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Main sol. 320

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Internal Solution-ID: S594

Equipment needed: Autoclave Filter Gassing station Hungate tubes Centrifuge
Compound Amount Unit Conc. [g/L] Conc. [mM]
K2HPO4 x 3 H2O 1.00 g 1 5.741
NH4Cl 1.00 g 1 18.695
KCl 0.50 g 0.5 6.707
MgSO4 x 7 H2O 0.50 g 0.5 2.029
Trypticase peptone
(BD BBL) 		0.50 g 0.5 -
Yeast extract 0.50 g 0.5 -
Clarified rumen fluid 20.00 ml - -
Sludge fluid , alternative
optional 		20.00 ml - -
Trace element solution SL-10 1.00 ml - -
Sodium resazurin
(0.1% w/v) 		0.50 ml 5.0e-4 -
L-Cysteine HCl x H2O 0.15 g 0.15 0.854
Na2CO3 1.00 g 1 9.435
Cellobiose 5.00 g 5 14.607
Cellulose , MN 301
optional 		10.00 g - -
Na2S x 9 H2O 0.15 g 0.15 0.625
Distilled water 1000.00 ml - -
1 Dissolve ingredients (except cysteine, carbonate, cellobiose and sulfide), bring medium to the boil, then cool to room temperature under 80% N2 and 20% CO2 gas mixture and add cysteine. Dispense under same gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. Add sulfide from a sterile anoxic stock solution prepared under 100% N2 gas and carbonate from a sterile anoxic stock solution prepared under 80% N2 and 20% CO2 gas mixture. Sterilize cellobiose separately by filtration under 100% N2 gas. Adjust pH of the complete medium to 7.0, if necessary.
2 Note: Some strains can be adapted to cellulose as substrate using 10.0 g/l cellulose powder MN 301 (MACHEREY-NAGEL).
320 CLOSTRIDIUM CELLULOVORANS MEDIUM