Medium for Chlorobium ferrooxidans (DSMZ Medium 29a)

[所属分类:培养基配方] [发布时间:2021-5-19] [发布人:网站管理员2] [阅读次数:] [返回]

Medium for Chlorobium ferrooxidans
(DSMZ Medium 29a)

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.................................................................................30.0mL
Solution C..................................................................................5.0mL
Solution D..................................................................................5.0mL
pH 6.8 at 25°C
Solution A:
Composition per 4.0L:
MgSO 4 ..........................................................................................2.5g
KH 2 PO 4 .........................................................................................1.7g
NH 4 Cl ...........................................................................................1.7g
KCl................................................................................................1.7g
CaCl 2 ·2H 2 O ................................................................................1.25g
Na-acetate...................................................................................0.82g
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
are for tubes 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 solution A to room temperature under a N 2  atmosphere with a
positive pressure of 0.05–0.1 atm (a manometer for low pressure will
be required). Saturate the cold medium with CO 2  by magnetic stirring
for 30 min under a CO 2 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 N 2 in an anaerobic jar.
Solution C:
Composition per 100.0mL:
Vitamin B 12 ................................................................................2.0mg
Preparation of Solution C:  Add vitamin B 12 to distilled/deionized
water and bring volume to 100.0mL. Mix thoroughly. Sparge under
100% N 2  gas for 3 min. Filter sterilize. Store under N 2  gas.
Solution D:
Composition per liter:
FeCl 2 ·4H 2 O...................................................................................1.5g
H 3 BO 3 ....................................................................................300.0mg
CoCl 2 ·6H 2 O...........................................................................190.0mg
MnCl 2 ·4H 2 O ..........................................................................100.0mg
ZnCl 2 ........................................................................................70.0mg
Na 2 MoO 4 ·2H 2 O.......................................................................36.0mg
NiCl 2 ·6H 2 O..............................................................................24.0mg
CuCl 2 ·2H 2 O ...............................................................................2.0mg
HCl (25% solution)....................................................................7.7mL
Preparation of Solution D: Add FeCl 2 ·4H 2 O to 10.0mL of HCl so-
lution. Mix thoroughly. Add distilled/deionized water and bring vol-
ume to 1.0L. Add remaining components. Mix thoroughly. Sparge with
100% N 2 . Autoclave for 15 min at 15 psi pressure–121°C.
Solution E:
Composition per 100.0mL:
NaHCO 3 ........................................................................................4.2g
Preparation of Solution E : Add NaHCO 3 to distilled/deionized
water and bring volume to 100.0mL. Mix thoroughly. Sparge with
100% CO 2 until saturated. Filter sterilize under 100% CO 2 into a ster-
ile, gas-tight 100.0mL screw-capped bottle.
Solution F:
Composition per 100.0mL:
FeSO 4 ..........................................................................................25.0g
Preparation of Solution F : Add FeSO 4 to distilled/deionized water
and bring volume to 100.0mL. Mix thoroughly. Sparge with 100% CO 2
until saturated. Filter sterilize under 100% CO 2 into a sterile, gas-tight
100.0mL screw-capped bottle.
Preparation of Medium:  Add solutions B, C, D, and E to solution
A through one of the screw-cap openings against a stream of either N 2
gas or, better, a mixture of 95% N 2 and 5% CO 2 while the medium is
magnetically stirred. Adjust the pH of the medium with sterile HCl or
Na 2 CO 3 solution (2M solutions) to pH 6.8. 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 N 2  /CO 2 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.
Use: For the cultivation of Chlorobium ferrooxidans.