Yeast Busters: Stopping Fungus in its Tracks with Antifungal Medicines Project HELP

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soccersalau
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Yeast Busters: Stopping Fungus in its Tracks with Antifungal Medicines Project HELP

Post by soccersalau »

Hi.


I am doing this project with a bit of change and wondered if I could use oregano oil as an antifungfal. If so what would I do for the concentrations/ how would I do it. My other question is do the azole antifungals perform the best, because according to my research they have the lowest resistance.

Thanks
donnahardy2
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Re: Yeast Busters: Stopping Fungus in its Tracks with Antifungal Medicines Project HELP

Post by donnahardy2 »

Hi,

I think you are doing a variation of this excellent project from this website:

https://www.sciencebuddies.org/science- ... p012.shtml

Your idea of using oregano for the project is an excellent idea. If you search for, “is oregano an antifungal,l?” you will find many links that report oregano oil as being an antifungal (and antibacterial, anti=inflammatory, anti-parasitic, etc.) Please note that none of the sources are from the scientific literature.

You can help with this situation with your science project by doing a carefully controlled experiment and measuring the antifungal properties of oregano. The world obviously needs some scientific investigation on this topic.

It’s hard to tell what concentration of oregano to use. Here is a website that refers to a scientific paper on the subject, however the link to the actual details of the paper does not work. This site does mention that the active ingredients in oregano oil are carvacrol and thymol, so it would be great if you could find these substances available to test separately.
ttps://www.thecandidadiet.com/oregano-oil.

To find out what concentration to use,please do some more searching and see if you can find the reference that was mentioned, or any other references that are published. Also look for any information on the non-scientific sites and look for a recommended concentration. With a science project, you want to read about what others have done before, and then design an experiment based on past research. I looked briefly through several sites and did not find any useful details. It sounds like everyone is using undiluted oregano oil.

If you cannot find the information, then I recommend trying undiluted oregano oil and then do dilutions of 1:2 and 1:4. If you find the 1:4 dilution is effective, then do another experiment with additional dilutions.

What controls are you going to use in your experiment? You will need both a negative and a positive control.
I hope this helps. Please post again if you have any questions.

Donna Hardy
soccersalau
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Re: Yeast Busters: Stopping Fungus in its Tracks with Antifungal Medicines Project HELP

Post by soccersalau »

Thank you very much for the response.

What do you mean by a negative control?
'
For my positive control I will do an environment in which the yeasts are by themselves. I will then measure the carbon dioxide produced from the fermentation that occurs and compare it to when I add an antifungal to look at the effect of the antifungal.

If I were to use oregno oil couldn't i just use the oil without diluting because if I were to apply this to the public, most people only use orgegno oil. I also believe that both carvacrol and thymol do not have antifungal properties, but when they are together the antifungal property comes into play.

Can you also please explain the dilating part because I really do not understand. (especially the mathematical part)

How do I create a hypothesis considering the fact that oregano is not a scientifically tested substance? How do I know the fungal resistance to it? Should I look for the fungal resistance to carvacrol and thymol and base my hypothesis off of this?

Based off of my research, azole antifungals will perform the best due to the fact that they have very low fungal resistance. Is this correct?

Latly, how do I keep the water at a constant temperature when I am creating the dilutions?


Thanks,
soccersalau
donnahardy2
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Re: Yeast Busters: Stopping Fungus in its Tracks with Antifungal Medicines Project HELP

Post by donnahardy2 »

Hi soccersalau,

You have great questions.

A negative control is always included in a well-designed experiment, and for your experiment, would be a sample with no yeast. Including a negative control and doing your experiment in duplicate (or repeating it to find out if results are reproducible) would impress the science fair judges. You would expect to see no response with the negative control; if you did see some activity, you would know something was wrong.

