All media and dilution water shall be sterilized in the autoclav at 15 lbs. (120° C.) for 15 minutes after the pressure reaches 15 lbs. All air must be forced out of the autoclav before the pressure is allowed to rise. As soon as possible after sterilization the media shall be removed from the autoclav and cooled rapidly. Rapid and immediate cooling of gelatin is imperative. Media shall be sterilized in small containers, and these must not be closely packed together. No part of the medium shall be more than 2.5 cm. from the outside surface of the glass. All glassware shall be sterilized in the dry oven at 170° C. for at least 1½ hours. c. _Nutrient Broth. To Make One Liter._ 1. Add 3 grams of beef extract and 5 grams of peptone to 1,000 cc. of distilled water. 2. Heat slowly on a 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 filter paper until clear. 5. Distribute in test-tubes, 10 cc. to each tube. 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] Footnote G: The solution of the gelatin will be facilitated by allowing it to soak in the cold one-half hour before heating.