[Histonet] Problem with Schiff's reagent

Pamela Marcum pmarcum <@t> vet.upenn.edu
Tue Feb 17 10:22:08 CST 2009

Frances is using the classic formula for Schiff's Reagent.  I have done the
activated charcoal step for a few hours and overnight if we started too late
in the day.  Both times should work very well.  Thank goodness we can now
buy from good vendors that ship good Schiff's.  I have been bright pink more
than once, and it is not my favorite colour.  We use the same vendor she
uses by the way.  

Pamela A Marcum
University of Pennsylvania 
School of Veterinary Medicine
Comparative Orthopedic Laboratory (CORL)
382 W Street Rd
Kennett Square PA 19438

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Swain,
Frances L
Sent: Tuesday, February 17, 2009 10:32 AM
To: pbrunko <@t> siue.edu; histonet <@t> lists.utsouthwestern.edu
Subject: RE: [Histonet] Problem with Schiff's reagent

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
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
for making our own Schiff's reagent, I cannot get the final solution to
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
yellow) and very active Schiff's reagent.

But now I cannot seem to get the filtrate to be clear.  Even after 10
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

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

Anyone have any troubleshooting suggestions?  I don't know the chemistry
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 E. Brunkow, PhD
Department of Biological Sciences
Southern Illinois University Edwardsville
Edwardsville, IL    USA
SIUE Web Mail

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