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Imaging bacteria help

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Shane van Breda

Imaging bacteria help

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Hi everyone,

I have two questions about imaging bacteria (specifically Tuberculosis or any
Mycobacterium).

1.) I am interested in the mycolic acids of the bacteria. Does anyone know of
a specific fluorescent dye (with specific wavelengths) or any anti - mycolic
acid antibodies for immunolabelling?

2.) What would be the best way to fix the bacteria to slides from broth? They
need to fixed and dead since they are pathogenic. But I would like to view
whole cells and not sections from resin.

Thanks very much for your input.

Shane

Tim Feinstein-2

Re: Imaging bacteria help

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Hi Shane,

When studying toxic algae, we would often affix them to lysine-coated slides for antibody staining by spotting 50-100 ul of fixed cells in a square or circle drawn with a wax pencil, and then place the slides in a special holder and spin them in a large swinging-bucket benchtop centrifuge. You may have to spin bacteria longer or faster but the same principle should apply. Cannot help with your first question.

cheers,

TF

Timothy Feinstein, PhD
Visiting Research Associate
Laboratory for GPCR Biology
Dept. of Pharmacology & Chemical Biology
University of Pittsburgh, School of Medicine
BST W1301, 200 Lothrop St.
Pittsburgh, PA 15261

On Jan 21, 2013, at 11:27 AM, Shane van Breda wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi everyone,
>
> I have two questions about imaging bacteria (specifically Tuberculosis or any
> Mycobacterium).
>
> 1.) I am interested in the mycolic acids of the bacteria. Does anyone know of
> a specific fluorescent dye (with specific wavelengths) or any anti - mycolic
> acid antibodies for immunolabelling?
>
> 2.) What would be the best way to fix the bacteria to slides from broth? They
> need to fixed and dead since they are pathogenic. But I would like to view
> whole cells and not sections from resin.
>
> Thanks very much for your input.
>
> Shane

Rob Palmer

Re: Imaging bacteria help

In reply to this post by Shane van Breda

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*****

Shane - what exactly are you trying to do? It is unlikely that you
will be able to differentiate between different mycolic acid
structures using anything other than very well characterized
antibodies together with immunoTEM. If you are trying to distinguish
cells that have mycolic acids from those that do not, there are
probably easier ways unrelated to mycolic acids. What do you want to
do with your bacteria from broth? Simply drying washed cells onto a
slide coated with polylysine ought to do the trick. I think you
understand that these cells are very small compared to most eukaryotic
cells and, depending on what exactly you want to see, EM or image-
processing of confocal/digital-decon images may be the way to go. You
can contact me off-list if you'd like to discuss in detail.

On Jan 21, 2013, at 11:27 AM, Shane van Breda wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi everyone,
>
> I have two questions about imaging bacteria (specifically
> Tuberculosis or any
> Mycobacterium).
>
> 1.) I am interested in the mycolic acids of the bacteria. Does
> anyone know of
> a specific fluorescent dye (with specific wavelengths) or any anti -
> mycolic
> acid antibodies for immunolabelling?
>
> 2.) What would be the best way to fix the bacteria to slides from
> broth? They
> need to fixed and dead since they are pathogenic. But I would like
> to view
> whole cells and not sections from resin.
>
> Thanks very much for your input.
>
> Shane

Shane van Breda

Re: Imaging bacteria help

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*****

Thanks for your reply Rob.

I apologize if I wasn't clear enough. To explain, I am doing an in vivo experiment in a 96 well micro titer plate, where I incubate my cells with various antibiotics. The one antibiotic acts on the cell wall (we think the mycolic acids but could be the cytoplasmic membrane). So I would like to view the disruption of the cells when treated with the antibiotic.

I use the cells directly from the plate (250ul total volume). There are very few cells due to the incubation with the antibiotic.

I would like to use confocal as I can view intact cells (if I do immuno work I will permeablize my cells if needed) and that I can the some 3D imaging. The cells are 1um in size.

