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After fixation bone and other calcified tissues can be treated with an acid solution to remove the calcium content.

Formic acid:
Drain the fixative from the tissue and replace it with 10% aqueous formic acid.  After a reasonable length of time (depending on the tissue size and type) test for the presence of calcium using the method below.

Hydrochloric acid:
For very urgent samples a solution of 10% hydrochloric acid may be used instead but there is some danger of tissue damage.

Commercial fluids:
Commercial decalcifying solutions can be obtained which will decalcify tissue more rapidly than 10% formic acid without damaging the sample.

Testing for remaining calcium in decalcifying tissue.

  1. Take approximately 3ml of decalcification solution from the tissue container and place it in a test tube.
  2. Test with a piece of litmus paper and neutralize the fluid by adding strong ammonia solution drop by drop until the paper just turns blue.  If the litmus turns too blue add more decalcification fluid and try again.
  3. Add an equal quantity (3ml) of saturated ammonium oxalate shake the tube and leave it for ten minutes.
  4. If after ten minutes there is any precipitate at all (slight cloudiness) there is still calcium present and the specimen needs further decalcification.

EDTA - Ethylenediaminetetracetic acid (Hilleman and Lee 1953):
EDTA disodium salt - 5.5g
Distilled water - 90ml
Formalin - 10ml
This is a gentle, non-acid decalcifying solution which has the advantage of not damaging the tissue but is slow acting.  Calcium is removed by chelation; expect the process to take from 1 - 8 weeks at room temperature depending on the size of the sample.  Judging when the decalcification is complete is more difficult than with acid solutions as the ammonium oxalate test cannot be used.  X-raying the sample is the best way to ensure that all traces of calcium have been removed.

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