Basal Medium Salt solution (See Below) 20 ml Trace element solution (See Below) 1 ml Se/Wo solution (See Below) 1 ml Resazurin solution (See Below) 0.25 ml Sodium acetate 0.41 g DI Water 956.5 ml Boil and cool down to room temperature under N2, then add the following components: Na2S 0.2 mmol L-cysteine 0.2 mmol Autoclave medium at 121oC and after the medium has cooled to room temperature add the following stock solutions: NaHCO3 (See Below) 1 ml MOPS (See Below) 20 ml Ti(III)NTA(See Below) 20 ml 3-vitamin solution(See Below) 0.5 ml 6-vitamin solution (See Below) 1 ml Vitamin B12 solution (See Below) 1 ml Dispense anaerobically under 100% nitrogen into appropriate vessel. Salt solution NaCl 50.0 g MgCl2 . 6H2O 20.5 g NH4Cl 13.5 g KCl 26.0 g CaCl2 . 2H2O 0.75 g KH2PO4 10.0 g DI Water 1000 ml Store at room temperature. Trace element solution HCl (25% w/w) 10 ml FeCl2 . 4H2O 1.5 g H3BO3 6.0 mg CoCl2 . 6H2O 190.0 mg MnCl2 . 4H2O 100.0 mg ZnCl2 70.0 mg Na2MoO4 . 2H2O 36.0 mg NiCl2 . 6H2O 24.0 mg CuCl2 . 2H2O 2.0 mg DI Water 1000 ml Store at room temperature in the dark. Se/Wo solution NaOH 0.5 g Na2SeO3 . 5H2O 6.0 mg Na2WO4 . 2H2O 8.0 mg DI Water 1000 ml Store at room temperature in the dark. Resazurin solution Resazurin 0.1 g DI Water 100 ml Stock solutions (add aseptically) NaHCO3 1M autoclaved MOPS, 1M pH adjusted to 7.45, filter sterilized Ti(III)NTA, 25mM, filter sterilized 3-vitamin solution Folic acid 1.0 mg Riboflavin 2.5 mg DL-6,8-thioctic 2.5 mg DI Water 250.0 ml Filter sterilize: store at 4oC in the dark. 6-vitamin solution Biotin 1.0 mg 4-aminobenzoic acid 5.0 mg Pantothenate1.0 mg Pyridoxamine 25.0 mg Nicotinic acid 10.0 mg Thiamine 2.0 mg DI Water 100.0 ml Filter sterilize: store at 4oC in the dark. Vitamin B12 solution Vitamin B12 2.5 mg DI Water 100.0 ml Filter sterilize: store at 4oC in the dark.
