NH4Cl 0.50 g KCl 0.20 g MgCl2 x 6 H2O 0.10 g KH2PO4 0.20 g NaCl 1.00 g Trace element solution SL-10 1.00 ml Selenite-tungstate solution 1.00 ml Na2CO3 3.00 g NaHCO3 10.00 g Sulfur (powdered) 10.00 g Yeast extract 0.10 g Na-acetate 2.50 g Distilled water 1000.00 ml Dissolve ingredients (ecxept carbonate, hydrogencarbonate, sulfur, yeast extract and acetate), then sparge medium with 100% N2 gas for 30 - 45 min to make it anoxic. Add solid carbonate and bicarbonate, adjust pH to 9.0 - 9.2, and dispense under 100% N2 gas atmosphere into anoxic Hungate-type tubes or serum vials containing already the appropriate amount of sulfur. Sterilize medium by autoclaving at 110°C for 20 min. Add yeast extract and acetate from sterile anoxic stock solutions prepared under 100% N2 gas. Trace element solution SL-10 (from medium 320) HCl (25%) 10.00 ml 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 990.00 ml First dissolve FeCl2 in the HCl, then dilute in water, add and dissolve the other salts. Finally make up to 1000.00 ml. Selenite-tungstate solution (from medium 385) NaOH 0.50 g Na2SeO3 x 5 H2O 3.00 mg Na2WO4 x 2 H2O 4.00 mg Distilled water 1000.00 ml
