CULTURAL TECHNIQUE 15 



Preparation of agar. Infuse 500 grains of meat in 500 cc. 

 of distilk'tl \vater. Strain the infusion. Weigh the vessel, and 

 bring weight of infusion to 500 grams. Add 10 grams of pep- 

 tone. Warm over a water bath until peptone is dissolved. 

 Heat 500 cc. of water in a weighed vessel until it is boiling 

 rapidly. Add 15 grams of thread agar to the boiling water, 

 continue the heating with constant stirring until the agar is 

 completely dissolved, restore loss, and allow the whole to cool 

 to 55 C. The agar may also be dissolved by heating hi the 

 autoclave for ten or fifteen minutes at 120 C. To the 500 

 grams of meat infusion, which should have a temperature of 

 45-50 C., add the agar solution, keeping the temperature 

 below 60 C. Bring weight of mixture to 10QO grams. Titrate 

 and bring reaction to + 1 per cent. Heat thirty minutes over 

 boiling water. Boil two minutes over a free flame, stirring 

 constantly, as the agar is very likely to burn. Eestore weight. 

 Filter as in the case of gelatin. Test reaction, and adjust if 

 necessary. Eecord final reaction. Tube and sterilize. 



The filtering of agar is likely to be difficult unless the 

 medium has been correctly prepared. The points to be noted 

 are : (1) to obtain a perfect solution of the agar by prolonged 

 boiling, or heating in the autoclave; (2) to produce a firm 

 coagulation of the proteid matter by subsequent heating until 

 the mass of the medium appears clear. The removal of the 

 fine turbidity in agar can only be accomplished through its 

 collection by the coagulating proteid, which is readily re- 

 moved by filtration. If an attempt is made to filter before 

 the medium is in proper condition, it will result in loss of 

 time and material. If a heavy granular coagulum does not 

 form upon prolonged heating, allow the medium to stand 

 until the next day, break up the mass with a stirring rod, 

 and melt over boiling water. This will usually result in a 



