Handbook of Microbiological Media, Fourth Edition part 110 pptx

Methanobacterium II Medium 1085 use inject, for each 5.0mL medium, 0.2mL sterile NaHCO 3 solution, 0.05mL sterile Na 2 S·9H 2 O solution, and 0.05mL sterile methanol so- lution. The pH should be 7.0. Use: For the cultivation of Methanosarcina barkeri DSM 10131 and Methanosarcina mazei=Methanococcus mazei (Methanosarcina fri- sia) DSM 10132. Methanobacterium II Medium (DSMZ Medium 825) Composition per 1065.0mL: Yeast extract 1.0g NH 4 Cl 1.0g NaCl 0.6g Cysteine-HCl·H 2 O 0.5g Sodium acetate 0.5g K 2 HPO 4 0.3g KH 2 PO 4 0.3g MgCl 2 ·6H 2 O 0.2g CaCl 2 ·2H 2 O 0.1g KCl 0.1g Resazurin 0.5mg NaHCO 3 solution 40.0mL Na-formate solution 15.0mL Trace elements solution 10.0mL Vitamin solution 10.0mL Na 2 S·9H 2 O solution 10.0mL pH 7.0 ± 0.2 at 25°C Na-Formate Solution: Composition per 10.0mL: Na-formate 5.0g Preparation of Na-Formate Solution: Add Na-formate to dis- tilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N 2 . Autoclave for 15 min at 15 psi pressure–121°C. Trace Elements Solution: Composition per liter: MgSO 4 ·7H 2 O 3.0g Nitrilotriacetic acid 1.5g NaCl 1.0g MnSO 4 ·2H 2 O 0.5g CoSO 4 ·7H 2 O 0.18g ZnSO 4 ·7H 2 O 0.18g CaCl 2 ·2H 2 O 0.1g FeSO 4 ·7H 2 O 0.1g NiCl 2 ·6H 2 O 0.025g KAl(SO 4 ) 2 ·12H 2 O 0.02g H 3 BO 3 0.01g Na 2 MoO 4 ·4H 2 O 0.01g CuSO 4 ·5H 2 O 0.01g Na 2 SeO 3 ·5H 2 O 0.3mg Preparation of Trace Elements Solution: Add nitrilotriacetic acid to 500.0mL of distilled/deionized water. Dissolve by adjusting pH to 6.5 with KOH. Add remaining components. Add distilled/deionized water to 1.0L. Mix thoroughly. Vitamin Solution: Composition per liter: Pyridoxine-HCl 10.0mg Thiamine-HCl·2H 2 O 5.0mg Riboflavin 5.0mg Nicotinic acid 5.0mg D-Ca-pantothenate 5.0mg p-Aminobenzoic acid 5.0mg Lipoic acid 5.0mg Biotin 2.0mg Folic acid 2.0mg Vitamin B 12 0.1mg Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 80% H 2 + 20% CO 2 . Filter sterilize. Na 2 S·9H 2 O Solution: Composition per 10.0mL: Na 2 S·9H 2 O 0.3g Preparation of Na 2 S·9H 2 O Solution: Add Na 2 S·9H 2 O to dis- tilled/deionized water and bring volume to 10.0mL. Sparge with N 2 . Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Store an- aerobically. NaHCO 3 Solution: Composition per 100.0mL: NaHCO 3 10.0g Preparation of NaHCO 3 Solution: Add NaHCO 3 to distilled/de- ionized water and bring volume to 100.0mL. Mix thoroughly. Auto- clave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Must be prepared freshly. Preparation of Medium: Prepare and dispense medium under 80% N 2 + 20% CO 2 gas mixture. Add components, except Na-formate solu- tion, NaHCO 3 solution, and Na 2 S·9H 2 O solution, to 1.0L distilled/de- ionized water. Mix thoroughly. Sparge with 80% N 2 + 20% CO 2 . Dispense 5.0mL aliquots into Hungate tubes under 80% H 2 + 20% CO 2 gas mixture. Autoclave for 15 min at 15 psi pressure–121°C. Prior to use inject, for each 5.0mL medium, 0.2mL sterile NaHCO 3 solution, 0.05mL sterile Na 2 S·9H 2 O solution, and 0.075mL sterile Na-formate solution. The pH should be 7.0. Use: For the cultivation of Methanobacterium formicicum DSM 10111. Methanobacterium II Medium (DSMZ Medium 825) Composition per 1075.0mL: Yeast extract 1.0g NH 4 Cl 1.0g NaCl 0.6g Cysteine-HCl·H 2 O 0.5g Sodium acetate 0.5g K 2 HPO 4 0.3g KH 2 PO 4 0.3g MgCl 2 ·6H 2 O 0.2g CaCl 2 ·2H 2 O 0.1g KCl 0.1g Resazurin 0.5mg NaHCO 3 solution 40.0mL Na-formate solution 15.0mL Trace elements solution 10.0mL Vitamin solution 10.0mL Na 2 S·9H 2 O solution 10.0mL Trypticase™ solution 10.0mL pH 7.0 ± 0.2 at 25°C Trypticase™ Solution: Composition per 10.0mL: Trypticase™ 1.0g © 2010 by Taylor and Francis Group, LLC 1086 Methanobrevibacter curvatus Medium Preparation of Trypticase™ Solution: Add Trypticase™ to dis- tilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave under 100% N 2 for 15 min at 15 psi pressure–121°C. Cool to room temperature. Na-Formate Solution: Composition per 10.0mL: Na-formate 5.0g Preparation of Na-Formate Solution: Add Na-formate to dis- tilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N 2 . Autoclave for 15 min at 15 psi pressure–121°C. Trace Elements Solution: Composition per liter: MgSO 4 ·7H 2 O 3.0g Nitrilotriacetic acid 1.5g NaCl 1.0g MnSO 4 ·2H 2 O 0.5g CoSO 4 ·7H 2 O 0.18g ZnSO 4 ·7H 2 O 0.18g CaCl 2 ·2H 2 O 0.1g FeSO 4 ·7H 2 O 0.1g NiCl 2 ·6H 2 O 0.025g KAl(SO 4 ) 2 ·12H 2 O 0.02g H 3 BO 3 0.01g Na 2 MoO 4 ·4H 2 O 0.01g CuSO 4 ·5H 2 O 0.01g Na 2 SeO 3 ·5H 2 O 0.3mg Preparation of Trace Elements Solution: Add nitrilotriacetic acid to 500.0mL of distilled/deionized water. Dissolve by adjusting pH to 6.5 with KOH. Add remaining components. Add distilled/deionized water to 1.0L. Mix thoroughly. Vitamin Solution: Composition per liter: Pyridoxine-HCl 10.0mg Thiamine-HCl·2H 2 O 5.0mg Riboflavin 5.0mg Nicotinic acid 5.0mg D-Ca-pantothenate 5.0mg p-Aminobenzoic acid 5.0mg Lipoic acid 5.0mg Biotin 2.0mg Folic acid 2.0mg Vitamin B 12 0.1mg Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 80% H 2 + 20% CO 2 . Filter sterilize. Na 2 S·9H 2 O Solution: Composition per 10.0mL: Na 2 S·9H 2 O 0.3g Preparation of Na 2 S·9H 2 O Solution: Add Na 2 S·9H 2 O to dis- tilled/deionized water and bring volume to 10.0mL. Sparge with N 2 . Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Store an- aerobically. NaHCO 3 Solution: Composition per 100.0mL: NaHCO 3 10.0g Preparation of NaHCO 3 Solution: Add NaHCO 3 to distilled/de- ionized water and bring volume to 100.0mL. Mix thoroughly. Auto- clave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Must be prepared freshly. Preparation of Medium: Prepare and dispense medium under 80% N 2 + 20% CO 2 gas mixture. Add components, except Trypticase™ so- lution, Na-formate solution, NaHCO 3 solution, and Na 2 S·9H 2 O solu- tion, to 1.0L distilled/deionized water. Mix thoroughly. Sparge with 80% N 2 + 20% CO 2 . Dispense 5.0mL aliquots into Hungate tubes un- der 80% H 2 + 20% CO 2 gas mixture. Autoclave for 15 min at 15 psi pressure–121°C. Prior to use inject, for each 5.0mL medium, 0.2mL sterile NaHCO 3 solution, 0.05mL sterile Trypticase™ solution, 0.05mL sterile Na 2 S·9H 2 O solution, and 0.075mL sterile Na-formate solution. The pH should be 7.0. Use: For the cultivation of Methanobacterium oryzae DSM 11106. Methanobrevibacter curvatus Medium (DSMZ Medium 734) Composition per 1010.0mL: NaCl 1.0g KCl 0.5g Casamino acids 0.5g Yeast extract 0.5g MgCl 2 ·6H 2 O 0.4g NH 4 Cl 0.3g KH 2 PO 4 0.2g Na 2 SO 4 0.15g CaCl 2 ·2H 2 O 0.1g Resazurin 0.5mg NaHCO 3 solution 40.0mL Dithionite solution 10.0mL Trace elements solution SL-10 1.0mL Selenite-tungstate solution 1.0mL Seven vitamin solution 1.0mL pH 7.4 ± 0.2 at 25°C NaHCO 3 Solution: Composition per 40.0mL: NaHCO 3 5.8g Preparation of NaHCO 3 Solution: Add NaHCO 3 to distilled/de- ionized water and bring volume to 40.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Must be prepared freshly. Selenite-Tungstate Solution Composition per liter: NaOH 0.5g Na 2 WO 4 ·2H 2 O 4.0mg Na 2 SeO 3 ·5H 2 O 3.0mg Preparation of Selenite-Tungstate Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 100% N 2 . Filter sterilize. Dithionite Solution Composition per 10.0mL: Na-dithionite 2.0mg Preparation of Dithionite Solution: Add Na-dithionite to dis- tilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N 2 . Filter sterilize. Trace Elements Solution SL-10: Composition per liter: FeCl 2 ·4H 2 O 1.5g CoCl 2 ·6H 2 O 190.0mg © 2010 by Taylor and Francis Group, LLC Methanobrevibacter curvatus Medium 1087 MnCl 2 ·4H 2 O 100.0mg ZnCl 2 70.0mg Na 2 MoO 4 ·2H 2 O 36.0mg NiCl 2 ·6H 2 O 24.0mg H 3 BO 3 6.0mg CuCl 2 ·2H 2 O 2.0mg HCl (25% solution) 10.0mL Preparation of Trace Elements Solution SL-10: Add FeCl 2 ·4H 2 O to 10.0mL of HCl solution. Mix thoroughly. Add distilled/deionized water and bring volume to 1.0L. Add remaining components. Mix thor- oughly. Sparge with 80% N 2 + 20% CO 2 . Autoclave for 15 min at 15 psi pressure–121°C. Seven Vitamin Solution: Composition per liter: Pyridoxine hydrochloride 300.0mg Thiamine-HCl·2H 2 O 200.0mg Nicotinic acid 200.0mg Vitamin B 12 100.0mg Calcium pantothenate 100.0mg p-Aminobenzoic acid 80.0mg D(+)-Biotin 20.0mg Preparation of Seven Vitamin Solution: Add components to dis- tilled/deionized water and bring volume to 1.0L. Sparge with 100% N 2 . Mix thoroughly. Filter sterilize. Preparation of Medium: Prepare and dispense medium under 80% H 2 + 20% CO 2 gas atmosphere. Add components, except NaHCO 3 solu- tion, seven vitamin solution, selenite-tungstate solution, and trace ele- ments solution SL-10, to distilled/deionized water and bring volume to 947.0mL. Mix thoroughly. Adjust pH to 7.6. Sparge with 80% H 2 + 20% CO 2 . Autoclave for 15 min at 15 psi pressure–121°C. Aseptically and an- aerobically add 40.0mL NaHCO 3 solution, 1.0mL selenite-tungstate solution, 1.0mL seven vitamin solution, and 1.0mL trace elements so- lution SL-10. Mix thoroughly. Aseptically and anaerobically distribute into sterile tubes or bottles. Adjust pH to 7.6. Prior to inoculation add di- thionite solution (0.1mL per 10mL medium) as reductant. Use: For the cultivation of Methanobrevibacter curvatus. Methanobrevibacter curvatus Medium (DSMZ Medium 734) Composition per 1022.0mL: NaCl 1.0g KCl 0.5g Casamino acids 0.5g Yeast extract 0.5g MgCl 2 ·6H 2 O 0.4g NH 4 Cl 0.3g KH 2 PO 4 0.2g Na 2 SO 4 0.15g CaCl 2 ·2H 2 O 0.1g Resazurin 0.5mg Rumen fluid, bovine, clarified 400mL NaHCO 3 solution 40.0mL Dithionite solution 10.0mL MOPS buffer 10.0mL Nutrient supplement solution 2.0mL Trace elements solution SL-10 1.0mL Selenite-tungstate solution 1.0mL Seven vitamin solution 1.0mL pH 7.2 ± 0.2 at 25°C NaHCO 3 Solution: Composition per 40.0mL: NaHCO 3 5.8g Preparation of NaHCO 3 Solution: Add NaHCO 3 to distilled/de- ionized water and bring volume to 40.