# 375: METHANOHALOBIUM MEDIUM ## Main sol. 375 250 g NaCl 0.33 g NH4Cl 0.33 g KH2PO4 0.33 g KCl 0.33 g CaCl2 x 2 H2O 0.33 g MgCl2 x 6 H2O 4 g MgSO4 x 7 H2O 0.5 ml Sodium resazurin (0.1% w/v) 1.5 g Na2CO3 1 ml **Trace element solution SL-10** 0.05 g Yeast extract (OXOID) 5 g Trimethylamine-HCl 0.5 g Na2S x 9 H2O 1000 ml Distilled water 1. Dissolve ingredients except carbonate, trace elements, yeast extract, trimethylamine and sulfide. Sparge medium with 80% N2 and 20% CO2 gas mixture for at least 30 - 45 min to make it anoxic, then dispense under same gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. Add trace elements, yeast extract, trimethylamine and sulfide from sterile anoxic stock solutions prepared under 100% N2 gas and carbonate from a sterile anoxic stock solution prepared under 80% N2 and 20% CO2 gas mixture. Adjust pH of complete medium to 7.4 with an anoxic sterile solution of 5% w/v Na2CO3, if necessary. 2. Note: A white precipitate that forms after autoclaving of the mineral base will dissolve again after storage at room temperature for 2 - 3 days. ## Trace element solution SL-10 10 ml HCl (25%) 1.5 g FeCl2 x 4 H2O 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 990 ml Distilled water 1. First dissolve FeCl2 in the HCl, then dilute in water, add and dissolve the other salts. Finally make up to 1000.00 ml.