KH2PO4 0.50 g NaCl 1.00 g (NH4)2SO4 0.50 g MgSO4 x 7 H2O 0.10 g CaCl2 x 2 H2O 0.10 g K2HPO4 0.50 g Clarified rumen fluid 300.00 ml Sludge fluid, alternative 300.00 ml Sodium resazurin (0.1% w/v) 0.50 ml Na2CO3 2.50 g Cellobiose 5.00 g L-Cysteine HCl x H2O 0.25 g Na2S x 9 H2O 0.25 g Distilled water 700.00 ml 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. Sludge fluid (from medium 119) Yeast extract 4.00 g Sludge 1000.00 ml Add 0.4% yeast extract to sludge from an anaerobic digester, and after gassing with nitrogen gas for a few minutes incubate it at 37°C for 24 hours. Then centrifuge the sludge at 13000 g and autoclave the resulting, clear supernatant in screw-capped vessels under nitrogen gas. The sludge fluid can be stored at 8-12°C in the dark. Clarified rumen fluid (from medium 1310) Rumen fluid from cow or sheep (obtained from fistulated animals or abattoir refuse) is filtered through muslin, autoclaved at 121°C for 15 min and then centrifuged at 27,000 g for 20 min. The supernatant is made anoxic by sparging with 100% N2 gas for 15 min, dispensed under same gas atmosphere into anoxic serum vials to 30% of volume and thenstored frozen at -20°C.
