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Problems in Histopathological Technique
Prepared by
ROY ELLIS
IMVS Division of Pathology
The Queen Elizabeth Hospital
Woodville Road, Woodville, South Australia 5011
Email: roy.ellis@imvs.sa.gov.au
Did you know that the pH of tissue and nuclei within the body is within the range 7.6 - 7.8?
What this means is that when you fix tissue the pH of the fixative should remain as close as possible to the pH of tissue if radical changes to tissue structure are not to occur.
Since formalin is the most common fixative in regular use formalin is the fixative I have chosen to discuss. Unbuffered concentrated formalin has a pH in the range 3.0-4.0.
It varies because concentrated formalin contains the polymers, carbonyl formaldehyde and the monomer methylene glycol and usually some condensation product in the form of the solid paraformaldehyde. Paraformaldehyde is often visible as a white precipitate in the bottle. The proportions of each in the final product can vary thus affecting the final pH also depolymerisation of the polymers is an ongoing process which can also alter the pH.
10% formalin neutralised, note the term neutralised - not buffered, because there is a difference, neutralised with calcium carbonate has a pH of 7.0 whereas if magnesium carbonate is used the pH is 7.6. Also if you use magnesium carbonate instead of calcium carbonate you avoid calcium artefacts in the tissue. Formalin buffered with sodium chloride, or sea water, for marine histology also has a pH of 7.6. Whilst neutral buffered formalin has a pH of 7.
pH is very important because the further a fixative is from the pH of body tissues the more distortion you can get in the tissue at the cellular level. Buffering your fixative is also better than neutralising your fixative because if large tissues are fixed the pH of the solution should be maintained and then things like the formation of formalin pigment in acid fixatives is avoided.
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© Roy C. Ellis 2002