Zinc formalin

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From:"Anatech Ltd." <anatech@net-link.net>
Date:Mon, 21 Jun 1999 12:12:19 -0400
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A comprehensive review of zinc formalin up to 1992 appears in the following

RW Dapson, 1993.  Fixation for the 1990's: a review of needs and
accomplishments.  Biotechnic & Histochem 68:75-82.

The mechanism appears to be that zinc ions hold macromolecules in their
native conformation via coordinate bonds, preventing the damaging
crosslinkages that formaldehyde alone would create.  The result is greatly
enhanced immunopreservation:  rarely is antigen retrieval necessary, and
primary antibodies can be diluted 2-10 fold greater than usual.

The original use of zinc formalin (by P. Banks and coworkers) was to
prevent nuclear bubbling artifact in tissues fixed for less than 24-48
hours.  Because nuclear morphology is so sharply defined, zinc formalin is
frequently used as a replacement for B-5.

Today there are several distinct varieties of zinc formalin, but all share
the advantages mentioned above.  The original formlula for zinc formalin is
1% zinc sulfate heptahydrate in 10% unbuffered formalin.  It tends to
precipitate in processors, as the zinc is not soluble in the 70% alcohol
used in the first dehydration station.  This can cause lines to plug if
routine acidic rinses are not performed.  Neutral or alkaline water will
not dissolve the precipitate.  Some processor manufacturers do not want
zinc formalin on their machines because of this.  The zinc inside the
specimens also precipitates (you cannot see it), and causes difficulties in

Anatech Ltd. developed an unbuffered zinc sulfate formula that does not
precipitate in 70% alcohol (it will precipitate if you go directly from it
to 80% or higher alcohol), and can be used in all processors.  Tissues are
not crunchy, and in fact customers often remark that they are easier to cut
than those fixed in NBF.

All unbuffered zinc formalins are acidic (pH varies from about 3 up to
about 4.5).  Formalin pigment will develop below about pH 5.3, faster as pH
gets lower.  The vast majority of our customers using unbuffered zinc
formalin do not report formalin pigment artifact (which frankly surprised
us), and I can only assume that it is because exposure time is too short
for it to happen.  Given comparable pH levels, acidic zinc formalin and
acidic (unbuffered) formalin will behave similarly regarding pigment

Buffered zinc formalin is also available, but again, some formulations
precipitate badly in processors.  Anatech's product (Z-Fix) is freely
soluble in alcohol, and can be made as an alcoholic buffered zinc formalin
for those of you who like the added benefits (speed, penetration of fat)
that alcoholic formalin provides.

Most zinc formalin solutions do not corrode metal any faster than
formaldehyde will.  Remember that formaldehyde is rather corrosive to
nearly all steel, including most forms of stainless (316 and 410 stainless
are safe).  I suspect that Pearl Gervais' cassette lids are made of some
other stainless steel; 316 and 410 are expensive and notoriously difficult
to mill.

One group of zinc formalin solutions is highly corrosive, however, and that
is made with zinc chloride.  Faster than its cousins and nearly as
aggressive as mercuric chloride, it has been used (not by us) as a B-5
replacement.  It can overfix tissues, just like B-5 does, and absolutely
must not be put in an enclosed processor.  There are very effective rapid,
zinc-based B-5 replacements that do not contain the chloride salt.

Anatech's products are availble only in the US and Canada.  Shandon Lipshaw
has a processor-compatible zinc formalin (unbufferd) that is available
worldwide.  I believe that Richard-Allan's zinc formalin product line is
available only in the US (Joan, correct me if I err here!).

Sorry for the lengthy answer: I wanted to cover everyone's questions and


Richard W. Dapson, Ph.D.
1020 Harts Lake Road
Battle Creek, MI  49015
800-262-8324 or 616-964-6450
Fax 616-964-8084
E-mail anatech@net-link.net

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