RE: Fading of eosin

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From:"Gary W. Gill" <>,
Date:Sat, 04 Sep 1999 11:09:24 -0500
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By now, perhaps you have learned the cause.  I suspect you may have found
that something was indeed changed, but the change was not appreciated.  In
general and for the benefit of subscribers, consider the following
systematic troubleshooting recommendations:

*	Be sure the eosin solution is working.  Stain a control tissue, the type
is irrelevant, in eosin only, and process routinely.  You might even want to
stain a buccal smear, rinse, and examine wet.  In other words, do the least
possible to the stained tissue to eliminate unexpected source of variation
and have the most confidence in your evaluation.  Always run a control
tissue as a quality assurance measure with every new batch of stain.

	If you're using readymade eosin, the vendor may have misformulated it.
Such mistakes don't occur frequently, but they do occur.  I know of several
from first-hand experience as a consultant.

*	If too little stain is present, whether in the control or routine tissues,
then either too little is getting in, or enough is getting in, but too much
is getting out.

*	If too little eosin is getting in, possible reasons include:

	If you're making the stain from scratch, correct for dye content variation.
	If you're making the stain from scratch and the eosin powder is "old", be
sure the dye content is 90% or greater.  Older lots that contain between
80-89% dye content sometimes contained unidentified salts that decreased
stain uptake, which is why the Biological Stain Commission increased the
minimum dye content requirement for eosin to 90%.
	Stain is insufficiently concentrated.  Increase the concentration.
	Dissolve in 70% ethanol.
	Include 0.5 mL glacial acetic acid per 100 mL stain.
	Stain longer.  Do the equivalent of bracketing photographic exposures:
double the staining time (e.g., 30 sec, 1, 2, 4, 8 min) to quickly find the
time that's right for you.  Very thin sections require longer staining times
than do thicker sections, simply because there's less there.

*	If too much eosin is getting out, possible reasons include:
	Inappropriate rinses.  An alkaline rinse, for example, will quickly undo
the staining.
	Rinsing too long (unlikely).
	Oddball causes (e.g., xylene substitutes [someone recently noted pale light
green staining in Pap EA stains])
	Water in xylene.  Have you checked for water droplets with the substage
condenser aperture diaphragm closed to exaggerate the appearance?
	Mounting medium related.  A few mounting media fade dyes substantially,
even in the dark.

Gary W. Gill

-----Original Message-----
From: []
Sent: August 04, 1999 3:22 PM
Subject: Fading of eosin

Have any of you in histoland had a problem with pale or fading eosin in the
H&E stain when you have changed nothing in your procedure.  If so, and you
solved the problem - what was the cause?


Freida Carson

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