Category Archives: D2O adaptation

D2O adaptation, Microorganisms, Water Type Effects on Organism Growth, Yeast

Day 1 Time Trials: Results

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Via figshare:

Yeast growth for adaptation to D2O: Day 1. Anthony Salvagno. figshare.
Retrieved 22:04, Sep 27, 2012 (GMT)
http://dx.doi.org/10.6084/m9.figshare.96151

In that uploaded data set, I’ve included the graph for all the specimens and one specific for the D2O growth. There was not much growth today for D2O so compared to the other two samples it’s hard to see, hence the “zoom-in” plot.

To test if there is growth, I’m leaving that sample in the incubator overnight and will give it another scan then.

D2O adaptation, Microorganisms, Water Type Effects on Organism Growth, Yeast

Yeast Growth in D2O

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After getting the starter cultures up and running and getting the time trial experiment running, there is another experiment to contend with. Since this whole project is about getting yeast to adapt to D2O, I’m going to track the growth of the yeast over long times (several days vs just one day). This experiment is going to be a challenge as YPD evaporates and the yeast settles in the test tube, but I’ll try to make it work.

Below is a spreadsheet of the yeast growth over time, starting with today’s 24 hour measurement.

Be sure to check it daily to see what is going on!

D2O adaptation, Water Type Effects on Organism Growth, Yeast

Yeast Adaption Time Trials: Setup

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Yesterday’s starter cultures finally started to grow so I can move ahead as planned. For now there isn’t much difference between these experiments and the time trials I did in May (see D2O1 and DDW1 categories). The only difference is that I’ll be comparing the growth amongst data sets over time and I’m also doing a long term growth sample. I’ll explain in my next post.

Here is my setup:

  1. Put 9ml of DI, DDW, and D2O YPD in 3 test tubes respectively (each in it’s own).
  2. Add 1ml of each yeast sample from the starter cultures.
  3. Starting at t=0 remove 400ul and put that in a semi-micro cuvette for nanodrop analysis. Do this every 60min.
  4. Incubate the cultures at 30C and 150rpm

Simple!

D2O adaptation, Water Type Effects on Organism Growth, Yeast

Starter Cultures Try 3!

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Gosh this yeast is tough to get started. It just can’t get going in liquid YPD. I finally got a few colonies to appear after 2 days on YPD plates. I inoculated a few of those colonies in some new liquid YPD (DI, DDW, and D2O) so hopefully tomorrow I’ll be cooking meth! LOL, just kidding… or am I?

I wonder if meth with D’s instead of H’s would affect the high you get… hmmmm….

D2O adaptation, Microorganisms, Water Type Effects on Organism Growth, Yeast

The SciFund Experiment: Yeast Adaptation to D2O

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It has taken me quite a while, but I’m finally at the point where I can begin the experiments that my SciFund quest funded. To refresh your memory, here is my project proposal:

Water is arguably the most important molecule in the universe. It’s a simple molecule that is composed of two hydrogen atoms and one oxygen. But did you know there are different types of water molecules? Every element has alternate forms known as isotopes, and hydrogen is no different. A common isotope of hydrogen, known as deuterium (D) which is twice the mass of hydrogren, can bond with oxygen to make heavy water (D2O).

Immediately after heavy water was first purified from naturally occurring water in the early 1930s, it was discovered that most organisms cannot survive in pure heavy water. It was also shown that increased (but not toxic) levels of heavy water significantly affect many systems in these same organisms, like fertility, metabolism, temperature regulation, and many more, all of which are essential for healthy organism function. Interestingly, on a cellular level the increased mass of heavy water may affect chemical processes. Not many studies have been performed in this area, unfortunately, because many experimenters ignore the effects of water even though it is by far the most abundant molecule in these experiments.

Because of the presence of deuterium in naturally occurring water, life may have evolved essential uses for deuterium. I plan to study the effects of heavy water on E. coli and S. cerevisiae (baker’s yeast). I will be growing cultures of these microbes in water with varying amounts of heavy water (from 0% to 99.9%) and comparing characteristics between the cultures, looking for effects in growth, development, appearance, and other physical differences.

But that isn’t specific enough to describe what I actually intend to do with the money. The full explanation of the experiment is way cooler!

Initially I’m going to continue the experiments from April and May that compared the growth of yeast grown in DI water, DDW, and D2O. Each time I did a time trial experiment I would start from scratch to show that yeast grows much slower in D2O. This time I’m going to carry over the yeast each day. Over time I hope to show that the yeast is adapting to growing in D2O and begins to develop at a similar rate to how it develops in DI water and DDW.

Once that happens I’ll be doing two experiments.

First I’ll be looking for phenotype differences between yeast grown in H2O vs yeast grown in D2O. I’ll be looking for shape, size, motility, etc of the yeast using the lab’s light microscopes.

In the second experiment, I’ll flip the script on the yeast. Once I get the yeast to adapt to D2O, I would like to determine if H2O is as harmful to D2O adapted yeast as D2O is to H2O yeast (aka natural yeast). I’ll do this by growing the D2O adapted yeast in DDW/DI water and recording the growth. No matter what happens with this experiment, the results will be interesting.

