BIOLOGICAL PROPERTIES 527 



from the Institut Pasteur easily became asporogenic in the hands of these investi- 

 gators although they failed when they worked with a bacterium isolated by themselves 

 from a case of human anthrax. 



B. The bichromate method of Chamberland and Roux. Bichromate of 

 potassium is used in the same way as the carbolic acid in the previous experi- 

 ment but in different proportions. A solution of 1 in 2000 in broth is the 

 best. 



C. Other methods. The following may be placed on record here but none 

 of them have up till the present given satisfactory results. The method of 

 von Behring depends upon the Use of rosolic and hydrochloric acids ; that 

 of Phisalix is based upon the application of heat (successive cultures at 

 42'5C.). More recently Phisalix and also Bormans have advised repeated 

 sub-cultivation on horse or dog serum ; Phisalix also recommends collodion 

 sac cultures in the peritoneal cavities of dogs and other animals. 



2. Virulence. Attenuation. Pasteur's vaccination. 



Virulence. In man one attack of anthrax confers an absolute immunity. 

 As a rule, anthrax is fatal to domestic animals, but Pasteur found that cows 

 which had recovered from an attack of the disease were able to resist without 

 ill-effect the subsequent inoculation of a very virulent organism. It had been 

 observed also that in the district of La Beauce some of the sheep were immune 

 to anthrax, and by way of explanation the suggestion had been made that 

 these immune animals had already suffered from an abortive attack of the 

 disease. From these facts Pasteur inferred that if he could infect animals 

 with a mild form of anthrax, he would be able to render them immune to 

 the epizootic disease. 



Attenuation. -To demonstrate the truth of this inference an attenuated 

 virus had to be prepared. But since the virulence of the bacterium is retained 

 intact and perpetuated by the spore which originates in it the bacillus had to 

 be prevented from forming spores. The method by which this was and is 

 still done is as follows : 



1. Sow a flask of broth with anthrax blood and incubate at 42*5 C. The 

 organism grows but does jaot form spores. 



The virulence of the bacillus is at first considerable but soon diminishes 

 and after about 8-10 days is harmless on inoculation to guinea-pigs and 

 rabbits. This attenuation is due to the combined action of air and heat 

 on the micro-organism. 



If the attenuated culture be inoculated into a sheep the animal suffers 

 from a very mild attack of anthrax and, after it has recovered, it will be found 

 to be capable of resisting the inoculation of a fully virulent organism. 



That is to say, the inoculation of an attenuated virus confers an immunity 

 upon the animal inoculated. 



2. If the attenuated bacillus be sown in a flask and incubated at 33-37 C. 

 it will again form spores but the virulence will be that of the bacillus in which 

 they develop ; in this way the virulence is fixed and may be indefinitely 

 perpetuated. 



Restitution of the virulence. The virulence of an attenuated bacillus which has 

 become saprophytic can be restored by passage through suitable animals. 



For instance, take a bacillus which fails to kill an adult mouse, and inoculate it 

 into a mouse which has just been born. The latter dies in 2 or 3 days. Inoculate 

 a little of the blood of this first animal into a second mouse 3 days old and after it 

 is dead inoculate some of its blood into a mouse 6 days old. Inoculate the blood of 

 the latter into an adult mouse then some of the blood of the adult mouse into a 

 young guinea-pig and so on through an adult guinea-pig, a rabbit, a sheep and a bovine 

 animal : finally a fully- virulent organism will be recovered. 



