Peptone-free nematode growth medium improves culture stability and physiological resilience in Caenorhabditis elegans

Nematode growth medium (NGM) has been the standard culture medium for Caenorhabditis elegans (C. elegans) for over five decades and contains peptone to support proliferation of the bacterial food source, Escherichia coli OP50. However, the increasing use of paraformaldehyde (PFA)-killed OP50 in studies of metabolism, aging, and host-microbe interactions raise the question of whether peptone remains necessary once bacterial proliferation has been eliminated. Here, we systematically compared conventional and peptone-free NGM using PFA-killed OP50 as the bacterial food source. Peptone removal completely prevented residual bacterial proliferation and markedly reduced fungal contamination, thereby improving culture stability. Although worms cultured on peptone-free NGM exhibited reduced food preference and pharyngeal pumping, they maintained normal growth, reproduction, embryonic viability, lifespan, and chemotactic behavior. Peptone-free NGM also reduced basal intracellular reactive oxygen species (ROS) and lipid accumulation, accompanied by downregulation of genes involved in oxidative stress responses and lipogenesis. In addition, worms displayed enhanced locomotor activity and significantly greater resistance to oxidative, ultraviolet, and thermal stress. Collectively, these findings demonstrate that peptone is dispensable when PFA-killed OP50 is used as the bacterial food source. Peptone-free NGM therefore represents a simple, inexpensive, and practical refinement of the conventional culture system for C. elegans studies employing PFA-killed bacterial diets.