3. On Cutting unprepared Animal Tissues.—For the cutting of fresh animal tissues several plans may be followed. Thus, if a section of only very limited area be required, it may be obtained by snipping a piece off the tissue with a pair of bent scissors, which, for this purpose, are so made that the blades are curved on the flat (Carpenter). If this be carefully performed it will be found that a large portion of the section (particularly at the circumference) so obtained will be sufficiently thin for examination. If a larger section be desired, an attempt may be made to cut it with a very sharp scalpel or razor, the blade of which whilst in use must be kept flooded with water, or spirit, the latter of which is to be preferred. Recourse may also be had to Valentin’s knife. This consists of two long, narrow blades, running parallel to each other, the distance at which the blades are held apart, and which, of course, determines the thickness of the section, being regulated by means of a fine screw passing through both blades. A milled head attached to this screw gives a ready means of opening or closing the blades, so as to bring them to the desired degree of approximation. The method of using the knife is very simple. After having “set” the blades at the desired distance apart by means of the milled head, the tissue to be cut is held in the left hand immersed in a basin of water. The knife is now steadily and with a rapid motion drawn through the tissue, care being taken that the cut is made in such a manner that the blades move from heel to point. By slightly separating the blades and gently shaking them in the water, the section at once becomes disengaged. After use, the blades must be thoroughly dried, when they may be smeared with some oil which does not readily oxidize. For this purpose, a very suitable oil is that known as “Rangoon.”[[1]] Though it has been deemed advisable briefly to describe the preceding methods of cutting unhardened tissues, it will be found that for the purposes of the ordinary microscopical student sections so obtained are of very little value. They are always of very limited dimensions, seldom of uniform thickness, and often so extremely friable as to render it very difficult and frequently impossible to submit them with safety to such further treatment as is necessary to fit them for being mounted as permanent objects. This method of section-cutting, however, is not without its uses, for by its means the medical practitioner is provided with a simple and ready method of roughly investigating the structure of morbid tissues, whilst to the general student it furnishes an easy means of making a cursory examination of certain substances, in order that he may determine whether it be worth his while to subject them to some of those various processes of hardening hereafter to be described.
4. Preparation of Vegetable Tissues.—Let us now pass to a brief consideration of the methods usually adopted for preparing the various objects for easy section. In the case of vegetable tissues, not only do we, as a rule, find their texture of too great density to be readily cut in their natural condition, but they also contain much resinous and starchy matter, of which it is highly desirable to get rid. In order to do this we first cut the substance (say a stem or root) into small pieces, which are to be placed in water for three or four days, by which time all the soluble gummy matters will have disappeared. The pieces are now transferred to a wide-necked bottle, containing methylated spirit,[[2]] which, in the course of a few days, will dissolve out all the resin, etc. Many kinds of woody tissue are by these processes reduced to a fit condition for immediate cutting; others, however, are so hard as to render it necessary to give them another soaking for some hours in water, to bring them to a sufficient degree of softness to cut easily. If the wood (as in some few refractory cases will happen) be still too hard for section, a short immersion in warm, or if necessary, in boiling water, will not fail effectually to soften it. The treatment of such members of the vegetable division as require peculiar methods, will be found described in future pages.