I am currently busy optimizing TEM and SEM protocols as well.

So I am not interested in a single mycolic acid, but just to use a specific technique to aid me in visualizing the disruption in membrane integrity of MTB i.e., mycolic acids/ cytoplasmic membrane etc

I have also considered some AFM work recently.

Thanks for your help.

On 22 Jan 2013, at 15:46, Rob Palmer <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Shane - what exactly are you trying to do? It is unlikely that you will be able to differentiate between different mycolic acid structures using anything other than very well characterized antibodies together with immunoTEM. If you are trying to distinguish cells that have mycolic acids from those that do not, there are probably easier ways unrelated to mycolic acids. What do you want to do with your bacteria from broth? Simply drying washed cells onto a slide coated with polylysine ought to do the trick. I think you understand that these cells are very small compared to most eukaryotic cells and, depending on what exactly you want to see, EM or image-processing of confocal/digital-decon images may be the way to go. You can contact me off-list if you'd like to discuss in detail.
>
> On Jan 21, 2013, at 11:27 AM, Shane van Breda wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Hi everyone,
>>
>> I have two questions about imaging bacteria (specifically Tuberculosis or any
>> Mycobacterium).
>>
>> 1.) I am interested in the mycolic acids of the bacteria. Does anyone know of
>> a specific fluorescent dye (with specific wavelengths) or any anti - mycolic
>> acid antibodies for immunolabelling?
>>
>> 2.) What would be the best way to fix the bacteria to slides from broth? They
>> need to fixed and dead since they are pathogenic. But I would like to view
>> whole cells and not sections from resin.
>>
>> Thanks very much for your input.
>>
>> Shane
>
> Robert J. Palmer Jr., Ph.D.
> Microbial Receptors Unit
> Laboratory of Cell and Developmental Biology
> Natl Inst Dental Craniofacial Res - Natl Insts Health
> Bldg 30, Room 310
> 30 Convent Drive
> Bethesda MD 20892
> ph 301-594-0025
> fax 301-402-0396

nicoletta fucà

Re: Imaging bacteria help

In reply to this post by Rob Palmer

________________________________
Da: Rob
*****
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*****

I'm interested as well.

________________________________
Da: Rob Palmer <[hidden email]>
A: [hidden email]
Inviato: Martedì 22 Gennaio 2013 14:46
Oggetto: Re: Imaging bacteria help

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Shane - what exactly are you trying to do? It is unlikely that you will be able to differentiate between different mycolic acid structures using anything other than very well characterized antibodies together with immunoTEM. If you are trying to distinguish cells that have mycolic acids from those that do not, there are probably easier ways unrelated to mycolic acids. What do you want to do with your bacteria from broth? Simply drying washed cells onto a slide coated with polylysine ought to do the trick. I think you understand that these cells are very small compared to most eukaryotic cells and, depending on what exactly you want to see, EM or image-processing of confocal/digital-decon images may be the way to go. You can contact me off-list if you'd like to discuss in detail.

On Jan 21, 2013, at 11:27 AM, Shane van Breda wrote:

Deanne Veronica Catmull

Re: Imaging bacteria help

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Have you thought about using BacLight live/dead stain (Molecular Probes/Invitrogen)? You will be able to determine the percentage of cells with ruptured membranes using this stain along with a good analysis package on your computer. As long as you set up all the appropriate controls you can get a good idea of the extent your antibiotic is affecting the cells. To look at specific cell structures though, I would follow the advice of others and utilize higher resolution techniques.

Kind regards,
Deanne.

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of nicoletta fucà
Sent: Wednesday, 23 January 2013 2:49 AM
To: [hidden email]
Subject: Re: Imaging bacteria help

________________________________
Da: Rob
*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

I'm interested as well.

