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Main sol. 1743

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Internal Solution-ID: S3607

Equipment needed: Autoclave Filter Gassing station Serum bottle
Compound Amount Unit Conc. [g/L] Conc. [mM]
NaCl 27.50 g 27.201 465.448
MgCl2 x 6 H2O 5.38 g 5.321 26.175
MgSO4 x 7 H2O 6.78 g 6.706 27.209
KCl 0.72 g 0.712 9.553
NH4Cl 0.50 g 0.495 9.246
CaCl2 x 2 H2O 1.40 g 1.385 9.419
KH2PO4 0.05 g 0.049 0.363
Trace element solution 10.00 ml - -
NaHCO3 1.70 g 1.682 20.016
Wolin's vitamin solution (10x) 1.00 ml - -
Distilled water 1000.00 ml - -
1 Dissolve ingredients (except vitamins), then sparge medium with 80% N2 and 20% CO2 gas mixture to make it anoxic and until a pH of 6.5 is reached. Dispense medium under same gas atmosphere into anoxic serum vials to 20% of their volume and autoclave. After autoclaving add vitamins from a sterile stock solution prepared under 100% N2 gas atmosphere and sterilized by filtration. Prior to inoculation add 5% (v/v) sterile H2 and 5% (v/v) sterile air to the headspace by using syringes. Check pH of the complete medium and adjust pH to 6.5, if necessary. Incubate without shaking.
2 Note: After growth has started cell density of the culture can be increased by replenishing H2 and air every two days.
1743 GHIORSEA MEDIUM