ISOLATION OF B. TYPHOSUS 721 



Both l found that caffeine in broth would retard B. coli, but 

 allow B. typhosus to multiply. The method was further elaborated 

 by Hoffmann and Ficker, 2 who converted the water itself into a 

 nutrient medium by the addition of 1 per cent, of nutrose, 0-5 per 

 cent, caffeine, and 0-001 per cent, of krystal violet. The mixture 

 is incubated at 37 C. for not more than twelve to thirteen hours, 

 at the end of which time the typhoid bacilli should have multiplied 

 to such an extent as to permit of direct isolation by plating, the 

 B. coli being inhibited. While caffeine may materially help, it 

 cannot be entirely relied on to eliminate B. coli and allied forms. 



Loffler found that malachite green (No. 120 Hoechst) in the 

 proportion of about 1 in 5,000 in media inhibits the growth of 

 B. coli while still permitting the growth of B. typhosus. The dye 

 may be added either to liquid or to solid media. The medium 

 recommended by Loffler 3 is composed of 3 per cent, agar made 

 with meat infusion, with 1 per cent, nutrose, and containing in 

 every 100 c.c. 2-2-5 c.c. of a 1 per cent, solution of malachite green. 

 On this medium the B. typhosus grows in twenty-four hours as 

 delicate, slightly crinkled colonies, surrounded by a colourless 

 zone (due to alkali formed by the bacilli). Thus it is possible to 

 detect one colony of B. typhostis among 300 to 600 colonies of 

 other bacteria. As a medium for " enriching " i.e. for specially 

 advancing the growth of the B. typhosus Loffler recommended a 

 15 per cent, gelatin, prepared with beef-juice and peptone, and 

 containing per 100 c.c. 3 c.c. of doubly normal phosphoric acid 

 and 2 c.c. of 2 per cent, malachite-green solution. With the 

 suspected matter, firstly, one series of malachite -gelatin plates is 

 prepared and incubated at 25 C. for twenty to twenty-four hours ; 

 secondly, a tube of malachite gelatin is inoculated and incutiated 

 at 37 C. for twelve to twenty-four hours ; from this a second tube 

 is inoculated and incubated at 37 C., and then plated out on 

 malachite gelatin and incubated at 25 C. The colonies of B. 

 typhosus are well marked after twenty to twenty-four hours, as 

 large as a pin's head, transparent, highly refractile, light grey and 

 granular. Their shape is circular or oval, and they show charac- 

 teristic offshoots resembling a bone-corpuscle or the body of an 



1 Hyg. Rundschau, xiii, 1003, p. 489. 



2 Ibid., xiv, 1904, p. 1. 



3 Deutsch. med. Woch., 1906, No. 8. 



M.B. 46 



