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Dissolve ingredients of solution A, adjust pH to 7.0, then sparge medium with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Dispense under the same gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. Solution B is filter-sterilized and then equilibrated with 80% N2 and 20% CO2 gas mixture for at least 15 min. Solution C is prepared under 100% N2 gas and sterilized by filtration. Solutions D, E, F and G are autoclaved under 100% N2 gas. To complete the medium, appropriate amounts of the solutions B to G are added to solution A in the sequence indicated. Adjust pH of complete medium to 7.2, if necessary.
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