CHAPTER IV.

_SECRETION AND COLLECTION OF VENOM IN SNAKES._

_Non-poisonous_ as well as _poisonous_ snakes possess _parotid_ and _upper labial_ glands capable of secreting venom. In the former the organs of inoculation are wanting, but we shall see later on that the toxic secretion of their glands is just as indispensable to them as to the snakes of the second category for the purpose of enabling them to digest their prey.

For the morphological, histological, and physiological demonstration of the existence of these glands in harmless reptiles we are indebted to Leydig (1873), whose discovery has since been confirmed and extended by the researches of Phisalix and Bertrand, Alcock, L. Rogers, and L. Lannoy.

The parotids of Grass Snakes are mixed glands of the sero-mucous type. The serous tubes are situate almost exclusively in the posterior portion of the gland. As we proceed towards the anterior portion, we find that these serous tubes are interspersed with others which are exclusively mucous or sero-mucous, and they become entangled with those of the upper labial gland, properly so-called. The substance of the gland is divided into several lobes by bands of connective tissue; the tubes are separated by _septa_ of the same tissue, in extremely delicate layers (Lannoy).

In poisonous snakes these glands are much more developed, especially in their hinder portions, which sometimes assume enormous dimensions. They may attain the size of a large almond (_Crotalus_, _Naja_), and they then occupy the spacious chamber already described (Chap. I., p. 10), which is situated behind the eye on each side of the skull.

Each gland is surrounded by a thick capsule of fibrous tissue, two prolongations of which, the one anterior, the other posterior, keep it in its place beneath the _masseter_ muscle. A portion of the latter is inserted in the capsule itself, in such a way that when the snake closes its jaws to bite, the gland is forcibly compressed and the contained liquid is squeezed into its excretory duct.

Between the muscle and the envelope of the gland there is a serous pouch, which enables the one to slide over the other.

The excretory duct runs along the outer side of the upper jaw, and opens by a slit at the base of the poison-fang, with which it inosculates at right angles in a little muscular mass forming a _sphincter_.

In the normal position of repose, the poison-fang is always concealed by a gingival fold of mucous membrane, in the substance of which are buried a few fibres of the tendon of the internal pterygoid muscle. When the latter contracts, the tooth is almost completely exposed, and the efferent duct of the gland then assumes an oblique position, which allows of the direct discharge of the venom through the canal which runs along the greater portion of the length of the tooth.

When the poison-fangs are folded back in their sheath, the poisonous secretion can escape freely into the buccal cavity by the slit situated at the base of the fangs.

At the moment when the animal is about to bite, when it throws back its head and opens its jaws, directing its fangs forwards, the muscles that come into action (_masseters_, _temporals_, and _pterygoids_) compress the glands on each side, and cause the venom to be expelled in a sudden jet, as if by a sort of ejaculatory process. In the case of certain species the venom may be projected to a distance of more than a yard.

The quantity of venom secreted by the glands varies greatly, according to the length of time which has elapsed since the animal took its last meal, and in accordance with a number of other conditions not very easy to determine.

The Common Viper of Europe yields scarcely 10 centigrammes of poison, while an adult Indian Cobra may excrete more than 1 gramme.

* * * * *

Freshly collected venom is a syrupy liquid, citron-yellow or slightly opalescent white in colour.

When dried rapidly _in vacuo_ or in a desiccator over calcium chloride, it concretes in cracked translucent lamellæ like albumin or gum arabic, and thus assumes a crystalloid aspect. In this condition it may be kept indefinitely, if protected from light, air, and moisture. It dissolves again in water just as readily as albumin or dried serums.

I regularly weighed the dry residue from eleven bites made on a watch-glass by two _Naja haje_, received at my laboratory from Egypt at the same time, and placed in the same case. Both snakes were approximately of equal length, 1,070 millimetres. Throughout the entire course of the experiment, which lasted _one hundred and two days_, neither of them took any food, but they drank water and frequently bathed.

The results that I obtained are shown in the table on next page.

It will be seen that in one hundred and two days, an adult _Naja haje_ is capable of producing on an average 0·632 gramme of liquid venom, equal to a mean weight of 0·188 gramme of dry extract; and we may conclude that 1 gramme of _liquid_ gives 0·336 gramme of _dry_ venom.

In Australia it has been found by MacGarvie Smith, of Sydney, that _Pseudechis porphyriacus_ yields at each bite a quantity of venom varying from 0·100 gramme to 0·160 gramme (equal to 0·024 gramme to 0·046 gramme of dry venom), and that a _Hoplocephalus curtus_ (Tiger Snake) yields 0·065 gramme to 0·150 gramme of liquid venom, with 0·017 gramme to 0·055 gramme of dry residue. In all the experiments of this physiologist, the proportion of dry residue varied from 9 to 38 per cent. of the liquid venom excreted by the reptile.

