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Main sol. J230

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Internal Solution-ID: S3907

Equipment needed: Autoclave Anaerobic workbench
Compound Amount Unit Conc. [g/L] Conc. [mM]
KH2PO4 0.227 g 0.227 1.665
K2HPO4 0.348 g 0.347 1.994
NH4Cl 0.500 g 0.499 9.329
MgSO4 x 7 H2O 0.500 g 0.499 2.025
CaCl2 x 2 H2O 0.250 g 0.25 1.697
NaCl 2.250 g 2.246 38.424
FeSO4 x 7 H2O 2.000 mg 0.002 0.007
Yeast extract
(BD-Difco) 		2.000 g 1.996 -
Casitone
(BD-Difco) 		2.000 g 1.996 -
Trace vitamins 10.000 ml - -
FeCl2 solution 1.000 ml - -
Trace element solution 1.000 ml - -
NaHCO3 0.850 g 0.848 10.098
Methanol 10.000 ml - -
L-Cysteine HCl x H2O 0.300 g 0.299 -
Na2S x 9 H2O 0.300 g 0.299 1.247
Resazurin 1.000 mg 1.0e-3 0.004
Distilled water 980.000 ml - -
1 Mix ingredients, except NaHCO3, methanol, cysteine x HCl and Na2S x 9H2O, and bring to a boil for 5-10 sec. Cool down under a stream of N2-CO2 (80:20), and add NaHCO3 and methanol to the medium. Dispense the medium into suitable culture vessels (e.g., 20 ml of the medium in 120 ml serum bottles) under a stream of N2-CO2 (80:20) and seal with butyl rubber stoppers. After autoclaving, stand the medium overnight. Separately autoclave cysteine x HCl and Na2S x 9H2O as 5% solutions, respectively, under a N2 atmosphere. Prior to inoculation, aseptically and anaerobically add the cysteine x HCl and Na2S x 9H2O solutions.
J230 METHANOSARCINA MEDIUM