D2O adaptation, Microorganisms, Water Type Effects on Organism Growth, Yeast

Starter Cultures part 2

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Yesterday’s setup didn’t work so well. I’m not sure what prevents the yeast from growing. I’ll have to make a glycerol stock of some yeast colonies so I have a supply of always working yeast. Anyways, today I made a bunch of new starter batches following the same protocol from yesterday. Crossing my fingers.

D2O adaptation, Microorganisms, Water Type Effects on Organism Growth, Yeast

YPD setup and Starter Culture prep

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This is going to be a hyper detailed post because I’m unable to work in the lab today (soccer injury) and so Steve will be filling in. So pardon me while I write the tiniest of details about the lab so Steve can access everything I need him to:

Making YPD:

  1. Supplies:
    1. You will need 3 beakers. The beakers are stored above the autoclave and there may be more in the cabinet below and to the right of the sink. You’ll want beakers that can hold well over 100ml because when you stir you may spill some.
    2. You will also need 3 bottles with caps for long term storage of the prepared YPD. Those are also above the autoclave.
    3. While you are here, the aluminum foil is in the drawer under the autclave, you’ll need some.
    4. The hotplate stirrer is in the front of the lab near the sink and microcentrifuge.
    5. YPD broth mixture is on the wet lab bench with all the powder chemicals right behind Kiney’s computer.
    6. The scale is in the front of the lab next to the PCR machine, and there are mini weigh boats around there too (aolong with regular sized weigh boats).
    7. Scoops and scoopulas are near the sink in the back. There are two cups one is labeled for dirty scoops and the other for clean. You’ll need a clean one.
    8. Medium gloves are in the top left most drawer opposite the autoclave (and under the seed growth station).
    9. Syringes and syringe filters are in the right most lower cabinet opposite the Kiney computer. There are some really big syringes (60ml I think) and filters. Use 1 filter per squeeze.
    10. Stir bars are magnetized to the bench right about eye level near the powder chemicals. You’ll need three, and you might as well grab a magnet too so you can get the stirbars back.
    11. New bottles of DDW are kept in the desicator next to Nadia’s bench. New bottles of D2O are kept next to the powder chemicals. You will need one of each.
    12. Sigma DI bottled water is above the seed growth station. You will need 100ml of this.
    13. The autopipetter is somewhere in the lab. That thing gets around quite a bit. It is either somewhere around Nadia’s bench, somewhere in the front of the lab (maybe in it’s holder near the pipette tips), or near my bench hanging above or near the laptop. Tips are in the front of the lab next to the peeper PC.
    14. Sharpie – these things are everywhere in the lab. If you can’t find one there should be like 3 sitting on my bench.
  2. Measure the amount of DDW and D2O in each bottle and put in beakers. Cover with aluminum foil. And measure 100ml of DI water and put into the third beaker.
  3. Calculate the amount of YPD needed based on what it says on the YPD bottle. From this post here, it looks like you need 5g per 100ml of water. And the D2O should yield about 90ml of water.
  4. Add the ypd to the beakers with water (and make sure you keep track of which beaker has which water) and one by one, using the stirrer, mix until dissolved.
  5. Filter this water into the bottles. It takes kinda a while to get full dissolving so while one is mixing you can syringe and filter an already stirred water into a bottle.
  6. Label the water type.

You’ve just made YPD in different water types! Yay! Ok now to make the starter cultures.

  1. Gather supplies:
    1. yeast – in the fridge on the right side (and maybe near the bottom) is a rack of PCR tubes with yeast starters in it. It should say yeast on the caps, but it may be hard to read.
    2. test tubes – are above the autoclave in a blue test tube holder. There should be 2 holders and one should be empty. You’ll need 3 test tubes.
    3. prepared ypd – you just made this!
    4. disposable inoculation loops – these are in the drawer under the laptop. There are two colors, green and blue and each is a different size. I can’t remember which one is smaller, but you’ll need the smaller one because the larger one won’t fit in the PCR tube.
    5. ypd plates – might as well streak some yeast while we’re at it. These are on the top most part of the bench above the seed growth station. Take out one or two if you please.
    6. aluminum foil – again, since you just used this it should be accessible to you.
    7. incubator/shaker – in the chase near the hole in the lab, where the banana picture was made
  2. Put 10ml of each water type into a test tube and label it. Sharpies abound on my bench so you should be able to find one.
  3. Use the inoculating loop to inoculate the yeast in the liquid media. One loop per tube and swoosh around! Fun right?! (BTW, the ?! combo is called an interrobang and there is an interesting wikipedia article on this.)
  4. With the ypd plate, streak some cells from the starter tube onto here. And since there will be a lot of liquid left in the tube, I like to just poor the tube onto another ypd plate to ensure something starts growing. Paranoia is a bitch.
  5. Bring your new colonies to the incubator, make sure the incubator is set for 30C and about 100rpm. According to this post I set it for 125rpm because I can.

Yay! You have made fire! Now just leave it til tomorrow when hopefully I can return to do the next phase of experiments.