Yeast busters : stopping fungus in its tract

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Yeast busters : stopping fungus in its tract

Postby scifiboi65 » Mon Jan 07, 2013 8:14 pm

hi,
I am trying to find which antifungal is most effective against yeast. Even though I keep all my variable constant like temperature, water used, quantity of sugar and yeast used, the CO2 produced with yeast alone is differtent each time. I tried doing 3 trials at the same time....still get different results even though I did the same thing to all the bottles at the same time.
When I tried with antifungals: The amount of CO2 produced is more than than the amount of CO2 produced with just yeast. I tried it with different antifungals and each time the CO2 produced is random and usually more than the control( with just yeast)
Does this mean that the antifungals dont work on the bakers yeast? Is it somehow causing more CO2 to be produced? and How?
What am I doing wrong?
Please help!!!!
I have to submit this project this week and still have not able do finish the experiment as I thought.
Please help!!!!
thank you.
scifiboi65
 
Posts: 11
Joined: Thu Jan 03, 2013 5:13 pm
Occupation: student
Project Question: Testing natural and over the counter antifungals through collecting amounts of carbon dioxide from yeast by water displacement.
Project Due Date: 1/14/13
Project Status: I am conducting my experiment

Re: Yeast busters : stopping fungus in its tract

Postby Teisha » Wed Jan 09, 2013 8:50 am

Hello scifiboi65,

As you noted, it is important to make sure that all the variables are constant so that they do not affect how the yeast grows in the different conditions. The variable that is probably easiest to change by accident would be the temperature of the water, so I'd recommend keeping a particularly close eye on that.

Next I would recommend making sure that you are using enough antifungal agent. It can be a bit tricky to make the dilutions -- you may want to confirm that the dilutions you are using are correct by going over step 8 and the Technical Note in the "Testing Different Antifungal Medicines" section again. The concentrations you test should be 0.1 ug/mL and 10 ug/mL of antifungal agent. You may also want to re-check the percentage of antifungal agent in the creams (for example, clotrimazole and terabinafine will probably be at 1%, whereas undecylenic acid will probably be at 25%). You may also want to make sure that the antifungal creams are fresh and not expired. Also be sure that the antifungal cream is not sticking to the measuring spoons during any of the dilution steps -- that cream can be pretty sticky. If you check all of this and it looks correct, you may want to try slightly higher concentrations of antifungal agents, if you have time.

You may also want to try doing one trial at a time so you can more closely check your setup as you do it. Along these lines, you may also want to try doing multiple experiments with the same gas collection apparatus -- it is possible there may be some variation between the different collection setups you made. (For example, there could be a small leak in one of the setups that is causing a decrease in CO2 yield, or a different length of tubing in the setups may also affect your results.) Did you see certain trends when using one collection setup compared to a different one? For example, are you doing all of the control trials using one collection setup, and always getting low CO2 yields with that setup?

Ultimately, you are trying to test a hypothesis. If you think you are doing everything correctly, and it looks like your results do not support your hypothesis, then that may be the conclusion for your project. Maybe your results indicate that the antifungal agents do not kill the baker's yeast in your setup -- you could say that you think additional studies should be done to confirm this result. Alternatively, you could say that you think additional studies should be done using a higher concentration of antifungal agent, and that maybe the concentration you used was too low to kill the yeast.

I hope that helps!
Teisha
 
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Re: Yeast busters : stopping fungus in its tract

