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.

20. Absolute Alcohol.—As we purpose mounting the sections which have just been stained, in Canada balsam, we will briefly consider the preliminary treatment to which they must be submitted before this can be effected. The object of this is to abstract from the tissue all its water, for if any moisture be permitted to remain in the section it will, when mounted in balsam, become obscured and surrounded by a kind of opalescent halo, due to the imperfect penetration of the balsam into the only partially dehydrated tissue. The old-fashioned plan of dehydration was simple exposure to the air. The method now generally adopted is to bring about the same result by means of absolute alcohol. This fluid has such a strong affinity for water that tissues submitted to its influence are rapidly and effectually deprived of any water they may contain. Absolute alcohol in small quantity may be obtained from the druggist at about sixpence per ounce. It will be necessary for the student to provide himself with a little of this agent, say about two ounces, the method of using which will very shortly be explained. Absolute alcohol must be kept in a bottle with a very accurately-fitting stopper, in order to prevent its absorbing moisture from the air. For our purpose such a bottle, having a neck as wide as possible, is to be selected.

21. Clove Oil.—After being thoroughly dehydrated the sections may, in special instances (§ 48), be at once mounted in balsam; but, as a general rule, it will be found necessary (particularly in the case of animal sections) to treat them with some clarifying agent, in order to remove the cloudiness and opacity which is (in part) due to their previous immersion in alcohol. For this purpose turpentine, or any of the essential oils, may be used: of these, oil of cloves is to be specially recommended. It is rather expensive, ranging from sixpence to one shilling per ounce: but, as a drop or two will be sufficient for preparing each slide, only a small quantity—say half an ounce or an ounce—need be procured. The most convenient vessel in which to keep the oil is one of the small test bottles used by watchmakers. These bottles are provided with a glass cap to exclude dust, and the stopper is prolonged into a glass rod, which dips into the bottle. The use of this rod and the method of employing the oil will be explained shortly.

22. Canada Balsam, as ordinarily met with, is a thick resinous balm of great viscidity, but readily rendered perfectly fluid by the application of heat. Formerly, sections were mounted in this medium in its pure state, but owing to the annoyance which was so constantly being experienced from the tenacity with which intruding air-bubbles were held by the viscous medium, this plan of mounting is rapidly falling out of use.^[14] It is now usual to employ the balsam in a diluted condition, the two chief diluents being chloroform and benzole. As balsam, however, often contains more or less moisture, it is desirable to drive this off before adding the diluent. A very convenient way of doing so is to expose some pure balsam to the heat of a cool oven for several hours, when the balsam will be found to have assumed a hard, vitreous character. It should now be broken into small pieces, these put into a bottle, and some methylated chloroform added, which in a little while will completely dissolve the hardened balsam. More chloroform is then to be added, until a solution is obtained sufficiently thin to run through filtering-paper. A glass spirit lamp must now be procured, having a capacity of about two ounces, and provided with a cap. Into the wick-holder of this (which must be made of porcelain) a hollow glass tube is to be so fitted that its end dips into the lamp to within about a third of the bottom. (See Fig.) The thin chloroform-balsam is now to be filtered into this lamp, very fine filtering-paper—through which a little chloroform has first been passed—being used for the purpose. When the lamp is full it must (deprived of its cap) be put in a warm place until sufficient of the chloroform has evaporated to leave behind it a fluid of the consistence of thin syrup.

23. Mounting in Balsam.—Let us now return to our sections which, it will be remembered, were left in methylated spirit (§ 19). These we will now mount in balsam, and although, of course, any number may be proceeded with at the same time, yet to avoid confusion, in the following directions one section only will be spoken of. This section then is, with the perforated spoon, to be transferred to the bottle of absolute alcohol, where it may remain for about an hour—considerably less time is actually required, but as from constant use the spirit becomes weakened, it is as well to be on the safe side. It must now be removed to the centre of a clean glass slip, and here the plain end of the spoon comes into use. If this be employed for effecting the transfer, it will be found that when the section is being removed from the alcohol it will bring along with it a small pool of the spirit. A slight touch of the needle applied to the edge of the section will cause it to float from the spoon on to the slide, at the same time carrying the pool of alcohol with it, in which it will gently spread itself out upon the slide without the faintest risk of injury. The superfluous spirit is now to be drained off, and just as the section is becoming glazed and sodden-looking (not dry) we must, by means of the long glass stopper (§ 21), apply to it a large drop of clove oil. The oil, however, should not be placed on the section, but be allowed to drop on to the slide near to its margin. By gently tilting the slide the oil will gradually insinuate itself beneath the section and slowly ascend through it to the surface. The slide should now be covered with a bell-glass (or wine-glass), and about two minutes allowed for the oil thoroughly to saturate the section. As much as possible of the superfluous oil must then be drained off, and the remainder removed with blotting paper. By means of the glass rod a small quantity of chloroform-balsam is now taken from the spirit lamp which contains it, and allowed gently to fall upon the section, which must then be covered with a thin glass circle in the manner previously described (§ 16). When the object is very fragile, it is a good plan, after draining off the clove oil, to apply the cover directly upon the section, and then to place a drop of the balsam near to the edge of the cover. This, by capillary attraction, will speedily diffuse itself beneath the cover, flowing over and surrounding the object, without in the slightest degree disturbing its position. If, during the process of mounting, any air-bubbles arise, we may view their development with equanimity, being well assured that as the chloroform evaporates they too will quickly disappear. When the mounting is completed, the slide should be roughly labelled and placed on a warm mantel-piece for a few days to dry.

24. Finishing the Slide.—In the course of two or three days it will be advisable to take an old penknife, and after heating the blade in the flame of a spirit lamp, gently to run the point of it round the margin of the cover, so as to remove any excess of balsam which may have oozed from beneath it. In a few days more, any remaining balsam may be carefully scraped away with a cold knife. All remaining traces of balsam are then to be removed from around the cover by means of a rag just moistened with methylated spirit, or, what is better, with a mixture of equal parts of spirit and æther, after which the slide is to be thoroughly washed in cold water. The slide is now in reality finished, but, in order to give it a smart appearance, it is usual, with the assistance of the turn-table, to run a ring of colored varnish round the covering glass. A very useful varnish for the purpose is the white zinc cement. To prepare this, dissolve an ounce of gum dammar in an ounce of turpentine by the aid of heat. Take one dram of oxide of zinc and an equal quantity of turpentine; rub them up together in a mortar, adding the turpentine drop by drop, so as to form a creamy mixture perfectly free from lumps or grit. One fluid ounce of the dammar solution previously made must now gradually be added, the mixture being kept constantly stirred (Frey). The cement, when made, should be strained through a piece of fine muslin, previously wetted with turpentine, into a small wide-necked bottle, which, instead of having a cork or stopper, should be covered with a loose metal cap. Instead of a bottle, the varnish may be kept in one of the collapsible tubes used by artists; but though this plan is highly recommended by many, it is not without its disadvantages. If the varnish becomes thick by keeping, a few drops of turpentine or benzole well stirred in will soon reduce it to a suitable consistence for use.