Re: Molar ratio of Ca2+ for Alizarin stain

From:"J. A. Kiernan"

On Fri, 27 Jul 2001, Montague, Donna C wrote:
> To: 'Histonet' 
> We are using Alizarin red stain as a colorimetric indicator of Ca++ in
> ... Further, by looking up the chemical structure in Conn's, I surmise 
> the equivalence ratio would be 1 mole Alizarin per 2 moles Calcium. 
> Do you concur?

No. The traditional tale is 2 alizarin to 1 calcium. With a coordination
number of 4, a Ca2+ ion is supposed to bind to suitably spaced oxygens
on 2 alizarin molecules, its charge being neutralized because on each
alizarin molecule one of the oxygens is an ionized phenol group. BUT
an in vitro study by Lievremont & 2 al (1982) Acta Anat. 114:268-280
found metal:dye approx= 1:1 in precipitates. A precipitate is not
necessarily the same as a histochemical staining, where a large
excess of the reagent is provided, followed by washing.
Another consideration is that alizarin red S that you buy isn't
100% of that dye. The Biological Stain Commission certifies batches
of this dye on the strength of performance. They haven't found a
suitable assay of total dye content for this one.

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

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