tion of toxin-antitoxin mixtures.

ACTIVE IMMUNISATION.

The immunisation of the rabbit against the Diplococcus pneumoniae may be
instanced as an example of the general methods of immunisation of
laboratory animals.

1. Take a full grown rabbit weighing not less than 1200 to 1500 grammes
(large rabbits of 2000 grammes and over are the most suitable for
immunising experiments). Observe weight and temperature carefully during
the few days occupied in the following steps.

2. Inoculate a small rabbit intraperitoneally with one or two loopfuls
of a twenty-four-hour-old blood agar cultivation of a _virulent_ strain
of Diplococcus pneumoniae.

Death should follow within twenty-four hours, and in any case will not
be delayed beyond forty-eight hours.

3. Under aseptic precautions, at the post-mortem, transfer a loopful of
heart blood to an Erlenmeyer flask containing 50 c.c. sterile nutrient
broth. Incubate at 37 deg. C. for twenty-four hours.

4. Prepare also several blood agar cultures from the heart blood of the
rabbit, label them all O.C. (original culture). After twenty-four hours
incubation at 37 deg. C. place an india-rubber cap over the plugged mouth
of the tube of all but one of these cultures and paint the cap with Canada
balsam or shellac varnish, dry, and replace in the hot incubator.

This will prevent evaporation, and cultures thus sealed will remain
unaltered in virulence for a considerable time.

5. Make a fresh subcultivation on blood agar from the uncapped O.C.
cultivation and after twenty-four hours incubation at 37 deg. C.
determine the minimal lethal dose of this strain upon a series of mice
(see page 316).

6. Suspend the flask containing the twenty-four-hour-old broth culture
(step 3) in the water-bath at 60 deg. C. for one hour. Cool the flask
rapidly under a stream of cold water.

7. Determine the sterility of this (?) killed cultivation by
transferring one cubic centimetre to each of several tubes of nutrient
broth, and incubate at 37 deg. C. for twenty-four hours. If growth of
Diplococcus pneumoniae occurs, again heat culture in water-bath at 60
deg. C. for one hour and again test for sterility.

8. Inject the selected rabbit intravenously (see page 363) with 2 c.c.
of the killed cultivation, and inject a further 10 c.c. into the
peritoneal cavity.

During the next few days the animal will lose some weight and perhaps
show a certain amount of pyrexia.

9. When the temperature and