Examine the cultivations for the various products of bacterial metabolism after forty-eight hours' growth, and ~never omit to examine "control" (uninoculated) tube or flask of medium from the same batch, kept for a similar period under identical conditions~. If the results are negative, test further cultivations at three days, five days, and ten days. ~1. Enzyme Production.~-- (A) _Proteolytic Enzymes._--(Convert proteins into proteose, peptone and further products of hydrolysis; e. g., B. pyocyaneus.) _Media Required_: Blood-serum and milk-serum which have been carefully filtered through a porcelain candle. _Reagents Required_: Ammonium sulphate. Thirty per cent. caustic soda solution. Copper sulphate, 0.5 per cent. aqueous solution. One per cent. acetic acid solution. Millon's reagent. Glyoxylic acid solution. Concentrated sulphuric acid. METHOD.-- 1. Prepare cultivations in bulk (50 c.c.) in a flask and incubate. 2. Make the liquid faintly acid with acetic acid, then boil. (This precipitates the unaltered proteins.) 3. Filter. 4. Take 10 c.c. of the filtrate in a test-tube and add 1 c.c. of the caustic soda, then add the copper sulphate drop by drop. Pink colour which becomes violet with more copper sulphate = proteose and peptone. 5. Saturate the rest of the filtrate with ammonium sulphate. Precipitate = proteose. 6. Filter and divide the filtrate into three parts a, b and c. a. Repeat the copper sulphate test, using excess of caustic soda to displace the ammonia from the ammonium sulphate. Pink colour = peptone. b. Boil with Millon's reagent. Red colour = tyrosine. c. Add glyoxylic acid solution and run in concentrated sulphuric acid. Violet ring at upper level of acid = tryptophane. Both the tyrosine and tryptophane may be either in the free state or in combination as polypeptid or peptone. (B) _Diastase._--(Converts starch into sugar; e. g., B. subtilis.) _Medium Required_: Inosite-free bouillon. _Reagents Required_: Starch. Thymol. Fehling's solution. METHOD.-- 1. Prepare tube cultivation and incubate. 2. Prepare a thin starch paste and add 2 per cent. thymol to it. 3. Mix equal parts of the cultivation to be tested and the starch paste, and place in the incubator at 37°C. for six to eight hours. 4. Filter. Test the filtrate for sugar. Boil some of the Fehling's solution in a test-tube. Add the filtrate drop by drop until, if necessary, a quantity has been added equal in amount to the Fehling's solution employed, keeping the mixture at the boiling-point during the process. Yellow or orange precipitate = sugar. (C) _Invertase._--(Convert saccharose into a mixture of dextrose and lævulose e. g., B. fluorescens liquefaciens.) _Medium Required_: Inosite-free bouillon. _Reagents Required_: Cane sugar, 2 per cent. aqueous solution. Carbolic acid. METHOD.--