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Medium No.
MMJYP2 Medium
  Yeast extract 4 g
  Bacto Tryptone (Difco) 4 g
  Sodium pyruvate 0.2 g
  NaHCO3 1 g
  Na2S 0.5 g
  MJ synthetic seawater for MMJYP2 medium * 1 L
  pH 6.5  
    Mix ingredients except for pyruvate, sulfide and bicarbonate. Dispense the medium into suitable culture vessels under a stream of N2/CO2 (80/20), seal with butyl rubber stoppers, and autoclave at 121°C for 15 min. After cooling to room temperature, aseptically and anaerobically add the filter-sterilized bicarbonate and pyruvate to the medium, and the pH is adjusted to 6.5 by adding of sterile pH-adjusting agent with a syringe without opening the vessel. Prior to inoculation, aseptically add the stelile neutralized sulfide solution, and pressurize the vessels to 350 kPa with N2/CO2.
    *MJ synthetic seawater for MMJYP2 medium
      NaCl 25 g
      K2HPO4 0.14 g
      CaCl2·2H2O 0.7 g
      NH4Cl 0.25 g
      MgSO4·7H2O 3.4 g
      MgCl2·6H2O 4.2 g
      KCl 0.5 g
      Balch's trace mineral solution** 10 ml
      Distilled water 990 ml
    **Balch's trace mineral solution
      Nitrilotriacetic acid (NTA) 1.5 g
      MgSO4·7H2O 3 g
      MnSO4·2H2O 0.5 g
      NaCl 1 g
      FeSO4·7H2O 0.1 g
      CoSO4·7H2O 0.18 g
      CaCl2·2H2O 0.1 g
      ZnSO4·7H2O 0.18 g
      CuSO4·5H2O 0.01 g
      KAl(SO4)2·12H2O 0.02 g
      H3BO3 0.01 g
      Na2MoO4·2H2O 0.01 g
      NiCl2·6H2O 0.025 g
      Na2SeO3·5H2O 0.3 mg
      Distilled water 1 L
    First dissolve nitrilotriacetic acid and adjust pH to 6.5 with KOH, then add minerals. Final pH 7.0 (with KOH).