Yeast Growth Issues

[ericjang]

Hi, to whom may concern

I am doing a project on raising yeast cells and exposing them to UV radiation, followed by analysis of their changes in DNA patterns. I am having a lot of technical difficulties raising these yeast cells though. I am in possession of petri dishes, YPD agar, and baker's yeast. I have tried various setup procedures, but none of them seem to work right. My current setup consists of a thermostat-regulated aquarium, with a sealed plastic box surrounded by a bath of warm water. Inside the box are petri dishes of upside-down agar plates (to avoid condensation dripping). I turn the dishes upside down after the yeast has soaked into the agar. However, over the past few days I have not seen any growth in the yeast. Does anybody have any ideas as to how I can modify my experiment procedure?

Any help would be greatly appreciated.

Thank you so much!
Eric Jang

[adance]

Hi Eric,

Let's see if we can work out what's going on here.

What temperature is you aquarium/incubator at? (By the way, that's a creative technique you're using--not one I've come across before!) Do you have a thermometer inside the box so you know what the yeast are exposed to? You probably want something like 30 degrees Celsius. Lower temperatures are OK too, but will just take a bit longer to grow.

Where did you get your starter culture from? Was it dry or a liquid? One important factor to consider is innoculating your cultures from steadily growing cells. If the cells were already at the end of their growth cycle--called stationary phase--it may take longer for them to get growing again.

Here's a link to a project similar to what you're describing: http://www.scienceteacherprogram.org/bi ... hah04.html
It sounds like you already have most of the information included in here, but I include it in case there's anything that would be new to you.

[ericjang]

Hi Amber,

Thank you so much for your quick reply!

I am setting the aquarium to about 29 C(85 F), so I am pretty sure the optimum temperature is not the problem.

I grew my yeast cells directly from a store-bought package (standard active, dry yeast). I submerged about a teaspoon of yeast cells in a petri dish of lukewarm water and added about a teaspoon of ordinary table sugar. After about 3 minutes, I transfered some of the liquid broth into a YPD agar dish, and then placed the dish into the environment. I didn't know about the stationary phase of yeast, but I do know that yeast undergoes cryptobiosis in the lack of water, so it may take a few days for them to start multiplying. However, after 5 days of wait, I so no change at all in the yeast. I also just recently found out about turning the agar dish upside down to prevent condensation from forming, so I have yet to see those results.

Thank you so much for the link! It was very helpful to me to know that I have all the materials I need, in theory, but I think what may be going wrong is that I am letting the agar sit for a full day before turning upside down. Also, i think I am adding too high of a concentration into the agar plate, as much as covering the entire dish in a "pool" of yeast+sugar medium. However, for the purpose of my experiment, I am not particularly concerned with the counting of colonies, but is it okay if I just use a 10^-1 yeast solution for my experiment?

Thank you for your help! I will let you know if I run into more difficulties

Eric Jang

[adance]

Hi Eric,

Sounds like you're on the right track.

Your culture plan sounds about right. You should spread 50-100 microliters (microliter=1/1000 milliliters) of liquid on the plates. Usually one would use a sterile tool, like a bent glass rod dipped in ethanol and flamed, to spread the culture evenly. Then you'll want to wait 5-30 minutes for the liquid to absorb. It doesn't need to be a big pool. And yes,you're right, you want to turn the plates over, otherwise drops of condensation can interfere with a nice lawn.

Without knowing the original concentration of your yeast solution, I couldn't even guess if a 1:10 dilution is correct. Scientists often try a few dilutions to see which grows best for their purposes.

I would recommend getting a thermometer to put into the tub with the plates. I don't know how well heat transfers from water, through plastic, to air, so I'm not positive the temperature of your water will be the same as the yeast are experiencing.

Make sure to also include a control plate--one that you don't add any yeast to. This will (1) give you something to compare to, so you can see how the yeast grow and (2) allow you to confirm your plates aren't contaminated with anything else that grows in the incubator.

Good luck with the next round. Sometimes it takes a few tries to get an experimental protocol to work properly.

