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

D2O Adaptation Day 41

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Today’s measurement:

  • D2O yeast (Gen 32) – 3.212 at 24h
  • D2O yeast (gen 33) – 0.675 at 0h

Since I’m still awaiting the arrival of my DDW I can’t get a starter culture of that going yet, so I’m just going to keep adapting the yeast in D2O:

  • 9ml of D2O YPD and 1ml of culture from generation 32.
  • Incubate at 30C

I also had to make a fresh batch of D2O YPD:

  1. measure D2O (92ml in this bottle)
  2. add 4.6g of YPD
  3. stir
  4. using a syringe filter the YPD (don’t autoclave)

Hopefully by tomorrow I’ll have my DDW so I can prepare for a time trial by the end of the week.

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

Day 36 Time Trials: Setup

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Yesterday I created starter cultures for today’s time trial experiment. Today’s goal is to analyze the growth of the (potentially) D2O adapted yeast and compare it to the growth of non-adapted yeast grown in D2O, 50% D2O, and DDW.

I started by measuring the growth (absorbance) overnight from 6 samples of yeast grown in:

  1. DDW (from adaptation experiments) – 3.075
  2. 50% D2O (adapted from adaptation experiments) – 3.079
  3. 99% D2O (adapted from adaptation experiments) – 2.950
  4. DDW (from glycerol stock) – 1.177
  5. 50% D2O (non-adapted from glycerol stock) – 0.556
  6. 99% D2O (non-adapted from glycerol stock) – 0.041

I created the time trial cultures as follows:

  1. 9ml of each water type in test tubes for a total of 6 samples (2 of each water type)
    • DDW
    • 50% D2O (4.5ml DDW, 4.5ml 99% D2O)
    • 99% D2O
      • because the non-adapted yeast in 99% D2O grew in such a small quantity I am just using the entire culture as a starter culture
  2. For the non-adapted samples I added 1ml of each culture to each water type, except for the 99% D2O sample which I explained above (3 samples total)
  3. For the adapted samples I added 0.4ml of each culture to each water type (3 samples total)
  4. Measure the samples hourly via the nanodrop in semi-micro cuvettes filled to 400ul.

The reason for the different amounts of culture in each sample is to get the 0h absorbance reading as similar as possible. This way it will be easier to compare growth rates. Here are the starting absorbance values:

  1. DDW (from adaptation experiments) – 0.259
  2. 50% D2O (adapted from adaptation experiments) – 0.222
  3. 99% D2O (adapted from adaptation experiments) – 0.228
  4. DDW (from glycerol stock) – 0.160
  5. 50% D2O (non-adapted from glycerol stock) – 0.059
  6. 99% D2O (non-adapted from glycerol stock) – 0.031 (remeasured)

It is interesting to note that the non-adapted yeast grown in 99% D2O struggled to grow as a starter culture, like I’ve witnessed countless times (ok maybe like 10 times). But the adapted yeast grew just as well as the DDW yeast under the same conditions.Further proof of adaptation? I think it helps the case.

Recall the difference between the sets of samples is that 3 samples were inoculated from already existing cultures and the other 3 were inoculated from glycerol stocks. Both were inoculated the same way otherwise (ie with an inoculating loop).

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

D2O Adaptation Day 36

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Results:

  • DDW yeast growth – 3.240 at 24h
  • 50% D2O yeast (gen 29) – 3.044 at 24h
  • 99% D2O yeast (gen 29) – 3.207 at 24h
  • 50% D2O yeast (gen 30) – 0.629 at 0h
  • 99% D2O yeast (gen 30) – 0.664 at 0h

And the setup for day 37:

  • 9ml of YPD of each water type (50% D2O (4.5ml of DDW, 4.5ml of D2O) and 99% D2O) with 1ml of culture from previous generation.
D2O adaptation, Microorganisms, Water Type Effects on Organism Growth, Yeast

D2O Adaptation Day 35

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Back on track here are the Day 35 results:

  • DDW Yeast – 3.111 at 24h
  • 50% D2O yeast (gen 28) – 2.969 at 24h
  • 99% D2O yeast (gen 28) – 3.007 at 24h
  • DDW Yeast – 1.192 at 0h
  • 50% D2O yeast (gen 29) – 0.606 at 0h
  • 99% D2O yeast (gen 29) – 0.806 at 0h

I set up the next day’s experiment with some added twists. I have a total of 9 samples for tomorrow, 3 samples of each water type:

  1. yeast in 99% DDW YPD
  2. yeast in 99% D2O YPD
  3. yeast in 50% D2O YPD

The first set of samples (1 of each type) is prepared as I normally prepare an experiment: 9ml of ypd with 1ml of ypd with culture.

