We did not buffer 15% formic acid v/v in distilled water. We have also used 10% formic acid v/v in distilled water. The HCl/formic acid mixture is not buffered either. and is a fast decalcifying solution, more so than 10% or 15% formic acid. The bone actually buffers itself to a certain extent since it contains calcium and other phosphate salts. We always make up decalcifying solutions with distilled water, and the stock formic acid we purchase is 90% formic acid, not 100% formic acid (although the latter is available from Sigma and other companies). If you calculate the true concentration of formic acid made up from the 90% formic acid stock solution, you will have slightly less than a true 15% formic acid component.
Buffered formic acid, sometimes called acidic buffers, will have a concentration of approximately 4.5%. Curiosity about formic acid concentration drove me to calculate the concentration - revealing a mild decalcifier with buffering salt that is excellent for immunohistochemical purposes. Caveat: one can still overexpose the bone to acid with these buffered acids.
Many commercial buffered formic acid decalcifiers are made up from original, old recipes, i.e. sodium formate with formic acid and/or sodium citrate/formic acid. It is a good idea to read the MSDS to see content and hopefully, concentrations. You can find these mixtures in histotechnology textbooks, one is Kristensen's acid decalcifier, a classic. We rarely bought it commercially, choosing to make it up when needed. Convenience is everything in a busy laboratory though.
Linda Jenkins had another excellent endpoint test that relied on viewing the sample, it was much simpler than weighing, xraying or chemical testing, and was accurate. Hopefully she will provide the method, although I have it buried in a file somewhere.
Gayle M. Callis
----- Original Message -----
To: 'Gayle Callis'
Sent: Thursday, January 03, 2008 4:00 PM
Subject: RE: [Histonet] immunocal
I also do a lot of veterinary decalcifying. When you use the 15% formic
acid or the HCl/formic acid, is it buffered with sodium citrate or do you
make it in distilled water (i.e. 15ml formic acid into 85ml DH2O)?
Mass Histology Service
[mailto:firstname.lastname@example.org] On Behalf Of Gayle Callis
Sent: Thursday, January 03, 2008 5:20 PM
To: Michele Wich; email@example.com
Subject: Re: [Histonet] immunocal
You can use 15% formic acid providing the bone is totally fixed, and another
good decalcifier is 4% hydrochloric acid/8% formic acid - one we have used
with great success for huge sheep distal femur slabs. Endpoint testing was
done daily, and we did not leave the bones in decalcifying solution over a
weekend IF the endpoint was close to completion. Overexposure aka over
decalcification is not a good thing for any bones, and staining will be
terrible if this happens.
Gayle M. Callis
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