96 well plate contamination issue - getting contamination in random wells (Oct/29/2010 )

Hi friends,


I am facing a very strange problem. I am doing 96 well plate assay using multichannel pippette and getting contamination in random wells after incubating overnight or 48h. If this is from contaminated tips, then it should be in one complete row or column since I am using multichannel pippette. It is not from media since all wells are not contaminated. It is not from cells since some blank wells (with media only) have contamination. It is not from formulation since not all the triplicate have contamination. I have started to think it may be the particular packet of 96 well plates. I opened another packet and used but had same problem. Now I think it may the particular lot that may be bad. Has this occurred with anyone?

Please share your views or suggest me what is going wrong.


Thanks a lot for your help.
Mamcy 10

It is very likely that your medium is contaminated at a low level. The low level and as the distribution of bacteria in the medium is likely to be not homogenous, meaning tha some wells get a higher innoculum that shows up after the short incubation.

Maybe the lid was lifted off the plate outside of the sterile hood.

Are you using antibiotics?

We aren't and had a similar thing occur in our lab about 2 months ago. Some possible sources may be if you have new people who joined the lab-- if their techniques are slightly less than sterile- there might be your source; if the environment has changed (that is, your heating has come on, or air conditioning) that might be a source.
If you have people walking in front of the hood, and therefore disturbing the air flow-- that's another source.
There are just an infinite number of possible sources.
I would clean your hood well with some phenol and then let it air out over a weekend prior to using it. Then, remake your media (just to be safe). Next, take a good long look at what you are using: tubes, pipets, tips... anything that is shared amongst other people or common stock that you take and use.