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
Testing for
completeness of decalcification
Questions.
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
(kiernan[AT]uwo.ca)