Differentiation of the eosin occurs best in 70% alcohol and, to a lesser extent, in higher-grade alcohols; therefore, adding 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. Carryover 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.
