EDTA combined with nitric acid

From:Gayle Callis

You wrote: 

Hi histonetters
i need your help 
i put some teeth in EDTA for decalcification for two months, and then to
accelerate decalcification: I put them in acid nitric 5% for four weeks, i
embedded them in paraffine, teeth are too yelow now, and too hard, what do
you think about this, could you give me some advice, or any protocole please 
thank you very much in advance
Myriam Baali

If you wanted to speed up decalcification, you should have started in
Nitric acid to begin with.  Nitric acid, in particular, old nitric acid
will turn bone/teeth yellow, and it should be avoided. There are some good
nitric acid mixtures, Perenyi's (Lillie's book), or use 5% nitric acid - is
very rapid, but the endpoint must be determined to know when
decalcification is completed. 

EDTA, tetrasodium salt, can be be used up to 14% in water, pH adjusted to
7.6 would probably have been faster than EDTA at pH 7 since it functions in
relationship to it's pH.  Higher pH decalcifies faster than lower, more
acidic pH but you should not go above pH 8, or alkaline sensitive protein
linkages can be damaged.  Go into Histonet archives, this has been
discussed at length many times before your query.  

It would be better to decalcify your teeth in just some acid solution to
speed up the rate of calcium removal.  As for hard teeth, large or very
dense bones/teeth benefit from extended processing into paraffin, longer
infiltration in paraffin may help along with good long soaks in ice water
to help get the section and using a harder paraffin for infiltration and
embedding (Tissue Prep 2).  I don't think I would have mixed two
decalcification methods, use either one or the other i.e. either EDTA OR an
acid solution. 

Just leaving a tooth in a decalcifying solution for a long time does not
provide good decalcification, overexposure to acid MUST be avoided.
Decalcification endpoint tests should be done to know when the calcium is
removed, tooth can be then be rinsed and processed. 

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

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

email: gcallis@montana.edu

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