URGENT PLEASE REPLY Science project - what effect do ultraviolet rays have on yeast colony growth

[hadinakh1]

Hi, to anyone who is reading this I have a question regarding my science project I will be doing for a huge task (kind of like a science fair but not really).

I have chosen the question for my project - What effect do ultraviolet rays have on yeast colony growth? I'm in year 10 and I know this project I have chosen can be complex, but I can also keep it to a year 10 level and not go too deep.

Anyways, I don't have a lot of information on this topic I believe. I have researched many websites, online articles and past experiments and have learnt about this project, and I also have studied genetics in school. But I don't think I have the clearest picture.

If anybody was willing to give any necessary information about this project, or background information or just pointers, it would be really appreciated! I just need a path to follow and the opinion of an expert.

Note: The websites I've viewed and research I've done have led me to many questions so here are some of them (please reply thanks!)
- what kind of experiment would I do? should I use different times of UV flashlights on one sample of UV sensitive yeast, then UV flashlight on wild type yeast to compare the two and find the effect of UV rays on yeast colony growth?
- how would I measure the yeast colonies if they are too small or too much to count?
- how would I keep the project to a pretty moderate year 10 level and not go too deep or complex?
- my experiment seems a bit hard and I don't know if I will apply the best effort when I can't really understand it or do it efficiently. how can I fix this?
- it's currently winter where I am so I don't think I can experiment my question by putting yeast outside (because there is literally no sun at all). so should I use UV flashlights? where can I find that?
- the project I've chosen was from a science buddies recommended project for me. they said that we can purchase the equipment necessary for the project from a website called Carolina biological. if I get this equipment can I still modify the experiment to make it my own?

[17eugenekim]

Hi there,

Sounds like you've got a solid start on planning out this project. I did a similar project on fungal growth in high school, so hopefully I can provide some insights on your questions.

First: for a broad guide on project design, check our our Science Buddies guide page: https://www.sciencebuddies.org/science- ... ience-fair

Don't be discouraged by the complexity of the project! Try to break it down in your mind step by step. Start with why are you doing this project? What is the project hoping to understand? What variables of yeast growth are you interested in? What are your hypotheses and expected results?

Developing your methods is based on what you want to test. Let's say we think that more UV exposure will reduce yeast growth. What aspect of UV exposure should we test? Specifics can include UV lamp intensity, UV wavelength, duration of UV exposure. Type of yeast can be another variable, if you can acquire multiple types. The important thing is that you clearly indicate which variable(s) you tested, and that you have a way of measuring different levels of it. I'd say these are all feasible for a "year 10 level" project.

Continuing the example: let's say we want to only look at duration of UV on one type of yeast. Then we can start forming experimental groups. Some background research can go into this, but let's just say we make three groups: one that is always under UV, one that gets UV for half the day, and one that doesn't get UV (control). Then we count yeast growth at pre-defined intervals, let's say every day at noon for two weeks. Then, with our data of three groups across 14 days, we can plot them and assess what the data says. Again these are all arbitrary examples that I'm coming up with to help illustrate how to build a project plan. All the specifics are up to you and what you want to test.


It sounds like your first step right now is going to be figuring out what your variables are. But below are some answers for your questions on methods:

Hopefully the yeast colonies won't be hard to count. There's a visual guide here: https://www.sciencebuddies.org/science- ... gar-plates Usually you'll just get spots to count. This assumes you're using agar plates of some kind, which you can buy online. Make sure to use sterile technique and don't get the plates contaminated when using them.

UV lamps should be purchasable in various shops. There are UV "blacklight" bulbs and flashlights, sometimes used for cleaning to make stains visible. There are full-spectrum "sunlight" bulbs that are designed to give off UV as well as visible light, sometimes used in pet care. There are sterilizer lamps used for germicidal purposes. There are some UV lights used in beauty/skincare, since UV is used in tanning and drying nail polish. These would all fit under the broad "UV light" category, which is also good for how different UV wavelengths may have different effects. I don't know what your budget is but definitely don't feel the need to get all of them. Decide what your variables are first. Also make sure they actually give off UV light (look up what wavelengths that would be) - some cheap bulbs barely do, and just tint their bulbs purple. Oh, and avoid looking directly at UV light.