________________________________
Da: Rob Palmer <[hidden email]>
A: [hidden email]
Inviato: Martedì 22 Gennaio 2013 14:46
Oggetto: Re: Imaging bacteria help

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Shane - what exactly are you trying to do? It is unlikely that you will be able to differentiate between different mycolic acid structures using anything other than very well characterized antibodies together with immunoTEM. If you are trying to distinguish cells that have mycolic acids from those that do not, there are probably easier ways unrelated to mycolic acids. What do you want to do with your bacteria from broth? Simply drying washed cells onto a slide coated with polylysine ought to do the trick. I think you understand that these cells are very small compared to most eukaryotic cells and, depending on what exactly you want to see, EM or image-processing of confocal/digital-decon images may be the way to go. You can contact me off-list if you'd like to discuss in detail.

On Jan 21, 2013, at 11:27 AM, Shane van Breda wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi everyone,
>
> I have two questions about imaging bacteria (specifically Tuberculosis
> or any Mycobacterium).
>
> 1.) I am interested in the mycolic acids of the bacteria. Does anyone
> know of a specific fluorescent dye (with specific wavelengths) or any
> anti - mycolic acid antibodies for immunolabelling?
>
> 2.) What would be the best way to fix the bacteria to slides from
> broth? They need to fixed and dead since they are pathogenic. But I
> would like to view whole cells and not sections from resin.
>
> Thanks very much for your input.
>
> Shane

Shane van Breda

Re: Imaging bacteria help

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Awesome Deanne!

I will look into this now. It will work nicely with my TEM and SEM analysis.

Thanks,

Shane

On 23 Jan 2013, at 2:11, Deanne Veronica Catmull <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Have you thought about using BacLight live/dead stain (Molecular Probes/Invitrogen)? You will be able to determine the percentage of cells with ruptured membranes using this stain along with a good analysis package on your computer. As long as you set up all the appropriate controls you can get a good idea of the extent your antibiotic is affecting the cells. To look at specific cell structures though, I would follow the advice of others and utilize higher resolution techniques.
>
> Kind regards,
> Deanne.
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of nicoletta fucà
> Sent: Wednesday, 23 January 2013 2:49 AM
> To: [hidden email]
> Subject: Re: Imaging bacteria help
>
> ________________________________
> Da: Rob
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> I'm interested as well.
>
>
> ________________________________
> Da: Rob Palmer <[hidden email]>
> A: [hidden email]
> Inviato: Martedì 22 Gennaio 2013 14:46
> Oggetto: Re: Imaging bacteria help
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Shane - what exactly are you trying to do? It is unlikely that you will be able to differentiate between different mycolic acid structures using anything other than very well characterized antibodies together with immunoTEM. If you are trying to distinguish cells that have mycolic acids from those that do not, there are probably easier ways unrelated to mycolic acids. What do you want to do with your bacteria from broth? Simply drying washed cells onto a slide coated with polylysine ought to do the trick. I think you understand that these cells are very small compared to most eukaryotic cells and, depending on what exactly you want to see, EM or image-processing of confocal/digital-decon images may be the way to go. You can contact me off-list if you'd like to discuss in detail.
>
> On Jan 21, 2013, at 11:27 AM, Shane van Breda wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Hi everyone,
>>
>> I have two questions about imaging bacteria (specifically Tuberculosis
>> or any Mycobacterium).
>>
>> 1.) I am interested in the mycolic acids of the bacteria. Does anyone
>> know of a specific fluorescent dye (with specific wavelengths) or any
>> anti - mycolic acid antibodies for immunolabelling?
>>
>> 2.) What would be the best way to fix the bacteria to slides from
>> broth? They need to fixed and dead since they are pathogenic. But I
>> would like to view whole cells and not sections from resin.
>>
>> Thanks very much for your input.
>>
>> Shane
>
> Robert J. Palmer Jr., Ph.D.
> Microbial Receptors Unit
> Laboratory of Cell and Developmental Biology Natl Inst Dental Craniofacial Res - Natl Insts Health Bldg 30, Room 310
> 30 Convent Drive
> Bethesda MD 20892
> ph 301-594-0025
> fax 301-402-0396

nicoletta fucà

Re: Imaging bacteria help

In reply to this post by Deanne Veronica Catmull

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

I use to work with BacLight live/dead stain for assessing bacteria viability but still, if bacteria flow onto the slide or coverslip (for inverted microscope), taking good images to use for quantitative analysisis is hard. As Shane said, it is necessary to fix bacteria onto the slides using a non destructive method. Any suggestions?

