MS Medium for Methanogens

[所属分类:培养基配方] [发布时间:2021-6-9] [发布人:网站管理员2] [阅读次数:] [返回]

MS Medium for Methanogens

山东拓普生物工程有限公司 培养基配方 http://www.topbiol.com

Composition per 408.8mL:

Agar..............................................................................................8.0g
NaHCO 3 ........................................................................................2.4g
L -Cysteine-sulfide reducing agent...........................................16.0mL
Mineral solution 1....................................................................15.0mL
Mineral solution 2....................................................................15.0mL
Sodium formate (20% solution).................................................6.0mL
Yeast extract-soybean casein solution .......................................4.0mL
Sodium acetate (25% solution)..................................................4.0mL
Wolfe’s vitamin solution............................................................4.0mL
Wolfe’s mineral solution............................................................4.0mL
FeSO 4 ·7H 2 O (0.2% solution).....................................................0.4mL
Resazurin (0.1% solution) .........................................................0.4mL
pH 7.0 ± 0.2 at 25°C
L -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  L -Cysteine-Sulfide Reducing Agent: Add dis-
tilled/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 pre-
pared Na 2 S solution. Renew gassing for several minutes. Cap with rub-
ber stoppers. Distribute into 8.0mL/18.0mm Hungate tubes.
Mineral Solution 1:
Composition per liter:
K 2 HPO 4 .........................................................................................6.0g
Preparation of Mineral Solution 1: Add K 2 HPO 4 to distilled/de-
ionized water and bring volume to 1.0L. Mix thoroughly.
Mineral Solution 2:
Composition per liter:
NaCl............................................................................................12.0g
KH 2 PO 4 .........................................................................................6.0g
(NH 4 ) 2 SO 4 .....................................................................................6.0g
MgSO 4 ·7H 2 O................................................................................2.6g
CaCl 2 ·2H 2 O.................................................................................0.16g
Preparation of Mineral Solution 2: Add components to distilled/
deionized water and bring volume to 1.0L. Mix thoroughly.
Yeast Extract-Soybean Casein Solution:
Composition per 100.0mL:
Yeast extract................................................................................20.0g
Pancreatic digest of casein..........................................................20.0g
Preparation of Yeast Extract-Soybean Casein 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.
Wolfe’s Vitamin Solution:
Composition per liter:
Pyridoxine·HCl........................................................................10.0mg
Thiamine·HCl ............................................................................5.0mg
Riboflavin ..................................................................................5.0mg
Nicotinic acid.............................................................................5.0mg
Calcium pantothenate ................................................................5.0mg
p-Aminobenzoic acid.................................................................5.0mg
Thioctic acid ..............................................................................5.0mg
Biotin .........................................................................................2.0mg
Folic acid....................................................................................2.0mg
Cyanocobalamin .....................................................................100.0μg
Preparation of Wolfe’s Vitamin Solution: Add components to
distilled/deionized water and bring volume to 1.0L. Mix thoroughly.
Preparation of Medium: Add components to distilled/deionized
water and bring volume to 408.8mL. Gently heat and bring to boiling
under 80% N 2 + 20% CO 2 . Continue boiling until resazrin turns col-
orless, indicating reduction. Adjust pH to 7.0. Anaerobically distrib-
ute into tubes under 80% N 2 + 20% CO 2 . Cap with rubber stoppers
and aluminum crimp closures. Autoclave for 15 min at 15 psi pres-
sure–121°C.
Use:  For the cultivation and maintenance of Methanobacterium ther-
moautotrophicum, Methanobacterium wolfei, Methanobrevibacter
smithii, Methanogenium bourgense, and Methanogenium species.