FW: Hematoxylin pH

From:"Monson, Frederick C."

Morning Barbara,
	Your question brings back memories of long-ago discussions of what
the protocol for Hematoxylin staining could tell 'us' about the chemical
nature of the dye.
	Most Hematoxylin protocols with which you are probably familiar have
post-staining steps called "differentiation" and "stabilization" that are
performed with acidic and basic solutions of one type or another
	The biological stain, called Hematoxylin, C.I. 75290 IS an acid-base
indicator, having a primary inflection at around pH 6 (0.5% in ethanol).
Below this pH, its color is yellow.  Above pH 6-7, its color is described as
violet(blue is an alternative). In the pH range of 1-1.0, its color shifts
from Yellow to Red. (Ref:  Long's "Biochemists' Handbook",  van Nostrand,
1961[I finally have a copy of this wonderful book after searching for 25
years.]   It would appear reasonable to expect that this molecule would be
"violet-blue" at any pH above 6, not correcting for the effects of all of
the salts in the staining preparations.
	I too have often wondered what the pH was of my self-prepared
Ehrlich's Acid Hematoxylin preparation, but I have NEVER risked a pH
electrode for the job.  Thus, I have used pH paper strips in the mid-range
and taken the color of the paper just above the upper limit of the dye color
on the paper to represent the pH of the formulation.  That pH, as I
estimated it, with my male-limited capacity to "see" shades of red, was
always between pH=5 and 5.? (again, salt effects!?!?).
	It should be noted that the acid differentiation used in Hematoxylin
protocols operates on the dye-metal complex (Lake) which obviously is NOT
stable at acid pH.  For whatever mechanistic reason this coincides with the
violet-yellow transition that remains in the active heating byproduct of
Hematoxylin oxidation (see Kiernan, Histological and Histochemical Methods,
Arnold, 1999.  
	In closing, since H&E staining is so widespread in histology, and
since fading of archival materials is always a matter of importance, many of
the older preparations of mounting media recommend the use of clean(!)
marble chips to insure that acid byproducts do not accumulate while the
mountant preps languish on the shelf or in the working bottles on the
laboratory bench.  I have prepared my own dammar for 30 years (from defatted
crystals of the resin) and my Hematoxylin slides from the 1960's remain
	Finally, I must add a comment about your use of the word "heme" to
represent Hematoxylin "batches".  To chemists and biochemists, the word
"heme" conjures data about a molecule that has nothing to do with
Hematoxylin or its derivatives or formulations.  Using it in the way you
have will introduce a disconcerting pause into any conversation with
chemists, biochemists or any who know/remember the difference.  Something
like that happens to me when I hear a chairperson of a department of Cell
Biology give an hour talk on "The Structure of the Cell Nuculus[Sick!], or
hearing the President of the United States giving a news conference about
the nucular[double-Sick!!!] weapons of mass destruction in the hands of mad
tyrants.  I only add these last, because I don't want you to think that your
abbreviation of Hematoxylin is really a big deal in the scheme of things.  I
would still vote for you for President, for example.

Knowledge is power.  That's why the military wanted first contact with E.T.


Fred Monson

Frederick C. Monson, PhD
Center for Advanced Scientific Imaging
Mail to Geology
West Chester University of Pennsylvania
Schmucker II Science Center, Room SS024
South Church Street and Rosedale Avenue
West Chester, PA, 19383
Phone:  610-738-0437
eMail:  fmonson@wcupa.edu
An FEI (Quanta 400 and Technai 12), 
Oxford INCA Energy 400, and 
Olympus FV-300 Shop.

-----Original Message-----
From: barbalb@itsa.ucsf.edu [mailto:barbalb@itsa.ucsf.edu]
Sent: Monday, January 13, 2003 5:41 PM
To: histonet@pathology.swmed.edu
Subject: hematoxylin pH

Hi all,
Got a question from a pathologist (academic).  He asked about the pH of
hematoxylin.  I assume it's acidic because of the acids we've all added
to our home made heme batches, but I've never pH'd a batch of hematoxylin 
and find no reference to the pH in the few texts I've already consulted.

What do you all have to say?

Barbara Albert
UCSF Medical Center
San Francisco

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