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Stringy rapidly growing contaminants in primary culture

mice fibroblasts

Best Answer SusieQ, 07 July 2014 - 10:14 PM

Hi Ahyi,

 

I've seen these deposits before, but I've never had the experience that my cells started dying or anything out of the oridinary, so after intricate thinking and discussing it with my coworkers, I dismissed them as dust or debris (perhaps unjustified). I myself have no experience with primary cell culture, however, here's my two cents:

 

- Is your medium discolored and/or cloudy? This may incidate a contimatination of some kind (bacterial, most likely).

- How much serum do you use in your medium, if at all? I've been told and I've also seen for myself that, sometimes, high percentages of serum can cause coagulated protein deposits, looking like tiny strings or ribbons. I've no clue as to whether it's a certain kind of protein that causes this, or whether it's protein at all, but I got rid of this by filtering the (horse)serum before adding it to the medium. It didn't seem to harm my cultures afterwards.

- Does your colleague from whom you received that second batch of cells have this kind of problem as well, or do any of them know what this can be?

 

If you want to be sure whether it's a biological contaminant you can remove the medium that contains this phenomenon and reincubate it in a clean flask without cells. If it's then still propagating, it may be an organism (if you want to be really really sure, you could do a nucleus stain, like DAPI). If it's a protein deposit originating from added serum to the medium, these can already appear after you've warmed up your medium. You can see them if you hold the bottle up against the light, they're kind of floating around.

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#1 Ahyi

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Posted 07 July 2014 - 08:28 PM

I am 3 months into a new job and have been isolating/ immortalizing primary mouse intestinal fibroblasts by serial passage.

Up until P6-7 all was well till I noticed these (see attached images) stringy contaminants.

 

Protocol - ... PBS wash x2 while changing medium (FBS, Pen/Strept, 2-3 days), freeze cells (stock) after every 2 passages. Spli

Cells started dying overnight.

I discarded my precious stock of near-immortalised cell lines plus medium and received new cells from a colleague.

Noticed same objects after 24h.

Cultured my fresh medium, and trypsin stock - there they were!

My big challenge is what to do next.

I have frozen vials that I can re-propagate and continue to immortalise but I`m so afraid the contaminants will reoccur.

 

The images are attached.

Please help as I don`t know what else to do and this has given me insomnia for days now + I need this job. I've read through the posts here and am uploading the images.

 

Thanks in advance!

Ahyiコピー ~ au voleur!.jpg b3.jpg au voleur!2.jpg



#2 SusieQ

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Posted 07 July 2014 - 10:14 PM   Best Answer

Hi Ahyi,

 

I've seen these deposits before, but I've never had the experience that my cells started dying or anything out of the oridinary, so after intricate thinking and discussing it with my coworkers, I dismissed them as dust or debris (perhaps unjustified). I myself have no experience with primary cell culture, however, here's my two cents:

 

- Is your medium discolored and/or cloudy? This may incidate a contimatination of some kind (bacterial, most likely).

- How much serum do you use in your medium, if at all? I've been told and I've also seen for myself that, sometimes, high percentages of serum can cause coagulated protein deposits, looking like tiny strings or ribbons. I've no clue as to whether it's a certain kind of protein that causes this, or whether it's protein at all, but I got rid of this by filtering the (horse)serum before adding it to the medium. It didn't seem to harm my cultures afterwards.

- Does your colleague from whom you received that second batch of cells have this kind of problem as well, or do any of them know what this can be?

 

If you want to be sure whether it's a biological contaminant you can remove the medium that contains this phenomenon and reincubate it in a clean flask without cells. If it's then still propagating, it may be an organism (if you want to be really really sure, you could do a nucleus stain, like DAPI). If it's a protein deposit originating from added serum to the medium, these can already appear after you've warmed up your medium. You can see them if you hold the bottle up against the light, they're kind of floating around.



#3 bob1

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Posted 08 July 2014 - 12:11 AM

I agree with SusieQ, probably not a living contaminant

 

They look like plastic fibers from the tissue culture vessels.  If they are fungi (they certainly aren't yeast of bactria), then they should actively grow to form fluffy colonies over a number of days.

 

Culture the medium alone and in the presence of some cells you can afford to waste (if it is fungus).



#4 Tabaluga

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Posted 08 July 2014 - 02:11 AM

Agree with the above posts. Looka like something I had too, which was simply fuzz. But in my case it didn't affect the cells, so are you sure there is no other reason for the cells' death ?

Il dort. Quoique le sort fût pour lui bien étrange,
Il vivait. Il mourut quand il n'eut plus son ange;
La chose simplement d'elle-même arriva,
Comme la nuit se fait lorsque le jour s'en va.

 


#5 mlomonaco

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Posted 08 July 2014 - 09:05 AM

I too believe these are serum precipitates.  In my experience they arise from improper thawing of frozen serum (ie., too fast and too hot).  thaw over 1-2 days in 4C.  Also,  homemade heat in activation is rarely performed correctly and causes precipitates and loss of essential factors.  Primary cells may be sensitive to this.  Good luck!



#6 mlomonaco

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Posted 08 July 2014 - 09:09 AM

I always culture my primary cells in commercially prepared and pre-qualified media.  The extra expense is worth it.



#7 Ahyi

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Posted 08 July 2014 - 08:25 PM

Hi Ahyi,

 

I've seen these deposits before, but I've never had the experience that my cells started dying or anything out of the oridinary, so after intricate thinking and discussing it with my coworkers, I dismissed them as dust or debris (perhaps unjustified). I myself have no experience with primary cell culture, however, here's my two cents:

 

- Is your medium discolored and/or cloudy? This may incidate a contimatination of some kind (bacterial, most likely).

