I find that fungizone was effective against different types of fungus. I also recently found the problem was with the autoclave interestingly enough. So make sure your filters and bottles are also not the source of contamination. But yes, you can use Fisher Scientific ventilated T75 flasks. Hope that helps!

It's sometimes very difficult to eliminate contamination, especially with fungi. Is the last option, but you would have to discard everything you can discard, that this fungus. And autoclave glassware and other lab materials. Something like a quarantine.

Thanks for your answers. The culture is at air liquid interface and the molds grow on the surface of the epithelium. This is not prevented by fungizone in the lower compartment of the transwell insert... To polarize cells I need to grow them in transwell. Thus I should need a gas permeable box large enough to accomodate a 24 well plate with the filter inserts.

I use the same incubators for bovine embryo development. The whole culture from IVM to blastocyst takes 9 days and I never had contamination.

First I would thoroughly disinfect the incubator. We have used formaldehyde and Ozone successfully. It will lower the spore count significantly and you may be able to get your cells to grow before contamination hits again.

Did you try adding apmhotericin B to the culture media? A concentration of 1ug/mL should be enough.

Hi Lucas, how did you treat with formaldehyde? Does it require technical assistance? I know that it is quite laborious and residual vapors may be toxic also for cultures.

Erkin, I use amphotericin B in the medium but the surface of the epithelium in air liquid interface conditions is not exposed to media.

Dusting the inner part of the incubator with contact fungicide like Mancozeb can be an option........

formaldeheyde is not simple adn it is dangerous. Somebody has to know how to do it safely. read this http://www.phac-aspc.gc.ca/publicat/lbg-ldmbl-04/ch8-eng.php

This is a profilactic measure. Once you did it and vapors are ventilated cells can be cultured again (you cannot culture anything during the disinfection of course) .

This is a tough question. I culture epithelia for up to a year. I like all the comments so far. Fumigation is a good start, but as noted by Lucas, should be done professionally. The problem is also the room. Once mold gets into the ceiling and air vents it is impossible to remove and your battle will be constant. My problems were resolved when I move from an old lab to new construction, but I work hard to keep mold out. Besides all the comments so far, don't store anything in your hood and bring in only what you need for the current procedure. Everything you bring in disrupts the laminar flow that makes the hood work. Wipe everything you bring in to the hood with ethanol, including your gloved hands!. Change your UV light every six months. You only need to use it a few hours each night. Even though it appears to be on, old bulbs no longer produce germicidal wave lengths. Put a sticky mat on the floor of the entrance to minimize dust brought in by peoples shoes (these are found online and come in layer of 30 sheets). Your idea of a gas permeable box might work. A pricer but perhaps more effective solution might be a chamber from Biospherix. These were intended for regulating O2 levels, but they have their own filters and are easy to clean. They sit in your incubator so that you can coexist with other users. Hope some of this helps.

Thanks Lucas ands Lawrence for your clear and useful answers. Lawrence, how do you routinely sterilize your incubators? Ethanol should not be fully effective with molds. Do you culture epithelia on inserts?

Yes, we culture on Transwell and Snapwell inserts. We use ethanol and put a few crystals of dichromate in the water tray.

Also, you might look at http://www.wak-chemie.net/.

Thanks Lawrence!

May I suggest looking for a hidden spot that act as a source of fungi? In one occasion we got fungi below the rubber that seals the incubator glass door and this affected all incubators.

Thanks Ahmed! I'll check it.

I agree with most of Lawrence's points. Whenever I train people to grow cells for long periods of time i.e. months, I always suggest that they grow their cells in filtered flasks. These have 0.2uM filters which act as a barrier to fungal spores and allows gaseous exchange. Growing cells long term in plates and dishes is not recommended because they have no barrier to the fungal spores. You can fumigate your incubator but within weeks the fungal spores will re-contaminate your CO2 Incubator form the room environment. There are a number of things that you CAN DO YOURSELF that will reduce fungal spore contamination of CO2 Incubators:

Only use NON-FAN assisted CO2 Incubators....fans are fantastically efficient at spreading fungal spores AND fantastically difficult to clean

Purchase incubators that have a high temperature decontamination cycle...this makes it easier to keep on top of fungal/bacterial contamination.

Change the water in the CO2 Incubator on a weekly basis.

Wipe all TC plates, flasks and dishes in and out of the CO2 Incubator. Leaving TC media on the outside of these vessels gives a rich environment for fungal spores.

I have not used UV light now for 30 years. It does not work and causes TC workers to be lazy and not clean their "environment".

Regularly clean all surfaces in your TC rooms. All TC should be done in side rooms away from the general lab areas. It is the TC workers who are responsible for their work so they should have a vested interest in keeping their environment clean.

Reduce any unwanted traffic in TC areas...this will reduce the spread of dust and dirt in these areas i.e. Make sure you have all your pipettes and consumables ready to use in the TC cabinet so that you are not constantly getting up and down form the cabinet.

Use a separate coat for TC work

Change the water in your waterbath on a weekly basis. Mould and fungus contamination cannot be controlled just by putting a chemical into the waterbath. Again laziness if you do not change the water regularly

I hope some of this helps.

Hi Naele,

"Wipe all TC plates, flasks and dishes in and out of the CO2 Incubator. " Do you wipe with 70% etoh? Are cells affected by etoh vapors?

Dear Roberto,

Yes wipe the outside of all flasks/dishes and plates........and no the cells are not affected by etoh vapours. How do I know this:-

Cell viability when tested is between 99and 100%

We look at oxygen consumption rates in intact cells, a very sensitive method of looking at cellular health.

When we subculture our cells we put tremendous stresses on them i.e. working in ambient temperatures, enzymatic treatments, centrifugation, low CO2 levels etc. A little etoh vapour is nothing

Sorry but I forgot to mention about Fungizone....DO NOT USE IT IN CELL CULTURE MEDIA. It is a very dirty compound containing amphotericin B. In our studies with Nitric Oxide production in Porcine Endothelial cells (Nature, vol 327, No. 6122 pp524-526, 11th June 1987), we found that using this compound to reduce fungal contamination (primary cells from the abattoir)completely inhibited NO release.