Directional drift of bacteria over time

[Avi Jacob]

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Was wondering if anyone came across this issue.
One of my users images auto-florescent bacteria over time to observe the
motility.

Many of his cells are semi-adherent yet are still mobile and this is what
he tracks.

However, there also are many cells that move across the FoV, in same
direction. It seems as though each experiment will have it's own direction.
We cannot figure out why they do that. They should be moving in a localized
Browning motion  manner, not flowing across the FoV.

We came up with a couple hypotheses. 1. The dish is not exactly straight.
2. Phototaxis. So we made sure was is complete darkness, and as well as
possible that the stage and dish were straight. We still got the motion.
Or we figured that there might be some sort of flow within the dish -  but
why?

Anyone have an explanation?
Thanks.
Avi

--
Avi Jacob, Ph.D.
Head of Light Microscopy
http://tinyurl.com/BIU-Microscopy

[rjpalmer]

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What bacterium are you using?  If you are using motile cells (the only
kind for which you might record clear changes in position on the
substratum), you have no way of predicting the motion unless you have a
chemoattractant and, in that case, you will still see directional changes
(run/tumble).  It should be quite clear whether your cells a swimming or
are floating along.  If they are not swimming, they are inactive and
potentially ³dead² (another topic altogether).  Over what time period are
the experiments taking place?  Have you considered that cells may detach
from the substratum?  In that case, you have now way of predicting the
direction.  If your cells are not swimming, they are likely being carried
by some sort of convection current.

Robert J. Palmer Jr., Ph.D.

Room 207, Building 30
30 Convent Drive
National Institute of Dental and Craniofacial Research
National Institutes of Health
Bethesda MD 20892

301-594-0025





On 7/31/14, 7:59 AM, "Avi Jacob" <[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.
>*****
>
>Was wondering if anyone came across this issue.
>One of my users images auto-florescent bacteria over time to observe the
>motility.
>
>Many of his cells are semi-adherent yet are still mobile and this is what
>he tracks.
>
>However, there also are many cells that move across the FoV, in same
>direction. It seems as though each experiment will have it's own
>direction.
>We cannot figure out why they do that. They should be moving in a
>localized
>Browning motion  manner, not flowing across the FoV.
>
>We came up with a couple hypotheses. 1. The dish is not exactly straight.
>2. Phototaxis. So we made sure was is complete darkness, and as well as
>possible that the stage and dish were straight. We still got the motion.
>Or we figured that there might be some sort of flow within the dish -  but
>why?
>
>Anyone have an explanation?
>Thanks.
>Avi
>
>--
>Avi Jacob, Ph.D.
>Head of Light Microscopy
>http://tinyurl.com/BIU-Microscopy

[jerie]

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Avi,

if you add fluorescent beads, do these float in the same direction? What
kind of sample holder or chamber are you using?

Regards,  Jens

http://br.linkedin.com/pub/jens-rietdorf/6/4a3/189/
Skype jens.Rietdorf
Am 31.07.2014 09:03 schrieb "Avi Jacob" <[hidden email]>:

> *****
> 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.
> *****
>
> Was wondering if anyone came across this issue.
> One of my users images auto-florescent bacteria over time to observe the
> motility.
>
> Many of his cells are semi-adherent yet are still mobile and this is what
> he tracks.
>
> However, there also are many cells that move across the FoV, in same
> direction. It seems as though each experiment will have it's own direction.
> We cannot figure out why they do that. They should be moving in a localized
> Browning motion  manner, not flowing across the FoV.
>
> We came up with a couple hypotheses. 1. The dish is not exactly straight.
> 2. Phototaxis. So we made sure was is complete darkness, and as well as
> possible that the stage and dish were straight. We still got the motion.
> Or we figured that there might be some sort of flow within the dish -  but
> why?
>
> Anyone have an explanation?
> Thanks.
> Avi
>
> --
> Avi Jacob, Ph.D.
> Head of Light Microscopy
> http://tinyurl.com/BIU-Microscopy
>

