# 397: FERULATE MEDIUM ## Main sol. 397 882 ml **Solution A** 30 ml **Solution B** 1 ml **Solution D** 10 ml **Solution E** 10 ml **Solution F** 10 mM Sodium ferulate 1. Add and dissolve ingredients of solution A and sparge medium with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Dispense medium under the same gas atmosphere into anoxic Hungate-type tubes and autoclave. Solution B is prepared under 80% N2 and 20% CO2 gas atmosphere and autoclaved. Solutions E and F are autoclaved under 100% N2 gas atmosphere. 2. Solution D is prepared under 100% N2 gas and sterilized by filtration. To complete the medium appropriate amounts of solutions B to F are added to the sterile solution A in the sequence as indicated. 3. Note: Some cultures are shipped in semi-solid medium which stimulates growth at the beginning. For agar stabs 3.00 g/l agar are added to the complete medium from a sterile anoxic stock solution (2% w/v). Upon receipt add anoxically 1 - 2 ml of the recommended freshly prepared liquid medium to the agar tube and incubate for 3 - 5 days . After incubation transfer 0.5 ml of the resulting cell suspension in the liquid phase to tubes with liquid medium. 4. Add sodium ferulate to 10 mM final concentration from filter-sterilized anaerobic stock solution. ## Solution A 0.5 g KH2PO4 0.33 g MgCl2 x 6 H2O 0.4 g NaCl 0.4 g NH4Cl 0.05 g CaCl2 x 2 H2O 1 ml **Trace element solution SL-10** 1 ml **Selenite-tungstate solution** 50 ml **Clarified rumen fluid**, clarified 1 g Trypticase peptone (BD BBL) 0.5 ml Sodium resazurin (0.1% w/v) 830 ml Distilled water ## Solution B 1.5 g Na2CO3 30 ml Distilled water ## Solution D 1 ml **Seven vitamins solution** ## Solution E 0.3 g L-Cysteine HCl x H2O 10 ml Distilled water ## Solution F 0.3 g Na2S x 9 H2O 10 ml Distilled water ## Trace element solution SL-10 10 ml HCl (25%) 1.5 g FeCl2 x 4 H2O 70 mg ZnCl2 100 mg MnCl2 x 4 H2O 6 mg H3BO3 190 mg CoCl2 x 6 H2O 2 mg CuCl2 x 2 H2O 24 mg NiCl2 x 6 H2O 36 mg Na2MoO4 x 2 H2O 990 ml Distilled water 1. First dissolve FeCl2 in the HCl, then dilute in water, add and dissolve the other salts. Finally make up to 1000.00 ml. ## Selenite-tungstate solution 0.5 g NaOH 3 mg Na2SeO3 x 5 H2O 4 mg Na2WO4 x 2 H2O 1000 ml Distilled water ## Seven vitamins solution 100 mg Vitamin B12 80 mg p-Aminobenzoic acid 20 mg D-(+)-biotin 200 mg Nicotinic acid 100 mg Calcium pantothenate 300 mg Pyridoxine hydrochloride 200 mg Thiamine-HCl x 2 H2O 1000 ml Distilled water ## 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.