# 1092: THERMOSULFIDIBACTER MEDIUM ## Main sol. 1092 30 g Sea Salt (SIGMA) 1 ml **Wolfe's mineral elixir** 0.5 ml Sodium resazurin (0.1% w/v) 3 g Sulfur (powdered) 2.5 g NaHCO3 2 g Yeast extract 2 ml **Wolin's vitamin solution (10x)** 0.5 g Na2S x 9 H2O 1000 ml Distilled water 1. Dissolve ingredients (except sulfur, bicarbonate, yeast extract, vitamins and sulfide) and sparge medium with 80% H2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Dispense medium under same gas atmosphere into anoxic serum vials (e.g., Balch-type tubes) containing already the appropriate amount of sulfur, only to 30% of their volume to allow for a large headspace. Autoclave at 110°C for 20 min. Add yeast extract, vitamins and sulfide form sterile anoxic stock solutions prepared under 100% N2 gas and bicarbonate from a sterile anoxic stock solution prepared under 80% N2 and 20% CO2 gas mixture. The vitamin solution should be sterilized by filtration. Adjust pH of the complete medium to 7.0-7.2. 2. After inoculation pressurize vessels with sterile 80% H2 and 20% CO2 gas mixture to 2 bar overpressure. ## Wolfe's mineral elixir 30 g MgSO4 x 7 H2O 5 g MnSO4 x H2O 10 g NaCl 1 g FeSO4 x 7 H2O 1.8 g CoCl2 x 6 H2O 1 g CaCl2 x 2 H2O 1.8 g ZnSO4 x 7 H2O 0.1 g CuSO4 x 5 H2O 0.18 g AlK(SO4)2 x 12 H2O 0.1 g H3BO3 0.1 g Na2MoO4 x 2 H2O 2.8 g (NH4)2Ni(SO4)2 x 6 H2O 0.1 g Na2WO4 x 2 H2O 0.1 g Na2SeO4 1000 ml Distilled water 1. First adjust pH to 1.0 with diluted H2SO4, then add and dissolve the salts. ## Wolin's vitamin solution (10x) 20 mg Biotin 20 mg Folic acid 100 mg Pyridoxine hydrochloride 50 mg Thiamine HCl 50 mg Riboflavin 50 mg Nicotinic acid 50 mg Calcium D-(+)-pantothenate 1 mg Vitamin B12 50 mg p-Aminobenzoic acid 50 mg (DL)-alpha-Lipoic acid 1000 ml Distilled water