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Name: Anonymous
Status: Educator
Age: 50s
Location: N/A
Country: N/A
Date: N/A 


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?



Replies:
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


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


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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