Hi all,

I'm looking for the expert opinion from a chemist, since I am only a biologist.

I have a set of samples made in the following manner: many litters of seawater were flocculated/precipitated/coagulated adding FeCl3. The flocculation reaction was allowed to stand for 1h and then was filtrated using a pore size around 1 μm. So those filters carry a film of flocculated iron that trapped small particles like viruses and other microbes. Filters were folded, placed inside a tube with a bit of water (so they do not get dried) and stored in the dark at 4C. The samples were taken between 5 and 9 years ago.

To have access to what was trapped in the flocculant (microbes, viruses, nucleic acids, etc.), I have to reduce and dissolve the iron using buffers with reducing agents. I tried 2 different "resuspension" buffers:

- Buffer 1: 0.25 M Tris, 0.1 M MgCl2, 0.1 M ascorbic acid, 0.2 M EDTA, final pH 6.

- Buffer 2: 0.2 M trisodium citrate, 0.05 M MgCl2, final pH 6.

After many assays using freshly made flocculated samples (with microbial isolates), both buffers work decently, recovering a good % of the initial microbes trapped in the flocculant, with an evidently higher efficiency of the second buffer. Nevertheless, when I applied these to the old “real samples” (stored as described above for several years), the resuspension of the iron flocculant is more difficult. Even after adding 40-50 mL of buffer to a 1/4 of those filters and letting an overnight incubation, there are persistent brownie-black chunks of solid iron particles persistent. Even if you filter or centrifuge the suspensions to get rid of the iron particles, there are tiny “colloidal” particles persisting in solution. So I guess that I have still non-dissolved Fe3+/ferric oxide particles from the flocculant, so the buffers/methods to reduce/dissolve the flocculated samples are still far from 100% efficiency. Notice that the reduction/dissolution of this iron is critical since making the iron stay completely dissolved in solution is the first step to separate it from the “biological fraction” of the flocculant (microbes, viruses, etc.) by further filtration, ultracentrifugation, etc. - keep in mind that polyvalent metals such as iron, magnesium, calcium, etc. promote base-hydrolysis reaction which leads to fragmentation of nucleic acids.

So how would you address this problem?

What is changing between the old real samples and freshly-made samples? What changes with AGED ferric oxide?

Are there any other (mineral?) components in seawater that could be affecting to this process?

Should I change the resuspension buffer? Carry out the reaction in another manner? I read in the bibliography that Titanium-Citrate-EDTA buffer is pretty efficient. What do you think of this?

Thank you so much in advanced!

Guillermo