What is the source of contamination in my cell culture? - (Nov/28/2017 )

Hello,

My cell culture got contamination a while ago. Two three cell lines which different media were using to feed them were contaminated. All of the cell cultures were stopped. I cleaned the incubator, our safety hood flow was adjusted by company technician, all of pipettes were cleaned. Everything in our lab was cleaned with 70% isopropranol. I did not have any issue for over a month and now, it is back again. Even, when I feed the media, I see this white particles showing up after a few hours. Attached pictures show my cell culture condition 24 hours after feeding with fresh media.

I would appreciate any help or comments. I have not tested for mold, yeast, or bacteria. but I suspect that it is mold.

Thanks

-Hamed.R-

Hello:

I've felt your frustration at getting seemingly random repeated contamination in different cell culture lines at different times. There's a common element that is causing the contamination, but it may not be obvious.

You didn't mention it in your post, but aliquoting every component that goes into a bottle of media may help eliminate the problem. When you open a new bottle of FBS, BSA, antibiotics, amino acids etc. that you would put into your complete media, aliquot and store (4 degrees C or freeze) each component, then just use one aliquot per bottle of growth media. When you make up a bottle of complete media, aliquot that as well (100 ml in sterile bottles). This way, you reduce the chance of contaminating a whole bottle of media, FBS is not cheap these days.

Keep a dedicated lab coat only for cell culture work and change your lab coat often (have it cleaned every few weeks). The spores are likely being brought into the hood by you, either on your coat or any equipment you bring into the hood. Spray down everything with 70% ethanol before putting it into the hood. Don't talk to people when you do work, you may be misting microdroplets from your mouth into the hood. Close the door if your hood is in a separate room, air currents from people moving around the room can bring contaminants into the hood.

As bad as it sounds, being paranoid when doing cell culture can be a good thing. Examine every object you use or wear and think "How can this mess up my cell culture?" Think of what it may carry and how to clean it before using the object for cell culture.

Good luck!

-Leishman001-

The above advice is excellent.

However, I was wondering - can you provide a better resolution image of the "dots". From what I can see, what you have here are dead cells, probably as a result of overgrowth of the cells in the dish, or death while seeding. For a mold/fungus to show up within a few hours it would have to be growing at a tremendous rate with a high inoculum, which, apart from in yeasts, is very uncommon. Yeasts are very easy to identify under the microscope - they bud, so you will get a parent with a smaller version of itself sticking off the side.

If you or someone in your lab happens to homebrew beer or bake bread, these are great sources of yeasts, so the baker/brewer should be very careful in their cell culture work, and it would be a good idea for them to not be involved in preparing/aliquoting lab stocks of media and associated reagents.

Hair also harbours a wide range of yeasts - this is one of the many reasons long hair should be tied back/up when working in the lab.

-bob1-