# 666: SUCCINICLASTICUM MEDIUM ## Main sol. 666 400 ml **Clarified rumen fluid** 0.23 g K2HPO4 0.23 g KH2PO4 0.45 g NaCl 0.45 g (NH4)2SO4 0.06 g CaCl2 x 2 H2O 0.09 g MgSO4 x 7 H2O 5 mg Indigocarmine 6.4 g NaHCO3 5 g Disodium succinate 5 g Yeast extract 0.3 g L-Cysteine HCl x H2O 0.3 g Na2S x 9 H2O 600 ml Distilled water 1. Dissolve ingredients (except bicarbonate, succinate, yeast extract, cysteine and sulfide), 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 succinate, yeast extract, cysteine and sulfide from sterile anoxic stock solutions prepared under 100% N2 gas atmosphere. Adjust pH of complete medium to 6.7 - 6.8, if necessary. 2. Note: Supplementing the medium with 1.50 g/l agar stimulates growth of strains after resuscitation from ampoules. ## Clarified rumen fluid 1. 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.