12. Employment of Microtome.—The paraffine being sufficiently hard, we will clamp the microtome on to the table, and seat ourselves on a chair of convenient height before it. To our right stand the basin of water, razor, and section-knife; the beaker of spirit to the left, and a cloth on our knee. A few turns of the microtome screw having brought the paraffine to the surface, a thick slice is to be cut off, and this repeated until the imbedded tissue comes into view. This preliminary work had best be done with the razor, as it is needless to subject our section-knife to unnecessary wear and tear. By a fractional revolution of the screw the tissue is now slightly elevated, and with the pipette held in the left hand, a large drop of spirit is to be let fall upon its surface. The section-knife, grasped firmly but lightly in the right hand, is to be laid flat upon the cutting-plate of the machine, so as to occupy the diagonal position shown in the figure. Two fingers of the left hand are now laid gently upon the back of the blade, so as to give it an equable support, whilst the knife with a rapid motion is pushed in the combined direction of forwards and to the left, so that the blade in cutting the tissue will pass through it from point to heel. Thus it will be observed that the stroke of the knife is from the operator—a far easier and more effective mode of cutting than the reverse plan. The blade of the knife, having the section just cut, either floating in a small pool of spirit on its surface or adhering thereto, must now be immersed in the basin of water, when by a little very gentle agitation of the knife the section will be floated off. And now we shall find the great practical value of immersing the tissue in gum before imbedding, for no sooner is the section disengaged from the knife than the thin film of gum which separates the paraffine from it becomes dissolved, and the section will be observed gradually to subside to the bottom, leaving the paraffine floating upon the surface. After carefully wiping the knife from all shreds of paraffine, the microtome screw must again be partially revolved, more spirit applied to the tissue, and another section being cut, it must be transferred to the water as before, and so on, until a sufficient number of sections have been obtained. As to how thin the sections should be cut, no general directions can be given; each case must be regulated by its own conditions. The denser the tissue, the thinner should the section be; whilst certain substances of loose and spongy texture do not require the sections to be particularly thin—it may be said, however, in a general way that sections, and especially animal ones, cannot be cut too thin so long as they remain perfect and entire. If Professor Rutherford’s microtome (as made by Gardner) be employed, the head of the screw will be found to be graduated into divisions of slightly unequal value; the sections will therefore be marked by corresponding variations of thickness, so that amongst a number cut, there must be many of the exact thickness to meet the requirements of any individual case.
DIAGRAM SHOWING DIAGONAL POSITION OF KNIFE IN COMMENCING TO MAKE A SECTION.
13. Staining Agents.—Before proceeding to mount the sections which have just been cut, it will be very advisable that they should be submitted to the action of some staining fluid, in order to render more clear and distinct their minute structure. Organic substances possess the property of being able to absorb various colouring matters from their solution, and to incorporate such colour into their own texture. This power of attraction is not, however, possessed by all substances indiscriminately, or to an equal extent. Some possess it in a high degree, while others appear to be nearly, if not entirely, devoid of such power. Hence it follows, that if we immerse an organic tissue (one of our sections, for instance) of complex structure, in a suitable staining fluid, the tissue will not become stained in an even and uniform manner throughout, but the several portions of it will receive varying depths of colour in accordance with the varying attractive power of its several constituents. By this means we are enabled in stained sections to discriminate by their difference of shade, minute and delicate structures, which in the unstained condition it would be difficult and often impossible to differentiate. For the purpose of section-staining there are many agents in use, the most generally suitable being carmine, logwood (§ 19), and aniline blue (§ 27); whilst for special purposes chloride of gold (§ 28), pyrogallate of iron (§ 28), and several others are all of much value.
14. Carmine Staining.—In the case of animal sections, carmine is, as a rule, to be selected, giving as it does most satisfactory and beautiful results. Tissues may be stained with carmine by two different plans: in the first, a strong solution is used, and the tissue subjected to its action for a very short period only, whilst in the latter only very weak solutions are employed, the time of immersion being considerably prolonged. The rapid method, however, is not to be recommended, for the strong carmine acts so powerfully upon the tissue as to give the various elements comprising it no time, as it were, to exercise their power of quantitive selection, but involves the whole in one uniform degree of shadeless colour. By adopting the gradual method much better results are obtained, each portion of the tissue being now at liberty to acquire its own particular shade. Amongst the various formulæ for the preparation of carmine fluid, none can be so safely followed as that devised by Dr. Lionel Beale. It runs thus:—Place ten grains of the finest carmine in a test tube, add thirty minims of strong liquor ammonia, boil, add two ounces of distilled water, and filter; then add two ounces of glycerine, and half an ounce of rectified spirit—this solution ought to be kept in a well stoppered bottle. The best vessels in which to stain sections are small jars of white porcelain, capable of holding about two fluid ounces, and furnished with lids—they are much preferable to beakers or watch glasses, for owing to the white background which they afford it is very easy to watch how the staining is proceeding. The carmine solution which we have just described is both too strong and of too great density to be used in its pure state. It will, therefore, require to be diluted with distilled water before use—the most useful degree of dilution being attained by adding one part of stain to seven of water. Sections may be placed in this solution for twenty-four hours, in which time they will usually be found to have acquired a sufficient depth of colour. If, however, the tissue be unusually difficult to stain, the time of immersion may be doubled, or still further prolonged, without detriment to the section.
