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Main sol. 274

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Internal Solution-ID: S539

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 2.75 g 2.715 13.353
MgSO4 x 7 H2O 3.40 g 3.356 13.617
NH4Cl 1.50 g 1.481 27.683
CaCl2 x 2 H2O 0.14 g 0.138 0.94
K2HPO4 0.14 g 0.138 0.793
NaCl 6.31 g 6.229 106.588
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 -
NaHCO3 5.00 g 4.936 58.755
Na-acetate x 3 H2O 4.15 g 4.097 -
Sodium resazurin
(0.1% w/v) 		0.50 ml 4.9e-4 0.002
MOPS
(3-(N-morpholino) propane-sulfonic acid) 		20.93 g 20.661 98.736
Wolin's vitamin solution (10x) 1.00 ml - -
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 MOPS, vitamins, cysteine and sulfide in a total volume of 890 ml. The MOPS buffer is dissolved separately in 90 ml of distilled water, the pH is adjusted to 7.0 with 2 M KOH, then add the buffer to the salt solution. Sparge medium with 80% H2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic, then dispense medium in anoxic Hungate-type tubes under the same gas atmosphere to 30% of their volume 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 7.0, if necessary.
2 After inoculation add sterile 80% H2 and 20% CO2 gas mixture to 1 bar overpressure.
274 METHANOLACINIA MEDIUM