Methanogenium aggregans Medium (DSMZ Medium 321)

[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]

Methanogenium aggregans Medium
(DSMZ Medium 321)

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-formate....................................................................................5.0g
Na 2 CO 3 .........................................................................................1.5g
Na-acetate .....................................................................................1.0g
Trypticase™..................................................................................1.0g
Yeast extract..................................................................................1.0g
NH 4 Cl ...........................................................................................1.0g
K 2 HPO 4 ·3H 2 O...............................................................................0.4g
MgCl 2 ·6H 2 O..................................................................................0.4g
Resazurin ...................................................................................1.0mg
Mineral solution.......................................................................50.0mL
Cysteine solution......................................................................10.0mL
Na 2 S·9H 2 O solution.................................................................10.0mL
Trace elements solution ...........................................................10.0mL
Sludge fluid................................................................................5.0mL
pH 6.8 ± 0.2 at 25°C
Mineral Solution:
Composition per liter:
NaCl............................................................................................12.0g
KH 2 PO 4 .........................................................................................6.0g
(NH 4 ) 2 SO 4 .....................................................................................6.0g
MgSO 4 ·7H 2 O................................................................................2.6g
CaCl 2 ·2H 2 O.................................................................................0.16g
Preparation of Mineral Solution:  Add components to distilled/
deionized water and bring volume to 1.0L. Mix thoroughly.
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.
Cysteine Solution:
Composition per 10.0mL:
L -Cysteine·HCl·H 2 O.....................................................................0.2g
Preparation of Cysteine Solution:  Add  L -cysteine·HCl·H 2 O to
distilled/deionized water and bring volume to 10.0mL. Mix thorough-
ly. Sparge with 100% N 2 . Autoclave for 15 min at 15 psi pressure–
121°C.
Na 2 S·9H 2 O Solution :
Composition per 10.0mL:
Na 2 S·9H 2 O....................................................................................0.2g
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 10.0mL. Mix thoroughly.
Autoclave under 100% N 2 for 15 min at 15 psi pressure–121°C. Cool
to room temperature.
Sludge Fluid:
Composition per 500.0mL:
Yeast extract..................................................................................2.0g
Sludge....................................................................................500.0mL
Preparation of Sludge Fluid:  Add yeast extract to sludge from an
anaerobic digester. Gas with nitrogen gas for a few minutes. Incubate
at 37°C for 24 hours. Centrifuge the sludge at 13,000 x g. Autoclave for
15 min at 15 psi pressure–121°C. Place the resulting, clear supernatant
in screw-capped vessels under nitrogen gas. The sludge fluid can be
stored at room temperature in the dark.
Preparation of Medium: Prepare and dispense medium under an
oxygen-free 80% H 2 + 20% CO 2  gas mixture. Add components, except
sludge fluid, cysteine solution, and Na 2 S·9H 2 O solution, to distilled/
deionized water and bring volume to 975.0mL. Mix thoroughly. Sparge
with 80% H 2 + 20% CO 2 . Autoclave for 15 min at 15 psi pressure–
121°C. Cool to 25°C while sparging with 80% H 2 + 20% CO 2 . Asepti-
cally and anaerobically add 5.0mL sterile sludge fluid, 10.0mL of ster-
ile cysteine solution, and 10.0mL of sterile Na 2 S·9H 2 O solution. Mix
thoroughly. Aseptically and anaerobically distribute into sterile tubes
or flasks.
Use:  For the cultivation of Methanocorpusculum aggregans (Methano-
genium aggregans).