TREASURY DEPARTMENT STANDARD oll 
fished and transplanted into a lactose-broth fermentation tube, which shall be 
incubated at 37° C. for forty-eight hours. 
For the purposes 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 Baczllus 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 (0). 
The formation of gas, occupying 10 per cent or more of closed arm of fer- 
mentation tube, in lactose peptone broth fermentation tube, inoculated with 
colony fished from twenty-four-hour lactose litmus agar or Endo’s medium plate. 
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 | c.c. portion, and one 0.1 ¢.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 Asso- 
ciation, as set forth in Standard Methods of Water Analysis (A. P. H. A., 1912). 
This standard has been applied to the examination of water from 
trains by Bartow (1916), Creel (1914), Hanford (1916), and to water 
from boats by Cobb, Williams and Letton (1916). Bartow confirmed 
the presence of B. colz in 83 per cent of the samples examined. Creel 
found that an anaerobic bacillus which formed gas was responsible for 
gas in the presumptive test in 91 out of 421 samples. Such data indicate 
the significance of the confirmatory test. More data is required before 
it may be determined whether this method is the best. Letton (1917) 
has later stated that the requirement of ‘‘ not more than 100 colonies 
per cubic centimeter on agar” of the Treasury Department standard 
is very lenient. He regards the limit of permissible B. cols (not more 
than two per 100 c.c.) as not too low. 
Lactose Bile in Water Analysis. Lactose bile was advocated for 
some time for use in the presumptive test for B. co. It was supposed 
to inhibit other bacteria and a few of the weaker types of B. colz. For 
that reason it was deemed superior for the presumptive test than the 
ordinary carbohydrate broths. Jordan (1915) found that the typical 
as well as the typical B. coli were inhibited. The work of Cummings 
(1917) for the United States Public Health Service is interesting in this 
connection. He found, in his stream pollution studies on the Potomac 
