Dr. Jakaria,

You mentioned that there is a bad smell in the incubator. What kind of smell is it?

1. Mildew or mold? Make sure*** the incubator door seals and chamber walls around the inside of the door opening are clean of black mold. Make sure that the condensation/drip gutter on the inside of the door is clean... inside the gutter and underneath too.

2. Fermenting yeast? Yeast in chamber. Is anyone in the same lab or a neighboring lab growing yeast?

3..Foul/ sewage? E. coli or bacterial problem. Anybody nearby growing bacterial cultures?

4. Chemical odor? There should be no chemical odor inside incubator. If there is, identify the odor and either clean the incubator thoroughly or find and remove the source.

If this incubator was ever used to grow yeast or bacteria, then it needs a VERY thorough cleaning. If this is a shared incubator and another investigator is growing yeast or bacterial cultures in this incubator, then that is the problem. As soon as you see contamination in any culture in the incubator, it is best to immediately scrub out the incubator and discard ALL cultures, not just obviously contaminated cultures. As soon as you notice contamination problems in an incubator, you must immediately mount a very vigorous attack on the problem and maintain precautions in future or the problem will recur.

You mentioned a HEPA filter. Are there syringe type filters connected by tubing on the CO2 lines? Check the fan that circulates air inside the incubator for dirt or other foreign matter and clean. Replace any syringe type or other filters if possible, flush or replace tubing.

You did not mention how you are cleaning the interior of the incubator. We find the best thing to use is 70% ethanol with a few drops of detergent added. After cleaning, allow the incubator door to remain open for an hour or so to disperse residual fumes. Bleach or other cleaning agents may leave chemical residues that can damage your cultures.

What kind of culture vessels are you using? We find that the best types of vessels to use for preventing contamination problems are closed filter top flasks. Petri type culture plates, loosely capped flasks, or multiwell plates are too easily contaminated.

If you continue to have problems, always check your cultures in an inverted scope, even if they are dead, to see if the contamination is or is not present inside of your culture vessels and what kind of contamination is present (bacteria, yeast, other). After examining them, seal them up and discard them. Get them away from the incubator and out of the lab if possible.

Contamination problems in a tissue culture lab can be very frustrating and difficult to resolve. They don’t necessarily start inside incubator, but that’s the best place to begin looking for them. However, they can come from anywhere in the lab. Since contaminated cultures essentially shut down your research effort, it is of prime importance to find the source of the problem, resolve it, and then remain vigilant to prevent recurrence.

Good luck!

RW

Based on the information you provided, it is likely that water container in the cell culture incubator, which maintains the humidity, has contamination. This could be minimized by sterilizing the water tray through autoclaving and adding an inhibitor to water (see below) to prevent any future growth of any organism.

https://uk.vwr.com/store/product/3218295/disinfectant-for-incubators-incuwater-clean

Dear Dr. Choubey,

Thanks for your answer. Last time, I filled autoclaved water and also used inhibitor.

Hi

Why your cells die? because of contamination?

If you face with a incubator contamination, you have to raise up the temp to 60 degree for one hour.

Dear Dr. Mordadi,

Thanks for your advice. I am not sure about whether cells were died because of contamination or not. Last time, I used fresh media and cells were contamination free. Moreover, could you tell me about HEPA filter? Do you think the problem was due to HEPA filter dysfunction?

Your welcome

when you have incubator contamination problem like a fungal source even you use fresh media or other things spores in incubator are alive yet. so first step is clean that and raise up temp at list for twice. if didnt work and you be sure about other things then you can check HEPA that need a person with special staff and experience from manufacture.

I hope it be useful

good luck

Dr. Jakaria,

You mentioned that there is a bad smell in the incubator. What kind of smell is it?

1. Mildew or mold? Make sure*** the incubator door seals and chamber walls around the inside of the door opening are clean of black mold. Make sure that the condensation/drip gutter on the inside of the door is clean... inside the gutter and underneath too.

2. Fermenting yeast? Yeast in chamber. Is anyone in the same lab or a neighboring lab growing yeast?

3..Foul/ sewage? E. coli or bacterial problem. Anybody nearby growing bacterial cultures?

4. Chemical odor? There should be no chemical odor inside incubator. If there is, identify the odor and either clean the incubator thoroughly or find and remove the source.

If this incubator was ever used to grow yeast or bacteria, then it needs a VERY thorough cleaning. If this is a shared incubator and another investigator is growing yeast or bacterial cultures in this incubator, then that is the problem. As soon as you see contamination in any culture in the incubator, it is best to immediately scrub out the incubator and discard ALL cultures, not just obviously contaminated cultures. As soon as you notice contamination problems in an incubator, you must immediately mount a very vigorous attack on the problem and maintain precautions in future or the problem will recur.

You mentioned a HEPA filter. Are there syringe type filters connected by tubing on the CO2 lines? Check the fan that circulates air inside the incubator for dirt or other foreign matter and clean. Replace any syringe type or other filters if possible, flush or replace tubing.

You did not mention how you are cleaning the interior of the incubator. We find the best thing to use is 70% ethanol with a few drops of detergent added. After cleaning, allow the incubator door to remain open for an hour or so to disperse residual fumes. Bleach or other cleaning agents may leave chemical residues that can damage your cultures.

What kind of culture vessels are you using? We find that the best types of vessels to use for preventing contamination problems are closed filter top flasks. Petri type culture plates, loosely capped flasks, or multiwell plates are too easily contaminated.

If you continue to have problems, always check your cultures in an inverted scope, even if they are dead, to see if the contamination is or is not present inside of your culture vessels and what kind of contamination is present (bacteria, yeast, other). After examining them, seal them up and discard them. Get them away from the incubator and out of the lab if possible.

Contamination problems in a tissue culture lab can be very frustrating and difficult to resolve. They don’t necessarily start inside incubator, but that’s the best place to begin looking for them. However, they can come from anywhere in the lab. Since contaminated cultures essentially shut down your research effort, it is of prime importance to find the source of the problem, resolve it, and then remain vigilant to prevent recurrence.

Good luck!

RW

Dear Dr. Walter,

Thank you so much for your excellent reply. I am trying to find causes and solution as per your direction.

Hi Jakaria,

Just wondering if you were able to find solution to your problem?

Dear Ms. Sharma,

I just followed suggestions of all experts.

Hi Jakaria

I hope you have found out the causes of contamination. Only fresh culture medium does not work, cells may be contaminated by PBS buffer and Trypsin solution. If you could not confirm the reasons behind the contamination, it's better to use all freshly prepared reagents. Best luck.

Thanks a lot, sir! @ Md. Rafikul Islam

Parameters are to be considered:

The enclosure is contaminated, either by distilled water which is unsuitable

(it must be sterilized before adding it to the enclosure) add a decontaminant intended for incubators

Renew the distilled water once a week in your case

Either that your boxes, plates of cultures present leaks of medium which favors the growth of bacteria, fungies...

Arrival of CO2 is not protected by a filter

Do not neglect the cleanliness of the room where the incubator is installed

Is the incubator used by several people, different samples?

Dear Dr. Mahfoud, Yes, I followed every steps that you have mentioned here. We share incubator for different cell lines. Problem had been solved and incubator is working properly.