inal pH: 7.3 - 7.6 Final volume: 1003 ml Solution A 892.00 ml Solution B 10.00 ml Solution C 2.00 ml Solution D 35.00 ml Solution E 20.00 ml Solution F 40.00 ml Solution G 3.00 ml Solution H 1.00 ml 1. Sparge solution A with 100% N2 gas for 30 - 45 min to make it anoxic, then distribute under same gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. Solutions B and H are autoclaved separately under 100% N2 gas. Solutions C, E, F and G are prepared under 100% N2 gas and sterilized by filtration. Solution D is autoclaved under 80% N2 and 20% CO2 gas atmosphere. 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 complete medium to 7.3 - 7.6, if necessary. 2. Note: Before inoculation, the medium must be completely reduced, i.e. resazurin must be colorless. The addition of 10 - 20 mg sodium dithionite per liter (e.g. from 5% (w/v) solution, freshly prepared under N2 and filter-sterilized, see medium 829) can help to reduce the medium. For DSM 24537: Adjust pH of complete medium to 8.5. Solution A Na2SO4 0.70 g KH2PO4 0.20 g NH4Cl 0.25 g NaCl 0.25 g MgCl2 x 6 H2O 0.40 g KCl 0.50 g CaCl2 x 2 H2O 0.15 g Yeast extract 2.00 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 890.00 ml Solution B Potassium phosphate buffer (0.1 M, pH 7.5) 10.00 ml Solution C Wolin's vitamin solution (10x) 1.00 ml Seven vitamins solution 1.00 ml Solution D Na2CO3 1.75 g Distilled water 35.00 ml Solution E Na-pyruvate 4.50 g Distilled water 20.00 ml Solution F Na2-fumarate 6.40 g Distilled water 40.00 ml Solution G FeSO4 x 7 H2O 30.00 mg H2SO4 (0.1 N) 3.00 ml Solution H L-Cysteine HCl x H2O 50.00 mg Distilled water 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 Seven vitamins solution (from medium 503) Vitamin B12 100.00 mg p-Aminobenzoic acid 80.00 mg D-(+)-biotin 20.00 mg Nicotinic acid 200.00 mg Calcium pantothenate 100.00 mg Pyridoxine hydrochloride 300.00 mg Thiamine-HCl x 2 H2O 200.00 mg Distilled water 1000.00 ml
