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

[deleted-372076]

Hi,

I am doing a project on: "Yeast Busters: Stopping Fungus in its Tracks with Antifungal Medicines". I was wondering how I would conduct the experiment if I am testing the control, two antifungal medications, and tea tree oil all at one time?

Thank you,
Santha

[deleted-372076]

Hi,

I was wondering how I would conduct the experiment if I am testing the control, two antifungal medications, and tea tree oil all at one time?

Thank you,
Santha

[deleted-372076]

Hi,

I am doing a project on stopping or prevent the growth of fungus.
Project name: Yeast Busters: Stopping Fungus in its Tracks with Antifungal Medicines
Going back to my previous question, would I just place 4 graduated cylinders in one bucket? And would I have to do 4 bottles a day as it takes that much time for the epoxy cement to seal? Also, I am planning to do each agent (1 control, 2 antifungal medications, and using tea tree oil) 10 times (40 bottles) so that means I would take around 10 days to complete the experiment?

Thank you,
Santha123

[MadelineB]

Hi Santha123,
Congratulations on the design of your experiment! Having controls and different test agents plus taking the time to test each situation 10 times should provide you with very good data!

As for your questions:
(1) would I just place 4 graduated cylinders in one bucket?

No, you only want to have 1 graduated cylinder in the bucket, since you need to measure the CO2 displaced separately for each of your test situations.

(2) And would I have to do 4 bottles a day as it takes that much time for the epoxy cement to seal?

If I understand the procedures correctly, you will only one cap with the tubing expoxied in place. You can then wash that cap and tubing after each test, so that you can use that cap and tubing for each of your test bottles. Look at the yeast bottle on the right in Figure 2:
https://www.sciencebuddies.org/science- ... #procedure

You will need to use a 500 mL bottle to set up each test situation and do each test separately, but using just the one cap with the tubing attached.

The procedure suggests that it will take 30-60 minutes to measure the CO2 collected for each test situation. If you can do 4 tests in a day, then it will take 10 days to complete your measurements.

You might find it easier to set up the yeast for each antifungal once and do 5 replicates for that situation, so it will help if you have 5 bottles. You might not have time to do 10 in one day!

I hope this helped to answer your questions and please be sure to let us know if you have more questions!

[deleted-372076]

Hi MadelineB,

Thank you so much for responding! Since it takes about 1 day for the epoxy cement to dry how will I test 60 bottles? (I actually have to find a high and low concentration mix for the antifungal medications). Is there an alternative which takes less time to dry? Some say one hour to dry but others say 24 hours? Is it ok if I let it dry for one hour?

Please correct me if I am wrong.
1) First I set up the gas collection apparatus by filling a bucket 1/3 full and inverting a graduated cylinder.
***Do I put the tube completely inside the graduated cylinder?
2)Then seal the tube to the bottle cap (2cm deep).
3) Open the bottle cap and add the yeast (control).
4) Then close the cap and observe. Wait 30 min and measure how much water is left in the graduated cylinder inside the bucket.
5) clean everything and start over for each new experiment?

***Also since I applied the cement to one bottle cap, can't I just replace the bottle and use the cap with the tube in it to save time? Then I would just add water into the tub and place the graduated cylinder and the tube (the other end) inside it?

-OR-

Would this be better?
Day 1- All 10 trials of the control (using same bottle cap)
Day 2- All 10 trials of Antifungal 1(high concentration) (using same bottle cap)
Day 3- All 10 trials of Antifungal 1(low concentration) (using same bottle cap)
Day 4- All 10 trials of Antifungal 2(high concentration) (using same bottle cap)
Day 5- All 10 trials of Antifungal 2(low concentration) (using same bottle cap)
Day 6 - All 10 trials of tea tree oil (using same bottle cap)
**Each experiment would run for 30 minutes and I would use the same bottle cap throughout?
Thank you so much,
Santha123

[MadelineB]

Hi Santha123,
You are very wise to take the time now to make sure you understand how to set up this experiment.

Let me answer your question about the epoxy and bottle cap - Yes, my understanding from reading the procedures is that you need only prepare just bottle cap with the tube. I would follow the directions and definitely let the epoxy dry for 24 hours.

Once you have that bottle cap, follow your list of 5 steps for each test situation, except you only do 2) once, prior to all of the tests.

You ask if you should put the tube completely inside the graduated cylinder?

From my reading of the procedures, I think you need only feed the tube so the last couple of inches are inside the graduated cylinder. The procedures suggest using a short length of tubing (see figure 2).

