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 1. Solution A is sparged with 80% N2 and 20% CO2 gas mixture to reach a pH below 6 (at least 30 min), then distributed under the same gas atmosphere in anoxic Hungate-type tubes or serum vials and autoclaved. Solutions C and E are autoclaved separately under 100% N2 gas. Solution B is autoclaved under 80% N2 and 20% CO2 gas atmosphere. Solution D is prepared under 100% N2 gas atmosphere and sterilized by filtration. To complete the medium appropriate amounts of solutions B to E are added to the sterile solution A in the sequence as indicated. Final pH of the medium should be 7.1 - 7.4. 2. Note: Addition of 10 - 20 mg sodium dithionite per liter (e.g. from 5% (w/v) solution, freshly prepared under N2 and filter-sterilized) may stimulate growth at the beginning. For transfers use 5 - 10% (v/v) inoculum. For DSM 9120, DSM 10141, DSM 17456, DSM 19275: Supplement medium with 1.00 g/l yeast extract added to the autoclaved medium from a sterile anoxic stock solution. For DSM 11974: Supplement medium with 1.00 ml/l seven vitamins solution (see medium 503) added to the autoclaved medium from an anoxic stock solution sterilized by filtration. For DSM 14728: Supplement medium with 0.50 g/l Na-acetate added to the autoclaved medium from a sterile anoxic stock solution. For DSM 14982: Supplement medium with 5.00 g/l Na2S2O3 x 5 H2O added to the autoclaved medium from an anoxic stock solution sterilized by filtration. For DSM 16109, DSM 17464: Supplement medium with 2.00 g/l Casamino acids (DIFCO) and 2.00 g/l Trypticase peptone (BD BBL) added to the autoclaved medium from sterile anoxic stock solutions. Solution A Na2SO4 3.00 g KH2PO4 0.20 g NH4Cl 0.30 g NaCl 21.00 g MgCl2 x 6 H2O 3.00 g KCl 0.50 g CaCl2 x 2 H2O 0.15 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-L-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 0.40 g Distilled water 10.00 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 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. 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
