Final pH: 5.9 Final volume: 1011 ml NH4Cl 1.00 g KCl 0.10 g MgSO4 x 7 H2O 0.20 g NaCl 0.80 g KH2PO4 0.10 g CaCl2 x 2 H2O 0.02 g Modified Wolin's mineral solution 10.00 ml Sodium resazurin (0.1% w/v) 0.50 ml Yeast extract 1.00 g NaHCO3 1.00 g D-Fructose 5.00 g Wolin's vitamin solution (10x) 1.00 ml L-Cysteine HCl x H2O 0.30 g Na2S x 9 H2O 0.30 g Distilled water 1000.00 ml Dissolve ingredients (except bicarbonate, yeast extract, fructose, vitamins, and reducing agents), sparge medium with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic and adjust pH to 5.0. Dispense medium under the same gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave at 121°C for 15 min. Add yeast extract, fructose, vitamins (sterilized by filtration), cysteine, and sulfide from sterile stock solutions prepared under a 100% N2 gas atmosphere and bicarbonate from a sterile stock solution prepared under an 80% N2 and 20% CO2 gas atmosphere. Adjust the pH of the complete medium to 5.9! For DSM 13641: Replace fructose with 5.00 g/l sucrose added to the autoclaved medium from a sterile anoxic stock solution and increase amount of NaHCO3 to 2.50 g/l. Adjust pH of complete medium to 6.9! For DSM 115981: Omit Yeast extract. 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
