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Main sol. 1110

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Internal Solution-ID: S2231

Equipment needed: Filter Hungate tubes Gassing station Water bath
Compound Amount Unit Conc. [g/L] Conc. [mM]
NaCl 20.00 g 19.763 338.173
MgCl2 x 6 H2O 3.00 g 2.964 14.581
CaCl2 x 2 H2O 0.15 g 0.148 1.008
KCl 0.50 g 0.494 6.627
NH4Cl 0.25 g 0.247 4.618
KH2PO4 0.20 g 0.198 1.452
Trace element solution SL-10 1.00 ml - -
Selenite-tungstate solution 1.00 ml - -
Sodium resazurin
(0.1% w/v) 		0.50 ml 4.9e-4 0.002
Sulfur
(powdered) 		5.00 g 4.941 154.06
Na2CO3 1.50 g 1.482 13.985
Wolin's vitamin solution (10x) 1.00 ml - -
Na2S x 9 H2O 0.50 g 0.494 2.057
Distilled water 1000.00 ml - -
1 Dissolve ingredients (except sulfur, carbonate, vitamins and sulfide), then sparge medium with 80% H2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Dispense medium under same gas atmosphere into anoxic Hungate-type tubes or serum vials (to 20% of volume) that contain already the appropriate amount of sulfur. For sterilization heat cultivation vessels in a water bath to 90 - 100°C for 1 - 2 hours on each of three successive days. Add vitamins and sulfide from sterile anoxic stock solutions prepared under 100% N2 gas and carbonate from a sterile anoxic stock solution prepared under 80% N2 and 20% CO2 gas mixture. The vitamin solution should be sterilized by filtration. Adjust the pH of the complete medium to 7.0.
2 After inoculation, pressurize culture vials to 1 bar overpressure with sterile 80% H2 and 20% CO2 gas mixture.
1110 NAUTILIA PROFUNDICOLA MEDIUM