Yeast Busters: Stopping Fungus in its Tracks with Antifungal Medicines
AbstractHave you ever wondered how antibiotics and other medicines are able to stop dangerous infections? How do such medications kill microorganisms without in general harming the person the microorganisms are infecting? Because many different types of microorganisms can infect us, we have had to develop an amazing number of ways to deal with these harmful microbes. Fungal infections can be particularly dangerous, but we have developed many different antifungal medications that can usually deal with these infections. But how do antifungal medications work, and how effective are they? In this science project, you will test how well different common antifungal medications can stop the growth of baker's yeast, a harmless variety of fungus often used in baking.
Determine how different antifungal medications slow or stop the growth of fungus.
Teisha Rowland, PhD, Science Buddies
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Last edit date: 2018-04-26
Left untreated, fungal infections can lead to serious medical conditions. Consequently, it is important to know what kind of antifungal medicine, and how much, to take to kill a certain type of fungus. Some of the most common fungal infections are athlete's foot, nail infections, and yeast infections. The fungus group includes molds, yeast, mushrooms, and more. Fungi actually make up a kingdom of organisms separate from plants, animals, and bacteria.
Different antifungal agents work in different ways to kill fungus. Two of the most common antifungal agents used in nonprescription antifungal medicines are azoles and allylamines. Azole and allylamine both work by disrupting the fungus' ability to make ergosterol, which is a chemical compound important for the fungus to make a strong cell membrane. Without a strong cell membrane, the fungal cells could become leaky and die. Ergosterol is not in plant or animal cells, which makes it a good compound to target if you only want to kill the fungus without hurting infected plants or animals (including people).
Azoles and allylamines disrupt the fungus' ability to make ergosterol in different ways, as shown in the molecular pathway diagram in Figure 1. A molecular pathway is a series of chemical reactions that take place within a cell. In this diagram, arrows pointing down indicate a chemical reaction. This means that squalene is normally converted to lanosterol during a chemical reaction aided by the enzyme squalene epoxidase. An enzyme is something that helps a chemical reaction take place. The lanosterol is then converted to ergosterol with the help of the enzyme lanosterol 14α-demethylase. Ergosterol is used by the fungal cells to create a functional cell membrane. The sideways "T"s in the diagram show how a chemical reaction can be stopped. Allylamines stop the function of the squalene epoxidase enzyme, thus blocking the fungus from making lanosterol. Azoles stop the function of the enzyme lanosterol 14α-demethylase, thus blocking the fungus from making ergosterol.
Figure 1. Two groups of common antifungal agents, azoles and allylamines, work in different ways to disrupt the fungus' ability to make ergosterol. Fungus needs ergosterol to make strong cell membranes.
A common nonprescription azole found in antifungal medications is called clotrimazole, while a common nonprescription allylamine is called terbinafine. Tolnaftate (also sold as Tinactin) is another common nonprescription antifungal agent, and it is thought to inhibit squalene epoxidase, like the allylamines. Nonprescription medicines, also called "over-the-counter" medicines, do not require a doctor's prescription to buy, so that anyone can go into a pharmacy and buy them, while a doctor's prescription is needed to buy prescription medicines.
Undecylenic acid, another common nonprescription antifungal agent, can be used to fight fungal infections as well as infections caused by other microorganisms. Undecylenic acid is derived from the castor bean, which contains toxic compounds. Scientists do not completely understand how undecylenic acid fights fungal infections, but they think that it interferes with the fungus' life cycle. Specifically, it may stop the fungus from reproducing (making more fungus).
Different types of fungus react to antifungal agents in different ways, and fungus can even become resistant to certain antifungals agents. For example, if a fungus starts making a large amount of the enzyme lanosterol 14α-demethylase, it may become resistant to azole because no matter how much of the azole is used, the fungus could still have enough lanosterol 14α-demethylase to be able to turn lanosterol to ergosterol.
In this science project, you will investigate how efficient different antifungal agents are at stopping fungus from growing. Baker's yeast is a type of fungus used in baking and can easily be grown at home. As yeasts grow and reproduce, they make carbon dioxide, which is what makes bread rise. Which antifungal agents are most efficient at stopping yeast from growing? What does this say about the relative importance of the mechanisms that these antifungal agents disrupt in the yeast?
