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The page below is a sample from the LabCE course Theoretical and Practical Aspects of Routine H&E Staining. Access the complete course and earn ASCLS P.A.C.E.-approved continuing education credits by subscribing online.

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Poor Eosin Differentiation

Differentiation of the eosin occurs best in 70% alcohol and to a lesser extent in higher grade alcohols; therefore, adding a 70% alcohol to the dehydration series or adjusting the time in the 70% alcohol will aid in obtaining the desirable three distinct shades of eosin.
The pH of the eosin is another possible cause of poor differentiation. Carry-over of the alkaline bluing solution into the eosin can occur if the slides are not rinsed adequately prior to the eosin. This increase in pH will interfere with the uptake of the dye into the tissue. Proper rinse time, adjusting the pH with the addition of acetic acid, and/or regular changes of fresh eosin will resolve this problem.
The image on the left is one shade of pale pink as opposed to three shades of pink in the image to the right. This was probably due to being over-differentiated in the alcohols.