Persistent contamination in cell culture - (Jan/28/2014 )

Hello I am a graduate student who has been having contamination issues for 3+ months now.  Specifically in human embryonic stem cell culture.  I have worked with these cells 2 years ago as an undergraduate and had no issues until recently.  I have gone ~2 weeks without contamination but it always comes back.  We have cleaned the incubators, hoods (recently re-certified), and I have thrown anything away that has been suspect.  I have had other students watch me to catch any errors but nothing is out of the ordinary.  The equipment I use like pipets, tips, etc. is all used by everyone but I am the only one experiencing contamination.  I tried different lab coats but now I do not wear one at all and spray diligently.  I scrub my arms with soap before putting on gloves and spray my arms with 70% ethanol.  Once in the hood I do not stick my arms all the way in...usually just wrists.

 

I feed these cells everyday with filtered media.  The ingredients for media is shared, like I said earlier, others haven't had any issues.  It takes less than 16 hours for me to get contamination where the media (sometimes) turns yellow and always turns murky.  The media becomes incredibly blurry and as the plate is being swirled you can see the murkyness mix.  The pictures I have attached are H9 aMHC GFP cells on a mouse embryonic fibroblast feeder layer and the very next day where the stem cells are dead and a lot of cell debris.  When contamination hits I usually thaw from a different vial/person/passage.  I can usually go 1-2 passes before I get contamination again.

 

Any help ID-ing this would be greatly appreciated.  Hopefully then I can figure out the source of the contamination.

 

If I can find a picture of the petri dish with the murky contamination I will upload.

 

Thank you!

Have you identified whether this is a yeast or bacterial contamination?  It's a little hard to say, but I would guess that it is bacterial, which is probably good news in that it is easier to get rid of bacteria than yeast.

 

Have you tried having someone getting a different vial of the same stock up in parallel with you to make sure that it isn't just contaminated stocks?

 

Usually I would recommend getting rid of everything you are currently using - literally everything, no matter the cost.  cleaning hoods and incubators, changing lab coats, tips, use micropipette filter tips straight out of the box, getting fresh media and supplements, fresh cell stocks (untouched prior to your arrival), change the filter in your auto-pipette and rinse the silicone stopper out with 70% ethanol and virkon/trigene, clean your micropipettes too (remove the tip ejector when using for cell culture).  Note that these should all be done at the same time, so as to eliminate cases where you have more than one thing contaminated.

 

You don't bake bread or brew beer by any chance?  If so contamination is reasonably common with yeasts from these processes, and you will either need to improve your sterile technique or give up the baking/brewing.

 

You said that you have had students check your technique - get someone like a senior tech to do so too - often students have not such great technique themselves and might not recognize something that could be done better.

 

I would also use a fresh closed-fronted lab coat (use it only for cell culture - keep a different one for general lab use, and keep them separate), with gloves pulled over the sleeves.  Bare arms are all well and good, but very very hard to sterilize, especially if you have any hair on your arms.

bob1 on Tue Jan 28 07:44:18 2014 said:

Have you identified whether this is a yeast or bacterial contamination?  It's a little hard to say, but I would guess that it is bacterial, which is probably good news in that it is easier to get rid of bacteria than yeast.

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You don't bake bread or brew beer by any chance?  If so contamination is reasonably common with yeasts from these processes, and you will either need to improve your sterile technique or give up the baking/brewing.

 

I’d like the irony if it was Lactococcus lactis.

We believe it is bacterial but haven't figured out what it is.  And no, I have not tried that but I have been given cells by someone and my plates would be contaminated while their cells are fine.  I have gotten rid of everything and have my own separate stock of DMEM, pennstrep, etc, just in case and also for the sake of other lab mates.  Do not bake bread or brew.  I guess I should have been more specific, post docs and senior phd students have watched me work.  I am in a lab at one of the UCs and we recently received free PPE so I got a total of 4 lab coats, 2 are rated for BSL2 and the other 2 are fire-resistant, however I have gotten contamination with all 4.  So, I started to use temporary lab coats that are thrown away after being opened in the packet.  

 

What is strange is the timing, I have worked with these cells 2 years ago but for some reason I keep getting hit every 2-3 times a month for these past 3 months.  I have been taking note when I get contamination but see no pattern.  Also, no one else is getting contamination even though we work in the same hoods, use the same tips, etc.  I have also been wearing a face mask in case my breathing is causing it.

 

Thanks for your ideas.  We will go back to the drawing board and see if there's another solution.

I guess that the incubator doesn't need to be decontaminated if everyone else is having no problem? You spray 70% ethanol on your gloves and rub it all around before starting? Ideally you'd clean everything in the hood this way before starting. I'm sure I'm the largest consumer of ethanol in our lab for this reason. There are a number of how-to videos on YouTube re: cell culture. Maybe one will feature something relevant that you're skipping? Lab coats should be laundered regularly and nothing should obscure or disrupt the air-flow of the LAF. Are you using antibiotics in your medium? I've hardly needed to culture without so I have no idea to what extent they're responsible for my maintenance of sterile cultures.

Yes, I am very diligent with the 70% ethanol and we regularly need to refill the bottles.  I make sure that the ethanol is in between my fingers as well.  Lab coats are laundered every month as we have a service come to the campus.  However, since I've been hit with contamination so often I don't wear them now until I can wash them again..which is next week.  Yes, we use penicillin streptomycin in our medium.  It is very frustrating as I haven't changed my techniques and I can't figure out where it is coming from.  I will continue to research on aseptic techniques to see if there is anything I need to change.

Do you wear (latex) gloves during culturing? And if not, you happen to have rings on your fingers or wear a wristwatch or anything of the kind?

SusieQ on Thu Jan 30 14:44:07 2014 said:

Do you wear (latex) gloves during culturing? And if not, you happen to have rings on your fingers or wear a wristwatch or anything of the kind?

 

What's wrong with nitrile?!

 

This is probably/hopefully not the case but I did read of a girl whose cultures were deliberately interfered with by a colleague who wanted to hinder her progress to make himself not look as bad in comparison (they filmed it with a secretly-placed camera and I think the story was in "Lab Times" (I'm not sure how well that magazine is distributed); not at my institution). I've no idea what your facilities are like but if it was me in this situation and I had no other explanation I'd thaw fresh cells using fresh medium in a completely different flow cabinet, using a different incubator in a different culture lab outside of normal working hours and without telling anyone. If this doesn't help and you're already wearing gloves, I don't know what else to say :(

I'd say - first identify the contamination: if it isn't contamination, but something that you are doing (e.g. maybe inactivating the feeder layer too much?) that could give you this result, then all the sterile technique in the world isn't going to work.

 

Take some of your contaminated medium and inoculate some fresh in a tube (get someone else in the lab to do this if you aren't sure of your medium - does that stay sterile?)  Incubate some of your medium alone too.  Streak some of the contamination out on some plates; I would start with YPD for yeast and TSA for bacteria.  Try and determine if something grows, and if it does, do a Gram stain and see what it looks like.  Often the type of bacteria will tell you the source.

Hi kiwicheese,

 

I have been having the same problem as well. I was wondering if you have figured out what the problem was? I have recently stopped having the contamination after using disposable gowns and changing the filter on the pipetter gun, in addition to using new medium as well. But I am worried I might be overlooking some factor causing the contamination.