# 525a: METHANOHALOPHILUS (MG+NaCl) MEDIUM ## Main sol. 525a 0.34 g KCl 2.75 g MgCl2 x 6 H2O 3.45 g MgSO4 x 7 H2O 0.25 g NH4Cl 0.14 g K2HPO4 x 3 H2O 0.14 g CaCl2 x 2 H2O 150 g NaCl 10 ml **Modified Wolin's mineral solution** 1 g Na-acetate 0.5 ml Sodium resazurin (0.1% w/v) 4 g NaHCO3 0.5 g Na2CO3 5 g Trimethylamine-HCl 1 ml **Wolin's vitamin solution (10x)** 0.5 g L-Cysteine HCl x H2O 0.5 g Na2S x 9 H2O 1000 ml Distilled water 1. Dissolve ingredients except carbonates, trimethylamine, vitamins, cysteine and sulfide, then sparge medium with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Dispense medium under same gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. Complete medium by adding sterile anoxic stock solutions of trimethylamine, vitamins (sterilized by filtration), cysteine and sulfide prepared under 100% N2 gas atmosphere and bicarbonate and carbonate from sterile anoxic stock solutions prepared under 80% N2 and 20% CO2 gas mixture. Adjust pH of complete medium to 6.9 - 7.0, if necessary. ## Modified Wolin's mineral solution 1.5 g Nitrilotriacetic acid 3 g MgSO4 x 7 H2O 0.5 g MnSO4 x H2O 1 g NaCl 0.1 g FeSO4 x 7 H2O 0.18 g CoSO4 x 7 H2O 0.1 g CaCl2 x 2 H2O 0.18 g ZnSO4 x 7 H2O 0.01 g CuSO4 x 5 H2O 0.02 g AlK(SO4)2 x 12 H2O 0.01 g H3BO3 0.01 g Na2MoO4 x 2 H2O 0.03 g NiCl2 x 6 H2O 0.3 mg Na2SeO3 x 5 H2O 0.4 mg Na2WO4 x 2 H2O 1000 ml Distilled water 1. First dissolve nitrilotriacetic acid and adjust pH to 6.5 with KOH, then add minerals. Adjust final to pH 7.0 with KOH. ## Wolin's vitamin solution (10x) 20 mg Biotin 20 mg Folic acid 100 mg Pyridoxine hydrochloride 50 mg Thiamine HCl 50 mg Riboflavin 50 mg Nicotinic acid 50 mg Calcium D-(+)-pantothenate 1 mg Vitamin B12 50 mg p-Aminobenzoic acid 50 mg (DL)-alpha-Lipoic acid 1000 ml Distilled water