Need Help ASAP

[deleted-653124]

Hey! I’m a student, and I’m trying to do a science fair project dealing with yeast on agar Petri dishes under different types of light. I was wondering if I can activate store-brought yeast and take a Q-tip and swab it on a normal regular agar dish instead of a YED dish? Will it still grow, and is it growing mold? If it grows mold, then I wasn’t understanding why it would grow mold. Can you guys please help me ASAP?
Thank you very much, Cassidy

[SciB]

Hey, Cassidy. Those are all great questions and I don't know the answers. The yeast you buy in the store is bread yeast and I think it will grow on plain nutrient agar.

What are you measuring? You think light will affect the growth? Why? Yeast does not need light. It is not photosynthetic like algae. What did you mean by 'types' of light? If you shined ultraviolet light on yeast, it might have a negative effect because UV can damage DNA.

If you want to work with something more responsive to light of different colors I would suggest chlorella which is a kind of alga that grows easily in solutions and is green and photosynthesizes to make oxygen that you can collect and measure.

Let me know what other questions you have.

Sybee

[deleted-653124]

I’m going to be measuring the growth of the yeast colonies on the agar plate... I chose different types of lights because sometimes before people start using the yeast they put it or place it somewhere that always deals with light in their homes... I was also very unsure about this world application, but if you can find anything else that would be easy to measure and use please help me... I researched about that algae but I found it hard to approach because I don’t have a science lab or anything, so I didn’t want to do something too difficult. Thank you!

[SciB]

Hi Cass,
You don't have to use a lab. You can do experiments in your kitchen with ordinary tools.
I suggested algae because I know that they do respond to light, but maybe yeast will too. You won't know unless you try it.

The one thing that you will have to test first is how much yeast to spread on the agar. If you put too much it will grow over the entire surface and there won't be any separate colonies. What you need to do is dilute the yeast first and then spread it on the plate.

Dilutions are easy. You just need some sterile distilled water (DW) which you can get in the grocery store. Now you will want to make a saline solution for the dilutions because if you use water the yeast cells may swell and burst. Get a glass jar that will hold at least 8 ounces, wash it and rinse it well then place the wet jar in the microwave and sterilize it by microwaving it on high for 1 minute. Sterilize the lid by pouring some rubbing alcohol in it, letting it stand for about a minute and pouring the alcohol down the drain.

Now put 8 ounces of sterile water into your sterile jar and put the lid on. Add 1/4 teaspoon (tsp) of ordinary table salt (non-iodized is best to use) and 1/8 tsp of baking soda (sodium bicarbonate) and swirl the liquid carefully until all the crystals dissolve.

For the dilutions you will need a glass eyedropper with a rubber squeeze bulb. Sterilize the dropper by sucking up a litlle rubbing alcohol and leaving it for a minute. Rinse the dropper with sterile DW and keep it sterile by not letting anything non-sterile touch it.

Now, to make dilutions of the yeast you will need some culture tubes to make the dilutions in. You can look for them online. I'm sure Amazon has some that would be acceptable, but we usually use Carolina Bio for supplies:

https://www.carolina.com/lab-tubes-tubi ... tx=on_site

Wash the tubes, rinse well and sterilize them in the microwave on high for one minute.

Fill one tube half full with your sterile saline and add a small amount of dry yeast--about as much as fits on the tip of a steak knife. Swirl the liquid gently until the yeast dissolves. Now set up 4 sterile glass culture tubes each filled about half full with saline. Using the sterile dropper, swirl the yeast solution and suck up a little. Put 3 drops of yeast solution in the first tube and label it 1:10. Rinse the dropper with sterile saline. Swirl the 1:10 tube and suck up some of the liquid and put 3 drops into the next tube and label it 1:100. Repeat this two more times, labeling the next tubes 1:1000 and 1:10,000.

That is the way to make dilutions of any single-celled organism either bacteria or yeast.

Now you are ready to spread the yeast on the agar plates. Remember, this is just a test to see how much yeast it takes to get separate colonies. Swirl each dilution tube, suck up some of the liquid with the dropper and put three drops on the agar and spread it carefully all over the surface using a sterile glass spreader. I can show you how to make one of those.

