Attenuation of the virus can also be secured by

5. Pa.s.sage through naturally resistant animals.

6. Exposure to desiccation.

7. Exposure to gaseous disinfectants.

8. By a combination of two or more of the above methods.

IMMUNISATION.

The further study of the pathogenetic powers of any particular bacterium involves the active immunisation of one or more previously normal animals. This end may be attained by various means; but it must be remembered that immunisation is not carried out by any hard and fast rule or by one method alone, but usually by a combination of methods adapted to the exigencies of each particular case. The ordinary methods include:

A. Active Immunisation.

I. By inoculation with dead bacteria (i. e., bacteria killed by heat; the action of ultra-violet rays, of chemical germicides, or by autolysis).

II. By the inoculation of attenuated strains of bacteria.

III. By the inoculation of living virulent bacteria (exalted in virulence if necessary).

B. Combined Active and Pa.s.sive Immunisation:

IV. By the inoculation of toxin-ant.i.toxin 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 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 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 sh.e.l.lac 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 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 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 C. for twenty-four hours. If growth of Diplococcus pneumoniae occurs, again heat culture in water-bath at 60 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 weight have again returned to normal--generally about seven days after the inoculation--again inject killed cultivation, this time giving a dose of 5 c.c. intravenously and 20 c.c. intraperitoneally. A temperature and weight reaction similar to, but less marked than that following the first injection will probably be observed, but after about a week's interval the animal will be ready for the next injection.

10. When ready to give the third injection prepare a fresh blood agar subculture from another O.C. tube and after twenty-four hours incubation prepare a minimal lethal dose (as determined in 5) and inject it subcutaneously into the rabbit's abdominal wall.

A slight local reaction will probably be observed as well as the weight and temperature reactions.

11. A week to ten days later inject a similar minimal lethal dose into the peritoneal cavity.

12. Observe the weight and temperature of the rabbit very carefully, and regulating the dates of inoculation by the animal's general condition, continue to inject living cultivations of the pneumococcus into the peritoneal cavity, gradually increasing the dose by multiples of ten.

13. At intervals of two months samples of blood may be collected from the posterior auricular vein and the serum tested for specific antibodies.

14. Under favourable conditions it will be found after some six months steady work that the rabbit may be injected intraperitoneally with an entire blood agar cultivation without any ill effects being apparent; and this characteristic--resistance to the lethal effects of large doses of the virus--is the sole criterion of _immunity_. Further, the serum separated from blood withdrawn from the animal about a week after an injection, if used in doses of .01 c.c., will protect a mouse against the lethal effects of at least ten minimal lethal doses of living pneumococci.

In the foregoing ill.u.s.tration it has been a.s.sumed that complete acquired active immunity has been conferred upon the experimental rabbit in consequence of the formation of antibody, specific to the diplococcus pneumoniac, sufficient in amount to ensure the destruction of enormous doses of the living cocci--the _antigen_ (that is the substance injected in response to which _antibody_ has been elaborated) in this particular case being the bacterial protoplasm of the pneumococcus with its endo-toxins.

But provided death does not immediately follow the injection of the antigen, specific antibody is always formed in greater or lesser amount; and in experimental work a sufficient amount of any required antibody can often be obtained without carrying the process of immunisation to its logical termination.

For instance, if the immunisation of a rabbit toward Bacillus typhosus is commenced on the lines already set out it will often be found, after a few injections of "killed" cultivation that the blood serum of the animal (even when diluted with several hundred times its volume of normal saline) contains specific agglutinin for B. typhosus--and if the sole object of the experiment has been the preparation of agglutinin the inoculations may well be stopped at this point, although the animal is not yet immune in the strict meaning of the word.

Again, antibodies may be formed in response to antigens other than infective particles--thus the injection into suitable animals of foreign proteins such as egg alb.u.min, heterologous blood sera or red blood discs from a different species of animal, will result in the formation of specific antibodies possessing definite affinities for their respective antigens.

The most important antibody of this latter type is Haemolysin, a substance that makes its appearance in the blood serum of an animal previously injected with washed blood cells from an animal of a different species. The serum from such an animal possesses the power of disintegrating red blood discs of the variety employed as antigen and causing the discharge of their contained haemoglobin, and is specific in its action to the extent of failing to exert any injurious effect upon the red blood cells of any other species of animal.

The action of this serum is due to the presence of two distinct bodies, complement and haemolysin.

_Complement_ (or alexine) is a thermo-labile readily oxidised body present in variable but unalterable amount in the normal serum of every animal. It is a substance which exerts a lytic effect upon all foreign matter introduced into the blood or tissues; but by itself is a comparatively inert body, and is only capable of exerting its maximum lytic effect in the presence of and in combination with a specific antibody, or immune body.

Complement is obtained (unmixed with antibody) by collecting fresh blood serum from any healthy normal (that is uninoculated) animal.

Guinea-pigs' serum is that most frequently employed for experimental work.

_Haemolysin_ (immune body, copula, sensitising body, amboceptor) is a _thermostable_ antibody formed in response to the injection of red cells which although in itself inert is capable of linking up complement present in the normal serum to the red cells of the variety used as antigen--a combination resulting in haemolysis.

Haemolysin is obtained by collecting fresh blood serum from a suitably inoculated animal and exposing it to a temperature of 56 C. (to destroy the thermo-labile complement) for 15 to 30 minutes before use. It is then referred to as _inactivated_, and is _reactivated_ by the addition of fresh normal serum--that is serum containing complement.

Haemolysin is of importance academically owing to the fact that many of the problems of immunity have been elucidated by its aid; but its present practical importance lies in the application of the _haemolytic system_ (that is haemolysin, corresponding erythrocyte solution and complement) to certain laboratory methods having for their object either the identification of the infective ent.i.ty or the diagnosis of the existence of infection.

For use in these laboratory methods of diagnosis it is most convenient to prepare haemolytic serum specific for human blood--whether the laboratory is isolated or attached to a large hospital. Ox blood, sheep blood or goat blood if readily obtainable, may however be used instead, and although the following method is directed to the preparation of human haemolysin the same procedure serves in all cases.

THE PREPARATION OF HaeMOLYTIC SERUM.