DIAONOSIS OF ASIATIC CHOLERA. 339 



several single colonies, and finally the test for the pres- 

 ence or absence of indol in these cultures. 



In all doubtful cases in which only a few curved 

 bacilli are present, or in which irregularities in either 

 the rate or mode of their development occurs, pure cul- 

 tures should be obtained by the agar-plate method and 

 the method of cultivation in peptone solution, as soon 

 as possible, and their virulence tested upon animals. 

 For this purpose cultures upon agar-agar from single 

 colonies must be made. From the surface of one of such 

 cultures a good sized wire-loopful should be scraped 

 and this broken up in about one cubic centimetre of 

 bouillon, and the suspension thus made injected by 

 means of a hypodermatic syringe directly into the peri- 

 toneal cavity of a guinea-pig of about 350 to 400 

 grammes weight. For larger animals more material 

 should be used. If the material injected is from a 

 fresh culture of the cholera organism toxic symptoms 

 at once begin to appear ; these have their most pro- 

 nounced expression in the lowering of temperature, 

 and if one follows this decline in temperature from time 

 to time with the thermometer it will be seen to be gradual 

 and continuous from the time of injection to the death 

 of the animal (Pfeiffer'), which occurs in from eighteen 

 to twenty-four hours after the operation. 



In general, this is the procedure employed in the 

 Institute for Infectious Diseases, at Berlin, under 

 Koch's direction. 



1 liOe. cit. 



