Science Buddies: "Ask an Expert"

I am conducting an experiment on what products will decrease mouth microbes. I will be testing gum, candy, toothpaste, and mouthwash. Studies show cinnamon is the most effective, but I will also be testing a few other flavors and possibly a few other brands. If anyone can help me with my science project, that would be great! Thank you!

Mollyh13,

What is it about your experiment that you need help with? How will you be measuring how well these products decrease mouth microbes? Will you be making repeated measurements on the same person, or on different people?

I am not sure how I should measure how well these products will decrease mouth microbes, which is what I need help with. I was going to make repeated measurements on the same person, but I am not sure if testing it on different people will make my project more scientifically accurate. Thank you for your time!

If you do decide to take repeated measurements on the same person, you probably want to do them on different days. If you can't do that, you want to randomize the order in which the four (or more) substances are presented. The reason for this is that if some of the products work too well, you may have trouble measuring anything after that.

As for measuring microbes, you could do a couple of things--take swabs and culture them on some medium like agar, which you'd need to do under the supervision of your teacher and which may involve special permissions because you may end up culturing some harmful bacteria. The other way I thought of is those little red pills that they use to show you how well you're actually brushing at the dentist's or in school--the ones that react with plaque and turns any areas where there's plaque red. You could have the person take one of these, score the amount of red in their mouth (as long as you think you can do this objectively--another good way would be to take digital photos of their mouths and compare the amount of red that way), then have them use the treatment (gum/candy/toothpaste/mouthwash), then have them eat another of these tablets and compare before and after photos.

If you did the latter, you'd definitely want to do the tests on different days if you used the same people. Also, I'm not sure where you can get those pills--I suggest you call up a dentist and ask if they have them or know where you can get them.

Thanks a lot, MelissaB! Those are really good ideas that I hadn't thought of. If anyone else has suggestions on anything pertaining to my project, please let me know!

Hi Molly,

I am doing a project similar to yours, except I am testing to see whether an herb inhibits the growth of e.coli cultures. Similar to what Melissa said, I am taking a strain of E.Coli and spreading across a sterilized petri dish with a nutrient medium - mine is LB Broth. I am then soaking filter paper with the herb and doing so in different concentrations. Testing different concentrations is very important because although I know that the herb I am using has anti-bacterial property, I don't know how to quantify my information. Quantitative analysis is very important in this kind of experiment. I am then placing the filter paper on the petri dish with the lawn application of E.Coli, and incubating it at 37 degrees celsius (Bacteria grows best at body temperature). After 24 hours, if there is no growth near my herb, called the zone of inhibition, then I know that at that particular concentration, the herb shows anti-bacterial activity. And then with the information you have of the different concentrations, you can do all sorts of comparative analyis.

If you are to follow with this method, you would also need positive and negative controls. This is very important. Here are some websites on pos/neg controls, plating, and zone of inhibition.

pos/neg control
http://www.ourstolenfuture.org/NewScien ... ntrols.htm
http://en.wikipedia.org/wiki/Experiment
zone of inhibition
http://www.newton.dep.anl.gov/askasci/m ... e00531.htm
plating
http://coep.pharmacy.arizona.edu/curric ... lating.pdf

there are of course different ways to plate bacteria, but the way I am doing it is a lawn application. Google these key words, and you'll get many hits.

I hope this helps,
Sareena

Thanks, Sareena! When I start my experimentation, I will keep quantitative analysis in mind. I will try using different concentrations as well. Thanks for the links, too. If anyone else has any suggestions, please let me know!

Hi Sareena,

Where did you find your e-coli? Or did you make it from raw chicken juice? Also, how did you pick out the herb that you did? I am testing essential oils against each other (should I just do one against another instead of 4?) and I'm having a hard time deciding as there are several that (claim to be) antibacterial in nature. Thanks.

Hi sunn,

Everything was acquired from my teacher - stock culture of the E.Coli strain I'm working with, agar, petri dishes, so on. If you want to test your project on E.Coli, and would like to conduct your experiment in your school lab, make sure the strain you are using is non-pathogenic. The strain I am using is E.Coli HB 101, which is frequently used in school labs. Talk to your mentor about acquiring a stock culture, and he or she will definitely know how to acquire it.

