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Acid-fast stains (AFS) remain indispensable in modern diagnostic microbiology; they are used for detecting mycobacteria (including Mycobacterium tuberculosis and Mycobacterium leprae), acid-fast parasites, and some acid-variable bacteria as well as in histopathology. Fluorescent AFS surpass brightfield AFS (Ziehl-Neelsen) in sensitivity, particularly when pathogen loads are low. However, latest evidence suggests that these stains target nucleic acids, whereas lipid-rich, intact cell walls merely prevent decolourisation; this evidence corrects the long-held assumption that AFS bind to mycolic acids. This mechanism explains morphological features, such as the characteristic beading in mycobacteria, with direct implications for training microscopists and advancing artificial intelligence-assisted image analysis. This mechanism also facilitates protocol enhancements, including the use of high-yield fluorochromes or novel approaches to reduce background fluorescence. The latest novel applications, such as the detection of a low number of Schistosoma spp eggs, exemplify the broader utility of AFS. Combined with artificial intelligence-based slide interpretation, these advances in understanding staining mechanisms and expanding diagnostic applications show that AFS remain an important diagnostic laboratory modality, with considerable potential for future improvements.
Hänscheid et al. (Wed,) studied this question.