Chapter VII: Part II (1)

25. _Special Methods._—Having in the preceding pages entered at some length into the general subject of section-cutting, it remains for us now to consider those special methods of preparation which the peculiarities of certain objects demand. In order to keep the bulk (and consequent price) of this manualette within due bounds, we shall, without further preface, proceed to the description of these methods, in doing which every endeavour will be made to employ such brevity of expression as may be consistent with perfect clearness of meaning. As the most convenient plan, the objects here treated of will be arranged in alphabetical succession.

26. _Bone._—Both transverse and longitudinal sections should be prepared, the former being the prettier and most interesting. After prolonged maceration in water, all fat, etc., must be removed and the bone dried, when as thin a slice as possible is to be cut off in the desired direction, by means of a very fine saw. If the section so obtained be placed upon a piece of smooth cork it may, with the aid of a fine file and the exercise of care, be further reduced in thickness. It is then to be laid upon a hone moistened with water, and being pressed gently and _evenly_ down upon it with the tip of the finger (protected, if necessary, by a bit of cork or gutta-percha), it must be rubbed upon the stone until the desired degree of thinness has been attained. Finally, in order to remove scratches and to polish the section, it should be rubbed upon a dry hone of very fine texture, or upon a strop charged with putty-powder. After careful washing in several waters the section must be allowed thoroughly to dry, when it may be mounted by the _dry method_ in the following manner:—A ring of gold-size must, by means of the turn-table, be drawn in the centre of a slide, and the slide put away in a warm place for several days (the longer the better), in order that the ring may become perfectly dry and hard. When this has been accomplished the section is to be placed in the centre of the ring, and a covering circle of the requisite size having been cleaned, this must have a _thin_ ring of gold-size applied round its margin. The cover is now to be placed in position and gently pressed down, a spring clip being employed, if necessary, to prevent it from moving. In about twenty-four hours another layer of the varnish should be applied, and the slide afterwards finished in the manner already described (§ 24). The above method is also applicable to the preparation of sections of _teeth_ and also of _fruit-stones_ and other hard bodies, which are incapable of being rendered soft enough for cutting.

As the process just described, however, is both troublesome and tedious, it is much better for ordinary purposes to have recourse to the _decalcifying method_, by which means sections in every way suitable for the examination of the essential structure of bone may be obtained with ease. To carry out this plan a piece of fresh bone should be cut into small pieces and placed in a solution made by dissolving 15 grains of pure chromic acid in 7 ounces of distilled water, to which 30 minims of nitric acid s. g. 1.420 are afterwards to be added. Here they should remain for three or four weeks, or until the bone has become sufficiently soft to cut easily, the fluid being repeatedly changed during the process. From this solution they must be transferred to methylated spirit for a few days, when a piece may be selected, imbedded in paraffine, and cut in the microtome (§ 12). Some of the sections should be mounted, unstained, in spirit. For this purpose a cell of gold-size, as above described, must first be prepared and filled _full_ of a mixture of spirit of wine one part, and distilled water three parts. Into this the section must be carefully placed and the cover applied, the same precautions for the exclusion of air-bubbles being taken which were recommended when speaking of mounting in glycerine (§ 16). When the cover is in position a ring of gold-size must be laid on, repeated when dry, and the slide afterwards finished in the ordinary manner. It will also be advisable to stain some of the sections with carmine (§ 14), or picro-carmine (§ 42), and mount them in glycerine. _Teeth_ may also be treated by the decalcifying method, but in this case it must be remembered that the enamel will dissolve away.

27. _Brain._—The best hardening fluid is that recommended by Rutherford, and is made by dissolving 15 grains of pure chromic acid and 31 grains of crystalized bichromate of potash in 43 ounces of distilled water. Small pieces of brain, which have previously been immersed for twenty-four hours in rectified spirit, should be placed in about a pint of this solution, where they must remain for five or six weeks, the fluid being repeatedly changed during the process. If by this time they are not sufficiently hard the induration must be completed in alcohol. Sections are easily cut in the microtome by the paraffine method (§ 12). These may advantageously be stained in a solution of aniline blue, made by dissolving 1-1/2 grain of aniline blue in 10 ounces of distilled water, and adding 1 drachm of rectified spirit (_Frey_). As this stain acts very rapidly two or three minutes’ immersion will generally be found long enough. The sections must then be mounted in balsam (§ 23).

