WATER 429 



9. Replace those tubes which show no signs of 

 growth in the incubator. Examine after another 

 period of twenty-four hours (total forty-eight hours 

 incubation) with reference to the same points. 



10. Remove culture tubes which show visible growth 

 from the Buchner's tubes, whether acid production 

 and gas formation are present or not. 



11. Examine all tubes which show growth by hang- 

 ing-drop preparations. Note such as show the presence 

 of chains of cocci. 



12. Prepare surface plate cultivations upon nutrose 

 agar from each tube that shows growth either macro- 

 scopically or microscopically, and incubate for twenty- 

 four hours aerobically at 37 C. 



13 . Examine the growth on the plates either with the 

 naked eye or with the help of a small hand lens. Prac- 

 tice will facilitate the recognition of colonies of the coli 

 group, the typhoid group and the paratyphoid group; 

 also those due to the growth of streptococci. The 

 investigation from this stage proceeds along two 

 divergent lines of enquiry the first being concerned 

 with the identity of the bacilli typhoid bacilli, the 

 second with that of the cocci. 



A. B. Coli and its allies. 



14. Pick off coliform or typhiform colonies; make 

 streak or smear subcultivations upon nutrient agar; 

 incubate aerobically for twenty-four hours at 37 C. 



15. Examine the growth in each tube carefully both 

 macroscopically and microscopically. If the growth is 

 impure, replate on nutrose agar, pick off colonies and 

 subcultivate again. When the growth in a tube is pure, 

 add 5 c.c. sterile normal saline solution or sterile broth, 

 and emulsify the entire surface growth with it. 



1 6. Utilise the emulsion for the preparation of a 

 series of subcultivations upon the media enumerated 

 below, using the ordinary loop to make the subcultures 

 upon solid media, but adding one-tenth of a cubic 



