Final pH: 7.0 - 7.2 Final volume: 1003 ml Solution A 952.00 ml Solution B 20.00 ml Solution C 20.00 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 in anoxic cultivation vials and autoclaved under the same gas atmosphere. Solution B is autoclaved separately under 80% N2 and 20% CO2 gas atmosphere. Solutions C and D are prepared under 100% N2 gas and filter-sterilized. Solution E is autoclaved under 100% N2 gas. 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 7.0 – 7.2. 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 of some strains at the beginning. For transfers use 5 - 10% inoculum. Incubate all strains in the dark. For DSM 2056: Replace pyruvate with 0.70 g/l Na-butyrate, 0.30 g/l Na-caproate, and 0.15g/l sodium octanoate. For DSM 3384: The vitamin solution of medium 120 is replaced with 1.0 ml/l of seven vitamins solution of medium 503. Replace pyruvate with 0.4 g/l Na-benzoate and add,after autoclaving, 1.0 g/l yeast extract from a sterile anoxic stock solution as a growth supplement. Initiation of growth requires the addition of freshly prepared sodium dithionite. For DSM 4661: Replace pyruvate with resorcinol. During growth, the culture is fed once with the same amount of resorcinol. For DSM 5091, DSM 9788: Replace pyruvate with 2.00 g/l malonic acid. For DSM 7044, DSM 7120, DSM 7467, DSM 12567, DSM 13228, DSM 23484: Supplement medium with 1.00 ml/l seven vitamins solution (see medium 503). Replace pyruvate with 0.40 g/l benzoate and 0.10 g/l yeast extract sterilized separately by filtration and add to the autoclaved medium from anoxic stock solutions. For DSM 8540: Replace pyruvate with 0.30 g/l p-hydroxybenzoate. For DSM 9705: Replace pyruvate with 1.00 g/l Na-glycolate sterilized by filtration. For DSM 12861, DSM 12883: Replace pyruvate with 1.00 g/l valeric acid. For DSM 12888: Replace pyruvate with 1.10 g/l Na-butyrate and 1.10 g/l caproic acid. For DSM 13036: Replace pyruvate with 1.00 g/l betaine. For DSM 14454: Naphthalene dissolved in heptamethylnonane can be used as substrate instead of pyruvate. For DSM 15576, DSM 16219: Replace pyruvate with 1.00 g/l Na-caprylate and 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 17291: Replace pyruvate with 3.00 g/l Casamino acids. For DSM 17477: Replace pyruvate with 1.90 g/l sodium glutamate monohydrate and 1.00g/l yeast extract. For DSM 21856: Replace pyruvate with 0.90 ml/l 1-butanol. For DSM 28890: Replace pyruvate with 0.50 g/l yeast extract and 0.68 g/l sodium formate. For DSM 100305, DSM 105015: Change amount of pyruvate to 2.20 g/l and add 0.10 g/l yeast extract. 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 1.00 g Distilled water 20.00 ml Solution C Na-pyruvate 2.50 g Distilled water 20.00 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
