100 



These steps are selected with reference to demonstrating the presence 

 in the samples examined of aerobic lactose-fermenting organisms. 



3. It is recommended, as a routine procedure, that in addition to 

 five 10 c. c. portions, one 1 c. c. portion, and one 0.1 c. c. portion of 

 each sample examined be planted in a lactose peptone broth fermentation 

 tube, in order to demonstrate more fully the extent of pollution in grossly 

 polluted samples. 



4. It is recommended that in the above-designated tests the culture 

 media and methods used shall be in accordance with the specifications 

 of the committee on Standard Methods of Water Analysis of the American 

 Public Health Association, as set forth in "Standard Methods of Water 

 Analysis" (A. P. H. A., 1912). 



II. English Procedure (After Savage). Add 0.1 and 1.0 c. c. 

 of water respectively to tubes of lactose bile salt broth in double tubes. 

 Add 10 c. c. to a similar tube, but containing lactose bile salt broth of 

 double strength. To the remainder in the bottle, after all the different 

 amounts of water have been withdrawn for the different parts of the 

 examination, add the contents (about 10 c. c.) of a tube of four times 

 strength neutral red broth. Replace the glass stopper. Four times 

 strength bile salt broth may be used, and, if the examination is for B. coli 

 alone, is preferable, but by using neutral red broth the mixture is also avail- 

 able for the examination for streptococci. 



If a 2-ounce sample is collected, the amount remaining in the bottle 

 will be about 30 c. c. If a large sample of water is collected, then 50 c. c. 

 should be added by sterile pipette to a tube of four times strength neutral 

 red broth large enough to hold the added water. 



The tubes are labeled, incubated at 37 C., and examined after 24 

 and after 48 hours. 



If the 0.1, 1.0, and 10 c. c. tubes show no gas after 48 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 color 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 in- 

 oculating 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. 



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 some other medium se- 

 lected. 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 recommended 

 for routine work, i. e.: 



(a) Gelatine slope (for morphology, motility, cultural appearance, 

 and liquefaction). 



