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Thiorhodococcus Medium (DSMZ Medium 28a)

[所属分类:培养基配方] [发布时间:2021-9-15] [发布人:网站管理员2] [阅读次数:] [返回]

Thiorhodococcus Medium

(DSMZ Medium 28a)

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

Composition per 5.0L:

Solution A.....................................................................................4.0L

Solution B ..............................................................................860.0mL

Solution E ..............................................................................100.0mL

Solution F.................................................................................20.0mL

Solution C ..................................................................................5.0mL

Solution D..................................................................................5.0mL

pH 7.3 at 25°C

Solution A:

Composition per 4.0L:

NaCl.......................................................................................... 100.0g

MgCl2·6H2O ................................................................................. 5.0g

MgSO4 .......................................................................................... 2.5g

Na2S2O3·5H2O .............................................................................. 2.5g

KH2PO4......................................................................................... 1.7g

NH4Cl ........................................................................................... 1.7g

KCl................................................................................................ 1.7g

CaCl2·2H2O ................................................................................ 1.25g

Preparation of Solution A: Add components to 4.0L distilled wa

ter. Mix thoroughly. Autoclave for 45 min at 15 psi pressure–121°C in

a 5-liter special bottle or flask with four openings at the top, together

with a teflon-coated magnetic bar. In this 5-liter bottle, two openings

for tubes are in the central, silicon rubber stopper; one is a short, gas

inlet tube with a sterile cotton filter, and the other is an outlet tube for

medium, which reaches the bottom of the vessel at one end and has, at

the other end, a silicon rubber tube with a pinch cock and a bell for

aseptic dispensing of the medium into bottles. The other two openings

have gas-tight screw caps; one of these openings is for the addition of

sterile solutions and the other serves as a gas outlet. After autoclaving,

cool to room temperature under a 100% N2 atmosphere with a positive

pressure of 0.05– 0.1 atm (a manometer for low pressure will be re

quired). Saturate the cold medium with CO2 by magnetic stirring for 30

min under a CO2 atmosphere of 0.05–0.1 atm.

Solution B:

Distilled water........................................................................860.0mL

Preparation of Solution B: Autoclave distilled water for 15 min at

15 psi pressure–121°C in a cotton-stoppered Erlenmeyer flask. Cool to

room temperature under an atmosphere of N2 in an anaerobic jar.

Solution C:

Composition per 100.0mL:

Vitamin B12 ................................................................................2.0mg

Preparation of Solution C: Add vitamin B12 to distilled/deionized

water and bring volume to 100.0mL. Mix thoroughly. Sparge under

100% N2 gas for 3 min. Filter sterilize Store under N2 gas.

Solution D:

Composition per liter:

Disodium ethylendiamine tetraacetate (Disodium EDTA) ........... 3.0g

FeSO4·7H2O................................................................................. 1.1g

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

CoCl2·6H2O ................................................................................ 0.19g

MnCl2·2H2O.............................................................................50.0mg

ZnCl2........................................................................................42.0mg

NiCl2·6H2O ..............................................................................24.0mg

Na2MoO4·2H2O ...................................................................... 18.0mg

CuCl2·2H2O ...............................................................................2.0mg

Preparation of Solution D: Add components to distilled/deionized

water and bring volume to 1.0L. Mix thoroughly. Autoclave for 15 min

at 15 psi pressure–121°C.

Solution E:

Composition per 100.0mL:

NaHCO3 ........................................................................................ 7.5g

Preparation of Solution E: Add NaHCO3 to distilled/deionized

water and bring volume to 100.0mL. Mix thoroughly. Sparge with

100% CO2 until saturated. Filter sterilize under 100% CO2 into a ster

ile, gas-tight 100.0mL screw-capped bottle.

Solution F:

Composition per 100.0mL:

Na2S·9H2O.................................................................................. 10.0g

Preparation of Solution F: Add Na2S·9H2O to distilled/deionized

water in a 250.0mL screw-capped bottle fitted with a butyl rubber sep

tum and bring volume to 100.0mL. Mix thoroughly. Sparge under

100% N2 gas for 3 min. Autoclave for 15 min at 15 psi pressure–

121°C. Cool to room temperature.

Neutralized Sulfide Solution:

Composition per 100.0mL:

Na2S·9H2O.................................................................................... 1.5g

Preparation of Neutralized Sulfide Solution: Add Na2S·9H2O

to distilled/deionized water in a 250.0mL screw-capped bottle fitted

with a butyl rubber septum and bring volume to 100.0mL. Add a mag

netic stir bar. Mix thoroughly. Sparge under 100% N2 gas for 3 min.

Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temper

ature. Adjust pH to about 7.3 with sterile 2M H2SO4. Do not open the

bottle to add H2SO4; use a sterile syringe. Stir the solution continuously

to avoid precipitation of elemental sulfur. The final solution should be

clear and yellow in color.

Preparation of Medium: Add solutions B, C, D, E, and F to solu

tion A through one of the screw-cap openings against a stream of either

N2 gas or, better, a mixture of 95% N2 and 5% CO2 while the medium

is magnetically stirred. Adjust the pH of the medium with sterile HCl

or Na2CO3 solution (2M solutions) to pH 7.3. Distribute the medium

aseptically through the medium outlet tube into sterile, 100mL bottles

(with metal caps and autoclavable rubber seals) using the positive gas

pressure (0.05–0.1 atm) of the N2 /CO2 gas mixture. Leave a small air

bubble in each bottle to meet possible pressure changes. The tightly

sealed, screw-cap bottles can be stored for several weeks or months in

the dark. During the first 24 hr, the iron of the medium precipitates in

the form of black flocs. No other sediment should arise in the otherwise

clear medium. Incubate in the light at 500–1,000 lux intensity. Feed pe

riodically with neutralized solution of sodium sulfide to replenish sul

fide and with other supplement solutions.

Use: For the cultivation of Thiorhodococcus minor.

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Thiorhodococcus Medium (DSMZ Medium 28a)