Contamination of cultured cells - (Nov/01/2007 )
Thankyou all for your advice, and I have already thrown away my cell culture 
. Now I am trying to find out how did it contaminated. One possible route of contamination is the medium, I am guessing that it is contaminated. However, I have checked the RPMI medium that I used. Just by looking at it, it isnt turbid or anything. It's perfectly clear. But the possible linking of the 2 continuous contamination that I can think of is due to the medium. Do you guys usually get a new bottle of medium when your cells are contaminated?
Yes I do. Only the smalest contamination in the medium is enough to contaminate your next cells. It is better to change the medium too.
What type of contamination is it? Fungal, bacterial, etc?
Usually to prevent having to discard the entire media bottle you can aliqout it in to smaller media bottles. Just be careful not to get it contaminated when you do so.
So today I went back to lab and found out my cell line was contaminated again. And I dont really know how to handle this situation. Coz this is already the second time in a roll ofcontamination, it happened as well last week. What do you guys usually do once your cell line was contaminated?Also, how to minimize the chance of contamination?
Thanks
How did you know that it was contaminated and with what and when? Use good aseptic techniques when handling your cells and labware.
I am new in here. also, I am new in lab life. I have contamination problem in my lab too, our incubator had yeast contamination before and my advisor explained that they did not disinfect after contamination. Now, I have to disinfect our cell culture lab, especially our incubator. But I do not know the disinfection protocols for incubator. how can I get this disinfection protocol ? I need your advise.. please help!!!
zefza
It depends on the incubator. Check your equipment manual for any instructions and if its not there post the make and model. It shouldn't be too difficult but it can be very time consuming and depending on the air/vent system of the incubator it may take several sweeps to get it clean.
thanks a lot. tomorrow I will check our manual and will post the make and model.
if your like us you keep the media in the frige - pop some in a small flask and stick it in the incubator - that will give you a truer sense of contamination
dom
hi,
I check the lab manual but I did not find any solution for my contamination. our incubator make is "SANYO O2/CO2 incubator, model no: MCO-175M " also, our biological safety cabinet make is "NUAIRE model no: NU:440-400E series 9" how can I get disinfection manuals for this type of incubator? thanks!!
if your like us you keep the media in the frige - pop some in a small flask and stick it in the incubator - that will give you a truer sense of contamination
dom
Hey, thanks for your advice but I have already pour away the medium. If I ever contaminate my cells again, I will try that to see if the medium is contaminated.
Any time I get contamination I assume everything is bad. I always figure better safe than sorry. Throw away the pbs you are working with, filter or throw away the media and trypsin..etc. Most incubators can be taken apart to some degree. Remove all the metal pieces (racks, side panels) you can and stick them in the autoclave. Wipe down everything with 70% EtOH and perhaps even change the filter in the incubator (especially for mold or fungus). Use lots of alcohol...my lab makes fun of me for the amount I use but I very rarely get contamination so I don't care what they think. Be very aware of your hands and what they've touched. Don't allow your arm or hand to cross over the top of the cells or an open bottle of media in the hood. I don't even chew gum or talk to people when I'm working in the hood. Some people would certainly say this is a bit extreme but there is nothing worse than a big experiment going down the drain (especially when you used expensive reagents) or being set back a week or so because you need to rethaw. I also try to keep minimally two stock flasks at all times. One thin and one heavy. That way, when I split the heavy flask and contaminate them, I have another stock flask which should be ok and ready to split. This way I almost always have cells ready for experiments as well. Otherwise this is simply a skill which takes time and experience and even the best tissue culture people have a contamination issue from time to time.
A couple of tips noone has mentioned yet:
-ethanol your gloves before you enter the hood and ethanol them every time you re-enter the hood, eg. after you've gone to get you media bottle from the fridge or something like that - hands are a nice easy source of contamination in your cell culture
-work in the back of the hood - the closer you are to the front of the hood, the closer you are to potential sources of contamination, i.e. air
-keep lids on bottles when not in use - some people leave their lids off in the hood assuming that the hood is completely devoid of microorganisms - keeping them on just helps reduce the chance of contamination
Good luck, Rob