Bacterial fixation issue

[Shane van Breda]

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Dear Members,

I am having problems with my method of fixation. I am attempting to fix my bacteria to
poly - l - lysine slides. My cells are grown in 98 microtiter plates (used for antimicrobial
investigations) and one well contains ~ 200 ul of 1 x 10^5 CFU/ml in media. I have
attempted centrifuging to obtain a pellet, but no luck. So my last attempt was to remove
the 200 ul of cells in the media and place them in fixative (2.5 % v/v FA and GA for AFM
and 4 % v/v FA for confocal in 1 x PBS). I place ~ 50 - 100 ul of the sample on a slide and
allow to fix to for ~ 30 min at room temperature. I then rinse with 1 x PBS and dH2O.

Almost no cells are being fixed to slide and I can't understand why?

If there are any suggestions, that would be great.

Would it be better to avoid this method of fixation and rather use heat? I thought I could
allow the solution to dry to the slide, then pass it through a flame?

Thanks

[Tobias Baskin]

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Shane,
            Might be the polylysine. How are you applying that? We have
found much better results if we put our slide or coverslip (whatever we
plan to polylysine coat) into a plasma for a minute. Immediately add
water and let evaporate to dryness, and then add the polylysine and
again let evaporate. This seems to make the coat really sticky. Of
course to try this you need a handy plasma generator -- but these are
often around in materials science labs or EM labs because they are used
to clean surfaces.

         Hope this helps,
                                 Tobias
On 5/27/14, 6:21 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
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Dear Members,
>
> I am having problems with my method of fixation. I am attempting to fix my bacteria to
> poly - l - lysine slides. My cells are grown in 98 microtiter plates (used for antimicrobial
> investigations) and one well contains ~ 200 ul of 1 x 10^5 CFU/ml in media. I have
> attempted centrifuging to obtain a pellet, but no luck. So my last attempt was to remove
> the 200 ul of cells in the media and place them in fixative (2.5 % v/v FA and GA for AFM
> and 4 % v/v FA for confocal in 1 x PBS). I place ~ 50 - 100 ul of the sample on a slide and
> allow to fix to for ~ 30 min at room temperature. I then rinse with 1 x PBS and dH2O.
>
> Almost no cells are being fixed to slide and I can't understand why?
>
> If there are any suggestions, that would be great.
>
> Would it be better to avoid this method of fixation and rather use heat? I thought I could
> allow the solution to dry to the slide, then pass it through a flame?
>
> Thanks
>

--
       __    ___   ^    ___   ___  Tobias I. Baskin
      /  \  /     / \  /      \     Professor
     /   / /     /   \ \       \     Biology Department
    / __/ /__   /___  \ \       \__   611 N. Pleasant St.
   /     /     /       \ \       \     University of Massachusetts
  /     /     /         \ \       \     Amherst, MA, 01003 USA
/     /___  /           \ \___/   \_____    413 - 545 - 1533
   www.bio.umass.edu/biology/baskin

[mmodel]

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We kept E coli in nutrient broth over glass coverslips for 3 hours and they became attached.

Other methods:

Chemical pretreatment of glass:
Vadillo-Rodríguez, V., Busscher, H.J., Norde, W., De Vries, J., Dijkstra, R.J., Stokroos, I.,
Van Der Mei, H.C., 2004. Comparison of atomic force microscopy interaction forces
between bacteria and silicon nitride substrata for three commonly used immobilization methods. Appl. Environ. Microbiol. 70, 5441-5446.

Cytocentrifugation:
Shanholtzer, C.J., Schaper, P.J., Peterson, L.R., 1982. Concentrated gram stain smears
prepared with a cytospin centrifuge. J. Clin. Microbiol. 16, 1052-1056

Treatment with azide:
Sjoblad, R.D., Doetsch, R.N., 1982. Adsorption of polarlyflagellated bacteria to surfaces.
Curr. Microbiol. 7, 191-194.

---------------------------------------

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Shane van Breda
Sent: Tuesday, May 27, 2014 6:22 AM
To: [hidden email]
Subject: Bacterial fixation issue

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Dear Members,

I am having problems with my method of fixation. I am attempting to fix my bacteria to poly - l - lysine slides. My cells are grown in 98 microtiter plates (used for antimicrobial
investigations) and one well contains ~ 200 ul of 1 x 10^5 CFU/ml in media. I have attempted centrifuging to obtain a pellet, but no luck. So my last attempt was to remove the 200 ul of cells in the media and place them in fixative (2.5 % v/v FA and GA for AFM and 4 % v/v FA for confocal in 1 x PBS). I place ~ 50 - 100 ul of the sample on a slide and allow to fix to for ~ 30 min at room temperature. I then rinse with 1 x PBS and dH2O.

Almost no cells are being fixed to slide and I can't understand why?

If there are any suggestions, that would be great.

Would it be better to avoid this method of fixation and rather use heat? I thought I could allow the solution to dry to the slide, then pass it through a flame?

Thanks