Methanosarcina Medium

[所属分类:培养基配方] [发布时间:2021-5-26] [发布人:网站管理员2] [阅读次数:] [返回]

Methanosarcina 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:

Agar ............................................................................................20.0g
NaCl............................................................................................2.25g
Yeast extract..................................................................................2.0g
Pancreatic digest of casein............................................................2.0g
NH 4 Cl ...........................................................................................0.5g
MgSO 4 ·7H 2 O................................................................................0.5g
K 2 HPO 4 .......................................................................................0.35g
CaCl 2 ·2H 2 O.................................................................................0.25g
KH 2 PO 4 .......................................................................................0.23g
FeSO 4 ·7H 2 O...............................................................................2.0mg
NaHCO 3 solution.....................................................................20.0mL
L -Cysteine-sulfide reducing agent...........................................20.0mL
Wolfe’s vitamin solution..........................................................10.0mL
Methanol..................................................................................10.0mL
Resazurin (0.025% solution) .....................................................4.0mL
Trace elements solution SL-6....................................................3.0mL
pH 6.8 ± 0.2 at 25°C
NaHCO 3 Solution:
Composition per 20.0mL:
NaHCO 3 .................................................................................850.0mg
Preparation of NaHCO 3 Solution:  Add NaHCO 3 to distilled/de-
ionized water and bring volume to 20.0mL. Mix thoroughly. Filter ster-
ilize. Gas with 100% CO 2 for 20 min.
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 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.
Trace Elements Solution SL-6:
Composition per liter:
H 3 BO 3 ...........................................................................................0.3g
CoCl 2 ·6H 2 O..................................................................................0.2g
ZnSO 4 ·7H 2 O.................................................................................0.1g
MnCl 2 ·4H 2 O ...............................................................................0.03g
Na 2 MoO 4 ·H 2 O............................................................................0.03g
NiCl 2 ·6H 2 O.................................................................................0.02g
CuCl 2 · 2H 2 O ................................................................................0.01g
Preparation of Trace Elements Solution SL-6: Add components
to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.
Adjust pH to 3.4.
Preparation of Medium: Add components, except NaHCO 3 solu-
tion,  L -cysteine-sulfide reducing agent, Wolfe’s vitamin solution, and
methanol, to distilled/deionized water and bring volume to 940.0mL.
Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool
under 80% N 2 + 20% CO 2 . Aseptically and anaerobically add the ster-
ile NaHCO 3 solution, the sterile  L -cysteine-sulfide reducing agent, the
sterile Wolfe’s vitamin solution, and filter-sterilized methanol. Mix
thoroughly. Adjust pH to 6.8. Aseptically and anaerobically distribute
into sterile tubes or flasks.
Use:  For the cultivation and maintenance of Bifidobacterium asteroides,
Methanosarcina barkeri, Methanosarcina species, and Methanosarcina
vacuolata.