Contamination in 96 well clonal selection plates - (May/06/2015 )

I have recently performed serial dilution of several different cell lines in 96-well plates to isolate single clones. After several weeks of growth, I started to notice bacterial contamination in one well, then a few more wells, of different plates, plated on different days. I'm trying to figure out how individual wells could have become contaminated after plating (I assume it was after because nothing showed up for weeks). More of the contaminated wells seemed to be on or near the perimeter of the plates. Is it possible for a contaminant to spread using the moisture which often forms inside the plate lid as a conduit?

The cells are growing in penn/strep as well as two selection antibiotics.

Short answer: yes, contamination can easily spread from well to well via the lid and aerosols generated when changing media.

Long answer: You probably have a low level contamination of your medium stock (or perhaps something like PBS) at such a level that you only have enough colony forming units to propagate effectively in some wells. However, as you are using pen/strep, which suppresses growth of bacteria (not fully killing them) it may be that your level of contamination is actually much higher than you are seeing now. Try making your medium without pen/strep and see if that shows up more.

In general contamination indicates that you should throw away the medium and other reagents you are currently using for cell culture... precious ones can be filter sterilized if needed. Any contaminated plates should also be thrown out, but if you are very very careful and confident of your culture skills, you may be able to passage some wells into a fresh plate - keep these on a separate plate from any uncontaminated passaged ones, and observe very closely for contamination.

We're having something similar... In our case, after some constant rains, a wall got humid: we found one of the culprits, a mite, inside a well. They're probably the ones carrying fungi and bacteria around.