Again, yes you can use the bottle cap and tube for all of your tests, but you will need to clean everything between each test. I can see that it would be easier to do the 10 replicates of each of your 6 tests (that is, 1 control, 5 tests) all on the same day. But that means that the differences you observe between your 6 test situations include a "day effect". So, although it means more work for you, you would have a "cleaner" experiment if you tested all 6 situations on each day. If you have time, you might do 2 replicates for each of the 6 test situations. Be sure to record the day and time and duration for each result!

I hope I've answered your questions about the set up. Please let us know if you have more questions.

[deleted-372076]

Hi MadelineB,

Thank you so much for clarifying all my doubts! Also when graphing my data, I was thinking about using either a bar graph or line graph. One for each experiment (1 control, 5 tests) and another combining all the results. Is one more advisable than the other?


Thank you,
Santha123

[MadelineB]

Hello Santha123,
In general, I think the line graph (or scatterplot) will provide more information about your results than a bar graph.

You might want to show the results for each antifungal and the controls on one graph, with different symbols to identify the controls, the low dose, and the high dose. To begin with, you could plot the result for each experiment versus the time (on the horizontal axis) you started that experiment. This would let you see if your results did vary with time.

It also occurred to me that you might want to do 2-3 control tests on each day. Then you could plot the control results versus day (horizontal axis) to see if those results varied by day. If you made this plot as you get the results, then you could see quickly if the control results varied by day. If that happens, then you might need to rethink how you have set up the experiments, since ideally, the control results should not vary very much from day to day!

Let us know how your experiment proceeds, and by all means, let us know if you have additional questions!

[deleted-372076]

Hi MadelineB,
I was wondering what the difference between the low and high concentration of the antifungal medications are??? Both seem to have the same procedure... Thank you in advance for your response!

High Concentration:
"First, dilute your antifungal medicine tenfold. To do this, measure 1/8 tsp. of the 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."

Low Concentration:
"Use the 1000-fold dilution you made previously.
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."

And if they are that similar, can't I use one for my experiment rather than both of them??


Also, since the Tea Tree Oil is a natural ingredient, can't use the same amount of yeast as the oil. I don't think I can make a high - low concentration out of this product??


Thank you,
Santha123

[MadelineB]

Hello Santha123,
If you and I are looking at the same version of the project procedures, look carefully at the instructions in steps 9b i and 9b ii: this is your ten-fold dilution.

Look again at the instructions for Steps 9b iii - here you are using your ten-fold dilution to make a further dilution. Think about your answer to the question: "What is the final-fold dilution of antifungal agent that you are using?"

Work through the arithmetic described in the Technical Note. Now carefully read the instructions for making the low concentration (Step 9c). Do you now see that you have two different concentrations?

Let us know if this now makes sense or if you are still puzzled!

[deleted-372076]

Hello MadelineB,

Please correct me if I am wrong:
High: 1%
1) in a plastic cup add 1/8 tsp of the antifungal agent and 9/8 tsp of water. Mix well.
2) add 1/8 tsp of the solution you made in step 1 to the 1/4 cup water and sugar in the measuring cup.

Low: 1%
1) add 1/8 tsp of the antifungal, sugar, and water mixture to another cup of water and sugar.


In other words:
(high)A= 1/8 tsp (1/8 tsp fungal + 9/8 tsp water) + 1/4 water + sugar
(low)B= 1/8(A) + 1/4 water + sugar

25% is 1/8 tsp of fungal + 3 tsp water
Then similar step after you make the dilution as in the 1%


And do I have to make a new mixture every time or can I reuse the same solution in the plastic cup?

And when I make the high solution, it's convenient to make the low solution as well. My question is, will my results be different if I let one of them sit out until I'm ready to use them? Or that really doesn't matter and I can make my antifungal solutions beforehand and I'll add the yeast when I am ready to use that experiment?


Also, since the Tea Tree Oil is a natural ingredient, can I use the same of oil as the yeast??

[MadelineB]

Hello Santha123 and congratulations on your persistence!

I think you do not have the right steps. But before we get to the steps for making the concentrations, there are 2 important points discussed further down in the procedures. That reminds us to always carefully read all of the steps in the procedures before starting!

Point 1 is discussed in Step 9 d. You need to read the label on each of your antifungal agents to see the original concentration. Is that 1% for each of the agents? If not, then you will need to dilute the originals to 1% before attempting to make the concentrations!

