I have to grow some bacteria in minimum salt medium (pH 7-8) containing various copper concentrations ranging 1mM to 6mM. Whenever I add copper in the medium, blue colored precipitates (pppts) appear. I checked that copper is making ppts with phosphate buffer. then i checked that copper does not make ppts with tris. So I decided to replace phosphate buffering system with tris buffering system.
Can someone help me in determining what should be the concentration of tris in the medium. and if I have to change any other ingredient also in the same medium.
I"ll also welcome if someone suggests some other solution such as another medium. But that medium should not be complex, as in LB medium copper is not totally biologically available to the growing cells. I want some system in which whole copper present in the medium should be biologically available.
The recipie of the minimal salt medium, I am using at the moment, is:
Make M9 salts
To make M9 Salts aliquot 800ml H2O and add
- 64g Na2HPO4-7H2O
- 15g KH2PO4
- 2.5g NaCl
- 5.0g NH4Cl
- Stir until dissolved
- Adjust to 1000ml with distilled H2O
- Sterilize by autoclaving
Measure ~700ml of distilled H2O (sterile)
[*]Add 200ml of M9 salts
[*]Add 2ml of 1M MgSO4 (sterile)
[*]Add 20 ml of 20% glucose (or other carbon source)
[*]Add 100ul of 1M CaCl2 (sterile)
[*]Adjust to 1000ml with distilled H2O
[/list]Waiting for kind response