# J1033: ST65 MEDIUM ## Main sol. J1033 1. Mix components, except sulfur, bring to a boil and cool down under a CO2 (100%) gas stream. While gassing, add the following components to the medium: 18 g NaCl 4.33 g MgCl2 x 6 H2O 0.33 g NH4Cl 0.33 g KCl 0.165 g KH2PO4 0.033 g CaCl2 x 2 H2O 10 ml **Trace elements solution** 5 g Sulfur (powder) 1000 ml Distilled water 10 ml **Trace vitamins** 2 g NaHCO3 2. Bubble CO2 for 3 - 4 min and check pH to be 6.5 - 6.8. Readjust pH to the range if necessary. Autoclave under a CO2 gas atmosphere. Add the following solution to the medium (separately autoclaved under N2): 10 ml KNO3 (1 M) 3. Steam sulfur for 3 hr on each of 3 successive days. Aseptically and anaerobically distribute the medium into culture vessels containing appropriate amount of sulfur (e.g., 10 ml of the medium and 0.05 g sulfur in Hungate tubes) under a CO2 stream and seal with butyl rubber stoppers. ## Trace elements solution 78.4 mg Fe(NH4)2(SO4)2 x 6 H2O 23.8 mg CoCl2 x 6 H2O 16.6 mg NiCl2 x 6 H2O 2.42 mg Na2MoO4 x 2 H2O 5.49 mg Na2WO4 x 2 H2O 14.4 mg ZnSO4 x 7 H2O 0.17 mg CuCl2 x 2 H2O 9.45 mg Na2SeO4 0.62 mg H3BO3 19.8 mg MnCl2 x 4 H2O 0.92 mg SrSO4 0.5 mg CrK(SO4)2 x 12 H2O 2.41 mg AlCl3 x 6 H2O 2.08 mg BaCl2 14.2 mg Na2SiO3 x 9 H2O 11.9 mg KBr 16.6 mg KI 71 mg Na2SO4 1000 ml Distilled water ## Trace vitamins 2 mg Biotin 2 mg Folic acid 10 mg Pyridoxine hydrochloride 5 mg Thiamine HCl 5 mg Riboflavin 5 mg Nicotinic acid 5 mg Calcium pantothenate 0.1 mg Vitamin B12 5 mg p-Aminobenzoic acid 5 mg Lipoic acid 1000 ml Distilled water