Enhancing Reproducibility in Chemotaxis Assays for Caenorhabditis elegans

The ability of Caenorhabditis elegans (C. elegans) to navigate complex environments is essential for their survival. This natural behavior is commonly used in chemotaxis assays, which are important tools for studying the function of sensory neurons and neural circuits. Chemotaxis has been essential for discovering fundamental functions in neuronal signaling during the past decades. However, a lack of thoroughly optimized and standardized procedures can lead to variable results that can be difficult to interpret. To improve reproducibility, we optimized several aspects of chemotaxis protocols by testing different odorant concentrations, numbers of worms, and assay durations, as well as the preparation of chemotaxis plates and the washing procedures of worms. The usage of a 2-choice or a 4-choice assay was also evaluated. Our new protocol improves the clarity of results and simplifies worm counting. The protocol optimization is condensed into a 5-day step-by-step protocol that increases the reproducibility of chemotaxis in C. elegans. Compared to previously published chemotaxis protocols, the revised method reduces day-to-day variability using an improved and standardized assay design that ensures clear and reliable results. Several key components in the assay preparation and during the assay have been evaluated based on previous protocols, such as odor concentration, worm density, and assay length. By considering multiple factors that influence the worm's behavior, our optimized protocol enhances the reproducibility of chemotaxis assays in C. elegans, making them more reliable and accessible for studying phenotypes related to olfaction and neural circuit behavior. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Optimized Chemotaxis Assay for C. elegans.