Desulfonema ishimotoi Medium (DSMZ Medium 739)

[所属分类:培养基配方] [发布时间:2020-6-8] [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

Desulfonema ishimotoi Medium
(DSMZ Medium 739)

培养基配方
Composition per 1007.6mL:
Solution A.......................................................................850.0mL
Agar solution.....................................................................50.0mL
Solution C........................................................................50.0mL
Solution H........................................................................20.0mL
Solution D........................................................................10.0mL
Solution F (Vitamin solution).....................................................10.0mL
Solution I........................................................................10.0mL
Solution G (Artificial sediment)...................................................6.6mL
Solution E ........................................................................1.0mL
Solution B (Trace elements solution SL-10).........................................1.0mL
pH 6.9 ± 0.2 at 25°C
Solution A:
NaCl...............................................................................21.0g
MgCl2 ·6H2O........................................................................5.5g
Na2SO4 .............................................................................3.0g
CaCl2 ·2H2O ......................................................................1.35g
KCl.................................................................................0.5g
NH4Cl ..............................................................................0.3g
KH2PO4 .............................................................................0.2g
Resazurin .........................................................................0.5mg
Na2SeO3 ·5H2O.......................................................................3μg
Preparation of Solution A: Add components to distilled/deionized
water and bring volume to 800.0mL. Mix thoroughly.
Solution B (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 Solution B (Trace Elements Solution SL-10):
Add FeCl2 ·4H2O to 10.0mL of HCl solution. Mix thoroughly. Add dis-
tilled/deionized water and bring volume to 1.0L. Add remaining com-
ponents. Mix thoroughly. Sparge with 100% N2 . Autoclave for 15 min
at 15 psi pressure–121°C.
Solution C:
Composition per 100.0mL:
NaHCO3 ...........................................................................5.0g
Preparation of Solution C: Add NaHCO3 to distilled/deionized
water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize.
Flush with 80% N2  + 20% CO2 to remove dissolved oxygen.
Solution D:
Composition per 10.0mL:
Na-acetate·3H2O..................................................................2.5g
Isobutyric acid .................................................................0.18g
Na2 -succinate....................................................................0.1g
Preparation of Solution D: Add components to distilled/deionized
water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100%
N2 . Filter sterilize.
Solution E:
Composition per 10.0mL:
Na2 -succinate......................................................................1.0g
Preparation of Solution E: Add Na2 -succinate to distilled/deion-
ized water and bring volume to 10.0mL. Mix thoroughly. Sparge with
100% N2 . Autoclave for 15 min at 15 psi pressure–121°C.
Solution F (Vitamin Solution):
Composition per liter:
Pyridoxine-HCl....................................................................10.0mg
Vitamin B 12 ......................................................................5.1mg
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
Preparation of Solution F (Vitamin Solution): Add compo-
nents to distilled/deionized water and bring volume to 1.0L. Mix thor-
oughly. Filter sterilize. Sparge with 100% N2 .
Solution G (Artificial Sediment):
Composition per 6.6mL:
AlCl3 ·6H2O, 4.9% (w/v)............................................................5.0mL
Na2CO3 , 10.6% (w/v) ...............................................................1.6mL
Preparation of Solution G (Artificial Sediment): Combine
components. Mix thoroughly. Sparge with 100% N2 . Autoclave for 15
min at 15 psi pressure–121°C.
Solution H:
Composition per 20.0mL:
Rumen fluid, clarified.............................................................20.0mL
Preparation of Solution H: Sparge with 100% N2 . Autoclave for
15 min at 15 psi pressure–121°C.
Solution I:
Composition per 10.0mL:
Na2S·9H2O..........................................................................0.4g
Preparation of Solution I:  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.
Preparation of Medium: Gently heat solution A and bring to boil-
ing. Boil solution A for a few minutes. Cool to room temperature. Gas
with 80% N2  + 20% CO2 gas mixture to reach a pH below 6. Autoclave
for 15 min at 15 psi pressure–121°C. Add 50.0mL hot agar solution.
Mix thoroughly. Cool to 50°C. Sequentially add 1.0mL solution B,
100.0mL solution C, 10.0mL solution D, 1.0mL solution E, 10.0mL
solution F, 6.6mL solution G, 20.0mL solution H, and 10.0mL solution
I. Distribute anaerobically under 80% N2  + 20% CO2 into appropriate
vessels. The pH should be 6.9. Addition of 10–20mg sodium dithionite
per liter from a 5% (w/v) solution, freshly prepared under N2  and filter
sterilized, may stimulate growth.
Use: For the cultivation of Desulfonema ishimotoi.