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Thermophilic Methanosarcina 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 1021.0mL:

NaCl............................................................................................ 2.25g

Pancreatic digest of casein............................................................ 2.0g

Yeast extract.................................................................................. 2.0g

NaHCO3...................................................................................... 0.85g

MgSO4·7H2O................................................................................ 0.5g

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

K2HPO4 .................................................................................... 0.348g

CaCl2·2H2O ................................................................................ 0.25g

KH2PO4 .................................................................................... 0.227g

FeSO4·7H2O .............................................................................. 2.0mg

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

Rumen fluid, clarified..............................................................50.0mL

Methanol solution ....................................................................10.0mL

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

L-Cysteine·HCl·H2O solution ..................................................10.0mL

Na2S·9H2O solution.................................................................10.0mL

Trace elements solution SL-10 ..................................................1.0mL

pH 6.5–6.8 at 25°C

Methanol Solution:

Composition per 10.0mL:

Methanol....................................................................................5.0mL

Preparation of Methanol Solution: Add methanol to distilled/de

ionized water and bring volume to 10.0mL. Sparge with 100% N2. Au

toclave 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/

deionized water and bring volume to 1.0L. Mix thoroughly. Filter ster

ilize. Sparge with 80% N2 + 20% CO2.

L-Cysteine·HCl·H2O Solution:

Composition per 10.0mL:

L-Cysteine·HCl·H2O ..................................................................... 0.3g

Preparation of L-Cysteine·HCl·H2O Solution: Add L-cyste

ine·HCl·H2O to distilled/deionized water and bring volume to 10.0mL.

Mix thoroughly. Autoclave under 100% N2 for 15 min at 15 psi pres

sure–121°C.

Na2S·9H2O Solution:

Composition per 10.0mL:

Na2S·9H2O.................................................................................... 0.3g

Preparation of Na2S·9H2O Solution: Add Na2S·9H2O 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.

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 components. Mix thor

oughly. Sparge with 100% N2.

Preparation of Medium: Add components, except methanol solu

tion, L-cysteine·HCl·H2O solution, and Na2S·9H2O solution, to dis

tilled/deionized water and bring volume to 960.0mL. Mix thoroughly.

Sparge under 80% N2 + 20% CO2 for 3–4 min. Autoclave for 15 min

at 15 psi pressure–121°C. Aseptically and anaerobically add 20.0mL of

sterile vitamin solution, 10.0mL of sterile L-cysteine·HCl·H2O solu

tion, and 10.0mL of sterile Na2S·9H2O solution. Mix thoroughly.

Aseptically and anaerobically distribute into sterile screw-capped bot

tles under 80% N2 + 20% CO2.

Use: For the cultivation and maintenance of Methanosarcina thermo

phila.

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Thermophilic Methanosarcina Medium