Quantitative Electropharyngogram Assay of Anthelmintics

All EPG recordings were made in M9 buffer (Stiernagle, 2006 ), to which drugs, solvents or bacteria were added. Stocks of 5-Hydroxytryptamine creatinine sulfate complex (5HT, Sigma-Aldrich H7752; St. Louis, MO) were prepared in M9 at 40 mM, stored at −20 °C and diluted to desired concentrations in M9. Chip perfusion was initiated within 70 min of preparing a 5HT solution. Ivermectin (IVM; Sigma-Aldrich I8898) stocks (5 mM) were prepared in 100% DMSO and stored at −20 °C. The highest concentrations of DMSO that did not perturb EPG activity in control experiments were 0.2% (Fisher D-136) or 0.5% (Sigma-Aldrich Hybri-Max D2650) (data not shown) so these were the highest concentrations used in working solutions. Levamisole hydrochloride (LEV; Sigma 31,742) stocks (100 mM) were prepared in dH₂0 and stored at 4 °C until dilution on the day of use. Piperazine hexahydrate (PPZ; Sigma P7003) stocks (1M) were prepared in dH₂0 and stored at 4 °C until use. The pH of PPZ solutions was adjusted to 7.0 using HEPES buffer in M9 and titrating with 12 N HCl. Working solutions of all anthelmintic drugs were prepared from stocks and used on the same day. In some initial experiments, 0.005% Fast Green (Fisher F-99) was added to solutions to confirm uninterrupted perfusate flow (Lockery et al., 2012 (link)).