ALL hematoxylins require blueing, but not all require differentiation in
acid.  Progressive stains, such as Mayer's, do not; regressive ones, such as
Harris's, do.  Cells and tissue stained by aluminum-hematein (i.e., what we
ordinarily call "hematoxylin" for convenience)are initially red.  Blueing
begin so rapidly, however, that we don't see the red color.  To observe it,
first pass the stained preps into absolute alcohol, which is essentially
chemically inert relatively to any color change of hematoxylin, you'll see
the red color.  Indeed, in some staining methods, unblued hematoxylin is
used as a nuclear "counterstain."  Purple nuclei indicate partial blueing,
meaning a mixture of red and blue molecules.  Blue longer to complete the
color conversion.

Blueing can occur over a wide range of pHs.  All that's important is that
the pH of the solution be alkaline relative to the isoelectric point of
aluminum-hematein (which escapes me at the moment).  Blueing can begin at pH
5 and continue thru pH 10+.  The lower the pH, the longer the time, and
conversely.  Very high pHs can detach cells from slides, apparently by
hydrolysis.  There's no need to use a chemically defined blueing agent; tap
water alone for 1-2 minutes will  suffice.  Try it at various times until
the blue color equals that of a control prep blued routinely -- especially
in thick areas.  Tap water saves time and money.

Gary W. Gill

-----Original Message-----
From: Victor A. Tobias [mailto:tobiasv@vetmed.wsu.edu]
Sent: August 05, 1999 6:49 PM
To: Barry Rittman
Cc: histonet@pathology.swmed.edu
Subject: Re: eosin fading


I can't totally agree on the blueing theory, because we had a problem
with eosin fading many years ago and we only use Mayer's Hematoxylin, no
blueing required. Our first thought was that we had wrong dilutions of
alcohol after the eosin. That wasn't the problem, we just made up some
fresh eosin and everything was back to normal.

Victor