Aqueous Alum Hematoxylin Solution

Hematoxylin crystals..................... 1 gm.

Sat. aq. sol. ammonia alum................ 100 c.c.

Water.................................. 300 c.c.

Thymol................................. a crystal.

Dissolve the hematoxylin in a little water by the aid of heat. After the solutions have been mixed, expose to the light and air in an unstoppered bottle for about ten days. Then tightly cork.

Delafield's Hematoxylin

Hematoxylin crystals...................... 4 gm.

Alcohol (95 per cent.)...................... 25 c.c.

Sat. aq. sol. ammonia alum................. 400 "

Dissolve the hematoxylin in the alcohol, then add the alum solution. Expose the mixture to the air and light four to five days. Then filter and add:

Glycerin................................. 100 c.c.

Alcohol (95 per cent.)...................... 100 "

Expose to light and air for a couple of weeks, then filter and keep tightly corked. The solution lasts well and stains the more rapidly the older it gets.

Ehrlich's Acid Hematoxylin

Hematoxylin crystals.......... 2 gm.

Absolute alcohol.............. 60 c.c.

Glycerin.....................60

Water.......................60 "

Glacial acetic acid............. 3 " saturated with ammonia alum.

The solution is ripened in an uncorked bottle until it becomes deep red in color; requires a couple of weeks. If kept in well-stoppered bottle precipitates do not form and the solution retains its staining powers for years. Also does not overstain.

Mayer's Hematein

When hematein is used, ripening is unnecessary, but the results from such stains are not as satisfactory as when hematoxylin is used:

Hematein............................... 0.4 gm.

(Dissolve in a few drops of glycerin).

Alum................................... 5.0 "

Glycerin................................ 30.0 c.c.

Water.................................. 70.0 "

Hematoxylin Staining

The nuclei are stained blue. The older the solutions, the quicker they act and the deeper they stain. If the sections are overstained, the excess of color can be removed by placing them in hydrochloric acid alcohol until the proper color is obtained. The acid causes the blue to change to a brown, but the color is regained when the sections are placed in water. The acid should be thoroughly washed out; this can be hastened by using water to which an equal amount of a saturated watery solution of lithium carbonate has been added.

1. Stain three to ten minutes, according to age of stain.

2. Wash thoroughly.

3. Differentiate with acid alcohol, about thirty seconds if sections are overstained.

4. Wash thoroughly.

5. A counterstain, eosin, is usually employed.

6. Dehydrate, clear, and mount in balsam.

Alum Carmin

Carmin.................................. 1 gm.

Alum solution (5 per cent.)................. 100 c.c.

Boil for one-half to one hour and when cool filter. It stains the nuclei a violet red. There is no danger of overstaining and the color is not very easily removed in water or weak acid solutions. This preparation does not work well with objects that are difficult to stain.

The sections are placed:

1. In the stain for ten minutes to two hours.

2. Then washed thoroughly in distilled water.

3. Dehydrated in alcohol, cleared, and mounted.

Lithium Carmin

Carmin.......................... 2.5 to 5.0 gm.

Sat. sol. lithium carbonate......... 100.0 c.c.

Heat and filter. The nuclei are stained an intense red. Is well adapted for tissues that stain with difficulty. Any excess of color can be removed in acid alcohol. Is a good counterstain for tissues that have been injected with blue substances. Sections are placed:

1. In the stain for two to three minutes.

2. Washed in water.

3. Differentiated for one-half to one minute in acid alcohol; hydrochloric acid, 1; 70 per cent, alcohol, 100.

4. Washed thoroughly so as to remove the acid.

5. Dehydrated in alcohol, cleared, and mounted in balsam.

Picrolithium Carmin

Lithium carmin solution..................... 1 part.

Sat. watery sol. picric acid.................. 2 parts.

Sections are:

1. Stained three to five minutes.

2. Washed.

3. Differentiated two to three minutes acid alcohol.

4. Washed thoroughly.

5. Dehydrated in alcohol that has had a little picric acid added to it. 7. Cleared and mounted.

Nuclei are stained brownish red, and the protoplasm yellow.

Borax Carmin

Carmin................................ 0.5 gm.

Borax.................................. 2.0 "

Distilled water.......................... 100.0 c.c.

Mix and heat until boiling begins; should be stirred constantly; then add 4.5 parts of dilute acetic acid (0.5 per cent.) and let stand twenty-four hours; then filter.

This gives the same results as the lithium carmin except that the color is not intense.

Sections placed:

1. In stain for five to fifteen minutes.

2. Washed in water.

3. Differentiated one-half to one minute in acid alcohol solution.

4. Washed in water thoroughly to remove acid.

5. Dehydrated, cleared, and mounted. Bismarck Brown.

Either a 3 to 4 per cent, watery solution obtained by boiling and filtering. Or a concentrated alcoholic solution made in 40 per cent. alcohol, equal to 1 to 2 per cent. Sections are:

1. Stained five minutes.

2. Washed in alcohol or 1 per cent, hydrochloric acid alcohol.

3. Dehydrated, cleared, and mounted.

The nuclei are stained a deep brown; the protoplasm, a lighter color. Bacteria are an intense brown. Cannot over-stain. This method is especially adapted for micro-photographic work.

Gentian-Violet

Either a 1 per cent, watery or a 2 per cent, alcoholic solution may be used. Are likely to overstain.

Sections are:

1. Stained three to five minutes.

2. Washed in alcohol until they become a pale blue.

3. Then in absolute alcohol.

4. Cleared and mounted.

The nuclear staining is clearer if the sections are put for fifteen to thirty seconds in a 0.5 per cent, solution of acetic acid and then into the alcohol.

Safranin is usually employed after fixing in Flemming's solution to bring out karyokinetic figures.

Sections:

1. Stained one-half to twenty-four hours in a 1 per cent. watery solution of safranin.

2. Quickly washed in water.

3. Washed in absolute alcohol to which 5 to 10 drops of 1 per cent, hydrochloric acid alcohol have been added.

4. Washed in pure absolute alcohol until the section is a clear brown.

5. Cleared and mounted in alcohol.

The resting nuclei are pink, those undergoing mitotic changes are deep red.

Another method is:

Anilin oil................................. 2 c.c.

Water................................... 100 "

Safranin in excess.

Heat to 6o° C. and filter. The solution will last about two months. This forms stains almost immediately. The after-steps are as above.