Isolation of Microbes from Nematode Cuticles

Baiting of microbes from the rhizosphere or bulk soil suspensions on the cuticle of P. penetrans was done as previously described [25 (link)]. Briefly, 20,000 surface-sterilized nematodes were incubated in 5 mL of each microbial suspension in 15 mL tubes. The tubes were positioned horizontally on a shaker at a speed of 150 rpm at 20 ± 2 °C for 24 h. The nematodes with attached microbes were recovered on 5-µm sieves and washed with 20 mL of sterile tap water to remove loosely attached microbes. To isolate the attached bacterial strains, nematodes were plated on R2A media (Merck, Germany) supplemented with 10 mg L⁻¹ cycloheximide. The plates were incubated at 28 °C and bacterial strains were collected from the emerged colonies over a 2-week period. A portion of the nematodes with attached microbes was transferred to bead-beating tubes with Lysing Matrix E (MP Bio, Heidelberg, Germany) for DNA extraction.