latter, because under these conditions there is always formed, at the expense of the salts accompanying the venom, a sufficient quantity of chlorinated products (hypochlorites, chlorates, &c.), and a small amount of ozone, the oxidizing action of which is extremely powerful.
With alternating currents of high frequency, Phisalix, repeating the experiments that Arsonval and Charrin had performed upon diphtheria toxin, thought that he had succeeded in attenuating venom to the point of transforming it into vaccine.[11] But it has been shown by Marmier that this attenuation was simply the result of thermic actions. When, by means of a suitable arrangement, any rise of temperature was carefully avoided, no modification of toxicity was obtained.[12]
The influence of light, which has no effect upon venom preserved in a dry state, is, on the contrary, very marked upon venom in solution. Solutions of venom that are destined for physiological experiments should therefore not be employed without controls, if they be several days old. Apart from the fact that, if care be not taken to render them aseptic, they very soon become contaminated with the germs of all kinds of microbes, it is found that they gradually lose a large part of their activity, especially when they remain in contact with the air. By filtering them through a Chamberland candle and keeping them in the dark, in a refrigerator, in perfectly closed phials, they may be kept unimpaired for several months.
The addition of glycerine in equal parts to a concentrated solution of venom is also an excellent means of preservation.
Phisalix has shown that the emanations from radium attenuate and then destroy the virulence of Cobra- and also of Viper-venom.
“Dry Viper-venom, dissolved in aqua chloroformi in the proportion of 1 in 1,000, is put up in four tubes, three of which are irradiated, the first for six hours, the second for twenty hours, and the third for thirty-six hours. Three guinea-pigs, of equal weight, are inoculated with equal quantities of the irradiated venom; a control receives the non-irradiated venom. The latter dies in ten hours; the animal inoculated from the first tube dies in twelve hours; the one inoculated from the second tube in twenty hours, and the third proves resistant without any symptom of poisoning. A second inoculation produces a transitory lowering of the animal’s temperature by half a degree. At the end of four days it dies after inoculation with a lethal dose.”
The nature of the solvent exerts a great influence upon the action of the emanations from radium: if the same experiment be performed with venom dissolved in a 50 per cent. mixture of glycerine and water, the attenuation is merely relative after six hours.
Auguste Lumière and Joseph Nicolas, of Lyons, conceived the idea of studying the effect upon venom of the prolonged action of the intense cold produced by the evaporation of liquid air.[13] The Cobra-venom employed by these investigators was in solution at a strength of 1 in 1,000. It was submitted to the action of liquid air, partly for twenty-four hours and partly for nine days at -191° C. Its toxicity was in no way diminished.
Lastly, I must mention the recent researches of Hideyo Noguchi,[14] with reference to the photodynamic action of eosin and erythrosin upon the venoms of the Cobra, Vipera russellii, and Crotalus. It was found by the scientist in question that the toxicity of these various venoms is more or less diminished in the presence of these aniline colours, when the mixtures are insolated. Cobra-venom is the most resistant, just as it is in regard to the other physical or chemical agents. That of Crotalus, on the contrary, is the least stable.
CHAPTER VI.
THE PHYSIOLOGICAL ACTION OF SNAKE-VENOMS.
A.—Physiology of Poisoning in Man and in Animals Bitten by the Different Species of Poisonous Snakes.
(Colubridæ; Viperidæ; Hydrophiidæ.)
The bites of poisonous snakes produce very different effects according to the species of snake, the species to which the animal bitten belongs, and according to the situation of the bite. It is therefore necessary to take these various factors into account, in describing the symptoms of poisoning in different animals.
When the quantity of venom introduced into the tissues by the bite of the reptile is sufficient to produce fatal results—which is happily not always the case—the venom manifests its toxic action in two series of phenomena: the first of these is local and affects only the seat and surroundings of the bite; the second, or general series, is seen in the effects produced upon the circulation and nervous system.
It is remarkable to find how great is the importance of the local disorders when the venomous reptile belongs to the Solenoglypha group (Viperidæ), while it is almost nil in the case of the Proteroglypha (Colubridæ and Hydrophiidæ).
The effects of general intoxication, on the contrary, are much more intense and more rapid with the venom of Proteroglypha, than with that of Solenoglypha.
In considering the usual phenomena of snake-poisoning in man, we must therefore take this essential difference into account, and draw up separately a clinical description of the symptoms observed after a bite from a Cobra (Colubridæ), for instance, and another list of those that accompany a bite from Lachesis or Vipera berus (Viperidæ).
The bite of a Cobra, even of large size, is not very painful; it is characterized especially by numbness, that supervenes in the bitten part, rapidly extends throughout the body, and produces attacks of syncope and fainting. The patient soon experiences a kind of lassitude and irresistible desire to sleep; his legs scarcely support him; he breathes with difficulty and his respiration becomes of the diaphragmatic type.
By degrees the drowsiness and the difficulty of breathing become greater; the pulse, which at first is more rapid, becomes slower and gradually weaker, the mouth contracts, and there is profuse salivation, the tongue appears swollen, the eyelids remain drooping, and, after a few hiccoughs frequently accompanied by vomiting and involuntary emissions of urine or fæcal matter, the unfortunate victim falls into the most profound coma and dies. The pupils react to luminous impressions up to the last moment, and the heart continues to beat sometimes for two hours after respiration has ceased.
All this takes but a few hours, most frequently from two to six or seven, rarely more.
When the reptile by which the bite is inflicted is one of the Solenoglypha, such as a Lachesis for example, the seat of the bite immediately becomes very painful and red, then purple. The surrounding tissues are soon infiltrated with sanguinolent serosity. Sharp pains, accompanied by attacks of cramp, extend towards the base of the limb. The patient complains of intense thirst, and extreme dryness of the mouth and throat; the mucous membranes of the eyes, mouth, and genitalia become congested.
These phenomena often continue for a very long period, even for more than twenty-four hours, and are sometimes accompanied by hæmorrhages from the eyes, mouth, stomach, intestines, or bladder, and by more or less violent delirium.
If the quantity of venom absorbed be sufficient to cause death, the patient exhibits, a few hours after being bitten, stupor, insensibility, and then somnolence, with increasing difficulty of respiration, which ends by becoming stertorous. Loss of consciousness seems complete a good while before coma appears. Asphyxia then ensues, and the heart continues to beat for nearly a quarter of an hour after respiratory movements have entirely ceased.
In certain exceptional cases death is very rapid; it may supervene suddenly in a few minutes, even before the local phenomena have had time to manifest themselves; in this case the venom, having penetrated directly into a vein, has produced almost immediate coagulation of the blood, thus causing the formation of a generalized embolism.
If the venom be introduced in a highly vascular region, or directly into a vein, the result is almost invariably fatal. On the contrary, if the derm be scarcely broken, or if the clothing has acted as a protection, scarcely any absorption will take place. We are here confronted with the same factors of gravity as in the case of bites inflicted upon human beings by animals suffering from rabies.
In experiments we are able to eliminate all these factors, and to follow in an animal inoculated with a known quantity of venom the whole series of phenomena of poisoning, the intensity of which can be graduated. Let us see, then, how the various animals that it is possible to make use of in laboratories behave with regard to venoms of different origins.