KH2PO4 0.41 g Na2HPO4 0.43 g MgCl2 x 6 H2O 0.10 g CaCl2 x 2 H2O 0.11 g NaCl 0.30 g NH4Cl 0.30 g Trace element solution 1.00 ml Selenite-tungstate solution 1.00 ml Yeast extract 0.20 g Sodium resazurin (0.1% w/v) 0.50 ml Na2CO3 1.50 g D-Glucose 1.80 g Wolin's vitamin solution 2.00 ml Na2S x 9 H2O 0.30 g L-Cysteine HCl x H2O 0.50 g Distilled water 1000.00 ml Dissolve ingredients (except carbonate, glucose, vitamins and reducing agents) and sparge medium with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Dispense medium under same gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. Add glucose, vitamins, sulfide and cysteine from sterile anoxic stock solutions prepared under 100% N2 gas and carbonate from a sterile stock solution prepared under 80% N2 and 20% CO2 gas mixture. Adjust pH of the complete medium to 7.0-7.2, if necessary. Trace element solution (from medium 732) Na2-EDTA 0.50 g FeCl2 x 4 H2O 2.00 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 AlCl3 10.00 mg NiCl2 x 6 H2O 24.00 mg Na2MoO4 x 2 H2O 36.00 mg Distilled water 1000.00 ml First dissolve EDTA in distilled water, adjust pH to 7 using 2 N NaOH and add ferrous chloride. After ferrous chloride has dissolved add remaining compounds. Selenite-tungstate solution (from medium 385) NaOH 0.50 g Na2SeO3 x 5 H2O 3.00 mg Na2WO4 x 2 H2O 4.00 mg Distilled water 1000.00 ml Wolin's vitamin solution (from medium 141) Biotin 2.00 mg Folic acid 2.00 mg Pyridoxine hydrochloride 10.00 mg Thiamine HCl 5.00 mg Riboflavin 5.00 mg Nicotinic acid 5.00 mg Calcium D-(+)-pantothenate 5.00 mg Vitamin B12 0.10 mg p-Aminobenzoic acid 5.00 mg (DL)-alpha-Lipoic acid 5.00 mg Distilled water 1000.00 ml
