1. For staining nerve fibres.

Three methods (two of which are modifications of the first) are employed far more often than any others. By these methods the myelin coating is stained. Tissues must have been hardened previously for many weeks in MÜller’s fluid, or some other bichromate solution. They are then overstained in a solution of hÆmatoxyline, and the section treated with a suitable bleaching reagent, when the colour is discharged from all the tissue elements except the nerve fibres. This method displays not merely the nerve fibres in the white matter, but also the fine network in the grey matter of the brain and spinal cord. Degenerated fibres are left unstained and so degenerated tracts shew up as unstained spots on a dark background. The sections may be subsequently stained with alum carmine or eosine to shew the cells and neuroglia.

Weigert’s method.—The piece of cord to be cut after prolonged hardening in MÜller’s fluid is transferred without washing to absolute alcohol and dehydrated preparatory to embedding in celloidin (p.30). When sections are cut they are transferred at once to Weigert’s hÆmatoxyline solution:—

They are stained in this for twenty-four hours or longer, until they are quite black.

The staining will take place much more rapidly if the fluid be kept at 100°F. in the incubator. After staining they are transferred to Weigert’s differentiating solution:—

They are left in this solution for several hours, until the ground work becomes nearly decolourised.

Sections will sometimes stain more satisfactorily if they are treated, according to Weigert’s original directions, for a few hours with a half saturated solution of acetate of copper. If the hardening in MÜller’s fluid has been sufficiently prolonged, this step is usually superfluous.

Pal’s modification of Weigert’s method.—By this method quicker and more complete decolouration of the neuroglia, nerve cells, &c., is obtained. Sections are prepared in exactly the same way as in Weigert’s method and then transferred to Weigert’s hÆmatoxyline. Pal recommends that this solution be diluted to half the strength and a few drops of a saturated solution of lithium carbonate added. The writer finds the results equally good if the ordinary Weigert solution be employed.

When the sections have been thoroughly stained they are washed in distilled water and placed in a three-quarter per cent. solution of permanganate of potassium. The time required in this solution depends on the time the specimen has been in MÜller’s fluid. It should not be less than half a minute, and in very thoroughly hardened specimens, five minutes may be allowed with advantage. In this solution the sections will become of an opaque brown colour. They are washed in distilled water, and transferred to:—

Pal’s differentiating solution.

They are kept in this for one to five minutes, according to the depth of staining, until the white and grey matter are clearly defined and the brown colour is completely discharged. If the brown stain does not readily clear up, the section should be returned to the permanganate solution for about half a minute, and again treated with “Pal’s solution.” This manoeuvre may be repeated several times. As soon as the sections are thoroughly differentiated they are transferred one by one to a large vessel of water and thoroughly washed. The blue stain of the hÆmatoxyline becomes brighter during the washing process. The sections may be mounted at once, but more beautiful results will be obtained if they are stained in alum carmine for 24 hours. They should then be washed, dehydrated in alcohol, clarified in oil of bergamot, and mounted in Canada balsam.

The very prolonged hardening in MÜller’s fluid which is a necessary preliminary for this method led to the introduction of:—

SchÄfer’s modification of Pal’s method.—In this method hardening in MÜller’s fluid for three or four weeks is sufficient. The sections are made exactly as in the previous method, and transferred to Marchi’s fluid (p.24) for six hours. They are washed and stained all night in the following:—

The subsequent processes of differentiation, bleaching, &c., are exactly the same as in Pal’s method.

Osmic acid.—Employed with fresh and also with hardened specimens to demonstrate the medullary sheath. Much the best results are obtained with the former. The nerves, or small pieces of the central nervous system are placed in half to one per cent. solution of osmic acid as soon as possible after death and kept in the dark for about a week. The tissue must be very thoroughly washed in running water to remove all traces of osmic acid, and then stained for a couple of days in borax carmine to demonstrate the nuclei and axis cylinders. Sections may be made in gum, or the tissue may be teased with needles and then mounted in Farrant. Embedding in celloidin, and mounting in balsam are inadvisable, because the ether tends to dissolve out myelin, and the clarifying oil to render it too transparent.

2. Intra-muscular ramifications of nerves:—

Sihler’s chloral hÆmatoxyline method.—This method reveals the intra-muscular nerve-endings, and also brings into prominence the curious “muscle spindles” which Sherrington has shown to be connected with the posterior nerve roots, and which are believed by some to be the end organs subserving muscular sense.

