De-feet the Yeast 1

De-feet the Yeast

Tori Robinson

Dayton Regional STEM School

Honors Biology

February 2018

De-feet the Yeast 2

Table of Contents

Abstract 3

Introduction 4

Background 4

Materials & Methods 7

Results 8

Discussion 12

Conclusion 13

Acknowledgments 14

References 15

De-feet the Yeast 3

Abstract

Yeast infections are incredibly difficult to kill. That is an interesting prospect since yeast has such resistance against medicines and it is so hard to find something that can kill it. That’s why my research question is “What is the best known cure that takes the least amount of time, has the capacity to kill the yeast, and is also the safest to use?” My hypothesis is that tea tree oil will kill or remove the yeast by double the size that plain salt water does. For my experiment, first the yeast had to be grown in multiple agar plates. Then, after a few weeks of growing the yeast, there were three discs, which soaked in the selected solutions for this experiment, then they were placed on the yeast in separate agar plates. The removal of yeast was measured by measuring the diameter of the clear area the disc created. To make sure the medicine is killing the yeast, a control plate was put aside for the experiment where there was only the yeast on the agar plate. From all of the averaged of the data, calendula oil works effectively in removing yeast over time by killing about 3.57 cm area. The experiment refuted my hypothesis, but it’s intriguing that tea tree oil wasn’t able to kill the yeast, instead the tea tree oil allowed the yeast to grow back. This means that there are solutions can work briefly, but then allow the yeast to grow back in great amounts.

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De-feet the Yeast

Yeast is a known cooking product for many recipes like cinnamon rolls or bread (Baker’s Yeast,

2005). But that is just one species of yeast (Baker’s Yeast, 2005). Yeast also naturally occurs on our skin. In people who are immunocompromised these yeasts can become opportunistic and turn into an infection (CDC, n.d.-b). Sometimes, when there is a medicine proven to stop it, it can be either too harsh after a while or calm the infection down for a brief amount of time. The worst part about it is in August of 2017 the CDC concluded preliminary investigation on

Candida auris and in a clinical update of September 2017 stated that this yeast infection “is an emerging, multidrug-resistant yeast that causes invasive infections and is transmitted in healthcare settings” (CDC, 2017) (it is the latest hospital-acquired infection that people have to worry about). The death rates for this infection can be as high as 60 percent (Whyte, 2017). This was alarming, and made me very curious about yeast so I decided to see, for this experiment, what can kill the yeast and what is used against it. My research question is “What is the best easily available drug that takes the least amount of time and is the safest to use?” My hypothesis is that tea tree oil will kill or remove the yeast by double the size that plain salt water does.

Background

The experiment is to find the best way to kill yeast on a variety of factors. I tried to learn more about yeast and how yeast reacts to different elements. From this research, yeast is actually not just a simple baking ingredient. It is an organism called Sachhoromyces cerevisiane, and is also classified as an ascomycetes - a group that includes other popular genetic material or other ways known as a fungus (Baker’s Yeast, 2005). There are many different types of yeast (Baker’s Yeast,

2005). The yeast that we do not like is actually the yeasts that carry many diseases or even infectious conditions (Baker’s Yeast, 2005). An example of this is with some yeast infections like De-feet the Yeast 5

Candida albicans (CDC, n.d.-b). This yeast infection is the most common in human (CDC, n.d.- b). Many of the Candida try to at least infect the intestinal tract or even skin (CDC, n.d.-b). Many of Candida try to grow on the skin before trying to infect the body, but it usually depends on how and where it grows on the body that affects the yeast’s growth (CDC, n.d.-b). Yeast infections are fungal infections since yeast is classified as a fungus (Baker’s Yeast, 2005).

Most medicines used against yeast usually stop working or stop killing the yeast in general (Micronutrient, n.d.). The reason is that yeast has a survival response against medicines is to actually shut itself down (Micronutrient, n.d.). The yeast releases proteins in order to go dormant again until future resources are found so then it can continue to grow (Broach, 2012).

The resources that yeast waits for is water, temperature, acids, oxygen, nitrogen, and carbohydrates (Fraser, 2012) . If there is too much of these resources, yeast try to compact in on itself to save energy or try to grow outward to get more of that energy (Broach, 2012).

