I. Carmine and Picro-nigrosin.—1. Stain in neutral or lithium carmine.

2. Decolorize in acid alcohol.

3. Wash in water.

4. Absolute alcohol.

5. Aniline-gentian-violet, ten minutes.

6. Iodin solution, two to three minutes.

7. Absolute alcohol, so long as any color is discharged. 8. Saturated aqueous solution of picric acid and of nigrosin, five minutes.

9. Decolorize in a I per cent. alcoholic solution of oxalic acid.

10. Water, several minutes.

II. Absolute alcohol.

12. Oil of bergamot.

13. Balsam.

Nuclei, red; cell-protoplasm, light olive-green; connective tissue, dark olive-green; elastic fibers, bluish; bacteria and blastomycetes, violet.

II. Methylene-blue and Eosin in Borax Solution.Keep three solutions on hand :

(a) Saturated solution of methylene-blue in a saturated aqueous solution of borax.

(b) per cent. solution of "bluish eosin" in 70 per cent. alcohol.

(c) Saturated aqueous solution of borax.

For use mix together 2 parts of the filtered solution a, I of b, and 2 of c. The different steps of the staining process are as follows:

1. Absolute alcohol.

2. Staining solution, ten to twenty minutes.

3. Decolorize in a I per cent. solution of acetic acid.

4. Wash in water.

5. Absolute alcohol.

6. Xylol.

7. Xylol balsam.

Nuclei, blue; red blood-globules, cell-protoplasm, granules

of eosinophiles, connective tissue, etc., rose-red.

III. a. Malachite-green, Acid Fuchsin, and Nigrosin.—

2. Stain in 20 drops of above solution diluted with 10 c.c. of distilled water for twenty-four hours.

3. Decolorize in a per cent. aqueous solution of oxalic acid.

4. Wash in water.

5. Absolute alcohol. 6. Xylol balsam.

Resting nuclei, light red; protoplasm, reddish yellow. In the karyokinetic figures, nuclein green; fibrillæ of the achromatic spindle and of the mitoma, bright red; centrosome and polar bodies, red; the rest of the cell-body, a reddishyellow color.

III. b. Malachite-green, Acid Fuchsin, and Martius Yellow.

1. Stain in the solution without diluting, half an hour.

2. Absolute alcohol.

3. Xylol.

4. Xylol balsam.

Nuclei of resting and dividing cells, green; cell-protoplasm,
connective tissue, etc., rose-colored; "cancer-bodies," mainly
red, but in masses of them some are red and some green.
IV. Acid Fuchsin and Picro-nigrosin.-

Saturated alcoholic solution of acid fuchsin, 6 drops;
Martinotti's picro-nigrosin,

Distilled water,

8

IO C.C.

1. 70 per cent. alcohol.

2. Stain in the solution six hours.

3. Decolorize in dilute acetic acid.

4. Absolute alcohol.

5. Xylol.

6. Xylol balsam.

Resting nuclei, red; nuclein of karyokinetic figures, yellow : cell-protoplasm, dark olive-green; "cancer-bodies," mainly olive-gray, but some or portions of them may be ruby-red. V. Light Green (Lichtgrün) and Hematoxylin.— Ehrlich's acid hematoxylin,

Saturated solution of Lichtgrün in 70 per cent.
alcohol,

Distilled water,

1. Distilled water.

2. Stain in above mixture half an hour.

3. Wash thoroughly in several waters.

4. Alcohol.

5. Oil of bergamot.

6. Balsam.

15 c.c.

5

Nuclei, green;

"cancer-bodies" take the hematoxylin stain.

VI. Acid Fuchsin and Hematoxylin.—

Ehrlich's acid hematoxylin,

I per cent. solution of acid fuchsin in 70 per

15 c.c.

Nuclei, red; protoplasm, brick-red; "cancer-bodies" take the hematoxylin stain.

Orcein, a vegetable dye obtained from certain tinctorial lichens, is used mainly for staining elastic fibers. It is soluble in alcohol, and is employed either in a neutral or acid (HCI) alcoholic solution.

Iodin is the oldest of the histological stains, but is now but little used for that purpose.

The tincture of iodin, a saturated solution in alcohol, is

used for getting rid of the precipitate of mercury formed in tissues fixed in corrosive sublimate or in Zenker's fluid; a small amount of the tincture is added to the alcohol in which the tissue is preserved (see page 223).

Lugol's solution, a solution of iodin in water containing iodid of potash, is of varying strength. Iodin in this form is much used as a test for starch, amyloid, glycogen, and corpora amylacea. In Gram's stain and its modifications iodin produces some chemical change in the coloring material employed, in consequence of which, when appropriate decolorizers are used, the stain remains fast in certain structures, while from others it is easily entirely extracted.

The strength originally employed by Gram for his staining method was

Iodin,

Iodid of potash,
Water,

I gram;

2 grams;

300 c.c.

Weigert in his modification of this method employed a stronger solution:

Recently he has recommended the following strength both for fibrin and for neuroglia-fibers:

Iodid of potash,

Water,

5 grams

saturated with iodin.

100 C.C.

The only difference in the action of the various solutions probably is that the strong solution acts practically instantaneously, while the weaker solutions require some little time. Acid Alcohol (Orth's Discharging Fluid).—

Hydrochloric acid,

70 per cent. alcohol,

I C.C.;

99

Aniline Water (Aniline-oil Water).-Shake together 5

parts of aniline with 95 parts of water, and filter the resulting milky fluid. It should come through perfectly clear.

Carbolic-acid water is made in like manner by shaking together 5 c.c. of melted carbolic-acid crystals and 95 c.c. of water. The solution should be filtered.

Mayer's glycerin-albumin mixture for attaching paraffin sections to slides is composed of equal parts of the white of egg and of glycerin. The mixture should be thoroughly beaten and then filtered, or after standing for some time can be decanted. Add a little camphor or carbolic acid to prevent decomposition. Egg-albumin is dissolved by acids and alkalies, so that when such reagents are to be used the sections are best attached to the slide by some other substance. For this purpose Schällibaum's solution, of celloidin I part in 3 or 4 parts of oil of cloves, is often useful. Cover the slide with a thin layer of the solution. Arrange the sections in order on the slide and place it in the thermostat at 54° to 60° C. for several hours, or heat for a few seconds to half a minute over the flame until the oil of cloves runs together in drops. After cooling, remove the paraffin with xylol, pass through origanum oil to 95 per cent. alcohol, and proceed as with other paraffin sections.

Clearing Reagents.-The object of clearing reagents is to render certain tissue-elements more prominent than others. This result may be brought about by dilute acetic acid (2-5 100), which swells up the ground substance, so that nuclei, elastic fibers, fat, myelin, and micro-organisms are more distinct, or by alkalies, which destroy the cells and ground substance and leave only elastic fibers and bacteria but little changed. This method is used almost wholly for fresh tissues.

The same result is more commonly obtained by soaking the tissues in substances which by reason of their high index of refraction render the tissues more or less transparent. Any structure which it is desirable to study is usually previously stained and thus easily rendered prominent. This second method is most applicable to hardened tissues.

For soaking and clearing the tissues a variety of reagents