12 SHELLFISH CONTAMINATION FROM SEWAGE-POLLUTED WATERS. 
Liquid cultures—In order to determine the presence of gas-pro- 
ducing organisms, bile containing 1 per cent of peptone and 2 per 
cent of lactose is used, or 2 per cent dextrose fermentation tubes 
are inoculated with 10, 5, 1, 0.1, 0.01, and 0.001 ce quantities of the 
sample, provided no unusual pollution is suspected. With water 
from polluted or questionable sources 1 ce quantities are first used 
and then higher dilutions than are ordinarily employed. Fermen- 
tation tubes of the old style are being replaced by small inverted 
tubes, with one end closed. These are placed within a large test 
tube containing a fermentable medium. Tubes of this form require 
less space, and as a whole are more convenient for routine work. 
When desired, a large number of such tubes containing the ox-bile 
medium can be carried from place to place in making presumptive 
tests for colon organisms. By using 1 cc pipettes, graduated in 
tenths, the above-mentioned ox-bile medium may be inoculated with 
1 cc and 0.1 ce quantities of water or oyster liquor. These tubes 
can be incubated over a radiator and the general character of the 
material determined with a fair degree of accuracy by noting the 
presence of fermenting organisms in this medium. Such a test is of 
course only tentative, but 1s of service in fieldwork. 
OYSTERS. 
The liquor removed from shell oysters is cultured in the same 
manner as samples of water, except that 10 and 5 cc quantities are 
not used. With market shucked oysters, in which the bacterial 
count is likely to be much higher than in the shell stock, higher dilu- 
tions are generally necessary. 
Dilutions of water and oyster liquor are made by adding 1 cc of 
the sample to 9 ce of sterile water in a test tube or small Erlenmeyer 
flask, thus giving a dilution of 1:10. Dilutions of 1:100, 1:1000, etce., 
can be made by taking 1 cc of each lower dilution and adding to other 
flasks containing 9 cc of sterile water. Sterile normal salt solution 
is preferred by some workers. After making the dilutions each flask 
should be thoroughly agitated (twenty-five times) in order to break 
up masses of bacteria. With semisolid substances sterile glass shot 
may be added to the liquid for this purpose. 
IDENTIFICATION OF ORGANISMS. .- 
PURE CULTURES. 
The classifications of Chester ” and of Miquel *® were followed in 
identifying species herein described. Well-isolated colonies on bile 
salt agar were selected from plates containing colon-like organisms 
and subcultures made in termentation tubes. These cultures had 
been incubated for from twenty-four to forty-eight hours when ex- 
amined, and, if gas-producing, each culture was sown in the following 
alin 
