Coming up with a procedure and materials list

deleted-920666 · Post by **deleted-920666** » Wed Oct 21, 2020 9:17 pm

So what I want to do is a project about urinary tract infections. i want to see if anti-inflammatory ligands (specifically 2-acetoxybenzioc acid, indomethacin, tolfenamic acid, ibuprofen, and mefenamic acid) aid fluconazole in clear urinary tract infections caused by saccharomyces cerevisiae (household yeast). so pretty much i know that i would need to find the powder version of all these ligands and buy them, i just dont know how much i would need. i also know that i could need fluconazole. i dont know how much of that i will need. i will need yeast, i also dont know how much i would need. i plan on doing five trials for each anti-inflammatory. and i also plan on doing a control. one with yeast culture. one with yeast and fluconazole. i dont know how long the project will last, what the procedure will look like, and i dont know what materials i will need. i have been having trouble getting in contact with someone that does because of the coronavirus and everywhere being on lockdown. i know that i need agar, petri dishes. soo please let me know what you think:)

kgrivera · Post by **kgrivera** » Thu Oct 22, 2020 7:59 pm

Hello!

I see that you have a good idea of the different compounds that you would like to test, but are unsure about how to use them in your experiments. In order to find out how much of each compound you need, look up what the concentration of these drugs are for normal dosages or the concentrations in the blood after treatment with the drugs if you can find it. This will allow you to design an experiment where you are testing the susceptibility of yeast to concentrations of the drug they would be exposed to if someone actually took aspirin for example.

By the way, https://www.ncbi.nlm.nih.gov/pmc/ is a great resource for finding free access articles from biomedical and life sciences journals.

Based on the results of your searches, you will then be able to determine how much of each compound you actually need to purchase for these experiments.

I think the easiest procedure to follow to determine the effect on the yeast would be to spread the yeast culture on the agar plate at a high enough concentration that the yeast will form a lawn, then place filter paper discs treated with the drugs (soak the filter disks in the drug) on the plate. After the yeast grows, you will see a zone of inhibition on the plate, which will provide you with a quantitative measurement of the effectiveness of the different anti-inflammatory ligands.

Another consideration is the solubility of each of the compounds. Indomethacin for example, is not soluble in water at all. You might have to dissolve some of your compounds in other solutions, like ethanol in the case of indomethacin. If this is the case, then you should also include that solution by itself as control to make sure it is not having its own effect on the growth of the cells.

Feel free to ask me anymore questions! Hope this helps

deleted-920666 · Post by **deleted-920666** » Thu Oct 22, 2020 10:09 pm

it helps a lot! so one question how exactly would i grow the S. cerevisae on the agar plates using household yeast. i want this project to be a project that I could possibly work on at home in case im unable to access a lab. that is why i chose the strain of yeast used in food:)

deleted-920666 · Post by **deleted-920666** » Thu Oct 22, 2020 10:13 pm

also how would i incorporate the anti-fungal (im using fluconazole) into the procedure. the project is supposed to see which of the five anti-inflammatory drugs aid fluconazole in clearing the anti fungal. i also dont know if i should be testing to see which one clears it faster, because i am aware that the anti-fungal will clear the yeast, so maybe i could find which one clears the yeast faster. or i should see which one has a larger or smaller zone of inhibition. or should i see which one has less yeast colonies on the plate?

deleted-920666 · Post by **deleted-920666** » Sun Oct 25, 2020 11:49 am

So what I want to do is a project about urinary tract infections. i want to see if anti-inflammatory ligands (specifically 2-acetoxybenzioc acid, indomethacin, tolfenamic acid, ibuprofen, and mefenamic acid) aid fluconazole in clear urinary tract infections caused by saccharomyces cerevisiae (household yeast). so pretty much i know that i would need to find the powder version of all these ligands and buy them, i just dont know how much i would need. i also know that i could need fluconazole. i dont know how much of that i will need. i will need yeast, i also dont know how much i would need. i plan on doing five trials for each anti-inflammatory. and i also plan on doing a control. one with yeast culture. one with yeast and fluconazole. i dont know how long the project will last, what the procedure will look like, and i dont know what materials i will need. i have been having trouble getting in contact with someone that does because of the coronavirus and everywhere being on lockdown. i know that i need agar, petri dishes.

how exactly would i grow the S. cerevisae on the agar plates using household yeast. i want this project to be a project that I could possibly work on at home in case im unable to access a lab. that is why i chose the strain of yeast used in food:)

also how would i incorporate the anti-fungal (im using fluconazole) into the procedure. the project is supposed to see which of the five anti-inflammatory drugs aid fluconazole in clearing the anti fungal. i also dont know if i should be testing to see which one clears it faster, because i am aware that the anti-fungal will clear the yeast, so maybe i could find which one clears the yeast faster. or i should see which one has a larger or smaller zone of inhibition. or should i see which one has less yeast colonies on the plate?

brandimiller610 · Post by **brandimiller610** » Sun Oct 25, 2020 4:38 pm

Hi lylahgirl66,

I hope you are having a great day. Your project sounds very interesting thus far.

In terms of determining how much of each ligand you need and determining materials and a general experimental design, it is best to follow protocols from previously published papers. If you already have some, see what materials were used in the "Methods" section. If you don't have papers, you should do some quick Google searches to find some scholarly articles related to your project -- include keywords such as "yeast", "S. cerevisiae", "anti-inflammatory" and "urinary tract infection". Five trials for each anti-inflammatory is more than sufficient (in general, three replicates is the standard).

In general, you will need a stock culture of yeast, petri dishes (agar culture)/15 ml conical tubes (liquid culture), agar/liquid medium, fluconazole, and the anti-inflammatory ligands you want to test. You could choose to grow your yeast on agar plates or in liquid medium, depending on personal preference. Specific protocols for growing your strain of yeast can be found on the internet (atcc.org, Thermo Fisher Scientific, Sigma-Aldrich, etc.) -- these sheets will tell you the most optimal media to use, growth temperature, and the optimal growth period. When growing yeast (or any type of microorganisms), it is important to maintain sterile conditions. Therefore, access to a laboratory is highly recommended for growing yeast cultures; furthermore, you will need access to an incubator to grow your yeast in and a heat source to sterilize your working surface. However, if you have no other option but to work from home, I found this YouTube video: https://www.youtube.com/watch?v=-EKb6AcaEBg. The author seems to have a whole collection of similar videos regarding yeast; I hope you find them helpful! Here is another video about the maintenance of yeast in culture: https://www.jove.com/v/5095/yeast-maintenance.

To answer the questions regarding the anti-fungal (fluconazole), I recommend first using it as a positive control (meaning it should give you positive results). Your negative control would be the plate with no anti-fungal and no anti-inflammatory ligand. Once you determine which of the anti-inflammatories clears the yeast the fastest, in comparison to fluconazole, you could then maybe see how different concentrations of the anti-inflammatory drug affect the time in which fluconazole is able to clear the yeast. Another approach would be to measure the zones of inhibition for each drug and the anti-fungal after different amounts of time (i.e. you could have five different time points -- 0 hr, 4 hr, 8 hr, 12 hr, 24 hr). Counting colonies, in my opinion, would be the least feasible option.

I hope I have been able to help you answer your questions. If you have any more questions or something I have said is unclear, please feel free to follow-up. Good luck on your project!

--Brandi

MadelineB · Post by **MadelineB** » Mon Oct 26, 2020 9:47 pm

Hello lylahgirl66,

You will notice that I've merged your posts on this topic into a single thread. Science Buddies requests that you keep your posts together, since that helps the experts see your follow-up questions.

Thank you and good luck with your project.
Madeline