Counterstaining

Counterstaining is the application of a second stain after an initial staining step in microscopy to provide contrast and differentiate structures. The counterstain targets components that the primary stain does not color, or it colors them a contrasting shade, enabling clearer visualization of tissues, cells, or microorganisms.

In histology and cytology, a common example is the hematoxylin and eosin (H&E) stain. Hematoxylin binds to nucleic acids, coloring nuclei blue to purple, while eosin serves as the counterstain, staining cytoplasm, connective tissue, and other elements pink to red. This combination highlights cellular morphology and tissue architecture.

In microbiology, counterstaining is used in several differential stains. For example, the Gram stain begins with crystal violet, then after a decolorization step uses safranin as a counterstain to color Gram-negative bacteria pink, providing contrast against the purple Gram-positive cells. In endospore staining, malachite green is the primary stain and safranin acts as a counterstain to distinguish spores from vegetative cells. In Ziehl-Neelsen acid-fast staining, a methylene blue counterstain colors non–acid-fast organisms.

Counterstaining is also employed in more complex staining schemes, such as the Papanicolaou stain, which uses multiple counterstains to differentiate various cellular components in cytology preparations. The choice of counterstain depends on compatibility with the primary stain, tissue or organism type, and the desired level of contrast.