if it is a very watery
specimen, allow the film to dry, then spread a second and even a third
layer over the first.)

2. Fix by passing three times through the flame.

3. Stain in hot carbol-fuchsin (as in staining for spores) for five to
ten minutes. (This stains everything on the film.) Avoid over-heating.

4. Decolourise by dipping in sulphuric acid, 25 per cent. (This removes
stain from everything but acid-fast bacilli; e. g., tubercle, leprosy,
and smegma bacilli and the film turns yellow.)

5. Wash in water. (A pale red colour returns to the film).

6. Wash in alcohol till no more colour is discharged. (This often, but
not invariably, removes the stain from acid-fast bacilli other than
tubercle; e. g., smegma bacillus.)

7. Wash in water.

8. Counterstain in weak methylene-blue. (Stains non-acid-fast bacilli,
leucocytes, epithelial cells, etc.)

9. Wash in water, dry, and mount.

~Pappenheim's Method.~--

This method is supposed to differentiate between B. tuberculosis and
other acid-fast micro-organisms.

1. Prepare and fix film in the usual way.

2. Stain in carbol-fuchsin _without heat_ for three minutes.

3. Without previously washing in water treat the film with three or four
successive applications of corallin (Rosolic acid) solution.

Corallin 1 gramme
Methylene-blue
(saturated alcoholic solution) 100 c.c.
Glycerine 20 c.c.

4. Wash in water.

5. Dry and mount.

~Neisser's Method--Modified.~--(To demonstrate diphtheroid bacilli.)

_Stain I._--

Measure out and mix

Methylene-blue, saturated alcoholic solution 4.0 c.c.
Acetic acid, 5 per cent. aqueous solution 96.0 c.c.

Filter.

_Stain II._--

Weigh out

Neutral red 2.5 grammes

and dissolve in

Distilled water 1000 c.c.

Filter.

METHOD.--

1. Prepare and fix films in the usual way.

2. Pour stain I on the film and allow it to act for two minutes.

3. Wash thoroughly in water.

4. Treat with Lugol's iodine for ten seconds.

5. Wash thoroughly in water.

6. Pour stain II on to the film and allow it to act for thirty seconds.

7. Wash thoroughly in water.

8. Dry and mount.

NOTE.--The cultivation from which the films are prepared
must be upon blood-serum which has been incubated at 37 deg. C.
for from nine to eighteen hours.

The bacilli are stained a light red