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Thermophilic Methanothrix Medium

[所属分类:培养基配方] [发布时间:2021-9-7] [发布人:网站管理员2] [阅读次数:] [返回]

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

Composition per liter:

NH4Cl ........................................................................................... 0.5g

K2HPO4......................................................................................... 0.4g

MgCl2·6H2O.................................................................................. 0.1g

Resazurin ...................................................................................1.0mg

NaHCO3 solution .....................................................................20.0mL

Trace elements solution ..........................................................10.0mL

CaCl2·2H2O solution................................................................10.0mL

Sodium acetate solution ...........................................................10.0mL

Vitamin solution.......................................................................10.0mL

Coenzyme M solution..............................................................10.0mL

Na2S·9H2O solution ...................................................................5.0mL

pH 6.5 ± 0.2 at 25°C

NaHCO3 Solution:

Composition per 20.0mL:

NaHCO3........................................................................................ 1.0g

Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/de

ionized water and bring volume to 20.0mL. Mix thoroughly. Sparge

with 80% N2 + 20% CO2 for 15 min. Autoclave for 15 min at 15 psi

pressure–121°C.

Trace Elements Solution:

Composition per liter:

MgSO4·7H2O............................................................................... 3.0g

Nitrilotriacetic acid ....................................................................... 1.5g

NaCl.............................................................................................. 1.0g

MnSO4·2H2O............................................................................... 0.5g

CoSO4·7H2O............................................................................... 0.18g

ZnSO4·7H2O ............................................................................... 0.18g

CaCl2·2H2O .................................................................................. 0.1g

FeSO4·7H2O.................................................................................. 0.1g

NiCl2·6H2O............................................................................... 0.025g

KAl(SO4)2·12H2O....................................................................... 0.02g

CuSO4·5H2O............................................................................... 0.01g

H3BO3 ......................................................................................... 0.01g

Na2MoO4·2H2O ......................................................................... 0.01g

Na2SeO3·5H2O........................................................................... 0.3mg

Preparation of Trace Elements Solution: Add nitrilotriacetic

acid to approximately 500.0mL of distilled/deionized water. Dissolve

by adding KOH and adjust pH to 6.5. Add remaining components.

Bring volume to 1.0L with additional distilled/deionized water. Adjust

pH to 7.0 with KOH.

CaCl2·2H2O Solution:

Composition per 10.0mL:

CaCl2·2H2O .................................................................................. 0.1g

Preparation of CaCl2·2H2O Solution: Add CaCl2·2H2O to dis

tilled/deionized water and bring volume to 10.0mL. Mix thoroughly.

Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C.

Sodium Acetate Solution:

Composition per 10.0mL:

Sodium acetate.............................................................................. 3.3g

Preparation of Sodium Acetate Solution: Add sodium acetate to

distilled/deionized water and bring volume to 10.0mL. Mix thorough

ly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–

121°C.

Vitamin Solution:

Composition per liter:

Pyridoxine·HCl ........................................................................ 10.0mg

Calcium DL-pantothenate........................................................... 5.0mg

Lipoic acid ................................................................................. 5.0mg

Nicotinic acid............................................................................. 5.0mg

p-Aminobenzoic acid................................................................. 5.0mg

Riboflavin .................................................................................. 5.0mg

Thiamine·HCl ............................................................................ 5.0mg

Biotin ......................................................................................... 2.0mg

Folic acid ................................................................................... 2.0mg

Vitamin B12 ................................................................................ 0.1mg

Preparation of Vitamin Solution: Add components to distilled/de

ionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 80%

N2 + 20% CO2.

Coenzyme M Solution:

Composition per 10.0mL:

Coenzyme M............................................................................. 0.142g

Preparation of Coenzyme M Solution: Add coenzyme M to dis

tilled/deionized water and bring volume to 10.0mL. Mix thoroughly.

Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C.

Na2S·9H2O Solution:

Composition per 10.0mL:

Na2S·9H2O.................................................................................... 0.5g

Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to dis

tilled/deionized water and bring volume to 10.0mL. Mix thoroughly.

Sparge with 80% N2 + 20% CO2. Autoclave for 15 min at 15 psi pres

sure–121°C.

Preparation of Medium: Add components, except NaHCO3 solu

tion, CaCl2·2H2O solution, sodium acetate solution, vitamin solution,

coenzyme M solution, and Na2S·9H2O solution, to distilled/deionized

water and bring volume to 935.0mL. Gently heat and bring to boiling.

Continue boiling for 10 min. Cool to room temperature while sparging

with 80% N2 + 20% CO2. Gas the medium until the pH reaches 5.8. An

aerobically distribute the medium into serum bottles. Autoclave for 15

min at 15 psi pressure–121°C. Aseptically and anaerobically add

20.0mL of sterile NaHCO3 solution, 10.0mL of sterile CaCl2·2H2O so

lution, 10.0mL of sterile sodium acetate solution, 10.0mL of sterile vi

tamin solution, 10.0mL of sterile coenzyme M solution, and 5.0mL of

sterile Na2S·9H2O solution. Bring gas atmosphere in each bottle to

30% CO2.

Use: For the cultivation and maintenance of Methanothrix thermo

phila.

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Thermophilic Methanothrix Medium