16 GENERAL BACTERIOLOGY 



EXERCISE 8. PREPARATION OF AGAR (Ravenel). 



General Directions. 



Add 15 grams of agar-agar threads (finely chopped) to 500 cc. 

 of water and either (1) dissolve in autoclave by heating up to 120° 

 C, closing off gas and allowing to cool, or (2) boil until the agar-agar 

 is dissolved (about ^2 hour) and make up loss of water by evapora- 

 tion. While the agar is being dissolved proceed as follows : 



a. Same as in the preparation of bouillon (4 a). 



i. Add 500 cc. of distilled water. 



c. Same as bouillon (4 c). 



d. Add 10 gms. of peptone and 5 gms. of salt. 



e. Heat until peptone is dissolved. 



f. Neutralize. 



g. Cool to 60° C, add agar solution and mix (in case extract is 

 used it will be necessary to add an egg at this point) . 



h. Boil until albumin is coagulated and floats in the clear liquid 

 and restore weight. 



i. Test reaction. 



j. Add 0.5% normal hydrochloric acid. 



k. Filter as in case of gelatin. (7^.) 



I. Tube. 



m. Sterilize in steam for 15 minutes on three successive days or 

 in autoclave for 20 minutes at 120° C. 

 After the last sterilization place most of 

 the tubes in a sloping position to harden 

 (Fig. 6) , these are known as agar slopes. 

 Those solidified in an upright„EPsition, 

 frequently called "deep stick agar," Fig. 6. Method of sloping agar, 

 are used to make plate cultures. "^ 



References. A. 104-;. H. 47 ; M. & R. 38 ; McF. 185 ; P. 215 ; 

 P. B. C. 27 ; Journal of Applied Microscopy, 1898, 1 ; 106. 



Special Directions. Use meat extract, make 1 liter, fill 25 

 tubes and after last sterilization incline 20 of them. Place the 

 remainder in flasks and sterilize. 



