H&E stained sections of liver show very dark nuclei and some blue staining of the cytoplasm. This is most likely caused by:

• A. Inadequate differentiation of hematoxylin
• B. Over-differentiation of eosin
• C. Under-fixed tissue
• D. Excess eosin concentration

Answer: (A)

Very dark nuclei in H&E sections occur when hematoxylin is not adequately differentiated. Hematoxylin binds to nuclear DNA and RNA, giving a blue/purple color to the nuclei. During differentiation, acid alcohol is used to remove excess hematoxylin from non-nuclear structures. If this step is insufficient, too much hematoxylin remains bound in the chromatin, making nuclei appear very dark or almost black. The small amount of blue in the cytoplasm can happen because residual hematoxylin may also stain some cytoplasmic basophilic components before eosin fully counterstains with pink. Conversely, over-differentiation of hematoxylin would lighten the nuclei, and excessive eosin would intensify cytoplasmic pink without producing such dark nuclei. Under-fixed tissue can cause general staining artifacts but does not specifically explain the marked nuclear darkness.