There are many different types of drugs chosen to test. I decided to try over the counter medications, homeopathic remedies, and old wives tale cures. But before the experiment, I need to know more about them, specifically if they can cause me any harm. From the research that

Terbafine Hcl (Ahtlete’s Foot cream), tea tree oil, and Grapefruit Extract are very harmful to the body, especially the skin. Terbinafine Hcl and tea tree oil are known to be used against Athlete’s

Foot or other fungal infections or yeast infections on the skin (Web M.D., n.d.-e) (Web M.D., n.d.-d). Grapefruit extract though is known to be used for yeast infections, but it is more likely used to work against cancer and also a skin disease called Psoriasis (Web M.D., n.d.-b). Each one has harmful side effects in its own manner. Ahtlete’s Foot cream can have the side effects as follows: vision change, mental changes, unexplained bleeding/bruising, yellowing eyes, liver damage, signs of kidney problems, and abdominal pains (Web M.D., n.d.-e). Grapefruit Extract’s De-feet the Yeast 6 side effects are if taken by mouth can be very deadly (Web M.D., n.d.-b). Also, Grapefruit

Extract is taken a quart a day can be known to increase the chances of breast cancer (Web M.D., n.d.-b). Tea tree oil’s side effects are as follows: Skin irritation of any kind, can very unsafe if taken by mouth, and also rashes (Web M.D., n.d.-d). If taken by mouth, tea tree oil is known to cause confusion, inability to walk, unsteadiness, rashes, and also, in severe cases, comas (Web

M.D., n.d.-d). This drug should be used with caution and in an attentive manner.

From the research, Calendula oil, Oregano oil, and also Clotrimazole (Antifungal Cream) are the least harmful medicines being used. Antifungal cream is known to be used against fungal infections or ringworm (Walgreens, n.d.), but Oregano oil and Calendula oil are not used to go against fungal infections (Web M.D., n.d.-a) (Web M.D., n.d.-c). Oregano Oil is used for bleeding disorders and healing wounds, but it can be used to help against parasites (Web M.D., n.d.-c). Calendula Oil is used for menstrual periods and skin inflammation, but it can be used against some cancers and also ear infections (Web M.D., n.d.-a). Even though these are the least harmful, they can still cause some harm. Antifungal Cream can be known to cause some skin irritation and also is warned to stay away from contact from the eyes (Walgreens, n.d.).

Calendula Oil is known to cause allergic reactions and drowsiness if taken with surgery medication (Web M.D., n.d.-a). The side effects for Oregano oil are lowering blood sugar levels, stomach upsets, can cause allergic reactions, and might cause unintentional bleeding for those with a bleeding disorder (Web M.D., n.d.-c).

For this experiment, there must be some safety precautions put into place. Keep hands away from face or open wounds. If any symptoms are starting to show from one of these medicines, immediately go to a doctor for treatment.

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Materials and Methods

The materials needed for this science fair experiment are 10 agar plates, a beaker, distilled water, yeast, stirring rods, a scale, a petri dish, a pipette that has milliliters measurements on side, petri lids, and sugar to make up the agar plates with yeast on them. After that, you will need tea tree oil, Oregano oil, Isopropyl Alcohol, salt, water, Grapefruit seed extract, Calendula oil, Clotrimazole, Terbinafine Hcl, and some dawn soap. You will also need tweezers, a ruler, and an incubator.

The steps for beginning this process are simple. Before testing the yeast, the yeast has to grow on the agar plates to be tested. To do this, there has to be a yeast formula to put onto the agar plates for growing purposes. Measure out 10 ml of water into the beaker, 2 grams of yeast onto the petri dish’s lid, and then 2 grams of sugar onto the petri dish—use the scale to figure out exact measurements. Once everything is measured out, begin to mix things together. First, mix the sugar and water together with the popsicle stick. After the sugar is dissolved in the water, add the sugar water to the yeast and mix it together. Once everything is mixed together, use a pipette to measure out 1 ml of the mixture. In a zig-zag motion, slowly put the yeast mixture drops onto the agar plate. Do not turn the agar plate upside down after you put the drops on just yet, the mixture will still be a liquid. Repeat using the pipette several times until all of the agar plates have been used. Place into the incubator for 48 hours. Once the water is gone from mixture, put De-feet the Yeast 8 it upside down. Make sure to keep water that condenses onto the lid in the agar plate again to help the yeast grow.

After several weeks in the incubator, the yeast should have grown outward into different colonies on the plate. The next step would be to start killing the yeast. First, put a few drops of each oil/medicine in a plastic cup and put one disc into each solution -- for salt water, you’re going to have to put some water till it fills the bottom and measure out 1 grams of salt. While waiting, label each one as follows: tea tree oil, Oregano, Dawn Soap, Isopropyl Alcohol,

Calendula Oil, Athlete’s Foot Cream/Terbafine HCL, Antifungal Cream/Clotrimazole, Salt

Water, Grapefruit extract, and nothing. After 1 min in the solutions, put the disc with the specific solution on the disc onto a colony from the correct labeled agar plate and put the lid back on it.

