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2. Wash off in water.

3. Alcohol; imbed in celloidin.

4. Stain sections in a 1 per cent. solution of osmic acid half an hour in the dark.

5. Place in a 5 per cent. aqueous solution of pyrogallic acid for half an hour.

6. Differentiate in a per cent. aqueous solution of permanganate of potassium one to four minutes.

7. Remove brown color in 1 per cent. oxalic acid three to five minutes.

8. Alcohol, oil, balsam.

It is important to wash carefully in water between each of the staining steps.

Stains for Neuroglia-fibers. It is possible to obtain a differential stain of the neuroglia-fibers in man by three different methods, provided the first two of the following steps are complied with:

1. The tissue must be as fresh as possible. The best results are obtained with tissues placed in the fixing solution within one hour after death. After four to six hours the results are only fair; after twenty-four hours they are practically nil. The chemical property in the neuroglia-fibers on which the differential stain depends has undergone some chemical change or has disappeared. It is retained longest where the fibers are most numerous, as about the central canal.

2. Formaldehyde must be used as the fixative, either alone or combined with certain other reagents mentioned below. No other fixatives yet known penetrate so rapidly and preserve so well the chemical properties of the neuroglia-fibers. According to Weigert, who first fully recognized the value of formaldehyde in preserving neuroglia-fibers, the best strength to use is a 4 per cent. solution (i. e. a 10 per cent. solution of formaline, etc.). The pieces of tissue in which it is desired to stain the neuroglia-fibers must be cut very thin, never overcm. thick, and preferably thinner. With thicker pieces only the surface sections are of any value.

3. The tissues after fixation must be mordanted, so as to

render the staining more intense; without mordanting only a few of the fibers will stain.

4. Staining may be performed by modifications of Weigert's fibrin-stain or with phosphotungstic-acid hematoxylin.

The methods of staining neuroglia-fibers are given in the order of their publication. The second method (Weigert's) gives a more intense stain than the first, but has the drawback of staining degenerated nerve-fibers. The third method is not so selective as the other two, but is useful because it brings out all the tissue-elements. All of the methods are unsuccessful with the neuroglia-fibers of animals other than

man.

Differential Stain for Neuroglia-fibers (Mallory).—1. Fix in a 4 per cent. aqueous solution of formaldehyde four days

or more.

2. Place in a saturated aqueous solution of picric acid four to eight days.

Steps 1 and 2 may be combined by adding 10 parts of the 40 per cent. formaldehyde solution to 90 parts of the saturated aqueous solution of picric acid.

3. Transfer to a 5 per cent. aqueous solution of bichromate of ammonium for four to six days in the incubator at 37° C., or for three to four weeks at room-temperature. Change the solution on the second day.

4. Place directly in alcohol.

5. Imbed in celloidin.

6. Fasten sections to slide by means of ether-vapor.

7. Stain in aniline-gentian-violet fifteen to twenty minutes. 8. Wash off with normal salt solution.

9. Iodin solution, 1: 2: 100, for one minute, or a stronger solution for a few seconds.

10. Wash off with water.

II. Dry with filter-paper.

12. Decolorize in equal parts of aniline and xyloi.

13. Wash off thoroughly with xylol.

14. Xylol balsam.

The neuroglia-fibers, fibrin, nuclei, and, to some extent, the red blood-globules, are stained blue. The other tissue

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elements are colorless. By very faintly tinting the anilineand-xylol mixture with fuchsin, which is readily soluble in aniline, the other tissue-elements are easily brought out, but the finer neuroglia-fibers are likely to lose their blue color. Weigert's Method for Neuroglia-fibers.-A. Fix thin pieces of tissue, not overcm. thick, in a 4 per cent. solution of formaldehyde for at least four days.

B. Mordant in the following solution for four to five days in the incubator or for eight days at room-temperature:

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Boil the chrome alum in a covered dish (it turns green in color), turn out the gas, add the acetic acid and then the acetate of copper; stir until the latter is dissolved, then cool. The solution remains clear. If the directions and order of procedure are not followed exactly, a green precipitate will form.

(Steps 1 and 2 may be combined by adding 4 per cent. of formaldehyde to the above solution; change on the second day; harden eight days.)

C. Wash off in water; dehydrate in alcohol; imbed in celloidin.

D. Reduction of copper salt in sections:

1. Place the sections, which must not be over .02 mm. thick, in a per cent. aqueous solution of permanganate of potassium for ten minutes.

2. Wash off with water.

3. Decolorize and reduce for two to four hours in the following solution, carefully filtrated:

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to 90 c.c. of which are added just before using 10 c.c. of a

10 per cent. solution of sulphite of sodium.

The sections lose their color in a few minutes, but are best kept in the solution as long as above directed.

The sections can now be stained in the manner to be described, but the color of the fibers will be more intense if the following steps are added, and a slight yellowish contrast-stain is obtained for the ganglion and ependymal cells and for the larger nerve-fibers.

E. Further reduction of copper salt:

1. Wash twice in water.

2. Place sections in a carefully filtered saturated (5 per cent.) aqueous solution of chromogen over night.

3. Wash in water.

4. The sections are now ready for staining or may be preserved until wanted in

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1. Lift section from large dish of water on slide freshly cleaned with alcohol; blot with filter-paper (method recommended by Weigert for attaching sections to slide).

2. Stain in the following mixture:

Saturated solution of methyl-violet in

70-80 per cent. alcohol,

(saturated with aid of heat; decanted

when cold).

5 per cent. aqueous solution of oxalic

acid,

100 c.c.;

5

The oxalic acid is added to render the preparations more lasting. The staining is practically instantaneous.

3. Wash off with normal salt solution.

4. Iodin solution : 5 per cent. iodid-of-potassium solution saturated with iodin. It is simply poured on and then off, as the reaction is instantaneous.

5. Wash off with water and blot with filter-paper.

6. Decolorize thoroughly in equal parts of xylol and

aniline oil.

7. Wash repeatedly with xylol or the stain will not keep. 8. Canada balsam.

The sections keep better if exposed for from two to five days to diffuse light before being put away.

Differential Stain of Neuroglia-fibers by means of Phosphotungstic-acid Hematoxylin (Mallory).—1. Fix and mordant tissues in exactly the same way as in the first method given for neuroglia-fibers—viz. :

(a) Fix in 4 per cent. aqueous solution of formaldehyde

four days.

(b) Saturated aqueous solution of picric acid four days. (c) 5 per cent. aqueous solution of bichromate of ammonium four to six days in incubator or three to four weeks at room-temperature.

(d) Alcohol, celloidin, etc.

2. Stain sections in phosphotungstic-acid hematoxylin four to twenty-four hours.

3. Wash in water.

4. Alcohol.

5. Oleum origani cretici. 6. Xylol balsam.

Neuroglia-fibers, if properly preserved, deep blue; nuclei, blue; connective tissue, deep pink; axis-cylinders, light pink; myelin-sheaths, yellow if thoroughly mordanted with the bichromate of ammonium, otherwise with prolonged staining they may take on a greenish-blue color: protoplasm of ganglion-cells and the dendritic processes, purplish or bluish gray. It will sometimes be found advantageous to stain sections lightly in Van Gieson's mixture first, so that the axis-cylinders may have a deeper red stain, and the contrast in color between the dendritic processes and the neuroglia-fibers may be greater.

Degenerations of the Nervous System.-The same methods apply to the study of degenerations in nervous tissue that apply elsewhere, except in the demonstration of fat. Both myelin and fat reduce osmic acid, so that the ordinary test for fat in the hardened tissues fails. Marchi and Algeri, however, have shown that after myelin has been mor

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