[Histonet] Re. Decalcification with formic acid sodium citrate

Gayle Callis gayle.callis at bresnan.net
Sat Jul 25 12:24:19 CDT 2015

Merissa and Tim,  


This formic acid decalcifying solution is basically the classic Evans and
Krajian fluid (Sheehan and Hrapchak,   Theory and Practice of
Histotechnology, 2nd edition, P.92).  Shandon has added other ingredients
for some reason, and has kept those concentrations proprietary.   You really
don't need to add a surfactant or PVP emulsifier when making up this
decalcifying agent.   Simply use the classic recipe for successful
decalcification.   This is also referred to as buffered formic acid and in
some publications an "acidic buffer".  It is excellent if IHC is needed and
less damaging, obviously, than a strong mineral HCL acid decalcifiers.  


Sodium citrate crystals (a buffering salt) 10 g 

90% formic acid stock                            25 ml  

Distilled water                                        75 ml   


One can calculate the concentration of formic acid i.e. approx. 4.5% since
is it made from 90% formic acid stock.  


Don't bother with the surfactants or PVP.  


Enjoy an excellent in house formic acid decalcifying solution.  I also
suggest you read Sheehan and Hrapchak textbook chapter on bone as a way to
familiarize yourself with decalcifiying solutions that manufacturers now
supply with some modifications.  Some manufacturers will refer to these
methods but probably prefer not to do this since they want you to buy their
commercial product that is obviously a time saver with elimination of having
to store stock acid solutions.   The classic methods made in house are
excellent if you have time to make them up.   Formic acid with sodium
formate is another popular buffered formic acid.   I suggest you look for
another source/manufacturer of the your favorite decalcifier in question as
more than one company will make it.  Decal Corp, recently sold to Stat Lab,
could also be the source as Shandon isn't the only game in town.   Others
are Newcomer Supply, Poly Scientific.  Not having to make it up may remain
your preference. 


Gayle M. Callis 










Written by Tim and Merissa:   




     Water                                77-80       solvent

    Formic acid                      21-23       active ingredient

     Fluorad                              >1           surfactant  - a
wetting agent to make the solution wet the bone more easily

    Sodium citrate                   >1           emulsifier , buffer

     Polyvinyl pyrrolidone        >1           emulsifier 


They say less than one percent of the last three, but you really have no
idea whether that is 1%, .1% or .01%. It could be any of those.


But all those surfactants and emulsifiers are meant to keep the solution
viable for long periods on the shelf. When you make it fresh you don't
really need them.


You can either buy a different decalcifier, or make your own. Making your
own with just the water and acid will work just fine. 



Tim Morken

Pathology Site Manager, Parnassus 

Supervisor, Electron Microscopy/Neuromuscular Special Studies

Department of Pathology

UC San Francisco Medical Center


-----Original Message-----

From: M.O. via Histonet [mailto:
<http://lists.utsouthwestern.edu/mailman/listinfo/histonet> histonet at

Sent: Wednesday, July 22, 2015 1:24 PM

To:  <http://lists.utsouthwestern.edu/mailman/listinfo/histonet> histonet at

Subject: [Histonet] understanding reagents in decalcifier; making it


Hello Histonet


The supplier for our decalcifier, TBD-2 from Shandon, is having issues with
getting the product out and we will not be receiving it for at least another
month.  Our samples are piling up and I don't know what I should do, but
maybe I can make the decalcifier in-house.  I am wondering if I can make my
own based on the reagents they listed and their percentages and if certain
reagents are not actually necessary.


The samples we typically decalcify are mouse knees (decal time = 2 days),
mouse spines (3 days), human bone slabs about 7mm in thickness (7-12 days).
Fixation is in zinc buffered formalin, then decalcification, then 70% EtOH.
Our choice to use TBD-2 is due to the gentle decalcification for IHC and we
get GREAT results.


Composition of Shandon TBD-2 Decalcifier:

Component                Weight %

     Water                      77-80

Formic acid                 21-23

    Fluorad                       >1

Sodium citrate               >1

Polyvinyl pyrrolidone      >1


If you have any input on what reagents I should use and the percentages for
making a decalcifier myself, it would be much appreciated!


Thank you for you help,



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