After you have spread all the plates, labeled of course, put them in a warm (24C) place and leave them there until the colonies grow up. Make a note of the dilution that gave you the greatest number of separate colonies. When you count colonies you need to count about 100 to get an accurate result.

OK. That's it. Now you know how to grow yeast on agar and can now do the experiment with exposing the plates to different lights. Remember to do 3 plates for EACH treatment so you can average the results and get a statistical answer.

I hope this was helpful. I'm sure you will have more questions, so post again when you do.

Good luck!

Sybee

[deleted-653124]

Okay thank you so much! Now I understand dilution so much more than I did before! So I researched and it said that mold will grow on the agar plate, why is that? Also, since I’m exposing different kinds of light (incandescent, black light, fluorescent, and LED light) do you think I should try to get them at all the same wattages because I understand that all of them are different. Also, my plan to expose the dishes under the light is to put the dishes under each light for maybe 30 minutes and then I’ll gather them all together and put it under the incandescent light because that’s the most natural state for an incubator. Would that be okay and what’s the most natural wattage for the light of the incubator because I’m going to be making my own incubator at home with a cardboard box.

[SciB]

Hi Cass,
I'm glad my explanation of dilution helped. There are also a lot of videos about making dilutions and spreading bacteria on agar plates on YouTube that you can watch. Seeing someone do this is always better, I think, than just reading about it.

Try to find lights that give the illumination in lumens. You can't use wattage because different light sources provide different amounts of light per watt. For example, an 18W LED can provide the same lumens as a 75W incandescent. I'm not sure if 'black' light is measured in lumens. If you go to a pet store, like Petsmart or Petco, you can see what are called reptile bulbs that provide a certain amount of ultraviolet light that is apparently beneficial to them. You might want to try this lamp also.

I did a search for making a homemade incubator and this site used a 15 W incandescent bulb i a box to maintain a reasonably warm temp (80-95F) without getting it too warm: https://classroom.synonym.com/make-incu ... 39092.html

Bacteria, molds and fungi will all grow on nutrient agar. That's why you have to try and keep your dilution tubes, saline, dropper and yeast solution sterile to avoid contamination and overgrowth with undesirable microbes.

I don't know how much you will have to dilute the yeast in order to see separate colonies. You will just have to do the experiment to find out. Be sure you record everything in your lab notebook--don't trust to memory! Also, use your camera to take pics of what your are doing. You could do a video if you want to be able to explain the procedure as you go along. Just have your partner hold the camera while you are talking about the yeast or making dilutions and spreading the agar plates.

There is one technical problem that I foresee and that is the way you will have to expose the yeast to the light. I assume that you are using disposable plastic Petri dishes, right? Well, the plastic seems clear but it may absorb some light, especially in the ultraviolet (UV) range, so you really should do the light exposure with the lid off the dish. I know this also exposes the agar to bacteria, mold spores and wild yeasts and you may not want to do it. Alternatively, you can do some research online and find out just how transparent the plastic of the Petri dish is. If you have time, you can do your own experiment exposing the yeast on agar to light either through the plastic lid or with the lid taken off. Counting the colonies would tell you if the light effect was different with the lid off.

Let me know when you have more questions.

Sybee

[deleted-653124]

Thank you!! I’m doing my project right now... and I’m wondering is it okay if we use a sanitized Q-tip to spread the yeast on the agar plate?

[SciB]

Spreading microbes evenly on agar is always a problem and swabs don't work very well. That's why we use glass or plastic spreaders in the lab. You can use a large paper clip to make a spreader and because it is metal, you can sterilize it with rubbing alcohol that you burn off in a flame.

I can't easily explain what shape to bend the straightened-out paper clip into, so I will tell you to watch this video that shows the method for spreading bacteria on an agar plate using a disposable plastic spreader. This will show you the right moves and also what the spreader looks like so you can duplicate it with the paper clip: https://www.youtube.com/watch?v=jr4-ye50Za4

If this video doesn't provide enough information, you can watch others on the list and eventually you will get the idea how to do a good spread.

Let me know if you have more questions.

