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.
[11]. A crochet-needle holder made of bone, and which may be bought at the smallware dealers’ for about sixpence, makes an admirable handle for microscopical needles. At one extremity there is a small cavity, closed with a cap, for the storage of reserve needles, whilst the other end terminates in a metal tip, provided with a crucial slit and central perforation for the reception of the needle in actual use, and so arranged that, by means of a small screw-nut, needles of various sizes may be firmly held in position.
[12]. If square covers be employed, they may be fixed to the slide by a simple method much in vogue in Germany. A thin wax taper is to be lighted, and being partially inverted for a few seconds, the wax surrounding the wick will become melted. After the slide has been freed from excess of glycerine, a drop of this heated wax is allowed to fall upon each corner of the cover, and a line of the melted wax run along the margins of the cover between these points, so as perfectly to surround it. If a good coat of white zinc cement be subsequently laid over the wax a very durable, and not unornamental, line of union will have been formed.
METHOD OF APPLYING COVER.
17. Use of Freezing Microtome.—Our preceding consideration of the method of employing the microtome in conjunction with paraffine as an imbedding agent (§ 11), will have formed a very suitable introduction to the study of the somewhat more complicated process of imbedding the tissue in gum, for section in the freezing microtome. This method is of the utmost value to the practical histologist, for by its means he is enabled with ease to possess himself of perfect sections of several structures, the cutting of which, before the introduction of this process, was always a matter of difficulty and anxiety. The freezing microtome is especially valuable for the section of such substances as from their extreme delicacy are liable to be injured by being imbedded in paraffine—for instance, the delicate villi of the intestines becomes very frequently, by the use of paraffine, denuded of their epithelium, and the villi themselves not seldom become torn off or otherwise damaged. The great value of the method is also very well seen in the treatment of those tissues which, like the lung, are of such loose and spongy texture as to offer insufficient resistance to the knife unless their interstices have previously been filled up with some solid yet easily cut material. As the space at our command is strictly limited, we are precluded from entering as fully into this branch of section-cutting as the importance of the subject demands and our own inclination would lead us. To those who wish to become thoroughly conversant with the full value of this method we cannot do better than recommend the perusal of Professor Rutherford’s Practical Histology, 2d edition, than which, on the whole subject of physiological microscopy, no treatise with which we are acquainted is at once so plain, practical, and profound.
18. Employment of Freezing Microtome.—A very suitable object with which to demonstrate the method of using this form of microtome will be afforded us by a portion of intestine, say of the ileum of a cat or dog. Suppose we have some of this in methylated spirit—let us select a piece of about half an inch in length. Our first care will be to deprive this of its spirit; for so long as the tissue remains impregnated with alcohol it would, of course, be impossible to freeze it. We will, therefore, throw it into a large basinful of water, and leave it there for twenty-four hours, during which time it would be as well to change the water once or twice. We shall now require a strong solution of gum. This, which should have been made some time previously, may be prepared by placing a quantity, say three or four ounces, of ordinary gum arabic in a glass beaker, and adding sufficient water to cover it—the mixture must be stirred occasionally with a glass rod until solution has taken place, which will be in a few days. If necessary a little more water may be added, but so long as the gum will pour from vessel to vessel, it cannot well be made too strong. Mucilage, by keeping, is very apt to become sour and mouldy—this may be prevented by adding to each ounce of the water with which it is prepared about half a grain of salicylic acid. We now pour some of this mucilage into a small vessel—an egg cup will answer very well—and into it transfer the piece of ileum from the water. Here we must allow it to remain for a time sufficient to permit of its becoming thoroughly saturated with the gum, for which purpose some hours will be necessary. When this soaking has been accomplished we will prepare the microtome, which we will assume to be Rutherford’s. In the first place it will be necessary to remove the plug—which is to be done by turning the handle connected with the screw until the plug rises so high in its tube that it may be grasped with the fingers and removed, when it is to be well smeared all over with sperm oil and replaced. This is done to prevent any unpleasant adhesions taking place whilst the freezing is going on. We must next depress the plug, so as to convert the upper part of the tube into a kind of “well” of sufficient depth to hold our specimen. It will now be very advisable to look carefully into this well and observe whether the plug fits accurately into the tube (§ 8) for if there be any interval between the two it will give rise to much subsequent annoyance, as the gum penetrating this interstice will there become firmly frozen into irregular patches, which will so interfere with the even gliding of the plug within its tube as to cause the former to ascend in such an irregular and jerky manner as to be utterly destructive of all accuracy in the cutting. If this defect be observed, it may be at once remedied by dropping a small quantity of gently heated paraffine into the well, which will effectually close up any fissures. The microtome, by means of its clamping arrangement, must now be firmly attached to the table, and a suitable vessel be placed on the floor beneath it, so that it may catch the water which will issue from the waste-pipe of the apparatus. The next requirement is a supply of block ice and finely-powdered salt. A lump of the ice must be wrapped in a towel, and crushed into small pieces; these, by means of a large mortar, are to be further reduced to a very fine powder. Any attempt to hurry over this troublesome part of the operation will lead to future disappointment, for unless the ice be used in a very fine powder great delay (at least) in the freezing will be the result. With the aid of a small spoon the ice and salt are in alternate spoonsful to be conveyed into the freezing-box of the machine, great care being taken that the cavity under the cutting-plate and around the tube be thoroughly packed, after which the uncovered portion of the box should also be well filled. The well is