Final pH: 7.2 - 7.5 Final volume: 1026 ml NH4Cl 0.30 g K2HPO4 0.20 g MgCl2 x 6 H2O 0.40 g CaCl2 x 2 H2O 0.10 g KCl 0.50 g NaCl 1.00 g Yeast extract (BD Bacto) 5.00 g Trypticase peptone (BD BBL) 5.00 g Trace element solution SL-10 1.00 ml Sodium resazurin (0.1% w/v) 0.50 ml Na2CO3 2.00 g trans-Cinnamic acid (3% w/v, neutralized with NaOH) 25.00 ml L-Cysteine HCl x H2O 0.50 g Na2S x 9 H2O 0.50 g Distilled water 1000.00 ml Dissolve all ingredients except the carbonate, cinnamic acid, sulfide, and cysteine. Then, sparge the medium with an 80% N2 and 20% CO2 gas mixture for 30-45 minutes to make it anoxic-. Dispense medium under the same gas atmosphere into anoxic Hungatetype tubes or serum vials and autoclave. Add the neutralized cinnamic acid, sulfide, and cysteine from sterile anoxic stock solutions prepared under 100% N2 gas and carbonate from a sterile anoxic stock solution prepared under 80% N2 and 20% CO2 gas atmosphere. Adjust pH of complete medium to 7.2 - 7.5, if necessary. Trace element solution SL-10 (from medium 320) element solution SL-10 HCl (25%) (from medium 320) 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.