Sybee

[deleted-653124]

Thank you! So I'm trying to grow yeast on agar, and i don't know which type of agar that I should use that is easy to buy from online. I heard that YED should be used, but I can't easily get access to it because I'm a student... Do you know any safe agar petri dishes that can be easily brought that i can use? Maybe from Amazon would be amazing!

[SciB]

Hi Cass,
Good to hear from you again.

I would try growing the yeast on nutrient agar plates if you have them, otherwise Carolina Bio sells YED (yeast extract dextrose) agar that is specifically for growing yeast: https://www.carolina.com/yeast-genetics ... n=yed+agar

The problem is that the YED is in powder form so you will have to mix it in 500 mL of sterile water (distilled water from the store is usually sterile if unopened), boil it to dissolve the agar (Carolina sends it with instructions) and then pour it into plastic Petri dishes. 500 mL of YED agar is enough to pour 20 100 mm dishes using about 25 mL per dish. These sterile disposable plastic Petri dishes are available from Carolina Bio in sleeves of 20: https://www.carolina.com/lab-dishes/pet ... tx=on_site

I would use a 4 cup Pyrex glass measuring cup which has mL markings. This will easily hold 500 mL of sterile water plus the YED agar and it will be easy to pour the plates because of the pour spout.

I think the best method for heating the solution to dissolve the agar is to get a 3.5 to 4 quart metal pan which is large enough to hold the 4 cup glass measure. Put 500 mL of tap water into the cup, put it into the pan and then put water into the pan until the level comes up to about half an inch above the level of liquid in the measuring cup.

Remove the cup and discard the water. Put the pot containing the water on the stove and heat it until the water is almost boiling. Wash the measuring cup, rinse it ten times with hot tap water then a couple times with sterile water. Put about 500 mL of sterile water into the cup and put it into the pan on the stove.

Add the YED medium to the 500 mL of water and stir with a clean spoon. Heat the water in the pan until it is at the simmer but not boiling violently. Keep stirring the agar medium until it dissolves completely and becomes clear yellow. Cover the cup with a clean saucer and keep heating at the simmer for ten minutes longer to sterilize the agar medium.

Normally, in the lab, the agar would be sterilized in a steam autoclave under pressure. The pressure allows the temperature of the water to reach 121 degrees Celsius (250 F) which is hot enough to kill nearly all bacteria and fungi. The boiling in the pan that you do is enough to kill most microbes and is sufficient for the yeast experiments.

After the agar medium has been heated for 10 minutes, turn off the heat, move the pan to a cool part of the stove or a trivet and allow it to cool before pouring the plates. The agar starts to solidify about 50 degrees C so you want to pour the plates when the water in the pan reaches about 60 deg C. I am often impatient and start pouring the plates almost immediately and sometimes melt the plastic of the dish! https://bitesizebio.com/6938/how-to-mak ... very-time/

I can't tell you how to pour out exactly 25 mL per plate. We don't measure it. We just know from experience and looking at the depth of liquid in the dish when to stop pouring. In a 100 mm diameter Petri dish, you want the agar depth to be about 3 mm and from 500 mL of agar you should be able to pour 20 plates.

There are a lot of good videos that show how to make agar plates and you can watch these to get any details that I may have forgotten.

Store the plates upside down in the plastic sleeve that they came in. The reason the plates are stored upside down is because water will condense on the lid and you don't want it running down onto the agar and soaking it. That's another reason to let the agar cool before pouring. The hotter it is, the more water vapor will condense on the lids. If you are going to use the plates in a day or so, you can store them at room temperature, otherwise put them in the refrigerator at 4 deg C. When you take the plates out to use, keep them upside down and if you see a lot of water droplets on the lid, remove it and quickly shake the water off into the sink and replace the lid. The idea is that you don't want the agar surface to be too wet.

OK. I think that's all the info you need to pour the yeast plates. Post again when you have more questions.

Sybee

[deleted-653124]

Thank you! So I just ordered the nutrient agar plates... I’m trying to maintain the temperatures of the incubators, and since there are different types of lights that I’m using then there will be different temperatures of the lights... Is there a technique to maintain the temperatures of the incandescent light so it won’t over or under heat? My plan is to put the agar dishes under the different types of lights for an hour, and then take it out and put it under the incandescent light for it to grow... will that work?

