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Contamination doesn't grow


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#1 Kitty

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Posted 14 April 2010 - 01:04 AM

I'm having contamination in my cell culture. I'm not sure what type these are... The cells are dying off. Bacterial contaminations usually grow very rapidly. But this one doesn't seem to be growing or is growing at a very slow rate. It's more or less the same after 3 days. Does anyone know what this is and how to get rid of it ?

BTW I noticed the contamination right after thawing. Could it be introduced during the thawing process or it is from the media or ...?

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Edited by Kitty, 14 April 2010 - 01:17 AM.


#2 Salem

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Posted 14 April 2010 - 05:41 AM

Hi Kitty,
It doesn't look like a contamination, if you have a contamination you'll see the bacteria grow very fast in 24 hrs, anyway usually after thawing some cells die and this is common thing and vary according your preservation protocol, what you see is the particles of dead cell.
To get rid of them just be patient and keep culturing :(

#3 rhombus

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Posted 14 April 2010 - 06:53 AM

I'm having contamination in my cell culture. I'm not sure what type these are... The cells are dying off. Bacterial contaminations usually grow very rapidly. But this one doesn't seem to be growing or is growing at a very slow rate. It's more or less the same after 3 days. Does anyone know what this is and how to get rid of it ?

BTW I noticed the contamination right after thawing. Could it be introduced during the thawing process or it is from the media or ...?



To my eyes it looks like the cells have not survived the freezing process. It is common to get "Ghost cells" like these when the freezing has not worked:-

i) The cells have been in DMSO too long (in liquid phase) in the freezing down process.
ii) You have forgotten to add DMSO in the first place.
iii) You have added too much DMSO.
iv) You over trypsinised the cells when preparing the cells for freezing.

Again it is common to get "Cell Debris" after thawing the cells out. The cell suspension will contain cells that have been disrupted during freezing and you are observing cellular debris RATHER than bacterial contamination.

How do you freeze your cells down???
Have you another batch of cells that were frozen down that you can try?????


Hope this is useful

Kindest regards

Uncle Rhombus

#4 Dukey

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Posted 14 April 2010 - 09:12 AM

I'm having contamination in my cell culture. I'm not sure what type these are... The cells are dying off. Bacterial contaminations usually grow very rapidly. But this one doesn't seem to be growing or is growing at a very slow rate. It's more or less the same after 3 days. Does anyone know what this is and how to get rid of it ?

BTW I noticed the contamination right after thawing. Could it be introduced during the thawing process or it is from the media or ...?



To my eyes it looks like the cells have not survived the freezing process. It is common to get "Ghost cells" like these when the freezing has not worked:-

i) The cells have been in DMSO too long (in liquid phase) in the freezing down process.
ii) You have forgotten to add DMSO in the first place.
iii) You have added too much DMSO.
iv) You over trypsinised the cells when preparing the cells for freezing.

Again it is common to get "Cell Debris" after thawing the cells out. The cell suspension will contain cells that have been disrupted during freezing and you are observing cellular debris RATHER than bacterial contamination.

How do you freeze your cells down???
Have you another batch of cells that were frozen down that you can try?????


Hope this is useful

Kindest regards

Uncle Rhombus


Couldn't agree more with Rhombus on this one, these are dead cells and are very common after thawing. Sometimes it can appear that they "proliferate" but what actually happens is the cells float off and then accumulate and settle down in one place in the flask/plate (usually the middle). In a small well, this can look very alarming indeed and it is easy to mistake it for some kind of contamination. You can actually look for an old post of mine where I posed an almost identical question. My problem was a pH one; my incubator went crazy after a cylinder change and was only inputing 1% CO2, with obvious consequences. Cells are very sensitive to pH especially after thawing so that might be a place to look if you are sure of your everything else.

#5 Kitty

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Posted 14 April 2010 - 07:56 PM

Thank you for all your replies :) Unfortunately the cells are not growing or attaching to the flask.

I'm having contamination in my cell culture. I'm not sure what type these are... The cells are dying off. Bacterial contaminations usually grow very rapidly. But this one doesn't seem to be growing or is growing at a very slow rate. It's more or less the same after 3 days. Does anyone know what this is and how to get rid of it ?

BTW I noticed the contamination right after thawing. Could it be introduced during the thawing process or it is from the media or ...?



To my eyes it looks like the cells have not survived the freezing process. It is common to get "Ghost cells" like these when the freezing has not worked:-

i) The cells have been in DMSO too long (in liquid phase) in the freezing down process.
ii) You have forgotten to add DMSO in the first place.
iii) You have added too much DMSO.
iv) You over trypsinised the cells when preparing the cells for freezing.

