ACTIVE IMMUNISATION 323 



This will prevent evaporation, and cultures thus 

 sealed will remain unaltered in virulence for a consider- 

 able 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 3 1 6). 



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 cultiva- 

 tion 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 

 pneumonias 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. 



