The removal of calcium deposits is essential for good embedding procedure. Decalcification is usually carried out between the fixation and processing steps. Bone must obviously be processed in this way, but other tissues may also contain calcified areas. A variety of agents or techniques have been developed to decalcify tissues, each with advantages and disadvantages. Immersion in solutions containing mineral acids, organic acids, or EDTA are the predominant methods used. Electrolysis has also been tried.

Strong mineral acids such as nitric and hydrochloric acids are used with dense cortical bone because they will remove large quantities of calcium at a rapid rate. As might be expected, these strong acids also damage cellular morphology. Mineral acid decalcifiers are not recommended for delicate tissues such as bone marrow. Because they are not as aggressive, organic acids such as acetic and formic acid are better suited to bone marrow and other soft tissues. Organic acids act more slowly than mineral acids, and will require extended treatments to decalcify cortical bone. Formic acid in a 10% concentration is the best all-around decalcifier. Some commercial solutions combine formic acid with formalin to fix and decalcify tissues at the same time.

EDTA can remove calcium and is not as harsh as mineral or organic acids. The use of EDTA is limited by the fact that it penetrates tissue poorly and works slowly. It is also expensive in large amounts. Electrolysis has been tried in experimental situations where calcium had to be removed with the least tissue damage. Electrolysis is slow and not suited for routine daily use.

In order to achieve optimal results when processing calcified tissues, it is important to determine the point at which decalcification is complete.  While incomplete decalcification can lead to tissue distortions (and possibly a damaged microtome), over-decalcification causes problems with staining, in particular nuclear staining. The following protocol can be used to determine when decalcification is complete.

TESTING FOR DECALCIFICATION

1. Remove 5 ml of used decalcification fluid from the tissue processing vessel
2. Add ammonium hydroxide dropwise until the pH of the solution is neutral to pH paper.
3.  Add 5ml of saturated ammonium oxalate and shake well
4.  Allow to stand at room temperature for 30 minutes.

INTERPRETATION OF RESULTS

The formation of a precipitate (calcium hydroxide) after the addition of ammonium hydroxide indicates the presence of substantial amounts of calcium in the spent fluid.  The fluid should be changed and the tissue further decalcified.

Formation of a precipitate after ammonium oxalate addition shows less calcium.  If the solution remains clear for 30 minutes after addition of the oxalate, the tissue is essentially calcium free.

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