I wouldn't recommend making it from raw chicken juice; knowing the strain you are working with is very important for doing a science fair project.

As for the herb, I am using Turmeric powder, which is an herb used extensively in Indian medicine and cooking. I became interested in Indian medicine (called Ayurveda) after talking with my parents about the medicinal values this practice offers.

Choosing what essential oil to use can be tough. What I would suggest is research about each essential oil, and draw out the pros and cons of using each one in your experiment. Discuss with your mentor your notes about each oil, and base your decision off of that. When drawing out pros and cons, write down why using that particular oil would be important - applicability, or easy to work with, or very difficult to work with. Write down biological hazards as well. Make sure you keep this in your notebook! It could be valuable information later, if you wish to pursue this project further. I don't know exactly what you mean by 'testing essential oils "against each other,"' but if you would like to do that, what is the importance of doing it? Why is it important to compare the effects of different oils on E.Coli? Remember, judges always want to know the 'why' behind the 'what.'

I hope this helps.

Sincerely,
Sareena

Hi Sareena,

Thanks for your help. This is my sixth grade/ first science project so I'm not quite sure how it all works just yet. You brought up a good point of the "why" aspect. I will keep that in mind. My mom does not like any chemical disinfectants in the house as she gets dizzy and sick feeling and her eyes swell up, but she does have tea tree oil and thyme and puts them in spray bottles. I'm just wondering if they work to rid of bacteria (which essential oil works the best in the house when applied to hard surfaces. It might depend on the bacteria I imagine. Thanks again for your time in trying to help me. I'm thankful for that. Have fun with your science project. I'll try something like that when I get older and more experienced with this type of thing.

Hi sunn,

I think your project is a great idea! I really like how you are taking something that is used frequently in your home and incorporating it into your project. Science projects are really fun to do; I hope you enjoy yours.

If you need help figuring out the next steps of your project, just post your questions on this thread, and either me or another expert will help you.

Sincerely,
Sareena

I am just starting my research for my project. Does anyone have any good suggestions or good research websites? Thanks!

I'm having trouble finding good research sites/books/etc. for my project. I need help finding information on mouth microbes. Please help asap! Thanks, again!

Hi Sareena,
For plating bacteria, I used 8% house hold bleach on a disk for the positive control. then I put nothing on a blotter paper for the negative control. (Or should I have not put any blotter paper on it at all for the standard/negative control?) Anyway, the essential oils disks on the other plates have less bacteria on them than the positive control? Does that mean that my experiement failed (or can the vapors of the essential oils have something to do with it)? My bacteria has only been growing for five days and although the bacteria is less on my experiemental group, there's still not much bacteria overall. what would have been a better way to make sure the bacteria was all over my dishes so I could measure for the zone of inhibition? Thanks. this my first science project for sixth grade and I am a little confused.

Hi sunn,

I'm glad to see you've gotten really far with your project. Your negative control is right; you should see if the blotting paper is what is causing the killing. If your negative control has a zone, then something is wrong with your experiment.

As for looking at the zones for your substance, if your substance has less bacteria than your pos. control, it could be because its toxicity is stronger. Were your concentrations for all the disks in your experiment the same? Also, how did you go about measuring the amount of bacteria on the disks? Was it just an observation? These are all questions you should answer in your conclusion part. You should definitely measure the zone though; incorporate qualitative (the observations of bacteria on your disks) and quantitative (zone measures) in your experiment.

Plating bacteria can be difficult. Did you do a lawn application of the bacteria you are working with? Well, first, how was your setup? Was it: one petri dish, divided into quadrants, where one was labeled with a plus for the pos control, and the other a negative for the neg. control and there was either one or two quadrants left for your substance? Or did you use separate petri dishes for your experimental, positive, and negative controls? If it was the first choice, and your bacteria didn't spread across all the quadrants, your experiment might be faulty. You could either redo your experiment, or discuss your problems in your conclusion. This definitely depends on how much time you have. If your bacteria spread all across the dish, then a repeat is not necessary. This also applies if you did individual petri dishes for your controls and experimentals. If you could give me some more information on how you set up your experiment, that would be great.

Happy Holidays!

