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His method, as opposed to vague conceptions and a priori 

 speculations, went on fortifying itself day by day. Artificial 

 attenuation, that is, virus modified by the oxygen of air, which 

 weakens and abates virulence ; vaccination by the attenuated 

 virus those two immense steps in advance were announced by 

 Pasteur at the end of 1880. But would the same process apply 

 to the microbe of charbon? That was a great problem. The 

 vaccine of chicken-cholera was easy to obtain ; by leaving pure 

 cultures to themselves for a time in contact with air, they soon 

 lost their virulence. But the spores of charbon, very indiffer- 

 ent to atmospheric air, preserved an indefinitely prolonged 

 virulence. After eight, ten or twelve years, spores found in 

 the graves of victims of splenic fever were still in full virulent 

 activity. It was therefore necessary to turn the difficulty by 

 a culture process which would act on the filament-shaped bac- 

 teridium before the formation of spores. What may now be 

 explained in a few words demanded long weeks of trials, tests 

 and counter tests. 



In neutralized chicken broth, the bacteridium can no longer 

 be cultivated at a temperature of 45 C. ; it can still be culti- 

 vated easily at a temperature of 42 C. or 43 C., but the spores 

 do not develop. 



"At that extreme temperature," explains M. Chamberland, 

 " the bacteridia yet live and reproduce themselves, but they 

 never give any germs. Thenceforth, when trying the viru- 

 lence of the phials after six, eight, ten or fifteen days, we have 

 found exactly the same phenomena as for chicken-cholera. 

 After eight days, for instance, our culture, which originally 

 killed ten sheep out of ten , only kills four or five ; after ten or 

 twelve days it does not kill any ; it merely communicates to 

 animals a benignant malady which preserves them from the 

 deadly form. 



' ' A remarkable thing is that the bacteridia whose virulence 

 has been attenuated may afterwards be cultivated in a tempera- 

 ture of 30 C. to 35 C., at which temperature they give germs 

 presenting the same virulence as the filaments which formed 

 them." 



Bouley, who was a witness of all these facts, said, in other 

 words, that "if that attenuated and degenerated bacteridium 

 is translated to a culture medium in a lower temperature, fav- 

 ourable to its activity, it becomes once again apt to produce 

 spores. But those spores born of weakened bacteridia, will 



