Common objects of the microscope

by J. G. Wood

Section-Cutting—Staining

No method of examination can equal, for general applicability and usefulness, that of section-work. The relations of the parts to each other being preserved, it is possible to draw conclusions as to their actual relations which no other mode allows of, and we shall devote this concluding chapter to some account of the methods to be employed to this end.

The apparatus required is not necessarily complicated. Reduced to its elements, it consists essentially only of a razor to cut the sections and a dish to receive them. It but seldom happens, however, that the relations of the parts in sufficiently thin sections can be preserved by such a rough-and-ready method, and frequently the object to be cut is of such small dimensions as to render it impossible to deal with it in this way. It is therefore necessary to “imbed” it, so as to obtain a handle by which to hold it, in such a way that it shall be equally supported in all directions. Moreover, since the human hand can only in exceptional cases be brought to such a pitch of skill as to cut a series of sections, or even single ones, of the needful delicacy, some mechanical means of raising the object through a definite distance is highly desirable. The writer has cut many thousands of sections with the “free hand,” but the personal equation is a large one, and is not always the same in the same person. For single sections the method will, with practice, succeed very well, but some means of securing a number of sections of more or less the same thickness is usually required.

Let us deal with the imbedding first.

If it be desired to imbed a tissue which has merely been fixed with formalin, the block should be immersed in strong gum (made by saturating water with picked gum arabic, white and clean) for several days. It is then taken out and, without draining, transferred to the plate of a freezing microtome, and the sections cut from the frozen block, and mounted in glycerine at once.

This plan is of limited usefulness, since it allows of very little differentiation of the tissue elements, and that only optical.

To get the best results, some plan of staining must be adopted. Perhaps the simplest, and certainly a very excellent one, is as follows. After the tissue has been passed from the hardening, or fixing, fluid into the successive alcohols, as described, it is placed in the following solution. Take about forty grains of carmine and eighty grains of borax, dissolve in about an ounce of water, add to the mixture an ounce of methylated spirit, and let it stand for some time with frequent shaking; about a week will be sufficient, and the process of solution may be hastened by gentle warming at intervals. The clear upper portion is then poured off, and into this the block of tissue is dropped, and allowed to remain until thoroughly penetrated. Perhaps the best plan is to substitute the carmine solution for the 50 per cent. alcohol, and thus to make the staining a stage in the hardening process. From the carmine solution the tissue is transferred to 70 per cent. alcohol, to each ounce of which two drops of hydrochloric acid have been added, and after remaining in it for a day (with a piece of the usual size) is placed in 70 per cent. alcohol, in two successive quantities. Sections from this material now only require treatment with the carbolic acid and turpentine above mentioned to be fit for mounting and covering in balsam. We now proceed to indicate how the sections may be cut.

A mixture of wax and almond oil, in proportions varying with the heat of the weather, usually about equal ones, is prepared. The piece of tissue is freed from superfluous spirit by being placed on a bit of blotting-paper for a minute or two, and is then immersed in a quantity of the wax-and-oil mixture contained in a little box of paper or lead-foil. The tissue is held on the point of a needle, and lifted up and down until it is coated with the mixture, and, before solidification of the mass sets in, is lowered into the box and left to cool. The block now furnishes a handle, and this should be wrapped round with paper, the sections cut with the keenest possible razor, and as thin as possible, and placed in spirit as cut. From the spirit, which must be the strongest obtainable, they are placed in the clearing liquid, carbolic and turpentine, and then slid on to the slide, a drop of balsam placed on the section, and the cover over all. Of late years all sections of ordinary soft tissues, animal or vegetable, have been cut by one of the infiltration methods, in which the interstices of the tissue are filled up by some material which prevents the relations of the cells from being altered during the process of cutting. To enter fully into this matter would occupy too much space, and would serve no useful purpose, for the worker who requires to make use of such means will find it indispensable to obtain Bolles Lee’s Microtomist’s Vade Mecum, in which the whole matter is exhaustively treated.

The simple method above detailed will answer most ordinary purposes, provided that a few precautions be attended to. The chief are as follows. The outside of the block of tissue must be sufficiently dry for the wax-and-oil to adhere to it. The razor must be extremely sharp, and must be kept so by application to a Turkey stone during the section-cutting. The blade must be drawn across the tissue from heel to point, and kept wetted with spirit the whole time, so as to prevent any dragging of the section. The transference of the section to the slide must be effected by means of a section-lifter, which may be made by beating out a piece of stout copper wire to a thin flat blade; or a small palette-knife, or German-silver lifter, may be purchased for a few pence. The carbolic turpentine is best used by placing a little in a watch-glass, and floating the sections on to it by lifting them singly with the lifter, freeing them from superfluous spirit by draining on to blotting-paper, and allowing them to float on to the surface of the liquid in the watch-glass, so that the spirit may evaporate from above, and be replaced by the clearing agent from below The balsam solution should be thin, and the cover-glass must be allowed to settle down into place without pressure.

The question of staining sections is a very large one, and is becoming of daily increasing complexity.