The Elements of Bacteriological Technique Part 92

8. Flasks (75 c.c.) containing sterilised normal saline solution (or sterile bouillon).

9. Sterilised cotton-wool. Cotton-wool (absorbent) is packed loosely in a copper cylinder similar to that used for storing capsules, and sterilised in the hot-air oven.

10. Sterilised gauze. Gauze is sterilised in the same way as cotton-wool.

11. Sterilised silk and catgut for sutures. These are sterilised, as required, by boiling for some ten minutes in the water steriliser.

12. Flexible collodion (or compound tincture of benzoin).

13. Grease pencil.

14. Tie-on celluloid labels, to affix to the cages.

15. Razor.

16. Small pot of warm water.

17. Liquid soap. Liquid soap is prepared as follows: Measure out 100 grammes of soft soap and add to 500 c.c. of 2 per cent. lysol solution in a large gla.s.s beaker; dissolve by heating in a water-bath at about 90 C. Bottle and label "Liquid Soap."

18. In place of the liquid soap and razor it is sometimes convenient to use a Depilatory powder.

Barium sulphide 1 part Rice starch 3 parts

Dust the powder thickly over the area to be denuded of hair, sprinkle with water and mix into a thin paste _in situ_; allow the paste to act for three minutes, then sc.r.a.pe off with a bone spatula--the hair comes away with the paste and leaves a perfectly bare patch. This process is preferably carried out, the day previous to the operation.

~Material Utilised for Inoculation.~--The material inoculated may be either--

1. Cultures of bacteria--grown in fluid media, or on solid media.

2. Metabolic products of bacterial activity--e. g., toxins in solution.

3. Pathological products (fluid secretions and excretions, solid tissues).

~The Preparation of the Inoculum.~--

(a) _Cultivations in Fluid Media._--

1. Flame the plug of the culture tube.

2. Remove the plug and flame the mouth of the tube.

3. Slightly raise the lid of a sterile capsule, insert the mouth of the culture tube into the aperture and pour some of the cultivation into the capsule.

4. Remove the mouth of the culture tube from the capsule, replace the lid of the latter, flame the mouth of the tube, and replug.

5. Remove the syringe from the steriliser, squirt out the water from its interior, and allow to cool.

6. Raise the lid of the capsule sufficiently to admit the needle of the syringe and draw the required amount of the cultivation into the barrel of the syringe.

(Or, remove a definite measured quant.i.ty of the cultivation directly from the tube or flask by means of a sterile graduated pipette, discharge the measured amount into a sterile capsule, and fill into the syringe; or take up the required quant.i.ty of the cultivation directly into the graduated syringe from the tube or flask.)

[Ill.u.s.tration: FIG. 173.--Conical separatory funnel, fitted for injection of fluid cultivations.]

If it is necessary to introduce a large bulk of fluid into the animal, the cultivation should be transferred with aseptic precautions, to a sterile separatory funnel, preferably of the shape shown in figure 173, and graduated if necessary. This is supported on a retort stand and raised sufficiently above the level of the animal to be injected, so as to secure a good "fall." A piece of sterilised rubber tubing of suitable length, fitted with an injection needle and provided with a screw clamp, is now attached to the nozzle of the funnel and the operation completed according to the requirements of the particular case.

This method is quite satisfactory when the injection is made into the pleural or abdominal cavities or directly into a vein but if the injection has to be made into the subcutaneous tissue the "fall" may not be sufficient to force the fluid in. In this case it will be necessary to transfer the culture to a sterile wash-bottle and fasten a rubber hand bellows to the air inlet tube (interposing an air filter) and attach the tubing with the injection needle to the outlet tube (Fig.

174). By careful use sufficient force can be obtained to drive the injection in.

(b) _Cultivations on Solid Media (e. g., Sloped Agar)._--

1. By means of a sterile graduated pipette introduce a suitable small quant.i.ty of sterile bouillon (or sterile normal saline solution) into the culture tube.

[Ill.u.s.tration: FIG. 174.--Arrangement of pressure injection apparatus.]

2. With a sterile platinum loop or spatula sc.r.a.pe the bacterial growth off the surface of the medium, and emulsify it with the bouillon. It then becomes to all intents and purposes a fluid inoculum.

3. Pour the emulsion into a sterile capsule and fill the syringe therefrom.

(c) _Toxins._--Prepared by previously described methods (_vide_ page 318), are manipulated in a similar manner to cultivations in fluid media.

(d) _Pathological Products._--Fluid secretions, excretions, etc., such as serous exudation, pus, blood, etc., are treated as fluid cultivations; but if the material is very thick or viscous, a small quant.i.ty of sterile bouillon or normal saline solution may be used to dilute it, and thorough incorporation effected by the help of a sterile platinum rod.

Solid tissues, such as spleen, lymph glands, etc., may be divided into small pieces by sterile instruments and rubbed up in a sterilised agate mortar (using an agate pestle), with a small quant.i.ty of sterile bouillon, and the syringe filled from the resulting emulsion.

[Ill.u.s.tration: FIG. 175.--Holding rabbit for shaving.]

If it is desired to inoculate tissue _en ma.s.se_, remove from the material a small cube of 1 or 2 mm. and introduce it into a wound made by sterile instruments in a suitable situation, and occlude the wound by means of Michel's steel clips and a sealed dressing.

~Method of Securing Animals During Inoculation.~--

For the majority of inoculations, especially when no anaesthetic is administered, it is customary to employ an a.s.sistant to hold the animal (see Fig. 175).

If working single handed Voge's holder for guinea-pigs, is a useful piece of apparatus the method of using which is readily seen from the accompanying figures (Figs. 176, 177).

The instrument itself consists of a hollow copper cylinder, one end of which is turned over a ring of stout copper wire, and from this open end a slot is cut extending about half way along one side of the cylinder.

The opposite end is closed by a "pull-off" cap and is perforated around its edge by a row of ventilating holes, which correspond with holes cut in the rim of the cap. In the event of the animal resisting attempts to remove it from the holder backwards, this cap is taken off and the holder placed on the table and the guinea-pig allowed to walk out.

[Ill.u.s.tration: FIG. 176.--Taking guinea-pig's temperature.]

To provide for different-sized animals, two sizes of this holder will be found useful:

1. Length, 16 cm.; breadth, 6 cm.; size of slot, 8 cm. by 2.5 cm.

2. Length, 20 cm.; breadth, 8 cm.; size of slot, 10 cm. by 2.5 cm.

A convenient holder for mice and even small rats is shown in figure 178, the tail being securely held by the spring clip. Needless to say, the holder should be entirely of metal, and the wire cage detachable and easily renewed.

[Ill.u.s.tration: FIG. 177.--Voge's holder.]