Counterstained

Counterstained refers to a specimen that has been treated with a second dye after a primary stain to provide contrast between different cellular components or tissue elements. The counterstain binds to structures that the first dye does not adequately stain, enabling visualization of multiple features within the same sample.

In microbiology, counterstaining is a common step in differential stains. For example, in Gram staining, the primary stain crystal violet is followed by a decolorizing step and then a counterstain with safranin. This produces purple Gram-positive cells and pink or red Gram-negative cells, highlighting the difference in cell wall structure. In acid-fast staining, carbol fuchsin is the primary stain and a counterstain such as methylene blue or brilliant green is used after decolorization to color non–acid-fast organisms, aiding differentiation.

In histology, counterstaining is used to add contrast to tissue sections. Hematoxylin stains nuclei blue to purple, and a counterstain like eosin colors the cytoplasm and extracellular matrix pink, improving visibility and differentiation of tissue components. In fluorescence microscopy, counterstains such as DAPI or phalloidin provide contrasting signals to highlight nuclei or cytoskeletal structures, respectively.

Purpose and considerations: counterstains improve contrast, reveal additional features, and assist in identification or characterization of samples. Selection of a counterstain depends on its chemical compatibility with the initial stain, the target structure, and the desired contrast. Proper timing and washing steps are important to prevent overstaining or masking of textures.

Overall, counterstaining is a key technique in microscopy that enhances interpretability by adding complementary color information to differential staining protocols.