322 METHODS OF IDENTIFICATION AND STUDY 



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 neces- 

 sary). 



B. Combined Active and Passive Immunisation : 



IV. By the inoculation of toxin-antitoxin 

 mixtures. 



ACTIVE IMMUNISATION. 



The immunisation of the rabbit against the Diplococ- 

 cus pneumonias 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 experi- 

 ments). 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 

 3 7 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 3 7 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. 



