# J236: THERMOCLADIUM MEDIUM ## Main sol. J236 1000 ml **Modified Brock's salt base solution** 2.94 g Trisodium citrate x 2 H2O 0.5 g Yeast extract (BD-Difco) 10 ml **Trace vitamins** 1 mg Resazurin 0.5 g Na2S x 9 H2O 10 g Sulfur (powder) 1. Combine Modified Brock's salt base solution, trisodium citrate and resazurin, and adjust pH to 3.5 with 10 N H2SO4. Autoclave under a N2 atmosphere. Separately autoclave yeast extract and Na2S x 9H2O as 10% and 5% solutions, respectively, under a N2 atmosphere. Filter-sterilize Trace vitamins. Steam sulfur for 3 hr on each of 3 successive days. Aseptically mix components except Na2S x 9H2O in suitable culture vessels (e.g., 20 ml of the medium in 120 ml serum bottles) while gassing with H2-CO2 (80:20) gas mixture and seal with butyl rubber stoppers. Prior to inoculation, aseptically and anaerobically add the Na2S x 9H2O solutions and adjust pH to 4.0-4.5, if necessary. When the indicator (resazurin) turns to colorless, inoculate the culture and pressurize the vessels to 100 kPa H2-CO2 (80:20). ## Modified Brock's salt base solution 1.3 g (NH4)2SO4 0.28 g KH2PO4 0.25 g MgSO4 x 7 H2O 0.07 g CaCl2 x 2 H2O 2 mg FeCl3 x 6 H2O 1.8 mg MnCl2 x 4 H2O 4.5 mg Na2B4O7 x 10 H2O 0.22 mg ZnSO4 x 7 H2O 0.05 mg CuCl2 x 2 H2O 0.03 mg Na2MoO4 x 2 H2O 0.03 mg VOSO4 x n H2O 0.01 mg CoSO4 x 7 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