1. Introduction.—Many of the most interesting objects with which the microscopist has to deal, cannot be made to reveal their beauty or minute structure until they have been cut into slices or sections, of such a degree of thinness as to render them transparent, and thus permit of their examination by transmitted light, with objectives of varying power. Unfortunately, however, very few of the objects of this class are, in their natural condition, in a suitable state to be submitted to this method of procedure. Some are of such a soft and yielding nature that any attempt to cut them is an utter failure, for in place of a perfect section being obtained, nothing remains upon the knife but a mass of diffluent pulp; others, again, are of such density as to resist the action of any cutting instrument.

It is evident, therefore, that nothing can be done with such refractory materials until, by subjection to appropriate methods of preparation, they shall have been reduced to such a consistence as to render them suitable for cutting. How this is to be accomplished will depend entirely upon the physical and chemical nature of the substance to be operated upon. As the various objects differ so widely from each other in these respects, so must the methods of preparation suitable to each also vary. It is clear, therefore, that no general directions for attaining this end can be given which would be of any practical value. It is possible, however, and very convenient, to arrange the various objects into groups or classes, to the treatment of each of which certain general rules are applicable; but there will still remain a comparatively numerous series of objects whose individual peculiarities of structure will demand for them correspondingly special methods of preparation. When such objects come to be spoken of, the particular treatment most suitable to each will also be noticed.

2. On Cutting unprepared Vegetable Tissues.—There are some few substances, however, which may with more or less success be cut into sections whilst in their natural condition. Such objects are to be found in the vegetable world in certain kinds of leaves and allied structures, whilst in the animal kingdom they are principally represented by the various internal organs of man and the lower animals. Special directions are given in text-books for the preparation of sections of leaves and similar substances. For instance, it is recommended to lay the leaf, etc., on a piece of fine cork, and with a sharp knife to shave off thin slices, cutting down upon the cork. Another plan is to place the leaf, etc., between two thin layers of cork, and cut through the mass. No method, however, is at once so simple and successful as the process of imbedding in paraffine. To do this, it is necessary to make a paper mould by twisting a strip of stout writing paper round a ruler, and turning-in the paper over the end of the ruler. This mould, the height of which may vary from an inch to an inch and a half, should now be about half filled with melted paraffine mixture (§ 11), the leaf or other object plunged into it, and held in position by small forceps till the paraffine has become sufficiently solidified to yield it a support. More of the paraffine mixture is now poured in until the specimen is thoroughly imbedded; the whole is to be put away in a cold place for an hour or so, when the mass will be found sufficiently firm to be cut with ease. Sections may be made with a razor kept constantly wetted with water, or, if the preservation of colour be no object, methylated spirit may be employed for the purpose. As the subsequent treatment of such sections in no wise differs from that required by those cut in the microtome, we shall defer its consideration until that method of section has been described (§ 12).

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.

9. Æther Microtome.—A word here as to freezing microtomes, where the agent used is Æther. Such as have fallen under our notice have not answered the expectations we were justly entitled to form of them. That it is possible to freeze a piece of tissue by their use is undeniable, but it is, as a rule, at an expenditure of such a quantity of Æther (only the very best of which must be used) as to constitute it a very expensive proceeding. Another serious disadvantage they possess is, that if the supply of Æther be intermitted for only a very short time, the already frozen tissue thaws with great and most inconvenient rapidity.

10. Section-Knife.—Of not less importance than the microtome is the section-knife, to be used in conjunction with it. How perfect soever the former, and whatever the dexterity of the operator, unless he be provided with a suitable and well-made knife, he will never succeed in obtaining satisfactory results. As to the most desirable size of the knife, much difference of opinion seems to exist, section knives varying in this respect from a blade of extreme shortness to one which fell under our observation, in which the portentous length of thirteen inches was attained. What advantages were to be expected by prolonging the blade to this extravagant length, must remain an inscrutable mystery to all save its designer. Concerning the shape of the knife, it is frequently advised that the surface which has to glide along the cutting-plate of the microtome should be ground flat. A most unsuitable arrangement, as a very little actual experience of section-cutting will speedily demonstrate. After many unsuccessful attempts to obtain a really good and reliable section-knife, we determined to have one specially made, which, as it has proved everything that could be desired, merits a brief description. It is of the utmost importance that the blade be made of good and well-tempered steel, not only that it may be capable of receiving an edge of the most exquisite keenness, but also that it may retain it. The knife of which we speak (and which was made by Mr. Gardner, of South Bridge, Edinburgh) is furnished with a blade four inches long, and 7/8-inch broad, set into a square handle of boxwood, also four inches in length. The thickness of the blade at the back is not quite 1/4-inch, while both of its surfaces are slightly hollow ground. It is essentially necessary that the back and edge of the blade be strictly parallel to each other, otherwise the knife, when in use, will have such a tendency to tilt over as to render its management extremely difficult. It is very easy to discover if this condition be fulfilled, for if on carefully laying the flat of the blade upon a piece of level glass, every portion of both back and edge are found to be in close contact with it, the knife may in this respect be considered perfect. Every student who aspires to be a successful section-cutter should provide himself with a good Turkey oilstone, and learn to use it. He should also possess a razor strop, as it will be in constant requisition.^[7] It may here be remarked that though razors, as a rule, are unsuitable for use with the microtome from want of uniformity in the thickness of their blades, yet, if only a small object is to be cut—for instance, a thin root or stem—very good results may be obtained from their use, especially if one of the old-fashioned make, having a thick back and slightly concave surfaces, be employed.

