Solution A 870.00 ml Solution B 100.00 ml Solution C 10.00 ml Solution D 1.00 ml Solution E 2.00 ml Solution F 10.00 ml Solution G 15.00 ml 1. Sparge solution A with 80% H2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic, then dispense under same gas atmosphere into anoxic serum vials (e.g., 9 ml medium in 50 ml bottle). Pressurize closed bottles with 80% H2 and 20% CO2 gas mixture to 0.5 bar overpressure and autoclave. Solution B is autoclaved separately under 80% N2 and 20% CO2 gas atmosphere. Solutions C, D and F are autoclave under 100% N2 gas atmosphere. Solution E is prepared under 100% N2 gas atmosphere and sterilized by filtration. Prepare solution G by filling 13.5 ml hexadecane into a 50 ml serum bottle, then sparge with 100% N2 gas to make it anoxic and autoclave. Add 1.50 ml anoxic autoclaved tetrachloroethene to the sterile anoxic hexadecane solution by syringe. To complete the medium add appropriate amounts of solutions B to F to the sterile solution A in the sequence as indicated. The pH of the medium before inoculation should be at 7.2. 2. Add solution G only after inoculation of the medium! Solution A K2HPO4 0.65 g NaH2PO4 x H2O 0.17 g Peptone (BD Bacto) 0.10 g Na-acetate 0.46 g Sodium resazurin (0.1% w/v) 0.50 ml Distilled water 870.00 ml Solution B (NH4)2CO3 0.27 g NaHCO3 3.73 g Distilled water 100.00 ml Solution C CaCl2 x 2 H2O 0.11 g MgCl2 x 6 H2O 0.10 g Distilled water 10.00 ml
Solution D Trace element solution 1.00 ml Solution E Wolin's vitamin solution (10x) 1.00 ml Seven vitamins solution 1.00 ml Solution F Na2S x 9 H2O 0.30 g Distilled water 10.00 ml Solution G Hexadecane 13.50 ml Tetrachloroethene 1.50 ml Trace element solution (from medium 732) Na2-EDTA 0.50 g FeCl2 x 4 H2O 2.00 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 AlCl3 10.00 mg NiCl2 x 6 H2O 24.00 mg Na2MoO4 x 2 H2O 36.00 mg Distilled water 1000.00 ml First dissolve EDTA in distilled water, adjust pH to 7 using 2 N NaOH and add ferrous chloride. After ferrous chloride has dissolved add remaining compounds. 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
