I want to study Iron starvation in cyanobacteria Nostoc punctiforme. But I did not want to use iron chelator in culture media, because they can make some difficulty.
Hi Loknath,
if you don't want to use a chelator in solution then you have to reduce the iron concentration in your growth medium somehow and this can be difficult to do effectively and long term unless you have a very trace metal clean working environment. To make iron free media you can pass the solution through a chelating column (e.g. Chelex-100 or NOBIAS-chelate) to remove the iron, though this will also remove other trace elements such as Cu, Co, Mn and Zn which may also limit the growth of your organisms. Alternatively you can use chelating beads and filter the beads out later., though the same problem with removal of other trace metals will occur also. All the handling procedures would need to be performed under class 100 conditions (laminar flow HEPA filtered air) to prevent contamination and reintroduction of Fe into the culture media. Plasticware and glassware also needs to be trace metal clean - i.e. acid rinsed prior to use). Autoclaving is problematic normally as it can seriously contaminate for Fe and Zn. Sterilization of the culture media is best achieved then by either filtration or microwaving.
However if you want to have some well defined experimental conditions where the iron speciation is known then using chelators such as EDTA, IDA etc are probably the easiest way to run the experiments even in the presence of a small amount of Fe contaminants from reagents, lab ware etc. The use of a stronger chelator such as desferrioxamine B is also a common strategy to limit phytoplankton cultures or natural samples if inducing iron limitation is the sole aim.
Perhaps if you can explain more why you think there is a problem in using a chelator with these cyanobacteria?
Kind regards
Peter
In some experiments in order to have a very "fast" iron starvation, a chelator :(2-2' dipyridyl) has been used. However, more physiological conditions were to grow cells after three washing with a culture medium depleted of iron, in iron depleted medium. The iron starvation can be tested by the release of siderophores in the extrernal medium, and in cyanobacteria by the blue shift of the cell spectra (see the papers).
I would rinse your flasks over night in an iron chelator like EDTA. Then to a quick rinse in ethanol, oven bake and/or autoclave.
To remove metals from new glassware, we boil 35% nitric acid in the glassware during the day, then rinse with MilliQ water. We repeat this step for a total of three treatments. We then consider the glassware acid-washed and only use this glassware with solutions that have minimal levels of trace metals. This approach won't remove all metals but will reduce metal levels substantially.
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