11. Imbedding in Paraffine for Microtome.—Having described at some length the various instruments necessary for section-cutting, we will now consider how they are to be used. Let us endeavor to cut some sections—say of a piece of kidney—and in so doing we will adopt the “paraffine” method of imbedding. Ordinary paraffine, however, when used alone, is rather too hard for our purpose. In order, therefore, to bring it to a suitable consistence, it must be mixed with one-fifth its weight of common unsalted lard, a gentle heat applied, and the two thoroughly stirred together. A quantity of this should be prepared, so that it may always be ready when wanted—it is very conveniently kept in an ointment pot or preserve jar, the top of the latter being well covered, to keep out the dust. When it is intended to use this mixture for the purpose of imbedding, only just about the quantity required should be melted; for in doing this it is advisable to use as low a degree of heat as possible, not only to prevent injury to the tissue to be imbedded, but also that the paraffine when cooling may not undergo such an amount of contraction as to cause it to shrink from the sides of the microtome-tube. It is therefore a good plan to effect the melting in a water-bath, a simple kind of which, something after the fashion of a glue-pot, would be made for a few pence by any tinman.

The kidney which we are about to cut has, of course, gone through the process of hardening already described (§ 5), and is now preserved in spirit. A small piece, say half an inch square, is selected, removed with forceps, and placed on a bit of blotting paper, when the surface of the tissue will rapidly become dry (only the surface must be allowed to dry). It is the usual plan now to proceed at once to imbed it in the melted paraffine. This is a most undesirable step, and gives rise at a later stage of our proceedings to a great amount of trouble and annoyance, for after sections have been cut from a tissue so imbedded it will be found that portions of paraffine adhere to their edges with such tenacity that in the case of many of them there is no effectual method of removing the paraffine, short of soaking the sections in warm Æther; a very objectionable proceeding, for though the Æther will undoubtedly remove the paraffine, it will also dissolve out any fatty matters which the section itself may naturally contain. All this annoyance may be prevented by subjecting the tissue to a simple preparatory treatment before it is imbedded in the paraffine. For this purpose prepare a very weak solution of gum arabic in water—twenty grains to the ounce. Into this, by means of the forceps, dip for a few moments the already surface-dried tissue, taking special care not to squeeze it, or the pressure will cause the spirit from its interior to remoisten the surface, which would prevent the gum from adhering. We shall see the value of this a little later on. Remove the tissue from the solution on to blotting paper, when the superfluous gum will speedily drain off, and in two or three minutes the surface will have become quite glazy and dry. Having melted some paraffine mixture in the water-bath, the tissue held in the forceps must be plunged for an instant into the heated liquid and immediately withdrawn, when the crust of paraffine with which it is enveloped will promptly harden. Whilst this is taking place we may make ready the microtome. Having by means of the milled head or handle depressed the plug in the tube so as to leave a free opening about an inch deep at its upper end, we must pour in the melted paraffine, which by this time will have become a little cooler, until the cavity be about half filled. The prepared tissue must now be introduced, care being taken to place it in such a position that the sections may be cut in the desired direction. The tissue must, if necessary, be held in position with forceps or a needle point, till the imbedding material becomes hard enough to give it due support. It is here to be remembered that it will not be advisable to place the tissue in the centre of the tube—it will be much more easily cut if placed rather nearer to that edge of the tube which is situated next the operator in the act of cutting. More paraffine is to be slowly added, until the tissue is completely covered; even after this still more should be added, for it will be found that in cooling the paraffine shrinks so as to leave a cup-shaped depression in its centre, whereby portions of the tissue which were previously covered are again laid bare. The best method of preventing this is to use the paraffine at as low a temperature as possible, and to use plenty of it. The microtome, with its contents, must now be removed to a cool place, when the paraffine will soon become solidified. Whilst this is being accomplished we may make our further preparations. The first thing we require will be a large basin, full of freshly-filtered water, and provided with a cover. A small beaker of methylated spirit, with a dipping rod or pipette, will also be necessary. We must now see that the section-knife is in thorough order, to ensure which it will be advisable to give it a few turns on the strop. An ordinary razor will also be of service.

