Methanosarcina semesiae 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 semesiae Medium

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

Composition per 1214.0mL:

Solution A..............................................................................950.0mL
NaHCO 3 solution.....................................................................50.0mL
Solution E ................................................................................30.0mL
Na 2 S·9H 2 O solution.................................................................15.0mL
Solution D................................................................................10.0mL
Solution F.................................................................................10.0mL
Solution G................................................................................10.0mL
Trace elements solution...........................................................10.0mL
Vitamin solution.......................................................................10.0mL
Methanol solution......................................................................5.0mL
Solution B..................................................................................1.0mL
Solution C..................................................................................1.0mL
Seven vitamin solution ..............................................................1.0mL
Dithionite solution.....................................................................1.0mL
pH 7.2 ± 0.2 at 25°C
Solution A:
Composition per 950.0mL:
KH 2 PO 4 .........................................................................................1.4g
NH 4 Cl ...........................................................................................0.5g
MgCl 2 ·6H 2 O .................................................................................0.2g
CaCl 2 ·2H 2 O ................................................................................0.15g
Resazurin...................................................................................1.0mg
Preparation of Solution A:  Add components to distilled/deionized
water and bring volume to 950.0mL. Mix thoroughly. Sparge with 80%
N 2 + 20% CO 2 . Filter sterilize.
Solution B:
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 Solution B: Add FeCl 2 ·4H 2 O to 10.0mL of HCl so-
lution. Mix thoroughly. Add distilled/deionized water and bring vol-
ume to 1.0L. Add remaining components. Mix thoroughly. Sparge with
80% N 2 + 20% CO 2 . Autoclave for 15 min at 15 psi pressure–121°C.
Solution C:
Composition per liter:
NaOH............................................................................................0.5g
Na 2 WO 4 ·2H 2 O...........................................................................4.0mg
Na 2 SeO 3 ·5H 2 O...........................................................................3.0mg
Preparation of Solution C: Add components to distilled/deionized
water and bring volume to 1.0L. Mix thoroughly. Sparge with 100%
N 2 . Filter sterilize.
Solution D:
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 Solution D:  Add components to distilled/deionized
water and bring volume to 1.0L. Mix thoroughly. Sparge with 80% H 2
+ 20% CO 2 . Filter sterilize.
Solution E:
Composition per 100.0mL:
NaHCO 3 ........................................................................................5.0g
Preparation of Solution E:  Add NaHCO 3 to distilled/deionized
water and bring volume to 150.0mL. Mix thoroughly. Sparge with 80%
N 2 + 20% CO 2 . Filter sterilize.
Solution F:
Composition per 10.0mL:
Na 2 -maleate...................................................................................1.6g
Preparation of Solution F:  Add Na 2 -maleate to distilled/deionized
water and bring volume to 150.0mL. Mix thoroughly. Sparge with 80%
N 2 + 20% CO 2 . Filter sterilize.
Solution G :
Composition per 10.0mL:
Na 2 S·9H 2 O..................................................................................0.25g
Preparation of Solution G: Add Na 2 S·9H 2 O to distilled/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 tempera-
ture.
NaHCO 3 Solution:
Composition per 100.0mL:
NaHCO 3 ........................................................................................5.0g
Preparation of NaHCO 3 Solution:  Add NaHCO 3 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. Cool to
25°C.
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.
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% H 2 + 20% CO 2 . Filter sterilize.
Seven Vitamin Solution:
Composition per liter:
Pyridoxine hydrochloride ......................................................300.0mg
Thiamine-HCl·2H 2 O..............................................................200.0mg
Nicotinic acid.........................................................................200.0mg
Vitamin B 12 ............................................................................100.0mg
Calcium pantothenate ............................................................100.0mg
p-Aminobenzoic acid...............................................................80.0mg
D (+)-Biotin...............................................................................20.0mg
Preparation of Seven Vitamin Solution: Add components to dis-
tilled/deionized water and bring volume to 1.0L. Sparge with 100% N 2 .
Mix thoroughly. 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 20.0mL:
Na 2 S·9H 2 O....................................................................................0.6g
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 20.0mL. Mix thoroughly.
Autoclave under 100% N 2 for 15 min at 15 psi pressure–121°C. Cool
to room temperature.
Dithionite Solution
Composition per 10.0mL:
Na-dithionite...............................................................................0.25g
Preparation of Dithionite Solution: Add Na-dithionite to dis-
tilled/deionized water and bring volume to 10.0mL. Mix thoroughly.
Sparge with 100% N 2 . Filter sterilize.
Preparation of Medium: Prepare and dispense medium under 80%
N 2 + 20% CO 2 . Combine component solutions A-G; do not add
NaHCO 3 solution, Na 2 S·9H 2 O solution, trace elements solution, vita-
min solution, methanol solution, seven vitamin solution, and dithionite
solution. Mix thoroughly. Flush medium with 80% N 2 + 20% CO 2  for
5 min. Distribute into serum bottles. Autoclave for 15 min at 15 psi pres-
sure–121°C. Cool to 25°C. For every 10.0mL medium aseptically and
anaerobically add from sterile anaerobic solution: 0.5mL NaHCO 3 so-
lution, 0.1mL trace elements solution, 0.1mL vitamin solution, 0.01mL
seven vitamin solution, 0.01mL dithionite solution, 0.15mL
Na 2 S·9H 2 O solution, and 0.05mL methanol solution. Final pH should
be 7.0.
Use:  For the cultivation of Methanosarcina semesiae.