Re: formalin fixative Again!

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From:"R.Wadley" <>
Date:Fri, 16 Jul 1999 16:36:29 +1000
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	Dear John,

	My most available reference is Bancroft & Stevens, 1990, Theory & Practice
of Histological Techniques, 3rd Edn., pp33.

	Commercial formalin is ~35-40% formaldehyde gas in water, + numerous
inorganic & organic contaminants (depending on its quality).  In solution
the formaldehyde is present in (broadly) 3 forms; its monohydrate,
methylene glycol; in equilibrium with its monomeric formaldehyde; & formic
acid.  Some low molecular weight polymers are also present.

	As the solution ages the low molecular weight polymers gradually becaome
high molecular weight polymers.  This is the white gritty stuff that is
seen in old drums of bulk solution.  To slow this process commercial
reagents have 10-15% methanol included.  The increase in high molecular
weight polymers is matched by an increase in formic acid.

	Therefore, formalin doesn't go bad as much as its active constituent
becomes progressively polymerised & the solution becomes more acid.  The
effect on tissue antigens is 2 fold:  1; they get poorly fixed 2: depending
on their sensitivity to low pH they get wiped out.

	As to 4% buffered paraformaldehyde, I like to use mine within 7 days
maximum, even if its stored at 4C.  For imortant EM I make it fresh & toss
away any left over.  Paraformaldehyde solutions are 'cleaner' than
commercial preparations but importantly they lack methanol, this is good
for those sensitive antigens, but bad becuase the
polymerisation/acidification process moves along at a much faster rate.

	I'm sure I've got some more comprehensive references somewhere, if I
remember Iwill try & find them.

	PS, formaldehyde is an irritant, it is also an allergen, potentially
carcinogenic, highly flammable as a gas, & unless I dip my nose in it I
can't even smell it.



At 11:20 AM 7/15/99 -0500, you wrote:
>Dear Folks
>This is a probably a general irritant BUT can someone please explain what
>it is about formalin fixatives that goes bad and what the chemistry
>actually is and what the effects on tissue antigens are?
>I've read several discussions but never one that was really satisfying.
>I'm sure that no one knows most of what is really going on but what are
>the best guesses.
>In my case, I suspect I've been too cavalier with my 4% buffered
>parafomalin fixative.
>I'd appreciate any good references on the topic as well as personal
>knowledge, witnessing, or general venting.
>Yours in a fix
>John Carroll Dennis
>Anatomy, Physiology, and Pharmacology
>109 Greene Hall
>Auburn University, AL  36849

R. Wadley, B.App.Sc. M.L.S, Grad.Dip.Sc.MM
Laboratory Manager
Cellular Analysis Facility
School of Microbiology & Immunology
UNSW, New South Wales, Australia, 2052
Ph (BH) 	+61 (2) 9385 3517
Ph (AH)	+61 (2) 9555 1239
Fax 	+61 (2) 9385 1591

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