NH4Cl 0.50 g MgSO4 x 7 H2O 0.50 g CaCl2 x 2 H2O 0.25 g NaCl 2.25 g FeSO4 x 7 H2O (0.1% w/v in 0.1 N H2SO4) 2.00 ml Trace element solution SL-10 1.00 ml Selenite-tungstate solution 1.00 ml Yeast extract 2.00 g Casitone 2.00 g Betaine x H2O 6.70 g Sodium resazurin (0.1% w/v) 0.50 ml K2HPO4 0.35 g KH2PO4 0.23 g Na2CO3 1.00 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 phosphates, carbonate, vitamins, cysteine and sulfide) 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 phosphates, vitamins (sterilized by filtration), cysteine and sulfide to the medium after autoclaving from sterile stock solutions prepared under 100% N2 gas and carbonate from a sterile anoxic stock solution prepared under 80% N2 and 20% CO2 gas mixture. Adjust pH of complete medium to pH 7.0, if necessary. Trace element solution SL-10 (from medium 320) HCl (25%) 10.00 ml FeCl2 x 4 H2O 1.50 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 NiCl2 x 6 H2O 24.00 mg Na2MoO4 x 2 H2O 36.00 mg Distilled water 990.00 ml First dissolve FeCl2 in the HCl, then dilute in water, add and dissolve the other salts. Finally make up to 1000.00 ml. 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 (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
