Re: [Histonet] Bouin's alternative/substitution question - Rather long reply

From:John Kiernan

Dear Jason,

After 6 days nobody has posted a Histonet answer
to your two questions. This is not surprising. The
product "Bouin's fixative substitute" is on
the vendor's website, and elsewhere on the Web
too, but with no indication of what it contains.
Without this knowledge, who could take the risk of
using the product?

Bouin's fixative contains three ingredients, each
with a function that has been fairly well
understood since Bouin published the recipe in
1897. The advantages and limitations of Bouin's
fluid are also well documented, and are familiar
to anyone who examines Bouin-fixed and
otherwise-fixed preparations. If the substitute
does not contain formaldehyde or picric acid, what
chemicals replace the actions of both these
compounds? I think we should be told!

Davidson's fixative, mentioned in one reply, is
one of several AFA or FAA
(alcohol-formalin-acetic) mixtures, with an
alcohol content (30%) lower than most others in
this class. It has an interesting and incompletely
known history; evidently Davidson never published
the mixture himself. Check out the Histosearch
archives; also

Another use of Bouin's fluid is pre-treating
sections of formaldehyde-fixed tissue before
staining by a trichrome method (Masson, Mallory,
Heidenhain, Cason, Gomori, Gabe etc). The
pre-treatment enhances the different coloration of
collagen fibres and cytoplasm (especially that of
smooth muscle). This effect is equally achievable
with an aqueous solution of picric acid. Other
compounds might achieve the same effect, but have
their advantages passed the test of peer reviewed
publication? Nothing in an AFA/Davidson mixture
has this priming action for trichrome stains.

For what it's worth, my advice is to use Bouin's
fluid as a fixative (if you are happy with the
results). If you have to do nucleic acid
histochemistry, change the routine fixative to
neutral buffered formaldehyde, an AFA or even a
simple formaldehyde-free alcohol-acetic mixture
such as Clarke's or Carnoy's.

For really critical work requiring preservation of
intracellular structure at the oil-immersion level
of resolution in paraffin sections it is difficult
to avoid mercuric chloride, osmium tetroxide and
potassium dichromate, if organelles and thin
collagenous structures are to be stained with
traditional methods that use dyes. For
immunohistochemistry, HgCl2, OsO4 and K2Cr2O7 are
best avoided, but picric acid has a long and
honourable history of being IHC-friendly. (This
may have nothing to do with its fixative action. I
could expound  but won't.)

Bottom 2 lines:
1. Know your fixative, and don't use any mixture
with undisclosed ingredients.
2. A wrong diagnosis could easily result from
inappropriate fixation. If this comes to
judgement: a pathologist might be scolded, but a
technician (less expensive lawyer) is likely to be
fired (scapegoat).

John Kiernan
Anatomy, UWO
London, Canada
--------------------------------------- wrote:
> We are removing Bouin's fixative from our laboratory and we are looking
> for an alternative.  Has anyone had experience in using the Bouin's
> Fixative Substitution distributed by IMEB or something similar from
> another vendor?  From reviewing the Histonet  archives I see that most
> people use Davidson's fixative but I just wanted to see if anyone had any
> personal experience with these substitutions.
> Thanks in advance,
> Jason
> Jason D. Burrill
> Manager, Histology
> Charles River Laboratories
> 251 Ballardvale Street
> Wilmington, MA 01887
> ph:   978-658-6000 ext 1652
> fax:  978-988-8793
> E-mail:
> _______________________________________________
> Histonet mailing list

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