Note:—When a number of pieces of tissue are blocked at the same time, either in celloidin or paraffin, they may be tagged with paper labels fastened to the tissue with a drop of gum tragacanth or gum arabic. As the gum is not soluble in any of the infiltrating and imbedding media the labels remain attached until the block is ready for cutting.
CHAPTER XXIII.
SECTION-CUTTING.
Fixed and hardened tissues may be sectioned on a microtome without freezing or imbedding, or they may be cut on a freezing-microtome; or, having been imbedded in either celloidin or paraffin, they may be sectioned on any form of microtome suited to the purpose desired. The choice of the microtome depends, therefore, upon the character of the work. For ordinary purposes the small sliding microtomes furnished with a crank are preferable, as they are more easily kept in order and can be used for rapid cutting. For serial paraffin sections the Minot automatic rotary microtome is advised. Especial types of microtomes can be obtained for the cutting of large sections, particularly for brain-sections. The Bardeen freezing-microtome I have already recommended as the most practical instrument for freezing work. In all cases it is absolutely necessary that the instruments be in good order and that they work with precision. The microtome knife must be carefully honed and stropped. A heavy, rigid, biconcave knife-blade should be used, and a good hone and strop are absolute necessities. In honing, the knife is drawn from heel to toe with cutting edge forward; while, in stropping, the motion is reversed, the back of the knife being forward, and the motion from toe to heel. The knife during the honing should be fitted in a knife-holder, and the back of the blade protected from the hone by a knife-back. The knife-blade must be kept free from grease and dirt, and nothing must be permitted to touch its edge. When many sections are cut at one sitting frequent stropping is necessary. When the cutting is finished the knife should be removed from the microtome, carefully cleaned and dried, and placed in its box. The slide and other bearings of the microtome should be well-oiled with the best microtome oil, and kept free from dust.
1. Cutting of Fixed Tissues Without Imbedding or Freezing. It occasionally becomes necessary in pathologic work to cut tissues directly upon the microtome without freezing or imbedding. This can be done satisfactorily in the case of very firm substances, such as amyloid liver and spleen, etc. The sectioning is done in the wet, using 80 per cent alcohol, as in celloidin cutting. A large celloidin knife should be used, and the blade should be flooded in alcohol. A large piece of the tissue, or, better, several pieces, are clamped in the object holder with only a small layer of tissue above the holder. The knife-blade is placed at the least possible angle to the pieces of tissue. To obtain sections of 15-20 microns in thickness the object-holder is raised each time about 25-30 microns. The sections will vary in thickness, some thin, others very thick.
2. Sectioning of Frozen Fixed Tissues. Tissues fixed in formol, Müller’s fluid or Orth’s fluid may be frozen directly on the freezing-microtome without previous washing. For other fixations previous washing is necessary. Tissues in alcohol must have the alcohol thoroughly washed out before freezing. Celloidin blocks may also be cut by freezing after the removal of alcohol. The fixed tissues cut to the proper size are placed on the object-holder of the freezing-microtome in a drop of saturated gum-arabic and the freezing carried out as directed above. (See Page [214].) As the freezing causes little or no damage to fixed tissue, the frozen sections may be transferred directly to alcohol, or floated out on the dilute molasses or sugar-dextrin solution, and stained separately or in sheets, according to the directions given in the next chapter.
3. Sectioning of Celloidin Blocks. The celloidin blocks are fastened in the object-holder of the microtome, and the knife-blade (a longer and broader one than for paraffin-sectioning is advisable) set nearly parallel with the longitudinal axis of the microtome, so that the cutting-edge, striking the block at a very slight angle, will be utilized for the greater part of its length in cutting entirely across the surface of the block. Both object and knife-blade must be kept constantly wet with 80 per cent alcohol; a broad camel’s-hair brush or a drip-bottle can be used for this purpose. The sections, as they are cut, are transferred from the knife to 80 per cent alcohol by sweeping the finger or brush from above toward the edge of the blade. Care must be taken not to strike the brush against the edge of the blade, and this can be avoided by using the brush always with a downward stroke. As the sections are cut they are guided onto the knife-blade by means of a fine-pointed camel’s-hair brush, if the sections show any tendency to curl. They should be smoothed out at once on the knife, and then transferred to the dish of 80 per cent alcohol. Sections 7-10µ thick are easily cut in celloidin, if the process of imbedding has been carefully carried out.
To prepare serial-sections from celloidin blocks the sections as they are cut must be arranged in their order on the knife-blade and thence transferred in this order to a slide or glass-plate, to which they are either fastened so that they cannot become loose during the staining process, or they are fastened together by a film of celloidin and stained in one piece. To fasten the celloidin sections to the slide Mallory and Wright suggest the cutting of the block in 95 per cent alcohol, and the arrangement of the sections in their order on the knife, whence they are drawn on to a dry, clean and numbered slide. Ether vapor from a half-full bottle of ether is then poured over the slide, slowly, to flatten out the sections and fasten down the frilled edges. The slides are then transferred to 80 per cent alcohol to harden the celloidin, and they are kept in this solution until ready for staining. Celloidin sections may also be fastened to the slide by albumin glycerin (equal parts white of egg and glycerin with a crystal of phenol or thymol). The celloidin sections, as they are cut, may be transferred to a dry gelatinized slide or plate (16 grms. gelatin in 300 cc. warm water), and are then covered with a thin celloidin; the slide is then placed in water at 50°C., the gelatin dissolves and the celloidin film floats off; the latter is then stained as one section. Serial sections of celloidin-blocks may also be arranged upon slides or plates covered with a coating of the Schmorl-Obregia sugar-dextrin solution or diluted New Orleans black molasses; the slide or plate is flooded with absolute alcohol; drained; a thin celloidin is then poured over it; it is immersed in warm water and the celloidin-sheet containing the sections is liberated, and is then stained as a whole or cut into strips as desired. The molasses method is the cheapest and simplest method, and much to be preferred to the Weigert closet-paper method, by which the sections are arranged upon a strip of moist closet-paper which is either held upon the slide or kept upon a piece of blotting-paper wet with 80 per cent alcohol. A slide or glass-plate is covered with a thin layer of celloidin and the strip of paper containing the sections is laid upon the celloidin surface, the sections down, so that these stick to the slide as the paper is carefully peeled off. The preparation is dried with filter-paper and a thin layer of celloidin poured over the slide and sections. The celloidin is then hardened in 80 per cent alcohol and the sections stained on the slide; or the celloidin film is removed by immersion in warm water, and then treated as one section. Celloidin films may be marked with a brush dipped in a water solution of methylene-blue. This should be done as soon as they are made. Langhans’ method is advised for the remounting of serial sections from tissues stained in bulk and imbedded in celloidin. The sections are cut in origanum-alcohol (1 part absolute alcohol to 3 parts origanum oil) and placed on a slide in a layer of origanum oil, blotted and mounted in balsam. Should sections become milky in the oil, renew the latter until they are cleared.
