861: DESULFOFRIGUS MEDIUM Final pH: 7.0 - 7.2 Final volume: 1004 ml Solution A 952.00 ml Solution B 30.00 ml Solution C 10.00 ml Solution D 1.00 ml Solution E 10.00 ml Solution F 1.00 ml 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. For DSM 12341: Replace Na-DL-lactate with 1.50 g/l Na-acetate. For DSM 12344: Replace Na-DL-lactate with 1.50 g/l Na-propionate. Solution A NaCl 20.00 g Na2SO4 4.00 g KH2PO4 0.20 g NH4Cl 0.25 g MgCl2 x 6 H2O 3.00 g CaCl2 x 2 H2O 0.15 g KBr 0.09 g KCl 0.50 g Trace element solution SL-10 1.00 ml Selenite-tungstate solution 1.00 ml Sodium resazurin (0.1% w/v) 0.50 ml Distilled water 950.00 ml Solution B Na2CO3 1.50 g Distilled water 30.00 ml Solution C Na-DL-lactate 2.50 g Distilled water 10.00 ml Solution D Wolin's vitamin solution (10x) 1.00 ml Solution E Na2S x 9 H2O 0.30 g Distilled water 10.00 ml Solution F Na-dithionite solution (5% w/v) 1.00 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. Selenite-tungstate solution (from medium 385) NaOH 0.50 g Na2SeO3 x 5 H2O 3.00 mg Na2WO4 x 2 H2O 4.00 mg Distilled water 1000.00 ml Wolin's vitamin solution (10x) (from medium 120) Biotin 20.00 mg Folic acid 20.00 mg Pyridoxine hydrochloride 100.00 mg Thiamine HCl 50.00 mg Riboflavin 50.00 mg Nicotinic acid 50.00 mg Calcium D-(+)-pantothenate 50.00 mg Vitamin B12 1.00 mg p-Aminobenzoic acid 50.00 mg (DL)-alpha-Lipoic acid 50.00 mg Distilled water 1000.00 ml Na-dithionite solution (5% w/v) (from medium 829) NaOH (1 N) 0.50 ml Na2S2O4 0.50 g Distilled water 9.50 ml Add 1 N NaOH to distilled water and make the solution anoxic by sparging with 100% N2 gas. Then dissolve the Na-dithionite and filter-sterilize the solution into anoxic Hungate tubes. The final pH of the solution should be between 9 and 10. Store the prepared solution in a dark and cool place. Prepare only small amounts of stock solution, as Na -dithionite decomposes rapidly.
