Hi Pooja,

The small strand-like things are fungal hyphae. Once you see those you might as well throw the cells out. The hyphae will form a large fungal mass in a day or two.

Which cells do you see the hyphae in (MEFs or stem cells)? Are you harvesting these cells yourself? I use an antibiotic/antimycotic with my cells since I harvest the stem cells myself.

It sounds like the contamination is coming from the cells since you've cleaned/changed all the possible sources of outside contamination. Just as a test, put some media in a flask (no cells) like you would normally do when performing cell culture and then put it in the incubator. If the source is coming from the hood or incubator you should see it. If not, its coming from your cells.

Hi Tom,

Thanks for your reply.I had left the MEFs in culture for 4-5 days and the strand size, amount in media, or cell death did not increase in this duration of time, so I was thinking if it was some FBS residue...?

I am seeing these in the MEFs, though my stem cells get contaminated very often as well- which we harvest in house, ourselves. We only use 1% Pen-Strep in MEF media and ESC media.

I have tried culturing only media and serum in different incubators too.. some incubators showed infection while others didn't. I cleaned everything, got rid of media.. started everything a fresh.. was infection free for a 2-3 weeks before it came back again. I have to mention that I am not the only one using the hood or incubators.. so its difficult to control other people's work culture. But my colleague, using the same MEF stock also keeps developing fungal infection. We culture cells in petri- dishes. Other people using flasks, and other cell type don't seem to develop infection in the same incubator.

Could flasks be better than petri-dishes in filtering infection-might sound lame, but do you think this could be the reason?

thanks.

Hi Pooja,

Do you filter your media after making it up (basal media+FBS+additives)? That would probably eliminate any large particulates in the serum.

I would definitely recommend using the antibiotic/antimycotic solution (Pen-Strep + Amphotericin B) which you can get through most vendors. When you harvest your tissues make sure you put them in the buffer with antibiotic/antimycotic and all subsequent buffers/media during the stem cell isolation. Also, try and not let your animals sit too long after you sacrifice them before harvesting the tissues. The longer you wait the more potential contamination will will have.

I understand it is difficult when you have multiple people using the same hood/incubators. Not everyone is as meticulous about cleaning/wiping down surfaces. Do you put anything in the water (for humidity) inside the incubator to suppress microbial growth? Once when I had a contamination problem that was the source of it. After I cleaned it I didn't have a problem again.

I think using flasks is a better option than petri-dishes since there is a filter in the cap of the flask which should keep contamination from getting in there. Petri-dishes are really designed to keep out contamination. Even multi-well plates are better than petri-dishes because there is not the over-hanging lid.

Hi Tom,

We've had discussions about filtering media before- but it always comes to an inconclusive conclusion that you'd never be sure which ingredients you might be filtering out.. So I don't filter anything so far..

Would you say its wise to introduce another antibiotic in midst of an on-going study? It could potentially introduce alterations within the cells and might influence epigenetic studies..?

Do you harvest or flush your embryos also in the presence of pen-strep? I read that some people double the concentration of pen-strep while deriving MEFs, but wouldn't that make the cells more vulnerable when the concentration is lowered?

I add Aqua Resist to the water baths and incubator water trays.. though we have had fungus growing in its presence too recently..

Hi Pooja,

I have always filtered my media/FBS and have not noticed any difference in the growth of the cells but that is a legitimate concern.

Amphotericin B is a antifugal not an antibitotic. My guess is that it shouldn't have any effect on the cells since so many people use it but who knows for sure. Maybe do a comparison of cells with and without the Amphotericin B in the media and see how they do.

I don't work with embryos. I works with stem cells derived from adipose tissue and bone marrow. But after harvesting I do wash the tissue in 5X Pen-Strep/Amphotericin B and then do the isolation in 2X Pen-Strep/Amphotericin B. After isolation the cells are grown/expanded in 1X Pen-Strep/Amphotericin B. This works well to stop fungal contamination although occassionally I do get a random flask/well that has fungal growth in it. No idea why though.

Fungus growing in the incubator water trays is probably the source of the contamination and its getting into your petri-dishes just due to the air flow in the incubator since the lids do not fit tightly.

Thanks Tom. This is quite helpful. I've tried isolating stem cells from adipose tissue once in a Summer School in Jena.. it wasn't a very pleasant experience! ;)

But I would get the antifungal chemical, and see if things improve. Anything that I could include in the water trays, that's specifically for fungus? And have you ever professionally/ commercially gotten your hood or incubator decontaminated/ cleaned? How often would you run the decon. cycle in your incubators (if at all you do)..?

does UV act against such infections? In previous labs we always considered UV as very vital- before and after work. But here, it doesn't seem to matter at all.. What's your take on it?

You can try adding something called Sporicidin which might help with the fungal growth but I'm sure how well it will do with spores. The only thing that kills spores effectively is autoclaving.

I have not had my hood/incubators professionally cleaned. I just do it myself. I take out all of the removable hardware I can from the incubator and autoclave it and wipe the inside of the incubator down with 10% bleach followed by 70% EtOH. I usually wipe everything down with 70% when I fill the water trays in the incubator and thoroughly clean the water trays themselves. Obviously this depends on how frequently you have to fill the trays. 

I never use the UV in the hood whereas other labs have it on all the time when they are not using it. The problem with UV is it will break down anything plastic/rubber you have in the hood. Also to be really effective you should remove everything from the hood every time you turn the UV on. That would be a pain. Plus I'm not sure how well UV kills spores. Are the hoods you use ever turned off or are they on all the time? 

I usually just thoroughly clean the hood with 10% bleach followed by 70% EtOH one a month and that seems to work fine. Since you have so many people using the hood you can set up a schedule to bleach/EtOH it once a week?