I would be happy to send anyone interested diagrams and explanation of the
reaction.  John Koski worked this out for EM Science years ago when he
developed Harleco's Schiff's Reagent.  It pretty well answers Peggy's
question.

E-mail me at rkline@emscience with your fax number or address.

Rande Kline HT (ASCP)
Technical Services
EM Science
800-222-0342x443




lpwenk@netquest.com (Wenk, Lee & Peggy) on 10/30/98 05:54:42 AM

To:   histonet@pathology.swmed.edu
cc:
Subject:  THE CHEMISTRY OF SCHIFF




Have a question that I've never been able to find the answer, and
it bugs me. Please reply directly to me, as I'm the one with
the itch.

Why do we make Schiff reagent a leuko dye? In other words,
why doesn't the basic fuchsin stain the glycogen/neutral mucins/
few acid mucins without being contorted into a colorless
compound? Why, after we wash the slides after staining, does the
dye still stick to the component, but it wouldn't bind before?

I learned that the sulfur from the sodium metabisulfite
rearranges the chromophores of the basic fuchsin, making the
basic fuchsin colorless. The HCl lowers the pH allowing this new
colorless structure to be stable. The charcoal is to remove
any excess basic fuchsin and an acridine compound impurity that
arises in the making of basic fuchsin. Then the basic fuchsin
binds to 2 aldehydes created with the periodic acid and
glycogens/mucins. Then, according to what I was taught/read,
washing in water raises the pH, making the leuko structure
unstable, rearranging the chromophores back to their (nearly)
original positions, and, voila, we get magenta.

As long as that is correct (and I know someone(s) will let me
know if it is not), my question is still . . . why doesn't
the basic fuchsin bind to the aldehydes of glycogen/etc.
WITHOUT having to be rearranged? And why does it remain
bound to the glycogen/etc. AFTER it has been rearranged back?
If it couldn't bind before, why can it bind after?

Again, this is purely "Got To Know the Chemical Reason Itch".
But I cannot find an explanation in about a dozen books.

Please help, Histonetters!

--
Peggy A. Wenk, HTL (ASCP)
Anatomic Pathology
Wm. Beaumont Hospital
3601 W. 13 Mile Rd.
Royal Oak, MI 48073-6769