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Must be prepared freshly. Selenite-Tungstate Solution: Composition per liter: NaOH 0.5g Na 2 WO 4 ·2H 2 O 4.0mg Na 2 SeO 3 ·5H 2 O 3.0mg Preparation of Selenite-Tungstate Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 100% N 2 . Filter sterilize. Dithionite Solution: Composition per 10.0mL: Na-dithionite 2.0mg Preparation of Dithionite Solution: Add Na-dithionite to dis- tilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N 2 . Filter sterilize. Trace Elements Solution SL-10: Composition per liter: FeCl 2 ·4H 2 O 1.5g CoCl 2 ·6H 2 O 190.0mg MnCl 2 ·4H 2 O 100.0mg ZnCl 2 70.0mg Na 2 MoO 4 ·2H 2 O 36.0mg NiCl 2 ·6H 2 O 24.0mg H 3 BO 3 6.0mg CuCl 2 ·2H 2 O 2.0mg HCl (25% solution) 10.0mL Preparation of Trace Elements Solution SL-10: Add FeCl 2 ·4H 2 O to 10.0mL of HCl solution. Mix thoroughly. Add distilled/deionized water and bring volume to 1.0L. Add remaining components. Mix thor- oughly. Sparge with 80% N 2 + 20% CO 2 . Autoclave for 15 min at 15 psi pressure–121°C. Seven Vitamin Solution: Composition per liter: Pyridoxine hydrochloride 300.0mg Thiamine-HCl·2H 2 O 200.0mg Nicotinic acid 200.0mg Vitamin B 12 100.0mg Calcium pantothenate 100.0mg p-Aminobenzoic acid 80.0mg D(+)-Biotin 20.0mg Preparation of Seven Vitamin Solution: Add components to dis- tilled/deionized water and bring volume to 1.0L. Sparge with 100% N 2 . Mix thoroughly. Filter sterilize. Nutrient Supplement Solution: Composition per liter: Pancreatic digest of gelatin 5.0g Beef extract 3.0g Preparation of Nutrient Supplement Solution: Add compo- nents to distilled/deionized water and bring volume to 1.0L. Mix thor- oughly. Sparge with 100% N 2 . Autoclave for 15 min at 15 psi pressure– 121°C. Cool to room tempterature. © 2010 by Taylor and Francis Group, LLC 1088 Methanobrevibacter curvatus Medium MOPS Buffer: Composition per 10.0mL: MOPS [3-(N-morpholino) propane sulfonic acid] 2.1g Na-acetate 0.3g EDTA 0.1g Preparation of MOPS Buffer: Add components to distilled/de- ionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N 2 . Adjust to pH 7.2. Filter sterilize. Preparation of Medium: Prepare and dispense medium under 80% H 2 + 20% CO 2 gas atmosphere. Add components, except clarified bo- vine rumen fluid, MOPS buffer, nutrient supplement solution, NaHCO 3 solution, seven vitamin solution, selenite-tungstate solution, and trace elements solution SL-10, to distilled/deionized water and bring volume to 547.0mL. Mix thoroughly. Adjust pH to 7.2. Sparge with 80% H 2 + 20% CO 2 . Autoclave for 15 min at 15 psi pressure–121°C. Aseptically and anaerobically add 400.0mL sterile clarified bovine rumen fluid, 10.0mL MOPS buffer, 2.0mL nutrient supplement solution, 40.0mL NaHCO 3 solution, 1.0mL selenite-tungstate solution, 1.0mL seven vi- tamin solution, and 1.0mL trace elements solution SL-10. Mix thor- oughly. Aseptically and anaerobically distribute into sterile tubes or bottles. Adjust pH to 7.2. Prior to inoculation add dithionite solution (0.1mL per 10mL medium) as reductant. Use: For the cultivation of Methanobrevibacter curvatus DSM 11111 (strain RFM-2). Methanobrevibacter curvatus Medium (DSMZ Medium 734) Composition per 1020.0mL: NaCl 1.0g KCl 0.5g Casamino acids 0.5g Yeast extract 0.5g MgCl 2 ·6H 2 O 0.4g NH 4 Cl 0.3g KH 2 PO 4 0.2g Na 2 SO 4 0.15g CaCl 2 ·2H 2 O 0.1g Resazurin 0.5mg Rumen fluid, bovine, clarified 200mL NaHCO 3 solution 40.0mL Dithionite solution 10.0mL MOPS buffer 10.0mL Trace elements solution SL-10 1.0mL Selenite-tungstate solution 1.0mL Seven vitamin solution 1.0mL pH 7.7 ± 0.2 at 25°C NaHCO 3 Solution: Composition per 40.0mL: NaHCO 3 5.8g Preparation of NaHCO 3 Solution: Add NaHCO 3 to distilled/de- ionized water and bring volume to 40.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Must be prepared freshly. Selenite-Tungstate Solution: Composition per liter: NaOH 0.5g Na 2 WO 4 ·2H 2 O 4.0mg Na 2 SeO 3 ·5H 2 O 3.0mg Preparation of Selenite-Tungstate Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 100% N 2 . Filter sterilize. Dithionite Solution: Composition per 10.0mL: Na-dithionite 2.0mg Preparation of Dithionite Solution: Add Na-dithionite to dis- tilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N 2 . Filter sterilize. Trace Elements Solution SL-10: Composition per liter: FeCl 2 ·4H 2 O 1.5g CoCl 2 ·6H 2 O 190.0mg MnCl 2 ·4H 2 O 100.0mg ZnCl 2 70.0mg Na 2 MoO 4 ·2H 2 O 36.0mg NiCl 2 ·6H 2 O 24.0mg H 3 BO 3 6.0mg CuCl 2 ·2H 2 O 2.0mg HCl (25% solution) 10.0mL Preparation of Trace Elements Solution SL-10: Add FeCl 2 ·4H 2 O to 10.0mL of HCl solution. Mix thoroughly. Add distilled/deionized water and bring volume to 1.0L. Add remaining components. Mix thor- oughly. Sparge with 80% N 2 + 20% CO 2 . Autoclave for 15 min at 15 psi pressure–121°C. Seven Vitamin Solution: Composition per liter: Pyridoxine hydrochloride 300.0mg Thiamine-HCl·2H 2 O 200.0mg Nicotinic acid 200.0mg Vitamin B 12 100.0mg Calcium pantothenate 100.0mg p-Aminobenzoic acid 80.0mg D(+)-Biotin 20.0mg Preparation of Seven Vitamin Solution: Add components to dis- tilled/deionized water and bring volume to 1.0L. Sparge with 100% N 2 . Mix thoroughly. Filter sterilize. MOPS Buffer: Composition per 10.0mL: MOPS [3-(N-morpholino) propane sulfonic acid] 2.1g Na-acetate 0.3g EDTA 0.1g Preparation of MOPS Buffer: Add components to distilled/de- ionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N 2 . Adjust to pH 7.7. Filter sterilize. Preparation of Medium: Prepare and dispense medium under 80% H 2 + 20% CO 2 gas atmosphere. Add components, except clarified bo- vine rumen fluid, MOPS buffer, NaHCO 3 solution, seven