Metronidazole bioassay with increased sensitivity

The bioassay of metronidazole using Clostridium butyricum incorporated in agar plates detected concentrations higher than 1.0 microgram/ml. Gentamicin in the sample did not affect the growth of the target organism or the inhibition by metronidazole. Penicillin in the sample could be eliminated by the incorporation of penicillinase in the agar or by using as the target organism a surface inoculum of the penicillin-resistant Bacteroides fragilis. Increasing duration and temperature of aerobic prediffusion before allowing the growth of the strictly anaerobic Cl. butyricum increased the diameters of inhibition by metronidazole, but did not affect the threshold of detectability. The incorporation of metronidazole in the agar increased the sensitivity of the assay ten- to 100-fold.