al volatile substances--ethyl alcohol, aldehyde, acetone.

Aromatic products--indol, phenol.

Soluble pigments.

Test the power of reducing (a) colouring matters, (b) nitrates to
nitrites.

Investigate the gas production--H_{2}S, CO_{2}, H_{2}. Estimate the
ratio between the last two gases.

Prepare all cultivations for these methods of examination under
_optimum_ conditions, previously determined for each of the organisms it
is intended to investigate, as to

(a) Reaction of medium;
(b) Incubation temperature;
(c) Atmospheric environment;

and keep careful records of these points, and also of the age of the
cultivation used in the final examination.

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