Accurate bacterial inactivation methods are essential for nutritional and microbiota studies in Caenorhabditis elegans, to determine whether the observed effects arise from nutrients provided by ingested bacteria or from active symbiotic interactions. However, some inactivation methods alter bacterial palatability, complicating conclusions about their direct impact. We aimed to identify an effective method for inactivating the bacterial strain Escherichia coli OP50, the standard food source for most C. elegans experiments, that preserves normal behavior and physiology in C. elegans. We compared heat inactivation (65 °C for 35 min) with 0.5% paraformaldehyde (PFA) inactivation. Worms fed PFA-inactivated bacteria showed no food aversion, and maintained wild-type pharyngeal pumping levels, fertility rates, and lipid accumulation, closely resembling the behavior and physiology of worms fed alive E. coli OP50. In contrast, heat‑inactivated bacteria elicited strong food avoidance, reduced pumping activity, activation of the mitochondrial unfolded protein response (UPR), decreased lipid stores and fertility, and increased survival relative to the other groups. These findings demonstrate that 0.5% PFA inactivation more accurately preserves C. elegans physiological and behavioral traits than heat inactivation, making it a more suitable method for microbiota and nutritional studies. mt
