Microtomy
MICROTOMY (Gr. TOM; Tipveiv, to cut), the term applied to the preparation of minute sections of organic tissue for the microscope. In 1875 the methods were yet in their infancy; their development has enabled observers to achieve the most exact study of minute anatomy, in the case of small objects, which without these methods could only be investigated by the unsatisfactory process of focusing with the microscope through the solid object.
It is not necessary here to detail at length the wet method of preparing sections. Briefly, the tissue is soaked in a solution of gum, or of gum and syrup, and after being frozen by ether spray, or by a mixture of ice and salt, is cut into sections either by the Rutherford, Cathcart or some similar section-cutter, or by apparatus which can be fitted to the more modern types of microtome referred to below. This method, which is to-day used mainly by pathologists, has two main disadvantages: the prolonged action of watery fluids on the tissues, and the impossibility of getting ribbons, each section having to be picked up separately.
The general processes of the dry method employed in zoological and botanical microtomy are, up to a certain point, practically identified with those used for the preservation of animals and their tissues for other branches of microscopic work. In the first place the tissues must be killed; in the second, they must be fixed, i.e. the protoplasm must be set or coagulated as far as possible in the condition in which it appears in life; and in the third, they must be hardened, i.e. in most cases dehydrated. Killing may be effected by asphyxiation or narcotization (nicotine, cocaine, chloral hydrate, etc.) in special cases, but is generally achieved by fixing reagents, of which corrosive sublimate and other chlorides, picric, acetic, osmic and chromic acids, alone or in combination, chromates and strong alcohol are the most usual. These serve to a great extent also as hardening agents, but alcohol, used after them, completes this process effectively, and when not too strong (70%) is the best storage fluid. The second set of processes relates to the staining, without which transparent sections are almost invisible. The stains are divisible into general stains, which dye the tissue practically uniformly and indifferently; and selective stains, which have affinity for special tissues or cell elements. Of the latter group some fasten on nuclei, others only on the chromatin of the nuclei; some on connective tissues, others on muscle fibres and so on. It is probable that the action of all these selective stains is produced by definite chemical combination with compounds originally present in, generated in, or introduced into the tissue selected. The most generally useful stains for ordinary work belong either to the cochineal series (borax- carmine, carmalum, etc.), or to the logwood series (haematoxylin, haemalum, iron haematoxylin, etc.); in both of these great improvements have been introduced of late years by Dr Paul Mayer. The activity of these stains apparently depends upon the presence of alumina or of some similar base. For more special researches, such as cytology, neuropathology, neurohistology, and so forth, greater dependence is placed on the coal-tar colours, the name of which is legion. Some of these, such as safranine or gentian violet, are regressive stains; that is to say, the tissues are overstained uniformly, and the superfluous colouring matter washed out either by alcohol or by weak hydrochloric acid from the unselected parts. Others, such as methyl green, are progressive that is, the colour is brought up to the pitch required and the reaction promptly stopped. The coal-tar stains can be used singly, or in combinations of two or three. Some of the best, unfortunately, are not permanent. A third group of stains is furnished by such reagents as silver nitrate, gold chloride, and the like (impregnation stains), which can be made not only to stain, but also to deposit a fine metallic precipitate on certain structures. In the case of small and delicate objects, the staining is done in the mass before any further preparation for sections, but with larger animals, or large pieces of resistant tissue, the stain is applied to the sections only. The processes so far mentioned are applicable to many branches of microscopic work.
When preparing tissues for sections the first step is complete dehydration, generally effected by bringing the object into absolute alcohol. It is then transferred to one of a group of reagents, which are miscible with absolute alcohol, but would form an emulsion with water, and are solvents of the embedding medium. The embedding mass in most general use is paraffin wax, melting at a temperature of 54 to 60 C., according to the character of the object and the thickness of section required. The object is transferred from absolute alcohol to benzol, chloroform, cedar oil, or similar fluid to the melted paraffin; the fluid diffuses and evaporates, leaving the tissues to be completely permeated by the paraffin. This process can be greatly hastened by the use of a partial vacuum. When impregnation is complete the paraffin is cooled rapidly, so as to assume a homogeneous non-crystalline condition, and the tissue thus comes to form part of a block of soft but tenacious material, which protects it from damage by air or damp, and can be readily cut by a razor. The block is then trimmed to the form of a triangle or rectangle, and fixed by a clamp or by local melting in the holder of the microtome.
