K2HPO4 0.40 g NH4Cl 1.00 g Yeast extract 2.00 g Trypticase 2.00 g MgCl2 x 6 H2O 1.00 g CaCl2 x 2 H2O 0.40 g Modified Wolin's mineral solution 10.00 ml Sodium resazurin (0.1% w/v) 0.50 ml Na2CO3 1.50 g Na2S x 9 H2O 0.30 g Distilled water 1000.00 ml Dissolve ingredients except carbonate and sulfide, then sparge medium with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Dispense medium under same gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. Add carbonate after autoclaving from an anoxic stock solution prepared under 80% N2 and 20% CO2 gas atmospher and sulfide from a sterile anoxic stock solution autoclaved under 100% N2 gas. Adjust pH of the complete medium to pH 7.0. Modified Wolin's mineral solution (from medium 141) Nitrilotriacetic acid 1.50 g MgSO4 x 7 H2O 3.00 g MnSO4 x H2O 0.50 g NaCl 1.00 g FeSO4 x 7 H2O 0.10 g CoSO4 x 7 H2O 0.18 g CaCl2 x 2 H2O 0.10 g ZnSO4 x 7 H2O 0.18 g CuSO4 x 5 H2O 0.01 g AlK(SO4)2 x 12 H2O 0.02 g H3BO3 0.01 g Na2MoO4 x 2 H2O 0.01 g NiCl2 x 6 H2O 0.03 g Na2SeO3 x 5 H2O 0.30 mg Na2WO4 x 2 H2O 0.40 mg Distilled water 1000.00 ml First dissolve nitrilotriacetic acid and adjust pH to 6.5 with KOH, then add minerals. Adjust final to pH 7.0 with KOH.
