Lifespan Assay of C. elegans Under Bacterial Stress

Experimental procedures and data analysis were performed as previously described (33 (link), 125 (link)). C. elegans wild-type Bristol strain N2 worms were grown at 22°C on nematode growth medium (NGM) agar plates using E. coli OP50 as the nutrient. Untreated or treated bacteria (10⁹ CFU mL⁻¹) were spread onto NGM solidified agar plates before incubation at 37°C overnight. The plates were cooled to room temperature for 4 h, and 20 to 30 L4-synchronized worms were plated and incubated at 22°C in a humid environment to prevent plate drying. Worm survival was scored daily for 32 days using an Axiovert S100 optical microscope (Zeiss, Oberkochen, Germany) equipped with a digital camera (DXM 1200F; Nikon Instruments, Melville, NY). Four independent experiments per condition were performed, and all worms from each condition were used for the survival assay. The Kaplan-Meier method was used to calculate the nematode survival, and the significance of survival differences was tested using a log-rank test (Prism software, version 4.0; GraphPad Software, San Diego, CA). As a control to ascertain similar growth on NGM plates between treated and untreated bacteria, the NGM agar plates were entirely scraped every 5 days for enumeration on LB agar plates.