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Is this contamination or otherwise? - (Jun/25/2009 )

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Been seeing some weird things in my mamalian cell cultures recently. These are perfectly round bubbles that seem to attach to cell remnants... I don't know if I am paranoid or whatelse, but if I look into the 'bubble' I swear I see black specks moving around. I have seen these in cultures that had bacterial contamination, and from my point of view it feels like the bacteria is 'incubating' in the bubble before exploding out of it.

I have shown it to my supervisor and a couple of other people and they think that those bubbles are just apoptotic cells but I am not entirely convinced!

-tyrosine-

Might be anything.... show a pic!

-Minna-

Might be anything.... show a pic!




We don't have a camera to take a photo! And I haven't seem to be able to find any on the interenet that looks vaguely familliar, except for this thread:



Reply wasn't helpful though...
Attached File

-tyrosine-

if you dont trust your cells - nuke em - its the safest option

apoptotic cells would always have been present (unless you added something new) - a sharp rise would also mean problems

nuke em

dom

-Dominic-

tyrosine on Jun 25 2009, 11:10 PM said:

Been seeing some weird things in my mamalian cell cultures recently. These are perfectly round bubbles that seem to attach to cell remnants... I don't know if I am paranoid or whatelse, but if I look into the 'bubble' I swear I see black specks moving around. I have seen these in cultures that had bacterial contamination, and from my point of view it feels like the bacteria is 'incubating' in the bubble before exploding out of it.

I have shown it to my supervisor and a couple of other people and they think that those bubbles are just apoptotic cells but I am not entirely convinced!


How has the pH of the culture changed? Are you using phenol-red media? If it is bacteria contamination, the pH will drastically lower (if you are using phenol red media, it will turn yellow) within 48 hours depending on your culture volume.

-XD88-

tyrosine on Jun 26 2009, 08:10 AM said:

Been seeing some weird things in my mamalian cell cultures recently. These are perfectly round bubbles that seem to attach to cell remnants... I don't know if I am paranoid or whatelse, but if I look into the 'bubble' I swear I see black specks moving around. I have seen these in cultures that had bacterial contamination, and from my point of view it feels like the bacteria is 'incubating' in the bubble before exploding out of it.

I have shown it to my supervisor and a couple of other people and they think that those bubbles are just apoptotic cells but I am not entirely convinced!



The link you posted shows nothing?

In that forum post, there is a link, but this one does not work?


Anyway, download and look at file I have uploaded, is it somthing like that? (the little black dots that move inside the cells itself?)

or?


check the movie here:
movie

You might need to download the correct codec for it.
I think you can download one here: codec

Let me know if this is a similar thing.

-pito-

XD88 on Jul 4 2009, 06:13 AM said:

How has the pH of the culture changed? Are you using phenol-red media? If it is bacteria contamination, the pH will drastically lower (if you are using phenol red media, it will turn yellow) within 48 hours depending on your culture volume.


Hi XD88, we do use phenol-red media, and the media seemed to change colour a little (been using 6-well plates, so there was relatively less media in the wells compared to a flask). It's not yellow though, and the media is still clear and not turbid. I also don't see precipitate at the bottom, which I have been told is a hallmark of bacterial contamination.

I have tried to culture these cells using all possible 'combinations' of medium i.e. with AB/with serum, without AB/with serum, without AB/without serum and so far I haven't seen anything suspicious. It has been 2 days so if I were meant to see anything it would have been obvious by now?

-tyrosine-

pito on Jul 4 2009, 06:37 AM said:

The link you posted shows nothing?

In that forum post, there is a link, but this one does not work?


Anyway, download and look at file I have uploaded, is it somthing like that? (the little black dots that move inside the cells itself?)

or?


check the movie here:
movie

You might need to download the correct codec for it.
I think you can download one here: codec

Let me know if this is a similar thing.


Hi pito, thanks for your video. I think the link no longer works but I posted it because it seemed to have an exact description of what I was seeing. My cells did not look like the ones in your video. The 'moving black dots' are in a perfect, circular bubble instead of the long rod-like structures. The bubbles also seem to be stuck on the flask rather than motile. I initially thought that it could be nothing till I saw similar moving black dots outside of those bubbles too... that was what raised my concern.

If it helps I am working with HCT116 cells.

-tyrosine-

Offcourse it didnt look like the cells I posted, those were yeast cells :P

I doubt it is contamination then if you do not seem to be able to incubate those cells.
However: if those are yeast cells, it might take a bit longer then 2 days before you see anything.
Maybe you should try another medium.

and your co-workers that think that those are Vapoptotic cells can be right.

-pito-

tyrosine on Jul 4 2009, 10:59 AM said:

XD88 on Jul 4 2009, 06:13 AM said:

How has the pH of the culture changed? Are you using phenol-red media? If it is bacteria contamination, the pH will drastically lower (if you are using phenol red media, it will turn yellow) within 48 hours depending on your culture volume.


Hi XD88, we do use phenol-red media, and the media seemed to change colour a little (been using 6-well plates, so there was relatively less media in the wells compared to a flask). It's not yellow though, and the media is still clear and not turbid. I also don't see precipitate at the bottom, which I have been told is a hallmark of bacterial contamination.

I have tried to culture these cells using all possible 'combinations' of medium i.e. with AB/with serum, without AB/with serum, without AB/without serum and so far I haven't seen anything suspicious. It has been 2 days so if I were meant to see anything it would have been obvious by now?


So if the media is not bright orange/yellow, that means your culture pH is OK and it's probably not bacterial contamination if it is even contaminated (2 days is definitely sufficient, with well plates and flasks, 24 hours is sufficient because bacteria grow extremely fast). Do you see any big white string-like stuff floating around in the culture? If you do, you may check for yeast contamination.
How is the cell growth -- if suspension cells, take out a little bit from one of your wells and check out the viability -- this should give you a good idea how your culture is performing. If it's adherence cells, has the % confluence increased?
If the cells are viable and growing, then your culture is fine.
Have you tried changing the media, by the way? It might just be something from your plates. If you changed media and the stuff is still there, then you know it's either from the cells or your media.
Hope I'm helping!

-XD88-
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