The first automatic microtome suitable for cutting a block of tissue into a continuous series of sections was made in 1883 in the university workshops of Cambridge, from a design by W. H. Caldwell and R. Threlfall. Only a single machine was made, but in 1884 twelve machines were made by the Cambridge Scientific Instrument Company from a design by Caldwell. Since then numerous excellent and simpler forms of microtome have been evolved. Some of these have distinct advantages over others, but with microtomes as with other tools the success of the results depends very largely on the manipulator, for every one works best with his accustomed instrument. In one type of microtome the razor is attached at one end only to a heavy block, sliding backwards and forwards in a horizontal V-groove; the paraffin block is fed to this either up a vertical guide (Schanze, Reichert, etc.) or up an inclined plane (Thoma-Jung). In another type the razor is firmly clamped at both ends, to diminish vibration, and the paraffin block advances to it at the end of a long lever on trunnion bearings (Cambridge rocker) or up a vertical guide (Minot types).
In the selection of a microtome, apart from its steadiness, rigidity, accuracy of workmanship, and so forth, it must be borne in mind that, in general, simplicity of working parts means longer life, and that an elaborate " automatic " mechanism, by which a single movement is translated into several in different directions, not only complicates the machine, but robs the operator of those alterations of pace, rigidity, pressure, etc., which are often necessitated by the varying texture in different parts of the object cut. For general use by less skilful students in a laboratory, price, simplicity and rapidity of work recommend the rocking microtome of the Cambridge Scientific Instrument Company, but it tends to fail at large or hard objects. For the all-round work of an investigator, its simplicity and finish have made Jung's sliding microtome with the Naples improvements deservedly popular for many years; it can be fitted with special apparatus for cutting celloidin and frozen objects, and it can be relied upon to cut any tissue, however difficult; but it cannot be worked as rapidly as some others, nor produce long ribbons of large objects. For this latter purpose the Minot-Becker, Minot-Zimmermann and ReinholdGilltay have been strongly recommended; these, however, are all of more complicated construction, with corresponding liability to uneven wear and damage; they are highly " automatic," leaving nothing but pace under control of the operator, and they are (particularly the last) expensive.
[In 1910 the Cambridge Scientific Instrument Company issued a new microtome designed primarily for cutting larger sections than was possible in their earlier forms, which respectively dealt with sections 12X20 mm. and 30 mm. in diameter; the new instrument cuts sections measuring 150X120 mm. (6X4! in.) embedded in paraffin or celloidin and of a thickness varying from 0-002 to 0-06* mm., each division of the scale being equal to 0-002 mm. and the total distance of automatic feed being 21 mm. The construction and action of the instrument can be understood by referring to the figure; a detailed description is given, since the same principles are utilized to a greater or less extent in all sliding microtomes.
Large Sliding Microtome.