[ericjang]

Hi Amber,

I managed to solve the yeast growth issue over the past few days! Thank you so much for your help! I also managed to get some more information from a very experienced lab specialist from Schmahl Science Workshop.
For the record, I would like to post my procedure for anybody who may run into a similar problem in the future.

I acquired some YPD broth in addition to the YPD agar. I discovered that while sugar water can serve as an adequate food source for the yeast, YPD broth contains the nutrients/amino acids needed to actually allow the yeast to divide. Also, the yeast dish I was working with earlier actually was still alive; however, the YPD agar was completely depleted because I spread too much yeast on in the first place.

Here is my procedure for growing yeast

- Scrape a small sample of yeast from the original "lawn" and swirl it around in a flask of 50 ml of YPD broth.
- Allow the yeast to grow in that solution for 18-24 hours in the correct temperature environment.
-The next day, pipette 50 microliters of the solution into agar plates. Wait 5 or so minutes, then turn the plates upside down.
-Let the plates sit for another 24 hours in the correct environment
- The next day, the yeast should have resulted in significant growth.


I also ran into another issue with part of my procedure: my experiment involves extracting pure DNA from the yeast, but I am unable to get ahold of the complex reagents and equipment needed to do so. I have the following materials:

Meat tenderizer
Detergent
Salt
Distilled Water
Ethanol
Table-top centrifuge

My current procedure is a modified version of a spooling lab, but with the centrifuge, I should be able to purify the DNA further. I am concerned, however, that some parts of my experiment are unnecessary (e.g. mashing the cells with a plastic bag).I have copied my extraction procedure below:

1.) Remove 5 ml of yeast from each dish by scraping off a thin layer of cells from each dish with the wire scraper. Place these in separate plastic bags. (For the first extraction, use 5 ml of yeast directly from the liquid broth).
2.) For each of the 12 batches, Add 5 ml detergent/water solution.
3.) Mash each bag thoroughly until mixture is liquefied.
4.) Pour mixture into a beaker, add 2 ml of meat tenderizer/water solution, and stir to mix. Add one milligram of salt and continue mixing.
5.) Place 1.5 ml of mixture into a microfuge tube.
6.) Spin the mixture in the microfuge for 2 minutes, with a tube of water on the opposite end as a counterbalance. Make sure to keep the hinge of the microfuge facing away from the rotor, as standard of procedure.
7.) Transfer the lighter layer of DNA into a new tube, and discard the protein precipitate.
8.) Pour about 1.5 ml of ice-cold ethanol carefully down the side of the tube to form a layer. It should double the volume of the solution. Invert a couple times to mix the solution.
9.) Spin the microfuge tube in the microfuge for another 2 minutes so that the DNA precipitates at the bottom of the tube.
10.) Siphon off the excess liquid

I am not sure if you are experienced with DNA extraction protocols, but I was wondering if you knew anybody who can help me out with this part of my experiment?

Thank you so much!
Eric Jang

[adance]

That's great that your yeast are growing. Thanks for posting the protocol!

I have extracted lots of DNA (from bacteria)--but always using professional kits--so am not perfectly familiar with the protocol you describe. It should work, though, it's definitely possible to isolate DNA with simple materials. However here are my thoughts:

1) Are you planning to get 5 ml of solid yeast off of plates? That would take a LOT of plates (and be way more cells that you probably need to get DNA). Maybe you mean scrape the yeast into 5 ml of liquid? Are you going to be doing gel electrophoresis with your isolated DNA? Then a plate's worth is PLENTY of DNA.

3) What you're doing here is lysing the yeast--popping them open so the DNA (and protein and other yeast "guts") can spill out. The detergent is a amphiphilic molecule--it has parts that bind lipids (which form the yeast's membrane) and parts that bind water. So it gets into the membrane, disrupts it, and pops the cells. Mooshing cells in a bag is not a procedure I've seen before. (I'm used to flipping the tube upside down a few times and letting it sit for about 5 min.) I found another protocol that suggests putting it in a blender (http://www.accessexcellence.org/AE/ATG/ ... yeast1.php). You should see that your solution changes and gets all sticky and goopy.

