[ozana maria]

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? 

 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!


Ozana Petraru - Master's degree  Microbial and cell biotechnology

Faculty of Biology

"Al. I. Cuza" of  Iasi University
Romania

[SunilNagpal]

Hi Ozana,
Apologies for the delayed response. Your query was assigned to a subject matter expert at IIT Delhi, but being out for a conference she has not been able to respond. So, I thought to try my knowledge of the long left wet lab.?

Well, following could be a logical flow for troubleshooting :
a) My dyes are working fine or not (use a different batch of dyes). If that's not a problem then:

b) Resuspending in 2 batches: One in PBS, and other in Medium broth itself (to ascertain whether there was a problem with PBS or not).
If that's not a problem then:

c) OD monitoring before and after incubation period (to ascertain that there was no problem with growth conditions).
If that's not a problem then:

d) I am using sufficient active inoculum or not.

Looking at the protocol, it's indeed intriguing that you are getting this problem. In all its probability, it seems a problem of step a or b.

Hope it helps.

Do let us know what was the issue once you figure it out.

Cheers