96 



through a layer of cotton placed in the neck of the funnel, 

 and it' the filtrate is not fairly clear, heat it to the boil- 

 ing-point and filter again through the same cotton filter. 

 The nutrient agar thus obtained should be (1) transparent 

 or semi-transparent ; (2) slightly alkaline in reaction, and 

 (3) should not coagulate or cloud on heating. 



To 100 c. c. of the filtered agar add 6 to 7 c. c. of pure 

 glycerine, mix thoroughly; lest the reaction again to see if 

 alkaline, and then fill into sterilized test-tubes=^///cer^e 

 agar. 



To 100 c. c. of the filtered agar add 2 per cent, of glu- 

 cose ; warm till dissolved, then fill into sterilized test- 

 tubes to a height of about 1| to 2 \\\U\\QS= glucose ayar. 



Transfer the remainder of the agar to sterilized test- 

 tubes^ ordinary agar. 



Sterilize the bouillon and agar tubes l>y heating in 

 steam sterilizer, 4- hour each day, for three consecutive 

 days. At the end of the third heat place the agar tubes, 

 while still liquid, in a slightly inclined position, so that 

 the agar reaches to within 1 to H inches of the plug, and 

 allow it to solidify. 



Instead of preparing an extract of fresh meat it is 

 sometimes more convenient to employ commercial meat 

 extract, such as Liebig's. In that case 2.5 g. of Liebig's 

 extract with the usual amount of peptone and common 

 salt is added to 100U c. c. of water. To this solution or 

 bouillon the ordinary proportion of agar or gelatin is 

 added, and the nutrient media are otherwise prepared in 

 exactly the same manner as already given. 



When a perfectly transparent agar is desired it is, as a 

 rule, necessary to filter though paper. Thiscan be accom- 

 plished most rapidly by placing a filter-stand with funnel 

 and plaited filter, slightly moistened, in a steam sterilizer. 

 When the funnel is thoroughly heated the boiling agar 

 solution is transferred to the filter. 



