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Anti fungal Assay of Saccharomyces Cerevisiae HELP

Posted: Sun Mar 24, 2019 5:59 am
by ymahmoud
I am trying to conduct an anti fungal assay of the antifungal properties of several essential oils against baker's yeast, Saccharomyces Cerevisiae. I want to investigate cinnamon oil, lavender oil, and tea tree oil in particular. So far I have been using cinnamon oil for my trial and have tried:
- Using the disk diffusion assay by using Whatman disks soaked in cinnamon oil (just dipped into the container of oil) and this caused complete inhibition of yeast.
- Agar well diffusion assay (making 6mm holes in the agar) with 10, 20, 30, and 40 microlitres placed in 6mm diameter wells. This didn't really work and I saw no inhibition for some reason (does oil not diffuse well in agar?)
Now I'm wondering:
- Should I use different amounts in microlitres (20, 30, etc) of the oil and just load them on each disk and see if there are results?
- Should I make a solution where I have maybe 20 microlitres with 30 microlitres of solvent and load that in the disk. i.e, do the disks have to be soaked to work, or is the small amount of oil on its own enough?
- If I use a solvent, which one should I use? I know ethanol can dissolve the oil but will this be an interfering variable since ethanol is toxic at high levels to yeast? I used ethanol in another experiment where I dissolved cinnamon powder and loaded the disks with that and I saw clear zones of inhibition, should I worry that this is due to ethanol rather than cinnamon powder?
- If not ethanol, what other cheap and available solvents can I use?

Re: Anti fungal Assay of Saccharomyces Cerevisiae HELP

Posted: Wed Apr 03, 2019 3:59 pm
by AmyCowen
Hi - I am sorry your post has not received a reply yet. Is this for a K-12 student science project you are doing?

Science Buddies

Re: Anti fungal Assay of Saccharomyces Cerevisiae HELP

Posted: Fri Apr 05, 2019 3:44 pm
by MS15
This is an interesting project and hope you will find my suggestions useful.
First, let's talk about solvents. You are correct about the fact that you will need an organic solvent (like ethanol or acetone) for oils. Now as you already identified, these solvents can themselves affect the growth of microorganisms, including yeast. So in this case, what you can do is have a 'control' experiment alongside the different amounts/concentrations of the oil you are testing. Let's say you want to try 10, 20, 30, 40 microlitres of a certain oil. Lets keep the total volume of the liquid to be administered to the disks, constant (say 50 microlitres). So we will make individual solutions with 10 microlitres of oil and 40 of solvent, 20 of oil and 30 of solvent and so on... Last will be a disk which will have 50 microlitres of only the solvent - this is your control experiment where you are testing if the solvent alone is responsible for the growth inhibition you observe.

Now let's say, you realize that your solvent (I would prefer ethanol over acetone, chloroform etc.) alone is killing the cells and you want to test the effect of the oil without any solvent. I would suggest in that case, to first spot the different amounts of the oil on individual disks and let the disks air dry. This will ensure that each disk has only as much amount as you want. Whereas by dipping it in an oil you lose control over how much oil is on each disk which depends on external factors like the oil's viscosity and also can be quite random disk to disk. You may want to place the disks on a clean plastic sheet while spotting and air-drying rather than on tissue/paper napkins which may soak up some of the oil, changing the amount on each disk.

Once the experiment is done, finally you can measure the radius (or diameter) of the zone of inhibition starting at the center of your disk and plot that against the amount/concentration of oil. It would result in what we call a dose-response curve and that would be a really cool way to represent your data!
Good luck with the experiments!

Re: Anti fungal Assay of Saccharomyces Cerevisiae HELP

Posted: Sun Apr 28, 2019 12:51 pm
by ymahmoud
THANK YOU! I will definitely try to plot a dose-dependent curve. How many trials would you suggest and how many different amounts/concentrations? Also, I'm not sure what kind of statistical analysis I should do, can you help with that? Again, thank you for your time! :D

Re: Anti fungal Assay of Saccharomyces Cerevisiae HELP

Posted: Tue Apr 30, 2019 4:25 pm
by MS15
You're very welcome!
For good practice (and meaningful results), it is best to do the experiment at least 3 independent times if possible. I would also try to include 4-5 concentrations (including the control with '0' concentration) so you can plot a good dose response curve.
Regarding statistical analysis, one option is to do a one-way ANOVA (analysis of variance) which will basically allow you to compare mean values (say the mean values of the diameter of zone of inhibition, for each concentration as a group between your three replicate experiments) and determine if there are statistically significant differences between groups (i.e. between different concentrations or between the control and individual concentrations). Hope this is not too confusing. Let me know if it is.

Best wishes for your project!