Standard solutions of Pb(NO3)2, Cd(NO3)2, and Cr(NO3)3 of 1000 mg/l were purchased from CHEM-LAB (Belgium). Solutions were sterilized by filtration through 0.45μm Millipore bacterial filters (Advantec, Co. Ltd. Tokyo, Japan) and used for the preliminary screening of heavy metal-resistant actinomycetes isolates by the agar well diffusion method35 . Briefly, wells (8 mm diameter) were punched in ISP2 agar media, seeded with each actinomycete isolate via sterile borers. To each well, 100 μl of the standard metal solution, at various concentrations (30–500 mg/l), was loaded and incubated at 30 °C for 7 days. The diameter of growth inhibition around each well was measured (in millimeters) and the Minimum Inhibitory Concentration (MIC) of each metal, that prevents visible growth of actinomycetes, was recorded13 (link). The actinomycete isolate with the highest metal-resistance ability was selected for further studies.
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