Make samples from sera on Agar works to detect the source of contamination in mo - (Sep/29/2016 )

Hi everyone,

I have a problem with weird contamination in my fibroblast culture for almost 2 months. I am doing cell culture almost 10 years so I recognize alliens in culture, current one is bacteria. However, this is a type I have never seen. If a culture contaminated, all flasks you feed at the same time contaminated. But mine is new fashion. Let me say what I mean. I did primary culture from mice, I frozen cells in clean condition at passage 1. Then I started to do some experiments and thawed a vial to a 96 well plate and a 6 well plate. Result: 6 well plate is contaminated, 96 well plate very nice. Then I thawed new one to 2 x T75 flasks. Result: one is contaminated. Then I thawed another one to 2 x T75 flasks and one 96 well plate. Result: 96 well plate is very fine, T75 flasks are contaminated. I used two different brands of flasks for subculture, both contaminated. I had another brand for the primary culture which was clean. All contaminations occur after 48 hours not 24 hours (I am used to see the day after contamination). OK my questions here if anyone can help: 

1. Is that possible to have contaminated plastic ware (flasks) batch from different country's companies at the same time? (If anyone matematician or statistician can be more sure of the answer)

2. I put samples of DMEM, FBS, trypsin, pen-strep etc into incubator to incubate them for 3 days to see any turbiditiy. But I also think that take some amount of these samples spread onto agar plate and see bacteria culture is there?

Thanks for your answers in advance. Please HELP :(

P.S. I work very careful and clean. I wipe all things out with EtOH, sprayed  hood and incubator, clean and changing water in bath etc. 

SELCEN 

It sounds like you have a problem with the cells themselves - probably a low level contamination of the cells, such that when you split the cells, some contaminated ones enter only some of the vessels, while others remain fine. This would be unusual, but is not unheard of. It is especially the case when you use antibiotics in the cell culture medium, as these can mask low-level contaminations, and usually don't actually kill bacteria, just suppress growth (medical professionals have a saying 20%  of the cure is the antibiotics, the rest is the immune system). It might be also that your antibiotics are getting old and are not as effective as they once were.

It is very unlikely that the flasks are contaminated, this is extremely rare as these are mass produced on sterile lines (including gamma irradiation after production) with extensive quality control.

Taking some of your samples and spreading on a plate is a good measure to take. It would also pay to check who has been doing cell culture around you recently - perhaps you have a new person who's sterile technique isn't as good as it could be.

Dear Bob,

Thanks for the reply. My primary culture took 7 days in incubator with media changing each two days. It was very good culture just before freezing as main stock. I could not attach microscopy images here 

But may be I contaminated them while freezing process ! :( 

Anyways. If I find the exact answer, let you all know :)