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 (a) hyaline—articular and costal; () yellow fibro-cartilage—epiglottis, or external ear; (?) 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.

30. Fat.—Adipose tissue may be hardened in alcohol, cut in paraffine, and mounted in glycerine. If the tissue has been injected the sections may be mounted in balsam, and are then very beautiful objects, showing the capillary network encircling the fat cells.

31. Hair.—Longitudinal sections are readily made by splitting the hair with a sharp razor. It is more difficult to cut the hair transversely. This, however, may easily be done in the following manner. The hairs having previously been well soaked in Æther to remove all fatty matters, a sufficient number of them must be selected to form a bundle about the thickness of a crow quill. This bundle, after being tied at each extremity with a bit of thread, is to be immersed for several hours in strong gum (§ 18,) to which a few drops of glycerine have been added. On removal, the bundle must be suspended by means of a thread attached to one end of it, in a warm place until sufficiently hard, when it is to be imbedded and cut in paraffine (§ 12). Each section, as cut, is to be floated off the knife into methylated spirit. From this it is with the aid of the spoon (§ 14) to be transferred to a slide, the spirit tilted off, a drop of absolute alcohol added, when, after a minute or two, this also is to be drained off, the section treated with clove oil, and the mounting completed as described in § 23.

32. Horn varies very much in consistence, in some instances having a cartilaginous character, whilst in others it is almost bony. In the latter case, sections will have to be ground down in the manner explained when speaking of bone (§ 26). Where the texture is less dense, recourse may be had to prolonged steeping in hot or boiling water; in some cases it will be necessary to continue the immersion for several hours. When sufficiently soft the piece of horn may, by means of bits of soft wood, be firmly wedged into the tube of the microtome, and sections cut with a razor, or what is better, with a broad and very sharp chisel. The sections are to be put between glass slips, held together by American clips (or pegs), and put away for two or three days in order to become thoroughly dry. After well soaking in good turpentine or benzole, they must be transferred to slides, the superfluous turpentine drained off, and chloroform-balsam added, etc. (§ 23). Sections of horn should, of course, be cut in different directions, but for examination with the polariscope those cut transversely yield by far the most magnificent results. Hoofs, whalebone, and allied structures should also be treated by the above method.

33. Intestine.—The method to be pursued with sections has already been described (§ 18). The ileum, however, is a very pretty object when a portion of it is so mounted as to show the villi erect. To do this it is necessary to cement to the slide, by marine glue, a glass cell of sufficient depth. This should have been prepared some time beforehand, so that the cement may be perfectly dry and hard. The cell is now to be filled with turpentine, and the piece of ileum (having been previously passed through methylated spirit and absolute alcohol into turpentine) is gently placed into it, having the villi uppermost; pour some pure and rather fluid balsam on the object at one end, and gradually incline the slide, so as to allow the turpentine to flow out at the opposite side of the cell, till it is full of balsam. Then take a clean cover, and having placed upon it a small streak of balsam from one end to the other, allow it gradually to fall upon the cell, so as to avoid the formation of air-bubbles (§ 17), and finish the slide in the usual manner.^[15] Or, the intestine may be dried, and mounted dry, in a cell with a blackened bottom, for examination as an opaque object.

34. Liver.—Small pieces of liver may be very successfully hardened by immersion in alcohol, beginning with weak spirit and ending with absolute alcohol. Cut and mount as usual.

35. Lung must be prepared in chromic acid (§ 5). For the cutting of sections the freezing microtome (§ 18) is of especial value, and should, therefore, be used. If, however, the student be not provided with this instrument, he must proceed as follows. A small piece of lung, previously deprived of all spirit, is to be immersed until thoroughly saturated in solution of gum (§ 18). A small mould of bibulous paper (§ 2), only just large enough to receive the piece of tissue, having been prepared and filled with the mucilage, the specimen is to be transferred to it. The mould, with its contents, is now to be placed in a saucer, into which a mixture of about 6 parts of methylated spirit and 1 part of water (SchÄfer) is to be poured until the fluid reaches to within about a third of the top of the paper mould. In the course of several hours the surface of the mucilage will begin to whiten and solidify. As soon as this occurs more dilute spirit must be poured into the saucer, until the mould is completely submerged. In a day or two the gum will be found to have acquired a suitable consistence for cutting, when it must be removed from the spirit, the paper mould peeled off, and the mass imbedded and cut in paraffine, the sections being afterwards treated as if they had been obtained by the freezing method (§ 18). If the solidification of the gum should proceed too slowly, a few drops of pure spirit may be added to the contents of the saucer. If, on the other hand, the gum should become overhard, it will be necessary to put into the saucer a few drops of water, and repeat this until the required consistence be obtained.

36. Muscle.—Harden in chromic acid, and cut in paraffine. Transverse sections may be made to show the shape of the fibrils. Longitudinal sections will only be required in the case of injected tissues, when such sections will be found very elegant, showing, as they do, the elongated meshes of capillaries running between and around the muscular fasciculi. Mount in glycerine or balsam. To see the transverse striÆ characteristic of voluntary muscle, a very good plan is to take a bit of pork (cooked or fresh), and by means of needles to teaze it out into the finest possible shreds. If these be examined in water or glycerine, the markings will be shown very perfectly.

