BACILLUS ANTHRACIS. 421 



nutrient bouillon in about twenty test-tubes, which 

 are immediately placed in an incubator that is care- 

 fully regulated to maintain a temperature of 42.5° C. 

 There should not be a fluctuation of over 0.1° C. 



After about a week a tube is removed from the incu- 

 bator on each successive day and its virulence tested at 

 once on animals. The degree of attenuation experienced 

 by the cultures grown under these circumstances is deter- 

 mined by tests upon rabbits, guinea-pigs, and mice. The 

 first culture removed may or may not kill rabbits, the 

 most resistant of the three animals used for tlie test, 

 while it will certainly kill the guinea-pigs and mice; after 

 another two or three days rabbits will no longer succumb 

 to inoculation with the culture last removed from the 

 incubator, while no diminution will as yet be noticed 

 in its pathogenesis for the other two species. After 

 four to seven days more a culture may be encountered 

 that kills only mice, the guinea-pigs escaping; while 

 ultimately, if the experiment be continued, a degree of 

 attenuation may be reached in which the organism has 

 not even the power of killing a mouse, though it still 

 retains its vitality. Investigation of these attenuations 

 shows them to possess all the characteristics of enfeebled 

 anthrax bacillus; they grow slowly and less vigorously 

 when transplanted; they do not form spores while 

 under a high temperature; and microscopically they 

 present evidences of degeneration. AYhen introduced 

 beneath the skin of animals they disseminate but 

 slightly beyond the site of inoculation, and do not, as 

 a rule, cause the general septicaemia that occurs in sus- 

 ceptible animals after inoculation with normal cultures 

 of this organism. In the practical employment of these 

 attenuated cultures for protective purposes two vaccines 



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