Small Molecule Nematicides Screening Assay: A Medium Throughput Method to Screen Potential Nematicides Against Ditylenchus dipsaci

To prepare the assay plates, pour autoclaved distilled water into a sterile trough, and dispense 40 microliters of distilled water from the trough into each well of a flat-bottom, 96-well plate with a multichannel pipette. Add chemicals from the 96-well chemical stock plates to the assay plates, by pinning three times into the chemical plate. Then, transfer the pins 10 times into the assay plate. Blot onto paper in front of the cleaning solution.

To count the number of nematodes from the collection, first, resuspend the collection, and then, pipette 5 microliters of the solution using low-retention tips onto a slide. Count the number of nematodes in 5 microliters using a dissection microscope. Then, adjust the concentration to two worms per microliter using sterile distilled water.

Next, add 10 microliters of the sample to each well of the 96-well plates with a multichannel pipette and a trough. Wrap the plates with a damp paper towel and place them in a box. Then, add an extra damp paper towel to stabilize and ensure minimal movement of the plates, and affix on a sticky pad in a 20-degree Celsius shaking incubator set to 200 rpm.

Observe the plates on day 5 under a dissecting microscope. Count the number of mobile and total D. dipsaci in DMSO solvent controls and drug-treated wells.

If the worms are immobile, add 2 microliters of 1 molar sodium hydroxide to a final concentration of 40 millimolar to the well to stimulate movement. Calculate the proportion of mobile worms. In the D. dipsaci screens, wells that reproducibly yielded 0% mobile worms are categorized as strong hits.