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

Iodine 1 gramme

Iodide of potassium 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 substituted 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.

Modified Gram-Weigert Method.—(To demonstrate trichophyta in hair.)

1. Soak the hairs in ether for ten minutes to remove the fat.

2. Stain thirty minutes in a tar-like solution of aniline gentian violet (prepared by adding 15 drops of the alcoholic solution of gentian violet to 3 drops of aniline water).

3. Dry the hairs between pieces of blotting paper.

4. Treat with perfectly fresh iodine solution.

5. Again dry between blotting paper.

6. Treat with aniline oil to remove excess of stain. (If necessary, add a drop or two of nitric acid to the oil.)

7. Again treat with aniline oil.

8. Treat with aniline oil and xylol, equal parts.

9. Clear with xylol.

10. Mount in xylol balsam.

To obtain the best differentiation the preparation should be repeatedly examined microscopically (with a 1/6-inch objective) between steps 5 and 9, as the actual time involved varies with different specimens.

Ziehl-Neelsen's Method.—(To demonstrate tubercle and other acid-fast bacilli.)

1. Smear a thin, even film of the specimen on the cover-slip by means of the platinum loop. (In the case of sputum, 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°C. for from nine to eighteen hours.

The bacilli are stained a light red by the neutral red, which contrasts well with the two or three black spots, situated at the poles and occasionally one in the centre representing protoplasmic aggregations (? metachromatic granules) stained by the acid methylene-blue.

Wheal and Chown (Oxford) Method.—(To demonstrate actinomyces.)

1. Stain briefly with Ehrlich's hæmatoxylin (until nuclei are faint blue after washing with tap water).

2. Wash in tap water.

3. Stain in hot carbol-fuchsin (as for tubercle bacilli) for five to ten minutes.

4. Wash in tap water.

5. Decolourise with Spengler's picric acid alcohol. This is prepared by mixing:

Alcohol, absolute 20 c.c.

Picric acid, saturated aqueous solution 10 c.c.

Distilled water 10 c.c.

During the progress of steps 1-5 the preparation must be repeatedly examined microscopically with the 1/6-inch objective.

When properly differentiated the clubs appear brilliant red on greenish ground.

6. Dehydrate in alcohol.

7. Clear in xylol.

8. Mount in xylol balsam.

This method serves equally well for films and for sections.