The object to be cut, having been embedded in a suitable preparation A, is fixed to a wooden block which is attached by clamps to the object-holder B. The object-holder is provided with mechanism by means of which the height of the block is determined; this is effected by mounting the holder in a cup-shaped socket at the extremity of a brass pillar E, which can be raised or lowered and fixed in any position by a clamp. In addition, the direction in which a section is cut can be varied by adjusting the four screws, one of which is shown at C, which orientate the block. The objectholder and feeding mechanism are carried on a sliding carriage which rests at three points on two guides in the frame N, NI of the instrument; and in order to secure easy running the necessary lubrication of the bearing surfaces is provided for by a groove in which oil is placed. The motion of the carriage in either direction is effected by the handle G, connected to a system of levers H, which, being constructed on geometrical principles, prevent any side-play and ensure a uniform motion. The arrangement for determining the thickness of the section cut consists of a stop-pin, which, operating through the ratchet M, causes a toothed wheel to revolve, which in turn raises the pillar K; the amount of the motion can be read off by an index. On the return stroke of the sliding carriage the stop-pin is again actuated in such a manner that just before the knife R reaches the object-holder the mechanism depresses this part of the instrument so that the knife is not fouled ; and after its passage the object-holder is raised to the position appropriate for taking the next section. The knife R is rigidly set in two heavy brass clamps adjustable by the screws S, and these clamps are attached to the frame of the instrument by the screws T. The angle which the cutting edge makes with the frame is also adjustable, and by means of a small angular scale engraved on the knife-holders any setting can be easily determined or repeated. The knife is flat on one side and hollow-ground on the other. In using the microtome it is essential that the cutting edge of the knife points towards the end of the instrument where the handle is placed; the hollow-ground face should be uppermost, and the flat surface should not be exactly horizontal but slightly inclined so that the lower facet of the cutting edge is parallel to the frame. As to the relation of the position of the knife to the direction of motion, it is the usual practice, when paraffin sections are to be taken, to have the cutting edge at right angles to the motion; when, on the other hand, celloidin preparations are being cut, the knife must be set obliquely across the frame, an angle of 30 being convenient. This oblique setting is also recommended for paraffin sections. In addition it must be remembered that celloidin preparations always require lubricating when being cut, and it is also necessary to keep both the knife and the preparation constantly moistened with either 80 % alcohol or with cedar-wood oil.] The sections, when cut by the microtome with the knife straight and the two sides of the rectangular paraffin block parallel to it, in most cases can be got off in a continuous ribbon, each sticking to its predecessor. This very desirable result generally can be insured by a coating of softer paraffin; but if the object be large, or brittle, or of varying texture, it is safer to cut the sections singly from a triangular block with an oblique knife. The sections or ribbon are often not quite flat, but rolled, creased or compressed; they must be flattened before being attached to the slide. It is possible to carry out these two processes simultaneously by covering the carefully cleaned slide with plenty of a very dilute solution of Mayer's glycerine and albumen, and laying the sections on the fluid and the slide on a hot-plate; as the water becomes warm the sections flatten out, and as it evaporates they settle down on the slide, and are held there by the albumen (many other methods are in use). The slide is then warmed to melt the paraffin, and plunged into benzol, or some similar fluid, which removes the paraffin; thence into absolute alcohol, which dehydrates and coagulates the albumen. If the tissue has not been stained en bloc the sections can now be stained on the slide. After staining they are fully dehydrated, rendered transparent by oil of. cloves, and mounted in xylol-dammar or Canada balsam. W. Giesbrecht was the first to fix sections on the slide, using a solution of shellac in creasote in 1881; and also in the same year and in the laboratory of the Naples aquarium, W. H. Caldwell first cut and fixed ribbons of sections.
For ordinary work the paraffin method excels all others for rapidity, certainty and cleanliness; but for large and hard objects, or crumbling tissues (such as ova with a large quantity of yolk), some manipulators prefer to embed in celloidin. By this method, after dehydration, the tissue is soaked in a mixture of absolute alcohol and ether; thence transferred either to increasingly strong solutions of celloidin in the same mixture or to a thin solution which is then boiled down till strong. The celloidin mass is then hardened: at first, if necessary, by drying; afterwards by a bath of chloroform or its vapour. It can then be cut in the microtome, either wet, or (if previously cleaned with cedar oil) dry like a paraffin block. The method is more tedious and more messy than the paraffin process; but amongst its advantages must be reckoned that little or no heat is required, and that the embedding mass is transparent, though it does not allow of such thin sections as paraffin.
The above accounts present an outline of the complex processes employed to-day, by which, on the one hand, sections 30 ju in thickness may be made through the entire human brain; and, on the other, organisms invisible to the naked eye may be cut into a long ribbon of consecutive sections i n (one-thousandth of a millimetre) thick, every minutest fragment being retained in its proper place.
The standard book on the subject is Bolles Lee's Microtomist 's vade-mecum. Other works are G. Mann, Methods and Theory of Physiological Histology (Oxford, 1902), and A. Flatters, Methods in Microscopical Research (London, 1905). (G. H. Fo.)
Note - this article incorporates content from Encyclopaedia Britannica, Eleventh Edition, (1910-1911)
