BACTERIOLOGICAL EXAMINATION OF WATER 445 



our counts represent a section through the true bacterial flora which 

 fairly represents the quick-growing sewage forms. Comparability is 

 the vitally essential factor. 



The temperature at which the bacteria develop is of great impor- 

 tance, and they should be protected from light. The access of oxygen 

 whicli prevents the growth of anaerobes must also not be forgotten. 

 As a rule, the plate cultures are developed for three days at room tem- 

 perature, and for eighteen hours at incubator temperature. Some 

 bacteria do not develop colonies in three days, but these are neglected. 

 The number of bacteria growing at room temperature is usually five 

 to ten times as many as at 35 C. 



The glucose broth is placed at 37 C. for the development of the colon 

 bacilli. The fermentation tubes not showing gas are recorded and usually 

 discarded. Those showing gas are suspected to contain colon bacilli. 

 To a number of tubes containing melted litmus-lactose agar at about 

 44 C. are added 1, 0.1, and 0.01 loop of the culture fluid. Plates are 

 poured and the whole placed in the incubator. The bacillus coli fer- 

 ments lactose and thus produces acid ; so that if colon bacilli are present 

 we have a number of red colonies on a blue field. Later, if many colon 

 bacilli were present, the whole medium becomes acid. At forty-eight 

 hours, on account of alkali being produced, the blue color returns. 



If after inspection red colonies are seen, four or five are picked and 

 planted into glucose bouillon and other media. For the characteristics 

 of the colon bacilli the Massachusetts State Board of Health uses six 

 media gelatin, lactose agar, dextrose broth, milk, nitrate solution, and 

 peptone solution. 



Significance of the Colon Bacillus. The colon test has been received 

 by the majority of engineers and practical sanitarians with great satis- 

 faction, and has been applied with confidence to the examination, not 

 only of water, but of shell-fish and other articles of food as well. On 

 the other hand, some have denied its value. Bacteriologists have found 

 bacilli like certain members of the colon group in apparently unpol- 

 luted well-water. The discovery that most animals have colon bacilli 

 apparently identical in the usual characteristics studied with those of 

 man has complicated matters. Thus a fresh hillside stream may be 

 loaded with colon bacilli from the washings of horse or cow manure 

 put on the fields through which it runs, or polluted by a stray cow or 

 horse. Swine, hens, birds, etc., may contaminate in unsuspected WHYS. 

 Up to the present there is no conclusive evidence that colon bacilli 

 increase for any considerable length of time anywhere except in the 

 intestines of the higher vertebrates, and they are widely distributed 

 in nature mainly because the fecal discharges of man and animals are 

 a common thing on the soil. When the colon bacillus is present so as 

 to be isolated from 1 c.c. of water in a series of tests, it is reasonable 

 proof of pollution and the conditions should be investigated. Ten 

 colon bacilli in 1 c.c. indicates serious pollution, and very likely a 

 dangerous one. \Vinslow reports that in only two out of fifty-eight 

 samples of presumably non-polluted waters did he get colon bacilli in 



