564 APPLICATION OF METHODS OF BACTERIOLOGY 



through the circulation, through the air-passages, through 

 the alimentary tract, or, as we have just seen, through the 

 subcutaneous tissues. 



Protective Inoculation. The most noteworthy application 

 of artificially prepared living vaccines to the protection of 

 animals from infection is seen in connection with anthrax 

 in sheep and in bo vines. 



By a variety of procedures the virulent anthrax bacterium 

 may be in part or totally robbed of its pathogenic properties. 

 It is through the very mild constitutional disturbance 

 caused in animals vaccinated with such weakened cultures 

 that protection is often afforded against the severer, fre- 

 quently fatal, form of the infection. 



Without reviewing the various methods that have been 

 employed for attenuating the virulence of this organism to 

 a degree suitable for protective vaccination, it will suffice 

 to say that the most satisfactory results have been obtained 

 by the classical method of Pasteur. This comprehends the 

 long-continued cultivation (ten to thirty days) at a tem- 

 perature of from 42 to 43 C. In this procedure the spore- 

 free, virulent bacterium anthracis, obtained directly from 

 the blood of a recently dead animal, is brought at once into 

 sterile nutrient bouillon in about twenty test-tubes, which 

 are immediately placed in an incubator that is carefully 

 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 incubator 

 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 determined by 

 tests upon rabbits, guinea-pigs, and mice. The first culture 

 removed may or may not kill rabbits, the most resistant 



