On Mon, 27 Sep 1999 Rose_Bellantoni@integra-ls.com wrote:

> I am in need of a recipe for a citrate-phosphate buffer solution that is
> used with a saturated aqueous ammonium oxalate solution for end point
> determination of EDTA decalcified specimens.  I have a article that
> describes this method but it neglects to give the amounts needed to make up
> the buffer.
> 
> This procedure calls for 0.5 ml of spent EDTA solution to be mixed with 1.0
> ml of citrate-phosphate buffer and 2.5 ml of saturated aqueous amonium
> oxalate.  If calcium is present in the test tube, a cloudy white precipate
> will form.  A clear solution after 20 minutes indicates the absence of
> detectable calcium in the decalcifying solution.

   You do not mention the pH of the buffer. (Phosphate-citrate
   works from 2.6 to 7.8). For maximum sensitivity of the
   oxalate test on EDTA solutions the pH should be adjusted 
   to 3.2-3.6 before adding the ammonium oxalate solution. 
   If you use a buffer (probably not needed; HCl does the job
   adequately), be sure to check the pH _after_ mixing with the
   Na2EDTA solution, which is self-buffering for a higher pH range
   (about 6 to 8 I think; reference not to hand!)
 
   The pH adjustment for EDTA is  different from testing an acid 
   decalcifier, when you make the solution alkaline before mixing 
   with ammonium oxalate.

   Some authorities say you should wait for 30 minutes for
   precipitation of calcium oxalate. I don't know why you
   need to wait more than one or two minutes, but then there
   are lots of things I don't know. Perhaps someone with more
   experience of decalcifying could tell us if long-delayed
   precipitation really happens when doing the oxalate test,
   and if it does, why? 

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