- How much serum do you use in your medium, if at all? I've been told and I've also seen for myself that, sometimes, high percentages of serum can cause coagulated protein deposits, looking like tiny strings or ribbons. I've no clue as to whether it's a certain kind of protein that causes this, or whether it's protein at all, but I got rid of this by filtering the (horse)serum before adding it to the medium. It didn't seem to harm my cultures afterwards.

- Does your colleague from whom you received that second batch of cells have this kind of problem as well, or do any of them know what this can be?

 

If you want to be sure whether it's a biological contaminant you can remove the medium that contains this phenomenon and reincubate it in a clean flask without cells. If it's then still propagating, it may be an organism (if you want to be really really sure, you could do a nucleus stain, like DAPI). If it's a protein deposit originating from added serum to the medium, these can already appear after you've warmed up your medium. You can see them if you hold the bottle up against the light, they're kind of floating around.

Thanks SusieQ for the prompt response.

I'm relieved to hear someone's seen this before :) 

 

- My medium is not cloudy / discoloured

- I use 10%FBS

- My colleagues cells didn't show the same phenomenon

- One colleague thinks it's debris / fibre , another (the colleague I'm working with), says contaminant so I discarded all my cells. (Kept the medium though)

-  I did reincubate the medium and trypsin - both showed same phenomenon.

 

So I checked the medium against the light & saw these 'deposits' (crystals?) swirling around. I also remember not thawing the FBS for as long as I normally would. I will note that as mlomonaco suggested below.

I do think these are some deposits and not live organisms. Only, in my panic not to 'infect' my colleagues' cells I hastily discarded my near-immortalised cells.

Glad for the help SusieQ.

 

Ahyi



#8 Ahyi

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Posted 08 July 2014 - 08:29 PM

I agree with SusieQ, probably not a living contaminant

 

They look like plastic fibers from the tissue culture vessels.  If they are fungi (they certainly aren't yeast of bactria), then they should actively grow to form fluffy colonies over a number of days.

 

Culture the medium alone and in the presence of some cells you can afford to waste (if it is fungus).

Hi bob1,

I agree. They appeared in culture of medium alone and in another batch of cells.

 

Appreciate the help on here! Thanks.



#9 Ahyi

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Posted 08 July 2014 - 08:30 PM

I too believe these are serum precipitates.  In my experience they arise from improper thawing of frozen serum (ie., too fast and too hot).  thaw over 1-2 days in 4C.  Also,  homemade heat in activation is rarely performed correctly and causes precipitates and loss of essential factors.  Primary cells may be sensitive to this.  Good luck!

I agree.

Thanks. I will note the thawing process next time.

Grateful for the support!



#10 Ahyi

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Posted 08 July 2014 - 08:31 PM

Agree with the above posts. Looka like something I had too, which was simply fuzz. But in my case it didn't affect the cells, so are you sure there is no other reason for the cells' death ?

Same here, just not sure about the cell death. I will repeat the culture and see.

Thanks!



#11 bob1

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Posted 09 July 2014 - 01:46 PM

 

I agree with SusieQ, probably not a living contaminant

 

They look like plastic fibers from the tissue culture vessels.  If they are fungi (they certainly aren't yeast of bactria), then they should actively grow to form fluffy colonies over a number of days.

 

Culture the medium alone and in the presence of some cells you can afford to waste (if it is fungus).

Hi bob1,

I agree. They appeared in culture of medium alone and in another batch of cells.

 

Appreciate the help on here! Thanks.

 

The important thing here is - do they increase in number or size of the threads over time.  If not, then it isn't contamination.



#12 Ahyi

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Posted 09 July 2014 - 09:15 PM

 

 

I agree with SusieQ, probably not a living contaminant

 

They look like plastic fibers from the tissue culture vessels.  If they are fungi (they certainly aren't yeast of bactria), then they should actively grow to form fluffy colonies over a number of days.

 

Culture the medium alone and in the presence of some cells you can afford to waste (if it is fungus).

Hi bob1,

I agree. They appeared in culture of medium alone and in another batch of cells.

 

Appreciate the help on here! Thanks.

 

The important thing here is - do they increase in number or size of the threads over time.  If not, then it isn't contamination.

 

After overnight culture I can say the length increased but not number  (?could be my subjective view though), I had to stop the culture for colleagues to clean out incubator to avoid cross contamination.



#13 Ahyi

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Posted 13 July 2014 - 11:55 PM

 

 

 

I agree with SusieQ, probably not a living contaminant

 

They look like plastic fibers from the tissue culture vessels.  If they are fungi (they certainly aren't yeast of bactria), then they should actively grow to form fluffy colonies over a number of days.

 

Culture the medium alone and in the presence of some cells you can afford to waste (if it is fungus).

Hi bob1,

I agree. They appeared in culture of medium alone and in another batch of cells.

 

Appreciate the help on here! Thanks.

 

The important thing here is - do they increase in number or size of the threads over time.  If not, then it isn't contamination.

 

After overnight culture I can say the length increased but not number  (?could be my subjective view though), I had to stop the culture for colleagues to clean out incubator to avoid cross contamination.

 

Hi bob1,

 

After 72h culture there was no increase in length or size. So it isn't contamination. My guess is fibres (plastic or the tissue for wipe down maybe)

Thanks!







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