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Pfennig’s Medium 2 with Salt

山東拓普生物工程有限公司 培養(yǎng)基配方 http://qp8008.cn

Composition per 5000.0mL:

Solution A............................................................................4000.0mL
Solution B..............................................................................860.0mL
Solution E ..............................................................................100.0mL
Solution F.................................................................................30.0mL
Solution C (Vitamin B 12 solution) .............................................5.0mL
Solution D (Trace elements solution SL-10B)...........................5.0mL
pH 6.8 ± 0.2 at 25°C
Solution A:
Composition per 4000.0mL:
NaCl............................................................................................50.0g
CaCl 2 ·2H 2 O.................................................................................1.25g
KH 2 PO 4 .........................................................................................1.7g
NH 4 Cl ...........................................................................................1.7g
KCl................................................................................................1.7g
MgSO 4 ......................................................................................... 2.5g
Preparation of Solution A : Add components to distilled/deionized
water and bring volume to 4.0L. Mix thoroughly. Adjust pH to 6.0.
Dispense into a 5L flask with four openings at the top (two openings
are in a central silicon rubber stopper and two openings are gas-tight
screw caps). Add a teflon-coated magnetic stir bar to the flask. Auto-
clave for 45 min at 15 psi pressure–121°C. Cool to room temperature
under 100% N 2 at 0.05–0.1 atm pressure (use a manometer to measure
low pressure).
Solution B:
Composition per 860.0mL:
Distilled/deionized water.......................................................860.0mL
Preparation of Solution B : Add 860.0mL of distilled/deionized wa-
ter to a cotton-stoppered flask. Autoclave for 20 min at 15 psi pressure–
121°C. Cool to room temperature under 100% N 2 in an anaerobic jar.
Solution C (Vitamin B 12 Solution):
Composition per 5.0mL:
Vitamin B 12 ................................................................................1.0mg
Preparation of Solution C (Vitamin B 12 Solution) : Add vita-
min B 12 to distilled/deionized water and bring volume to 5.0mL. Mix
thoroughly. Filter sterilize.
Solution D (Trace Elements Solution SL-10B):
Composition per liter:
FeCl 2 ·4H 2 O...................................................................................1.5g
CoCl 2 ·6H 2 O................................................................................0.19g
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
H 3 BO 3 ........................................................................................6.0mg
CuCl 2 ·2H 2 O...............................................................................2.0mg
HCl (25% solution)..................................................................10.0mL
Preparation of Solution D (Trace Elements Solution SL-10B):
Add FeCl 2 ·4H 2 O to 10.0mL of HCl solution. Mix thoroughly. Add dis-
tilled/deionized water and bring volume to 1.0L. Add remaining com-
ponents. Mix thoroughly.
Solution E:
Composition per 100.0mL:
NaHCO 3 ........................................................................................7.5g
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:
Na 2 S·9H 2 O................................................................................. 10.0g
Preparation of Solution F : Add Na 2 S·9H 2 O to distilled/deionized
water and bring volume to 100.0mL. Mix thoroughly. Dispense into a
screw-capped bottle. Sparge with 100% N 2 for 3–4 min. Autoclave for
15 min at 15 psi pressure–121°C.
Preparation of Medium: Saturate cooled solution A under 100%
CO 2 at 0.05–0.1 atm pressure for 30 min with magnetic stirring. Add
860.0mL of solution B, 5.0mL of solution C, 5.0mL of solution D,
100.0mL of solution E, and 20.0mL of solution F through one of the
screw-cappped openings under 95% N 2  and 5% CO 2 with magnetic
stirring. Adjust pH to 6.8 with sterile 2M HCl or sterile 2M Na 2 CO 3  so-
lution. Aseptically and anaerobically distribute the medium through
the medium outlet tube into sterile 100.0mL bottles under 95% N 2  and
5% CO 2 at 0.05–0.1 atm pressure. Leave a small gas bubble in each
bottle to accommodate pressure changes. After 24 hr, the iron in the
medium will precipitate out of solution as black flocs.
Use: For the cultivation and maintenance of Chlorobium phaeobacte-
roides, Chlorobium vibrioforme, Pelodictyon luteolum, Pelodictyon
phaeum, and Prosthecochloris aestuarii.