1. Prepare tube cultivations and incubate. 2. Add 2 per cent. of carbolic acid to the sugar solution. 3. Mix equal quantities of the carbolised sugar solution and the cultivation in a test-tube; allow the mixture to stand for several hours. 4. Filter. Test the filtrate for reducing sugar as in the preceding section. (D) _Rennin and "Lab" Enzymes._--(Coagulate milk independently of the action of acids; e. g., B. prodigiosus.) _Media Required_: Inosite-free bouillon. Litmus milk. METHOD.-- 1. Prepare tube cultivations and incubate. 2. After incubation heat the cultivation to 55° C. for half an hour, to sterilise. 3. By means of a sterile pipette run 5 c.c. of the cultivation into each of three tubes of litmus milk. 4. Place in the cold incubator at 22° C. and examine each day for ten days. Absence of coagulation at the end of that period will indicate absence of rennin ferment formation. Fermentation Reactions. As tested upon carbohydrate substances and organic salts. _Media Required_: Peptone water containing various percentages (generally 2 per cent.) of each of the substances referred to under "sugar" media (page 177), also tubes of peptone water containing 1 per cent. respectively of each of the following: Organic salts: Sodium citrate, formate, lactate, malate, tartrate. METHOD.-- 1. Prepare tube cultivations in each of the above media. 2. Observe from day to day up to the expiration of ten days if necessary. 3. Note growth, reaction, gas production. 2. Acid Production. (a) _Quantitative._-- _Medium Required_: Sugar (glucose) bouillon of known "optimum" reaction. _Apparatus and Reagents Required_: As for estimating reaction of media (_vide_ page 150). METHOD.-- 1. Prepare cultivation in bulk (100 c.c.) in a flask; also "control" flask of medium from same batch. 2. After suitable incubation, heat both flasks in the steamer at 100° C. for thirty minutes to sterilise. 3. Determine the _titre_ of the medium in "inoculated" and "control" flasks as described in the preparation of nutrient media (_vide_ page 151). 4. The difference between the titre of the medium in the two flasks gives the total acid production of the bacterium under observation in terms of normal NaOH. NOTE.--If the growth is very heavy it may be a difficult matter to determine the end-point. The cultivation should then be filtered through a Berkefeld filter candle previous to step 2, and the filtrate employed in the titration. (b) _Qualitative_ (of all the organic acids present).-- _Medium Required_: Sugar (glucose or lactose) bouillon as in quantitative examination.