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Main sol. 1597

Internal Solution-ID: S3296

Equipment needed: Gassing station Autoclave Hungate tubes Filter
Compound Amount Unit Conc. [g/L] Conc. [mM]
Solution A 750 ml - -
Solution B 250 ml - -
Solution C 1 ml - -
Solution D 1 ml - -
Solution E 5 ml - -
Solution F 3 ml - -
Solution G 5 ml - -
Solution H 10 ml - -
Solution I 1 ml - -
Solution J 10 ml - -
1 Sterilize solutions A and B in closed thick-walled screw-top bottles (e.g., SCHOTT) for 20 min at 120°C. The pH of solution A after sterilization should be 10. There is some precipitate forming that settles at the bottom after 3 - 4 days. It is best to remove precipitates by decantation before using solution A for medium preparation.
2 Combine solution A with solution B and sparge medium with 100% N2 gas for at least 30 - 45 min to make it anoxic, then dispense under same gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. Solutions C to J are sterilized separately under 100% N2 gas. Vitamins and coenzyme M should be sterilized by filtration. To complete the medium appropriate amounts of solutions C to J are added to the combined sterile solutions A and B in the sequence as indicated. Final pH of the medium should be 9.5.
3 Note: Addition of 10 - 20 mg sodium dithionite per liter (e.g. from 5% (w/v) solution, freshly prepared under N2 and filter-sterilized) may stimulate growth at the beginning. For transfers use 5 - 10% inoculum.