When testing the effect of a substance in a biological system, it is a better practice to test different concentrations to show that there is an effect due to dosage of the product. The Wikipedia article on this subject is pretty good; you can also search for other sites that explain why you should measure dose effect:

https://en.wikipedia.org/wiki/Dose%E2%8 ... lationship

One important principle of a science project is that there should be just one independent variable. If you are testing oregano oil that has a mixture of carvacrol and thymol, there is always more than one independent variable so the experiment is not ideal.

If you can compare carvacrol, thymol, and oregano oil and show that there is an antifungal effect only with the mixture of compounds, your experiment would be amazing. If you have scientific sources that document that the two compounds are not effective separately, you can use this information to explain why you are not including the carvacrol and thymol separately.

Making dilutions: You will need to pick an oil that does not have antifungal activity; perhaps olive oil or canola oil could be used. You need to verify that the oil used for dilution will not affect yeast growth and does not contain carvacrol or thymol. If you use undiluted oregano oil and mix one milliliter of oregano oil and one mL of olive oil; this will make a 1:2 dilution. If you take one mL of the 1:2 dilution and add one mL of olive oil, this will make a 1:4 dilution. Using 3 dilutions is a minimum; using five dilutions would make the experiment outstanding. However, I don't know what dilutions would be best to use, so it might be best to do a preliminary experiment with a couple of dilutions and then repeat the experiment with more dilutions.

Please refer to the serial dilution section of this link for additional explanation:

http://abacus.bates.edu/~ganderso/biolo ... tions.html

You are doing the definitive scientific experiment on this topic, so you need a project question and a hypothesis. The lack of scientific data on this subject makes a scientific experiment with measurable results essential to finding out if oregano oil really does have antifungal properties.

Your question could be something like, “Does oregano oil have antifungal properties?” Your hypothesis could be. “Oregano oil has been used to treat fungal infections in humans. If I add oregano oil to a yeast culture, then I will be able to measure the anti-fungal activity.”

Think about this and put it in your own words. This is really important because it will keep you on track to design your experiment. I recommend reading the science buddies link on writing a hypothesis for more background information to help you with this step:
https://www.sciencebuddies.org/science- ... esis.shtml

I hope this helps. Please keep asking questions until you understand exactly what you need to do.

Donna
soccersalau
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Re: Yeast Busters: Stopping Fungus in its Tracks with Antifungal Medicines Project HELP

Post by soccersalau »

Thank you very much for your response!

The part that I am still not understanding is why I am creating the dilution. Is it so that I have a acurate results and so that I am focusing on a certain percentage of the substance ( I understand the dosage effect)?

When creating the dilutions, I understand that testing the substance at different concentrations is important ,but if the box say 20% concentration, that should mean that there 1/5 of the medication in every __ I can't really grasp this concept. Even though the website gave examples can you please give an example that directly correlates to the experiment that shows all of the math? (especially how the numbers are coming)

I did research on carvacrol and thymol and found that although carvacol and thymol have some antifungal properties apart, they show the strongest fungicidal effect against all of the Candida isolates. Based off of this I have decided to just use the oil and dilute it. The question then arises, how do I make sure that both carvacrol and thymol are in the solution. Also why can't I use water to dilute it
soccersalau
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Re: Yeast Busters: Stopping Fungus in its Tracks with Antifungal Medicines Project HELP

Post by soccersalau »

And as always thank you for taking the time to answer my questions. I really do appreciate it!
donnahardy2
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Re: Yeast Busters: Stopping Fungus in its Tracks with Antifungal Medicines Project HELP

Post by donnahardy2 »

Hi,

You have excellent questions.

In biological systems, different substances can cause a beneficial effect at one concentration and a toxic effect at another concentration. This dual effect is called hormesis. Please read the brief Wikipedia article on this phenomenon; this is an important principle for you to understand.

https://en.wikipedia.org/wiki/Hormesis

Oregano could have a similar effect, but you will not know unless you test different concentrations.
Another more practical reason for you to consider doing dilutions is that at the science fair, if there are two similar projects entered and one project includes dilutions to show the dosage effect and one does not, then the project with the dilutions would be considered the best project. Including dilutions would make the experimental design better.

