The anaerobic conditions likewise favor the multiplication of intestinal bacteria, and also their fermentative activity. The temperature 42° C. destroys ordinary water bacteria and inhibits the growth of many ordinary mesophilic bacteria. 5. Pipette 25 c.c. of double strength bile salt broth into flask 6, and 50 c.c. double strength bile salt broth into flask 7. 6. Pipette 25 c.c. water sample into flask 6, and 50 c.c. water sample into flask 7. 7. Incubate the two flasks aerobically at 42°C. 8. After twenty-four hours incubation note in each culture: a. The presence or absence of visible growth. b. The reaction of the medium as indicated by the colour change, if any, the litmus has undergone. c. The presence or absence of gas formation, as indicated by a froth on the surface of the medium, and the collection of gas in the inner "gas" tube. 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 hanging-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 macroscopically 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. Practice 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. 16. 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 centimetre of the emulsion to each of the fluid media by means of a sterile pipette.