SECTION OF MICROTOME-TUBE SHOWING ARRANGEMENT (A) TO PREVENT “RISE” OF PARAFFINE.
9. Æther Microtome.—A word here as to freezing microtomes, where the agent used is æther. Such as have fallen under our notice have not answered the expectations we were justly entitled to form of them. That it is possible to freeze a piece of tissue by their use is undeniable, but it is, as a rule, at an expenditure of such a quantity of æther (only the very best of which must be used) as to constitute it a very expensive proceeding. Another serious disadvantage they possess is, that if the supply of æther be intermitted for only a very short time, the already frozen tissue thaws with great and most inconvenient rapidity.
10. Section-Knife.—Of not less importance than the microtome is the section-knife, to be used in conjunction with it. How perfect soever the former, and whatever the dexterity of the operator, unless he be provided with a suitable and well-made knife, he will never succeed in obtaining satisfactory results. As to the most desirable size of the knife, much difference of opinion seems to exist, section knives varying in this respect from a blade of extreme shortness to one which fell under our observation, in which the portentous length of thirteen inches was attained. What advantages were to be expected by prolonging the blade to this extravagant length, must remain an inscrutable mystery to all save its designer. Concerning the shape of the knife, it is frequently advised that the surface which has to glide along the cutting-plate of the microtome should be ground flat. A most unsuitable arrangement, as a very little actual experience of section-cutting will speedily demonstrate. After many unsuccessful attempts to obtain a really good and reliable section-knife, we determined to have one specially made, which, as it has proved everything that could be desired, merits a brief description. It is of the utmost importance that the blade be made of good and well-tempered steel, not only that it may be capable of receiving an edge of the most exquisite keenness, but also that it may retain it. The knife of which we speak (and which was made by Mr. Gardner, of South Bridge, Edinburgh) is furnished with a blade four inches long, and 7/8-inch broad, set into a square handle of boxwood, also four inches in length. The thickness of the blade at the back is not quite 1/4-inch, while both of its surfaces are slightly hollow ground. It is essentially necessary that the back and edge of the blade be strictly parallel to each other, otherwise the knife, when in use, will have such a tendency to tilt over as to render its management extremely difficult. It is very easy to discover if this condition be fulfilled, for if on carefully laying the flat of the blade upon a piece of level glass, every portion of both back and edge are found to be in close contact with it, the knife may in this respect be considered perfect. Every student who aspires to be a successful section-cutter should provide himself with a good Turkey oilstone, and learn to use it. He should also possess a razor strop, as it will be in constant requisition.[[7]] It may here be remarked that though razors, as a rule, are unsuitable for use with the microtome from want of uniformity in the thickness of their blades, yet, if only a small object is to be cut—for instance, a thin root or stem—very good results may be obtained from their use, especially if one of the old-fashioned make, having a thick back and slightly concave surfaces, be employed.
11. Imbedding in Paraffine for Microtome.—Having described at some length the various instruments necessary for section-cutting, we will now consider how they are to be used. Let us endeavor to cut some sections—say of a piece of kidney—and in so doing we will adopt the “paraffine” method of imbedding. Ordinary paraffine, however, when used alone, is rather too hard for our purpose. In order, therefore, to bring it to a suitable consistence, it must be mixed with one-fifth its weight of common unsalted lard, a gentle heat applied, and the two thoroughly stirred together. A quantity of this should be prepared, so that it may always be ready when wanted—it is very conveniently kept in an ointment pot or preserve jar, the top of the latter being well covered, to keep out the dust. When it is intended to use this mixture for the purpose of imbedding, only just about the quantity required should be melted; for in doing this it is advisable to use as low a degree of heat as possible, not only to prevent injury to the tissue to be imbedded, but also that the paraffine when cooling may not undergo such an amount of contraction as to cause it to shrink from the sides of the microtome-tube. It is therefore a good plan to effect the melting in a water-bath, a simple kind of which, something after the fashion of a glue-pot, would be made for a few pence by any tinman.
The kidney which we are about to cut has, of course, gone through the process of hardening already described (§ 5), and is now preserved in spirit. A small piece, say half an inch square, is selected, removed with forceps, and placed on a bit of blotting paper, when the surface of the tissue will rapidly become dry (only the surface must be allowed to dry). It is the usual plan now to proceed at once to imbed it in the melted paraffine. This is a most undesirable step, and gives rise at a later stage of our proceedings to a great amount of trouble and annoyance, for after sections have been cut from a tissue so imbedded it will be found that portions of paraffine adhere to their edges with such tenacity that in the case of many of them there is no effectual method of removing the paraffine, short of soaking the sections in warm æther; a very objectionable proceeding, for though the æther will undoubtedly remove the paraffine, it will also dissolve out any fatty matters which the section itself may naturally contain. All this annoyance may be prevented by subjecting the tissue to a simple preparatory treatment before it is imbedded in the paraffine. For this purpose prepare a very weak solution of gum arabic in water—twenty grains to the ounce. Into this, by means of the forceps, dip for a few moments the already surface-dried tissue, taking special care not to squeeze it, or the pressure will cause the spirit from its interior to remoisten the surface, which would prevent the gum from adhering. We shall see the value of this a little later on. Remove the tissue from the solution on to blotting paper, when the superfluous gum will speedily drain off, and in two or three minutes the surface will have become quite glazy and dry. Having melted some paraffine mixture in the water-bath, the tissue held in the forceps must be plunged for an instant into the heated liquid and immediately withdrawn, when the crust of paraffine with which it is enveloped will promptly harden. Whilst this is taking place we may make ready the microtome. Having by means of the milled head or handle depressed the plug in the tube so as to leave a free opening about an inch deep at its upper end, we must pour in the melted paraffine, which by this time will have become a little cooler, until the cavity be about half filled. The prepared tissue must now be introduced, care being taken to place it in such a position that the sections may be cut in the desired direction. The tissue must, if necessary, be held in position with forceps or a needle point, till the imbedding material becomes hard enough to give it due support. It is here to be remembered that it will not be advisable to place the tissue in the centre of the tube—it will be much more easily cut if placed rather nearer to that edge of the tube which is situated next the operator in the act of cutting. More paraffine is to be slowly added, until the tissue is completely covered; even after this still more should be added, for it will be found that in cooling the paraffine shrinks so as to leave a cup-shaped depression in its centre, whereby portions of the tissue which were previously covered are again laid bare. The best method of preventing this is to use the paraffine at as low a temperature as possible, and to use plenty of it. The microtome, with its contents, must now be removed to a cool place, when the paraffine will soon become solidified. Whilst this is being accomplished we may make our further preparations. The first thing we require will be a large basin, full of freshly-filtered water, and provided with a cover. A small beaker of methylated spirit, with a dipping rod or pipette, will also be necessary. We must now see that the section-knife is in thorough order, to ensure which it will be advisable to give it a few turns on the strop. An ordinary razor will also be of service.