5. Apply thick celloidin to the tube-capsule joint, the opposite end of the capsule, and the line of junction of the capsule with its cap; dry thoroughly.

6. With a teat pipette fill the capsule (through the attached tube) with hot water, and stand the capsule in a beaker of boiling water for a few minutes to melt the gelatine.

7. Remove the solution of gelatine from the interior of the celloidin case with a pipette.

8. Fill the sac with nutrient broth and place it, _gla.s.s tube downward_, in a tube containing sufficient sterile nutrient broth to cover the sac to the depth of 1 cm. Plug the tube and sterilise in the steamer in the usual manner.

9. To prepare the sac for use, empty it out of the broth tube into a sterile gla.s.s dish.

10. Grasp the tube near its junction with the sac in the jaws of sterile forceps, and with a teat pipette remove sufficient of the contained broth to leave a small s.p.a.ce in the sac. Introduce the inoculum in the form of an emulsion by means of another pipette.

11. Still holding the tube in the forceps, draw it out and seal off near the sac in the blowpipe flame.

12. When cool wash the sac in sterile water, then transfer to a tube of nutrient broth and incubate over night to determine its impermeability to bacteria.

13. If the broth outside the sac remains sterile, insert the sac in the peritoneal cavity of the experimental animal.

~5. Intracranial.~--(_Anaesthetic, A. C. E._)

[Ill.u.s.tration: FIG. 186.--Guarded trephine.]

_Trephines and Surgical Engine._--The most useful instrument for intracranial operations upon animals is the small nasal trephine (Curtis) having a tooth cutting circle of 7 mm. The addition of an adjustable collar guard--secured by a screw--prevents accidental laceration of the dura mater or brain substance[13] (Fig. 186). This size is suitable for monkeys, dogs, cats and large rabbits. Other smaller sizes which will be found useful for guinea pigs and other small animals cut circles of 6 and 4 mm.; for very small animals--young guinea pigs and rats--a small dental drill or screw will make a sufficiently large hole to admit the syringe needle. The trephine can be set in ordinary metal handles and rotated by hand, but a surgical engine of some kind is much preferable on the score of rapidity and safety to the animal. The Guy's electrical Dental engine[14] (Fig. 187) which can be connected to a lamp socket or wall plug, and is operated by a foot switch, although inexpensive is eminently satisfactory.

NOTE.--A fine dental drill attached to the dental engine renders the manufacture of aluminium handles needles (see page 71) quite an easy matter.

(a) _Subdural._

1. Anaesthetise the animal and secure it to the operating table, dorsum uppermost.

2. Shave a portion of the scalp immediately in front of the ears.

[Ill.u.s.tration: FIG. 187.--Guy's electrical dental engine.]

3. Mark out with a sharp scalpel a crescentic flap of skin muscle, etc., convexity forward, commencing 0.5 cm. in front of the root of one ear and terminating at a similar spot in front of the other ear. Reflect the marked flap.

4. Make a corresponding incision through the periosteum and raise it with a blunt dissector.

5. With a small trephine (diameter 6 mm.) remove a circular piece of bone from the parietal segment. The centre of the trephine hole should be at the intersection of the median line and a line joining the posterior canthi (Fig. 188).

6. Introduce the inoculum by means of a hypodermic syringe, perforating the dura mater with the needle and depositing the material immediately below this membrane, at the same time taking care to avoid injuring the sinuses.

7. Turn back the flap of skin and secure it in position with Michel's steel clips.

8. Dress with sterile gauze and wool and seal the dressing with collodion.

9. Label, etc.

(b) _Intracerebral._--This inoculation is performed precisely as for subdural save in step 6 the needle after perforating the dura mater is pushed onward into the substance of one or other cerebral hemispheres before the contents are ejected.

[Ill.u.s.tration: FIG. 188.--Intracranial inoculation of rabbit. The circle indicates the situation of the trephine hole.]

~6. Intraocular.~--

(a) _Fluid Inoculum._--(_Anaesthetic, cocaine._)

1. Instil a few drops of a sterile solution of cocaine, and repeat the instillation in two minutes.

2. Five minutes later have the animal firmly held by an a.s.sistant as in intravenous injection (see Fig. 189), the head being steadied by the a.s.sistant's hands.

3. Select two needles to accurately fit the same syringe and sterilise.

4. Attach one needle to the syringe and take up the required dose of inoculum and remove the needle.

5. Steady the eye with fixation forceps; then pierce the cornea with the other syringe needle and allow the aqueous to escape through the needle.

6. Without removing the needle from the cornea attach the syringe and make the injection into the anterior chamber.

7. Irrigate the conjunctival sac with sterile saline solution.

8. Label, etc.

(b) _Solid Inoculum._--(_Anaesthetic, A. C. E._)

1. Anaesthetise the animal and secure it firmly to the operating table.

2. Irrigate the conjunctival sac thoroughly with sterile saline solution.

3. Make an incision through the upper quadrant of the cornea into the anterior chamber by means of a triangular keratome.

4. Separate the lips of the corneal wound with a flexible silver spatula; seize the solid inoculum in a pair of iris forceps, introduce it through the corneal wound, and deposit it on the anterior surface of the iris; withdraw the forceps.

5. Again irrigate the sac and the surface of the cornea.

6. Release the animal from the operating table.

7. Label, etc.

~7. Intrapulmonary.~--

_Fluid Inoculum._--(_Anaesthetic, none._)

1. Have the animal firmly held by an a.s.sistant. (In this case the foreleg of the selected side is drawn up by the a.s.sistant and held with the ear of that side.)

2. Shave carefully in the axillary line and disinfect the denuded skin.

3. Thrust the needle of the syringe boldly through the fifth or sixth intercostal s.p.a.ce into the lung tissue.