Copper Acetate Manufacturers(WSDTY) proposed growth media and culturing conditions lists all strains tested in this work were Copper Acetate grown anaerobically in minimal freshwater medium as described previously with a lowered phosphate concentration and an alternate trace element mixture: the basal medium consisted of 100 μM KH2PO4, 5.61 mM NH4Cl, 0.68 mM CaCl2, and 2 mM MgSO4. After autoclaving, the medium was cooled under 20% CO2–80% N2 gas. After cooling, 30 mM sterile NaHCO3, a vitamin mixture [0.29 μM 4-aminobenzoic acid, 41 nM d-(+)-biotin, 0.81 μM nicotinic acid, 0.21 μM Ca-(+)-pantothenate, 0.41 μM pyridoxamine dihydrochloride, 0.29 μM thiamine dichloride, 1.32 μM riboflavin)], B12 (0.1 mg/ml), and a trace element mixture were added. The cooled medium was adjusted to pH 7 with 1 M Na2CO3 and was stored in an anoxic glove box under a 5% H2–15% CO2–80% N2 atmosphere. Acetate was added before inoculation (final concentration, 10 mM). For routine aerobic growth, YPS-MOPS medium (3 g/liter yeast extract, 3 g/liter peptone, 10 mM succinate, 20 mM morpholinepropanesulfonic acid [MOPS] [pH 7]) was used. Anaerobic cultures were grown in sealed Balch tubes at 30C in a light incubator, and aerobic cultures were grown in a dark shaking incubator.
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