Re: Bone Marrow Trephines

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From:Barry Rittman <>
To:histology <>
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I am assuming that the words decalcification and demineralization haven't been
patented by some company!

I don't like using formalin or glutaraldehyde in decalcification mixtures. They
slow down the process of demineralization considerably and providing that the
tissue is adequately fixed to start with offer no real advantages. I think that
this may be your major probem. Bone marrow trephines should not take very long to

The best way to prepare EDTA is to start with 10% disodium EDTA in the buffer,
adjust the pH, then make up to final volume and recheck then pH.
When you adjust the pH to say 7.2 or so you actually end up with a mixture of
di-sodium EDTA and tri-sodium EDTA.  Using tetra-sodium EDTA as a starting point
doesn't make much sense as the sodium groups that are available for replacement by
calcium depend on the pH.
If for example you start with EDTA acid itself and adjust the pH with NaOH, the
EDTA will go slowly into solution.  At pH 3.45 the solution contains monosodium
salt, at pH 4.65 the disodium salt, at pH 8.1 the trisodium salt and at pH 10.84
the tetrasodium salt.  This means that at pH 7.3 there is a mixture of disodium
and trisodium EDTA in the solution. While it would be nice to have 4 available
sodiums for replacement, the high pH precludes this.
Hope this helps

Buttigieg Carmen at MOH wrote:

> Thanks to all for your help
> We never used to check th pH of the EDTA before. I then checked what was being
> used and found that the pH was 5.1. I threw it out immediately. Then I started
> to prepare a fresh solution using the tris/HCl buffer and pHed it to 7.0.
> My original formula contained 10% Formalin. Should I still include it?
> Any help would be appreciated.
> Carmen
> St. Luke's Hospital
> Malta

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