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Main sol. 924b

Internal Solution-ID: S1901

Equipment needed: Autoclave Hungate tubes Filter Gassing station
Compound Amount Unit Conc. [g/L] Conc. [mM]
NaCl 1.00 g 1 17.112
MgCl2 x 6 H2O 1.00 g 1 4.919
KCl 0.50 g 0.5 6.707
CaCl2 x 2 H2O 0.40 g 0.4 2.721
K2HPO4 0.40 g 0.4 2.297
NH4Cl 1.00 g 1 18.695
Modified Wolin's mineral solution 10.00 ml - -
Yeast extract
(OXOID)
2.00 g 2 -
Trypticase peptone
(BD BBL)
2.00 g 2 -
Sodium resazurin
(0.1% w/v)
0.50 ml 5.0e-4 -
NaHCO3 4.00 g 4 47.615
Na-formate 6.80 g 6.8 99.989
Na-acetate 1.60 g 1.6 19.504
Wolin's vitamin solution 10.00 ml - -
L-Cysteine HCl x H2O 0.25 g 0.25 1.423
Na2S x 9 H2O 0.25 g 0.25 1.041
Distilled water 1000.00 ml - -
1 Dissolve ingredients (except bicarbonate, formate, acetate vitamins, and reducing agents), then sparge medium with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Add solid bicarbonate, adjust pH to 6.8 - 7.0, dispense medium under 80% N2 and 20% CO2 gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. Add formate, acetate, vitamins (sterilized by filtration), cysteine and sulfide from sterile anoxic stock solutions prepared under 100% N2 gas. The pH of the complete medium should be 7.2.
924b METHANOSARCINA MB MEDIUM (1 g/l NaCl)