to act for ten minutes.

5. Wash in water.

6. Decolourise in sulphuric acid, 5 per cent. aqueous
solution, for five seconds.

7. Wash in water.

8. Counterstain with Kuehne's carbolic methylene-blue for
one or two minutes.

9. Wash in water.

10. Dry and mount.

(Spores red, bacilli blue.)

~4. Abbott's Method.~--

1. Prepare and fix films in the usual manner.

2. Pour Loeffler's alkaline methylene-blue on the film; warm
cautiously over the flame till steam rises and allow the hot
steam to act for one to five minutes.

3. Wash thoroughly in water.

4. Decolourise in nitric acid, 2 per cent. alcoholic
(alcohol 80 per cent.) solution.

5. Wash thoroughly in water.

6. Counterstain in eosin, 1 per cent. aqueous solution.

7. Wash.

8. Dry and mount.

(Spores blue, bacilli red.)

DIFFERENTIAL METHODS OF STAINING.

~Gram's Method.~--This method depends upon the fact that the protoplasm of
some bacteria permits aniline gentian violet and Lugol's iodine
solution, when applied consecutively, to enter into a chemical
combination which results in the formation of a new blue-black pigment,
only very sparingly soluble in absolute alcohol. Such organisms are said
to "stain by Gram," or to be "Gram positive."

1. Prepare a cover-slip film and fix in the usual way.

2. Stain in aniline gentian violet three to five minutes. Filter as much
aniline water on to the cover-slip as it will hold; then add the
smallest quantity of alcoholic solution of gentian violet which suffices
to saturate the aniline water and form a "bronze scum" upon its
surface--if too much of the alcoholic gentian violet is added the
alcohol present redissolves this scum.

To prepare aniline water, pour 4 or 5 c.c. aniline oil into
a stoppered bottle and add distilled water, 100 c.c. Shake
vigourously and filter immediately before use. The excess of
oil sinks to the bottom of the bottle and may be used again.

3. Wash in water.

4. Treat with Lugol's iodine solution until the film is black or dark
brown.

To do this treat with iodine solution for a few seconds, wash in water,
and examine the film over a piece of white filter paper. Note the
colour. Repeat this process until the film ceases to darken with the
fresh application of iodine solution.

Lugol's solution is prepared by dissolving

Iodi