Hi Jhoti,

We recently experienced fungal contamination in our incubators. We treated the incubators in the following way and we do not have problems anymore:

1) take out all the parts of the incubator, screws included

2) wash all the parts with soap, also the inside of the incubator, and rinse carefully with water

3)spray all the parts with special anti-fungi spray, screws included (we used Biocidal ZF), take care to spray also the inner parts of the incubator that are less accessible, let dry.

4) spray all the parts with ethanol and let dry

5) place all the removable parts in a dry heat oven (that goes up to 200 C) and sterilize for at least 4 hours - plastic parts should not be included in this step

6) mount all the pieces back in the incubator

7) fill the waterbath with autoclaved water

Check weekly if the water in the waterbath looks clean. If the contamination is still detected repeat the procedure.

I hope this helps.

Cheers

I the water is the trouble, you could add some copper sulfate. Definitely autoclave the water before putting it in the incubator.

Some older type incubators can be a real hassle: the ventilator is nearly impossible to clean and an ideal place for fungal or yeast spores to attach. If this is the case, the only real solution is an incubator with a self-autoclaving routine.

We place the cleaned water container in a laminar flow hood that has a UV lamp for 10 minutes, before refilling it with autoclaved water that then has the following germicidal solution added at a rate of 3 mL per litre.

STOCK SOLUTION

- 50 ml Ethanol

- 50 ml Glycerol

- 3.5 g Sorbic Acid

Store in the dark at 4C for 6-8 months max

I have stainless steel pans filled with distilled water that I place in our incubators. I change out the pans weekly by replacing them with sterilized pans and fresh distilled water. You also want to check the CO2 lines coming into the incubator. I attach a 0.2 u filter in the tubing between the CO2 source and the incubator. For cleaning, I have found 70% ethanol to be good for decomtaminating an incubator. I remove everything, clean all the surfaces with 70% ETOH, and then add a pan of 70% ETOH in the incubator and let it go overnight. The next day, I remove the ETOH and replace the cleaned, sterilized components.

Luca's advice about disassembling the incubator and cleaning the parts is good. Wear rubber gloves. Make sure you spay anti-fungal disinfectant into all the cracks and crevices, but not into electrical parts like the fan motor. Be sure to wipe up the excess disinfectant with sterile gauze/cloth, and allow the bare inside of the incubator to dry out to remove fumes. You may find it easier to autoclave the parts instead of using a dry heat oven. As long as plastic parts are made of polypropylene, they can be steam sterilized. Also include a dilute solution of disinfectant in the water pan (use autoclaved DI water).

One of the people in my lab used to leave dead cultures in the incubator, and then fungus took over. Once fungus sporulates, the spores get into places that can't be disinfected, including the culture room. We had to hire professionals to fumigate everything. this is how bad it can get. Preventative vigilance is better than any remedial suggestion mentioned above.

Lastly, the most important factor is good technique. No amount of antibiotics or antimycotics can substitute.

I agree with much of the above. Fungal infection can be very difficult to shift once it is present. In my lab we fumigate annually as a matter of routine, along with regular cleaning with virkon and 70% EtOH. Although copper sulphate is useful for cleaningthe water, it will corrode steel lining of standard incubators.

We routinely clean the incubator in similar ways as previously mentioned and add a diluted benzalkonium chloride rinse prior to 80% ETOH. This has not rusted the incubator and has controlled growth contaminations very well.

Excellent! I highly appreciate all of your suggestions. I think i should try autoclaving the removable parts, and i should definitely purchase some anti-bacterial sprays. Thanks so much for your help!

@ Julia Powers: does this germicidal solution work effectively? will any fumes be liberated and affect my cultures?

I must add that the ultraviolet light and germicidal solution are used in addition to regular thorough cleaning of the incubator with Vircon and 70% alcohol. The germicidal solution does not release fumes aside from an initial small amount of vaporised alcohol- at a much lower concentration than that used to clean the interior of the incubator.

Also check the filter inside Your incubator - if it is autacleavable (and usually it is) then autoclave it, if not, then exchange it for fresh one. Some fungi can nicely grow on a filters and most people forget about that.

the problem with your cell culture contamination are.... your hands and the way you touch the material before cell culture. I have the same problem each time I have a new trainnee in the lab. But it you still want to clean your Co2 incubator, wash it 2 times with cetrimid or CTAB (yes the detergent used to isolate DNA) and the final wash leave it, don't do a final wash with water. let it dry in the incubator. I add also a spun of Cetrimid or CTAB to the water.

I might add that we always place a UV-lamp in the incubator for 24 h....after the whole cleaning protocol (cleaning+desinfection) has been completed...work wonders!!!

What about your laminar flow cabinet? May be the problem is not the incubator but hood.

We clean our incubators by wiping them down with 1% virkon and immediately therafter with 70% ethanol. You cannot leave Virkon on metal surfaces for more than 10 min because this causes corrosion. Water trays are also emptied and cleaned regularly and filled up with water and 1 tbsp copper sulfate to prevent growth. Our incubators have a decontamination programme built-in which will heat it up, but this reduces the overall lifelength of the product wherefore we prefer routine cleaning.

