42 TTBERCULIX, ITS HISTORY, PREPARATION AND USE. 



Having prepared a stock of the above material, it is trans- 

 ferred to a number of 50 cc Erlenmyer's conical shaped flasks 

 which have been previously ehemically cleaned, plugged with 

 cotton wool and sterlised. 



It is necessary that in order to obtain as large a surface of 

 the fluid exposed to the air, that only 20 cc of the nutrient 

 media be placed in each flask. 



The flasks and their contents are sterilized on four succes- 

 sive days for 20 minutes at each operation, in 

 a steam sterilizer at 212 degrees Fahr., or for 30 minutes on two 

 successive days in an autoclave or steam digestor, under a pres- 

 sure of 27 lb. on the S(]uare inch. On removal they are allowed 

 to cool down, when each flask is ready to be inoculated wnth the 

 pure culture of the tubercle bacillus, which to perform for the 

 first time is an exti'ernely difficult and tedious operation. Night 

 after night, for many weeks at a time, I have worked away by 

 myself in the laboratory in order to perfect this operation. 



If we take a trace of the growth from the surface of an 

 Agar Agar culture of tubercle baccilli, and place it into the flask, it 

 gradually sinks to the bottom of the fluid ; if this flask of inocu- 

 lated media is placed in an incubator we have to wait many 

 months before we see a perceptible increase in the growth of the 

 colony of tubercle bacilli, the reason being that the organisms 

 have not had free access to oxygen, which is absolutely essential 

 for their rapid development. Although this specific bacillus is 

 recognised as one of the slowest growing organisms, we have suc- 

 ceeded in obtaining most luxuriant cultivations by growing it on 

 the surface of this fluid media. In the first place this is only 

 accomplished l)y inoculating from an old and very dry Agar 

 Agar ciilture, a very large numl^er of flasks, special care being 

 ■exercised to make the minute traces of growth of bacilli float. 

 After the flasks have been in the incubator for about three weeks, 

 it is possible to find out of 100 flasks, perhaps one flask with an 

 extremely delicate film, almost imperceptible to the naked eye 

 floating over a small portion of the surface fluid. Should a 

 microscopical examination of this film after staining with special 

 aniline dyes result in showing nothing but a pure culture of the 

 iubercle baciUus, immediate steps are taken to transfer by 



