Decalcifying large bones

From:Gayle Callis

You wrote: 

I use formic acid with sodium formate or sodium citrate for decalcification 
of bone in the laboratory.  This procedure is excellent and I have not 
problem staining with Mayer's hematoxylin.  My new boss want to use HCL or 
Nitric acid to decal large bone specimen because formic acid is too slow.  I 
need procedure for using these strong acids and how do you monitor the end 
result.  I will using this method to decal mandible etc.  Thank again for any 
help that is given.

First is decalcification endpoint determinations:  

1.  Xray is the most sensitive, if you have a FAXITRON available, you will
have     excellent results - 
2.  Chemical endpoint determination is popular, you can buy a kit from
Polysciences or make up your own reagents, a bit more time consuming but it
is better to take the time than overexpose bone to acids, ANY acids
including formic. This method is found in many histotechnic books and
probably Histonet archives. 
3.  There is a weight gain/weight loss method, but is requires analytical
balance in order to weigh in mg, originally used for testing nitric acid,
but you need to do a chemical test at the end.  

There are several recipes for decalcifying large bones with strong mineral
acids, or you can do some other tweaks. 

For HCl methods, buy a commercial decalcifier, save time and mixing of
corrosive acids.  I prefer an HCl decalcifier with no more than 5% HCl in
the solution, it is still fast, but not so fast that I can't control it.
There is no law to say you can't dilute a commercial, highly concentrated
(10 - 12%)  HCl decalcifier to 5%!  We also published some results using
RDO diluted 1:1, a concentrated HCl, and had excellent staining results,
plus it was still fast.  

My favorite for large bones (1 cm thick slabs of distal femurs, sheep, and
large dog bones) is 8% formic acid/4% HCl, mix just before use, this is
very fast, and gives excellent results, endpoint testing is always done. DO
NOT increase the HCl concentration with this decalcifier, it will do large
bones very nicely.    

By increasing formic acid concentration to 15%, you will speed up the
decalcification rate.  This is 15 mls stock formic acid in 85 mls distilled
water, and one could try 20% formic acid.  Use endpoint determination.  

Nitric acid works well, but must be carefully controlled, there are several
methods in histotechnic books.  It is very rapid and also available
commercially. One nitric acid methods contains alcohol - put into acid
solution to slow down rate of decalcification.  Also, do not use OLD,
yellow nitric acid, it creates some ugly artifact, discoloration.

During decalcification, suspend bones in the decalcifier to completely
surround the sample with decalcifier.  If you do the chemical test, DO NOT
STIR - you need to collect an aliquot from bottom of container where
removed calcium settles. 

To stop decalcification, you can water rinse briefly, then put bone into
70% ethanol - basically the first dehydrant in processing or back into NBF
to hold until processing.  

If you get weak staining with your Mayers after strong, mineral acids - try
Richard Allan Hematoxylin 1 or Gill II or III for 10 minutes, rinse and
blue,  to overcome the effects of acid decalcification. 

Good luck
Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology - Marsh Lab
Montana State University - Bozeman
19th and Lincoln St
Bozeman MT 59717-3610

406 994-6367 (voice mail)
406 994-4303 (FAX)


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