AO and PI

Posted by mmodel on
URL: http://confocal-microscopy-list.275.s1.nabble.com/no-subject-tp7586114p7586115.html

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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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From: Confocal Microscopy List <[hidden email]> on behalf of Ozana Maria Petraru <[hidden email]>
Sent: Wednesday, December 7, 2016 5:12 PM
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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