[Christophe Lamy]

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Temperature gradient?
Christophe

On 31.07.2014 14:25, jens rietdorf 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.
> *****
>
> Avi,
>
> if you add fluorescent beads, do these float in the same direction? What
> kind of sample holder or chamber are you using?
>
> Regards,  Jens
>
> http://br.linkedin.com/pub/jens-rietdorf/6/4a3/189/
> Skype jens.Rietdorf
> Am 31.07.2014 09:03 schrieb "Avi Jacob" <[hidden email]>:
>
>> *****
>> 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.
>> *****
>>
>> Was wondering if anyone came across this issue.
>> One of my users images auto-florescent bacteria over time to observe the
>> motility.
>>
>> Many of his cells are semi-adherent yet are still mobile and this is what
>> he tracks.
>>
>> However, there also are many cells that move across the FoV, in same
>> direction. It seems as though each experiment will have it's own direction.
>> We cannot figure out why they do that. They should be moving in a localized
>> Browning motion  manner, not flowing across the FoV.
>>
>> We came up with a couple hypotheses. 1. The dish is not exactly straight.
>> 2. Phototaxis. So we made sure was is complete darkness, and as well as
>> possible that the stage and dish were straight. We still got the motion.
>> Or we figured that there might be some sort of flow within the dish -  but
>> why?
>>
>> Anyone have an explanation?
>> Thanks.
>> Avi
>>
>> --
>> Avi Jacob, Ph.D.
>> Head of Light Microscopy
>> http://tinyurl.com/BIU-Microscopy
>>

[Sam Swift]

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*****

I'd be inclined to check whether it's something the bacteria are doing or
something the microscope/prep is doing.  If you kill them and repeat the
experiment, they should stop moving if it's down to swimming rather than
floating.  If it is floating, and it's a uniform direction but the
direction can change from experiment to experiment, I'd have thought it
was down to the sample drying out.  Solid material suspended in liquid
will generally dry to leave an annulus, which is due to the leading edge
of the droplet being pinned and the solid material moving to the edge by a
form of capillary action as it dries.  (See "Capillary flow as the cause
of ring stains from dried liquid drops" Robert D. Deegan, Olgica Bakajin,
Todd F. Dupont, Greg Huber, Sidney R. Nagel, and Thomas A. Witten, in
Nature 389, 827 (1997))




On 31/07/2014 12:59, "Avi Jacob" <[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.
>*****
>
>Was wondering if anyone came across this issue.
>One of my users images auto-florescent bacteria over time to observe the
>motility.
>
>Many of his cells are semi-adherent yet are still mobile and this is what
>he tracks.
>
>However, there also are many cells that move across the FoV, in same
>direction. It seems as though each experiment will have it's own
>direction.
>We cannot figure out why they do that. They should be moving in a
>localized
>Browning motion  manner, not flowing across the FoV.
>
>We came up with a couple hypotheses. 1. The dish is not exactly straight.
>2. Phototaxis. So we made sure was is complete darkness, and as well as
>possible that the stage and dish were straight. We still got the motion.
>Or we figured that there might be some sort of flow within the dish -  but
>why?
>
>Anyone have an explanation?
>Thanks.
>Avi
>
>--
>Avi Jacob, Ph.D.
>Head of Light Microscopy
>http://tinyurl.com/BIU-Microscopy

The University of Dundee is a registered Scottish Charity, No: SC015096

[Alfred Bahnson]

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*****

Avi,

Your colleague is viewing circulatory motion within the well from heat and
convection - I'm very confident of this.  In our heated chambers, the flow
is generally across the bottom of the well from an inner side toward an
outer side of the plate, where heat causes the medium along the outer wall
to rise toward the surface.  There may be a slight cooling from evaporation
at the surface, and this cooler layer would then flow toward the cooler
inner wall and then down to the bottom to complete the circuit.  The rate
of circulation is very slow in real time, but it shows up as a current that
sweeps debris and non-adherent cells across the viewfield in time-lapse
image sets.

