For development rate assays, P. pacificus, C. elegans, Rhabditophanes sp., and A. sudhausi were grown on OP50 at 20 °C. Nematode eggs were bleached, washed with M9 several times and allowed to hatch in M9 medium for 20 h in the absence of food to cause juvenile arrest at the J2 development stage. Once synchronized, juvenile larvae were filtered through two Millipore 20 μm filters and around 30–60 juvenile (J2) animals were transferred to NGM plates (supplemented with/without 500 nM vitamin B12) spotted in 50 μl of the desired test bacterial strain. Nematodes were subsequently allowed to develop on test bacteria for the following time periods: P. pacificus 57 h at 20 °C, C. elegans and Rhabditophanes sp. 45 h at 20 °C and A. sudhausi for 144 h at RT. Following this, worms were categorized into groups based on the development of the vulva and germ line using 0.63× objective of ZEISS SteREO Discovery V12 following previously established protocols [38 (link)].
20 μm filters
Manufactured by Merck Group
20 μm filters are laboratory equipment designed to remove particles larger than 20 micrometers in diameter from liquid samples. These filters are used to purify and clarify solutions by selectively retaining larger suspended solids while allowing the passage of smaller molecules and compounds.
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2 protocols using 20 μm filters
1
Nematode Development Assay Protocol
2
Quantifying Predatory Behavior in Pristionchus
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Corpse assays facilitated rapid quantification of predatory behavior and were conducted as previously described (15 (link), 25 (link)). Briefly, to generate substantial quantities of larvae for use as prey, Pristionchus strains were maintained on E. coli OP50 bacteria until freshly starved, resulting in an abundance of young larvae. These plates were washed with M9 buffer, passed through two Millipore 20-μm filters and centrifuged at 377g to form a concentrated larval pellet of juvenile animals. Excess buffer was removed, and 1 μl of worm pellet was deposited onto a 6-cm NGM-unseeded assay plates. This resulted in roughly 3000 prey larvae on each assay plate. Assay plates were left for a minimum of 1 hour to allow larvae to distribute evenly over the plate. Young adult Pristionchus predators were screened for the required mouth form and transferred to empty NGM plates for 30 min to remove any excess bacteria from their bodies. Subsequently, 20 Pristionchus predators were added to assay plates and allowed to feed for 24 hours before removal, and the plates were scored for the presence of corpses.
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