6. MERCURIC CHLORIDE. This is used most commonly in the form of a concentrated water solution (mercuric chloride 7.5 grms., sodium chloride 0.5 grm., glacial acetic acid 5 cc., water 100.0 cc.), or as Zenker’s solution (mercuric chloride 5.0 grms., sodium sulphate 1.0 grm., potassium bichromate 2.5 grms., water 100 cc.; dissolve by heating, add 5 cc. glacial acetic acid just before using. The use of a 5 per cent formol solution instead of acetic acid is recommended). The pieces of tissue should not be thicker than 5 mm. Fix 6-24 hours, then wash 24 hours in running water, and after-harden in alcohol. Should the sections show mercuric precipitates they should be treated with Lugol’s solution for 30-60 minutes, then washed in a dilute solution of lithium carbonate and thoroughly washed out in water and alcohol. Much better stains can be obtained by this treatment of the sections with Lugol’s. The use of iodine in the alcohol during the process of after-hardening is not advisable because of the action of the iodine upon the albuminates of mercury. A 5 per cent solution of sublamine in distilled water has recently been recommended by Klingmüller and Veiel. Fix 1-3 hours, wash and after-harden in alcohol. Precipitates are not formed, and good staining-results are obtained.
Advantages. The mercuric chloride solutions preserve well the red blood-cells, mitotic figures and finer details of cell-structure, and permit the staining of bacteria and animal parasites in the sections. Certain especial staining methods (Mallory’s reticulum-stain, etc.) can be used only after mercuric chloride fixation, while many others (Heidenhain’s iron-hæmatoxylin, Biondi-Heidenhain triple stain, etc.) give best results after this fixation. For ordinary work the saturated mercuric chloride solution is preferable to Zenker’s, as the latter does not give good results with the commonly-used hæmatoxylin stains.
Disadvantages. More troublesome and expensive; require thorough washing and subsequent removal of precipitates, and affect (Zenker’s particularly) certain stains.
7. OSMIC ACID. Osmic acid is used alone in a 1 per cent solution, or in such combinations as Flemming’s solution (chromic acid 1 per cent. sol. 15 cc., 1 per cent osmic acid 4 cc., glacial acetic acid 1 cc.), Hermann’s solution (same as Flemming’s, with 15 cc. of a 1 per cent platinic chloride substituted for the chromic acid), Altmann’s solution (5 per cent potassium bichromate solution 50 cc., 2 per cent osmic acid solution 50 cc.), Marchi’s solution (Müller’s fluid 2 parts, 1 per cent osmic acid solution 1 part), and that of Pianese (1 per cent sodium-chloroplatinate 15 cc., 2 per cent osmic acid 5 cc., ¼ per cent chromic acid 5 cc., formic acid 1 drop). The pieces of tissue must be very thin, as osmic acid penetrates very slightly. Fix 6-24 hours in the dark, and wash thoroughly in running water, and after-harden in graded alcohols. These solutions have but limited use in pathology, and are used chiefly for the study of fat (oleates) and mitotic figures. Flemming’s and Hermann’s solutions are the best for the study of mitotic figures, the latter bringing out plasma details more clearly. Marchi’s fluid is used especially for the study of nerve-degeneration, and Altmann’s for the demonstration of Altmann’s granules. The method of Pianese is used for the demonstration of cell-inclusions. The osmic-acid mixtures are all expensive, penetrate poorly, cause precipitates, and affect greatly the staining-power of the tissues, so that it becomes necessary to use certain stains (safranin, carbol fuchsin, aniline gentian violet, etc.) as counterstains.
8. PICRIC ACID. A saturated water solution of picric acid is usually employed. Fix 12-24 hours, and wash in alcohol, not water, and after-harden in graded alcohols. Preserves mitotic figures, fine details of cell-structure, and is very good for bone and calcified tissues, as it decalcifies and fixes at the same time.
Numerous modifications and combinations of the above methods have been proposed such as Flemming’s chrom-acetic solution, Rawitz’s chromic-picric-nitric fluid, Rabl’s chrom-formic mixture, Burckhardt’s chrom-osmic-nitric solution, Merkel’s fluid (chromic acid-platinic chloride), Carnoy’s mixture (glacial acetic acid 1, absolute alcohol 6, chloroform 3), and many others. They have a limited use in pathologic work.
In the judgment of a section as to its fixation the following points may be of service: in alcohol fixation the red blood cells are haemolyzed, and there is much shrinking; with formol fixation the red cells stain copper-red with eosin; in mercuric chloride fixations the red cells stain rose-red with eosin, and pigment precipitates are present; in bichromate fixation the red cells preserve their natural color, and fat cells show a brownish color; osmic-acid fixation is shown by the black color of the oleates, and the failure of the tissue to stain by ordinary stains.
CHAPTER XXI.
DECALCIFICATION.
Bone and tissue containing deposits of lime must be decalcified before they can be sectioned on the microtome. The decalcification should be carried out after fixation and before the after-hardening in alcohol. Some reagents may combine decalcification with fixation, but this is satisfactory only when the amount of lime-salts is relatively small. Fresh tissues should not be put into any of the stronger acid decalcifying fluids, as they alter unfixed cells so that the staining-power is lost and the fine histologic details destroyed. The fixed tissue cut into small pieces is put into the decalcifying reagent, which is used in large amount and must be frequently changed. It is left in the decalcifying fluid until the calcium salts are removed, as shown by tests with needle or scalpel. The tissue must not be left in the fluid after decalcification is attained, as the staining-power is affected by all decalcifying reagents; it is therefore necessary to make frequent tests in order to judge of the progress of the decalcifying process. After decalcification the tissue should be washed in running water for 24 hours, and then after-hardened in alcohol. Alkaline solutions may be used to remove the acid before washing. Sections of decalcified tissue always stain slowly, and it is advisable to remove any acid remaining in the tissues by soaking the sections in a saturated water solution of lithium carbonate before staining. Numerous formulæ for decalcifying fluids have been recommended; a few of the best methods only are given here.
1. Combined Fixation and Decalcification. Picric acid or Müller’s fluid may be used for this purpose when the amount of lime-salts contained in the tissues is very small. The process is slow.