Part of science is just doing things, seeing if they work, refining things to make them work better, rinse and repeat. I didn't really know what I was getting myself into either, but don't be afraid to fail. You can fail at every step of the way and still do good science, as long as you look back and figure out why you failed.

Hope that helps.

[hadinakh1]

Hi 17eugenekim,

Thank you so much for your reply and the really helpful information you gave! This definitely will stick with me.

I'm doing this project because I want to learn the dangerous impacts of UV (or simply sunlight) on humans - but obviously using yeast as a model organism. I'm hoping to understand the effect UV rays place on yeast, which will help me understand UV light effects on humans. I haven't just yet created a hypothesis and formed an idea of the expected results, that's part 2 (doing the experiment) of my science assignment - I'm currently on part 1 (forming a question, doing background research etc).

I think I will use the 'duration of UV exposure' aspect of UV exposure. I do believe that longer the UV exposure, yeast will reduce growth. I feel like that is a good variable, and I don't know if I will look into different types of yeast. Do you think this is a good variable?

here is what my first ideas of variables may be:
- different durations of UV exposure (using full-spectrum UV light bulbs) - independent variable
- DNA-repair-deficient yeast (dependant variable)
- 3 experimental groups: one agar plate of yeast under UV all day, one agar plate of yeast under UV half of the day, and one agar plate of yeast that doesn't get yeast
- the control variable would be the agar plate of yeast that doesn't get any UV

The example of an experiment you gave me is a good way of finding the answer to my science project question. I think I will take that further. The science buddies version of this project also has an entire experiment explained in it. It comes with a package with sterile equipment and DNA-repair-deficient yeast among others, could I use this to accompany my experiment? I don't want to follow it word for word but the package comes with many convenient things that significantly contribute to my project.

I did some research and I saw that UV lamps with stands are often used in labs. I'll see if my school's labs have one - if not, I'll buy one of those 'full spectrum' UV light bulbs hopefully with a stand!

Anyway, thank you again for the informative information and the recommended websites!

[17eugenekim]

That's a lot of good background and planning! Sounds like a very workable project.

In terms of the materials in the project idea (this one?: https://www.sciencebuddies.org/science- ... #materials), yes I think it's perfectly fine to use the kit and just change your methods a bit. All the equipment will probably be stuff you need to do any variant of this project. That's part of the reason why we have those outlines in the first place—not just for people to take word for word (which is still good!), but for people to be inspired and start asking the questions they really want to answer.

I would clarify your dependent variable to make sure we know exactly what you are measuring. I am assuming it would be colony counts as we discussed earlier. Also, consider your use of the two types of yeast from the kit; why would someone be interested in results from both a DNA-repair-deficient strain and a wildtype strain? If you don't think it's interesting to compare, which one are you going to pick to experiment on, and why? If you do think it's interesting (why?), make sure you're subjecting both strains to the same conditions so that the comparisons are valid. You may need to do a little background work on DNA repair and UV light to flesh out why it might matter, in which case let us know if you're having difficulties.
Based on the yeast strain, you may find some further tuning adjustments to the procedure. For instance, when just using the repair-deficient strain, it may be unnecessary to shed UV light on them all day (why?).

Keep up the good work!

[hadinakh1]

Hi 17eugenekim,

I think I will modify my experiment by only experimenting on a wild-type yeast such as saccharomyces cerevisiae. I chose this type of yeast to experiment on because it is easier and less complex to just study the comparison of results in one type of yeast that has not been modified. It is unnecessary to also test the DNA-repair-deficient yeast as the effect can be easily detected. It's more 'testable' to use the normal strain of yeast (wild-type).

This makes my dependent variable the wild-type yeast strain (or the count of colonies like you said). So as a summary here is kind of like a rough outline of what my experiment could be (including variables):

I will be testing 3 experimental groups of wild-type yeast strain under different durations of UV exposure.
- group 1: yeast on agar plate (1) will be under UV all-day
- group 2: yeast on agar plate (2) will be under UV half the day
- group 3: yeast on an agar plate (3) will not get any UV I will count yeast growth at a pre-defined time for two weeks.

