PROTECTIVE INOCULATIONS. 277 



or three large punctures under the skin, upon the internal surface of the ear, 

 suffice for a successful inoculation. 



' ' The vaccine prepared in this way should be used at once, or at least 

 not later than the day after it has been prepared. Experience has shown me 

 that it is then quite as harmless and quite as efficacious as Pasteur's vaccine." 



In the preparation of an attenuated virus by this method Chauveau 

 insists upon attention to the following points : 



' ' The first rule to follow, and the principal one, is to practise the heating 

 in such a manner that all parts of the anthrax blood are raised to the re- 

 quired temperature and withdrawn from it at the same instant. When the 

 quantity of blood to be transformed to a vaccine is too great, all parts are 

 not uniformly acted upon by the very short exposure to heat ; the virulent 

 agents in the deeper layers may, in that case, preserve all of their activity, 

 and cause a fatal infection. To avoid this it is best to enclose the blood in 

 little cylindrical pipettes, 1 mm. in diameter. The extremity of these 

 pipettes is sealed, and the portion which contains the blood is immersed in a 

 considerable quantity of water maintained at the proper temperature. At 

 the end of the proper time they are taken from the hot bath and plunged into 

 cold water. 



' ' Another rule should be rigorously observed if one wishes to be sure of 

 success. The blood should be collected under conditions which make it sure 

 that the virulent agents introduced into the tubes all have the same vitality, 

 the same activity, and that they are impressed in the same degree by the 

 heating. This is the case when we take the blood from a guinea-pig just 

 dead, after having survived from thirty-six to forty-eight hours an inocula- 

 tion with very active virus. Before introducing the blood into the pipettes 

 it should be allowed to coagulate, and the coagula should be broken and 

 crushed in order to obtain a defibrinated blood, which is always very rich in 

 virulent bacilli." 



In a subsequent communication (February 26th, 1883), Chauveau 

 admits that the application of this method is somewhat difficult and 

 delicate when blood is employed, and states that it is far more satis- 

 factory to use pure cultures, which may be attenuated in the same 

 way. He prefers to cultivate the bacillus in a bouillon made from 

 the flesh of a chicken, and to start his culture by adding to this bouil- 

 lon a drop of blood from an animal just dead from anthrax. The cul- 

 ture is left for twenty hours in an incubating oven at a temperature of 

 43 0. During this time there is an abundant development of the 

 bacillus, and the culture is ready to be subjected to the attenuating 

 action of a higher temperature. This is accomplished by exposure 

 to a temperature of 47 C. for a period of one, two, three, or four 

 hours, according to the degree of attenuation desired. After three 

 hours' exposure the attenuated culture no longer kills guinea-pigs. 

 In a later communication (March 5th, 1883) Chauveau states that he 

 has ascertained by experiment that the degree of attenuation produced 

 by this method is maintained in subsequuent cultures made at 43 C., 

 from the attenuated culture thus obtained. 



Another method of attenuating the virulence of anthrax cultures is 



