A fraction of the bacteria will remain attached to the particles that are trapped on the GFC. To get the maximum bacteria out of the GFC you can put the filter in a centrifuge tube containing a buffer solution (keep the buffer composition as simple as possible), vortex the tube with the filter inside, centrifuge, and filter the supernatant through 0.22 micron (filter membrane for bacteria). You can use the same tricks for the membrane filter too, and compare the final results. Still you may not get the actual picture as there are bacteria those reduce bivalent Hg to gaseous Hg (0).

Dear Buenaflor,

it is in fact a very important and yet unresolved question that you are addressing. Surely some bacteria are attached to particles which are retained on filters. If you are not interested in a particular genus or species, you can find out how much bacteria is retained on the filters (you can either dye filters directly or wash bacteria off, then dye and count it under microscope ) and correct your Hg measurements for the portion of adsorbed cells. For that you will need cell count of your final filtrate too.

As for other options, gradient centrifugation might help you in distinguishing cells from various organic molecules, which are rich in Hg and still pass through both of your filters. If you find a resin, which would specifically retain cells you can separate them from the rest of the matrix. Don't forget that low-weight organic molecules can be adsorbed as well.

During my PhD I had great trouble with filtering seawater and it turned out that a lot of filters retain or release Hg. This is important for total Hg measurements in your bacteria. On the other hand, applying too much pressure or vacuum to your filters might break the cells and release methylating agents from inside them, which would create artefacts if you are interested in speciation and MeHg.

I hope this helps a bit. Best of luck with your work!

Arne

First, you should make process of clarification with filtration (different pore diameters), that depends on the particle size of your sediments. Once you have removed the sediments that are bigger than the bacteria, you should filter to retain the bacteria and wash with enough rise solution to remove the sediments adhered to the bacteria surface.

The you have clean bacteria to determine the Hg inside

Yes, you will lose a lot of bactéria by simply filtering out the sediments, but it is impossible to catch them all. Just be sure to always use the same strategy if you want to compare results. Shaking vigorously a sediment sample in sterile buffer and trying to recover bacteria from the water phase by centrifugation might help you recover more of them than using a filter (0.4 um?), but i don't know how much Hg or biomass is in the sediment and therefore how much can you dilute the sample. What is your metal detection/quantification equipment?

I would need more informarion on the purpose of quantifying Hg in the bacteria. Many bacterial species reduce (volatilize) Hg, which makes it difficult to measure real accumulation.

Thank you @Arne. You really hit the nail in the head and opened several options. What resin would you recommend which have lower ion exchange and not affect mercury speciation? Does GFC filter retain or release Hg?

@Jaysankar, I am working on marine bacteria so I have to adjust the salinity. What buffer would you suggest?

@Carlos. Thanks for your thought provoking insight. I want to quantify the Hg in bacteria to know how muchis transferred from the sediment to the next trophic level and determine how much Hg is sequestered by certain microbial biomass/ background. I intended at first to isolate mercury resistant or utilizing bacteria but culturing them require addition of mercury in the medium which may be difficult for me to quantify. Yest, It’s difficult to measure real accumulation of metals in microorganisms, because many bacteria volatilize, some adsorb in their wall and some uptake inside the cell and i need to know also the initial mercury background to undertand.

Thank you @Edelberto and @ Romeu for your comments and suggestions. I really appreciated them. Please feel free to add some comments you might think of later.

@BJ, please make artificial seawater, sterilize and use it for your work...

@Jaysankar thanks you.

@Buenaflor,

GFC filters themselves do usually not affect Hg content much, since they are made of borosilicate glass fibers. On the other hand, the filtration device itself or the housing of the filter may have some effect. Before mass production of disposable filters began, researchers usually used glass filters, which were acid-washed and no Hg could contaminate the sample. I have never done that, but it is supposed to be very effective (and also time consuming).

During work with 197Hg radioisotope (also called radiotracer) and we used to separate Hg2+ form from MeHg by means of DOWEX (mesh 100-200) resin. The resin unfortunately retained lots of organic material too, but was very useful for working with clean solutions. If you need more detailed info, I will be happy to provide it.

One more thing, my coworker and I did a preliminary experiment with pure bacterial culture, to which we added 197Hg2+ radioisotope. Within15 minutes, almost all of the tracer was adsorbed onto the cells. There was negligible amount left in the growth medium. This is only to emphasize that really most of Hg will be adsorbed to one surface or another. We could not determine the inside concentration with our method, though.

Es posible que utilizando la propiedades fííscas o químicas de la sustancia encuentres la forma de separar el mercurio, ej. La solubilidad, el punto de ebullción entre otros.

La solubilidad, el punto de ebullción entre otros. Sírvanse explicar. ¿Esto no afecta a la especiación de mercurio?

@Arne Thanks. I will check on it. How about standard 0.45um filters poly filters?

It depends heavily on the manufacturer. All I can say for certain is this: take a standard solution, filter it through and measure the filtrate. You will have your answer immediately :)

This is an addition to what Arne has tried to explain. This is like why we add internal standard while extracting organic/inorganic pollutants using different extraction methods. Recovery of the internal standard and/or its signature in the extracted material gives us the idea about the effectiveness of the method. You can same way take any filter paper and check it for your standard (Hg containing) solution and then your samples. It will be quite a close call, if not the same to same (because matrix of the samples are different from that of the standard solution). But this is what we can do at the best. I have used this technique for all my analyses (most of then were from marine water and marine bacteria). Good luck.