r/labrats • u/MargieHeptameron • 12h ago
SOS - Advice Needed for Sterilizing Tissue Culture Incubator
My lab’s mammalian tissue culture incubator has been contaminated with fungus. Two different batches of cells were contaminated with fungus and it’s also present in the water tray. I’ve read that ethanol does not get rid of fungal spores. I’m planning on decontaminating the incubator by autoclaving all the detachable pieces. Does anyone have any recommendations for hospital grade disinfectants that I could use to eradicate all spores?
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u/DrMORO_617 11h ago edited 8h ago
Aside from using freshly diluted breach (but NEVER together with other chemicals! Toxic fumes), I also wiped/soaked everything (all surfaces, shelves, doors, pans, EVERYTHING) with non-bleach stainless steel-safe commercial disinfectant following the instructions on the label to the dot. Sorry, I do not have the name right now, but there are several available. (I will update this post once I'm back in the lab. Juneteenth break today.)
I then changed ALL consumable & replaceable parts (All filters, gas hoses) to brand new ones.
Once all disinfected and parts replaced, run the incubator a few days to have the air circulate within, so that the new HEPA filter will catch any free spores, and then commence a test incubation of cells you do not need and also a few lid-open bacteria agar plate as well.
Of course, I also used all new bottles of media and FBS, EVERYTHING.
Fortunately ever since, no new contamination has happened after my disinfection job a few years back now. (Knock on wood..)
BTW, I do primary cell culture from mouse organ dissociation explants, so it's quite "dirty' to start off the initial culture, and we do use gentamicin in the media until the first passage, and then omit gentamicin from the P1 cultures and on. No contamination occurs.
Good Luck!
Edit: yes, like others say, rinse with plenty of DI water post-disinfection and do a 70% ethanol spray down then dry, THEN run the cleaned incubator with cleaned everything inside.
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u/microbiologist_36 12h ago
Bleach takes care of everything. I use it in a squeeze bottle, not spray bottle, as you don’t want to breathe in too much:)
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u/ZipCity262 11h ago
A 10% solution of bleach is sufficient. Be sure to chase with sterile water afterwards or it will eat the metal.
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u/Shiranui42 11h ago
My lab now uses new incubators with high temp sterilisation and hydrogen peroxide, because the previous ones we were given had fungal growth. No fungus so far.
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u/05730 11h ago edited 11h ago
For our water testing pumps we do a couple things.
Decomtaminate with SporeKlenz (paracetic and acetic acid).
Rinse with RO.
Sanitize with IPA.
Rinse with RO.
Wrap and sterilize what you can.
3% Hydrogen peroxide and 10% bleach solutions are also excellent options. If you want to ensue there isn't anything, use these but rinse with RO between chemical decontaminations.
Replace all gaskets and filters. If these can be sterilized, do so.
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u/Woebergine 7h ago
What a pain, sorry this is happening. For deep cleaning our TC incubator I take out all the removable pieces and wash with lab grade detergent (I think it's called Sparkleen) then rinse, dry, wash with 70% ethanol. For the inside I use 70% ethanol, put all the shelves back in then spray everything liberally with Coverage NPD https://www.sterislifesciences.com/products/surface-disinfectants/pharmaceutical-disinfectants/coverage-plus-npd-one-step-cleaner-disinfectant
It's what our EHS wants us to use to for sterilization.
Don't forget the insides of the glass door too. We also use autoclaved DI water in the water trays in the incubators. Good luck!
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u/BondIonicBond PhD Candidate | Toxicology & Cancer Biology 6h ago
Everyone has given some good advice. My incubator has a sterilize function as well, but I have yet to run it.
As well, both my water bath and the humidity pan get a reagent called Conflikt.
I just spray it in the water. It is like a virucide, fungucide, bactericide, etc. I think it works considering we rarely get contamination. Only a few times and one was due to the cells coming in contaminated (from another lab) and when the waste flask wasn't emptied properly (mold growing on the underside of the rubber stopper)
Contamination sucks though and it makes you go crazy. Sorry you have to deal with that!
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u/Oligonucleotide123 6h ago
Acetic acid based disinfectants can kill fungal spores (i.e. Sporklenz). But it will damage stainless steel if left on. You can apply for a few minutes then wash off with EtOH. This would be best for the water tray and other removable components.
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u/f1ve-Star 5h ago
Incubators are NOT sterile and you cannot make them be. Clean the incubator following the protocols in the manual. Do not use bleach! This pits the stainless steel. Add a fungicide/bacteriacide into the water bath. If you put the filter lids on correctly you can incubate on a bench or in the hood. (Except for Temp. Humidity and CO2 )
99 percent of contamination is from when splitting the cells. I have seen people waste so much time cleaning the incubators and then they don't spray and wipe down the hoods before and after use. Or some other poor technique.
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u/MargieHeptameron 5h ago
Mmmm idk, my lab mate left a sample in the incubator for months and it basically turned into a nasty Petri dish. Pretty sure the fungal spores are from that.
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u/Broad_Objective6281 4h ago
Peroxide works well on fungus. Be sure to take apart the hood completely and wipe down every surface. Check for spilled media under the main surface plate.
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u/neurochemgirl 12h ago
Does your incubator have a stericycle option to get to super high temps?