ATTENUATION OF ORGANISMS. 657 



to be a very suitable means ; the temperature may vary 

 between 42 and 55 C,, and the organisms must be ex- 

 posed to it for a longer time the lower the temperature 

 employed. Thus according to Toussaint a temperature 

 of 55 C. was able to attenuate the bacilli of an anthrax 

 cultivation in ten minutes ; according to Chauveau a tem- 

 perature of 52 C. must be employed for fifteen minutes, of 

 50 C. for twenty minutes, of 47 C. for one to four hours 

 after previous exposure to 42 to 43 C. for twenty hours ; 

 according to Pasteur and Koch a temperature of 43 C. 

 must be employed for six days, a temperature of 42 C. 

 for about twenty-eight to thirty days, in order to obtain a 

 complete loss of the pathogenic action ; where the action 

 is continued above thirty days death of the bacilli occurs. 



According to Pasteur's recommendations, which have Most suitable 

 been more precisely worked out by Koch, the following m 

 is the method best suited for obtaining attenuated an- 

 thrax bacilli : the cultivations are made in neutralised 

 chicken broth, which is placed in a moderately deep layer 

 in Erlenmeyer's flasks. These flasks are then placed in 

 a D'Arsonval's thermostat kept at 42 C. In spite of 

 careful regulation the temperature varies somewhat in 

 the interior ; and hence the individual vessels frequently 

 show a somewhat varying degree of attenuation. At 

 times, also, spore formation may take place, though as a 

 rule this does not occur at 42 C. ; if it does take place the 

 attempt at attenuation fails. Hence it must always be 

 borne in mind that some of the cultivations may not have 

 succeeded. From eight days onwards the vessels must 

 be tested daily ; and the degree of attenuation of each 

 specimen is ascertained either by sowing the material in 

 a vessel containing chicken-broth and kept at 37 C., so 

 that spores are soon formed ; or by inoculating a mouse 

 with the specimen, and after its death making culti- 

 vations from the spleen, and thus obtaining spores. The 

 cultivations or spores so obtained preserve for a long 

 time that degree of virulence at which the material had 

 arrived. 



After exposure to a temperature of 42 C. for ten days Various 

 the anthrax bacilli are so attenuated that rabbits and attenuation 



42 