5. Preparation of Animal Tissues.—Animal tissues differ from one another so greatly, both in consistence and in chemical composition, as well as in their degree of natural hardness, that no general rules can be given which would be applicable to the preparation of the whole class. Such as are of any considerable degree of hardness, as horn and kindred structures, must be treated much in the same manner as the denser varieties of wood, viz., by more or less prolonged immersion in water—cold, hot, or boiling. Those which are of extreme hardness, as bones and teeth, can be cut only by following certain special methods, full details of which will be found in the Second Part of this work (§ 26). Many, and indeed the vast majority of animal tissues, offer a direct contrast in point of hardness to those we have just been considering. All the internal organs of the body are, when freshly removed, of much too soft a nature to permit, when in their unprepared condition, of easy or perfect cutting. It is upon bringing them to that critical degree of hardness, which is often so difficult to attain, that the chief secret of successful section-cutting depends; for unless the hardening process has been carried up to, but not beyond, a given point, which varies with different tissues, the operator, however dexterous, will fail to obtain satisfactory sections. For, if the hardening has fallen short of this critical point, he is, to some extent, in the same position as if he were dealing with unhardened tissues; whilst, if this point has been exceeded, the tissue will have become so brittle as to crumble before the knife. For the purpose of hardening animal tissues, the student has at his command two principal agents, namely, alcohol and chromic acid, each of which possesses advantages of its own, but the use of each of which is also attended by its own inconveniences. Thus, by the use of alcohol, there is very much less risk of overhardening the specimen than if chromic acid had been employed. Alcohol, however, though a capital indurating agent in some instances, does not answer so well in many others. Chromic acid is, therefore, to be preferred for general use. It is, however, a very delicate agent to manage, for unless the greatest care be taken it is exceedingly likely to overharden tissues submitted to its action, and when this happens the specimen becomes utterly useless for cutting, as there is no known means of removing the extreme brittleness which it has acquired. By taking the precautions now to be given, this overhardening may generally be avoided. Let us harden a portion of some viscus, say the kidney, for instance. Suppose we cut from the organ five or six small pieces (from half to three-quarters of an inch square, not larger). These must be placed in a mixture of equal parts of methylated spirit and water for three days, at the end of which period they may be transferred to a solution of chromic acid, made by dissolving twenty grains of the pure acid in sixteen ounces of distilled water. The solution should be kept in a wide-necked bottle furnished with a glass stopper. At the expiration of seven days, pour off the solution and replace it by fresh. At the end of another week, carefully examine the immersed tissues, and by means of a sharp razor see if they have acquired the necessary degree of hardness to allow of a section of moderate thinness being made. If so, remove the pieces and put them into a stoppered bottle containing from six to eight ounces of methylated spirit. If, however, the hardening be found not to be sufficiently advanced, the chromic acid solution is to be poured off and again replaced by fresh. It will now be necessary to examine the tissues at intervals of about two days, until they are found to be sufficiently hard, when they must be transferred to the spirit. Under no circumstances, however, should they be permitted to remain in the chromic acid longer than the end of the third week, and though they should at this time appear not to have undergone sufficient induration, yet it will be advisable to transfer them to the methylated spirit, which in a short time will safely complete the process of hardening, without any risk being run of the tissue becoming ruinously brittle. It will be noticed, that when the specimens have been transferred to spirit, the latter will in a day or two become of a deep yellow color, whilst a thick flocculent deposit falls to the bottom of the bottle. The tissues should then be removed, the bottle emptied and well washed, and, being refilled with clean spirit, the preparations are again to be replaced. This may occasionally be repeated, until the spirit becomes and remains perfectly bright and clear. The specimens are then ready for section.
6. Special Methods of Hardening.—The brain (§ 27), spinal cord (§ 43), liver (§ 34), and several other organs, etc., require special methods of hardening, details of which will be found in the paragraph devoted to each. In the case of injected preparations, the best plan is to harden them in alcohol from the outset, beginning with weak spirit, and gradually increasing the strength as the hardening proceeds. When the object has been injected with Prussian blue, a few drops of hydrochloric acid should be added to the alcohol to fix the color.
It may here be observed, that specimens of morbid tissues require, as a general rule, a shorter immersion in chromic acid solution than healthy tissues do. A very small degree of overhardening speedily renders them brittle and useless. They should, therefore, be removed from the acid medium at the end of ten days or a fortnight, and their further hardening carried on by means of alcohol.
7. Cutting Hardened Tissues by Hand.—Our material being now reduced to a fit condition for cutting, let us proceed to consider the several methods by which this may be effected. The readiest and most simple plan, if the piece be large enough, is to hold it in the left hand, and, having brought the surface to a perfect level by cutting off several rather thick slices, endeavor to cut a thin section by the aid of a very sharp razor, the blade of which must be kept well flooded with spirit. As in the use of Valentin’s knife, so here, great care must be taken steadily to draw the blade across the tissue, every effort being made to avoid pushing the knife, else the section will be torn off, instead of being cut. Though this method[[3]] is of very great importance for many purposes, yet a considerable degree of manipulative skill is required to enable the operator to obtain anything like perfect sections by its means, and, unfortunately, this skill is acquired by very few persons indeed, even after much practice. If the piece which it is desired to cut be too small to be conveniently held in the hand, it may be imbedded in paraffine in the manner already described (§ 3). A very simple imbedding agent, and one of the greatest practical value, is a strong solution of gum arabic, which, upon being dehydrated either by ordinary drying or the action of alcohol, soon acquires such a degree of hardness as to permit it (with the imbedded tissue) to be cut with ease. As this method of imbedding, however, is most frequently resorted to where, by its means, special difficulties have to be overcome, a full description of the process (§ 35) will be deferred until such special cases come to be spoken of.