vitamin solu- tion, selenite-tungstate solution, and trace elements solution SL-10, to distilled/deionized water and bring volume to 747.0mL. Mix thoroughly. Adjust pH to 7.7. Sparge with 80% H 2 + 20% CO 2 . Autoclave for 15 min at 15 psi pressure–121°C. Aseptically and anaerobically add 200.0mL sterile clarified bovine rumen fluid, 10.0mL MOPS buffer, 240.0mL NaHCO 3 solution, 1.0mL selenite-tungstate solution, 1.0mL seven vi- tamin solution, and 1.0mL trace elements solution SL-10. Mix thor- oughly. Aseptically and anaerobically distribute into sterile tubes or bottles. Adjust pH to 7.7. Prior to inoculation add dithionite solution (0.1mL per 10mL medium) as reductant. © 2010 by Taylor and Francis Group, LLC Methanocalculus pumilus Medium 1089 Use: For the cultivation of Methanobrevibacter curvatus DSM 11111 DSM 11139 (strain RFM-1) Methanocalculus halotolerans Medium (DSMZ Medium 905) Composition per 1010.0mL: NaCl 50.0g NH 4 Cl 1.0g Na-acetate 0.5g Yeast extract 0.5g Trypticase™ 0.5g K 2 HPO 4 0.3g KH 2 PO 4 0.3g KCl 0.17g Resazurin 0.5mg Magnesium chloride solution 30.0mL NaHCO 3 solution 20.0mL Trace elements solution 10.0mL Calcium chloride solution 10.0mL Na 2 S·9H 2 O solution 10.0mL L-Cysteine solution 10.0mL pH 7.2-7.6 at 25°C Magnesium Chloride Solution: Composition per 30.0mL: MgCl 2 ·6H 2 O 3.2g Preparation of Magnesium Chloride Solution: Add MgCl 2 ·6H 2 O to distilled/deionized water and bring volume to 30.0mL. Mix thoroughly. Sparge with 100% N 2 . Autoclave for 15 min at 15 psi pressure–121°C. Calcium Chloride Solution: Composition per 10.0mL: CaCl 2 ·2H 2 O 0.6g Preparation of Calcium Chloride Solution: Add CaCl 2 ·2H 2 O to distilled/deionized water and bring volume to 10.0mL. Mix thor- oughly. Sparge with 100% N 2 . Autoclave for 15 min at 15 psi pressure– 121°C. L-Cysteine Solution: Composition per 10.0mL: L-Cysteine·HCl·H 2 O 0.5g Preparation of L-Cysteine Solution: Add L-cysteine·HCl·H 2 O to distilled/deionized water and bring volume to 10.0mL. Mix thorough- ly. Sparge with 100% N 2 . Autoclave for 15 min at 15 psi pressure– 121°C. Na 2 S·9H 2 O Solution: Composition per 10.0mL: Na 2 S·9H 2 O 0.3g Preparation of Na 2 S·9H 2 O Solution: Add Na 2 S·9H 2 O to dis- tilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N 2 . Autoclave for 15 min at 15 psi pressure–121°C. NaHCO 3 Solution: Composition per 20.0mL: NaHCO 3 2.0g Preparation of NaHCO 3 Solution: Add NaHCO 3 to distilled/de- ionized water and bring volume to 20.0mL. Mix thoroughly. Sparge with 80% N 2 + 20% CO 2 . Autoclave for 15 min at 15 psi pressure– 121°C. Cool to 25°C. Must be prepared freshly. Trace Elements Solution: Composition per liter: MgSO 4 ·7H 2 O 3.0g Nitrilotriacetic acid 1.5g NaCl 1.0g MnSO 4 ·2H 2 O 0.5g CoSO 4 ·7H 2 O 0.18g ZnSO 4 ·7H 2 O 0.18g CaCl 2 ·2H 2 O 0.1g FeSO 4 ·7H 2 O 0.1g NiCl 2 ·6H 2 O 0.025g KAl(SO 4 ) 2 ·12H 2 O 0.02g H 3 BO 3 0.01g Na 2 MoO 4 ·4H 2 O 0.01g CuSO 4 ·5H 2 O 0.01g Na 2 SeO 3 ·5H 2 O 0.3mg Preparation of Trace Elements Solution: Add nitrilotriacetic acid to 500.0mL of distilled/deionized water. Dissolve by adjusting pH to 6.5 with KOH. Add remaining components. Add distilled/deionized water to 1.0L. Mix thoroughly. Preparation of Medium: Prepare and dispense medium under sparge with 80% N 2 + 20% CO 2 . Add components, except NaHCO 3 so- lution, magnesium chloride solution, calcium chloride solution, L- cysteine-HCl·H 2 O solution, and Na 2 S·9H 2 O solution, to distilled/de- ionized water and bring volume to 950.0mL. Mix thoroughly. Gently heat and bring to boiling. Boil for 3 min. Cool to room temperature while sparging with 80% N 2 + 20% CO 2 . Add 10.0mL L-cysteine-HCl·H 2 O solution and 20.0mL NaHCO 3 solution. Mix thoroughly. Adjust pH to 7.5. Distribute into anaerobic tubes or bottles. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically and anaerobically add per liter, 30.0mL magnesium chloride solution, 10.0mL calcium chloride solu- tion, and 10.0mL Na 2 S·9H 2 O. Mix thoroughly. After inoculation pres- surize vessels with 80% H 2 + 20% CO 2 gas mixture to 1 bar overpressure and add sulfide from a sterile, anaerobic stock solution. The final pH of the medium should be 7.2–7.6. Use: For the cultivation of Methanocalculus halotolerans. Methanocalculus pumilus Medium (DSMZ Medium 892) Composition per 1080.0mL: NaCl 10.0g Yeast extract 2.0g Trypticase™ 2.0g NH 4 Cl 0.9g K 2 HPO 4 0.4g MgCl 2 ·6H 2 O 0.36g Resazurin 0.5mg Na 2 CO 3 solution 50.0mL Na 2 S·9H 2 O solution 15.0mL L-Cysteine-HCl·H 2 O solution 15.0mL Vitamin solution 10.0mL Trace elements solution 10.0mL L-Cysteine Solution: Composition per 15.0mL: L-Cysteine·HCl·H 2 O 0.5g Preparation of L-Cysteine Solution: Add L-cysteine·HCl·H 2 O to distilled/deionized water and bring volume to 15.0mL. Mix thorough- ly. Sparge with 100% N 2 . Autoclave for 15 min at 15 psi pressure– 121°C. © 2010 by Taylor and Francis Group, LLC 1090 Methanococcoides Medium Na 2 CO 3 Solution: Composition per 50.0mL: Na 2 CO 3 5.0g Preparation of Na 2 CO 3 Solution: Add Na 2 CO 3 to distilled/de- ionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 80% N 2 + 20% CO 2 . Filter sterilize. Na 2 S·9H 