Fluorescence problem

[ozana]

Heloo!

I am performing a membrane integrity test on Staphylococcus 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?

I ve used I3 filter (blue) to to excite both fluorochromes and also i ve tried the mixture on Bacillus subtilis (it did not work). I attached some pictures: S. aureus AO+PI (when the mixture work), S. aureus AO-PI DIC 1 and S. aureus AO-PI FLUO 1 (when the mixture did not work) - photos pair. I used DIC and FLUO to the same microscopic field to highlight that there are red cells and some green cells with a poor fluorescence and some cells with any fluorescence at all.

http://imgur.com/vMi3rT7 - S. aureus AO+PI (when the mixture did not work)

http://imgur.com/r9CizfE - AO-PI DIC
http://imgur.com/SP6WXaX S. aureus AO+PI (when the mixture work)

Thank you!