# J1211: METHANOCALDOCOCCUS LEIGHII MEDIUM ## Main sol. J1211 22 g NaCl 2.7 g MgCl2 x 6 H2O 3.4 g MgSO4 x 7 H2O 0.5 g NH4Cl 0.33 g KCl 0.14 g K2HPO4 0.14 g CaCl2 x 2 H2O 9.5 mg FeSO4 x 7 H2O 10 ml Trace mineral solution (see below) 10 ml **Trace vitamins** 1.4 g Sodium acetate x 3 H2O 15.12 g PIPES 1 g NaHCO3 1 mg Resazurin 1000 ml Distilled water 1. Mix components, except NaHCO3, and adjust pH to 6.0. Bring to a boil for 5-10 sec, cool down under a stream of H2-CO2 (80:20, v/v) and add NaHCO3 to the medium. Dispense the medium into suitable culture vessels (e.g., 5 ml in Balch type tubes) under the same gas mixture, seal with butyl rubber stoppers and autoclave. After cooling, add per liter the following solution (autoclaved and stored under N2): 10 ml Na2S x 9 H2O (5%) 2. Trace mineral solution: 2.1 g Trisodium citrate x 2 H2O 3 g MgSO4 x 7 H2O 0.5 g MnSO4 x n H2O 0.1 g CoSO4 x 7 H2O 0.1 g ZnSO4 x 7 H2O 0.1 g Na2MoO4 x 2 H2O 0.025 g NiCl2 x 6 H2O 0.02 g Na2SeO3 0.01 g CuSO4 x 5 H2O 0.01 g AlK(SO4)2 0.01 g H3BO3 0.01 g VOSO4 x n H2O 3.3 mg Na2WO4 x 2 H2O 1000 ml Distilled water 3. Pressurize the inoculated vessels to 200 kPa H2-CO2 (80:20, v/v). 4. Comment: For growth by nitrogen-fixation, remove NH4Cl from the medium and cultivate under a H2-N2-CO2 (60:20:20, v/v/v, 200 kPa) gas mixture. 5. Dissolve trisodium citrate and adjust pH to 6.5. Then proceed to add minerals. ## Trace vitamins 2 mg Biotin 2 mg Folic acid 10 mg Pyridoxine hydrochloride 5 mg Thiamine HCl 5 mg Riboflavin 5 mg Nicotinic acid 5 mg Calcium pantothenate 0.1 mg Vitamin B12 5 mg p-Aminobenzoic acid 5 mg Lipoic acid 1000 ml Distilled water