394 MANUAL OF BACTERIOLOGY. 



a pure culture, death usually results, apparently from the toxic 

 substances contained in the culture. Pfeiffer was the first to 

 show that an animal may be made immune from cholera by 

 repeated small doses of cultures which have been heated 

 in order to kill the organism. He also showed, in the same 

 connection, that when living comma bacilli are introduced 

 in the peritoneum of an immune animal they first clump 

 together and are then rapidly destroyed and disintegrated 

 (see page 228); furthermore, that a drop of the peritoneal 

 fluid added to a hanging-drop culture of the cholera spirillum 

 produces the same effect. This is now called Pfeiffer's phe- 

 nomenon, and is the underlying principle of all agglutination 

 reactions, such as the Gruber-Widal typhoid test. 



It seems probable, from the results so far obtained, that it is practicable 'to 

 use injections of dead cultures upon human beings with safety, and in this 

 way so protect healthy persons from cholera during an epidemic.* 



Haffkinef elaborated a special method which goes by his name. He found 

 that by passage through guinea-pigs the virulence of the culture could 

 be exalted to a constant maximum point which he calls his "virus fixe." This is 

 attained by a passage through 20 or 30 guinea pigs. The guinea-pigs are all 

 injected into the peritoneal cavity, starting by inoculating a guinea-pig with 

 a dose larger than is necessary to kill the animal, and inoculating the subsequent 

 animals of the series with the peritoneal exudate of the dead animals. The 

 exudate must be exposed to the air for 15 hours before it is injected into the 

 next guinea-pig, otherwise it is found to be not so virulent, this exposure con- 

 trary to usual experience enhancing the virulence. Small guinea-pigs give less 

 abundant but more concentrated exudate. This so-called "virus fixe" is 

 cultivated in beef-broth at 39 C. in a current of air, and kept in this manner 

 until cultures made daily on agar fail to show any growth. The last culture 

 which grows injected into a guinea-pig subcutaneously does not produce any 

 necrosis, and it also prevents the necrosis of the skin produced by "virus fixe" 

 when so injected. Haffkine inoculated himself with the attenuated culture 

 described, and 6 days later with the "virus fixe;" he then proceeded to inject a 

 large number of persons, and finally to try its inoculations upon more than 

 100,000 persons in localities in India, with apparently somewhat favorable re- 

 sults. The inoculation does not modify the course of the disease in those cases 

 in which an inoculated person contracts the disease. The percentage of death 

 is as great among such persons as among uninoculated persons who contract 

 the disease. 



Although a positive demonstration that the spirillum of 

 Koch is the cause of cholera is lacking, as far as the exact 



*Strong. American Medicine. August 15, 1903. 



fBull. de VInst. Past. Vol. IV., Nos. 17 and 18. 1906. pp. 697-705, and 

 737-747- 