Sincerely,
Sareena

Hi Sareena, Thank you for taking the time to respond to my questions! All my essential oils used were full strength, from the bottle. I put three drops (bottles have measured droppers, about .15ml for each disk) and put them as best as I could in the middle of separate petri dishes that had been swabbed with bacteria in three directions. Therefore, I used a total of 18 petri dishes> three dishes for each oil, three for the positive control, and three for the negative control. For the positive control, I didn't use full strength though (I used chlorox bleach) as I used the concentration 8%, that was recommended for household cleaning. The biggest problem I need to mention was the bacteria collection. I wanted to test the oils as a surface disinfectant, but I wanted the bacteria to be as controlled as possible. Being in sixth grade, I didn't think I'd need to know what kind of bacteria I was growing. I researched and found that chicken can contain three or four common types of bacteria. I ended up using canned natural chicken broth, spread evenly over 18 separate tiles that had been cleaned 48 hours beforehand (with bleach)<<<<is that where my problem of not enough bacteria came in as there may have been bleach residue? I wanted a clean slate to start with, along with some regular household "air" bacteria. Anyway, I let the broth sit there for over 8 hours. Maybe I should have waited longer. I was thinking that I should have ordered some of that E.Coli k-12, but I didn't know how to deal with the tube and any possible diluting or methods. As for measuring, I have been counting the bacteria colonies. There are not enough to measure zone of inhibition. ,,,What should I do here?My colony counts range from 0 to 14, so as you can, I don't really have a clear zone. Looking back, I probably should have used the bleach full strength because that is the positive control and those three dishes have different yucky shapes in them. My essential oil dishes are mostly small white spheres or larger cream spheres. One of my bleach disks has a dish with bacteria that look like mold, along with long jagged looking stuff. How close in colony count should the dishes be in order to know the experiment is accurate? My oil trials are pretty consistent with each other. It's my controls that are so much different. I dropped one control dish and it has more bacteria than the other two dishes. So there you have it. I used separate petri plates and my oils are more consistent as far as colony counts than my controls. (Am I in trouble here?) Maybe I need to wait a few days to really find out, but that will be Christmas! Thanks for reading and thanks again in advance. I hope that one day I can be as helpful as you, to someone who is just starting out like me. Science is my favorite subject. It rocks!

Hi sunn,

So just to summarize:
1. essential oils full strength, three drops in the middle of separate peri dishes.
-You didn't mention if you used disks or not. In order for your experiment to work, disks would be necessary.

2. 18 petri dishes were used, 3 different essential oils, 3 positive controls, and three negative controls. 2 trials.
-It's good that you chose to do two trials instead of just one; that way you have more data to analyze.

3. You spread chicken broth over disinfected tiles and let it sit 8 hours. You chose chicken broth because you researched and found out that chicken broth has 3 to 4 different types of bacteria.
-This could possibly be your problem; I'll discuss this later in this post.

4. You have been counting bacterial colonies, but there are not enough to measure a zone of inhibition.
-What was your streaking method? Did you spread that bacteria over the entire plate? Or did you streak over one quadrant, and then dilute your streaking? If this sounds confusing, here's a link with images to clarify my question:

http://www.bmb.leeds.ac.uk/mbiology/ug/ ... leming.jpg
-this picture shows the bacteria spread across the entire petri dish, and you can see a zone of inhibition - a bacterial lawn.

http://www.microbiologyonline.org.uk/gr ... onella.gif
-this picture shows the bacteria streak across the plate. If you used this method, or something like this method, it is likely that your results will not be reliable.

In an experiment, you should definitely try to maintain consistency in everything except what you are experimenting. You used different concentrations of bleach and substance, which might be a problem when you are trying to analyze your data. For example, when you are comparing the effects of an essential oil to bleach, how do you know if it is the bleach that was more powerful, or that it was the concentration of the bleach that inhibited the growth of culture there? When you control concentrations, it allows you to do comparative analysis. If you would like to do this experiment again, maybe you could try different concentrations of essential oil - 20%, 50%, 75%; in your conclusion, you could state at which concentration was the zone size the greatest.

