ACTIVE IMMUNITY. 465 



when cultures were kept for a long time in ordinary conditions, 

 they gradually lost their virulence, and that when sub-cultures 

 were made, the diminished virulence persisted. Such attenuated 

 cultures could be used for protective inoculation. He considered 

 the loss of virulence to be due to the action of the oxygen of the 

 air, as he found that in tubes sealed in the absence of oxygen 

 the virulence was not lost. Haffkine attenuated cultures of the 

 cholera spirillum by growing them in a current of air (p. 421). 



(2) The virulence of an organism for a particular animal may 

 be lessened by passing the organism through the body of another 

 animal. Duguid and Burdon Sanderson found that the virulence 

 of the anthrax bacillus for bovine animals was lessened by being 

 passed through guinea-pigs, the disease produced in the ox by 

 inoculation from the guinea-pig being a non-fatal one. This dis- 

 covery was confirmed by Greenfield, who found that the bacilli 

 cultivated from guinea-pigs preserved their property in cultures, 

 and could therefore be used for protective inoculation of cattle. 

 A similar principle was applied in the case of swine plague by 

 Pasteur, who found that if the organism producing this disease 

 was inoculated from rabbit to rabbit, its virulence was increased 

 for rabbits but was diminished for pigs. Organisms which had 

 been passed through a series of rabbits produced in the pig 

 illness, but not death, and protection for at least a year resulted. 

 The method of vaccination against smallpox depends upon the 

 same principle. 



(3) Many organisms become diminished in virulence when 

 grown at an abnormally high temperature. The method of 

 Pasteur, already described (p. 315), for producing immunity in 

 sheep against anthrax bacilli, depends upon this fact. A virulent 

 organism may also be attenuated by being exposed to an elevated 

 temperature which is insufficient to kill it. Toussaint at an early 

 date obtained protective inoculation against anthrax by means of 

 cultures which had been exposed for a certain time to a tempera- 

 ture of 55° C, though it is possible that in some cases the bacilli 

 were really killed, and immunity resulted from the chemical sub- 

 stances in the bacilli or produced by them. 



(4) Still another method may be mentioned, namely, the 

 attenuation of the virulence by growing the organism in the 

 presence of weak antiseptics. Chamberland and Roux, for ex- 

 ample, succeeded in attenuating the anthrax bacillus by grow- 