2 O Solution: Composition per 15mL: Na 2 S·9H 2 O 0.5g Preparation of Na 2 S·9H 2 O Solution: Add Na 2 S·9H 2 O to dis- tilled/deionized water and bring volume to 15.0mL. Mix thoroughly. Autoclave under 100% N 2 for 15 min at 15 psi pressure–121°C. Cool to room temperature. Trace Elements Solution: Composition per liter: MgSO 4 ·7H 2 O 3.0g Nitrilotriacetic acid 1.5g NaCl 1.0g MnSO 4 ·2H 2 O 0.5g CoSO 4 ·7H 2 O 0.18g ZnSO 4 ·7H 2 O 0.18g CaCl 2 ·2H 2 O 0.1g FeSO 4 ·7H 2 O 0.1g NiCl 2 ·6H 2 O 0.025g KAl(SO 4 ) 2 ·12H 2 O 0.02g H 3 BO 3 0.01g Na 2 MoO 4 ·4H 2 O 0.01g CuSO 4 ·5H 2 O 0.01g Na 2 SeO 3 ·5H 2 O 0.3mg Preparation of Trace Elements Solution: Add nitrilotriacetic acid to 500.0mL of distilled/deionized water. Dissolve by adjusting pH to 6.5 with KOH. Add remaining components. Add distilled/deionized water to 1.0L. Mix thoroughly. Vitamin Solution: Composition per liter: Pyridoxine-HCl 10.0mg Thiamine-HCl·2H 2 O 5.0mg Riboflavin 5.0mg Nicotinic acid 5.0mg D-Ca-pantothenate 5.0mg p-Aminobenzoic acid 5.0mg Lipoic acid 5.0mg Biotin 2.0mg Folic acid 2.0mg Vitamin B 12 0.1mg Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 80% H 2 + 20% CO 2 . Filter sterilize. Preparation of Medium: Add components, except Na 2 CO 3 solu- tion, Na 2 S·9H 2 O solution, and L-cysteine solution, to distilled/deion- ized water and bring volume to 1.0L. Mix thoroughly. Sparge with 100% N 2 for 30 min. Autoclave for 15 min at 15 psi pressure–121°C. Asepti- cally distribute 5.0mL aliquots into Hungate tubes under 100% N 2 . Aseptically and anaerobically add per 5.0mL medium 0.25mL Na 2 CO 3 solution, 0.075mL Na 2 S·9H 2 O solution, and 0.075mL L-cysteine solu- tion. Mix thoroughly. Replace N 2 atmosphere with atmosphere of 80% H 2 + 20% CO 2 . Repeat atmosphere replacement several times with overpressurization. The initial pH of 9.0 will decrease over a 30 min period to 7.3–7.5. After inoculation use atmosphere of 80% H 2 + 20% CO 2 to 1.5 bar overpressure. Use: For the cultivation of Methanocalculus pumilus. Methanococcal Complex Medium See: Methanococcus McC Medium Methanococcoides Medium Composition per liter: NaCl 18.0g NaHCO 3 5.0g MgCl 2 ·6H 2 O 4.0g MgSO 4 ·7H 2 O 3.45g Trimethylamine·HCl 3.0g Trypticase™ 2.0g Yeast extract 2.0g Sodium acetate 1.0g L-Cysteine·HCl 0.5g Na 2 S·9H 2 O 0.5g KCl 0.335g NH 4 Cl 0.25g CaCl 2 ·2H 2 O 0.14g K 2 HPO 4 0.14g Fe(NH 4 ) 2 (SO 4 ) 2 ·7H 2 O 2.0mg Resazurin 1.0mg Trace elements solution 10.0mL Vitamin solution 10.0mL pH 7.0 ± 0.2 at 25°C Trace Elements Solution: Composition per liter: MgSO 4 ·7H 2 O 3.0g Nitrilotriacetic acid 1.5g NaCl 1.0g MnSO 4 ·2H 2 O 0.5g CoSO 4 ·7H 2 O 0.18g ZnSO 4 ·7H 2 O 0.18g CaCl 2 ·2H 2 O 0.1g FeSO 4 ·7H 2 O 0.1g NiCl 2 ·6H 2 O 0.025g KAl(SO 4 ) 2 ·12H 2 O 0.02g CuSO 4 ·5H 2 O 0.01g H 3 BO 3 0.01g Na 2 MoO 4 ·2H 2 O 0.01g Na 2 SeO 3 ·5H 2 O 0.3mg Preparation of Trace Elements Solution: Add nitrilotriacetic acid to 500.0mL of distilled/deionized water. Adjust pH to 6.5 with KOH. Add remaining components. Adjust pH to 7.0. Add distilled/de- ionized water to 1.0L. Vitamin Solution: Composition per liter: Pyridoxine·HCl 10.0mg Calcium DL-pantothenate 5.0mg Lipoic acid 5.0mg Nicotinic acid 5.0mg p-Aminobenzoic acid 5.0mg Riboflavin 5.0mg Thiamine·HCl 5.0mg Biotin 2.0mg Folic acid 2.0mg Vitamin B 12 0.1mg © 2010 by Taylor and Francis Group, LLC Methanococcus jannaschii Medium 1091 Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Adjust pH to 7.0. Mix thor- oughly. Preparation of Medium: Prepare the medium anaerobically under 80% H 2 + 20% CO 2 . Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 80% H 2 + 20% CO 2 . Autoclave for 15 min at 15 psi pressure–121°C. Use: For the cultivation and maintenance of Methanococcoides meth- ylutens. Methanococcus deltae Medium Composition per liter: NaCl 35.0g NaHCO 3 5.0g MgCl 2 ·6H 2 O 4.0g NH 4 Cl 2.7g Sodium acetate 2.5g L-Cysteine·HCl 0.3g K 2 HPO 4 0.3g KH 2 PO 4 0.3g Na 2 S·9H 2 O 0.3g MgSO 4 ·7H 2 O 0.13g Resazurin 1.0mg (NH 4 ) 2 SO 4 0.3mg Trace elements solution 10.0mL Vitamin solution 10.0mL L-Cysteine·HCl solution 10.0mL Na 2 S·9H 2 O solution 10.0mL pH 6.9 ± 0.2 at 25°C Trace Elements Solution: Composition per liter: MgSO 4 ·7H 2 O 3.0g Nitrilotriacetic acid 1.5g NaCl 1.0g MnSO 4 ·2H 2 O 0.5g CoSO 4 ·7H 2 O 0.18g ZnSO 4 ·7H 2 O 0.18g CaCl 2 ·2H 2 O 0.1g FeSO 4 ·7H 2 O 0.1g NiCl 2 ·6H 2 O 0.025g KAl(SO 4 ) 2 ·12H 2 O 0.02g CuSO 4 ·5H 2 O 0.01g H 3 BO 3 0.01g Na 2 MoO 4 ·2H 2 O 0.01g Na 2 SeO 3 ·5H 2 O 0.3mg Preparation of Trace Elements Solution: Add nitrilotriacetic acid to 500.0mL of distilled/deionized water. Adjust pH to 6.5 with KOH. Add remaining components. Adjust pH to 7.0. Add distilled/de- ionized water to 1.0L. Vitamin Solution: Composition per liter: Pyridoxine·HCl 10.0mg Calcium DL-pantothenate 5.0mg Lipoic acid 5.0mg Nicotinic acid 5.0mg p-Aminobenzoic acid 5.0mg Riboflavin 5.0mg Thiamine·HCl 5.0mg Biotin 2.0mg Folic acid 2.0mg Vitamin B 12 0.1mg Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Adjust pH to 7.0. Mix thor- oughly. L-Cysteine·HCl Solution: Composition per 10.0mL: L-Cysteine·HCl 0.3g Preparation of L-Cysteine·HCl Solution: Add L-cysteine·HCl