growing a biofilm in a microscope-mounted incubator - bubble problems

[Vernita Gordon]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hello,

We are trying to grow a bacterial biofilm in a flow cell.  Flow is driven at
3.75 mL/hr by a peristaltic pump, from a reservoir in a one-liter bottle.
 The bottle is warmed on a hot plate to 35 C and the pump is at room
temperature.  The flow cell and a bubble trap are inside an incubator
enclosure that is mounted on the confocal microscope and kept at 30 C.  We
are trying to do long-term imaging, to watch the growth of this biofilm over
several days.

My question for this list is, we are having lots of difficulties with
bubbles forming in the flow cell and destroying the biofilm.  We have tried
all the tricks we can think of to get rid of them.  These include placing
the medium at 37 C immediately after autoclaving and holding it there until
we're ready to use it, reducing the number of connections, and gluing tubing
to the bubble trap and the flow cell to try to make a better seal.  None of
these have gotten rid of the bubbles.

Have any of you worked with a similar system?  Do you have any ideas that we
haven't thought of for preventing these bubbles, or at least preventing them
from entering the flow cell?

We're in the department of Physics at UT Austin, if any of you have
suggestions for people nearby I should talk to as well.

thanks,

Vernita

[O'Toole, P]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hi Vernita

We describe our approach for time-lapse imaging of biofilms on our webpages
and recently published in Journal of Microscopy (2009, Volume 235, Pt 2,
128-137)

http://www.york.ac.uk/biology/technology-facility/imaging-cytometry/ic-guides/imaging-biofilms/

I hope that this helps.

Best

Peter

Dr Peter O'Toole
Head of Imaging and Cytometry
Technology Facility
Department of Biology (Area 15)
University of York
YORK
YO10 5DD

Tel : [phone]
Fax : [phone]
email : [hidden email]
www.york.ac.uk/biology/tf

EMAIL DISCLAIMER http://www.york.ac.uk/docs/disclaimer/email.htm

----- Original Message -----
From: "Vernita Gordon" <[hidden email]>
To: <[hidden email]>
Sent: Tuesday, December 14, 2010 5:39 PM
Subject: growing a biofilm in a microscope-mounted incubator - bubble
problems

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hello,
>
> We are trying to grow a bacterial biofilm in a flow cell.  Flow is driven
> at
> 3.75 mL/hr by a peristaltic pump, from a reservoir in a one-liter bottle.
> The bottle is warmed on a hot plate to 35 C and the pump is at room
> temperature.  The flow cell and a bubble trap are inside an incubator
> enclosure that is mounted on the confocal microscope and kept at 30 C.  We
> are trying to do long-term imaging, to watch the growth of this biofilm
> over
> several days.
>
> My question for this list is, we are having lots of difficulties with
> bubbles forming in the flow cell and destroying the biofilm.  We have
> tried
> all the tricks we can think of to get rid of them.  These include placing
> the medium at 37 C immediately after autoclaving and holding it there
> until
> we're ready to use it, reducing the number of connections, and gluing
> tubing
> to the bubble trap and the flow cell to try to make a better seal.  None
> of
> these have gotten rid of the bubbles.
>
> Have any of you worked with a similar system?  Do you have any ideas that
> we
> haven't thought of for preventing these bubbles, or at least preventing
> them
> from entering the flow cell?
>
> We're in the department of Physics at UT Austin, if any of you have
> suggestions for people nearby I should talk to as well.
>
> thanks,
>
> Vernita
>

[rjpalmer]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

>We are trying to grow a bacterial biofilm in a flow cell.  Flow is driven at
>3.75 mL/hr by a peristaltic pump, from a reservoir in a one-liter bottle.
>  The bottle is warmed on a hot plate to 35 C and the pump is at room
>temperature.  The flow cell and a bubble trap are inside an incubator

Is this a commercial flowcell setup or have you constructed it
yourself?  Glass/plastic/metal?

>enclosure that is mounted on the confocal microscope and kept at 30 C.

How is the chamber heated?

>These include placing
>the medium at 37 C immediately after autoclaving and holding it there until
>we're ready to use it,

May not not good for your bacteria - pH of the medium may be wrong
unless you let it cool down completely prior to warming it up again.
But not likely to be the source of your bubble problem.  At low flow
rates, small periods of time at a temp different (especially colder)
than that in the flow cell can encourage bubble formation.  If the
reservoir and upstream line are at a temp different than the
flowcell, you might have a problem.  This includes tubing hanging in
the air or running along the table between the reservoir and the
heated chamber.

>Have any of you worked with a similar system?  Do you have any ideas that we
>haven't thought of for preventing these bubbles, or at least preventing them
>from entering the flow cell?

Do you ever see bubbles anywhere in the line up to the flowcell?  Do
you know for a fact that bubbles enter the bubble trap?  What about
your pump?  Big-bore peristaltic pumps are not good for this kind of
work, especially long-term, because they have few rollers (means more
peristalsis) and big tubes (means more potential for slippage).  No
commercial interest, but Watson-Marlow make nice pumps for this type
of work.  Also, you can put the pump on the downstream side and
elevate the reservoir - pump is then a meter rather than a pump.

Feel free to call and discuss.  As far as "face time", give Marvin
Whiteley a call (Mol Genetics Micro, 512 471 5493).  Give him my
regards.

