CO2 grade for on-scope incubator?

[Peter Werner]

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At our facility, we have a spinning-disk confocal system, with an
incubator that surrounds the entire stage and substage area. The
question I have concerns the type of CO2 I should be using for the
incubator. I was told by someone from the UC Berkeley Biological Imaging
Facility that I should be careful about the grade of carbon dioxide that
I use - apparently, it should be high purity and as low as possible in
H2O. The reason is, even if the cell cultures themselves will be fine
with a lower purity of CO2, CO2 forms carbonic acid in reaction with
water, and the acidified water content in the chamber atmosphere can be
damaging to the instrument over the long term, hence, the less H2O mixed
in with the CO2, the better. If this is the case, that would imply that
"Instrument Grade" or "Bone Dry" grades would be what I need to use.

I was wondering if anybody else knew of this recommendation and followed
this practice. It makes sense to me why one would want to minimize
carbonic acid formation in environments the confocal system is exposed
to. On the other hand, it does entail some expense, not so much in terms
of the gas itself, but in terms of special cylinders for high-purity
CO2, which must be rented, or purchased at $500+ per gas cylinder.

Let me know,
Peter G. Werner
Instructional Assistant/Lab Technician, Microscopy, Merritt College
SEM/AFM Lab Technician, Ohlone College
[hidden email]

[Kurt Thorn]

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Something seems weird here, since a live cell incubator needs to be
humidified to keep the cells from drying out. Typically, only the area
around the sample is kept in a 5% CO2 atmosphere, and this is also
humidified to close to 100%, so any water in the CO2 tank is negligible
compared to the amount of water added by the humidification system.

Even without this, unless you're in the desert, the air is often 50% or
greater relative humidity, so as soon as you mix the CO2 with air,
there's a fair amount of water present....

Kurt

On 7/22/2014 4:42 PM, Peter Werner 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.
> *****
>
> At our facility, we have a spinning-disk confocal system, with an
> incubator that surrounds the entire stage and substage area. The
> question I have concerns the type of CO2 I should be using for the
> incubator. I was told by someone from the UC Berkeley Biological
> Imaging Facility that I should be careful about the grade of carbon
> dioxide that I use - apparently, it should be high purity and as low
> as possible in H2O. The reason is, even if the cell cultures
> themselves will be fine with a lower purity of CO2, CO2 forms carbonic
> acid in reaction with water, and the acidified water content in the
> chamber atmosphere can be damaging to the instrument over the long
> term, hence, the less H2O mixed in with the CO2, the better. If this
> is the case, that would imply that "Instrument Grade" or "Bone Dry"
> grades would be what I need to use.
>
> I was wondering if anybody else knew of this recommendation and
> followed this practice. It makes sense to me why one would want to
> minimize carbonic acid formation in environments the confocal system
> is exposed to. On the other hand, it does entail some expense, not so
> much in terms of the gas itself, but in terms of special cylinders for
> high-purity CO2, which must be rented, or purchased at $500+ per gas
> cylinder.
>
> Let me know,
> Peter G. Werner
> Instructional Assistant/Lab Technician, Microscopy, Merritt College
> SEM/AFM Lab Technician, Ohlone College
> [hidden email]
>
>

--
Kurt Thorn
Director, Nikon Imaging Center
http://nic.ucsf.edu/blog/

[Loralei Dewe-3]

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It seems to me that if this is a problem use the culture medium that has
phenol red so you can see the pH change occuring.   In all my years, I've
never had to order specific CO for microscopy.

Loralei
 On Jul 22, 2014 4:45 PM, "Peter Werner" <[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.
> *****
>
> At our facility, we have a spinning-disk confocal system, with an
> incubator that surrounds the entire stage and substage area. The question I
> have concerns the type of CO2 I should be using for the incubator. I was
> told by someone from the UC Berkeley Biological Imaging Facility that I
> should be careful about the grade of carbon dioxide that I use -
> apparently, it should be high purity and as low as possible in H2O. The
> reason is, even if the cell cultures themselves will be fine with a lower
> purity of CO2, CO2 forms carbonic acid in reaction with water, and the
> acidified water content in the chamber atmosphere can be damaging to the
> instrument over the long term, hence, the less H2O mixed in with the CO2,
> the better. If this is the case, that would imply that "Instrument Grade"
> or "Bone Dry" grades would be what I need to use.
>
> I was wondering if anybody else knew of this recommendation and followed
> this practice. It makes sense to me why one would want to minimize carbonic
> acid formation in environments the confocal system is exposed to. On the
> other hand, it does entail some expense, not so much in terms of the gas
> itself, but in terms of special cylinders for high-purity CO2, which must
> be rented, or purchased at $500+ per gas cylinder.
>
> Let me know,
> Peter G. Werner
> Instructional Assistant/Lab Technician, Microscopy, Merritt College
> SEM/AFM Lab Technician, Ohlone College
> [hidden email]
>

[Steffen Dietzel]

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Am 23.07.2014 02:30, schrieb Loralei Dewe:
> It seems to me that if this is a problem use the culture medium that has
> phenol red so you can see the pH change occuring.

