Hi,
I am having bacterial contamination in cell culture. I see these wriggling at high magnification and as black balls at low magnification. My problem is, I see these only after I start splitting the cells. When taken from liq nitrogen, it seems to be fine, but dont know what is happening after that. I tested my medium, by keeping it in the incubator and nothing grew in it even after 4- 5 days. I threw away the old medium, trypsin and everything I use, cleaned the incubator with alcohol, but still not able to get rid of it. Please help me out of this .
Thanks in advance
It seems that your frozen stocks may be contaminated. You may only see if after splitting not because of splitting, but because it was happens to be the time for the infection to get to a limit that you can see it.
Are you sure the laminar cabinet is sterilized. Why not trying to leave an empty bacterial agar (w/o antibiotics) dish for 2 hrs, and continue to grow either in 37 C incubator overnight and see if anything grows in it?
If the cells are 'precious' and you want to try and recover the population that is infected with bacteria, but apparently resistant to the standard tissue culture complement of antiobiotics, try using cephoxitan sulfate (mefoxin). It's likely to be available in your hospital pharmacy and works quite well in eradicating resistant strains of bacteria that plague cell cultures. We typically carry in the media at 50 ug/ml and replenish on cell feeding. Once the infection is eradicated, you can switch back to the standard pen-strep mixtures used. Hope this helps!
Hi Jaya,
First of all, I would recommend that you streak some of your culture onto sterile LB agar like others have suggested above. Culture for 48 h at 37C to see if anything grows. Check for mycoplasma contamination, too (use PCR kit).
What culture medium are you using? Generally, if your cultures are contaminated you'll observe a change in the color and transparency of the media. Also, bacterial contamination tends to give off a foul smell, and you would notice that when opening the incubator (except for mycoplasma). If your medium has FBS, you may observe dots or debris under the microscope. Also, there may be cell debris, as mentioned above by Aravinth.
I would also suspect that your stocks are contaminated. If that's the case, you'd have to try to use a different stock to run your experiments.
Now, about cleaning the incubator.. You must autoclave all the removable pieces, shelves in autoclave-safe bags (121C for at least 30 min). Then, wipe the incubator with 5% bleach (if you had mold contamination, the ethanol will not eliminate it). To avoid rusting of the stainless-steel, wipe with 70% ethanol afterwards. Always make sure that there is water in the incubator and add a small amount of copper sulfate to prevent microbial growth. Autoclaving and cleaning the incubators should be routinely done.
I would also recommend that you clean the cell culture hood with 5% bleach followed by 70% ethanol.
Hope this helps,
Sandra
Thank you Aravinth and Sandra. I will try it out and see what happens.
I think the black balls appearing in the flasks are not bacteria it might be a yeast. One probability is that the frozen culture itself might have contamination.you please check that and pay some attention to the environment of the culture room.
all the best
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