4. Observe during life or until the completion of twenty-eight days, and in the event of death occurring during that period, make a complete post-mortem examination.

5. Repeat the experiment at least once. In the event of a positive result estimate the minimal lethal dose of "killed" culture for each of the species of animals experimented upon.

(b) Extracellular or Soluble Toxins:

1. Filter the cultivation through a porcelain filter candle (Berkfeld) into a sterile filter flask, arranging the apparatus as in the accompanying figure (Fig. 135).

Kant

竞意

Fig. 135.-Apparatus arranged for toxin filtration.

2. Inoculate mice, rats, guinea-pigs, and rabbits subcutaneously with that quantity of toxin corresponding to 1 per cent. of the body-weight of each respectively, and observe, if necessary, until the completion of one month.

3. Inoculate a "control" tube of bouillon with a similar quantity and incubate.

4. In the event of a fatal termination make complete and careful post-mortem examinations.

5. Repeat the experiments and, if the results are. positive, ascertain the minimal lethal dose of toxin for each of the susceptible animals.

XV. EXPERIMENTAL INOCULATION OF

ANIMALS.

THE animals generally employed in the study of the pathogenic properties of the various micro-organisms are:

Mouse.
Rat.

Guinea-pig.

Rabbit.

Pigeon.
Fowl.

Preparation. Before inoculation, the experimental animals should be carefully examined, to avoid the risk of employing diseased animals; the weight should be recorded and the rectal temperature taken.

Weighing. The larger animals are most conveniently weighed in a decimal scale provided with a metal cage for their reception instead of the ordinary pan (Fig. 136). Mice and rats are weighed in a modification of the letter balance, weighing to 250 grammes, which has a conical wire cage substituted for its pan (Fig. 137).

The weight of inoculated animals should be observed and recorded each day, at precisely the same hour, during the entire period of observation, preferably before the morning feeding.

Temperature. To take the rectal temperature of any of the laboratory animals, the animal should be firmly held by an assistant and the bulb of an ordinary clinical thermometer, well greased with vaseline, introduced just within the sphincter ani. Allow it to remain in this position for a few seconds, and then push it on gently and steadily

until the entire bulb and part of the stem, as far as the constriction, have passed into the rectum. Three to five minutes later, the time varying, of course, with the sensibility of the thermometer used, withdraw the instrument and take the reading.

Daily, if not more frequently, observations should be made of the temperature of inoculated animals during the entire period they are under observation.

Fig. 137.-Mouse scales.

9 89 90 100

Cages. During the period which elapses between inoculation and death, or complete recovery, the experimental animals must be kept in suitable receptacles that can easily be kept clean and which can be readily disinfected.

The mouse is usually stored in a glass jar (Fig. 138) 11 cm. high and 11 cm. in diameter, closed by a wire gauze top which is weighted with lead or fastened to the mouth of the jar by a bayonet catch. A matter of great convenience is a small oblong label 5 cm. by

2.5 cm., sand-blasted on the side of the cylinder, as marks made upon this with an ordinary lead pencil

Fig. 138.-Mouse jar.

show up well and only require

the use of a damp cloth to remove them (Fig. 138).

The rat is kept under observation in a glass jar similar, but larger, to that used for the mouse.

These jars are sterilised after use either by chemical reagents or by autoclaving.

The rabbit and the guineapig are confined in cages of suitable size, made entirely of metal (Fig. 139). The sides and top and bottom are of woven wire work; beneath the cage is a movable metal tray filled with sawdust, for the reception of the excreta. The cage as a whole is raised from the ground on short legs. The sides,

etc., are generally hinged so that the cage packs up flat, for convenience of storing and also of sterilising. The ordinary rat cage, a rectangular wire-work

box, 30 cm. from front to back, 20 cm. wide, and 14 cm. high, makes an excellent cage for guinea-pigs if fitted with a shallow zinc tray,35 by 24 cm., for it to

stand upon.

These cages are sterilised after use either by autoclaving or spraying with formalin.

The tray which receives the dejecta should be cleaned out and supplied with fresh sawdust each day, and the soiled sawdust, remains of food, etc., should be cremated.

As animal inoculation is purely a surgical operation, the necessary instruments will be similar to those employed by the surgeon, and, like them, must be sterile. In the performance of the inoculation strict attention must be paid to asepsis and suitable precautions adopted to guard against accidental contamination of the material to be introduced into the animal. addition, the hands of the operator should be carefully disinfected.

In

The list of apparatus used in animal inoculations given below comprises practically everything needed for any inoculation. Needless to remark, all the apparatus will never be required for any one inoculation.

Apparatus Required for Animal Inoculation:

1. Water steriliser (vide page 38). It is also convenient to have a second water steriliser, similar but smaller (23 by 7 by 5 cm.), for the sterilisation of the syringes.

Fig. 140.-Hypodermic syringe with finger rests.

2. Injecting syringe. The best form is one of the ordinary hypodermic pattern, fitted with finger rests, but with the leather washers and the packing of the piston replaced by those made of asbestos (Fig. 140). The instrument must be easily taken to pieces, and