12 SHELLFISH CONTAMINATION FROM SEWAGE-POLLUTED WATKKS. 



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 cc quantities of the 

 sample, provided no unusual pollution is suspected. With water 

 from polluted or questionable sources 1 cc 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 cc 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 is 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 t>e 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 cc of sterile water in a test tube or small Erlenmeyer 

 flask, thus giving a dilution of 1 : 10. Dilutions of 1 : 100, 1 : 1000, etc., 

 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 12 and of Miquel 46 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 lermentation tubes. These cultures liad 

 been incubated for from twenty-four to forty-eight hours when ex 

 amined, and, if gas-producing, each culture was sown in the following 



