Treatment of folded sections.—The folding may be due:—

(1.) To the section having creased through being cut with a knife whose surface was not perfectly smooth. This is best remedied by placing the section in methylated spirit for a minute, and then transferring it to a bowl of clean water, when the section will rapidly rise to the top, and spread itself out flat on the surface of the water, in consequence of the alcohol rapidly diffusing out at the edges into the surrounding water.

(2) To the section containing a large amount of fat, as in those of the skin and subcutaneous tissue. The fat may be removed from the fat cells without materially altering the appearance of the section. This is done by dehydrating the section in alcohol, and then transferring to a watch glass containing ether or chloroform to extract the fat. The tissue should be washed free from ether in the alcohol and then transferred to the bowl of water, and allowed to float out. This process does not interfere with subsequent staining operations.

Mounting Media.

Farrant’s solution:—

In making this solution the best gum arabic must be used, and only the clearest pieces of this. “Powdered gum acacia” should be avoided, as though it looks white it often yields a brown mucilage, and besides is frequently adulterated with starch, &c.

The glycerine and water should be mixed and the gum arabic added. The mixture should be allowed to stand for some weeks, with frequent stirring until the whole of the gum is dissolved. Then allow it to stand for a week or two longer in order that the dirt may subside, and the bubbles rise to the top. The scum should be removed and the clear fluid decanted from the sediment into a “Balsam bottle” (p.58) containing a few drops of a saturated solution of arseniate of sodium and a small lump of camphor.

If properly made it is an extremely useful mounting reagent. It does not clarify the tissues too much, and in consequence of its containing gum it dries at the edges and cements the cover-glass more or less firmly in a week or two. If this is not the case the medium contains too much glycerine and more gum must be added to compensate for this. This drying at the edge prevents any further evaporation while the glycerine keeps the section permanently moist.

The camphor and arseniate of sodium prevent the formation of fungi. Sections preserve their original appearance in this medium for many years. After a long time they are apt to become a little cloudy and granular.

Unstained sections should always be mounted in Farrant’s medium, as the Canada balsam process renders them quite transparent. It is suitable for almost any tissue stained or unstained, but sections of the nervous centres require to be mounted in Canada balsam, owing to the opacity of myelin when mounted in glycerine.

Canada balsam solution:—The medium is made thus:—

The ordinary Canada balsam which is of a treacly consistence is heated gently in a water bath for some hours, to drive off turpentine and other volatile oils. It is then allowed to cool to a yellow vitreous mass. Take of

Leave till dissolved, stirring occasionally.

Unless the solution be perfectly clear, it must be filtered through a very thin paper, previously wetted with xylol. If the medium be too thick more xylol should be added, if too thin, the xylol should be allowed to evaporate until the medium is of the consistence of a thin syrup.

If the medium is made too thin much annoyance will be caused by its evaporating at the edge of the cover-glass, leaving an air-space, which will increase daily until the section is left quite dry. This should be remedied by putting another drop of balsam at the edge of the coverslip and allowing it to run in and displace the air. A ring of cement should be put on as early as possible afterwards.

The bottle in which the balsam is preserved must be very carefully dried before being filled and then rinsed out with absolute alcohol, and afterwards with xylol. Turpentine or benzol are often used instead of xylol in the preparation of the medium, and in the same proportion, but the latter is less apt to dissolve out the aniline colours from the sections.

To mount sections in Canada balsam they must be transferred first to a watch glass containing absolute alcohol or an alcoholic solution of some staining reagent, e.g., eosine (p.72) and left in it, no attempt being made to spread it out, until it is perfectly dehydrated, i.e., in about two minutes. It should then be transferred to the clarifying oil on a mounted needle, or on a section lifter, which must be perfectly dry as any spot of moisture that gets on to the section will resist the clarifying action of the oil, and will cause unsightly opaque areas when the section is mounted. Even breathing on the section on its way to the clarifying agent will prevent uniformity of clearing. Should white spots appear in the section while in the oil it must be taken out with as little oil as possible, and again dehydrated in absolute alcohol.

The process of clarifying must be performed in some medium in which Canada balsam is readily soluble, and which is also readily miscible with alcohol. Those most frequently employed are oil of cloves, xylol, oil of bergamot, oil of cedar, and origanum oil. The first named has always been much used because of its agreeable odour, its cheapness, and the ease with which it can be obtained. But it has the disadvantage of dissolving out many important staining reagents, especially eosine and the various aniline colours. In addition as it dissolves celloidin, sections cut in this medium tend to fall to pieces when transferred to oil of cloves, and one of the other oils (which have no solvent action on celloidin) should always be employed with celloidin sections. Oil of bergamot is the most generally useful, but rather expensive. Where there are special reasons for employing other dehydrating agents, they will be indicated in the special directions for particular staining methods in Chapters VI. and VII.

As soon as the section is plunged into the oil, the alcohol rapidly diffuses out, so that the edges of the section fly out with it, and the section floats quite flat on the surface of the oil. When it is completely clarified (in about a minute), as shown by its sinking in the oil, it should be transferred to the slide by the section lifter, and the oil drained off. Excess of oil may be removed by pressing gently on the section with a flat piece of filter paper folded several times. If carefully performed this manoeuvre will not injure the section, but it requires practice.

If the tissue is very delicate, and likely to be injured by changing from one vessel to another, or if it is larger than the section lifter will conveniently carry, it should be floated out on a glass slide, and, as much water as possible having been removed by blotting paper, should be dehydrated by adding a little alcohol from a pipette once or twice. Most of the alcohol should then be removed by tilting the slide, and before the remainder has evaporated, some oil of cloves or bergamot should be added from another pipette. The section will float on the oil at first, but the latter will gradually come through and appear on the top of the section. When this occurs the clarification is complete, and the oil may be run off by tilting the slide and the section mounted in Canada balsam.

Cementing of cover-glasses.—The cover-glasses may be cemented down to prevent their shifting and spoiling the specimen. If the cover-glass be circular, a Shadbolt’s turntable should be used. It consists simply of a horizontal heavy brass disc, rotating easily on a pivot. There are a number of circles traced on the disc concentrically. The slide is then fixed on the disc by means of the clips, so that the circumference of the cover-glass corresponds to one of the circles. The disc is then rotated and the cement applied to the edge of the cover-glass with a brush.

Many materials are employed. The most suitable are:—(1) Canada balsam, which is almost colourless and transparent and looks very neat. (2) Gold size. (3) Marine glue.

When these are dry a finished appearance may be given to the slide by laying on a ring of zinc white. This is made as follows:—

Preservation of sections.—They should be kept flat, and preserved from both light and dust. Very useful cardboard trays are now sold by almost all dealers in boxes made to contain twenty-four dozen slides for about eight shillings, or suitable cabinets may be constructed by a carpenter.