________________________________
Da: Deanne Veronica Catmull <[hidden email]>
A: nicoletta fucà <[hidden email]>; "[hidden email]" <[hidden email]>
Inviato: Mercoledì 23 Gennaio 2013 1:11
Oggetto: RE: Imaging bacteria help

Have you thought about using BacLight live/dead stain (Molecular Probes/Invitrogen)? You will be able to determine the percentage of cells with ruptured membranes using this stain along with a good analysis package on your computer. As long as you set up all the appropriate controls you can get a good idea of the extent your antibiotic is affecting the cells. To look at specific cell structures though, I would follow the advice of others and utilize higher resolution techniques.

Kind regards,
Deanne.

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of nicoletta fucà
Sent: Wednesday, 23 January 2013 2:49 AM
To: [hidden email]
Subject: Re: Imaging bacteria help

________________________________
Da: Rob
*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

I'm interested as well.

________________________________
Da: Rob Palmer <[hidden email]>
A: [hidden email]
Inviato: Martedì 22 Gennaio 2013 14:46
Oggetto: Re: Imaging bacteria help

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Shane - what exactly are you trying to do? It is unlikely that you will be able to differentiate between different mycolic acid structures using anything other than very well characterized antibodies together with immunoTEM. If you are trying to distinguish cells that have mycolic acids from those that do not, there are probably easier ways unrelated to mycolic acids. What do you want to do with your bacteria from broth? Simply drying washed cells onto a slide coated with polylysine ought to do the trick. I think you understand that these cells are very small compared to most eukaryotic cells and, depending on what exactly you want to see, EM or image-processing of confocal/digital-decon images may be the way to go. You can contact me off-list if you'd like to discuss in detail.

On Jan 21, 2013, at 11:27 AM, Shane van Breda wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi everyone,
>
> I have two questions about imaging bacteria (specifically Tuberculosis
> or any Mycobacterium).
>
> 1.) I am interested in the mycolic acids of the bacteria. Does anyone
> know of a specific fluorescent dye (with specific wavelengths) or any
> anti - mycolic acid antibodies for immunolabelling?
>
> 2.) What would be the best way to fix the bacteria to slides from
> broth? They need to fixed and dead since they are pathogenic. But I
> would like to view whole cells and not sections from resin.
>
> Thanks very much for your input.
>
> Shane

Jeremy Adler-4

Re: Imaging bacteria help

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

could you work with bacteria in a gel - it would immobilise them.

do you need to use a confocal - given the size of bacteria there will
be little out of focus light.

