Much information may be obtained from unstained sections, and in most cases one section should be examined unstained, but the specimens mounted in this way are so transparent that it is difficult to study the details of the tissue. They are therefore usually prepared by treating them with some staining reagent, not merely to render them less transparent, but also to “differentiate” the elements of the section, by staining one part more deeply than another, or of a different colour. Thus hÆmatoxyline stains the nuclei and rapidly growing parts of the tissue, leaving the formed material, as a rule, much more lightly tinted. Methyl violet again stains healthy tissues blue, and parts affected with waxy degeneration a red-violet colour. By combining stains also much differentiation of the tissue elements may be obtained. Sections should be stained with several reagents, as their effect on individual specimens varies a good deal.

The following are the most useful stains for general purposes:—

Logwood.—This or its purified principle hÆmatoxyline is the most useful general stain. The hÆmatoxyline itself is preferable, giving more constant results, and less diffuse staining.

For general staining purposes the following formula will be found to give excellent results:—
HÆmatoxyline. Schuchardt’s formula.—

Add (a) to (b) drop by drop and with constant agitation. Keep for some days exposed to diffuse daylight until its colour is so deep that it will not transmit the light. It should then be filtered, and a crystal of thymol added. It will not give very satisfactory staining reactions at first, and should be allowed to ripen at least a month or six weeks before using. It improves as a dye with every month that it is kept. Whenever hÆmatoxyline has been made up with alum as in the above formula, an abundant reddish-brown precipitate forms after some time. This in no way interferes with the activity of the solution, but it must always be filtered before being used.

Barrett’s formula.—Introduced by Dr. W. H. Barrett, of Belfast. It gives almost as good results as the above. It is made from ordinary English extract of logwood, and is considerably cheaper.

The extract should be dried, and finely powdered, and then extracted with absolute alcohol for several days.

Filter and add slowly to

The strength of the solution will vary with different samples of logwood and must be estimated by trial. This solution is comparatively cheap and is useful for class purposes.

Ehrlich’s hÆmatoxyline.—This very useful nuclear stain is made as follows:—

Add (a) slowly to (b) with constant agitation.

Allow to ripen in sunlight for two months before using. It may be employed as a rapid stain undiluted but far better results are obtained by using a weak solution, a few drops to a watch-glass full of distilled water, and staining slowly for from half an hour to two hours. The solution improves by keeping. If after a time the staining becomes diffuse it is an indication that the acetic acid has evaporated, and a few drops more should be added.

Kleinenberg’s hÆmatoxyline.—This formula differs from the previous one in being an alcoholic solution. The calcium chloride is added because it “sets up diffusion currents between the alcohol in the material to be stained and the alcoholic staining solution, so enabling the latter to penetrate more rapidly” (Squire). It is much used in staining embryonic specimens in bulk before embedding in paraffin, and was strongly recommended for that purpose by Foster and Maitland Balfour.

Various formulÆ have been given from time to time. That advised by Squire (Methods and FormulÆ, p.25) can be accurately made up without much difficulty.

Mix and add

Allow it to stand and any excess of calcium sulphate, &c., to separate. Filter and add

A little thymol should be added as a preservative.

In making up these solutions care must be taken that only distilled water is used, and that all the vessels employed have been previously rinsed out with it, otherwise precipitation of the hÆmatoxyline will occur.

Should sections be overstained in hÆmatoxyline, this may be remedied by washing it in a half per cent. solution of acetic acid, until sufficient of the stain is discharged, but the staining is more diffuse than if the happy mean had been hit in the first instance.

HÆmatoxyline stains the nuclei of the cells a beautiful violet colour, and also tints, more or less lightly, the cell protoplasm and the fibrous elements. It also stains the axis cylinders of nerves, and is much used in special staining of the nerve centres as will be described later, (pp.88–91).

The stain is permanent. Sections may be mounted either in Farrant’s solution, or in Canada balsam, the latter being preferable.

Eosine.—Much more satisfactory results are obtained from the commercial eosine (an amorphous orange powder used in dyeing and in the manufacture of red ink), than from the pure crystalline form.

It may be used as an aqueous solution ( 1/30 per cent.) or as a solution in absolute alcohol ( 1/15 per cent.). Sections stained in the former should be rapidly passed through a one per cent. solution of acetic acid in order to “fix” the stain, and then washed in distilled water.

It is a very transparent stain, and the most delicate details of a section stained with it are perfectly visible.

It stains the nucleus but slightly, while it stains the cell protoplasm and fibrous tissues and especially muscular tissues a beautiful rose colour.

It will be seen, therefore, that it stains those parts which are left unstained by hÆmatoxyline, and vice versÂ. This complementary action is applied in the following method.

Double staining with eosine and hÆmatoxyline.—Sections having been stained in hÆmatoxyline in the ordinary way, are washed in distilled water, and dehydrated in a solution (about 1 in 1500) of eosine in absolute alcohol. They should remain in this for about two minutes, and then be passed through oil of cloves and mounted in Canada balsam in the ordinary way.

