Fixative question Formalin/Glutaraldehyde

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From:"Gregory Lloyd" <> (by way of histonet)
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Colleen wrote:

What is the difference between paraformaldehyde and glutaraldehyde,
chemically speaking. I recently used a fixative that was part of each for an
EM project and noticed quite a difference in how they worked on the tissue.

Colleen Forster
U of MN, Dept. of Neurology

Hi Colleen,

I'm not Jon but i"m sure my answers will match closely to what he has to
First, formalin (Paraformaldehyde with water added) is a mono-aldehyde  This
means that there is one aldehyde group attached at one end of the molecule.
This end is what reacts with the tissue (bonds to the tissue).  When
multiple molecules are close to each other loose hydrogen bonding occurs,
this effectively holds the tissue together. The product is referred to as
the formation of methylene bridges.  The methylene bridging that occurs is
what is holding the tissue together, the bonding to the tissue prevents
autolysis.  All this occurs fairly rapidly - at about 1 millimeter per hour.
Secondly glutaraldehyde is a dialdehyde, meaning two aldehyde groups, one at
each end.  The reaction of the aldehyde groups on this molecule is the same
as in formalin but, because there are two aldehyde groups on the molecule,
bonding is much stronger and at a higher definition (more tissue elements
fixed and better preservation).  Consequentially fixation is much slower
with this fixative with a rate of about 1mm per every two to three hours.

Glutaraldehyde is a better fixative for EM purposes because of the
preservation of tissue elements. BUT you must use very small pieces of
tissue (about 1mm cubed).  Glutaraldehyde would not be a good choice for
routine tissue preparation in the histology lab because of the rate of
fixation.  Thus most routine histology labs use formalin.
Formalin on the other hand is not the best fixative for EM because it does
not have very good preservation qualities, especially at the resolution that
EM requires.

I've had to pull the dust off from 15 years ago, I hope this rather lengthy
answer (unintended) answers your inquiry.  Wishing you good luck,

Cameron Lloyd, MLT
U of Alberta Hospital, Anatomical Pathology.
Edmonton, Alberta, Canada.

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