blotting-paper, exerting an even but not great pressure with the ball of the thumb. The section will adhere to the slide.1 7. Without allowing the section to become too dry, pour onto it immediately a small quantity of absolute alcohol sufficient to cover the section and adjacent parts of the slide. After a few seconds drain off the alcohol. 8. Flow from a bottle quickly and evenly over the section and adjacent surface of the slide a thin solution of celloidin in equal parts of absolute alcohol and ether, drain off immediately, blow the breath briskly once or twice on the surface of the section, and immerse the slide immediately in water for a few seconds. Thus a thin film of celloidin is formed, which fastens the section to the slide. The solution of celloidin should be almost watery in consistence, and so thin that it will form drops readily without stringing. If it is too thin, however, it will not hold the specimen on the slide, and if it is too thick, the layer on the slide will become white when the slide is immersed in water. The film of celloidin on the slide should be so thin as to be almost invisible. 9. Stain the section by any of the usual methods applied to sections affixed to the slide. The thin layer of celloidin offers no obstruction to the staining of the section. The staining fluid may be heated on the slide, as in the staining of tubercle bacilli with fuchsin, without loosening the section. The most satisfactory staining of these sections for general purposes is with Delafield's hematoxylin, followed by eosin. The various methods for staining bacteria in tissues are applicable to these sections. 10. Dehydrate by flooding the section and adjacent portions of the slide with absolute alcohol. This will remove most of the celloidin. It will not, however, loosen the section unless its action is unusually prolonged. II. Clear by flooding the preparation and adjacent parts 1 If the tissue contains much mucoid material or if it has not been thoroughly fixed, the section may stick to the blotter and not to the slide. This difficulty will be overcome by covering the section with absolute alcohol for a few seconds just before blotting it, draining off, and then proceeding as indicated in this step. of the slide with oil of origanum. If parts of the section refuse to clear, apply alcohol again and then more oil of origanum. When clear, drain off the oil, blot with a piece of dry blotting-paper, and mount in balsam. When mounted, none of the celloidin will be visible if the proper technique has been employed. The staining fluids, alcohol, and oil of origanum are conveniently applied from drop bottles. Boiling. Boiling precipitates the soluble albumin in tissues as a granular material which can be readily recognized. The method is used particularly for the demonstration of albumin in renal diseases and in edema of the lungs. By means of boiling the quickest permanent mounts of tissues can be obtained. The method is not advocated on account of the shrinkage caused by the heat, but will sometimes be found useful. Small pieces of tissue not over 1.5 cm. in diameter should be dropped into the boiling water for one-half to two minutes; cool quickly in cold water, and make frozen sections, or put into 80 per cent. alcohol. Any stain can be used; methylene-blue will be found excellent. Harden tissues six to eight weeks. Change the fluid daily during the first week; once a week thereafter. Ordinary tissues are then washed in running water overnight before being placed in alcohol. Nervous tissue is transferred directly from the fluid to the alcohol. This famous hardening solution seems destined before long to give way entirely to better fixatives. It hardens tissues slowly, evenly, and with little or no shrinkage, but it is a poor nuclear fixative, and does not encourage any great variety of stains. The sulphate of sodium seems to serve absolutely no function. For ordinary tissues it will undoubtedly be replaced by Zenker's or Orth's fluid, both of which fix very quickly, besides having all its good qualities. For nervous tissues formaldehyde followed by other solutions of the chrome salts is a great deal quicker and better. Tellyesniczky has recently recommended the following mixture, which has met with considerable favor, and which may be regarded as an improved Müller's fluid: Fix thin sections for one to two days; thicker sections longer. Wash out thoroughly in running water. Dehydrate in graded alcohols. Marchi's Fluid. Müller's fluid, Osmic acid, I per cent. aqueous solution, 2 parts; I part. Place small pieces of tissue in the mixture for five to eight days, wash thoroughly in running water, and harden in alcohol. For its