28. _Cartilage._—The method to be employed in the preparation of cartilage will entirely depend upon the nature of the staining agent, to the action of which the sections are to be submitted. Thus, if the elegant _gold method_ is to be followed, it is necessary that the cartilage should be perfectly fresh; whilst if any of the other staining agents are to be employed the tissue may have been previously preserved in alcohol. An excellent object on which to demonstrate the gold process is to be found in the articular cartilage of bone. It is a very easy matter to obtain from the butchers the foot of a sheep which has just been killed. The joint is to be opened, and the bones dissociated, when they will be seen to have their extremities coated with a white glistening membrane—this is the _articular cartilage_. Exceedingly thin slices must be at once cut from it, and as only small sections are required, a sharp razor may be used for the purpose, the blade being either dry or simply wetted with distilled water. The sections as cut are to be transferred to a small quantity of a half per cent. solution of chloride of gold in a watch glass. Chloride of gold may be purchased in small glass tubes hermetically sealed, each tube containing 15 grains, and costing about 2s. If, however, the student requires only a small quantity of the staining fluid he need not be even at this small expense, for as photographers for the requirements of their art always keep on hand a standard solution of chloride of gold of the strength of one per cent., a little of this may readily be obtained, and diluted to the required degree. After the sections have been exposed to the action of the staining fluid for about ten minutes they may be transferred to a small beaker of distilled water, and exposed to diffused light for about twenty-four hours, when they must be mounted in glycerine (§ 16).

Sections of cartilage may also be examined, without being stained, in which case the field of the microscope should be only very feebly illuminated. Or carmine staining (§ 14) may be resorted to—these sections show well in glycerine, or if the staining be made very deep, even Canada balsam may be employed, and with fair results.

Microscopists are indebted to Dr. Frances Elizabeth Hoggan for the description of a new method of staining, which we have found especially suited to the treatment of cartilage. The agent employed is _iron_, and the process, which is very simple, is as follows. Two fluids are necessary—(1) tincture of steel; (2) a two per cent. solution of pyrogallic acid in alcohol. A little of the former is to be poured into a watch glass, and into this the sections, after having been previously steeped in alcohol for a few minutes, are to be placed. In about two minutes the iron solution is to be poured away and replaced by solution No. 2. In the course of a minute or two the desired depth of colour will have been produced, when the sections are to be removed, washed in distilled water, and mounted in glycerine. The results obtained by this process are very beautiful, the colour produced being a very fine neutral tint, of delightful softness. The process also answers admirably in the case of morbid tissues, and we have now in our possession some sections of ulcerated cartilage tinged by the iron method, in which the minute changes resulting from the ulcerative disintegration are brought out with wonderful distinctness.

As the structure of cartilage differs according to its purpose and situation, the student will find his time profitably employed in a careful examination of the following forms (α) _hyaline_—articular and costal; (β) yellow fibro-cartilage—epiglottis, or external ear; (γ) _cellular_—ear of mouse. Sections of the _intervertebral_ ligaments should also be made, in which the different kinds of cartilage may be examined side by side with each other.

29. _Coffee Berry_ affords sections of great beauty. The _unroasted_ berry should be soaked for hours or days in cold water until sufficiently soft; then imbedded in paraffine, and cut in the microtome (§ 12), the section being made in the direction of the long axis of the berry. Put up in glycerine, or stain rather strongly with carmine, and mount in balsam. The same method of treatment may also be applied to other hard berries or _seeds_.

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.

Footnote 15:

Ralf.

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.

Footnote 16:

“Practical Histology,” 2d edit. p. 173.

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.

Footnote 17:

It is well, in connection with these papillæ, to bear in mind a fact
pointed out by _Frey_, namely, that the tips of the fingers frequently
become, _post-mortem_, the seat of extensive natural injections;
hence, in sections from this region, we frequently obtain good views
of distended capillaries without having been at the trouble of
previously injecting them.—_Frey_, “Microscopical Technology.”

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.

Footnote 18:

See a paper by Mr. Styles in the “Pharmaceutical Journal,” also
“Monthly Microscopical Journal” for August, 1875. [For a very
exhaustive paper on this subject by the late Dr. Beatty, of Baltimore,
Md., see “American Journal of Microscopy” for June, 1876.

