Mix ingregients except for vitamin and cysteine-HCl, dispense the medium into suitable culture vessels, and autoclave under a N₂/CO₂ atmosphere (80/20). Separately autoclave concentraed cysteine-HCl solution under a N₂ atmosphere in tightly colsed vessels. Prior to inoculation, add the filter-sterile vitamin and autoclaved cysteine-HCl solutions.

*Vitamin solution

Adjust pH to 7.0 with NaOH.

**Trace element solution

First dissolve NTA and adjust pH to 6.5 with NaOH, and then add minerals. Final pH is 7.0.