37. Orange-peel, common object though it be, is not to be despised by the microscopist. Transverse sections must be prepared by the gum method (§ 35). These sections are not to be subjected to the action of alcohol (as this would destroy the colour), but after drying between glass slides they must be soaked in turpentine and mounted in balsam. We shall then have a good view of the large globular glands whose office it is to secrete that essential oil upon which the odor of the orange depends.

38. Ovary may be prepared in the same manner as liver (§ 34). Sections, which are to be cut in paraffine, may be stained with carmine, and mounted in glycerine or balsam. Apart from all scientific value, we know of no slide for the microscope which, even as a mere object of show, surpasses in beauty a well-prepared section of injected ovary, showing the wondrous Graafian vesicles, surrounded by their meandering capillaries.

39. Porcupine Quill.—Soften in hot water, cut in paraffine, and mount in balsam. Much (in our opinion too much) lauded as an object for the polariscope.

40. Potato.—From the large amount of water which it contains thin sections cannot be cut from the potato in its natural state. It must, therefore, be partially desiccated, either by immersion in methylated spirit for a few days or by exposure to the air. Sections may then readily be obtained by imbedding and cutting in paraffine. Such sections mounted in balsam are very beautiful, the starch being seen in sitÚ, whilst if polarized light be employed each granule gives out its characteristic black cross.

41. Rush is to be prepared and cut as orange-peel (§ 37). Transverse sections of this “weed” furnish slides of the most exquisite beauty.

42. Skin.—To prepare skin for section a piece is to be selected which, after having been boiled for a few seconds in vinegar, must be stretched out on a bit of flat wood, and being maintained in position by pins be allowed to remain until thoroughly dry. Then imbed in paraffine, and cut exceedingly thin transverse sections. These may be stained in carmine, but more beautiful results are obtained if picro-carmine be employed. Sections of skin, when stained by this agent are much increased both in beauty and instructiveness; for the several constituents of the tissue becoming tinged with different colours are readily distinguishable from each other, whilst the contrast of colouring forms a pleasing picture to the eye. The method of preparing picro-carmine is very simple, though it sometimes yields a solution not altogether satisfactory. The best formula with which we are acquainted is that given by Rutherford,^[16] and if due care be taken in following it out failure will generally be avoided. “Take 100 c.c. of a saturated solution of picric acid. Prepare an ammoniacal solution of carmine, by dissolving 1 gramme in a few c.c. water, with the aid of excess of ammonia and heat. Boil the picric acid solution on a sand bath, and when boiling add the carmine solution. Evaporate the mixture to dryness. Dissolve the residue in 100 c.c. water, and filter. A clear solution ought to be obtained; if not, add some more ammonia, evaporate, and dissolve as before.” Sections may be exposed to the action of this fluid for a period varying from fifteen to thirty minutes, then rapidly washed in water, and mounted in glycerine. They may also be mounted in balsam, care being taken in that case to shorten as much as possible the period of their immersion in alcohol, so that no risk may be run of the picric acid stain being dissolved out.

If it is intended to study the structure of the skin with anything like thoroughness, portions must of course be examined from different localities, in order that its several varieties and peculiarities may be observed. Thus the sudoriforous, or sweat glands, may be found in the sole of the foot, whilst the sebaceous glands are to be sought in the skin of the nose. The papillÆ are well represented at the tips of the fingers,^[17] whilst the structure of the shaft of the hair, together with that of the follicle within which its root is enclosed, as also the muscles by which it is moved, are to be studied in sections of skin from the scalp or other suitable locality.

43. Spinal Cord.—The spinal cord, say of a cat or a dog (or if procurable, of man), after being cut into pieces about half an inch in length, may be hardened in the usual chromic acid fluid (§ 5). As it is peculiarly liable to overharden and become uselessly brittle, the process must be carefully watched. Its further treatment is the same as that of brain. These sections may be stained very satisfactorily by the ink process, for communicating details of which we are indebted to the kindness of Dr. Paul, of Liverpool. The agent usually employed is Stephenson’s blue-black ink, which, for this purpose, must be quite fresh. As in the case of carmine, two methods of staining may be adopted—either rapid, by using concentrated solutions, or more prolonged, according to degree of dilution. For the reasons previously given (§ 14), slow methods of staining are always to be preferred, as yielding the most beautiful results, yet, for the purposes of preliminary investigation, it is often convenient to have recourse to the quick process. To carry out the latter plan, an ink solution of the strength 1 in 5—10 parts of water is to be freshly prepared, and the sections exposed to its action for a few minutes. For gradual staining the dilution must be carried to 1 in 30—50, and the time of immersion prolonged to several hours, the sections being occasionally examined during the staining, so that they may be removed just as they have acquired the desired tint. When a satisfactory coloration has been obtained, the preparations should be mounted in dammar or balsam (§ 23). One advantage of this method of staining is, that definition is almost as good by artificial light as by day.

