Dehalobacter restrictus Medium (DSMZ Medium 732)

[所属分类:培养基配方] [发布时间:2020-6-2] [发布人:] [阅读次数:] [返回]

山东拓普生物工程有限公司

Shandong Tuopu Biol-Engineering Co.,Ltd

Dehalobacter restrictus Medium
(DSMZ Medium 732)

培养基配方
Composition per 1056mL:
Solution A..................................................................900.0mL
Solution B..................................................................100.0mL
Solution G...................................................................15.0mL
Solution C...................................................................10.0mL
Solution E ..................................................................10.0mL
Solution F...................................................................10.0mL
Solution H...................................................................10.0mL
Solution D....................................................................1.0mL
pH 7.2 ± 0.2 at 25°C
Solution A:
Composition per liter:
K2HPO4 .......................................................................0.653g
Na-acetate ...................................................................0.460g
NaH2PO4 ·H2O.................................................................0.173g
Peptone.........................................................................0.1g
Resazurin .....................................................................0.5mg
Preparation of Solution A: Add components to distilled/deionized
water and bring volume to 1.0 L. Mix thoroughly. Gently heat and
bring to boiling. Cool to room temperature under 80% N2 + 20% CO2
gas. Distribute 9ml volumes into 50mL serum bottles under 80% N2 +
20% CO2 gas. Pressurize closed bottles with N2 + CO2 gas to 0.5 bar
overpressure. Autoclave for 15 min at 15 psi pressure–121°C.
Solution B:
Composition per 100.0mL:
NaHCO3 .......................................................................3.730g
NH4HCO3 ......................................................................0.443g
Preparation of Solution B: Add components to distilled/deionized
water and bring volume to 100.0mL in bottles. Mix thoroughly. Flush
solution with 80% N2 + 20% CO2 gas. Close bottles. Autoclave for 15
min at 15 psi pressure–121°C.
Solution C:
Composition per 10.0mL:
MgCl2 ·6H2O ..................................................................0.12g
CaCl2 ·2H2O ..................................................................0.11g
Preparation of Solution C: Add components to distilled/deionized
water and bring volume to 10.0mL in bottles. Mix thoroughly. Flush
solution with 100% N 2 for 20 min. Close bottles. Autoclave for 15 min
at 15 psi pressure–121°C.
Solution D:
Composition per 10.0mL:
Na2 -EDTA.......................................................................5.0mg
FeCl2 ·4H2O....................................................................5.0mg
AlCl3 ..........................................................................0.1mg
Trace elements solution SL-10..................................................10.0mL
Trace Elements Solution SL-10:
Composition per liter:
FeCl2 ·4H2O.....................................................................1.5g
CoCl2 ·6H2O..................................................................190.0mg
MnCl2 ·4H2O .................................................................100.0mg
ZnCl2 .........................................................................70.0mg
Na2MoO4 ·2H2O.................................................................36.0mg
NiCl2 ·6H2O...................................................................24.0mg
H3BO3 ..........................................................................6.0mg
CuCl2 ·2H2O....................................................................2.0mg
HCl (25% solution).............................................................10.0mL
Preparation of Trace Elements Solution SL-10: Add
FeCl2 ·4H2O 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% N2 . Autoclave for 15 min at
15 psi pressure–121°C.
Preparation of Solution D: Add Na2 -EDTA, FeCl2 ·4H2O, and
AlCl3 to 10.0mL trace solution SL-10 in a Hungate bottle. Mix thor-
oughly. Flush solution with 100% N2 for 20 min. Close bottles. Auto-
clave for 15 min at 15 psi pressure–121°C.
Solution E:
Composition per liter:
Vitamin solution................................................................900.0mL
Seven vitamin solution .........................................................100.0mL
Vitamin Solution:
Composition per liter:
Pyridoxine-HCl...................................................................10.0mg
Thiamine-HCl·2H2O................................................................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.
Seven Vitamin Solution:
Composition per liter:
Pyridoxine hydrochloride ......................................................300.0mg
Thiamine-HCl·2H2O.............................................................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% N2 .
Mix thoroughly.
Preparation of Solution E: Combine 900.0mL vitamin solution
and 100.0mL seven vitamin solution. Mix thoroughly. Sparge with
100% N2 . Filter sterilize.
Solution F:
Composition per 10.0mL:
Na2S·9H2O........................................................................0.3g
Preparation of Solution F:  Add Na2S·9H2O to distilled/deionized
water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100%
N2 . Autoclave for 15 min at 15 psi pressure–121°C.
Solution G:
Hexadecane.......................................................................45.0mL
Tetrachloroethene.................................................................5.0mL
Hexadecane:
Hexadecane.......................................................................45.0mL
Preparation of Hexadecane:  Add hexadecane to a 100mL serum
bottle. Sparge with 100% N2 . Autoclave for 15 min at 15 psi pressure–
121°C.
Tetrachloroethene:
Tetrachloroethene................................................................10.0mL
Preparation of Tetrachloroethene:  Add tetrachloroethene to a
10mL serum bottle. Sparge with 100% N2 . Autoclave for 15 min at 15
psi pressure–121°C.
Preparation of Solution G:  Using a syringe, aseptically inject
5.0mL sterile tetrachloroethene to the 45.0mL sterile hexadecane in the
100mL serum bottle. Sparge with 100% N2 . Autoclave for 15 min at 15
psi pressure–121°C.
Solution H:
Composition per 10.0mL:
Na-lactate........................................................................2.5g
Preparation of Solution H:  Add Na-lactate to distilled/deionized
water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100%
N2 . Autoclave for 15 min at 15 psi pressure–121°C.
Preparation of Medium:  Add 9mL sterile solution A to a 50mL
sterile bottle. Then add by injection 1.0mL sterile solution B, 0.1mL
sterile solution C, 0.01mL sterile solution D, 0.1mL sterile solution E,
0.1mL sterile solution F, and 0.1mL solution H. Inoculate the culture
into the medium. Then add by injection 0.15mL sterile solution G.
Use: For the cultivation of Sulfurospirillum halorespirans DSM
13726.