Possible Explanations for Cell Nuclei Being Too Dark or Too Light in a Hematoxylin and Eosin Stain

Nuclear staining with hematoxylin may be too light or too dark. This can often be traced back to a problem in the slide preparation.

Parameter	Too Dark	Too Light
hematoxylin solution	too concentrated	too dilute; solution too old
time in hematoxylin solution	too long	too short
differentiation of stain	inadequate	excessive
pH of hematoxylin solution	incorrect (should be around 2.5)
deparaffinization	NA	incomplete
fixation	NA	poor
processing	NA	poor
water used for rinsing	NA	tap water with iron, sulfur or chlorine

Parameter	Too Dark	Too Light
hematoxylin solution	too concentrated	too dilute; solution too old
time in hematoxylin solution	too long	too short
differentiation of stain	inadequate	excessive
pH of hematoxylin solution	incorrect (should be around 2.5)
deparaffinization	NA	incomplete
fixation	NA	poor
processing	NA	poor
water used for rinsing	NA	tap water with iron, sulfur or chlorine

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