Here is the protocol in use at The Jackson Laboratory for producing mineral oil for use in in vitro fertilizations and embryo culture:
1. In a sterile bottle add 1000mL of oil and 100mL tissue culture medium.
2. Stir oil/medium for two days at room temperature.
3. Let oil/medium settle for one day.
4. Decant medium the next day.
5. Filter the oil using a 0.8ĂL filter (Fisher #09-740-30), aliquot into 250 ml units.
The oil is stable for two months at room temp.
Use it to layer over cells in medium, to prevent evaporation. It will exchange gases, allowing CO2 to maintain the proper pH (we have verified this using a pH indicator, and moving dishes in and out of CO2 incubators.
James Denegre, Ph.D.
Associate Research Scientist
Manager, Light Microscopy
The Jackson Laboratory
Bar Harbor, ME 04609
207.288.6321
-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On Behalf Of Armstrong, Brian
Sent: Thursday, February 26, 2009 11:46 AM
To: [log in to unmask]
Subject: Re: CO2, humidity, temp sensors and mu\ineral oil
Hi James, can you elaborate on your technique " equilibrate the mineral
oil with the medium"?
Thanks,
Brian D Armstrong PhD
Light Microscopy Core Manager
Beckman Research Institute
City of Hope
Dept of Neuroscience
1450 E Duarte Rd
Duarte, CA 91010
626-256-4673 x62872
http://www.cityofhope.org/research/support/Light-Microscopy-Digital-Imag
ing/Pages/default.aspx
-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]]
On Behalf Of James Denegre
Sent: Thursday, February 26, 2009 8:10 AM
To: [log in to unmask]
Subject: Re: CO2, humidity, temp sensors and mu\ineral oil
In our lab we equilibrate the mineral oil with the medium to prevent
uptake of components, and have successfully imaged under mineral oil for
five continuous days.
James Denegre, Ph.D.
Associate Research Scientist
Manager, Light Microscopy
The Jackson Laboratory
Bar Harbor, ME 04609
207.288.6321
-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]]
On Behalf Of Beat Ludin
Sent: Wednesday, February 25, 2009 5:05 AM
To: [log in to unmask]
Subject: Re: CO2, humidity, temp sensors
Over time, the mineral oil overlay will basically void the medium of
all lipophilic components (vitamin E, for instance). This may or may
not be a problem, but you need to be aware of it and do the proper
controls.
If you want to check for evaporation, just weigh your sample before
and after the imaging.
Beat
At 23:23 12-02-2009, you wrote:
>My Guess is still evaporation of media is killing your
>cells.. Overlay the cell media in each compartment with mineral
>oil. It stops all evaporation but allows CO2 gas exchange to occur
>to maintain pH.. I do this with all my chamber systems and it works
>great. No need to humidify flowing air or gas.
>
>
>Joe Goodhouse
>Confocal Core Lab Manager
>Dept. of Molecular Biology
>Princeton University
>609-258-5432
>
>Visit us at
><http://www.molbio/>http://www.molbio1.princeton.edu/facility/confocal/
>
>
>
>----------
>From: Confocal Microscopy List
>[mailto:[log in to unmask]] On Behalf Of Paul Herzmark
>Sent: Thursday, February 12, 2009 4:36 PM
>To: [log in to unmask]
>Subject: Re: CO2, humidity, temp sensors
>
>
>I knew I would not get away with out details!
>
>I have an Invivo (company) plexiglass incubator box around my
>inverted microscope. 5% CO2 is piped inside to a small space (about
>50 ml) around the multiwell-Labtech (company)-coverslip. The small
>space is created by a glass plate with a frame around it that goes
>over the coverslip.The CO2 passes into the small space via special
>tubing.The tubing goes under incubator-warmed water where it is
>supposed to pick up water vapor without bubbling.
>
>1. The whole incubator, with most of the microscope is warmed to 37
degrees
>2. Only the small space surrounding the cells is humidified and has
CO2.
>
>
>
>
>On Thu, Feb 12, 2009 at 11:30 AM, Goodhouse, Joseph G.
><<mailto:[log in to unmask]>[log in to unmask]> wrote:
>Paul,
> Could you describe in detail your heating system. Do you
> overlay your cells with mineral oil? This prevents evaporation
> which will alter your salt concentration.
>
> I use a Recordable Temp sensor from Fisher Scientific. Cat No
> 15-007-26. Inexpensive and works well.
>
>
>Joe Goodhouse
>Confocal Core Lab Manager
>Dept. of Molecular Biology
>Princeton University
>609-258-5432
>
>Visit us at
><http://www.molbio/>http://www.molbio1.<http://princeton.edu/facility/c
onfocal/>princeton.edu/facility/confocal/
>
>
>
>
>----------
>From: Confocal Microscopy List
>[mailto:[log in to unmask]] On Behalf Of Paul Herzmark
>Sent: Thursday, February 12, 2009 2:24 PM
>To:
<mailto:[log in to unmask]>[log in to unmask]
U
>Subject: CO2, humidity, temp sensors
>
>I have a cell dying problem in my microscope when I take pictures of
>them overnight or longer. I suspect the humidity is failing and I
>want to monitor that, CO2 and temperature.(It is not photodamage.
>Cells die in areas away from the objective.)
>
>Can anyone suggest a supplier of recording sensors that I can get
>next to the well slides on my stage?
>
>Thank you!
>
>Paul Herzmark
>Specialist
><mailto:[log in to unmask]>[log in to unmask]
>
>Department of Molecular and Cell Biology
>479 Life Science Addition
>University of California, Berkeley
>Berkeley, CA 94720-3200
>(510) 643-9603
>(510) 643-9500 fax
>
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