to distilled/deionized water and bring volume to 10.0mL. Mix thorough- ly. Autoclave under 100% N 2 for 15 min at 15 psi pressure–121°C. Na 2 S·9H 2 O Solution: Composition per 10.0mL: Na 2 S·9H 2 O 0.3g Preparation of Na 2 S·9H 2 O Solution: Add Na 2 S·9H 2 O to dis- tilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N 2 . Autoclave for 15 min at 15 psi pressure–121°C. Preparation of Medium: Prepare and dispense medium under 80% H 2 + 20% CO 2 . Add components, except L-cysteine·HCl solution and Na 2 S·9H 2 O solution, to distilled/deionized water and bring volume to 980.0mL. Mix thoroughly. Anaerobically distribute into tubes or flasks fitted with butyl rubber stoppers. Autoclave for 15 min at 15 psi pres- sure–121°C. Anaerobically add 10.0mL of sterile L-cysteine·HCl solu- tion and 10.0mL of sterile Na 2 S·9H 2 O solution to each liter of medium or, using a syringe, inject the appropriate amount of sterile L- cysteine·HCl solution and sterile Na 2 S·9H 2 O solution into individual tubes containing medium. Use: For the cultivation and maintenance of Methanococcus deltae. Methanococcus jannaschii Medium Composition per liter: NaCl 30.0g MgSO 4 ·7H 2 O 3.40g MgCl 2 ·2H 2 O 2.7g NaHCO 3 1.0g Na 2 S·9H 2 O 0.5g KCl 0.33g NH 4 Cl 0.25g CaCl 2 ·2H 2 O 0.14g K 2 HPO 4 0.14g Fe(NH 4 ) 2 (SO 4 ) 2 ·6H 2 O 0.01g Resazurin 1.0mg Na 2 SeO 3 ·5H 2 O 0.5mg NiCl 2 ·6H 2 O 0.5mg Trace elements solution 10.0mL Vitamin solution 10.0mL L-Cysteine·HCl solution 10.0mL Na 2 S·9H 2 O solution 10.0mL pH 6.0 ± 0.2 at 25°C Trace Elements Solution: Composition per liter: MgSO 4 ·7H 2 O 3.0g Nitrilotriacetic acid 1.5g NaCl 1.0g MnSO 4 ·2H 2 O 0.5g CoSO 4 ·7H 2 O 0.18g ZnSO 4 ·7H 2 O 0.18g CaCl 2 ·2H 2 O 0.1g © 2010 by Taylor and Francis Group, LLC 1092 Methanococcus jannaschii Medium FeSO 4 ·7H 2 O 0.1g NiCl 2 ·6H 2 O 0.025g KAl(SO 4 ) 2 ·12H 2 O 0.02g CuSO 4 ·5H 2 O 0.01g H 3 BO 3 0.01g Na 2 MoO 4 ·2H 2 O 0.01g Na 2 SeO 3 ·5H 2 O 0.3mg Preparation of Trace Elements Solution: Add nitrilotriacetic acid to 500.0mL of distilled/deionized water. Adjust pH to 6.5 with KOH. Add remaining components. Adjust pH to 7.0. Add distilled/de- ionized water to 1.0L. Vitamin Solution: Composition per liter: Pyridoxine·HCl 10.0mg Calcium DL-pantothenate 5.0mg Lipoic acid 5.0mg Nicotinic acid 5.0mg p-Aminobenzoic acid 5.0mg Riboflavin 5.0mg Thiamine·HCl 5.0mg Biotin 2.0mg Folic acid 2.0mg Vitamin B 12 0.1mg Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Adjust pH to 7.0. Mix thor- oughly. L-Cysteine·HCl Solution: Composition per 10.0mL: L-Cysteine·HCl 0.5g Preparation of L-Cysteine·HCl Solution: Add L-cysteine·HCl to distilled/deionized water and bring volume to 10.0mL. Mix thorough- ly. Autoclave under 100% N 2 for 15 min at 15 psi pressure–121°C. Na 2 S·9H 2 O Solution: Composition per 10.0mL: Na 2 S·9H 2 O 0.3g Preparation of Na 2 S·9H 2 O Solution: Add Na 2 S·9H 2 O to dis- tilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N 2 . Autoclave for 15 min at 15 psi pressure–121°C. Preparation of Medium: Prepare and dispense medium under 80% H 2 + 20% CO 2 . Add components, except L-cysteine·HCl solution and Na 2 S·9H 2 O solution, to distilled/deionized water and bring volume to 980.0mL. Mix thoroughly. Anaerobically distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Anaerobically add 10.0mL of sterile L-cysteine·HCl solution and 10.0mL of sterile Na 2 S·9H 2 O solution to each liter of medium or, using a syringe, inject the appropriate amount of sterile L-cysteine·HCl solution and sterile Na 2 S·9H 2 O solution into individual tubes containing medium. Use: For the cultivation and maintenance of Methanococcus species. Methanococcus jannaschii Medium Composition per 1020.0mL: PIPES (piperazine-N,N´- bis[2-ethanesulfonic acid]) buffer 15.12g MgCl 2 ·6H 2 O 4.3g MgSO 4 ·7H 2 O 3.4g NaCl 3.0g NH 4 Cl 0.25g K 2 HPO 4 0.14g CaCl 2 ·2H 2 O 0.14g KCl 0.33g Minerals solution 10.0mL Na 2 S 2 O 3 solution 10.0mL β-Mercaptoethanol solution 10.0mL SeO 2 solution 1.0mL pH 7.0 ± 0.2 at 25°C Minerals Solution: Composition per liter: Nitrilotriacetic acid 4.5g FeCl 2 ·4H 2 O 0.4g CoCl 2 ·2H 2 O 0.17g MnCl 2 ·4H 2 O 0.1g ZnCl 2 0.1g NaMoO 4 ·6H 2 O 36.0mg CaCl 2 ·H 2 O 27.0mg Preparation of Minerals Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Na 2 S 2 O 3 Solution: Composition per 10.0mL: Na 2 S 2 O 3 ·5H 2 O 0.63g Preparation of Na 2 S 2 O 3 Solution: Add Na 2 S 2 O 3 ·5H 2 O to dis- tilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N 2 . Autoclave for 15 min at 15 psi pressure–121°C. β-Mercaptoethanol Solution: Composition per 10.0mL: β-Mercaptoethanol 0.39g Preparation of β-Mercaptoethanol Solution: Add β-mercapto- ethanol to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N 2 . Autoclave for 15 min at 15 psi pres- sure–121°C. SeO 2 Solution: Composition per 100.0mL: SeO 2 0.011g Preparation of SeO 2 Solution: Add SeO 2 to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Preparation of Medium: Prepare and dispense medium under 100% N 2 . Add components, except Na 2 S 2 O 3 solution and β-mercapto- ethanol solution, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Continue boiling