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Ozana Maria Petraru

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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

mmodel

AO and PI

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!

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

LISTSERV 16.0 - CONFOCALMICROSCOPY List at LISTS.UMN.EDU

lists.umn.edu

[hidden email]: listserv archives. confocalmicroscopy

Heloo!

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

Lloyd Donaldson

Re: AO and PI

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

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of MODEL, MICHAEL
Sent: Thursday, December 08, 2016 1:23 PM
To: [hidden email]
Subject: AO and PI

Ozana,

What exactly stopped working?

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!

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

LISTSERV 16.0 - CONFOCALMICROSCOPY List at LISTS.UMN.EDU

lists.umn.edu

[hidden email]: listserv archives. confocalmicroscopy

Heloo!

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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Ozana Maria Petraru

Răspuns: AO and PI

In reply to this post by mmodel

Acridine orange is an intercalating dye that can permeate both live and dead cells. AO will stain all cells to generate green fluorescence. Propidium iodide can only enter dead cells with poor membrane intergrity => all live cells fluoresce green and all dead cells fluoresce red. I can not use DAPI because I do not have a UV filter.

 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.

Thank you!

 Ozana Petraru, Iasi, Romania

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Imgur: The most awesome images on the Internet

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Imgur: The most awesome images on the Internet

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În Joi, 8 Decembrie 2016 2:23:36, "MODEL, MICHAEL" <[hidden email]> a scris:

Ozana,

What exactly stopped working?

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!

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

LISTSERV 16.0 - CONFOCALMICROSCOPY List at LISTS.UMN.EDU

lists.umn.edu

[hidden email]: listserv archives. confocalmicroscopy

Heloo!

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

mmodel

Re: Răspuns: AO and PI

Hi Ozana,

Your bright field (it's not DIC) looks strange, it should be uniform without bright spots. I would start by checking your microscope, make sure you can obtain good and clear brightfield and fluorescent images of something that is definitely fluorescent (a drop of fluorescein, for example)

Mike Model