Composition per 410.8mL:Agar ..............................................................................................8.0gNaCl..............................................................................................3.6gNaHCO 3 ........................................................................................2.0gComplete salts solution..........................................................200.0mLCysteine-sulfide reducing agent...............................................16.0mLWolfe’s mineral solution............................................................4.0mLVitamin solution.........................................................................4.0mLYeast extract-Trypticase solution...........................................4.0mLSodium acetate (25% solution)..................................................2.0mLFe(NH 4 ) 2 SO 4 (0.2% solution)....................................................0.4mLResazurin (0.1% solution)..........................................................0.4mLpH 7.0 ± 0.2 at 25°CComplete Salts Solution:Composition per liter:MgSO 4 ·7H 2 O................................................................................6.9gMgCl 2 ·6H 2 O..................................................................................5.5gKCl..............................................................................................0.67gNH 4 Cl ...........................................................................................0.5gCaCl 2 ·2H 2 O.................................................................................0.28gK 2 HPO 4 .......................................................................................0.28gPreparation of Complete Salts Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.Cysteine-Sulfide Reducing Agent:Composition per 400.0mL:L- Cysteine·HCl·H 2 O......................................................................5.0gNa 2 S (12.5% solution).............................................................40.0mLNaOH (1N solution).................................................................30.0mLPreparation of Cysteine-Sulfide Reducing Agent: Add distilled/deionized water to a 500.0mL round-bottomed flask. Add freshly prepared NaOH solution. Gently heat and bring to boiling under 100% N 2 . Remove gassing probe. Add L -cysteine·HCl·H 2 O. Add freshly prepared Na 2 S solution. Renew gassing for several minutes. Cap with rubber stoppers. Distribute into 8.0mL/18mm Hungate tubes.Yeast Extract-Trypticase Solution:Composition per 100.0mL:Yeast extract................................................................................20.0gPancreatic digest of casein..........................................................20.0gPreparation of Yeast Extract-Trypticase Solution: Add components to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly.Wolfe’s Mineral Solution:Composition per liter:MgSO 4 ·7H 2 O................................................................................3.0g Nitrilotriacetic acid.......................................................................1.5g NaCl..............................................................................................1.0gMnSO 4 ·H 2 O..................................................................................0.5gFeSO 4 ·7H 2 O..................................................................................0.1gCoCl 2 ·6H 2 O..................................................................................0.1gCaCl 2 .............................................................................................0.1gZnSO 4 ·7H 2 O.................................................................................0.1gCuSO 4 ·5H 2 O...............................................................................0.01gAlK(SO 4 ) 2 ·12H 2 O.......................................................................0.01gH 3 BO 3 .........................................................................................0.01gNa 2 MoO 4 ·2H 2 O..........................................................................0.01gPreparation of Wolfe’s Mineral 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.Vitamin Solution:Composition per liter:Pyridoxine-HCl........................................................................10.0mgThiamine-HCl·2H 2 O..................................................................5.0mgRiboflavin..................................................................................5.0mgNicotinic acid.............................................................................5.0mgD -Ca-pantothenate .....................................................................5.0mgp-Aminobenzoic acid.................................................................5.0mgLipoic acid.................................................................................5.0mgBiotin.........................................................................................2.0mgFolic acid ...................................................................................2.0mgVitamin B 12 ................................................................................0.1mgPreparation 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 cysteine-sulfide reducing agent, to distilled/deionized water and bring volume to 410.8mL. Mix thoroughly. Adjust pH to 7.0. Gently heat and bring to boiling under 80% N 2 + 20% CO 2 . Add cysteine-sulfide reducing agent. Continue boiling until resazurin turns colorless, indicating reduction. Distribute anaerobically into culture tubes with aluminum crimp seals. Autoclave for 15 min at 15 psi pressure–121°C.Use: For the cultivation and maintenance of Methanococcus deltae, Methanococcus vannielii, Methanococcus voltae, Methanogenium cariaci, Methanogenium marisnigri, and Methanogenium olentangyi. 下一篇 返回