counterstain

A counterstain is a secondary stain applied after an initial stain to provide contrast and differentiate structures within a specimen. By binding to different cellular components than the primary stain, the counterstain helps reveal morphology and relationships that might be obscure if only the first stain were used. The choice of counterstain depends on the staining protocol, the specimen, and the desired diagnostic or research information.

In microbiology, counterstains are commonly used to distinguish types of cells after a decolorization step. For example, in the Gram stain, crystal violet is followed by a decolorizer and a counterstain such as safranin; Gram-positive cells retain the primary stain, while Gram-negative cells take up the counterstain and appear pink. In acid-fast staining, a counterstain like methylene blue colors non-acid-fast cells after the primary stain is applied and the decolorization step.

In histology and pathology, counterstains enhance tissue architecture. Hematoxylin stains nuclei blue or purple, and eosin serves as a counterstain to color cytoplasm and extracellular matrix pink, improving contrast for morphological assessment. In immunohistochemistry, a nuclear or cytoplasmic counterstain (e.g., hematoxylin) provides a contrasting background that helps interpret specific labeling.

In fluorescence microscopy, fluorescent counterstains such as DAPI or propidium iodide color additional cellular components and permit multi-channel visualization. Important considerations include stain compatibility with the primary dye, optimal timing, and potential over- or under-staining, which can affect contrast and interpretation.

Limitations include color overlap, nonspecific binding, and the need to balance brightness of the primary stain with the counterstain to avoid obscuring key structures.