A steady direction isn't always the case, and we've seen situations where
the direction shifts drastically over time.  Many of us have made the
mistake of interpreting this as biological motion of some sort.  It was a
significant problem when tracking non-adherent hematopoietic cells and
studying motility effects of cytokines and chemokines.  We discovered an
effective "cure" that you might try; see US patent number: US6821747
B2.  Basically,
you add methyl cellulose at a low concentration.  The concentrations
described in the patent are almost hopelessly confused*.  Perhaps the best
place to start is by purchasing a base methylcellulose medium from Stem
Cell Technologies (MethoCult H4100) and then adding this to the bacterial
cultures either by making a uniform solution or by adding a small (1 -
10ul) volume of the base methylcellulose medium to the well without mixing
so that it settles into a thin layer on the bottom surface.  My sense of
how this works is that the slightly increased viscosity drastically lowers
the flow rate within the immediate vicinity of the culture surface, maybe
within 10um of the surface where the cells reside.  The main body of the
circulating medium continues to carry small out-of-focus particles of
debris somewhat above the culture surface.

We did also find that non-adherent hematopoietic cells tend to roll
downhill, and at first we may have been confused by the two counteracting
processes - cells rolling downward versus being swept toward the outside
edge by the circulating medium.  But we consistently found single
hematopoietic stem cells resting against the same corner of the wells after
overnight incubation from the time of seeding.  So under the assumption
that the plate bottoms were slightly tilted, we built a tilt-able platform
for the microscope, hoping that it would help us locate single cells after
seeding.  I can show videos where we corralled stem cells into one corner
of multiple wells in a 96-well plate and then tilted the whole microscope
assembly toward the opposite corner.  The cells in all the wells
simultaneously traveled across to the new lowest corner.

*Most of the confusion arises from not being able to distinguish whether
the % methylcellulose refers to the true methylcellulose concentration or
the concentration of the MethoCult base medium, which starts at 2.6%
methylcellulose.  Even I have trouble with this, and I wrote it!

Al Bahnson


On Thu, Jul 31, 2014 at 7:59 AM, Avi Jacob <[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.
> *****
>
> Was wondering if anyone came across this issue.
> One of my users images auto-florescent bacteria over time to observe the
> motility.
>
> Many of his cells are semi-adherent yet are still mobile and this is what
> he tracks.
>
> However, there also are many cells that move across the FoV, in same
> direction. It seems as though each experiment will have it's own direction.
> We cannot figure out why they do that. They should be moving in a localized
> Browning motion  manner, not flowing across the FoV.
>
> We came up with a couple hypotheses. 1. The dish is not exactly straight.
> 2. Phototaxis. So we made sure was is complete darkness, and as well as
> possible that the stage and dish were straight. We still got the motion.
> Or we figured that there might be some sort of flow within the dish -  but
> why?
>
> Anyone have an explanation?
> Thanks.
> Avi
>
> --
> Avi Jacob, Ph.D.
> Head of Light Microscopy
> http://tinyurl.com/BIU-Microscopy
>

[Avi Jacob]

*****
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*****

Thanks all for your input.

1. The set up is a Zeiss Observer with full enclosure incubator box.
2. Cell are in 6 well glass bottom plate.
3. Time is live capture over 30 min.

He has not tried beads - a great idea. This would be a good idea for this
setup in any case, irrespective of this specific experiment.

If I had to guess, it seems to me now that it is probably indeed some kind
of convection. Even though the medium should be heated homogeneously, as
the entire enclosure is heated, I suppose there can certainly still be some
sort of gradient. This never occurred to me as an issue.

