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. 2. =Trichloracetic Acid.= Fix tissues in 10% formalin and decalcify in trichloracetic acid 90 cc., formol (40 per cent formaldehyde) 10 cc. Change frequently. Decalcification is rapid, the tissue is but little changed and the staining-power not affected. 3. =Concentrated Sulphurous Acid.= Fix in 10 per cent formol; decalcify in concentrated sulphurous acid for 24 hours or longer if necessary. Wash thoroughly in alkaline water. This is a very good and rapid method; the staining-power is but little affected. 4. =Haug’s Solution.= (Pure nitric acid 3-9 cc., absolute alcohol 70 cc., sodium chloride 0.25 grm., water 30 cc.). For tissues fixed in mercuric chloride. 5. =Phloroglucin.= (Phloroglucin 1 grm., pure nitric acid 10 cc., distilled water 50 cc.). The solution must be carefully dissolved over the flame in a hood. Decalcification is rapid, and the tissue is protected from the acid by the phloroglucin. 6. =Ebner’s Fluid.= (Hydrochloric acid 5 cc., sodium sulphate 5 grms., alcohol 500 cc., water 1,000 cc.).