Special Staining Methods for Sections

Double-staining Carmine and Gram-Weigert.—

1. Prepare the section for staining as above, sections 1 to 3.

2. Stain in lithium carmine (Orth's) or picrocarmine for ten to thirty minutes, in a porcelain staining pot (Fig. 76).

3. Wash in picric acid solution until yellow. At this stage cell nuclei are red, protoplasm is yellow, and bacteria are colourless.

Picric acid solution is prepared by mixing

Picric acid, saturated aqueous solution 40 c.c.

Hydrochloric acid 1 c.c.

Alcohol (90 per cent.) 160 c.c.

4. Wash in water.

5. Wash in alcohol.

6. Stain in aniline gentian violet.

7. Wash in iodine solution till dark brown or black.

8. Wash in water.

9. Dip in absolute alcohol for a second.

10. Decolourise with aniline oil till no more colour is discharged.

11. Wash with aniline oil, 2 parts, xylol, 1 part.

12. Clear with xylol.

13. Mount in xylol balsam.

Alternative Gram-Weigert Method for Sections.—

1. Fix paraffin section on slide and prepare for staining in the usual manner.

2. Stain in alum carmine for about fifteen minutes.

3. Wash thoroughly in water.

4. Filter aniline gentian violet solution on to the section on the slide and allow to stain about twenty-five minutes.

5. Wash thoroughly in water.

6. Treat with Lugol's iodine until section ceases to become any blacker.

7. Wash thoroughly in water.

8. Treat with a mixture of equal parts of aniline oil and xylol until no more colour comes away.

9. Wash thoroughly with xylol.

10. Decolourise and dehydrate rapidly with absolute alcohol until there remains only a very faint bluish tint.

11. Clear with xylol.

12. Mount in xylol balsam.

(Then fibrin and hyaline tissue are stained deep blue, whilst bacteria which "stain Gram" appear of a deep blue-violet colour.)

Unna-Pappenheim Method.—

Stain.—

Weigh out and mix

Methylene green 0.15 gramme

Pyronin 0.25 gramme

and dissolve in

Carbolic acid 0.5 per cent. aqueous solution 78 c.c.

Measure out

Alcohol 2.5 c.c. }

Glycerine 20.0 c.c. } and add to the stain.

Method.—

1. Place tissue in the above stain for ten minutes.

2. Differentiate and dehydrate with absolute alcohol.

3. Clear in xylol.

4. Mount in xylol balsam.

To Demonstrate Capsules.—

1. MacConkey's Method.—Stain precisely as for cover-slip films (vide page 100).

2. Friedländer's Method.—

Stain.—

Gentian violet, saturated alcoholic solution 50 c.c.

Acetic acid, glacial 10 c.c.

Distilled water 100 c.c.

Method.—

1. Prepare the sections for staining, secundum artem.

2. Stain sections in the warm (e. g., in the hot incubator) for twenty-four hours.

3. Wash with water.

4. Decolourise lightly with acetic acid, 1 per cent.

5. Dehydrate rapidly with absolute alcohol.

6. Clear with xylol.

7. Mount in xylol balsam.

To Demonstrate Acid-fast Bacilli.—

1. Prepare the sections for staining in the usual way.

2. Stain with hæmatin solution ten to twenty seconds, to obtain a pure nuclear stain; then wash in water.

3. Stain with carbolic fuchsin twenty to thirty minutes at 47°C.; then wash in water.

4. Treat with aniline hydrochlorate, 2 per cent. aqueous solution, for two to five seconds.

5. Decolourise in 75 per cent. alcohol till section appears free from stain—fifteen to thirty minutes.

6. Dehydrate with absolute alcohol.

7. Clear very rapidly with xylol.

8. Mount in xylol balsam.

To Demonstrate Spirochætes in Tissues.

Piridin Method (Levaditi).—

1. Cut slices of tissue 1 mm. thick.

2. Fix in 10 per cent. formalin solution for twenty-four hours.

3. Wash in water for one hour.

4. Place in 96 per cent. alcohol for twenty-four hours.

5. Measure into a dark green or amber bottle 100 c.c. silver nitrate solution 1 per cent., and 10 grammes pyridin puriss. Transfer slices of tissue to this. Stopper and keep at room temperature three hours, then in thermostat at 50° C. for four to six hours.

6. Wash quickly in 10 per cent. pyridin solution.

7. Reduce silver by transferring slices of tissue to following solution for forty-eight hours.

Pyrogallic acid 4 grammes

Acetone 10 c.c.

Pyridin puriss 15 grammes

Distilled water 100 c.c.

8. Wash well in water.

Take through alcohols of increasing strength up to absolute, keeping in each strength for twenty-four hours.

9. Clear, embed, cut very thin sections, mount, remove paraffin, again clear and mount in xylol balsam.

The spirochætes if present are black and show up against the pale yellow color of the background.

Weak carbol fuchsin, neutral red or toluidin blue can also be used to stain the background if desired, after the removal of the paraffin in step 9.

To Demonstrate Protozoa in Sections (Leishman).—

Reagents required:

Leishman's Polychrome stain.

Acetic acid 1 in 1500 aqueous solution.

Caustic soda 1 in 7000 aqueous solution.

Distilled water.

1. Mount section, remove paraffin and take into distilled water as usual (vide page 121).

2. Drain off the excess of water.

3. Cover the section with diluted Leishman (1 part stain, 2 parts distilled water) and allow to act for five to ten minutes (until tissue appears a deep blue).

4. Decolourise with acetic acid solution until only the nuclei appear blue (examine the section wet, with low power objective).

5. If the eosin colour is too well marked treat with the caustic soda solution until the desired tint is obtained (as seen with the 1/6-inch objective).

6. Wash with distilled water.

7. Rapidly dehydrate with alcohol.

8. Clear with xylol.

9. Mount in xylol balsam.