546 APPLICATION OF METHODS OF BACTERIOLOGY 



of curved or spiral organisms pure cultures should be made 

 for subsequent identification. 



In abortive cases of cholera the organisms may be present 

 in the intestinal canal in very small numbers, and micro- 

 scopic examination is not, therefore, of so much assistance. 

 In these cases the adoption of one or the other of the fore- 

 going methods is imperative. 



In the foregoing suggested plans it will be observed that 

 plates are not made in the usual way. The reason for this 

 is the cholera spirillum being markedly aerobic develops 

 much more readily on the surface than in the depths of the 

 medium. For the same reason the material taken for 

 plating from the enriching media should always be from 

 the surfaces, without the tubes or flasks having been shaken. 



It being desirable to have the colonies isolated from one 

 another the plates should be relatively dry, that is, there 

 should be no collection of moisture on their surfaces that 

 would cause the colonies to become confluent. After pour- 

 ing, the plates should always be kept in a dust-free incubator 

 with their lids off until all excess of moisture is evaporated. 

 All colonies of curved rods should be isolated in pure culture 

 in peptone solution, and after twenty to twenty-four hours 

 at 37 to 38 C. such cultures should be tested for the 

 presence of indol. After giving positive indol reaction 

 should be regarded as probably cholera spirilla. 



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 cultures should be 

 obtained as soon as possible and their virulence tested upoi 

 animals. For this purpose* cultures upon agar-agar from 

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

 such cultures a large wire-loopful should be scraped and 