now to be filled with the strong gum to within a little distance of its top, and a piece of sheet gutta-percha (such as shoe soles are made of) being applied over the well, and kept in position by a weight, we must wait until the freezing commences. In a short time we shall notice that the gum has acquired a thick muddy appearance. The tissue must now, by means of the forceps, be transferred to the well, and there placed in such a position that the sections, when cut, shall run in the desired direction. After more gum has, if necessary, been added, so as completely to cover the tissue, the well is again to be covered, and attention given to the freezing-box. As the mixture which this contains becomes melted, it must constantly be renewed, care being at the same time taken that the mouth of the discharge-pipe be kept quite free, otherwise water accumulating in the box, the freezing mixture will degenerate into a useless puddle. When the gum becomes sufficiently hard to cut, this must be done much in the same manner as if paraffine had been used (§ 12). In this case, however, no fluid will be required, or must be used, to wet the knife with, and especial care must be taken that in disengaging the sections from the knife into the water they be not torn. These sections often adhere very tenaciously to the blade, but if a little patience be exercised the water will soon float them off in safety—much more safely than if any attempt be made to liberate them prematurely. There is one circumstance connected with the use of the freezing microtome which is rather annoying. The moisture of the breath and atmosphere is apt to become condensed on the cutting-plate, and here, mixed with accidental smears of gum, it becomes frozen into a jagged and irregular sheet of ice, which not only seriously interferes with the smooth play of the knife, but also constitutes a real peril to its edge. As this evil cannot be avoided, all we can do is, by constant wiping, to keep the cutting-plate clean and free from this accumulation. This is best done with a bit of soft rag just moistened with spirit, but this must not come into contact with any portion of the cylinder of frozen gum, else it will instantly thaw it. When using the freezing microtome it is always advisable to wear an apron, otherwise our clothes may receive considerable damage from the constant splashing of the salt water, as it falls from the waste-pipe into the vessel beneath it. After use, the microtome must be well washed in plenty of cold water till every trace of salt be removed, for if any of this remain it will quickly corrode the brass-work of the instrument. The plug and screw, as also the section-knife, should be well smeared with Rangoon oil before the machine is put away.
19. Logwood Staining.—The employment of logwood as a staining agent is now becoming very general. It acts much in the same manner as carmine, but the violet color which it produces is by many thought to be of a more soft and agreeable character than that due to the action of carmine. A valuable and very convenient property also which it possesses is that it stains tissues very rapidly, and this without interfering with that differential kind of coloration (§ 14) upon which the chief value of all staining processes depends. A simple method of preparing the logwood fluid is to mix an aqueous solution of extract of logwood with a solution of alum (1 to 8) till the deep impure red colour has become violet, and then to filter the mixture (Frey). This will stain sections in about half an hour. This stain, though here mentioned for the ease with which it may be made is, as a rule, very inferior to a fluid prepared directly from hæmatoxylon, the alkaloid or active principle of logwood. As, however, it is difficult and troublesome to make the solution in this manner,[[13]] it will be advisable for the student to purchase, ready prepared, such small quantity of the dye as he may require. Small bottles may be obtained for a few pence of Mr. Martindale, 10, New Cavendish Street, London, and from repeated trials of this solution we can recommend it as producing excellent results. It is a very strong fluid, and requires to be diluted before use. The degree to which the dilution must be carried cannot, however, be very accurately indicated, for all staining fluids of this nature possess the very undesirable property of becoming decomposed by age. After the fluid has been kept for some time, a portion of the colouring matter is thrown out of solution, and becomes deposited upon the sides and bottom of the vessel in which it is contained, hence the older the preparation, the weaker it will have become. As the time required for staining with logwood is but short, it is desirable that all the sections should begin to be submitted to its action at the same time, otherwise some will become more deeply stained than others. A good plan is to fill a small porcelain jar (§ 14) with filtered water, and into this transfer the sections. Whilst they are settling well down to the bottom, a mixture must be prepared of half a drachm of Martindale’s solution (fresh) to one ounce of distilled water, and everything got in readiness for its immediate filtration. The water is now very gently to be poured off the sections, and if care be exercised this may be done in such a manner as to leave them undisturbed at the bottom, after removing almost every drop of water. The diluted logwood fluid must now be immediately filtered upon the sections, so that they may run no risk of becoming dry. In the present instance the staining may be allowed to proceed for about thirty minutes, and this will be found a convenient time for the immersion of the general run of animal sections. If the logwood fluid be not quite fresh, either a little more of it will have to be added to the water, or the time of immersion must be prolonged until the desired depth of color has been produced. It is well whilst the staining is going on gently to shake the vessel occasionally, so that the sections may not remain in a heap at the bottom, but all be as fully as possible exposed to the action of the dye. When the staining is judged to be complete, the logwood solution must be gently poured off, leaving the stained sections at the bottom of the jar, when they should be quickly covered with methylated spirit, which will fix the colour. We shall now be able to see if the coloration obtained be perfectly satisfactory. If not deep enough, it is very easy again to submit them to the action of the dye for a few minutes longer. If on the other hand, and as more frequently happens, the coloration should be too deep, the excess of colour may readily be removed by transferring the sections for a short time into some diluted acetic acid prepared by adding five drops of the glacial acid to an ounce of water. The action of this should be carefully watched, and when the colour has been reduced to the desired tint the sections may be retransferred to the methylated spirit.