PART II.
25. Special Methods.—Having in the preceding pages entered at some length into the general subject of section-cutting, it remains for us now to consider those special methods of preparation which the peculiarities of certain objects demand. In order to keep the bulk (and consequent price) of this manualette within due bounds, we shall, without further preface, proceed to the description of these methods, in doing which every endeavour will be made to employ such brevity of expression as may be consistent with perfect clearness of meaning. As the most convenient plan, the objects here treated of will be arranged in alphabetical succession.
26. Bone.—Both transverse and longitudinal sections should be prepared, the former being the prettier and most interesting. After prolonged maceration in water, all fat, etc., must be removed and the bone dried, when as thin a slice as possible is to be cut off in the desired direction, by means of a very fine saw. If the section so obtained be placed upon a piece of smooth cork it may, with the aid of a fine file and the exercise of care, be further reduced in thickness. It is then to be laid upon a hone moistened with water, and being pressed gently and evenly down upon it with the tip of the finger (protected, if necessary, by a bit of cork or gutta-percha), it must be rubbed upon the stone until the desired degree of thinness has been attained. Finally, in order to remove scratches and to polish the section, it should be rubbed upon a dry hone of very fine texture, or upon a strop charged with putty-powder. After careful washing in several waters the section must be allowed thoroughly to dry, when it may be mounted by the dry method in the following manner:—A ring of gold-size must, by means of the turn-table, be drawn in the centre of a slide, and the slide put away in a warm place for several days (the longer the better), in order that the ring may become perfectly dry and hard. When this has been accomplished the section is to be placed in the centre of the ring, and a covering circle of the requisite size having been cleaned, this must have a thin ring of gold-size applied round its margin. The cover is now to be placed in position and gently pressed down, a spring clip being employed, if necessary, to prevent it from moving. In about twenty-four hours another layer of the varnish should be applied, and the slide afterwards finished in the manner already described (§ 24). The above method is also applicable to the preparation of sections of teeth and also of fruit-stones and other hard bodies, which are incapable of being rendered soft enough for cutting.
As the process just described, however, is both troublesome and tedious, it is much better for ordinary purposes to have recourse to the decalcifying method, by which means sections in every way suitable for the examination of the essential structure of bone may be obtained with ease. To carry out this plan a piece of fresh bone should be cut into small pieces and placed in a solution made by dissolving 15 grains of pure chromic acid in 7 ounces of distilled water, to which 30 minims of nitric acid s. g. 1.420 are afterwards to be added. Here they should remain for three or four weeks, or until the bone has become sufficiently soft to cut easily, the fluid being repeatedly changed during the process. From this solution they must be transferred to methylated spirit for a few days, when a piece may be selected, imbedded in paraffine, and cut in the microtome (§ 12). Some of the sections should be mounted, unstained, in spirit. For this purpose a cell of gold-size, as above described, must first be prepared and filled full of a mixture of spirit of wine one part, and distilled water three parts. Into this the section must be carefully placed and the cover applied, the same precautions for the exclusion of air-bubbles being taken which were recommended when speaking of mounting in glycerine (§ 16). When the cover is in position a ring of gold-size must be laid on, repeated when dry, and the slide afterwards finished in the ordinary manner. It will also be advisable to stain some of the sections with carmine (§ 14), or picro-carmine (§ 42), and mount them in glycerine. Teeth may also be treated by the decalcifying method, but in this case it must be remembered that the enamel will dissolve away.
27. Brain.—The best hardening fluid is that recommended by Rutherford, and is made by dissolving 15 grains of pure chromic acid and 31 grains of crystalized bichromate of potash in 43 ounces of distilled water. Small pieces of brain, which have previously been immersed for twenty-four hours in rectified spirit, should be placed in about a pint of this solution, where they must remain for five or six weeks, the fluid being repeatedly changed during the process. If by this time they are not sufficiently hard the induration must be completed in alcohol. Sections are easily cut in the microtome by the paraffine method (§ 12). These may advantageously be stained in a solution of aniline blue, made by dissolving 1-1/2 grain of aniline blue in 10 ounces of distilled water, and adding 1 drachm of rectified spirit (Frey). As this stain acts very rapidly two or three minutes’ immersion will generally be found long enough. The sections must then be mounted in balsam (§ 23).
28. Cartilage.—The method to be employed in the preparation of cartilage will entirely depend upon the nature of the staining agent, to the action of which the sections are to be submitted. Thus, if the elegant gold method is to be followed, it is necessary that the cartilage should be perfectly fresh; whilst if any of the other staining agents are to be employed the tissue may have been previously preserved in alcohol. An excellent object on which to demonstrate the gold process is to be found in the articular cartilage of bone. It is a very easy matter to obtain from the butchers the foot of a sheep which has just been killed. The joint is to be opened, and the bones dissociated, when they will be seen to have their extremities coated with a white glistening membrane—this is the articular cartilage. Exceedingly thin slices must be at once cut from it, and as only small sections are required, a sharp razor may be used for the purpose, the blade being either dry or simply wetted with distilled water. The sections as cut are to be transferred to a small quantity of a half per cent. solution of chloride of gold in a watch glass. Chloride of gold may be purchased in small glass tubes hermetically sealed, each tube containing 15 grains, and costing about 2s. If, however, the student requires only a small quantity of the staining fluid he need not be even at this small expense, for as photographers for the requirements of their art always keep on hand a standard solution of chloride of gold of the strength of one per cent., a little of this may readily be obtained, and diluted to the required degree. After the sections have been exposed to the action of the staining fluid for about ten minutes they may be transferred to a small beaker of distilled water, and exposed to diffused light for about twenty-four hours, when they must be mounted in glycerine (§ 16).
Sections of cartilage may also be examined, without being stained, in which case the field of the microscope should be only very feebly illuminated. Or carmine staining (§ 14) may be resorted to—these sections show well in glycerine, or if the staining be made very deep, even Canada balsam may be employed, and with fair results.
Microscopists are indebted to Dr. Frances Elizabeth Hoggan for the description of a new method of staining, which we have found especially suited to the treatment of cartilage. The agent employed is iron, and the process, which is very simple, is as follows. Two fluids are necessary—(1) tincture of steel; (2) a two per cent. solution of pyrogallic acid in alcohol. A little of the former is to be poured into a watch glass, and into this the sections, after having been previously steeped in alcohol for a few minutes, are to be placed. In about two minutes the iron solution is to be poured away and replaced by solution No. 2. In the course of a minute or two the desired depth of colour will have been produced, when the sections are to be removed, washed in distilled water, and mounted in glycerine. The results obtained by this process are very beautiful, the colour produced being a very fine neutral tint, of delightful softness. The process also answers admirably in the case of morbid tissues, and we have now in our possession some sections of ulcerated cartilage tinged by the iron method, in which the minute changes resulting from the ulcerative disintegration are brought out with wonderful distinctness.
As the structure of cartilage differs according to its purpose and situation, the student will find his time profitably employed in a careful examination of the following forms (α) hyaline—articular and costal; (β) yellow fibro-cartilage—epiglottis, or external ear; (γ) cellular—ear of mouse. Sections of the intervertebral ligaments should also be made, in which the different kinds of cartilage may be examined side by side with each other.
29. Coffee Berry affords sections of great beauty. The unroasted berry should be soaked for hours or days in cold water until sufficiently soft; then imbedded in paraffine, and cut in the microtome (§ 12), the section being made in the direction of the long axis of the berry. Put up in glycerine, or stain rather strongly with carmine, and mount in balsam. The same method of treatment may also be applied to other hard berries or seeds.