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Persistent fungal contamination - (Aug/19/2009 )

Hello everyone,

I have recently started transfection in 293Ts to make virus that will eventually make a stable cell line. However, our tissue culture room has had a persistent fungal contamination problem for about a year now, and I am at my wits end. My cells keep dying and I was wondering if anyone could give me pointers as to what might be going on and what to do about it. Here are the facts:

-In the cell culture dish, I can see hyphae-like stringy things floating in the medium

-When I see the contamination, I make new media and add fugizone. The fungus goes away for a little bit (about a week or so), but once the fungizone has degraded, it comes back.

-We have tried sterilizing the incubator completely (it has a built-in sterilization protocol), but the fungus returns after a few days.


We have only recently started to change out the incubator pan on a regular basis (the reason we didn't before is a long story...basically there were months during which we never used out tissue culture room, and as such, we are particularly bad about taking care of it, but I've convinced the PI that this needs to be a priority, so we've started to pay more attention). The most frustrating part of this problem is that we have tried shutting down the room and sterilizing the incubator, etc, only to see the fungus return!

I know its not an issue of my technique, because I have worked in cell culture environments before with only having contamination once or twice. We are super careful about everything (wear gloves, labcoats, use disposable sterile pipets, don't pass over openings of any kind, and make sure not to cross contaminate or make a mess with media).

What might be going on? Where else should I be focusing my efforts to keep this fungus out? Is it possible that there are some fungal spores that the sterilization just will not kill (and if this is the case, am I screwed)? Could there be other sources of contamination that I am not thinking of (hood, counter, etc)? We have a dishwasher spray the counter and floor down with Vikron every week, but maybe this isn't helping. Also, do you guys spray your flasks down with EtOH before putting them into the incubator, hood, etc? I would rather not resort to fungizone to keep the fungus at bay, for obvious reasons (plus, its not working that well and I think the fungizone is killing my 293Ts when I try to transfect them). I would really just like to get rid of it permanently.

Thanks everyone, any help and comments would be great.

-mgmtech-

Hi mgmtech,

Fungus is insidious. Once it gets a foothold, eradication can be difficult. Here are my suggestions:

Prepare a 1% stock solution of copper sulfide, and dilute to a 0.1% working solution. Use this working solution to wipe down all surfaces of the inside of the incubator and to fill the humidity pan. The residue from the copper will continue to deter fungal growth, even after amphotercin degrades. Copper sulfate will, however, cause stainless steel surfaces to rust over time, so once the fungus goes away, stop using it.

Tear down the incubator completely. Remove the pans, the fan cover, the brackets from the walls, etc. Autoclave everything that is autoclavable. Wipe down fan blades with 70% EtOH. Wipe everything down with the copper sulfate solution and reassemble the incubator. Wipe down the inside and outside of the doors, and the gaskets. If you can remove and replace the gaskets without ruining them, clean under the gaskets as well. Change the air filters. You may need to do this monthly until the fungus goes away.

Change the water jacket. I don't know why this works, but it helped us when our fungal problems were out of control. According to our incubator's owner's manual, the water jacket is supposed to be changed twice yearly. Fill with RO water (not DI). Do not add antifungal solutions to the water jacket, as this will probably void your warranty.

Watch for "dust" on any incubator chamber surfaces--this is mold. Watch for excessive condensation. Sometimes mold likes to hide in the crevices of flask lids. Wipe down the flask to remove any drips after changing medium. Try changing your medium twice weekly to keep the ampho fresh until the fungus is controlled.

And yes, you should ALWAYS disinfect the outside of your flasks, medium bottles, etc. before placing them in the hood. Start the hood ~20 minutes before you use it, to allow dust and contaminants to get trapped by the HEPA filter. Disinfect the hood completely prior to use. Make sure the items are still damp with disinfectant before putting them in the hood. Disinfect your hands everytime you go in and out of the hood. Once you're in the hood, stay in the hood...Minimizing the number of times you go in and out will decrease the chance of dragging contaminants into the hood.

