and distil.

3. Collect 50 c.c. distillate, from each, in a Nessler glass.

4. Add 1 c.c. Nessler reagent to each glass by means of a clean pipette.

Yellow colour = ammonia.

The depth of colour is proportionate to the amount present.

~4. Alcohol, etc., Production.~--Divide the distillate "A" obtained in the
course of a previous experiment (_vide_ page 282, step 3) into four
portions and test for the production of alcohol, acetaldehyde, acetone.

1. Add Lugol's iodine, then a little NaOH solution, and stir with a
glass rod till the colour of the iodine disappears.

Pale-yellow crystalline precipitate of iodoform, with its characteristic
smell, appearing in the cold, indicates acetaldehyde, or acetone;
appearing only on warming indicates alcohol.

The precipitate may be absent even when the odour is pronounced.

2. Add Schiff's reagent.

Violet or red colour = aldehyde.

3. To 10 c.c. of solution add 2.5 c.c., 25 per cent. sulphuric acid, and
a crystal or two of potassium bichromate and distil. Reduction of the
bichromate to a green colour and a distillate, which smells of
acetaldehyde and reacts with Schiff's reagent, shows the presence of
alcohol in the original liquid.

4. Add a few drops of sodium nitroprusside solution, make alkaline with
ammonia, then saturate with ammonium sulphate crystals. Acetone gives
little colour on the addition of ammonia, but after the addition of
ammonium sulphate a deep permanganate colour, which takes ten minutes to
reach its full intensity. Aldehyde gives a carmine red unaltered by
ammonium sulphate.

~5. Indol Production.~--

_Media Required_:

Inosite-free bouillon (_vide_ page 183).
Or peptone water (_vide_ page 177).

_Reagents Required_:

Potassium persulphate, saturated aqueous solution.
Paradimethylamino-benzaldehyde solution. This is prepared by mixing:

Paradimethylamino-benzaldehyde 4 grammes
Absolute alcohol 380 c.c.
Hydrochloric acid, concentrated 80 c.c.

METHOD.--

Prepare several test-tube cultivations of the organism to be tested, and
incubate.

Test for indol by means of the Rosindol reaction in the following
manner. (If the culture has been incubated at 37 deg. C., it must be
allowed to cool to the room temperature before applying the test.)

1. Remove 2 c.c. of the cultivation by means of a sterile pipette and
transfer to a clean tube, then,

2. Add 2 c.c. paradime