Terms and Concepts
- Fungal infections
- Molecular pathway
- Undecylenic acid
- How is ergosterol related to both allylamine and azole antifungal agents?
- How do allylamine and azole antifungal agents work differently to stop fungal infections?
- What is the name of a common azole found in antifungal medicines? What about a common allylamine?
- How do scientists think undecylenic acid stops fungal infections?
- How might you be able to measure whether yeast is growing? Hint: Think about what yeast produces that makes bread rise.
- Which type of antifungal agents (allylamines, azoles, or undecylenic acid) do you think will be most effective at stopping yeast from growing? Why, based on their different molecular mechanisms, do you think this is so?
These resources are a good place to start gathering information about fungal infections and antifungal medications:
- U.S. National Library of Medicine, National Institutes of Health: Bookshelf. (1996). Chapter 76 antifungal agents. Retrieved September 28, 2011, from http://www.ncbi.nlm.nih.gov/books/NBK8263/
- Drugs.com. (n.d.). Azole antifungals. Retrieved September 28, 2011, from http://www.drugs.com/drug-class/azole-antifungals.html
- Thorne Research, Inc. (2002, February). Undecylenic acid. Retrieved October 29, 2013, from http://www.altmedrev.com/publications/7/1/68.pdf
The range of antifungal agent concentrations suggested for this science project are based on these scientific studies/reports:
- Sud, I., and D. S, Feingold. (1981, July). Heterogeneity of action mechanisms among antimycotic imidazoles. Antimicrobial Agents and Chemotherapy. Published online. Retrieved October 4, 2011, from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC181635/pdf/aac00007-0077.pdf
- Tiballi, R. N. et al. (1995, December). Saccharomyces cerevisiae infections and antifungal susceptibility studies by colorimetric and broth macrodilution methods. Diagnostic Microbiology and Infectious Disease. Published online. Retrieved October 4, 2011, from http://www.sciencedirect.com/science/article/pii/0732889395001883
- University of Adelaide: Mycology Online. (2011). Antifungal susceptibility testing. Retrieved October 4, 2011, from https://mycology.adelaide.edu.au/laboratory/
For tips on growing microorganisms such as yeast, see this Science Buddies resource:
- Science Buddies. (1999). Why won't my cultures grow? Retrieved September 28, 2011, from http://www.sciencebuddies.org/science-fair-projects/project_ideas/microbio_cultureproblems.pdf
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Materials and Equipment
- Graduated cylinder (at least 100 mL). Alternatively an empty 500 mL plastic bottle may be used. Tip: Use 3 if you are doing the three separate trials at the same time.
- Plastic tubing, approximately three times as long as the height of the plastic tub or bucket used. (For a 10 inch tall bucket, you will need 30 inches of tubing.)
- Thermometer to measure water temperature
- Dry yeast. Alternatively this can be found in the baking aisle of the grocery store. Tip: Buying a whole jar is probably more economical than individual packets.
- Disposable gloves. Can be purchased at a local drug store or pharmacy. If you are allergic to latex, use vinyl or polyethylene gloves.
- Plastic tub or bucket
- Optional: Plastic wrap
- Packing tape
- Empty 500 mL plastic bottles, such as bottled water containers (7) Tip: Use 21 if you are doing your three separate trials all at the same time.
- A cap that fits all bottles
- Drill or a nail and a hammer
- Elmer's® super fast epoxy cement or a silicone sealant that works with plastics
- Permanent marker
- Measuring teaspoon (that can measure 1 tsp., ½ tsp., and 1/8 tsp.)
- Measuring cup (that can measure 1 cup and ¼ cup)
- Warm water, typically 110°F-115°F, but consult the recommendations on your yeast package.
- Different antifungal medicines (3), each using a different type of antifungal agent. If it is not labeled on the front of the packaging, look under the "active ingredient" section on the back of the package to see what antifungal agent is being used in the medicine. Antifungal agents commonly found in nonprescription antifungal medicines are clotrimazole, terbinafine, tolnaftate, and undecylenic acid.
