Mix ingredients except for bicarbonate, fructose, vitamin solution, cysteine-HCl and Na₂S·9H₂O, sparge medium with N₂/CO₂ (80/20) gas and autoclave at 121°C for 15 min in Hungate-type tubes of serum vials. Concentrated fructose, cysteine-HCl and sodium sulfide solutions are sterilized separately under a N₂ atmosphere in tightly closed vessels. Bicarbonate solution are also sterilized under an N₂/CO₂ (80/20) gas atmosphere in tightly closed vesseles. Prior to inoculation, add bicarbonate, fructose, filter-sterile vitamin solution, cysteine-HCl and sodium sulfide solutions. Adjust the pH to 7.5 - 8.0 by adding a sterile anoxic stock solution of sodium carbonate (5% w/v).

For autotrophic growth, D-fructose is omitted and a gas atmosphere of 80% H2 and 20% CO₂ is used.

First dissolve nitrilotriacetic acid and adjust pH to 6.5 with KOH, then add minerals. Adjust final to pH 7.0 with KOH.