Quoting nicoletta fucà <[hidden email]>:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> I use to work with BacLight live/dead stain for assessing bacteria
> viability but still, if bacteria flow onto the slide or coverslip
> (for inverted microscope), taking good images to use for
> quantitative analysisis is hard. As Shane said, it is necessary to
> fix bacteria onto the slides using a non destructive method. Any
> suggestions?
>
>
> ________________________________
> Da: Deanne Veronica Catmull <[hidden email]>
> A: nicoletta fucà <[hidden email]>;
> "[hidden email]" <[hidden email]>
> Inviato: Mercoledì 23 Gennaio 2013 1:11
> Oggetto: RE: Imaging bacteria help
>
> Have you thought about using BacLight live/dead stain (Molecular
> Probes/Invitrogen)? You will be able to determine the percentage of
> cells with ruptured membranes using this stain along with a good
> analysis package on your computer. As long as you set up all the
> appropriate controls you can get a good idea of the extent your
> antibiotic is affecting the cells. To look at specific cell
> structures though, I would follow the advice of others and utilize
> higher resolution techniques.
>
> Kind regards,
> Deanne.
>
> -----Original Message-----
> From: Confocal Microscopy List
> [mailto:[hidden email]] On Behalf Of nicoletta fucà
> Sent: Wednesday, 23 January 2013 2:49 AM
> To: [hidden email]
> Subject: Re: Imaging bacteria help
>
> ________________________________
> Da: Rob
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> I'm interested as well.
>
>
> ________________________________
> Da: Rob Palmer <[hidden email]>
> A: [hidden email]
> Inviato: Martedì 22 Gennaio 2013 14:46
> Oggetto: Re: Imaging bacteria help
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Shane - what exactly are you trying to do? It is unlikely that you
> will be able to differentiate between different mycolic acid
> structures using anything other than very well characterized
> antibodies together with immunoTEM. If you are trying to
> distinguish cells that have mycolic acids from those that do not,
> there are probably easier ways unrelated to mycolic acids. What do
> you want to do with your bacteria from broth? Simply drying washed
> cells onto a slide coated with polylysine ought to do the trick. I
> think you understand that these cells are very small compared to
> most eukaryotic cells and, depending on what exactly you want to
> see, EM or image-processing of confocal/digital-decon images may be
> the way to go. You can contact me off-list if you'd like to discuss
> in detail.
>
> On Jan 21, 2013, at 11:27 AM, Shane van Breda wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Hi everyone,
>>
>> I have two questions about imaging bacteria (specifically Tuberculosis
>> or any Mycobacterium).
>>
>> 1.) I am interested in the mycolic acids of the bacteria. Does anyone
>> know of a specific fluorescent dye (with specific wavelengths) or any
>> anti - mycolic acid antibodies for immunolabelling?
>>
>> 2.) What would be the best way to fix the bacteria to slides from
>> broth? They need to fixed and dead since they are pathogenic. But I
>> would like to view whole cells and not sections from resin.
>>
>> Thanks very much for your input.
>>
>> Shane
>
> Robert J. Palmer Jr., Ph.D.
> Microbial Receptors Unit
> Laboratory of Cell and Developmental Biology Natl Inst Dental
> Craniofacial Res - Natl Insts Health Bldg 30, Room 310
> 30 Convent Drive
> Bethesda MD 20892
> ph 301-594-0025
> fax 301-402-0396
>

Jeremy Adler
IGP
Rudbeckslaboratoriet
Daghammersköljdsväg 20
751 85 Uppsala
Sweden

0046 (0)18 471 4607

Rob Palmer

Re: Imaging bacteria help

In reply to this post by Deanne Veronica Catmull

*****
To join, leave or search the confocal microscopy listserv, go to:
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*****

Yes, this is a good approach if you want a rough assessment of
viability. It is dependent on the type of bacterium (i.e., may
require some tweaking over the standard protocol) and may not work
well with bacteria that do not have typical cell walls. The
manufacturer has reported successful use of the stain for M. phlei.
It is important to remember that both stains must be assessed
simultaneously almost every cell incorporates at least a bit of both
dyes. Also, the test cannot differentiate between cells that have a
low membrane potential and those that have no membrane potential.
This is why you need good controls if you want to talk about
viability. Dependent on what you want to say, you may have to culture
the bacteria post-treatment. Shane, you have stated that the bacteria
must be dead prior to placement on the slide. If you fix them, all
bets are off with the Baclight product - it requires cellular
activity. After all, that is why you would use that product anyway.
Rob

