Purification and Detection of Bacterial Endospores: Current Methods and Challenges

Bacterial endospores are highly resistant, dormant forms that pose persistent challenges in food safety, environmental microbiology, and industrial hygiene. Accurate evaluation of endospore resistance, physiology, and inactivation depends on both purification and detection methods; however, these processes are typically examined independently, limiting methodological consistency and contributing to variability across studies. In this review, current approaches for endospore purification and detection are critically examined, including washing-based methods, density gradient centrifugation, enzymatic treatments, culture-based enumeration, molecular assays, flow cytometry, and emerging biosensor technologies. In addition, these methods are compared using metrics such as purity, recovery yield, sensitivity, and specificity, and their advantages and limitations are summarized to clarify performance. It is further proposed that endospore purification and detection should be considered as a single, end-to-end analytical workflow and optimized accordingly. Purification strategies influence sample cleanliness and aspects of endospore quality, including viability, structural integrity, and physiological state, which affect detection performance and quantitative accuracy. Based on this integrated perspective, a conceptual framework linking purification efficiency to detection outcomes is presented, along with practical considerations for method selection across relevant application contexts. Finally, gaps in standardization are identified, and future research directions are outlined to improve reproducibility and cross-study comparability in endospore-related studies.