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Position: Discussion forums > Bioscience biotechniques discussion forums > Cell biology and histology methods forum
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contamination-HELP
Posted by: icypineg (IP Hidden, New member, 6)
Date: April 23, 2006 07:55PM
Hi All,
I have been thawing some of the previously stored stable cell lines of HepG2 cells. I m having a real weird problem with this, hope somebody can help me with this! I thaw these cells out and seed them to T-25s, Change the medium the very next day. When I change the medium I use MEM medium containing pyruvate, sodium bicarbonate with FBS and Pen-strep. I m not using selection medium though they are stable lines as the previous person worked on that specifically says not to use selection medium till the cells become confluent and ready for subculture. I change the medium every alternate day, but I see cells are contaminated with bacteria after 8-10 days of growth and when the cells are nearing to 80% confluency, till then these cells seem to show no signs of contamination at all! I m not able to reason that out, is it possible that every time I am thawing a fresh vial it can get contaminated only after 8-10 days due to handling problems, or is it that frozen stock itself or that particular batch can be contaminated? till now I have been using the same batch of cells thawed on the same day! Should I try to another batch of cells? Any suggestions regarding this is most welcome, I m confused!! Thanks in advance Geetha
Re: contamination-HELP
Posted by: mitolab (IP Hidden, Senior member, 89)
Date: April 25, 2006 07:22AM
How often you split cells? 8-10days seems pretty long unless you seed cells very sparse in the begining. If it is too confluent it may be cell debris from dead cells which look like contamination. Long time culture in dishes also increases chances of contamination and you may want to use flask with filter cap. Sure you should try another batch if you suspect they are contaminated.
Re: contamination-HELP
Posted by: icypineg (IP Hidden, New member, 6)
Date: April 25, 2006 06:03PM
Hi,
In this particular case I m not at all splitting the cells! These cells are stable cell lines which are made and frozen by the previous worker of the lab. I thaw them out, put them in antibiotic free media for two days. Then I change the media every alternate day replacing them with antibiotic media (G418). Cells stored are really very sparse and there are also lots of floaters seen when I change the media for the first time. So here I m not passaging the cells at all. This contamination appears only after few days of changing media, thats what is bothering me! Its not cell debris coz I can see bacteria happily multiplying after some days. Thanks for the suggestions, I have thawed fresh batch of cells let me see if at all it works! About the flasks, till now I have been using it, but this lab really dont work with filter capped ones, I have submitted a request let me see.
Re: contamination-HELP
Posted by: icypineg (IP Hidden, New member, 6)
Date: April 25, 2006 06:03PM
Hi,
In this particular case I m not at all splitting the cells! These cells are stable cell lines which are made and frozen by the previous worker of the lab. I thaw them out, put them in antibiotic free media for two days. Then I change the media every alternate day replacing them with antibiotic media (G418). Cells stored are really very sparse and there are also lots of floaters seen when I change the media for the first time. So here I m not passaging the cells at all. This contamination appears only after few days of changing media, thats what is bothering me! Its not cell debris coz I can see bacteria happily multiplying after some days. Thanks for the suggestions, I have thawed fresh batch of cells let me see if at all it works! About the flasks, till now I have been using it, but this lab really dont work with filter capped ones, I have submitted a request let me see.
Re: contamination-HELP
Posted by: dkhurtado (IP Hidden, New member, 5)
Date: June 7, 2006 11:33AM
I sounds like that batch of HepG2 are contaminated. Did anyone freeze down and earlier batch, to your knowledge? The fact you get a lot of floaters indicates a big problem with the culture. It
was probably frozen down contaminated. If you used antibiotic free media past the initial 2 days, you would see the contamination a lot sooner than 8-10 days.
Re: contamination-HELP
Posted by: icypineg (IP Hidden, New member, 6)
Date: June 7, 2006 05:12PM
Hi,
Thanks for the suggestions, yes I checked it out that whole batch was contaminated I guess. I was not able to get another batch of frozen cells (Guess they were sceptical about handing over fresh batch!!). Just that I had started fresh in this laboratory I wasnt sure whats happening. Now I have started the experiments with another batch of cells. But thanks a ton for the suggestion.
Re: contamination-HELP
Posted by: Antoine (IP Hidden, New member, 2)
Date: November 20, 2006 08:18AM
I heard from somebody that high confluency can help having contaminations, but I can't find a good reason, if you avoid getting contaminated stuff in your flasks(!), how this can make sense. Is there somebody that can bring an hypothesis?
Antoine
Re: contamination-HELP
Posted by: allureg11 (IP Hidden, New member, 1)
Date: November 20, 2006 07:15PM
Hey, this doesnt make sense at all...no offense to whoever said that to you, but how is it possible? contamination can occur even when the cells arent confluent, as far as my knowledge goes I dont think the confluency and contamination are relative at all!!
Re: contamination-HELP
Posted by: Antoine (IP Hidden, New member, 2)
Date: November 24, 2006 07:45AM
That didn't make any sense to me too, but, as a scientist that is humble enough to doubt (even about something quite evident), I wanted to verify if it's possibly because some people still beleive in spontaneous generation! And the hypothesis start to be confirmed!!!
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