overstaining

Overstaining is a condition in which a specimen is stained too intensely, resulting in colors that are darker or more saturated than intended. In histology and cytology, overstaining reduces contrast between cellular components and can obscure diagnostic details, leading to misinterpretation. It is most commonly encountered during slide staining procedures such as hematoxylin and eosin (H&E) staining, Gram staining, and special stains, where precise timing and destaining are critical.

In Gram staining, overstaining can cause Gram-negative bacteria to retain crystal violet, making them appear Gram-positive, while in H&E, excessive staining with hematoxylin or eosin can darken nuclei or cytoplasm excessively.

Causes include prolonged stain exposure, high stain concentration, inadequate or excessive destaining, inadequate washing, old reagents, thick tissue sections, and improper pH. Effects include reduced contrast, masking fine structures such as nuclei and cytoplasmic details, and potential misclassification of cells or organisms.

Prevention and troubleshooting involve optimizing staining times, using fresh reagents, adjusting concentrations, balancing destaining steps, checking section thickness, verifying pH, and including appropriate controls. If overstaining is detected, one may attempt brief destaining or rehydration steps followed by re-staining with shortened times, or, in some cases, starting anew with a properly prepared slide.