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.

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NOTES.

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NOTE A., Page 14.

RANGOON oil is not found in this market. It is a variety of naphtha or petroleum found at Rangoon, in Asia, and contains a variable percentage of solid paraffin. Kerosene with a very little paraffin dissolved in it, would undoubtedly answer the same purpose.

Good sperm oil or tallow (free from salt) will serve quite as well. It is scarcely necessary to caution the reader that all oily matter should be removed from the knife before it is used for cutting sections. This is best effected by dipping it in hot water and carefully wiping it.

NOTE B., Page 15.

METHYLATED spirit is alcohol which contains a small percentage of methyl. This renders it unfit for use in the manufacture of liquors or even chloroform, but does not injure it as a preservative fluid. The scientific men of Great Britain have the privilege of obtaining this fluid free from duty, and consequently they make very general use of it. In this country the student of science has no such privilege, and must use the more expensive alcohol. This however is but of a piece with other restrictions placed upon scientific pursuits in this country, where men like Marshall Jewell and Hannibal Hamlin have influence enough to pass laws which prohibit the exchange by mail of microscopic specimens, which pass freely in every other civilized country.

True methyl alcohol, or methylated spirits, is not to be had in this country, and therefore whenever this liquid is directed to be used in English books the American microscopist must substitute good alcohol. What is sold for “methyl alcohol” is wood spirit or wood naphtha, and will not serve the purposes of the microscopist, though it answers well enough for spirit lamps, making varnishes, etc.

NOTE C., Page 20.

THE method described in the text is generally known as the “free-hand” method, and although it is not generally employed for the production of cabinet specimens or slides that are intended for sale, yet it possesses so many advantages that every student should endeavor to acquire the art of cutting sections without the use of the microtome. For purposes of study such sections are quite equal to the best of those usually found on sale, and for the encouragement of beginners we may add that we have seen sections cut by the free-hand method which for size, thinness and accuracy, excelled any that we have ever seen produced with the microtome. Indeed Schäffer distinctly affirms that no microtome can equal the skilled hand in the production of thin sections.

NOTE D., Page 22.

THE mere existence of a binding screw should be no objection, and where bushings are employed it is sometimes necessary. But as the author well observes, a binding screw cannot be used to secure histological preparations.

NOTE E., Page 22.

A STILL better system is where the section-knife is held in a frame which slides on the plate so that the edge is kept just above the surface. In this way the edge of the knife is never dulled by grinding on glass or metal. A common mistake in this form, however, is to raise the cutting edge too far above the surface of the plate. This gives rise to great irregularities.

NOTE F., Page 22.

AS the size of the sections which it may be desired to cut, varies within wide limits, the best microtomes made in this country are furnished with as many as three tubes or “bushings,” fitting one within the other, and in this way the hole may be partially filled up and thus reduced in size. This enables us to obtain a range of from one inch and a quarter, suitable for large histological preparations, to one-eighth of an inch for such objects as hair sections.

NOTE G., Page 37.

KNIVES FOR CUTTING SECTIONS.

SUCCESS in cutting sections depends more upon the excellence of the knife that is used than upon any other one point, and therefore the art of keeping the knife in good order is one which should be acquired by every microscopist, even at the cost of considerable time, study and labor. As a stepping stone to the acquisition of this art, there are certain general principles which must be thoroughly understood before we can hope for success in practice. These principles are simple, and when once clearly stated, quite obvious, but they are frequently overlooked.

Cutting instruments act in two ways—either as simple wedges or as a series of wedges, the latter being generally known as the saw-like action. It is frequently stated in school philosophies that the edges of _all_ cutting instruments are toothed like a saw, and that they act like a saw; this is evidently not the case with many, such as the axe, the chisel, the plane, etc., and it is not even true of several to which it is frequently applied, such as the razor, the penknife, etc. The action can be said to be “saw-like” only when the teeth act as independent wedges and remove a small portion of the substance acted upon, the portion being known as _saw-dust_. Now in cutting sections of wood with a sharp razor, where but a single stroke is used to carry the cutting edge across a section, say half an inch in diameter, the edge of the razor does not act like a saw; none of the material is removed except the section itself; in other words, there is no saw-dust. That the edge of the knife or razor is not even, is very easily proved by means of the microscope, but it will be found that the notches and projections form a series of lancet-shaped knifelets, each one of which acts just as does the blade of a penknife when drawn over a piece of wood or a quire of paper. Now if we lay a quire of paper on a board and draw the edge of a penknife over it, we will cut down through the paper, not by sawing but by a moving wedge. It is precisely in this way that the little lancet-shaped teeth on the edge of a razor act when the latter is used to cut a section.

