# 291: PYROBACULUM NEUTROPHILUM MEDIUM ## Main sol. 291 1.3 g (NH4)2SO4 0.28 g KH2PO4 0.25 g MgSO4 x 7 H2O 0.07 g CaCl2 x 2 H2O 0.02 g FeCl3 x 6 H2O 10 ml **Allen's trace element solution** 1000 ml Distilled water 1. Prepare medium, then add per liter: 8 g Sulfur (powdered) 0.4 mg Resazurin 2. Adjust pH to 6.5 with NaOH, bring to boil for 5 min, then cool to room temperature under H2 + CO2 (80% + 20%) gas mixture. Add 0.85 g NaHCO3 per liter and equilibrate the pH to c. 6.5 by further gassing. 3. Dispense in serum bottles (10 ml/30 ml bottle), seal and heat the bottles for one hour at 85°C on each of three successive days. 4. Before inoculation, add from anaerobic stock solutions 0.02% yeast extract (for mixotrophic growth only) and 0.01 ml Na-dithionite solution (250 mg Na-dithionite/10 ml dist. water, filter-sterilized). After inoculation, pressurize to 1 bar H2 + CO2 overpressure. ## Allen's trace element solution 180 mg MnCl2 x 4 H2O 450 mg Na2B4O7 x 10 H2O 22 mg ZnSO4 x 7 H2O 5 mg CuCl2 x 2 H2O 3 mg Na2MoO4 x 2 H2O 3 mg VOSO4 x 2 H2O 1 mg CoSO4 x 7 H2O 1000 ml Distilled water 1. Adjust pH of final solution to 2 with 1 N HCl.