P.S.  The air in the lab has been tested and found to contain 3 genuses of fungi.  The company that did the work said the numbers of fungi found were very low, but this could be the source.  Our CO2 incubator is old and doesn't have an air filter.  We haven't had this problem to this extent in other labs and I've done cell culture for 40 years.  I don't think it's our technique!  We are all very careful.  Medium is pre-cultured to make sure it is clean, and some of it is sterile filtered.

It is extremely difficult to eradicate fungal contamination in the lab. I used to work with fungi and everybody else wanted to kill me. But you can suppress the fungal growth in your cultures if you add amphotericin B. http://en.wikipedia.org/wiki/Amphotericin_B

Depending on the cells you use you can add more or less of the amphotericin. Please check for toxicity by titrating your amphotericin and using a cell count or cell activity assay such as Alamar Blue.

Hi Earl I have heard (anecdotally) that bacteria can be transmitted from one vial to another in LN2 storage although  have never personally seen this happen. Also to get it happen so often the LN2 would probably have to be dense with fungal spores (?).

Not entirely sure how you wold test if this is the case.

I had a fungal contamination of my incubator last year which was a devil to get rid of so I sympathise with your plight.

Sorry I can't help!

Gary

In order to decontaminate the lab, fumigate it with a paraformaldehyde 'bomb'. This involves vacating and sealing the lab overnight and requires an extraction system to clear the toxic fumes. It is a serious process that mustn't be taken lightly! But it will get rid of any air-borne contamination.

It will be much more difficult to ensure that there are no spores in your liquid nitrogen storage. The only way to be sure to get rid of it is to store the cells in another room after fumigation and to reintroduce them gradually to the fumigated lab, carefully monitoring them for contamination.

The easiest and most important issue to address is the lack of filters. You can easily attach syringe filters to the tubing that leads from the CO2 supply. You might have to work a bit harder to adapt a filter to the air input, but it will be important if you want to overcome the contamination issue.

Make sure that your water bath where you are warming media has clean water in it.  I believe this was a source of persistent contamination in our lab a few years ago, even though we sprayed down the media bottles with ethanol before putting them in the  hood. 

Yes, I was using a waterbath with a lid but condensation from the lid was dripping down onto the bottles and I was getting contamination. I removed the lid and no more contamination (just had to top up the water more often).

I would also recommend using SigmaClean Water Bath Treatment. Just add a couple of drops every time you top up or replace water in the water bath or incubator and it will keep contamination away.

http://www.sigmaaldrich.com/catalog/product/sigma/s5525?lang=fi®ion=FI

Thanks, Everyone!  I don't use a water bath to warm the medium.  I put the bottles in fresh warm tap water in an ice bucket, then dump the water after use, so it doesn't sit around.  I can install a filter on the CO2 line. There is no air supply except room air (The incubator is 30 years old). 

I don't know if the Environmental Health Dept. would allow us to fumigate the lab, as the formaldehyde would be exhausted to the outside air.  They keep close tabs on the formaldehyde in the gross anatomy lab nearby.  The anatomy lab may be the source of the mold spores, as they've had trouble with mold growing on the cadavers.

Dear Earl,

When I was a postdoc we had an incubator with a water jacket that became infected with fungus and yeast. It was impossible to celan it. Even with professional cleaning. We actually threw it away eventually.

Now I have one with a hepa filter for air and a basin with  (sterile) water in the bottom of the incubator. In that I always have som copper and a little bit of detergent which help with bacteria and perhaps fungus.

So the incubator may be a problem for you?

In order to be extra safe (we do not use antibiotics) we also have the culture dishes in big plastic sterile assay trays with lids. That gives an extra barrier from all these things.

All people always carry around yeast on their skin, in their hair and in their clothes. I always put up my hair, use gloves, put alcohol on my arms, remove my watch and put on a new lab coat when I go into the cell culture room. It may sund like a lot of precausons, but we have had the same problems as you are having and it just would not stop.

I think that I am overly precausious sometimes, but it has worked and we have not had a problem for a long time.

Thanks!  We also use copper sulfate in a metal water pan in the incubator.  Sometimes we have used a Lysol detergent, but it did not help much.  The incubator is water jacketed, and I've used a quaternary ammonium compound as a disinfectant.  The increase in mold and yeast coincided with an involuntary move of my lab, so I have inquired about moving the lab to another floor.  We use gloves and treat them with fresh 10% bleach, then 70% alcohol.

I am in stem cell lab, too.

We frequently decontaminate our incubators and water bath. If important culture is contaminated, we use Primocin to suppress yeast and fungi. 

We have used Normocin in the last few months.  It's supposed to kill yeast and mold, but it did not!  What is Primocin?  I will look into it.  Thanks!

yeah, Me too had used Normocin and found nothing was fantastic. Just wasted. Do not buy it. Complained to supplier but no responses. 

I turned out that our problem is that I had forgotten to put a disinfectant into the water jacket when we moved the lab.  A colleague's lab gave me some Quatricide.  As soon as we added it, the frequent mold/yeast contamination ended!  Thanks to all who replied and for valuable advice.

I am using primocin, it does not kill fungus, someone told me to use normocin, but as I see here it does not help either. Cleaning company cleaned it with hydrogen, but it did not help.

I know that formaldehyde helps, but it is not allowed in my current work place. usually we used to leave PFA solution for 24-48 hours inside the incubator. It will help for sure, but you may need to sure it is allowed.

We had a serious fungal infection over the summer, that we could not get rid off even after several autoclaving and washings. I actually think we have had the same fungi for half a year in the incubator. I think it is gone now.

The first thing we did was take just everything out of the incubator that we could take out. The other times there were some things that needed to be screwed off and I had been hesitant to remove them, but that was removed. This included the propeller and everything. The propeller was washed in soap and water and then immersed in 70% alcohol.The rest of the loose parts were autoclaved in a real autoclave.