I recently experienced a problem with cell culture. The media in the T75 flasks turns cloudy. What maybe the reasons and how to resolve the problem. Im working with A549 and Hela cancer cell lines, which are adhesive cell lines. I have never encountered this problem before.
Please advise, thank you!
dear Mer Mercurate
the only reason i could think of is contamination. just check for bacterial contamination.
cloudy as colleagues above suggested could be due to contamination. however, i would like to add that check the flask to see if they are over confluent. hela cells grow like weed and they might be over confluent. as suggested above, we cannot rule out the possibility of contamination. freezing media for storing cells easily gets contamination. if it is a freshly thawed cells do check other stock of cells for possibility of contamination
cheers
the cloudy media probably is mean contamination in your medium. You can check it under the microscope, the contamination of bacteria. then you should to check to where the contamination from, probably from your medium (stock), FBS (i hope no from FBS), or your stock cells. Maybe you can add the antibiotics, if the problem is bacteria contaminate (http://www.atcc.org/products/all/30-2300.aspx). when you found that the problem due to contamination bacteria, please clean your incubator,laminar,all equipment before your start again.
good luck. -dee
Thank you all for your time. I gonna check the laminar flow of the hood today. I will report back after checking. I suspect the laminar flow of hood.
Besides contamination, the media can also become cloudy if the serum was added and the media is several months old. Or if it was left at room temperature for too long. In this case, the cloudiness would be the serum going bad. Always store your media at 4C to preserve the serum.
Dear David Molkentine,
May I check with you that serum should be store at 4oC or -20oC as when i collected from lab supplies, they are kept at -20oC. Thank you!
i normally aliquot my serum into 50ml tubes and keep them at -20. so for every 500ml of media that i use i thaw the serum at 37 and then filter it and add to the media. i do filter both the serum and antibiotics together, should in case i need to add some antibiotics to my media.
i hope this helps
Mer Mercurate,
Yes serum should be stored at -20C, but after you add it to your media it is stored at 4C since its inside your media.
Thank you David Molkentine and thanks you all for your reply. I checked the laminar flow of my hood today using dry ice. The flow appears to be okay, no turbulence and the working area under the hood is protected. The culture media, however, still turns cloudy. I am monitoring the situation. Tried to wash the cell with PBS and media couple of time before adding new media. See if it help.
At first it was suspected that i somehow mixed up the cell lines. But the new T75 flask i just thaw also got problem.
Hi!
Did you try just putting your media (no cells) in a flask, and incubate this to see if it comes from the media or the cells?
i guess Karl suggestion is right. if the media still turns cloudy then it could be from your media if not then your cells. may be that batch of cells were frozen with the same freezing media. so should one vial gets contaminated the others will also.
sure the cloudy media mean bacterial contamination and you can examine under microscope you will find black small rod like shapes in the media
sadly it means bacterial contamination, start again, and look for videos on aceptic work in cell culture, there is plenty and it will help for this not to happen again
If it looks a bit like a strawberry milkshake, it's probably a bacterial contamination! I would recommend just disposing of the culture as soon as possible and doing a fresh thaw. There's not much point in treating the contamination. Depending on the severity of the situation, your incubator may also need a clean.
Agree with Karl and Adam....Go for the Karl suggestion...may be it is coming from your media or may be you need to do fumigation to clean your incubator.
Thank you all, very much for your reply.
I decided to start over withi this cell line clean the hood. I am monitoring and so far it seems to be okay.
Many thanks for your very useful comments/suggestions.
- Badges
- Science method