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Dissolve the ingredients (except carbonates, sulfide, formate, and vitamins) and sparge medium with 100% N2 gas for 30 - 45 min. to make it anoxic. Add and dissolve carbonates and sulfide, then adjust pH to 9.0 - 9.5, dispense under 100% N2 gas atmosphere into Hungate-type tubes or serum vials and autoclave. Before use add formate and vitamins (sterilized by filtration) from sterile anoxic stock solutions prepared under 100% N2 gas.
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