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Main sol. 1053a

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Internal Solution-ID: S2130

Equipment needed: Filter Autoclave Hungate tubes Gassing station
Compound Amount Unit Conc. [g/L] Conc. [mM]
NaCl 20.00 g 19.782 338.508
K2HPO4 0.09 g 0.089 0.511
KH2PO4 0.07 g 0.069 0.509
CaCl2 0.80 g 0.791 7.13
NH4Cl 0.25 g 0.247 4.623
NaNO3 1.00 g 0.989 11.637
MgSO4 x 7 H2O 4.00 g 3.956 16.052
MgCl2 x 6 H2O 3.00 g 2.967 14.596
KCl 0.33 g 0.326 4.378
Fe2(SO4)3 x H2O 0.01 g 0.01 -
Modified Wolin's mineral solution 10.00 ml - -
Sodium resazurin
(0.1% w/v) 		0.50 ml 4.9e-4 0.002
Na2S2O3 x 5 H2O 3.00 g 2.967 11.956
NaHCO3 1.00 g 0.989 11.774
Wolin's vitamin solution (10x) 1.00 ml - -
Distilled water 1000.00 ml - -
1 Dissolve ingredients (except thiosulfate, bicarbonate and vitamins), then sparge medium with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Adjust pH to 6.8 with NaOH, then dispense under same gas atmosphere into anoxic Hungate-type tubes or serum vials up to a volume of 20% and autoclave. Add thiosulfate, vitamins and bicarbonate to the autoclaved medium from sterile anoxic stock solutions. The stock solutions of thiosulfate and vitamins are sterilized by filtration and stored under 100% N2 gas, whereas the stock solution of bicarbonate is prepared under 80% N2 and 20% CO2 gas mixture and autoclaved. Adjust pH of the complete medium to 6.5.
2 After inoculation add 1% (v/v) sterile O2 gas (e.g., by adding 1.50 ml sterile air to 5 ml medium in a Balch-type tube) and pressurize vials to 2 bar overpressure with sterile 80% N2 and 20% CO2 gas mixture.
1053a THIOPROFUNDUM LITHOTROPHICUM MEDIUM