On Jan 22, 2013, at 7:11 PM, Deanne Veronica Catmull wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Have you thought about using BacLight live/dead stain (Molecular
> Probes/Invitrogen)? You will be able to determine the percentage of
> cells with ruptured membranes using this stain along with a good
> analysis package on your computer. As long as you set up all the
> appropriate controls you can get a good idea of the extent your
> antibiotic is affecting the cells. To look at specific cell
> structures though, I would follow the advice of others and utilize
> higher resolution techniques.
>
> Kind regards,
> Deanne.
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]
> ] On Behalf Of nicoletta fucà
> Sent: Wednesday, 23 January 2013 2:49 AM
> To: [hidden email]
> Subject: Re: Imaging bacteria help
>
> ________________________________
> Da: Rob
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> I'm interested as well.
>
>
> ________________________________
> Da: Rob Palmer <[hidden email]>
> A: [hidden email]
> Inviato: Martedì 22 Gennaio 2013 14:46
> Oggetto: Re: Imaging bacteria help
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Shane - what exactly are you trying to do? It is unlikely that you
> will be able to differentiate between different mycolic acid
> structures using anything other than very well characterized
> antibodies together with immunoTEM. If you are trying to
> distinguish cells that have mycolic acids from those that do not,
> there are probably easier ways unrelated to mycolic acids. What do
> you want to do with your bacteria from broth? Simply drying washed
> cells onto a slide coated with polylysine ought to do the trick. I
> think you understand that these cells are very small compared to
> most eukaryotic cells and, depending on what exactly you want to
> see, EM or image-processing of confocal/digital-decon images may be
> the way to go. You can contact me off-list if you'd like to discuss
> in detail.
>
> On Jan 21, 2013, at 11:27 AM, Shane van Breda wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Hi everyone,
>>
>> I have two questions about imaging bacteria (specifically
>> Tuberculosis
>> or any Mycobacterium).
>>
>> 1.) I am interested in the mycolic acids of the bacteria. Does anyone
>> know of a specific fluorescent dye (with specific wavelengths) or any
>> anti - mycolic acid antibodies for immunolabelling?
>>
>> 2.) What would be the best way to fix the bacteria to slides from
>> broth? They need to fixed and dead since they are pathogenic. But I
>> would like to view whole cells and not sections from resin.
>>
>> Thanks very much for your input.
>>
>> Shane
>
> Robert J. Palmer Jr., Ph.D.
> Microbial Receptors Unit
> Laboratory of Cell and Developmental Biology Natl Inst Dental
> Craniofacial Res - Natl Insts Health Bldg 30, Room 310
> 30 Convent Drive
> Bethesda MD 20892
> ph 301-594-0025
> fax 301-402-0396

Kilgore, Jason-2

Re: Imaging bacteria help vendor reply

In reply to this post by Jeremy Adler-4

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

**vendor reply**

Regarding immobilization, a common method is to pull down the bacteria onto a black filter paper, using a vacuum filter. This filter paper can then be mounted on a slide and imaged using the mounting oil in the kit (assuming you are using Molecular Probes product L7007 or L7012).

Cheers,

Jason

Jason A. Kilgore
Technical Application Scientist
Molecular Probes Labeling and Detection Technologies
Cells Systems Division

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-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Jeremy Adler
Sent: Wednesday, January 23, 2013 2:33 AM
To: [hidden email]
Subject: Re: Imaging bacteria help

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could you work with bacteria in a gel - it would immobilise them.

do you need to use a confocal - given the size of bacteria there will
be little out of focus light.

Quoting nicoletta fucà <[hidden email]>:

Jeremy Adler
IGP
Rudbeckslaboratoriet
Daghammersköljdsväg 20
751 85 Uppsala
Sweden

0046 (0)18 471 4607

Shane van Breda

Re: Imaging bacteria help

In reply to this post by Rob Palmer

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Unfortunately I cant use the live/dead stain as I need to work with dead
fixed cells and I dont have acess to a confocal in a biosafety level 3 lab.

The DeltaVision OMX 3D SIM system sounds promising. I am in Pretoria, South
Africa. I am really unsure if such a system even exists in my country. It
might be possible for me to send my slides over? Ill have to think about
this one. I can just imagine the border control questions!

The main issue is finding out a way to visualise the disruption in membrane
integrity and to figure out what the antibiotic is doing to the membrane.
Its slowly looking like I will be focusing on TEM, SEM and AFM.

