In our lab, Sanyo-MCO-175 water jacket Co2 incubator was used for cell culture maintenance. Past 3 month we were faced contamination problem in our cell lines (A549 and MCF7). So I was checked all possibility way to prevent contamination but I didn’t eradicate bacteria (rod and spherical shaped).
1. I was checked Co2 cylinder and Co2 incubator connection, no external filter attachment. If I will insert 0.22µ filter means, there is a possibility to remove bacterial contamination.
2. Sanyo-MCO-175 water jacket Co2 incubator contain in build attachment of 0.3µ filter.
3. Please suggest me to eradicate those problems.
I dont think a filter is needed. Contamination happens for various reasons, and especially in monsoon. Here are some tips which helped us:
1. Cleaning of the culture room must be done with same broom, mop, bucket etc..This must not be used for other rooms! Cleaning of room by only 1 person wearing cap, mask, slippers is important. All people working in culture room must also wear cap, mask, slipper etc. This sounds too simple-but it really matters!
2. The metal parts of CO2 incubator must be cleaned with 70% ethanol regularly. Water pan and its contents included...copper rod placed here helps as antibacterial.
3. If a sink for water or AC is inside culture room--more chances of contamination!! The AC and drain from sink must be cleaned. It maybe better to stop using the sink-that is what we did, and it really helped. Take all dirty items outside.
Ok-Hope this helps!
1. An in-line filter can easily be added but check that it does not adversely affect Co2 pressure.
2. what decontamination procedures have you used - ethanol? H202? Heat? Detergent? Disinfectant (Trigene etc) (some incubators come with potential for heat decontamination. What is the max temp your incubator can go to?)
3 Have you checked that your cell source is not contaminated from the stocks?
4. Do you have a water tray to maintain humidity or is it a dry incubator? If it has a water tray, have you added a disinifectant/ detergent to the water to prevent bacterial growth?
5 Check the seals carefully to make sure that they are not damaged or harbouring growth of any kind.
Dear Lan Teo,
1. In eppendorf Galaxy in line filter is available(0.22 micron filter).
2. isopropanol and temp.max = 50 to 55.
3. Yes. i was checked after 6 hours of retrieval mother stock culture in bright field microscope, i was observed clear medium without bacteria.
4. yes i have water tray to maintain humidity. yes. 3.50 gram of copper sulfate (disinfectant).
5. yes, NO damage.
In our lab, Previously we were used eppendorf Galaxy dry incubator for cell culture. cells are grown well but in this sanyo water jacket make lot of problem, We didn't get single passage.
Galaxy motherboard was damaged by ant at the month of january. So we used this sanyo after 2 years.
Before we used sanyo, hole room and sanyo were fumigated with formaldehyde and then i was retrieved A549 cell from our mother stock . 1st- 2 day - i didn't get any contamination; 3rd day i saw rod and spiral shaped bacteria with tribute medium in yellowish color.
After that i didn't retrieve cells. So i think to fit external 0.2 micron filter on behalf of internal sanyo 0.3 micron filter.
Its correct Dr. Lan Teo.
Dear Venil N Sumantran,
Already i am in your way my friend. i did all your suggestion. still my cultures are contaminated.
Your contamination controls seem to be OK. You could try leaving the incubator at 55C o/n - it will kill some bacteria but not spores. You only looked at your mother stocks after 6h and saw nothing, but got contamination after 3d. This suggests very low level of contamination or a very slow growing contaminant. Try growing the sealed cultures outside the incubator (+ a media only control) for several days to check that it is not a media or infected sample issue (i.e to exclude the incubator as a problem) - this is to grow bacteria , not cells. Incubate media only flask in the incubator. Do your flasks have an inbuilt filter in the cap to exclude any air-borne contaminants?
A filter is good practice, but I doubt your CO2 is the source of the contamination. After all, your cultures are not in open containers in the incubator. Frankly, most TC contamination is usually operator related, during plating, feeding, or preparing reagents.
Are you using any "home made" reagents or culture materials, such as pipettes or plates? If so, how are you sterilizing those? Maybe the sterilization is not adequate.
Have you thrown out everything, cleaned thoroughly, and then restarted with all fresh materials? It's possible you have contamination in your frozen stocks.
Dr. Peter has given very good suggestions. I can add the following:
Is it media or cell stocks..??? you can narrow down by placing media (without antibiotics) in incubator for few days..that will help.
If media are clean, you can see if some other cell line grows without contamination.
If media and other cell line do well- you know the problem lies with your batch of A549 and MCF7. Then, its best to start with new stocks of cells.
Hope this helps!
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