Long-Term Culture of Individual <em>Caenorhabditis elegans</em> on Solid Media for Longitudinal Fluorescence Monitoring and Aversive Interventions

SICKO: Systematic Imaging ofCaenorhabditisKilling Organisms

“…Recently, we reported a new method for long-term cultivation of C. elegans that is conducive to longitudinal fluorescent imaging of C. elegans on solid nematode growth media (NGM), as an alternative to microfluidics devices. 11 SICKO combines this cultivation technique with systematic image collection and processing of individual C. elegans colonized with fluorescently labeled bacteria to capture the dynamic nature of C. elegans host-microbe interactions across lifespan and the impact on host health and survival. SICKO experiments begin by age-synchronizing a population of C. elegans using standard techniques and initially maintaining animals in group culture on petri plates under standard conditions (NGM seeded with E. coli food; Fig.…”

“…(b) Confocal image of C. elegans following bacterial exposure [step ii] and wash [step iii] displaying an advanced infection in the gut. (c) Custom multi-well plate providing isolated single-worm culture environment for C. elegans that is compatible with fluorescent imaging (see Espejo et al 11 for preparation protocol). (e) Sample GFP fluorescent images capture daily for a single animal demonstrating progressing of E. coli OP50 infection throughout life.…”

“…1a[iv], c ), the preparation of which we recently published. 11 Finally, fluorescent images of each isolated, free-crawling worms is captured daily ( Fig. 1a[v] ) and analyzed to quantify the area and intensity of the fluorescent bacteria in the gut ( Fig.…”

“…[i] Age-synchronized worms arc harvested and allowed to grow to adulthood under standard culture conditions, [ii] Adult worms are challenged with pathogen expressing a fluorescent protein at an age and for an exposure period defined by the researcher and optimized for the pathogen of interest, [iii] Following the exposure period, worms are transferred to plates seeded with non-fluorescent bacteria and allowed to crawl for 16 hours to expel unattached live bacteria from the gut and cuticle, [iv] Worms are transferred to a culture environment that isolates individual animals on solid NGM seeded with non-fluorescent bacterial food, [v] Survival is scored and fluorescent images capture for each animal daily, [vi] Images are processed to quantify daily infection intensity and area, (b) Confocal image of C. elegans following bacterial exposure [step ii] and wash [step iii] displaying an advanced infection in the gut. (c) Custom multi-well plate providing isolated single-worm culture environment for C. elegans that is compatible with fluorescent imaging (see Espejo et al 11 for preparation protocol), (e) Sample GFP fluorescent images capture daily for a single animal demonstrating progressing of E. coil OP50 infection throughout life, (f) Heatmap of composite SICKO infection and survival data. Each row represents the infection state of a single animal.…”

“…At the end of the 16 hour incubation, randomly selected animals are transferred to individual wells of single-worm multi-well culture environments, prepared as previously described. 11 Individual agar pads contain non-fluorescent OP50 and gentamycin to prevent colonizing growth during the observation period, and to continue selecting for fluorescent bacteria adherent to the C. elegans gut. The number of animals transferred, the number remaining alive on the plate, and the number that died on the plate are all recorded for calculation of SICKO coefficient later.…”