Mouth Microbes Science Project

[sunn]

[sunn]

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!

[Sareena Avadhany]

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

[sunn]

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!

[Sareena Avadhany]

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

[sunn]

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!

[Sareena Avadhany]

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

[mollyh13]

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!

[MelissaB]

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!

[Sareena Avadhany]

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

[sunn]

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.

[Sareena Avadhany]

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

[sunn]

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.

[Sareena Avadhany]

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

[sunn]

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!

[Sareena Avadhany]

Hi sunn,

I am glad I could be of help, and am delighted that your project has turned out really really well.

I'm sure your presentation is going to look great; definitely put creative finishes on your board to captivate your judges.

When is the competition day?

Great job!

Have a nice weekend as well.

Sincerely,
Sareena

[sunn]

Hi Sareena,

I have to do my presentation in exactly one month, so I have a little time although it will go by fast. I don't know what to expect as I have never done this before. But it will be fine. I don't know what you mean by "creative finishes," but by the way I feel right now, I don't know if I will have enough time to do extra things for the board this year as I am soooo burnt out (I got sick again) and I have other homework. I am so happy for your help because all the info has gottne me on track, plus I will be way ahead of the game next time, although I do not know what other grades or classes where science experiments are assigned. I am delirious right now so I better go, but have a nice week and thanks again, Sareena. Please excuse any errors.

[Sareena Avadhany]

Hi sunn,

Definitely get better soon. In terms of putting your board together, a month would be plenty of time. All you would need is a typed version of everything you have written in your notebook. This is when notebooks come real handy; instead of rewriting all of the parts of the scientific method again, just use most of it from your notebook.

What I mean by creative finishes is basically make your board look attractive. For example, a friend of mine in ninth grade, who did very well in science fairs, drew small caricatures related to his project, decorated his board with them, added color and showed many colorful diagrams and drawings of his work. This all adds to impress the judges; presentation is a very important part of scientific work, and showing effort in making your project creative is impressive. However, if you feel this is not possible due to your circumstances, I wouldn't worry about it. The main stuff (research, quesiton, hypothesis, etc.) is what is important; decorations on your board are definitely not as important.

I'm very happy to hear that you will be participating in another science competition next year; I am sure it will be a very successful project. I believe next year you are qualified to apply to Discover Channel Youth Scientist Challenge. If your regional science fair is associated with the Intel International Science and Engineering Fair (ISEF), then you would be able to compete for this award in your regional, and if selected you are able to advance to the next level. I think you can even apply this year. Here is the URL: http://school.discovery.com/sciencefaircentral/dysc/. This is a very prestigious award, and from what my friends who were participants have told me, a great experience!

I hope you feel better sunn.

Hope this helps!

Sincerely,
Sareena