Regards,

Shane

----- Original Message -----
From: "Rob Palmer" <[hidden email]>
To: <[hidden email]>
Sent: Wednesday, January 23, 2013 5:49 PM
Subject: Re: Imaging bacteria help

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Yes, this is a good approach if you want a rough assessment of
viability. It is dependent on the type of bacterium (i.e., may
require some tweaking over the standard protocol) and may not work
well with bacteria that do not have typical cell walls. The
manufacturer has reported successful use of the stain for M. phlei.
It is important to remember that both stains must be assessed
simultaneously almost every cell incorporates at least a bit of both
dyes. Also, the test cannot differentiate between cells that have a
low membrane potential and those that have no membrane potential.
This is why you need good controls if you want to talk about
viability. Dependent on what you want to say, you may have to culture
the bacteria post-treatment. Shane, you have stated that the bacteria
must be dead prior to placement on the slide. If you fix them, all
bets are off with the Baclight product - it requires cellular
activity. After all, that is why you would use that product anyway.
Rob

On Jan 22, 2013, at 7:11 PM, Deanne Veronica Catmull wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Have you thought about using BacLight live/dead stain (Molecular
> Probes/Invitrogen)? You will be able to determine the percentage of cells
> with ruptured membranes using this stain along with a good analysis
> package on your computer. As long as you set up all the appropriate
> controls you can get a good idea of the extent your antibiotic is
> affecting the cells. To look at specific cell structures though, I would
> follow the advice of others and utilize higher resolution techniques.
>
> Kind regards,
> Deanne.
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email] ]
> On Behalf Of nicoletta fucà
> Sent: Wednesday, 23 January 2013 2:49 AM
> To: [hidden email]
> Subject: Re: Imaging bacteria help
>
> ________________________________
> Da: Rob
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> I'm interested as well.
>
>
> ________________________________
> Da: Rob Palmer <[hidden email]>
> A: [hidden email]
> Inviato: Martedì 22 Gennaio 2013 14:46
> Oggetto: Re: Imaging bacteria help
>
> *****
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> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Shane - what exactly are you trying to do? It is unlikely that you will
> be able to differentiate between different mycolic acid structures using
> anything other than very well characterized antibodies together with
> immunoTEM. If you are trying to distinguish cells that have mycolic
> acids from those that do not, there are probably easier ways unrelated to
> mycolic acids. What do you want to do with your bacteria from broth?
> Simply drying washed cells onto a slide coated with polylysine ought to
> do the trick. I think you understand that these cells are very small
> compared to most eukaryotic cells and, depending on what exactly you want
> to see, EM or image-processing of confocal/digital-decon images may be
> the way to go. You can contact me off-list if you'd like to discuss in
> detail.
>
> On Jan 21, 2013, at 11:27 AM, Shane van Breda wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Hi everyone,
>>
>> I have two questions about imaging bacteria (specifically Tuberculosis
>> or any Mycobacterium).
>>
>> 1.) I am interested in the mycolic acids of the bacteria. Does anyone
>> know of a specific fluorescent dye (with specific wavelengths) or any
>> anti - mycolic acid antibodies for immunolabelling?
>>
>> 2.) What would be the best way to fix the bacteria to slides from
>> broth? They need to fixed and dead since they are pathogenic. But I
>> would like to view whole cells and not sections from resin.
>>
>> Thanks very much for your input.
>>
>> Shane
>
> Robert J. Palmer Jr., Ph.D.
> Microbial Receptors Unit
> Laboratory of Cell and Developmental Biology Natl Inst Dental
> Craniofacial Res - Natl Insts Health Bldg 30, Room 310
> 30 Convent Drive
> Bethesda MD 20892
> ph 301-594-0025
> fax 301-402-0396

Robert J. Palmer Jr., Ph.D.
Microbial Receptors Unit
Laboratory of Cell and Developmental Biology
Natl Inst Dental Craniofacial Res - Natl Insts Health
Bldg 30, Room 310
30 Convent Drive
Bethesda MD 20892
ph 301-594-0025
fax 301-402-0396

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