If we further examine the edge of the razor, we will find that the bottoms of the notches are comparatively blunt; if therefore we press the razor directly forward we soon press these blunt portions of the general edge against the material to be cut, and the resistance becomes so great that the material is actually crushed or torn. This would not happen if we had an absolutely perfect edge—one without any notches. This being practically unattainable, however, we must endeavor to make up for it by a sawing motion, (though not a sawing action) at the same time seeking to avoid as much as possible those saw-like features which render this motion necessary. We have dwelt at length upon this point because so much has been written comparing the edge of a razor to that of a saw, that some persons actually advise us to give the edges of our knives and razors a saw-like character, under the impression that by so doing they will work better. This is a fallacy; the best work will be obtained from the smoothest and most perfect edge, provided the latter is thin enough.

If the material of which our cutting instruments are made would allow it, that is to say if it was perfectly hard, perfectly rigid and incapable of being crushed or torn apart, the proper shape of a cutting instrument would be a wedge of the most acute form possible. But since the steel which we use is limited in its hardness and its power of resisting crushing and bending influences, the blades of our knives must have a certain strength or thickness, depending upon the character of the material to be cut. For very hard substances the knife must be stout and the edge ground to a comparatively obtuse angle; for moderately soft substances, such as wood, the angle may be more acute, while for very soft tissues the thinnest blade and the sharpest edge are most suitable.

The various angles to which cutting tools of different kinds are ground are shown in the accompanying illustrations, which explain themselves.

In the diagrams just given, the sides of the wedge are carried in straight lines to the very edge. In practice, however, this is never done, there being two strong objections to such a form. In the first place if the sides were as shown in the figures, every time the tool was ground or set, the entire side would have to be ground off, and this would involve great labor. But another difficulty, quite as great, would occur in the use of the instrument, for since the wedge keeps growing constantly and proportionately thicker, the friction between the sides of the wedge and the material would rapidly become very great. Therefore most cutting edges are ground to a second angle, much more obtuse than the first, and it is this second angle which forms the true cutting edge. And it is obvious that the mass of material required to carry out the ultimate angle in any case is wholly unnecessary, for if the tool be strong enough at _a b_ (Fig. 2) it will certainly be strong enough at _c d_. An extreme illustration of this principle is shown in the cold chisel used for cutting iron, the edge of which is shown in Fig. 2, and the angle of 80° to which it is ground is carried out by dotted lines. The angle which the sides of the chisel (not the sides of the extreme cutting edge however) make with each other is 25°.

There are two methods by which this change of angle may be made, one of which is shown in the cold chisel just figured, and the other in the razor of which a section is shown in figures 3 and 4. In the case of the cold chisel it will be seen that the tool is first formed to a thin straight wedge which is afterwards changed to one that is much more blunt. This answers very well where the ultimate angle is comparatively large or blunt, as is the case with penknives, table-knives, carving-knives, etc. But where the ultimate angle is very small this plan does not answer well, and the method shown in Fig. 3 is generally adopted. Here the ultimate angle is such that lines touching the extreme edge and the back of the blade are perfectly straight and form the actual cutting angle. In this case, therefore, the relief is obtained by hollowing out the sides of the blade, and this is done to various extents, the extremes being shown in figures 3 and 4. Fig. 3 shows a section of a razor ground on a stone 12 inches in diameter, which is as large as is generally used for this purpose. Fig. 4 is a section of a razor ground on a four-inch stone—the smallest in general use. This method of changing the angle is of course substantially the same as the first, merely differing in the mechanical device used, but it affords this important advantage that in the subsequent honing and stropping processes the back of the razor forms a perfect guide by which the ultimate angle may be determined. This is not the case with the cold chisel or the carving-knife, in both of which cases the eye and hand alone determine the cutting angle, which is therefore apt to become irregular or even rounded—the worst form of all. This will be more easily understood from the following engravings where A, Fig. 5, shows a penknife blade, as applied to an oilstone for the purpose of giving it the final edge. The angle here shown is considerably greater than that generally used, but the principle is the same. It is evident that if, in moving the blade back and forth on the stone we allow it to rock or change the angle which the blade makes with the stone’s surface, the edge will become rounded as shown in Fig. 6—a form which for delicate work is useless.

