PREPARATION OF MEDIA 91 



closed water bath and boil for 20 minutes, then cool it to a 

 temperature between 45 and 50 C., add the white of three 

 eggs and thoroughly mix in the liquid agar. This is easily 

 accomplished by pouring it a number of times from one 

 beaker to another. It is well to add a small quantity of 

 Coignet Fils wine clarifier. When the egg albumen is dis- 

 solved, the agar is returned to the water bath and boiled 

 vigorously until the white of the egg is firmly coagulated. 

 This usually takes about 30 minutes. The rapidity and com- 

 pleteness with which the coagulation takes place depends 

 somewhat upon the reaction. If too alkaline, a firm coagu- 

 lation is not formed. Filter the agar immediately, while 

 hot, through ordinary filter paper which has been moistened 

 with boiling water. A layer of moistened absorbent cotton 

 can be used in place of the paper. A hot- water funnel is. 

 recommended by some but we have not found it necessary. 

 Distribute the filtrate in small and large tubes or flasks, as- 

 desired. Sterilize, label and store the agar in the same man- 

 ner as the bouillon. 



Preparation of glycerine agar. Take the desired quan- 

 tity of the agar previously prepared and add 5% of pure 

 glycerin. Thoroughly mix it with the agar, after which dis- 

 tribute in tubes. Sterilize, label and store as ordinary agar. 



Preparation of acid agar. This is prepared in the same 

 manner as ordinary agar, by using acid bouillon. 1 



The preparation of nutrient gelatin. Take a flask of 

 bouillon containing 1000 cc. and pour it into a small agate 

 iron dish, add 100 grams of sheet gelatin and heat, with fre- 

 quent stirring, in a water bath, until the gelatin is dissolved. 

 Allow it to cool to a temperature between 45 and 50 C. and 

 then add the white of three eggs and mix it thoroughly by- 

 stirring, or, better, by pouring the gelatin many times from 

 one flask or beaker to another. After the egg albumen is 

 completely diffused, return the liquid gelatin to the large 

 covered water bath and boil until the egg albumen is firmly 



*Acid bouillon is bouillon that has not been neutralized by NaOH 

 solution. For accuracy it should be titrated to the acidity required. 



