592 APPLICATION OF METHODS OF BACTERIOLOGY 



of the three animals used for the 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 diminu- 

 tion 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 escap- 

 ing; 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 

 bacteria; they grow slowly and less vigorously when trans- 

 planted; they do not form spores when exposed to a high 

 temperature; and microscopically they present evidences 

 of degeneration. When introduced beneath the skin of 

 animals they disseminate but slightly beyond the site of 

 inoculation, and do not, as a rule, cause the general septicemia 

 that occurs in susceptible animals inoculated with normal 

 cultures of this organism. In the practical employment of 

 these attenuated cultures for protective purposes two 

 vaccines are employed. These were designated by Pasteur 

 as "first" and "second" vaccines. The "first" is the one 

 that killed only the mice in the preliminary tests; while 

 the " second" is that which killed both mice and guinea-pigs, 

 but failed to kill the rabbit. When larger animals, such as 

 sheep or cattle, are to be protected by vaccination with 

 these vaccines, a subcutaneous inoculation of about 0.3 c.c. 

 of the first vaccine is usually given. This should be prac- 

 tically without noticeable effect, causing neither rise of 

 body-temperature nor other constitutional or local symp- 

 toms. After a period of about two weeks the second vaccine 