Regarding dead or alive, that is a control I will advise him to do. Though
he thinks that they are not dead. Still.
Regarding methyl cellulose, I wonder if this might affect the motile
adherent/ semi-adherent cells too. Ill need to discuss this idea with him.

His cells lack flagella but have pili. I am not a bacteria person, but I
wonder why many of his cells do adhere and move on the glass and many
don't. Assuming they are indeed all alive.

Thanks,
Avi




On Thu, Jul 31, 2014 at 4:28 PM, Alfred Bahnson <[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.
> *****
>
> Avi,
>
> Your colleague is viewing circulatory motion within the well from heat and
> convection - I'm very confident of this.  In our heated chambers, the flow
> is generally across the bottom of the well from an inner side toward an
> outer side of the plate, where heat causes the medium along the outer wall
> to rise toward the surface.  There may be a slight cooling from evaporation
> at the surface, and this cooler layer would then flow toward the cooler
> inner wall and then down to the bottom to complete the circuit.  The rate
> of circulation is very slow in real time, but it shows up as a current that
> sweeps debris and non-adherent cells across the viewfield in time-lapse
> image sets.
>
> A steady direction isn't always the case, and we've seen situations where
> the direction shifts drastically over time.  Many of us have made the
> mistake of interpreting this as biological motion of some sort.  It was a
> significant problem when tracking non-adherent hematopoietic cells and
> studying motility effects of cytokines and chemokines.  We discovered an
> effective "cure" that you might try; see US patent number: US6821747
> B2.  Basically,
> you add methyl cellulose at a low concentration.  The concentrations
> described in the patent are almost hopelessly confused*.  Perhaps the best
> place to start is by purchasing a base methylcellulose medium from Stem
> Cell Technologies (MethoCult H4100) and then adding this to the bacterial
> cultures either by making a uniform solution or by adding a small (1 -
> 10ul) volume of the base methylcellulose medium to the well without mixing
> so that it settles into a thin layer on the bottom surface.  My sense of
> how this works is that the slightly increased viscosity drastically lowers
> the flow rate within the immediate vicinity of the culture surface, maybe
> within 10um of the surface where the cells reside.  The main body of the
> circulating medium continues to carry small out-of-focus particles of
> debris somewhat above the culture surface.
>
> We did also find that non-adherent hematopoietic cells tend to roll
> downhill, and at first we may have been confused by the two counteracting
> processes - cells rolling downward versus being swept toward the outside
> edge by the circulating medium.  But we consistently found single
> hematopoietic stem cells resting against the same corner of the wells after
> overnight incubation from the time of seeding.  So under the assumption
> that the plate bottoms were slightly tilted, we built a tilt-able platform
> for the microscope, hoping that it would help us locate single cells after
> seeding.  I can show videos where we corralled stem cells into one corner
> of multiple wells in a 96-well plate and then tilted the whole microscope
> assembly toward the opposite corner.  The cells in all the wells
> simultaneously traveled across to the new lowest corner.
>
> *Most of the confusion arises from not being able to distinguish whether
> the % methylcellulose refers to the true methylcellulose concentration or
> the concentration of the MethoCult base medium, which starts at 2.6%
> methylcellulose.  Even I have trouble with this, and I wrote it!
>
> Al Bahnson
>
>
> On Thu, Jul 31, 2014 at 7:59 AM, Avi Jacob <[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.
> > *****
> >
> > Was wondering if anyone came across this issue.
> > One of my users images auto-florescent bacteria over time to observe the
> > motility.
> >
> > Many of his cells are semi-adherent yet are still mobile and this is what
> > he tracks.
> >
> > However, there also are many cells that move across the FoV, in same
> > direction. It seems as though each experiment will have it's own
> direction.
> > We cannot figure out why they do that. They should be moving in a
> localized
> > Browning motion  manner, not flowing across the FoV.
> >
> > We came up with a couple hypotheses. 1. The dish is not exactly straight.
> > 2. Phototaxis. So we made sure was is complete darkness, and as well as
> > possible that the stage and dish were straight. We still got the motion.
> > Or we figured that there might be some sort of flow within the dish -
>  but
> > why?
> >
> > Anyone have an explanation?
> > Thanks.
> > Avi
> >
> > --
> > Avi Jacob, Ph.D.
> > Head of Light Microscopy
> > http://tinyurl.com/BIU-Microscopy
> >
>

[Gareth Howell-2]

Avi,

Is there a motorised stage on your system? If so it might be drifting. Or if your sample isn't secured on the stage it may be drifting.

