1
Dissolve ingredients (except bicarbonate, thiosulfate and vitamins), then sparge medium with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Dispense medium under same gas atmosphere into anoxic Hungate-type tubes or serum vials up to a volume of 20% and autoclave. Add bicarbonate, thiosulfate and vitamins to the autoclaved medium from sterile anoxic stock solutions. Solutions of vitamins and thiosulfate are sterilized by filtration and stored under 100% N2 gas, whereas the solution of bicarbonate is prepared under 80% N2 and 20% CO2 gas mixture and autoclaved. Adjust pH of the complete medium to 6.7.
2
After inoculation add sterile air in an amount that is equivalent to a volume of 50% of the headspace.
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