# 861: DESULFOFRIGUS MEDIUM ## Main sol. 861 952 ml **Solution A** 30 ml **Solution B** 10 ml **Solution C** 1 ml **Solution D** 10 ml **Solution E** 1 ml **Solution F** 1. Solution A is sparged with 80% N2 and 20% CO2 gas mixture to reach a pH below 6 (at least 30 - 45 min), then dispensed under the same gas atmosphere into serum vials (use at least bottles of 50 ml volume filled with 30 ml medium) and autoclaved. Solutions B and F are prepared under 80% N2 and 20% CO2 gas atmosphere and sterilized. Sterile solutions C, D and E are prepared under 100% N2 gas. Solutions D and F are 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. Final pH of the medium should be 7.0 - 7.2. 2. Note: For transfer of cultures use at least 10% (v/v) inoculum. ## Solution A 20 g NaCl 4 g Na2SO4 0.2 g KH2PO4 0.25 g NH4Cl 3 g MgCl2 x 6 H2O 0.15 g CaCl2 x 2 H2O 0.09 g KBr 0.5 g KCl 1 ml **Trace element solution SL-10** 1 ml **Selenite-tungstate solution** 0.5 ml Sodium resazurin (0.1% w/v) 950 ml Distilled water ## Solution B 1.5 g Na2CO3 30 ml Distilled water ## Solution C 2.5 g Na-DL-lactate 10 ml Distilled water ## Solution D 1 ml **Wolin's vitamin solution (10x)** ## Solution E 0.3 g Na2S x 9 H2O 10 ml Distilled water ## Solution F 1 ml **Na-dithionite solution (5% w/v)** ## 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 ## 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.