Methanocorpusculum Medium (DSMZ Medium 279)

[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]

Methanocorpusculum Medium
(DSMZ Medium 279)

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-acetate.....................................................................................4.0g
NaHCO 3 ........................................................................................4.0g
Na-formate....................................................................................2.0g
Yeast extract..................................................................................1.0g
KH 2 PO 4 .........................................................................................0.5g
MgSO 4 ·7H 2 O................................................................................0.4g
NaCl..............................................................................................0.4g
NH 4 Cl ...........................................................................................0.4g
CaCl 2 ·2H 2 O ................................................................................0.05g
FeSO 4 ·7H 2 O..............................................................................0.002g
Resazurin ..................................................................................0.001g
NiCl 2 ·6H 2 O..............................................................................24.0mg
Sludge fluid..............................................................................50.0mL
Fatty acid mixture....................................................................20.0mL
L -Cysteine solution..................................................................10.0mL
Na 2 S·9H 2 O...............................................................................10.0mL
Trace elements solution SL-10 ..................................................1.0mL
pH 6.7–7.0
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 hrs. 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.
Fatty Acid Mixture:
Composition per 20.0mL:
Valeric acid ...................................................................................0.5g
Isovaleric acid...............................................................................0.5g
α-Methylbutyric acid....................................................................0.5g
Isobutyric acid...............................................................................0.5g
Distilled water..........................................................................20.0mL
Preparation of Fatty Acid Mixture:  Add components to 20.0mL
distilled/deionized water. Mix thoroughly.
Trace Elements Solution SL-10:
Composition per liter:
FeCl 2 ·4H 2 O...................................................................................1.5g
CoCl 2 ·6H 2 O ...........................................................................190.0mg
MnCl 2 ·4H 2 O...........................................................................100.0mg
ZnCl 2 ........................................................................................70.0mg
Na 2 MoO 4 ·2H 2 O .......................................................................36.0mg
NiCl 2 ·6H 2 O..............................................................................24.0mg
H 3 BO 3 ........................................................................................6.0mg
CuCl 2 ·2H 2 O ...............................................................................2.0mg
HCl (25% solution)..................................................................10.0mL
Preparation of Trace Elements Solution SL-10: Add FeCl 2 ·4H 2 O
to 10.0mL of HCl solution. Mix thoroughly. Add distilled/deionized
water and bring volume to 1.0L. Add remaining components. Mix thor-
oughly. 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....................................................................................0.5g
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 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.
L -Cysteine Solution:
Composition per 10.0mL:
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 10.0mL. Mix thorough-
ly. Sparge with 100% N 2 . Autoclave for 15 min at 15 psi pressure–
121°C.
Preparation of Medium: Prepare and dispense medium under 80%
H 2 + 20% CO 2 gas atmosphere. Add components, except  L -cysteine so-
lution, Na 2 S·9H 2 O solution, and trace elements solution SL-10, to dis-
tilled/deionized water and bring volume to 920.0mL. Mix thoroughly.
Adjust pH to 6.8. Sparge with 80% H 2 + 20% CO 2 . Autoclave for 15 min
at 15 psi pressure–121°C. Aseptically and anaerobically add 10.0mL  L -
cysteine solution, 10.0mL Na 2 S·9H 2 O solution, and 1.0mL trace ele-
ments solution SL-10. Mix thoroughly. Aseptically and anaerobically
distribute into sterile tubes or bottles. After inoculation, flush and re-
pressurize the gas head space of culture bottles with sterile 80% H 2 +
20% CO 2 to 1 bar overpressure. Alternately, the medium without  L -
cysteine solution, Na 2 S·9H 2 O solution, and trace elements solution SL-
10 can be distributed to tubes anaerobically prior to autoclaving. After
autoclaving in tubes the appropriate volumes of the individual solu-
tions can be injected through the stoppers so that the final concentra-
tions of the medium are achieved.
Use: For the cultivation of Methanococcoides spp. and Methanolobus
bombayensis.