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Main sol. 291

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Internal Solution-ID: S5597

Equipment needed: Water bath Serum bottle Filter Gassing station
Compound Amount Unit Conc. [g/L] Conc. [mM]
(NH4)2SO4 1.30 g 1.3 9.838
KH2PO4 0.28 g 0.28 2.058
MgSO4 x 7 H2O 0.25 g 0.25 1.014
CaCl2 x 2 H2O 0.07 g 0.07 0.476
FeCl3 x 6 H2O 0.02 g 0.02 0.074
Allen's trace element solution 10.00 ml - -
Distilled water 1000.00 ml - -
1 Prepare medium, then add per liter:
Sulfur
(powdered) 		8.00 g 8 249.454
Resazurin 0.40 mg 4.0e-4 0.002
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.
291 PYROBACULUM NEUTROPHILUM MEDIUM