Contamination issue in cell culture - (Nov/16/2004 )

Hi all

I've been isolating and culturing neonatal rat cardiac myocytes for a while now. Every now and then we experience a period when cells, from several subsequent isolations, die. We do not see moving particles, but it looks like the cells just fall apart in thousands of little pieces. The microbiology department tested the media for several contaminants but they couldnt find anything. Two weeks ago it started again and it drives me crazy!! After several perfect isolations cells die again, without any visual sign of a contamination. Again we see that cells suddenly die, preferably after putting them on serum free media overnight. We used fresh solutions, new sterilized media, but for some reason we cannot get rid of it. I attached a picture of dead cells. Has anyone seen this before?

thanks!

Well I never got around to posting those other pictures because I wanted to get rid of the cells out of my lab to remove the source. You CAN win against this 'bug'. The answer is just age old mycoplasma is the contaminant.

'GOOD Technique,' as some of the avid posters to this forum who throw around the word without clarifying or offering further help, should include culturing cells WITHOUT ANY ANTIBIOTICS. Why? Because when you DO get contaminants from a sleeve hitting a bottle neck etc, you rarely just get one kind. Usually one just grows faster. In other words the Penn/Strep kills the bacteria with a cell wall that you WOULD have seen and thrown out the culture, but because you never saw them grow up you miss out noticing the mycoplasma that came along with them on your sleeve. The Antibiotics simply MASK slower growing resistant bacteria. Moreover, mycoplasma can survive 4-6 days, without liquid, on surfaces on the hood just waiting for a culture to infect. Something not everyone knows is that standard 0.2uM 'sterilizing' filters don't filter out mycoplasma. They're too small. Only autoclaving or passing serum etc through a 0.1uM filter will do as they range in size from 0.15uM to 0.2uM. A good kit to check for mycoplasma is the Mycosensor PCR kit from Stratagene. Pretty sensitive and has internal controls.


The best work, around assuming you have no other cell stocks to go back to or suspect the stocks to be contaminated as well, is to do the Following:

1) check your serum, take a sample of pure serum and just try and grow it like a mini prep culture for like two weeks. If it gets murky then your serum is at fault

2) dispose of all your current media, it's probably contaminated anyway which is why it keeps coming back. Remake your fresh media AFTER you do the remaining steps.

3) THOROUGHLY clean your tissue culture ROOM. Autoclave EVERYTHING that can be autoclaved, bleach-bath everything else followed by 70% ethanol. This includes the damn little fan and housing that stirs the air inside your incubator and the water pan, water bath for cells, etc. Also disassemble the TC hood and clean the same way under the work area (you'll be disgusted with what grows under there) and all the hood pieces.

4)wipe down the insides of your disassembled incubator with 10% bleach followed by water then 70% ethanol. You WILL corrode your incubator if you don't rinse well after the bleach.

5) put Bath Clear (from Fisher) in the water baths to prevent further growth and change baths every week to two weeks (70% treating all parts you take it out and put it back in cabinet, but bleach not necessary anymore).

6) restart your lines in your newly made media as normal but WITHOUT antibiotics, however you will add Cipro to them at 10ug/mL to the media for two weeks. ***Cipro will NOT always work, it depends on the mycoplasma being susceptible but will work ~70% of the time. You may want to try Tetracyclines or some others if Cipro fails.***

After that, culture as normal without ANY antibiotics. This isn't surefire, but it's better than just floating in $hit creek and being told after the fact to have better technique.

Dear all,
I really hope that someone can give me some suggestions for the contamination problems I have faced for the past 3 weeks.

All this while, I prepared my media by dissolving the medium powder in autoclaved UHQ water and filtered through autoclavable filtration unit with 0.2um filter membrane. I didn't face any problem when I first prepared it. But, the contamination problem started for my second preparation. After each filtration of the media, I aliquoted 2mL of the media into a 6 wells plate and incubated at 37oC, 5% CO2. After overnight incubation, I could see cocci in the media. For your information, the aliquoted media contained no FBS or antibiotics.

The following are what I have done to troubleshoot the problem.
1. I suspected my filtration unit and bottles were not properly washed. Therefore, I soaked them overnight in 7X detergent with a small V of bleaching agent added, though I know that I not supposed to use bleaching agent for tissue culture work glassware. I even used disposable filtration unit instead of the reusable one.

