bacteria contamination problem - (Sep/21/2006 )

hello everyone, I just found this site, and it's terrific.

Our lab is suffering a sporadic contamination problem.

It occurs only in amp selective media

It occurs without any discernable pattern

The contaminant has no plasmid

The contaminant smells 'yeasty'-I used to work in a brewery, and it reminds me of that

The contaminant does not seem to be able to grow in carb media, unless it is transferred from a contaminated amp selective media aliquot into the carbenecillin.

any thoughts?


thanks!

My many and varied thoughts on this sporadic problem...

might the plating area be near or under an air vent, 10metres from an open window, near a door. Has everyone been washing their labcoats... because if it is anything like where I work... my street clothes are cleaner. Is there a no" talking while I am plating cells" rule? Splatter from a person talking can and will contaminate the plates.

Might the amp vial be contaminated? Do you filter sterile the Amp stocks into autoclaved containers (just checking). Are all the unused bags of plate closed tightly (I use a clip), or is the half used bag left open?

Are the finished plates kept in their own sealed sleaves? Or left open to the elements. is the cold room/refrigerator clean?

Hmm... that is all I think off at the moment. I am sure somebody can continue.

Thanks for your ideas regarding the problem.

We have filtered the amp salt thorough .2 micron filter. We have also diluted it in ethanol.

We work with a cDNA library, and we are growing up cDNAs and spotting them on Whatman cards.

The problem occurs occasionally when the cDNAs are being amplified prior to spotting on whatman cards, so the thinking is that it is a media problem, possibly occuring in the common cold room where it is stored.

Of course the one time I set up a controlled experiment with freshly autoclaved media, some stored in the cold room, and some stored in our fridge, no contamination occurred for several weeks.


Perhaps I should just set up a permanent experiment. (laughs to self)

Of course I can't let the "contaminant" know what I'm up to!

you may have a contaminant in your comeptent cells...?

We had problems with a water bath -- you might check to see that it has CuSO4 inhibitors.

So the E.coli with the plasmids are picked from 96 well plates, of which we have thousands. They are amplified overnight in the selective media, and never come into contact with our competent cells.