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培养基配方

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Thermofilum pendens 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:

Sulfur, powdered......................................................................... 10.0g

(NH4)2SO4 .................................................................................... 1.3g

KH2PO4....................................................................................... 0.28g

MgSO4·7H2O.............................................................................. 0.25g

CaCl2·2H2O ................................................................................ 0.07g

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

FeCl3·6H2O................................................................................. 0.02g

Na2B4O7·10H2O......................................................................... 4.5mg

MnCl2·4H2O .............................................................................. 1.8mg

ZnSO4·7H2O ............................................................................ 0.22mg

CuCl2·2H2O ............................................................................. 0.05mg

Na2MoO4·2H2O ....................................................................... 0.03mg

VOSO4·2H2O........................................................................... 0.03mg

CoSO4·7H2O............................................................................ 0.01mg

Yeast extract solution...............................................................20.0mL

Sucrose solution.......................................................................20.0mL

Polar lipid fraction ............................................................6.0–12.0mL

pH 5.2 ± 0.2 at 25°C

Yeast Extract Solution:

Composition per 20.0mL:

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

Preparation of Yeast Extract Solution: Add yeast extract to dis

tilled/deionized water and bring volume to 20.0mL. Mix thoroughly.

Gently heat and bring to boiling. Boil for a few minutes. Sparge with

100% N2. Do not autoclave.

Sucrose Solution:

Composition per 20.0mL:

Sucrose.......................................................................................... 2.0g

Preparation of Sucrose Solution: Add sucrose to distilled/deion

ized water and bring volume to 20.0mL. Mix thoroughly. Filter steril

ize. Sparge with 100% N2.

Preparation of Sulfur: Add 10.0g of powdered sulfur to a flask and

sterilize by steaming for 3 hr on 3 consecutive days.

Polar Lipid Fraction:

Composition per 20.0mL:

Thermoproteus tenax cells (wet weight)..................................... 10.0g

Chloroform ............................................................................500.0mL

Acetone..................................................................................500.0mL

Methanol................................................................................500.0mL

TA buffer solution....................................................................80.0mL

Chloroform/methanol 1:1 (v/v)................................................20.0mL

TA Buffer Solution:

Composition per 100.0mL:

Tris·HCl ...................................................................................... 0.79g

β-mercaptoethanol ...................................................................... 0.78g

NH4Cl ....................................................................................... 0.118g

EDTA........................................................................................ 0.029g

Preparation of TA Buffer Solution: Add components to distilled/

deionized water and bring volume to 100.0mL. Mix thoroughly.

Preparation of Polar Lipid Fraction: Add 10.0g (wet weight) of

Thermoproteus tenax cells to 20.0mL of TA buffer solution. Mix thor

oughly. Sonicate for 2 min. Centrifuge at 20,000 rpm for 20 min. Re

suspend pellet in 20.0mL of fresh TA buffer solution. Recentrifuge at

20,000 rpm for 20 min. Again resuspend pellet in 20.0mL of fresh TA

buffer solution. Recentrifuge at 20,000 rpm for 20 min. Resuspend pel

let in 20.0mL of fresh TA buffer solution. Centrifuge at 5,000 rpm for

5 min. Decant the supernatant solution and discard the pellet. Extract

the supernatant solution twice with 20.0mL of chloroform/methanol

(1:1) each time. Chromatograph the extract on a SIL-LC (325 mesh) si

licic acid column (20cm × 2cm) using 500.0mL of chloroform, fol

lowed by 500.0mL of acetone, followed by 500.0mL of methanol. The

methanol fraction is further purified by DEAE chromatography using

chloroform/methanol 1:1 and methanol.

Preparation of Medium: Prepare and dispense medium under

100% N2. Add components, except yeast extract solution, sucrose so

lution, sulfur, and polar lipid fraction, to distilled/deionized water and

bring volume to 950.0mL. Mix thoroughly. Sparge with 100% N2. An

aerobically distribute into tubes or bottles. Autoclave for 15 min at 15

psi pressure–121°C. Aseptically and anaerobically add to 1.0L of me

dium 20.0mL of sterile yeast extract solution, 20.0mL of sterile sucrose

solution, 10.0g of sterile sulfur, and 6.0–12.0mL of sterile polar lipid

fraction. Mix thoroughly.

Use: For the cultivation and maintenance of Thermofilum pendens.

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Thermofilum pendens Medium