[Histonet] Problem with Schiff's reagent

[pbrunko <@t> siue.edu]

Good Morning!

I'm a newcomer to the list, and I'll start by pointing out that I'm not a
histologist by any means.  I am a freshwater ecologist, and we're trying to
study mucus secretion behavior in freshwater snails.

Last Spring and last summer, we developed a process whereby we could visualize
snail mucus trails on glass slides using a periodic acid-Schiff's reagent
staining technique.  But now, in following the same protocol as used last year
for making our own Schiff's reagent, I cannot get the final solution to filter
out clear.

Recipe I'm using:
900 ml boiling water
10 grams basic fuchsin
25 ml concentrated HCl acid (12 M)
40 grams sodium metabisulfite
(this is essentially Sigma Aldrich's ratios, I think)

Let this sit for 24 to 72 hours, take 100 ml aliquot, add 0.75 - 1.0 gram
ground activated charcoal, stir for 10 minutes, filter through filter paper
then through GF/C glass fiber filter.  Last summer I got nice, clear (slightly
yellow) and very active Schiff's reagent.

But now I cannot seem to get the filtrate to be clear.  Even after 10 minutes
exposure to ground activated charcoal and filtering, the filtrate remains
bright orange to dark red and it does not seem to stain mucus trails very well.

All the reagents are the same as those used last summer (i.e., less than 7
months old; although the HCl is a bottle several years old from a different
lab).

Anyone have any troubleshooting suggestions?  I don't know the chemistry very
well, but the sodium metabisulfite is used for "decoloring" the initial
solution, right?  So is the metabisulfite not working for some reason now??

Any help/suggestions would be greatly appreciated.

Cheers -

Paul

Paul E. Brunkow, PhD
Department of Biological Sciences
Southern Illinois University Edwardsville
Edwardsville, IL    USA
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