Then I can compare the results and find out what it says.

Anyway, thank you for the information its been super helpful!

[17eugenekim]

Excellent! That seems like a great revised plan with clear justifications, and a good place to start the experiments. Glad I could be of assistance, and if you run into further questions down the line, let me know!

[hadinakh1]

hi 17eugenekim, I have a few other questions now that i am upt o creating my experimental procedure for my project on 'what is the "What effect do ultraviolet rays have on yeast colony growth?". So far, heres what I've come up with for my plan:

Hypothesis: Petri dishes of saccharomyces cerevisiae exposed to an increased duration of UV light will have fewer colonies than petri dishes of saccharomyces cerevisiae which are left unexposed.

independent variable: The duration of UV exposure, measured in hours
dependent variable: Quantity of colonies of yeast, measured by counting, at a predefined time interval
control variables: Strength of UV light
- Same type and amount of agar on each petri dish
- Same type and amount of yeast in each petri dish
- Same size of petri dishes
- distance between yeast plates and UV lamp

3 experimental groups
group 1: petri dish/agar plate of wild-type yeast exposed to UV for 1 hour
Group 2: petri dish/agar plate of wild-type yeast exposed to UV for 30 minutes
Group 3: petri dish/agar plate of wild-type yeast left unexposed
Observe results at predefined intervals (??)

Materials:
- wild-type yeast culture (Saccharomyces cerevisiae)
- petri dishes with YED agar medium
- UV yeast experiment kit from carolina biological (i might order this because it has everything i already need but i dont need the uv sensitive yeast)
- sterile toothpicks
- pipettes (??)
- sterile distilled water (??)
- disposable gloves
- sterile culture tubes (what is this used for??)
- permanent marker
- aluminium foil
- plastic wrap
- UV lamp

procedure (first draft):
streaking the plates -
1. make sure working area is clean and disinfected and wear gloves
2. place out one of the agar plates with YED medium, make sure its covered and wrapped in foil/plastic wrap so it doesn't absorb any light and that nothing else gets in the plate.
3. quickly open the lid of the plate and set the lid aside - wrap in plastic wrap if in a sunny area because it can absorb light
4. take a sterile toothpick and 'dip' it into an area where you see yeast growing in the tube
5. spread the yeast on the toothpick onto the plate in a zig zag pattern
6. take a new sterile toothpick and make another zig zag pattern through the first one to separate the yeast cells
7. place the lid on the plate and label with permanent marker: exposed #1, <date>, s.cerevisiae
8. wrap the plate again in aluminium foil to protect it from the light.
9. let the yeast grow for two days in a dark area at room temperature
10. repeat this procedure for the two other agar plates but for the control plate label it 'control, <date>, s.cerevisiae'

exposing the yeast to UV light -
1. remove the lid from the 'exposed #1' labelled plate and immediately cover it in plastic wrap so it doesn't absorb light
2. place the plate about a forearms distance from the UV lamp stand (about 40 cm) and let it be exposed to the UV for an hour.
3. after exposing the first plate, remove the plastic wrap and place the lid on
4. wrap the plate in aluminium foil and let it sit for two days at room temperature in a dark area
5. repeat this same procedure for the 'exposed #2' plate but expose for 30 minutes.

questions i have:
1. How long should I expose my experimental groups 1 and 2 to UV to get good results? What duration would be good enough to get results?
2. Are germicidal lamps a good UV lamp to use when exposing the yeast to UV?
3. Can i buy petri dishes that already have YED agar in it prepared for me to use instantly? Or do i have to buy YED medium and prepare it and pour it into petri dishes?
4. Should i observe my yeast colonies quantity every hour?
5. Would my experiment require serial dilutions for the yeast? Im not sure what serial dilutions are and what the purpose is for the experiment nor do i know how to do it. Wouldn't the serial dilution act as a variable and affect the results so we wouldn’t know which variable affected the results?
6. Should i buy the dna damage experiment package from carolina biological or should i buy the equipment separately? The carolina biological does come with many things necessary such as wild-type yeast, sterile equipment, but also has things i wouldn’t need like pipettes??
7. What are pipettes and what are their purpose in this experiment?
8. What are sterile culture tubes used for and should I use them?? I don't think i would need them because my yeast would be in the agar plates the entire experiment
9. What should I write on my three agar plates of yeast? For example, for experimental group 1 can i write something like ‘exposed #1, <date>, <duration of exposure>, s.cerevisiae’. Then for experimental group 2 i would say ‘exposed #2, <date>, <duration of exposure>, s.cerevisiae’. And for experimental group 3 i would say ‘control, <date>, s.cerevisiae’. Does this seem right?
10. What safety/risk factors do i need to be aware of, hence risk assessment? Especially when I'm handling the living yeast and UV lamp.
11. How far do i need to place the yeast plates from the uv lamps?

