Solution A 972.00 ml Solution B 30.00 ml Solution C 1.00 ml Solution D 10.00 ml Solution E 3.00 ml Sparge solution A with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic, distribute under same gas atmosphere 8.9 ml medium per Hungate-type tube and autoclave. Solution B is autoclaved separately under 80% N2 and 20% CO2 gas atmosphere. Solution C is prepared under 100% N2 gas atmosphere and sterilized by filtration. Autoclave solutions D and E under 100% N2 gas. To complete the medium add appropriate amounts of the solutions B to E to the sterile solution A. Adjust pH of complete medium to 7.0 - 7.2, if necessary. Solution A Mineral solution 50.00 ml Trace element solution SL-10 1.00 ml Yeast extract 2.00 g Na-vanillate 2.00 g Clarified rumen fluid 300.00 ml Sodium resazurin (0.1% w/v) 0.50 ml Distilled water 620.00 ml Adjust pH to 6.9 before autoclaving. Solution B Na2CO3 1.50 g Distilled water 30.00 ml Solution C Seven vitamins solution 1.00 ml Solution D L-Cysteine HCl x H2O 0.30 g Distilled water 10.00 ml Solution E Na2S x 9 H2O 90.00 mg
Distilled water 3.00 ml Mineral solution (from medium 335) KH2PO4 10.00 g MgCl2 x 6 H2O 6.60 g NaCl 8.00 g NH4Cl 8.00 g CaCl2 x 2 H2O 1.00 g Distilled water 1000.00 ml Trace element solution SL-10 (from medium 320) HCl (25%) 10.00 ml FeCl2 x 4 H2O 1.50 g ZnCl2 70.00 mg MnCl2 x 4 H2O 100.00 mg H3BO3 6.00 mg CoCl2 x 6 H2O 190.00 mg CuCl2 x 2 H2O 2.00 mg NiCl2 x 6 H2O 24.00 mg Na2MoO4 x 2 H2O 36.00 mg Distilled water 990.00 ml First dissolve FeCl2 in the HCl, then dilute in water, add and dissolve the other salts. Finally make up to 1000.00 ml. 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. Seven vitamins solution (from medium 503) Vitamin B12 100.00 mg p-Aminobenzoic acid 80.00 mg D-(+)-biotin 20.00 mg Nicotinic acid 200.00 mg Calcium pantothenate 100.00 mg Pyridoxine hydrochloride 300.00 mg Thiamine-HCl x 2 H2O 200.00 mg Distilled water 1000.00 ml