Using chicken broth could go both ways - positive or negative. On the positive side, you used a product bought by millions of families. When you tested essential oils on chicken broth, your results could be very informative. On the other hand, isolating one bacteria could help you analyze your data better. You might not find a zone of inhibition because you are looking at 4 different species of bacteria. What if one bacteria could not grow in the presence of an oil, but another kind could? It would be too difficult to analyze because there would be no way to completely distiguish four types of bacteria on your plate. There definitely could have been residue on the tiles, or you didn't leave it long enough.

Counting colonies could be one way, but the most effective is the zone of inhibition. To measure the zone, you would just need a ruler, or a more specific instrument, like a caliper, to obtain accurate results.

I hope all of this helps. The experimental part of your project is important, but what is the most important part is how you analyze your data, what you could have done better, what were your strengths, and what were your weaknesses. Incorporate all that you mentioned in your post, and consider the suggestions I gave you. This all goes in your discussion area. If you would like, repeat your experiment - since you have learned so much from this time, your next experiment will yield better results that you could thoroughly analyze.

I'm very impressed that you have looked at so many aspects of your project. When you are in 9th grade - not too far away - I would really encourage you to become a student volunteer. Science Buddies needs passionate students like you to help other students with their science projects.

As for now, enjoy the holidays! You've worked really hard, and you deserve a break.

Sincerely,
Sareena

Thanks, Sareena

One more thing before I let you go enjoy the holidays. I used paper disks and did 3 trials for each. I streaked the entire plates in three different directions, but I can see that my zig zags were about 3mm apart, so it may not have been close enough. I understand what you're trying to say about keeping the concentrations of the oils and bleach the same. I also understand what you were trying to say about the bacteria. Thanks again. I will do what you say and take a break (I've been home sick all week, but I've been trying to get this in gear). More importantly, I will let you take a break after all my questions so no need to respond. Have an awesome Christmas and Happy New Year with lots of fun!. AND THANKS AGAIN!

Hi sunn,

From what you have described about your plating techniques, it looks like you followed the streaking method, where your inoculation was diluted. If you would like to do a lawn application, or discuss this in your conclusion, use a pipette, where you transfer your chicken broth onto the agar plate - and then apply 2 drops to the plate, and then using the inoculation loop spread it entirely over the plate, making sure the loop reaches the ends of your plate, and rotate the plate clockwise while still inoculating the plate.

I hope you feel better, sunn. Thank you for the holiday wishes, and have a great Christmas and New Year as well!

Sincerely,
Sareena

Hi Sareena,
Hope you are having a fun holiday season. I had some extra dishes and experimented with them, putting the chicken broth directly in the plate, like you suggested. However, no matter what I do, the chicken broth does not seem to grow much bacteria. (This broth contains tumeric!) Therefore, I think that I will try to order some E.coli k-12 and repeat the experiment if I have time. If I want to make a lawn application, can I just spread in one direction? (I'm only planning to order two tubes. This project is going to get more expensive for my parents than I thought.) Plus, I need to get more agar and dishes. Last time, the agar had globs. I was afraid to get the agar too hot and have it almost boil. So I had to start over with new plates and agar. (Could that ruinthe agar if it gets too hot?) Back to the application, what is the best way to make it even? Do I just take the swab and dip it in the E.coli tube and swab the plate? (do I need to use a pipette?) Is there any effect if the agar plate is not turned over with the agar on top? I ask this becasue I am putting my disk on there with the liquid and I'm thinking that the liquid will soak in the agar much better if the plate is turned the other way. Now, in the case that I am not able to start over, I was thinking that I could measure the closest visible colony of bacteria from the edge of each disk and take the average. (The average of the most visible colony farthest away from the disk would be the most effective oil.) Looking at the measurements, they are consistent in each group. Would that make any sense or should I call my results inconclusive if I was looking for the zone of inhibition? (Should I change my measurment method in the procedure list?)Thanks again in advance! I know I have a lot of quastion here. sorry. do what you can. I am determined to see some growths of inhibition and I guess it shows. Have a Happy New Year!

Hi sunn,

I am definitely enjoying the holiday season, and I hope you are too!

As for your experiment with chicken broth, this would be a great discussion to explore in your conclusion statement. Talk about what is happening with bacteria growth, why it might be growing wrong - and different solutions to each problem. Remember, judges really like that you are observing and analyzing everything in your experiment. Turmeric could even be the problem - since it is an anti-bacterial, it could be preventing growth on the dishes.

