1
Add and dissolve ingredients of solution A, adjust pH to 7.2, then sparge medium with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Distribute medium 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 is prepared under 100% N2 gas and sterilized by filtration. Solutions D, E and F are autoclaved under 100% N2 gas. To complete the medium, appropriate amounts of solutions B to F are added to the sterile solution A. Adjust pH of complete medium to 7.2, if necessary.
2
Note: Use 25 - 50% inoculum. Growth can be followed by the clearing of stearate. Maintain cultures at 25°C.
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