A single colony of E. coli OP50 bacteria is inoculated in lysogenic buffer (LB) and cultured overnight (18 h) in a 37 °C shaking incubator. The bacterial culture is then aliquoted into Erlenmeyer flasks and 32% paraformaldehyde (PFA) is added to the aliquots to bring the final PFA concentration down to 0.5%. The PFA-treated bacterial aliquots are then mixed in the 37 °C shaking incubator for 1 h to allow for even mixing and sufficient exposure to PFA. The aliquots are transferred to 50 mL conical tubes and centrifuged at RCF (relative centrifugal force) of approximately 3000 × g for 20 min and washed with 25 mL of LB five times. The aliquots are concentrated 10-fold for lifespan assays and 5-fold for other downstream applications and seeded on the NGM plates.