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Dissolve ingredients (except bicarbonate and vitamins), then sparge medium with 100% N2 gas for 30 - 45 min to make it anoxic. Adjust pH to 7.0 and dispense medium under same gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. Add vitamins from an anoxic stock solution prepared under 100% N2 gas and sterilized by filtration and bicarbonate from a sterile anoxic stock solution prepared under 80% N2 and 20% CO2 gas atmosphere. The pH of the complete medium should be 7.0.
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