Nematode growth medium agar with 10 ml 0.1% Tergitol was supplemented with 0.01 mM of EPA as described. Synchronised L2 C.elegans nematodes were grown for 24 h on NGM agar plates with or without 0.01 mM EPA, seeded with E.coli OP50 until they reached L3 stage. Thereafter, the L3 nematodes were carefully harvested and washed three times with sterile M9 buffer. L3 nematodes were transferred to fresh plates and pre-treated for 24 h with 17-octadecynoic acid (17-ODYA) or 6-(2-propargyloxyphenyl)hexanoic acid (PPOH) (Cayman Chemicals) until they reached L4 stage. The compounds were added directly to the bacterial food at a final concentration of 0.05 mM [37 (link), 38 (link)]. In all cases, solvent controls were included. L4 nematodes were infected with C.albicans and 60 nematodes were transferred into 2 ml of liquid medium (80% M9 buffer, 20% BHI, 90 μg/ml kanamycin) in a single well of a six-well tissue culture plate and incubated at 25 °C. Hyphal formation was monitored after 24 h of incubation. In addition, nematodes exposed to EPA and 17-ODYA were monitored for survival by scoring them as either alive or dead. If nematodes did not show any movement in response to mechanical stimulation, they were considered dead and thus removed from liquid medium assay [25 (link)].
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