NUTRIENT A OAR- A GAR. 89 



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 nutrient 

 agar-agar by the beginner is far too frequently a tedious 

 and time-taking experience. This is due mainly to 

 lack of patience and to deviation from the rules laid 

 down for the preparation of this medium. If the 

 directions given below for the preparation of nutrient 

 agar-agar be strictly observed, no difficulty whatever 

 should be encountered. Many methods are recom- 

 mended for its preparation; almost every worker has 

 some slight modification of his own. 



The methods that have given us the best results, and 

 from which we have no good grounds for departing, 

 are as follows: 



Prepare the bouillon in the usual way. Agar-agar 

 reacts neutral or very slightly alkaline, so that the 

 bouillon may be neutralized before the agar-agar is 

 added. Then add finely chopped or powdered agar- 

 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 side of the vessel at which the level of 

 the fluid stands; if a litre of medium is being made, 

 add about 250 c.c. to 300 c.c. more of water and 

 allow the mass to boil slowly, occasionally stirring, 

 over a free flame, for from one and one-haK to two 

 hours ; or, in other words, until the excess of water 

 — i. e., the 250 or 300 c. c. that were added — has 

 evaporated. Care must be taken 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 volume of 1 litre is 