[MadelineB]

Hello Hadinakh1,

I merged your most recent post with your previous posts so that the expert who has been helping you can more easily see your new post. Thanks, Madeline, Moderator

[17eugenekim]

Thanks Madeline!

Hi hadinakh1,

Good to see you've made some awesome progress on this project. And in the future you can keep adding posts under this thread and I will be notified all the same.

I'm going to have to take the questions out of order, so bear with me.

Regarding serial dilutions:
The reason why the original project guide calls for serial dilutions is because you'll want to dilute the yeast down to a concentration where you can feasibly count colonies. Going straight from yeast to agar gives you massive blooms of colonies, making it difficult to count. Doing a set of serial dilutions gives you a range of concentrations to work with. Once you find a concentration that works for you (gives countable colonies, ideally somewhere in the double digits), you can just use that one for all trials and throw out the rest.
To illustrate this, check out this image of serial dilution results: https://www.phys.ksu.edu/gene/photos/sd.html (It's bacteria, not yeast, but it gets the same point across.)
You don't want to count out colonies from plates 1-3. Somewhere between 4-5 is ideal. 6 has too few to find significant differences between experiment groups. You would write down which concentration you liked, and use that for the rest of the experiment.
This serial dilution step is why the kit calls for pipettes, sterile water, and sterile tubes.
Also note that the yeast will come in dry form, meaning you'll have to put it in sterile water anyways before you start streaking plates.
I'd recommend doing serial dilutions. Consider it like a preliminary step where you're checking if the procedure will actually work. If you want, you could probably do a bunch of serial dilution tests on a single plate by dividing it into sections, kind of like the picture here: https://www.cs.montana.edu/webworks/pro ... ge002.html
(They use drops of diluted culture instead of streaking. I'd recommend streaking since it's what you'll be doing on the full plates)

Regarding UV exposure:
Because this is the dependent variable, it's tough to say exactly what durations would be best for showing results. The project guide here says just a few minutes of sun exposure, but that's for UV-sensitive mutants. My guess is that, for your wild-type (WT) strains, 30 & 60 mins should be a good place to start. If they end up being too similar, you could adjust, like trying 15 & 45 to compare. Hopefully you'll have enough materials that doing another run won't be an issue.
Distance from UV lamp shouldn't be a huge deal so long as you keep it consistent and write it down, in case you want to troubleshoot or do calculations. I don't know what style/strength of lamp you'll be working with, but as long as the plates are reasonably close, it should be fine.
I see you've modified the procedure so that the two-day incubation comes before the UV exposure. Can I ask why that is? It would make more sense to me to incubate after UV.
Yeast grows decently fast, but you could probably get away with checking and counting every 2-4 hours instead of every hour. That said, more data points is rarely ever a bad thing. Whatever interval you choose, just try to keep it consistent.

Regarding risk & safety:
Yeast is a microorganism, so the usual applies there. Sterile technique protects both you and your experiments. Avoid inhaling yeast from your equipment; heck, wear a mask! (You have one around, right?)
After doing any work with yeast, disinfect the area and wash your hands.
When you're done with yeast, disinfect or bleach the plates. You don't want to throw live biocontamination into the trash.
With UV, don't look at the light, because your eyes can get damaged. Try to avoid UV exposure on your skin.

