Decalcification

DECALCIFICATION (from Latin de - negative particle, and calx - lime), removal of lime. In histological technique, in order to make organs or tissues containing calcium salts suitable for cutting on a microtome, it is necessary to first extract the lime from them by treating the cut (or sawn) pieces with special decalcifying mixtures containing various acids. During decalcification, the following rules are observed: 1. Pieces before D. should be previously fixed (e.g. in formalin, alcohol, etc.). This can be omitted only if the decalcifying mixture contains fixing substances. After fixation, the pieces should be well washed in water. 2. Pieces should be as small as possible, since otherwise the penetration of liquid to the central parts of the piece will occur too slowly and the peripheral parts may be damaged by the prolonged action of the acid. On the contrary, one should take as large a quantity of decalcifying liquid as possible and frequently agitate it. 3. Pieces should be previously defatted by passing them through alcohols of ascending strength, otherwise crystals of fatty acids may form, which will spoil the preparation. 4. Pieces are subjected to D. for as long as is required for complete removal of lime. If they are left too long, the structure and stainability of the tissues may change due to prolonged exposure to the acid; therefore one should test as often as possible by pricking with a needle or making an incision with a knife to determine whether the lime has been removed or not. The slightest obstacles or crunching indicate that D. is not complete. 5. Upon completion of D., pieces should be thoroughly washed in water to remove the acid. It is even better after washing in water to neutralize the acid by leaving the pieces for a day in a 5% solution of ordinary potassium alum [ALK^SO^-^ttjO] or in a 5% solution of lithium sulfate, after which they should be again washed in running water for 24 hours. 6. After washing, the pieces can be cut on a freezing microtome or embedded in celloidin or paraffin (celloidin is preferable). If after embedding in celloidin it is found that not all lime has been removed, decalcification can be performed on the embedded pieces. 7. Before staining, sections are recommended to be passed through a saturated aqueous solution of lithium carbonate or through an alcohol made alkaline with ammonia. As decalcifying fluids, solutions of nitric, sulfuric, sulfurous, trichloroacetic, hydrochloric, formic and other acids are used. The following fluids are most common. 1. Nitric acid is used depending on the density of the object in a 2-10% aqueous solution. It should be remembered that the percentage is calculated not by volume but by weight. According to the Russian pharmacopoeia, Acidum nitricum purum (sp. gr. 1.200) contains 32.5% pure nitric acid; therefore to obtain a 5% solution, one should take 15.5 cm³ of acid and add 84.5 cm³ of water. In Germany, nitric acid with sp. gr. 1.414 is available, i.e. containing 68% pure acid by weight. Sometimes substances that prevent tissue swelling are added to the aqueous solution, such as: alum, formalin or alcohol. The mixture with alum is prepared as follows: take a half-diluted saturated (5%) solution of alum and prepare a 2-10% solution of nitric acid. For the formalin mixture, take a 10% solution of formalin and prepare a 5-10% solution of nitric acid. Of the alcoholic solutions, the most commonly used are Thoma's fluid: 5 parts of 96% alcohol and 1 part nitric acid, and Haug's fluid: pure nitric acid (sp. gr. 1.200) - 30-90 cm³, pure alcohol (96°) - 725 cm³, distilled water - 300 cm³, sodium chloride - 2.5 g. 2. Sulfurous acid (ac. sulfurosum) is used as a saturated aqueous solution (the usual commercial preparation), which contains about 5% acid. Pre-fixation in 5-10% formalin is possible but not necessary, as sulfurous acid itself acts as a fixative. The action is rapid, swelling is weak, and moreover it decreases upon washing. For simplicity of handling, reliability of action, availability, cheapness and weak swelling, the sulfurous acid preparation is one of the best decalcifying agents. 3. Hydrochloric acid causes significant swelling and more strongly changes the structure of tissues, especially nuclei. Usually a 3-10% aqueous or starting solution is used, i.e. 12-40 cm³ per 100 parts of mixture. The most commonly used mixture is Ebner's. Saturated cold aqueous solution of sodium chloride - 100 cm³, distilled water - 100 cm³, pure hydrochloric acid - 4 cm³. During D., add 1-2 cm³ of hydrochloric acid to this solution daily. 4. Of the organic acids, formic acid is most commonly used. It causes significant tissue swelling but does not harm staining. It should be used for decalcifying dense bones and teeth. It is used in a 20-25% solution or even (for decalcifying teeth) in its pure form. Since subsequent washing in water further affects tissue swelling, it is better after D. to transfer the pieces directly to alcohol, changing it until the acidic reaction disappears. It is better to use formic acid mixed half and half with 10% formalin. When combined with gold gilding, it gives well-stained preparations, especially suitable for teeth (dentin) in this combination. Recipe: 1% solution of gold chloride - 6 parts, pure concentrated formic acid - 3 parts. 5. Weak decalcifying properties are possessed by Müller's fluid, Flemming's fluid and a saturated solution of picric acid. When using these fluids, preliminary fixation is not required. Decalcification lasts several weeks. Zilkens proposed the following mixture for teeth and dense bones: nitric acid (25%) - 18 cm³, pure formalin (40% formaldehyde) - 8 cm³, chloral hydrate (saturated aqueous solution) - 8 cm³, anti-formin (20%) - 10 cm³, distilled water - 120 cm³, sodium chloride - 8 g. For spongy bones and generally for tissues with low lime content: nitric acid (25%) - 15 cm³, pure formalin - 8 cm³, chloral hydrate (saturated aqueous solution) - 5 cm³, anti-formin (20%) - 5 cm³, distilled water - 100 cm³, sodium chloride - 8 g. This fluid decalcifies extremely quickly (1-2 days) and excellently preserves in the tissues the ability to be stained.