Best
Gareth
Twitter: @ghowell2812

[Chris Tully-2]

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*****

Regarding thermal convection and uniformity. I highly recommend that you try to borrow a thermal imaging camera and point it at these samples during or immediately after the experiment. I suspect that you will find a fairly non-uniform temperature gradient.

I am basing that guess on my experiences trying to hold a sample at a constant temperature over about 2 hours. My samples were 1mm hydrogel rods inside glass tubes. I started with a small camber roughly equivalent to a 50 mm Petri dish and found that even though I was pumping water through the dish from a large stirred water bath the temperature in my sample chamber varied wildly - I am assuming that this was due to evaporation. The second generation sample holder was a plexi glass box with a lid over the sample and large reservoirs on either end. Again I found higher than acceptable temperature fluctuations. I did not finally get clean data until I built an aluminum block with a chamber in the middle for my sample and buffer, and channels around it for pumping water through. My best guess at why this worked is that with my sample in a sealed chamber with no water flowing through it, I eliminated evaporation. Also by making the aluminum block about 250 x 250 x 35 mm and upgrading my water bath I was able to take advantage of the thermal conductivity of the aluminum and the fact that a large block of aluminum has a significant thermal mass. Also I designed the channels to bring the hot water from the bath closest to the sample chamber and then wrapping out toward the edges. I also had an RTD temperature probe measuring the temperature by my sample.

Now, why is all of this relevant to a discussion of imaging bacteria? Remember that I was looking at 1 mm diameter rods, and let me add that I was looking for 300 microns or less change in diameter if these rods. Every one of the rigs I described above was used on a microscope! My only wish for that system is that I had purchased an inverted microscope because my last sample holder was rather heavy for the stage of my Nikon E800!

If I were to do this again I would use an inverted microscope. In the case of bacteria in a dish I would replace the plexi glass bottom I used with cover glass. I might also design my block to replace the stage!

Chris Tully, M.S., Image Analysis Expert
t 240.475.9753 f 419.831.0527 | [hidden email]

Sent from my iPhone please excuse typos.

