For bacteria you can use use Grams stain, that has both stainer crystal violet and developer, ethanol and iodine, You will get different color, you you need differentiation staining for which eosin or safrinin  are best, these are used for staining algae and fungi. You should usedifferent staining methods that allow distinguishing all microorganisms (different fluorescences) than collect them for further use.

Hi Caroline, do you remember me from phyllosphere 9 in Corvallis?

You can give FISH a go. Bacteria, algae, and fungi can be well separated using fluorescence in situ hybridisation using fluorescence microscopy or flow cytometry. I propose to fix your samples in EtOH but process them asap (on the same day). After reading up on FISH you will understand why. Alternatively you can try and fix the samples using paraformaldehyde, but you might miss gram positive bacteria then. Depending on later questions, you can then use more specific FISH probes to look into specific taxa and phyla. Algae have actually a quite pronounced autofluorescence, so there is no probe needed to pick them up.

Just go ahead and ask if anything is too unclear, I can refer you to some useful reviews and protocol collections, but I guess you will find them yourself in no time.

Best of luck! Mitja

Thanks Mitja! my question is: can it work if you want to differentiate all microorganisms at the same time. Ok you can have different probes targeting different genes, but you may need different fluorescences too to distinguish them anyway. You have a doc for that? Yes I agree for algae.

Hi Caroline, you can use several fluorescent tags at the same time, yes. What kind of detector do you have? I suppose you have a microscope with several fluorescence channels that would switch every other millisecond? Otherwise, in flow cytometry, fluorescence channels are organised so that they are detected sequentially so that always works. You will definitely always need several several fluorescence channels.

Check out: http://www.sciencedirect.com/science/article/pii/S0580951701300466

and check out http://www.microbial-ecology.net/probebase/ there you will find most of the 16S/ 18S-targeted probes and sequences that you will need.

Maybe if you give more specifics about what kind of system you are using it will be easier to help.

Cheers, Mitja

Hi Benjamin, the etoh fixation works very well for gram positive bacteria, but for gram negative bacteria it often just offers short term fixation. On some surfaces, i found that it still works very well but in solution one should expect rapid degradation of gram negative cell walls. To be on the save side processing the samples within a day should be OK but of course one can always test if the cells are more stable than expected. Problem is, if you would use paraformaldehyde fixation, which is really great for gram negative bags, gram positive one might be rendered impermeable to fish probes.

Mitja, just thinking about that fixation step.

Imagine I just want a particle count and that I don't want to differenciate microorganisms, is paraformaldehyde the best method to use for fixation?

Also, if I cannot pass the samples to the flow cytometer right away, how should I store the fixed samples? in a freezer with glycerol for exemple? -4°C? And how long?

Your help is very appreciated!

Hi Caroline, if you just want to perform particle counts PFA fixation would be the way to go, yes. If you fixed your samples in pfa first you can store them for very long periods of time @ -20 in 50% etoh. Pfa will stabilize the cell walls and membranes first thus etoh will be no problem anymore.

What about the concentration of PFA? Do you have any protocol to advise to me for PFA fixation? Cheers,

Hi Caroline, the usual procedure that I am using is to add 3 volumes of 4% PFA in PBS to your sample, incubate it o/n or at least 3 hours at 4 °C. Wash the sample twice with 1 volume PBS. Resuspend in an appropriate volume of PBS (possibly smaller volumes) before you add another volume of 100% EtOH to reach a 50% EtOH solution to store your sample in. Storing should be done at -20 °C and depending on the sample they might be good for a couple of months, to several years. I think a PFA fixation protocol is also outlined in the paper I mention above.

Cheers!