Need Help ASAP

[CassL07]

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

[CassL07]

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?

[CassL07]

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

[CassL07]

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...

[CassL07]

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

[CassL07]

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

[CassL07]

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

[CassL07]

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

[CassL07]

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...