Recurrent yeast contamination - (Oct/15/2014 )

Hi all,

I have previously worked with cell culture (3 years ago - HeLa cells) and had no problems with contamination. However, I have just started a project with various cell lines and have been experiencing bad yeast contamination. The contamination sometimes occurs in only a few cell lines (T47D, SK-MEL-28, A431). I have done various testing with the medium, Trypsin and PBS and discarded any contaminated solutions. Even when spliting my cells into multiple flasks, 1-2 of 4 flasks gets contaminated. I have had my supervisor observe my technique to determine if that was the cause, but he couldn't identify any specific cause for contamination. I have even resorted to using a face mask to prevent breathing into the laminar flow and to keep my hair out of the way. My cell culture work went well for 4 weeks before the contamination occured again. Recently, I have even started using a flame in the hood but still managed to have contamination in my flasks (1 during splitting and 2 during revival of cell freezer stocks, though I am not sure now if my freezer stock is contaminated). I tested all my solutions before any culture work and found no contamination. Please help if you have any advice on how to proceed with my cell culture work. I can't see how I am causing the contamination of the cells. I am so frustrated that I cant proceed with my work!

Thanks in advance

-kiavn-

Hi kiavn,

Assuming you haven't already done this: it's worthwhile to clean the entire flow cabinet, the incubator you keep your cells in, the waterbath you use to warm your medium, pipettes and anything else you're using during culturing thoroughly. The source may not be you or your disposables, but maybe it's coming from the equipment: water in the incubator or the water in the waterbath (which can get pretty nasty over time if not cleaned on a regular basis).

Besides this, is there anyone else in your group or on your workfloor who works in the same flow cabinet with anything yeast related?

Hi SusieQ,

There are number of people who use the flow cabinet, waterbath and some of them use the incubator as well. So far, none of them have this problem, only me. So I don't belive it is from that.

From the erratic nature of it, it sounds like you have a low level contamination, which might (might) be in the stocks. If other people are using those same stocks and not having this problem, then it won't be in the stocks (unless they are using an antifungal, which they shouldn't for routine culture!).  If it is in your stocks, I hope that you have some older, non-contaminated stocks that you can thaw out...but before you do this make sure that you clean everything thoroughly. By which I mean literally everything that you use for TC, including taking your micropipettes apart and cleaning those and changing filters in your pipette gun.  Also, change your lab coat/gown - these can hold contaminating fungi quite easily.

Don't just discard any contaminated solutions, throw out everything that you have that is open and get new ones. Low level contaminations are hard to pick up with conventional testing methods, so don't be so sure that you have got rid of the contaminated ones. The only exception to this would be stocks of chemicals/drugs that are precious (if you have contaminated them, then you are out of luck, you'll have to get more). If possible filter sterilize those that can be filter sterilized.  If you make your own medium, get someone else to prepare it for a while, and see if that helps.

If you brew beer or make bread, you might need to stop for a while, this is one of the more common routes for yeast contamination.

Hi bob1,

Thanks for the advice. I am planing to use new stocks of cells as my supervisor also suspects that I may have had low level contamination in my cells. Hopefully I wil sort out this problem soon.  

When u clean every thing, don`t forget to clean ur lab-coat.

Sometimes we just forget to clean it, since we wear only in lab, so u think it will not be contaminated.

May be ur sleeve is the reason for contamination

Best of luck

Hi kiavn,

Any luck with the contamination problem? I've been thinking it over and I agree with bob1, if the contamination is that persistent then maybe you should restart all the cells that are having these issues. Hopefully they aren't extremely valuable cell lines and this will solve your problem. Try ordering new cells, restart them in freshly prepared medium with new antibiotics and serum. Maybe it's also good to introduce a lab-clean with your coworkers (if they have time). Thoroughly clean everything (again) and see if this helps.

Hi SusieQ,

Well, i have got new batches of the cells and have been working with them for 2 weeks now. I have not had any major contamination issues with them so far, especially after changing the filter of the pipette gun. But I'm not quite sure if that was the reason.