The tissue should be put into the fixing fluid as soon as possible after its removal from the body. It must not be allowed to dry. There should be an abundance of the fixing solution, 25-50 times the volume of the object to be fixed. The tissue should never be put into a dry vessel and the fixing fluid poured upon it; the vessel should first be filled with the fixing solution and the tissue then dropped into the latter. A slight agitation of the fluid will prevent the sticking of the tissue to the bottom or sides of the vessel. The size of the pieces must be adapted to the penetrating power of the fixing reagent, but as a rule the pieces should not be more than 2-3 cms. in thickness, and for some reagents 0.5 cm. is as thick as they can safely be. The reagents used should be changed when they become cloudy or discolored. The used solution may be filtered and used again, but some reagents can be used but once. Alcohol may be saved for redistillation. In the case of some reagents (mercuric chloride, chromic acid, osmic acid, etc.), the time limits of the fixation should not be exceeded, as over-fixation will ruin the staining-power of the tissues. Alcohol is practically the only solution in which tissues may be left indefinitely, but even with it there are certain limitations. As a general rule the time required for fixation may be shortened by keeping the reagents at incubator-temperature. As the different fixing reagents vary so greatly as to their especial advantages and disadvantages these will be considered separately. Only the best and most commonly used methods are here included.
1. ACETONE. A water-free acetone is employed by placing pure dried white copper sulphate in the bottom of the bottle or vessel in which the fixation-process is carried on. Several layers of filter-paper are put over the copper sulphate to keep the tissues from touching it, and the acetone is then poured into the vessel. As soon as the copper-sulphate becomes blue it must be again fused. It is only by this method of constant dehydration that acetone can be employed to any advantage as a fixing agent; if fused copper sulphate is not employed the amount of acetone necessary to fix well is so great that the method becomes too troublesome and expensive to be recommended. But with the simple copper-sulphate method of constant dehydration acetone becomes the cheapest, most rapid and one of the best fixing reagents. The use of alcohol is avoided, and the period of infiltration in xylol and in paraffin shortened. For very quick work the entire process of fixation, hardening and dehydration may be achieved by the use of acetone alone, small pieces of tissue being fixed ½-2 hours in acetone and then transferred directly to soft paraffin. For ordinary work pieces of tissue 0.5 cm. thick are put into acetone over fused copper sulphate for 20-60 minutes. A judgment of the degree of fixation can be obtained by pressing the tissue lightly between the fingers; if it is of uniform consistence, and does not give as if the inner portions were softer than the surface, the fixation is complete. From the acetone the tissues may be brought directly into xylol for 5-10 minutes, until they obtain a cloudy transparency, thence into paraffin for 15-30 minutes and then blocked. The whole process of fixation, imbedding, cutting and staining can be carried out in 30 minutes. Acetone may be combined with formol, alcohol or any of the other fixing agents, but when it is desired to use any one of these for some especial purpose it is better to fix first with the desired reagent and then to use acetone for the dehydration-process alone, instead of alcohol. We have found that formol-fixation followed by acetone dehydration gives excellent results for general pathologic work. Formol-acetone (acetone 100 cc., formol 10 cc.) may be found to have advantages. The quick fixation in water-free acetone causes less contraction than fixation with absolute alcohol; fixation with graded acetone-solutions causes practically none.
Advantages. It is the cheapest and quickest method. It penetrates well and causes little contraction of the tissues, shortens the time in xylol and paraffin and makes more easy the cutting of dense fibrous structures. Cell-division figures are as well preserved as by alcohol fixation, and the staining of bacteria in the tissues can be carried out as well after acetone-fixation as after alcohol. It preserves lecithin, hence can be used for the fixation of nerve-tissues. When combined with formol (formol-acetone) the red blood-cells are well-preserved and take a brilliant eosin stain.
Disadvantages. The disadvantages are practically the same as with alcohol fixation but not so marked. Fat is dissolved, cell-division figures are not so well-preserved as with mercuric chloride and Flemming’s solution, and the blood-cells not so well preserved as with formol and mercuric chloride.
2. ALCOHOL. Absolute, 95-96 per cent alcohol, or graded alcohols (70, 95 per cent. and absolute) may be used for fixation. For this purpose the stronger alcohols are preferable, as the weaker solutions do not fix quickly enough. On the whole the use of 95-96 per cent is to be advised. The pieces of tissue must not be thick. Plenty of alcohol should be used and it should be changed several times during the process of fixation, which for larger pieces requires several days. For the last change absolute should be used. Very small bits such as uterine curettings can be fixed in one hour, by using three changes of absolute alcohol. Since alcohol both fixes and hardens it has been generally used, but it is a relatively poor fixative. For after-hardening it is indispensable. Absolute alcohol may be made from 96 per cent by the use of fused copper sulphate. To test the strength of alcohol mix a few drops with pure water-free xylol; if no sediment appears when viewed against a dark background the alcohol is absolute or practically so. Many of the disadvantages of alcohol fixation can be obviated by the use of formol-alcohol (95 per cent alcohol 100 cc., formalin 10 cc.).
Advantages. Cheapness, quickness, and ease of method. Can be used for quick diagnostic work. Penetrates well, and can be used for large pieces of tissue. Preserves glycogen, is especially good for the staining of bacteria in sections, and the majority of stains work well with alcohol-fixation.
Disadvantages. Causes much shrinking and loss of finer details; preserves cell-division figures not at all or poorly; destroys the red blood cells; dissolves fat and other chemic products: does not permit of the use of certain specific staining methods (nerve-tissues); causes excessive hardness of fibrous and elastic tissues and makes cutting difficult.
3. CHROMIC ACID AND SALTS. Chromic acid is rarely used alone in pathologic work, but is a constituent of Flemming’s solution (see below). Its salts are employed in the form of:—
A. Müller’s Fluid (Potassium bichromate 25.0 grms., sodium sulphate 10.0 grms., water 1,000.0 cc.) This was formerly the favorite fixing solution, but is now used chiefly for the eye and nervous tissues, either alone, or after formol fixation, or mixed with formol. Large pieces may be used, even an entire brain, but the process requires months or even a year for the best results. Even small pieces take several weeks. The process may be hastened in the incubator. The solution should be changed whenever it becomes cloudy. When fixation is complete the fixed and hardened tissue may be cut directly on the freezing-microtome or after-hardened and dehydrated in alcohol or acetone when it is to be imbedded. The dehydration in alcohol should be carried out in the dark and without previous washing of the tissue in the case of nervous tissue. Greenish or brownish chrome precipitates appear if the dehydration takes place in the light; but for ordinary material this precipitate can be avoided by washing the fixed material in running water for 24 hours before transferring to alcohol. Moulds grow luxuriantly in Müller’s fluid, but may be inhibited by the use of pieces of camphor, thymol, etc.
Advantages. Cheap, penetrates well, causes little shrinking, permits special nerve-stains, preserves red blood-cells, gives beautiful results with ordinary stains, preserves fat.