ime, before use.

(b) _Stain._--

Alum, saturated aqueous solution 25 c.c.
Gentian violet, saturated alcoholic solution 5 c.c.

Filter.

This stain must be freshly prepared.

METHOD.--The cultivations employed should be smear agar cultures, twelve
to eighteen hours old if incubated at 37 deg. C, twenty-four to thirty
hours if incubated at 22 deg. C.

1. Remove a very small quantity of the growth by means of the platinum
spatula.

2. Emulsify it with a few cubic centimetres of distilled water in a
watch-glass, by gently moving the spatula to and fro in the water. Do
not rub up the growth on the side of the watch-glass. Some workers
prefer to use tap water, others employ normal saline solution, but
distilled water gives the best emulsion.

3. Spread a thin film of the emulsion on a newly flamed cover-slip,
using no force, but rather _leading_ the drop over the cover-slip with
the platinum loop.

4. Allow the film to dry in the air, properly protected from falling
dust.

5. Fix by passing thrice through the Bunsen flame, holding the
cover-slip whilst doing so by one corner between the finger and thumb.

6. Pour on the film as much of the mordant as the cover-glass will hold.
Grasp the cover-slip with the forceps and hold it, high above the flame,
until steam rises. Allow the steaming mordant to remain in contact with
the film two minutes.

7. Wash well in water and dry carefully.

8. Pour on the film as much of the stain as the cover-glass will hold.
Steam over the flame as before for two minutes.

9. Wash well in water.

10. Dry and mount.

~2. "Pitfield" Original Method.~--

(a) _Mordant._--

Tannic acid 1 gramme
Water 10 c.c.

(b) _Stain._--

Saturated aqueous solution of alum 10 c.c.
Saturated alcoholic solution of gentian violet 1 c.c.
Distilled water 5 c.c.

Mix equal parts of a and b before using.

1. Prepare and fix the film in the manner described above.

2. Boil the mixture and immerse the cover-slip in it, whilst
still hot, for one minute.

3. Wash in water.

4. Examine in water; if satisfactory, dry and mount in
Canada balsam.

~3. MacCrorrie's Method.~--

_Mordant-Stain._--

Measure out and mix.

Night blue, saturated alcoholic solution 10 c.c.
Potash alum, saturated aqueous solution