If you would like to redo the experiment with E.Coli, you definitely need to apply a lawn application. The way I have inoculated petri dishes with a bacterial lawn is to take a pipette, dip it into your culture tube, and apply 1-2 drops of it on your dish. Then using a sterile inoculation loop, streak the plate in a zig zag motion, making sure you touch the ends of the plate. Once finished, close the dish cap to prevent contamination, rotate it clockwise, and redo the experiment. Keep doing this until you have completed 360 degrees. The transfer pipette allows more bacteria to be inoculated. Using just a sterile loop might not be sufficient.

I don't think you need two tubes of E.Coli; one tube is sufficient. How is your order? Is it already in broth? My teacher has ordered E.Coli cultures - stock cultures. And the stock culture is then placed in LB broth. Once cultured in the broth, the sample was extracted and inoculated on a LB agar dish. If you would like to do an E.Coli test, definitely test it at school or in a lab. If this is not possible, discuss this as an extension project in your conclusion, and maybe you could continue it. I have encountered problems in my project and solutions that I really wanted to utilize but couldn't because of limitations. I was disappointed, but I think it is really important to state in your conclusion your ideas, even though they are not feasible as of now.

As for preparing agar, follow these instructions on sciencebuddies:
https://www.sciencebuddies.org/mentorin ... Agar.shtml
This web page gives a great list of different agars and which one is the best. Using LB agar is suggested. You should microwave the broth until it becomes a liquid - I don't think waiting till it boils is necessary. Wait till the agar hardens, and place the dishes in the fridge upside down. This is a very important step, because it stops condensation from dripping onto the agar and helping the movement of bacteria to different colonies. When incubating dishes, place them upside down as well.

Looking at zones of inhibition can be tricky. So is your measurements based on how far the bacteria is from the substance? The bacteria probably grew in a spot because it was inoculated there, not because of substance. However, if there was no growth near or on top of the substance, definitely talk about that. You could measure the closest colony from the substance. From what I have gathered, it seems the data yielded has resulted in an inconclusive conclusion. Chicken broth has 3-4 different kinds of bacteria, and your bacterial streaking could have been another problem. If you find very small zones of inhibition around your substances, your conclusion could be supportive of your hypothesis.

I hope this helps, sunn. Definitely update me on your project!
Happy New Year
Sincerely,
Sareena

I have just started conducting my experiment. I tested my three brands of gum: Big Red, Winterfresh, and Trident. I had two other people and myself use Agent Cool Blue plaque detecting rinse for 30 seconds. I then took a picture of the inside of our mouths. We each chewed one brand of gum for 20 minutes. We then used the Agent Cool Blue plaque detecting rinse for 30 seconds. I then took another picture of the inside of our mouths to see the difference. I am planning to do the same with my three brands of toothpaste and my three brands of mouthwashes. I need some feedback please! I want to make sure I am doing my experiment accurately. ASAP! Thank you!

Hi,

I do have one suggestion: have all three people in your study use all three brands of gum. With your current set-up, if you see that one brand of gum decreases plaque relative to the other two (for example), you won't know if it's the gum or something about how that person chews gum. It is good that you're looking at it both before and after.

Have you checked to be sure you can tell the different colors on the photographs? This is something you want to check ASAP if you haven't already...many a science project has failed because people took it on faith that the photographs they took would reflect what they actually saw with their own eyes.

Otherwise, it sounds interesting!

Hi molly13,

Your project is very interesting. Like Melissa said, I am glad that you are looking at the before and after.

This is actually a post that has been running on a different project to yours. Next time you would like to ask a question, if you could please create another topic thread, that would be appreciated. Doing so maintains an organized list of topics, so we don't have more than one project on one thread. It also helps Science Buddies Experts to help students.

Good luck on your project!

Sincerely,
Sareena

Hi and Happy New Year Sareena!
I had all these ideas about redoing my experiment, but now I don't have the time to repeat becasue of all my tons of other homework. However, I will keep in mind about what you said and try not to be too disappointed. I may even do this experiment on my own later or even this summer as I am interested in finding some more answers. Anyway, I have one small thing that I am a bit confused about: the responding variable. If I am measuring the bacteria inhibition distances (the closest colony on each plate to the edge of the disk), is my responding variable "the growth/amount of bacteria" or "the bacteria inhibition distance?" Also, should I keep my results simple and to the point on the display board or can I elaborate and discuss (or do I do this in the write-up in the binder)?
Wow, can you believe it? That's about all I have to ask right now! You've helped me so much that I am at a loss for questions! Thank you again for everything! I really am thankful for your help.