44. Sponge may readily be cut after being tightly compressed between two bits of cork; or its interstices may be filled up by immersion either in melted paraffine (§ 11) or in strong gum (§ 18), and then cut as usual.

45. Stomach requires no special method of hardening (chromic acid). Sections should always, when practicable, be cut in the freezing microtome. In default of this, proceed in the manner as directed for lung (§ 35). Both vertical and horizontal sections will, of course, be required. If the preparation has been injected, the latter are particularly beautiful. Stain with carmine or aniline blue (§ 27), and mount—if for very close study, in glycerine—if injected and for a “show” slide, use balsam.

46. Tongue.—Harden in chromic acid, imbed and cut transverse sections in paraffine. As, however, the paraffine is apt to get entangled amongst the papillÆ, whence it is afterwards with difficulty dislodged, it will be as well before imbedding to soak the tongue in strong gum for a few minutes, and afterwards immerse in alcohol till the gum becomes hardened, so that the delicate papillÆ may thus be protected from the paraffine by a surface-coating of gum. The best staining agent is picro-carmine (§ 42). Sections of cat’s tongue near the root, when thus stained, furnish splendid objects. Sections should also be made of the taste-bulbs, found on the tongues of rabbits. These are small oval prominences, situated one on each side of the upper surface of the tongue near its root. They should be snipped off with scissors, and vertical sections made in the direction of their long axis. Stain with carmine or picro-carmine, and mount in glycerine or balsam.

47. Vegetable Ivory.—After prolonged soaking in cold water may readily be cut in the microtome. The sections should be mounted in balsam, and though not usually regarded as polariscopic objects, nevertheless, when examined with the selenite, yield very good colours.

48. Wood.—Shavings of extreme thinness may be cut from large pieces or blocks of timber, by means of a very sharp plane. In this way very good sections may be procured of most of the common woods, as oak, mahogany, “glandular wood” of pine, etc. Where however, the material to be operated upon takes the form of stems, roots, etc., of no great thickness, they should, after having been reduced to a suitable consistence (§ 4), be imbedded in paraffine, and cut in the microtome. Before imbedding it must not be forgotten to immerse the wood to be cut in weak gum-water (§ 11), this precaution being of great importance, especially in the case of stems, etc., the bark of which is at all rough and sinuous. If the sections are to be mounted unstained, they are usually put up in weak spirit (§ 26). A very general method also of dealing with this class of objects is to mount them dry (§ 26). This plan, however, cannot be recommended, for however thin the sections may be, the outlines, when this process is adopted, always present a disagreeable black or blurred appearance. To avoid this we may have recourse to Canada balsam, but the ordinary method of employing it must be slightly modified, a drop of chloroform being substituted for the clove oil (§ 23), otherwise this latter agent will cause the section to become so transparent as to render minute details of structure difficult to recognize. A better plan, perhaps, is to stain the section with carmine or logwood, and mount in balsam by the ordinary process. The best course to follow, however, especially in the case of transverse sections, is the double staining method.^[18] For this purpose the sections in the first place must be subjected to the action of a solution of chloride of lime (1/4 oz. to a pint of water) until they become thoroughly bleached. They must then be soaked in a solution of hyposulphite of soda (one drachm to four ounces of water) for an hour, and after being washed for some hours, in several changes of water, are to be transferred for a short time to methylated spirit. Some red staining fluid is now to be prepared by dissolving half a grain of Magenta crystals in one ounce of methylated spirit. A little of this solution being poured into a small vessel of white porcelain (§ 14), the sections are to be immersed in the dye for about thirty minutes. They are now to be removed, and after rapid rinsing in methylated spirit to remove all superfluous colour, they must be placed in a blue staining fluid made by dissolving half a grain of aniline blue in one drachm of distilled water, adding ten minims of dilute nitric acid and afterwards sufficient methylated spirit to make two fluid ounces. The sections must be permitted to remain in this solution for a very short time only, one to three minutes being generally sufficient, for as the action of the dye is very energetic, it will, if too long exposure be allowed, completely obliterate the previous coloration by the magenta. After being again rapidly rinsed in methylated spirit, as much of this as possible must be drained off, and the sections put into oil of cajeput, whence, in an hour, they may be transferred to spirits of turpentine, and after a short soaking, mounted in balsam.

If the student will carefully carry out the above process, his trouble will be amply repaid by the beautiful results obtained, for by its means he may, with ease, prepare for himself a series of slides of such value as to constitute a worthy addition to his cabinet.

The preceding list by no means represents all the objects, sections of which will be found interesting to the microscopic student. Such was not its purpose—had it been so, the enumeration might have been prolonged almost indefinitely. The end in view was to bring under the notice of the reader only those substances the cutting of which is accompanied by difficulty; and even of this class the space at our disposal has been so limited that we have been unwillingly compelled to pass over many, and dwell only on such as possess a typical character.