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Sparge solution A with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic, distribute under same gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. Solution B is autoclaved separately under 80% N2 and 20% CO2 gas atmosphere. Solution C and D are prepared under 100% N2 gas and sterilized by filtration. Solution E is autoclaved under 100% N2 gas. To complete the medium appropriate amounts of solutions B to E are added to the sterile solution A in the sequence as indicated. Adjust pH of complete medium to 7.2 - 7.4, if necessary.
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