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Main sol. 905

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Internal Solution-ID: S1859

Equipment needed: Autoclave Hungate tubes Gassing station
Compound Amount Unit Conc. [g/L] Conc. [mM]
NH4Cl 1.00 g 0.99 18.51
K2HPO4 0.30 g 0.297 1.705
KH2PO4 0.30 g 0.297 2.183
KCl 0.17 g 0.168 2.258
NaCl 50.00 g 49.505 847.107
Na-acetate 0.50 g 0.495 6.035
Yeast extract
(OXOID) 		0.50 g 0.495 -
Trypticase
(BD BBL) 		0.50 g 0.495 -
Modified Wolin's mineral solution 10.00 ml - -
Sodium resazurin
(0.1% w/v) 		0.50 ml 5.0e-4 0.002
NaHCO3 2.00 g 1.98 23.572
L-Cysteine HCl x H2O 0.50 g 0.495 2.819
CaCl2 x 2 H2O 0.60 g 0.594 4.041
MgCl2 x 6 H2O 3.20 g 3.168 15.584
Na2S x 9 H2O 0.30 g 0.297 1.237
Distilled water 1000.00 ml - -
1 Dissolve ingredients (except bicarbonate, cysteine, calcium chloride, magnesium chloride and sulfide), then sparge medium with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Add and dissolve solid bicarbonate and cysteine, then adjust pH to 7.5 with 10 N KOH. Dispense medium under 80% N2 and 20% CO2 gas atmosphere into anoxic Hungate-type tubes or serum vials (to 30% of volume) and autoclave. Add calcium chloride, magnesium chloride and sulfide from sterile anoxic stock solutions prepared under 100% N2 gas. The pH of the complete medium should be between 7.2 and 7.6.
2 After inoculation pressurize vessels with sterile 80% H2 and 20% CO2 gas mixture to 1 bar overpressure.
905 METHANOCALCULUS HALOTOLERANS MEDIUM