Abstract
We investigated the bacterial food digestion and accumulation in wild-type adult Caenorhabditis elegans (C. elegans) worms that have fed on either non-pathogenic RFP-expressing Escherichia coli (E. coli) OP50 or pathogenic-RFP-expressing Pseudomonas aeruginosa (P. aeruginosa) PAO1 during the first 4 days of adulthood. Once the worms had completed their planned feeding cycles, they were loaded on microfluidic chips, where they were fixed to allow high-resolution z-stack fluorescence imaging of their intestines utilizing a Spinning Disk Confocal Microscope (SDCM) equipped with a high-resolution oil-immersion objective (60×). IMARIS software was used to visualize and analyze the obtained images, resulting in the production of three-dimensional constructs of the intestinal bacterial load. We discovered two distinct patterns for the bacteria-derived fluorescence signal in the intestine: (i) individual fluorescent spots, originating from intact bacteria, were present in the fluorescent E. coli-OP50-fed worms, and (ii) individual fluorescent spots (originating from intact bacteria) were dispersed in large regions of diffuse fluorescence (RDF), originating from disrupted bacteria, in fluorescent P. aeruginosa-PAO1-fed worms. We performed a semi-automated single-worm-resolution quantitative analysis of the intestinal bacterial load, which showed that the intestinal bacterial load generally increases with age of the worms, but more rapidly for the fluorescent P. aeruginosa-PAO1-fed worms.