Again it is common to get "Cell Debris" after thawing the cells out. The cell suspension will contain cells that have been disrupted during freezing and you are observing cellular debris RATHER than bacterial contamination.

How do you freeze your cells down???
Have you another batch of cells that were frozen down that you can try?????


Hope this is useful

Kindest regards

Uncle Rhombus


The cell were frozed by at another facility. I'm not sure of their freezing procedure. But it was frozen about 2-3 months ago.

It sort of vibrates. Hence I thought it was bacterial contam. Can cell debris move ???

The first vial I thawed, the cells looked very healthy but would not attach or grow even after 1 week. This is the second vial I thawed (the pic in my post). This time the cells seem to be dying off and have not attached either ;) The puzzling thing is neither the cells nor the 'black dots' seem to be growing.

BTW I'm working with RIN 5F cells (rat beta cells).

Edited by Kitty, 14 April 2010 - 07:59 PM.


#6 Dukey

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Posted 15 April 2010 - 11:41 AM

Thank you for all your replies :P Unfortunately the cells are not growing or attaching to the flask.

I'm having contamination in my cell culture. I'm not sure what type these are... The cells are dying off. Bacterial contaminations usually grow very rapidly. But this one doesn't seem to be growing or is growing at a very slow rate. It's more or less the same after 3 days. Does anyone know what this is and how to get rid of it ?

BTW I noticed the contamination right after thawing. Could it be introduced during the thawing process or it is from the media or ...?



To my eyes it looks like the cells have not survived the freezing process. It is common to get "Ghost cells" like these when the freezing has not worked:-

i) The cells have been in DMSO too long (in liquid phase) in the freezing down process.
ii) You have forgotten to add DMSO in the first place.
iii) You have added too much DMSO.
iv) You over trypsinised the cells when preparing the cells for freezing.

Again it is common to get "Cell Debris" after thawing the cells out. The cell suspension will contain cells that have been disrupted during freezing and you are observing cellular debris RATHER than bacterial contamination.

How do you freeze your cells down???
Have you another batch of cells that were frozen down that you can try?????


Hope this is useful

Kindest regards

Uncle Rhombus


The cell were frozed by at another facility. I'm not sure of their freezing procedure. But it was frozen about 2-3 months ago.

It sort of vibrates. Hence I thought it was bacterial contam. Can cell debris move ???

The first vial I thawed, the cells looked very healthy but would not attach or grow even after 1 week. This is the second vial I thawed (the pic in my post). This time the cells seem to be dying off and have not attached either :lol: The puzzling thing is neither the cells nor the 'black dots' seem to be growing.

BTW I'm working with RIN 5F cells (rat beta cells).


That's funny I am working with a pancreas line too. I would seriously check the pH, pancreas cells are very sensitive to oxidative damage and if the pH gets to high (>pH 7.8), they will really struggle, especially when thawed.

#7 bob1

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Posted 15 April 2010 - 04:43 PM

The cell were frozed by at another facility. I'm not sure of their freezing procedure. But it was frozen about 2-3 months ago.

It sort of vibrates. Hence I thought it was bacterial contam. Can cell debris move ???

The first vial I thawed, the cells looked very healthy but would not attach or grow even after 1 week. This is the second vial I thawed (the pic in my post). This time the cells seem to be dying off and have not attached either :o The puzzling thing is neither the cells nor the 'black dots' seem to be growing.

BTW I'm working with RIN 5F cells (rat beta cells).

Cell debris can vibrate by a process called Brownian motion (look it up).

Are you sure these are not suspension cells?

#8 Kitty

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Posted 15 April 2010 - 06:04 PM

That's funny I am working with a pancreas line too. I would seriously check the pH, pancreas cells are very sensitive to oxidative damage and if the pH gets to high (>pH 7.8), they will really struggle, especially when thawed.


Thank you. For the 2nd thawing I used media (RPMI 1640, powder) that was freshly prepared. I'll rechecked on the pH. It seems fine.

Cell debris can vibrate by a process called Brownian motion (look it up).

Are you sure these are not suspension cells?


Oh I see. BTW how do I distingush between bacteria (and other contam.) and cell debris? I was perhaps misguidedly told that anything that moves is a bacteria :o

Yup these are definitely adherent cells... I checked it again at ATCC website. The cells are supposed to attach and grow. By now it should look like the attached pic.

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#9 jongf

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Posted 22 June 2010 - 05:50 AM

Kitty.

i recently began working with RIN-5 cells and experienced the exact same problem.
Did you find out what it was?
i could use the advice.
thanks

jonathan




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