SECTION SPOON.
Having prepared and filtered some of this dilute solution, say an ounce, let us proceed to stain with it those sections which we left in the basin of water (§ 12). Here we are at once met by a practical difficulty. How are the sections to be transferred from one vessel to the other? This is ordinarily effected by means of a soft camel’s-hair pencil. It is a method, however, open to grave objections, for the sections so curl around the brush, and get entangled amidst its hairs, that, notwithstanding every care, valuable sections not unfrequently become torn during transit. Every difficulty at once vanishes if we substitute for the brush a small implement, which any one can readily make for himself. All that is necessary is to take a strip of German-silver, or copper, of the thickness of stout cardboard, and about seven inches in length by five-eighths of an inch in breadth. The sharp angles are to be filed off and the edges carefully smoothed, whilst at a distance of five-eighths of an inch from each extremity the end must be turned up so as to form an angle of about 35°. One end must be left plain, whilst the other, with the aid of a punch or drill, is to be pierced with five holes about the thickness of a stocking needle[[8]] (see Figure). If we now dip the perforated end of this spoon into the water containing the sections, and gently agitate it, the sections will rise from the bottom and float about. The spoon is now brought under one of them, and being steadily lifted up the water flows downwards through its apertures, and the section smoothly spreading itself out upon the spoon, may be gently lifted out of the water, and on the spoon being dipped into the staining fluid the section at once floats off. By this simple means sections, however large, thin or delicate, may with ease be conveyed from one fluid to another, with the utmost certainty of their not being injured during the process. The sections having been in the carmine fluid for about twenty-four hours, as much of the liquor as is possible must be gently poured off, and its place supplied by a freshly-filtered mixture of five drops of glacial-acetic acid to one ounce of water, when in a few moments the carmine will become permanently fixed in the tissue, and the process of staining be complete.
[8]. Dr. Klein describes a kind of “lifter,” made by bending some German-silver wire, but as no drawing accompanies his description, it is not easy to form a clear idea as to the form of this instrument. In the recent and philosophical work of Schäfer, a lifter is figured, which consists of a wire stem, having attached to its end a spade-like blade. It will be observed that the spoon described in the text differs from this lifter in having one end perforated, and in this consists the real value of the implement.
15. Mounting Media.—The further treatment of the stained sections will entirely depend upon the nature of the medium in which it is intended to mount them. There are a variety of fluids in use for this purpose, the principal being dilute alcohol (§ 26), dammar, or Canada balsam (§ 22-23), and glycerine. These, however, cannot be used indiscriminately, each possessing certain special properties which render it suitable for use with particular classes of objects only. Thus, weak spirit, having no tendency to increase the transparency of objects, can advantageously be used with such only as are already perfectly transparent. It is also more suitable for the preservation of vegetable tissues (when the retention of colour is no object) than animal, since with the latter it has a tendency after a while to cause a kind of granular disintegration, which ultimately destroys much of the usefulness of the preparation. Dammar and Canada balsam, on the other hand, possess very great refractive power, so that they are of great service in mounting objects which require their transparency to be much increased. For this reason they are not well adapted to the preservation of very delicate or transparent tissues (unless previously stained), the minute details of which become almost entirely obliterated when mounted in them. The chief advantage possessed by these resinous media is, that tissues mounted in them undergo no alteration, even after the lapse of many years. Glycerine, in respect of its clarifying powers, occupies an intermediate position between spirit and balsam, being much more refractive than the former, infinitely less so than the latter. It is, therefore, of very great value for the preservation of such tissues as possess a medium degree of transparency, and which would become obscured if mounted in spirit, or have their outlines rendered indistinct if preserved in balsam. It is of the utmost value for mounting unstained anatomical sections which, when put up in this medium, reveal such minute details of structure as would readily have escaped observation had any other agent been employed. It may also be used with stained sections, but in this case the sections should be of extreme thinness, otherwise the refractive power of the glycerine will be insufficient to render them thoroughly transparent. The great drawback to the use of glycerine is the extreme difficulty experienced in preventing its escape from beneath the covering glass, for it unfortunately possesses such great penetrating power that no cement hitherto devised can be thoroughly depended upon for withstanding its solvent action for any considerable length of time.[[9]] Attention to the instructions presently to be given (§ 16) will, however, reduce this risk of leakage to a minimum. In the use of glycerine Dr. Carpenter’s caution must ever be borne in mind, viz., that, as carbonate of lime is in time dissolved by glycerine, this agent ought never to be employed for the preservation of objects containing such salt.