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APPENDIX A. ROUTINE METHODS OF WATER ANALYSIS 

 AND THE COLON INDEX. 



Although the American Public Health Association has been issuing 

 standard methods of water analysis for some fifteen years, there is still 

 a marked lack of uniformity in the methods employed in different labora- 

 tories. Thus Norton (1918), in a tabulation of 23 laboratories, found 

 that 13 employed lactose broth, 8 lactose bile, 1 lactose and dextrose broth, 

 and 1 lactose agar and dextrose broth for primary inoculation or prelim- 

 inary enrichment. The most commonly employed routine methods are 

 given here. 



I. The Treasury Department Standard for the Examination 

 of Water on Interstate Common Carries. The following method for 

 the examination of water on Interstate common carriers has been formu- 

 lated by a committee of prominent sanitarians. The permissible limits 

 of bacteriological impurity are stated as follows: 



1. The total number of bacteria developing on standard agar plates, 

 incubated 24 hours at 37 C., shall not exceed 100 per cubic centimeter; 

 provided, that the estimate shall be made from not less than two plates, 

 showing such numbers and distribution of colonies as to indicate that the 

 estimate is reliable and accurate. 



2. Not more than one out of five 10 c. c. portions of any sample ex- 

 amined shall show the presence of organisms of the Bacillus coli group when 

 tested as follows: 



(a) Five 10 c. c. portions of each sample tested shall be planted, 

 each in a fermentation tube containing not less than 30 c. c. of lactose 

 peptone broth. These shall be incubated 48 hours at 37 C. and observed 

 to note gas formation. 



(b) From each tube showing gas, occupying more than five per- 

 cent of the closed arm of the fermentation tube, plates shall be made 

 after 48 hours' incubation, upon lactose litmus agar or Endo's medium. 



(c) When plate colonies resembling B. coli develop upon either of 

 these plate media within 24 hours, a well-isolated characteristic colony 

 shall be fished and transplanted into a lactose-broth fermentation tube, 

 which shall be incubated at 37 C. for 48 hours. 



For the purpose of enforcing any regulations which may be based 

 upon these recommendations the following may be considered sufficient 

 evidence of the presence of organisms of the Bacillus coli group. 



Formation of gas in fermentation tube containing original sample of 

 water (a). 



Development of acid-forming colonies on lactose-litmus-agar plates 

 or bright red colonies on Endo's medium plates, when plates are prepared 

 as directed above under (b). 



The formation of gas, occupying 10 percent or more of closed arm of 

 fermentation tube, in lactose peptone broth fermentation tube inoculated 

 with colony fished from 24 hour lactose litmus agar or Endo's medium 

 plate. 



