Bacteria Science Fair Questions - A few basic and complex questions about bacteria (Nov/04/2005 )

You might consider buying prepared, pre-sterilized agar plates, like these. Nutrient agar is a suitable replacement for L-agar for E. coli.

Or you could go with something like this, and get the sterilized swabs as well.

If you get the pre-poured plates, I'm sure you can buy sterile swabs at a drugstore (probably in the first aid section).

How do you plan to spot your test chemicals on the plate? You might also grab a bottle of 70% ethanol or isopropanol (I don't know if they sell ethanol in drugstores) and a glass eyedropper. You can use the ethanol to sterilize any instruments you use (like the eyedropper), either by soaking it in the alcohol, or by dipping it in and then passing it through a flame (under supervision, of course).

So, I'm not sure how you're going to proceed.. Here's how I guess I'd do it -- get the MicroLIVE cultures, follow the directions to get them growing, incubate them overnight.

Mark the bottom of your plates (not the top, but the outside bottom of the plate on the side that actually has the media in it) with small letters or numbers using a Sharpie (drug store again) indicating where you are going to drop your chemicals (the letters and/or numbers will allow you to keep a key of what went where; i.e. plate 1, drop A = Listerine mouth wash (full strength), plate 1, drop B = Dow bathroom cleaner (full strength), etc.).

Then, using a sterile Q-tip, paint a solid lawn of bacteria on your plates. Next, spot the plates with your test chemicals (perhaps at various dilutions) and the key you created above, dropping the appropriate substance right on the number or letter you wrote on the plate (you'll be able to see it through the agar) using your sterilized eyedropper.

Incubate the plates covered and upside down overnight so the lawns can grow up. The next day, measure the zone of clearing around each spot.

Controls -- one plate on which nothing (or perhaps sterile water; again the drug store) is spotted but bacteria are spread, and another plate that has no bacteria on it, but is incubated overnight (sterility check for the plates -- nothing should grow). You might have a third plate with no bacteria on it, but just a drop of each of your test chemicals, and incubate that, too -- this will show there are no bacteria in your test chemicals.

Is this about what you were thinking?

BTW, I'm just rattling this in of the top of my head, so if anybody sees I've left something out, or can see a better way to proceed, feel free to jump in...

Oh, and sorry, also - do I need to stain my bacteria if I'm not going to look at them under a microscope? I probably will, but does it take a lot of skill and work, or do you just drop some Methylane Blue into the bacteria culture. When it comes to staining bacteria, I'm lost. Please give me some simple tips and instructions to help me stain my bacteria cultures (15 of them, and if I even need to stain them). Thank you! crossbow1313@hotmail.com

If you start with a swab dipped in an overnight liquid culture, your lawn of bacteria will defintely be grown by the next day, and the zones of clearing will thus be visible ('cause there won't be any lawn there).

To swab the plates, I would dip the Q-tip in the culture and draw a squggily line down the center of the plate, twirling the Q-tip as you go. Then, starting at the top of the plate, go back and forth horizontally from the top of the plate all the way to the bottom. Turn the plate 45 degrees, and do it again (full horizontial back-and-forth strokes the entire width of the plate from top to bottom). Keep turning the plate about 45 degrees and re-painting it until you've turned the plate all the way around, 45 degrees at a time. This will insure you have an even lawn of bacteria.

The other (easier) way to do it is with an alcohol-sterilized glass spreader. You put an amount (0.1 ml maybe?) of the overnight liquid culture on the plate and spread it with the glass spreader until it is evenly distributed. I don't know if you have a spreader, though.

I was thinking you'd be able to spot multiple spots per plate (sort of like they've done here, but you'd be using drops), but I don't know how big your zones will be (because I don't know what chemicals you'll be trying, or how sensitive E. coli is to them, or how far the will diffuse through the agar). You don't want the zones to converge, obviously.

Unfortunately, this is a quick experiment -- it'll not take too much time. You could spice it up a bit by trying the same chemicals against two different organisms, perhaps a Gram negative vs a Gram positive (E. coli versus Bacillus subtilis or Bacillus thuringiensis, or E. coli versus Staphylococcus epidermidis). You could also generate more data if you test your chemicals at vaious dilutions -- then you could come up with a MIC (minimum inhibitory concentration) like they do at hospitals when testing for antibiotic sensitivity.

You could also try to distigush between chemicals that are bacteriostatic versus those that are bacteriocidal. If you carefully use the sterile non-cotton-tipped end of a Q-tip to sample the zone of clearing, and then streak that sample to another plate and incubate that plate overnight, bacteria will grow if the chemical producing the cleared zone was bacteriostatic, but not if it was bacteriocidal.

As for staining, I'm not sure what information you want to get from staining the cells and looking at them under the microscope, but if you do want to do that, a Gram stain would be best.

Whew! I'm pretty tapped out! Maybe some other forumers have some ideas?

Just to add another tip to the already much detailed answer from Homebrew.

To sterilize media or Q-Tips a regular pressure cooker works fine. You just put all your liquids in bottles loosely closed (do not close them tighly otherwise the steam can build pressure in an make the bottle explode) and your Q tips in boxes that are not airtight and wrapped in aluminium foil. Put some water (not a lot, 2 or 3 cm depth is probably enough) at the bottom of the pressure cooker and heat. Once it starts whistling (the security valve is letting some air go out) keep it running for 15 to 20 minutes then turn the heat off and let pressure and temperature go down slowly. Do not remove the security valve if there are glass bottle, they will explode (it is not that important if you only have Q-Tip boxes ).

Metal and glassware can also be sterilized by dry heat (in your oven) if you keep them over 350ºC for at least 8 hours.

Patrick

Thanks! Some great ideas! Yes I think I will compare how the disinfects/antibacterials affect E. coli to Bacillus subtilis, gram negative vs. gram postive, it sounds like a great idea, and would take more time...... however I barely know anything about gram negative and postive and what it means... so if anyone could fill me in that'd be great!

Look here for an explanation of Gram negative, and at the links within that page for Gram positive, how to do the staining, etc.

Note that "Gram" should always be capitalized, as it's the name of the inventor of the staining procedure, Hans Christian Gram (1853-1938).

Expanding the experiment this way is a good choice -- you're likely to find some chemicals that are effective against one but not the other, because of differences in the cell walls of these bacteria. This will also allow you to use a staining procedure, and the microscope...

Good luck! Let us know how it goes!

So basically, the main difference between Gram negative and positive is in the cell walls, I basically get it.
But still there is this question I have, which is .... will one tube of the MicroLIVE bacteria cultures be enough for 15 full lawns of bacteria(E. coli)?