But that is not something to be universally recommended, since phenol
red is fluorescent and can cause quite a background. I'd restrict its
use to testing purposes. Or a small volume right next to your actual sample.

Steffen


--
------------------------------------------------------------
Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Walter-Brendel-Zentrum für experimentelle Medizin (WBex)
Head of light microscopy

Mail room:
Marchioninistr. 15, D-81377 München

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[Craig Brideau]

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I agree that special CO is unnecessary. As Kurt says, the atmospheric water
is probably higher anyway. The one thing we have done in the past is swap
out carbogen (5% CO2 balance O2) for a 5% CO2 balance air mixture, because
we found 95% O2 was harsh on a particular sample.

Craig Brideau


On Tue, Jul 22, 2014 at 6:30 PM, Loralei Dewe <[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.
> *****
>
> It seems to me that if this is a problem use the culture medium that has
> phenol red so you can see the pH change occuring.   In all my years, I've
> never had to order specific CO for microscopy.
>
> Loralei
>  On Jul 22, 2014 4:45 PM, "Peter Werner" <[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.
> > *****
> >
> > At our facility, we have a spinning-disk confocal system, with an
> > incubator that surrounds the entire stage and substage area. The
> question I
> > have concerns the type of CO2 I should be using for the incubator. I was
> > told by someone from the UC Berkeley Biological Imaging Facility that I
> > should be careful about the grade of carbon dioxide that I use -
> > apparently, it should be high purity and as low as possible in H2O. The
> > reason is, even if the cell cultures themselves will be fine with a lower
> > purity of CO2, CO2 forms carbonic acid in reaction with water, and the
> > acidified water content in the chamber atmosphere can be damaging to the
> > instrument over the long term, hence, the less H2O mixed in with the CO2,
> > the better. If this is the case, that would imply that "Instrument Grade"
> > or "Bone Dry" grades would be what I need to use.
> >
> > I was wondering if anybody else knew of this recommendation and followed
> > this practice. It makes sense to me why one would want to minimize
> carbonic
> > acid formation in environments the confocal system is exposed to. On the
> > other hand, it does entail some expense, not so much in terms of the gas
> > itself, but in terms of special cylinders for high-purity CO2, which must
> > be rented, or purchased at $500+ per gas cylinder.
> >
> > Let me know,
> > Peter G. Werner
> > Instructional Assistant/Lab Technician, Microscopy, Merritt College
> > SEM/AFM Lab Technician, Ohlone College
> > [hidden email]
> >
>

[Armstrong, Brian]

Hi Peter, I agree with the consensus here. I think that our CO2 costs around $3 (US$) per small bottle. You should be careful however with the pressure you deliver to the mixing unit as high pressures can damage the sensor unit. This usually happens the upon the initial installation. To avoid this you can open your flow with the regulator attached and adjust the flow rate prior to attaching it to your system. Also important is having a good filter on the line.
Best of luck,

Brian D Armstrong PhD
Associate Research Professor
Director, Light Microscopy Core
Beckman Research Institute
City of Hope
Dept of Neuroscience
1450 E Duarte Rd
Duarte, CA 91010
626-256-4673 x62872



-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Craig Brideau
Sent: Wednesday, July 23, 2014 10:53 AM
To: [hidden email]
Subject: Re: CO2 grade for on-scope incubator?

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I agree that special CO is unnecessary. As Kurt says, the atmospheric water
is probably higher anyway. The one thing we have done in the past is swap
out carbogen (5% CO2 balance O2) for a 5% CO2 balance air mixture, because
we found 95% O2 was harsh on a particular sample.

Craig Brideau


On Tue, Jul 22, 2014 at 6:30 PM, Loralei Dewe <[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.
> *****
>
> It seems to me that if this is a problem use the culture medium that has
> phenol red so you can see the pH change occuring.   In all my years, I've
> never had to order specific CO for microscopy.
>
> Loralei
>  On Jul 22, 2014 4:45 PM, "Peter Werner" <[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.
> > *****
> >
> > At our facility, we have a spinning-disk confocal system, with an
> > incubator that surrounds the entire stage and substage area. The
> question I
> > have concerns the type of CO2 I should be using for the incubator. I was
> > told by someone from the UC Berkeley Biological Imaging Facility that I
> > should be careful about the grade of carbon dioxide that I use -
> > apparently, it should be high purity and as low as possible in H2O. The
> > reason is, even if the cell cultures themselves will be fine with a lower
> > purity of CO2, CO2 forms carbonic acid in reaction with water, and the
> > acidified water content in the chamber atmosphere can be damaging to the
> > instrument over the long term, hence, the less H2O mixed in with the CO2,
> > the better. If this is the case, that would imply that "Instrument Grade"
> > or "Bone Dry" grades would be what I need to use.
> >
> > I was wondering if anybody else knew of this recommendation and followed
> > this practice. It makes sense to me why one would want to minimize
> carbonic
> > acid formation in environments the confocal system is exposed to. On the
> > other hand, it does entail some expense, not so much in terms of the gas
> > itself, but in terms of special cylinders for high-purity CO2, which must
> > be rented, or purchased at $500+ per gas cylinder.
> >
> > Let me know,
> > Peter G. Werner
> > Instructional Assistant/Lab Technician, Microscopy, Merritt College
> > SEM/AFM Lab Technician, Ohlone College
> > [hidden email]
> >
>

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[Teng-Leong Chew-2]

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Hi Peter,

Kurt is right. You need humidity to minimize evaporation from
the culture medium or that will drastically alter the osmolarity
of the medium very quickly. That¹s why there is a tray of water
at the bottom of your regular cell culture incubator. Apparently
the effect from the carbonic acid level is miniscule.