application to degenerated nerve-fibers see page 348. DECALCIFICATION. TISSUES which are to be decalcified should be sawn with a fine hair-saw into thin slices, so that they will decalcify quickly. It is usually desirable to saw the tissue into pieces of proper size for imbedding in celloidin. Very dense bone. ought not to be over 2 or 3 mm. thick; softer tissues do not need to be thinner than 4 to 6 mm. In cutting sections after decalcifying and imbedding it is necessary to throw away the first half-dozen sections or so, because the tissue is so lacerated to a slight depth by the movement of small fragments of bone in the process of sawing as to be useless for microscopic purposes. The extent of the decalcification can be tested at any time by thrusting a needle into the tissue. The following steps in the decalcification of tissues must be carefully borne in mind. 1. The tissues must first be thoroughly hardened. The three most useful reagents for this purpose are alcohol and Zenker's and Orth's fluids. After the two latter reagents the tissues must have been washed thoroughly in water and placed in alcohol for at least twenty-four hours. They will then be ready for decalcification. 2. The decalcifying fluid must be used in large amounts, and, if necessary, be frequently changed. 3. After decalcification the tissues must be thoroughly washed in running water for twenty-four hours to get rid of every trace of the acid. 4. The tissues finally must be hardened again in alcohol. Of the various agents used for decalcifying bone, nitric, hydrochloric, chromic, picric, trichloracetic acids, etc., the most important is nitric acid. It acts quickly, without swelling the tissues or attacking injuriously the tissue-elements, and does not interfere to any marked degree with any subsequent staining process. Red blood-globules will be found uninjured in tissues hardened in Zenker's fluid even after remaining four days in 5 per cent. nitric acid. This acid is used in dilute solution alone or in combination with phloroglucin. Directions for Using Nitric Acid.-1. Decalcify in large quantities of a 5 per cent. aqueous solution of nitric acid, changing the solution every day for one to four days. 2. Wash twenty-four hours in running water to remove every trace of acid. 3. Harden in 80 per cent., and then 95 per cent., alcohol. Imbed in celloidin. According to Schaffer, it is best to transfer the tissue directly from the nitric acid to a 5 per cent. solution of alum for twenty-four hours before placing in running water, so as to avoid any possibility of the tissue swelling. Phloroglucin and Nitric Acid.—Phloroglucin is not a decalcifying agent, but is added to nitric acid to protect the tissues while allowing a stronger solution of the acid to be used than would otherwise be possible. The solution is prepared by dissolving I gram of phloroglucin in 10 c.c. of nitric acid. Solution takes place quickly, with generation of considerable heat. The fluid is reddish brown at first, but becomes light yellow in the course of twenty-four hours. Dilute with 100 c.c. of a 10 per cent. solution of nitric acid. This gives nearly a 20 per cent, solution of nitric acid. The process of decalcification in this fluid is extremely rapid; a few hours only, as a rule, are required. It is not advisable to dilute the solution by the simple addition of water, but by the use of less acid, because the phloroglucin must be present to the amount of 1 per cent. or it will not protect the tissues so well. The following slower-acting solution may be found useful: I; 5; 70; 30. A rather deep single stain with alum-hematoxylin (either aqueous solution or Delafield's) will usually be found to give the best results with tissues decalcified with nitric acid. It is very important to leave the sections after staining in a large dish of water overnight, otherwise the stain will not be so sharp and clear. Sulphurous Acid.—A saturated solution-about 5 per cent. is used. It works very quickly and causes little swelling. The tissues should be carefully washed out in running water as after nitric acid. The stock solution rapidly grows weak through evaporation if the bottle in which it comes is not kept tightly corked. Trichloracetic Acid.-A 5 per cent. solution of this acid has lately been recommended for the decalcification of bone and teeth. It acts more slowly than nitric acid, and seems to possess no advantages over it. Tissues must be washed out in running water, as after nitric acid. IMBEDDING PROCESSES. SECTIONS of hardened tissues can be cut with a razor by hand, or with a microtome knife after fastening the specimen in the microtome clamp either directly or between pieces of |