10 c.c.
Tannin, 10 per cent. aqueous solution 10 c.c.

NOTE.--The addition of gallic acid, 0.1 to 0.2 gramme, may
improve the solution, but is not necessary.

METHOD.--

1. Prepare and fix the films as above.

2. Pour some of the mordant-stain on the film and warm
gently, high above the flame, for two minutes (or place in
the "hot" incubator for a like period).

3. Wash thoroughly in water.

4. Dry and mount.

~4. Loeffler's Method.~--

(a) _Mordant._--

Tannic acid, 20 per cent. aqueous solution 10 c.c.
Ferrous sulphate, saturated aqueous solution 5 c.c.
Haematoxylin solution 3 c.c.
Carbolic acid, 1 per cent. aqueous solution 4 c.c.

This solution must be freshly prepared.

_Haematoxylin solution_ is prepared by boiling 1 gramme
logwood

with 8 c.c. distilled water, filtering and replacing the loss from
evaporation.

_Alternative Mordant_ (Bunge's Mordant).--

Tannic acid, 20 per cent. aqueous solution 10 c.c.
Ferrous sulphate, saturated aqueous solution 5 c.c.
Fuchsin, saturated alcoholic solution 1 c.c.

(b) _Stain._--

Weigh out
Methylene-blue }
Or methylene-violet } 4 grammes
Or fuchsin }

and dissolve in

Aniline water, freshly saturated and filtered 100 c.c.

METHOD.--

1. Prepare and fix films as above.

2. Pour the mordant on to the film and warm cautiously over the flame
till steam rises; keep the mordant gently steaming for one minute.

3. Wash well in distilled water till no more colour is discharged; if
necessary, wash carefully with absolute alcohol.

4. Filter a few drops of the stain on to the film, warm as before, and
allow the steaming stain to act for one minute.

5. Wash well in distilled water.

6. Dry and mount.

NOTE.--The flagella of some organisms can be demonstrated better by
means of an alkaline stain or an acid stain--a point to be determined
for each. Speaking generally, those bacilli which give rise to an acid
reaction in the culture medium require an alkali; those which form
alkali in cultivation require an acid. According to requirements,
therefore, Loeffler recommends the addition of sodium hydrate, 1 per
cent. aqueous solution, 1 c.c.; or an equal quantity of an exactly
comparable solution of sulphuric acid.

~5. Va