Figure 1. Overview of SICKO workflow and data output.

(a) Flow chart of SICKO workflow. [i] C. elegans are age-synchronized as eggs and allowed to grow to adulthood. [ii] Adult worms are challenged with fluorescently labeled bacteria or other microbes. [iii] Following challenge, worms are allowed to crawl on media seeded with non-fluorescent bacteria for 16 hours to wash bacteria from intestine and external surfaces that are not part of an adherent colony. [iv] Individual animals are plated on wells within a single-worm culture environment and [v] imaged daily. [vi] Images are analyzed and compiled. (b) Representative confocal image of an individual C. elegans harboring an intestinal E. coli labeled with GFP following challenge and wash. (c) Image of a single-worm culture environment. Worms are housed on individual wells of a Terasaki tray filled with NGM and seeded with bacteria. Wells are surrounded by an aversive barrier of palmitic acid to prevent fleeing. The Terasaki tray is mounted inside a single-well tray on a custom 3D printed adapter. The space around the Terasaki tray is filled with saturated water absorbing crystals, and the single well tray is wrapped in parafilm to prevent the wells from drying out. (d) Representative images of a single C. elegans harboring a GFP labeled E. coli intestinal colony over time. Images were taken using the GFP channel on a fluorescent stereoscope and processed with the SICKO software. GFP-labeled E. coli is shown in white. (f) Representative heatmap representation of data from a population of C. elegans challenged with fluorescently labeled bacteria. Each row is a single animal and each column is a day. Blue and yellow boxes indicate colony size. Red boxes indicate dead worms and black boxes indicate censored data. Rows above the white line represent animals harboring a bacteria colony, while rows below the white line represent animals without a colony.