Archived tissue viability when stored in 10% formalin

From:Gayle Callis

Poor tissue is probably because the formalin has oxidized and pH is very
acid. It might be worth a pH check to test pH.   Tissues stored for a long
time should have the formalin changed regularly to maintain proper pH. This
has happened to us, but with bovine bone, the bone was actually decalfied
after many years!!  

Here is a discussion I had with Dr. Fred Monson about formalin degradation.
 He enlightened me on the chemical nature of formalin oxidation, a
wonderful lesson in what happens to formalin.  


The inquiry about old bones stored in formalin for 5 years.  Unless the pH
of the formalin was maintained at pH 7, i.e. changed to maintain fresh NBF,
you may encounter problems. Formalin tends to break down to formic acid over
time, the pH of "NBF" becomes acidic, and bone can also become 
decalcified. We had bovine bones stored for many years without changing
NBF, and when trying to do undecalcified PMMA method, the bone literally
twisted apart in the plastic block.  We should have stored the bone in 70%
alcohol IF we wanted undecalcified bone.   

If NBF has been allowed to drop below 6.8 or lower, nuclear staining may
also be damaged by this lower pH. You can try so see what happens, but be
prepared for some changes.  Heavy duty cortical bone may withstand storage
better than trabecular bone and soft tissues.  You might have to adjust
your staining to have optimal results.  
Gayle Callis 

Fred Monson reply: 

I agree with every word, but would slightly amplify one, just FYI and for
future reference.

The single-carbon sequence is as follows:

1.	CH4		methane,
2.	CH3OH		methanol,
3.	CH2O		methanal (or formaldehyde), and
4.	CHOOH		methanoic acid (or formic acid).

There are some rare variations on this sequence, e.g., #3 can be a diol by
having the =C pick up an H+ to give a second -OH.  This diol is often
stipulated as the "active" form of formaldehyde.

Each step (1-4) represents an oxidation, not a "breakdown".

Frederick C. Monson, PhD
Center for Advanced Scientific Imaging
Mail to Geology
West Chester University of Pennsylvania
Schmucker II Science Center, Room SS024
South Church Street and Rosedale Avenue
West Chester, PA, 19383
Phone:  610-738-0437

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)


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