Postby scifiboi65 » Wed Jan 09, 2013 10:07 am

Hi Teisha,
I have checked and made sure all the variables are constant especially the temperature.
I used the 10ug/ml concentration of different antifungals first. I was very carefull that there was no medicine sticking to the spoon and the medicines used are new and are 1%.
I checked the setup and it seems fine. I used the same setup for both the control and the antifungal seperately. I have also done both control and antifulgal eperiment at the same time using the same water and amount of yeast.
I am getting the same amount CO2 with the control( just yeast). That part looks good.
But when I add the antifungal, It is consistantly increasing the amount of CO2 produced. I tried adding a huge amount of antifungal just to check if higher concentration would kill the yeast. It is producing more CO2.
What does the increase in CO2 mean?
I am confused. Is there any mechanism that is causing the increase in CO2 production with antifungal creams. I thought maybe the inactive ingredients are causing this but when I tried with natural antifungals also, there was a considerable increase in CO2 production. But the foam prodused on top of the solution was very less.
Can you tell me what the foam that forms on top of the yeast solution consists of. Does the amount foam mean anything?
Has this project been done by anyone? What were the results and were they able to prove the hypothesis? Please help me in finding the required information that I can use to finish the project. Thanks
scifiboi65
 
Posts: 11
Joined: Thu Jan 03, 2013 5:13 pm
Occupation: student
Project Question: Testing natural and over the counter antifungals through collecting amounts of carbon dioxide from yeast by water displacement.
Project Due Date: 1/14/13
Project Status: I am conducting my experiment

Re: Yeast busters : stopping fungus in its tract

Postby Teisha » Wed Jan 09, 2013 2:04 pm

Hi scifiboi65,
It looks like you are taking the right steps, and I am not sure why your experiment would not be working.

One thing I thought I'd ask about -- are you running the different samples for the same amount of time? They should each be run for as long as you run the control (starting from when you first add the yeast).

Another possible problem is that there could be different amounts of air stuck in the tube and/or collection bottle when you start the experiments -- this is just something to check.

Yet another possibility is that somehow extra surgar (such as from the measuring cups) is getting into your antifungal trial samples, but I doubt this is the case.

Which antifungals are you using? Which "natural antifungals" did you try? Are you seeing the exact same results for all of the antifungals? What do your yields look like, with the controls and with the antifungal agents added?

The yeast is consuming the sugar and creating carbon dioxide (the bubbles) and ethanol alcohol. Carbon dioxide gas travels up, making the bubbles. I would expect there to be a lot of foam if a lot of CO2 is produced, so your results with the natural antifungals are a bit odd to me.

This project has been tested by Science Buddies and worked in our hands. I hope that some of this information may help.
Teisha
 
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Re: Yeast busters : stopping fungus in its tract

Postby scifiboi65 » Wed Jan 09, 2013 3:16 pm

Hello Teisha,
Thank you for all the suggestions you gave. I took my mom's help and tried out the experiment again today.
I am double checked all my controls. Only thing I changed is (1/2 tsp yeast and 1/4 tsp sugar ) instead of ( 1 tsp of yeast and 1/2 tsp sugar)
All my experiments are run for 45 minutes. but I did wait till 60 minutes to check if it made a difference. it is only increasing the amount of CO2 produced with antifungals.
Here are my results
control : 125 ml CO2
clotrmazole : 200 ml of CO2
tolnaftate : 225ml
terbinafine 260ml
garlic : 175ml
neem : 225ml

All the antifungals caused an increase in CO2. Is the inactive ingredients causing the extra CO2 somehow? or are they changing the PH of the solution somehow?I'm using EQUATE brand of antifungals. The inactive ingredients listed are benzyl alcohol, cetyl alcohol, cetyl palmitate, isopropyl myristate, polysorbate 60 sodium hydroxide sorbitan monostearate, stearyl alcohol.
thank you so much for your help. I need to finish this project by this week.
scifiboi65
 
Posts: 11
Joined: Thu Jan 03, 2013 5:13 pm
Occupation: student
Project Question: Testing natural and over the counter antifungals through collecting amounts of carbon dioxide from yeast by water displacement.
Project Due Date: 1/14/13
Project Status: I am conducting my experiment

Re: Yeast busters : stopping fungus in its tract

Postby scifiboi65 » Wed Jan 09, 2013 3:27 pm

I was also wondering if particular types of suger could affect the outcome. I am using organic sugar. Is this okay?
scifiboi65
 
