Blood Stains

When watery solutions of medicinal methylene blue and water soluble eosins are mixed a precipitate is formed which is soluble only in alcohol, and solutions of this precipitate impart a peculiar reddish-purple colour to chromatin. This compound was first used by Romanowsky to demonstrate malarial parasites, but various modifications are now employed for staining blood films generally, and also for bacteria and protozoa. The best modifications of the original Romanowsky are those of Jenner and Leishman—Jenner being most suitable for the histological study of the blood, and Leishman for the demonstration of protozoa.

Jenner's Stain.—

A. Weigh out:

Eosin aqueous yellow 6.0 grammes

Dissolve in

Distilled water (non-alkaline) 250 c.c.

This will make a thick solution.

B. Weigh out:

Methylene blue (medicinally pure) Hoechst 5.0 grammes

Dissolve in

Distilled water (non-alkaline) 250 c.c.

1. Add B to A very slowly, stirring all the time. A viscous precipitate forms which frequently loses its viscosity when heat is applied. (This explains the necessity of mixing slowly).

2. Evaporate slowly in a porcelain basin, stirring occasionally, on a water bath at 55° C. When a paste begins to form scrape and break up occasionally. (On no account must the paste be allowed to fuse.)

3. Grind the resulting mass into an amorphous powder.

4. Weigh out:

Amorphous powder 0.5 grammes

Dissolve in

Methylic alcohol (Merck's puriss, for analysis) 100 c.c.

Allow time for true solution. (About three days is sufficient.)

Method.—

1. Prepare film, dry, but do not fix.

2. Flood the unfixed film with the stain, allow it to act for 3 minutes (the methylic alcohol of the stain fixes the film).

3. Pour off the stain and wash in distilled water until the film presents a pink colour.

4. Dry and mount.

Leishman's Stain.—

A. Weigh out:

Methylene blue (medicinal) 1 gramme

Dissolve in

Sodium carbonate, 0.5 per cent. aqueous solution 100 c.c.

Keep at 65° C. for 12 hours in either a hot incubator or a water-bath; then stand in dark place at room temperature (20°C.) for ten days.

B. Weigh out:

Eosin, extra B. A. 0.1 gramme

Dissolve in

Distilled water 100 c.c.

1. Mix the two solutions A and B in equal volumes, and allow the mixture to stand for 12 hours with occasional stirring.

2. Filter, and collect precipitate on filter paper.

3. Wash precipitate thoroughly with distilled water, and dry.

4. Weigh out 0.15 gramme of the dried precipitate; rub up in a mortar with 5 c.c. of methylic alcohol (Merck's puriss, for analysis).

Allow undissolved powder to settle, then decant the supernatant fluid to a clean 100 c.c. measuring cylinder.

5. Add further 5 c.c. alcohol to sediment in mortar and repeat the process, and so on until all the sediment has been dissolved.

6. Now make up the fluid in the measuring cylinder to 100 c.c. by the addition of more methylic alcohol.

Method.—

1. Prepare film, dry, but do not fix.

2. Flood the unfixed film with stain, allow it to act 30 seconds.

3. Add double the volume of distilled water to the stain on the film, and mix with glass rod or platinum loop.

4. Allow this diluted stain to act five minutes.

5. Wash off with distilled water.

6. Leave some water on film for thirty seconds to intensify the colour contrasts.

7. Dry and mount.