Imaging BSL2 samples in a core facility

[Smith, Benjamin E.]

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Hey Microscopists,
   I have a user who wants to image unfixed MRSA on our confocal microscope.  Before, I had them fix the cells in glutaraldehyde before bringing the samples over to remove any pathogenicity.  However, they are now concerned that the fixative might be interfering with their compound, and so would like to try imaging live cells.  

  I have worked with BSL2 specimens before, but never in the scope of a confocal core facility.  What special considerations should be involved, especially concerning sealing slides and avoiding cross contamination of the microscope.  I definitely would like to avoid needing to sterilize any contaminated objectives at all costs.

Thanks,
   Ben Smith

Benjamin E. Smith, Ph.D.
Samuel Roberts Noble Microscopy Laboratory
Research Scientist, Confocal Facility Manager
University of Oklahoma
Norman, OK 73019
E-mail: [hidden email]
Voice   405-325-4391
FAX  [phone]
http://www.microscopy.ou.edu/

[Mel Symeonides]

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Hello Ben,

We set up a protocol with our biosafety officer here to image live
HIV-infected cells (BSL2+) on a facility microscope. The cells are
contained in a multiwell glass-bottom plate with the lid taped to the
plate to prevent any chance of a spill. The plates we use are from In
Vitro Scientific (www.invitrosci.com), but there are many glass-bottom
vessels you could use that could be taped shut (e.g. Nunc Lab-Tek
chamber slides, MatTek dishes, Ibidi microslides etc.). The outside of
the plate was decontaminated with 70% ethanol, and was transported from
the lab to the microscope facility inside a box which was then placed
inside a tertiary container (e.g. an Igloo cooler). There was never any
contact between contaminated fluid or material and any part of the
microscope. This of course assumes that you don't need to use an
immersion objective.

There are of course pathogen-specific considerations here, e.g. whether
there is a chance of aerosol transmission and how that might be
contained, what culture conditions are required, etc. I suggest you get
in touch with your institute's biosafety officer to compile a protocol,
it should be possible to figure something out.

Best regards,
Mel


On 3/17/2015 4:02 PM, Smith, Benjamin E. 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.
> *****
>
> Hey Microscopists,
>     I have a user who wants to image unfixed MRSA on our confocal microscope.  Before, I had them fix the cells in glutaraldehyde before bringing the samples over to remove any pathogenicity.  However, they are now concerned that the fixative might be interfering with their compound, and so would like to try imaging live cells.
>
>    I have worked with BSL2 specimens before, but never in the scope of a confocal core facility.  What special considerations should be involved, especially concerning sealing slides and avoiding cross contamination of the microscope.  I definitely would like to avoid needing to sterilize any contaminated objectives at all costs.
>
> Thanks,
>     Ben Smith
>
> Benjamin E. Smith, Ph.D.
> Samuel Roberts Noble Microscopy Laboratory
> Research Scientist, Confocal Facility Manager
> University of Oklahoma
> Norman, OK 73019
> E-mail: [hidden email]
> Voice   405-325-4391
> FAX  [phone]
> http://www.microscopy.ou.edu/
>

--
Menelaos Symeonides
University of Vermont
Cell & Molecular Biology Graduate Program
Department of Microbiology and Molecular Genetics
318 Stafford Hall
95 Carrigan Dr
Burlington, VT 05405
[hidden email]
Phone: [phone]

[Craig Brideau]

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I second Mel's comments. We used a transport container to carry lentivirus
samples from our cell culture room to our imaging room as part of the
protocol we worked out with our biosafety officer. They will be your best
resource. For reference, this is the container we used, although it may or
may not be appropriate for your regulations:

https://www.spectrumchemical.com/OA_HTML/lab-supplies-products_Thermo-Scientific-Nalgene-Bio-Transport-Carrier-PC-967-18316_306522.jsp?minisite=10020&respid=22372&phrase=Test%20Tube%20Clamp

Craig

On Tue, Mar 17, 2015 at 4:13 PM, Menelaos Symeonides <[hidden email]>
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.
> *****
>
> Hello Ben,
>
> We set up a protocol with our biosafety officer here to image live
> HIV-infected cells (BSL2+) on a facility microscope. The cells are
> contained in a multiwell glass-bottom plate with the lid taped to the plate
> to prevent any chance of a spill. The plates we use are from In Vitro
> Scientific (www.invitrosci.com), but there are many glass-bottom vessels
> you could use that could be taped shut (e.g. Nunc Lab-Tek chamber slides,
> MatTek dishes, Ibidi microslides etc.). The outside of the plate was
> decontaminated with 70% ethanol, and was transported from the lab to the
> microscope facility inside a box which was then placed inside a tertiary
> container (e.g. an Igloo cooler). There was never any contact between
> contaminated fluid or material and any part of the microscope. This of
> course assumes that you don't need to use an immersion objective.
>
> There are of course pathogen-specific considerations here, e.g. whether
> there is a chance of aerosol transmission and how that might be contained,
> what culture conditions are required, etc. I suggest you get in touch with
> your institute's biosafety officer to compile a protocol, it should be
> possible to figure something out.
>
> Best regards,
> Mel
>
>
>
> On 3/17/2015 4:02 PM, Smith, Benjamin E. 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.
>> *****
>>
>> Hey Microscopists,
>>     I have a user who wants to image unfixed MRSA on our confocal
>> microscope.  Before, I had them fix the cells in glutaraldehyde before
>> bringing the samples over to remove any pathogenicity.  However, they are
>> now concerned that the fixative might be interfering with their compound,
>> and so would like to try imaging live cells.
>>
>>    I have worked with BSL2 specimens before, but never in the scope of a
>> confocal core facility.  What special considerations should be involved,
>> especially concerning sealing slides and avoiding cross contamination of
>> the microscope.  I definitely would like to avoid needing to sterilize any
>> contaminated objectives at all costs.
>>
>> Thanks,
>>     Ben Smith
>>
>> Benjamin E. Smith, Ph.D.
>> Samuel Roberts Noble Microscopy Laboratory
>> Research Scientist, Confocal Facility Manager
>> University of Oklahoma
>> Norman, OK 73019
>> E-mail: [hidden email]
>> Voice   405-325-4391
>> FAX  [phone]
>> http://www.microscopy.ou.edu/
>>
>>  --
> Menelaos Symeonides
> University of Vermont
> Cell & Molecular Biology Graduate Program
> Department of Microbiology and Molecular Genetics
> 318 Stafford Hall
> 95 Carrigan Dr
> Burlington, VT 05405
> [hidden email]
> Phone: [phone]
>