Methanosphaerula Medium (DSMZ Medium 1094)

[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]

Methanosphaerula Medium
(DSMZ Medium 1094)

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

Composition per liter:

Solution A.....................................................................................1.0L
Solution B..................................................................................1.0mL
Solution C..............................................................................12.55mL
Solution D................................................................................20.0mL
Solution E ................................................................................10.0mL
Solution F.................................................................................10.0mL
Solution G................................................................................10.0mL
Solution H................................................................................10.0mL
pH 6.9 ± 0.2 at 25°C
Solution A :
Composition per liter:
NH 4 Cl .....................................................................................26.8mg
KH 2 PO 4 ...................................................................................13.6mg
KCl ............................................................................................1.5mg
Preparation of Solution A:  Add components to distilled/deionized
water and bring volume to 1.0L. Mix thoroughly.
Solution B :
Composition per liter:
Na 2 EDTA ...............................................................................37.229g
KAl(SO 4 ) 2 ·12H 2 O.....................................................................3.446g
CaCl 2 ·2H 2 O ..............................................................................2.336g
MgSO 4 ·7H 2 O............................................................................1.556g
FeCl 2 ·4H 2 O...............................................................................1.344g
CoCl 2 ·6H 2 O................................................................................0.24g
ZnCl 2 .........................................................................................0.075g
MnSO 4 ·2H 2 O............................................................................0.026g
NiCl 2 ·6H 2 O...............................................................................0.024g
NaMoO 4 ·2H 2 O..........................................................................0.024g
H 3 BO 3 .......................................................................................0.019g
CuSO 4 .......................................................................................0.009g
Preparation of Solution B:  Add components to distilled/deionized
water and bring volume to 1.0L. Mix thoroughly.
Solution C :
Composition per 12.55mL:
TRIS-HCl, 1.0M, pH 8.0 ...........................................................7.2mL
Sodium nitrilotriacetic acid, 0.5M.............................................4.8mL
TiCl 3 , 15% in HCl....................................................................0.55mL
Preparation of Solution C:  Combine solutions. Mix thoroughly.
Filter sterilize. Store under N 2  gas atmosphere.
Solution D :
Composition per 20.0mL:
MES .........................................................................................3.905g
NaOH ...........................................................................................0.8g
Preparation of Solution D:  Add components to distilled/deionized
water and bring volume to 20.0mL. Mix thoroughly. Adjust to pH 7.5
with NaOH. Filter sterilize. Store under N 2  gas atmosphere.
Solution E :
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 E:  Add components to distilled/deionized
water and bring volume to 1.0L. Mix thoroughly. Sparge with 100%
N 2 . Filter sterilize. Store under N 2  gas atmosphere.
Solution F :
Composition per 10.0mL:
2-Mercaptoethanesulfonic acid (coenzyme M) .......................0.082g
Preparation of Solution F:  Add components to distilled/deionized
water and bring volume to 10.0mL. Mix thoroughly. Sparge with N 2
gas. Autoclave for 15 min at 15 psi pressure–121°C. Store under N 2  gas
atmosphere.
Solution G :
Composition per 10.0mL:
Na-acetate ..................................................................................0.68g
Preparation of Solution G:  Add Na-acetate to distilled/deionized
water and bring volume to 10.0mL. Mix thoroughly. Sparge with N 2
gas. Autoclave for 15 min at 15 psi pressure–121°C. Store under N 2  gas
atmosphere.
Solution H :
Composition per 10.0mL:
Na 2 S·9H 2 O...............................................................................0.96mg
Preparation of Solution H: Add Na 2 S·9H 2 O to distilled/deionized
water and bring volume to 10.0mL. Mix thoroughly. Sparge with N 2
gas. Filter sterilize. Store in sealed vial under N 2  gas atmosphere.
Preparation of Medium: Add 1.0mL of solution B to 1.0L of solu-
tion A. Gently heat and bring to boiling. Boil for 1 min. Cool to room
temperature under 80% N 2  + 20% CO 2  gas mixture. Dispense under
same gas atmosphere into culture vessels. Autoclave for 15 min at 15
psi pressure–121°C. Aseptically add solutions C to H in the sequence
indicated and let medium equilibrate overnight. After inoculation pres-
surize vials to 1 bar overpressure with 80% H 2  + 20% CO 2  gas mixture.
The final pH should be 5.7.
Use: For the cultivation of Methanosphaerula spp.