A piece of muscle is taken as soon as possible after death, or from an amputated limb, and slices cut about one-tenth of an inch thick with the freezing microtome. Transfer for twenty-four hours to the following solution:—

The tissues swell up in this fluid and become translucent and gelatinous in appearance. They are now placed in pure glycerine until saturated as shown by their sinking to the bottom of the dish. This usually takes several days. They may now be stained in the following solution:—

They may be left in this from three days to a week with little fear of overstaining. Portions may then be teased with needles, and mounted in glycerine, or the stained tissue may be pressed out into a sufficiently thin layer by squeezing it forcibly between two glass slides.

Motor and sensory nerve endings.—These are best stained by the chloride of gold method (p.82).

Specimens must be taken from the body immediately after death. The method is therefore useless for the post-mortem room, but may be used for tissues removed by operation. Small pieces of tissue must be employed and must be stained in bulk, sections being made subsequently.

For motor nerve endings the muscle of a frog or human muscle from a limb just amputated may be taken. Specimens should be prepared after staining by teasing in preference to making sections. Mount in Farrant.

Sensory nerve endings may be conveniently studied in the cornea of a recently killed frog or rabbit, or in a freshly extirpated human eye. Tactile end organs may be studied in the lip or finger tips, taste buds in the papilla foliata of the rabbit’s tongue, and Pacini’s corpuscles are well seen in the mesentery of a thin cat.

3. Staining nerve cells.

Bevan Lewis’s aniline blue-black method:—This method is the best for demonstrating the wealth of nerve cells in the fresh cerebral cortex. The solution of aniline blue-black should be of the strength of 1 in 400, about a grain to the ounce. A piece of the cerebral cortex with pia mater attached, should be removed as soon as possible after death by parallel cuts about 1/8inch apart, and perpendicular to the surface of the convolution, placed on the plate of the freezing microtome and just frozen—not too hard or the tissue will be brittle and will also injure the edge of the razor. As soon as a good section is obtained the razor should be plunged into a large bowl of cold water to detach the section, which is at once floated on a glass slide, and osmic acid solution, 1/4per cent. allowed to flow over it from a pipette. This will fix the tissue elements in about two minutes. The section is again floated off into the bowl of water and thoroughly washed to free it from the osmic acid. It is then stained either on the slide, or in a watch-glass, with the aniline blue-black solution for an hour in the cold, or half-an-hour if the solution is slightly warmed. The dye is thoroughly washed away with distilled water, excess of moisture wiped off the slide with blotting paper, and the section allowed to dry under a glass bell jar. It is not practicable to dehydrate by means of alcohol as it would cause sudden shrinking of the tissues. When the section is dry a drop of Canada balsam is applied and it is covered with alcohol in the usual way. The nerve cells and their processes are stained a deep slate colour, as are the nuclei of the connective tissue cells, while the ground work of the neuroglia is faintly stained and of a neutral grey tint. This method gives beautiful results both with normal and morbid specimens.

Various other aniline dyes, indulin, methylene blue, gentian violet, have been employed in the same way, but none of them give such good or uniform results as aniline blue-black.

Hardened specimens may also be stained with aniline blue-black, but the results are not to be compared with those obtained by the fresh method. The stain is usually diffuse, but this can be improved by placing the sections for 1/2 –2 minutes in a 2 per cent. solution of chloral hydrate in distilled water after staining.

Golgi’s metallic stains for nerve cells.

Golgi introduced the methods of producing a metallic deposit of mercury or silver in the nerve cell, revealing both the cell and its processes. This method has been very fruitful in discoveries, especially in the hands of Ramon y Cajal, KÖllicher, Van Gehucten and others. It gives best results with embryonic tissues. To ensure good results it is important that the tissue be removed immediately after death. Sections of brains removed some hours after death usually give disappointing results.

There are several methods now in vogue, all slight modifications of Golgi’s original methods.

