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Main sol. 520

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Internal Solution-ID: S1155

Equipment needed: Filter Autoclave Hungate tubes Gassing station
Compound Amount Unit Conc. [g/L] Conc. [mM]
(NH4)2SO4 1.30 g 1.296 9.809
KH2PO4 1.50 g 1.496 10.99
K2HPO4 x 3 H2O 2.90 g 2.891 16.6
FeSO4 x 7 H2O
(0.1% w/v in 0.1 N H2SO4) 		1.25 ml 0.001 0.004
Trace element solution SL-10 1.00 ml - -
Yeast extract 2.00 g 1.994 -
Sodium resazurin
(0.1% w/v) 		0.50 ml 5.0e-4 0.002
MgCl2 x 6 H2O 0.20 g 0.199 0.981
CaCl2 x 2 H2O 75.00 mg 0.075 0.509
Cellobiose 6.00 g 5.982 17.476
Cellulose , MN 301
optional 		10.00 g 9.97 -
Na2CO3 1.50 g 1.496 14.11
L-Cysteine HCl x H2O 0.50 g 0.499 2.838
Distilled water 1000.00 ml - -
1 Dissolve ingredients except magnesium chloride, calcium chloride, cellobiose, cysteine and carbonate, then sparge medium with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Dispense medium under the same gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. After autoclaving add magnesium chloride, calcium chloride and cellobiose from anoxic stock solutions prepared under 100% N2 gas and carbonate from a sterile anoxic stock solution prepared under 80% N2 and 20% CO2. Cellobiose has to be sterilized by filtration. Prior to inoculation add cysteine from a sterile anoxic stock solution prepared under 100% N2 gas and adjust pH to 7.2.
2 Note: Some strains can be adapted to cellulose as substrate using 10.00 g/l cellulose powder MN 301 (MACHEREY-NAGEL).
520 RUMINOCLOSTRIDIUM CELLULOLYTICUM (CM3) MEDIUM