1
Dissolve ingredients of solution A, adjust pH to 5.0 with 0.1 N HCl and sparge with 80% H2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Dispense solution A under same gas atmosphere into anoxic Hungate-type tubes or serum vials (e.g., 20 ml medium in 50 ml bottles) and autoclave. Solutions B - F are prepared under 100% N2 gas atmosphere and sterilized by filtration. Prior to inoculation complete the medium by adding appropriate amounts of solutions B to F to the sterile solution A in the sequence as indicated.
2
Adjust pH of the complete medium to 5.1 with a sterile anoxic solution of 1 N HCl. After inoculation pressurize vials to 1 bar overpressure with sterile 80% H2 and 20% CO2 gas mixture.
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