[deleted-653124]

[quote="CassL07"]Thank you! So I just ordered the nutrient agar plates... I’m trying to maintain the temperatures of the incubators, and since there are different types of lights that I’m using then there will be different temperatures of the lights... Is there a technique to maintain the temperatures of the incandescent light so it won’t over or under heat? I’m scared that since I have a lot of lights on and running for a long time, will it burn or cause a fire? How do I prevent that from happening? My plan is to put the agar dishes under the different types of lights for an hour, and then take it out and put it under the incandescent light for it to grow... will that work?

[SciB]

Hi Cass,

Let's be clear on one thing--well several things--yeast is NOT a plant, it is a fungus like mushrooms and mold. Plants have chlorophyll and need light energy to make sugars. Yeast does NOT. It eats the available sugars on fruits, vegies and other things.

There is really only one way that light could affect baker's yeast that I know of and that would be by mutating DNA. Radiation such as from the sun's ultraviolet can cause skin cancer by mutating DNA in skin cells.

What kinds of light were you planning to use?

Yeast will grow at temperatures from 65 to 95 F, but faster at the higher temperatures. Don't you have a thermometer? Put the thermometer in the box and turn on the light, wait 30 minutes or so and read the temperature. What is the temperature of the air in the room where you are going to do the experiment? If it is cold, like 60 F, then you may have to put the yeast plates somewhere else to grow where it is warmer.

Don't worry about the lights starting a fire. You can use a 40 watt incandescent light and that would not heat up the area that much. LEDs and fluorescent lights don't give off that much heat.

How long are you planning to keep the lights on the yeast? What is your control? Darkness all the time?

What is your dependent variable--what are you measuring? Colonies? I think I mentioned in one of my previous posts about making dilutions of the yeast in liquid so you can get individual colonies. If you just swab yeast on agar you will get smears that all look the same and you won't have anything to measure. Go to youtube and watch some videos on making dilutions for plating on agar so you will understand what i mean. Most of the videos will be about diluting bacterial cells, but it works the same way for yeast which is a single-celled fungus.

If you don't understand all this, look it up online. There are many good lectures and tutorials that explain everything about doing experiments with yeast and bacteria. You can educate yourself on the internet. You don't have to wait for a teacher to tell you something. Look it up yourself.

Good luck!

Sybee

[deleted-653124]

For the streaking of the colonies, is it better to streak it or to use the spreader and do it in circular motions instead of the zig-zag motion?

[SciB]

You have to visualize an even film of liquid spread over the surface of the agar--that's the goal. Use the spreader after sterilizing it briefly in a candle flame.

If you look at some of the videos on spreading bacteria, you will see that there is not one way to spread the cells evenly. When I do it I push the spreader all over the surface including the edges of the dish. It's a combination of circular and straight motions but the one thing you have to be careful of is digging into the agar surface with the spreader. You have to hold it sort of loosely, and move it gently.

When you bend the paper clip to make the spreader, be sure that you turn up the end because that is the part that is most likely to dig into the agar.

Practice is the best thing for doing good spreads. After you have done 20 plates, you will be an expert.

Sybee

[deleted-653124]

So it’s been two days, and I’ve been waiting for my agar plates (the different diltutions) to grow but it hasn’t grown anything.... I don’t know if I should add more yeast because when I was doing the dilutions in the culture tubes after the 1:10 the liquid didn’t seem yellow or little brown... it looked like almost liquid water... since I already did everything and now waiting for the agar plates to grow, are there any suggestions that I can do for my plates to grow because nothing grew yet?

[SciB]

Sorry to hear of the problems, but I'm afraid that's pretty normal for science experiments.

Well, in order to answer your Q about why no yeast growth i need to ask you some Qs. First, what temp are you incubating the plates at? Too cold, or too hot and the cells won't grow and may die. If you have the plates in a cool place [12-15 degrees Celsius] the yeast may not make colonies. The plates have to be keep at 20-30 C.

How did you do the dilutions? How much of a dilution did you make? How much of the yeast solution did you put on each plate?Do the plates appear moist or dry?

