THE DIAGNOSIS OF ASIATIC CHOLERA. 471 



become confluent. To obviate this he recommends that 

 the agar-agar be poured into the plates and the water 

 allowed to separate from the surface at the temperature 

 of the incubator before they are used. It is wise, there- 

 fore, when one is liable to be called on for such work 

 as this, to keep a number of sterilized plates of agar- 

 agar in the incubator ready for use, just as sterilized 

 tubes of the media are always ready at hand. The 

 advantage of using the agar plates is the higher tem- 

 perature at which they can be kept, and consequently 

 a more favorable condition for the development of the 

 colonies. 



As soon as isolated colonies appear they should 

 be examined microscopically for the presence of bac- 

 teria having the morphology of the one we are seek- 

 ing, and as soon as such is detected gelatin plates 

 and cultures in peptone solution (for the indol reac- 

 tion) should be made. The peptone culture from the 

 original material should be examined microscopically 

 from hour to hour after the sixth hour that it has 

 been in the incubator. The material taken for ex- 

 amination 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 

 considerable numbers in the upper layers of the 

 fluid agar-agar plates and fresh peptone cultures 

 should be made from them. In from ten to twelve 

 hours at 37° C the colonies will develop on the agar- 

 agar plates to a size sufficient for recognition by micro- 

 scopic examination, and from this examination an 

 opinion can usually be formed. This opinion should 

 always be controlled by cultures in the peptone solution 

 made from each of several single colonies, and finally 



