There are chemical additives from the major bio companies like VWR and Fisher you can add to water baths, either cell culture or heated baths.  The are often called clearing agents.  We have a solution from Spectrum that we have had forever.  The catalog is 105535 (I think some of the numbers are partially missing).   Fisher even has a convienient bar that suction cups to the water bath and lasts for several months. 

Copper is also very effective against microbes especially bacteria.  A final concentration of 0.1% copper sulfide in autoclaved water for the baths can also inhibit microbe growth and is probably the cheapest solution.  Try to replace monthly.  Just be careful when making the concentrated stock as it is an exothermic reaction.  I have also been in labs that will autoclave pennies and throw them into the water bath.  When they become coated just scrub and autoclave again.  There are also copper plated basins for tissue culture incubators.

I would make sure to thoroughly wash all the shelving and autoclave them.  Make sure they are completly cool before trying to grow cells though.  The heat can kill cells especially temperature sensitive cells.  Spray the shelves with ethanol before placing back into the incubator.  Also, wipe down the inside surfaces with ethanol .  This will help give a fresh start to the incubator and hopefully minimize contamination. 

As far as the cultures themselves, I recommend Gibco's antibiotic antimycotic solution.  It is stable for 3 days at 37 C so if you need to culture a plate longer than that I would change media.  Also, be extra careful when working in the hood.  Try to avoid moving your hands over the open cultures and keep track of where the pipette tip is.  Many people accidently touch the back wall or even have them outside the hood while juggiling multiple things. 

If you have sloppy lab mates, make sure all spilled media is cleaned up before working.  And I would recommend talking to your PI and lab mates nicely about how important keeping the cell culture area clean.   Just for your own piece of mind it can be a good idea to spray down all pipettors just in case someone forgot and always spray down the inside.  Check to see if there are vents nearby that may be causing air drafts.   This can mess with the biosaftey cabinet's air flow and increase the risk of contamination.  Also, make sure biohazardous waste is regularly disposed of.  If media inside of tubes or conicals are thrown into the waste, then try to make sure to cap them.   The anti-microbial compounds like Pen/Strep will breakdown over time and grow nasty things.  And if it is a shared tissue culture resource for the department be extra careful, people are sometimes not very careful when they know it will be hard to track the problem back to them. 

If it is a problem from a newly acquired cell line either from a company or collaborator, it may be worth asking for a stock frozen on a different date. 

(If I gave you a bunch of information you already knew I was not trying to be insulting. I'm not sure all of what you know and I've found it safer just to assume people don't know much just in case, even seasoned scientists.  We switch around and learn so many different things in are careers that sometimes people assume we know an important detail and we don't.  I have had my fair share of mistakes because I was unfamiliar with an important detail that was assumed I already knew.)

Hope some of this helps,

Christina

There are chemical additives from the major bio companies like VWR and Fisher you can add to water baths, either cell culture or heated baths.  The are often called clearing agents.  We have a solution from Spectrum that we have had forever.  The catalog is 105535 (I think some of the numbers are partially missing).   Fisher even has a convienient bar that suction cups to the water bath and lasts for several months. 

Copper is also very effective against microbes especially bacteria.  A final concentration of 0.1% copper sulfide in autoclaved water for the baths can also inhibit microbe growth and is probably the cheapest solution.  Try to replace monthly.  Just be careful when making the concentrated stock as it is an exothermic reaction.  I have also been in labs that will autoclave pennies and throw them into the water bath.  When they become coated just scrub and autoclave again.  There are also copper plated basins for tissue culture incubators.

I would make sure to thoroughly wash all the shelving and autoclave them.  Make sure they are completly cool before trying to grow cells though.  The heat can kill cells especially temperature sensitive cells.  Spray the shelves with ethanol before placing back into the incubator.  Also, wipe down the inside surfaces with ethanol .  This will help give a fresh start to the incubator and hopefully minimize contamination. 

As far as the cultures themselves, I recommend Gibco's antibiotic antimycotic solution.  It is stable for 3 days at 37 C so if you need to culture a plate longer than that I would change media.  Also, be extra careful when working in the hood.  Try to avoid moving your hands over the open cultures and keep track of where the pipette tip is.  Many people accidently touch the back wall or even have them outside the hood while juggiling multiple things. 

If you have sloppy lab mates, make sure all spilled media is cleaned up before working.  And I would recommend talking to your PI and lab mates nicely about how important keeping the cell culture area clean.   Just for your own piece of mind it can be a good idea to spray down all pipettors just in case someone forgot and always spray down the inside.  Check to see if there are vents nearby that may be causing air drafts.   This can mess with the biosaftey cabinet's air flow and increase the risk of contamination.  Also, make sure biohazardous waste is regularly disposed of.  If media inside of tubes or conicals are thrown into the waste, then try to make sure to cap them.   The anti-microbial compounds like Pen/Strep will breakdown over time and grow nasty things.  And if it is a shared tissue culture resource for the department be extra careful, people are sometimes not very careful when they know it will be hard to track the problem back to them. 

If it is a problem from a newly acquired cell line either from a company or collaborator, it may be worth asking for a stock frozen on a different date. 

(If I gave you a bunch of information you already knew I was not trying to be insulting. I'm not sure all of what you know and I've found it safer just to assume people don't know much just in case, even seasoned scientists.  We switch around and learn so many different things in are careers that sometimes people assume we know an important detail and we don't.  I have had my fair share of mistakes because I was unfamiliar with an important detail that was assumed I already knew.)

Hope some of this helps,

Christina

We use copper laminated/coated shelves in our incubators. However contamination shows up from time to time. In these cases I found that the molds grow on top of the plastic flask and dishes so we also weep the plastic gear with antimicotic solutions. This reduces the risk of fungal contamination a lot.

Jason,

If you use copper solutions, please do not allow them to go "down the drain".  These are faunal pollutants and regulated by federal and state laws.

In my experience, once you have decontaminated your incubator, look to the following causes of mold contamination: (1) Each time you open your incubator you are creating eddy currents which drive the local environment into your incubator.  This is particularly significant if your incubator door is near the floor but, also check the top of the incubator. (2) It is important to use medical quality CO2 and a pre-filter to charge your incubator. (3) The door gaskets can harbor mold spores especially, under the gasket in the grommet space.  (4)  You also need to determine if the air handling units in your lab are moving "dirty" air when you access your incubators.  (5) Finally, I once had a colleague who was an aficionado of gourmet bread who inadvertently transferred mold spores from the kitchen to the culture room.

Please let us all learn of your success in solving this problem.

Michael

http://www.cymitquimica.com/en/producto-quimico/09-WAK-ZF-1

This is a really good disinfectant we have been using without any problems!

we use 10% virkon and 70%Etoh to clean and add treatments to water baths, but even with prevention methods, you still need to keep on top of cleaning, giving the whole incubator a proper clean once every 3 months or so, if your contamination is more frequent then michael and christina have given very good pointers

Guys and gals, thank you so much. I cleaned everything thoroughly, and autoclaved the shelves. We do add a disinfectant to our water bath. The water has no sign of contamination. DO these disinfectants evaporate and combat airborne spores in other parts of the incubator as well?