Ozana
Is it possible the acridine orange is quenching due to the concentration being too high. Try diluting 10-fold or 100-fold or staining for a shorter
time. This often happens when staining plant cell walls with this type of dye although I have not seen it with bacteria.
Regards –
Lloyd Donaldson
Scion, New Zealand
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Sent: Thursday, December 08, 2016 1:23 PM
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Subject: AO and PI
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Ozana,
What exactly stopped working?
Try killing all bacteria with heat, check that PI works. DAPI for example, doesn't stain every cell type, and I don't know about AO. If all you bacteria are alive and AO for some reason stopped working
you might not see anything.
What else:
Make sure there are not too many bacteria so the dye doesn't get depleted (that's unlikely though).
Check your microscope settings.
Try other cells.
Post pictures, that might help.
Good luck!
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Heloo!
I am performing a
membrane integrity test on
Saphylococcus aureus using a mixture of two fluorochromes: propidium iodide and acridine orange. The cells
grow in a MHB medium, washed and resuspended in PBS pH-7,2.
Acridine orange and propidium iodide were added to 100 µl aliquots of these cells,
followed by incubation in the dark for 15 minutes.
In the past this method worked, so the cells with damaged membrane were stained in red and the cells with intact membrane in green. In the past two months this double staining does not work anymore, and I do not know why?!!!! The cells are no longer staind in green. In the best case the cells have a low fluorescence.
I ve rebuilt stock solutions but nothing improved.
It can be the PBS?
Do you have any idea how to solve or find the problem?
Thank you!
Ozana Petraru - Master's degree Microbial and cell biotechnology
Faculty of Biology
"Al. I. Cuza" of Iasi University
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