Methanosarcina Thermophilic Medium (DSMZ Medium 164)

[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 Thermophilic Medium
(DSMZ Medium 164)

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

Composition per 1051.0mL:

NaCl............................................................................................2.25g
Yeast extract..................................................................................2.0g
Casitone........................................................................................2.0g
NH 4 Cl ...........................................................................................0.5g
MgSO 4 ·7H 2 O................................................................................0.5g
K 2 HPO 4 .....................................................................................0.348g
CaCl 2 ·2H 2 O ................................................................................0.25g
KH 2 PO 4 .....................................................................................0.227g
FeSO 4 ·7H 2 O..............................................................................0.002g
Resazurin..................................................................................0.001g
Sludge fluid..............................................................................50.0mL
Methanol solution....................................................................10.0mL
Vitamin solution.......................................................................10.0mL
NaHCO 3  solution.....................................................................10.0mL
L -Cysteine solution..................................................................10.0mL
Na 2 S·9H 2 O solution.................................................................10.0mL
Trace elements solution SL-10..................................................1.0mL
pH 6.5–6.8 at 25°C
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 hr. 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.
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% N 2 + 20% CO 2 . Filter sterilize.
Methanol Solution:
Composition per 100.0mL:
Methanol..................................................................................50.0mL
Preparation of Methanol Solution:  Add methanol to distilled/de-
ionized water and bring volume to 100.0mL. Mix thoroughly. Sparge
with 100% N 2 . Autoclave for 15 min at 15 psi pressure–121°C.
Na 2 S·9H 2 O Solution:
Composition per 100.0mL:
Na 2 S·9H 2 O....................................................................................3.0g
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 100.0mL. 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.
NaHCO 3 Solution:
Composition per 100.0mL:
NaHCO 3 ........................................................................................8.5g
Preparation of NaHCO 3 Solution:  Add NaHCO 3 to distilled/de-
ionized water and bring volume to 100.0mL. Mix thoroughly. Auto-
clave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Must be
prepared freshly.
Trace Elements Solution SL-10:
Composition per liter:
FeCl 2 ·4H 2 O...................................................................................1.5g
H 3 BO 3 ....................................................................................300.0mg
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
CuCl 2 ·2H 2 O ...............................................................................2.0mg
HCl (25% solution)....................................................................7.7mL
Preparation of Trace Elements Solution SL-10: Add 1.5g of
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 compo-
nents. Mix thoroughly. Sparge with 100% N 2 . Autoclave for 15 min at
15 psi pressure–121°C.
L -Cysteine Solution:
Composition per 10.0mL:
L -Cysteine·HCl·H 2 O.....................................................................0.3g
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%
N 2 + 20% CO 2 gas atmosphere. Add components, except vitamin solu-
tion, NaHCO 3 solution, methanol solution,  L -cysteine-HCl·H 2 O solu-
tion, Na 2 S·9H 2 O solution, and trace elements solution SL-10, to
distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Ad-
just pH to 6.5. Sparge with 80% N 2 + 20% CO 2 . Autoclave for 15 min at
15 psi pressure–121°C. Aseptically and anaerobically add 10.0mL vita-
min solution, 10.0mL NaHCO 3 solution, 10.0mL methanol solution,
10.0mL  L -cysteine-HCl·H 2 O solution, 10.0mL Na 2 S·9H 2 O solution,
and 1.0mL trace elements solution SL-10. Mix thoroughly. Aseptically
and anaerobically distribute into sterile tubes or bottles. After inocula-
tion, flush and repressurize the gas head space of culture bottles with
sterile 80% H 2 + 20% CO 2 to 1 bar overpressure. Alternately, the me-
dium without vitamin solution, NaHCO 3 solution, methanol solution,
L -cysteine-HCl·H 2 O solution, Na 2 S·9H 2 O solution, and trace elements
solution SL-10 can be distributed to tubes anaerobically prior to auto-
claving. After autoclaving in tubes the appropriate volumes of the in-
dividual solutions can be injected through the stoppers so that the final
concentrations of the medium are achieved.
Use: For the cultivation of Methanosarcina thermophila.