Re: Colour of decalcifying fluid

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From:"J. A. Kiernan" <> (by way of Marvin Hanna)
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On Wed, 19 Jan 2000, A. F. Brandwood wrote:

> method of decalcification. In the past we used 5% Sulphosalicylic acid only
> to decalcify bone and the fluid turned purple with some of the bones.We now
> start off with 10% Nitric acid for a maximum of 48 hours and if the bone is
> not decalcified we transfer it to 5% Sulphosalicylic acid until
> decalcification is complete and we still get the purple colour with some
> bones.

    According to the Merck Index, sulphosalicylic acid forms
    a violet complex with ferric iron, and is used in a
    colorimetric assay for Fe3+.

    Possibly your 10% nitric acid treatment is releasing
    "masked" iron from your bones, and the iron is then
    chelated by the sulphosalicylic acid to form a
    soluble coloured complex. If the specimens have spent
    several weeks in non-neutralized formaldehyde prior to
    decalcification, some of the haemoglobin will have
    been converted to formalin pigment, from which iron
    could be released by acid hydrolysis. Just a
    suggestion; I don't think you mentioned the fixative
    or the time.

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

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