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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. Solutions C and E are autoclaved under 100% N2 gas. Solution D is prepared under 100% N2 gas and sterilized by filtration. 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 7.5 - 7.8 with a sterile anoxic stock solution of Na2CO3, if necessary.
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