

CULTURAL CHARACTERISTICS 253 



B. Crystal violet (Hochst), - 1 gram. 



Absolute alcohol, - 10 c.c. 



Distilled water, - 300 



1. Stain for 1 second or longer in a mixture made just before use of 2 parts 

 of A and 1 part of B. 



2. Wash rapidly in water. 



3. Counterstain for 3 seconds in cresoidin solution. 



Cresoidin, - 1 part. 



Warm water, - - 300 parts. 



When dissolved, filter. 



4. Wash in water. ] 



(7) For staining the polar bodies Epstein recommends pyronin. Stain 

 for 20 seconds in pyronin', wash, treat with Gram's solution for 10 minutes, 

 dry and mount. The polar bodies are stained bright red, the bacilli pale red. 



Note. The diphtheria bacillus becomes decolourized very quickly when mounted 

 in balsam. For permanent preparations the following method is recommended. 



1. Stain in dilute Ziehl's fuchsin for 13 minutes. Wash in water. 



2. Stain in the same manner with Roux's blue. Wash, dry, and mount. 



2. Cultural characteristics. 



A. Conditions of growth. The diphtheria bacillus will grow at all tempera- 

 tures between 20 C. and 40 C. but not above 42 C., the optimum being 

 35-37 C. The bacillus is aerobic : some growth however takes place under 

 anaerobic conditions, but it is poor and the organism rapidly loses its vitality. 



B. Media. 1. Broth. Peptonized- veal-broth gives a /better growth than 

 beef-broth. 



After incubating at 37 C. for 12-24 hours small white masses of growth 

 will be seen adhering to the sides of the flask and later a film forms on the 

 surface : if examined microscopically this film will be found to consist of 

 tangled masses of bacilli many of the latter being club-shaped. Finally, 

 a deposit forms at the bottom of the tube leaving the supernatant fluid 

 clear. The best way to obtain rapidly a luxuriant growth is to sow the 

 bacillus in a flat flask and pass a current of air over the growth during 

 incubation. 



For this purpose select a flask similar to that (Fern- 

 bach's) shown in fig. 168. Fill it with broth through 

 the central vertical tubulure until the level of the fluid 

 reaches to just below the openings of the lateral tubu- 

 lures : plug all three openings with wool, sterilize in 

 the autoclave, sow through the vertical tubulure, 

 replace the wool plug and cover it with an india-rubber 

 cap. Now attach one of the lateral tubes to a water 

 pump and draw a slow current of air through the flask ; 

 the air entering by the other tube sweeps in a con- 

 tinuous stream over the surface of the broth. 



When the diphtheria bacillus is grown in broth the reaction of the latter 

 first becomes acid, but after a few days this initial acidity is converted into 

 an alkaline reaction accompanied by a precipitate of ammonio-magnesium 

 phosphate. In media containing glycerin the acid reaction is very marked 

 and persistent, and the bacilli rapidly die in such media. 



Martin's broth (p. 33) is better than ordinary broth and in it growth is 

 very luxuriant, the medium never becomes acid and the virulence of the 

 bacilli is maintained for a long time. 



2. Coagulated serum. On coagulated serum which is the best medium for 

 the diphtheria bacillus growth is very rapid. 



