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From:Gayle Callis <uvsgc@msu.oscs.montana.edu>
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Comments and suggestions on the following decalcifying fluid.  This is a
modification of the Richmond Gelfand method  (approx 4% - 8% formic acid
plus 4-8% HCL- I believe that is correct with some differences in acid
concs, and found in Brain's book) with a formic/hydrochloric acid mixture.
It is fast, compared to most 4 - 10% formic acid mixtures.  However purpose
of alcohol in any acid decalcification mixture will slow down the
ionization of calcium, and was probably done so to help prevent the protein
hydrolysis that is so common with strong acids (HCl and nitric), besides
slowing down the rate of decalcification and preventing tissue swelling.
Lillie also has an excellent discussion of this process. 

I never noticed tissue swelling with the above decalcifier, but I did with
excess washing (overnight) and had to make sure the rat/mouse bones are

In the past, I have used 70% alcohol rinses to STOP decalcification, a
recommendation made by Culling years ago and would not advise going back to
aqueous NBF after decalcification is completed in an alcoholic decalcifying
solution.  It sort of defeats the purpose, one can wash the bones in 70% to
remove decalcifier, stop the decalcification process, store the bones in
70% or proceed with processing IN 70% or higher concentration of alcohol,
you have already done the first dehydration step.

You can also slow down this decalcifying solutions action by reducing the
concentration of HCl to 4%, and still use 8% formic acid, without any
alcohol.  It is still recommended that decalcification endpoint testing be
done with ANY decalcification procedure.  

Test and change decalcifying solution DAILY or more often, acids are

>I worked routinely with rats. We also processed tibia and elbows
>in our standart program.
>We used a simple mixture of 30ml Formic acid
>                            10ml HCL
>                            60ml 70% Ethanol
>after 1 to 1 1/2 days you wash your elbows in 70% Ethanol or 10% Formaldehyd
>to prevent tissue from swelling.
>To speed up your decalcification you might chance your solution sometimes!
>Hope it helps,
>Joachim Siegmund /BTA
>105 Lakeshore Ct.
>Milledgeville, GA 31061
>Email: joachim.siegmund@gmx.net
>       jo_siegmund@web.de
>-----Ursprungliche Nachricht-----
>Von: Ann Thornton [mailto:pathat@wnmeds.ac.nz]
>Gesendet: Wednesday, July 05, 2000 4:03 PM
>An: histonet@pathology.swmed.edu
>Betreff: Decalcification of rats elbows
>Hi all,
>I work in a research lab, and one of our researchers would like to get in
>contact with histologists who specialize in bone work.  He is working on
>rats elbows, and it is important that, when decaling and cutting, none of
>the cartilage is removed from the bone.
>I would appreciate advise from all of you experts out there.
>Ann Thornton
>Departmental Manager
>Department of Pathology and Molecular Medicine
>Wellington School of Medicine
>New Zealand
Gayle Callis
Veterinary Molecular Biology
Montana State University
Bozeman MT 59717-3610
406 994-4705
406 994-4303

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