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Dissolve the ingredients (except carbonate, bicarbonate, vitamins, formate and sulfide,) and sparge medium with 100% N2 gas for 30 - 45 min to make it anoxic. Add and dissolve carbonate and bicarbonate, adjust pH to 8.8 - 9.0, then dispense under 100% N2 gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. Before use add vitamins (sterilized by filtration), formate and sulfide from sterile anoxic stock solutions prepared under 100% N2 gas.
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