# J425: A1 MARINE MEDIUM ## Main sol. J425 100 mM **Poorly crystalline iron(III) oxide** 20 g NaCl 0.77 g KCl 0.25 g NH4Cl 0.1 g KH2PO4 0.2 g MgSO4 x 7 H2O 10 ml **Trace vitamins** 10 ml **Trace mineral solution** 10 ml **Acetate solution 41.0 g/L** 20 ml **APM salts solution** 1. Add components except acetate and APM salts solutions to distilled water, bring volume to 1.0 L and adjust pH to 6.8. Distribute the medium into culture vessels under a N2-CO2 (4:1, v/v) gas mixture, sparge with the same gas mixture for 10 - 15 min, seal with butyl rubber stoppers and autoclave. Separately autoclave acetate and APM salts solutions under a N2 gas atmosphere and add to the medium anaerobically. ## Acetate solution 41.0 g/L 41 g Sodium acetate 1000 ml Distilled water ## APM salts solution 21.2 g MgCl2 x 6 H2O 3.04 g CaCl2 x 2 H2O 1000 ml Distilled water ## Poorly crystalline iron(III) oxide 1. Dissolve 108 g FeCl3 in 1.0 L distilled water. Adjust pH to 7.0 with 10 N NaOH. Allow the suspension to sit for 30 min, then readjust pH to 7.0. Centrifuge the suspension at 5,000 x g for 20 min and pour off supernatant. Wash the precipitate with distilled water by resuspending in distilled water followed by the centrifugation. Repeat the washing at least 5 times, then, resuspend in 1.0 L distilled water. Use the Fe(III) suspension as 0.67 M solution. More precisely, check the concentration of Cl- to be less than 1 mM and determine the concentration of Fe(III) by using hydroxylamine as described by Lovley and Phillips (Appl. Environ. Microbiol. 54, 751-757, 1986). ## 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 ## Trace mineral solution 1. Use Trace minerals of Medium No. 151 supplemented with 0.025 g (final) Na2MoO4 x 2H2O, 0.024 g NiCl2 x 6H2O and 0.025 g Na2WO4 x 2H2O (per liter). 2. Comment: For strain JCM 12378, inoculate with a 10% inoculum.