Simple and Accessible Egg-Hatching Assays in Caenorhabditis elegans: Adaptable Formats for Drug Screening

Egg-hatching assays (EHA) are widely used to evaluate the effects of chemical compounds on early developmental stages of nematodes, particularly in anthelmintic research. However, existing methodologies vary substantially in format, complexity, and reproducibility, and are often presented as fixed protocols rather than adaptable experimental frameworks. In this study, we establish a simple, accessible, and flexible platform for egg-hatching assays in Caenorhabditis elegans by integrating and directly comparing three experimental configurations: (i) controlled incubation in microcentrifuge tubes followed by seeding on agar plates, (ii) direct exposure on drug-containing agar plates, and (iii) multiwell-based liquid assays. Two representative compounds were used to illustrate the assay: one that inhibits egg hatching and one that has no detectable effect. Each configuration provides distinct advantages in terms of experimental control, throughput, and accessibility. The microcentrifuge tube-based approach enables precise control of drug exposure and supports quantitative pharmacological analyses. Agar-based assays offer a simplified and low-cost alternative suitable for continuous exposure and educational settings. Multiwell formats enable scalable, standardized screening consistent with modern drug discovery pipelines. By directly comparing these formats, we show how methodological choices influence assay reproducibility, drug exposure dynamics, and biological interpretation, particularly in relation to stage-specific factors such as the protective role of the eggshell and developmental variation in molecular target expression. Overall, this work establishes egg-hatching assays in C. elegans as a versatile and scalable platform adaptable to diverse applications, including anthelmintic screening, toxicological assessment, developmental studies, and teaching laboratories, thereby facilitating broader and more rational use of this methodology.