Test the cultivation and the control for ammonia in the following manner:

2. To each flask add 2 grammes of calcined magnesia, then connect up with condensers and distil.

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

4. Add 1 c.c. Nessler reagent to each gla.s.s 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 gla.s.s 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 p.r.o.nounced.

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 pota.s.sium 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_:

Pota.s.sium 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 37C., 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. paradimethylamino-benzaldehyde solution.

3. Add 2 c.c. pota.s.sium persulphate solution.

The presence of indol is indicated by the appearance of a delicate rose-pink colour throughout the mixture which deepens slightly on standing.

Indol is tested for in many laboratories by the ordinary nitrosoindol reaction which, however, is not so delicate a method as that above described. The test is carried out as follows:

1. Remove the cotton-wool plug from the tube, and run in 1 c.c. pure concentrated sulphuric acid down the side of the tube by means of a sterile pipette. Place the tube upright in a rack, and allow it to stand, if necessary, for ten minutes.

A rose-pink or red colour at the junction of the two liquids = indol (_plus a nitrite_).

2. If the colour of the medium remains unaltered, add 2 c.c.

of a 0.01 per cent. aqueous solution sodium nitrite, and again allow the culture to stand for ten minutes.

Red colouration = indol.

NOTE.--In place of performing the test in two stages as given above, 2 c.c. concentrated _commercial_ sulphuric, hydrochloric, or nitric acid (all of which hold a trace of nitrite in solution), may be run into the cultivation. The development of a red colour within twenty minutes will indicate the presence of indol.

~5a. Phenol Production.~--

_Medium Required_:

Nutrient bouillon.

_Reagents Required_:

Hydrochloric acid, concentrated.

Millon's reagent.

Ferric chloride, 1 per cent. aqueous solution.

METHOD.--

1. Prepare cultivation in a Bohemian flask containing at least 50 c.c.

of medium, and incubate.

Test for phenol in the following manner:

2. Add 5 c.c., 25 per cent. sulphuric acid to the cultivation and connect up the flask with a condenser.

3. Distil over 15 to 20 c.c. Divide the distillate into three portions a, b and c.

4. Add to (a) 0.5 c.c. Millon's reagent and boil.

Red colour = phenol.