I am adding filter sterilized heavy metals to autoclaved LB agar to see bacterial growth on it. Now, if I add the metal when the LB agar is hot, can this still reduce the heavy metal. Should I wait till LB agar cools? At what temperature should I add the metal?
Reason:
I did this with two metal salts, namely K2Cr2O7 and HgCl2, and I have seven bacterial strains (yet to be identified). As I remember, I added K2Cr2O7 to hot molten LB agar ( not that hot though) and all the strains showed good growth up to the metal concentration of 200 mg/L and some strains up to 350 mg/L.
But, I added HgCl2 to molten LB agar which was not hot, (temperature slightly more than necessary to maintain molten state) and none of the strain grew, even at 50 mg/L. In both cases, 5 ul of 1 day old culture was put on surface of solidified agar. Now, I think Cr is more toxic to bacteria than mercury. But with my results, I am thinking that K2Cr2O7 might have been reduced so bacteria could tolerate, it but not the mercury.
Can this happen?
The total metal concentration cannot be reduced by the hot agar (I would not recommend this with organic chemicals). However, there are several factors that influence the metal toxicity in agar. (1) Biovailability of the metals can be influenced be the agar-matrix. A part of the metal is bound by the agar, so that you find only a reduced amount of freely dissolved (bioavailable) metal. The binding might be different for the two metals. (2) The susceptibility of the bacteria towards the metals is different. It is plausible that Hg is more toxic than Cr.
I recommend to add the heavy metal solution into the agar medium cold as much as possible. For example, when the agar medium is liquid and very hot, put it into a incubator or a water bath at 60C... So it will be at the temperature limit before to coming back solid and then you can make your plate.
The reason for that is not to avoid to reduce metal (the heavy metal will be fine) but because the heavy metal steams are very dangerous for the health !!!! And less hot medium... less heavy metal steam...
Hi Vipin;
It apears that you are trying to cultivate bacteria that grow by reducing metals such as chromate. If this is the case, I am not sure that a rich medium such as LB is really the best choice, since you are very likely to grow bacterial colonies on the LB alone, irrespective of the presence or absence of your metal of interest. You are probably better off using a liquid enrichment with a a mineral salts solution, single carbon source (such as acetate), and your oxidized metal (e.g., chromate) as the only electron acceptor. Add a small amount of inoculum, such as metals-contaminated soil, and monitor for cell growth in the flasks under anoxic conditions. If you see growth, add a small amount of the liquid to a fresh flask of the media. You are much more likely to isolate bacteri capable of reducing chromate or some other metals that can be used as electron acceptors under these conditons.
It is possible for a bacterium to be resistant to Cr(VI) and not to Hg . Heavy metal resistance is emanated from genes dwelling in plasmids. These genes give different levels of tolerance to each bacterium. So your results are nothing but acceptable.
I would recommend that you dissolve the K2Cr2O7 to LB agar that is not hot (40C). If the solution is hotter then you cause structural alternation to Cr(VI) and reduce it to Cr(III). That is why your bacteria tolerate higher concentrations (i used to have the same problem with my bacteria). K2Cr2O7 should be dissolved in lukewarm LB agar (just before you pour it in petri plates). In order to achieve higher K2Cr2O7 stability you should store it at -20C either in ddH2O solution or in LB broth solution.
Hi, I do not know why you use HgCl2! We use HgCl2 0.1% for 15 second to steralize seeds which kill all microorganisms because it it very toxic. And you want to cultivate bacteria on it. It is beter to use another heavy metal.
Thank You all!
Sebastian Höss: I will consider the influence of agar matrix on bio-availability of metals
Marie-Mathilde Perrineau: That health issue I will take care from now onwards. I was not aware of it.
Paul Hatzinger: I will be using mineral salt solution and different carbon sources also also because I will be working on recovery of metals from industrial effluent where bacteria cant find nutrients.
Konstantinos Tsigaridas: Is storing at -20 C recommende? I stored at 4 c. Genes on Lasmids can be good reason for my result.
Dr Anwar Mohammad: I will try with other metals also and discuss it here.
hello vipin by adding the metals to any hot media including LB agar does not reduce the metal concentration. You will get growth of microbes which are tolerant to the concerned metals. Add the media at 45 degree condition as its normally acceptable. If any quiries regarding the bioremediation of heavy metals u always welcome to ask any question as am also pursing my phd in the same area and working as microbiology lecture in Karnatak Science College, dharwad, Karnataka India.
No, it would not. Even if you autoclave after the addition of heavy metals there would not be any loss. I am saying this because I did tests for heavy metal resistive bacterial strains using the method where I autoclaved the media after the addition of heavy metals. Even in that case, Cadmium-resistive strains could not be isolated, meaning that cadmium levels stood the same to not allow the microbe to grow.
Storing at -20 C is recommended as Cr(VI) remains stable. If you store it in 4 C in a water or medium solution your will see after a week that solution's color has changed, becoming dark red. Substantially, -20 C storing slows down the natural Cr(VI) to CrIII degradation.
In my experience HgCl2 could be more toxic than K2Cr2O7, for example at my lab, the MIC for a group of strains is 30-300 ppm of K2Cr2O7 and 10-50 ppm of HgCl2; also it´s recomended to test you media plating a heavy metal tolerant reference strain as a positive control for growth.
hi Vipin
LB medium is an organic medium therefore metals can easily form complexes reducing the toxicity. You should use a basal mineral medium or any inorganic medium when you are working with metal related studies
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