III. WASHING.
Thorough washing after staining is necessary after nearly all stains. The washing should usually be done by soaking the sections in several changes of distilled water, although tap-water, alcohol, alum-water, and other solutions may be used to intensify the staining effects. When this is done a final washing in distilled water or alcohol is usually necessary. Differentiating fluids should always be thoroughly removed from the section before mounting. Sections should not be allowed to lie in wash-water that is colored by the stain; as soon as the wash-water becomes colored it should be replaced by fresh. When sections are left lying in the wash-water for some time the vessel containing them should be covered to prevent the settling of dust on the sections, as it is practically impossible to remove from the latter dust or other precipitates that may become attached to them. Some stains give better results after long washing; others are easily washed out if the sections are left standing in the wash-water. The time-limits of washing will depend upon the character of the stain employed.
IV. DEHYDRATION.
Dehydration of the sections is usually produced by passing them through two alcohols, 80 per cent and absolute, or 80 per cent and 95 per cent. For certain clearing reagents (xylol) it is necessary to use absolute alcohol. When carbol-xylol is used as a clearing reagent absolute alcohol is not necessary, and 95 per cent can be used instead for the second dehydrating solution. Usually a minute in each alcohol is sufficient for the dehydration of single sections. If dehydration with alcohol is not desirable because of its action on the stain it is possible to dehydrate and clear in xylol by repeatedly blotting the section with absorbent paper, covering the section several times with xylol and then blotting. The section should never be allowed to become perfectly dry. Dehydration with alcohol may also be avoided by staining paraffin sections without removing the paraffin, drying in the incubator or over the flame, removing the paraffin in xylol, and mounting. Imperfect dehydration is shown by the presence of white spots or a milky cloud in the section when it is put into the clearing fluid.
V. CLEARING.
After dehydration, sections must be cleared in some solvent of balsam before they can be mounted in the latter medium. When 95 per cent alcohol has been used for the final dehydration the sections may be completely dehydrated and cleared at the same time by the use of carbol-xylol (xylol, 3 parts; melted crystals of carbolic acid, 1 part; add melted carbolic acid to the xylol to prevent formation of crystals). The sections (on the slide or cover-slips, in celloidin sections or films) are transferred from the alcohol, draining or blotting off excess of the latter, into the carbol-xylol, and left until perfectly clear. This can be most easily determined by viewing the sections against a dark background. Carbol-xylol cannot be used for sections treated with aniline stains. These are dehydrated in 95 per cent alcohol, and the final dehydration and clearing accomplished by repeatedly placing xylol upon the slide and blotting it out until the sections are transparent. Turpentine, chloroform, benzine, toluol, the oils of bergamot, cloves, thyme, lavender, origanum cretici, and cedarwood, aniline oil, and various mixtures of these oils are also used as clearing agents. The majority of these will clear from 95 per cent alcohol, but not so readily as carbol-xylol; they have individual disadvantages of taking out the eosin, affecting aniline colors, dissolving celloidin, making sections brittle, slow action, clinging odor, etc. Chloroform and benzine may be used for clearing osmic-acid preparations; oil of turpentine is also good for clearing sections stained with kresyl-echt-violett, and Wright’s blood-slain. With but few exceptions carbol-xylol and xylol meet all requirements better than any other clearing reagents. There is but one disadvantage in the case of carbol-xylol; some of the phenols in the market cause a fading of eosin and hæmatoxylin stains. DeWitt has shown that this fault can be corrected by redistillation, stopping the distillation as soon as the temperature begins to rise above the constant boiling point of the phenol; or the carbol-xylol that fades the stains can also be corrected by supersaturating it with a mixture of sodium bicarbonate one part, and sodium-potassium tartrate two parts. Sections kept in xylol or carbol-xylol should be protected from dust and evaporation; it is not a good plan to keep them in these solutions for more than 24 hours.
VI. MOUNTING.
Permanent mounts are made in glycerin, potassium acetate, lævulose, glycerin gelatin, balsam, damar or colophonium. For celloidin and paraffin sections a solution of Canada balsam in xylol is most commonly used for mounting. Celloidin sections (celloidin films are best cut into strips and single sections when in the carbol-xylol; the wheel-shaped paper-cutter used by paper-hangers is the best instrument for this purpose) are lifted onto the slide from the clearing-fluid; folds or wrinkles in the celloidin are straightened or removed by cutting the celloidin at right angles to the section in order to relieve the tension, and the section is then blotted firmly against the slide by means of a pad of absorbent paper. The greatest care should be taken to prevent wrinkling, folding or turning over of the edge of the section. As soon as the pad is removed a drop of balsam is placed upon the section and the cover-glass put over it. There should be just enough balsam used to fill the space between cover-slip and slide, so that air-bubbles are not formed. The balsam must not be so thin that the cover-glass will float about on the liquid, or so thick that it does not spread well. In the latter case warming the slide may cause it to spread more readily, but care must be taken not to injure the stain by over-heating. Paraffin sections on the slide are similarly blotted and covered with balsam and cover-glass; those on cover-slips are blotted between folds of absorbent paper and immediately placed with section-side downward upon a drop of balsam that has been put upon the slide.
Xylol-damar may be used in place of Canada balsam; it is cheaper and colorless, but it tends to become cloudy. Colophonium is the cheapest of the three and has but little color; it is highly recommended by many workers. In a xylol-solution it may be used for aniline stains; a chloroform solution is advisable for the mounting of osmic-acid preparations; while a solution in turpentine and shellac is recommended for Weigert’s neuroglia method, Wright’s blood-stain, and other special staining methods.
Glycerin, potassium acetate, laevulose and glycerin-gelatin are used for the preservation of amyloid-, mucin- and fat-stains, as well as for other preparations that do not permit the use of alcohol and xylol. Glycerin-gelatin is probably the best medium for this purpose. It is made according to Kaiser by soaking 7 grms. of gelatin for 2 hours in 42 cc. distilled water, then adding 50 grms. glycerin and 1 grm. carbolic acid; the mixture is warmed 10-15 minutes, stirring constantly, and filtered while hot. It is also made by taking water, 200 cc.; gelatin, 20 grms.; powdered white shellac, 2 grms.; Farrant’s solution (gum arabic, glycerin, solutio acidi arsenicosi conc., aa. 30.0 grms.), dissolve by warming, and filter while warm. To mount in this medium the section is placed on the slide, and blotted with absorbent paper. A drop of warm glycerin-gelatin is then placed on it and the cover-slip affixed. The drop spreads evenly beneath the cover-glass and becomes solid as it cools. Mounts in glycerin, glycerin-gelatin, potassium acetate and laevulose must be cemented around the borders of the cover-slip with asphalt, wax, paraffin, gold size, etc., using a brush or glass rod for this purpose.