Pseudomonas contamination

HARDIES at THORIN.UTHSCSA.EDU HARDIES at THORIN.UTHSCSA.EDU
Tue Dec 27 11:04:42 EST 1994


Kit Tam wrote:

> Hi! Can anyone give me a suggestion on how to avoid the
> contamination of my YT plates (50ug/ml Ampicillin added)
> after I plated the transformants onto them.  I did not have
> this problem before but it occurs quite often recently even
> though I steriled everything.  I wonder if there is a vector
> which carrys an antibiotic gene that can kill or suppress the
> growth of Pseudomonas ( I suspect this is the contaminant ).
> Thank you.

Hi Kit,

It's imperative that you eliminate the contamination through sterile
technique.  Other methods of suppressing it leave you terribly
vulnerable to some awful experimental artifacts.  Here are some
tips that I hope will help:

1) With all transformations do a mock transformed negative control
(including everything but the DNA).  As long as this problem
continues, also just incubate an untreated plate.  This will tell you
if the plates are contaminated to start with, or if it's in your
reagents or the DNA itself.  The DNA can be cleaned by phenol
extraction/ etoh precipitation; the reagents by filtration.

2) If the plates come up contaminated without treatment, then your
autoclave is not doing the job.  You should make some amp- plates and
incubate them to check this out.  A typical problem with the autoclave
is that people just don't autoclave long enough.  The more fluid you
put in it, the longer you have to autoclave.  Also check that it's
really reaching the prescribed temperature and holding it throughout
the run.

3) If you're using liquid media, say for an outgrowth, then never
store it at 4C to suppress it growing up.  If it grows up at room
temp. that tells you it's not sterile and you have to improve your
technique.  After you use a bottle, set the rest on the shelf to see
if it grows up.  If so, you have to do a better job of flaming
utensils, etc.  Aliquot the media into several small bottles and don't
use a bottle for more than one experiment.  Learn to get the cap off,
the pipet in and out of the bottle, and the cap back on without
setting the cap on the table.  If you do touch the cap to the table,
flame it before you replace it, and don't leave it off any longer than
necessary.  

4) Never extend the shank of a Pipetman into a sterile tube; you can't
possibly keep it sterile.  Either use small tubes so only the sterile
tip goes in, or use a 0.1 ml flamed glass pipet.  Autoclave the pipet
tips yourself, and keep the lid of the box closed as much as is
practical.

5) If your plates are picking up contamination during manipulations
after the original experiment, then the best defense it to keep the
cover on them as much as possible.  Learn to tilt up the cover and
slide the loop in underneath to pick colonies.  Pseudomonas usually
comes from waterbaths.  Look around for a grungy water bath that for
some reason is generating an aerosole and clean it out.  Pick a site
for your experiment that is away from obvious aerosole generators,
like centrifuges, shaking water baths, sparging units, air
conditioning vents, etc.  Set a drug free plate out open in the work
space for a short time and then incubate it to see how much airborne
contamination you're picking up in the area.  Pay particular attention
to where ever you're drying your plates, since they are partially open
for a long time.  Dry them upside down with the bottom part propped up
on the edge of the lid.  Beware of incubators with a circulating fan
and a tray of grungy water in the bottom.  You don't want water in the
incubator if you're trying to dry plates anyway.

6)  It's considered good technique to preclean the area with 70% EtoH
and glove up.  Frankly, these procedures only keep you from
contaminating your lunch with your experiment, not the other way
around, since the desk and the gloves don't stay sterile; so if you
touch a sterile surface with one of them, it's contaminated anyway. 
If you do touch a sterile implement to a nonsterile surface, just
throw it out and get a new one; don't risk the experiment.

I hope something in here helps.
Steve Hardies, Dept. of Biochem., Univ. of Texas HSC at San Antonio
Hardies at thorin.uthscsa.edu




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