VAEIATIONS IN TYPHOID BACILLI 277 



Media and technique 



In our experiments the media used and technique employed were as 

 follows : 



(a) Meat infusion broth. Meat infusion inoculated with Bad. coli, 

 incubated about twenty-four hours at 37°C., autoclaved and filtered. 

 To this was added 1 per cent pepton and 0.5 per cent sodium chloride. 



(b) Nutrose broth. Instead of meat infusion 0.25 per cent nutrose 

 was used. Both broth media were autoclaved for jBfteen minutes at 15 

 pounds' pressure, and the reaction was adjusted to pH 7.0 or pH 7.1, 



(c) Media containing sugars. In order to diminish the risk of decom- 

 posing the sugars during sterilization, they were dissolved in sterile 

 distilled water, and heated in the autoclave for ten minutes at 10 

 pounds' pressure. 



The sterilized sugar solution was added in the proportion of 1 per cent 

 to the sterile broth together with 5 cc. of sterilized litmus or 5 cc. of 

 2 per cent phenol red and 1.2 cc. of decolorized 1 per cent aqueous 

 solution of china blue (Morishima, 1917) per 100 cc. of the broth. Then 

 the medium was transferred to small test tubes, and allowed to stand 

 at least twenty-four hours at 37°C. in an incubator and for twenty-four 

 hours at room temperature before being used. 



For plates meat infusion agar (2 per cent) containing 1 per cent of 

 the sugar was used. Decolorized china blue was then added in the 

 proportion given above for the fluid medium. The reaction of all media 

 mentioned above was adjusted to pH = 7.0 or pH = 7.1 by means of 

 phenol red. 



The stock cultures were transferred to agar slopes and incubated over 

 night. Then pepton water tubes (1 per cent pepton 0.5 per cent salt 

 solution, reaction pH 7.0) were inoculated from the slant cultures. 

 After the latter had been incubated over night, one loopful of the pepton 

 water growth was transferred to each tube of sugar medium; agglutina- 

 tion tests with the pepton cultures were also carried out. 



In plating cultures on Endo or any other plates, one or two loopfuls 

 of bacterial suspensions were usually streaked close to the margin of the 

 Petri dish. The plate was divided into five parts by lines drawn on its 

 bottom. From the first streak made with the loop, the suspension 

 was spread over one-fifth of the surface; from the border of this area 

 over the next third, and then from the last border over the remaining 

 surface. By this method the distribution of bacteria was found to be 

 satisfactory (Morishima, 1917). They were then kept in the incuba- 