I hope this advice helps with your problem.

lab rat

-lab rat-

Thanks for your advice! I am determined to get rid of this.

lab rat on Aug 19 2009, 03:02 PM said:

Hi mgmtech,

Fungus is insidious. Once it gets a foothold, eradication can be difficult. Here are my suggestions:

Prepare a 1% stock solution of copper sulfide, and dilute to a 0.1% working solution. Use this working solution to wipe down all surfaces of the inside of the incubator and to fill the humidity pan. The residue from the copper will continue to deter fungal growth, even after amphotercin degrades. Copper sulfate will, however, cause stainless steel surfaces to rust over time, so once the fungus goes away, stop using it.

Tear down the incubator completely. Remove the pans, the fan cover, the brackets from the walls, etc. Autoclave everything that is autoclavable. Wipe down fan blades with 70% EtOH. Wipe everything down with the copper sulfate solution and reassemble the incubator. Wipe down the inside and outside of the doors, and the gaskets. If you can remove and replace the gaskets without ruining them, clean under the gaskets as well. Change the air filters. You may need to do this monthly until the fungus goes away.

Change the water jacket. I don't know why this works, but it helped us when our fungal problems were out of control. According to our incubator's owner's manual, the water jacket is supposed to be changed twice yearly. Fill with RO water (not DI). Do not add antifungal solutions to the water jacket, as this will probably void your warranty.

Watch for "dust" on any incubator chamber surfaces--this is mold. Watch for excessive condensation. Sometimes mold likes to hide in the crevices of flask lids. Wipe down the flask to remove any drips after changing medium. Try changing your medium twice weekly to keep the ampho fresh until the fungus is controlled.

And yes, you should ALWAYS disinfect the outside of your flasks, medium bottles, etc. before placing them in the hood. Start the hood ~20 minutes before you use it, to allow dust and contaminants to get trapped by the HEPA filter. Disinfect the hood completely prior to use. Make sure the items are still damp with disinfectant before putting them in the hood. Disinfect your hands everytime you go in and out of the hood. Once you're in the hood, stay in the hood...Minimizing the number of times you go in and out will decrease the chance of dragging contaminants into the hood.

I hope this advice helps with your problem.

lab rat

-mgmtech-

I'd not count in copper sulfate to do much against fungi and 70% ethanol is not going to do that much either, esp. vs. fungal spores. Blowing up and reconstructing your equipment and work systems is pretty dramatic and may do more to spread contamination than eliminate, esp. as you don't know if everything in your world is contaminated.
I'm not aware of fungi/fungal spores that survivie autoclave sterilization.

What is the fungal contaminant? Is the contamination due to what appears to be a single isolate?

Suggest you attempt to track down the vehicle and circumstances contamination. Add some settle plates to your work processes (including what you consider to be your adequate sterile techniques), media prep, culture manipulation and incubation etc.

-GeorgeWolff-

I have no idea what kind of fugus it is, but it does look like the same kind every time I see it flare up again. We know exactly where it came from. We culture a lot of exotic primate cell lines that we purchase from the Coriell cell bank. We noticed the fungus in a sealed flask of fibroblasts they had sent us (sealed, with no porous membrane, so obviously it did not come from us). Unfortunately, by the time we noticed this, we had already purchased 2 or 3 other times from them and the same fungus had already been growing in our lab for quite some time, so we went ahead and used the cell line. Plus, its not like primate cell lines are as easy to get by as 293Ts or HeLas...special permits are required, and of course, they are a lot pricier.

Coriell refuses to acknowledge that the contamination came from them, but mysteriously, they have also agreed to send us two of the lines again at no charge...pretty infuriating...

Do you need any kind of special settle plates, or are plain YPD plates good enough for this?

-mgmtech-

Assume it grows on YPD - just use that medium.
Worst case - Neuropsora crassa. It's a fast growing pinkish mycelial fungus that plays hell in the bakery industry and occasionally in labs.
(see: http://www.apsnet.org/online/feature/colle...age/dish2sm.jpg ).

More of an issue in mycology labs are book mites that track the fungus around.

-GeorgeWolff-