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Sparge solution A with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic, dispense under same gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. Solutions B, C, F, G, and H are autoclaved separately under 100% N2 gas. Solution D is autoclaved under 80% N2 and 20% CO2 gas atmosphere and autoclaved. Solution E is prepared under 100% N2 gas atmosphere and sterilized by filtration. To complete the medium appropriate amounts of solutions B to H are added to the sterile solution A in the sequence as indicated. Final pH of the medium should be checked and adjusted to 7.6 with a sterile anoxic stock solution of Na2CO3 (5% w/v), if necessary.
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