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Main sol. 716

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Internal Solution-ID: S1467

Equipment needed: Autoclave Gassing station Filter Serum bottle
Compound Amount Unit Conc. [g/L] Conc. [mM]
K2HPO4 x 3 H2O 0.30 g 0.299 1.717
KH2PO4 0.22 g 0.219 1.612
(NH4)2SO4 0.22 g 0.219 1.66
NaCl 1.00 g 0.997 17.06
MgSO4 x 7 H2O 0.09 g 0.09 0.364
CaCl2 x 2 H2O 0.06 g 0.06 0.407
FeSO4 x 7 H2O
(0.1% w/v in 0.1 N H2SO4) 		2.00 ml 0.002 0.007
NiCl2 x 6 H2O
(0.1% w/v) 		0.20 ml 2.0e-4 0.002
Modified Wolin's mineral solution 1.00 ml - -
Sodium resazurin
(0.1% w/v) 		0.50 ml 5.0e-4 0.002
KNO3 1.00 g 0.997 9.861
Na2CO3 1.50 g 1.496 14.11
DL-Dithiothreitol 0.30 g 0.299 1.938
Distilled water 1000.00 ml - -
1 Dissolve ingredients (except nitrate, carbonate, and dithiothreitol), then sparge medium with 80% H2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Dispense medium under the same gas atmosphere into anoxic serum vials (e.g., 10 ml medium in 25 ml Balch-type tubes) and autoclave. Before use, add nitrate from a sterile anoxic stock solution prepared under 100% N2 gas and carbonate from a sterile anoxic stock solution prepared under 80% N2 and 20% CO2 gas mixture. Before inoculation add dithiothreitol from an anoxic stock solution sterilized by filtration under 100% N2 gas and adjust the pH of the medium to 7.2.
2 After growth has started pressurize vials to 0.5 bar overpressure with a sterile 80% H2 and 20% CO2 gas mixture.
716 AMMONIFEX DEGENSII MEDIUM