# J198: JCM MEDIUM No. 198 ## Main sol. J198 0.75 g KH2PO4 0.75 g K2HPO4 0.36 g MnCl2 x 4 H2O 1 g NH4Cl 4 g NaHCO3 9 ml **Trace mineral solution** 10 ml **Trace vitamins** 0.5 g Na2S x 9 H2O 0.5 g L-Cysteine HCl x H2O 1 mg Resazurin 1000 ml Distilled water 1. Mix ingredients, except NaHCO3, Trace vitamins, Na2S x 9H2O and L-cysteine x HCl x H2O, and autoclave under a H2-CO2 (80:20) atmosphere. Filter-sterilize NaHCO3 as 8% solution and Trace vitamins while gassing with N2. Separately autoclave Na2S x 9H2O and L-cysteine x HCl x H2O as 5% solutions under a N2 atmosphere. Aseptically and anaerobically add the NaHCO3 solution and Trace vitamins to the medium under a H2-CO2 (80:20) atmosphere. Prior to inoculation, add sterile the Na2S x 9H2O and L-cysteine x HCl solutions, and adjust pH to 7.4. Pressurize inoculated bottles to 200 kPa H2-CO2 (80:20). ## Trace mineral solution 4.5 g Nitrilotriacetic acid 0.4 g FeCl2 x 4 H2O 0.1 g MnCl2 x 4 H2O 0.17 g CoCl2 x 6 H2O 0.1 g ZnCl2 0.02 g CaCl2 x 2 H2O 0.019 g H3BO3 0.01 g Na2MoO4 x 2 H2O 0.03 g NiCl2 x 6 H2O 1000 ml Distilled water 1. Adjust pH to 7.0 with 1 N KOH. ## 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