Hi, I want to grow bacteria (E.coli) in liquid media, in the presence of lead ions (Pb2+). Every media I've tried so far (LB, M9, Davis and several more) has reacted with the lead (I'm using 1M stock Pb(NO3)2 in ddH2O) and created white precipitant. How can I overcome this problem?
Quick question; are you adding the lead solution prior to autoclave sterilization, or is there the option to add the lead after steam sterilization via a filter sterilized method?
Well, there goes that idea! Obviously, some of your Pb is oxidizing, but it may be that you have to find a way to measure the available lead vs. that which has precipitated. This is a problem if you want to use Pb precipitate as a measure of microbial lead oxidation. I found one other recipe that might be of help, but it does not mention your problem specifically, thus it may not be of help;
Bioinformatics and Biomedical Engineering , 2009. ICBBE 2009. 3rd International
June 2009
Author(s): Xu-mei Wang
Coll. of Water Sci., Beijing Normal Univ., Beijing, China
Hong-qi Wang ; Nan Sheng ; Xiu-chun Tong ; Yin-yin Geng
Page(s): 1 - 3
Let me know if you need access to this paper.
Well, I've tried the medium from the article (using Glucose instead of cane sugar) and It did not work. White precipitant forms instantly on adding the lead.
I'm starting to get desperate.
Thank you anyway.
Please let me know if you have any more ideas.
Are you using this media simply to screen for lead tolerance? You might be able to get E. coli to grow in media down to a pH of ~4 I believe. That lower pH may keep more of the Pb in solution.
I'm trying to screen some mutants for lead tolerance, and compair them to the wildtype. If I'l lower the PH with HCl, won't the lead react with the Chloride ions? (PbCl2 is poorly soluble)
Pb(NO3)2 is not stable in solutions with neutral pH, at this pH it precipitates as Pb hydroxyde;
you need to use Pb complex with chelator, for example, lead acetate
in this case you need to recalculate how much of Pb cations contains this salt
At neutral pH almost all heavy metals form barely soluble compounds. Phosphates are usually the biggest problem in media due its high concentration and I doubt this may be a case of oxidation.
In this case, Cl- can be a problem too, though PbCl2 is supposed to have a solubility slightly over 16mM. Try to mix only the solutions containing Cl and Pb up to the final volume and check if they precipitate at the desired pH. If nothing happens immediately, leave it for a couple of hours at least, some times it can take a while.
If it remains soluble I'd say that phosphates are the problem. Try to change the P source to a organic one (eg. glycerophosphate), it shouldn't precipitate because.
Alternatively you can just go for EDTA or any other chelator. I've seen it in articles even when authors determine MIC for heavy metals.
Thank you everyone. I had no luck yet, but the EDTA idea sounds good.
Xabier, can you add a link to one of those articles?
Thank you.
Daniel
Check these, describe media for lead-resistance
http://link.springer.com/article/10.1007%2Fs002489900145?LI=true#page-1
http://jb.asm.org/content/183/19/5651.full
http://onlinelibrary.wiley.com/doi/10.1111/j.1574-6968.1996.tb08610.x/abstract
Search by "lead resistance", there are many other useful references
Adding a chelator is a trick to seemingly keep a metal ion in solution, but actually you're tricking yourself as the bioavailability of the metal will be much lower and hence the exposure concentration you're trying to reach will not be correct. No guarantee for success, but I would try a minimal medium, with only one carbon source and only vitamins and aminoacids that are required by the specific strain you are using. This list you can easily find through the strain name and genotype. The first time you put your coli's on this medium they will grow much slower as they, coming from a rich medium, need to get there metabolic machinery in place. After one or two passes they will again grow a bit faster. If it works, you will also get a more accurate exposure concentration.
What about filter sterilization of the medium instead of autoclaving?
Hello Daniel,
Have you come over the problem of participation by adding EDTA?
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