> On Aug 1, 2014, at 2:48 AM, Avi Jacob <[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.
> *****
>
> Thanks all for your input.
>
> 1. The set up is a Zeiss Observer with full enclosure incubator box.
> 2. Cell are in 6 well glass bottom plate.
> 3. Time is live capture over 30 min.
>
> He has not tried beads - a great idea. This would be a good idea for this
> setup in any case, irrespective of this specific experiment.
>
> If I had to guess, it seems to me now that it is probably indeed some kind
> of convection. Even though the medium should be heated homogeneously, as
> the entire enclosure is heated, I suppose there can certainly still be some
> sort of gradient. This never occurred to me as an issue.
>
> Regarding dead or alive, that is a control I will advise him to do. Though
> he thinks that they are not dead. Still.
> Regarding methyl cellulose, I wonder if this might affect the motile
> adherent/ semi-adherent cells too. Ill need to discuss this idea with him.
>
> His cells lack flagella but have pili. I am not a bacteria person, but I
> wonder why many of his cells do adhere and move on the glass and many
> don't. Assuming they are indeed all alive.
>
> Thanks,
> Avi
>
>
>
>
>> On Thu, Jul 31, 2014 at 4:28 PM, Alfred Bahnson <[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.
>> *****
>>
>> Avi,
>>
>> Your colleague is viewing circulatory motion within the well from heat and
>> convection - I'm very confident of this.  In our heated chambers, the flow
>> is generally across the bottom of the well from an inner side toward an
>> outer side of the plate, where heat causes the medium along the outer wall
>> to rise toward the surface.  There may be a slight cooling from evaporation
>> at the surface, and this cooler layer would then flow toward the cooler
>> inner wall and then down to the bottom to complete the circuit.  The rate
>> of circulation is very slow in real time, but it shows up as a current that
>> sweeps debris and non-adherent cells across the viewfield in time-lapse
>> image sets.
>>
>> A steady direction isn't always the case, and we've seen situations where
>> the direction shifts drastically over time.  Many of us have made the
>> mistake of interpreting this as biological motion of some sort.  It was a
>> significant problem when tracking non-adherent hematopoietic cells and
>> studying motility effects of cytokines and chemokines.  We discovered an
>> effective "cure" that you might try; see US patent number: US6821747
>> B2.  Basically,
>> you add methyl cellulose at a low concentration.  The concentrations
>> described in the patent are almost hopelessly confused*.  Perhaps the best
>> place to start is by purchasing a base methylcellulose medium from Stem
>> Cell Technologies (MethoCult H4100) and then adding this to the bacterial
>> cultures either by making a uniform solution or by adding a small (1 -
>> 10ul) volume of the base methylcellulose medium to the well without mixing
>> so that it settles into a thin layer on the bottom surface.  My sense of
>> how this works is that the slightly increased viscosity drastically lowers
>> the flow rate within the immediate vicinity of the culture surface, maybe
>> within 10um of the surface where the cells reside.  The main body of the
>> circulating medium continues to carry small out-of-focus particles of
>> debris somewhat above the culture surface.
>>
>> We did also find that non-adherent hematopoietic cells tend to roll
>> downhill, and at first we may have been confused by the two counteracting
>> processes - cells rolling downward versus being swept toward the outside
>> edge by the circulating medium.  But we consistently found single
>> hematopoietic stem cells resting against the same corner of the wells after
>> overnight incubation from the time of seeding.  So under the assumption
>> that the plate bottoms were slightly tilted, we built a tilt-able platform
>> for the microscope, hoping that it would help us locate single cells after
>> seeding.  I can show videos where we corralled stem cells into one corner
>> of multiple wells in a 96-well plate and then tilted the whole microscope
>> assembly toward the opposite corner.  The cells in all the wells
>> simultaneously traveled across to the new lowest corner.
>>
>> *Most of the confusion arises from not being able to distinguish whether
>> the % methylcellulose refers to the true methylcellulose concentration or
>> the concentration of the MethoCult base medium, which starts at 2.6%
>> methylcellulose.  Even I have trouble with this, and I wrote it!
>>
>> Al Bahnson
>>
>>
>>> On Thu, Jul 31, 2014 at 7:59 AM, Avi Jacob <[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.
>>> *****
>>>
>>> Was wondering if anyone came across this issue.
>>> One of my users images auto-florescent bacteria over time to observe the
>>> motility.
>>>
>>> Many of his cells are semi-adherent yet are still mobile and this is what
>>> he tracks.
>>>
>>> However, there also are many cells that move across the FoV, in same
>>> direction. It seems as though each experiment will have it's own
>> direction.
>>> We cannot figure out why they do that. They should be moving in a
>> localized
>>> Browning motion  manner, not flowing across the FoV.
>>>
>>> We came up with a couple hypotheses. 1. The dish is not exactly straight..
>>> 2. Phototaxis. So we made sure was is complete darkness, and as well as
>>> possible that the stage and dish were straight. We still got the motion.
>>> Or we figured that there might be some sort of flow within the dish -
>> but
>>> why?
>>>
>>> Anyone have an explanation?
>>> Thanks.
>>> Avi
>>>
>>> --
>>> Avi Jacob, Ph.D.
>>> Head of Light Microscopy
>>> http://tinyurl.com/BIU-Microscopy
>>

