Methanocalculus pumilus Medium (DSMZ Medium 892)

[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]

Methanocalculus pumilus Medium
(DSMZ Medium 892)

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

Composition per 1080.0mL:

NaCl............................................................................................10.0g
Yeast extract..................................................................................2.0g
Trypticase™..................................................................................2.0g
NH 4 Cl ...........................................................................................0.9g
K 2 HPO 4 .........................................................................................0.4g
MgCl 2 ·6H 2 O ...............................................................................0.36g
Resazurin...................................................................................0.5mg
Na 2 CO 3  solution.......................................................................50.0mL
Na 2 S·9H 2 O solution.................................................................15.0mL
L -Cysteine-HCl·H 2 O solution..................................................15.0mL
Vitamin solution.......................................................................10.0mL
Trace elements solution...........................................................10.0mL
L -Cysteine Solution:
Composition per 15.0mL:
L -Cysteine·HCl·H 2 O.....................................................................0.5g
Preparation of  L -Cysteine Solution:  Add  L -cysteine·HCl·H 2 O to
distilled/deionized water and bring volume to 15.0mL. Mix thorough-
ly. Sparge with 100% N 2 . Autoclave for 15 min at 15 psi pressure–
121°C.
Na 2 CO 3 Solution:
Composition per 50.0mL:
Na 2 CO 3 .........................................................................................5.0g
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 . Filter sterilize.
Na 2 S·9H 2 O Solution :
Composition per 15mL:
Na 2 S·9H 2 O....................................................................................0.5g
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 15.0mL. Mix thoroughly.
Autoclave under 100% N 2 for 15 min at 15 psi pressure–121°C. Cool
to room temperature.
Trace Elements 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
CoSO 4 ·7H 2 O...............................................................................0.18g
ZnSO 4 ·7H 2 O...............................................................................0.18g
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
H 3 BO 3 .........................................................................................0.01g
Na 2 MoO 4 ·4H 2 O ..........................................................................0.01g
CuSO 4 ·5H 2 O...............................................................................0.01g
Na 2 SeO 3 ·5H 2 O...........................................................................0.3mg
Preparation of Trace Elements 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 to 1.0L. Mix thoroughly.
Vitamin Solution:
Composition per liter:
Pyridoxine-HCl........................................................................10.0mg
Thiamine-HCl·2H 2 O..................................................................5.0mg
Riboflavin ..................................................................................5.0mg
Nicotinic acid.............................................................................5.0mg
D -Ca-pantothenate......................................................................5.0mg
p-Aminobenzoic acid.................................................................5.0mg
Lipoic acid .................................................................................5.0mg
Biotin .........................................................................................2.0mg
Folic acid....................................................................................2.0mg
Vitamin B 12 ................................................................................0.1mg
Preparation of Vitamin Solution:  Add components to distilled/
deionized water and bring volume to 1.0L. Mix thoroughly. Sparge
with 80% H 2 + 20% CO 2 . Filter sterilize.
Preparation of Medium: Add components, except Na 2 CO 3  solu-
tion, Na 2 S·9H 2 O solution, and  L -cysteine solution, to distilled/deion-
ized water and bring volume to 1.0L. Mix thoroughly. Sparge with 100%
N 2 for 30 min. Autoclave for 15 min at 15 psi pressure–121°C. Asepti-
cally distribute 5.0mL aliquots into Hungate tubes under 100% N 2 .
Aseptically and anaerobically add per 5.0mL medium 0.25mL Na 2 CO 3
solution, 0.075mL Na 2 S·9H 2 O solution, and 0.075mL  L -cysteine solu-
tion. Mix thoroughly. Replace N 2  atmosphere with atmosphere of 80%
H 2 + 20% CO 2 . Repeat atmosphere replacement several times with
overpressurization. The initial pH of 9.0 will decrease over a 30 min
period to 7.3–7.5. After inoculation use atmosphere of 80% H 2 + 20%
CO 2  to 1.5 bar overpressure.
Use: For the cultivation of Methanocalculus pumilus.