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Contamination in HTC116 culture. - (May/28/2013 )

Hello everyone.

I am experiencing this recurring and rather annoying issue with HCT116. These cotton/wool-like particles floating in the medium regularly show up after 2 days, and whenever I start a new batch of cells. They look like yeasts to me, but I'm pretty new to the field so any help would be much appreciated. So has any of you guys ever seen some of those?

I don't think that the incubator is infected, as I've been always growing HeLa and never had any problem. So technically it should't be my aseptic technique either.

Many thanks

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-Evan V-

Yeasts typically look like very big bacteria, but you can see them budding and the culture vessel will smell yeasty (like bread). I don't think these are yeasts.

They could be filamentous fungi, especially if they look like cotton balls floating on or in the medium. Fungi like this are very hard to get rid of by treating with antifungals - you would need several months worth of treatment - so you will be better off getting up some fresh tubes or getting an uncontaminated stock in.

The source could be anywhere - waterbaths are a common source, but so are people's clothes, anywhere that is damp and warm really. Incubators are a favourite of the fungi as they are typically perfect for growth. Cold rooms and fridges also grow them though, so be aware of where you are storing reagents and how clean those locations are. The water traps at the back of fridges commonly have fungi too. Ethanol will not kill fungal spores or even most growing fungi.

Your only solution really is to make sure that EVERYTHING is cleaned, including changing labcoats, and getting fresh stocks of all reagents.