Methanospirillum hungatei JMA 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]

Methanospirillum hungatei JMA 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:

Sodium acetate..............................................................................2.5g
NH 4 Cl ...........................................................................................1.9g
Mineral solution 1....................................................................75.0mL
Mineral solution 2....................................................................75.0mL
Wolfe’s mineral solution..........................................................10.0mL
Vitamin solution.......................................................................10.0mL
L -Cysteine·HCl·H 2 O solution ..................................................10.0mL
Na 2 S·9H 2 O solution.................................................................10.0mL
FeSO 4 ·6H 2 O solution.................................................................6.0mL
Na 2 CO 3 solution.........................................................................4.8mL
Resazurin ...................................................................................1.0mL
NiCl 2 ·6H 2 O solution..................................................................0.1mL
FeSO 4 ·6H 2 O Solution:
Composition per 100.0mL:
FeSO 4 ·6H 2 O..................................................................................0.2g
Preparation of FeSO 4 ·6H 2 O Solution:  Add FeSO 4 ·6H 2 O to dis-
tilled/deionized water and bring volume to 100.0mL. Mix thoroughly.
NiCl 2 ·6H 2 O Solution:
Composition per liter:
NiCl 2 ·6H 2 O...................................................................................1.2g
Preparation of NiCl 2 ·6H 2 O Solution:  Add NiCl 2 ·6H 2 O to dis-
tilled/deionized water and bring volume to 1.0L. Mix thoroughly.
Mineral Solution 1:
Composition per liter:
K 2 HPO 4 .......................................................................................39.0g
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:
KH 2 PO 4 .......................................................................................24.0g
(NH 4 ) 2 SO 4 ....................................................................................6.0g
MgSO 4 ·7H 2 O................................................................................1.2g
CaCl 2 ·2H 2 O ................................................................................0.79g
NaCl............................................................................................0.59g
Preparation of Mineral Solution 2:  Add components to distilled/
deionized water and bring volume to 1.0L. 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 ·2H 2 O................................................................................0.5g
CoCl 2 ·6H 2 O..................................................................................0.1g
ZnSO 4 ·7H 2 O.................................................................................0.1g
CaCl 2 ·2H 2 O ..................................................................................0.1g
FeSO 4 ·7H 2 O..................................................................................0.1g
NiCl 2 ·6H 2 O...............................................................................0.025g
KAl(SO 4 ) 2 ·12H 2 O.......................................................................0.02g
CuSO 4 ·5H 2 O...............................................................................0.01g
H 3 BO 3 .........................................................................................0.01g
Na 2 MoO 4 ·2H 2 O..........................................................................0.01g
Na 2 SeO 3 ·5H 2 O...........................................................................0.3mg
Preparation of Wolfe’s Mineral Solution: Add nitrilotriacetic
acid to 500.0mL of distilled/deionized water. Adjust pH to 6.5 with
KOH. Add remaining components one at a time. Add distilled/deion-
ized water to 1.0L. Adjust pH to 6.8.
Vitamin Solution:
Composition per liter:
Calcium  DL -pantothenate..............................................................5.0g
Vitamin B 12 ...................................................................................0.1g
Pyridoxine·HCl........................................................................10.0mg
p-Aminobenzoic acid.................................................................5.0mg
Lipoic acid.................................................................................5.0mg
Nicotinic acid.............................................................................5.0mg
Riboflavin..................................................................................5.0mg
Thiamine·HCl............................................................................5.0mg
Biotin.........................................................................................2.0mg
Folic acid ...................................................................................2.0mg
Vitamin B 12 ................................................................................0.4mg
Preparation of Vitamin Solution:  Add components to distilled/
deionized water and bring volume to 1.0L. Mix thoroughly. Sparge
with 100% N 2 . Filter sterilize.
Na 2 CO 3 Solution:
Composition per liter:
Na 2 CO 3 .......................................................................................0.84g
Preparation of Na 2 CO 3 Solution:  Add Na 2 CO 3 to distilled/de-
ionized water and bring volume to 10.0mL. Mix thoroughly. Sparge
with 80% N 2 + 20% CO 2 . Autoclave for 15 min at 15 psi pressure–
121°C.
Na 2 S·9H 2 O Solution:
Composition per 10.0mL:
Na 2 S·9H 2 O....................................................................................0.2g
Preparation of Na 2 S·9H 2 O Solution:  Add Na 2 S·9H 2 O to dis-
tilled/deionized water and bring volume to 10.0mL. Mix thoroughly.
Sparge with 100% N 2 . Autoclave for 15 min at 15 psi pressure–121°C.
Before use, neutralize to pH 7.0 with sterile HCl.
L -Cysteine·HCl·H 2 O Solution:
Composition per 10.0mL:
L -Cysteine·HCl·H 2 O ...................................................................0.22g
Preparation of  L -Cysteine·HCl·H 2 O Solution:  Add  L -cyste-
ine·HCl·H 2 O to distilled/deionized water and bring volume to 10.0mL.
Mix thoroughly. Sparge with 100% N 2 . Autoclave for 15 min at 15 psi
pressure–121°C.
Preparation of Medium: Prepare and dispense medium under 80%
N 2 + 20% CO 2 . Add components, except Na 2 CO 3 solution,  L -
cysteine·HCl solution, and Na 2 S·9H 2 O solution, to distilled/deionized
water and bring volume to 975.2mL. Mix thoroughly. Gently heat and
bring to boiling. Cool while sparging with 80% N 2 + 20% CO 2 . Auto-
clave for 15 min at 15 psi pressure–121°C. Aseptically and anaerobi-
cally add 4.8mL of sterile Na 2 CO 3 soltuion, 10.0mL of sterile  L -
cysteine·HCl solution, and 10.0mL of sterile Na 2 S·9H 2 O solution. Mix
thoroughly. Aseptically and anaerobically distribute into sterile tubes
or bottles.
Use:  For the cultivation of Methanospirillum hungatei.