RE: [Histonet] RE: Eosin too pink

From:"Rittman, Barry R"

If I were in your shoes I would try a weaker solution of esoin and if necessary prolong the times.
I am also a believer in timing the stages of staining, as one perons two dips equals another persons three dips and so on.
One would think that considering the amount of time that we all have been doing H and Es, that there should be a published standard image that we all could agree upon.  This ain't gonna happen as the amount of staining with eosin that is regarded as desirable is generally a matter of  personal taste. When stainig for our pathologist many moons ago, we stained deeply with eosin, when staning for the researcher the eosin was extermely weak. I am not convinced that either of them had a good grasp of the finer points of staining but they were the customers.
I think that in talking about eosin staining, it should be borne in mind that the type of staining  with aqueous and with alcoholic eosin solutions can differ considerably.
Here in the states people have generally used alcoholic solutions of eosin. This gives a more rapid stain but in my opinion does not give the range of hues that can be obtained with aqueous solutions.
I am used to using a solution of an aqueous solution, a mixture of eosin Y and eosin B  (4 part to 1 part). Staining with this for 5 minutes and then carrying out most of the differentiation  in tap water followed by a rapid dehydration. This used for all tissue except bone where a much more dilute solution for longer times is best.
I believe that this provides a range of hues to the eosin staining that allows significantly better differentiation of structures. Heavy eosin staining tends to lose this differntiation.
The only time that I used to stain more heavily with esoin was for photographing sections, and that is not necessary anymore due to the improved imaging techniques and manipulations that are easy to carry out.
I think that as opinions are bound to differ, what we need is  to compare what individuals regard as a good H and Es.
How about sending in photos of what you regard as "good H and Es" to the Histonet picture site?
I suggest that there be a limited number of tissues so that we do not end up with a logistic nightmare.
How about liver, small intestine, uterus, skeletal muscle and skin?
I think that for bone this might be best via the Hard Tissue Communique.
Just my opinion.


From: on behalf of Mark Adam Tarango
Sent: Sat 1/14/2006 8:03 PM
Subject: [Histonet] RE: Eosin too pink

So is he complaining that the eosin is too strong or just too pink?  It seems to me like the more water you have in the alcohol after Eosin or if you use a water rinse after eosin it gets's more orange when you have a higher percentage of alcohol in the alcohol after eosin(just seems that way to me anyway).  If it's just too strong, dilute it down with alcohol.  I hardly get any specimens, so I don't like changing the Eosin often, i just keep adding alcohol to it when it gets low and crusted around the edges..... still seems to work fine....

Mark T.

> Sent: Friday, January 13, 2006 10:42 AM
> To:
> Subject: [Histonet] Eosin too pink
> Hi List,
>  We are currently doing our H & E stain by hand and using the
> Richard-Allan 7211 Hematoxylin and their Eosin Y.  Our pathologist is
> still complaining of slides being TOO PINK.  I have cut the eosin step
> down to just 5 dips from 15 dips, and still too pink.  The Eosin is very
> rich.
> This is our protocol:
> Hydrate to water like normal (thru Clear Rite 3, 100% and 95% Flex)
> Hematoxylin- 2 mins
> Rinsing in water until the water runs clear
> Clarifier II- 30 dips
> running water -30 dips
> Richard Allan Bluing - 30 dips
> running water - 30 dips 
> 95% Flex - 10 dips
> Eosin-Y- 5 quick dips
> Thru 95%, 100% etc to xylene
> Any suggestions on a possible method change or a more mild Eosin.
> Complaint seems to solely "TOO PINK".  I have done some experimenting w/
> cutting the eosin w/ 80% Flex- and it does calm it down a bit but, the
> Hemo looks more purply then blue, almost periwinkle so not sure if that
> will be better or not- we still need to have him review the test slides.
> Any suggestions ?
> Helayne Parker
> Histology Section Head
> Skaggs Community Health Center
> Branson, Missouri

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