4. Sectioning of Paraffin Blocks. The paraffin block trimmed to the proper proportions, leaving a rim of paraffin about 2-3 mm. wide all about the tissue, is fastened to the wooden block by melting the under side of the paraffin by drawing a hot knife across it, and then immediately pressing the block upon the wood. The wooden block is then clamped into the object-holder. If desired the paraffin block can be fixed directly to the metal plate of the object-holder in the same manner. The block is raised until the level of the edge of the knife is reached. The knife is placed transversely or at a slight angle, and the cutting done with a relatively small portion of the edge. The paraffin block may be breathed upon to warm slightly the upper layer when a hard paraffin is used, and the knife is then drawn carefully through the block, guiding the section onto the knife-blade by means of a fine-pointed camel’s-hair brush held in the left hand. It is preferable, I think, to have a wider rim of paraffin at one corner of the block and to place the block with that corner at a slight angle to the knife, so that the edge of the blade will first engage the block at that point. The tip of the camel’s-hair brush moistened in water catches the corner of the section as the knife begins to cut, and pushes it over onto the blade, thus holding the section flat and preventing curling. When entirely cut through, the section is removed from the knife by the brush, still holding it at the corner first touched, or if necessary it is again moistened and applied to the upper side of the section, lifting the latter off the knife. The block is trimmed down to the proper level by cutting thick sections first, then sections of the desired thickness when the level of the entire block is reached. The sections are then transferred, with their shiny side down, just as they come off the knife, to a slide, sheet of paper, warm water, warm molasses or sugar-dextrin solution, or 70 per cent alcohol, and treated further according to the directions given in the next chapter. The presence of ridges on the cut surface of the block is an indication that the knife needs stropping. The knife must cut the paraffin, not scrape it. Crumbling of the paraffin is the result of imperfect infiltration during the imbedding process; water, alcohol, aniline oil or xylol may be left in the tissue, the paraffin may contain oil, or the cooling process may have been delayed. Curling of the sections can be prevented by using a sharp knife and slightly warming the surface of the block by breathing upon the block, use of a warm knife or spatula, heat-focus, etc. For ordinary work paraffin sections 5-7µ thick are easily obtained; for especial work 1-2µ sections may be obtained by especial care in imbedding, using graded paraffins and finally imbedding in a 56°C. paraffin. The pieces of tissue should be small, and the sections may be cut with a knife wet with water or alcohol. Serial paraffin sections are easily obtained; in fact, paraffin imbedding is by far the best method for serial cutting. To cut ribbons of sections upon the ordinary slide microtome the block must be clamped into the object-holder so that the edge of the block facing the knife, as well as the opposite side of the block, is parallel with the knife-edge. The knife should be placed at right angles to the microtome. If the paraffin has the right consistency the edges of the sections as they are cut will adhere, and a ribbon of serial sections will be pushed up over the knife. This ribbon can be cut into pieces of the desired length and mounted according to the directions given in the next chapter. The Minot automatic rotary microtome is especially well adapted for the cutting of ribbon sections. Failure of sections to adhere to each other is usually the result of too hard consistence of the paraffin, and this can be remedied by warming slightly, according to the method given above. If the paraffin is too soft the sections fold together. The block may be cooled in ice-water, or the sections may be cut with a cooled knife. Paraffin knives so constructed that they may be cooled by a stream of ice-water are supplied by dealers in microtomes. The conditions essential for successful paraffin cutting are perfect infiltration and imbedding, sharp clean knives and a certain degree of skill in manipulation that can only be secured by practice.
CHAPTER XXIV.
THE PREPARATION OF MOUNTED SECTIONS.
Sections of fresh material, unimbedded or imbedded tissues must be treated by a series of processes before they are finally permanently mounted and ready for use. These processes in general are: preparation for staining, staining, differentiation, washing, dehydration, clearing and mounting. The general procedure will be modified to some extent by the character of the tissue, manner of preparation (fixed or unfixed, imbedded or unimbedded, unstained or stained), character of stain (affected by alcohol, xylol, etc.), and the mounting agent (glycerin, balsam, damar, colophonium). Two or more of these steps may be combined in one; the same agent may differentiate, dehydrate and clear. Several stains may be combined in one solution, or it may be necessary to use them in succession. The very greatest variation is possible in pathologic technique; in fact, practically every laboratory worker modifies methods according to the light of his individual experience. The really important thing is to be master of the method, and not allow the method to control the situation. One of the greatest attractions about laboratory work is the infinite possibility of variation and improvement of methods and the invention of new ones.