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Main sol. 321

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Internal Solution-ID: S596

Equipment needed: Hungate tubes Autoclave Gassing station Centrifuge
Compound Amount Unit Conc. [g/L] Conc. [mM]
NH4Cl 1.0 g 1 18.695
K2HPO4 x 3 H2O 0.4 g 0.4 2.297
MgCl2 x 6 H2O 0.4 g 0.4 1.968
Minerals solution 50.0 ml - -
Na-formate 5.0 g 5 73.522
Na-acetate 1.0 g 1 12.19
Trypticase peptone
(BD BBL) 		1.0 g 1 -
Yeast extract
(OXOID) 		1.0 g 1 -
Modified Wolin's mineral solution 10.0 ml - -
Sludge fluid 5.0 ml - -
Clarified rumen fluid , alternative
optional 		5.0 ml - -
Sodium resazurin
(0.1% w/v) 		0.5 ml 5.0e-4 0.002
Na2CO3 1.5 g 1.5 14.153
L-Cysteine HCl x H2O 0.2 g 0.2 1.139
Na2S x 9 H2O 0.2 g 0.2 0.833
Distilled water 935.0 ml - -
1 Dissolve ingredients except carbonate, cysteine and sulfide, then sparge medium with 80% H2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Dispense medium under same gas atmosphere into anoxic Hungate-type tubes or serum vials to 30% of their volume and autoclave. Add cysteine and sulfide from sterile anoxic stock solutions prepared under 100% N2 gas and carbonate from a sterile anoxic stock solution prepared under 80% N2 and 20% CO2 gas mixture. Prior to use check pH of complete medium and adjust to 6.8 - 7.0, if necessary.
2 After inoculation add sterile 80% H2 and 20% CO2 gas mixture to 2 bar overpressure.
321 METHANOCORPUSCULUM AGGREGANS MEDIUM