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 x 10(4) trophozoites per well (300 microl), good growth was obtained after sealing the plate with an air-tight adhesive tape and incubation at 37 degrees C for 72 h in modified TYI-S-33 medium. Viable burdens were quantified using the formazan dyes MTT (100 microg/well) and XTT (20 microg/well) and the fluorescent substrate resazurin (2.5 microg/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 x 10(4) trophozoites. Inhibitory concentrations (IC(50)) of several anti-giardial reference drugs were within the range of published values: metronidazole 2.25 microM, tinidazole 1.75 microM, albendazole 0.10 microM, furazolidone 2.00 microM and quinacrine 0.32 microM. 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.
Bénéré
Ely
E
Laboratory for Microbiology, Parasitology and Hygiene, Faculty of Pharmaceutical, Biomedical and Veterinary Sciences, Antwerp University, Groenenborgerlaan 171, B-2020 Antwerp, Belgium.
da Luz
Raquel Andreia Inocêncio
RA
Vermeersch
Marieke
M
Cos
Paul
P
Maes
Louis
L
D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D000981:Antiprotozoal Agents / Q000494:pharmacology; D003470:Culture Media / Q000737:chemistry; D046508:Culture Techniques / Q000379:methods*; D005562:Formazans / Q000378:metabolism; D005872:Giardia / Q000187:drug effects / Q000254:growth & development* / Q000378:metabolism; D020128:Inhibitory Concentration 50; D008826:Microbial Sensitivity Tests / Q000379:methods*; D050296:Microbial Viability; D010078:Oxazines / Q000378:metabolism; D010274:Parasitology / Q000379:methods*; D013778:Tetrazolium Salts / Q000378:metabolism; D053058:Trophozoites / Q000187:drug effects / Q000254:growth & development* / Q000378:metabolism; D014966:Xanthenes / Q000378:metabolism
D000900:Anti-Bacterial Agents; D000981:Antiprotozoal Agents; D003470:Culture Media; D005562:Formazans; D010078:Oxazines; D013778:Tetrazolium Salts; D014966:Xanthenes; C005843:resazurin; C079782:MTT formazan; C059088:1,5-bis(2-methoxy-4-nitro-5-sulfophenyl)-3-((phenylamino)carbonyl)formazan