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Thermococcus chitinophagus Medium (DSMZ Medium 766)

[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]

Thermococcus chitinophagus Medium

(DSMZ Medium 766)

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

Composition per liter:

Chitin, purified.............................................................................. 4.0g

(NH4)2SO4 .................................................................................... 0.5g

KH2PO4......................................................................................... 0.5g

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

(NH4)2Ni(SO4)2..........................................................................0.3mg

Na2WO4·2H2O .........................................................................0.15mg

Na2SeO4 ...................................................................................0.15mg

Synthetic seawater .................................................................485.0mL

Trace elements solution SL-6 ..................................................15.0mL

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

NaHCO3 solution .....................................................................10.0mL

pH 6.7 ± 0.2 at 25°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.

Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C. Cool

to room temperature.

NaHCO3 Solution:

Composition per 10.0mL:

NaHCO3 ........................................................................................ 0.2g

Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/de

ionized water and bring volume to 10.0mL. Mix thoroughly. Sparge

with 80% N2 + 20% CO2. Filter sterilize.

Trace Elements Solution SL-6:

Composition per liter:

MnCl2·4H2O.................................................................................. 0.5g

H3BO3 ........................................................................................... 0.3g

CoCl2·6H2O .................................................................................. 0.2g

ZnSO4·7H2O ................................................................................ 0.1g

Na2MoO4·2H2O ......................................................................... 0.03g

NiCl2·6H2O ................................................................................. 0.02g

CuCl2·2H2O ................................................................................ 0.01g

Preparation of Trace Elements Solution SL-6: Add components

to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.

Autoclave for 15 min at 15 psi pressure–121°C.

Synthetic Seawater:

Composition per liter:

NaCl ........................................................................................ 23.477g

MgCl2·6H2O.............................................................................. 4.981g

Na2SO4...................................................................................... 3.917g

CaCl2 .......................................................................................... 1.12g

KCl.........................................................................................664.0mg

NaHCO3 .................................................................................192.0mg

H3BO3 ......................................................................................26.0mg

SrCl2........................................................................................24.0mg

KBr.............................................................................................6.0mg

NaF.............................................................................................3.0mg

Preparation of Synthetic Seawater: Add components to dis

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

sterilize.

Preparation of Purified Chitin: Cool 200.0mL of 37% HCl to 4°C.

Add 20.0g chitin (practical grade from crab shells) to the cooled HCl.

Mix thoroughly. Stir for 60 min at 4°C. Pour the suspension into 1 liter

of distilled water, pre-cooled to 4°C. Filter through filter paper. Wash

the residue five times with 500.0mL distilled water. Resuspend in 1.0L

of distilled water. Neutralize the suspension with 10.0mL of 5M KOH

to achieve a final pH of 6.5. Filter and wash with 3.0L of distilled water

to remove KCl. Allow to air dry.

Preparation of Medium: Add components, except NaHCO3 solu

tion and Na2S·9H2O solution, to distilled/deionized water and bring

volume to 980.0mL. Gently heat and bring to boiling. Boil for 5 min.

Cool to 25°C while sparging with 100% N2. Add 10.0mL NaHCO3 so

lution. Adjust pH to 6.7. Distribute the medium into Hungate tubes un

der an atmosphere of 100% N2. Autoclave for 15 min at 15 psi pressure–

121°C. Cool to room temperature. Reduce the medium by adding

10.0mL Na2S·9H2O solution. Aseptically and anaerobically distribute

to sterile tubes or bottles.

Use: For the cultivation of Thermococcus chitinophagus.

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Thermococcus chitinophagus Medium (DSMZ Medium 766)