fluorescence problem

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ozanamaria
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fluorescence problem

#1 Post by ozanamaria » Wed Dec 07, 2016 9:45 pm

Heloo!
I am performing a membrane integrity test on Saphylococus 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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hkv
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Re: fluorescence problem

#2 Post by hkv » Wed Dec 07, 2016 9:54 pm

Are you certain you have the correct filters and cubes installed? If you have verified and reverified your process and the chemicals involved I should try to rule out possible misconfiguration of the equipment used.
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gekko
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Re: fluorescence problem

#3 Post by gekko » Thu Dec 08, 2016 1:34 am

I agree with hkv. And since it was working before and I assume you have not changed anything in the hardware, could it be that you changed something in your preparation/staining procedure without realizing it (in which case a detailed review should reveal any problems), but also could it be that your fluorescent stains lost their activity (so remixing a new working solution from them would not help)? You ask about PBS: that would be the cheapest thing to replace with fresh solution. Good luck.

ozanamaria
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Re: fluorescence problem

#4 Post by ozanamaria » Sun Dec 11, 2016 3:12 pm

I remade all the stock solution , including the PBS, AO and IP. 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)
Ozana

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Re: fluorescence problem

#5 Post by zzffnn » Sun Dec 11, 2016 3:43 pm

Ozana,

You should probably discuss and trouble-shoot each and every possible change that you introduced, with your coworkers or mentor in your lab.

It is not an easy task to trouble shoot an experiment, even in professional environment. I doubt any amateur microscopists here can tell you anything meaningful, unless (s)he has done the exact same thing. I bet less than 3 people here have even handled AO, PI and a pro fluorescence scope.

If I have to guess, I would say your fluorochromes are not working anymore. Buy or borrow brand new fresh fluorochromes and try again, without changing anything else.

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Re: fluorescence problem

#6 Post by apochronaut » Mon Dec 12, 2016 4:40 pm

Have you checked the hardware? The DIC image is terrible....blurry and of low contrast and the fluorescence image that did not work, seems to be fluorescing but again the image is blurry and clouded. I can just make out a lot of very weak signals, across the field. I assume the instrument has multiple users?

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hkv
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Re: fluorescence problem

#7 Post by hkv » Mon Dec 12, 2016 9:59 pm

I stand behind my first post. Check you equipment configuration for misalignment. The DIC image does not show any DIC characteristics. You must adjust your gear and see if things improve.

What is the white spots in your image? Is the condenser mounted correctly?

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