Ask A Scientist© Molecular Biology Archive

status      educator
age         50s

Question -  Is there a way to make bacterial culture media using
sterile plastic petri dishes? I can only afford those in my school, and
would like to smear samples from various surfaces onto the media, to
determine bacterial contamination - e.g. before using soap and after.
I've tried, but I keep getting contamination AND lots of condensation. I
don't have an autoclave. Any ideas?
You can get te same effect as an autoclave by using a pressure cooker. You
can get one ceaply at any hardware store or KMart, Target, etc. I have an
autoclave andI often only autoclave one bottle anyway! Read the directions
carefully, bu basically its a pot with a special sealable lid. It has a
weight that sits over a valve and this is the way you alter the pressure.
You put your media bottle in the pot with the specified amount of water and
seal the lid (according to the directions!) and you heat it on the stove.
Boiling only kills a certain amount of bacteria. Those that form spores need
to be killed under pressure. The bacteria you are seeing on your plates are
probably those that are spore formers. As far as condensation goes, that is
normal. You can cut down on this by storing your plates upside down in a
refrigerator and taking them out of the fridge a few hours before use to
allow the condensation to dissipate before you use them. Good luck.


Van Hoeck
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Home brewers use this trick to sterilize glassware to avoid bacterial
contamination in their beer:

Wash all your glassware as thoroughly as possible. I'd stick to bottles or
flasks (as opposed to something like a saucepan) so you minimize the
opportunity for airborne contamination. Cover all openings (for instance
the top of a bottle) with aluminum foil to avoid letting dust and other
stuff from the air float in. If you use a container at least three times
the volume of media you want to make, you should be able to put in the
components and the water (maybe a bit extra water so you have room to boil
it down to the volume you want). If you boil it hard (covered with foil) on
the stove for 15 minutes or so, you should probably kill anything that you
don't want living in there.

To reduce condensation on the plates once they're poured, leave them on the
benchtop at room temperature overnight, covers on, before storing in the
fridge. Storing and growing them inverted also helps any condensation to
fall on the lid instead of on the media.

Christine Ticknor
Ph.D. Candidate
Yale University
New Haven, Connecticut
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Since you say you have tried, but you get
contamination and condensation, I guess I don't have
to explain which media to use and how to prepare it.
The problem of contamination is (partly) because you
don't have an autoclave. Boiling is not sufficient to
kill off spores. A pressure cooker would do the job
and these are not too expensive. Prepare your medium
with the agar according to instructions. Cook it at
the highest pressure possible for 20 min. Use a glass
bottle that is not completely closed, the air should
be able to escape. After cooling down to 80 or 90 C,
shake the bottle well to completely dissolve the agar.
Be careful with this, the liquid can start boiling
again. Then let it cool to 60 or 50 C. That is one
trick to prevent condensation: don't poor the plates
too hot. After pooring each plate, close the lid
immediately and let it solidify completely. Then you
can turn the plates upside down, and open them less
than half, using their lid as support, to let them dry
in this tilted position. It would go best in an airing
cupboard, but a clean bench would also be ok. Let them
dry for 30 min. Then you can seal them in plastic
bags, store them in a fridge and use within 7 days.
Always keep the plates upside down untill you use
them. This should be sufficient to get sterile, dry
plates. Before use, acclimatize the plates. When you
see condense water, repeat the drying as above.
Inoculated plates are cultured upside down, to prevent
any condensation water from smearing the growth.

Trudy Wassenaar
===========================================================
Yes....it is quite routine to do so...you need to plate sterile hot (still
liquid medium into the dishes in a sterile or asceptic hood. The procedure
is in most micro lab texts

PF
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