This month's question from the Molecular Biology Forums (online at molecularbiology.forums.biotechniques.com) comes from the “Cell Culture” section. Entries have been edited for concision and clarity. Mentions of specific products and manufacturers have been retained from the original posts, but they do not represent endorsements by, or the opinions of, BioTechniques.How can I safely clean a tissue culture incubator contaminated with bacillus? (Thread 20015)
Q We recently had a rod-shaped bacterial contamination in one of our incubators. We are planning to throw out all of the cells and sterilize the incubator.
I have thought of autoclaving the removable parts of the incubator, but I would have to take the parts out in the open air when they leave the autoclave and return to the tissue culture room. Is autoclaving really essential or valuable?
I might also spray the incubator with IMS or wash it with Virkon. Will either of these leave spots on the incubator? What is the best way to use them?
After cleaning, how long should I wait before it is safe to start cultures again? How can I be sure the contamination is gone and that there is no residual cleaning chemical left that could harm my cells?
A1 If the contaminating bugs are uniform in appearance, you probably have a single source of contamination. Try to find the source before you take the incubator apart. It may be anything, even the Pen-Strep stock solution. Gram-positive, spore-forming bacteria may even survive in your trypsin working solution.
Check your water bath. You might find the same bacteria there. If it has a metal tray, you can bake it at 180°C for 5 h.
To autoclave your incubator racks, wrap them loosely in aluminum foil or brown paper beforehand and unwrap them only when you put them back. Make sure your cycle is long enough to kill off spores and don't put any liquids in the same load.
To sanitize your incubator (if it's not a self-sterilizing one), you should only use isopropanol. Surface disinfectants can leave a residue that might harm your cells and feed the next bug.
Flasks with filter lids are safer than dishes in a questionable incubator.
A2 Cultures can become contaminated in liquid N2. Check your frozen stocks and make sure the vials are above the level of the liquid phase in N2 storage.
A3 Instead of autoclaving the removable parts, you could use the UV light in your tissue culture hoods.
Make sure to throw out all the media you were using at the time of contamination in case that was the source.
A4 Autoclaving is the only effective way to clean the incubator. Autoclave all the shelves, side panels, and water pans. Replace the fan gasket and plastic fan. Seal the parts in double plastic trash bags before taking them out of the TC room and wipe down every possible surface with ethanol before reopening the autoclaved bags.
A5 Try the Mycoplasma Disinfection Kit II from Promokine.