Posts: 11
Joined: Thu Jan 03, 2013 5:13 pm
Occupation: student
Project Question: Testing natural and over the counter antifungals through collecting amounts of carbon dioxide from yeast by water displacement.
Project Due Date: 1/14/13
Project Status: I am conducting my experiment

Re: Yeast busters : stopping fungus in its tract

Postby Teisha » Wed Jan 09, 2013 4:25 pm

Hello Scifiboi65,

You have probably checked this already, but just in case you have not, I have a few more suggestions that may help:

* Make sure the antifungal cream is thoroughly mixed into the liquid that you use to dilute the cream, and that it is thoroughly mixed with the yeast. (If the cream is not mixed well, it may not affect most of the yeast.)
* In the CO2 collection cylinder/bottle, make sure you are measuring the amount of air (CO2) that has displaced the liquid, and not the amount of liquid left. (This is step 6 in the "Testing Different Antifungal Medicines" section.) If you are reading the amount of liquid in the bottle, this would give you the opposite results, which you are reporting. I do not think it would affect anything, but what are you using as a CO2 collection container? Are you using a graduated cylinder or a 500 mL plastic bottle?
* If you are using too much yeast, this could certainly affect your results -- with too much yeast, there may no longer be enough antifungal agent to kill a significant percentage of the yeast. The Procedure recommends using 1/2 tsp sugar in 1/4 cup warm water with 1 tsp yeast. For this project, it is important to use the exact amounts listed in the Procedure.

Do the bottles with antifungal agent start bubbling before the control bottle? This is another way to compare the yeast activity in the different conditions.

Also, the CO2 yields you are reporting seem a bit high overall based on Science Buddies testing. This could just be variation in testing, but it could suggest that you may be using too much yeast or sugar, or not enough antifungal agent. But it sounds like you have checked all of that thoroughly.

I do not think the inactive ingredients you listed should increase yeast growth, and using organic sugar should not matter.
Teisha
 
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Joined: Mon Jan 07, 2013 1:21 pm
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Re: Yeast busters : stopping fungus in its tract

Postby scifiboi65 » Wed Jan 09, 2013 9:57 pm

Hi Teisha,
Thank you for all the suggestions you gave me.I checked all the things you mentioned.
I thoroughly mixed the antifungal(1/8 tsp in 9/8 tsp water) And used 1/8 tsp of this mixture in the 1/4 cup of water used to mix yeast.
the temperature I am using is 120F and the brand I am using is EQUATE brand of antifungals.
I am measuring the amount of CO2 produced and not the amount of water left. I am using a 500ml measuring cylinder.
Amount of yeast used till now: I used 1/2 tsp of yeast and 1/4 tsp of organic sugar in all the experiments. If I used 1 tsp yeast with 1/2 tsp sugar the amount of CO2 produced was greater than my measuring cylinder. I made sure both the bottle with control and the one with antifungal used the same water at the same temperature and at the same time. I did the experiment with both of them at the same time using 2 measuring cylinders in the bucket. I did this for all the antifungals each time I did the experiment along with the control so as not to get any errors.
Results seen: I am consistantly getting around 125 ml of CO2 with the control and I am consistantly getting around 200 and more of CO2 with antifungals and naturals. Also the foam seen is so much more in the bottle containing the antifungal than the control. Except with the natural antifungal, the foam was less.
I really dont know how to proceed from here. I have done this experiment so many times for the last 2 weeks and still getting more co2 with antifungals.
Is something from the inactive ingredients reacting with the minerals in sugar to cause extra CO2 . Or if the brand I amusing is not good , then atleast I should get the same CO2 as the control. Why am I getting more CO2? I does not make sense to me.

