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Main sol. 666a

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Internal Solution-ID: S1403

Equipment needed: Filter Autoclave Hungate tubes Gassing station Centrifuge
Compound Amount Unit Conc. [g/L] Conc. [mM]
Clarified rumen fluid 400.00 ml - -
K2HPO4 0.23 g 0.23 1.321
KH2PO4 0.23 g 0.23 1.69
NaCl 0.45 g 0.45 7.7
(NH4)2SO4 0.45 g 0.45 3.405
CaCl2 x 2 H2O 0.06 g 0.06 0.408
MgSO4 x 7 H2O 0.09 g 0.09 0.365
Agar 1.00 g 1 2.973
Indigocarmine 5.00 mg 0.005 0.011
NaHCO3 6.40 g 6.4 76.184
Cellobiose 2.50 g 2.5 7.304
Yeast extract 5.00 g 5 -
L-Cysteine HCl x H2O 0.30 g 0.3 1.708
DL-Dithiothreitol
(DTT) 		0.30 g 0.3 1.944
Distilled water 600.00 ml - -
1 Dissolve ingredients (except bicarbonate, cellobiose, yeast extract, cysteine and DTT), bring medium to the boil, then cool to room temperature under 100% CO2 gas atmosphere. Add the bicarbonate and equilibrate the medium with the CO2 gas to pH 6.8. Distribute under 100% CO2 gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. Thereafter, add cellobiose, yeast extract, cysteine and DTT from sterile anoxic stock solutions prepared under 100% N2 gas atmosphere. Cellobiose has to be sterilzed by filtration. Adjust pH of complete medium to 6.7 - 6.8, if necessary.
666a CELLULOSILYTICUM MEDIUM