Re: Imaging bacteria help

Posted by Shane van Breda on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Imaging-bacteria-help-tp7579528p7579545.html

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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:

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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:
>
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>> To join, leave or search the confocal microscopy listserv, go to:
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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
>
> Robert J. Palmer Jr., Ph.D.
> Microbial Receptors Unit
> Laboratory of Cell and Developmental Biology
> Natl Inst Dental Craniofacial Res - Natl Insts Health
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