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Copper Acetate Manufacturers(WSDTY) proposed Copper Acetate growth curves and preparation of metal stocks.To better characterize the effects of metals on R. palustris, cultures were inoculated into media containing metals, and growth was monitored. Measuring growth in the presence of metals provided a simple and reproducible way to measure the inhibitory effects of metals and has been used in other toxicity studies (15, 27). Growth curve experiments were performed in 10 ml of the media listed above, using sealed 25-ml Balch tubes. For growth assays, metals were added to individual tubes before inoculation. R. palustris and R. capsulatus from stationary anaerobic cultures were inoculated into an anoxic chamber with a 1:200 inoculum for growth curve measurement. E. coli cultures were started by diluting an overnight anaerobic minimal-medium culture 1:100, and S. oneidensis was diluted 1:50 from an anaerobic LB culture. Fe(II), Cu(II), Ni(II), and Co(II) stocks were prepared by adding FeCl2 (98% pure [Sigma] or 99.9% pure), CuCl2, NiCl2, or CoCl2 (Fisher) to nanopurified water under anoxic conditions.

Cu(I) was prepared under anoxic conditions from CuCl (Alfa Aesar) as a 100 μM stock in 1 M NaCl, with heating to 37C to aid dissolution. Bacterial culture growth was monitored by measuring the optical densities of Balch tubes on a Spectronic 20D+ spectrophotometer at 660 nm for R. palustris and R. capsulatus and at 600 nm for E. coli and S. oneidensis.

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