培養基配方
28. PFENNIG'S MEDIUM I
Solution A:
CaCl2 x 2 H2O 0.25 g
Yeast extrakt 0.25 g
Distilled water 460.00 ml
Fill 10x 46ml in 100 ml screw-cap bottles. Bubble with N2/CO2 and autoclave 121°C 15 min.
(For marine or estuarine isolates add 100.0 g NaCl to this solution and increase the MgSO4 x 7 H2O to 15.0 g).
Solution B:
Na2S x 9 H2O 2.00 g
Distilled water 135.00 ml
Prepare in a screw-cap bottle, bubble with N2 to replace air, close tightly and autoclave.
Solution C:
NaHCO3 1.50 g
H2O 50.00 ml
Bubble with CO2 and filter sterilize into sterile, gas-tight, 100 ml screw-cap bottle.
Solution D
Resazurin (0,1%) 0.5 ml
Distilled water 450.00 ml
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.
Cool to room temperature under an atmosphere of N2/CO2 in an ice bath.
Solution E:
Ammonium chloride 0.35 g
Ammonium acetate 0.25 g
Pyruvic acid sodium salt 0.25 g
Dextrose 0.25 g
MgSO4x7H2O 0.50 g
KCL 0.35 g
KH2PO4 0.35 g
Trace element solution SL-12 B 1.00 ml
Distilled water 25 ml
Filter sterilize into sterile, gas-tight, 100 ml screw-cap bottle.
Solution F:
Vitamin B12 0.01 g
Distilled water 100.00 ml
Filter sterilized
Trace element solution SL-12 B:
Distilled water 1000.00 ml
Na2-EDTA 3.00 g
FeSO4 x 7 H2O 1.10 g
CoCl2 x 6 H2O 190.00 mg
MnCl2 x 2 H2O 50.00 mg
ZnCl2 42.00 mg
NiCl2 x 6 H2O 24.00 mg
Na2MoO4 x 2 H2O 18.00 mg
H3BO3 300.00 mg
CuCl2 x 2 H2O 2.00 mg
Adjust pH to 6.0.
Mix solution D, C and E. Bubble with CO2 in an ice bath under sterile conditions.
Fill 50 ml in each bottle of solution A. Before using add 4 ml solution B and 0.1 ml
solution F.
Adjust the pH with filter-sterilised 1M Na2CO3 to 7.1-7.3.
Fill in sterile, N2 gassed screw-cap tubes under N2 gas.
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. Feed periodically with
neutralized 3% solution of sodium sulfide to replenish sulfide and with other supplement solutions (see Ref. 3365).
Neutralized sulfide solution:
Distilled water 100.00 ml
Na2S x 9 H2O 3.00 g
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.
Appearance of a yellow colour indicates the drop of pH to about 8. The solution should be stirred continuously to avoid precipitation of elemental sulfur. The final solution
should be clear and is yellow in colour.
