Science Fair Bacteria Cultures

[Guest denizen]

I'm helping my daughter with her science fair project which is to be a test of the effectiveness of various mouthwashes that are sold in grocery stores.

 

To do this, we want to culture various swabs from the mouth (we've decided to use her brother for the sample... in multiple agar (soy tryptic) petri dishes.

 

After the culture is established, a drop of each mouth wash will be applied to the culture and labeled.

 

The effects of each mouthwash will be documented with slides made from samples at each point in time including a description of the initial effect of the mouthwash on the culture along with a slide preparation using a gram stain kit followed by observation to determine recovery time for each culture.

 

My question is what are we missing and what could we be doing that we are not in order to improve our technique and our results...

 

Thank you in advance for any help.

[Glider]

You might add a control sample (e.g. exactly the same as the others, but using a drop of distilled water instead of maouthwash).

[Guest denizen]

Thank you...of course I should have thought of that but it didn't occur to me..

[Cookie]

One thing you could do, which actually is more like what is the usual accepted protocol for antimicrobial testing, is this:

 

Establish the cultures - make sure there's an even lawn of growth over the plate (usually it's better to do an agar overlay, if you are able, but if all you have are prepared solid agar plates, then just make sure you plate your swabs evenly).

 

Soak small (~8mm diameter) filter paper disks with each of the mouth washes. Let them dry.

 

Place each of the disks on the culture plate, leaving enough room between disks so that the zones of inhibition do not overlap (i.e. no more than 4-6 disks/plate, evenly spaced).

 

Add one small drop of water to the top of each disk (this will help the mouthwash diffuse from the disk and into the agar, where it can act on your bacteria).

 

Incubate the plates for a period of time, and then measure the zones of clearing around each of the disks.

 

For more info about this, check out the Kirby Bauer antimicrobial screening technique. It's the method of choice in antimicrobial testing labs.

 

Also, be sure to include enough replicates (i.e. do everything in duplicate at least) as that will make your results more convincing to the judges.

 

As for Gram staining - feel free; if you are able to take pictures of the slides, it's a great visual for a science fair presentation. However, it is probably not the best way to be quantitative in an experiment like this, because counting under the microscope using a normal microscope slide isn't very accurate. You need something called a hemocytometer, and they can be pretty expensive if you're only going to use it once. With the Kirby Bauer method, the zones of inhibition around the disk provide a direct quantitative measure of antimicrobial activity.

 

If you divide the zone of inhibition of each mouth wash by the zone of inhibition produced by a disk soaked just in water (your control), then you can calculate a relative index of antimicrobial activities, which allows you to easily rank the results.

 

Anyway - just a thought. I do a lot of this sort of thing, so if you have any more questions, please fire away.

 

Cookie

[Guest denizen]

This was the sort of response I was hoping for...is there a place where I can find the filter paper disks you mention?

 

Perhaps I can make them out of a coffee paper filter or something similar...anyway, i'll figure something out! Thank you very much for your response!

[Glider]

Good protocol Cookie. Would you use the same method if you wanted to test the degree to which mouthwash inhibited bacterial growth, rather than killed existing bacteria? I.e. many mouth washes claim to inhibit plaque-causing bacteria for 12 hours, so if you wanted to test that, would you still use an established culture?

[Cookie]

With regard to filter paper disks - you can use ordinary filter paper (which you should be able to buy at any science supply place - wherever you got the agar plates should have filter paper). Just use a hole punch to make the disks. Coffee filters might work but they're a bit too thin. Ideally you want something that's pretty absorbant and will hold the liquid in one place so it diffuses out at a controlled rate. Maybe some heavy duty paper towel disks?

 

About Glider's question - yes, you'd expect that the bacteria would begin to grow back in toward the disk after the inhibitory effect has worn off. I think the main antibacterial additive to mouth wash is the alcohol, and bacteria will readily metabolize it, so it will gradually decrease below its inhibitory concentration over time. You could use an established culture and kill two birds with one stone - first, you'd see the bacteriocidal effect near the disk, then later you'd see the culture start growing back in from the edge of the inhibition zone.

 

Alternatively, you could apply the mouth wash to the plate directly (better done if you're pouring your own plates or can do an agar overlay) and then plate the cultures on top and see if it stops them from establishing themselves. That one's a little harder to be quantitative with though.

 

Cookie

[DeoxyriboNucleicAcid]

Hmmm, I did some lab work at Stonybrook in a similar experiment. Maybe you could use antibiotics instead of mouthwash. Perhaps, if you can get your hands on it, you could also use genetically altered e-coli bacteria (that wont hurt you) and test the medicine in that. Then try exchanging the antibiotics, so for example, one is resistant to A, and not resistant to B. After exposed to B, this is resistant to A for remaining bacteria is resistant to the stronger (or some other trait) B. This may sound like a bunch of mindless babble to you. it partially is because im rushing, but anyhow, good luck, keep us posted on results .

[Glider]

that makes sense. Thanks.