Regarding materials:
Germicidal lamps are great! Those will probably use UV-C light, the "strongest" (highest energy, shortest wavelength) UV light classification. You'll want UV-C for WT yeast. (Check to make sure what kind of UV you're getting.)
Prepared agar plates should be fine, and helps with keeping things consistent. People use medium because it's usually cheaper.

A few other things:
When streaking with a toothpick, avoid stabbing/damaging the surface of the agar as much as possible.
Labeling plates in the way that you've proposed sounds fine. A tip: label the BOTTOM of your plates! Labeling lids makes less sense because lids can easily be misplaced or exchanged. This goes double for dividing plates into sectors (lids spin around).

That was a lot of info at once but I hope it made sense. Don't hesitate to ask further.

Hope that helps!

[hadinakh1]

Thank you Madeline!

Hi 17eugenekim, thank you for the reply and all of that information, its been very helpful so far. I've concluded a lot of things since your post and I find many things that i found confusing make sense now, so I appreciate that alot.

So, I understand the entire concept of serial dilutions now so I will be using it in my experiment. I will obviously make a bunch of dilutions and choose one that is the best and suitable to use for the rest of the experiment to keep things consistent. One question I have about this though is if in the picture you had referenced, would you happen to know what concentration plates numbers 4-5 would be? they seem like good concentrations to use for the yeast to be well enough to count. Since I am doing the serial dilutions, I most likely will buy the UV effects of yeast kit on carolina biological because it has majority of the materials i would need.

I've concluded that I will be running a few trials of the entire experiment, as well as using many petri dishes/pipettes(maybe??)/culture tubes etc so I would need a lot of materials to sustain everything. Should I buy a kit for more than one student off carolina biological to have enough materials or would this be too pricey along with the prepared agar plates and UV lamp. I've also been researching that prepared agar plates are too expensive for what they should be so to take some of the expense away I could just get the YED medium and prepare it myself.

Also, I might just do a bunch of serial dilutions on a single plate to prevent myself from using many plates and not having enough for the actual experiment. I would label the bottom of the plate into divisions of the serial dilutions and choose a concentration. Also note: should i write down the serial dilutions as a preliminary step in my method/procedure so that my teacher who's marking will know too?

Regarding the UV exposure, i have decided that I will expose them for 15 and 45 minutes so that the results aren't too similar and I don't have that risk of running another trial. I will write down the distance from the UV lamp too, but is there any specific measurement (in centimetres preferably) to use, especially with a strong lamp like a UV-C germicidal lamp?

About the incubation, I was following what the method/procedure was saying on the science buddies website version of the experiment I'm doing (https://www.sciencebuddies.org/science- ... #procedure). I understand now more that it would be more logical to have the two-day incubation period after the UV exposure, and I'm sure I did write that in my procedure in my last post. But, one question I do have, doesn't the yeast need to grow for a little bit before the UV exposure to an extent that we can see the colonies? Or do we just prepare the plates of yeast, let it sit for a bit (not as long as a 2 day incubation) and then straight away expose it? I'm a bit confused on this. Like, it makes more sense to me to let it grow before UV exposure, as well as letting it grow after UV exposure. If I'm wrong, correct me please.

During the incubation/resting/growing time after the UV exposure, I'd check in intervals to write down my observations of the colonies of yeast at around 2-4 hours as you suggested. Could you consider a specific duration if thats alright? Like maybe every 2 hours or 3. I would like it to be not too many data points as I think it'll just get confusing for me.

For the safety and risk; I will be wearing a mask, frequently washing my hands and disinfecting the area, and disinfecting the materials once I'm done with them. I'll also wear safety glasses and cover any exposed parts of my body to prevent UV damage. I'll also ensure I add these all to my risk assessment.

I've researched germicidal lamps on many websites such as amazon, carolina biologica and fischer supplier. I've noticed that the lamps I find on amazon are for the majority hand-held and could be fakes just to make profit for the high demand of disinfectant lights. However, some of them are cheap compared to the science suppliers which are wayyyy too overpriced. So, I could buy a good reliable looking one from amazon and use a cardboard box with a square cutout at the top to place the handheld light on. Then, i can place the plates underneath the box and let it be exposed. What do you think of this so far?

