Methanospirillum hungatei SAM Medium

[所属分类:培养基配方] [发布时间:2021-5-27] [发布人:网站管理员2] [阅读次数:] [返回]

Methanospirillum hungatei SAM 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:

Na 2 CO 3 .........................................................................................2.63
Sodium acetate·3H 2 O....................................................................2.5g
(NH 4 ) 2 SO 4 ..................................................................................0.45g
K 2 HPO 4 .........................................................................................0.3g
KH 2 PO 4 .......................................................................................0.18g
MgSO 4 ·9H 2 O..............................................................................0.12g
CaCl 2 ·2H 2 O ................................................................................0.06g
NaCl............................................................................................0.05g
Na 2 CO 3  solution.......................................................................20.0mL
Trace minerals solution............................................................10.0mL
Vitamin solution.......................................................................10.0mL
L -Cysteine/Na 2 S solution.........................................................10.0mL
FeSO 4 ·7H 2 O solution.................................................................5.0mL
Resazurin solution .....................................................................0.2mL
pH 7.1 ± 0.2 at 25°C
Na 2 CO 3 Solution:
Composition per liter:
Na 2 CO 3 .....................................................................................100.0g
Preparation of Na 2 CO 3 Solution:  Bring 1.0L of distilled/deion-
ized water to boiling. Cool to room temperature while sparging with
80% H 2 + 20% CO 2 . Add Na 2 CO 3 . Distribute into serum bottles fitted
with butyl rubber stoppers and aluminum seals. Do not grease stoppers.
Autoclave for 15 min at 15 psi pressure–121°C. Store at room temper-
ature in an anaerobic chamber.
Trace Minerals Solution:
Composition per liter:
Nitrolotriacetic acid......................................................................1.5g
MnSO 4 ·2H 2 O................................................................................0.5g
Na 2 MoO 4 ·2H 2 O..........................................................................0.24g
Na 2 WO 4 ·2H 2 O..........................................................................0.165g
Na 2 SeO 3 ......................................................................................0.15g
CoCl 2 ·6H 2 O..................................................................................0.1g
NiCl 2 ·6H 2 O...................................................................................0.1g
ZnSO 4 ·7H 2 O.................................................................................0.1g
AIK(SO 4 ) 2 ·12H 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. Mix thoroughly. Add distilled/de-
ionized water to 1.0L. Adjust pH to 7.0.
Vitamin Solution:
Composition per liter:
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.
L -Cysteine/Na 2 S Solution:
Composition per liter:
L -Cysteine·HCl............................................................................1.25g
Na 2 S·7H 2 O..................................................................................1.25g
Preparation of  L -Cysteine/Na 2 S Solution:  Gently heat and
bring 75.0mL of distilled/deionized water to boiling. Add  L -
cysteine·HCl. Adjust pH to 10.0 with 3N NaOH. Add Na 2 S·9H 2 O. Mix
thoroughly. Gently heat and bring to boiling. Cool to room temperature
while sparging with 100% N 2 . Bring volume to 100.0mL with distilled/
deionized water. Gently heat and bring to boiling. Cool to 60°C while
sparging with 100% N 2 . Anaerobically distribute into serum bottles.
Autoclave for 15 min at 15 psi pressure–121°C.
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.
Resazurin Solution:
Composition per 10.0mL:
Resazurin .................................................................................10.0mg
Preparation of Resazurin Solution:  Add resazurin to distilled/
deionized water and bring volume to 10.0mL. Mix thoroughly.
Preparation of Medium: Prepare and dispense medium under 80%
H 2 + 20% CO 2 . Add components, except Na 2 CO 3 , Na 2 CO 3  solution,
and L -cysteine/Na 2 S solution, to distilled/deionized water and bring
volume to 1.0L. Mix thoroughly. Adjust pH to 7.0. Gently heat and
bring to boiling. Continue boiling for 3 min. Cool to 60°C while sparg-
ing with 80% H 2 + 20% CO 2 . Add Na 2 CO 3  and L -cysteine/Na 2 S solu-
tion. Mix thoroughly. Anaerobically distribute 10.0mL volumes into
anaerobic tubes. Autoclave for 20 min at 15 psi pressure–121°C. Asep-
tically and anaerobically add 0.2mL of sterile Na 2 CO 3  solution to each
tube. Mix thoroughly. Adjust pH to 7.1.
Use:  For the cultivation of Methanospirillum hungatei.