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Main sol. 141b

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Internal Solution-ID: S244

Equipment needed: Filter Autoclave Hungate tubes Gassing station
Compound Amount Unit Conc. [g/L] Conc. [mM]
KCl 0.34 g 0.336 4.502
MgCl2 x 6 H2O 4.00 g 3.949 19.423
MgSO4 x 7 H2O 3.45 g 3.406 13.818
NH4Cl 0.25 g 0.247 4.614
CaCl2 x 2 H2O 0.14 g 0.138 0.94
K2HPO4 0.14 g 0.138 0.793
NaCl 6.00 g 5.923 101.352
Modified Wolin's mineral solution 10.00 ml - -
Fe(NH4)2(SO4)2 x 6 H2O
(0.1% w/v) 		2.00 ml 0.002 -
Na-acetate 1.00 g 0.987 12.034
Yeast extract
(OXOID) 		2.00 g 1.974 -
Trypticase peptone
(BD BBL) 		2.00 g 1.974 -
Sodium resazurin
(0.1% w/v) 		0.50 ml 4.9e-4 0.002
Wolin's vitamin solution (10x) 1.00 ml - -
NaHCO3 5.00 g 4.936 58.755
L-Cysteine HCl x H2O 0.50 g 0.494 2.81
Na2S x 9 H2O 0.50 g 0.494 2.055
Distilled water 1000.00 ml - -
1 Dissolve ingredients (except bicarbonate, vitamins, cysteine and sulfide), sparge medium with 80% H2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Add and dissolve bicarbonate and adjust pH to 7.0, then dispense medium under 80% H2 and 20% CO2 gas atmosphere into anoxic Hungate-type tubes or serum vials to 30% of their volume and autoclave. After sterilization add cysteine and sulfide from sterile anoxic stock solutions autoclaved under 100% N2 gas atmosphere. Vitamins are prepared under 100% N2 gas atmosphere and sterilized by filtration. Adjust pH of final medium to 6.8 - 7.0.
2 For incubation use sterile 80% H2 and 20% CO2 gas mixture at two atmospheres of pressure.
3 Note: If the medium is being used without overpressure then adjust pH with a small amount of sterile anoxic 1 N HCl, if necessary.
141b METHANOCULLEUS SP. MEDIUM