--
Robert J. Palmer Jr., Ph.D.
Natl Inst Dental Craniofacial Res - Natl Insts Health
Oral Infection and Immunity Branch
Bldg 30, Room 310
30 Convent Drive
Bethesda MD 20892
ph 301-594-0025
fax [phone]

[Vernita Gordon]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Thanks, Robert.  When is a good time for you for me to call?  (I have indeed
contacted Marvin but he is travelling right now)

On 14 December 2010 12:30, Robert J. Palmer Jr.
<[hidden email]>wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
>  We are trying to grow a bacterial biofilm in a flow cell.  Flow is driven
>> at
>> 3.75 mL/hr by a peristaltic pump, from a reservoir in a one-liter bottle.
>>  The bottle is warmed on a hot plate to 35 C and the pump is at room
>> temperature.  The flow cell and a bubble trap are inside an incubator
>>
>
> Is this a commercial flowcell setup or have you constructed it yourself?
>  Glass/plastic/metal?
>
>
>  enclosure that is mounted on the confocal microscope and kept at 30 C.
>>
>
> How is the chamber heated?
>
>
>  These include placing
>> the medium at 37 C immediately after autoclaving and holding it there
>> until
>> we're ready to use it,
>>
>
> May not not good for your bacteria - pH of the medium may be wrong unless
> you let it cool down completely prior to warming it up again. But not likely
> to be the source of your bubble problem.  At low flow rates, small periods
> of time at a temp different (especially colder) than that in the flow cell
> can encourage bubble formation.  If the reservoir and upstream line are at a
> temp different than the flowcell, you might have a problem.  This includes
> tubing hanging in the air or running along the table between the reservoir
> and the heated chamber.
>
>
>  Have any of you worked with a similar system?  Do you have any ideas that
>> we
>> haven't thought of for preventing these bubbles, or at least preventing
>> them
>> from entering the flow cell?
>>
>
> Do you ever see bubbles anywhere in the line up to the flowcell?  Do you
> know for a fact that bubbles enter the bubble trap?  What about your pump?
>  Big-bore peristaltic pumps are not good for this kind of work, especially
> long-term, because they have few rollers (means more peristalsis) and big
> tubes (means more potential for slippage).  No commercial interest, but
> Watson-Marlow make nice pumps for this type of work.  Also, you can put the
> pump on the downstream side and elevate the reservoir - pump is then a meter
> rather than a pump.
>
> Feel free to call and discuss.  As far as "face time", give Marvin Whiteley
> a call (Mol Genetics Micro, 512 471 5493).  Give him my regards.
>
> --
> Robert J. Palmer Jr., Ph.D.
> Natl Inst Dental Craniofacial Res - Natl Insts Health
> Oral Infection and Immunity Branch
> Bldg 30, Room 310
> 30 Convent Drive
> Bethesda MD 20892
> ph 301-594-0025
> fax [phone]
>

[rjpalmer]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

You can call tomorrow 10-4, Monday 8-11 or Tues 1-3:30.

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>Thanks, Robert.  When is a good time for you for me to call?  (I have indeed
>contacted Marvin but he is travelling right now)
>
>On 14 December 2010 12:30, Robert J. Palmer Jr.
><[hidden email]>wrote:
>
>>  *****
>>  To join, leave or search the confocal microscopy listserv, go to:
>>  http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>  *****
>>
>>   We are trying to grow a bacterial biofilm in a flow cell.  Flow is driven
>>>  at
>>>  3.75 mL/hr by a peristaltic pump, from a reservoir in a one-liter bottle.
>>>   The bottle is warmed on a hot plate to 35 C and the pump is at room
>>>  temperature.  The flow cell and a bubble trap are inside an incubator
>>>
>>
>>  Is this a commercial flowcell setup or have you constructed it yourself?
>>   Glass/plastic/metal?
>>
>>
>>   enclosure that is mounted on the confocal microscope and kept at 30 C.
>>>
>>
>>  How is the chamber heated?
>>
>>
>>   These include placing
>>>  the medium at 37 C immediately after autoclaving and holding it there
>>>  until
>>>  we're ready to use it,
>>>
>>
>>  May not not good for your bacteria - pH of the medium may be wrong unless
>>  you let it cool down completely prior to warming it up again. But not likely
>>  to be the source of your bubble problem.  At low flow rates, small periods
>>  of time at a temp different (especially colder) than that in the flow cell
>>  can encourage bubble formation.  If the reservoir and upstream line are at a
>>  temp different than the flowcell, you might have a problem.  This includes
>>  tubing hanging in the air or running along the table between the reservoir
>>  and the heated chamber.
>>
>>
>>   Have any of you worked with a similar system?  Do you have any ideas that
>>>  we
>>>  haven't thought of for preventing these bubbles, or at least preventing
>>>  them
>>>  from entering the flow cell?
>>>
>>
>>  Do you ever see bubbles anywhere in the line up to the flowcell?  Do you
>>  know for a fact that bubbles enter the bubble trap?  What about your pump?
>>   Big-bore peristaltic pumps are not good for this kind of work, especially
>>  long-term, because they have few rollers (means more peristalsis) and big
>>  tubes (means more potential for slippage).  No commercial interest, but
>>  Watson-Marlow make nice pumps for this type of work.  Also, you can put the
>>  pump on the downstream side and elevate the reservoir - pump is then a meter
>>  rather than a pump.
>>
>>  Feel free to call and discuss.  As far as "face time", give Marvin Whiteley
>>  a call (Mol Genetics Micro, 512 471 5493).  Give him my regards.
>>
>>  --
>>  Robert J. Palmer Jr., Ph.D.
>>  Natl Inst Dental Craniofacial Res - Natl Insts Health
>>  Oral Infection and Immunity Branch
>>  Bldg 30, Room 310
>>  30 Convent Drive
>>  Bethesda MD 20892
>>  ph 301-594-0025
>>  fax [phone]
>>

--
Robert J. Palmer Jr., Ph.D.
Natl Inst Dental Craniofacial Res - Natl Insts Health
Oral Infection and Immunity Branch
Bldg 30, Room 310
30 Convent Drive
Bethesda MD 20892
ph 301-594-0025
fax [phone]