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Main sol. 140

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Internal Solution-ID: S239

Equipment needed: Hungate tubes Filter Autoclave Gassing station Centrifuge
Compound Amount Unit Conc. [g/L] Conc. [mM]
KH2PO4 0.50 g 0.5 3.674
NaCl 1.00 g 1 17.112
(NH4)2SO4 0.50 g 0.5 3.784
MgSO4 x 7 H2O 0.10 g 0.1 0.406
CaCl2 x 2 H2O 0.10 g 0.1 0.68
K2HPO4 0.50 g 0.5 2.871
Clarified rumen fluid 300.00 ml - -
Sludge fluid , alternative
optional 		300.00 ml - -
Sodium resazurin
(0.1% w/v) 		0.50 ml 5.0e-4 -
Na2CO3 2.50 g 2.5 23.588
Cellobiose 5.00 g 5 14.607
L-Cysteine HCl x H2O 0.25 g 0.25 1.423
Na2S x 9 H2O 0.25 g 0.25 1.041
Distilled water 700.00 ml - -
1 Dissolve ingredients (except carbonate, cellobiose and reducing agents), bring medium to the boil, then cool to room temperature under 100% CO2 gas atmosphere. Use either clarified rumen fluid or sludge fluid as supplement. Dispense medium under same gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. Add cellobiose, sulfide and cysteine from sterile anoxic stock solutions prepared under 100% N2 gas and carbonate from a sterile anoxic stock solution prepared under 80% N2 and 20% CO2 gas mixture. Cellobiose has to be sterilized by filtration. Adjust pH of complete medium to 6.8, if necessary.
140 RUMINICLOSTRIDIUM MEDIUM