Experiments Using Non-Sterile Plates

Often scientists and kids figure out ways to do experiments with non-sterile plates. There are lots of things you can do with non-sterile plastic (or glass) petri plates or plastic 98 hole trays. See below for a way we reused plastic trays at Cal Tech.

If you can work with non-sterile equipment, you may save lots of time. Working with non-sterile equipment helps you do hundreds or thousands of experiments in one day. High Throughput Screening is the big news in pharamacetial screening today. Can you use HTS in your science project? You probably can if you are clever enough to figure out ways to do things.

Warning: Do not use these methods for pathogenic microbes. There is much you can do with yeast and other food organisms. There is no reason for amateurs to work with pathogens!

Reusing Plastic Petri Plates.

The standard dish is 100 x 15 mm. Most of them are used once and discarded. I save mine and try to figure ways to reuse them. Also in my plant micropropagation lab I use a special dish which is 100 mm dia and 50 mm tall and very expensive. Plastic dishes are made of polystyrene because it is crystal clear, but it crumples when autoclaved. The factory uses Cobalt-60 gamma radiation to sterilize them after they are packed in tough sealed platic bags.

I sterilize them in 10% Chlorox (undiluted it is 5.25 % sodium hypochlorite). Make this by adding one volume of Chlorox to 9 parts tap water. This 10% Chlorox is used in homes, farms, and labs to sterilize many things, but it will corrode iron and many other metals.

If leave the bleach on the plates, your experiments may be killed unless there is enough dilution. Therefore I dip them in sterile autoclaved water or rubbing alcohol which evaporates faster. However for most uses the wet Petri plates are fine.

In my micropropagation lab, I fill the wet plates with sterile agar which was autoclaved in bottles or jars and plant my strawberry plants. Growth takes weeks, but very few dishes become contaminated. How long must you soak them to kill all the bacteria? Killing is a logarithmic function. You will never kill every organism in a million plates. I suggest you do a test. I let them soak 20 miuntes and I have no problems. Therefore less soaking would sterilize most plates. I handle them with sterile forceps. When not using the forceps, I stand them in 70% iso-propanol--bleach will corrode metals. You could use 70% iso-propanol to sterilze your plates, but bleach is cheaper. Of course I wash the plastic items before sterilizing them with 10% Chlorox. .

The above method is for reusing dishes for semi aseptic work. I do not use this method when working with my important pure cultures because a occasional plate may not be sterilized. The amount of contamination will depend upon your skill. First plan your work and then work your plan. To find problems ahead of time, do a practice run first using tapwater to be sure I have all my tools ready.

Is it possible to sterilize petri plates by steaming them. You could place the dishes upside down in a steamer and allow steam to pass thru them for one hour. I have not tried this. I do not not know whether the plates would melt. If not, this method should be useful for some work. Do use the method for pure culture or aseptic work.

Some petri dishes are made of polycarbonate and can be autoclaved like glass dishes. I have never seen any of these dishes.

Using Bacterial Lawns in Reused Dishes (Phage Work)

This is very clever, it does not require sterilized Petri plates. I use this method in high volumes in my high school classroom. We begin with washed and dry plastic petri dishes. Drying kills most bacteria but the dry dishes are not sterile. Near the end of the hour, kids pour bottom agar into the dishes for the next class. They leave the lids off so drying and faster gelling occurs. By the time the second class is ready for them, the bottom agar (need not be sterile) is solid. A kid then adds 3 drops of overnight bacteria to 3 mL of top agar at 45C mixes and pours onto the plate of solid bottom agar. That makes a layer of top agar about 1 mm thick (thickness is not important). We let it cool 5 minutes or so until the top layer has gelled. Now if we incubate the plate for 18 hours or more, we get a solid lawn of bacteria. Can you think of any clever ways to use such plates which are going to develop lawns of bacteria unless you interfere? Notice that we did not have to use sterile anything because 3 drops of overnight bacteria is 4,000 million bacteria and a few bacteria from the dust of the air will have no effect. (We don't even cover the plates; leaving them open helps them dry). In fact, a few million stray bacteria would have no effect. To test whether I am correct, you might try adding a speck of soil or sour milk and see if you can see any effect (but milk will be an awful lot of bacteria--should have an effect).

Bottom agar is any kind of agar that is suitable for the bacterium you are using. Top agar is bottom agar with a little extra water if you are going to work with phages. 0.7 % agar is ideal for top agar in most instances. Undiluted bottom agar (10 grams agar per liter) is fine in many instances.

What can you do with these freshly poured plates? You could punch holes with a soda straw and put various dilutions of bleach or other disinfectants in separate holes. Then incubate overnight. The plate which contains a bit of soil dispersed in the top agar, may contain 1 to 3 mm clear spots the next day where phages (bacterial viruses) created a furious epidemic destroying every bacterial cell that was not resistant.

My favorite method is to use a paper cutter to make paper strips 5 mm wide and dip these in "stuff" and streak these on the plate. You can easily test a dozen or two dozen "stuffs" on the plate. Be sure you use a paper strip for only one kind of "stuff". Good "stuffs" to test are diluted bleach, disinfectants, antibiotics, and any other kinds of bacteria you have. You could test cosmetics, weed killers, anything. You expect bleach to kill the lawn, therefore try various dilutions 1:1 serial dilutions are a good idea. A very good "stuff" is the liquid medium in which a mold or streptomycete was growing. If the mold make an antibiotic active against your bacterium, you will find clear areas around the antibiotic because the antibiotic inhibited grow of the bacterium. The most famous strain of Penicellium crysogenum was isolated from a rotten orange. Or just cut a piece of agar out of the molds culture tube and Splat it onto the lawn.

Use of Non-sterile Trays in Phage Research at CalTech

At California Institute of Technology we had some large plastic trays about 8 x 10 inches which had 8 rows of 12 depressions. Each of these depressions was about 2 cm diameter and 7 mm deep. We reused these trays may years. We cut ridged flexible sheeting into 8 x 10 pieces and laid it on top of the tray for a cover. That allowed us to stack the trays about 10 trays high. Some days I filled 30 trays. That was about 3,000 experiments in one day!! You can accomplish a lot when you do that many assays in one day! I encourage you to try similar BIG SCIENCE. Most kids do one or two dishes. Just think how impressed the judge will be if you did lots of work. Go to our Phage Site for more about such experiments.

Revision #3 1998 Jan 23
Written by Harold Eddleman, Ph. D., President, Indiana Biolab, 14045 Huff St., Palmyra IN 47164
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