I need to perform some experiments with Staphylococcus epidermidis, which is an S2 organism in Germany. Unfortunately the flow cytometry setup in our lab is not in S2 level. I must kill the cells and fix them in such a way that they retain intracellular fluorescence and morphology. Does anyone know a protocol for such fixation? Will formalin treatment work in this case? Can I just expose the suspended cells to UV and expect the fluorescence to be intact? Some inputs would be helpful.
Dear Vihang Ghalsasi,
there is a simple and safe protocol to fix bacterial cells which is often used (in e.g. FISH): Add 3 volumes of 4% paraformaldehyde in 200mM sodium Phosphate buffer (pH 7.0) to one volume of bacterial cells (in e.g. PBS). Mix gently, incubate for 3 h at room temperature. Then check for sterility (by e.g. plating 100µl....).
Fixed cells can be stored at 4°C for some weeks. For Details see: Amann RI, Krumholz L, Stahl DA: Fluorescent-oligonucleotide probing of whole cells for determinative, phylogenetic, and environmental studies in microbiology. J Bacteriol 1990, 172:762-770
Kind regards Wolf
Thank you very much Ben and Wolf. That was really helpful. I see that in this paper, cells were fixed first and later stained with fluorescent dyes. In my case, cells are expressing the fluorescent protein. Will this fixation keep the fluorescence intact? I am also concerned about the morphology of cells. If formaldehyde makes cross links with membrane proteins and clumps all cells together, it will not be measurable by flow cytometry. Please comment on this.
I know this is too late for you Vihang, but my answer is for others who might have the same question.
If you have only bacteria growing in liquid culture, incubation for 10 min at RT in only 0.5% final PFA is actually enough to kill them. This may change depending on your particular PFA, so just do a trial yourself by incubating your bacteria with different final conc of PFA and plate it all to see what's enough to kill. I have successfully fixed my fluorescent BSL-2 Salmonella under these conditions for FACS experiments and did not observe clumps - also you can always gate out clumps. I have obtained usually
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