Bacteria contamination in yeast two hybrid screening

Schmidt.Thorsten at gmx.de Schmidt.Thorsten at gmx.de
Thu Nov 11 15:10:55 EST 1999


Hi,

I have a serious problem and hope that you could help me:

I am conducting a yeast two hybrid screen using
the GAL 4-system and the Y190 yeast strain).

My problem is that almost all of my plates (80 !) of
my yeast screening seem to be contaminated with bacteria!!!

The plates are now incubating for 6 days and several yeast cultures
(approx. 0.5 mm diameter)have
formed all over the plates, as expected. But all over the plates (in the
area not covered by yeast colonies)a light "smear" is visible.

I streaked over a part of some plates and streaked the cells
on a slide.

Under the microscope many healthy yeast are visible as well
as some yeast with clear bacteria IN it (they are moving in the yeasts
cells) as well as single bacteria
cells and some clusters of bacteria forming around lysed yeast cells.

After that,I picked single yeast colonies, they are looking healthy and
hardly unaffected.

What can I do now?

Do I have to throw all these 80 (!) plates away and start a new
screening?

Or could I still use them?

Will the bacteria seriously affected the results of the screening?

Could I somehow "spray" any antibiotic on the plates?

Could I (to prevent this problem in future screenings)
just add antibiotics (Pen/Strep or something else)
to the yeast SC-media plates? If yes at which concentration?
Will the antibiotic affect the yeast or the results of the screening?

Normally, I would like to incubate the plates for another four days
before
performing the beta-Galactosidase-Assay.

Should I wait these days or should I proceed as fast as possible? I
would
imagine that the E.coli might have overtaken the
complete plates and killed all yeasts if I will wait longer.

Do you have experiences with that?

Thank you so much in advance for your answer!

Everything would help!

                  Thorsten Schmidt



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