What is the source of your yeast? are you sure that it is alive? You can test it by putting a little of the yeast powder into some water with sugar [1 tsp sugar to 1/4 c water]--sucrose, that is, aka table sugar. if the yeastie beasties are alive they will start eating the sugar and making carbon dioxide gas that you will see as bubbles in the solution.

That's all the possibilities I can think of right now. Let me know the answers to the Qs and I may be able to suggest something else for you to try.

Sybee

[deleted-653124]

Thank you! Actually, the colonies did end up growing! The 1:10 and the 1:100 didn’t have clear results, but the 1:1000 and the 1:10000 grew pretty clear. With this case, I don’t know which dilution is best to use for my experiment... Also, for my actual experiment instead of salt and baking soda, should I use water and sugar instead to activate the yeast for the dilution? Or should I just leave it as salt and baking soda mixed with water and yeast which I did for the dilutions?

[deleted-653124]

Also, how do you dispose the agar plates?

[SciB]

Hi Cass,
Glad to hear that your yeast colonies finally appeared. The lower dilutions were probably just one uniform layer of yeast so no separate colonies to see on the agar. For good statistical measurements you need to be able to count about 200 colonies per plate, so choose the dilution that will give you the number.

The nutrient agar has the sugar glucose (also called 'dextrose') in it so you don't have to add sugar to the yeast solution--but on the other hand, it does no harm and it might get them started a little quicker.

For plate disposal, fill a bucket half full with 10% Clorox (12 ounces of Clorox plus 116 ounces of water--approx.) and put the plates and agar and yeast in there and let them sit for several hours or overnight. Yeast itself is harmless to humans, but there could be some bad bacteria or fungi growing on the nutrient agar and you want to make sure they are all dead. The sodium hypochlorite in Clorox will kill nearly all bacteria, viruses and fungi. It is also VERY irritating to eyes and skin, so be sure to wear safety glasses and disposable gloves when making and handling the Clorox solution. It is a household chemical, but if you splash some into your eyes it would be harmful and you would need to rinse with water very thoroughly.

Your eyes are pretty important to the rest of your life and it certainly is not worth risking them. I even wear safety glasses outside when i'm gardening and doing yard work because I have had branches hit me in the face and stones from the lawn mower fly up. Being safe is not wimpy--it's smart!

Good luck!

Sybee

[deleted-653124]

Thank you very much! For my actual experiment, I actually noticed some black smudges on the agar... what is that supposed to be? And it’s also on the black light group... for my dilutions this didn’t occur...

[deleted-653124]

For my dilutions, I’m pretty sure it’s yeast colonies because it was yellow with dots, but for my actual experiment, I’m noticing black smudges which could be mold growing on the black light group which is supposed to be UV light... The black light was at about 100 degrees Fahrenheit which I thought would for sure kill the cells but it didn’t? Please help! Thank you!

[SciB]

Hello again.

I hope the lids of your Petri dishes are securely taped onto the bottoms. Don't open the plates! You may expose yourself to the spores or aerosols or dangerous pathogens, and that is a BAD idea.

There are several types of fungus such as Aspergillus (https://en.wikipedia.org/wiki/Aspergillus_niger) and Stachybotrys (https://en.wikipedia.org/wiki/Stachybotrys) that are black or dark colored and that is probably what you are seeing on the agar as their spores are commonly floating around in the air. These living spores can cause serious lung infections--that's why I said don't open the plates. Take your photos through the clear plastic lids.

Yeast can grow at temperatures around 100F and the surface of the agar was probably cooler than that.

Keep posting and let me know what you discover.

Sybee

[deleted-653124]

I put two pieces of tape onto each side of the agar plates and didn’t open any of them... But I’m confused because I don’t understand why did my dilutions grew yeast colonies, but for my actual experiment, it grew mold instead. Are there reasons for that? Also, probably the black light that I used didn’t emit UV rays, and the temperatures for that was pretty high around 100 degrees Fahrenheit. That was probably why my black light group grew first and maybe bigger than the other groups. But the one thing I’m concerned about is why did it grow yeast colonies and then now it’s mold?!? Please help, and thank you so much!

