Methanobacteria Medium

[所属分类:培养基配方] [发布时间:2021-5-21] [发布人:网站管理员2] [阅读次数:] [返回]

Methanobacteria Medium

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

Composition per liter:

Mineral solution 2....................................................................50.0mL
Sodium carbonate solution.......................................................50.0mL
Mineral solution 1....................................................................25.0mL
L -Cysteine-sulfide reducing agent...........................................20.0mL
Wolfe’s mineral solution..........................................................10.0mL
Wolfe’s vitamin solution..........................................................10.0mL
Resazurin (0.025% solution)......................................................4.0mL
pH 7.2 ± 0.2 at 25°C
Mineral Solution 1:
Composition per liter:
K 2 HPO 4 .........................................................................................6.0g
Preparation of Medium: Add K 2 HPO 4 to distilled/deionized 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................................................................................2.4g
CaCl 2 ·2H 2 O...................................................................................1.6g
Preparation of Mineral Solution 2: Add components to distilled/
deionized water and bring volume to 1.0L. Mix thoroughly.
Sodium Carbonate Solution:
Composition per 100.0mL:
Na 2 CO 3 .........................................................................................8.0g
Preparation of Sodium Carbonate Solution: Add Na 2 CO 3 to
distilled/deionized water and bring volume to 100.0mL. Mix thorough-
ly.
L -Cysteine-Sulfide Reducing Agent:
Composition per 20.0mL:
L- Cysteine·HCl·H 2 O .....................................................................0.3g
Na 2 S·9H 2 O....................................................................................0.3g
Preparation of  L -Cysteine-Sulfide Reducing Agent: Add  L-
cysteine·HCl·H 2 O to 10.0mL of distilled/deionized water. Mix thor-
oughly. In a separate tube, add Na 2 S·9H 2 O to 10.0mL of distilled/de-
ionized water. Mix thoroughly. Gas both solutions with 100% N 2 and
cap tubes. Autoclave both solutions for 15 min at 15 psi pressure–
121°C using fast exhaust. Cool to 50°C. Aseptically combine the two
solutions under 100% N 2 .
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 and bring volume 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.
Filter sterilize.
Preparation of Medium: Add components, except vitamin solu-
tion and  L -cysteine-sulfide reducing agent, to distilled/deionized water
and bring volume to 970.0mL. Mix thoroughly. Autoclave for 15 min
at 15 psi pressure–121°C. Cool under 80% N 2 + 20% CO 2 . Aseptically
add the sterile vitamin solution and then the sterile  L -cysteine-sulfide
reducing agent. Adjust the pH to 7.2. Distribute aseptically and anaer-
obically into sterile tubes.
Use:  For the cultivation and maintenance of Acetogenium kivui, Meth-
anobacterium formicicum, Methanobacterium thermoautotrophicum,
and Methanobrevibacter arboriphilicus.