APPENDIX 469 



eight hours examine the plate with a lens, and pick out the minute 

 colonies, streptococci, and sub-culture in broth, and incubate at 37° C. 

 Stain by Gram's method, and, if necessary, further sub-culture. 



(/) Sewagfe Orgcanisms and the organisms indicative of surface 

 pollution should also be examined for. If they be present in the water, 

 it may be taken as proved that such water has been recently polluted, 

 and shoiild be condemned. Crude sewage generally contains in 1 c.c. : 

 (a) 1 to 10 million bacteria ; (6) 100,000 B. coli (or closely allied forms) ; 

 (c) 100 spores of B. enterilidis sporogenes ; and (d) 1000 streptococci 

 (Houston). Further, so minute a quantity as -^^-^ of a c.c. of crude 

 sewage is usually sufficient to produce "gas" in a gelatine "shake" 

 culture in twenty-four hours at 20° C, and the inoculation of animals 

 with crude sewage always leads to a local reaction and not uncommonly 

 results in death. These three organisms, B. coli, B. enteritidis sporogenes, 

 and streptococci have been termed the "microbes of indication." These 

 bacteria are wholly, or relatively, absent from pure water, and their 

 presence, at all events in considerable numbers, must be taken as 

 indicating recent animal pollution.* B. coli is a most accurate measure of 

 intestinal pollution, and far greater information as regards the sewage 

 pollution of water can be gathered by its estimation, than by simply 

 counting the total number of organisms present in water. It is an 

 intestinal parasite, and tends to perish in other media.f When it is 

 present in a small stream, contamination from houses can be traced. J 



Thresh has suggested the following scheme of examination of a 

 water as one furnishing the minimum amount of information which will 

 enable anyone to say positively that a water is faecally contaminated : — 

 (1) The detection of the presence of organisms of intestinal type ; (2) 

 the isolation and identification of B. coli; and (3) the detection of the 

 presence of spores of B. enteritidis sporogenes. The process he recom- 

 mends is as follows : — («) Make bUe-salt broth cultures with 1, 5, 10, 

 and 20 c.c. of the water to be examined. After twenty-four hours the 

 tube containing the smallest quantity of water showing acid and gas 

 formation is selected for further examination. If after forty-eight hours 

 there is no such reaction, no further examination is made. (6) Two or 

 three loopfuls of the culture are added to 10 c.c. of sterilised water, and 

 a loopful of the solution is spread over a plate of bile-salt-lactose- 

 peptone agar containing neutral red and made faintly alkaUne to litmus. 

 This plate is incubated for twenty-fours at 37° C, and the colonies pro- 

 duced carefully examined. As the B. coli communis ferments lactose 

 with the production of acid, any colonies of this organism will be of a 

 red colour and be surrounded by a haze, formed by the precipitation of 

 the bile acids. As this haze may not be apparent at the end of twenty- 

 four hours, types of all the red colonies are taken for the further 

 examination, (c) Each colony so selected is used to inoculate a tube of 

 lactose-peptone-bile-salt-litmus solution, and after twenty-four hours' 

 incubation, if acid and gas is produced, the growth is examined micro- 



* Second Report of liuyal Commission on Sewage Disposal, 1902, pp. 26 and 27. 

 t ll)id., p. 99. ■ J Ibid., p. 109. 



