ong as acid comes over
(distilled water being occasionally added to the distilling flask) the
distillate can be measured and 50 c.c. used for titration. This will
give the amount of volatile acid formation.

9. The second part of the filtrate "B" (see page 282) should be examined
for lactic, oxalic, succinic, benzoic, salicylic, gallic and tannic
acids, as follows:

~Ether Soluble Acids.~--

1. Evaporate to a thin syrup, acidify strongly with phosphoric acid.

2. Extract with five times its volume of ether by agitation in a
separatory funnel.

3. Evaporate the ethereal extract to a thin syrup.

4. Add 100 c.c. water and mix thoroughly.

5. To a small portion of this solution add slight excess of sodium
carbonate, evaporate to dryness on the water-bath, dissolve in 5-10 c.c.
pure sulphuric acid, add 2 drops saturated copper sulphate solution,
place in a test-tube and heat in a boiling water-bath for 2 minutes,
cool, add 2 or 3 drops of the alcoholic thiophene and warm gently.

Cherry red colour = lactic acid.

If a brown colour is produced on the addition of sulphuric acid, another
sample should be taken and boiled with animal charcoal before
evaporating.

6. If lactic acid is definitely present, prepare zinc lactate by boiling
part of the solution of the ether extract with excess of zinc carbonate,
filtering and evaporating to crystallise. The crystals so obtained have
a characteristic form, and if dried at 110 deg. C, should contain 26.87
per cent. of zinc.

7. Test a portion of the rest of the solution of the ether extract for
oxalic acid (page 282, step 5). Carefully neutralise the remainder and
add ferric chloride solution.

Red brown gelatinous precipitate = succinic acid.

Buff precipitate = benzoic acid, and other acids related to benzoic
acid.

Violet colour = salicylic acid.

Inky black colour or precipitate = gallic acid or tannic acid.

For further identification the melting-points of the crystalline acids,
and the percentage of silver in their silver salts should be determined.

~3. Ammonia Production.~--

_Medium Required_:
Nutrient bouillon.

_Reagent Required_:
Nessler reagent.

METHOD.--

1. Prepare cultivation in bulk (100 c.c.) in a 250 c.c. flask and
incubate together with a control flask.

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