1
Solution A is sparged with 80% N2 and 20% CO2 gas mixture to reach a pH below 6 (at least 30 – 45 min), then dispensed under the same gas atmosphere into serum vials (use at least bottles of 50 ml volume filled with 30 ml medium) and autoclaved. Solutions B and F are prepared under 80% N2 and 20% CO2 gas atmosphere and sterilized. Sterile solutions C, D and E are prepared under 100% N2 gas. Solutions D and F are sterilized by filtration. To complete the medium appropriate amounts of solutions B to F are added to the sterile solution A in the sequence as indicated. Final pH of the medium should be 7.0 - 7.2.
2
Note: For transfer of cultures use at least 10% (v/v) inoculum.
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