THE NEW ENGLAND STATES. 27 



procedure for dilutions of 1 to 100 and 1 to 1,000, the four dilutions 

 used in making an examination of ordinary water. If the water is 

 badly contaminated higher dilutions may be required. Melted agar 

 kept at about 43 C. is poured into each plate containing a dilution, 

 which plate is gently rotated and shaken so as to thoroughly mix 

 the contents. Allow the plates to cool rapidly before incubation. 



In addition to plating 1 cc, 0.1 cc, 0.01, cc, and 0.001 cc quantities 

 in two agar plates for each dilution (one set being incubated at 25 

 the other at 37 C.), ox-bile fermentation tubes are also inoculated 

 with 10 cc, 5 cc, 1 cc, 0.1 cc, 0.01 cc, and 0.001 cc, quantities, 

 for ordinary water. Should it appear, however, that the sample 

 was from a polluted source, the quantities used for this purpose 

 would range from 1 cc, to 0.0001 cc, or higher dilutions as found to 

 be necessary. 



INCUBATION. 



The standard methods recognize two incubation temperatures, one 

 for gelatin at 20 C. (corresponding to room temperature), the other 

 for agar at 37 C. (blood heat). This laboratory incubates all plate 

 cultures for from three to four days, when the maximum number of 

 colonies appears before making final counts. 



TESTS FOR B. COLI. 



From the ox-bile fermentation tubes showing the presence of gas- 

 producing organisms in the two highest dilutions, plate cultures are 

 made on the MacConkey's bile salt agar for the isolation of pure cul- 

 tures. Generally five plates are prepared from each fermentation 

 tube by using a single platinum loop-full of the mixed culture, then, 

 without flaming the needle during the interval, five dilutions are 

 made by passing the needle from one tube to another of the melted 

 agar held at about 43 C. The tubes are agitated and the contents 

 poured into five sterile plates. These plate cultures should be made 

 within 24 to 36 hours after the appearance of gas, since the organ- 

 isms are then most active and more easily developed. Difficulty 

 may be experienced in recovering B. coli in old sugar cultures unless 

 they are rejuvenated. Often pure cultures of B. coli develop from 

 the ox-bile fermentation tubes. The presence of gas in the ox-bile 

 tubes is considered as only a presumptive test for B. coli. 



DIAGNOSTIC CHARACTERS. 



The tests for B. coli are considered positive only when the organ- 

 ism possesses the following characteristics: 



(1) Morphology typical. Non-spore-bearing bacillus, relatively small and thick, 

 with rounded ends. The size, arrangement, and form may vary slightly according to 

 the age of the culture and the medium employed. Atypical forms under abnormal 

 conditions may develop typical types under favorable circumstances. 



