1
Sparge solution A with 80% H2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic, then dispense under same gas atmosphere into anoxic serum vials (e.g., 20 ml medium in 100 ml bottles) and autoclave. Solutions B and C are autoclaved separately under 80% N2 and 20% CO2 gas mixture. Solution D is prepared under 100% N2 gas atmosphere and sterilized by filtration. Solution E is autoclaved under 100% N2 gas atmosphere.
To complete the medium appropriate amounts of solutions B to E are added to solution A. Adjust pH of the complete medium to 6.5, if necessary.
2
After inoculation pressurize vials to 1 bar overpressure with sterile 80% H2 and 20% CO2 gas mixture.
3
Note: Solution A has to be prepared freshly. It cannot be stored for a long period of time.
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