348 PUBLIC HEALTH BACTERIOLOGY 



three weeks old. For cultivation at room temperature a 

 choice may be made from the following : Distilled-water 

 gelatin, nutrient gelatin, distilled-water agar, nutrient agar, 

 and gelatin agar. At blood heat use agar or gelatin agar. 

 Both agar and gelatin media should be used in any one test. 

 Polluted water gives more colonies on nutrient gelatin than 

 on distilled-water gelatin ; unpolluted water gives more with 

 distilled-water gelatin. 



The size of the plates, the amount of medium to be used in 

 plating, and the amounts of sample to be added to the media 

 are all specified. Plates should be 10 c.m. in diameter ; 10 c.c. 

 of medium should be used ; and for ordinary waters, 0-2 c.c., 

 0-3 c.c., and 0-5 c.c. of sample should be added to gelatin 

 media, and o-i c.c. and i-o c.c. of sample to agar media. The 

 sample should be thoroughly shaken before removing these 

 amounts, and the tubes should be thoroughly mixed by rota- 

 tion before plating. Duplicates should be put up. In an 

 unknown water, additional plates of dilutions (ten and one 

 hundred fold) should be made. The colonies should be counted 

 by the naked eye, and preferably by daylight. A lens may be 

 used for doubtful colonies. The time of counting should be : 



For gelatin plates, at the end of 72 hours (3 days) ; 



For agar plates, after 40 to 48 hours. 



The gelatin plates should be inspected daily, in case counting 

 becomes necessary earlier from liquefaction of the medium. 



Search for B. Coli. MacConkey's broth is recommended 

 to be used, the sample to be added directly to the medium, 

 and not first concentrated by filtration. 



Isolation of B. Coli. If indications of the presence of 

 B. coli are got, then the organism must be isolated, cultivated, 

 and identified. This is advised to be done by making surface 

 cultures on plates of either : 



(a.) Litmus lactose agar (reaction +10) ; 

 or (6.) Bile-salt lactose agar (MacConkey's) ; 

 or (c.) Nutrose lactose agar (Drigalski and Conradi) ; 

 or (d.) Ordinary nutrient gelatin. 



They consider (c) to be the best medium of all. Agar media 

 save time. 



Identification and Tests. Having obtained coli-like colonies 

 on plates made from the preliminary cultivations of the water 

 in MacConkey's broth, subcultures must be made to identify 

 the organism. The following subcultures should at least be 

 made : 



(a.) Surface Agar, on which the abundant growth 

 enables subcultures, etc., to be easily made if 

 required. 