Here is what I am going to do tonight.
I change 3 things 1] I use regular tap water instead of filtered water 2] I use regular white sugar instead of organic sugar 3] I use 1 tsp of yeast and 1/2 tsp sugar and do the experiment again tonight. I doubt I will see any change. I'll note the readings and post as soon as I finish.

thank you so much for your help. Please let me know if you think of anything else that will help me in this project.
scifiboi65
 
Posts: 11
Joined: Thu Jan 03, 2013 5:13 pm
Occupation: student
Project Question: Testing natural and over the counter antifungals through collecting amounts of carbon dioxide from yeast by water displacement.
Project Due Date: 1/14/13
Project Status: I am conducting my experiment

Re: Yeast busters : stopping fungus in its tract

Postby donnahardy2 » Thu Jan 10, 2013 9:26 am

Hi Scifiboi65,

Teisha has provided you with excellent advice on this project, and I have some additional comments that I think will help.

Don't worry, you have a great science fair project here. When you do a science experiment, results are empirical. That means you obtained the correct answer because that's what happened. If you obtain unexpected results, as you have done repeatedly, this suggests that you need more information to explain the results. But you still have the right answer for your display board and research report. You will just have a more interesting discussion section compared to other projects where the expected results were obtained.

This experiment should work without a problem, and there is no apparent reason why an antifungal would cause Baker's yeast to grow more compared to the control Here are some questions that might explain your results:

1. Did you use the same batch and quantity of Baker's yeast for your control and samples?
2. What antifungal and brand did you use, and what are the ingredients other than the antifungal?
3. Was your experimental set-up identical for the controls and samples (you have already verified the temperature is the same).
4. What did you dilute the antifungal agent in? Did you add the same to your control.
5. Did you do a control sample that contained antifungal only and no yeast?
6. Was the extra carbon dioxide released very soon after you set up the experiment, or did it appear after a few hours?

Possible explanation: Carbon dioxide can be released by a chemical reaction if a sample contains carbonate. Yeast will produce acid as they grow and the acid will react with carbonate ions to release carbon dioxide. If your antifungal agent contains carbonate ions, then this may have released carbon dioxide unrelated to cell metabolism.

Another possible explanation: Perhaps the antifungal contained a nutrient source that the yeast were able to utilize. Getting the list of inert ingredients would help confirm if this might be possible.

You can check for carbonate by adding some vinegar to your antifungal and any other components that you added to the sample and look for bubbles.

Please post again with answers to the questions and I'll probably be able to think of something else. Go ahead and write up your results as they are, however, since your project is due. .

Donna Hardy
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Re: Yeast busters : stopping fungus in its tract

Postby donnahardy2 » Thu Jan 10, 2013 9:54 am

Hi Scifiboi65,

Thanks for posting the information on the brand of antifungals you are working with and finding out about the inert ingredients. I had read your previous posts yesterday, and did not see that information when I was writing my initial reply. Teisha is really good at troubleshooting and I think her questions have eliminated some possible reasons for your unexpected results.

Can you post all of your data? Do you know how to do a student's t-test to compare results? If not, I can show you how to do a statistical test to verify that your results are significantly different (or not).

I believe that your results show a very significant difference between the controls and the antifungal samples. I do not believe that the approximately 25 to 100% increase in carbon dioxide could not be coming from experimental error, especially if you have repeated the results.

The alcohols should all inhibit the growth of the Baker's yeast, especially the benzyl alcohol and none of the ingredients, except the sodium hydroxide, could be the source of the extra carbon dioxide. Sodium hydroxide will absorb carbon dioxide from the air and will release it when the pH is lowered to below 7. So perhaps the sodium hydroxide used in the formulation of the antifungals contained high levels of carbonates that outgassed into your sample. This would explain your results.

I agree with Teisha; using organic sugar would not affect your results. The type of sugar used is one of your controlled parameters.

Again, I would concentrate on writing up your results and presenting them as they are. A bar graph would work well for your data. And, you have a reasonable explanation of what may have happened and you can explain what you would do differently if you had time to repeat the experiment. This is actually a really great project, and you may have discovered something new.


