Figure 1.

Workflow diagram for the Onchocerca gutturosa adult worm in vitro 5-day motility/MTT assay. (A) Nuchal ligament connective tissue from naturally infected cattle; (B) Tissue in culture medium placed under a dissecting microscope (40× magnification) to isolate worms using fine forceps; (C) Isolated worm floating on tissue culture medium, worm length approximately 2 cm; (D) Worms were maintained individually in each well of a 24-well plate containing 2 mL culture medium, at 37 °C under an atmosphere of 5% CO₂ in air; this was replaced after 24 h with culture medium containing the test drug at a final concentration of 1.25 × 10⁻⁵ M. Worm motility was determined microscopically every 24 h up to 120 h; (E) Worm viability was then assessed by the transfer of each worm to the well of a 48-well plate containing 0.5 mg/mL MTT, incubation at 37 °C for 30 min; (F) Each worm was transferred to a well in a 96-well plate containing 200 µL dimethylsulfoxide to solubilize the formazan. After 1 h at 23–26 °C, the plate was gently agitated and formazan formation measured using absorbance (490 nm) on an ELISA reader (Dynatech, Willenhall, UK).