124 POPULAR SCIENCE MONTHLY. 



In trying to obviate this result I found, perhaps contrary 

 to expectations, that the exudation liquid should be exposed to the air 

 for a few hours before it is injected into a subsequent animal. This 

 result was contradictory to the effect which Pasteur had found to be 

 exercised by atmospheric oxygen on the virulence of microbes, and it 

 requires at least some provisional explanation. The microbes of cholera 

 differ from a certain number of other microbes in that they stand in 

 need of a free and abundant access of air for growing and multiplying 

 quite satisfactorily. They are deprived of this condition in the peri- 

 toneal cavity of an animal. It is possible, therefore, that a certain 

 opposition between the maintenance or development of virulence on the 

 one hand, and a lowering in vitality on the other, takes place while 

 they are cultivated there, and a respite must be given them between 

 each successive 'passage' through the Guinea pig by leaving them for 

 a time in the free atmosphere. Be that explanation true or not, the 

 result is that under such conditions the successive animals inoculated 

 with the virus do succumb, and even in a shorter and shorter time, after 

 the inoculation, the microbe apparently undergoing under such a treat- 

 ment a progressive increase in virulence. A similar development up to a 

 certain stage was observed by Pasteur when transferring the rabies 

 virus from rabbit to rabbit. The last difficulty that presented itself was 

 the following: The exudation liquid which is found in the peritoneal 

 cavity post mortem varies in quantity; sometimes it is inconveniently 

 large and diluted; sometimes, on the contrary, so scanty that it becomes 

 difficult to collect and transfer it to another animal. I found that this 

 variation stands in connection with the size of the animal, so that a 

 diluted exudation fluid can be concentrated by injecting it into a small 

 animal, while a too much concentrated exudate is rendered more dilute 

 by transferring it to an animal of a larger size. 



Thus, by the initial use of more than a fatal dose, by alternating 

 cultivation in an animal with exposure to air, and by attention to the 

 size of the animal employed, a material was obtained which, as men- 

 tioned, increased in intensity from the first and proved fatal to animals 

 in a shorter and shorter time after inoculation. Later the virus reached 

 a stage when it killed a Guinea pig of three hundred and fifty 

 grammes weight in eight hours. After that, in each further inocula- 

 tion the time of eight hours remained stationary, showing that the virus 

 has reached the condition of a 'virus fixe/ These experiments were 

 conducted by me in the Pasteur Institute, in 1889 to 1893, simultane- 

 ously on the cholera microbe and on the bacillus of typhoid. The two 

 exhibited a number of common features in their nature, and the results 

 as above detailed for the cholera microbe were found valid for the 

 typhoid bacillus also. 



Starting from the Virus fixe' obtained as above, a method of double 