Donna Hardy
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Re: Yeast busters : stopping fungus in its tract

Postby scifiboi65 » Thu Jan 10, 2013 10:31 am

Hi Donna,
A quick question.
When the experiment was tested by science buddies...did they use regular yeast or rapid rise yeast. We are using Fleischmann's active dry yeast.
I'll post a detail reply about all the suggestions you gave and what we did in the next hour.
thanks.
scifiboi65
 
Posts: 11
Joined: Thu Jan 03, 2013 5:13 pm
Occupation: student
Project Question: Testing natural and over the counter antifungals through collecting amounts of carbon dioxide from yeast by water displacement.
Project Due Date: 1/14/13
Project Status: I am conducting my experiment

Re: Yeast busters : stopping fungus in its tract

Postby scifiboi65 » Thu Jan 10, 2013 12:42 pm

Hi Donna and Teisha,
Thank you both for all the information.
I did follow all the things you said but each time the CO2 produced is higher with the antifungal. I tried to eliminate the effect of inactive ingredients by doing the experiment without yeast and I did not get any CO2 at all. So there should be another mechanism where the antifungal is making the yeast grow/ inactive ingredients causing the CO2 with yeast.The CO2 released is continuous I have data recorded every 15 minutes. It is consistantly above the control at every reading. At a lower concentration , the antifungal is somehow causing an increase in fungal activity.

I'll check for the carbonate this evening ..I neeed to get vinegar.

This morning in my last attempt, I tried to use 1 tsp of antifungal(2% miconozole...desenex powder)in 1 tsp water and mixed well and added the whole thing in sugar solution. I dont know the exact concentration but roughly used a huge amount to see if it was killing any yeast.
Finally for the first time I saw a decrease in the CO2 produced. here is the data
Time CO2 produced with antifungal CO2 prodcued with control
15 mins 17ml 28ml
30 mins 60ml 75ml
45 mins 100ml 128ml
60 mins 140ml 180ml

So when I used higher concentration, it showed reduction in CO2.
I want to use higher concentration for all the antifungals now and see what happens and will post again.
Can you help me how to convert the concentrations at a higher level,,If I use a tsp of antifungal in 1tsp of water and mix it with the solution , what will my concentration of the antifungal be? What if I want to try the 2% or the 25% antifungals.

Please let me know if someone else is doing this experiment in the future and getting the same problewm as I have.

Thank you so much again.
scifiboi65
 
Posts: 11
Joined: Thu Jan 03, 2013 5:13 pm
Occupation: student
Project Question: Testing natural and over the counter antifungals through collecting amounts of carbon dioxide from yeast by water displacement.
Project Due Date: 1/14/13
Project Status: I am conducting my experiment

Re: Yeast busters : stopping fungus in its tract

Postby donnahardy2 » Thu Jan 10, 2013 4:22 pm

Hi Scifiboi65,

I don't know what type of yeast was used for testing. Maybe Teisha knows. When you set up an experiment, are you using the same lot of yeast for all of the samples. There can be differences in the viability of the yeast from batch to batch, so when setting up an experiment, it would be important to use the same lot of yeast for the control and all samples.

Do you have a method for measuring pH? Yeast grow optimally at a slightly acidic pH, so if the antifungals are lowering the pH at all, it could provide better growth conditions compared to the control, which is not buffered.. That could also explain the reason for your earlier results.

Your latest results look great! The antifungals are apparently inhibiting the growth of the yeast at the higher concentrations. One more experiment is a good idea because it will confirm that the effect of the concentration of the antifungal. However, after that you have to stop experimenting and start writing. This is a potential prize-winning project, but it will be evaluated based on your board. The science fair judges will be interested in knowing if you understand the science behind your project, if you used a well designed experiment, and if your evaluation of the results matches the actual outcome. So save the whole weekend for the project board. If you win, you can do some more experiments to completely solve the problem.

