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162 - RHODOSPIRILLACEAE MEDIUM (modified)
Distilled water1050.00ml
Trace element solution SL-61.00ml
Vitamin B12 solution (10 mg in 100 ml H2O)0.40ml
CaCl2 x 2 H2O0.05g
NH4Cl0.40g
NaCl0.40g
MgSO4 x 7 H2O0.40g
KH2PO40.50g
Fe(III)citrate solution (0.1% in H2O)5.00ml
(NH4)-acetate0.50g
Na2-succinate1.00g
Ethanol0.50ml
Yeast extract0.30g
Adjust pH to 6.8. Boil the medium under a stream of nitrogen gas for few minutes and distribute 45 ml medium into 50 ml screw-capped bottles (already flushed with nitrogen gas). Bubble each bottle with nitrogen gas and close immediately with a rubber septum and screw tight. Autoclave at 121°C for 15 min. Sterile syringes are used to inoculate and remove samples. Incubate in the light using a tungsten lamp. For brown and other oxygen sensitive Rhodospirillaceae add 300 mg of L-cysteine (0.03% end concentration) to the boiling medium and readjust the pH to 6.8 or to the prepared medium in bottles inject neutralized sulfide solution (0.005 to 0.01% end concentration). The medium has been modified according to reference DSM 3365.
 
Neutralized sulfide solution
Na2S x 9 H2O1.50g
Distilled water100.00ml
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 7.3 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 continously to avoid precipitation of elemental sulfur. The final solution should be clear and is yellow in colour.
 
Trace element solution SL-6
Distilled water1000.00ml
Na2MoO4 x 2 H2O0.03g
NiCl2 x 6 H2O0.02g
CuCl2 x 2 H2O0.01g
CoCl2 x 6 H2O0.20g
H3BO30.30g
MnCl2 x 4 H2O0.03g
ZnSO4 x 7 H2O0.10g
 
 

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