Re: CONFOCALMICROSCOPY Digest - 20 Sep 2010 to 21 Sep 2010 (#2010-22)

[David Coder-2]

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There are lots of questions to be answered to arrive at an optimal solution.

First, you probably don't need to sterilize the entire apparatus.

What kind of cell chamber are you using, and what kind of environmental
exposure do the cells/medium have while the incubation/perfusion chamber is
on the microscope stage?

What are potential sources of contamination? How is the humidified CO2/air
delivered? Is the water sterile? How often is the tubing sterilized? How
often are the sterile filters (at least 0.2um) replaced? Is the airflow
moving over contaminated surfaces bringing contaminants into the medium?
Once you have sterilized the components that come in contact with the
sample, you need to prevent re-contamination.

What is the contaminant: fungal or bacterial? The type of microbe will
dictate the best--least severe but  effective--means of decontamination.

What parts can be sterilized? The materials used--plastics, aluminum,
adhesives, coatings etc.--can be incompatible with some decontamination
procedures. Common methods-- UV irradiation, autoclaving, ethylene oxide,
formaldehye or glutaraldehyde, acids/bases, H2O2 vapor--can work but are
ruled out for some materials. When using noxious chemical sterilization,
you need to assure that sufficient traces are removed such that you do not
affect cell physiology. One of the more effective and least noxious means is
H2O2 vapor. (It's been used to sterilize satellites going to Mars.) What do
the manufacturers of the various components recommend?

Alternatively, will the cells/experimental conditions tolerate increased
levels of anti-bacterials and/or anti-fungals? If so, the occasional
contaminant can be averted without heroic attempts to clean everything.

Finally, does contamination happen at random or only with some experiments?
Some people are better at aseptic techniques than others.

==============
David M. Coder, PhD
Consultant in Cytometry
Irvine, CA
Cell phone: [phone]
Skype: dave.coder
Email: [hidden email]

On Tue, Sep 21, 2010 at 10:01 PM, CONFOCALMICROSCOPY automatic digest system
<[hidden email]> wrote:

> Date:     Wed, 22 Sep 2010 00:01:37 -0500
> Reply-To: Confocal Microscopy List <[hidden email]>
>
> Date:    Tue, 21 Sep 2010 16:25:46 -0400
> From:    Stephen Lockett <[hidden email]>
> Subject: decontaminating large incubation chambers on optical microscopes
> MIME-Version: 1.0
> Content-Type: text/plain; charset="us-ascii"; format=flowed
>
>
> Dear Confocal Microscopists,
>
> I have two Olympus microscope systems, one is a confocal (FV1000) and
> the other is a TIRF (TIRF3) and both have large incubation chambers
> that surround the stage, objective lenses etc.  The incubation
> chambers are from Precision Plastics and provide heat, humidity and
> CO2 to the sample and generally work well for long term experiments
> lasting several days.  However, occasionally we experience
> contamination in the samples and we suspect the contamination resides
> in the incubation chamber, in the tubing carrying the warm, moist air
> in and out of the chamber or in the humidifier itself.  Do you have
> any suggestions about how best to decontaminate the chamber and
> associated components.  Many thanks.
>
> Sincerely,
>
>
> Stephen Lockett, Ph.D.,
> Principal Scientist,
> Director, Optical Microscopy and Analysis Laboratory,
> Rm 104A, Building 538,
> P.O. Box B
> (For Fedex, use Building 1050, Boyles Street)
> National Cancer Institute - Frederick / SAIC - Frederick,
> Fort Detrick,
> Frederick,
> MD 21702, USA
> Office: 301 846 5515
> Mobile: [phone]
>
>