THERAPEUTIC IMMUNIZATION IN MAN 485 



ated organisms, applied similar" methods to cholera. First experi- 

 menting with guinea pigs, he soon applied his method to human 

 beings, inoculating them with small quantities of living broth cul- 

 tures of cholera spirilla. In many of his experiments he gave, at the 

 first injection, 8 drops of a fresh broth culture, following this after 

 8 days with 0.5 c. c. of a similar culture. There is no reason why 

 Ferran's method should not have yielded excellent results. How- 

 ever, it is stated that he worked with impure cultures, and other 

 observers, notably Mkati and Eietsch, van Ermengen, da Lara, and 

 others, failed to obtain encouragement in their subsequent investiga- 

 tion of this method of vaccination. 



The method which Haffkine 90 worked out some years after Fer- 

 ran'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 cul- 

 ture, 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 



90 Haffkine. The Lancet, February, 1893; Brit. Med. Journ., December, 

 1895. 



