# 346: DESULFALLAS MEDIUM ## Main sol. 346 966 ml **Solution A** 20 ml **Solution B** 11 ml **Solution C** 10 ml **Solution D** 1 ml **Solution E** 1 ml **Solution F** 1 ml **Solution G** 1.7 ml **Solution H** 1. Sparge solution A with 100% N2 gas for 30 - 45 min to make it anoxic. Thereafter, dispense under same gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. Solution B is autoclaved separately under 80% N2 and 20% CO2 gas atmosphere. Anoxic stock solutions C, D, E, F, and H are prepared under 100% N2 gas atmosphere and solution G under 80% N2 and 20% CO2 gas atmosphere. Filter sterilize solutions D, F and G. To complete the medium appropriate amounts of solutions B to H are added to the sterile solution A in the sequence as indicated. Adjust pH of the complete medium to 7.2 - 7.5, if necessary. ## Solution A 3 g Na2SO4 0.2 g KH2PO4 0.3 g NH4Cl 0.15 g CaCl2 x 2 H2O 0.4 g MgCl2 x 6 H2O 1 g NaCl 1 ml **Trace element solution SL-10** 0.5 ml Sodium resazurin (0.1% w/v) 965 ml Distilled water ## Solution B 1 g NaHCO3 20 ml Distilled water ## Solution C 1 ml Ethanol 10 ml Distilled water ## Solution D 0.7 g Na-benzoate 10 ml Distilled water ## Solution E 1 ml **Clarified rumen fluid** ## Solution F 1 ml **Wolin's vitamin solution (10x)** ## Solution G 1 ml **Na-dithionite solution (5% w/v)** ## Solution H 50 mg Na2S x 9 H2O 1.7 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. ## 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. ## Wolin's vitamin solution (10x) 20 mg Biotin 20 mg Folic acid 100 mg Pyridoxine hydrochloride 50 mg Thiamine HCl 50 mg Riboflavin 50 mg Nicotinic acid 50 mg Calcium D-(+)-pantothenate 1 mg Vitamin B12 50 mg p-Aminobenzoic acid 50 mg (DL)-alpha-Lipoic acid 1000 ml Distilled water ## Na-dithionite solution (5% w/v) 50 g NaHCO3 50 g Na2S2O4 1000 ml Distilled water 1. Dissolve NaHCO3 in water and make the solution anoxic by sparging with 80% N2 and 20% CO2 gas mixture. Then dissolve the Na-dithionite and filter sterilize the solution into anoxic Hungate tubes. Store the prepared solution in the dark and refrigerated. Prepare only small amounts of stock solution, as Na-dithionite decomposes rapidly.