Hi,
has someone ever done characterization of filamentous bacteria from frozen samples?
I would like to do the identification of the floc from frozen samples while ago but I haven't found any references about. I know the "standard" procedure working with fresh sludge but I don't know how the frozen process could interfere with the analytical results.
Thanks in advance.
Dear Beatriz, it is a very interesting question, which I do not believe has been addressed so far. I would use snap freezing/cryofixation by immersion in e.g., liquid propane at −
190 ◦ C; then transferring sample to a freeze-etch unit and freeze-dry sample at − 90 ◦ C at 10 − 7 mbar for an hour or two.
Prior to cryofixation, I would do gentile sonication simillar to that used for FISH analysis.
You may find it important/relevant that different filamentous bacteria residing in different parts of the floc may have different degrees of effects on ecosystem services, e.g., floc settling velocity and rheology (see more on this in the paper attached).
good luck, Benedek
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Hi Benedek,
Thanks for your answer :) Samples are frozen at -80. For the moment I've placed them overnight at 4 degrees before doing the microscopy. But It would really helpful to hear from other people how has been in the same situation as me, as I've never read about doing morphological analysis from frozen samples (always from fresh samples within the 24 first hours since sample was taken).
Regards,
How far would you like to classify the filaments? I know that at industry level they usually use 'de visu' classifications that don't necessarily match the taxonomy.
You could start by observing the filaments with an optical microscope to evaluate if they are prokaryotic or eukaryotic. Unless it came from anaerobic conditions, it would be bacteria most likely. I would use molecular techniques for the ID. If you are familiar with FISH, you could try probes used in samples from similar environments. Otherwise I would go with some sequencing...
Hi Xabier and thanks for your answer.
I work with industrial wastewater. The first step is the microscopy before to think in moving to other analysis as FISH or sequencing. I would like to know if someone has done morphological studies from frozen samples, as usually you might work with fresh samples.
Regards,
Bea.
Not an expert in "morphology of frozen samples" but in general, cell-walled organisms conserve quite well the morphology, at least from the optical microscopy point of view.
For electron microscopy, your samples might not be so suitable though. But even so, I think it would be too laborious. I guess you didn't snap-freeze your samples and even if you do cryo-SEM or similar, I wouldn't be very confident due to the slow freezing process.
Easy way, extract DNA, do amplicon sequencing and try to correlate the taxa in the results with known filamentous bugs. Off course, after confirming they aren't fungi.
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