This swells up and fixes the capsule and enables it to take the stain.

3. Blow off the acetic acid by the aid of a pipette.

4. Immerse in aniline gentian violet, five to thirty seconds.

5. Wash in water.

6. Dry and mount.

~4. Ribbert's Method.~--

_Stain._--

Measure out and mix:

Acetic acid, glacial 12.5 c.c.

Alcohol, absolute 50.0 c.c.

Distilled water 100.0 c.c.

Warm to 36 C. (e. g., in the "hot" incubator) and saturate with dahlia. Filter.

METHOD.--

1. Prepare and fix films in the usual manner.

2. Cover the film with the stain and allow it to act for one or two seconds only.

3. Wash thoroughly in water.

4. Dry and mount.

~To Demonstrate Flagella.~

~1. Muir's Modified Pitfield.~--This is the best method and gives the most reliable results, for not only is the percentage of successful preparations higher than with any other, but the bacilli and flagella retain their relative proportions.

(a) ~Mordant.~--

Tannic acid, 10 per cent. aqueous solution 10 c.c.

Corrosive sublimate, saturated aqueous solution 5 c.c.

Alum, saturated aqueous solution 5 c.c.

Carbolic fuchsin (Ziehl) 5 c.c.

Mix thoroughly.

A precipitate forms which must be allowed to settle for a few hours.

Decant off the clear fluid into tubes and centrifugalise thoroughly.

This solution is at its best some four or five days after manufacture; it keeps for about a couple of weeks, but must be re-centrifugalised each time, 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 37C, twenty-four to thirty hours if incubated at 22C.

1. Remove a very small quant.i.ty of the growth by means of the platinum spatula.

2. Emulsify it with a few cubic centimetres of distilled water in a watch-gla.s.s, by gently moving the spatula to and fro in the water. Do not rub up the growth on the side of the watch-gla.s.s. 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 pa.s.sing 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-gla.s.s 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-gla.s.s 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.