Point 2 is discussed in Step 10 a. This says that you need to be sure to use the same water temperature for each experiment, since the yeast activity is temperature dependent! I think this point addresses your question about whether you can make up multiple samples of your concentrations ahead of time. You will want the drug solution to be the same temperature when you add the yeast. I'm also thinking you want the solutions to be warm so that the yeast dissolves completely as possible for each experiment. Note that the procedure also suggests that you record the time and the temperature when you start each experiment (I think "start" means when you add the yeast and hook up the bottle to the gas collector).

Now, to your questions about the concentrations! My interpretation of the procedures is that the high dose concentration is made by following Steps 9 b i, 9 b ii and 9 b iii. It might help you to follow those steps if you make a table and label 4 rows with those 3 steps, and then write down the drug concentration achieved at each step. At the end of those 3 steps, you have prepared the high dose concentration.

Then, you will use some of the high dose concentration and follow Step 9 c to make your low dose concentration. Again, you might find it helpful to make a table to record the concentration you achieve at each step.

I will try to answer your question about the Tea tree oil is the next post!

[MadelineB]

Hi Santha123,

So this is part 2 of my reply to your last set of questions!

You asked "since the Tea Tree Oil is a natural ingredient, can I use the same of oil as the yeast??"

I'm thinking you might of intended to ask about the same volume of oil?

You might want to do some background research in order to determine the range of concentrations of Tea Tree oil which might be active as an antifungal. I think it will be informative to test a high and a low dose.

I hope that you are keeping a lab notebook and recording all of these questions and answers! This learning process is every bit as important as the actual experiments!

Let us know how you're doing and if you have more questions!

[deleted-372076]

Hi MadelineB,
I am using terbinafine and undecylenic acid products. There is 1% of terbinafine in one product and 25% undecylenic acid in the other product.

To make sure that the undecylenic acid product is equal to 1% concentration I should add 1/8 tsp of the product to 3tsp of water.

9BI (1%): add 1/8 of the product to 9/8 tsp of water= (ten fold mix)
9BII (1%): mix well
9BIII (1%): add 1/8 tsp of the tenfold mix to 1/4 cup water and sugar
Finally, you have a 1000 fold mix

9CI (1%): use 1/8 tsp of the 1000fold mix and add it to 1/4 cup water and sugar.

*Even to make a low concentration, you first need to make the high concentration.

9BI (25%): After you made the 1% concentration, add 1/8 tsp of that product to 9/8 tsp water = (tenfold mix)
9BII (25%): mix well
9BIII (25%): add 1/8 of the tenfold mix to 1/4 cup water and sugar
Finally, you have a 1000 fold mix

9CI (25%): use 1/8 tsp of the 1000fold mix and add it to 1/4 cup water and sugar.


After you make the high or low concentration, add the yeast, cap it, and wait 30min.


For the tea tree oil, there was no specific agent but, it did say it is used to treat fungus. Also, I think it would be easier to add the same amount of oil as fungus.

Thanks,
Santha

[MadelineB]

Hello Santha,

Congratulations again for your perseverance!

I'm a bit puzzled by the (25%) in your second set of 9b steps. Do those steps use the 1% solution of the agent which you get by diluting the original 25%?

And, I am still unclear what the concentrations of Tea Tree oil you plan to use. Does the bottle of your TTO list the percentages of the active ingredients?

As part of your research, have you looked for online publications reporting the antifungal properties of TTO? You could google PuBMed antifungal Tea Tree Oil. You might find information regarding the concentrations of TTO which had antifungal activity.

I think you will want to use that sort of information to decide which concentrations to test.

Also, I remember that you were planning to replicate each treatment 10 times. You might want to start with 3 replicates of each, and plot your data to see if your high dose and low dose are clearly producing different results!

[deleted-372076]

Hi Madeline,

The procedure gave an example for a concentration greater than 1% (e.g. 25%) to dilute it to make it a 1% solution and then to follow steps 9b and 9c. But are the rest of my understanding of the procedure correct?

The Oil that I am using does not specify the active antifungal ingredients with percent, but it does say that it's a "fungus fighter."

These are the ingredients listed:
Soybean Oil, Tea Tree Oil, Safflower Oil, Vitamin E, Carrot Extract, Aloe Vera Extract, Rose Hips Oil, Peanut Oil, Sweet Almng Oil, Methylparaben (And) Fragrance, D And C Red #17. D And C Yellow 311

Methylparaben is a fungicide so I think this is the main antifungal ingredient?

and here is the link for the product (TTO):
https://www.walmart.com/ip/Hollywood-Be ... z/10313104

Thanks,
Santha

[MadelineB]

Hello Santha,

Thank you for the link to the Walmart information for your bottle of Tea Tree oil.

