Final pH: 6.8 Final volume: 1000 ml dehydrated Wilkins-Chalgren medium (Oxoid CM0643) 33.00 g Sodium resazurin (0.1% w/v) 0.50 ml L-Cysteine HCl 0.30 g D-Glucose 5.00 g Na2CO3 1.00 g Distilled water 1000.00 ml 1. Dissolve 33.0 g/l dehydrated Wilkins-Chalgren medium (Oxoid CM0643) in distilled water and add 0.5 ml/l Na-resazurin solution (0.1% w/v), bring to the boil and cool to room temperature while sparging with 80% N2 and 20% CO2 gas mixture. Add L-cysteine-HCL x H2O (0.3 g/l), dispense the medium into anoxic Hungate-type tubes or serum vials and autoclave. After autoclaving supplement medium with 5.0 g/l D-glucose added from a sterile anoxic stock solution sterilized by filtration. Adjust pH of medium to 6.8 with a sterile anoxic stock solution of Na2CO3 (5% w/v) prepared under 80% N2 and 20% CO2 gas atmosphere. 2. Note: In case resazurin is not colourless addition of 10 - 20 mg sodium dithionite per liter (e.g. from 5% (w/v) solution, freshly prepared under N2 and filter-sterilized) can be used to completely reduce the medium. For DSM 14428, DSM 100335: Replace D-glucose with 5.0 g/l cellobiose prepared under 100% N2 gas atmosphere and sterilized by filtration. For DSM 14924, DSM 22607: Adjust pH of medium after autoclaving to 7.4 using a sterile anoxic stock solution of Na2CO3 (5% w/v) prepared under 80% N2 and 20% CO2 gas atmosphere. For DSM 15176, DSM 15567: Omit D-glucose. For DSM 15248: Adjust pH of medium to 6.0. For DSM 23669: Supplement medium after autoclaving with 0.2 g/l dithiothreitol (DTT) added from a filter-sterilized anoxic stock solution and adjust pH of complete medium to 7.2. For DSM 28816: Replace D-glucose with 3.2 g/l Na2-fumarate prepared under 100% N2 gas and sterilized by filtration. Adjust pH of complete medium to 7.2. For DSM 117481: Instead of Na-resazurin add 0,002 ml/ml Phenosafranin (Stock solution 1,25 mg/ml in H2O). After heating add 0,010 ml/ml Cystein-DTT (DL-Dithiothreitol)-solution (0,05 g/ml Cystein, 0,02 g/ml DTT). For DSM 118555: Instead of Na-resazurin add 0,002 ml/ml Phenosafranin (Stock solution 1,25 mg/ml in H2O). After heating add 0,010 ml/ml Cystein-DTT (DL-Dithiothreitol)-solution(0,05 g/ml Cystein, 0,02 g/ml DTT).