for 3 min. Cool to room temperature while sparging with 100% N 2 . Adjust pH to 7.0 with KOH. Anaerobically distribute 100.0mL volumes into an- aerobic bottles. Autoclave for 15 min at 15 psi pressure–121°C. Aseptical- ly and anaerobically add 1.0mL of sterile Na 2 S 2 O 3 solution and 1.0mL of sterile β-mercaptoethanol solution to each bottle. Mix thoroughly. Prior to inoculation, flush each bottle with 80% H 2 + 20% CO 2 . Use: For the cultivation of Methanococcus jannaschii. Methanococcus McC Medium Composition per 1100.0mL: NaHCO 3 5.0g Yeast extract 2.0g L-Cysteine·HCl·H 2 O 0.5g General salts solution 500.0mL NaCl solution 75.0mL Na 2 S·9H 2 O solution 20.0mL K 2 HPO 4 solution 10.0mL © 2010 by Taylor and Francis Group, LLC Methanococcus McN Medium 1093 Trace minerals solution 10.0mL Sodium acetate solution 10.0mL Iron stock solution 5.0mL Resazurin solution 1.0mL General Salts Solution: Composition per liter: MgSO 4 ·7H 2 O 6.9g MgCl 2 ·6H 2 O 5.5g NH 4 Cl 1.0g KCl 0.67g CaCl 2 ·2H 2 O 0.28g Preparation of General Salts Solution: Add components to dis- tilled/deionized water and bring volume to 1.0L. Mix thoroughly. NaCl Solution: Composition per 100.0mL: NaCl 29.3g Preparation of NaCl Solution: Add NaCl to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Na 2 S·9H 2 O Solution: Composition per 100.0mL: NaOH 1 pellet Na 2 S·9H 2 O 2.5g Preparation of Na 2 S·9H 2 O Solution: Bring 100.0mL of dis- tilled/deionized water to boiling. Cool to room temperature while sparging with 100%N 2 . Dissolve 1 pellet of NaOH in the anaerobic wa- ter. Weigh out a little more than 2.5g of Na 2 S·9H 2 O. Briefly rinse the crystals in distilled/deionized water. Dry the crystals by blotting on pa- per towels or filter paper. Add 2.5g of washed Na 2 S·9H 2 O crystals to 100.0mL of anaerobic NaOH solution. Distribute into serum bottles fit- ted with butyl rubber stoppers and aluminum seals. Do not grease stop- pers. Pressurize to 60kPa with 100% N 2 . Autoclave for 15 min at 15 psi pressure–121°C. Store at room temperature in an anaerobic chamber. K 2 HPO 4 Solution: Composition per 100.0mL: K 2 HPO 4 1.4g Preparation of K 2 HPO 4 Solution: Add K 2 HPO 4 to distilled/de- ionized water and bring volume to 100.0mL. Mix thoroughly. Trace Minerals Solution: Composition per liter: Nitrilotriacetic acid 1.5g Na 2 WO 4 ·2H 2 O 1.0g Fe(NH 4 ) 2 (SO 4 ) 2 ·6H 2 O 0.2g Na 2 SeO 3 0.2g Na 2 MoO 4 ·2H 2 O 0.1g Mn 4 ·2H 2 O 0.1g Zn 4 ·7H 2 O 0.1g NiCl 2 ·7H 2 O 0.025g CuSO 4 ·5H 2 O 0.01g Preparation of Trace Minerals Solution: Add nitrilotriacetic acid to 500.0mL of distilled/deionized water. Adjust pH to 6.5 with KOH. Add remaining components. Add distilled/deionized water to 1.0L. Adjust pH to 7.0. Sodium Acetate Solution: Composition per 100.0mL: Sodium acetate·3H 2 O 13.6g Preparation of Sodium Acetate Solution: Add sodium ace- tate·3H 2 O to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Iron Stock Solution: Composition per 100.0mL: Fe(NH 4 ) 2 (SO 4 ) 2 ·6H 2 O 0.2g Preparation of Iron Stock Solution: Add Fe(NH 4 ) 2 (SO 4 ) 2 ·6H 2 O to 5.0mL of distilled H 2 O containing 2 drops of concentrated HCl. Mix thoroughly. When the Fe(NH 4 ) 2 (SO 4 ) 2 ·6H 2 O has dissolved, bring the volume to 100.0mL with distilled/deionized water. Resazurin Solution: Composition per 10.0mL: Resazurin 10.0mg Preparation of Resazurin Solution: Add resazurin to distilled/ deionized water and bring volume to 10.0mL. Mix thoroughly. Preparation of Medium: Prepare and dispense medium under 80% H 2 + 20% CO 2 . Add components, except NaHCO 3 and Na 2 S·9H 2 O so- lution, to distilled/deionized water and bring volume to 1080.0mL. Mix thoroughly. Gently heat and bring to boiling. Continue boiling for 3 min. Cool to room temperature while sparging with 80% H 2 + 20% CO 2 . Add NaHCO 3 . Mix thoroughly. Anaerobically distribute 9.8mL volumes into anaerobic tubes. Autoclave for 15 min at 15 psi pressure– 121°C. Aseptically and anaerobically add 0.2mL of sterile Na 2 S·9H 2 O solution to each tube. Mix thoroughly. Use: For the cultivation of Methanococcus species. Methanococcus McN Medium Composition per 1100.0mL: NaHCO 3 5.0g L-Cysteine·HCl·H 2 O 0.5g General salts solution 500.0mL NaCl solution 75.0mL Na 2 S·9H 2 O solution 20.0mL K 2 HPO 4 solution 10.0mL Trace minerals solution 10.0mL Iron stock solution 5.0mL Resazurin solution 1.0mL General Salts Solution: Composition per liter: MgSO 4 ·7H 2 O 6.9g MgCl 2 ·6H 2 O 5.5g NH 4 Cl 1.0g KCl 0.67g CaCl 2 ·2H 2 O 0.28g Preparation of General Salts Solution: Add components to dis- tilled/deionized water and bring volume to 1.0L. Mix thoroughly. NaCl Solution: Composition per 100.0mL: NaCl 29.3g Preparation of NaCl Solution: Add NaCl to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Na 2 S·9H 2 O Solution: Composition per 100.0mL: Na 2 S·9H 2 O 2.5g NaOH 1 pellet Preparation of Na 2 S·9H 2 O Solution: Bring 100.0mL of dis- tilled/deionized water to boiling. Cool to room temperature while © 2010 by Taylor and Francis Group, LLC 1094 Methanococcus McNail Medium sparging with 100%N 2 . Dissolve 1 pellet of NaOH in the anaerobic wa- ter. Weigh out a little more than 2.5g