NH4Cl 1.50 g Na2HPO4 0.60 g KCl 0.10 g Na-acetate 0.82 g Modified Wolin's mineral solution 10.00 ml NaHCO3 2.50 g Na2-fumarate 8.00 g Wolin's vitamin solution (10x) 1.00 ml Distilled water 1000.00 ml Dissolve ingredients (except bicarbonate, fumarate and vitamins), sparge medium with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Add bicarbonate and equilibrate the medium with 80% N2 and 20% CO2 gas mixture to reach a pH of 6.8, then dispense medium under the same gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. After autoclaving add fumarate and vitamins from anoxic stock solutions prepared under 100% N2 gas atmosphere and sterilized by filtration. Check medium pH and adjust to 6.8, if necessary. 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. W'olins 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