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.

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.

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.

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.

16. Mounting in Glycerine.—To illustrate the method of using this medium we will mount our present sections in glycerine. In the first place we shall require a deep watch-glass, which is to be half filled with glycerine diluted with an equal amount of distilled water. By means of the spoon, one or more sections may be transferred into this, either directly from the acetic acid solution (§ 14), or if, since cutting, they have been preserved in spirit, they should first undergo a short immersion in a large vessel full of water. The watch-glass should now be covered with an inverted wine-glass, and put away for some hours, in order that the sections may become thoroughly saturated with the dilute glycerine. When this has been accomplished, a slide must be cleaned, and one of the sections, with the aid of the unpierced end of the spoon, be transferred to its centre.^[10] As the kind of section with which we are now dealing is, or ought to be, of extreme thinness, no cell (§ 26) is necessary. After tilting up one end of the slide, so as to drain off as much of the weak glycerine as possible, a drop of Price’s best glycerine must, with a glass rod or pipette, be allowed to fall gently upon the section, so as to avoid the formation of air-bubbles. If any of these, however, should be produced, they must be removed with the point of a needle set in a wooden handle,^[11] and the slide then covered with a small bell-glass (or wine-glass). A circular cover is now to be cleaned with a soft handkerchief, and after gently blowing from it any adhering fibres of lint, etc., it will be advisable to hold the side of the glass which is to come into contact with the preparation close to the mouth, and breathe upon it, so as to cover it with moisture. The cover held between the thumb and forefinger of the left hand must now be applied by its edge near to the margin of the preparation, and the surface of the cover directed in an inclined manner over it. Beneath the overhanging edge of the cover the point of the needle, held in the right hand, is now to be inserted (see Figure). By gently lowering the needle, the cover will come into gradual contact with the slide, driving before it a minute wave of glycerine, in which any air-bubbles that may have become developed are usually carried off. A very considerable degree of tact, however, is required to perform this little operation, simple as it may appear, for the retreating wave of glycerine not unfrequently floats out the section, either wholly or partially, from beneath the cover. Air-bubbles, also (the bÊtes noires of this process), are exceedingly likely to arise. When this happens the best plan to adopt is, by means of the needle point, gently to raise and remove the cover, apply another drop of glycerine to the section, and cover with a fresh piece of thin glass. It will now be necessary to remove any superfluous glycerine which may have collected around and near the cover. The great bulk must be wiped away by means of a camel’s-hair pencil, slightly wetted between the lips, any remaining stickiness being removed with a bit of blotting paper which has been slightly damped. With a very small camel’s-hair pencil, charged with solution of gelatine, a ring must be made round the margin of the cover, of sufficient breadth to take in a small tract of both cover and slide. As this cement is perfectly miscible with glycerine, it readily unites with any of that fluid which may ooze from beneath the cover, and which, in the case of any of the ordinary varnishes, would act as a fatal obstacle to perfect adhesion. To make the cement, take half an ounce of Nelson’s opaque gelatine, put in a small beaker, add sufficient cold water to cover it, and allow the mixture to remain until the gelatine has become thoroughly soaked. The water is now poured off, and heat applied until the gelatine becomes fluid, when three drops of creosote should be well stirred in, and the fluid mixture transferred to a small bottle to solidify. Before use, this compound must be rendered liquid by immersing the bottle containing it in a cup of warm water. When the ring of gelatine has become quite set and dry (which will not take long), every trace of glycerine must be carefully removed from the cover and its neighborhood, by gently swabbing these parts with a large camel’s-hair pencil dipped in methylated spirit. After drying the slide a ring of Bell’s microscopical cement may be applied over the gelatine, and, when this is dry, another coat is to be laid on. If it be desired to give the slide a neat and tasteful appearance, it is a very easy matter, by means of the turn-table, to lay on a final ring of Brunswick black or white zinc cement (§ 24). Every care has now been taken to render our preparation permanent; but, to make assurance doubly sure, it will be well to follow Dr. Carpenter’s advice, and, every year or so, to lay on a thin coating of good gold-size.^[12]

10. The appearance of a slide is vastly improved if the preparation be placed exactly in its centre. This may readily be done in the following manner:—Take some very finely-powdered Prussian blue, and rub it up in a mortar with a little of the weak gum solution (§ 11), so as to form a thin blue pigment. A quantity of this should be made, so as always to be at hand. A slide having been cleaned, the best surface is to be selected, and on the reverse side, by means of the self-centring turn-table, a small circle is to be drawn with a camel’s-hair pencil, charged with the pigment. In the centre of this ring, but on the opposite side of the slide, the section is to be placed, when it of course will occupy a position exactly central. When the slide comes to be finished, the blue ring may easily be removed with a wet rag.