Fluorescent acid-fast stains for diagnosing mycobacteria and beyond: back to the future?

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.