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. 6. Sterilize in the autoclav at 15 lbs. (120° C.) for 15 minutes after the pressure reaches 15 lbs. f. _Nutrient Agar. To Make One Liter._ 1. Add 3 grams of beef extract, 5 grams of peptone and 12 grams of agar, dried for one-half hour at 105° C. before weighing, to 1,000 cc. 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 cc. to each tube, or in larger containers, as desired. 6. Sterilize in the autoclav at 15 lbs. (120° C.) for 15 minutes after the pressure reaches 15 lbs. g. _Litmus or Azolitmin Solution._ The standard litmus solution shall be a 2 per cent. aqueous solution of reagent litmus. Powder the litmus, add to the water and boil for five minutes. The solution usually needs no correction in reaction and may be at once distributed in flasks or test-tubes and sterilized as is culture media. It should give a distinctly blue plate when 1 cc. is added to 10 cc. of neutral culture medium in a Petri dish. The standard azolitmin solution shall be a 1 per cent. solution of Kahlbaum’s azolitmin. Add the azolitmin powder to the water and boil for five minutes. The solution may need to be corrected in reaction by the addition of sodium hydrate solution so that it will be approximately neutral and will give a distinctly blue plate when 1 cc. is added to 10 cc. of neutral culture medium in a Petri dish. It may be distributed in flasks or test-tubes and sterilized as is culture media. h. _Litmus-lactose-agar._ Litmus-lactose-agar shall be prepared in the same manner as nutrient agar with the addition of 1 per cent. of lactose just before sterilization. The reaction 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. One cc. of sterilized litmus or azolitmin solution shall be added to each 10 cc. of the medium just before it is poured into the Petri dish, or the mixture may be made in the dish itself. i. _Endo’s Medium._[209][214][215] _To Make One Liter._