141.METHANOGENIUMMEDIUM(H2CO2).PDFVIP

141. METHANOGENIUM MEDIUM (H2/CO2) KCl MgCl2 x 6 H2O MgSO4 x 7 H2O NH4Cl CaCl2 x 2 H2O K2HPO4 NaCl Trace element solution (see below) Fe(NH4)2(SO4)2 x 6 H2O solution (0.1% w/v) Na-acetate Yeast extract (OXOID) Trypticase peptone (BD BBL) Na-resazurin solution (0.1% w/v) NaHCO3 Vitamin solution (see below) L-Cysteine-HCl x H2O Na2S x 9 H2O Distilled water 0.34 4.00 3.45 0.25 0.14 0.14 18.00 10.00 2.00 1.00 2.00 2.00 0.50 5.00 10.00 0.50 0.50 1000.00 g g g g g g g ml ml g g g ml g ml g g ml Dissolve ingredients (except bicarbonate, vitamins, cysteine and sulfide), sparge medium with 80% H2 and 20% CO2 gas mixture for 30 – 45 min to make it anoxic. Add and dissolve bicarbonate and adjust pH to 7.0, then dispense medium under 80% H2 and 20% CO2 gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. After sterilization add cysteine and sulfide from sterile anoxic stock solutions autoclaved under 100% N2 gas. Vitamins are prepared under 100% N2 gas atmosphere and sterilized by filtration. Adjust pH of complete medium to 6.8 – 7.0, if necessary. For incubation use sterile 80% H2 and 20% CO2 gas mixture at two atmospheres of pressure. Note: If the medium is being used without overpressure then adjust pH with a small amount of sterile anoxic 1 N HCl, if necessary. Continued on next page ? 2014 DSMZ GmbH - All rights reserved Trace element solution: Nitrilotriacetic acid 1.50 g MgSO4 x 7 H2O 3.00 g MnSO4 x H2O 0.50 g NaCl 1.00 g FeSO4 x 7 H2O CoSO4 x 7 H2O 0.10 g 0.18 g CaCl2 x 2 H2O 0.10 g ZnSO4 x 7 H2O 0.18 g CuSO4 x 5 H2O 0.01 g KAl(SO4)2 x 12 H2O 0.02 g H3BO3 0.01 g Na2MoO4 x 2 H2O NiCl2 x 6 H2O 0.01 g 0.03 g Na2SeO3 x 5 H2O 0.30 mg Na2WO4 x 2 H2O 0.40 mg Distilled water 1000.00 ml First dissolve nitrilotriacetic acid and adjust pH to 6.5 with KOH, then add minerals. Adjust final to pH 7.0 with KOH. Vitamin solution: Biotin Folic acid Pyridoxine-HCl Thiamine-HCl x 2 H2O Riboflavin Nicotinic acid D-Ca-pantothenate Vitamin B12 p-Aminobenzoic acid Lipoic