488 INFECTION AND RESISTANCE 



tan's experiments also depended upon the injection of living cul- 

 tures, but Haffkine attempted, by a rather elaborate technique, to 

 produce two separate vaccines, one attenuated, the other enhanced in 

 virulence. Attenuation was accomplished by growing the cholera 

 spirilla at a temperature of 39 C in broth over the surface of which 

 a constant stream of sterile air was passed. Under these conditions 

 the first crop of cholera organisms died rapidly, but Haffkine prac- 

 ticed reinoculation into new broth flasks before complete death of the 

 original culture had taken place; after a series of generations of 

 cultivation in this way he obtained cultures which produced merely 

 temporary and slight edema when injected under the skin of guinea 

 pigs. This weakened virus was used for the first inoculation. 



He enhanced the virulence of cholera cultures with the purpose 

 of producing a strain of maximum potency comparable to virus fixe. 

 His procedure was as follows: 



a. Giving an animal an intraperitoneal injection of cholera 

 spirilla larger than the fatal dose. 



b. Taking out the peritoneal exudate and exposing it for a few 

 hours to the air. 



c. Injecting this exudate into another animal and treating the 

 resulting peritoneal exudate in the same way. 



After a series of such animal passages he claims to have obtained 

 a virus of great virulence, and this is his second and stronger vaccine. 



In applying the method to human beings Haffkine planted the 

 cholera spirilla upon agar slants of the standard size, emulsified the 

 growths in sterile water, and injected 1/5 to 1/20 c. c. of such a 

 culture, using first the weak vaccine and five days later a more 

 virulent culture. 



Beginning his work as early as 1893, Haffkine and others vac- 

 cinated as many as 40,000 people in India. On the whole, the results 

 obtained were very encouraging. It is a question, however, whether 

 or not his method is unnecessarily complicated. In the light of our 

 more recent knowledge concerning cholera immunity it seems likely 

 that the importance which Haffkine attached to the virulence of the 

 cholera culture used for injection was exaggerated, and we have 

 reason to believe that simple immunization with killed cultures may 

 produce results fully as efficacious. After all, we could not expect, 

 at least at present, to produce by active artificial immunization an 

 immunity as permanent as that which results from an attack of the 

 disease. Concerning the reasons for the acquisition of such perma- 

 nent immunity we have as yet little knowledge. Even Haffkine's 

 method of inoculation with living virus does not, by his own estima- 

 tion, last longer than possibly two years. It is therefore likely that 

 prophylactic immunization in cholera is efficacious by reason of the 

 appearance in the blood serum of the specific bactericidal and opsonic 

 substances by which the small numbers of cholera organisms enter- 



