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Dissolve ingredients (except calcium chloride, bicarbonate, glucose, vitamins and sulfide), then sparge medium with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Dispense medium under same gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. After autoclaving add calcium chloride, glucose, vitamins and sulfide from sterile anoxic stock solutions prepared under 100% N2 gas and bicarbonate from a sterile stock solution prepared under 80% N2 and 20% CO2 gas mixture. The vitamin solution should be sterilized by filtration. Adjust pH of the complete medium to 7.4, if necessary.
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