NUTRIENT AGAR-AGAB. ' 65 



retained in the flask into which it has been filtered or 

 decanted off into sterilized test-tubes, it should be steril- 

 ized in the steam sterilizer on three successive days, for 

 fifteen minutes each day — the mouth of the flask or the 

 test-tubes containing it having been previously closed 

 with cotton plugs. 



Nutrient Agar-agar. — The preparation of the 

 nutrient agar-agar by the beginner is, as a rule, a some- 

 what tedious and time-taking experience. This is owing 

 mainly to lack of patience and failure to adhere strictly 

 to the rules laid down for the preparation of this medium. 

 Many methods are recommended for its preparation ; 

 almost every worker has some slight modification of his 

 own. 



The method which has given the best results in our 

 hands, and from which there are no grounds for devi- 

 ating, is as follows : 



Prepare the bouillon in the usual way. Agar-agar 

 reacts neutral, so that the bouillon may be neutralized 

 before the agar is added. Then add finely-chopped 

 agar in the proportion of 1 to 1.5 per cent. Place 

 the mixture in a porcelain-lined iron vessel and make a 

 mark on the sides of the vessel at which the level of the 

 fluid stands, add about 250 c.c. of water and allow the 

 mass to boil slowly, occasionally stjrring, over a free 

 flame for three or four hours. Care must be given that 

 it does not boil over the sides of the vessel. From time 

 to time observe if the fluid has fallen below the mark of 

 its original level ; if it has, add water until its original 

 volume is restored. At the end of the time given re- 

 move the flame and place the vessel containing the mix- 

 ture in a large dish of cold water ; stir the agar con- 

 tinously until it has cooled down to about 68°-70° C, 



