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Main sol. 666b

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Internal Solution-ID: S6394

Equipment needed: Autoclave Hungate tubes Gassing station Centrifuge
Compound Amount Unit Conc. [g/L] Conc. [mM]
Clarified rumen fluid 400.00 ml - -
K2HPO4 0.23 g 0.23 1.321
KH2PO4 0.23 g 0.23 1.69
NaCl 0.45 g 0.45 7.7
(NH4)2SO4 0.45 g 0.45 3.405
CaCl2 x 2 H2O 0.06 g 0.06 0.408
MgSO4 x 7 H2O 0.09 g 0.09 0.365
Indigocarmine 5.00 mg 0.005 0.011
NaHCO3 6.40 g 6.4 76.184
Yeast extract 5.00 g 5 -
L-Cysteine HCl x H2O 0.30 g 0.3 1.708
Na2S x 9 H2O 0.30 g 0.3 1.249
Distilled water 600.00 ml - -
1 Dissolve ingredients (except bicarbonate, cysteine, and sulfide), bring the medium to a boil, then cool to room temperature under 80% H2 and 20% CO2 gas atmosphere. Add the bicarbonate and equilibrate the medium to pH 6.8. Distribute under the same gas atmosphere into anoxic Hungate-type tubes or serum vials to 30% of their volume and autoclave. After that, add cysteine and sulfide from sterile anoxic stock solutions prepared under a 100% N2 gas atmosphere. Adjust the pH of the complete medium to 6.7 - 6.8, if necessary.
2 After inoculation add sterile 80% H2 and 20% CO2 gas mixture to 2 bar of overpressure.
666b METHANOMICROBIUM MOBILE MEDIUM