Make sure to have one dish with no solution or medicine on it. The rest of the agar plates should have a disc with medicine on it. Repeat disc steps to get discs 2 and 3, but those one separate colonies as the first one and other one. Label each agar plate with that specific medicine. Put those off to the side in a 32 degrees Celsius room. Daily, check on the yeast and measure the diameter of how much yeast is killed. Measure this by turning the agar plate upside down or checking the bottom of the plate to see where there is a clear circle and measure that diameter.

Continue this process for two weeks and compile the data.

Results

The data was on multiple days over two weeks, but there were days where the data could not be taken. That can be seen with where there is no data about that day. There are some samples that are going into the negatives because instead of killing the yeast, the solution helped it to grow a little bit. That can be seen for the Athlete’s Foot cream, Antifungal Cream, Grapefruit Extract, and tea tree oil. The lowest negative reading from the ones above came from Athlete’s Foot De-feet the Yeast 9 cream with a reading of -0.23 cm. This is also is one of the many outliers in the data points. The other outliers in my data points are 1.35 cm, 1.06 cm, and 0.91 cm for Calendula Oil, 1.1 cm and

1.03 cm for tea tree oil, and 1.1 cm for Grapefruit extract. These are all affected the by either changing the data from being very high or changing the data points to read some very low readings. The rest of the data is not far from the standard deviation which is 0.44 cm. There are also some readings like Isopropyl Alcohol, Dawn Soap, and Salt water that had readings of zero meaning that they could not reduce the yeast. The rest of the data can be seen below.

Average Yeast Removed versus Time

Day 1 Day 5 One Week Two Weeks

Athlete’s Foot cream 0.40 cm 0.05 cm -0.07cm -0.23 cm (Terbinafine HCl)

Antifungal cream 0.40 cm 0.45 cm 0.17 cm 0.07 cm (Clotrimazole)

Isopropyl Alcohol 0.00 cm 0.00 cm 0.13 cm 0.03 cm

Grapefruit Extract 0.70 cm 1.10 cm 0.65 cm 0.17 cm

Dawn Soap 0.70 cm 0.45 cm 0.00 cm 0.00 cm

Calendula Oil 0.90 cm 1.35 cm 0.97 cm 1.07 cm De-feet the Yeast 10

Salt Water 0.00 cm 0.00 cm 0.00 cm 0.00 cm

Oregano 0.80 cm 0.80 cm 0.88 cm 0.80 cm

Tea Tree oil 1.03 cm 1.10 cm 0.30 cm -0.03 cm

Table 1. Average amount of diameter of yeast removal.

Average Yeast Removed versus Time

1.6

1.4

1.2

1

0.8

0.6

0.4

0.2 Diamter Diamter Yeast of (cm) Killed 0

-0.2

-0.4 Day 1 Day 5 One Two Time week weeks

Athlete's Foot Cream Antifungal Cream Isopropyl Alcohol Grapefruit Extract

Dawn Soap Calendula Oil Salt Water Oregano

Teatree oil Control (Nothing)

Figure 1. Average yeast reduction over time.

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Figure 2. Set up for starting to grow the yeast.

Figure 3. The yeast grown De-feet the Yeast 12

Discussion

The data did not follow the expected results since there were so many different things wrong in the data. An example can be seen with some of the materials used to kill the yeast. I had expected the medicines used against fungal infections to be able to kill some of the yeast, but for some of the discs, such as tea tree oil, the yeast grew more in. That should not have happened if the medicines are proven to work against the yeast. I have no idea why this happened, but my theory for this is that maybe there was something that might have gone wrong when mixing the medicines with the discs.

That was not the only thing that changed, too. There were also data points that were outliers compared to the rest of the data. An example can be seen with the Grapefruit Extract.

For the first disc, the data read into the negatives. A difference of about 0.4 is understandable, not a difference of 1 centimeter. Another thing that might have changed these results is the fact that I had problems with the tea tree oil in the beginning. At the beginning, I did not know that tea tree oil could melt plastic, so that ended up with changed time because I had to save the agar plates from being melted.

If I could change a few things about this project, I would have liked to prevent the tea tree oil accident from happening. If I could have done that again, I would have put the oil into a glass beaker rather than a plastic cup like I did when I was soaking the other two disks. I would also like to change how I treated the yeast. I would have like to add a little bit of water to them once every couple of days. That way, the agar would not have dried up. I would have also liked to change the amount of time the yeast spent in the incubator. I kept them in there for almost four weeks, but that seemed to have dried up the yeast. I think it would have been a lot better to take De-feet the Yeast 13 them out after around three weeks. The last thing I would have liked to change was the days I got the data. I wish I could have done it every other day unlike every week since there were many snow days for school, meaning I could not get data I needed for my experiment.

