Final pH: 7.5 - 8.0 Final volume: 1021 ml NH4Cl 1.00 g KH2PO4 0.33 g K2HPO4 0.45 g MgSO4 x 7 H2O 0.10 g Modified Wolin's mineral solution 20.00 ml Yeast extract 2.00 g Sodium resazurin (0.1% w/v) 0.50 ml NaHCO3 10.00 g D-Fructose 10.00 g Wolin's vitamin solution (10x) 1.00 ml L-Cysteine HCl x H2O 0.50 g Na2S x 9 H2O 0.50 g Distilled water 1000.00 ml 1. Dissolve ingredients except bicarbonate, fructose, vitamins, cysteine, and sulfide, bring to the boil, and cool to room temperature under 80% N2 and 20% CO2 gas mixture. Add bicarbonate (solid) and equilibrate the medium with the gas until a pH of around 7.4 is reached. Then distribute under the same gas atmosphere in anoxic Hungate-type tubes or serum vials and autoclave. Before use adjust the pH to 7.5 - 8.0 by adding a sterile anoxic stock solution of sodium carbonate (5% w/v) prepared under 80% N2 and 20% CO2 gas mixture and add fructose, vitamins (sterilized by filtration), cysteine, and sulfide from anoxic sterile stock solutions prepared under 100% N2. 2. Note: For autotrophic growth D-fructose is omitted and a gas atmosphere of 80% H2 and 20% CO2 is used. For DSM 4132: Use D-fructose as substrate at a final concentration of 1.00 g/l. 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. Wolin's vitamin solution (10x) (from medium 120) Biotin 20.00 mg Folic acid 20.00 mg Pyridoxine hydrochloride 100.00 mg Thiamine HCl 50.00 mg Riboflavin 50.00 mg Nicotinic acid 50.00 mg Calcium D-(+)-pantothenate 50.00 mg Vitamin B12 1.00 mg p-Aminobenzoic acid 50.00 mg (DL)-alpha-Lipoic acid 50.00 mg Distilled water 1000.00 ml
