DIAGNOSIS OF ASIATIC CHOLERA. 357 



cally from hour to hour after the sixth hour that they 

 have been in the incubator. The material taken for 

 examination should always come from near the surface 

 of the fluid, and care should be taken not to shake the 

 tube. As soon as comma bacilli are detected in anything 

 like considerable numbers in the upper layers of the 

 fluid, agar-agar plates and fresh peptone cultures should 

 be made from them. The colonies will develop on the 

 agar-agar plates at 37 C. in from ten to twelve hours 

 to a size sufficient for recognition by microscopic ex- 

 amination, and from this examination an opinion can 

 usually be given. This opinion should always be con- 

 trolled by cultures in the peptone solution made from 

 each of several single colonies, and finally the test 

 for the presence 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-agar-plate method 

 and by 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 hypodermic syringe directly into the per- 

 itoneal 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 & fresh 

 culture of the cholera organism toxic symptoms at once 

 begin to appear ; these have their most pronounced ex- 

 pression in the lowering of temperature, and if one fol- 



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