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 quant.i.ty of alcoholic solution of gentian violet which suffices to saturate the aniline water and form a "bronze sc.u.m" upon its surface--if too much of the alcoholic gentian violet is added the alcohol present redissolves this sc.u.m.

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

Iodine 1 gramme Iodide of pota.s.sium 3 grammes In distilled water 300 c.c.

5. Wash in water.

6. Wash with alcohol until no more colour is discharged and the alcohol runs away clear and colourless.

The following mixture may be subst.i.tuted for absolute alcohol as a decolouriser

Acetone 10 c.c.

Absolute alcohol 100 c.c.

7. Wash in water.

8. Counterstain very lightly with aqueous solution of Neutral Red. Other counterstains may be used such as dilute eosin, dilute fuchsin, or vesuvin.

NOTE.--This section may be omitted when dealing with films prepared from pure cultivations.

9. Wash in water.

10. Dry and mount.

~Gram-Claudius Method.~--

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

2. Stain in methyl violet, 1 per cent. aqueous solution for three to five minutes.

3. Treat with two lots picric acid, saturated aqueous solution.

4. Wash in water and dry.

5. Decolourise with clove oil.

6. Wash off clove oil with xylol.

7. Mount in xylol balsam.

~Gram-Weigert Method.~--

1-5. Proceed as for the corresponding sections of Gram's method (_quod vide_).

6. Dry in the air.

7. Wash in aniline oil, 1 part, xylol, 2 parts, until no more colour is discharged.

8. Wash in xylol.

9. Mount in xylol balsam.