If the blade be laid down flat as at B, Fig. 5, the labor of wearing away the superfluous metal will be enormous. But if the blade be hollow as Fig. 7, (where the hollow is, for clearness of illustration, slightly exaggerated) then the ultimate angle may be formed quickly and accurately, there being no danger of the angles being changed on account of want of skill on the part of the operator.

In some cases one side is left quite flat and the other side is ground to two or sometimes three angles, as in the carpenter’s chisel, a section of which is shown in Fig. 8, where _a a_ shows the angle made by the body of the blade; _b b_ the angle of the bevel formed by the grindstone, and _c c_ the angle of the cutting edge which is formed by the oilstone. In this case the flat side A B forms a straight guide and enables the workman, in cutting, to make straight, clean work. In sharpening such a tool on the oilstone the flat side is always laid flat on the oilstone and merely smoothed off,[19] and the skill of the workman is chiefly shown in the accuracy with which he forms the angle _c_ B _c_. The dexterity with which skilled workmen can effect this by the hand alone is astonishing, but in the hands of those who have had but little practice the edge infallibly becomes rounded as shown in Fig. 6. Therefore wherever a knife with a flat side is used, no attempt should ever be made to grind this flat side on the hone. Such an attempt will almost always result in an edge so irregular that it will be almost impossible to cut a good section with it. But if the under side of the knife be left truly flat, we will have the very best tool that can be had for use with the microtome. Unfortunately, however, it is very difficult to get a knife with a truly flat surface, that is, one that is perfectly “out of wind” as mechanics say. As a general rule the flat sides of knives are not true planes but irregularly curved surfaces; consequently when laid on a plane surface, such as the table of a microtome, they rock, and the edge is in contact with the table at some points and separated from it at others, and the points which are in contact change as the knife is moved diagonally across the table. This makes the section irregular and worthless. But a really good knife with a perfectly true flat side is such a valuable tool that a good deal of effort may be profitably expended in getting it, and when once obtained never let the flat side be touched by a grindstone, or more than touched by a hone. It is not absolutely necessary, however, that the sides of a delicate knife should be curved, provided the knife is used for cutting very soft materials. An angle of 10° or even 5° gives a pretty stout blade when carried back three-quarters of an inch, as may be seen by examining Fig. 1, and the guiding action of the very thick back may be obtained by means of an artificial guide, applied like the brass backs of tenon saws. Such an arrangement is shown in Fig. 9, and has been found very effectual.

Footnote 19:

Holtzapffel tell us that chisels that are required for paring across
the end grain of moderately soft wood are considered to hang better to
the work when they have a very slight keen burr or wire edge thrown up
on the face or flat side of the tool. But this does not apply to
section knives.

When the knife is to be honed, the back is applied and fastened by means of two or three set screws. When laid on the hone, the edge of the knife and the lower surface of the back form the guide and regulate the cutting angle. In this way we can use a broad, thin blade and yet secure great accuracy in honing it. Such a blade has this advantage also, that it is not so liable to be rounded and thus injured by stropping as one in which the relief is obtained by hollowing out the sides.

Such, then, are the general principles which govern the construction of cutting tools, including the section knives used by microscopists. We will now give a few practical directions for selecting a knife and putting and keeping it in order.

In selecting a knife for cutting sections, regard must be had to the texture of the material that is to be cut. To attempt to cut delicate sections of soft tissues with the stout knives which are suitable for cutting sections of woody fibre would be to destroy the sections, while to reverse the operation and cut sections of wood with thin, delicate knives would result in the ruin of the knife. We have seen a most excellent knife seriously injured by an attempt to cut material that was too hard for it. The knife was very thin, and had proved most excellent in cutting sections of such material as kidney, liver, etc. An attempt was made, against our protest, to cut a section of an apple-shoot, the wood of which was mature. Before the knife had gone half way across, it bent, dug into the wood and broke, leaving an ugly gap in the middle. Experienced section-cutters know this very well, but young microscopists are not so familiar with the facts just stated, and the point is too important to be overlooked. Those, therefore, who devote themselves to microscopical studies, or who expect to make sections of materials of several kinds, differing in hardness, etc., must provide themselves with knives of different degrees of strength.

