34 THE GROUSE DISEASE chap. 



more or less extensive necrosis of the skin and sub- 

 cutaneous tissue at and about the seat of inoculation is 

 noticed. 



As stated just now, the majority of the mice 

 inoculated with a recent broth culture (3-5 minims) 

 die. Gelatine cultures less than 10 days old, and 

 potato- or Agar cultures less than 4 or 5 days old, 

 used as a slightly milky salt mixture, prove viru- 

 lent : old cultures of gelatine and old cultures of 

 Agar are apt to show less virulence, this may 

 become almost lost with age. In the former case a 

 similar proportion of the mice succumb, but later than 

 after inoculation with recent cultures ; in the latter 

 no death follows on inoculation of even large doses. 

 In order to ensure virulence I use recent broth 

 cultures. These do not fail, no matter whether they 

 are sub-cultures of old or recent cultures on gelatine, 

 Agar, or broth, I have tested in this respect cultures 

 that have been carried on in sub-cultures for many 

 generations — in gelatine for more than three years 

 and a half; and I find that when a broth sub-culture 

 is made of an old gelatine culture, say one or several 

 months to half a year old, and is inoculated into 

 mice, it proves virulent. The highest degree of 

 virulence is obtained by making a sub-culture in 

 alkaline broth to which a piece of boiled white of 

 egg is added (the whole being sterilised before use). 