Here’s an article reporting results of a research study where the scientists did study the effect of dosage effect, but there were some scientists who claimed the experiment was flawed because some important parameters were not included in the study. This topic is unrelated to your project, but it illustrates the importance of designing the best possible experiment.

https://www.scientificamerican.com/arti ... e-effects/

If you cannot obtain carvacrol and thymol separately, make sure that you explain that you decided to use oregano oil because of the reports from the previous studies, and be sure you include the citations in your bibliography. In general, testing a mixture of substances without testing them separately is not considered a good experimental design.

Can you obtain some oregano oil that the manufacturer has tested the concentration of carvacrol and thymol? This would allow you to report the concentration of these substances that you actually tested. If not, then in your conclusion, be sure to mention that you would have used known concentrations of the active components if it had been possible.

Oils have a lower density compared to water and they are immiscible with water (they will not mix together), so you will need to use another oil to dilute the oregano oil. An organic solvent such as methanol or a detergent would allow the oregano oil to mix with water, but these would be toxic to the yeast cells, so not suitable for your experiment. Be sure to include one sample of your diluent in your controls to show that it is not toxic.

http://www.letstalkscience.ca/programs- ... t-mix.html

Thymol and carvacrol are small molecules that contain lots of carbon and hydrogen atoms and they both have a low solubility in water. Molecules with lots of carbons and hydrogens are hydrophobic (water hating). The OH (hydroxyl group present on each molecule allows mixing with water. The bibliography included in the Wikipedia articles for these two compounds will be useful for your background discussion, and including the structure of the molecules on your

https://en.wikipedia.org/wiki/Thymol
ttps://en.wikipedia.org/wiki/Carvacrol

More questions?

Donna
soccersalau
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Re: Yeast Busters: Stopping Fungus in its Tracks with Antifungal Medicines Project HELP

Post by soccersalau »

Hi Ms.Hardy

Thank you for your timely responses. I hope I am not pestering you. I spent the day researching and came to the conclusion that distributors of oregano oil are not 100% sure of the concentrations of thymol or carvarcol, rather the say that they assure a minimum of a percent in their product. I believe that the best course of action would be to order undiluted oil and dilute it.


Please correct me if I am wrong. The goal of dilating in this project is to get the substances at certain concentrations (1%) so that we can have accurate results. It is also so that I know exactly what is inside and how it is affecting the yeasts.

When diluting the oregano oil, what am I trying to get 1% of? Is it the thymol and carvarcol?

I decided to make my question, " what is the effect of antifungals on fungal growth"
I will then discuss how each antifungal affected the yeast growth. I will then say which is the best to use based off of the results.

http://www.healthy-oil-planet.com/buyin ... egano.html
soccersalau
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Re: Yeast Busters: Stopping Fungus in its Tracks with Antifungal Medicines Project HELP

Post by soccersalau »

And as always thank you very much! Your aid is very appreciated!
donnahardy2
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Re: Yeast Busters: Stopping Fungus in its Tracks with Antifungal Medicines Project HELP

Post by donnahardy2 »

Hi occersalau .

You are not pestering me at all; this is my job as a volunteer for Science Buddies. it’s very important to me to make sure you have all of the information that you need to do an excellent science project.

You were wise to investigate the possibility of finding oregano oil with measured concentrations of thymol or carvacrol, but it sounds like manufacturers don’t do the testing.

The best way to handle this situation in a science fair, where you know the judges prefer quantitative measurements is to explain that you tried to find the information, but it was just not available. Then explain what you would do to measure these analytes (if you had more time and resources).

Do a little more background reading when you have time and find out how you would assay the active compounds. Include a statement in your conclusion about what you would do differently if you were to repeat the project in the future. This will inform the judges that you know that quantitative results are important.

The purpose of diluting the oil is to allow you to test a range of concentrations of the active ingredients. In pharmacology applications, compounds can be toxic in one concentration and therapeutic in a different concentration. If you test only undiluted oregano oil, you won’t know if it’s the best concentration for inhibiting yeast growth. Maybe, the 1:2 dilution is the optimum concentration. If you do a series of dilutions, you should see an optimum concentration for yeast inhibition, and decreasing activity as the concentration decreases. You will be testing the dose-response relationship.

