Category Archives: D2O adaptation

D2O Adaptation Day 51

No results today :( I accidentally disposed of the samples from yesterday after inoculating the samples for today. And there is no data today because the inoculation used an inoculating loop, so the absorbance wouldn’t be noticeable. But the setup is 10ml of YPD (3 samples, two DDW YPD and one D2O YPD) with an inoculation (using the loop) from the previous generation. The reason for the 3 samples is because I want to see if after a few generations, the D2O adapted yeast revert back to the wt yeast, so in addition to a sample of DDW yeast and D2O yeast, I’m doing the D2O adapted yeast in DDW. We’ll see what happens.

D2O Adapted Yeast Colony Morphology

via figshare:

D2O Adapted Yeast Colony Morphology. Anthony Salvagno. figshare.
Retrieved 21:36, Nov 15, 2012 (GMT)
http://dx.doi.org/10.6084/m9.figshare.97601

These images and the final comparison image (and ALL the original raw files from the camera) are available via the figshare link above. These images were also taken after this post, ie the conditions are the same.

Images were acquired at 10x magnification and the scale is 1um/px. The largest image is 1959×1925 px (the larger D2O yeast on normal agar) and the smallest image is 1462x1749px (the D2O yeast on D2O).

As you can see there is quite the interesting phenomenon here. The interesting thing is that there is something morphologically different about the D2O adapted yeast. The reason for that thinking is because the yeast colony retains its brainy shape when placed on normal media, as compared to the wt yeast on the normal agar (smooth circle). Before I make any bold claims about what may be happening here, I need to read some papers about yeast morphology.

Also if anyone wants a glycerol stock of this strain of yeast to run some tests on it, I’d be happy to send it to you. I personally don’t have the means to perform any advanced tests so any experimentation is welcomed!

I’d love to hear what you all think!

D2O Adaptation Day 50: D2O Yeast Time Trials – Setup

After some really interesting results from some microscope observation yesterday (to be uploaded shortly), I’m doing some different time trials today. The setup is the same, but today I’m switching the growth media. D2O yeast will be grown in DDW YPD and wt yeast will be grown in D2O. Here’s the setup:

  1. Add 9ml of YPD to 3 test tubes:
    • D2O YPD
    • D2O YPD
    • DDW YPD
  2. Add 1ml of culture to each test tube:
    • 1ml of D2O yeast to D2O YPD
    • 1ml of D2O yeast to DDW YPD
    • 1ml of DDW yeast to D2O YPD
  3. Incubate at 30C and shake
  4. Measure in nanodrop every hour

Yesterday I noticed that the D2O yeast grew more on normal solid media than DDW yeast did (and also more than D2O yeast on D2O solid media). I wanted to get an hourly analysis of this growth. Unfortunately I didn’t have enough made DDW YPD and don’t have time to make a new batch because of timing of the measurements with this afternoon’s activities. So for now I’ll compare this to every other DDW growth experiment. And next week run the trial again with DDW yeast growing in DDW.

I also want to do some microscope analysis of the yeast in the different media because of the observations I made last night. Like I said that data will be up shortly.

 

D2O Adaptation Day 50

Wow, 50 days of incubating yeast! Results:

  • D2O yeast gen 39 – 3.065 at 24h
  • D2O yeast gen 40 – 0.739 at 0h
  • DDW yeast – 2.971 at 24h
  • DDW yeast (next gen) – 1.175

And the setup for gen 40 was 9ml of D2O YPD with 1ml of culture from generation 39. Setup for the DDW yeast was 9ml of DDW YPD with 1ml of DDW yeast culture.

 

Basic Observations of the D2O Adapted Yeast compared to WT Yeast

After having been adapting wild type yeast to D2O for the past 49 days, I’ve been making some basic observations regarding their growth. Initially wild type (wt) yeast exhibits slowed growth in D2O. Usually the first 24 hours there is relatively little growth and they yeast takes about 72 hours to reach the maximum growth (cell number) as yeast grown in DI water or DDW. But there are also some differences between adapted yeast and wt yeast. I’ve yet to quantify most of these but I’ve noticed some things:

Yeast grown in liquid YPD media. On the left is D2O adapted yeast grown in D2O YPD. On the right is wild type yeast grown in DDW. Yeast was grown at 30C and shaken (not stirred) for 24 hours.

The first of which is the aggregation of the cells in the mediums. D2O adapted yeast (grown in D2O) seem to be less prone to aggregation/clumping. In the image above, there is a pretty high cell count (observed by the cloudy appearance of the medium) in the test tube on the left. There is some settling of cells (which would indicated the cells are clumping), but compared to the test tube on the right (wt yeast grown in DDW) that’s really not a problem. It is important to note that when both samples are resuspended their absorbance are almost identical (3.256 for D2O yeast vs 3.249 for DDW yeast).

We’ve also noticed a difference in color between the cells. It’s not yet sure whether the cells are in face a different color or if this is related to the aggregation characteristic.

Obviously tests will need to be done, and I’m going to get on it. Most of which will be microscope analysis, but hopefully I can speak with some friends over at Cancer Research to perform some advanced tests on the cells.

I’ve also been trying to determine if my yeast cells are officially D2O adapted, and my latest experiment may finally put my own skepticism to rest.

Yeast grown on YPD agar made with 99.9% D2O. On the left is wild type yeast, and on the right is yeast adapted to D2O. The yeast was incubated for 24 hours at 30C.

After 24 hours of incubation, wt yeast grown in D2O greatly struggles (left sample) while my potentially adapted yeast grows like crazy. That looks super conclusive, especially because the same culture of wt yeast grows really well on normal YPD agar plates:

Yeast grown on YPD agar plates (commercial, made with DI water). On the left is wild type yeast and on the right is D2O adapted yeast. The yeast was incubated at 30C for 24 hours.

But interestingly enough, the D2O adapted yeast appears to grow on H2O media much better than wt yeast. If anything I would have assumed the same growth, but the results above are ridiculous. Tempering excitement for a minute, more tests will need to be done, and tomorrow I’ll have some (hopefully) more conclusive data.

With that said, I can’t help but be excited for the future of the project. Let’s see what tomorrow brings!

D2O Adaptation Day 49

Results:

  • D2O Yeast gen 38 – 3.256 at 24h
  • D2O yeast gen 39 – 0.763 at 0h
  • DDW yeast – 3.249 at 24h
  • DDW yeast (next gen) – 1.026 at 0h

Tomorrow I’ll be doing another time trial experiment, now that the issues of the lab have been put behind us. The setup for tomorrow is the same as usual: 9ml of YPD with 1ml of culture from the previous generation.