# 1003: HYDROGEN-OXYDIZING MEDIUM ## Main sol. 1003 1000 ml Water 1. Place 1000 ml of anaerobic water (CO2 water) in a flask. Add the following compounds: 7 g MgSO4 x 7 H2O 2 g NaS2O3 0.4 g CaCl2 x 2 H2O 0.48 g KCl 0.78 g MgCl2 1.95 g MES 2. Add solutions A, B, and Trace mineral solution (see recipes below): 2 ml **Solution A (100x solution)** 1.5 ml **Solution B (500x solution)** 10 ml **Trace mineral solution (100x)** 3. pH the media to 6.0 and gas the flask containing the media with CO2 for 20 min at least. Gas first culture tube in rack and place 5 ml of media in previous tube. Place stopper on loaded culture tube, crimp tube cap onto stopper. Autoclave the medium for 20 min., 121°C. Add 1 ml of O2 to each tube before inoculation. After the inoculation the tubes are pressurized with H2 (138 KP). ## Solution A (100x solution) 100 g NH4Cl 100 g MgCl2 x H2O 40 g CaCl2 x 2 H2O 1000 ml Distilled water 1. Adjust the pH to 4 with HCl. ## Solution B (500x solution) 200 g K2HPO4 x 3 H2O 1000 ml Distilled water ## Trace mineral solution (100x) 500 mg Na-EDTA x 2 H2O 150 mg CoCl2 x 6 H2O 100 mg MnCl2 x 4 H2O 100 mg FeSO4 x 7 H2O 100 mg ZnCl2 40 mg AlCl3 x 6 H2O 30 mg Na-tungstate x 2 H2O 20 mg CuCl 20 mg NiSO4 x 6 H2O 10 mg Se-acid 10 mg HBO3 10 mg Na2MoO4 x 2 H2O 1000 ml Distilled water 1. Adjust the pH of the solution to 3.0 with HCl.