of Na 2 S·9H 2 O. Briefly rinse the crystals in distilled/deionized water. Dry the crystals by blotting on pa- per towels or filter paper. Add 2.5g of washed Na 2 S·9H 2 O crystals to 100.0mL of anaerobic NaOH solution. Distribute into serum bottles fit- ted with butyl rubber stoppers and aluminum seals. Do not grease stop- pers. Pressurize to 60kPa with 100% N 2 . Autoclave for 15 min at 15 psi pressure–121°C. Store at room temperature in an anaerobic chamber. K 2 HPO 4 Solution: Composition per 100.0mL: K 2 HPO 4 1.4g Preparation of K 2 HPO 4 Solution: Add K 2 HPO 4 to distilled/de- ionized water and bring volume to 100.0mL. Mix thoroughly. Trace Minerals Solution: Composition per liter: Nitrilotriacetic acid 1.5g Na 2 WO 4 ·2H 2 O 1.0g Fe(NH 4 ) 2 (SO 4 ) 2 ·6H 2 O 0.2g Na 2 SeO 3 0.2g Na 2 MoO 4 ·2H 2 O 0.1g Mn 4 ·2H 2 O 0.1g Zn 4 ·7H 2 O 0.1g NiCl 2 ·7H 2 O 0.025g CuSO 4 ·5H 2 O 0.01g Preparation of Trace Minerals Solution: Add nitrilotriacetic acid to 500.0mL of distilled/deionized water. Adjust pH to 6.5 with KOH. Add remaining components. Add distilled/deionized water to 1.0L. Adjust pH to 7.0. Iron Stock Solution: Composition per 100.0mL: Fe(NH 4 ) 2 (SO 4 ) 2 ·6H 2 O 0.2g Preparation of Iron Stock Solution: Add Fe(NH 4 ) 2 (SO 4 ) 2 ·6H 2 O to 5.0mL of distilled H 2 O containing 2 drops of concentrated HCl. Mix thoroughly. When the Fe(NH 4 ) 2 (SO 4 ) 2 ·6H 2 O has dissolved, bring the volume to 100.0mL with distilled/deionized water. Resazurin Solution: Composition per 10.0mL: Resazurin 10.0mg Preparation of Resazurin Solution: Add resazurin to distilled/ deionized water and bring volume to 10.0mL. Mix thoroughly. Preparation of Medium: Prepare and dispense medium under 80% H 2 + 20% CO 2 . Add components, except NaHCO 3 and Na 2 S·9H 2 O so- lution, to distilled/deionized water and bring volume to 1080.0mL. Mix thoroughly. Gently heat and bring to boiling. Continue boiling for 3 min. Cool to room temperature while sparging with 80% H 2 + 20% CO 2 . Add NaHCO 3 . Mix thoroughly. Anaerobically distribute 9.8mL volumes into anaerobic tubes. Autoclave for 15 min at 15 psi pressure– 121°C. Aseptically and anaerobically add 0.2mL of sterile Na 2 S·9H 2 O solution to each tube. Mix thoroughly. Use: For the cultivation of Methanococcus species. Methanococcus McNail Medium Composition per 1100.0mL: NaHCO 3 5.0g L-Leucine 1.0g L-Isoleucine 0.5g L-Cysteine·HCl·H 2 O 0.5g General salts solution 500.0mL NaCl solution 75.0mL Na 2 S·9H 2 O solution 20.0mL K 2 HPO 4 solution 10.0mL Trace minerals solution 10.0mL Sodium acetate solution 10.0mL Pantoyllactone solution 10.0mL Iron stock solution 5.0mL Resazurin solution 1.0mL General Salts Solution: Composition per liter: MgSO 4 ·7H 2 O 6.9g MgCl 2 ·6H 2 O 5.5g NH 4 Cl 1.0g KCl 0.67g CaCl 2 ·2H 2 O 0.28g Preparation of General Salts Solution: Add components to dis- tilled/deionized water and bring volume to 1.0L. Mix thoroughly. NaCl Solution: Composition per 100.0mL: NaCl 29.3g Preparation of NaCl Solution: Add NaCl to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Na 2 S·9H 2 O Solution: Composition per 100.0mL: NaOH 1 pellet Na 2 S·9H 2 O 2.5g Preparation of Na 2 S·9H 2 O Solution: Bring 100.0mL of dis- tilled/deionized water to boiling. Cool to room temperature while sparging with 100%N 2 . Dissolve 1 pellet of NaOH in the anaerobic wa- ter. Weigh out a little more than 2.5g of Na 2 S·9H 2 O. Briefly rinse the crystals in distilled/deionized water. Dry the crystals by blotting on pa- per towels or filter paper. Add 2.5g of washed Na 2 S·9H 2 O crystals to 100.0mL of anaerobic NaOH solution. Distribute into serum bottles fit- ted with butyl rubber stoppers and aluminum seals. Do not grease stop- pers. Pressurize to 60kPa with 100% N 2 . Autoclave for 15 min at 15 psi pressure–121°C. Store at room temperature in an anaerobic chamber. K 2 HPO 4 Solution: Composition per 100.0mL: K 2 HPO 4 1.4g Preparation of K 2 HPO 4 Solution: Add K 2 HPO 4 to distilled/de- ionized water and bring volume to 100.0mL. Mix thoroughly. Trace Minerals Solution: Composition per liter: Nitrilotriacetic acid 1.5g Na 2 WO 4 ·2H 2 O 1.0g Fe(NH 4 ) 2 (SO 4 ) 2 ·6H 2 O 0.2g Na 2 SeO 3 0.2g Na 2 MoO 4 ·2H 2 O 0.1g Mn 4 ·2H 2 O 0.1g Zn 4 ·7H 2 O 0.1g NiCl 2 ·7H 2 O 0.025g CuSO 4 ·5H 2 O 0.01g Preparation of Trace Minerals Solution: Add nitrilotriacetic acid to 500.0mL of distilled/deionized water. Adjust pH to 6.5 with KOH. Add remaining components. Add distilled/deionized water to 1.0L. Adjust pH to 7.0. © 2010 by Taylor and Francis Group, LLC . Anaerobically add 10.0mL of sterile L-cysteine·HCl solu- tion and 10.0mL of sterile Na 2 S·9H 2 O solution to each liter of medium or, using a syringe, inject the appropriate amount of sterile L- cysteine·HCl. Anaerobically add 10.0mL of sterile L-cysteine·HCl solution and 10.0mL of sterile Na 2 S·9H 2 O solution to each liter of medium or, using a syringe, inject the appropriate amount of sterile L-cysteine·HCl. 2.5g Preparation of Na 2 S·9H 2 O Solution: Bring 100.0mL of dis- tilled/deionized water to boiling. Cool to room temperature while sparging with 100%N 2 . Dissolve 1 pellet of NaOH in the anaerobic