Cell culture contamination, antibiotic recommendation, any recommd on Normocin - (Jan/28/2007 )
Hi,
I recently experiencing contamination with my cell culture after going for so long without any issue. The problem is bacterial, the culture media would turn yellow and CLOUDY. Yellow media does not worry me as much because I work with HepG2 cells and they tend to acidify the media overnight, but the cloudiness is the clear and sure sign. I can see the round wiggling bacterial cells at 400x magnification. I need to 'SAVE' my cultures because these are lines that I spent year(s) to select and screened.
My media does contain pen/strep, and G418 as a selection. I have tried putting cipro into the media, but that did not help. The cultures would just be fine for a while, but 1 or 2 flaskes would just instantly turn cloudy overnight after a fresh change of media. I try to be very careful/paranoid with pippeting technique to avoid any touching of any surface and I change my pippets, even when 2 flasks are of the same cell line that was split the previous day.
I have noticed that of the contaminated flasks, the more confluent the flasks, the more cloudy/more bacteria in them. So it seems that the growth/severity of bacteria level is connected to the confluency of cells.
I don't know how the contamination occured, or whether it is systematic. But I get only random contaminations of 1 or 2 flasks out of the 10-15 flasks in a batch. As I said, I tried putting cipro in the media, but that did not stop the problem.
I came across another thread about using Normocin to stop/kill bacterial contamination, and it seems promising as a way to rescue contaminated cultures. Has anybody have extensive experience with this product? and does it work as advertised? What other products are out there that you can recommend?
Thanks.
why don't you use peni/strep?
I am not sure but if hyou can culture your cells in a medium with strep/peni then culture again after one day only
u may reduce chances of getting the contamination
from what you described and with the few i know it seems to be like the cell line is already contaminated try to go one or steps back
try to remember at which step u strated having the contamination then if possible restart at that step? 
I am not sure but if hyou can culture your cells in a medium with strep/peni then culture again after one day only
u may reduce chances of getting the contamination
from what you described and with the few i know it seems to be like the cell line is already contaminated try to go one or steps back
try to remember at which step u strated having the contamination then if possible restart at that step?
I said I have and use pen/strep, and I do change the media on a daily basis. But 1 or 2 random flasks just turn yellow overnight without any prior suspicion. Trust me, only if I know when/how the contamination occured, it would not be a problem. I have gone back to the original cryopreserve samples.
I need a way to 'rescue' these cultures, I am just safely assuming that this problem is in all of my cultures, as a precaution, I want to treat everything as contaminated.
ooh sorry i misunderstood u
in this case I really don't know what to do
good luck! hope someone who have experience on that will help!
Hi Biocell,
Have you sorted your contamination problem? I am having the same problem currently. I have cultured this cell line for more than a year without any problem. Untill early this year, I found my cells are contaiminated. At the begining I suspected it's yeast contamination. Because I use antibiotics peni/strep all the time. I wouldn't think it's bacterial of fungi contaminaiton. Everytime, immediately after I thawed the cells, they are fine. From day 3 or 4, medium became turbid and there are some visible round dots in flask. I centrifuged the solution and noticed the cell pellet are bigger than normal and there are "yellow powder" looking stuff on top of the cell pellet. My colleague suggested it's yeast contamination. But now after I read your discussion and other information online. I doubt that it might be bacterial contamination. If so, it would be a serioius problem. Do you have any good suggestion?
I wouldn't think it's cell source problem as I alwasy thawed the same batch and they were all OK. I first found contamination about 3 weeks ago. I immediately cleaned cell culture lab, incubator, cell culture hood and water bath etc. I ordered new medium, FCS and anti yeast antibiotics. I started again and for the past 3 weeks, cells seem fine. But.............yesterday I found contamination again. Why?????? I am very frustrated at the moment. I have done everything I could. But it just didn't work. Could anyone help me?
Many thanks
Sun
Hi Sun and Biocell,
Do you have access to help from a bacteriologist? A colleague doing bacteriology can help you with some supplies: Petri plates, a plate turntable, some antibiotic disks, an incubator for bacterial cultures (keep bacterial cultures out of your eukaryotic incubators).
You might try plating out some media onto a rich bacterial growth agar like TGYE (tryptone glucose yeast extract ) to see if you get colonies -- to speed the process up, plate onto a few different agar media to see what you contaminant bugs like to eat. Just grow them in an incubator for a day or two and see which plates turn turbid.
Then get some antibiotic disks (commercially available disks, about 4 or 5 mm in diameter, impregnated with antibiotics). Inoculate some fresh plates of your contaminant bugs' favorite medium (ideally right from your contaminated cultures), as determined from the previous incubations. Inoculate the fresh plates with your contaminant bacteria so that the bacteria are distributed smoothly across the surface of the plates; smearing the inoculum across each plate with a serilized glass "hockey stick" works well, especially if you have a petri plate turntable; alternatively, your bacteriologist friend may have a different favorite method such as putting the bugs into some liquid medium and layering the liquid atop the agar (a small volume will soak right in). Place the antibiotic disks onto the surface of the freshly-inoculated plate. Incubate for a day or two and see what antibiotic has most effectively inhibited the growth of the bacteria. Try some of this antibiotic in a split of your cell culture to see whether your cultured eukaryotic cells tolerate the antibiotic. Spike the culture with some of your infected cultures and see if the antibiotic knocks back the bacterial growth.
This is a reasonably cheap set of experiments but it is a bunch of labor and money if you have to make all the media and order the supplies yourself (e.g. you don't need a whole package of antibiotic disks, just a few). If there is a microbiology course taught at your institution, most of these supplies will be available for the student labs -- make a deal.
Good luck! If you test the contaminant's antibiotic susceptability first, you will likely be able to knock back the contamination with the least number of chemical insults to your eukaryotic culture.
- Jon
This may sound weird, but is your hood sterile?
We were having the same issue for the past 3 months, and we found out that all of the cells we were carrying had a low grade infection. If course, we never noticed it until transfecting our cells, because macrophages, well, like to eat. The problem was traced to our hood; the membrane encasing the blower had a hole burnt in it.
Try leaving a sterile petri dish of antibiotic-free media open on the work area of the hood for about 15-20 minutes. After a night in the warm room, you should (hopefully) be able to see if your hood is or isn't the guilty party.
Good luck on this. Losing cells (and therefore data) is painful.
Hi
This may sound different but are you working alone or some new person is coming along with you. I use to have the contamination problem when i take a new person to work along with me in the cell culture. One of the problem is he is to talk to much and check your hepa filter of the Biosaftey cabinets also.
These are very small to here but cause a great load on the work.