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Main sol. J447

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Internal Solution-ID: S4195

Equipment needed: Filter Autoclave Anaerobic workbench
Compound Amount Unit Conc. [g/L] Conc. [mM]
CaCl2 x 2 H2O 0.150 g 0.148 1.008
MgCl2 x 6 H2O 3.000 g 2.964 14.581
NH4Cl 0.250 g 0.247 4.618
KH2PO4 0.200 g 0.198 1.452
KCl 0.500 g 0.494 6.627
NaCl 20.000 g 19.763 338.173
Trace element solution 1.000 ml - -
Selenite-tungstate solution 1.000 ml - -
Sodium acetate 0.082 g 0.081 0.988
Yeast extract
(BD-Difco) 		1.000 g 0.988 -
Trace vitamins 10.000 ml - -
Vitamin B12 0.050 mg 4.9e-5 3.6e-5
NaHCO3 2.500 g 2.47 29.407
Resazurin 1.000 mg 9.9e-4 0.004
Na2S x 9 H2O 0.500 g 0.494 2.057
Distilled water 1000.000 ml - -
1 Mix ingredients except NaHCO3 and Na2S x 9H2O, and bring to a boil for 5 min, then, cool down under a stream of a H2-CO2 (80:20, v/v) gas mixture. Distribute the medium into culture vessels (e.g., 20 ml of the medium in 120 ml serum bottles) under the same gas mixture, seal with butyl rubber stoppers and autoclave. Filter-sterilize NaHCO3 as an 8% solution while gassing with N2-CO2 (80:20, v/v). Separately autoclave 5% Na2S x 9H2O solution under a N2 atmosphere. Aseptically and anaerobically add the NaHCO3 solution to the medium and readjust pH to 7.6 - 7.8 with a sterile 1% Na2CO3 solution (stocked anaerobically). Prior to inoculation, add the sterile Na2S x 9H2O solution. Pressurize inoculated bottles to 200 kPa H2-CO2 (80:20, v/v).
J447 METHANOBACTERIUM ADHERENS MEDIUM