Anyways, sorry for the long replies but I had to fit in a few more questions ! I appreciate all of the responses and the help you've given me so far, I literally have done my whole project based on what you have told me.

[17eugenekim]

I have no idea what concentrations to use to get something like plates 4-5. I also don't know what concentration you will start with from your yeast stock. This is partly why you'll need to do a serial dilution test. According to the guide, best guess would be somewhere between 1:1000-1:10000 based on the yeast from the kit, but again, no clue till we try it.

I don't think you'll need more than one kit, but it's your call. The kit should come with surplus of everything (it's designed for 4 students), the only possible exception being petri dishes to make agar plates. You might need to buy more, but getting those separately will definitely save you a lot of money than buying two kits.

Serial dilution is still part of your procedure. I called it "preliminary" to help clarify what it's doing for your project. I'm sorry if that was confusing.

What made you decide on 15 and 45 minutes? The numbers I'm giving are purely hypothetical. I only proposed 15 & 45 so that, if you did 30 & 60 the first time, you would effectively have data from 15, 30, 45, & 60 minutes across two trials. These are just examples. I have never performed this experiment and do not know what to expect.

Likewise with the lamp, as long as it's consistent, the exact number is not important. You mentioned you want to put the lamp in a cut-out box. Just measure to make sure the box(es) for each trial are fairly similar.

What you're doing with UV exposure is you are damaging the yeast's ability to reproduce and make colonies. When you first put yeast on the plates, there are already microscopic yeast cells ready to make colonies (what we call "viable" cells). Damaging these parental cells with UV is the basis of the project. If you damaged them after they already grew into colonies, it would be hard to tell what exactly is the effect of UV damage.

For observation, I offered a range because I don't know what you should expect. You'll have to pick something to start and might need to adjust based on how things go. You could even do every 6 hours if you feel like 2-4 is too many.

Don't get too bogged down in the details. A lot of things will come to you as you go. And be prepared to troubleshoot.

Things are looking great so far - I wish you luck!

[hadinakh1]

Thanks for the reply!

I understand majority of everything now and I do understand that I shouldn't be detailing every single thing, I've always been like that with details. I understand I have to troubleshoot when doing my experiment, it's pretty much inevitable but I'll make sure I still do good science.

One thing that has changed since my last post, I have realised I can't buy the kit from Carolina Biological as it has to ship overseas and it is asking for a quote summary with all of my details. I find this a bit dodgy why it would be asking for a quote, so I don't think it is safe to buy from there. I can get some of my materials from my school lab if they have some such as the pipets, test tubes, maybe even a UV light, petri dishes (if not, i can buy it online), sterile water, and sterile toothpicks. I'm not sure what I will do with the agar medium because the experiment recommends YED agar and I can't get that anywhere besides Carolina Biological (unfortunate). I also don't know how i'm going to get the wild-type yeast culture? I can obviously buy a UV light from online, and I can buy types of agar online but not YED specifically.

I chose the 15 and 45 minutes to compare the results so that they are not too similar, but I get where you're coming from so I may start off with 30 mins to have results from 15, 30 etc mins. Regarding the diy UV light box, ill make sure to write measurements down and use the same box for every trial/experimental group. Heres a photo on a similar idea: https://www.livelarq.com/blog/the-diy-b ... ld-objects.

Thanks for the advice and information!

[17eugenekim]

That's too bad! I don't know much about their business so I can't say whether they're dodgy or not, but I'm glad you're looking for decent alternatives. YED agar happens to be the type of agar they recommend (there's tons of types of medium), but any type of nutrient agar you can get should work for growing yeast. The most basic "general-purpose nutrient agar" is fine for many fungi: https://www.sciencebuddies.org/science- ... gar-plates

Getting yeast culture should also be possible, since a lot of people buy yeast to make bread or brew beer from home. I just can't promise it'll be cheap. But these starter cultures should be viable for the experiment. I'm sure there are some differences in purity or something between food-oriented and lab-oriented cultures on the market, but I can't imagine that obstructing your project significantly. At best it's something worth noting in your later discussions or reflections on results.

Hope that helps!

