1
Dissolve ingredients except thiosulfate, bicarbonate, and vitamins, then sparge medium with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to remove dissolved oxygen and to saturate the solution with CO2. Dispense solution under 80% N2 and 20% CO2 gas atmosphere in vials suitable for anaerobic cultures (e.g., Balch-type tubes) to 30% of volume. Close vials with butyl rubber septa and autoclave. Add vitamins from a sterile anoxic stock solution prepared under 100% N2 gas and bicarbonate from a sterile anoxic stock solution prepared under 80% N2 and 20% CO2 gas mixture. The vitamin solution should be sterilized by filtration. Adjust the pH of the complete medium to 6.7 - 7.0, if necessary.
2
After inoculation, add 1 bar overpressure of sterile 100% H2 gas.
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