# J431: BN3 MEDIUM ## Main sol. J431 1000 ml **Basal medium** 1 ml **Trace element solution** 1 ml **Selenite-tungstate solution** 10 ml **Trace vitamins** 5 �g Vitamin B12 3.6 g Glucose 0.5 g Yeast extract (BD-Difco) 1.5 g Tryptone (BD-Difco) 20 g Sulfur (powder) 3.4 g NaHCO3 0.5 mg Resazurin 0.36 g Na2S x 9 H2O 1. Autoclave Basal medium supplemented with Trace element solution, Selenite-tungstate solution and resazurin under N2-CO2 (4:1, v/v). Separately autoclave glucose, yeast extract and tryptone (10% each) and add to the medium. Filter-sterilize Trace vitamins and NaHCO3 (8%) solution and add to the medium. Steam sulfur for 3 hr on each of 3 successive days. Distribute the medium into culture vessels containing sulfur under a N2-CO2 (4:1, v/v) gas mixture and seal with butyl rubber stoppers. Before inoculation, reduce the medium with 5% Na2S x 9H2O solution (autoclaved under a N2 atmosphere). Readjust pH to 7.2, if necessary. ## Basal medium 1 g NaCl 0.4 g MgCl2 x 6 H2O 0.075 g CaCl2 x 2 H2O 0.25 g NH4Cl 0.2 g KH2PO4 0.5 g KCl 1000 ml Distilled water ## Trace element solution 987 ml Distilled water 12.5 ml HCl (25%) 2.1 g FeSO4 x 7 H2O 30 mg H3BO3 100 mg MnCl2 x 4 H2O 190 mg CoCl2 x 6 H2O 24 mg NiCl2 x 6 H2O 2 mg CuCl2 x 2 H2O 144 mg ZnSO4 x 7 H2O 36 mg Na2MoO4 x 2 H2O ## Selenite-tungstate solution 0.4 g NaOH 6 mg Na2SeO3 x 5 H2O 8 mg Na2WO4 x 2 H2O 1000 ml Distilled water ## Trace vitamins 2 mg Biotin 2 mg Folic acid 10 mg Pyridoxine hydrochloride 5 mg Thiamine HCl 5 mg Riboflavin 5 mg Nicotinic acid 5 mg Calcium pantothenate 0.1 mg Vitamin B12 5 mg p-Aminobenzoic acid 5 mg Lipoic acid 1000 ml Distilled water