CH. XVI] VIBRIO AND SPIRILLUM 429 



the guinea-pig) stands in no definite relation to the severity 

 of the cholera case from which they are derived. 



Haffkine has on the other hand shown that by successive 

 transference from guinea-pig to guinea-pig of the peritoneal 

 exudation produced in the first of the series by intraperi- 

 toneal injection of a fatal dose of cholera (Agar) culture, 

 after as many as twenty and more transferences the cultures 

 of cholera vibrios obtained from the peritoneal fluid of the last 

 guinea-pig reach a high degree of virulence, so much so 

 that minute quantities of such a culture injected intraperi- 

 toneally are capable of causing fatal general septicemic 

 infection of the guinea-pig. 



It has been shown by Sabolotny (Central, f. Bakt. u. 

 Paras, vol. xv. p. 150) that the marmot is particularly sus- 

 ceptible to subcutaneous injection with the vibrio, acute 

 septicaemic infection and death being the result. 



Similarly also for the guinea-pig the virulence of a given 

 stock of vibrios can be materially increased by adding to 

 the culture medium potassium nitrate, or even a larger pro- 

 portion of sodium chloride. 



The same holds good for the degrees of virulence shown 

 by the cholera (Agar) cultures when injected intraperi- 

 toneally into guinea-pigs. Of some varieties ~ of an 

 Agar culture is sufficient to produce a fatal result in a 

 guinea-pig of 300 grammes weight in twenty to twenty-four 

 hours, while of others as much as \ or even \oi a. culture 

 tube is required. The slanting surface of nutrient Agar is 

 inoculated over its whole extent, then incubated at 37" C. 

 for forty-eight hours. By this time the whole surface (six 

 centimetres by two) is covered with a translucent gray film 

 of growth ; to the culture tube are then added four, five, 

 or six cc. of sterile bouillon, and by means of a sterile plati- 

 num loop the growth is rubbed completely down into the 