[rjpalmer]

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If your cells lack flagella but have pili, they are capable of crawling
across the surface (referred to as ³twitching motility²).  In 30 min, I
would expect some movement to be detected, but these sorts of twitching
motility expts typically progress over hours.  Maybe they are really fast
crawlersŠ.  These cells are not good swimmers so they may well appear
non-motile in the bulk liquid as opposed to the biofilm, and this makes me
even more convinced that you are seeing convection in the bulk phase.
However, this need not be a big problem if you are concentrating on
biofilm cells.  You should however know roughly how many cells detach
(this could easily be a result of convection), and an assessment of
viability in the detached cells would be prudent.  Otherwise it seems you
are moving in the right direction (pardon the pun).

Robert J. Palmer Jr., Ph.D.

Room 207, Building 30
30 Convent Drive
National Institute of Dental and Craniofacial Research
National Institutes of Health
Bethesda MD 20892

301-594-0025





On 8/1/14, 2:48 AM, "Avi Jacob" <[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.
>*****
>
>Thanks all for your input.
>
>1. The set up is a Zeiss Observer with full enclosure incubator box.
>2. Cell are in 6 well glass bottom plate.
>3. Time is live capture over 30 min.
>
>He has not tried beads - a great idea. This would be a good idea for this
>setup in any case, irrespective of this specific experiment.
>
>If I had to guess, it seems to me now that it is probably indeed some kind
>of convection. Even though the medium should be heated homogeneously, as
>the entire enclosure is heated, I suppose there can certainly still be
>some
>sort of gradient. This never occurred to me as an issue.
>
>Regarding dead or alive, that is a control I will advise him to do. Though
>he thinks that they are not dead. Still.
>Regarding methyl cellulose, I wonder if this might affect the motile
>adherent/ semi-adherent cells too. Ill need to discuss this idea with him.
>
>His cells lack flagella but have pili. I am not a bacteria person, but I
>wonder why many of his cells do adhere and move on the glass and many
>don't. Assuming they are indeed all alive.
>
>Thanks,
>Avi
>
>
>
>
>On Thu, Jul 31, 2014 at 4:28 PM, Alfred Bahnson <[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.
>> *****
>>
>> Avi,
>>
>> Your colleague is viewing circulatory motion within the well from heat
>>and
>> convection - I'm very confident of this.  In our heated chambers, the
>>flow
>> is generally across the bottom of the well from an inner side toward an
>> outer side of the plate, where heat causes the medium along the outer
>>wall
>> to rise toward the surface.  There may be a slight cooling from
>>evaporation
>> at the surface, and this cooler layer would then flow toward the cooler
>> inner wall and then down to the bottom to complete the circuit.  The
>>rate
>> of circulation is very slow in real time, but it shows up as a current
>>that
>> sweeps debris and non-adherent cells across the viewfield in time-lapse
>> image sets.
>>
>> A steady direction isn't always the case, and we've seen situations
>>where
>> the direction shifts drastically over time.  Many of us have made the
>> mistake of interpreting this as biological motion of some sort.  It was
>>a
>> significant problem when tracking non-adherent hematopoietic cells and
>> studying motility effects of cytokines and chemokines.  We discovered an
>> effective "cure" that you might try; see US patent number: US6821747
>> B2.  Basically,
>> you add methyl cellulose at a low concentration.  The concentrations
>> described in the patent are almost hopelessly confused*.  Perhaps the
>>best
>> place to start is by purchasing a base methylcellulose medium from Stem
>> Cell Technologies (MethoCult H4100) and then adding this to the
>>bacterial
>> cultures either by making a uniform solution or by adding a small (1 -
>> 10ul) volume of the base methylcellulose medium to the well without
>>mixing
