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Dissolve ingredients (except bicarbonate and pyruvate) and sparge medium with 100% N2 gas for 30 - 45 min to make it anoxic. Then, add and dissolve bicarbonate, adjust pH to 8.5 - 9.0, dispense under 100% N2 gas atmosphere into anoxic Hungate-type tubes or serum vials, and autoclave. After sterilization, add pyruvate from a sterile, anoxic stock solution that has been prepared under a 100% N2 gas atmosphere and sterilized by filtration. Adjust pH of complete medium to 8.5 - 9.0, if necessary.
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