Methanocalculus halotolerans Medium (DSMZ Medium 905)

[所属分类:培养基配方] [发布时间:2021-5-24] [发布人:网站管理员2] [阅读次数:] [返回]

Methanocalculus halotolerans Medium
(DSMZ Medium 905)

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

Composition per 1010.0mL:

NaCl............................................................................................50.0g
NH 4 Cl ...........................................................................................1.0g
Na-acetate .....................................................................................0.5g
Yeast extract..................................................................................0.5g
Trypticase™..................................................................................0.5g
K 2 HPO 4 .........................................................................................0.3g
KH 2 PO 4 .........................................................................................0.3g
KCl..............................................................................................0.17g
Resazurin ...................................................................................0.5mg
Magnesium chloride solution...................................................30.0mL
NaHCO 3  solution .....................................................................20.0mL
Trace elements solution ...........................................................10.0mL
Calcium chloride solution........................................................10.0mL
Na 2 S·9H 2 O solution.................................................................10.0mL
L -Cysteine solution..................................................................10.0mL
pH 7.2-7.6 at 25°C
Magnesium Chloride Solution:
Composition per 30.0mL:
MgCl 2 ·6H 2 O..................................................................................3.2g
Preparation of Magnesium Chloride Solution:  Add
MgCl 2 ·6H 2 O to distilled/deionized water and bring volume to 30.0mL.
Mix thoroughly. Sparge with 100% N 2 . Autoclave for 15 min at 15 psi
pressure–121°C.
Calcium Chloride Solution:
Composition per 10.0mL:
CaCl 2 ·2H 2 O...................................................................................0.6g
Preparation of Calcium Chloride Solution:  Add CaCl 2 ·2H 2 O
to distilled/deionized water and bring volume to 10.0mL. Mix thor-
oughly. 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 .....................................................................0.5g
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.
Na 2 S·9H 2 O Solution:
Composition per 10.0mL:
Na 2 S·9H 2 O....................................................................................0.3g
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 10.0mL. Mix thoroughly.
Sparge with 100% N 2 . Autoclave for 15 min at 15 psi pressure–121°C.
NaHCO 3 Solution:
Composition per 20.0mL:
NaHCO 3 ........................................................................................2.0g
Preparation of NaHCO 3 Solution:  Add NaHCO 3 to distilled/de-
ionized water and bring volume to 20.0mL. Mix thoroughly. Sparge
with 80% N 2 + 20% CO 2 . Autoclave for 15 min at 15 psi pressure–
121°C. Cool to 25°C. Must be prepared freshly.
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.
Preparation of Medium: Prepare and dispense medium under
sparge with 80% N 2 + 20% CO 2 . Add components, except NaHCO 3 so-
lution, magnesium chloride solution, calcium chloride solution,  L -
cysteine-HCl·H 2 O solution, and Na 2 S·9H 2 O solution, to distilled/de-
ionized water and bring volume to 950.0mL. Mix thoroughly. Gently
heat and bring to boiling. Boil for 3 min. Cool to room temperature while
sparging with 80% N 2 + 20% CO 2 . Add 10.0mL  L -cysteine-HCl·H 2 O
solution and 20.0mL NaHCO 3 solution. Mix thoroughly. Adjust pH to
7.5. Distribute into anaerobic tubes or bottles. Autoclave for 15 min at
15 psi pressure–121°C. Aseptically and anaerobically add per liter,
30.0mL magnesium chloride solution, 10.0mL calcium chloride solu-
tion, and 10.0mL Na 2 S·9H 2 O. Mix thoroughly. After inoculation pres-
surize vessels with 80% H 2 + 20% CO 2 gas mixture to 1 bar
overpressure and add sulfide from a sterile, anaerobic stock solution.
The final pH of the medium should be 7.2–7.6.
Use: For the cultivation of Methanocalculus halotolerans.