I have been trying to perform FISH/DAPI staining of bacteria in water samples for cell counts and identification of certain bacterial populations. The issue I am having is that several cells show high autofluorescence in all channels I am using for the probes so I cannot be confident of the signal I obtain with my probes.
I have tried using pre-treatments with UV exposure (I read this can potentially damage the ribosome and affect the hybridization) and using Sudan Black B (although this was used in tissues, not planktonic cells) with little success.
I was wondering if what protocol/method you know or have used to reduce or at least minimize this issue in water samples? Thanks!
what FISH method are you using? is it for DNA or RNA? what antigen retrieval method do you use?
Dear Carlos, I have no experience in tge kind of samples you are assaying. There are some points however that you may find useful. Provided you are using water samples, it is possible you are handling a lot of photosynthetic bacteria in them. Photosynthetic pigments will of course be absorbing light in several portions of the spectrum and emmiting at some others. Getting rid of them through a selective culture step could help you. In the old times, people would get rid of some autofluorescence issues by incubating 5min with 1% evans blue. This would switch much of the greenish autofluo into the red channel, thus allowing them using Fitc-conjugated stuff.
There is still another option which is performing a lambda scanning of your unstained preparation and finding out which portions of the spectra do not autofluoresce. Once identified, you can choose some quantum dots as fluorophores with very narrow absorption and emmission spectra. Hope these comments would help you.
Best,
Daniel
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