When I checked the link that you gave to me, I see they also have "Humco 100% Pure Australian Tea Tree Oil."

I suggest that you use a product which tells you the concentration of the Tea Tree oil. Then, you will need to do a literature search to find out the concentrations that researchers have used, so you know which concentrations to test as the high and which to test as the low concentration. The judges who review your project will want to know that information. Also, remember that you will be putting in a lot of work to measure the outcome, so it would be nice to know what the concentration is that yields the result!

Let us know if you have more questions!

[deleted-372076]

Hi Madeline,

I found a really good scholar page :https://www.google.com/patents/US8333981.
It states how useful tea tree oil is and gives examples of the amount used, but not the dilution. (.06 and .10)

Since the tea tree oil is 100%, I was wondering if I could add 12 tsp to 1/8 of the oil? (25% = 3 tsp)
Then I would do the steps for the low and high concentration.

Just making sure, as far as we have discussed, my understanding of the project procedure is correct?

Thank you so much again for helping all this time. Now I can start my project with a great understanding and no confusion!!!


Thanks,
Santha

[MadelineB]

Hello Santha,
It sounds like you now have a bottle of 100% Tea Tree oil. That will make things easier for you!

However, you will still need to look at the scientific literature to find the starting dilutions for your high dose.

Try googling using a search something like "pub med antifungal effect of tea tree oil."

This should show you open access scientific papers in Pub Med Central so you can read the actual journal papers. You want to find the concentrations of Tea Tree oil reported as "minimum fungicidal concentration."

This is information that you should include in your research report to justify your choice of concentrations to test.

Your patience and persistence will pay off, since the judges review your written report with as much interest as your results!

Let us know if you have more questions.

[deleted-372076]

Hi Madeline,

I found a website (http://www.ncbi.nlm.nih.gov/pubmed/15140856) which clearly states the amount used (1%), so doesn't that mean that I need to dilute it to a 1% concentration( 1/8 tsp oil + 12tsp water)??



Thanks,
Santha

[deleted-372076]

Hi Madeline,

So as far as we have discussed, my understanding of the procedure is good right??? I'm just making sure because I don't want any complications when conducting my experiment.

Thanks,
Santha

[MadelineB]

Hello Santha,
Again, congratulations on your persistence in working through the subtle points of this experiment.

Also, thank you for the link to the journal article on the antifungal effects of Tea Tree oil. Those authors report that Tea Tree oil altered the permeability and membrane fluidity of the fungus Candida albicans. However, those authors did not study the antifungal activity of Tea Tree oil. That is, they did not measure how effective Tea Tree oil was in stopping the growth of fungus. This might seem like a subtle difference, but your project is studying the effect of Tea Tree oil in stopping the growth of yeast. So you need to find a journal article that reports the antifungal activity of Tea Tree oil so you will be able to use the reported minimum concentration of Tea Tree oil which killed their target fungus ("minimum lethal concentration (MLC)" or "minimum inhibitory concentration (MIC)."

To answer the second part of your question - assuming that you want to get a 1% concentration from the 100% concentration, you would want to do two ten-fold dilutions (sometimes called "serial dilutions"), use step 9 (b) (i) for the first ten-fold, and then repeat that step, using 1/8 tsp of the ten-fold dilution.

But remember, that you may find the journal articles report a lower concentration for the MLC or MIC. You will want to use the lower concentration, which might be as low as 0.25%, or maybe even lower!

Let us know what you find and if you have more questions.

[deleted-372076]

Hi Madeline,

I found this website (http://www.webmd.com/vitamins-supplemen ... ientid=113). It only states the concentration, but not the amount to use.

This website says to use 4-5 drops of the oil (http://www.goodhealthacademy.com/how-to ... il-fungus/)

This website says 1/2 tsp oil + 1/2 cup water (http://homeremediesforlife.com/tea-tree ... infection/)

This website says "the MICs of tea tree oil is between 0.125 and 2%" (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3264233/)

This website says "Reported MICs range from 0.12% to 2%" (https://www.drugs.com/npp/tea-tree-oil.html)

This website also says that "fungicidal concentrations generally range from 0.12 to 2%" (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1360273/)


***Overall 1% is in the range (which will keep all of my concentrations the same.) So, to make the tea tree oil a 1% concentration, I should add 1/4 tsp oil and 18/8 tsp water. Then follow steps 9b and so on.

Thank you so much in advance,
Santha

[MadelineB]

Hooray Santha,
Congratulations on your persistence and patience!

