Fluorescent dye for bacteria

[Emma King-2]

Hi All,

I am looking to fluorescently label bacteria in human skin samples
(cryosections, frozen not yet fixed) for imaging using a confocal
microscope.

Ideally we would like to determine the localisation of all bacteria within
the tissue and be able to tell if they are alive or dead. Obviously we
would like to specifically label the bacteria without also staining the
tissue.

All help much appreciated.

Cheers,
Emma

School of Biomedical Sciences
Medical School, Univeristy of Nottingham

email: [hidden email]

[Susan Anderson]

HI Emma
There is a live dead bacterial stain available from Invitrogen which has
been used reliably by us in the past. It works in a similar way to the
live dead stain for eukaryotic cells
Susan

Dr Susan I Anderson
Lecturer in Pathology Education
Room 4028
School of Graduate Entry Medicine and Health
University of Nottingham Medical School
Derby City General Hospital
Uttoxeter Road
DERBY DE22 3DT
[phone] (ext 24613 internal)


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Emma King
Sent: 23 June 2010 13:45
To: [hidden email]
Subject: Fluorescent dye for bacteria

Hi All,

I am looking to fluorescently label bacteria in human skin samples
(cryosections, frozen not yet fixed) for imaging using a confocal
microscope.

Ideally we would like to determine the localisation of all bacteria
within
the tissue and be able to tell if they are alive or dead. Obviously we
would like to specifically label the bacteria without also staining the
tissue.

All help much appreciated.

Cheers,
Emma

School of Biomedical Sciences
Medical School, Univeristy of Nottingham

email: [hidden email]
This message has been checked for viruses but the contents of an attachment
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[Mark Auty]

Emma

You could try the BacLight livedead kit from Invitrogen; it's based on
Syto9 & propidium iodide. It may also stain cell nuclei (if present) and
some proteins but usually with lower fluorescence intensity than
bacteria.

Regards
Mark


Dr Mark Auty
Manager, National Food Imaging Centre
Moorepark Food Research Centre
Teagasc Moorepark
Fermoy, Co Cork
Ireland
tel: [phone]
fax: [phone]
[hidden email]


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Emma King
Sent: 23 June 2010 13:45
To: [hidden email]
Subject: Fluorescent dye for bacteria

Hi All,

I am looking to fluorescently label bacteria in human skin samples
(cryosections, frozen not yet fixed) for imaging using a confocal
microscope.

Ideally we would like to determine the localisation of all bacteria
within
the tissue and be able to tell if they are alive or dead. Obviously we
would like to specifically label the bacteria without also staining the
tissue.

All help much appreciated.

Cheers,
Emma

School of Biomedical Sciences
Medical School, Univeristy of Nottingham

email: [hidden email]
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[Emma King-2]

Hi again,

Thanks for your responses.

My user has tried the BacLight kit but says there is a strong
red 'background' in her samples. I think this is probably a combination
of PI also labelling the nuclei of the cells in the tissue and her sample
prep (I haven't actually seen the images myself). Does anyone have
any experience as to how we could reduce 'background' by tweaking
the sample prep?

Is the SYTO9 dye bacteria specific and doesn't stain eukaryotic cells?

Cheers,
Emma

[Deanne Veronica Catmull]

Make sure you do not use PBS with this dye as it will create a strong
background (I think you will find this in the technical notes). I have
also found that if you do not wash the media from the preparation
properly before staining, it can cause this kind of "background" due to
auto fluorescent components in the media.

Kind regards,
Deanne.

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Emma King
Sent: Wednesday, 23 June 2010 11:31 PM
To: [hidden email]
Subject: Re: Fluorescent dye for bacteria

Hi again,

Thanks for your responses.

My user has tried the BacLight kit but says there is a strong
red 'background' in her samples. I think this is probably a combination
of PI also labelling the nuclei of the cells in the tissue and her
sample
prep (I haven't actually seen the images myself). Does anyone have
any experience as to how we could reduce 'background' by tweaking
the sample prep?

Is the SYTO9 dye bacteria specific and doesn't stain eukaryotic cells?

Cheers,
Emma

[JOEL B. SHEFFIELD]

Hi

Syto 9 will certainly stain nuclei in eukaryotes.  It works by binding to DNA.

Joel

On Wed, Jun 30, 2010 at 8:26 PM, Deanne Veronica Catmull <[hidden email]> wrote:

Make sure you do not use PBS with this dye as it will create a strong
background (I think you will find this in the technical notes). I have
also found that if you do not wash the media from the preparation
properly before staining, it can cause this kind of "background" due to
auto fluorescent components in the media.

Kind regards,
Deanne.

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Emma King
Sent: Wednesday, 23 June 2010 11:31 PM
To: [hidden email]
Subject: Re: Fluorescent dye for bacteria

Hi again,

Thanks for your responses.

My user has tried the BacLight kit but says there is a strong
red 'background' in her samples. I think this is probably a combination
of PI also labelling the nuclei of the cells in the tissue and her
sample
prep (I haven't actually seen the images myself). Does anyone have
any experience as to how we could reduce 'background' by tweaking
the sample prep?

Is the SYTO9 dye bacteria specific and doesn't stain eukaryotic cells?

Cheers,
Emma

--


Joel B. Sheffield, Ph.D
Department of Biology
Temple University
Philadelphia, PA 19122
Voice: 215 204 8839
e-mail: [hidden email]
URL:  http://astro.temple.edu/~jbs