- Optional: Toothpicks
- Optional: Fork
- Lab notebook
- Graph paper
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For health and safety reasons, science fairs regulate what kinds of biological materials can be used in science fair projects. You should check with your science fair's Scientific Review Committee before starting this experiment to make sure your science fair project complies with all local rules. Many science fairs follow Intel® International Science and Engineering Fair (ISEF) regulations. For more information, visit these Science Buddies pages: Projects Involving Potentially Hazardous Biological Agents and Scientific Review Committee. You can also visit the webpage ISEF Rules & Guidelines directly.
Setting up the Gas Collection Apparatus
In the first part of this procedure, you will be setting up the gas collection apparatus. In the process of growing, yeast produce carbon dioxide (CO2), and if the number of living yeast decreases, the amount of CO2 produced will also decrease. Consequently, measuring CO2 production is a good way to determine how much yeast is alive relative to the other conditions you test. The gas collection apparatus that you set up here will show you how much CO2 is being produced by the yeast.
- Read about the antifungal agents you picked to test in your experiment and find out how they stop fungal infections.
- Collect CO2 from the yeast by displacing water trapped in an inverted graduated cylinder, as shown in Figure 2. Here is how to set it up:
- Fill the plastic tub (or bucket) about a third with water.
- Fill the graduated cylinder with water.
- If the tub is big enough, fill the graduated cylinder by tipping it on its side inside the tub. Allow any bubbles to escape by tilting the cylinder up slightly, while keeping it under water. Keeping the opening of the cylinder under water, turn it upside down and attach it to the side of the tub with packing tape.
- If the tub is not big enough, fill the graduated cylinder completely and cover the top tightly with plastic wrap. Quickly invert the cylinder and place the opening in the tub beneath the surface of the water. Remove the plastic wrap, being careful not to let any water out of the cylinder. Attach the cylinder to the side of the tub with packing tape. There should be no air inside the graduated cylinder.
- The graduated cylinder should now be upside down, full of water, and have its opening under the surface of the water in the tub. It is ready to trap CO2 produced by the yeast.
Figure 2. To create your gas collection apparatus, first fill a bucket or tub about one-third full with water. Then fill a graduated cylinder completely full with water and invert it in the bucket, making sure that there is no air inside the graduated cylinder, and then tape it to the bucket's side. Next, feed the empty end of the plastic tubing from the yeast bottle (on the right) into the opening at the bottom of the graduated cylinder.
- Next, bring the CO2 from the yeast to your gas collection apparatus, as shown in Figure 2.
- Make a hole in the bottle cap, just big enough to insert the plastic tubing. Use a drill or a nail and a hammer. Get help from an adult if needed.
- Insert a piece of plastic tubing through the hole in the cap so that it sticks out about 2 centimeters (cm) on the inside of the cap, as shown in Figure 3. This will be the tube for collecting CO2. It should remain above the surface of the yeast solution in the bottle.
- Seal the tube to the cap with epoxy or silicone sealant so that it is airtight. Allow the epoxy or silicone to cure completely before conducting your experiment.
- After the epoxy or silicone has completely cured, which should take approximately one day at most, check to make sure the tube and cap are sealed together with an airtight seal or you may have a leak that could affect your results. If there is a leak, apply more epoxy or silicone sealant and let it completely cure again.
- After the epoxy or silicone has completely cured, attach the cap with the tubing to one of your empty 500 mL bottles.
- Put the other end of the tubing inside the inverted graduated cylinder, as shown in Figure 2. Any CO2 produced by the yeast will bubble up inside the cylinder, where it will be trapped and displace the water in the cylinder.
- Try to use as short a piece of tubing as you can to connect the yeast container to the inverted cylinder so that a lot of CO2 does not get trapped in the tubing.
- There should be water in the tubing as soon as it is submerged under the water. The CO2 gas will push some water out of the tubing before the graduated cylinder starts to fill with CO2 gas.
- You will be measuring how much CO2 is produced by seeing how much water is displaced from the graduated cylinder.
- You can test your gas collection apparatus by removing the cap from the bottle and blowing gently into the tube. The bubbles you create should be captured inside the cylinder. (You will need to refill the cylinder completely with water before starting your experiment.)
- When your gas collection apparatus is ready, you can start the actual experiment.
