50 BACTERIOLOGY. 



upon fowl-cholera, observed that after non-fatal cases the disease 

 either did not recur, or the severity of a subsequent attack was in 

 inverse proportion to the severity of the first attack. It occurred 

 to him to endeavour to obtain the virus of this disease in a form 

 which would provoke a mild attack of the disease, and thus give 

 protection against the virulent form. This attenuation or miti- 

 gation of the virus was successfully attained by allowing cultiva- 

 tions of the microbe in chicken-broth to remain with a lapse 

 of several months between the carrying on of successive cultiva- 

 tions in fresh media. The new generations which were then 

 obtained were found to have diminished in virulence, and ultimately 

 a virus was obtained which produced only a slight disorder ; on 

 recovery the animal was found to be proof against inoculation with 

 virulent matter. The explanation given by Pasteur of this change 

 was, that prolonged contact with the oxygen of the air was the 

 influence which diminished the virulence, and he endeavoured to 

 prove this by showing that when broth was inoculated in tubes 

 which could be sealed up, so that only a small quantity of air 

 was accessible to the microbe, the virulence of the cultures was 

 retained. 



Toussaint investigated the possibility of attenuating the virus of 

 anthrax. Sheep injected with 3 cc. of defibrinated blood, con- 

 taining anthrax bacilli, which had been exposed to 55 C. for ten 

 minutes, recovered, and were afterwards insusceptible. Pasteur 

 subsequently argued that this method did not admit of practical 

 application, because difficulties would arise in dealing with infective 

 blood in quantity, and artificial cultivations started from this blood 

 could not be relied upon, as they proved sometimes as virulent as 

 ever. 



Pasteur endeavoured to apply the same method for obtaining 

 an attenuated virus of anthrax, as he had successfully employed 

 in fowl-cholera. A difficulty was soon encountered, for in culti- 

 vations of this bacillus, with free access of air, spore-formation 

 readily takes place, and the spores are well known to have an 

 extraordinary power of retaining their virulence. Pasteur found 

 that the bacilli ceased to develop at 45 C., and he believed that 

 spore-formation ceased at 42 to 43 C C., the bacilli continuing to 

 develop by fission only,. The cultivations were, therefore, kept at 

 this temperature, and at the end of eight days the bacilli were 

 found to have lost their virulence, and were quite inert when 

 inoculated in guinea-pigs, sheep, or rabbits. This total destruction 

 was, however, preceded by a gradual mitigation, so that a virus 



