f peptone water containing 1 per cent. respectively of each of
the following:

Organic salts: Sodium citrate, formate, lactate, malate,
tartrate.

METHOD.--

1. Prepare tube cultivations in each of the above media.

2. Observe from day to day up to the expiration of ten days if
necessary.

3. Note growth, reaction, gas production.

2. Acid Production.

(a) _Quantitative._--

_Medium Required_:
Sugar (glucose) bouillon of known "optimum" reaction.

_Apparatus and Reagents Required_:
As for estimating reaction of media (_vide_ page 150).

METHOD.--

1. Prepare cultivation in bulk (100 c.c.) in a flask; also "control"
flask of medium from same batch.

2. After suitable incubation, heat both flasks in the steamer at 100
deg. C. for thirty minutes to sterilise.

3. Determine the _titre_ of the medium in "inoculated" and "control"
flasks as described in the preparation of nutrient media (_vide_ page
151).

4. The difference between the titre of the medium in the two flasks
gives the total acid production of the bacterium under observation in
terms of normal NaOH.

NOTE.--If the growth is very heavy it may be a difficult
matter to determine the end-point. The cultivation should
then be filtered through a Berkefeld filter candle previous
to step 2, and the filtrate employed in the titration.

(b) _Qualitative_ (of all the organic acids present).--

_Medium Required_:
Sugar (glucose or lactose) bouillon as in quantitative examination.

_Reagents Required_:
Hydrochloric acid, concentrated.
Hydrochloric acid, 25 per cent.
Sulphuric acid, concentrated (pure).
Phosphoric acid, concentrated solution.
Ammonia.
Ammonium sulphate.
Baryta water.
Sodium carbonate, saturated aqueous solution.
Absolute alcohol.
Ether.
Calcium chloride.
Calcium chloride solution.
Zinc carbonate.
Copper sulphate saturated aqueous solution.
Alcoholic thiophene solution (0.15 c.c. in 100 c.c.).
Animal charcoal.
Five per cent. sodium nitroprusside solution.
Potassium bichromate.
Schiff's reagent.
Arsenious oxide.
Ferric chloride, 4 per cent. aqueous solution.
Silver nitrate, 1 per cent. aqueous solution.
Lugol's iodine.
Ten per cent. caustic soda solution.
Hard paraffin wax (melting-point about 52 deg. C.).

METHOD.--

1. Prepare cultivation in bulk (500