For common work, good razors are as good as anything, provided they can be obtained with straight edges. Where razors are not suitable, recourse must be had to the surgical instrument maker, though we are sorry to say that there are but few in this country that know how to forge, temper and grind a decent knife. Most of our dealers in instruments do not make the instruments they sell; they import the goods they sell with their names stamped on, and thus get a reputation as manufacturers; a special order they are unable to fill respectably. There are some exceptions, but of the majority of dealers what we have written above is true.

The points which specially demand attention in a knife for cutting sections are these: 1. Quality of the steel used; 2. Temper; 3. Form of the blade.

Of the quality of the material of which a knife is made, nothing can be determined except by actual trial. The old tests of staining with acids, examining with the microscope, etc., are worthless, or at least too crude and uncertain to be of any practical value. Color changes with the degree of polish that is produced, and, in short, there is no reliable guide. The purchaser must depend entirely upon the reputation of the manufacturer. There is plenty of good steel to be found; the trouble lies with the cutlers. They are careless and in haste, and as a consequence they burn the steel or fail to work it sufficiently, and the result is a useless tool.

The steel may be of the very best quality, however, and well forged, and yet the knife may fail from being badly tempered—too soft or too hard. If too soft, the edge is soon dulled; the knife requires to be frequently honed, and the time wasted in keeping it in order is a serious drawback. If too hard, it is impossible to give it a keen edge, for the metal crumbles away as soon as it is honed or stropped very thin, and the edge becomes ragged and dull. Good steel, well forged, may be so tempered that it will neither crumble nor become rapidly dull.

Much may be learned on this point from careful inspection of the edge, and trial on a piece of horn, such as an old razor handle. When drawn over a clean piece of horn once or twice the edge of a soft knife is completely dulled; if well-tempered it should scarcely lose its keenness. Again, when laid flat on the thumb nail and pressed, the edge ought to bend up without breaking or crumbling, and at once, when the pressure is removed, resume its original shape. The extent to which extreme hardness and durability, or toughness, may be combined, is well shown in the famous Toledo sword-blades. One of these blades will shear through an iron nail without having its edge perceptibly dulled, and yet so tough and springy is it that it may be coiled up into a hoop of several folds without breaking or receiving any permanent set. If we could only get section knives of stuff like this, it would be a pleasure to work with them.

After all, however, the only test of these two points, material and temper, is a fair trial in actual practice. Of the form of the blade, however, it is easy to judge, and there are a few points which are frequently overlooked and which give rise to errors that are attributed to other causes.

In the text we are told that “It is essentially necessary that the back and edge of the blade be strictly parallel to each other.” The author undoubtedly knew what the correct form should be, but the definition he has given is not a correct mathematical statement of the conditions involved. These are as follows:

_The edge must form a straight line, and both the edge and the under side of the back must lie in the same plane._

Let us consider these conditions somewhat carefully.

Since the hole in which the material to be cut is imbedded, is always circular, and since the blade is generally pressed forcibly against the table of the microtome, it is obvious that if the edge of the knife be curved it will sink into the hole to a greater or less depth according as it is further from the centre of the hole or nearer to it. To explain this let us refer to the greatly exaggerated drawing given in figure 10 where the edge of the knife, _a_, _b_, is shown in two different positions covering the hole of the microtome. The least degree of reflection will show that as the knife passes towards the centre from _a b_ to _aʹ bʹ_, it will have a tendency to turn on the back as on a hinge, and the middle of the edge will sink deeper into the hole as it approaches the centre of the latter. The section will therefore vary in thickness and prove useless. This difficulty can be obviated only by the use of a straight edge like _e f_, which will always lie in the same plane.

If the knife were perfectly rigid, and the curved edge and back lay entirely in the same plane, this difficulty would not occur to any great extent. But since a curved edge rarely lies wholly in the plane of the back of the knife, and since the blades of most knives, especially those thin ones used for cutting sections of soft tissues, yield a little to pressure, and since this pressure is never exactly the same, it is impossible to prevent irregularities from occurring.