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 aldehyde stage.
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 solution.
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 oxidation times.
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
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