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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" <[log in to unmask]>
To: <[log in to unmask]>
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:[log in to unmask] ] 
> On Behalf Of nicoletta fucà
> Sent: Wednesday, 23 January 2013 2:49 AM
> To: [log in to unmask]
> Subject: Re: Imaging bacteria help
>
> ________________________________
> Da: Rob
> *****
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>
> I'm interested as well.
>
>
> ________________________________
> Da: Rob Palmer <[log in to unmask]>
> A: [log in to unmask]
> 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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