~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. _Friedlander'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 haematin 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 47C.; 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 Spirochaetes 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 spirochaetes 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).