The second set of samples (1 of each type) is 9ml of each water type with a culture inoculated from the Day 34 samples. By this I mean instead of adding 1ml I added whatever the inoculating sticks can hold and swooshed it around in the sample. The goal of this is to see how much yeast can grow in 24 hours from a small number of cells. In my original starting culture samples, the cells in D2O would grow astronomically slower than in DDW and my assumption is that if my cells are truly adapted the difference in growth won’t be so drastic.

The third set of sample (1 of each type) is 9ml of each water with a culture inoculated from glycerol stocks I made of samples from the first week of experiments. All the samples are inoculated from DDW grown yeast. The purpose of this set is so that I can do a time trial of the (potentially) adapted yeast compared to nonadapted yeast grown in D2O and with DDW yeast. And if that experiment reveals that my yeast is adapted to D2O, I would like to compare yeast grown in D2O to yeast adapted to D2O and to yeast grown in and adapted to H2O.

Exciting times ahead!

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

D2O Adaptation Day 34

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I was way out of it yesterday. I completely forgot to write up my Day 34 adaptation results! Rest assured I did the experiment and documented the results and set up the next day’s experiment, but just missed the recording function. Anyways here is what you are clamoring for:

  • 50% D2O Yeast (Gen 27) – 3.01 at 24h
  • 99% D2O Yeast (Gen 27) – 3.078 at 24h
  • 50% D2O Yeast (Gen 28) – 0.293 at 0h
  • 99% D2O Yeast (Gen 28) – 0.357 at 0h

I also set up the experiment for Day 35 and added a special guest: DDW YPD (1.362 at 0h), mostly because I had the time trial experiment from the day before and I wanted to compare another 24 hour growth to DDW adapted yeast. So the day 35 results will feature 3 samples:

  • Yeast grown in 99% DDW
  • Yeast grown in 50% D2O
  • Yeast grown in 99% D2O
D2O adaptation, Microorganisms, Water Type Effects on Organism Growth, Yeast

D2O Yeast Adaptation Day 33: Time Trials Setup

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In addition to the Day 33 daily adaptation I’m going to run an hourly experiment to see if the yeast has adapted or evolved or changed. I’ll be looking for a growth rate that is faster than in previous trials, and more comparable to yeast growth in DDW YPD. Here is my setup:

  1. Prepare the YPD in test tubes, I’m doing final volumes of 10ml:
    • 95% DDW – 9ml of DDW YPD
    • 35% D2O – 6ml of DDW YPD, 3ml of D2O YPD
    • 50% D2O – 4.5ml of DDW YPD, 4.5ml of D2O YPD
    • 65% D2O – 3ml of DDW YPD, 6ml of D2O YPD
    • 99% D2O – 9ml of D2O YPD
  2. Add yeast:
    • add 1ml of yeast grown in 50% D2O YPD to all test tubes except the 99% D2O YPD
    • add 1ml of yeast grown in 99% D2O YPD to the 99% D2O YPD test tube
  3. Incubate at 30C
  4. Measure in Nanodrop hourly starting at t=0. The starting absorbance values are:
    • 95% DDW – 0.511
    • 35% D2O – 0.512
    • 50% D2O – 0.505
    • 65% D2O – 0.634
    • 99% D2O – 0.439
D2O adaptation, Microorganisms, Water Type Effects on Organism Growth, Yeast

D2O Adaptation Day 33

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Unbeknownst to most of the world, I’ve had a secret agent specially trained to carry out my experiments while I’ve been away. Before I left for Seattle, I trained my lab colleague Nadia to do the 24 hour yeast adaptation experiments. She’s been keeping the experiment up to date and posting the results in the Google Doc I set up back on Day 1. THANK YOU NADIA!!! You can see the updates at that link or just read down:

This morning is my first day in the lab since my departure and I’m picking up right where I left off, the 27th Generation of yeast in the table above are my first recorded values based on Nadia’s efforts.

