I‘m investigating the epithiel cell interaction with bacteria. So I co-cultured the gram + ,gram - with epithiel cell. The bacteria were stained with SYTO9 or hexidium iodide separately. The staining time were 15 minutes and wash with PBS for 3 times to remove remaining dye. After co-culture, I utilized 4% PFA for fixation.The final step was mounting. However, can’t observe the fluorescent light of of bacteria under confocal microscope and phase contrast microscope. While I can see the bacter is under bright light field. And the stained bacteria also can be found and emit fluorescents.
I‘m wondering is there any step I made wrong?
Dear Tzu Hsuan Li,
Under bright field illumination, you don't observe fluorescent light.
As your staining is SYTO 9, it is excited at ~488nm (GFP-like) and emits at ~510nm (http://www.thermofisher.com/order/catalog/product/S34854).
Maybe it is just a matter of parameters. On your confocal microscope, you have several parameters to adjust: laser wavelength and power, gain of your PMT/camera, pinhole diameter, and light path. I think that you can choose GFP parameters for laser wavelength and light path. Other ones depend on your sample.
Finally, your cover slip must be on the objective-side of course and your sample thickness < 100µm.
If nothing works, maybe your staining didn't work... ?
Dear Tzu Hsuan Li,
As Laetitia Huducek said, you should adjust all the parameters on your confocal microscope. Something ultra important before you observe your samples is know if the mounting medium is antifade and if the amount of it is enough, because if the mounting medium is poor is difficult to observe the sample at the confoco. Another thing to consider is the magnification that you use, commonly to observe bacteria is necessary use 63x, or 100x objectives. Remember always clean your sample and your objective before to use them and as a last recommendation try to dye the bacteria with DAPI, maybe the SYTO9 did not work.
Best wishes.
Hi. There are a couple different problems. First, the nucleic acid stains, by binding to the nucleic acids, will affect the nucleic acid functionality. Thus it is not a good choice to use them for tracking purposed due to viability issues. Also, nucleic acid dyes do not bind covalently to the nucleic acids, and thus will have an off-rate leading to loss over time.
Instead, I recommend you use CFDA SE or the CellTracker dyes, which bind covalently to proteins (and thus are retained long term). in the bacteria and haven't been shown to be disruptive to cell function. Example paper for CFDA SE usage: Pubmed ID 11010903. Example paper for use of CellTracker Orange CMTMR: 10802143.
Also, if this is being used for phagocytosis assays, you might want to look into the pHrodo Red or pHrodo Green bacterial labeling kits. These dyes are virtually non-fluorescent at neutral pH (outside of cells), but upon being phagocytosed (and thus in the low-pH environment of a phagosome , and the dye is covalently bound to the bacterial surface (and thus won't be lost).
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