Quantifying Pathogen Infection in C. elegans

N. parisii spores were prepared as previously described [46 (link)], and Orsay virions were prepared as described previously [9 (link)]. For pathogen load analysis (dx.doi.org/10.17504/protocols.io.waiface), synchronized L1 worms were plated with a mixture of OP50 bacteria and 5 x 10⁵N. parisii spores or a 1:20 dilution of Orsay virus filtrate, and then incubated at 25°C for either 30 hours (N. parisii) or 18 hours (Orsay virus) before fixing with paraformaldehyde. Fixed worms were stained with individual FISH probes conjugated to the red Cal Fluor 610 dye (Biosearch Technologies) targeting either N. parisii ribosomal RNA or Orsay virus RNA. N. parisii pathogen load was measured with the COPAS Biosort machine (Union Biometrica). Orsay virus infection was assayed visually using the 10x objective on a Zeiss AxioImager M1 microscope. In feeding measurement assays (dx.doi.org/10.17504/protocols.io.v6ne9de), plates were set up as for pathogen infection with the addition of fluorescent beads (Fluoresbrite Polychromatic Red Microspheres, Polysciences Inc.). Worms were fixed in paraformaldehyde after 30 minutes and red fluorescence signal was measured with the COPAS Biosort machine (Union Biometrica).