Methanosarcina Nitrogen-Fixing 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]

Methanosarcina Nitrogen-Fixing Medium

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

Composition per 1005.0mL:

NaH 2 PO 4 .....................................................................................1.38g
K 2 HPO 4 .......................................................................................0.34g
MgCl 2 ·2H 2 O..................................................................................0.1g
Yeast extract..................................................................................0.1g
CaCl 2 ·2H 2 O.................................................................................0.05g
Resazurin ..................................................................................0.001g
Trace elements solution ...........................................................10.0mL
Methanol....................................................................................2.0mL
Na 2 S·9H 2 O solution...................................................................2.0mL
NaHCO 3  solution .......................................................................1.0mL
pH 7.0 ± 0.2 at 25°C
Trace Elements Solution:
Composition per liter:
Nitrilotriacetic acid.......................................................................4.5g
FeCl 2 ·7H 2 O...................................................................................0.4g
H 3 BO 3 .........................................................................................0.19g
CoCl 2 ·6H 2 O ................................................................................0.17g
MnCl 2 ·4H 2 O..................................................................................0.1g
ZnCl 2 .............................................................................................0.1g
NiCl 2 ·6H 2 O.................................................................................0.02g
Na 2 MoO 4 ·6H 2 O ..........................................................................0.01g
Preparation of Trace Elements Solution:  Add components to
distilled/deionized water and bring volume to 1.0L. Mix thoroughly.
Sparge with 100% N 2 . Adjust pH to 7.0 with 10N NaOH. Autoclave for
15 min at 15 psi pressure–121°C.
Preparation of Methanol:  Sparge 2-propanol with 100% N 2 . Filter
sterilize.
Na 2 S·9H 2 O Solution:
Composition per 10.0mL:
Na 2 S·9H 2 O....................................................................................2.0g
NaOH............................................................................................0.1g
Preparation of Na 2 S·9H 2 O Solution:  Add Na 2 S·9H 2 O and
NaOH to distilled/deionized water and bring volume to 10.0mL. Mix
thoroughly. Sparge with 100% N 2 . Autoclave for 15 min at 15 psi pres-
sure–121°C. Before use, neutralize to pH 7.0 with sterile HCl.
NaHCO 3 Solution:
Composition per 10.0mL:
NaHCO 3 ........................................................................................1.0g
Preparation of NaHCO 3 Solution:  Add NaHCO 3 to distilled/deion-
ized 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.
Preparation of Medium: Prepare and dispense medium under
100% N 2 . Add components, except methanol, Na 2 S·9H 2 O solution,
and NaHCO 3 solution, to distilled/deionized water and bring volume to
1.0L. Mix thoroughly. Adjust pH to 7.5. Sparge with 100% N 2 . Auto-
clave for 15 min at 15 psi pressure–121°C. Aseptically and anaerobi-
cally add 2.0mL of sterile methanol, 2.0mL of sterile Na 2 S·9H 2 O
solution, and 2.0mL of sterile NaHCO 3 solution. Mix thoroughly. Ad-
just pH to 7.0 by adding sterile anaerobic 10N NaOH. Aseptically and
anaerobically distribute into sterile tubes or bottles.
Use: For the cultivation of nitrogen-fixing Methanosarcina species.