2. I suspected the autoclave machine didn't work, so I used another autoclave machine.

3. I UVed the biohazard hood 30 minutes, wiped it with 70% EtOH, and UVed again for 30 minutes again before I used the hood for filtering my media.

4. Instead of using single layer of filter membrane, I used double layers.

5. I have added a filter from my filtration unit connection to the pump.

6. I suspected the UHQ water system did work properly, so I shifted to freshly collected dH2O.

7. I suspected my aseptic techniques, so I asked another colleague of mine who have more experience in tissue culture works to prepare the media for me.

I have tried all the above mentioned steps, but the problem remains. My experiments are stucked at this point.

I did try to streak the suspected contaminated media on plain LB agar plates, but I couldn't isolate any colony from there.

Can anyone tell me what other possible troubleshooting steps that I can do just to solve this headache of mine? I just don't understand why the media contamination only happened to mine.

I am wondering if it is possible that the powder media itself is contaminated? or Can it be the biohazard hood that I am using is contaminated? or the incubator is contaminated?

I am extremely frustrated now. Maybe I am cursed.

Hi virus_fan,

May be the pipette you use to aliquot the media?
May be the cell lines from the frozen stock is contaminated?

Just wondering did you check the bottle of media after a few days??


Hope this help.

Why do you have to make up your medium from powder? If you buy it in liquid form it's already filtered and tested...

Dear all,
I really hope that someone can give me some suggestions for the contamination problems I have faced for the past 3 weeks.

All this while, I prepared my media by dissolving the medium powder in autoclaved UHQ water and filtered through autoclavable filtration unit with 0.2um filter membrane. I didn't face any problem when I first prepared it. But, the contamination problem started for my second preparation. After each filtration of the media, I aliquoted 2mL of the media into a 6 wells plate and incubated at 37oC, 5% CO2. After overnight incubation, I could see cocci in the media. For your information, the aliquoted media contained no FBS or antibiotics.

The following are what I have done to troubleshoot the problem.
1. I suspected my filtration unit and bottles were not properly washed. Therefore, I soaked them overnight in 7X detergent with a small V of bleaching agent added, though I know that I not supposed to use bleaching agent for tissue culture work glassware. I even used disposable filtration unit instead of the reusable one.

2. I suspected the autoclave machine didn't work, so I used another autoclave machine.

3. I UVed the biohazard hood 30 minutes, wiped it with 70% EtOH, and UVed again for 30 minutes again before I used the hood for filtering my media.

4. Instead of using single layer of filter membrane, I used double layers.

5. I have added a filter from my filtration unit connection to the pump.

6. I suspected the UHQ water system did work properly, so I shifted to freshly collected dH2O.

7. I suspected my aseptic techniques, so I asked another colleague of mine who have more experience in tissue culture works to prepare the media for me.

I have tried all the above mentioned steps, but the problem remains. My experiments are stucked at this point.

I did try to streak the suspected contaminated media on plain LB agar plates, but I couldn't isolate any colony from there.

Can anyone tell me what other possible troubleshooting steps that I can do just to solve this headache of mine? I just don't understand why the media contamination only happened to mine.

I am wondering if it is possible that the powder media itself is contaminated? or Can it be the biohazard hood that I am using is contaminated? or the incubator is contaminated?

I am extremely frustrated now. Maybe I am cursed.

Hi virus_fan
as you said..they seems to be cocci..maybe they can be yeast...if so, if they have sporified, you have to decontaminate your hood and especially your incubator... Check the medium..and better if you use the liquid ones, already checked and decontaminated
Hope this helps!

I used Metranidazole (0,0000010M) for three days and it worked. This bug is intracellular parasite just like Protozoas...
Good luck

Hi,
I have had two contaminations in mammalian cell culture. Attached is one picture of the contaminants taken under 400x. Can anybody help me to identify what species are them? I got the contamination only after I transfected cells with lipofectamine and OPTI-MEM. Other people in the lab used the same reagents for transfection never get it. And my un-transfected cells never have it. It can rarely be seen 24 hrs after transfection. But after 48hrs, there are a lot in most transfected wells. The contaminants are round and PenStrep-resistant. They form chain-like or branch-like structures, show a little bit movement and they also seem to bud. Most of them are in the medium. We suspect that they might be yeast, but they are too small.

[attachment=3917:Conta3.JPG]

I had this once. It was from our water bath. Not sure yeasts or bacteria.
But the key is to find out where it came from to avoid the next incident. You are not going to make use of it anyway.