5. Dehydrate in 95 per cent. alcohol.

6. Oleum origani cretici.

7. Canada balsam.

Ribbert's Method for Connective-tissue Fibrillæ.-I. Fixation in alcohol is preferable.

2. Place sections in a 10 per cent. solution of phosphomolybdic acid for five to thirty seconds (use glass or platinum needles).

3. Wash quickly in water.

4. Stain five minutes or less in phosphomolybdic acid hematoxylin. (For this purpose the solution can be used after it is twenty-four hours' old.)

5. Water.

6. Alcohol, oil, Canada balsam.

Fibrillæ deep blue; other tissue-elements grayish-green; contrast-stains incompatible. The method is recommended for staining the finest fibers of connective tissue.

Unna's Method for the Connective-tissue Fibrillæ.-I. Harden in alcohol.

2. Stain in the concentrated solution of polychrome methylene-blue five minutes.

3. Wash in water.

4. Decolorize, differentiate, and stain in a 1 per cent. solution of orcein in absolute alcohol fifteen minutes.

5. Wash in absolute alcohol.

6. Oil of bergamot.

7. Balsam.

Nuclei, dark blue; protoplasm, pale blue; elastic and connective-tissue fibers, deep orcein red; smooth muscle-fibers, bluish; mastzellen granules, red: protoplasm of plasma cells, deep blue.

ELASTIC FIBERS.

Elastic fibers are not affected by dilute caustic soda or potash or by acids. They show a marked affinity for osmic acid, staining with greater rapidity than most other tissueelements. They also show much affinity for certain aniline dyes, such as Victoria blue and safranin, but much depends.

on the exact sort used. Phosphotungstic-acid hematoxylin and Unna's orcein method will be found to give very satisfactory results.

A. Staining of Elastic Fibers with Phosphotungsticacid Hematoxylin (Mallory) (see page 243).—Harden in alcohol. The results after other fixatives do not seem so good.

1. Stain sections in phosphotungstic-acid hematoxylin twenty-four hours.

2. Wash in water.

3. Alcohol.

4. Oil, Canada balsam.

Nuclei and elastic fibers, blue; connective tissue, pink.

B. Unna's Orcein Method for Elastic Fibers.-Unna's latest method of using orcein is as follows, and can be highly recommended:

1. Stain sections in the following solution:

Orcein (Grübler),

Hydrochloric acid,

Absolute alcohol,

I;

I;

100.

Place the sections in a dish and pour over them enough of the solution to cover them. Warm gently in an incubator or over a small flame for ten to fifteen minutes until the solution thickens, or leave in the solution at room-temperature over night.

2. Wash off thoroughly in dilute alcohol (70 per cent.). 3. Wash in water to get rid of all the acid and to fix the color.

4. Alcohol.

5. Oil.

6. Balsam.

The washing in water is not absolutely essential.

Elastic fibers are stained of a deep silky-brown color, connective tissue a pale brown. If it is desirable to have only the clastic fibers stained, wash for a few seconds in I per cent. hydrochloric-acid alcohol before washing in water. The nuclei can be brought out by staining in Unna's poly

chrome methylene-blue solution after washing the sections in water.

C. Another method of Unna's is the following: 1. Harden in alcohol or Flemming's solution.

2. Stain first, if desired, in vesuvin.

3. Wash in water.

4. Stain twenty-four hours in

5. Two to three seconds in 25 per cent. nitric acid.

6. Decolorize in weak acetic acid.

7. Dehydrate quickly in absolute alcohol.

8. Xylol, Canada balsam.

D. Herxheimer's Method.-1. Harden in Müller's fluid;

alcohol and other fixatives are not so good.

2. Stain three to five minutes in—

3. Decolorize for from five to twenty seconds in the officinal solution of chlorid of iron.

4. Wash in water.

5. Alcohol, oil, Canada balsam.

Elastic fibers, blue black and deep black; the surrounding tissue, pale blue to blue.

THE CENTRAL NERVOUS SYSTEM.

In the preservation of the central nervous system the special structures which require consideration are the ganglion-cells, including both the dendritic and the axis-cylinder processes, the myelin sheaths, and the neuroglia-fibers. No one fixing reagent is suited for the best preservation of all of them, unless possibly it be formaldehyde.

The main fixing fluids for the nervous system until within a very short time have been various solutions of the chrome salts, particularly of bichromate of potassium, either alone or in combination with sulphate of sodium, as in the wellknown Müller's fluid. The chief objections to the chrome salts as fixatives are that they penetrate and harden very slowly, and do not preserve properly either the ganglioncells or the neuroglia-fibers. On the other hand, they probably preserve the axis-cylinders as well as any reagent we yet know, and are invaluable for their property of entering into some chemical combination with myelin, in consequence of which it is possible to obtain by the method originated by Weigert a differential stain of the myelin sheaths.

The new fixing reagent, formaldehyde, seems likely to find. its greatest use histologically as a fixative of the central nervous system. It penetrates and hardens up to a certain degree with great rapidity. It also preserves in certain structures the special chemical properties on which certain differential stains depend. Small pieces of nervous tissue are properly fixed in the standard solution (4 per cent. solution of formaldehyde gas) in four days. A whole brain will be so hardened in ten days to two weeks that thin serial sections can be made through it without fear of the slices altering their shape in the least. The process could undoubtedly be hastened by injecting the arteries.

It must be borne in mind, however, that for most purposes formaldehyde must be followed by other reagents before the structures and their chemical properties preserved by it are properly fixed so that they will not be altered when transferred to alcohol. In other words, formaldehyde may be looked upon as a very quick preliminary fixing reagent. The hardening of brains entire in it is not recommended, except in certain cases-for instance, of cysts, hemorrhages, or occasionally of tumors-where the gross lesions and the tracts or structures affected by them are of more importance than the finer histological changes. For the proper preservation of ganglion-cells and of neuroglia-fibers very small pieces must be taken and fixed by the special methods.

given; but if the main object is to trace system-degenerations, much larger pieces, or even the whole brain, may be taken, because the myelin-sheaths change comparativly very slowly after death.

The stains for the central nervous system may be divided into two classes, general and differential. For nearly all of them preliminary fixation in formaldehyde is advisable or possible. This renders the immediate preservation of nervous tissue very simple, and at the same time allows a variety of mordanting and staining methods to be used later.

The staining of the various histological elements of the central nervous system and the fixing reagents best suited for each of them will be considered under the following headings:

General Stains.

Protoplasmic granules;

Stains for ganglion-cells, Dendritic and axis-cylinder processes;

Axis-cylinders and their terminal processes.

Stains for the myelin-sheath.
Stains for the neuroglia-fibers.

General Stains.-General stains include the ordinary nuclear stains, with or without a contrast-stain, and certain diffuse single or combined stains which color the nuclei, the cell-protoplasm, including to a varying extent the dendritic processes of the ganglion-cells, the axis-cylinders, and the neuroglia-fibers. The different stains vary somewhat in regard to the structures which they bring out most prominently.

The best fixation for the general stains is, on the whole, that used for mordanting the myelin-sheaths; in other words, formaldehyde combined with or followed by a chrome salt. (see page 316). Occasionally the fixatives employed for other tissues are indicated. Alum-hematoxylin, followed by eosin, is always a useful stain. The eosin if deep enough brings out fairly well both the dendrites and the axiscylinders. The basic aniline dyes, especially methylene-blue, find their greatest use in Nissl's method of staining the protoplasmic granules of the ganglion-cells.