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Main sol. 892

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Internal Solution-ID: S1842

Equipment needed: Filter Autoclave Hungate tubes Gassing station
Compound Amount Unit Conc. [g/L] Conc. [mM]
KH2PO4 0.40 g 0.396 2.907
NH4Cl 0.90 g 0.89 16.643
MgCl2 x 6 H2O 0.36 g 0.356 1.751
NaCl 10.00 g 9.891 169.254
Na-acetate 0.50 g 0.495 6.029
Yeast extract
(OXOID) 		2.00 g 1.978 -
Trypticase peptone
(BD BBL) 		2.00 g 1.978 -
Trace element solution 10.00 ml - -
Sodium resazurin
(0.1% w/v) 		0.50 ml 4.9e-4 0.002
Na2CO3 5.00 g 4.946 46.662
Wolin's vitamin solution (10x) 1.00 ml - -
Na2S x 9 H2O 0.50 g 0.495 2.059
L-Cysteine HCl x H2O 0.50 g 0.495 2.816
Distilled water 1000.00 ml - -
1 Dissolve ingredients except carbonate, vitamins, cysteine and sulfide, then sparge medium with 80% H2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Add and dissolve carbonate, adjust pH to 7.2 then dispense medium under 80% H2 and 20% CO2 gas atmosphere into anoxic Hungate-type tubes or serum vials to 30% of their volume and autoclave. Add vitamins (sterilized by filtration), cysteine and sulfide from sterile anoxic stock solutions prepared under 100% N2 gas. Prior to use check pH of complete medium and adjust to 7.3 - 7.5, if necessary.
2 After inoculation add sterile 80% H2 and 20% CO2 gas mixture to 1.5 - 2 bar overpressure.
892 METHANOCALCULUS PUMILUS MEDIUM