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Mix ingredients of Solution A, adjust pH to 7.2, bring to a boil for a few minutes and cool down to room temperature under a stream of N2-CO2 (4:1, v/v) gas mixture. Distribute the solution into culture vessels under the same gas mixture, seal with butyl rubber stoppers and autoclave. Filter-sterilize Solution B and equilibrate by gassing with the same gas mixture for at least 15 min. Autoclave Solutions C and D under a N2 atmosphere. Add appropriate amounts of Solutions B, C and D to Solution A to complete the medium.
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