306 WATER HYGIENE 
3. Make a lactose-litmus-agar of Endo’s medium plate from the 
smallest portion of each sample showing gas. Incubate plate 
at 37° C. 
Note. In case the smallest portion in which gas has been formed 
shows less than 10 per cent (10%) of gas it 1s well to make a 
plate also from the next largest portion so that in case the 
smallest portion gives a negative end result, it may still be 
possible to demonstrate B. col: in the next larger dilution. 
Fourth Day. 
1. Examine Endo’s medium on lactose-litmus-agar plates If typical 
colonics have developed, select two and transfer cach to a 
lactose-broth-fermentation tube and an agai slant, both of 
which are to be incubated at 37° C. 
2. If no typical B. colz colonies are found, incubate the plates another 
twenty-four hours. 
Fofith Day. 
1. Select at least two colonies, whether typical or not, from the 
Endo’s medium or lactose-litmus-agar plates which have been 
incubated an additional twenty-four hours; transfer each to a 
lactose-broth-fermentation tube and an agar slant, and com- 
plete the test as for typical colonies. 
2. Examine-lactose-broth fermentation tubes inoculated from plates 
on the previous day. ‘Tubes in which gas has been formed may 
be discarded after the result has been recorded. ‘Those in 
which no gas has formed should be incubated an additional 
twenty-four hours. 
Sixth. Day. 
1. Examine lactose-broth-fermentation tubes reincubated the previ- 
ous day. 
2. Examine microscopically agar slants corresponding to lactose-fer- 
mentation tubes inoculated from plate colonies and showing 
gas formation. 
Methyl Red Test. As sanitary knowledge increased and it became 
apparent that B. cole or B. coli-like bacteria were present on substances 
which had not received fecal pollution, studies were made to detect 
those B. cols which came from fecal sources. To this end Rogers (1915) 
and his co-workers found that the gas ratio separated B. colt of fecal 
origin from B. cols of non-fecal origin. B. col¢ from bovine feces fer- 
mented dextrose with the formation of equal volumes of carbon dioxide 