Figure 3. To bring the CO2 from the yeast bottle to your gas collection apparatus, make a hole in the bottle cap and insert the tubing about 2 cm through the top of the cap and into the side that will be inside the bottle. Seal the tubing to the cap airtight with epoxy or silicone sealant and allow it to cure.
Testing Different Antifungal Medicines
In the second part of this procedure, you will be testing how effective different antifungal agents are on yeast. After following the steps in the "Setting up Your Gas Collection Apparatus" section, you will be ready to test different antifungal agents. First you will grow yeast and collect the CO2 produced without adding any antifungal agents. This will be your control. Then you will test two different concentrations of each antifungal agent.
- Using the permanent marker, label each of the bottles with the name of the antifungal agent you will be testing, along with the concentration that you will test. Label an extra bottle "control."
- Dissolve ½ teaspoon (tsp.) of sugar in ¼ cup of warm water (110°F to 115°F, or whatever temperature the yeast package recommends). When the sugar is fully dissolved, add 1 tsp. of yeast (this is about the same amount as half a packet of yeast), mix well, and pour into the appropriate bottle. Tip: It may help to use a fork to mix in the yeast. Be sure to note the actual temperature of the water in your lab notebook.
- Cap the bottle tightly using the cap with the tubing sealed in it. Make sure the open end of the collection tube is still inside the submerged gas-collecting graduated cylinder. Note the starting time in your lab notebook.
- Within five to ten minutes, the yeast should start foaming, and soon you should see bubbles collecting in the graduated cylinder. Note the time when you first start seeing bubbles in your lab notebook.
- Collect the CO2 (somewhere between 30 and 60 minutes should be good, but you can try adjusting this). Use the same amount of time for all of your tests.
- When the time is up, note how much CO2 was collected by observing how much water was displaced from the graduated cylinder.
- If you used a bottle instead of a graduated cylinder to collect the CO2, you can figure this out by marking where the CO2 level is on the bottle using a permanent marker, and then measuring the amount of water that is needed to fill up the bottle to that mark. Record the amount of water in your lab notebook.
- Remove and set aside the bottle with the yeast solution. You can rinse it out now, or observe it on a protected surface for a while longer before rinsing it out. Caution: Pouring too much yeast down the sink may be harmful to pipes and septic systems, so you may want to dispose of it in another way, such as by composting it or carefully pouring it into the trash.
- Set up the collection apparatus again using a fresh bottle for the yeast, repeating the section titled "Setting up Your Gas Collection Apparatus."
Repeat steps 2 to 8, now testing the antifungal agents, modifying the steps as detailed. You will be testing each antifungal agent at two different concentrations, a low concentration and a high concentration. The concentration range that you are testing is based on concentrations used in scientific studies listed in the Bibliography. You will be testing each antifungal agent and concentration one at a time (unless you decide to set up more than one gas collection apparatus).
- In step 2, add the antifungal medicine to the warm water with dissolved sugar before adding the yeast.
- Prepare two different concentrations of each antifungal medicine. First, check the "active ingredient" box on the antifungal medication's packaging and note what percentage of antifungal agent is in the medication. If you are using a medication that has the antifungal agent at a concentration of 1%, dilute it using the following steps to generate your high test concentration:
- First, dilute your antifungal medicine tenfold. To do this, measure 1/8 tsp. of antifungal medicine and mix this with 9/8 tsp. water (that is, 1 tsp. plus another 1/8 tsp.). Make sure you get all of the antifungal medicine off the measuring spoon. Using a toothpick may help. (Remember to wear latex gloves to protect your hands.)
- Mix this tenfold dilution very well. You may want to use a fork to vigorously stir, or whisk, the dilution. Mix it until almost all of the tiny pieces of antifungal medicine are not visible (although there may still be a small number visible).
- Next, measure 1/8 tsp. of this tenfold dilution and add it to the ¼ cup solution of warm water and sugar (as made in step 2). Mix well. Then add the yeast and mix well again. What is the final-fold dilution of antifungal agent that you are using?
- Because you performed a tenfold dilution in step 9bi, and a 100-fold dilution in step 9biii, you will be using a 1000-fold dilution for these tests. See the Technical Note for calculations used to determine the concentration of the antifungal solution you just made.