I think it would be better to report the actual concentration of the antifungals, not use 2% or 25%. Do the product labels have the concentration in mg or micrograms per gram? If so, then please post this information explain how you prepared each sample. The directions call for 1/2 tsp sugar in 1/4 warm water plus 1 tsp yeast. The antifungal is supposed to be dissolved in the water before you add the sugar and yeast. Is this what you did? And, what quantity of the antifungal did you use in the 1/4 cup water?

I had another idea. What was your water source? If you used tap water that contains chlorinated compounds, that might slightly inhibit the growth of the yeast. If you used different amount of water used in samples and controls, it might help explain the results. If you used distilled water, then disregard this comment.

Let us know what happens.


Donna Hardy
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Re: Yeast busters : stopping fungus in its tract

Postby scifiboi65 » Thu Jan 10, 2013 5:55 pm

Hi Donna,
Thank you so much for all the help and encouragement.
I am using the same yeast for all the experiments(same jar). I checked all the controls so many times by now.
That is good point about the pH. I will check the ph also from now on and will post them.

In the beginning I used filtered water, organic sugar. From yesterday I am using regular tap water and white sugar. It still is doing the same.
Today I am trying all the antifungals at a higher concentration and noting down the results. Hope fully they will work as they should. Will post them once I finish the experiment tonight.

I need some help from you
A] Please advise me how to put the results in a graph. One at lower concentration and one at higher concentration.
Also the procedure says to add the ntifungal after the sugar is mixed. I did not mix the antifungal first. Maybe I will try that and see.

B] Please advise me on how to calculate the concentration if
1] I am using 1/8 tsp of AF in 9/8 tsp of water and using the whole thing in the solution. I am thinking it is 1% of th antifungal so 1/100 of dilution. So what will be the amount of Antifungal in ug?
2] If I am using 1tsp of the antifungal in the 1/4 cup solution..what will be the concentration of antifungal?

thanks.
scifiboi65
 
Posts: 11
Joined: Thu Jan 03, 2013 5:13 pm
Occupation: student
Project Question: Testing natural and over the counter antifungals through collecting amounts of carbon dioxide from yeast by water displacement.
Project Due Date: 1/14/13
Project Status: I am conducting my experiment

Re: Yeast busters : stopping fungus in its tract

Postby scifiboi65 » Thu Jan 10, 2013 10:20 pm

Hi Donna and Teisha,
I checked the pH --- it is the same for both the solutions. So that is not causing the increase.
I found one thing. I tried to keep both the solutions in beakers outside and see the level of foam produced .
I saw that the control has a lot of foam in 10 -15 minutes than the one with antifungal. This means that antifungal is reducing the amount of yeast. Then I am curious as to why it is producing more CO2 in the bottle? Is it because it is anaerobic metabolism in the bottle and aerobic metabolism in the beaker? Or some how it is producing more CO2 even though it is killing th yeast(less foam)
When you pour the solutions in the bottles and close with cap with a tube, what type of metabolism occurs - aerobic or anaerobic?.
Please let me know if you have any ideas.?

Also I am noticing that if I measure in the first few minutes the antifungal is working and producing less CO2 and by 15 minutes it is producing more CO2 or some other gas also than control. Since I was only measuring for 15 min, 30 min, 45 min....I may have missed the initial difference in th first few minutes. Maybe the antifungal is working in the beginning and some how it is making the yeast grow more later on. Since Feischmann yeast is supposed to be better than red star brand ( that was used by sceincebuddies) maybe it was producing a lot of CO2 and it is becoming difficult to see the difference after 15 minutes. I'll try one more trial tomorrow morning with the red star brand and see if what I am guessing is correct.
If you think of any ideas please let me know ..I will focus on my chart as you said.
thanks.
scifiboi65
 
Posts: 11
Joined: Thu Jan 03, 2013 5:13 pm
Occupation: student
Project Question: Testing natural and over the counter antifungals through collecting amounts of carbon dioxide from yeast by water displacement.
Project Due Date: 1/14/13
Project Status: I am conducting my experiment

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