109 



at 105 C. before weighing, to 1,000 c. c. of distilled water. Boil over a 

 water bath until all agar is dissolved, and then make up the loss by 

 evaporation. 



2. Cool to 45 C. in a cold water bath, then warm to 65 C. in the 

 same bath, without stirring. 



3. Make up lost weight and adjust the reaction to a faint pink with 

 phenol red, or if the phenolphthalein titration is used, and the reaction 

 is not already between +0.5 and +1, adjust to +1. 



4. Filter through cloth and cotton until clear. 



5. Distribute in test-tubes, 10 c. c. to each tube, or in larger contain- 

 ers, as desired. 



6. Sterilize in the autoclave at 15 pounds (120 C.) for 15 minutes 

 after the pressure reaches 15 pounds. 



B. Nutrient Gelatin (A. P. H. A., 1920). 1. Add 3 grams of 

 beef extract and 5 grams of peptone to 1,000 c. c. of distilled water and add 

 100 grams of gelatin dried for one-half hour at 105 C. before weighing. 



2. Heat slowly on a steam bath to 65 C until all gelatin is dissolved.* 



3. Make up lost weight and adjust the reaction to a faint pink with 

 phenol red, or if the phenolphthalein titration is used, and the reaction 

 is not already between +0.5 and +1, adjust to +1. 



4. Filter through cloth and cotton until clear. 



5. Distribute in test-tubes, 10 c. c. to each tube, or in large con- 

 tainers as desired. 



6. Sterilize in the autoclave at 15 pounds (120 C.) for 15 minutes 

 after the pressure reaches 15 pounds. 



C. Nutrient Broth. (A. P. H. A., 1920). 1. Add 3 grams of 

 beef extract and 5 grams of peptone to 1,000 c. c. of distilled water. 



2. Heat slowly on steam bath to at least 65 C. 



3. Make up lost weight and adjust the reaction to a faint pink with 

 phenol red, or if the phenolphthalein titration is used, and the reaction is 

 not already between +0.5 and +1, adjust to +1. 



4. Cool to 25 C. and filter through paper until clear. 



5. Distribute in test-tubes, 10 c. c. to each tube. 



6. Sterilize in the autoclave at 15 pounds (120 C.) for 15 minutes 

 after the pressure reaches 15 pounds. 



D. Media for Indol Test. (A. P. H. A., 1920). To 1,000 c. c. 

 of distilled water add 0.3 gram tryptophane, 5 grams dipotassium hydro- 

 gen phosphate (K 2 HP0 4 ), and 1 gram peptone. Heat until ingredients 

 are thoroughly dissolved, tube (6 to 8 c. c.), and sterilize in autoclave for 

 15 minutes after the pressure reaches 15 pounds. Some American peptones 

 are standardized to contain a uniform amount of tryptophane. If such 

 peptone is used the tryptophane in the above formula may be omitted and 

 the peptone increased to 5 grams. 



E. Litmus Milk. (After Prescott and Winslow). The milk to be 

 used as a culture medium shall be as fresh as possible, "Certified Milk" 

 being ordinarily the best obtainable in city laboratories. It shall be placed 



*The solution of the gelatin will be facilitated by allowing it . to soak in the cold 

 one-half hour before heating. 



