(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 37C. 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 quant.i.ty 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 laevulose e. g., B. fluorescens liquefaciens.)

_Medium Required_: Inosite-free bouillon.

_Reagents Required_: Cane sugar, 2 per cent. aqueous solution.

Carbolic acid.

METHOD.--

1. Prepare tube cultivations and incubate.

2. Add 2 per cent. of carbolic acid to the sugar solution.

3. Mix equal quant.i.ties 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.