You don¹t need the bone dry CO2.

Regards,
Leong

--
Teng-Leong Chew, PhD
Director, Advanced Imaging Center
Howard Hughes Medical Institute at Janelia
Ashburn, VA 20147







On 7/22/14, 7:42 PM, "Peter Werner" <[hidden email]> wrote:

>*****
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>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>Post images on http://www.imgur.com and include the link in your posting.
>*****
>
>At our facility, we have a spinning-disk confocal system, with an
>incubator that surrounds the entire stage and substage area. The
>question I have concerns the type of CO2 I should be using for the
>incubator. I was told by someone from the UC Berkeley Biological Imaging
>Facility that I should be careful about the grade of carbon dioxide that
>I use - apparently, it should be high purity and as low as possible in
>H2O. The reason is, even if the cell cultures themselves will be fine
>with a lower purity of CO2, CO2 forms carbonic acid in reaction with
>water, and the acidified water content in the chamber atmosphere can be
>damaging to the instrument over the long term, hence, the less H2O mixed
>in with the CO2, the better. If this is the case, that would imply that
>"Instrument Grade" or "Bone Dry" grades would be what I need to use.
>
>I was wondering if anybody else knew of this recommendation and followed
>this practice. It makes sense to me why one would want to minimize
>carbonic acid formation in environments the confocal system is exposed
>to. On the other hand, it does entail some expense, not so much in terms
>of the gas itself, but in terms of special cylinders for high-purity
>CO2, which must be rented, or purchased at $500+ per gas cylinder.
>
>Let me know,
>Peter G. Werner
>Instructional Assistant/Lab Technician, Microscopy, Merritt College
>SEM/AFM Lab Technician, Ohlone College
>[hidden email]

[Cole, Richard W (HEALTH)]

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Not to belabor the point but.

I have been using stage incubators for ~30 years without any "high purity" gases. I use both the type that requires premixed, i.e. 5% and the type that mixes at the scope and have never seen any effect on the scopes


Cheers

Rich


Richard Cole
Research Scientist V
Director: Advanced Light Microscopy & Image Analysis Core
Wadsworth Center
 
Research Assistant Professor
Dept. of Biomedical Sciences
School of Public Health State University of New York

P.O. Box 509 Albany N.Y. 12201-0509
518-474-7048 Phone
518-473-2895 Fax

Website www.wadsworth.org/cores/alm/index.htm

[jerie]

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Dear Peter,

EMBL operates such incubators for 10+ years using technical grade C02
without any problems. We have even saturated the air in the incubator with
water in order to counteract evaporation of culture media for long time
lapse acquisitions. You can use pre-mixed gas, as mentioned, and it is
better to mix CO2 with air or with N2. A low partial pressure of O2 helps
avoiding photobleaching and facilitates photo-switching of some GFPs.

Kind regards, Jens



Visiting Scientist @ Center for Technological Development in Health (CDTS),
Oswaldo Cruz Foundation (Fiocruz), Ministry of Health, Rio de Janeiro,
Brazil.
http://br.linkedin.com/pub/jens-rietdorf/6/4a3/189/
Skype jens.rietdorf


On Tue, Jul 22, 2014 at 8:42 PM, Peter Werner <[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.
> *****
>
> At our facility, we have a spinning-disk confocal system, with an
> incubator that surrounds the entire stage and substage area. The question I
> have concerns the type of CO2 I should be using for the incubator. I was
> told by someone from the UC Berkeley Biological Imaging Facility that I
> should be careful about the grade of carbon dioxide that I use -
> apparently, it should be high purity and as low as possible in H2O. The
> reason is, even if the cell cultures themselves will be fine with a lower
> purity of CO2, CO2 forms carbonic acid in reaction with water, and the
> acidified water content in the chamber atmosphere can be damaging to the
> instrument over the long term, hence, the less H2O mixed in with the CO2,
> the better. If this is the case, that would imply that "Instrument Grade"
> or "Bone Dry" grades would be what I need to use.
>
> I was wondering if anybody else knew of this recommendation and followed
> this practice. It makes sense to me why one would want to minimize carbonic
> acid formation in environments the confocal system is exposed to. On the
> other hand, it does entail some expense, not so much in terms of the gas
> itself, but in terms of special cylinders for high-purity CO2, which must
> be rented, or purchased at $500+ per gas cylinder.
>
> Let me know,
> Peter G. Werner
> Instructional Assistant/Lab Technician, Microscopy, Merritt College
> SEM/AFM Lab Technician, Ohlone College
> [hidden email]
>