# 732: DEHALOBACTER RESTRICTUS MEDIUM (TCE) ## Main sol. 732 870 ml **Solution A** 100 ml **Solution B** 10 ml **Solution C** 1 ml **Solution D** 2 ml **Solution E** 10 ml **Solution F** 15 ml **Solution G** 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 0.65 g K2HPO4 0.17 g NaH2PO4 x H2O 0.1 g Peptone (BD Bacto) 0.46 g Na-acetate 0.5 ml Sodium resazurin (0.1% w/v) 870 ml Distilled water ## Solution B 0.27 g (NH4)2CO3 3.73 g NaHCO3 100 ml Distilled water ## Solution C 0.11 g CaCl2 x 2 H2O 0.1 g MgCl2 x 6 H2O 10 ml Distilled water ## Solution D 1 ml **Trace element solution** ## Solution E 1 ml **Wolin's vitamin solution (10x)** 1 ml **Seven vitamins solution** ## Solution F 0.3 g Na2S x 9 H2O 10 ml Distilled water ## Solution G 13.5 ml Hexadecane 1.5 ml Tetrachloroethene ## Trace element solution 0.5 g Na2-EDTA 2 g FeCl2 x 4 H2O 70 mg ZnCl2 100 mg MnCl2 x 4 H2O 6 mg H3BO3 190 mg CoCl2 x 6 H2O 2 mg CuCl2 x 2 H2O 10 mg AlCl3 24 mg NiCl2 x 6 H2O 36 mg Na2MoO4 x 2 H2O 1000 ml Distilled water 1. 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) 20 mg Biotin 20 mg Folic acid 100 mg Pyridoxine hydrochloride 50 mg Thiamine HCl 50 mg Riboflavin 50 mg Nicotinic acid 50 mg Calcium D-(+)-pantothenate 1 mg Vitamin B12 50 mg p-Aminobenzoic acid 50 mg (DL)-alpha-Lipoic acid 1000 ml Distilled water ## Seven vitamins solution 100 mg Vitamin B12 80 mg p-Aminobenzoic acid 20 mg D-(+)-biotin 200 mg Nicotinic acid 100 mg Calcium pantothenate 300 mg Pyridoxine hydrochloride 200 mg Thiamine-HCl x 2 H2O 1000 ml Distilled water