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Main sol. 795a

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Internal Solution-ID: S1623

Equipment needed: Autoclave Filter Gassing station Serum bottle Water bath
Compound Amount Unit Conc. [g/L] Conc. [mM]
Synthetic seawater (2 x conc.) 500.00 ml - -
NH4Cl 1.25 g 1.233 23.046
KH2PO4 0.50 g 0.493 3.623
NiCl2 x 6 H2O
(0.1% w/v) 		3.00 ml 0.003 0.023
Modified Wolin's mineral solution 10.00 ml - -
Na-formate 0.20 g 0.197 2.9
Tryptone
(BD Bacto) 		1.00 g 0.986 -
Yeast extract
(OXOID) 		1.00 g 0.986 -
Sodium resazurin
(0.1% w/v) 		0.50 ml 4.9e-4 0.002
NaHCO3 2.00 g 1.972 23.479
Wolin's vitamin solution (10x) 1.00 ml - -
Sulfur , powdered 10.00 g 9.862 307.513
Na2S x 9 H2O 0.50 g 0.493 2.053
Distilled water 500.00 ml - -
1 Dissolve ingredients (except bicarbonate, vitamins, sulfur, and sulfide), then sparge medium with 80% H2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic and adjust pH to 5.5 with H2SO4. Dispense under the same gas atmosphere in suitable culture vessels (e.g. 20 ml of the medium in 100 ml serum bottles) and autoclave. Steam sulfur for 3 hours on each of 3 successive days (see medium 35). Aseptically mix the sterilized sulfur with the sterile medium while retaining anoxic conditions. Add vitamins from an anoxic stock solution prepared under 100% N2 gas atmosphere and sterilized by filtration and bicarbonate from a sterile anoxic stock solution prepared under 80% N2 and 20% CO2 gas mixture. Prior to inoculation add sulfide from a sterile anoxic stock solution prepared under 100% N2 gas atmosphere. Check pH and adjust to 5.5, if necessary.
2 After inoculation pressurize vials to 1 bar overpressure with sterile 80% H2 and 20% CO2 gas mixture.
795a LEBETIMONAS MEDIUM