Need Help Identifying Contamination - (Oct/08/2008 )
Hello,
I think my cells are contaminated, but I'm not sure. I was hoping someone with more experience could help me identify the problem. My cells are a primary culture of gill cells from a fish. I grow these cells in flasks which I coat with 10ug/cm2 collagen I (I cannot get the cells to attach to the flask without collagen). About 1 day post seeding, the cells form a very confluent layer on the bottom of the flasks (see first attached picture).
In the last few weeks, however, all the cells that I've cultured become elongated, detach and die after 2 days post seeding (see second attached picture). (In the past, I've been able to keep cells going for up to 6 weeks). In dying flasks, the media does not become cloudy, but the pH becomes slightly basic.
In addition to dead cells and debris, I've noticed these odd spheres floating in the media. The spheres appear to contain vibrating black dots (see third attached picture, circle). Furthermore, dying cells have dark dots all over them, which do not appear to move (see fourth attached picture, arrow) and I've also observed amoeboid-like microorganisms slowly moving via pseudopodia between my dying cells (see fourth attached picture, circle), though only a few of them.
I culture these cells in L15 media containing 5uL/mL gentamicin sulfate and 20 uL/mL penicillin-streptomycin solution. I have also stained dying flasks with DAPI to quickly check for mycoplasmas, but I didn't see any "extra staining".
The incubator that I am using had been in a mold-infested cold room for about 1 year before it was moved into our lab, where it had been sitting for another year unused before I started using it. My supervisor assured me that it should be fine if I just cleaned the inside with 70%ethanol (which I did), but I'm not convinced. Do you think my contamination may be mold, even though I've never seen fungal hyphae in my flasks? Also, I can't visibly see any mold in incubator.
Do you have any idea what may be killing my cells and do you have any ideas for decontaminating the incubator?
I appreciate any suggestions!
Thank you!
Hello,
It might not necessarily be caused by contamination. To me it doesn't look like bacteria or fungal contamination. Once we had a similar case of mysterious cell death. I even suspected of sabotage. It turned out the the CO2 sensor was broken and CO2 concentration went wild in the incubator. After we had it calibrated everything returned to normal.
Hi pcrman,
Thank you for your reply!
I use Leibovitz's L-15 which does not require a CO2 environment, so I don't use CO2 with the incubator.
From reading previous postings on this site, I get the impression that the best thing to do is start over (i.e. chuck out contaminated cells, decontaminate equipment and use new media and reagents).
Several people have suggested decontaminating incubators using methanol or formaldehyde fumigation. Has anyone ever actually done this themselves? If so, could please let me know how you did it and what precautions I would need to take in order to ensure it is done safely?
Thanks!
Arghh!!
The alarm went off on my incubator and it says the HEPA filter needs to be replaced...sigh...why do these things happens all at once! lol
Sorry, just venting my frustration! 
[quote name='pixienoodles' date='Oct 9 2008, 05:24 PM' post='153219']
Arghh!!
The alarm went off on my incubator and it says the HEPA filter needs to be replaced...sigh...why do these things happens all at once! lol
Sorry, just venting my frustration!
I would not worry too much as Hepa filters do not keep incubators clean. To me your pictures show no contamination, but it is always difficult with images, it is advisable to get an experienced TC person to have a look.
Regards
Rhombus