Silver nitrate method.—Small pieces not more than a quarter of an inch cube, are transferred straight from the body to a large quantity of Marchi’s fluid (p.24) and kept in it for about a week, or longer in the case of adult specimens. On removal from Marchi’s solution the tissue should be washed for a few seconds in distilled water, and then placed in a large quantity of a 3/4 per cent. solution of nitrate of silver solution in distilled water for at least a week. The lump of tissue becomes of a brick red colour owing to a coating of silver chromate. On removal from the silver solution the tissue should be washed in methylated spirit for a few minutes and the incrustation of silver chromate brushed off. Sections may be cut in gum and celloidin; or they may be fixed on a cork with celloidin or spirit varnish and cut without embedding: very thin sections are not required. Dehydrate in alcohol, clear in xylol, and mount in balsam. Goodall advises a mixture of pyridine and xylol for clearing, and mounts in strong xylol-dammar solution, without a cover-glass.

Very careful attention to details and much practice are required before uniformly good results can be obtained. The results are extremely beautiful and well repay the labour expended on them. The cells and their processes appear black on a yellowish ground.

A method has been employed for deepening the colour of the stain, but the writer has no experience of it. Kallus (Zeitsch. f. Wiss. Mikr., 1893, 477) dilutes an ordinary hydrokinone developing solution (prepared as for developing an ordinary photographic plate) with about ten times its volume of distilled water. Just before using a third part of absolute alcohol is added. Sections which have been through the silver process when placed in it become grey or black in a few minutes, and, after washing in methylated spirit, are transferred to a 20 per cent. aqueous solution of hyposulphite of soda for a couple of minutes and then washed very thoroughly in distilled water for twenty-four hours. They are then dehydrated and mounted in balsam.

Buckley’s modification of the silver method.—Described in Brain, Winter number, 1895.

The method is applicable to specimens that have been hardened in MÜller’s fluid. Thin slices are cut in the usual way, and then immersed in

for three to five days. Excess of bichromate is removed from the sections by blotting paper, and they are transferred to the freshly prepared staining mixture:—

which must not be filtered. Stain for several days.

The sections should be cut at once after removal from the staining solution. It is claimed that the minute details of structure of the cell processes are better shewn by this method.

Corrosive sublimate method.—This method is similar in its mode of action to the last, mercury being deposited in the cell instead of silver. It is rather less certain and requires more practice. It seldom stains uniformly. One cell will be found exquisitely stained while those in its vicinity are unaffected.

Small pieces of cortex are hardened for several weeks in MÜller’s fluid, or other bichromate solution, and are then transferred direct to a one-half per cent. aqueous solution of corrosive sublimate, in which they should be left from three to six weeks. Shorter periods will only give disappointing and inconstant results. Sections should be cut, if possible, in gum. They may be mounted in Farrant, or dehydrated and mounted in balsam. Tal has proposed to render the effect sharper by transforming the deposit of mercury into mercuric sulphide, by treating the sections with a solution of sulphide of sodium, which he prepares by saturating a ten per cent. solution of caustic soda with sulphuretted hydrogen and then adding an equal quantity of fresh soda solution. They are stained in this for a few minutes and then thoroughly washed.

By this method the pyramidal cells and their delicate processes appear as black opaque objects on a light ground. The neuroglia cells with their fine delicate processes are often also beautifully stained.

Nissl’s aniline method.—This method is complementary to Golgi’s method. The latter impregnates the cell rendering it opaque and shewing its form with great definiteness.

Nissl’s method stains the protoplasm without greatly reducing its transparency and allows us to study details of cell structure. Small portions of tissue, removed as soon as possible after death, are hardened in alcohol. Sections are then cut, preferably in gum, as celloidin is inconvenient owing to its staining so deeply with aniline dyes.

Sections are transferred from alcohol to a one-half per cent. aqueous solution of methylene blue, which is heated in a watch glass till it steams freely, but short of the boiling point. Stain for about a quarter of an hour and allow to cool. Transfer the sections to a mixture containing one part of aniline oil and ten of absolute alcohol, and move them about till no more colour comes away. Transfer the section to a slide with a section lifter, drain, and dry well by pressing folded filter paper carefully on the section. Allow some origanum oil to flow over the section and remove excess of this by pressure with blotting paper. Moisten with benzine,1 and add a drop of colophonium resin dissolved in benzine. The slide is warmed cautiously till the benzine is driven off and the colophonium liquefied by heat alone, and then the cover-glass is applied.

Magenta and other aniline dyes may also be employed in a similar manner.