Solution A: Mineral solution (see below) 50.00 ml Trace element solution SL-10 (see medium 320) 1.00 ml Yeast extract 2.00 g Crotonic acid 5.00 g Vitamin solution (see medium 503) 1.00 ml Resazurin 1.00 mg Distilled water 840.00 ml Adjust pH to 6.9 before autoclaving. Solution B: NaHCO3 5.00 g Distilled water 100.00 ml Solution C: L-Cysteine-HCl x H2O 0.24 g Distilled water 10.00 ml Solution D: Na2S x 9 H2O 78.00 mg Distilled water 1.00 ml Prepare solution A under 80% N2 and 20% CO2 gas mixture, distribute 8.9 ml per Hungate-type tube under same gas atmosphere and autoclave. Filter sterilize NaHCO3 solution and gas for 20 min with 80% N2 and 20% CO2 gas mixture. Autoclave cysteine and sulfide under 100% N2 gas. Complete medium by adding the solutions B, C, and D with a syringe to solution A. Adjust final pH of medium to 7.0 - 7.5. Mineral solution: KH2PO4 10.0 g MgCl2 x 6 H2O 6.6 g NaCl 8.0 g NH4Cl 8.0 g CaCl2 x 2 H2O 1.0 g Distilled water 1000.0 ml
