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Pfennig's Medium 2 Modified for Green Sulfur Bacteria
(DSMZ Medium 29)

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

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
Neutralized sulfide solution................................................ As needed
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
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
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: a short, gas-inlet
tube with a sterile cotton filter; and 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 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
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
H 3 BO 3 ....................................................................................300.0mg
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 ........................................................................................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 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% N 2  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:
Na 2 S·9H 2 O....................................................................................1.5g
Preparation of Neutralized Sulfide Solution : Add Na 2 S·9H 2 O
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% N 2 gas for 3 min.
Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temper-
ature. Adjust pH to about 6.8 with sterile 2M H 2 SO 4 . Do not open the
bottle to add H 2 SO 4 ; 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 solution B, C, D, E, and F 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 solution) to pH 6.8. Distribute the medium asep-
tically through the medium outlet tube into sterile, 100mL bottles (with
metal caps and autoclavable rubber seals) using the positive gas pres-
sure (0.05–0.1 atm) of the N 2  /CO 2 gas mixture. Leave a small air bub-
ble 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 using a tungsten lamp. Feed peri-
odically with neutralized solution of sodium sulfide to replenish sulfide
and with other supplement solutions.
Use: For the cultivation of green sulfur bacteria.