# J1095: BICARBONATE BUFFERED MEDIUM ## Main sol. J1095 0.53 g Na2HPO4 x 2 H2O 0.41 g KH2PO4 0.3 g NH4Cl 0.3 g NaCl 1 ml **FeCl2 solution** 1 ml **Trace element solution** 0.5 ml **Selenite-tungstate solution** 0.1 g Yeast extract 0.5 mg Resazurin 930 ml Distilled water 1. Mix components and autoclave under a N2-CO2 (4:1, v/v) gas mixture. After cooling, add the following solutions (autoclaved or *filter-sterilized): 1 ml MgCl2 x 6 H2O (10%) 1 ml CaCl2 x 2 H2O (10%) 10 ml **Trace vitamins** (*) 50 ml NaHCO3 (*, 8%) 10 ml **Substrate Solution** 2. Aseptically and anaerobically distribute the medium into culture vessels (e.g., 50 ml of the medium in 120 ml serum bottle) under a N2-CO2 (4:1, v/v) gas mixture, and seal with butyl rubber stoppers. Prior to use, add per liter the following solution (autoclaved and stored under a N2 atmosphere): 10 ml Na2S x 9 H2O (5%) ## Substrate Solution 1. For strain JCM 30818, 1 M glycerin solution; 2. For strain JCM 31247, 1 M trisodium citrate solution; 3. For strain JCM 31248, 1 M maltose solution. ## FeCl2 solution 10 ml HCl (7.7 M, 25%) 1.5 g FeCl2 x 4 H2O 990 ml Distilled water ## Trace element solution 70 mg ZnCl2 100 mg MnCl2 x 4 H2O 6 mg H3BO3 190 mg CoCl2 x 6 H2O 2 mg CuCl2 x 2 H2O 24 mg NiCl2 x 6 H2O 36 mg Na2MoO4 x 2 H2O 1000 ml Distilled water ## Selenite-tungstate solution 0.4 g NaOH 6 mg Na2SeO3 x 5 H2O 8 mg Na2WO4 x 2 H2O 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