[hadinakh1]

Hi 17eugenekim,

I have a few more questions about my procedure, I've finally made my first 'draft' and I wanted to see your opinion if it is good and suitable. Note: the procedure is super long sorry

Preliminary Steps (note - make sure you write down any important numbers, observations, or just anything you find important in you logbook) -
Preparing Agar Plates -
1. Put on gloves and a lab coat.
2. Add 100mL of distilled water to a graduated cylinder
3. Place a wayboat onto a weight scale and measure out 5 grams of nutrient agar powder.
4. Add the powder to a flask, then add about 75mL of the distilled water to the flask.
5. Stir the flask well but gently for a minute or so.
6. After, add the remaining 25 mL of water to the flask.
7. Stir the solution until all of the visible clumps have dissolved.
8. Wrap the top of the flask in aluminium foil and tape, label the medium name on the tape.
9. Sterilise the agar. --------- (this has a dash because I don't know how to sterilise the agar flask/bottle and I'm not sure if my school lab has an autoclave?)
10. Once sterilised, stir the media very gently, and allow it to cool to 50ºC.
11. Place the petri dishes in a stack.
12. Start by pouring the media at the bottom of the stack, lifting up the entire stack as you pour your media. Make sure you fill the plates enough so the bottom is covered. Fill 15 plates with agar.
13. Once the media has cooled, turn the plates upside down to prevent moisture condensing on the agar surface. Wrap them in aluminium foil and store them in a plastic bag until you're ready to use them.
Making the serial dilutions -
14. Weigh the dry yeast package, then suspend contents into 100mL of distilled water in a beaker. Stir well for 5-10 minutes.
15. Weigh empty packages of yeast and determine the dry weight of the yeast added, write this down in your logbook.
16. Prepare 4 other test tubes all with 9mL of sterile distilled water. Label the tubes 1 through to 4 with their dilution factor i.e 1st tube is 10^-1, 2nd tube is 10^-2, next is 10^-3 and 4th tube is 10^-4.
17. Draw 1mL of the yeast solution into a sterile pipet.
18. Add the 1mL to the first test tube to make it a volume of 10mL. This makes an initial dilution of 10^-1. Properly mix the dilution for a minute or two.
19. Discard the pipet and grab a fresh sterile one.
20. Now, 1 ml of mixture is taken from the 10-1 dilution and is emptied into the second tube. The second tube now has a total dilution factor of 10-2.
21. The same process is then repeated for the remaining tube, taking 1 ml from the previous tube and adding it to the next 9 ml diluents.
22. As four tubes are used, the final dilution for the yeast cells will be 10^-4 (1 in 10,000).
23. Wrap the top of the tubes with aluminium foil and tape, label the tape with the yeast type name.
Spreading the diluted yeast solutions onto an agar plate -
24. Prepare a petri dish of agar growth medium.
25. Label the bottom of the plate into 4 even squares with a permanent marker, with their dilution factors labelled (i.e 10^-1). Label the type of yeast name and date as well.
26. Open the lid of the petri dish just enough to pipet the solution, use a pipet to draw 1mL of one of the solutions to the appropriately labelled square and use a sterile toothpick to spread the yeast cells in that square. Note: after pipetting one solution at a time, quickly close the lid of the plate so it does not get contaminated.
27. Repeat this same process (in step 3) for the rest of the solutions in their appropriately labelled square.
28. Turn the plate upside down (lid facing down) and wrap it in aluminium foil. Let the plate sit at room temperature for two days to grow. This is to see which dilution factor produces enough colonies suitable enough to count.
29. Wrap the test tube with aluminium foil and tape labelled with the yeast type name, store them somewhere dry and clean of other contaminants.
Experiment -
Streaking -
1. After the serial dilution plate has grown and you have chosen a dilution factor that produces well separated colonies that are good enough to count (choose a diluent factor that has colonies around the double digits), it is time to use that dilution to streak agar plates.
2. Place out the dilution test tube you are going to use, a new agar plate lid facing down, sterile toothpicks and sterile pipettes on the table
3. Prepare a bunsen burner.
4. Take off the foil on the test tube and pass the neck of the tube over the bunsen burner flame 2-3 times to sterilise it.
5. Use a sterile pipette to draw 0.250 mL of the solution without the pipet touching the sides of the tube.
6. Pass the neck of the tube over the bunsen burner again another 2-3 times and place the tube on the table with foil placed back over the top.
7. Open the lid plate and add the 0.250mL of the solution, quickly grab a sterile toothpick and smother the solution across the agar. Be careful to use the toothpick gently on the agar to not damage it.
8. Repeat this same process for the remaining two agar plates.
9. Label 2 of the 3 agar plates with the date, yeast type name, dilution factor, initials of your name, and exposed. Label the remaining agar plate with the date, yeast type name, dilution factor, initials of your name, and controlled. Look at the following as an example:
10. Wrap all three agar plates with aluminium foil to protect it from contaminants and the sun.
Exposing the Yeast to UV light (note - make sure the exposing area is a human-free zone and is a dry and clean place) -
11. Clean your work area with a disinfectant such as bleach, soap, or wipes. Wash hands and put on gloves. Wear your protective glasses and make sure your covering your whole body.
12. Place the two exposed agar plates wrapped in foil on your table.
13. Remove the lid from one of the exposed labelled plates and immediately cover the lid in plastic wrap as it may absorb UV light.
14. Place the plate in the UV exposure box and turn on the UV lamp. Turn on your timer for one hour and let it be exposed for that duration. Be quick to not be in the room too long while the UV light is on, avoid looking directly at the light as well.
25. After exposing the yeast, remove the plastic wrap and replace with the lid. Wrap the plate in aluminium foil to prevent UV absorption.
16. Place the remaining exposed plate in the UV exposure box and turn on the UV lamp. Turn on your timer for 30 minutes and let it be exposed for that duration. Once again, be quick not to be in the room for too long and avoid looking directly at the light.
17. After exposing the yeast, remove the plastic wrap and replace with the lid. Wrap the plate in aluminium foil.
18. Wrap the plates in aluminum foil to protect them from light and let them sit at room temperature for two days.
Post-Experiment -
Analysing the results -
1. Unwrap the plates and count the number of colonies on each. Each colony is formed from a single yeast cell.
2. Graph the number of colonies for each plate. Put the number of colonies on the y-axis and the treatment and dilution on the x-axis.
3. Calculate the percentage of cells killed by UV light. See Equation 1, below. Compare colony counts from plates with the same dilution.
Equation 1: 100 × ( 1 - colonies on exposed plates/colonies on control plate) = % killed. Graph the percentage of yeast killed by exposure to the sunlight.