[SciB]

Well, I wish i had a good answer to your dilemma, Cass. If I were there to look at your plates and see the set-up, I might be able to find an answer.

I am a little confused about what you meant by this: "For my dilutions, I’m pretty sure it’s yeast colonies because it was yellow with dots, but for my actual experiment..."

You should be using the yeast dilution for your actual experiment. Can you explain this better. There's something I am not understanding.

Mold spores are in the air and could land on some of your plates but not others. I don't think there's a connection between the light and the mold. If you want a lamp that emits UV, go to a pet supply store and get a UVB reptile light: https://www.petsmart.com/reptile/enviro ... and-lamps/. This might work better than a 'black' light. You need to be careful with UV as it can burn the retina of your eyes and cause DNA damage. UVB is not that harmful but there are some germicidal UV lamps that can be dangerous to your eyes and skin--just so you know.

I hope you are less confused now; but post again with more questions and I will continue to help.

Sybee

[deleted-653124]

For my experiment with all the different kinds of lights, I used the 1:1,000 dilution... When I was testing the dilutions and seeing which one grew the best, the 1:1,000 dilution grew best, and it grew actual yeast colonies. But for my experiment with all the different types of lights, the 1:1,000 grew mold instead of yeast colonies... and it grew mold on all the plates too under the different lights. So for the dilutions, it grew yeast colonies, and for some reason, my actual experiment grew mold instead of yeast colonies. Nothing was changed between the methods but just placed under different types of lights for my actual experiment. Thank you!

[SciB]

Thanks for the explanation--now I see what you meant.

My question would be--what yeast culture did you use for the actual experiment? The same one that you used for the dilution? Did the yeast solution contain sugar? How long was the time between when you did the dilution test and the light experiment?

The dry yeast powder is in a dormant state so is not changing much with time; but when you put it into a solution, especially one containing sugar, the yeast will ferment all the sugar and eventually turn it into vinegar or alcohol, neither of which is good for the yeast.

The correct way to do the light experiment would have been to make a fresh dilution of the yeast and spread that on the plate. If that IS what you did then I have no explanation for why you got mold and no yeast colonies. All i can suggest is try it again with a freshly prepared dilution of your yeast and try to keep everything as sterile as possible. Work in a clean area away from drafts and sterilize your spreader before each use [just make sure it cools off before you use it!].

Sybee

[deleted-653124]

So for my actual experiment, I used a brand new pack of yeast (the same brand) and made a fresh new dilution and just used the dilution that I needed... For the dilution, I just used baking soda and salt for the solution... there were no sugar added... but you said something about alcohol, and maybe when I sterilized the spreaders there were still alcohol on it and it didn’t dry yet... is that what maybe caused mold to grow? Also, for the disposable, should I open the lid and then put it in a bucket or trash bag with the Clorox and then after a day dump the liquid out and then throw away the plates?

[SciB]

OK. So much for my fermentation theory. Yeast MAKES alcohol. That is where it comes from--not the spreader. The correct way to sterilize the spreader is to dip it into rubbing alcohol and then pass it through a flame and wait until the alcohol burns off and the wire cools down. There will be no alcohol left on the spreader.

How long did you let the plates sit after you spread them before you saw the yeast colonies? Was it the same time for the black mold? I'm trying to think of some explanation for your results.

One thing that was different between your dilution test and the light experiment was, of course, the light. When you did the yeast dilution test, where did you put the plates? When you did the light test, how long did you expose the plates to lights and how close were the plates to the lights?

If you have two agar plates left I would try the the experiment again. Make a fresh yeast dilution, spread the same amount on each plate, put one in the dark and the other under a fluorescent or LED light at the same distance from the plate and for the same length of time as you did before. Do you get black mold on the plate exposed to light but not the one held in the dark (at the same temperature if possible)?

Let me know what happens.

Sybee

[deleted-653124]

While the dilutions were growing, I let the other agar plates sit in room temperature for about a week when the dilutions were growing... after that, I did a fresh dilution and then used the rest of the agar plates. Now I’m thinking, maybe there were still alcohol on the spreader so maybe that’s why it caused molding?? Sadly, I don’t have any more plates left over...