572 APPLICATION OF METHODS OF BACTERIOLOGY 



Steam at 100 C., for three hours to insure complete solution 

 of the agar-agar. 



Decant, or filter through cotton, and distribute in 100 

 c.c. flasks. 



Sterilize by steam at 100 C. for an hour and a half. 



For use mix one volume of a with 5 volumes of 6, the 

 latter having been completely liquefied by steam. When 

 thoroughly mixed pour into Petri dishes, to a depth of about 

 3 mm. in each dish, and allow to solidify. When the medium 

 is solid, the dishes may be placed in the incubator with the 

 covers partly removed until the condensed vapor has eva- 

 porated. The medium should be comparatively dry before 

 attempting to use it. When dry the plates so prepared may 

 be stored in dust proof receptacles at 15 C. The plates may 

 now be inoculated from the surface of the Benedict medium. 

 This is best done by transferring a loopful of the Benedict 

 culture to the surface of the solid alkaline egg-glucose- 

 agar and distributing it over the surface with a sterile, bent- 

 glass spreader. When thus inoculated the plates are placed 

 in the incubator at 37-38 C. until colonies develop. 



On this medium the cholera, and the cholera-like spirilla 

 grow luxuriantly, while the other bacteria are to some extent 

 restrained. 



The colonies of the cholera vibrios, and of those other 

 vibrios that closely resemble it, when well developed on 

 this medium, i. e., after about twenty hours at 37^38 C., 

 are, to the naked eye, more opaque than those of other 

 bacteria; under the low power lens they seem as if in the 

 depths of the medium, are more or less hazy, are surrounded 

 by an indistinct halo or fringe which may be in turn 

 surrounded by a clear zone. All such colonies should be 

 examined microscopically and from all that are composed 



