Histology FAQ

Staining, Histochemistry and Histotechnology

(Frequently Asked Questions)


Dr. John A. Kiernan
Department of Anatomy and Cell Biology
The University of Western Ontario
London, Canada



FAQ Home > Processing, Decalcifying, Embedding


Testing for completeness of decalcification


How should I test for complete decalcification? Is the same method OK after either formic acid or EDTA?

Answer 2.

The ammonium oxalate test is simple. Take a 5 ml sample of used decalcifying fluid. Neutralize it by adding drops of strong ammonia (ammonium hydroxide); avoid the fumes! When the solution turns litmus blue (pH above 7), add 5 ml of saturated aqueous solution of ammonium oxalate (about 3%; stable stock solution). Wait for half an hour. If there is no precipitate, the last change of decalcifying fluid was free of calcium ions.

According to Eggert & Germain (1979) you can use the ammonium oxalate test on EDTA. Rosen (1981) said the sensitivity was higher if you lowered the pH to 3.2-3.6 before doing the test (instead of neutralizing to pH 7 as done with an acid decalcifier).

Eggert FM, Germain JP 1979. Histochemistry 39: 215-224.
Rosen AD 1981. End-point determination in EDTA decalcification using ammonium oxalate. Stain Technology 56: 48-49.

John A. Kiernan,
Department of Anatomy & Cell Biology,
The University of Western Ontario,
LONDON, Canada N6A 5C1