WATER 35 



If the OT, ro, and 10 c.c. tubes show no gas after forty- 

 eight hours, it can be assumed that B. coli is absent in these 

 amounts. Then, in every case, the larger amount (i.e. the 

 30 c.c. in the bottle) should be examined for this organism. The 

 alteration of the red colour to yellow, with the presence of 

 fluorescence, is an indication of the probable presence of B. coli. 



If gas is present in the tubes containing smaller amounts, use 

 the one showing gas in the tube with the least quantity of added 

 water for inoculating plates of solid media. In this way it can 

 be definitely ascertained whether B. coli is present or absent in 

 50 c.c. or less, and if present, approximately in what numbers. 



In certain cases it is of value to ascertain the presence or 

 absence of B. coli in 500 or 1000 c.c. of the water. 



This may be done by filtering the water through a porcelain 

 filter, as described for the examination of B. enteritidis sporogenes 

 (Chapter II), and using the filter brushings, emulsified in a 

 little water, to add to tubes of bile salt broth. This method is, 

 however, cumbersome and unsatisfactory. 



A far better procedure is to convert the water sample itself 

 into a nutrient medium by the addition of four times strength 

 broth, incubate at 37 C. for twenty-four hours, and then either 

 inoculate suitable plates direct, or, what is preferable, add I c.c. 

 by sterile pipette to a tube of bile salt broth, and incubate this 

 for one or two days. If any B. coli were present in the original 

 bulk of water, the preliminary incubation in the water broth 

 would have allowed them to multiply sufficiently to be at least 

 present in I c.c. of the sample, an amount of fluid readily 

 examined. 



The composition of the media used is given in the Appendix. 



To isolate the B. coli group organism a trace of the positive 

 tube selected is distributed over the surface of a plate containing 

 neutral red lactose bile salt agar (L.B.A.), fuchsin agar or other 

 medium selected. L.B.A. is recommended as most suitable. 

 Several colonies should be subcultivated and worked out. 



Subcultivation upon or in the following five media is recom- 

 mended for routine work, i.e. : 



(a) Gelatine slope (for morphology, motility, cultural ap- 

 pearance, and liquefaction). 



32 