8. Microtome.—Although the preceding plans may be sufficient to answer all his requirements, if the student wishes to obtain only one or two sections of small dimensions, of a given object, if he requires a number of such sections he will find these methods fail him, for even though by practice he may have attained to considerable aptitude in the use of the knife, it will still unquestionably happen that the vast majority of his sections will be more or less imperfect. If, therefore, it be desired to procure a number of perfect sections, of equable thickness and large area, it is absolutely necessary to resort to the use of some form or other of microtome, or section-cutter. This instrument, in its simplest form, merely consists of a stout brass tube closed at one end, and being by the other fixed at right angles into a smooth plate of metal. A plug or disk of brass, accurately fitting the interior of the tube, is acted upon by a fine threaded screw piercing the base of the tube, and by means of which the plug, and any object it may support, can be elevated at pleasure. The object by this means being made gradually to rise out of the tube, sections are cut from it by simply gliding a sharp knife along the smooth cutting-plate, and hence across the specimen. Any intelligent worker in brass would make an instrument of this kind at a very small cost, and although perhaps it might lack the finish of an instrument bought at the optician’s, it would, if accurately made, do its work as well as the most complicated and expensive. If, however, the student resolves to purchase a microtome, there are a variety of forms in the market from which he may choose. A few hints may perhaps be of service in enabling him to make a judicious selection. At the outset we may say that unless the student intends to devote himself solely to the production of sections of wood, etc., he ought not to procure one of those forms of microtome known as wood section-cutters, in which the object to be cut is held in position in the tube by means of a binding screw which pierces its side.[[4]] Although these machines are all that can be desired for cutting hard bodies, they are not so suitable for soft ones. The chief points to be attended to in selecting a microtome are, (1) that the cutting-plate of the instrument be made of glass, or in default of this, of very hard metal of the most perfect smoothness;[[5]] (2) that the diameter of the tube be neither too large nor too small—it ought not to be less than 5/8-in., or greater than 1 inch;[[6]] (3) that the screw, which should be fine and well cut, be provided with a graduated head; (4) that there be some kind of index by which fractional portions of a revolution of the screw may be measured; and (5) that the plug fit the tube of the microtome so accurately that when melted paraffine, gum, or other imbedding agent be poured into it, it may not find its way between the plug and side of the tube (§ 18). It often happens in cutting tissues imbedded in paraffine, that the pressure of the knife causes the cylinder of the imbedding agent to twist round in the tube of the machine, and so cause considerable difficulty and annoyance. This evil is usually met by running a deep groove across the upper surface of the plug, and into this the paraffine sinks, and so is prevented from rotating. It will be found, moreover, that another difficulty of a kindred, though much more serious character, will frequently be encountered. During section the paraffine has a tendency not only to rotate, but also to become loosened from the subjacent plug, and to rise in the tube of the microtome. When this happens the power to cut sections of uniform thickness has completely gone, for some will now be found to be many times thicker than others; in fact, the irregularity in this respect soon becomes so monstrous as to render it useless to prolong the sitting. In the ordinary run of microtomes no provision seems to have been made to meet this difficulty, and for this reason many instruments, of otherwise great merit, have their efficiency seriously impaired. Fortunately, this imperfection is easily remedied, all that is required being that the upper surface of the plug should be furnished with some kind of projection, having at its summit a table-like expansion, as shown at A in the figure. The imbedding paraffine, by penetrating beneath and around this, becomes firmly attached to the plug, and thus all risk of its rising is effectually avoided. If the student wishes to secure a really first-class instrument, none can be so confidently recommended as the freezing microtome of Professor Rutherford. In addition to its being the best instrument for carrying out the freezing method (§ 18), this machine is equally effective for cutting tissues imbedded in paraffine, or any of the other agents used for that purpose; indeed, whatever work a microtome can do, this one will perform.