General Salts Medium for Estuarine Methanogens

[所属分类:培养基配方] [发布时间:2021-3-10] [发布人: ] [阅读次数:] [返回]

General Salts Medium for Estuarine Methanogens

培养基配方

Composition per 410.8mL:
Agar ..............................................................................................8.0g
NaCl..............................................................................................3.6g
NaHCO 3 ........................................................................................2.0g
Complete salts solution..........................................................200.0mL
Cysteine-sulfide reducing agent...............................................16.0mL
Wolfe’s mineral solution............................................................4.0mL
Vitamin solution.........................................................................4.0mL
Yeast extract-Trypticase™ solution...........................................4.0mL
Sodium acetate (25% solution)..................................................2.0mL
Fe(NH 4 ) 2 SO 4 (0.2% solution)....................................................0.4mL
Resazurin (0.1% solution)..........................................................0.4mL
pH 7.0 ± 0.2 at 25°C
Complete Salts Solution:
Composition per liter:
MgSO 4 ·7H 2 O................................................................................6.9g
MgCl 2 ·6H 2 O..................................................................................5.5g
KCl..............................................................................................0.67g
NH 4 Cl ...........................................................................................0.5g
CaCl 2 ·2H 2 O.................................................................................0.28g
K 2 HPO 4 .......................................................................................0.28g
Preparation 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......................................................................5.0g
Na 2 S (12.5% solution).............................................................40.0mL
NaOH (1N solution).................................................................30.0mL
Preparation 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................................................................................20.0g
Pancreatic digest of casein..........................................................20.0g
Preparation 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..............................................................................................1.0g
MnSO 4 ·H 2 O..................................................................................0.5g
FeSO 4 ·7H 2 O..................................................................................0.1g
CoCl 2 ·6H 2 O..................................................................................0.1g
CaCl 2 .............................................................................................0.1g
ZnSO 4 ·7H 2 O.................................................................................0.1g
CuSO 4 ·5H 2 O...............................................................................0.01g
AlK(SO 4 ) 2 ·12H 2 O.......................................................................0.01g
H 3 BO 3 .........................................................................................0.01g
Na 2 MoO 4 ·2H 2 O..........................................................................0.01g
Preparation 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........................................................................10.0mg
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 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.