This method gives extremely useful and beautiful results with almost all tissues, and is superior to picrocarmine for differentiating the tissue elements. Thus, the nuclei are stained violet, the cell protoplasm a much paler and warmer violet, the fibrous tissues pink, and red blood corpuscles orange or brick red.

The alcoholic solution of eosine is also used as a contrast stain after staining for micro-organisms with blue or violet dyes.

Carmine.—It is made as follows:—

Rub the carmine with a little water in a mortar, add the ammonia, when the liquid will turn black. Gradually add the rest of the water, rubbing it up all the time. It should be bottled, allowed to stand for a few days, and then filtered, and a piece of camphor put in the bottle.

Lithium carmine resembles closely ammonia carmine in its staining effects. It is usually a matter of individual preference which is employed.

Dissolve and filter.

Sections may be sufficiently stained in either of these fluids in from three to five minutes, but more satisfactory results are to be obtained by diluting with twenty times the bulk of distilled water, and leaving sections to stain for twenty-four hours.

After staining in carmine the sections must be passed through a half per cent. solution of acetic acid, in order to fix the carmine in the tissues, as otherwise the water will dissolve the stain out.

Borax carmine—

Dissolve with the aid of heat and add slowly to (b).

Allow to stand for a fortnight. Filter, and add a lump of camphor.

To use it, place sections, or the tissue in bulk, in it for from four to twenty-four hours, according to size, and then transfer to alcohol (seventy per cent.) containing a drop to the ounce of hydrochloric acid, for twenty-four hours, and then wash thoroughly in water. The tissue may then be placed in gum if it is to be frozen, dehydrated in alcohol if paraffin or celloidin is to be employed.

Its advantage is that it is very diffusible, and so can be used to stain tissues in bulk. It takes a considerable time to stain sufficiently deeply, but there is little fear of overstaining.

It stains nerve-cells and axis cylinders brightly, and also the connective tissue, bringing a sclerosed patch out very prominently.

Alum carmine:—

Boil for twenty minutes. Filter. Add a few drops of carbolic acid.

In using this reagent it should be filtered into a watch glass, and the sections placed in it for at least an hour. There is no fear of overstaining, and they may be left all night. After they have been stained they must be thoroughly washed in water to remove the alum, otherwise numerous crystals of it will be seen in the field when the section is mounted. Sections may be mounted in Farrant’s solution or in Canada balsam. The staining effect improves very much after the section has been kept a few days.

If desired its staining action may be complemented by dehydrating it in an alcoholic solution, either of eosine (1 in 1500) or of picric acid, and then clearing up in oil of cloves, and mounting in Canada balsam.

By itself it gives a stain very like that of hÆmatoxyline, only warmer. It picks out the nuclei and axis cylinders of nerves, stains cell protoplasm slightly, and the fibrous elements scarcely at all.

It may be used for the same purposes as hÆmatoxyline. The colour is less attractive, and not so deep as that of the latter, but as it does not overstain sections, even when left in it for a week, it is a very convenient stain for general purposes.

It is particularly useful as a contrast stain for sections of brain and spinal cord, after the Weigert-Pal hÆmatoxylin process (p.88).

Ammonia-picrocarmine was formerly very largely used as a staining reagent. Its place has now to a large extent been taken by lithio-picrocarmine.

In its preparation the best carmine must be used.

It is made as follows:—

Dissolve with gentle heat, and add

Bring the mixture to the boiling point, and then place in a shallow vessel, covered with a glass plate, and leave it in full sunlight for a month or more. Filter, bottle, and add six drops of carbolic acid to each ounce of the mixture. It will keep indefinitely and improves with age. It requires filtering from time to time, as a gelatinous crimson mud tends to deposit from the solution.

Lithio-picrocarmine.—Prepared as follows:—

Dissolve, and add

Add a few drops of carbolic acid to each ounce.

It should stand a day or two in sunlight and then be filtered. It improves by keeping.

It should be kept in a stoppered bottle with a glass rod fused into the stopper.

When sections are to be stained they are to be floated out on a clean glass slide as described on page 55. The slide should then be tilted to allow the water to drain off, and superfluous moisture round the section removed by a soft rag, or blotting paper. A drop or two of the stain should then be transferred to the slide, which should be left lying quite flat for about ten minutes. Unless the room is very warm it is advisable to heat the slide very gently over a spirit lamp, as this causes the tissues to stain more brightly and more rapidly.

The excess of the picrocarmine should be allowed to run off the slide, and the latter wiped. Some of the stain should, however, be left on the section, as its effects go on increasing, and are often not fully seen until a few weeks have elapsed. They should be mounted in Farrant’s medium. As a rule those mounted in Canada balsam do not give such good results. Should there be special reasons for using this medium, as in mounting spinal cord sections, &c., they should be dehydrated after staining in picrocarmine in an alcoholic solution of picric acid (one part of a saturated alcoholic solution to five of alcohol), before clarifying in oil of cloves, as otherwise the alcohol will dissolve out the picric acid, and much of the differential staining effect will be lost. The nuclei should be stained a bright crimson, the protoplasm of the cells yellow, or a dull pink, the fibrous elements a bright pink, red corpuscles green, and all dead material, e.g., caseous matter, bright yellow. It also stains nerve-cells, and the axis cylinders of nerve fibres very brightly. It is, however, a rather uncertain dye. The results are most brilliant in the case of fresh sections.

