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Main sol. 120a

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Internal Solution-ID: S215

Equipment needed: Autoclave Hungate tubes Filter Gassing station
Compound Amount Unit Conc. [g/L] Conc. [mM]
K2HPO4 0.35 g 0.342 1.962
KH2PO4 0.23 g 0.225 1.651
NH4Cl 0.50 g 0.488 9.128
MgSO4 x 7 H2O 0.50 g 0.488 1.981
CaCl2 x 2 H2O 0.25 g 0.244 1.661
NaCl 2.25 g 2.197 37.599
FeSO4 x 7 H2O solution (0.1% w/v) 2.00 ml - -
Trace element solution SL-10 1.00 ml - -
Yeast extract
(OXOID) 		2.00 g 1.953 -
Casitone
(BD BBL) 		2.00 g 1.953 -
Sodium resazurin
(0.1% w/v) 		0.50 ml 4.9e-4 0.002
NaHCO3 0.85 g 0.83 9.881
Methanol
(50% v/v) 		20.00 ml 15.469 482.765
Wolin's vitamin solution (10x) 1.00 ml - -
L-Cysteine HCl x H2O 0.30 g 0.293 1.668
Na2S x 9 H2O 0.30 g 0.293 1.22
Distilled water 1000.00 ml - -
1 Dissolve ingredients (except bicarbonate, vitamins, methanol, cysteine and sulfide) and sparge medium with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Then add and dissolve bicarbonate, adjust pH to 6.5 and dispense medium under 80% N2 and 20% CO2 gas atmosphere into anoxic Hungate-type tubes or serum vials to 30% of their volume and autoclave. Methanol (50% v/v stock solution) and the reducing agents are each autoclaved separately under 100% N2 gas atmosphere as concentrated solutions in tightly closed tubes. Vitamins are prepared under 100% N2 gas atmosphere and sterilized by filtration. Appropriate volumes of the solutions are injected into the sterile medium with hypodermic syringes. Adjust pH of the complete medium to 6.5 - 6.8, if necessary.
120a METHANOSARCINA BARKERI MEDIUM