A Staggered L1 Plating Strategy Enables Daily Availability of Synchronized Caenorhabditis elegans L4 Worms

Caenorhabditis elegans (C. elegans) is a non-parasitic roundworm, widely used as an animal model in biological research due to its genetic tractability, short lifecycle, and suitability for high-throughput experimental studies. Synchronization is commonly employed strategy to obtain age-matched C. elegans populations, particularly at the L4 stage for experimental studies. However, the developmental interval required for L1 larvae to reach the L4 stage limits flexibility for spontaneous experiments. Furthermore, conventional synchronization protocols become time-consuming, labor-intensive, and repetitive when multiple experimental replicates are required over consecutive days. Here, we present a staggered L1 plating strategy that enables the daily availability of synchronized L4 worms. In this approach, a large worm population is bleached to obtain growth-arrested L1 larvae, which are then plated in fractions onto NGM plates seeded with paraformaldehyde-killed OP50 at 0, 1, 2, 3, 4, 5, and 6 days post-hatching. Our results show no significant differences in body length, body volume, brood size, embryonic viability, or locomotion (body length per second [BLPS] and body bends per second [BBPS]) among worms plated at different time points. Together, these findings demonstrate that this method provides a simple, efficient, and reproducible strategy for ensuring daily access to synchronized L4 populations, thereby facilitating both routine and spontaneous experiments.