Osmic acid is invaluable for staining fatty particles in the cells.

For ordinary use the one per cent. stock solution (p.21) should be diluted with ten times its bulk of distilled water, and sections stained in it all night in a dark cupboard, or the watch glass containing them may be placed inside a small box.

The sections must be washed thoroughly in plenty of water. If desired they may be stained subsequently in picrocarmine or methyl violet if waxy degeneration also be present. Sections should be mounted in Farrant’s solution, as Canada balsam usually gives disappointing results.

It demonstrates the most minute fatty particles in degenerating cells, &c., staining them black. It may be employed to demonstrate the globules of fat blocking up the vessels in fat embolism.

It stains the myelin sheaths of nerves black, and will be again referred to when speaking of methods of staining the spinal cord.

Nitrate of silver is employed for staining the intercellular cement of epithelial cells. It stains this substance a deep black, while the rest of the tissue takes on a brown colour. It is used as a half per cent. solution in distilled water, and kept in a stoppered bottle carefully covered up with brown paper. To use it take some epithelial tissue, e.g., the omentum from a recently killed animal, or a section of some epithelial tumour, immediately after excision. Wash thoroughly in distilled water to remove all chlorides, and then place in a watch glass containing the silver solution. Keep this in the dark for half an hour and then wash thoroughly in plenty of water. The section should be mounted in glycerine or Farrant’s medium and kept from the light or it will become too darkly stained.

Chloride of gold is employed to demonstrate the peripheral terminations of nerves. It can only be employed within the first half hour after the tissue has been removed from the living body. The pieces of tissue must be small and may be stained in bulk, sections being subsequently made.

A half per cent. solution in distilled water is employed. The tissue is transferred to this on its removal from the body, until it becomes lemon coloured. It is then exposed in a one per cent. solution of acetic acid to a strong light until it assumes a purplish tinge, which takes from two hours to two days. Sections should be mounted in Farrant’s medium. It stains the cells of the tissue, and nerve cells reddish purple, and nerve fibrils, especially the terminal ones, rather more violet. This is very well seen in the cornea.

It is useful sometimes for clinical purposes to excise a portion of muscular tissue and examine the nerve endings by this method. Unfortunately the stain is somewhat uncertain in its action. Better results are obtained by Sihler’s chloral hÆmatoxyline method (p.92).

Methyl violet.—A very satisfactory solution may be obtained ready made in the “telegraphen tinte,” prepared by Leonhardi, of Dresden, as recommended by Woodhead. It may also be used as a one per cent. solution in distilled water, a few drops of carbolic acid being added to prevent the growth of fungi.

It is a very useful selective stain. It gives two reactions, red violet, and blue violet. Thus it stains the matrix of hyaline cartilage blue violet, but the cells red violet. It has also a most important pathological application, as it picks out any parts which have undergone “waxy” or “lardaceous” degeneration, staining them red violet, but the rest of the section blue violet.

About ten drops of a one per cent. solution should be filtered into a watchglassful of water, and the sections stained for about five minutes. They must then be passed through a half per cent. solution of acetic acid and washed thoroughly for some time in a large quantity of water till no more colour comes away.

If these steps are not taken with care, the dye will diffuse out after the section has been mounted, blurring all details and spoiling the appearance of the section.

Sections may be mounted in Farrant’s solution (to which a spot of formic acid may be added): if mounted in Canada balsam the sections must be overstained as both the alcohol and oil of cloves rapidly dissolve out the dye.

Safranine.—Employed as a freshly made saturated solution in aniline oil water warmed to 60°C. (140°F.). Filter into a watch glass. Stain for not more than a minute. Dehydrate in alcohol which will remove much of the stain, clarify in oil of cloves or origanum oil. Mount in balsam.

Another method is to stain for about ten minutes, and then leave for a minute in Gram’s iodide solution. The sections are then washed in alcohol, dehydrated, clarified in oil of cloves, and mounted in balsam. By these methods the stain is withdrawn except from certain elements, e.g., those undergoing colloid or calcareous degeneration.

A quarter per cent. watery solution is sometimes employed as it stains nucleoli and actively dividing nuclei very brightly, while the rest of the cell is stained faintly. It may be employed to study karyokinesis in the cells of a rapidly growing cancer.

Ehrlich-Biondi stain.—This stain has been much employed for staining specimens of blood, for studying karyokinesis, and for investigations on the supposed parasitic bodies found in cancer cells.

It is prepared by mixing saturated aqueous solutions of the following aniline dyes, slowly and with constant agitation:—

finally add

Filter from the copious precipitate which forms. The solution must be made up frequently as it does not keep well.

Sections may be stained rapidly for half an hour or an hour, but better results are obtained by diluting the fluid with twenty volumes of water, and staining all night. Sections should be washed in water and then passed rapidly through absolute alcohol and xylol and mounted in Canada balsam.