Final pH: 6.8 - 7.0 Final volume: 1013 ml KCl 0.34 g MgCl2 x 6 H2O 4.00 g MgSO4 x 7 H2O 3.45 g NH4Cl 0.25 g CaCl2 x 2 H2O 0.14 g K2HPO4 0.14 g NaCl 18.00 g Modified Wolin's mineral solution 10.00 ml Fe(NH4)2(SO4)2 x 6 H2O (0.1% w/v) 2.00 ml Na-acetate 1.00 g Yeast extract (OXOID) 2.00 g Trypticase peptone (BD BBL) 2.00 g Sodium resazurin (0.1% w/v) 0.50 ml Wolin's vitamin solution 10.00 ml NaHCO3 5.00 g L-Cysteine HCl x H2O 0.50 g Na2S x 9 H2O 0.50 g Distilled water 1000.00 ml 1. Note: If the medium is being used without overpressure then adjust pH with a small amount of sterile anoxic 1 N HCl, if necessary. 2. 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 6.5, then dispense medium under 80% H2 and 20% CO2 gas atmosphere into anoxic Hungate-type tubes or serum vials to 30% of their volume 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. 3. For incubation use sterile 80% H2 and 20% CO2 gas mixture at two atmospheres of pressure. For DSM 1498, DSM 22353: Adjust pH to 6.5. For DSM 2373: Increase the amount of trypticase to 6.00 g/l. For DSM 4103: Supplement medium with 10 ml of a methanol solution (50% v/v) added to the autoclaved medium from a sterile anoxic stock solution. For DSM 4254: Add a filter-sterilized, anoxic solution of L-histidine to a final concentration of 80.00 mg/l. For DSM 7268, DSM 7466, DSM 14266: Use only one atmosphere overpressure of sterile 80% H2 and 20% CO2 gas mixture. For DSM 15219, DSM 18860, DSM 21220: Adjust pH to 7.5. For DSM 15558: Supplement medium after autoclaving with 0.50 g/l coenzyme M (2-mercaptoethanesulfonic acid) added from a filter-sterilized anoxic stock solution prepared under 100% N2 gas. Adjust pH to 6.5 and use only one atmosphere overpressure of sterile 80% H2 and 20% CO2 gas mixture. For DSM 16458: Supplement medium after autoclaving with 0.50 g/l coenzyme M (2-mercaptoethanesulfonic acid) added from a filter-sterilized anoxic stock solution prepared under 100% N2 gas. Adjust pH to 7.5 and use only one atmosphere overpressure of sterile 80% H2 and 20% CO2 gas mixture. For DSM 22026: Adjust pH to 7.5 - 8.0. Modified Wolin's mineral solution (from medium 141) 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 0.10 g CoSO4 x 7 H2O 0.18 g CaCl2 x 2 H2O 0.10 g ZnSO4 x 7 H2O 0.18 g CuSO4 x 5 H2O 0.01 g AlK(SO4)2 x 12 H2O 0.02 g H3BO3 0.01 g Na2MoO4 x 2 H2O 0.01 g NiCl2 x 6 H2O 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. Wolin's vitamin solution (from medium 141) Biotin 2.00 mg Folic acid 2.00 mg Pyridoxine hydrochloride 10.00 mg Thiamine HCl 5.00 mg Riboflavin 5.00 mg Nicotinic acid 5.00 mg Calcium D-(+)-pantothenate 5.00 mg Vitamin B12 0.10 mg p-Aminobenzoic acid 5.00 mg (DL)-alpha-Lipoic acid 5.00 mg Distilled water 1000.00 ml
