114 THE GROUSE DISEASE chap. 



This broth culture proved, therefore, also useless for 

 protective inoculation. 



The next attempts were directed towards attenu- 

 ating virulent broth cultures by heat : ( i ) by exposing 

 broth cultures incubated at 37° C. from 24 to 48 hours 

 to a temperature of 50 C. for 10 minutes. Of such a 

 culture about ^ of a cubic centimetre was injected 

 subcutaneously into each of ten fowls. All these 

 fowls became ill with fowl enteritis, eight died, two 

 survived. These two were then re-inoculated with 

 virulent broth culture, and proved refractory. 



(2) Of broth culture exposed to 50" C. for 20 

 minutes, about \ oi 2. cc. was injected into each of 

 eleven fowls. All became ill, four died, seven survived. 

 These seven were also re-inoculated and proved 

 refractory. 



(3) Of broth culture exposed to 55^ C. for 15 

 minutes about l of a cc. was injected into each of 

 eight fowls. All these fowls were quiet on the 6th, 7th, 

 and 8th days, were off their food, but had no diarrhoea ; 

 seven survived and became well again, ^ but the eighth 

 was found dead on the 8th day with enlarged spleen ; 

 cultivations made of the heart's blood proved that the 

 animal had died of fowl enteritis. This series seemed, 

 therefore, a considerable step in advance of the former. 



1 Also these seven fowls were re-inoculated with \irulent broth 

 culture, but proved refractory. 



