Contamination issue in cell culture - (Nov/16/2004 )
I have the same problem with my primary neuron culture.
What are they? How to deal with them?
Did you solve this problem?
Thanks
Hi,
Same problem. I'm doing some primary culture and also cell lines. My advise, just get rid of them. Don't try to wash because it just impossible to wash the contaminants.
Just thought I'd join in...
Same problem here with EC and SMC, random cultures will get this debris. Mostly with my SMC cultures...sometimes they grow fine with the small black/blueish slightly-moving speckles, sometimes they don't. I wondered myself it is was from the FBS I was using or the gelatin/fibronectin I use to coat my flasks, but it doesn't seem to be.
I would chuck them out. I had the same problem with primary type II cells for a while and washing or antibiotics didn't help. Just started all over again and it cured the problem.
Check your FCS. We have something like that. It forms after a week or so in FCS alone. Not in HS or DMEM or PBS. According to a one-time test they are weakly stained with a Mycobacteria test.
We have those as well. And so do our neighbouring labs. It ivery peculiar since it appears to disappear and reappear. THey do not change pH, but from a certain number they begin to inhibit cell growth (I work with several breast carcinoma cell lines and my lab fellow work with keratinocytes and melanocytes). We have tried filtration of the medium with 0.22micron filters but we are not sure it works, since they seem to reappear. We have tracked them down to the FBS and FCS, and it appears that they simply swarm there unharmed. What is even more surprising is that they survive the mammalian freezing procedures and don't mind being cryopreserved in liquid nitrogen. Super parasite.
The sad thing is that even if I throw away my cells, I believe my frozen cells are infected as well. I just can't do as my fellow researchres and ignore them...
And so do I. I have the same problem in culturing neural stem cell , PC12 and C2C12 when using bovine serum(not FBS/FCS) form invitrogen. Could any one tell me what is happening ? There are many strange particles ( some like sprinke,spot in the bottom, otherwise it is like a linear or Y shape wrinkle penetrated NSC resulting in cell dead). I have tried all ways for washing out the particles(eg. 100 micom filters, PBS wash, low speed centrifugation and others...etc) but none of them seem work. I have no time for spending on this issue and my graduate report is coming on may ....Help !! Help !! Thanks !!
I have been experiencing similar problems since the last couple of weeks. I am working with MCF-7 cell lines that I had got from another lab three months back. Evreything was fine till around two weeks back when I started seeing tiny black specs between cell colonies. No pH changes, no turbidity of media. The specs stated increasing in number- strongly adherent, slow growing. I got help from our Department Microbiologist and plated the contaminated cells and media on agar plates and tryptic Soy broth. Sure enough they showed colonies on blood agar, chocolate agar and Tryptic Soy agar after 3-4 days with very strong repulsive smell from these plates. The broths look clear. The microbiologist believes that they are Acinetobacter species- slow growing,Gram negetive, multi drug resistant & could be found in water, soil and is the only Gram negetive bacteria found as a part of normal skin flora!!
I tried Penicillin Strep treatment but they seem to be resistant. Now they have spread to another cell line that I am using- Embryonic Rat cardiomyocytes that I had recently obtained from ATCC.
I have noticed most of them remain adherent to the flask even after trypsinising the cell cuture.
I have no clue what to do to eliminate them from my lab before I start a new batch.
Please advice.
drmvk2000
I tried Penicillin Strep treatment but they seem to be resistant. Now they have spread to another cell line that I am using- Embryonic Rat cardiomyocytes that I had recently obtained from ATCC.
I have just started working with cell cultures (COS-1) and have noticed the same thing. At first I thought it was just cell debris floating around, but after reading through this thread I'm beginning to think otherwise. I suppose the fact that it may be part of normal skin flora would explain the widespread nature of this problem. Also, my media contains pen/strep. I was going to make up a new batch of it tomorrow because I thought it was bad, but if you experienced pen/strep resistance then maybe I have the same thing. Perhaps I'll try to culture it this week and see if I get similar results.
Does it cloud up your media too?
-Hank
The media looks fine. no ph changes , no turbidity. I incubated the media in a tube for more than a week & still its clear. Probably the bacterial load was very less & also maybe they grow very slow.
We have decided to dump all our stocks & start all over again.
Any suggestions as to how to decontaminate the hood & incubator would be most appreciated
drmvk2000