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DSMZ

DSMZ 28 . PFENNIG'S MEDIUM I

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* Medium for Thiorhodococcus drewsii DSM 15006

Plus 2% NaCl

Growth conditions: 7-14 days anaerobic in light 30 °C
Equipment needed: Autoclave Filter Ice bath
Final pH: 7.1 - 7.3
Select a strain (optional):

ml
Compound Amount Unit
Solution A 460 ml
1 Aliquot Solution A into 100 mL screw-cap bottles, filled with 46 mL each. Bubble with N2/CO2 and autoclave at 121°C for 15 min (as decribed below).
2 Prepare the following solutions (resazurin, bicarbonate and Pfennig's heterotrophic salts) and sterilize as given below.
Resazurin solution 450 ml
Bicarbonate solution 50 ml
Pfennig's heterotrophic salts solution 26 ml
3 Add bicarbonate solution and Pfennig's heterotrophic salts to the resazurin (complete volumina, i.e. 50 mL bicarbonate solution and 26 mL Pfennig's heterotrophic salts solution). Bubble with CO2 in an ice bath under sterile conditions.
4 Before use, add 4 ml sulfide solution (1.5%) and 0.1 ml Vitamin B12 solution to each 100 mL bottle.
5 Fill 50 ml of this mixture to each bottle of solution A (46 mL + 50 mL).
Sulfide solution, 1.5% 40 ml/l
Vitamin B12 solution 1 ml/l
6 Feed the actively growing culture periodically with neutralized 3% solution of sodium sulfide (use 1 -3 mL/100 mL depending on strain and cultivation stage) to replenish sulfide and with other supplement solutions (see Ref. 3365).
7 If needed, aliquot into sterile, N2 gassed screw-cap tubes under N2 gas.
8 During the first 24 h, the iron of the medium precipitates in the form of black flocks. No other sediment should arise in the otherwise clear medium.
9 Adjust the pH with filter-sterilised 1M Na2CO3 to 7.1-7.3.
Neutralized sulfide solution 3% (w/v) 10 ml
NaCl 20 g/l

Solution A #

460
Compound Amount Unit
CaCl2 x 2 H2O 0.25 g
Yeast extract 0.25 g
Distilled water 460.00 ml
1 Aliquot Solution A into 100 mL screw-cap bottles, filled with 46 mL each. Bubble with N2/CO2 and autoclave at 121°C for 15 min.

Sulfide solution, 1.5% #

135
Compound Amount Unit
Na2S x 9 H2O 2 g
Distilled water 135 ml
1 Prepare in a screw-cap bottle, bubble with N2 to replace air, close tightly and autoclave.

Bicarbonate solution #

50
Compound Amount Unit
NaHCO3 1.5 g
H2O 50.0 ml
1 Bubble with CO2 and filter sterilize into sterile, gas-tight, 100 ml screw-cap bottle.

Resazurin solution #

450
Compound Amount Unit
Resazurin
(0,1%) 		0.5 ml
Distilled water 450.0 ml
1 Autoclave in a cotton-stoppered Erlenmeyer flask with an outlet tube for medium, connected to a glass outlet at the bottom of the vessel 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.
2 Cool to room temperature under an atmosphere of N2/CO2 in an ice bath.

Pfennig's heterotrophic salts solution #

26
Compound Amount Unit
Ammonium chloride 0.35 g
Ammonium acetate 0.25 g
Pyruvic acid sodium salt 0.25 g
Dextrose 0.25 g
MgSO4 x 7 H2O 0.50 g
KCl 0.35 g
KH2PO4 0.35 g
Trace element solution SL-12 B 1.00 ml
Distilled water 25.00 ml
1 Filter sterilize into sterile, gas-tight, 100 ml screw-cap bottle.

Vitamin B12 solution #

100
Compound Amount Unit
Vitamin B12 0.01 g
Distilled water 100.00 ml
1 Filter sterilized

Trace element solution SL-12 B #

1000
Compound Amount Unit
Na2-EDTA 3.0 g
FeSO4 x 7 H2O 1.1 g
CoCl2 x 6 H2O 190.0 mg
MnCl2 x 2 H2O 50.0 mg
ZnCl2 42.0 mg
NiCl2 x 6 H2O 24.0 mg
Na2MoO4 x 2 H2O 18.0 mg
H3BO3 300.0 mg
CuCl2 x 2 H2O 2.0 mg
Distilled water 1000.0 ml
1 Adjust pH to 6.0.

Neutralized sulfide solution 3% (w/v) #

100
Compound Amount Unit
Na2S x 9 H2O 3 g
Distilled water 100 ml
1 The sulfide solution is prepared in a 250 ml screw-capped bottle with a butyl rubber septum and a magnetic stirrer. The solution is bubbled with nitrogen gas, closed and autoclaved for 15 min. at 121°C. After cooling to room temperature the pH is adjusted to about 7.0 by adding of sterile 2 M H2SO4 drop-wise with a syringe without opening the bottle.