6. Sterilize in the autoclav at 15 lbs. (120° C.) for 15 minutes after the pressure reaches 15 lbs.

d. Sugar Broths.

Sugar broths shall be prepared in the same general manner as nutrient broth with the addition of 0.5 per cent. of the required carbohydrate just before sterilization. The removal of muscle sugar is unnecessary as the beef extract and peptone are free from any fermentable carbohydrates. The reaction of sugar broths shall be a faint pink with phenol red or, if on titration with phenolphthalein the reaction is not already between neutral and +1, adjust to neutral. Sterilization shall be in the autoclav at 15 lbs. (120° C.) for 15 minutes after the pressure reaches 15 lbs., provided the total time of exposure to heat is not more than one-half hour; otherwise a 10 per cent. solution of the required carbohydrate shall be made in distilled water and sterilized at 100° C. for 1½ hours, and this solution shall be added to sterile nutrient broth in amounts sufficient to make a 0.5 per cent. solution of the carbohydrate and the mixture shall then be tubed and sterilized at 100° C. for 30 minutes, or it is permissible to add by means of a sterile pipette directly to a tube of sterile neutral broth enough of the carbohydrate to make the required 0.5 per cent. The tubes so made shall be incubated at 37° C. for 24 hours as a test for sterility.

e. Nutrient Gelatin. To Make One Liter.

1. Add 3 grams of beef extract and 5 grams of peptone to 1,000 cc. 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.[[G]]

[G]. The solution of the gelatin will be facilitated by allowing it to soak in the cold one-half hour before heating.

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 cc. to each tube, or in larger containers as desired.