[Histonet] Problem with Schiff's reagent

[Swain, Frances L]

Sorry Paul I replied to Renee in stead of you.  I purchase my Schiff's from PolyScientific R & D.  When I made my Schiff's Used essentially the same recipe that you do.  I made mine in a flask stored it in the dark for 24 hours.  I then added activated charcoal to excess and let it sit in the dark for at least 2-3 hours.  I then filtered it through filter paper that to which charcoal was added to the filter paper before I pour the Schiff's in it to clear.  It will usually clear up if it has a slightly yellow tinge you can put it in the refrigerator in a tightly capped bottle overnight and it should become clear.  It is important to store your Schiff's in the refrigerator when you are not usuing it and use a brown bottle.  You can test your Schiff's to see if it is viable by dropping a drop of your Schiff's in 50 ml of 37-40% Formaldehyde.  If it turns purplish pink it is still good if it turns any other color.  Do not use it. 

Frances L. Swain HT(ASCP) A. A. S.
Special Procedures Technician
Department of Orthopaedic Surgery
Center for Orthopaedic Research
Barton Research Building 2R28
4301 West Markham Street
Little Rock AR 72205
(501) 686-8739 PHONE
(501) 686-8987 FAX
swainfrancesl <@t> uams.edu email

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of pbrunko <@t> siue.edu
Sent: Saturday, February 14, 2009 11:54 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Problem with Schiff's reagent


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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