Since the reported MICs are in a range like .1% to 2%, you might want to pick a high concentration and also a low concentration. I realize that you started out thinking to just test one concentration of the TTO, but test a low and a high fits in with the concentrations of the other antifungals that you are testing. Makes for a more complete experiment!

Best of luck!

[deleted-372076]

Hi Madeline,

So I finally finished my experiment. I calculated how much the yeast rose by measuring it in terms of water. I measured how much water it is equivalent to, but I'm not sure what the numbers mean. I'm unable to send you the picture but here are my data:

I also have an outlier (OH Trial 1), but other than that the amount of water for all 3 trials seem relatively the same. I'm not sure what these numbers means in terms of if the experiment worked or not? Also when I am doing a t-test, I am comparing each other experiment (TH...) with the control and for the test, I'm not sure which sign to use (greater or less)? The numbers for the most part, are greater but I'm not sure in this case if greater is better?

Trial 1(mL)
Control 155
TH 232.5
TL 235
UH 242.5
UL 190
OH 397.5
OL 167.5


Trial 2(mL)
Control 227.5
TH 260
TL 240
UH 215
UL 240
OH 210
OL 145


Trial 3(mL)
Control 230
TH 255
TL 250
UH 235
UL 235
OH 135
OL 135

Thank you,
Santha

[MadelineB]

Hello Santha,
Thank you for posting your data!

Before you start thinking about testing the differences between the different concentrations, I would suggest that you look at the data as 3 replicates for the control, and for each antifungal.

That is, first, look at the 3 replicates for the control: Rep 1: 155, Rep 2: 227.5, Rep 3: 230. There seems to be a bit too much variability across your 3 replicates just for the control. With that much variability in the control, it can be difficult to make sensible comparisons between the antifungal agents and concentrations.

I wonder if the water temperature was the same for each of the replicates? Did you collect the CO2 at the same time for each replicate? I know the project gives a range, but you need to select a time, and use the same time and temperature for each replicate. Did you record the time and the temperature in your lab book for each replicate? Did you make sure that all of the air was out of the water before you began collecting the CO2?

I know that it can be frustrating trying to do each of the steps exactly the same for each of the trials, but do not despair!

I think it would be very useful to repeat the measurements for the control for 3 more times, trying very hard to keep the temperature the same, record your measurements at exactly the same time.

I will see if another expert here at Science Buddies has experience with this project and can offer some more advice.

So don't get discouraged! Science is doing lots of picky details!!

[deleted-372076]

Hi Madeline,

I kept the timer for 30 min for every trial and 110F for every trial. I do see that trial 1 is off but 2 and 3 are relatively similar. The thing that I observed for each trial is that at least 2 are very similar and one is kinda off. It could be that since I have to make the mix for each experiment every time, during that process the water might have cooled down, but even if it did, I reheated it to make sure it was 110F before combining everything.


-Santha

[MadelineB]

Hello Santha,
Thank you for your patience and persistence.

I'm a bit confused when you say "for each trial is that at least 2 are very similar and one is kinda off" ...

Are you looking at the results for each trial? Or the results for each treatment group for the 3 trials?

If you haven't already done this, I suggest you make a table with a row for each treatment group (think of "control" as its own treatment group) and three columns, one column for each trial. You could also make a graph, with the horizontal axis indicating the treatment group and the vertical axis showing the values observed for the 3 trials for each treatment group.

Now think about which results would be expected to be most similar across the 3 trials.

And which group of 3 results will be your baseline, to be used for comparing the effects of treatments?

Here's a link to the section on Data Analysis and Graphs in the Science Buddies Project Guide.
https://www.sciencebuddies.org/science- ... l#overview

That might help you examine your results.

Be sure to let us know if you have more questions.

Madeline

[deleted-372076]

Hi Madeline,

The deadline for my project is approaching very soon and I don't know what the numbers mean. I am doing more trials as you have suggested. I have to write a junior reseach paper and for that I need things like results, conculsion, analysis and ect. Is it better if there is more CO2 in the control or the antifungal treatments?


Thank you,
Santha Rani

[MadelineB]

Hi Santha,
I think that the background and procedures sections of this project will help you to understand the role of CO2 in measuring the antifungal activity. See if you can answer the list of questions given in the background section.

https://www.sciencebuddies.org/science- ... background

https://www.sciencebuddies.org/science- ... #procedure

Remember that your written report is an important part of your science project. You might find ideas for organizing your report from the Science Buddies Project Guide.

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

Let us know if you have more questions.