Part 97

7. Clarify in oil of cloves, oil of cedar, or beechwood creosote, ten minutes.

8. Mount in balsam.

9. Label and study.

Delafield’s hematoxylon is prepared as follows:

Hematoxylon crystals 4 grams Alcohol, 95 per cent 25 c.c. Saturated aqueous solution of ammonia alum 400 c.c.

Add the hematoxylon dissolved in the alcohol to the alum solution, and expose in an unstoppered bottle to the light and air for three or four days. Filter and add:

Glycerin 100 c.c. Alcohol, 95 per cent. 100 c.c.

Allow the solution to stand in the light until the color is sufficiently dark; then filter, and keep in a tightly stoppered bottle. The solution keeps well and is extremely powerful. So long as it is good the solution has a purplish tinge. If time permits, it would be wise to combine the alum, the hematoxylin, and the water, and to ripen the solution for two or three weeks before adding the other ingredients, which have a tendency to prevent oxidation.

The acid alcohol is made as follows:

Absolute alcohol 70 c.c. Distilled water 30 c.c. Mix.

Above solution 99 c.c. Hydrochloric acid 1 c.c.

Eosin is sold in two forms, that soluble in water and that soluble in alcohol. The eosin soluble in water is preferred, because with it a greater degree of diffusion in stain can be obtained.

Keep on hand a saturated aqueous solution and dilute with water as needed. The strength of the solution to be used varies somewhat with the tissue and the reagent in which it is to be fixed; but usually the strength should be between ⅒ and ½ per cent. when eosin is used after hematoxylin. The diluted solutions should contain 25 per cent. of alcohol, otherwise they will not keep well. When eosin is used before an aniline dye, such as methylene blue, a 5 per cent. or even a saturated solution should be used.

=To Stain Sections of Liver for the Study of Kupffer Cells.=—To bring out this reaction Keys suggests the following technic:

Fix small blocks of the fresh tissue of spleen or liver for eighteen to twenty-four hours in Müller’s fluid plus 5 per cent. mercuric sublimate. Imbed in paraffin and section to 4 microns. Fix sections to slide, and stain twenty to forty minutes with acid carmine. Wash, and transfer to equal parts of a 2 per cent. aqueous solution of potassium ferrocyanid and of a 2 per cent. aqueous solution of hydrochloric acid. Remove after three to ten minutes, wash in distilled water, and pass quickly through a 0.5 per cent. aqueous erythrosin solution. Dehydrate in alcohol, clarify in xylol, and mount in Canada balsam.

=To Prepare Anatomical Specimens for a Museum.=—The Keiserling method gives the best results, since by this method the tissues retain their normal color. The three steps are as follows:

1. Place the specimen in the following solution and leave from one to seven days, the length of time depending upon the size of the specimen.

Formalin 200 c.c. Potassium acetate 30 grams Potassium nitrate 15 grams Water 1000 c.c.

2. Pass the specimen through each of the following solutions, leaving it in each twenty-four hours or until the normal color is obtained. The specimen should be removed from alcohol as soon as color is attained. If it is left in the alcohol too long it will again lose some of its color.

Alcohol 40 per cent. Alcohol 60 per cent. Alcohol 80 per cent. Alcohol full strength

3. Place the specimen in the following permanent solution, label, and place in museum.

Glycerin 40 c.c. Potassium acetate 40 grams Distilled water 400 c.c.

A small piece of thymol must be placed on the top of the liquid in each jar, or mold will develop and spoil the specimen.

=To Make Specimens Transparent.=—Specimens may be rendered transparent by the method of Spalteholz.

The steps are essentially as follows:

I. Preparation of the fresh tissue. If any parts are to be made conspicuous, as blood-vessels, or the lymphatic system, they must be injected with an insoluble, unbleachable substance. Spalteholz recommended carmine or methylene blue, and carbon. I have found Higgin’s ordinary black carbon ink excellent for this purpose. The system or systems are injected with this substance while the tissue is perfectly fresh.

II. Fixation. The tissues are fixed preferably in 10 per cent. formalin. The length of time for fixation depends on the size of the tissue. In formalin it requires from eight hours upward to fix completely.

III. Rinsing. Running tap water for five or ten minutes accomplishes the rinsing. If it is impracticable to pass directly into the bleaching fluid, the tissues may be kept temporarily in 60 per cent. or 70 per cent. alcohol. They should then be rinsed again thoroughly in water when ready to bleach.