I am trying to quantify bacteria based on the fluorescence emitted by GFP expressing bacteria. I want to quench any background fluorescence from the media to make sure that my fluorescence data really reflect the no. of GFP expressing bacteria and not for the media in which bacteria are grown. Any suggestions greatly appreciated. Thanks
I have the same question. Currently, I measure the fluorescence of the growth media (LB / Amp in my case) and subtract it from the sample measurement. I am finding that the media has a strong green flourescence signal making it difficult to use for detecting weak GFP signals from bacteria. My latest question is, should one be using the media as a blank to standardize the sample data since the bacteria change the properties of the media when the consume the nutrients in the broth itself?
Why not try doing the experiment in minimal medium, LB has lots of compounds that both absorb and fluoresce at many wavelengths. Or harvest an aliquot of cells and resuspend in buffer for the measurement.
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