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Dissolve ingredients except carbonates, magnesium chloride, trace elements, lactate, vitamins and sulfide, then sparge medium with 100% N2 gas for 30 - 45 min to make it anoxic. Then add and dissolve carbonates, adjust pH to 9.0, dispense under 100% N2 gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. After sterilization add magnesium chloride, trace elements, lactate, vitamins and sulfide from sterile anoxic stock solution prepared under 100% N2 gas. The vitamin solution should be sterilized by filtration. Adjust pH of complete medium to 9.0 - 9.5, if necessary.
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