attaching pseudomonas bacteria to cover slips

[Vernita Gordon]

Hello,

I am trying to attach pseudomonas bacteria to glass coverslips for
microscopy.  For my application, the bacteria need to attach with very high
efficiency - ie, all the bacteria that reach the glass surface need to stick
there.  I have tried incubating the glass in fibrinogen, fibronectin, and the
Sigma poly-L-lysine solution reccomended for cell attachment.  None of these
achieve anywhere near the attachment efficiency I need.  Does anyone know
how to do this, or have suggestions for approaches or protocols?

thank you,

Vernita

[Boswell, Carl A - (cboswell)]

Celltak works well as an adherent substrate
http://www.bdbiosciences.com/discovery_labware/products/display_product.php?keyID=227.
It's derived from the adhesive that mussles use to hold themselves onto
surfaces in the rocky intertidal.
Carl

Carl A. Boswell, Ph.D.
Molecular and Cellular Biology
University of Arizona
520-954-7053
FAX [phone]
----- Original Message -----
From: "Vernita Gordon" <[hidden email]>
To: <[hidden email]>
Sent: Friday, December 12, 2008 4:18 PM
Subject: attaching pseudomonas bacteria to cover slips


Hello,

I am trying to attach pseudomonas bacteria to glass coverslips for
microscopy.  For my application, the bacteria need to attach with very high
efficiency - ie, all the bacteria that reach the glass surface need to stick
there.  I have tried incubating the glass in fibrinogen, fibronectin, and
the
Sigma poly-L-lysine solution reccomended for cell attachment.  None of these
achieve anywhere near the attachment efficiency I need.  Does anyone know
how to do this, or have suggestions for approaches or protocols?

thank you,

Vernita

[Tobias Baskin]

Vernita,

        I have used Poly-ethylene-imine. its sticky as blazes. I have heard that living plant cells don't like it (while they are alive). I believe I used a 1% aqueous solution, spread a drop on the coverslips and let it evaporate. I am traveling right now, don't have my notes, perhaps you can find info about it? If you want to pursue this further I might be able to dredge up some other stuff for you.

        Good luck,

                Tobias

Hello,

I am trying to attach pseudomonas bacteria to glass coverslips for
microscopy.  For my application, the bacteria need to attach with very high
efficiency - ie, all the bacteria that reach the glass surface need to stick
there.  I have tried incubating the glass in fibrinogen, fibronectin, and the
Sigma poly-L-lysine solution reccomended for cell attachment.  None of these
achieve anywhere near the attachment efficiency I need.  Does anyone know
how to do this, or have suggestions for approaches or protocols?

thank you,

Vernita

    _      ____          __   ____   

     /  \   /          / \    /   \ \        Tobias I. Baskin

    /   /  /          /   \   \      \         Biology Department

   /_ /   __      /__ \   \       \__    611 N. Pleasant St.

  /      /          /       \   \       \        University of Massachusetts

 /      /          /         \   \       \          Amherst, MA, 01003

/      / ___   /           \   \__/  \ ____

www.bio.umass.edu/biology/baskin

Voice: 413 - 545 - 1533 Fax: 413 - 545 - 3243

[Carol Heckman]

Vernita-
The best way might be to use an antibody against LPS, if they have that, or some other external coat component.  You can stick an antibody to glass as a monolayer, once you have the glass surface molecularly clean.  This can be done by putting the slides in dilute acid then rinsing with copious amounts of clean (Millipore) water and ethanol.
Carol
Center for Microscopy & Microanalysis
Bowling Green State University
________________________________________
From: Confocal Microscopy List [[hidden email]] On Behalf Of Tobias Baskin [[hidden email]]
Sent: Friday, December 12, 2008 9:53 PM
To: [hidden email]
Subject: Re: attaching pseudomonas bacteria to cover slips

Vernita,
        I have used Poly-ethylene-imine. its sticky as blazes. I have heard that living plant cells don't like it (while they are alive). I believe I used a 1% aqueous solution, spread a drop on the coverslips and let it evaporate. I am traveling right now, don't have my notes, perhaps you can find info about it? If you want to pursue this further I might be able to dredge up some other stuff for you.

        Good luck,
                Tobias


Hello,

I am trying to attach pseudomonas bacteria to glass coverslips for
microscopy.  For my application, the bacteria need to attach with very high
efficiency - ie, all the bacteria that reach the glass surface need to stick
there.  I have tried incubating the glass in fibrinogen, fibronectin, and the
Sigma poly-L-lysine solution reccomended for cell attachment.  None of these
achieve anywhere near the attachment efficiency I need.  Does anyone know
how to do this, or have suggestions for approaches or protocols?

thank you,

Vernita


    _      ____          __   ____
     /  \   /          / \    /   \ \        Tobias I. Baskin
    /   /  /          /   \   \      \         Biology Department
   /_ /   __      /__ \   \       \__    611 N. Pleasant St.
  /      /          /       \   \       \        University of Massachusetts
 /      /          /         \   \       \          Amherst, MA, 01003
/      / ___   /           \   \__/  \ ____
www.bio.umass.edu/biology/baskin
Voice: 413 - 545 - 1533 Fax: 413 - 545 - 3243

[rjpalmer]

What strain of Pa are you using?  How do you prepare the cells? What
happens to the glass after you "adhere" the bacteria?  In general,
you should not have to coat glass to get adherence - biofilm
formation occurs best with living cells in a low-nutrient medium.

Rob Palmer

>Hello,
>
>I am trying to attach pseudomonas bacteria to glass coverslips for
>microscopy.  For my application, the bacteria need to attach with very high
>efficiency - ie, all the bacteria that reach the glass surface need to stick
>there.  I have tried incubating the glass in fibrinogen, fibronectin, and the
>Sigma poly-L-lysine solution reccomended for cell attachment.  None of these
>achieve anywhere near the attachment efficiency I need.  Does anyone know
>how to do this, or have suggestions for approaches or protocols?
>
>thank you,
>
>Vernita

--
Robert J. Palmer Jr., Ph.D.
Natl Inst Dental Craniofacial Res - Natl Insts Health
Oral Infection and Immunity Branch
Bldg 30, Room 310
30 Convent Drive
Bethesda MD 20892
ph 301-594-0025
fax [phone]

[Kevin Braeckmans]

Hi,

No practical experience, but you could have a look at the Stovall Flow Cell
(http://www.slscience.com/flowcell.html) which was specifically designed for
growing biofilms in a microscopy compatible chamber.

Best regards,

Kevin

> -----Oorspronkelijk bericht-----
> Van: Confocal Microscopy List [mailto:[hidden email]]
> Namens Vernita Gordon
> Verzonden: zaterdag 13 december 2008 0:19
> Aan: [hidden email]
> Onderwerp: attaching pseudomonas bacteria to cover slips
>
> Hello,
>
> I am trying to attach pseudomonas bacteria to glass coverslips for
> microscopy.  For my application, the bacteria need to attach with very
> high
> efficiency - ie, all the bacteria that reach the glass surface need to
> stick
> there.  I have tried incubating the glass in fibrinogen, fibronectin,
> and the
> Sigma poly-L-lysine solution reccomended for cell attachment.  None of
> these
> achieve anywhere near the attachment efficiency I need.  Does anyone
> know
> how to do this, or have suggestions for approaches or protocols?
>
> thank you,
>
> Vernita