5 replies to this topic

[Soumble]

Hello every one


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

Waiting for kind response


Soumble

[ascacioc]

also it can be the case that copper sulphate (sulphate from the MgSO₄) precipitates. Also blue. Another minimal media I would recommend is MD5052:

http://www.ncbi.nlm....pubmed/15915565

At least this media adds trace elements such as Cu (even though 0.4 uM is not even close to what you need)

The paper above is also a good overview of why every component is needed in the media.

Andreea

[Soumble]

Hello every one


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

Waiting for kind response


Soumble

[Soumble]

ascacioc, on 30 August 2012 - 05:38 AM, said:

also it can be the case that copper sulphate (sulphate from the MgSO₄) precipitates. Also blue. Another minimal media I would recommend is MD5052:

http://www.ncbi.nlm....pubmed/15915565

At least this media adds trace elements such as Cu (even though 0.4 uM is not even close to what you need)

The paper above is also a good overview of why every component is needed in the media.

Andreea

Thank you Dear  Andreea for replying me.

A few things, I must tell you are:

1.sulphate from the MgSO₄ is nothing to do as I am adding copper in form of copper sulfate solution.
2. I tried to check its solubility in different ingredients of minimal salt medium. ppts appeared when copper was added in phosphate buffer only. It is phosphate that is making problem, I think so. I dont know the ppts color and solubility of CuPO4.

[ascacioc]

Cu₃(PO₄)₂ is also blue but more greenish

check the article I recommended for other media. It describes quite a few media. Even though phosphate is a crucial component of all the media; compare the concentrations of different salts; in the same article they also give several media with different strengths of phosphate; maybe if you use one with lower amounts of phosphate you can get it working.

PS: Please do not post the same question in different forums; if someone replies here and someone replies on the other forum, we end up having the same conversation in parallel. Usually, people hit new content button when they reply to entries.

[El Crazy Xabi]

Heavy metals and circumneutral or alkaline pH don't match very well. And having phosphates, which usually are quite reactive with them doesn't help.

Cu solubility at that pH is around 1 mg/L and if it forms a phosphate, it is reduced to the order of pg/L.

If the phosphate is a problem, using an "exotic" source of P (you have to add some P anyway besides the buffer) like a glycerophosphate can be a solution. But you will be in a problem by pH-dependant solubility limit