A new quantitative in vitro microculture method for Giardia duodenalis trophozoites

A reliable, rapid and low-cost method for drug sensitivity determination of Giardia duodenalis trophozoites (WB-strain) was developed in a 96-well plate. Using a standard inoculum of 5 × 104 trophozoites per well (300 μl), good growth was obtained after sealing the plate with an air-tight adhesive tape and incubation at 37 °C for 72 h in modified TYI-S-33 medium. Viable burdens were quantified using the formazan dyes MTT (100 μg/well) and XTT (20 μg/well) and the fluorescent substrate resazurin (2.5 μg/well). Prior removal of the culture medium is required since it causes spontaneous reduction of the substrate. Resazurin proved to be far superior to MTT and XTT with a level of sensitivity of about 3 × 104 trophozoites. Inhibitory concentrations (IC50) of several anti-giardial reference drugs were within the range of published values: metronidazole 2.25 μM, tinidazole 1.75 μM, albendazole 0.10 μM, furazolidone 2.00 μM and quinacrine 0.32 μM. The broad-spectrum antibiotics chloramphenicol, rifampicin, penicillin G + streptomycin and gentamycin were devoid of any inhibitory activity and are considered suitable for decontamination during excystation experiments.