RE: H & E Staining Method

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From:"Gary W. Gill" <>
To:"Wenk, Lee & Peggy" <>, <Edna_J_Gonzalez/>
Date:Sat, 21 Aug 1999 09:53:03 -0500
Content-Type:text/plain; charset="iso-8859-1"

All hematoxylins are acidic, but not all are acidic because acetic acid has
been added.  The mordant itself lowers the pH (i.e., Lewis acid [?]).
Harris hematoxylin has been used in at least four different formulations:
full-strength, half-strength, with and without acetic acid.  Full-strength
without acetic acid is strongest; half-strength with acetic acid is least
strong.  Acetic acid separates the mordant from the hematein, and so reduces
the working concentration of aluminum-hematein, the combination that
produces the ultimate blue color, and that for convenience we call

If acetic acid is added to excess, as I've done experimentally, the solution
reverts to the color of oxidized hematoxylin (i.e., hematein [amber in 25%
ethylene glycol {Gill's 'hematoxylin solvent}]) and does not stain at all.
When adding acetic acid to a purple hematoxylin formulation until the color
becomes a "deep cherry red", one is simply adjusting the working
aluminum-hematein concentration to approximately the same starting point
from batch to batch.  The eye is serving as a qualitative spectrophotometer.
A hematoxylin formulation with acetic acid, therefore, stains less
vigorously therefore than the same formulation without acetic acid.

pH per se is less important than the consequences it has on the working
concentration of Al-hematein.

Gary W. Gill

-----Original Message-----
From: Wenk, Lee & Peggy []
Sent: August 20, 1999 5:18 AM
To: Edna_J_Gonzalez/
Cc: HistoNet Server
Subject: Re: H & E Staining Method

I have two question. What is non-acidified Harris hematoxylin?
Why is it being used for this project? (No sarcasm intended.
I just don't happen to know about non-acidified hematoxylin.)

Aren't all hematoxylins used in the H&E stain acidified, i.e.,
pH'ed with acetic acid? For our Mayer and Gill, we like the
pH around 2.4-2.5. What is the best pH for Harris hematoxylin?
Would there be a difference in pH if the ripening agent
is mercuric oxide vs. sodium iodate?

Could this be part of the problem with overstaining? Too
high of a pH, making is more non-specific - staining
nucleoplasm and cytoplasm in addition to the chromatin
material? Out of curiosity, are your slides picking up
a lot of blue color too, which would indicate too high
of a pH?

I guess I have more than 2 questions. Oh, well.


Peggy A. Wenk, HTL(ASCP)
William Beaumont Hospital
Royal Oak, MI 48073

Edna_J_Gonzalez/ wrote:
> I am working with pig skin and doing H&E regressive staining using Harris
> Hematoxylin (non-acidified). The problem is that the hematoxylin is too
> dark and I can't differentiate what I need, especially it is too dark in
> the basal layer and the epidermis of the skin. I need a H&E Regressive
> Method that will be lighter. Currently I am staining with hematoxylin for
> 10 minutes, which I think is too much, but this is what one of the methods
> say. I know there are different H&E regressive methods (with different
> times for Hematoxylin). Any suggestions?
> Edna Gonzalez
> PowderJect Vaccines

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