By following steps 9bi to 9biii, you created a 1:1000 dilution of the antifungal agent, or an antifungal solution that is at a concentration of 10 µg/mL. This concentration can be calculated by doing the following measurement conversions and calculations.
In step 9bi, you used 1/8 tsp. of antifungal medicine. The conversions can be used to find out what 1/8 tsp. equals in grams (g).
Measurement conversions:1/8 tsp. = 0.62 mL = 0.02 fluid ounces (oz.) = 0.59 grams (g) (for water)
We make the assumption that water and the antifungal medicine have similar weights. This is reasonable, because the medicine contains a lot of water.
This means that 1/8 tsp. of antifungal medicine weighs approximately 0.59 g. Consequently, you used 0.59 g of antifungal medicine in step 9bi. This was in a total volume of 10/8 tsp., so in step 9biii when you used 1/8 tsp., you were using 1/10 of the 0.59 g, which is 0.059 g. 0.059 grams equals 59 milligrams (mg).
Because the medicine has the antifungal agent at a concentration of 1%, this means that the 59 mg of medicine you used only has 1% antifungal agent, or 0.59 mg antifungal agent. 0.59 mg equals 590 micrograms (µg).
In step 9biii, this 590 µg antifungal agent was diluted into ¼ cup water, and ¼ cup equals 59 mL. If you divide 590 µg by 59 mL, you get 10 µg/mL, the final concentration of antifungal agent.
- To generate your low test concentration, use the following steps:
- Use the 1000-fold dilution you made in step 9b.
- Measure out 1/8 tsp. of the 1000-fold dilution and add it to the ¼ cup solution of warm water and sugar (as made in step 2). Mix well. Then add the yeast and mix well again. What is the final-fold dilution of antifungal agent that you are using? What is the final concentration of antifungal agent?
- If you are using an antifungal medicine that has a concentration of antifungal agent greater than 1%, you will need first to dilute the medicine so that it is at a 1% concentration, then follow steps 9b and 9c.
- For example, if the antifungal medicine is made up of 25% of antifungal agent, you will need to make a 25-fold dilution. You can do this by mixing 1/8 tsp. of the antifungal agent with 3 tsp. of warm water.
- In step 4, you may not see foaming or the formation of bubbles. Why do you think this happens? Note in your lab notebook whether you see foaming or bubbling for each condition tested.
- Run at least three separate trials for each antifungal agent concentration tested, and three separate trials of the yeast without any antifungal medicine added.
- Make sure to use the same water temperature each time you make a solution, because yeast activity is temperature dependent.
- Multiple trials help scientists make sure that their results are accurate and reproducible.
- Calculate the average volume of the CO2 produced for each amount of antifungal agent tested and write this in your lab notebook.
- Make a graph of your results.
- Write the different antifungal medicines and concentrations tested on the x-axis (the horizontal axis).
- Plot the corresponding average volume of CO2 produced on the y-axis (the vertical axis).
- Which antifungal medicine was most effective at stopping the yeast from growing? Did any completely stop antifungal growth? Which was least effective? How do you justify your reasoning? Why do you think one worked better than another? Did any of the antifungal medicines have the same effect at both the higher and lower concentrations tested?
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- There are many substances and essential oils that are thought to have some antifungal properties, such as tea tree oil, zinc (a dietary supplement), citronella oil, and several others. Read more about these other antifungal substances and use this experimental procedure as the basis to test which of these other antifungal substances are most effective. Are any of them more effective at stopping yeast growth than the antifungal agents you already tested in this experiment?
- You may have found in this experiment that some antifungal medicines are more effective at stopping yeast growth than others, when they are all used at the same concentration. But what is the lowest concentration at which each antifungal medicine needs to be used to completely stop yeast growth? Is it different for each antifungal medicine? You can investigate this using the same procedure.
- When you first did this experiment, if you found that the higher concentration completely stopped yeast growth for one of the antifungal medicines, but the lower concentration did not, try a concentration in between these two concentrations. If this in-between concentration still completely stops yeast growth, try a concentration in between this new one and the low concentration tested. Keep going until it does not completely stop yeast growth.
- This experiment uses yeast to test how effective different antifungal medicines are, but you can do many other experiments easily at home using yeast. Here are some additional Science Buddies projects and resources that use yeast:
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