The reasoning sounds right to me.
I might suggest using 0.5% periodic acid (such as in the PAS stain) for 5-10
minutes as the oxidizer for the GMS stain, instead of the chromic acid.
Since the picric acid has started the carbohydrate oxidation, and the
chromic acid is then overoxidizing the remaining carbohydrates, then using a
mild oxidizer such as periodic acid might do the trick. Usually, periodic
acid is NOT used in the GMS stain, as not enough background is overoxidized.
The PASM/Jones stain used to demonstrate basement membranes in kidney
biopsies is the same silver methenamine solution as in the GMS, but periodic
acid is used instead of chromic acid. Our kidney biopsies are fixed in DB,
which is an alcoholic-Bouins, which does contain picric acid. But the
PASM/Jones stain continues to demonstrate basement membrane. If a GMS is
done on the kidney biopsy, the basement membrane is not demonstrated.
So I think using 0.5% periodic acid for 5-10 minutes, rinsing in d. water,
and then going into the GMS methenamine silver solution should work.
Peggy A. Wenk, HTL(ASC)SLS
William Beaumont Hospital
Royal Oak, MI 48073
[mailto:firstname.lastname@example.org] On Behalf Of Paul
Sent: Saturday, February 16, 2008 2:53 AM
To: email@example.com; HistoNet Server
Subject: [Histonet] GMS and Bouin's fixation
Logically, there may be a conflict between fixing tissues in Bouin's and
successful demonstration of fungi using a methemamine silver technique.
This is my reasoning:
Bouin has long been the recommended fixative for glycogen and proteoglygans
which are best demonstrated by the PAS reaction. In the PAS, carbohydrate
groups are oxidised by periodic acid to form aldehydes, which in turn react
with Schiff reagent to produce the magenta colour. Periodic acid, as a 1%
solution at room temperature, will oxidize these groups only as far as the
The methenamine silver is essentially a modification of that same concept,
but using chromic acid and an unstable silver solution in place of the
periodic acid and Schiff reagent. In the methenamine silver technique, the
carbohydrates in the capsule surrounding the fungal elements are oxidized to
form aldehydes which reduce the silver solution to produce visible deposits
of silver. Prolonged treatment with chromic acid will over-oxidize the
carbohydrates to carboxyl groups which are non-reactive with the silver
There may well be a reaction between the picric acid in Bouin's fixative and
carbohydrates. Picric acid is a potent oxidizer and may begin the oxidation
of the carbohydrate groups in the fungal capsule. When the sections are
further oxidized by chromic acid, the fungal walls become over-oxidized and
form non-reactive carboxyl groups.
It may be worth trying a much shorter chromic acid treatment on Bouin's
fixed tissues to see if this will leave the carbohydrates in the fungi at
the aldehyde stage.
Most methenamine silver techniques suggest a 60 minutes treatment in 5%
chromic acid at room temperature. In the case of Bouin-fixed tissues, I
would suggest running a trial using a range of chromic acid times from 10-40
minutes to see if the fungi are still reactive with one of the shorter
I would be very interested to know if this solves your problem.
> Hello all, has anyone ever experienced Bouin's fixed tissue?preventing
> the methenamine from staining fungus?? Thank you,
> Alice Neumann MD
> Western Wyoming Pathology
> Jackson, WY 83001
> Cell: 307-413-4042
> Work: 307-733-6418
> Home: 307-734-4410
> Fax: 307-734-0885
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