Methanohalophilus euhalobius Medium

[所属分类:培养基配方] [发布时间:2021-5-25] [发布人:网站管理员2] [阅读次数:] [返回]

Methanohalophilus euhalobius Medium

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

Composition per 1010.0mL:

NaCl............................................................................................60.0g
MgCl 2 ·6H 2 O..................................................................................2.4g
Yeast extract..................................................................................2.0g
KCl................................................................................................1.0g
Disodium EDTA ...........................................................................0.5g
NH 4 Cl ...........................................................................................0.5g
KH 2 PO 4 .........................................................................................0.4g
Resazurin ...................................................................................0.5mg
Trimethylamine solution..........................................................50.0mL
NaHCO 3  solution .....................................................................40.0mL
CaCl 2  solution..........................................................................20.0mL
Na 2 S·9H 2 O solution.................................................................15.0mL
L -Cysteine·HCl solution...........................................................15.0mL
Wolfe’s mineral solution..........................................................10.0mL
Wolfe’s vitamin solution............................................................5.0mL
pH 6.8–7.0 at 25°C
Trimethylamine Solution:
Composition per 50.0mL:
Trimethylamine·HCl...................................................................10.0g
Preparation of Trimethylamine Solution:  Add trimethy-
lamine·HCl to distilled/deionized water and bring volume to 50.0mL.
Mix thoroughly. Sparge with 100% N 2 . Autoclave for 15 min at 15 psi
pressure–121°C.
NaHCO 3 Solution:
Composition per 40.0mL:
NaHCO 3 ........................................................................................4.0g
Preparation of NaHCO 3 Solution:  Add NaHCO 3 to distilled/de-
ionized water and bring volume to 40.0mL. Mix thoroughly. Sparge
with 80% N 2 + 20% CO 2 . Autoclave for 15 min at 15 psi pressure–
121°C.
CaCl 2 Solution:
Composition per 20.0mL:
CaCl 2 ·2H 2 O...................................................................................2.0g
Preparation of CaCl 2 Solution:  Add CaCl 2 ·2H 2 O to distilled/de-
ionized water and bring volume to 20.0mL. Mix thoroughly. Sparge
with 80% N 2 + 20% CO 2 gas mixture. Autoclave for 15 min at 15 psi
pressure–121°C.
Na 2 S·9H 2 O Solution:
Composition per 20.0mL:
Na 2 S·9H 2 O....................................................................................0.6g
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 20.0mL. 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 Solution:
Composition per 20.0mL:
L -Cysteine·HCl·H 2 O.....................................................................0.6g
Preparation of  L -Cysteine·HCl Solution:  Add  L -cyste-
ine·HCl·H 2 O to distilled/deionized water and bring volume to 20.0mL.
Mix thoroughly. Sparge with 100% N 2 . Autoclave for 15 min at 15 psi
pressure–121°C.
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. Add distilled/deionized water to
1.0L. Adjust pH to 6.8.
Wolfe’s Vitamin Solution:
Composition per liter:
Pyridoxine·HCl........................................................................10.0mg
p-Aminobenzoic acid.................................................................5.0mg
Lipoic acid.................................................................................5.0mg
Nicotinic acid.............................................................................5.0mg
Riboflavin..................................................................................5.0mg
Thiamine·HCl............................................................................5.0mg
Calcium  DL -pantothenate...........................................................5.0mg
Biotin.........................................................................................2.0mg
Folic acid ...................................................................................2.0mg
Vitamin B 12 ................................................................................0.1mg
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: Prepare and dispense medium under 80%
N 2 + 20% CO 2 gas mixture. Add components, except trimethylamine so-
lution, NaHCO 3  solution, CaCl 2  solution solution,  L -cysteine·HCl solu-
tion, and Na 2 S·9H 2 O solution, to distilled/deionized water and bring
volume to 860.0mL. Mix thoroughly. Adjust pH to 6.8–7.0. Sparge with
80% N 2 + 20% CO 2 gas mixture. Autoclave for 15 min at 15 psi pres-
sure–121°C. Aseptically and anaerobically add 50.0mL of sterile trim-
ethylamine solution, 40.0mL of sterile NaHCO 3  solution, 20.0mL of
sterile CaCl 2  solution, 15.0mL of sterile  L -cysteine·HCl solution, and
15.0mL 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 Methanohalophilus euhalobius.