Try reading through the following link on dose-response relationships from the Merck Manual; there’s a professional version that shows how to graph results and a consumer version.

https://www.merckmanuals.com/profession ... ationships

If you still don’t understand why you should do dilutions, then just go ahead and do the experiment with dilutions, and I think it will make sense when you see your results.

I like your title, except I think you should change “antifungals” to either “oregano oil” or “thymol and carvacrol.” Your experiment will be measuring the antifungal properties of the oregano oil.

Donna
soccersalau
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Re: Yeast Busters: Stopping Fungus in its Tracks with Antifungal Medicines Project HELP

Post by soccersalau »

Alright thank you very much!

Since my science experiment is not only based on oregano. I have three other different kinds of anitfungals that I will be using. From the research I have done and what is needed to be done, it seems that the oregano oil by itself will take a majority of the time allotted to me for the completion of my experiment. Because I am doing a timed oral presentation, I am slightly worried that in my presentation I will spend to much time talking about just the oregano. If it talk to little, the judges may not fully understand my experiment. If I talk about it too much, I am faced with the dilemma of not being able to discuss some of the other things in my experiment.

I guess my question is: is doing all the research and putting the rest of my project in jeopardy worth it? Could I just add oregano oil as an extension of the project for next year?

Your opinion on this will be greatly appreciated.

Thanks,
soccersalau
donnahardy2
Former Expert
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Re: Yeast Busters: Stopping Fungus in its Tracks with Antifungal Medicines Project HELP

Post by donnahardy2 »

Hi soccersalau,

When you talk to the science fair judges, you will give them a one sentence summary of your project (an overview), and then fill in a few details. You will use your project board to lead them through your experiment so they will understand what you did and you won’t get bogged down with too much information about oregano. When you are getting ready for the fair, we can review what you are going to say to the judges and I’m sure it will be a brilliant presentation.

For now, just focus on getting the quantitative data that the science fair judges will be expecting. You can work on other antifungals this year, and save the oregano oil for next year if you prefer. Your experiment should have one independent variable and a dependent variable that you can measure. You will run samples in duplicate, or do the complete experiment and then repeat it. All other parameters other than the independent variable should be controlled, such as time, temperature, size of sample, and age of the yeast culture.

I would recommend including as many samples as you can manage comfortably. If you are testing a commercial preparation that presumably has an optimized dose, then you can skip the dilutions. Perhaps you could test several products and include one oregano oil sample so you can get some experience for next year’s project.
What other products will you be testing?

Donna
soccersalau
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Re: Yeast Busters: Stopping Fungus in its Tracks with Antifungal Medicines Project HELP

Post by soccersalau »

Thank you Ms.Hardy,

Sorry about the late response. I will be testing tolnafate, terabafine, and clotrimazole (all creams).

Thanks

Soccersalau
donnahardy2
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Re: Yeast Busters: Stopping Fungus in its Tracks with Antifungal Medicines Project HELP

Post by donnahardy2 »

Hi Soccersalau,

I'm sorry for my late response also. Thanks for the list of products you will be testing. I think that the chemistry is different for all of these products, so this should make a a good project and you will definitely have some results that you can graph and analyze. Please let me know if you have any other questions at this point.

Good luck! Let me know what happens.

Donna
soccersalau
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Re: Yeast Busters: Stopping Fungus in its Tracks with Antifungal Medicines Project HELP

Post by soccersalau »

Hi Ms. Hardy!

While researching I noticed that I was not able to read the results accurately. I timed the environments for 40 minutes. After this I would go to my graduated cylinder to measure the amount of Carbon Dioxide produced, but I couldn't see past the water surface. I tried using a flashlight, but I still was unable to see. Also there were times when the whole cylinder was empty except the water surface and about 5 mL below it. Am I doing something wrong?
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