Acid-fastness in microbiology: The versatility of Ziehl–Neelsen staining technique, its modifications, and integration of artificial intelligence – A brief review

Abstract

The Ziehl–Neelsen (ZN) staining technique, first developed in the late 19 th century, remains a cornerstone in microbiological identification of acid–fast bacilli (AFB). This review explores the historical development, principle, and procedure of the ZN staining method and also highlights its role in diagnosing tuberculosis (TB) and related infections. In addition to standard (hot) ZN staining, numerous modifications have been introduced to accommodate diverse clinical specimens and organisms. The review also discusses alternative techniques such as auramine fluorochrome staining and their comparative advantages. A significant recent advancement is the integration of artificial intelligence (AI) into AFB detection workflows, offering improved accuracy, reduced workload, and faster diagnostics through automated image analysis. Studies demonstrate high sensitivity and specificity of AI-based tools in identifying AFB in ZN-stained slides, suggesting strong potential for implementation in resource-limited settings. As global health efforts intensify against TB and related diseases, the ZN staining method, accompanied by digital innovation, continues to evolve as a pivotal diagnostic tool.