Bacterial biofilm extracellular matrix labelling?

[Christian Elowsky-2]

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I'm looking for input on directly labeling the ECM of bacterial biofilms.  I'm only concerned with the ECM as I'm able to modify the bacterial labels as needed.  Any pointers are appreciated, as a primarily plant biologist, these methods are quite alien.  Thanks!

Christian



Christian Elowsky, Ph.D.
Department of Agronomy and Horticulture
Restorative|Relator|Learner|Analytical|Individualization

[Palmer, Robert J (NIH/NIDCR) [E]]

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Hello Christian -

IMHO, no great universally applicable approach exists.  I do believe confocal microscopy is critical for getting good images.
The most specific approach involves lectins and a recent article demonstrates in a highly multiplexed manner how these can be used.

Lawrence, John R.; Winkler, Marcus; Neu, Thomas R.
FRONTIERS IN MICROBIOLOGY   Volume: 9     Article Number: 2186   Published: SEP 19 2018


Less specific (and, in a certain sense, at least as informative) approaches have recently been reviewed.

Confocal microscopy imaging of the biofilm matrix
By: Schlafer, Sebastian; Meyer, Rikke L.
JOURNAL OF MICROBIOLOGICAL METHODS   Volume: 138   Special Issue: SI   Pages: 50-59   Published: JUL 2017


Good luck!  Happy to chat off-line.

Rob Palmer






On 10/17/18, 10:36 AM, "Christian Elowsky" <[hidden email]> wrote:

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    I'm looking for input on directly labeling the ECM of bacterial biofilms.  I'm only concerned with the ECM as I'm able to modify the bacterial labels as needed.  Any pointers are appreciated, as a primarily plant biologist, these methods are quite alien.  Thanks!
   
    Christian
   
   
   
    Christian Elowsky, Ph.D.
    Department of Agronomy and Horticulture
    Restorative|Relator|Learner|Analytical|Individualization
   

[Angela Kurz]

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Hi,

Do you want to label the ECM in mammalian cells or in plants?
And which microscope are you working with?
It’s worth trying to get the laser reflection signal by putting the detector close to the laser (don’t use HyDs).
You will mostly visualize collagen fibres, similar to the SHG signal you see in 2Photon microscopy.
Otherwise I would recommend an antibody against pan-laminin,  collagen-I or collagen-IV - depending on what you exactly want to see.

Cheers,
Angela


DR. ANGELA R.M. KURZ
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> On 18 Oct 2018, at 1:35 am, Christian Elowsky <[hidden email]> wrote:
>
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> *****
>
> I'm looking for input on directly labeling the ECM of bacterial biofilms.  I'm only concerned with the ECM as I'm able to modify the bacterial labels as needed.  Any pointers are appreciated, as a primarily plant biologist, these methods are quite alien.  Thanks!
>
> Christian
>
>
>
> Christian Elowsky, Ph.D.
> Department of Agronomy and Horticulture
> Restorative|Relator|Learner|Analytical|Individualization

[Christian Elowsky-2]

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Angela,

I'm working in three very different systems, but in all three the biofilms are forming on inorganic surfaces.  From several very kind suggestions off-list, I believe the labs will be attempting dextran labeling of the extracellular matrix.  I'll keep your suggestions in mind for subsequent work as bacterial biofilms appear to be quite popular research questions in our core.  As we are not attached to a medical school, we never know what is going to walk through the door.  Thanks!

Christian


 

-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Angela Kurz
Sent: Sunday, October 21, 2018 6:22 PM
To: [hidden email]
Subject: Re: Bacterial biofilm extracellular matrix labelling?

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Hi,

Do you want to label the ECM in mammalian cells or in plants?
And which microscope are you working with?
It’s worth trying to get the laser reflection signal by putting the detector close to the laser (don’t use HyDs).
You will mostly visualize collagen fibres, similar to the SHG signal you see in 2Photon microscopy.
Otherwise I would recommend an antibody against pan-laminin,  collagen-I or collagen-IV - depending on what you exactly want to see.

Cheers,
Angela


DR. ANGELA R.M. KURZ
Research Officer | DFG Postdoctoral Research Fellow | Immune Imaging Program | Centenary Institute

CENTENARY INSTITUTE
Mail | Locked Bag 6 | Newtown | NSW | 2042 | Australia Courier | Building 93 Royal Prince Alfred Hospital | Missenden Road | Camperdown | NSW | 2050 | Australia

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> On 18 Oct 2018, at 1:35 am, Christian Elowsky <[hidden email]> wrote:
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> *****
>
> I'm looking for input on directly labeling the ECM of bacterial biofilms.  I'm only concerned with the ECM as I'm able to modify the bacterial labels as needed.  Any pointers are appreciated, as a primarily plant biologist, these methods are quite alien.  Thanks!
>
> Christian
>
>
>
> Christian Elowsky, Ph.D.
> Department of Agronomy and Horticulture
> Restorative|Relator|Learner|Analytical|Individualization

[Farid Jalali]

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Hi Christian, it has been several years since I did this, but check out calcofluor white (CW) for labelling bacterial ECM. There is quite a lot of literature relevant to its use, Gram positivity/negativity may be something to consider. It will excite with a 405 nm laser, crosstalk/bleedthrough may be an issue depending on what else you are staining or expressing in the biofilm. Spectral scanning/separation/unmixing may be required, I’ve successfully separated CW and DAPI with relative ease using a spectral scan and post-processing.  It’s use with reagents that are emitting in the green or greater seems very feasible without having to use a spectral confocal. Good luck.

Cheers.
Farid.

Sent from my iPhone

> On Oct 17, 2018, at 7:35 AM, Christian Elowsky <[hidden email]> wrote:
>
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> *****
>
> I'm looking for input on directly labeling the ECM of bacterial biofilms.  I'm only concerned with the ECM as I'm able to modify the bacterial labels as needed.  Any pointers are appreciated, as a primarily plant biologist, these methods are quite alien.  Thanks!
>
> Christian
>
>
>
> Christian Elowsky, Ph.D.
> Department of Agronomy and Horticulture
> Restorative|Relator|Learner|Analytical|Individualization

[daj1u06]

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There are some interesting EM based papers on the effect of different fixatives on bacterial ECM (many fixes remove most of it!) and the use of compounds like alcian blue or ruthenium red to try to preserve it. Our biofilm folks routinely spike PFA with alcian blue.

Check out High-resolution Visualization of the Microbial Glycocalyx with Low-voltage Scanning Electron Microscopy: Dependence on Cationic Dyes
Stanley L. Erlandsen, Christopher J. Kristich, Gary M. Dunny, and Carol L. Wells
J Histochem Cytochem 52(11) 1427-1435