Hi and Happy New Year sunn,

I'm glad that you are thinking about continuing your project over the summer.

A responding variable is something that is reacting to something you are changing - which is your manipulative variable. In simple terms, responding variable is your dependent variable and your manipulative variable is your independent variable. So to help you answer your question, fill in the blanks: ______ depends on _________. So you are trying to see whether the essential oils had any effect on the bacterial growth of chicken broth. In this experiment, what is in your control that you can change? And what is not in your control, therefore you are expecting it to respond to your changes? One of your answer choices is correct, but you should definitely think about why that choice is the dependent variable, and fit it in with the (dep)_____ depends on (ind)_____

If this is confusing, I can clarify for you.

When displaying your results, do so with charts, graphs (if possible), pictures and drawings. Remember, your experiment is qualitative and quantitative. So you are not only looking at zone sizes, or in your case number of colonies, but also describing the shape and size of these colonies. Your observations in description format should be included as well. Sometimes pictures do not capture the important parts of your data that you want to analyze, so draw them in great detail with color.

With pictures, graphs and drawings, write a small description on the bottom about what you observed with the graph, picture, or drawing. This would be part of your data results, where you convert all your data into a readable format (pics, graphs, drawings) and write below the figure what you observe in the results. In your discussion, include what went right and wrong, what you could have done better, extension projects and an explanation as to why your results turned out as they did. In your conclusion, relate your findings back to your hypothesis. Did your conclusion support or refute your hypothesis? Or was it inconclusive?

With regards to your data results, I think simple and to the point on the board makes things easy to read and understand; the judges won't be overwhelmed with the writing and can focus on what's important. If you are going to explain your project to your judges, definitely elaborate and discuss more in detail. Your discussion should be in great detail on the board, because this is one of the most important parts of your lab. Deifnitely include everything you do with your project in your lab binder.

I hope this helps sunn!

Keep me posted.

Sincerely,
Sareena

Hi again!
I'm a little confused about the responding variable thing. If a responding variable is something that is reacting to something I am changing, I would say that the growth of bacterial would be the responding variable. so I could think "the growth of bacteria depends on the essential oil used" However, I could also think "the bacteria inhibition distance to the disk depends on the essential oil.":roll: Oh oh I am a little stumped on this one. Thanks in advance for any clarification. Have a nice short week of school. Take care Sareena.

Hi sunn,

Now that I am looking over both possibilities, you are completely right. I apologize for making you decide between your two responding variables. For my experiment, growth of bacteria and size of zone essentially are the same, because I was expecting no growth around the disk, therefore a zone of inhibition. Since my experiment is very similar to yours, I stated the latter - the zone sizes - because that was what I was looking for.

Like you said, either state: The growth of bacteria around the disk depends on the essential oil, or, The size of the zone of inhibition depends on the essential oil.

In your case, think about what you were looking for. Were you going to measure a zone of inhibition, or measure the growth of bacteria around the disk? What were you expecting? This part is a component of your experimental set-up, so I believe you are writing this as if you had not conducted your experiment yet. In that mindframe, what would you write?

When you decide, write an explanation after each to show why each one is your variable. If you chose zones, then state that essential oils inhibit the growth of bacteria, therefore no growth around the disk is to be expected. If you choose growth of bacteria, you could state that essential oils show some anti-bacterial properties due to your research, but you are unsure of whether it stops all species of bacteria. Remember, you did use chicken broth, which contains 4 different kinds of bacteria.

Great that you caught my mistake!

Keep up the wonderful work.

Sincerely,
Sareena

Hi Sareena,

No problem about any mistakes! You have been SO helpful that you can make a few more and be fine by me. All of this makes me use my brain even more. I am deciding to use the zone of inhibition as my responding variable. Now for the tough part: getting my things typed up and put on the board neatly and straight as possible. Thank you once again and have a nice weekend!