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Main sol. 1597

Internal Solution-ID: S3296

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.

Compound

Amount

Unit

Conc. [g/L]

Conc. [mM]

Solution A

750

Solution B

250

Solution C

Solution D

Solution E

Solution F

Solution G

Solution H

Solution I

Solution J

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.

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Main sol. 1597

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Main sol. 1597

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