Science Buddies: "Ask an Expert"

I am doing a project in which I am required to measure the efficiency of the bacterial removal that toothpaste and mouthwash have on the mouth after use. The following problem is what ive bumped into. How do you measure efficiency?
I have thought of keeping a record of the amount of bacteria present in a petri dish, and comparing it to the amount of bacteria present after a combination of mouthwash and toothpaste have been mixed, poured, and stirred within the dish.

Which would be the most efficient way to measure the amount of bacteria present before and after in the dish?

Hi Nicoloco, I think you're on the right track! Here are my thoughts. First, you'll need at least 6 petri dishes with agar gel in them. I'm hoping your teacher has provided these.

POSITIVE CONTROL:
You'll need a positive control, meaning 'how many bacteria are there if I do nothing'. You'd get this by swabbing your teeth without brushing or using mouth wash.
What I mean by swabbing: rub your teeth with a q tip. Then rub your swab on agar in a petri dish. Rub it all over the surface, to spread out the bacteria. Do not leave any visible globs of bacteria. You have to have agar, which is bacteria food, or nothing will grow. Immediately close the dish, so no other bacteria get onto your agar. This will give you a baseline to compare the later plates to.

NEGATIVE CONTROL:
You should also set up a negative control dish: one with agar, but where you don't rub any bacteria on it. Open it for maybe 30 seconds, then close it and leave it in the same space as your other plates, and watch to see if anything grows on it.

This will tell you how many colonies you got from the air, and you'll subtract this from the colony counts you get on all other plates.

EXPERIMENTAL PLATES:
Now for your experimental plates:
1) use just toothpaste before swabbing your teeth
2) use just mouthwash pre-swab
3) Use both products, one after the other
4) Use both again, in the opposite order (these last 2 plates will tell you if using both is more effective than using 1 product on its own, and if the order you use them in is important)

Collecting bacteria for each plate needs to be done on the same teeth/areas of your mouth each time, at the same time of day each time - I'd suggest morning before food.
So to keep all of your variables consistent, this will take 5 days to do all 5 plates listed above. (Including the positive control in the first paragraph).

Put the plate in a warm spot , lid down. RECORD the time you start each plate.

DATA COLLECTION:
Bacteria colonies will grow as white or pink dots. Each dot is called a CFU - a colony forming unit. Assuming you're using a 10 cm dish, you ideally want to see between 20 and 200 CFUs when you count. Check your plates every 2 hours. At each time point, record how many CFUs you see. If there aren't at least 20, leave the plate for 2 more hours and count again. Go until you count at least 20 CFUs. If you have time, I would keep taking time points until you have around 200 colonies, or until they start to grow together and are difficult to count. (Which ever comes first.) If every two hours is hard to do, pick a time interval and stick to it.

This will ensure you catch multiple time points for comparison. If a plate doesn't grow 20 colonies after 3 days, you can stop. This goes for the negative control plate as well. (When you need to go to bed, put your plate in the fridge, wrapped in saran wrap. This should stop growth. In the morning take off the wrap, put it back in its warm spot, and keep collecting data.)

HOW TO COUNT BACTERIA:
To check for growth do not open the dish. Look at the bottom of the plate, not through the lid. you should be able to see bacterial colonies through the agar. You may need to hold it up to a bright light to see them. If you want examples of what this looks like, google 'bacteria colonies on agar pictures'.

These guys can be hard to count - so looking through the agar (not through the lid), take a sharpie and make a tiny dot on the plastic exactly where you see each colony. After you are sure they're all marked, count your dots. Record your number of CFUs, and the time point. Now, get some good pictures. Take pics through the agar if you can see the colonies well enough. At your last time point, whenever that is, you can open the dish and take direct pictures of the bacteria. You can do this because these live in your mouth, and aren't going to give you an infection.

NOTES ON CONTROL PLATE:
For your positive control plate, you're going to get a lot of colonies, fast. They may start growing together before you can count them, so maybe check every hour.
If they're still hard to count just take good pictures, and record how long they'd been growing. Do your best to count, and then you can record the CFU number as 'more than __'.

EXPERIMENT EXTENSION:
The plate you're suggesting with toothpaste and mouthwash in one plate would tell you a slightly different thing - not how many are removed, but how many are actually killed. I think that's really interesting data to have, but if you run out of time I'd skip it.

So for these I'd take a swab, get toothpaste on it and smear a very thin layer all over your agar. I'd say to use less than a pea size glob of toothpaste for an entire 10cm dish, or less if your dishes are smaller.You should barely be able to see the toothpaste, and be able to easily see light through it, so you can count CFUs. Then you'll close the dish and let the toothpaste dry for 10 min or so. Then swab the plate with bacteria from your teeth. Do another one where you apply a thin layer of mouthwash first, and another where you mix the two and rub them on the agar.

If you have time, do another experimental plate where you brush your teeth, but don't use toothpaste. This tells you how much removal is happening just because of the physical scraping.


WHAT IF I GET COLORFUL OR FUZZY COLONIES?
These are fungal - go ahead and keep track of them as well as you can

I know this is a lot; let me know what questions you have!

Hello LilGreen!
Thnak you for all the info.
Some things have changed since i posted that question.
My experiment is now: what effect does Cetylpyridinium Chloride have on non-pathogenic e-coli.

Nice, that is a much more defined question. Do you have a hypothesis? Or an experimental design you'd like to discuss? I'm here if you do!