After that , now, here are my questions I have:
1. when I'm letting the serial dilution plate grow, the test tubes with the dilution solutions in them have to go somewhere but I don't know where. Should I refrigerate them? obviously I will wrap it in foil and tape labelled with its name to keep it sterile, but I'm not sure where to store it for the meantime. It has to be somewhere where it can stay sterile and useable for at least 1 or 2 days depending on the yeast growth

2. should I add diagrams to my procedure to make things clearer? I've already made illustrations of how to label plates but I couldn't copy it into this text. Any key places I should make a diagram?

And I think thats about it, thanks!

[17eugenekim]

Excellent - a very meticulously planned procedure! I personally can't speak for the details on things like precise numbers and measurements, but from what I can tell, you've got a solid plan so far.

1) Probably put them in a fridge. I was thinking if you should discard those tubes you used to streak your serial dilution test plates, then re-make one using whatever dilution factor you found was optimal. But this does change your material usage. Refrigerating yeast is a viable way of storing them, especially if it's only a few days. Wrap the tubes tightly in foil or plastic before refrigerating. Unless you want to keep them for much longer (i.e. months), don't freeze them.

2) Diagrams are awesome and help get the point across in fewer words. Create diagrams or attach pictures wherever you think the audience would benefit; if you end up with more visuals than necessary, you can always throw them out of your presentation/poster/etc later. If you're spending too many sentences describing what something looks like, that might be good place for a visual. It may also be a good idea to take pictures of incubated plates, not only to demonstrate the procedure but also to remember what they looked like in case you throw them out too early and forget to count or something.