>> so that it settles into a thin layer on the bottom surface.  My sense of
>> how this works is that the slightly increased viscosity drastically
>>lowers
>> the flow rate within the immediate vicinity of the culture surface,
>>maybe
>> within 10um of the surface where the cells reside.  The main body of the
>> circulating medium continues to carry small out-of-focus particles of
>> debris somewhat above the culture surface.
>>
>> We did also find that non-adherent hematopoietic cells tend to roll
>> downhill, and at first we may have been confused by the two
>>counteracting
>> processes - cells rolling downward versus being swept toward the outside
>> edge by the circulating medium.  But we consistently found single
>> hematopoietic stem cells resting against the same corner of the wells
>>after
>> overnight incubation from the time of seeding.  So under the assumption
>> that the plate bottoms were slightly tilted, we built a tilt-able
>>platform
>> for the microscope, hoping that it would help us locate single cells
>>after
>> seeding.  I can show videos where we corralled stem cells into one
>>corner
>> of multiple wells in a 96-well plate and then tilted the whole
>>microscope
>> assembly toward the opposite corner.  The cells in all the wells
>> simultaneously traveled across to the new lowest corner.
>>
>> *Most of the confusion arises from not being able to distinguish whether
>> the % methylcellulose refers to the true methylcellulose concentration
>>or
>> the concentration of the MethoCult base medium, which starts at 2.6%
>> methylcellulose.  Even I have trouble with this, and I wrote it!
>>
>> Al Bahnson
>>
>>
>> On Thu, Jul 31, 2014 at 7:59 AM, Avi Jacob <[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.
>> > *****
>> >
>> > Was wondering if anyone came across this issue.
>> > One of my users images auto-florescent bacteria over time to observe
>>the
>> > motility.
>> >
>> > Many of his cells are semi-adherent yet are still mobile and this is
>>what
>> > he tracks.
>> >
>> > However, there also are many cells that move across the FoV, in same
>> > direction. It seems as though each experiment will have it's own
>> direction.
>> > We cannot figure out why they do that. They should be moving in a
>> localized
>> > Browning motion  manner, not flowing across the FoV.
>> >
>> > We came up with a couple hypotheses. 1. The dish is not exactly
>>straight.
>> > 2. Phototaxis. So we made sure was is complete darkness, and as well
>>as
>> > possible that the stage and dish were straight. We still got the
>>motion.
>> > Or we figured that there might be some sort of flow within the dish -
>>  but
>> > why?
>> >
>> > Anyone have an explanation?
>> > Thanks.
>> > Avi
>> >
>> > --
>> > Avi Jacob, Ph.D.
>> > Head of Light Microscopy
>> > http://tinyurl.com/BIU-Microscopy
>> >
>>

[Patrick Van Oostveldt]

*****
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*****

I guess on evaporation.

Patrick Van Oostveldt


Sent from my iPad

> On 31-jul.-2014, at 13:59, Avi Jacob <[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.
> *****
>
> Was wondering if anyone came across this issue.
> One of my users images auto-florescent bacteria over time to observe the
> motility.
>
> Many of his cells are semi-adherent yet are still mobile and this is what
> he tracks.
>
> However, there also are many cells that move across the FoV, in same
> direction. It seems as though each experiment will have it's own direction.
> We cannot figure out why they do that. They should be moving in a localized
> Browning motion  manner, not flowing across the FoV.
>
> We came up with a couple hypotheses. 1. The dish is not exactly straight.
> 2. Phototaxis. So we made sure was is complete darkness, and as well as
> possible that the stage and dish were straight. We still got the motion.
> Or we figured that there might be some sort of flow within the dish -  but
> why?
>
> Anyone have an explanation?
> Thanks.
> Avi
>
> --
> Avi Jacob, Ph.D.
> Head of Light Microscopy
> http://tinyurl.com/BIU-Microscopy