Some experiment I would like to suggest after this would be to test out the lifetime of yeast and what environment is the best place for it to grow in. I can possibly try a different yeast besides the cooking yeast. There is the bad type of yeast that I can use more to compare to other disease or just yeast. I can also try more types of medicines to see if the yeasts responds to other things. Another test I can try is do is putting fresh medicine/oil on the yeast colonies to see what happens if they are used repeatedly.

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Conclusions

My hypothesis, “tea tree oil will kill or remove the yeast by double the size that plain salt water does” is proven to be slightly plausible. It turns out that tea tree oil did kill yeast better than salt water, but not by double. In the data I collected, the salt water was incapable of killing any of the yeast. At the beginning of the tea tree oil, it seemed to be killing the yeast by about by about 0.1333 difference between the closest rival calendula oil. At the end though, tea tree oil was no longer killing the yeast and it was allowing the yeast to grow to where the diameter was about 0.3 centimeter. This was a surprising part of my experiment. Tea tree oil is actually recommended and used against many yeast infections like Athlete’s foot and Candidiasis.

The best one to use to kill yeast over time would be the calendula oil. Not only does it remove the yeast, it removed almost seven times more area than the Isopropyl Alcohol. A problem with calendula oil for some people is that there are those out there that are allergic to calendula oil. Hopefully though, I can tell others about this information who are having problems with fungal infections so they can have the best medicine/oil to fight against fungal infections on their skin. De-feet the Yeast 15

Acknowledgment

I would like to say thank you to Ms. Campbell for helping me with my experiment when

I had no idea how to start or run a science fair project, and all her help with my first science fair project. (Also for helping me create the yeast through many difficulties and struggles.)

I would also like to thank some of my friends and family who have participated in science fair before, as they helped me to understand how to do some things for science fair and they critiqued my report.

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References

Baker’s Yeast and Its Life Cycle. (2005 August 19). Retrieved from:

https://www.phys.ksu.edu/gene/a1.html

Broach J. R. (2012). Nutritional control of growth and development in yeast. Retrieved from:

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3430547/

Fraser A. (2012). Describe how to prevent yeast growth in food. Retrieved from:

http://www.foodsafetysite.com/educators/competencies/general/yeast/yst3.html

CDC. (2017). General information about candida auris. Retrieved from

https://www.cdc.gov/fungal/diseases/candidiasis/candida-auris-qanda.html

CDC. (n.d.-a). Candidiasis. Retrieved from

https://www.cdc.gov/fungal/diseases/candidiasis/index.html

CDC. (n.d.-b). Invasive Candidiasis Risk & Prevention. Retrieved from

https://www.cdc.gov/fungal/diseases/candidiasis/invasive/risk-prevention.html

Micronutrient Infusion Services. (n.d.). Why is candida so hard to kill? Retrieved from

http://www.ahctherapies.com/candida-therapy/candida-hard-kill/

Walgreens Clotrimazole Antifungal. (n.d.). Retrieved from

https://www.drugs.com/otc/325650/walgreens-clotrimazole-antifungal.html

WebMD. (n.d.-a). Calendula. Retrieved from https://www.webmd.com/vitamins-

supplements/ingredientmono-235-

CALENDULA.aspx?activeIngredientId=235&activeIngredientName=CALENDULA

WebMD. (n.d.-b). Grapefruit. Retrieved from https://www.webmd.com/vitamins-

supplements/ingredientmono-946-

GRAPEFRUIT.aspx?activeIngredientId=946&activeIngredientName=GRAPEFRUIT De-feet the Yeast 17

WebMD. (n.d.-c). Oregano. Retrieved from https://www.webmd.com/vitamins-

supplements/ingredientmono-644-

OREGANO.aspx?activeIngredientId=644&activeIngredientName=OREGANO

WebMD. (n.d.-d). Tea tree oil. Retrieved from https://www.webmd.com/vitamins-

supplements/ingredientmono-113-

tea%20tree%20oil.aspx?activeingredientid=113&activeingredientname=tea%20tree%20o

il

WebMD. (n.d.-e). Terbinafine HCl. Retrieved from https://www.webmd.com/drugs/2/drug-

6188-8262/terbinafine-hcl-oral/terbinafine-oral/details

Whyte C. (2017). Deadly, drug-resistant Candida yeast infection spreads in the US. Retrieved

from https://www.newscientist.com/article/2125283-deadly-drug-resistant-candida-yeast-

infection-spreads-in-the-us/