A _Lachesis lanceolatus_ (Fer-de-lance) from Martinique, of medium size, when both of its glands were squeezed, furnished me with 0·320 gramme of liquid venom, and 0·127 gramme of dry extract.

+---------+----------+-------------------+-------------------+ | | | _NAJA HAJE_ I. | _NAJA HAJE_ II. | |Number of| Date | WEIGHT OF VENOM | WEIGHT OF VENOM | | bite | +---------+---------+---------+---------+ | | | Fresh | Dry | Fresh | Dry | +---------+----------+---------+---------+---------+---------+ | | | Gramme | Gramme | Gramme | Gramme | | 1 | April 20 | 0·119 | 0·031 | -- | -- | | 2 | “ 23 | -- | -- | 0·151 | 0·043 | | 3 | May 14 | 0·124 | 0·035 | -- | -- | | 4 | “ 21 | -- | -- | 0·132 | 0·037 | | 5 | “ 28 | -- | -- | 0·091 | 0·019 | | 6 | June 2 | 0·127 | 0·039 | -- | -- | | 7 | “ 19 | -- | -- | 0·121 | 0·043 | | 8 | July 1 | -- | -- | 0·078 | 0·026 | | 9 | “ 2 | 0·122 | 0·048 | -- | -- | | 10 | “ 25 | -- | -- | 0·111 | 0·034 | | 11 | “ 26 | 0·079 | 0·021 | -- | -- | +---------+----------+---------+---------+---------+---------+ | | Totals | 0·581 | 0·174 | 0·684 | 0·202 | +---------+----------+---------+---------+---------+---------+

Two large _Cerastes_ vipers, from Egypt, yielded me, one 0·123 gramme, the other 0·085 gramme of liquid venom, which, after desiccation, left respectively 0·027 gramme and 0·019 gramme of dry residue.

Under the same conditions, a magnificent _Crotalus confluentus_ (Mottled Rattle-Snake), for which I was indebted to the kindness of Mr. Retlie, of New York, yielded, two months after reaching my laboratory, 0·370 gramme of liquid venom and 0·105 gramme of dry extract in _a single bite_.

The total quantity of liquid venom that I found contained in the two glands of the same reptile, when extirpated after death, and after the snake had been in the laboratory for five months, amounted to 1·136 gramme, which gave 0·480 gramme of dry extract.

We see, therefore, that the proportion of dry residue, including albumin, salts, the _débris_ of leucocytes, and the toxic substance, oscillates between 20 and 38 per cent. Its strength varies with the length of time that has elapsed since the snake’s last bite or last meal.

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From the _histological standpoint_, the process of the secretion of venom, in the cells of the glands, may be divided into two stages:--

(_a_) A stage of nuclear elaboration.

(_b_) A stage of cytoplasmic elaboration.

These two stages are superposed and successive.

In addition to the passive exchanges between the nucleus and the cytoplasm, the nuclear mass actively participates in the secretion. This participation is rendered evident:--

(1) By the difference of chromaticity in the granules of chromatin.

(2) By the emission of formed granules into the cytoplasm, granules which are spherical and of equal bulk, with the chromatic reactions of differentiated intranuclear chromatin.

(3) By the exosmosis of the dissolved nuclear substance, accessorily formed in an ergastoplasmic shape.

These formations constitute, on the one hand, the granules of _venogen_; on the other, the ergastoplasmic venogen. In the poison-cell of _Vipera aspis_, and in the serous cell of the parotid glands of _Tropidonotus natrix_ (Grass Snake) the venogen is elaborated chiefly in granular form.

On entering the perinuclear cytoplasm, the granule of venogen and the ergastoplasmic venogen may either disappear immediately, as happens in periods of cellular stimulation, or else continue to exist for some time within the cell, indicating a period of saturation by the elaborated material.

During cytoplasmic activity the granule of venogen and the ergastoplasmic venogen disappear.

Nuclear elaboration and cytoplasmic elaboration constitute two different cycles of secretion. The effect of the nuclear cycle is to furnish the cytoplasm with the elements necessary for the work of secretion properly so-called. Cytoplasmic elaboration is not confined to the basal protoplasm, but takes place throughout the entire cell: it is especially active in the perinuclear cytoplasm.

The granule of venogen is distinguished from the granule of elaborated venom by its affinity for Unna’s blue, safranin, and fuchsin. The granule of venom has an affinity for eosin; it is never excreted in granular form, but after intracellular dissolution.

Venogen is never met with in the lumen of the gland-tube.[6]

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 of anything. In this way the most powerful snake is perfectly under control.

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 artificial feeding in the manner previously described (see p. 17), for they very often refuse to feed themselves.

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 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.