4) The tenderizer contains proteases (http://www.wisegeek.com/how-do-meat-ten ... s-work.htm)--enzymes that chop up protein. This will get the protein out of the way so you can get your DNA.

6) So at this point the protein is solid (precipitate), but the DNA is still dissolved. You will take out the DNA-containing liquid, leaving the protein pellet in the tube.

The ethanol will cause the DNA to precipitate--solidify out of the solution.

10) So now your DNA is in the pellet at the bottom of the tube. Sometimes scientists repeat steps 8-10 to get the DNA more pure. You will want to resuspend your DNA in liquid or buffer for use in your experiments, I imagine. But first you want to remove all the ethanol. You can do this with a vacuum, or, more simply, just leave the tubes open in a warm, dry place until all the ethanol evaporates and you just have a dry DNA pellet left.

The best thing to do is try the protocol with just one sample first to make sure it works. Then do your big experiment.

Good luck!

[ericjang]

Hi Amber,

Thank you so much for your advice! I will address your points one by one to avoid confusion.

1.) Yes, I was suspecting that 5 ml of solid might be too overly abundant: suspending some cells in a liquid solution first might be a good idea. What kind of liquid should I use? Water or the YPD growth broth?

3.) Thank you for the suggestion! I was a little concerned about mooshing the cells in a bag because that procedure does not seem very common to me.

10.) What liquid/buffer should I use to suspend the yeast cells? If I am preparing a restriction digest for gel electrophoresis, would I need to still suspend the pellet, or can I add it directly to the other reagents? (see below)

Add restriction enzymes to DNA samples to break up DNA into fragments. (2µl 10x Buffer, 13µl H2O, 4µl DNA, 1µl Enzyme in an eppendorf tube). For mass-digestion, prepare one large batch of buffer, water, and enzyme by mixing ingredients together and mixing by using the pipette to suck up and down (when you put the enzyme in after the first two ingredients). Then, distribute the solutions into tubes and with new tips, add the DNA on top of that. Incubate in 37°C water for about an hour.

Thank you for your fast reply!

Eric Jang

[adance]

Hi Eric,

Happy to help!

1) Hmm. Depends on what reagents you have access to. Here's a protocol with a recipe for "lysis buffer:" http://www.musc.edu/BCMB/ceramide/protocols/0002.html Triton and SDS are detergents, which you're taking care of with your dish soap, so won't be necessary. Tris is what keeps the liquid at the right pH. But the protocol we were looking at yesterday (the one that suggested the blender) just suggests dissolving the yeast in water. So I wouldn't be afraid to just use water at this stage.

3) Indeed! I'd like to see someone write a scientific paper including the line, "The cells were placed in a plastic baggie and squished around for a while." Doesn't have quite a "scientific" ring to it.

10) You could definitely resuspend your dried DNA pellet in the restriction enzyme mix. But, that's the route for when you're quite confident of your protocol. Otherwise, suppose something goes wrong with your restriction digest and you've used up all your DNA--now you're stuck growing up the yeast again. So it might be wise to save some DNA at this stage. You can just resuspend it in 10-100 ul distilled water.

I am not sure how much DNA you'll get, so keep in mind your pellet may be a big white streak, or it may be a near-invisible pucker at the bottom of the microfuge tube. Don't worry--a little DNA goes a long way.

Happy DNA making!

[adance]

I was just thinking about your blender vs baggie issue further.

There are, potentially, reasons not to use a blender.

1) If you put dish soap into a blender--you are going to get a lot of suds.
2) The blades could cut, or shear, the DNA. DNA is very long fragile strands that could get cut up. I'm not sure if this would affect your experiment or not.

You might want to try both methods and see which works best. As an alternative to the baggie, since it feels so unscientific, you could put the solution in a clean container with a lid and gently turn it upside down and rightside up several times.

[ericjang]

Hi Amber,

Thanks for the info! I have decided to post a separate thread on yeast DNA extraction, as this the original problem of this thread has been resolved.

I will post the information we have discussed so far on the thread.

Thanks!
Eric