Here are the results in typical fashion:

  • 50% yeast (Gen 26) – 2.934 at 24h
  • 99% (gen 26) – 2.862 at 24h
  • 50% (gen 27) – 0.536 at 24h
  • 99% (gen 27) – 0.442 at 24h

The experiment was setup in typical fashion:

  1. 9ml of 50% mix of DDW YPD and D2O YPD; 9ml of 99% D2O YPD
  2. 1ml of 50% DDW YPD yeast added to 9ml of 50% D2O YPD; 1ml of 99% D2O yeast added to the 9ml of 99% D2O YPD
  3. put in incubator at 30C
D2O adaptation, Microorganisms, Water Type Effects on Organism Growth, Yeast

D2O Adaptation Day 13

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I am leaving town for the next 2.5 weeks. So in my absence, Nadia is going to be taking over the experiment. I’ve trained her in the art of yeast inoculation and culture growing, and the science of nanodropping. Now she is ready to carry the burden of mentoring millions of little beings and all that entails.

Today we made the 7th generation of cells via the usual method of inoculating 1ml of the previous generation of cells in 9ml of new batches for 99% D2O and 50% D2O (which is an equal mixture of DDW and D2O). Here are the cell measurements for today:

  • 50% D2O (6th Gen) – 3.131 at 24h
  • 50% D2O (7th Gen) – 0.970 at 0h
  • 99% D2O (6th Gen) – 2.503 at 24h
  • 99% D2O (7th Gen) -0.331 at 0h

 

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

D2O Adaptation Day 12

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So the Balloon Fiesta was this weekend and I didn’t get an opportunity to get in lab. But it didn’t much matter because the results I got Thursday and Friday of last week were not consistent/reliable. Out of that frustration I did an ONS no no and didn’t write any posts. That and I had no time because I was working on my SACNAS poster. Regardless, I hold myself accountable and make no excuses (except for the ones I mentioned above).

I’ll post my results later this afternoon from those days. I thought there were issues with the nanodrop, turns out evaporation from my blank cuvette was the culprit. A while ago I measured an appropriate volume to fill my cuvettes so I don’t waste too much yeast. I discovered that 400ul worked well. Thus I filled all my cuvettes to that volume. Additionally the blank cuvette gets for extended periods of time. After about a week or so the volume reduces to around 300ul which I discovered the nanodrop has a difficult time reading at that volume. So therein lies my issues from last week. Today I made a new blank cuvette to take readings from.

On Friday I innoculated some culture from the Thursday (Day 8) samples. Today I measured the results of that growth (72h later) and innoculated new cultures. Here are the measurements:

  • 50% D2O (5th Gen) – 3.145 at 72h
  • 99% D2O (5th Gen) – 2.958 at 72h
  • 50% D2O (6th Gen) – 1.101 at 0h
  • 99% D2O (6th Gen) – 0.669 at 0h

As I said, I’ll publish the failed results later this afternoon. I have a presentation on Open Notebook Science to give at 1:30 for the PNMGC. Wish me luck!

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

D2O Adaptation Day 8

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I setup new cultures for 99% D2O and 50% D2O making this the 4th generation of yeast in D2O. Yesterday I ran out of D2O YPD and had to deal with a smaller batch for the 50% D2O yeast. Today I made some new 99% D2O YPD and inoculated 1ml of each yeast-water type in 9ml of each YPD type. Then I took some measurements.

Today’s Absorption measurements are:

  • D2O (Generation 3) – 2.351 at 24h
  • 50% D2O (gen 3) – 2.65 at 24 h
  • D2O (gen 4) – 0.041 at 0h
  • 50% D2O (gen 4) – 0.419 at 0h

It was interesting to find that the 24h batch of 99% D2O yeast had an absorbance of 2.351 for 2 reasons; 1) that’s a good amount for 24h from what I’ve been observing and 2) because my 1:10 dilution resulted in a much different absorption than 50% D2O yeast (note that they started at a similar value).

When I tried to analyze by eye it was apparent there was a difference in absorbance but it definitely didn’t look 10x less clear. But I did notice that the cells in 50% D2O seemed to be pretty uniformly “dissolved” (I don’t really know what to call that). Meanwhile, the cells in 99% D2O seemed to form larger clumps and the average “particle” size (I’m guessing they are colony clumps) was larger than the clumps in 50% D2O.

I’ll have to analyze this under a microscope and report observations, something that will have to wait until tomorrow or next week.