275: TREPONEMA SUCCINIFACIENS MEDIUM Final pH: 7.4 Final volume: 1000 ml CaCl2 x 2 H2O 0.10 g MgSO4 x 7 H2O 0.10 g KH2PO4 0.50 g K2HPO4 0.50 g NaCl 1.00 g Yeast extract (Oxoid) 0.50 g Peptone (BD Bacto) 0.50 g (NH4)2SO4 0.50 g Clarified rumen fluid 300.00 ml Sodium resazurin (0.1% w/v) 0.50 ml L-Cysteine HCl x H2O 0.50 g Na2CO3 1.50 g D-Glucose 10.00 g Distilled water 700.00 ml Dissolve ingredients except for cysteine, carbonate, and glucose, and sparge medium with 80% N2 and 20% CO2 gas atmosphere to make it anoxic. Add cysteine and adjust pH to 7.2 - 7.4 by adding and dissolving solid Na2CO3. Then distribute the medium under the same gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. After autoclaving add glucose from a sterile anoxic stock solution prepared under a 100% N2 gas atmosphere. The final pH of the medium should be at 7.4. 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 then stored frozen at -20°C.
