Hi experts,
I need your help in identifying this kind of contamination. It has been a pain in my experiment to see this unforeseen event. This appeared first during my transfection experiments. Now it is affecting my cell cultures. Please see the video below.
We tried to eliminate the possibilities where this contamination came from? From media, FBS, plasmid, transfection reagent, materials or handling issue.
These are H4 astrocytes, but it seems that HEK cells can also get this contamination. Could you help me identify this contamination? And how can I solve this problem? If you have any questions, please let me know. Thanks in advance.
When in doubt...bleach.
In all seriousness, those buggers look both motile and big...so I'm thinking protist or similar. You can try amphotericin, pretty sure it's toxic to protists. if you use too much it'll do a number on your mammalian cells though. (hence, bleach, its cheaper)
Might be a stupid suggestion, but did you properly clean your laminar flowhood after your contamination? Did you clean your incubators? Most important thing in my opinion is finding the source of the contamination first.... Might your frozen aliquots be contaminated?
I completely agree with dear Thomas.
First things is first, as you said you must check source of contamination, it is best and cheaper way, and Thomas suggestions is very good for starting, many scientist forgot these simple details.
Thanks for your answers. However, I've been meticulous on aseptic techniques; I have been doing cell cultures for a significant amount of time. I just want to know what kind of contamination this is, based on other people's experience, so that I know how to avoid them and how to be more careful. By identifying them (hopefully, some one experienced the same), I can quickly determine the possible source of contamination and avoid them. Granted I become EXTREMELY cautious for cell culture work. Still, this happens (hopefully not), I can single out that maybe my cryopreserved stocks might be the contaminated (which I did not prepare). Can I treat them with antibiotics/bleach/chemical to disinfect my cryopreserved cell stocks?
You think I don't use Pen/Strep? No offense, but can I have some other suggestions please?
Firstly, do these appear in freshly resuscitated cells from LiN2? If they do, then there's a possibility your LiN2 system is compromised - do you store in liquid or gas phase - if former, then you have increased chance of contaminants spreading between stocks. If this is the case - it's sometimes easier to start from scratch and re-order new cells or ask for gifts from other groups.
I'm guessing you've changed all your media to fresh unopened ones - and this includes all additives you add in (like fresh pen/strep - not a new aliquot of current stock)? You can think about filtering your media through 0.2um filters as you prepare them - for FBS you prob want a a filter for sera to prevent clogs or non-specific binding of stuff to filter membrane).
Next is to look at your technique - how do you culture and how do you discard waste media? We use a vacusafe system from Integra Biosciences which keeps the waste pot away from the hood - we had contamination events previously when keeping waste pots in the hood and people pouring or pipetting their waste media into it - causing aerosolation of the contaminants. Also bleach the waste pot often with hypochlorite - but make sure if you're using it in the hood afterwards to give it a good washout as the remnant chlorine gas can affect cell cultures if not rinsed out. What is your disinfection procedure pre and post work? Just sticking on a UV light will not cut it - but I'm guessing you use a disinfectant spray? If in doubt bleach your hood/worktops!
Some bacterial contaminants are resistant to pen/strep so you could try gentamycin, kanamycin or neomycin in your media (and hope your cell line survives). Think about anti-mycotics like Fungizone too.
Hope it helps!
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