Methanococcus voltae BD Medium

[所属分类:培养基配方] [发布时间:2021-5-24] [发布人:网站管理员2] [阅读次数:] [返回]

Methanococcus voltae BD Medium

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

Composition 1003.0mL:

L -Leucine....................................................................................50.0g
NaCl............................................................................................18.0g
Sodium panthothenate...................................................................5.0g
MgSO 4 ·7H 2 O..............................................................................3.48g
MgCl 2 ·6H 2 O................................................................................2.75g
Sodium acetate·3H 2 O....................................................................1.0g
KCl..............................................................................................0.34g
NH 4 Cl .........................................................................................0.26g
CaCl 2 ·2H 2 O.................................................................................0.14g
K 2 PO 4 ..........................................................................................0.14g
L -Isoleucine...................................................................................0.1g
L -Cysteine/Na 2 S solution.........................................................17.5mL
Trace minerals solution............................................................10.0mL
Vitamin solution.......................................................................10.0mL
Na 2 CO 3  solution.........................................................................6.0mL
FeSO 4 ·7H 2 O solution.................................................................4.5mL
Trace Minerals Solution:
Composition per liter:
Nitrilotriacetic acid.......................................................................1.5g
MnSO 4 ·7H 2 O................................................................................0.5g
NiCl 2 ·6H 2 O.................................................................................0.12g
CoCl 2 ·6H 2 O ..................................................................................0.1g
FeSO 4 ·7H 2 O..................................................................................0.1g
Resazurin ......................................................................................0.1g
ZnSO 4 ·7H 2 O.................................................................................0.1g
AIK(SO 4 ) 2 ·5H 2 O.........................................................................0.01g
CuSO 4 ·5H 2 O...............................................................................0.01g
H 3 BO 3 .........................................................................................0.01g
Preparation of Trace Minerals 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:
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 ................................................................................0.5mg
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.
L -Cysteine/Na 2 S Solution:
Composition per liter:
L -Cysteine·HCl ...........................................................................1.25g
Na 2 S·9H 2 O..................................................................................1.25g
Preparation of  L -Cysteine/Na 2 S Solution:  Add  L -cysteine·HCl
and Na 2 S·9H 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. Before use, neutralize to pH 7.0 with sterile
HCl.
Na 2 CO 3 Solution:
Composition per 100.0mL:
Na 2 CO 3 .........................................................................................1.0g
Preparation of Na 2 CO 3 Solution:  Add Na 2 CO 3 to distilled/de-
ionized water and bring volume to 100.0mL. Mix thoroughly. Sparge
with 80% N 2 + 20% CO 2 . Autoclave for 15 min at 15 psi pressure–
121°C.
FeSO 4 ·7H 2 O Solution:
Composition per 100.0mL:
FeSO 4 ·7H 2 O..................................................................................0.1g
Preparation of FeSO 4 ·7H 2 O Solution:  Add FeSO 4 ·7H 2 O to dis-
tilled/deionized water and bring volume to 100.0mL. Mix thoroughly.
Filter sterilize. Sparge with 100% N 2 .
Preparation of Medium: Prepare and dispense medium under 80%
H 2 + 20% CO 2 . Add components, except  L -cysteine/Na 2 S solution,
Na 2 CO 3  solution, and FeSO 4 ·7H 2 O solution, to distilled/deionized wa-
ter and bring volume to 975.0mL. Mix thoroughly. Gently heat and
bring to boiling while sparging with 80% H 2 + 20% CO 2 . Cool to room
temperature while sparging with 80% H 2 + 20% CO 2 . Add 17.5mL of
L -cysteine/Na 2 S·9H 2 O solution and 6.0mL of Na 2 CO 3  solution. Anaer-
obically dispense into tubes or bottles in 10.0mL aliquots under an at-
mosphere of 80% H 2 + 20% CO 2 . Seal with butyl rubber stoppers
secured with aluminum crimp seals. Autoclave for 15 min at 15 psi
pressure–121°C. Immediately prior to inoculation, aseptically and an-
aerobically add 0.05mL of sterile FeSO 4 ·7H 2 O solution to each tube.
Use:  For the cultivation of Methanococcus voltae.