For cleaning incubator all these previous suggestions work well. Some incubator have heating system to disinfect it. additionally U can add anti-fungal agents in water in incubator tray.

To check your safety cabinet, put media in 6-well plate, open the lid and leave it in hood for 5-10 minutes. Then place the plate for incubation, if growth is there, then clean the filters of Ur safety cabinet. If no growth then Ur safety cabinet have no problem.

@ Fernando Cardoso- is it safe to wipe the inner mouth of a flask with a bit of 70% alcohol if media accidentally drops on it? or should i wash it off with sterile PBS instead?

My laminar flow is regularly cleaned and i use the UV light twice every week. I've purchased the Virkon spray, and have taken to cleaning my incubator and water tray twice weekly and decontaminating it the using UV light as well. I hope this work, fingers crossed!! Thanks so much for all your suggestions and tips.

firstly, take out your flasks and plates.

after that wash it with 70% alcohol. finally, if you have UV lamb put it inside the incubator and close the incubator for 15 min. it will become sterile.

regards

@Jhoti Somanah I never fo that, I prefer to change the cells to another flask.

The medium is sterile so what I do, when that happens is to collected the medium in the inner mouth of a flask with micropipet with a sterile 200ul tip.

Hi Somanah

Before you use, clean with ethyl alchol (over 70%), subsequently run UV closed system (with water system) min 24 hours. It is important that you should routine check UV lamb and follow lifetime for effective sterilization. Also Hepa filters are important for contamination, so you should check or replace with new ones.

I usually do it this way, I have never encountered contamination so far.

Best wishes

Hello, Jhoti.

Here are some steps that I do to maintain our incubator:

1. Make sure we have at least 2 litres of autoclaved water before cleaning the incubator.

2. Remove all the inner part of the incubator, wash it with soap and water.

3. Autoclave those parts after washing.

3. While waiting for the autoclaved, I wiped down the inside of the incubator with 70% ethanol.

4. Clean the water pan with soap and water, wiped it down with 70% ethanol, then add the autoclaved water. Then add a pinch of sodium dodecyl sulfate (SDS) to the water

5. After autoclaved is done, let it cool off a bit before setting it back into the incubator because it will be too hot. Then when they are a bit cooler, before setting it back into the incubator, wipe it down with 70% ethanol again.

I usually do this once a year, but for the daily/weekly/monthly maintenance, I just make sure the filter still clean, there are enough water, wipe the wall and cabinet of the incubator with 70% ethanol once a while. :)

I think a qualified technician from the company you purchased your incubator from should clean/replace the HEPA filter for you.

Jhoti,

Some incubators employ continuous UV irradiation to stop humidifying pan water from becoming a vector for contamination. The UV light in this case is directed towards the humidity pan (and away from your cultures) by a back plenum. The UV light doesn’t harm the cultures and is constantly decontaminating the air flowing through your incubator, as well as the water in your humidity pan. It’s a great way to prevent this type of recurring growth. Here's some background info:http://us.panasonic-healthcare.com/wp-content/uploads/Active-Background_41614.pdf

Persistent contamination is a nightmare scenario. Somewhere during your careful preparations for cell culture, one or possibly several steps may introduce a contaminant. As the source can be something quite small, we recommend making a long checklist of possible sources of contamination. A dirty lab coat, loose hair, and even talking are common contamination sources. 

The cleaning procedure might also be a possible source of contamination. High-heat decontamination cycles may not eliminate certain species of microbes, which may quickly proliferate if conditions allow. An alternative sterilization process is 10 minutes H2O2 vapor followed by 2 hours of UV irradiation. The UV irradiation reduces the H2O2 to sterile water. This method also allows the shelving to be cleaned in place. 

Here's a list of the species that H2O2 decontamination has been shown to be effective against: http://us.panasonic-healthcare.com/wp-content/uploads/H2O2-Efficacy-Proof_41614.pdf

Panasonic sells H2O2 vapor sterilized incubators: http://us.panasonic-healthcare.com/wp-content/uploads/Fact-Sheet_41614.pdf

can anyone give tips on avoidance of epithelial primary culture contamination by fibroblasts ? Is there any chemical available which selectively inhibits fibroblasts sparing epithelia

Quick Question? Any need to wrap the shelves in aluminum foil when autoclaving... best way to clean copper shelves?

You an use aluminium foil to wrap. But the paper is best.

Why don't you try fumigating your incubator once in a week?. Place a clean petri plate on a PVC sheet and place it inside the incubator. Now add 15 grams of potassium permanganate and add 20 to 30 ml of formaldehyde. Immediately close the door of the incubator and set the temperature to 37. Close all the windows of your lab and get out as soon as possible. Don't forget to Label the door outside with fumigation under process. Next day allow enough ventilation inside your lab and of your incubator. Wear gloves during the entire process.

Wash two times with cetrimid, tha last one leave there, don't clean, it will dry and it will last for a long time.

Does anyone use 5-Chloro-2-methyl-4-isothiazolinone for incubator sterilization?