A rapid agar-diffusion test for quantifying the toxic effects of copper on microorganisms

The rapid agar diffusion test for quantifying the toxic effects of copper on microorganisms was elaborated. The assay was run in standard Petri dishes containing Czapek Solution Agar (Difco). Immediately after inoculation with the organism tested 3.2 mm diameter wells were punched in the agar. Wells were filled (20 microliters) with distilled water (control) and a range of solutions containing 128 to 4096 ppm Cu2+ (as CuSO4). After incubation (2-5 days, 26 degrees C) the diameters of Cu-inhibition zones were measured. Cu-sensitivity indices (ICu's) were calculated according to the formula: ICu = a2/(3.2-b), where: a--slope of the linear regression equation: log [Cu] (x)--inhibition zone diameter (y), b--intercept of the same equation. Additionally for characterization of Cu-sensitivity of the organism tested the predicted highest non-inhibitory Cu concentration (10c) was calculated, where: c = (3.2-b)/a. Both ICu's and adjusted average diameters of Cu-inhibition zones characterized well the Cu-sensitivity in organisms tested, as indicated by the statistical analyses. ICu's correlated very well (negative correlation) with 50% effective doses (Ed50's) of Cu2+ for mycelial growth of Cylindrocarpon destructans. The applicability of the elaborated assay in ecotoxicological studies was discussed.