Solution A 952.00 ml Solution B 30.00 ml Solution C 1.00 ml Solution D 1.00 ml Solution E 10.00 ml Solution F 10.00 ml Solution A is sparged with 80% N2 and 20% CO2 gas mixture to reach a pH below 6 (at least 30 -45 min), then distributed under the same gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclaved. Solution B is autoclaved separately under 80% N2 and 20% CO2 gas atmosphere. Solutions C, E and F are autoclaved under 100% N2 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 F are added to the sterile solution A in the sequence as indicated. Final pH of the medium should be 7.2 - 7.4. Solution A NaCl 15.00 g MgCl2 x 6 H2O 2.30 g KCl 0.50 g NH4Cl 0.30 g KH2PO4 0.20 g CaCl2 x 2 H2O 0.03 g Selenite-tungstate solution 1.00 ml Yeast extract 1.00 g 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 Trace element solution SL-10 1.00 ml Solution D Wolin's vitamin solution (10x) 1.00 ml Solution E Starch 1.00 g Distilled water 10.00 ml Solution F Na2S x 9 H2O 0.30 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
