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Main sol. 506

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Internal Solution-ID: S1111

Equipment needed: Hungate tubes Filter Autoclave Gassing station
Compound Amount Unit Conc. [g/L] Conc. [mM]
Na-acetate x 3 H2O 2.50 g 2.473 -
(NH4)2SO4 0.45 g 0.445 3.368
K2HPO4 0.29 g 0.287 1.647
KH2PO4 0.18 g 0.178 1.308
MgSO4 x 7 H2O 0.12 g 0.119 0.482
CaCl2 x 2 H2O 0.06 g 0.059 0.404
NaCl 0.05 g 0.049 0.846
Modified Wolin's mineral solution 10.00 ml - -
Sodium resazurin
(0.1% w/v) 		0.50 ml 4.9e-4 0.002
Na2CO3 0.50 g 0.495 4.666
Wolin's vitamin solution (10x) 1.00 ml - -
L-Cysteine HCl x H2O 0.25 g 0.247 1.408
Na2S x 9 H2O 0.25 g 0.247 1.03
Distilled water 1000.00 ml - -
1 Dissolve ingredients except carbonate, vitamins, cysteine and sulfide, then sparge medium with 80% H2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Add and dissolve carbonate, then dispense medium into anoxic Hungate-type tubes or serum vials to 30% of their volume under 80% H2 and 20% CO2 gas atmosphere and autoclave. Add vitamins (sterilized by filtration), cysteine and sulfide from sterile anoxic stock solutions prepared under 100% N2 gas. Prior to use check pH of complete medium and adjust to 5.8 - 6.0, if necessary.
2 Note: After growth has started and the culture is becoming turbid add sterile 80% H2 and 20% CO2 gas mixture to 0.5 - 1 bar overpressure.
506 METHANOBACTERIUM ESPANOLAE MEDIUM