Solution A 951.00 ml Solution B 3.00 ml Solution C 40.00 ml Solution D 10.00 ml Sparge solution A with 100% N2 gas for 30 - 45 min to make it anoxic. Dispense solution under same gas atmosphere into anoxic Hungate-type tubes or serum vials and sterilize by autoclaving. Solutions B and D are autoclaved separately under 100% N2 gas atmosphere. Solution C is autoclaved under 80% N2 and 20% CO2 gas atmosphere. The medium is completed before use by adding appropriate amounts of solutions B, C and D to solution A. Adjust pH of complete medium to 7.2, if necessary. Solution A KH2PO4 1.00 g NH4Cl 0.30 g MgSO4 x 7 H2O 1.00 g MgCl2 x 6 H2O 2.00 g NaCl 20.00 g CaCl2 x 2 H2O 0.10 g Na2SO4 1.77 g Trace element solution SL-11 1.00 ml Na2-DL-malate 2.66 g Yeast extract 0.50 g Sodium resazurin (0.1% w/v) 0.50 ml Distilled water 950.00 ml Adjust pH to 6.0 with 2 N NaOH. Solution B Ethanol 0.30 ml Distilled water 2.70 ml Solution C NaHCO3 1.85 g Distilled water 40.00 ml Solution D Na2S x 9 H2O 0.30 g Distilled water 10.00 ml Trace element solution SL-11 (from medium 722) Na2-EDTA x 2 H2O 5.20 g FeCl2 x 4 H2O 1.50 g ZnCl2 70.00 mg MnCl2 x 4 H2O 100.00 mg H3BO3 6.00 mg CoCl2 x 6 H2O 190.00 mg CuCl2 x 2 H2O 2.00 mg NiCl2 x 6 H2O 24.00 mg Na2MoO4 x 2 H2O 36.00 mg Distilled water 1000.00 ml Dissolve EDTA in 800 ml distilled water, adjust pH to 7 using 2 N NaOH and add ferrous chloride. After ferrous chloride has dissolved add other compounds. Finally adjust pH to 6.0 and bring volume to 1000 ml.
