1
Solution A is sparged with 80% N2 and 20% CO2 gas mixture to reach a pH below 6 (at least 30 min), then distributed under the same gas atmosphere in anoxic Hungate-type tubes or serum vials and autoclaved. Solutions C and E are autoclaved separately under 100% N2 gas. Solution B is autoclaved under 80% N2 and 20% CO2 gas atmosphere. Solution D is prepared under 100% N2 gas atmosphere and sterilized by filtration. To complete the medium appropriate amounts of solutions B to E are added to the sterile solution A in the sequence as indicated. Final pH of the medium should be 7.1 - 7.4.
2
Note: Addition of 10 - 20 mg sodium dithionite per liter (e.g. from 5% (w/v) solution, freshly prepared under N2 and filter-sterilized) may stimulate growth at the beginning. For transfers use 5 - 10% (v/v) inoculum.
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