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Dissolve components except NaHCO3, Vitamin solution, Thiamine solution, Vitamin B12 solution, sodium lactate and Na2S x 9H2O in 900 ml distilled water and adjust pH to 7.0. Bring to a boil and cool down under a N2-CO2 (4:1, v/v) gas stream. Distribute the medium into culture vessels (e.g., 9 ml of the medium in Hungate tubes) under a N2-CO2 (4:1, v/v) gas mixture, seal with butyl rubber stoppers and autoclave. Separately autoclave 0.2 M sodium lactate under a N2 atmosphere. After cooling, add 1/9 volume of the sodium lactate solution, and filter-sterilized 8% NaHCO3 solution, Vitamin solution, Thiamine solution and Vitamin B12 solution to the medium. Readjust pH to 7.0, if necessary. Prior to use, reduce the medium with Na2S x 9H2O (3% solution, autoclaved and stocked under N2).
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