The Elements of Bacteriological Technique Part 24

~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 pa.s.sing 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 s.m.e.g.m.a 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., s.m.e.g.m.a 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.