392 BACTERIOLOGY. 



face 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 

 examination, 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 occur, 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 ani- 

 mals. 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 centi- 

 metre of bouillon, and the suspension thus made injected 

 by means of a hypodermic 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 



